A novel TaqMAMA assay for allelic discrimination of immunoproteasome subunit PSMB8 in pediatric patients.

BACKGROUND: A more recent improvement in MAMA was made when the allele selectivity of MAMA primers was combined with the 5' fluorogenic exonuclease (TaqMan) assay. This strategy referred to as TaqMAMA. METHODS: A SNP rs9357155 at the PSMB8 locus (GenBank access number NM_148919.3.) on chromosome 6 showing a C/T transition in position 32842071, has been chosen as model in this study. RESULTS: Tested assays have provided to be linear over two log10 of magnitude and have efficiencies close to the average of 80.3%. Average interference limit values of TaqMAMA have been considered around the 0.6%. Coefficient of variation (CV) for PSMB8c and t were approximatively 1%. CONCLUSIONS: Prevalidation procedure described in this paper can be applied, not only to the allele quantification, but also to many other real time PCR applications.

HERV-K and W expression in peripheral mononuclear cells of children with Henoch-Schönlein purpura and relation with TLR activation.

BACKGROUND: HERVs expression seems impaired in several diseases, ranging from autoimmune to neoplastic disorders. However, various stimuli may re-activate HERVs transcription. Henoch-Schönlein purpura is the most common cause of vasculitis in children. The role of microbial antigens in the pathogenesis of HSP remains elusive. Toll-like receptors (TLRs) are the first line of defense for the host to initiate an immune and inflammatory response. We aimed to investigate HERV, K and W expression in peripheral mononuclear cells of children with HSP and relation with TLRs activation. METHODS: The study enrolled 63 children affected by HSP. We used RT-PCR to detect GAPDH in the peripheral blood mononuclear cells samples to normalize the data. Subsequently, the viral pol gene HERVs and TLRs transcripts in the same samples were assessed. RESULTS: HERV K and W was expressed in all samples analyzed but the level of expression was much lower in the HSP that in HC P=0.0006 and P=0.0004 for HERV-K and -W respectively. TLR2 was hyper-expressed in 39 vs. 63 (62%) of the HSP, TLR3 in 23 vs. 63 (42%), TLR4 in 42 vs. 63 (66%) and TLR9 in 32 vs. 63 (52%). The different expression was statistically significant only for TLR4 (P=0.0276) no for TLR2,3 and 9 (P=0.1251; 0.3964 and 0.1882 respectively). Spearman's test demonstrated no correlation between the low expression of HERV-K and HERV-W and high expression of TLRs. CONCLUSIONS: The results of the present study demonstrated that mRNA expression of HERV-K and -W and TLRs did not directly correlated.

The switch from proteasome to immunoproteasome is increased in circulating cells of patients with fast progressive immunoglobulin A nephropathy and associated with defective CD46 expression.

The proteasome to immunoproteasome (iPS) switch consists of ß1, ß2 and ß5 subunit replacement by low molecular weight protein 2 (LMP2), LMP7 and multicatalytic endopeptidase-like complex-1 (MECL1) subunits, resulting in a more efficient peptide preparation for major histocompatibility complex 1 (MHC-I) presentation. It is activated by toll-like receptor (TLR) agonists and interferons and may also be influenced by genetic variation. In a previous study we found an iPS upregulation in peripheral cells of patients with immunoglobulin A nephropathy (IgAN). We aimed to investigate in 157 IgAN patients enrolled through the multinational Validation Study of the Oxford Classification of IgAN (VALIGA) study the relationships between iPS switch and estimated glomerular filtration rate (eGFR) modifications from renal biopsy to sampling. Patients had a previous long follow-up (6.4 years in median) that allowed an accurate calculation of their slope of renal function decline. We also evaluated the effects of the PSMB8/PSMB9 locus (rs9357155) associated with IgAN in genome-wide association studies and the expression of messenger RNAs (mRNAs) encoding for TLRs and CD46, a C3 convertase inhibitor, acting also on T-regulatory cell promotion, found to have reduced expression in progressive IgAN. We detected an upregulation of LMP7/ß5 and LMP2/ß1 switches. We observed no genetic effect of rs9357155. TLR4 and TLR2 mRNAs were found to be significantly associated with iPS switches, particularly TLR4 and LMP7/ß5 (P < 0.0001). The LMP7/ß5 switch was significantly associated with the rate of eGFR loss (P = 0.026), but not with eGFR at biopsy. Fast progressors (defined as the loss of eGFR >75th centile, i.e. -1.91 mL/min/1.73 m2/year) were characterized by significantly elevated LMP7/ß5 mRNA (P = 0.04) and low CD46 mRNA expression (P < 0.01). A multivariate logistic regression model, categorizing patients by different levels of kidney disease progression, showed a high prediction value for the combination of high LMP7/ß5 and low CD46 expression.

