RESUMO
Cytomegalovirus (CMV) infection is associated with poor kidney transplant outcomes. While innate and adaptive immune cells have been implicated in its prevention, an in-depth characterization of the in vivo kinetics of multiple cell subsets and their role in protecting against CMV infection has not been achieved. Here, we performed high-dimensional immune phenotyping by mass cytometry, and functional assays, on 112 serially collected samples from CMV seropositive kidney transplant recipients. Advanced unsupervised deep learning analysis was used to assess immune cell populations that significantly correlated with prevention against CMV infection and anti-viral immune function. Prior to infection, kidney transplant recipients who developed CMV infection showed significantly lower CMV-specific cell-mediated immune (CMI) frequencies than those that did not. A broad diversity of circulating cell subsets within innate and adaptive immune compartments were associated with CMV infection or protective CMV-specific CMI. While percentages of CMV (tetramer-stained)-specific T cells associated with high CMI responses and clinical protection, circulating CD3+CD8midCD56+ NK-T cells overall strongly associated with low CMI and subsequent infection. However, three NK-T cell subsets sharing the CD11b surface marker associated with CMV protection and correlated with strong anti-viral CMI frequencies in vitro. These data were validated in two external independent cohorts of kidney transplant recipients. Thus, we newly describe the kinetics of a novel NK-T cell subset that may have a protective role in post-transplantation CMV infection. Our findings pave the way to more mechanistic studies aimed at understanding the function of these cells in protection against CMV infection.
Assuntos
Infecções por Citomegalovirus , Transplante de Rim , Células T Matadoras Naturais , Humanos , Transplante de Rim/efeitos adversos , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/prevenção & controle , Infecções por Citomegalovirus/virologia , Infecções por Citomegalovirus/sangue , Pessoa de Meia-Idade , Masculino , Feminino , Adulto , Células T Matadoras Naturais/imunologia , Citomegalovirus/imunologia , Citomegalovirus/isolamento & purificação , Citometria de Fluxo , Imunofenotipagem , Idoso , Imunidade CelularRESUMO
Focal segmental glomerulosclerosis (FSGS) is one of the leading causes of kidney failure and it is characterized by a high rate of recurrence after kidney transplant. Moreover, FSGS recurrence is worsened by an increased risk of graft failure. Common therapies for FSGS recurrence mostly consist of plasma exchange treatments, also for prolonged time, and rituximab, with variable efficacy. We report 5 cases of early FSGS recurrence after kidney transplant, resistant to plasma exchange and rituximab treatment that subsequently resolved after combined therapy with rituximab and daratumumab. All cases were negative for genetic FSGS. The combined treatment induced a complete response in all the cases and was well tolerated. We also performed a comprehensive flow cytometry analysis in 2 subjects that may suggest a mechanistic link between plasma cells and disease activity. In conclusion, given the lack of viable treatments for recurrent FSGS, our reports support the rationale for a pilot trial testing the safety/efficacy profile of combined rituximab and daratumumab in posttransplant FSGS recurrence.
Assuntos
Glomerulosclerose Segmentar e Focal , Humanos , Rituximab/uso terapêutico , Glomerulosclerose Segmentar e Focal/tratamento farmacológico , Glomerulosclerose Segmentar e Focal/etiologia , Recidiva , Anticorpos Monoclonais/uso terapêuticoRESUMO
BACKGROUND: Systemic Lupus Erythematosus (SLE) has a strong genetic susceptibility, but little is known about the impact of diet on disease severity. The Western diet is typically deficient in magnesium (Mg), and given the immunomodulatory effects of Mg, we hypothesized that the low Mg intake increases disease risk and that increasing Mg intake would reduce severity of murine lupus. Here, we placed 12-week old MRL/lpr female lupus mice on a normal (Mg500) or a high (Mg2800) Mg diet for 9 weeks. Urine and blood were collected during the study for quantification of urinary albumin, BUN, anti-dsDNA antibodies, and immune phenotyping. RESULTS: MRL/lpr lupus mice on high Mg2800 diet had significantly fewer skin lesions and less severe skin histology score, and reduced levels of pathogenic anti-dsDNA antibodies, compared with the Mg500 group (143.8±75.0 vs. 47.4±36.2 × 106U/ml; P < 0.05). The high Mg2800 group had a nearly two-fold increase in the percentage of CD4+FOXP3+ Treg cells compared to controls (19.9±5.4 vs. 11.4±5.5%; P < 0.05). Treg percentages inversely correlated with the concentration of anti-dsDNA. None of the mice developed arthritis during the observation period and there were no significant differences in weight, proteinuria, BUN or kidney histology. CONCLUSION: In conclusion, oral supplementation of Mg has a protective effect in a murine lupus model and may represent an inexpensive and safe adjuvant in the treatment of SLsE.
