Temporal and long-term gut microbiota variation in allergic disease: A prospective study from infancy to school age.

BACKGROUND: Compositional changes in the early-life gut microbiota have been implicated in IgE-associated allergic diseases, but there is lack of longitudinal studies. We examined gut microbiota development from infancy to school age in relation to onset of IgE-associated allergic diseases. At 8 years of age, we also examined the relationship between gut microbiota and T-cell regulation, estimated as responses to polyclonal T-cell activation. METHODS: Stool samples were collected from 93 children at 4, 6, 13 months, and 8 years of age. The gut microbiota was profiled using 16S rRNA gene sequencing. Peripheral blood was drawn from all children, and mononuclear cells were polyclonally activated. Levels of IL-10 and FOXP3 mRNA copies were determined using real-time quantitative reverse transcriptase-PCR. RESULTS: At 8 years of age, 21 children were diagnosed with IgE-associated allergic disease and 90% displayed allergic comorbidity. Seventy-two children were nonallergic and nonsensitized. Statistical tests with multiple testing corrections demonstrated temporal underrepresentation of Ruminococcus and consistent underrepresentation of Bacteroides, Prevotella, and Coprococcus in allergic compared to nonallergic children from infancy to school age. The gut microbiota of the allergic 8-year-olds was enriched in Bifidobacterium and depleted of Lactobacillus, Enterococcus, and Lachnospira. In allergic 8-year-olds, Faecalibacterium correlated with IL-10 mRNA levels (rs  = 0.49, Padj  = 0.02) with the same trend for FOXP3 (rs  = 0.39, Padj  = 0.08). CONCLUSIONS: We identified both temporal and long-term variation in the differential abundance of specific bacterial genera in children developing IgE-associated allergic disease. Improved dietary interventions aiming at expanding immune-modulatory taxa could be studied for prevention of allergic disease.

Probiotics in primary prevention of allergic disease--follow-up at 8-9 years of age.

BACKGROUND: Long-term effects of probiotics in primary prevention of allergic disease need further evaluation. We previously reported a reduced cumulative incidence of infant eczema by feeding Lactobacillus paracasei ssp paracasei F19 (LF19) during weaning. Therefore, we assessed effects of LF19 on the prevalence of allergic disease at school age. METHODS: In a double-blind placebo-controlled trial infants were randomized to daily intake of cereals with (n = 89) or without LF19 10(8) CFU (n = 90) from 4-13 months of age. At age 8-9, we evaluated the prevalence of allergic disease (eczema, allergic rhinitis, asthma, and food allergy) by clinical examination and validated questionnaires. IgE sensitization was assessed by skin prick test (inhalant allergens) and specific IgE levels (food allergens). Lung function was evaluated by a spirometry reversibility test. Fractional exhaled nitric oxide (FENO ) was measured. RESULTS: Of 171 children that completed the intervention, 121 were assessed at age 8-9. In the probiotic group, 15/59 (25%) were diagnosed with any allergic disease vs 22/62 (35%) in the placebo group [OR (95% CI) 0.62 (0.28-1.36)]. Corresponding numbers for IgE-associated allergic disease were 9/53 (17%) vs 12/59 (20%) [0.80 (0.31-2.09)]. Median (25th-75th percentile) FENO was 9 (8-12) in the probiotic vs 8 (7-12) ppb in the placebo group (P > 0.05). There was no effect of LF19 on lung function measures (P > 0.05). CONCLUSIONS: There was no long-term effect of LF19 on any diagnosed allergic disease, airway inflammation or IgE sensitization. This suggests delayed eczema onset but to fully examine long-term benefits a larger study population had been needed.

Lymph node tissue kallikrein-related peptidase 6 mRNA: a progression marker for colorectal cancer.

