Posts Tagged ‘interleukins’

The Role of Exosomes in Metabolic Regulation

Author: Larry H. Bernstein, MD, FCAP


On 9/25/2017, Aviva Lev-Ari, PhD, RN commissioned Dr. Larry H. Bernstein to write a short article on the following topic reported on 9/22/2017 in


We are publishing, below the new article created by Larry H. Bernstein, MD, FCAP.



During the period between 9/2015  and 6/2017 the Team at Leaders in Pharmaceutical Business Intelligence (LPBI)  has launched an R&D effort lead by Aviva Lev-Ari, PhD, RN in conjunction with SBH Sciences, Inc. headed by Dr. Raphael Nir.

This effort, also known as, “DrugDiscovery @LPBI Group”  has yielded several publications on EXOSOMES on this Open Access Online Scientific Journal. Among them are included the following:


QIAGEN – International Leader in NGS and RNA Sequencing, 10/08/2017

Reporter: Aviva Lev-Ari, PhD, RN


cell-free DNA (cfDNA) tests could become the ultimate “Molecular Stethoscope” that opens up a whole new way of practicing Medicine, 09/08/2017

Reporter: Aviva Lev-Ari, PhD, RN


Detecting Multiple Types of Cancer With a Single Blood Test (Human Exomes Galore), 07/02/2017

Reporter and Curator: Irina Robu, PhD


Exosomes: Natural Carriers for siRNA Delivery, 04/24/2017

Reporter: Aviva Lev-Ari, PhD, RN


One blood sample can be tested for a comprehensive array of cancer cell biomarkers: R&D at WPI, 01/05/2017

Curator: Marzan Khan, B.Sc


SBI’s Exosome Research Technologies, 12/29/2016

Reporter: Aviva Lev-Ari, PhD, RN


A novel 5-gene pancreatic adenocarcinoma classifier: Meta-analysis of transcriptome data – Clinical Genomics Research @BIDMC, 12/28/2016

Curator: Tilda Barliya, PhD


Liquid Biopsy Chip detects an array of metastatic cancer cell markers in blood – R&D @Worcester Polytechnic Institute, Micro and Nanotechnology Lab, 12/28/2016

Reporters: Tilda Barliya, PhD and Aviva Lev-Ari, PhD, RN


Exosomes – History and Promise, 04/28/2016

Reporter: Aviva Lev-Ari, PhD, RN


Exosomes, 11/17/2015

Curator: Larry H. Bernstein, MD, FCAP


Liquid Biopsy Assay May Predict Drug Resistance, 11/16/2015

Curator: Larry H. Bernstein, MD, FCAP


Glypican-1 identifies cancer exosomes, 10/31/2015

Curator: Larry H. Bernstein, MD, FCAP


Circulating Biomarkers World Congress, March 23-24, 2015, Boston: Exosomes, Microvesicles, Circulating DNA, Circulating RNA, Circulating Tumor Cells, Sample Preparation, 03/24/2015

Reporter: Aviva Lev-Ari, PhD, RN


Cambridge Healthtech Institute’s Second Annual Exosomes and Microvesicles as Biomarkers and Diagnostics Conference, March 16-17, 2015 in Cambridge, MA, 03/17, 2015

Reporter: Aviva Lev-Ari, PhD, RN


The newly created think-piece on the relationship between regulatory functions of Exosomes and Metabolic processes is developed conceptually, below.


The Role of Exosomes in Metabolic Regulation

Author: Larry H. Bernstein, MD, FCAP

We have had more than a half century of research into the genetic code and transcription leading to abundant work on RNA and proteomics. However, more recent work in the last two decades has identified RNA interference in siRNA. These molecules may be found in the circulation, but it has been a challenge to find their use in therapeutics. Exosomes were first discovered in the 1980s, but only recently there has been a huge amount of research into their origin, structure and function. Exosomes are 30–120 nm endocytic membrane-bound extracellular vesicles (EVs)(1-23) , and more specifically multiple vesicle bodies (MVBs) by a budding process from invagination of the outer cell membrane that carry microRNA (miRNA), and have structures composed of protein and lipids (1,23-27 ). EVs are the membrane vesicles secreted by eukaryotic cells for intracellular communication by transferring the proteins, lipids, and RNA under various physiologic conditions as well as during the disease stage. EVs also act as a signalosomes in many biological processes. Inward budding of the plasma membrane forms small vesicles that fuse. Intraluminal vesicles (ILVs) are formed by invagination of the limiting endosomal membrane during the maturation process of early endosome.

EVs are the MVBs secreted that serve in intracellular communication by transferring a cargo consisting of proteins, lipids, and RNA under various physiologic conditions (4, 23). Exosome-mediated miRNA transfer between cells is considered to be necessary for intercellular signaling and exosome-associated miRNAs in biofluids (23). Exosomes carry various molecular constituents of their cell of origin, including proteins, lipids, mRNAs, and microRNAs (miRNAs) (. They are released from many cell types, such as dendritic cells (DCs), lymphocytes, platelets, mast cells, epithelial cells, endothelial cells, and neurons, and can be found in most bodily fluids including blood, urine, saliva, amniotic fluid, breast milk, hydrothoracic fluid, and ascitic fluid, as well as in culture medium of most cell types.Exosomes have also been shown to be involved in noncoding RNA surveillance machinery in generating antibody diversity (24). There are also a vast number of long non-coding RNAs (lncRNAs) and enhancer RNAs (eRNAs) that accumulate R-loop structures upon RNA exosome ablation, thereby, resolving deleterious DNA/RNA hybrids arising from active enhancers and distal divergent eRNA-expressing elements (lncRNA-CSR) engaged in long-range DNA interactions (25). RNA exosomes are large multimeric 3′-5′ exo- and endonucleases representing the central RNA 3′-end processing factor and are implicated in processing, quality control, and turnover of both coding and noncoding RNAs. They are large macromolecular cages that channel RNA to the ribonuclease sites (29). A major interest has been developed to characterize of exosomal cargo, which includes numerous non-randomly packed proteins and nucleic acids (1). Moreover, exosomes play an active role in tumorigenesis, metastasis, and response to therapy through the transfer of oncogenes and onco-miRNAs between cancer cells and the tumor stroma. Blood cells and the vascular endothelium is also exosomal shedding, which has significance for cardiovascular,   neurologicological disorders, stroke, and antiphospholipid syndrome (1). Dysregulation of microRNAs and the affected pathways is seen in numerous pathologies their expression can reflect molecular processes of tumor onset and progression qualifying microRNAs as potential diagnostic and prognostic biomarkers (30).

