Low Prevalence of Pathogenic or Likely Pathogenic Variants in a US Cohort Evaluated for Genetic Causes of Atypical Hemolytic Uremic Syndrome (aHUS) (POSTER #16)
Mitchell G. Springer1, Juliana Perez Botero1,2, Stefanie N. Dugan1, Jamie McCreery1, Ruchika Sharma1,2, Valerie Trapp-Stamborski1, Mia J. Sullivan1, Sean Tracey1 and Kenneth D. Friedman1,2
1Versiti Blood Center of Wisconsin, Milwaukee, USA
2Medical College of Wisconsin, Milwaukee, USA
Background: Uncontrolled activation of the complement system underlies the majority of atypical hemolytic uremic syndrome (aHUS) cases. Older reports indicated positive genetic findings in 55% to 0% of cases; a recent study downgraded 10 of 61 variants reported in Human Gene Mutation Database (HGMD) from pathogenic to likely benign and reported finding pathogenic or likely pathogenic variants in approximately 1 of 5 of cases. We report our 5-year experience providing specialty reference laboratory aHUS genetic evaluation.
Methods: 384 patients were tested from February 2014 to September 2019 with a 15-gene panel (ADAMTS13, C3, C4BPA, C4BPB, CFB, CFH, CFHR1, CFHR3, CFHR4, CFHR5, CFI, DGKE, LMNA, MCP, and THBD) utilizing massively parallel sequencing supplemented with Sanger sequencing for variant confirmation and multiplex ligation-dependent probe amplification (MLPA) analysis for deletion/duplication detection in CFH and CFHR1/3/4/5 genes. Pathogenicity was classified following published best practices and then 2015 American College of Medical Genetics and Genomics (ACMG) guidelines: pathogenic, likely pathogenic, and variants of uncertain significance (VUS) were reported.
Results: Sequencing identified a total of 211 reportable variants in 143 (37.2%) of 384 patients: 23 (10.9%) were classified as pathogenic, 6 (2.8%) as likely pathogenic, and 182 (86.3%) as VUS. MLPA detected homozygous deletion of CFHR1 (conferring factor H-autoantibody development risk) in 30 (7.8%) patients.
Conclusions: Detection rate of pathogenic or likely pathogenic sequence variants was 13.8%, lower than in older published reports. Contributors to this discrepancy may include advances in human genetics and variant interpretation guidelines, and lower accuracy of aHUS diagnosis in clinical care versus in well-characterized research cohorts. Disseminating the limitations and implications of aHUS genetic testing may prevent overutilization.
Proteomics of Complement in Thrombotic Microangiopathy (POSTER #17)
Sanjeev Sethi1, Lilian Monteiro P. Palma2, Benjamin Madden3, and M. Cristine Charlesworth3
1Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA
2Pediatric Nephrology, State University of Campinas (UNICAMP), Brazil
3Medical Genome Facility, Proteomics Core, Mayo Clinic, Rochester, MN, USA
Background: Thrombotic microangiopathy (TMA) is a clinical-pathological entity that results from different pathophysiological mechanisms. Distinguishing atypical hemolytic uremic syndrome (aHUS) from secondary causes of TMA is a challenge. A comprehensive and thorough description of complement proteins including complement burden in different causes of TMA has not been described.
Material and Methods: Through laser microdissection and mass spectrometry (MS/MS), glomeruli were dissected (Figure 1) and glomerular complement protein profile was performed in a case of STEC-HUS (Escherichia coli), severe hypertension-, drug-induced, aHUS-associated TMA.
Results: C3 was the most the abundant complement protein. MS/MS also identified large spectral counts (in decreasing order) of C4, C9, C6, C7, and C8 in all cases (Figure 2). The spectral counts of complement proteins were much higher (3- to 4-fold) in aHUS and drug-induced TMA compared with STEC-HUS and hypertension-associated TMA. In addition, higher spectral counts of complement regulating proteins FH, FHR-5, FHR-1, and FHR-2 were detected in aHUS and drug-induced TMA compared with STEC- and hypertension-associated TMA.
Discussion: Complement proteins presented the highest spectral counts among 1500 to 2000 proteins identified in TMA. The identification of C4 indicates a role for classical and/or lectin pathway (even in aHUS). Terminal complement proteins were also identified and may likely contribute to glomerular injury. A limitation is the small number of cases. MS/MS of a larger series is ongoing.
Conclusions: Significant complement activation is present in TMA as evidenced by large spectral counts of complement and complement-regulating proteins. The burden of complement deposition appears to be different according to TMA cause.
In the kidney biopsies of patients presenting different causes of Thrombotic Microangiopathy, 10 micron thick formalin-fixed paraffin sections were obtained and mounted on a special PEN membrane laser slide (Panel A); glomeruli were microdissected using a Zeiss Palm MicroBeam microscope the to reach approximately 250 to 500,000 M2 per case (Panel B).
Mass spectrometry of complement proteins in kidney biopsies according to the cause of Thrombotic Microangiopathy. Total spectral counts estimate the abundance of the protein in the tissue.
Clinical Value of Urinary Complement Biomarkers in Autoimmune Glomerulonephritis (POSTER #18)
Background: Complement activation plays a central role in the mechanisms of injury of autoimmune glomerular diseases. Urinary excretion of complement biomarkers could indicate relevant activated pathogenic pathways, parallel disease activity, and add clinical value beyond proteinuria.
Material and Methods: We performed a prospective observational cohort of 83 patients including focal segmental glomerulosclerosis, membranous nephropathy, IgA nephropathy, lupus nephritis, and Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). We measured at different time points proteinuria, urinary C4a (classical and lectin pathway), Bb (alternative pathway), and sC5b-9 (terminal cascade) expressed as creatinine ratios. We assessed remission status as currently defined for each disease. For AAV, we assessed renal Birmingham Vasculitis Activity Score (BVAS) score after 6 months of treatment.
Results: At baseline, urinary excretion of sC5b-9 was present in each individual (4.28, interquartile range [IQR] = 0.84-2.96 µg/mmol creatinine) and correlated with the initial proteinuria (P < .05 for each disease). Urinary C4a and Bb were mostly absent. In those who obtained clinical remission (Table 1), we observed a 92% reduction in urinary sC5b-9 levels, which was greater than the 69% reduction observed in proteinuria (P = .02 by Wilcoxon signed rank test). The same pattern occurred in each disease group and reached statistical significance for MN (P = .05) and AAV (P = .03).
Discussion: In active autoimmune glomerular diseases, urinary sC5b-9 is measurable in all individuals and correlates with the initial proteinuria. In those who obtained a clinical remission, the urinary sC5b-9 reduction was greater than the decline observed with proteinuria, suggesting earlier and more precise variations in sC5b-9.
Conclusions: sC5b-9 could be a more sensitive marker of remission in glomerular diseases and a useful clinical tool to monitor immunologic activity and guide treatment.
Changes in Urinary sC5b-9 and Proteinuria Seen With Remission for Each Glomerular Disease Group.
