Induced regulatory T cells are phenotypically unstable and do not protect mice from rapidly progressive glomerulonephritis.

Regulatory T (Treg) cells are a suppressive CD4+ T-cell subset. We generated induced Treg (iTreg) cells and explored their therapeutic potential in a murine model of rapidly progressive glomerulonephritis. Polyclonal naive CD4+ T cells were cultured in vitro with interleukin-2 (IL-2), transforming growth factor-ß1, all-trans-retinoic acid and monoclonal antibodies against interferon-γ and IL-4, generating Foxp3+ iTreg cells. To enhance their suppressive phenotype, iTreg cultures were modified with the addition of a monoclonal antibody against IL-12p40 or by using RORγt-/- CD4+ T cells. Induced Treg cells were transferred into models of delayed-type hypersensitivity and experimental glomerulonephritis. The iTreg cells exhibited comparable surface receptor expression and in vitro suppressive ability to natural Treg cells, but did not regulate antigen-specific delayed-type hypersensitivity or systemic inflammatory immune responses, losing Foxp3 expression in vivo. In glomerulonephritis, transferred iTreg cells did not prevent renal injury or modulate systemic T helper type 1 immune responses. Induced Treg cells cultured with anti-IL-12p40 had an enhanced suppressive phenotype in vitro and regulated dermal delayed-type hypersensitivity in vivo, but were not protective against renal injury, losing Foxp3 expression, especially in the transferred cells recruited to the kidney. Use of RORγt-/- CD4+ T cells or iTreg cells generated from sensitized CD4+ Foxp3- cells did not regulate renal or systemic inflammatory responses in vivo. In conclusion, iTreg cells suppress T-cell proliferation in vitro, but do not regulate experimental glomerulonephritis, being unstable in this inflammatory milieu in vivo.

Interleukin-17RA Promotes Humoral Responses and Glomerular Injury in Experimental Rapidly Progressive Glomerulonephritis.

BACKGROUND/AIMS: Interleukin (IL)-17A and IL-17F are proinflammatory cytokines, which signal through a receptor complex consisting of IL-17RA and IL-17RC subunits. We sought to define the role of IL-17RA expression by leukocytes and stromal cells in nephritogenic immunity and injury in experimental glomerulonephritis. METHODS: Glomerulonephritis was induced in wild-type and IL-17RA-deficient (IL-17RA-/-) mice by sheep anti-mouse glomerular basement membrane globulin. Renal injury and immune responses were assessed at day 21. Glomerulonephritis was induced in bone marrow (BM) chimeric mice, with either BM or tissue cell (TC) deficiency of IL-17RA. To assess humoral responses, WT and IL-17RA-/- mice were sensitized to sheep globulin and euthanized 10 days later. RESULTS: IL-17RA-/- mice had reduced glomerular crescent formation, neutrophils and macrophages compared to wild-type mice, while nephritic BM-TC+ mice developed less glomerular segmental necrosis. IL-17RA expression was required in both BM and TC for maximal systemic interferon-γ expression. Antigen-specific humoral immune responses were impaired in the absence of IL-17RA. Compared to BM+TC+ mice, glomerular IgG and C3 deposition was reduced in BM+TC- and BM-TC+ mice, respectively. Humoral immunity was also impaired in BM- and TC-deficient chimeras. BM+TC- mice had fewer B cells expressing CXCR5, while IL-17RA-/- mice had abnormal germinal centre development after immunization, with reduced follicular B cell and follicular helper T-cell CXCR5 expression, explaining the impaired humoral immunity. CONCLUSION: IL-17RA contributes to experimental glomerulonephritis, with IL-17RA expression on both leukocytes and stromal cells being required for the full expression of nephritogenic humoral immunity.

Development and function of Foxp3(+) regulatory T cells.

Regulatory T cells (Tregs) have been recognized as having a major role in maintaining peripheral tolerance and preventing and limiting autoimmune and chronic inflammatory diseases. Tregs derive from the thymus and also develop peripherally. In this review, we discuss recent progress in our understanding of the basic mechanisms involved in Treg development and function in protecting against autoimmunity in the periphery, including thymic selection, peripheral induction and the many mechanisms of Treg suppression. Specifically in kidney disease, Tregs have been shown to play a role in limiting injury and may potentially have a therapeutic role.

