key: cord-0074332-puibl2j8 authors: nan title: Abstracts of the 2nd Toronto Complement Conference: 2021 November 11-13 date: 2022-01-26 journal: Can J Kidney Health Dis DOI: 10.1177/20543581211069286 sha: 07c25c4ec8d4bb745845ecba81434a4f6044c2b4 doc_id: 74332 cord_uid: puibl2j8 nan To investigate the role of FH in macrophages, bone marrow derived macrophage cultures were established from wildtype and FH deficient (FH-/-) mice. Different techniques such as Gene expression analysis, Sea horse etc for analyzing the cells. Results: Wildtype(WT) macrophages in culture express FH and have a significantly higher M1/M2 ratio than FH-/-macrophages. FH deficiency reduced phagocytic activity in macrophages. Seahorse analysis showed reduced basal mitochondrial respiration, ATP production and maximal respiratory capacity in FH deficient macrophages, which is not affected by serum treatment or C3a and C5a, proteins generated by complement activation. Absence of FH showed a marked inability to form stable lamellipodia necessary for directional locomotion. Nanostring gene expression analysis revealed that the FH dependent gene changes in mouse macrophages include upregulation of inflammatory genes such as CX3CR, CXCR, IL-6 and TNFa, while genes such as antifibrotic MMP19 and antiproliferative BMP6 are reduced, which was substantiated by qRT-PCR. Conclusion: For the first time, our studies show the functional relevance of intrinsic FH in macrophages. Background: Epidermolysis bullosa acquisita (EBA) is a rare blistering skin disease induced by autoantibodies directed against type VII collagen (COL7) [1, 2] . Transfer of antibodies against murine COL7 (mCOL7) into mice mimics the effector phase of EBA and results in a subepidermal blistering phenotype [3, 4] . Activation of the complement system and especially the C5a/C5aR1 axis and subsequent activation of neutrophils are critical for EBA pathogenesis [5, 6] . However, the role of the second C5a receptor, C5aR2, in the pathogenesis of EBA is still elusive. We sought to delineate the functional relevance of C5aR2 in the effector phase of EBA by injecting anti-mCOL7 antibodies into C5aR2-deficient (C5ar2 -/-) and wildtype control mice. To decipher the molecular mechanisms mediated by C5aR2, neutrophils from C5ar2 -/-, C5ar1 -/and wild-type control mice were activated in vitro and then their biological response was analyzed. Results: In our mouse model, C5ar2 -/mice showed an attenuated disease phenotype, similar to C5ar1 -/mice, suggesting a pathogenic contribution of C5aR2. In addition, neutrophils from C5ar2 -/mice exhibited significantly lower C5a-mediated activation and migration in vitro. Both functions are completely absent when C5ar1 -/neutrophils are activated. Discussion: Our in vitro-data suggest that C5aR2 might influences C5a-mediated neutrophil activation and migration by enhancing C5aR1 signaling. This is further supported Figure 1 . GT103 (200 µg/mL) + NHS (10%) treatment of H460 tumor cells causes increased C3b/iC3b. Heat inactivated NHS and nonspecific IgG3 control antibody were used as negative controls. Cells were treated for 1 hr at 37°C. Each condition was done in triplicate. NHS + GT103 vs NHS + IgG3 p value = 0.02 by our in vivo-study, where C5aR2 deficiency ameliorated disease development. An effect that leads to complete disease protection when C5aR1 is missing [5, 6] . Conclusion: Collectively, we demonstrate here a proinflammatory contribution of C5aR2 to the pathogenesis of (auto-)antibody-induced tissue damage in experimental EBA, which might be mediated by enhancing C5aR1 signaling. Background: C3 glomerulopathy (C3G) is a rare progressive kidney disease attributed to dysregulation of the complement alternative pathway (AP). We describe baseline biomarker and clinical characteristics of C3G patients in two phase II studies of the oral factor D inhibitor danicopan (ALXN2040). Conclusions: Fluid phase biomarker assessments and correlations confirmed systemic terminal pathway activation associated with AP dysregulation in C3G patients. Ba and FD were strongly correlated with eGFR, confounding their use as markers of complement activation. Additional characterizations including autoantibodies and genetic variants will contribute further to our characterization of C3G pathophysiology. Background: TMAs can show a wide spectrum of findings in renal biopsies. The role of nephropathology in the context of genetics and laboratory findings in the diagnosis, determination of etiology, prediction of therapy response and prognosis is unclear. Although clinicopathological registries could address these questions, large-scale nephropathology annotation seems too costly and prone to variation. In this proof-of-concept study, we propose a novel deep learning network architectures allowing multimodal data integration from the three decisive tissue compartments and the four routine nephropathological stainings. Material and methods: 25 renal biopsies