Modulation of mucosal immunity in a murine model of food-induced intestinal inflammation

Background Hypersensitivity or uncontrolled responses against dietary antigens can lead to inflammatory disorders like food allergy and current models reflect a variety of causes but do not reveal the detailed modulation of gut immunity in response to food antigens after breakdown in mucosal tolerance. Objective To develop and characterize a murine model for food-induced intestinal inflammation and to demonstrate the modulation of gut immune response by dietary allergenic antigens. Methods C57BL/6 mice were sensitized with peanut proteins, challenged with peanut seeds and their sera and gut segments were collected for subsequent analyses. Results Sensitization and challenged with peanut seeds led to alterations in gut architecture with inflammatory response characterized by oedema in lamina propria and cell infiltrate composed mainly by eosinophils, mast cells, phagocytes, natural killer and plasma cells, together with low percentage of gamma delta(+) and CD4(+)CD25(+)Foxp3(+) cells in Peyer`s patches. These animals also presented high levels of specific IgE and IgG1 in sera and modulation of mucosal immunity was mediated by increased expression of GATA-3, IL-4, IL-13 and TNF-alpha in contrast to low IFN-gamma in the gut. Conclusion A murine model for food-induced intestinal inflammation was characterized in which modulation of gut immunity occurs by peanut antigens in consequence of T-helper type 2 (Th2) allergic response and failure of regulatory mechanisms necessary for mucosa homeostasis, resembling food allergy. This work shed some light on the understanding of the pathogenesis of gastrointestinal disorders and intolerance in the gut and supports the development of therapies for food-related enteropathies like food allergy, focusing on gut-specific immune response.

The involvement of CD4(+)CD25(+) T cells in the acute phase of Trypanosoma cruzi infection

The infection with Trypanosoma cruzi leads to a vigorous and apparently uncontrolled inflammatory response in the heart. Although the parasites trigger specific immune response, the infection is not completely cleared out, a phenomenon that in other parasitic infections has been attributed to CD4(+)CD25(+) T cells (Tregs). Then, we examined the role of natural Tregs and its signaling through CD25 and GITR in the resistance against infection with T. cruzi. Mice were treated with mAb against CD25 and GITR and the parasitemia, mortality and heart pathology analyzed. First, we demonstrated that CD4(+)CD25(+)GITR(+)Foxp3(+) T cells migrate to the heart of infected mice. The treatment with anti-CD25 or anti-GITR resulted in increased mortality of these infected animals. Moreover, the treatment with anti-GITR enhanced the myocarditis, with increased migration of CD4(+), CD8(+), and CCR5(+) leukocytes, TNF-alpha production, and tissue parasitism, although it did not change the systemic nitric oxide synthesis. These data showed a limited role for CD25 signaling in controlling the inflammatory response during this protozoan infection. Also, the data suggested that signaling through GITR is determinant to control of the heart inflammation, parasite replication, and host resistance against the infection. (C) 2008 Elsevier Masson SAS. All rights reserved.

Experimental autoimmune encephalomyelitis evolution was not modified by multiple infections with Strongyloides venezuelensis

P>According to the hygiene hypothesis, the increased incidence of allergic and autoimmune diseases in developed countries is mainly explained by the decreased contact between the human population and certain environmental agents as lactobacillus, mycobacteria and helminths. In this study, we evaluated the effect of multiple infections with Strongyloides venezuelensis on the development of experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Multiple infections before EAE induction were not able to change the evolution of the disease. No alterations were observed in weight loss, clinical score and inflammation intensity at the central nervous system. The presence of significant levels of parasite-specific IgG1 but not IgG2b suggested a Th2 polarization. However, the percentage and absolute number of CD4+CD25+Foxp3+ T cells were not changed, being their levels in the spleen and lymph nodes of infected rats comparable to the ones found in normal animals. These results suggest that a Th2-polarized response without concomitant expansion of Foxp3+ regulatory T cells was not able to modify EAE progression. Even though these results do not threaten the hygiene hypothesis, they suggest that this paradigm might be an oversimplification. They also emphasize the need of a study to compare the immunoregulatory ability associated with different helminth spp.

Patients with oral squamous cell carcinoma are characterized by increased frequency of suppressive regulatory T cells in the blood and tumor microenvironment

Oral squamous cell carcinoma (OSCC) is a cancerous lesion with high incidence worldwide. The immunoregulatory events leading to OSCC persistence remain to be elucidated. Our hypothesis is that regulatory T cells (Tregs) are important to obstruct antitumor immune responses in patients with OSCC. In the present study, we investigated the frequency, phenotype, and activity of Tregs from blood and lesions of patients with OSCC. Our data showed that > 80% of CD4(+)CD25(+) T cells isolated from PBMC and tumor sites express FoxP3. Also, these cells express surface Treg markers, such as GITR, CD45RO, CD69, LAP, CTLA-4, CCR4, and IL-10. Purified CD4(+)CD25(+) T cells exhibited stronger suppressive activity inhibiting allogeneic T-cell proliferation and IFN-gamma production when compared with CD4(+)CD25(+) T cells isolated from healthy individuals. Interestingly, approximately 25% of CD4(+)CD25(-) T cells of PBMC from patients also expressed FoxP3 and, although these cells weakly suppress allogeneic T cells proliferative response, they inhibited IFN-gamma and induced IL-10 and TGF-beta secretion in these co-cultures. Thus, our data show that Treg cells are present in OSCC lesions and PBMC, and these cells appear to suppress immune responses both systemically and in the tumor microenvironment.

