Abstracts of the 3rd Annual Toronto Complement Conference

Author Index Page Clinical Research and Case Reports
01 Allen, C.; Goulding, A.; Reich, H.; Barth, D.; Patriquin, C.; Jauhal, A.
  Complement-mediated TMA in Pregnancy – Case Report. 2 02 Ferra-Neto, O.A.; Barros-Silva, G. E.; Oliveira-Cruz, M.; Maronese, N.
  The uncommon coexistence of Atypical Hemolytic Uremic Syndrome and Acute Diffuse Glomerulonephritis - Case Report. 2 03 Gogorza, M.J.; Spizzirri A.P.; Chaparro, S.; Lombardi, L.; De Rose, E.; Denis, I.; Pereyra, P.; Raffetto, F.; Santibañez; Cobeñas, C.; Suarez, A.
  Perianal involvement in STEC-HUS: an uncommon manifestation of hemorrhagic colitis. 3 04 Rho, H.; Yang, K.; Chow, S.; Patriquin, C.
  Searching for Service and Accessibility Excellence in Paroxysmal
  Nocturnal Hemoglobinuria in Canada: The EPIC Program. 3 Basic Research
05 Bohorquez-Hernandez, A.; Rostam, N.; Howard, C.; Ortiz-Sandoval, C.G.; Campbell, C.J.; Licht, C.
  Complement system mediates COVID-19 endothelial cell injury, modifying cell permeability 5 06 Bruno, V.; Ortiz-Sandoval, C.G.; Moran, S.; Bowen, E.E.; Cattran, D.; Licht, C. 
  The role of complement in primary membranous nephropathy. 6 07 Diatlov, D.; Bohorquez-Hernandez, A.; Jackson, M.; Cheong, M.; Kirby-Allen, M.; Licht, C.
  Investigating the role of the complement system in paediatric sickle cell disease. 7 08 Hannan, F.; Jurkiewicz, M.; Thiessen, J.D.; Huang, S.HS.; Patriquin, C.; Pavenski, K.
  Investigating Thrombotic Thrombocytopenic Purpura etiology on neurocognitive function. 8 09 Ramos, M.V.; Fiorentino, G.; Santiago, A.; Abelleyro, M.; Rossetti, L.; Exeni, R.; De Brasi, C.; Sandra Palermo, M.
  Assessment of Interleukin-10 promoter variant (-1082A/G) and cytokine production in Hemolytic Uremic Syndrome patients. 9

01

COMPLEMENT-MEDIATED TMA IN PREGNANCY CASE REPORT

Allen, Celine; Goulding, Anna; Reich, Heather; Barth, David; Patriquin, Christopher; Jauhal, Arenn.

Rationale: Pregnancy-associated thrombotic microangiopathy (TMA) comprises a spectrum of primary and secondary conditions. Although pre-eclampsia/HELLP is the predominant cause of pregnancy-TMA, atypical Hemolytic uremic syndrome (aHUS), also known as complement mediated thrombotic microangiopathy (CM-TMA), is an uncommon cause of TMA in pregnancy. Description of treatment and outcomes with eculizumab are limited to <50 case reports.

Presenting concerns of the patient: 28-year-old female with a significant medical history who is presenting hours after the uncomplicated vaginal delivery of her first child with microangiopathic hemolytic anemia (MAHA), thrombocytopenia, elevated liver enzymes and elevated serum creatinine.

Diagnoses: Prior to delivery, there were no signs of hypertension or proteinuria that would suggest evolving pre-eclampsia. The initial diagnosis was that of acute fatty liver disease of pregnancy and pre-eclampsia. As the renal injury continued to worsen without improvement in anemia and thrombocytopenia, aHUS became the predominant differential. ADAMTS13 and anti-phospholipid antibody syndrome (APLAS) screen were negative. Serum C3 was found to be low. Genetic complement screen was negative.

Intervention: Treatment included anti-C5 inhibition with eculizumab. After receiving weekly induction doses, they were transitioned to and remain on eculizumab every 2 weeks.

