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Introduction: Hepatitis E virus (HEV) can cause chronic infection (≥3 months) and cirrhosis in immunocompromised patients, especially kidney transplant recipients. Low alanine aminotransferase (ALT) levels and high HEV intrahost diversity have previously been associated with evolution toward chronicity in these patients. We hypothesized that additional clinical and viral factors could be associated with the risk of chronic HEV infection. Methods: We investigated a series of 27 kidney transplant recipients with HEV infection, including 20 patients with chronic hepatitis E. Results: High tacrolimus trough concentration at diagnosis was the most relevant marker associated with chronic hepatitis E (9.2 vs. 6.4 ng/ml, P = 0.04). Most HEV genetic changes selected during HEV infection were compartmentalized between plasma and feces. Conclusion: This compartmentalization highlights the diversity and complexity of HEV replication compartments. Tacrolimus trough concentration at diagnosis of HEV infection could allow an early identification of patients at high risk of chronic hepatitis E and guide treatment initiation.
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Recent evidence showed that in response to elevated sodium dietary intakes, many body tissues retain Na+ ions for long periods of time and can reach concentrations up to 200 mM. This could modulate the immune system and be responsible for several diseases. However, studies brought contrasted results and the effects of external sodium on human dendritic cell (DC) responses to danger signals remain largely unknown. Considering their central role in triggering T cell response, we tested how NaCl-enriched medium influences human DCs properties. We found that DCs submitted to high extracellular Na+ concentrations up to 200 mM remain viable and maintain the expression of specific DC markers, however, their maturation, chemotaxis toward CCL19, production of pro-inflammatory cytokines and ROS in response to LPS were also partially inhibited. In line with these results, the T-cell allostimulatory capacity of DCs was also inhibited. Finally, our data indicate that high NaCl concentrations triggered the phosphorylation of SGK1 and ERK1/2 kinases. These results raised the possibility that the previously reported pro-inflammatory effects of high NaCl concentrations on T cells might be counterbalanced by a downregulation of DC activation.
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Lipopolissacarídeos , Cloreto de Sódio , Humanos , Cloreto de Sódio/farmacologia , Cloreto de Sódio/metabolismo , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/metabolismo , Diferenciação Celular , Cloretos/metabolismo , Cloretos/farmacologia , Células Dendríticas , Citocinas/metabolismo , Sódio/metabolismo , Sódio/farmacologia , Células CultivadasRESUMO
AIMS: Basiliximab, an anti-CD25 chimeric monoclonal antibody, is approved in prevention of acute kidney transplant rejection. This study aims at investigating target-mediated pharmacokinetics of basiliximab. METHODS: Data from the IDEALE study, where 16 kidney transplant patients were treated with 2 40- or 80-mg basiliximab injections, were reanalysed. Basiliximab pharmacokinetics was described using a population 2-compartment target-mediated drug disposition model with the quasi-steady-state approximation. RESULTS: Volume of distribution was significantly higher in males (P = .029). Estimated baseline target antigen (CD25) level was lower is patients cotreated with cyclosporine (P = .026). CONCLUSION: This analysis allows the first description of the target-mediated nonlinear elimination of basiliximab. Our results suggest that cyclosporine cotreatment is associated with decreased target level and that an optimized dosing regimen may improve basiliximab effects.
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Transplante de Rim , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Basiliximab , Ciclosporina , Quimioterapia Combinada , Rejeição de Enxerto/prevenção & controle , Humanos , Imunossupressores , Masculino , Proteínas Recombinantes de FusãoRESUMO
The SCN4B gene, coding for the NaVß4 subunit of voltage-gated sodium channels, was recently found to be expressed in normal epithelial cells and down-regulated in several cancers. However, its function in normal epithelial cells has not been characterized. In this study, we demonstrated that reducing NaVß4 expression in MCF10A non-cancer mammary epithelial cells generated important morphological changes observed both in two-dimensional cultures and in three-dimensional cysts. Most notably, the loss of NaVß4 induced a complete loss of epithelial organisation in cysts and increased proteolytic activity towards the extracellular matrix. Loss of epithelial morphology was associated with an increased degradation of ß-catenin, reduced E-cadherin expression and induction of mesenchymal markers N-cadherin, vimentin, and α-SMA expression. Overall, our results suggest that Navß4 may participate in the maintenance of the epithelial phenotype in mammary cells and that its downregulation might be a determining step in early carcinogenesis.