Exosomes are secreted by many cells like B lymphocytes and dendritic cells of hematopoietic and non-hematopoietic origin viz. platelets, Schwann cells, neurons, mast cells, cytotoxic T cells, oligodendrocytes, intestinal epithelial cells were also found to be releasing exosomes (4). They are engaged in complex functions like persuading immune response as the exosomes secreted by antigen presenting cells activate T cells (4). They all have a common set of proteins e.g. Rab family of GTPases, Alix and ESCRT (required for transport) protein and they maintain their cytoskeleton dynamics and participate in membrane fusion. However, they are involved in retrovirus disease pathology as a result of recruitment of the host`s endosomal compartments in order to generate viral vesicles, and they can either spread or limit an infection based on the type of pathogen and its target cells (5).

Upon further consideration, it is understandable how this growing biological work on exosomes has enormous significance for laboratory diagnostics (1, 3, 5, 6, 11, 14, 15, 17-20, 23,30-41) . They are released from many cell types, such as dendritic cells (DCs), lymphocytes, platelets, mast cells, epithelial cells, endothelial cells, and neurons, and can be found in most bodily fluids including blood, urine, saliva, amniotic fluid, breast milk, thoracic and abdominal effusions, and ascitic fluid (1). The involvement of exosomes in disease is broad, and includes: cancer, autoimmune and infectious disease, hematologic disorders, neurodegenerative diseases, and cardiovascular disease. Proteins frequently identified in exosomes include membrane transporters and fusion proteins (e.g., GTPases, annexins, and flotillin), heat shock proteins (e.g., HSC70), tetraspanins (e.g., CD9, CD63, and CD81), MVB biogenesis proteins (e.g., alix and TSG101), and lipid-related proteins and phospholipases. The exosomal lipid composition has been thoroughly analyzed in exosomes secreted from several cell types including DCs and mast cells, reticulocytes, and B-lymphocytes (1). Dysregulation of microRNAs of pathways observed in numerous pathologies (5, 10, 12, 21, 27, 35, 37) including cancers (30), particularly, colon, pancreas, breast, liver, brain, lung (2, 6, 17-20, 30, 33-36, 38, 39). Following these considerations, it is important that we characterize the content of exosomal cargo to gain clues to their biogenesis, targeting, and cellular effects which may lead to identification of biomarkers for disease diagnosis, prognosis and response to treatment (42).

We might continue in pursuit of a particular noteworthy exosome, the NLRP3 inflammasome, which is activated by a variety of external or host-derived stimuli, thereby, initiating an inflammatory response through caspase-1 activation, resulting in inflammatory cytokine IL-1b maturation and secretion (43).
Inflammasomes are multi-protein signaling complexes that activate the inflammatory caspases and the maturation of interleukin-1b. The NLRP3 inflammasome is linked with human autoinflammatory and autoimmune diseases (44). This makes the NLRP3 inflammasome a promising target for anti-inflammatory therapies. The NLRP3 inflammasome is activated in response to a variety of signals that indicate tissue damage, metabolic stress, and infection (45). Upon activation, the NLRP3 inflammasome serves as a platform for activation of the cysteine protease caspase-1, which leads to the processing and secretion of the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18. Heritable and acquired inflammatory diseases are both characterized by dysregulation of NLRP3 inflammasome activation (45).
Receptors of innate immunity recognize conserved moieties associated with either cellular damage [danger-associated molecular patterns (DAMPs)] or invading organisms [pathogen-associated molecular patterns (PAMPs)](45). Either chronic stimulation or overwhelming tissue damage is injurious and responsible for the pathology seen in a number of autoinflammatory and autoimmune disorders, such as arthritis and diabetes. The nucleotide-binding domain leucine-rich repeat (LRR)-containing receptors (NLRs) are PRRs are found intracellularly and they share a unique domain architecture. It consists of a central nucleotide binding and oligomerization domain called the NACHT domain that is located between an N-terminal effector domain and a C-terminal LRR domain (45). The NLR family members NLRP1, NLRP3, and NLRC4 are capable of forming multiprotein complexes called inflammasomes when activated.

The (NLRP3) inflammasome is important in chronic airway diseases such as asthma and chronic obstructive pulmonary disease because the activation results, in pro-IL-1β processing and the secretion of the proinflammatory cytokine IL-1β (46). It has been proposed that Activation of the NLRP3 inflammasome by invading pathogens may prove cell type-specific in exacerbations of airway inflammation in asthma (46). First, NLRP3 interacts with the adaptor protein ASC by sensing microbial pathogens and self-danger signals. Then pro-caspase-1 is recruited and the large protein complex called the NLRP3 inflammasome is formed. This is followed by autocleavage and activation of caspase-1, after which pro-IL-1β and pro-IL-18 are converted into their mature forms. Ion fluxes disrupt membrane integrity, and also mitochondrial damage both play key roles in NLRP3 inflammasome activation (47). Depletion of mitochondria as well as inhibitors that block mitochondrial respiration and ROS production prevented NLRP3 inflammasome activation. Futhermore, genetic ablation of VDAC channels (namely VDAC1 and VDAC3) that are located on the mitochondrial outer membrane and that are responsible for exchanging ions and metabolites with the cytoplasm, leads to diminished mitochondrial (mt) ROS production and inhibition of NLRP3 inflammasome activation (47). Inflammasome activation not only occurs in immune cells, primarily macrophages and dendritic cells, but also in kidney cells, specifically the renal tubular epithelium. The NLRP3 inflammasome is probably involved in the pathogenesis of acute kidney injury, chronic kidney disease, diabetic nephropathy and crystal-related nephropathy (48). The inflammasome also plays a role in autoimmune kidney disease. IL-1 blockade and two recently identified specific NLRP3 inflammasome blockers, MCC950 and β-hydroxybutyrate, may prove to have value in the treatment of inflammasome-mediated conditions.

