The Cellular Location of Self-antigen Determines the Positive and Negative Selection of Autoreactive B Cells

Systemic autoimmune disease is frequently characterized by the production of autoantibodies against widely expressed intracellular self-antigens, whereas B cell tolerance to ubiquitous and highly expressed extracellular antigens is strictly enforced. To test for differences in the B cell response to intracellular and extracellular self-antigens, we sequestered a tolerogenic cell surface antigen intracellularly by addition of a two amino acid endoplasmic reticulum (ER) retention signal. In contrast to cell surface antigen, which causes the deletion of autoreactive B cells, the intracellularly sequestered self-antigen failed to induce B cell tolerance and was instead autoimmunogenic. The intracellular antigen positively selected antigen-binding B cells to differentiate into B1 cells and induced large numbers of IgM autoantibody-secreting plasma cells in a T-independent manner. By analyzing the impact of differences in subcellular distribution independently from other variables, such as B cell receptor affinity, antigen type, or tissue distribution, we have established that intracellular localization of autoantigen predisposes for autoantibody production. These findings help explain why intracellular antigens are targeted in systemic autoimmune diseases

Randomized trial on the effect of an oral spleen tyrosine kinase inhibitor in the treatment of IgA nephropathy

Introduction We reported increased spleen tyrosine kinase (SYK) expression in kidney biopsies of patients with IgA nephropathy (IgAN) and that inhibition of SYK reduces inflammatory cytokines production from IgA stimulated mesangial cells. Methods This study was a double-blind, randomised, placebo-controlled phase 2 trial of fostamatinib (an oral SYK inhibitor) in 76 patients with IgAN. Patients were randomised to receive placebo, fostamatinib at 100 mg or 150 mg twice daily for 24 weeks on top of maximum tolerated dose of renin-angiotensin system inhibitors (RASi). The primary end point was reduction of proteinuria. Secondary endpoints included change from baseline in eGFR and kidney histology. Results While we could not detect significant reduction in proteinuria with fostamatinib overall, in a pre-determined subgroup analysis, there was a trend for dose-dependent reduction in median proteinuria (from baseline to 24 weeks by 14%, 27% and 36% in the placebo, fostamatinib 100 mg and 150 mg groups respectively) in patients with baseline urinary protein to creatinine ratios (UPCR) more than 1000 mg/g. Kidney function (eGFR) remained stable in all groups. Fostamatinib was well tolerated. Side effects included diarrhea, hypertension and increased liver enzymes. Thirty-nine patients underwent repeat biopsy showing reductions in SYK staining associated with therapy at low dose (-1.5 v 1.7 SYK+ cells/glomerulus in the placebo group, p<0.05). Conclusions There was a trend towards reduction in proteinuria with fostamatinib in a predefined analysis of high risk patients with IgAN despite maximal care, as defined by baseline UPCR greater than 1000 mg/g. Further study may be warranted

The Oxford IgA nephropathy clinicopathological classification is valid for children as well as adults

To study the predictive value of biopsy lesions in IgA nephropathy in a range of patient ages we retrospectively analyzed the cohort that was used to derive a new classification system for IgA nephropathy. A total of 206 adults and 59 children with proteinuria over 0.5 g/24 h/ 1.73 m2and an eGFR of stage-3 or better were followed for a median of 69 months. At the time of biopsy, compared with adults children had a more frequent history of macroscopic hematuria, lower adjusted blood pressure, and higher eGFR but similar proteinuria. Although their outcome was similar to that of adults, children had received more immunosuppressants and achieved a lower follow-up proteinuria. Renal biopsies were scored for variables identified by an iterative process as reproducible and independent of other lesions. Compared with adults, children had significantly more mesangial and endocapillary hypercellularity, and less segmental glomerulosclerosis and tubulointerstitial damage, the four variables previously identified to predict outcome independent of clinical assessment. Despite these differences, our study found that the cross-sectional correlation between pathology and proteinuria was similar in adults and children. The predictive value of each specific lesion on the rate of decline of renal function or renal survival in IgA nephropathy was not different between children and adults

The Oxford classification of IgA nephropathy: pathology definitions, correlations, and reproducibility

Pathological classifications in current use for the assessment of glomerular disease have been typically opinion-based and built on the expert assumptions of renal pathologists about lesions historically thought to be relevant to prognosis. Here we develop a unique approach for the pathological classification of a glomerular disease, IgA nephropathy, in which renal pathologists first undertook extensive iterative work to define pathologic variables with acceptable inter-observer reproducibility. Where groups of such features closely correlated, variables were further selected on the basis of least susceptibility to sampling error and ease of scoring in routine practice. This process identified six pathologic variables that could then be used to interrogate prognostic significance independent of the clinical data in IgA nephropathy (described in the accompanying article). These variables were (1) mesangial cellularity score; percentage of glomeruli showing (2) segmental sclerosis, (3) endocapillary hypercellularity, or (4) cellular/ fibrocellular crescents; (5) percentage of interstitial fibrosis/ tubular atrophy; and finally (6) arteriosclerosis score. Results for interobserver reproducibility of individual pathological features are likely applicable to other glomerulonephritides, but it is not known if the correlations between variables depend on the specific type of glomerular pathobiology. Variables identified in this study withstood rigorous pathology review and statistical testing and we recommend that they become a necessary part of pathology reports for IgA nephropathy. Our methodology, translating a strong evidence-based dataset into a working format, is a model for developing classifications of other types of renal disease