Assuntos
Anticorpos Antinucleares , Lúpus Eritematoso Sistêmico , Magnésio , Linfócitos T Reguladores , Animais , Lúpus Eritematoso Sistêmico/imunologia , Feminino , Camundongos , Anticorpos Antinucleares/imunologia , Anticorpos Antinucleares/sangue , Magnésio/administração & dosagem , Linfócitos T Reguladores/imunologia , Modelos Animais de Doenças , Administração Oral , Camundongos Endogâmicos MRL lpr , Autoanticorpos/imunologia , Autoanticorpos/sangue , Pele/patologia , Pele/imunologia , Pele/efeitos dos fármacos , Dermatopatias/imunologia , Dermatopatias/tratamento farmacológico , Dermatopatias/patologiaRESUMO
BACKGROUND: Although high-affinity IgG auto- and alloantibodies are important drivers of kidney inflammation that can result in ESKD, therapeutic approaches that effectively reduce such pathogenic antibodies remain elusive. Erythropoietin (EPO) has immunomodulatory functions, but its effects on antibody production are unknown. METHODS: We assessed the effect and underlying mechanisms of EPO/EPO receptor (EPOR) signaling on primary and secondary, T cell-dependent and T-independent antibody formation using in vitro culture systems, murine models of organ transplantation and lupus nephritis, and mice conditionally deficient for the EPOR expressed on T cells or B cells. RESULTS: In wild-type mice, recombinant EPO inhibited primary, T cell-dependent humoral immunity to model antigens and strong, polyclonal stimuli, but did not alter T-independent humoral immune responses. EPO also significantly impaired secondary humoral immunity in a potent allogeneic organ transplant model system. The effects required T cell, but not B cell, expression of the EPOR and resulted in diminished frequencies of germinal center (GC) B cells and T follicular helper cells (TFH). In vitro and in vivo experiments showed that EPO directly prevented TFH differentiation and function via a STAT5-dependent mechanism that reduces CD4+ T cell expression of Bcl6. In lupus models, EPO reduced TFH, GC B cells, and autoantibody production, and abrogated autoimmune glomerulonephritis, demonstrating clinical relevance. In vitro studies verified that EPO prevents differentiation of human TFH cells. CONCLUSIONS: Our findings newly demonstrate that EPO inhibits TFH-dependent antibody formation, an observation with potential implications for treating antibody-mediated diseases, including those of the kidney.
