Respiratory health among office workers in Malaysia and endotoxin and (1,3)-ß-glucan in office dust.

OBJECTIVE: To examine the associations between endotoxin and (1,3)-ß-glucan concentrations in office dust and respiratory symptoms and airway inflammation among 695 office workers in Malaysia.METHODS: Health data were collected using a questionnaire, sensitisation testing and measurement of fractional exhaled nitric oxide (FeNO). Indoor temperature, relative air humidity (RH) and carbon dioxide (CO2) were measured in the offices and settled dust was vacuumed and analysed for endotoxin and (1,3)-ß-glucan concentrations. Associations were analysed by two level multiple logistic regression.RESULTS: Overall, 9.6% of the workers had doctor-diagnosed asthma, 15.5% had wheeze, 18.4% had daytime attacks of breathlessness and 25.8% had elevated FeNO (≥25 ppb). The median levels in office dust were 11.3 EU/mg endotoxin and 62.9 ng/g (1,3)-ß-glucan. After adjusting for personal and home environment factors, endotoxin concentration in dust was associated with wheeze (P = 0.02) and rhinoconjunctivitis (P = 0.007). The amount of surface dust (P = 0.04) and (1,3)-ß-glucan concentration dust (P = 0.03) were associated with elevated FeNO.CONCLUSION: Endotoxin in office dust could be a risk factor for wheeze and rhinoconjunctivitis among office workers in mechanically ventilated offices in a tropical country. The amount of dust and (1,3)-ß-glucan (a marker of indoor mould exposure) were associated with Th2 driven airway inflammation.

Antifungal susceptibilities, biofilms, phospholipase and proteinase activities in the Candida rugosa complex and Candida pararugosa isolated from tertiary teaching hospitals.

PURPOSE: Non-albicansCandida species have emerged as fungal pathogens that cause invasive infections, with many of these species displaying resistance to commonly used antifungal agents. This study was confined to studying the characteristics of clinical isolates of the C. rugosa complex and C. pararugosa species. METHODOLOGY: Seven isolates of the C. rugosa complex and one isolate of C. pararugosa were obtained from two tertiary referral hospitals in Malaysia. Their antifungal susceptibilities, biofilm, proteinase, phospholipase, esterase and haemolysin activities were characterized. Biofilms were quantified using crystal violet (CV) and tetrazolium (XTT) reduction assays at 1.5, 6, 18, 24, 48 and 72 h.Results/Key findings. The E-test antifungal tests showed that both species have elevated MICs compared to C. albicans and C. tropicalis. The highest biomass was observed in one of the C. rugosa isolates (0.237), followed by C. pararugosa (0.206) at 18 h of incubation. However, the highest bioactivity was observed in the C. rugosa ATCC 10571 strain at 24 h (0.075), followed by C. pararugosa at 48 h (0.048) and the same C. rugosa strain at 24 h (0.046), with P<0.05. All isolates exhibited high proteinase activity (+++) whereas six isolates showed very strong esterase activity (++++). All the isolates were alpha haemolytic producers. None of the isolates exhibited phospholipase activity. CONCLUSION: Elevated MICs were shown for the C. rugosa complex and C. pararugosa for commonly used antifungal drugs. Further studies to identify virulence genes involved in the pathogenesis and genes that confer reduced drug susceptibility in these species are proposed.

Metabolic adaptation via regulated enzyme degradation in the pathogenic yeast Candida albicans.

The virulence of Candida albicans is dependent upon fitness attributes as well as virulence factors. These attributes include robust stress responses and metabolic flexibility. The assimilation of carbon sources is important for growth and essential for the establishment of infections by C. albicans. Previous studies showed that the C. albicans ICL1 genes, which encode the glyoxylate cycle enzymes isocitratelyase are required for growth on non-fermentable carbon sources such as lactate and oleic acid and were repressed by 2% glucose. In contrast to S. cerevsiae, the enzyme CaIcl1 was not destabilised by glucose, resulting with its metabolite remaining at high levels. Further glucose addition has caused CaIcl1 to lose its signal and mechanisms that trigger destabilization in response to glucose. Another purpose of this study was to test the stability of the Icl1 enzyme in response to the dietary sugars, fructose, and galactose. In the present study, the ICL1 mRNAs expression was quantified using Quantitative Real Time PCR, whereby the stability of protein was measured and quantified using Western blot and phosphoimager, and the replacing and cloning of ICL1 ORF by gene recombination and ubiquitin binding was conducted via co-immuno-precipitation. Following an analogous experimental approach, the analysis was repeated using S. cerevisiaeas a control. Both galactose and fructose were found to trigger the degradation of the ICL1 transcript in C. albicans. The Icl1 enzyme was stable following galactose addition but was degraded in response to fructose. C. albicans Icl1 (CaIcl1) was also subjected to fructose-accelerated degradation when expressed in S. cerevisiae, indicating that, although it lacks a ubiquitination site, CaIcl1 is sensitive to fructose-accelerated protein degradation. The addition of an ubiquitination site to CaIcl1 resulted in this enzyme becoming sensitive to galactose-accelerated degradation and increases its rate of degradation in the presence of fructose. It can be concluded that ubiquitin-independent pathways of fructose-accelerated enzyme degradation exist in C. albicans.