Outcomes: Liver enzymes had improved prior to initiation of eculizumab. There was normalization of hematological parameters after initiation of eculizumab therapy. They currently remain dialysis dependent, but recently urine output has returned.

Teaching Points: Diagnosis of aHUS in pregnancy is one of exclusion. Development of MAHA, thrombocytopenia, and kidney injury in the post-partum period, with negative serologies for TTP/APLAS, and failure to improve after 24-72 hours should raise the suspicion of aHUS. Prompt diagnosis is needed for timely initiation of anti-complement therapy. This is one of the first descriptions in North America of aHUS presenting post-partum treated with eculizumab.

02

THE UNCOMMON COEXISTENCE OF ATYPICAL HEMOLYTIC UREMIC SYNDROME AND ACUTE DIFFUSE GLOMERULONEPHRITIS - CASE REPORT

Ferra Neto, Oreste A. ¹; Barros Silva, Gyl E.²; Oliveira Cruz, Mauren¹; Maronese, Natalia¹

¹Hospital Maria Aparecida Pedrossian, Brazil.

²University Hospital of Universidade Federal do Maranhão, Brazil.

Disclosure of relevant financial relationship: The following study has no financial relationships, neither do the authors.

Purpose of the study: To describe a rare case of association between acute diffuse glomerulonephritis (GNDA) and atypical hemolytic uremic syndrome (aHUS) in a 10-year-old patient.

Methods: The following study is a case report, with clinical description of the patient from admission, tests performed, treatment applied and clinical follow-up. The data were taken from the medical records and the biopsy were performed by the UFMA pathology team.

Results: A 10-year-old female patient with convulsive history and residual skin lesions, started vomiting which evolved to edema, hematuria, oliguria and decreased renal function. An initial diagnosis of acute diffuse glomerulonephritis (GNDA), was made, evolving to acute renal failure requiring dialysis and corticosteroid therapy, considering a possible rapidly progressive glomerulonephritis. During treatment, the patient evolved worsening renal function associated with thrombocytopenia and hemolytic anemia. A renal biopsy was performed, identifying GNDA exudative and proliferative phase with signs of thrombotic microangiopathy (MAT) and mild to moderate tubular necrosis, therefore, allowing the diagnosis of aHUS. Plasma therapy was initially started, it was suspended due to the patient’s reaction. We opted to start therapy with Eculizumab, which allowed the suspension of dialysis, because of significant improvement in renal function and hematological parameters after 4 doses.

Conclusion: After using it for one month of induction, the patient was released from dialysis therapy, remaining stable from aHUS for approximately 24 months with complete recovery of renal function, with no new recurrences since then. This proves that the in-depth study of pathophysiology, and its correlation with pharmacotherapy, allows the development of medications of important clinical use. Allowing discontinuation of renal replacement therapy and a better quality of life for the patient.

03

PERIANAL INVOLVEMENT IN STEC-HUS: AN UNCOMMON MANIFESTATION OF HEMORRHAGIC COLITIS

Gogorza, Maria J.; Spizzirri, A.P.; Chaparro, S.; Lombardi, L.; De Rose, E.; Denis, I.; Pereyra, P.; Raffetto, F.; Santibañez; Cobeñas, C.; Suarez, A.

Hospital de Niños Sor Maria Ludovica. Servicio de Nefrología, La Plata, Argentina.

Introduction: STEC-HUS is the leading cause of acute kidney injury in children under 5 years in Argentina. Acute morbidity and mortality are related to extrarenal compromise, mainly CNS and bowel disease. Hemorrhagic colitis involves transverse, ascending and descending colon and less frequently ileum and sigmoid. Rectal compromise is scarcely reported. Additionally, extrarenal disease may affect other systems as cardiac, ophthalmological and pancreatic tissue.

Objective: To present a severe case of STEC-HUS with perianal involvement as an unusual manifestation of hemorrhagic colitis.

