To investigate the inflammatory cytokine production of human epithelial cell lines stimulated with uropathogenic E. coli strains showing 3 different adherence patterns, differential expression of inflammatory cytokine (IL-1a, IL-lB, IL-8, TNFa, and TGFB) mRNA were detected by RT-PCR. IL-1a, IL-1B, IL-8, and TGFB mRNAs constitutively expressed in epithelial cell lines, but not TNFa. The expression of IL-1a and IL-1B mRNA was increased in J-82 cells stimulated with E. coli strains showing DA, LA, or AggA pattern. The expression of IL-8 mRNA was increased, whereas TGFj3 mRNA was decreased in J-82 cells stimulated with E. coli strain showing AggA.pattern. Treatment with crude bacterial adhesins (CBA) isolated from E. coli strains showing DA or LA pattern increased IL-la, IL-lB, IL-S, and TGFj3 mRNA expressions in J-82 cells and HeLa cells. IL-la, IL-lB, and TGFB mRNA expressions were decreased in epitheUal cells stimulated with CBA from E. coli strain showing AggA pattern, whereas IL-8 mRNA expression was significantly increased. The expressions of cytokine mRNAs showed little differences between epithelial ceRs used, but great differences between CBA from DA or LA and AggA strain. LPS stimulation was little changed cytokine mRNA expressions in epithelial cells. This study suggests that cytokine gene expression of epithelial cells by the bacterial stimulation mainly depends on the bacterial adhesins recognized by the respective receptors of epithelial cells.
Adhesins, Bacterial ; Epithelial Cells* ; Gene Expression ; HeLa Cells ; Humans ; Interleukin-8 ; RNA, Messenger ; Uropathogenic Escherichia coli*

In our previous report an anti-HBsAg human monoclonal antibody was generated using antibody phage display library technique. Using pComb3 filamentous phagemid vector, Fab molecule was expressed in fusion form to a phage coat protein in the periplasm of E. coli. A clone of HBsAg binder was selected after panning and designated as B7. In order to select the clones with higher affinity and to examine which chain contributes most to the affinity of B7, the light and heavy chain of B7 was sequentially deleted and replaced with new library. HBsAg-binders were selected and tested by EIA (enzyme immunoassay). It was revealed that the affinity of B7 depends only on the heavy chain of Fd. B7 Fd was constructed without light chain and specificity and affinity was further confirmed by western blot analysis. This human monoclonal Fd antibody was found to react with d antigenic determinant of HBsAg as the original clone did. The nucleotide sequence analysis revealed that VH of B7 could be classified into Kabat's subgroup II and human IgG heavy chain family IV. The CH1 of B7 was IgG1.
Bacteriophages* ; Base Sequence ; Blotting, Western ; Clone Cells ; Hepatitis B Surface Antigens ; Humans ; Immunoglobulin G ; Periplasm ; Sensitivity and Specificity

Genes encoding sequence-specific DNA binding proteins have been isolated by screening cDNA libraries constructed in rgt11 expression vector with recognition site DNAs. We isolated a rgt11 recombinant human cDNA clone, designated to C2, using a DNA probe consisted of heptamer of the perfect palindrome (PP; GGGGATTCCCC) of enhancer A (Enh A) of HLA dass I promoter. Sequencing analysis showed that this clone contained a partial cDNA homologous to NF-kB2. Lysogenic E. coli containing the C2 was generated and crude cell extract was prepared. Immunoblot using anti-B-galactosidase antibody showed that this lysogenic E. coli expressed B-galactosidase fusion protein. Electrophoretic mobility shift assay (EMSA) and DNase I footprinting assay were done using crude cell extract and their patterns were compared with nuclear protein extracted from an EBV transformed B lymphoblastoid cell line (BLCL). EMSA showed that crude cell extract prepared from E. coli lysogen speci5cally bound to the PP of Enh A region of HLA class I gene. DNase I footprinting assay showed that the binding sequence of this recombinant B-galactosidase fusion protein was identical to that of nuclear protein extracted from a BLCL. Our data indicate that a Agt11 recombinant cDNA clone was isolated from a human cDNA library using the PP of Enh A of the HLA class I promoter and this clone encoded a B-galactosidase fusion protein capable of binding to the PP and belongs to a NF-xB subunit.
Cell Line ; Clone Cells ; Deoxyribonuclease I ; DNA ; DNA, Complementary* ; DNA-Binding Proteins ; Electrophoretic Mobility Shift Assay ; Gene Library ; Genes, MHC Class I ; Herpesvirus 4, Human ; Humans* ; Mass Screening ; Nuclear Proteins

