| 1. |
- Bas, Anna, 1973-
(författare)
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Extrathymic T cell receptor gene rearrangement in human alimentary tract
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- T lymphocytes regulate the initiation, duration, and magnitude of adaptive immune responses and function as effector cells in cell mediated immunity. To become immunologically competent they must generate functional antigen receptors. This process takes place in the thymus and requires somatic recombination of T cell receptor (TCR) genes. It is mediated by the endonucleases recombination activating gene-1 (RAG1) and RAG2. Although the thymus regresses at puberty, T cells are present throughout life implying that other tissues must provide the proper milieu for T cell development. This thesis describes extrathymic T cell maturation in man. RAG1, RAG2, and the preTα-chain (pTα), which is exclusively utilized in developing T cells, were used as markers for TCR gene rearrangement. Two new exons (1A and 1B) encoding sequences in the 5’ untranslated region (5’UTR) of mRNA were discovered in the human RAG1 gene. The previously described 5’UTR exon (renamed 1C) was located between the new exons and exon 2, the latter containing the entire coding sequence. We found that small intestinal lymphocytes of the T cell lineage expressed the new exons in three different splice forms. RAG1 mRNA containing the 1C exon was not expressed in small intestinal lymphocytes. In contrast, splice forms containing the 1A exon were not expressed in thymocytes. RAG1 and pTα mRNA expressing lymphocytes were seen both within the epithelium and in lamina propria. Thymocyte-like CD2+CD7+CD3-, CD4+CD8+, CD1a+, and IL7-R+ lymphocytes were identified in the small intestinal mucosa. CD2+CD7+CD3- cells had the highest expression levels of mRNA for RAG1 and pTα, suggesting that the small intestinal mucosa is indeed a site for T cell maturation. Small intestinal T lymphocytes were also shown to kill via the Fas/FasL pathway in a TCR/CD3 independent manner and via the perforin/granzyme pathway in a TCR/CD3 dependent manner. The Fas/FasL-mediated cytotoxicity may reflect an ongoing selection process of extrathymically maturated T cells.The nasopharyngeal tonsil is the major inductive site for immune reactions against inhaled antigens. Previous demonstration of RAG1 expression in tonsillar B cells was interpreted as antigen driven receptor revision. The present study confirms the expression of RAG1 in B cells. We also found that RAG1, RAG2, and pTa mRNAs were expressed in lymphocytes of the T cell lineage. A small population of cells with the immature phenotype CD2+CD7+CD3- was demonstrated. This population had the highest expression levels of mRNA for RAG1, RAG2, pTα and terminal deoxynucleotidyl transferase. All four splice-forms of RAG1 mRNA were expressed. RAG1 and pTα mRNA expressing cells were mainly located in the proximity of the surface epithelium and in the outer rim of the follicles. These results suggest that the nasopharyngeal tonsil is a site where extrathymic T cell development and antigen driven TCR revision are occurring in parallel.Celiac disease (CD) is a small intestinal enteropathy characterized by permanent intolerance to gluten. Gluten reactive intestinal T cells are central in the pathogenesis and CD can be regarded as a failure to maintain tolerance to this food antigen. Expression of the RAG1 1A/2 splice form was significantly decreased in small intestinal T cell subsets of CD patients suggesting that impaired TCR gene rearrangement could contribute to failure of maintain tolerance in CD.Together, these findings show that both small intestinal and nasopharyngeal tonsillar lymphocytes of T cell lineage have the molecular machinery for antigen receptor rearrangement and that thymocyte-like lymphocytes are present in both tissues. Thus these organs are likely sites of T lymphocyte ontogeny as well as for secondary T cell receptor rearrangement in man.