Standard and immunoproteasome expression in pediatric tonsillectomy patients.

BACKGROUND: Pro-inflammatory cytokines including TNF-α and IFN-γ, play an important role during the recurrent tonsillitis illness and proteasome and immunoproteasome stimulation. METHODS: mRNA expression analysis of proteasome and immunoproteasome subunits was performed by PBMC whole blood isolation from children tonsillectomy patients. Total RNA was extracted from PBMC and retro-transcribed in cDNA. A real-time PCR with TaqMan probe was then performed. RESULTS: PBMC of children with tonsillectomy showed, a significantly increased expression of proteasome constitutive subunit ß2 encoding (1.52±0.61 vs. 0.88±0.5, P<0.0001) compared to healthy controls (HC). The same results were observed for immunoproteasome inducible subunit LMP2 (2.55±1.95 vs. 0.73±0.49, P<0.001), LMP7 (1.94±1.18 vs. 0.79±0.41, P<0.001), MECL1 (1.97±1.20 vs. 0.79±0.41, P<0.001). No differences were observed for proteasome subunit ß1. Conversely a significantly decreased expression of proteasome constitutive subunit ß5 encoding mRNA (0.96±0.39 vs. 1.28±0.65, P=0.0291) was observed. CONCLUSIONS: In children with recurrent tonsillitis was observed an increased expression of ß2, the corresponding iPS, ß1i and ß5i compared to healthy controls. Contrariwise ß5 subunit mRNA expression was significantly decreased.

Defective gene expression of the membrane complement inhibitor CD46 in patients with progressive immunoglobulin A nephropathy.

BACKGROUND: Complement is thought to play a role in immunoglobulin A nephropathy (IgAN), though the activating mechanisms are unknown. This study focused on the gene expression of CD46 and CD55, two key molecules for regulating C3 convertase activity of lectin and alternative complement pathways at a cellular level. METHODS: The transcriptional expression in peripheral white blood cells (WBCs) of CD46 and CD55 was investigated in 157 patients enrolled by the Validation of the Oxford Classification of IgAN group, looking for correlations with clinical and pathology features and estimated glomerular filtration rate (eGFR) modifications from renal biopsy to sampling. Patients had a previous median follow-up of 6.4 (interquartile range 2.8-10.7) years and were divided into progressors and non-progressors according to the median value of their velocity of loss of renal function per year (-0.41 mL/min/1.73 m2/year). RESULTS: CD46 and CD55 messenger RNA (mRNA) expression in WBCs was not correlated with eGFR values or proteinuria at sampling. CD46 mRNA was significantly correlated with eGFR decline rate as a continuous outcome variable (P = 0.014). A significant difference was found in CD46 gene expression between progressors and non-progressors (P = 0.013). CD46 and CD55 mRNA levels were significantly correlated (P < 0.01), although no difference between progressors and non-progressors was found for CD55 mRNA values. The prediction of progression was increased when CD46 and CD55 mRNA expressions were added to clinical data at renal biopsy (eGFR, proteinuria and mean arterial blood pressure) and Oxford MEST-C (mesangial hypercellularity, endocapillary hypercellularity, segmental glomerulosclerosis, tubular atrophy/interstitial fibrosis, presence of any crescents) score. CONCLUSIONS: Patients with progressive IgAN showed lower expression of mRNA encoding for the complement inhibitory protein CD46, which may implicate a defective regulation of C3 convertase with uncontrolled complement activation.

Toll-like receptors are essential for the control of endogenous retrovirus expression in Idiopathic Nephrotic Syndrome.

BACKGROUND: Idiopathic nephrotic syndrome (INS) in children is a continuum of clinical disorders characterized by severe proteinuria, dyslipidemia, hypoalbuminemia, and hypercoagulability. It has been hypothesized that toll-like receptors (TLRs) in peripheral blood mononuclear cell might be involved in INS disease. Infections appear to be involved in the onset of INS. The aim of this study was to investigate the expression and function of TLRs in PBMC of INS patients and the relation with two human endogenous retrovirus (HERV), K and W expression. METHODS: The study enrolled 37 children at the exordium and without treatments of minimal-change disease (MCD), the most common single form of nephrotic syndrome (INS) and 20 healthy controls (HC). Relative quantification of target genes expression in patients compared with normal samples was performed with the ΔΔCt method. RESULTS: HERV K and W were expressed in all samples analyzed but the level of expression was much lower in the INS that in HC P<0.0001 and P=0.0002 for HERV-K and -W, respectively. TLR2 was hyperexpressed in 10 vs. 17 (58.8%) of the INS, TLR3 in 3 vs. 17 (17.6%), TLR4 in 12 vs. 17 (70.6%) and TLR9 in 9 vs. 17 (52.9%) (Figure 2). The different expression were statistically significance in the case of TLR2, 3 and 4 (P=0.0183, 0.0218 and 0.0034, respectively) no for TLR9 (P=0.2010). Statistical analysis demonstrated a significance correlation between the low expression of HERV-K and HERV-W and high expression of TLRs. We obtain a statistically significance P<0.0001 considering all TLRs expression in comparison with HERV expression. CONCLUSIONS: We described a down regulation of pol gene of HERV-W and HERV-K mediated by TLRs.

