Mitogenic and morphogenic effects of a bovine salivary gland extract on astrocytes and fibroblasts.

The presence of mitogenic and morphogenic activity in extracts of bovine salivary (parotid) glands is reported. The crude and partially purified extracts stimulated cultured rat cerebellar cells (astrocytes) and skin fibroblasts to undergo morphogenesis. The incorporation of [3H]thymidine was also stimulated in astrocytes, skin fibroblasts, and established fibroblastic cell lines. Growth-promoting activity was also demonstrated. The expression of maximum mitogenic activity in skin fibroblast cultures, but not kidney fibroblast cultures, required the presence of serum. The biological activity had an apparent native molecular weight greater than 230,000, was heat-sensitive, trypsin-resistant, and slightly sensitive to the action of papain.

Typing of sequential bacterial isolates by pulsed-field gel electrophoresis.

We typed 39 sets of multiple bacterial isolates of the same species from patients by pulsed-field gel electrophoresis of genomic DNA (PFGE). Isolates were cultured from different sites or over a 2-week or longer interval. Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae were tested. Excluding E. cloacae, 28 of 32 sets of isolates (87%) demonstrated only identical or highly related PFGE types. Four of the seven sets of E. cloacae showed different types. For species other than E. cloacae, our results suggest that patients are usually colonized and infected with a single strain of these bacterial pathogens. Unlike all of the other tested species, E. cloacae PFGE typing differences suggested the presence of multiple strains causing colonization and infection.

Species-specific binding of transformed Ah receptor to a dioxin responsive transcriptional enhancer.

The Ah receptor (AhR) mediates many, if not all, of the toxic and biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related halogenated aromatic hydrocarbons. Although wide variations in species sensitivity to these compounds have been observed, numerous biochemical and physiochemical characteristics of the AhR appear similar among species. We have examined the ability of cytosolic AhR, from a variety of species (rat, rabbit, guinea pig, hamster, mouse, cow, sheep, fish, chicken and human), to transform and bind to its cognate DNA recognition sequence, the dioxin responsive enhancer (DRE), to evaluate the importance of these events in species variations in TCDD responsiveness. Gel retardation analysis using a murine DRE oligonucleotide has revealed that cytosolic AhR from a wide variety of species can transform in vitro and bind to the DRE and demonstrates that all of the factors necessary for AhR transformation and DNA binding are present in cytosol. In addition, DNA-binding analysis using a series of mutant DRE oligonucleotides has indicated no apparent species- or ligand-dependent, nucleotide-specific difference in AhR binding to the DRE. These studies support a highly conserved nature of the DRE and AhR (at least in DNA binding) and imply that a sequence closely related to the murine consensus DRE sequence is responsible for conferring AhR-dependent, TCDD responsiveness in each of these species.

Assessment of analysis by gender in the Cochrane reviews as related to treatment of cardiovascular disease.

BACKGROUND: Cardiovascular disease (CVD) is the leading killer of women in the United States, yet medical care is often based on evidence from clinical trials performed predominantly with men. Numerous studies show that CVD risk factors, clinical presentation, treatment, and treatment outcomes can vary between men and women. METHODS: The Cochrane Library maintains a large database of critically appraised evidence including meta-analyses of clinical trials, called Systematic Reviews. There were 30 Systematic Reviews pertaining to the treatment of CVD published collectively by the Cochrane Heart Group, Hypertension Group, and Peripheral Vascular Diseases Groups at the time of our study. We examined these 30 Systematic Reviews and the great majority of the clinical trials used for their meta-analyses for inclusion of women and gender-based data analyses. Women comprised only 27% of the pooled population of 258 clinical trials. RESULTS: Of those trials that included both men and women (n = 196), only 33% examined outcomes by gender. In trials that performed a gender-based analysis, 20% reported significant (p < 0.05) differences in cardiovascular-related outcomes by gender. CONCLUSIONS: We conclude that (1) there are not enough large-scale clinical trials or meta-analyses concerning CVD in women to determine if their medical treatment should differ from that of men, (2) all clinical trials relating to CVD treatment should have significantly more female participants, and gender-based analyses should be performed, as currently recommended for National Institutes of Health (NIH)-sponsored research by the NIH Revitalization Act of 1993, and (3) the Cochrane Library would be a more useful tool for the evidence-based healthcare of women if the Systematic Reviews used all available gender-specific information in their analyses.

Factors influencing the interaction of Candida albicans with fibroblast cell cultures.

The interaction of Candida albicans clinical isolates with primary and established fibroblast cultures was studied. The intent was to determine whether yeast adherence and invasion of nonendothelial cell monolayer cultures could be quantitated reproducibly and whether this system could be used for future studies on yeast pathogenesis. Our results demonstrated that specific interactions between the yeast cells and fibroblasts only occurred at 37 degrees C and correlated with the germination process. Fluorescent-antibody staining indicated that invasion or tight associations between the germinating yeast cells and mammalian cells occurred after less than 3 h of incubation. Yeast adherence was estimated radiometrically and trypsin-resistant interaction with individual mammalian cells (infection) was measured microscopically after inoculated monolayer cells were detached with trypsin. We demonstrated that both types of association were time dependent at 37 degrees C; neither was affected by the concentration of glucose used to grow the yeast cells. Primary and established fibroblast cell lines were equally susceptible to infection, but primary cells appeared to have more yeast-binding sites. Fibroblasts maintained in confluent culture for an extended period of time also appeared to have more binding sites, and while not quantitatively more susceptible to infection, the older cells were more susceptible to infection-related cell death. An established kidney epithelial cell line (MDCK) was not susceptible to either type of yeast interaction, indicating that the yeast-fibroblast associations were specific.

