Archive for the ‘Bone marrow derived cells’ Category

Medical Informatics View

Chapter 1 Statement of Inferential    Second Opinion

Realtime Clinical Expert Support

Gil David and Larry Bernstein have developed, in consultation with Prof. Ronald Coifman, in the Yale University Applied Mathematics Program, a software system that is the equivalent of an intelligent Electronic Health Records Dashboard that provides empirical medical reference and suggests quantitative diagnostics options.


Keywords: Entropy, Maximum Likelihood Function, separatory clustering, peripheral smear, automated hemogram, Anomaly, classification by anomaly, multivariable and multisyndromic, automated second opinion

Abbreviations: Akaike Information Criterion, AIC;  Bayes Information Criterion, BIC, Systemic Inflammatory Response Syndrome, SIRS.


Background: The current design of the Electronic Medical Record (EMR) is a linear presentation of portions of the record by services, by diagnostic method, and by date, to cite examples.  This allows perusal through a graphical user interface (GUI) that partitions the information or necessary reports in a workstation entered by keying to icons.  This requires that the medical practitioner finds the history, medications, laboratory reports, cardiac imaging and EKGs, and radiology in different workspaces.  The introduction of a DASHBOARD has allowed a presentation of drug reactions, allergies, primary and secondary diagnoses, and critical information about any patient the care giver needing access to the record.  The advantage of this innovation is obvious.  The startup problem is what information is presented and how it is displayed, which is a source of variability and a key to its success.

Intent: We are proposing an innovation that supercedes the main design elements of a DASHBOARD and utilizes the conjoined syndromic features of the disparate data elements.  So the important determinant of the success of this endeavor is that it facilitates both the workflow and the decision-making process with a reduction of medical error. Continuing work is in progress in extending the capabilities with model datasets, and sufficient data because the extraction of data from disparate sources will, in the long run, further improve this process.  For instance, the finding of  both ST depression on EKG coincident with an elevated cardiac biomarker (troponin), particularly in the absence of substantially reduced renal function. The conversion of hematology based data into useful clinical information requires the establishment of problem-solving constructs based on the measured data.

The most commonly ordered test used for managing patients worldwide is the hemogram that often incorporates the review of a peripheral smear.  While the hemogram has undergone progressive modification of the measured features over time the subsequent expansion of the panel of tests has provided a window into the cellular changes in the production, release or suppression of the formed elements from the blood-forming organ to the circulation.  In the hemogram one can view data reflecting the characteristics of a broad spectrum of medical conditions.

Progressive modification of the measured features of the hemogram has delineated characteristics expressed as measurements of size, density, and concentration, resulting in many characteristic features of classification. In the diagnosis of hematological disorders proliferation of marrow precursors, the domination of a cell line, and features of suppression of hematopoiesis provide a two dimensional model.  Other dimensions are created by considering the maturity of the circulating cells.  The application of rules-based, automated problem solving should provide a valid approach to the classification and interpretation of the data used to determine a knowledge-based clinical opinion. The exponential growth of knowledge since the mapping of the human genome enabled by parallel advances in applied mathematics that have not been a part of traditional clinical problem solving.  As the complexity of statistical models has increased the dependencies have become less clear to the individual.  Contemporary statistical modeling has a primary goal of finding an underlying structure in studied data sets.  The development of an evidence-based inference engine that can substantially interpret the data at hand and convert it in real time to a “knowledge-based opinion” could improve clinical decision-making by incorporating multiple complex clinical features as well as duration of onset into the model.

An example of a difficult area for clinical problem solving is found in the diagnosis of SIRS and associated sepsis.  SIRS (and associated sepsis) is a costly diagnosis in hospitalized patients.   Failure to diagnose sepsis in a timely manner creates a potential financial and safety hazard.  The early diagnosis of SIRS/sepsis is made by the application of defined criteria (temperature, heart rate, respiratory rate and WBC count) by the clinician.   The application of those clinical criteria, however, defines the condition after it has developed and has not provided a reliable method for the early diagnosis of SIRS.  The early diagnosis of SIRS may possibly be enhanced by the measurement of proteomic biomarkers, including transthyretin, C-reactive protein and procalcitonin.  Immature granulocyte (IG) measurement has been proposed as a more readily available indicator of the presence of granulocyte precursors (left shift).  The use of such markers, obtained by automated systems in conjunction with innovative statistical modeling, provides a promising approach to enhance workflow and decision making.   Such a system utilizes the conjoined syndromic features of disparate data elements with an anticipated reduction of medical error.  This study is only an extension of our approach to repairing a longstanding problem in the construction of the many-sided electronic medical record (EMR).  In a classic study carried out at Bell Laboratories, Didner found that information technologies reflect the view of the creators, not the users, and Front-to-Back Design (R Didner) is needed.

Costs would be reduced, and accuracy improved, if the clinical data could be captured directly at the point it is generated, in a form suitable for transmission to insurers, or machine transformable into other formats.  Such data capture, could also be used to improve the form and structure of how this information is viewed by physicians, and form a basis of a more comprehensive database linking clinical protocols to outcomes, that could improve the knowledge of this relationship, hence clinical outcomes.



How we frame our expectations is so important that it determines the data we collect to examine the process.   In the absence of data to support an assumed benefit, there is no proof of validity at whatever cost.   This has meaning for hospital operations, for nonhospital laboratory operations, for companies in the diagnostic business, and for planning of health systems.

In 1983, a vision for creating the EMR was introduced by Lawrence Weed,  expressed by McGowan and Winstead-Fry (J J McGowan and P Winstead-Fry. Problem Knowledge Couplers: reengineering evidence-based medicine through interdisciplinary development, decision support, and research. Bull Med Libr Assoc. 1999 October; 87(4): 462–470.)   PMCID: PMC226622    Copyright notice





They introduce Problem Knowledge Couplers as a clinical decision support software tool that  recognizes that functionality must be predicated upon combining unique patient information, but obtained through relevant structured question sets, with the appropriate knowledge found in the world’s peer-reviewed medical literature.  The premise of this is stated by LL WEED in “Idols of the Mind” (Dec 13, 2006): “ a root cause of a major defect in the health care system is that, while we falsely admire and extol the intellectual powers of highly educated physicians, we do not search for the external aids their minds require”.  HIT use has been focused on information retrieval, leaving the unaided mind burdened with information processing.



The data presented has to be comprehended in context with vital signs, key symptoms, and an accurate medical history.  Consequently, the limits of memory and cognition are tested in medical practice on a daily basis.  We deal with problems in the interpretation of data presented to the physician, and how through better design of the software that presents this data the situation could be improved.  The computer architecture that the physician uses to view the results is more often than not presented as the designer would prefer, and not as the end-user would like.  In order to optimize the interface for physician, the system would have a “front-to-back” design, with the call up for any patient ideally consisting of a dashboard design that presents the crucial information that the physician would likely act on in an easily accessible manner.  The key point is that each item used has to be closely related to a corresponding criterion needed for a decision.  Currently, improved design is heading in that direction.  In removing this limitation the output requirements have to be defined before the database is designed to produce the required output.  The ability to see any other information, or to see a sequential visualization of the patient’s course would be steps to home in on other views.  In addition, the amount of relevant information, even when presented well, is a cognitive challenge unless it is presented in a disease- or organ-system structure.  So the interaction between the user and the electronic medical record has a significant effect on practitioner time, ability to minimize errors of interpretation, facilitate treatment, and manage costs.  The reality is that clinicians are challenged by the need to view a large amount of data, with only a few resources available to know which of these values are relevant, or the need for action on a result, or its urgency. The challenge then becomes how fundamental measurement theory can lead to the creation at the point of care of more meaningful actionable presentations of results.  WP Fisher refers to the creation of a context in which computational resources for meeting the challenges will be incorporated into the electronic medical record.  The one which he chooses is a probabilistic conjoint (Rasch) measurement model, which uses scale-free standard measures and meets data quality standards. He illustrates this by fitting a set of data provided by Bernstein (19)(27 items for the diagnosis of acute myocardial infarction (AMI) to a Rasch multiple rating scale model testing the hypothesis that items work together to delineate a unidimensional measurement continuum. The results indicated that highly improbable observations could be discarded, data volume could be reduced based on internal, and increased ability of the care provider to interpret the data.


Classified data a separate issue from automation

 Feature Extraction. This further breakdown in the modern era is determined by genetically characteristic gene sequences that are transcribed into what we measure.  Eugene Rypka contributed greatly to clarifying the extraction of features in a series of articles, which set the groundwork for the methods used today in clinical microbiology.  The method he describes is termed S-clustering, and will have a significant bearing on how we can view hematology data.  He describes S-clustering as extracting features from endogenous data that amplify or maximize structural information to create distinctive classes.  The method classifies by taking the number of features with sufficient variety to map into a theoretic standard. The mapping is done by a truth table, and each variable is scaled to assign values for each: message choice.  The number of messages and the number of choices forms an N-by N table.  He points out that the message choice in an antibody titer would be converted from 0 + ++ +++ to 0 1 2 3.

Even though there may be a large number of measured values, the variety is reduced by this compression, even though there is risk of loss of information.  Yet the real issue is how a combination of variables falls into a table with meaningful information.  We are concerned with accurate assignment into uniquely variable groups by information in test relationships. One determines the effectiveness of each variable by its contribution to information gain in the system.  The reference or null set is the class having no information.  Uncertainty in assigning to a classification is only relieved by providing sufficient information.  One determines the effectiveness of each variable by its contribution to information gain in the system.  The possibility for realizing a good model for approximating the effects of factors supported by data used for inference owes much to the discovery of Kullback-Liebler distance or “information”, and Akaike found a simple relationship between K-L information and Fisher’s maximized log-likelihood function. A solid foundation in this work was elaborated by Eugene Rypka.  Of course, this was made far less complicated by the genetic complement that defines its function, which made  more accessible the study of biochemical pathways.  In addition, the genetic relationships in plant genetics were accessible to Ronald Fisher for the application of the linear discriminant function.    In the last 60 years the application of entropy comparable to the entropy of physics, information, noise, and signal processing, has been fully developed by Shannon, Kullback, and others,  and has been integrated with modern statistics, as a result of the seminal work of Akaike, Leo Goodman, Magidson and Vermunt, and unrelated work by Coifman. Dr. Magidson writes about Latent Class Model evolution:


The recent increase in interest in latent class models is due to the development of extended algorithms which allow today’s computers to perform LC analyses on data containing more than just a few variables, and the recent realization that the use of such models can yield powerful improvements over traditional approaches to segmentation, as well as to cluster, factor, regression and other kinds of analysis.

Perhaps the application to medical diagnostics had been slowed by limitations of data capture and computer architecture as well as lack of clarity in definition of what are the most distinguishing features needed for diagnostic clarification.  Bernstein and colleagues had a series of studies using Kullback-Liebler Distance  (effective information) for clustering to examine the latent structure of the elements commonly used for diagnosis of myocardial infarction (CK-MB, LD and the isoenzyme-1 of LD),  protein-energy malnutrition (serum albumin, serum transthyretin, condition associated with protein malnutrition (see Jeejeebhoy and subjective global assessment), prolonged period with no oral intake), prediction of respiratory distress syndrome of the newborn (RDS), and prediction of lymph nodal involvement of prostate cancer, among other studies.   The exploration of syndromic classification has made a substantial contribution to the diagnostic literature, but has only been made useful through publication on the web of calculators and nomograms (such as Epocrates and Medcalc) accessible to physicians through an iPhone.  These are not an integral part of the EMR, and the applications require an anticipation of the need for such processing.

Gil David et al. introduced an AUTOMATED processing of the data available to the ordering physician and can anticipate an enormous impact in diagnosis and treatment of perhaps half of the top 20 most common causes of hospital admission that carry a high cost and morbidity.  For example: anemias (iron deficiency, vitamin B12 and folate deficiency, and hemolytic anemia or myelodysplastic syndrome); pneumonia; systemic inflammatory response syndrome (SIRS) with or without bacteremia; multiple organ failure and hemodynamic shock; electrolyte/acid base balance disorders; acute and chronic liver disease; acute and chronic renal disease; diabetes mellitus; protein-energy malnutrition; acute respiratory distress of the newborn; acute coronary syndrome; congestive heart failure; disordered bone mineral metabolism; hemostatic disorders; leukemia and lymphoma; malabsorption syndromes; and cancer(s)[breast, prostate, colorectal, pancreas, stomach, liver, esophagus, thyroid, and parathyroid].

Extension of conditions and presentation to the electronic medical record (EMR)

We have published on the application of an automated inference engine to the Systemic Inflammatory Response (SIRS), a serious infection, or emerging sepsis.  We can report on this without going over previous ground.  Of considerable interest is the morbidity and mortality of sepsis, and the hospital costs from a late diagnosis.  If missed early, it could be problematic, and it could be seen as a hospital complication when it is not. Improving on previous work, we have the opportunity to look at the contribution of a fluorescence labeled flow cytometric measurement of the immature granulocytes (IG), which is now widely used, but has not been adequately evaluated from the perspective of diagnostic usage.  We have done considerable work on protein-energy malnutrition (PEM), to which the automated interpretation is currently in review.  Of course, the

cholesterol, lymphocyte count, serum albumin provide the weight of evidence with the primary diagnosis (emphysema, chronic renal disease, eating disorder), and serum transthyretin would be low and remain low for a week in critical care.  This could be a modifier with age in providing discriminatory power.


Chapter  3           References


The Cost Burden of Disease: U.S. and Michigan. CHRT Brief. January 2010. @www.chrt.org

The National Hospital Bill: The Most Expensive Conditions by Payer, 2006. HCUP Brief #59.


Rudolph RA, Bernstein LH, Babb J: Information-Induction for the diagnosis of

myocardial infarction. Clin Chem 1988;34:2031-2038.

Bernstein LH (Chairman). Prealbumin in Nutritional Care Consensus Group.

Measurement of visceral protein status in assessing protein and energy malnutrition: standard of care. Nutrition 1995; 11:169-171.

Bernstein LH, Qamar A, McPherson C, Zarich S, Rudolph R. Diagnosis of myocardial infarction: integration of serum markers and clinical descriptors using information theory. Yale J Biol Med 1999; 72: 5-13.


Kaplan L.A.; Chapman J.F.; Bock J.L.; Santa Maria E.; Clejan S.; Huddleston D.J.; Reed R.G.; Bernstein L.H.; Gillen-Goldstein J. Prediction of Respiratory Distress Syndrome using the Abbott FLM-II amniotic fluid assay. The National Academy of Clinical Biochemistry (NACB) Fetal Lung Maturity Assessment Project.  Clin Chim Acta 2002; 326(8): 61-68.


Bernstein LH, Qamar A, McPherson C, Zarich S. Evaluating a new graphical ordinal logit method (GOLDminer) in the diagnosis of myocardial infarction utilizing clinical features and laboratory data. Yale J Biol Med 1999; 72:259-268.


Bernstein L, Bradley K, Zarich SA. GOLDmineR: Improving models for classifying patients with chest pain. Yale J Biol Med 2002; 75, pp. 183-198.

Ronald Raphael Coifman and Mladen Victor Wickerhauser. Adapted Waveform Analysis as a Tool for Modeling, Feature Extraction, and Denoising. Optical Engineering, 33(7):2170–2174, July 1994.


R. Coifman and N. Saito. Constructions of local orthonormal bases for classification and regression. C. R. Acad. Sci. Paris, 319 Série I:191-196, 1994.


Chapter 4           Clinical Expert System

Realtime Clinical Expert Support and validation System

We have developed a software system that is the equivalent of an intelligent Electronic Health Records Dashboard that provides empirical medical reference and suggests quantitative diagnostics options. The primary purpose is to gather medical information, generate metrics, analyze them in realtime and provide a differential diagnosis, meeting the highest standard of accuracy. The system builds its unique characterization and provides a list of other patients that share this unique profile, therefore utilizing the vast aggregated knowledge (diagnosis, analysis, treatment, etc.) of the medical community. The main mathematical breakthroughs are provided by accurate patient profiling and inference methodologies in which anomalous subprofiles are extracted and compared to potentially relevant cases. As the model grows and its knowledge database is extended, the diagnostic and the prognostic become more accurate and precise. We anticipate that the effect of implementing this diagnostic amplifier would result in higher physician productivity at a time of great human resource limitations, safer prescribing practices, rapid identification of unusual patients, better assignment of patients to observation, inpatient beds, intensive care, or referral to clinic, shortened length of patients ICU and bed days.

The main benefit is a real time assessment as well as diagnostic options based on comparable cases, flags for risk and potential problems as illustrated in the following case acquired on 04/21/10. The patient was diagnosed by our system with severe SIRS at a grade of 0.61 .


The patient was treated for SIRS and the blood tests were repeated during the following week. The full combined record of our system’s assessment of the patient, as derived from the further Hematology tests, is illustrated below. The yellow line shows the diagnosis that corresponds to the first blood test (as also shown in the image above). The red line shows the next diagnosis that was performed a week later.









As we can see the following treatment, the SIRS risk as a major concern was eliminated and the system provides a positive feedback for the treatment of the physician.


Method for data organization and classification via characterization metrics.

Our database organized to enable linking a given profile to known profiles. This is achieved by associating a patient to a peer group of patients having an overall similar profile, where the similar profile is obtained through a randomized search for an appropriate weighting of variables. Given the selection of a patients’ peer group, we build a metric that measures the dissimilarity of the patient from its group. This is achieved through a local iterated statistical analysis in the peer group.

We then use this characteristic metric to locate other patients with similar unique profiles, for each of whom we repeat the procedure described above. This leads to a network of patients with similar risk condition. Then, the classification of the patient is inferred from the medical known condition of some of the patients in the linked network. Given a set of points (the database) and a newly arrived sample (point), we characterize the behavior of the newly arrived sample, according to the database. Then, we detect other points in the database that match this unique characterization. This collection of detected points defines the characteristic neighborhood of the newly arrived sample. We use the characteristic neighbor hood in order to classify the newly arrived sample. This process of differential diagnosis is repeated for every newly arrived point.   The medical colossus we have today has become a system out of control and beset by the elephant in the room – an uncharted complexity. We offer a method that addresses the complexity and enables rather than disables the practitioner.  The method identifies outliers and combines data according to commonality of features.



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Graft-versus-Host Disease

Writer and Curator: Larry H. Bernstein, MD, FCAP 



This piece is a follow up to the article on allogeneic transfusion reactions, which extends into transplantation and transplantation outcomes for hematological diseases, both malignant and nonmalignant. The safety of transfusions in Western countries has improved substantially, and the causes for transfusion mishaps has been reduced to unexpected infectious sources, uncommon immune incompatibilities, and errors in processing the blood products.  The greatest risk is incurred in platelet transfusions because of the short shelf-life of the product, and the time needed for testing prior to release.  This portion of the review is concerned with Graft-versus-Host Disease, which is unique to transfusion and transplanting of blood. In other transplantation, there is graft failure because of host versus graft incompatibility or complications.  The reverse order applies to blood.  In this case, on the contrary, the transfused or grafted donor tissue becomes a pursuer after the recipients hematopoietic cells.

Peter Brian Medawar: Father of Transplantation

Thomas E. Starzl, M.D., PH.D., F.A.C.S.
J Am Coll Surg. 1995 Mar; 180(3): 332–336

Most of the surgical specialities can be tracked to the creative vision of a surgeon. Transplantation is an exception. Here, the father of the field is succinctly defined in the dictionary as: “Peter Brian Medawar: a Brazilian born British Zoologist who at the age of 45 shared a 1960 Nobel Prize for his work on acquired immunologic tolerance”. Medawar was mysteriously overwhelming to many colleagues and observers, even when he was young. He was the son of a Lebanese father and an English mother—tall, athletic, abnormally handsome, hypnotically articulate in public, and politely cordial in his personal relations. In September 1969, at the age of 54, he had the first of a series of strokes. These crippled him physically but not in spirit. Although I saw Medawar often professionally and privately over a 22 year period, before and after he was disabled, this sporadic exposure was not enough to understand him. My sense is that no one did, except perhaps Jean, his wife for nearly 50 years.

Medawar’s dazzling personality before and great courage after his strokes was inspirational, but his fame was based on the unique achievement in 1953 captured by the terse dictionary mention of “acquired immunologic tolerance.” The roots leading to this accomplishment had fed on the blood of war. More than 12 years earlier, the recently wed zoologist Medawar—24 years of age and fresh from graduate studies at Oxford University—was assigned to
the service of the British surgeon, Dr. Thomas Gibson, to determine if skin allografts could be used to treat casualties from the Battle of Britain. First,
in human studies with Gibson, and then with simple and logical rabbit experiments, Medawar showed that rejection of the skin was an immunologic phenomenon. This later was shown  to be analogous to the cell-mediated delayed hypersensitivity that confers immunity to diseases such as tuberculosis. The principal evidence in the early studies was that repetitive grafts from the same donor were rejected more rapidly with each successive attempt —the sensitization and donor specificity confirming an earlier clinical observations by Emil Holman of Stanford in skin-grafted burn victims. Once it was established that rejection was an immune reaction, strategies began to evolve to weaken the recipient immune system. By 1953, total body irradiation and adrenal cortical steroids had been shown to delay skin rejection. However, this immunosuppressive effect was either minor if the animals survived, or lethal to the recipient if the grafts were spared.


In the resulting atmosphere of nihilism about clinical applications, a three and one-half page article by Billingham, Brent, and Medawar in the October 3, 1953 issue of Nature describing acquired tolerance, came as a blinding beacon of hope. The three men had learned that donor splenocytes could be engrafted by their intravenous infusion into immunologically immature mice in utero or perinatally. When these inoculated recipients matured, they could accept skin and other tissues from the donor (but from no other) mouse strain. The immune system of the recipients had been populated by the immunocytes of the donor, meaning that they were now chimeras. The race was on to convert this principle to humans. However, the dark side of their accomplishment soon was revealed by the two younger members of Medawar’s team, Billingham and Brent and by the Dane, Simonsen. The engrafted donor cells could turn the tables and reject the defenseless recipient unless the tissue match was a good one. This was the dreaded graft versus host disease (GVHD) in which transplanted donor cells attacked the recipient skin, gastrointestinal tract, lungs, liver, and the bone marrow itself. Medawar’s dream of 1953 was suddenly a nightmare. Or was it?

On the contrary, the work took a straight line to clinical application, after the demonstration by Prehn and Main that similar tolerance could be induced in adult mice rendered immunologically defenseless by total body irradiation before splenocyte (or later bone marrow) infusion. The recipient conditioning, known as cytoablation, also could be accomplished with myelotoxic drugs. However, as Billingham, Brent, and Medawar had predicted, donor specific tolerance could be induced in humans without GVHD only if there was a good tissue (HLA) match. In 1968, 15 years after the epic Billingham, Brent and Medawar publication, Robert Good and Fritz Bach reported the first two successful human bone marrow transplants. Both recipients of well matched bone marrow from blood relatives are still alive. This was a triumph in which the principal clinicians were internists, as summarized 25 years later in the acceptance speech by the 1990 Nobel Laureate Donnall Thomas.

The growth of bone marrow and whole organ transplantation

The growth of bone marrow and whole organ transplantation


The growth of bone marrow (right) and whole organ transplantation (left) from the seed planted by Peter Medawar during World War II. GVHD, Graft versus host disease.

Immunological Tolerance: Medawar Nobel Acceptance Lecture

“Immunological tolerance” may be described as a state of indifference or non-reactivity towards a substance that would normally be expected to excite an immunological response. The term first came to be used in the context of tissue transplantation immunity, i.e. of the form of immunity that usually prohibits the grafting of tissues between individuals of different genetic make-up; and it was used to refer only to a non-reactivity caused by exposing animals to antigenic stimuli before they were old enough to undertake an immunological response. For example, if living cells from a mouse of strain CBA are injected into an adult mouse of strain A, the CBA cells will be destroyed by an immunological process, and the A-line mouse that received them will destroy any later graft of the same origin with the speed to be expected of an animal immunologically forearmed. But if the CBA cells are injected into a foetal or newborn A-line mouse, they are accepted; more than that, the A-line mouse, when it grows up, will accept any later graft from a CBA donor as if it were its own. I shall begin by using the term “immunological tolerance” in the rather restricted sense that is illustrated by this experiment, and shall discuss its more general usage later on.

The experiment I have just described can be thought of as an artificial reproduction of an astonishing natural curiosity, the phenomenon of red-cell chimerism in certain dizygotic twins. The blood systems of twin cattle before birth are not sharply distinct from each other, as they are in most other twins; instead, the blood systems make anastomoses with each other, with the effect that the twins can indulge in a prolonged exchange of blood before birth. In 1945, R.D. Owen2 made the remarkable discovery that most twin cattle are born with, and may retain throughout life, a stable mixture – not necessarily a fifty-fifty mixture – of each other’s red cells; it followed, then, that the twin cattle must have exchanged red-cell precursors and not merely red cells in their mutual transfusion before birth. This is the first example of the phenomenon we came to call immunological tolerance; the red cells could not have “adapted” themselves to their strange environment, because they were in fact identified as native or foreign by those very antigenie properties which, had an adaptation occurred, must necessarily have been transformed. A few years later R.E. Billingham and I3, with the help of three members of the scientific staff of the Agricultural Research Council, showed that most dizygotic cattle twins would accept skin grafts from each other, and that this mutual tolerance was specific, for skin transplanted from third parties was cast off in the expected fashion.

Some properties of the tolerant state

The main points that emerged from our analysis of the tolerant state were these. In the first place, tolerance must be due to an alteration of the host, not to an antigenic adaptation of the grafted cells, for grafts newly transplanted in adult life have no opportunity to adapt themselves, and the descendants of the cells injected into foetal or newborn animals can be shown by N.A. Mitcbison’s methods to retain their antigenic power10. Once established, the state of tolerance is systemic; if one part of the body will tolerate a foreign graft, so will another; we found no evidence that a tolerated graft builds up a privileged position for itself within its own lymphatic territory. The stimulus that is responsible for instating tolerance is an antigenic stimulus – one which, had it been applied to older animals, would have caused them to become sensitive or immune. A plural stimulus can induce plural tolerance; the donor will usually contain several important antigens that are lacking in the recipient, and long-lasting tolerance must imply tolerance of them all. The state of tolerance is specific in the sense that it will discriminate between one individual and another, for an animal made tolerant of grafts from one individual will not accept grafts from a second individual unrelated to the first; but it will not discriminate between one tissue and another from the same donor.

Tolerance and auto-immunity: 50 years after Burnet.

Martini A1, Burgio GR
Eur J Pediatr. 1999 Oct;158(10):769-75.

