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Posts Tagged ‘Doctor of Philosophy’


Hebrew University’s Professor Haim Sompolinksy and Columbia University Prof. Larry Abbott Win First New $100,000 Mathematical Neuroscience Prize

 
Curator: Aviva Lev-Ari, PhD, RN

Professor Haim Sompolinsky of The Hebrew University of Jerusalem has been awarded the 1st Annual Mathematical Neuroscience Prize by Israel Brain Technologies (IBT), a non-profit organization committed to advancing Israel’s neurotechnology industry and establishing the country as a global hub of brain technology innovation.

Professor Sompolinsky, who pioneered the field of computational neuroscience, is the William N. Skirball Professor of Neuroscience at The Hebrew University’s Edmond and Lily Safra Center for Brain Sciences (ELSC).

ELSC is one of the most ambitious neuroscience centers in the world, providing a multi-disciplinary environment where theorists, computer scientists, cognitive psychologists and biologists collaborate to revolutionize brain science.

IBT’s $100,000 Mathematical Neuroscience Prize, awarded at the 1st annual BrainTech Israel 2013 Conference in Tel Aviv, honors researchers worldwide who have significantly advanced our understanding of the neural mechanisms of perception, behavior and thought through the application of mathematical analysis and theoretical modeling.

Professor Sompolinsky specializes in building mathematical models that describe the collective behavior and the informational processing in neural circuits in the brain. The principles that emerge from Professor Sompolinsky’s work contribute to our understanding of the system-wide failures that take place in brain diseases, from epilepsy to psychiatric disorders.

According to Sompolinsky, “Computational neuroscience is a vibrant and ambitious field that uses mathematical theories and models to cope with the most daunting challenges — from answering fundamental questions about the brain and its relation to the mind to answering questions posed by the quest to heal the brain’s debilitating diseases.”

Also winning a $100,000 Mathematical Neuroscience Prize was Professor Larry Abbott, Bloor Professor of Theoretical Neuroscience at Columbia University, who developed models ranging from the level of neurons and synapses to large-scale networks, and showed how plasticity mechanisms that change the properties of neural circuits can maintain their proper operation and allow them to change during the learning process.

Nobel Laureate Professor Bert Sakmann, inaugural Scientific Director of the Max Planck Florida Institute, presented the awards at the conference. “This prize honors the founders of mathematical neuroscience, and is a milestone because it gives due recognition to this field,” said Sakmann.

“This prize recognizes leaders in the important field of mathematical neuroscience, whose advances support our ultimate quest to find new solutions for the betterment of all humankind,” said Miri Polachek, Executive Director of IBT.

In the future, the Prize Selection Committee will consist of previous prize winners, including Sompolinsky and Abbott.

IBT’s BrainTech Israel 2013 Conference is exploring developments in brain technology and their commercialization through a “meeting of the minds” among government leaders, entrepreneurs, researchers, leading companies and investors from Israel and around the world.

Inspired by the vision of Israeli President Shimon Peres and building on Israel’s position as a global technology powerhouse, IBT aims to make Israel both the “Startup Nation” and the “Brain Nation.”  IBT is also focused on increasing collaboration between the Israeli neurotechnology ecosystem and its counterparts around the world. IBT is led by a team of technology entrepreneurs and life science professionals and is advised by a panel of renowned academic, industry and public sector representatives including two Nobel Prize Laureates.

 SOURCE

From: AFHU <AFHU@mail.vresp.com>
Date: Tue, 15 Oct 2013 18:04:16 +0000
To: <avivalev-ari@alum.berkeley.edu>

Download CV

please use the above link to download a PDF copy of my CV

Professor of Physics, Racah Institute of Physics
William N. Skirball Professor of Neuroscience
The Interdisciplinary Center for Neural Computation
The Edmond and Lily Safra Center for Brain Sciences
The Hebrew University
Jerusalem, 91904, Israel
(t) 972-2-658-4563; (f) 972-2-658-4440
haim@fiz.huji.ac.il

Personal Information
Born:  Copenhagen, Denmark, 1949
Israeli citizen: 1951
Married with five children

RESEARCH:

Sompolinsky’s research goal is to uncover the fundamental principles of the organization, the dynamics and the function of the brain, viewing the brain through multiscale lenses, spanning the molecular, the cellular, and the circuit levels. To achieve this goal, Sompolinsky has developed new theoretical approaches to computational neuroscience based on the principles and methods of statistical physics, and physics of dynamical and stochastic systems. This new field, Neurophysics, builds in part on Sompolinsky’s earlier work on critical phenomena, random systems, spin glasses, and chaos. His research areas cover theoretical and computational investigations of cortical dynamics, sensory processing, motor control, neuronal population coding, long and short-term memory, and neural learning. The highlights of his research include theories and models of local cortical circuits, visual cortex, associative memory, statistical mechanics of learning, chaos and excitation-inhibition balance in neuronal networks, principles of neural population codes, statistical mechanics of compressed sensing and sparse coding in neuronal systems, and the Tempotron model of spike time based neural learning. He also studies the neuronal mechanisms of volition and the impact of physics and neuroscience on the foundations of human freedom and agency.

http://elsc.huji.ac.il/sompolinsky/biocv

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Israeli, US Profs win 1st annual Mathematical Neuroscience Prize

$100,000 prizes awarded for outstanding work in human brain modeling at BrainTech Israel 2013 Conference in Tel Aviv.

From left, Nobel Laureate Prof. Bert Sakmann; Hebrew University of Jerusalem Prof. Haim Sompolinsky; Columbia University Prof. Larry Abbott; and Dr. Rafi Gidron, founder and chairman of Israel Brain Technologies, at BrainTech Israel 2013. Sompolinsky won IBT’s inaugural Mathematical Neuroscience Prize.From left, Nobel Laureate Prof. Bert Sakmann; Hebrew University of Jerusalem Prof. Haim Sompolinsky; Columbia University Prof. Larry Abbott; and Dr. Rafi Gidron, founder and chairman of Israel Brain Technologies, at BrainTech Israel 2013. Sompolinsky won IBT’s inaugural Mathematical Neuroscience Prize.

Hebrew University of Jerusalem Prof. Haim Sompolinsky and Columbia University Prof. Larry Abbott are the winners of the 1st Annual Mathematical Neuroscience Prize by Israel Brain Technologies (IBT). The two $100,000 prizes were awarded at the 1st annual BrainTech Israel 2013 Conference in Tel Aviv.

Prof. Haim Sompolinsky (photo: Hebrew University)

IBT’s Mathematical Neuroscience Prize honors researchers worldwide who have significantly advanced our understanding of the neural mechanisms of perception, behavior and thought through the application of mathematical analysis and theoretical modeling.

Prof. Sompolinsky is considered a pioneer in the field of computational neuroscience. He specializes in building mathematical models that describe the collective behavior and the informational processing in neural circuits in the brain. His work helps researchers understand the system-wide failures that take place in brain diseases, from epilepsy to psychiatric disorders.

“Computational neuroscience is a vibrant and ambitious field that uses mathematical theories and models to cope with the most daunting challenges – from answering fundamental questions about the brain and its relation to the mind to answering questions posed by the quest to heal the brain’s debilitating diseases,” said Sompolinsky.

Meanwhile, Prof. Abbott won for showing how plasticity mechanisms that change the properties of neural circuits can maintain their proper operation and allow them to change during the learning process.

Inspired by the vision of Israeli President Shimon Peres, IBT was set up to advance Israel’s neurotechnology industry and establish the country as a global hub of brain technology innovation.

“This prize recognizes leaders in the important field of mathematical neuroscience, whose advances support our ultimate quest to find new solutions for the betterment of all humankind,” said Miri Polachek, Executive Director of IBT.

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The immune response

The immune response (Photo credit: Wikipedia)

Confined Indolamine 2, 3 dioxygenase (IDO) Controls the Hemeostasis of Immune Responses for Good and Bad

Curator: Demet Sag, PhD, CRA, GCP

ABSTRACT:

The immune response mechanism is the holy grail of the human defense system for health.   IDO, indolamine 2, 3-dioxygenase, is a key gene for homeostasis of immune responses and producing an enzyme catabolizing the first rate-limiting step in tryptophan degradation metabolism. The hemostasis of immune system is complicated.  In this review we will discuss properties of IDO such as basic molecular genetics, biochemistry and genesis. IDO belongs to globin gene family to carry oxygen and heme.

The main function and genesis of IDO comes from the immune responses during host-microbial invasion and choice between tolerance and immunegenity. In addition IDO has a role in vascular tone as well.  In human there are three kinds of IDOs, which are IDO1, IDO2, and TDO, with distinguished mechanisms and expression profiles. , IDO mechanism includes three distinguished pathways: enzymatic acts through IFNgamma, non-enzymatic acts through TGFbeta-IFNalpha/IFNbeta and moonlighting acts through AhR/Kyn. These mechanisms and their relation with various health and disease will be presented. Overall our purpose is to find a method to manipulate IDO to correct/fix/modulate immune responses for clinical applications.

Our focus is on cancer prevention with DCvax.  The first study proving the connection between IDO and immune response came from, a very natural event, a protection of pregnancy in human. This led to discover that high IDO expression is a common factor in cancer tumors. Thus, attention promoted investigations on IDO’s role in various disease states, immune disorders, transplantation, inflammation, women health, mood disorders.  Many approaches, vaccines and adjuvants are underway to find new immunotherapies by combining the power of DCs in immune response regulation and specific direction of siRNA.  As a result, with this unique qualities of IDO, DCs and siRNA, we orchestrated a novel intervention for immunomodulation of IDO by inhibiting with small interference RNA, called siRNA-IDO-DCvax.  Proven that our DCvax created a delay and regression of tumor growth without changing the natural structure and characterization of DCs in melanoma and breast cancers in vivo.

_____________________________________________________________________________

IDO is a key homeostatic regulator and confined in immune system mechanism for the balance between tolerance and immunity.  This gene encodes indoleamine 2, 3-dioxygenase (IDO) – a heme enzyme (EC=1.13.11.52) that catalyzes the first rate-limiting step in tryptophan catabolism to N-formyl-kynurenine and acts on multiple tryptophan substrates including D-tryptophan, L-tryptophan, 5-hydroxy-tryptophan, tryptamine, and serotonin (1; 2; 3; 4).

The basic genetic information describes indoleamine 2, 3-dioxygenase 1 (IDO1, IDO, INDO) as an enzyme located at Chromosome 8p12-p11 (5; 6) that active at the first step of the Tryptophan catabolism.    The cloned gene structure showed that IDO contains 10 exons ad 9 introns (7; 8) producing 9 transcripts.  After alternative splicing only five of the transcripts encode a protein but the other four does not make protein products, three of transcripts retain intron and one of them create a nonsense code (7).  Based on IDO related studies 15 phenotypes of IDO is identified, of which, twelve in cancer tumor models of lung, kidney, endometrium, intestine, two in nervous system, and one HGMD- deletion.

The specific cellular location of IDO is in cytosol, smooth muscle contractile fibers and stereocilium bundle. The expression specificity shows that IDO is present very widely in all cell types but there is an elevation of expression in placenta, pancreas, pancreas islets, including dendritic cells (DCs) according to gene atlas of transcriptome (9).  Expression of IDO is common in antigen presenting cells (APCs), monocytes (MO), macrophages (MQs), DCs, T-cells, and some B-cells. IDO present in APCs (10; 11), due to magnitude of role play hierarchy and level of expression DCs are the better choice but including MOs during establishment of three DC cell subset, CD14+CD25+, CD14++CD25+ and CD14+CD25++ may increase the longevity and efficacy of the interventions.

IDO is strictly regulated and confined to immune system with diverse functions based on either positive or negative stimulations. The positive stimulations are T cell tolerance induction, apoptotic process, and chronic inflammatory response, type 2 immune response, interleukin-12 production (12).  The negative stimulations are interleukin-10 production, activated T cell proliferation, T cell apoptotic process.  Furthermore, there are more functions allocating fetus during female pregnancy; changing behavior, responding to lipopolysaccharide or multicellular organismal response to stress possible due to degradation of tryptophan, kynurenic acid biosynthetic process, cellular nitrogen compound metabolic process, small molecule metabolic process, producing kynurenine process (13; 14; 15).   IDO plays a role in a variety of pathophysiological processes such as antimicrobial and antitumor defense, neuropathology, immunoregulation, and antioxidant activity (16; 17; 18; 19).

Active site of IDO–PI complex. (A) Stereoview of the residues around the heme of IDO viewed from the side of heme plane. The proximal ligand H346 is H-bonded to wa1. The 6-propionate of the heme contacts with wa2 and R343 Nε. The wa2 is H-bonded to wa1, L388 O, and 6-propionate. Mutations of F226, F227, and R231 do not lose the substrate affinity but produce the inactive enzyme. Two CHES molecules are bound in the distal pocket. The cyclohexan ring of CHES-1 (green) contacts with F226 and R231. The 7-propionate of the heme interacts with the amino group of CHES-1 and side chain of Ser-263. The mutational analyses for these distal residues are shown in Table 1. (B) Top view of A by a rotation of 90°. The proximal residues are omitted. (http://www.pnas.org/content/103/8/2611/F3.expansion.html)

Active site of IDO–PI complex. (A) Stereoview of the residues around the heme of IDO viewed from the side of heme plane. The proximal ligand H346 is H-bonded to wa1. The 6-propionate of the heme contacts with wa2 and R343 Nε. The wa2 is H-bonded to wa1, L388 O, and 6-propionate. Mutations of F226, F227, and R231 do not lose the substrate affinity but produce the inactive enzyme. Two CHES molecules are bound in the distal pocket. The cyclohexan ring of CHES-1 (green) contacts with F226 and R231. The 7-propionate of the heme interacts with the amino group of CHES-1 and side chain of Ser-263. The mutational analyses for these distal residues are shown in Table 1. (B) Top view of A by a rotation of 90°. The proximal residues are omitted. (http://www.pnas.org/content/103/8/2611/F3.expansion.html)

Molecular genetics data from earlier findings based on reporter assay results showed that IDO promoter is regulated by ISRE-like elements and GAS-sequence at -1126 and -1083 region (20).

Two cis-acting elements are ISRE1 (interferon sequence response element 1) and interferon sequence response element 2 (ISRE2).    Analyses of site directed and deletion mutation with transfected cells demonstrated that introduction of point mutations at these elements decreases the IDO expression. Removing ISRE1 decreases the effects of IFNgamma induction 50 fold and deleting ISRE1 at -1126 reduced by 25 fold (3). Introducing point mutations in conserved t residues at -1124 and -1122 (from T to C or G) in ISRE consensus sequence NAGtttCA/tntttNCC of IFNa/b inducible gene ISG4 eliminates the promoter activity by 24 fold (21).

ISRE2 have two boxes, X box (-114/1104) and Y Box 9-144/-135), which are essential part of the IFNgamma response region of major histocompatibility complex class II promoters (22; 23).  When these were removed from ISRE2 or introducing point mutations at two A residues of ISRE2 at -111 showed a sharp decrease after IFNgamma treatment by 4 fold (3).  The lack of responses related to truncated or deleted IRF-1 interactions whereas IRF-2, Jak2 and STAT91 levels were similar in the cells, HEPg2 and ME180 (3). Furthermore, 748 bp deleted between these elements did not affect the IDO expression, thus the distance between ISRE1 and ISRE2 elements have no function or influence on IDO (3; 24)

There are three types of IDO in human genome:

IDO was originally discovered in 1967 in rabbit intestine (25). Later, in 1990 the human IDO gene is cloned and sequenced (7).  However, its importance and relevance in immunology was not created until prevention of allocation of fetal rejection and founding expression in wide range of human cancers (26; 27).  There are three types of IDO, pro-IDO like, IDO1, and IDO2.  In addition, another enzyme called TDO, tryptophan 2, 3, dehydrogenase solely degrade L-Trp at first-rate limiting mechanism in liver and brain.

 

IDO1 mechanism is the target for immunotherapy applications. The initial discovery of IDO in human physiology is protection of pregnancy (1) since lack of IDO results in premature recurrent abortion (28; 26; 29).   The initial rate-limiting step of tryptophan metabolism is catalyzed by either IDO or tryptophan 2, 3-dioxygenase (TDO).

Structural studies of IDO versus TDO presenting active site environments, conserved Arg 117 and Tyr113, found both in TDO and IDO for the Tyr-Glu motif, but His55 in TDO replaced by Ser167b in IDO (30; 2). As a result, they are regulated with different mechanisms (1; 2) (30).

 The short-lived TDO, about 2h, responds to level of tryptophan and its expression regulated by glucorticoids (31; 32).  Thus, it is a useful target for regulation and induced by tryptophan so that increasing tryptophan induces NAD biosynthesis. Whereas, IDO is not activated by the level of Trp presence but inflammatory agents with its interferon stimulated response elements (ISRE1 and ISRE2) in its (33; 34; 35; 36; 3; 10) promoter.

TDO promoter contains glucorticoid response elements (37; 38) and regulated by glucocorticoids and other available amino acids for gluconeogenesis. This is how IDO binds to only immune response cells and TDO relates to NAD biosynthesis mechanisms.

Furthermore, TDO is express solely in liver and brain (36).  NAD synthesis (39) showed increased IDO ubiquitous and TDO in liver and causing NAD level increase in rat with neuronal degeneration (40; 41).  NAM has protective function in beta-cells could be used to cure Type1 diabetes (40; 42; 43). In addition, knowledge on NADH/NAD, Kyn/Trp or Trp/Kyn ratios as well as Th1/Th2, CD4/CD8 or Th17/Threg are equally important (44; 40).

The third type of IDO, called IDO2 exists in lower vertebrates like chicken, fish and frogs (45) and in human with differential expression properties. The expression of IDO2 is only in DCs, unlike IDO1 expresses on both tumors and DCs in human tissues.  Yet, in lower invertebrates IDO2 is not inhibited by general inhibitor of IDO, D-1-methyl-tryptophan (1MT) (46).

Recently, two structurally unusual natural inhibitors of IDO molecules, EXIGUAMINES A and B, are synthesized (47).  LIP mechanism cannot be switch back to activation after its induction in IDO2 (46). Crucial cancer progression can continue with production of IL6, IL10 and TGF-beta1 to help invasion and metastasis.  Inclusion of two common SNPs affects the function of IDO2 in certain populations.  SNP1 reduces 90% of IDO2 catalytic activity in 50% of European and Asian descent and SNP2 produce premature protein through inclusion of stop-codon in 25% of African descent lack functional IDO2 (Uniport).

The Origin of IDO:

A: Structure of human IDO2 gene and transcripts. Complete coding region is 1260 bps encoding a 420 aa polypeptide. Alternate splice isoforms lacking the exons indicated are noted. Hatch boxes represent a frameshift in the coding region to an alternate reading frame leading to termination. Black boxes represent 3' untranslated regions. Nucleotide numbers, intron sizes, and positioning are based on IDO sequence files NW_923907.1 and GI:89028628 in the Genbank database. (reference: http://atlasgeneticsoncology.org/Genes/IDO2ID44387ch8p11.html)

A: Structure of human IDO2 gene and transcripts. Complete coding region is 1260 bps encoding a 420 aa polypeptide. Alternate splice isoforms lacking the exons indicated are noted. Hatch boxes represent a frameshift in the coding region to an alternate reading frame leading to termination. Black boxes represent 3′ untranslated regions. Nucleotide numbers, intron sizes, and positioning are based on IDO sequence files NW_923907.1 and GI:89028628 in the Genbank database.
(reference: http://atlasgeneticsoncology.org/Genes/IDO2ID44387ch8p11.html)

Knowing the evolutionary steps will helps us to identify how we can manage the regulator function to protect human health in cancer, immune disorders, diabetes, and infectious diseases.   Bacterial IDO has two types of IDOs that are group I and group II IDO (48)These are the earliest version of the IDO, pro-IDO like, proteins with a quite complicated function.  Each microorganism recognized by a specific set of receptors, called Toll-Like Receptors (TLR), to activate the IDO-like protein expression based on the origin of the bacteria or virus (49; 35).  

Thus, the genesis of human IDO originates from gene duplication of these early bacterial versions of IDO-like proteins after their invasion interactions with human host.  IDO1 only exists in mammals and fungi.  Fungi also has three types of IDO; IDOa, IDO beta, and IDO gamma (50) with different properties than human IDOs, perhaps multiple IDO is necessary for the world’s decomposers.

All globins, haemoglobins and myoglobins, destined to evolve from a common ancestor that is only 14-16kDa (51) length. Binding of a heme and being oxygen carrier are central to the enzyme mechanism of this family.  Globins are classified under three distinct origins; a universal globin, a compact globin, and IDO-like globin (52).  IDO like globin widely distributed among gastropodic mollusks (53; 51).

The indoleamine 2, 3-dioxygenase 1–like “myoglobin” (Myb) was discovered in 1989 in the buccal mass of the abalone Sulculus diversicolor (54).  The conserved region between Myb and IDO-like Myb existed for at least 600 million years (53).  Even though the splice junction of seven introns was kept intact, the overall homolog region between Myb and IDO is only about 35%.  No significant evolutionary relationship is found between them after their amino acid sequence of each exon is compared to usual globin sequences. This led the hint that molluscan IDO-like protein must have other functions besides carrying oxygen, like myoglobin.   Alignment of S. cerevisiae cDNA, mollusk and vertebrate IDO–like globins show the key regions for controlling IDO or myoglobin function (55). These data suggest that there is an alternative pathways of myoglobin evolution.  In addition, understanding the diversity of globin may help to design better protocols for interventions of diseases.

B: Amino acid alignment of IDO and IDO2. Amino acids determined by mutagenesis and the crystal structure of IDO that are critical for catalytic activity are positioned below the human IDO sequence. Two commonly occurring SNPs identified in the coding region of human IDO2 are shown above the sequence which alter a critical amino acid (R248W) or introduce a premature termination codon (Y359stop).

B: Amino acid alignment of IDO and IDO2. Amino acids determined by mutagenesis and the crystal structure of IDO that are critical for catalytic activity are positioned below the human IDO sequence. Two commonly occurring SNPs identified in the coding region of human IDO2 are shown above the sequence which alter a critical amino acid (R248W) or introduce a premature termination codon (Y359stop).

The Immune Cells and IDO in DCs:

DCs are the orchestrator of the immune response (56; 57; 58) with list of functions in uptake, processing, and presentation of antigens; activation of effector cells, such as T-cells and NK-cells; and secretion of cytokines and other immune-modulating molecules to direct the immune response. The differential regulation of IDO in distinct DC subsets is widely studied to delineate and correct immune homeostasis during autoimmunity, infection and cancer and the associated immunological outcomes.

Genesis of antigen presenting cells (APCs), eventually the immune system, require migration of monocytes (MOs), which is originated in bone marrow. Then, these MOs move from bloodstream to other tissues to become macrophages and DCs (59; 60). Initiation of immune response requires APCs to link resting helper T-cell with the matching antigen to protect body. DCs are superior to MQs and MOs in their immune action model. When DCs are first described (61) and classified, their role is determined as a highly potent antigen-presenting cell (APC) subset with 100 to 1000-times more effective than macrophages and B-cells in priming T-cells. Both MQs and monocytes phagocytize the pathogen, and their cell structure contains very large nucleus and many internal vesicles. However, there is a nuance between MQ and DCs, since DCs has a wider capacity of stimulation, because MQs activates only memory T cells, yet DCs can activate both naïve and memory T cells.

DCs are potent activators of T cells and they also have well controlled regulatory roles. DC properties determine the regulation regardless of their origin or the subset of the DCs.  DCs react after identification of the signals or influencers for their inhibitory, stimulatory or regulatory roles, before they express a complex repertoire of positive and negative cytokines, transmembrane proteins and other molecules. Thus, “two signal theory” gains support with a defined rule.

The combination of two signals, their interaction with types of cells and time are critical. In short, specificity and time are matter for a proper response.  When IDO mRNA expression is activated with CTL40 ligand and IFNgamma, IDO results inhibition of T cell production (4).  However, if DCs are inhibited by 1MT, an inhibitor of IDO, the response stop but IgG has no affect (10).  In addition, if the stimulation is started by a tryptophan metabolite, which is downstream of IDO, such as 3-hydroxyantranilic or quinolinic acids, it only inhibits Th1 but not Th2 subset of T cells (62). Furthermore, inclusion of signal molecules, such as Fas Ligand, cytochrome c, and pathways also differ in the T cell differentiation mechanisms due to combination, time and specificity of two-signals.  The co-culture experiments are great tool to identify specific stimuli in disease specific microenvironment (63; 12; 64) for discovering the mechanism and interactions between molecules in gene regulation, biochemical mechanism and physiological function during cell differentiation.

As a result, the simplest differential cell development from the early development of DCs impact the outcome of the data. For example, collection of MOs from peripheral blood mononuclear cells (PBMCs) with IL4 and GM-CSF leads to immature DCs (iDCs). On next step, treatment of iDCs with tumor necrosis factor (TNF) or other plausible cytokines (TGFb1, IFNgamma, IFNalpha,  IFNbeta, IL6 etc.) based on the desired outcome differentiate iDCs  into mature DCs (mDCs). DCs live only up to a week but MOs and generated MQs can live up to a month in the given tissue. B cells inhibit T cell dependent immune responses in tumors (65).

Mechanisms of IDO:

IDO mechanism for immune response

The dichotomy of IDO mechanism lead the discovery that IDO is more than an enzyme as a versatile regulator of innate and adaptive immune responses in DCs (66; 67; 68). Meantime IDO also involve with Th2 response and B cell mediated autoimmunity showing that it has three paths, short term (acute) based on enzymatic actions, long term (chronic) based on non-enzymatic role, and moonlighting relies of downstream metabolites of tryptophan metabolism (69; 70).

IFNgamma produced by DC, MQ, NK, NKT, CD4+ T cells and CD8+ T cells, after stimulation with IL12 and IL8.  Inflammatory cytokine(s) expressed by DCs produce IFNgamma to stimulate IDO’s enzymatic reactions in acute response.  Then, TDO in liver and tryptophan catabolites act through Aryl hydrocarbon receptor induction for prevention of T cell proliferation. This mechanism is common among IDO, IDO2 (expresses in brain and liver) and TDO (expresses in liver) provide an acute response for an innate immunity (30). When the pDCs are stimulated with IFNgamma, activation of IDO is go through Jak, STAT signaling pathway to degrade Trp to Kyn causing Trp depletion. The starvation of tryptophan in microenvironment inhibits generation of T cells by un-read t-RNAs and induce apoptosis through myc pathway.  In sum, lack of tryptophan halts T cell proliferation and put the T cells in apoptosis at S1 phase of cell division (71; 62).

 T-reg, regulatory T cells; Th, T helper; CTLA-4, cytotoxic T lymphocyte-associated antigen 4; TCR, T cell receptor; IDO, indoleamine 2,3-dioxygenase. (refernece: http://www.pnas.org/content/101/28/10398/suppl/DC)

T-reg, regulatory T cells; Th, T helper; CTLA-4, cytotoxic T lymphocyte-associated antigen 4; TCR, T cell receptor; IDO, indoleamine 2,3-dioxygenase. (refernece: http://www.pnas.org/content/101/28/10398/suppl/DC)

The intermediary enzymes, functioning during Tryptophan degradation in Kynurenine (Kyn) pathway like kynurenine 3-hydroxylase and kynureninase, are also induced after stimulation with liposaccaride and proinflammatory cytokines (72). They exhibit their function in homeostasis through aryl-hydrocarbon receptor (AhR) induction by kynurenine as an endogenous signal (73; 74).  The endogenous tumor-promoting ligand of AhR are usually activated by environmental stress or xenobiotic toxic chemicals in several cellular processes like tumorigenesis, inflammation, transformation, and embryogenesis (Opitz ET. Al, 2011).

Human tumor cells constitutively produce TDO also contributes to production of Kyn as an endogenous ligand of the AhR (75; 27).  Degradation of tryptophan by IDO1/2 in tumors and tumor-draining lymph nodes occur. As a result, there are animal studies and Phase I/II clinical trials to inhibit the IDO1/2 to prevent cancer and poor prognosis (NewLink Genetics Corp. NCT00739609, 2007).

Systemic inflammation (like in sepsis, cerebral malaria and brain tumor) creates hypotension and IDO expression has the central role on vascular tone control (63).  Moreover, inflammation activates the endothelial coagulation activation system causing coagulopathies on patients.  This reaction is namely endothelial cell activation of IDO by IFNgamma inducing Trp to Kyn conversion. After infection with malaria the blood vessel tone has decreases, inflammation induce IDO expression in endothelial cells producing Kyn causing decreased trp, lower arterial relaxation, and develop hypotension (Wang, Y. et. al 2010).  Furthermore, existing hypotension in knock out Ido mice point out a secondary mechanism driven by Kyn as an endogenous ligand to activate non-canonical NfKB pathway (63). Another study also hints this “back –up” mechanism by a significant outcome with a differential response in pDCs against IMT treatment.  Unlike IFN gamma conditioned pDC blocks T cell proliferation and apoptosis, methyl tryptophan fails to inhibit IDO activity for activating naïve T cells to make Tregs at TGF-b1 conditioned pDCs (77; 78).

The second role of the IDO relies on non-enzymatic action as being a signal molecule. Yet, IDO2 and TDO are devoid of this function. This role mainly for maintenance of microenvironment condition. DCs response to TGFbeta-1 exposure starts the kinase Fyn induce phosphorylation of IDO-associated immunoreceptor tyrosine–based inhibitory motifs (ITIMs) for propagation of the downstream signals involving non-canonical (anti-inflammatory) NF-kB pathway for a long term response.

When the pDCs are conditioned with TGF-beta1 the signaling (68; 77; 78) Phospho Inositol Kinase3 (PIK-3)-dependent and Smad independent pathways (79; 80; 81; 82; 83) induce Fyn-dependent phosphorylation of IDO ITIMs.  A prototypic ITIM has the I/V/L/SxYxxL/V/F sequence (84), where x in place of an amino acid and Y is phosphorylation sites of tyrosines (85; 86).  Smad independent pathway stimulates SHP and PIK3 induce both SHP and IDO phosphorylation. Then, formed SHP-IDO complex can induce non-canonical (non-inflammatory) NF-kB pathway (64; 79; 80; 82) by phosphorylation of kinase IKKa to induce nuclear translocation of p52-Relb towards their targets.  Furthermore, the SHP-IDO complex also may inhibit IRAK1 (68).  SHP-IDO complex activates genes through Nf-KB for production of Ido1 and Tgfb1 genes and secretion of IFNalpha/IFNbeta.  IFNa/IFNb establishes a second short positive feedback loop towards p52-RelB for continuous gene expression of IDO, TGFb1, IFNa and IFNb (87; 68).  However, SHP-IDO inhibited IRAK1 also activates p52-RelB.  Nf-KB induction at three path, one main and two positive feedback loops, is also critical.  Finally, based on TGF-beta1 induction (76) cellular differentiation occurs to stimulate naïve CD4+ T cell differentiation to regulatory T cells (Tregs).  In sum, TGF-b1 and IFNalpha/IFNbeta stimulate pDCs to keep inducing naïve T cells for generation of Treg cells at various stages, initiate, maintain, differentiate, infect, amplify, during long-term immune responses (67; 66).

Moonlighting function of Kyn/AhR is an adaptation mechanism after the catalytic (enzymatic) role of IDO depletes tryptophan and produce high concentration of Kyn induce Treg and Tr1 cell expansion leading Tregs to use TGFbeta for maintaining this environment (67; 76). In this role, Kyn pathway has positive-feedback-loop function to induce IDO expression.

