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GeoMx Digital Spatial Profiling (Nanostring) is a commercial spatial transcriptomics method to selectively analyze regions of interest within intact tissue sections. We show that decalcification with ethylene-diamine-tetra-acetic (EDTA) variably attenuates probe counts, while probes that are more resistant to this effect consequently appear overexpressed after quantile normalization. By determining the undisclosed full-length target sequences of probes used in the human whole transcriptome panel, hereby updating target transcripts and genes, we find that the gene-promiscuity of probes is an important factor that determines sensitivity to EDTA incubation., (© 2024. The Author(s).)

ANCA-associated vasculitides (AAV) are rare autoimmune diseases causing inflammation and damage to small blood vessels. New autoantibody biomarkers are needed to improve the diagnosis and treatment of AAV patients. In this study, we aimed to profile the autoantibody repertoire of AAV patients using in-house developed antigen arrays to identify previously unreported antibodies linked to the disease per se, clinical subgroups, or clinical activity. A total of 1743 protein fragments representing 1561 unique proteins were screened in 229 serum samples collected from 137 AAV patients at presentation, remission, and relapse. Additionally, serum samples from healthy individuals and patients with other type of vasculitis and autoimmune-inflammatory conditions were included to evaluate the specificity of the autoantibodies identified in AAV. Autoreactivity against members of the kinesin protein family were identified in AAV patients, healthy volunteers, and disease controls. Anti-KIF4A antibodies were significantly more prevalent in AAV. We also observed possible associations between anti-kinesin antibodies and clinically relevant features within AAV patients. Further verification studies will be needed to confirm these findings.

Small extracellular vesicles (sEV) purified from blood have great potential clinically as biomarkers for systemic disease; however interpretation is complicated by release of sEV ex vivo after blood taking. To quantify the problem and devise ways to minimise it, we characterised sEV in paired serum, plasma and platelet poor plasma (PPP) samples from healthy donors. Immunoblotting showed twofold greater abundance of CD9 in sEV fractions from fresh serum than from fresh plasma or PPP. MACSPlex confirmed this, and showed that proteins expressed on platelet sEV, either exclusively (CD41b, CD42a and CD62P) or more widely (HLA-ABC, CD24, CD29 and CD31) were also twofold more abundant; by contrast non-platelet proteins (including CD81) were no different. Storage of plasma (but not serum) increased abundance of platelet and selected leukocyte sEV proteins to at least that of serum, and this could be recapitulated by activating cells in fresh plasma by Ca 2+ , an effect abrogated in PPP. This suggests that a substantial proportion of sEV in serum and stored plasma were generated ex vivo, which is not the case for fresh plasma or PPP. Thus we provide strategies to minimise ex vivo sEV generation and criteria for identifying those that were present in vivo., Competing Interests: The authors declare no conflict of interest., (© 2023 The Authors. Journal of Extracellular Biology published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Autophagy is a central mechanism for maintaining cellular homeostasis in health and disease as it provides the critical energy through the breakdown and recycling of cellular components and molecules within lysosomes. One of the three types of autophagy is chaperone-mediated autophagy (CMA), a degradation pathway selective for soluble cytosolic proteins that contain a targeting motif related to KFERQ in their amino acid sequence. This motif marks them as CMA substrate and is, in the initial step of CMA, recognised by the heat shock protein 70 (Hsc70). The protein complex is then targeted to the lysosomal membrane where the interaction with the splice variant A of the lysosomal-associated membrane protein-2 (LAMP-2A) results in its unfolding and translocation into the lysosome for degradation. Altered levels of CMA have been reported in a wide range of pathologies including many cancer types that upregulate CMA as part of the pro-tumorigenic phenotype, while in aging a decline is observed and associated with a decrease of LAMP-2 expression. The potential of altering CMA to modify a physiological or pathological process has been firmly established through genetic manipulation in animals and chemical interference with this pathway. However, its use for therapeutic purposes has remained limited. Compounds used to target and modify CMA have been applied successfully to gain a better understanding of its cellular mechanisms, but they are mostly not specific, also influence other autophagic pathways and are associated with high levels of toxicity. Here, we will focus on the molecular mechanisms involved in CMA regulation as well as on potential ways to intersect them, describe modulators successfully used, their mechanism of action and therapeutic potential. Furthermore, we will discuss the potential benefits and drawbacks of CMA modulation in diseases such as cancer.