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 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 donor's 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.

Can tonsillectomy modify the innate and adaptive immunity pathways involved in IgA nephropathy?

The benefits of tonsillectomy in IgA nephropathy (IgAN) are still debated. Tonsillectomy may remove pathogen sources and reduce the mucosal associated lymphoid tissue (MALT), limiting degalactosylated IgA1 (deGal-IgA1) production, which is considered to be the initiating pathogenetic event leading to IgA glomerular deposition. In the European network VALIGA, 62/1147 IgAN patients underwent tonsillectomy (TxIgAN). In a cross-sectional study 15 of these patients were tested and compared to 45 non-tonsillectomized IgAN (no-TxIgAN) and healthy controls (HC) regarding levels of deGal-IgA1, and markers of innate immunity and oxidative stress, including toll-like receptors (TLR)2, 3, 4 and 9 mRNAs, proteasome (PS) and immunoproteasome (iPS) mRNAs in peripheral blood mononuclear cells (PBMC), and advanced oxidation protein products (AOPP). Levels of deGal-IgA1 were lower in TxIgAN than in no-TxIgAN (p = 0.015), but higher than in HC (p = 0.003). TLR mRNAs were more expressed in TxIgAN than in HC (TLR4, p = 0.021; TLR9, p = 0.027), and higher in TxIgAN than in no-TxIgAN (p ≤ 0.001 for TLR2, 4, 9). A switch from PS to iPS was detected in PBMC of TxIgAN in comparison to HC and it was higher than in no-TxIgAN [large multifunctional peptidase (LMP)2/ß1, p = 0.039; LPM7/ß5, p < 0.0001]. The levels of AOPP were significantly higher in TxIgAN than HC (p < 0.001) and no-TxIgAN (p = 0.033). In conclusion, the activation of innate immunity via TLRs and ubiquitin-proteasome pathways and the pro-oxidative milieu were not affected by tonsillectomy, even though the levels of aberrantly galactosylated IgA1 were lower in patients with IgAN who had tonsillectomy. The residual hyperactivation of innate immunity in tonsillectomized patients may result from extra-tonsillar MALT.

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.

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

BACKGROUND: Henoch-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. METHODS: This 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). RESULTS: The expression of mRNAs encoding for TLR4 in both HSP and pIgAN was higher than in controls (HC) and in both diseases FoxP3mRNA and TGF-ß1mRNA expression was significantly lower than in HC. A switch from PS to iPS (LMP2/ß1) was detected only in PBMC of HSP and it correlated with the level of TLR2mRNA, which was selectively increased only in children with HSP. CONCLUSION: Children 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.

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.

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.

Effect of glucose degradation products, glucose-containing dialysate and icodextrin on AQP1 and eNOS expression in cultured endothelial cells.

BACKGROUND: Aquaporin-1 (AQP1) and endothelial NO synthase (eNOS) expression on the endothelium of peritoneal vessels modulates ultrafiltration during peritoneal dialysis (PD) by different mechanisms. Protracted eNOS activation may, in the long term, be deleterious for peritoneal functioning. We aimed at examining the effect of peritoneal dialysis solutions (PDSs) and glucose degradation products (GDPs) on the expression of AQP1 and eNOS in cultured endothelial cells. METHODS: An endothelial cell line (t End.1) was incubated for 24 hours with 2 GDPs (2-furaldehyde [Fur] or methylglyoxal [MGly] at concentrations found in traditional PDSs) or with a different PDS (1.36% glucose, 3.86% glucose and 7.5% icodextrin) in Transwell culture devices. AQP1 and eNOS gene expression were detected by reverse transcriptase polymerase chain reaction. RESULTS: Fur and MGly at concentrations reported in traditional PDSs (Fur 0.8 microM; MGly 35 microM) significantly up-regulated eNOS mRNA and tended to down-regulate AQP1 mRNA in cultured endothelial cells. Glucose-based PDS as well as icodextrin PDS significantly up-regulated basal AQP1 and eNOS mRNA. The effect of 3.86% glucose PDS on AQP1 was significantly higher than that of icodextrin. CONCLUSIONS: In cultured endothelial cells, all PDSs triggered both AQP1 and eNOS in a likely feedback mechanism. GDPs stimulated e-NOS expression only, and this effect might favor PD ultrafiltration failure in the long term.