A surface antigen of Orientia tsutsugamushi activates human monocyte-derived dendritic cells via nuclear factor-kB & p38 mitogen-activated protein kinase pathways

Background & objectives: Scrub typhus is a chigger-borne disease caused by Orientia tsutsugamushi. The immunological reactions to O. tsutsugamushi infection are not completely understood. In this study, we investigated the response of dendritic cells (DCs) to a major 56-kDa scrub typhus antigen Sta56. Methods: Monocyte-derived human DCs were incubated with different concentrations of recombinant Sta56 and analyzed for maturation based on phagocytic capacity, the ability to induce T-cell proliferation, expression of surface markers, cytokine secretion and activation of toll-like receptor (TLR)-dependent signalling pathways. Results: Treatment of DCs with Sta56 induced cell surface expression of CD80, CD83, CD86 and MHC Class II increased the production of interleukin-12 (IL-12) p40, IL-12 p70 and IL-10 and decreased DC phagocytic capacity. Furthermore, Sta56 increased the ability of DCs to activate T-cell proliferation and interferon-? secretion. TLR4-specific antibodies neutralized Sta56-elicited effects on DC maturation, suggesting direct interaction between Sta56 and TLR4. Moreover, Sta56 activated nuclear factor (NF)-?B and p38 mitogen-activated protein kinase (MAPK) signalling as evidenced by decrease in Sta56-induced cytokine production and surface marker expression by specific inhibitors helenalin and SB203580, respectively, and increase in I?B? and p38 phosphorylation and NF-?B-DNA binding. Interpretation & conclusions: Our results showed that the surface antigen of O. tsutsugamushi activated DCs through interaction with TLR4 and activation of MAPK and NF-?B signalling, suggesting Sta56 as a potential candidate molecule for the development of vaccine against scrub typhus.

Associação entre esclerose múltipla e alelos HLA-DRB1 em uma população miscigenada de Salvador, Ba, Brasil / Association between multiple sclerosis and HLA-DRB1 alleles in an admixed population of Salvador, Ba, Brazil

Introdução: a esclerose múltipla é uma doença que afeta preferencialmente o sistema nervoso central de mulheres jovens, causandolhes graus variáveis de incapacidades física e cognitiva. Etiologicamente associa fatores ambientais, biológicos, sócio-econômicos e genéticos, como por exemplo genes do MHC classe II, especialmente os alelos HLA-DRB1*. Objetivo: determinar a frequência dos alelos HLA DRB1* em portadores de esclerose múltipla atendidos no centro de referência do C.H.U.P.E.S, UFBA, no período de outubro de 2014 a abril de 2015 e associá-las a variáveis clínico-demográficas. Metodologia: estudo do tipo caso-controle, aprovado pelo comitê de ética da Faculdade de medicina da Universidade Federal da Bahia (CAAE: 3517134.0.0000.5577), que envolveu uma amostra de conveniência composta por 97 indivíduos, cujos dados clínico-demográficos foram coletados através de questionário desenvolvido para a pesquisa. A genotipagem dos alelos HLA-DRB1* foi realizada através da técnica HLA-DR SSO GenotypingTest. Resultados: a análise quantitativa revelou perfil genotípico do tipo HLA-DRB1*15 (20,5%), em mulheres (83,0%), das raças/etnias negra ou parda (75,0%), com faixa etária entre 30 e 39 anos (28,0%). Houve predomínio da forma clínica surtoremissiva da doença (76,0%), dentre os doentes com idade mais avançada (55,0%), sem permanência de sequela clínica (70,0%) e que usavam algum tipo de Interferon (58,0%). A análise qualitativa indicou maiores frequências, na forma progressiva de esclerose múltipla dos grupos alélicos HLA-DRB1*12 (22,0%), e dos alelos HLA-DRB1*13 (12,6%)e HLA-DRB1*15 (22,0%) naqueles indivíduos com a forma surtoremissiva. Negros e pardos demonstraram maior prevalência do alelo HLA-DRB1*15 (24,0%), enquanto que nos brancos houve maior prevalência do alelo HLA-DRB1*07 (20,0%). Conclusão: forte associação entre as frequências alélicas, esclerose múltipla e as variáveis raça/etnia e forma clínica da doença.