BACKGROUND: A most important characteristic feature for poor prognosis in colorectal cancer (CRC) is the presence of lymph node metastasis. Determination of carcinoembryonic antigen (CEA) mRNA levels in lymph nodes has proven powerful for quantification of disseminated tumour cells. Here, we investigate the utility of human tissue kallikrein-related peptidase 6 (KLK6) mRNA as a progression biomarker to complement CEA mRNA, for improved selection of patients in need of adjuvant therapy and intensified follow-up after surgery. METHODS: Lymph nodes of pTNM stage I-IV CRC- (166 patients/503 lymph nodes) and control (23/108) patients were collected at surgery and analysed by quantitative RT-PCR. RESULTS: Lymph node KLK6 positivity was an indicator of poor outcome (hazard ratio 3.7). Risk of recurrence and cancer death increased with KLK6 lymph node levels. Patients with KLK6 lymph node levels above the 90th percentile had a hazard ratio of 6.5 and 76 months shorter average survival time compared to patients with KLK6 negative nodes. The KLK6 positivity in lymph nodes with few tumour cells, that is, low CEA mRNA levels, also indicated poor prognosis (hazard ratio 2.8). CONCLUSION: In CRC patients, lymph node KLK6 positivity indicated presence of aggressive tumour cells associated with poor prognosis and high risk of tumour recurrence.

Probiotic effects on T-cell maturation in infants during weaning.

BACKGROUND: We previously reported that feeding the probiotic Lactobacillus paracasei ssp. paracasei F19 (LF19) during weaning reduced the cumulative incidence of eczema. OBJECTIVE: To investigate the impact of feeding LF19 on T-cell maturation. METHODS: One hundred and seventy-nine healthy, term infants with no prior allergic manifestations were randomized to daily intake of cereals with (n = 89) or without (n = 90) the addition of LF19 10(8 ) colony forming units per serving from 4 to 13 months of age. Venous blood was drawn at 5.5 and 13 months of age. We used the cytokine response to polyclonal T-cell stimulation by anti-CD3 plus anti-CD28 monoclonal antibodies, and in vitro stimulation with the vaccine tetanus toxoid (TT) as measures of global adaptive immunity and capacity to raise a specific T-cell response, respectively. Expression levels of IL-2, IFN-γ, IL-4, IL-17A and IL-10 messenger RNAs (mRNAs) were used as proxies for general T-cell stimulation and naive Th0 cells, Th1-, Th2-, Th17- and T regulatory lineages. RESULTS: There was no difference between the two groups at 5.5 months of age. At 13 months, the polyclonal IL-2 response was higher in the placebo group (P < 0.05), whereas the IFN-γ/IL-2 (P < 0.01) and IL-17A/IL-2 (P < 0.05) ratios after polyclonal stimulation were higher in the probiotic group, as was the TT-specific IL17-A response (P < 0.001). In both groups, the IFN-γ and IL-4 responses increased from 5.5 to 13 months upon both polyclonal and specific stimulation (P < 0.01), whereas the IL-10 response remained low (P > 0.05). CONCLUSION AND CLINICAL RELEVANCE: Our findings suggest modest effects by probiotics on T-cell maturation following 9 months of probiotic intake. Future studies should address if specific probiotics may drive immune development with possible preventive effects on the development of allergic disease.

Chronic colitis induces expression of ß-defensins in murine intestinal epithelial cells.

Anti-microbial peptides are important effectors in innate immunity. In the gut they defend against pathogens, shape the commensal microbiota and probably control intestinal homeostasis. Ulcerative colitis (UC), but not Crohn's disease, shows increased expression of inducible ß-defensins (hBD-2, hBD-3 and hBD-4) in colonic epithelial cells. Does inducible defensin production precede the chronic intestinal inflammation characteristic of UC, or is it a consequence of the T cell-driven chronic inflammation? The aim was to analyse defensin mRNA and protein expression in colonic epithelial cells in two colitis mouse models resembling UC, the interleukin (IL)-2(-/-) mouse and the dextran sulphate sodium (DSS)-induced colitis mouse. Defensin mRNA was assayed by in situ hybridization and quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). Defensin peptide was assayed by immunohistochemistry. Mouse ß-defensin 3 (mBD-3, orthologue to hBD-2) was up-regulated strongly in colonic epithelium of 15-week-old IL-2(-/-) mice and DSS-induced colitis mice with chronic bowel inflammation, but not in apparently healthy IL-2(-/-) 5-week-old mice, IL-2(+/-) 15-week-old mice or in acute stage DSS mice. Up-regulation was seen both at the mRNA- and at the protein level (only mBD-3 investigated). IL-17, but not several other cytokines, including interferon (IFN)-γ, induced mBD-3 mRNA expression in mouse colon carcinoma cells. The mRNA expression level of the constitutively expressed α-defensin, cryptdin-4, was up-regulated marginally in acute stage DSS-colitis mice and in IL-2(-/-) mice before signs of colitis. Inducible ß-defensin expression in colonic epithelium is the consequence of the chronic bowel inflammation caused by activated T cells releasing cytokines including IL-17.