Fifty years ago Sir F. Macfarlane Burnet published his first fundamental contribution to the theory of immune tolerance he perfected 10 years later. Since then an impressive amount of new information on the function of the immune system has been gathered. As any original meaningful theory, Burnet’s hypothesis on the development of immune tolerance has undergone extensive modifications to take into account all these new findings. An improved understanding of the mechanisms of tolerance has led to new possibilities for the treatment of auto-immune diseases.

Clonal Selection

Clonal selection theory is a scientific theory in immunology that explains the functions of cells (lymphocytes) of the immune system in response to specific antigens invading the body. The concept was introduced by an Australian doctor Frank Macfarlane Burnet in 1957 in an attempt to explain the formation of a diversity of antibodies during initiation of the immune response. The theory has become a widely accepted model for how the immune system responds to infection and how certain types of B and T lymphocytes are selected for destruction of specific antigens.

The theory states that in a pre-existing group of lymphocytes (specifically B cells), a specific antigen only activates (i.e. selection) its counter-specific cell so that particular cell is induced to multiply (producing its clones) for antibody production. In short the theory is an explanation of the mechanism for the generation of diversity of antibody specificity. The first experimental evidence came in 1958, when Gustav Nossal and Joshua Lederberg showed that one B cell always produces only one antibody. The idea turned out to be the foundation of molecular immunology, especially in adaptive immunity.

The fundamental contribution of Robert A. Good to the discovery of the crucial role of thymus in mammalian immunity

Domenico Ribatti
Immunology. 2006 Nov; 119(3): 291–295.

Robert Alan Good was a pioneer in the field of immunodeficiency diseases. He and his colleagues defined the cellular basis and functional consequences of many of the inherited immunodeficiency diseases. His was one of the groups that discovered the pivotal role of the thymus in the immune system development and defined the separate development of the thymus-dependent and bursa-dependent lymphoid cell lineages and their responsibilities in cell-mediated and humoral immunity.  Keywords: bursa of Fabricius, history of medicine, immunology, thymus

Robert Alan Good (May 21, 1922 – June 13, 2003) was an American physician who performed the first successful human bone marrow transplant

Robert A. Good began his intellectual and experimental queries related to the thymus in 1952 at the University of Minnesota, initially with pediatric patients. However, his interest in the plasma cell, antibodies and the immune response began in 1944, while still in Medical School at the University of Minnesota in Minneapolis, with his first publication appearing in 1945.

Idiopathic Acquired Agammaglobulinemia Associated with Thymoma (1953)

  • a markedly deficient ability to produce antibodies and significant deficits of all or most of the cell-mediated immunities
  • in no instance did removal of the thymic tumour restore immunological function or correct the protein deficit

Good syndrome: thymoma with immunodeficiency

  • increased susceptibility to bacterial infections by encapsulated organisms and opportunistic viral and fungal infections
  • immunodeficiencies, leukopenia, lymphopenia and eosinophylopenia
  • severely hypogammaglobulinemic rather than agammaglobulinemic

Good and others found that the patients lacked all of the subsequently described immunoglobulins. These patients were found not to have plasma cells or germinal centers in their hematopoietic and lymphoid tissues. They possessed circulating lymphocytes in normal numbers.

Speculation on the reason for immunological failure following neonatal thymectomy has centered on the thymus as a source of cells or humoral factors essential to normal lymphoid development and immunological maturation.

The bursa of Fabricius and the thymus are ‘central lymphoid organs’ in the chicken, essential to the ontogenetic development of adaptive immunity in that species. Studies by Papermaster and co-workers in Good’s laboratory34,35 indicated that bursectomy in the newly hatched chicks did not completely abolish immunological potential in the adult animal but rather produced a striking quantitative reduction insufficient to eliminate the homograft reaction. The failure of thymectomy in newly hatched chicks to alter the immunological potential of the maturing animal probably only reflected the participation of the bursa of Fabricius in the development of full immunological capacity.

Bursectomized and irradiated birds were completely devoid of germinal centers, plasma cells and the capacity to make antibodies yet they had perfectly normal development of thymocytes and lymphocytes elsewhere in the body that mediated cellular immune reactions. On the other hand, thymectomized and irradiated animals were deficient in lymphocytes that mediated cellular immunity as assessed by skin graft rejection, delayed-type hypersensitivity and graft versus host assays, but they still produced germinal centers, plasma cells and circulating immunoglobulins.


Graft vs Host Disease

Graft-versus-host disease (GVHD) is a complication that can occur after a stem cell or bone marrow transplant. With GVHD, the newly transplanted donor cells attack the transplant recipient’s body.

Graft-versus-host disease (GVHD) is a common complication following an allogeneic tissue transplant. It is commonly associated with stem cell or bone marrow transplant but the term also applies to other forms of tissue graft. Immune cells (white blood cells) in the tissue (the graft) recognize the recipient (the host) as “foreign“. The transplanted immune cells then attack the host’s body cells. GVHD can also occur after a blood transfusion if the blood products used have not been irradiated or treated with an approved pathogen reduction system.



GVHD may occur after a bone marrow or stem cell transplant in which someone receives bone marrow tissue or cells from a donor. This type of transplant is called allogeneic. The new, transplanted cells regard the recipient’s body as foreign. When this happens, the newly transplanted cells attack the recipient’s body.

GVHD does not occur when someone receives his or her own cells during a transplant. This type of transplant is called autologous.

Before a transplant, tissue and cells from possible donors are checked to see how closely they match the person having the transplant. GVHD is less likely to occur, or symptoms will be milder, when the match is close. The chance of GVHD is:

  • Around 30 – 40% when the donor and recipient are related
  • Around 60 – 80% when the donor and recipient are not related

There are two types of GVHD: acute and chronic. Symptoms in both acute and chronic GVHD range from mild to severe.

  • Acute GVHD usually happens within the first 6 months after a transplant.
  • Chronic GVHD usually starts more than 3 months after a transplant, and can last a lifetime.

Bone marrow transplant

A bone marrow transplant is a procedure to replace damaged or destroyed bone marrow with healthy bone marrow stem cells.  Stem cells are immature cells in the bone marrow that give rise to all of your blood cells.

There are three kinds of bone marrow transplants:

  • Autologous bone marrow transplant: The term auto means self. Stem cells are removed from you before you receive high-dose chemotherapy or radiation treatment. The stem cells are stored in a freezer (cryopreservation). After high-dose chemotherapy or radiation treatments, your stems cells are put back in your body to make (regenerate) normal blood cells. This is called a rescue transplant.
  • Allogeneic bone marrow transplant: The term allo means other. Stem cells are removed from another person, called a donor. Most times, the donor’s genes must at least partly match your genes. Special blood tests are done to see if a donor is a good match for you. A brother or sister is most likely to be a good match. Sometimes parents, children, and other relatives are good matches. Donors who are not related to you may be found through national bone marrow registries.
  • Umbilical cord blood transplant: This is a type of allogeneic transplant. Stem cells are removed from a newborn baby’s umbilical cord right after birth. The stem cells are frozen and stored until they are needed for a transplant. Umbilical cord blood cells are very immature so there is less of a need for matching. But blood counts take much longer to recover.

Before the transplant, chemotherapy, radiation, or both may be given. This may be done in two ways:

  • Ablative (myeloablative) treatment: High-dose chemotherapy, radiation, or both are given to kill any cancer cells. This also kills all healthy bone marrow that remains, and allows new stem cells to grow in the bone marrow.
  • Reduced intensity treatment, also called a mini transplant: Patients receive lower doses of chemotherapy and radiation before a transplant. This allows older patients, and those with other health problems to have a transplant.

Histocompatibility antigen:

  • A histocompatibility antigen blood test looks at proteins called human leukocyte antigens (HLAs). These are found on the surface of almost all cells in the human body. HLAs are found in large amounts on the surface of white blood cells. They help the immune system tell the difference between body tissue and substances that are not from your own body.


Induction of transplantation tolerance in haploidenical transplantation under reduced intensity conditioning: The role of ex-vivo generated donor CD8+ T cells with central memory phenotype

Eran Ophir, Y Eidelstein, E Bachar-Lustig, D Hagin, N Or-Geva, A Lask, , Y Reisner
Best Practice & Research Clinical Haematology 24 (2011) 393–401

Haploidentical hematopoietic stem cell transplantation (HSCT) offers the advantage of readily available family member donors for nearly all patients. A ‘megadose’ of purified CD34þ hematopoietic stem cells is used to overcome the host’s residual immunity surviving the myeloablative conditioning, while avoiding severe GVHD. However, the number of CD34+ cells that can be harvested is insufficient for overcoming the large numbers of host T cells remaining after reduced intensity conditioning (RIC). Therefore, combining a ‘megadose’ of CD34+ HSCT with other tolerizing cells could potentially support and promote successful engraftment of haploidentical purified stem cell transplantation under a safer RIC. One approach to address this challenge
could be afforded by using Donor CD8 T cells directed against 3rd-party stimulators, bearing an ex-vivo induced central memory phenotype (Tcm). These Tcm cells, depleted of GVH reactivity, were shown to be highly
efficient in overcoming host T cells mediated rejection and in promoting
fully mismatched bone-marrow (BM) engraftment, in HSCT murine models.
This is likely due to the marked lymph node homing of the Tcm, their strong proliferative capacity and prolonged persistence in BM transplant recipients. Thus, combining anti 3rd-party Tcm cell therapy with a ‘megadose’ of purified CD34+ stem cells, could offer a safer RIC protocol for attaining hematopoietic chimerism in patients with hematological diseases and as a platform for organ transplantation or cell therapy in cancer patients.

Induction of tolerance in organ recipients by hematopoietic stem cell transplantation

Eran Ophir, Yair Reisner
International Immunopharmacology 9 (2009) 694–700

The use of hematopoietic stem cell transplantation (HSCT) for the establishment of mixed chimerism represents a viable and attractive approach for generating tolerance in transplantation biology, as it generally leads to durable immune tolerance, enabling the subsequent engraftment of organ transplants without the need for a deleterious continuous immunosuppressive therapy. However, in order to apply HSCT to patients in a manner that enables long term survival, transplant-related mortality must be minimized by eliminating the risk for graft-versus-host-disease (GVHD) and by reducing the toxicity of the conditioning protocol. T-cell depleted bone marrow transplants (TDBMT) have been shown to adequately eliminate GVHD. However, even in leukemia patients undergoing supralethal conditioning, mismatched TDBMT are vigorously rejected. This barrier can be overcome through the modulatory activity of CD34 cells, which are endowed with veto activity, by the use of megadose stem cell transplants. In mice, megadoses of Sca+linhematopoietic stem cells can induce mixed chimerism following sub-lethal conditioning. Nevertheless, the number of human CD34 cells that can be harvested is not likely to be sufficient to overcome rejection under reduced intensity conditioning (RIC), which might be acceptable in recipients of organ transplantation. To address this challenge, we investigated a novel source of veto cells, namely anti 3rd-party cytotoxic T cells (CTLs) which are depleted of GVH reactivity, combined with megadoses of purified stem cells and a RIC protocol. This approach might provide a safer modality for the induction of durable chimerism.

Intrinsic unresponsiveness of Mertk/B cells to chronic graft-versus-host disease is associated with unmodulated CD1d expression

Wen-Hai Shao, Y Zhen, FD Finkelman, RA Eisenberg, PL Cohen
Journal of Autoimmunity 39 (2012) 412e419

Activation and migration of marginal zone B (MZB) cells into follicular (FO) regions of the spleen has been proposed as one of the mechanisms that regulate the development of autoreactive B cells. The mer receptor tyrosine kinase (Mertk) mediates apoptotic cell clearance and regulates activation and cytokine secretion. In the well-studied class II chronic GVH model of bm12 cells into B6 hosts, we observed that Mertk deficient B6 mice did not generate autoantibodies in response to this allogeneic stimulus. We posited that Mertk is important in MHC-II-mediated B cell signaling. In the present study, we show that B cells from Mertk-/- mice but not WT B6 mice exhibited decreased calcium mobilization and tyrosine phosphorylation when stimulated by MHC-II cross-linking. The finding that Mertk was important for class II signaling in B cells was further supported by the preponderance of a-allotype autoantibodies in cGVH in RAG-KO mice reconstituted with a mixture of bone marrow from Mertk-/-mice (b-allotype) and C20 mice (a-allotype). MZB cells from Mertk-/-  mice were unable to down regulate surface CD1d expression and subsequent inclusion in the MZ, associated with significantly lower germinal center responses compared to MZB cells from WT. Moreover, Mertk-/- mice treated with an anti-CD1d down regulating antibody responded significantly to bm12 cells, while no response was observed in Mertk-/- mice treated with control antibodies. Taken together, these findings extend the role of Mertk to include CD1d down regulation on MZB cells, a potential mechanism limiting B cell activation in cGVH.

Galectin-9 ameliorates acute GVH disease through the induction of T-cell apoptosis

Kazuki Sakai, Eri Kawata, Eishi Ashihara, Yoko Nakagawa, et al.
Eur. J. Immunol. 2011. 41: 67–75 http://dx.doi.org:/10.1002/eji.200939931

Galectins comprise a family of animal lectins that differ in their affinity for β-galactosides. Galectin-9 (Gal-9) is a tandem-repeat-type galectin that was recently shown to function as a ligand for T-cell immunoglobin domain and mucin domain-3 (Tim-3) expressed on terminally differentiated CD41 Th1 cells. Gal-9 modulates immune reactions, including the induction of apoptosis in Th1 cells. In this study, we investigated the effects of Gal-9 in murine models of acute GVH disease (aGVHD). First, we demonstrated that recombinant human Gal-9 inhibit MLR in a dose-dependent manner, involving both Ca21 influx and apoptosis in T cells. Next, we revealed that recombinant human Gal-9 significantly inhibit the progression of aGVHD in murine BM transplantation models. In conclusion, Gal-9 ameliorates aGVHD, possibly by inducing T-cell apoptosis, suggesting that gal-9 may be an attractive candidate for the treatment of aGVHD.


GVHD Prevention: An Ounce Is Better Than a Pound

Pavan Reddy, Gerard Socie, Corey Cutler, Daniel Weisdorf
Biol Blood Marrow Transplant 18:S17-S26, 2012  http://dx.doi.org:/10.1016/j.bbmt.2011.10.034

The pathophysiology of acute graft-versus-host disease (aGVHD) is known to involve donor T cells responding to host histoincompatible allo-antigens presented by the host antigen presenting cells (APCs) and the subsequent induction of pro-inflammatory cytokines and cellular effectors that cause target organ damage. In a more general sense, GVHD can be considered as an immune response against foreign antigens that has gone awry. Similar to all immune responses, GVHD, can be understood as a process that consists of (A) triggers, (B) sensors, (C) mediators, and (D) effectors of GVHD.

Like all immune responses, certain triggers are critical for induction of acute graft-versus-host disease (aGVHD). These include: (1) Disparities between histocompatibility antigens: antigen disparity can be at the level of major histocompatibility complex (MHC), that is, MHC mismatched or at the level of minor histocompatibility antigens (miHA), that is, MHC matched but miHA mismatched. The severity of aGVHD is directly related to the degree of M HC mismatch. In bone marrow transplants (BMT) that are MHC matched but miHA disparate, donor T cells still recognize MHC-peptide derived from the products of recipient polymorphic genes, the miHAs.

Damage induced by conditioning regimens and underlying diseases: under most circumstances, the initiation of an adaptive immune response is triggered by the innate immune response. The innate immune system is triggered by certain exogenous and endogenous molecules. This is likely the case in the induction of aGVHD. Pattern recognition receptors such as Toll-like receptors (TLR), nucleotide-binding oligomerization domain containing 2 (NOD2) play an essential role in innate immunity and in initiating the cellular signaling pathways that activate cytokine secretion, such as NF-kB. Some of their ligands, such as lipopolysaccharide, CpG, and MDP2, which is recognized by TLR-4, TLR-9, and NOD2, respectively, are released by the preparative regimens and contribute to the induction and enhancement of allo-T cell responses. In this way, the conditioning regimens amplify the secretion of proinflammatory cytokines like interleukin (IL)-1, tumor necrosis factor (TNF)-α,  IL-6, and other interferon family members in a process described as a ‘‘cytokine storm.’’

The triggers that initiate an immune response have to be sensed and presented. APCs might be considered the sensors for aGVHD. The APCs sense the DAMPs, present the MHC disparate or miHA disparate protein, and provide the critical secondary (costimulatory) and tertiary (cytokine) signals for activation of the alloreactive T cells, the mediators of aGVHD. APCs sense allo-disparity through MHC and peptide complexes. Dendritic cells (DCs) are the most potent APCs and the primary sensors of allo-disparity.

APCs provide the critical costimulation signals for turning on the aGVHD process. The interaction between the MHC/allopeptide complex on APCs and the T cell receptor of donor T cells along with the signal via T cell costimulatory molecules and their ligands on APCs is required to achieve T cell activation, proliferation, differentiation, and survival and the in vivo blockade of positive costimulatory molecules (such as CD28, ICOS, CD40, CD30, etc.), or inhibitory signals (such as PD-1 and CTLA-4) mitigate or exacerbate aGVHD, respectively.

Evidence suggests that alloreactive donor T cells consist of several subsets with different stimuli responsiveness, activation thresholds, and effector functions.

The allo-antigen composition of the host determines which donor T cells subsets differentiate and proliferate. As mentioned previously, in the majority of HLA-matched HCT, aGVHD may be induced by either or both CD41 and CD81 subsets responses to miHAs. The repertoire and immunodominance of the GVHD-associated peptides presented by MHC class I and class II molecules has not been defined. Donor naive CD62L1 T cells are the primary alloreactive T cells that drive the GVHD reaction while the donor effector memory CD62L2 T cells do not. Interestingly, donor regulatory T cells (Tregs) expressing CD62L are also critical to the regulation of GVHD. We now know that it is possible to modulate the alloreactivity of na€ıve T cells by inducing anergy with costimulation blockade, deletion via cytokine modulation, or mixed chimerism. Donor effector memory T cells that are nonalloreactive do not induce GVHD, yet are able to transfer functional memory. In contrast, memory T cells that are alloreactive can cause severe GVHD.

The effector phase that leads to GVHD target organ damage is a complex cascade that involves cytolytic cellular effectors such as CD8 cytotoxic T lymphocytes (CTLs), CD4 T cells, natural killer cells, and inflammatory molecules such as IL-1β, TNF-α, IFN-ϒ, IL-6, and reactive oxygen species. The cellular effectors require cell-cell contact to kill the cells of the target tissues via activation of perforin granzyme, Fas-FasL (CD95-CD95L), or TNFR TRAIL pathways. Other CTLs killing mechanisms such as TWEAK, and LTβ/LIGHT pathways have also been implicated in GVHD. It is important to note that
CTL pathways are essential for GVL effects as well.

All of the above aspects of the biology of aGVHD have been summarized in the mold of a normal immune response. Although this allows for accessing the biology of GVHD, it is important to note that GVHD is a complicated systemic process with as yet still many unknowns and is not a simplified, linear, or cyclical process.

Kinetics of CD4+ and CD8+ T-cell subsets in graft-versus-host reaction (GVHR) in ginbuna crucian carp Carassius auratus langsdorfii

Yasuhiro Shibasakia, H Todaa, Isao kobayashib, T Moritomoa, T Nakanishia
Developmental and Comparative Immunology 34 (2010) 1075–1081

We have previously demonstrated the presence of graft-versus-host reaction (GVHR) in fish employing a model system of clonal triploid ginbuna and tetraploid ginbuna-goldfish hybrids. To elucidate the role of CD8+ T cells in the induction of GVHR, we investigate the kinetics of CD4+ and CD8+ T-cell subsets in GVHR along with the pathological changes associated with GVH disease (GVHD) in ginbuna. GVHR was not induced with a leukocyte fraction lacking CD8+ T cells separated by magnetic cell sorting. Ploidy and immunofluorescence analysis revealed that CD4+ and CD8+  T cells from sensitized donors greatly

increased in the host trunk kidney, constituting more than 80% of total cells 1–2 weeks after donor cell injection, while those from non-sensitized donors constituted less than 50% of cells present. The increase of CD4+ T cells was greater and more rapid than that of CD8+ T cells. The number of donor CD4+ and CD8+ T cells was highest in trunk kidney followed by spleen. Increases in donor CD4+ and CD8+ T cells were also found in liver and PBL, although the percentages were not as high. Pathologic changes similar to those in human and murine acute GVHD were observed in the lymphoid organs as well as target organs such as skin, liver and intestine, including the destruction of cells and tissues and massive leukocyte infiltration. The pathologic changes became more severe with the increase of CD8+ T cells. These results suggest that donor-derived CD8+ T cells play essential roles for the induction of acute GVHR/D in teleosts as in mammals.

Fludarabine and Exposure-Targeted Busulfan Compares Favorably with Busulfan/Cyclophosphamide-Based Regimens in Pediatric Hematopoietic
Cell Transplantation: Maintaining Efficacy with Less Toxicity

I.H. Bartelink, E.M.L. van Reij, C.E. Gerhardt, E.M. van Maarseveen, et al
Biol Blood Marrow Transplant 20 (2014) 345e353

Busulfan (Bu) is used as a myeloablative agent in conditioning regimens before allogeneic hematopoietic cell transplantation (allo-HCT). In line with strategies explored in adults, patient outcomes may be optimized by replacing cyclophosphamide (Cy) with or without melphalan (Mel) with fludarabine (Flu). We compared outcomes in 2 consecutive cohorts of HCT recipients with a nonmalignant HCT indication, a myeloid malignancy, or a lymphoid malignancy with a contraindication for total body irradiation (TBI). Between 2009 and 2012, 64 children received Flu + Bu at a target dose of 80-95 mg-h/L, and between 2005 and 2008, 50 children received Bu targeted to 74-80 mg-h/L þ Cy. In the latter group, Mel was added for patients with myeloid malignancy (n = 12). Possible confounding effects of calendar time were studied in 69 patients receiving a myeloablative dose of TBI between 2005 and 2012. Estimated 2-year survival and event-free survival were 82% and 78%, respectively, in the FluBu arm and 78% and 72%, respectively, in the BuCy (Mel) arm (P,  not significant). Compared with the BuCy (Mel) arm, less toxicity was noted in the FluBu arm, with lower rates of acute (noninfectious) lung injury (16% versus 36%; P < .007), veno-occlusive disease (3% versus 28%; P < .003), chronic graft-versus-host disease (9% versus 26%; P < .047), adenovirus infection (3% versus 32%; P < .001), and human herpesvirus 6 infection reactivation (21% versus 44%; P < .005). Furthermore, the median duration of neutropenia was shorter in the FluBu arm (11 days versus 22 days; P < .001), and the patients in this arm required fewer transfusions. Our data indicate that Flu (160 mg/m2) with targeted myeloablative Bu (90 mg-h/L) is less toxic than and equally effective
as BuCy (Mel) in patients with similar indications for allo-HCT.

Fibrotic and Sclerotic Manifestations of Chronic Graft-versus-Host Disease

Carrie L. Kitko, Eric S. White, Kristin Baird
Biol Blood Marrow Transplant 18:S46-S52, 2012

Chronic graft-versus-host disease (cGVHD) is a common cause of morbidity
and mortality following allogeneic stem cell transplantation (HCT), with approximately 50% to 60% of long-term HCT survivors developing one or more manifestations of the disorder. Although acute GVHD is typically limited to skin, liver, and gastrointestinal involvement, virtually every organ is at risk for the development of cGVHD. Although the pathophysiology of cGVHD remains poorly understood, some of the most severe organ manifestations are linked by end-organ fibrosis. In particular, fibrotic cutaneous and bronchiolar changes, resulting in scleroderma-like changes and bronchiolitis obliterans syndrome (BOS), respectively, are two of the most devastating outcomes for these patients. Both sclerotic GVHD (ScGVHD) and BOS have been reported in 5% to 15% of patients with cGVHD.

Many of the manifestations of cGVHD share clinical characteristics seen in nontransplant conditions, including systemic sclerosis or pulmonary fibrosis. Thus, understanding the pathophysiology underlying these related conditions may help identify potential mechanisms and ultimately new therapeutic options for patients with cGVHD.

Tyrosine kinase inhibitors (TKIs) have been shown to inhibit two different profibrotic pathways (transforming growth factor β [TGF-β] and platelet-derived growth factor [PDGF]) in various mouse models of fibrotic disease and offer a possible novel treatment approach for cGVHD patients suffering from severe sclerosis. Likewise, overexpression of TNF-α has been shown to induce fibrogenesis in experimental hepatocellular disease and has been linked with human scleroderma-associated interstitial pulmonary fibrosis and profibrotic responses in human osteoarthritic hip joint fibroblasts. The use of TNF antagonists has been examined in some clinical situations associated with fibrosis, suggesting they may also be of some benefit to patients with cGVHD; however, this must first be prospectively tested.

Table. Proposed Modifications to NIH BOS Clinical Definition

  • Absence of infection (no change)
  • Another cGVHD manifestation in another organ (no change)
  • FEV1 <75% predicted (no change) or >10% decline from pre-HCT value (modification)
  • Signs of Obstruction
  • FEV1/SVC ratio <0.7 (modification), or
  • RV >120% predicted (no change), or
  • RV/TLC >120% (modification), and
  • HRCT with evidence of air trapping (no change)

SVC indicates slow vital capacity; RV, residual volume; TLC, total lung capacity; HRCT, high-resolution computed tomography

Figure (not shown)
Effect of etanercept on survival in post-HCT patients with subacute lung injury. (A) Overall 5-year survival by pulmonary function testing defect. Patients with an obstructive defect (solid line) had a 5-year survival of 67% compared with 44% in those with a restrictive lung defect (dashed line) (P 5 .19). (B) Overall 5-year survival by response to therapy. Patients who responded to etanercept therapy (solid line) had a 5-year survival of 90% compared with 55% in patients who failed to respond (dashed line) (P 5.07). (Figures reprinted with permission, Biol Blood and Marrow Trans).

Extensive, sclerotic skin changes with superficial or deep subcutaneous or fascial involvement are seen in approximately 4% to 13% of patients with cGVHD and can be a life-threatening manifestation. ScGVHD of the skin includes several cutaneous presentations characterized by inflammation and progressive fibrosis of the dermis and subcutaneous tissues. These changes can resemble morphea, systemic sclerosis, or eosinophilic fasciitis and may or may not occur in the setting of concurrent overlying epidermal GVHD. When severe, ScGVHD can result in contractures, severe wasting, and chest wall restriction.