Analysis of swine leukocyte antigen class I gene profiles and porcine endogenous retrovirus viremia level in a transgenic porcine herd inbred for xenotransplantation research

Molecular characterization of swine leukocyte antigen (SLA) genes is important for elucidating the immune responses between swine-donor and human-recipient in xenotransplantation. Examination of associations between alleles of SLA class I genes, type of pig genetic modification, porcine endogenous retrovirus (PERV) viral titer, and PERV subtypes may shed light on the nature of xenograft acceptance or rejection and the safety of xenotransplantation. No significant difference in PERV gag RNA level between transgenic and non-transgenic pigs was noted; likewise, the type of applied transgene had no impact on PERV viremia. SLA-1 gene profile type may correspond with PERV level in blood and thereby influence infectiveness. Screening of pigs should provide selection of animals with low PERV expression and exclusion of specimens with PERV-C in the genome due to possible recombination between A and C subtypes, which may lead to autoinfection. Presence of PERV-C integrated in the genome was detected in 31.25% of specimens, but statistically significant increased viremia in specimens with PERV-C was not observed. There is a need for multidirectional molecular characterization (SLA typing, viremia estimation, and PERV subtype screening) of animals intended for xenotransplantation research in the interest of xeno-recipient safety.

Isolation, culture, characterization and cryopreservation of stem cells derived from amniotic mesenchymal layer and umbilical cord tissue of bovine fetuses / Isolamento, cultura, caracterização e criopreservação de células tronco derivadas de camada amniótica mesenquimal e de tecido do cordão umbilical de fetuses bovinos

Stem cells are undifferentiated cells with a high proliferation potential. These cells can be characterized by their in vivo ability to self-renew and to differentiate into specialized cell lines. The most used stem cell types, in both human and veterinary fields, are the mesenchymal stem cells (MSC) derived from bone marrow and adipose tissue. Nowadays, there is a great interest in using stem cells derived from fetal tissues, such as amniotic membrane (AM) and umbilical cord tissue (UCT), which can be obtained non-invasively at delivery time. Due to the scarcity of studies in bovine species, the aim of this study was to isolate, characterize, differentiate and cryopreserve MSC derived from the mesenchymal layer of amniotic membrane (AM), for the first time, and umbilical cord tissue (UCT) of dairy cow neonates after assisted delivery (AD) and from fetus at initial third of pregnancy (IT) obtained in slaughterhouse. Cells were isolated by enzymatic digestion of the tissue fragments with 0.1% collagenase solution. Six samples of AM and UCT at delivery time and six samples of AM and UCT at first trimester of pregnancy were subjected to morphology evaluation, imunophenotype characterization, in vitro osteogenic, adipogenic and chondrogenic differentiation and viability analysis after cryopreservation. All samples showed adherence to plastic and fibroblast-like morphology. Immunocytochemistry revealed expression of CD 44, NANOG and OCT-4 and lack of expression of MHC II in MSC from all samples. Flow cytometry demonstrated that cells from all samples expressed CD 44, did not or low expressed CD 34 (AM: IT-0.3%a, AD-3.4%b; UCT: 0.4%, 1.4%) and MHC II (AM: IT-1.05%a, AD-9.7%b; UCT: IT-0.7%a, AD-5.7%b). They were also capable of trilineage mesenchymal differentiation and showed 80% viability after cryopreservation. According to the results, bovine AM and UCT-derived cells, either obtained at delivery time or from slaughterhouse, are a painless and non-invasive source of MSC and can be used for stem cell banking.(AU)

As células tronco mesenquimais (CTMs) estão presentes na maioria dos tecidos adultos e possuem grande capacidade de multiplicação. Quando cultivadas in vitro são capazes de se auto renovar e dar origem a novos tipos celulares. As células tronco mais utilizadas, tanto na medicina humana como na medicina veterinária são as células tronco mesenquimais derivadas da medula óssea e do tecido adiposo. Atualmente, uma grande tendência para a utilização de CTMs obtidas de tecidos fetais, como a membrana amniótica (MA), matriz extravascular do cordão umbilical (TCU) e sangue do cordão umbilical (SCU) pode ser observada, já que estas fontes podem ser colhidas no momento do parto por uma técnica não invasiva. Dessa forma, o objetivo deste estudo foi isolar, caracterizar, diferenciar e criopreservar CTMs obtidas de MA e TCU de fetos bovinos colhidos no momento do parto e de fetos do terço inicial da gestação em abatedouro-frigorífico. As células foram recuperadas por meio de digestão enzimática tecidual, realizada com solução de colagenase 0,1%. Foram colhidas amostras de MA e TCU no momento do parto (n=6) e de MA e TCU no terço inicial de gestação (n=6), as quais foram submetidas às análises morfológicas, imunofenotípica por imunocitoquímica e citometria de fluxo, diferenciações in vitro nas linhagens osteogênica, adipogênica e condrogênica e ainda, avaliação da viabilidade após a criopreservação por citometria de fluxo. Todas as amostras dos diferentes grupos demonstraram adesão ao plástico e morfologia fibroblastóide. No ensaio imunocitoquímico todas as amostras foram imunomarcadas para CD44, NANOG e Oct-4, com ausência de marcação para MHC II. Na análise imunofenotipica por citometria de fluxo, todas as amostras apresentaram marcação para CD44, ausência de marcação para ou baixíssima expressão de CD34 (MA: TI-0,3%a, PA-3.4%b; TCU: TI-0,4%, PA-1.4%) e nula ou baixa expressão de MHC II (MA: TI-1.5%a, PA-9.7%b; UCT: TI-0.7%a, PA-5.7%b. Apresentaram também capacidade de diferenciação in vitro nas três linhagens mesodermais e quando analisadas pós criopreservação por citometria de fluxo, todas as amostras apresentaram viabilidade de 80%. Estes resultados indicam que MA e TCU, obtidos tanto no momento de parto como em abatedouro, de fetos bovinos podem ser utilizados como fonte não invasiva e indolor de CTMs e possibilitam a formação de bancos de armazenamento de células.(AU)