TABLE 3- Kyn induced Genes

Table 2: Kyn induced genes based on the only microarray analysis (based on Opitz et. al 2011 data)
  Upregulators Phenotype Location
 Upregulators MYC Oncogene myc
avian myelocytomatosis viral oncogene homolog
protooncogene homologous to myelocytomatosis virus
INDIRECTLY MANIPULATED TARGET
NfKB complex Inappropriate activation of NF-kappa-B has been linked to inflammatory events associated with autoimmune arthritis, asthma, septic shock, lung fibrosis, glomerulonephritis, atherosclerosis, and AIDS. In contrast, complete and persistent inhibition of NF-kappa-B has been linked directly to apoptosis, inappropriate immune cell development, and delayed cell growth. 10q24.32POSSIBLE INDIRECTLY MANIPULATEDTARGET   4q24 
  Downregulators
       

 

ALDH1A3(ALDEHYDE DEHYDROGENASE 1 FAMILY, MEMBER A3) An unique ALDH isozyme in human saliva 15q26.3
ARNT2,ARYL HYDROCARBON RECEPTOR NUCLEAR TRANSLOCATOR 2 Member of a novel transcription factor family consisting of a conserved basic helix-loop-helix (bhlh) structural motif contiguous with a PAS domain. Members of this family include PER, the aryl hydrocarbon receptor,SIM1,and HIF1A. 15q25.1
C2CD2 Myogenesis in C2C12 mouse myoblasts by DUX4 and inhibited zebrafish development past gastrulation or caused severe developmental abnormalities in the surviving embryos. 4q35.2
CDC42EP2,CDC42 EFFECTOR PROTEIN 2 A small RHO gtpase, regulates the formation of F-actin-containing structures through its interaction with several downstream effector proteins.  11q13.1
 CDH1,CADHERIN 1;  Uvomorulin, a specific calcium ion-dependent cell adhesion molecule, expresses its adhesive function during the preimplantation stage of development and in epithelial cells,Endometrial carcinoma, somatic, Ovarian carcinoma, somatic, Gastric cancer, familial diffuse, with or without cleft lip and/or palate, Breast cancer, lobular, Prostate cancer, susceptibility to. 16q22.1
CENPACENTROMERIC PROTEIN A;  Centromeric proteins, see CENPB 2p23.3
CREB3L2cAMP RESPONSE ELEMENT-BINDING PROTEIN 3-LIKE 2; Member of the old astrocyte specifically induced substance (OASIS) DNA binding and basic leucine zipper dimerization (bzip) family of transcription factors, which includes CREB3 and CREB4. 7q33
CYP1B1,CYTOCHROME P450, SUBFAMILY I, POLYPEPTIDE 1
Glaucoma 3A, primary open angle, congenital, juvenile,Or adult onset, Peters anomaly 231300
2p22.2
EGR; Discovered first as a putative G0/G1 switch regulatory gene in human blood lymphocyte cultures and named G0S30 (Forsdyke, 1985). Sequence analysis of the murine gene predicted a protein with 3 DNA-binding zinc fingers POSSIBLE TARGET
EGR1;EARLY GROWTH RESPONSE 1 Displays FOS-like induction kinetics in fibroblasts, epithelial cells, and lymphocyte. EGR1 is also known as KROX24. Or nerve growth factor-induced clone A (NGFIA). 5q31.2(Sukhatme et al., 1988).
EREG;EPIREGULIN Functions as a tumor growth-inhibitory factor inducing morphologic changes and exhibits low affinity for the EGF receptor. Found on hela,on human epidermoid carcinoma A431 cells. Toyoda et al. (1995), Toyoda et al. (1997)
GPR115; G PROTEIN-COUPLED RECEPTOR 115 Expression in pregnant uterus, breast, and genitourinary tract. 6p12.3fredriksson et al. (2002) POSSIBLE target
HK2; HEXOKINASE 2 Hexokinase (EC 2.7.1.1) catalyzes the first step in glucose metabolism, using ATP for the phosphorylation of glucose to glucose-6-phosphate. Four different types of hexokinase, designated HK1, HK2, HK3, and HK4 (encoded by different genes, are present in mammalian tissues. 2p12
HTT; HUNTINGTON DISEASE  Huntington disease (HD) is caused by an expanded trinucleotide repeat (CAG)n, encoding glutamine, in the gene encoding Huntington. An autosomal dominant progressive neurodegenerative disorder with a distinct phenotype characterized by chorea, dystonia, incoordination, cognitive decline, and behavioral difficulties. 4p16.3
IGFBP4; INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN 4 Insulin-like growth factor binding proteins (igfbps), such as IGFBP4, are involved in the systemic and local regulation of IGF activity. Igfbps contain 3 structurally distinct domains each comprising approximately one-third of the molecule.). 17q21.2(Kiefer et al., 1992
IL1A; INTERLEUKIN 1-ALPHA IL1A is 1 of 2 structurally distinct forms of IL1, the other being IL1B (147720). The IL1A and IL1B proteins are synthesized by a variety of cell types, including activated macrophages, keratinocytes, stimulated B lymphocytes, and fibroblasts, and are potent mediators of inflammation and immunity 2q13(Lord et al., 1991).
IL1B; INTERLEUKIN 1-BETA {Gastric cancer risk after H. Pylori infection} 2q13
IL6INTERFERON, BETA-2; IFNB2
B-
CELL DIFFERENTIATION FACTOR, B-CELL STIMULATORY FACTOR 2; BSF2,  HEPATOCYTE STIMULATORY FACTOR; HSF,HYBRIDOMA GROWTH FACTOR; HGF 
Crohn disease-associated growth, failure}, {Diabetes, susceptibility to}, {Kaposi sarcoma, susceptibility to}, {Intracranial hemorrhage in brain, Cerebrovascular malformations, susceptibility to}, {Rheumatoid arthritis, systemic juvenile}. 7p15.3 POSSIBLE COSTIMULATION TARGET
IL8SMALL INDUCIBLE CYTOKINE SUBFAMILY B, MEMBER 8; SCYB8, MONOCYTE-DERIVED NEUTROPHIL CHEMOTACTIC FACTOR,
NEUTROPHIL-ACTIVATING PEPTIDE 1;
NAP1
GRANULOCYTE CHEMOTACTIC PROTEIN 1; GCP1
CHEMOKINE, CXC MOTIF, LIGAND 8; CXCL8
 
A member of the CXC chemokine family. These small basic heparin-binding proteins are proinflammatory and primarily mediate the activation and migration of neutrophils into tissue from peripheral blood. 4q13.3 (Hull et al., 2001). POSSIBLE  COSTIMULATION TARGET
ITGAE;INTEGRIN, ALPHA-ECD103 ANTIGEN
HUMAN MUCOSAL LYMPHOCYTE ANTIGEN 1, ALPHA SUBUNIT
Integrins are a family of cell surface adhesion molecules that play a major role in diverse cellular and developmental processes including morphogenesis, hemostasis, leukocyte activation, cellular adhesion, and homing.Immune responses at mucosal sites are mediated by lymphocytes associated with mammary glands and the gastrointestinal, genitourinary, and respiratory tracts. Cerf-Bensussan et al. (1987),Parker et al. (1992)
JUN
kiaa1644;TRIL;  TLR4 INTERACTOR WITH LEUCINE-RICH REPEATS TRIL is a component of the TLR4 complex and is induced in a number of cell types by lipopolysaccharide (LPS) 7p14.3(Carpenter et al., 2009).
LDO C1LLEUCINE ZIPPER, DOWNREGULATED IN CANCER 1; LDOC1 Contains a leucine zipper-like motif in its N-terminal region and a proline-rich region that shares marked similarity to an SH3-binding domain.  Northern blot analysis detected ubiquitous expression of LDOC1 in normal tissues, with high expression in brain and thyroid and low expression in placenta, liver, and leukocytes.  LDOC1 was expressed in 6 of 7 human breast cancer cell lines examined, but, with only 1 exception, was not expressed in any pancreatic or gastric cancer cell lines examined.  Fluorescence microscopy analysis demonstrated that the LDOC1 protein is located predominantly in the nucleus. Xq27.1 Nagasaki et al. (1999) COSTIMULATION TARGET
MID1; MIDLINE 1 Midline 1 ring finger gene
midin
finger on x and y, mouse, homolog of; fxyOpitz gbbb syndrome, type I
 xp22.2
mir-124; MICRO RNA 124-1 Lagos-Quintana et al. (2002) cloned mouse mir124a.Northern blot analysis showed that mir124a was highly expressed in mouse brain, but not in any other mouse tissues examined.Suh et al. (2004) cloned human mirna124a from embryonic stem cells. The mature mirna124a sequence is UUAAGGCACGCGGUGAAUGCCA.Sempere et al. (2004) found that mirna124 was preferentially expressed in brain. Chromosome 8
mir-290 Both of the major editing sites in pri-mir-376 rnas (+4 and +44) are located within the functionally critical 5-prime-proximal ‘seed’ sequences, critical for the hybridization of mirnas to targets, of mir-376, suggesting that edited mature mir-376 rnas may target genes different from those targeted by the unedited mir-376 rnas. Their results suggested that a single A-I base change is sufficient to redirect silencing mirnas to a new set of targets.Editing of mir-376 appears to be one of the mechanisms that ensure tight regulation of uric acid levels in select tissues such as the brain cortex. MICRO RNA 376-B; MIRN376B Kawahara et al. (2007)POSSIBLE COSTIMULATION TARGET
mir548
RB1;RB1 GENE Bladder cancer, somatic, Osteosarcoma, somatic, Retinoblastoma, Retinoblastoma, trilateral, Small cell cancer of the lung, somatic. 13q14.2Dryja et al. (1984)
RELA; V-REL AVIAN RETICULOENDOTHELIOSIS VIRAL ONCOGENE HOMOLOG A NUCLEAR FACTOR KAPPA-B, SUBUNIT 3; NFKB3
TRANSCRIPTION FACTOR NFKB3
NFKB, p65 SUBUNIT
NUCLEAR FACTOR OF KAPPA LIGHT CHAIN
GENE ENHANCER IN B CELLS 3Activated NFKB complex translocates into the nucleus and binds DNA at kappa-B-binding motifs such as 5-prime GGGRNNYYCC 3-prime or 5-prime HGGARNYYCC 3-prime (where H is A, C, or T; R is an A or G purine; and Y is a C or T pyrimidine). 
11q13.1   POSSIBLE CO-TIMULATION TARGET
SERPINB2;SERPIN PEPTIDASE INHIBITOR,Clade B (Ovalbumin), Member 2Plasminogen Activator Inhibitor, Type 2; Pai2
Planh2
Monocyte Arginine-Serpin
Monocyte-Derived Plasminogen Activator Inhibitor
Urokinase Inhibitor
The specific inhibitors of plasminogen activators have been classified into 4 immunologically distinct groups: PAI1 type PA inhibitor from endothelial cells; PAI2 type PA inhibitor from placenta, monocytes, and macrophages; urinary inhibitor; and protease-nexin-I.Plasminogen activator inhibitor-2 is also known as monocyte arg-serpin because it belongs to the superfamily of serine proteases in which the target specificity of each is determined by the amino acid residue located at its reactive center; i.e., met or val for elastase, leu for kinase, and arg for thrombin. 18q21.33
SH3RF1; SH3 DOMAIN-CONTAINING RING FINGER PROTEIN 2 Chen et al. (2010) cloned SH3RF2, which they called HEPP1. The deduced 186-amino acid protein contains a PP1-binding motif (KTVRFQ). Northern blot analysis detected 1.24- and 0.68-kb HEPP1 transcripts in heart and testis only. 5q32
STC2;STANNIOCALCIN-RELATED PROTEIN Northern blot analysis revealed that STC2 is expressed as multiple transcripts in several human tissues, with the strongest expression in skeletal muscle and heart. No entry??
TAF9; TAF9 RNA POLYMERASE II, TATA BOX-BINDING PROTEIN-ASSOCIATED FACTOR, 32-KD The tafs are required for activated rather than basal transcription and serve to mediate signals between various activators and the basal transcriptional machinery. 5q13.2
TGFB1; TRANSFORMING GROWTH FACTOR, BETA-1
Camurati-Engelmann disease 131300
{Cystic fibrosis lung disease, modifier of}TGFB is a multifunctional peptide that controls proliferation, differentiation, and other functions in many cell types. TGFB acts synergistically with TGFA (190170) in inducing transformation. It also acts as a negative autocrine growth factor. Dysregulation of TGFB activation and signaling may result in apoptosis. Many cells synthesize TGFB and almost all of them have specific receptors for this peptide. TGFB1, TGFB2 (190220), and TGFB3 (190230) all function through the same receptor signaling systems.
19q13.2
  TIPARP; TCDD-INDUCIBLE POLY(ADP-RIBOSE) POLYMERASE Amplified and upregulated in head and neck squamous cell carcinoma (HNSCC). The N-terminal part of the TPH domain contains a CCCH-type zinc finger. 3q25.31Katoh and Katoh (2003)  Redon et al. (2001)
TOP2A DNA topoisomerase II, resistance to inhibition of, by amsacrine.  DNA topoisomerases (EC 5.99.1.3) are enzymes that control and alter the topologic states of DNA in both prokaryotes and eukaryotes.There are about 100,000 molecules of topoisomerase II per hela cell nucleus, constituting about 0.1% of the nuclear extract. In a human leukemia cell line, HL-60/AMSA, Hinds et al. (1991) found that resistance to inhibition of topoisomerase II by amsacrine and other intercalating agents was dueTo a single base change, AGA (arginine) to AAA (lysine). 17q21.2
TP53; P53
TRANSFORMATION-RELATED PROTEIN 53;
TRP53Osteosarcoma, Choroid plexus papilloma, Breast cancer,Adrenal cortical carcinoma, Colorectal cancer, Hepatocellular carcinoma, Li-Fraumeni syndrome, Nasop haryngeal carcinoma, Pancreatic cancer, {Glioma susceptibility 1}, {Basal cell carcinoma 7} 
The transcription factor p53 responds to diverse cellular stresses to regulate target genes that induce cell cycle arrest, apoptosis, senescence, DNA repair, or changes in metabolism. In addition, p53 appears to induce apoptosis through nontranscriptional cytoplasmic processes.Activity of p53 is ubiquitously lost in human cancer either by mutation of the p53 gene itself or by loss of cell signaling upstream or downstream 17p13.1 POSSIBLE TARGET FOR CO-STIMULATION
TP73; p53-RELATED PROTEIN p73; p73
TRP73, MOUSE, HOMOLOG OF
The p53 gene (TP53; 191170) is the most frequently mutated tumor suppressor in human cancers. The ability of p53 to inhibit cell growth is due, at least in part, to its ability to bind to specific DNA sequences and activate transcription of target genes, such as that encoding cell cycle inhibitor p21(Waf1/Cip1) 1p36.32
ZIC2; ZIC FAMILY MEMBER 2  Brown et al. (1998) reported that the human ZIC2 gene, a homolog of the Drosophila ‘odd-paired’ (opa) gene, maps to the region of chromosome 13 associated with holoprosencephaly (HPE5; 609637). Have zinc finger domain.Holoprosencephaly-5Holoprosencephaly is the most common structural anomaly of the human brain and is one of the anomalies seen in patients with deletions and duplications of chromosome 13 13q32.3

In T cells, tryptophan starvation induces Gcn2-dependent stress signaling pathway, which  initiates uncharged Trp-tRNA binding onto ribosomes. Elevated GCN2 expression stimulates elF2alfa phosphorylation to stop translation initiation (88). Therefore, most genes downregulated and LIP, an alternatively initiated isoform of the b/ZIP transcription factor NF-IL6/CEBP-beta (89).  This mechanism happens in tumor cells based on Prendergast group observations. As a result, not only IDO1 propagates itself while producing IFNalpha/IFNbeta, but also demonstrates homeostasis choosing between immunegenity by production of TH17or tolerance by Tregs. This mechanism acts like a see-saw. Yet, tolerance also can be broken by IL6 induction so reversal mechanism by SOC-3 dependent proteosomal degradation of the enzyme (90).  All proper responses require functional peripheral DCs to generate mature DCs for T cells to avoid autoimmunity (91)

Niacin (vitamin B3) is the final product of tryptophan catabolism and first molecule at Nicotinomic acid (NDA) Biosynthesis.  The function of IDO in tryptophan and NDA metabolism has a great importance to develop new clinical applications (40; 42; 41).  NAD+, biosynthesis and tryptophan metabolisms regulate several steps that can be utilize pharmacologically for reformation of healthy physiology (40).

IDO for protection in Microbial Infection with Toll-like Receptors

The mechanism of microbial response and infectious tolerance are complex and the origination of IDO based on duplication of microbial IDO (49).  During microbial responses, Toll-like receptors (TLRs) play a role to differentiate and determine the microbial structures as a ligand to initiate production of cytokines and pro-inflammatory agents to activate specific T helper cells (92; 93; 94; 95). Uniqueness of TLR comes from four major characteristics of each individual TLR by ligand specificity, signal transduction pathways, expression profiles and cellular localization (96). Thus, TLRs are important part of the immune response signaling mechanism to initiate and design adoptive responses from innate (naïve) immune system to defend the host.

TLRs are expressed cell type specific patterns and present themselves on APCs (DCs, MQs, monocytes) with a rich expression levels (96; 97; 98; 99; 93; 100; 101; 102; 87). Induction signals originate from microbial stimuli for the genesis of mature immune response cells.  Co-stimulation mechanisms stimulate immature DCs to travel from lymphoid organs to blood stream for proliferation of specific T cells (96).  After the induction of iDCs by microbial stimuli, they produce proinflammatory cytokines such as TNF and IL-12, which can activate differentiation of T cells into T helper cell, type one (Th1) cells. (103). Specific TLR stimulation links innate and acquired responses through simple recognition of pathogen-associated molecular patterns (PAMPs) or co-stimulation of PAMPs with other TLR or non-TLR receptors, or even better with proinflammatory cytokines.   Some examples of ligand- TLR specificity shown in Table1, which are bacterial lipopeptides, Pam3Cys through TLR2 (92; 104; 105), double stranded (ds) RNAs through TLR3 (106; 107), lipopolysaccharide (LPS) through TLR4, bacterial flagellin through TLR5 (108; 109), single stranded RNAs through TLR7/8 (97; 98), synthetic anti-viral compounds imiquinod through TLR 7 and resiquimod through TLR8, unmethylated CpG DNA motifs through TLR9 (Krieg, 2000).

The specificity is established by correct pairing of a TLR with its proinflammatory cytokine(s), so that these permutations influence creation and maintenance of cell differentiation. For example, leading the T cell response toward a preferred Th1 or Th2 response possible if the cytokines TLR-2 mediated signals induce a Th2 profile when combined with IL-2 but TLR4 mediated signals lean towards Th1 if it is combined with IL-10 or Il-12, (110; 111)  (112).

TLR ligand TLR Reference
Lipopolysaccharide, LPS TLR4 (96).  (112).
Lipopeptides, Pam3Cys TLR2 (92; 104; 105)
Double stranded (ds) RNAs TLR3 (106; 107)
Bacterial flagellin TLR5 (108; 109)
Single stranded RNAs TLR7/8 (97; 98)
Unmethylated CpG DNA motifs TLR9 (Krieg, 2000)
Synthetic anti-viral compounds imiquinod and resiquimod TLR7 and TLR8 (Lee J, 2003)

Furthermore, IL6 stimulated DCs relieve the suppression of effector T cells by regulatory T cells (113).  The modification of IDO+ monocytes towards specific subset of T cell activation with specific TLRs are significantly important (94).  The type of cell with correct TLR and stimuli improves or decreases the effectiveness of stimuli. Induction of IDO in monocytes by synthetic viral RNAs (isRNA) or CMV was possible but not in monocyte derived DCs or TLR2 ligand lipopeptide Pam3Cys.  Single- stranded RNA ligands target TLR7/8 in monocytes derive DCs only (Lee J, 2003).  These futures of TLRs important during design of experiments to target and improve the efficacy.

Double-Edged Sword of IDO: The Good and The Bad for Clinical intervention and Developments

High expression level of IDO has a positive impact during pregnancy (29; 28; 114), transplants (115; 116; 117; 118; 119), infectious diseases (96). On the other hand, high IDO expression leads the system to a tolerance state is negative during autoimmune-disorders (120; 121; 122), tumors of cancer (123; 124; 117; 121; 125; 126; 127) (127), and mood disorders (46).

Prevention of allogeneic fetal rejection is possible by tryptophan metabolism (26) by rejecting at lack of IDO but allocating with abundant IDO  (29; 28; 114). The plasticity of  mammary and uterus during reproduction may hold some more answers to prevent GVHD and tumors of cancer with good understanding of IDO and tryptophan mechanism (129; 130). These studies lead to find “the natural regulation mechanism” for protecting the transplants from graft versus host disease GVHD (128) and getting rid of tumors. After allogeneic bone marrow transplants the risk of solid tumor development increased about 80% among 19,229 patients,  even with a greater risk if patients are under 18 years old (117).  The adaptation of tolerance against host mechanism is connected to the IDO expression (131).   During implantation and early pregnancy IDO has a role by making CD4+CD25+Foxp3+ regulatory T cells (Tregs) and expressing in DCs and  MQs  (114; 132; 133).  Clonal deletion mechanism prevents mother to react with paternal products since female mice accepted the paternal MHC antigen-expressing tumor graft during pregnancy and rejected three weeks after delivery (134). CTLA-4Ig gene therapy alleviates abortion through regulation of apoptosis and inhibition of spleen lymphocytes (135).

AutoImmune Disorders:

The balance of IDO expression becomes necessary to prevent overactive immune response self-destruction, so modulation in tryptophan and NDA metabolisms maybe essential.  When splenic IDO-expressing CD11b (+) DCs from tolerized animals applied, they suppressed the development of arthritis, increased the Treg/Th17 cell ratio, and decreased the production of inflammatory cytokines in the spleen (136).   The role of Nicotinamide prevention on type 1 diabetes and ameliorates multiple sclerosis in animal model presented with activities of  NDAs stimulating GPCR109a to produce prostaglandins to induce IDO expression, then these PGEs and PGDs converted to the anti-inflammatory prostaglandin, 15d-PGJ(2) (137; 138; 139).  Thus, these events promote endogenous signaling mechanisms involving the GPCRs EP2, EP4, and DP1 along with PPARgamma. (137).

IDO (indoleamine 2,3-dioxygenase) and IDO2 control a tryptophan catabolism signaling pathway. (a) From tryptophan starvation to LIP activation. By catabolizing the essential amino acid tryptophan, IDO and IDO2 generate kynurenines and other reaction products that can modulate T-cell immunity as well as a local microenvironment that is starved for tryptophan. Little is known as yet of the precise mechanistic effects of the tryptophan catabolites generated. Elaboration of the starvation condition triggers a stress response in local T cells through Gcn2, which responds to amino-acid deprivation by phosphorylating the translation initiation factor eIF2alpha, leading to a blockade of most translation initiation with the exception of certain factors such as LIP involved in mediating responses to the stress. (b) LIP is a dominant negative isoform of the immune regulatory b/ZIP transcription factor NF-IL6, also known as CEBPbeta. LIP is an alternately translated isoform of the transcription factor NF-IL6/CEBPbeta implicated in regulating proliferation and immune response. Starvation responses switch NF-IL6/CEBPbeta expression from LAP isoforms to the LIP isoform through the use of a downstream translation start site in the mRNA. Encoding only a b/ZIP dimerization domain, LIP functions as a 'natural' dominant negative molecule that disrupts NF-IL6/CEBPbeta function by competing with LAP isoforms for binding to target gene promoters. Both IDO and IDO2 can switch on LIP, but subsequent restoration of tryptophan levels will only switch it off in the case of IDO, offering a possible mechanism for distal propagation of immune suppression away from the local tumor microenvironment (Figure 5). NF-IL6/CEBPbeta target genes with relevance to the function of IDO include the immune suppressive cytokines IL-6, IL-10 and TGF-beta, which may be upregulated as a result of LIP induction. (http://www.nature.com/onc/journal/v27/n28/fig_tab/onc200835f3.html)

IDO (indoleamine 2,3-dioxygenase) and IDO2 control a tryptophan catabolism signaling pathway. (a) From tryptophan starvation to LIP activation. By catabolizing the essential amino acid tryptophan, IDO and IDO2 generate kynurenines and other reaction products that can modulate T-cell immunity as well as a local microenvironment that is starved for tryptophan. Little is known as yet of the precise mechanistic effects of the tryptophan catabolites generated. Elaboration of the starvation condition triggers a stress response in local T cells through Gcn2, which responds to amino-acid deprivation by phosphorylating the translation initiation factor eIF2alpha, leading to a blockade of most translation initiation with the exception of certain factors such as LIP involved in mediating responses to the stress. (b) LIP is a dominant negative isoform of the immune regulatory b/ZIP transcription factor NF-IL6, also known as CEBPbeta. LIP is an alternately translated isoform of the transcription factor NF-IL6/CEBPbeta implicated in regulating proliferation and immune response. Starvation responses switch NF-IL6/CEBPbeta expression from LAP isoforms to the LIP isoform through the use of a downstream translation start site in the mRNA. Encoding only a b/ZIP dimerization domain, LIP functions as a ‘natural’ dominant negative molecule that disrupts NF-IL6/CEBPbeta function by competing with LAP isoforms for binding to target gene promoters. Both IDO and IDO2 can switch on LIP, but subsequent restoration of tryptophan levels will only switch it off in the case of IDO, offering a possible mechanism for distal propagation of immune suppression away from the local tumor microenvironment (Figure 5). NF-IL6/CEBPbeta target genes with relevance to the function of IDO include the immune suppressive cytokines IL-6, IL-10 and TGF-beta, which may be upregulated as a result of LIP induction. (http://www.nature.com/onc/journal/v27/n28/fig_tab/onc200835f3.html)

Modulating the immune response at non-canonical at canonocal pathway while keeping the non-canonical Nf-  KB intact may help to mend immune disorders. As a result, the targeted blocking in canonical at associated  kinase IKKβ and leaving non-canonocal Nf-kB pathway intact, DCs tips the balance towards immune supression.  Hence, noncanonical NF-κB pathway for regulatory functions in DCs required effective IDO induction, directly or  indirectly by endogenous ligand Kyn and negative regulation of proinflammatory cytokine production.

As a result, this may help to treat autoimmune diseases such as rheumatoid arthritis, type 1 diabetes,        inflammatory bowel disease, and multiple sclerosis, or allergy or transplant rejection. While the opposite action  needs to be taken during prevention of tumors, that is inhibition of non-canonical pathway.  Inflammation    induces not only relaxation of veins and lowering blood pressure but also stimulate coagulopathies that worsen  the microenvironment and decrease survival rate of patients after radio or chemotherapies .

Viable tumor environment. Tumor survival is dependent upon an exquisite interplay between the critical functions of stromal development and angiogenesis, local immune suppression and tumor tolerance, and paradoxical inflammation. TEMs: TIE-2 expressing monocytes; “M2” TAMs: tolerogenic tumor-associated macrophages; MDSCs: myeloid-derived suppressor cells; pDCs: plasmacytoid dendritic cells; co-stim.: co-stimulation; IDO: indoleamine 2,3-dioxygenase; VEGF: vascular endothelial growth factor; EGF: epidermal growth factor; MMP: matrix metaloprotease; IL: interleukin; TGF-β: transforming growth factor-beta; TLRs: toll-like receptors.  (reference: http://www.hindawi.com/journals/cdi/2012/937253/fig1/)

Viable tumor environment. Tumor survival is dependent upon an exquisite interplay between the critical functions of stromal development and angiogenesis, local immune suppression and tumor tolerance, and paradoxical inflammation. TEMs: TIE-2 expressing monocytes; “M2” TAMs: tolerogenic tumor-associated macrophages; MDSCs: myeloid-derived suppressor cells; pDCs: plasmacytoid dendritic cells; co-stim.: co-stimulation; IDO: indoleamine 2,3-dioxygenase; VEGF: vascular endothelial growth factor; EGF: epidermal growth factor; MMP: matrix metaloprotease; IL: interleukin; TGF-β: transforming growth factor-beta; TLRs: toll-like receptors. (reference: http://www.hindawi.com/journals/cdi/2012/937253/fig1/)

Cancer:

Generating tumor vaccines and using adjuvants underway (140).   Comparison of clinical correlation and genetic responses in several studies hopes to diagnose and target the system for cancer therapies (127; 141; 131).  The recent surveys on IDO expression and human cancers show that IDO targeting is a candidate for cancer therapy since IDO expression recruiting Tregs, downregulating MHC class I and creating negative immune microenvironment for protection of development of tumors (125; 27; 142).  Inhibition of IDO expression can make advances in immunotherapy and chemotherapy fields (143; 125; 131; 144).  IDO has a great importance on prevention of cancer development (126).    There are many approaches to create the homeostasis of immune response by Immunotherapy.  However, given the complexity of immune regulations, immunomodulation is a better approach to correct and relieve the system from the disease.  Some of the current IDO targeted immunotherapy or immmunomodulations are with RNA technology for cancer prevention (145; 146; 147; 148; 149; 150) or applied on human or animals  (75; 151; 12; 115; 152; 9; 125) or chemical, (153; 154) or  radiological (155).  The targeted cell type in immune system generally DCs, monocytes (94), T cells (110; 156) and neutrophils (146; 157). On this paper, we will concentrate on DCvax on cancer treatments.

IDO and the downstream enzymes in tryptophan pathway produce a series of immunosuppressive tryptophan     metabolites that may lead into Tregs proliferation or increase in T cell apoptosis (62; 16; 27; 158), and some can   affect NK cell function (159).  The interesting part of the mechanism is, even without presence of IDO itself,    downstream enzymes of IDO in the kynurenine tryptophan degradation still show immunosuppressive outcome   (160; 73) due to not only Kyn but also TGFbeta stimulated long term responses. DC vaccination with IDO is    plausible (161) due to its power in immune response changes and longevity in the bloodstream for reversing  the system for Th17 production (162).

Taking advantage of the DC’s central role and combining with enhancing molecules for induction of immunity may overcome tolerogenic DCs in tumors of cancers (163; 164). The first successful application of DC vaccine used against advanced melanoma after loading DCs with tumor peptides or autologous cell lysate in presence of adjuvants keyhole limpet hematocyanin (KLH) (165).  Previous animal and clinical studies show use of DCs against tumors created success (165; 166; 167) as well as some problems due to heterogeneity of DC populations in one study supporting tumor growth rather than diminishing (168).

DC vaccination applied onto over four thousand clinical trial but none of them used siRNA-IDO DC vaccination method. Clinical trials evaluating DCs, loaded ex vivo with purified TAAs as anticancer immunotherapeutic interventions, also did not include IDO (Table from (169). This data is coming from 30 clinical trials, 3 of which discontinued, evaluating DCs loaded ex vivo with TAAs as an anticancer immunotherapy for 12 types of cancer [(AML(1), Breast cancer (4), glioblastoma (1), glioma (2), hepatocellular carcinoma (1), hematological malignancies (1), melanoma (6), neuroblastoma sarcoma (2), NSCLC (1), ovarian cancer (3), pancreatic cancer (3), prostate cancer (10)] at phase I, II or I/II.

Tipping the balance between Treg and Th17 ratio has a therapeutic advantage for restoring the health.  This is shown in ovarian cancer by DC vaccination with adjuvants (161).  Rebalancing of the immune system towards immunogenicity may restore Treg/Th17 ratio (162; 170) but it is complicated. The stimulation of IL10 and IL12 induce Tregs produce less Th17 while inhibiting CTL activation and its function (76; 171; 172).  When animals were pre-treated with anti-TGFbeta before vaccination, elevation in the plasma levels of IL-15 for tumor specific T cell survival in (173; 174) ovarian cancer studies was observed.   After human papilloma virus infection, the system present an increase of IL12 (175).  Opposing signal mechanism downregulates the TGFbeta to activate CTL and Th1 population with IL12 and IL15 expression (162; 173).  The effects of IL17 on antitumor properties observed by unique subset of CD4+ T cells (176) called also CD8+ T cells secrete even more IL17 (177).

Use of cytokines as adjuvants during vaccination may improve the efficacy of vaccination, since cancer vaccines, unlike infections vaccines, applied after the infection or disease started against the established adoptive immune response.  It is an almost common practice to use adjuvants to improve efficacy in immunotherapy as a combination therapy (178). Enhancing cancer vaccine efficacy via modulation of the microenvironment is another solution, if only know who are the players.  For example, changing intercellular Ca signaling in T cells is necessary to convert them to Tregs.    Several molecules can be used to initiate and lengthen the activity of intervention to stimulate IDO expression without compromising the mechanism (179) because of the positive feedback loops.  The system is complicated so generally induction is completed ex-vivo stimulation of DCs in cell lysates, or in whole tumor lysates, to create the microenvironment and natural stimulatory agents. Introduction of molecules as an adjuvants on genetic regulation on modulation of DCs are critical, because order and time of the signals, specific location/ tissue, and heterogeneity of personal needs (174; 138; 180). These studies demonstrated that IL15 with low TGFb stimulates CTL and Th1, whereas elevated TGFb with IL10 increases Th17 and Tregs in cancer microenvironments.

For example Ret-peptide antitumor vaccine contains an extracellular fragment of Ret protein and Th1 polarized immunoregulator CpG oligonucleotide (1826), with 1MT, a potent inhibitor of IDO, brought a powerful as well as specific cellular and humoral immune responses in mice (152).  The main idea of choosing Ret is to produce vaccine in ret related carcinomas because ret fulfilled two requirements, first choosing patients self-antigens for cancer therapy with a non-mutated gene, and second, there is no evidence of genetic mutations in Ret amino acids 64-269.