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| 2. |
- Bitar, Aziz, 1984-
(författare)
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Vibrio cholerae modulates the immune defense of human gut mucosa
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The key function of innate immunity is to sense danger signals and initiate effective responses as a defense mechanism against pathogens. Simultaneously, effector responses must be regulated to avoid excessive inflammation with resulting tissue damage. microRNAs (miRNAs), are small endogenous molecules, that has recently gained attention as important regulatory elements in the human inflammation cascade. The control over host miRNA expression may represent a previously uncharacterized molecular strategy exploited by pathogens to mitigate innate host cell responses.Vibrio cholerae is a Gram-negative bacterium that colonizes the human small intestine and causes life-threatening secretory diarrhea, essentially mediated by cholera toxin (CT). It is considered a non-invasive pathogen and does not cause clinical inflammation. Still, cholera is associated with inflammatory changes of the small intestine. Furthermore, CT-negative strains of V. cholerae cause gastroenteritis and are associated with extra-intestinal manifestations, suggesting that other virulence factors than CT are also involved in the pathogenesis.The innate immune response to V. cholerae is poorly investigated and the potential role of miRNA in cholera had not been studied before. Therefore, this thesis explores the role of intestinal epithelial cells in response to V. cholerae infection with a focus on regulatory miRNA as a potential contributor to the pathogenesis. The in vivo material was small intestinal biopsies from patients suffering from V. cholerae infection. As an in vitro model for V. cholerae attack on intestinal epithelium, we used tight monolayers of T84 cells infected with V. cholerae and their released factors. We analyzed changes in levels of cytokines, immunomodulatory microRNA and their target genes.We showed that miRNA-146a and miRNA-155 reached significantly elevated levels in the intestinal mucosa at acute stages of disease in V. cholerae infected patients and declined to normal levels at the convalescent stage. Low-grade inflammation was identified at the acute stage of V. cholerae infection, which correlated with elevated levels of regulatory miRNA. Furthermore, outer membrane vesicles (OMVs) released by the bacteria were shown to induce miR-146a and live bacteria induced miR-155 in intestinal epithelial cells. In addition, OMVs decreased epithelial permeability and caused mRNA suppression of pro-inflammatory cytokines, including immune cell attractant IL-8 and CLL20, and the inflammasome markers IL-1b and IL-18. These results propose that V. cholerae regulates the host expression of miRNA during infection and may set the threshold for activation of the intestinal epithelium.Moreover, we showed that V. cholerae also harbors inflammatory-inducing capabilities, by secreting a pore-forming toxin, Vibrio cholerae cytolysin (VCC). By using genetically modified strains as well as soluble protein challenge experiments, VCC was found solely responsible for the increased epithelial permeability and induction of several pro-inflammatory cytokines in intestinal epithelial cells. In contrast to OMVs, VCC displayed strong upregulation of the pro-inflammatory cytokines IL-8, TNF-a, CCL20 and IL-1b and IRAK2, a key signaling molecule in the IL-1 inflammasome pathway. This suggest that VCC is an important virulence factor in the V. cholerae pathogenesis, particularly in CT-negative strains. Furthermore, we showed that the bacterium could control the inflammatory actions of VCC by secreting the PrtV protease, which degraded VCC and consequently abolished inflammation. In summary, we showed that V. cholerae harbors immunomodulating capabilities, both at the gene level, through induction of host regulatory miRNA, and at the protein level, through secretion of VCC and PrtV. These strategies may be relevant for V. cholerae to promote survival in the gut and cause successful infections in the human host.
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| 3. |
- Fahlgren, Anna, 1972-
(författare)
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Defence capabilities of human intestinal epithelial cells