Characterization of CD4(+)CD25(+) natural regulatory T cells in the inflammatory infiltrate of human chronic periodontitis

Periodontitis is an infectious disease, where putative periodontopathogens trigger chronic inflammatory and immune responses against periodontal structures, in which an unbalanced host response is also determinant to the disease outcome. It is reasonable to assume that patient susceptibility to periodontal tissue destruction could be determined by the balance between the response against periodontopathogens and regulatory mechanisms of these events mediated by suppressive T cells. In the present study, we identified and characterized natural regulatory T cells ( Tregs) in the inflammatory infiltrate of human chronic periodontitis ( CP) with emphasis on phenotypic analyses that were carried out to address the participation of Tregs in CP. Results showed that patients with CP presented increased frequency of T lymphocytes and CD4(+)CD25(+) T cells in the inflammatory infiltrate of gingival tissues. These cells exhibited the phenotypic markers of Tregs such as forkhead box p3 ( Foxp3), CTLA- 4, glucocorticoidinducible TNFR, CD103, and CD45RO and seemed to be attracted to the inflammation site by the chemokines CCL17 and CCL22, as their expression and its receptor CCR4 were increased in CP patients. Moreover, besides the increased detection of Foxp3 mRNA, diseased tissues presented high expression of the regulatory cytokines IL-10 and TGF-beta. In addition, the inflammatory infiltrate in CP biopsies was composed of CD25(+)Foxp3(+) and CD25(+)TGF-beta(+) cells, thus corroborating the hypothesis of the involvement of Tregs in the pathogenesis of CP. Finally, these results indicate that Tregs are found in the chronic lesions and must be involved in the modulation of local immune response in CP patients.

CCR5-dependent regulatory T cell migration mediates fungal survival and severe immunosuppression

Paracoccidioidomycosis, a debilitating pulmonary mycosis, is caused by the dimorphic fungus Paracoccidioides brasiliensis. The infection results in the formation of granulomas containing viable yeast cells that are the fungal sources for disease reactivation. Because CD4(+)CD25(+) regulatory T cells (Tregs) are in the lesions of patients with paracoccidioidomycosis, the migration of Treg cells is dependent on the axis chemokine-chemokine receptors, and CCR5 ligands are produced in P. brasiliensis-induced lesions, we investigated the role of CCR5 in the control of the infection. The results showed that CCR5(-/-) mice are more efficient in controlling fungal growth and dissemination and exhibited smaller granulomas than wild-type (WT) mice. In the absence of CCR5, the percentage of CD4(+)CD25(+) T cells expressing Foxp3, glucocorticoid-induced TNFR (GITR), CD103, CD45(low), and CTLA-4 in the granulomas was significantly decreased. Interestingly, P. brasiliensis infection resulted in an absence of T cell proliferation in response to Con A in WT but not CCR5(-/-) mice that was abrogated by anti-CTLA-4 mAb and anti-GITR mAb. Moreover, the adoptive transfer of CD4(+)CD25(+) but not CD4(+)CD25(-) T cells from infected WT to infected CCR5(-/-) mice resulted in a significant increase in fungal load. Overall, CCR5 is a key receptor for the migration of Treg cells to the site of P. brasiliensis infections leading to down-modulation of effector immune response and the long-term presence of the fungus in the granulomas. Thus, a tight control of Treg cell migration to the granulomatous lesions could be an important mechanism for avoiding exacerbation and reactivation of the disease.

Comprehensive gene expression profiling in lungs of mice infected with Mycobacterium tuberculosis following DNAhsp65 immunotherapy

Background The continued increase in tuberculosis (TB) rates and the appearance of extremely resistant Mycobacterium tuberculosis strains (XDR-TB) worldwide are some of the great problems of public health. In this context, DNA immunotherapy has been proposed as an effective alternative that could circumvent the limitations of conventional drugs. Nonetheless, the molecular events underlying these therapeutic effects are poorly understood. Methods We characterized the transcriptional signature of lungs from mice infected with M. tuberculosis and treated with heat shock protein 65 as a genetic vaccine (DNAhsp65) combining microarray and real-time polymerase chain reaction analysis. The gene expression data were correlated with the histopathological analysis of lungs. Results The differential modulation of a high number of genes allowed us to distinguish DNAhsp65-treated from nontreated animals (saline and vector-injected mice). Functional analysis of this group of genes suggests that DNAhsp65 therapy could not only boost the T helper (Th)1 immune response, but also could inhibit Th2 cytokines and regulate the intensity of inflammation through fine tuning of gene expression of various genes, including those of interleukin-17, lymphotoxin A, tumour necrosis factor-cl, interleukin-6, transforming growth factor-beta, inducible nitric oxide synthase and Foxp3. In addition, a large number of genes and expressed sequence tags previously unrelated to DNA-therapy were identified. All these findings were well correlated with the histopathological lesions presented in the lungs. Conclusions The effects of DNA therapy are reflected in gene expression modulation; therefore, the genes identified as differentially expressed could be considered as transcriptional biomarkers of DNAhsp65 immunotherapy against TB. The data have important implications for achieving a better understanding of gene-based therapies. Copyright (C) 2008 John Wiley & Sons, Ltd.

X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy

To determine whether human X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome (IPEX; MIM 304930) is the genetic equivalent of the scurfy (sf) mouse, we sequenced the human ortholog (FOXP3) of the gene mutated in scurfy mice (Foxp3), in IPEX patients. We found four non-polymorphic mutations. Each mutation affects the forkhead/winged-helix domain of the scurfin protein, indicating that the mutations may disrupt critical DNA interactions.