Development of clinical trials for patients with cGVHD is difficult because of the complexity and heterogeneity of disease, variable approaches to treatment, and the lack of standardized assessments of disease. In particular, the study of ScGVHD lacks universally accepted measures of disease burden and response. Investigators have used several measures to assess ScGVHD involvement including body surface area, magnetic resonance imaging, ultrasound, and range-of-motion measurements. Additionally, investigators have tried to apply the Rodnan score, the standardmeasure for skin involvement in scleroderma. Thus far, none of these measures has proven
to be completely reliable in the setting of ScGVHD, and it is likely that multiple measures will need to be integrated into the assessment of ScGVHD.

Imatinib mesylate (Gleevec in the US; Glivec in Europe, Australia, and Latin America, marketed by Novartis) is a TKI that has biological activity against both PDGF and TGF-β signaling pathways. Both cytokines have been implicated in the pathogenesis of several fibrosing diseases, including hepatic, renal, and lung, as well as in scleroderma, a disease that closely resembles ScGVHD. In addition, stimulatory antibodies specific for the PDGF receptor (PDGFR) were identified in a series of 39 patients with extensive cGVHD with higher levels detected in those patients with skin involvement. Similar stimulatory antibodies targeting PDGFR have been reported in patients with scleroderma, suggesting an important therapeutic target for these fibrosing conditions. Imatinib mesylate has particularly potent activity against PDGF and is FDA approved in the United States for the treatment of several disorders associated with aberrant PDGFR signaling. The side effect profile of the drug is well established in non-HCT patients, which is helpful in the setting of a therapy for allogenic HCT patients, many of whom have multiorgan system symptoms and possible dysfunction and who will require ongoing immunosuppressive therapy.

Through the efforts of the Chronic GVHD Consortium, led by Stephanie Lee at the Fred Hutchinson Cancer Research Center, there is a multicenter, ongoing prospective evaluation of the NIH diagnostic and assessment tools. This effort has already resulted in several publications that have further refined essential criteria for cGVHD evaluation, including organ-specific manifestations such as BOS and ScGVHD. Currently, the Consortium is conducting a multicenter prospective clinical trial of fluticasone propionate, azithromycin, and montelukast for the treatment of BOS (ClinicalTrials.gov NCT01307462); a separate trial of imatinib versus rituximab for treatment of ScGVHD is also enrolling subjects (ClinicalTrials.gov NCT01309997).

Although cGVHD remains a significant problem for many long-term survivors of HCT, critical advances in cGVHD research and treatment can be achieved by cooperative group efforts such as those put forth by the Chronic GVHD Consortium and the Clinical Trials Network.

Hematopoietic stem cell transplantation (HSCT): An approach to autoimmunity

Carmen Alaez, Mariana Loyola, Andrea Murguıa, Hilario Flores, et al.
Autoimmunity Reviews 5 (2006) 167– 179

HSCT provides the opportunity to replace a damaged tissue. It is the most important treatment for high risk hematologic malignant and nonmalignant disorders. An important challenge in the identification of matched donors/patients is the HLA diversity. The Mexican Bone Marrow Registry (DONORMO) has nowadays N5000 donors. The prevalent alleles are Amerindian, Mediterranean (Semitic and Spanish genes) and African. In theory, it is possible to find 11% of 6/6 A–B–DR low resolution matches for 70% of patients with Mexican ancestry. We contributed with 39 unrelated, cord blood and autologous HSCT for patients with malignant, genetic and autoimmune disorders. Overall disease survival was 50% (2–7 years) depending on the initial diagnosis, conditioning, disease evolution or other factors. Clinical studies using autologous and unrelated HSC are performed on patients with refractory autoimmune diseases producing mixed results: mainly, T1D, RA, MS, SLE. Improvement has been observed in skin damage and quality of life in SLE and systemic sclerosis. Disease stabilization in 2/3 of MS patients. However, in RA and T1D, initial benefits have been followed by eventual relapse. With growing clinical experience and protocol improvement, treatment-related mortality is decreasing. Proof efficacy will be achieved by comparing HSCT with standard therapy in autoimmunity.

Monoclonal Antibody-Mediated Targeting of CD123, IL-3 Receptor α Chain, Eliminates Human Acute Myeloid Leukemic Stem Cells

Liqing Jin, Erwin M. Lee, Hayley S. Ramshaw, Samantha J. Busfield, et al.
Cell: Stem Cell 5, 31–42, July 2, 2009

Leukemia stem cells (LSCs) initiate and sustain the acute myeloid leukemia (AML) clonal hierarchy and possess biological properties rendering them resistant to conventional chemotherapy. The poor survival of AML patients raises expectations that LSC-targeted therapies might achieve durable remissions. We report that an anti-interleukin-3 (IL-3) receptor α chain (CD123)-neutralizing antibody (7G3) targeted AML-LSCs, impairing homing
to bone marrow (BM) and activating innate immunity of nonobese diabetic/ severe-combined immunodeficient (NOD/SCID) mice. 7G3 treatment profoundly reduced AML-LSC engraftment and improved mouse survival.
Mice with preestablished disease showed reduced AML burden in the BM
and periphery and impaired secondary transplantation upon treatment, establishing that AMLLSCs were directly targeted. 7G3 inhibited IL-3-mediated intracellular signaling of isolated AML CD34+ CD38[1] cells in vitro and reduced their survival. These results provide clear validation for therapeutic monoclonal antibody (mAb) targeting of AML-LSCs and for translation of in vivo preclinical research findings toward a clinical application.

Many Days at Home during Neutropenia after Allogeneic Hematopoietic Stem Cell Transplantation Correlates with Low Incidence of Acute Graft-versus-Host Disease

Olle Ringdén, Mats Remberger, Katarina Holmberg, Charlotta Edeskog, et al.
Biol Blood Marrow Transplant 19 (2013) 314e320

Patients are isolated in the hospital during the neutropenic phase after allogeneic hematopoietic stem cell transplantation. We challenged this by allowing patients to be treated at home. A nurse from the unit visited and checked the patient. One hundred forty-six patients treated at home were compared with matched hospital control subjects. Oral intake was intensified from September 2006 and improved (P < .002). We compared 4 groups: home care and control subjects before and after September 2006. The cumulative incidence of acute graft-versus-host disease (GVHD) of grades II to IV was 15% in the “old” home care group, which was significantly lower than that of 32% to 44% in the other groups (P <.03). Transplantation-related mortality, chronic GVHD, and relapse were similar in the groups. The “new” home care patients spent fewer days at home (P < .002). In multivariate analysis, GVHD of grades 0 to I was associated with home care (hazard ratio [HR], 2.46; P <.02) and with days spent at home (HR, .92; P < .005) but not with oral nutrition (HR, .98; P = .13). Five year survival was 61% in the home care group as compared with 49% in the control subjects (P < .07). Home care is safe. Home care and many days spent at home were correlated with a low risk of acute GVHD.

Impact on Outcomes of Human Leukocyte Antigen Matching by Allele-Level Typing in Adults with Acute Myeloid Leukemia Undergoing Umbilical Cord Blood Transplantation

Jaime Sanz, Francisco J. Jaramillo, Dolores Planelles, Pau Montesinos, et al.
Biol Blood Marrow Transplant 20 (2014) 106e110

This retrospective study analyzed the impact of directional donor-recipient human leukocyte antigen (HLA) disparity using allele-level typing at HLA-A, -B, -C, and -DRB1 in 79 adults with acute myeloid leukemia (AML) who received single-unit umbilical cord blood (UCB) transplant at a single institution. With extended high resolution HLA typing, the donor-recipient compatibility ranged from 2/8 to 8/8. HLA disparity showed no negative impact on nonrelapse mortality (NRM), graft-versus-host (GVH) disease or engraftment. Considering disparities in the GVH direction, the 5-year cumulative incidence of relapse was 44% and 22% for patients receiving an UCB unit matched > 6/8 and < 6/8, respectively (P <.04). In multivariable analysis, a higher HLA disparity in the GVH direction using extended high-resolution typing (Risk ratio [RR] 2.8; 95% confidence interval [CI], 1.5 to 5.1; P ¼.0009) and first complete remission at time of transplantation (RR 2.1; 95% CI, 1.2 to 3.8; P < .01) were the only variables significantly associated with an improved disease-free survival. In conclusion, we found that in adults with AML undergoing single-unit UCBT, an increased number of HLA disparities at allele-level typing improved disease-free survival by decreasing the relapse rate without a negative effect on NRM.

HLA mismatch direction in cord blood transplantation: impact on outcome and implications for cord blood unit selection
Cladd E. Stevens, C Carrier, C Carpenter, D Sung, and A Scaradavou

Blood. 2011; 118(14):3969-3978

Donor-recipient human leukocyte antigen mismatch level affects the outcome of unrelated cord blood (CB) transplantation. To identify possible “permissive” mismatches, we examined the relationship between  direction of human leukocyte antigen mismatch (“vector”) and transplantation outcomes in 1202 recipients of single CB units from the New York Blood Center National Cord Blood Program treated in United States Centers from 1993-2006. Altogether, 98 donor/patient pairs had only unidirectional mismatches: 58 in the graft-versus-host (GVH) direction only (GVH-O) and 40 in the host-versus-graft or rejection direction only (R-O). Engraftment was faster in patients with GVH-O mismatches compared with those with 1 bidirectional mismatch (hazard ratio [HR] = 1.6, P < .003). In addition, patients with hematologic malignancies given GVH-O grafts had lower transplantation-related mortality (HR = 0.5, P < .062), overall mortality (HR = 0.5, P < .019), and treatment failure (HR = 0.5, P < .016), resulting in outcomes similar to those of matched CB grafts. In contrast, R-O mismatches had slower engraftment, higher graft failure, and higher relapse rates (HR = 2.4, P < .010). Based on our findings, CB search algorithms should be modified to identify unidirectional mismatches. We recommend that transplant centers give priority to GVH-O-mismatched units over other mismatches and avoid selecting R-O mismatches, if possible.

Mutation of the NPM1 gene contributes to the development of donor cell–derived acute myeloid leukemia after unrelated cord blood transplantation
for acute lymphoblastic leukemia

G Rodríguez-Macías, C Martínez-Laperche, J Gayoso, V Noriega, .., Ismael Buño
Human Pathology (2013) 44, 1696–1699

Donor cell leukemia (DCL) is a rare but severe complication after allogeneic stem cell transplantation. Its true incidence is unknown because of a lack of correct recognition and reporting, although improvements in molecular analysis of donor-host chimerism are contributing to a better diagnosis of this complication. The mechanisms of leukemogenesis are unclear, and multiple factors can contribute to the development of DCL. In recent years, cord blood has emerged as an alternative source of hematopoietic progenitor cells, and at least 12 cases of DCL have been reported after unrelated cord blood transplantation. We report a new case of DCL after unrelated cord blood transplantation in a 44-year-old woman diagnosed as having acute lymphoblastic leukemia with t(1;19) that developed acute myeloid leukemia with normal karyotype and nucleophosmin (NPM1) mutation in donor cells. To our knowledge, this is the first report of NPM1 mutation contributing to DCL development.

Graft-versus-leukemia in the bone marrow
Blood, 23 JAN 2014; 123(4)

63-year-old female with relapsed acute myeloid leukemia (AML) after allogeneic stem cell transplantation reached CR2 after re-induction therapy followed by consolidation with donor lymphocyte infusions: 3 x 107/kg and 3 x 108/kg after 1 and 2.5 months, respectively. No signs of graft-versus-host disease were observed at this time. At 5 months follow-up, her blood count deteriorated: hemoglobin: 6.9 mmol/L, thrombocytes: 58 x 109/L and leukocytes: 1.37 x 109/L. Bone marrow aspirate was not evaluable. Bone marrow trephine biopsy showed relapse AML with hypercellularity in the H&E staining (340 objective lens, panel A) and 20% CD341 blast cells without any signs of maturation (panel B). Also, a high number of CD3 positive T cells (panel C) was noted, intermingling with the CD34 positive blasts, both staining positively with CD43 (panel D). Only supportive care was given. However, normalization of the blood count was observed in the following months and she developed graft-versus-host disease of the lung, which was treated with ciclosporin and prednisone. A bone marrow aspirate performed 3 months after relapse showed a third remission with 0.8% myeloid blasts. In retrospect, one could therefore consider the picture of the bone marrow trephine biopsy at the second relapse as graft-versus-leukemia in the bone marrow.

GVL- panel A

GVL- panel A

GVL - panel B

GVL – panel B

GVL - panel C

GVL – panel C

GVL - panel D

Long-Term Outcomes of Alemtuzumab-Based Reduced-Intensity Conditioned Hematopoietic Stem Cell Transplantation for Myelodysplastic Syndrome and Acute Myelogenous Leukemia Secondary to Myelodysplastic Syndrome

Victoria T. Potter, Pramila Krishnamurthy, Linda D. Barber, ZiYi Lim, et al.
Biol Blood Marrow Transplant 20 (2014) 111e117

Allogeneic hematopoietic stem cell transplantation (HSCT) with reduced-intensity conditioning (RIC) offers a potential cure for patients with myelodysplastic syndrome (MDS) who are ineligible for standard-intensity regimens. Previously published data from our institution suggest excellent outcomes at 1 yr using a uniform fludarabine, busulfan, and alemtuzumab-based regimen. Here we report long-term follow-up of 192 patients with MDS and acute myelogenous leukemia (AML) secondary to MDS (MDS-AML) transplanted with this protocol, using sibling (n = 45) or matched unrelated (n = 147) donors. The median age of the cohort was 57 yr (range, 21 to 72 yr), and median follow-up was 4.5 yr (range, 0.1 to 10.6 yr). The 5-yr overall survival (OS), event-free survival, and nonrelapse mortality were 44%, 33%, and 26% respectively. The incidence of de novo chronic graft-versus-host disease (GVHD) was low at 19%, illustrating the efficacy of alemtuzumab for GVHD prophylaxis. Conversely, the 5-yr relapse rate was 51%. For younger patients (age <50 yr), the 5-yr OS and relapse rates were 58% and 39%, respectively. On multivariate analysis, advanced age predicted significantly worse outcomes, with patients age >60 yr having a 5-yr OS of 15% and relapse rate of 66%. Patients receiving preemptive donor lymphocyte infusions had an impressive 5-yr OS of 67%, suggesting that this protocol may lend itself to the incorporation of immunotherapeutic strategies. Overall, these data demonstrate good 5-yr OS for patients with MDS and MDS-AML undergoing alemtuzumab-based RIC-HSCT. The low rate of chronic GVHD is encouraging, and comparative studies with other RIC protocols are warranted.

Natural killer cell activity influences outcome after T cell depleted stem cell transplantation from matched unrelated and haploidentical donors

Peter Lang, Matthias Pfeiffer,  Heiko-Manuel Teltschik, Patrick Schlegel, et al.
Best Practice & Research Clinical Haematology 24 (2011) 403–411

Lytic activity and recovery of natural killer (NK) cells was monitored in pediatric patients with leukemias (ALL, AML, CML, JMML) and myelodysplastic syndromes after transplantation of T cell depleted stem cells from matched unrelated (n = 18) and mismatched related (haploidentical, n = 29) donors. CD34+ selection with magnetic microbeads resulted in 8 x 103/kg residual T cells. No post-transplant immune suppression was given. NK cells recovered rapidly after transplantation (300 CD56+/mL at day 30, median), whereas T cell recovery was delayed (median: 12 CD3+/mL at day 90). NK activity was measured as specific lysis of K 562 targets several times (mean: 3 assays per patient). Four temporal patterns of lytic activity could be differentiated: consistently low, consistently high, decreasing and increasing activity. Patients with consistently high or increasing activity had significantly lower relapse probability than patients with consistently low or decreasing levels (0.18 vs 0.73 at 2 years, p < 0.05). The subgroup of patients with ALL showed similar results (0.75 vs 0.14 at 2 years, p < 0.05). Speed of T cell recovery had no influence. These data suggest that both achieving and maintaining a high level of NK activity may contribute to prevent relapse. Since NK activity could be markedly increased by in vitro stimulation with Interleukin 2 (IL-2), in vivo administration should be considered.

Graft-versus-host disease: Pathogenesis and clinical manifestations of graft-versus-host disease

Sharon R. Hymes, Amin M. Alousi,  and Edward W. Cowen
J Am Acad Dermatol  2012; 66: 515.e1-18.

  • Graft-versus-host disease is the primary cause of morbidity and nonerelapse related mortality in patients who undergo allogeneic hematopoietic cell transplantation.
  • Acute graft-versus-host disease manifests as a skin exanthem, liver dysfunction, and gastrointestinal involvement.
  • Chronic graft-versus-host disease of the skin is remarkably variable in its clinical presentation.
  • Chronic graft-versus-host disease is a multisystem disorder that may affect nearly any organ; the most common sites are the skin, oral mucosa, and eyes.

Key points

  • Allogeneic transplantation is in widespread use for hematologic malignancies, but is also increasingly used for marrow failure syndromes, immunodeficiencies, and other life-threatening conditions
  • Graft-versus-host disease is the primary cause of morbidity and nonerelapse related mortality after allogeneic hematopoietic cell transplantation
  • Minimizing graft-versus-host disease without losing the graft-versus-tumor effect is an area of active research
  • The skin is the most common organ affected in patients with graft-versus-host disease

Outcomes of Thalassemia Patients Undergoing Hematopoietic Stem Cell Transplantation by Using a Standard Myeloablative versus a Novel Reduced-Toxicity Conditioning Regimen According to a New Risk Stratification

Usanarat Anurathapan, S Pakakasama, P Mekjaruskul, N Sirachainan, et al.
Biol Blood Marrow Transplant 20 (2014) 2056e2075

Improving outcomes among class 3 thalassemia patients receiving allogeneic hematopoietic stem cell transplantations (HSCT) remains a challenge. Before HSCT, patients who were > 7 years old and had a liver size > 5 cm constitute what the Center for International Blood and Marrow Transplant Research defined as a very high risk subset of a conventional high-risk class 3 group (here referred to as class 3 HR). We performed HSCT in 98 patients with related and unrelated donor stem cells. Seventy-six of the patients with age < 10 years received the more conventional myeloablative conditioning (MAC) regimen (cyclophos-phamide, busulfan,  + fludarabine); the remaining 22 patients with age > 10 years and hepatomegaly (class 3 HR), and in several instances additional comorbidity problems, underwent HSCT with a novel reduced-toxicity conditioning (RTC) regimen (fludarabine and busulfan). We then compared the outcomes between these 2 groups (MAC versus RTC). Event-free survival (86% versus 90%) and overall survival (95% versus 90%) were not significantly different between the respective groups; however, there was a higher incidence of serious treatment-related complications in the MAC group, and although we experienced 6 graft failures in the MAC group (8%), there were none in the RTC group. Based on these results, we suggest that (1) class 3HRthalassemia patients can safely receive HSCT with our novel RTC regimen and achieve the same excellent outcome as low/standard-risk thalassemia patients who received the standard MAC regimen, and further, (2) that this novel RTC approach should be tested in the low/standard-risk patient population.

Pharmacological Immunosuppression Reduces But Does Not Eliminate the Need for Total-Body Irradiation in Nonmyeloablative Conditioning Regimens for Hematopoietic Cell Transplantation

Marco Mielcarek, Beverly Torok-Storb, Rainer Storb
Biol Blood Marrow Transplant 17: 1255-1260 (2011)

In the dog leukocyte antigen (DLA)-identical hematopoietic cell transplantation (HCT) model, stable marrow engraftment can be achieved with total-body irradiation (TBI) of 200 cGy when used in combination with postgrafting immunosuppression. The TBI dose can be reduced to 100 cGy without compromising engraftment rates if granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells (G-PBMC) are infused with the marrow. T cell-depleting the G-PBMC product abrogates this effect. These results were interpreted to suggest that the additional T cells provided with G-PBMC facilitated engraftment by overcoming host resistance.We therefore hypothesized that the TBI dose may be further reduced to 50 cGy by augmenting immunosupression either by (1) tolerizing or killing recipient T cells, or (2) enhancing the graft-versus-host (GVH) activity of donor T cells. To test the first hypothesis, recipient T cells were activated before HCT by repetitive donor-specific PBMC infusions followed by administration of methotrexate (MTX) (n 5 5), CTLA4-Ig (n = 4), denileukin diftitox (Ontak; n = 4), CTLA4-Ig 1 MTX (n = 8), or 5c8 antibody (anti-CD154) 1 MTX (n = 3). To test the second hypothesis, recipient dendritic cells were expanded in vivo by infusion of Flt3 ligand given either pre-HCT (n = 4) or pre- and post-HCT (n = 5) to augment GVH reactions. Although all dogs showed initial allogeneic engraftment, sustained engraftment was seen in only 6 of 42 dogs (14% of all dogs treated in 9 experimental groups). Hence, unless more innovative pharmacotherapy can be developed that more forcefully shifts the immunologic balance in favor of the donor, noncytotoxic immunosuppressive drug therapy as the sole component of HCT preparative regimens may not suffice to ensure sustained engraftment.

Pretransplant Immunosuppression followed by Reduced-Toxicity Conditioning and Stem Cell Transplantation in High-Risk Thalassemia: A Safe Approach to Disease Control

Usanarat Anurathapan, S Pakakasama, P Rujkijyanont, N Sirachainan, et al.
Biol Blood Marrow Transplant 19 (2013) 1254e1270

Patients with class 3 thalassemia with high-risk features for adverse events after high-dose chemotherapy with hematopoietic stem cell transplantation (HSCT) are difficult to treat, tending to either suffer serious toxicity or fail to establish stable graft function. We performed HSCT in 18 such patients age 7 years and hepatomegaly using a novel approach with pretransplant immunosuppression followed by a myeloablative reduced-toxicity conditioning regimen (fludarabine and i.v. busulfan [Flu-IV Bu]) and then HSCT. The median patient age was 14 years (range, 10 to 18 years). Before the Flu-IV Bu þ antithymocyte globulin conditioning regimen, all patients received 1 to 2 cycles of pretransplant immunosuppression with fludarabine and dexamethasone. Thirteen patients received a related donor graft, and 5 received an unrelated donor graft. An initial prompt engraftment of donor cells with full donor chimerism was observed in all 18 patients, but 2 patients developed secondary mixed chimerism that necessitated withdrawal of immunosuppression to achieve full donor chimerism. Two patients (11%) had acute grade III-IV graft-versus-host disease, and 5 patients had limited chronic graft-versus-host disease. The only treatment-related mortality was from infection, and with a median follow-up of 42 months (range, 4 to 75), the 5-year overall survival and thalassemia-free survival were 89%. We conclude that this novel sequential immunoablative pretransplant-ation conditioning program is safe and effective for patients with high-risk class 3 thalassemia exhibiting additional comorbidities.

Profiling antibodies to class II HLA in transplant patient sera

Curtis McMurtrey, D Lowe, R Buchli, S Daga, D Royer, A Humphrey, et al.
Human Immunology 75 (2014) 261–270

Immunizing events including pregnancy, transfusions, and transplantation promote strong alloantibody responses to HLA. Such alloantibodies to HLA preclude organ transplantation, foster hyperacute rejection, and contribute to chronic transplant failure. Diagnostic antibody-screening assays detect alloreactive antibodies, yet key attributes including antibody concentration and isotype remain largely unexplored. The goal here was to provide a detailed profile of allogeneic antibodies to class II HLA. Methodologically, alloantibodies were purified from sensitized patient sera using an HLA-DR11 immunoaffinity column and subsequently categorized. Antibodies to DR11 were found to fix complement, exist at a median serum concentration of 2.3 lg/mL, consist of all isotypes, and isotypes IgG2, IgM, and IgE were elevated. Because multimeric isotypes can confound diagnostic determinations of antibody concentration, IgM and IgA isotypes were removed and DR11-IgG tested alone. Despite removal of multimeric isotypes, patient-to patient antibody concentra-tions did not correlate with MFI values. In conclusion, allogeneic antibody responses to DR11 are comprised of all antibody isotypes at differing proportions, these combined isotypes fix complement at nominal serum concentrations, and enhancements other than the removal of IgM and IgA multimeric isotypes may be required if MFI is to be used as a means of determining anti-HLA serum antibody concentrations in diagnostic clinical assays.

Reduced-intensity conditioning and HLA-matched hemopoietic stem-cell transplantation in patients with chronic granulomatous disease: a prospective multicenter study

Tayfun Güngör, P Teira, M Slatter, G Stussi, P Stepensky, D Moshous, et al.
Lancet 2014; 383: 436–48

Background In chronic granulomatous disease allogeneic hemopoietic stem-cell transplantation (HSCT) in adolescents and young adults and patients with high-risk disease is complicated by graft-failure, graft-versus-host disease (GVHD), and transplant-related mortality. We examined the effect of a reduced-intensity conditioning regimen designed to enhance myeloid engraftment and reduce organ toxicity in these patients.       Methods This prospective study was done at 16 centers in ten countries worldwide. Patients aged 0–40 years with chronic granulomatous disease were assessed and enrolled at the discretion of individual centers. Reduced-intensity conditioning consisted of high-dose fludarabine (30 mg/m² [infants <9 kg 1∙2 mg/kg]; one dose per day on days –8 to –3), serotherapy (anti-thymocyte globulin [10 mg/kg, one dose per day on days –4 to –1; or thymoglobulin 2·5 mg/kg, one dose per day on days –5 to –3]; or low-dose alemtuzumab [<1 mg/kg on days –8 to –6]), and low-dose (50–72% of myeloablative dose) or targeted busulfan administration (recommended cumulative area under the curve: 45–65 mg/L × h). Busulfan was administered mainly intravenously and exceptionally orally from days –5 to –3. Intravenous busulfan was dosed according to weight-based recommendations and was administered in most centers (ten) twice daily over 4 h. Unmanipulated bone marrow or peripheral blood stem cells from HLA-matched related donors or HLA-9/10 or HLA-10/10 matched unrelated-donors were infused. The primary endpoints were overall survival and event-free survival (EFS), probabilities of overall survival and EFS at 2 years, incidence of acute and chronic GVHD, achievement of at least 90% myeloid donor chimerism, and incidence of graft failure after at least 6 months of follow-up. Findings 56 patients (median age 12∙7 years; IQR 6·8–17·3) with chronic granulomatous disease were enrolled from June 15, 2003, to Dec 15, 2012. 42 patients (75%) had high-risk features (ie, intractable infections and autoinflammation), 25 (45%) were adolescents and young adults (age 14–39 years). 21 HLA-matched related-donor and 35 HLA-matched unrelated-donor transplants were done. Median time to engraftment was 19 days (IQR 16–22) for neutrophils and 21 days (IQR 16–25) for platelets. At median follow-up of 21 months (IQR 13–35) overall survival was 93% (52 of 56) and EFS was 89% (50 of 56). The 2-year probability of overall survival was 96% (95% CI 86∙46–99∙09) and of EFS was 91% (79∙78–96∙17). Graft-failure occurred in 5% (three of 56) of patients. The cumulative incidence of acute GVHD of grade III–IV was 4% (two of 56) and of chronic graft-versus-host disease was 7% (four of 56). Stable (≥90%) myeloid donor chimerism was documented in 52 (93%) surviving patients. Interpretation This reduced-intensity conditioning regimen is safe and efficacious in high-risk patients with chronic granulomatous disease.