Disease type
Remission obtained (%)
Reduction of proteinuria at remission (%)
Reduction of sC5b-9 at remission (%)
FSGS
25
76 (69-82)
87 (39-94)
IgAN
40
60 (56-70)
79 (34-96)
LN
69
71 (61-93)
93 (67-98)
MN
43
80 (68-87)
95 (91-98)
AAV
71
68 (57-83)
89 (85-98)
Inhibition of the Classical Pathway of Complement With Sutimlimab in Chronic Immune Thrombocytopenic Patients Without Adequate Response to 2 or More Prior Therapies (POSTER #19)
Catherine M. Broome1, Alexander Röth2, David Kuter3, Marie Scully4, Roy Smith5, Jennifer Wang6, Caroline Reuter6, and William Hobbs6
1Division of Hematology, MedStar Georgetown University Hospital, Washington, DC, USA
2Department of Hematology, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Germany
3Division of Hematology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
4Department of Haematology, University College London Hospitals, Cardiometabolic Program, National Institute for Health Research UCLH-UCL Biomedical Research Center, London, UK
5Division of Hematology/Oncology, University of Pittsburgh Medical Center, PA, USA
6Sanofi, Waltham, MA, USA
Background: Classical complement pathway (CP) activation is reported in 50% of patients with immune thrombocytopenia (ITP). Sutimlimab (formerly BIVV009), a targeted CP inhibitor, was assessed in patients with ITP.
Methods: An open-label Phase 1 trial (NCT03275454) included adult patients with chronic, severe ITP and inadequate response to ≥2 prior therapies. Patients received sutimlimab on Day (D) 0, 7, then biweekly for up to 21 weeks (Part A), followed by washout and re-treatment (Part B).
Results: All results are as of December 14, 2018. Seven patients were treated in Part A (mean age = 44.9 years; 85.7% were women). Mean platelet count (PC) increased from 27.9 × 109/L (baseline) to 81.3 × 109/L at D1 and was maintained at >50 × 109/L throughout Part A (Figure 1). By D14, 57% of patients had achieved a response (PC > 50 × 109/L, measured >7 days apart). CH50 levels decreased on sutimlimab initiation and coincided with a PC increase. Re-treatment efficacy was observed in patients continuing in Part B (n = 4). Safety results are for Part A only. Six patients experienced 30 adverse events (AEs) and 2 patients experienced 3 serious AEs with no AE-related discontinuations.
Discussion: Sutimlimab results in rapid, sustained PC increase in patients with ITP. Washout kinetics demonstrate thrombocytopenia reoccurring on sutimlimab discontinuation and resolving with re-treatment.
Conclusions: This is the first clinical evidence that CP plays a role in thrombocytopenia in a subset of patients with ITP. Results suggest ≥1 pathophysiologic explanation for the heterogeneity of ITP and provide rationale for continued evaluation of CP inhibition in ITP treatment.
Mean platelet counts (×109/L) over time in patients receiving sutimlimab.
Complement Activation Causes Major Metabolic and Energetic Changes on Endothelial Cells (POSTER #20)
Carolina Ortiz1,*, Magdalena Riedl1,*, Valentina Bruno1, and Christoph Licht1,2,3
1Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, ON, Canada
2Division of Nephrology, The Hospital for Sick Children, Toronto, ON, Canada
3University of Toronto, ON, Canada
*Co-first authors.
Background: Complement dysregulation on vascular endothelial cells (ECs) causes EC injury and leads to thrombotic microangiopathy (TMA), caused by the formation of membrane attack complexes (MAC, C5b-9) on the plasma membrane of these cells. Although much is known about the role of complement in EC pathology, the consequences and mechanisms have not been studied in detail at a cellular level.
Results: We examined the effects of complement-mediated EC damage in experiments using human blood outgrowth endothelial cells (BOECs) derived from healthy donors, which were sensitized to surface attack by complement factors present in normal human serum. Initial experiments showed that induction of cell surface C5b-9 formation on BOECs resulted in (1) an abrupt rise in intracellular Ca2+, indicative of plasma membrane leakage; (2) a decrease in cell–cell contacts; (3) the loss of cytoskeletal re-arrangement; and (4) subsequent impaired cell motility. However, endothelial C5b-9 formation did not result in cellular necrosis or apoptosis, or impaired proliferation, indicating the capability of ECs to evade complement attack, and suggesting a defect in EC energy homeostasis as likely cause for the observed functional defects. Indeed, preliminary results showed that complement activation caused a drop in intracellular adenosine triphosphate (ATP) levels and mitochondrial membrane potential. Of note, all effects were reversible following discontinuation or blockage of complement activation (removal of serum and exposure of BOECs to C5-depleted or heat inactivated serum samples, respectively), indicating that ECs were still viable, but in an arrested energetic state.
Discussion and Conclusions: Together, these observations point toward the activation of survival mechanisms in ECs to sustain complement attack, including the re-sealing of the plasma membrane attack complex (MAC)-induced pores and the activation of autophagy. Our most recent data show the activation of the autophagy machinery, as seen by the degradation of the markers LC3 and p62. Future experiments will not only shed light on additional survival pathways (ie, inhibition of apoptosis—programmed cell death—and endoplasmic reticulum (ER) stress induction—halting protein expression) ECs may use to repair and withstand membrane damage but may also determine the tipping point, at which compensatory mechanisms are exhausted and cell death is the consequence.
Cyclosporin-Induced Endothelial Injury and Complement Activation Is Caused by Impaired CFH Binding to the Glycocalyx (POSTER #21)
Chia Wei Teoh1,2,3, Carolina G. Ortiz Sandoval4, Magdalena Riedl3, Valentina Bruno1, Jun Li4, Jee Woo Park4, Lisa A. Robinson1,3,4, and Christoph Licht1,3,4
1Division of Nephrology, The Hospital for Sick Children, Toronto, ON, Canada
2Transplant & Regenerative Medicine Centre, The Hospital for Sick Children, Toronto, ON, Canada
3Department of Paediatrics, University of Toronto, ON, Canada
4Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, ON, Canada
Introduction: Calcineurin inhibitors (CNIs) are associated with nephrotoxicity, endothelial cell (EC) dysfunction and thrombotic microangiopathy (TMA). Evolving evidence suggests a central role for complement dysregulation in the pathogenesis of CNI-induced TMA. However, the exact mechanism of CNI-induced complement-mediated injury remains unknown.
Methods: In an in-vitro model utilizing Blood Outgrowth endothelial cells (BOECs) from healthy donors, we evaluated the effects of cyclosporine (CsA) on EC injury, complement activation (C3c, C9) and regulation (CD46, CD55, CD59 and complement factor H [CFH]) on EC surfaces, and on the EC glycocalyx, utilizing flow cytometry, Western blot, and immunofluorescence imaging. Functional activity of CFH was assessed via CFH co-factor assay. Co-immunoprecipitation of Angiopoietin-2 (Angpt-2), Angiopoietin-1 (Angpt-1), and Tie2 was assessed by Western blot.