Contribution of intestinal epithelial cells to innate immunity of the human gut--studies on polarized monolayers of colon carcinoma cells.

The aim was to establish an in vitro model for studies of innate defence mechanisms of human intestinal epithelium. Ultrastructural characterization and determination of mRNA expression levels for apical glycocalyx and mucous components showed that polarized, tight monolayers of the colon carcinoma cell lines T84 and Caco2 acquire the features of mature- and immature columnar epithelial cells, respectively. Polarized monolayers were challenged with non-pathogenic Gram+ and Gram- bacteria from the apical side and the proinflammatory cytokines interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) from the basolateral side. Immune responses were estimated as changes in mRNA expression levels for the mucous component mucin-2 (MUC2), the glycocalyx components carcinoembryonic antigen (CEA), CEA-related cell adhesion molecule-1 (CEACAM1), CEACAM6, CEACAM7 and MUC3, the antimicrobial factors human beta-defensin-1 (hBD1), hBD2, hBD3 and lysozyme, the chemokine IL-8 and the cytokines IL-6 and TNF-alpha. Tight monolayer cells were generally unresponsive to bacterial challenge, but increased their hBD2 levels when challenged with Bacillus megaterium. T84 cells also increased their TNF-alpha levels upon bacterial challenge. Tight monolayer cells responded to cytokine challenge suggesting awareness of basolateral attack. TNF-alpha induced significantly increased levels of IL-8 and TNF-alpha itself in both cell lines suggesting recruitment and activation of immune cells in the underlying mucosa in vivo. Cytokine challenge also increased levels of CEACAM1, which includes two functionally different forms, CEACAM1-L and CEACAM1-S. In T84 cells, IFN-gamma was selective for CEACAM1-L while TNF-alpha upregulated both forms. Increased CEACAM1 expression may influence epithelial function and communication between epithelial cells and intraepithelial lymphocytes.

Aberrant extrathymic T cell receptor gene rearrangement in the small intestinal mucosa: a risk factor for coeliac disease?

BACKGROUND: Coeliac disease is a small intestine enteropathy caused by permanent intolerance to wheat gluten. Gluten intake by patients with coeliac disease provokes a strong reaction by intestinal intraepithelial lymphocytes (IELs), which normalises on a gluten-free diet. AIM: To investigate whether impaired extrathymic T cell maturation and/or secondary T cell receptor (TCR) gene recombination in IELs are features of coeliac disease which could contribute to the failure of establishing tolerance to gluten. METHODS: Expression levels of the four splice-forms of recombination activating gene-1 (RAG1) mRNA and preT alpha-chain (preTalpha) mRNA were determined in IEL-subsets of children with coeliac disease and controls. Frequencies of RAG1 expressing IELs were determined by immunomorphometry. RESULTS: In controls, the RAG1-1A/2 splice-form selectively expressed outside the thymus, was dominant and expressed in both mature (TCR(+)) and immature (CD2(+)CD7(+)TCR(-)) IELs ( approximately 8 mRNA copies/18S rRNA U). PreTalpha was expressed almost exclusively in CD2(+)CD7(+)TCR(-) IELs ( approximately 40 mRNA copies/18S rRNA U). By contrast, RAG1 and preTalpha mRNA levels were low in patients with coeliac disease compared to controls, both with active disease and with inactive, symptom-free disease on a gluten-free diet (p values <0.01 for mature and <0.05 for immature IELs). Similarly, the frequencies of RAG1+ IELs were significantly lower in patients with coeliac disease compared to controls (p<0.001). CONCLUSIONS: Patients with coeliac disease appear to have an impaired capacity for extrathymic TCR gene rearrangement. This is an inherent feature, which probably plays a pivotal role in the failure to efficiently downregulate the T cell response to gluten.

Basal lymphoid aggregates in ulcerative colitis colon: a site for regulatory T cell action.