Regulatory Cells, Cytokine Pattern and Clinical Risk Factors for Asthma in Infants and Young Children with Recurrent Wheeze

Several risk factors for asthma have been identified in infants and young children with recurrent wheeze. However, published literature has reported contradictory findings regarding the underlying immunological mechanisms. OBJECTIVES: This study was designed to assess and compare the immunological status during the first 2 years in steroid-naive young children with >or= three episodes of physician-confirmed wheeze (n=50), with and without clinical risk factors for developing subsequent asthma (i.e. parental asthma or a personal history of eczema and/or two of the following: wheezing without colds, a personal history of allergic rhinitis and peripheral blood eosinophilia >4%), with age-matched healthy controls (n=30). METHODS: Peripheral blood CD4(+)CD25(+) and CD4(+)CD25(high) T cells and their cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), GITR and Foxp3 expression were analysed by flow cytometry. Cytokine (IFN-gamma, TGF-beta and IL-10), CTLA-4 and Foxp3 mRNA expression were evaluated (real-time PCR) after peripheral blood mononuclear cell stimulation with phorbol 12-myristate 13-acetate (PMA) (24 h) and house dust mite (HDM) extracts (7th day). RESULTS: Flow cytometry results showed a significant reduction in the absolute number of CD4(+)CD25(high) and the absolute and percentage numbers of CD4(+)CD25(+)CTLA-4(+) in wheezy children compared with healthy controls. Wheezy children at a high risk of developing asthma had a significantly lower absolute number of CD4(+)CD25(+) (P=0.01) and CD4(+)CD25(high) (P=0.04), compared with those at a low risk. After PMA stimulation, CTLA-4 (P=0.03) and Foxp3 (P=0.02) expression was diminished in wheezy children compared with the healthy children. After HDM stimulation, CTLA-4 (P=0.03) and IFN-gamma (P=0.04) expression was diminished in wheezy children compared with healthy children. High-risk children had lower expression of IFN-gamma (P=0.03) compared with low-risk and healthy children and lower expression of CTLA-4 (P=0.01) compared with healthy children. CONCLUSIONS: Although our findings suggest that some immunological parameters are impaired in children with recurrent wheeze, particularly with a high risk for asthma, further studies are needed in order to assess their potential as surrogate predictor factors for asthma in early life.

T cell Maturation and Regulatory T Cell Differentiation:From the Thymus to the Periphery

Immunological tolerance, that is, the failure to mount an immune response to an otherwise immunogenic molecule, is one of the fundamental questions in immunology. The fact that lymphocytes express antigen receptors that are generated randomly and have the potential to recognize any conceivable antigen, adds another puzzle to the physiology of immunological tolerance. The other side of the coin, the general absence of immune responses to self antigens, is ensured by a tight regulation and several selection steps during T and B cell differentiation. One of these processes is the differentiation of regulatory T cells (Treg). While developing in the thymus, T cell clones bearing receptors with high affinity/avidity to antigens present at the time of differentiation may be eliminated by apoptosis or, alternatively, express Foxp3 and become Treg. Treg are key players in the regulation of immunological tolerance since humans and mice with complete loss of function variants of this gene develop fatal autoimmune conditions early in life.(...)