Assuntos
Formação de Anticorpos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Eritropoetina/farmacologia , Imunidade Humoral/efeitos dos fármacos , Células T Auxiliares Foliculares/fisiologia , Animais , Linfócitos B/imunologia , Contagem de Linfócito CD4 , Células Cultivadas , Eritropoetina/genética , Eritropoetina/metabolismo , Feminino , Humanos , Masculino , Camundongos , Fosforilação , Receptores da Eritropoetina/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Células T Auxiliares Foliculares/imunologia , Células T Auxiliares Foliculares/metabolismo , Linfócitos T Reguladores/imunologiaRESUMO
OBJECTIVE: Autoimmune hepatitis (AIH) is a chronic liver disease associated with impaired regulatory T cell (Treg) number and function. Erythropoietin (EPO) is a kidney-produced erythropoietic hormone that has known immune-modulating effects, including Treg induction. Whether EPO administration increases Treg in patients with AIH is unknown. METHODS: We treated six stable AIH patients with a single 1000 IU dose of EPO and comprehensively characterized changes in Treg overall and in Treg subsets before and at 4 and 12 weeks after treatment using mass cytometry (CyTOF) combined with an unbiased clustering approach (Phenograph) based on 22 Treg-relevant cell-surface markers. RESULTS: EPO was well-tolerated and no patients showed significant changes in hematological parameters, liver enzymes, or IgG levels from baseline to 12 weeks following EPO administration. Total Treg and Treg/CD8+ T cell ratios significantly increased at 4 weeks and returned to baseline levels at 12 weeks after EPO injection. We identified 17 Treg subsets of which CD4+CD25HICD127NEG HLADR+ Treg had the highest increase and the most favorable Treg/CD8+ ratio upon EPO treatment. At 12 weeks after EPO administration, the HLADR+ Treg subset also returned to values comparable to those at baseline. Ex vivo assays documented that Treg were functional and the ones isolated at 12 weeks after EPO injection were significantly more suppressive than the ones isolated at baseline. In Treg-depleted assays, EPO did not show a significant effect on IFN-γ+, IL-2+, and IL-17+ CD4+ T cells. CONCLUSION: In stable AIH patients, EPO increases overall Treg and particularly those expressing the high function marker HLA-DR. These results provide the rationale for future studies testing the hypothesis that EPO or EPO analogues improve outcomes of AIH patients by increasing Treg.
Assuntos
Eritropoetina/administração & dosagem , Hepatite Autoimune/tratamento farmacológico , Hepatite Autoimune/etiologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Adulto , Idoso , Biomarcadores , Citocinas/metabolismo , Duração da Terapia , Feminino , Hepatite Autoimune/diagnóstico , Hepatite Autoimune/metabolismo , Humanos , Imunomodulação/efeitos dos fármacos , Imunofenotipagem , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Linfócitos T Reguladores/metabolismo , Resultado do TratamentoRESUMO
Whether kidney transplant recipients are capable of mounting an effective anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) adaptive immune response despite chronic immunosuppression is unknown and has important implications for therapy. Herein, we analyzed peripheral blood cell surface and intracellular cytokine phenotyping by flow cytometry along with serum antibody testing in 18 kidney transplant recipients with active coronavirus disease 2019 (COVID-19) infection and 36 matched, transplanted controls without COVID-19. We observed significantly fewer total lymphocytes and fewer circulating memory CD4+ and CD8+ T cells in the COVID-19 subjects. We also showed fewer anergic and senescent CD8+ T cells in COVID-19 individuals, but no differences in exhausted CD8+ T cells, nor in any of these CD4+ T cell subsets between groups. We also observed greater frequencies of activated B cells in the COVID-19 patients. Sixteen of 18 COVID-19 subjects tested for anti-SARS-CoV-2 serum antibodies showed positive immunoglobulin M or immunoglobulin G titers. Additional analyses showed no significant correlation among immune phenotypes and degrees of COVID-19 disease severity. Our findings indicate that immunosuppressed kidney transplant recipients admitted to the hospital with acute COVID-19 infection can mount SARS-CoV-2-reactive adaptive immune responses. The findings raise the possibility that empiric reductions in immunosuppressive therapy for all kidney transplant recipients with active COVID-19 may not be required.
Assuntos
COVID-19/epidemiologia , Imunidade Humoral , Hospedeiro Imunocomprometido , Transplante de Rim/efeitos adversos , Pandemias , Insuficiência Renal/epidemiologia , Transplantados , Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , Comorbidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Insuficiência Renal/cirurgia , Estudos Retrospectivos , SARS-CoV-2 , Estados Unidos/epidemiologiaRESUMO
BACKGROUND: Polymorphisms in genes modulating xenobiotics metabolism, in particular the ABCC2 c.3972C > T single nucleotide polymorphism (SNP) at exon 28, have been suggested to increase primary liver cancer (PLC) risk. Conversely, the occurrence of PLCs in Wilson disease patients is a rare event, in contrast with the occurrence observed in other chronic liver diseases. Here we report the clinical case of five siblings carrying the ABCC2 c.3972C > T SNP; three of them were affected by Wilson disease and two brothers with Wilson disease also developed PLCs. METHODS: The presence of the ABCC2 c.3972C > T SNP was assessed by Sanger sequencing and the exposure of PLC risk factors by standardized questionnaires. RESULTS: Notably, PLCs occurred only in the two brothers with the ABCC2 c.3972C > T SNP and Wilson disease who resulted exposed to asbestos and cigarette smoking, but not in the other siblings with the ABCC2 c.3972C > T SNP, alone or in association with Wilson disease, not exposed to these carcinogens and/or to other known risk factors for PLCs. CONCLUSIONS: These findings suggest that ABCC2 c.3972C > T SNP and WD, also in association, may not represent a sufficient condition for PLC development, but that co-occurrence of further host/exogenous risk factors are needed to drive this process, reinforcing the notion that liver carcinogenesis is the result of a complex interplay between environmental and host genetic determinants. Due to the sporadic cases of this study and the paucity of data currently available in literature on this issue, future investigations in a larger population are needed to confirm our findings.