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Células Epiteliais/metabolismo , Glândulas Mamárias Animais/citologia , Subunidades Proteicas/metabolismo , Subunidade beta-4 do Canal de Sódio Disparado por Voltagem/metabolismo , Animais , Linhagem Celular , Polaridade Celular , Regulação para Baixo , Células Epiteliais/citologia , Feminino , Humanos , Mesoderma/metabolismo , Fenótipo , Proteólise , beta Catenina/metabolismoRESUMO
Implantable cardioverter-defibrillators (ICD) are meant to fight life-threatening ventricular arrhythmias and reduce overall mortality. Ironically, life-saving shocks themselves have been shown to be independently associated with an increased mortality. We sought to identify myocardial changes at the protein level immediately after ICD electrical shocks using a proteomic approach. ICD were surgically implanted in 10 individuals of a healthy male sheep model: a control group (N = 5) without any shock delivery and a shock group (N = 5) with the delivery of 5 consecutive shocks at 41 J. Myocardial tissue samples were collected at the right-ventricle apex near to the lead coil and at the right ventricle basal free wall region. Global quantitative proteomics experiments on myocardial tissue samples were performed using mass spectrometry techniques. Proteome was significantly modified after electrical shock and several mechanisms were associated: protein, DNA and membrane damages due to extreme physical conditions induced by ICD-shock but also due to regulated cell death; metabolic remodeling; oxidative stress; calcium dysregulation; inflammation and fibrosis. These proteome modifications were seen in myocardium both "near" and "far" from electrical shock region. N-term acetylated troponin C was an interesting tissular biomarker, significantly decreased after electrical shock in the "far" region (AUC: 0.93). Our data support an acute shock-induced myocardial tissue injury which might be involved in acute paradoxical deleterious effects such as heart failure and ventricular arrhythmias.
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Cardioversão Elétrica , Miocárdio/patologia , Proteômica , Animais , Masculino , Modelos Animais , Miocárdio/metabolismo , Ovinos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The acquisition of invasive capacities by carcinoma cells, i.e. their ability to migrate through and to remodel extracellular matrices, is a determinant process leading to their dissemination and to the development of metastases. these cancer cell properties have often been associated with an increased Rho-ROCK signalling, and ROCK inhibitors have been proposed for anticancer therapies. In this study we used the selective ROCK inhibitor, Y-27632, to address the participation of the Rho-ROCK signalling pathway in the invasive properties of SW620 human colon cancer cells. Contrarily to initial assumptions, Y-27632 induced the acquisition of a pro-migratory cell phenotype and increased cancer cell invasiveness in both 3- and 2-dimensions assays. This effect was also obtained using the other ROCK inhibitor Fasudil as well as with knocking down the expression of ROCK-1 or ROCK-2, but was prevented by the inhibition of NaV1.5 voltage-gated sodium channel activity. Indeed, ROCK inhibition enhanced the activity of the pro-invasive NaV1.5 channel through a pathway that was independent of gene expression regulation. In conclusions, our evidence identifies voltage-gated sodium channels as new targets of the ROCK signalling pathway, as well as responsible for possible deleterious effects of the use of ROCK inhibitors in the treatment of cancers.
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Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Invasividade Neoplásica/patologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Amidas/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Renal resistive index (RI) predicts mortality in renal transplant recipients, but we do not know whether this is true in diabetic patients. The objective of this study was to analyse the long-term predictive value of RI for death with a functioning graft (DWFG) in renal transplant recipients with or without pre-transplant diabetes. METHODS: We conducted a retrospective study in 1800 renal transplant recipients between 1985 and 2017 who were followed for up to 30 years (total observation period: 14 202 patient years). Donor and recipient characteristics at time of transplantation and at 3 months were reviewed. The long-term predictive value of RI for DWFG and the age-RI and arterial pressure-RI relationships were assessed. RESULTS: A total of 284/1800 (15.7%) patients had diabetes mellitus before transplantation. RI was <0.75 in 1327/1800 patients (73.7%). High RI was associated with a higher risk of DWFG in non-diabetic patients [hazard ratio (HR) = 3.39, 95% confidence interval 2.50-4.61; P < 0.001], but not in patients with pre-transplant diabetes (HR = 1.25, 0.70-2.19; P = 0.39), even after multiple adjustments. There was no interaction between diabetes and age. In contrast, there was an interaction between RI and pulse pressure. CONCLUSION: Our study indicates that RI is not a predictor of DWFG in diabetic renal transplant recipients, in contrast to non-diabetic recipients. These findings could be due to a different age-RI or pulse pressure-RI relationship.
