Investigating hepatitis C virus heterogeneity in a high prevalence setting using heteroduplex tracking analysis

Hepatitis C virus (HCV) infection is very common among chronic hemodialysis patients. In the past, blood transfusion appeared to be the primary risk factor; however evidence of nosocomial HCV transmission in the hemodialysis setting has recently been reported. This report describes a molecular investigation of HCV isolates obtained from a population of 670 patients attending six different Seattle-King County based hemodialysis centers in order to identify potential common source infections. 733 serum specimens were collected from hemodialysis patients in 1992 and 1996, and were tested for HCV antibodies and RNA. Overall, 115 of 670 (17%) patients were positive for HCV RNA, and thus were considered actively infected by HCV. HCV genotype was determined in all cases by restriction fragment length polymorphism, and 93 patients were found to be infected by HCV genotype 1. HCV envelope genes were amplified from the 93 patients with genotype 1 infection, and were studied in further detail by heteroduplex tracking analysis (HTA) using genotype 1a and 1b specific probes derived from the envelope 1 (E1) and envelope 2 (E2) genes. Genetic relatedness between pairs of HCV envelope genes was estimated by calculating the degree of gel shift relative to homoduplex controls. Nucleotide sequencing and phylogenetic analysis was used to confirm genetic relatedness detected by HTA. When HTA was performed using the E1 gene probe, 12 apparently related infections were detected; 10 of 12 (83%) of these infections were confirmed as truly related using the gold standard method of nucleotide sequencing plus phylogenetic analysis. Using an E2 gene probe, 24 infections were apparently related, but only six (25%) were confirmed by sequencing. As a control, 41 envelope genes, which were unrelated by HTA, were sequenced; 0 of 41 (0%) were truly related. In summary, HTA provides a rapid and effective molecular technique for screening HCV genetic relatedness in population-based studies, and should prove valuable in future studies of HCV molecular epidemiology.     

Noradrenergic mechanisms in the central amygdalar nucleus and gastric stress ulcer formation in rats

Microinjections of noradrenaline (NA, 0.3, 3.0 and 30.0 micrograms) into the central amygdalar nucleus (CEA) produced dose-related attenuations of cold restraint (3 h at 4 degrees C) induced gastric ulcer formation in rats. On the other hand, stress ulcer aggravating effects were seen with beta-adrenoceptor antagonist, propranolol (10 micrograms) but not with the alpha-adrenoceptor antagonist, prazosin (1 and 10 micrograms). Moderate enhancements of gastric stress lesions were also seen with the NA release inhibitor clonidine (1 microgram) and the neurotoxin DSP-4 (25 micrograms). Further, pretreatment of rats with intra-amygdalar (i.am.) propranolol but not prazosin, antagonized and reversed the gastric cytoprotective effects of NA. The results indicate that beta-adrenoceptor-mediated NAergic mechanisms at the level of the CEA are important for the maintenance of gastric mucosal integrity during immobilization stress.     

Altered patterns of migration of cytokine-producing T lymphocytes in skin-grafted naive or immune mice following in vivo administration of anti-VCAM-1 or -ICAM-1.

Naive or preimmunized (to B10.BR or BALB.k) C3H/HeJ mice received skin grafts from multiple minor histoincompatible B10.BR or BALB.k mice following antigen-specific portal venous (p.v.) pretransplant transfusion, a protocol known to produce prolongation of graft survival in naive animals. In addition, groups of mice received intravenous (i.v.) infusion following transplantation with a mixture of monoclonal antibodies (mAb) to vascular adhesion molecule-1L: very late activation antigen-4 (VCAM-1:VLA-4) or intracellular adhesion molecule-1:lymphocyte function-associated antigen-3 (ICAM-1:LFA-1). Cells were harvested from different tissues of the grafted mice at various times post grafting. RNA was extracted and analysed, using polymerase chain reaction, for expression of different cytokines potentially involved in the regulation of graft rejection [interleukin-2 (IL-2), IL-4, IL-6, IL-10, tumour necrosis factor-alpha, interferon-gamma and transforming growth factor-beta]. In addition, using limiting dilution analysis, we investigated the frequency of allo-specific and third-party reactive cells producing IL-2 and IL-4 in vitro in different tissues of grafted mice following these treatments. The mAb treatment protocol which produced optimum increases in graft survival in naive versus immune mice was different, with anti-LFA-1:ICAM-1 superior for naive mice compared with anti-VLA-4:VCAM-1, and vice versa for immune animals. However, in each case, increased survival was associated with increases local to the graft in the frequency of occurrence of antigen-specific type-2 cytokine-producing cells.     

Prevention of mother-to-child transmission of HIV--what next?