Results: CsA resulted in a dose- and time-dependent enhancement of EC complement deposition and EC death. CsA (10 µg/ml for 24 hours) led to upregulation of CD46, CD55, and CD59 on EC surface. CsA led to Angpt-2-mediated breakdown of the EC glycocalyx, which was mitigated by Angpt-1. This EC glycocalyx breakdown led to decrease in CFH surface binding and surface cofactor activity.
Conclusions: Our findings confirm a role for complement in CsA-induced EC injury and suggest Angpt-2-mediated glycocalyx abolishment, induced by CsA, as a mechanism leading to complement alternative pathway dysregulation via decreased CFH surface binding. Insights into this mechanism may provide a potential therapeutic target that might lead to improved patient outcomes which is subject to further studies. It might also apply to other TMAs, in which a role for complement has so far not been recognized.
Moderated Case Discussions
C3 Glomerulonephritis—Can Therapy be Individualized? (POSTER #22)
Jocelyn S. Garland1,2 and Sarah M. Moran1,2
1Department of Medicine, Queen’s University, Kingston, ON, Canada
2Glomerulonephritis Specialty Clinic, Kingston Health Sciences Centre, ON, Canada
Background: A 31-year-old woman was diagnosed as membranoproliferative glomerulonephritis (MPGN) in 2006 (aged 19 years). Treatment with prednisone had no effect. She was treated with an angiotensin receptor blocker. She was assessed in 2018 at Kingston Health Sciences GN clinic. Albumin was 21 g/L, C3 was 0.15 (0.8-1.9), C4 was normal 0.13 (0.13-0.4), and proteinuria was severe (12 g per day).
Results: Repeat kidney biopsy in 2019 showed C3GN, with MPGN pattern, 4/34 globally sclerotic glomeruli, moderate interstitial fibrosis. Immunofluorescence: C3 3+; IgM 1+; C1Q negative. No dense deposits on electron microscopy. Complement function studies demonstrated elevated C5b-9 level, low C5, C5 nephritic factor (C5Nef) 1+, and C4 nephritic factor (C4Nef) 4+. C3 nephritic factor was negative. Noris et al (Blood, September 2014) have demonstrated that most C3GN patients have fluid-phase complement activation, versus activated complement on the cell surface. Nevertheless, C5Nef and C4Nef prompted testing for C5b-9 deposition on endothelial cells (Milan, Italy). Results were positive on activated endothelial cells: 160% (normal < 150%).
Discussion: Overall, complement function testing suggested targeting terminal complement pathway for treatment might be of value. After 3 months of eculizumab, proteinuria has decreased (urine albumin-to-creatinine ratio [ACR] = 600 mg/mmol) and creatinine is 85 versus 101 µmol/L, and albumin is 33 g/L (versus 21). C3 remains low.
Conclusions: This C3GN case is unusual given the cell surface activation of complement, and classical and terminal complement pathway involvement. Complement function studies are crucial to help aid the localization of complement pathway defect, and to individualize treatment for C3GN patients.
Nov 2018
Reference range
Result
Interpretation
CH50
Normal > 70 U Eq/mL
16 U Eq/mL
Low
APFA (alternative)
Normal 50%-130%
0%
Abolished
C3 NeF
Negative < 20%
17%
Negative
C4 Nef
Negative < 20%
92% 4+
Positive
C5 Nef
Negative < 20%
27% 1+
Positive
C3
Normal 0.9-1.8 g/L
0.2 g/L
Low
C4
Normal 0.15-0.57 g/L
0.15 g/L
Normal
C5
Normal 10-21 mg/dL
6.2 mg/dL
Low
C5b-9
Normal <0.3 mg/L
2.2 mg/L
High
C5b-9 deposits
Normal < 150%
Resting: 138%
Normal
C5b-9 deposits
Normal < 150%
Activated: 160%
High
Fresh Frozen Plasma as the Main Treatment for a C3 Glomerulopathy Case (POSTER #23)
Sara Rodriguez Lopez1, Anwar Al-Omairi1, Catherine Morgan1, and Abdullah Alabbas1
1Department of Pediatrics, Division of Nephrology, Stollery Children’s Hospital, University of Alberta, Edmonton, Canada
Background: C3 glomerulopathy (C3G) includes a group of rare nephritides with heterogeneous outcome and no specific treatment.
Case Description: A 11-year-old man presented 3 years ago with hematuria, proteinuria, and acute kidney injury (AKI), diagnosed with postinfectious glomerulonephritis (PIGN) as per clinical presentation and pathology findings. Complete recovery with conservative management. Normal C3/C4. Represented 6 months later with the same picture. Initial biopsy was reviewed and diagnosis changed to C3G. C3 nephritic factor not elevated. Genetic testing confirmed heterozygosity for C3 and CFH missense variants of uncertain significance (C3 c.463A>C, P.(Lys155Gln) and CFH c.172T>G, P.(Ser58Ala)). Since then, recurrent episodes of gross hematuria, proteinuria, and AKI occurred, treated with fresh frozen plasma (FFP) with resolution of the abnormalities in between flares. Short courses of steroids used on 2 occasions due to severe AKI on one and persistent proteinuria and mildly elevated creatinine on another despite infusions of FFP. Currently, normal kidney function, persistent microscopic hematuria, no proteinuria.
Discussion: Classical presentation simulating PIGN although normal C3. Typical course with recurrent episodes of hematuria, proteinuria, and AKI, but that fully recovered after FFP infusions on most occasions. Immunosuppressants not required to date except for 2 short courses of steroids. This favorable outcome so far may be secondary to a more benign disease as mutations considered of uncertain significance or due to our quick intervention with FFP infusions at the beginning of the episodes.
Conclusions: C3G treated with FFP infusions at the beginning of disease may provide good disease control while limiting immune suppression.
Poster Presentations
POSTER #1: Unraveling the Role of Neutrophils and Nets in Patients With Shiga Toxin–Producing Escherichia coli Hemolytic Uremic Syndrome
Wouter J. C. Feitz1,2,*, S. Suntharalingham2,*, Nicole C. A. J. van de Kar1, N. Palaniyar3, Lambert P. J. W. van den Heuvel1, and Christoph Licht2,4
1Department of Pediatric Nephrology, Amalia Children’s Hospital, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands
2Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, ON, Canada
3Department of Biochemistry, University of Toronto, ON, Canada
4Division of Nephrology, The Hospital for Sick Children, Toronto, ON, Canada
*Both authors contributed equally to the study
Background: The hemolytic uremic syndrome (HUS) is characterized by hemolytic anemia, thrombocytopenia, and acute renal failure. In children, HUS is mainly caused by infections with Shiga toxin (Stx)-producing Escherichia coli (STEC-HUS) causing damage of the endothelium. We hypothesized that neutrophil activation—in particular, the formation of neutrophil extracellular traps (NETs)—is triggered by Shiga toxin 2a (Stx2a).