Case report: A 2 y-o girl was admitted with abdominal pain and bloody diarrhea in the previous 3 days, adding lethargy and anuria during the last 36 hours. Her physical exam revealed altered mental status and pallor. The perianal and perineum area showed a non indurate nor fluctuating dark reddish lesion, with devitalized anal mucosae and bloody fetid stools. The laboratory findings included anemia (hemoglobin 6.9 g/dl; hematocrit 18 %) with schistocytes; platelets 150,000/mm³; WBC 38,200/mm³ with immature cells; urea 1.71 g/l; creatinine 2.31 mg/dl; lactic dehydrogenase 4567 U/l; C-reactive protein 144.5 mg/l; sodium 120 mEq/l; potasium 4.5 mEq/l; Cl 97 mEq/l; pH 7.34; bicarbonate 10.9 mEq/l; BE -12; albumin 24 g/l; glucose 94 mg/l. Stool culture revealed VTX1/VTX2 Shiga toxin producing enterotoxigenic E. coli and enterohemorrhagic E. coli. She was prescribed antibiotics. Exploratory laparoscopy showed distension and petechiae along descending colon and sigmoid. An ileostomy was performed 10 cm far from ileocecal valve. She required 9 days of hemodialysis, parenteral nutrition for 7 days and 6 RBC transfusions. She also exhibited endocrine pancreatic failure with transient insulin requirement. At funduscopic examination, bilateral retinal hemorrhages were found. Cardiac involvement was evidenced as pericardial effusion, volume overload and repolarization disorder. At discharge, her kidney function was normal, the perianal and perineum lesion recovered completely and the ileostomy was permeable.

Conclusion: Our patient presented severe extrarenal disease, with hemorrhagic colitis including the infrequent perianal and perineal involvement, CNS, pancreatic, cardiac and ocular disease and biochemical markers of severity such as hyponatremia, hypoalbuminemia and leukocytosis. Our objective was to highlight the infrequent perineal and perianal disease and the favorable outcome despite of the presence of clinical and biochemical risk factors of poor outcome.

04

SEARCHING FOR SERVICE AND ACCESSIBILITY EXCELLENCE IN PAROXYSMAL NOCTURNAL HEMOGLOBINURIA IN CANADA: THE EPIC PROGRAM

Rho, Hayeong¹; Yang, Kelsey¹; Chow, Signy^1,2; Patriquin, Christopher^1,2,3

¹University of Toronto, Toronto, ON, Canada.

²Division of Medical Oncology and Hematology, Sunnybrook Health Sciences Center, Toronto, ON, Canada.

³University Health Network, Toronto, ON, Canada.

Disclosure of relevant financial relationships: Hayeong Rho: No disclosure. Kelsey Yang: No disclosure.

Signy Chow: Alexion, Site Investigator for Clinical Trial. Christopher Patriquin: Alexion, Consultancy, Honoraria and Speakers Bureau; AstraZeneca Rare Disease, Consultancy, Honoraria and Speakers Bureau; Apellis, Consultancy, Honoraria and Membership on an entity’s Board of Directors or advisory committees; Biocryst, Honoraria; Sanofi, Honoraria.

Introduction and purpose: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, life-threatening disease, characterized by uncontrolled complement-mediated intravascular hemolysis, thrombosis, and marrow failure. Due to its nonspecific presentation such as fatigue or dyspnea often without hemoglobinuria, patients may experience a delay in accurate diagnosis, which leads to a significant impact on quality of life (QoL) and survival. At any point in the patient’s journey, they may experience delays or poor accessibility to care. Key challenges in PNH are its identification, diagnosis, and subsequent appropriate and timely initiation of treatment.

Methods: The Canadian PNH Network (CPNHN) use the “CATCH criteria” to suspect diagnosis of PNH: Cytopenias, Aplastic anemia/myelodysplasia, Thrombosis, Coombs’-negative hemolysis, and Hemoglobinuria. This screening tool was hypothesized to be a useful tool for real-world practitioners. In this study, we aimed to: (1) identify opportunities and gaps during the journey of a patient with suspected or confirmed PNH, referred to our pilot sites: University Health Network and Sunnybrook Health Sciences Centre; (2) characterize time to diagnosis and treatment initiation, specifically considering CATCH criteria; and (3) create a process map.

