| 1. |
- Bas, Anna, 1973-
(author)
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Extrathymic T cell receptor gene rearrangement in human alimentary tract
- 2003
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Doctoral thesis (other academic/artistic)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. |
- Fahlgren, Anna, 1972-
(author)
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Defence capabilities of human intestinal epithelial cells
- 2003
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Doctoral thesis (other academic/artistic)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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| 3. |
- Ou, Gangwei, 1958-
(author)
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Human intestinal epithelial cells in innate immunity : interactions with normal microbiota and pathogenic bacteria
- 2009
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Doctoral thesis (other academic/artistic)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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| 4. |
- Bitar, Aziz, 1984-
(author)
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Vibrio cholerae modulates the immune defense of human gut mucosa
- 2018
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Doctoral thesis (other academic/artistic)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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| 5. |
- Lundqvist, Carina
(author)
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Human intraepithelial lymphocytes : a comparative study of phenotype, morphology, and functional properties of intraepithelial lymphocytes in gut and oral mucosa
- 1995
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Doctoral thesis (other academic/artistic)abstract
- Human intraepithelial lymphocytes (IEL) constitute a unique cell population situated in the first line of defense of the alimentary tract. Here they are continuously exposed to a massive antigenic load of high complexity. However, different conditions prevail along the alimentary tract. In small intestine food antigens dominate whereas bacterial antigens are abundant in large intestine. The oral cavity is exposed to an enormous variety of antigens from the microflora as well as food constituents. The abundance and selective localization of lymphocytes in the surface epithelium of these challenged tissues implicate important roles for IEL in immune protection.IEL in normal human jejunum, ileum and colon as well as in normal and chronically inflamed gingiva were studied in situ and after isolation, with regard to phenotype, ultrastructure, cytokine mRNA expression and response to T-cell mitogens. Furthermore, an isolation technique was developed which yielded highly purified, functionally active IEL and enterocytes from the same sample.Intestinal IEL were situated in the basal part of the epithelium, often in small clusters and in close contact with adjacent lymphocytes and epithelial cells. They had an irregular shape with long processes and some had pseudopodium-like extensions penetrating the basement membrane. This indicates cell co-operation within the epithelium, as well as transmigration of IEL to underlying tissues. Freshly isolated IEL expressed several cytokines (IL-1β, IL-8, IL-2, TNF-α and IFN-γ) and in vitro activation induced expression of IL-2, IL-10, IFN-γ, TNF-α, TNF-β and TGF-β1, suggesting that IEL are involved in cell mediated cytotoxicity and suppressor cell activities.γδ T cells showed preferential homing to the epithelium both in gingiva and in intestine. They constituted the major lymphocyte population in normal gingiva and on average 30% of IEL at all levels of the intestine. Gingival as well as intestinal γδ IEL showed preferential usage of Vδ1Vγ8, suggesting common reactivity patterns along the alimentary tract. Intestinal γδ IEL and γδ IEL in normal gingiva were CD4-CD8-. In contrast, γδ IEL in chronically inflamed gingiva were predominantly CD8+ and showed induced expression of CD45RO. This indicates that γδ IEL participate in anti-bacterial immune responses in mucosa. Intestinal and gingival γδ IEL displayed ultrastructural features of cytolytic effector cells, e.g. electron-dense cytoplasmic granules and multivesicular bodies. They also expressed cytokines indicative of cell mediated-cytolytic effector functions. γδ IEL from inflamed gingiva expressed IFN-γ, TNF-α, TGF-β1 and IL-6 mRNA while intestinal γδ IEL expressed IL-2, IFN-γ and TNF-α.Intraepithelial αβ T cells were rare in gingiva while they constituted the major population of intestinal IEL. The phenotype of αβ IEL varied at different levels of the intestine. Thus, CD8+αβ IEL dominated in jejunum while cells with the unusual T-cell phenotype, CD4-CD8- TCR αβ+, constituted a major population of colonic IEL. CD4+ αβ IEL were equally represented, as a minor population, at all three levels of the gut. Intestinal αβ IEL had the same cytokine profile as γδ IEL. Taken together, these data suggest that αβ IEL are involved in immunoregulatory responses to luminal antigens.IEL with thymocyte-like phenotyped (CD2+TCR/CD3-, CD1+TCR/CD3-, CD1+TCRαβ+ and CD1+TCRγδ+) were present in jejunal epithelium. Furthermore, recombination activating gene-1 (RAG-1) mRNA was expressed in CD2+TCR/CD3- and CD3+/TCR- jejunal IEL. RAG-1 was not expressed in colonic IEL. Thus, the epithelium of small intestine is a site for extrathymic T cell maturation in humans.
