Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 88
Filter
1.
Br Med Bull ; 131(1): 109-118, 2019 09 19.
Article in English | MEDLINE | ID: mdl-31583398

ABSTRACT

BACKGROUND: Clostridium difficile infection is transmitted via spores, and the disease is mediated via secreted toxins. It represents a significant healthcare problem, and clinical presentation can range from asymptomatic carriage to life-threatening pseudomembranous colitis. SOURCES OF DATA: publications in the field, with a focus on recent developments and concepts. AREAS OF AGREEMENT: infection control measures, antibiotic stewardship and current management of the initial episode of C. difficile infection. AREAS OF CONTROVERSY: selection and sequence of interventions for the management of recurrent C. difficile infection; management of persistent carriers of toxigenic C. difficile in patients at high risk of subsequent C. difficile infection. GROWING POINTS: use of faecal microbiota transplantation for recurrent C. difficile infection. AREAS TIMELY FOR DEVELOPING RESEARCH: role of specific microbiota-mediated interventions and vaccination in the treatment and prevention of C. difficile infection.


Subject(s)
Clostridioides difficile , Clostridium Infections/prevention & control , Anti-Bacterial Agents/therapeutic use , Bacterial Vaccines , Clostridium Infections/transmission , Drug Therapy, Combination , Feces/microbiology , Humans , Infection Control/methods , Inflammatory Bowel Diseases/microbiology , Recurrence , Risk Factors
2.
Clin Exp Immunol ; 178(1): 28-39, 2014 Oct.
Article in English | MEDLINE | ID: mdl-24828022

ABSTRACT

The aim of our studies was to investigate the expression of Toll-like receptor (TLR)-2 and TLR-4 (and in some studies TLR-5) in myofibroblasts and small and large intestinal crypt epithelial cells from control patients and those affected by Crohn's disease and ulcerative colitis. Isolated and disaggregated crypt epithelial cells and monolayers of myofibroblasts were used for studies by reverse transcription-polymerase chain reaction (RT-PCR), real-time RT-PCR, flow cytometry, immunocytochemistry and Western blot analysis. Compared to control cells, crypt epithelial cells isolated from active ulcerative colitis and Crohn's disease colonic mucosal samples showed significantly higher expression of TLR-2 and TLR-4 transcripts and protein (on the cell surface). There was also enhanced expression of TLR-4 in crypt cells from ileal Crohn's disease. Expression of TLR-2 and TLR-4 transcripts in crypt epithelial cells isolated from inflamed mucosa of distal ulcerative colitis did not differ significantly from such cells obtained from the normal proximal colon. Crypt epithelial cells with side population characteristics (putative stem cells) also expressed transcripts and protein for TLR-2, TLR-4 and TLR-5. Colonic myofibroblast expression of these TLRs was much weaker than in crypt epithelial cells. In conclusion, enhanced TLR-2 and TLR-4 expression by crypt epithelial cells in active inflammatory bowel disease likely reflects greater ability to respond to microbial products. Results from our studies using mucosal samples from patients with distal ulcerative colitis suggest that the enhanced expression of these TLRs could be constitutive. TLR-2, TLR-4 and TLR-5 expression by stem cells imply ability to respond to distinct bacterial products.


Subject(s)
Colon/metabolism , Ileum/metabolism , Inflammatory Bowel Diseases/metabolism , Intestinal Mucosa/metabolism , Myofibroblasts/metabolism , Stem Cells/metabolism , Toll-Like Receptors/biosynthesis , Adult , Aged , Case-Control Studies , Colitis, Ulcerative/metabolism , Colitis, Ulcerative/pathology , Colon/cytology , Crohn Disease/metabolism , Epithelial Cells/metabolism , Epithelial Cells/pathology , Humans , Ileum/cytology , Inflammatory Bowel Diseases/pathology , Intestinal Mucosa/pathology , Middle Aged , Stem Cells/pathology , Toll-Like Receptors/metabolism
3.
Scand J Immunol ; 74(3): 264-271, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21595735