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Diabetes Mellitus/fisiopatologia , Rejeição de Enxerto/mortalidade , Transplante de Rim/efeitos adversos , Rim/fisiopatologia , Mortalidade Prematura/tendências , Pressão Sanguínea , Feminino , Rejeição de Enxerto/etiologia , Rejeição de Enxerto/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida , Doadores de Tecidos , TransplantadosRESUMO
Sterile inflammation is a key determinant of myocardial reperfusion injuries. It participates in infarct size determination in acute myocardial infarction and graft rejection following heart transplantation. We previously showed that P2Y11 exerted an immunosuppressive role in human dendritic cells, modulated cardiofibroblasts' response to ischemia/reperfusion in vitro and delayed graft rejection in an allogeneic heterotopic heart transplantation model. We sought to investigate a possible role of P2Y11 in the cellular response of cardiomyocytes to ischemia/reperfusion. We subjected human AC16 cardiomyocytes to 5 h hypoxia/1 h reoxygenation (H/R). P2Y11R (P2Y11 receptor) selective agonist NF546 and/or antagonist NF340 were added at the onset of reoxygenation. Cellular damages were assessed by LDH release, MTT assay and intracellular ATP level; intracellular signaling pathways were explored. The role of P2Y11R in mitochondria-derived ROS production and mitochondrial respiration was investigated. In vitro H/R injuries were significantly reduced by P2Y11R stimulation at reoxygenation. This protection was suppressed with P2Y11R antagonism. P2Y11R stimulation following H2O2-induced oxidative stress reduced mitochondria-derived ROS production and damages through PKCε signaling pathway activation. Our results suggest a novel protective role of P2Y11 in cardiomyocytes against reperfusion injuries. Pharmacological post-conditioning targeting P2Y11R could therefore contribute to improve myocardial ischemia/reperfusion outcomes in acute myocardial infarction and cardiac transplantation.
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Miócitos Cardíacos/efeitos dos fármacos , Proteína Quinase C-épsilon/metabolismo , Receptores Purinérgicos P2/efeitos dos fármacos , Traumatismo por Reperfusão/prevenção & controle , Transdução de Sinais , Trifosfato de Adenosina/administração & dosagem , Cardiotônicos/farmacologia , Transplante de Coração , Humanos , Infarto do Miocárdio/prevenção & controle , Miócitos Cardíacos/enzimologia , Oxigênio/metabolismo , Agonistas do Receptor Purinérgico P2/farmacologiaRESUMO
Depleting induction therapy is recommended in sensitized kidney transplant recipients (KTRs), though the detrimental effect of nondonor-specific anti-HLA antibodies is not undeniable. We compared the efficacy and safety of basiliximab and rabbit anti-thymocyte globulin (rATG) in sensitized KTRs without pre-existing donor-specific antibodies (DSAs). This monocentric retrospective study involved all sensitized KTR adults without pre-existing DSAs (n = 218) who underwent transplantation after June 2007. Patients with basiliximab and rATG therapy were compared for risk of biopsy-proven acute rejection (BPAR) and a composite endpoint (BPAR, graft loss and death) by univariate and multivariate analysis. Patients with basiliximab (n = 60) had lower mean calculated panel reactive antibody than those with rATG (n = 158; 23.7 ± 24.2 vs. 63.8 ± 32.3, P < 0.0001) and more often received a first graft (88% vs. 54%, P < 0.0001) and a transplant from a living donor (13% vs. 2%, P = 0.002). Risks of BPAR and of reaching the composite endpoint were greater with basiliximab than rATG [HR = 3.63 (1.70-7.77), P = 0.0009 and HR = 1.60 (0.99-2.59), P = 0.050, respectively]. Several adjustments did not change those risks [BPAR: 3.36 (1.23-9.16), P = 0.018; composite endpoint: 1.83 (0.99-3.39), P = 0.053]. Infections and malignancies were similar in both groups. rATG remains the first-line treatment in sensitized KTR, even in the absence of pre-existing DSAs.
