C3 Deposition Research Articles

33841 Pregnancy dermatosis clinical scoring system: How to predict between pemphigoid gestationis and polymorphic eruption of pregnancy

Pre-bullous pemphigoid gestationis (PG) and polymorphic eruption of pregnancy (PEP) may be difficult to differentiate due to similar features of urticated papules and plaques. The gold standard for diagnosis requires direct immunofluorescence (DIF) with presence or lack of linear C3 deposition at the dermoepidermal junction in PG and PEP, respectively. We aimed to develop a clinical scoring system based on clinical history and examination to stratify patient risk for PG and to therefore help guide risk of neonatal rash, prematurity, and low birth weight while awaiting DIF results, in areas in which DIF is inaccessible, or when the patient declines biopsy.

Livedo racemosa in lymphocytic vasculitis.

A 24-year-old female presented with a 10-year history of mottled patches on the limbs. These were asymptomatic and persisted on rewarming. She was otherwise well with no past history or constitutional symptoms. Examination revealed reticulate erythematous to cyanotic patches forming incomplete rings, consistent with livedo racemosa. Histology showed a lymphocytic vasculitis with 1+ granular deposits of C3, IgM and fibrin on direct immunofluorescence. Clinical systemic review, antiphospholipid, hypercoagulability and autoimmune workups were unremarkable. This was consistent with the diagnosis of lymphocytic thrombophilic arteritis, a medium vessel vasculitis typically without systemic involvement. This presents with livedo racemosa, which is differentiated from livedo reticularis by the presence of larger broken rings that do not reverse with warming. Unlike livedo reticularis which may be physiological, livedo racemosa is always pathological. Critically, a skin biopsy should include the centre of the ring in order to sample the feeding artery.

Absence of NC14A Domain of COLXVII/BP180 in Mice Results in IL-17‒Associated Skin Inflammation

The deletion of exon 18 from Col17a1 in transgenic ΔNC14A mice results in the absence of the NC14Adomain. NC14A corresponds to the human NC16A domain, the immunodominant epitope in bullous pemphigoid. Before the age of 1 year, 84% of ΔNC14A mice have developed severe itch and skin erosion. Further characterization of mice with mutated CoLXVII (Bp180) revealed acanthosis; subepidermal blistering; and inflammatory cell infiltrates, especially neutrophils, eosinophils, and mast cells in the lesional skin. Direct immunofluorescence analysis detected linear complement C3, IgG, and/or IgA deposition in the dermo‒epidermal junction of symptomatic ΔNC14A mice. Elevated gene expression of IL-17‒associated cytokines was detected in the lesional skin. An increased proportion of dendritic cells, myeloid-derived suppressor cells, and NK cells and a decrease of T cells were found in both the spleen and lymph nodes of symptomatic ΔNC14A mice. The proportions of B cells and regulatory T cells were increased in lymph nodes. An 8-week treatment with an anti‒IL-17A decreased the expression of Il6, Il23a, and Cxcl1 in the nonlesional skin. Our results suggest that the absence of the NC14A domain of CoLXVII in mice causes an autoimmune response against the cutaneous basement membrane and manifests as an IL-17‒associated inflammation in the skin.

THE IMPORTANCE OF GLOMERULAR C3 ACCUMULATION IN ELDERLY PATIENTS WITH PRIMARY MEMBRANOUS NEPHROPATHY

Objective: The purpose of this study was to investigate the impact of glomerular C3 accumulation density on clinical, histopathological parameters and outcomes in elderly (>60 years) individuals with primary membranous nephropathy (PMN). Material and Methods: In this study, we examined the patients (n=105) in two groups according to the C3 staining density in kidney biopsy samples as low intensity (C3: 1+; LI group) and high intensity (C3: 2+ or C3: 3+; HI group). The primary endpoint of our study was the end-stage renal disease, and the secondary endpoints were the development of partial remission (PR) or complete remission (CR). Results: At the end of the follow-up (mean 30.6 months), more patients achieved the primary endpoint, and fewer patients achieved the secondary endpoints in the HI group compared to the LI group. (p=0.015 and p=0.016, respectively). Moreover, the glomerular filtration rate (eGFR) was lower (p<0.001), and proteinuria was higher in the HI group (p=0.018). Kaplan-Meier survival analysis revealed that renal survival (p=0.031) was lower in the HI group compared to the LI group. In the multivariate logistic regression analyses, no predictive parameters could be detected for the endpoints. Conclusion: Intense glomerular C3 deposition in elderly (>60 years) patients with PMN may be related to poor clinical outcomes.

