Effects of rapamycin on the epithelial-to-mesenchymal transition of human peritoneal mesothelial cells.

The preservation of the peritoneal membrane is crucial for long-term survival in peritoneal dialysis. Epithelial-to-mesenchymal transition (EMT) is a process demonstrated in mesothelial cells (MC), responsible for negative peritoneal changes and directly related to PD. EMT enables neovascularization and fibrogenic capabilities in MC. Vascular endothelial growth factor (VEGF) is the mediator for neo-vascularization. Rapamycin is a potent immunosuppressor with antifibrotic action in renal allografts and has a demonstrated anti-VEGF effect. We performed this study with the hypothesis that rapamycin may regulate the EMT of MC. MC from human omentum were cultured. When mesothelial cells reached confluence, some of them were stimulated with r-TGF-beta (1 ng/mL) to induce EMT, co-administered with rapamycin (0.2, 2, 4, 20 and 40 nM). Other groups of cells received similar doses of rapamycin or r-TGF-beta, separately. Cells were analyzed at 6, 24, 48 hours and 7 days. As markers of EMT we included alfa -SMA, E-cadherin and snail nuclear factor by quantitative RT-PCR. EMT markers and regulators demonstrated the following changes with rapamycin: E-cadherin (a protective gene for EMT) increased 2.5-fold relative to controls under 40 nM, at 24h. Importantly, rapamycin inhibited snail expression induced by TGF-beta at 6h, whereas TGF-beta increased snail 10-fold. At day 7, rapamycin showed no anti-EMT properties. An important decrease in alfa -SMA expression by MC after rapamycin addition was observed. In conclusion, rapamycin shows a mild protective effect on EMT, as it increases E-cadherin and decreases alfa -SMA expression. Consequently, rapamycin might partially regulate the epithelial-to-mesenchymal transition of mesothelial cells.

Embryonic thymic epithelium naturally devoid of APCs is acutely rejected in the absence of indirect recognition.

Transplants of tissues depleted of passenger leukocytes are upon in vitro culture usually accepted in allogeneic recipients. Accordingly, fully allogeneic embryonic thymic epithelium was suggested to be poorly immunogenic. However, this tissue is capable of inducing donor-specific tolerance to peripheral tissues, when restoring T cell development in nude mice, through the production of regulatory cells. In the present work, adult immunocompetent allogeneic recipients were grafted with embryonic tissues isolated at stages before hemopoietic colonization or even before the establishment of circulation. Allogeneic thymic epithelium of day 10 embryos and heart primordium of day 8 embryonic donors were always rejected. Acute rejection of the thymic anlagen takes place in less than 12 days, with maximal CD4(+) and CD8(+) T cell infiltrates at 10 days post-transplant. In addition, a significant infiltrate of NK1.1(+) cells is observed, although without any essential role in this process. Furthermore, recipients lacking the indirect pathway of Ag presentation to CD4(+) T cells do not reveal any significant delay in rejection, even when CD8(+) T cells are also eliminated. Thus, our experimental approach reveals acute allograft rejection in the absence of all known pathways of naive T cell activation and therefore unveils a novel graft rejection mechanism that should be mediated by direct recognition of parenchymal cells. Given the importance of dendritic cells in naive T cell activation, it is likely that cross-reactive memory T cells may also drive rejection.

Variance in fibronectin binding and fnb locus polymorphisms in Staphylococcus aureus: identification of antigenic variation in a fibronectin binding protein adhesin of the epidemic CMRSA-1 strain of methicillin-resistant S. aureus.