Assuntos
Carcinoma Hepatocelular/genética , Colangiocarcinoma/genética , Degeneração Hepatolenticular/genética , Cirrose Hepática/genética , Neoplasias Hepáticas/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Polimorfismo de Nucleotídeo Único , Amianto/efeitos adversos , Sequência de Bases , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/patologia , Colangiocarcinoma/etiologia , Colangiocarcinoma/patologia , Fumar Cigarros/efeitos adversos , Éxons , Feminino , Expressão Gênica , Interação Gene-Ambiente , Haplótipos , Degeneração Hepatolenticular/etiologia , Degeneração Hepatolenticular/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Cirrose Hepática/etiologia , Cirrose Hepática/patologia , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Proteína 2 Associada à Farmacorresistência Múltipla , Linhagem , Fatores de Risco , IrmãosRESUMO
Patients with autosomal dominant polycystic kidney disease (ADPKD), a genetic disease due to mutations of the PKD1 or PKD2 gene, show signs of complement activation in the urine and cystic fluid, but their pathogenic role in cystogenesis is unclear. We tested the causal relationship between complement activation and cyst growth using a Pkd1KO renal tubular cell line and newly generated conditional Pkd1-/- C3-/- mice. Pkd1-deficient tubular cells have increased expression of complement-related genes (C3, C5, CfB, C3ar, and C5ar1), while the gene and protein expression of complement regulators DAF, CD59, and Crry is decreased. Pkd1-/- C3-/- mice are unable to fully activate the complement cascade and are characterized by a significantly slower kidney cystogenesis, preserved renal function, and reduced intrarenal inflammation compared with Pkd1-/- C3+/+ controls. Transgenic expression of the cytoplasmic C-terminal tail of Pkd1 in Pkd1KO cells lowered C5ar1 expression, restored Daf levels, and reduced cell proliferation. Consistently, both DAF overexpression and pharmacological inhibition of C5aR1 (but not C3aR) reduced Pkd1KO cell proliferation. In conclusion, the loss of Pkd1 promotes unleashed activation of locally produced complement by downregulating DAF expression in renal tubular cells. Increased C5a formation and C5aR1 activation in tubular cells promotes cyst growth, offering a new therapeutic target.
Assuntos
Antígenos CD55 , Complemento C3 , Camundongos Knockout , Rim Policístico Autossômico Dominante , Animais , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/patologia , Rim Policístico Autossômico Dominante/metabolismo , Camundongos , Antígenos CD55/genética , Antígenos CD55/metabolismo , Complemento C3/genética , Complemento C3/metabolismo , Receptor da Anafilatoxina C5a/metabolismo , Receptor da Anafilatoxina C5a/genética , Modelos Animais de Doenças , Ativação do Complemento , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/metabolismo , Humanos , Proliferação de Células , Masculino , Linhagem Celular , Receptores de Complemento 3b/genética , Receptores de Complemento 3b/metabolismoRESUMO
The deposition of antipodocyte autoantibodies in the glomerular subepithelial space induces primary membranous nephropathy (MN), the leading cause of nephrotic syndrome worldwide. Taking advantage of the glomerulus-on-a-chip system, we modeled human primary MN induced by anti-PLA2R antibodies. Here we show that exposure of primary human podocytes expressing PLA2R to MN serum results in IgG deposition and complement activation on their surface, leading to loss of the chip permselectivity to albumin. C3a receptor (C3aR) antagonists as well as C3AR gene silencing in podocytes reduced oxidative stress induced by MN serum and prevented albumin leakage. In contrast, inhibition of the formation of the membrane-attack-complex (MAC), previously thought to play a major role in MN pathogenesis, did not affect permselectivity to albumin. In addition, treatment with a C3aR antagonist effectively prevented proteinuria in a mouse model of MN, substantiating the chip findings. In conclusion, using a combination of pathophysiologically relevant in vitro and in vivo models, we established that C3a/C3aR signaling plays a critical role in complement-mediated MN pathogenesis, indicating an alternative therapeutic target for MN.
