Asthma Prevention by Lactobacillus Rhamnosus in a Mouse Model is Associated With CD4+CD25+Foxp3+ T Cells

PURPOSE: Probiotic bacteria can induce immune regulation or immune tolerance in allergic diseases. The underlying mechanisms have been recently investigated, but are still unclear. The aim of this study was to evaluate the protective effects of the probiotic Lactobacillus rhamnosus (Lcr35) in a mouse model of asthma and to identify its mechanism of action. METHODS: Lcr35 was administered daily by the oral route at a dosage of 1x10(9) CFU/mouse in BALB/c mice for 7 days before the first sensitization. Clinical parameters and regulatory T (Treg) cells were examined. The role of CD4+CD25+Foxp3+ Treg cells was analyzed using a Treg cell-depleting anti-CD25 monoclonal antibody (mAb). RESULTS: Airway hyperresponsiveness, total IgE production, pulmonary eosinophilic inflammation, and splenic lymphocyte proliferation were suppressed after Lcr35 treatment. Th1 (IFN-gamma) and Th2 (IL-4, IL-5, and IL-13) cytokines in the serum were suppressed, and the percentage of CD4+CD25+Foxp3+ Treg cells in the spleen was significantly increased in the Lcr35 treatment group. Anti-CD25 mAb administration abolished the protective effects of Lcr35, indicating that CD4+ CD25+Foxp3+ Treg cells are essential in mediating the activity of Lcr35. CONCLUSIONS: Oral administration of Lcr35 attenuated the features of allergic asthma in a mouse model and induced immune regulation by a CD4+CD25+Foxp3+ Treg cell-mediated mechanism.

The Effects of Lactobacillus rhamnosus on the Prevention of Asthma in a Murine Model

PURPOSE: Lactobacilli are probiotic bacteria that are effective in the management of allergic diseases or gastroenteritis. It is hypothesized that such probiotics have immunoregulatory properties and promote mucosal tolerance. Our goal was to investigate whether Lactobacillus casei rhamnosus Lcr35 could inhibit airway inflammation in an ovalbumin (OVA)-induced murine model of asthma. METHODS: BALB/c mice aged 6 weeks were used in the present study. Lactobacillus casei rhamnosus Lcr35 was administered daily, starting 1 week prior to the first OVA sensitization (group 1) and 2 days before the first 1% OVA airway challenge (group 2). Mice that received only saline at both sensitization and airway challenge time points were used as negative controls (group 3), and those that had OVA-induced asthma were used as positive controls (group 4). Airway responsiveness to methacholine was assessed, and bronchoalveolar lavage (BAL) was performed. At the endpoint of the study, total IgE as well as OVA-specific IgE, IgG1 and IgG2a in serum was measured by enzyme-linked immunosorbent assay. Lung pathology was also evaluated. RESULTS: Airway hyperresponsiveness, total cell counts and the proportion of eosinophils in BAL fluid were significantly decreased in group 1 compared with group 4 (P<0.05). Total serum IgE levels were also significantly decreased in group 1 compared with group 4. Serum levels of OVA-specific IgE, IgG1 and IgG(2a) were not significantly influenced by treatment with Lcr35. There was significantly less peribronchial and perivascular infiltration of inflammatory cells in group 1 compared with group 4; however, there were no significant differences in methacholine challenge, BAL, serology or histology between groups 2 and 4. CONCLUSIONS: Oral treatment with Lcr35 prior to sensitization can attenuate airway inflammation and hyperreactivity in a mouse model of allergic airway inflammation. These results suggest that Lcr35 may have potential for preventing asthma.