Perfil imunol��gico de gr��vidas at��picas versus gr��vidas saud��veis

RESUMO: A preval��ncia das doen��as at��picas tem vindo a aumentar, em especial ao n��vel dos pa��ses ocidentalizados. V��rios fatores t��m sido apontados para justificar este aumento de preval��ncia,destacando-se o reduzido tamanho das fam��lias, o elevado uso de antibi��ticos, a melhoria das condi����es sanit��rias, bem como a diminui����o quer das infe����es de helmintas, quer da contamina����o orofecal. Alguns estudos t��m tamb��m avaliado a influ��ncia do ambiente pr��-natal no desenvolvimento de atopia e asma. Da an��lise da literatura, parece ineg��vel a import��ncia deste per��odo para o desenvolvimento do sistema imunit��rio. Neste ��mbito, a transmiss��o de atopia �� descend��ncia em mulheres at��picas, e concretamente com asma al��rgica, poder�� ser moldada desde este per��odo. A possibilidade de identificar marcadores de risco precoces para o desenvolvimento de atopia poder�� ser o primeiro passo para o desenvolvimento de estrat��gias de preven����o para os indiv��duos em risco. Este trabalho pretendeu abordar o sistema imunit��rio materno de forma a enriquecer a sua carateriza����o desde o terceiro trimestre da gravidez at�� ao fim do puerp��rio. Para al��m da explora����o de perfis celulares e citoc��nicos maternos (nos quais se incluiu sobretudo a avalia����o de diferentes popula����es de c��lulas T e B, com fun����es efetoras e reguladoras), foi tamb��m considerada a sua eventual rela����o com o desenvolvimento de atopia nas crian��as. Foram recrutadas 135 mulheres com crit��rios para serem inclu��das num dos 4 grupos do estudo: gr��vidas at��picas ��� GA (n=24), n��o gr��vidas at��picas ��� NGA (n=32), gr��vidas saud��veis ��� GS (n=44) e n��o gr��vidas saud��veis ��� NGS (n=35). Foram caraterizadas por Citometria de Fluxo popula����es de leuc��citos e linf��citos, com particular interesse nos perfis maturativos de linf��citos T e B, bem como nas subpopula����es de c��lulas T e B reguladoras. Foi ainda efetuada uma an��lise funcional, para avaliar a capacidade de produ����o de citocinas pelos linf��citos T e B. Foram igualmente avaliadas as concentra����es de citocinas s��ricas por ensaios imunoenzim��ticos. Estes par��metros imunol��gicos maternos foram acompanhados desde o terceiro trimestre de gesta����o, at�� depois do puerp��rio (primeiras 6 semanas p��s parto), e aos seis meses de idade, foi efetuada uma avalia����o cl��nica das crian��as. As mulheres n��o gr��vidas at��picas apresentaram contagens celulares mais elevadas para a generalidade das popula����es leucocit��rias e linfocit��rias (em rela����o a mulheres n��o gr��vidas saud��veis). Destaca-se ainda uma maior presen��a de eosin��filos nas mulheres NGA (p=0,0009; teste de Mann-Whitney U), que tinham igualmente os seus compartimentos linfocit��rios T e B mais ricos em c��lulas de mem��ria, em rela����o ��s mulheres NGS. Para os perfis de regula����o, verificou-se que as c��lulas T reguladoras se encontravam percentualmente aumentadas (p���0,003; teste de Mann-Whitney U), tal omo as c��lulas T produtoras de IL10 ap��s estimula����o (p���0,03; teste de Mann-Whitney U) em mulheres NGA. Tamb��m se observou uma maior express��o de Foxp3 (p=0,0002; teste de Mann-Whitney U), e ainda a diminui����o dos n��veis s��ricos de IFN-�� nas mulheres NGA (p=0,0019; teste de Mann-Whitney U), em rela����o a mulheres NGS. De um modo geral, as altera����es verificadas nos par��metros imunol��gicos de mulheres gr��vidas at��picas no terceiro trimestre da gravidez foram semelhantes ��s observadas em mulheres gr��vidas saud��veis. Comparadas com mulheres NGA, nas mulheres gr��vidas at��picas ocorreu uma altera����o substancial da f��rmula leucocit��ria, com um importante incremento de neutr��filos (p<0,0001; teste de Mann-Whitney U) e diminui����o dos valores das restantes popula����es leucocit��rias. A diminui����o nas contagens de linf��citos totais estendeu-se a grande parte das subpopula����es linfocit��rias caraterizadas. Nos compartimentos linfocit��rios T e B foi poss��vel observar uma diminui����o das subpopula����es de c��lulas de mem��ria. Verificou-se igualmente na gravidez uma menor express��o de Foxp3 em mulheres GA (p<0,0001; teste de Mann-Whitney U) e ainda menos c��lulas B CD24HiCD38Hi circulantes (p=0,0012; teste de Mann-Whitney U). Ocrreu ainda uma diminui����o relativa das c��lulas T CD4 produtoras de IFN-�� em mulheres GA (p���0,024; teste de Mann-Whitney U), e uma maior presen��a de c��lulas T CD8 produtoras de IL17 (p=0,0172; teste de Mann-Whitney U), em rela����o ao observado em mulheres NGA. Depois do puerp��rio, no compartimento T de mulheres do grupo GA, verificou-se um aumento das popula����es de c��lulas de mem��ria. Em compara����o com a gravidez, ap��s o puerp��rio o compartimento B, apresentou nas mulheres GA um aumento significativo da subpopula����o de c��lulas B de transi����o (p<0,0001; teste de Wilcoxon). Verificou-se, igualmente em mulheres GA ap��s o puerp��rio, uma maior express��o de Foxp3 nas c��lulas T reguladoras (p<0,0001; teste de Wilcoxon) e o aumento das popula����es de c��lulas T circulantes produtoras de IFN-�� (p���0,0234; teste de Wilcoxon). As modula����es das popula����es T e B desde a gravidez at�� depois do puerp��rio ocorreram de forma semelhante nas mulheres dos grupos GA e GS. Apesar de as mulheres GA manterem um perfil imunol��gico pr��ximo do das mulheres GS depois do puerp��rio, aconteceu tamb��m neste per��odo um processo de reaproxima����o ao perfil observado nas mulheres NGA. As mulheres GA com manifesta����es de risco para atopia na descend��ncia (comparadas com mulheres GA sem manifesta����es de risco para atopia na descend��ncia at�� aos 6 meses de vida) apresentaram uma maior propor����o de c��lulas T e menor propor����o de c��lulas B, percentagens mais elevadas de c��lulas T CD8 de mem��ria efetoras, de c��lulas B de transi����o e de c��lulas B CD24HiCD38Hi, e contagens mais baixas de c��lulas B de mem��ria. Na avalia����o destes par��metros como marcadores de risco para o desenvolvimento de atopia verificou-se que o par��metro com melhor desempenho foi a percentagem de c��lulas B de transi����o, com uma Odds-Ratio de 54,0 [IC 95%: 4,2-692,9; (p=0,0005)], sensibilidade de 90,0% [IC 95%: 55,5 ��� 99,8] e especificidade de 85,7% [IC 95%: 57,2 ��� 98,2]. Este estudo foi pioneiro em Portugal, e no mundo, no que se refere ao acompanhamento do compartimento linfocit��rio B circulante, abordando o seu perfil de matura����o, e em particular as c��lulas B com fun����es reguladoras, desde a gravidez at�� ao fim do puerp��rio, em mulheres at��picas e n��o at��picas. A este n��vel, encontram-se estudos na literatura a documentar a altera����o do compartimento B durante a gravidez. O presente trabalho reporta agora que altera����es, como a diminui����o do n��mero de c��lulas B em circula����o, s��o impostas tamb��m na mulher at��pica. Em suma, demonstrou-se a exist��ncia de um perfil imunol��gico carater��stico em mulheres at��picas, que sofre altera����es significativas durante a gravidez, tendendo os par��metros imunol��gicos a normalizar ap��s o puerp��rio. O compartimento T, para o qual a literatura �� mais rica em estudos e abordagens, demonstrou tamb��m neste trabalho oscila����es carater��sticas entre o per��odo pr�� e p��s-natal. Verificaram-se sobretudo varia����es nos compartimentos de c��lulas T de mem��ria, sem grandes altera����es ao n��vel das c��lulas Treg no que se refere �� sua presen��a em circula����o. Apenas a registar a menor express��o de Foxp3 nas c��lulas Treg durante a gesta����o observada em mulheres at��picas, tal como em mulheres saud��veis (como tamb��m j�� foi relatado em estudos anteriores). Apesar de muitos dos dados se encontrarem em concord��ncia com a literatura, quer no que se refere ��s subpopula����es de c��lulas de mem��ria, quer no que se refere ��s c��lulas Treg, tamb��m se encontram resultados discordantes, por exemplo documentando varia����es num��ricas nas c��lulas Treg em circula����o em mulheres at��picas e mulheres at��picas gr��vidas. A import��ncia de harmonizar protocolos e fen��tipos, parece crucial na abordagem de estudos futuros. Ao n��vel do risco para a atopia na descend��ncia de mulheres at��picas, acrescentou-se ainda a possibilidade de definir marcadores n��o invasivos para a crian��a, em particular as c��lulas B de transi����o. Estas c��lulas, cuja maior presen��a em circula����o no rec��m-nascido foi recentemente associada com manifesta����es al��rgicas subsequentes, s��o agora apontadas j�� na mulher at��pica, gr��vida do terceiro trimestre, como um elemento de risco para o desenvolvimento de atopia. Os marcadores de risco descritos, para al��m de facilmente poderem vir a ser englobados no ��mbito dos normais rastreios maternos durante a gravidez, apresentam ainda a vantagem da precocidade do diagn��stico, permitindo n��o s�� a possibilidade de preven����o p��s-natal, mas estendendo esta possibilidade ao per��odo gestacional.