Refinement of the Definition of Permissible HLA-DPB1 Mismatches with Predicted Indirectly ReCognizable HLA-DPB1 Epitopes

Kirsten A. Thus, MTA Ruizendaal, TA de Hoop, Eric Borst, et al.
Biol Blood Marrow Transplant 20 (2014) 1705e1710

Hematopoietic stem cell transplantation with HLA-DPB1emismatched donors leads to an increased risk of acute graft-versus-host disease (GVHD). Studies have indicated a prognostic value for classifying HLA-DPB1 mismatches based on T cell epitope (TCE) groups. The aim of this study was to determine the contribution of indirect recognition of HLA-DPe derived epitopes, as determined with the Predicted Indirectly ReCognizable HLA Epitopes (PIRCHE) method. We therefore conducted a retrospective single-center analysis on 80 patients transplanted with a 10/10 matched unrelated donor that was HLA-DPB1 mismatched. HLADPB1 mismatches that were classified as GVH nonpermissive by the TCE algorithm correlated to higher numbers of HLA class I as well as HLA class II presented PIRCHE (PIRCHE-I and -II) compared with permissive or host-versus-graft nonpermissive mismatches. Patients with acute GVHD grades II to IV presented significantly higher numbers of PIRCHE-I compared with patients without acute GVHD (P < .05). Patients were divided into 2 groups based on the presence or absence of PIRCHE. Patients with PIRCHE-I or -II have an increased hazard of acute GVHD when compared with patients without PIRCHE-I or -II (hazard ratio [HR], 3.19; 95% confidence interval [CI], 1.10 to 9.19; P <.05; and HR, 4.07; 95% CI, .97 to 17.19; P < .06, respectively). Patients classified as having an HLA-DPB1 permissive mismatch by the TCE model had an increased risk of acute GVHD when comparing presence of PIRCHE-I with absence of PIRCHE-I (HR, 2.96; 95% CI, .84 to 10.39; P < .09). We therefore conclude that the data presented in this study describe an attractive and feasible possibility to better select permissible HLA-DPB1 mismatches by including both a direct and an indirect recognition model.

Treosulfan-Thiotepa-FludarabineeBased Conditioning Regimen for
Allogeneic Transplantation in Patients with Thalassemia Major: A
Single-Center Experience from North India

Dharma Choudhary, SK Sharma, N Gupta,…, Satyendra Katewa
Biol Blood Marrow Transplant 19 (2013) 492e503

Hematopoietic stem cell transplantation (HSCT) is the definite treatment
for patients with thalassemia major. A busulfan (Bu) and cyclophosphamide
(Cy)ebased regimen has been the standard myeloablative chemotherapy,
but it is associated with higher treatment-related toxicity, particularly in
patients classified as high risk by the Pesaro criteria. Treosulfan-based
conditioning regimens have been found to be equally effective and less
toxic. Consequently, we analyzed the safety and efficacy of treosulfan/
thiotepa/fludarabine (treo/thio/flu)-based conditioning regimens for
allogeneic HSCT in patients with thalassemia major between February
2010 and September 2012. We compared those results retrospectively
with results in patients who underwent previous HSCT with a Bu/Cy/
antithymocyte globulin (ATG)ebased conditioning regimen. A treo/thio/
flu-based conditioning regimen was used in 28 consecutive patients with
thalassemia major. The median patient age was 9.7 years (range, 2-18
years), and the mean CD34+ stem cell dose was 6.18 x 106/kg. Neutrophil
and platelet engraftment occurred at a median of 15 days (range, 12-23
days) and 21 days (range, 14-34 days), respectively. Three patients
developed veno-occlusive disease, 4 patients developed acute graft
versus-host disease (GVHD), and 2 patients had chronic GVHD. Treatment-
related mortality (TRM) was 21.4%. Two patients experienced secondary
graft rejection. We compared these results with results in patients who
underwent previous HSCT using a Bu/Cy/ATG-based conditioning regimen.
Twelve patients were treated with this protocol, at a median age of 7.2
years (range, 2-11 years). One patient had moderate veno-occlusive disease,
2 patients developed acute GVHD, 2 patients had chronic GVHD, and 2
patients experienced graft rejection. There was no TRM in this group. We
found no significant differences between the 2 groups (treo/thio/flu vs Bu/
Cy/ATG) in terms of the incidence of acute GVHD, chronic GVHD, TRM,
and graft failure, although a trend toward higher TRM was seen with the
treo/thio/flu regimen.

Graft-versus-Host Disease
James L.M. Ferrara, John E. Levine, Pavan Reddy, and Ernst Holler
Lancet. 2009 May 2; 373(9674): 1550–1561

The number of allogeneic hematopoietic cell transplantations (HCT)
continues to increase with more than 25,000 allogeneic transplantations
performed annually. The graft-versus leukemia/ tumor (GVL) effect during
allogeneic HCT effectively eradicates many hematological malignancies.
The development of novel strategies that use donor leukocyte infusions,
non-myeloablative conditioning and umbilical cord blood (UCB)
transplantation have helped expand the indications for allogeneic HCT
over the last several years, especially among older patients. Improvements
in infectious prophylaxis, immunosuppressive medications, supportive care
and DNA-based tissue typing have also contributed to improved outcomes
after allogeneic HCT. Yet the major complication of allogeneic HCT, graft-
versus-host disease (GVHD), remains lethal and limits the use of this
important therapy. Given current trends, the number of transplants from
unrelated donors is expected to double within the next five years,
significantly increasing the population of patients with GVHD. In this
seminar we review advances made in identifying the genetic risk
factors and pathophysiology of this major HCT complication, as well
as its prevention, diagnosis and treatment.

Non-HLA Genetics—Despite HLA identity between a patient and donor,
approximately 40% of patients receiving HLA-identical grafts develop
acute GVHD due to genetic differences that lie outside the HLA loci,
or “minor” histocompatibility antigens (HA). Some minor HAs, such as HY
and HA-3, are expressed on all tissues and are targets for both GVHD
and GVL. Other minor HAs, such as HA-1 and HA-2, are expressed most
abundantly on hematopoietic cells (including leukemic cells) and may
therefore induce a greater GVL effect with less GVHD. Polymorphisms
in both donors and recipients for cytokines that are involved in the
classical `cytokine storm’ of GVHD have been implicated as risk factors
for GVHD. Tumor Necrosis Factor (TNF)-α, Interleukin 10 (IL-10),
Interferon-γ (IFNγ) variants have correlated with GVHD in some, but
not all, studies. Genetic polymorphisms of proteins involved in innate
immunity, such as nucleotide oligomerization domain 2 and Keratin 18
receptors, have also been associated with GVHD.

Future strategies to identify the best possible transplant donor will
probably incorporate both HLA and non-HLA genetic factors. Skin is most
commonly affected and is usually the first organ involved, often coinciding
with engraftment of donor cells. The characteristic maculopapular rash is
pruritic and can spread throughout the body, sparing the scalp. In severe
cases the skin may blister and ulcerate. Apoptosis at the base of epidermal
rete pegs is a characteristic pathologic finding. Other features include
dyskeratosis, exocytosis of lymphocytes, satellite lymphocytes adjacent
to dyskeratotic epidermal keratinocytes, and a perivascular lymphocytic
infiltration in the dermis.

Gastrointestinal tract involvement of acute GVHD usually presents as
diarrhea but may also include vomiting, anorexia, and/or abdominal pain
when severe. The diarrhea of GVHD is secretory and often voluminous
(greater than two liters per day). Bleeding, which carries a poor prognosis,
occurs as a result of mucosal ulceration but patchy involvement of the
mucosa often leads to a normal appearance on endoscopy.

The incidence of the severity of acute GVHD is determined by the extent
of involvement of  three principal target organs. The overall grades are
classified as I (mild), II (moderate), III (severe) and IV (very severe). Severe
GVHD carries a poor prognosis, with 25% long term survival for grade III and
5% for grade IV. The incidence of acute GVHD is directly related to the
degree of mismatch between HLA proteins and ranges from 35-45% in
recipients of full matched sibling donor grafts to 60-80% in recipients of
one-antigen HLA mismatched unrelated donor grafts. The same degree
of mismatch causes less GVHD using UCB grafts and incidence of acute
GVHD is lower following the transplant of partially matched UCB units
and ranges from 35-65%.

Two important principles are important to consider regarding the
pathophysiology of acute GVHD. First, acute GVHD reflects exaggerated
but normal inflammatory mechanisms mediated by donor lymphocytes infused
into the recipient where they function appropriately, given the foreign
environment they encounter. Second, the recipient tissues that stimulate
donor lymphocytes have usually been damaged by underlying disease,
prior infections, and the transplant conditioning regimen. As
a result, these tissues produce molecules (sometimes referred to as
“danger” signals) that promote the activation and proliferation of donor
immune cells.  Based largely on experimental models, the development
of acute GVHD can be conceptualized in three sequential steps or phases:
(1) activation of the APCs; (2) donor T cell activation, proliferation,
differentiation and migration; and (3) target tissue destruction.

Alemtuzumab is a monoclonal antibody that binds CD52, a protein
expressed on a broad spectrum of leukocytes including lymphocytes,
monocytes, and dendritic cells. Its use in GVHD prophylaxis in a
Phase II trial decreased the incidence of acute and chronic GVHD
following reduced intensity transplant.98 In two prospective studies,
patients who received alemtuzumab rather than methotrexate showed
significantly lower rates of acute and chronic GVHD, but experienced
more infectious complications and higher rates of relapse, so that there
was no overall survival benefit. Alemtuzumab may also contribute to
graft failure when used with minimal intensity conditioning regimens.

An alternative strategy to TCD attempted to induce anergy in donor
T cells by ex vivo antibody blockade of co-stimulatory pathways prior
to transplantation. A small study using this approach in haploidentical
HCT recipients was quite encouraging, but has not yet been replicated.
Thus the focus of most prevention strategies remains  pharmacological
manipulation of T cells after transplant.

Administration of anti-T cell antibodies in vivo as GVHD prophylaxis
has also been extensively tested. The best studied drugs are anti-
thymocyte globulin (ATG) or antilymphocyte globulin (ALG) preparations.
These sera, which have high titers of polyclonal antibodies, are made
by immunizing animals (horses or rabbits) to thymocytes or lymphocytes,
respectively. A complicating factor in determining the role of these
polyclonal sera in transplantation is the observation that even different
brands of the same class of sera exert different biologic effects. However,
the side effects of ATG/ALG infusions are common across different
preparations and include fever, chills, headache, thrombocytopenia
(from cross-reactivity to platelets), and, infrequently, anaphylaxis. In
retrospective studies, rabbit ATG reduced the incidence of GVHD in
related donor HSCT recipients without appearing to improve survival.
In recipients of unrelated donor HSCT, addition of ALG to standard
GVHD prophylaxis effectively prevented severe GVHD, but did not
result in improved survival because of increased infections. In a long
term follow-up study, however, pretransplant ATG provided significant
protection against extensive chronic GVHD and chronic lung dysfunction.

As allogeneic transplantation becomes an increasingly attractive therapeutic
option, the need for novel approaches to GVHD has accelerated. The
number of patients receiving transplants from unrelated donors is
expected to double in the next five years, significantly increasing
the population of patients with GVHD. The advent of RIC regimens
has reduced transplant-related mortality and lengthened the period
during which acute GVHD may develop (many new cases present up
to day 200) and the need for close monitoring of patients in this period
has increased. Patients have often returned to the care of their primary
hematologists by this time, increasing the need for these physicians to
collaborate with transplant specialists in the management of GVHD and
its complications.

Identification of biomarkers for GVHD with diagnostic (and possibly
prognostic) significance may eventually make the treatment of GVHD
preemptive rather than prophylactic. The use of cellular component therapy,
such as regulatory T cells that have been expanded ex vivo. will also
enter clinical trials in the near future, but the extensive infrastructure
required for such cellular approaches will likely limit their use initially.

Immunomodulatory Effects of Palifermin (Recombinant Human
Keratinocyte Growth Factor) in 
an SLE-Like Model of Chronic
Graft-Versus-Host Disease

C. A. Ellison, Y. V. Lissitsyn, I. Gheorghiu & J. G. Gartner
Scandinavian Journal of Immunology 2011; 75, 69–76

Keratinocyte growth factor (KGF) promotes epithelial cell proliferation
and survival. Recombinant human KGF, also known as palifermin, protects
epithelial cells from injury induced by chemicals, irradiation and acute murine
graft versus-host disease (GVHD). Findings from our studies and others
have shown that palifermin also has immunomodulatory properties. In a
model of acute GVHD, we showed that it shifts the immune response
from one in which Th1 cytokines dominate to mixed Th1 and Th2 cytokine
profile. Using the DBA⁄ 2 fi (C57BL ⁄ 6 · DBA⁄ 2)F1-hybrid model of chronic,
systemic lupus erythematosus-like GVHD, we showed that palifermin
treatment is associated with higher levels of Th2 cytokines, the production
of anti-nuclear antibodies, cryoglobulinemia and the development of more
severe pathological changes in the kidney. The aim of our current study
was to gain a better understanding of the immunobiology of KGF by
further characterizing the palifermin-mediated effects in this model of
chronic GVHD. Because the pathological changes we observed resemble
those seen in thymic stromal lymphopoietin (TSLP) transgenic mice, we
had originally hypothesized that palifermin might augment TSLP levels.
Surprisingly, we did not observe an increase in thymic

TSLP mRNA expression in palifermin-treated recipients. We did, however,
observe some differences in the percentages of CD4+CD25+Foxp3+
regulatory T cells in the spleen at some time points in palifermin-treated
recipients. Most importantly, we found that TGFβ levels were higher in
palifermin-treated recipients early in the GVH reaction, raising the
possibility that KGF might indirectly induce the development of fibrosis
and glomerulonephritis through a pathway involving TGFβ.

Keratinocyte growth factor (KGF) is an epithelial cell growth factor that is
produced by both mesenchymal cells and intraepithelial cdT cells. It is
also known as fibroblast growth factor 7. Its receptor, (KGFR⁄FGF7R), an
alternatively spliced form of FGFR2 ⁄ bek, is found on epithelial cells in
the intestine, mammary glands, ovaries and urinary tract, and on
hepatocytes, keratinocytes and alveolar type II cells. Previously, it
was shown that recombinant human KGF, also known as palifermin,
can protect the lung, bladder or intestine from chemical- or irradiation-
induced injury. This has been attributed to the ability of KGF to reduce
oxidative damage and enhance DNA repair.

Our own studies have provided a better understanding of the immuno-
biological properties of KGF in pathologically distinct models of systemic
disease driven by intense immunological and inflammatory responses.
The acute GVHD that develops in the C57BL ⁄ 6 fi (C57BL ⁄ 6 · DBA⁄ 2)F1-
hybrid model is characterized by the activation of alloreactive donor T cells,
the production of Th1 cytokines and tissue injury in the skin, gastrointestinal
tract, liver, thymus and lung, where epithelia are present. Injury to the
intestinal mucosa permits the translocation of endotoxin into the system,
which, if untreated, leads to the development of endotoxemic shock. We
showed that palifermin treatment protects recipients from epithelial
cell injury, endotoxemia and morbidity in GVH mice. Palifermin also
shifts the immune response away from one that is predominated by Th1
cytokines towards a profile of mixed Th1 and Th2 cytokines, with a
preponderance of Th2 cytokines. The DBA⁄ 2 fi (C57BL ⁄ 6 · DBA⁄ 2)F1-
hybrid model of chronic GVHD is characterized by pathological changes
resembling those seen in systemic lupus erythematosus (SLE). Using this
model, we showed that palifermin treatment augments the production of Th2
cytokines such as IL-4, IL-5 and IL-13 and obviates IFN-c production. Both
untreated and palifermin-treated recipients developed pathological changes
in the kidney, but these changes were more severe in palifermin-treated
recipients. Some of the changes that developed in the palifermin-treated
recipients resemble those seen in thymic stromal lymphopoietin (TSLP)
transgenic mice. These similarities include the presence of ANA in the
sera, the development of cryoglobulinemia and the development of
glomerulonephritis featuring the deposition of immune complexes
consisting of IgG, IgA, IgM and C3 in the mesangium and the glomerular
capillaries. This led us to hypothesize that treating the recipient mice with
palifermin might induce TSLP expression in this model.

In this study, we were interested in determining whether palifermin
treatment was indeed associated with increased TSLP expression.
We were also interested in knowing whether palifermin treatment
changes the percentage of CD4+CD25+FoxP3+ cells in the spleen,
because palifermin treatment has been associated with increased
percentages of CD4+CD25+FoxP3+ cells in other studies including
our own. Lastly, we wished to study the effect of palifermin treatment
on TGFb levels, because this cytokine is known to play a pivotal role
in the development of glomerulonephritis.

We studied the histopathological changes to confirm that the pathological
changes seen in the kidney in this study were the same as those reported
by us previously.We examined kidney sections from both untreated and
palifermin-treated recipients. In these experiments, we were able to
reproduce findings from an earlier study that showed that palifermin-
treated recipients mice in this model of chronic GVHD develop a severe,
extracapillary proliferative glomerular nephritis characterized by epithelial
crescents and hyaline thrombi. These changes were associated with higher
levels of protein in the urine and the development of ascites, presumably
related to the development of nephrotic syndrome, as a consequence
of glomerular injury.

Pathological changes in the kidney

Pathological changes in the kidney. (A) shows a section from a BDF1-hybrid control
mouse that did not receive a graft. (B) shows increased epithelial cellularity within a
glomerulus from an untreated recipient with chronic graft-versus-host disease, on
day 50. No crescents were observed in sections from this group of recipients.
(C and D) show examples of pathological changes observed in kidneys from
palifermin-treated recipients on day 50. Arrows indicate examples of crescentic
glomerulonephritis and the development of protein casts within tubular lumena.
(E and F) show examples of the hyaline thrombi (arrows) seen in the glomeruli
in kidney sections from palifermin-treated recipients on day 50. All sections
were stained with haematoxylin and eosin except for that shown in (F), which
was stained with Masson Trichrome. The concentration of protein measured in
the urine is shown in the lower left corner of each photomicrograph. Original
magnification: ·200 (B–E) and ·400 (A and F).

TGFβ is a highly pleiotropic cytokine with three isoforms, TGFβ1, TGFβ2 and
TGFβ3 . Nearly, all cells have receptors for at least one of these isoforms,
but cells of the immune system primarily express TGFβ1. This cytokine
was implicated in the development of experimental glomerulonephritis in
experiments in which rats were treated with antiserum directed against
TGFβ1. The ability of palifermin to induce TGFβ release and reverse
limited airflow was demonstrated in a mouse model of emphysema. The
authors further showed that palifermin induced the release of TGFβ1
from primary cultures of mouse alveolar type 2 cells. Our results show
that palifermin treatment is associated with a rise in splenic TGFβ levels
during the first month of the GVH reaction. It is possible that by inducing
TGFβ production shortly after transplantation, palifermin treatment is able
to promote the development of the severe, crescentic glomerulonephritis
that we observed at later time points. As such, our findings raise the
possibility that endogenous KGF might play a role in the development
of glomerulonephritis and ⁄ or other autoimmune phenomena associated
with chronic GVHD and ⁄ or SLE.

T cells, murine chronic graft-versus-host disease and autoimmunity

Robert A. Eisenberg, Charles S. Via
Journal of Autoimmunity 39 (2012) 240e247

The chronic graft-versus-host disease (cGVHD) in mice is characterized by
the production of autoantibodies and immunopathology characteristic of
systemic lupus erythematosus (lupus). The basic pathogenesis involves
the cognate recognition of foreign MHC class II of host B cells by alloreactive
CD4 T cells from the donor. CD4 T cells of the host are also necessary for
the full maturation of host B cells before the transfer of donor T cells.
CD8 T cells play critical roles as well. Donor CD8 T cells that are highly
cytotoxic can ablate or prevent the lupus syndrome, in part by killing
recipient B cells. Host CD8 T cells can reciprocally downregulate donor
CD8 T cells, and thus prevent them from suppressing the autoimmune
process. Thus, when the donor inoculum contains both CD4 T cells and
CD8 T cells, the resultant syndrome depends on the balance of activities
of these various cell populations. For example, in one cGVHD model
(DBA/2 (C57BL/6xDBA/2)F1, the disease is more severe in females, as
it is in several of the spontaneous mouse models of lupus, as well as in
human disease. The mechanism of this female skewing of disease
appears to depend on the relative inability of CD8 cells of the female host
to downregulate the donor CD4 T cells that drive the autoantibody response.
In general, then, the abnormal CD4 T cell help and the modulating roles
of CD8 T cells seen in cGVHD parallel the participation of T cells in
genetic lupus in mice and human lupus, although these spontaneous
syndromes are presumably not driven by overt alloreactivity.

Systemic lupus erythematosus (SLE) is characterized by a spectrum of auto-
antibodies that targets multiple normal cellular components, particularly
nucleic acids or proteins that are physiologically bound to nucleic acids.
Although SLE is highly diverse in its manifestations, a common theme
is the loss of B cell tolerance to these cellular autoantigens. More than
for any other human condition, several spontaneously arising mouse
models for SLE have been described, beginning with the New Zealand
strains in 1959. These models are largely genetic. In some cases, an
individual gene such as fas or Yaa plays a major role in driving the loss
of tolerance. However, in general the genetic contribution is complex and
involves multiple loci, which are not yet fully defined.

Despite extensive investigations, the failures in immunoregulation that
underlie the genetic SLE models remain poorly understood. It is not known
for sure which B cell tolerance checkpoints are breached in a given model,
and why. The autoantibody response to DNA, Sm, and other autoantigens
resembles the normal response to exogenous antigens: it involves clonal
expansion, somatic mutation, and a pattern of isotype use characteristic of
a T-cell dependent immunization. Thus the cellular dynamics of the response
may be basically normal. Yet the B-cell repertoire is abnormally autoreactive.

In this review we wish to focus more on the role of the T cell in SLE. As
stated above, the loss of B cell tolerance in SLE does appear in general
to require the participation of T cells. Multiple T cells abnormalities have
been described in human and in murine SLE, although in most cases it is
not clear if these are primary or secondary manifestations. Nevertheless,
it is striking how difficult it has been to demonstrate definitively the specificity
of the T cells that provide help for autoantibody production.

The key cellular mechanism in the cGVHD that results in the loss of B cell
tolerance and the production of the autoantibodies typical of SLE is the
cognate interaction of CD4 T cells with an MHC class II determinant on
the B cell surface. A variety of protocols have achieved this interaction.
In general, either the donor/recipient strains are paired in such away
that they only differ at the MHC class II loci, or the CD4 cells are isolated
free of CD8 cells that would recognize MHC class I. If the allorecognition
involves both CD4 T cell interaction with MHC II and CD8 interaction with
MHC I, an acute GVHD occurs, which is immunosuppressive, rather than
immunostimulatory. The DBA/2 (C57BL/6 DBA/2)F1 (B6D2F1) and the
BALB/c (BALB/c A/J)F1 models are exceptions to this rule. The former
has been investigated extensively for a deficiency in CD8 cytotoxic

The MHC class II recognition may be at either the I-A or the I-E locus.
However, the autoantibody specificities seen and the degree of immuno-
pathology differ depending on the locus targeted. In one set of experiments,
F1 mice were bred between either B6 or coisogenic bm12 mice and
B10.A(2R) or B10.A(4R) MHC recombinant congenics. The MHC class II
of B6 is I-Ab, while that of bm12 is I-Abm12. These two alleles differ by
only three amino acids, which is sufficient for a full strength MLR (mixed
lymphocyte reaction) between the two strains. Otherwise B6 and bm12
are identical. B10.A(2R) and B10.A(4R) differ only by the expression of
I-E in the former strain, but not in the latter strain. Thus, donor/recipient
combinations could be employed that provided for allogeneic differs only
at I-A, only at I-E, or at both loci.

Results from Busser et al. delineate requirements for this MHC class II
recognition. Utilizing several transgenic mouse strains that express a
more or less constricted CD4 autoreactive repertoire, they showed that
a diverse repertoire was essential to the production of SLE autoantibodies
by MHC II recognition. On the other hand, the non-specific, early polyclonal
B cell activation phase of cGVHD occurred even with a limited CD4 repertoire.

Figure not shown. Chronic GVHD in bm12 C57BL/6 mice. The MHC of the
bm12 donor differs from the MHC of the C57BL/6 recipient just in three
amino acids in the I-A class II molecule. Thus donor CD4 T cells recognize
MHC IIþ B cells as foreign. Donor CD8 T cells see only self MHC I. All T
cells do not express MHC II. Polyclonal activation and specific lupus
autoantibody responses ensue..

Lupus can result from unchecked CD4 T cell cognate help to a polyclonal
population of B cells. CD8 T cells can downregulate this CD4 driven B-cell
hyperactivity through CD8 CTL effectors and can maintain remission,
possibly through memory CD8 T cells. Whether CD8 CTL actually prevent
lupus in normals and fail in lupus prone individuals is not known; however,
data from the P F1 model suggest that therapeutic induction of CD8 CTL
and possibly long term memory cells may be beneficial in preventing or
limiting disease expression. The potential major role played by either
IFNa and IL-21 in both lupus expression and CD8 CTL function remains
to be further defined, but already these cytokines are being targeted in
human or murine lupus.