Association Between IL-13 Polymorphism (-1512A/C) and Atopic Dermatitis in Korean Children / 소아알레르기및호흡기학회지

PURPOSE: Interleukin (IL) -13 plays a pivotal role in the induction of immunoglobulin E (IgE) and in the pathogenesis of atopic dermatitis (AD). Recently, an association was reported between the polymorphism of the IL-13 promoter region (-1512A/C) and atopic asthma. We investigated the association between the IL-13 single nucleotide polymorphism (SNP) in the promoter region (-1512A/C) and atopic dermatitis in Korean children with AD. METHODS: We enrolled 204 allergic AD, 92 non-allergic AD, and 116 non-atopic healthy children. Evaluated phenotypes of atopic dermatitis included total IgE, total eosinophil count, and eosinophil fraction. We used a PCR-RFLP method to identify IL-13 genotypes. RESULTS: The allele frequencies of the IL-13 promoter polymorphism (-1512A/C) did not differ statistically among the three groups. Children with one or two copies of risk alleles in the promoter region (-1512C) did not show any significant association with the clinical phenotypes of atopic dermatitis including total IgE, eosinophil phenotypes and SCORAD score in the allergic or non-allergic atopic dermatitis. CONCLUSION: These data suggest that the -1512A/C polymorphism of IL-13 gene may not be associated with neither the development nor the clinical phenotypes of atopic dermatitis in Korean children.

Increased Cutaneous Lymphocyte Antigen (CLA) +T Cells in the Peripheral Blood of Children with Severe Atopic Dermatitis / 소아알레르기및호흡기학회지

PURPOSE: Skin-homing T cells expressing cutaneous lymphocyte antigen (CLA) are known to be important in the pathogenesis of atopic dermatitis (AD). So far, there have been few reports on the peripheral lymphocyte subpopulations expressing CLA, especially in children with AD. METHODS: We investigated the peripheral blood lymphocyte subpopulations expressing CLA in children with severe AD andcontrol subjects to identify which proportions of circulating CLA+ T cells were expanded in atopic dermatitis. We assayed the peripheral blood lymphocyte subpopulation with flow cytometry in 15 children with severe chronic lichenified skin lesions and 12 control subjects who had no symptoms of atopic dermatitis. The expressions of peripheral blood CD4+CLA+ T cells and CD3+CLA+ T cells were significantly increased in children with AD compared with those in control subjects, whereas there was no significant difference of CD8+CLA+ T cells between the two groups. RESULTS: The expressions of CD3+ T cells, CD4+ T cells, and CD8+ T cells showed no significant differences between children with AD and control subjects. CONCLUSION: These findings suggest that circulating CD4+CLA+ T cells play an important role in the pathogenesis of chronic severe AD in children.

Changes in Peripheral Blood T Cells after Treatment of Cyclosporine in Children with Severe Atopic Dermatitis / 소아알레르기및호흡기학회지

BACKGROUND: Skin-homing T cells expressing cutaneous lymphocyte antigen (CLA) are known to be important in the pathogenesis of atopic dermatitis (AD). Cyclosporine is known as an effective treatment for severe atopic dermatitis, which controls the cytokine production from T cells and regulates the activation of T cells. There have been no reports about the changes of circulating CLA+ T cells after the treatment of cyclosporine in AD. The aim of this study was to evaluate the clinical outcome and the changes of CLA+ T cells after treatment of cyclosporine in childhood AD. METHODS: Ten children with severe AD were treated with cyclosporine (5 mg/kg/day) for six weeks. Clinical outcome was monitored by the SCORAD index. We assayed the peripheral blood T lymphocyte subpopulation including CLA+ T cells with flow cytometry. RESULTS: The SCORAD index decreased significantly after treatment (P< 0.05). CD4+CLA+ T cells and CD3+CLA+ T cells were significantly decreased after the treatment of cyclosporine. (P< 0.05) But CD3+ T cells, CD4+ T cells and CD8+ T cells were not changed. CONCLUSION: Cyclosporine is effective to control severe AD in children and decreases CD4+CLA+ T cells, which may be important in the pathogenesis of AD.

