Elisa Loiacono

Oxidative Stress and Galactose-Deficient IgA1 as Markers of Progression in IgA Nephropathy

BACKGROUND AND OBJECTIVES We assessed the activation of the oxidative stress pathway in patients with IgA nephropathy (IgAN), while evaluating the classic marker of the disease (galactose-deficient serum IgA1). DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Sera from 292 patients and 69 healthy controls from Italy and the United States were assayed for advanced oxidation protein products (AOPPs), free sulfhydryl groups on albumin (SH-Alb), and IgA1 with galactose-deficient hinge-region O-glycans (Gd-IgA1). Gd-IgA1 was detected by binding to Helix aspersa agglutinin (HAA) and expressed as total Gd-IgA1 or as degree of galactose deficiency relative to a standard Gd-IgA1 myeloma protein (%HAA). RESULTS Sera from IgAN patients showed higher levels of Gd-IgA1, %HAA, and AOPPs, but lower levels of SH-Alb in comparison to that from healthy controls. Serum levels of AOPPs significantly correlated with serum Gd-IgA1 and %HAA. The relationship between these biomarkers and clinical features at sampling and during follow-up was assessed in 62 patients with long-term follow-up. AOPPs and %HAA correlated with proteinuria at sampling and independently associated with subsequent proteinuria. Levels of AOPPs correlated with rate of decline in renal function after sampling. The combination of a high level of AOPPs and a high level of %HAA associated with decline in estimated GFR. CONCLUSIONS Serum levels of aberrantly glycosylated IgA1 are elevated and oxidative stress pathways are activated in patients with IgAN; the intensity of the stress correlated with expression and progression of the disease. We speculate that oxidative stress may modulate the nephrotoxicity of aberrantly glycosylated IgA1 in IgAN.

Aberrantly glycosylated IgA1 induces mesangial cells to produce platelet-activating factor that mediates nephrin loss in cultured podocytes

The reaction of mesangial cells with aberrantly glycosylated IgA1 has been implicated in the etiology of IgA nephropathy (IgAN). Tumor necrosis factor, which is assumed to mediate the interaction between mesangial cells and podocytes, also induces the expression of platelet-activating factor (PAF). In this study, we determined whether PAF affects the expression of nephrin (an adhesion molecule critical to glomerular permselectivity) and cytoskeletal F-actin organization in podocytes. We treated human mesangial cells with atypically glycosylated IgA1 either prepared in vitro or derived from the sera of patients with IgAN. We then prepared conditioned media from these cells and added them to cultured human podocytes in the presence of PAF receptor antagonists. Podocytes transfected to overexpress acetylhydrolase, the main catabolic enzyme of PAF, served as controls. Downregulation of nephrin expression and F-actin reorganization occurred when podocytes were cultured with mesangial cell-conditioned medium. Preincubation of podocytes with a PAF receptor antagonist prevented the loss and redistribution of nephrin. In control podocytes overexpressing acetylhydrolase, nephrin loss was abrogated. Our results suggest that atypically glycosylated IgA-induced PAF from mesangial cells is a mediator of podocyte changes, which, when more directly tested elsewhere, were found to be associated with proteinuria. Hence, it is possible that these in vitro findings may be relevant to the proteinuria of IgAN.

Saquinavir in steroid-dependent and -resistant nephrotic syndrome: a pilot study

BACKGROUND Some difficult cases of idiopathic nephrotic syndrome (NS) have been treated with a HIV protease inhibitor provided with proteasome-inhibiting activity. The objective of this study was to limit nuclear factor κB (NF-κB) activation which is up-regulated in these patients, aiming at decreasing proteinuria and prednisone need. METHODS Ten cases with long-lasting (up to 15 years) history of NS with steroid dependence (six cases, of which three with secondary steroid resistance) or resistance to steroids (four cases) unsuccessfully treated with multiple immunosuppressive drugs, accepted a treatment with the protease inhibitor saquinavir. p50/p65 NF-κB nuclear localization and immunoproteasome/proteasome messenger RNA (mRNA) were monitored in peripheral blood mononuclear cells (PBMCs). The effects of saquinavir on NF-κB nuclear localization in cultured PBMCs and in immortalized human podocytes were assessed. RESULTS After a median follow-up of 14.7 months (6-68.7), 1/4 primary steroid-resistant NS (SRNS) and 5/6 steroid-dependent NS or secondary SRNS became infrequent (5) or frequent (1) relapsers, with 63% prednisone reduction (from 25.3 to 8.4 mg/kg/month, P = 0.015). Saquinavir was effective in association with low doses of calcineurin inhibitors (cyclosporine 2 mg/kg/day or tacrolimus 0.01-0.06 mg/kg/day). No side effects were observed apart from transitory mild diarrhoea. In PBMCs, NF-κB was down-regulated, while MECL-1 immunoproteasome/beta2 proteasome mRNA ratio was reversed to normal values. In culture, saquinavir blunted NF-κB activation in human podocytes and in PBMCs. CONCLUSIONS In this pilot study, a HIV antiprotease drug reduced proteinuria and had a steroid-sparing effect in some multidrug-resistant/-dependent NS. This observation warrants further investigation.