It is not surprising that the T cells have been shown to have diverse roles in
the autoimmune cGVHD in mice. Donor CD4 T cells drive the host B cell
activation, while host CD4 T cells are required to mature these B cells prior
to their encounter with donor T cells. Donor CD4 T cells also help activate
donor CD8 T cells, which in turn can downregulate or even ablate the
autoimmune response. Donor CD4 T cells license host DC cells, which in
turn can interact with donor CD8 T cells. Host CD8 T cells can suppress
the activity of donor CD8 T cells, and thereby favor the development of
the lupus syndrome. Although the precise mechanisms of T cell participation
in spontaneous lupus are still being defined, it seems reasonable to probe
these syndromes in humans and in mice for T cell mechanism that have
been shown to participate in cGVHD, CD4-B cell interactions almost
certainly are central to the pathogenesis of spontaneous lupus, and they
have been a target of investigation for several decades. If we understood
the peptide specificity of the alloreactive CD4 T cells that drive the formation
of the characteristic lupus autoantibodies, we would have a much clearer
idea where to look for such epitopes in spontaneous disease. Much less
is known about the other T cell activities defined in cGVHD, particularly
those that involve CD8 T cells. This area should invite further detailed
investigation. For example, the striking role of CD8 T cells in the stronger
female disease in the DBA BDF1 model clearly demands that similar
mechanisms be sought for in spontaneous disease.

Understanding Chronic GVHD from Different Angles

Bruce Blazar, Eric S. White, Daniel Couriel
Biol Blood Marrow Transplant 18:S184-S188, 2012

Whereas acute graft-versus-host disease (aGVHD) rates have decreased
with more intensive GVHD preventive agents and use of single and double
umbilical cord blood units as a source of donor cells in adult recipients,
significant chronic GVHD (cGVHD) rates unexpectedly have remained high.
Moreover, granulocyte colony stimulating factor mobilized peripheral blood
stem cell grafts have been associated with an increased overall risk of
cGVHD. As such, cGVHD has emerged as a primary cause of morbidity
and mortality following allogeneic hematopoietic stem cell transplantation.
Progress in developing cGVHD interventional strategies has been hampered
by variable onset and clinical and pathological manifestations of cGVHD, now
better defined by the National Institutes of Health (NIH) consensus conference,
and a dearth of preclinical models that closely mimic the conditions in which
cGVHD is generated and manifested. Although the exact causes of cGVHD
remain unknown, higher antibody levels have been associated with auto-
immunity and implicated in cGVHD. Newly diagnosed patients with
extensive cGVHD had elevated soluble B cell activating factor levels and
anti-double-strand DNA antibodies were found, which was associated with
higher circulating levels of pregerminal center (GC) B cells and post-GC
plasmablasts. B cells from cGVHD patients were hyperresponsive to Toll-like
receptor-9 signaling and have up-regulated CD86 levels.

By using a Cy and low doses of donor T cells, aGVHD was avoided and
cGVHD with BO favored. Histologic changes were similar to the findings in
human cGVHD with peribronchiolar and perivascular cuffing and infiltration
of the airway epithelium. The liver had inflammation and lymphocytic
infiltration, along with collagen deposition. The parotid and submandibular
salivary glands displayed lymphocytic infiltrates in both the bone marrow
and cGVHD groups, likely because of transplantation conditioning.

Treatment of steroid refractory cGVHD patients with rituximab, a B cell–
depleting anti-CD20 monoclonal antibody, has shown a beneficial role in
resolution of the autoimmune disorders such as systemic lupus erythmatosus
and rheumatoid arthritis, andcGVHD, with overall response rates of 29%
to 36% for oral, hepatic, gastrointestinal, and lung cGVHD, and 60% for
cutaneous cGVHD in aggregate data from multiple trials. Thus, we recently
undertook studies to identify the presence of CD41 T helper cells and B2201
B cells in the airways of mice that had BO, tissue-specific antibodies from sera,
and alloantibody deposition in the lung and liver of cGVHD recipients. cGVHD
development was associated with IgG2c deposition in the lung and liver,
abrogated if the donor bone marrow was deficient in mature B cells or
incapable of producing antihost reactive IgG. Robust GC formation was
seen in mice with cGVHD. Alleviation of symptoms in mice that received
B cell–deficient bone marrow confirms the requirement of B cells for lung
dysfunction and inflammation and fibrosis in the lung and liver.

Given a role for IgG antibodies, allo- or auto-Ab binding to the cGVHD organs
could enable tissue destruction or the pathology could be defined by the
specific function of these secreted antibodies. Pathogenic antibody production
therefore is likely to be an important inducer of cGVHD, and targeting this
specific function of the B cells is an attractive strategy for cGVHD. Because
GC B cells display lower susceptibility to rituximab-mediated clearance, probably
because they reside in a nonoptimal environment for antibody-based depletion,
our observation that GC B cells are critical to the development of cGVHD
suggests that agents that are more effective at disrupting the GC might be
more clinically useful. Treatment with LTbR-Ig, a fusion protein that blocks
interactions between LTbR and its ligands, had a direct effect on the
symptoms of cGVHD, at least in part by blocking GC formation and suggest
that LTbR-Ig could be a potential clinical interventional strategy for prevention
and therapy of cGVHD.

Fibrosis is the end result of a number of inflammatory and other injurious events,
resulting in replacement of normal tissue with a dense extracellular matrix (ECM)
scar composed primarily of collagens. While some degree of tissue fibrosis is
considered protective (e.g. in the setting of cutaneous wound healing),
exaggerated or unrelenting ECM deposition with replacement of the normal
tissue architecture is considered pathologic. Fibroproliferative disorders as
a class involving multiple organs (e.g. cGVHD following hematopoietic stem
cell transplant [affecting up to 30% of recipients surviving more than 100 days,
scleroderma [estimated to affect 70,000 in the US], idiopathic pulmonary fibrosis
[estimated to affect 200,000 in the US], hepatic cirrhosis [estimated to affect
up to 400,000 in the US], and renal fibrosis due to diabetic nephropathy and
other causes [estimated to affect over 400,000 in the US]) are a major cause
of morbidity and mortality. Combined, these disorders alone are conservatively
estimated to affect approximately 1 in 300 persons in the United States. When
coupled with a host of other disorders in which tissue fibrosis contributes to
morbidity (e.g. fibroproliferative acute respiratory distress syndrome,
hypersensitivity pneumonitis, solid organ transplant rejection), that estimate
is likely to be much greater.

Wound healing occurs by a highly orchestrated, complex process that has
been well defined. In general, wound repair occurs in 4 stages which overlap
considerably: clotting/coagulation, inflammation, fibroproliferation, and tissue
remodeling. The initial injury leads to a local disruption of epithelial and
endothelial barriers resulting in the elaboration of inflammatory mediators and
extravasation of cells and plasma proteins that serve to achieve hemostasis
and provide a provisional fibrin-rich matrix for the influx of inflammatory and
other reparative cells. Simultaneously, platelet degranulation provides a local
“boost” of vasodilators, growth factors, and ECM proteins that aid in the wound
healing response. Inflammatory cell influx occurs next, with polymorphonuclear
leukocytes (PMNs) arriving first. Following PMN degranulation, mononuclear
cells (macrophages and lymphocytes) arrive next and, along with PMN derived
products, sterilize and remove foreign materials from the wound. This process
also results in the elaboration of cytokines and chemokines designed to
augment the inflammatory response, to promote angiogenesis (allowing for
enhanced nutrient and oxygen delivery to the wound bed), and to recruit
fibroblasts to the wound bed. Fibroblast recruitment and transdifferentiation to
myofibroblasts (or recruitment of already-differentiated myofibroblasts or
fibroblast precursors; this point is still controversial) marks the fibroproliferative
stage, with the result being the elaboration of ECM proteins (collagens,
fibronectins) to repair the tissue defect.

Vorinostat plus tacrolimus and mycophenolate to prevent graft-versus-host
disease after related-donor reduced-intensity conditioning allogeneic
stem-cell transplantation: a phase 1/2 trial

Sung Won Choi, T Braun, L Chang, JLM Ferrara, A Pawarode, et al.
Lancet Oncol 2014; 15: 87–95

Background Acute graft-versus-host disease (GVHD) remains a barrier to more
widespread application of allogeneic hemopoietic stem-cell transplantation.
Vorinostat is an inhibitor of histone deacetylases and was shown to attenuate
GVHD in preclinical models. We aimed to study the safety and activity of
vorinostat, in combination with standard immunoprophylaxis, for prevention of
GVHD in patients undergoing related-donor reduced-intensity conditioning
hemopoietic stem-cell transplantation. Methods Between March 31, 2009,
and Feb 8, 2013, we did a prospective, single-arm, phase 1/2 study at two
centers in the USA. We recruited adults (aged ≥18 years) with high-risk
hematological malignant diseases who were candidates for reduced-intensity
conditioning hemopoietic stem-cell transplantation and had an available 8/8
or 7/8 HLA matched related donor. All patients received a conditioning regimen
of fl udarabine (40 mg/m² daily for 4 days) and busulfan (3·2 mg/kg daily for
2 days) and GVHD immunoprophylaxis of mycophenolate mofetil (1 g three
times a day, days 0–28) and tacrolimus (0·03 mg/kg a day, titrated to a goal
level of 8–12 ng/mL, starting day –3 until day 180). Vorinostat (either 100 mg
or 200 mg, twice a day) was initiated 10 days before haemopoietic stem-cell
transplantation until day 100. The primary endpoint was the cumulative
incidence of grade 2–4 acute GVHD by day 100. This trial is registered with
ClinicalTrials.gov, number NCT00810602.
Findings 50 patients were assessable for both toxic effects and response;
eight additional patients were included in the analysis of toxic effects. All
patients engrafted neutrophils and platelets at expected times after
hemopoietic stem-cell transplantation. The cumulative incidence of grade
2–4 acute GVHD by day 100 was 22% (95% CI 13–36). The most common
non-hematological adverse events included electrolyte disturbances (n=15),
hyperglycemia (11), infections (six), mucositis (four), and increased activity
of liver enzymes (three). Non-symptomatic thrombocytopenia after
engraftment was the most common hematological grade 3–4 adverse
event (nine) but was transient and all cases resolved swiftly.
Interpretation Administration of vorinostat in combination with standard
GVHD prophylaxis after related-donor reduced-intensity conditioning
hemopoietic stem-cell transplantation is safe and is associated with a
lower than expected incidence of severe acute GVHD. Future studies
are needed to assess the effect of vorinostat for prevention of GVHD in
broader settings of hemopoietic stem-cell transplantation.

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Cell Research News – What’s to Follow?

Larry H. Bernstein, MD, FCAP, Reporter

Leaders in Pharmaceutical Intelligence



Stem Cell Research ‘Holy Grail’ Uncovered, Thanks to Zebrafish

By Estel Grace Masangkay

With help from the zebrafish, a team of Australian researchers has uncovered how
hematopoietic stem cells (HSC) renew themselves.

HSCs refers to stem cells present in the blood and bone marrow that are used 
for  the replenishment of the body’s supply of blood and immune cells – 

  • in transplants for leukemia and myeloma.
  • Stem cells have the potential to transform into vital cells

    including muscle, bone, and blood vessels.

Understanding how HSCs form and renew themselves has potential application in the
treatment of

  • spinal cord injuries
  • degenerative disorders
  • diabetes.

Professor Peter Currie, of the Australian Regen Med Institute at Victoria’s Monash
University, led a research team to discover a crucial part of HSC’s development. Using 
a high-resolution microscopy, Prof. Curie’s team 

  • caught zebrafish embyonic SCs on film as they formed. 
  • the researchers were studying muscle mutations in the aquatic animal.

“Zebrafish make ESCs in exactly the same way as humans do, but their embryos and
larvae develop free living, but the larvae are both free swimming and transparent, so one could see every cell in the body forming, including ESCs,” explained Prof. Currie.

The researchers noticed in films that a

  •  ‘buddy cell’ came along to help the ESCs form.

Called endotome cells, 

  • they aided pre-ESCs to turn into ESCs.  

Prof. Currie said that endotome cells act as helper cells for pre-ESCs , 

  • helping them progress to become fully fledged stem cells.

The team not only

  • identified some of the cells and signals 
  • required for ESC formation, but also 
  • pinpointed the genes required 
  • for endotome formation in the first place.

The next step for the researchers is to 

  • locate the signals present in the endotome cells 
  • that trigger ESC formation in the embryo. 

This may provide clues for developing

  • specific blood cells on demand for blood-related disorders. 

Professor Currie also pointed out the discovery’s potential for 

  • correcting genetic defects in the cell and 
  • transplanting them back in the body to treat disorders.

The team’s work was published in the international journal Nature.


Jell-O Like Biomaterial Could Hold Key to Cancer Cell Destruction

by Estel Grace Masangkay

Scientists from Penn State University reported that a biomaterial made of tiny 
molecules was able to attract and destroy cancer cells.

Professor Yong Wang and bioengineering faculty at Penn State, built the 
tissue-like biomaterial to accomplish what chemotherapy could not –

  • kill every cancer cell without leaving
  • the possibility of a recurrence.

Prof. Wang and team built polymers 

  • from tiny molecules called monomers. They
  • then wove the polymers into 3D networks 

called hydrogels. Hydrogel is soft and flexible, 
like Jell-O, and it contains a lot of water, and

  • can be safely put into the body, unlike 

other implants that the body often tries 

  • to get rid of through the immune response.

“We want to make sure the materials we are using are compatible in the body.”

The researchers 

  • attached aptamers to the hydrogels, 
  • which release bio-chemical signal-only molecules 
  • that draw in cancer cells. 

Once attracted, the cancer cells are entrapped in the Jell-O-like substance. 

What happens next is 

  • an oligonucleotide binds to the protein-binding site of the aptamer 
  • and triggers the release of anticancer drugs at the proper time.

“Once we trap the cancer cells, we can deliver anticancer drugs 

  • to that specific location to kill them. 

This technique would help avoid the need for systemic medications that kill not only cancer cells, but normal cells as well. Systemic chemotherapy drugs

  • make patients devastatingly sick and possibly 
  • leave behind cancer cells to wreak havoc another day

If our new technique has any side effects at all, it would be only local side 
effects and not whole-body systemic side effects,” explained Prof. Wang.

The initial results of the research were published by Prof. Wang in the 
Journal of the American Chemical Society in 2012. Prof. Wang also shared 
the latest results of his work at the Society for Biomaterials Meeting &
 Exposition in April this year.


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microRNA called miR-142 involved in the process by which the immature cells in the bone marrow give rise to all the types of blood cells, including immune cells and the oxygen-bearing red blood cells


Reporter: Aviva Lev-Ari, PhD, RN






Master Key




Hornstein group

Dr. Elik Chapnik, Natali Rivkin and Dr. Eran Hornstein
It takes only a tiny key to open a door wide or set large machinery in motion. Dr. Eran Hornstein of the Weizmann Institute’s Molecular Genetics Department and his team recently discovered such a key – one that unlocks the cellular machinery for producing mature blood cells. That key is a minuscule, hairpin-shaped RNA belonging to a class of RNA strands so small they had long been ignored. Even now, these so-called microRNAs are too often thought to be secondary to the cell’s major processes. The new findings suggest that microRNAs can also be master keys, putting several vital processes into motion at once.
In collaboration with Prof. Steffen Jung of the Immunology Department and his coworkers Dr. Elik Chapnik, Natali Rivkin and Dr. Alexander Mildner, Hornstein discovered that a microRNA called miR-142 was involved in the process by which the immature cells in the bone marrow give rise to all the types of blood cells, including immune cells and the oxygen-bearing red blood cells. In fact, an early hint for the importance of this microRNA had been documented years earlier, in 1989, as it plays a role in a type of B-cell leukemia. But back then, before the era of genomics and before the microRNA revolution, it was thought to be a protein-coding gene.


wt megakaryocyte

Wild type megakaryocyte
The Weizmann researchers looked at a broad lineage of myeloid cells – a group that includes the red blood cells and the platelets that make our blood clot. They were looking for cells in which miR-142 is instrumental, and that could provide a clear insight into its function. Their initial analysis pointed to megakaryocytes as the ideal experimental model.
Megakaryocytes are very large cells in the bone marrow that generate platelets by budding off bits of their internal cytoplasm. The development of megakaryocytes and their ability to function as they mature depends on a strong, malleable internal structure – the cytoskeleton. In a series of experiments on mouse megakaryocytes, the researchers found that miR-142 is essential to the proper formation of the primary building material of the cytoskeleton – actin fibers. When miR-142 activity was halted, the production of actin was deregulated, and the megakaryocytes were not able to mature and produce platelets.
KO blood cell

Megakaryocyte in which the microRNA mir-142 has been knocked out
Using a large array of techniques in their labs, the researchers were able to reveal the precise activities of miR-142. Their findings, which were recently published ineLife, show that miR-142 is, indeed, a master key that turns on and off a number of different cellular processes; these are crucial to actin production and regulation. To put it another way, microRNA-142 is a “hub” in the cellular network of pathways that keeps the cell growing, dividing, developing and functioning.
According to Hornstein, the impact of microRNA-142 and its mechanism may even go all the way back to the first blood cells in the embryo. In addition, miR-142 malfunctions are likely to show up in certain clotting disorders; but the findings hint that the same miRNA gene may be involved in any number of other blood diseases. Hornstein: “This model for blood cell development is very informative and fruitful. Together with Jung we have already characterized four different cell types in which this miRNA is influential, which is very exciting.”
The implications are clear for microRNA research, says Hornstein, helping cast microRNA in a new light: they can no longer be seen as mere helper molecules that “fine-tune” the cellular pathways; they are also key players with the power to direct the development of the cell.
Dr. Eran Hornstein’s research is supported by the Kekst Family Institute for Medical Genetics; the David and Fela Shapell Family Center for Genetic Disorders Research; the Crown Human Genome Center; the Yeda Sela Center; the Nella and Leon Benoziyo Center for Neurological Diseases; the Y. Leon Benoziyo Institute for Molecular Medicine; the Helen and Martin Kimmel Institute for Stem Cell Research; the Nathan, Shirley, Philip and Charlene Vener New Scientist Fund; the Julius and Ray Charlestein Foundation; the Celia Benattar Memorial Fund for Juvenile Diabetes; the Wolfson Family Charitable Trust; the Legacy Heritage Fund; the Adelis Foundation; the Minna-James-Heineman Stiftung; Dr. Sidney Brenner and Friends; Maria Halphen, France; and the estate of Fannie Sherr. Dr. Hornstein is the incumbent of the Helen and Milton A. Kimmelman Career Development Chair.

Prof. Steffen Jung’s research is supported by the Leir Charitable Foundations; the Leona M. and Harry B. Helmsley Charitable Trust; the Maurice and Vivienne Wohl Biology Endowment; the Adelis Foundation; Lord David Alliance, CBE; the Wolfson Family Charitable Trust; the estate of Olga Klein Astrachan; and the European Research Council.



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Epilogue: Volume 4 – Translational, Post-Translational and Regenerative Medicine in Cardiology

  • Larry H Bernstein, MD, FCAP, Author and Curator, Volume Four, Co-Editor
  • Justin Pearlman, MD, PhD, FACC, Content Consultant for Series A: Cardiovascular Diseases
  • Aviva Lev-Ari, PhD, RN, Co-Editor of Volume Four and Editor-in-Chief, BioMed e-Series


This completes Chapter 4 in two parts on the most dynamic developments in the regulatory pathways guiding cardiovascular dynamics and function in health and disease.  I have covered key features of these in two summaries, so I shall try to look further into important expected future directions and their anticipated implications.

1. Mechanisms of Disease

Signal Transduction: Akt Phosphorylates HK-II at Thr-473 and Increases Mitochondrial HK-II Association to Protect Cardiomyocytes

David J. Roberts, Valerie P. Tan-Sah, Jeffery M. Smith and Shigeki Miyamoto
J. Biol. Chem. 2013, 288:23798-23806.  http://dx.doi.org/ 10.1074/jbc.M113.482026

Backgound: Hexokinase II binds to mitochondria and promotes cell survival.
Results: Akt phosphorylates HK-II but not the threonine 473 mutant. The phosphomimetic T473D mutant decreases its dissociation from mitochondria induced by G-6P and increases cell viability against stress.
Conclusion: Akt phosphorylates HK-II at Thr-473, resulting in increased mitochondrial HK-II and cell protection.
Significance: The Akt-HK-II signaling nexus is important in cell survival.

HK-II Phosphorylation

HK-II Phosphorylation







It has been demonstrated that an increased level of HK-II at mitochondria is protective and is increased by protective interventions but decreased under stress.

It   has not  been fully determined   which  molecular  signals  regulate  the    level    of  HK-II at mitochondria.

Thr-473 in HK-II  is phosphorylated by Akt and this phosphorylation  leads to  increases  in  mitochondrial  HK-II binding  through inhibition  of  G-6P-dependent  dissociation, conferring resistance to oxidative stress  (Fig.     7).

Overexpression of  WTHK-II increases mitochondrial HK-II and confers protection against  hydrogen peroxide,  which  is enhanced significantly  in   HK-II   T473D-expressing  cells, whereas  NHK-II, lacking the ability to bind to mitochondria, does not confer protection.   Conversely,  mitochondrial  HK-II from mitochondria (Fig.6, and B) inhibits  the  IGF-1-mediated increase in mitochondrial HK-II and cellular protection.   Similar   dose-dependent  curves were obtained in mitochondrial   HK-II     against stress    (15–25).

Gene Expression and Genetic Variation in Human Atria

Honghuang Lin PhD, Elena V. Dolmatova MD, Michael P. Morley, PhD, Kathryn L. Lunetta PhD, David D. McManus MD, ScM, et al.
Heart Rhythm  2013   http://dx.doi.org/10.1016/j.hrthm.2013.10.051

Background— The human left and right atria have different susceptibilities to develop atrialfibrillation (AF). However, the molecular events related to structural and functional changes that
enhance AF susceptibility are still poorly understood.
Objective— To characterize gene expression and genetic variation in human atria.
Results— We found that 109 genes were differentially expressed between left and right atrial tissues. A total of 187 and 259 significant cis-associations between transcript levels and genetic
variants were identified in left and right atrial tissues, respectively. We also found that a SNP at a known AF locus, rs3740293, was associated with the expression of MYOZ1 in both left and right
atrial tissues.
Conclusion— We found a distinct transcriptional profile between the right and left atrium, and extensive cis-associations between atrial transcripts and common genetic variants. Our results
implicate MYOZ1 as the causative gene at the chromosome 10q22 locus for AF.

Long-Term Caspase Inhibition Ameliorates Apoptosis, Reduces Myocardial Troponin-I Cleavage, Protects Left Ventricular Function, and Attenuates Remodeling in Rats With Myocardial Infarction

Y. Chandrashekhar,  Soma Sen, Ruth Anway,  Allan Shuros,  Inder Anand,

J Am Col  Cardiol  2004; 43(2)   http://dx.doi.org/10.1016/j.jacc.2003.09.026

This study was designed to evaluate whether in vivo caspase inhibition can prevent myocardial contractile protein degradation, improve myocardial function, and attenuate ventricular remodeling.
Apoptosis is thought to play an important role in the development and progression of heart failure (HF) after a myocardial infarction (MI). However, it is not known whether inhibiting apoptosis can attenuate left ventricular (LV) remodeling and minimize systolic dysfunction.

A 28-day infusion of caspase inhibitor was administeredimmediately after an anterior MI. In addition, five sham-operated rats given the caspase inhibitor were compared with 17 untreated sham-operated animals to study effects in non-MI rats. Left ventricular function, remodeling parameters, and hemodynamics were studied four weeks later. Myocardial caspase 3 activation and troponin-I contractile protein cleavage were studied in the non-infarct, remote LV myocardium using Western blots. Apoptosis was assessed using immunohistochemistry for activated caspase-positive cells as well as the TUNEL method. Collagen volume was estimated using morphometry.

Caspase inhibition reduced myocardial caspase 3 activation. This was accompanied by less cleavage of troponin-I, an important component of the cardiac contractile apparatus, and fewer apoptotic cardiomyocytes. Furthermore, caspase inhibition reduced LV-weight-to- body-weight ratio, decreased myocardial interstitial collagen deposition, attenuated LV remodeling, and better preserved LV systolic function after MI.

Caspase inhibition, started soon after MI and continued for four weeks, preserves myocardial contractile proteins, reduces systolic dysfunction, and attenuates ventricular remodeling.

These findings may have important therapeutic implications in post-MI HF. J Am Col Cardiol 2004;43:295–301)

Precardiac deletion of Numb and Numblike reveals renewal of cardiac progenitors

Lincoln T Shenje,  Peter P Rainer , Gun-sik Cho , Dong-ik Lee , Weimin Zhong , Richard P Harvey , David A Kass , Chulan Kwon *,  et al.
eLife 2014.    http://dx.doi.org/10.7554/eLife.02164.001

Cardiac progenitor cells (CPCs) must control their number and fate to sustain the rapid heart growth during development, yet the intrinsic factors and environment governing these processes remain unclear. Here, we show that deletion of the ancient cell-fate regulator Numb (Nb) and its homologue Numblike (Nbl) depletes CPCs in second pharyngeal arches (PA2s) and is associated with an atrophic heart. With histological, fow cytometric and functional analyses, we fnd that CPCs remain undifferentiated and expansive in the PA2, but differentiate into cardiac cells as they exit the arch. Tracing of Nb- and Nbl-defcient CPCs by lineage-specifc mosaicism reveals that the CPCs normally populate in the PA2, but lose their expansion potential in the PA2. These fndings demonstrate that Nb and Nbl are intrinsic factors crucial for the renewal of CPCs in the PA2 and
that the PA2 serves as a microenvironment for their expansion.

2. Diagnostics and Risk Assessment

Classical and Novel Biomarkers for Cardiovascular Risk Prediction in the United States

Aaron R. Folsom
J Epidemiol 2013;23(3):158-162   http://dx.doi.org/10.2188/jea.JE20120157

Cardiovascular risk prediction models based on classical risk factors identified in epidemiologic cohort studies are useful in primary prevention of cardiovascular disease in individuals. This article briefly reviews aspects of
cardiovascular risk prediction in the United States and efforts to evaluate novel risk factors. Even though many novel risk markers have been found to be associated with cardiovascular disease, few appear to improve risk prediction
beyond the powerful, classical risk factors. A recent US consensus panel concluded that clinical measurement of certain novel markers for risk prediction was reasonable, namely,

  1. hemoglobin A1c (in all adults),
  2. microalbuminuria (in patients with hypertension or diabetes), and
  3. C-reactive protein,
  4. lipoprotein-associated phospholipase,
  5. coronary calcium,
  6. carotid intima-media thickness, and
  7. ankle/brachial index (in patients deemed to be at intermediate cardiovascular risk, based on traditional risk factors).

Diagnostic accuracy of NT-proBNP ratio (BNP-R) for early diagnosis of tachycardia-mediated cardiomyopathy: a pilot study

Amir M. Nia, Natig Gassanov, Kristina M. Dahlem, Evren Caglayan, Martin Hellmich, et al.
Clin Res Cardiol (2011) 100:887–896    http://dx.doi.org/10.1007/s00392-011-0319-y

Tachycardia-mediated cardiomyopathy (TMC) occurs as a consequence of prolonged high heart rate due to ventricular and supraventricular tachycardia. In animal models, rapid pacing induces severe biventricular remodeling with dilation and dysfunction [7]. On a cellular basis, cardiomyocytes exert fundamental morphological and functional roles.

