Detection of Histoplasma capsulatum DNA in human samples by real-time polymerase chain reaction

The main aim of our study was to determine the added value of real-time polymerase chain reaction (PCR) for the diagnosis of Histoplasma capsulatum in routine biologic practice. No amplification signal was observed with the 18 non-H. capsulatum strains used to test the specificity of the protocol. The sensitivity threshold of the real-time PCR assay was about 10 fg of H. capsulatum DNA per microliter, tested with a 10-fold serial dilution of the positive control. We analyzed 348 human samples submitted for the routine diagnosis of systemic mycosis. Real-time PCR using the TaqMan system was evaluated against direct microscopic examination and culture. Among the 341 samples without PCR inhibition (n = 7), 66 tested positive by culture, whereas 74 tested positive by real-time PCR. Sensitivity of the real-time PCR assay was estimated at 95.4% and specificity at 96.0% with respect to culture, widely considered to be the gold standard method; however, the molecular approach in fact produced better sensitivity and specificity results. Moreover, for the 38 samples that tested negative by direct examination but positive by culture, the culture method took a mean of 31 days longer than the PCR method to generate results. The protocol presented here may be very useful for improving routine histoplasmosis diagnosis.     

Antiviral nucleoside analogs phosphorylation by nucleoside diphosphate kinase

The reaction of NDP kinase was studied in vitro with several antiviral derivatives, using kinetic steady state and presteady state analysis. The enzyme is highly efficient with natural nucleotides but most of the analogs are slow substrates. The catalytic efficiency, also related to the affinity of the analog, is mainly dependent on the presence of a 3'-OH group on the ribose moiety.     

Comparison of the in vitro activity of amoxycillin + clavulanic acid and ampicillin + sulbactam combinations against 50 Haemophilus influenzae strains producing beta lactamase

We compared the ability of two beta lactamase inhibitors, clavulanic acid and sulbactam, to reduce the minimal inhibitory concentrations (MICs) respectively of amoxycillin and ampicillin against fifty strains of Haemophilus influenzae producing a beta lactamase. The reduction of MICs of aminopenicillins was studied using low concentrations of beta lactamase inhibitors (0.125 to 0.5 mg/l). Moreover, the synergic effect of each association was evaluated against a heavy bacterial inoculum (10(8) cfu/ml). The combination of amoxycillin + clavulanic acid was more synergic on these strains than the association of ampicillin + sulbactam.     

In vitro quantitative model of catheter infection during simulated parenteral nutrition.

We developed a quantitative in vitro model of catheter infection. Colonization was initiated by inoculating the catheter lumen with a small number of bacteria (approximately 5 x 10(3) viable organisms). Then the inoculated catheters were used for simulated total parenteral nutrition therapy consisting of infusions for 9 h a day, and bacteria were counted in the effluent fluid against time, enabling us to monitor catheter colonization quantitatively. Bacterial colonization of prosthetic devices is a progressive process, as evidenced by the slow day-to-day increase of bacterial release seen here. On the other hand, bacterial strains of various representative species exhibited significant differences in their ability to infect catheters. These results suggest that the in vitro model presented here is a reliable tool for monitoring the degree of catheter colonization under standardized conditions and could be used for further studying the main factors of catheter-related sepsis or the treatment of this information.     

Impaired resistance to Listeria monocytogenes in mice chronically exposed to cadmium.

It is shown in this work that resistance to Listeria monocytogenes is greatly impaired in C57BL/6 mice chronically exposed to cadmium (Cd) chloride. Animals received 0.5 mg/kg Cd by an intraperitoneal route three times a week during a 4-week period and were then infected with L. monocytogenes. Susceptibility to this pathogenic bacteria was not due to a defect of the specific immune response, since mice developed normal levels of anti-Listeria T cell-mediated immunity and did not show any impairment of macrophage activation. In fact, bacterial growth in organs was rapid in Cd-exposed mice during the early phase of infection, suggesting an impairment of non-specific defence mechanisms. Experimental data indicate that the susceptibility to L. monocytogenes might be due to a defect of macrophage recruitment in sites of infection during the early phase of the host response.     

Semaphorin3a Promotes Advanced Diabetic Nephropathy

The onset of diabetic nephropathy (DN) is highlighted by glomerular filtration barrier abnormalities. Identifying pathogenic factors and targetable pathways driving DN is crucial to developing novel therapies and improving the disease outcome. Semaphorin3a (sema3a) is a guidance protein secreted by podocytes. Excess sema3a disrupts the glomerular filtration barrier. Here, using immunohistochemistry, we show increased podocyte SEMA3A in renal biopsies from patients with advanced DN. Using inducible, podocyte-specific Sema3a gain-of-function (Sema3a⁺) mice made diabetic with streptozotocin, we demonstrate that sema3a is pathogenic in DN. Diabetic Sema3a⁺ mice develop massive proteinuria, renal insufficiency, and extensive nodular glomerulosclerosis, mimicking advanced DN in humans. In diabetic mice, Sema3a⁺ exacerbates laminin and collagen IV accumulation in Kimmelstiel-Wilson-like glomerular nodules and causes diffuse podocyte foot process effacement and F-actin collapse via nephrin, αvβ3 integrin, and MICAL1 interactions with plexinA₁. MICAL1 knockdown and sema3a inhibition render podocytes not susceptible to sema3a-induced shape changes, indicating that MICAL1 mediates sema3a-induced podocyte F-actin collapse. Moreover, sema3a binding inhibition or podocyte-specific plexinA1 deletion markedly ameliorates albuminuria and abrogates renal insufficiency and the diabetic nodular glomerulosclerosis phenotype of diabetic Sema3a⁺ mice. Collectively, these findings indicate that excess sema3a promotes severe diabetic nephropathy and identifies novel potential therapeutic targets for DN.     