Autophagosomes derived from tumor cells are referred to as defective ribosomal products in blebs (DRibbles). DRibbles mediate tumor regression by stimulating potent T-cell responses and, thus, have been used as therapeutic cancer vaccines in multiple preclinical cancer models (49). It has been found that DRibbles could induce a rapid differentiation of monocytes and DC precursor (pre-DC) cells into functional APCs (49). Consequently, DRibbles could potentially induce strong innate immune responses via multiple pattern recognition receptors. This explains why DRibbles might be excellent antigen carriers to induce adaptive immune responses to both tumor cells and viruses. This suggests that isolated autophagosomes (DRibbles) from antigen donor cells activate inflammasomes by providing the necessary signals required for IL-1β production.

The Hsp90 system is characterized by a cohort of co-chaperones that bind to Hsp90 and affect its function (50). The co-chaperones enable Hsp90 to chaperone structurally and functionally diverse client proteins. Sahasrabudhe et al. (50) show that the nature of the client protein dictates the contribution of a co-chaperone to its maturation. The study reveals the general importance of the cochaperone Sgt1 (50). In addition to Hsp90, we have to consider Hsp60. Adult cardiac myocytes release heat shock protein (HSP)60 in exosomes. Extracellular HSP60, when not in exosomes, causes cardiac myocyte apoptosis via the activation of Toll-like receptor 4. the protein content of cardiac exosomes differed significantly from other types of exosomes in the literature and contained cytosolic, sarcomeric, and mitochondrial proteins (21).

A new Protein Organic Solvent Precipitation (PROSPR) method efficiently isolates the EV repertoire from human biological samples. Proteomic profiling of PROSPR-enriched CNS EVs indicated that > 75 % of the proteins identified matched previously reported exosomal and microvesicle cargoes. In addition lipidomic characterization of enriched CNS vesicles identified previously reported EV-specific lipid families and novel lipid isoforms not previously detected in human EVs. The characterization of these structures from central nervous system (CNS) tissues is relevant to current neuroscience, especially to advance the understanding of neurodegeneration in amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD) and Alzheimer’s disease (AD)(15). In addition, study of EVs in brain will enable characterization of the degenerative posttranslational modifications (DPMs) occurring in those proteins.
Neurodegenerative disease is characterized by dysregulation because of NLRP3 inflammasome activation. Alzheimer’s disease (AD) and Parkinson’s disease (PD), both neurodegenerative diseases are associated with the NLRP3 inflammasome. PD is characterized by accumulation of Lewy bodies (LB) formed by a-synuclein (aSyn) aggregation. A recent study revealed that aSyn induces synthesis of pro-IL-1b by an interaction with TLR2 and activates NLRP3 inflammasome resulting in caspase-1 activation and IL-1b maturation in human primary monocytes (43). In addition mitophagy downregulates NLRP3 inflammasome activation by eliminating damaged mitochondria, blocking NLRP3 inflammasome activating signals. It is notable that in this aberrant activation mitophagy downregulates NLRP3 inflammasome activation by eliminating damaged mitochondria, blocking NLRP3 inflammasome activating signals (43).


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NIH Considers Guidelines for CAR-T therapy: Report from Recombinant DNA Advisory Committee

Reporter: Stephen J. Williams, Ph.D.

In the mid to late 1970’s a public debate (and related hysteria) had emerged surrounding two emerging advances in recombinant DNA technology;

  1. the development of vectors useful for cloning pieces of DNA (the first vector named pBR322) and
  2. the discovery of bacterial strains useful in propagating such vectors

As discussed by D. S, Fredrickson of NIH’s Dept. of Education and Welfare in his historical review” A HISTORY OF THE RECOMBINANT DNA GUIDELINES IN THE UNITED STATES” this international concern of the biological safety issues of this new molecular biology tool led the National Institute of Health to coordinate a committee (the NIH Recombinant DNA Advisory Committee) to develop guidelines for the ethical use, safe development, and safe handling of such vectors and host bacterium. The first conversations started in 1974 and, by 1978, initial guidelines had been developed. In fact, as Dr. Fredrickson notes, public relief was voiced even by religious organizations (who had the greatest ethical concerns)

On December 16, 1978, a telegram purporting to be from the Vatican was hand delivered to the office of Joseph A. Califano, Jr., Secretary of Health, Education,

and Welfare. “Habemus regimen recombinatum,” it proclaimed, in celebration of the

end of a long struggle to revise the NIH Guidelines for Research Involving

Recombinant DNA Molecules

The overall Committee resulted in guidelines (2013 version) which assured the worldwide community that

  • organisms used in such procedures would have limited pathogenicity in humans
  • vectors would be developed in a manner which would eliminate their ability to replicate in humans and have defined antibiotic sensitivity

So great was the success and acceptance of this committee and guidelines, the NIH felt the Recombinant DNA Advisory Committee should meet regularly to discuss and develop ethical guidelines and clinical regulations concerning DNA-based therapeutics and technologies.

A PowerPoint Slideshow: Introduction to NIH OBA and the History of Recombinant DNA Oversight can be viewed at the following link:

Please see the following link for a video discussion between Dr. Paul Berg, who pioneered DNA recombinant technology, and Dr. James Watson (Commemorating 50 Years of DNA Science):

The Recombinant DNA Advisory Committee has met numerous times to discuss new DNA-based technologies and their biosafety and clinical implication including:

A recent Symposium was held in the summer of 2010 to discuss ethical and safety concerns and discuss potential clinical guidelines for use of an emerging immunotherapy technology, the Chimeric Antigen Receptor T-Cells (CART), which at that time had just been started to be used in clinical trials.

Considerations for the Clinical Application of Chimeric Antigen Receptor T Cells: Observations from a Recombinant DNA Advisory Committee Symposium Held June 15, 2010[1]

Contributors to the Symposium discussing opinions regarding CAR-T protocol design included some of the prominent members in the field including:

Drs. Hildegund C.J. Ertl, John Zaia, Steven A. Rosenberg, Carl H. June, Gianpietro Dotti, Jeffrey Kahn, Laurence J. N. Cooper, Jacqueline Corrigan-Curay, And Scott E. Strome.

The discussions from the Symposium, reported in Cancer Research[1]. were presented in three parts:

  1. Summary of the Evolution of the CAR therapy
  2. Points for Future Consideration including adverse event reporting
  3. Considerations for Design and Implementation of Trials including mitigating toxicities and risks

1. Evolution of Chimeric Antigen Receptors

Early evidence had suggested that adoptive transfer of tumor-infiltrating lymphocytes, after depletion of circulating lymphocytes, could result in a clinical response in some tumor patients however developments showed autologous T-cells (obtained from same patient) could be engineered to express tumor-associated antigens (TAA) and replace the TILS in the clinical setting.

