RESUMO
Type 1 diabetes (T1D) and celiac disease (CeD) are common autoimmune diseases in children where the pathophysiology is not fully characterized. The autoimmune process involves a complex scenario of both inflammatory and regulatory features. Galectin-1 (GAL-1) has a wide range of biological activities e.g. interaction with immune cells. We examined the relationship between GAL-1 and soluble immune markers and T-cell subsets in a cohort of children with T1D and/or CeD relative to healthy children. GAL-1, together with several soluble immune markers [e.g. interleukins (IL)], tumor necrosis factor (TNF), acute phase proteins, and matrix metalloproteinases (MMP) were measured in sera from children with T1D and/or CeD by fluorochrome (Luminex) technique using children without these diseases as a reference. Subgroups of T cells, including T-regulatory (Treg) cells, were analysed by flow cytometry. Association between GAL-1, pro-inflammatory markers, and Treg cells differed depending on which illness combination was present. In children with both T1D and CeD, GAL-1 correlated positively with pro-inflammatory markers (IL-1ß, IL-6, and TNF-α). Composite scores increased the strength of correlation between GAL-1 and pro-inflammatory markers, Th1-associated interferon (IFN)-γ, and T1D-associated visfatin. Contrary, in children diagnosed with exclusively T1D, GAL-1 was positively correlated to CD25hi and CD25hiCD101+ Treg cells. For children with only CeD, no association between GAL-1 and other immune markers was observed. In conclusion, the association observed between GAL-1, soluble immune markers, and Treg cells may indicate a role for GAL-1 in the pathophysiology of T1D and, to some extent, also in CeD.
Assuntos
Benzamidas , Doença Celíaca , Diabetes Mellitus Tipo 1 , Tirosina , Criança , Humanos , Biomarcadores/metabolismo , Doença Celíaca/patologia , Galectina 1/metabolismo , Linfócitos T Reguladores , Fator de Necrose Tumoral alfa/metabolismo , Tirosina/análogos & derivadosRESUMO
Our purpose was to characterize the pattern of B cell subsets in children with a combined diagnosis of type 1 diabetes (T1D) and celiac disease (C) since children with single or double diagnosis of these autoimmune diseases may differ in peripheral B cell subset phenotype patterns. B cells were analyzed with flow cytometry for the expression of differentiation/maturation markers to identify transitional, naive, and memory B cells. Transitional (CD24hiCD38hiCD19+) and memory Bregs (mBregs; CD24hiCD27+CD19+, CD1d+CD27+CD19+, and CD5+CD1d+CD19+) were classified as B cells with regulatory capacity. Children with a combined diagnosis of T1D and C showed a pattern of diminished peripheral B cell subsets. The B cells compartment in children with combined diagnosis had higher percentages of memory B subsets and Bregs, including activated subsets, compared to children with either T1D or C. Children with combined diagnosis had a lower percentage of naive B cells (CD27-CD19+; IgD+CD19+) and an increased percentage of memory B cells (CD27+CD19+; IgD-CD19+). A similar alteration was seen among the CD39+ expressing naive and memory B cells. Memory Bregs (CD1d+CD27+CD19+) were more frequent, contrary to the lower percentage of CD5+ transitional Bregs in children with a combined diagnosis. In children with either T1D or C, the peripheral B cell compartment was dominated by naive cells. Differences in the pattern of heterogeneous peripheral B cell repertoire subsets reflect a shifting in the B cell compartment between children with T1D and/or C. This is an immunological challenge of impact on the pathophysiology of these autoimmune diseases.