Methods: Neutrophils were stimulated with Stx2a. The activation marker CD11b was studied by immunofluorescence (IF) microscopy and release of NETs was determined by SYTOX green and IF. Reactive oxygen species (ROS) assays were used to study different pathways involved. Second, we isolated neutrophils from STEC-HUS patients and studied the release of NETs and the different pathways involved.
Results: (1) Neutrophils upregulate CD11b expression and release more DNA after treatment with Stx2a. (2) Neutrophils produce more cytosolic ROS after treatment with Stx2a. (3) Neutrophils release NETs by the NOX-dependent pathway after stimulation with Stx2a in a dose-dependent matter. (4) Neutrophils from patients in the acute phase of STEC-HUS release less DNA by the NOX-dependent pathway (5) Neutrophils from patients in the acute phase of STEC-HUS show less production of ROS by the NOX-dependent pathway. (6) Neutrophils of STEC-HUS patients are more prone to undergo spontaneous NETosis.
Discussion and Conclusions: It has been published that NETs attract platelets to form clots. Thrombus formation is one of the main causes of kidney failure in patients with STEC-HUS. More research about the interaction between neutrophils, NETs, platelets, and Stx would gain more knowledge about the underlying pathophysiology of STEC-HUS.
POSTER #2: Outcome of Anti-Complement Factor H–Associated Hemolytic Uremic Syndrome in a Resource-Limited Setting
Karalanglin Tiewsoh1, Amit Rawat2, Srinivasavaradan Govindarajan1, Raja Ramachandran3, and Lesa Dawman1
1Division of Pediatric Nephrology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
2Division of Allergy Immunology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
3Department of Nephrology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
Background: Anti-complement factor H (AFH)-associated HUS is a common cause of pediatric acute kidney injury (AKI) in India.
Aims: To understand the outcome of AFH-HUS treated with plasma exchange (PLEX) and immunosuppression in terms of hypertension, eGFR, and proteinuria.
Methods: Twenty-five children with AFH-HUS (2015-2018) were followed for ≥12 months. At each visit, blood pressure, hematological, and renal parameters were documented. Chronic kidney disease (CKD) risk stratification and staging were done according to KDIGO and KDOQI, respectively. After PLEX (n = 19), 17 children received 6 doses of cyclophosphamide and maintenance immunosuppression using azathioprine (n = 10) or mycophenolate mofetil (n = 4). Appropriate statistical tests were used.
Results: Mean age was 7.6 ± 2.6 years, with a sex ratio of 4:1. Twenty-four children had hypertension with median of 3 drugs at presentation. On follow-up, only 3 children (20%) were on 1 (1, 2) anti-hypertensive drug. Median AFH level was 187 AU/mL (134 387). Mean minimum hemoglobin and platelet was 56 ± 10 g/L and 43 × 103 cells/L (IQR = 22-66), which after remission had no drop during follow-up. Median 7 cycles (2, 14) of PLEX was performed. Twenty percent (n = 5) did not attain remission, 20% (n = 5) had lost to follow-up, and 20% (n = 5) died during therapy. On follow-up, risk of CKD markedly reduced from 80% at 3 months to 26% at 1 year. Forty percent children had CKD Stage 2 at 12 months (median eGFR = 96.7 (78.3-109.6) mL/min/1.73 m2).
Conclusions: Although our children had undergone fewer sessions of PLEX than recommended, they received adequate immunosuppression and had a good outcome. We suggest adequate immunosuppression for such patients in developing countries where PLEX and eculizumab is not freely available.
POSTER #3: A Case Report of a Child With Sepsis-Induced Multiorgan Failure and Massive Complement Consumption Treated With a Short Course of Eculizumab: A Case of Crosstalk Between Coagulation and Complement?
Slobodan Galic1, Dorottya Csuka2, Zoltán Prohászka2, Daniel Turudic1, Petra Dzepina1, and Danko Milosevic1,3
1University Hospital Centre Zagreb, Croatia
2Research Laboratory, MTA-SE Research Group of Immunology and Hematology, 3rd Department of Internal Medicine, Hungarian Academy of Sciences and Semmelweis University, Budapest, Hungary
3School of Medicine, University of Zagreb, Croatia
Background: A 14-month-old boy was presented with a severe, rapidly progressing, life-threatening disease with sudden onset of fever, hematemesis, hematuria, and bloody diarrhea alongside fast-spreading hematomas and general corporeal edema. A Clostridium difficile sepsis-induced thrombotic microangiopathy was a presumable diagnosis.
Methods: A positive C difficile bacteria strain in coproculture, clinical, and laboratory tests affirming disseminated intravascular coagulation (DIC) and global complement activation and consumption were key finding that lead to diagnosis. The patient was treated with antibiotics (metronidazole, vancomycin), plasmapheresis, dialysis, methylprednisolone, mycophenolate mofetil, and eculizumab.
Results: Save chronic renal failure, the child is in fair overall condition in a 2-year follow-up.
Discussion: In rare cases of sepsis with massive complement consumption, a case-sensitive eculizumab therapy may be at least considered for complement reduction after resolution of life-threatening multiorgan failure.
Conclusions: This drug can be administered only after sepsis-induced disease is under control. After massive complement consumption is resolved, a withdrawal of eculizumab is recommended to prevent triggering of second sepsis reactivation.
POSTER #4: Cytomegalovirus (CMV)-Associated De Novo Thrombotic Microangiopathy (TMA) in Renal Transplant Recipient: A Case Report
Rehab Fatani1, Pooja Singhal1, Rodrigo Garcia1, Venkat Sainaresh Vellanki2, Istvan Musci2, and Carmen Avila-Casado1
1Department of Pathology, Toronto General Hospital, University of Toronto, ON, Canada
2Department of Nephrology, Toronto General Hospital, University of Toronto, ON, Canada
Background: Cytomegalovirus (CMV) nephritis is an extremely rare condition. We are reporting a case of kidney transplant recipient with systemic thrombotic microangiopathy (TMA) secondary to CMV viremia.
Case Description: A 41-year-old woman, 3 months postrenal transplant, had low-grade fever, nausea, generalized weakness, and easy fatigability. She is known to have hypertension and systemic lupus erythematosus (SLE) with pretransplant biopsy-proven Class IV lupus nephritis. On examination, she was febrile. Serum creatinine was high. Urine microscopy revealed 1+ albumin, occasional granular casts, and red blood cells/corpuscles (RBCs), but no RBC casts. She had low hemoglobin and significant thrombocytopenia. Lactate dehydrogenase was increased and had decreased haptoglobin. The peripheral smear showed fragmented RBCs. Other lab workup results were within normal limits. CMV titer was elevated in the blood. Renal allograft biopsy revealed CMV nephritis and TMA. Recovery was successfully achieved by managing the disease and controlling the infection with the following: IV ganciclovir, plasma exchange (PE) along with post-PE CMV immunoglobin and then maintenance therapy with periodic monitoring of CMV titers. After 14 months, she got an episode of acute rejection and was progressed to severe interstitial fibrosis. Within 4 months, diagnosis of end-stage renal disease was established, and she underwent graft nephrectomy, which showed features of chronic TMA.