Results: A total of 19 participants were identified. 17 charts were reviewed, and 15 patients were available for 30-minute individual interviews. Median age at diagnosis was 40 (SD ± 16.7), with varied symptoms at presentation (Figure 1). Median number of healthcare providers seen prior to diagnosis and/or referral was 6 (SD ± 4.9; Figure 2), and time from symptom onset to referral was 149.5 months (SD ± 60). Baseline demographic data are presented in Table 1. The most common CATCH criteria at presentation were hemoglobinuria, macrocytic anemia as explained by signs of hemolysis, and thrombocytopenia (Table 2). Flow cytometry data revealed elevated proportions of granulocytes (85.9% ± 16.9), monocytes (84.7% ± 17.4), and type III RBC (20.8% ± 18.5). From the interviews, reflecting on the COVID-19 pandemic, the participants reported little to no change in quality of care they had received, and some endorsed the convenience of virtual consultations. They suggested: improving community physician awareness and education on PNH, assistance with finances, and minimizing the uncertainty surrounding medication and clinical trial availability.

Discussion and Conclusion: In this study, we have characterized the diverse patient journey followed at our centers. The variability and delay in assessment for PNH may be attributed to the diverse backgrounds of the participants, their first presentation at a different country, the year of symptom presentation, and availability of high-sensitivity flow cytometry. We evaluated the humanistic factors such as QoL of these patients living with PNH. As PNH is rare, the study was limited by the small sample size. Some patients had their initial diagnosis and care outside Canada and some initial investigations and notes could not be accessed.

We aim to expand our work across Canada, which will increase the cohort size and capture the impact of geographical differences. Following this, we plan to provide recommendations for diagnostic and treatment benchmarks to colleagues across the country, introduce the CATCH criteria, and subsequently evaluate the impact of these knowledge translation strategies with comparison to our initial cohort.

05

COMPLEMENT SYSTEM MEDIATES COVID-19 ENDOTHELIAL CELL INJURY, MODIFYING CELL PERMEABILITY

Bohorquez-Hernandez, Arlette¹; Rostam, Niyousha¹; Howard, Claire¹; Ortiz-Sandoval, Carolina G.¹; Campbell, Christopher J.²; Licht, Christoph^1,3

¹Cell Biology Program, Research Institute, The Hospital for Sick Children, Toronto, ON, Canada.

²Paragon Ventures Inc, Vancouver, BC, Canada.

³Division of Nephrology, The Hospital for Sick Children, Toronto, ON, Canada.

Background: Coronavirus Disease 2019 (COVID-19), a coronavirus (SARS-CoV-2)-mediated respiratory disease, has become a global pandemic with more than one hundred million cases worldwide, presenting with varying degrees of severity, the majority (80%) experience a mild course with flu-like symptoms and recover without specific treatment. About 20%, require hospitalization, and of those 2-5% require ICU admission with ventilation. COVID-19 has resulted in about 4.1 million cases and 285,000 deaths worldwide as of date (72,000 and 5,000 in Canada, respectively).

The complement system (CS) is a key component of the innate immune response. The CS activation involves a series of enzymatic transformations, where C3b acts as a mediator. The final step of complement activation is the formation of the membrane attack complex (MAC, C5b-9) on the cell’s plasma membrane (PM). Several pathologies like thrombotic, inflammatory, autoimmune and age-related diseases are associated with abnormal complement activation.

While the underlying mechanisms contributing to COVID-19 pathogenesis remain poorly understood, there is evidence linking the activation of the CS and endothelial injury to organ damage and complications that increase the risk of mortality in COVID-19. Thus, we hypothesize that vascular endothelial injury resulting from complement activation contributes to COVID-19-associated endothelial activation and injury.

For this study, clinical information, and sera from SARS-CoV-2 positive patients with mild (non hospitalized) and severe COVID-19 (deceased) were obtained from the Canadian COVID-19 Prospective Cohort Study (CANCOV) to identify complement activation and endothelial cell (EC) injury.

Aim: To determine the role of complement system activation on endothelial cell damage in COVID-19 patients based on disease severity.

