RESUMO
Antibody-mediated allograft rejection (AMR) causes more kidney transplant failure than any other single cause. AMR is mediated by antibodies recognizing antigens expressed by the graft, and antibodies generated against major histocompatibility complex (MHC) mismatches are especially problematic. Most research directed towards the management of clinical AMR has focused on identifying and characterizing circulating donor-specific HLA antibody (DSA) and optimizing therapies that reduce B-cell activation and/or block antibody secretion by inhibiting plasmacyte survival. Here we describe a novel set of reagents and techniques to allow more specific measurements of MHC sensitization across different animal transplant models. Additionally, we have used these approaches to isolate and clone individual HLA-specific B cells from patients sensitized by pregnancy or transplantation. We have identified and characterized the phenotypes of individual HLA-specific B cells, determined the V(D)J rearrangements of their paired H and L chains, and generated recombinant antibodies to determine affinity and specificity. Knowledge of the BCR genes of individual HLA-specific B cells will allow identification of clonally related B cells by high-throughput sequence analysis of peripheral blood mononuclear cells and permit us to re-construct the origins of HLA-specific B cells and follow their somatic evolution by mutation and selection.
Assuntos
Subpopulações de Linfócitos B/imunologia , Rejeição de Enxerto/imunologia , Antígenos HLA/imunologia , Memória Imunológica , Isoanticorpos/sangue , Animais , Especificidade de Anticorpos , Linhagem Celular , Células Cultivadas , Evolução Clonal , Células Clonais , Feminino , Rearranjo Gênico do Linfócito B , Genes Reporter , Antígenos de Histocompatibilidade/biossíntese , Antígenos de Histocompatibilidade/imunologia , Humanos , Imunoglobulina G/imunologia , Indicadores e Reagentes , Isoanticorpos/imunologia , Ativação Linfocitária , Macaca mulatta , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , RNA Guia de Cinetoplastídeos/genética , Transplante de Pele , Especificidade da Espécie , Organismos Livres de Patógenos Específicos , Recombinação V(D)J , Microglobulina beta-2/antagonistas & inibidores , Microglobulina beta-2/genéticaRESUMO
The present study aimed to investigate the differential expression and clinical significance of histone methyltransferase G9a, histone H3K9me2 and histone H3K9me1 in human brain glioma and adjacent tissue samples. It also aimed to observe the effect and mechanism of BIX01294, as an inhibitor of methyltransferase G9a, on the proliferation, apoptosis, methylation of H3K9 and H3K27, and the acetylation in U251 glioma cells in vitro. The differential expression of methyltransferase G9a, histone H3K9me2 and histone H3K9me1 in in human brain glioma and adjacent tissues were analyzed by immunohistochemistry, a growth curve of U251 cells following treatment with BIX01294 was determined using the MTT assay. In addition, the apoptosis percentage of U251 cells was analyzed by TUNEL assay and the expression levels of apoptosisassociated proteins, including Bcell lymphoma 2 (Bcl2), Bcl2associated X protein (Bax), caspase9 and caspase3, and the acetylation of histones, including H3K27me1, H3K27me2 and H3 in U251 were analyzed by western blot following BIX01294 treatment. The positive rate of G9a in glioma tissues was 86% (43/50), which was significantly different from 42% (21/50) in adjacent tissues (P<0.01). The positive rate of H3K9me2 in glioma tissues was 82% (41/50), which was significantly different from 38% (19/50) in adjacent tissues (χ²=18.38; P<0.01). The expression of G9a and H3K9me2 were associated with the World Health Organization (WHO) glioma grade. The positive rate of H3K9me1 in glioma tissues was 54% (27/50) and 44% (22/50) in adjacent tissues, though this result was not significantly different (χ²=1.21, P>0.05). BIX01294 inhibited the proliferation of U251, downregulated expression of Bcl2, and upregulated expression of Bax, caspase3 and caspase9, and induced apoptosis of U251. BIX01294 downregulated H3K9me1, H3K9me2, H3K27me1 and H3K27me2, however, it did not affect the acetylation of H3K9me3 and H3. High expression of G9a and H3K9me2 in glioma tissue samples was associated with the WHO grade, which indicated that G9a and H3K9me2 may promote generation and development of glioma. BIX01294 inhibited proliferation and induced apoptosis of glioma cells, changes in methylation of H3K9 and H3K27 resulting in conformational changes of chromosome may be an underlying mechanism. BIX01294 may be a potential novel therapeutic agent in the treatment of glioma.
