Low-dose N-acetylcysteine protects rats against endotoxin-mediated oxidative stress, but high-dose increases mortality.

We evaluated the effect of the antioxidant N-acetylcysteine (NAC) on oxidative stress, lung damage, and mortality induced by an endotoxin (lipopolysaccharide, or LPS) in the rat. Continuous intravenous infusion of 275 mg NAC/kg in 48 h, starting 24 h before LPS challenge, decreased hydrogen peroxide (H2O2) concentrations in whole blood (p < 0.01). This decrease was accompanied by fewer histologic abnormalities of the lung and decreased mortality (p < 0.025), compared with rats receiving LPS alone. N-Acetylserine, which has no sulfhydryl group, did not protect rats against LPS toxicity. Improved survival was not associated with an increase in pulmonary reduced glutathione, nor with inhibition of serum tumor necrosis factor (TNF) activity. In vitro, TNF production and DNA binding of nuclear factor kappa B (NF-kappaB) in human Mono Mac 6 cells was only inhibited at concentrations of NAC above 20 mM. High-dose NAC treatment (550 and 950 mg/kg in 48 h) decreased lung GSH (p < 0.05) and resulted in a significantly smaller number of surviving animals when compared with the low-dose NAC group (p < 0.025). In vitro, NAC increased hydroxyl radical generation in a system with Fe(III)-citrate and H2O2 by reducing ferric iron to its catalytic, active Fe2+ form. We conclude that low-dose NAC protects against LPS toxicity by scavenging H2O2, while higher doses may have the opposite effect.

Elevated ocular levels of vascular endothelial growth factor in patients with von Hippel-Lindau disease.

BACKGROUND: Von Hippel Lindau disease (VHL) is a rare autosomal dominant inherited disorder characterized by highly vascularized tumors in various organs. The abundant presence of endothelial cells in VHL tumors strongly suggest a role of the VHL tumor suppressor gene in the regulation of angiogenesis. Recently, in vitro studies have shown that the VHL tumor suppressor gene regulates the expression of vascular endothelial growth factor (VEGF). We investigated whether VHL patiens have increased levels of VEGF in their body fluids. PATIENTS AND METHODS: The concentration of VEGF was measured in fluid of the anterior chamber of the eye, serum, urine, and fluid from renal cysts of VHL patients and unaffected individuals by ELISA. In addition, levels of basic fibroblast growth factor (bFGF), interleukin-8 (IL-8) and endothelin-1 (ET-1) were measured in urine and serum of VHL patients and control subjects. RESULTS: In 80% of the VHL patients VEGF was detectable in aqueous fluid of the anterior chamber of their eyes. A strong positive correlation (r = 0.90) was found between the age of VHL patients and ocular VEGF concentrations. At comparable age, VEGF levels in ocular fluid of VHL patients were significantly higher (P < 0.001) than in unaffected subjects. No correlation was found between VEGF concentration and the presence of retinal angiomas. A 10 and 16 fold increase of VEGF concentration was seen in fluid from two independent VHL-related cysts as compared with VEGF serum levels of the same patient. The mean concentration of VEGF in serum of VHL patients (n = 15) (319 +/- 84 pg/ml) was higher than in matched controls (238 +/- 68 pg/ml; P = NS). The mean concentration of VEGF in urine of VHL patients (128 +/- 36 pg/ml) was lower than in matched controls (183 +/- 25 pg/ml; P = NS). Concentrations of VEGF did not correlate with the presence of VHL-related tumors. No differences were observed between concentrations of bFGF, IL-8 and ET-1 in serum and urine of VHL patients and matched controls. CONCLUSIONS: These findings support a role for the VHL tumor suppressor gene in the in vivo regulation of VEGF.

Dimethylthiourea protects rats against gram-negative sepsis and decreases tumor necrosis factor and nuclear factor kappaB activity.

