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1.
J Virol ; 94(7)2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-31915285

RESUMO

While the role of CD8+ T cells in the control of herpes simplex virus 1 (HSV-1) infection and disease is gaining wider acceptance, a direct involvement of effector CD4+ T cells in this protection and the phenotype and function of HSV-specific human CD4+ T cell epitopes remain to be fully elucidated. In the present study, we report that several epitopes from the HSV-1 virion tegument protein (VP11/12) encoded by UL46 are targeted by CD4+ T cells from HSV-seropositive asymptomatic individuals (who, despite being infected, never develop any recurrent herpetic disease). Among these, we identified two immunodominant effector memory CD4+ TEM cell epitopes, amino acids (aa) 129 to 143 of VP11/12 (VP11/12129-143) and VP11/12483-497, using in silico, in vitro, and in vivo approaches based on the following: (i) a combination of the TEPITOPE algorithm and PepScan library scanning of the entire 718 aa of HSV-1 VP11/12 sequence; (ii) an in silico peptide-protein docking analysis and in vitro binding assay that identify epitopes with high affinity to soluble HLA-DRB1 molecules; and (iii) an ELISpot assay and intracellular detection of gamma interferon (IFN-γ), CD107a/b degranulation, and CD4+ T cell carboxyfluorescein succinimidyl ester (CFSE) proliferation assays. We demonstrated that native VP11/12129-143 and VP11/12483-497 epitopes presented by HSV-1-infected HLA-DR-positive target cells were recognized mainly by effector memory CD4+ TEM cells while being less targeted by FOXP3+ CD4+ CD25+ regulatory T cells. Furthermore, immunization of HLA-DR transgenic mice with a mixture of the two immunodominant human VP11/12 CD4+ TEM cell epitopes, but not with cryptic epitopes, induced HSV-specific polyfunctional IFN-γ-producing CD107ab+ CD4+ T cells associated with protective immunity against ocular herpes infection and disease.IMPORTANCE We report that naturally protected HSV-1-seropositive asymptomatic individuals develop a higher frequency of antiviral effector memory CD4+ TEM cells specific to two immunodominant epitopes derived from the HSV-1 tegument protein VP11/12. Immunization of HLA-DR transgenic mice with a mixture of these two immunodominant CD4+ T cell epitopes induced a robust antiviral CD4+ T cell response in the cornea that was associated with protective immunity against ocular herpes. The emerging concept of developing an asymptomatic herpes vaccine that would boost effector memory CD4+ and CD8+ TEM cell responses is discussed.


Assuntos
Antígenos Virais/imunologia , Linfócitos T CD4-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Memória Imunológica , Ceratite Herpética/imunologia , Proteínas Virais/imunologia , Adulto , Idoso , Animais , Infecções Assintomáticas , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/virologia , Simulação por Computador , Feminino , Antígenos HLA-DR/genética , Haplótipos , Humanos , Epitopos Imunodominantes/imunologia , Interferon gama/imunologia , Ceratite Herpética/prevenção & controle , Proteína 1 de Membrana Associada ao Lisossomo/imunologia , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Fenótipo , Adulto Jovem
2.
J Virol ; 94(7)2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-31915281

RESUMO

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that encodes many proteins to modulate the host immune response. Extensive efforts have led to the elucidation of multiple strategies employed by HCMV to effectively block NK cell targeting of virus-infected cells and the major histocompatibility complex (MHC) class I-primed CD8+ T cell response. However, viral regulation of the MHC class II-mediated CD4+ T cell response is understudied in endogenous MHC class II-expressing cells, largely because the popular cell culture systems utilized for studying HCMV do not endogenously express MHC class II. Of the many cell types infected by HCMV in the host, myeloid cells, such as monocytes, are of particular importance due to their role in latency and subsequent dissemination throughout the host. We investigated the impact of HCMV infection on MHC class II in Kasumi-3 cells, a myeloid-progenitor cell line that endogenously expresses the MHC class II gene, HLA-DR. We observed a significant reduction in the expression of surface and total HLA-DR at 72 h postinfection (hpi) and 120 hpi in infected cells. The decrease in HLA-DR expression was independent of the expression of previously described viral genes that regulate the MHC class II complex or the unique short (US) region of HCMV, a region expressing many immunomodulatory genes. The altered surface level of HLA-DR was not a result of increased endocytosis and degradation but was a result of a reduction in HLA-DR transcripts due to a decrease in the expression of the class II transactivator (CIITA).IMPORTANCE Human cytomegalovirus (HCMV) is an opportunistic herpesvirus that is asymptomatic for healthy individuals but that can lead to severe pathology in patients with congenital infections and immunosuppressed patients. Thus, it is important to understand the modulation of the immune response by HCMV, which is understudied in the context of endogenous MHC class II regulation. Using Kasumi-3 cells as a myeloid progenitor cell model endogenously expressing MHC class II (HLA-DR), this study shows that HCMV decreases the expression of HLA-DR in infected cells by reducing the transcription of HLA-DR transcripts early during infection independently of the expression of previously implicated genes. This is an important finding, as it highlights a mechanism of immune evasion utilized by HCMV to decrease the expression of MHC class II in a relevant cell system that endogenously expresses the MHC class II complex.