Methodology: Complement activation was induced using an established protocol. Briefly, primary human umbilical vein endothelial cells (HUVECs) were treated with adenosine diphosphate (ADP), followed by treatment with healthy control or COVID-19 patient serum. To confirm the biological activity of complement, we measured the deposition of C5b-9 and C3b on the PM of HUVECs via immunofluorescence (IF). In addition, an assay by means of a transwell model was used to measure the changes in permeability. Briefly, HUVECs were seeded on the top part of the insert, allowing them to form a monolayer. Monolayer integrity was assessed by imaging cells on the day of the experiment, before and after incubation with the sera.

Results: Complement activation caused increased C5b-9 deposition on the PM of cells treated with patient sera compared to those treated with sera from healthy donors, with further increased deposition when cells are treated with deceased patient sera. As for C3b deposition, there was no difference between groups.

The activation of complement induces an increase in cell permeability. Cellular permeability is significantly lower in those cells treated with non-hospitalized COVID-19 patient sera than deceased patients.

Conclusion: There is a differential biological activity of complement on endothelial cells depending on the severity of COVID-19, this complement activation leads to an increase on cell permeability. While more research needs to be done, these results contribute to a better understanding of the consequences of complement activation on endothelia and help elucidate the effects of complement activation on COVID-19.

06

THE ROLE OF COMPLEMENT IN PRIMARY MEMBRANOUS NEPHROPATHY

Bruno, Valentina^1,2,3; Ortiz-Sandoval, Carolina G.³; Moran, Sarah⁴; Bowen, Emily E. ⁵; Cattran, Daniel⁶; Licht, Christoph^1,2,3

¹Division of Nephrology, The Hospital for Sick Children, Toronto, ON, Canada

²Department of Paediatrics, University of Toronto, Toronto, ON, Canada

³Research Institute, Cell Biology Program, The Hospital for Sick Children, Toronto, ON, Canada

⁴Cork University Hospital, Cork, Ireland

⁵University of Bristol, Bristol, United Kingdom

⁶Toronto General Hospital, Research Institute, University Health Network, Toronto, ON, Canada

The authors have no relevant financial interests to disclose.

Introduction and aim of the study: Primary membranous nephropathy (PMN) is the leading cause of nephrotic syndrome in adults and a common cause of end-stage kidney disease (ESKD). The Heymann’s nephritis mouse model of PMN shows that proteinuria is complement-mediated. However, the pathogenetic role of complement in human PMN remains unclear. We aim to demonstrate that complement is active in PMN, leading to both structural and functional effects on podocytes.

Methods: An in-vitro model of immortalized human podocytes (Moin Saleem, Bristol, UK) was used for all experiments. Complement deposition (C3b and C5b9) and actin cytoskeleton re-arrangements were determined by immunofluorescence (IF) following exposure to serum from eight PMN patients, selected from the Toronto Glomerulonephritis Registry. As positive control, podocytes were pre-sensitized with anti-CD59 and exposed to 50% normal human serum (NHS) to obtain complement deposition on the cell surface. Subsequently, changes in intracellular calcium levels were monitored using a fluorescent dye (Fluo8-AM), acquiring images every 20 seconds (up to 10 minutes) by confocal microscopy. Calcium effects on mitochondrial membrane potential were measured by flow cytometry using tetramethylrhodamine, methyl ester (TMRM) dye. Changes of intracellular ATP following complement activation on podocytes were measured by bioluminescence using EnzyLight ADP/ATP Ratio Assay Kit. Wound healing assays were performed to study functional effects on podocyte migration.

Results: Incubation with 50% PMN serum led to deposition of both C3b and C5b9 on the surface of cultured podocytes, which was significantly higher compared to podocytes medium (p < 0.0005) or 50%NHS (p < 0.005), used as controls. No difference was noted compared to the positive control (anti-CD59 + 50% NHS) in terms of C5b9 deposition.

Complement activation induced intracellular calcium increase, loss of mitochondrial membrane potential, intracellular ATP depletion and actin cytoskeleton disruption, and ultimately resulted in impaired podocyte migration. Functional consequences of complement activation on podocytes were reversible via inhibition of the terminal or the alternative complement pathway (by using anti-C5 or Factor B inhibitor, respectively) (Figures 1-4).