The anti-inflammatory effects of Hedyotis diffusa Willd on SLE with STAT3 as a key target

Ethnopharmacological relevanceHedyotis diffusa Willd, also named Scleromitrion diffusum (Willd.) R.J. Wang, is one medical herb, which has been traditionally used by the She nationality in China. And H. diffusa represents a beneficial effect on Systemic lupus erythematosus (SLE) treatment in clinic. Aim of the studyThe underlying mechanisms of the protective effects of H. diffusa on SLE remain unclear. In this study, we treated MRL/lpr mice with H. diffusa water extract (HDW) to assess its therapeutic effects and verified its regulating signalling pathway through cytological experiments. Materials and methodsIn the present study, the constituents of HDW were analysed through ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and SCIEX OS software. The protective activity and underlying mechanisms were studied in a MRL/lpr lupus mouse model. The blood cells, autoantibodies, metabolites and the cytokines in serum were identified with a hematology analyzer, specific ELISA kit, GC/MS system and cytometric assays. The histological and immunohistochemical analysis were engaged in the morphologic, and the expression and translocation of the crucial protein observation. The dual luciferase reporter assay was applied to identifying the regulative activity of HDW. The transcription and translation expression of the protein was studied by real-time PCR and Western blot assays. The network pharmacology analysis was employed to predict the IL-6/STAT3 pathway regulators and the screen the STAT3 inhibitors in HDW. ResultsThe results revealed the capability of HDW to attenuate the production of autoantibodies, secretion of inflammatory cytokines (IL-6 and IFN-γ), and suppressed the IgG and C3 deposition, the development of glomerular lesions in MRL/lpr mice. Serum metabolomics study showed the improvement in serum metabolites, especially aminoacyl-tRNA biosynthesis, by HDW. IL-6 was clarified to be highly associated with the significantly changed metabolites in network analysis. We further demonstrated the effects of HDW on the IL-6/STAT3 pathway in vivo and in vitro. ConclusionsThis study suggested that HDW exerts a therapeutic effect in SLE model mice by suppressing the IL-6/STAT3 pathway.

Alternative Complement Pathway Inhibition With Iptacopan for the Treatment of C3 Glomerulopathy-Study Design of the APPEAR-C3G Trial

IntroductionComplement 3 glomerulopathy (C3G) is a rare kidney disease characterized by dysregulation of the alternative pathway (AP) of the complement system. About 50% of patients with C3G progress to kidney failure within 10 years of diagnosis. Currently, there are no approved therapeutic agents for C3G. Iptacopan is an oral, first-in-class, potent, and selective inhibitor of factor B, a key component of the AP. In a Phase II study, treatment with iptacopan was associated with a reduction in proteinuria and C3 deposit scores in C3G patients with native and transplanted kidneys, respectively.MethodsAPPEAR-C3G (NCT04817618) is a randomized, double-blind, and placebo-controlled Phase III study to evaluate the efficacy and safety of iptacopan in C3G patients, enrolling 68 adults with biopsy-confirmed C3G, reduced C3 (<77 mg/dl), proteinuria ≥1.0 g/g, and estimated glomerular filtration rate (eGFR) ≥30 ml/min per 1.73 m2. All patients will receive maximally tolerated angiotensin-converting enzyme inhibitor/angiotensin receptor blocker and vaccination against encapsulated bacteria. Patients with any organ transplantation, progressive crescentic glomerulonephritis (GN), monoclonal gammopathy of undetermined significance, or kidney biopsy with >50% interstitial fibrosis/tubular atrophy, will be excluded. Patients will be randomized 1:1 to receive either iptacopan 200 mg twice daily or placebo for 6 months, followed by open-label treatment with iptacopan 200 mg twice daily for all patients for 6 months. The primary objective is to evaluate the efficacy of iptacopan versus placebo on proteinuria reduction urine protein:creatinine ratio (UPCR) (24 h urine). Key secondary endpoints will assess kidney function measured by eGFR, histological disease total activity score, and fatigue.ConclusionThis study aims to demonstrate the clinical benefits of AP inhibition with iptacopan in C3G.

Divergent complement system activation in two clinically distinct murine models of multiple sclerosis.