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are severe inflammatory disorders that often involve focal necrotizing glomerulonephritis (FNGN) and consequent glomerular scarring, interstitial fibrosis, and chronic kidney disease. Robust murine models of scarring in FNGN that may help to further our understanding of deleterious processes are still lacking. Here, we present a murine model of severe FNGN based on combined administration of antibodies against the glomerular basement membrane (GBM) and myeloperoxidase (MPO), and bacterial lipopolysaccharides (LPS), that recapitulates acute injury and was adapted to investigate subsequent glomerular and interstitial scarring. Hematuria without involvement of other organs occurs consistently and rapidly, glomerular necrosis and crescent formation are evident at 12 days, and consequent glomerular and interstitial scarring at 29 days after initial treatment. Using mass-spectrometric proteome analysis, we provide a detailed overview of matrisomal and cellular changes in our model. We observed increased expression of the matrisome including collagens, fibronectin, tenascin-C, in accordance with human AAV as deduced from analysis of gene expression microarrays and tissue staining. Moreover, we observed tissue infiltration by neutrophils, macrophages, T cells and myofibroblasts upon injury. Experimental inhibition of CXCR4 using AMD3100 led to a sustained histological presence of fibrin extravasate, reduced chemokine expression and leukocyte activation, but did not markedly affect ECM composition. Altogether, we demonstrate an adapted FNGN model that enables the study of matrisomal changes both in disease and upon intervention, as exemplified via CXCR4 inhibition., Competing Interests: Declaration of competing interests The authors declare no competing financial interests., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Renal allograft rejection can be prevented by immunological tolerance, which may be associated with de novo formed lymphatic vessels in the donor kidney after transplantation in man. A suitable mouse model of renal allograft rejection in which lymphangiogenesis can be deliberately induced in the graft is critical for elucidating the mechanisms responsible for the association between attenuated transplant rejection and abundance of lymphatic vessels. Here we describe the development of a novel mouse model of rapid renal transplant rejection in which transgenic induction of lymphangiogenesis in the immune-incompatible graft greatly extends its survival time. Thus, our novel approach may facilitate exploitation of lymphangiogenesis in the grafted organ., (Copyright © 2019 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Background: GATA3 is a dual-zinc finger transcription factor that regulates gene expression in many developing tissues. In the kidney, GATA3 is essential for ureteric bud branching, and mice without it fail to develop kidneys. In humans, autosomal dominant GATA3 mutations can cause renal aplasia as part of the hypoparathyroidism, renal dysplasia, deafness (HDR) syndrome that includes mesangioproliferative GN. This suggests that GATA3 may have a previously unrecognized role in glomerular development or injury., Methods: To determine GATA3's role in glomerular development or injury, we assessed GATA3 expression in developing and mature kidneys from Gata3 heterozygous ( + /-) knockout mice, as well as injured human and rodent kidneys., Results: We show that GATA3 is expressed by FOXD1 lineage stromal progenitor cells, and a subset of these cells mature into mesangial cells (MCs) that continue to express GATA3 in adult kidneys. In mice, we uncover that GATA3 is essential for normal glomerular development, and mice with haploinsufficiency of Gata3 have too few MC precursors and glomerular abnormalities. Expression of GATA3 is maintained in MCs of adult kidneys and is markedly increased in rodent models of mesangioproliferative GN and in IgA nephropathy, suggesting that GATA3 plays a critical role in the maintenance of glomerular homeostasis., Conclusions: These results provide new insights on the role GATA3 plays in MC development and response to injury. It also shows that GATA3 may be a novel and robust nuclear marker for identifying MCs in tissue sections., (Copyright © 2019 by the American Society of Nephrology.)

Extracellular vesicles, released from cells, are important for intercellular communication. They are heterogeneous but fall into two broad categories based on origin and function: microvesicles formed by outward budding from the plasma membrane; and exosomes that originate as intraluminal vesicles in multivesicular endosomes that fuse with the plasma membrane to release them. Extracellular vesicles generally and exosomes in particular have powerful effects on specific immune responses, and recent advances highlight their potential therapeutic uses. Dendritic cells (DC) that have internalized antigen release exosomes that express MHC class II molecules loaded with antigenic peptides, co-stimulatory molecules and intact antigen. Depending on the setting, these stimulate CD4 T-cell proliferation either directly or only in the context of accessory antigen naïve DC. Here, we discuss the reasons for this; and review current knowledge about the loading of antigen, class II and other cargo into exosomes released by DC and other professional antigen-presenting cells in the context of advances in exosome biology more generally., (© 2018 The Authors Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of Australasian Society for Immunology Inc.)