Heterogeneous Nuclear Ribonucleoprotein A2B1 Exerts a Regulatory Role in Lipopolysaccharide-stimulated 38B9 B Cell Activation

Major histocompatibility complex (MHC) class II molecules, which are recognized for their primary function of presenting an antigen to the T cell receptor, are involved in various signaling pathways in B cell activation. We identified heterogeneous nuclear ribonucleoprotein (hnRNP) A2B1 as an MHC class II molecule-associated protein involved in MHC class II-mediated signal transduction in lipopolysaccharide (LPS)-stimulated 38B9 B cells. Although the function of hnRNP A2B1 in the nucleus is primarily known, the level of hnRNP A2B1 in the cytoplasm was increased in LPS-stimulated 38B9 cells, while it was not detected in the cytoplasm of non-treated 38B9 cells. The silencing of hnRNP A2B1 expression using siRNA disturbed B cell maturation by regulation of mitogen-activated protein kinase signaling, NF-κB activation, and protein kinase B activation. These results suggest that hnRNP A2B1 is associated with MHC class II molecules and is involved in B cell activation signaling pathways in LPS-stimulated 38B9 cells.

Tolerancia inmune al tratamiento con toxina botulínica tipo A

Actualmente la neurotoxina botulínica tipo A es utilizada en el tratamiento de diversos trastornos relacionados con hiperactividad muscular localizada y para aplicación cosmética. La neurotoxina botulínica tipo A está conformada por un núcleo proteico de 150 kDa asociada a un complejo de hasta 6 proteínas auxiliares no tóxicas. En la respuesta al tratamiento crónico con neurotoxina botulínica tipo A se ha descrito una disminución entre los periodos de aplicación de la toxina, lo cual se puede expresar como una disminución del efecto de la neurotoxina botulínica tipo A. Se buscó identificar el mecanismo inmunológico, así como las principales moléculas participantes en el desarrollo de la tolerancia inmune hacia la neurotoxina botulínica tipo A. Se llevó a cabo una búsqueda sistematizada en las siguientes bases de datos: PubMed, Imbiomed y Scielo. Tomando en consideración artículos de investigación básica y clínica publicados después del año 2000, con especial interés en las publicaciones realizadas en los últimos 3 años. La disminución entre los periodos de aplicación de la neurotoxina botulínica tipo A, es generada por tolerancia inmune mediada principalmente por la acción biológica del complejo de histocompatibilidad tipo II, antígeno CD19 y la interleucina-6. Provocando un aumento en las dosis requeridas para mantener un tratamiento efectivo con neurotoxina botulínica tipo A, esto no significa una dependencia a la toxina botulínica. Significando una disminución en la efectividad de un tratamiento de enfermedades cronicas.

Nowadays, botulinum toxin Type A is used for various disease treatment with hyperactivity muscular located and cosmetic using. botulinum toxin Type A is conformed to a protein nucleus of 150 kDa associated to a complex of 6 auxiliary non-toxic proteins. In the response to chronic treatment with botulinum toxin Type A a decrease of interlude time for toxin application has been described, which can be expressed as an effect decrease of botulinum toxin Type A. Immune mechanism was identified by a search, as well as main molecules involved in development of tolerance immune versus botulinum toxin Type A. Systematic research was performed in data bases as: Pubmed, Scielo, Imbiomed. Basics and clinical research articles published after 2000 were consulted. Focusing in publications with 4 or less years since their publication. The reduction of periodicity of dosage of BoNTA is induced by immune tolerance, it is principally mediated by major histocompatibility, antigen CD19 and interleukin-6. The immune tolerance induce an increases of dosage required for establish an effective treatment with botulinum toxin Type A; however, this does not mean a botulinum toxin dependence. Development of immune tolerance to treatments with BoNTA induces a low effectiveness of chronic treatment and evidence the necessity to researching new treatments that reduce the immune tolerance of botulinum toxin Type A. Meaning an effectivity reduces of a treatment to chronic diseases.