Assuntos
Infecções por Citomegalovirus/imunologia , Antígenos HLA-DR/genética , Células Progenitoras Mieloides/imunologia , Proteínas Nucleares/genética , Transativadores/genética , Ativação Transcricional , Apresentação do Antígeno , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Linhagem Celular , Citomegalovirus , Endocitose , Fibroblastos/imunologia , Fibroblastos/virologia , Regulação da Expressão Gênica , Humanos , Células Progenitoras Mieloides/virologia
3.
J Am Soc Nephrol ; 31(2): 365-373, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31919107

RESUMO

BACKGROUND: Primary membranoproliferative GN, including complement 3 (C3) glomerulopathy, is a rare, untreatable kidney disease characterized by glomerular complement deposition. Complement gene mutations can cause familial C3 glomerulopathy, and studies have reported rare variants in complement genes in nonfamilial primary membranoproliferative GN. METHODS: We analyzed whole-genome sequence data from 165 primary membranoproliferative GN cases and 10,250 individuals without the condition (controls) as part of the National Institutes of Health Research BioResource-Rare Diseases Study. We examined copy number, rare, and common variants. RESULTS: Our analysis included 146 primary membranoproliferative GN cases and 6442 controls who were unrelated and of European ancestry. We observed no significant enrichment of rare variants in candidate genes (genes encoding components of the complement alternative pathway and other genes associated with the related disease atypical hemolytic uremic syndrome; 6.8% in cases versus 5.9% in controls) or exome-wide. However, a significant common variant locus was identified at 6p21.32 (rs35406322) (P=3.29×10-8; odds ratio [OR], 1.93; 95% confidence interval [95% CI], 1.53 to 2.44), overlapping the HLA locus. Imputation of HLA types mapped this signal to a haplotype incorporating DQA1*05:01, DQB1*02:01, and DRB1*03:01 (P=1.21×10-8; OR, 2.19; 95% CI, 1.66 to 2.89). This finding was replicated by analysis of HLA serotypes in 338 individuals with membranoproliferative GN and 15,614 individuals with nonimmune renal failure. CONCLUSIONS: We found that HLA type, but not rare complement gene variation, is associated with primary membranoproliferative GN. These findings challenge the paradigm of complement gene mutations typically causing primary membranoproliferative GN and implicate an underlying autoimmune mechanism in most cases.


Assuntos
Complemento C3/imunologia , Glomerulonefrite Membranoproliferativa/genética , Sequenciamento Completo do Genoma , Fator Nefrítico do Complemento 3/análise , Feminino , Glomerulonefrite Membranoproliferativa/etiologia , Antígenos HLA-DQ/genética , Antígenos HLA-DR/genética , Humanos , Masculino , Sorogrupo
4.
BMC Ophthalmol ; 19(1): 214, 2019 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-31699055

RESUMO

BACKGROUND: The common presence of human leukocyte antigen (HLA)-DRB1*04 in Vogt-Koyanagi-Harada (VKH) disease is well known. The aim of this study was to investigate the relationship between visual prognosis and HLA-DRB1*04 alleles during systemic corticosteroid therapy in patients with VKH disease. METHODS: This retrospective case series included 57 eyes from 29 consecutive patients with treatment-naïve VKH disease who received systemic corticosteroid therapy. Visual acuity, sex, refractive error, central retinal thickness (CRT), central choroidal thickness (CCT), and duration from onset to treatment were measured at initial and final visits. Mean values of parameters were compared with each visit. Genotyping was performed by polymerase chain reaction amplification with sequence-specific primer. RESULTS: Linear regression showed significant differences in logMAR best-corrected visual acuity between the three groups of homozygotes, heterozygotes, and normal subjects at baseline (p < 0.01), at 3 months after treatment (p < 0.01). There was no significant differences at 6 months after treatment (p = 0.257). No significant differences were detected between the three groups in age, sex, refractive error, CRT, CCT, or duration from onset to treatment. CONCLUSION: Alleles of HLA-DRB1*04 might affect visual prognosis and be related to early response after initiation of treatment in VKH disease.


Assuntos
Antígenos HLA-DR/genética , Síndrome Uveomeningoencefálica , Acuidade Visual/fisiologia , Corticosteroides/uso terapêutico , Adulto , Corioide/patologia , Feminino , Cadeias HLA-DRB1 , Teste de Histocompatibilidade , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Regressão , Retina/patologia , Estudos Retrospectivos , Síndrome Uveomeningoencefálica/tratamento farmacológico , Síndrome Uveomeningoencefálica/genética , Síndrome Uveomeningoencefálica/fisiopatologia
6.
Hum Immunol ; 80(12): 966-975, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31604581