Table 2- IDO Clinical Trials

1

Clinical Trials From Clinicaltrials.Gov

Title And Details Of The Study

NCT Number Recruitment Condition Primary Completion Date Sponsor/Collaborator Phase orObservation. Intervention Type
2 IDO Inhibitor Study For Relapsed Or Refractory Solid Tumors NCT00739609

Terminated

Breast Cancer|Lung Cancer|Melanoma|Pancreatic Cancer|Solid Tumors October 2012 NewlLink enetics Corp. 1 CHEM
3  IDO2 Genetic Status Informs The Neoadjuvant Efficacy Of Chloroquine (CQ) In Brain Metastasis Radiotherapy NCT01727531

Recruiting

Brain Metastasis Dec. 2020 Main Line Health NA CHEM
4 Peptide Vaccine And Temozolomide For Metastatic Melanoma Patients NCT01543464

Recruiting

Malignant Melanoma September 2014 Newlink Genetics Corporation 2 CHEM
5 A Phase 1/2 Randomized, Blinded, Placebo Controlled Study Of Ipilimumab In Combination With INCB024360 Or Placebo In Subjects With Unresectable Or Metastatic Melanoma NCT01604889

Recruiting

Metastatic Melanoma August 2014 Incyte Corporation 1/2 BIOLINH+CHEM
6 A Phase 2 Study Of The IDO Inhibitor INCB024360 Versus Tamoxifen For Subjects With Biochemical-Recurrent-Only EOC, PPC Or FTC Following Complete Remission With First-Line Chemotherapy NCT01685255

Recruiting

April 2014 Incyte Corporation 2 CHEM
7   Different Injection Number Of The Same Dose Of Botulinum Toxin A On Overactive Bladder Syndrome NCT01657409

Recruiting

Overactive Bladder March 2014 Buddhist Tzu Chi General Hospital 2 CHEM
8  Study On The Effect Of Intravenous Ascorbic Acid On Intraoperative Blood Loss In Women With Uterine MyomaInterventions: Drug: Ascorbic Acid NCT01715597

Recruiting

Uterine Leiomyoma January 2014 Seoul National University Hospital 3 CHEM
9 1-Methyl-D-Tryptophan In Treating Patients With Metastatic Or Refractory Solid Tumors That Cannot Be Removed By Surgery NCT00567931

Recruiting

Unspecified Adult Solid Tumor, Protocol Specific September 2013 National Cancer Institute (NCI) 1 CHEM
10  Properties Of Mesenchymal Stem Cells In Lung Transplant NCT01668576

Recruiting

Lung Transplantation August 2013 Emory University OBS BIOL
11  The Effects Of Medical Clowns In Children Undergoing Blood Tests NCT01396876

Recruiting

Pain And Anxiety Reduction July 2012 Tel-Aviv Sourasky Medical Center NA BIOL
12   Saline Injection – Assisted Anesthesia In Eyelid Surgery NCT01239498

Recruiting

Blepharoptosis October 2011 Sheba Medical Center 4 CHEMBIOL
13   Effects Of The Consumption Of California Walnuts On Cardiovascular HealthInterventions:            Dietary Supplement: Walnuts NCT01235390

Recruiting

Cardiovascular Disease|Immune Health October 2011 University Of California, Davis|California Walnut Commission 1 FOODALERGY-WALNUT
14  Pomegranate To Reduce Maternal And Fetal Oxidative Stress And Improve Outcome In Pregnancies Complicated With Preterm Premature Rupture Of The Membranes NCT01584323

Recruiting

Preterm Premature Rupture Of Membranes|Pregnant State 2013 Rambam Health Care Campus NA FOODSUP
1 Phase II INCB024360 Study For Patients With Myelodysplastic Syndromes (MDS) NCT01822691

Not Yet Recruiting

Myelodysplastic Syndromes September 2015 H. Lee Moffitt Cancer Center And Research Institute|Incyte Corporation 2 CHEM-BIOL?
2   Title:  Schizophrenia Imaging NCT01655472

Not Yet Recruiting

Foetal Differences Between Healthy And Schizophernic Parents July 2014 Tel-Aviv Sourasky Medical Center IMAGEN
3  C11 AMT Positron Emission Tomography (PET) Imaging In Patients With Metastatic Invasive Breast Cancer NCT01302821

Not Yet Recruiting

Breast Cancer April 2014     H. Lee Moffitt Cancer Center And Research Institute|National Cancer Institute (NCI) NA BIOLDCAV-P53MTRADIA
4   Sonographic Evaluation Of Visceral Fat After Bariatric Surgery NCT01285791

Not Yet Recruiting

Morbid Obesity April 2012 Hadassah Medical Organization OBS BIOL CELL
5 How Our Immune System Can Help Fight Cancer NCT01042847

Not Yet Recruiting

Ovarian Cancer January 2011 Winthrop UniversityHospita EVALNA POLY.BIOL
6  Title: Study Of The Long-Term Effect Of Frequent Anti-VEGF Dosing On Retinal Function In Patients With Neovascular AMD NCT00533689

Not Yet Recruiting

Electrophysiology 2013 NA  Tel-Aviv Sourasky Medical Center NAEYE BIOL
7  Microbial Surveillance In Children Hospitalized For Cardiovascular Surgery NCT00426894

Not Yet Recruiting

Cardiac Surgery|Perioperative Prophylaxis 2013 NA Hadassah Medical Organization OBS
8 Study Of Chemotherapy In Combination With IDO Inhibitor In Metastatic Breast Cancer NCT01792050

Not Recruitinbut  Active  G

Metastatic Breast Cancer January 2015 Newlink Genetics Corporation 2 CHEM
9 A Dose-Escalation Study In Subjects With Advanced Malignancies NCT01195311

Not BUT  Active Recruiting

Advanced Malignancies November 2012 Incyte Corporation 1 CHEM DOSE
SP  Title:   Mesalamine To Reduce T Cell Activation In HIV Infection NCT01090102

Enrolling By Invitation

HIV Infections|Sexually Transmitted Diseases|Immune System Diseases|Lentivirus Infections|Acquired Immunodeficiency Syndrome January 2013 UC, San Francisco|California HIV/AIDS Research Program|Salix Pharmaceuticals 4 CHEM
1 Study Of Indoleamine 2,3-Dioxygenase Activity, Serum Levels Of Cytokines, BDNF, BH4 And NCT00919295

Completed

Fibromyalgia Syndrome October 2011 Mahidol University|University Of Texas|University Of Wuerzburg 2 CHEM.
2 Diagnosis Of Posttraumatic Stress Disorder Following Primary Rhegmatogenous Retinal Detachment NCT01233908

Completed

Stress Disorders, Post-Traumatic|Retinal Detachment September 2010 Sheba Medical Center OBSEYE BIOL
3 Comparison Of DCT, ORA And GAT In Eyes After Penetrating Keratoplasty NCT00834782

Completed

Corneal Transplantation December 2009 Sheba Medical Center 4
4 Disturbances Of Kynurenine Pathway Of Trytophan Metabolism In Schizophrenia: A Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) Study NCT00573300

Completed

Schizophrenia May 2009 North Suffolk Mental Health Association OBSV. BIOL
5 Effect Of Biological Therapy On Biomarkers In Patients With Untreated Hepatitis C, Metastatic Melanoma, Or Crohn Disease NCT00897312

Completed

Melanoma August 2008 Vanderbilt-Ingram Cancer Center|National Cancer Institute (NCI) OBSV.  BIOL-CHEM
6 A Prospective Comparative Study Of Induction Of Labor With A Cervical Ripening Double Balloon Vs Foley Catheter NCT00501033

Completed

Induction Of Labor|Cesarean|Endometritis February 2008 Western Galilee Hospital-Nahariya NA DEVICE
7 Tryptophan, Serotonin And Kynurenine In Septic Shock NCT00684736

Completed

Shock, Septic April 2007 Central Hospital, Versailles OBS KYN
8 Imatinib Mesylate In Treating Patients With Metastatic Breast Cancer NCT00045188

Completed

Male Breast Cancer|Recurrent Breast Cancer|Stage IV Breast Cancer July 2004 National Cancer Institute (NCI) 2 CHEM
9 IDO Peptid Vaccination For Stage III-IV Non Small-Cell Lung Cancer Patients. NCT01219348

Completed

NSCLC|Lung Cancer NA IDO Peptide Vaccinantio 1
10  Indoleamine 2,3-Dioxygenase (IDO) Activity In Patients With Chronic Lymphocytic Leukemia (CLL) NCT01397916

Completed

CLL NA Tampere University Hospital NA
11   Tryptophan Metabolism In Kidney Disease NCT00758537

Completed

Chronic Kidney Disease NA Charite University, Berlin, Germany OBS BIOLTRP LEVELS
12 Observational To Investigate The Efficacy Of CRESTOR 5mg In Reaching LDL-C Target Goals In Patients Who Are At High Risk For A Cardiovascular Event NCT00347217

Completed

HypercholesteremiaCardiovascular NA Astrazeneca 4 CHEM
13 The Association Between Delivery Method And Maternal Rehospitalization NCT00501501

Completed

Hospitalization NA Western Galilee Hospital-Nahariya OBS BIOL
14 Uterine Flora During Elective And Urgent Cesarean Sections NCT00500019

Completed

Endometritis NA Western Galilee Hospital-Nahariya OBS BIOL
15   Title: Pilot, Proof-Of-Concept Study Of Sublingual Tizanidine In Children With Chronic Traumatic Brain Injury (TBI) NCT00287157

Completed

 Traumatic Brain Injury NA Teva GTC 1B CHEM

Another example came from demonstration of proliferating hemangiomas, benign endothelial tumors and often referred as hemangiomas of infancy appearing at head or neck, expresses IDO and slowly regressed as a result of immune mediated process. Large scale of genomic analysis shows insulin like growth factor 2 as the key regulator of hematoma growth (Ritter et al. 2003).

We set out to develop new technology with our previous expertise in immunotherapy and immunomodulation (181; 182; 183; 184), correcting Th17/Th1 ratio (185), and siRNA technology (186; 187).  We developed siRNA-IDO-DCvax. Patented two technologies “Immunomodulation using Altered DCs (Patent No: US2006/0165665 A1) and Method of Cancer Treatments using siRNA Silencing (Patent No: US2009/0220582 A1). In melanoma cancer DCs were preconditioned with whole tumor lysate but in breast cancer model pretreatment completed with tumor cell lysate before siRNA-IDO-DCvax applied. Both of these studies presented a success without modifying the autanticity of DCs but decreasing the IDO expression to restore immunegenity by delaying tumor growth in breast cancer (147) and in melanoma (188).  Thus, our DCvax specifically interfere with IDO without disturbing natural structure and content of the DCs in vivo.  Thus, we showed that DCvax can carry on this technology to clinical applications.   Furthermore, our method of intervention is more sophisticated since it has a direct interaction mechanism with ex-vivo DC modulation without creating long term metabolism imbalance in Trp/Kyn metabolite mechanisms with corrective and non-invasive actioins.

There are several reasons for us to combine DCs with siRNA technology for making DCvax.  First, prevention of tumor development studies targeting non-enzymatic pathway initiated by pDCs conditioned with TGFbeta is specific to IDO1 (189). Second, IDO upregulation in antigen presenting cells allowing metastasis show that most human tumors express IDO at high levels (123; 124).  Third, tolerogenic DCs secretes several molecules some of them are transforming growth factor beta (TGFb), interleukin IL10), human leukocyte antigen G (HLA-G), and leukemia inhibitory factor (LIF), and non-secreted program cell death ligand 1 (PD-1 L) and IDO, indolamine 2.3-dioxygenase, which promote tumor tolerance. Thus, we took advantage of DCs properties and IDO specificity to prevent the tolerogenicity with siRNA-IDO DC vaccine in both melanoma and breast cancer.  Fourth, IDO expression in DCs makes them even more potent against tumor antigens and create more T cells against tumors. IDOs are expressed at different levels by both in broad range of tumor cells and many subtypes of DCs including monocyte-derived DCs (10), plasmacytoid DCs (142), CD8a+ DCs (190), IDO compotent DCs (17), IFNgamma-activated DCs used in DC vaccination.  These DCs suppress immune responses through several mechanisms for induction of apoptosis towards activated T cells (156) to mediate antigen-specific T cell anergy in vivo (142) and for enhancement of Treg cells production at sites of vaccination with IDO-positive DCs+ in human patients (142; 191; 192; 168; 193; 194).  If DCs are preconditioned with tumor lysate with 1MT vaccination they increase DCvax effectiveness unlike DCs originated from “normal”, healthy lysate with 1MT in pancreatic cancer (195).  As a result, we concluded that the immunesupressive effect of IDO can be reversed by siRNA because Treg cells enhance DC vaccine-mediated anti-tumor-immunity in cancer patients.

Gene silencing is a promising technology regardless of advantages simplicity for finding gene interaction mechanisms in vitro and disadvantages of the technology is utilizing the system with specificity in vivo yet improved(186; 196).  siRNA technology is one of the newest solution for the treatment of diseases as human genomics is only producing about 25,000 genes by representing 1% of its genome. Thus, utilizing RNA opens the doors for more comprehensive and less invasive effects on interventions. Thus this technology is still improving and using adjuvants.  Silencing of K-Ras inhibit the growth of tumors in human pancreatic cancers (197), silencing of beta-catenin in colon cancers causes tumor regression in mouse models (198), silencing of vascular endothelial growth factor (VGEF) decreased angiogenesis and inhibit tumor growth (199).   Combining siRNA IDO and DCvax from adult stem cell is a novel technology for regression of tumors in melanoma and breast cancers in vivo. Our data showed that IDO-siRNA reduced tumor derived T cell apoptosis and tumor derived inhibition of T cell proliferation.  In addition, silencing IDO made DCs more potent against tumors since treated or pretreated animals showed a delay or decreased the tumor growth (188; 147).

Clinical Trials:

First FDA approved DC-based cancer therapies for treatment of hormone-refractory prostate cancer as autologous cellular immunotherapy (163; 164).  However, there are many probabilities to iron out for a predictive outcome in patients.  Clinical trials report shows 38 total studies specifically IDO related function on cancer (16), eye (3), surgery (2), women health (4), obesity (1), Cardiovascular (2), brain (1), kidney (1), bladder (1), sepsis shock (1), transplant (1),  nervous system and behavioral studies (4), HIV (1).  Among these only 22 of which active, recruiting or not yet started to recruit, and 17 completed and one terminated. Most of these studies concentrated on cancer by the industry, Teva GTC ( Phase I traumatic brain injury), Astra Zeneca (Phase IV on efficacy of CRESTOR 5mg for cardiovascular health concern), Incyte corporation (Phase II ovarian cancer), NewLink Genetics Corporation (Phase I breast/lung/melanoma/pancreatic solid tumors that is terminated; Phase II malignant melanoma recruiting, Phase II active, not recruiting metastatic breast cancer, Phase I/II metastatic melanoma, Phase I advanced malignancies), and Salix Corp-UC, San Francisco and HIV/AIDS Research Programs (for HIV Phase IV enrolling by invitation).  Most of these studies based on chemotherapy but there are few that use biological methods completed study with  IDO vaccine peptide vaccination for Stage III-IV non-small-cell lung cancer patients (NCT01219348), observational study on effect of biological therapy on biomarkers in patients with untreated hepatitis C, metastasis melanoma, or Crohn disease by IFNalpha and chemical (ribavirin, ticilimumab (NCT00897312), polymorphisms of patients after 1MT drug application in treating patients with metastatic or unmovable refractory solid tumors by surgery (NCT00758537), IDO expression analysis on MSCs (NCT01668576), and not yet recruiting intervention with adenovirus-p53 transduced dendric cell vaccine , 1MT , radiation, Carbon C 11 aplha-methyltryptophan (NCT01302821).

Among the registered clinical trials some of them are not interventional but  observational and evaluation studies on Trp/Kyn ratio (NCT01042847), Kyn/Trp ratio (NCT01219348), Kyn levels (NCT00897312, NCT00573300),  RT-PCR analysis for Kyn metabolism (NCT00573300, NCT00684736, NCT00758537), and intrinsic IDO expression of mesenchymal stem cells in lung transplant with percent inhibition of CD4+ and CD8+ T cell proliferation toward donor cells (NCT01668576), determining polymorphisms (NCT00426894). These clinical trials/studies are immensely valuable to understand the mechanism and route of intervention development with the data collected from human populations.

 

Future Actions for Molecular Diagnosis and Targeted Therapies:

Current survival or response rate is around 40 to 50 % range.  By using specific cell type, selected inhibition/activation sequence based on patient’s genomic profile may improve the efficacy of clinical interventions on cancer treatments.

Targeted therapies for specific gene regulation through signal transduction are necessary but there are few studies with genomics based approach.  On the other hand, there are surveys, observational or evaluations (listed in clinical trials section) registered with www.clinicaltrials.gov that will provide a valuable short-list of molecules.  Preventing stimulation of Ido1 as well as Tgfb-1gene expression by modulating receptor mediated phosphorylation between TGFb/SMAD either at Mad-Homology 1 (MH1) or Mad-Homology 1 (MH2) domains is possible (79; 82; 80). Within Smads there is a conserved Mad-Homology 1 (MH1) domain, which is a DNA binding module contains tightly bound Zinc atom. So the zinc can be targeted with a small molecule adjuvant.  Smad MH2 domain is also well conserved as one the most diverse protein-signal interacting molecule during signal transduction due to two important Serine residues located extreme distal C-termini at Ser-Val-Ser in Smad 2 or at pSer-X-PSer in RSmads (80).  Kyn activated orphan G protein–coupled receptor, GPR35 with unknown function with a distinct expression pattern that collides with IDO sites since its expression at high levels of the immune system and the gut (63) (200; 63).

 

The first study to connect IDO with cancer shows that group (75) so direct targeting to regulate IDO expression is another method.  It is best to act through modulating ISREs in its promoter with RNA-peptide combination technology. Indirectly, IDO can be regulated through Bin1 gene expression control over IDO since Bin1 is a negative regulator of IDO and prevents IDO expression.  IDO is under negative genetic control of Bin1, BAR adapter–encoding gene Bin1 (also known as Amphiphysin2). Bin1 functions in cancer suppression, because attenuation of Bin1 observed in many human malignancies (141; 201; 202; 203; 204; 205; 206).  Absence of Bin1, null Bin1-/- mice studies, upregulates IDO through STAT1- and NF-kB-dependent in tumor cells to escape from T cell–dependent antitumor immunity.   Detailed molecular genetics studies showed that alternative spicing of Bin1 creates tumor suppressor affect.  Its activities also depends on these spliced outcome, such as Exon 10, in muscle. On the other hand, alternative spliced Exon12A contributes brain cell differentiation (209; 210).  In turn Exon 13 in mice has importance in role for regulating growth. When Bin1 is deleted or mutated C2C12 myoblasts interrupted due to its missing Myc, cyclinD1, or growth factor inhibiting genes like p21WAF1 (207; 208).  Thus, myc becomes a natural target and biomarker as well.

Myc is a target at the junction between IDO gene interaction and Trp metabolism.  Bin1 interacts with Myc either early-dependent on Myc or late-independent on Myc, meaning Myc is not present. This gene regulation also interfered by the long term signaling mechanism related to moonlighting pathway (73; 74).  Hence, Trp/Kyn, Kyn/Trp, Th1/Th17 ratios are important to be observed in patients peripheral blood. These direct and indirect gene interactions place Bin1 to function in cell differentiation (211; 212; 205).

Moonlighting maintains the tolerance. The key factor is in this pathway is Kyn so by reviewing one of the microarray analysis for Kyn affect is critical. This data showed that there are 25 genes affected by Kyn, two of which are upregulated and 23 of them downregulated (100). The list of genes and additional knowledge based on previous intervention methods are a good place to start creating a diagnostics panel as a biomarker to monitor outcomes of given immunotherapies. The short list of candidates are as an adjuvant or co-stimulation agents are myc, NfKB at IKKA, C2CD2, CREB3L2, GPR115, IL2, IL8, IL6, and IL1B, mir-376 RNA, NFKB3, TGFb, RelA, and SH3RF1. From the preivos studies we can also add LIP, Fox3P, CTLA-4, Bin1 and IMPACT to the list.  In addition, specific use of TLR, conserved sequences of IDO across its homologous structures and ISREs of IDO or glucorticoid response elements of TDO are great direct targets to modulate the mechanism. Furthermore, some of the signaling pathway molecules CCR6, CCR5, RORgammat, Jak, STAT, IRFs, MH1 and MH2 domains of Smads may add a value.

Endothelial cell coagulation activation mechanism and pDC maturation or immigration from lymph nodes to bloodstream should marry to control not only IDO expression but also genesis of preferred DC subsets. Stromal mesenchymal cells are activated by this modulation at vascular system and interferes with metastasis of cancer. First, thrombin (human factor II) is a well regulated protein in coagulation hemostasis has a role in cell differentiation and angiogenesis. Protein kinase activated receptors (PARs), type of GPCRs, moderate the actions. Second, during hematopoietic response endothelial cells produce hematopoietic growth factors (213; 214) to revise the vascular structure.  Third, components of bone marrow stroma cells include monocytes, adipocytes, and mesenchymal stem cells (215), which are addressing occurrence of coagulapathologies, DIC, bleeding, thrombosis, and penalizing patients response rate towards therapies and decreasing survival rates specially in breast, lung, prostate cancers.

Both silencing IDO in DCs and reinstallinig antitumor immunity by inhibiting tumor-derived immunosupressive molecule IDO through RNA inference combined with our specialization in stem cell technology created a novel method with a success in vivo.  This data suggests that IDO siRNA DCvax can provide a clinical intervention to increase survival rate and prevention of cancer.

Personal genomic profiles are powerful tool to improve efficacy in immunotherapies so considering the influence of age (young vs. adult) and state of immune system (innate vs. adopted or acquired immunity) are important as well.

CONCLUSION


IDO has a confined function in immune system through complex interactions to maintain hemostasis of immune responses. The genesis of IDO stem from duplication of bacterial IDO-like genes.  Inhibition of microbial infection and invasion by depleting tryptophan limits and kills the invader but during starvation of tryptophan the host may pass the twilight zone since tryptophan required by host’s T cells.  Thus, the host cells in these small pockets adapt to new microenvironment with depleted tryptophan and oxygen poor conditions. Hence, the cell metabolism differentiates to generate new cellular structures, like nodules and tumors under the protection of constitutively expressed IDO in tumors, DCs to inhibit T cell proliferation. On the other hand, having a dichotomy in IDO function can be a potential limiting factor that means is that IDO’s impact on biological system could be variable at many levels based on target cells, IDO’s capacity, pathologic state of the disease and conditions of the microenvironment.  This complexity requires a very close monitoring to analyze the outcome and to prevent conspiracies over the data since some previous studies generated paradoxical results.  Healthcare cost of current therapies through chemotherapies, radiotherapies is very high and provide low efficacy.  Clinical interventions of immunotherapies require control of more than one system, such as coagulation and vascular biology manipulations for a higher efficacy and survival rate in cancer patients. Our siRNA and DC technologies based on stem cell modulation will provide at least prevention of cancer development and hopefully prevention in cancer.

References

1. Biochemistry of tryptophan in health and disease. BenderDA. 1983, Mol Aspects Med , pp. 6:101–197.

2. Molecular insights into substrate recognition and catalysis by indolamine 2,3-dioxygenase. Forouhar, F., Anderson, R., Mowat, C.F, et al. 2006, PNAS, pp. vol. 104, no:2, 473-478.

3. Importance of the Two Interferon-stimulated Response Element. Konan KV, Taylor, MW. 1996, J. Biol. Chem.-, pp. 19140-5.

4. induction of indolamine 2,3 dioxygenase: A mechanism of the anti-tumor activity of interferon gamma. Ozaki, Y., Edelstein, M.P., Duch, D.S. 1998, PNAS USA., pp. vol:85, 1242-1246.

5. Localization of the human indoleamine 2,3-dioxygenase (IDO) gene to the pericentromeric region of human chromosome 8. . Burkin, D. J., Kimbro, K. S., Barr, B. L., Jones, C., Taylor, M. W., Gupta, S. L. 1993, Genomics , pp. 17: 262-263.

6. Localization of indoleamine 2,3-dioxygenase gene (INDO) to chromosome 8p12-p11 by fluorescent in situ hybridization. Najfeld, V., Menninger, J., Muhleman, D., Comings, D. E., Gupta, S. L. 1993, Cytogenet. Cell Genet. , pp. 64: 231-232.

7. Molecular cloning, sequencing and expression of human interferon-gamma-inducible indoleamine 2,3-dioxygenase cDNA. . Dai, W., Gupta, S. L. 1990, Biochem. Biophys. Res. Commun. , pp. 168: 1-8.

8. Gene structure of human indoleamine 2,3-dioxygenase. Kadoya, A., Tone, S., Maeda, H., Minatogawa, Y., Kido, R. 1992, Biochem. Biophys. Res. Commun. , pp. 189: 530-536.

9. A gene atlas of th emouse and human protein-encoding transcriptomes. Andrew I. Su, Tim Wiltshire, Serge Batalov , Hilmar Lapp , Keith A. Ching , David Block, Jie Zhang , Richard Soden , Mimi Hayakawa , Gabriel Kreiman , Michael P. Cooke , John R. Walker , and John B. Hogenesch. 2004, PNAS, pp. vol. 101, no. 166062-6067 (http://dx.doi.org:/10.1073/pnas.0400782101).

10. Indoleamine 2,3-dioxygenase production by human dendritic cells results in the inhibition of T cell proliferation. Hwu P, Du MX, Lapointe R, Do M, Taylor MW, Young HA. 2000, J. Immunol, pp. 164:3596–3599.

11. Inhibition of T cell proliferation by acrophage tryptophan catabolism. Munn, D.H. et al. 1999, J. Exp. Med., p. 189:1363.

12. HeLa cells cocultured with peripheral blood lymphocytes acquire an immuno-inhibitory phenotype through up-regulation of indoleamine 2,3-dioxygenase activity. Logan, G. J., Smyth, C. M. F., Earl, J. W., Zaikina, I., Rowe, P. B., Smythe, J. A., Alexander, I. E. 2002, Immunology, pp. 105:478-487.

13. Indoleamine 2,3-Dioxygenase – Is It an Immun Suppressor? Soliman H, Mediaville-Varela M, Antonia S. 2010, Cancer J. , pp. 16:354-359.

14. Targeting the immunoregulatory indoleamine 2,3-dioxygenase pathway in immunotherapy. Johnson BA, III, Baban B, Mellor AL. 2009, Immunotherapy. , pp. 645–661.

15. Indoleamine 2,3-dioxygenase and regulation of T cell immunity. AL., Mellor. 2005, Biochem Biophys Res Commun. , pp. 338(1):20–24.

16. Fallarino, F., Grohmann, U., Hwang, K. W., Orabona, C., Vacca, C., Bianchi, R., Belladonna, M. L., Fioretti, M. C.Modulation of tryptophan catabolism by regulatory T cells. Fallarino, F., Grohmann, U., Hwang, K. W., Orabona, C., Vacca, C., Bianchi, R., Belladonna, M. L., Fioretti, M. C., Alegre, M.-L., Puccetti, P. 2003, Nature Immun., pp. 4: 1206-1212.

17. CTLA-4-Ig regulates tryptophan catabolism in vivo. Grohmann, U., Orabona, C., Fallarino, F., Vacca, C., Calcinaro, F., Falorni, A., Candeloro, P., Belladonna, M. L., Bianchi, R., Fioretti, M. C., Puccetti, P. 2002, Nature Immun. , pp. 3: 1097-1101.

18. Reverse signaling through GITR ligand enables dexamethasone to activate IDO in allergy. Grohmann, U., Volpi, C., Fallarino, F., Bozza, S., Bianchi, R., Vacca, C., Orabona, C., Belladonna, M. L., Ayroldi, E., Nocentini, G., Boon, L., Bistoni, F., Fioretti, M. C., Romani, L., Riccardi, C., Puccetti, P. 2007, Nature Med., pp. 13:579-586.

19. Cells expressing indoleamine 2,3-dioxygenase inhibit T cell responses. Mellor, A. L., Keskin, D. B., Johnson, T., Chandler, P., Munn, D. H. 2002, J. Immun. , pp. 168: 3771-3776.

20. Chon, SY, Hassanain, HH, Piine, R., and Gupta, SL. 1995, J. Interferon Cytokine Res. , pp. 15, 517-526.

21. Levy, ED, KEsler, DS, Pine, R., Reich, N, and Darnell, JE.Jr et al. 1988, Genes Dev, pp. 2,383-393.

22. Benoist, C. and Manthis, D. 1990, Annu. Rev of Immunol., pp. 8, 681-715.

23. Dorn, A, Durand, B., Marling, C., Meur, M.L., Beoist, C., and Mathis, D. 1987, PNAS USA, pp. 34, 6249-6253.

24. Konan, K.V. Ph.D. Thesis. Transcriptional Regulation of the Indolamine 2,3-oxygenase Gene. s.l. : Indiana University, Bloominigton, 1995.

25. Tryptophan pyrrolase of rabbit intestine: D- and L–tryptophan cleaving enzyme or enzymes. Yamamoto, S., and Hayashi, O. 1967, J Biol Chem, pp. 242: 5260-5266.

26. Prevention of allogeneic fetal rejection by tryptophan catabolism. Munn, DH, Zhou M, Attwood JT, Bondarev I, Conway SJ, Marshall B, Brown C, Mellor AL. 1998, Science, pp. 281:1191–3.

27. Evidence for a tumoral immune resistance mechanismbased on tryptophan degradation by indoleamine 2,3-dioxygenase. Uyttenhove, C. et al. 2003, Nature Med. 9,, pp. 1269–1274 .

28. Pregnancy: success and failure within the Th1/Th2/Th3 paradigm. Raghupathy, R. 2001., Seminars in Immunology, pp. Volume 13, Issue 4, Pages 219–227.

29. Why is the fetal allograft not rejected? Davies, C. J. March 2007 , J ANIM SCI , pp. vol. 85 no. 13 suppl E32-E35 .

30. Exploring the mechanism of tryptoophan 2,3-dioxygenase. Thackray, S., Mowat, C.G., Chapman, K. 2008, Biochem. Society Transaction., pp. 36, 1120-1123.

31. The new life of a centenarian: signalling functions of NAD(P). Berger F, Ramírez-Hernández MH, Ziegler M. 2004, Trends Biochem Sci , pp. 29:111–118 .

32. Biochemistry of tryptophan in health and disease. DA, Bender. 1983, Mol Aspects Med, pp. 6:101–197.

33. Poliovirus induces indoleamine-2,3-dioxygenase and quinolinic acid synthesis in macaque brain. Heyes MP, Saito K, Jacobowitz D, Markey SP, Takikawa O, Vickers JH. 1992, FASEB J., pp. 6:2977–2989.

34. Sanni LA, Thomas SR, Tattam BN, Moore DE, Chaudhri G, Stocker R, Hunt NH 1998Dramatic changes in oxidative tryptophan metabolism along the kynurenine pathway in experimental cerebral and noncerebral malaria. . Sanni LA, Thomas SR, Tattam BN, Moore DE, Chaudhri G, Stocker R, Hunt NH. 1998, Am J Pathol, pp. 152:611–619.

35. Induction of pulmonary indoleamine 2,3-dioxygenase by intraperitoneal injection of bacterial lipopolysaccharide. . Yoshida R, Hayaishi O. 1978, Proc Natl Acad Sci USA , pp. 75:3998–4000.

36. Induction of indoleamine 2,3-dioxygenase in mouse lung during virus infection. . Yoshida R, Urade Y, Tokuda M, Hayaishi O. 1979, Proc Natl Acad Sci USA , pp. 76:4084–4086.

37. Induction of pulmonary indoleamine 2,3-dioxygenase by intraperitoneal injection of bacterial lipopolysaccharide. Yoshida R, Hayaishi. 1978, PNAS USA, pp. 3998-4000.

38. Sequence of human 2,3-dioxygenase (TDO2): presence of a glucorticoid response-like element composed of a GTT repeat and intronic CCCCT repeat. Comings DE, Muhleman D, Dietz G, Sherman M, Forest. 1995, Genomics, pp. 29:390-396165.

39. Studies on the biosynthesis of Nicotinamide adenine inucleotide. II.Arole of picolinic carboxylase in the Biosynthesisofnicotinamideadeninedinucleotidefromtryptophan in mammals. Ikeda M, Tsuji H, Nakamura S, Ichiyama A, Nishizuka Y, HayaishiO. 1965, J. Biol. Chem. , pp. 240: 1395-1401.

40. The Secret Life of NAD+: An Old Metabolite Controlling New Metabolic Signaling Pathways. Houtkooper R.H., Carles Cantó C. , Wanders, R.J. and Auwerx, J. 2010, Endocrine Reviews , pp. vol. 31 no. 2 194-223,  http://dx.doi.org:/10.1210/er.2009-0026.

41. Stimulation of Nicotinamide adenine dinucleotide biosynthetic pathways delays axonal degeneration after axotomy. Sasaki Y, Araki T, Milbrandt J. 2006, J Neurosci , pp. 26: 8484–8491.

42. European Nicotinamide Diabetes Intervention Trial (ENDIT): a randomised controlled trial of intervention before the onset of type 1 diabetes. Gale EA, Bingley PJ, Emmett CL, CollierT. 2004, Lancet., pp. 363:925–931.

43. Safety of high-dose nicotinamide: a review. Knip M, Douek IF, Moore WP, Gillmor HA, McLean AE, Bingley PJ, Gale EA. 2000, Diabetologia, pp. 43:1337–1345.

44. Large supplements of nicotinic acid and nicotinamide increase tissue NAD and poly(ADP-ribose) levels but do not affect diethylnitrosamine-induced altered hepatic foci in Fischer-344 rats. JacksonTM, Rawling JM, Roebuck BD, Kirkland JB. 1995, J Nutr , p. 125:1455.

45. Characterization and evolution of vertebrate indelamine 2,3-dihydrogenases IDOs from monotremes and marsupials. Yuasa, HJ, Ball, HJ, Ho, YF, Austin, CJ, et al. 2009, Comp. Biochem. Physiol. B. Biochem.. Mol. Biol., pp. 153 (2): 137-144.

46. Novel tryptophan catabolic enzyme IDO2 is the preferred biochemical target of the antitumor indolamine 2,3-dihydrogenase inhibitor compound D-1 methyl-tryptophan. Metz, R., Duhadaway, JB, Kamasani, U, Laury-Kleintop, L., Muller, AJ, Prendergast, GC. 2007, Cancer Res., pp. 67 (15): 7082-7087.

47. Total synthesis of exiguamines A and B inspired by catechollamine chemistry. Sofiyev, V, Lumb, JP, Volgraf, M., Trauner, D. 2012, Chemistry., pp. 18 (16): 4999-5005.

48. Molecular evolution of bacterial indolamine 2,3-dioxygenase. Yuasa, H J, Ushigoe, A, Ball, HJ. 2011, Gene., pp. 484 (1) : 22-31.

49. Infectious tolerance and the long-term acceptance of transplant tissue. Waldman, H., Adams, E., Fairchild, P., and Cobbold, S. 2006, J. Immunol., pp. 212:301-313.

50. Molecular evolution and characterizationof fungal indolamine 2,3-dioxygenases. Yuasa, HJ and Ball, HJ. 2012, J. Mol. Eval., pp. 72 (2): 160-168.

51. convergent evolution. The gene structure of Sulculus 41 kDa myoglobin is homologous with tht of human indolamine dioxygenase. Suzuki, T, Imai, K. 1996, Biochim. Biophys. Acta., pp. 1308(1):41-48.

52. Evolutionof myoglobin. Suzuki, T., Imai, K. 1998, Cell Mol Life Sci, pp. 54(9):979-1004.

53. A myoglobin evolved from indolamine 2,3-dioxygenase, trtptophan-degrading enzyme. Suzuki, T., Kawamichi, H., Imai, K. 1998, Comp Biochem Phisiol. Mol. Biol., pp. 121(2):117-128.

54. Do molluscs possess indolamine 2,3-dioxygenase? Yuasa, HJ and Suzuki, T. 2005, Comp. Biochem. Physiol. B. Biochem. Mol. Biol. , pp. (3) 445-454.

55. Comparison studies of the indolamine dioxygenase-like myoglobin from the abalone Sulculus diversicolor. Suzuki, T., Imai, K. 1997, Comp. Biohem. Phsiol B Biochem Mol Biol, pp. 117 (4)599-604.

56. Orchestration of the immune response by dendritic cells. Buckwalter MR, Albert ML. 2009, Curr Biol., pp. 19(9):355–361.

57. Dendritic cells and the control of immunity. Banchereau J, Steinman RM. 1998, Nature., pp. 245–52.

58. IDO expression by dendritic cells: tolerance and tryptophan catabolism. . Munn DH, Mellor AL. 2004, Nat Rev Immunol. , pp. 762–74.