The fnbA and fnbB genes of Staphylococcus aureus 8325-4 encode fibronectin (Fn) binding proteins FnBPA and FnBPB, which promote adherence to host tissues. Each adhesin contains three copies of a repeated D motif that binds Fn and is a target for vaccine development. In this study, we assess variability within the Fn-binding domain of the FnBP adhesins and evaluate factors that promote variance in Fn binding among clinical isolates. Based on variation in the number of fnb genes or the number of D motifs, we identified five polymorphism groups. S. aureus 8325-4 and 91% of methicillin-resistant S. aureus (MRSA) isolates belong to polymorphism group I, with two fnb genes and three copies of the D motif. Polymorphism group II contained one fnb gene with only two D motifs and was associated with the epidemic CMRSA-4 strain, which exhibited high protease activity and low Fn binding. Polymorphism group III was unique to the epidemic CMRSA-1 strain, defined by the presence of a fourth D motif that exhibited antigenic variation within a conserved sequence that is essential for Fn binding. However, the sequence of the D motifs was otherwise highly conserved among the other polymorphism groups. Variation in Fn binding among MRSA isolates was inversely related to protease activity but not to the number of fnb genes or the number of D motifs. Therefore, the fnb locus is polymorphic in a small number of strains, but this does not contribute to variation in Fn binding. The antigenic variation that was observed only in the epidemic CMRSA-1 strain may have evolved in response to a host immune response encountered during successive cycles of colonization, transmission, and infection in the nosocomial environment.

Enterohemorrhagic Escherichia coli induces apoptosis which augments bacterial binding and phosphatidylethanolamine exposure on the plasma membrane outer leaflet.

Enterohemorrhagic Escherichia coli (EHEC) is a gastrointestinal pathogen that causes watery diarrhea and hemorrhagic colitis and can lead to serious and even fatal complications such as hemolytic uremic syndrome. We investigated the ability of EHEC to kill host cells using three human epithelial cell lines. Analysis of phosphatidylserine expression, internucleosomal cleavage of host cell DNA and morphological changes detected by electron microscopy changes revealed evidence of apoptotic cell death. The rates and extents of cell death were similar for both verotoxin-producing and nonproducing strains of EHEC as well as for a related gastrointestinal pathogen, enteropathogenic E. coli (EPEC). The induction of apoptosis by bacterial attachment was independent of verotoxin production and greater than that produced by a similar treatment with verotoxin alone. Expression of phosphatidylethanolamine, previously reported to bind EHEC and EPEC, was also increased on apoptotic cells but with little correlation to phosphatidylserine expression. Phosphatidylethanolamine levels but not phosphatidylserine levels on dying cells correlated with EHEC binding. Cells treated with phosphatidylethanolamine-containing liposomes also showed increased EHEC binding. These results suggest that bacterial induction of apoptosis offers an advantage for bacterial attachment by augmenting outer leaflet levels of the phosphatidylethanolamine receptor.

Synthetic peptide immunogens elicit polyclonal and monoclonal antibodies specific for linear epitopes in the D motifs of Staphylococcus aureus fibronectin-binding protein, which are composed of amino acids that are essential for fibronectin binding.

A fibronectin (Fn)-binding adhesin of Staphylococcus aureus contains three tandem 37- or 38-amino-acid motifs (D1, D2, and D3), which function to bind Fn. Plasma from patients with S. aureus infections contain antibodies that preferentially recognize ligand induced binding sites in the D motifs and do not inhibit Fn binding (F. Casolini, L. Visai, D. Joh, P. G. Conaldi, A. Toniolo, M. Höök, and P. Speziale, Infect. Immun. 66:5433-5442, 1998). To eliminate the influence of Fn binding on antibody development, we used synthetic peptide immunogens D1(21-34) and D3(20-33), which each contain a conserved pattern of amino acids that is essential for Fn binding but which cannot bind Fn without N- or C-terminal extensions. The D3(20-33) immunogen promoted the production of polyclonal antibodies that were 10-fold more effective as inhibitors of Fn-binding to the D3 motif than antibodies obtained by immunizing with an extended peptide D3(16-36), which exhibits functional Fn binding. The D3(20-33) immunogen also facilitated the production of a monoclonal antibody, 9C3, which was highly specific for the epitope SVDFEED, and abolished Fn binding by the D3 motif. When mixed with polyclonal anti-D1(21-34) immunoglobulin G, 70% inhibition of Fn binding to the three tandem D motifs was achieved compared to no more than 30% inhibition with either antibody preparation alone. Therefore, by immunizing with short synthetic peptides that are unable to bind Fn, we have effectively stimulated the production of antibodies specific for epitopes comprised of amino acids that are essential for Fn binding. Although these epitopes occur within a conserved pattern of amino acids that is required for Fn binding, the antibodies recognized specific linear epitope sequences and not a conserved structure common to all repeated motifs.