with a histopathological diagnosis of TMA and n=25 mimickers of TMA (ANCA-associated necrotizing arteritis, severe benign nephrosclerosis, cryoglobulinemic vasculitis, Bevacizumab-plugs) from two institutions were digitized. A total of 1366 arteries, 9561 arterioles and 7112 glomeruli from HE, PAS, trichrome and silver stainings at a 40x scan magnification were loaded into a novel network architecture including transformer and perceiver operations to train a binary classifier of TMA vs. Mimicker with five-fold internal cross-validation. Results: So far, without hyperparameter tuning, we achieved a binary diagnostic accuracy of 82% through five-fold internal cross-validation. Discussion: Our novel architecture can be trained to integrate histopathological findings from arteries, arterioles and glomeruli to render a diagnosis of TMA across the four common nephropathological stainings. This novel architecture could be expanded with further input channels like tubulointerstitial tiles, EM images, genetic sequences and laboratory data to address open questions regarding TMAs. Together with powerful segmentation models this architecture enables the composition and examination of large clinicopathological registries. Dos Background: Atypical Hemolytic Uremic Syndrome (aHUS) is a complement-mediated thrombotic microangiopathy. Pathophysiological mechanism involves uncontrolled complement activation due to a genetic or acquired anomaly coupled with a triggering event. We report a case of aHUS recurrence following COVID-19 vaccination. Whole blood (EDTA) was collected and processed with CD46-PE, CD45-PerCP, isotype control-PE markers. Staining was measured through median fluorescence intensity and expressed as CD46/isotype ratio. Sanger sequencing was used for identification of variants in CD46 gene. All the participants provided informed written consent. Results: Proband (P) is a 39-year-old woman admitted for nausea, vomiting, epigastric pain and haematuria, three days after first dose of ChAdOx1 nCov-19 vaccine. Laboratory testing showed MAHA (Hb:8.8g/dL, Ht:26%), thrombocytopenia (80x10 9 /mm 3 ) and acute kidney injury (Cr:2.15mg/dL, Ur:92mg/dL). P and three of her siblings have experienced recurrent TMA episodes since childhood. In 2019, genetic study from P's sister (S) identified two heterozygous variants in CD46, one pathogenic (Glu179Gln) and one of uncertain significance (Cys94Tyr). We demonstrated that P carries the same variants and observed a 50% decrease of CD46 expression in both P and S ( fig.1 ). Platelet transfusion, corticosteroids and 9 sessions of plasmapheresis contributed to rapid recovery of P. Discussion: Glu179Gln was reported to increase CD46 expression on granulocytes in aHUS patient and to reduce C4b cofactor activity 1 . We observed that combination of Glu179Gln and Cys94Tyr was associated with low levels of CD46 on cell surface. This case report supports the evidence of COVID-19 vaccine as a precipitating event for aHUS recurrence. A. Flow cytometric analysis using phycoerythrin (PE)-conjugated anti-CD46 was performed 24 hours after blood collection for the three samples. B. Staining was measured through median fluorescence intensity (MFI) and reference intervals (RI) were previously calculated using ratio CD46 MFI/isotype MFI in 20 control individuals. CD46 expression from P and S is decreased by approximatively 50% compared with median of reference samples. Background/aims: Factor H (FH) is a critical regulator of the alternative complement pathway in both mice and men. Therefore, it is not surprising that FH abnormalities are associated with renal diseases. Macrophages (Mφs) play a critical role in FH dependent immune complex glomerulonephritis (ICGN), but the mechanism remains unclear. Methods: In this study, ICGN was induced by immunizing 8-wk-old FH knockout (Cfh −/− ) mice daily for 5 wk with 4 mg horse spleen apoferritin given ip. To determine the role of Mφs in this setting, during the last two weeks of apoferritin treatment, one group of mice received clodronate that selectively depletes Mφs in mice, while the other group received saline. Kidney pathology and function was assessed using different techniques. Results: Gene expression analysis of kidneys Cfh −/− mice with ICGN revealed that a number of Mφ related genes were significantly elevated, compared to the kidneys from control mice. In mice, monocytes/Mφs were significantly reduced in circulation by seven days after clodronate administration. Chlodronate treated, ICGN mice had significantly lower levels of both serum creatinine (p<0.05) and BUN (p<0.02), compared to PBS/ICGN mice. In line with BUN and creatinine, histopathological kidney injury is significantly reduced. Chlodronate treatment did not significantly change C3 and IgG deposits, but reduced laminin in the kidney. The expression of Mφ inflammatory mediators such as ICAM, MMP9 and iNOS were reduced with chlodronate treatment. Our results indicate an