Assuntos
Glomerulonefrite Membranosa , Síndrome Nefrótica , Podócitos , Animais , Humanos , Camundongos , Albuminas , Glomerulonefrite Membranosa/genética , Glomérulos Renais/patologia , Síndrome Nefrótica/patologia , Podócitos/patologiaRESUMO
Background: Administration of recombinant erythropoietin (EPO), a kidney-produced hormone with erythropoietic functions, has been shown to have multiple immunoregulatory effects in mice and humans, but whether physiological levels of EPO regulate immune function in vivo has not been previously evaluated. Methods: We generated mice in which we could downregulate EPO production using a doxycycline (DOX)-inducible, EPO-specific silencing RNA (shEPOrtTAPOS), and we crossed them with B6.MRL-Faslpr/J mice that develop spontaneous lupus. We treated these B6.MRL/lpr shEPOrtTAPOS with DOX and serially measured anti-dsDNA antibodies, analyzed immune subsets by flow cytometry, and evaluated clinical signs of disease activity over 6 months of age in B6.MRL/lpr shEPOrtTAPOS and in congenic shEPOrtTANEG controls. Results: In B6.MRL/lpr mice, Epo downregulation augmented anti-dsDNA autoantibody levels and increased disease severity and percentages of germinal center B cells compared with controls. It also increased intracellular levels of IL-6 and MCP-1 in macrophages. Discussion: Our data in a murine model of lupus document that endogenous EPO reduces T- and B-cell activation and autoantibody production, supporting the conclusion that EPO physiologically acts as a counterregulatory mechanism to control immune homeostasis.
Assuntos
Doenças Autoimunes , Eritropoetina , Nefropatias , Animais , Humanos , Camundongos , Imunidade , Rim , Camundongos Endogâmicos MRL lprRESUMO
Immunoglobulin A nephropathy(IgAN) is the most common primary glomerulonephritis worldwide. The working model for the pathogenesis of IgAN involves a multistep process starting from the production of galactose-deficient and polymeric immunoglobulin A-1 (gd-IgA1) that enters systemic circulation from gut-associated lymphoid tissue (GALT). Galactose-deficient IgA are targeted by endogenous IgG, leading to the formation of circulating immune complexes that deposit in the mesangium and resulting in glomerular inflammation. Disease onset and relapses are often associated with gut infections, supporting the hypothesis that the gut plays an important pathogenic role. In the presence of microbial pathogens or food antigens, activated dendritic cells in the gut mucosa induce T cell dependent and independent B cell differentiation into IgA secreting plasma cells. In IgAN patients, this promotes the systemic release of mucosal gd-IgA1. Not all bacterial strains have the same capacity to elicit IgA production, and little is known about the antigen specificity of the pathogenic gd-IgA1. However, efficacy of treatments targeting gut inflammation support a pathogenic link between the bowel immune system and IgAN. Herein, we review the evidence supporting the role of gut inflammation in IgAN pathogenesis.