When heart failure and tachycardia occur simultaneously, a useful diagnostic tool for early discrimination of patients with benign tachycardia-mediated  cardiomyopathy (TMC) versus major structural heart disease  (MSHD) is not available. Such a tool is required to prevent unnecessary and wearing diagnostics in patients with reversible TMC. Moreover, it could lead to early additional diagnostics and therapeutic approaches in patients with  MSHD.

A total of 387 consecutive patients with supraventricular arrhythmia underwent assessment.  Of these patients, 40 fulfilled the inclusion criteria
with a resting heart rate C100 bpm and an impaired left ventricular ejection fraction \40%. In all patients, successful electrical cardioversion was performed. At baseline, day 1 and weekly for 4 weeks, levels of NT-proBNP and echocardiographic parameters were evaluated.

NT-proBNP ratio (BNP-R) was calculated as a quotient of baseline NT-proBNP/follow-up NT-proBNP. After 4 weeks, cardiac catheterization was performed to identify patients with a final diagnosis of TMC versus MSHD.

Initial NT-proBNP concentrations were elevated and consecutively decreased after cardioversion in all patients studied. The area under the ROC curve for BNP-R to detect TMC was 0.90 (95% CI 0.79–1.00; p \ 0.001) after 1 week  and 0.995 (95% CI 0.99–1.00; p \ 0.0001) after 4 weeks. One week after cardioversion already, a BNP-R cutoff C2.3 was useful for TMC diagnosis indicated by an accuracy of 90%, sensitivity of 84% and specificity of 95%.

BNP-R was found to be highly accurate for the early diagnosis of TMC.

Omega-3 Index and Cardiovascular Health

Clemens von Schacky
Nutrients 2014; 6: 799-814;  http://dx. doi.org/10.3390/nu602099

Fish, marine oils, and their concentrates all serve as sources of the two marine omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), as do some products from algae.
To demonstrate an effect of EPA + DHA on heart health, a number of randomized, controlled intervention studies with clinical endpoints like overall mortality or a combination of adverse cardiac events were conducted in populations with elevated cardiovascular risk. One early intervention study with oily fish, rich in EPA + DHA, and some early studies with fish oil or fish oil concentrate or even purified EPA at doses ranging between 0.9 and 1.8 g/day indeed demonstrated effects in terms of fewer sudden cardiac deaths, fatal or non-fatal myocardial infarctions, or a combination of adverse cardiac events.

Recent meta-analyses found no significant benefits on total mortality, cardiovascular mortality, and other adverse cardiac or cardiovascular events [13–18]. This is in contrast to findings in epidemiologic studies, where intake of EPA + DHA had been found to correlate generally with an up to 50% lower incidence of adverse cardiac events [18,19], and in even sharper contrast to epidemiologic studies based on levels of EPA + DHA, demonstrating e.g., a 10-fold lower incidence of sudden cardiac death associated with high levels of the
fatty acids, as compared to low levels.

This seemingly contradictory evidence has led the American Heart Association to recommend “omega-3 fatty acids from fish or fish oil capsules (1 g/day) for cardiovascular disease risk reduction” for secondary prevention, whereas the European Society for Cardiology recommends “Fish at least twice a week, one of which to be oily fish”, but no supplements for cardiovascular prevention.

A similar picture emerges for atrial fibrillation: In epidemiologic studies, consumption of EPA + DHA or higher levels of EPA + DHA were associated with lower risk for developing atrial fibrillation, while intervention studies found no effect. Pertinent guidelines do not mention EPA + DHA. A similar picture also emerges for severe ventricular rhythm disturbances.

Why is it that trial results are at odds with results from epidemiology? What needs to be done to better translate the epidemiologic findings into trial results? The current review will try to shed some light on this  issue, with a special consideration of the Omega-3 Index.

Recent large trials with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the cardiovascular field did not demonstrate a beneficial effect in terms of reductions of clinical endpoints like

  • total mortality,
  • sudden cardiac arrest or
  • other major adverse cardiac events.

Pertinent guidelines do not uniformly recommend EPA + DHA for cardiac patients. In contrast,

  • in epidemiologic findings, higher blood levels of EPA + DHA were consistently associated with a lower risk for the endpoints mentioned.

The following points argue for the use of erythrocytes: erythrocyte fatty acid
composition has a low biological variability, erythrocyte fat consists almost exclusively of phospholipids, erythrocyte fatty acid composition reflects tissue fatty acid composition, pre-analytical stability, and other points.  In 2004, EPA + DHA in erythrocyte fatty acids were defined as the Omega-3 Index and suggested as a risk factor for sudden cardiac death [39]. Integral to the definition was a specific and standardized analytical procedure, conforming the quality management routinely implemented in the field of clinical chemistry.

The laboratories adhering to the HS-Omega-3 Index methodology perform regular proficiency testing, as mandated in routine Clinical Chemistry labs. So far, the HS-Omega-3 Index is the only analytical procedure used in several laboratories. A standardized analytical procedure is a prerequisite to generate the data base necessary to transport a laboratory parameter from research into clinical routine. Moreover, standardization of the analytical procedure is the first important criterion for establishing a new biomarker for cardiovascular risk set forth by the American Heart Association and the US Preventive Services Task Force.

Because of low biological and analytical variability, a standardized analytical procedure, a large database and for other reasons,

  • blood levels of EPA + DHA are frequently assessed in erythrocytes, using the HS-Omega-3 Index methodology.

Table 1. Mean HS-Omega-3 Index values in various populations, Mean (±standard deviation (SD)). Please note that in every population studied, a lower value was found to be associated with a worse condition than a higher value. References are given, if not, unpublished, n = number of individuals measured.

All levels of fatty acids are determined by the balance of substance entering the body and those leaving the body. Neither a recent meal, even if rich in EPA + DHA, nor severe cardiac events altered the HS-Omega-3 Index. However, while long-term intake of EPA + DHA, e.g., as assessed with food questionnaires, was the main predictor of the HS-Omega-3 Index, long-term intake explained only 12%–25% of its variability. A hereditary component of 24% exists. A number of other factors correlated positively (+) or negatively (−), like age (+), body mass index (−), socioeconomic status (+), smoking (−), but no other conventional cardiac risk factors. More factors determining the level of the HS-Omega-3 Index, especially regarding efflux remain to be  defined. Therefore, it is impossible to predict the HS-Omega-3 Index in an individual, as it is impossible to predict the increase in the HS-Omega-3 Index in an individual in response to a given dose of EPA + DHA. In Table 2, current evidence is presented on the relation of the HS-Omega-3 Index to CV events.

The HS-Omega-3 Index has made it possible to reclassify individuals from intermediate cardiovascular risk into the respective high risk and low risk strata, the third criterion for establishing a new biomarker for CV  risk.

A low Omega-3 Index fulfills the current criteria for a novel cardiovascular risk factor.

Increasing the HS-Omega-3 Index by increased intake of EPA + DHA in randomized controlled trials improved a number of surrogate parameters for cardiovascular risk:

  1. heart rate was reduced,
  2. heart rate variability was increased,
  3. blood pressure was reduced,
  4. platelet reactivity was reduced,
  5. triglycerides were reduced,
  6. large buoyant low-density lipoprotein (LDL)-particles were increased and
  7. small dense LDL-particles were reduced,
  8. large buoyant high-density lipoproteins (HDL)2 were increased,
  9. very low-density lipoprotein (VLDL1) + 2 was reduced,
  10. pro-inflammatory cytokines (e.g., tumor necrosis factor alpha, interleukin-1β, interleukins-6,8,10 and monocyte chemoattractant protein-1) were reduced,
  11. anti-inflammatory oxylipins were increased.

Importantly, in a two-year randomized double-blind angiographic intervention trial, increased erythrocyte EPA + DHA

  • reduced progression and increased regression of coronary lesions, an intermediate parameter.

Taken together, increasing the HS-Omega-3 Index improved surrogate and intermediate parameters for cardiovascular events. A large intervention trial with clinical endpoints based on the HS-Omega-3 Index remains to be conducted. Therefore, the fourth criterion, proof of therapeutic consequence of determining the HS-Omega- Index, is only partially fulfilled.


Neutral results of intervention trials can be explained by issues of bioavailability and trial design that surfaced after the trials were initiated.

In the future, incorporating the Omega-3 Index into trial designs by

  1. recruiting participants with a low Omega-3 Index and
  2. treating them within a pre-specified target range (e.g., 8%–11%),
  3. will make more efficient trials possible and
    • provide clearer answers to the questions asked than previously possible.


3. Stem Cells and Regenerative Biology

Adult Stem Cells Reverse Muscle Atrophy In Elderly Mice   http://www.science20.com/profile/news_staff

Bioengineers at the University of California, Berkeley in a new study published in Nature say they have identified two key regulatory pathways that control how well adult stem cells repair and replace damaged tissue. They then tweaked how those stem cells reacted to those biochemical signals to revive the ability of muscle tissue in old mice to repair itself nearly as well as the muscle in the mice’s much younger counterparts. Irina Conboy, an assistant professor of bioengineering and an investigator at the Berkeley Stem Cell Center and at the California Institute for Quantitative Biosciences (QB3), led the research team conducting this study. Because the findings relate to adult stem cells that reside in existing tissue, this approach to rejuvenating degenerating muscle eliminates the ethical and medical complications associated with transplanting tissues grown from embryonic stem cells. The researchers focused on

  • the interplay of two competing molecular pathways that control the stem cells,

which sit next to the mature, differentiated cells that make up our working body parts. When the mature cells are damaged or wear out, the stem cells are called into action to begin the process of rebuilding.

old muscle tissue is left with

old muscle tissue is left with













“We don’t realize it, but as we grow our bodies are constantly being remodeled,” said Conboy. “We are constantly falling apart, but we don’t notice it much when we’re young because we’re always being restored. As we age, our stem cells are prevented, through chemical signals, from doing their jobs.” The good news, the researchers said, is that

  • the stem cells in old tissue are still ready and able to perform their regenerative function
  • if they receive the appropriate chemical signals.

Studies have shown that when old tissue is placed in an environment of young blood, the stem cells behave as if they are young again. “Conversely, we have found in a study published last year that even young stem cells rapidly age when placed among blood and tissue from old mice,” said Carlson, who will stay on at UC Berkeley to expand his work on stem cell engineering.

  • Adult stem cells have a receptor called Notch that, when activated,
  • tells them that it is time to grow and divide
  • stem cells also have a receptor for the protein TGF-beta
  • that sets off a chain reaction activatingthemoleculepSmad3 and
    • ultimately producing cyclin-dependent kinase (CDK) inhibitors, which regulate the cell’s ability to divide.
  • activated Notch competeswithactivatedpSmad3 for
    • binding to the regulatory regions of the same CDK inhibitors in the stem cell

“We found that Notch is capable of physically kicking off pSmad3 from the promoters for the CDK inhibitors within the stem cell’s nucleus, which tells us that a precise manipulation of the balance of these pathways would allow the ability to control stem cell responses.” Notch and TGF-beta are well known in molecular biology, but Conboy’s lab is the first to connect them to the process of aging, and the first to show that they act in opposition to each other within the nucleus of the adult stem cell. Aging and the inevitable march towards death are, in part, due to the progressive decline of Notch and the increased levels of TGF-beta , producing a one-two punch to the stem cell’s capacity to effectively rebuild the body, the researchers said.

The researchers disabled the “aging pathway” that tells stem cells to stop dividing by using an established method of RNA interference that reduced levels of pSmad3. The researchers then examined the muscle of the different groups of mice one to five days after injury to compare how well the tissue repaired itself. As expected,

  •  muscle tissue in the young mice easily replaced damaged cells with new, healthy cells. In contrast,
  • the areas of damaged muscle in the control group of old mice were characterized by fibroblasts and scar tissue. However,
  • muscles in the old mice whose stem cell “aging pathway”had been dampened showed levels of cellular regeneration that were
    • comparable to their much younger peers, and that were 3 to 4 times greater than those of the group of “untreated” old mice.

Adult Stem Cells To Repair Damaged Heart Muscle


In the first trial of its kind in the world, 60 patients who have recently suffered a major heart attack will be injected with selected stem cells from their own bone marrow during routine coronary bypass surgery. The Bristol trial will test

  • whether the stem cells will repair heart muscle cells damaged by the heart attack,
  • by preventing late scar formation and hence impaired heart contraction.

“ Cardiac stem cell therapy aims to repair the damaged heart as it has the potential to replace the damaged tissue.” We have elected to use a very promising stem cell type selected from the patient’s own bone marrow. This approach ensures no risk of rejection or infection. It also gets around the ethical issues that would result from use of stem cells from embryonic or foetal tissue.

In this trial (known as TransACT), all patients will have bone marrow harvested before their heart operation. Then either stem cells from their own bone marrow or a placebo will be injected into the patients’ damaged hearts during routine coronary bypass surgery. The feasibility and safety of this technique has already been demonstrated. As a result of the chosen double blind placebo-controlled design, neither the patients nor the surgeon knows whether the patient is going to be injected with stem cells or placebo. This ensures that results are not biased in any way, and is the most powerful way to prove whether or not the new treatment is effective.

Research of Stem Cells Repair Damaged Heart

By Kelvinlew Minhan | March 26th 2008

Under highly specific growth conditions in laboratory culture dishes, stem cells

  • can be coaxed into developing as new cardiomyocytes and vascular endothelial cells (Kirschstein and Skirboll, 2001).

Discoveries that have triggered the interest in the application of adult stem cells to heart muscle repair in animal models have been made by researchers in the past few years (Kirschstein and Skirboll, 2001). One  study demonstrated that cardiac tissue can be regenerated in the mouse heart attack model through the introduction of adult stem cells from mouse bone marrow (Kirschstein and Skirboll, 2001). These cells were transplanted into the marrow of irradiated mice approximately 10 weeks before the recipient mice were subjected to heart attack thru tying off different major heart blood vessel, the left anterior descending (LAD) coronary artery. The survival rate was 26 percent at two to four weeks after the induced cardiac injury (Kirschstein and Skirboll, 2001). Another study of the region surrounding the damaged tissue in surviving mice showed the presence of donor-derived cardiomyocytes and endothelial cells (Kirschstein and Skirboll, 2001).

  • the mouse hematopoietic stem cells transplanted into the bone marrow had migrated to the border part of the damaged area, and differentiated into several types of tissue for cardiac repair.

Regenerating heart tissue through stem cell therapy



A groundbreaking study on repairing damaged heart tissue through stem cell therapy has given patients hope that they may again live active lives. An international team of Mayo Clinic researchers and collaborators has done it by discovering a way to regenerate heart tissue.

“It’s a paradigm shift,” says Andre Terzic, M.D., Ph.D., director of Mayo Clinic’s Center for Regenerative Medicine and senior investigator of the stem cell trial. “We are moving from traditional medicine, which addresses the symptoms of disease to cure disease.” Treating patients with cardiac disease has typically involved managing heart damage with medication.  In collaboration with European researchers, Mayo Clinic researchers have discovered a novel way to repair a damaged heart. In Mayo Clinic’s breakthrough process,
  • stem cells are harvested from a patient’s bone marrow.
  •  undergo a laboratory treatment that guides them into becoming cardiac cells,
  • which are then injected into the patient’s heart in an effort to grow healthy heart tissue.
The study is the first successful demonstration in people of the feasibility and safety of transforming adult stem cells into cardiac cells. Beyond heart failure, the Mayo Clinic research also is a milestone in the emerging field of regenerative medicine, which seeks to fully heal damaged tissue and organs.

Creating a heart repair kit

Process of converting bone marrow cells to heart cells
This image shows the process used in the clinical trials to repair damaged hearts. Cardioprogenitor cells is another term for cardiopoietic cells, those that were transformed into cardiac cells.
Stem cells transforming to cardiac tissue
Transformation: The cardiopoietic cells on the left react to the cardiac environment, cluster together with like cells and form tissue.
 Mayo Clinic researchers pursued this research, inspired by an intriguing discovery. In the early 2000s, they analyzed stem cells from 11 patients undergoing heart bypass surgery. The stem cells from two of the patients had an unusually high expression of certain transcription factors — the proteins that control the flow of genetic information between cells. Clinically, the two patients appeared no different from the others, yet their stem cells seemed to show unique capacity for heart repair.
That observation drove them to  determine how to convert  nonreparative stem cells to become reparative. Doing so required determining precisely how the human heart naturally develops, at a subcellular level. That painstaking work was led by Atta Behfar, M.D., Ph.D., a cardiovascular researcher at Mayo Clinic in Rochester, Minn. With other members of the Terzic research team, Dr. Behfar identified hundreds of proteins involved in the process of heart development (cardiogenesis). The researchers then set out to identify which of these proteins are essential in driving a stem cell to become a cardiac cell. Using computer models,
  • they simulated the effects of eliminating proteins one by one from the process of heart development.
  • That method yielded about 25 proteins.
    • The team then pared that number down to 8 proteins that their data indicated were essential.
The research team was then able to develop the lab procedure that guides stem cells to become heart cells.
The treated stem cells were dubbed cardiopoietic, or heart creative. A proof of principle study about guided cardiopoiesis, whose results were published in the Journal of the American College of Cardiology in 2010, demonstrated that animal models with heart disease that had been injected with caridiopoietic cells had improved heart function compared with animals injected with untreated stem cells. Hailed as “landmark work,” by the journal’s editorial writer, the study showed it was indeed possible to teach stem cells to become cardiac cells. Stem cells from each patient in the cardiopoiesis group were successfully guided to become cardiac cells. The treated cells were injected into the heart wall of each of those patients without apparent complications.
“Ihis newprocessofcardiopoiesiswas achieved in 100 percent of cases, with a very good safety profile,” Dr.Terzic says. “We are enabling the heart toregainitsinitial structure and function,” Dr.Terzic says, “and we will not stop here.” The clinicaltrialfindingsareexpectedto be published in the Journal of the American College of Cardiology in 2013.  Meanwhile, research to improve the injection process and effectiveness is underway.

Stem Cells from Humans Repair Heart Damage in Monkeys

GEN News Highlights  May1, 2014

GPCR Insights Brighten Drug Discovery Outlook

Ken Doyle, Ph.D.

GEN Apr 15, 2014 (Vol. 34, No. 8)

Recent years have seen major advances in understanding the structure-function relationships of G protein-coupled receptors (GPCRs). This large superfamily of transmembrane receptors comprises over 800 members in humans.

GPCRs regulate a wide variety of physiological processes including

  • sensation (vision, taste, and smell),
  • growth,
  • hormone responses, and
  • regulation of the immune and
  • autonomic nervous systems.

Their involvement in multiple disease pathways makes GPCRs attractive targets for drug discovery efforts.

These multifaceted proteins will be the subject of “GPCR Structure, Function and Drug Discovery,” a Global Technology Community conference scheduled to take place May 22–23 in Boston. The conference is expected to cover a broad range of topics including biased signaling, membrane protein structures, GPCR signaling dynamics, computational approaches to disease.

According to Bryan Roth, M.D., Ph.D., Michael Hooker Distinguished Professor at the University of North Carolina, Chapel Hill,

  • drugs that can selectively target various downstream GPCR pathways hold the most promise.

Dr. Roth’s laboratory studies approximately 360 different GPCRs with therapeutic potential using massively parallel screening methods. His research focuses on “functional selectivity,” which he describes as

  • “the ligand-dependent selectivity for certain signal transduction pathways in one and the same receptor.”

Dr. Roth notes that structural data have demonstrated that GPCRs exist in multiple conformations: “The structures of the 5-hydroxytryptamine 2B receptor and the recent high-resolution delta-opioid receptor structure have provided evidence for conformational rearrangements that contribute to functional selectivity.” Drugs that take advantage of this selectivity by preferentially stabilizing certain conformations may have unique therapeutic utility.

“Generally, we look at G protein versus arrestin-based signaling, although it’s also possible to examine how drugs activate one G protein-mediated signaling pathway versus another.


fluorescently tagged Arrestin and GPRC of interest

fluorescently tagged Arrestin and GPRC of interest








  • β-Arrestins constitute a major class of intracellular scaffolding proteins that regulate GPCR signaling by preventing or enhancing the binding of GPCRs to intracellular signaling molecules. Laura Bohn, Ph.D., associate professor at Scripps Florida,  studies the roles that β-arrestins play in GPCR-mediated signaling.
  • a particular β-arrestin can play multiple, tissue-specific roles—shutting down the signaling of a receptor in one tissue while activating signaling in another.
  • different ligands can direct GPCR signaling to different effectors, which could result in different physiological effects,” comments Dr. Bohn. “Our challenge is in determining what signaling pathways to harness to promote certain effects, while avoiding others.”
Arrestin binding to active GPCR kinase (GRK)-phosphorylated GPCRs blocks G protein coupling

Arrestin binding to active GPCR kinase (GRK)-phosphorylated GPCRs blocks G protein coupling












Using Designer Proteins

The multifunctional signaling abilities of β-arrestins has prompted large-scale study of their properties. Vsevolod Gurevich, Ph.D., professor of pharmacology at Vanderbilt University, studies

  1. the structure,
  2. function, and
  3. biology of arrestin proteins.

β-arrestins have three main functions.

  1. First, they prevent the coupling of GPCRs to G proteins, thereby blocking further G protein-mediated signaling (a process known as desensitization).
  2. Second, the binding of a GCPR releases the β-arrestin’s carboxy-terminal “tail” and promotes internalization of the receptor.
  3. Third, receptor-bound β-arrestins bind other signaling proteins, resulting in a second wave of arrestin-mediated signaling.

Dr. Gurevich’s laboratory studies β-arrestin biology through the use of three types of specially designed mutants—

  1. enhanced phosphorylation-dependent,
  2. receptor-specific, and
  3. signaling-biased mutants.

an enhanced mutant of visual β-arrestin-1 partially compensates for defects of rhodopsin phosphorylation in vivo,

“Several congenital disorders are caused by mutant GPCRs that cannot be normally phosphorylated because they have lost GPCR kinase (GRK) sites. Enhanced super-active arrestins have the potential to compensate for these defects, bringing the signaling closer to normal.”

  • Dr. Gurevich explains the strategy involved in creating designer β-arrestins: “We identify residues critical for individual β-arrestin functions by mutagenesis, using limited structural information as a guide.
  • We also work on getting more structural information. In collaboration with different crystallographers, we solved the crystal structures of all four vertebrate β-arrestin subtypes in the basal state, as well as the structure of the arrestin-1-rhodopsin complex.”
  • Dr. Gurevich believes that designer β-arrestins “are the next step in research and therapy, moving way beyond what small molecules can achieve.
  • The difference in capabilities between redesigned signaling proteins, including β-arrestins, and conventional small molecule drugs is about the same as that between airplanes and horse-driven carriages.”
  • Dr. Gurevich observes that redesigned signaling proteins face considerable obstacles in terms of gene delivery, but that the efforts are worth it. “Using designer signaling proteins, we can tell the cell what to do in a language it cannot disobey,” asserts Dr. Gurevich.

Synthesis and Antihypertensive Screening of Novel Substituted 1,2- Pyrazoline Sulfonamide Derivatives

Avinash M. Bhagwat , Anilchandra R. Bha , Mahesh S. Palled , Anand P. Khadke , Anuradha M. Patil, et al.

Am. J. PharmTech Res. 2014; 4(2).    http://www.ajptr.com/ 

Angiotensin II receptor antagonists, also known as angiotensin receptor blockers , AT1-receptor antagonists or sartans, are a group of pharmaceuticals which modulate the renin-angiotensin-aldosterone system. Their main use is in hypertension, diabetic nephropathy and congestiveheart failure. These substances are AT1-receptor antagonists which

  • block the activationof angiotensin II AT1 receptors.

Blockade of AT1 receptors directly causes

1 vasodilation,

2 reduces secretion of vasopressin,

3 reduces production and secretion of aldosterone, amongst other actions –

4 the combined effect of which is reduction of blood pressure.

Irbesartan is a safe and effectiveangiotensin II receptor antagonist with an affinity for the AT1 receptor that is more than 8,500times greater than its affinity for AT2 receptor. This agent has a higher bioavailability (60-80%) than other drugs in its class . In both Losartan and Irbesartan structures imidazole moiety is being present. A structure analog of losartan and Irbesartan are designed by incorporating the heterocycles like pyrazoline group. We felt it would be interesting to explore the possibilities of 1,2-pyrazoline derivatives for Angiotensin II receptor antagonistic activity.

The Irbesartan structure was a modified Losartan structure, which had all the identity of a Losartan molecule but with groups that would fit the hydrophobic cavity with a tetramethylene group and an alkyl side chain that would fit in the pocket in the AT1 receptor. The hydroxyl methyl group of Losartan being replaced with carbonyl group of Irbesartan. With a view to introduce a hydrogen bonding interaction with AT1 receptor, these structures were further modified with a view of retaining both hydrogen bonding characteristics and as well as lipophilic groups. Losartan and Irbesartan structure contains a diphenyl molecule & imidazole ring.

In Losartan and Irbesartan diphenyl molecule is attached to the nitrogen of the imidazole ring. It is interesting to to see the activity of compounds containing two phenyl rings attached at two different positions namely3,5 position of 1, 2-pyrazoline ring. The sulphonamide derivatives known for its diuretics activity which reduces renal hypertension. We use to synthesize sulphonamide and pyrazoline in one molecule to check its possible Angiotensin II receptor antagonist property. For this reason chalcones were synthesized reacted with hydrazine hydrate to yield the corresponding 1,2-pyrazoline derivatives which further condensed with sulphanilamide and formaldehyde by mannich condensation reaction.

Acute Toxicity Study (LD50)

This study was carried out in order to establish the therapeutic and toxic doses of the newly synthesized 1,2 pyrazoline derivatives. To establish LD50 of these compounds the method described by Miller & Tainter was employed.