Assuntos
Azepinas/administração & dosagem , Metilação de DNA/genética , Glioma/tratamento farmacológico , Antígenos de Histocompatibilidade/biossíntese , Histona-Lisina N-Metiltransferase/biossíntese , Quinazolinas/administração & dosagem , Adulto , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/genética , Glioma/patologia , Antígenos de Histocompatibilidade/genética , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/genética , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/biossínteseRESUMO
Recent studies have shown that epigenomic modifications are significantly associated with neuronal differentiation. Many neuronal specific genes contain the repressor element-1 (RE-1), which recruits epigenetic modulators, such as the histone methyltransferase G9a and interrupts the expression of neuronal genes in non-neuronal cells. This study investigated the functional role of G9a during neuronal differentiation of human bone marrow mesenchymal stem cells (BM-MSCs). Human BM-MSCs treated with the G9a inhibitor BIX01294 showed an increased expression of various neuronal-lineage genes. Using genomic sequence analysis, we identified RE-1 consensus sequences in the proximal region of several neuronal-specific genes. Chromatin immunoprecipitation (ChIP) assay results have showed that H3K9me2 (dimethylation of lysine 9 on histone 3) occupancy at RE-1-containing sequences from neuronal-specific genes was significantly decreased in BIX01294-MSCs. When BIX01294-MSCs were differentiated with neuronal induction medium, cells differentiated more effectively into neuron-like cells, complete with a cell body and dendrites. Expression of neuronal-specific genes containing the RE-1 sequences was significantly increased in differentiated BIX01294-MSCs, as confirmed by immunocytochemical staining and immunoblotting. Thus, this study shows that BIX01294 pretreated human BM-MSCs can be effectively differentiated into neuron-like cells by induced expression of neuronal-specific genes containing RE-1 sequences.
Assuntos
Diferenciação Celular/fisiologia , Antígenos de Histocompatibilidade/biossíntese , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/biossíntese , Células-Tronco Mesenquimais/metabolismo , Neurônios/metabolismo , Proteínas Repressoras/biossíntese , Azepinas/farmacologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Quinazolinas/farmacologia , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologiaRESUMO
AIMS: Histone methyltransferase G9a has been primarily understood as a co-repressor of gene expression, but it has been shown that G9a also positively regulates nuclear receptor-mediated transcription. MCM7, a critical component of the DNA replication licensing complex, is amplified and overexpressed in a variety of human malignancies. The objectives of the present study were to study the relationship between the expression of G9a and MCM7 and the pathological grade, clinical stage and prognosis of oesophageal squamous cell carcinoma (OSCC). METHODS AND RESULTS: We collected 139 formalin-fixed and paraffin-embedded tissues from patients with OSCC and surveyed them by tissue microarray-based immunohistochemical staining. Associations between the expression of MCM7 and G9a and clinicopathological parameters and prognosis of OSCC were examined. From tissue microarray immunohistochemistry staining results, we found that nuclear staining intensity for MCM7 and G9a was associated with histological grade (both P < 0.001), tumour depth (P = 0.050, 0.034), lymph node metastasis (P = 0.001, 0.009) and tumour stage (P < 0.001, =0.003). G9a expression was correlated with that of MCM7. G9a overexpression independently predicted poor cancer-specific survival in OSCC (hazard ratio 0.05, 95% confidence interval 0.006-0.417, P = 0.006) and MCM7 (hazard ratio 0.05, 95% confidence interval 0.013-0.441, P = 0.004). OSCC patients whose tumours showed double-positive expression of G9a and MCM7 (G9a(+) MCM7(+) ) had much shorter survival than those from either the G9a or MCM7 low expression groups (G9a(-) MCM7(-) , G9a(+) MCM7(-) , G9a(-) MCM7(+) ). CONCLUSIONS: MCM7 and G9a may serve as effective prognostic factors and could also be used as biomarkers for predicting various clinical outcomes of OSCCs in the Chinese population.
Assuntos
Biomarcadores Tumorais/análise , Carcinoma de Células Escamosas/patologia , Neoplasias Esofágicas/patologia , Antígenos de Histocompatibilidade/biossíntese , Histona-Lisina N-Metiltransferase/biossíntese , Componente 7 do Complexo de Manutenção de Minicromossomo/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Carcinoma de Células Escamosas/mortalidade , Neoplasias Esofágicas/mortalidade , Carcinoma de Células Escamosas do Esôfago , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico , RNA Interferente Pequeno , Análise Serial de TecidosRESUMO
Methylation and demethylation of histone H3 lysine 9 (H3K9) play a role in the transcriptional regulation of several cancer-related genes and are closely associated with malignant tumor behavior. A novel study has recently demonstrated that SETDB1, a member of the H3K9 methyltransferases, accelerates tumor formation significantly in a zebrafish melanoma model. However, the expression of H3K9 methyltransferases including SETDB1 and demethylases has not been systematically examined in samples of human melanoma. Here, we used immunohistochemistry to examine the expression of the H3K9 methyltransferases, EHMT2 and SETDB1, and a H3K9 demethylase, LSD1, in 67 patients with melanoma. Overexpression of EHMT2, SETDB1, and LSD1 was observed in 14 (21%), 38 (57%), and 53 (79%) of the 67 patients, respectively. A significant relationship was observed between overexpression of EHMT2 or SETDB1 and aggressive tumor behavior such as lymph node metastasis and/or distant metastasis (P < 0.05), whereas no significant relationship was evident for LSD1 immunoreactivity. Univariate log-rank tests demonstrated that patients with melanoma overexpressing EHMT2 had a poorer outcome (P < 0.001), whereas overexpression of SETDB1 or LSD1 had no prognostic impact. These results suggest that overexpression of EHMT2 might be a prognostic marker in patients with melanoma.