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| 6. |
- Sjöberg, Veronika, 1980-
(author)
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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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Doctoral thesis (other academic/artistic)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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| 7. |
- West, Christina, 1969-
(author)
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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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Doctoral thesis (other academic/artistic)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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| 8. |
- Ohlsson, Lina, 1981-
(author)
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Biomarker mRNAs for staging and prognosis of colorectal cancer
- 2011
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Doctoral thesis (other academic/artistic)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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| 9. |
- Pietz, Grzegorz, 1983-
(author)
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Innate immunity of human intestinal epithelium in childhood celiac disease : influences from celiac disease associated bacteria and dietary oats
- 2017
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Doctoral thesis (other academic/artistic)abstract
- Background & Aims: Celiac disease (CD) is a chronic inflammatory small-bowel enteropathy caused by permanent intolerance to gliadin in wheat gluten, and related proteins in ray and barley. It is disputed whether CD patients tolerate oats. The only treatment of CD is lifelong gluten-free diet (GFD). Only individuals that carry the HLA-DQ2 and/or DQ8 alleles, and eat gluten can develop CD. Dysbiosis in the gut microbiota is a suggested risk factor for CD. T cells in small intestinal mucosa, including intraepithelial lymphocytes (IELs), are known to be important in the pathogenesis of CD. In contrast, the role of intestinal epithelial cells (IECs) is poorly understood. In this thesis we investigated the role of IECs in the immune pathology of CD from duodenal mucosa of children with CD, clinical controls and treated CD. We also investigated the role of CD associated bacteria and oats supplemented GFD on the mucosal immune system.Results: A new CD-associated bacterium, Prevotella jejuni, was isolated and characterized. It is a saccharolytic and proteolytic anaerobe. More than 25 defense-related genes, including IRF1, SPINK4, ITLN1, OAS2, CIITA, HLA-DMB, HLA-DOB, PSMB9, TAP1, BTN3A1, and CX3CL1, were upregulated in IECs in active CD. In two in vitro models for intestinal epithelium, small intestine enteroids and T84 polarized tight monolayers, we showed that 70% of these genes were upregulated by interferon (IFN)-γ via the IRF1 pathway. IRF1 was also upregulated by the CD-associated bacteria P. jejuni and Actinomyces gravenitzii. IECs expressed the NLRP6/8 inflammasome yielding CASP1 and biologically active interleukin (IL)-18, which induces IFN-γ in IELs. P. jejuni bound the intestinal epithelial cell lines T84, Caco2, HT29, and INT407, while Lachnoanaerobaculum umeaense preferentially bound Caco2. P. jejuni caused decreased transepithelial resistance over tight monolayers, while L. umeaense caused an increase. P. jejuni upregulated mRNAs for the detoxification molecules CYP1A1, CYP1A2, CYP1B1, and TIPARP, the chemokines CX3CL1, CXCL1, and CXCL10, the sialyltranserase ST3GAL4, and the inflammation promoting protein S100A3 in tight monolayers. L. umeaense upregulated the chemokines CCL20 and CXCL10, and down-regulated TLR2. In a randomized, double-blinded intervention trial comparing two study-groups, standard GFD and oat-containing GFD, we found that mRNAs for several immune effector molecules and tight junction proteins were only reduced in patients receiving GFD, but not in a substantial fraction of patients on GFD with oats. The down-regulatory cytokines IL-10 and TGF-β1, the cytotoxicity-activating NK-receptors NKG2C and NKG2E, and the tight junction protein claudin-4 remained elevated in the study group on GFD with oats.Conclusions: IECs are far from inactive in CD. A key factor in the epithelial reaction in CD appears to be over-expression of IRF1 in IECs. Dual activation of IRF1 and IRF1-regulated genes, both directly by P. jejuni and indirectly by IFN-γ via the IL-18-inflammasome, would drastically enhance the inflammatory response and lead to the pathological situation seen in active CD. P. jejuni harms the intestinal epithelium, i.e., it is a likely risk factor for CD, while L. umeaense strengthen barrier function and local immunity, possibly acting as a protective. A fraction of CD patients should avoid oats in the diet.
