Search PubMed⌕ Search

Biomedical subjects

S P Borriello

Publications and source records attributed to S P Borriello.

At least 19 recordsLinked to original sources
PubMed2004

Colonic IgA producing cells and macrophages are reduced in recurrent and non-recurrent Clostridium difficile associated diarrhoea.

BACKGROUND: In Clostridium difficile associated diarrhoea (CDAD), histological changes in the colonic mucosa range from minimal inflammation to pseudomembranous colitis (PMC). The disease also recurs in a considerable proportion of patients. AIM: To investigate mucosal immune system cells in colonic biopsies of patients with CDAD. METHODS: Colonic biopsies were obtained from 12 control patients with diarrhoea, six patients with CDAD and minimal inflammation, and 10 patients with CDAD with pseudomembranous colitis (samples obtained from areas with and without inflammatory exudate). Immunohistochemical studies were performed using antibodies to T cells (CD3), macrophages (CD68), B/plasma cells (CD79alpha), and to IgA, IgM, and IgG. Labelled cells in lamina propria were quantified. RESULTS: In contrast to T cells, there were significant reductions in B/plasma cell and macrophage counts in all biopsies from patients with CDAD compared with controls (p<0.001). Studies using anti-immunoglobulin antibodies showed significant reductions in IgA producing cells in CDAD biopsies (p<0.05), with the greatest reduction in samples from patients with PMC. In contrast, there was a significant increase (p<0.05) in IgG producing cells in CDAD biopsies. Only patients with PMC relapsed. In these patients, B/plasma cell and IgA producing cell counts (in biopsies with and without inflammatory exudates) were significantly lower (p<0.01) in mucosal samples from those who subsequently relapsed (five) than those who did not. CONCLUSIONS: A selective reduction in mucosal IgA producing cells and macrophages is associated with colonic disease in C difficile infected patients. Severe reduction in colonic IgA producing cells may predispose to recurrence of CDAD.

Adolescent↗
PubMed2003

Evaluation of Helicobacter species in inflammatory bowel disease.

BACKGROUND: Bacteria have been implicated in the pathogenesis of inflammatory bowel disease. Helicobacter species have been shown to cause colitis in animal models and have been identified in human diarrhoeal illness and Crohn's disease. AIM: To determine whether Helicobacter species are present in human inflammatory bowel disease tissue. METHODS: Thirty patients undergoing colonoscopy for clinical reasons were studied. Nine had Crohn's disease, 11 had ulcerative colitis and 10 had histologically normal colons. Tissue was snap-frozen at -70 degrees C. DNA was extracted and examined by five different polymerase chain reaction (PCR) assays that were either genus or species specific for Helicobacter. RESULTS: Analyses of colonic biopsies by two Helicobacter genus-specific PCR assays, two H. pylori-specific assays and a PCR assay designed to amplify fragments of 'H. heilmannii'-like organisms demonstrated that product was not generated by any test. Internal control PCR demonstrated that PCR results for the five assays were not negative due to the presence of residual substances inhibitory to PCR. CONCLUSIONS: Helicobacter species were not identified in this study, using multiple PCRs to eliminate the problems of non-specific cross-reaction. This suggests that Helicobacter species do not play a role in the pathogenesis of inflammatory bowel disease.

Adult↗
PubMed2003

Safety of probiotics that contain lactobacilli or bifidobacteria.

Lactobacilli and bifidobacteria are extremely rare causes of infection in humans, as are probiotics based on these organisms. This lack of pathogenicity extends across all age groups and to immunocompromised individuals. Strains used for new probiotics should be chosen from the commensal flora of humans and should not carry intrinsic resistance to antibiotics that would prevent treatment of a rare probiotic infection. Vigilance regarding the detection of possible rare cases of infection due to probiotics should be maintained, and isolates should be sent to reference centers for molecular characterization and confirmation.

Bifidobacteriales Infections↗
PubMed2000

Neisseria meningitidis expressing transferrin binding proteins of Actinobacillus pleuropneumoniae can utilize porcine transferrin for growth.