----------------------------ABSTRACT: The prevalence of atopic diseases has been increasing, especially in Westernized countries. Several factors have been suggested to justify this increase in prevalence, as the small size of families, the high use of antibiotics, the improvement in sanitation conditions, as well as the reduction of both helminth infections, and orofecal contamination. A few studies have adressed the influence of prenatal environment on the development of atopy and asthma. From literature, it seems undeniable the importance of the prenatal period for the development of the immune system. In this context, the transmission of atopy to the progeny in atopic women, and specifically in women with allergic asthma, can be modulated from this period on. The ability to detect early risk markers for the development of atopic diseases may be the first step in the development of prevention strategies for individuals at risk. This study aimed to approach the maternal immune system in order to enrich its characterization from the third trimester of pregnancy until the end of the puerperium period. In addition to the evaluation of the maternal cellular profiles (in which, mostly, diferente populations of T and B cells with effector and regulatory functions were included) and citokines, the relation between these profiles and the development of atopy in the progeny was also assessed. 135 women were recruited for this study, and fullfiled the inclusion criteria necessary to be included in one of the four groups preset: atopic pregnant women - GA (n = 24), atopic nonpregnant women - NGA (n = 32), healthy pregnant women - GS (n = 44) and healthy nonpregnant women - NGS (n = 35). Populations of leukocytes and lymphocytes, and particularty maturation profiles of T and B lymphocytes, as well as subpopulations of T and B cells with regulatory functions, were characterized by flow cytometry. Functional assays were also performed, to assess the ability of cytokine production by T and B lymphocytes. Serum cytokine concentrations were assessed as well by enzymatic immunoassays. These maternal imune parameters were monitored since the third trimester of pregnancy until the end of the puerperium period (first six weeks after delivery). A clinical evaluation of all the newborn children was performed at the age of six months. Non-atopic pregnant women presented higher cell counts for most leukocyte and lymphocyte populations (compared to healthy non-pregnant women). We should also highlight the increased presence of eosinophils in NGA women (p = 0,0009; Mann-Whitney U test). Again compared to NGS women, NGA women showed increased memory cells within the circulating T and B lymphocyte compartments. Considering the regulatory profiles, NGA women presented higher percentages of regulatory T cells (p���0,003; Mann-Whitney U test) and IL10 producing T cells after stimulation (p���0,03; Mann Whitney U), as well as increased expression of Foxp3 (p = 0,0002; Mann-Whitney U test), and also decreased serum levels of IFN-�� (p = 0,0019; test Mann-Whitney U test) compared to NGS women. In general, the changes observed in immune parameters of atopic pregnant women in the third trimester of gestation were similar to those observed in healthy pregnant women. Comparing pregnant and non-pregnant atopic women, an important change in leukocyte subsets was observed, with a significant increase of neutrophils (p <0,0001; Mann-Whitney U test) and the consequent diminution of the remaining leukocyte populations in the GA group. The decrease in total lymphocyte counts was extended to most of the lymphocyte subsets characterized. It was possible to detect a decrease in memory cell subsets within the T and B lymphocyte compartments, also. During pregnancy, a lower expression of Foxp3 was reported in GA women (p <0,0001; Mann-Whitney U test) and, besides, lesser CD24HiCD38Hi B cells were present in circulation in these women, compared to NGA women (p = 0,0012; Mann-Whitney U test). There was still a decrease in the percentages of IFN-��-producing CD4 T cells in GA women (p���0,024; Mann-Whitney U test) and a greater presence of IL17-producing CD8 T cells (p = 0,0172; Mann-Whitney U test), compared to the levels observed in NGA women. At the end of the puerperium, there was an increase in memory cell subpopulations within the T cell compartment of GA women. Compared with the pregnancy evaluation, after puerperium, the B cell compartment showed a significant increase in the transitional subpopulation (p<0,0001; Wilcoxon test), in GA women. Moreover, after puerperium, GA women exhibited a greater expression of Foxp3 in Treg cells (p <0,0001; Wilcoxon test) and there was an increase in circulating IFN-��-producing T cells (p���0,0234; Test Wilcoxon). The modulations of T and B cell subpopulations from pregnancy until the end of puerperium were similar in women of GA and GS groups. Although at the end of puerperium, GA women still kept an immune profile close the one observed in GS women, at this time point, there were also signs of rapprochement between the immune profiles observed in women of GA and NGA groups. GA women with atopic manifestations in the offspring (compared to GA women without atopic manifestations in the offspring at the age of 6 months) presented higher proportions of T cells and lower proportions of B cells, higher percentages of effector memory CD8 T cells, transitional B cells and CD24HiCD38Hi B cells, and, finally, lower absolute counts of memory B cells. In the evaluation of these parameters as risk markers for the development of atopy, the parameter which presented the best performance was the percentage of transitional B cells, with an Oddsratio of 54,0 [95% CI: 4,2 to 692,9; (p = 0,0005)], sensitivity of 90,0% [95% CI: 55,5 to 99,8] and a specificity of 85,7% [95% CI: 57,2 to 98,2]. This study was a pioneer in Portugal, and in the world, in what concerns the monitoring of the circulating B cell compartment, addressing not only the maturation profile, but, in particular, B cells with regulatory functions, from pregnancy untill after puerperium, in atopic and non-atopic women. Literature presents evidence of a typical change in circulating B cells during pregnancy. This study now reports that changes, such as the decrease in the number of circulating B cells,/ are also imposed by pregnancy in atopic woman. In brief, it demonstrated the existence of a characteristic immune profile in atopic women, which undergoes significant alterations during pregnancy, tending to normalize after the puerperium. As for the T cell compartment, for which the literature is richer in studies and approaches, this study also showed characteristic fluctuations between the pre- and postnatal periods. There were variations mostly in the memory subsets within the T cell compartment, without major changes in regulatory T cells regarding their presence in circulation. Only the expression of Foxp3 in Treg cells presented lower levels during pregnancy, in both atopic and healthy women (as previously reported in other studies). Although much of the data now reported are in agreement with literature, regarding either memory cell subsets or regulatory T cells, there are also conflicting results, for example documenting changes in the numbers of regulatory T cells circulating in atopic pregnant and atopic non-pregnant women. The importance of harmonizing protocols and phenotypes seems crucial for the establishement of future studies. Considering the risk for atopy in the offspring of atopic women, this study added the possibility to define non-invasive markers for the child, in particular transitional B cells. These cells, whose greater presence in circulation in newborns has recently been associated with subsequent allergy development, are here identified in atopic pregnant women in the third trimester of gestation as a risk factor in the development of atopy in their progeny. The risk factors described, besides having the capacity to easily become integrated within the normal maternal screening protocols during pregnancy, also have the advantage of an early diagnosis, allowing not only the possibility of postnatal prevention but extending this possibility to the prenatal period.

