Glomerular targets of autoimmunity in human membranous nephropathy are poorly understood. Here, we used a combined proteomic approach to identify specific antibodies against podocyte proteins in both serum and glomeruli of patients with membranous nephropathy (MN). We detected specific anti-aldose reductase (AR) and anti-manganese superoxide dismutase (SOD2) IgG 4 in sera of patients with MN. We also eluted high titers of anti-AR and anti-SOD2 IgG 4 from microdissected glomeruli of three biopsies of MN kidneys but not from biopsies of other glomerulonephritides characterized by IgG deposition (five lupus nephritis and two membranoproliferative glomerulonephritis). We identified both antigens in MN biopsies but not in other renal pathologies or normal kidney. Confocal and immunoelectron microscopy (IEM) showed co-localization of anti-AR and anti-SOD2 with IgG 4 and C5b-9 in electron-dense podocyte immune deposits. Preliminary in vitro experiments showed an increase of SOD2 expression on podocyte plasma membrane after treatment with hydrogen peroxide. In conclusion, our data support AR and SOD2 as renal antigens of human MN and suggest that oxidative stress may drive glomerular SOD2 expression.

Renal targets of autoimmunity in human lupus nephritis (LN) are unknown. We sought to identify autoantibodies and glomerular target antigens in renal biopsy samples from patients with LN and determine whether the same autoantibodies can be detected in circulation. Glomeruli were microdissected from biopsy samples of 20 patients with LN and characterized by proteomic techniques. Serum samples from large cohorts of patients with systemic lupus erythematosus (SLE) with and without LN and other glomerulonephritides were tested. Glomerular IgGs recognized 11 podocyte antigens, with reactivity varying by LN pathology. Notably, IgG2 autoantibodies against a-enolase and annexin AI were detected in 11 and 10 of the biopsy samples, respectively, and predominated over other autoantibodies. Immunohistochemistry revealed colocalization of a-enolase or annexin AI with IgG2 in glomeruli. High levels of serum anti-a-enolase (.15 mg/L) IgG2 and/or anti-annexin AI (.2.7 mg/L) IgG2 were detected in most patients with LN but not patients with other glomerulonephritides, and they identified two cohorts: patients with high anti-a-enolase/low anti-annexin AI IgG2 and patients with low anti-a-enolase/high anti-annexin AI IgG2. Serum levels of both autoantibodies decreased significantly after 12 months of therapy for LN. Anti-a-enolase IgG2 recognized specific epitopes of a-enolase and did not cross-react with dsDNA. Furthermore, nephritogenic monoclonal IgG2 (clone H147) derived from lupus-prone MRL-lpr/lpr mice recognized human a-enolase, suggesting homology between animal models and human LN. These data show a multiantibody composition in LN, where IgG2 autoantibodies against a-enolase and annexin AI predominate in the glomerulus and can be detected in serum.

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SUMMARYAlthough morphological criteria for apoptosis are in general reliable, no systematic comparison of the techniques employed thus far has yet been performed. In this study, using confocal laser microscopy, we compared the performance of annexin V-FITC and calcein-AM for early detection of apoptosis in living adherent cells. Experiments were carried out on two distinct cell lines, PC 12 and NIH3T3, endowed with different shape and adhesion properties. The apoptotic process was followed for a prolonged period in the same cells of a predetermined field by means of a special flow chamber. Our results show that both probes allowed the detection of apoptotic cells in either cell line. However, some cells that clearly exhibited apoptotic changes on calcein visualization were annexin-negative. In NIH3T3 cells, annexin negativity of apoptotic cells was correlated with the preservation of cell shape and adhesion properties. These findings show that, at least in PC12 and NIH3T3 cells, annexin might be less sensitive than calcein-AM for early apoptosis detection and, for NIH3T3 cells, suggest that phosphatidilserine exposure is in some way linked to changes in cell shape and/or adhesion to culture substrate.

Objective Rheumatoid arthritis (RA) is associated with accelerated atherosclerosis. The reduction in cardiovascular risk that is induced by methotrexate (MTX) and anti–tumor necrosis factor α agents in RA is considered secondary to their anti‐inflammatory action, but their effects on serum lipoprotein function and foam cell formation are unknown. The reduced capacity of high‐density lipoprotein (HDL) to promote cell cholesterol efflux and the increased serum cell cholesterol‐loading capacity (CLC) demonstrated in RA may contribute to foam cell development. The aim of this study was to investigate the influence of MTX and adalimumab treatment on serum cholesterol efflux capacity (CEC) and CLC in RA patients and to study the in vitro effects of the two drugs on macrophage cholesterol handling. Methods Sera from RA patients treated with MTX (n = 34) or with adalimumab and MTX (n = 22) obtained before treatment, after 6 weeks of treatment, and after 6 months of treatment were analyzed for CEC and CLC by radioisotopic and fluorometric techniques, respectively. The influence of MTX and adalimumab on macrophage cholesterol efflux and uptake was evaluated in vitro using human THP‐1–derived macrophages. Results MTX treatment was associated with increases in serum HDL, low‐density lipoprotein, and total cholesterol levels and with ATP‐binding cassette G1–mediated and scavenger receptor class B type I (SR‐BI)–mediated increases in CEC; MTX treatment was not associated with modifications in CLC. Adalimumab treatment was associated with increases in serum HDL levels, a transient increase in SR‐BI–mediated CEC, a transient decrease in ATP‐binding cassette A1–mediated CEC, and a significant reduction in CLC; in addition, adalimumab reduced macrophage cholesterol uptake in vitro. Conclusion Antiatherosclerotic activity asso‐ciated with MTX and adalimumab may be mediated by beneficial and complementary effects on lipoprotein functions and on macrophage cholesterol handling. As a whole, these mechanisms may oppose foam cell formation.

