A canine model of septic shock: balancing animal welfare and scientific relevance

A shock canine pneumonia model that permitted relief of discomfort with the use of objective criteria was developed and validated. After intrabronchial Staphylococcus aureus challenge, mechanical ventilation, antibiotics, fluids, vasopressors, sedatives, and analgesics were titrated based on algorithms for 96 h. Increasing S. aureus (1 to 8 x 10(9) colony-forming units/kg) produced decreasing survival rates (P = 0.04). From 4 to 96 h, changes in arterial-alveolar oxygen gradients, mean pulmonary artery pressure, IL-1, serum sodium levels, mechanical ventilation, and vasopressor support were ordered based on survival time [acute nonsurvivors (< or =24 h until death, n = 8) > or = subacute nonsurvivors (>24 to 96 h until death, n = 8) > or = survivors (> or =96 h until death, n = 22) (all P < 0.05)]. In the first 12 h, increases in lactate and renal abnormalities were greatest in acute nonsurvivors (all P < 0.05). Compared with survivors, subacute nonsurvivors had greater rises in cytokines and liver enzymes and greater falls in platelets, white cell counts, pH, and urine output from 24 to 96 h (all P < 0.05). Importantly, these changes were not attributable to dosages of sedation, which decreased in nonsurvivors [survivors vs. nonsurvivors: 5.0 +/- 1.0 vs. 3.8 +/- 0.7 ml x h(-1) x (fentanyl/midazolam/ medetomidine)(-1); P = 0.02]. In this model, the pain control regimen did not mask changes in metabolic function and lung injury or the need for more hemodynamic and pulmonary support related to increasing severity of sepsis. The integration into this model of both specific and supportive titrated therapies routinely used in septic patients may provide a more realistic setting to evaluate therapies for sepsis.     

Effect of progestins on serum hormones,semen production,and agonistic behavior in the gerenuk (Litocranius walleri walleri)

The use of progestins to suppress endogenous testosterone production to reduce agonistic behavior and prevent semen production was studied in gerenuk. Five male gerenuk (20 months to 3 years of age), housed as a bachelor group, were treated with 3 monthly injections of medroxyprogesterone acetate (MPA; 2.5–20 mg/kg), followed by a melengestrol acetate implant (MGA; 0.3 g/kg) for 2 months. Blood samples collected monthly were assayed for serum testosterone and cortisol using enzyme‐linked immunoassays. Quantitative behavioral data were collected for 30 min 3/week starting 1 month before treatment. Body weight, testes volume, and semen traits were measured before treatment, after MPA treatment, and after MGA treatment. Results showed lower (P<0.05) mean serum testosterone concentrations after MPA (4.34 pg/ml) and MGA (5.02 pg/ml) treatment compared to pre‐treatment values (65.9 pg/ml) in four of five gerenuk. The remaining sub‐adult gerenuk had low testosterone initially (4.9 pg/ml) that did not decrease further with treatment (1.4 and 7.8 pg/ml for MPA and MGA, respectively). Mean serum cortisol concentrations decreased markedly after treatment with MPA (6.0±3.7 ng/ml) and MGA (0.8±0.3 ng/ml). Cortisol concentrations were regained rapidly post‐treatment (42.8±4.8 ng/ml) and were not significantly different from the pre‐treatment value (60.6±12.6 ng/ml; P>0.05). The mean incidence of combined aggressive/dominant behaviors (horning, sparring, supplanting, threat) was not different before and after treatment. Body weight, total numbers of spermatozoa produced per ejaculate, percent motility, and percent normal spermatozoa declined maximally 8 months after treatment. Mean testes volume decreased (P<0.05) after MGA treatment (10.53 cm³vs. 11.96 cm³ pre‐treatment). Elevated hepatic enzymes and bile acids were seen in three of five animals after progestin treatment and anorexia was noted in two males after MGA implant removal, however two of three males had elevated liver enzymes before progestin treatment began. Results show that reducing serum testosterone concentration does not seem to modify agonistic behavior in bachelor gerenuk groups. Zoo Biol 26:245–257, 2007. © 2007 Wiley‐Liss, Inc.     