ABSTRACT

Colitis due to Clostridium difficile infection is mediated by secreted toxins A and B and is characterized by infiltration by cells from the systemic circulation. The aim of our study was to investigate interactions between fluorescently labelled toxin A and peripheral blood monocytes, neutrophils and lymphocytes. Purified toxin A was labelled with Alexa Fluor® 488 (toxin A(488)) and incubated with isolated human peripheral blood mononuclear cells or washed whole blood cells for varying time intervals at either 37 or 4 °C/ice. The ability of trypan blue to quench cell surface-associated (but not cytoplasmic) fluorescence was also investigated. At 37 °C, toxin A(488) -associated fluorescence in monocytes peaked at 1 h (majority internalized), with subsequent loss associated with cell death. In contrast to monocytes, binding of toxin A(488) in neutrophils was greater on ice than at 37 °C. Studies using trypan blue suggested that over 3 h at 37 °C, most of the toxin A(488)-associated fluorescence in neutrophils remained at the cell surface. Over 48 h (37 °C and ice/4 °C), there was minimal toxin A(488)-associated fluorescence in lymphocytes. These studies suggest major differences in interactions between toxin A and circulating cells that infiltrate the mucosa during colonic inflammation in C. difficile infection.


Subject(s)
Bacterial Toxins/metabolism , Clostridioides difficile , Enterotoxins/metabolism , Gastric Mucosa/immunology , Leukocytes, Mononuclear/metabolism , Lymphocytes/metabolism , Neutrophils/metabolism , Clostridioides difficile/pathogenicity , Flow Cytometry , Fluorescent Dyes , Gastric Mucosa/microbiology , Humans , Lymphocyte Count , Staining and Labeling
4.
Infect Immun ; 79(4): 1623-30, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21245273

ABSTRACT

Colonic inflammation in Clostridium difficile infection is mediated by released toxins A and B. We investigated responses to C. difficile toxins A and B by isolated primary human colonic myofibroblasts, which represent a distinct subpopulation of mucosal cells that are normally located below the intestinal epithelium. Following incubation with either purified toxin A or B, there was a change in myofibroblast morphology to stellate cells with processes that were immunoreactive for alpha-smooth muscle actin. Most of the myofibroblasts remained viable, with persistence of stellate morphology, despite exposure to high concentrations (up to 10 µg/ml) of toxin A for 72 h. In contrast, a majority of the toxin B-exposed myofibroblasts lost their processes prior to cell death over 24 to 72 h. At low concentrations, toxin A provided protection against toxin B-induced cell death. Within 4 h, myofibroblasts exposed to either toxin A or toxin B lost expression of the nonglucosylated form of Rac1, and there was also a loss of the active form of RhoA. Despite preexposure to high concentrations of toxin A for 3 h, colonic myofibroblasts were able to recover their morphology and proliferative capacity during prolonged culture in medium. However, toxin B-preexposed myofibroblasts were not able to recover. In conclusion, primary human colonic mucosal myofibroblasts are resistant to toxin A (but not toxin B)-induced cell death. Responses by colonic myofibroblasts may play an important role in mucosal protection, repair, and regeneration in colitis due to C. difficile infection.


Subject(s)
Bacterial Proteins/toxicity , Bacterial Toxins/toxicity , Clostridioides difficile/chemistry , Enterotoxins/toxicity , Myofibroblasts/pathology , Aged , Aged, 80 and over , Blotting, Western , Cell Death , Cell Line , Colon/microbiology , Colon/pathology , Enzyme-Linked Immunosorbent Assay , Female , Flow Cytometry , Humans , Immunohistochemistry , Male , Microscopy, Electron, Scanning , Microscopy, Phase-Contrast , Middle Aged
5.
Inflamm Bowel Dis ; 17(1): 213-20, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20848536

