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Tumor necrosis factor (TNF)-alpha induces pleiotropic cellular effects through a 55kDa, type 1 receptor (TNFR1) and a 75kDa type 2 receptor (TNFR2). Moreover, it participates in the pathogenesis of several CNS diseases, including demyelinating diseases. TNF-alpha receptors are differentially expressed and are regulated in many cell types. However, data regarding the TNF-alpha receptor expression and regulation in human astrocytes is limited to date. We investigated TNF- receptor expression, its regulation by cytokines, and its functional role in primary cultured human fetal astrocytes, which are the most abundant cellular population in the central nervous system and are known to be immunologically active. In this study, astrocytes were found to constitutively and predominantly transcribe, translate and shed TNFR1 rather than TNFR2, but TNFR2 expression was increased by adding TNF-alpha, IL-1, and IFN-gamma, but not by adding LPS. To determine the functional roles of TNFR1 and TNFR2 on TNF induction, we investigated NF-kappaB activation and TNF-alpha induction after neutralizing TNFR1 and TNFR2 by an antibody treatment. We found that NF-kappaB activation and TNF-alpha induction are blocked by TNFR1 neutralizing antibody treatments.

Purpose Candida albicans (C. albicans) and Proteus species are causative agents in a variety of opportunistic nosocomial infections, and their ability to form biofilms is known to be a virulence factor. In this study, the influence of co-cultivation with Proteus vulgaris (P. vulgaris) and Proteus mirabilis (P. mirabilis) on C. albicans biofilm formation and its underlying mechanisms were examined. Materials and methods XTT reduction assays were adopted to measure biofilm formation, and viable colony counts were performed to quantify yeast growth. Real-time reverse transcriptase polymerase chain reaction was used to evaluate the expression of yeast-specific genes (rhd1 and rbe1), filament formation inhibiting genes (tup1 and nrg1), and hyphae-related genes (als3, ece1, hwp1, and sap5). Results Candida biofilm formation was markedly inhibited by treatment with either living or heat-killed P. vulgaris and P. mirabilis. Proteus-cultured supernatant also inhibited Candida biofilm formation. Likewise, treatment with live P. vulgaris or P. mirabilis or with Proteus-cultured supernatant decreased expression of hyphae-related C. albicans genes, while the expression of yeast-specific genes and the filament formation inhibiting genes of C. albicans were increased. Heat-killed P. vulgaris and P. mirabilis treatment, however, did not affect the expression of C. albicans morphology-related genes. Conclusion These results suggest that secretory products from P. vulgaris and P. mirabilis regulate the expression of genes related to morphologic changes in C. albicans such that transition from the yeast form to the hyphal form can be inhibited.

Astrocytes are ubiquitous in the brain and have multiple functions. It is becoming clear that they play an important role in monitoring the neuromicroenvironment, information processing, and signaling in the central nervous system (CNS) in normal conditions and that they respond to CNS injuries. During the development of the CNS, astrocytes play a key role as a substrate for neuronal migration and axonal growth. To identify genes that could participate in astrocyte maturation, we used the differential display reverse transcription-PCR (DDRT-PCR) method. Human fetal astrocytes were cultured and total RNAs were isolated at intervals of 5 days for 50 days. Using 24 primer combinations, we identified a set of 18 candidate cDNAs deriving from the excised DDRT-PCR bands. DNA sequencing revealed 16 genes that have been described already. We found that RTP, TG, hTM-alpha, SPARC, TRIP7, and RPL7 genes were expressed increasingly, while HMGCR, RPL27a, NACA, NPM, and TARBP2 genes were expressed decreasingly, according to their culture stages. We also found two unidentified genes, A3 and C8, which were expressed differently in culture stages; the former was expressed decreasingly and the latter increasingly. These two genes were found in the same amount in genomic DNA from various human cells such as astrocytes, astrocytoma, trophoblasts and lymphocytes. The A3 gene was found only in human genomic DNA, but not in rat (ATr5), mouse (RAW264.7), or monkey (Vero) cells, whereas the C8 gene was found in human genomic DNA and monkey cells, but not in rat or mouse cells. We analysed these two genes for identification. There was >92% nucleotide sequence identity between the A3 gene (3,626 bp) and the Homo sapiens general transcription factor 3 (GTF3), and >96% nucleotide sequence identity between the C8 gene (2,401 bp) and the transmembrane receptor Unc5h2. These findings suggest that these two genes may participate in some functional roles within the cells.

