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We have recently shown that transforming growth factor-beta (TGF beta) acts in an autocrine manner to maintain the beating rate of neonatal rat cardiac myocytes cultured in serum-free medium on cardiac fibroblast matrix. Interleukin-1 beta (IL-1 beta) suppresses the myocyte-beating rate, and TGF beta antagonizes this effect. We now show that TGF beta and IL-1 beta also have antagonistic effects on the secretion of nitric oxide (NO) by these myocytes, and that NO secretion, the activity of NO synthase (NOS), and expression of the inducible form of NOS correlate inversely with the effects of these two agents on the beating rate. Western blot analysis shows that treatment of myocytes with TGF beta antagonizes the induction of NOS after treatment with IL-1 beta. Release of NO, induced by IL-1 beta, is dependent upon the availability of the substrate, L-arginine, and is suppressed by a competitive inhibitor, NG-monomethyl-L-arginine. L-Arginine (0.25 mM) also suppresses, and NG-monomethyl-L-arginine (0.5 mM) enhances the myocyte-beating rate. Treatment with IL-1 beta, but not TGF beta, increases cellular cGMP, presumably by activation of guanylate cyclase by NO. Methylene blue, an inhibitor of guanylate cyclase, reverses the suppression of beating caused by IL-1 beta. Bacterial lipopolysaccharide, present in the serum-free medium, is a coinducer of NO secretion. The suppressive effects of NO on the beating rate can be overcome by altering either the set of cytokines employed to induce NO or the matrix on which the myocytes are cultured, demonstrating that additional parameters are also involved in regulation of the beating rate.

Upon activation macrophages kill or inhibit growth of both intracellular and extracellular parasites as well as malignant host cells (1–6; for review: 7–11). A number of secretory products of the macrophage, such as reactive oxygen intermediates (ROI), reactive nitrogen intermediates (RNI), tumor necrosis factor a (TNF) and interleukin 1 (IL-1) are believed to mediate — directly or indirectly — the tumoricidal and microbicidal capacity of the macrophage (11). In addition to their effector function macrophages play an important role in the activation of other components of the immune system, such as Thelper cells, cytotoxic T-cells and natural killer cells (12).

Sepsis is defined by the systemic response to an infection, governed by dynamic interactions between the tissues, immune cells and inflammatory mediators. We used the Immunetrics platform to build a large-scale mathematical model that encompasses these biological components. The model incorporates a virtual clinician, an automated system to examine simulated patients' status at clinically relevant intervals and administer standard of care interventions as necessary, thereby altering the dynamics of the disease state. The model reproduces many characteristics of systemic response to an infection, including the time course of cytokines, coagulation factors, clinical markers, early and late organ failure, and early versus late deaths.

Results: Mortality was 14% (76 patients). Increases in pressures were associated with small but statistically significant decreases in HR complexity; associations between RR and HR complexity were not significant (Table 1). Decreased HR complexity remained a significant explanatory variable in logistic regression, after incorporating ventilator parameters (Table 2). Conclusions: 1. Pressure, but not rate, of mechanical ventilation may be important in interpreting integer HR complexity.

Recombinant mouse interleukin 10 (IL-10) was exceedingly potent at suppressing the ability of mouse peritoneal macrophages (m phi) to release tumor necrosis factor alpha (TNF-alpha). The IC50 of IL-10 for the suppression of TNF-alpha release induced by 0.5 microgram/ml lipopolysaccharide was 0.04 +/- 0.03 U/ml, with as little as 1 U/ml suppressing TNF-alpha production by a factor of 21.4 +/- 2.5. At 10 U/ml, IL-10 markedly suppressed m phi release of reactive oxygen intermediates (ROI) (IC50 3.7 +/- 1.8 U/ml), but only weakly inhibited m phi release of reactive nitrogen intermediates (RNI). Since TNF-alpha is a T cell growth and differentiation factor, whereas ROI and RNI are known to inhibit lymphocyte function, it is possible that m phi exposed to low concentrations of IL-10 suppress lymphocytes. m phi deactivated by higher concentrations of IL-10 might be permissive for the growth of microbial pathogens and tumor cells, as TNF-alpha, ROI, and RNI are major antimicrobial and tumoricidal products of m phi. IL-10's effects on m phi overlap with but are distinct from the effects of the two previously described cytokines that suppress the function of mouse m phi, transforming growth factor beta and macrophage deactivation factor. Based on results with neutralizing antibodies, all three m phi suppressor factors appear to act independently.