ABSTRACT

BACKGROUND: Wnt signaling regulates intestinal epithelial stem cell function. Wnt ligands bind Frizzled (Fz) receptors and low-density lipoprotein-receptor-related protein (LRP) 5 and 6. Secreted Frizzled-related protein (SFRP) and Dickkopf families inhibit Wnt signaling. Our aim was to study expression of Wnt family of genes in isolated intestinal myofibroblasts and crypt epithelial cells. METHODS: Myofibroblasts were isolated from normal colonic and small intestinal mucosal samples and those affected by ulcerative colitis (UC) and Crohn's disease. Expression of the Wnt family of genes was studied by polymerase chain reaction (PCR) array. Epithelial proliferation was studied using IEC-6 cells. RESULTS: Most of the myofibroblast isolates expressed Wnt2, Wnt5A, Wnt5B, Fzd1, Fzd2, Fzd4, Fzd6, Fzd7, Fzd8, LRP6, Dickkopf1, and SFRP1. Compared to myofibroblasts isolated from normal colonic mucosal samples, real-time reverse transcription-PCR studies (using additional isolates) showed significantly reduced expression of SFRP1 in UC myofibroblasts (3.34-fold reduction, P < 0.01). Recombinant SFRP1 inhibited proliferation of IEC-6 epithelial cells. In colonic crypt epithelial cells, expression of Wnt ligands and their inhibitors was generally either absent or very weak. By contrast, all the crypt epithelial preparations expressed Fzd1, Fzd5, Fzd7, Fzd8, and LRP6. CONCLUSIONS: Human intestinal myofibroblasts expressed a number of Wnt ligands, their receptors, and inhibitors. In contrast, colonic crypt epithelial cells predominantly expressed Wnt receptors. Compared to myofibroblasts isolated from normal colonic mucosa, those affected by UC showed significantly reduced expression of SFRP1. Since reduced SFRP1 expression has been associated with malignancy, low myofibroblast expression of this Wnt inhibitor may be implicated in increased risk of cancer in UC.


Subject(s)
Colon/metabolism , Epithelial Cells/metabolism , Gene Expression Profiling , Inflammatory Bowel Diseases/genetics , Intercellular Signaling Peptides and Proteins/genetics , Intestinal Mucosa/metabolism , Membrane Proteins/genetics , Myofibroblasts/metabolism , Wnt Proteins/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Blotting, Western , Cell Proliferation , Cells, Cultured , Colon/pathology , Epithelial Cells/pathology , Female , Humans , Inflammatory Bowel Diseases/metabolism , Inflammatory Bowel Diseases/pathology , Intercellular Signaling Peptides and Proteins/metabolism , Intestines/pathology , Male , Membrane Proteins/metabolism , Middle Aged , Myofibroblasts/pathology , Prognosis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Wnt Proteins/metabolism , Young Adult
6.
Cell Prolif ; 42(6): 731-50, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19788585

ABSTRACT

Recently, there has been resurgence of interest in the question of small intestinal stem cells, their precise location and numbers in the crypts. In this article, we attempt to re-assess the data, including historical information often omitted in recent studies on the subject. The conclusion we draw is that the evidence supports the concept that active murine small intestinal stem cells in steady state are few in number and are proliferative. There are two evolving, but divergent views on their location (which may be more related to scope of capability and reversibility than to location) several lineage labelling and stem cell self-renewing studies (based on Lgr5 expression) suggest a location intercalated between the Paneth cells (crypt base columnar cells (CBCCs)), or classical cell kinetic, label-retention and radiobiological evidence plus other recent studies, pointing to a location four cell positions luminally from the base of the crypt The latter is supported by recent lineage labelling of Bmi-1-expressing cells and by studies on expression of Wip-1 phosphatase. The situation in the human small intestine remains unclear, but recent mtDNA mutation studies suggest that the stem cells in humans are also located above the Paneth cell zone. There could be a distinct and as yet undiscovered relationship between these observed traits, with stem cell properties both in cells of the crypt base and those at cell position 4.


Subject(s)
Intestine, Small/cytology , Stem Cells/cytology , Animals , Biomarkers , Cell Differentiation , Humans , Immunohistochemistry , Mice
7.
Cell Prolif ; 42(3): 394-403, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19397593