Assuntos
Antígenos de Histocompatibilidade/biossíntese , Histona Desmetilases/biossíntese , Histona-Lisina N-Metiltransferase/biossíntese , Melanoma/enzimologia , Proteínas Metiltransferases/biossíntese , Neoplasias Cutâneas/enzimologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Melanoma/mortalidade , Melanoma/patologia , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/patologiaRESUMO
Leishmania parasites are able to interfere with host immune responses on many levels, as T cell responses balance, as observed in the murine model of infection. In the present study, we analyzed genes expression in both host and parasite during the progression of infection. Host genes associated to T-lymphocytes responses, MHC classes I and II, as well as parasite enzymes genes, cysteine-proteinases (CP) B and C, were examined in mice along evolution of infection by Leishmania (Leishmania) amazonensis. Murine strains with distinct levels of susceptibility to infection presented different patterns of MHC genes expression: MHC class I genes tend to have higher expression levels in CBA mice, whereas MHC class II genes expression predominates in BALB/c mice. CPB genes expression in the parasites was shown to predominate over CPC in both mice strains tested. Understanding genes expression patterns during infection may lead to new and more efficient treatments for leishmaniasis.
Assuntos
Expressão Gênica , Interações Hospedeiro-Patógeno , Leishmania mexicana/genética , Leishmania mexicana/patogenicidade , Animais , Cisteína Proteases/biossíntese , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Antígenos de Histocompatibilidade/biossíntese , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBARESUMO
We have studied the effect of radioresistant host cells in inducing tolerance and adaptation of the MHC recognition repertoire of donor-derived NK cells in stem cell allotransplanted (allo-SCT) rats. Sub-lethally irradiated PVG.1AV1 rats (RT1(av1)) were transplanted with bone marrow from fully MHC-mismatched allotype-marked PVG.7B (RT1(c)) rats; MHC-identical PVG (RT1(c)) controls were transplanted in parallel. In the PVG.7B â PVG.1AV1 allogeneic chimeras, NK cells were donor derived and showed partial tolerance toward host cells. Allogeneic chimeras failed to efficiently reject PVG.1AV1 cells by an NK-mediated mechanism in vivo (allogeneic lymphocyte cytotoxicity), and IL-2-cultured NK cells derived from these chimeras showed diminished cytolytic activity against PVG.1AV1 cells in vitro. There were corresponding changes in the phenotype and function of the highly alloreactive Ly49i2(+) NK cells, which are specifically inhibited by a donor MHC class I ligand, RT1-A1(c). The ligand-negative host MHC haplotype apparently induced expression of a second uncharacterized inhibitory MHC receptor responsible for the partial tolerance toward host-derived cells, along with a modest increase in Ly49i2 receptor levels. The host MHC haplotype did not induce a general hyporesponsiveness in Ly49i2(+) NK cells, which showed normal activation responses in a panel of MHC congenic strains. The data suggest that the MHC constitution of radiation-resistant host cells can have permanent, albeit not fully tolerogenic, effects on the development of a functional NK repertoire following allo-SCT.
Assuntos
Transplante de Medula Óssea/imunologia , Células Matadoras Naturais/imunologia , Tolerância a Radiação/imunologia , Tolerância ao Transplante , Animais , Antígenos Ly/biossíntese , Antígenos de Histocompatibilidade/biossíntese , Complexo Principal de Histocompatibilidade/imunologia , Ratos , Receptores Imunológicos/biossíntese , Células Estromais/imunologia , Células Estromais/efeitos da radiaçãoRESUMO
Immunotherapy for leukemia is a promising targeted strategy to eradicate residual leukemic cells after standard therapy, in order to prevent relapse and to prolong the survival of leukemia patients. However, effective anti-leukemia immune responses are hampered by the weak immunogenicity of leukemic cells. Therefore, much effort is made to identify agents that could increase the immunogenicity of leukemic cells and activate the immune system. Synthetic agonists of Toll-like receptor (TLR)7 and TLR8 are already in use as anticancer treatment, because of their ability to activate several immune pathways simultaneously, resulting in effective antitumor immunity. However, for leukemic cells little is known about the expression of TLR7/8 and the direct effects of their agonists. We hypothesized that TLR7/8 agonist treatment of human acute myeloid leukemia (AML) cells would lead to an increased immunogenicity of AML cells. We observed expression of TLR7 and TLR8 in primary human AML cells and AML cell lines. Passive pulsing of primary AML cells with the TLR7/8 agonist R-848 resulted in increased expression of MHC molecules, production of proinflammatory cytokines, and enhanced allogeneic naïve T cell-stimulatory capacity. These effects were absent or suboptimal if R-848 was administered intracellularly by electroporation. Furthermore, when AML cells were cocultured with allogeneic PBMC in the presence of R-848, interferon (IFN)-gamma was produced by allogeneic NK and NKT cells and AML cells were killed. In conclusion, the immunostimulatory effect of the TLR7/8 agonist R-848 on human AML cells could prove useful for the design of TLR-based immunotherapy for leukemia.