Lack of association between alopecia areata and HLA class I and II in a southeastern Brazilian population

Abstract: Background: Alopecia areata (AA) is a common disorder of unknown etiology that affects approximately 0.7% to 3.8% of patients among the general population. Currently, genetic and autoimmune factors are emphasized as etiopathogenic. Studies linking Human Leukocyte Antigens (HLA) to AA have suggested that immunogenetic factors may play a role in the disease's onset/development. Objectives: To investigate an association between AA and HLA class I/II in white Brazilians. Methods: Patients and control groups comprised 33 and 112 individuals, respectively. DNA extraction was performed by column method with BioPur kit. Allele's classification was undertaken using the PCR-SSO technique. HLA frequencies were obtained through direct counting and subjected to comparison by means of the chi-square test. Results: Most patients were aged over 16, with no familial history, and developed partial AA, with no recurrent episodes. Patients showed a higher frequency of HLA-B*40, HLA-B*45, HLA-B*53 and HLA-C*04 compared with controls, although P was not significant after Bonferroni correction. Regarding HLA class II, only HLA-DRB1*07 revealed statistical significance; nevertheless, it featured more prominently in controls than patients (P=0.04; Pc=0.52; OR=0.29; 95%; CI=0.07 to 1.25). P was not significant after Bonferroni correction. Conclusions: The development of AA does not seem to be associated with HLA in white Brazilians, nor with susceptibility or resistance. The studies were carried out in populations with little or no miscegenation, unlike the Brazilian population in general, which could explain the inconsistency found.

Detection of Foreign Antigen-specific CD4+Foxp3+ Regulatory T Cells by MHC Class II Tetramer and Intracellular CD154 Staining

The unrestricted population of CD4+Foxp3+ regulatory T (Treg) cells, which have been known to control the expression of autoimmune diseases and protective immunity to inflammatory reactions, has led to greater appreciation of functional plasticity. Detecting and/or isolating Ag-specific CD4+Foxp3+ Tregs at the single cell level are required to study their function and plasticity. In this study, we established and compared both MHC class II tetramer and intracellular CD154 staining, in order to detect CD4+Foxp3+ Treg specific for foreign Ag in acute and chronic infections with lymphocytic choriomeningitis virus (LCMV). Our results revealed that MHC class II tetramer staining showed a lower detection rate of LCMV GP66-77-specific CD4+ T cells because most of MHC class II tetramers were unbound and unstable when combined staining was performed with intracellular cytokines. In contrast, intracellular CD154 staining was revealed to be easier and simple for detecting LCMV GP66-77-specific CD4+ T cells, compared to MHC class II tetramer staining. Subsequently, we employed intracellular CD154 staining to detect LCMV GP66-77-specific CD4+Foxp3+ Tregs using Foxp3GFP knock-in mouse, and found that LCMV GP66-77-specific CD4+Foxp3+ Tregs and polyclonal CD4+Foxp3+ Tregs showed differential expansion in mice infected with LCMV Arms or Cl13 at acute (8 and 13 days pi) and chronic phases (35 days pi). Therefore, our results provide insight into the valuable use of intracellular CD154 staining to detect and characterize foreign Ag-specific CD4+Foxp3+ Treg in various models.

HLA among Brazilian patients with spontaneous chronic urticaria and positive autologous serum skin test / HLA em brasileiros com urticária crônica espontânea e teste cutâneo do soro autólogo positivo

