Relative carriage rates of nuclear dehydrogenating clostridia in two populations of different colorectal cancer risk

Carriage of nuclear dehydrogenating clostridia has been associated with colon cancer and implicated in its aetiology. This study has compared the carriage of these organisms in a British population at high risk for the development of colon cancer with a low risk Nigerian population. Clostridia were found in all of the stools from both populations. Nuclear dehydrogenating clostridia were only found in the stools of the British subjects (32%). These results support the suggestion that the carriage rate of nuclear dehydrogenating clostridia in a population is related to the risk of colon cancer.     

Iron regulation of Serratia marcescens hemolysin gene expression.

The hemolytic activity of Serratia marcescens was examined as a function of iron availability. Restriction of iron by the nonmetabolizable chelator 2,2'-dipyridyl or the iron-binding protein transferrin produced a marked increase in hemolytic activity. The hemolytic activity of S. marcescens is determined by two adjacent genes, 5'-shlB-shlA-3', where shlA encodes the hemolysin which requires the ShlB protein for activity. A gene fusion between the promoter-proximal portion of shlA and phoA, the Escherichia coli alkaline phosphatase gene, was subcloned into a medium-copy-number vector, and the recombinant plasmid was introduced into S. marcescens. The expression of shlA was measured as a function of alkaline phosphatase activity, which increased threefold under iron-restricted conditions. Removal of the 5' noncoding region upstream of shlB in the fusion vector resulted in a 10-fold decrease in alkaline phosphatase activity under iron-sufficient conditions, with no effect of iron limitation on this residual activity. This suggested that the site mediating iron regulation of shlA expression occurs upstream of shlB. Consistent with this, we observed iron-regulated synthesis of the ShlB protein in Western immunoblots of isolated outer membranes. The hemolysin determinant was subsequently expressed on a medium-copy-number vector in fur+/fur isogenic strains of E. coli K-12, where a 10-fold-higher activity was observed in the mutant strain compared with the wild type. A sequence exhibiting some homology to the Fur-binding consensus sequence was identified upstream of the shlB coding region, overlapping the -35 region of a putative promoter.     

Monoclonal antibodies against Pseudomonas aeruginosa outer membrane antigens: isolation and characterization.

Hybridomas secreting monoclonal antibodies specific for Pseudomonas aeruginosa outer membrane antigens were isolated. One of the antibodies was highly specific for the O antigen of the lipopolysaccharide of International Antigen Typing Scheme serotype 5 strains, reacting only weakly with a serotype 17 strain and failing to react with the outer membranes of strains representing 15 other serotypes. This monoclonal antibody was able to agglutinate heat-killed bacterial cells as well as lipopolysaccharide-coated sheep erythrocytes. Two other monoclonal antibodies were able to interact with the outer membranes of strains representing all 17 serotypes, although they were unable to agglutinate heat-killed bacterial cells. One of these was shown to be specific for the major outer membrane lipoprotein H2. The antigenic site against which this monoclonal antibody reacted was present in the outer membranes of two Pseudomonas fluorescens strains, two Pseudomonas putida strains, a Pseudomonas anguilliseptica strain, and an Azotobacter vinelandii strain, but not in the outer membranes of five other bacterial species.     

Development of hatching blastocysts from immature human oocytes following in-vitro maturation and fertilization using a co-culture system

Recently, in-vitro maturation (IVM) of immature human oocytes recovered from non-stimulated follicles has been applied in the treatment of infertility. However, in previous reports, very few embryos cultured in conventional medium have reached the expanded blastocyst stage following in-vitro maturation and fertilization (IVM/IVF). The objective of this study was to investigate whether the developmental competence of human embryos following IVM/IVF could be enhanced by the use of a human ampullary cell co-culture system. Immature human oocytes were aspirated from small follicles at Caesarean section and then cultured in medium containing human menopausal gonadotrophin for 36 to 48 h, followed by insemination. Zygotes were randomly cultured either in conventional culture medium alone or in the co-culture system. Of 48 embryos cultured in conventional medium alone, all arrested at the 2-16- cell stage on day 3 after insemination. Of 46 embryos cultured in the co-culture system, 26 embryos (56.5%) arrested at the 2-16-cell stage. Six embryos (13%) developed to the morula stage. Fourteen embryos (30.4%) developed to expanded blastocysts and two blastocysts were hatching on day 7 after insemination. We conclude that co-culture significantly enhances the development of blastocysts in embryos resulting from IVM/IVF.      