Podocyte-Specific VEGF-A Gain of Function Induces Nodular Glomerulosclerosis in eNOS Null Mice

VEGF-A and nitric oxide are essential for glomerular filtration barrier homeostasis and are dysregulated in diabetic nephropathy. Here, we examined the effect of excess podocyte VEGF-A on the renal phenotype of endothelial nitric oxide synthase (eNOS) knockout mice. Podocyte-specific VEGF₁₆₄ gain of function in eNOS^−/− mice resulted in nodular glomerulosclerosis, mesangiolysis, microaneurysms, and arteriolar hyalinosis associated with massive proteinuria and renal failure in the absence of diabetic milieu or hypertension. In contrast, podocyte-specific VEGF₁₆₄ gain of function in wild-type mice resulted in less pronounced albuminuria and increased creatinine clearance. Transmission electron microscopy revealed glomerular basement membrane thickening and podocyte effacement in eNOS^−/− mice with podocyte-specific VEGF₁₆₄ gain of function. Furthermore, glomerular nodules overexpressed collagen IV and laminin extensively. Biotin-switch and proximity ligation assays demonstrated that podocyte-specific VEGF₁₆₄ gain of function decreased glomerular S-nitrosylation of laminin in eNOS^−/− mice. In addition, treatment with VEGF-A decreased S-nitrosylated laminin in cultured podocytes. Collectively, these data indicate that excess glomerular VEGF-A and eNOS deficiency is necessary and sufficient to induce Kimmelstiel-Wilson–like nodular glomerulosclerosis in mice through a process that involves deposition of laminin and collagen IV and de-nitrosylation of laminin.Vascular glomerular endothelial factor-A (VEGF-A) is essential for the development and maintenance of normal glomerular structure and function.¹ Podocytes are the most important source of glomerular VEGF-A.^1–4 Glomerular VEGF-A plays a critical role in the pathogenesis of diabetic nephropathy.^5–7 Transgenic mice with podocyte VEGF₁₆₄ gain of function develop a glomerular phenotype indistinguishable from early diabetic nephropathy, in the context of normal blood glucose and normal systemic VEGF-A.⁵ In the setting of type 1 diabetes, plasma VEGF-A increases but nodular glomerulosclerosis develops only in mice with podocyte VEGF₁₆₄ gain of function, demonstrating that local rather than systemic VEGF excess is critical for the progression of diabetic glomerulopathy to advanced disease.⁶Nitric oxide (NO) is a product of arginine oxidation: L arginine+O₂ → citrulline+NO, catalyzed by NO synthase (NOS). The major source of endogenous NO, NOS isoforms (neuronal NOS, inducible NOS, and endothelial NOS),^8–10 are expressed in the kidney.^11–14 VEGF-A activates endothelial NOS (eNOS), inducing NO generation, which stimulates soluble guanylate cyclase, thereby causing vasodilatation. VEGF-A activates eNOS via phosphatidylinositol-3-kinase/Akt.¹⁵ Signals downstream from VEGF-A and NO stimulate endothelial cell proliferation and migration in human endothelium, regulate endothelial integrity, and contribute to angiogenesis.^16–21 In diabetes, low NO bioavailability is associated with high VEGF-A levels.^7,8,22–25 Nakagawa et al. called this process “uncoupling of VEGF to NO,” connecting mechanistically the advanced nephropathy with the relationship between VEGF and NO in the kidney.²⁶ Experimental diabetes induced in eNOS knockout (KO) mice resulted in severe diabetic nephropathy: nodular glomerulosclerosis, decreased GFR, and hypertension, associated with increased VEGF mRNA renal expression.^26,27 Consistent with these findings, db/db mice treated with l-arginine and sepiapterin had improved albuminuria and glomerular basement membrane (GBM) thickness, associated with reversed eNOS dimerization and phosphorylation, suggesting that improving eNOS activity delays the progression of diabetic nephropathy.²⁸ However, the mechanisms whereby excess VEGF-A and eNOS insufficiency lead to advanced diabetic nephropathy remain unclear.At the cellular level, binding of NO to soluble guanylate cyclase leads to increased cyclic guanosine monophosphate (cGMP) production and activation of protein kinase G, phosphodiesterases, and cGMP-gated ion channels. However, extensive evidence demonstrates that NO exerts multiple biologic functions through cGMP–independent S-nitrosylation of proteins.^29–32 S-Nitrosylation is a reversible, covalent addition of NO to thiol groups on specific cysteine from proteins, forming nitroso-protein (SNO).^30–32 Nitrosylation induces redox-based conformational changes in target proteins that modulate signaling and function.³² Altered protein S-nitrosylation has been demonstrated in pulmonary, hematologic, neurologic, and cardiovascular diseases, as well as in cancer, preeclampsia, and diabetes.^31,33We hypothesized that deficient S-nitrosylation of specific proteins mediates the glomerular phenotype resulting from eNOS deletion and excess VEGF-A in vivo. Here we examined the effects of increased podocyte VEGF₁₆₄ in eNOS KO mice and evaluated whether S-nitrosylation is mechanistically involved in the ensuing glomerular phenotype. Our findings indicate that podocyte VEGF₁₆₄ gain of function in eNOS null mice is sufficient to induce nodular glomerulosclerosis, massive proteinuria, and renal failure in the absence of diabetic milieu. Podocyte VEGF₁₆₄ gain of function decreases glomerular laminin S-nitrosylation in eNOS null mice, linking this post-translational modification to nodular glomerulosclerosis.