Assuntos
Glomerulonefrite por IGA , Complexo Antígeno-Anticorpo , Galactose , Humanos , Imunoglobulina A , Imunoglobulina G , Inflamação , RimRESUMO
Background: Belatacept (Bela) was developed to reduce nephrotoxicity and cardiovascular risk that are associated with the chronic use of Calcineurin inhibitors (CNIs) in kidney transplant recipients. The use of Bela with early steroid withdrawal (ESW) and simultaneous CNI avoidance has not been formally evaluated. Methods: At 3 months post-transplant, stable kidney transplant recipients with ESW on Tacrolimus (Tac) + mycophenolate (MPA) were randomized 1:1:1 to: 1) Bela+MPA, 2) Bela+low-dose Tac (trough goal <5 ng/mL), or 3) continue Tac+MPA. All patients underwent surveillance graft biopsies at enrollment and then at 12, and 24 months post-transplant. Twenty-seven recipients were included; 9 underwent conversion to Bela+MPA, 8 to Bela+low-dose Tac and 10 continued Tac+MPA. Serial blood samples were collected for immune phenotyping and gene expression analyses. Results: The Bela+MPA arm was closed early due to high rate of biopsy proven acute rejection (BPAR). The incidence of BPAR was 4/9 in Bela+MPA, 0/8 in Bela+low dose Tac and 2/10 in Tac+MPA, P= 0.087. The Bela+low-dose Tac regimen was associated with +8.8 mL/min/1.73 m2 increase in eGFR compared to -0.38 mL/min/1.73 m2 in Tac+MPA, P= 0.243. One graft loss occurred in the Bela+MPA group. Immunophenotyping of peripheral blood monocyte count (PBMC) showed that CD28+CD4+ and CD28+CD8+ T cells were higher in Bela+MPA patients with acute rejection compared to patients without rejection, although the difference did not reach statistical significance. Conclusions: Our data indicate that, in steroid free regimens, low-dose Tac maintenance is needed to prevent rejection when patients are converted to Bela, at least when the maneuver is done early after transplant.
Assuntos
Abatacepte , Inibidores de Calcineurina , Imunossupressores , Transplante de Rim , Humanos , Abatacepte/administração & dosagem , Abatacepte/uso terapêutico , Calcineurina , Inibidores de Calcineurina/efeitos adversos , Antígenos CD28 , Linfócitos T CD8-Positivos , Imunossupressores/administração & dosagem , Imunossupressores/uso terapêutico , Leucócitos Mononucleares , Ácido Micofenólico/administração & dosagem , Ácido Micofenólico/uso terapêutico , Esteroides , Tacrolimo/administração & dosagem , Tacrolimo/uso terapêutico , Substituição de MedicamentosRESUMO
Erythropoietin (EPO) has multiple nonerythropoietic functions, including immune modulation, but EPO's effects in transplantation remain incompletely understood. We tested the mechanisms linking EPO administration to prolongation of murine heterotopic heart transplantation using WT and conditional EPO receptor-knockout (EPOR-knockout) mice as recipients. In WT controls, peritransplant administration of EPO synergized with CTLA4-Ig to prolong allograft survival (P < 0.001), reduce frequencies of donor-reactive effector CD8+ T cells in the spleen (P < 0.001) and in the graft (P < 0.05), and increase frequencies and total numbers of donor-reactive Tregs (P < 0.01 for each) versus CTLA4-Ig alone. Studies performed in conditional EPOR-knockout recipients showed that each of these differences required EPOR expression in myeloid cells but not in T cells. Analysis of mRNA isolated from spleen monocytes showed that EPO/EPOR ligation upregulated macrophage-expressed, antiinflammatory, regulatory, and pro-efferocytosis genes and downregulated selected proinflammatory genes. Taken together, the data support the conclusion that EPO promotes Treg-dependent murine cardiac allograft survival by crucially altering the phenotype and function of macrophages. Coupled with our previous documentation that EPO promotes Treg expansion in humans, the data support the need for testing the addition of EPO to costimulatory blockade-containing immunosuppression regimens in an effort to prolong human transplant survival.
