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.

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.

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.

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.

Specificity and affinity of monoclonal antibodies against carcinoembryonic antigen.

The binding specificities of 52 well-characterized monoclonal antibodies (Mabs) against carcinoembryonic antigen (CEA) from 12 different research groups were studied by immunohistochemistry and immuno flow cytometry. In addition, the binding constant for the interaction between Mab and CEA was determined by a solution-phase assay. Cryostat sections of colon carcinoma and normal colon, stomach, liver, pancreas, and spleen were studied by immunohistochemistry. Peripheral blood granulocytes, monocytes, and lymphocytes were assayed by immuno flow cytometry. The Mabs used here have previously been classified into five essentially nonoverlapping epitope groups (GOLD 1-5) (Cancer Res., 49: 4852-4858, 1989). Most Mabs cross-reacted with different normal tissues, ranging from highly cross-reactive Mabs (positive reaction with 8 of 9 discriminating tissues) to relatively specific Mabs (positive reaction with 1 of 9 discriminating tissues). Five Mabs (10%) were specific, reacting only with colon carcinoma, normal colon mucosa, and normal gastric foveola. There was a correlation between epitope group and binding specificity. Mabs with a high degree of CEA specificity almost exclusively belonged to epitope groups 1, 2, and 3, while highly cross-reactive Mabs belonged to epitope groups 4 and 5. There was no correlation between antibody specificity and affinity for CEA. Specific Mabs with high as well as low affinity were found.

Isolation of functionally active intraepithelial lymphocytes and enterocytes from human small and large intestine.

A mild purification method has been developed for the isolation of human intraepithelial lymphocytes (IEL) and enterocytes from the same individual. The isolation procedure includes mechanical disruption of the mucosal layer, treatment with reducing agent and sedimentation followed by Percoll gradient centrifugation. Finally, epithelial cells are removed from the IEL fraction using magnetic beads coated with the anti-epithelial antigen monoclonal antibody (mAb) BerEP4. Leucocytes are removed from the enterocyte fraction using magnetic beads coated with mAbs directed against common leucocyte antigen (CD45). Using this procedure IEL and enterocytes have been isolated from apparently normal jejunal, ileal and colonic tissue specimens. Recoveries of IEL were 7 x 10(5), 4 x 10(5) and 1 x 10(5)/cm2 mucosa from jejunum, ileum and colon respectively. 1-2 x 10(6) enterocytes/cm2 mucosa were recovered from small intestine while the corresponding value for colonic biopsies was approximately 2 x 10(5) enterocytes/cm2. The IEL fraction was pure as judged by the low percentages of B cells, macrophages and BerEP4 positive cells (less than 4%) present in the purified fraction. The enterocyte fraction contained less than 2% CD45+ cells. The two cell fractions were viable and expanded in vitro. Enterocytes expanded spontaneously while IEL required initial stimulation with mitogens. The isolation procedure described here will make it possible to study the function of human IEL, interactions between IEL and enterocytes and the role of both cell types in local immunity.

Allergen induced cytokine profiles in type I allergic individuals before and after immunotherapy.

Allergen immunotherapy (IT) involves subcutaneous injections of increasing doses of specific allergen over a period of time. It is recognised as highly effective in the treatment of patients with allergic rhinitis. However, the specific immunological mechanisms by which IT achieves its effect have not been fully elucidated. Recent studies, have shown that the clinical effects following IT of allergic individuals is concomitant with a reduced production of IL-4 by allergen specific CD4+ T-cells. The aim of the present study was to gain better knowledge about the immunological mechanisms by which IT exerts its beneficial effects. For this purpose, peripheral blood mononuclear cells (PBMC) from ten individuals receiving birch allergen or placebo in an IT-study performed in a double-blind manner, were analysed for IL-4, IFN-gamma, IL-5 and IL-10 mRNA expression at the onset of the study and during the pollen season, during treatment. Both spontaneous and in vitro allergen-induced cytokine mRNA expression was analysed using reverse transcriptase-polymerase chain reaction (RT-PCR). Spontaneous expression of IL-4 mRNA could be detected in most of the allergic patients, but not in healthy donors. The IT-treated patients showed a decrease in the spontaneous expression of IL-4 mRNA during the pollen season as compared to at the onset of the study, while in patients receiving placebo the IL-4 mRNA expression increased or remained unchanged. Similar results were obtained after in vitro stimulation with allergen. This was in contrast to the results for IFN-gamma, which was readily detected in both patient groups with no significant differences between the groups at either timepoint. IL-5 was shown to be increased during the pollen season in both groups and thereby presumably not affected by allergen IT. Taken together, these observations suggest that the cytokine profiles in circulating T lymphocytes change as a consequence of allergen IT.

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.

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.

Structure of the human uvula.

Eleven uvular biopsies were investigated for their morphology, the presence of mast cells and the distribution of hyaluronan and its major ligand CD44. Three microanatomical sites--surface epithelium, subepithelial area and area of glands--were examined. The oral side of the uvula was covered by a 15-20 cell thick layer of keratinized/parakeratinized surface epithelium, firmly anchored to the underlying connective tissue by connective tissue papillae. The width of the intercellular spaces in the epithelium increased toward the basal lamina, a location that exhibited intense hyaluronan and anti-CD44 staining. Most mast cells were located in the vicinity of blood vessels, at which sites there was high staining intensity of hyaluronan. Tissue mast cells could also be observed in the connective tissue septa enclosing the acini. Glands and muscle fibres became more sparse from the proximal part of the uvula to the distal end, while the amount of connective tissue increased. The localization and architecture of connective tissue elements and mast cells are consistent with the ability of the uvula to resist mechanical stresses and to develop oedema and fibrosis, respectively.