59. Monocyte and Macrophage. Gordon, S. and Taylor, P.R. 2005, NATURE REVIEWS | IMMUNOLOGY , pp. vol:5, 953-964.

60. Blood monocytes consist of two principal subsets with distinct migratory properties. Geissmann F, Jung S, Littman DR. 2003, Immunity. , pp. 19:71–82.

61. Identification of a novel cell type in peripheral lymphoid organs of mice. I Morphology, quantitation, tissue distribution. . Steinman RM, Cohn ZA. 1973, J Exp Med., pp. 137(5):1142–1162.

62. T cell apoptosis by tryptophan catabolism. Fallarino F, Grohmann U, Vacca C, Bianchi R, Orabona C, Spreca A, Fioretti MC, Puccetti P. 2002, Cell Death Differ , pp. 9:1069–1077.

63. Kynurenine is a novel endothelium derived relaxing factor produced during inflammation. Wang, et al. 2010, Nat. Med., pp. 16(3): 279-285.

64. Activation of the noncanonical NF-kB pathway by HIV controls a Dendritic cell immunoregulatory phenotype. Manches, O. Fernandez, V.M.,, Plumas, J., Chaperot, L., and Bhardwaj, N. 2012, PNAS, pp. vol: 109, 14122-14127.

65. B cells inhibit induction of T cell-dependent tumor immunity. Qin, Z., Richter, G., Schuler, T., Ibe, S., Cao, X, Blakenstein, T. 1998, Nat. Med, p. 4:627.

66. Different partners, Opposite Outcmes: A new perspective of immunobiology of Indolamine 2,3 dioxygenase. Orabona, C., Pallotta, M.T., Grohman, U. 2012, Molecular Medicine., pp. 18:834-842.

67. Indolamine 2,3-dioxygenase: From catalyst to signaling function. Fallarino, F., Grohman, U., and Puccetti, P. 2012, Eurepean J. of Immunol. , pp. 42:1932-1937.

68. IDO: more than an enzyme. Chen, W. 2011, Nature Immonology, pp. 809-811.

69. Indolamine2,3-dehydrogenase in lung dendritic cells promotes Th2 responses and allergic inflammation. Xu, H., Oriss, T.B., Fei, M., Henry, A.C., Melgert, B.N., Chen, L., Mellor, A.L. 2008, PNAS USA, pp. 105: 6690-6695.

70. The immunoregulatory enzyme IDO paradoxically drives B-cellmediated autoimmunity. Scott, G.N., DuHadaway, J., Pigott, E., Ridge, N., Prendergast, G.C., Muller, A.J., Mandik-Nayak, L. 2009, J. Immunol., pp. 182:7509-7517.

71. Tryptophan deprivation sensitizes activated T cells to apoptosis prior to cell division. Lee GK, Park HJ, Macleod M, Chandler P, Munn DH, Mellor AL. 2002, Immunology , pp. 107:452–460.

72. Enzymology of NAD+ homeostasis in man. . Magni G, Amici A, Emanuelli M, Orsomando G, Raffaelli N, Ruggieri S. 2004, Cell Mol Life Sci , pp. 61:19–34.

73. Kynurenine pathway enzymes in dendritic cells initiate tolerogenesis in the absence of functional IDO. . Belladonna ML, Grohmann U, Guidetti P, Volpi C, Bianchi R, Fioretti MC, Schwarcz R, Fallarino F, Puccetti P. 2006, J Immunol. , pp. ;177:130–7.

74. An indogenous tumour promoting ligand of the human aryl hydrocarbon receptor. Opitz, et. al. 2011, pp.  http://dx.doi.org:/10.1038/nature10491,.

75. Inhibition of indoleamine 2,3-dioxygenase, animmunoregulatorytarget of the cancer suppression gene Bin1, potentiates cancer chemotherapy. Muller, A. J. et al. 2005, Nature Med. , pp. 11, 312–319 .

76. TGF-b; a master of all T cell trades. Li, M.O., Fravell, R.A. 2008, Cell. , pp. 134: 392-404.

77. Palotta, M.T. et al. 2011, Nat. Immunol., pp. 12:870-878.

78. Chen, W. et al. 2003, J. Exp. Immunol., p. 198: 1875.

79. Smads: transcriptional activators of TGF-beta responses. . Derynck R, Zhang Y, Feng XH. 1998, Cell , pp. 95 (6): 737–40.
http://dx.doi.org:/10.1016/S0092-8674(00)81696-7.PMID 9865691. .

80. Smad transcription factors. Massagué J, Seoane J, Wotton D. 2005, Genes Dev, pp. 19 (23): 2783–810.
http://dx.doi.org:/10.1101/gad.1350705. PMID .

81. A structural basis for mutational inactivation of the tumour suppressor Smad4. Shi Y, Hata A, Lo RS, Massagué J, Pavletich NP. 1997, Nature., pp. 388 (6637): 87–93.  http://dx.doi.org:/10.1038/40431. PMID 9214508.

82. Promoting bone morphogenetic protein signaling through negative regulation of inhibitory Smads. Itoh F, Asao H, Sugamura K, Heldin CH, ten Dijke P, Itoh S. 2001, EMBO J., pp. 20 (15): 4132–   http://dx.doi.org:/10.1093/emboj/20.15.4132. PMC 149146. PMID 11483516.

83. SMAD_Signaling_Network. http://www.sabiosciences.com. [Online] 2013. http://www.sabiosciences.com/pathway.php?sn=SMAD_Signaling_Network.

84. Immune inhibitory receptors. Revetch, J.V., and Lanier, L.L. 2000, Science., pp. 290:84-89.

85. Soc3 drives proteasomal degradation of indolamine 2,3-dioxygenase (IDO) and antagonizes IDO-dependent tolerogenesis. Orabona, C., Pallotta, M., Volpi, C., et al. 2008, PNAS USA, pp. 105: 20828-20833.

86. Cutting edge; silencing supressor of cytokine signaling3 expression in dendritic cells turns CD28-Ig from immune adjuvant to supressant. Orabona, C.,, Belladonna, M.L., et all. 2005, J. Immunol., pp. 174: 6582-6586.

87. Molecular signatures of T-cell inhibition in HIV-1 infection. Larsson, M., Shankar. E.M, Che, K.F., Ellegard, R., Barathan, M., Velu, V., and Kamarulzaman, A. 2013, Retrovirology, p. 10:31.

88. TGF-beta and CD4+CD25+ regulatory cells. Huber, S. and Schramn, C. 2006, Front. Bioscie., pp. 11:1014-1023.

89. Immune Escape as a fundemental trait of cancer; focus on IDO. Prendergast, G.C. 2008, Oncogene., pp. 27, 3889-3900.

90. Il-6 inhibits the tolerogenic functionof CD8+ dendritic cells expressing indolamine 2,3-dioxygenase. Grohman, U., Fallarino, F., et al. 2001, J. Immunol., pp. 167:708-714.

91. Avoiding horror autotoxicus: Th eimportance of dentritic cells in peripheral T cell tolerance. Steinman, R.M., and Nussenzweig, M.C. 2002, PNAS, pp. no:1, 351-358.

92. Dendritic-cell function in Toll-like receptor- and MyD88-knockout mice . Kaisho, T., Akira, S. 2001, Trends Immunol , pp. 22,78-83.

93. Innate sensing of self and non-self RNAs by Toll-like receptors. Sioud, M. 2006., Trends Mol Med., pp. 12:67–76.

94. Impaired expression of indoleamine 2, 3-dioxygenase in monocyte-derived dendritic cells in response to Toll-like receptor-7/8 ligands. Furset, G., Fløisand, Y. and Sioud, M. 2008, Immunology., pp. 123(2): 263–271, http://dx.doi.org:/10.1111/j.1365-2567.2007.02695.x.

95. Toll-;ike receptor 9 mediated induction of the immunorepressor pathway of tryptophan metabolism. Fallarino, F., and Puccetti, P. 2006, Eur. J. of Imm., pp. 36:8-11.

96. Toll-like receptors and host defense against microbial pathogens: bringing specificity to the innate immune system. . Netea MG, der Graaf C, Van der Meer JWM, Kullberg BJ. 2004, J Leukoc Biol. , pp. 75:749–55.

97. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. . Heil F, Hemmi H, Hochrein H, et al. 2004, Science. , pp. 303:1526–9.

98. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. . Diebold SS, Kaisho T, Hemmi H, Akira S, Reis e Sousa C. 2004., Science. , pp. 303:1529–31. .

99. The role of CpG motifs in innate immunity. Krieg, A.M. 2000., Curr Opin Immunol., pp. 12:35–43.

100. Anendogenous tumour-promoting ligand of the human aryl hydrocarbon receptor. Opitz, C.A., Litzenburger, U.M., Sahm, F., Ott,M., Tritschler, I., Trump, S. 2011, Nature, pp. vol 478; 197-203.

101. Impaired impression of Indolamine 2,3-deoxygenase in monocyte derived DCs in response to TLR-7/8. Furset, G., Floisand, Y., Sioud, M. 2007, Immunology, pp. 263-271.

102. Activationof the noncanonical NF-kB pathway by HIV controls a Dendritic cell immunoregulatory phenotype. Manches, O. Fernandez, V.M.,, Plumas, J., Chaperot, L., and Bhardwaj, N. 2012, PNAS, pp. vol: 109, 14122-14127.

103. Regulation of dendritic cell numbers and maturation by lipopolysaccharide in vivo . de Smedt, T., Pajak, B., Muraille, E., Lespagnard, L., Heinen, E., De Baetselier, P., Urbain, J., Leo, O., Moser, M. 1996, J. Exp. Med., pp. 184,1413-1424.

104. Subsets of dendritic cell precursors express different Toll-like receptors and respond to different microbial antigens . Kadowaki, N., Ho, S., Antonenko, S., de Waal Malefyt, R., Kastelein, R. A., Bazan, F., Liu, Y-J. 2001, J. Exp. Med., pp. 194,863-869 .

105. TRAF6 is a critical factor for dendritic cell maturation and development . Kobayashi, T., Walsh, P. T., Walsh, M. C., Speirs, K. M., Chiffoleau, E., King, C. G., Hancock, W. W., Caamano, J. H., Hunter, C. A., Scott, P., Turka, L. A., Choi, Y. 2003, Immunity , pp. 19,353-363 .

106. Activation of interferon regulatory factor-3 via toll-like receptor 3 and immunomodulatory functions detected in A549 lung epithelial cells exposed to misplaced U1-snRNA. Sadik CD, Bachmann M, Pfeilschifter J, Mühl H. 2009, Nucleic Acids Res. , pp. 37(15):5041-56. http://dx.doi.org:/10.1093/nar/gkp525. Epub 2009 Jun 18.

107. Triggering of the dsRNA sensors TLR3, MDA5, and RIG-I induces CD55 expression in synovial fibroblasts. Karpus ON, Heutinck KM, Wijnker PJ, Tak PP, Hamann J. 2012, PLoS One., p. 7(5):e35606.  http://dx.doi.org:/10.1371/journal.pone.0035606. Epub 2012 May 10.

108. The structure of the TLR5-flagellin complex: a new mode of pathogen detection, conserved receptor dimerization for signaling. Lu J, Sun PD. 2012, Sci Signal., p. 5(216):pe11.   http://dx.doi.org:/10.1126/scisignal.2002963. .

109. Flagellin/Toll-like receptor 5 response was specifically attenuated by keratan sulfate disaccharide via decreased EGFR phosphorylation in normal human bronchial epithelial cells. Shirato K, Gao C, Ota F, Angata T, Shogomori H, Ohtsubo K, Yoshida K, Lepenies B, Taniguchi N. 2013, Biochem Biophys Res Commun., pp. doi:pii:S0006-291X(13)00779-1. http://dx.doi.org:/10.1016/j.bbrc.2013.05.009. [Epub ahead of print].

110. Differential induction of interleukin-10 and interleukin-12 in dendritic cells by microbial Toll-like receptor activators and skewing of T-cell cytokine profiles Infect. Qi, H., Denning, T. L., Soong, L. 2003, Immun. , pp. 71,3337-3342 .

111. Thoma-Uszynski, S., Kiertscher, S. M., Ochoa, M. T., Bouis, D. A., Norgard, M. V., Miyake, K., Godowski, P. J., Roth, M. D.Activation of Toll-like receptor 2 on human dendritic cells triggers induction of IL-12, but not IL-10 . Thoma-Uszynski, S., Kiertscher, S. M., Ochoa, M. T., Bouis, D. A., Norgard, M. V., Miyake, K., Godowski, P. J., Roth, M. D., Modlin, R. L. 2000, J. Immunol. , pp. 165,3804-3810.

112. Toll-like receptor 2 (TLR2) and TLR4 differentially activate human dendritic cells . Re, F., Strominger, J. L. 2001, J. Biol. Chem. , pp. 276,37692-37699.

113. Pasare, C., Medzhitov, R. (2003) Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells. Pasare, C., Medzhitov, R. 2003, Science , pp. 299,1033-1036 .

114. What is the role of regulatory T cells in the success of implantation and early pregnancy? Saito, S., Shima, T., Nakashima, A., Shiozaki, A., Ito, M., Sasaki, Y. 2007, J Assist Reprod Genet, pp. 24: 379-386.

115. Sleeping Beauty-based gene therapy with indoleamine 2,3-dioxygenase inhibits lung allograft fibrosis. . Liu H, Liu L, Fletcher BS, Visner GA. 2006, FASEB J, pp. 20:2384-2386. .

116. Indoleamine 2,3-dioxygenase expression in transplanted NOD Islets prolongs graft survival after adoptive transfer of diabetogenic splenocytes. Alexander AM, Crawford M, Bertera S, et al. 2002, Diabetes. , pp. 51(2):356–365.

117. Solid Cancers after Bone Marrow Transplantatioin. Curtis, R.E., Rowlings, P.A., Deeg, J., Schirer, D.A. et al. 1997, The New England Journal of Medicine., pp. 336, No: 13: 897-904.

118. More ADO about IDO; GVHD (commentary). Curti, A., Trabanelli, S., Lemoli, M. 2008, Blood, p. 2950.

119. Jasperson, et al, . 2008, Blood, p. 3257.

120. Tolerance, DCs and tryptophan: much ado about IDO. Grohmann U, Fallarino F, Puccetti P. 2003, Trends Immunol, pp. 24:242-248.

121. Evidence for a tumoral immune resistance mechanism based on tryptophan degradation by indoleamine 2,3-dioxygenase. Uyttenhove C, Pilotte L, Théate I, Stroobant V, Colau D, Parmentier N, et al. 2003, Nat Med , pp. 9:1269–74.

122. Indoleamine 2,3-dioxygenase is a critical regulator of acute graft-versus-host disease lethality. Lisa K. Jasperson, Christoph Bucher, Angela Panoskaltsis-Mortari, Patricia A. Taylor, Andrew L. Mellor, David H. Munn, and Bruce R. Blazar. 2008., Blood., pp. 111:3257-3265.

123. The metabolism of tryptophan. 2. The metabolism of tryptophan in patients suffering from cancer of the bladder. . Boyland, E. & Willliams, D.C. 1956, Biochem. J., pp. 64, 578−582 .

124. Tryptophan metabolism in carcinoma of the breast. . Rose, D. 1967, Lancet , pp. 1, 239−241 .

125. Inhibitors of indoleamine-2,3-dioxygenase for cancer therapy: can we see the wood for the trees? . Löb S, Königsrainer A, Rammensee HG, Opelz G, Terness P. 2009;, Nat Rev Cancer , pp. 9:445–52.  http://dx.doi.org:/10.1158/1078-0432.CCR-11-1331.

126. The hallmarks of cancer. . Hanahan, D. & Weinberg, R.A. 2000., Cell., pp. 100, 57−70.

127. Indoleamine 2,3-Dioxygenase Expression in Human Cancers: Clinical and Immunologic Perspectives. Godin-Ethier, J., Hanafi,L.A., Piccirillo,C.A. and Lapointe, R. 2011, Clin Cancer Res, pp. 17; 6985,  http://dx.doi.org:/10.1158/1078-0432.CCR-11-1331.

128. Dendritic cell modification as a route to inhibiting corneal graft rejection by the indirect pathway of allorecognition. Khan A, Fu H, Tan LA, Harper JE, Beutelspacher SC, Larkin DF, Lombardi G, McClure MO, George AJ. 2013, Eur J Immunol., pp. 43(3):734-46.  http://dx.doi.org:/10.1002/eji.201242914. Epub 2013 Jan 18.

129. Possible role of the ‘IDO-AhR axis’ in maternal-foetal tolerance. . Hao K, Zhou Q, Chen W, Jia W, Zheng J, Kang J, Wang K, Duan T. 2013, Cell Biol Int., pp. 37(2):105-8. doi: 10.1002/cbin.10023. Epub 2013 Jan 2.

130. Implication of indolamine 2,3 dioxygenase in the tolerance toward fetuses, tumors, and allografts. . Dürr S, Kindler V. 2013, J Leukoc Biol. , pp. 93(5):681-7. doi: 10.1189/jlb.0712347. Epub 2013 Jan 16.

131. Evidence for a tumoral immune resistance mechanism based on tryptophan degradation by indoleamine 2,3-dioxygenase. Uyttenhove C, Pilotte L, Théate I, Stroobant V, Colau D, Parmentier N, et al. 2003, Nat Med, pp. 9:1269–74.

132. NAturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Sagaguchi, S. 2004, Annu. Rev. of Immunol., pp. 22: 531-562.

133. Regulatory T cells in transplantation tolerance. Wood, K.J., zZSakaguchi, S.,. 2003, Nat. Rev. Immunol., pp. 3; 199-210.

134. The cell awareness of paternal alloantigens during pregnancy. Tafuri, A., Alferink, J., Hammerling, G.J., Arnold, B. 1995, Science, pp. 270; 630-3.

135. Adenovirus mediated CTLA4Ig transgene therapy alleviates abortion by inhibiting spleen lymphocyte proliferation and regulating apoptosis in the feto-placental unit. Li W, Li B, Li S. 2013, J Reprod Immunol. , pp. 97(2):167-74.

136. A distinct tolerogenic subset of splenic IDO(+)CD11b(+) dendritic cells from orally tolerized mice is responsible for induction of systemic immune tolerance and suppression of collagen-induced arthritis. Park MJ, Park KS, Park HS, Cho ML, Hwang SY, Min SY, Park MK, Park SH, Kim HY. 2012, Cell Immunol. , pp. 278(1-2):45-54. http://dx.doi.org:/10.1016/j.cellimm.2012.06.009. Epub 2012 Jul 10.

137. Pharmacological targeting of IDO-mediated tolerance for treating autoimmune disease. Penberthy, W.T. 2007, Curr. Drug Metab., pp. 8:(3):245-266.

138. Indoleamine 2,3-dioxygenase expression in transplanted NOD Islets prolongs graft survival after adoptive transfer of diabetogenic splenocytes. Alexander AM, Crawford M, Bertera S, et al. 2002, Diabetes. , pp. 51(2):356–365.

139. Heme oxygenase-1 plays an important protective role in experimental autoimmune encephalomyelitis. . Liu Y, Zhu B, Luo L, Li P, Paty DW, Cynader MS. 2001., NeuroReport. , pp. 12(9):1841–1845. .

140. Tumor vaccines in 2010: need for integration. Koos, D., Josephs, SF, Alexandrescu, DT et al. 2010, Cell Immunol, pp. 263: 138-147.

141. BIN1 is a novel MYC-interacting protein with features of a tumor suppressor. . Sakamuro, D., Elliott, K., Wechsler-Reya, R. & Prendergast, G.C. 1996, Nat. Genet. , pp. 14, 69−77. .

142. Expression of Indolamine 2,3-dioxygenase by plasmacytoid dendritic cells in tumor draining nodes. Munn, S.H., Sharma, M.D., Hou, D., Baban, B. et al. 2004, J. Clin. Invest. , pp. 114: 280-290.

143. Indoleamine 2,3-Dioxygenase Expression in Human Cancers: Clinical and Immunologic Perspectives. Jessica Godin-Ethier, Laïla-Aïcha Hanafi, Ciriaco A. Piccirillo, and Réjean Lapointe. 2011 , Clin Cancer Res, pp. 17; 6985,   http://dx.doi.org:/10.1158/1078-0432.CCR-11-1331.

144. Potential regulatory function of human dendritic cells expressing indoleamine 2,3-dioxygenase. . Munn, D.H. et al. 2002, Science 297, 1867−1870, pp. 297, 1867−1870 .

145. An HDAC inhibitor enhances cancer therapeutic efficiency of RNA polymerase III promoter-driven IDO shRNA. Yen MC, Weng TY, Chen YL, Lin CC, Chen CY, Wang CY, Chao HL, Chen CS, Lai MD. 2013, Cancer Gene Ther. , p.  http://dx.doi.org:/10.1038/cgt.2013.27. [Epub ahead of print].

146. Systemic delivery of Salmonella typhimurium transformed with IDO shRNA enhances intratumoral vector colonization and suppresses tumor growth. Blache CA, Manuel ER, Kaltcheva TI, Wong AN, Ellenhorn JD, Blazar BR, Diamond DJ. 2012, Cancer Res. , pp. 72(24):6447-56.
http://dx.doi.org:/10.1158/0008-5472.CAN-12-0193. Epub 2012 Oct 22.

147. Silencing IDO in dendritic cells: a novel approach to enhance cancer immunotherapy in a murine breast cancer model. Zheng X, Koropatnick J, Chen D, Velenosi T, Ling H, Zhang X, Jiang N, Navarro B, Ichim TE, Urquhart B, Min W. 2013, Int J Cancer., pp. 132(4):967-77. http://dx.doi.org:/10.1002/ijc.27710. Epub 2012 Jul 20.

148. Immunosuppressive CD14+HLA-DRlow/neg IDO+ myeloid cells in patients following allogeneic hematopoietic stem cell transplantation. Mougiakakos D, Jitschin R, von Bahr L, Poschke I, Gary R, Sundberg B, Gerbitz A, Ljungman P, Le Blanc K. 2013, Leukemia. , pp. 27(2):377-88.
http://dx.doi.org:/10.1038/leu.2012.215. Epub 2012 Jul 25.

149. Upregulated expression of indoleamine 2, 3-dioxygenase in primary breast cancer correlates with increase of infiltrated regulatory T cells in situ and lymph node metastasis. Yu J, Sun J, Wang SE, Li H, Cao S, Cong Y, Liu J, Ren X. 2011, Clin Dev Immunol. , p. 11:469135.
http://dx.doi.org:/10.1155/2011/469135. Epub 2011 Oct 24.

150. Skin delivery of short hairpin RNA of indoleamine 2,3 dioxygenase induces antitumor immunity against orthotopic and metastatic liver cancer. Huang TT, Yen MC, Lin CC, Weng TY, Chen YL, Lin CM, Lai MD. 2011, Cancer Sci. , pp. 102(12):2214-20. http://dx.doi.org:/10.1111/j.1349-7006.2011.02094.x. .

151. Indoleamine 2,3-dioxygenase expression in transplanted NOD Islets prolongs graft survival after adoptive transfer of diabetogenic splenocytes. . Alexander AM, Crawford M, Bertera S, et al. 2002, Diabetes. , pp. 51(2):356–365.

152. Prevention of Spontaneous Tumor Development in a ret Transgenic Mouse Model by Ret Peptide Vaccination with Indoleamine 2,3-Dioxygenase Inhibitor 1-Methyl Tryptophan. Zeng, J., Cai, S., Yi, Y., et al. 2009, Cancer Res., pp. 69: 3963-3970, http://dx.doi.org:/10.1158/0008-5472.CAN-08-2476.

153. Medicinal electronomics bricolage design of hypoxia-targeting antineoplastic drugs and invention of boron tracedrugs as innovative future-architectural drugs. Hori H, Uto Y, Nakata E. 2010, Anticancer Res. , pp. 30(9):3233-42. .

154. Synthesis of 4-cyano and 4-nitrophenyl 1,6-dithio-D-manno-, L-ido- and D-glucoseptanosides possessing antithrombotic activity. Bozó E, Gáti T, Demeter A, Kuszmann J. 2002, Carbohydr Res. , pp. 3;337(15):1351-65.

155. Radiopharmaceuticals XXVII. 18F-labeled 2-deoxy-2-fluoro-d-glucose as a radiopharmaceutical for measuring regional myocardial glucose metabolism in vivo: tissue distribution and imaging studies in animals. Gallagher BM, Ansari A, Atkins H, Casella V, Christman DR, Fowler JS, Ido T, MacGregor RR, Som P, Wan CN, Wolf AP, Kuhl DE, Reivich M. 1977, J Nucl Med. , pp. 18(10):990-6.

156. Tryptophan deprivation sensitizes activated T cells to apoptosis prior to cell division. Lee GK, Park HJ, Macleod M, Chandler P, Munn DH, Mellor AL. 2002, Immunology, pp. 107:452–460.

157. Induction of indoleamine 2,3-dioxygenase by uropathogenic bacteria attenuates innate responses to epithelial infection. Loughman JA, Hunstad DA. 2012 , J Infect Dis. , pp. 205(12):1830-9. http://dx.doi.org:/10.1093/infdis/jis280.

158. Inhibition of allogeneic T cell proliferation by indoleamine 2,3-dioxygenase-expressing dendritic cells: mediation of suppression by tryptophan metabolites. . Terness, P., et al. 2002, J. Exp. Med.196:447–457., pp. 196:447–457.

159. The tryptophan catabolite L-kynurenine inhibits the surface expression of NKp46- and NKG2D-activating receptors and regulates NK-cell function. . Chiesa, M.D., et al. 2006, Blood. , pp. 108:4118–4125.38.

160. Differential effects of the tryptophan metabolite 3-hydroxyanthranilic acid on the proliferation of human CD8+ T cells induced by TCR triggering or homeostatic cytokines. Weber, W.P., et al. 2006, Eur. J. Immunol. , pp. 36:296-304.

161. Dendritic cell vaccination against ovarian cancer–tipping the Treg/TH17 balance to therapeutic advantage? Cannon MJ, Goyne H, Stone PJ, Chiriva-Internati M. 2011, Expert Opin Biol Ther. , pp. 11(4):441-5.  http://dx.doi.org:/10.1517/14712598.2011.554812. .

162. Phenotype, distribution, generation, and functional and clinical relevance of Th17 cells in the human tumor environments. . Kryczek I, Banerjee M, Cheng P, et al. 2009, Blood., pp. 114:1141–1149. .

163. The use of dendritic cells in cancer immunitherapy. Schuler, G., Schuker-Turner, B., Steinman, RM,. 2003, Curr. Opin. Immunol., pp. 15: 138-147.

164. Clinical applications of dentritic cell vaccines. Morse, MA, Lyerly, HK. 2000, Curr. Opin. Mol Ther., pp. 2:20-28.

165. Vaccination of melanoma patients with peptide or tumor lysate-pulsed dendritic cells. Nestle, FO, Alijagic, S., Gillet, M. et al. 1998, Nat. Med., pp. 4: 328-332.

166. Dentritic cell based tumor vaccination in prostate and renal cell cancer: a systamatic review. Draube, A., Klein-Gonzales, Matheus, S et al. 2011, Plos One, p. 6:e1881.

167. [Online] http://www.fda.gov/BiologicsBloodVaccines/CellularGeneTherapy-Products/ApprovedProducts/ucm210215.htm..

168. Dendritic cell based antitumor vaccination: impact of functional indolamine 2,3-dioxygenase expression. Wobster, m., Voigt, H., Houben, R. et al. 2007, Cancer Immunol Immunother, pp. 56:1017-1024.

169. [Online] oncoimmunology.2012 October1; 1(17):1111-1134, doi: 10.4161/onci.21494.

170. Interleukins 1beta and 6 but not transforming growth factor-beta are essential for the differentiation of interleukin 17-producing human T helper cells. Acosta-Rodriguez EV, Napolitani G, Lanzavecchia A, Sallusto F. 2007 , Nat Immunol. , pp. 8(9):942-9. .

171. IFNgamma promotes generationof Il-10 secreting CD4+ T cells that suppress generationof CD8responses in an antigen-experienced host. Liu, X.S., Leerberg, J., MacDonald, K., Leggatt, G.R., Frazer, I.H. 2009, J. Immunol., pp. 183: 51-58.

172. Antigen, in the presence of TGF-beta, induces up-regulationof FoxP3gfp+ in CD4+ TCR transgenic T cells that mediate linked supressionof CD8+ T cell responses. . Kapp, J.A., Honjo, K., Kapp, L.M., Goldsmith, K., Bucy, R.P. 2007, J. Immunol., pp. 179: 2105-2114.

173. Opposing effects of TGF-beta and IL-15 cytokines control the number of short lived effecctor CD8+ T cells. Sanjabi, S, Mosaheb, M.M., Flavell, R.A. 2009, Immunity., pp. 31; 131-144.

174. Synergestic enhancement of CD8+ T cell mediated tumor vaccines efficacy by an anti-tumor forming growth factor-beta monoclonal antibody. . Terabe, M., Ambrosino, E., Takaku, S. et al. 2009, Clin. Cancer Res., pp. 15; 6560-9.

175. IL-12 enhances CTL synapse formationand induces self-reactivity. Markinewicz, MA, Wise, EL, Buchwald, ZS et al. 2009, J. Immunol., pp. 182: 1351-1362.

176. Tumor specific Th17-polarized cells eradicate large established melanoma. Muranski, P., Boni, A., Antony, PA, et al. 2008, Blood, pp. 112; 362-373.

177. Type17 CD8+ T cells dispplay enhanced antitumor immunity. Hinrichs, C.S., Kaiser, A., Paulos, C.M., et al. 2008, Blood., pp. 112:362-373.

178. Marying Immunotherapy with Chemotherapy: Why Say IDO? Muller, AJ, and Prendergrast, GC. 2005, Cancer Research, pp. 65: 8065-8068.

179. Enhancing Cancer Vaccine efficacy via Modulationof the Tumor Environment. Disis, ML. 2009, Clin Cancer Res, pp. 15: 6476-6478.

180. Systemic inhibition of transforming growth factor beta 1 in glioma bearing mice improves the therapeutic efficacy of glioma-associated antigen peptide vaccines. Ueda, R., Fujita, M., Zhu, X., et al. 2009, Clin. Cancer res., pp. 15: 6551-9.

181. Immune modulation by silencing IL-12 productionin dendritic cells using smal interfering RNA. Hill, JA, Ichim, TE, Kusznieruk, KP, et al. 2003, J. Immunol, pp. 171:809-813.

182. Immune modulation and tolerance induction by RelB-silenced dentritic cells through RNA interference. Li, M. Zang, X, Zheng, X, et al. 2007, J. Immunol, pp. 178: 5480-7.

183. RNAi mediated CD40-CD54 interruption promotes tolerance in autoimmune arthritis. . Zheng, X., Suzuki, M., Zhang, X., et al. 2010, Arthritis Res. Ther., p. 12:R13.

184. Dendritic cells genetically engineered to express Fas ligand induce donor-specific hyporesponsiveness and prolong allograft survival. Min, WP. Gorczynki, R., huang, XY et al. 2000, J. Immunol., pp. 164:161-167.

185. LF15-0195 generates tolerogenic dendritic cells by supressionof NF-kappaB signaling through inhibitionof IKK activity. . Yang, J., Bernier, SM, Ichim, TE, et al. 2003, J Leukoc. Biol., pp. 74: 438-447.

186. RNA interfrence: A potent tool for gene specific therapeutics. . Ichim, TE, Li, M., Qian, H., Popov, HI, Rycerz, K., Zheng, X., White, D., Zhong, R., and Min, WP. 2004, Am. J. Transplant, pp. 4:1227-1236.

187. A novel in vivo siRNA delivery system specifically targeting dendritic cells and silencing CD40 genes for immunomodulation. Zheng, X., Vladau, C., Zhang, X. et al. 2009, Blood, pp. 113:2646-2654.

188. Reinstalling Antitumor Immunity by Inhibiting Tumor derived ImmunoSupressive Molecule IDO through RNA interference. Zheng, X et al. 2006, Int. Journal of Immunology., pp. 177:5639-5646.

189. Roles of TGFbeta in metastasis. Padua, D., Massague, J. 2009, Cell Res., pp. 19;89-102.

190. Functional expression of indolamine2,3-dioxygenase by murine CDalpha+dendritic cells. Fallarino, F., Vacca, C, Orabona, C et al. 2002, Int Immunol., pp. 14:65-8.

191. Indolamine2,3-dioxygenase controls conversion of Fox3+ Tregs to TH17-like cells in tumor draining lymph nodes. Sharma, MD, Hou, DY, Liu, Y et al. 2009, Blood, pp. 113: 6102-11.

192. IDO upregulates regulatory T cells via tryptoophan catabolite and supresses encephalitogenic T cell responses in experimental autoimmune encephalomyelitis. Yan, Y, Zhang, GX, Gran, B et al. 2010, J Immunol, pp. 185; 5953-61.

193. IDO activates regulatory T cells and blocks their conversion into Th-17-like T cells. Baban, B, Chandler, PR, Sharma, MD et al. 2009, J Immunol, pp. 183; 2475-83.

194. Enhancement of vaccine-mediated antitumor immunity in cancer patients after depletionof regulatory T cells. Dannull, J., Farrand, KJ, Mathews, SA, et al. 2005, J Clin Invest, pp. 115: 3623-33.

195. 1-MT enhances potency of tumor cell lysate pulled dentritic cells against pancreatic adenocarcinoma by downregulating percentage of Tregs. Li, Y, Xu, J, Zhou, H. et al. 2010, J Huazhong Univ Sci Technol Med Sci , pp. 30: 344-8.

196. siRNA mediated antitumorigenesis for drug target validation and therapeutics. Lu, PY, Xie, FY and Woodle, MC. 2003, Curr Opin Mol. Ther., pp. 5:225-234.

197. Stable supression of tumorigenicity by virus-mediated RNA interference. Brumellkamp, TR, Bernards, R, Agami, R. 2002, Cancer Cell, pp. 2; 243-247.