ABSTRACT

OBJECTIVES: Infection of mice with the parasite Trichinella spiralis leads to small intestinal inflammation, characterized by changes in mucosal architecture and subpopulations of epithelial cells. This model has been used to explore changes in the epithelial proliferative cell population and expression of transforming growth factor-beta (TGF-beta). MATERIALS AND METHODS: Histochemical and immunohistochemical studies were undertaken in duodenal samples. Location and number of Ki-67-positive cells were assessed using Score and Wincrypts program. Changes in mRNA transcripts were studied by real-time RT-PCR. RESULTS: T. spiralis infection induced an increase in total number of proliferative (Ki-67-positive) cells per half crypt on day 2 post-infection. Transcription of Math1, a transcription factor required for secretory cell differentiation in the intestine, was up-regulated on days 6-18 post-infection. At these time points, numbers of Paneth cells at the crypt base were also increased and the epithelial proliferative zone was shifted up the crypt-villus axis. Transcription of TGF-beta isoforms within the small intestine was up-regulated on days 6 and 12 post-infection, but anti-TGF-beta antibody treatment had no effect on T. spiralis-induced changes in mucosal architecture or increase in Paneth/intermediate cells. CONCLUSIONS: T. spiralis infection promotes an initial increase in small intestinal epithelial proliferation and subsequent cell differentiation along the secretory cell lineage. The resulting increase in numbers of Paneth cells at the crypt base causes the proliferative zone to move up the crypt-villus axis. Further studies are required to determine the significance of an increase in the expression of TGF-beta transcripts.


Subject(s)
Intestinal Mucosa/pathology , Intestine, Small/parasitology , Stem Cells/pathology , Trichinella spiralis/pathogenicity , Trichinellosis/pathology , Animals , Base Sequence , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Proliferation , DNA Primers , Female , Immunohistochemistry , Mice , Mice, Inbred BALB C , Neutralization Tests , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/immunology
8.
Postgrad Med J ; 85(1001): 152-62, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19351643

ABSTRACT

The main purpose of this article is to review recent developments in the management of acute and recurrent Clostridium difficile-associated disease, with consideration of existing and new antibiotic and non-antibiotic agents for treatment. Details of the current developmental stage of new agents are provided and the role of surgery in the management of severe disease is discussed. Infection control measures considered comprise prudent use of antimicrobials, prevention of cross-infection and surveillance. Other topics that are covered include the recent emergence of an epidemic hypervirulent strain, pathogenesis, clinical presentation and approaches to rapid diagnosis and assessment of the colonic disease.


Subject(s)
Anti-Bacterial Agents/therapeutic use , Clostridioides difficile , Enterocolitis, Pseudomembranous/drug therapy , Communicable Disease Control/organization & administration , Cross Infection/prevention & control , Enterocolitis, Pseudomembranous/diagnosis , Enterocolitis, Pseudomembranous/surgery , Humans , Probiotics/therapeutic use , Risk Factors , Secondary Prevention
9.
Am J Physiol Cell Physiol ; 296(2): C296-305, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19073897

ABSTRACT

Colonic epithelial stem cells are believed to be located at the crypt base where they have previously been shown to express musashi-1. The colonic stem cell niche, which includes extracellular matrix and myofibroblasts (together with other cell types), is likely to be important in maintaining the function of the progenitor cells. The aims of our studies were to characterize stem cells in isolated and disaggregated human colonic crypt epithelial cells and investigate their interactions with monolayers of primary human colonic myofibroblasts. In unfractionated preparations of disaggregated colonic crypts, musashi-1 positive cells preferentially adhered to colonic myofibroblasts, despite the presence of excess blocking anti-beta(1)-integrin antibody. These adherent epithelial cells remained viable for a number of days and developed slender processes. Cells with side population characteristics (as demonstrated by ability to expel the dye Hoechst 33342) were consistently seen in the isolated colonic crypt epithelial cells. These side population cells expressed musashi-1, beta(1)-integrin, BerEP4, and CD133. Sorted side population crypt epithelial cells also rapidly adhered to primary colonic myofibroblasts. In conclusion, in preparation of isolated and disaggregated human colonic crypts, cells with stem cell characteristics preferentially adhere to primary human colonic myofibroblasts in a beta(1)-integrin-independent fashion.