Tackling stain variability using CycleGAN-based stain augmentation

Background: Considerable inter- and intra-laboratory stain variability exists in pathology, representing a challenge in development and application of deep learning (DL) approaches. Since tackling all sources of stain variability with manual annotation is not feasible, we here investigated and compared unsupervised DL approaches to reduce the consequences of stain variability in kidney pathology. Methods: We aimed to improve the applicability of a pretrained DL segmentation model to 3 external multi-centric cohorts with large stain variability. In contrast to the traditional approach of training generative adversarial networks (GAN) for stain normalization, we here propose to tackle stain variability by data augmentation. We augment the training data of the pretrained model by the stain variability using CycleGANs and then retrain the model on the stain-augmented dataset. We compared the performance of i/ the unmodified pretrained segmentation model with ii/ CycleGAN-based stain normalization, iii/ a feature-preserving modification to ii/ for improved normalization, and iv/ the proposed stain-augmented model. Results: The proposed stain-augmented model showed highest mean segmentation accuracy in all external cohorts and maintained comparable performance on the training cohort. However, the increase in performance was only marginal compared to the pretrained model. CycleGAN-based stain normalization suffered from encoded imperceptible information into the normalizations that confused the pretrained model and thus resulted in slightly worse performance. Conclusions: Our findings suggest that stain variability can be tackled more effectively by augmenting data by it than by following the commonly used approach of normalizing the stain. However, the applicability of this approach providing only a rather slight performance increase has to be weighted against an additional carbon footprint

Cytokine gene polymorphisms predict acute graft rejection following renal transplantation.

Background: The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) has been implicated in the pathogenesis of acute rejection, while animal models suggest a role for interleukin-10 (IL-10) in promoting graft survival. It has also been shown that polymorphisms in the TNFA gene promoter (position -308) and in the IL-10 gene promoter (position -1082) correlate with differential production of these cytokines in vitro. The aim of this study was to determine whether TNF-alpha and IL-10 gene polymorphisms influence the incidence and severity of acute rejection in the first six months following renal transplantation. Methods: The cytokine genotypes of 115 consecutive first cadaveric kidney allograft recipients and their donors were screened. The rejection episodes (REs) were defined clinically and confirmed histologically where possible and further classified according to severity (RS), namely steroid-resistant or responsive REs. The genotypes were then correlated with the REs and RS. Results: The recipient TNF-alpha high producer genotype and IL-10 high producer genotype were significantly associated with multiple REs (>/=2) in human leukocyte antigen (HLA)-DR mismatched transplants (P = 0.0047 and P = 0.045, respectively), whereas only the TNF-alpha high producer genotype was associated with steroid-resistant REs (P = 0.025). When recipient cytokines were analyzed together, the TNF-alpha high/IL-10 high producer genotype had the worst prognosis, whereas TNF-alpha low/IL-10 low producer genotype was protective. Conclusions: We conclude that recipient TNF-alpha and IL-10 gene polymorphisms are determinants of REs and RS following kidney transplantation. Routine screening of these gene polymorphisms may have a clinical role in identifying patients at risk of multiple REs and severe rejections

The MEST score provides earlier risk prediction in IgA nephropathy

The Oxford Classification of IgA nephropathy (IgAN) includes the following four histologic components: mesangial (M) and endocapillary (E) hypercellularity, segmental sclerosis (S) and interstitial fibrosis/tubular atrophy (T). These combine to form the MEST score and are independently associated with renal outcome. Current prediction and risk stratification in IgAN requires clinical data over 2 years of follow-up. Using modern prediction tools, we examined whether combining MEST with cross-sectional clinical data at biopsy provides earlier risk prediction in IgAN than current best methods that use 2 years of follow-up data. We used a cohort of 901 adults with IgAN from the Oxford derivation and North American validation studies and the VALIGA study followed for a median of 5.6 years to analyze the primary outcome (50% decrease in eGFR or ESRD) using Cox regression models. Covariates of clinical data at biopsy (eGFR, proteinuria, MAP) with or without MEST, and then 2-year clinical data alone (2-year average of proteinuria/MAP, eGFR at biopsy) were considered. There was significant improvement in prediction by adding MEST to clinical data at biopsy. The combination predicted the outcome as well as the 2-year clinical data alone, with comparable calibration curves. This effect did not change in subgroups treated or not with RAS blockade or immunosuppression. Thus, combining the MEST score with cross-sectional clinical data at biopsy provides earlier risk prediction in IgAN than our current best methods

The MEST score provides earlier risk prediction in lgA nephropathy

The Oxford Classification of IgA nephropathy (IgAN) includes the following four histologic components: mesangial (M) and endocapillary (E) hypercellularity, segmental sclerosis (S) and interstitial fibrosis/tubular atrophy (T). These combine to form the MEST score and are independently associated with renal outcome. Current prediction and risk stratification in IgAN requires clinical data over 2 years of follow-up. Using modern prediction tools, we examined whether combining MEST with cross-sectional clinical data at biopsy provides earlier risk prediction in IgAN than current best methods that use 2 years of follow-up data. We used a cohort of 901 adults with IgAN from the Oxford derivation and North American validation studies and the VALIGA study followed for a median of 5.6 years to analyze the primary outcome (50% decrease in eGFR or ESRD) using Cox regression models. Covariates of clinical data at biopsy (eGFR, proteinuria, MAP) with or without MEST, and then 2-year clinical data alone (2-year average of proteinuria/MAP, eGFR at biopsy) were considered. There was significant improvement in prediction by adding MEST to clinical data at biopsy. The combination predicted the outcome as well as the 2-year clinical data alone, with comparable calibration curves. This effect did not change in subgroups treated or not with RAS blockade or immunosuppression. Thus, combining the MEST score with cross-sectional clinical data at biopsy provides earlier risk prediction in IgAN than our current best methods