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Anticorpos/imunologia , Soro Antilinfocitário/uso terapêutico , Basiliximab/uso terapêutico , Rejeição de Enxerto , Transplante de Rim/efeitos adversos , Adulto , Idoso , Animais , Biópsia , Feminino , Sobrevivência de Enxerto , Humanos , Falência Renal Crônica/cirurgia , Transplante de Rim/mortalidade , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Coelhos , Estudos Retrospectivos , Risco , Doadores de Tecidos , Condicionamento Pré-Transplante/métodos , Resultado do TratamentoRESUMO
High renal resistive index (RI) is observed in diabetes and is associated with poor patient survival, but whether it is primarily due to renal vascular resistance or systemic vascular alterations is unclear. The respective impact of kidney transplant from diabetic donors or to diabetic recipients on RI would shed some light on this issue. The objective of the study was to analyze the impact of donor and recipient diabetes on RI in order to understand the respective impact of the kidney and the vascular environment. The authors conducted a retrospective study in 1827 renal transplant recipients who received a kidney between 1985 and 2017, and had Doppler measurements at 3 months after transplant. Donor and recipient characteristics at the time of transplant and at 3 months were reviewed. Both donor diabetes and recipient diabetes were associated with RI in univariate analysis, but only recipient diabetes remained significantly associated in stepwise multivariate analyses (effect estimate on RI: +0.03 ± 0.005, P < 0.001). These findings were confirmed when RI was expressed as a binary variable using a cutoff of 0.75 (OR = 2.50 [1.77, 3.54], P < 0.001). Other determinants of RI were recipient characteristics (age, sex, systolic and diastolic blood pressure, and duration of dialysis). Donor characteristics were not associated with RI. Our results suggest that high RI observed in diabetic recipients shortly after transplant is primarily due to the new vascular environment, rather than to characteristics of the transplanted kidney. Therefore, RI reflects systemic rather than intra-renal changes.
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Diabetes Mellitus/fisiopatologia , Transplante de Rim/efeitos adversos , Rim/fisiopatologia , Resistência Vascular/fisiologia , Adulto , Idoso , Algoritmos , Pressão Sanguínea/fisiologia , Creatinina/sangue , Função Retardada do Enxerto/fisiopatologia , Feminino , França/epidemiologia , Taxa de Filtração Glomerular/fisiologia , Humanos , Rim/irrigação sanguínea , Rim/diagnóstico por imagem , Transplante de Rim/mortalidade , Masculino , Pessoa de Meia-Idade , Proteinúria/urina , Diálise Renal/estatística & dados numéricos , Estudos Retrospectivos , Doadores de Tecidos/estatística & dados numéricos , Transplantados/estatística & dados numéricos , Ultrassonografia Doppler/métodosRESUMO
Cardiac fibroblasts are important regulators of myocardial structure and function. Their implications in pathological processes such as Ischemia/Reperfusion are well characterized. Cardiac fibroblasts respond to stress by excessive proliferation and secretion of pro-inflammatory cytokines and other factors, e.g. ATP, leading to purinergic receptors activation. P2Y11 receptor (P2Y11R) is an ATP-sensitive GPCR playing an immunomodulatory role in human dendritic cells (DC). We hypothesized that P2Y11R stimulation modulated the pro-inflammatory responses of human cardiac fibroblasts (HCF) to Hypoxia/Reoxygenation (H/R) mainly by acting on their secretome. P2Y11R stimulation in HCF at the onset of reoxygenation significantly limited H/R-induced proliferation (-19%) and pro-inflammatory cytokines and ATP secretion (-44% and -83% respectively). Exposure of DC to HCF secretome increased their expression of CD83, CD25 and CD86, suggesting a switch from immature to mature phenotype. Under LPS stimulation, DC had a pro-inflammatory profile (high IL-12/IL-10 ratio) that was decreased by HCF secretome (-3,8-fold), indicating induction of a tolerogenic profile. Moreover, P2Y11R inhibition in HCF led to high IL-12 secretion in DC, suggesting that the immunomodulatory effect of HCF secretome is P2Y11R-dependant. HCF secretome reduced H/R-induced cardiomyocytes death (-23%) through RISK pathway activation. P2Y11R inhibition in HCF induced a complete loss of HCF secretome protective effect, highlighting the cardioprotective role of P2Y11R. Our data demonstrated paracrine interactions between HCF, cardiomyocytes and DC following H/R, suggesting a key role of HCF in the cellular responses to reperfusion. These results also demonstrated a beneficial role of P2Y11R in HCF during H/R and strongly support the hypothesis that P2Y11R is a modulator of I/R injury.