Conclusions: Complement deposition leads to a defect in cell energy metabolism impairing both structure and function of podocytes in PMN. Effects can be reversed by inhibition of the terminal or alternative complement pathway.

Further studies are needed to fully understand the consequences of complement activation on the podocyte energy machinery and the rationale for the use of complement inhibitors in PMN. Our research may identify novel molecular treatment targets with the potential of improved patient outcomes and quality of life.

07

INVESTIGATING THE ROLE OF THE COMPLEMENT SYSTEM IN PAEDIATRIC SICKLE CELL DISEASE

Diatlov, Daniel^1,2; Bohorquez-Hernandez, Arlette²; Jackson, Melanie³; Cheong, Melina³; Kirby-Allen, Melanie³; Licht, Christoph^2,4

¹University of Toronto Institute of Medical Science, Toronto, ON, Canada

²The Hospital for Sick Children Research Institute, Cell Biology Program, Toronto, ON, Canada

³The Hospital for Sick Children Division of Haematology/Oncology, Toronto, ON, Canada

⁴The Hospital for Sick Children Division of Nephrology, Toronto, ON, Canada

Introduction: Sickle cell disease (SCD) is one of the most common hereditary red blood cell (RBC) disorders in the world, with an estimated 300,000 infants born with the disease annually. In SCD, a mutation in the gene for β-globin results in rigid sickled RBCs that can form blockages in the micro-vessels within organs, such as the kidney, leading to RBC lysis, endothelial cell (EC) damage, ischemia/reperfusion injury, and extremely painful vaso-occlusive crises (VOC). SCD can give rise to a variety of renal manifestations such as hyperfiltration, microalbuminuria, and acute kidney injury. Approximately 16-18% of overall mortality in patients with SCD is ascribed to kidney disease. The complement system – a critical part of the innate immune system - is involved in a myriad of kidney and vascular disorders, and emerging research points to the involvement of complement in SCD as well, potentially contributing to sickle cell nephropathy (Fig. 1A).

Methods: Paediatric patients with SCD (HbSS or HbS/β0) were enrolled during hospital admission with diagnosed VOC or acute chest crisis (ACS) not caused by infection. Patient serum was collected during hospital admission (crisis) and during follow-up (steady state). Complement activity was measured using the WIESLAB Complement System Screen (Svar Life Science). Immunofluorescence (IF) imaging was used to measure the deposition of C3b and C5b-9 on the surface of ECs exposed to patient serum.

Results: There is equal classical and alternative pathway activity during disease steady state and crisis, with an interesting trend showing elevated MBL pathway activity during crisis compared to steady state (Fig. 1B). IF assay data shows significantly elevated deposition of C3b and C5b-9 proteins on ECs when comparing crisis samples and healthy controls, with a trend suggesting a potential difference in complement deposition between steady state and crisis patients (Fig. 1C).

Conclusion: Our preliminary data shows complement is active in SCD, resulting in elevated C3b and C5b-9 deposition on ECs during SCD crisis. Future work will focus on further quantifying complement activity, and assessing the functional consequences of this on the surface of ECs.

08

INVESTIGATING THROMBOTIC THROMBOCYTOPENIC PURPURA ETIOLOGY ON NEUROCOGNITIVE FUNCTION

Hannan, Fahad¹; Jurkiewicz, Michael; Thiessen, Jonathan D.; Huang, Susan HS; Patriquin, Christopher; Pavenski, Katerina.

¹Department of Medical Biophysics, Western University, London, ON, Canada

Study Purpose: Using myelin water imaging (MWI), a specific MRI technique for detecting white matter damage, may aid in understanding the structural changes caused by thrombotic thrombocytopenic purpura (TTP) on the brain, as patients in remission from TTP have an increased risk of depression and neurocognitive decline despite treatment.

