Dynamical modeling of pro- and anti-inflammatory cytokines in the early stage of septic shock.

A dynamical model of the pathophysiological behaviors of IL18 and IL10 cytokines with their receptors is tested against data for the case of early sepsis. The proposed approach considers the surroundings (organs and bone marrow) and the different subsystems (cells and cyctokines). The interactions between blood cells, cytokines and the surroundings are described via mass balances. Cytokines are adsorbed onto associated receptors at the cell surface. The adsorption is described by the Langmuir model and gives rise to the production of more cytokines and associated receptors inside the cell. The quantities of pro and anti-inflammatory cytokines present in the body are combined to give global information via an inflammation level function which describes the patient's state. Data for parameter estimation comes from the Sepsis 48 H database. Comparisons between patient data and simulations are presented and are in good agreement. For the IL18/IL10 cytokine pair, 5 key parameters have been found. They are linked to pro-inflammatory IL18 cytokine and show that the early sepsis is driven by components of inflammatory character.

Presence of accessory abductor digiti minimi muscle in two cadavers.

During routine cadaveric dissection, accessory hypothenar muscles were incidentally discovered in two cadavers, both males, aged 86 and 92. Both muscles originated from the palmaris longus tendon in the distal portion of the forearm and were identified as accessory abductor digiti minimi (AADM) muscles, based on their association with abductor digiti minimi. While AADM is a common variant in the antebrachium, it is less typical for them to originate from the palmaris longus tendon. The presence of such an AADM could complicate surgical procedures requiring resection of the palmaris longus tendon. Moreover, the surrounding neurovasculature - namely the ulnar nerve as it passes through the ulnar canal between the pisiform and hook of the hamate - could be compressed by contractions of an AADM with such a proximal origin. This can manifest as ulnar neuropathies resulting in pain, weakness, or protracted flexion of the fourth and fifth digits (ulnar claw). Our description of these muscles adds to previous accounts of variation of the palmaris longus and abductor digiti minimi muscles while considering potential clinical implications.

Evaluation of IL12B as a candidate type I diabetes susceptibility gene using data from the Type I Diabetes Genetics Consortium.

As part of its efforts to identify genes affecting the risk of type I diabetes (T1D), the Type I Diabetes Genetics Consortium commissioned an extensive survey of variants associated with genes reported earlier to have an association with disease susceptibility. In this report, we present the analysis of a set of single-nucleotide polymorphisms (SNPs) within and flanking the IL12B gene, which encodes the p40 subunit of the cytokines interleukin (IL)-12 and IL-23. No SNP showed individually significant association in the population as a whole. Nevertheless, subjects stratified according to genotype at the earlier reported SNP in the IL12B 3'UTR, rs3212227, confirmed small, but significant, differences in age of disease onset with a relative hazard=0.88 (P=0.005). The protective effect of rs3212227 allele 2 was gender specific (P=0.004 overall and P=0.0003 when unaffected siblings were considered). Among females, the 2.2 genotype was more protective, with relative hazard=0.75. We conclude that while there was no major effect of IL12B polymorphisms on T1D susceptibility in the entire study group, they have an impact on a subset of at-risk individuals.

Arrangement of domains, and amino acid residues required for binding of vascular cell adhesion molecule-1 to its counter-receptor VLA-4 (alpha 4 beta 1).