Multiple sclerosis (MS) is a neurological disease featuring neuroinflammation and neurodegeneration in young adults. So far, most research has focused on the peripheral immune system, which appears to be the driver of acute relapses. Concurrently, the mechanisms underlying neurodegeneration in the progressive forms of the disease remain unclear. The complement system, a molecular component of the innate immunity, has been recently implicated in several neurological disorders, including MS. However, it is still unknown if the complement proteins detected in the central nervous system (CNS) are actively involved in perpetuating chronic inflammation and neurodegeneration. To address this knowledge gap, we compared two clinically distinct mouse models of MS: 1) proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (rEAE) resembling a relapsing-remitting disease course, and 2) Theiler’s murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) resembling a progressive disease. Real-time PCR was performed in the spinal cord of rEAE mice, TMEV-IDD mice and age-matched sham controls to quantify gene expression for a broad range of complement components. In both experimental models, we found significantly increased expression of complement factors, such as C1q, C3, CfB, and C3aR. We showed that the complement system, specifically the classical complement pathway, was associated with TMEV-IDD pathogenesis, as the expression of C1q, C3 and C3aR1 were all significantly correlated to a worse disease outcome (all P≤0.0168). In line with this finding, C1q and C3 deposition was observed in the spinal cord of TMEV-IDD mice. Furthermore, C1q deposition was detected in spinal cord regions characterized by inflammation, demyelination, and axonal damage. Conversely, activation of the classical complement cascade seemed to result in protection from rEAE (C1q: P=0.0307). Interestingly, the alternative pathway related to a worse disease outcome in rEAE (CFb: P=0.0006). Overall, these results indicate potential divergent roles for the complement system in MS. The chronic-progressive disease form is more reliant on the activation of the classic complement pathway, while protecting from acute relapses. Conversely, relapsing MS appears more likely affected by the alternative pathway. Understanding the functions of the complement system in MS is critical and can lead to better, more targeted therapies in the future.

Complement factor B inhibitor LNP023 improves lupus nephritis in MRL/lpr mice

BackgroundLupus nephritis (LN) is the most common complication of systemic lupus erythematosus (SLE), and the abnormal activation of the alternative complement pathway is associated with the pathogenesis of LN. As an inhibitor of complement factor B (CFB) in the alternative pathway, LNP023 has been used in the treatment of a variety of renal diseases with abnormal complement system involvement, such as paroxysmal nocturnal hemoglobinuria, IgA nephropathy, and membranous nephropathy. The aim of our study was to explore whether LNP023 improved LN in MRL/lpr mice by inhibiting the activation of the alternative complement pathway. MethodsThe mice were divided into a normal control group (Normal group) (n = 6), MRL/lpr model group (n = 6), and LNP023 group (n = 6). The LNP023 group was administered LNP023 for 2 weeks by gavage; the MRL/lpr model group was administered saline for 2 weeks by gavage; and the Normal group was administered saline for 2 weeks by gavage. External signs, renal pathology, renal function, renal immune complex and complement deposition, serum anti-dsDNA, serum ANA concentration, and the expression of core complement factors in the alternative complement pathway were analyzed in the 3 groups of animals. The core targets of LNP023 in the treatment of LN were screened using network pharmacology. The pathogenicity of the core targets in LN was verified by analyzing the mRNA expression of the core targets in the peripheral blood mononuclear cells (PBMCs) of normal individuals, SLE patients, and LN patients. The mRNA and protein expression of core targets in the Normal group, MRL/lpr group, and LNP023 group were analyzed to verify whether LNP023 exerted it LN therapeutic effect through the regulation of core targets. ResultsCompared with the MRL/lpr group, the LNP023 group had reduced lupus-like signs, improved renal function, decreased serum anti-dsDNA and ANA concentrations, and reduced renal IgM, IgG, IgG1, C1q, C3, and C4 deposition. Renal pathology showed that LNP023 attenuated pathological damage in the kidneys of MRL/lpr mice. Compared with the MRL/lpr model group, the treatment group had no crescent formation, less immune deposition, no nuclear fragmentation, and less inflammatory cell infiltration. The expression of complement proteins C3, C3b, CR1, CFB, and C5b-9 in kidney tissues and liver was decreased, and the expression of C5 was increased. Network pharmacology screening indicated that AKT, TNF-α, MDM2, UBC, STST3, ESR1, and TP53 were core targets of LNP023 in the treatment of LN. Compared with that in the Normal group, the mRNA expression of the core target in the SLE and LN groups was different; compared with the MRL/lpr group, the LNP023 treatment group showed different mRNA and protein expression levels of AKT, TNF-α, and STST3. ConclusionLNP023 improves LN in MRL/lpr mice. The mechanism is as follows: LNP023 binds to CFB to inhibit the activation of the alternative complement pathway. LNP023 treatment for LN may also play a role in regulating the protein expression of AKT, TNF-α, and STST3.

