SS Patients Research Articles

Digital image analysis of intraepithelial B-lymphocytes to assess lymphoepithelial lesions in salivary glands of Sjögren's syndrome patients.

Salivary glands of primary SS (pSS) patients characteristically harbour periductal infiltrates, in which lymphoepithelial lesions (LELs) can develop. LELs are composed of hyperplastic ductal epithelium with infiltrating lymphocytes and may assist in the challenging diagnostic process of pSS. As manual identification of LELs remains difficult, we aimed to identify LELs by using an objective digital image analysis (DIA) algorithm that detects intraepithelial lymphocytes. A virtual triple-staining technique developed for this study was used to count intraepithelial lymphocytes in consecutive slides stained for CD3 (T-lymphocytes), high-molecular-weight cytokeratin (hmwCK) (striated ducts) and CD20 (B-lymphocytes) in labial and parotid gland biopsies in a diagnostic cohort of 109 sicca patients. Patients were classified as having pSS or non-SS according to the ACR-EULAR classification criteria. T-lymphocytes were detected in almost all analysed ducts of pSS and non-SS sicca patients, whereas intraepithelial B-lymphocytes were present in 59-68% of labial and parotid gland biopsies of pSS patients, against only 2-3% of patients classified as non-SS. Intraepithelial B-lymphocytes were found in almost all striated ducts with hyperplasia (LELs). Remarkably, ∼25% of analysed striated ducts without hyperplasia of pSS patients also contained B-lymphocytes (precursor-LELs). Furthermore, presence of intraepithelial B-lymphocytes was associated with clinical parameters of pSS (i.e. serology). The presence of intraepithelial B-lymphocytes in salivary gland biopsies of sicca patients is a clear indicator of pSS and can be used as an objective alternative to LEL scoring. Therefore, identification of B-lymphocyte-containing ducts should be added to the diagnostic histopathological work-up of patients suspected of pSS.

Anti-HMGB1 antibody is a potential characteristic autoantibody for Sj\xf6gren's syndrome

Sjögren's syndrome (SS) is a common chronic inflammatory autoimmune disease that affects about 0.33–0.77% population in China. The positive for antinuclear antibodies (ANA) is one of the key features of SS, which shows a nuclear fine speckled (AC-4) pattern in an indirect immunofluorescent antibody test (IIFT). About 70% of ANA-positive SS patients have detectable anti-SS-A and/or SS-B antibodies, which indicates that other autoantibodies may present in SS patients. The anti-HMGB1 antibodies in 93 SS patients and 96 healthy controls were investigated with in-house developed ELISA and immunoblotting, and the locations of HMGB1 and fluorescent pattern of anti-HMGB1 antibody were investigated with IIFT. The contribution of anti-HMGB1 antibody in ANA-IF was evaluated with Cas9-induce HMGB1 knockout B16 cells. The anti-HMGB1 antibody level is higher in SS patients (9.96 ± 5.55 RU/ml) than in healthy controls (4.9 ± 1.4 RU/ml). With ROC curve analysis, when taking 8 RU/ml as the cutoff value, the sensitivity, specificity, and the area under the curve were 64.5%, 96.9%, and 0.83, respectively. A total of 18 patients (20.7%) with nuclear fine speckled (AC-4) pattern in ANA-IF test were anti-HMGB1 antibody positive only. With commercial antibody, anti-HMGB1 antibody showed the same nuclear fine speckled (AC-4) pattern. The serum from ANA-IF (+), SS-A (−), and SS-B (−) SS patients showed nuclear fine speckled (AC-4) pattern in wildtype B16 cells, but no fluorescence in HMGB1 knockout B16 cells. Anti-HMGB1 antibody may be one of the characteristic autoantibodies of SS in addition to anti-SS-A and SS-B. The detection of anti-HMGB1 antibody can provide more laboratory evidence for clinical diagnosis of SS.

Integrative analysis of datasets of proteins in Sjogren's syndrome patients

This study aims to compare reported proteins expressed in controls and patients with SS in tear, saliva and serum from proteins identified in papers published between 2000 and 2020, to obtain a systematic comparison of proteins expressed in SS patients to find proteins that can be used as biomarkers in SS. Differentially expressed proteins from Sjögren's patients identified in the studies were classified by body fluid (saliva, serum, tear and saliva from healthy patients). Protein pairing was carried out in RStudio, using the previously created data framework, PANTHER and REACTOME 7.O. were used to explore the type of proteins associated with SS and enriched metabolic pathways. Five differentially expressed proteins were found in saliva, tear, and serum. Finally, this study suggests antithrombin III and E-cadherin. as biomarkers in SS, because these proteins are differentially expressed in plasma, blood, and tear, and that neutrophil degranulation pathway as a target for SS.

Is Detection of Relapse by Surveillance Imaging Associated With Longer Survival in Patients With Rhabdomyosarcoma?