198. Small interferring RNAs directed against beta-catenin inhibit the in vitro and in vivo growth of colon cancer cells. Verma, UN, Surabhi, RM, Schmaltieg, A., Becerra, C., Gaynor, RB. 2003, Clin. Cancer. Res., pp. 9:1291-1300.

199. siRNA mediated inhibition of vascular endothelial growth factor severely limits tumor resistance to antiangiogeneic thromboposdin-1 and slows tumor vascularization and growth. Filleur, S., Courtin, A, Ait-Si-Ali, S., Guglielmi, J., Merel, C., Harel-Bellan, A., CLezardin, P., and Cabon, F. 2003, Cancer Res, pp. 63; 3919-3922.

200. Kynurenic acid as a ligand for orphan G protein-coupled receptor GPR35. . Wang, J., et al. 2006, J. Biol.Chem. , pp. 281:22021–22028.

201. Bin1 functionally interacts with Myc in cells and inhibits cell proliferation by multiple mechanisms. Elliott, K. et al. 1999, Oncogene , pp. 18, 3564−3573 .

202. Mechanism for elimination of a tumor suppressor: aberrant splicing of a brain-specific exon causes loss of function of Bin1 in melanoma. . Ge, K. et al. 1999, Proc. Natl. Acad. Sci. USA, pp. 96, 9689−9694. .

203. Losses of the tumor suppressor Bin1 in breast carcinoma are frequent and reflect deficits in a programmed cell death capacity. Ge, K. et al. 2000, Int. J. Cancer , pp. 85, 376−383.

204. Loss of heterozygosity and tumor suppressor activity of Bin1 in prostate carcinoma. Ge, K. et al. 2000, Int. J. Cancer , pp. 86, 155−161.

205. Expression of a MYCN-interacting isoform of the tumor suppressor BIN1 is reduced in neuroblastomas with unfavorable biological features. . Tajiri, T. et al. 2003, Clin. Cancer Res., pp. 9, 3345−3355.

206. Targeted deletion of the suppressor gene Bin1/Amphiphysin2 enhances the malignant character of transformed cells. Muller, A.J., DuHadaway, J.B., Donover, P.S., Sutanto-Ward, E. & Prendergast, G.C. 2004, Cancer Biol. Ther. , p. 3.

207. Interactions of myogenic factors and the retinoblastoma protein mediates muscle commitment and cell differentiation. Gu, WJ., Scheniider,W., Condrolli,G., Kaushal,, S, Mahdavi,V., Nadal-Gnard, B. 1993, Cell, pp. 72; 309-324.

208. Structural analysis of the human BIN1 gene: evidence of tissue-specific transcriptional regualtion and alternate splicing. Wechsler-Reya, R, Sakamuro, J., Zhang, J., DuHadaway, J., and Predengast. 1998, J of Biol Chem.

209. A role for th ePutative Tuimor Supressor Bin1 in Muscle Differentiation. Wechsler-Reya, R., Elliott, KJ, Prendergast, GC. 1998, Molecular and Cellular Biology, p. 18 (1) :566.

210. The putative tumor repressor BIN1 is a short lived nuclear phosphoprotein whose localization is altered in malignant cells. Wechsler-Reya, R., Elliot, K., Herlyn, M., Prendergast, GC. 1997, Cancer Res, pp. 57: 3258-3263.

211. Transformation selective apoptosis by farnesyltransferase inhibitors requires Bin1. DuHadaway, J.B. et al. 2003, Oncogene, pp. 22, 3578−3588 (2003).

212. The c-Myc-interacting adapter protein Bin1 activates a caspase-independent cell death program. Elliott, K., Ge, K., Du, W. & Prendergast, G.C. 2000., Oncogene , pp. 19, 4669−4684.

213. Growth stimulation of human bone marrow cells in agar culture by vascular cells. Knudtzon, S., and Mortensen, BT. 1975, Blood, pp. 46 (6) 937-943.

214. Exogenous endothelial cells as accelerators of hematopoietic reconstitution. Mizer, C., Ichim, TE, Alexandrescu, DT, DAsanu, CA, Ramos, F., Turner, A., Woods, EJ, Bogon, V., Murphy, MP, Koos, D., and Patel, A. 2013, J. Translational Medicine, p. 10: 231.

215. Dissecting the bone marrow microenvironment . Torok-Storb, B. et al. 1999, Annals of New York Academy of Science, pp. 872: 164-170.

217. Yuasa, XX and Ball YY. 2011.

218. Possible role of the ‘IDO-AhR axis’ in maternal-foetal tolerance. Hao K, Zhou Q, Chen W, Jia W, Zheng J, Kang J, Wang K, Duan T. 2013, Cell Biol Int. , pp. 37(2):105-8. doi: 10.1002/cbin.10023. .

219. Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells. Pasare, C., Medzhitov, R. 2003, Science , pp. 299,1033-1036 .

220. Activation of Toll-like receptor 2 on human dendritic cells triggers induction of IL-12, but not IL-10. Thoma-Uszynski, S., Kiertscher, S. M., Ochoa, M. T., Bouis, D. A., Norgard, M. V., Miyake, K., Godowski, P. J., Roth, M. D., Modlin, R. L. 2000, J. Immunol. , pp. 165,3804-3810.

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aprotinin-sequence.Par.0001.Image.260

aprotinin-sequence.Par.0001.Image.260 (Photo credit: redondoself)

English: Protein folding: amino-acid sequence ...

Protein folding: amino-acid sequence of bovine BPTI (basic pancreatic trypsin inhibitor) in one-letter code, with its folded 3D structure represented by a stick model of the mainchain and sidechains (in gray), and the backbone and secondary structure by a ribbon colored blue to red from N- to C-terminus. 3D structure from PDB file 1BPI, visualized in Mage and rendered in Raster3D. (Photo credit: Wikipedia)

 

 

 

 

 

 

 

 

 

 

 

 

The Effects of Aprotinin on Endothelial Cell Coagulant Biology

Demet Sag, PhD*†, Kamran Baig, MBBS, MRCS; James Jaggers, MD, Jeffrey H. Lawson, MD, PhD

Departments of Surgery and Pathology (J.H.L.) Duke University Medical Center Durham, NC  27710

Correspondence and Reprints:

                             Jeffrey H. Lawson, M.D., Ph.D.

                              Departments of Surgery & Pathology

                              DUMC Box 2622

                              Durham, NC  27710

                              (919) 681-6432 – voice

                              (919) 681-1094 – fax

                              lawso006@mc.duke.edu

*Current Address: Demet SAG, PhD

                          3830 Valley Centre Drive Suite 705-223, San Diego, CA 92130

Support:

Word Count: 4101 Journal Subject Heads:  CV surgery, endothelial cell activationAprotinin, Protease activated receptors,

Potential Conflict of Interest:         None

Abstract

Introduction:  Cardiopulmonary bypass is associated with a systemic inflammatory response syndrome, which is responsible for excessive bleeding and multisystem dysfunction. Endothelial cell activation is a key pathophysiological process that underlies this response. Aprotinin, a serine protease inhibitor has been shown to be anti-inflammatory and also have significant hemostatic effects in patients undergoing CPB. We sought to investigate the effects of aprotinin at the endothelial cell level in terms of cytokine release (IL-6), tPA release, tissue factor expression, PAR1 + PAR2 expression and calcium mobilization. Methods:  Cultured Human Umbilical Vein Endothelial Cells (HUVECS) were stimulated with TNFa for 24 hours and treated with and without aprotinin (200KIU/ml + 1600KIU/ml). IL-6 and tPA production was measured using ELISA. Cellular expression of Tissue Factor, PAR1 and PAR2 was measured using flow cytometry. Intracellular calcium mobilization following stimulation with PAR specific peptides and agonists (trypsin, thrombin, Human Factor VIIa, factor Xa) was measured using fluorometry with Fluo-3AM. Results: Aprotinin at the high dose (1600kIU/mL), 183.95 ± 13.06mg/mL but not low dose (200kIU/mL) significantly reduced IL-6 production from TNFa stimulated HUVECS (p=0.043). Aprotinin treatment of TNFa activated endothelial cells significantly reduce the amount of tPA released in a dose dependent manner (A200 p=0.0018, A1600 p=0.033). Aprotinin resulted in a significant downregulation of TF expression to baseline levels. At 24 hours, we found that aprotinin treatment of TNFa stimulated cells resulted in a significant downregulation of PAR-1 expression. Aprotinin significantly inhibited the effects of the protease thrombin upon PAR1 mediated calcium release. The effects of PAR2 stimulatory proteases such as human factor Xa, human factor VIIa and trypsin on calcium release was also inhibited by aprotinin. Conclusion:  We have shown that aprotinin has direct anti-inflammatory effects on endothelial cell activation and these effects may be mediated through inhibition of proteolytic activation of PAR1 and PAR2. Abstract word count: 297

INTRODUCTION   Each year it is estimated that 350,000 patients in the United States, and 650,000 worldwide undergo cardiopulmonary bypass (CPB). Despite advances in surgical techniques and perioperative management the morbidity and mortality of cardiac surgery related to the systemic inflammatory response syndrome(SIRS), especially in neonates is devastatingly significant. Cardiopulmonary bypass exerts an extreme challenge upon the haemostatic system as part of the systemic inflammatory syndrome predisposing to excessive bleeding as well as other multisystem dysfunction (1). Over the past decade major strides have been made in the understanding of the pathophysiology of the inflammatory response following CPB and the role of the vascular endothelium has emerged as critical in maintaining cardiovascular homeostasis (2).

CPB results in endothelial cell activation and initiation of coagulation via the Tissue Factor dependent pathway and consumption of important clotting factors. The major stimulus for thrombin generation during CPB has been shown to be through the tissue factor dependent pathway. As well as its effects on the fibrin and platelets thrombin has been found to play a role in a host of inflammatory responses in the vascular endothelium. The recent discovery of the Protease-Activated Receptors (PAR), one of which through which thrombin acts (PAR-1) has stimulated interest that they may provide a vital link between inflammation and coagulation (3).

Aprotinin is a nonspecific serine protease inhibitor that has been used for its ability to reduce blood loss and preserve platelet function during cardiac surgery procedures requiring cardiopulmonary bypass and thus the need for subsequent blood and blood product transfusions. However there have been concerns that aprotinin may be pro-thrombotic, especially in the context of coronary artery bypass grafting, which has limited its clinical use. These reservations are underlined by the fact that the mechanism of action of aprotinin has not been fully understood. Recently aprotinin has been shown to exert anti-thrombotic effects mediated by blocking the PAR-1 (4). Much less is known about its effects on endothelial cell activation, especially in terms of Tissue Factor but it has been proposed that aprotinin may also exert protective effects at the endothelial level via protease-activated receptors (PAR1 and PAR2). In this study we simulated in vitro the effects of endothelial cell activation during CPB by stimulating Human Umbilical Vein Endothelial Cells (HUVECs) with a proinflammatory cytokine released during CPB, Tumor Necrosis Factor (TNF-a) and characterize the effects of aprotinin treatment on TF expression, PAR1 and PAR2 expression, cytokine release IL-6 and tPA secretion.  In order to investigate the mechanism of action of aprotinin we studied its effects on PAR activation by various agonists and ligands.

These experiments provide insight into the effects of aprotinin on endothelial related coagulation mechanisms in terms of Tissue Factor expression and indicate it effects are mediated through Protease-Activated Receptors (PAR), which are seven membrane spanning proteins called G-protein coupled receptors (GPCR), that link coagulant and inflammatory pathways. Therefore, in this study we examine the effects of aprotinin on the human endothelial cell coagulation biology by different-dose aprotinin, 200 and 1600units.  The data demonstrates that aprotinin appears to directly alter endothelial expression of inflammatory cytokines, tPA and PAR receptor expression following treatment with TNF.  The direct mechanism of action is unknown but may act via local protease inhibition directly on endothelial cells.  It is hoped that with improved understanding of the mechanisms of action of aprotinin, especially an antithrombotic effect at the endothelial level the fears of prothrombotic tendency may be lessened and its use will become more routine.  

METHODS Human Umbilical Vein Endothelial Cells (HUVECS) used as our model to study the effects of endothelial cell activation on coagulant biology. In order to simulate the effects of cardiopulmonary bypass at the endothelial cell interface we stimulated the cells with the proinflammatory cytokine TNFa. In the study group the HUVECs were pretreated with low (200kIU/mL) and high (1600kIU/mL) dosages of aprotinin prior to stimulation with TNFa and complement activation fragments. The effects of TNFa stimulation upon endothelial Tissue Factor expression, PAR1 and PAR2 expression, and tPA and IL6 secretion were determined and compared between control and aprotinin treated cells. In order to delineate whether aprotinin blocks PAR activation via its protease inhibition properties we directly activated PAR1 and PAR2 using specific agonist ligands such thrombin (PAR1), trypsin, Factor VIIa, Factor Xa (PAR2) in the absence and presence of aprotinin.

Endothelial Cell Culture HUVECs were supplied from Clonetics. The cells were grown in EBM-2 containing 2MV bullet kit, including 5% FBS, 100-IU/ml penicillin, 0.1mg/mL streptomycin, 2mmol/L L-glutamine, 10 U/ml heparin, 30µg/mL EC growth supplement (ECGS). Before the stimulation cells were starved in 0.1%BSA depleted with FBS and growth factors for 24 hours. Cells were sedimented at 210g for 10 minutes at 4C and then resuspended in culture media. The HUVECs to be used will be between 3 and 5 passages.

Assay of IL-6 and tPA production Levels of IL-6 were measured with an ELISA based kit (RDI, MN) according to the manufacturers instructions. tPA was measured using a similar kit (American Diagnostica).

  Flow Cytometry The expression of transmembrane proteins PAR1, PAR2 and tissue factor were measured by single color assay as FITC labeling agent. Prepared suspension of cells disassociated trypsin free cell disassociation solution (Gibco) to be labeled. First well washed, and resuspended into “labeling buffer”, phosphate buffered saline (PBS) containing 0.5% BSA plus 0.1% NaN3, and 5% fetal bovine serum to block Fc and non-specific Ig binding sites. Followed by addition of 5mcl of antibody to approx. 1 million cells in 100µl labeling buffer and incubate at 4C for 1 hour. After washing the cells with 200µl with wash buffer, PBS + 0.1% BSA + 0.1% NaN3, the cells were pelletted at 1000rpm for 2 mins. Since the PAR1 and PAR2 were directly labeled with FITC these cells were fixed for later analysis by flow cytometry in 500µl PBS containing 1%BSA + 0.1% NaN3, then add equal volume of 4% formalin in PBS. For tissue factor raised in mouse as monoclonal primary antibody, the pellet resuspended and washed twice more as before, and incubated at 4C for 1 hour addition of 5µl donkey anti-mouse conjugated with FITC secondary antibody directly to the cell pellets at appropriate dilution in labeling buffer. After the final wash three times, the cell pellets were resuspended thoroughly in fixing solution. These fixed and labeled cells were then stored in the dark at 4C until there were analyzed. On analysis, scatter gating was used to avoid collecting data from debris and any dead cells. Logarithmic amplifiers for the fluorescence signal were used as this minimizes the effects of different sensitivities between machines for this type of data collection.  

Intracellular Calcium Measurement

Measured the intracellular calcium mobilization by Fluo-3AM. HUVECs were grown in calcium and phenol free EBM basal media containing 2MV bullet kit. Then the cell cultures were starved with the same media by 0.1% BSA without FBS for 24 hour with or without TNFa stimulation presence or absence of aprotinin (200 and 1600KIU/ml). Next the cells were loaded with Fluo-3AM 5µg/ml containing agonists, PAR1 specific peptide SFLLRN-PAR1 inhibitor, PAR2 specific peptide SLIGKV-PAR2 inhibitor, human alpha thrombin, trypsin, factor VIIa, factor Xa for an hour at 37C in the incubation chamber. Finally the media was replaced by Flou-3AM free media and incubated for another 30 minutes in the incubation chamber. The readings were taken at fluoromatic bioplate reader. For comparison purposes readings were taken before and during Fluo-3AM loading as well.  

RESULTS Aprotinin reduces IL-6 production from activated/stimulated HUVECS The effects of aprotinin analyzed on HUVEC for the anti-inflammatory effects of aprotinin at cultured HUVECS with high and low doses.  Figure 1 shows that TNF-a stimulated a considerable increase in IL-6 production, 370.95 ± 109.9 mg/mL.   If the drug is used alone the decrease of IL-6 at the low dose is 50% that is 183.95 ng/ml and with the high dose of 20% that is 338.92 from 370.95ng/ml being compared value.  TNFa-aprotinin results in reduction of the IL-6 expression from 370.95ng/ml to 58.6 (6.4fold) fro A200 and 75.85 (4.9 fold) ng/ml, for A1600.  After the treatment the cells reach to the below baseline limit of IL-6 expression. Aprotinin at the high dose (1600kIU/mL), 183.95 ± 13.06mg/mL but not low dose (200kIU/mL) significantly reduced IL-6 production from TNF-a stimulated HUVECS (p=0.043).  Therefore, the aprotinin prevents inflammation as well as loss of blood.  

Aprotinin reduces tPA production from stimulated HUVECS Whether aprotinin exerted part of its fibrinolytic effects through inhibition of tPA mediated plasmin generation examined by the effects on TNFa stimulated HUVECS. Figure 2 also demonstrates that the amount of tPA released from HUVECS under resting, non-stimulated conditions incubated with aprotinin are significantly different. Figure 2 represents that the resting level of tPA released from non-stimulated cells significantly, by 100%, increase following TNF-a stimulation for 24 hours.  After application of aprotinin alone at two doses the tPA level goes down 25% of TNFa stimulated cells.  However, aprotinin treatment of TNF-a activated endothelial cells significantly lower the amount of tPA release in a dose dependent manner that is low dose decreased 25 but high dose causes 50% decrease of tPA expression (A200 p=0.0018, A1600 p=0.033) This finding suggests that aprotinin exerts a direct inhibitory effect on endothelial cell tPA production.

Aprotinin and receptor expression on activated HUVECS

TF is expressed when the cell in under stress such as TNFa treatments. The stimulated HUVECs with TNF-a tested for the expression of PAR1, PAR2, and tissue factor by single color flow cytometry through FITC labeled detection antibodies at 1, 3, and 24hs.

 

Tissue Factor expression is reduced:

Figure 3 demonstrates that there is a fluctuation of TF expression from 1 h to 24h that the TF decreases at first hour after aprotinin application 50% and 25%, A1600 and A200 respectively.  Then at 3 h the expression come back up 50% more than the baseline.  Finally, at 24h the expression of TF becomes almost as same as baseline.  Moreover, TNFa stimulated cells remains 45% higher than baseline after at 3h as well as at 24h.

PAR1 decreased:
Figure 4 demonstrates that aprotinin reduces the PAR1 expression 80% at 24h but there is no affect at 1 and 3 h intervals for both doses.

During the treatment with aprotinin only high dose at 1 hour time interval decreases the PAR1 expression on the cells. This data explains that ECCB is affected due to the expression of PAR1 is lowered by the high dose of aprotinin.

PAR2 is decreased by aprotinin:

  Figure 5 shows the high dose of aprotinin reduces the PAR2 expression close to 25% at 1h, 50% at 3h and none at 24h.  This pattern is exact opposite of PAR1 expression.  Figure 5 demonstrates the 50% decrease at 3h interval only.  Does that mean aprotinin affecting the inflammation first and then coagulation?

This suggests that aprotinin may affect the PAR2 expression at early and switched to PAR1 reduction later time intervals.  This fluctuation can be normal because aprotinin is not a specific inhibitor for proteases.  This approach make the aprotinin work better the control bleeding and preventing the inflammation causing cytokine such as IL-6.

Aprotinin inhibits Calcium fluxes induced by PAR1/2 specific agonists

  The specificity of aprotinin’s actions upon PAR studied the effects of the agent on calcium release following proteolytic and non-proteolytic stimulation of PAR1 and PAR2. Figure 6A (Figure 6) shows the stimulation of the cells with the PAR1 specific peptide (SFLLRN) results in release of calcium from the cells. Pretreatment of the cells with aprotinin has no significant effect on PAR1 peptide stimulated calcium release. This suggests that aprotinin has no effect upon the non-proteolytic direct activation of the PAR 1 receptor. Yet, Figure 6B (Figure 6) demonstrates human alpha thrombin does interact with the drug as a result the calcium release drops below base line after high dose (A1600) aprotinin used to zero but low dose does not show significant effect on calcium influx. Figure 7 demonstrates the direct PAR2 and indirect PAR2 stimulation by hFVIIa, hFXa, and trypsin of cells.  Similarly, at Figure 7A aprotinin has no effect upon PAR2 peptide stimulated calcium release, however, at figures 7B, C, and D shows that PAR2 stimulatory proteases Human Factor Xa, Human Factor VIIa and Trypsin decreases calcium release. These findings indicate that aprotinin’s mechanism of action is directed towards inhibiting proteolytic cleavage and hence subsequent activation of the PAR1 and PAR2 receptor complexes.  The binding site of the aprotinin on thrombin possibly is not the peptide sequence interacting with receptors.

Measurement of calcium concentration is essential to understand the mechanism of aprotinin on endothelial cell coagulation and inflammation because these mechanisms are tightly controlled by presence of calcium.  For example, activation of PAR receptors cause activation of G protein q subunit that leads to phosphoinositol to secrete calcium from endoplasmic reticulum into cytoplasm or activation of DAG to affect Phospho Lipase C (PLC). In turn, certain calcium concentration will start the serial formation of chain reaction for coagulation.  Therefore, treatment of the cells with specific factors, thrombin receptor activating peptides (TRAPs), human alpha thrombin, trypsin, human factor VIIa, and human factor Xa, would shed light into the effect of aprotinin on the formation of complexes for pro-coagulant activity.    DISCUSSION   There are two fold of outcomes to be overcome during cardiopulmonary bypass (CPB):  mechanical stress and the contact of blood with artificial surfaces results in the activation of pro- and anticoagulant systems as well as the immune response leading to inflammation and systemic organ failure.  This phenomenon causes the “postperfusion-syndrome”, with leukocytosis, increased capillary permeability, accumulation of interstitial fluid, and organ dysfunction.  CPB is also associated with a significant inflammatory reaction, which has been related to complement activation, and release of various inflammatory mediators and proteolytic enzymes. CPB induces an inflammatory state characterized by tumor necrosis factor-alpha release. Aprotinin, a low molecular-weight peptide inhibitor of trypsin, kallikrein and plasmin has been proposed to influence whole body inflammatory response inhibiting kallikrein formation, complement activation and neutrophil activation (5, 6). But shown that aprotinin has no significant influence on the inflammatory reaction to CPB in men.  Understanding the endothelial cell responses to injury is therefore central to appreciating the role that dysfunction plays in the preoperative, operative, and postoperative course of nearly all cardiovascular surgery patients.  Whether aprotinin increases the risk of thrombotic complications remains controversial.   The anti-inflammatory properties of aprotinin in attenuating the clinical manifestations of the systemic inflammatory response following cardiopulmonary bypass are well known(15) 16)  However its mechanisms and targets of action are not fully understood. In this study we have investigated the actions of aprotinin at the endothelial cell level. Our experiments showed that aprotinin reduced TNF-a induced IL-6 release from cultured HUVECS. Thrombin mediates its effects through PAR-1 receptor and we found that aprotinin reduced the expression of PAR-1 on the surface of HUVECS after 24 hours incubation. We then demonstrated that aprotinin inhibited endothelial cell PAR proteolytic activation by thrombin (PAR-1), trypsin, factor VII and factor X (PAR-2) in terms of less release of Ca preventing the activation of coagulation.  So aprotinin made cells produce less receptor, PAR1, PAR2, and TF as a result there would be less Ca++ release.    Our findings provide evidence for anti-inflammatory as well as anti-coagulant properties of aprotinin at the endothelial cell level, which may be mediated through its inhibitory effects on proteolytic activation of PARs.   IL6   Elevated levels of IL-6 have been shown to correlate with adverse outcomes following cardiac surgery in terms of cardiac dysfunction and impaired lung function(Hennein et al 1992). Cardiopulmonary bypass is associated with the release of the pro-inflammatory cytokines IL-6, IL-8 and TNF-a.  IL-6 is produced by T-cells, endothelial cells as a result monocytes and plasma levels of this cytokine tend to increase during CPB (21, 22). In some studies aprotinin has been shown to reduce levels of IL-6 post CPB(23) Hill(5). Others have failed to demonstrate an inhibitory effect of aprotinin upon pro-inflammatory cytokines following CPB(24) (25).  Our experiments showed that aprotinin significantly reduced the release of IL-6 from TNF-a stimulated endothelial cells, which may represent an important target of its anti-inflammatory properties. Its has been shown recently that activation of HUVEC by PAR-1 and PAR-2 agonists stimulates the production of IL-6(26). Hence it is possible that the effects of aprotinin in reducing IL-6 may be through targeting activation of such receptors.   TPA   Tissue Plasminogen activator is stored, ready made, in endothelial cells and it is released at its highest levels just after commencing CPB and again after protamine administration. The increased fibrinolytic activity associated with the release of tPA can be correlated to the excessive bleeding postoperatively. Thrombin is thought to be the major stimulus for release of t-PA from endothelial cells. Aprotinin’s haemostatic properties are due to direct inhibition of plasmin, thereby reducing fibrinolytic activity as well as inhibiting fibrin degradation.  Aprotinin has not been shown to have any significant effect upon t-PA levels in patients post CPB(27), which would suggest that aprotinin reduced fibrinolytic effects are not the result of inhibition of t-PA mediated plasmin generation. Our study, however demonstrates that aprotinin inhibits the release of t-PA from activated endothelial cells, which may represent a further haemostatic mechanism at the endothelial cell level.   TF   Resting endothelial cells do not normally express tissue factor on their cell surface. Inflammatory mediators released during CPB such as complement (C5a), lipopolysaccharide, IL-6, IL-1, TNF-a, mitogens, adhesion molecules and hypoxia may induce the expression of tissue factor on endothelial cells and monocytes. The expression of TF on activated endothelial cells activates the extrinsic pathway of coagulation, ultimately resulting in the generation of thrombin and fibrin. Aprotinin has been shown to reduce the expression of TF on monocytes in a simulated cardiopulmonary bypass circuit (28).

We found that treatment of activated endothelial cells with aprotinin significantly reduced the expression of TF after 24 hours. This would be expected to result in reduced thrombin generation and represent an additional possible anticoagulant effect of aprotinin. In a previous study from our laboratory we demonstrated that there were two peaks of inducible TF activity on endothelial cells, one immediately post CPB and the second at 24 hours (29). The latter peak is thought to be responsible for a shift from the initial fibrinolytic state into a procoagulant state.  In addition to its established early haemostatic and coagulant effect, aprotinin may also have a delayed anti-coagulant effect through its inhibition of TF mediated coagulation pathway. Hence its effects may counterbalance the haemostatic derangements, i.e. first bleeding then thrombosis caused by CPB. The anti-inflammatory effects of aprotinin may also be related to inhibition of TF and thrombin generation. PARs  

It has been suggested that aprotinin may target PAR on other cells types, especially endothelial cells. We investigated the role of PARs in endothelial cell activation and whether they can be the targets for aprotinin.  In recent study by Day group(30) demonstrated that endothelial cell activation by thrombin and downstream inflammatory responses can be inhibited by aprotinin in vitro through blockade of protease-activated receptor 1. Our results provide a new molecular basis to help explain the anti-inflammatory properties of aprotinin reported clinically.    The finding that PAR-2 can also be activated by the coagulation enzymes factor VII and factor X indicates that PAR may represent the link between inflammation and coagulation.  PAR-2 is believed to play an important role in inflammatory response. PAR-2 are widely expressed in the gastrointestinal tract, pancreas, kidney, liver, airway, prostrate, ovary, eye of endothelial, epithelial, smooth muscle cells, T-cells and neutrophils. Activation of PAR-2 in vivo has been shown to be involved in early inflammatory processes of leucocyte recruitment, rolling, and adherence, possibly through a mechanism involving platelet-activating factor (PAF)   We investigated the effects of TNFa stimulation on PAR-1 and PAR-2 expression on endothelial cells. Through functional analysis of PAR-1 and PAR-2 by measuring intracellular calcium influx we have demonstrated that aprotinin blocks proteolytic cleavage of PAR-1 by thrombin and activation of PAR-2 by the proteases trypsin, factor VII and factor X.  This confirms the previous findings on platelets of an endothelial anti-thrombotic effect through inhibition of proteolysis of PAR-1. In addition, part of aprotinin’s anti-inflammatory effects may be mediated by the inhibition of serine proteases that activate PAR-2. There have been conflicting reports regarding the regulation of PAR-1 expression by inflammatory mediators in cultured human endothelial cells. Poullis et al first showed that thrombin induced platelet aggregation was mediated by via the PAR-1(4) and demonstrated that aprotinin inhibited the serine protease thrombin and trypsin induced platelet aggregation. Aprotinin did not block PAR-1 activation by the non-proteolytic agonist peptide, SFLLRN indicating that the mechanism of action was directed towards inhibiting proteolytic cleavage of the receptor. Nysted et al showed that TNF did not affect mRNA and cell surface protein expression of PAR-1 (35), whereas Yan et al showed downregulation of PAR-1 mRNA levels (36). Once activated PAR1 and PAR2 are rapidly internalized and then transferred to lysosomes for degradation.

Endothelial cells contain large intracellular pools of preformed receptors that can replace the cleaved receptors over a period of approximately 2 hours, thus restoring the capacity of the cells to respond to thrombin. In this study we found that after 1-hour stimulation with TNF there was a significant upregulation in PAR-1 expression. However after 3 hours and 24 hours there was no significant change in PAR-1 expression suggesting that cleaved receptors had been internalized and replenished. Aprotinin was interestingly shown to downregulate PAR-1 expression on endothelial cells at 1 hour and increasingly more so after 24 hours TNF stimulation. These findings may suggest an effect of aprotinin on inhibiting intracellular cycling and synthesis of PAR-1.    

Conclusions   Our study has identified the anti-inflammatory and coagulant effects of aprotinin at the endothelial cell level. All together aprotinin affects the ECCB by reducing the t-PA, IL-6, PAR1, PAR 2, TF expressions. Our data correlates with the previous foundlings in production of tPA (7, (8) 9) 10), and  decreased IL-6 levels (11) during coronary artery bypass graft surgery (12-14). We have importantly demonstrated that aprotinin may target proteolytic activation of endothelial cell associated PAR-1 to exert a possible anti-inflammatory effect. This evidence should lessen the concerns of a possible prothrombotic effect and increased incidence of graft occlusion in coronary artery bypass patients treated with aprotinin. Aprotinin may also inhibit PAR-2 proteolytic activation, which may represent a key mechanism for attenuating the inflammatory response at the critical endothelial cell level. Although aprotinin has always been known as a non-specific protease inhibitor we would suggest that there is growing evidence for a PAR-ticular mechanism of action.  

REFERENCES

1.         Levy, J. H., and Tanaka, K. A. Inflammatory response to cardiopulmonary bypass. Ann Thorac Surg. 75: S715-720, 2003.

2.         Verrier, E. D., and Morgan, E. N. Endothelial response to cardiopulmonary bypass surgery. Ann Thorac Surg. 66: S17-19; discussion S25-18, 1998.

3.         Cirino, G., Napoli, C., Bucci, M., and Cicala, C. Inflammation-coagulation network: are serine protease receptors the knot? Trends Pharmacol Sci. 21: 170-172, 2000. 4.         Poullis, M., Manning, R., Laffan, M., Haskard, D. O., Taylor, K. M., and Landis, R. C. The antithrombotic effect of aprotinin: actions mediated via the proteaseactivated receptor 1. J Thorac Cardiovasc Surg. 120: 370-378, 2000.

5.         Hill, G. E., Alonso, A., Spurzem, J. R., Stammers, A. H., and Robbins, R. A. Aprotinin and methylprednisolone equally blunt cardiopulmonary bypass-induced inflammation in humans. J Thorac Cardiovasc Surg. 110: 1658-1662, 1995.

6.         Hill, G. E., Pohorecki, R., Alonso, A., Rennard, S. I., and Robbins, R. A. Aprotinin reduces interleukin-8 production and lung neutrophil accumulation after cardiopulmonary bypass. Anesth Analg. 83: 696-700, 1996. 7.         Lu, H., Du Buit, C., Soria, J., Touchot, B., Chollet, B., Commin, P. L., Conseiller, C., Echter, E., and Soria, C. Postoperative hemostasis and fibrinolysis in patients undergoing cardiopulmonary bypass with or without aprotinin therapy. Thromb Haemost. 72: 438-443, 1994.

8.         de Haan, J., and van Oeveren, W. Platelets and soluble fibrin promote plasminogen activation causing downregulation of platelet glycoprotein Ib/IX complexes: protection by aprotinin. Thromb Res. 92: 171-179, 1998.

9.         Erhardtsen, E., Bregengaard, C., Hedner, U., Diness, V., Halkjaer, E., and Petersen, L. C. The effect of recombinant aprotinin on t-PA-induced bleeding in rats. Blood Coagul Fibrinolysis. 5: 707-712, 1994.

10.       Orchard, M. A., Goodchild, C. S., Prentice, C. R., Davies, J. A., Benoit, S. E., Creighton-Kemsford, L. J., Gaffney, P. J., and Michelson, A. D. Aprotinin reduces cardiopulmonary bypass-induced blood loss and inhibits fibrinolysis without influencing platelets. Br J Haematol. 85: 533-541, 1993.

11.       Tassani, P., Augustin, N., Barankay, A., Braun, S. L., Zaccaria, F., and Richter, J. A. High-dose aprotinin modulates the balance between proinflammatory and anti-inflammatory responses during coronary artery bypass graft surgery. J Cardiothorac Vasc Anesth.14: 682-686, 2000.

12.       Asehnoune, K., Dehoux, M., Lecon-Malas, V., Toueg, M. L., Gonieaux, M. H., Omnes, L., Desmonts, J. M., Durand, G., and Philip, I. Differential effects of aprotinin and tranexamic acid on endotoxin desensitization of blood cells induced by circulation through an isolated extracorporeal circuit. J Cardiothorac Vasc Anesth. 16: 447-451, 2002.

