SARS-CoV-2 Humoral Immunity Persists Following Rituximab Therapy.

Long-term humoral immunity is mediated by short-lived plasma cells (replenished by memory B cells) and long-lived plasma cells. Their relative contributions are uncertain for immunity to SARS-CoV-2, especially given the widespread use of novel mRNA vaccines. Yet, this has far-reaching implications in terms of the need for regular booster doses in the general population and perhaps even revaccination in patients receiving B cell-depleting therapy. We aimed to characterise anti-SARS-CoV-2 antibody titres in patients receiving Rituximab following previous SARS-CoV-2 vaccination. We recruited 10 fully vaccinated patients (age: 16.9 ± 2.52 years) with childhood-onset nephrotic syndrome, not in relapse, receiving Rituximab for their steroid/calcineurin-inhibitor sparing effect. Antibodies to SARS-CoV-2 spike (S) and nucleocapsid (N) proteins were measured immediately prior to Rituximab and again ~6 months later, using the Roche Elecys® Anti-SARS-CoV-2 (S) assay. All ten patients were positive for anti-S antibodies prior to Rituximab, with six patients (60%) having titres above the upper limit of detection (>12,500 U/mL). Following Rituximab therapy, there was a reduction in anti-S titres (p = 0.043), but all patients remained positive for anti-S antibodies, with five patients (50%) continuing to have titres >12,500 U/mL. Six patients (60%) were positive for anti-N antibodies prior to Rituximab. Following Rituximab therapy, only three of these six patients remained positive for anti-N antibodies (p = 0.036 compared to anti-S seroreversion). Humoral immunity to SARS-CoV-2 is likely to be mediated in part by long-lived plasma cells.

Long-Term Efficacy and Safety of Repeated Rituximab to Maintain Remission in Idiopathic Childhood Nephrotic Syndrome: An International Study.

BACKGROUND: Long-term outcomes after multiple courses of rituximab among children with frequently relapsing, steroid-dependent nephrotic syndrome (FRSDNS) are unknown. METHODS: A retrospective cohort study at 16 pediatric nephrology centers from ten countries in Asia, Europe, and North America included children with FRSDNS who received two or more courses of rituximab. Primary outcomes were relapse-free survival and adverse events. RESULTS: A total of 346 children (age, 9.8 years; IQR, 6.6-13.5 years; 73% boys) received 1149 courses of rituximab. A total of 145, 83, 50, 28, 22, and 18 children received two, three, four, five, six, and seven or more courses, respectively. Median (IQR) follow-up was 5.9 (4.3-7.7) years. Relapse-free survival differed by treatment courses (clustered log-rank test P<0.001). Compared with the first course (10.0 months; 95% CI, 9.0 to 10.7 months), relapse-free period and relapse risk progressively improved after subsequent courses (12.0-16.0 months; HRadj, 0.03-0.13; 95% CI, 0.01 to 0.18; P<0.001). The duration of B-cell depletion remained similar with repeated treatments (6.1 months; 95% CI, 6.0 to 6.3 months). Adverse events were mostly mild; the most common adverse events were hypogammaglobulinemia (50.9%), infection (4.5%), and neutropenia (3.7%). Side effects did not increase with more treatment courses nor a higher cumulative dose. Only 78 of the 353 episodes of hypogammaglobulinemia were clinically significant. Younger age at presentation (2.8 versus 3.3 years; P=0.05), age at first rituximab treatment (8.0 versus 10.0 years; P=0.01), and history of steroid resistance (28% versus 18%; P=0.01) were associated with significant hypogammaglobulinemia. All 53 infective episodes resolved, except for one patient with hepatitis B infection and another with EBV infection. There were 42 episodes of neutropenia, associated with history of steroid resistance (30% versus 20%; P=0.04). Upon last follow-up, 332 children (96%) had normal kidney function. CONCLUSIONS: Children receiving repeated courses of rituximab for FRSDNS experience an improving clinical response. Side effects appear acceptable, but significant complications can occur. These findings support repeated rituximab use in FRSDNS.

Multicenter study on the genetics of glomerular diseases among southeast and south Asians: Deciphering Diversities - Renal Asian Genetics Network (DRAGoN).