Regulatory T cells in immune-mediated renal disease.

Regulatory T cells (Tregs) are CD4+ T cells that can suppress immune responses by effector T cells, B cells and innate immune cells. This review discusses the role that Tregs play in murine models of immune-mediated renal diseases and acute kidney injury and in human autoimmune kidney disease (such as systemic lupus erythematosus, anti-glomerular basement membrane disease, anti-neutrophil cytoplasmic antibody-associated vasculitis). Current research suggests that Tregs may be reduced in number and/or have impaired regulatory function in these diseases. Tregs possess several mechanisms by which they can limit renal and systemic inflammatory immune responses. Potential therapeutic applications involving Tregs include in vivo induction of Tregs or inducing Tregs from naïve CD4+ T cells or expanding natural Tregs ex vivo, to use as a cellular therapy. At present, the optimal method of generating a phenotypically stable pool of Tregs with long-lasting suppressive effects is not established, but human studies in renal transplantation are underway exploring the therapeutic potential of Tregs as a cellular therapy, and if successful may have a role as a novel therapy in immune-mediated renal diseases.

Endogenous Toll-Like Receptor 9 Regulates AKI by Promoting Regulatory T Cell Recruitment.

Toll-like receptor 9 (TLR9) enhances proinflammatory responses, but whether it can act in a regulatory capacity remains to be established. In experimental murine AKI induced by cisplatin, Tlr9(-/-) mice developed enhanced renal injury and exhibited fewer intrarenal regulatory T cells (Tregs) compared with genetically intact mice. A series of reconstitution and depletion studies defined a role for TLR9 in maintaining Treg-mediated homeostasis in cisplatin-induced AKI. When Rag1(-/-) mice were reconstituted with nonregulatory CD25(-) splenocytes from wild-type (WT) or Tlr9(-/-) mice, AKI was similarly enhanced. However, when Rag1(-/-) mice were reconstituted with CD4(+)CD25(+) regulatory cells, WT CD4(+)CD25(+) cells were more renoprotective and localized to the kidney more efficiently than Tlr9(-/-) CD4(+)CD25(+) cells. In Treg-depleted Foxp3(DTR) mice, reconstitution with naive WT CD4(+)CD25(+) cells resulted in less severe AKI than did reconstitution with Tlr9(-/-) Tregs. Tlr9(-/-) mice were not deficient in CD4(+)CD25(+) cells, and WT and TLR9-deficient Tregs had similar suppressive function ex vivo. However, expression of adhesion molecules important in Treg trafficking was reduced on peripheral CD4(+)CD25(+) cells from Tlr9(-/-) mice. In conclusion, we identified a pathway by which TLR9 promotes renal Treg accumulation in AKI.

Targeting IL-17 and IL-23 in Immune Mediated Renal Disease.

T helper (Th) cells belong to the adaptive immune system and provide an effective and antigen-specific means of host protection. Th17 cells are a subset of Th cells, characterized by the production of the inflammatory cytokines interleukin (IL)-17A (IL-17A) and IL-17F, which bind to a receptor complex comprised of IL-17RA and IL-17RC subunits. Th17 cells combat extracellular and fungal infections, but have been implicated in autoimmune diseases. In many autoimmune conditions, the dysregulated immune response involves several parts of the immune system, including autoantibodies, B and T cells. Targeted biological therapies are appealing, as they may prevent unwanted side effects in patients. There is evolving evidence that Th17 cells are important in the kidney, mediating injury in response to vascular or chemical insults to the renal tubules, and in autoimmune diseases of the glomerulus, either through a specific attack on the glomerular basement membrane or as part of a generalized systemic inflammatory disease. Therapies targeting IL-17A, IL-12p40 and IL-17RA are being explored in clinical trials or are being utilized in clinical practice for the treatment of other IL-17 mediated diseases, such as psoriasis. This review explores the current evidence that IL-17A and Th17 cells may be pathogenic in immune kidney disease, including anti-glomerular basement membrane disease, antineutrophil cytoplasmic antibody associated vasculitis and lupus nephritis, as well as in acute kidney injury. It will discuss the place that biological agents against IL-17A, IL-12p40 and IL-17RA may have in the treatment of these conditions.