Hemidesmosomal Reactivity and Treatment Recommendations in Immune Checkpoint Inhibitor-Induced Bullous Pemphigoid-A Retrospective, Monocentric Study.

Immune checkpoint inhibitors (ICI) induce T-cell-mediated antitumour responses. While ICI were initially successfully applied in metastasized melanoma, they are now approved for several tumour entities. Numerous autoimmune disorders have been reported to occur as adverse events of the treatment, among them bullous pemphigoid (BP), with less than 1% of the patients experiencing ICI-induced BP. This number is higher than the estimated prevalence of autoimmune bullous diseases in the general population of Germany, which lies around 0.05%. We here describe our cohort of eight patients, who developed a bullous pemphigoid under or shortly after ICI treatment. Half of them had a severe subtype (as shown by BPDAI >57) and showed a median onset of ICI-BP after 10 months of ICI initiation. Six patients had a palmar and/or plantar involvement, while oral involvement occurred in one case. All patients had linear epidermal IgG depositions in split skin in the indirect immunofluorescence. In four out of five biopsies available for direct immunofluorescence, linear IgG and C3 depositions were detected at the basement membrane, while one patient showed linear IgM staining. Moderate to high levels of FLBP180 autoantibodies were found in seven of eight cases. The disease can still be active after ICI discontinuation, while rituximab might be required for remission. Finally, four tumour samples were stained histochemically for collagen XVII (BP180), but no enhanced expression was found.

Upregulation of HIF-1α contributes to complement activation in transplantation-associated thrombotic microangiopathy.

Transplantation-associated thrombotic microangiopathy (TA-TMA) is a severe complication of haematopoietic stem cell transplantation (HSCT). Complement activation is involved in the development of TA-TMA. However, the underlying mechanism is unclear. Therefore, 21 samples of TA-TMA and 1:1 matched controls were measured for hypoxia-inducible factor-1α (HIF-1α) and complement protein. The mechanism was investigated both in vitro and in vivo. In this study, we found that levels of HIF-1α were significantly higher in TA-TMA patients than that in non-TA-TMA controls. Upregulation of HIF-1α induced an increase in membrane-bound complement C3 and dysfunction of human umbilical vein endothelial cells (HUVECs) in vitro. Increasing HIF-1α in vivo led to C3 and C5b-9 deposition in the glomerular endothelial capillary complex, thrombocytopenia, anaemia, and increased serum lactate dehydrogenase (LDH) levels in wild-type (WT) but not in C3-/- mice subjected to HSCT. High platelet aggregation in peripheral blood and CD41-positive microthrombi in the kidney were also found in dimethyloxallyl glycine (DMOG)-treated mice, recapitulating the TA-TMA phenotype seen in patients. Comprehensive analysis, including DNA array, luciferase reporter assay, chromatin immunoprecipitation (ChIP)-seq, and quantitative polymerase chain reaction (PCR), revealed that HIF-1α interacted with the promoter of complement factor H (CFH) to inhibit its transcription. Decreased CFH led to complement activation in endothelial cells.

Predictive prognostic value of glomerular C3 deposition in IgA nephropathy.

IgAN is the most common primary glomerulonephritis worldwide. However, the pathogenesis of IgAN remains unknown. Currently, there is evidence that C3 deposition plays a role in disease development. This study aimed to investigate clinical, pathological features, and prognosis of adult IgAN patients with C3 deposition, as well as explore the role of complement activation in disease progression. A total of 821 patients with biopsy-proven IgAN were included in this study. Patients were divided into three different groups according to their C3 deposition intensity. Clinical and pathological characteristics were compared between groups. Logistic analysis was used to estimate the relationship between C3 deposition and the Oxford scoring system. Univariate and multivariate Cox proportional hazard regression models were used to analyze the effect of the presence ofC3 deposits on the prognosis of patients with IgA nephropathy. Kaplan-Meier survival analysis was used to evaluate the cumulative incidence of renal progression between groups. Patients with C3 deposition exhibited more severe clinical and pathological features and had a higher score according to the Oxford scoring system. With the increasing intensity of C3 deposition, patients present more hematuria, crescents, heavier interstitial inflammatory cell infiltration and a higher score on segmental sclerosis lesions. Logistic regression identified a positive relationship between C3 deposition and histopathology. Univariate and multivariate Cox regression indicated thatC3 deposition was an independent risk factor for IgANseverity. The Kaplan-Meier survival curves indicated that patients with positive C3 deposition had a worse prognosis compared to those without C3 deposition. Patients with positive glomerular C3 deposition presented with more severe clinical and histopathological characteristics and a higher score on the Oxford scoring system. With the increasing intensity of C3 deposition, IgAN patients were more likely to present with high level ofmicroscopic hematuria, fibrous crescents, interstitial inflammatory cell infiltration, and a higher score on segmental sclerosis lesions. C3 deposition at the time of renal biopsy is likely an independent risk factor for IgA nephropathyseverity and progression.