Phase-partitioning with Triton X-114 (TX114) was applied to the TSP antigen, which may be preferentially associated with the cell wall of M. tuberculosis. The hydrophilic protein components of the TSP antigen were successfully separated from integral hydrophobic macromolecules. To further characterize and examine the cellular immune response of the aqueous fraction of the TSP antigen (TSPa), the in vitro properties of the antigen were measured by lymphoproliferation; surface expression of IL-2 Ra on T lymphocytes was analyzed by flow cytometry; and the cytokine mRNA expression pattern was determined by RT-PCR. Significant lymphoproliferative responses to the TSPa antigen were observed in healthy tuberculin reactive donors after a 5 day in vitro stimulation. TSPa treatment of PBMCs from healthy tuberculin positive subjects for 5 days resulted in progressive augmentation of IFN-r, II 2, and IL-2Ra mRNA expression, as measured by RT-PCR, but considerably reduced IL-4 mRNA expression. In addition, the TSPa antigen stimulated more IL-12 p40 mRNA production than did the PPD antigen, and graduaBy suppressed IL- 10 mRNA expression. Moreover, the CD3' T cells of tuberculin positive subjects displayed a profound increase in their expression of the II 2Ru protein (39.0%) in response to the TSPa antigen. Proliferation was correlated with IL-2 and IL-2Ra mRNAs, but not correlated with distinct IFN-r or IL-12 p40 mRNA production. These findings strongly suggest that the TSPa antigen preferentially evokes the generation of a Thl-like immune response in healthy tuberculin reactors.
Cell Wall ; Flow Cytometry ; Humans* ; Immunity, Cellular* ; Interleukin-12 ; Interleukin-2 ; Interleukin-4 ; Mycobacterium tuberculosis* ; Mycobacterium* ; Neptune ; RNA, Messenger ; T-Lymphocytes ; Tissue Donors ; Tuberculin ; Tuberculosis

Enterotoxigenic B. pagilis (ETBF) strains which produce a 20 kDa zinc metalloprotease toxin (BFI) have been associated with diarrheal disease of animals and young children. Using B. pngilis toxin gene (bfi) from strain 86-5443-2-2 (piglet isolate) as a probe, the gene was identified in 74/77 human and animal ETBF strains but only 2/97 non-toxigenic B. fragilis (NTBF) strains. The region flanking bp was mapped with several restriction enzymes and 8 resriction fragments aacent to bft were used to probe colony blots of 77 KTBF and 97 NTBF strains. All 74 bft-positive ETBF strains hybridized to the 8 probes spanning a ca. 18 kb chromosomal region; however, this 18 kb region was absent in the 3 ETBF strains lacking p, and 47 of the 97 (48%) NTBF strains lacked the entire 18 kb region. Of note, the 2 NTBF strains containing btf did not have a ca. 12 kb region upstream of btfp. A ca. 9 kb fragment flanking the btf gene has been sequenced. Analysis of this data revealed several open reading frames (ORF) of which 3 are of particular interest (ORFs 1, 2 and 3). ORF1 and ORF3 encode proteins with significant homology to mobilization proteins, and ORF2 encodes a protein with significant homology to metalloprotease proteins, but only 50% similarity and 30% identity to BFf. These results suggest: 1) the btf genes are flanked by at least 18 kb of DNA largely unique to ETBF strains indicating a putative pathogenic island, 2) another metalloprotease protein present in ETBF strains may contribute to the pathogenicity and variable virulence of these diarrheagenic strains and 3) the pathogenic island may be mobiTized among different Bacteroides strains, and possibly among different species of intestinal bacteria.
Animals ; Bacteria ; Bacteroides fragilis* ; Bacteroides* ; Child ; DNA ; Genomic Islands* ; Humans ; Open Reading Frames ; Virulence* ; Zinc

Treatment of rainbow trout macrophages with glycyrrhizin (GL), an aqueous extract of licorice (Glycyrrhiza glabra), enhanced their respiratory burst activity. Maximal effects were seen using concentrations of 10-100 ug/ml. GL also modulated trout lymphocytes, increasing proliferation responses to the mitogen phytohemagglutinin two-fold over a range of GL concentrations. In addition, GL elicited the release of a macrophage activating factor (MAF) kom head kidney leukocytes, as assessed by the ability of generated supernatants to increase respiratory burst activity of target macrophages. MAF activity was most apparent using 100 ug/ml GL to induce MAF release and a 48 h incubation period with the target macrophages. Finally, GL was shown to enhance the release oF MAF in response to the mitogen concanavalin A. The results suggest that GL might modulate the innate defences in fish.
Concanavalin A ; Glycyrrhiza ; Glycyrrhizic Acid* ; Head Kidney ; Leukocytes* ; Lymphocytes ; Macrophages ; Oncorhynchus mykiss* ; Oncorhynchus* ; Respiratory Burst ; Trout