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Traumatismo por Reperfusão Miocárdica/genética , Miocárdio/metabolismo , Receptores Purinérgicos P2/genética , Traumatismo por Reperfusão/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Hipóxia/genética , Hipóxia/patologia , Fatores Imunológicos/metabolismo , Interleucina-12/genética , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Comunicação Parácrina/genética , Receptores Purinérgicos P2/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologiaRESUMO
We aimed to determine the role of cytomegalovirus (CMV)-infected donor cells in the development of a CMV-specific immune response in kidney transplant recipients. We assessed the CMV pp65-specific immune response by using interferon-É£ ELISPOT and dextramers in peripheral blood mononuclear cells from 115 recipients (D+R- 31, D+R + 44, D-R + 40) late after transplantation (mean 59 ± 42 months). Receiving a kidney from a D+ donor resulted in a higher number of IFN-É£-producing anti-CMV T cells (P = .004). This effect disappeared with the absence of shared HLA class I specificities between donors and recipients (P = .430). To confirm the role of donor cells in stimulating the expansion of newly developed CMV-specific CD8+ T cells after transplantation, we compared the number of HLA-A2-restricted CMV-specific CD8+ T cells in primo-infected recipients who received an HLA-A2 or non-HLA-A2 graft. The median of anti-CMV pp65 T cells restricted by HLA-A2 was very low for patients who received a non-HLA-A2 graft vs an HLA-A2 graft (300 [0-14638] vs. 17972 [222-85594] anti-CMV pp65 CD8+ T cells/million CD8+ T cells, P = .001). This adds new evidence that CMV-infected kidney donor cells present CMV peptides and drive an inflation of memory CMV-specific CD8+ T cells, likely because of frequent CMV replications within the graft.
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Linfócitos T CD8-Positivos/imunologia , Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Genes MHC Classe I/imunologia , Rejeição de Enxerto/etiologia , Transplante de Rim/efeitos adversos , Leucócitos Mononucleares/imunologia , Doadores de Tecidos , Antígenos Virais/imunologia , Estudos Transversais , Infecções por Citomegalovirus/virologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/imunologia , Fosfoproteínas/imunologia , Estudos Retrospectivos , Linfócitos T Citotóxicos/imunologia , Proteínas da Matriz Viral/imunologiaRESUMO
BACKGROUND: Cytomegalovirus (CMV) has a role in chronic rejection and graft loss in kidney transplant (KTx) and lung transplant (LTx) recipients. In addition, donor CMV seropositivity is an independent risk factor for renal graft loss. The anti-CMV response might modulate this risk. Expression of programmed cell death 1 (PD-1), a receptor involved in viral-specific T-cell exhaustion, is influenced by a single nucleotide polymorphism called PD-1.3 (wild-type allele G, variant allele A). METHODS: We performed a retrospective study to assess the impact of PD-1.3 on graft outcome in donor CMV seropositive (D+) and donor CMV seronegative (D-) KTx and LTx. We also performed a case-control study to evaluate the anti-CMVpp65 response according to genotype. RESULTS: PD-1.3 was determined in 1,119 KTx and 181 LTx. In 481 D+ KTx, A allele carriers (24%) experienced significantly less graft failure compared with GG carriers (p = 0.001). Multivariate analysis showed that this association was independent of donor and recipient age, acute rejection episodes, and number of human leukocyte antigen mismatches (hazard ratio, 0.381; 95% confidence interval, 0.209-0.696; p = 0.002). Analysis in 85 D+ LTx showed similar results: A allele carriers had better survival (hazard ratio, 0.302; 95% confidence interval, 0.128-0.716; p = 0.006) and greater 6-month forced expiratory volume (71% ± 17% vs 54% ± 16%, p = 0.001). In D- recipients, PD-1.3 did not affect KTx or LTx outcome. Finally, AA recipients had a stronger anti-CMVpp65 T-cell response than matched GG recipients (p = 0.003). CONCLUSIONS: The A variant allele in PD-1.3 single nucleotide polymorphism improved graft survival in kidney and lung transplant recipients receiving grafts from CMV-positive donors.