Assuntos
Eritropoetina , Linfócitos T Reguladores , Abatacepte , Aloenxertos , Animais , Epoetina alfa , Eritropoetina/genética , Eritropoetina/metabolismo , Camundongos , Células MieloidesRESUMO
B cell depleting therapies permit immunosuppressive drug withdrawal and maintain remission in patients with frequently relapsing nephrotic syndrome (FRNS) or steroid-dependent nephrotic syndrome (SDNS), but lack of biomarkers for treatment failure. Post-depletion immune cell reconstitution may identify relapsing patients, but previous characterizations suffered from methodological limitations of flow cytometry. Time-of-flight mass cytometry (CyTOF) is a comprehensive analytic modality that simultaneously quantifies over 40 cellular markers. Herein, we report CyTOF-enabled immune cell comparisons over a 12-month period from 30 children with SDNS receiving B cell depleting therapy who either relapsed (n = 17) or remained stable (n = 13). Anti-CD20 treatment depleted all B cells subsets and CD20 depleting agent choice (rituximab vs ofatumumab) did not affect B cell subset recovery. Despite equal total numbers of B cells, 5 subsets of B cells were significantly higher in relapsing individuals; all identified subsets of B cells were class-switched. T cell subsets (including T follicular helper cells and regulatory T cells) and other major immune compartments were largely unaffected by B cell depletion, and similar between relapsing and stable children. In conclusion, CyTOF analysis of immune cells from anti-CD20 antibody treated patients identifies class-switched B cells as the main subset whose expansion associates with disease relapse. Our findings set the basis for future studies exploring how identified subsets can be used to monitor treatment response and improve our understanding of the pathogenesis of the disease.
Assuntos
Subpopulações de Linfócitos B/imunologia , Síndrome Nefrótica/imunologia , Adolescente , Adulto , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Antígenos CD20/imunologia , Criança , Pré-Escolar , Feminino , Humanos , Switching de Imunoglobulina , Fatores Imunológicos/farmacologia , Fatores Imunológicos/uso terapêutico , Contagem de Linfócitos , Depleção Linfocítica , Masculino , Espectrometria de Massas , Síndrome Nefrótica/tratamento farmacológico , Recidiva , Rituximab/farmacologia , Rituximab/uso terapêutico , Subpopulações de Linfócitos T/imunologia , Adulto JovemRESUMO
Individuals with kidney failure are at increased risk of cardiovascular events, as well as infections and malignancies, but the associated immunological abnormalities are unclear. We hypothesized that the uremic milieu triggers a chronic inflammatory state that, while accelerating atherosclerosis, promotes T cell exhaustion, impairing effective clearance of pathogens and tumor cells. Clinical and demographic data were collected from 78 patients with chronic kidney disease (CKD) (n = 42) or end-stage kidney disease (ESKD) (n = 36) and from 18 healthy controls (HC). Serum cytokines were analyzed by Luminex. Immunophenotype of T cells was performed by flow cytometry on peripheral blood mononuclear cells. ESKD patients had significantly higher serum levels of IFN-γ, TNF-α, sCD40L, GM-CSF, IL-4, IL-8, MCP-1, and MIP-1ß than CKD and HC. After mitogen stimulation, both CD4+ and CD8+ T cells in ESKD group demonstrated a pro-inflammatory phenotype with increased IFN-γ and TNF-α, whereas both CKD and ESKD patients had higher IL-2 levels. CKD and ESKD were associated with increased frequency of exhausted CD4+ T cells (CD4+KLRG1+PD1+CD57-) and CD8+ T cells (CD8+KLRG1+PD1+CD57-), as well as anergic CD4+ T cells (CD4+KLRG1-PD1+CD57-) and CD8+ T cells (CD8+KLRG1-PD1+CD57-). Although total percentage of follicular helper T cell (TFH) was similar amongst groups, ESKD had reduced frequency of TFH1 (CCR6-CXCR3+CXCR5+PD1+CD4+CD8-), but increased TFH2 (CCR6-CXCR3-CXCR5+PD1+CD4+CD8-), and plasmablasts (CD3-CD56-CD19+CD27highCD38highCD138-). In conclusion, kidney failure is associated with pro-inflammatory markers, exhausted T cell phenotype, and upregulated TFH2, especially in ESKD. These immunological changes may account, at least in part, for the increased cardiovascular risk in these patients and their susceptibility to infections and malignancies.