Assuntos
Adjuvantes Imunológicos/farmacologia , Imidazóis/farmacologia , Leucemia Mieloide Aguda/imunologia , Células T Matadoras Naturais/imunologia , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistas , Adulto , Idoso , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Citocinas/biossíntese , Citocinas/imunologia , Feminino , Antígenos de Histocompatibilidade/biossíntese , Antígenos de Histocompatibilidade/imunologia , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-Idade , Receptor 7 Toll-Like/biossíntese , Receptor 8 Toll-Like/biossíntese , Células Tumorais Cultivadas , Adulto JovemRESUMO
The complement system is important for protection from invading pathogens, removal of waste products and guidance of the immune response. Furthermore, complement can be also targeted to cancer cells. However, membrane-bound inhibitors over-expressed by certain types of tumor cells restrict the cytotoxic activity of complement. Herein we report that non-small cell lung cancer (NSCLC) cells produce soluble complement inhibitors factor I (FI) and C4b-binding protein (C4BP). FI is a serine protease capable of degrading the activated complement components C3b and C4b, whilst C4BP acts as its cofactor. Furthermore, NSCLC cells express membrane-bound regulators and shed membrane cofactor protein (MCP), which shares cofactor function with C4BP. Secretion of FI from NSCLC cells was higher than previously reported for any non-hepatic source and FI produced by these cells could efficiently support cleavage of C3b and C4b. In vitro functional assays revealed that additional FI significantly decreased C3 deposition and complement-dependent lysis, particularly when cofactors were added. Our results demonstrate that soluble inhibitors produced by NSCLC cells may provide further protection from complement beyond the level ensured by membrane-bound inhibitors and, as such, contribute to the aggressive phenotype of these lung cancer cells.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/imunologia , Fator I do Complemento/biossíntese , Antígenos de Histocompatibilidade/biossíntese , Neoplasias Pulmonares/imunologia , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Ativação do Complemento/imunologia , Complemento C3b/metabolismo , Complemento C4b/metabolismo , Proteína de Ligação ao Complemento C4b , Fator I do Complemento/genética , Citotoxicidade Imunológica , Regulação Neoplásica da Expressão Gênica , Antígenos de Histocompatibilidade/genética , Antígenos de Histocompatibilidade/metabolismo , Humanos , Neoplasias Pulmonares/genética , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , SolubilidadeRESUMO
Tumors often target dendritic cells (DCs) to evade host immune surveillance. DC injury is reported in many rodent and human tumors but seldom in tumors of other mammals. Canine transmissible venereal tumor (CTVT), a unique and spontaneous cancer transmitted by means of viable tumor cells. CTVT causes manifold damage to monocyte-derived DCs. This cancer provides an in vivo model of cancer to study the role of monocyte-derived DCs during spontaneous regression. Using flow cytometry and real-time reverse-transcription polymerase chain reactions, we compared the expression of surface molecules on monocyte-derived DCs between normal dogs and dogs with CTVT. These markers were CD1a, CD83, costimulatory factors (CD40, CD80, and CD86), and major histocompatability complex classes I and II. In immature DCs (iDCs) and lipopolysaccharide-treated mature DCs (mDCs), the surface markers were mostly downregulated during tumoral progression and regression. The tumor lowered endocytic activity of iDCs, as reflected in dextran uptake, and decreased allogeneic mixed lymphocyte reactions of mDCs. In addition, it decreased the number of monocytes in the peripheral blood by 40%. The tumor substantially impaired the efficiency with which DCs were generated from monocytes and with which mDCs were generated from iDCs. We also found that progression-phase CTVT supernatants that were cultured for 48 h and that contained protein components killed both monocytes and DCs. Additionally, DC numbers were significantly lower in the draining lymph nodes in CTVT dogs than in normal dogs. In conclusion, CTVT caused devastating damage to monocyte-derived DCs; this might be one of its mechanisms for evading host immunity. Reestablishment of monocyte-derived DC activity by the host potentially might contribute to spontaneous tumoral regression. These findings provide insight into the extent of tumoral effects on host immune systems and responses. This information is useful for developing cancer immunotherapies.