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Antígeno B7-H1/genética , Infecções por Citomegalovirus/diagnóstico , Citomegalovirus/isolamento & purificação , Rejeição de Enxerto/genética , Polimorfismo Genético , Adulto , Estudos de Casos e Controles , Intervalos de Confiança , Feminino , Sobrevivência de Enxerto , Humanos , Transplante de Rim/efeitos adversos , Transplante de Rim/métodos , Transplante de Pulmão/efeitos adversos , Transplante de Pulmão/métodos , Masculino , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais , Estudos Retrospectivos , Estudos Soroepidemiológicos , Doadores de Tecidos , TransplantadosRESUMO
BACKGROUND: An efficient strategy for programming dendritic cells (DCs) for cancer immunotherapy is the optimization of their maturation so that they can efficiently stimulate cancer-specific T cell responses. Interleukin (IL)-4 has appeared as an essential cytokine, widely used in vitro with granulocyte macrophage-colony stimulating factor (GM-CSF) to differentiate monocytes into immature DCs (iDC) and to prevent macrophage formation. Conflicting data have been published regarding the effect of IL-4 on functional DC maturation. To further understand IL-4's effects on DC maturation and function in vitro, we choose the most commonly used maturation factor tumor necrosis factor (TNF)-α. METHODS: Human monocyte-derived iDC were treated for 48 h with GM-CSF and TNF-α in the presence (IL-4(+)-DC) or absence (IL-4(-)-DC) of IL-4 and functions of both DC populations were compared. RESULTS: On mixed lymphocyte reaction assay, IL-4(+)-DC were less potent than IL-4(-)-DC at inducing the proliferation of allogeneic CD4(+) T cells and the proportion of activated T cells expressing CD69 and/or CD25 was smaller. Interleukin-4 reduced the cell-surface expression of TNF-α-induced DC maturation markers CD83, CD86, HLA-DR and CD25 and generated a heterogeneous population of DCs. IL-4(+)-DC secreted less IL-12 and more IL-10 than IL-4(-)-DC following activation by soluble CD40L, and IL-4(+)-DC-activated T cells secreted lesser amounts of T helper (Th) 1 cytokines (IL-2 and interferon-γ). Importantly, IL-4 impaired the in vitro migratory capacity of DCs in response to CCL21 and CCL19 chemokines. This effect was related to reduced expression of CCR7 at both mRNA and protein levels. CONCLUSION: Interleukin-4 used with GM-CSF and TNF-α during the maturation of DCs in vitro impaired DC functions and disturbed the maturation effect of TNF-α. Finally, our study reinforces the view that the quality of the DC maturation stimulus, which regulates DC migration and cytokine production, may be a decisive feature of the immunogenicity of DCs.
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Diferenciação Celular/efeitos dos fármacos , Células Dendríticas/citologia , Fator de Necrose Tumoral alfa/farmacologia , Biomarcadores/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quimiocinas/farmacologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Humanos , Interleucina-12/metabolismo , Interleucina-4/metabolismo , Ativação Linfocitária/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CCR7/genética , Receptores CCR7/metabolismo , Células Th1/efeitos dos fármacosRESUMO
High concentrations of extracellular ATP (eATP) resulting from cell damage may be found during an ischemia/reperfusion (I/R) episode at the site of injury. eATP activates purinergic receptors in dendritic cells (DCs) and may inhibit inflammation. This immunosuppressive activity could be of interest in the field of I/R, which is an inflammatory condition involved in myocardial infarction, stroke, and solid organ transplantation. However, the specific purinergic receptor responsible for this effect remains to be identified. In this study, we report that eATP induced maturation of human monocyte-derived DCs. Additionally, eATP inhibited IL-12 production whereas IL-10 levels remained unchanged in activated DCs. These effects were prevented by the P2Y11R antagonist NF340. Interestingly, a 5-h hypoxia prevented the effects of eATP on cytokine production whereas a 1-h hypoxia did not affect the eATP-mediated decrease of IL-12 and IL-6. We showed a time-dependent downregulation of P2Y11R at both mRNA and protein levels that was prevented by knocking down hypoxia-inducible factor-1α. In this study, we showed an immunosuppressive role of P2Y11R in human DCs. Additionally, we demonstrated that the time-dependent downregulation of P2Y11R by hypoxia orientates DCs toward a proinflammatory phenotype that may be involved in post-I/R injuries as observed after organ transplantation.