13.       Dehoux, M. S., Hernot, S., Asehnoune, K., Boutten, A., Paquin, S., Lecon-Malas, V., Toueg, M. L., Desmonts, J. M., Durand, G., and Philip, I. Cardiopulmonary bypass decreases cytokine production in lipopolysaccharide-stimulated whole blood cells: roles of interleukin-10 and the extracorporeal circuit. Crit Care Med. 28: 1721-1727, 2000.

14.       Greilich, P. E., Brouse, C. F., Rinder, C. S., Smith, B. R., Sandoval, B. A., Rinder, H. M., Eberhart, R. C., and Jessen, M. E. Effects of epsilon-aminocaproic acid and aprotinin on leukocyte-platelet adhesion in patients undergoing cardiac surgery. Anesthesiology. 100: 225-233, 2004.

15.       Mojcik, C. F., and Levy, J. H. Aprotinin and the systemic inflammatory response after cardiopulmonary bypass. Ann Thorac Surg. 71: 745-754, 2001.

16.       Landis, R. C., Asimakopoulos, G., Poullis, M., Haskard, D. O., and Taylor, K. M. The antithrombotic and antiinflammatory mechanisms of action of aprotinin. Ann Thorac Surg. 72: 2169-2175, 2001.

17.       Asimakopoulos, G., Kohn, A., Stefanou, D. C., Haskard, D. O., Landis, R. C., and Taylor, K. M. Leukocyte integrin expression in patients undergoing cardiopulmonary bypass. Ann Thorac Surg. 69: 1192-1197, 2000.

18.       Landis, R. C., Asimakopoulos, G., Poullis, M., Thompson, R., Nourshargh, S., Haskard, D. O., and Taylor, K. M. Effect of aprotinin (trasylol) on the inflammatory and thrombotic complications of conventional cardiopulmonary bypass surgery. Heart Surg Forum. 4 Suppl 1: S35-39, 2001.

19.       Asimakopoulos, G., Thompson, R., Nourshargh, S., Lidington, E. A., Mason, J. C., Ratnatunga, C. P., Haskard, D. O., Taylor, K. M., and Landis, R. C. An anti-inflammatory property of aprotinin detected at the level of leukocyte extravasation. J Thorac Cardiovasc Surg. 120: 361-369, 2000.

20.       Asimakopoulos, G., Lidington, E. A., Mason, J., Haskard, D. O., Taylor, K. M., and Landis, R. C. Effect of aprotinin on endothelial cell activation. J Thorac Cardiovasc Surg. 122: 123-128, 2001.

21.       Butler, J., Chong, G. L., Baigrie, R. J., Pillai, R., Westaby, S., and Rocker, G. M. Cytokine responses to cardiopulmonary bypass with membrane and bubble oxygenation. Ann Thorac Surg. 53: 833-838, 1992.

22.       Hennein, H. A., Ebba, H., Rodriguez, J. L., Merrick, S. H., Keith, F. M., Bronstein, M. H., Leung, J. M., Mangano, D. T., Greenfield, L. J., and Rankin, J. S. Relationship of the proinflammatory cytokines to myocardial ischemia and dysfunction after uncomplicated coronary revascularization. J Thorac Cardiovasc Surg. 108: 626-635, 1994.

23.       Diego, R. P., Mihalakakos, P. J., Hexum, T. D., and Hill, G. E. Methylprednisolone and full-dose aprotinin reduce reperfusion injury after cardiopulmonary bypass. J Cardiothorac Vasc Anesth. 11: 29-31, 1997.

24.       Ashraf, S., Tian, Y., Cowan, D., Nair, U., Chatrath, R., Saunders, N. R., Watterson, K. G., and Martin, P. G. “Low-dose” aprotinin modifies hemostasis but not proinflammatory cytokine release. Ann Thorac Surg. 63: 68-73, 1997.

25.       Schmartz, D., Tabardel, Y., Preiser, J. C., Barvais, L., d’Hollander, A., Duchateau, J., and Vincent, J. L. Does aprotinin influence the inflammatory response to cardiopulmonary bypass in patients? J Thorac Cardiovasc Surg. 125: 184-190, 2003.

26.       Chi, L., Li, Y., Stehno-Bittel, L., Gao, J., Morrison, D. C., Stechschulte, D. J., and Dileepan, K. N. Interleukin-6 production by endothelial cells via stimulation of protease-activated receptors is amplified by endotoxin and tumor necrosis factor-alpha. J Interferon Cytokine Res. 21: 231-240, 2001.

27.       Ray, M. J., and Marsh, N. A. Aprotinin reduces blood loss after cardiopulmonary bypass by direct inhibition of plasmin. Thromb Haemost. 78: 1021-1026, 1997.

28.       Khan, M. M., Gikakis, N., Miyamoto, S., Rao, A. K., Cooper, S. L., Edmunds, L. H., Jr., and Colman, R. W. Aprotinin inhibits thrombin formation and monocyte tissue factor in simulated cardiopulmonary bypass. Ann Thorac Surg. 68: 473-478, 1999.

29.       Jaggers, J. J., Neal, M. C., Smith, P. K., Ungerleider, R. M., and Lawson, J. H. Infant cardiopulmonary bypass: a procoagulant state. Ann Thorac Surg. 68: 513-520, 1999.

30.       Day, J. R., Taylor, K. M., Lidington, E. A., Mason, J. C., Haskard, D. O., Randi, A. M., and Landis, R. C. Aprotinin inhibits proinflammatory activation of endothelial cells by thrombin through the protease-activated receptor 1. J Thorac Cardiovasc Surg. 131: 21-27, 2006.

31.       Vergnolle, N. Proteinase-activated receptor-2-activating peptides induce leukocyte rolling, adhesion, and extravasation in vivo. J Immunol. 163: 5064-5069, 1999.

32.       Vergnolle, N., Hollenberg, M. D., Sharkey, K. A., and Wallace, J. L. Characterization of the inflammatory response to proteinase-activated receptor-2 (PAR2)-activating peptides in the rat paw. Br J Pharmacol. 127: 1083-1090, 1999.

33.       McLean, P. G., Aston, D., Sarkar, D., and Ahluwalia, A. Protease-activated receptor-2 activation causes EDHF-like coronary vasodilation: selective preservation in ischemia/reperfusion injury: involvement of lipoxygenase products, VR1 receptors, and C-fibers. Circ Res. 90: 465-472, 2002.

34.       Maree, A., and Fitzgerald, D. PAR2 is partout and now in the heart. Circ Res. 90: 366-368, 2002.

35.       Nystedt, S., Ramakrishnan, V., and Sundelin, J. The proteinase-activated receptor 2 is induced by inflammatory mediators in human endothelial cells. Comparison with the thrombin receptor. J Biol Chem. 271: 14910-14915, 1996.

36.       Yan, W., Tiruppathi, C., Lum, H., Qiao, R., and Malik, A. B. Protein kinase C beta regulates heterologous desensitization of thrombin receptor (PAR-1) in endothelial cells. Am J Physiol. 274: C387-395, 1998.

37.       Shinohara, T., Suzuki, K., Takada, K., Okada, M., and Ohsuzu, F. Regulation of proteinase-activated receptor 1 by inflammatory mediators in human vascular endothelial cells. Cytokine. 19: 66-75, 2002.

FIGURES

Figure 1: IL-6 production following TNF-a stimulation Figure 1

Figure 2:  tPA production following TNF-a stimulation Figure 2

Figure 3:  Tissue Factor Expression on TNF-a stimulated HUVECS Figure 3

Figure 4:  PAR-1 Expression on TNF-a stimulated HUVECS Figure 4

Figure 5:  PAR-2 Expression on TNF-a stimulated HUVECS Figure 5

Figure 6:  Calcium Fluxes following PAR1 Activation Figure 6

Figure 7:  Calcium Fluxes following PAR2 Activation Figure 7

 

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Treatment Options for Left Ventricular Failure  –  Temporary Circulatory Support: Intra-aortic balloon pump (IABP)Impella Recover LD/LP 5.0 and 2.5, Pump Catheters (Non-surgical) vs Bridge Therapy: Percutaneous Left Ventricular Assist Devices (pLVADs) and LVADs (Surgical) 

Author: Larry H Bernstein, MD, FCAP
And
Curator: Justin D Pearlman, MD, PhD, FACC

 

UPDATED on 12/2/2013 – HeartMate II – LVAD

http://www.nytimes.com/2013/11/28/business/3-hospital-study-links-heart-device-to-blood-clots.html?pagewanted=1&_r=0&emc=eta1

Hospital Studies Link Heart Device to Clots

David Maxwell for The New York Times

Dr. Randall Starling, right, said that he could only speculate about the reason for the rapid rise in early blood clots.

By 
Published: November 27, 2013

Doctors at the Cleveland Clinic began to suspect in 2012 that something might be wrong with a high-tech implant used to treat patients with advanced heart failure like former Vice President Dick Cheney.

Thoratec Corportation

The HeartMate II is a left ventricular assist device, which contains a pump that continuously pushes blood through the heart.

The number of patients developing potentially fatal blood clots soon after getting the implant seemed to be rising. Then early this year, researchers completed a check of hospital records and their concern turned to alarm.

The data showed that the incidence of blood clots among patients who got the device, called the HeartMate II, after March 2011 was nearly four times that of patients who had gotten the same device in previous years. Patients who developed pump-related clots died or needed emergency steps like heart transplants or device replacements to save them.

“When we got the data, we said, ‘Wow,’ ” said Dr. Randall C. Starling, a cardiologist at Cleveland Clinic.

On Wednesday, The New England Journal of Medicineposted a study on its website detailing the findings from the Cleveland Clinic and two other hospitals about the device. The HeartMate II belongs to a category of products known as a left ventricular assist device and it contains a pump that continuously pushes blood through the heart.

The abrupt increase in pump-related blood clots reported in the study is likely to raise questions about whether its manufacturer, Thoratec Corporation, modified the device, either intentionally or accidentally. By March, the Cleveland Clinic had informed both Thoratec and the Food and Drug Administration about the problems seen there, Dr. Starling said.

Officials at Thoratec declined to be interviewed. But in a statement, the company, which is based in Pleasanton, Calif., said that the HeartMate II had been intensively studied and used in more 16,000 patients worldwide with excellent results. It added that the six-month survival rate of patients who received the device had remained consistently high.

“Individual center experience with thrombosis varies significantly, and Thoratec actively partners with clinicians at all centers to minimize this risk,” the company said in a statement.

Thoratec and other cardiologists also pointed to a federally funded registry that shows a smaller rise in the rate of blood clots, or thrombosis, among patients getting a HeartMate II than the one reported Wednesday by the three hospitals. In the registry, which is known as Intermacs, the rate of pump-related blood clot associated with the HeartMate II rose to about 5 percent in devices implanted after May 2011 compared with about 2 percent in previous years.

The data reported on Wednesday in The New England Journal of Medicine found rates of clot formation two months after a device’s implant had risen to 8.4 percent after March 2011 from 2.2 percent in earlier years. Researchers also suggested in the study that the Intermacs registry might not capture all cases of pump-related blood clots, such as when patients gets emergency heart transplants after a clot forms.

Not only did the rate of blood clots increase, but the clots also occurred much sooner than in the past, according to the study. After March 2011, the median time before a clot was 2.7 months, compared with 18.6 months in previous years. In addition to the Cleveland Clinic, the report on Wednesday included data from Duke University and Washington University in St. Louis.

All mechanical heart implants are prone to producing blood clots that can form on a device’s surface. And experts say that the rate of blood clot formation can be affected by a variety of factors like changes in the use of blood-thinning drugs or the health of a patient.

In a telephone interview, Dr. Starling described the Thoratec officials as cooperative, adding that they have been looking into the problem since March to understand its cause. He said that he could only speculate about the reason for the rapid rise in early blood clots but believed it was probably device-related.

“My belief is that it is something as subtle as a change in software that affects pump flow or heat dissipation near a bearing,” said Dr. Starling, who is a consultant to Thoratec.

Asked about his comments, Thoratec responded that it had yet to determine the reason for even the smaller rise in blood clots seen in the federally funded database. “We have performed extensive analysis on HeartMate II and have not identified any change that would cause the increase observed in the Intermacs registry,” the company said.

In a statement, the F.D.A. said that it was reviewing the findings of the study. “The agency shares the authors concerns about the possibility of increased pump thrombosis,” the F.D.A. said in a statement.

The fortunes of Thoratec, which has been a favorite of Wall Street investors, may depend on its ability to find an answer to the apparent jump in pump-related blood clots. Over the last two years, the company’s stock has climbed from about $30 a share to over $43 a share. In trading Wednesday, Thoratec stock closed at $42.12 a share, up 61 cents. (The New England Journal of Medicine article was released after the stock market closed.)

The HeartMate II has been a lifesaver for many patients like Mr. Cheney in the final stages of heart failure, who got his device in 2010, sustaining them until they get a heart transplant or permanently assisting their heart. Dr. Starling said that he planned to keep using the HeartMate II in appropriate patients at the Cleveland Clinic because those facing death from heart failure had few options.

But the company has also been pushing to expand the device’s use beyond patients who face imminent death from heart failure. For example, the F.D.A. approved a clinical trial for patients with significant, but less severe, heart failure to receive a HeartMate II to compare their outcomes with patients who take drugs for the same condition. Researchers at the University of Michigan Medical Center who are leading the trial said on Wednesday that, based on the lower rates of blood clots seen in the Intermacs registry, they are planning to move forward with the trial.

Dr. Starling and researchers at the Cleveland Clinic tried this spring to get The New England Journal of Medicine to publish a report about the findings at that hospital, but the publication declined, saying the data might simply represent the experience of one facility. As a result, Dr. Starling contacted Duke University and Washington University for their data. When analyzed, it mirrored events at the Cleveland Clinic, he said.

The problems seen with the HeartMate II at the three hospitals were continuing as recently as this summer, when researchers paused the collection of data to prepare Wednesday’s study. The study also noted that a preliminary analysis of data provided by a fourth hospital, the University of Pennsylvania, showed the same pattern of blood clot formation, but that the data had been submitted too late for full analysis.

 SOURCE

 

This article presents the following four Sections:

I.     Impella LD – ABIOMED, Inc.

II.   IABP VS. Percutaneous LVADS

III. Use of the Impella 2.5 Catheter in High-Risk Percutaneous Coronary Intervention

IV.  PROTECT II Study – Experts Discussion

This account is a vital piece of recognition of very rapid advances in cardiothoracic interventions to support cardiac function mechanically by pump in the situation of loss of contractile function and circulatory output sufficient to sustain life, as can occur with the development of cardiogenic shock.  This has been mentioned and its use has been documented in other portions of this series.   On the one hand, PCI has a long and steady history in the development of interventional cardiology. This necessitated the availability of thoracic-surgical operative support. The situation is changed, and is more properly, conditional.

I. Impella LD – ABIOMED, Inc.

This micro-axial blood pump can be inserted into the left ventricle via open chest procedures. The Impella LD device has a 9 Fr catheter-based platform and a 21 Fr micro-axial pump and is  inserted through the ascending aorta, across the aortic and mitral valves and into the left ventricle.  It requires minimal bedside support and a 9 Fr single-access point  requires no priming outside the body.

Impella.LD_

Impella Recover LD/LP 5.0

The Impella Recover miniaturized impeller pump located within a catheter. The Impella Recover LD/LP 5.0 Support System has been developed to address the need for ventricular support in patients who develop heart failure after heart surgery (called cardiogenic shock) and who have not responded to standard medical therapy. The system is designed to provide immediate support and restore hemodynamic stability for a period of up to 7 days. Used as a bridge to therapy, it allows time for developing a definitive treatment strategy.

The Pump

The Impella Recover LD 5.0 showing implantation via direct placement into the left ventricle.
 Insert B – location in LV
imeplla-LD-video
The Impella Recover system is a miniaturized impeller pump located within a catheter. The device can provide support for the left side of the heart using either the
  • Recover LD 5.0 (implanted via direct placement into the left ventricle) or the
  • Recover LP 5.0 LV (placed percutaneously through the groin and positioned in the left ventricle).
The microaxial pump of the Recover LP/LD 5.0 can pump up to 4.5 liters per minute at a speed of 33,000 rpm. The pump is located at the distal end of a 9 Fr catheter.

II.   IABP VS. Percutaneous LVADS

An intra-aortic balloon pump (IABP) remains the method of choice for mechanical assistance1 in patients experiencing LV failure because of its

  • proven hemodynamic capabilities,
  • prompt time to therapy, and
  • low complication rates.

Percutaneous left ventricular assist devices (pLVADs), such as described above, represent an emerging option for partial or total circulatory support2 and several studies have compared the and efficacy of these devices with intra-aortic balloon pump (IABP) (IABP.)

Despite some randomized controlled trials demonstrating better hemodynamic profiles for pLVADs compared with IABP, there is no difference in  30-day survival or trend toward a reduced 30-day mortality rate associated with pLVADs.  Patients treated with pLVADs tended to have a
  • higher incidence of leg ischemia and
  • device related bleeding.3
Further, no differences have been detected in the overall use of
  • positive inotropic drugs or
  • vasopressors in patients with pLVADs.4,5
However, pLVADs may increase their use for patients not responding to
  • PCI,
  • fluids,
  • inotropes, and
  • IABP
Therefore, the decision making process on how to treat requires an integrated stepwise approach. A pLVAD might be considered on the basis of
  • anticipated individual risk,
  • success rates, and for
  • postprocedural events.6

Potential Algorithm for Device Selection during High-Risk PCI

PADS_HRPCI cardiac assist device selection

Potential Algorithm for Device Selection during Cardiogenic Shock
device_selection_CS
Until an alternative modality, characterized by improved efficacy and safety features compared with IABP, is developed, IABP remains the cornerstone of temporary circulatory support.2

Device Comparison for Treatment of Cardiogenic Shocktraditional intra-aortic balloon therapy with Impella 2.5 percutaneous ventricular assist device

 
1. Percutaneous LVADs in AMI complicated by cardiogenic shock. H Thiele, et al. EHJ 2007;28:2057-2063
2. Cardiogenic shock current concepts and improving outcomes. H R Reynolds et al. Circulation 2008 ;117 :686-697
3. Percutaneous left ventricular assist devices vs. IABP counterpulsation for treatment of cardiogenic shock. J M Cheng, et al. EHJ doi:10.1093/eurheart/ehp292
4. A randomized clinical trial to evaluate the safety and efficacy of a pLVAD vs. IABP for treatment of cardiogenic shock caused by MI. M Seyfarth, et al. JACC 2008;52:1584-8
5. A randomized multicenter clinical study to evaluate the safety and efficacy of the tandem heart pLVAD vs. conventional therapy with IABP for treatment of cardiogenic shock.
6. Percutaneous LVADs in AMI complicated by cardiogenic shock. H Thiele, et al. EHJ 2007;28:2057-2063

III. Use of the Impella 2.5 Catheter in High-Risk Percutaneous Coronary Intervention

Brenda McCulloch, RN, MSN
Sutter Heart and Vascular Institute, Sutter Medical Center, Sacramento, California
Crit Care Nurse 2011; 31(1): e1-e16    http://dx.doi.org/10.4037/ccn2011293
Abstract
The Impella 2.5 is a percutaneously placed partial circulatory assist device that is increasingly being used in high-risk coronary interventional procedures to provide hemodynamic support. The Impella 2.5 is able to unload the left ventricle rapidly and effectively and increase cardiac output more than an intra-aortic balloon catheter can. Potential complications include bleeding, limb ischemia, hemolysis, and infection. One community hospital’s approach to establishing a multidisciplinary program for use of the Impella 2.5 is described.
Patients who undergo high-risk percutaneous coronary intervention (PCI), such as procedures on friable saphenous vein grafts or the left main coronary artery, may have an intra-aortic balloon catheter placed if they require hemodynamic support during the procedure. Currently, the intra-aortic balloon pump (IABP) is the most commonly used device for circulatory support. A newer option that is now available for select patients is the Impella 2.5, a short-term partial circulatory support device or percutaneous ventricular assist device (VAD).
In this article, I discuss the Impella 2.5, review indications and contraindications for its use, delineate potential complications of the Impella 2.5, and discuss implications for nursing care for patients receiving extended support from an Impella 2.5. Additionally, I share our experiences as we developed our Impella program at our community hospital. Routine management of patients after PCI is not addressed.

IABP Therapy: Background

  • decreases after-load,
  • decreases myocardial oxygen consumption,
  • increases coronary artery perfusion, and
  • modestly enhances cardiac output.1,2
The IABP cannot provide total circulatory support. Patients must have some level of left ventricular function for an IABP to be effective.
Optimal hemodynamic effect from the IABP is dependent  on:
  • the balloon’s position in the aorta,
  • the blood displacement volume,
  • the balloon diameter in relation to aortic diameter,
  • the timing of balloon inflation in diastole and deflation in systole, and
  • the patient’s own blood pressure and vascular resistance.3,4

Impella 2.5 Catheter – ABIOMED, Inc.

Effect
  • reduces myocardial oxygen consumption,
  • improves mean arterial pressure, and
  • reduces pulmonary capillary wedge pressure.2

The Impella 2.5 has been used for

  • hemodynamic support during high-risk PCI and for
  • hemodynamic support of patients with
  1. myocardial infarction complicated by cardiogenic shock or ventricular septal defect,
  2. cardiomyopathy with acute decompensation,
  3. postcardiotomy shock,
  4. off-pump coronary artery bypass grafting surgery, or
  5. heart transplant rejection and
  6. as a bridge to the next decision.9
The Impella provides a greater increase in cardiac output than the other IABP provides. In one trial5 in which an IABP was compared with an Impella in cardiogenic shock patients, after 30 minutes of therapy, the cardiac index (calculated as cardiac output in liters per minute divided by body surface area in square meters) increased by 0.5 in the patients with the Impella compared with 0.1 in the patients with an IABP.
Unlike the IABP, the Impella does not require timing, nor is a trigger from an electrocardiographic rhythm or arterial pressure needed (Table 1). The device received 510(k) clearance from the Food and Drug Administration in June 2008 for providing up to 6 hours of partial circulatory support. In Europe, the Impella 2.5 is approved for use up to 5 days. Reports of longer duration of therapy in both the United States and Europe have been published.8,9
Table IABT vs Impella

Clinical Research and Registry Findings

Abiomed has sponsored several trials, including PROTECT I, PROTECT II, RECOVER I, RECOVER II, and ISAR-SHOCK.
The PROTECT I study was done to assess the safety and efficacy of device placement in patients undergoing high-risk PCI.10

Twenty patients who had

  • poor ventricular function (ejection fraction =35%) and had
  • PCI on an unprotected left main coronary artery or the
  • last remaining patent coronary artery or graft.

The device was successfully placed in all patients, and the duration of support ranged from 0.4 to 2.5 hours. Following this trial, the Impella 2.5 device received its 510(k) approval from the Food and Drug Administration.

The ISAR-SHOCK trial was done to evaluate the safety and efficacy of the Impella 2.5 versus the IAPB in patients with cardiogenic shock due to acute myocardial infarction.5 Patients were randomized to support from an IABP (n=13) or an Impella (n=12).

The trial’s primary end point of hemodynamic improvement was defined as improved cardiac index at 30 minutes after implantation.

  1. Improvements in cardiac index were greater with the Impella (P=.02).
  2. The diastolic pressure increased more with Impella (P=.002).
  3. There was a nonsignificant difference in the MAP (P=.09), as was the use of inotropic agents and vasopressors similar in both groups of patients.

Device Design: Impella 2.5 Catheter

The Impella 2.5 catheter contains a nonpulsatile microaxial continuous flow blood pump that pulls blood from the left ventricle to the ascending aorta, creating increased forward flow and increased cardiac output. An axial pump is one that is made up of impellar blades, or rotors, that spin around a central shaft; the spinning of these blades is what moves blood through the device.13

The Impella 2.5 catheter has 2 lumens. A tubing system called the Quick Set-Up has been developed for use in the catheterization laboratory. It is a single tubing system that bifurcates and connects to each port of the catheter. This arrangement allows rapid initial setup of the console so that support can be initiated quickly. When the Quick Set-Up is used, the 10% to 20% dextrose solution used to purge the motor is not heparinized. One lumen carries fluid to the impellar blades and continuously purges the motor to prevent the formation of thrombus. The proximal port of this lumen is yellow. The second lumen ends near the motor above the level of the aortic valve and is used to monitor aortic pressure.
The components required to run the device are assembled on a rolling cart and include the power source, the Braun Vista infusion pump, and the Impella console. The Impella console powers the microaxial blood pump and monitors the functioning of the device, including the purge pressure and several other parameters. The console can run on a fully charged battery for up to 1 hour.

Placement of the Device

The Impella 2.5 catheter is placed percutaneously through the common femoral artery and advanced retrograde to the left ventricle over a guidewire. Fluoroscopic guidance in the catheterization laboratory or operating room is required. After the device is properly positioned, it is activated and blood is rapidly withdrawn by the microaxial blood pump from the inlet valve in the left ventricle and moved to the aorta via the outlet area, which sits above the aortic valve in the aorta.
If the patient tolerates the PCI procedure and hemodynamic instability does not develop, the Impella 2.5 may be removed at the end of the case, or it can be withdrawn, leaving the arterial sheath in place, which can be removed when the patient’s activated clotting time or partial thromboplastin time has returned to near normal levels. For patients who become hemodynamically unstable or who have complications during the PCI (eg, no reflow, hypotension, or lethal arrhythmias), the device can remain in place for continued partial circulatory support, and the patient is transported to the critical care setting.

Potential Complications of Impella Therapy

The most commonly reported complications of Impella 2.5 placement and support include

  • limb ischemia,
  • vascular injury, and
  • bleeding requiring blood transfusion.6,9
Hemolysis is an inherent risk of the axial construction, and results in transfusions.5,10
Hemolysis can be mechanically induced when red blood cells are damaged as they pass through the microaxial pump. Other potential complications include
  • aortic valve damage,
  • displacement of the distal tip of the device into the aorta,
  • infection, and
  • sepsis.
  • Device failure, although not often reported, can occur.
Patients on Impella 2.5 support who may require
  • interrogation of a permanent pacemaker or
  • implantable cardioverter defibrillator
present an interesting situation. In order for the interrogator to connect with the permanent pacemaker or implantable cardioverter defibrillator, the Impella console must be turned off for a few seconds while the signal is established. As soon as the signal has been established, Impella support is immediately restarted.

Impella 2.5 Console Management

The recommended maximum performance level for continuous use is P8. At P8, the flow rate is 1.9 to 2.6 L/min and the motor is turning at 50000 revolutions per minute. When activated, the console is silent. No sound other than alarms is audible during Impella support, unlike the sound heard with an IABP. Ten different performance levels ranging from P0 to P9 are available. As the performance level increases, the flow rate and number of revolutions per minute increase. At maximum performance (P9), the pump rotates at 50000 revolutions per minute and delivers a flow rate of 2.1 to 2.6 L/min. P9 can be activated only for 5-minute intervals when the Impella 2.5 is in use.

IV.  PROTECT II Study – Experts Discussion

the use of the Impella support device and the intraortic balloon pump for high-risk percutaneous coronary intervention
 
DR. SMALLING: Well, the idea about the PROTECT trial is that it would show that using the Impella device to support high-risk angioplasty was not inferior to utilizing the balloon pump for the same patient subset. Ejection fraction’s were in the 30%–35% range. Supposedly last remaining vessel or left main disease or left-main plus three-vessel disease and low EF; so I think that was the screening for entry into the trial.
major adverse cardiac event endpoints
  1. Acute myocardial infarction,
  2. mortality,
  3. bleeding,
mortality was the same. Their endpoints really didn’t show that much difference. In subgroup analysis, they felt that they Impella may have had a little advantage over balloon pump.
DR. KERN: So do you think this study would tip the interventionalist to move in one direction or the other for high-risk angioplasty?
DR. SMALLING: That’s an interesting concept, you know? One has to get to: What is really a high-risk angioplasty. I think you and I are both old enough to remember that back in the mid-’80s, we determined that high-risk angioplasty was a patient with an ejection fraction of 25% or less, with a jeopardy score over 6. The EFs were a little higher. And, I guess, based on our prior experience with other support devices — like, for instance, CPS and then, later on, the Tandem Heart — there really was not an advantage of so-called more vigorous support systems. And so, the balloon pump served as well.
DR. SMALLING:
Those of us that have looked carefully at what it can really do, I think it may get one liter a minute at most, maybe more.1-6 But I think, for all intents and purposes, it doesn’t support at a very vigorous level. So I think personally, if I had someone I was really worried about, I would opt for something more substantial like, for instance, a Tandem Heart device.
DR. KERN: I think this is a really good summary of the study and the. Are there any final thoughts for those of us who want to read the PROTECT II study when it comes out?
DR. SMALLING: We have to consider a $20,000, $25,000 device. Is that really necessary to do something that we could often do without any support at all, or perhaps with a less costly device like a balloon pump.
DR. KERN: We’re going to talk for a few minutes about the PROTECT II study results that were presented here in their form. And Ron, I know you’ve been involved with following the work of the PROTECT II investigators. Were you a trial site for this study?
DR. WAKSMAN: No, actually, we were not, but we have a lot of interest in high-risk PCI and using devices to make this safe — mainly safe — and also effective. We were not investigators, but we did try to look, based on the inclusion/exclusion criteria, on our own accord with the balloon pump. If you recall, this study actually was comparing balloon time to the Impella device for patients who are high-risk PCI.
The composite endpoint was very complicated. They added like probably nine variables there, which is unusual for a study design. … They basically estimated that the event rate on the balloon pump would be higher than what we thought it should be. So we looked at our own data, and we found out that the actual — if you go by the inclusion/exclusion criteria and their endpoints — the overall event rate in the balloon pump would be much lower than they predicted and built in their sample size.
DR. KERN: And, so, the presentation of the PROTECT II trial, was it presented as a positive study or a negative study.
DR. WAKSMAN: Overall the study did not meet the endpoint. So the bottom line, you can call it the neutral study, which is a nice way to say it.
if you go and do all those analyses, you may find some areas that you can tease a P value, but I don’t think that this has any scientific value, and people should be very careful. We’re not playing now with numbers or with statistics, this is about patient care. You’re doing a study — the study, I think, has some flaws in the design to begin with — and we actually pointed that out when we were asked to participate in the study. But if the study did not meet the endpoint, then I think all those subanalyses, subgroups, you extract from here, you add to there, and you get a P value, that means nothing. So we have to be careful when we interpret this, other than as a neutral study that you basically cannot adopt any of the … it did not meet the hypothesis, that’s the bottom line.

A first-in-man study of the Reitan catheter pump for circulatory support in patients undergoing high-risk percutaneous coronary intervention.

Smith EJ, Reitan O, Keeble T, Dixon K, Rothman MT.
Department of Cardiology, London Chest Hospital, United Kingdom.
Catheter Cardiovasc Interv. 2009 Jun 1;73(7):859-65.
http://dx.doi.org/10.1002/ccd.21865.

To investigate the safety of a novel percutaneous circulatory support device during high-risk percutaneous coronary intervention (PCI).

BACKGROUND:

The Reitan catheter pump (RCP) consists of a catheter-mounted pump-head with a foldable propeller and surrounding cage. Positioned in the descending aorta the pump creates a pressure gradient, reducing afterload and enhancing organ perfusion.

METHODS:

Ten consecutive patients requiring circulatory support underwent PCI; mean age 71 +/- 9; LVEF 34% +/- 11%; jeopardy score 8 +/- 2.3. The RCP was inserted via the femoral artery. Hemostasis was achieved using Perclose sutures. PCI was performed via the radial artery. Outcomes included in-hospital death, MI, stroke, and vascular injury. Hemoglobin (Hb), free plasma Hb (fHb), platelets, and creatinine (cre) were measured pre PCI and post RCP removal.

RESULTS:

The pump was inserted and operated successfully in 9/10 cases (median 79 min). Propeller rotation at 10,444 +/- 1,424 rpm maintained an aortic gradient of 9.8 +/- 2 mm Hg.  Although fHb increased,

  • there was no significant hemolysis (4.7 +/- 2.4 mg/dl pre vs. 11.9 +/- 10.5 post, P = 0.04, reference 20 mg/dl).
  • Platelets were unchanged (pre 257 +/- 74 x 10(9) vs. 245 +/- 63, P = NS).
  • Renal function improved (cre pre 110 +/- 27 micromol/l vs. 99 +/- 28, P = 0.004).

All PCI procedures were successful with no deaths or strokes, one MI, and no vascular complications following pump removal.

14F RCP first in man mechanical device post PCI LVEF 25% JS 10

CONCLUSIONS:

The RCP can be used safely in high-risk PCI patients.

(c) 2009 Wiley-Liss, Inc.  PMID: 19455649

Todd J. Brinton, MD and Peter J. Fitzgerald, MD, PhD, Chapter 14: VENTRICULAR ASSIST TECHNOLOGIES

http://www.sis.org/docs/2006Yearbook_Ch14.pdf

Other related articles published on this Open Access Online Scientific Journal include the following:

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A coronary angiogram that shows the LMCA, LAD and LCX. (Photo credit: Wikipedia)

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English: Figure A shows the structure and bloo...

English: Figure A shows the structure and blood flow in the interior of a normal heart. Figure B shows two common locations for a ventricular septal defect. The defect allows oxygen-rich blood from the left ventricle to mix with oxygen-poor blood in the right ventricle. (Photo credit: Wikipedia)

 

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Phrenic Nerve Stimulation in Patients with Cheyne-Stokes Respiration and Congestive Heart Failure

Writer: Larry H Bernstein, MD, FCAP

and

Curator: Aviva Lev-Ari, PhD, RN

Transvenous Phrenic Nerve Stimulation in Patients With Cheyne-Stokes Respiration and Congestive Heart Failure:A Safety and Proof-of-Concept Study

Xi-Long Zhang, MD; Ning Ding; Hong Wang; Ralph Augostini; Bing Yang; Di Xu; Weizhu Ju; Xiaofeng Hou; Xinli Li; Buqing Ni, PhD; Kejiang Cao; Isaac George; Jie Wang, MD, PhD; Shi-Jiang Zhang
Chest. 2012; 142(4):927-934. doi:10.1378/chest.11-1899
Text Size: A A A

Background:  Cheyne-Stokes respiration (CSR), which often occurs in patients with congestive heart failure (CHF), may be a predictor for poor outcome. Phrenic nerve stimulation (PNS) may interrupt CSR in patients with CHF. We report the clinical use of transvenous PNS in patients with CHF and CSR.