Association of Histologic Parameters with Outcome in C3 Glomerulopathy and Idiopathic Immunoglobulin-Associated Membranoproliferative Glomerulonephritis.

C3 glomerulopathy and idiopathic Ig-associated membranoproliferative GN are kidney diseases characterized by abnormal glomerular complement C3 deposition. These conditions are heterogeneous in outcome, but approximately 50% of patients develop kidney failure within 10 years. To improve identification of patients with poor prognosis, we performed a detailed analysis of percutaneous kidney biopsies in a large cohort of patients. Using a validated histologic scoring system, we analyzed 156 native diagnostic kidney biopsies from a retrospective cohort of 123 patients with C3 glomerulopathy and 33 patients with Ig-associated membranoproliferative GN. We used linear regression, survival analysis, and Cox proportional hazards models to assess the relationship between histologic and clinical parameters with outcome. Frequent biopsy features were mesangial expansion and hypercellularity, glomerular basement membrane double contours, and endocapillary hypercellularity. Multivariable analysis showed negative associations between eGFR and crescents, interstitial inflammation, and interstitial fibrosis/tubular atrophy. Proteinuria positively associated with endocapillary hypercellularity and glomerular basement membrane double contours. Analysis of second native biopsies did not demonstrate associations between immunosuppression treatment and improvement in histology. Using a composite outcome, risk of progression to kidney failure associated with eGFR and proteinuria at the time of biopsy, cellular/fibrocellular crescents, segmental sclerosis, and interstitial fibrosis/tubular atrophy scores. Our detailed assessment of kidney biopsy data indicated that cellular/fibrocellular crescents and interstitial fibrosis/tubular atrophy scores were significant determinants of deterioration in kidney function.

Glomerular deposition of galactose-deficient IgA1-containing immune complexes via glomerular endothelial cell injuries.

ABSTRACTBackgroundGalactose-deficient immunoglobulin A1 (Gd-IgA1) plays a crucial role in the development of IgA nephropathy (IgAN). However, the pathological role of Gd-IgA1-containing immune complexes (ICs) and the mechanism of deposition in the mesangial region remain unclear.MethodsTo examine the deposition of Gd-IgA1-containing ICs in the mesangial region through glomerular endothelial cell injury, we evaluated the alteration of renal microvascular endothelial glycocalyx in nude mice injected with Gd-IgA1-IgG ICs. Human renal glomerular endothelial cells (HRGECs) were used to assess the potential capacity of Gd-IgA1-IgG ICs to activate endothelial cells.ResultsNude mice injected with Gd-IgA1-containing ICs showed podocyte and endothelial cell injuries, with IgA, IgG and C3 depositions in glomerular capillaries and the mesangium. Moreover, albuminuria and hematuria were induced. Real-time glycocalyx imaging showed that renal microvascular glycocalyx was decreased immediately after injection of Gd-IgA1-containing ICs and then mesangial IgA deposition was increased. After coculture of Gd-IgA1-containing ICs with HRGECs, messenger RNA expression levels of endothelial adhesion molecules and proinflammatory mediators were upregulated significantly.ConclusionGd-IgA1-IgG ICs had a high affinity for glomerular endothelial cells, which resulted in glomerular filtration barrier dysfunction mediated by glycocalyx loss. Furthermore, Gd-IgA1-IgG ICs accelerated the production of adhesion factors and proinflammatory cytokines in glomerular endothelial cells. The glomerular endothelial cell injury induced by Gd-IgA1-containing ICs may enhance the permeability of Igs in the mesangial region and subsequent inflammatory responses in the pathogenesis of IgAN.

P1530: PERITRANFUSIONAL C1-INHIBITOR IN PATIENTS WITH SEVERE COMPLEMENT-MEDIATED AUTOIMMUNE HEMOLYTIC ANEMIA: AN OPEN LABEL PHASE 2 TRIAL.