Assuntos
Células Dendríticas/imunologia , Oxigênio/farmacologia , Receptores Purinérgicos P2/imunologia , Trifosfato de Adenosina/farmacologia , Hipóxia Celular , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Terapia de Imunossupressão , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-12/antagonistas & inibidores , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Cultura Primária de Células , Antagonistas Purinérgicos/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/imunologia , Receptores Purinérgicos P2/genética , Transdução de Sinais , Fatores de Tempo , Transcrição GênicaRESUMO
Human dendritic cells (hDCs) produce IL-2 and express IL-2R α-chain (CD25), but the role of IL-2 in DC functions is not well defined. A recent study suggested that the main function of CD25 on hDCs was to transpresent IL-2 to activate T lymphocytes. Our results demonstrate the expression of the three chains of the IL-2R on hDCs and that IL-2 induces STAT5 phosphorylation. Interestingly, use of inhibitors of p-STAT5 revealed that IL-2 increases LPS-induced IFN-γ through STAT5 phosphorylation. Finally, we report that IL-2 increases the ability of hDCs to activate helpless CD8(+) T cells, most likely because of IL-2-triggered IFN-γ synthesis, as we previously described. For the first time, to our knowledge, we disclose that IL-2 induces monocyte-derived hDC's functional maturation and activation through IL-2R binding. Interestingly, our study suggests a direct effect of anti-CD25 mAbs on hDCs that may contribute to their clinical efficacy.
Assuntos
Células Dendríticas/imunologia , Interferon gama/imunologia , Interleucina-2/imunologia , Fator de Transcrição STAT5/imunologia , Anticorpos/farmacologia , Células Dendríticas/citologia , Feminino , Humanos , Subunidade alfa de Receptor de Interleucina-2/antagonistas & inibidores , Subunidade alfa de Receptor de Interleucina-2/imunologia , Lipopolissacarídeos/farmacologia , Masculino , Monócitos/citologia , Monócitos/imunologia , Fosforilação/efeitos dos fármacos , Fosforilação/imunologiaRESUMO
Regulatory T cells (Treg) play a crucial role in controlling immunity and transplant rejection. Two main groups of Treg have been described: antigen-induced Treg (iTreg) and natural Treg (nTreg). The ways to induce and the mechanisms of action of Treg subsets remained ill defined, particularly for their effects on CD8(+) T cells. CD8(+) T cells are major agents in the rejection of allografts; the aim of this study is to investigate the effects exerted on CD8(+) T cells by human CD4(+) iTreg induced by mycophenolic acid-treated dendritic cells. iTreg suppress the proliferation of CD8(+) T cells by allogeneic cell-cell interaction with mature dendritic cells and irrespectively of the TCR specificity of the CD8(+) T cells and cell-cell contact of iTreg with CD8(+) T cells. In our model, this suppression is independent of the action of IL-10 and TGF-ß1. iTreg were able to modify phenotype and inhibited IFN-γ and TNF-α secretion by CD8(+) T cells. Most interestingly, iTreg inhibit the synthesis of perforin and of granzymes A and B by CD8(+) T cells and impaired their cytotoxicity against allogeneic targets. In summary, our study showed the involvement of iTreg in the down-regulation of cytotoxic responses mediated by CD8(+) T cells in an allospecific context. Following studies that have shown the existence of a regulation control exerted by iTreg on CD4(+) T cells and dendritic cells, this work ultimately shows that this regulation can reach CD8(+) T-cell functions.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/efeitos dos fármacos , Ácido Micofenólico/farmacologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Antígenos CD4/metabolismo , Comunicação Celular , Diferenciação Celular , Células Cultivadas , Técnicas de Cocultura , Citotoxicidade Imunológica , Células Dendríticas/imunologia , Granzimas , Humanos , Terapia de Imunossupressão , Interferon gama/metabolismo , Ativação Linfocitária , Perforina/metabolismo , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Imunologia de Transplantes , Fator de Necrose Tumoral alfa/metabolismoRESUMO
To describe long-term CD4+ T-cell reconstitution after rabbit antithymocyte globulin (rATG) treatment and identify predictive factors following kidney transplantation. A single-center retrospective study analyzed lymphocyte subsets in rATG-treated kidney transplant recipients (1986-2009). 589 patients were analyzed (maximum follow-up 21 years). A comparator group (n=298) received an anti-IL-2 receptor monoclonal antibody. CD4+ T-cell lymphopenia (<200/mm3) was present in 48.5%, 9.2%, 6.7%,2.0%, and 0% of patients at one, three, five, 10, and 20 years post-transplant, respectively. CD4+ T-cell count increased during the first 10 years but remained below the pretransplant count even after 20 years. At 1, 3, and 6 months post-transplant, mean CD4+ T-cell count was significantly lower in patients with CD4+ T-cell lymphopenia at 12 months versus patients without lymphopenia. On multivariate analyses, significant independent predictors for long-term impaired CD4 T-cell reconstitution were recipient age, pretransplant CD4+ T-cell count, 12-month CD4+ T-cell count, and tacrolimus or MMF therapy. Recipient age>40 years was identified as a cutoff point. CD4+ T-cell reconstitution following rATG treatment remains impaired even after 21 years. Most risk factors for long-term impaired CD4+ T-cell reconstitution may be evaluated pretransplant or are modifiable post-transplant.