The effects of amphotericin B on the interaction of Candida albicans with fibroblast cultures.

Sublethal amounts of amphotericin B inhibited the interaction of Candida albicans with cultured fibroblasts. Different C. albicans clinical isolates exhibited varying degrees of sensitivity to the drug, but those isolates that were the most infective in control cultures appeared to be the most resistant to amphotericin B mediated infection inhibition. Although amphotericin B inhibited germ tube formation at the sublethal concentration of 0.3 microgram/mL, lower concentrations inhibited infection without preventing germination. The extent of this latter activity varied with the isolate and amphotericin B concentration and appeared to be related to sublethal effects on germinated yeasts. While amphotericin B effectively prevented new fibroblast infection, it did not dissociate those yeasts which had established an infection before its addition.

Conditions affecting the amphotericin B mediated inhibition of Candida albicans attachment to cell cultures.

Sublethal amounts of amphotericin B inhibited the attachment of Candida albicans to cultured mammalian cells. The extent of inhibition was influenced by the concentration of serum and the growth phase of the yeasts used to inoculate the cell cultures. Yeasts which were in their exponential phase of growth or had formed germ tubes were the most sensitive to amphotericin B. Equivalent amounts of amphotericin B inhibited yeast-mammalian cell interactions to different degrees depending upon the culture's tissue origin.

Cell surface specific immunoglobulin inhibits alpha factor mediated morphogenesis in Saccharomyces cerevisiae.

Immunoglobulins raised from Saccharomyces cerevisiae a and alpha mating type cell envelope preparations inhibited alpha factor mediated morphogenesis of the a cell without inhibiting normal cell division. The Ig responsible for this inhibition was absorbed to both a and alpha whole cells and heat-killed cells, indicating that the immunoglobulin binding sites were exposed on the cell surface and not mating type specific. Additionally, alpha factor mediated cell cycle arrest was not affected by the immunoglobulin preparations, implying that the immunoglobulin was not preventing alpha factor from binding to its receptor.

Assessment of bacterial cross-transmission as a cause of infections in patients in intensive care units.

The prevalence of possible cross-transmission of selected bacteria (Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Pseudomonas aeruginosa, Staphylococcus aureus, and enterococci) among infected patients was evaluated in five intensive care units (ICUs) over 6 months. A total of 284 isolates from clinical specimens were typed by plasmid profile analysis (E. coli, K. pneumoniae, and E. cloacae), restriction endonuclease analysis of plasmid DNA (S. aureus), and/or pulse-field gel electrophoresis of chromosomal DNA (P. aeruginosa, enterococci, S. aureus, and other bacteria without plasmid DNA). By typing criteria, only 13% of the 177 isolates obtained after > 2 days in an ICU were classified as possibly cross-transmitted. Many patients whose cultures yielded bacteria of an identical type may have been the sources rather than the recipients of these organisms. Episodes of possible cross-transmission were scattered among all ICUs, usually affected only two patients, and were associated with most bacterial species. These data suggest that endemic bacterial cross-transmission in ICUs is relatively infrequent and that cross-transmitted bacteria are not common causes of endemic ICU-related nosocomial infections.

In vivo stability and discriminatory power of methicillin-resistant Staphylococcus aureus typing by restriction endonuclease analysis of plasmid DNA compared with those of other molecular methods.

We evaluated test discriminatory power and DNA type alterations among methicillin-resistant Staphylococcus aureus strains by testing 199 sequential isolates from 39 patients collected over 30 to 228 days. Isolates were typed by one or three different methods (restriction endonuclease analysis of plasmid DNA [REAP] with or without pulsed-field gel electrophoresis of genomic DNA [PFGE] and immunoblotting [IB]). REAP was highly discriminatory compared with PFGE and IB. However, the initial isolates from 4 of the 39 patients lacked detectable plasmid DNA and could not be typed by REAP. Typing of individual patient isolates showed that a different REAP type was identified only once every 138 days. Among 25 comparisons, seven sequential isolate pairs demonstrating REAP differences were also different by PFGE and IB. This likely represented the presence of more than one strain. Eighteen other pairs with REAP differences were identical or related to one another by PFGE and IB typing, and 17 of these differences were likely caused by a single genetic alteration within the same strain or clone. The rate of PFGE differences explicable by single genetic alterations among sequential isolates identical by REAP was similar to the overall rate for REAP differences in the whole collection. We conclude that REAP and PFGE typing differences explicable by single genetic alterations are relatively infrequent but not rare. These isolates should be examined by alternative typing systems to further support or refute clonality.