Homologous recombination was used to generate a number of mutants of serogroup B Neisseria meningitidis B16B6 with the following characteristics: (i) an inability to bind human or porcine transferrin because of loss of both transferrin binding proteins (Tbp) A and B [strain B16B6(Str(r))/tbpA(-)B(-)] and (ii) an ability to bind porcine transferrin but not human transferrin [strain B16B6(Str(r))/tbpA(ap)B(ap)] due to replacement of the meningococcal Tbp with the Tbp of Actinobacillus pleuropneumoniae. During construction of the B16B6(Str(r))/tbpA(ap)B(ap) strain, transformants expressing only TbpA or TbpB of A. pleuropneumoniae were isolated [strains B16B6(Str(r))/tbpA(ap)B(-) and B16B6(Str(r))/tbpA(-)B(ap)]. Expression of the A. pleuropneumoniae Tbp in N. meningitidis B16B6 was iron regulated and expressed under the control of the meningococcal promoter. The relative abilities of the meningococcal transformants to bind porcine transferrin were in the order B16B6(Str(r))/tbpA(ap)B(ap) > B16B6(Str(r))/tbpA(ap)B(-) > B16B6(Str(r))/tbpA(-)B(ap). Of these transformants, only B16B6(Str(r))/tbpA(ap)B(ap) could grow in the presence of porcine transferrin as the sole iron source, achieving a growth rate similar to that of the B16B6 parent strain in the presence of human transferrin.

Actinobacillus pleuropneumoniae↗
PubMed1999

Identification and characterization of TspA, a major CD4(+) T-cell- and B-cell-stimulating Neisseria-specific antigen.

In search for novel T-cell immunogens involved in protection against invasive meningococcal disease, we screened fractionated proteins of Neisseria meningitidis (strain SD, B:15:P1.16) by using peripheral blood mononuclear cells (PBMCs) and specific T-cell lines obtained from normal individuals and patients convalescing from N. meningitidis infection. Proteins of iron-depleted meningococci produced higher PBMC proliferation indices than proteins of iron-replete organisms, indicating that iron-regulated proteins are T-cell immunogens. Insoluble proteins of the iron-depleted cells, which produced better T-cell stimulation than soluble ones, were fractionated by using sodium dodecyl sulfate-polyacrylamide gels and recovered as five fractions (F1 to F5) corresponding to decreasing molecular weight ranges. The proteins were purified (by elution and precipitation) or electroblotted onto nitrocellulose membranes (dissolved and precipitated) before use in further T-cell proliferation assays. One of the fractions (F1), containing high-molecular-mass proteins (>130 kDa), consistently showed the strongest T-cell proliferation responses in all of the T-cell lines examined. F1 proteins were subdivided into four smaller fractions (F1A to F1D) which were reexamined in T-cell proliferation assays, and F1C induced the strongest responses in patients' T-cell lines. Rabbit polyclonal antibodies to F1C components were used to screen a genomic expression library of N. meningitidis. Two major clones (C1 and C24) of recombinant meningococcal DNA were identified and fully sequenced. Sequence analysis showed that C24 (1,874 bp) consisted of a single open reading frame (ORF), which was included in clone C1 (2, 778 bp). The strong CD4(+) T-cell-stimulating effect of the polypeptide product of this ORF (named TspA) was confirmed, using a patient T-cell line. Immunogenicity for B cells was confirmed by showing that convalescent patients' serum antibodies recognized TspA on Western blots. Additional genetic sequence downstream of C24 was obtained from the meningococcal genomic sequence database (Sanger Centre), enabling the whole gene of 2,761 bp to be reconstructed. The DNA and deduced amino acid sequence data for tspA failed to show significant homology to any known gene, except for a corresponding (uncharacterized) gene in Neisseria gonorrhoeae genome sequences, suggesting that tspA is unique to the genus Neisseria. The DNA and deduced amino acid sequence of the second ORF of clone C1 showed significant homology to gloA, encoding glyoxalase I enzyme, of Salmonella typhimurium and Escherichia coli. Thus, we have identified a novel neisserial protein (TspA) which proved to be a strong CD4(+) T-cell- and B-cell-stimulating immunogen with potential as a possible vaccine candidate.

Animals↗
PubMed1998

A novel flow cytometric assay for quantitating adherence of Helicobacter pylori to gastric epithelial cells.