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Summary – Volume 4, Part 2: Translational Medicine in Cardiovascular Diseases

Summary – Volume 4, Part 2:  Translational Medicine in Cardiovascular Diseases

Author and Curator: Larry H Bernstein, MD, FCAP


We have covered a large amount of material that involves

  • the development,
  • application, and
  • validation of outcomes of medical and surgical procedures

that are based on translation of science from the laboratory to the bedside, improving the standards of medical practice at an accelerated pace in the last quarter century, and in the last decade.  Encouraging enabling developments have been:

1. The establishment of national and international outcomes databases for procedures by specialist medical societies

Stent Design and Thrombosis: Bifurcation Intervention, Drug Eluting Stents (DES) and Biodegrable Stents
Curator: Aviva Lev-Ari, PhD, RN

On Devices and On Algorithms: Prediction of Arrhythmia after Cardiac Surgery and ECG Prediction of an Onset of Paroxysmal Atrial Fibrillation
Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC

Mitral Valve Repair: Who is a Patient Candidate for a Non-Ablative Fully Non-Invasive Procedure?
Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC and Article Curator: Aviva Lev-Ari, PhD, RN

Cardiovascular Complications: Death from Reoperative Sternotomy after prior CABG, MVR, AVR, or Radiation; Complications of PCI; Sepsis from Cardiovascular Interventions
Author, Introduction and Summary: Justin D Pearlman, MD, PhD, FACC and Article Curator: Aviva Lev-Ari, PhD, RN

Survivals Comparison of Coronary Artery Bypass Graft (CABG) and Percutaneous Coronary Intervention (PCI) /Coronary Angioplasty
Larry H. Bernstein, MD, Writer And Aviva Lev-Ari, PhD, RN, Curator

Revascularization: PCI, Prior History of PCI vs CABG
Curator: Aviva Lev-Ari, PhD, RN

Outcomes in High Cardiovascular Risk Patients: Prasugrel (Effient) vs. Clopidogrel (Plavix); Aliskiren (Tekturna) added to ACE or added to ARB
Reporter and Curator: Aviva Lev-Ari, PhD, RN

Endovascular Lower-extremity Revascularization Effectiveness: Vascular Surgeons (VSs), Interventional Cardiologists (ICs) and Interventional Radiologists (IRs)
Curator: Aviva Lev-Ari, PhD, RN

and more

2. The identification of problem areas, particularly in activation of the prothrombotic pathways, infection control to an extent, and targeting of pathways leading to progression or to arrythmogenic complications.

Cardiovascular Complications: Death from Reoperative Sternotomy after prior CABG, MVR, AVR, or Radiation; Complications of PCI; Sepsis from Cardiovascular Interventions Author, Introduction and Summary: Justin D Pearlman, MD, PhD, FACC and Article Curator: Aviva Lev-Ari, PhD, RN

Anticoagulation genotype guided dosing
Larry H. Bernstein, MD, FCAP, Author and Curator

Stent Design and Thrombosis: Bifurcation Intervention, Drug Eluting Stents (DES) and Biodegrable Stents
Curator: Aviva Lev-Ari, PhD, RN

The Effects of Aprotinin on Endothelial Cell Coagulant Biology
Co-Author (Kamran Baig, MBBS, James Jaggers, MD, Jeffrey H. Lawson, MD, PhD) and Curator

Outcomes in High Cardiovascular Risk Patients: Prasugrel (Effient) vs. Clopidogrel (Plavix); Aliskiren (Tekturna) added to ACE or added to ARB
Reporter and Curator: Aviva Lev-Ari, PhD, RN

Pharmacogenomics – A New Method for Druggability  Author and Curator: Demet Sag, PhD

Advanced Topics in Sepsis and the Cardiovascular System at its End Stage    Author: Larry H Bernstein, MD, FCAP

3. Development of procedures that use a safer materials in vascular management.

Stent Design and Thrombosis: Bifurcation Intervention, Drug Eluting Stents (DES) and Biodegrable Stents
Curator: Aviva Lev-Ari, PhD, RN

Biomaterials Technology: Models of Tissue Engineering for Reperfusion and Implantable Devices for Revascularization
Author and Curator: Larry H Bernstein, MD, FACP and Curator: Aviva Lev-Ari, PhD, RN

Vascular Repair: Stents and Biologically Active Implants
Author and Curator: Larry H Bernstein, MD, FACP and Curator: Aviva Lev-Ari, RN, PhD

Drug Eluting Stents: On MIT’s Edelman Lab’s Contributions to Vascular Biology and its Pioneering Research on DES
Author: Larry H Bernstein, MD, FACP and Curator: Aviva Lev-Ari, PhD, RN

MedTech & Medical Devices for Cardiovascular Repair – Curations by Aviva Lev-Ari, PhD, RN

4. Discrimination of cases presenting for treatment based on qualifications for medical versus surgical intervention.

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

Coronary Reperfusion Therapies: CABG vs PCI – Mayo Clinic preprocedure Risk Score (MCRS) for Prediction of in-Hospital Mortality after CABG or PCI
Writer and Curator: Larry H. Bernstein, MD, FCAP and Curator: Aviva Lev-Ari, PhD, RN

ACC/AHA Guidelines for Coronary Artery Bypass Graft Surgery Reporter: Aviva Lev-Ari, PhD, RN

Mitral Valve Repair: Who is a Patient Candidate for a Non-Ablative Fully Non-Invasive Procedure?
Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC and Article Curator: Aviva Lev-Ari, PhD, RN

5.  This has become possible because of the advances in our knowledge of key related pathogenetic mechanisms involving gene expression and cellular regulation of complex mechanisms.

What is the key method to harness Inflammation to close the doors for many complex diseases?
Author and Curator: Larry H Bernstein, MD, FCAP

CVD Prevention and Evaluation of Cardiovascular Imaging Modalities: Coronary Calcium Score by CT Scan Screening to justify or not the Use of Statin
Curator: Aviva Lev-Ari, PhD, RN

Richard Lifton, MD, PhD of Yale University and Howard Hughes Medical Institute: Recipient of 2014 Breakthrough Prizes Awarded in Life Sciences for the Discovery of Genes and Biochemical Mechanisms that cause Hypertension
Curator: Aviva Lev-Ari, PhD, RN

Pathophysiological Effects of Diabetes on Ischemic-Cardiovascular Disease and on Chronic Obstructive Pulmonary Disease (COPD)
Curator:  Larry H. Bernstein, MD, FCAP

Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))
Reviewer and Co-Curator: Larry H Bernstein, MD, CAP and Curator: Aviva Lev-Ari, PhD, RN

Notable Contributions to Regenerative Cardiology  Author and Curator: Larry H Bernstein, MD, FCAP and Article Commissioner: Aviva Lev-Ari, PhD, RD

As noted in the introduction, any of the material can be found and reviewed by content, and the eTOC is identified in attached:



This completes what has been presented in Part 2, Vol 4 , and supporting references for the main points that are found in the Leaders in Pharmaceutical Intelligence Cardiovascular book.  Part 1 was concerned with Posttranslational Modification of Proteins, vital for understanding cellular regulation and dysregulation.  Part 2 was concerned with Translational Medical Therapeutics, the efficacy of medical and surgical decisions based on bringing the knowledge gained from the laboratory, and from clinical trials into the realm opf best practice.  The time for this to occur in practice in the past has been through roughly a generation of physicians.  That was in part related to the busy workload of physicians, and inability to easily access specialty literature as the volume and complexity increased.  This had an effect of making access of a family to a primary care provider through a lifetime less likely than the period post WWII into the 1980s.

However, the growth of knowledge has accelerated in the specialties since the 1980’s so that the use of physician referral in time became a concern about the cost of medical care.  This is not the place for or a matter for discussion here.  It is also true that the scientific advances and improvements in available technology have had a great impact on medical outcomes.  The only unrelated issue is that of healthcare delivery, which is not up to the standard set by serial advances in therapeutics, accompanied by high cost due to development costs, marketing costs, and development of drug resistance.

I shall identify continuing developments in cardiovascular diagnostics, therapeutics, and bioengineering that is and has been emerging.

1. Mechanisms of disease

REPORT: Mapping the Cellular Response to Small Molecules Using Chemogenomic Fitness Signatures 

Science 11 April 2014:
Vol. 344 no. 6180 pp. 208-211

Abstract: Genome-wide characterization of the in vivo cellular response to perturbation is fundamental to understanding how cells survive stress. Identifying the proteins and pathways perturbed by small molecules affects biology and medicine by revealing the mechanisms of drug action. We used a yeast chemogenomics platform that quantifies the requirement for each gene for resistance to a compound in vivo to profile 3250 small molecules in a systematic and unbiased manner. We identified 317 compounds that specifically perturb the function of 121 genes and characterized the mechanism of specific compounds. Global analysis revealed that the cellular response to small molecules is limited and described by a network of 45 major chemogenomic signatures. Our results provide a resource for the discovery of functional interactions among genes, chemicals, and biological processes.


Laura Zahn
Sci. Signal. 15 April 2014; 7(321): ec103.   http://dx.doi.org/10.1126/scisignal.2005362

In order to identify how chemical compounds target genes and affect the physiology of the cell, tests of the perturbations that occur when treated with a range of pharmacological chemicals are required. By examining the haploinsufficiency profiling (HIP) and homozygous profiling (HOP) chemogenomic platforms, Lee et al.(p. 208) analyzed the response of yeast to thousands of different small molecules, with genetic, proteomic, and bioinformatic analyses. Over 300 compounds were identified that targeted 121 genes within 45 cellular response signature networks. These networks were used to extrapolate the likely effects of related chemicals, their impact upon genetic pathways, and to identify putative gene functions

Key Heart Failure Culprit Discovered

A team of cardiovascular researchers from the Cardiovascular Research Center at Icahn School of Medicine at Mount Sinai, Sanford-Burnham Medical Research Institute, and University of California, San Diego have identified a small, but powerful, new player in thIe onset and progression of heart failure. Their findings, published in the journal Nature  on March 12, also show how they successfully blocked the newly discovered culprit.
Investigators identified a tiny piece of RNA called miR-25 that blocks a gene known as SERCA2a, which regulates the flow of calcium within heart muscle cells. Decreased SERCA2a activity is one of the main causes of poor contraction of the heart and enlargement of heart muscle cells leading to heart failure.

Using a functional screening system developed by researchers at Sanford-Burnham, the research team discovered miR-25 acts pathologically in patients suffering from heart failure, delaying proper calcium uptake in heart muscle cells. According to co-lead study authors Christine Wahlquist and Dr. Agustin Rojas Muñoz, developers of the approach and researchers in Mercola’s lab at Sanford-Burnham, they used high-throughput robotics to sift through the entire genome for microRNAs involved in heart muscle dysfunction.

Subsequently, the researchers at the Cardiovascular Research Center at Icahn School of Medicine at Mount Sinai found that injecting a small piece of RNA to inhibit the effects of miR-25 dramatically halted heart failure progression in mice. In addition, it also improved their cardiac function and survival.

“In this study, we have not only identified one of the key cellular processes leading to heart failure, but have also demonstrated the therapeutic potential of blocking this process,” says co-lead study author Dr. Dongtak Jeong, a post-doctoral fellow at the Cardiovascular Research Center at Icahn School of  Medicine at Mount Sinai in the laboratory of the study’s co-senior author Dr. Roger J. Hajjar.

Publication: Inhibition of miR-25 improves cardiac contractility in the failing heart.Christine Wahlquist, Dongtak Jeong, Agustin Rojas-Muñoz, Changwon Kho, Ahyoung Lee, Shinichi Mitsuyama, Alain Van Mil, Woo Jin Park, Joost P. G. Sluijter, Pieter A. F. Doevendans, Roger J. :  Hajjar & Mark Mercola.     Nature (March 2014)    http://www.nature.com/nature/journal/vaop/ncurrent/full/nature13073.html


“Junk” DNA Tied to Heart Failure

Deep RNA Sequencing Reveals Dynamic Regulation of Myocardial Noncoding RNAs in Failing Human Heart and Remodeling With Mechanical Circulatory Support

Yang KC, Yamada KA, Patel AY, Topkara VK, George I, et al.
Circulation 2014;  129(9):1009-21.
http://dx.doi.org/10.1161/CIRCULATIONAHA.113.003863              http://circ.ahajournals.org/…/CIRCULATIONAHA.113.003863.full

The myocardial transcriptome is dynamically regulated in advanced heart failure and after LVAD support. The expression profiles of lncRNAs, but not mRNAs or miRNAs, can discriminate failing hearts of different pathologies and are markedly altered in response to LVAD support. These results suggest an important role for lncRNAs in the pathogenesis of heart failure and in reverse remodeling observed with mechanical support.

Junk DNA was long thought to have no important role in heredity or disease because it doesn’t code for proteins. But emerging research in recent years has revealed that many of these sections of the genome produce noncoding RNA molecules that still have important functions in the body. They come in a variety of forms, some more widely studied than others. Of these, about 90% are called long noncoding RNAs (lncRNAs), and exploration of their roles in health and disease is just beginning.

The Washington University group performed a comprehensive analysis of all RNA molecules expressed in the human heart. The researchers studied nonfailing hearts and failing hearts before and after patients received pump support from left ventricular assist devices (LVAD). The LVADs increased each heart’s pumping capacity while patients waited for heart transplants.

In their study, the researchers found that unlike other RNA molecules, expression patterns of long noncoding RNAs could distinguish between two major types of heart failure and between failing hearts before and after they received LVAD support.

“The myocardial transcriptome is dynamically regulated in advanced heart failure and after LVAD support. The expression profiles of lncRNAs, but not mRNAs or miRNAs, can discriminate failing hearts of different pathologies and are markedly altered in response to LVAD support,” wrote the researchers. “These results suggest an important role for lncRNAs in the pathogenesis of heart failure and in reverse remodeling observed with mechanical support.”

‘Junk’ Genome Regions Linked to Heart Failure

In a recent issue of the journal Circulation, Washington University investigators report results from the first comprehensive analysis of all RNA molecules expressed in the human heart. The researchers studied nonfailing hearts and failing hearts before and after patients received pump support from left ventricular assist devices (LVAD). The LVADs increased each heart’s pumping capacity while patients waited for heart transplants.

“We took an unbiased approach to investigating which types of RNA might be linked to heart failure,” said senior author Jeanne Nerbonne, the Alumni Endowed Professor of Molecular Biology and Pharmacology. “We were surprised to find that long noncoding RNAs stood out.

In the new study, the investigators found that unlike other RNA molecules, expression patterns of long noncoding RNAs could distinguish between two major types of heart failure and between failing hearts before and after they received LVAD support.

“We don’t know whether these changes in long noncoding RNAs are a cause or an effect of heart failure,” Nerbonne said. “But it seems likely they play some role in coordinating the regulation of multiple genes involved in heart function.”

Nerbonne pointed out that all types of RNA molecules they examined could make the obvious distinction: telling the difference between failing and nonfailing hearts. But only expression of the long noncoding RNAs was measurably different between heart failure associated with a heart attack (ischemic) and heart failure without the obvious trigger of blocked arteries (nonischemic). Similarly, only long noncoding RNAs significantly changed expression patterns after implantation of left ventricular assist devices.


Decoding the noncoding transcripts in human heart failure

Xiao XG, Touma M, Wang Y
Circulation. 2014; 129(9): 958960,  http://dx.doi.org/10.1161/CIRCULATIONAHA.114.007548 

Heart failure is a complex disease with a broad spectrum of pathological features. Despite significant advancement in clinical diagnosis through improved imaging modalities and hemodynamic approaches, reliable molecular signatures for better differential diagnosis and better monitoring of heart failure progression remain elusive. The few known clinical biomarkers for heart failure, such as plasma brain natriuretic peptide and troponin, have been shown to have limited use in defining the cause or prognosis of the disease.1,2 Consequently, current clinical identification and classification of heart failure remain descriptive, mostly based on functional and morphological parameters. Therefore, defining the pathogenic mechanisms for hypertrophic versus dilated or ischemic versus nonischemic cardiomyopathies in the failing heart remain a major challenge to both basic science and clinic researchers. In recent years, mechanical circulatory support using left ventricular assist devices (LVADs) has assumed a growing role in the care of patients with end-stage heart failure.3 During the earlier years of LVAD application as a bridge to transplant, it became evident that some patients exhibit substantial recovery of ventricular function, structure, and electric properties.4 This led to the recognition that reverse remodeling is potentially an achievable therapeutic goal using LVADs. However, the underlying mechanism for the reverse remodeling in the LVAD-treated hearts is unclear, and its discovery would likely hold great promise to halt or even reverse the progression of heart failure.


Efficacy and Safety of Dabigatran Compared With Warfarin in Relation to Baseline Renal Function in Patients With Atrial Fibrillation: A RE-LY (Randomized Evaluation of Long-term Anticoagulation Therapy) Trial Analysis

Circulation. 2014; 129: 951-952     http://dx.doi.org/10.1161/​CIR.0000000000000022

In patients with atrial fibrillation, impaired renal function is associated with a higher risk of thromboembolic events and major bleeding. Oral anticoagulation with vitamin K antagonists reduces thromboembolic events but raises the risk of bleeding. The new oral anticoagulant dabigatran has 80% renal elimination, and its efficacy and safety might, therefore, be related to renal function. In this prespecified analysis from the Randomized Evaluation of Long-Term Anticoagulant Therapy (RELY) trial, outcomes with dabigatran versus warfarin were evaluated in relation to 4 estimates of renal function, that is, equations based on creatinine levels (Cockcroft-Gault, Modification of Diet in Renal Disease (MDRD), Chronic Kidney Disease Epidemiology Collaboration [CKD-EPI]) and cystatin C. The rates of stroke or systemic embolism were lower with dabigatran 150 mg and similar with 110 mg twice daily irrespective of renal function. Rates of major bleeding were lower with dabigatran 110 mg and similar with 150 mg twice daily across the entire range of renal function. However, when the CKD-EPI or MDRD equations were used, there was a significantly greater relative reduction in major bleeding with both doses of dabigatran than with warfarin in patients with estimated glomerular filtration rate ≥80 mL/min. These findings show that dabigatran can be used with the same efficacy and adequate safety in patients with a wide range of renal function and that a more accurate estimate of renal function might be useful for improved tailoring of anticoagulant treatment in patients with atrial fibrillation and an increased risk of stroke.

Aldosterone Regulates MicroRNAs in the Cortical Collecting Duct to Alter Sodium Transport.

Robert S Edinger, Claudia Coronnello, Andrew J Bodnar, William A Laframboise, Panayiotis V Benos, Jacqueline Ho, John P Johnson, Michael B Butterworth

Journal of the American Society of Nephrology (Impact Factor: 8.99). 04/2014;     http://dx. DO.org/I:10.1681/ASN.2013090931

Source: PubMed

ABSTRACT A role for microRNAs (miRs) in the physiologic regulation of sodium transport in the kidney has not been established. In this study, we investigated the potential of aldosterone to alter miR expression in mouse cortical collecting duct (mCCD) epithelial cells. Microarray studies demonstrated the regulation of miR expression by aldosterone in both cultured mCCD and isolated primary distal nephron principal cells.

Aldosterone regulation of the most significantly downregulated miRs, mmu-miR-335-3p, mmu-miR-290-5p, and mmu-miR-1983 was confirmed by quantitative RT-PCR. Reducing the expression of these miRs separately or in combination increased epithelial sodium channel (ENaC)-mediated sodium transport in mCCD cells, without mineralocorticoid supplementation. Artificially increasing the expression of these miRs by transfection with plasmid precursors or miR mimic constructs blunted aldosterone stimulation of ENaC transport.

Using a newly developed computational approach, termed ComiR, we predicted potential gene targets for the aldosterone-regulated miRs and confirmed ankyrin 3 (Ank3) as a novel aldosterone and miR-regulated protein.

A dual-luciferase assay demonstrated direct binding of the miRs with the Ank3-3′ untranslated region. Overexpression of Ank3 increased and depletion of Ank3 decreased ENaC-mediated sodium transport in mCCD cells. These findings implicate miRs as intermediaries in aldosterone signaling in principal cells of the distal kidney nephron.


2. Diagnostic Biomarker Status

A prospective study of the impact of serial troponin measurements on the diagnosis of myocardial infarction and hospital and 6-month mortality in patients admitted to ICU with non-cardiac diagnoses.

Marlies Ostermann, Jessica Lo, Michael Toolan, Emma Tuddenham, Barnaby Sanderson, Katie Lei, John Smith, Anna Griffiths, Ian Webb, James Coutts, John hambers, Paul Collinson, Janet Peacock, David Bennett, David Treacher

Critical care (London, England) (Impact Factor: 4.72). 04/2014; 18(2):R62.   http://dx.doi.org/:10.1186/cc13818

Source: PubMed

ABSTRACT Troponin T (cTnT) elevation is common in patients in the Intensive Care Unit (ICU) and associated with morbidity and mortality. Our aim was to determine the epidemiology of raised cTnT levels and contemporaneous electrocardiogram (ECG) changes suggesting myocardial infarction (MI) in ICU patients admitted for non-cardiac reasons.
cTnT and ECGs were recorded daily during week 1 and on alternate days during week 2 until discharge from ICU or death. ECGs were interpreted independently for the presence of ischaemic changes. Patients were classified into 4 groups: (i) definite MI (cTnT >=15 ng/L and contemporaneous changes of MI on ECG), (ii) possible MI (cTnT >=15 ng/L and contemporaneous ischaemic changes on ECG), (iii) troponin rise alone (cTnT >=15 ng/L), or (iv) normal. Medical notes were screened independently by two ICU clinicians for evidence that the clinical teams had considered a cardiac event.
Data from 144 patients were analysed [42% female; mean age 61.9 (SD 16.9)]. 121 patients (84%) had at least one cTnT level >=15 ng/L. A total of 20 patients (14%) had a definite MI, 27% had a possible MI, 43% had a cTNT rise without contemporaneous ECG changes, and 16% had no cTNT rise. ICU, hospital and 180 day mortality were significantly higher in patients with a definite or possible MI.Only 20% of definite MIs were recognised by the clinical team. There was no significant difference in mortality between recognised and non-recognised events.At time of cTNT rise, 100 patients (70%) were septic and 58% were on vasopressors. Patients who were septic when cTNT was elevated had an ICU mortality of 28% compared to 9% in patients without sepsis. ICU mortality of patients who were on vasopressors at time of cTNT elevation was 37% compared to 1.7% in patients not on vasopressors.
The majority of critically ill patients (84%) had a cTnT rise and 41% met criteria for a possible or definite MI of whom only 20% were recognised clinically. Mortality up to 180 days was higher in patients with a cTnT rise.


Prognostic performance of high-sensitivity cardiac troponin T kinetic changes adjusted for elevated admission values and the GRACE score in an unselected emergency department population.

Moritz BienerMatthias MuellerMehrshad VafaieAllan S JaffeHugo A Katus,Evangelos Giannitsis

Clinica chimica acta; international journal of clinical chemistry (Impact Factor: 2.54). 04/2014;   http://dx.doi.org/10.1016/j.cca.2014.04.007

Source: PubMed

ABSTRACT To test the prognostic performance of rising and falling kinetic changes of high-sensitivity cardiac troponin T (hs-cTnT) and the GRACE score.
Rising and falling hs-cTnT changes in an unselected emergency department population were compared.
635 patients with a hs-cTnT >99th percentile admission value were enrolled. Of these, 572 patients qualified for evaluation with rising patterns (n=254, 44.4%), falling patterns (n=224, 39.2%), or falling patterns following an initial rise (n=94, 16.4%). During 407days of follow-up, we observed 74 deaths, 17 recurrent AMI, and 79 subjects with a composite of death/AMI. Admission values >14ng/L were associated with a higher rate of adverse outcomes (OR, 95%CI:death:12.6, 1.8-92.1, p=0.01, death/AMI:6.7, 1.6-27.9, p=0.01). Neither rising nor falling changes increased the AUC of baseline values (AUC: rising 0.562 vs 0.561, p=ns, falling: 0.533 vs 0.575, p=ns). A GRACE score ≥140 points indicated a higher risk of death (OR, 95%CI: 3.14, 1.84-5.36), AMI (OR,95%CI: 1.56, 0.59-4.17), or death/AMI (OR, 95%CI: 2.49, 1.51-4.11). Hs-cTnT changes did not improve prognostic performance of a GRACE score ≥140 points (AUC, 95%CI: death: 0.635, 0.570-0.701 vs. 0.560, 0.470-0.649 p=ns, AMI: 0.555, 0.418-0.693 vs. 0.603, 0.424-0.782, p=ns, death/AMI: 0.610, 0.545-0.676 vs. 0.538, 0.454-0.622, p=ns). Coronary angiography was performed earlier in patients with rising than with falling kinetics (median, IQR [hours]:13.7, 5.5-28.0 vs. 20.8, 6.3-59.0, p=0.01).
Neither rising nor falling hs-cTnT changes improve prognostic performance of elevated hs-cTnT admission values or the GRACE score. However, rising values are more likely associated with the decision for earlier invasive strategy.


Troponin assays for the diagnosis of myocardial infarction and acute coronary syndrome: where do we stand?

Arie Eisenman

ABSTRACT: Under normal circumstances, most intracellular troponin is part of the muscle contractile apparatus, and only a small percentage (< 2-8%) is free in the cytoplasm. The presence of a cardiac-specific troponin in the circulation at levels above normal is good evidence of damage to cardiac muscle cells, such as myocardial infarction, myocarditis, trauma, unstable angina, cardiac surgery or other cardiac procedures. Troponins are released as complexes leading to various cut-off values depending on the assay used. This makes them very sensitive and specific indicators of cardiac injury. As with other cardiac markers, observation of a rise and fall in troponin levels in the appropriate time-frame increases the diagnostic specificity for acute myocardial infarction. They start to rise approximately 4-6 h after the onset of acute myocardial infarction and peak at approximately 24 h, as is the case with creatine kinase-MB. They remain elevated for 7-10 days giving a longer diagnostic window than creatine kinase. Although the diagnosis of various types of acute coronary syndrome remains a clinical-based diagnosis, the use of troponin levels contributes to their classification. This Editorial elaborates on the nature of troponin, its classification, clinical use and importance, as well as comparing it with other currently available cardiac markers.

Expert Review of Cardiovascular Therapy 07/2006; 4(4):509-14.   http://dx.doi.org/:10.1586/14779072.4.4.509 


Impact of redefining acute myocardial infarction on incidence, management and reimbursement rate of acute coronary syndromes.