Method: A 3T MRI scanner (Biograph mMR, Siemens Healthineers, Erlangen, Germany) with a 32-channel head-only receive coil was used to acquire MRI in 19 TTP patients 30 days after remission and 6 healthy controls. Standard-of-care (SOC) MRI, including T1-weighted, T2-weighted, diffusion-weighted, and susceptibility-weighted images, were read by a radiologist to assess gross pathology and determine regions of interest (ROI). A multi-component driven equilibrium single pulse observation of T1 and T2 (mcDESPOT) MRI sequence was acquired to generate T1 relaxation, T2 relaxation, and myelin maps. Additional testing included a cognitive assessment and depression test to correlate with MRI findings.

Results: Significant T1 was found in the cingulate cortex (Figure 1A). Significant T2 was found in the following lobes: frontal, parietal, and temporal lobes (Figure 1B). No significant MWF increases/decreases were found. Decreased scores less than 1 and 2 standard deviations (STD) were found in a variety of cognitive-related tests when compared to a cohort of >10,000 age-matched, sex-matched controls. From depression scores, more than 50% of patients reported experiencing the following symptoms: concentration difficulties (89%), inner tension (67%), lassitude (61%), and reported sadness (56%). Of the 19 patients in the study, 11 showed no signs of depression, 7 showed mild symptoms, and 1 patient did not take the test.

Figure 1.

T1 (A) and T2 (B) relaxation times between patients and controls for each lobe.

Conclusions: Multiple regions in the brain have significantly increased T2 relaxation time while the cingulate cortex also had a significantly increased T1 relaxation time. T1 and T2 depend on a variety of tissue structure and biochemistry, thus pathologies that alter these properties reflect a change in T1 and T2. In particular, an increase in T1 and T2 can be indicative of either neuroinflammation, white matter edema, axonal loss, or early signs of demyelination. Each of these pathologies can play a role in impacting neurocognitive function which could be associated with decreased cognitive scores and depressive symptoms as seen in the patient group. However, results from this study alone are not enough to conclude which pathology is present and further techniques are required ascertain which pathology is present.

09

ASSESSMENT OF INTERLEUKIN-10 PROMOTER VARIANT (-1082A/G) AND CYTOKINE PRODUCTION IN HEMOLYTIC UREMIC SYNDROME PATIENTS

Ramos, Maria V.¹; Fiorentino, Gabriela^1,2; Santiago, Adriana²; Abelleyro, Martin³; Rossetti, Liliana³; Exeni, Ramon²; De Brasi, Carlos.³; Palermo, Marina S.¹

¹Laboratorio de Patogénesis e Inmunología de los Procesos Infecciosos. Instituto de Medicina Experimental - Academia Nacional de Medicina, Buenos Aires, Argentina.

²Hospital del Niño Prof Dr Ramon Exeni, San Justo, Argentina.

³Laboratorio de Genetica Molecular de la Hemofilia, Instituto de Medicina Experimental - Academia Nacional de Medicina, Buenos Aires, Argentina.

Background: Hemolytic Uremic Syndrome (HUS) produces acute renal failure mainly in children, caused by Shiga toxin-producing E. coli and inflammatory response. Despite anti-inflammatory mechanisms are triggered, the implication of them in HUS is scarce. Interleukin-10 (IL-10) regulates in vivo inflammation and the interindividual differences in its expression are related to genetic variants. Particularly, the single nucleotide polymorphism (SNP) rs1800896 -1082 (A/G) located in the IL10 promoter, regulates cytokine expression.

Methods: Plasma and peripheral blood mononuclear cells (PBMC) were obtained from HUS and healthy children peripheral blood. IL-10 levels were quantified by ELISA and SNP -1082 (A/G) was analysed by Allele Specific-PCR.

Results: The circulating IL-10 was increased in HUS, but peripheral blood mononuclear cells (PBMC) from these patients exhibited a lower capacity to secrete this cytokine compared to healthy children. We observed that circulating IL-10 was almost two-fold higher in HUS patients with allele -1082G in comparison to genotype AA. Moreover, there was a relative enrichment of genotypes GG/AG in HUS patients with severe renal failure.

Conclusion: Our results suggest a possible contribution of SNP -1082 (A/G) to renal HUS severity that should be further evaluated in a larger cohort.