Adherence may be an important virulence factor for Helicobacter pylori. Current methods available for quantitation of adherence are time consuming and liable to observer error. A new direct technique for fluorescent labelling of bacteria has been developed to quantitate adherence of H. pylori to epithelial cells by fluorescence activated cell sorting (FACS). Type strains of H. pylori, H. mustelae, H. cinaedi and H. fennelliae were grown microaerobically in broth culture for 24 h and fluorescently labelled by incubation with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) at 37 degrees C. After washing to remove excess CFDA-SE, bacteria were co-incubated (ratio 10:1) with gastric epithelial cells at 37 degrees C for up to 24 h. After washing to remove non-adherent bacteria, epithelial cells were detached with EDTA (2 mM) and fixed with formaldehyde for flow cytometry. Adherence was quantitated both in terms of the proportion of cells with adherent H. pylori and as the mean number of adherent bacteria per cell. All H. pylori strains adhered to gastric-type epithelial cells. The proportion of cells with bound bacteria varied from 40-99% and the number of bacteria per cell from 1-50, both of which correlated with microscopy (r = 0.6, and r = 0.8 respectively, n = 35). Time course studies demonstrated saturation of binding by H. pylori within 90 min. For H. mustelae, H. cinaedi and H. fennelliae the proportion of cells with bound bacteria varied from 5-15% and the mean number of bacteria per cell was < 4. Binding of H. pylori to epithelial cells could be partly blocked by pre-incubation with polyclonal anti-sera or using oligosaccharides against potential binding epitopes of gastric mucus. Fluorescent labelling of H. pylori with CFDA-SE in combination with flow cytometry provides a quick, specific, and sensitive method to quantitate in vitro the adherence of H. pylori.

Antibodies, Bacterial↗
PubMed1998

Multicentre evaluation of a commercial test for the rapid diagnosis of Clostridium difficile-mediated antibiotic-associated diarrhoea.

An immunoassay for the detection of Clostridium difficile toxin A in stool samples (Clearview C. DIFF A; Unipath, UK) was evaluated against the cell cytotoxicity assay using 407 stool samples from patients suspected to have, or considered at risk of, antibiotic-associated diarrhoea. Of the samples tested, 98 were positive and 280 were negative by both tests (sensitivity 83.1%, specificity 96.9%). Following resolution of the 29 discrepant results, the sensitivity and specificity of the immunoassay were 91% and 98%, respectively, and the sensitivity for the cell cytotoxicity assay was calculated as 91.5%, with a specificity of 99%. The Clearview C. DIFF A test proved to be a rapid simple assay for the detection of Clostridium difficile toxin A in stool samples. The test was equally suited to single or batch testing, required minimal sample handling, and provided results within 30 min of applying the sample to the test unit.

Animals↗
PubMed1998

Pathogenesis of Clostridium difficile infection.

Clostridium difficile produces two major toxins referred to as toxins A and B. These are thought to be primarily responsible for the virulence of the bacterium and the major contributors to the pathogenesis of antibiotic-associated gastrointestinal disease. The molecular organization and control of expression of toxins A and B is now starting to be understood, and the cellular mechanism of action of both toxins, glucosylation of Rho family proteins, has been discovered. Other factors, such as production of proteolytic and hydrolytic enzymes, expression of fimbriae and flagella, chemotaxis and adhesion to gut receptors, and production of capsule, may all play a part in pathogenesis by facilitating colonization or by directly contributing to tissue damage, or both. Differential expression between strains of various combinations of these colonization and virulence factors may explain the apparent variability in virulence of C. difficile strains.

Animals↗
PubMed1998

Differential binding of apo and holo human transferrin to meningococci and co-localisation of the transferrin-binding proteins (TbpA and TbpB).

Apo-transferrin (apo-hTf) and holo-transferrin (holo-hTf) were separately conjugated to 15-nm colloidal gold. Iron-restricted Neisseria meningitidis strain SD (B:15:P1.16) bound up to three-fold more holo-hTf than apo-hTf (p <0.001). The ability of meningococcal mutants lacking either transferrin-binding protein A (TbpA) or TbpB to discriminate between apo-hTf and holo-hTf was also investigated. There was no significant difference between the amount of gold-labelled apo-transferrin bound by the isogenic TbpA mutant (expressing TbpB) and the parent strain, whereas an isogenic TbpB mutant (expressing TbpA) bound significantly less gold-labelled apo-hTf. The isogenic TbpA and TbpB mutants and the parent strain all bound significantly more holo-hTf than apo-hTf, whereas the double 'knock-out' mutant failed to bind hTf irrespective of the iron-loading. In the isogenic mutants, TbpB was more effective in binding either apo- or holo-hTf than TbpA. Monoclonal antibodies against TbpA and TbpB were used to co-localise the transferrin-binding proteins on strain SD. The ratio of TbpA:TbpB was approximately 1:1. TbpA and TbpB were occasionally observed in close proximity to each other, but the two proteins were generally quite separate, which may indicate that they do not usually form a complex to act as a transferrin receptor.