Phosphatidylethanolamine recognition promotes enteropathogenic E. coli and enterohemorrhagic E. coli host cell attachment.

Using both solid phase and liposome aggregation assays, we screened a variety of glycolipids and phospholipids and found that EHEC and EPEC bind specifically and in a dose-dependent manner to PE. This binding was consistently observed whether the lipid was immobilized on a thin layer chromatography plate, in a microtitre well or incorporated into a unilamellar vesicle suspended in aqueous solution. There was no evidence of binding to other phospholipids such as phosphatidylcholine (PC) or phosphatidylserine (PS). Bacterial binding to two epithelial cell lines also correlated with the level of outer leaflet PE and was reduced following preincubation with anti-PE. The PE-binding phenotype of EPEC appeared to correlate with the bundle-forming pilus (bfp) genotype of a number of clinical isolates. These results provide evidence of a receptor role for PE in the adhesion of EHEC and EPEC to host cells.

Characterization of an acidic-pH-inducible stress protein (hsp70), a putative sulfatide binding adhesin, from Helicobacter pylori.

The in vitro glycolipid binding specificity of the gastric pathogen Helicobacter pylori is altered to include sulfated glycolipids (sulfatides) following brief exposure of the organism to acid pH typical of the stomach. This change is prevented by anti-hsp70 antibodies, suggesting that hsp70 may be a stress-induced surface adhesin, mediating sulfatide recognition. To facilitate investigation of the role of hsp70 in attachment, we have cloned and sequenced the H. pylori hsp70 gene (dnaK). The hsp70 gene was identified by probing a cosmid DNA library made from H. pylori 439 with a PCR amplicon generated with oligonucleotides synthesized to highly conserved regions of dnaK. The 1.9-kb H. pylori hsp70 gene encodes a product of 616 amino acids. Primer extension analysis revealed a single transcription start site, while Northern blot analysis established that hsp70 was preferentially induced by low pH rather than by heat shock. The ability of H. pylori to alter its glycolipid binding specificity following exposure to low pH by upregulating hsp70 and by expressing hsp70 on the bacterial surface may provide a survival advantage during periods of high acid stress.

Inhibition of Helicobacter pylori and Helicobacter mustelae binding to lipid receptors by bovine colostrum.

Helicobacter pylori, the etiologic agent of chronic-active gastritis and duodenal ulcers in humans, and Helicobacter mustelae, a gastric pathogen in ferrets, bind to phosphatidylethanolamine (PE), a constituent of host gastric mucosal cells, and to gangliotetraosylceramide (Gg4) and gangliotriaosylceramide (Gg3). The effect of a bovine colostrum concentrate (BCC) on the interaction of H. pylori and H. mustelae to their lipid receptors was examined. BCC blocked attachment of both species to Gg4, Gg3, and PE. Partial inhibition of binding was observed with native bovine and human colostra. BCC lacked detectable antibodies (by immunoblotting) to H. pylori surface proteins (adhesins). However, colostral lipid extracts contained PE and lyso-PE that bound H. pylori in vitro. These results indicate that colostrum can block the binding of Helicobacter species to select lipids and that binding inhibition is conferred, in part, by colostral PE or PE derivatives. Colostral lipids may modulate the interaction of H. pylori and other adhesin-expressing pathogens with their target tissues.

Characterization of prosomes in human lymphocyte subpopulations and their presence as surface antigens.

Prosomes, also called "multicatalytic proteinase" (MCP) or "proteasomes," are a new type of ubiquitous RNP particle present in some archeobacteria and in all eukaryotic cells tested from yeast to human. They were discovered as subcomplexes of untranslated messenger-ribonucleoproteins (mRNP) and later found to have a MCP activity putatively involved in antigen processing. Being composed of variable sets of characteristic proteins and associating small RNAs (pRNA), families of individual "mosaic" prosome particles seem to characterize the differentiation type and physiological state of individual cells and tissues. Here, prosomes from human lymphocytes, isolated and characterized biochemically and by Western blot analysis, were found to differ in their subunit composition compared to other human prosomes. Surprisingly, prosomal antigens were discovered at the outer surface of blood cells monitored by flow cytometry with monoclonal antibodies to individual prosomal proteins. It was observed that human T and B lymphocytes have variable and characteristic prosomal antigens at their surface according to their CD classification. Interestingly, the lymphocyte subpopulations most strongly labeled by the anti-p25K and anti-p27K mAbs were the NK and B cells.