FMS-like tyrosine kinase 3 ligand treatment does not ameliorate experimental rapidly progressive glomerulonephritis.

Fms-like tyrosine kinase 3-ligand (FL) is a growth factor that may expand dendritic cell and regulatory T cell populations. We hypothesised that FL-induced regulatory T cells would protect mice from experimental rapidly progressive glomerulonephritis. To determine if FL was able to enhance regulatory T cell populations, C57BL/6 mice received 10 days of daily intraperitoneal injections of either FL or phosphate buffered saline. To induce accelerated autologous-phase anti-mouse glomerular basement membrane glomerulonephritis, mice were sensitized to sheep globulin 4 days prior to the induction of glomerulonephritis with sheep anti-mouse glomerular basement membrane globulin, and experiments ended 10 days later. FL was administered before, throughout and during the sensitization phase of this glomerulonephritis model. Renal disease and systemic immunity to the nephritogenic antigen were assessed. FL increased regulatory T cell and plasmacytoid dendritic cell proportions within spleen and lymph nodes. FL administration prior to glomerulonephritis did not protect mice from renal injury. When FL was given throughout the model, FL treated mice had reduced survival, with more interstitial neutrophils and glomerular CD11c+ cells than controls. Systemic immune responses showed increased IL-17A production from splenocytes, with more CD11c+ cells, but reduced plasmacytoid dendritic cell proportions in spleen and lymph nodes, despite increased regulatory T cell proportions. Under homeostatic conditions, FL expanded regulatory T cell and plasmacytoid dendritic cell populations, but FL enhanced systemic inflammatory responses and conventional dendritic cell populations when given during experimental glomerulonephritis, suggesting selective attempts to suppress pathogenic immunity by dendritic cell manipulation may be harmful.

HLA-DQA1 and PLCG2 Are Candidate Risk Loci for Childhood-Onset Steroid-Sensitive Nephrotic Syndrome.

Steroid-sensitive nephrotic syndrome (SSNS) accounts for >80% of cases of nephrotic syndrome in childhood. However, the etiology and pathogenesis of SSNS remain obscure. Hypothesizing that coding variation may underlie SSNS risk, we conducted an exome array association study of SSNS. We enrolled a discovery set of 363 persons (214 South Asian children with SSNS and 149 controls) and genotyped them using the Illumina HumanExome Beadchip. Four common single nucleotide polymorphisms (SNPs) in HLA-DQA1 and HLA-DQB1 (rs1129740, rs9273349, rs1071630, and rs1140343) were significantly associated with SSNS at or near the Bonferroni-adjusted P value for the number of single variants that were tested (odds ratio, 2.11; 95% confidence interval, 1.56 to 2.86; P=1.68×10(-6) (Fisher exact test). Two of these SNPs-the missense variants C34Y (rs1129740) and F41S (rs1071630) in HLA-DQA1-were replicated in an independent cohort of children of white European ancestry with SSNS (100 cases and ≤589 controls; P=1.42×10(-17)). In the rare variant gene set-based analysis, the best signal was found in PLCG2 (P=7.825×10(-5)). In conclusion, this exome array study identified HLA-DQA1 and PLCG2 missense coding variants as candidate loci for SSNS. The finding of a MHC class II locus underlying SSNS risk suggests a major role for immune response in the pathogenesis of SSNS.

Glomerulonephritis Induced by Heterologous Anti-GBM Globulin as a Planted Foreign Antigen.

The glomerulonephritides are diseases characterized by immune-mediated glomerular inflammation. Most severe and rapidly progressive forms of glomerulonephritis feature the participation of injurious leukocytes that localize to glomeruli. This unit describes classical models of rapidly progressive glomerulonephritis in mice, induced by injecting heterologous globulin (raised in sheep) that binds to the glomerular basement membrane. These models have been particularly useful in defining the participation of effector leukocytes in severe glomerular disease. In these models, injury typically occurs in two phases. In the initial, heterologous phase, injury is mediated by the globulin bound within the glomerulus acting as an antibody. The later, autologous phase of injury is mediated by the host's adaptive immunity to the heterologous globulin now functioning as a planted foreign antigen within glomeruli. As autologous phase injury is driven by immunity to sheep globulin, assessment of antigen-specific systemic immunity to sheep globulin is critical when using this model.