Antibodies, Monoclonal↗
PubMed1998

Effect of Clostridium difficile toxin A on human colonic lamina propria cells: early loss of macrophages followed by T-cell apoptosis.

We have previously shown that Clostridium difficile toxin A induces detachment of human colonic epithelial cells from the basement membrane and subsequent cell death by apoptosis. Because these cells require adhesion-dependent signalling from the extracellular matrix for survival, their detachment from the basement membrane by other means also induces apoptosis. The role of toxin A in the induction of apoptosis therefore remains to be determined. In addition, sensitivities to C. difficile toxin A of lamina propria lymphocytes, macrophages, and eosinophils, which lie below the surface epithelium, are not known. In contrast to epithelial cells, these lamina propria cells do not require adhesion-dependent signalling from the extracellular matrix for survival, and this may allow the mechanisms of toxin A-induced cell death to be further investigated. The aim of this study was to investigate the effect of purified C. difficile toxin A on human colonic lamina propria T cells, macrophages, and eosinophils. We show that C. difficile toxin A induces loss of viability in isolated colonic lamina propria cell preparations containing the three different cell types in a dose- and time-dependent fashion. Exposure to high concentrations of the toxin led to loss of macrophages within 72 h. T-lymphocyte and eosinophil cell death was prominent at later time points and occurred by apoptosis. Exposure to toxin A also induced the production of tumor necrosis factor alpha by the isolated colonic lamina propria cells. However, the presence of neutralizing antibodies to this cytokine did not influence C. difficile toxin A-induced T-cell apoptosis. Moreover, purified T cells also underwent apoptosis following exposure to toxin A, implying that apoptosis occurred as a consequence of a direct interaction between T cells and the toxin. Our studies suggest that C. difficile toxin A is capable of suppressing human colonic mucosal immune responses by inducing early loss of macrophages followed by T-cell apoptosis.

Apoptosis↗
PubMed1998

Nonspecific binding of Clostridium difficile toxin A to murine immunoglobulins occurs via the fab component.

Clostridium difficile toxin A binds nonspecifically to a mouse monoclonal antibody (MAb) immunoglobulin G3 lambda chain [IgG3(lambda)], through the Fab component. This binding, which is retained even after boiling the MAb, is temperature dependent, with more toxin bound at 4 than 37 degrees C (P = 0.0024). The nonspecific binding was decreased by incubation of the IgG3 lambda MAb with alpha- or beta-galactosidase (P = 0.0001 and 0.029, respectively), indicating that toxin A binds to a carbohydrate moiety on the Fab. However, binding was not blocked by the Bandeiraea simplicifolia lectin BS-1, indicating that a terminal alpha-galactose may not be involved. Binding was also not affected by competitive assays with Lewis X antigen. The dependence on carbohydrate moieties in nonspecific binding was also shown for two other MAbs, IgA(kappa) and IgM(lambda), with demonstration of a significant reduction in binding with alpha-galactosidase (P = 0.0001 and 0.0002, respectively) but not beta-galactosidase (P = 0.27 and 0.25, respectively).

Animals↗
PubMed1998

Heterogeneity in responses by primary adult human colonic epithelial cells to purified enterotoxin of Bacteroides fragilis.

BACKGROUND: Enterotoxigenic strains of Bacteroides fragilis (ETBF) have been implicated in diarrhoeal illness in livestock and children, but their role in adult human colonic disease is unknown. AIMS: To investigate responses by primary adult human colonic epithelial cells to purified B fragilis toxin (BFT). METHODS: BFT was purified from culture supernatant of a highly toxigenic strain of ETBF. Morphological changes to primary colonic epithelial cells, in response to purified BFT, were studied in organ culture of colonic biopsy specimens from 15 adults. RESULTS: BFT induced epithelial cell cytotoxicity in colonic biopsy specimens from 12/15 subjects. The BFT induced morphological changes were characterised by epithelial cell rounding, separation from adjacent cells, and detachment from the basement membrane. In severely affected specimens, almost all the epithelial cells were affected. There was heterogeneity between subjects in the rate at which BFT induced epithelial cell cytotoxicity occurred. Furthermore, in colonic biopsy specimens from three subjects, exposure to BFT did not induce any significant morphological changes to epithelial cells. CONCLUSION: BFT is capable of inducing cytotoxicity in primary adult human colonic epithelial cells. Such an effect of ETBF derived BFT on epithelial cells in the colon in vivo would be expected to lead to mucosal inflammation and diarrhoea. Heterogeneity in responses by primary colonocytes probably reflects the outcome of host-BFT interactions. Such interactions in vivo could determine the occurrence of colonic disease in some individuals but not others.