Assuntos
Células Dendríticas/citologia , Células Dendríticas/metabolismo , Monócitos/citologia , Monócitos/metabolismo , Evasão Tumoral/imunologia , Tumores Venéreos Veterinários/imunologia , Animais , Antígenos CD/biossíntese , Diferenciação Celular/fisiologia , Sobrevivência Celular/fisiologia , Modelos Animais de Doenças , Progressão da Doença , Doenças do Cão , Cães , Regulação para Baixo , Citometria de Fluxo , Antígenos de Histocompatibilidade/biossíntese , Humanos , Teste de Cultura Mista de Linfócitos , Linfócitos do Interstício Tumoral , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tumores Venéreos Veterinários/fisiopatologiaRESUMO
The product of the human major histocompatibility (HLA) class I allele HLA-B*1402 only differs from that of allele HLA-B*1403 at amino-acid position 156 of the heavy chain (Leu in HLA-B*1402 and Arg in HLA-B*1403). However, both subtypes are known to be differentially associated with the inflammatory rheumatic disease ankylosing spondylitis (AS) in black populations in Cameroon and Togo. HLA-B*1402 is not associated with AS, in contrast to HLA-B*1403, which is associated with this disease in the Togolese population. The products of these alleles can present peptides with Arg at position 2, a feature shared by a small group of other HLA-B antigens, including HLA-B*2705, the prototypical AS-associated subtype. Complexes of HLA-B*1402 with a viral peptide (RRRWRRLTV, termed pLMP2) and a self-peptide (IRAAPPPLF, termed pCatA) were prepared and were crystallized using polyethylene glycol as precipitant. The complexes crystallized in space groups P2(1) (pLMP2) and P2(1)2(1)2(1) (pCatA) and diffracted synchrotron radiation to 2.55 and 1.86 A resolution, respectively. Unambiguous solutions for both data sets were obtained by molecular replacement using a peptide-complexed HLA-B*2705 molecule (PDB code 1jge) as a search model.
Assuntos
Antígenos Virais/química , Autoantígenos/química , Regulação da Expressão Gênica , Antígenos HLA-B/química , Antígenos HLA-B/genética , Antígenos de Histocompatibilidade/genética , Antígenos de Histocompatibilidade/isolamento & purificação , Fragmentos de Peptídeos/química , Antígenos Virais/biossíntese , Antígenos Virais/genética , Autoantígenos/genética , Autoantígenos/isolamento & purificação , Cristalografia por Raios X , Antígenos HLA-B/biossíntese , Antígenos HLA-B/isolamento & purificação , Antígenos de Histocompatibilidade/biossíntese , Antígenos de Histocompatibilidade/química , Humanos , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/genéticaRESUMO
The expression of MHC class I, MHC class II and the chemokines IP-10, MIP-1alpha, RANTES, fractalkine and I-TAC has been analyzed after intra-vaginal treatment with three synthetic toll-like receptors (TLR) agonists-double-stranded RNA (poly I:C), imiquimod and CpG-rich oligonucleotides (CpG-ODN). These compounds act mainly through TLR3, TLR7 and TLR9, respectively. CpG-ODN induced an accumulation of leucocytes in the vagina, and a strong up-regulation of MHC class I expression on both leucocytes and epithelial cells. Imiquimod and poly I:C induced a weak MHC class I up-regulation in the epithelium but not in the lamina propria. Neither treatment had any profound effect on expression of MHC class II on epithelial cells but poly I:C and to a lesser extent CpG-ODN, up-regulated MHC class II staining intensity which, in the case of CpG-ODN, treatment, was associated with a strong accumulation of CD11c-positive dendritic cells. All three treatments induced an early (8h) but transient IP-10 response. Imiquimod and CpG-ODN, but not poly I:C induced an early MIP-1alpha response which remained for at least 7 days in CpG-ODN-treated animals but not in imiquimod-treated mice. Poly I:C and CpG-ODN, but not imiquimod, induced significant levels of RANTES at different time-points post-treatment. None of the treatments induced any significant changes in the levels of fractalkine, I-TAC or IFN-alpha. These studies have implications for the manipulation of the genital immune response and also improving the outcome of vaginal immunotherapy.