Antiepileptic medications increase osteoporosis risk in male fabry patients: bone mineral density in an Australian cohort.

BACKGROUND: Fabry disease (FD) is an inherited X-linked lysosomal storage disease with widespread clinical manifestations. Small prospective studies have shown increased osteopenia and osteoporosis in male FD patients. Limited information however exists about bone metabolism and osteoporosis risk factors within this group. We reviewed osteoporosis risk factors within our cohort. METHODS: A retrospective analysis of bone mineral density (BMD) results and fracture incidence in 44 patients (22 males and 22 females) was undertaken. Dual X-ray absorptiometry scans were performed at the lumbar spine, hip and femoral neck. The impact of risk factors including renal function, antiepileptic drug (AED), analgesia and vitamin D levels were assessed. RESULTS: Male FD patients had low T scores at all sites (spine -1.2 ± 1.06, hip -1.6 ± 0.9, femoral neck -2.23 ± 1.01). Female T scores showed more typical distribution (spine -0.07 ± 1.47, hip 0.02 ± 1.14, femoral neck -0.49 ± 1.31). A higher incidence of osteopenia and/or osteoporosis occurred in males versus females (spine 46.9% versus 31.8%, hip 75.5% versus 18.2% and femoral neck 86.4% versus 45.5%). Multiple regression analysis showed a 50.8% (p < 0.001) reduction in femoral neck BMD with AED usage, after adjustment for age, gender and renal function. Non-traumatic fractures occurred in 27.3% males over 205 patient-years versus 4.6% in females over 149 patient-years, p = 0.095. CONCLUSIONS: Low bone density was highly prevalent in male patients with increased incidence of non-traumatic fractures. AED usage significantly reduces BMD. Treatment to prevent BMD deterioration will depend on determining the bone turnover status.

Circulating precursor CCR7(lo)PD-1(hi) CXCR5⁺ CD4⁺ T cells indicate Tfh cell activity and promote antibody responses upon antigen reexposure.

Follicular B helper T (Tfh) cells support high affinity and long-term antibody responses. Here we found that within circulating CXCR5⁺ CD4⁺ T cells in humans and mice, the CCR7(lo)PD-1(hi) subset has a partial Tfh effector phenotype, whereas CCR7(hi)PD-1(lo) cells have a resting phenotype. The circulating CCR7(lo)PD-1(hi) subset was indicative of active Tfh differentiation in lymphoid organs and correlated with clinical indices in autoimmune diseases. Thus the CCR7(lo)PD-1(hi) subset provides a biomarker to monitor protective antibody responses during infection or vaccination and pathogenic antibody responses in autoimmune diseases. Differentiation of both CCR7(hi)PD-1(lo) and CCR7(lo)PD-1(hi) subsets required ICOS and BCL6, but not SAP, suggesting that circulating CXCR5⁺ helper T cells are primarily generated before germinal centers. Upon antigen reencounter, CCR7(lo)PD-1(hi) CXCR5⁺ precursors rapidly differentiate into mature Tfh cells to promote antibody responses. Therefore, circulating CCR7(lo)PD-1(hi) CXCR5⁺ CD4⁺ T cells are generated during active Tfh differentiation and represent a new mechanism of immunological early memory.

Carpal tunnel syndrome in fabry disease.

Carpal tunnel syndrome (CTS) is a common peripheral mononeuropathy affecting up to 4% of the general population, typically women in late middle age. The incidence in patients with Fabry disease (FD) is unclear, but may affect 25% of patients with this X-linked lysosomal storage disease. We report three cases of CTS in young Caucasian male patients with classical FD, who developed CTS symptoms with supportive nerve conduction study (NCS) findings. Two patients had bilateral CTS and two had evidence of concurrent ulnar nerve neuropathy on NCS, suggesting a systemic process contributed to nerve compression. All were receiving enzyme replacement therapy (ERT) and had a moderate burden of FD complications. It is possible that an increase in connective tissue in the intracarpal canal in FD patients may be incited by injury to fibroblasts, via either accumulation of globotriaosylceramide (GL3) or local ischaemia through endothelial injury. The former hypothesis may be a more plausible explanation for the development of CTS, as histology of the flexor retinaculae from our patients has demonstrated fibroblasts with characteristic vacuolation and excessive myxomatous stroma, despite endothelial clearance of GL3 in these patients receiving ERT. CTS should not be overlooked in FD patients and young patients presenting with CTS should be evaluated for an underlying systemic or genetic disorder. Surgical carpal tunnel decompression was effective in our patients, already troubled by long-standing acroparesthesia, in providing sustained relief of symptoms.