Immunotherapeutic Effects of CTLA4Ig Fusion Protein on Murine EAE and GVHD

BACKGROUND: CTLA4 (CD152), which is expressed on the surface of T cells following activation, has a much higher affinity for B7 molecules comparing to CD28, and is a negative regulator of T cell activation. In contrast to stimulating and agonistic capabilities of monoclonal antibodies specific to CTLA-4, CTLA4Ig fusion protein appears to act as CD28 antagonist and inhibits in vitro and in vivo T cell priming in variety of immunological conditions. We've set out to confirm whether inhibition of the CD28-B7 costimulatory response using a soluble form of human CTLA4Ig fusion protein would lead to persistent inhibition of alloreactive T cell activation. METHODS: We have used CHO-dhfr cell-line to produce CTLA4Ig fusion protein. After serum free culture of transfected cell line we purified this recombinant molecule by using protein A column. To confirm characterization of fusion protein, we carried out a series of Western blot, SDS-PAGE and silver staining analyses. We have also investigated the efficacy of CTLA4Ig in vitro such as mixed lymphocyte reaction (MLR) & cytotoxic T lymphocyte (CTL) response and in vivo such as experimental autoimmune encephalomyelitis (EAE), graft versus host disease (GVHD) and skin-graft whether this fusion protein could inhibit alloreactive T cell activation and lead to immunosuppression of activated T cell. RESULTS: In vitro assay, CTLA4Ig fusion protein inhibited immune response in T cell-specific manner: 1) Human CTLA4Ig inhibited allogeneic stimulation in murine MLR; 2) CTLA4Ig prevented the specific killing activity of CTL. In vivo assay, human CTLA4Ig revealed the capacities to induce alloantigen-specific hyporesponsiveness in mouse model: 1) GVHD was efficiently blocked by dose-dependent manner; 2) Clinical score of EAE was significantly decreased compared to nomal control; 3) The time of skin-graft rejection was not different between CTLA4Ig treated and control group. CONCLUSION: Human CTLA4Ig suppress the T cell-mediated immune response and efficiently inhibit the EAE, GVHD in mouse model. The mechanism of T cell suppression by human CTLA4Ig fusion protein may be originated from the suppression of activity of cytotoxic T cell. Human CTLA4Ig could not suppress the rejection in mouse skin-graft, this finding suggests that other mechanism except the suppression of cytotoxic T cell may exist on the suppression of graft rejection.

Inhibitory effects of CTLA4-Ig fusion protein on the proliferation of T cell and the antibody production of B cell / 천식및알레르기

BACKGROUND: Atopic asthma is characterized by activation of Th2-type T cells in the bronchial mucosa. Several reports have suggested an important role for costimulation through the CD28/CTLA4 (cytotoxic T lymphocyte-associated antigen 4)-B7 (CD80/CD86) pathway in allergen activation of T cells in animal models of allergen-induced asthma, because B7-CD28/ CTLA4 interaction can promote the differentiation and development of the Th2 lymphocyte subset. OBJECTIVE: In the present study, we intended to investigate a potential role of humanized CTLA4-Ig on the inhibition of T and B cell activation by blocking B7/CD28 interactions. METHOD: For this purpose we produced humanized CTLA4-Ig fusion protein by transfection to CHO cell and examined its inhibitory effects for activated T and B cell responses. We evaluated the inhibitory effect of MLR (mixed lymphocyte reaction) and con A-stimulated T cell proliferation. And we assayed wheather B cell was inhibited by stimulation of costimulatory signal in LPS-induced B cell response and PFC assay. RESULT: In vitro assay, humanized CTLA4-Ig fusion protein inhibited T cell-specific immune response in dose-dependent manner: CTLA4-Ig inhibited allogeneic stimulation in murine MLR, and the proliferation of T cell by the stimulation of Con A. But CTLA4-Ig did not inhibit directly the proliferative response of B cell by the stimulation of LPS. In addition, in vivo assay, CTLA4-Ig inhibited the production of antibody from B cell, which was presented by plaque-forming cell (PFC) assay. CONCLUSION: These findings suggest that humanized CTLA4-Ig is effective to inhibit the proliferation of activated T cell directly by blocking B7/CD28 costimulation. And humanized CTLA4-Ig influences antibody-producing capacity of B cell indirectly by regulating T cell.