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The epithelial cells lining the intestinal mucosa separate the underlying tissue from components of the intestinal lumen. Innate immunity mediated by intestinal epithelial cells (IECs) provides rapid protective functions against microorganisms. Innate immunity also participates in orchestrating adaptive immunity. Key components in innate defence are defensins.To study the production of defensins and how it is affected by intestinal inflammation IECs were isolated from the small and large intestines of patients suffering from ulcerative colitis (UC), Crohn´s disease (MbC), celiac disease (CD), and from controls, and analyzed by quantitative RT-PCR (qRT-PCR) and immunoflow cytometry. Defensin expressing cells were also studied by in situ hybridization and immunohistochemistry.Normally, only small intestinal Paneth cells express human α-defensin 5 (HD-5) and HD-6. In UC colon IECs, HD-5, HD-6, and lysozyme mRNAs were expressed at high levels. In Crohn´s colitis colon the levels of HD-5 and lysozyme mRNAs were also increased although not to the same extent as in UC. No increase was detected in MbC with ileal localization. Metaplastic Paneth cell differentiation in UC colon was primarily responsible for the expression of the antimicrobial components. Human β-defensin 1 (hBD-1) mRNA was more abundant in large than in small intestine of controls, and remained unchanged in UC and MbC. hBD-2 mRNA was barely detectable in normal intestine and was induced in UC IECs but not in MbC IECs. mRNAs for the recently discovered hBD-3 and hBD-4, were detected in IECs from both small and large intestine. Both hBD-3 and hBD-4 mRNA were significantly increased in IECs of UC patients but not of MbC patients. Bacteria and IL-1β induced hBD-2 but not hBD-1 mRNA in colon carcinoma cell lines. IFN-γ, but not TNF-α or IL-1β, augmented hBD-3 expression in these cells, while none of the agents induced hBD-4. High antimicrobial activity of IECs in UC may be a consequence of changes in the epithelial lining, which permit the adherence of microorganisms.Unexpectedly, in situ hybridization revealed expression of hBD-3 and hBD-4 mRNAs by numerous lamina propria cells in colonic tissue from UC patients. These cells were identified as plasma cells (CD138+). hBD-3 and hBD-4 mRNAs were also demonstrated in the plasmacytoma cell line U266. This is the first demonstration of defensins in plasma cells.The four prominent constituents of the intestinal glycocalyx, carcinoembryonic antigen (CEA), CEA cell adhesion molecule 1 (CEACAM1), CEACAM6 and CEACAM7 all seem to play a critical role in innate defence of the intestinal mucosa by trapping and expelling microorganisms at the epithelial surface. The inducibility of these molecules in colonic epithelial cell lines was analyzed by qRT-PCR, immunoflow cytometry, and immunoelectron microscopy. IFN-g but not bacteria, LPS, TNF-α, or IL-1β modified the expression of CEA, CEACAM1 and CEACAM6. None of these agents modified CEACAM7 expression. IFN-γ was shown to have two effects: a direct effect on CEACAM1 transcription, and promotion of cell differentiation resulting in increased CEA and CEACAM6 and decreased CEACAM7 expression.Scanning electron microscopy of jejunal biopsies from children with CD revealed the presence of rod shaped bacteria in ~40% of patients with active CD, but only in 2% of controls. 19% of treated CD patients still had adhering bacteria. Presence of bacteria is not due to lack of antimicrobial factors. In fact, HD-5, HD-6, and lysozyme mRNA levels were significantly increased in IECs of patients with active CD. hBD-1 and hBD-2 were unchanged. Lack of induction of hBD-2 may reflect disturbed signalling in IECs of CD patients. Analysis of CEA and CEACAM1 mRNA/protein expression showed no differences between CD patients and controls. Analysis of the mucins MUC2 and MUC3 revealed significantly increased MUC2 levels in active disease and unchanged MUC3. Immunohistochemistry demonstrated goblet cell metaplasia as well as staining of the apical portion of absorptive cells. Glycosylation status of proteins was studied by lectin histochemistry. Goblet cells in the mucosa of CD patients were stained by the lectin UEAI. This was not seen in controls. The lectin PNA stained the glycocalyx of controls but not that of CD patients. Thus, unique carbohydrate structures of the glycocalyx/mucous layer are likely discriminating features of CD patients and may allow bacterial binding.We conclude that the intestinal epithelium is heavily involved in the innate defence of the mucosa and that its reactive pattern is affected by intestinal inflammation.Keywords: human intestinal mucosa; epithelial cells; innate immunity; defensin; ulcerative colitis; Crohn´s disease; celiac disease; glycoαcalyx; mucin
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| 4. |
- Forsberg, Göte, 1957-
(författare)
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Innate and adaptive immunity in childhood celiac disease