The lysosome-associated membrane protein (LAMP) family includes the dendritic cell endocytic receptors DC-LAMP and CD68, as well as LAMP-1 and LAMP-2. In this study we identify LAMP-1 (CD107a) and LAMP-2 (CD107b) on the surface of human monocyte-derived dendritic cells (MoDC) and show only LAMP-2 is internalized after ligation by specific Abs, including H4B4, and traffics rapidly but transiently to the MHC class II loading compartment, as does Ag conjugated to H4B4. However, pulsing MoDC with conjugates of primary (keyhole limpet hemocyanin; KLH) and recall (Bet v 1) Ags (H4B4*KLH and H4B4*Bet v 1) induced significantly less CD4 cell proliferation than pulsing with native Ag or Ag conjugated to control mAb (ISO*KLH and ISO*Bet v 1). In H4B4*KLH-pulsed MoDC, the duration of KLH residence in MHC class II loading compartments was significantly reduced, as were surface HLA-DR and DR-bound KLH-derived peptides. Paradoxically, MoDC pulsed with H4B4*KLH, but not the other KLH preparations, induced robust proliferation of CD4 cells separated from them by a transwell membrane, indicating factors in the supernatant were responsible. Furthermore, extracellular vesicles from supernatants of H4B4*KLH-pulsed MoDC contained significantly more HLA-DR and KLH than those purified from control MoDC, and KLH was concentrated specifically in exosomes that were a uniquely effective source of Ag in standard T cell proliferation assays. In summary, we identify LAMP-2 as an endocytic receptor on human MoDC that routes cargo into unusual Ag processing pathways, which reduces surface expression of Ag-derived peptides while selectively enriching Ag within immunogenic exosomes. This novel pathway has implications for the initiation of immune responses both locally and at distant sites., (Copyright © 2017 by The American Association of Immunologists, Inc.)

FimH-mediated adhesion of Escherichia coli to bladder epithelium is a prerequisite for urinary tract infections. FimH is also essential for blood-borne bacterial dissemination, but the mechanisms are poorly understood. The purpose of this study was to assess the influence of different FimH mutations on bacterial adhesion using a novel adhesion assay, which models the physiological flow conditions bacteria are exposed to. We introduced 12 different point mutations in the mannose binding pocket of FimH in an E. coli strain expressing type 1 fimbriae only (MSC95-FimH). We compared the bacterial adhesion of each mutant across several commonly used adhesion assays, including agglutination of yeast, adhesion to mono- and tri-mannosylated substrates, and static adhesion to bladder epithelial and endothelial cells. We performed a comparison of these assays to a novel method that we developed to study bacterial adhesion to mammalian cells under flow conditions. We showed that E. coli MSC95-FimH adheres more efficiently to microvascular endothelium than to bladder epithelium, and that only endothelium supports adhesion at physiological shear stress. The results confirmed that mannose binding pocket mutations abrogated adhesion. We demonstrated that FimH residues E50 and T53 are crucial for adhesion under flow conditions. The coating of endothelial cells on biochips and modelling of physiological flow conditions enabled us to identify FimH residues crucial for adhesion. These results provide novel insights into screening methods to determine the effect of FimH mutants and potentially FimH antagonists., Competing Interests: Compliance with ethical standards Funding The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement numbers 261382 (INTRICATE) and number 238756 (TranSVIR), and was supported by the Wiener Wissenschafts-, Forschungs- und Technologiefonds (WWTF) project no. LS09-075 and the Austrian Science Fund (FWF) special research program SFB F35-24 to TS. Conflict of interest The authors declare no conflict of interest. Ethics approval This article does not contain any studies with human participants or animals performed by any of the authors. Informed consent This study did not involve human subjects or samples; therefore, no informed consent was required.

Mammalian tissues contain networks of mononuclear phagocytes (MPh) that sense injury and orchestrate the response to it. In mice, this is affected by distinct populations of dendritic cells (DC), monocytes and macrophages and recent studies suggest the same is true for human skin and intestine but little is known about the kidney. Here we describe the analysis of MPh populations in five human kidneys and show they are highly heterogeneous and contain discrete populations of DC, monocytes and macrophages. These include: plasmacytoid DC (CD303+) and both types of conventional DC-cDC1 (CD141+ cells) and CD2 (CD1c+ cells); classical, non-classical and intermediate monocytes; and macrophages including a novel population of CD141+ macrophages clearly distinguishable from cDC1 cells. The relative size of the MPh populations differed between kidneys: the pDC population was bi-modally distributed being less than 2% of DC in two kidneys without severe injury and over 35% in the remaining three with low grade injury in the absence of morphological evidence of inflammation. There were profound differences in the other MPh populations in kidneys with high and low numbers of pDC. Thus, cDC1 cells were abundant (55 and 52.3%) when pDC were sparse and sparse (12.8-12.5%) when pDC were abundant, whereas the proportions of cDC2 cells and classical monocytes increased slightly in pDC high kidneys. We conclude that MPh are highly heterogeneous in human kidneys and that pDC infiltration indicative of low-grade injury does not occur in isolation but is part of a co-ordinated response affecting all renal DC, monocyte and macrophage populations.