Carísi A Polanczyk, Samir Schneid, Betina V Imhof, Mariana Furtado, Carolina Pithan, Luis E Rohde, Jorge P Ribeiro

ABSTRACT: Although redefinition for acute myocardial infarction (AMI) has been proposed few years ago, to date it has not been universally adopted by many institutions. The purpose of this study is to evaluate the diagnostic, prognostic and economical impact of the new diagnostic criteria for AMI. Patients consecutively admitted to the emergency department with suspected acute coronary syndromes were enrolled in this study. Troponin T (cTnT) was measured in samples collected for routine CK-MB analyses and results were not available to physicians. Patients without AMI by traditional criteria and cTnT > or = 0.035 ng/mL were coded as redefined AMI. Clinical outcomes were hospital death, major cardiac events and revascularization procedures. In-hospital management and reimbursement rates were also analyzed. Among 363 patients, 59 (16%) patients had AMI by conventional criteria, whereas additional 75 (21%) had redefined AMI, an increase of 127% in the incidence. Patients with redefined AMI were significantly older, more frequently male, with atypical chest pain and more risk factors. In multivariate analysis, redefined AMI was associated with 3.1 fold higher hospital death (95% CI: 0.6-14) and a 5.6 fold more cardiac events (95% CI: 2.1-15) compared to those without AMI. From hospital perspective, based on DRGs payment system, adoption of AMI redefinition would increase 12% the reimbursement rate [3552 Int dollars per 100 patients evaluated]. The redefined criteria result in a substantial increase in AMI cases, and allow identification of high-risk patients. Efforts should be made to reinforce the adoption of AMI redefinition, which may result in more qualified and efficient management of ACS.

International Journal of Cardiology 03/2006; 107(2):180-7. · 5.51 Impact Factor   http://www.sciencedirect.com/science/article/pii/S0167527305005279


3. Biomedical Engineerin3g

Safety and Efficacy of an Injectable Extracellular Matrix Hydrogel for Treating Myocardial Infarction 

Sonya B. Seif-Naraghi, Jennifer M. Singelyn, Michael A. Salvatore,  et al.
Sci Transl Med 20 February 2013 5:173ra25  http://dx.doi.org/10.1126/scitranslmed.3005503

Acellular biomaterials can stimulate the local environment to repair tissues without the regulatory and scientific challenges of cell-based therapies. A greater understanding of the mechanisms of such endogenous tissue repair is furthering the design and application of these biomaterials. We discuss recent progress in acellular materials for tissue repair, using cartilage and cardiac tissues as examples of application with substantial intrinsic hurdles, but where human translation is now occurring.

 Acellular Biomaterials: An Evolving Alternative to Cell-Based Therapies

J. A. Burdick, R. L. Mauck, J. H. Gorman, R. C. Gorman,
Sci. Transl. Med. 2013; 5, (176): 176 ps4    http://stm.sciencemag.org/content/5/176/176ps4

Acellular biomaterials can stimulate the local environment to repair tissues without the regulatory and scientific challenges of cell-based therapies. A greater understanding of the mechanisms of such endogenous tissue repair is furthering the design and application of these biomaterials. We discuss recent progress in acellular materials for tissue repair, using cartilage and cardiac tissues as examples of applications with substantial intrinsic hurdles, but where human translation is now occurring.

Instructive Nanofiber Scaffolds with VEGF Create a Microenvironment for Arteriogenesis and Cardiac Repair

Yi-Dong Lin, Chwan-Yau Luo, Yu-Ning Hu, Ming-Long Yeh, Ying-Chang Hsueh, Min-Yao Chang, et al.
Sci Transl Med 8 August 2012; 4(146):ra109.   http://dx.doi.org/ 10.1126/scitranslmed.3003841

Angiogenic therapy is a promising approach for tissue repair and regeneration. However, recent clinical trials with protein delivery or gene therapy to promote angiogenesis have failed to provide therapeutic effects. A key factor for achieving effective revascularization is the durability of the microvasculature and the formation of new arterial vessels. Accordingly, we carried out experiments to test whether intramyocardial injection of self-assembling peptide nanofibers (NFs) combined with vascular endothelial growth factor (VEGF) could create an intramyocardial microenvironment with prolonged VEGF release to improve post-infarct neovascularization in rats. Our data showed that when injected with NF, VEGF delivery was sustained within the myocardium for up to 14 days, and the side effects of systemic edema and proteinuria were significantly reduced to the same level as that of control. NF/VEGF injection significantly improved angiogenesis, arteriogenesis, and cardiac performance 28 days after myocardial infarction. NF/VEGF injection not only allowed controlled local delivery but also transformed the injected site into a favorable microenvironment that recruited endogenous myofibroblasts and helped achieve effective revascularization. The engineered vascular niche further attracted a new population of cardiomyocyte-like cells to home to the injected sites, suggesting cardiomyocyte regeneration. Follow-up studies in pigs also revealed healing benefits consistent with observations in rats. In summary, this study demonstrates a new strategy for cardiovascular repair with potential for future clinical translation.

Manufacturing Challenges in Regenerative Medicine

I. Martin, P. J. Simmons, D. F. Williams.
Sci. Transl. Med. 2014; 6(232): fs16.   http://dx.doi.org/10.1126/scitranslmed.3008558

Along with scientific and regulatory issues, the translation of cell and tissue therapies in the routine clinical practice needs to address standardization and cost-effectiveness through the definition of suitable manufacturing paradigms.




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Summary of Translational Medicine – e-Series A: Cardiovascular Diseases, Volume Four – Part 1

Summary of Translational Medicine – e-Series A: Cardiovascular Diseases, Volume Four – Part 1

Author and Curator: Larry H Bernstein, MD, FCAP


Curator: Aviva Lev-Ari, PhD, RN


Part 1 of Volume 4 in the e-series A: Cardiovascular Diseases and Translational Medicine, provides a foundation for grasping a rapidly developing surging scientific endeavor that is transcending laboratory hypothesis testing and providing guidelines to:

  • Target genomes and multiple nucleotide sequences involved in either coding or in regulation that might have an impact on complex diseases, not necessarily genetic in nature.
  • Target signaling pathways that are demonstrably maladjusted, activated or suppressed in many common and complex diseases, or in their progression.
  • Enable a reduction in failure due to toxicities in the later stages of clinical drug trials as a result of this science-based understanding.
  • Enable a reduction in complications from the improvement of machanical devices that have already had an impact on the practice of interventional procedures in cardiology, cardiac surgery, and radiological imaging, as well as improving laboratory diagnostics at the molecular level.
  • Enable the discovery of new drugs in the continuing emergence of drug resistance.
  • Enable the construction of critical pathways and better guidelines for patient management based on population outcomes data, that will be critically dependent on computational methods and large data-bases.

What has been presented can be essentially viewed in the following Table:


Summary Table for TM - Part 1

Summary Table for TM – Part 1




There are some developments that deserve additional development:

1. The importance of mitochondrial function in the activity state of the mitochondria in cellular work (combustion) is understood, and impairments of function are identified in diseases of muscle, cardiac contraction, nerve conduction, ion transport, water balance, and the cytoskeleton – beyond the disordered metabolism in cancer.  A more detailed explanation of the energetics that was elucidated based on the electron transport chain might also be in order.

2. The processes that are enabling a more full application of technology to a host of problems in the environment we live in and in disease modification is growing rapidly, and will change the face of medicine and its allied health sciences.


Electron Transport and Bioenergetics

Deferred for metabolomics topic

Synthetic Biology

Introduction to Synthetic Biology and Metabolic Engineering

Kristala L. J. Prather: Part-1    <iBiology > iBioSeminars > Biophysics & Chemical Biology >

http://www.ibiology.org Lecturers generously donate their time to prepare these lectures. The project is funded by NSF and NIGMS, and is supported by the ASCB and HHMI.
Dr. Prather explains that synthetic biology involves applying engineering principles to biological systems to build “biological machines”.

Dr. Prather has received numerous awards both for her innovative research and for excellence in teaching.  Learn more about how Kris became a scientist at
Prather 1: Synthetic Biology and Metabolic Engineering  2/6/14IntroductionLecture Overview In the first part of her lecture, Dr. Prather explains that synthetic biology involves applying engineering principles to biological systems to build “biological machines”. The key material in building these machines is synthetic DNA. Synthetic DNA can be added in different combinations to biological hosts, such as bacteria, turning them into chemical factories that can produce small molecules of choice. In Part 2, Prather describes how her lab used design principles to engineer E. coli that produce glucaric acid from glucose. Glucaric acid is not naturally produced in bacteria, so Prather and her colleagues “bioprospected” enzymes from other organisms and expressed them in E. coli to build the needed enzymatic pathway. Prather walks us through the many steps of optimizing the timing, localization and levels of enzyme expression to produce the greatest yield. Speaker Bio: Kristala Jones Prather received her S.B. degree from the Massachusetts Institute of Technology and her PhD at the University of California, Berkeley both in chemical engineering. Upon graduation, Prather joined the Merck Research Labs for 4 years before returning to academia. Prather is now an Associate Professor of Chemical Engineering at MIT and an investigator with the multi-university Synthetic Biology Engineering Reseach Center (SynBERC). Her lab designs and constructs novel synthetic pathways in microorganisms converting them into tiny factories for the production of small molecules. Dr. Prather has received numerous awards both for her innovative research and for excellence in teaching.









II. Regulatory Effects of Mammalian microRNAs

Calcium Cycling in Synthetic and Contractile Phasic or Tonic Vascular Smooth Muscle Cells

Current Basic and Pathological Approaches to
the Function of Muscle Cells and Tissues – From Molecules to HumansLarissa Lipskaia, Isabelle Limon, Regis Bobe and Roger Hajjar
Additional information is available at the end of the chapter
1. Introduction
Calcium ions (Ca ) are present in low concentrations in the cytosol (~100 nM) and in high concentrations (in mM range) in both the extracellular medium and intracellular stores (mainly sarco/endo/plasmic reticulum, SR). This differential allows the calcium ion messenger that carries information
as diverse as contraction, metabolism, apoptosis, proliferation and/or hypertrophic growth. The mechanisms responsible for generating a Ca signal greatly differ from one cell type to another.
In the different types of vascular smooth muscle cells (VSMC), enormous variations do exist with regard to the mechanisms responsible for generating Ca signal. In each VSMC phenotype (synthetic/proliferating and contractile [1], tonic or phasic), the Ca signaling system is adapted to its particular function and is due to the specific patterns of expression and regulation of Ca.
For instance, in contractile VSMCs, the initiation of contractile events is driven by mem- brane depolarization; and the principal entry-point for extracellular Ca is the voltage-operated L-type calcium channel (LTCC). In contrast, in synthetic/proliferating VSMCs, the principal way-in for extracellular Ca is the store-operated calcium (SOC) channel.
Whatever the cell type, the calcium signal consists of  limited elevations of cytosolic free calcium ions in time and space. The calcium pump, sarco/endoplasmic reticulum Ca ATPase (SERCA), has a critical role in determining the frequency of SR Ca release by upload into the sarcoplasmic
sensitivity of  SR calcium channels, Ryanodin Receptor, RyR and Inositol tri-Phosphate Receptor, IP3R.
Synthetic VSMCs have a fibroblast appearance, proliferate readily, and synthesize increased levels of various extracellular matrix components, particularly fibronectin, collagen types I and III, and tropoelastin [1].
Contractile VSMCs have a muscle-like or spindle-shaped appearance and well-developed contractile apparatus resulting from the expression and intracellular accumulation of thick and thin muscle filaments [1].
Schematic representation of Calcium Cycling in Contractile and Proliferating VSMCs

Schematic representation of Calcium Cycling in Contractile and Proliferating VSMCs


Figure 1. Schematic representation of Calcium Cycling in Contractile and Proliferating VSMCs.

Left panel: schematic representation of calcium cycling in quiescent /contractile VSMCs. Contractile re-sponse is initiated by extracellular Ca influx due to activation of Receptor Operated Ca (through phosphoinositol-coupled receptor) or to activation of L-Type Calcium channels (through an increase in luminal pressure). Small increase of cytosolic due IP3 binding to IP3R (puff) or RyR activation by LTCC or ROC-dependent Ca influx leads to large SR Ca IP3R or RyR clusters (“Ca -induced Ca SR calcium pumps (both SERCA2a and SERCA2b are expressed in quiescent VSMCs), maintaining high concentration of cytosolic Ca and setting the sensitivity of RyR or IP3R for the next spike.
Contraction of VSMCs occurs during oscillatory Ca transient.
Middle panel: schematic representa tion of atherosclerotic vessel wall. Contractile VSMC are located in the media layer, synthetic VSMC are located in sub-endothelial intima.
Right panel: schematic representation of calcium cycling in quiescent /contractile VSMCs. Agonist binding to phosphoinositol-coupled receptor leads to the activation of IP3R resulting in large increase in cytosolic Ca calcium pumps (only SERCA2b, having low turnover and low affinity to Ca depletion leads to translocation of SR Ca sensor STIM1 towards PM, resulting in extracellular Ca influx though opening of Store Operated Channel (CRAC). Resulted steady state Ca transient is critical for activation of proliferation-related transcription factors ‘NFAT).
Abbreviations: PLC – phospholipase C; PM – plasma membrane; PP2B – Ca /calmodulin-activated protein phosphatase 2B (calcineurin); ROC- receptor activated channel; IP3 – inositol-1,4,5-trisphosphate, IP3R – inositol-1,4,5- trisphosphate receptor; RyR – ryanodine receptor; NFAT – nuclear factor of activated T-lymphocytes; VSMC – vascular smooth muscle cells; SERCA – sarco(endo)plasmic reticulum Ca sarcoplasmic reticulum.


Time for New DNA Synthesis and Sequencing Cost Curves

By Rob Carlson

I’ll start with the productivity plot, as this one isn’t new. For a discussion of the substantial performance increase in sequencing compared to Moore’s Law, as well as the difficulty of finding this data, please see this post. If nothing else, keep two features of the plot in mind: 1) the consistency of the pace of Moore’s Law and 2) the inconsistency and pace of sequencing productivity. Illumina appears to be the primary driver, and beneficiary, of improvements in productivity at the moment, especially if you are looking at share prices. It looks like the recently announced NextSeq and Hiseq instruments will provide substantially higher productivities (hand waving, I would say the next datum will come in another order of magnitude higher), but I think I need a bit more data before officially putting another point on the plot.




Illumina’s instruments are now responsible for such a high percentage of sequencing output that the company is effectively setting prices for the entire industry. Illumina is being pushed by competition to increase performance, but this does not necessarily translate into lower prices. It doesn’t behoove Illumina to drop prices at this point, and we won’t see any substantial decrease until a serious competitor shows up and starts threatening Illumina’s market share. The absence of real competition is the primary reason sequencing prices have flattened out over the last couple of data points.

Note that the oligo prices above are for column-based synthesis, and that oligos synthesized on arrays are much less expensive. However, array synthesis comes with the usual caveat that the quality is generally lower, unless you are getting your DNA from Agilent, which probably means you are getting your dsDNA from Gen9.

Note also that the distinction between the price of oligos and the price of double-stranded sDNA is becoming less useful. Whether you are ordering from Life/Thermo or from your local academic facility, the cost of producing oligos is now, in most cases, independent of their length. That’s because the cost of capital (including rent, insurance, labor, etc) is now more significant than the cost of goods. Consequently, the price reflects the cost of capital rather than the cost of goods. Moreover, the cost of the columns, reagents, and shipping tubes is certainly more than the cost of the atoms in the sDNA you are ostensibly paying for. Once you get into longer oligos (substantially larger than 50-mers) this relationship breaks down and the sDNA is more expensive. But, at this point in time, most people aren’t going to use longer oligos to assemble genes unless they have a tricky job that doesn’t work using short oligos.

Looking forward, I suspect oligos aren’t going to get much cheaper unless someone sorts out how to either 1) replace the requisite human labor and thereby reduce the cost of capital, or 2) finally replace the phosphoramidite chemistry that the industry relies upon.

IDT’s gBlocks come at prices that are constant across quite substantial ranges in length. Moreover, part of the decrease in price for these products is embedded in the fact that you are buying smaller chunks of DNA that you then must assemble and integrate into your organism of choice.

Someone who has purchased and assembled an absolutely enormous amount of sDNA over the last decade, suggested that if prices fell by another order of magnitude, he could switch completely to outsourced assembly. This is a potentially interesting “tipping point”. However, what this person really needs is sDNA integrated in a particular way into a particular genome operating in a particular host. The integration and testing of the new genome in the host organism is where most of the cost is. Given the wide variety of emerging applications, and the growing array of hosts/chassis, it isn’t clear that any given technology or firm will be able to provide arbitrary synthetic sequences incorporated into arbitrary hosts.

 TrackBack URL: http://www.synthesis.cc/cgi-bin/mt/mt-t.cgi/397


Startup to Strengthen Synthetic Biology and Regenerative Medicine Industries with Cutting Edge Cell Products

28 Nov 2013 | PR Web

Dr. Jon Rowley and Dr. Uplaksh Kumar, Co-Founders of RoosterBio, Inc., a newly formed biotech startup located in Frederick, are paving the way for even more innovation in the rapidly growing fields of Synthetic Biology and Regenerative Medicine. Synthetic Biology combines engineering principles with basic science to build biological products, including regenerative medicines and cellular therapies. Regenerative medicine is a broad definition for innovative medical therapies that will enable the body to repair, replace, restore and regenerate damaged or diseased cells, tissues and organs. Regenerative therapies that are in clinical trials today may enable repair of damaged heart muscle following heart attack, replacement of skin for burn victims, restoration of movement after spinal cord injury, regeneration of pancreatic tissue for insulin production in diabetics and provide new treatments for Parkinson’s and Alzheimer’s diseases, to name just a few applications.

While the potential of the field is promising, the pace of development has been slow. One main reason for this is that the living cells required for these therapies are cost-prohibitive and not supplied at volumes that support many research and product development efforts. RoosterBio will manufacture large quantities of standardized primary cells at high quality and low cost, which will quicken the pace of scientific discovery and translation to the clinic. “Our goal is to accelerate the development of products that incorporate living cells by providing abundant, affordable and high quality materials to researchers that are developing and commercializing these regenerative technologies” says Dr. Rowley


Life at the Speed of Light


NHMU Lecture featuring – J. Craig Venter, Ph.D.
Founder, Chairman, and CEO – J. Craig Venter Institute; Co-Founder and CEO, Synthetic Genomics Inc.

J. Craig Venter, Ph.D., is Founder, Chairman, and CEO of the J. Craig Venter Institute (JVCI), a not-for-profit, research organization dedicated to human, microbial, plant, synthetic and environmental research. He is also Co-Founder and CEO of Synthetic Genomics Inc. (SGI), a privately-held company dedicated to commercializing genomic-driven solutions to address global needs.

In 1998, Dr. Venter founded Celera Genomics to sequence the human genome using new tools and techniques he and his team developed.  This research culminated with the February 2001 publication of the human genome in the journal, Science. Dr. Venter and his team at JVCI continue to blaze new trails in genomics.  They have sequenced and a created a bacterial cell constructed with synthetic DNA,  putting humankind at the threshold of a new phase of biological research.  Whereas, we could  previously read the genetic code (sequencing genomes), we can now write the genetic code for designing new species.

The science of synthetic genomics will have a profound impact on society, including new methods for chemical and energy production, human health and medical advances, clean water, and new food and nutritional products. One of the most prolific scientists of the 21st century for his numerous pioneering advances in genomics,  he  guides us through this emerging field, detailing its origins, current challenges, and the potential positive advances.

His work on synthetic biology truly embodies the theme of “pushing the boundaries of life.”  Essentially, Venter is seeking to “write the software of life” to create microbes designed by humans rather than only through evolution. The potential benefits and risks of this new technology are enormous. It also requires us to examine, both scientifically and philosophically, the question of “What is life?”

J Craig Venter wants to digitize DNA and transmit the signal to teleport organisms


2013 Genomics: The Era Beyond the Sequencing of the Human Genome: Francis Collins, Craig Venter, Eric Lander, et al.


Human Longevity Inc (HLI) – $70M in Financing of Venter’s New Integrative Omics and Clinical Bioinformatics




Where Will the Century of Biology Lead Us?

By Randall Mayes

A technology trend analyst offers an overview of synthetic biology, its potential applications, obstacles to its development, and prospects for public approval.

  • In addition to boosting the economy, synthetic biology projects currently in development could have profound implications for the future of manufacturing, sustainability, and medicine.
  • Before society can fully reap the benefits of synthetic biology, however, the field requires development and faces a series of hurdles in the process. Do researchers have the scientific know-how and technical capabilities to develop the field?

Biology + Engineering = Synthetic Biology

Bioengineers aim to build synthetic biological systems using compatible standardized parts that behave predictably. Bioengineers synthesize DNA parts—oligonucleotides composed of 50–100 base pairs—which make specialized components that ultimately make a biological system. As biology becomes a true engineering discipline, bioengineers will create genomes using mass-produced modular units similar to the microelectronics and computer industries.

Currently, bioengineering projects cost millions of dollars and take years to develop products. For synthetic biology to become a Schumpeterian revolution, smaller companies will need to be able to afford to use bioengineering concepts for industrial applications. This will require standardized and automated processes.

A major challenge to developing synthetic biology is the complexity of biological systems. When bioengineers assemble synthetic parts, they must prevent cross talk between signals in other biological pathways. Until researchers better understand these undesired interactions that nature has already worked out, applications such as gene therapy will have unwanted side effects. Scientists do not fully understand the effects of environmental and developmental interaction on gene expression. Currently, bioengineers must repeatedly use trial and error to create predictable systems.

Similar to physics, synthetic biology requires the ability to model systems and quantify relationships between variables in biological systems at the molecular level.

The second major challenge to ensuring the success of synthetic biology is the development of enabling technologies. With genomes having billions of nucleotides, this requires fast, powerful, and cost-efficient computers. Moore’s law, named for Intel co-founder Gordon Moore, posits that computing power progresses at a predictable rate and that the number of components in integrated circuits doubles each year until its limits are reached. Since Moore’s prediction, computer power has increased at an exponential rate while pricing has declined.

DNA sequencers and synthesizers are necessary to identify genes and make synthetic DNA sequences. Bioengineer Robert Carlson calculated that the capabilities of DNA sequencers and synthesizers have followed a pattern similar to computing. This pattern, referred to as the Carlson Curve, projects that scientists are approaching the ability to sequence a human genome for $1,000, perhaps in 2020. Carlson calculated that the costs of reading and writing new genes and genomes are falling by a factor of two every 18–24 months. (see recent Carlson comment on requirement to read and write for a variety of limiting  conditions).

Startup to Strengthen Synthetic Biology and Regenerative Medicine Industries with Cutting Edge Cell Products


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


Synthesizing Synthetic Biology: PLOS Collections


Capturing ten-color ultrasharp images of synthetic DNA structures resembling numerals 0 to 9


Silencing Cancers with Synthetic siRNAs


Genomics Now—and Beyond the Bubble

Futurists have touted the twenty-first century as the century of biology based primarily on the promise of genomics. Medical researchers aim to use variations within genes as biomarkers for diseases, personalized treatments, and drug responses. Currently, we are experiencing a genomics bubble, but with advances in understanding biological complexity and the development of enabling technologies, synthetic biology is reviving optimism in many fields, particularly medicine.

BY MICHAEL BROOKS    17 APR, 2014     http://www.newstatesman.com/

Michael Brooks holds a PhD in quantum physics. He writes a weekly science column for the New Statesman, and his most recent book is The Secret Anarchy of Science.

The basic idea is that we take an organism – a bacterium, say – and re-engineer its genome so that it does something different. You might, for instance, make it ingest carbon dioxide from the atmosphere, process it and excrete crude oil.

That project is still under construction, but others, such as using synthesised DNA for data storage, have already been achieved. As evolution has proved, DNA is an extraordinarily stable medium that can preserve information for millions of years. In 2012, the Harvard geneticist George Church proved its potential by taking a book he had written, encoding it in a synthesised strand of DNA, and then making DNA sequencing machines read it back to him.

When we first started achieving such things it was costly and time-consuming and demanded extraordinary resources, such as those available to the millionaire biologist Craig Venter. Venter’s team spent most of the past two decades and tens of millions of dollars creating the first artificial organism, nicknamed “Synthia”. Using computer programs and robots that process the necessary chemicals, the team rebuilt the genome of the bacterium Mycoplasma mycoides from scratch. They also inserted a few watermarks and puzzles into the DNA sequence, partly as an identifying measure for safety’s sake, but mostly as a publicity stunt.

What they didn’t do was redesign the genome to do anything interesting. When the synthetic genome was inserted into an eviscerated bacterial cell, the new organism behaved exactly the same as its natural counterpart. Nevertheless, that Synthia, as Venter put it at the press conference to announce the research in 2010, was “the first self-replicating species we’ve had on the planet whose parent is a computer” made it a standout achievement.

Today, however, we have entered another era in synthetic biology and Venter faces stiff competition. The Steve Jobs to Venter’s Bill Gates is Jef Boeke, who researches yeast genetics at New York University.

Boeke wanted to redesign the yeast genome so that he could strip out various parts to see what they did. Because it took a private company a year to complete just a small part of the task, at a cost of $50,000, he realised he should go open-source. By teaching an undergraduate course on how to build a genome and teaming up with institutions all over the world, he has assembled a skilled workforce that, tinkering together, has made a synthetic chromosome for baker’s yeast.


Stepping into DIYbio and Synthetic Biology at ScienceHack

Posted April 22, 2014 by Heather McGaw and Kyrie Vala-Webb

We got a crash course on genetics and protein pathways, and then set out to design and build our own pathways using both the “Genomikon: Violacein Factory” kit and Synbiota platform. With Synbiota’s software, we dragged and dropped the enzymes to create the sequence that we were then going to build out. After a process of sketching ideas, mocking up pathways, and writing hypotheses, we were ready to start building!

The night stretched long, and at midnight we were forced to vacate the school. Not quite finished, we loaded our delicate bacteria, incubator, and boxes of gloves onto the bus and headed back to complete our bacterial transformation in one of our hotel rooms. Jammed in between the beds and the mini-fridge, we heat-shocked our bacteria in the hotel ice bucket. It was a surreal moment.

While waiting for our bacteria, we held an “unconference” where we explored bioethics, security and risk related to synthetic biology, 3D printing on Mars, patterns in juggling (with live demonstration!), and even did a Google Hangout with Rob Carlson. Every few hours, we would excitedly check in on our bacteria, looking for bacterial colonies and the purple hue characteristic of violacein.

Most impressive was the wildly successful and seamless integration of a diverse set of people: in a matter of hours, we were transformed from individual experts and practitioners in assorted fields into cohesive and passionate teams of DIY biologists and science hackers. The ability of everyone to connect and learn was a powerful experience, and over the course of just one weekend we were able to challenge each other and grow.

Returning to work on Monday, we were hungry for more. We wanted to find a way to bring the excitement and energy from the weekend into the studio and into the projects we’re working on. It struck us that there are strong parallels between design and DIYbio, and we knew there was an opportunity to bring some of the scientific approaches and curiosity into our studio.



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