important role for macrophages in the pathogenesis of FH dependent ICGN, and implicate macrophages as a potential therapeutic target. Background: Renal cortical necrosis is a rare cause of acute kidney injury (AKI) characterized by diffuse or patchy ischemic coagulation necrosis of cortex. One of important cause is Acute Thrombotic Microangiopathy (TMA). It is very crucial to diagnose & treat it early to avoid morbidity & mortality. Here we report a case of TMA with cortical infarct with poor response to plasmapheresis & steroid, treated with Intravenous Immunoglobulin (IVIG). A 30-year female, presented with RPGN with anuria with with no extrarenal features of organ involvement. She found to have severe progressive anemia with mild thrombocytopenia with raised serum LDH, mild indirect hyperbilirubinemia with normal coagulation parameters with low C3, normal C4, immunological marker & APLA negative. Renal Biopsy done suggestive of acute TMA with cortical necrosis. Her urine output improved with 5 sessions of Plasmapheresis (PLEX), pulse steroid, followed by oral steroid. But her Renal parameters & anemia continued to worsening and she remained dialysis dependent. She received IVIG, to which she responded and currently she is off dialysis & hematological & renal parameters improving. Her complement gene analysis report is awaited. Discussion: Anuria with dialysis dependency are bad prognostic markers for case of TMA with cortical necrosis, if there is delay in diagnosis or treatment. In a resource poor setting, we should avoid delay in treatment. In this case refractory to treatment to standard therapy got good response to IVIG. In a case of Acute TMA with cortical infarct IVIG may help if there is partial or failure to response to standard therapy. Background: Atypical haemolytic uraemic syndrome (aHUS) is a rare disease that manifests as complementmediated thrombotic microangiopathy (TMA), which can lead to severe organ damage. Some patients with aHUS may present with malignant hypertension (MHT); both conditions can result in TMA. The objective of this analysis was to characterise patients with aHUS and MHT. In this analysis, patients from the Global aHUS Registry (NCT01522183) were included if they were diagnosed with MHT and were followed ≥90 days after initial aHUS symptom presentation or diagnosis date. Demographics and clinical characteristics were evaluated (table) . Results: Seventy-one of 1903 registry patients were included in the analysis. Sixty-nine percent of patients were reported to have MHT at around the same time as aHUS diagnosis (+/-2 months), while 11% and 13% experienced MHT before and after aHUS diagnosis, respectively. aHUS triggering conditions were reported in 6/71 patients (8%) (Table) . were the most commonly reported extra-renal manifestations. Eight patients (11%) had a reported family history of aHUS and 40 patients (56%) had a complement pathogenic variant or an anti-CFH-antibody. Thirty-three patients (46%) had a kidney transplant; of these, 20 were prescribed eculizumab in the peri-or post-transplant period. In this analysis of patients with aHUS and MHT, the observed high prevalence of pathogenic variants in complement genes or anti-CFH antibodies, alongside the high proportion of patients with extrarenal manifestations and/or requiring kidney transplant, indicate a high severity of presentation and poor prognosis of aHUS associated with MHT. Background: sC5b-9 reflects complement cytolytic function. Its role as a biomarker of complement activation in atypical HUS has not been elucidated. We report 2 cases of 6 and 7 year-old girls with atypical HUS attributed to single nucleotide polymorphisms in C3 (rs2230199) and CFH (rs800292) gene in the first case and in C3 (rs2230199) gene in the second case. Eculizumab therapy was initiated in both patients. sC5b-9 levels were measured, and HUS relapse episodes were recorded. Results: sC5b-9 level was high at initial HUS presentation in both patients (333 ng/ml and 342 ng/ml respectively, normal range <245 ng/ml) and fell after eculizumab initiation (201 ng/ml and 275 ng/ml respectively). In the first patient, therapy was postponed at 3 and at 13 months after disease onset, leading to high sC5b-9 levels (913 ng/ml and 1020 ng/ ml respectively), but clinical HUS relapse was recorded only during the first therapy postponement. In all cases, sC5b-9 levels fell to normal range after therapy re-initiation. In the second patient, therapy was postponed at 5 months after disease onset, leading to clinical HUS relapse, without rise in sC5b-9 levels (231 ng/ml). Discussion: sC5b-9 levels rise and reduction after eculizumab postponement and re-initiation suggest that longitudinal sC5b-9 changes reflect complement activation. Nevertheless, we observed a variability of sC5b-9 levels during HUS relapse at both inter-individual and intra-individual level. Conclusions: sC5b-9 may be useful for monitoring complement activation in atypical