Pauci-immune focal necrotizing GN (piFNGN) is usually associated with ANCAs that are thought to be pathogenic. However, 10%-15% of patients are ANCA negative and the cause of their injury is unknown. We previously reported a high frequency of autoantibodies to human lysosome-associated membrane protein-2 (hLAMP-2) in ANCA-associated piFNGN, and have now investigated whether the same is true in ANCA-negative patients. Of 11 patients, 8 (73%) had anti-hLAMP-2 antibodies detected by ELISA and confirmed by immunoblotting and indirect immunofluorescence. The autoantibodies from all 8 patients bound to native LAMP-2 purified from human glomeruli and recombinant hLAMP-2 expressed in ldlD cells, both with molecular masses of 110 kD. However, in contrast to anti-LAMP-2 antibodies from ANCA-positive patients, these antibodies from ANCA-negative patients failed to bind the more complexly glycosylated native neutrophil hLAMP-2 (190 kD). Treatment with the deglycosylating enzyme, endo-β-galactosidase, reduced the mass of neutrophil hLAMP-2 to 110 kD and enabled autoantibody binding. Similarly, pretreating neutrophils with endo-β-galactosidase or neuraminidase converted ANCA assay results from negative to positive. Finally, IgG from LAMP-2-positive ANCA-negative patients bound specifically to normal human kidney sections and to human glomerular endothelial cells in culture. In conclusion, in patients with ANCA-negative piFNGN, we have identified autoantibodies to hLAMP-2 that bind native glomerular but not neutrophil hLAMP-2, suggesting a role in pathogenesis.

Macrophages respond to their microenvironment and develop polarized functions critical for orchestrating appropriate inflammatory responses. Classical (M1) activation eliminates pathogens while alternative (M2) activation promotes regulation and repair. M1 macrophage activation is strongly associated with suppressor of cytokine signalling 3 (SOCS3) expression in vitro, but the functional consequences of this are unclear and the role of SOCS3 in M1-macrophage polarization in vivo remains controversial. To address these questions, we defined the characteristics and function of SOCS3-expressing macrophages in vivo and identified potential mechanisms of SOCS3 action. Macrophages infiltrating inflamed glomeruli in a model of acute nephritis show significant up-regulation of SOCS3 that co-localizes with the M1-activation marker, inducible nitric oxide synthase. Numbers of SOCS3(hi) -expressing, but not SOCS1(hi) -expressing, macrophages correlate strongly with the severity of renal injury, supporting their inflammatory role in vivo. Adoptive transfer of SOCS3-short interfering RNA-silenced macrophages into a peritonitis model demonstrated the importance of SOCS3 in driving production of pro-inflammatory IL-6 and nitric oxide, while curtailing expression of anti-inflammatory IL-10 and SOCS1. SOCS3-induced pro-inflammatory effects were due, at least in part, to its role in controlling activation and nuclear accumulation of nuclear factor-κB and activity of phosphatidylinositol 3-kinase. We show for the first time that SOCS3 also directs the functions of human monocyte-derived macrophages, including efficient M1-induced cytokine production (IL-1β, IL-6, IL-23, IL-12), attenuated signal transducer and activator of transcription 3 activity and ability of antigen-loaded macrophages to drive T-cell responses. Hence, M1-associated SOCS3 was a positive regulator of pro-inflammatory responses in our rodent models and up-regulated SOCS3 is essential for effective M1-macrophage activation and function in human macrophages., (© 2013 The Authors. Immunology published by John Wiley & Sons Ltd.)

The kidneys are frequently targeted by pathogenic immune responses against renal autoantigens or by local manifestations of systemic autoimmunity. Recent studies in rodent models and humans have uncovered several underlying mechanisms that can be used to explain the previously enigmatic immunopathology of many kidney diseases. These mechanisms include kidney-specific damage-associated molecular patterns that cause sterile inflammation, the crosstalk between renal dendritic cells and T cells, the development of kidney-targeting autoantibodies and molecular mimicry with microbial pathogens. Conversely, kidney failure affects general immunity, causing intestinal barrier dysfunction, systemic inflammation and immunodeficiency that contribute to the morbidity and mortality of patients with kidney disease. In this Review, we summarize the recent findings regarding the interactions between the kidneys and the immune system.

Purpose of Review: This review critically analyses the data implicating antibodies to lysosome associated membrane protein-2 (hLAMP-2) in ANCA-associated vasculitis (AAV). It addresses recent controversies over prevalence of anti-hLAMP-2 antibodies as well as their potential for diagnosis and monitoring disease activity., Recent Findings: Anti-hLAMP-2 antibodies were first described in the 1990s and have become the focus of intense clinical interest in the past 4 years. This followed the demonstration of their very high prevalence in untreated patients presenting with AAV but absence when patients were in remission. The data also demonstrated molecular mimicry between hLAMP-2 and the bacterial protein FimH. The same group later confirmed the original findings and showed the anti-hLAMP-2 autoantibodies have different kinetics to those recognising myeloperoxidase and proteinase-3 and are less likely to be detectable when the disease is in remission. By contrast, a different group reported a lower prevalence of anti-hLAMP-2 antibodies in AAV and questioned their relevance to pathogenesis. Critical analysis of these studies suggests that the differences are largely attributable to selection criteria of the AAV patients studied and the assays used., Summary: Anti-hLAMP-2 antibodies are frequently found in AAV but attempts to define their consequences have been frustrated by lack of generally available assays for them.