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Celiac disease (CD) is an inflammatory small-bowel enteropathy caused by a permanent intolerance to wheat gluten and related proteins in rye and barley. Even though the disease originate from the small intestine the clinical symptoms varies in affected individuals and are often different in small children compared to adolescents and adults. Susceptibility to develop the disease is strongly associated with certain genetic factors i.e. HLADQ2/DQ8 but it is undoubtedly that additional inherited and environmental factors are involved. As specific T lymphocyte reactions are central in the pathogenesis of CD, six key cytokine messenger RNA levels in intestinal intraepithelial and lamina propria T lymphocytes (IEL, LPL), retrieved from small intestinal biopsies, were determined by using quantitative real-time reverse transcription polymerase chain reaction (RTPCR). Levels of cytokines, small secreted proteins which mediate and regulate immunity, in children with active disease were compared with that of treated children and controls. Interferon (IFN)-γ and interleukin (IL)-10 were also determined at the protein level by immunohistochemistry. Active celiac disease was characterized by distortions in cytokine expression, with highly significant increases of IFN-γ and IL-10 but no concomitant increases in tumor necrosis factor α (TNF-α), transforming growth factor β1 (TGFβ1), or IL-2 and no induction of IL-4. A marked shift of IFN-γ and IL-10 production from LPLs to IELs was characteristic of active celiac disease, and as many as one fourth of the IELs expressed IFN-γ. IELs in treated, symptom-free celiac patients still had increased IFN-γ levels compared with controls. In CD, gluten intake seems to cause an overreaction in IELs, with uncontrolled production of IFN-γ and IL-10 which may cause both recruitment of more IELs and a leaky epithelium, leading to a vicious circle with amplified immune activity and establishment of the intestinal lesion. In order to determine different IEL subsets contribution of the produced cytokines, γδIELs, CD4+αβIELs, and CD8+αβIELs as well as CD94+CD8+αβIELs and CD94CD8+αβIELs of children with active CD and children with no food-intolerance were analyzed for cytokine mRNA expression levels by RT-PCR. In active CD, CD8+αβIELs had the highest expression levels of IFN-γ- and IL-10 mRNA and constituted the cellular source for almost all IFN-γ and a large fraction of the IL-10. Expression levels of these two cytokines correlated and were higher in CD94-CD8+αβIELs than CD94+CD8+αβIELs CD4+αβIELs had the highest expression levels of TNF-α and despite the small number of this cell subset they contributed with half of the small amounts of this cytokine. Interestingly, TNF-α levels correlated with IL-10 in CD4+αβIELs. γδIELs had the lowest expression levels of IFN-γ, TNF-α, IL-10, and TGF-β1. Essentially no IL-2 mRNA was detected in the three IEL subpopulations. “Classical” CD8+CD94-αβT cells in the epithelial compartment are responsible for most of the excessive production of proinflammatory IFN-γ. The question whether an impaired extrathymic T cell maturation and/or capacity for secondary T cell receptor (TCR) gene recombination in iIELs is a contributing factor to CD was addressed. Expression levels of recombination activating gene-1 (RAG1) and the pre T α-chain (preTα) mRNAs were determined in IEL T cell lineage subsets of children with CD and controls. In controls, RAG1 was expressed in both mature (TCRγδ+ and TCRαβ+) and immature (CD2+CD7+TCR-) IELs while preTα was expressed preferentially in immature IELs. The RAG1 splice form selectively expressed outside thymus (RAG1 1A/2) as well as preTα were significantly decreased in CD patients both in active and inactive disease suggesting a deteriorated capacity of de novo TCR gene rearrangement in local T cell development and / or of secondary TCR gene rearrangement during editing or antigen-driven revision. This may lead to an imbalance between thymus- and gut derived T lymphocytes in the intestinal mucosa with consequent inefficient regulation of T cell responses against food antigens. Innate or nonspecific immunity is the first line, immediate defense against pathogens mediated by the epithelial cells in the intestine (IECs). As certain adaptive immune reaction in CD mimics that of intestinal infections, aberrant innate immune reaction could be a contributing factor to CD. Therefore jejunal biopsies were screened for bacteria and the innate immune status of the epithelium was investigated. Bacteria were freqently (40%) associated with the mucosa of children with active but also treated disease (20%) compared to controls (2%). Lack of antimicrobial factors such as mucins, proteins forming protective biofilm on the IECs, defensins and lysozym, peptides and enzymes with antibacterial effects, could not explain the presence of bacteria. If anything, mucin-2 (MUC2), α-defensins, HD-5, HD-6, and lysozyme mRNA levels were increased in epithelial cells in active CD, returning to normal levels in treated CD. Their expression levels correlated to the IFN-γ mRNA levels in IELs. Analysis of beta defensins, hBD-1 and hBD-2 as well as carcinoembryonic antigen (CEA) cell adhesion molecule 1a (CECAM1a), glycoproteins in the glycocalyx with ability to bind micro organisms, were not affected by the disease. Lectin staining by histochemistry revealed that goblet cells were stained by UEA1 in CD both active and treated but not in controls. The opposite pattern was seen for the lectin PNA where staining was seen in controls in the glycocalyx layer but not in CD. Thus altered glycocalyx/mucous layer may promote bacterial adhesion in CD.