BACKGROUND: An association between class I and II alleles of the major histocompatibility complex and idiopathic chronic urticaria has previously been observed in different populations, but there are still no studies on Brazilian populations in this regard. OBJECTIVE: The involvement of the major histocompatibility complex classes I and II (loci A, B and DR) in Brazilian patients with idiopathic chronic urticaria and a positive autologous serum skin test was investigated and compared with a healthy population group. METHODS: DNA was extracted from the blood of 42 patients with idiopathic chronic urticaria and major histocompatibility complex classes I and II alleles were determined using the polymerase chain reaction and a laboratory test for oligonucleotide hybridization using a single-filament probe. The frequencies of these alleles in patients with chronic urticaria were compared with the frequencies in 1000 genetically unrelated voluntary blood donors from the same region of Brazil. The diagnosis of idiopathic chronic urticaria was based on the patients' clinical history and routine laboratory tests. Only the patients with positive autologous serum skin test were selected. The allele distribution resulted from the patient and control groups were analyzed using odds ratios and 95% confidence intervals. RESULTS: No statistically significant differences were found between the positive autologous serum skin test patients with chronic urticaria and the control group. CONCLUSIONS: We found that in this population group, there was no specific association between the HLA alleles studied and chronic urticaria. We believe that further population studies are needed in order to investigate the possible existence of this association.

FUNDAMENTOS: A associação entre os alelos do MHC classe I e II e a urticária crônica idiopática tem sido previamente constatada em diferentes populações, sendo que na população brasileira ainda não existem estudos a este respeito. OBJETIVOS: Foi estudado o envolvimento do MHC classe I e II (locci A, B e DR) em pacientes brasileiros com urticária crônica idiopática e teste cutâneo do soro autólogo positivo, comparando-se com um grupo populacional saudável. MÉTODOS: O DNA foi extraído do sangue de 42 pacientes com urticária crônica idiopática e o MHC classe I e II determinado por reação em cadeia da polimerase e teste laboratorial de hibridização de oligonucleotídeo com sonda de filamento único. A freqüência destes alelos em pacientes com urticária crônica idiopática foi comparada com a de 1000 doadores de sangue voluntários e geneticamente não relacionados, da mesma região do Brasil. O diagnóstico de urticária crônica idiopática foi baseado na história clínica do paciente e exames laboratoriais de rotina; foram selecionados apenas os pacientes com teste cutâneo do soro autólogo positivo. O resultado da distribuição alélica entre o grupo de pacientes e o grupo controle foi analisado através do odds rate com o cálculo do intervalo de confiança de 95% (95% IC). RESULTADOS: Não foram encontradas diferenças com significância estatística entre os pacientes com urticária crônica teste cutâneo do soro autólogo positivos e o grupo controle. CONCLUSÕES: Verificamos que neste grupo populacional estudado não houve associação específica entre os alelos HLA estudados e a urticária crônica; acreditamos na necessidade de outros estudos populacionais, para podermos verificar a possível existência desta associação.

Vitamin C Up-regulates Expression of CD80, CD86 and MHC Class II on Dendritic Cell Line, DC-1 Via the Activation of p38 MAPK

Vitamin C is an essential water-soluble nutrient which primarily exerts its effect on host defense mechanisms and immune homeostasis, but the mechanism related to immune-potentiation is poorly understood. Since dendritic cells (DCs) are known as a potent antigen presenting cell (APC) that could enhance the antigen specific immune responses, we investigate the effects of vitamin C on activation of DCs and its related mechanism by using dendritic cell lines, DC-1. First, we found that there was no damage on DC-1 by 2.5 mM of vitamin C. In the presence of vitamin C, the expression of CD80, CD86, and MHC molecules was increased, but it was decreased by the pre-treatment of SB203580, p38 MAPK-specific inhibitor. We confirmed the phosphorylation of p38 MAPK was increased by the treatment of vitamin C. Taken together, these results suggest that vitamin C could enhance the activity of dendritic cells via the up-regulation of the expression of CD80, CD86, and MHC molecules and the activation of p38 MAPK is related to this process.

Introduction of the CIITA gene into tumor cells produces exosomes with enhanced anti-tumor effects

Exosomes are small membrane vesicles secreted from various types of cells. Tumor-derived exosomes contain MHC class I molecules and tumor-specific antigens, receiving attention as a potential cancer vaccine. For induction of efficient anti-tumor immunity, CD4+ helper T cells are required, which recognize appropriate MHC class II-peptide complexes. In this study, we have established an MHC class II molecule-expressing B16F1 murine melanoma cell line (B16F1-CIITA) by transduction of the CIITA (Class II transactivator) gene. Exosomes from B16-CII cells (CIITA-Exo) contained a high amount of MHC class II as well as a tumor antigen TRP2. When loaded on dendritic cells (DCs), CIITA-Exo induced the increased expression of MHC class II molecules and CD86 than the exosomes from the parental cells (Exo). In vitro assays using co-culture of immunized splenocytes and exosome-loaded DCs demonstrated that CIITA-Exo enhanced the splenocyte proliferation and IL-2 secretion. Consistently, compared to B16-Exo, CIITA-Exo induced the increased mRNA levels of inflammatory cytokines such as TNF-alpha, chemokine receptor CCR7 and the production of Th1-polarizing cytokine IL-12. A tumor preventive model showed that CIITA-Exo significantly inhibited tumor growth in a dose-dependent manner. Ex vivo assays using immunized mice demonstrated that CIITA-Exo induced a higher amount of Th1-polarized immune responses such as Th1-type IgG2a antibodies and IFN-gamma cytokine as well as TRP2-specific CD8+ T cells. A tumor therapeutic model delayed effects of tumor growth by CIITA-Exo. These findings indicate that CIITA-Exo are more efficient as compared to parental Exo to induce anti-tumor immune responses, suggesting a potential role of MHC class II-containing tumor exosomes as an efficient cancer vaccine.