Subject(s)
Cell Communication , Cell Transdifferentiation , Colon/metabolism , Epithelial Cells/metabolism , Fibroblasts , Intestinal Mucosa/metabolism , Stem Cells/metabolism , AC133 Antigen , Antigens, CD/metabolism , Biomarkers/metabolism , Biomarkers, Tumor/metabolism , Cell Adhesion , Cell Separation , Cell Survival , Cells, Cultured , Coculture Techniques , Colon/cytology , Glycoproteins/metabolism , Humans , Immunohistochemistry , Integrin beta1/metabolism , Intestinal Mucosa/cytology , Nerve Tissue Proteins/metabolism , Peptides/metabolism , RNA-Binding Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Time Factors
11.
Clin Exp Immunol ; 153(3): 439-47, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18505424

ABSTRACT

Clostridium difficile induces mucosal inflammation via secreted toxins A and B and initial interactions between the toxins and intestinal epithelial cells (which lead to loss of barrier function) are believed to be important in disease pathogenesis. Secreted toxin-specific antibodies may inhibit such interactions. Using the Caco-2 epithelial cell line, we have investigated the use of an anti-toxin A monoclonal antibody (ATAA) in providing protection against toxin A-mediated disruption of epithelial barrier function (assessed by measurement of transepithelial electrical resistance and luminal to basolateral flux of labelled dextran). In contrast to free antibody, ATAA conjugated to sepharose beads was more effective in neutralizing the activity of purified toxin A. Sepharose bead-conjugated ATAA was subsequently used to investigate the contribution of toxin A in epithelial injury mediated by C. difficile supernatant samples (containing toxins A, B and other products). Loss of barrier function mediated by apical application of supernatant samples of reference and epidemic 027 strains of C. difficile was abrogated by neutralization of toxin A. However, this was not the case when the supernatant samples were applied to the basal surface of epithelial monolayers. In conclusion, our studies have shown that (i) sepharose bead-conjugated ATAA is more effective in neutralizing toxin A than free antibody and (ii) when the apical (luminal) surface of epithelial monolayers is exposed to the secretory products of reference and 027 strains of C. difficile, toxin A is required for the initial injury that leads to loss of barrier function.


Subject(s)
Antibodies, Monoclonal/pharmacology , Clostridioides difficile/pathogenicity , Enterotoxins/toxicity , Epithelial Cells/drug effects , Immunotoxins/pharmacology , Intestines/drug effects , Animals , Bacterial Toxins , Caco-2 Cells , Chlorocebus aethiops , Humans , Intestinal Mucosa/drug effects , Sepharose/pharmacology , Vero Cells/immunology
12.
Gut ; 57(6): 850-60, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18252748

ABSTRACT

The main purpose of this article is to review recent developments in the management of acute and recurrent Clostridium difficile-associated disease, with consideration of existing and new antibiotic and non-antibiotic agents for treatment. Details of the current developmental stage of new agents are provided and the role of surgery in the management of severe disease is discussed. Infection control measures considered comprise prudent use of antimicrobials, prevention of cross-infection and surveillance. Other topics that are covered include the recent emergence of an epidemic hypervirulent strain, pathogenesis, clinical presentation and approaches to rapid diagnosis and assessment of the colonic disease.


Subject(s)
Clostridioides difficile/pathogenicity , Enterocolitis, Pseudomembranous/therapy , Anti-Bacterial Agents/therapeutic use , Disease Outbreaks , Enterocolitis, Pseudomembranous/diagnosis , Enterocolitis, Pseudomembranous/epidemiology , Humans , Recurrence , Risk Factors , Virulence
13.
J Clin Pathol ; 60(9): 1029-34, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17412868