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| 5. |
- Ohlsson, Lina, 1981-
(författare)
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Biomarker mRNAs for staging and prognosis of colorectal cancer
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mesenteric lymph node (ln) metastasis is the single most important prognostic characteristic in colorectal cancer (CRC). The ln status is used for staging and is a decisive selection criterion for postoperative adjuvant therapy. However, it is difficult to accurately determine ln status by routine histopathology (H&E). Thus, ~25% of CRC patients, who by H&E are considered to lack tumor cells in their lns, i.e. stage I+II, die from CRC.To explore the utility of biomarker mRNA analysis for staging and prognosis of CRC, lns were collected at surgery and mRNA levels for fourteen biomarkers, including carcinoembryonic antigen (CEA), kallikrein 6 (KLK6), cytokeratin 20 (CK20), guanylyl cyclase C (GCC), CEACAM1-S, CEACAM6 and mucin 2 (MUC2), were determined by quantitative RT-PCR with RNA copy standards. Results were compared to routine H&E analysis.The biomarkers were analyzed for capacity to detect disseminated tumor cells in lns. mRNA levels were determined in CRC- and control lns, primary tumor, normal colon, immune cells and fibroblasts. Lack of expression in immune cells and fibroblasts and high and homogenous expression in primary tumors showed to be the determining factors. CEA fulfilled these criteria best, followed by KLK6, CK20, GCC, and MUC2.Utility of the biomarker mRNAs for staging and prognosis was examined in 174 CRC patients. CEA was the best predictor of disease-free survival time after surgery with a 71 months difference between CEA(+) and CEA(-) patients and a hazard ratio of 5.1 for risk of recurrence for CEA(+) patients. CEA, CK20 and MUC2 were more sensitive than H&E in that these biomarkers identified patients who succumbed from recurrent CRC although H&E analysis had failed to detect the disseminated tumor cells. Combined analysis of CEA and MUC2 mRNAs improved prediction of outcome. Patients with high risk for recurrence had low MUC2/CEA ratios.KLK6 mRNA was identified as a potential progression marker by genome-wide microarray analysis of gene expression. It was found to be ectopically expressed in CRC tumor cells. KLK6(+) lns was an indicator of poor prognosis (hazard ratio 3.7). Notably, the actual level was of importance for outcome. The higher the KLK6 mRNA levels the greater the risk of recurrence. At the 90thpercentile the hazard risk ratio for KLK6(+) patients was 5.6. KLK6 positivity in lns with low numbers of tumor cells, as indicated by low CEA mRNA levels, indicated poor prognosis (hazard ratio 2.8). Thus, KLK6 adds prognostic information to CEA analysis.Increased levels of mRNA for the proinflammatory cytokine interferon- and the down-regulatory cytokine interleukin-10 in lns of CRC patients suggested ongoing immune reactions against the infiltrating tumor cells. Elevated TGF-1 levels correlated weakly with survival, suggesting protection by the antiproliferative effect of TGF-1 in sporadic cases.CEA mRNA was the best single biomarker for staging and prediction of disease-free survival time and risk of recurrence after surgery. In addition to CEA, KLK6 positivity and low MUC2/CEA ratio correlate with poor prognosis. Thus, CEA, MUC2 and KLK6 mRNAs form a strong "trio" for staging and prediction of outcome for CRC patients.