Assuntos
Doenças Autoimunes , Subpopulações de Linfócitos B , Linfócitos B Reguladores , Doença Celíaca , Diabetes Mellitus Tipo 1 , Criança , Humanos , Diabetes Mellitus Tipo 1/metabolismo , Antígenos CD19/metabolismo , Citometria de Fluxo , Doenças Autoimunes/metabolismoRESUMO
Several key factors can affect the outcome of immunological studies; isolation/cryopreservation can possibly alter T, B, NK, and T-regulatory (Treg) cell marker expression patterns. Blood samples from 50 blood donors supplemented with Na-heparin or K2EDTA were handled within 4 and 24 h after blood sampling. PBMC were isolated with different density gradients. Flow cytometric analysis of intracellular and extracellular CD markers was performed on blood samples freshly isolated PBMC, and PBMC was thawed 6 and 12 mo post-cryopreservation for the purpose of identifying B, NK, Th, T-cytotoxic, and Treg cells. No differences were observed in the percentages for CD3+, CD3+CD4+, CD3+CD8+, CD19+, or CD56+CD16+ cells within 24 h of sampling regardless of which supplement or isolation techniques were used. Differentiated (diff) CD4+ cells were in general less affected by isolation and cryopreservation than diff CD8+ cells. Terminally diff effector CD4+ and CD8+ cells were not affected by either isolation of lymphocytes or cryopreservation. In contrast, naive and early-diff effector memory CD4+ and CD8+ cells were affected by isolation and cryopreservation. The percentages of Treg cells defined as CD4+CD25hi expressing CD101 or CD129, CD4+CD25hiCD127-, and CD4+CD25hiCD127-FOXP3+, respectively, remained stable after isolation and cryopreservation. Subsets expressing CD127, with or without FOXP3, were not affected by isolation/cryopreservation. Subsets expressing CD39, contrary to CD45RA, on CD4+CD25+CD127- cells with or without FOXP3 were not affected by either isolation or cryopreservation. In conclusion, subsets of CD4+, CD8+, and CD25hi lymphocytes are in general not influenced by isolation and long-term cryopreservation.
Assuntos
Antígenos CD4/imunologia , Antígenos CD8/imunologia , Separação Celular/métodos , Criopreservação/métodos , Subpopulações de Linfócitos/imunologia , Adolescente , Adulto , Idoso , Feminino , Humanos , Subpopulações de Linfócitos/citologia , Masculino , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: The use of statins has shown several anti-inflammatory actions, including modulatory effects on T cells in vitro. Since the effects on human T cells in vivo are less clarified, our aim was to investigate the effects of simvastatin on human T cells in vivo and ex vivo. METHODS AND RESULTS: A randomized, double-blind, placebo-controlled study design was applied. Eighty volunteers with mild to moderate hypercholesterolemia received either simvastatin 40 mg or placebo for 6 weeks. The serum levels of C-reactive protein (CRP) were significantly reduced by simvastatin. The proportions of CD4+ and CD8+ T cell subsets expressing early (CD25) or late (HLA-DR) activation markers, as assessed by flow cytometry, were not changed by simvastatin. However, simvastatin tended to increase the density of HLA-DR and L-selectin per CD8+ T cell. The T helper(h)1/Th2 response was evaluated by stimulatory assays followed by intra-cellular staining of interferon-gamma and interleukin-4. Simvastatin treatment did not affect the Th1 response but the results indicated a potential to suppress Th2. CONCLUSION: Simvastatin treatment resulted in a few discrete changes as regards peripheral T cells. However, the findings do not provide evidence that simvastatin-induced anti-inflammatory actions are related to any significant modulatory effects on human T cells in clinically healthy men with hypercholesterolemia.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Sinvastatina/farmacologia , Linfócitos T/efeitos dos fármacos , Adulto , Apolipoproteínas/sangue , Proteína C-Reativa/metabolismo , Concanavalina A/farmacologia , Método Duplo-Cego , Enterotoxinas/farmacologia , Antígenos HLA-DR/sangue , Humanos , Hipercolesterolemia/sangue , Hipercolesterolemia/tratamento farmacológico , Hipercolesterolemia/imunologia , Subunidade alfa de Receptor de Interleucina-2/sangue , Selectina L/sangue , Lipídeos/sangue , Masculino , Pessoa de Meia-Idade , Subpopulações de Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Several lines of evidence indicate that increased inflammatory activity in peripheral blood is associated with the acute coronary syndrome. Systemic inflammation in clinically stable conditions of coronary artery disease has been less studied. We examined cytokine profiles in 20 patients who had acute coronary syndrome, 45 who had angiographically verified coronary artery disease and stable angina pectoris, and 45 healthy controls. Circulating levels of C-reactive protein, interleukin-1 receptor antagonist, interleukin-2 receptor, interleukin-6, interleukin-10, and interleukin-18 were determined. Subpopulations of peripheral immune cells, including neutrophil-platelet aggregates, were analyzed by 3-color flow cytometry using a panel of monoclonal antibodies. Patients who had acute coronary syndrome and stable angina pectoris had significantly higher levels of C-reactive protein, interleukin-6, and interleukin-1 receptor antagonist than did controls, whereas levels of interleukin-2 receptor, interleukin-10, and interleukin-18 were similar across groups. Patients had significantly more neutrophils, and the numbers of neutrophil-platelet aggregates were particularly large in patients who had stable angina pectoris. High levels of C-reactive protein, interleukin-6, and interleukin-1 receptor antagonist in patients were significantly related to numbers of neutrophils and neutrophil-platelet aggregates but not to other immune cell subpopulations. The data suggest that the interaction between neutrophils and platelets is an important component of proinflammatory activity seen in peripheral blood of stable and unstable forms of coronary artery disease.