The role of the tumour microenvironment and complex cellular interactions has attracted interest in responses to primary chemotherapy. Of particular interest are tumour-infiltrating T cells and tumour-infiltrating macrophages (TIMs). We evaluated TIMs and their key activation markers in patients with breast cancer undergoing primary chemotherapy related to response and survival. One hundred and ninety nine patients with large or locally advanced breast cancers received primary chemotherapy. Clinical data, histopathological responses to chemotherapy and survival were examined related to infiltrating cells in tumour microenvironments: cluster of differentiation (CD)3 (pan T cell); CD4 (helper T cells); CD8 (cytotoxic T cells); CD25 (activated T cells); CD68, suppressor of cytokine signalling (SOCS)1, SOCS3 (macrophages); and CD11c and CD205 (dendritic). In tumours demonstrating better responses to chemotherapy, there were significantly fewer CD4(+) T-helper cells than a poorer response (p < 0.05). There were increased numbers of SOCS3 expressing macrophages (pro-inflammatory) in tumours with complete pathological responses compared with no response to chemotherapy (p < 0.05). There was no association between SOCS1 expressing macrophages (anti-inflammatory) and tumour response. Multivariate analysis revealed that factors indicating better survival were receiving anthracycline plus docetaxel (ExpB = 1.166; p = 0.006), better pathological chemotherapy response (ExpB = 0.309; p = 0.009) and a low macrophage SOCS1 expression (ExpB = 13.465; p = 0.044). This study highlights the heterogeneity of TIMs and provides further insight into complex interactions within tumours. The results emphasise the importance of characterising activation status of infiltrating macrophages and provides proof of principle for using macrophage SOCS protein expression as a survival predictor. The apparent impact of macrophage subsets on overall survival underlines the therapeutic potential of manipulating macrophage activation in cancer.

Background: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis is a severe condition encompassing two major syndromes: granulomatosis with polyangiitis (formerly known as Wegener's granulomatosis) and microscopic polyangiitis. Its cause is unknown, and there is debate about whether it is a single disease entity and what role ANCA plays in its pathogenesis. We investigated its genetic basis., Methods: A genomewide association study was performed in a discovery cohort of 1233 U.K. patients with ANCA-associated vasculitis and 5884 controls and was replicated in 1454 Northern European case patients and 1666 controls. Quality control, population stratification, and statistical analyses were performed according to standard criteria., Results: We found both major-histocompatibility-complex (MHC) and non-MHC associations with ANCA-associated vasculitis and also that granulomatosis with polyangiitis and microscopic polyangiitis were genetically distinct. The strongest genetic associations were with the antigenic specificity of ANCA, not with the clinical syndrome. Anti-proteinase 3 ANCA was associated with HLA-DP and the genes encoding α(1)-antitrypsin (SERPINA1) and proteinase 3 (PRTN3) (P=6.2×10(-89), P=5.6×10(-12,) and P=2.6×10(-7), respectively). Anti-myeloperoxidase ANCA was associated with HLA-DQ (P=2.1×10(-8))., Conclusions: This study confirms that the pathogenesis of ANCA-associated vasculitis has a genetic component, shows genetic distinctions between granulomatosis with polyangiitis and microscopic polyangiitis that are associated with ANCA specificity, and suggests that the response against the autoantigen proteinase 3 is a central pathogenic feature of proteinase 3 ANCA-associated vasculitis. These data provide preliminary support for the concept that proteinase 3 ANCA-associated vasculitis and myeloperoxidase ANCA-associated vasculitis are distinct autoimmune syndromes. (Funded by the British Heart Foundation and others.).

The involvement of autoantibodies to human lysosome-associated membrane protein-2 (hLAMP-2) in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis is controversial because of the absence of confirmatory data subsequent to the initial reports of their high prevalence in this disease. We characterized three assays for anti-hLAMP-2 antibodies: ELISA and Western blotting assays using unglycosylated recombinant hLAMP-2 expressed in Escherichia coli, and an indirect immunofluorescence assay using stably transfected ldlD cells that expressed glycosylated full-length hLAMP-2 on the plasma membrane. The assays detected autoantibodies to hLAMP-2 in human sera reproducibly and with comparable sensitivity and the assays gave the same results in 80.5% of the test panel of 40 selected positive and negative sera. In untreated patients at presentation, the frequencies of autoantibodies to LAMP-2 were 89%, 91%, and 80%, respectively, among three groups of patients with ANCA-associated vasculitis from Vienna, Austria (n=19); Groningen, the Netherlands (n=50) and Cambridge, United Kingdom (n=53). Prevalence of LAMP-2 autoantibodies was similar in both those with myeloperoxidase-ANCA and proteinase 3-ANCA. Furthermore, we detected LAMP-2 autoantibodies in two ANCA-negative patients. LAMP-2 autoantibodies rapidly became undetectable after the initiation of immunosuppressive treatment and frequently became detectable again during clinical relapse. We conclude that when robust assays are used, circulating autoantibodies to hLAMP-2 can be detected in most European patients with ANCA-associated vasculitis. Large-scale prospective studies are now needed to determine whether they are pathogenic or merely an epiphenomenon.