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| 10. |
- Yeung, Moorix Mo-Wai
(author)
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Specific and nonspecific immune mechanisms in human gut : a comparative study of normal and ulcerative colitis intestine
- 1996
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Doctoral thesis (other academic/artistic)abstract
- The intestine, with its large mucosal surface area, digests and absorbs food nutrients and maintains a beneficial microbial flora in the colon. Local protective immune responses against intestinal pathogens ensure the survival of the individual. These immune reactions are both specific and non-specific in nature. The intestinal epithelium is single-layered and constantly renewed with differentiating epithelial cells moving from the crypt to the luminal surface. Intraepithelial lymphocytes (IEL) are interspersed between the epithelial cells. Ulcerative colitis (UC) is a life-threatening chronic inflammation affecting colon.In this study three molecules belonging to the carcinoembryonic antigen (CEA) family, namely CEA proper, nonspecific cross-reacting antigen 50/90 (NCA 50/90) and biliary glycoprotein (BGP), were found to be specifically localized to the apical surface of colonic epithelial cells. Full expression of these molecules occurs when the cells reach the upper 1/3 of the crypts and are maintained on the mature cells at the luminal surface. Ultrastructurally, CEA, NCA50/90 and BGP are localized to microvesicles and microfilaments of the fuzzy coat/glycocalyx as well as to the microvilli of the epithelial cells. Their unique localization and documented bacterial binding capacities suggest that they have a role in innate immunity.Functional analysis of IEL in normal jejunum, ileum and colon revealed that IEL are in vivo activated T-lymphocytes expressing mRNA for the cytokines IL-1β, IL-2, IL-8, IFNγ and TNFα and that jejunal IEL have T-cell receptor (TCR)/CD3 dependent cytolytic capacity. As many as 10% of IEL actively produce IFNγ. CD4+TCRαβ+IEL, CD8+TCRαβ+IEL and CD4-CD8-TCRαβ+IEL all had a TH1/cytotoxic cytokine profile. IEL could be further stimulated in vitro to express IL-10, TNFβ and TGFβ1, to proliferate and to secrete IFNγ. Thus, active protection and/or regulation of the epithelium via cell-mediated immune reactions are prominent in the gut.UC colon was characterized by a marked lymphocyte infiltration in the lamina propria, 10-50 times the normal level. Most lymphocytes were present in follicle-like cell aggregates containing both T- and B-cells. An unexpected finding was that γδT-cells constituted about 15% of the cells in the aggregates. Such cells are only found intraepithelially in normal gut. γδT-cells of both the intestinal- (TCR-Vδ1/Vγ8) and blood type (TCR-Vδ2/Vγ9) were seen. T-cells in UC colon were activated but nonproliferating and had a down-regulated TCR/CD3 complex. RT-PCR and quantitative immunohistochemistry for cytokine mRNA (n=11) and protein respectively, revealed that the T-cells in UC colon did not produce IL-2, in marked contrast to T-cells in normal colon and to ileal T-cells from UC patients. This was a selective defect since TNFα, IFNγ, IL-1β, IL-8 and TGFβ1 were similarly expressed in normal colon. No TH2 cytokines were seen. Lamina propria leukocytes in UC colon expressed IL-6, a cytokine not found in normal colon. The epithelial cells of UC colon were activated expressing MHC class II antigens, heat shock proteins and the co-stimulatory molecule B7.1/CD80.Our study demonstrates that UC is an immunological disease. The immunopathological picture seen in UC colon probably reflects an inappropriate down-regulation of local immune responses perhaps due to a selective loss of the key cytokine IL-2 in a situation of extreme antigenic stress.