Recombinant Actinobacillus actinomycetemcomitans cytolethal distending toxin proteins are required to interact to inhibit human cell cycle progression and to stimulate human leukocyte cytokine synthesis

It has recently been discovered that Actinobacillus actinomycetemcomitans, an oral bacterium causing periodontitis, produces cytolethal distending toxin (CDT), a cell cycle-modulating toxin that has three protein subunits: CdtA, CdtB, and CdtC. In this study, we have cloned and expressed each toxin gene from A. actinomycetemcomitans in Escherichia coli and purified the recombinant Cdt proteins to homogeneity. Individual Cdt proteins failed to induce cell cycle arrest of the human epithelial cell line HEp-2. The only combinations of toxin proteins causing cell cycle arrest were the presence of all three Cdt proteins and the combination of CdtB and CdtC. A similar experimental protocol was used to determine if recombinant Cdt proteins were able to induce human peripheral blood mononuclear cells (PBMCs) to produce cytokines. The individual Cdt proteins were able to induce the synthesis by PBMCs of interleukin-1beta (IL-1beta), IL-6, and IL-8 but not of tumor necrosis factor alpha, IL-12, or granulocyte-macrophage colony-stimulating factor, with CdtC being the most potent and CdtB being the least potent cytokine inducer. There was evidence of synergism between these Cdt proteins in the stimulation of cytokine production, most markedly with gamma interferon, which required the minimum interaction of CdtB and -C to stimulate production.     

Detection of chromosomes and estimation of aneuploidy in human spermatozoa using fluorescence in-situ hybridization

The development and application of fluorescence in-situ hybridization (FISH) has opened the way for comprehensive studies on numerical chromosome abnormalities in human spermatozoa. FISH can be rapidly applied to large numbers of spermatozoa and thus overcomes the major limitation of karyotyping spermatozoa after penetration of zona-free hamster oocytes. The simultaneous hybridization of two or more chromosome-specific probes to spermatozoa and subsequent detection of the bound probes using different fluorescent detection systems enables two or more chromosomes to be localized simultaneously in the same spermatozoon and provides a technique for undertaking reasonable estimates of aneuploidy. The most commonly used probes are those which bind to the centromeric region of specific chromosomes. Most studies to date have concentrated on estimating aneuploidy in spermatozoa from normospermic men, although reports are beginning to appear on aneuploidy in spermatozoa from subfertile and infertile men. Multi- probe FISH studies have generally reported disomy (hyperhaploidy) estimates of 0.05-0.2% per chromosome. There is preliminary evidence that some chromosomes such as X, Y and 21 are predisposed towards higher rates of non-disjunction during spermatogenesis. There are also suggestions of inter-donor variability in aneuploidy frequencies for specific chromosomes, although this requires confirmation in larger studies. While FISH is clearly a powerful technique that has many applications in reproductive medicine, it must also be realized that it does have limitations and the technology itself is still evolving and has yet to be fully validated on spermatozoa.      

Adverse effects of early dexamethasone in extremely-low-birth-weight infants. National Institute of Child Health and Human Development Neonatal Research Network

BACKGROUND: Early administration of high doses of dexamethasone may reduce the risk of chronic lung disease in premature infants but can cause complications. Whether moderate doses would be as effective but safer is not known. METHODS: We randomly assigned 220 infants with a birth weight of 501 to 1000 g who were treated with mechanical ventilation within 12 hours after birth to receive dexamethasone or placebo with either routine ventilatory support or permissive hypercapnia. The dexamethasone was administered within 24 hours after birth at a dose of 0.15 mg per kilogram of body weight per day for three days, followed by a tapering of the dose over a period of seven days. The primary outcome was death or chronic lung disease at 36 weeks' postmenstrual age. RESULTS: The relative risk of death or chronic lung disease in the dexamethasone-treated infants, as compared with those who received placebo, was 0.9 (95 percent confidence interval, 0.8 to 1.1). Since the effect of dexamethasone treatment did not vary according to the ventilatory approach, the two dexamethasone groups and the two placebo groups were combined. The infants in the dexamethasone group were less likely than those in the placebo group to be receiving oxygen supplementation 28 days after birth (P=0.004) or open-label dexamethasone (P=0.01), were more likely to have hypertension (P<0.001), and were more likely to be receiving insulin treatment for hyperglycemia (P=0.02). During the first 14 days, spontaneous gastrointestinal perforation occurred in a larger proportion of infants in the dexamethasone group (13 percent, vs. 4 percent in the placebo group; P=0.02). The dexamethasone-treated infants had a lower weight (P=0.02) and a smaller head circumference (P=0.04) at 36 weeks' postmenstrual age. CONCLUSIONS: In preterm infants, early administration of dexamethasone at a moderate dose has no effect on death or chronic lung disease and is associated with gastrointestinal perforation and decreased growth.