Regulaci��n de la funci��n de macr��fagos por Cryptococcus neoformans y Cryptococcus gattii y su participaci��n en la generaci��n de la inmunidad adaptativa antif��ngica. Regulation of macrophage function by Cryptococcus neoformans and Cryptococcus gattii and their role in the antifungal adaptative immunity.

La criptococosis es causada por la inhalaci��n de levaduras encapsuladas de Cryptococcus neoformans o Cryptococcus gattii. Representa una de las tres infecciones graves por oportunistas en pacientes con SIDA y existe aproximadamente un 6 por ciento de incidencia de criptococosis cl��nica en pacientes con transplante de ��rganos s��lidos. Estas dos especies difieren la fisiopatogenia durante la infecci��n. El factor de virulencia principal de Cryptococcus sp. es la presencia del polisac��rido capsular, glucuronoxilomanano (GXM), de alto peso molecular, que es continuamente secretado por las levaduras. Los macr��fagos son c��lulas centrales en la respuesta innata al hongo, los cuales deben ser activados por linfocitos T helper 1 para un eficiente control de la infecci��n. Sin embargo, estas c��lulas tambi��n son suceptibles al parasitismo intracelular, permitiendo la infecci��n persistente y la diseminaci��n a sitios extrapulmonares. Este proyecto propone investigar la capacidad de levaduras de C. neoformans, C. gattii y de los polisac��ridos capsulares para modular la respuesta proinflamatoria de los macr��fagos. Queremos estudiar si el tratamiento de macr��fagos con levaduras o polisac��rido puede inducir perfiles supresores de la respuesta protectiva T helper 1, tales como linfocitos T helper 2 o T reguladores, favoreciendo la sobrevida intracelular del hongo. Adem��s, pensamos que C. neoformans o C. gattii podr��an inducir un activaci��n diferencial de macr��fagos lo que condicionar��a la respuesta adaptativa, lo que podr��a explicar las diferencias en la fisiopatogenia de estas dos especies. Procedimientos experimentales -Microorganismos y obtenci��n de GXM: se trabajar�� con C. neoformans variedad grubii, cepa ATCC 62067 y C. gattii serotipo B, cepa NIH112B. Se obtendr��n polisac��ridos capsulares (GXM) de C. neoformans y C. gattii por precipitaci��n con etanol y y acomplejamiento selectivo con CTAB. - Obtenci��n de macr��fagos murinos y cultivos celulares: se obtendr��n macr��fagos por lavados peritoneales y/o alveolares de ratones BALB/c. Los macr��fagos se cultivar��n por 24 h en ausencia o presencia de levaduras muertas o vivas (sin opsonizar u opsonizadas) de C. neoformans o C. gattii o en presencia de GXM purificado. -Objetivo 1. Estudio de la modulaci��n de las propiedades proinflamatorias de Mac: en sobrenadantes de los cultivos se medir��n las citoquinas por ELISA de captura y en lisados celulares, la expresi��n de las enzimas (iNOS, arginasa, IDO) por western blot. Se analizar�� por citometr��a de flujo la expresi��n de MCHII y mol��culas CD80, CD86, CD40, CTLA-4. -Objetivo 2. Estudios in vitro de la capacidad de macr��fagos tratados con levaduras o GXM para inducir linfocitos Th1, Th2 o Treg: los macr��fagos preincubados con GXM o levaduras, se incubar��n con linfocitos aut��logos estimulados con anti-CD3. Se medir�� la proliferaci��n celular y el perfil de citoquinas por citomtr��a de flujo. C��lulas T CD4+ CD25- ser��n purificadas de suspenciones espl��nicas de ratones normales. Luego las c��lulas ser��n incubadas con macr��fagos (sin tratar o tratados con levaduras o GXM) y estimulados con anti-CD3. Se analizar�� la proliferaci��n celular con CFSE y expresi��n de CD4, CD25 y Foxp3 . - Objetivo 3. Estudios in vivo de la capacidad de levaduras o GXM para inducir linfocitos Th1, Th2 o Treg . Rol de los macr��fagos in vivo: Los ratones ser��n inyectados con 100000 levaduras o con 200 ��g de GXM puro v��a endovenosa y luego de 7, 14, 30 y 40 d��as se evaluar��n las poblaciones celulares de bazo, por citometr��a de flujo usando marcaciones simult��neas para CD4, CD8, CD25, Foxp3 y citoquinas intracelulares. Para investigar la participaci��n in vivo de los macr��fagos, se depletaran estas c��lulas inyectando los animales con PBS-liposomas o clodronato (DMDP)-liposomas por v��a endovenosa o inhalatoria (200- 300 ��l por rat��n). Luego de 24 h, los animales se infectar��n con levaduras o inocular��n con GXM y se evaluar��n los perfiles de c��lulas T espl��nicos o de n��dulos linfaticos.