The renal mononuclear phagocytic system, conventionally composed of macrophages (Mø) and dendritic cells (DCs), plays a central role in health and disease of the kidney. Overlapping definitions of renal DCs and Mø, stemming from historically separate research tracks and the lack of experimental tools to specifically study the roles of these cells in vivo, have generated confusion and controversy, however, regarding their immunologic function in the kidney. This brief review provides an appraisal of the current state of knowledge of the renal mononuclear phagocytic system interpreted from the perspective of immunologic function. Physical characteristics, ontogeny, and known functions of the main subsets of renal mononuclear phagocytes as they relate to homeostasis, surveillance against injury and infection, and immune-mediated inflammatory injury and repair within the kidney are described. Gaps and inconsistencies in current knowledge are used to create a roadmap of key questions to be answered in future research.

Macrophages become activated by their environment and develop polarized functions: classically activated (M1) macrophages eliminate pathogens but can cause tissue injury, whereas alternatively activated (M2) macrophages promote healing and repair. Mechanisms directing polarized activation, especially in vivo, are not understood completely, and here, we examined the role of SOCS proteins. M2 macrophages activated in vitro or elicited by implanting mice i.p. with the parasitic nematode Brugia malayi display a selective and IL-4-dependent up-regulation of SOCS1 but not SOCS3. Using siRNA-targeted knockdown in BMDM, we reveal that the enhanced SOCS1 is crucial for IL-4-induced M2 characteristics, including a high arginase I:iNOS activity ratio, suppression of T cell proliferation, attenuated responses to IFN-γ/LPS, and curtailed SOCS3 expression. Importantly, SOCS1 was essential in sustaining the enhanced PI3K activity that drives M2 activation, defining a new regulatory mechanism by which SOCS1 controls M2 polarization. By contrast, for M1 macrophages, SOCS1 was not only an important regulator of proinflammatory mediators (IL-6, IL-12, MHC class II, NO), but critically, for M1, we show that SOCS1 also restricted IL-10 secretion and arginase I activity, which otherwise would limit the efficiency of M1 macrophage proinflammatory responses. Together, our results uncover SOCS1, not only as a feedback inhibitor of inflammation but also as a critical molecular switch that tunes key signaling pathways to effectively program different sides of the macrophage balance.

Background: Idiopathic membranous nephropathy is a major cause of the nephrotic syndrome in adults, but its etiologic basis is not fully understood. We investigated the genetic basis of biopsy-proven cases of idiopathic membranous nephropathy in a white population., Methods: We performed independent genomewide association studies of single-nucleotide polymorphisms (SNPs) in patients with idiopathic membranous nephropathy from three populations of white ancestry (75 French, 146 Dutch, and 335 British patients). The patients were compared with racially matched control subjects; population stratification and quality controls were carried out according to standard criteria. Associations were calculated by means of a chi-square basic allele test; the threshold for significance was adjusted for multiple comparisons (with the Bonferroni method)., Results: In a joint analysis of data from the 556 patients studied (398 men), we identified significant alleles at two genomic loci associated with idiopathic membranous nephropathy. Chromosome 2q24 contains the gene encoding M-type phospholipase A(2) receptor (PLA(2)R1) (SNP rs4664308, P=8.6×10(-29)), previously shown to be the target of an autoimmune response. Chromosome 6p21 contains the gene encoding HLA complex class II HLA-DQ alpha chain 1 (HLA-DQA1) (SNP rs2187668, P=8.0×10(-93)). The association with HLA-DQA1 was significant in all three populations (P=1.8×10(-9), P=5.6×10(-27), and P=5.2×10(-36) in the French, Dutch, and British groups, respectively). The odds ratio for idiopathic membranous nephropathy with homozygosity for both risk alleles was 78.5 (95% confidence interval, 34.6 to 178.2)., Conclusions: An HLA-DQA1 allele on chromosome 6p21 is most closely associated with idiopathic membranous nephropathy in persons of white ancestry. This allele may facilitate an autoimmune response against targets such as variants of PLA2R1. Our findings suggest a basis for understanding this disease and illuminate how adaptive immunity is regulated by HLA.