ABSTRACT

BACKGROUND: Roux-en-Y gastric bypass surgery provides a novel human model to investigate small bowel mucosal innate immunity, in which there is loss of gastric acid-mediated protection against orally-acquired microorganisms. AIM: To study changes in jejunal mucosal immunoreactivity of human defensin (HD)-5, an antimicrobial peptide normally produced by Paneth cells. METHODS: Mucosal samples were obtained from 18 female patients (24-54 years), from the same segment of jejunum during and after gastric bypass surgery. Samples were used for bacterial culture and immunohistochemistry using anti-HD-5 antibody. The number of immunoreactive cells per crypt and villus were determined and expressed as mean (SD). RESULTS: No bacteria were cultured from any of the perioperative jejunal samples but colonies of bacteria normally present in the pharynx were identified during culture of all postoperative jejunal biopsy specimens (1->100 colonies). Paneth cell numbers per crypt were unchanged after gastric bypass (4.16 (0.71) vs 4.24 (0.78)). However, following surgery, there was an increase in HD-5-positive intermediate cells per crypt (0.25 (0.41) vs 1.12 (0.66), p<0.01), HD-5 staining enterocytes per crypt (0.03 (0.09) vs 1.38 (1.10), p<0.01), HD-5 staining material in the crypt lumen (crypt lumens: 5.0% (10.9%) vs 68.1% (27.9%), p<0.01) and HD-5 immunoreactivity coating the luminal surface of villus enterocytes (villi sampled: 15.0% (31.0%) vs 67.5% (42.0%), p<0.01). CONCLUSIONS: Bacteria normally resident in the pharynx were present in the proximal jejunal mucosa following Roux-en-Y gastric bypass surgery. After gastric bypass, there was increased secretion of HD-5 and an increase in HD-5 expressing intermediate cells and enterocytes in the crypt. The increase in HD-5 expression in the jejunal mucosa following gastric bypass surgery is likely to be secondary to exposure to orally-acquired microorganisms.


Subject(s)
Gastric Bypass , Intestinal Mucosa/metabolism , Jejunum/metabolism , alpha-Defensins/metabolism , Adult , Bacteria/growth & development , Bacteria/isolation & purification , Cell Count , Female , Humans , Immunoenzyme Techniques , Intestinal Mucosa/microbiology , Jejunum/microbiology , Microscopy, Immunoelectron , Middle Aged , Muramidase/metabolism , Paneth Cells/pathology , Pharynx/microbiology , Postoperative Period
15.
Scand J Immunol ; 61(6): 575-84, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15963053

ABSTRACT

Intestinal macrophage responses to luminal bacteria and their constituents are important in mucosal inflammatory responses. We investigated the responses of intestinal macrophages to free lipopolysaccharide (LPS) and Escherichia coli. Macrophages were isolated from normal terminal ileum and colon by allowing them to migrate out of the lamina propria of mucosal samples denuded of epithelial cells. Following exposure to free LPS or fluorescein-labelled E. coli, responsiveness was studied by intracellular expression of tumour necrosis factor-alpha (TNF-alpha). CD14, CD33, CD68, TLR2 and TLR4 expression was studied by fluorescence-activated cell sorter (FACS). TLR and NOD2 expression was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR). CD14 was expressed by 36.5 +/- 4.0% of the macrophages obtained following migration out of the lamina propria. These cells also expressed TLR2, TLR4 and NOD2. Of cells exposed to free LPS or those that had taken up E. coli, a greater proportion of CD14(+) than CD14(-) macrophages expressed intracellular TNF-alpha. Moreover, a greater proportion of macrophages (CD14(+) and CD14(-)) demonstrated responses to E. coli than free LPS. In conclusion, a proportion of macrophages obtained following migration out of the lamina propria of normal terminal ileal and colonic mucosal samples express CD14, TLR2 and TLR4. These cells respond to free LPS and E. coli, as demonstrated by the expression of TNF-alpha.


Subject(s)
Escherichia coli , Lipopolysaccharides , Macrophages/immunology , Tumor Necrosis Factor-alpha/biosynthesis , Aged , Cell Separation , Cells, Cultured , Colon/immunology , Flow Cytometry , Humans , Ileum/immunology , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Macrophages/microbiology , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Middle Aged , Nod2 Signaling Adaptor Protein , Phagocytosis , RNA, Messenger/analysis , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Toll-Like Receptor 2 , Toll-Like Receptor 4 , Toll-Like Receptors
16.
Infect Immun ; 73(3): 1625-34, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15731062

ABSTRACT

In this study we investigated the in vitro responses of peripheral blood mononuclear preparations and purified monocytes to Clostridium difficile toxin A. In contrast to the responses of T and B cells, exposure to toxin A led to a rapid loss of monocytes in a time- and dose-dependent fashion (the majority of cells were lost within 24 h of exposure to >100 ng of toxin per ml). Transmission electron microscopy, flow cytometry, and fluorescence microscopy after propidium iodide and Hoechst staining showed that cell death in purified preparations of monocytes following exposure to 100 and 1,000 ng of toxin A per ml occurred by apoptosis. Further studies showed that 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazole-carbocyanine iodide aggregates were retained within toxin A-exposed monocyte mitochondria, but cytochrome c was released, suggesting that the apoptotic cascade was triggered in the absence of mitochondrial permeability transition. There was also an increase in caspase-3 activity in toxin A-stimulated monocytes. Following exposure to very high concentrations of toxin A (30 microg/ml), monocyte cell death was predominantly of the necrotic type, with rapid extracellular release of lactate dehydrogenase. These studies demonstrated that C. difficile toxin A has a cell-specific effect, in which monocytes exhibit greater susceptibility than lymphocytes and their death is induced in a concentration-dependent manner.