Regulatory T cells seem to play a central role in maintaining immune tolerance in the gut mucosa. Previously we have shown that interleukin (IL)-10 is produced at high levels in the inflamed colonic tissue of ulcerative colitis (UC) patients. The cellular source was CD4+ T cells, suggesting local activation of regulatory T cells. The present study was performed to determine whether the frequency of regulatory T cells is increased in UC colon and whether they are present in the basal lymphoid aggregates, the prominent microanatomical structure in UC colon. Colonic tissue specimens from UC and control patients were analysed for frequencies of lamina propria lymphocytes expressing the regulatory T cell markers forkhead box protein 3 (FoxP3), CD25 and glucocorticoid-induced tumour necrosis factor receptor family-related gene (GITR) as well as CD28, CD4 and CD3 by using marker specific reagents in immunomorphometry. Two-colour immunohistochemistry was used for detection of CD25/IL-10, FoxP3/IL-10 and CD25/FoxP3 double-positive cells. GITR+ and FoxP3+ cells were present in normal colon mucosa, although at a relatively low frequency, and were located preferentially within the solitary follicles. UC was associated with significantly increased frequencies of CD25+, GITR+ and FoxP3+ lamina propria lymphocytes both within the basal lymphoid aggregates and in the lamina propria outside. Many of the CD25+ cells co-expressed FoxP3 as well as IL-10, suggesting that these are indeed IL-10 secreting regulatory T cells, activated in an attempt to counteract the inflammation. Increased frequency of regulatory T cell subtypes seems insufficient to control the disease activity in UC.

Biomarker selection for detection of occult tumour cells in lymph nodes of colorectal cancer patients using real-time quantitative RT-PCR.

Accurate identification of lymph node involvement is critical for successful treatment of patients with colorectal carcinoma (CRC). Real-time quantitative RT-PCR with a specific probe and RNA copy standard for biomarker mRNA has proven very powerful for detection of disseminated tumour cells. Which properties of biomarker mRNAs are important for identification of disseminated CRC cells? Seven biomarker candidates, CEA, CEACAM1-S/L, CEACAM6, CEACAM7-1/2, MUC2, MMP7 and CK20, were compared in a test-set of lymph nodes from 51 CRC patients (Dukes' A-D) and 10 controls. Normal colon epithelial cells, primary tumours, and different immune cells were also analysed. The biomarkers were ranked according to: (1) detection of haematoxylin/eosin positive nodes, (2) detection of Dukes' A and B patients, who developed metastases during a 54 months follow-up period and (3) identification of patients with Dukes' C and D tumours using the highest value of control nodes as cutoff. The following properties appear to be of importance; (a) no expression in immune cells, (b) relatively high and constant expression in tumour tissue irrespective of Dukes' stage and (c) no or weak downregulation in tumours compared to normal tissue. CEA fulfilled these criteria best, followed by CK20 and MUC2.

beta-Defensin-3 and -4 in intestinal epithelial cells display increased mRNA expression in ulcerative colitis.

mRNA expression of two recently described human beta-defensins (hBD-3 and hBD-4) in epithelial cells of normal small and large intestine and the impact of chronic intestinal inflammation on their expression levels was investigated. Intestinal specimens from patients with ulcerative colitis (UC), Crohn's disease (CD) and controls with no history of inflammatory bowel disease were studied. hBD-3 and hBD-4 mRNAs were determined in freshly isolated epithelial cells by real-time quantitative reverse transcription-polymerase chain reaction (QRT-PCR) and by in situ hybridization. The effect of proinflammatory cytokines on hBD-3 and hBD-4 mRNA expression in colon carcinoma cells was also investigated. Purified epithelial cells of normal small and large intestine expressed both hBD-3 and hBD-4 mRNA, with higher expression levels of hBD-3 mRNA. In situ hybridization revealed higher levels of mRNA expression in the crypt- compared to the villus/luminal-compartment. Interferon (IFN)-gamma, but not tumour necrosis factor (TNF)-alpha or IL-1beta, augmented hBD-3 mRNA expression. None of these agents stimulated hBD-4 expression. Colonic epithelial cells from patients with UC displayed a significant increase in hBD-3 and hBD-4 mRNA compared to epithelial cells of controls. In contrast, small intestinal epithelial cells from CD patients did not show increased expression levels compared to the corresponding control cells. Moreover, Crohn's colitis did not show increased expression of hBD-4 mRNA, while the data are inconclusive for hBD-3 mRNA. We conclude that the chronic inflammatory reaction induced in the colon of UC patients enhances hBD-3 and hBD-4 mRNA expression in the epithelium, whereas in CD this is less evident.