Candida albicans exhibits the ability to grow in either a yeast or a mycelia form in response to different environmental factors. The mycelia form, found in infected tissues, is important as a virulence factor in the adherence of the organism to the host epithelium. In vitro, the morphological transition can be induced by environmental shifts in the growing conditions, or by a variety of exogenous factors, including ambient pH, nutritional status and temperature. The differential-display reverse transcription polymerase chain reaction (DDRT-PCR) is a powerful technique for comparing gene expression between cell types, stages of development or differentiation. Hyphae related genes were identified and characterized using a PCR-based differential display. Candida albicans formed a germ tube when cultured in rabbit serum, RPMI 1640 medium or 39 degrees C-YPD medium. We gained 21 cDNA bands showing a different expression pattern from that of the uninduced culture. DNA was extracted from the same location of the isolated bands, and PCR was performed under the same conditions, which reamplified the PCR product, showing the specific expression patterns according to the culture conditions. We cloned 18 germ tube-related cDNA clones (inserts average size is 80 - 700 bp) and sequenced them. The nucleotide sequences of the 18 clones were identified through in the present study from GenBank, and were found to have the accession number (AF405213-AF405230). We could not find any nucleotide sequence having a high homology with these clones. This study could form a part of the projects in the search for genes related to the germ tube formation of C. albicans.

Purpose Candida albicans is an opportunistic pathogen that is commonly found in human microflora. Biofilm formation (BF) is known as a major virulence factor of C. albicans. The aim of this study was to examine the influence of bacterial presence on biofilm formation of C. albicans. Materials and Methods The BF of Candida was investigated when it was co-cultured with C. albicans (C. albicans 53, a yeast with a low BF ability, and C. albicans 163, a yeast with high BF ability) and bacteria. BF was assessed with XTT reduction assay. A scanning electron microscope was used to determine the structure of the biofilm, and real-time reverse transcriptase polymerase chain reaction was used to amplify and quantify hyphae-associated genes. Results Co-culturing with two different types of bacteria increased the BF value. Co-culturing with C. albicans 53 and 163 also increased the BF value compared to the value that was obtained when the C. albicans was cultured individually. However, co-culturing with bacteria decreased the BF value of C. albicans, and the BF of C. albicans 163 was markedly inhibited. The expression of adherence and morphology transition related genes were significantly inhibited by co-culturing with live bacteria. Conclusion Bacteria have a negative effect on the formation of biofilm by C. albicans. This mechanism is the result of the suppression of genes associated with the hyphae transition of C. albicans, and bacteria particles physically affected the biofilm architecture and biofilm formation.

From the culture filtrates of C. albicans, C. tropicalis and C. parapsilosis, proteinases were purified using a series of chromatographic steps consisting of DEAE-Sepharose, Sephacryl S-200 and size-exclusion HPLC which removed contaminating mannoproteins and extraneous proteins. Anti-Candida proteinase antibodies in sera from mice infected with various Candida species were detected using ELISA for serodiagnosis of candidiasis. Three proteinases were blotted by homologous and heterologous anti-proteinase antisera on Western blot analysis. All sera from six Candida species-infected mice were reactive with proteinases of C. albicans, C. tropicalis, and C. parapsilosis, although C. glabrata, C. guilliermondii, and C. krusei did not secrete proteinase. The seroreactivities of proteinase with sera from mice infected with homologous C. albicans and C. tropicalis were higher than those with sera from heterologous Candida species-infected mice. These results suggest that three proteinases have at least one common epitope, but its application for diagnosis of candidiasis should be considered with limits of specificity.

Epidemiologic studies were conducted on 31 cases of Salmonella group E infection detected in 2000 through a laboratory-based pathogen surveillance in Gangwon Province, Korea. Data were collected on the environmental exposures and the patients' foods, including the brand(s) of milk consumed before the onset of diarrhea. The patients' medical records were also reviewed. All of the patients were infants under 10 months of age except one 7-year old child. Surprisingly, all of the infants were fed with infant formulas from Company A, although two infants were fed with infant formulas from both Company A and Company B. Antimicrobial susceptibility test and pulsed-field gel electrophoresis (PFGE) were performed in 25 out of 31 isolates from the patients and in 1 isolate from an opened packet of infant formula collected from the home of an infected infant. All of the 26 isolates were Salmonella enterica serovar London. They showed a single PFGE pattern, and all of the isolates were susceptible to the 18 antibiotics tested. The causative agent of the salmonella outbreaks in the Gangwon Province and its surrounding areas was Salmonella London, and the highly likely source of the infection was infant formula from Company A.