Clinical trials in systemic sclerosis: lessons learned and outcomes

The pathogenesis of systemic sclerosis (SSc) is complex and largely unclear. The clinical heterogeneity of the disease and its progression over a number of years makes the choice of endpoints in the design of clinical trials difficult. The overwhelming need in this disease is to diagnose it early and identify those patients who will benefit most from early, aggressive treatment that potentially can alter the clinical disease course. To achieve this, innumerable challenges must be overcome. This article reviews data from recent clinical trials and the lessons derived from retrospective observational studies, databases, and patient registries. Taken together, these observations will help to improve our understanding of the diverse clinical course of SSc and permit refinement of existing outcome measures for the design of future clinical trials, in which the likelihood of observing a positive treatment effect with the drugs at our disposal will be maximized.     

Analytical lessons learned from selected therapeutic protein drug comparability studies

The successful implementation of process and product changes for a therapeutic protein drug, both during clinical development and after commercialization, requires a detailed evaluation of their impact on the protein's structure and biological functionality. This analysis is called a comparability exercise and includes a data driven assessment of biochemical equivalence and biological characterization using a cadre of analytical methodologies. This review focuses on describing analytical results and lessons learned from selected published therapeutic protein comparability case studies both for bulk drug substance and final drug product. An overview of the currently available analytical methodologies typically used is presented as well as a discussion of new emerging analytical techniques. The potential utility of several novel analytical approaches to comparability studies is discussed including distribution and stability of protein drugs in vivo, and enhanced evaluation of higher-order protein structure in actual formulations using hydrogen/deuterium exchange mass spectrometry, two-dimensional nuclear magnetic resonance fingerprinting or empirical phase diagrams. In addition, new methods for detecting and characterizing protein aggregates and particles are presented as these degradants are of current industry-wide concern. The critical role that analytical methodologies play in elucidating the structure–function relationships for therapeutic protein products during the overall assessment of comparability is discussed.     

VTEC: lessons learned from British outbreaks

Important Escherichia coli O157 outbreaks in England and Scotland since 1982-83 are reviewed. The scientific lessons learned from them are described and their legal consequences outlined. The light shed by them on relationships between law and science is discussed, and questions of blame are analysed in the context of Reason's 'resident pathogen' metaphor and Vaughan's study of the 1986 Challenger Space Shuttle disaster.     

Mitochondrial efficiency: lessons learned from transgenic mice

Metabolic research has, like most areas of research in the life sciences, been affected dramatically by the application of transgenic technologies. Within the specific area of bioenergetics it has been thought that transgenic approaches in mice would provide definitive proof for some longstanding metabolic theories and assumptions. Here we review a number of transgenic approaches that have been used in mice to address theories of mitochondrial efficiency. The focus is largely on genes that affect the coupling of energy substrate oxidation to ATP synthesis, and thus, mice in which the uncoupling protein (Ucp) genes are modified are discussed extensively. Transgenic approaches have indeed provided proof-of-concept in some instances, but in many other instances they have yielded results that are in contrast to initial hypotheses. Many studies have also shown that genetic background can affect phenotypic outcomes, and that the upregulated expression of genes that are related to the modified gene often complicates the interpretation of findings.     

Insights into the MCM functional mechanism: lessons learned from the archaeal MCM complex

The helicase function of the minichromosome maintenance protein (MCM) is essential for genomic DNA replication in archaea and eukaryotes. There has been rapid progress in studies of the structure and function of MCM proteins from different organisms, leading to better understanding of the MCM helicase mechanism. Because there are a number of excellent reviews on this topic, we will use this review to summarize some of the recent progress, with particular focus on the structural aspects of MCM and their implications for helicase function. Given the hexameric and double hexameric architecture observed by X-ray crystallography and electron microscopy of MCMs from archaeal and eukaryotic cells, we summarize and discuss possible unwinding modes by either a hexameric or a double hexameric helicase. Additionally, our recent crystal structure of a full length archaeal MCM has provided structural information on an intact, multi-domain MCM protein, which includes the salient features of four unusual β-hairpins from each monomer, and the side channels of a hexamer/double hexamer. These new structural data enable a closer examination of the structural basis of the unwinding mechanisms by MCM.     