Cytolytic capabilities of lamina propria and intraepithelial lymphocytes in normal and chronically inflamed human intestine.

Cell-mediated lymphocyte cytotoxicity in ileum and colon of patients with ulcerative colitis (UC), Crohn's disease (CD) and controls was investigated. Frequencies of cells expressing perforin and Fas-ligand (FasL) were determined by immunomorphometry. mRNA expression of perforin, granzyme B and FasL in T cells and subsets was assayed by reverse transcriptase-polymerase chain reaction. Cytotoxicity of intraepithelial and lamina propria lymphocytes was analysed without ex vivo activation in three functional assays: (1) anti-CD3-dependent T-cell receptor (TCR)-/CD3-mediated redirected cytotoxicity, (2) Fas-/FasL-mediated TCR-/CD3-independent cytotoxicity and (3) natural killer (NK) cell cytotoxicity. Inflammation in ileum of CD patients caused increased frequency of perforin-expressing cells and enhanced perforin-dependent TCR-/CD3-mediated cytotoxicity. In contrast, lymphocytes in the inflamed colon of UC or Crohn's colitis patients did not display this cytotoxicity nor did lymphocytes of normal colon. Normal colon lymphocytes showed spontaneous Fas-/FasL-mediated cytotoxicity. This activity was retained but not enhanced in inflamed UC colon. In contrast, a significant increase of FasL-expressing cells was seen in situ. Inflammation did not induce NK cell activity in colonic lymphocytes. Intestinal lymphocytes comprise effectors active in two different cytolytic processes. 'Classical' cytotoxic T lymphocytes in small intestine and lymphocytes executing TCR-/CD3-independent FasL-/Fas-mediated killing of unknown biological role present throughout the intestinal mucosa. Ongoing normal cytolytic processes seem to be enhanced by chronic inflammation.

Utility of the housekeeping genes 18S rRNA, beta-actin and glyceraldehyde-3-phosphate-dehydrogenase for normalization in real-time quantitative reverse transcriptase-polymerase chain reaction analysis of gene expression in human T lymphocytes.

The accuracy of 18S rRNA, beta-actin mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as indicators of cell number when used for normalization in gene expression analysis of T lymphocytes at different activation stages was investigated. Quantitative real-time reverse transcriptase-polymerase chain reaction was used to determine the expression level of 18S rRNA, beta-actin mRNA, GAPDH mRNA and mRNA for six cytokines in carefully counted samples of resting human peripheral blood mononuclear cells (PBMCs), intestinal lymphocytes and PBMCs subjected to polyclonal T-cell activation. The 18S rRNA level in activated and resting PBMCs and intestinal lymphocytes was essentially the same, while the levels of beta-actin and GAPDH mRNAs fluctuated markedly upon activation. When isolated gammadeltaTCR(+), CD4(+) and CD8(+) subpopulations were studied, 18S rRNA levels remained unchanged after 21 h of activation but increased slightly after 96 h. In contrast, there was a 30-70-fold increase of GAPDH mRNA/cell in these cell populations upon activation. Cytokine analysis revealed that only normalization to 18S rRNA gave a result that satisfactorily reflected their mRNA expression levels per cell. In conclusion, 18S rRNA was the most stable housekeeping gene and hence superior for normalization in comparative analyses of mRNA expression levels in human T lymphocytes.

Interferon-gamma tempers the expression of carcinoembryonic antigen family molecules in human colon cells: a possible role in innate mucosal defence.

Four carcinoembryonic antigen-related cell adhesion molecule (CEACAM)s, i.e. CEA, CEACAM1, CEACAM6 and CEACAM7, are localized to the apical glycocalyx of normal colonic epithelium and have been suggested to play a role in innate immunity. The expression of these molecules in colon carcinoma cells was studied at the mRNA and protein levels after treatment with interferon-gamma (IFN-gamma), interleukin-1beta, live bacteria or lipopolysaccharide. The colon carcinoma cell lines LS174T and HT-29 were studied in detail using real-time quantitative reverse transcriptase-polymerase chain reaction, immunoflow cytometry and immunoelectron microscopy. IFN-gamma, but not the other agents, modified expression of CEA, CEACAM1 and CEACAM6. None of the agents upregulated CEACAM7 expression. Two expression patterns were seen. HT-29 cells, which initially showed low quantities of mRNAs and proteins, displayed marked upregulation of both mRNAs and proteins. LS174T cells transcribed stable high levels of mRNA before and after treatment. Additionally, IFN-gamma induced increased cell surface expression of CEA, CEACAM1 and CECAM6. IFN-gamma has two important effects on the expression levels of the CEA family molecules in colon epithelial cells: direct upregulation of CEACAM1 and promotion of cell differentiation resulting in increased expression of CEA and CEACAM6 and decreased expression of CEACAM7.