However there were some problems noticed.

  • Problem: HLA restriction of T-cells. Solution: genetically engineer T-cells to redirect T-cell specificity to surface TAAs
  • Problem: 1st generation vectors designed to engineer T-cells to recognize surface epitopes but engineered cells had limited survival in patients.   Solution: development of 2nd generation vectors with co-stimulatory molecules such as CD28, CD19 to improve survival and proliferation in patients

A summary table of limitations of the two types of genetically-modified T-cell therapies were given and given (in modified form) below

                                                                                                Type of Gene-modified T-Cell

Limitations aβ TCR CAR
Affected by loss or decrease of HLA on tumor cells yes no
Affected by altered tumor cell antigen processing? yes no
Need to have defined tumor target antigen? no yes
Vector recombination with endogenous TCR yes no

A brief history of construction of 2nd and 3rd generation CAR-T cells given by


Differences between  second- and third-generation chimeric antigen receptor T cells. (Adapted by permission from the American Association for Cancer Research: Lee, DW et al. The Future Is Now: Chimeric Antigen Receptors as New Targeted Therapies for Childhood Cancer. Clin Cancer Res; 2012;18(10); 2780–90. doi:10.1158/1078-0432.CCR-11-1920)

Constructing a CAR T Cell (from

The first efforts to engineer T cells to be used as a cancer treatment began in the early 1990s. Since then, researchers have learned how to produce T cells that express chimeric antigen receptors (CARs) that recognize specific targets on cancer cells.

The T cells are genetically modified to produce these receptors. To do this, researchers use viral vectors that are stripped of their ability to cause illness but that retain the capacity to integrate into cells’ DNA to deliver the genetic material needed to produce the T-cell receptors.

The second- and third-generation CARs typically consist of a piece of monoclonal antibody, called a single-chain variable fragment (scFv), that resides on the outside of the T-cell membrane and is linked to stimulatory molecules (Co-stim 1 and Co-stim 2) inside the T cell. The scFv portion guides the cell to its target antigen. Once the T cell binds to its target antigen, the stimulatory molecules provide the necessary signals for the T cell to become fully active. In this fully active state, the T cells can more effectively proliferate and attack cancer cells.

2. Adverse Event Reporting and Protocol Considerations

The symposium had been organized mainly in response to two reported deaths of patients enrolled in a CART trial, so that clinical investigators could discuss and formulate best practices for the proper conduct and analysis of such trials. One issue raised was lack of pharmacovigilence procedures (adverse event reporting). Although no pharmacovigilence procedures (either intra or inter-institutional) were devised from meeting proceedings, it was stressed that each institution should address this issue as well as better clinical outcome reporting.

Case Report of a Serious Adverse Event Following the Administration of T Cells Transduced With a Chimeric Antigen Receptor Recognizing ERBB2[2] had reported the death of a patient on trial.

In A phase I clinical trial of adoptive transfer of folate receptor-alpha redirected autologous T cells for recurrent ovarian cancer[3] authors: Lana E Kandalaft*, Daniel J Powell and George Coukos from University of Pennsylvania recorded adverse events in pilot studies using a CART modified to recognize the folate receptor, so it appears any adverse event reporting system is at the discretion of the primary investigator.

Other protocol considerations suggested by the symposium attendants included:

  • Plan for translational clinical lab for routine blood analysis
  • Subject screening for pulmonary and cardiac events
  • Determine possibility of insertional mutagenesis
  • Informed consent
  • Analysis of non T and T-cell subsets, e.g. natural killer cells and CD*8 cells

3. Consideration for Design of Trials and Mitigating Toxicities

  • Early Toxic effectsCytokine Release Syndrome– The effectiveness of CART therapy has been manifested by release of high levels of cytokines resulting in fever and inflammatory sequelae. One such cytokine, interleukin 6, has been attributed to this side effect and investigators have successfully used an IL6 receptor antagonist, tocilizumab (Acterma™), to alleviate symptoms of cytokine release syndrome (see review Adoptive T-cell therapy: adverse events and safety switches by Siok-Keen Tey).


Below is a video form Dr. Renier Brentjens, M.D., Ph.D. for Memorial Sloan Kettering concerning the finding he made that the adverse event from cytokine release syndrome may be a function of the tumor cell load, and if they treat the patient with CAR-T right after salvage chemotherapy the adverse events are alleviated..

Please see video below:

http link:

  • Early Toxic effects – Over-activation of CAR T-cells; mitigation by dose escalation strategy (as authors in reference [3] proposed). Most trials give billions of genetically modified cells to a patient.
  • Late Toxic Effectslong-term depletion of B-cells . For example CART directing against CD19 or CD20 on B cells may deplete the normal population of CD19 or CD20 B-cells over time; possibly managed by IgG supplementation

 Please look for a Followup Post concerning “Developing a Pharmacovigilence Framework for Engineered T-Cell Therapies”


  1. Ertl HC, Zaia J, Rosenberg SA, June CH, Dotti G, Kahn J, Cooper LJ, Corrigan-Curay J, Strome SE: Considerations for the clinical application of chimeric antigen receptor T cells: observations from a recombinant DNA Advisory Committee Symposium held June 15, 2010. Cancer research 2011, 71(9):3175-3181.
  2. Morgan RA, Yang JC, Kitano M, Dudley ME, Laurencot CM, Rosenberg SA: Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Molecular therapy : the journal of the American Society of Gene Therapy 2010, 18(4):843-851.
  3. Kandalaft LE, Powell DJ, Jr., Coukos G: A phase I clinical trial of adoptive transfer of folate receptor-alpha redirected autologous T cells for recurrent ovarian cancer. Journal of translational medicine 2012, 10:157.