Acidic pH changes receptor binding specificity of Helicobacter pylori: a binary adhesion model in which surface heat shock (stress) proteins mediate sulfatide recognition in gastric colonization.

The gastric pathogen helicobacter pylori is one of a number of bacteria which bind specifically to gangliotetraosylceramide, gangliotriaosylceramide, and phosphatidylethanolamine in vitro at neutral pH. Since this organism encounters an acid pH during initial infection of the stomach, we have monitored the effect of pH on receptor binding specificity and found induction of specific binding to sulfoglycolipids (sulfatide) following brief treatment at low pH. We have previously shown that heat shock proteins (hsps) bind to sulfatide, and the suspicion that this was a stress-induced response is supported by the fact that a similar change in H. pylori binding specificity was observed if the organisms were briefly exposed to heat shock treatment. Following the stress stimulus, the change in glycolipid binding specificity was prevented by the inclusion of inhibitors of protein synthesis or by incubation with anti-hsp antibodies. Expression of hsps in the surface extract and surface reactivity with anti-hsp antibodies correlated with the change in glycolipid binding specificity. Despite the presence of high levels of H. pylori cell surface urease activity which may neutralize the microenvironmental pH, the acid-induced change in binding specificity was enhanced in the presence of urea. These studies suggest that cell surface hsps mediate sulfatide recognition by this organism under stress conditions. A binary receptor model is proposed for gastric colonization by H. pylori.

Comparison of Helicobacter mustelae and Helicobacter pylori adhesion to eukaryotic cells in vitro.

BACKGROUND & AIMS: Bacterial adhesion to mucosal surfaces is an important pathogenic mechanism for Helicobacter-induced gastritis. The aims of this study were to compare binding of selected Helicobacter mustelae and Helicobacter pylori strains to lipids extracted from HEp-2, Chinese hamster ovary, human embryonic lung cells, and ferret gastrointestinal tissues as well as to intact tissue culture cells and to analyze the fatty acids of the receptor. METHODS: Thin-layer chromatography overlay binding and a receptor-based immunoassay detected adhesion of bacteria to commercial lipids and to individual species within the lipid extracts. H. mustelae binding to tissue culture cells was performed by whole cell bacterial adhesion assay. RESULTS: H. mustelae and H. pylori both bound to phosphatidylethanolamine and lysophosphatidylethanolamine. Adhesion of H. mustelae to intact eukaryotic cells correlated with the amount of phosphatidylethanolamine. Binding of helicobacters was greater to lipids derived from ferret antrum compared with colon (P < 0.05). Biochemical analysis suggested that heterogeneity in fatty acid composition of phosphatidylethanolamine could influence the degree of Helicobacter binding. CONCLUSIONS: Adhesion of Helicobacter strains correlates with the quantity of phosphatidylethanolamine present in the epithelial cell and with the differences in the fatty acid profile of the lipid.

Salmonella typhimurium gyrA mutations associated with fluoroquinolone resistance.

Spontaneous quinolone-resistant mutants obtained from Salmonella typhimurium Su694 were screened for mutations by direct DNA sequencing of an amplified PCR gyrA fragment. Substitutions Ser-83-->Phe (Ser83Phe), Ser83Tyr, Asp87Tyr, and Asp87Asn and double mutation Ala67Pro-Gly81Ser, which resulted in decreased sensitivities to ciprofloxacin, enoxacin, pefloxacin, norfloxacin, ofloxacin, and nalidixic acid, were found. The levels of resistance to quinolones for each mutant were determined.

Cytolocation of prosome antigens on intermediate filament subnetworks of cytokeratin, vimentin and desmin type.