Multinational studies have reported monogenic etiologies in 25%-30% of children with steroid-resistant nephrotic syndrome. Such large studies are lacking in Asia. We established Deciphering Diversities: Renal Asian Genetics Network (DRAGoN) and aimed to describe the genetic and clinical spectrums in Asians. We prospectively studied a cohort of 183 probands with suspected genetic glomerulopathies from South and Southeast Asia, of whom 17% had positive family history. Using multi-gene panel sequencing, we detected pathogenic variants in 26 (14%) probands, of whom one-third had COL4A4 or COL4A5 variants (n = 9, 5%). Of those with COL4A5 defects, only 25% had features suggestive of Alport syndrome. Besides traditional predictors for genetic disease (positive family history and extrarenal malformations), we identified novel predictors, namely older age (6.2 vs. 2.4 years; p = 0.001), hematuria (OR 5.6; 95% CI 2.1-14.8; p < 0.001), and proteinuria in the absence of nephrotic syndrome (OR 4.6; 95% CI 1.8-11.8; p = 0.001) at first manifestation. Among patients who first presented with proteinuria without nephrotic syndrome, the genetic diagnostic rates were >60% when a second risk factor (positive family history or extrarenal manifestation) co-existed. The genetic spectrum of glomerulopathies appears different in Asia. Collagen IV genes may be included in sequencing panels even when suggestive clinical features are absent.

Low regulatory T-cells: A distinct immunological subgroup in minimal change nephrotic syndrome with early relapse following rituximab therapy.

Rituximab is an important second line therapy in difficult nephrotic syndrome (NS), especially given toxicity of long-term glucocorticoid or calcineurin inhibitor (CNI) use. However, clinical response to rituximab is heterogenous. We hypothesized that this was underpinned by immunological differences amongst patients with NS. We recruited a cohort of 18 subjects with glucocorticoid-dependent or glucocorticoid-resistant childhood-onset minimal change NS who received rituximab either due to CNI nephrotoxicity, or due to persistent glucocorticoid toxicity with inadequate response to cyclophosphamide or CNIs. Immunological subsets, T-cell activation assays and plasma cytokines were measured at baseline and 6-months post-rituximab. Time to relapse was bifurcated: 56% relapsed within one year ("early relapse"), while the other 44% entered remission mainly lasting ≥3 years ("sustained remission"). At baseline, early relapse compared to sustained remission group had lower regulatory T-cells (Tregs) [2.94 (2.25, 3.33)% vs 6.48 (5.08, 7.24)%, P<0.001], PMA-stimulated IL-2 [0.03 (0, 1.85)% vs 4.78 (0.90, 9.18)%, P=0.014] and IFNγ [2.22 (0.18, 6.89)% vs 9.47 (2.72, 17.0)%, P=0.035] levels. Lower baseline Treg strongly predicted early relapse (ROC-AUC 0.99, 95% CI 0.97-1.00, P<0.001). There were no differences in baseline plasma cytokine levels. Following rituximab, there was significant downregulation of Th2 cytokines in sustained remission group (P=0.038). In particular, IL-13 showed a significant decrease in sustained remission group [-0.56 (-0.64, -0.35)pg/ml, P=0.007)], but not in the early relapse group. In conclusion, early relapse following rituximab is associated with baseline reductions in Treg and T-cell hyporesponsiveness, which suggest chronic T-cell activation and may be useful predictive biomarkers. Sustained remission, on the other hand, is associated with downregulation of Th2 cytokines following rituximab.

IL-13-driven alterations in hepatic cholesterol handling contributes to hypercholesterolemia in a rat model of minimal change disease.