Dissecting phosphorylation networks: lessons learned from yeast

Protein phosphorylation continues to be regarded as one of the most important post-translational modifications found in eukaryotes and has been implicated in key roles in the development of a number of human diseases. In order to elucidate roles for the 518 human kinases, phosphorylation has routinely been studied using the budding yeast Saccharomyces cerevisiae as a model system. In recent years, a number of technologies have emerged to globally map phosphorylation in yeast. In this article, we review these technologies and discuss how these phosphorylation mapping efforts have shed light on our understanding of kinase signaling pathways and eukaryotic proteomic networks in general.     

Transport of defense compounds from source to sink: lessons learned from glucosinolates

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Origin of resistance to Imatinib mesylate: lessons learned from this experience

For drug development and pharmaceutical research, targeting the molecular abnormalities is considered as a new challenge. A number of diseases including cancer are linked to perturbation of tyrosine kinase (TK). Imatinib (Glivec or Gleevec, Novartis), the most potent inhibitor of c-abl TK, was recently developed. This molecule has been approved in the treatment of chronic myeloid leukemia (CML). However, emergence of clinical resistance regarding a low rate of CML patients leads to intensive research. In the current article, we discuss the data and the mechanism of the resistance phenomenon. This review illustrates the important requirement to transfer back the information from patient to laboratory in order to improve future drug design.     

Regulation of immunity to malaria: valuable lessons learned from murine models

A major advance in immunology has been the establishment of a framework for analysing how certain immune responses dominate following exposure to a particular pathogen or antigen. CD4(+) T helper (Th) cells can be separated into two major subsets which mediate qualitatively distinct cell-mediated (Th1) and humoral (Th2) immune responses. Immunity to most pathogens can be broadly categorized into a predominant protective response of either type. A characteristic of murine malarias is that primary infections with asexual erythrocytic parasites (the pathogenic stage of the malaria life cycle) generate a host protective immune response with a broad spectrum of Th1- and Th2-type CD4(+) T-cell involvement and so can be examined as models of the interaction of Th1 and Th2 cells during an immune response to an infectious agent. Andrew Taylor-Robinson here describes recent events in the dissection of the mechanisms responsible for the generation of protective immunity to Plasmodium chabaudi chabaudi and other experimental malarias in mice.     

Dangerous exercise: lessons learned from dysregulated inflammatory responses to physical activity.

Exercise elicits an immunological "danger" type of stress and inflammatory response that, on occasion, becomes dysregulated and detrimental to health. Examples include anaphylaxis, exercise-induced asthma, overuse syndromes, and exacerbation of intercurrent illnesses. In dangerous exercise, the normal balance between pro- and anti-inflammatory responses is upset. A possible pathophysiological mechanism is characterized by the concept of exercise modulation of previously activated leukocytes. In this model, circulating leukocytes are rendered more responsive than normal to the immune stimulus of exercise. For example, in the case of exercise anaphylaxis, food-sensitized immune cells may be relatively innocuous until they are redistributed during exercise from gut-associated circulatory depots, like the spleen, into the central circulation. In the case of asthma, the prior activation of leukocytes may be the result of genetic or environmental factors. In the case of overuse syndromes, the normally short-lived neutrophil may, because of acidosis and hypoxia, inhibit apoptosis and play a role in prolongation of inflammation rather than healing. Dangerous exercise demonstrates that the stress/inflammatory response caused by physical activity is robust and sufficiently powerful, perhaps, to alter subsequent responses. These longer term effects may occur through as yet unexplored mechanisms of immune "tolerance" and/or by a training-associated reduction in the innate immune response to brief exercise. A better understanding of sometimes failed homeostatic physiological systems can lead to new insights with significant implication for clinical translation.     