Discussion: CMV can be associated with TMA in a kidney transplant recipient. The mechanism and prognosis could be unique in SLE patients.
Conclusions: For kidney allograft long-term survival, infection-induced TMA should be well controlled. Evaluation of renal allograft dysfunction needs a comprehensive workup and a high index of suspicion regarding atypical presentation.
POSTER #5: Shiga Toxin as a Cause of Recurrent Thrombotic Microangiopathy in a Patient With Atypical Hemolytic Uremic Syndrome
Kevin Yau1, Christopher Patriquin2, and Asad Merchant1
1Division of Nephrology, University of Toronto, ON, Canada
2Division of Hematology, University of Toronto, ON, Canada
Background: Atypical hemolytic uremic syndrome (a-HUS) is a form of thrombotic microangiopathy (TMA) characterized by complement pathway dysregulation. It is typically distinguished from Shiga toxin–producing Escherichia coli–associated HUS (STEC-HUS) by the absence of Shiga toxin and from thrombotic thrombocytopenic purpura by detectable ADAMTS13 activity.
Case Description: A 75-year-old white man presented with a 1 week history of nonbloody diarrhea, fatigue, and oliguria. He had an episode of TMA 4 years prior requiring temporary hemodialysis and plasma exchange (PLEX). Genetic testing by Sanger sequencing at the time was negative, but his TMA was presumed to be a-HUS precipitated by mirabegron. His current investigations showed hemoglobin of 126, lactate dehydrogenase (LDH) of 560 (upper limit of normal [ULN] of 220), platelets of 125, haptoglobin <0.03, creatinine of 403 μmol/L (previous baseline of 101 μmol/L), C3 of 0.87, C4 of 0.16, and ADAMTS13 activity of 63%. Recurrent a-HUS was suspected, and the patient was initiated on hemodialysis and PLEX. Of note, he was again taking mirabegron. Stool cultures were negative, however, polymerase chain reaction (PCR) for Shiga toxin (Stx) was positive for the Stx1 and Stx2 genes. Renal function and hematologic abnormalities resolved with PLEX. The patient declined treatment with eculizumab. Repeat genetic testing for an expanded gene panel was performed with results currently pending.
Discussion: This case suggests that exposure to Stx in an adult patient with a likely underlying complement abnormality may precipitate the development of recurrent TMA. Furthermore, it suggests that testing positive for Shiga Toxin does not necessarily exclude a diagnosis of a-HUS. Given the mirabegron usage on both presentations, a drug-induced TMA remains a possibility.
POSTER #6: First Report of the Atypical Hemolytic Uremic Syndrome Conducted by Rare Diseases Committee of the Brazilian Society of Nephrology
Vaisbich1 and de Andrade LGM1 on behalf of COMDORA—Brazilian Society of Nephrology and coworkers
1Brazilian Society of Nephrology, São Paulo, Brazil
Background: Atypical hemolytic uremic syndrome (aHUS) is a severe genetic rare disease. Studies enrolling a large population are difficult, and registries are a powerful tool to overcome this obstacle. Brazilian population is an attractive group because it is characterized by different ancestors and a high rate of miscegenation. This is the first report of the aHUS Brazilian Registry conducted by the Rare Diseases Committee of the Brazilian Society of Nephrology.
Methods: The registry is available at www.COMDORA-SBN.org.br and was approved by UNESP ethical board. This report includes demographic and clinical characteristics, diagnosis criteria, and treatment.
Results:Table 1 shows the main characteristics of 64 patients (26 men). Data show the predominance of men in early infancy and women among patients > 2 years and adults. Renal involvement and hypertension were frequently observed. Diarrhea was observed in 6 patients. aHUS was associated with pregnancy (n = 5), SLE (n = 1), malign hypertension (n = 4), and infections (n = 9). The number of children with all TMA criteria was higher than adults. Twenty patients were renal transplanted at diagnosis and in 12 of 20 patients taking a TMA-related drug, the drug was withdrawn, but TMA persisted. Twenty-five patients had a complement-related pathogenic variant detected. Eculizumab was the preferred treatment (62 of 64 patients). One patient died before Eculizumab. Some data were unavailable for some patients.
Conclusions: Brazilian Registry gave information about aHUS in this special population. The registry has to be improved to avoid missing data. Compared with registries from other regions, there are some differences.
At diagnosis
< 2 years(N = 14)
2-18 years(N = 17)
>18 years(N = 33)
Total(N = 64)
P value
Age (years) (mean ± SD)
0.8 ± 0.5
9.2 (5.1)
31.5 (7.1)
18.8 (14.6)
<.001
Sex (M/F)
11 (78.6%) 3 (21.4%)
5 (29.4%) 12 (70.6%)
10 (30.3%) 23 (69.7%)
26 (40.6%) 38 (59.4%)
.005
Positive familial history
1 (7.1%)
1 (5.9%)
4 (12.1%)
6 (9.4%)
.73
First episode of TMA
11 (78.6%)
15 (88.2%)
24 (72.7%)
50 (78.1%)
.45
Previous renal status: CKD 5
3 (27.3%)
5 (41.7%)
12 (57.1%)
20 (45.5%)
.29
Clinical and lab data
Increased serum creatinine (n/total)
13/14 (92.9%)
15/16 (93.8%)
31/32 (96.9%)
59/62 (95.2%)
.80
Hematuria positive (n/total)
10/14 (71.4%)
9/16 (56.2%)
13/29 (44.8%)
32/59 (54.2%)
.25
Proteinuria positive (n/total)
12/(85.7%)
14/(87.4%)
28/28 (100%)
54/58 (93.1%)
.33
Hb (g/dL)
6.1 ± 1.7
6.8 ± 1.2
8.0 ± 2.4
7.3 ± 2.1
.007
Positive schistocytes (n/total)
14/14 (100.0%)
12/14 (85.7%)
21/30 (70.0%)
47/58 (81.0%)
.13
Platelets (/mm3)
45.441 ± 19.206
69.066 ± 61.182
101.290 ± 58.766
80.792 ± 57.501
.002
LDH median (range)
1.855 (507-17.717)
2026 (555-10.573)
1000 (180-15.184)
1510 (180-17.717)
.006
Haptoglobin (mg/dL), median (range)
12.5 (7-30)
11 (6-30)
25 (2-339)
13 (2-339)
.22
Low C3 (n/total)
17/38 (44.7%)
Treatment
No dialysis (n/ total)
3/10 (30.0%)
6/14 (42.9%)
7/23 (30.4%)
16/47 (34.0%)
.83
Days from diagnosis to First dose of eculizumab, median (range)
15 (2-240)
30 (2-120)
42 (1-2721)
26 (1-2721)
.20
Plasmapheresis
0 (0.0%)
3 (20.0%)
14 (42.4%)
17 (27.9%)
.01
Note. TMA = thrombotic microangiopathy.