Transcriptional profiling of CD133(+) cells in coronary artery disease and effects of exercise on gene expression

Background aimsBone marrow (BM)-derived progenitor cells are under investigation for cardiovascular repair but may be altered by disease. Our aim was to identify differences in gene expression in CD133⁺ cells of patients with coronary artery disease (CAD) and healthy controls, and determine whether exercise modifies gene expression.MethodsCD133⁺ cells were flow-sorted from 10 CAD patients and four controls, and total RNA was isolated for microarray-based gene expression profiling. Genes that were found to be differentially regulated in patients were analyzed further to investigate whether exercise had any normalizing effect on CD133⁺ cells in CAD patients following 3 months of an exercise program.ResultsImprovement in effort tolerance and increases in the number of CD133⁺ cells were observed in CAD patients after 3 months of exercise. Gene expression analysis of the CD133⁺ cells identified 82 differentially expressed genes (2-fold cut-off, 25% false-discovery rate and % present calls) in patients compared with controls, of which 59 were found to be up-regulated and 23 down-regulated. These genes were found to be involved in carbohydrate metabolism, cell cycle, cellular development and signaling, and molecular transport. Following completion of the exercise program, gene expression patterns resembled those of controls in seven of 10 patients.ConclusionsAlterations in gene expression of BM-derived CD133⁺ progenitor cells were found in CAD patients, which in part may be normalized by exercise.     

Most nuclear systemic autoantigens are extremely disordered proteins: implications for the etiology of systemic autoimmunity

Patients with systemic autoimmune diseases usually produce high levels of antibodies to self-antigens (autoantigens). The repertoire of common autoantigens is remarkably limited, yet no readily understandable shared thread links these apparently diverse proteins. Using computer prediction algorithms, we have found that most nuclear systemic autoantigens are predicted to contain long regions of extreme structural disorder. Such disordered regions would generally make poor B cell epitopes and are predicted to be under-represented as potential T cell epitopes. Consideration of the potential role of protein disorder may give novel insights into the possible role of molecular mimicry in the pathogenesis of autoimmunity. The recognition of extreme autoantigen protein disorder has led us to an explicit model of epitope spreading that explains many of the paradoxical aspects of autoimmunity – in particular, the difficulty in identifying autoantigen-specific helper T cells that might collaborate with the B cells activated in systemic autoimmunity. The model also explains the experimentally observed breakdown of major histocompatibility complex (MHC) class specificity in peptides associated with the MHC II proteins of activated autoimmune B cells, and sheds light on the selection of particular T cell epitopes in autoimmunity. Finally, the model helps to rationalize the relative rarity of clinically significant autoimmunity despite the prevalence of low specificity/low avidity autoantibodies in normal individuals.     

