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1.
J Am Soc Nephrol ; 27(5): 1517-33, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26567246

RESUMO

The phospholipase A2 receptor (PLA2R1) is the major autoantigen in idiopathic membranous nephropathy. However, the value of anti-PLA2R1 antibody titers in predicting patient outcomes is unknown. Here, we screened serum samples from 50 patients positive for PLA2R1 for immunoreactivity against a series of PLA2R1 deletion mutants covering the extracellular domains. We identified reactive epitopes in the cysteine-rich (CysR), C-type lectin domain 1 (CTLD1), and C-type lectin domain 7 (CTLD7) domains and confirmed the reactivity with soluble forms of each domain. We then used ELISAs to stratify 69 patients positive for PLA2R1 by serum reactivity to one or more of these domains: CysR (n=23), CysRC1 (n=14), and CysRC1C7 (n=32). Median ELISA titers measured using the full-length PLA2R1 antigens were not statistically different between subgroups. Patients with anti-CysR-restricted activity were younger (P=0.008), had less nephrotic range proteinuria (P=0.02), and exhibited a higher rate of spontaneous remission (P=0.03) and lower rates of renal failure progression (P=0.002) and ESRD (P=0.01) during follow-up. Overall, 31 of 69 patients had poor renal prognosis (urinary protein/creatinine ratio >4 g/g or eGFR<45 ml/min per 1.73 m(2) at end of follow-up). High anti-PLA2R1 activity and epitope spreading beyond the CysR epitope were independent risk factors of poor renal prognosis in multivariable Cox regression analysis. Epitope spreading during follow-up associated with disease worsening (n=3), whereas reverse spreading from a CysRC1C7 profile back to a CysR profile associated with favorable outcome (n=1). We conclude that analysis of the PLA2R1 epitope profile and spreading is a powerful tool for monitoring disease severity and stratifying patients by renal prognosis.


Assuntos
Autoanticorpos/imunologia , Epitopos/imunologia , Glomerulonefrite Membranosa/imunologia , Receptores da Fosfolipase A2/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico
2.
J Vis Exp ; (97)2015 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-25867523

RESUMO

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and neurotransmitter loading in synaptic vesicles. This acidification is described to be mediated by proton ATPases, coupled to ClC chloride transporters. Highly-conserved electroneutral protons transporters, the Na+/H+ exchangers (NHE) 6, 7 and 9 are also expressed in these compartments. Mutations in their genes have been linked with human cognitive and neurodegenerative diseases. Paradoxically, their roles remain elusive, as their intracellular localization has prevented detailed functional characterization. This manuscript shows a method to solve this problem. This consists of the selection of mutant cell lines, capable of surviving acute cytosolic acidification by retaining intracellular NHEs at the plasma membrane. It then depicts two complementary protocols to measure the ion selectivity and activity of these exchangers: (i) one based on intracellular pH measurements using fluorescence video microscopy, and (ii) one based on the fast kinetics of lithium uptake. Such protocols can be extrapolated to measure other non-electrogenic transporters. Furthermore, the selection procedure presented here generates cells with an intracellular retention defective phenotype. Therefore these cells will also express other vesicular membrane proteins at the plasma membrane. The experimental strategy depicted here may therefore constitute a potentially powerful tool to study other intracellular proteins that will be then expressed at the plasma membrane together with the vesicular Na+/H+ exchangers used for the selection.


Assuntos
Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Endossomos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Transporte de Íons , Prótons , Trocadores de Sódio-Hidrogênio/química
3.
Gut ; 64(7): 1072-81, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25298539

RESUMO

OBJECTIVE: Under both physiological and pathological conditions, bone volume is determined by the rate of bone formation by osteoblasts and bone resorption by osteoclasts. Excessive bone loss is a common complication of human IBD whose mechanisms are not yet completely understood. Despite the role of activated CD4(+) T cells in inflammatory bone loss, the nature of the T cell subsets involved in this process in vivo remains unknown. The aim of the present study was to identify the CD4(+) T cell subsets involved in the process of osteoclastogenesis in vivo, as well as their mechanism of action. DESIGN: CD4(+) T cells were studied in IL10-/- mice and Rag1-/- mice adoptively transferred with naive CD4(+)CD45RB(high) T cells, representing two well-characterised animal models of IBD and in patients with Crohn's disease. They were phenotypically and functionally characterised by flow cytometric and gene expression analysis, as well as in in vitro cocultures with osteoclast precursors. RESULTS: In mice, we identified bone marrow (BM) CD4(+) T cells producing interleukin (IL)-17 and tumour necrosis factor (TNF)-α as an osteoclastogenic T cell subset referred to as Th17 TNF-α(+) cells. During chronic inflammation, these cells migrate to the BM where they survive in an IL-7-dependent manner and where they promote the recruitment of inflammatory monocytes, the main osteoclast progenitors. A population equivalent to the Th17 TNF-α(+) cells was also detected in patients with Crohn's disease. CONCLUSIONS: Our results highlight the osteoclastogenic function of the Th17 TNF-α(+) cells that contribute to bone loss in vivo in IBD.


Assuntos
Doenças Ósseas/fisiopatologia , Células da Medula Óssea/fisiologia , Doenças Inflamatórias Intestinais/fisiopatologia , Osteoclastos/fisiologia , Subpopulações de Linfócitos T/fisiologia , Células Th17/fisiologia , Imunidade Adaptativa/fisiologia , Animais , Doenças Ósseas/imunologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular , Doença de Crohn/imunologia , Doença de Crohn/fisiopatologia , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Doenças Inflamatórias Intestinais/imunologia , Interleucina-7/fisiologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Osteoclastos/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Células Th17/imunologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/fisiologia
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