Targeted Mutagenesis in Pathogenic Leptospira Species: Disruption of the LigB Gene Does Not Affect Virulence in Animal Models of Leptospirosis

The pathogenic mechanisms of Leptospira interrogans, the causal agent of leptospirosis, remain largely unknown. This is mainly due to the lack of tools for genetically manipulating pathogenic Leptospira species. Thus, homologous recombination between introduced DNA and the corresponding chromosomal locus has never been demonstrated for this pathogen. Leptospiral immunoglobulin-like repeat (Lig) proteins were previously identified as putative Leptospira virulence factors. In this study, a ligB mutant was constructed by allelic exchange in L. interrogans; in this mutant a spectinomycin resistance (Spc(r)) gene replaced a portion of the ligB coding sequence. Gene disruption was confirmed by PCR, immunoblot analysis, and immunofluorescence studies. The ligB mutant did not show decrease virulence compared to the wild-type strain in the hamster model of leptospirosis. In addition, inoculation of rats with the ligB mutant induced persistent colonization of the kidneys. Finally, LigB was not required to mediate bacterial adherence to cultured cells. Taken together, our data provide the first evidence of site-directed homologous recombination in pathogenic Leptospira species. Furthermore, our data suggest that LigB does not play a major role in dissemination of the pathogen in the host and in the development of acute disease manifestations or persistent renal colonization.

Comparison of the efficacy of two commercially available media for culturing one-cell embryos in the in vitro fertilization mouse model

Objective: To examine the effects of two commercial media on the development of mouse ova fertilized in vitro to the blastocyst stage. Design: Animal model. Setting: Academic institution. Animal(s): Eight-week old, superovulated mice. Intervention(s): One-cell embryos cultured in vitro up to the blastocyst stage in potassium-enriched simplex optimized medium (KSOM) or G1/G2 medium. Main Outcome Measure(s): Blastocyst and hatching rates, total cell number count, and proportion of allocation of cells to the inner cell mass (ICM) and trophectoderm (TE). Result(s): The percentage of zygotes that developed to the blastocyst stage 96 and 120 hours after insemination was statistically significantly higher in the KSOM group. The percentage of blastocysts that partially or completely hatched by day 5 of culture was 84% and 71% for the KSOM and G1/G2 groups, respectively, showing a statistically significant difference between the groups. The mean number of ICM cells was 11.7 +/- 4.0 and 9.2 +/- 5.2 for the zygotes cultured in KSOM and G1/G2 media, respectively, revealing a statistically significantly higher cell number in the ICM of blastocysts derived from culture in KSOM medium. The ICM/TE ratio in the blastocysts cultured in KSOM or G1/G2 media was similar in both groups. Conclusion(s): Commercially available KSOM medium is superior to sequential G1/G2 media for culturing one-cell embryos up to the blastocyst stage in the mouse IVF model.

EFFECTS OF A POTENT PEROXYNITRITE DECOMPOSITION CATALYST IN MURINE MODELS OF ENDOTOXEMIA AND SEPSIS

Excessive free-radical production due to various bacterial components released during bacterial infection has been linked to cell death and tissue injury. Peroxynitrite is a highly reactive oxidant produced by the combination of nitric oxide (NO) and superoxide anion, which has been implicated in cell death and tissue injury in various forms of critical illness. Pharmacological decomposition of peroxynitrite may represent a potential therapeutic approach in diseases associated with the overproduction of NO and superoxide. In the present study, we tested the effect of a potent peroxynitrite decomposition catalyst in murine models of endotoxemia and sepsis. Mice were injected i.p. with LPS 40 mg/kg with or without FP15 [Fe(III) tetrakis-2-(N-triethylene glycol monomethyl ether) pyridyl porphyrin] (0.1, 0.3, 1, 3, or 10 mg/kg per hour). Mice were killed 12 h later, followed by the harvesting of samples from the lung, liver, and gut for malondialdehyde and myeloperoxidase measurements. In other subsets of animals, blood samples were obtained by cardiac puncture at 1.5, 4, and 8 h after LPS administration for cytokine (TNF-alpha, IL-1 beta, and IL-10), nitrite/nitrate, alanine aminotransferase, and blood urea nitrogen measurements. Endotoxemic animals showed an increase in survival from 25% to 80% at the FP15 doses of 0.3 and 1 mg/kg per hour. The same dose of FP15 had no effect on plasma levels of nitrite/nitrate. There was a reduction in liver and lung malondialdehyde in the endotoxemic animals pretreated with FP15, as well as in hepatic myeloperoxidase and biochemical markers of liver and kidney damage (alanine aminotransferase and blood urea nitrogen). In a bacterial model of sepsis induced by cecal ligation and puncture, FP15 treatment (0.3 mg/kg per day) significantly protected against mortality. The current data support the view that peroxynitrite is a critical factor mediating liver, gut, and lung injury in endotoxemia and septic shock: its pharmacological neutralization may be of therapeutic benefit.