Apoptosis is a crucial mechanism for the selective elimination of mammalian cells and is involved in many physiological and pathological processes. The heightened awareness of the importance of apoptosis has increased the need for rapid and quantitative methods for measurement of apoptotic changes. Recently, 7-amino-actinomycin D (7-AAD) has been introduced as a valuable fluorescent dye for assessing apoptosis by flow cytometry. When the cells are stained with 7-AAD in the concentration of 10 - 20 ug/ml, live cells are not stained (7-AAD ) and early apoptotic cells are weakly stained (7-AAD ) while late apoptotic or dead cells are stained brightly (7-AAD). On scattergram of forward angle light scatter vs. 7-AAD fluorescence, the three populations can be discriminated not only between each other but also from cell debris or clumps. The apoptotic cells, defined as 7- AAD cells, were demonstrated as apoptotic by morphological observation of the sorted cells. The 7-AAD cell fraction was also demonstrated to be parallel with subdiploid fraction of cells stained with PL However, 7-AAD cells, whose definition is based on the alteration of membrane integrity, have never been demonstrated to be subdiploid fraction by simultaneous DNA staining. Here we directly demonstrate that 7-AAD cells, defined on the scattergram of forward angle light scatter vs. 7-AAD fluorescence, are subdiploid fraction by staining with DNA dye whose fluorescence is collected after 530/30 band pass filter (FL-1). We also demonstrate the effects of 7-AAD concentration, fixation of cells, and proliferation of cells, on the fluorescence pattern, for reference during assessment of apoptosis by simple and rapid method for flow cytometric analysis of cells stained with 7- AAD. We also present a flow cytometric analysis of cells stained with 7-AAD Eor sequential change in apoptotic fraction, with concurrent dual-color immunophenotyping.
Apoptosis* ; DNA ; Flow Cytometry* ; Fluorescence ; Fluorescent Antibody Technique* ; Immunophenotyping ; Membranes ; Pathologic Processes

Interaction with Candida albicans (C. albicans) causes secretion of a variety of cytokines by macrophages. Although macrophages and neutrophils are thought to be major effector cell types in inflammation, fibroblasts have also been shown to participate in a wide array of inflammatory reactions. The patterns of cytokine gene (IL-4, IL-6, IL-10) expression have been examined in NIH 3T3 fibroblasts (NIH 3T3) and thioglycollate-elicited murine peritoneal macrophages (macrophages) in response to Candida spp. (C. albicans and C. tropicalis) and compared with NIH 3T3 and macrophage cells stimulated with lipopolysaccharide (LPS) by using RT-PCR. Active and heat inactivated (100'C, 30min) C. albicans and C. tropicalis were used at 1:10 (macrophages or NIH 3T3: yeasts ratio) concentration as a stimulators. Active and inactivated forms of Candidn spp. induced same patterns of cytokine gene expression on macrophages or NIH 3T3 cells. IL-6 mRNA was induced on both macrophages and fibroblasts, but IL-4 mRNA was not induced on both of them. Howerver, IL-10 mRNA expression was observed differently in that it was expressed in macrophages but not in fibroblasts. C. albicans-induced cytokine mRNA expression were same to C. tropicalis-induced one. C. albicans or C. tropicalis-induced cytokines gene expression (IL-4, IL-6, IL-10) showed same patterns to LPS-induced one. These observation provide that, although C. albicans and C. tropicalis have different pathogenic activity, they can induce the expression of Th2 cell-derived cytokine genes with same patterns, and IL-10 gene expression may be in a cell type specific manner. But further experiment with various kinds of fibroblasts is needed to determine cell type-specific manner in C. albicans or C. tropicalis-induced IL-10 gene expression.
Animals ; Candida albicans ; Candida* ; Cytokines ; Fibroblasts* ; Gene Expression* ; Hot Temperature ; Inflammation ; Interleukin-10 ; Interleukin-4 ; Interleukin-6 ; Macrophages ; Macrophages, Peritoneal* ; Mice* ; Neutrophils ; NIH 3T3 Cells ; RNA, Messenger ; Yeasts

No abstract available.
Humans*

No abstract available.
Gelatin* ; Hemorrhagic Fever with Renal Syndrome*