Assuntos
Adjuvantes Imunológicos/farmacologia , Quimiocinas/biossíntese , Antígenos de Histocompatibilidade/biossíntese , Indutores de Interferon/farmacologia , Oligodesoxirribonucleotídeos/farmacologia , Poli I-C/farmacologia , Receptores Toll-Like/agonistas , Vagina/metabolismo , Aminoquinolinas/farmacologia , Animais , Antígeno CD11c/imunologia , Antígeno CD11c/metabolismo , Quimiocinas/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Antígenos de Histocompatibilidade/imunologia , Imiquimode , Leucócitos/citologia , Leucócitos/imunologia , Leucócitos/metabolismo , Ligantes , Camundongos , Fatores de Tempo , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologia , Vagina/citologia , Vagina/imunologiaRESUMO
Induction of donor-specific tolerance using embryonic stem (ES) cells followed by transplantation of ES cell-derived tissues from the same allogeneic strain could theoretically engender successful transplantation without immunosuppression. We sought to induce tolerance using bona fide murine ES cells in immunocompetent mice. ES cells were evaluated for the expression of markers restricted to undifferentiated cells [stage-specific embryonic antigen-1 (SSEA-1) and OCT-4] and the ability to form teratomas in immunodeficient mice. BALB/cByJ mice underwent intraportal inoculation with YC5-EYFP ES cells (129 strain; R1-derived) or saline followed by transplantation with 129X1/SvJ, CBA/J, or BALB/cByJ nonvascularized, neonatal cardiac grafts. Mice were sacrificed at graft failure and underwent histologic evaluation of transplanted grafts and lymphoid organs. ES cells and early differentiated progeny underwent real time (RT)-PCR and fluorescence-activated cell sorting (FACS) analysis to detect major histocompatibility complex (MHC) gene transcription and antigen expression. ES cells expressed markers restricted to undifferentiated cells while maintaining the ability to form teratomas in immunodeficient mice. No prolongation of allograft survival or evidence of lymphoid chimerism was observed in immunocompetent recipient mice despite hepatic teratoma formation. MHC class I, class II, and nonclassical antigens were undetectable on ES cells and early differentiated progeny despite the presence of mRNA transcripts. Class I expression was strongly upregulated upon exposure to gamma-interferon. Intraportal inoculation with murine ES cells does not produce lymphoid chimerism or induce donor-specific unresponsiveness to neonatal cardiac grafts in unmanipulated immunocompetent hosts. However, specific differentiated cell types such as ES cellderived dendritic cells, or alternate routes of ES cell administration, may be effective. ES cells appear to have immune privilege, allowing them to form teratomas in immunocompetent mice.
Assuntos
Antígenos/imunologia , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Antígenos de Histocompatibilidade/biossíntese , Tolerância Imunológica/imunologia , Sistema Porta/imunologia , Animais , Animais Recém-Nascidos , Citometria de Fluxo , Regulação da Expressão Gênica , Transplante de Coração , Antígenos de Histocompatibilidade/análise , Antígenos de Histocompatibilidade/genética , Antígenos de Histocompatibilidade Classe I/análise , Antígenos de Histocompatibilidade Classe I/biossíntese , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe II/análise , Antígenos de Histocompatibilidade Classe II/biossíntese , Antígenos de Histocompatibilidade Classe II/genética , Fígado/citologia , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Células-Tronco Pluripotentes/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de SobrevidaRESUMO
Salmonellae have evolved several mechanisms to evade host clearance. Here, we describe the influence on bacterial immune escape of the effector protein SopB, which is translocated into the cytosol through a type III secretion system. Wild-type bacteria, as well as the sseC and aroA attenuated mutants exerted a stronger cytotoxic effect on dendritic cells (DC) than their SopB-deficient derivatives. Cells infected with the double sseC sopB, phoP sopB and aroA sopB mutants also exhibited higher expression of MHC, CD80, CD86 and CD54 molecules, and showed a stronger capacity to process and present an I-E(d)-restricted epitope from the influenza hemagglutinin (HA) to CD4+ cells from TCR-HA transgenic mice in vitro. The incorporation of an additional mutation into the sopB locus of the attenuated sseC, phoP and aroA mutants resulted in the stimulation of improved humoral and cellular immune responses following oral vaccination. The obtained results define a new potential immune escape strategy of this important pathogen, and also demonstrate that this mechanism can be subverted to optimize the immune responses elicited using Salmonella as a live vaccine carrier.
Assuntos
Proteínas de Bactérias/fisiologia , Vacinas contra Salmonella/imunologia , Salmonella enterica/imunologia , Animais , Antígenos CD/biossíntese , Proteínas de Bactérias/genética , Linfócitos T CD4-Positivos/imunologia , Sobrevivência Celular , Células Dendríticas/microbiologia , Epitopos/imunologia , Feminino , Deleção de Genes , Hemaglutininas Virais/imunologia , Antígenos de Histocompatibilidade/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Salmonella enterica/patogenicidade , Vacinas Tíficas-Paratíficas , Vacinas Sintéticas/imunologiaRESUMO
Interleukin-1 (IL-1beta) beta and major histocompatibility complex (MHC) play an important role during pregnancy. Expression of non-classical class MHC II RT1-DM antigen and classical class MHC I RT1-A antigen induced by IL-1beta was examined by Northern blotting, Western blotting and immunohistochemistry. IL-1beta treatment significantly increased the expression of RT1-A and RT1-DM in early and mid pregnancy. In late pregnancy, expression of RT1-DM significantly increased in uteri and decreased in placenta. Immunohistochemical studies indicated that, in early pregnancy, RT1-DM protein mainly localized to luminal and glandular uterine epithelium, and RT1-A was present in deciduas basalis, outer layer of luminal epithelium and glandular epithelium. During mid and late pregnancy, RT1-DM was present in maternal blood vessels and syncytiotrophoblast of labyrinthine zone, and RT1-A was present in maternal blood vessels and trophoblastic epithelium of the labyrinthine layers. These findings show that exogenous IL-1beta affects expression of RT1-DM and RT1-A and does not affect the localization of corresponding molecules during pregnancy.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Antígenos de Histocompatibilidade/biossíntese , Interleucina-1/fisiologia , Prenhez/metabolismo , Animais , Northern Blotting , Western Blotting , Feminino , Imuno-Histoquímica , Placenta/metabolismo , Gravidez , RNA/isolamento & purificação , Ratos , Ratos Sprague-Dawley , Útero/metabolismoRESUMO
