| 1. |
- Johansson, Malin E V, 1971, et al.
(author)
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Normalization of Host Intestinal Mucus Layers Requires Long-Term Microbial Colonization
- 2015
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In: Cell Host & Microbe. - : Elsevier BV. - 1931-3128 .- 1934-6069. ; 18:5, s. 582-592
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Journal article (peer-reviewed)abstract
- The intestinal mucus layer provides a barrier limiting bacterial contact with the underlying epithelium. Mucus structure is shaped by intestinal location and the microbiota. To understand how commensals modulate gut mucus, we examined mucus properties under germ-free (GF) conditions and during microbial colonization. Although the colon mucus organization of GF mice was similar to that of conventionally raised (Convr) mice, the GF inner mucus layer was penetrable to bacteria-sized beads. During colonization, in which GF mice were gavaged with Convr microbiota, the small intestine mucus required 5 weeks to be normally detached and colonic inner mucus 6 weeks to become impenetrable. The composition of the small intestinal microbiota during colonization was similar to Convr donors until 3 weeks, when Bacteroides increased, Firmicutes decreased, and segmented filamentous bacteria became undetectable. These findings highlight the dynamics of mucus layer development and indicate that studies of mature microbe-mucus interactions should be conducted weeks after colonization.
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| 2. |
- Jabbar, Karolina S., et al.
(author)
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Association between Brachyspira and irritable bowel syndrome with diarrhoea
- 2021
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In: Gut. - : BMJ. - 0017-5749 .- 1468-3288. ; 70, s. 1117-1129
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Journal article (peer-reviewed)abstract
- Objective: The incidence of IBS increases following enteric infections, suggesting a causative role for microbial imbalance. However, analyses of faecal microbiota have not demonstrated consistent alterations. Here, we used metaproteomics to investigate potential associations between mucus-resident microbiota and IBS symptoms. Design: Mucus samples were prospectively collected from sigmoid colon biopsies from patients with IBS and healthy volunteers, and their microbial protein composition analysed by mass spectrometry. Observations were verified by immunofluorescence, electron microscopy and real-Time PCR, further confirmed in a second cohort, and correlated with comprehensive profiling of clinical characteristics and mucosal immune responses. Results: Metaproteomic analysis of colon mucus samples identified peptides from potentially pathogenic Brachyspira species in a subset of patients with IBS. Using multiple diagnostic methods, mucosal Brachyspira colonisation was detected in a total of 19/62 (31%) patients with IBS from two prospective cohorts, versus 0/31 healthy volunteers (p<0.001). The prevalence of Brachyspira colonisation in IBS with diarrhoea (IBS-D) was 40% in both cohorts (p=0.02 and p=0.006 vs controls). Brachyspira attachment to the colonocyte apical membrane was observed in 20% of patients with IBS and associated with accelerated oro-Anal transit, mild mucosal inflammation, mast cell activation and alterations of molecular pathways linked to bacterial uptake and ion-fluid homeostasis. Metronidazole treatment paradoxically promoted Brachyspira relocation into goblet cell secretory granules-possibly representing a novel bacterial strategy to evade antibiotics. Conclusion: Mucosal Brachyspira colonisation was significantly more common in IBS and associated with distinctive clinical, histological and molecular characteristics. Our observations suggest a role for Brachyspira in the pathogenesis of IBS, particularly IBS-D. © Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.
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| 3. |
- Lagergård, Teresa, 1946, et al.
(author)
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Formaldehyde treatment increases the immunogenicity and decreases the toxicity of Haemophilus ducreyi cytolethal distending toxin.
- 2007
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In: Vaccine. - : Elsevier BV. - 0264-410X. ; 25:18, s. 3606-14
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Journal article (peer-reviewed)abstract
- Haemophilus ducreyi cytolethal distending toxin (HdCDT) is a tripartite AB toxin, which causes DNA damage in affected cells. We investigated the effects of formaldehyde on the chemical, biological, and immunological properties of the HdCDT complex, which was purified by immobilizing the glutathione S-transferase (GST)-CdtB fusion protein, followed by binding of the CdtA and CdtC recombinant proteins. The HdCDT was treated with increasing concentrations of formaldehyde in the presence of lysine. The treatment of HdCDT at 1 and 0.1 mg protein/ml with 320 and 80 mM of formaldehyde, respectively, resulted in the complete abrogation of cytotoxic activity, loss of DNase activity, and loss of binding capacity to HeLa cells. The toxoid showed protein bands of 75-150 kDa in SDS-PAGE, composed of the three cross-linked CDT components detected by immunoblotting. Three doses of 10 microg protein/mouse of the formaldehyde-treated HdCDT elicited toxin-neutralizing antibodies at titers about 200 times higher than those elicited by the native toxin. The described methodology may be applied to produce immunogenic toxoids from other CDTs, which might be used as candidate components in vaccines against CDT-producing bacteria, including H. ducreyi.