HUS in some patients but cannot predict HUS relapse at both inter-individual and intra-individual level Background: The concomitant incidence of shiga-toxin Escherichia Coli (STEC) infection and atypical HUS has been rarely reported. A 6-year-old girl was presented with HUS without vomiting or diarrhea. At 5 th day of hospitalization, peritoneal dialysis therapy was started and lasted 20 days. During hospitalization, the patient presented pulmonary oedema and transient right ventricular diastolic dysfunction, necessitating an ICU stay for 2 days. Results: At admission, fresh frozen plasma infusion therapy was initiated due to atypical clinical presentation and continued for 20 days. Of note, shiga-toxin 1 was detected at stool-PCR test, despite the absence of gut symptoms. ADAMTS13 activity was normal. Initial serum C5b-9 level was high (333 ng/ml, normal range<245 ng/ml). Further complement analysis revealed single nucleotide polymorphisms at both C3 (rs2230199) and complement factor H (rs800292) genes. Due to the observed complement activation dysregulation, eculizumab therapy was initiated and serum C5b-9 levels fell to normal range (201 ng/ml). The patient presented HUS relapse 3 months later, concomitantly to rise in C5b-9 levels (913 ng/ml), due to transient therapy postponement, which was rapidly resolved after eculizumab infusion. Serum C5b-9 levels gradually fell to normal range (159 ng/ml). Discussion: In our case, asymptomatic STEC infection was associated with atypical HUS, suggesting that shiga-toxin may trigger uncontrolled complement activation in pediatric patients with inherited complement gene deficits. Conclusion: Complement activity analysis needs to be included in the initial diagnostic work-up of HUS, even at the presence of documented STEC infection. Riedl Khursigara, M 1 , Radhakrishnan, S 1,4 , Noone, D 1,4 , Chami R 2 , Tjon, J 3 , Parekh, R 1,4 , langlois, V 1,4 , licht, C 1,4 , Teoh, CW 1,4 Background: C3 glomerulonephritis (C3GN) is caused by complement alternative pathway dysregulation and is characterized by progression to ESRD. Terminal complement blockade via eculizumab has successfully been used especially in patients with elevated C5b-9 levels. Results: We describe a 6 year old boy with C3GN, who presented with nephrotic syndrome, severe hypertension (on 4 anti-hypertensive medications) and acute kidney injury. Complement C3 level was 0.12 g/L (0.8-1.5) with pos C3NeF and elevated C5b-9 levels (2135, <239ng/ml). Despite 6 months of treatment with prednisone and MMF, he had ongoing nephrotic syndrome and worsening kidney function. He commenced on standard Eculizumab dosing for aHUS. Despite 6 months of therapy, he had persistent severe hypertension, nephrotic syndrome requiring weekly albumin/furosemide infusions and worsening kidney function. Complement C3 levels remained low with elevated C5b-9 levels, suggesting sub-optimal terminal complement inhibition due to urinary loss. We confirmed sub-therapeutic plasma concentrations of eculizumab as free plasma eculizumab levels were low on day 7 (9, >99 ug/ml) and undetectable on days 10 and 14 post-infusion. On the other hand, Eculizumab was detected in the urine, indicating urinary loss. He was subsequently increased to weekly eculizumab infusions and MMF dosing was adjusted using MPA AUC measurements. Since then, his kidney function, C5b-9 levels (297 ng/ml) and nephrotic syndrome improved significantly (see Figure) , leading to discontinuation of albumin/furosemide and anti-hypertensive medications. (see Figure) . Use of pharmacokinetic studies can aid in individualized eculizumab treatment in those with C3GN who failed to respond to standard dosing, especially in patients with significant proteinuria as Eculizumab can be lost in the urine. Overview of kidney function, proteinuria, C3 levels and MPA AUC since patient presented (Feb 2020). Vertical lines indicate start of Eculizumab Q14d and Eculizumab Q7d. Patient received a prednisone taper within first months and was started on MMF, which was increased to target an AUC >30mg*h/L. The probands with high anti CFH titres signified severe disease,we resorted to PLEX and cyclophosphamide 3 therapy in both but one expired eventually.MMF started as maintainence therapy in the other. Conclusion: Therefore, early diagnosis of pathogenic variants and initiation of PLEX along with immunosuppressive therapy can result in delaying ESRD in complement mediated diseases & also can help in shaping up future transplants in appropriate time. TILs from CMT-167 tumors treated with GT103 or IgG2 control antibody. The anchor genes between samples, identified using the FindIntegrationAnchor function, were used as a basis for Seurat to perform integration of the samples. The data were scaled before running Principal Component Analysis (PCA). Cluster identification and annotation were performed using a shared nearest neighbor modularity optimization-based clustering algorithm and