Modulaci��n de la activaci��n de c��lulas dendr��ticas por ant��genos de Fasciola hepatica y ligandos para receptores Toll: Potencial terap��utico en respuestas anti-inflamatorias. Fasciola hepatica antigens and TLR ligands modulate dendritic cells activation: therapeutic potential in anti-inflammatory responses.

Antecedentes: En nuestro laboratorio hemos demostrado que ant��genos (Ags) de Fasciola hepatica inducen en c��lulas dendr��ticas murinas (CD), diferentes propiedades tolerog��nicas como la incapacidad por si mismos de inducir la maduraci��n de las c��lulas, la resistencia a la maduraci��n por ligandos de TLR, el incremento en la producci��n de IDO y tambi��n la capacidad de esta estas c��lulas de dirigir la respuesta inmune hacia un perfil Th2 y T reg. Por otra parte ha sido bien documentado que CD con caracter��sticas tolerog��nicas, ya sea inmaduras o semimaduras, son ��tiles para reducir respuestas inflamatorias excesivas tales como las que ocurren en enfermedades autoinmunes. Adem��s hemos demostrado que CD tratadas con Ags del par��sito en conjunto con un ligando Toll (CpG-ODN) producen altos niveles de citoquinas anti-inflamatorias (IL-10 y TGF-���) bajos de citoquinas proinflamatorias (TNF, IL-6, IL-12). Hip��tesis: El fenotipo semimaduro alcanzado en las CDpodr��a ser utilizado para reducir la inflamaci��n en un modelo de enfermedad autoinmune en donde existe una exacerbada respuesta Th1 y Th17, ya que la producci��n elevada de IL-10 y TGF-��� podr��a inhibir o controlar estas respuestas de manera directa o a trav��s de la inducci��n de c��lulas T regulatorias. Objetivos: En este proyecto nosotros proponemos la inmunizaci��n de animales susceptibles (ratones DBA1/j), al desarrollo de artritis inducida por col��geno (AIC) con CD tratadas con Ags de F. hepatica en conjunto con CpG-ODN para reducir los s��ntomas cl��nicos de la enfermedad. Materiales a utilizar: En nuestro laboratorio hemos desarrollado un modelo de artritis inducida por col��geno (AIC) mediante dos inmunizaciones de ratones DBA1/j con col��geno tipo II bovino y adyuvante de Freund. El modelo permiti�� establecer un ��ndice cl��nico mediante la hinchaz��n en las patas de los animales. Doce d��as posteriores a la primera inmunizaci��n los animales ser��n inyectados con CD tratadas con: 1. PBS, 2.Extracto total de F.hepatica (TE) + CII, 3. CpG + CII, 4. TE+CpG+CII Se realizar�� la observaci��n macrosc��pica diaria, a partir de los 7 d��as de la 2a inmunizaci��n Luego del sacrificio las articulaciones de las patas se preparar��n para realizar un an��lisis histol��gico. Se detectar�� en suero los niveles de anticuerpos IgG1 (perfil Th2) y de IgG2a (perfil Th1) mediante la t��cnica de ELISA. Se detectar�� tambi��n el perfil de citoquinas en los n��dulos drenantes por la t��cnica de ELISA y adicionalmente la poblaci��nes celulares de c��lulas T regulatorias (Treg) CD4+CD25+Foxp3 o c��lulas Tr1. Resultados esperados: Pensamos que el tratamiento de los animales que desarrollan AIC con CD semimaduras (por el tratamiento con TE y CpG), ser��n capaces de migrar a los ��rganos linfaticos y secretar TGF-be(inductora de c��lulas T reg), IL-10 (inductoras de c��lulas Tr1), IDO inhibitoria de la respuesta de Li T y promotor de c��lulas T reg, tambi��n podr��a generarse una respuesta Th2 (por la presencia de ant��genos del par��sito), y estas respuestas aisladas o en forma sin��rgica podr��an inhibir las respuestas de tipo Th17 y Th1 asociadas a la patolog��a en esta enfermedad. Importancia del proyecto: En el desarrollo de la artritis existe un aumento de la inmunidad mediada por c��lulas, asi como de la respuesta inmune humoral hacia componentes de la matriz del cart��lago. El tratamiento convencional de la artritis recae en general en el uso de inmunosupresores no-espec��ficos, los cuales poseen una variedad de efectos adversos y la inhibici��n de la respuesta inflamatoria no es espec��fica. En este proyecto proponemos el uso de CD tratadas con ant��genos del helminto F. hepatica y CpG ligando Tol que capacita a estas c��lulas para generar una respuesta adaptativa de tipo regulatoria, ��til en la inhibici��n de las respuestas inflamatorias como la que ocurre durante la progresi��n de artritis reumatoidea en un modelo experimental en ratones. We have shown that F. hepatica Ags-treated dendritic cells (DC) together with a TLRl ligand (CpG-ODN) produce high levels of anti-inflammatory cytokines (IL-10 and TGF-Beta) and low of proinflammatory cytokines (TNF, IL-6, IL -12). Hypothesis: The semimature phenotype achieved by DC, could be used to reduce inflammation in a model of autoimmune disease. The high production of IL-10 and TGF-Beta by these cells could directly or through the induction of T reg cells inhibit the inflammatory response. Objective: In this project we propose the immunization of DBA1 / j mice, susceptible to the development of collagen-induced arthritis (CIA) with F. hepatica-treated DC in conjunction with CpG-ODN to reduce clinical signs of disease. Materials: In our laboratory, we developed the CIA model by two immunizations of DBA1 / j mice with bovine type II collagen and Freund's adjuvant. The model allowed to stablish a clinical index by swelling in the legs of animals. Twelve days after the first immunization the animals are injected with DC treated with: 1. PBS 2. F.hepatica Extract (TE) + CII, 3. CpG + CII, 4. TE + CpG + CII Macroscopic observation will take place daily from 7 days of the 2nd immunization. After sacrifice the joints of the legs will be prepared for histological analysis. Serum levels of IgG1 antibodies (Th2 profile) and IgG2a (Th1 profile) will be detected by ELISA. It will also detected the cytokine profile in draining lymph nodes by ELISA and additionally the cell populations of regulatory T cells (Treg) CD4 + CD25 + Foxp3 or Tr1 cells. Expected results: We believe that the treatment of animals that had developed CIA with DC will be able to migrate to lymphatic organs and secrete TGF-B (T reg cell-inducing), IL-10 (inducing Tr1 cells), IDO (inhibitory of T cells and inducing of T reg cells) could alone or in synergy inhibit Th17-type responses and Th1 associated with the pathology in this disease.