POSTER #7: Short Course of Eculizumab May be Effective in Dialysis-Dependent Transplantation-Associated Thrombotic Microangiopathy After Hematopoietic Stem Cell Transplantation
Tomasz Jarmoliński1,2, Monika Rosa1, Anna Puziewicz-Zmonarska3, and Krzysztof Kałwak1
1Department of Pediatric Bone Marrow Transplantation, Oncology and Hematology, Wroclaw Medical University, Poland
2Department of Pediatrics and Pediatric Neurology, District Hospital, Gorzów Wielkopolski, Poland
3Department of Pediatric Nephrology, Wroclaw Medical University, Poland
Background: Allogeneic hematopoietic stem cell transplantation (alloHSCT) could induce several complications. The most frequent viral infections and graft-versus-host disease (GvHD) sometimes lead to thrombotic microangiopathy (TMA). It is associated with significant morbidity and mortality with the risk of death reaching 90%. Effective prevention and treatment have not been discovered, yet. Recently, attempts at using antibody against C5 have been made.
Case Description: A 19-years-old girl with acute myeloid leukemia underwent 2 alloHSCTs from 10/10 Human Leucocyte Antigen (HLA)-matched sister. After the second hematopoietic stem cell transplantation (HSCT), severe acute steroid-resistant Grade 4 GvHD occurred. Despite treatment with high doses of steroids, mycophenolate mofetil, biological therapy, and extracorporeal photopheresis, the patient developed TMA with acute kidney injury and the need for renal replacement therapy. The concentration of Complement Component 3 and activity of ADAMTS13 were normal, infection with Escherichia coli 0157H7 was excluded. Due to failure of all ordered therapies and severity of the condition, an attempt was taken to use eculizumab. Two 900 mg doses of eculizumab (Soliris®) were administered at interval of 2 weeks, which resulted in the improvement of renal function and amelioration of hemolysis and thrombocytopenia. Dialysis therapy was finished after 5 weeks and then a third dose of the drug was administered. Eighteen months later, the patient is alive and well, with limited chronic GvHD. eGFR remains stable at 40 to 46 mL/min/1.73 m2 and mild hypertension requires treatment with Angiotensin Converting Enzyme Inhibition (ACEI) and furosemide.
Conclusions: Even a short course of eculizumab can be sufficient in controlling the TMA after HSCT, provided that the TMA triggering factors are well controlled.
POSTER #8: TMA in Pregnancy Before 20 Weeks Gestation: Is This Preeclampsia or Primary TMA?
Jocelyn S. Garland1,2, Graeme N. Smith2, and Sarah M. Moran3
1Department of Medicine, Queen’s University, Kingston, ON, Canada
2Department of Obstetrics and Gynecology, Queen’s University, Kingston, ON, Canada
3Glomerulonephritis Specialty Clinic, Kingston Health Sciences Centre, ON, Canada
Background: A 28-year-old woman with CKD from diabetes mellitus (DM) type 1, G3P1A1 presents to MCKC clinic April 2018, 8 weeks pregnant. Creatinine is 200 µmol/L, and proteinuria is severe (uACR = 300 mg/mmol). Previous pregnancy (2016) is through C-section delivery at 26 weeks (preeclampsia [PE] and acute kidney injury).
Results: Referred to KHSC GN/Pregnancy clinic at 9 weeks: ASA = 162 mg OD; labetolol for blood pressure (BP). At 14 weeks, Creatinine = 233, urine albumin-to-creatinine ratio (UACR) = 600 and hemoglobin = 66, undetectable haptoglobin, LDH is normal, platelet = 143, no fragments on blood film, and ADAMTS13 is normal. Secondary TMA testing was negative. At 27 weeks, patient experienced anuria and required acute dialysis. At 28 weeks, patient undergone C-section (severe BP) and delivered a healthy baby girl, 1260 g. Kidney function recovers briefly, but then requires peritoneal dialysis. Live related kidney transplant occurs October 30, 2019, uncomplicated, and maintained on prednisone, mycophenolate mofetil, and tacrolimus.
Discussion: Atypical hemolytic uremic syndrome (aHUS) genetic testing was negative. Complement function testing for C5b-9 deposition on endothelial cells (Table 1) (pre- and postkidney transplant) are abnormally increased, suggesting complement mediated TMA (aHUS). Results were received after kidney transplant had occurred, thus close monitoring for TMA recurrence was recommended.
Conclusions: The cause of TMA in pregnancy can be difficult to diagnose. Not all TMA in pregnancy is PE, and pregnancy may be a trigger for TMA syndromes. Testing for complement activation may be useful in determining primary versus secondary TMA and aid in the risk stratification for TMA recurrence for dialysis patients preparing for kidney transplantation.
Reference range
Result
Interpretation
Pretransplant: October 2019
C5b-9 deposits
Normal < 150%
Resting: 160%
Increased
C5b-9 deposits
Normal < 150%
Activated: 200%
Increased
Posttransplant: November 2019
C5b-9 deposits
Normal < 150%
Resting: 143%
Borderline normal
C5b-9 deposits
Normal < 150%
Activated: 160%
Increased
POSTER #9: Diagnosis of Upshaw-Schulman Syndrome in Context of Kidney Transplant Rejection
Louis Marois1, Maxime Paquin1, Géraldine Goss1, Hugo Chapdelaine1, and Guilhem Cros1
1Clinique Immunodéficience Primaire de l’adulte, Institut de Recherches Cliniques de Montréal, Centre Hospitalier de l’Université de Montréal (CHUM), ON, Canada
Background: Upshaw-Schulman syndrome corresponds to the congenital form of thrombotic thrombocytopenic purpura, a rare autosomal recessive disease.
Case Description: We evaluate a 43-year-old woman in the context of kidney transplant rejection. She had a medical history of interstitial nephropathy with distal renal tubular acidosis since childhood. She has had bicytopenia for a long time with normal bone marrow investigations. She underwent a renal transplant at 33 years old after several years of dialysis. After an episode of bilobar pneumonia at age of 40 years, she rejected her kidney transplant and had to resume dialysis since. Renal biopsy demonstrated glomerulitis with membranoproliferative appearance and positive immunofluorescence labeling including C1q and C4d granular deposits. Its cause assessment revealed a decrease in C4 complement fraction and undetectable ADAMTS13 activity (FRET VWF73: Dosage 0). No anti-ADAMTS13 IgG was detected, and other investigations were normal (H factor, Anti-H factor, C100, C5b9). Genetic analysis was performed using thrombotic microangiopathies panel of 37 genes including ADAMTS13. Two variants were found in the ADAMTS13 gene: (1) c.3178C > T p.Arg1060Trp was previously reported as likely pathogenic mutation and (2) c.634G > A p.Glu212Lys was a variant of unknown significance. A replacement therapy with plasma and plasmapheresis was proposed.