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| 6. |
- Ou, Gangwei, 1958-
(författare)
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Human intestinal epithelial cells in innate immunity : interactions with normal microbiota and pathogenic bacteria
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Rod-shaped bacteria were previously shown to be associated with the small intestinal epithelium of children with celiac disease (CD). Using culture-dependent and independent methods, we characterized the microbiota of small intestine in children with CD and controls. The normal microbiota constitutes an unique organ-specific biofilm. Dominant bacteria are Streptococcus, Neisseria, Veillonella, Gemella, Actinomyces, Rothia and Haemophilus. Altogether 162 Genus Level Operational Taxonomic Units (GELOTU) of six different phyla were identified in a total of 63 children. In biopsies collected during 2004- 2007 we did not find major differences in the microbiota between CD patients and controls. However, in biopsies collected earlier from children born during the “Swedish CD epidemic” and demonstrated to have rod-shaped bacteria by electron microscopy, we found that unclassified-Clostridales and Prevotella species were associated with CD. These anaerobic, rod-shaped bacteria showed marked affinity for the intestinal epithelium. Changes in breast-feeding practice and/or regiments for introduction of gluten containing food probably affect the composition of the bacterial flora in small intestine. We hypotesize that these bacteria contribute to contraction of CD.An in vitro model for studies of immune mechanisms of the intestinal epithelium was established. Polarized tight monolayers of the human colon carcinoma cell lines, T84 and Caco2, were developed by culture in a two-chamber system. The two cell lines showed the features of mature- and immature columnar epithelial cells respectively. Polarized monolayers were challenged with bacteria and proinflammatory cytokines. Immune responses were estimated as quantitative changes in mRNA expression levels of a secreted mucin (MUC2), glycocalyx components (CEACAMs, MUC3), antimicrobial factors and cytokines (IFN-g, TNF-a, IL-6 and IL-8). Tight monolayer cells were more resistant to bacterial attack than ordinary tissue culture cells and only B. megaterium induced the defensin, hBD2. Tight monolayer cells responded to cytokine challenge suggesting awareness of basolateral attack. TNF-a induced markedly increased levels of IL-8 and TNF- a itself in both cell lines suggesting recruitment and activation of immune cells. Cytokine challenge also increased levels of CEACAM1, which includes two functionally different forms, CEACAM1-L and CEACAM1-S. In T84 cells, IFN-g was selective for CEACAM1- L while TNF-a upregulated both forms. Increased CEACAM1 expression may influence epithelial function and communication between epithelial cells and intraepithelial lymphocytes.As a pathogenic enteric bacterium, Vibrio cholerae secretes cholera toxin that is the major factor of cholera diarrhea. However, some strains of O1 serogroup lacking the cholera toxin still cause enterocolitis and most V. cholerae vaccines candidates exhibit reactogenicity in clinical trails. An extracellular metalloprotease PrtV was characterized. It was associated with killing of bacteria predators such as the nematode Caenorhabditis elegans. Its role in human intestine was addressed by using the T84 tight monolayer in vitro model. We found that Vibrio Cholera Cytolysin (VCC), a pore-forming toxin, induces an inflammatory response in intestinal epithelial cells that includes increased epithelial permeability and induction of IL-8 and TNF-a and hence could be responsible for enterocolitis. The inflammatory response was abolished by PrtV thus VCC is indeed an autologous substrate for PrtV. In protein rich environment PrtV degradation of VCC was inhibited, suggesting that the magnitude of the inflammatory response is modulated by the milieu in the small intestine. Thus, VCC is likely to be part of the pathogenesis of cholera diarrhea and the causative agent of enteropathy in V. cholerae strains lacking the cholera toxin.
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| 7. |
- Sjöberg, Veronika, 1980-
(författare)
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Immune response of the small intestinal mucosa in children with celiac disease : impact of two environmental factors, resident microbiota and oats
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Celiac disease (CD) is an immune-mediated enteropathy caused by permanent intolerance to dietary gliadin in wheat gluten and related prolamines in barley and rye. The pathogenesis of CD is still unknown and several different environmental factors have been associated with CD, such as dysbiosis of the microflora. In this translational study we investigated the immune status and the interplay of T-cells and Tregs in the mucosa of children with CD and controls, as well as the immune status in treated CD patients, provoked by either dietary oats, CD associated bacteria or gluten.The major findings in the studies were: First, indicators of extrathymic T-cells maturation (ETCM), i.e., the RAG1 enzyme required for recombination of the T cell receptor (TCR) genes and the preTα-surrogate chain in the immature TCR, were both expressed at lower levels in CD patients compared to controls. In addition, IELs expressing RAG1 were less abundant in CD patients compared to controls. The levels of these two indicators stayed low in treated CD patients as well, suggesting that impaired capacity of ETCM is an inherent feature of CD patients. Second, IL-17A, a cytokine involved in both inflammation and anti-bacterial responses was increased in active CD. The major cellular source was CD8+IELs. Furthermore, ex vivo challenge of biopsies from treated CD patients with gluten and with CD-associated bacteria induced an IL-17A response. The CD-associated bacteria also influenced the magnitude of the IL-17A response to gluten. Third, we investigated the effect of dietary oats on local immune status in the intestinal mucosa by comparing CD patients receiving GFD with and without oats. 22 different mRNAs for immunity effector molecules and tight junction proteins were analyzed. We found that expression of two down-regulatory cytokines, two activating NK-receptors and the tight-junction protein claudin-4 normalized in patients on a standard GFD while they did not normalize in patients on a GFD with oats. Fourth, we analyzed the expression level of mRNAs for chemokines, cytotoxic effector molecules, NK-receptors and their ligands in IELs and epithelial cells. Expression levels of several of these genes follow disease activity, suggesting massive recruitment of immune cells by both cell types accompanied by increased IEL-mediated cytotoxicity in the epithelium of inflamed mucosa.In this thesis we have identified three potential risk factors for development of CD: 1) an inherent lower level of ETCM in the small intestinal mucosa than in controls. This could lead to decreased generation of regulatory T cells and less capacity to tolerate gluten and adapt to the local milieu in the mucosa. 2) Dysbiosis of the resident microbiota with increased IL-17A production that could promote local inflammation and immune cell infiltration as well as antibacterial reactions. 3) Dietary oats may provoke a local immune response in a sub-population of CD patients. These patients should probably avoid oats in their GFD but larger studies are needed.