Assuntos
Soro Antilinfocitário/efeitos adversos , Linfócitos T CD4-Positivos/imunologia , Transplante de Rim , Depleção Linfocítica/efeitos adversos , Adulto , Animais , Anticorpos Monoclonais/uso terapêutico , Soro Antilinfocitário/uso terapêutico , Basiliximab , Feminino , Humanos , Contagem de Linfócitos , Depleção Linfocítica/métodos , Linfopenia/etiologia , Linfopenia/imunologia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Infecções Oportunistas/etiologia , Infecções Oportunistas/imunologia , Coelhos , Receptores de Interleucina-2/antagonistas & inibidores , Proteínas Recombinantes de Fusão/uso terapêutico , Estudos Retrospectivos , Fatores de RiscoRESUMO
Rabbit antithymocyte globulin (rATG; Thymoglobulin(®)) is currently used to prevent acute rejection in kidney transplantation. The dose and regimen of rATG have not been optimized. Moreover, the impact of different treatment regimens on T-cell phenotype reconstitution remains unknown. We conducted a prospective randomized study of 17 renal transplant patients to determine the pharmacokinetics of total and active (bound to human cells) rATG and T-cell phenotype reconstitution after rATG administration. Patients received rATG at a total dose of 6mg/kg, administered either as 1.5mg/kg/day on days 0-3 (Group 1, n=8) or 3mg/kg on days 0 and 3 (Group 2, n=9). All patients received tacrolimus, mycophenolate mofetil and steroids. Blood samples were assayed for total rATG by enzyme linked immunosorbent assay and active rATG by flow cytometry. Maximum concentrations and terminal half-lives were similar between the two groups but at month 3 Group 1 had significantly lower values for total rATG (concentration was 6.2±1.1µg/mL versus 10.2±2.9µg/mL in Group 2, p=0.027) and total rATG dose-normalized AUC (374±83dayg/mL versus 508±149dayg/mL in Group 2, p=0.046). Time to sub-therapeutic levels (<1µg/mL) of active rATG was significantly shorter in Group 1 (18.75±6.9days versus 20±7.5days in Group 2, p<0.001). rATG induced significant depletion followed by slow reconstitution of CD3(+), CD4(+) and CD8(+) cells, with no marked differences between groups. B-cell count was unaffected, whereas CD3(-)CD56(+) NK-cell depletion was observed in both groups. rATG induced a significant decrease in the proportion of naïve CD4(+) T-cells, which plateaued after month 1 in Group 1 and after month 6 in Group 2. The proportion of central memory CD4(+) T-cells increased to a similar extent in both groups (Group 1: 38±18% at baseline, 74±23% at one year, p=0.009; Group 2: 32±14% at baseline, 65±14% at one year, p=0.001). In conclusion, our results suggest that the dosing regimen for rATG induction influences pharmacokinetic parameters without affecting the quality of immune reconstitution.
Assuntos
Soro Antilinfocitário/farmacologia , Animais , Feminino , Rejeição de Enxerto/prevenção & controle , Humanos , Imunidade Inata/efeitos dos fármacos , Memória Imunológica/efeitos dos fármacos , Transplante de Rim , Masculino , Pessoa de Meia-Idade , Coelhos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
A large body of evidence has been accumulated from experimental models in the past decade to support the critical role of Foxp3-expressing regulatory T cells (Tregs) in the suppression of alloimmune responses. This has prompted transplant clinicians to investigate whether Foxp3 analysis might be used as an immunodiagnostic tool for better assessment of the significance of graft infiltrate and to predict its impact on graft outcome. However, conflicting results have emerged from these studies and may have generated more confusion than clarification. Foxp3 expression has been antagonistically correlated with either good or poor prognosis. We discuss here how methodological issues and specific clinical settings may have accounted for the discrepancies between the results of these studies. Depending on many factors, including the techniques used, the method of sampling normalization, the extent of intra-graft inflammation, the immunosuppressive regimen and the depletion or repletion of T lymphocyte compartment, the significance of Foxp3 expression may vary. We propose here the conditions to be fulfilled in order to use Foxp3 analysis as a relevant biomarker for graft outcome assessment. Far from challenging the key role of Tregs in dampening alloimmune responses, this review highlights the need for technical harmonization and standards.