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| 11. |
- Forsberg, Göte, 1957-
(author)
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Innate and adaptive immunity in childhood celiac disease
- 2006
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Doctoral thesis (other academic/artistic)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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| 12. |
- Hollén, Elisabet, 1966-
(author)
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Coeliac Disease in Childhood : On the Intestinal Mucosa and the Use of Oats
- 2006
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Doctoral thesis (other academic/artistic)abstract
- Celiaki, eller glutenintolerans, är en av våra vanligaste kroniska sjukdomar i barnaåren. Sjukdomen orsakar en kraftig inflammation i tunntarmens slemhinna efter intag av glutenhaltig föda hos personer med ärftlig benägenhet att utveckla celiaki. En frisk tarm är kraftigt veckad för att öka ytan för upptag av näringsämnen. Ytan består dessutom av åtskilliga fingerliknande utskott, s.k. villi, och mellan villi finns kryptorna där celldelning och celldifferentiering sker. Villi och kryptor kantas av epitelceller, enterocyter, vilkas uppgift är att ta upp näring från tarminnehållet samt att utgöra en selektiv barriär mellan den yttre och inre miljön i tarmen. Den typiska tarmskadan vid celiaki karakteriseras av avsaknad av villi och kraftigt förlängda kryptor, och både näringsupptaget och barriärfunktionen är dessutom störda. Den enda behandling som finns att tillgå vid celiaki är en livslång glutenfri diet. De skadliga proteinerna i vetegluten kallas gliadin, och det finns liknande proteiner i råg, korn, och havre. I havre kallas proteinet avenin. Möjligheten att använda havre vid celiaki har diskuterats flitigt, men numera anses det riskfritt för majoriteten av både barn och vuxna att använda havre i den glutenfria dieten.Målet med den här avhandlingen var att undersöka hur barn med celiaki reagerar på havre i kosten. Detta studerades med avseende på antikroppar mot avenin samt med en metod som mäter halten av kväveoxid- (NO-) produkter i urinen. Ett andra mål var att studera tunntarmens struktur vid olika stadier av celiaki.I den första studien undersökte vi om celiakibarn har antikroppar i serum mot avenin. Vi fann att så var fallet och att nivåerna var signifikant högre än hos friska kontrollbarn. När barnen sattes på glutenfri kost sjönk antikroppsnivåerna, för att öka igen när gluten återinfördes i kosten. Blodproverna till den här studien togs innan debatten om havre kom igång, vilket gör att vi tror att de olika dieterna även speglar ett sant intag av havre. Studien visade också att det inte var någon korsreaktion mellan antikroppar mot avenin och gliadin.Vi använde sedan vår metod för att mäta antikroppar mot avenin i en randomiserad studie där havre gavs till barn med nydiagnostiserad celiaki. Barnen fick antingen en vanlig glutenfri diet eller en med tillsats av specialhavre. Antikroppsnivåerna sjönk markant redan efter tre månader i båda grupperna, och vid studietidens slut, efter ca ett år, hade alla utom ett par patienter återfått normala nivåer. Samma barn studerades även med avseende på NO-produkter i urinen. NO är en kortlivad molekyl som fungerar som budbärare i och mellan celler, och produktionen av den ökar markant vid en inflammation. Tidigare studier har visat att barn med obehandlad celiaki har extremt höga halter av NO-produkter i urinen. I vår studie sjönk även dessa värden signifikant efter tre månader, och det var ingen skillnad mellan grupperna. Efter ett år hade dock fyra barn i havregruppen och ett barn i den grupp som fick vanlig glutenfri kost, fortfarande extremt höga nivåer av NO-produkter.Dessa båda studier styrker den kliniska uppfattningen att de flesta barn med celiaki kan tåla havre, men de visar också att man bör följa upp de celiakibarn som kompletterar sin glutenfria kost med havre eftersom vissa barn verkar ha kvarstående tecken på inflammation i tarmen.I tarmbiopsier från barn med olika stadier av celiaki studerades förekomst och lokalisering av occludin och claudiner, proteiner som är viktiga för att upprätthålla barriärfunktionen i tarmen. Vi fann ett ökat uttryck av occludin vid obehandlad celiaki, vilket vi tror speglar den ökade celldelning och de förändrade barriäregenskaper som man ser vid aktiv celiaki. Resultaten tyder även på att uttrycket av claudin 1-5 inte tycks påverkas av kosten hos barn med celiaki.