Over-expression of interleukin 10 in mucosal T cells of patients with active ulcerative colitis.

Ulcerative colitis (UC), a chronic inflammatory bowel disease, exhibits pronounced increase of T lymphocytes in the inflamed mucosa. To understand the role of intestinal T lymphocytes in the pathogenesis of UC their cytokine production in the mucosa was analysed. Intestinal T lymphocytes of UC, Crohn's disease and control patients were analysed for cytokine mRNA levels by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) directly after isolation without in vitro stimulation. Frequencies of cytokine positive cells were determined in UC and control colon by immunomorphometry. T lymphocytes in normal colon expressed interleukin (IL)-2, interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta1, but not IL-4, IL-5 or IL-10. In UC, a highly significant increase in IL-10 mRNA levels in T lymphocytes and an increased frequency of IL-10 positive cells was seen in colon. IL-10 mRNA levels were also elevated in T lymphocytes of the non-inflamed ileum and correlated with disease activity at both locations. CD4+ T lymphocytes were the major source of IL-10 mRNA. IL-2, IFN-gamma and TNF-alpha mRNA levels were decreased in colonic T lymphocytes, and virtually no IL-2, IFN-gamma, TNF-alpha or TGF-beta positive cells were detected in basal lymphoid aggregates. However, scattered IL-10 positive cells were found here. Lamina propria outside the aggregates contained IL-10-, IFN-gamma, TNF-alpha and TGF-beta but not IL-2 positive cells. T cells of UC patients did not express IL-4 or IL-5. Taken, together the data suggest a generalized activation of IL-10 producing CD4+ T cells along the intestine of UC patients. The local environment seems to determine the biological consequences of elevated IL-10.

Increased expression of antimicrobial peptides and lysozyme in colonic epithelial cells of patients with ulcerative colitis.

The impact of chronic inflammation on the expression of human alpha-defensins 5 and 6 (HD-5, HD-6), beta-defensins 1 and 2 (hBD-1, hBD-2) and lysozyme in epithelial cells of small and large intestine was investigated. Intestinal specimens from 16 patients with ulcerative colitis (UC), 14 patients with Crohn's disease (CD) and 40 controls with no history of inflammatory bowel disease were studied. mRNA expression levels of the five defence molecules were determined in freshly isolated epithelial cells by real-time quantitative RT-PCR. Specific copy standards were used allowing comparison between the expression levels of the different defensins. HD-5 and lysozyme protein expression was also studied by immunohistochemistry. Colonic epithelial cells from patients with UC displayed a significant increase of hBD-2, HD-5, HD-6 and lysozyme mRNA as compared to epithelial cells in controls. Lysozyme mRNA was expressed at very high average copy numbers followed by HD-5, HD-6, hBD-1 and hBD-2 mRNA. HD-5 and lysozyme protein was demonstrated in metaplastic Paneth-like cells in UC colon. There was no correlation between hBD-2 mRNA levels and HD-5 or HD-6 mRNA levels in colon epithelial cells of UC patients. Colonic epithelial cells of Crohn's colitis patients showed increased mRNA levels of HD-5 and lysozyme mRNA whereas ileal epithelial cells of Crohn's patients with ileo-caecal inflammation did not. Chronic inflammation in colon results in induction of hBD-2 and alpha-defensins and increased lysozyme expression. hBD-1 expression levels in colon remain unchanged in colitis. The high antimicrobial activity of epithelial cells in chronic colitis may be a consequence of changes in the epithelial lining, permitting adherence of both pathogenic bacteria and commensals directly to the epithelial cell surface.

Differential expression of vasoactive intestinal polypeptide receptor 1 and 2 mRNA in murine intestinal T lymphocyte subtypes.