Subject(s)
Apoptosis , Bacterial Toxins/toxicity , Clostridioides difficile/pathogenicity , Enterotoxins/toxicity , Monocytes/drug effects , Cell Death , Cell Line , Clostridioides difficile/metabolism , Diarrhea/microbiology , Enterocolitis, Pseudomembranous/microbiology , Feces/chemistry , Feces/microbiology , Flow Cytometry , Humans , Microscopy, Electron, Transmission , Microscopy, Fluorescence , Monocytes/pathology , Necrosis
17.
J Clin Pathol ; 57(9): 973-9, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15333661

ABSTRACT

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.


Subject(s)
B-Lymphocytes/immunology , Clostridioides difficile , Diarrhea/microbiology , Enterocolitis, Pseudomembranous/immunology , Intestinal Mucosa/immunology , Macrophages/physiology , Adolescent , Aged , Aged, 80 and over , Case-Control Studies , Cell Count , Colon , Diarrhea/immunology , Humans , Immunoglobulin A/analysis , Immunoglobulin G/analysis , Immunoglobulin M/analysis , Immunohistochemistry/methods , Male , Middle Aged , Prospective Studies , Recurrence
18.
Best Pract Res Clin Gastroenterol ; 18(2): 241-53, 2004 Apr.
Article in English | MEDLINE | ID: mdl-15123067

ABSTRACT

Intestinal epithelial cells are unique in that they represent the only host cells that are constantly interacting with a very large bacterial population in the lumen. The single monolayer of epithelial cells consists of subpopulations with distinct functions that include protection against luminal microorganisms. Although the microbial flora remains to be fully characterized, its normal relationship with the host intestinal epithelial cells appears to be predominantly symbiotic or commensal. The molecular complexity of the epithelial-microbial relationship has been shown in studies that have examined the establishment of the resident bacteria in germ-free mice. Recent work has also demonstrated the ability of resident bacteria to enhance epithelial protective responses. The mechanisms by which epithelial cells may avoid pro-inflammatory responses to resident microorganisms, while retaining the capacity to respond to pathogens, are also being characterized.


Subject(s)
Enterobacteriaceae/physiology , Epithelial Cells/microbiology , Epithelial Cells/physiology , Enterocytes/microbiology , Enterocytes/physiology , Humans , Intestinal Mucosa/microbiology , Intestinal Mucosa/physiology , Symbiosis/physiology
19.
Gut ; 53(5): 673-7, 2004 May.
Article in English | MEDLINE | ID: mdl-15082585

ABSTRACT

OBJECTIVES: Clostridium difficile associated diarrhoea (CDAD) is a hospital acquired infection in which optimal methods for diagnosis and the scale of the problem in the community remain to be determined. In hospitalised patients with CDAD, we aimed to (i) study patients in whom the onset of diarrhoea was in the community and (ii) investigate the role of bedside flexible sigmoidoscopy in diagnosis. METHODS: Patients with CDAD (onset in hospital or community) were studied prospectively. In those with diarrhoea of unknown aetiology, flexible sigmoidoscopy was compared with stool assay for C difficile cytotoxin. RESULTS: Of 136 patients with CDAD (which was associated with antibiotic exposure in 96%), diarrhoea started in the community in 38 (28%; majority in own home) and while an inpatient in 98 (72%). The majority with CDAD onset in the community had been hospitalised over the preceding 12 months (86.8% v 57.1% in the hospital onset group; p<0.001). In 56 patients with pseudomembranous colitis at sigmoidoscopy, the stool C difficile cytotoxin test was negative in 29 (52%) but toxigenic C difficile was isolated from all of nine stool samples cultured. Of patients with pseudomembranous colitis, 30.4% relapsed over the subsequent 57.7(4.2) days. CONCLUSIONS: In a significant proportion of hospitalised patients with CDAD, diarrhoea started in the community. However, the majority of these had been hospital inpatients previously when they may have acquired C difficile, with the subsequent onset of diarrhoea in the community following exposure to antibiotics. Flexible sigmoidoscopy is superior to the stool C difficile cytotoxin test in a subgroup of patients with pseudomembranous colitis. Sigmoidoscopy should therefore be considered in all hospitalised patients with diarrhoea in whom the stool test for C difficile cytotoxin and enteric pathogens is negative.