Toll-like receptors, immunoproteasome and regulatory T cells in children with Henoch–Schönlein purpura and primary IgA nephropathy

BackgroundHenoch–Schönlein purpura (HSP) nephritis and primary IgA nephropathy (pIgAN) present with glomerular IgA deposits, but differ with regard to clinical features. The suspected involvement of different immune system pathways is largely unknown.MethodsThis study was aimed at investigating some of the immunological features including Toll-like receptors (TLR), proteasome (PS)/immunoproteasome (iPS) switch, and the regulatory T cell system (Treg/Th17 cells) in 63 children with HSP with/without renal involvement and in 25 with pIgAN. Real-time PRC (Taqman) was used to quantify mRNA levels in peripheral blood mononuclear cells (PBMC).ResultsThe expression of mRNAs encoding for TLR4 in both HSP and pIgAN was higher than in controls (HC) and in both diseases FoxP3mRNA and TGF-β1mRNA expression was significantly lower than in HC. A switch from PS to iPS (LMP2/β1) was detected only in PBMC of HSP and it correlated with the level of TLR2mRNA, which was selectively increased only in children with HSP.ConclusionChildren with HSP and pIgAN present with similar signs of engagement of the innate immunity and regulatory T cell depression. The increased immunoproteasome switch, which correlated with TLR2 activation, may suggest an innate immunity pathway peculiar to HSP vasculitic presentation. This research area also deserves further investigation for possible therapeutic applications.

Evaluation of IFN-γ polymorphism +874 T/A in patients with recurrent tonsillitis by PCR Real Time Mismatch Amplification Mutation Assay (MAMA Real Time PCR)

Interferon gamma (IFN-γ) is an important cytokine that plays a crucial role in the balance between normal and pathological immune response. Defect of IFN-γ can give a predisposition to infectious disease, autoimmune pathologies and tumours. Different polymorphisms in this gene have been described, in particular the single nucleotide polymorphism (SNP)+874∗T/A that may affect IFN-γ gene expression. Several techniques can be used for the detection of SNPs. In this work two PCR Real Time assays were developed, an Amplification Refractory Mutation System (ARMS) and a Mismatch Amplification Mutation Assay (MAMA). Twenty-seven samples from patients (tonsillectomy) and 85 from donors blood bank were considered. As a result, 78/85 controls (91.7%) and 25/27 patients (92.6%) were heterozygosis, considering the ARMS-PCR; 55/85 (64.7%) and 14/27 (51.9%) were heterozygosis using MAMA-PCR assay. Fourteen of 85 (16.5%) and 8/27 (29.6%) were homozygosis A, 16/85 (18.8%) and 5/27 (18.5%) presented homozygosis T, taking into account the MAMA-PCR. There are statistically difference between the two assay with p<0.0001 at Chi-square test. Our preliminary data suggest that tonsillectomy patients had a statistical trend to possess the low IFN-γ polymorphism when compared with control subject (p=0.3) but is not statistically significant. In conclusion the Real time MAMA-PCR assay has several advantages over other SNP identification techniques such as rapidity, reliability, easily to perform in one working day and applicable in clinical molecular diagnostic laboratories, although sequencing remains the gold standard.

Development of a degenerated TaqMan real‐time Q‐PCR for detection of bacteria‐free DNA in dialysis fluid

Bacterial‐derived DNA fragments (BDNAs) have been shown to be present in a dialysis fluid, to pass through dialyzer membranes, and to induce interleukin 6 (IL‐6) in mononuclear cells. DNA fragments are thought to be derived from microorganisms inhabiting hemodialysis water and fluid. The primary aim of the present study was to develop two degenerated TaqMan real‐time quantitative‐PCR (Q‐PCR) for detection of a broad range of bacterial DNA that specifically detect 16S ribosomal DNA (rDNA) (862 and 241 bp) and evaluate the efficiency of the Bellco Selecta resin to capture the BDNAs in the dialysis fluid. For this purpose, we decided to compare measurements of unfragmented samples (9.8 × 105 Escherichia coli genome) with artificially fragmented DNA samples. We assessed two broad‐range real‐time PCR targeting bacterial 16S rRNA genes for detection of fragmented and unfragmented bacterial DNA in the dialytic fluid and demonstrated that Bellco Selecta resin is capable of retaining these types of bacterial DNA.

Evaluation of UL99 transcript as a target for antiviral treatment efficacy.

Human cytomegalovirus (HCMV) is a virus belonging to the Beta Herpes virus family. Its genome contains many different genes clustered in immediate early, early and late genes. This last cluster includes UL99, a late gene that encodes for a tegument protein called pp28. In immunocompetent patients, HCMV infection occurs asymptomatically, while its reactivation in immunocompromised patients can be a cause of pneumonia, retinitis and gastrointestinal diseases. To prevent or to contrast HCMV infection, several drugs (such as Ganciclovir, Acyclovir, Foscarnet) are available, and their efficiency is evaluated by HCMV DNA load monitoring, as also for antiviral resistance onset that may occur after the therapy. In this study is described the development of a Real Time PCR for the detection and quantification of UL99 transcript and the clearance of this target compared to HCMV DNA, both in vitro and in vivo on bronchoalveolar lavage samples.