RESUMO
The standard of care (SoC) for Type 1 diabetes (T1D) today is much the same as it was in the early 1920s, simply with more insulin options-fast-acting, slow-acting, injectable, and inhalable insulins. However, these well-tolerated treatments only manage the symptoms and complications, but do nothing to halt the underlying immune response. There is an unmet need for better treatment options for T1D that address all aspects of the disease. For decades, we have successfully treated T1D in preclinical animal models with immune-modifying therapies that have not demonstrated comparable efficacy in humans. The path to bringing such options to the clinic will depend on the implementation and standard inclusion of biomarkers of immune and therapeutic efficacy in T1D clinical trials, and dictate if we can create a new SoC that treats the underlying autoimmunity as well as the symptoms it causes.
Assuntos
Biomarcadores/análise , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/terapia , Imunoterapia , Animais , Autoanticorpos/análise , Autoanticorpos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Modelos Animais de Doenças , Humanos , Sensibilidade e Especificidade , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Assessing messenger RNA (mRNA) and microRNA levels in peripheral blood cells may complement conventional parameters in clinical practice. Working with small, precious samples requires optimal RNA yields and minimal RNA degradation. Several procedures for RNA extraction and complementary DNA (cDNA) synthesis were compared for their efficiency. The effect on RNA quality of freeze-thawing peripheral blood cells and storage in preserving reagents was investigated. In terms of RNA yield and convenience, quality quantitative polymerase chain reaction signals per nanogram of total RNA and using NucleoSpin and mirVana columns is preferable. The SuperScript III protocol results in the highest cDNA yields. During conventional procedures of storing peripheral blood cells at -180°C and thawing them thereafter, RNA integrity is maintained. TRIzol preserves RNA in cells stored at -20°C. Detection of mRNA levels significantly decreases in degraded RNA samples, whereas microRNA molecules remain relatively stable. When standardized to reference targets, mRNA transcripts and microRNAs can be reliably quantified in moderately degraded (quality index 4-7) and severely degraded (quality index <4) RNA samples, respectively. We describe a strategy for obtaining high-quality and quantity RNA from fresh and stored cells from blood. The results serve as a guideline for sensitive mRNA and microRNA expression assessment in clinical material.
Assuntos
Células Sanguíneas/química , Células Sanguíneas/metabolismo , Coleta de Amostras Sanguíneas/métodos , MicroRNAs/isolamento & purificação , Preservação Biológica/métodos , RNA Mensageiro/isolamento & purificação , Algoritmos , Células Sanguíneas/citologia , Análise Química do Sangue/métodos , Análise Química do Sangue/normas , Preservação de Sangue/métodos , Preservação de Sangue/normas , Coleta de Amostras Sanguíneas/normas , Calibragem , Técnicas Genéticas/normas , Guanidinas , Humanos , MicroRNAs/análise , MicroRNAs/metabolismo , Fenóis , Reação em Cadeia da Polimerase/métodos , Preservação Biológica/normas , Estabilidade de RNA/fisiologia , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Padrões de ReferênciaRESUMO
The generation of effector CD8(+) T cells with lytic capacity is crucial for tumor control. Dendritic cells (DCs) provide important signals to promote naive CD8(+) T cell priming and activation of effector T cells. Here, we report that the Notch pathway has an important role in both these processes in human CD8(+) T cells. Activated monocyte-derived DCs express Notch ligands Jagged1 and Delta-like4, whereas naive CD8(+) T cells express Notch2. The role for Notch signaling in CD8(+) T cell priming was determined using an ex-vivo model system in which tumor antigen-specific primary CD8(+) T cell responses were measured. Inhibition of Notch using γ-secretase inhibitors or soluble Delta-like4-Fc during activation reduced expansion of antigen-specific CD8(+) T cells, which was mirrored by decreased frequencies of interferon (IFN)γ-, tumor necrosis factor-α-, and granzymeB-producing CD8(+) T cells. Moreover, T cells primed when Notch signaling was prevented are functionally low-avidity T cells. In addition, Notch partially regulates established effector T cell function. Activation-induced Notch signaling is needed for IFNγ release but not for cytolytic activity. These data indicate that Notch signaling controls human CD8(+) T cell priming and also influences effector T cell functions. This may provide important information for designing new immunotherapies for treatment of cancer.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Ativação Linfocitária/imunologia , Receptor Notch1/imunologia , Receptor Notch2/imunologia , Transdução de Sinais/imunologia , Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Comunicação Celular/imunologia , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Interferon gama/metabolismo , Antígeno MART-1/imunologia , Antígeno MART-1/metabolismo , Monócitos/citologia , Monócitos/imunologia , Monócitos/metabolismo , Neoplasias/imunologia , Neoplasias/prevenção & controle , Receptor Notch1/metabolismo , Receptor Notch2/metabolismoRESUMO
Ever since the first successful kidney transplantation, the occurrence of acute rejection has been a dominant risk factor for adverse graft outcome, as it is associated with reduced graft survival and the development of chronic transplant dysfunction. Although the majority of acute renal allograft rejection episodes can be adequately treated with glucocorticoid therapy, 25 to 30% of the rejection episode cannot be reversed with glucocorticoids alone. At present, the diagnosis of steroid resistance primarily relies on post-transplantation follow-up of clinical parameters reflecting renal allograft function. However, it remains difficult to predict the response to the response to antirejection treatment. Prediction of steroid resistance could prevent unnecessary exposure to high-dose corticosteroid therapy and avoid the development and progression of irreversible nephron. This impact of steroid-refractory rejection on graft integrity stresses the need for tools to assess the response to AR treatment in an early stage. Here, we discuss our current understanding of resistance to anti-rejection treatment with glucocorticoids, and provide an overview of biomarkers for the detection and/or prediction of steroid resistance in kidney transplantation.