Assuntos
Insuficiência Renal/imunologia , Células T Auxiliares Foliculares/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Citocinas/imunologia , Feminino , Humanos , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-IdadeRESUMO
INTRODUCTION: Bipolar disorder (BD) is a highly heritable and disabling mental illness, commonly associated with substance abuse, being alcohol abuse the most frequent. Comorbid BD and substance abuse disorders are often associated with high levels of health service utilization and destabilization of the course of illness resulting in poor treatment outcomes. Although recent genome-wide association studies have detected a number of risk genes for BD, the data is still sparse and inconclusive for those genes that may contribute to the increased risk of comorbid alcohol abuse (AA) in BD. The primary aim of the present study was to investigate the effects of 46 single-nucleotide polymorphisms (SNPs) within eight genes on different phenotypes of BD patients, such as comorbid alcohol abuse. We further assessed clinical variables associated with AA. METHODS: One-hundred fifty-eight BD I and II patients were enrolled in a naturalistic cohort study. Genomic DNA of 92 patients was extracted from whole blood using standard procedures and 46 tag SNPs in eight genes of interest (ANK, CACNA1C, CACNB2, FKBP5, GRM7, ITIH3, SYNE1 and TCF4) were genotyped. RESULTS: Seventy-one patients out of 158 (45%) satisfied diagnostic criteria for comorbid AA. Among 46 SNPs analyzed, the only SNP associated with comorbid AA was rs1034936 polymorphism in the CANCA1C gene. This polymorphism was also associated with lifetime cocaine abuse, manic switch and current atypical antipsychotics. CONCLUSIONS: Our findings suggest a role of rs1034936 CACNA1C gene variant in BD-AA group. Despite their preliminary nature, the present results may provide new insight on mechanisms underlying AA in BD.
Assuntos
Alcoolismo/genética , Transtorno Bipolar/genética , Polimorfismo de Nucleotídeo Único , Adulto , Alcoolismo/epidemiologia , Transtorno Bipolar/epidemiologia , Estudos de Coortes , Feminino , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Fatores de Risco , Transtornos Relacionados ao Uso de SubstânciasRESUMO
Introduction: Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common inherited disorders in humans and the majority of patients carry a variant in either PKD1 or PKD2. Genetic testing is increasingly required for diagnosis, prognosis, and treatment decision, but it is challenging due to segmental duplications of PKD1, genetic and allelic heterogeneity, and the presence of many variants hypomorphic or of uncertain significance. We propose an NGS-based testing strategy for molecular analysis of ADPKD and its phenocopies, validated in a diagnostic setting. Materials and Methods: Our protocol is based on high-throughput simultaneous sequencing of PKD1 and PKD2 after long range PCR of coding regions, followed by a masked reference genome alignment, and MLPA analysis. A further screening of additional 14 cystogenes was performed in negative cases. We applied this strategy to analyze 212 patients with a clinical suspicion of ADPKD. Results and Discussion: We detected causative variants (interpreted as pathogenic/likely pathogenic) in 61.3% of our index patients, and variants of uncertain clinical significance in 12.5%. The majority (88%) of genetic variants was identified in PKD1, 12% in PKD2. Among 158 distinct variants, 80 (50.6%) were previously unreported, confirming broad allelic heterogeneity. Eleven patients showed more than one variant. Segregation analysis indicated biallelic disease in five patients, digenic in one, de novo variant with unknown phase in two. Furthermore, our NGS protocol allowed the identification of two patients with somatic mosaicism, which was undetectable with Sanger sequencing. Among patients without PKD1/PKD2 variants, we identified three with possible alternative diagnosis: a patient with biallelic mutations in PKHD1, confirming the overlap between recessive and dominant PKD, and two patients with variants in ALG8 and PRKCSH, respectively. Genotype-phenotype correlations showed that patients with PKD1 variants predicted to truncate (T) the protein experienced end-stage renal disease 9 years earlier than patients with PKD1 non-truncating (NT) mutations and >13 years earlier than patients with PKD2 mutations. ADPKD-PKD1 T cases showed a disease onset significantly earlier than ADPKD-PKD1 NT and ADPK-PKD2, as well as a significant earlier diagnosis. These data emphasize the need to combine clinical information with genetic data to achieve useful prognostic predictions.