HCV infection after liver transplantation is associated with lower levels of alloreactive CD4+CD25+CD45RO+IL-7R+high+ T cells

Aim: Expression of IL-7R discriminates alloreactive CD4 T cells (Foxp3 negative), from IL-7Rlow regulatory CD4 T cells (Foxp3 positive). Chronic hepatitis C virus infection (HCV) reduces expression of IL-7R on T cells thus promoting persistence of infection. The aim of this study was to analyze the effect of HCV infection on the expression of IL-7R of activated CD4+ T cells in liver transplant patients. Patients and methods: We analyzed PBMC from liver transplant recipients for the expression of CD4, CD25, FoxP3, IL-7R (24 HCV negative and 29 HCV-chronically infected). We compared these data with non-transplanted individuals (52 HCV-chronically infected patients and 38 healthy donors). Results: In HCV-infected liver transplant recipients, levels of CD4+CD25+CD45RO+IL-7R+ T cells were significantly reduced (10.5+/-0.9%) when compared to non-HCV-infected liver transplant recipients (17.6+/-1.4%) (P&lt;0.001), while both groups (HCV-infected and negative transplant recipients) had significantly higher levels than healthy individuals (6.6+/-0.9%) (P&lt;0.0001). After successful antiviral therapy (sustained antiviral response), 6 HCV-infected transplant recipients showed an increase of CD4+CD25+CD45RO+IL-7R+ T cells, reaching levels similar to that of non-HCVinfected recipients (10.73+/-2.63% prior therapy versus 21.7+/-6.3% after clearance of HCV). (P&lt;0.05) In 4 non-responders (i.e. HCVRNA remaining present in serum), levels of CD4+CD25+CD45RO+IL-7R+ T cells remained unmodified during and after antiviral treatment (11.8+/- 3.3% versus 11.3+/-3.3% respectively). Conclusions: Overall, these data indicate that CD4+CD25+CD45RO+IL-7R+ T cells appear to be modulated by chronic HCV infection after liver transplantation. Whether lower levels of alloreactive T cells in HCV-infected liver transplant recipients are associated with a tolerogenic profile remains to be studied.

An alteration in the levels of populations of CD4+ Treg is in part responsible for altered cytokine production by cells of aged mice which follows injection with a fetal liver extract.

We have shown previously that a fetal sheep liver extract (FSLE) containing significant quantities of fetal ovine gamma globin chain (Hbgamma) and LPS injected into aged (&gt;20 months) mice could reverse the altered polarization (increased IL-4 and IL-10 with decreased IL-2 and IFNgamma) in cytokine production seen from ConA stimulated lymphoid cells of those mice. The mechanism(s) behind this change in cytokine production were not previously investigated. We report below that aged mice show a &gt;60% decline in numbers and suppressive function of both CD4(+)CD25(+)Foxp3(+) Treg and so-called Tr3 (CD4(+)TGFbeta(+)), and that their number/function is restored to levels seen in control (8-week-old) mice by FSLE. In addition, on a per cell basis, CD4(+)CD25(-)Treg from aged mice were &gt;4-fold more effective in suppression of proliferation and IL-2 production from ConA-activated lymphoid cells of a pool of CD4(+)CD25(-)T cells from 8-week-old mice than similar cells from young animals, and this suppression by CD25(-)T cells was also ameliorated following FSLE treatment. Infusion of anti-TGFbeta and anti-IL-10 antibodies in vivo altered Treg development following FSLE treatment, and attenuated FSLE-induced alterations in cytokine production profiles.