The development of personalized medicine is a primary objective of the medical community and increasingly also of funding and registration agencies. Modeling is generally perceived as a key enabling tool to target this goal. Agent-Based Models (ABMs) have previously been used to simulate inflammation at various scales up to the whole-organism level. We extended this approach to the case of a novel, patient-specific ABM that we generated for vocal fold inflammation, with the ultimate goal of identifying individually optimized treatments. ABM simulations reproduced trajectories of inflammatory mediators in laryngeal secretions of individuals subjected to experimental phonotrauma up to 4 hrs post-injury, and predicted the levels of inflammatory mediators 24 hrs post-injury. Subject-specific simulations also predicted different outcomes from behavioral treatment regimens to which subjects had not been exposed. We propose that this translational application of computational modeling could be used to design patient-specific therapies for the larynx, and will serve as a paradigm for future extension to other clinical domains.

finding the equivalent of a wild-type thermodynamic state of an existing gene segment in other (unknown) instantiations of the biologic system, in both noncoding and coding gene regions. In coding regions, TT processes amino acid sequences in a codonbased 3-letter code with additional information about choice of codon alternatives. Differences in TT (mutant-wt, case-control) were used to assess objectives 1 and 2 for the genes coding for human CD14, TLR4, tumor necrosis factor (TNF), and activated protein C (APC). Results: Comparison of calculated TT profiles of the human CD14, TLR4, TNF, and APC genes with 16 species–based conservation profiles (ENCODE http://genome.ucsc.edu/) validated the evolutionary relevance of TT profiles, showing a significant correlation of interspecies conservation to low thermodynamically tolerant regions. Network analysis of context similarities captured by TT descriptors in TNF revealed a hierarchical block structure of this network that directly identifies precursor and mature protein segments, structural domains in the mature protein structure, and noncontiguous regions that are in direct contact in the folded TNF. For CD14 and APC, TT analysis gave insights with regard to protein-protein interaction sites in the oligomeric structure, and the correlation of structural periodicity to the periodicity of the TT characterization of the coding sequence. Thermodynamic tolerance profiles were used for interspecies comparisons of human-mouse (H-M) pairs, based on the assumption that optimal alignment of sequence H and M is the alignment in which the difference between TT(H) and TT(M) is minimal. This approach was used to obtain optimal unbiased differential TT potentials for H and M paralogs that were compared with various aspects of function. Conclusions: Thermodynamic tolerance analysis is a novel method that appears applicable to various types of analysis of inflammationrelated genes. This approach of using networks of TT-based similarities captured in corresponding adjacency matrices allows for the combination of both local and global impact of a given SNP at a particular locus. Differences between wild-type and mutant coding sequence TT descriptors in and adjacency matrices capturing networks of TT-based similarities are useful for the single sequence–based quantitative characterization of structural or functional impact of a mutation on a given gene.

whereas a 10-fold increase in k2 results in similar primary and secondary signaling peaks. Conclusions: Our simulations suggest that a simplified model of LPS/TLR4 signaling can account for complex preconditioning phenomena without invoking a specific signaling inhibition mechanism, but rather based on the dynamics of the signaling response itself, as well as the timing and magnitude of the LPS stimuli.

The interaction between the complex pattern of cytokine release and remote organ dysfunction after trauma is incompletely understood. The aim of this study was to investigate the pattern of cytokine release and its association with the evolution of remote organ dysfunction after bilateral femur fracture. Male C57/BL6 mice were euthanized at six different time points (1-6 h) after bilateral femur fracture. Serum cytokine concentrations were measured with the Luminex multiplexing platform, and serum alanine aminotransferase levels were measured with the Vitros 950 Chemistry System. Hepatic and pulmonary myeloperoxidase activity was determined with an enzyme-linked immunosorbent assay kit. Permeability changes of the lung were assessed via bronchoalveolar lavage, and those of the liver via assessment of edema formation. Serum TNF-alpha was unchanged in the fracture group throughout the experiment. Serum IL-6 and keratinocyte levels peaked at 5 h postinjury, whereas IL-10 levels peaked at 2 and 6 h. A brief IL-1beta peak was observed at 3 h after fracture. Hepatic and pulmonary myeloperoxidase activity was significantly elevated within 1 h after trauma. Hepatic permeability was significantly increased within 2 h, and pulmonary permeability was significantly increased within 6 h after injury. Serum alanine aminotransferase levels peaked at 3 and 5 h postinjury. The pattern of serum IL-6, keratinocyte, IL-10, and IL-1beta release was dynamic, whereas no significant elevations in TNF-alpha were observed. The early hepatic and pulmonary infiltration of polymorphonuclear cells occurred in the absence of significantly elevated serum cytokine levels, suggesting that either early minor changes with an unbalance in inflammatory mediators or locally produced cytokines may initiate this process.