Inhibitor and ion binding sites on the gastric H,K-ATPase

The gastric H,K-ATPase catalyzes electroneutral exchange of H(+) for K(+) as a function of enzyme phosphorylation and dephosphorylation during transition between E(1)/E(1)-P (ion site in) and E(2)-P/E(2) (ion site out) conformations. Here we present homology modeling of the H,K-ATPase in the E(2)-P conformation as a means of predicting the interaction of the enzyme with two known classes of specific inhibitors. All known proton pump inhibitors, PPIs, form a disulfide bond with cysteine 813 that is accessible from the luminal surface. This allows allocation of the binding site to a luminal vestibule adjacent to Cys813 enclosed by part of TM4 and the loop between TM5 and TM6. K(+) competitive imidazo-1,2alpha-pyridines also bind to the luminal surface of the E(2)-P conformation, and their binding excludes PPI reaction. This overlap of the binding sites of the two classes of inhibitors combined with the results of site-directed mutagenesis and cysteine cross-linking allowed preliminary assignment of a docking mode for these reversible compounds in a position close to Glu795 that accounts for the detailed structure/activity relationships known for these compounds. The new E(2)-P model is able to assign a possible mechanism for acid secretion by this P(2)-type ATPase. Several ion binding side chains identified in the sr Ca-ATPase by crystallography are conserved in the Na,K- and H,K-ATPases. Poised in the middle of these, the H,K-ATPase substitutes lysine in place of a serine implicated in K(+) binding in the Na,K-ATPase. Molecular models for hydronium binding to E(1) versus E(2)-P predict outward displacement of the hydronium bound between Asp824, Glu820, and Glu795 by the R-NH(3)(+) of Lys791 during the conformational transition from E(1)P and E(2)P. The site for luminal K(+) binding at low pH is proposed to be between carbonyl oxygens in the nonhelical part of the fourth membrane span and carboxyl oxygens of Glu795 and Glu820. This site of K(+) binding is predicted to destabilize hydrogen bonds between these carboxylates and the -NH(3)(+) group of Lys791, allowing the Lys791 side chain to return to its E(1) position.     

Equol is a novel anti-androgen that inhibits prostate growth and hormone feedback

Equol (7-hydroxy-3[4'hydroxyphenyl]-chroman) is the major metabolite of the phytoestrogen daidzein, one of the main isoflavones found abundantly in soybeans and soy foods. Equol may be an important biologically active molecule based on recent studies demonstrating that equol can modulate reproductive function. In this study, we examined the effects of equol on prostate growth and LH secretion and determined some of the mechanisms by which it might act. Administration of equol to intact male rats for 4-7 days reduced ventral prostate and epididymal weight and increased circulating LH levels. Using binding assays, we determined that equol specifically binds 5alpha-dihydrotestosterone (DHT), but not testosterone, dehydroepiandrosterone, or estrogen with high affinity. Equol does not bind the prostatic androgen receptor, and has a modest affinity for recombinant estrogen receptor (ER) beta, and no affinity for ERalpha. In castrated male rats treated with DHT, concomitant treatment with equol blocked DHT's trophic effects on the ventral prostate gland growth and inhibitory feedback effects on plasma LH levels without changes in circulating DHT. Therefore, equol can bind circulating DHT and sequester it from the androgen receptor, thus altering growth and physiological hormone responses that are regulated by androgens. These data suggest a novel model to explain equol's biological properties. The significance of equol's ability to specifically bind and sequester DHT from the androgen receptor have important ramifications in health and disease and may indicate a broad and important usage for equol in the treatment of androgen-mediated pathologies.     

Biological roles of nontypeable Haemophilus influenzae type IV pilus proteins encoded by the pil and com operons

We previously demonstrated that one or more products of the genes in the pil and com gene clusters of the opportunistic human respiratory pathogen nontypeable Haemophilus influenzae (NTHI) are required for type IV pilus (Tfp) biogenesis and function. Here, we have now demonstrated that the pilABCD and comABCDEF gene clusters are operons and that the product of each gene is essential for normal pilus function. Mutants with nonpolar deletions in each of the 10 pil and com genes had an adherence defect when primary human airway cells were used as the target. These mutants were also diminished in their ability to form a biofilm in vitro and, additionally, were deficient in natural transformation. Collectively, our data demonstrate that the product of each gene within these operons is required for the normal biogenesis and/or function of NTHI Tfp. Based on the similarity of PilA to other type IV pilins, we further predicted that the product of the pilA gene would be the major pilin subunit. Toward that end, we also demonstrated by immunogold labeling and mass spectrometry that PilA is indeed the majority type IV pilin protein expressed by NTHI. These new observations set the stage for experiments designed to dissect the function of each of the proteins encoded by genes within the pil and com gene clusters. The ability to characterize individual proteins with vital roles in NTHI colonization or pathogenesis has the potential to reduce the burden of NTHI-induced diseases through development of a Tfp-derived vaccine or a pilus-directed therapeutic.