RESUMO

BACKGROUND: HLA mismatching is a well known risk factor for worst outcomes in kidney transplantation. METHODS: In the present study, HLA antigen and eplet mismatches were determined in 151 living donor-recipient pairs transplanted between 2007 and 2014 and rejection episodes and graft survival were evaluated. RESULTS: We found that high HLA-II eplet mismatch load (EpMM ≥ 13, versus low EpMM ≤ 5), was an independent predictor of AMR (adjusted HR = 14.839; P = 0.011), while HLA-II AgMM was not. We also showed that HLA-II EpMM load was a significant better predictor of AMR than AgMM (c-statistic = 0.064; P = 0.023). After discriminating HLA-II into HLA-DR and HLA-DQ loci we demonstrated that high versus low eplet mismatch load for HLA-DR (T3 ≥ 6 versus T = 0-1, p = 0.013) and HLA-DQ (T3 ≥ 7 versus T = 0-1, p = 0.009) are independent predictors for AMR. HLA-II EpMM increased discrimination performance of the classical HLA-II AgMM risk model (IDI, 0.061, 95%CI: 0.005-0.195) for AMR. Compared with AgMM, HLA-II eplet model adequately reclassified 13 of 17 patients (76.5%) with AMR and 92 of 134 patients (68.7%) without AMR (cfNRI, 0.785, 95%CI: 0.300-1.426). CONCLUSIONS: Our study evidences that eplet-based matching is a refinement of the classical HLA antigen mismatch analysis in LDKT and is a potential biomarker for personalized assessment of alloimmune risk.


Assuntos
Rejeição de Enxerto/genética , Antígenos HLA-DQ/genética , Antígenos HLA-DR/genética , Isoanticorpos/metabolismo , Transplante de Rim , Doença Aguda , Idoso , Idoso de 80 Anos ou mais , Seguimentos , Rejeição de Enxerto/diagnóstico , Antígenos HLA-DQ/imunologia , Antígenos HLA-DR/imunologia , Histocompatibilidade/genética , Teste de Histocompatibilidade , Humanos , Imunidade Humoral , Doadores Vivos , Pessoa de Meia-Idade , Medicina de Precisão , Prognóstico , Risco
7.
Endocr Regul ; 53(2): 71-82, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31517622

RESUMO

OBJECTIVE: The development of obesity and its metabolic complications is associated with dysregulation of various intrinsic mechanisms, which control basic metabolic processes through changes in the expression of numerous regulatory genes. METHODS: The expression level of HLA-DRA, HLA-DRB1, HLA-G, HLA-F, and NFX1 genes as well as miR-190b was measured in the blood of obese adolescents without signs of resistance to insulin and with insulin resistance in comparison with the group of relative healthy control individuals without signs of obesity. RESULTS: It was shown that obesity without signs of insulin resistance is associated with upregulation of the expression level of HLA-DRA and HLA-DRB1 genes, but with down-regulation of HLA-G gene expression in the blood as compared to control group of relative healthy adolescents. At the same time, no significant changes were observed in the expression level of HLA-F and NFX1 genes in the blood of this group of obese adolescents. Development of insulin resistance in obese individuals leads to significant down-regulation of HLA-DRA, HLA-DRB1, HLA-G, and HLA-F gene expressions as well as to up-regulation of NFX1 gene as well as microRNA miR-190b in the blood as compared to obese patients without signs of insulin resistance. CONCLUSIONS: Results of this study provide evidence that obesity affects the expression of the subset of genes related to immune response in the blood and that development of insulin resistance in obese adolescents is associated with strong down-regulation of the expressions of HLA-DRA, HLA-DRB1, HLA-F, and HLA-G genes, which may be contribute to the development of obesity complications. It is possible that transcription factor NFX1 and miR-190b participate in downregulation of HLA-DRA gene expression in the blood of obese adolescents with insulin resistance.


Assuntos
Imunidade Inata/genética , Resistência à Insulina/fisiologia , Obesidade Pediátrica , Adolescente , Células Sanguíneas/metabolismo , Estudos de Casos e Controles , Feminino , Regulação da Expressão Gênica , Antígenos HLA-DR/genética , Antígenos HLA-G/genética , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Masculino , Obesidade Pediátrica/sangue , Obesidade Pediátrica/genética , Obesidade Pediátrica/imunologia , Obesidade Pediátrica/metabolismo , Proteínas Repressoras/genética
8.
An Bras Dermatol ; 94(3): 287-292, 2019 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-31365656

RESUMO

BACKGROUND: Renal transplant recipients are submitted to immunosuppression to avoid graft rejection, which makes them susceptible to various conditions. Furthermore, these individuals present malignant tumors more frequently than the general population, including nonmelanoma skin cancer. The individual genetic basis that acts in the pathogenesis of cutaneous cancer may present a protection or susceptibility factor for disease development. One of these factors is the HLA complex. OBJECTIVE: To investigate HLA alleles association to the occurrence of nonmelanoma skin cancer in renal transplant recipients from São Paulo State. METHODS: A total of 213 patients (93 renal transplant recipients with nonmelanoma skin cancer and 120 renal transplant recipients without nonmelanoma skin cancer) were evaluated by retrospective and cross-sectional study. Epidemiological, clinical and HLA typing data were found in databases. HLA class I (A, B) and class II (DR) alleles were compared to establish their association with nonmelanoma skin cancer. RESULTS: Comparing renal transplant recipients with and without nonmelanoma skin cancer, the HLA-B*13 allele was associated with higher risk of developing nonmelanoma skin cancer while B*45 and B*50 alleles were associated with protection. STUDY LIMITATIONS: The HLA A, B and DR alleles identification for the kidney transplantation routine is done by low and medium resolution techniques that do not allow discrimination of specific alleles. CONCLUSION: The involvement of HLA alleles in nonmelanoma skin cancer in renal transplant recipients was confirmed in this study. Renal transplant recipients with HLA-B*13 showed higher risk for developing a skin cancer (OR= 7.29) and should be monitored for a long period of time after transplantation.