Probiotic Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 exhibit strong antifungal effects against vulvovaginal candidiasis-causing Candida glabrata isolates.

AIMS: This study investigates the antagonistic effects of the probiotic strains Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 against vulvovaginal candidiasis (VVC)-causing Candida glabrata. METHODS AND RESULTS: Growth inhibitory activities of Lact. rhamnosus GR-1 and Lact. reuteri RC-14 strains against C. glabrata were demonstrated using a spot overlay assay and a plate-based microtitre assay. In addition, these probiotic lactobacilli strains also exhibited potent candidacidal activity against C. glabrata, as demonstrated by a LIVE/DEAD yeast viability assay performed using confocal laser scanning microscopy. The metabolic activities of all C. glabrata strains were completely shut down in response to the challenges by the probiotic lactobacilli strains. In addition, both probiotic lactobacilli strains exhibited strong autoaggregation and coaggregation phenotypes in the presence of C. glabrata, which indicate that these lactobacilli strains may exert their probiotic effects through the formation of aggregates and, thus the consequent prevention of colonization by C. glabrata. CONCLUSIONS: Probiotic Lact. rhamnosus GR-1 and Lact. reuteri RC-14 strains exhibited potent antagonistic activities against all of the tested C. glabrata strains. These lactobacilli exhibited antifungal effects, including those attributed to their aggregation abilities, and their presence caused the cessation of growth and eventual cell death of C. glabrata. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to report on the antagonistic effects of these probiotic lactobacilli strains against the non-Candida albicans Candida (NCAC) species C. glabrata.

Basement membrane formation by malignant mouse keratinocyte cell lines in organotypic culture and transplants: correlation with degree of morphologic differentiation.

Six malignant C3H mouse epidermal cell lines (HEL-30, HEL-37, HELP I, HELP IV, HD II, H3L), with different capacities for epidermal differentiation, were analyzed for their organized growth behavior and basement membrane (BM) formation in organotypical cultures in vitro and after transplantation into syngeneic mice. Expression and deposition of five BM components (type IV collagen, laminin, bullous pemphigoid antigen, fibronectin, heparan sulfate proteoglycan) were determined on frozen sections by indirect immunofluorescence. Additionally, synthesis and secretion of BM components by the line HEL-30 (in submersed cultures) were identified by metabolic labeling and immunoprecipitation. Morphologic differentiation features and formation of a structured BM were studied by electron microscopy. All cell lines were tumorigenic and invasive but nevertheless able to synthesize BM constituents in vitro and in vivo, although pronounced variations in the expression and the polarity and continuity of deposition were found. Irrespective of the amount of BM components synthesized, none of the cell lines formed a structured BM in organotypical cultures in vitro. After transplantation the production of BM components was improved and the newly formed epithelia were separated from the mesenchyme by a structured BM. The formation of BM occurred whether the epithelial cells were in immediate contact with the mesenchyme or separated by a 1 to 2 mm thick native collagen gel. Deposition of BM constituents and formation of BM structures occurred both at the superficial epithelium and around invading cell cords. The studies clearly demonstrated that malignant epidermal cells maintain their capacity to synthesize BM components. The extent of production and the polarity of deposition of the constituents and the quality of BM formation were usually higher in well differentiated cell lines and obviously correlated well with their preserved degree of differentiation. Comparable to normal keratinocytes, formation of structured BM requires interaction with living mesenchyme but occurs independently of direct epidermal-mesenchymal contact.