Gene copy number of complement component C4, which varies among individuals, may determine the intrinsic strength of the classical complement pathway. Presuming a major role of complement as an effector in transplant rejection, we hypothesized that C4 genetic diversity may partially explain the variation in allograft outcomes. This retrospective study included 1969 deceased-donor kidney transplants randomly selected from the Collaborative Transplant Study DNA bank. We determined recipient and donor gene copy number of total C4, C4 isotypes (C4A and C4B), and C4 gene length variants (C4L and C4S) by quantitative real-time PCR analysis. Groups defined according to recipient C4 gene copy number (low, intermediate, and high) had similar 10-year allograft survival. Genotypic groups showed comparable rates of graft dysfunction, treatment for rejection, immunological graft loss, hospitalization for infection, malignant disease, and death. Similarly, separate analyses of C4A, C4B, C4L, and C4S; combined evaluation of donor and recipient C4 genotype; or analysis of recipients with higher risk for rejection did not reveal considerable outcome effects. In conclusion, we did not demonstrate that C4 gene copy number associates with transplant outcome, and we found no evidence that the resulting variation in the strength of classical complement activation influences susceptibility to rejection.

Demographic and family studies support the existence of a genetic contribution to the pathogenesis of IgA nephropathy, but results from genetic association studies of candidate genes are inconsistent. To systematically survey common genetic variation in this disease, we performed a genome-wide analysis in a cohort of patients with IgA nephropathy selected from the UK Glomerulonephritis DNA Bank. We used two groups of controls: parents of affected individuals and previously genotyped, unaffected, ancestry-matched individuals from the 1958 British Birth Cohort and the UK Blood Service. We genotyped 914 affected or family controls for 318,127 single nucleotide polymorphisms (SNPs). Filtering for low genotype call rates and inferred non-European ancestry left 533 genotyped individuals (187 affected children) for the family-based association analysis and 244 cases and 4980 controls for the case-control analysis. A total of 286,200 SNPs with call rates >95% were available for analysis. Genome-wide analysis showed a strong signal of association on chromosome 6p in the region of the MHC (P = 1 × 10(-9)). The two most strongly associated SNPs showed consistent association in both family-based and case-control analyses. HLA imputation analysis showed that the strongest association signal arose from a combination of DQ loci with some support for an independent HLA-B signal. These results suggest that the HLA region contains the strongest common susceptibility alleles that predispose to IgA nephropathy in the European population.

This review provides an overview of the current understanding of the biology of monocytes and macrophages. It focuses on four rapidly advancing areas that underpin recent conceptual advances, namely: (1) the bone marrow origins of monocytes and macrophages, (2) monocyte heterogeneity, (3) the early inflammatory consequences of tissue injury, and (4) current concepts of macrophage activation and their limitations.

Background: IgA nephropathy is a major cause of end-stage renal disease worldwide. Its aetiology is poorly understood but there is good evidence for a major genetic component, although to date, no gene has been conclusively identified. We describe a new UK multicentre DNA collection assembled to investigate this. A Japanese genome-wide analysis recently reported that common genetic variation in immunoglobulin mu-binding protein 2 (IGHMBP2) was associated with IgA nephropathy. We sought to replicate this using the new UK collection, and through an independent parallel analysis of a Han Chinese population., Methods: In the UK collection, haplotype-tagging (tag) single-nucleotide polymorphisms (SNPs) and haplotypes were analysed in a case-control study (349 cases, 605 controls) and family-based analysis (162 complete and 23 partially complete family trios), which was performed using the transmission disequilibrium test. In parallel, 663 cases of IgA nephropathy and 663 controls from a Chinese population were analysed: coding and flanking regions of the gene were re-sequenced in a subset, and SNP and haplotype association analysis was performed in the whole collection using the identified tagSNPs and all the coding and exonic flanking SNPs., Results: Case-control studies in UK and Chinese populations, and family-based tests in the UK population provided no evidence for association between variation in IGHMBP2 and IgA nephropathy. The A allele of SNP G34448A was not present in the UK collection. It was present but not associated with the disease in the Chinese population., Conclusion: Variation in IGHMBP2 does not confer significant susceptibility to IgA nephropathy in UK Caucasian or Chinese Han populations.

The aetiology of anti-neutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis has not been well defined. Here we review two factors which may play a role in the pathogenesis of the disease: genetics and infection. In particular, we discuss the role of autoantibodies to LAMP-2, which may arise following infection with Gram-negative bacteria, and may contribute to the development of ANCA-associated systemic vasculitis in genetically susceptible individuals.