FindNeighbors function with 50 principal components, followed by the FindClusters function with the Louvain algorithm using a 0.5 resolution. Background: C3 Glomerulopathy (C3G) has been has been re classified as a glomerular complement mediated disease, with predominant C3 deposits almost a decade ago. Mutations and risk haplotypes in several complement system components and circulating autoantibodies result the loss of control of the alternative pathway has been described in patients with C3G. i , ii High incidence of disease recurrence after transplantation is reported -between 30 and 77%, including graft failure in 17-50% of cases. Modalities of preventing and treating C3G recurrence after kidney transplantation includeplasma exchange, Rithuximab, Eculizumab with various reports regarding success. iii , iv Disease recurrence rate after transplantation in childhood onset C3G is still unknown. The aim of our study was to retrospectively describe our cohort of childhood onset C3G transplanted patients, including treatment modalities and outcomes. Methods: Retrospective cohort study, data regarding patients diagnosed with C3G as children or adolescents and underwent kidney transplantation between 2015-2020 was collected, including complement workup, treatment modalities and outcomes. Results: Five patients underwent kidney transplantation, in four (80%) disease recurrence was diagnosed. Two patients improved after Eculizumab treatment (both had factor H antibodies), one patient reached graft failure 9 months after transplantation despite treatment with Eculizumab, one patient showed histologic signs of disease recurrence without clinical signs. Conclusions: C3G recurrence rate after kidney transplantation in patients diagnosed as children or adolescents may be higher than described in the literature, treatment was successful only in part of the patients, new treatments are needed in order to safely transplant C3G patients and avoid disease recurrence. Molecular mechanisms of complement evasion: learning from staphylococci and meningococci Insights Into the Structure-Function Relationships of Dimeric C3d Fragments Complement factor H autoantibodies are associated with early stage NSCLC Changing picture of renal cortical necrosis in acute kidney injury in developing country Complement in Secondary Thrombotic Microangiopathy Making the Correct Diagnosis in Thrombotic Microangiopathy: A Narrative Review Thrombotic microangiopathies assessment: mind the complement Intravenous gamma globulin for thrombotic microangiopathy of unknown etiology Genetics and complement in atypical HUS Acquired and genetic complement abnormalities play a critical role in dense deposit disease and other C3 glomerulopathies Haemolytic uraemic syndrome Human complement factor H is a novel diagnostic marker for lung adenocarcinoma Complement factor H autoantibodies are associated with early stage NSCLC Complement Factor H Antibodies from Lung Cancer Patients Induce Complement-Dependent Lysis of Tumor Cells, Suggesting a A Therapeutic Antibody for Cancer, Derived from Single Human B Cells C3 glomerulopathy: consensus report Clinical findings, pathology, and outcomes of C3GN after kidney transplantation Primary disease recurrence-effects on paediatric renal transplantation outcomes The overexpression of complement factor H (CFH) is associated with poor prognosis in lung cancers (1), and we have shown earlier that patients with early stage lung cancer had significantly higher incidence of anti-CFH antibodies than those with late-stage disease (2) . A therapeutic antibody, GT103 was developed by isolating and cloning antigen specific B cells from patients expressing autoantibodies to complement factor H. GT103 was shown promote complement activation, kill tumor cells in vitro by complement CDC and inhibits tumor growth in vivo (3, 4) . We report here that GT103 also exerts an immune modulatory effect within the tumor micro-environment. Tumor growth studies were done using the syngeneic CMT167 mouse lung cancer model. Immune profiling of tumors from IgG2 control and GT103treated mice was done using flow cytometry. Genome wide analysis of tumors was performed using bulk and single cell RNA sequencing.Results: Genome wide analysis and B cell depletion studies suggest that GT103 activates B-cell receptor signaling. Further, ScRNA-seq demonstrated that not only total intratumoral CD4+, CD8+ T and B lymphocytes were elevated in GT103-treated mice but Tregs, monocytic MDSCs and neutrophils with an immunosuppressive profile were reduced in GT103-treated tumors.Discussion: In addition to increasing the influx and activation of immune stimulatory T and B cells, GT103 also limits immune suppressive cell populations like Tregs, and monocytic MDSC and neutrophils in the tumor micro-environment.Conclusion: GT103 treatment creates a favorable immune microenvironment and induces anti-tumor immunity thus limiting the growth of CMT167 lung tumors.