Conclusions: This case illustrates the importance of the cause diagnosis before the renal transplant and the challenging management of the Upshaw-Shulman syndrome patients.
POSTER #10: The Effect of Complement in a Gaucher IPSC-Derived Macrophage Model
J. Serfecz1 and R. Feldman1
1Department of Microbiology and Immunology, University of Maryland, Baltimore, USA
Background: Gaucher disease (GD) is an autosomal recessive lysosomal disorder caused by bi-allelic mutations that reduce or eliminate the activity of the lysosomal enzyme β-glucocerebrosidase (GCase). Deficient GCase activity promotes excessive accumulation of cytotoxic lipids and cytokine expression in macrophages. Complement inhibitors are currently being considered as a novel therapy to suppress this inflammatory signaling; however, the mechanisms that connect complement as a driver of systemic macrophage-mediated inflammation in Gaucher disease remain incomplete.
Methods: iPSC-derived monocyte and macrophage cultures were validated by positive immunofluorescence staining and flow cytometry of CD14, CD163, and CD68 lineage markers. NFkB immunoblots, enzyme-linked immunosorbent assay (ELISA), and real-time polymerase chain reaction (RT-qPCR) were performed on iPSC-derived macrophages exposed to C5a to monitor cytokine expression and production. We will also investigate whether GD macrophages are more GCase deficient when exposed to C5a.
Results: Our results indicate that GD macrophages have a 3- to 5-fold increase in expression of inflammatory genes and cytokine production in the presence of C5a alone, or in the presence of C5a and lipopolysaccharide (LPS), when compared with the control. In addition, GD macrophages are not as efficient at complement-dependent phagocytosis in the presence of C5-depleted complement serum.
Discussion: The presence of C5a, in particular, leads to a greater increase in the cytokine production in Gaucher macrophages when compared with the control. We seek to understand this link between GCase deficiency and complement over stimulation by monitoring C5aR binding and signaling in GD.
Conclusions: This research provides further support for complement antagonists as a treatment option for GD and other lysosomal disorders.
POSTER #11: Functional Consequences of Complement Activation on Vascular Endothelial Cells: Results of a Pilot RNA SEQ Study
Magdalena Riedl1,2, Erin Jacobs1,2, Carolina Ortiz1, Alan Zhou1, Valentina Bruno1,2,3, and Christoph Licht1,2,3
1Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, ON, Canada
2University of Toronto, ON, Canada
3Division of Nephrology, The Hospital for Sick Children, Toronto, ON, Canada
Background: Complement dysregulation on vascular endothelial cells (ECs) causes EC injury and leads to thrombotic microangiopathy (TMA). We report on the results of an unbiased RNA seq approach to investigate complement-induced responses of ECs.
Methods: Complement activation on ECs was induced via sensitization on blood outgrowth endothelial cells (BOECs) from healthy individuals. RNA was isolated 1 hour after treatment. A stranded paired end library RNA seq library was used. Data quality was assessed using FastQC software, and adapters were trimmed with Trim Galore software. Manual analysis of regulated genes was carried out with a cut-off of fold change >|1.5|. Gene function was determined based on literature analysis. Functional assays included neutrophil adhesion in a microfluidic chamber, wound healing assay, measurement of transendothelial resistance, and staining for VE-Cadherin.
Results: RNA seq revealed 3 major findings: (1) upregulation of genes involved in leukocyte adhesion (eg, ICAM, E-selectin), (2) regulation of genes collectively resulting in loss of cytoskeletal re-arrangement (eg, down regulation of RhoA activators and upregulation of ROCK 1 inhibitors), and (3) downregulation of genes involved in cell adhesion (eg, VE-Cadherin). Functional relevance of these findings was confirmed by demonstration of complement-induced (1) EC neutrophil adhesion (microfluidic chamber), (2) defective EC migration (wound closure), and (3) decreased trans-endothelial resistance and VE-Cadherin. All these effects were reversible when complement activation was blocked.
Conclusions: Complement activation on ECs results in an array of molecular responses. Detailed mechanism and their link to the clinical phenotype of TMA remains to be elucidated in future research.
POSTER #12: Skeletal Muscle Injury and Repair: A Role for Complement
Erin Jacobs1,2, Carolina Ortiz1, and Christoph Licht1,2,3,4
1Cell Biology, Research Institute, The Hospital for Sick Children, Toronto, ON, Canada
2Institute of Medical Science, University of Toronto, ON, Canada
3Division of Nephrology, The Hospital for Sick Children, Toronto, ON, Canada
4Department of Paediatrics, University of Toronto, ON, Canada
Background: Skeletal muscle contains a highly dynamic microenvironment, including a stem cell niche, in which immune cells are activated to promote endogenous repair. The role of the complement system in this context is currently unclear.
Objective: The objective of this study is to determine the role of complement in skeletal muscle tissue injury and repair utilizing both in vitro and in vivo models. We hypothesize that complement exacerbates skeletal muscle injury and promotes repair through stem cell activation.
Methods: This study uses 3-dimensional tissue culture and barium chloride chemical injury for in vitro experiments. Muscle is examined in conditions with and without serum exposure. In vivo experiments use barium chloride injury in complement deficient mice and regeneration is analyzed over a 28-day time course. Single myofibers are isolated to analyze stem cell activation.
Results: The results from in vitro experiments show that skeletal muscle cells (1) have an intracellular source of C3, that is released and deposited on the surface on injury and (2) activate the membrane attack complex (MAC) on addition of serum. Furthermore, results show the functional benefit of complement blockade at the levels of C3 and MAC in reducing cell death. The in vivo experiment has shown that skeletal muscle regeneration is reduced in complement-deficient mice and that complement deficiency results in increased quiescent satellite cells.
Conclusions: In summary, these results shed light on a new mechanism involved in muscle injury and there is translation potential utilizing complement blocking treatment strategies to maintain muscle integrity.
POSTER #13: Elucidating the Effects of Complement Stress in Neutrophil Extracellular Traps (NETs) Formation in C3 Glomerulopathy
Samuel E. Suntharalingham1,2, Carolina Ortiz2, Wouter J. Feitz2, Nades Palaniyar1,2, and Christoph Licht2,3
1University of Toronto, ON, Canada
2The Hospital for Sick Children, Toronto, ON, Canada
3Cell Biology, University of Toronto, ON, Canada
Background: Neutrophil extracellular traps (NETs) have been recently implicated in several disease states, which is mostly attributed to their pro-inflammatory and prothrombotic properties. Neutrophils carry complement proteins and, when activated, release these complement proteins and contribute to their activation via NETs. By improving our understanding of the interaction of the complement system and neutrophils and their respective response to produce these NETs, we will be able to better understand how neutrophils and NETs play a role in complement-mediated diseases such as C3 glomerulopathy (C3G), in which both neutrophils and complement are expected to contribute to disease pathogenesis.