Assuntos
Proteína C-Reativa/análise , Doença da Artéria Coronariana/imunologia , Interleucinas/sangue , Neutrófilos/metabolismo , Agregação Plaquetária/fisiologia , Angina Pectoris/imunologia , Angina Pectoris/metabolismo , Angina Instável/imunologia , Angina Instável/metabolismo , Estudos de Casos e Controles , HDL-Colesterol/sangue , Doença da Artéria Coronariana/metabolismo , Citometria de Fluxo , Humanos , Proteína Antagonista do Receptor de Interleucina 1 , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/imunologia , Infarto do Miocárdio/metabolismo , Receptores de Interleucina-1/antagonistas & inibidores , Receptores de Interleucina-2/sangue , Proteínas Recombinantes/sangue , Sialoglicoproteínas/sangue , Linfócitos T Auxiliares-Indutores/metabolismo , Triglicerídeos/sangueRESUMO
An age-related increase of DNA damage/mutation has been previously reported in human lymphocytes. The high copy number and mutation rate make the mtDNA genome an ideal candidate for assessing damage and to act as a potential biomarker of ageing. In the present study, two assays were developed to evaluate the level of mtDNA(4977) and the accumulation of point mutations with age. A competitive polymerase chain reaction (PCR) methodology incorporating three primers was used to detect and quantify the levels of mtDNA(4977) and a novel heteroduplex reference strand conformational analysis (RSCA) technique was used to analyse the accumulation of point mutations. The assays were applied to an in vitro model of T cell ageing and ex vivo DNA samples from an elderly cohort of subjects and a younger control group. The mtDNA(4977) was detected in all the DNA samples examined but only a very low concentration was observed and no age-related increase or accumulation was observed. No accumulation of point mutations was identified using RSCA within the T cell clones as they were aged or the ex vivo lymphocytes from the elderly cohort. A higher level of variation was observed within the ex vivo DNA samples, verifying the high resolution of RSCA and its ability to identify different mtDNA species, although no correlation with age was observed. The low level of mtDNA damage observed with respect to the ex vivo lymphocyte DNA samples within this study may be due in part to the high turnover of blood cells/mtDNA, which may inhibit the accumulation of genetically abnormal mtDNA that may play a role in immunosenescence. A similar explanation may also apply to the in vitro model of T cell ageing if the vast majority of the cells are replicating rather than entering senescence.
Assuntos
Envelhecimento/genética , Dano ao DNA , DNA Mitocondrial , Linfócitos T , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular , Células Clonais , Análise Mutacional de DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ácidos Nucleicos Heteroduplexes , Reação em Cadeia da Polimerase/métodos , Linfócitos T/citologiaRESUMO
The results of previous work from our laboratories have suggested that free radical damage to T cells as they age may contribute to the age-related decline in the T cell-mediated immune response. The aims of this investigation were to assess the efficiency of in vivo antioxidant capacity through determining the antioxidant capacity of plasma using the ferric reducing ability of plasma assay, and to assess the levels and types of DNA damage (as a measure of in vivo antioxidant efficiency) using the alkaline comet assay and two enzymatic modifications of the comet assay, in peripheral blood mononuclear cells (PBMCs) from nonagenarian subjects drawn from the Swedish NONA Immune Study. The results obtained were compared with those from middle-aged (40-60 years) controls to identify potential anti-immunosenescent effects of in vivo antioxidants. The results revealed a significantly higher plasma antioxidant capacity in NONA subjects compared to controls, and these results support a relationship between longevity and intact immune function, which may be underpinned by antioxidant defences which reduce free radical damage to PBMC, thus helping to maintain cell function. The NONA subjects were found to have similar levels of DNA damage in their PBMCs to those found in middle aged controls.