The core-binding factor leukemias: lessons learned from murine models

The recent development of murine models of core-binding factor leukemias has provided important insights into the underlying molecular pathology of this common subtype of acute myeloid leukemia. Evidence from these models supports the idea that acute myeloid leukemia 1/core-binding factor beta-subunit (AML1/CBFbeta) has a critical role in the control of the self-renewal capacity of hematopoietic stem cells and their progeny. Moreover, the accumulated data demonstrate that the expression of translocation-encoded AML1 or CBFbeta fusion proteins are insufficient by themselves to induce a full leukemic phenotype. The models that have been developed should prove to be of value for defining the range of mutations that can cooperate with AML1/CBFbeta fusion proteins, and for assessing novel therapies targeted toward the pathways that are altered by the expression of these fusion proteins.     

Protozoa and pathogenic bacteria: lessons learned from Legionella pneumophila

Bacterivorous protozoa and bacteria have been in co-existence since the origin of life. This co-existence has led unequivocally to the evolution of many different co-interactions. Most bacteria are ingested and digested, but many escape ingestion for various reasons. Others are ingested but evade digestion, and a few, notoriously Legionella pneumophila , even have the capacity of multiplying within the protozoan host. The aims of this study were to elucidate the interactions of various multi-drug-resistant Staphylococcus aureus (MRSA) strains, Listeria monocytogenes sv4b, and Escherichia coli K12 with the amoeba, Acanthamoeba polyphaga . To evaluate the interactions, we set up co-cultures in Neffs' amoebic saline, at a multiplicity of invasion (MOI) of 1:100 of amoeba to bacteria, and a temperature of 37°C, although the effects of MOI and temperature were also assessed. Survival of bacteria and amoeba was checked at regular intervals, coupled with microscopy. It was discovered under our test conditions, that E. coli was ingested and digested by A. polyphaga , but in contrast, L. monocytogenes , had the capacity to flourish in the presence of A. polyphaga . We also report, for the first time, that all six MRSA isolates tested, survived and replicated in association with A. polyphaga , in comparison to conditions where amoebae were absent. Indeed, we also have evidence suggesting that increases in MRSA, in the presence of A. polyphaga , may be attributable to intracellular survival and replication. These findings have profound implications for the hospital environment, where Acanthamoeba sp., are also commonly isolated. In conclusion, this study illustrates the significance of protozoa as vehicles augmenting the survival of MRSA and L. monocytogenes in the environment.     

Immunology of fungal infections: lessons learned from animal models

The continuing AIDS epidemic coupled with increased usage of immunosuppressive drugs to prevent organ rejection or treat autoimmune diseases has resulted in an increase in individuals at risk for acquiring fungal diseases. These concerns highlight the need to elucidate mechanisms of inducing protective immune responses against fungal pathogens. Consequently, several experimental models of human mycoses have been developed to study these diseases. The availability of transgenic animal models allows for in-depth analysis of specific components, receptors, and signaling pathways that elicit protection against fungal diseases. This review focuses on recent advances in our understanding of immune responses to fungal infections gained using animal models.     

Protein folding diseases and neurodegeneration: lessons learned from yeast

Budding yeast Saccharomyces cerevisiae has proven to be a valuable model organism for studying fundamental cellular processes across the eukaryotic kingdom including man. In this respect, complementation assays, in which the yeast protein is replaced by a homologous protein from another organism, have been very instructive. A newer trend is to use the yeast cell factory as a toolbox to understand cellular processes controlled by proteins for which the yeast lacks functional counterparts. An increasing number of studies have indicated that S. cerevisiae is a suitable model system to decipher molecular mechanisms involved in a variety of neurodegenerative disorders caused by aberrant protein folding. Here we review the current knowledge gained by the use of so-called humanized yeasts in the field of Huntington's, Parkinson's and Alzheimer's diseases.