Basal neurons of the vomeronasal organ of the mouse express a superfamily of about 120 pheromone receptors, named V2Rs, that are grouped in four families, A, B, C, and D, according to sequence homology. Family-A, -B, and -D V2Rs are expressed as one receptor gene per cell, but we previously reported their co-expression with family-C V2Rs. Here, we show that basal neurons can be further grouped according to the combinatorial expression of different V2Rs. Altogether, these findings suggest that in each basal neuron a transcriptional program is active for expressing a combination of two compatible receptors and for excluding, at the same time, the expression of all other V2Rs. Further analyses revealed non-random combinations of co-expression between family-C V2Rs and genes of the class Ib major histocompatibility complex. Thus, each basal neuron of the vomeronasal organ represents a highly qualified sensory unit for detecting very specific combinations of pheromonal cues.

Background: It is unclear whether in late life serum thyroid-stimulating hormone (TSH) predicts risk of developing cognitive impairment. Objective: This study investigated the prospective relationship of serum TSH with the risk of developing mild cognitive impairment (MCI), Alzheimer’s disease (AD) and vascular dementia (VaD) in an elderly cohort with a 4-year follow-up. Methods: Data are for 660 subjects aged 65 years and older from an Italian population-based cohort who were cognitively normal at an extensive assessment in 1999/2000 and underwent follow-up assessment in 2003/2004. Serum TSH was measured at baseline. Multinomial logistic models adjusted for sociodemographic and cardiovascular risk factors were used to investigate the association of serum TSH (both as a tertile and continuous log-transformed variable) with risk of incident MCI, AD and VaD diagnosed according to international criteria. Results: Over 3.8 ± 0.7 years of follow-up, there were 149 incident MCI cases (77 with impairment of memory and 72 with impairment of nonmemory domains) and 86 incident dementia cases (53 with AD, 28 with VaD). No association between baseline TSH and risk of developing any MCI subtype or AD was found. The highest TSH tertile had a threefold higher increased risk of VaD (OR: 3.25, 95% CI: 1.01–10.77, p = 0.048) compared to the lowest tertile. Risk of VaD increased about 60% for each 1 SD increase in log-transformed TSH (OR: 1.61, 95% CI: 1.06–2.44, p = 0.025). Conclusions: In this elderly cohort, baseline TSH was not related to the risk of developing MCI or AD, but high TSH was associated with an increased risk of VaD. These results suggest further need for research using larger samples to examine the role of TSH as a predictor of VaD and the role of thyroid autoimmunity in vascular cognitive impairment.

The lymphocytic ionotropic purinergic P2X receptors (P2X1R-P2X7R, or P2XRs) sense ATP released during cell damage-activation, thus regulating T-cell activation. We aim to define the role of P2XRs during islet allograft rejection and to establish a novel anti-P2XRs strategy to achieve long-term islet allograft function. Our data demonstrate that P2X1R and P2X7R are induced in islet allograft-infiltrating cells, that only P2X7R is increasingly expressed during alloimmune response, and that P2X1R is augmented in both allogeneic and syngeneic transplantation. In vivo short-term P2X7R targeting (using periodate-oxidized ATP [oATP]) delays islet allograft rejection, reduces the frequency of Th1/Th17 cells, and induces hyporesponsiveness toward donor antigens. oATP-treated mice displayed preserved islet grafts with reduced Th1 transcripts. P2X7R targeting and rapamycin synergized in inducing long-term islet function in 80% of transplanted mice and resulted in reshaping of the recipient immune system. In vitro P2X7R targeting using oATP reduced T-cell activation and diminished Th1/Th17 cytokine production. Peripheral blood mononuclear cells obtained from long-term islet-transplanted patients showed an increased percentage of P2X7R+CD4+ T cells compared with controls. The beneficial effects of oATP treatment revealed a role for the purinergic system in islet allograft rejection, and the targeting of P2X7R is a novel strategy to induce long-term islet allograft function.