Nitric Oxide and the Cardiovascular Systemedited by J.H. Loscalzo and J.A. VitaHumana Press, 2000. $175.00 (hardback) (vii + 616 pages)ISBN 0 89603 620 0The massive increase in the amount of research into the roles of nitric oxide (NO) in myriad biological processes is staggering, so much so, that it is essentially impossible to write all-encompassing review articles in this field. Recent years have seen several books devoted to the roles of NO in defined physiological or pathological processes, and one of the most recent, Nitric Oxide and the Cardiovascular System, is no exception.Although NO is without a doubt important in all aspects of physiology, in 1998 the Nobel Committee decided that NO was primarily a molecule of importance in the vasculature. A reader of this book will certainly come away with the impression that this deceptively simple molecule has a very complex chemistry, is a vasodilator key to the function of endothelial cells, modulates the function of cardiac myocytes, and might improve the function of transplanted organs. Furthermore, it is clear that novel strategies involving chemical NO donors and gene therapy are being suggested as possible solutions to intractable problems such as restenosis post-angioplasty or post-stenting, transplant rejection, acute respiratory distress syndrome and atherosclerosis.Nitric Oxide and the Cardiovascular System is organized into three broad sections: ‘Biology of Nitric Oxide’, ‘Cardiovascular Pathophysiology of Nitric Oxide’ and ‘Nitric Oxide in Cardiovascular Therapeutics’. The first section starts with the chemistry that underlies much of the fascinating biology of NO and is followed by a discussion of the cellular and molecular biology of nitric oxide synthases (NOS), the enzymes that produce NO in animals. These chapters are de rigueur in any book about NO and provide the basic information for those new to the NO field in addition to some recent updates. The book then begins its true focus on the cardiovascular effects of NO, with a chapter on signal transduction mediated by NO, which discusses the classical (and Nobel-Prize-winning) cGMP pathway stimulated by NO and more recent studies on nitrosylation of target signal transduction proteins. Subsequent chapters in the first section deal with the regulation of gene expression by NO, the roles of NO in apoptosis, the modulation of ion channels by NO, the control of vasomotor and platelet functions by NO, and the effects of NO on cardiac myocytes and fibroblasts.The second section, ‘Cardiovascular Pathophysiology of Nitric Oxide’, contains discussions of the relevance of NO to endothelial dysfunction, hypertension, atherosclerosis, thrombosis, heart failure, shock, stroke and ischemia–reperfusion injury. From these chapters it is clear that cardiovascular disease, the bane of Western civilization in the 21st century, might be associated directly with dysregulation of NO production. This assessment leads logically to the third section, ‘Nitric Oxide in Cardiovascular Therapeutics’, which contains chapters on therapies for restenosis, acute respiratory failure, tissue preservation for transplantation and atherosclerosis. These diseases might be amenable to therapy with chemical NO donors of various classes (administered directly or coated on implantable biomaterials), inhaled NO, l-arginine (the substrate for NOS), NOS gene therapy and/or antioxidants.The book might have been laid out more logically along the lines of the chemistry of NO (comprising the biological chemistry chapter as well as the chapters on nitrovasodilators, S-nitrosothiols and diazeniumdiolates), the cellular and molecular biology of NO, and the involvement of NO in cardiovascular pathophysiology and therapeutics. Such a division would lead the reader from the basic to the applied chemistry, to the in vitro effects of NO, and finally to whole animals and clinical studies. This minor quibble aside, Nitric Oxide and Cardiovascular Therapeutics is a well-written, comprehensive tome that represents the state of the art in this field.