Assuntos
Antígenos HLA/genética , Transplante de Rim/efeitos adversos , Neoplasias Cutâneas/genética , Adulto , Idoso , Alelos , Brasil/epidemiologia , Estudos de Casos e Controles , Estudos Transversais , Feminino , Predisposição Genética para Doença/genética , Antígenos HLA-A/genética , Antígenos HLA-B/genética , Antígenos HLA-DR/genética , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Neoplasias Cutâneas/epidemiologia , Neoplasias Cutâneas/etiologia , Transplantados
9.
Hum Immunol ; 80(10): 855-862, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31285077

RESUMO

High mortality in pregnant women is a characteristic of hepatitis E virus (HEV) infection. Role of monocytes/T cells in HEV infection during pregnancy is still unclear. We compared CD14+monocytes and CD4+T cells by flow-cytometry in hepatitis-E patients including 13 pregnant (Antenatal care, ANC), 25 non-ANC patients and respective controls (12 and 20). Non-ANC-patients showed significantly higher frequency of monocytes with increased expression of CD80, CD86 and HLA-DR than control individuals (p < 0.001). Healthy pregnancy was associated with increased frequency of monocytes with higher CD80 expression and lower levels of HLA-DR (p < 0.05) compared to non-ANC controls. ANC-patients exhibited elevated levels of monocytes (p < 0.01) with higher expression of CD80 (p < 0.001) and reduced levels of HLA-DR and CD86 (p < 0.05) when compared with non-ANC patients. TLR2 and TLR4 surface expression on monocytes was higher in non-ANC-patients (p < 0.00) and lower in the ANC-patients (p < 0.01). Healthy-ANCs exhibited lower TLR4 expression on monocytes (p < 0.05). HEV infection did not change the frequency of CD4+ and CD4+CD28+T cells in patients' group (p > 0.05). Compared to respective controls, CD137+ and CD152+CD4+T cells were higher (p < 0.05) in both patients' categories. Higher levels of CD152+CD4+T cells (p < 0.001) was noted in healthy pregnant women. Among patients' groups, the CD4+T cells and their subpopulation were not different (p > 0.05). We found higher and reduced levels of circulating inflammatory cytokines (IL12, TNFα, IL6 and IL8; miliplex-assay) in non-ANC and ANC-patients respectively. In conclusion, on contrary to the classical activation of CD14+monocytes in the non-ANC-patients, impaired response was evident in the ANC-patients while the CD4+T cell populations were similar in the patient groups.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Vírus da Hepatite E/imunologia , Hepatite E/genética , Monócitos/imunologia , Fenótipo , Adulto , Antígeno B7-1/genética , Antígeno B7-1/metabolismo , Antígeno B7-2/genética , Antígeno B7-2/metabolismo , Citocinas/sangue , Feminino , Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Hepatite E/patologia , Humanos , Índia , Receptores de Lipopolissacarídeos/genética , Receptores de Lipopolissacarídeos/metabolismo , Masculino , Gravidez , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Adulto Jovem
10.
Int J Mol Sci ; 20(11)2019 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-31181776

RESUMO

BACKGROUND: Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system. The cause of multiple sclerosis is unknown but there are several evidences that associate the genetic basis of the disease with environmental causes. An important association between viral infection and development of MS is clearly demonstrated. Viruses have a strong impact on innate immune cells. In particular, myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs), are able to respond to viruses and to activate the adaptive immune response. METHODS: In this study we mimic viral infection using synthetic single-strand RNA, Resiquimod, and we compared the response of both DC subsets derived from healthy donors and MS patients by characterizing the expression of costimulatory molecules on the DC surface. RESULTS: We found that pDCs from MS patients express higher levels of OX40-L, HLA-DR, and CD86 than healthy donors. Moreover, we found that blood cells from MS patients and healthy donors upon Resiquimod-stimulation are enriched in a subpopulation of pDCs, characterized by a high amount of costimulatory molecules. CONCLUSION: Overall, these results indicate that activation of pDCs is enhanced in MS, likely due to a latent viral infection, and that costimulatory molecules expressed on pDCs could mediate a protective response against the viral trigger of autoimmunity.