Other posts on this site on Immunotherapy and Cancer include

Report on Cancer Immunotherapy Market & Clinical Pipeline Insight

New Immunotherapy Could Fight a Range of Cancers

Combined anti-CTLA4 and anti-PD1 immunotherapy shows promising results against advanced melanoma

Molecular Profiling in Cancer Immunotherapy: Debraj GuhaThakurta, PhD

Pancreatic Cancer: Genetics, Genomics and Immunotherapy

$20 million Novartis deal with ‘University of Pennsylvania’ to develop Ultra-Personalized Cancer Immunotherapy

Upcoming Meetings on Cancer Immunogenetics

Tang Prize for 2014: Immunity and Cancer

ipilimumab, a Drug that blocks CTLA-4 Freeing T cells to Attack Tumors @DM Anderson Cancer Center

Juno’s approach eradicated cancer cells in 10 of 12 leukemia patients, indicating potential to transform the standard of care in oncology

Read Full Post »

Ulcerative colitis

Ulcerative colitis (Photo credit: Wikipedia)

Tofacitinib, an Oral Janus Kinase Inhibitor, in Active Ulcerative Colitis

Reporter: Larry Bernstein, MD

This is an overview of a recently published article about a new treatment for ulcerative colitis. It also reviews the use of a class of drug in inflammatory conditions, and introduces the problem of sepsis.

Tofacitinib, an Oral Janus Kinase Inhibitor, in Active Ulcerative Colitis.
WJ Sandborn, S Ghosh, J Panes, I Vranic, C Su, for the Study A3921063 Investigators
N Engl J Med 2012; 367:616-624 August 16, 2012


Ulcerative colitis  is a chronic inflammatory disease of the colon that belongs to a group of diseases lumped together as Inflammatory Bowel Disease (IBD). There is a distinction to be made between Crohn’s disease, which may be limited to the small intestine (regional enteritis), the terminal ileum, or a portion of the transverse colon, and ulcerative colitis.

In ulcerative colitis the inflammation is limited to the mucosa and submucosa, but in Crohn’s disease there is a deep penetration of the intestinal wall (fistula) that may extend to the peritoneum causing abscess, scarring, peritonitis and possibly volvulus, obstruction and gangrenous bowel, which necessitate surgical resection. IBD tends to occur in children and young adults, repeats in families, and requires dietary management (fluid intake, Metamucil, restriction of fiber) . It is characterized by abdominal pain, diarrhea, bleeding, weight loss, and episodic fever, but also may be associated with joint pain.
Conservative medical treatment focuses on suppressing the immune response using 5-ASA, azathioprine, 6-mercaptopurine. If severe, biologic therapy is used to treat patients with severe Crohn’s disease that does not respond to any other types of medication, such as a TNF (tumor necrosis factor) inhibitor which can have secondary effects, and they are not universally effective. The importance of immunity can’t be understated, it involves a large portion of immune system and primitive Toll-like receptors (TLRs) that trigger signaling pathways. TLRs represent an important mechanism by which the host detects a variety of microorganisms that colonize in the gut. Endothelial and epithelial cells, and resident macrophages are potent producers of inflammatory cytokines, interleukins, IL-1, IL-6, and TNF-α, which are distinguished from another set that is treated in this study. In addition, there is a balance that has to be achieved between suppression and upregulation in treatment, which is referred to as immunomodulation.
The opposite of immunosuppression is upregulation It is cental to recent advances in chemotherapy of melanolma, small cell carcinoma and NSCCL of lung, and treatment resistant prostate cancer. An example is ipilimumab, whic upregulates cytotoxic T-cells to destroy cancer cells, but it has runaway destructive effects on the GI tract.

This study investigates the use of tofacitinib (CP-690,550), an oral inhibitor of Janus kinases 1, 2, and 3 with in vitro functional specificity for kinases 1 and 3 over kinase 2, which is expected to block signaling involving gamma chain–containing cytokines including interleukins 2, 4, 7, 9, 15, and 21. These cytokines are integral to lymphocyte activation, function, and proliferation.

The mechanism of drug action

Jak 1 and 3 inhibitor, which is targeted at blocking signaling involving gamma chain–containing cytokines including interleukins 2, 4, 7, 9, 15, and 21. The result would be to block signaling involving (gamma chains)–suppressing “lymphokines” 2, 4, 7, 9, 15, and 21. The lymphocyte pool is regional, being the antibody mediated immune system of the Bursa of Fabricius (B-lymphocytes, as opposed to the thymic derived T-cells) that form the largest immune organ extending the length of the intestines and the stomach.  The family transmission suggests an epigenetic event.

  • Gastrointestinal Tract
  • Oropharynx – Tonsils
  • Distal small intestine (ilieum) – Peyer’s Patches
  • Appendix, cecum

However, this classification of the lymphocytes has much greater complexity than I indicate.  The so called B-cells have receptors that recognize foreign antigen, but the T-cells have similar receptors and are tied to both the innate and the adaptive immune response.  Lymphocytes are the predominant cells of the immune system, but macrophages and plasma cells are present also.  Lymphocytes circulate, alternating between the circulatory blood stream and the lymphatic channels.  The end result of the immune reaction is the production of specific antibodies and antigen-reactive cells. These cells are called lymphocytes and are found in the blood and in the lymphoid system.

See Appendix

Trial features: double-blind, placebo-controlled, phase 2 trial; Patients were randomly assigned to receive tofacitinib at a dose of 0.5 mg, 3 mg, 10 mg, or 15 mg or placebo twice daily for 8 weeks.
Study goal: evaluated the efficacy of tofacitinib in 194 adults with moderately to severely active ulcerative colitis.

Primary outcome: a clinical response at 8 weeks, defined as an absolute decrease from baseline in the score on the Mayo scoring system for assessment of ulcerative colitis activity (possible score, 0 to 12, with higher scores indicating more severe disease) of 3 or more and a relative decrease from baseline of 30% or more with an accompanying decrease in the rectal bleeding subscore of 1 point or more or an absolute rectal bleeding subscore of 0 or 1.
Results and conclusion: The primary outcome, clinical response at 8 weeks, occurred in 32%, 48%, 61%, and 78% of patients receiving tofacitinib at a dose of 0.5 mg (P=0.39), 3 mg (P=0.55), 10 mg (P=0.10), and 15 mg (P<0.001), respectively, as compared with 42% of patients receiving placebo.
Clinical remission (defined as a Mayo score ≤2, with no subscore >1) at 8 weeks occurred in 13%, 33%, 48%, and 41% of patients receiving tofacitinib at a dose of 0.5 mg (P=0.76), 3 mg (P=0.01), 10 mg (P<0.001), and 15 mg (P<0.001), respectively, as compared with 10% of patients receiving placebo. Three patients treated with tofacitinib had an absolute neutrophil count of less than 1500.
Patients with moderately to severely active ulcerative colitis treated with tofacitinib were more likely to have clinical response and remission than those receiving placebo. (Funded by Pfizer; number, NCT00787202.)
Commentary: The study is only phase 2, and it is also limited to disease of the descending colon. The next phase will be necessary to determine the effect on a larger population at the selected dose, and will be necessary to determine both the size of the effect and identify unexpected adverse effects. We also have to keep in mind that the success of the study would limit the treatment to a subset of patients with IBD.