Background: Complement-mediated autoimmune hemolytic anemia (AIHA) is characterized by destruction of red blood cells (RBCs) due to autoantibodies that initiate complement activation via the classical pathway. This may result in fulminant intra- and extravascular hemolysis with need for RBC transfusions, of which the efficacy can be compromised by the antibodies reacting with donor RBCs. Hemolysis induces a pro-inflammatory response that can contribute to hypercoagulability, as seen in patients with other complement-mediated hemolytic disorders such as paroxysmal nocturnal hemoglobinuria. C1-inhibitor (C1-INH; Cinryze) is an endogenous plasma regulator of the classical and lectin complement pathway approved for patients with hereditary angioedema. In a single case report and in vitro, we observed that C1-INH administration attenuated complement deposition on RBCs and subsequent hemolysis. Therefore, we hypothesized that peritransfusional administration of C1-INH in complement AIHA would reduce complement activation and hemolysis and thus improve transfusion outcome. Aims: To assess the efficacy and safety of intravenous C1-INH in patients with transfusion-dependent complement-mediated AIHA on parameters for hemolytic activity, complement activation and inflammation. Methods: We conducted a prospective, single center, phase 2 open label trial. Patients with AIHA and confirmed C3c/d-positive direct antiglobulin test, baseline hemoglobin (Hb) of ≤8 g/dL and an indication for ≥ two RBC transfusion units were included after informed consent. Treatment consisted of four peritransfusional doses of C1-INH intravenously (6000, 3000, 2000 and 1000 U) with 12 hour (hr) intervals. Blood samples at 0, 12, 24, 36, 48, 72 hrs, 7 and 14 days were analyzed for hemolytic parameters, RBC opsonization and markers of complement activation and systemic inflammation (nucleosomes and human neutrophil elastase-α1-antitrypsin complexes (HNE-α1-ATc)). Safety was monitored and scored (CTCAE version 4.0). Results: Ten patients were included in the study. At baseline, we found increased complement C3d, IgG and IgM deposition on RBCs compared to healthy controls (p=0.002, 0.005 and 0.002). Circulating C3b/c levels, nucleosomes and HNE-α1-ATc were increased compared to healthy controls (p<0.001, p=0.046 and p=0.001). C1-INH administration resulted in an increase of C1-INH antigen and -activity, peaking at 48 hrs (Fig. A). At 48 hrs after first administration, treatment significantly reduced C3d deposition on RBCs (-21%, p=0.016), but not systemic complement activation as shown by C3b/c levels (-14.96 nM, p=0.874; Fig. B). With declining C1-INH levels, C3 deposition increased again. Shortly after transfusion, Hb increased (1.59 g/dL at 12 h; p=0.008), but at 48 hrs, hemolytic and inflammation parameters did not improve (LDH -169.5 U/L, p=0.572; bilirubin total -4.306 µmol/L, p=0.987; Hb -1.217 g/dL; p=0.056; nucleosomes -17%, p=0.938; HNE-α1-ATc +21%;, p=0.297; Fig. C, D). No serious adverse events were reported. Image:Summary/Conclusion: Peritransfusional C1-INH in AIHA temporarily reduced local complement activation as evidenced by C3d deposition on RBCs, but did not affect hemolysis or systemic complement activation. Increased Hb levels at later timepoints were observed, but this effect might be confounded by additional treatments and transfusions. In conclusion, we found no (clinically) significant effect of C1-INH on hemolysis and systemic complement activation. This may be explained by the dosing of C1-INH, which possibly was too low to counter the severe complement-mediated AIHA and additional transfusion effects.

Bullous pemphigoid diagnosis: the role of routine formalin-fixed paraffin-embedded skin tissue immunochemistry

The gold standard for diagnosing bullous pemphigoid (BP) is the detection of linear deposition of IgG and/or C3 at the dermoepidermal junction using direct immunofluorescence (DIF). Because DIF has several disadvantages, primarily the requirement for frozen specimens, we assessed the diagnostic value of immunohistochemical (IHC) staining for BP detection. Eighty-eight patients with bullous lesions were included in this study. IHC staining for C3d, C4d, and IgG was performed on 88 samples, which included specimens from patients with DIF-confirmed BP (n = 43), clinicopathologically suspected BP with negative DIF results (n = 9), and other bullous diseases (n = 36). Diagnosis based on positive results for C3d, C4d, or IgG in IHC staining detected 86% of DIF-confirmed BP cases. The sensitivity of IHC staining for the detection of DIF-confirmed BP cases and clinicopathologically suspected BP cases was similar to that of DIF (80.8% vs. 84.3%), but the specificity was higher (83.3% vs. 75.0%). Five of the nine clinicopathologically suspected BP cases were diagnosed using IHC staining. Thus, IHC staining of routine biopsy material could be an alternative method for diagnosing BP. IHC staining has considerable diagnostic potential, especially in cases with a high suspicion of BP, but negative or suboptimal DIF results.Please check and confirm the author names and initials are correct. Author 2: Given name: [Chul Hwan] Family name: [Kim], Author 3: Given name: [Yoo Jin] Family name: [Lee].Checked it.