Assuntos
Glucocorticoides/uso terapêutico , Rejeição de Enxerto/diagnóstico , Transplante de Rim , Doença Aguda , Animais , Biomarcadores/metabolismo , Resistência a Medicamentos , Diagnóstico Precoce , Rejeição de Enxerto/tratamento farmacológico , Sobrevivência de Enxerto , Humanos , Prognóstico , Medição de RiscoRESUMO
BACKGROUND: Steroid-resistant acute rejection is a risk factor for inferior renal allograft outcome. METHODS: From 873 kidney transplant recipients (1995-2005), 108 patients with a first rejection episode were selected for study using strict inclusion criteria and clinical endpoint definition. We aimed to predict response to corticosteroid treatment using gene expression of 65 transcripts. These reflect cytokines, chemokines, and surface and activation markers of various cell types including T cells, macrophages, B cells, and granulocytes. Steroid resistance (40% of the patients) was defined as requirement for antithymocyte globulin treatment within 2 weeks after corticosteroid treatment. RESULTS: None of the clinical and histomorphologic parameters showed a significant association with response to treatment. Univariate logistic regression analysis resulted in 11 messenger RNA markers, including T-cell-related transcripts CD25, lymphocyte activation gene-3, Granzyme B, and interleukin-10, and macrophage-specific transcripts mannose receptor and S100 calcium-binding protein A9, which significantly discriminated steroid resistant from steroid-responsive rejections (P<0.05). In multivariate logistic regression, the combination of T-cell activation markers CD25:CD3e ratio (odds ratio, 8.7; confidence interval, 2.4-31.2) and lymphocyte activation gene-3 (odds ratio, 3.3; confidence interval, 1.4-7.7) represented the best predictive model for steroid response (P<0.0001). Specificity and sensitivity were 78% and 60%, respectively. After internal stratified 10-fold cross-validation, the model remained significant. Inclusion of clinical variables into the model with molecular variables did not enhance prediction. CONCLUSIONS: Differences in intragraft expression profiles reflect variability in the response to antirejection treatment. In acute rejection, molecular markers, particularly those reflecting T-cell activation, offer superior prognostic value compared with conventional parameters.
Assuntos
Corticosteroides/uso terapêutico , Marcadores Genéticos , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto/efeitos dos fármacos , Imunossupressores/uso terapêutico , Transplante de Rim/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Doença Aguda , Biópsia , Resistência a Medicamentos/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/genética , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Países Baixos , Razão de Chances , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Fatores de Tempo , Resultado do TratamentoRESUMO
Type 1 diabetes is a common metabolic disorder accompanied by an increased secretion of glucocorticoids and cognitive deficits. Chronic excess of glucocorticoids per se can evoke similar neuropathological signals linked to its major target in the brain, the hippocampus. This deleterious action exerted by excess adrenal stress hormone is mediated by glucocorticoid receptors (GRs). The aim of the present study was to assess whether excessive stimulation of GR is causal to compromised neuronal viability and cognitive performance associated with the hippocampal function of the diabetic mice. For this purpose, mice had type 1 diabetes induced by streptozotocin (STZ) administration (170 mg/kg, i.p.). After 11 days, these STZ-diabetic mice showed increased glucocorticoid secretion and hippocampal alterations characterized by: (1) increased glial fibrillary acidic protein-positive astrocytes as a marker reacting to neurodegeneration, (2) increased c-Jun expression marking neuronal activation, (3) reduced Ki-67 immunostaining indicating decreased cell proliferation. At the same time, mild cognitive deficits became obvious in the novel object-placement recognition task. After 6 days of diabetes the GR antagonist mifepristone (RU486) was administered twice daily for 4 days (200 mg/kg, p.o.). Blockade of GR during early type 1 diabetes attenuated the morphological signs of hippocampal aberrations and rescued the diabetic mice from the cognitive deficits. We conclude that hippocampal disruption and cognitive impairment at the early stage of diabetes are caused by excessive GR activation due to hypercorticism. These signs of neurodegeneration can be prevented and/or reversed by GR blockade with mifepristone.