The central nervous system (CNS) has been referred to as the "immunological privileged site". However, it is now clear that the privileged status of the CNS is a result of a balance between immune privilege and effective response. In vitro, human bone marrow mesenchymal stem cells (MSCs) have the ability to differentiate into neurons. Based on this biological attribute we gain the possibility by means of using MSCs as the donors to develop a future cell therapy in clinical application. But using MSCs as donor cells inevitably raises the question as to whether these donor cells would be immunogenic, and if so, would they be rejected after transplantation. To investigate this, human MSCs were cultured in vitro and induced to differentiate along neuronal lineage. The expression of human leukocyte antigen (HLA) class I and class II molecules and the co-stimulatory protein CD80 were increased on the surface of MSCs in the course of neuronal differentiation. But neither of the co-stimulatory proteins, CD40 or CD86, was expressed. After IFN-gamma exposure, the expression of the HLA molecules was further enhanced, but the co-stimulatory proteins were unaffected. MSCs that had been differentiated along neuronal lineage were not capable of inducing the proliferation of peripheral blood lymphocytes (PBLs). Even after IFN-gamma exposure, PBLs remained unresponsive. Furthermore, MSCs differentiated along neuronal lineage suppressed the proliferation of PBLs induced by allogeneic PBLs and mitogens. The mechanisms involved in the immunosuppression may be related to the effect of soluble factors and cell-cell interactions of neuronal differentiated MSCs and PBLs. From the above data we suggested that the low immunogenicity and immunomodulatory function of MSCs in the course of neuronal differentiation in vitro, which will be helpful to further investigation in order to establish the new way for future medical application.
Assuntos
Diferenciação Celular/imunologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/imunologia , Neurônios/citologia , Neurônios/imunologia , Antígeno B7-2/biossíntese , Western Blotting , Células da Medula Óssea , Antígenos CD40/biossíntese , Proliferação de Células , Antígenos de Histocompatibilidade/biossíntese , Humanos , Teste de Cultura Mista de Linfócitos , Linfócitos/imunologiaRESUMO
The enzyme type 8 17beta-hydroxysteroid dehydrogenase (17beta-HSD) selectively catalyzes the conversion of estradiol (E2) to estrone (E1). To obtain detailed information on the sites of action of type 8 17beta-HSD, we have studied the cellular localization of type 8 17beta-HSD mRNA in mouse tissues using in situ hybridization. In the ovary, hybridization signal was detected in granulosa cells of growing follicles and luteal cells. In the uterus, type 8 17beta-HSD mRNA was found in the epithelial (luminal and glandular) and stromal cells. In the female mammary gland, the enzyme mRNA was seen in ductal epithelial cells and stromal cells. In the testis, hybridization signal was observed in the seminiferous tubule. In the prostate, type 8 17beta-HSD was detected in the epithelial cells of the acini and stromal cells. In the clitoral and preputial glands, labeling was detected in the epithelial cells of acini and small ducts. The three lobes of the pituitary gland were labeled. In the adrenal gland, hybridization signal was observed in the three zones of the cortex, the medulla being unlabeled. In the kidney, the enzyme mRNA was found to be expressed in the epithelial cells of proximal convoluted tubules. In the liver, all the hepatocytes exhibited a positive signal. In the lung, type 8 17beta-HSD mRNA was detected in bronchial epithelial cells and walls of pulmonary arteries. The present data suggest that type 8 17beta-HSD can exert its action to downregulate E2 levels in a large variety of tissues.
Assuntos
17-Hidroxiesteroide Desidrogenases/biossíntese , Antígenos de Histocompatibilidade/biossíntese , Oxirredutases/biossíntese , RNA Mensageiro/biossíntese , Animais , Feminino , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de ÓrgãosRESUMO
A feature of the tolerance that has been described in experimental models is that it can be transferred by CD4+ T cells to a naive recipient. Described is a novel approach to induce indirect pathway regulatory T cells in a rat model that exploits the natural processing and presentation of major histocompatability complex (MHC) molecules as peptide by the MHC class II molecules of the same cell. Dendritic cells (DC) coexpressing donor (AUG) and recipient (LEW) MHC molecules were rendered tolerogenic by treatment with dexamethasone. After injection into LEW animals followed by a single low dose of CTLA4-Ig, T cells were rendered unresponsive to indirectly presented AUG alloantigens, but retained direct pathway responsiveness to fully allogeneic AUG cells. The T cells from the DC-injected rats were unresponsive to (LEW x AUG)F1 stimulator cells, suggesting the presence of indirect pathway regulatory cells whose activity depended on the presence of LEW MHC molecules. Depletion of CD25+ cells from the responder population led to a marked increase in proliferation, and the T cells from the DC-injected rats inhibited the response of naive LEW T cells to (LEW x AUG)F1, but not to AUG, stimulator cells, further indicating indirect pathway-mediated regulation. Most importantly, pretreatment of LEW rats with the dexamethasone-treated DC led to the indefinite survival of AUG kidney grafts after a short course of cyclosporin to inhibit the early direct pathway response. Similarly treated AUG DC had no effect, confirming the privileged status of F1 cells in the induction of indirect pathway regulation.