The thiol-containing compound dimethylthiourea (DMTU) is a known protectant in various models of oxidant-mediated tissue damage. Protective effects of DMTU have also been reported in studies on endotoxin-induced (LPS-induced) tissue injury. DMTU may exert this protective effect by reducing oxidative stress. In this study we investigated the effect of DMTU on survival, oxidative stress, and tumor necrosis factor (TNF) activity in two rat models of gram-negative bacterial sepsis. Intraperitoneal injection of 500 mg DMTU/kg protected against the lethal effects of intraperitoneally injected LPS (5 mg/kg) and live Salmonella typhimurium (3.3 x 10(10) CFU/kg). LPS injection resulted in oxidative stress, as indicated by an elevated concentration of hydrogen peroxide (H(2)O(2)) in normal and carbon monoxide-treated deproteinized blood. We also observed increased H(2)O(2) levels in animals injected with live Salmonella typhimurium. Although DMTU improved survival in both models, H(2)O(2) concentrations were not affected by it. This is consistent with our in vitro observation that DMTU is a weak H(2)O(2) scavenger. Serum TNF activity, however, was substantially decreased by DMTU, and this was associated with a reduced activation of nuclear factor kappaB in the peritoneal cells of LPS-treated rats. In addition, LPS-induced TNF production in vitro by rat peritoneal macrophages was inhibited by DMTU (p < 0.05). These results suggest that the protective effect of DMTU in gram-negative bacterial sepsis may be the result of a reduction in TNF activity. DMTU does not exert this effect by H(2)O(2) scavenging but may inactivate toxic H(2)O(2) metabolites.

Multivalent binding of toxin A from Clostridium difficile to carbohydrate receptors.

A specific monoclonal antibody against toxin A from Clostridium difficile was generated that did not show thermolabile binding. Nonspecific murine monoclonal antibodies bound toxin A at 4 degrees C, but less effectively at 37 degrees C. Nonspecific human monoclonal antibodies did not bind to toxin A at 4 degrees C. Cytotoxic properties of purified toxin A were not inhibited by Bandeiraea simplicifolia lectin. This points to a carbohydrate moiety on the cell surface and a multivalent nonspecific carbohydrate binding ligand on toxin A.

Monoclonal antibodies that react with live Listeria spp.

Seven monoclonal antibodies (MAbs) against Listeria spp. that were reactive with live Listeria spp. were developed. Two of these MAbs (55-8 and 55-37) were members of the immunoglobulin M class, and all other MAbs were members of the immunoglobulin G class. MAb 55-23 reacted with 148 of 157 strains tested. MAb 34-51 reacted with serotype 1/2a, 1/2b, and 1/2c strains and exhibited a scattered reaction pattern with strains belonging to other serotypes. MAb 55-44 reacted with all of the strains belonging to serotype 4b tested. MAb 55-4 reacted with all of the serotype 1/2a isolates tested, although reactivity with other isolates also was observed. The other MAbs exhibited scattered reaction patterns. No correlation of reactivity pattern with serotype was found. Marked differences were observed between the reactivities of MAbs as determined by a magnetic immunoluminescence assay and a whole-cell enzyme-linked immunosorbent assay. Only MAb 55-23 exhibited minor reactivity with three Streptococcus spp. isolates, while no reactivity was observed with six Bacillus spp. strains, one Escherichia coli strain, and one Citrobacter sp. strain. In Western blots (immunoblots) MAbs 55-23, 55-44, and 34-9 exhibited reactivity; all other MAbs were negative in this assay.

Detection of Listeria monocytogenes in cheese with the magnetic immuno-polymerase chain reaction assay.

A new detection system, the magnetic immuno-polymerase chain reaction (PCR) assay (MIPA) has been developed to detect Listeria monocytogenes in food. This method separates Listeria cells from PCR-inhibitory factors present in enrichment broths containing food samples by using magnetic beads coated with specific monoclonal antibodies (MAbs). The separated bacteria were lysed, and the supernatant containing the bacterial DNA was subjected to the PCR. Detection of L. monocytogenes in three naturally contaminated cheese samples with two different MAbs and PCR primers specific for the gene encoding the delayed-hypersensitivity factor showed that with MAb 55 all three samples were positive whereas with MAb A two samples were positive. A further improvement of the method was obtained by using a PCR step based on the listeriolysin O gene. A MIPA employing MAb 55 and the listeriolysin O gene primer set detected L. monocytogenes after 24 h of culture in Listeria Enrichment Broth samples from Port Salut artificially contaminated with 40 CFU/25 g. We could detect 1 CFU of L. monocytogenes per g of cheese after a second enrichment for 24 h in Fraser broth. The analysis time including both enrichments is approximately 55 h.

Monoclonal antibodies that detect live salmonellae.

Nine immunoglobulin G and nine immunoglobulin M murine monoclonal antibody-producing hybridomas reactive with live Salmonella bacteria were obtained from several fusions of immune spleen cells and Sp2/0 myeloma cells. The antibodies were selected by the magnetic immunoluminescence assay. The monoclonal antibodies were reactive with serogroups A, B, C1, C2, D, E, and K and Salmonella choleraesuis subsp. diarizonae. Each monoclonal antibody proved to be reactive with a distinct serotype. Clinical isolates belonging to these Salmonella serogroups could be detected. Reactivity with non-Salmonella bacteria proved to be minor.