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| 4. |
- Pelaseyed, Thaher, 1979, et al.
(author)
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The mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system
- 2014
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In: Immunological Reviews. - : Wiley. - 0105-2896 .- 1600-065X. ; 260:1, s. 8-20
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Research review (peer-reviewed)abstract
- The gastrointestinal tract is covered by mucus that has different properties in the stomach, small intestine, and colon. The large highly glycosylated gel-forming mucins MUC2 and MUC5AC are the major components of the mucus in the intestine and stomach, respectively. In the small intestine, mucus limits the number of bacteria that can reach the epithelium and the Peyer's patches. In the large intestine, the inner mucus layer separates the commensal bacteria from the host epithelium. The outer colonic mucus layer is the natural habitat for the commensal bacteria. The intestinal goblet cells secrete not only the MUC2 mucin but also a number of typical mucus components: CLCA1, FCGBP, AGR2, ZG16, and TFF3. The goblet cells have recently been shown to have a novel gate-keeping role for the presentation of oral antigens to the immune system. Goblet cells deliver small intestinal luminal material to the lamina propria dendritic cells of the tolerogenic CD103+ type. In addition to the gel-forming mucins, the transmembrane mucins MUC3, MUC12, and MUC17 form the enterocyte glycocalyx that can reach about a micrometer out from the brush border. The MUC17 mucin can shuttle from a surface to an intracellular vesicle localization, suggesting that enterocytes might control and report epithelial microbial challenge. There is communication not only from the epithelial cells to the immune system but also in the opposite direction. One example of this is IL10 that can affect and improve the properties of the inner colonic mucus layer. The mucus and epithelial cells of the gastrointestinal tract are the primary gate keepers and controllers of bacterial interactions with the host immune system, but our understanding of this relationship is still in its infancy.
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| 5. |
- Wising, Catharina, 1973, et al.
(author)
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Induction of apoptosis/necrosis in various human cell lineages by Haemophilus ducreyi cytolethal distending toxin.
- 2005
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In: Toxicon : official journal of the International Society on Toxinology. - : Elsevier BV. - 0041-0101. ; 45:6, s. 767-76
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Journal article (peer-reviewed)abstract
- We investigated the impact of highly purified Haemophilus ducreyi cytolethal distending toxin (HdCDT) on the apoptosis and necrosis of various human cells; including myeloid cells, epithelial cells, keratinocytes, and primary fibroblasts. The levels of apoptosis and necrosis induced in these cells were compared to those induced by HdCDT in human T cells and in the Jurkat T cell line. Levels of caspase-3 activity were measured, and membrane changes like phosphatidylserine (PS) translocation was evaluated after double-staining with the fluorescein isothiocyanate (FITC)-labeled annexin V and propidium iodide (PI) using flow cytometry. HdCDT induced various degrees of apoptosis and necrosis in dose- and time-dependent manners in cells of various lineages. Early and late apoptosis (annexin V-stained cells) were induced in more than 90% of T cells and monocytes after treatment with 100 ng/ml HdCDT for 24 and 48 h, respectively. The corresponding numbers for epithelial cells, keratinocytes, and fibroblasts were 26-32% after treatment with 100 ng/ml HdCDT for 48 h. HdCDT appears to eliminate effectively by inducing apoptosis those cells that are involved in immune responses. Epithelial cells, keratinocytes and fibroblasts, which are important for the healing of chancroid ulcers, are eliminated by apoptosis or necrosis after contact with HdCDT, albeit slower and to a lesser extent than T cells.
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| 6. |
- Wising, Catharina, 1973, et al.
(author)
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The cytolethal distending toxin of Haemophilus ducreyi aggravates dermal lesions in a rabbit model of chancroid
- 2005
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In: Microbes Infect. - : Elsevier BV. - 1286-4579. ; 7:5-6, s. 867-74
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Journal article (peer-reviewed)abstract
- Haemophilus ducreyi, the etiologic agent of the sexually transmitted disease chancroid, produces a cytolethal distending toxin (HdCDT) that inhibits cultured cell proliferation, leading to cell death. A rabbit model of dermal infection was used to investigate the roles of H. ducreyi bacteria and HdCDT in the development, clinical appearance, and persistence of infection. A non-toxin producing H. ducreyi strain, and for comparison purposes a non-capsulated Haemophilus influenzae strain, were inoculated intradermally, with and without co-administration of purified HdCDT. Co-administration of HdCDT resulted in significant aggravation of H. ducreyi-induced inflammatory lesions, and development of ulcers in rabbit skin. Less pronounced inflammatory lesions and lack of epithelial eruption were observed after inoculation with H. influenzae. Histopathological sections of the H. ducreyi-induced lesions, in both the presence and absence of HdCDT, showed dense infiltrates of the same type inflammatory cells, with the exception of a prominent endothelial cell proliferation noted in sections from lesions caused by H. ducreyi and toxin. Signs of chronic inflammation with involvement of T cells, macrophages, eosinophils, and granuloma formation were observed after H. ducreyi inoculation both with and without toxin. In conclusion, H. ducreyi causes a pronounced, chronic inflammation with involvement of T cells and macrophages, and in combination with HdCDT production of ulcers in the rabbit model. These pathogenic mechanisms may promote the development and persistence of chancroid ulcers.