Regulation of Innate Immunity via MHC Class II-mediated Signaling; Non-classical Role of MHC Class II in Innate Immunity

MHC class II has long been known to play a classical role in antigen presentation and to act as a signal transducer capable of inducing the adaptive immunity needed to produce pathogen specific antibodies. However, it has recently been revealed that MHC class II can also promote the activation of Toll-like receptor mediated signaling by functioning as an adapter. This means that in addition to its classical function of adaptive immunity, MHC class II also plays an intriguing role in the mechanisms that regulate innate immunity. That being the case, queries inevitably arise regarding the fact that many pathogens have tried to control the induction of MHC class II so as to escape the host immune response. Liu et al (Nat Immunol 2011;12:416-424) demonstrated that intracellular MHC class II interacted with Btk, and that this activated Btk promoted TLR signaling via Myd88 and TRIF. The results of this study provide insight regarding the possibility of a novel role for MHC class II, which was heretofore regarded solely as a classical molecule involved in adaptive immune responses, as a regulator of innate immune responses.

Adenovirus-mediated MHC class II transactivator mutant in gene therapy of mouse experimental autoimmune thyroiditis / 第二军医大学学报

Objective: To observe the therapeutic effect of major histocompatibility complex (MHC) class II transactivator mutant (C II TAm) for gene therapy of mouse experimental autoimmune thyroiditis (EAT) and explore the possible mechanisms. Methods: Thirty-one healthy female CBA/J mice were randomly divided into 4 groups, namely EAT model group(n= 8), C II TAm therapy group(n=9), GFP control group (n=9), and normal control group(n=5). Animals in the first 3 groups were immunized with porcine thyroglobulin (pTg) and complete or incomplete Freud's adjuvant (CFA/IFA) to establish EAT model; mice in the C II TAm therapy group and GFP control group were also treated by intravenous recombinant adenovirus Ad-CMV-C II TAm and Ad-GFP, respectively, while those in the EAT model group were injected with equal volume of normal saline. Mice in the normal control group received no special treatment. All mice were sacrificed on the 29th day after the first immunization. The thyroid pathological changes were examined using H-E staining; the expression of MHC II molecules in the thyroid was examined using immunohistochemical staining; the spleen lymphocyte proliferation and IFN-γ secretion stimulated by pTg were examined in their culture supernatant; the titer of plasma anti-pTg autoantibody was assayed by ELISA; and the expression of inducible costimulator (ICOS) on CD4+ T cells in both peripheral blood and spleen was analyzed by flow cytometry. Results: H-E staining showed that the infiltration index of thyroid lymphocyte in the C II TAm therapy group (0.3±0.5) was significantly lower than that in the EAT model group (1.4±0.4) and the GFP control group (1.5±0.2, both P<0.01). Immunohistochemical staining showed diffused expression of MHC II molecules in the thyroid of the EAT model group and GFP control group, compared to very weak expression in the C II TAm therapy group and the negative expression in the normal control group. The lymphocyte stimulation index (SI) against 80 μg/ml pTg in the C II TAm therapy group was significantly lower than that in the EAT model group and the GFP control group(P<0.05). The IFN-γ secretion in the culture supernatants showed a similar difference as SI in all the groups (P<0.01). The titer of plasma anti-pTg autoantibody in the C II TAm therapy group was significantly lower than those in the EAT model group and the GFP control group (both P<0.01). The positive rate of ICOS on CD4+ T cells in the C II TAm therapy group was significantly lower than that in the EAT model group and the GFP control group(both P<0.01). Conclusion: Ad-CMV-C II TAm recombinant adenovirus can inhibit the MHC II molecule expression in the thyroid of EAT mouse and the proliferation of self-reactive T cells, attenuate the inflammatory cells infiltration in the thyroid, and decrease the titer of plasma anti-pTg autoantibody, indicating that C II TA mutants might have therapeutic effect for EAT.