Methods:  Nineteen patients with CHF and CSR were enrolled. A single stimulation lead was placed at the junction between the superior vena cava and brachiocephalic vein or in the left-side pericardiophrenic vein. PNS stimulation was performed using Eupnea System device (RespiCardia Inc). Respiratory properties were assessed before and during PNS. PNS was assessed at a maximum of 10 mA.

Results:  Successful stimulation capture was achieved in 16 patients. Failure to capture occurred in three patients because of dislocation of leads. No adverse events were seen under maximum normal stimulation parameters for an overnight study. When PNS was applied following a series of central sleep apneic events, a trend toward stabilization of breathing and heart rate as well as improvement in oxygen saturation was seen. Compared with pre-PNS, during PNS there was a significant decrease in apnea-hypopnea index (33.8 ± 9.3 vs 8.1 ± 2.3, P = .00), an increase in mean and minimal oxygen saturation as measured by pulse oximetry (89.7% ± 1.6% vs 94.3% ± 0.9% and 80.3% ± 3.7% vs 88.5% ± 3.3%, respectively, all P = .00) and end-tidal CO2 (38.0 ± 4.3 mm Hg vs 40.3 ± 3.1 mm Hg, P = .02), but no significant difference in sleep efficiency (74.6% ± 4.1% vs 73.7% ± 5.4%, P = .36).

Conclusions:  The preliminary results showed that in a small group of patients with CHF and CSR, 1 night of unilateral transvenous PNS improved indices of CSR and was not associated with adverse events.

Trial registry:  ClinicalTrials.gov; No.: NCT00909259; URL: www.clinicaltrials.gov

http://journal.publications.chestnet.org/article.aspx?articleid=1215995

Transvenous phrenic nerve stimulation in patients with Cheyne-Stokes respiration and congestive heart failure: a safety and proof-of-concept study

Zhang Xi-Long; Ding N, Wang H, Augostini R, Yang B.
CHEST 2012; 142(4):927–934
Trial registry: ClinicalTrials.gov; No.: NCT00909259; URL: http://www.clinicaltrials.gov
http://dx.doi.org/10.1378/chest.11-1899

Introduction

Cheyne-Stokes respiration (CSR), a condition characterized by a cyclic pattern of waxing and waning ventilation interposed by central apneas or hypopneas, may affect up to 40% of patients with congestive heart failure (CHF).  Whether CSR is related to significantly increased morbidity and mortality 2 or has no impact on long-term survival in patients with CHF is controversial. Nevertheless, several investigators have reported that CSR might be an independent prognostic index of poor outcome in patients with CHF, so that Cheyne-Stokes respiration (CSR), which often occurs in patients with congestive heart failure (CHF), may be a predictor for poor outcome. CSR in patients with CHF is believed to be associated with a hypersensitivity to arterial CO 2 during sleep. The key pathophysiologic mechanism leading to all forms of periodic breathing is the oscillation of blood CO 2 level below and above the apneic threshold.  Clinically, these pathophysiologic changes may translate to sleep fragmentation, excessive daytime sleepiness, reduced exercise capacity, and, possibly, ventricular arrhythmias.
Current treatment options for CSR include medications, positive airway pressure devices such as adapt servo-ventilation, or oxygen therapy. Although all these therapies have shown benefi t in some patients, none has shown a consistent benefi t of suffi cient clinical magnitude to reduce mortality and morbidity. In the current study, we explored the initial feasibility, safety, and possible effects of unilateral, transvenous, synchronized PNS on CSR in 19 patients with CHF . This novel treatment resulted in a marked reduction of minute ventilation and possible improvement of CSR. The authors here suggest that phrenic nerve stimulation (PNS) may interrupt CSR in patients with CHF.

Study Population

 Nineteen patients with CHF and CSR were enrolled.  All study patients (N 5 19) had received a diagnosis of CSR and chronic CHF and were hospitalized in The First Affiliated Hospital of Nanjing Medical University (Nanjing, China). Of them, 12 with rheumatic cardiac valve disease were waiting forcardiac surgery, and seven (fi ve with dilated cardiomyopathy and two with hypertensive heart disease) were enrolled from the cardiology ward because of severe heart failure.
The inclusion criteria were aimed at identifying patients with symptoms or a diagnosed condition indicative of CSR who would tolerate the testing procedure. The patients continued on their standard medical regimen during participation, and in the case of an adverse event, medical treatment was at the discretion of the investigator. The inclusion criteria were as follows: (1) both patient and direct family member willingness to sign a Patient Ethics Committee-approved informed consent, (2) age > 18 years, (3) index CSR of > 15 times/h, (4) history of CHF with a left ventricle ejection fraction < 45%, and (5) ability to tolerate the study procedure and remain clinically stable for the duration of the study. Exclusion criteria were as follows: (1) baseline oxygen saturation <  90% on a stable FiO2 ; (2) evidence of phrenic nerve palsy; (3) temperature > 38.0°C; (4) inability to place stimulation lead (eg, coagulopathy, distorted anatomy, etc); (5) current enrollment in another clinical study that may confound the results of the present study; (6) no informed consent; (7) pregnancy or of childbearing potential without a negative pregnancy test within 10 days of the study procedure; (8) pacemaker, implantable cardioverter defibrillator, or cardiac resynchronization device; (9) severe COPD; (10) a history of myocardial infarction within 6 months prior to the study; and (11) unstable angina.

Study Design

 This short-term, prospective, open-label, nonrandomized feasibility study consisted of a treatment-only cohort in which each patient served as his or her own control. After patients were screened and enrolled in the study, PNS leads were placed through an interventional procedure for observation of 1 night only. During the 1-night study, we examined whether PNS caused pain, arousal during sleep, arrhythmia, changes in BP, and changes in either normal breathing or sleep apnea. We also examined the impact of PNS on central, obstructive, and mixed sleep apnea. Alterations in sleep apnea and hypopnea events were compared before and during PNS. “Before stimulation” was defined as the number of sleep apnea and hypopnea events occurring during a segment of 10 min just before delivery of PNS and served as the control for the effects of PNS. The total number of the 10-min segments before PNS, the total number of sleep apnea and hypopnea events occurred during the sum of the 10-min time were calculated,  then averaged (total number of sleep apnea and hypopnea events/total hours of the 10-min segments from all patients) and presented as the apnea-hypopnea index (AHI) for statistical analysis. AHI during PNS were also calculated and compared with AHI prior to PNS.

Sleep Study and Monitored Parameters

 All participants underwent a nocturnal, in-laboratory polysomnography (Embla S4500 PSG Amplifi er; Natus Medical Inc) and were monitored for at least 7 h overnight. The standard polysomnography recorded the EEG, bilateral electrooculograms, submental  electromyogram, ECG, chest and abdominal movement using respiratory effort bands, body position, nasal airflow using a pressure sensor, and oxygen saturation as measured by pulse oximetry (Sp o 2 ).
EEG, sleep staging, and arousals were monitored and scored using 30 epochs according to the method of Rechtschaffen and Kales. Classification of apnea and hypopnea was described by standard methodologies. CSR was identified as a special kind of CSA behaving as a cyclic pattern of periods of hyperventilation with waxing and waning tidal volumes alternating with periods of central hypopnea/apnea .

Lead Placement and PNS

A single stimulation lead was placed at the junction between the superior vena cava and brachiocephalic vein or in the left-side pericardiophrenic vein. PNS stimulation was performed using Eupnea System device (RespiCardia Inc). Respiratory properties were assessed before and during PNS. PNS was assessed at a maximum of 10 mA.

Results

Successful stimulation capture was achieved in 16 patients. Failure to capture occurred in three patients because of dislocation of leads. No adverse events were seen under maximum normal stimulation parameters for an overnight study.  No new arrhythmias, muscle contractions, arterial BP variations, pain, or unpleasant sensations were observed once PNS was delivered during sleep for these patients. It was confirmed that the catheter could be secured adequately to obtain consistent predictable stimulation thresholds without arousal from sleep. During occurrence of CSR, intermittent PNS signals were first confirmed to be successfully captured in 16 patients. When PNS was applied following a series of central sleep apneic events, a trend toward stabilization of breathing and heart rate.  An improvement in oxygen saturation and elevation of end-tidal CO2 was observed as longer continuous stimulation was performed. The period of stable breathing lasted as long as 10 to 20 min in some patients after stimulation.  They found that when electric connection to the nerve was consistent, stimulation resulted in a reduced hyperventilation followed by the reduction or elimination of CSR.
Compared with pre-PNS, during PNS there was a significant decrease in apnea-hypopnea index (33.8 ± 9.3 vs 8.1 ± 2.3, P = .00), an increase in mean and minimal oxygen saturation as measured by pulse oximetry (89.7% ± 1.6% vs 94.3% ± 0.9% and 80.3% ± 3.7% vs 88.5% ± 3.3%, respectively, all P = .00) and end-tidal CO2 (38.0 ± 4.3 mm Hg vs 40.3 ± 3.1 mm Hg, P = .02), but no significant difference in sleep efficiency (74.6% ± 4.1% vs 73.7% ± 5.4%, P = .36).

Discussion

CSR is characterized by cyclical oscillations of respiration and apnea. The incidence of CSR ranges from 10% to 20% in patients with stable CHF and up to 40% to 50% of all patients with New York Heart Association functional class III?IV CHF.  Nocturnal breathing alterations in patients with CHF are believed to be due to hypersensitivity to CO 2 during sleep. Breathing is controlled by a negative feedback system in which an increase in Pa co 2 stimulates breathing, whereas a decrease in Pa co 2 inhibits breathing. Normally, Pa co 2 is maintained within a narrow range. Patients with CHF and CSA have a more brisk ventilatory response to CO 2 than those without CSA.
The preliminary results showed that in a small group of patients with CHF and CSR, 1 night of unilateral transvenous PNS improved indices of CSR and was not associated with adverse events.
The study was performed using temporary catheters or leads in the right-side brachiocephalic vein, SVC, or left-side pericardiophrenic vein to transvenously stimulate the hemidiaphragm through either the leftside or the right-side phrenic nerve. To consistently stimulate the phrenic nerve using acceptable and safe current levels ( < 10 mA), the stimulation electrode needs to be within 2 to 5 mm from the phrenic nerve.  This type of stimulation caused significantly improved respiratory parameters in patients with CHF and further support that oscillation of CO 2 level in the blood below and above the apneic threshold is a central mechanism leading to the CSR pattern of breathing. Stabilization of CO 2 levels through PNS produced a regular breathing pattern, improvement in oxygen saturation, and fewer apneic events.
Dr.  Isaac George: contributed to data evaluation and drafting of the manuscript.

Related articles

Other related articles published on this Open Access Online Scientific Journal include the following:

Implantable Synchronized Cardiac Assist Device Designed for Heart Remodeling: Abiomed’s Symphony

Aviva Lev-Ari, PhD, RN, 7/11/2012

https://pharmaceuticalintelligence.com/2012/07/11/implantable-synchronized-cardiac-assist-device-designed-for-heart-remodeling-abiomeds-symphony/

Biomaterials Technology: Models of Tissue Engineering for Reperfusion and Implantable Devices for Revascularization

Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/5_04_2013/bernstein_lev-ari/Bioengineering_of_Vascular_and_Tissue_Models

Foreseen changes in Guideline of Treatment of Cardiogenic Shock with Intra-aortic Balloon counterPulsation (IABP)

Evidence for Overturning the Guidelines in Cardiogenic Shock

Clinical Indications for Use of Inhaled Nitric Oxide (iNO) in the Adult Patient Market: Clinical Outcomes after Use, Therapy Demand and Cost of Care

Aviva Lev-Ari, PhD, RN, 6/3/2013

Aviral Vatsa PhD MBBS, 1/4/2013

Clinical Trials Results for Endothelin System: Pathophysiological role in Chronic Heart Failure, Acute Coronary Syndromes and MI – Marker of Disease Severity or Genetic Determination?

Aviva Lev-Ari, PhD, RN, 10/19/2013

https://pharmaceuticalintelligence.com/2012/10/19/clinical-trials-results-for-endothelin-system-pathophysiological-role-in-chronic-heart-failure-acute-coronary-syndromes-and-mi-marker-of-disease-severity-or-genetic-determination/

Diagnosis of Cardiovascular Disease, Treatment and Prevention: Current & Predicted Cost of Care and the Promise of Individualized Medicine Using Clinical Decision Support Systems

Justin Pearlman MD ME PhD MA FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/05/15/diagnosis-of-cardiovascular-disease-treatment-and-prevention-current-predicted-cost-of-care-and-the-promise-of-individualized-medicine-using-clinical-decision-support-systems-2/

Visualisation of Cheyne-Stokes respiration

Visualisation of Cheyne-Stokes respiration (Photo credit: Wikipedia)

Cheyne-Stokes respiration

Cheyne-Stokes respiration (Photo credit: Wikipedia)

Cheyne-Stokes respiration

Cheyne-Stokes respiration (Photo credit: Wikipedia)

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Transcatheter Aortic Valve Replacement (TAVR): Postdilatation to Reduce Paravalvular Regurgitation During TAVR with a Balloon-expandable Valve

Reviewer: Larry H Bernstein, MD, FCAP

and

Curator: Aviva Lev-Ari, PhD, RN

This report is one in a series on advances in cardiovascular surgery.  This report particularly focuses on the safety and efficacy of transcatheter aortic valve replacement (TAVR), a major study carried out at Columbia University Medical Center, involving reduction of paravalvular regurgitation post TAVI.

Circ Cardiovasc Interv. 2013 Feb;6(1):85-91. doi: 10.1161/CIRCINTERVENTIONS.112.971614. Epub 2013 Jan 22.

Efficacy and safety of postdilatation to reduce paravalvular regurgitation during balloon-expandable transcatheter aortic valve replacement.

Daneault BKoss EHahn RTKodali SWilliams MRGénéreux PParadis JMGeorge IReiss GRMoses JWSmith CRLeon MB.

Source

Columbia University Medical Center/New York-Presbyterian Hospital and the Cardiovascular Research Foundation, New York, NY 10032, USA.

Abstract

BACKGROUND:

Paravalvular regurgitation (PVR) is common after transcatheter aortic valve replacement (TAVR) and may be associated with adverse outcomes. Postdilatation (PD) has been proposed to treat PVR without being formally studied. We performed a study to evaluate the safety and efficacy of PD after balloon expandable TAVR.

METHODS AND RESULTS:

Consecutive cases of TAVR were reviewed for clinical outcomes. Procedural transesophageal echocardiography imaging was reviewed for a subgroup of consecutive patients. PVR areas seen on a short-axis view were measured immediately after deployment, after PD, and at the completion of the study. Stent dimensions measured using angiography and the Paieon’s C-THV system pre- and post-PD were compared. Between May 2007 and November 2011, 259 patients underwent TAVR at our institution. PD was performed in 106 patients (41%). These patients had larger annulus, lower cover-index; more often had transfemoral access and implantation of a 26 mm valve. There was a nonsignificant greater rate of cerebrovascular events in PD patients. There was no significant difference in major aortic injury and permanent pacemaker implantation rates between groups. TTE studies were reviewed in 58 patients (35 with PD and 23 without PD). PD patients had larger PVR areas immediately after deployment (40.3±17.1 versus 15.4±14.2 mm(2); P<0.0001). There was significant reduction in PVR area attributable to PD (21.7±9.3 mm(2); P<0.0001). Spontaneous regression of PVR was seen in both groups. PD increased stent dimensions.

CONCLUSIONS:

This study demonstrates the efficacy of PD at reducing PVR in patients with greater than mild PVR after balloon-expandable TAVR.

PMID: 23339841

Efficacy and Safety of Postdilatation to Reduce Paravalvular Regurgitation During Balloon-Expandable Transcatheter Aortic Valve Replacement

Daneault R, Koss E, Hahn RT, Kodali S, Williams MR, et al.
Circ Cardiovasc Interv. 2013;6:85-91. http://dx.doi.org/10.1161/circinterventions.112.971614

Transcatheter aortic valve replacement (TAVR) has emerged as a new alternative treatment for patients with severe aortic stenosis, who are at high risk or deemed inadequate candidates for conventional surgical aortic valve replacement. Paravalvular regurgitation (PVR) is common after transcatheter aortic valve replacement (TAVR) reported in 80% to 96% of TAVR cases Moreover, moderate and severe degrees of regurgitation are associated with worse clinical outcomes While the risk factors are known and include: smaller cover index, annulus eccentricity, and the degree and distribution of leaflet calcifications, postdilatation (PD) of balloon expandable valves after implantation, including transcatheter heart valve (THV) traumatic aorta injury, cerebrovascular embolus, and conduction block may outweigh the potential benefits from reduction in aortic regurgitation. Therefore, these investigators performed a study to evaluate the safety and efficacy of PD after balloon expandable TAVR.

What Is Known

• Significant paravalvular regurgitation after transcatheter aortic valve replacement is associatedwith increased mortality.
• Calcifications, undersized prosthesis, and malposition are causes of paravalvular regurgitation.

Study Design

Procedural and in-hospital outcomes for all consecutive patients treated between May 2007 and November 2011 with Edwards SAPIEN THV (Edwards Lifescience, Irvine, CA) as part of the PARTNER and PARTNER 2 trials were reviewed both prospectively and retrospectively. Information on PD was collected retrospectively from chart and imaging review for the period between 2007 and August 2010, and prospectively after August 2010. PD was performed in cases where PVR was qualitatively more than mild, by transesophageal echocardiography (TEE), immediately after THV implantation. There were 259 patients who underwent TAVR. PD was performed in 106 patients (41%). Procedural transesophageal echocardiography imaging was reviewed for a subgroup of consecutive patients. PVR areas seen on a short-axis view were measured immediately after deployment, after PD, and at the completion of the study. Stent dimensions measured using angiography and the Paieon’s C-THV system pre- and post-PD were compared, and TTE studies were reviewed in 58 patients (35 with PD and 23 without PD).

Endpoints

Neurological events were defined using valve academic research consortium definitions.14 Cover-index is defined as: 100×([THV diameter–TEE annulus diameter]/THV diameter).3 Clinical end points for the current analysis included 30-day mortality, in-hospital stroke or transient ischemic attack, procedural related major aortic injury (aortic dissection, aortic wall hematoma, or annulus/aortic rupture) and need for new permanent pacemaker during the index hospitalization. Echocardiographic end points included spontaneous reduction of PVR [difference between PVR1 and PVR3 in the non-PD group (PD−) and difference between PVR2 and PVR3 in the PD group (PD+)], and reduction of PVR attributable to PD
(PVR1−PVR2) in the PD+. Angiographic end points included additional expansion of IF, OF, and minimal diameters of stents after PD.

Results and Clinical Outcomes

No valve embolization occurred during PD. No patient required implantation of a second THV after PD. Multiple PD was performed in 4 cases. There was no statistically significant
difference between the 2 groups in the incidence of neurological events, although they were more frequent in patients with PD. Permanent pacemaker implantation during the index hospitalization was not significantly different between the 2 groups. Major aortic injuries were rare and occurred at a similar rate between both groups with no aortic annulus rupture in either group.

These (PD) patients had larger annulus, lower cover-index; more often had transfemoral access and implantation of a 26 mm valve. There was a nonsignificant greater rate of cerebrovascular events in PD patients. There was no significant difference in major aortic injury and permanent pacemaker implantation rates between groups.
PD patients had larger PVR areas immediately after deployment (40.3±17.1 versus 15.4±14.2 mm2; P<0.0001). There was significant reduction in PVR area attributable to PD (21.7±9.3 mm2; P<0.0001). Spontaneous regression of PVR was seen in both groups.
PD increased stent dimensions. There was a significant increase in the OF, IF, and minimal diameters after PD of 26 mm valves. The changes were not statistically significant for the 23 mm valves. There was a greater expansion in the IF and OF diameters compared with the minimal diameter.

Discussion

This study is the second that demonstrates the efficacy of PD at reducing postdeployment PVR in patients with greater than mild PVR after balloon-expandable TAVR. Moreover, judicious use of PD for greater than mild PVR is not associated with excess morbidity or mortality, although some concerns regarding cerebral embolism deserve comment. When it occurs, PVR is a significant cause of nonstructural prosthetic valve dysfunction. The anatomic positioning and resultant physiology of THV, however, are different from surgical valves. After surgical aortic valve replacement, most commonly PVR is attributable to infection, suture dehiscence, or fibrosis and calcification of the native annulus, resulting in inadequate contact or gaps between the sewing ring and annulus. Because THVs do not have a sewing ring traditional dehiscence cannot occur. For balloon-expandable THV, significant PVR most commonly results from incomplete prosthesis apposition to the native annulus.

What the Study Adds

• Additional postdilatation can reduce the magnitude of paravalvular regurgitation.
• Spontaneous regression of paravalvular regurgitation occurs within minutes after transcatheter aortic valve replacement.
• Postdilatation may be associated with increased risk of cerebrovascular events.

Other TAVR related articles published on this Open Access Online Scientific Journal include the following:

Lev-Ari, A. 2/12/2013 Clinical Trials on transcatheter aortic valve replacement (TAVR) to be conducted by American College of Cardiology and the Society of Thoracic Surgeons

https://pharmaceuticalintelligence.com/2013/02/12/american-college-of-cardiologys-and-the-society-of-thoracic-surgeons-entrance-into-clinical-trials-is-noteworthy-read-more-two-medical-societies-jump-into-clinical-trial-effort-for-tavr-tech-f/

  

Lev-Ari, A. 8/13/2012 Coronary Artery Disease – Medical Devices Solutions: From First-In-Man Stent Implantation, via Medical Ethical Dilemmas to Drug Eluting Stents https://pharmaceuticalintelligence.com/2012/08/13/coronary-artery-disease-medical-devices-solutions-from-first-in-man-stent-implantation-via-medical-ethical-dilemmas-to-drug-eluting-stents/

 

Lev-Ari, A. 7/18/2012 Percutaneous Endocardial Ablation of Scar-Related Ventricular Tachycardia

https://pharmaceuticalintelligence.com/2012/07/18/percutaneous-endocardial-ablation-of-scar-related-ventricular-tachycardia/

 

Lev-Ari, A. 6/22/2012 Competition in the Ecosystem of Medical Devices in Cardiac and Vascular Repair: Heart Valves, Stents, Catheterization Tools and Kits for Open Heart and Minimally Invasive Surgery (MIS)

https://pharmaceuticalintelligence.com/2012/06/22/competition-in-the-ecosystem-of-medical-devices-in-cardiac-and-vascular-repair-heart-valves-stents-catheterization-tools-and-kits-for-open-heart-and-minimally-invasive-surgery-mis/

Lev-Ari, A. 6/19/2012 Executive Compensation and Comparator Group Definition in the Cardiac and Vascular Medical Devices Sector: A Bright Future for Edwards Lifesciences Corporation in the Transcatheter Heart Valve Replacement Market

https://pharmaceuticalintelligence.com/2012/06/19/executive-compensation-and-comparator-group-definition-in-the-cardiac-and-vascular-medical-devices-sector-a-bright-future-for-edwards-lifesciences-corporation-in-the-transcatheter-heart-valve-replace/

 

Lev-Ari, A. 6/22/2012 Global Supplier Strategy for Market Penetration & Partnership Options (Niche Suppliers vs. National Leaders) in the Massachusetts Cardiology & Vascular Surgery Tools and Devices Market for Cardiac Operating Rooms and Angioplasty Suites

https://pharmaceuticalintelligence.com/2012/06/22/global-supplier-strategy-for-market-penetration-partnership-options-niche-suppliers-vs-national-leaders-in-the-massachusetts-cardiology-vascular-surgery-tools-and-devices-market-for-car/

 We reported on the following Medical Devices News:

Cardiac Surgery Theatre in China vs. in the US: Cardiac Repair Procedures, Medical Devices in Use, Technology in Hospitals, Surgeons’ Training and Cardiac Disease Severity”    https://pharmaceuticalintelligence.com/2013/01/08/cardiac-surgery-theatre-in-china-vs-in-the-us-cardiac-repair-procedures-medical-devices-in-use-technology-in-hospitals-surgeons-training-and-cardiac-disease-severity/

Acute Chest Pain/ER Admission: Three Emerging Alternatives to Angiography and PCI    https://pharmaceuticalintelligence.com/2013/03/10/acute-chest-painer-admission-three-emerging-alternatives-to-angiography-and-pci/

FDA Pending 510(k) for The Latest Cardiovascular Imaging Technology
https://pharmaceuticalintelligence.com/2013/01/28/fda-pending-510k-for-the-latest-cardiovascular-imaging-technology/

PCI Outcomes, Increased Ischemic Risk associated with Elevated Plasma Fibrinogen not Platelet Reactivity
https://pharmaceuticalintelligence.com/2013/01/10/pci-outcomes-increased-ischemic-risk-associated-with-elevated-plasma-fibrinogen-not-platelet-reactivity/

The ACUITY-PCI score: Will it Replace Four Established Risk Scores — TIMI, GRACE, SYNTAX, and Clinical SYNTAX
https://pharmaceuticalintelligence.com/2013/01/03/the-acuity-pci-score-will-it-replace-four-established-risk-scores-timi-grace-syntax-and-clinical-syntax/

Coronary artery disease in symptomatic patients referred for coronary angiography: Predicted by Serum Protein Profiles
https://pharmaceuticalintelligence.com/2012/12/29/coronary-artery-disease-in-symptomatic-patients-referred-for-coronary-angiography-predicted-by-serum-protein-profiles/

Ablation Devices Market to 2016 – Global Market Forecast and Trends Analysis by Technology, Devices & Applications
https://pharmaceuticalintelligence.com/2012/12/23/ablation-devices-market-to-2016-global-market-forecast-and-trends-analysis-by-technology-devices-applications/

Heart Renewal by pre-existing Cardiomyocytes: Source of New Heart Cell Growth Discovered
https://pharmaceuticalintelligence.com/2012/12/23/heart-renewal-by-pre-existing-cardiomyocytes-source-of-new-heart-cell-growth-discovered/

To Stent or Not? A Critical Decision
https://pharmaceuticalintelligence.com/2012/10/23/to-stent-or-not-a-critical-decision/

Transcatheter Aortic-Valve Replacement for Inoperable Severe Aortic Stenosis

https://pharmaceuticalintelligence.com/2012/09/03/transcatheter-aortic-valve-replacement-for-inoperable-severe-aortic-stenosis/

New Definition of MI Unveiled, Fractional Flow Reserve (FFR)CT for Tagging Ischemia

https://pharmaceuticalintelligence.com/2012/08/27/new-definition-of-mi-unveiled-fractional-flow-reserve-ffrct-for-tagging-ischemia/

New Drug-Eluting Stent Works Well in STEMI
https://pharmaceuticalintelligence.com/2012/08/22/new-drug-eluting-stent-works-well-in-stemi/

Expected New Trends in Cardiology and Cardiovascular Medical Devices
https://pharmaceuticalintelligence.com/2012/08/17/expected-new-trends-in-cardiology-and-cardiovascular-medical-devices/

English: This is a video clip from a living, b...

English: This is a video clip from a living, beating pig heart that was prepared in the laboratory as a working Langendorf preparation. The heart was arrested, connected to the perfusion system and restarted. The working fluid was oxygenated balanced saline solution. (Photo credit: Wikipedia)

English: Phonocardiograms from normal and abno...

English: Phonocardiograms from normal and abnormal heart sounds (Photo credit: Wikipedia)

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Synthetic Biology: On Advanced Genome Interpretation for Gene Variants and Pathways: What is the Genetic Base of Atherosclerosis and Loss of Arterial Elasticity with Aging

Curator: Aviva Lev-Ari, PhD, RN

UPDATED on 11/6/2018

Which biological systems should be engineered?

To solve real-world problems using emerging abilities in synthetic biology, research must focus on a few ambitious goals, argues Dan Fletcher, Professor of bioengineering and biophysics, and chair of the Department of Bioengineering at the University of California, Berkeley, USA. He is also a Chan Zuckerberg Biohub Investigator.
Start Quote

Artificial blood cells. Blood transfusions are crucial in treatments for everything from transplant surgery and cardiovascular procedures to car accidents, pregnancy-related complications and childhood malaria (see go.nature.com/2ozbfwt). In the United States alone, 36,000 units of red blood cells and 7,000 units of platelets are needed every day (see go.nature.com/2ycr2wo).

But maintaining an adequate supply of blood from voluntary donors can be challenging, especially in low- and middle-income countries. To complicate matters, blood from donors must be checked extensively to prevent the spread of infectious diseases, and can be kept for only a limited time — 42 days or 5 days for platelets alone. What if blood cells could be assembled from purified or synthesized components on demand?

In principle, cell-like compartments could be made that have the oxygen-carrying capacity of red blood cells or the clotting ability of platelets. The compartments would need to be built with molecules on their surfaces to protect the compartments from the immune system, resembling those on a normal blood cell. Other surface molecules would be needed to detect signals and trigger a response.

In the case of artificial platelets, that signal might be the protein collagen, to which circulating platelets are exposed when a blood vessel ruptures5. Such compartments would also need to be able to release certain molecules, such as factor V or the von Willebrand clotting factor. This could happen by building in a rudimentary form of exocytosis, for example, whereby a membrane-bound sac containing the molecule would be released by fusing with the compartment’s outer membrane.

It is already possible to encapsulate cytoplasmic components from living cells in membrane compartments6,7. Now a major challenge is developing ways to insert desired protein receptors into the lipid membrane8, along with reconstituting receptor signalling.

Red blood cells and platelets are good candidates for the first functionally useful synthetic cellular system because they lack nuclei. Complex functions such as nuclear transport, protein synthesis and protein trafficking wouldn’t have to be replicated. If successful, we might look back with horror on the current practice of bleeding one person to treat another.

Micrograph of red blood cells, 3 T-lymphocytes and activated platelets

Human blood as viewed under a scanning electron microscope.Credit: Dennis Kunkel Microscopy/SPL

Designer immune cells. Immunotherapy is currently offering new hope for people with cancer by shaping how the immune system responds to tumours. Cancer cells often turn off the immune response that would otherwise destroy them. The use of therapeutic antibodies to stop this process has drastically increased survival rates for people with multiple cancers, including those of the skin, blood and lung9. Similarly successful is the technique of adoptive T-cell transfer. In this, a patient’s T cells or those of a donor are engineered to express a receptor that targets a protein (antigen) on the surface of tumour cells, resulting in the T cells killing the cancerous cells (called CAR-T therapies)10. All of this has opened the door to cleverly rewiring the downstream signalling that results in the destruction of tumour cells by white blood cells11.

What if researchers went a step further and tried to create synthetic cells capable of moving towards, binding to and eliminating tumour cells?

In principle, untethered from evolutionary pressures, such cells could be designed to accomplish all sorts of tasks — from killing specific tumour cells and pathogens to removing brain amyloid plaques or cholesterol deposits. If mass production of artificial immune cells were possible, it might even lessen the need to tailor treatments to individuals — cutting costs and increasing accessibility.

To ensure that healthy cells are not targeted for destruction, engineers would also need to design complex signal-processing systems and safeguards. The designer immune cells would need to be capable of detecting and moving towards a chemical signal or tumour. (Reconstituting the complex process of cell motility is itself a major challenge, from the delivery of energy-generating ATP molecules to the assembly of actin and myosin motors that enable movement.)

Researchers have already made cell-like compartments that can change shape12, and have installed signalling circuits within them13. These could eventually be used to control movement and mediate responses to external signals.

Smart delivery vehicles. The relative ease of exposing cells in the lab to drugs, as well as introducing new proteins and engineering genomes, belies how hard it is to deliver molecules to specific locations inside living organisms. One of the biggest challenges in most therapies is getting molecules to the right place in the right cell at the right time.

Harnessing the natural proclivity of viruses to deliver DNA and RNA molecules into cells has been successful14. But virus size limits cargo size, and viruses don’t necessarily infect the cell types researchers and clinicians are aiming at. Antibody-targeted synthetic vesicles have improved the delivery of drugs to some tumours. But getting the drug close to the tumour generally depends on the vesicles leaking from the patient’s circulatory system, so results have been mixed.

Could ‘smart’ delivery vehicles containing therapeutic cargo be designed to sense where they are in the body and move the cargo to where it needs to go, such as across the blood–brain barrier?

This has long been a dream of those in drug delivery. The challenges are similar to those of constructing artificial blood and immune cells: encapsulating defined components in a membrane, incorporating receptors into that membrane, and designing signal-processing systems to control movement and trigger release of the vehicle’s contents.

The development of immune-cell ‘backpacks’ is an exciting step in the right direction. In this, particles containing therapeutic molecules are tethered to immune cells, exploiting the motility and targeting ability of the cells to carry the molecules to particular locations15.

A minimal chassis for expression. In each of the previous examples, the engineered cell-like system could conceivably be built to function over hours or days, without the need for additional protein production and regulation through gene expression. For many other tasks, however, such as the continuous production of insulin in the body, it will be crucial to have the ability to express proteins, upregulate or downregulate certain genes, and carry out functions for longer periods.

Engineering a ‘minimal chassis’ that is capable of sustained gene expression and functional homeostasis would be an invaluable starting point for building synthetic cells that produce proteins, form tissues and remain viable for months to years. This would require detailed understanding and incorporation of metabolic pathways, trafficking systems and nuclear import and export — an admittedly tall order.

It is already possible to synthesize DNA in the lab, whether through chemically reacting bases or using biological enzymes or large-scale assembly in a cell16. But we do not yet know how to ‘boot up’ DNA and turn a synthetic genome into a functional system in the absence of a live cell.