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| 7. |
- Wising, Catharina, 1973
(author)
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The cytolethal distending toxin of Haemophilus ducreyi. Purification and biological activity
- 2005
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Doctoral thesis (other academic/artistic)abstract
- Haemophilus ducreyi is a Gram-negative bacterium that causes the sexually transmitted disease (STD) chancroid. The disease is endemic in many developing countries, and occurs as restricted outbreaks in the industrialized part of the world. Chancroid as other STDs represents a risk factor for the transmission of HIV and this has given renewed attention to this disease. The disease is characterised by soft, slow healing ulcers on the external genitals, frequently accompanied by unilateral dissemination to regional lymph nodes and the formation of buboes. The mechanism of pathogenesis in chancroid is not clear. H. ducreyi possesses several potential virulence factors, which could increase its persistence in the human host by avoiding host defence mechanisms and/or cause tissue damage. The focus of this thesis is the cytolethal distending toxin of H. ducreyi (HdCDT). It belongs to a novel family of toxins (CDTs), which are all composed of three components, CdtA, CdtB, and CdtC. The toxin has the ability to induce DNA double strand breaks, and thereby cause cell cycle arrest and death of target cells, a unique mechanism of action among bacterial toxins. The overall aim of the present thesis was to investigate the role of HdCDT in the pathogenesis of chancroid. The specific aims were; to purify and characterise the HdCDT components and the holotoxin, to study HdCDT intoxication and cell death of various human cells, and to study the pathologic activity of HdCDT in a rabbit model of H. ducreyi infection. The different HdCDT components and the holotoxin were purified from E. coli recombinants expressing the components individually. The purity of the protein preparations was evaluated by SDS-PAGE Coomassie blue staining and immunoblotting. The toxicity of the HdCDT components in different combinations was evaluated using a cytotoxicity assay and by cell cycle analysis using flow cytometry. The HdCDT-induced cell death was investigated using two apoptosis methods; a colorimetric caspase-3 assay and a method detecting phosphatidyl serine in the cell membrane using flow cytometry. The pathologic effects of the HdCDT components and the holotoxin in vivo were evaluated in rabbit skin. Animals were inoculated intradermally with a non-toxin producing H. ducreyi strain and for comparison a Haemophilus influenzae strain, with and without the addition of purified HdCDT. We found that HdCDT can be reconstituted from individually expressed HdCDT components. The reconstituted holotoxin was successfully purified and formed a three-component complex. The HdCdtB alone was shown to have deoxyribonuclease (DNase) activity in vitro. HdCDT induced apoptosis in more than 90% of T cells and monocytes, whereas epithelial cells, keratinocytes, and fibroblasts were less affected; apoptosis and necrosis were noted for about 26-32% and 6-12%, respectively. Purified HdCDT caused inflammation, but did not cause ulcer formation in rabbit skin alone. HdCDT delivered together with H. ducreyi bacteria resulted in significant aggravation of inflammatory lesions and development of ulcers in rabbit skin. A histological picture of chronic skin lesions was observed, which underlines the persistent nature of H. ducreyi infection. Production of HdCDT may be a strategy for H. ducreyi bacterium to hamper the immune response, and to delay the healing process, and thereby cause a persistent infection with characteristic ulcers.
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| 8. |
- Wising, Catharina, 1973, et al.
(author)
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Toxic activity of the CdtB component of Haemophilus ducreyi cytolethal distending toxin expressed from an adenovirus 5 vector
- 2010
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In: APMIS. - : Wiley. - 1600-0463 .- 0903-4641. ; 118:2, s. 143-149
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Journal article (peer-reviewed)abstract
- The Haemophilus ducreyi cytolethal distending toxin (HdCDT) catalytic subunit CdtB has DNase-like activity and mediates DNA damage after its delivery into target cells. We constructed a replication-deficient adenovirus type 5 (Ad5) vector expressing CdtB and investigated the toxic properties of this vector on HeLa cells. Ad5CdtB caused loss of cell viability, morphologic changes, and cell cycle arrest, findings similar to HdCDT intoxication. This confirmed that CdtB is responsible for the toxicity of the holotoxin when expressed in cells following transduction by an adenoviral vector, and indicated a possible potential of this novel strategy in studies of activity of intracellular products and in gene therapy of cancer.
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