Effect of reduced agalsidase Beta dosage in fabry patients: the Australian experience.

BACKGROUND: In Australia, enzyme replacement therapy (ERT) for Fabry Disease (FD), both Agalsidase alfa (Replagal, Shire HGT) and beta (Fabrazyme, Genzyme), is funded and monitored through a specific government program. Agalsidase beta supply has been rationed by Genzyme since 2009 due to manufacturing issues. Consequently, the Australian Fabry Disease Advisory Committee has treated patients on Agalsidase beta at 50% of their usual dose from mid-2009, with a further reduction to 30% for some patients from late 2009. AIM: To determine the clinical effect of Agalsidase beta dose reduction in the Australian FD patient cohort. METHODS: A questionnaire assessing FD symptoms was administered to 40 patients on long-term ERT. Clinical data from The Fabry Registry for patients receiving Agalsidase alfa or beta, for at least 2 years prior to the time of enforced Agalsidase beta dose reduction, were reviewed. Disease burden and quality of life (QOL) were graded using the Disease Severity Scoring System, Mainz Severity Score Index, Brief Pain Inventory and Short Form 36 Health Survey at 2 years before dose reduction, at the time of dose reduction and at the most recent clinical review following dose reduction. RESULTS: Disease severity and QOL scores did not change between the ERT groups. Males on Agalsidase beta reported lower energy levels after dose reduction, while no change was reported by females on either product or by males on a stable dose of Agalsidase alfa. CONCLUSION: This study suggests that energy levels in male patients worsen after dose reduction of Agalsidase beta.

Microbiology and outcomes of peritonitis in Australian peritoneal dialysis patients.

We analyzed data from the Australia and New Zealand Dialysis and Transplant Registry for 1 October 2003 to 31 December 2008 with the aim of describing the nature of peritonitis, therapies, and outcomes in patients on peritoneal dialysis (PD) in Australia. At least 1 episode of PD was observed in 6639 patients. The overall peritonitis rate was 0.60 episodes per patient-year (95% confidence interval: 0.59 to 0.62 episodes), with 6229 peritonitis episodes occurring in 3136 patients. Of those episodes, 13% were culture-negative, and 11% were polymicrobial. Gram-positive organisms were isolated in 53.4% of single-organism peritonitis episodes, and gram-negative organisms, in 23.6%. Mycobacterial and fungal peritonitis episodes were rare. Initial antibiotic therapy for most peritonitis episodes used 2 agents (most commonly vancomycin and an aminoglycoside); in 77.2% of episodes, therapy was subsequently changed to a single agent. Tenckhoff catheter removal was required in 20.4% of cases at a median of 6 days, and catheter removal was more common in fungal, mycobacterial, and anaerobic infections, with a median time to removal of 4 - 5 days. Peritonitis was the cause of death in 2.6% of patients. Transfer to hemodialysis and hospitalization were frequent outcomes of peritonitis. There was no relationship between center size and peritonitis rate. The peritonitis rate in Australia between 2003 and 2008 was higher than that reported in many other countries, with a particularly higher rate of gram-negative peritonitis.

Elevated serum concentrations of troponin T in acute stroke: what do they mean?

Elevations in serum troponin T in acute stroke have been suggested as an early marker of a poor outcome. A prospective, case-control study was undertaken to define characteristics associated with elevations in troponin T concentrations. Consecutive admissions to the Royal Adelaide stroke unit were assessed. Stroke outcome was determined using the modified Rankin scale. Elevated serum troponin T was seen in 12/109 (11%) of patients with stroke and was associated with more severe stroke, larger lesion volume and a worse outcome. However, as a prognostic indicator, elevations in troponin T had lower sensitivity for predicting death or dependence at discharge than the National Institute of Health Stroke Scale. Troponin T levels are elevated in a significant proportion of patients with acute stroke, principally those with large infarcts affecting the territory supplied by the middle cerebral artery but their value as a prognostic indicator remains uncertain.