Mother-to-child transmission (MTCT) of the human immunodeficiency virus (HIV) is a global problem. HIV can be transmitted from mother-to-child at various stages of pregnancy including in utero, intrapartum and during breastfeeding. A number of interventions have, therefore, been aimed at effectively providing alternatives to breastfeeding and limiting the risk of newborn infection during delivery, by using caesarian section as the mode of delivery and administering antiretroviral (ARV) drugs prepartum and peripartum. However, these approaches are not always possible in developing countries and the use of ARV drugs, in particular nevirapine, zidovudine and zidovudine/lamivudine, have been investigated in both developing countries and developed countries. The studies have involved the administration of various ARV prophylaxis regimens to HIV-infected pregnant women perinatally, either as monotherapy or in various combinations. In some studies, infants have also received ARV prophylaxis. Although studies have enrolled different populations and utilized various ARV drugs and regimens, encouraging reductions in the MTCT rates have been reported. These interventions have raised concerns regarding the development of ARV-resistant HIV strains. Mutations that confer resistance to nevirapine have been detected in pregnant women who received this drug, but the emergence of these mutations was not associated with an increased risk of transmission of HIV-1 to their infants. Studies are ongoing to determine if the presence of these mutations has implications for the subsequent administration of nevirapine, either to prevent MTCT of HIV-1 or for the mother's own health. Effective interventions that can reduce MTCT of HIV are now available worldwide. However, a number of issues remain to be resolved, particularly methods to reduce the transmission of the virus during breastfeeding and to deliver effective treatment for the mothers' own HIV infection.     

Corticosterone influences on Mammalian neonatal sensitive-period learning

Infant rats exhibit sensitive-period odor learning characterized by olfactory bulb neural changes and odor preference acquisitions critical for survival. This sensitive period is coincident with low endogenous corticosterone (CORT) levels and stress hyporesponsivity. The authors hypothesized that low corticosterone levels modulate sensitive-period learning. They assessed the effects of manipulating CORT levels by increasing and removing CORT during (Postnatal Day 8) and after (Postnatal Day 12) the sensitive period. Results show that (a) exogenous CORT prematurely ends sensitive-period odor-shock-induced preferences; (b) adrenalectomy developmentally extends the sensitive period as indicated by odor-shock-induced odor-preference learning in older pups, whereas CORT replacement can reinstate fear learning; and (c) CORT manipulation modulates olfactory bulb correlates of sensitive-period odor learning in a manner consistent with behavior.     

High level of unequal meiotic crossovers at the origin of the 22q11. 2 and 7q11.23 deletions

Interstitial chromosomal deletions at 22q11.2 and 7q11.23 are detected in the vast majority of patients affected by CATCH 22 syndromes and the Williams-Beuren syndrome, respectively. In a group of 15 Williams- Beuren patients, we have shown previously that a large number of 7q11.23 deletions occur in association with an interchromosomal rearrangement, indicative of an unequal crossing-over event between the two homologous chromosomes 7. In this study, we show that a similar mechanism also underlies the formation of the 22q11.2 deletions associated with CATCH 22. In eight out of 10 families with a proband affected by CATCH 22, we were able to show that a meiotic recombination had occurred at the critical deleted region based on segregation analysis of grandparental haplotypes. The incidences of crossovers observed between the closest informative markers, proximal and distal to the deletion, were compared with the expected recombination frequencies between the markers. A significant number of recombination events occur at the breakpoint of deletions in CATCH 22 patients (P = 2.99x10(-7)). The segregation analysis of haplotypes in three- generation families was also performed on an extended number of Williams-Beuren cases (22 cases in all). The statistically significant occurrence of meiotic crossovers (P = 4.45x10(-9)) further supports the previous findings. Thus, unequal meiotic crossover events appear to play a relevant role in the formation of the two interstitial deletions. The recurrence risk for healthy parents in cases where such meiotic recombinations can be demonstrated is probably negligible. Such a finding is in agreement with the predominantly sporadic occurrence of the 22q11.2 and 7q11. 23 deletions. No parent-of-origin bias was observed in the two groups of patients with regard to the origin of the deletion and to the occurrence of inter- versus intrachromosomal rearrangements.      

Problems in the long-term assessment of risks from drugs used in pregnancy maintenance

Progestogens and oestrogens used to maintain pregnancy in casesof threatened abortion could have side-effects on the motheror fetus. The difficulties in interpreting epidemiological studieson the effects of sex hormones on pregnancy are discussed inrelation to the combination of hormones employed, their purpose,time of usage and dosage. ^*Based on a paper presented at the Conference on Post-MarketingSurveillance of Drugs Used in Fertility Regulation, organizedby the International Committee for Research in Reproduction,Leuven 17–19 October 1985.     

Investigation of an outbreak of antibiotic-associated colitis by various typing methods.

During an outbreak of diarrheal disease due to Clostridium difficile in a surgical ward, 16 C. difficile isolates were cultured from fecal samples of 15 patients. Agarose gel electrophoresis for the detection of plasmid DNA, crossed immunoelectrophoresis for the detection of extracellular antigens and toxins, polyacrylamide gel electrophoresis for analyses of soluble proteins, assays for cytotoxicity, and a comparison of susceptibility to antimicrobial agents were employed. At least 12 of the 16 isolates were shown to be phenotypically the same strain. These findings suggest that in a hospital setting, diarrhea and pseudomembranous colitis caused by C. difficile can be of nosocomial origin and that they can spread from patient to patient.