Circulating factors have been implicated in the pathogenesis of minimal change disease (MCD), and may have direct effects on cholesterol metabolism. This study investigated the pathogenesis of hypercholesterolemia in an IL-13 overexpression rat model of MCD prior to the onset of proteinuria, so as to establish the direct contribution of IL-13, especially with regard to hepatic cholesterol handling. In this model of MCD, the temporal relationship between hypercholesterolemia and proteinuria was first identified. Plasma proprotein convertase subtilisin/kexin type 9 (Pcsk9) and liver ATP-binding cassette sub-family G member 5 (Abcg5) were measured using ELISA. Liver Ldlr and liver X receptor alpha (Lxra) were quantified with Western blot. Abcg5-mediated cholesterol efflux in IL-13-stimulated rat primary hepatocytes was measured using taurocholate as cholesterol acceptor. The role of Lxra was validated using a luciferase assay in Lxre-luciferase-transfected IL-13-stimulated hepatocytes. IL-13-transfected rats developed hypercholesterolemia prior to proteinuria, with 35% of rats hypercholesterolemic but only 11% proteinuric by Day 20 (P = 0.04). These pre-proteinuric hypercholesterolemic rats showed elevations in total and LDL-cholesterol, but not hypertriglyceridemia or hepatic steatosis. The hypercholesterolemia was associated with increased hepatic Pcsk9 synthesis and enhanced circulating Pcsk9 levels, which correlated strongly with plasma total cholesterol (r = 0.73, P<0.001). The hypercholesterolemia was also contributed by decreased Abcg5 expression and activity, due to reduced Lxra expression. Lxra expression correlated with plasma total cholesterol levels (r = -0.52, P = 0.01), and overexpression of pLxra in rat hepatocytes abrogated the IL-13-mediated down-regulation of Lxre-driven gene expression. In conclusion, we have shown that IL-13 induced changes in hepatic cholesterol handling in a cytokine-induced rat model of MCD, resulting in hypercholesterolemia which can precede the onset of proteinuria.

Persistent Dengue Infection in an Immunosuppressed Patient Reveals the Roles of Humoral and Cellular Immune Responses in Virus Clearance.

Detailed understanding of the roles of humoral and cellular immune responses in sterilizing dengue virus (DENV) infection in humans is required to inform effective vaccine development. We report an unusual case of persistent DENV infection in a lymphopenic renal transplant recipient who was therapeutically immunosuppressed to prevent organ rejection. Following resolution of symptomatic dengue, this patient remained positive for DENV3 RNA in the blood for 4 months and viruric up to 9 months post-infection despite demonstrable levels of serum neutralizing antibodies throughout this period. Full resolution of DENV infection instead coincided with recovery of CD8+ T cell counts during reversal from lymphopenia. Taken collectively, our observations suggest a role for cellular immunity in sterilizing DENV infection in humans. Any dengue vaccine should thus be able to induce both humoral and cellular immunity that respectively prevent symptomatic infection and enable effective viral clearance.

MeSsAGe risk score: tool for renal biopsy decision in steroid-dependent nephrotic syndrome.

BACKGROUND: A lack of consensus exists as to the timing of kidney biopsy in children with steroid-dependent nephrotic syndrome (SDNS) where minimal change disease (MCD) predominates. This study aimed at examining the applicability of a biomarker-assisted risk score model to select SDNS patients at high risk of focal segmental glomerulosclerosis (FSGS) for biopsy. METHODS: Fifty-five patients with SDNS and biopsy-proven MCD (n = 40) or FSGS (n = 15) were studied. A risk score model was developed with variables consisting of age, sex, eGFR, suPAR levels and percentage of CD8+ memory T cells. Following multivariate regression analysis, total risk score was calculated as sum of the products of odds ratios and corresponding variables. Predictive cut-off point was determined using receiver operator characteristics (ROC) curve analysis. RESULTS: Plasma suPAR levels in FSGS patients were significantly higher, while percentage of CD45RO+CD8+CD3+ was significantly lower than in MCD patients and controls. ROC analysis suggests the risk score model with threshold score of 16.7 (AUC 0.84, 95% CI 0.72-0.96) was a good predictor of FSGS on biopsy. The 100% PPV cut-off was >24.0, while the 100% NPV was <13.3. CONCLUSION: A suPAR and CD8+ memory T cell percentage-based risk score model was developed to stratify SDNS patients for biopsy and for predicting FSGS.

Novel role of Vav1-Rac1 pathway in actin cytoskeleton regulation in interleukin-13-induced minimal change-like nephropathy.