Interaction of the vascular cell adhesion molecule (VCAM-1) with its counter-receptor very late antigen-4 (VLA-4) (integrin alpha 4 beta 1) is important for a number of developmental pathways and inflammatory functions. We are investigating the molecular mechanism of this binding, in the interest of developing new anti-inflammatory drugs that block it. In a previous report, we showed that the predominant form of VCAM-1 on stimulated endothelial cells, seven-domain VCAM (VCAM-7D), is a functionally bivalent molecule. One binding site requires the first and the other requires the homologous immunoglobulin-like domain. Rotary shadowing and electron microscopy of recombinant soluble VCAM-7D molecules suggests that the seven Ig-like domains are extended in a slightly bent linear array, rather than compactly folded together. We have systematically mutagenized the first domain of VCAM-6D (a monovalent, alternately spliced version mission domain 4) by replacing 3-4 amino acids of the VCAM sequence with corresponding portions of the related ICAM-1 molecule. Specific amino acids, important for binding VLA-4 include aspartate 40 (D40), which corresponds to the acidic ICAM-1 residue glutamate 34 (E34) previously reported to be essential for binding of ICAM-1 to its integrin counter-receptor LFA-1. A small region of VCAM including D40, QIDS, can be replaced by the similar ICAM-1 sequence, GIET, without affecting function or epitopes, indicating that this region is part of a general integrin-binding structure rather than a determinant of binding specificity for a particular integrin. The VCAM-1 sequence G65NEH also appears to be involved in binding VLA-4.

Phytoestrogens: adverse effects on reproduction in California quail.

Phytoestrogens, largely formononetin and genistein, are produced in the leaves of stunted desert annuals in a dry year. When ingested by California quail, these compounds apparently inhibit reproduction and prevent the production of young that will not have adequate food. In a wet year, forbs grow vigorously and phytoestrogenic substances are largely absent. Quail then breed prolifically and the abundant seed crop carries the enlarged population through the winter.

Gender-stratified analysis of DLG5 R30Q in 4707 patients with Crohn disease and 4973 controls from 12 Caucasian cohorts.

BACKGROUND: DLG5 p.R30Q has been reported to be associated with Crohn disease (CD), but this association has not been replicated in most studies. A recent analysis of gender-stratified data from two case-control studies and two population cohorts found an association of DLG5 30Q with increased risk of CD in men but not in women and found differences between 30Q population frequencies for males and females. Male-female differences in population allele frequencies and male-specific risk could explain the difficulty in replicating the association with CD. METHODS: DLG5 R30Q genotype data were collected for patients with CD and controls from 11 studies that did not include gender-stratified allele counts in their published reports and tested for male-female frequency differences in controls and for case-control frequency differences in men and in women. RESULTS: The data showed no male-female allele frequency differences in controls. An exact conditional test gave marginal evidence that 30Q is associated with decreased risk of CD in women (p = 0.049, OR = 0.87, 95% CI 0.77 to 1.00). There was also a trend towards reduced 30Q frequencies in male patients with CD compared with male controls, but this was not significant at the 0.05 level (p = 0.058, OR = 0.87, 95% CI 0.74 to 1.01). When data from this study were combined with previously published, gender-stratified data, the 30Q allele was found to be associated with decreased risk of CD in women (p = 0.010, OR = 0.86, 95% CI 0.76 to 0.97), but not in men. CONCLUSION: DLG5 30Q is associated with a small reduction in risk of CD in women.

Weight loss and reduced body mass index: a critical issue in children with multiorgan chronic graft-versus-host disease.

Weight loss and malnutrition are major problems in patients with chronic graft-versus-host disease (GVHD). In adults, low body mass index (BMI) is a predictor for mortality; however, weight loss and BMI have not been studied in pediatric chronic GVHD. A retrospective study on 18 children with extensive chronic GVHD was completed. Median age at SCT was 12.3 (range 0.6-23) years; age at chronic GVHD diagnosis was 12.5 (1-23) years. Patients with multiorgan involvement had a mean maximal decrease in BMI of 20.9% and most dropped below 10th percentile in expected weight-for-age. This change in BMI not only indicates a significant decrease in weight but often a plateau in stature. In contrast, patients with one organ system involved had a mean maximal decrease in BMI of 5% and did not fall below 10th percentile. All patients with multiorgan involvement required salvage therapy beyond steroids and CSA. Three patients died due to complications of chronic GVHD. Weight loss and malnutrition (as reflected by a decrease in BMI) are clinically significant issues in children with multisystem chronic GVHD. Weight loss is likely another systemic manifestation of the disease and may contribute, along with other factors such as increased immunosuppression and infection, to increased mortality in this group.

In situ DMSO hydration measurements of HTS compound libraries.