Methods: Neutrophils freshly isolated from healthy controls (HC) (men, aged 18-24 years) and C3G stable patients with ongoing low C3 levels (women and men, aged 7-16 years) were used. Experiments were done at least threefold (N = 3). We either applied our established protocol of complement activation via the use of a sensitizing antibody (monoclonal anti-human CD59 antibody) in combination with 50% NHS using HC neutrophils or investigated patient-derived neutrophils incubated in autologous serum or serum-free media. Complement deposition (C3b; C5b-9) on neutrophils was detected via immunofluorescence (IF) and flow cytometry (FC). NETosis was detected using SYTOX™ Green assay and IF.
Results: We found that complement activation of neutrophils resulted in (1) surface deposition of C3b and C5b-9; (2) appearance of citrullinated Histone 3 (cit-H3) and myeloperoxidase (MPO) release; (3) the stepwise completion of NETosis after the transfer of neutrophils into serum-free media (SFM). In addition, we found that C3G patient–derived neutrophils (1) were positive for surface complement deposition; (2) were positive for cit-H3; and (3) completed NETosis after transfer from autologous serum to SFM. Finally, HC neutrophils (1) when incubated with C3G patient-derived serum showed cit-H3 formation, and (2) showed NETosis when transferred into SFM.
Conclusions: Complement activation of neutrophils induces NETosis in a step-wise fashion with cit-H3 formation in serum (“priming”) and full NETosis in SFM. These findings can be interpreted as resemblance of an intravascular versus an extravascular environment, suggesting that neutrophils are primed intravascularly and committed to full NETosis only extravascularly.
POSTER #14: Investigating the Role of Neutrophil and Complement Cross Talk in Atypical Hemolytic Syndrome (aHUS) in Single Acute Crisis Patient
Samuel E. Suntharalingham1,2, Carolina Ortiz2, Nades Palaniyar1,2, and Christoph Licht2,3
1University of Toronto, ON, Canada
2The Hospital for Sick Children, Toronto, ON, Canada
3Cell Biology, University of Toronto, ON, Canada
Background: Neutrophil extracellular traps (NETs) have been recently implicated in several disease states, which is mostly attributed to their pro-inflammatory and prothrombotic properties. Neutrophils carry complement proteins and, when activated, release these complement proteins and contribute to their activation via NETs. Primary atypical hemolytic uremic syndrome (aHUS) is a rare complement-mediated kidney disease that manifests unpredictably. Current understanding pinpoints an underlying issue with the regulation of complement. Here, we propose that neutrophils and the production of NETs may help explain, at least partially, the manifestation and pathogenesis of aHUS
Methods: Neutrophils freshly isolated from healthy controls (HC) (men, aged 18-42 years old) and from a single aHUS patient from various follow-up points were used. Experiments were done at least 3-fold (N = 3). Fifty percent human serum derived from the aHUS patient collected at various time points was used to challenge HC neutrophils. The patient-derived neutrophils at each time of isolation was also incubated in autologous patient serum was also used. Complement deposition (C3b; C5b-9) on neutrophils was detected via immunofluorescence (IF) and flow cytometry (FC). NETosis was detected using SYTOX™ Green assay and IF.
Results: We found that complement activation of neutrophils was found in the aHUS patient at all time points of collection, as well as challenging HC neutrophils with aHUS-derived serum from any time point, and resulted in (1) surface deposition of C3b and C5b-9; (2) appearance of citrullinated Histone 3 (cit-H3) and myeloperoxidase (MPO) release. Neutrophils derived from the aHUS patient as well as HC neutrophils treated with aHUS-derived serum was shown have elevated levels of cleaved caspase-3, a marker of apoptosis. HC neutrophils treated with aHUS-derived serum as well as aHUS-derived neutrophils generally showed elevated levels of NETosis markers than HC treated with healthy human serum.
Conclusions: Neutrophils derived from the aHUS patient have shown high levels of activated complement on its surface. This was also emulated by treating HC neutrophils with aHUS-derived serum. A correlation of higher levels of activation of complement before patient treatment versus after treatment is readily apparent. This trend is also similar in which levels of cleaved-caspase 3 and cit-H3 expression in neutrophils, markers of apoptosis and NOX-independent NETosis respectively. This may show demonstrate a finding of overlapping signaling of apoptosis and NETosis, recently labeled as apoNETosis in the novel context of aHUS.
POSTER #15: Lower C4 Copy Number of Total C4 Gene, C4B Gene, and C4BL Gene in Children With Pediatric Acute-Onset Neuropsychiatric Syndrome (PANS)
Agnes Kalinowski1,*, Jennifer Frankovich2,*, Justin Lee3, Haley Hedin3, Reenal Pattini4, Hanna Ollila1,5, Emmanuel Mignot1, Doug Levinson1, Susan Swedo6, Tanya Murphy7,8, Avis Chan2, Margo Thiemann1, and Alexander Urban1,4
1Department of Psychiatry and Behavioral Sciences, Stanford University, CA, USA
2Department of Pediatric Rheumatology, Stanford University, CA, USA
3Quantitative Sciences Unit, Stanford University, CA, USA
4Department of Genetics, Stanford University, CA, USA
5University of Helsinki, Finland
6Pediatrics and Developmental Neuroscience Branch, National Institutes of Health, Bethesda, MD, USA
7University of South Florida, Tampa, USA
8Johns Hopkins Medicine, Baltimore, MD, USA
*Contributed equally to this work.
Background: Pediatric acute-onset neuropsychiatric syndrome (PANS) is characterized primarily by sudden-onset obsessive compulsive disorder (OCD). During PANS disease flares, patients have high levels of the C4 split product, which are consistent with activation of this pathway. As there is emerging evidence for copy number variations in the C4 gene that presents with neuropsychiatric symptoms, we hypothesized that patients with PANS have a vulnerability for this illness due to aberrant copy numbers of the C4 gene.
Methods: We have characterized the complex genotype of the C4 gene-complex using digital-droplet polymerase chain reaction (PCR) for 165 patients with PANS and 211 healthy controls. The specific copy numbers of each of these genes was compared between the PANS and healthy control cohorts using a Fisher exact test.
Results: Overall, the patient cohort had fewer total number of copies of the C4 gene in their genome compared with the healthy controls (P = .03), specifically in C4BL gene copies (P < .001). No statistically significant difference in the number of C4A genes was observed (P = .23) (Figure 1).
Discussion and Conclusions: We show, for the first time, a lower copy number of complement genes in patients who meet strict clinical criteria for PANS compared with healthy controls. Fewer copy numbers of the C4 gene appear to be attributed to lower copy numbers of the long form of the C4B gene. These findings will need to be confirmed in an independently collected cohort as well as explored in patients with different ethnic backgrounds.
Frequency of C4 gene copy number in respective cohort. Healthy controls are shown in blue and the PANS patient cohort is in red. Patient cohort has fewer high copy numbers of C4 genes, specifically in the C4BL form suggesting low copy numbers produce a vulnerability for the illness.
