Development of single-cycle replicable human immunodeficiency virus 1 mutants.

Non-infectious but antigenic human immunodeficiency virus 1 (HIV-1) particles are essential tool for the research on many topics associated with this virus. Here we report the construction of plasmid containing the HIV-1 genome mutated in the pol gene, which was co-transfected with plasmids expressing the pol gene products reverse transcriptase (RT) and integrase (IN), and the glycoprotein G of vesicular stomatitis virus (VSV-G). The virions produced in HEK 293 T cells were antigenic, but able to replicate only for one cycle, e.g. first generation single-cycle replicable (SCR) virions. The presence of VSV-G in the envelope of these virions had to ensure a wider spectrum of susceptible cell types for the replication of SCR. Replication of the first generation SCR virions in HEK 293T, MT-2, and mouse spleen cells was examined by p24-capture ELISA, syncytium formation assay, and electron microscopy (EM). HEK 293T and MT-2 cell lines showed a similar replication capacity, while primary cultures of mouse spleen cells were much less effective. The infection of MT-2 cells with the first generation of SCR virions yielded the second generation SCR virions, which were non-infectious. Summing up, the HIV-1 SCR virions represent the useful tool for HIV-1 research facilitating a better biological safety. Moreover, considering their antigenic composition and limited replication, SCR virions may be a promising candidate for the vaccine studies.

An efficient procedure for purification of recombinant human ß heat shock protein 90.

BACKGROUND AND THE PURPOSE OF THE STUDY: Heat Shock Protein 90 (Hsp90) is typically the most abundant chaperone in the eukaryotic cell cytoplasm, and its expression is essential for loading immunogenic peptides onto major histocompatibility complex molecules for presentation to T-cells. Therefore, it may act as a good candidate as an adjuvant molecule in vaccine technology. METHODS: Initially the human Hsp90ß gene was cloned into the heat inducible expression vector pGP1-2 and then the recombinant protein was isolated by ion exchange chromatography. After intradermal injection of confirmed purified band of protein to rabbits and isolation of the serum IgG antibody, for its affinity purification, the rabbit's purified Hsp90 specific IgG was coupled to the cyanogen bromide-activated Sepharose 4B. RESULTS: The recovery of the purified protein of interest by affinity chromatography was 50%. CONCLUSION: This research enabled purification of human heat shock protein by a laboratory prepared column chromatography.

Beta-human chorionic gonadotropin in cervicovaginal secretions and preterm delivery.

OBJECTIVES: To determine whether concentrations of beta-HCG in cervicovaginal secretions could predict spontaneous preterm birth (SPB) in asymptomatic high risk pregnancies. METHODS: A cohort study was undertaken with cervicovaginal samples collected from 540 pregnant women between 20 to 28 weeks of gestation. Levels of beta-HCG were measured by ELISA test. RESULTS: There was 3.2-fold increase in cervicovaginal beta-HCG concentrations among patients with SPB vs. term delivery. A single cervicovaginal beta-HCG > 77.8 mIU/ml, between 20 and 28 weeks' gestation, identified patients with subsequent SPB vs. term delivery with sensitivity of 87.5% (95% CI: 47.4-97.9) and a specificity of 97% (95% CI: 86.5-99.4) with positive and negative predictive values of 88.5% and 98%, respectively. Multiple logistic regression indicates that cervicovaginal beta-HCG level > 77.8 mIU/ml was an independent predictor of SPB (adjusted odds ratio 19.97, 95% CI: 10.65-37.45). CONCLUSIONS: Cervicovaginal beta-HCG is a sensitive and specific predictor of patients with subsequent preterm delivery.

Garlic induces a shift in cytokine pattern in Leishmania major-infected BALB/c mice.

The regulation of T helper (Th)1- and Th2-type cytokine patterns is important in the final outcome of leishmaniasis in human and murine models. We examined the efficacy of garlic therapy or a combination of garlic and an antimonial drug (glucantime) in promoting healing and regulation of Th1/Th2 cytokine patterns in highly susceptible BALB/c mice infected with Leishmania major. Separate groups of infected mice received 20 mg/kg/day garlic, 60 mg/kg/day glucantime or a combination of the two, from day 30 after infection for 2 weeks. An enzyme-linked immunosorbant assay (ELISA) was performed on spleen cell culture supernatants for interferon(IFN)-gamma interleukin(IL)-2, IL-4 and IL-10. The results indicate that garlic therapy is more effective than the usual antileishmanial drug in curing the infection. Garlic-treated mice developed Th1-type cytokine responses. In contrast, glucantime therapy led to a Th2-type response in the control group with a lower level of IL-2. However, a combination of garlic and glucantime treatment was more effective than either treatment alone, and resulted in a Th1-type response similar to that which developed with garlic treatment. These results suggest that garlic extract in combination with an antimonial drug, may provide effective therapy against L. major. The immunomodulatory properties of garlic were elucidated in terms of shifting the cytokine response to a Th1-type pattern and therefore causing the protective response.