Subject(s)
Clostridioides difficile , Cross Infection/transmission , Diarrhea/microbiology , Enterocolitis, Pseudomembranous/transmission , Aged , Anti-Bacterial Agents/adverse effects , Community-Acquired Infections/chemically induced , Community-Acquired Infections/diagnosis , Community-Acquired Infections/transmission , Cross Infection/chemically induced , Cross Infection/diagnosis , Diarrhea/chemically induced , Enterocolitis, Pseudomembranous/chemically induced , Enterocolitis, Pseudomembranous/diagnosis , Feces/microbiology , Hospitalization , Humans , Prospective Studies , Recurrence , Sigmoidoscopy
20.
Aliment Pharmacol Ther ; 19(1): 79-88, 2004 Jan 01.
Article in English | MEDLINE | ID: mdl-14687169

ABSTRACT

BACKGROUND: Tumour necrosis factor-alpha is thought to be important in the pathogenesis of portal hypertension. Oxpentifylline (pentoxifylline) and thalidomide inhibit endotoxin-induced tumour necrosis factor-alpha production in vitro. AIMS: To assess the toxicity of oxpentifylline (pentoxifylline) and thalidomide in cirrhosis and their effect on the hepatic venous pressure gradient and tumour necrosis factor-alpha production. METHODS: In an open-label pilot study, 20 abstinent patients with stable alcoholic cirrhosis and oesophageal varices were recruited; 12 patients completed haemodynamic measurements before and after treatment with oxpentifylline (pentoxifylline) 1800 mg (n=6) or thalidomide 200 mg (n=6) daily for 2 weeks. Tumour necrosis factor-alpha production was assessed in ex vivo monocyte cultures stimulated with endotoxin. RESULTS: Thalidomide reduced the hepatic venous pressure gradient from 19.7 mmHg (9.3-23.5 mmHg) to 12.2 mmHg (4.7-19.5 mmHg) (P=0.03) without reducing the hepatic blood flow or altering systemic haemodynamic parameters. Thalidomide reduced ex vivo tumour necrosis factor-alpha production by approximately 50%. Oxpentifylline (pentoxifylline) had no significant effect on any of the parameters measured. Side-effects led to dose reduction or treatment withdrawal in 40% of patients. CONCLUSION: Thalidomide, but not oxpentifylline (pentoxifylline), reduces the hepatic venous pressure gradient in stable alcoholic cirrhotics, an effect that may be mediated by the inhibition of tumour necrosis factor-alpha production. The role of tumour necrosis factor-alpha inhibitory drugs in the therapy of portal hypertension should be investigated in a randomized controlled trial.


Subject(s)
Enzyme Inhibitors/therapeutic use , Hypertension, Portal/drug therapy , Liver Cirrhosis, Alcoholic/complications , Pentoxifylline/therapeutic use , Thalidomide/therapeutic use , Tumor Necrosis Factor-alpha/metabolism , Vasodilator Agents/therapeutic use , Adult , Aged , Blood Pressure/drug effects , Cardiac Output/drug effects , Drug Therapy, Combination , Enzyme Inhibitors/adverse effects , Female , Humans , Hypertension, Portal/etiology , Hypertension, Portal/metabolism , Liver Cirrhosis, Alcoholic/metabolism , Male , Middle Aged , Pentoxifylline/adverse effects , Pilot Projects , Thalidomide/adverse effects , Vasodilator Agents/adverse effects
SELECTION OF CITATIONS
SEARCH DETAIL
...