Assuntos
Células Dendríticas/efeitos dos fármacos , Imidazóis/farmacologia , Esclerose Múltipla Recidivante-Remitente/imunologia , Antígeno B7-2/genética , Antígeno B7-2/metabolismo , Células Dendríticas/imunologia , Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Humanos , Ligante OX40/genética , Ligante OX40/metabolismo
11.
Parasite Immunol ; 41(8): e12632, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31099071

RESUMO

Trypanosoma brucei gambiense, an extracellular eukaryotic flagellate parasite, is the main etiological agent of human African trypanosomiasis (HAT) or sleeping sickness. Dendritic cells (DCs) play a pivotal role at the interface between innate and adaptive immune response and are implicated during HAT. In this study, we investigated the effects of T gambiense and its excreted/secreted factors (ESF) on the phenotype of human monocyte-derived DCs (Mo-DCs). Mo-DCs were cultured with trypanosomes, lipopolysaccharide (LPS), ESF derived from T gambiense bloodstream strain Biyamina (MHOM/SD/82), or both ESF and LPS. Importantly, ESF reduced the expression of the maturation markers HLA-DR and CD83, as well as the secretion of IL-12, TNF-alpha and IL-10, in LPS-stimulated Mo-DCs. During mixed-leucocyte reactions, LPS- plus ESF-exposed DCs induced a non-significant decrease in the IFN-gamma/IL-10 ratio of CD4 + T-cell cytokines. Based on the results presented here, we raise the hypothesis that T gambiense has developed an immune escape strategy through the secretion of paracrine mediators in order to limit maturation and activation of human DCs. The identification of the factor(s) in the T gambiense ESF and of the DCs signalling pathway(s) involved may be important in the development of new therapeutic targets.


Assuntos
Células Dendríticas/imunologia , Monócitos/imunologia , Proteínas de Protozoários/imunologia , Trypanosoma brucei gambiense/imunologia , Tripanossomíase Africana/imunologia , Animais , Células Dendríticas/parasitologia , Feminino , Antígenos HLA-DR/genética , Antígenos HLA-DR/imunologia , Interações Hospedeiro-Parasita , Humanos , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Lipopolissacarídeos/imunologia , Camundongos , Monócitos/parasitologia , Proteínas de Protozoários/genética , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/parasitologia , Trypanosoma brucei gambiense/genética , Tripanossomíase Africana/genética , Tripanossomíase Africana/parasitologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
12.
J Immunol Methods ; 470: 59-63, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31082373

RESUMO

BACKGROUND: Flow cytometry is a powerful analytical technique. However, it requires time-consuming, multi-step sample procedure. A new protocol was developed to perform extracellular staining and red blood cell lysis in one step, using dry antibodies. Common markers of white blood cells as well as sepsis biomarkers were tested as a model for modulated antigen expression. METHODS: Peripheral blood was stained using the reference and the one-step methods. Recruitment and staining of CD3-, CD4-, CD8-, CD14-, and CD15-positive cells were analyzed. Then, protocol modifications were tested for optimization. Finally, the one-step method was evaluated on subjects in septic conditions, by measuring expressions of CD64 and of HLA-DR. RESULTS: No statistical differences were observed between methods when comparing the proportions of cells. The procedure was optimized by decreasing blood volume from 100 µL to 5 µL, lysis from 1 mL to 500 µL, and time from 30 to 15 min. In the blood samples from septic subjects, an increase of CD64 on neutrophils and a decrease of HLA-DR on monocytes were observed. CONCLUSIONS: The one-step method, described here-in, enables an accurate, streamlined flow cytometry sample preparation protocol. The simplified phenotyping procedure reduces training requirements and could help overcome logistic constraints in many flow cytometry applications.


Assuntos
Citometria de Fluxo/métodos , Antígenos HLA-DR/sangue , Receptores de IgG/sangue , Choque Séptico/diagnóstico , Coloração e Rotulagem/métodos , Idoso , Biomarcadores/sangue , Estudos de Casos e Controles , Feminino , Expressão Gênica , Antígenos HLA-DR/genética , Humanos , Masculino , Monócitos/metabolismo , Monócitos/patologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Receptores de IgG/genética , Choque Séptico/sangue , Choque Séptico/genética , Choque Séptico/mortalidade , Análise de Sobrevida
13.
An. bras. dermatol ; 94(3): 287-292, May-June 2019. tab
Artigo em Inglês | LILACS, Sec. Est. Saúde SP, SESSP-ILSLPROD, Sec. Est. Saúde SP, SESSP-ILSLACERVO, Sec. Est. Saúde SP | ID: biblio-1011110

RESUMO

Abstract: Background: Renal transplant recipients are submitted to immunosuppression to avoid graft rejection, which makes them susceptible to various conditions. Furthermore, these individuals present malignant tumors more frequently than the general population, including nonmelanoma skin cancer. The individual genetic basis that acts in the pathogenesis of cutaneous cancer may present a protection or susceptibility factor for disease development. One of these factors is the HLA complex. Objective: To investigate HLA alleles association to the occurrence of nonmelanoma skin cancer in renal transplant recipients from São Paulo State. Methods: A total of 213 patients (93 renal transplant recipients with nonmelanoma skin cancer and 120 renal transplant recipients without nonmelanoma skin cancer) were evaluated by retrospective and cross-sectional study. Epidemiological, clinical and HLA typing data were found in databases. HLA class I (A, B) and class II (DR) alleles were compared to establish their association with nonmelanoma skin cancer. Results: Comparing renal transplant recipients with and without nonmelanoma skin cancer, the HLA-B*13 allele was associated with higher risk of developing nonmelanoma skin cancer while B*45 and B*50 alleles were associated with protection. Study limitations: The HLA A, B and DR alleles identification for the kidney transplantation routine is done by low and medium resolution techniques that do not allow discrimination of specific alleles. Conclusion: The involvement of HLA alleles in nonmelanoma skin cancer in renal transplant recipients was confirmed in this study. Renal transplant recipients with HLA-B*13 showed higher risk for developing a skin cancer (OR= 7.29) and should be monitored for a long period of time after transplantation.


Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Idoso , Neoplasias Cutâneas/genética , Transplante de Rim/efeitos adversos , Antígenos HLA/genética , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/epidemiologia , Brasil/epidemiologia , Antígenos HLA-A/genética , Antígenos HLA-B/genética , Antígenos HLA-DR/genética , Estudos de Casos e Controles , Estudos Transversais , Estudos Retrospectivos , Predisposição Genética para Doença/genética , Alelos , Transplantados
14.
Clin Immunol ; 203: 125-133, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31028919

RESUMO

Intestinal epithelial cells (IECs) form a fundamental mucosal barrier and actively participate in tolerance and immunity against intestinal contents. Major histocompatibility complex class II (MHC II) and invariant chain (Ii) molecules are essential for adaptive immune response. MHC II deficiency often presents with gastrointestinal disorders. Intestinal biopsy samples revealed an absence of HLA-DR, Ii, and local immunoglobulins in both hematopoietic immune cells and IECs accompanied by a lack of faecal sIgA. After successful hematopoietic stem cell transplantation (HSCT) absent HLA-DR and Ii expression persisted in IECs and faecal stool analysis indicated inflammation and high microbial activity. We describe multifaceted disturbance of adaptive mucosal immunity in MHC II deficient patients suffering from enteropathy. HLA-DR and Ii expression on enterocytes is not restored by HSCT. This may account for increased susceptibility to enteric infections and intestinal inflammation leading to prolonged enteropathy reported in MHC II deficient patients.


Assuntos
Gastroenteropatias/imunologia , Antígenos HLA-DR/genética , Síndromes de Imunodeficiência/imunologia , Inflamação/imunologia , Mucosa Intestinal/imunologia , Imunidade Adaptativa , Adolescente , Criança , Pré-Escolar , Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Feminino , Gastroenteropatias/genética , Antígenos HLA-DR/metabolismo , Humanos , Síndromes de Imunodeficiência/genética , Lactente , Inflamação/genética , Masculino , Mutação/genética , Linhagem , Fatores de Transcrição/genética
15.
Glycoconj J ; 36(3): 185-197, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31016559

RESUMO

In this study, we assessed the potential of liposomes coated with a neoglycolipid containing α1-3,α1-6-mannotriose residues (Man3-DPPE; Manα1-6(Manα1-3)Manitol-DPPE) for in vitro activation and maturation of human mononuclear phagocytes. In response to treatment with Man3-DPPE-coated liposomes (Man3-OMLs), PMA-stimulated human THP-1 cells showed enhanced expression of CD40, CD80 and HLA-DR and secreted significant levels of IL-12p40. Among various linkages of Man2-DPPE-coated liposomes, only liposomes coated with Manα1-6Manitol-DPPE (α1-6Man2-DPPE) induced these cellular responses similarly to Man3-OML treatment. Liposomes coated with Manα1-6(Manα1-3)Manα1-6(Manα1-3)Manitol-DPPE (Man5-DPPE) failed to activate the cells. These results suggest that an unsubstituted α1-6Man branch bound to a mannitol unit at the reducing end in Man3-DPPE is required for in vitro activation of human mononuclear phagocytes. Man3-OML-induced IL-12p40 production was not inhibited by BAY11-7082, an inhibitor of the MyD88-dependent signaling network, suggesting that TLRs are not involved in activation of human mononuclear phagocytes by Man3-OMLs. Stimulation of inflammatory monocytes or monocyte-derived dendritic cells (moDCs) with Man3-OMLs also induced enhanced expression of co-stimulatory molecules, HLA-DR, and CCR7, and IL-12p40 production from both types of cells. In response to Man3-OML treatment, moDCs but not inflammatory monocytes produced bioactive IL-12p70, which was enhanced by CD40 ligation. Thus, Man3-OMLs can activate naïve human mononuclear phagocytes and lead human moDCs to a fully matured status in vitro to elicit CTLs and a Th1 response without addition of inflammatory cytokines or TLR agonists.


Assuntos
Glicolipídeos/farmacologia , Lipossomos/farmacologia , Monócitos/efeitos dos fármacos , Trissacarídeos/farmacologia , 1,2-Dipalmitoilfosfatidilcolina/química , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Glicolipídeos/química , Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Humanos , Interleucina-1/genética , Interleucina-1/metabolismo , Lipossomos/química , Monócitos/imunologia , Receptores CCR7/genética , Receptores CCR7/metabolismo , Trissacarídeos/química
16.
Cell Physiol Biochem ; 52(3): 397-407, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30845379