Sepsis Mortality in CIITA Deficient Mice is Associated with Excessive Release of High-mobility Group Box 1

BACKGROUND: Down regulation of major histocompatibility complex class II transactivator (CIITA) has been identified as a major factor of immunosuppression in sepsis and the level of CIITA expression inversely correlates with the degree of severity. However, it has not been fully elucidated whether the lower expression of CIITA is a cause of disease process or a just associated sign. Here we determined whether the CIITA deficiency decreased survival rate using murine sepsis model. METHODS: Major histocompatibility complex class II (MHC-II) deficient, CIITA deficient and wild type B6 mice were subjected to cecal ligation puncture (CLP) surgery. CIITA and recombination activation gene (RAG)-1 double deficient mice were generated to test the role of lymphocytes in CIITA-associated sepsis progression. RESULTS: Sepsis mortality was enhanced in CIITA deficient mice, not by impaired bacterial clearance resulted from CD4 T cell depletion, but hyper-inflammatory response such as excessive release of a pro-inflammatory cytokine, high-mobility group box 1 (HMGB1). CONCLUSION: Our results demonstrate that CIITA deficiency affects the course of sepsis via the unexpected function of CIITA, regulation of cytokine release.

Low expression of antigen-presenting and costimulatory molecules by lung cells from tuberculosis patients

Costimulatory and antigen-presenting molecules are essential to the initiation of T cell immunity to mycobacteria. The present study analyzed by immunocytochemistry, using monoclonal antibodies and alkaline phosphatase-anti-alkaline phosphatase method, the frequency of costimulatory (CD86, CD40, CD40L, CD28, and CD152) and antigen-presenting (MHC class II and CD1) molecules expression on human lung cells recovered by sputum induction from tuberculosis (TB) patients (N = 22) and non-TB controls (N = 17). TB cases showed a statistically significant lower percentage of HLA-DR+ cells than control subjects (21.9 ± 4.2 vs 50.0 ± 7.2 percent, P < 0.001), even though similar proportions of TB cases (18/22) and control subjects (16/17, P = 0.36) had HLA-DR-positive-stained cells. In addition, fewer TB cases (10/22) compared to control subjects (16/17) possessed CD86-expressing cells (P = 0.04; OR: 0.05; 95 percentCI = 0.00-0.51), and TB cases expressed a lower percentage of CD86+ cells (P = 0.04). Moreover, TB patients with clinically limited disease (£1 lobe) on chest X-ray exhibited a lower percentage of CD86-bearing cells compared to patients with more extensive lung disease (>1 lobe) (P = 0.02). The lower expression by lung cells from TB patients of HLA-DR and CD86, molecules involved in antigen presentation and activation of T cells, may minimize T cell recognition of Mycobacterium tuberculosis, fostering an immune dysfunctional state and active TB.

Overexpression of CIITA in T Cells Aggravates Th2-Mediated Colitis in Mice

The MHC class II transactivator (CIITA) is the master transcriptional regulator of genes involved in MHC class II restricted antigen presentation. Previously we suggested another role of CIITA in Th1/Th2 balance by demonstrating that forced expression of CIITA in murine T cells repressed Th1 immunity both in vitro and in vivo. However, the results were contradictory to the report that CIITA functioned to suppress the production of Th2 cytokine by CD4+T cells in CIITA deficient mice. In this study, we investigated the influence of constitutive expression of CIITA in T cells on Th2 immune response in vivo using murine experimental colitis model. In the dextran sodium sulfate-induced acute colitis, a disease involving innate immunity, CIITA transgenic mice and wild type control mice showed similar progression of the disease. However, the development of oxazolone-induced colitis, a colitis mediated by predominantly Th2 immune response, was aggravated in CIITA-transgenic mice. And, CD4+T cells from the mesenteric lymph node of CIITA-transgenic mice treated with oxazolone exhibited a high level of IL-4 secretion. Together, these data demonstrate that constitutive expression of CIITA in T cells skews immune response to Th2, resulting in aggravation of Th2-mediated colitis in vivo.

Delayed allograft rejection by the suppression of class II transactivator

We examined the effect of class II transactivator (CIITA) down-modulation on allograft rejection. To inhibit the function of CIITA, we constructed a series of CIITA mutants and found one exhibiting the dominant-negative effect on the regulation of major histocompatibility complex (MHC) class II expression. To test whether the CIITA dominant-negative mutant reduces immunogenecity, CIITA-transfected melanoma cells were injected into allogeneic host and assessed for immune evading activity against host immune cells. We demonstrated that the CIITA dominant-negative mutant allowed tumor nodules to develop earlier in the lung than control by this tumor challenge study. Furthermore, skin grafts deficient for CIITA also survived longer than wild-type in allogeneic hosts. Both the tumor challenge and skin graft studies suggest the inhibition of CIITA molecules in donor tissue would be beneficial to the control of allo-response.