Compounds used in high throughput screening (HTS) are typically dissolved in DMSO. These solutions are stored automation-friendly racks of wells or tubes. DMSO is hygroscopic and quickly absorbs water from the atmosphere. When present in DMSO compound solutions, water can accelerate degradation and precipitation. Understanding DMSO hydration in an HTS compound library can improve storage and screening methods by managing the impact of water on compound stability. A non-destructive, acoustic method compatible with HTS has been developed to measure water content in DMSO solutions. Performance of this acoustic method was compared with an optical technique and found to be in good agreement. The accuracy and precision of acoustic measurements was shown to be under 3% over the tested range of DMSO solutions (0% to 35% water by volume) and insensitive to the presence of HTS compounds at typical storage concentrations. Time course studies of hydration for wells in 384-well and 1536-well microplates were performed. Well geometry, fluid volume, well position and atmospheric conditions were all factors in hydration rate. High rates of hydration were seen in lower-volume fills, higher-density multi-well plates and when there was a large differential between the humidity of the lab and the water content of the DMSO. For example, a 1536-well microplate filled with 2microL of 100% DMSO exposed for one hour to a laboratory environment with approximately 40% relative humidity will absorb over 6% water by volume. Understanding DMSO hydration rates as well as the ability to reverse library hydration are important steps towards managing stability and availability of compound libraries.

A humanized form of a CD4-specific monoclonal antibody exhibits decreased antigenicity and prolonged plasma half-life in rhesus monkeys while retaining its unique biological and antiviral properties.

Certain monoclonal antibodies (MAbs) directed against CD4 can efficiently block HIV-1 replication in vitro. To explore CD4-directed passive immunotherapy for prevention or treatment of AIDS virus infection, we previously examined the biological activity of a nondepleting CD4-specific murine MAb, mu5A8. This MAb, specific for domain 2 of CD4, blocks HIV-1 replication at a post-gp120-CD4 binding step. When administered to normal rhesus monkeys, all CD4+ target cells were coated with antibody, yet no cell clearance or measurable immunosuppression occurred. However, strong anti-mouse Ig responses rapidly developed in all monkeys. In the present study, we report a successfully humanized form of mu5A8 (hu5A8) that retains binding to both human and monkey CD4 and anti-AIDS virus activity. When administered intravenously to normal rhesus monkeys, hu5A8 bound to all target CD4+ cells without depletion and showed a significantly longer plasma half-life than mu5A8. Nevertheless, an anti-hu5A8 response directed predominantly against V region determinants did eventually appear within 2 to 4 weeks in most animals. However, when hu5A8 was administered to rhesus monkeys chronically infected with the simian immunodeficiency virus of macaques, anti-hu5A8 antibodies were not detected. Repeated administration of hu5A8 in these animals resulted in sustained plasma levels and CD4+ cell coating with humanized antibody for 6 weeks. These studies demonstrate the feasibility of chronic administration of CD4-specific MAb as a potential means of treating or preventing HIV-1 infection.

Single nucleotide polymorphisms in human Paneth cell defensin A5 may confer susceptibility to inflammatory bowel disease in a New Zealand Caucasian population.

BACKGROUND: Human Paneth cell alpha-defensins, especially DEFA5, are involved in maintaining homeostasis of the human microbial microflora. Since breakdown of normal mucosal antibacterial defence occurs in inflammatory bowel disease (IBD), variants in the DEFA5 gene could be associated with IBD risk. SUBJECTS: A cohort of 25 patients with indeterminate colitis (IC), 405 with ulcerative colitis (UC), and 385 with Crohn's disease (CD), were compared with 201 control individuals from the Canterbury region in New Zealand. METHODS: A 15 kb haplotype block surrounding DEFA5 contained 35 HapMap markers which were polymorphic in Caucasians. Four markers (A-D) were selected to tag 27 of the 35 markers at r(2)>0.68, and were genotyped in DNA samples. RESULTS: Minor allele frequencies for all single nucleotide polymorphisms (SNPs) were somewhat elevated in patients. Subgroup analysis showed SNP A had odds ratio 1.44 in UC patients with pancolitis (95% C.I. 1.07-1.94), SNP B odds ratio 2.37 in CD patients with onset prior to 17 years age (95% C.I. 1.12-5.03), SNP C odds ratio 1.68 in UC patients with left colonic localisation (95% C.I. 1.12-2.52), and SNP D had odds ratio 1.56 in CD patients with one or more relatives with IBD (95% C.I. 1.03-2.35). Two two-marker haplotypes and one three-marker haplotype were associated with UC (p-values 0.025-0.05). CONCLUSIONS: The SNPs genotyped in our study were surrogates for common variants, and observed associations between these and IBD status are likely due to linkage disequilibrium with a functional common DEFA5 variant. Identifying such functional variants will be prioritized in subsequent work.