An Emerging Role for Neutrophil Extracellular Traps in IgA Vasculitis: A Mini-Review

Immunoglobulin A vasculitis (IgAV) is the most common systemic small vessel vasculitis in childhood. Its clinical manifestations are non-thrombocytopenic purpura, accompanied by gastrointestinal tract, joint, kidney and other organ system involvement. The pathogenesis of IgAV has not been fully elucidated. It may be related to many factors including genetics, infection, environmental factors, and drugs. The most commonly accepted view is that galactose-deficient IgA1 and the deposition of IgA and complement C3 in small blood vessel walls are key contributors to the IgAV pathogenesis. Extensive neutrophil extracellular traps (NETs) in the peripheral circulation and skin, kidney, and gastrointestinal tissue of patients with IgAV has been identified in the past two years and is associated with disease activity. This mini-review provides a possible mechanism for NETs involvement in the pathogenesis of IgAV.

A desirable transgenic strategy using GGTA1 endogenous promoter-mediated knock-in for xenotransplantation model

Pig-to-human organ transplantation is a feasible solution to resolve the shortage of organ donors for patients that wait for transplantation. To overcome immunological rejection, which is the main hurdle in pig-to-human xenotransplantation, various engineered transgenic pigs have been developed. Ablation of xeno-reactive antigens, especially the 1,3-Gal epitope (GalT), which causes hyperacute rejection, and insertion of complement regulatory protein genes, such as hCD46, hCD55, and hCD59, and genes to regulate the coagulation pathway or immune cell-mediated rejection may be required for an ideal xenotransplantation model. However, the technique for stable and efficient expression of multi-transgenes has not yet been settled to develop a suitable xenotransplantation model. To develop a stable and efficient transgenic system, we knocked-in internal ribosome entry sites (IRES)-mediated transgenes into the α 1,3-galactosyltransferase (GGTA1) locus so that expression of these transgenes would be controlled by the GGTA1 endogenous promoter. We constructed an IRES-based polycistronic hCD55/hCD39 knock-in vector to target exon4 of the GGTA1 gene. The hCD55/hCD39 knock-in vector and CRISPR/Cas9 to target exon4 of the GGTA1 gene were co-transfected into white yucatan miniature pig fibroblasts. After transfection, hCD39 expressed cells were sorted by FACS. Targeted colonies were verified using targeting PCR and FACS analysis, and used as donors for somatic cell nuclear transfer. Expression of GalT, hCD55, and hCD39 was analyzed by FACS and western blotting. Human complement-mediated cytotoxicity and human antibody binding assays were conducted on peripheral blood mononuclear cells (PBMCs) and red blood cells (RBCs), and deposition of C3 by incubation with human complement serum and platelet aggregation were analyzed in GGTA1 knock-out (GTKO)/CD55/CD39 pig cells. We obtained six targeted colonies with high efficiency of targeting (42.8% of efficiency). Selected colony and transgenic pigs showed abundant expression of targeted genes (hCD55 and hCD39). Knocked-in transgenes were expressed in various cell types under the control of the GGTA1 endogenous promoter in GTKO/CD55/CD39 pig and IRES was sufficient to express downstream expression of the transgene. Human IgG and IgM binding decreased in GTKO/CD55/CD39 pig and GTKO compared to wild-type pig PBMCs and RBCs. The human complement-mediated cytotoxicity of RBCs and PBMCs decreased in GTKO/CD55/CD39 pig compared to cells from GTKO pig. C3 was also deposited less in GTKO/CD55/CD39 pig cells than wild-type pig cells. The platelet aggregation was delayed by hCD39 expression in GTKO/CD55/CD39 pig. In the current study, knock-in into the GGTA1 locus and GGTA1 endogenous promoter-mediated expression of transgenes are an appropriable strategy for effective and stable expression of multi-transgenes. The IRES-based polycistronic transgene vector system also caused sufficient expression of both hCD55 and hCD39. Furthermore, co-transfection of CRISPR/Cas9 and the knock-in vector not only increased the knock-in efficiency but also induced null for GalT by CRISPR/Cas9-mediated double-stranded break of the target site. As shown in human complement-mediated lysis and human antibody binding to GTKO/CD55/CD39 transgenic pig cells, expression of hCD55 and hCD39 with ablation of GalT prevents an effective immunological reaction in vitro. As a consequence, our technique to produce multi-transgenic pigs could improve the development of a suitable xenotransplantation model, and the GTKO/CD55/CD39 pig developed could prolong the survival of pig-to-primate xenotransplant recipients.