Receptor activator of NF-kappaB ligand enhances the activity of macrophages as antigen presenting cells

Receptor activator of NFkappaB ligand (RANKL) is known as a key regulator of osteoclastogenesis. However, the fact that fibroblasts and periodontal ligament cells express RANKL in response to bacterial substances, suggests that RANKL may have evolved as a part of the immunity to infection. As RANKL increases the survival and activity of dendritic cells, it may have similar effects on macrophages. To address this issue, we studied the effect of RANKL on various functions of macrophages using mouse bone marrow derived macrophages. RANKL enhanced the survival of macrophages and up-regulated the expression of CD86. RANKL-treated macrophages showed increased allogeneic T cell activation and phagocytic activity compared to control cells. In addition, RANKL increased the expression of TNFalpha, MCP-1, and IL-6 but not of IL-10, IL-12, IFN-gamma, and iNOS. Collectively, RANKL augmented the activity of macrophages especially as antigen presenting cells, suggesting its new role in immune regulation.

Ischemia-Reperfusion Injury Enhances Dendritic Cell Infiltration and TLR2 Expression in Rat Kidneys / 대한신장학회잡지

BACKGROUND: It is well known that ischemia/reperfusion (I/R) injury enhances immunogenicity. But, its influence on innate immunity is still undetermined. This study was performed to evaluate whether I/R injury activates innate immunity in rat kidneys. METHODS: Sprague-Dawley rats were used. Ischemic injury was induced by clamping both renal arteries for 45 minutes. Sham operation was performed in a similar manner, except clamping the renal vessels. Rats were sacrificed on day 1, 3, 5, and 7 after I/R injury. Activation of innate immunity was evaluated in terms of toll-like receptor (TLR), dendritic cells and MHC class II antigen. TLR2 mRNA expression was detected by RT-PCR, and in situ hybridization. Dendritic cells and MHC class II antigens were detected with OX62 and OX6 monoclonal antibodies by immunohistochemistry. RESULTS: TLR2 mRNA was significantly increased in on day 3 and 5 after I/R injury (1 d: 120+/-2%, 3 d: 137+/-5%, 5 d: 173+/-5% 7 d: 120+/-8% vs. 100+/-11%, p<0.05 vs. sham), and in situ hybridization signal was observed on proximal, distal tubules, and interstitial cells. Compared to the sham-operated rat kidneys, number of dendritic cells significantly increased from day 1 to day 7 after I/R injury (1 d: 22.9+/-2.4, 3 d: 25.8+/-4.9, 5 d: 26.5+/-2.3, 7 d: 24.3+/-1.6 vs. 13.3+/-1.1, p<0.05 vs. sham) with peak value at day 5. Increased expression of MHC class II antigen was observed in the proximal tubules and interstitial cells in I/R injured rat kidney and there was maximal MHC class II protein level on day 3 after I/R injury. CONCLUSION: Ischemia-reperfusion injury itself can activate innate immunity on early period after injury.

Age-Related Changes of MHC Class II-immunoreactive Dendritic Cells in Rat Brain

PURPOSE: Dendritic cells are antigen presenting cells(APC) that express class II major histocompatibility complex gene products on their surface. Recently, it was proved that dendritic cells activate antitumor immunity for intracranial germ cell tumor. The aim of the present study is to investigate the age-related changes of MHC class II-immunoreactive dendritic cells in the rat brain. METHODS: Male rats(Sprague-Dawley) were sacrificed at 1 month, 12 months and 24 months after birth. Brains were removed and sliced in rat brain matrix. Brain slices were cryosectioned coronally at interaural 5.70-6.70 mm. Brain tissue sections were immunohistochemically reacted with monoclonal MHC class II antibody. RESULTS: MHC class II-immunoreactive dendritic cells were observed in choroid plexuses and white matter(corpus callosum, cerebral peduncle and external capsule). The number of MHC class II-immunoreactive dendritic cells was slightly increased with age. As age increases, shapes of MHC class II-immunoreactive dendritic cells became more complex and aggregated together. CONCLUSION: As age increases, MHC class II-immunoreactive dendritic cells in choroid plexuses and white matter of the brain became not only more complex in shape, but also increased in number to improve immunity.