Efficacy of Proposed Treatment:

  • it is effective at about 40% remission for 8 weeks compared to 10% for placebo, or an adjusted actual 30% for 8 weeks.
  • A much larger study needs to be done to see how well the dose holds up, as well as the dosing interval. There are two factors that will affect the t1/2 of the drug so that 1/2 dose could be replaced at the end of t1/2.
  • The dose of 15 mg was no better for clinical response.
  • I would think that the next trial might give a loading dose of 15 mg, and then 7 mg (better that 3 mg) would be replaced every t1/2.  But this is more complicated than usual.

I identified two steps, not one direct effect.

  • The inhibitor has to balance the production rate versus the removal rate of the T-cell population. The drug itself is not measured, only the effect. I know that albumin, the liver produced protein, has a half-life of removal of 21 days. Platelets are short shelf-life as well as rapid turnaround in plasma.
  •  I don’t know what is the local production and removal rate of lymphocytes in the gut. That would be the key determinant for dosing.

The following may shed some light on what has been discussed:

Common characteristics of the lymphoid system.

  • The lymphoid system involves organs and tissues where lymphocytic cells originate as lymphocyte precursors that mature and differentiate, and either lodge in the lymphoid organs or move throughout the body.
  • Precursor cells originate in the yolk sac, liver, spleen, or bursa of Fabricius (or its mammalian equivalent, the bone marrow) in an embryo or fetus.
  • Stem cells from bone marrow or embryonic tissues are deposited and mature into lymphocytes in the central or primary lymphoid organs, which include the thymus and the bursa or bone marrow. Upon maturation, the lymphocytes undergo further maturation toward immunocompetence and production of immunoglobulins or sensitized lymphocytes.

Adaptive immunity has 2 main classes:

  • Antibody-mediated – B Lymphocyte
  • Cell-mediated – T Lymphocyte

Lymph follicles are our point of reference:

  • Organized concentrations of Lymphocytes
  • No capsule, covered by epithelia
  • Nodules are unit structure seen in a node
  • Oval concentrations in meshwork of reticular cells

If pathogens initially evade constitutive defenses, they may yet be attacked by more specific inducible defenses. The inducible defenses are so-called because they are induced upon primary exposure to a pathogen or one of its products. The inducible defenses must be triggered in a host, take time to develop, and are a function of the immune response. The type of resistance thus developed in the host is called acquired immunity.

Three important features of the immunological system relevant to host defense and/or “immunity are:

1. Specificity. An antibody or reactive T cell will react specifically with the antigen that induced its formation; it will not react with other antigens. Generally, this specificity is of the same order as that of enzyme-substrate specificity or receptor-ligand specificity.

  • The specificity of the immune response is explained on the basis of the clonal selection hypothesis: during the primary immune response, a specific antigen selects a pre-existing clone of specific lymphocytes and stimulates exclusively its activation, proliferation and differentiation.

2.  Memory. The immunological system has a “memory”.

  • Once the immunological response has reacted to produce a specific type of antibody or reactive T cell, it is capable of producing more of the antibody or activated T cell more rapidly and in larger amounts.

3. Tolerance. An animal generally does not undergo an immunological response to its own (potentially-antigenic) components.

  • The animal is said to be tolerant, or unable to react to its own potentially-antigenic components.

Gene expression – CD28 signal transduction , λδ T repertoire and antigen reactivity

Efficient lymphokine gene expression appears to require both T-cell antigen receptor (TCR) signal transduction and an uncharacterized second or costimulatory signal. CD28 is a T-cell differentiation antigen that can generate intracellular signals that synergize with those of the TCR to increase T-cell activation and interleukin-2 (IL-2) gene expression.

  • These investigators examined the effect of CD28 signal transduction on granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3), and gamma interferon (IFN-gamma) promoter activity.
  • Stimulation of CD28 in the presence of TCR-like signals increases the activity of the GM-CSF, IL-3, and IFN-gamma promoters by three- to sixfold.
  • As previously demonstrated for the IL-2 promoter, the IL-3 and GM-CSF promoters contain distinct elements of similar sequence which specifically bind a CD28-induced nuclear complex.
  • Mutation of the CD28 response elements in the IL-3 and GM-CSF promoters abrogates the CD28-induced activity without affecting phorbol ester- and calcium ionophore-induced activity.
  • These studies indicate that the TCR and CD28-regulated signal transduction pathways, coordinately regulate the transcription of several lymphokines, and the influence of CD28 signals on transcription is mediated by a common complex.

Fraser JD, Weiss A.  Regulation of T-cell lymphokine gene transcription by the accessory molecule CD28. Mol Cell Biol. 1992 Oct;12(10):4357-63.

These investigators looked at the relevance λδ T repertoire and the antigen reactivity of clones isolated from CSF in multiple sclerosis (MS).

  • they found an increased percentage of V delta 1+ cells as compared to peripheral blood of the same donors.
  • Phenotypic analysis of cells from MS CSF with V gamma- and V delta-specific monoclonal antibodies (mAb) showed that the V delta 1 chain is most frequently associated with gamma chains belonging to the V gamma 1 family.
  • Sequence analysis of TCR genes revealed heterogeneity of junctional regions in both delta and gamma genes indicating polyclonal expansion. gamma delta clones were established and some recognized glioblastoma, astrocytoma or monocytic cell lines.
  • Stimulation with these targets induced serine esterase release and lymphokine expression characteristic of the TH0-like phenotype.
  • Remarkably, these tumor-reactive gamma delta cells were not detected in the peripheral blood using PCR oligotyping, but were found in other CSF lines independently established from the same MS patient.
  • in the CSF there is a skewed TCR gamma delta repertoire and suggest that gamma delta cells reacting against brain-derived antigens might have been locally expanded.