Analysis by double-label indirect immunofluorescence of PtK1 and HeLa cells had previously demonstrated that prosome* antigens form networks that superimpose on those of the intermediate filaments of the cytokeratin type. We show here that in PtK1 cells various prosomal antigens also reside to a variable extent on intermediate filaments subnetworks of the vimentin type. In proliferating human fibroblasts the prosome and vimentin networks were found to coincide, while in proliferating myoblasts of the C2.7 mouse myogenic cell line the prosomal antigens seem to superimpose on the intermediate filaments of the desmin type. Thus, the prosomes, which are RNP particles of variable composition and subcomplexes of untranslated mRNP, and carry a multicatalytic proteinase activity, seem to co-localize with the specific kind of cytoplasmic intermediate filament in relation to the cell type. These results, which generalize the previous data, are discussed in view of possible role(s) for prosomes in mRNA metabolism and/or intermediate filaments remodelling.

Universal method for the facile production of glycolipid/lipid matrices for the affinity purification of binding ligands.

Glycolipid recognition is a common motif in cellular physiology and bacterial pathogenesis. Such protein/lipid interactions are most conveniently demonstrated by the thin-layer chromatogram overlay. We have designed a simple affinity matrix for the purification of such glycolipid (or lipid) binding ligands based on the same principle, i.e., glycolipid (or lipid) adsorbed onto silica. The versatility of the procedure is demonstrated by the purification of several anti-glycolipid antibodies and anti-phosphatidyl ethanolamine (anti-PE) and the affinity purification of the Escherichia coli-derived verotoxin which binds to globotriaosyl ceramide.

Therapeutics used to alleviate peptic ulcers inhibit H. pylori receptor binding in vitro.

Treatment with bismuth-containing remedies has been long associated with the alleviation of minor gastric ailments. Bismuth salts have a potent antimicrobial activity, and are part of the current standard regime used to treat Helicobacter pylori infection. H. pylori is considered to be the major etiological factor in the development of peptic ulcer disease. Earlier efficacious treatments for peptic ulcer included the oral administration of Tween detergents. We have found that these agents have an inhibitory effect on H. pylori adhesion to the lipid species phosphatidylethanolamine (PE) and gangliotetraosylceramide (Gg4) shown previously to be receptors for H. pylori binding in vitro. H. pylori binding to PE and Gg4 was inhibited after a thirty minute preincubation with different bismuth compounds: bismuth subsalicylate > bismuth subgallate > bismuth carbonate > colloidal bismuth subcitrate > tripotassium dicitrato bismuthate. No inhibitory effect on H. pylori binding was observed when bismuth salts were added directly into the binding assay. No changes in bacterial morphology and motility were observed after the thirty minute incubation. Pretreatment with Tween detergents also inhibited H. pylori receptor binding by up to 80% at concentrations as low as 0.0001%. These results suggest that inhibition of H. pylori/host cell adhesion might play a role in efficacious treatment for this infection.

Silencer and enhancer elements located at the 3'-side of the chicken and duck alpha-globin-encoding gene domains.

Enhancer activities have been observed in DNA fragments up to 1.36 kb long located on the 3'-side of the cluster of the three alpha-type globin-encoding genes in duck [Kretsovali et al., C.R. Acad. Sci. Paris 307 (1988) 563-568] and chicken [Knezetic and Felsenfeld, Mol. Cell. Biol. 9 (1989) 893-901]. We report here the identification of a chicken silencer element placed upstream from the three GATA-1 sites which constitute the core enhancer element in both species. This silencer element can autonomously reduce the activity of promoters for thymidine kinase and alpha D globin. Band shifts and DNase I footprinting experiments using nuclear extracts from thermosensitive avian erythroblastosis virus-transformed chicken erythroblasts led to the delineation of three sites for DNA-binding proteins within the silencer element.

Receptor affinity purification of a lipid-binding adhesin from Helicobacter pylori.

Our previous work has shown that Helicobacter pylori specifically recognizes gangliotetraosylceramide, gangliotriaosylceramide, and phosphatidylethanolamine in vitro. This binding specificity is shared by exoenzyme S from Pseudomonas aeruginosa, and monoclonal antibodies against this adhesin prevent the attachment of H. pylori to its lipid receptors. We now report the use of a novel, versatile affinity matrix to purify a 63-kDa exoenzyme S-like adhesin from H. pylori which is responsible for the lipid-binding specificity of this organism.