Our established interleukin-13 (IL-13) overexpression rat model of minimal change-like nephropathy provided a platform to study the molecular signalling pathways in T-helper 2 (Th2) cytokine associated minimal change nephrotic syndrome (MCNS). We hypothesized that IL-13 may act directly on podocytes, causing podocyte foot process effacement and hence proteinuria in our rat model of minimal change-like nephropathy. The present study aimed firstly to delineate the glomerular 'gene signature' associated with IL-13-mediated dysregulation of podocyte-related proteins, and subsequently to investigate the role of the differentially regulated genes (DEGs) in IL-13-mediated podocyte injury. Glomerular transcriptional profile of IL-13-overexpressed rats showed characteristic features of podocyte injury with 87% of podocyte-related genes being significantly down-regulated. Gene expression of Vav1 was shown to be highly up-regulated in the glomeruli of IL-13-overexpressed rats and pathway analysis of the DEGs suggested a possible novel role of Vav1 in podocyte cytoskeleton remodelling. Immunofluorescence examination demonstrated glomerular expression of Vav1 in rats which co-localized with synaptopodin, confirming podocyte expression. However, positive staining for the phosphorylated form of Vav1 (p-Vav1) was only seen in IL-13-overexpressed rats. Moreover, in vitro IL-13 stimulation of human podocytes resulted in phosphorylation of Vav1. This was associated with Rac1 activation and actin cytoskeleton rearrangement, which was abrogated in Vav1 knockdown podocytes. In conclusion, we have demonstrated the role of Vav1-Rac1 pathway characterized by phosphorylation of Vav1, activation of Rac1 and the subsequent actin cytoskeleton rearrangement in IL-13-induced podocyte injury, possibly explaining the podocyte foot process effacement seen in our IL-13 overexpression rat model.

T Lymphocyte Activation Markers as Predictors of Responsiveness to Rituximab among Patients with FSGS.

BACKGROUND AND OBJECTIVES: Rituximab is used with variable success in difficult FSGS. Because B cell depletion significantly affects T cell function, we characterized T cell subsets in patients with FSGS to determine if an immunologic signature predictive of favorable response to rituximab could be identified. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Twenty-two consecutive patients with FSGS (median age =14.4 years old; range =6.2-25.0 years old) and age of onset of nephrotic syndrome 1-18 years old receiving rituximab for clinical indications between October of 2009 and February of 2014 were studied. Indications for rituximab were lack of sustained remission despite calcineurin inhibitors (CNIs) and mycophenolate in steroid-resistant patients and lack of steroid-sparing effect with cyclophosphamide and CNI or CNI toxicity in steroid-dependent patients. Exclusion criteria were infantile onset, known genetic mutations, and secondary causes. Rituximab (375 mg/m(2)) was given fortnightly up to a maximum of four doses. Immunologic subset monitoring was performed at baseline and regular intervals until relapse. Median follow-up duration postrituximab was 26.7 months (range =6.5-66.5 months). Baseline immunologic subsets were examined for association with rituximab response defined as resolution of proteinuria with discontinuation of prednisolone and CNI 3 months postrituximab. RESULTS: Twelve patients (54.5%) responded to rituximab. Mitogen-stimulated CD154(+)CD4(+)CD3(+) subset before rituximab was significantly lower in FSGS responders compared with nonresponders (54.9%±28.1% versus 78.9%±16.4%; P=0.03). IFN-γ(+)CD3(+) and IL-2(+)CD3(+) were similarly decreased in responders compared with nonresponders (0.6%±0.8% versus 7.5%±6.1%; P=0.003 and 0.2%±0.5% versus 4.0%±4.7%; P<0.01, respectively). Recovery of all three activation subsets occurred 6 months postrituximab treatment (CD154(+)CD4(+)CD3(+), 74.8%±17.2%; IFN-γ(+)CD3(+), 7.1%±7.7%; and IL-2(+)CD3(+), 7.9%±10.9%; P<0.01). Receiver-operating characteristic analysis using optimal cutoff values showed that activated CD154(+)CD4(+)CD3(+) <83.3% (area under the curve [AUC], 0.81; 95% confidence interval [95% CI], 0.61 to 1.00), IFN-γ(+)CD3(+)<2.5% (AUC, 0.90; 95% CI, 0.75 to 1.00), and IL-2(+)CD3(+)<0.3% (AUC, 0.78; 95% CI, 0.57 to 0.98) were good predictors of rituximab response. CONCLUSIONS: We have identified prognostic markers that define a subset of patients with FSGS bearing an immunologic signature representing hyporesponsiveness to T cell stimulation and therefore, who respond better to rituximab.