Nick S, Pileri P, Tongiani S, Uematsu Y, Kappos L, De Libero G. T cell receptor gamma delta repertoire is skewed in cerebrospinal fluid of multiple sclerosis patients: molecular and functional analyses of antigen-reactive gamma delta clones. Eur J Immunol. 1995 Feb;25(2):355-63. PMID: 1328852 [PubMed – indexed for MEDLINE] PMCID: PMC360359 Free PMC Article

B Cells and T Cells:  Addendum…/B/B_and_Tcells.htmlShareAIDS; Building the T-cell Repertoire; Gamma/Delta T Cells … T cells specific for this structure (i.e., with complementary TCRs) bind the B cell and; secrete lymphokines that: … Each chain has a variable (V) region and a constant (C) region.

Although mature lymphocytes all look pretty much alike, they are extraordinarily diverse in their functions. The most abundant lymphocytes are:

  • B lymphocytes (often simply called B cells) and
  • T lymphocytes (likewise called T cells).
  • B cells are produced in the bone marrow.
  •  The precursors of T cells are also produced in the bone marrow but leave the bone marrow and mature in the thymus (which accounts for their designation).
  • Each B cell and T cell is specific for a particular antigen. What this means is that each is able to bind to a particular molecular structure.

The specificity of binding resides in a receptor for antigen:

  • the B cell receptor (BCR) for antigen and
  • the T cell receptor (TCR) respectively.

Both BCRs and TCRs share these properties:

  • They are integral membrane proteins.
  • They are present in thousands of identical copies exposed at the cell surface.
  • They are made before the cell ever encounters an antigen.
  • They are encoded by genes assembled by the recombination of segments of DNA.

How antigen receptor diversity is generated.

  • They have a unique binding site.
  • This site binds to a portion of the antigen called an antigenic determinant or epitope.
    The binding, like that between an enzyme and its substrate depends on complementarity of the surface of the receptor and the surface of the epitope.
  • The binding occurs by non-covalent forces (again, like an enzyme binding to its substrate).

Successful binding of the antigen receptor to the epitope, if accompanied by additional signals, results in:

  • stimulation of the cell to leave G0 and enter the cell cycle.
  • Repeated mitosis leads to the development of a clone of cells bearing the same antigen receptor; that is, a clone of cells of the identical specificity.

BCRs and TCRs differ in:

  • their structure;
  • the genes that encode them;
  • the type of epitope to which they bind.

heavy (H) plus kappa (κ) or lambda (λ) chains for BCRs;

alpha (α) and beta (β) or gamma (γ) and delta (δ) chains for TCRs)

……is encoded by several different gene segments.

The genome contains a pool of gene segments for each type of chain. Random assortment of these segments makes the largest contribution to receptor diversity.

There are two types of T cells that differ in their TCR:

alpha/beta (αβ) T cells. Their TCR is a heterodimer of an alpha chain with a beta chain. Each chain has a variable (V) region and a constant (C) region. The V regions each contain 3 hypervariable regions that make up the antigen-binding site. [Link]

gamma/delta (γδ) T cells. Their TCR is also a heterodimer of a gamma chain paired with a delta chain.

The discussion that follows now concerns alpha/beta T cells. Gamma/delta T cells, which are less well understood, are discussed at the end [Link].

The TCR (of alpha/beta T cells) binds a bimolecular complex displayed at the surface of some other cell called an antigen-presenting cell (APC).

Most of the T cells in the body belong to one of two subsets. These are distinguished by the presence on their surface of one or the other of two glycoproteins designated:

  • CD8+ T cells bind epitopes that are part of class I histocompatibility molecules. Almost all the cells of the body express class I molecules.
  • CD4+ T cells bind epitopes that are part of class II histocompatibility molecules. Only specialized antigen-presenting cells express class II molecules.

These include:

  • dendritic cells
  • phagocytic cells like macrophages and
  • B cells!

Building the T-cell Repertoire

T cells have receptors (TCRs) that bind to antigen fragments nestled in MHC molecules. But,

  • all cells express class I MHC molecules containing fragments derived from self proteins;
  • many cells express class II MHC molecules that also contain self peptides.

This presents a risk of the T cells recognizing these self-peptide/self-MHC complexes and mounting an autoimmune attack against them. Fortunately, this is usually avoided by a process of selection that goes on in the thymus (where all T cells develop).


FDA approves Abbott Humira as Ulcerative Colitis therapy
PBR Staff Writer Published 01 October 2012
The USFDA has approved Abbott’s Humira (adalimumab) for the treatment of adult patients with moderate to severe Ulcerative Colitis (UC) when certain other medicines have not worked well enough.
Humira, which works by inhibiting tumour necrosis factor-alpha (TNF-alpha), was previously approved for the treatment of moderate to severe Crohn’s disease.

Abbott Global Pharmaceutical Research and Development senior vice president John Leonard said, “Since the first FDA approval of HUMIRA in late 2002, Abbott has continued to investigate the medication in multiple conditions with the goal of bringing this treatment option to more patients who may benefit from it.”

The approval was based on the data from two phase 3 studies, ULTRA 1 and ULTRA 2, both of which enrolled adult patients who had moderately to severely active UC despite concurrent or prior treatment with immunosuppressants.  This should have special significance in view of the past history, which may be explainable, but also keep in mind the serious risks of complications.

It is worthy of comment that anti-TNF treatment was previously rejected in trials for use in sepsis leading to Multiple Organ Dysfunction Syndrome and cardiovascular collapse (shock).  More recently an anti-Factor Xa drug, Xygris,  to prevent hypercoagulability only in severe sepsis was withdrawn.

Anti TNF for sepsis

1.   In a group of patients with elevated interleukin-6 levels, the mortality rate was 243 of 510 (47.6%) in the placebo group and 213 of 488 (43.6%) in the afelimomab group. Using a logistic regression analysis, treatment with afelimomab was associated with an adjusted reduction in the risk of death of 5.8% (p = .041) and a corresponding reduction of relative risk of death of 11.9%. Mortality rates for the placebo and afelimomab groups in the interleukin-6 test negative population were 234 of 819 (28.6%) and 208 of 817 (25.5%), respectively. In the overall population of interleukin-6 test positive and negative patients, the placebo and afelimomab mortality rates were 477 of 1,329 (35.9%)and 421 of 1,305 (32.2%), respectively.

Panacek EAMarshall JCAlbertson TEJohnson DH, at al.  Efficacy and safety of the monoclonal anti-tumor necrosis factor antibody F(ab’)2 fragment afelimomab in patients with severe sepsis and elevated interleukin-6 levelsCrit Care Med. 2004 Nov;32(11):2173-82.