FLOSS: flexible ordered subset analysis for linkage mapping of complex traits.

The FLOSS software package is a flexible framework for ordered subset analysis. FLOSS is specifically designed for use with the Merlin linkage analysis package, but FLOSS can be used with any linkage analysis software package that reports NPL Z-scores for each locus and family. When FLOSS is used with the Merlin linkage analysis package, one can use either non-parametric Z-scores or Kong and Cox linear allele sharing model LOD scores. Monte Carlo P-values are calculated using a permutation test with an efficient Besag-Clifford sequential stopping rule. FLOSS also has a flexible tool for assigning family covariate scores from Merlin input files. FLOSS includes user documentation and is written in Java for easy portability. The FLOSS source code is documented and designed to be extensible.

Assembly of GABAA receptors composed of alpha1 and beta2 subunits in both cultured neurons and fibroblasts.

GABAA receptors are believed to be pentameric hetero-oligomers, which can be constructed from six subunits (alpha, beta, gamma, delta, epsilon, and rho) with multiple members, generating a large potential for receptor heterogeneity. The mechanisms used by neurons to control the assembly of these receptors, however, remain unresolved. Using Semliki Forest virus expression we have analyzed the assembly of 9E10 epitope-tagged receptors comprising alpha1 and beta2 subunits in baby hamster kidney cells and cultured superior cervical ganglia neurons. Homomeric subunits were retained within the endoplasmic reticulum, whereas heteromeric receptors were able to access the cell surface in both cell types. Sucrose density gradient fractionation demonstrated that the homomeric subunits were incapable of oligomerization, exhibiting 5 S sedimentation coefficients. Pulse-chase analysis revealed that homomers were degraded, with half-lives of approximately 2 hr for both the alpha1((9E10)) and beta2((9E10)) subunits. Oligomerization of the alpha1((9E10)) and beta2((9E10)) subunits was evident, as demonstrated by the formation of a stable 9 S complex, but this process seemed inefficient. Interestingly the appearance of cell surface receptors was slow, lagging up to 6 hr after the formation of the 9 S receptor complex. Using metabolic labeling a ratio of alpha1((9E10)):beta2((9E10)) of 1:1 was found in this 9 S fraction. Together the results suggest that GABAA receptor assembly occurs by similar mechanisms in both cell types, with retention in the endoplasmic reticulum featuring as a major control mechanism to prevent unassembled receptor subunits accessing the cell surface.

The crystal structure of an N-terminal two-domain fragment of vascular cell adhesion molecule 1 (VCAM-1): a cyclic peptide based on the domain 1 C-D loop can inhibit VCAM-1-alpha 4 integrin interaction.

Vascular cell adhesion molecule 1 (VCAM-1) represents a structurally and functionally distinct class of immunoglobulin superfamily molecules that bind leukocyte integrins and are involved in inflammatory and immune functions. X-ray crystallography defines the three-dimensional structure of the N-terminal two-domain fragment that participates in ligand binding. Residues in domain 1 important for ligand binding reside in the C-D loop, which projects markedly from one face of the molecule near the contact between domains 1 and 2. A cyclic peptide that mimics this loop inhibits binding of alpha 4 beta 1 integrin-bearing cells to VCAM-1. These data demonstrate how crystallographic structural information can be used to design a small molecule inhibitor of biological function.