Nonhuman primate models and the failure of the Merck HIV-1 vaccine in humans

The adenovirus type 5 (Ad5)-based vaccine developed by Merck failed to either prevent HIV-1 infection or suppress viral load in subsequently infected subjects in the STEP human Phase 2b efficacy trial. Analogous vaccines had previously also failed in the simian immunodeficiency virus (SIV) challenge-rhesus macaque model. In contrast, vaccine protection studies that used challenge with a chimeric simian-human immunodeficiency virus (SHIV89.6P) in macaques did not predict the human trial results. Ad5 vector -based vaccines did not protect macaques from infection after SHIV89.6P challenge but did cause a substantial reduction in viral load and a preservation of CD4(+) T cell counts after infection, findings that were not reproduced in the human trials. Although the SIV challenge model is incompletely validated, we propose that its expanded use can help facilitate the prioritization of candidate HIV-1 vaccines, ensuring that resources are focused on the most promising candidates. Vaccine designers must now develop T cell vaccine strategies that reduce viral load after heterologous challenge.

Higgs pair production at the LHC in models with universal extra dimensions

In this Letter we study the process of gluon fusion into a pair of Higgs bosons in a model with one universal extra dimension. We find that the contributions from the extra top quark Kaluza-Klem excitations lead to a Higgs pair production cross section at the LHC that can be significantly altered compared to the Standard Model value for small values of the compactification scale. (C) 2007 Elsevier B.V. All rights reserved.

Crotamine toxicity and efficacy in mouse models of melanoma

Objectives: Selective anticancer cell activity for both cell-penetrating and cationic antimicrobial peptides has previously been reported. As crotamine possesses activities similar to both of these, this study investigates crotamine`s anticancer toxicity in vitro and in vivo. Research design and methods: In vitro cancer cell viability was evaluated after treatment with 1 and 5 mu g/ml of crotamine. In vivo crotamine cytotoxic effects in C57Bl/6J mice bearing B16-F10 primary cutaneous melanoma were tested, with two groups each containing 35 mice. The crotamine-treated group received 1 mu g/day of crotamine per animal, subcutaneously which was well tolerated; the untreated group received a placebo. Results: Crotamine at 5 mu g/ml was lethal to B16-F10, Mia PaCa-2 and SK-Mel-28 cells and inoffensive to normal cells. In vivo crotamine treatment over 21 days significantly delayed tumor implantation, inhibited tumor growth and prolonged the lifespan of the mice. Mice in the crotamine-treated group survived at significantly higher rates (n = 30/35) than those in the untreated group (n = 7/35) (significance calculated with the Kaplan-Meier estimator). The average tumor weight in the untreated group was 4.60 g but was only about 0.27 g in the crotamine-treated mice, if detectable. Conclusions: These data warrant further exploration of crotamine as a tumor inhibition compound.

Comparison of Two Models for Evaluation Histopathology of Experimental Renal Ischemia

Renal ischemia/reperfusion (I/R) injury is one of the frequent causes of acute renal failure (ARF) due to the complex, interrelated sequence of events, that result in damage to and death of kidney cells. Cells of the proximal tubular epithelium are especially susceptible to I/R injury, leading to acute tubular necrosis, which plays a pivotal role in the pathogenesis of ARE Several models have been explicated to assess morphological changes, including those of Jabonski et al. and Goujon et al. We compared the 2 models for histopathological evaluation of 30- or 120-minute periods of renal ischemia followed by 24-hour reperfusion in rats. Several changes were observed after application of the 2 models: proximal tubular cell necrosis, loss of brush border, vacuolization, denudation of tubular basement membrane as a consequence of flattening of basal cells, and presence of intratubular exfoliated cells in the lumen of proximal convoluted tubules at various stages of degeneration (karyorexis, kariopyknosis and karyolysis). Evaluating tubular lesions after 2 periods of experimental ischemia with light microscopy allowed us to conclude that the Goujon classification better characterized the main changes in cortical renal tubules after ischemia.