Adult↗
PubMed1998

Clostridium difficile-associated diarrhoea.

At our hospital, the number of cases of Clostridium difficile-associated diarrhoea increased from 29 in 1993 to 210 in 1995. The case notes of 110 patients with C difficile-associated diarrhoea during the first 6 months of 1995 were analysed retrospectively. The majority of the patients (106) had received antibiotics before the onset of diarrhoea; 46 had received three or more different antibiotics and 28 had received metronidazole. In 19 patients, the first stool sample after the onset of diarrhoea was negative for C difficile cytotoxin, with a mean delay of 8.2 days before a positive stool sample. We conclude that C difficile-associated diarrhoea was associated with the usage of multiple antibiotics, and that metronidazole did not protect against colonisation by C difficile. We also recommend that more than one stool sample should be tested for the C difficile cytotoxin.

Adult↗
PubMed1997

Clostridium difficile toxin A binding to human intestinal epithelial cells.

Clostridium difficile radiolabelled toxin A ([3H]-toxin A) bound to human duodenal and colonic epithelial cells isolated from endoscopic biopsies. Binding was greater at 4 degrees C than 37 degrees C, consistent with the thermal binding characteristic of toxin A to a carbohydrate moiety. At 37 degrees C colonic cells bound significantly more [3H]-toxin A than duodenal cells. The amount of [3H]-toxin A binding varied considerably between individuals. [3H]-toxin A was displaced by unlabelled toxin A by 50% for duodenal cells and 70% for colonic cells with 94.3 nM unlabelled toxin A. Low non-displacable binding was observed in some samples at 4 degrees C and 37 degrees C, suggesting that these cells came from individuals incapable of specifically binding toxin. Pre-treating cells with alpha- or beta-galactosidases to cleave terminal alpha- and beta-galactose residues reduced [3H]-toxin A binding. There was also a reduction in [3H]-toxin A binding after heat treating cells, which is suggestive of protein binding. The reduction in binding varied between individuals. The reduction of [3H]-toxin A binding, after the removal of beta-linked galactose units, implicates these as components of the receptor and adds credence to the idea that the Lewis X, Y and I antigens may be involved in toxin A binding to human intestinal epithelial cells. However, because the Lewis antigens do not possess terminal alpha-galactose units, the reduction in binding after alpha-galactosidase treatment suggests that other receptors may be involved in toxin A binding to some human intestinal cells. These data are the first demonstration of direct toxin A binding to human intestinal epithelial cells.

Bacterial Toxins↗
PubMed1996

Blocking of iron uptake from transferrin by antibodies against the transferrin binding proteins in Neisseria meningitidis.

Neisseria meningitidis, when cultured in iron-restricted environments, synthesises new outer-membrane proteins, many of which are necessary for their survival and growth. Some of these proteins e.g. transferrin-binding proteins 1 and 2 (Tbp1 and Tbp2), are required for the acquisition of iron from transferrin and are examples of important iron-regulated meningococcal surface antigens which are not expressed after growth in common laboratory media. The antigenicity and antigenic heterogeneity of these proteins have been extensively studied, and the bactericidal activity of antibodies directed to them have been studied. In this work we analysed the ability of such antibodies to inhibit transferrin binding and to block iron uptake from human transferrin. Antisera from mice immunized with either meningococcal outer membrane vesicles, purified Tbp1/2 complexes, or purified Tbp2, were incorporated in radiolabeled-iron uptake assays. Uptake was blocked by more than 80% in the homologous strains, but blocked much less efficiently in some heterologous strains, correlating well with inhibition of transferrin binding and with an inhibitory effect on bacterial growth. Inhibition of iron uptake from citrate was unaffected which suggests that this effect is due to antibodies against the components of the transferrin binding system, specially Tbp2. Our results support the importance of these proteins and their suitability to be considered in the development of effective vaccines against serogroup B meningococci.

Antibodies, Bacterial↗