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| 8. |
- West, Christina, 1969-
(författare)
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Feeding Lactobacillus paracasei ssp. paracasei strain F19 to infants during weaning : effects on adaptive immunity and gut microbial function
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Introduction: Gut microbial composition has been associated with immune-mediated diseases. Breastfeeding yields a microbiota rich in bifidobacteria and promotes colonization by lactobacilli. Bifidobacteria and lactobacilli are considered health-promoting and are used as probiotics, i.e. live microbial food supplements which when ingested in adequate amounts confer a beneficial effect on the host. During weaning the developing gut immune system is exposed to an increasing variety of antigens from both foods and gut microbiota.Aims: We aimed to determine if daily feeding of 1x108 colony-forming units (CFU) of the probiotic Lactobacillus paracasei ssp. paracasei strain F19 (LF19) to healthy term infants from 4 to 13 months of age could maintain some of the beneficial effects conferred by breastfeeding on gut microbial composition, with possible effects on gut microbial function, T cell function, Th1/Th2 immune balance and eczema incidence.Study design: Infants were randomized to daily intake of cereals with (n=89) or without LF19 (n=90) from 4-13 months of age. Clinical outcome measures were monitored by diaries and a questionnaire. Stool and blood samples were obtained at 4, 6½, 9, 13 and 5½, 6½, 12 and 13 months of age, respectively. Stool samples were analyzed for lactobacilli counts by conventional culture methods and the presence of LF19 was verified by randomly amplified polymerase chain reaction (RAPD-PCR). Fecal short-chain fatty acid (SCFA) pattern, a proxy for gut microbial function, was determined by gas-liquid chromatography. After polyclonal or specific activation of T cells, the cytokine mRNA expression levels [interleukin 2 (IL2), IFN-, IL4 and IL10] were determined on isolated mRNA by quantitative real time reverse transcriptase-PCR. Serum concentrations of total and specific IgE antibodies, Haemophilus influenzae type b, diphtheria and tetanus toxoid specific IgG antibodies were analyzed by enzyme immunoassay.Results: Feeding LF19 maintained high fecal lactobacilli counts during weaning. Persistent colonization with LF19 induced differences in the fecal SCFA pattern. The cumulative incidence of eczema was lower in the probiotic group, in conjunction with a higher IFN-γ/IL4 mRNA ratio in polyclonally activated T cells. Even though there was an effect by LF19 on Th1/Th2 immune balance, there was no effect on IgE sensitization. Infants in both groups increased their capacity to express both Th1 and Th2 cytokines during the second half of infancy but the expression was still lower than that of adults. Infants in the probiotic group had lower IL2 levels after polyclonal T cell activation at 13 months of age compared with infants in the placebo group. Infants fed LF19 did not have fewer infections, but had fewer days with antibiotic prescription compared with infants fed placebo. In addition, compared to placebo, persistent colonization by LF19 enhanced specific vaccine responses to protein antigens during the course of vaccination.Conclusions: We conclude that feeding LF19 was safe, based on no observed adverse effects in our study. Infants in both groups demonstrated maturation of adaptive immune responses during weaning. Adding probiotics in complementary foods during weaning reduced the risk of eczema by 50%, with a concomitant shift towards an enhanced Th1/Th2 ratio. The reduction of eczema might be explained by probiotic effects on both T cell-mediated immune responses and reinforced gut microbial function.
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