Since the early 2000s, biologists have achieved gene expression in synthetic compartments loaded with cytoplasmic extract17. And genetic circuits of increasing complexity (in which the expression of one protein results in the production or degradation of another) are now the subject of extensive research. Still to be accomplished are: long-lived gene expression, basic protein trafficking and energy production reminiscent of live cells.

End Quote

SOURCE

https://www.nature.com/articles/d41586-018-07291-3?utm_source=briefing-dy&utm_medium=email&utm_campaign=briefing&utm_content=20181106

 

UPDATED on 10/14/2013

Genetics of Atherosclerotic Plaque in Patients with Chronic Coronary Artery Disease

372/3:15 Genetic influence on LpPLA2 activity at baseline as evaluated in the exome chip-enriched GWAS study among ~13600 patients with chronic coronary artery disease in the STABILITY (STabilisation of Atherosclerotic plaque By Initiation of darapLadIb TherapY) trial. L. Warren, L. Li, D. Fraser, J. Aponte, A. Yeo, R. Davies, C. Macphee, L. Hegg, L. Tarka, C. Held, R. Stewart, L. Wallentin, H. White, M. Nelson, D. Waterworth.

Genetic influence on LpPLA2 activity at baseline as evaluated in the exome chip-enrichedGWASstudy among ~13600 patients with chronic coronary artery disease in the STABILITY (STabilisation of Atherosclerotic plaque By Initiation of darapLadIb TherapY) trial.

L. Warren1, L. Li1, D. Fraser1, J. Aponte1, A. Yeo2, R. Davies3, C. Macphee3, L. Hegg3,

L. Tarka3, C. Held4, R. Stewart5, L. Wallentin4, H. White5, M. Nelson1, D.

Waterworth3.

1) GlaxoSmithKline, Res Triangle Park, NC;

2) GlaxoSmithKline, Stevenage, UK;

3) GlaxoSmithKline, Upper Merion, Pennsylvania, USA;

4) Uppsala Clinical Research Center, Department of Medical Sciences, Uppsala University, Uppsala, Sweden;

5) 5Green Lane Cardiovascular Service, Auckland Cty Hospital, Auckland, New Zealand.

STABILITY is an ongoing phase III cardiovascular outcomes study that compares the effects of darapladib enteric coated (EC) tablets, 160 mg versus placebo, when added to the standard of care, on the incidence of major adverse cardiovascular events (MACE) in subjects with chronic coronary heart disease (CHD). Blood samples for determination of the LpPLA2 activity level in plasma and for extraction of DNA was obtained at randomization. To identify genetic variants that may predict response to darapladib, we genotyped ~900K common and low frequency coding variations using Illumina OmniExpress GWAS plus exome chip in advance of study completion. Among the 15828 Intent-to-Treat recruited subjects, 13674 (86%) provided informed consent for genetic analysis. Our pharmacogenetic (PGx) analysis group is comprised of subjects from 39 countries on five continents, including 10139 Whites of European heritage, 1682 Asians of East Asian or Japanese heritage, 414 Asians of Central/South Asian heritage, 268 Blacks, 1027 Hispanics and 144 others. Here we report association analysis of baseline levels of LpPLA2 to support future PGx analysis of drug response post trial completion. Among the 911375 variants genotyped, 213540 (23%) were rare (MAF < 0.5%).

Our analyses were focused on the drug target, LpPLA2 enzyme activity measured at baseline. GWAS analysis of LpPLA2 activity adjusting for age, gender and top 20 principle component scores identified 58 variants surpassing GWAS-significant threshold (5e-08).

Genome-wide stepwise regression analyses identified multiple independent associations from PLA2G7, CELSR2, APOB, KIF6, and APOE, reflecting the dependency of LpPLA2 on LDL-cholesterol levels. Most notably, several low frequency and rare coding variants in PLA2G7 were identified to be strongly associated with LpPLA2 activity. They are V279F (MAF=1.0%, P= 1.7e-108), a previously known association, and four novel associations due to I1317N (MAF=0.05%, P=4.9e-8), Q287X (MAF=0.05%, P=1.6e-7), T278M (MAF=0.02%, P=7.6e-5) and L389S (MAF=0.04%, P=4.3e-4).

All these variants had enzyme activity lowering effects and each appeared to be specific to certain ethnicity. Our comprehensive PGx analyses of baseline data has already provided great insight into common and rare coding genetic variants associated with drug target and related traits and this knowledge will be invaluable in facilitating future PGx investigation of darapladib response.

SOURCE

http://www.ashg.org/2013meeting/pdf/46025_Platform_bookmark%20for%20Web%20Final%20from%20AGS.pdf

Synthetic Biology: On Advanced Genome Interpretation for

  • Gene Variants and
  • Pathways,
  • Inversion Polymorphism,
  • Passenger Deletions,
  • De Novo Mutations,
  • Whole Genome Sequencing w/Linkage Analysis

What is the Genetic Base of Atherosclerosis and Loss of Arterial Elasticity with Aging?

In a recent publication by my colleague, Stephen J. Williams, Ph.D. on  5/15/2013 titled

Finding the Genetic Links in Common Disease:  Caveats of Whole Genome Sequencing Studies

https://pharmaceuticalintelligence.com/2013/05/15/finding-the-genetic-links-in-common-disease-caveats-of-whole-genome-sequencing-studies/

we learned that:

  • Groups of variants in the same gene confirmed link between APOC3 and higher risk for early-onset heart attack
  • No other significant gene variants linked with heart disease

APOC3 – apolipoprotein C-III – Potential Relevance to the Human Aging Process

Main reason for selection
Entry selected based on indirect or inconclusive evidence linking the gene product to ageing in humans or in one or more model systems
Description
APOC3 is involved in fat metabolism and may delay the catabolism of triglyceride-rich particles. Changes in APOC3 expression levels have been reported in aged mice [1754]. Results from mice suggest that FOXO1 may regulate the expression of APOC3 [1743]. Polymorphisms in the human APOC3 gene and promoter have been associated with lipoprotein profile, cardiovascular health, insulin (INS) sensitivity, and longevity [1756]. Therefore, APOC3 may impact on some age-related diseases, though its exact role in human ageing remains to be determined.

Cytogenetic information

Cytogenetic band
11q23.1-q2
Location
116,205,833 bp to 116,208,997 bp
Orientation
Plus strand

Display region using the UCSC Genome Browser

Protein information

Gene Ontology
Process: GO:0006869; lipid transport
GO:0016042; lipid catabolic process
GO:0042157; lipoprotein metabolic process
Function: GO:0005319; lipid transporter activity
Cellular component: GO:0005576; extracellular region
GO:0042627; chylomicron

Protein interactions and network

No interactions in records.

Retrieve sequences for APOC3

Promoter
Promoter
ORF
ORF
CDS
CDS

Homologues in model organisms

Bos taurus
APOC3_BOVI
Mus musculus
Apoc3
Pan troglodytes
APOC3

In other databases

AnAge
This species has an entry in AnAge

Selected references

  • [2125] Pollin et al. (2008) A null mutation in human APOC3 confers a favorable plasma lipid profile and apparent cardioprotection.PubMed
  • [1756] Atzmon et al. (2006) Lipoprotein genotype and conserved pathway for exceptional longevity in humansPubMed
  • [1755] Araki and Goto (2004) Dietary restriction in aged mice can partially restore impaired metabolism of apolipoprotein A-IV and C-IIIPubMed
  • [1743] Altomonte et al. (2004) Foxo1 mediates insulin action on apoC-III and triglyceride metabolismPubMed
  • [1754] Araki et al. (2004) Impaired lipid metabolism in aged mice as revealed by fasting-induced expression of apolipoprotein mRNAs in the liver and changes in serum lipidsPubMed
  • [1753] Panza et al. (2004) Vascular genetic factors and human longevityPubMed
  • [1752] Anisimov et al. (2001) Age-associated accumulation of the apolipoprotein C-III gene T-455C polymorphism C 

http://genomics.senescence.info/genes/entry.php?hgnc=APOC3

Apolipoprotein C-III is a protein component of very low density lipoprotein (VLDL). APOC3 inhibitslipoprotein lipase and hepatic lipase; it is thought to inhibit hepatic uptake[1] of triglyceride-rich particles. The APOA1, APOC3 and APOA4 genes are closely linked in both rat and human genomes. The A-I and A-IV genes are transcribed from the same strand, while the A-1 and C-III genes are convergently transcribed. An increase in apoC-III levels induces the development of hypertriglyceridemia.

Clinical significance

Two novel susceptibility haplotypes (specifically, P2-S2-X1 and P1-S2-X1) have been discovered in ApoAI-CIII-AIV gene cluster on chromosome 11q23; these confer approximately threefold higher risk ofcoronary heart disease in normal[2] as well as non-insulin diabetes mellitus.[3]Apo-CIII delays the catabolism of triglyceride rich particles. Elevations of Apo-CIII found in genetic variation studies may predispose patients to non-alcoholic fatty liver disease.

  1. ^ Mendivil CO, Zheng C, Furtado J, Lel J, Sacks FM (2009). “Metabolism of VLDL and LDL containing apolipoprotein C-III and not other small apolipoproteins – R2”.Arteriosclerosis, Thrombosis and Vascular Biology 30 (2): 239–45. doi:10.1161/ATVBAHA.109.197830PMC 2818784PMID 19910636.
  2. ^ Singh PP, Singh M, Kaur TP, Grewal SS (2007). “A novel haplotype in ApoAI-CIII-AIV gene region is detrimental to Northwest Indians with coronary heart disease”. Int J Cardiol 130 (3): e93–5. doi:10.1016/j.ijcard.2007.07.029PMID 17825930.
  3. ^ Singh PP, Singh M, Gaur S, Grewal SS (2007). “The ApoAI-CIII-AIV gene cluster and its relation to lipid levels in type 2 diabetes mellitus and coronary heart disease: determination of a novel susceptible haplotype”. Diab Vasc Dis Res 4 (2): 124–29. doi:10.3132/dvdr.2007.030PMID 17654446.

In 2013 we reported on the discovery that there is a

Genetic Associations with Valvular Calcification and Aortic Stenosis

N Engl J Med 2013; 368:503-512

February 7, 2013DOI: 10.1056/NEJMoa1109034

METHODS

We determined genomewide associations with the presence of aortic-valve calcification (among 6942 participants) and mitral annular calcification (among 3795 participants), as detected by computed tomographic (CT) scanning; the study population for this analysis included persons of white European ancestry from three cohorts participating in the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium (discovery population). Findings were replicated in independent cohorts of persons with either CT-detected valvular calcification or clinical aortic stenosis.

CONCLUSIONS

Genetic variation in the LPA locus, mediated by Lp(a) levels, is associated with aortic-valve calcification across multiple ethnic groups and with incident clinical aortic stenosis. (Funded by the National Heart, Lung, and Blood Institute and others.)

SOURCE:

N Engl J Med 2013; 368:503-512

Related Research by Author & Curator of this article:

Artherogenesis: Predictor of CVD – the Smaller and Denser LDL Particles

Cardiovascular Biomarkers

Genetics of Conduction Disease: Atrioventricular (AV) Conduction Disease (block): Gene Mutations – Transcription, Excitability, and Energy Homeostasis

Genomics & Genetics of Cardiovascular Disease Diagnoses: A Literature Survey of AHA’s Circulation Cardiovascular Genetics, 3/2010 – 3/2013

Hypertriglyceridemia concurrent Hyperlipidemia: Vertical Density Gradient Ultracentrifugation a Better Test to Prevent Undertreatment of High-Risk Cardiac Patients

Hypertension and Vascular Compliance: 2013 Thought Frontier – An Arterial Elasticity Focus

Personalized Cardiovascular Genetic Medicine at Partners HealthCare and Harvard Medical School

Genomics Orientations for Individualized Medicine Volume One

Market Readiness Pulse for Advanced Genome Interpretation and Individualized Medicine

We present below the MARKET LEADER in Interpretation of the Genomics Computations Results in the emerging new ERA of Medicine:  Genomic Medicine, Knome.com and its home grown software power house.

A second Case study in the  Advanced Genome Interpretation and Individualized Medicine presented following the Market Leader, is the Genome-Phenome Analyzer by SimulConsult, A Simultaneous Consult On Your Patient’s Diagnosis, Chestnut Hill, MA

 

2012: The Year When Genomic Medicine Started Paying Off

Luke Timmerman

An excerpt of an interesting article mentioning Knome [emphasis ours]…

Remember a couple of years ago when people commemorated the 10-year anniversary of the first draft human genome sequencing? The storyline then, in 200, was that we all went off to genome camp and only came home with a lousy T-shirt. Society, we were told, invested huge scientific resources in deciphering the code of life, and there wasn’t much of a payoff in the form of customized, personalized medicine.

That was an easy conclusion to reach then, when personalized medicine advocates could only point to a couple of effective targeted cancer drugs—Genentech’s Herceptin and Novartis’ Gleevec—and a couple of diagnostics. But that’s changing. My inbox the past week has been full of analyst reports from medical meetings, which mostly alerted readers to mere “incremental” advances with a number of genomic-based medicines and diagnostics. But that’s a matter of focusing on the trees, not the forest. This past year, we witnessed some really impressive progress from the early days of “clinical genomics” or “medical genomics.” The investment in deep understanding of genomics and biology is starting to look visionary.

The movement toward clinical genomics gathered steam back in June at the American Society of Clinical Oncology annual meeting. One of the hidden gem stories from ASCO was about little companies like Cambridge, MA-based Foundation Medicine and Cambridge, MA-based Knome that started seeing a surprising surge in demand from physicians for their services to help turn genomic data into medical information. The New York Times wrote a great story a month later about a young genomics researcher at Washington University in St. Louis who got cancer, had access to incredibly rich information about his tumors, and—after some wrestling with his insurance company—ended up getting a targeted drug nobody would have thought to prescribe without that information. And last month, I checked back on Stanford University researcher Mike Snyder, who made headlines this year using a smorgasbord of “omics” tools to correctly diagnose himself early with Type 2 diabetes, and then monitor his progress back into a healthy state–read the entire article

http://www.knome.com/knome-blog/2012-the-year-when-genomic-medicine-started-paying-off/

Knome and Real Time Genomics Ink Deal to Integrate and Sell the RTG Variant Platform on knoSYS™100 System

Partnership to bring accurate and fast genome analysis to translational researchers

CAMBRIDGE, MA –  May 6, 2013 – Knome Inc., the genome interpretation company, and Real Time Genomics, Inc., the genome analytics company, today announced that the Real Time Genomics (RTG) Variant platform will be integrated into every shipment of the knoSYS™100 interpretation system. The agreement enables customers to easily purchase the RTG analytics engine as an upgrade to the system. The product will combine two world-class commercial platforms to deliver end-to-end genome analytics and interpretation with superior accuracy and speed. Financial terms of the agreement were not disclosed.

“In the past year demand for genome interpretation has surged as translational researchers and clinicians adopt sequencing for human disease discovery and diagnosis,” said Wolfgang Daum, CEO of Knome. “Concomitant with that demand is the need for accurate and easy-to-use industrial grade analysis that meets expectations of clinical accuracy. The RTG platform is both incredibly fast and truly differentiating to customers doing family studies, and we are excited to add such a powerful platform to the knoSYS ecosystem.”

The partnership simplifies the purchasing process by allowing knoSYS customers to purchase the RTG platform directly from Knome sales representatives.

“The Knome system is a perfect complementary channel to further expand our commercial effort to bring the RTG platform to market,” said Steve Lombardi, CEO of Real Time Genomics. “Knome has built a recognizable brand around human clinical genome interpretation, and by delivering the RTG platform within their system, both companies are simplifying genomics to help customers understand human disease and guide clinical actions.”

About Knome

Knome Inc. (www.knome.com) is a leading provider of human genome interpretation systems and services. We help clients in two dozen countries identify the genetic basis of disease, tumor growth, and drug response. Designed to accelerate and industrialize the process of interpreting whole genomes, Knome’s big data technologies are helping to pave the healthcare industry’s transition to molecular-based, precision medicine.

About Real Time Genomics

Real Time Genomics (www.realtimegenomics.com) has a passion for genomics.  The company offers software tools and applications for the extraction of unique value from genomes.  Its competency lies in applying the combination of its patented core technology and deep computational expertise in algorithms to solve problems in next generation genomic analysis.  Real Time Genomics is a private San Francisco based company backed by investment from Catamount Ventures, Lightspeed Venture Partners, and GeneValue Ltd.

http://www.knome.com/knome-blog/knome-and-real-time-genomics-ink-deal-to-integrate-and-sell-the-rtg-variant-platform-on-knosys100-system/

Direct-to-Consumer Genomics Reinvents Itself

Malorye Allison

An excerpt of an interesting article mentioning Knome [emphasis ours]:

Cambridge, Massachusetts–based Knome made one of the splashiest entries into the field, but has now turned entirely to contract research. The company began providing DTC whole-genome sequencing to independently wealthy individuals at a time when the price was still sky high. The company’s first client, Dan Stoicescu, was a former biotech entrepreneur who paid $350,000 to have his genome sequenced in 2008 so he could review it “like a stock portfolio” as new genetic discoveries unfolded4. About a year later, the company was auctioning off a genome, with such frills as a dinner with renowned Harvard genomics researcher George Church, at a starting price of $68,000; at the time, a full-genome sequence came at the price of $99,000, indicating that the cost of genome sequencing has been plummeting steadily.

Now, the company’s model is very different. “We stopped working with the ‘wealthy healthy’ in 2010,” says Jonas Lee, Knome’s chief marketing officer. “The model changed as sequencing changed.” The new emphasis, he says, is now on using Knome’s technology and technical expertise for genome interpretation. Knome’s customers are researchers, pharmaceutical companies and medical institutions, such as Johns Hopkins University School of Medicine in Baltimore, which in January signed the company up to interpret 1,000 genomes for a study of genetic variants underlying asthma in African American and African Caribbean populations.

Knome is trying to advance the clinical use of genomics, working with groups that “want to be prepared for what’s ahead,” Lee says. “We work with at least 50 academic institutions and 20 pharmaceutical companies looking at variants and drug response.” Cancer and idiopathic genetic diseases are the first sweet spots for genomic sequencing, he says. Although cancer genomics has been hot for a while, a recent string of discoveries of Mendelian diseases5 made by whole-genome sequencing has lit up that field, too. Lee is also confident, however, that “chronic diseases like heart disease are right behind those.” The company also provides software tools. The price for its KnomeDiscovery sequencing and analysis service starts at about $12,000 per sample–read the entire article here.

http://www.knome.com/knome-blog/direct-to-consumer-genomics-reinvents-itself/

Regenesis: How Synthetic Biology Will Reinvent Nature and Ourselves

VIEW VIDEO

http://www.colbertnation.com/the-colbert-report-videos/419824/october-04-2012/george-church

 

Knome Software Makes Sense of the Genome

The startup’s software takes raw genome data and creates a usable report for doctors.

DNA decoder: Knome’s software can tease out medically relevant changes in DNA that could disrupt individual gene function or even a whole molecular pathway, as is highlighted here—certain mutations in the BRCA2 gene, which affects the function of many other genes, can be associated with an increased risk of breast cancer.

A genome analysis company called Knome is introducing software that could help doctors and other medical professionals identify genetic variations within a patient’s genome that are linked to diseases or drug response. This new product, available for now only to select medical institutions, is a patient-focused spin on Knome’s existing products aimed at researchers and pharmaceutical companies. The Knome software turns a patient’s raw genome sequence into a medically relevant report on disease risks and drug metabolism. The software can be run within a clinic’s own network—rather than in the cloud, as is the case with some genome-interpretation services—which keeps the information private.

Advances in DNA sequencing technology have sharply reduced the amount of time and money required to identify all three billion base pairs of DNA in a person’s genome. But the use of genomic information for medical decisions is still limited because the process creates such large volumes of data. Less than five years ago, Knome, based in Cambridge, Massachusetts, made headlines by offering what seemed then like a low price—$350,000—for a genome sequencing and profiling package. The same service now costs just a few thousand dollars.

Today, genome profiling has two main uses in the clinic. It’s part of the search for the cause of rare genetic diseases, and it generates tumor-specific profiles to help doctors discover the weaknesses of a patient’s particular cancer. But within a few years, the technique could move beyond rare diseases and cancer. The information gleaned from a patient’s genome could explain the origin of specific disease, could help save costs by allowing doctors to pretreat future diseases, or could improve the effectiveness and safety of medications by allowing doctors to prescribe drugs that are tuned to a person’s ability to metabolize drugs.

But teasing out the relevant genetic information from a patient’s genome is not trivial. To find the particular genetic variant that causes a specific disease or drug response can require expertise from many disciplines—from genetics to statistics to software engineering—and a lot of time. In any given patient’s genome, millions of places in that genome will differ from the standard of reference. The vast majority of these differences, or variants, will be unrelated to a patient’s medical condition, but determining that can take between 20 minutes and two hours for each variant, says Heidi Rehm, a clinical geneticist who directs the Laboratory for Molecular Medicine at Partners Healthcare Center for Personalized Genetic Medicine in Boston, and who will soon serve on the clinical advisory board of Knome. “If you scale that to … millions of variants, it becomes impossible.”

A software package like Knome’s can help whittle down the list based on factors such as disease type, the pattern of inheritance in a family, and the effects of given mutations on genes. Other companies have introduced Web- or cloud-based services to perform such an analysis, but Knome’s software suite can operate within a hospital’s network, which is critically important for privacy-concerned hospitals.

The greatest benefit of the widespread adoption of genomics in the clinic will come from the “clinical intelligence” doctors gain from networks of patient data, says Martin Tolar, CEO of Knome. Information about the association between certain genetic variants and disease or drug response could be anonymized—that is, no specific patient could be tied to the data—and shared among large hospital networks. Knome’s software will make it easy to share that kind of information, says Tolar.

“In the future, you could be in the situation where your physician will be able to pull the most appropriate information for your specific case that actually leads to recommendations about drugs and so forth,” he says.

http://www.technologyreview.com/news/428179/knome-software-makes-sense-of-the-genome/

An End-to-end Human Genome Interpretation System

The knoSYS™100 seamlessly integrates an interpretation application (knoSOFT) and informatics engine (kGAP) with a high-performance grid computer. Designed for whole genome, exome, and targeted NGS data, the knoSYS™100 helps labs quickly go “from reads to reports.”


 


Advanced Interpretation and Reporting Software

The knoSYS™100 ships with knoSOFT, an advanced application for managing sequence data through the informatics pipeline, filtering variants, running gene panels, classifying/interpreting variants, and reporting results.

knoSOFT has powerful and scalable multi-sample comparison features–capable of performing family studies, tumor/normal studies, and large case-control comparisons of hundreds of whole genomes.

Multiple simultaneous users (10) are supported, including technicians running sequence data through informatics pipeline, developers creating next-generation gene panels, geneticists researching causal variants, and production staff processing gene panels.

http://www.knome.com/knosys-100-overview/

Publications

View our collection of journal articles and genome research papers written by Knome employees, Knome board members, and other industry experts.

Publications by Knome employees and board members

The Top Two Axes of Variation of the Combined Dataset (MS, BD, PD, and IBD)

21 Aug 2012

Discerning the Ancestry of European Americans in Genetic Association Studies

Co-authored by Dr. David Goldstein, Clinical and Scientific board member for Knome

Author summary: Genetic association studies analyze both phenotypes (such as disease status) and genotypes (at sites of DNA variation) of a given set of individuals. … more

Pedigree and genetic risk prediction workflow

20 Aug 2012

Phased Whole-Genome Genetic Risk in a Family Quartet Using a Major Allele Reference Sequence

Co-authored by Dr. George Church and Dr. Heidi Rehm, Clinical and Scientific Board Members for Knome

Author summary: An individual’s genetic profile plays an important role in determining risk for disease and response to medical therapy. The development of technologies that facilitate rapid whole-genome sequencing will provide unprecedented power in the estimation of disease risk. Here we develop methods to characterize genetic determinants of disease risk and … more

20 Aug 2012

A Genome-Wide Investigation of SNPs and CNVs in Schizophrenia

Co-authored by Dr. David Goldstein, Clinical and Scientific board member for Knome

Author summary: Schizophrenia is a highly heritable disease. While the drugs commonly used to treat schizophrenia offer important relief from some symptoms, other symptoms are not well treated, and the drugs cause serious adverse effects in many individuals. This has fueled intense interest over the years in identifying genetic contributors to … more

fetchObject

20 Aug 2012

Whole-Genome Sequencing of a Single Proband Together with Linkage Analysis Identifies a Mendelian Disease Gene

Co-authored by Dr. David Goldstein, Clinical and Scientific board member for Knome

Author summary: Metachondromatosis (MC) is an autosomal dominant condition characterized by exostoses (osteochondromas), commonly of the hands and feet, and enchondromas of long bone metaphyses and iliac crests. MC exostoses may regress or even resolve over time, and short stature … more

19 Aug 2012

Exploring Concordance and Discordance for Return of Incidental Findings from Clinical Sequencing Co-authored by Dr. Heidi Rehm, Clinical and Scientific board member for Knome

Introduction: There is an increasing consensus that whole-exome sequencing (WES) and whole-genome sequencing (WGS) will continue to improve in accuracy and decline in price and that the use of these technologies will eventually become an integral part of clinical medicine.1–7 … more

Publications by industry experts and thought-leaders

22 Aug 2012

Rate of De Novo Mutations and the Importance of Father’s Age to Disease Risk

Augustine Kong, Michael L. Frigge, Gisli Masson, Soren Besenbacher, Patrick Sulem, Gisli Magnusson, Sigurjon A. Gudjonsson, Asgeir Sigurdsson, Aslaug Jonasdottir, Adalbjorg Jonasdottir, Wendy S. W. Wong, Gunnar Sigurdsson, G. Bragi Walters, Stacy Steinberg, Hannes Helgason, Gudmar Thorleifsson, Daniel F. Gudbjartsson, Agnar Helgason, Olafur Th. Magnusson, Unnur Thorsteinsdottir, & Kari Stefansson

Abstract: Mutations generate sequence diversity and provide a substrate for selection. The rate of de novo mutations is therefore of major importance to evolution. Here we conduct a study of genome-wide mutation rates by sequencing the entire genomes of 78 … more

15 Aug 2012

Passenger Deletions Generate Therapeutic Vulnerabilities in Cancer

Florian L. Muller, Simona Colla, Elisa Aquilanti, Veronica E. Manzo, Giannicola Genovese, Jaclyn Lee, Daniel Eisenson, Rujuta Narurkar, Pingna Deng, Luigi Nezi, Michelle A. Lee, Baoli Hu, Jian Hu, Ergun Sahin, Derrick Ong, Eliot Fletcher-Sananikone, Dennis Ho, Lawrence Kwong, Cameron Brennan, Y. Alan Wang, Lynda Chin, & Ronald A. DePinho

Abstract: Inactivation of tumour-suppressor genes by homozygous deletion is a prototypic event in the cancer genome, yet such deletions often encompass neighbouring genes. We propose that homozygous deletions in such passenger genes can expose cancer-specific therapeutic vulnerabilities when the collaterally … more

1 Jul 2012

Structural Diversity and African Origin of the 17q21.31 Inversion Polymorphism

Karyn Meltz Steinberg, Francesca Antonacci, Peter H Sudmant, Jeffrey M Kidd, Catarina D Campbell, Laura Vives, Maika Malig, Laura Scheinfeldt, William Beggs, Muntaser Ibrahim, Godfrey Lema, Thomas B Nyambo, Sabah A Omar, Jean-Marie Bodo, Alain Froment, Michael P Donnelly, Kenneth K Kidd, Sarah A Tishkoff, & Evan E Eichler

Abstract: The 17q21.31 inversion polymorphism exists either as direct (H1) or inverted (H2) haplotypes with differential predispositions to disease and selection. We investigated its genetic diversity in 2,700 individuals, with an emphasis on African populations. We characterize eight structural haplotypes … more

http://www.knome.com/publications/

knome’s Systems & Software

Technical specifications

Connections and communications

Two networks: 40-Gigabit Infiniband QDR via a Mellanox Switch for storage traffic and HP ProCurve switch for network traffic

High performance computing cluster

Four nodes, each node with two 8-core/16 thread, 2.4Ghz, 64 bit Intel® Xeon® E5-2660 processor with 20MB cache, 128GB of DDR3 ECC 1600 memory; 2x2TB SATA drives (7,200RPM)

Metadata server

2x2TB 3.5″ drives with 6GB/sec SATA, RAID 1 and 2x300GB SSD (RAID 1)

Object storage server

Lustre array: Two 12x4TB arrays of 12 3.5″ drives with 6GB/sec serial SATA channels, each OSS powered by a 6-core Intel Xeon 64-bit processor running at 20GHz with 32GB RAM.

knoSYS_server

96TB total, 64TB useable storage (redundancy for failure tolerance). Expandable 384TB total.

Data sources

Reference genome GRCh37 (HG19)

dbSNP, v137

Condel (SIFT and PolyPhen-2)

HPO

OMIM

Exome Variant server, with allelisms and allele frequencies

1000 Genomes, with allelisms and allele frequencies

Human Gene Mutation db (HGMD)

Phastcons 46, mammalian conservation

PhyloP

Input/output formats

Input formats: kGAP accepts Illumina FASTQ and VCF 4.1 files as inputs

Output formats: annotated VCF files

Electrical and operating requirements

Line voltage: 110V to 120V AC, 200-240V (single phase)

Frequency: 50Hz to 60Hz

Current: 30A, RoSH compliant

Connection: NEMA L5-30

Operating temperature: 50° to 95° F

UPS included

Maximum operating altitude: 10,000 feet

Power consumption: 2,800 VA (peak)

Size and weight

Height 49.2 Inches (1250 mm)
Width 30.7 Inches (780 mm)
Depth 47.6 Inches (1210 mm)
Weight 394 lbs (179 kg)

Noise generation and heat dissipation

Enclosure provides 28dB of acoustic noise reduction; system suitable for placing in working lab environment

7200w of active heat dissipation

Included in the package

knoSYS™100 hardware

Knome software: knoSOFT, kGAP

Operating system: Linux (CentOS 6.3)

http://www.knome.com/knosys-100-specifications/

Our research services group uses a set of advanced software tools designed for whole genome and exome interpretation. These tools are also available to our clients through our knomeBASE informatics service. In addition to various scripts, libraries, and conversion utilities, these tools include knomeVARIANTS and knomePATHWAYS.

knomeVARIANTS

Genome_software_knomeVARIANTS

knome VARIANTS is a query kit that lets users search for candidate causal variants in studied genomes. It includes a query interface (see above), scripting libraries, and data conversion utilities.

Users select cases and controls, input a putative inheritance mode, and add sensible filter criteria (variant functional class, rarity/novelty, location in prior candidate regions, etc.) to automatically generate a sorted short-list of leading candidates. The application includes a SQL query interface to let users query the database as they wish, including by complex or novel sets of criteria.

In addition to querying, the application lets users export subsets of the database for viewing in MS Excel. Subsets can be output that target common research foci, including the following:

  • Sites implicated in phenotypes, regardless of subject genotypes
  • Sites where at least one studied genome mismatches the reference
  • Sites where a particular set of one or more genomes, but no other genomes, show a novel variant
  • Sites in phenotype-implicated genes
  • Sites with nonsense, frameshift, splice-site, or read-through variants, relative to reference
  • Sites where some but not all subject genome were called

knomePATHWAYS

Genome_software_knomePATHWAYS

knomePATHWAYS is a visualization tool that overlays variants found in each sample genome onto known gene interaction networks in order to help spot functional interactions between variants in distinct genes, and pathways enriched for variants in cases versus controls, differential drug responder groups, etc.

knomePATHWAYS integrates reference data from many sources, including GO, HPRD, and MsigDB (which includes KEGG and Reactome data). The application is particularly helpful in addressing higher-order questions, such as finding candidate genes and protein pathways, that are not readily addressed from tabular annotation data alone.

http://www.knome.com/interpretation-toolkit/

Genome-Phenome Analyzer by SimulConsult

A Simultaneous Consult On Your Patient’s Diagnosis

Clinicians can get a “simultaneous consult” about their patient’s diagnosis using SimulConsult’s diagnostic decision support software.

Using the free “phenome” version, medical professionals can enter patient findings into the software and get an initial differential diagnosis and suggestions about other useful findings, including tests.  The database used by the software has > 4,000 diagnoses, most complete for genetics and neurology.  It includes all genes in GeneTests and all diseases in GeneReviews.  The information about diseases is entered by clinicians, referenced to the literature and peer-reviewed by experts.  The software takes into account pertinent negatives, temporal information, and cost of tests, information ignored in other diagnostic approaches.  It transforms medical diagnosis by lowering costs, reducing errors and eliminating the medical diagnostic odysseys experienced by far too many patients and their families.

http://www.simulconsult.com/index.html

Using the “genome-phenome analyzer” version, a lab can combine a genome variant table with the phenotypic data entered by the referring clinician, thereby using the full power of genome + phenome to arrive at a diagnosis in seconds.  An innovative measure of pertinence of genes focuses attention on the genes accounting for the clinical picture, even if more than one gene is involved.  The referring clinician can use the results in the free phenome version of the software, for example adding information from confirmatory tests or adding new findings that develop over time.  For details, click here.

http://www.simulconsult.com/genome/index.html

Michael M. Segal MD, PhD, Founder,Chairman and Chief Scientist.  Dr. Segal did his undergraduate work at Harvard and his MD and PhD at Columbia, where his thesis project outlined rules for the types of chemical synapses that will form in a nervous system.  After his residency in pediatric neurology at Columbia, he moved to Harvard Medical School, where he joined the faculty and developed the microisland system for studying small numbers of brain neurons in culture.  Using this system, he developed a simplified model of epilepsy, work that won him national and international young investigator awards, and set the stage for later work on the molecular mechanism of attention deficit disorder.  Dr. Segal has a long history of interest in computers, and patterned the SimulConsult software after the way that experienced clinicians actually think about diagnosis.  He is on the Electronic Communication Committee of the Child Neurology Society and the Scientific Program Committee of the American Medical Informatics Association.

http://www.simulconsult.com/company/management.html

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