The therapeutic effects of human embryonic stem cells-derived immunity-and-matrix regulatory cells on membranous nephropathy

BackgroundPrimary membranous nephropathy (MN) is a kidney-specific autoimmune disease. Human embryonic stem cells-derived immunity-and-matrix regulatory cells (hESC-IMRCs) have immunoregulatory functions. We hypothesized that hESC-IMRCs might have therapeutic effects on MN and be a potential treatment in clinical practice.MethodsRats of Heymann nephritis were injected with sheep anti-rat Fx1A serum. hESC-IMRCs were intravenously administrated upon the detection of proteinuria, with 6 × 106 cells (high-dose) or 3 × 106 cells (low-dose) in 1 ml every other day. Splenocytes and IMRCs were co-cultured at different times and ratios. Cell types and cytokines were detected by flow cytometry and enzyme-linked immunosorbent assay.ResultsThe urinary protein of rats with Heymann nephritis was reduced remarkably to a level comparable to negative controls, in both low-dose (45.6 vs. 282.3 mg/d, P < 0.001) and high-dose (35.2 vs. 282.3 mg/d, P < 0.001) hESC-IMRC treatment groups. IgG and C3 deposit, glomerular basement membrane thickness and foot process effacement were alleviated and the reduced podocin was recovered in the kidneys. The proportions of CD4 + CD25 + T cells in circulation and spleen were increased, and the circulating level of IL-10 was increased, after IMRC interventions. IL-17 and TNF-α were reduced after IMRCs treatments. IL-10 increased remarkably in the co-culture supernatant of lymphocytes and IMRCs at 48 h with ratio 10:1.ConclusionsThe intravenously delivered hESC-IMRCs alleviated proteinuria and kidney injuries of Heymann nephritis, by their immunosuppressive functions through regulatory T cells and IL-10. These pre-clinical results indicate that IMRCs worth careful consideration for human trials in the treatment of MN.

Bullous systemic lupus erythematosus with nephrotic syndrome in an adolescent girl: a case report

Bullous systemic lupus erythematosus is an uncommon autoimmune subepidermal vesiculobullous disease occurring in less than 5% of patients with systemic lupus erythematosus. The diagnosis of bullous systemic lupus erythematosus is based on clinical picture, histopathological findings, direct immunofluorescence and other immunologic tests. Importantly, establishing the diagnosis of bullous systemic lupus erythematosus requires the occurrence of systemic lupus erythematosus according to the American College of Rheumatology criteria. We report a case of bullous systemic lupus erythematosus in a 16-year-old Vietnamese girl, who demonstrated an acquired vesiculobullous eruption associated with nephrotic syndrome and fulfilled the European League Against Rheumatism/ American College of Rheumatology 2019 classification criteria of systemic lupus erythematosus. Clinical examination showed numerous tense, clear fluid-filled vesicles and bullae affecting the whole body with the predilection for the face, neck, chest, abdomen and bilateral inner thigh areas. Erosions were observed on the tongue and the buccal mucosa. Histopathologic examination showed subepidermal vesicle containing abundant neutrophils and perivascular mixed inflammatory cell infiltration. Microabscesses at tips of dermal papillae and the features of a leukocytoclastic vasculitis were not seen. Direct immunofluorescence of perilesional skin biopsies demonstrated linear deposition of IgG, IgA, IgM and C3 in the basement membrane zone. Linear immunoglobulin deposition displayed a u-serrated pattern with the predominant staining intensity of IgG. Serologic tests showed positive antinuclear antibody. Complement components assay revealed low levels of C3 (66.5 mg/dL) and C4 (3,9mg/dL). Proteinuria level was increased to 3.5g/24 hours. The final diagnosis of bullous systemic lupus erythematosus with nephrotic syndrome was made. The patient was treated with a high-dose oral glucocorticoid scheme and showed significant clinical improvement.

POS-032 Human PLA2R-antibodies induce membranous nephropathy in minipigs and activate complement

POS-032 Human PLA2R-antibodies induce membranous nephropathy in minipigs and activate complement