RESUMO

BACKGROUND/AIMS: TNF-α-mediated pro-inflammatory phenotypic change in monocytes is known to be implicated in the pathogenesis of metabolic inflammation and insulin resistance. However, the mechanism by which TNF-α-induces inflammatory phenotypic shift in monocytes is poorly understood. Since long-chain acyl-CoA synthetase 1 (ACSL1) is associated with inflammatory monocytes/macrophages, we investigated the role of ACSL1 in the TNF-α-driven inflammatory phenotypic shift in the monocytes. METHODS: Monocytes (Human monocytic THP-1 cells) were stimulated with TNF-α. Inflammatory phenotypic markers (CD16, CD11b, CD11c and HLA-DR) expression was determined with real time RTPCR and flow cytometry. IL-1ß and MCP-1 were determined by ELISA. Signaling pathways were identified by using ACSL1 inhibitor, ACSL1 siRNA and NF-κB reporter monocytic cells. Phosphorylation of NF-κB was analyzed by western blotting and flow cytometry. RESULTS: Our data show that TNF-α induced significant increase in the expression of CD16, CD11b, CD11c and HLA-DR. Inhibition of ACSL1 activity in the cells with triacsin C significantly suppressed the expression of these inflammatory markers. Using ACSL-1 siRNA, we further demonstrate that TNF-α-induced inflammatory markers expression in monocytic cells requires ACSL1. In addition, IL-1b and MCP-1 production by TNF-α activated monocytic cells was significantly blocked by the inhibition of ACSL-1 activity. Interestingly, elevated NF-κB activity resulting from TNF-α stimulation was attenuated in ACSL1 deficient cells. CONCLUSION: Our findings provide an evidence that TNF-α-associated inflammatory polarization in monocytes is an ACSL1 dependent process, which indicates its central role in TNF-α-driven metabolic inflammation.


Assuntos
Coenzima A Ligases/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/patologia , Fator de Necrose Tumoral alfa/farmacologia , Linhagem Celular , Quimiocina CCL2/análise , Coenzima A Ligases/antagonistas & inibidores , Coenzima A Ligases/genética , Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Humanos , Inflamação/metabolismo , Interleucina-1beta/análise , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , NF-kappa B/metabolismo , Fosforilação , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores de IgG/genética , Receptores de IgG/metabolismo , Triazenos/química , Triazenos/metabolismo
17.
Acta Derm Venereol ; 99(6): 602-609, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30848289

RESUMO

Dipeptidyl peptidase-4 inhibitors (DPP-4i or gliptins) increase the risk of developing bullous pemphigoid (BP). To clarify, whether gliptin-associated BP has special features, we analyzed the clinical, histopathological and immunological features of 27 BP patients, 10 of which previously used gliptin medication. Compared to those who had not previously received gliptins, subjects who had, showed higher BP180-NC16A ELISA (enzyme-linked immunosorbent assay) values, fewer neurological co-morbidities and shorter time to remission, but differences were not statistically significant. The HLA-DQB1*03:01 allele was more commonly present among the BP patients than the control population, but was not more common in those with gliptin history. To determine the effect of gliptins on the expression of the DPP-4/CD-26 protein we performed immunohistochemistry, which showed that the skin expression of DPP-4/CD-26 was increased in BP patients, but not affected by prior gliptin treatment. We conclude that DPP-4i medication is common among BP patients and prior gliptin treatment may be associated with some specific features.


Assuntos
Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/efeitos adversos , Penfigoide Bolhoso/induzido quimicamente , Penfigoide Bolhoso/metabolismo , Idoso , Idoso de 80 Anos ou mais , Autoantígenos/metabolismo , Epiderme/metabolismo , Feminino , Genótipo , Antígenos HLA-DQ/genética , Antígenos HLA-DR/genética , Humanos , Queratinócitos/metabolismo , Laminina/metabolismo , Masculino , Pessoa de Meia-Idade , Colágenos não Fibrilares/metabolismo , Penfigoide Bolhoso/imunologia , Penfigoide Bolhoso/patologia
18.
Nature ; 567(7746): 109-112, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30787439

RESUMO

Zoonotic influenza A viruses of avian origin can cause severe disease in individuals, or even global pandemics, and thus pose a threat to human populations. Waterfowl and shorebirds are believed to be the reservoir for all influenza A viruses, but this has recently been challenged by the identification of novel influenza A viruses in bats1,2. The major bat influenza A virus envelope glycoprotein, haemagglutinin, does not bind the canonical influenza A virus receptor, sialic acid or any other glycan1,3,4, despite its high sequence and structural homology with conventional haemagglutinins. This functionally uncharacterized plasticity of the bat influenza A virus haemagglutinin means the tropism and zoonotic potential of these viruses has not been fully determined. Here we show, using transcriptomic profiling of susceptible versus non-susceptible cells in combination with genome-wide CRISPR-Cas9 screening, that the major histocompatibility complex class II (MHC-II) human leukocyte antigen DR isotype (HLA-DR) is an essential entry determinant for bat influenza A viruses. Genetic ablation of the HLA-DR α-chain rendered cells resistant to infection by bat influenza A virus, whereas ectopic expression of the HLA-DR complex in non-susceptible cells conferred susceptibility. Expression of MHC-II from different bat species, pigs, mice or chickens also conferred susceptibility to infection. Notably, the infection of mice with bat influenza A virus resulted in robust virus replication in the upper respiratory tract, whereas mice deficient for MHC-II were resistant. Collectively, our data identify MHC-II as a crucial entry mediator for bat influenza A viruses in multiple species, which permits a broad vertebrate tropism.