Pauci-immune focal necrotizing glomerulonephritis (FNGN) is a severe inflammatory disease associated with autoantibodies to neutrophil cytoplasmic antigens (ANCA). Here we characterize autoantibodies to lysosomal membrane protein-2 (LAMP-2) and show that they are a new ANCA subtype present in almost all individuals with FNGN. Consequently, its prevalence is nearly twice that of the classical ANCAs that recognize myeloperoxidase or proteinase-3. Furthermore, antibodies to LAMP-2 cause pauci-immune FNGN when injected into rats, and a monoclonal antibody to human LAMP-2 (H4B4) induces apoptosis of human microvascular endothelium in vitro. The autoantibodies in individuals with pauci-immune FNGN commonly recognize a human LAMP-2 epitope (designated P(41-49)) with 100% homology to the bacterial adhesin FimH, with which they cross-react. Rats immunized with FimH develop pauci-immune FNGN and also develop antibodies to rat and human LAMP-2. Finally, we show that infections with fimbriated pathogens are common before the onset of FNGN. Thus, FimH-triggered autoimmunity to LAMP-2 provides a previously undescribed clinically relevant molecular mechanism for the development of pauci-immune FNGN.

On infiltrating inflamed tissue, macrophages respond to the local microenvironment and develop one of two broad phenotypes: classically activated (M1) macrophages that cause tissue injury and alternatively activated macrophages that promote repair. Understanding how this polarization occurs in vivo is far from complete, and in this study, using a Th1-mediated macrophage-dependent model of acute glomerulonephritis, nephrotoxic nephritis, we examine the role of suppressor of cytokine signaling (SOCS)1 and SOCS3. Macrophages in normal kidneys did not express detectable SOCS proteins but those infiltrating inflamed glomeruli were rapidly polarized to express either SOCS1 (27 +/- 6%) or SOCS3 (54 +/- 12%) but rarely both (10 +/- 3%). Rat bone marrow-derived macrophages incubated with IFN-gamma or LPS expressed SOCS1 and SOCS3, whereas IL-4 stimulated macrophages expressed SOCS1 exclusively. By contrast, incubation with IFN-gamma and LPS together suppressed SOCS1 while uniquely polarizing macrophages to SOCS3 expressing cells. Macrophages in which SOCS3 was knocked down by short interfering RNA responded to IFN-gamma and LPS very differently: they had enhanced STAT3 activity; induction of macrophage mannose receptor, arginase and SOCS1; restoration of IL-4 responsiveness that is inhibited in M1 macrophages; and decreased synthesis of inflammatory mediators (NO and IL-6) and costimulatory molecule CD86, demonstrating that SOCS3 is essential for M1 activation. Without it, macrophages develop characteristic alternatively activated markers when exposed to classical activating stimuli. Lastly, increased glomerular IL-4 in nephrotoxic nephritis inhibits infiltrating macrophages from expressing SOCS3 and was associated with attenuated glomerular injury. Consequently, we propose that SOCS3 is essential for development of M1 macrophages in vitro and in vivo.

Autoreactive T cells in patients with Goodpasture's disease are specific for epitopes in the Goodpasture antigen (the NC1 domain of the alpha3 chain of type IV collagen) that are rapidly destroyed during antigen processing to a degree that diminishes their presentation to T cells. We hypothesized that patients' autoreactive T cells exist because antigen processing prevents presentation of the self-epitopes they recognize, circumventing specific tolerance mechanisms. We predicted that autoreactive T cells specific for these peptides should also exist in healthy individuals, albeit at low frequency and in an unprimed state. We obtained blood from healthy unrelated donors and, using a panel of 45 alpha3(IV)NC1 peptides, identified alpha3(IV)NC1-specific T cells in all donors. Thirty-six of 45 peptides elicited a proliferative T cell response from at least one subject, and 6 of the peptides evoked a response in >50% of the individuals. This consistency was not caused by selectivity of HLA class II molecules because the donors expressed a diversity of HLA antigens, but was largely a result of the substrate-specificity of the endosomal proteases Cathepsin D and E. There was a significant correlation between high susceptibility to Cathepsin D digestion and the capacity to stimulate primary T cell responses (P = 0.00006). In summary, healthy individuals have low frequencies of unstimulated alpha3(IV)NC1-reactive T cells with similar specificities to the autoreactive T cells found in patients with Goodpasture disease. In both cases, existence of the alpha3(IV)NC1-reactive T cells can be accounted for by destructive processing.

Foreign body in the knee is associated with trauma to knee or deliberate self harm. We see them often in clinical practice. They come in all forms and shapes. Very rarely one can find a foreign body within a joint without obvious external scarring (e.g. needle). We have not come across anywhere in the literature of a large foreign body in the knee joint without a definitive history of injury where the external scar has healed so well to become inconspicuous. With this background it is even more difficult when the X-rays mimic anterior cruciate ligament (ACL) avulsion. This case report highlights the fact that diagnosis of a foreign body in the knee joint can sometimes be challenging and almost impossible when there is no history of any injury and when the X-ray mimics ACL avulsion.