Assuntos
Células Dendríticas/imunologia , Antígenos de Histocompatibilidade/biossíntese , Transplante de Rim/imunologia , Animais , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Dexametasona/farmacologia , Sobrevivência de Enxerto/efeitos dos fármacos , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Ratos EndogâmicosRESUMO
It has been suggested that class I and class II MHC are contributing factors for numerous diseases including autoimmune thyroid diseases, type 1 diabetes, rheumatoid arthritis, Alzheimer's disease, and multiple sclerosis. The class II trans-activator (CIITA), which is a non-DNA-binding regulator of class II MHC transcription, regulates the constitutive and inducible expression of the class I and class II genes. FRTL-5 thyroid cells incubated in the presence of IFN-gamma have a significantly higher level of cell surface rat MHC class II RTI.B. However, the IFN-gamma-induced RT1.B expression was suppressed significantly in cells incubated in the presence of thyrotropin. Thyrotropin (TSH) represses IFN-gamma-induced CIITA expression by inhibiting type IV CIITA promoter activity through the suppression of STAT1 activation and IFN regulatory factor 1 induction. This study found that TSH induces transcriptional activation of the STAT3 gene through the phosphorylation of STAT3 and CREB activation. TSH induces SOCS-1 and SOCS-3, and TSH-mediated SOCS-3 induction was dependent on STAT3. The cell line stably expressing the wild-type STAT3 showed a higher CIITA induction in response to IFN-gamma and also exhibited TSH repression of the IFN-gamma-mediated induction of CIITA. However, TSH repression of the IFN-gamma-induced CIITA expression was not observed in FRTL-5 thyroid cells, which stably expresses the dominant negative forms of STAT3, STAT3-Y705F, and STAT3-S727A. This report suggests that TSH is also engaged in immunomodulation through signal cross-talk with the cytokines in thyroid cells.
Assuntos
Proteínas de Transporte/fisiologia , Proteínas de Ligação a DNA/fisiologia , Regulação para Baixo/imunologia , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Nucleares , Proteínas/fisiologia , Proteínas Repressoras , Glândula Tireoide/metabolismo , Tireotropina/fisiologia , Transativadores/antagonistas & inibidores , Transativadores/biossíntese , Transativadores/fisiologia , Animais , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Bovinos , Linhagem Celular , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/genética , Antígenos de Histocompatibilidade/biossíntese , Humanos , Fator Regulador 1 de Interferon , Fator Gênico 3 Estimulado por Interferon , Interferon gama/genética , Interferon gama/farmacologia , Camundongos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Regiões Promotoras Genéticas/imunologia , Ligação Proteica/genética , Ligação Proteica/imunologia , Biossíntese de Proteínas , Proteínas/genética , Proteínas/metabolismo , Ratos , Elementos de Resposta/imunologia , Fator de Transcrição STAT3 , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Proteína 1 Supressora da Sinalização de Citocina , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina , Glândula Tireoide/citologia , Glândula Tireoide/imunologia , Transativadores/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
MHC class I expression by rats of the RT1(o), RT1(d), and RT1(m) MHC haplotypes was investigated. Identical, functional cDNAs were obtained from RT1(o) and BDIX (RT1(dv1)) rats for three MHC class I molecules. RT1-A1(o/d) and -A2(o/d) are closely related in sequence to other cloned rat class Ia genes that have been shown to map to the RT1-A region, while RT1-A3 degrees is highly homologous to a class I gene identified by sequencing an RT1-A(n) genomic contig and is named A3(n). Detailed analysis of the three molecules was undertaken using serology with mAbs, two-dimensional gel analysis of immunoprecipitates, and killing assays using cytotoxic T cells. Arguments are presented suggesting that A1 degrees is the principal MHC class Ia (classical) restricting element of this haplotype. A2 degrees, which is highly cross-reactive with A1 degrees, and A3 degrees probably play more minor or distinct roles in Ag presentation. Unexpectedly, cDNAs encoding exactly the same three molecules were cloned from rats of the RT1(m) haplotype, an MHC that until now was thought to possess unique class Ia genes. RT1(m) contains the TAP-B allele of the TAP transporter, and we present evidence that functional polymorphism in rat TAP has an even greater impact on the expression of RT1-A1 degrees and -A2 degrees than it does on RT1-A(a) in the established case of class I modification (cim). Historically, this led to the misclassification of RT1(m) class Ia molecules as separate and distinct.