Neuropeptides may exert a variety of effects on the immune cells at both systemic and mucosal immune sites. The immunoregulatory properties refer to the ability of physiological signals and pathways to influence various immune functions. Vasoactive intestinal polypeptide (VIP), a neuropeptide present in high concentration in gut, was studied for its production and receptor expression in intraepithelial and lamina propria T lymphocytes of mouse intestine. Using reverse transcription-polymerase chain reaction (RT-PCR) analysis, it was demonstrated that VIP receptor 1 (VIPR1) was constantly expressed in intraepithelial and lamina propria T lymphocytes from both small and large intestine. In contrast, VIPR2 was identified only in T cells from small intestine. Further studies on purified subpopulations of T lymphocytes indicated the existence of VIPR2 in CD8(+) T cells, but not CD4(+) and CD4CD8 double negative T cells, although all these three subpopulations displayed VIPR1. In addition, VIPR1 mRNA was detected in splenic T lymphocytes, but no signal was obtained for VIPR2 mRNA, even after stimulation of the cells with anti-CD3(epsilon)-chain mAb, phorbol 12-myristate 13-acetate (PMA) and/or VIP. The presence of VIP receptor(s) on intestinal T lymphocytes was supported by the detection of VIP on the cell surface using dual colour immunoflowcytometry. In-vitro treatment with VIP resulted in a tendency towards an increased size of the VIP immunoreactive T cell population and significantly enhanced the average immunofluorescence intensity of the surface labelling. This indicates that the receptors are partially occupied by locally produced VIP in vivo and that more peptide molecules can be bound on the lymphocytes when needed, released and accumulated in higher concentration at the action sites. We failed to detect the expression of VIP mRNA in T lymphocytes, from either intestine or spleen. These observations support that VIP may be an important immune modulator in gut acting through specific receptors on T lymphocytes. The differential mRNA expression of VIP receptor subtypes in cells with different phenotypes and in different immune compartments may suggest diverse regulatory roles of the neuropeptide in immune responses.

Both substance P and its receptor are expressed in mouse intestinal T lymphocytes.

Substance P (SP), one of the most prevalent neuropeptides in gut, has been reported to have potent immune modulatory effects as a proinflammatory agent. The synthesis of SP and SP receptor expression in intraepithelial and lamina propria T lymphocytes of mouse intestine was investigated. Using RT-PCR analysis, it was demonstrated that SP receptor mRNA was exclusively expressed in intraepithelial and lamina propria T lymphocytes as well as their purified CD4+, CD8+ and CD4-CD8-CD3+ subsets. Messenger RNAs (mRNAs) for the two precursors of SP, beta and gamma-preprotachykinin-A, were also detected. These results were consistent in lymphocytes from both epithelium and lamina propria of small and large intestines, although the frequencies and/or intensities of mRNA expression varied. However, none of the findings could be repeated in splenic T lymphocytes. Activation of splenocytes with anti-CD3epsilon-chain mAb and PMA did not induce expression of SP or its receptor mRNAs. Furthermore, both cytoplasmic and surface-bound SP was demonstrated in intestinal T lymphocytes using dual color immunocytochemistry and immunoflow cytometry. In vitro treatment with SP did not significantly change the size of the SP-immunoreactive T cell population, indicating the presence of SP receptor on intestinal T lymphocytes as well as in vivo binding of endogenously released SP. Our data suggest that SP production and SP receptor expression are distinctive for mouse intestinal mucosal immunity and that SP may act as a modulator of an ongoing controlled inflammation in normal gut, by acting through its specific receptor on T lymphocytes in an autocrine and/or paracrine pattern.

Characterisation of mucosal lymphoid aggregates in ulcerative colitis: immune cell phenotype and TcR-gammadelta expression.