Helicobacter mustelae and Helicobacter pylori bind to common lipid receptors in vitro.

Helicobacter pylori is a recently recognized human pathogen causing chronic-active gastritis in association with duodenal ulcers and gastric cancer. Helicobacter mustelae is a closely related bacterium with similar biochemical and morphologic characteristics. H. mustelae infection of antral and fundic mucosa in adult ferrets causes chronic gastritis. An essential virulence property of both Helicobacter species is bacterial adhesion to mucosal surfaces. The aim of this study was to determine whether H. mustelae binds to the same lipids shown previously to be receptors for H. pylori adhesion in vitro. By using thin-layer chromatography overlay and a receptor-based enzyme-linked immunosorbent assay, H. mustelae was found to bind the same receptor lipids as H. pylori, namely, phosphatidylethanolamine and gangliotetraosylceramide. In addition, both H. pylori and H. mustelae bound to a deacylplasmalogen phosphatidylethanolamine. In contrast to H. pylori, H. mustelae binding to receptors was unaffected by motility or viability. Murine monoclonal and bovine polyclonal antibodies against exoenzyme S, and exoenzyme S itself (from Pseudomonas aeruginosa), inhibited binding of H. mustelae to phosphatidylethanolamine and gangliotetraosylceramide. These findings show that H. mustelae binds in vitro to the same lipid receptors as H. pylori and suggest that the adhesion of H. mustelae to such species is mediated by preformed, surface-exposed adhesins which include an exoenzyme S-like protein.

Comparison of Helicobacter pylori and attaching-effacing Escherichia coli adhesion to eukaryotic cells.

Adhesion of Helicobacter pylori was reported previously to be morphologically identical to "attaching and effacing" Escherichia coli. Therefore, the aim of the present study was to define the adhesion phenotype of H. pylori LC-11 to HEp-2, KATO-III, HEL, and CHO tissue culture cells. By using both staining of F-actin with fluorescein-labeled phalloidin and ultrastructural analysis, diffuse bacterial adhesion to discrete microvillus-denuded regions of the plasma membrane was observed in each of the infected cell lines. However, strain LC-11 did not induce formation of F-actin adhesion pedestals on the eukaryotic cells. H. pylori was negative by colony blot hybridization with an E. coli attaching and effacing gene probe. Elevations in inositol triphosphates followed infection of HEp-2 cells with H. pylori (405% of control values +/- 147%; P < 0.05). To correlate the observed histopathology with expression of the H. pylori phosphatidylethanolamine receptor, a thin-layer chromatography overlay-binding assay was used to identify receptors in each of the cell lines. H. pylori adhered to eukaryotic cells regardless of the presence (HEp-2, KATO-III, and CHO cells) or absence (HEL cells) of the lipid receptor as detected under the assay conditions. However, in comparison to cell lines that possess the phosphatidylethanolamine receptor, HEL cells demonstrated less quantitative H. pylori binding. These findings suggest that mechanisms distinct from E. coli enteropathogens underlie the adhesion of H. pylori to mucosal surfaces. In addition to the phosphatidylethanolamine H. pylori receptor, another host factor(s) likely mediates the attachment of H. pylori to human eukaryotic cells.

Patterns of cytodistribution of prosomal antigens on the vimentin and cytokeratin networks of monkey kidney cells.

Prosomes were found as mRNA-associated ribonucleoprotein particles (RNP) and cofactors of untranslated (ribosome-) free mRNP. Previous data have shown the presence of prosomal networks in the cytoplasm of PtK1 and HeLa cells and their superposition onto the intermediate filaments (IF) of cytokeratin type but little if any of vimentin type. Here it is shown that in LLC-MK2 cells various prosomal antigens are present on both, vimentin and cytokeratin networks, individual prosomal antigens superposing to variable degrees onto the IF subnetworks. Some prosomal antigens in variable relative concentrations were also observed in the nuclei of these cells. We suggest the existence of prosomal subnetworks specific for each prosomal antigen superposing to a variable extent onto the IF of both types.