Assuntos
Quirópteros/virologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Especificidade de Hospedeiro , Vírus da Influenza A/imunologia , Vírus da Influenza A/fisiologia , Zoonoses/imunologia , Zoonoses/virologia , Animais , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Galinhas/genética , Galinhas/imunologia , Quirópteros/genética , Quirópteros/imunologia , Quirópteros/metabolismo , Feminino , Perfilação da Expressão Gênica , Antígenos HLA-DR/genética , Antígenos HLA-DR/imunologia , Antígenos HLA-DR/metabolismo , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Especificidade de Hospedeiro/genética , Especificidade de Hospedeiro/imunologia , Humanos , Masculino , Camundongos , Camundongos Knockout , Sistema Respiratório/virologia , Suínos/genética , Suínos/imunologia , Tropismo Viral/genética , Tropismo Viral/imunologia , Replicação Viral , Zoonoses/genética , Zoonoses/metabolismo
19.
BMC Infect Dis ; 19(1): 56, 2019 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-30651082

RESUMO

BACKGROUND: The mechanism behind HIV mediated immune activation remains debated, although the role of virus replication in this process is increasingly evident. Toll like Receptor 9 (TLR9) has been implicated in HIV mediated immune activation via sensing of viral CpG DNA. Polymorphisms in the TLR9 gene and promoter region including TLR9 1635A/G and 1486C/T have been found to be associated with multiple infectious diseases and cancers. METHODS: In the current study, we looked at the correlation of TLR9 polymorphisms 1635A/G and 1486C/T with key hallmarks of HIV disease in a cohort of 50 HIV infected patients. We analyzed CD4 counts, T cell immune activation characterized by upregulation of CD38 and HLA-DR and upregulation of plasma biomarkers of inflammation like LPS, sCD14, IL-6 and IP10 in the HIV patient cohort and compared it to healthy controls. RESULTS: We found that TLR9 1635AA genotype was associated with lower CD4 counts and significantly higher immune activation in both CD4+ and CD8+ T cells. Analysis of HIV associated plasma biomarkers including LPS, sCD14, IL-6 and IP10 revealed a strong correlation between IP10 and immune activation. Interestingly, IP10 levels were also found to be higher in HIV patients with the 1635AA genotype. Furthermore, the TLR9 1486C/T polymorphism that is in linkage disequilibrium with 1635A/G was weakly associated with lower CD4 counts, higher CD8 immune activation and higher IP10 levels. CONCLUSIONS: As TLR9 stimulation is known to induce IP10 production by dendritic cells, our findings provide new insights into HIV mediated immune activation and CD4 loss. TLR9 stimulation by viral CpG DNA may be important to HIV immunopathogenesis and the TLR9 polymorphisms 1635A/G and 1486C/T may be associated with disease progression.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , Polimorfismo Genético , Receptores de Citocinas/sangue , Receptor Toll-Like 9/genética , Adulto , Linfócitos T CD8-Positivos/imunologia , Estudos de Coortes , Estudos Transversais , DNA Viral , Feminino , Infecções por HIV/genética , Infecções por HIV/virologia , Antígenos HLA-DR/genética , Antígenos HLA-DR/imunologia , Humanos , Interleucina-6/sangue , Ativação Linfocitária/imunologia , Masculino , Receptores de Citocinas/genética , Replicação Viral
20.
Immunology ; 156(2): 187-198, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30408168

RESUMO

Major histocompatibility complex (MHC) genes are highly polymorphic, which makes each MHC molecule different regarding their peptide repertoire, so they can bind and present to T lymphocytes. The increasing importance of immunopeptidomics and its use in personalized medicine in different fields such as oncology or autoimmunity demand the correct analysis of the peptide repertoires bound to human leukocyte antigen type 1 (HLA-I) and HLA-II molecules. Purification of the peptide pool by affinity chromatography and individual peptide sequencing using mass spectrometry techniques is the standard protocol to define the binding motifs of the different MHC-I and MHC-II molecules. The identification of MHC-I binding motifs is relatively simple, but it is more complicated for MHC-II. There are some programs that identify the anchor motifs of MHC-II molecules. However, these programs do not identify the anchor motif correctly for some HLA-II molecules and some anchor motifs have been deduced using subjective interpretation of the data. Here, we present a new software, called PRBAM (Peptide Repertoire-Based Anchor Motif) that uses a new algorithm based on the peptide-MHC interactions and, using peptide lists obtained by mass spectrometry sequencing, identifies the binding motif of MHC-I and HLA-DR molecules. PRBAM has an easy-to-use interface, and the results are presented in graphics, tables and peptide lists. Finally, the fact that PRBAM uses a new algorithm makes it complementary to other existing programs.


Assuntos
Antígenos HLA-DR/genética , Antígenos de Histocompatibilidade Classe I/genética , Análise de Sequência de Proteína , Software , Motivos de Aminoácidos , Antígenos HLA-DR/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos
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