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| 13. |
- Olofsson, Katarina
(author)
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Immune cells in human pharyngeal and palatine tonsils and in the uvula : tissue distribution, cellular composition and functional properties
- 1999
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Doctoral thesis (other academic/artistic)abstract
- The adenoid (pharyngeal tonsil), the palatine tonsils and the uvula are strategically located at the entrance of the upper airodigestive tract. By virtue of their location they are incessantly exposed to inhaled and ingested antigens. Severe nasal obstruction, otitis media with effusion, recurrent tinsillitis and obstructive airways during sleep are conditions often due to diseases in these organs.To advance our knowledge about the etiology and pathogenesis of these diseases, we have compared immune cell composition, cytokine expression and microbial colonisation in adenoids from children with hypertrophic obstructive adenoid (HOA) and chronically infected adenoid (CIA). Similarly, we compared immune cell composition in palatine tonsils from children with idiopathic tonsillar hypertrophy and recurrent tonsillitis. Finally, we wanted to characterise the human uvula from an immunological point of view, which had previously not been done. Its composition and distribution of immune cells, its cytokine profile, connective tissue elements and ultrastructure was studied.When comparing adenoids from children with HOA and CIA, the most striking finding was their similarity. A cytokine profile that was independent of diagnosis but seemed characteristic for the adenoid emerged. T cell expression of IL-5 and TGF-β1 but not IL-4 suggested an ongoing humoral response driven by a "mucosal TH2" cell. αβ T cells also expressed TNF-α, IFN-γ and IL-2, indicating a concomitant cell mediated response. Cell mediated immune responses often reflect viral infection. In line with this, adenovirus DNA was found in 80% of the adenoid samples. Furthermore, IL-6, IL-8 and TNF-α expressed in the non-T cell fraction suggested that the tissue macrophages were activated. TNF-α, IFN-γ and TGF-β1 were expressed by γδ T cells. The following differences between HOA and CIA were however, noted: i) most intraepithelial lymphocytes were CD8+ γδ T cells in HOA, while CD8+ αβ T cells dominated intraepithelially in CIA; ii) the number of follicles was twice as high in CIA as in HOA; iii) there were signifacantly more granulocytes in the interfollicular area in CIA than in HOA; iv)IL-6 mRNA expressing γδ T cells were only found in HOA and v) there was a tendency of higher TNF-α mRNA levels in non-T cells of CIA compared to HOA. The following scenarios emerge: in CIA there appears to be an inadequate first line of defence, with a low frequency of intraepithelial γδ T cells and a high frequency of cytotoxic CD8+ αβ T cells eliminating infected epithelial cells. Togehter, these two conditions cause a "leaky" epithelium, allowing infiltration of microbes into the underlying tissue and subsequent recruitment of granulocytes and follicle formation initiated by activated macrophages. In HOA, activated intraepithelial γδ T cells appear to be involved in antimicrobial defence reactions and surveillance of the epithelium.The difference in leukocyte profiles between tonsils from patients subjected to surgery due to idiopathic tonsillar hypertrophy or recurrent tonsillitis was limited to the surface epithelium. CD8+ γδ T cells utilising the unusual combination Vδ1/Vγ9 in their T cell receptor constituted the majority of intraepithelial lymphocytes in both groups. However, the frequency of these cells was significantly higher in recurrent tonsillitis. These results suggest that CD8+ Vδ1/Vγ9+ γδ T cells are characteristic of palatine tonsils and selectively expanded in recurrent tonsillitis. These γδ T cells may be involved in clearing infectious bacteria at the surface of the tonsil.Tissue macrophages, αβ T cells, γδ T cells, mast cells and B cells constituted, in declining order, the immune cell populations in the uvula. No fillicle-like structures were present. Most T cells had a CD8+ CD28-TCR-αβ+ phenotype, suggesting a down-regulatory function. Production of the down-regulatory cytokine TGF-β was also noted. This is consistent with the hypothesis that the uvula contributes to the development of mucosal tolerance. Furthermore, the uvula seems to be protected from pathogens penetrating the internal milieu by a subepithelial barrier of γδ T cells and macrophages. TNF-α secreting immune cells were found at this location. TNF-α and TGF-β may cause tissue fibrosis, TNF-α indirectly by stimulating mast cells to release histamine. Tissue fibrosis in conjunction with water binding to hyaluronan present in the connective tissue is the most likely explanation for the observed enlargement of the uvula in patients with sleeping disorders.
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