2. No survival benefit was found for the total study population, but patients with increased circulating TNF concentrations at study entry appeared to benefit by the high dose anti-TNF antibody treatment. Increased interleukin (IL)-6 levels predicted a fatal outcome (p =.003), but TNF levels were not found to be a prognostic indicator. TNFlevels were higher (206.7 +/- 60.7 vs. 85.9 +/- 26.1 pg/mL; p <.001) and outcome was poor (41% vs. 71% survival; p =.007) in patients who were in shock at study entry when compared with septic patients not in shock.

Fisher CJ JrOpal SMDhainaut JFStephens S, et al. Influence of an anti-tumor necrosis factor monoclonal antibody on cytokine levels in patients with sepsis. The CB0006 Sepsis Syndrome Study Group.  Critical Care Medicine [1993, 21(3):318-327] (PMID:8440099)

3.  Large clinical trials involving anti-TNF-alpha MAb have proven to be less conclusive and less successful than clinicians had hoped. The International Sepsis Trial (INTERSEPT), reported by Cohen and Carlet,[14] was designed to assess the safety and efficacy of Bay x 1351, a murine MAb to recombinant human TNF-alpha in patients with sepsis. The INTERSEPT trial was an international, multicenter trial involving 564 patients, 420 of whom were in septic shock. The main study end point — 28-day survival — showed no significant benefit for the treatment group vs controls. Prospectively, the researchers identified 2 secondary variables: shock reversal and frequency of organ failure. Post-28-day survival, treatment groups showed a more rapid reversal of shock compared with placebo, as well as a significant delay in time to first organ failure. The researchers concluded that the anti-TNF-alpha antibody may have a role as adjunctive therapy, but that such a putative role requires more in the way of clinical trial confirmation.

In the TNF-alpha MAb Sepsis Study Group trial, also called the North American Sepsis Trial I (NORASEPT I), Abraham and associates[15] evaluated the efficacy and safety of an anti-TNF-alpha MAb in the treatment of patients with sepsis syndrome. A total of 994 patients in 31 hospitals were enrolled in a randomized, prospective, multicenter, double-blind, placebo-controlled clinical trial. Patients were stratified into shock/nonshock subgroups, then randomized to receive a single infusion of 15 mg/kg of anti-TNF-alpha MAb, 7.5 mg/kg of anti-TNF-alpha MAb, or placebo. The researchers found that among all infused patients, there was no difference in mortality among those receiving therapy and those on placebo. In septic shock patients (n = 478), however, there was a trend toward a reduction in all-cause mortality, which was most evident 3 days after infusion. At day 3, 25 of 162 patients treated with the 15 mg/kg dose died; 22 of 156 treated with 7.5 mg/kg died, but 44 of 160 placebo-treated patients died (15 mg/kg: 44% mortality reduction vs placebo, P = .01; 7.5 mg/kg: 48% reduction vs placebo, P = .004). However, at day 28, the reduction in mortality of shock patients was not significant for either dose of the anti-TNF-alpha MAb relative to placebo.

All studies of MAb against TNF in septic patients and found an absolute risk reduction of 3.5%. The most recently published clinical trial found an absolute reduction in mortality of 3.7%.

Of note, therapy with MAb against TNF has been proven efficacious for treatment of rheumatoid arthritis and is approved by the US Food and Drug Administration for this purpose.

New directions in research on severe sepsis. Human trials with TNF alpha.  Medscape.

4. Why the poor results with sepsis?

This would be sufficient for another discussion.  That can be left for another day.


Sepsis syndrome, or sepsis, is an adverse systemic response to infection that includes fever, rapid heartbeat and respiration, low blood pressure and organ dysfunction associated with compromised circulation.

LPS is a major constituent of Gram-negative bacterial cell walls (see section 3-0) and is essential for membrane integrity. The portion of LPS that causes shock is the innermost and most highly conserved phosphoglycolipid, lipid A. Lipid A is a phosphoglycolipid consisting of a core hexosamine disaccharide with ester- and amide-linked acylated fatty acid tails arranged in either asymmetric or symmetric arrays that anchor the structure in the membrane. It acts by potently inducing inflammatory responses that are life-threatening when systemic, and is known as bacterial endotoxin.  Mice deficient in any of the LPS receptor components are more
susceptible to Gram-negative bacterial infection but, at the same time, are less susceptible to the sepsis syndrome.

TLRs have a lethal function in the septic shock syndrome. The physiological function of signaling through phagocyte TLRs is to induce the release of the cytokines TNF, IL-1, IL-6, IL-8 and IL-12 and trigger the inflammatory response, which is critical to containing bacterial infection in the tissues. However, if infection disseminates in the blood, the widespread activation of phagocytes in the bloodstream is catastrophic. Increase in the numbers of circulating neutrophils, or neutrophilia, is driven by effects of colony stimulating factors, such as G-CSF.

Time course of sepsis. The clinical manifestations of sepsis are manifested by successive waves of the serum cytokine cascade. In humans injected with purified LPS, TNF rises almost immediately and peaks at 1.5 h; the sharp decline of TNF may be due to modulation by its soluble receptor sTNFR. A second wave of cytokines that peaks at 3 h activates the acute-phase response
in the liver, the systemic pituitary response (via IL-6 and IL-1), and the activation and chemotaxis of neutrophils (via IL-6, IL-8 and  G-CSF). Neutrophil activation results in the release of lactoferrin from neutrophil secondary granules; the activation of endothelial procoagulants with the rise of tissue plasminogen activator (t-PA). Pituitary-derived adrenocorticotropic hormone (ACTH)  and migration inhibition factor (MIF) peak at 5 h and coincide with peak levels of the regulatory cytokines IL-Ra and IL-10 that counteract the release or activity of inflammatory cytokines. Diffuse endothelial activation is shown by the appearance of soluble E-selectin that peaks at about 8 h and remains elevated for several days.

Susceptibility to LPS Toxicity in Gene Knockout Mice

High LPS; Low LPS/D-Gal



LPS recognition

phagocyte function

caspase 1
The proteins encoded by the deleted genes are listed. SR-A is scavenger receptor A; Hck and Fgr are Src-family kinases with an essential role in integrin-mediated migration of neutrophils out of the bloodstream.

The Immune Response to Bacterial InfectionSepsis Syndrome: Bacterial Endotoxin
Chapter 9-3.  2007. p 232-233. New Science Press Ltd

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