BACKGROUND AND AIMS: A histopathological feature considered indicative of ulcerative colitis (UC) is the so-called basal lymphoid aggregates. Their relevance in the pathogenesis of UC is, however, unknown. We have performed a comprehensive analysis of the immune cells in these aggregates most likely corresponding to the lymphoid follicular hyperplasia also described in other colitides. METHODS: Resection specimens of UC and normal colon were analysed by immunomorphometry, immunoflow cytometry, and immunoelectron microscopy, using a large panel of monoclonal antibodies. RESULTS: (1) In all cases of UC, colonic lamina propria contained numerous basal aggregates composed of lymphocytes, follicular dendritic cells, and CD80/B7.1 positive dendritic cells. (2) CD4(+)CD28(-) alphabeta T cells and B cells were the dominant cell types in the aggregates. (3) The aggregates contained a large fraction of cells that are normally associated with the epithelium: that is, gammadelta T cells (11 (7)%) and alpha(E)beta(7)(+) cells (26 (13)%). The gammadelta T cells used Vdelta1 and were CD4(-)CD8(-). Immunoelectron microscopy analysis demonstrated TcR-gammadelta internalisation and surface downregulation, indicating that the gammadelta T cells were activated and engaged in the disease process. (4) One third of cells in the aggregates expressed the antiapoptotic protein bcl-2. CONCLUSIONS: Basal lymphoid aggregates in UC colon are a consequence of anomalous lymphoid follicular hyperplasia, characterised by abnormal follicular architecture and unusual cell immunophenotypes. The aggregates increase in size with severity of disease, and contain large numbers of apoptosis resistant cells and activated mucosal gammadelta T cells. The latter probably colonise the aggregates as an immunoregulatory response to stressed lymphocytes or as a substitute for defective T helper cells in B cell activation. gammadelta T cells in the aggregates may be characteristic of UC.

Neuroendocrine changes in colon of mice with a disrupted IL-2 gene.

Neuroendocrine peptides have a variety of physiological functions in the gastrointestinal tract. This study was carried out to investigate the impact of IL-2 deficiency on the neuroendocrine system in normal colon, and the neuroendocrine changes during colonic inflammation. Mice with homozygous disrupted IL-2 gene (IL-2-/-) spontaneously developed a bowel disease with similarities to human ulcerative colitis. Different types of colonic endocrine cells and myenteric nerves were analysed in the IL-2-/- mice using immunomorphometry. The neuropeptide contents in the colonic tissues were determined by radioimmunoassay. Age-matched healthy IL-2+/- and IL-2+/+ mice served as controls and the colonic IL-2 levels were compared between these two groups of mice by ELISA. Our data showed that less than half the amount of IL-2 was synthesized in the colon of IL-2+/- mice compared with the IL-2+/+ wild-type mice. Two major differences in the neuroendocrine colon were found between the mice with an intact and disrupted IL-2 gene. One was age-related. The frequencies of various endocrine cells and myenteric nerves increased with age in the IL-2+/+ mice. However, no such increases were seen in the mice with a disrupted IL-2 gene. Instead, the volume densities of enteroglucagon, serotonin cells and substance P (SP), vasoactive intestinal polypeptide (VIP) and total myenteric nerves were lower in the older IL-2+/- and IL-2-/- mice compared with the wild type. The other was disease-related. Polypeptide YY (PYY) cells and tissue levels of PYY, SP and VIP were significantly decreased in the IL-2-/- mice during the course of bowel inflammation compared with the healthy IL-2+/- and IL-2+/+ controls. These findings indicate that colonic neuroendocrine alterations did occur in the mice with a disrupted IL-2 gene and diminished local IL-2 level, suggesting a role of IL-2 in the regulation of the neuroendocrine system and a prevalent interaction between the immune and neuroendocrine systems in normal colon. On the other hand, there were some changes that seemed to correlate with the bowel inflammatory process. They might be associated with the impaired function in inflamed gut and contribute to the development and/or prolongation of disease.

Human uvula: characterization of resident leukocytes and local cytokine production.

Upper airway infections often lead to macroscopic changes in the architecture of the uvula. Using immunomorphometric analysis, we investigated the frequency and distribution of immune cells and of cytokine-producing cells in uvular samples. Tissue macrophages, alphabeta T cells, gammadelta T cells, and B cells were, in declining order, the main cell populations. Gammadelta T cells and B cells exhibited reciprocal localization, with almost all gammadelta T cells residing in the vicinity of the epithelium, and all B cells in the glandular area. The presence of cells expressing the suppressor phenotype CD8+CD28- alphabeta T cells is suggested. Fifteen to twenty-five percent of the immune cells expressed the down-regulatory cytokine tumor growth factor beta. Most macrophages were located subepithelially, in the vicinity of the basal lamina. The composition and cytokine profile of leukocytes in the tissue suggest that the uvula may be a site, additional to the jejunal mucosa, for induction of mucosal tolerance to inhaled and ingested antigens. Concomitantly, the uvula appears to be protected from invasion of microbial pathogens by a subepithelial barrier of macrophages and gammadelta T cells.