We investigated whether surveillance imaging had an impact on post-relapse survival in patients with rhabdomyosarcoma (RMS). We hypothesized that relapse detected by imaging (group IM) would be associated with longer survival compared with relapse detected with a clinical sign or symptom (group SS). We performed an observational multi-institutional study in 127 patients with relapsed RMS comparing overall survival (OS) after relapse using Kaplan-Meier and Cox proportional hazards analyses. Relapse was detected in 60 (47%) group IM and 67 (53%) SS patients. Median follow-up in survivors was 4 years (range 1.0 to 16.7 y). Four-year OS rates were similar between group IM (28%, 95% confidence interval [CI]: 14%-40%) and SS (21%, 95% CI: 11%-31%) ( P =0.14). In multivariable analyses accounting for institution, age at diagnosis, time to relapse, risk group at diagnosis, and primary site, not receiving chemotherapy (hazard ratio [HR]: 6.8, 95% CI: 2.8-16.6), radiation (HR: 3, 95% CI: 1.7-5.3), or surgery (HR: 2.8, 95% CI: 1.6-4.8) after relapse were independently associated with poor OS. These results on whether surveillance imaging provides survival benefit in patients with relapsed RMS are inconclusive. Larger studies are needed to justify current surveillance recommendations. Chemotherapy, radiotherapy and surgery to treat recurrence prolong OS.

Sézary Syndrome: Different Erythroderma Morphological Features with Proposal for a Clinical Score System.

Sézary syndrome is a rare subtype of cutaneous T-cell lymphoma characterized by erythroderma, peripheral lymphadenopathies, and circulating atypical cerebriform T-cells. To date, no definite staging system has been developed for these patients. In this retrospective analysis of the archive of the Dermatological Clinic of the University of Turin, Italy, erythrodermic SS patients were classified according to clinical records and photographs into three main presentations: erythematous, infiltrated, or melanodermic. The pattern of erythroderma was found to be associated with disease outcome, as better survivals were recorded in patients with erythematous and infiltrative erythroderma. Patients in the melanodermic group, though less represented in our investigation, seemed to show a worse trend in survival. According to this preliminary evidence, a new prognostic classification, with a revised score specific for Sézary syndrome patients, can be proposed to usefully integrate the current staging system. The correlation displayed in our research will be hopefully confirmed by prospective studies with larger cohorts, with the aim of identifying significant prognostic features in this subset of cutaneous T-cell lymphoma patients.

The respiratory manifestations in patients with primary Sjögren's syndrome: is interstitial lung disease related to disease activity?

Sjögren's syndrome is a systemic, autoimmune disease and can affect many organs and systems. In this study, we aimed to evaluate the respiratory manifestations, and the association between interstitial lung disease (ILD) and disease activity in primary SS (pSS) patients. The study design was retrospective cross-sectional, and 151 patients followed up with a diagnosis of pSS between 2004 and 2019 were included in the study. Demographic and clinical data, laboratory results, chest radiographs and thorax computed tomography (CT) results were obtained from patient files and hospital imaging system. Thorax CT was requested from all patients with respiratory complaints and abnormalities in physical examination and pulmonary function test. Disease activity was calculated with EULAR pSS disease activity index (ESSDAI) and clinical European League Against Rheumatism SS Disease Activity Index (clin-ESSDAI). In our study, 97% of pSS patients were female, and the mean age was 55.9 ± 12 years, disease onset age was 45.5 ± 12.1 years, disease duration was 10.7 (1-38) years. According to CT findings of 120 patients, 35% had nodules, and 13.3% had ILD (62.5% nonspecific interstitial pneumonia, 25% lymphocytic interstitial pneumonia, 12.5% usual interstitial pneumonia). Bronchiectasis, emphysema, sequelae fibrotic changes, and pleural thickening was found in 3.3%, 5.8%, 15.8%, and 1.7% of patients, respectively. It was observed that there was a significant relationship between the presence of ILD and persistent cough, mediastinal LAP, low DLCO, high ESSDAI and clin-ESSDAI scores reflecting disease activity. The most common pulmonary manifestation in our patients was ILD. ILD was observed more frequently in patients with moderate and severe disease activity. Some of the ILD patients were diagnosed while they were asymptomatic. Even if they are asymptomatic, it is important to follow up the patients with physical examination, spirometry, DLCO and thorax CT.

Assessment of Oxidative Stress Indicators among Patients with Sickle Cell Anaemia versus Non Sickle Cell Iron Deficiency Anaemia: A Case-control Study

Introduction: In sickle cell anaemia, haemolysed iron is reutilised, moreover, iron stores are increased owing to the blood transfusions, hence, iron deficiency is less common in sickle cell anaemia. But iron decompartmentalisation, due to unstable haemoglobin, is thought to be the cause for oxidative stress. Hence, iron deficiency anaemia and sickle cell anaemia need to be compared and studied to provide better treatment and awareness. Aim: To compare the oxidative stress, antioxidants and iron indices among patients with Sickle Cell Anaemia (SCA) and Iron Deficiency Anaemia (IDA). Materials and Methods: A case-control study was conducted at Jawaharlal Nehru Medical College, Wardha, Maharashtra, India, from January 2016 to December 2018. Total 240 SCA patients {120 SS (homozygous disease) and 120 AS (heterozygous trait)}, 120 patients with IDA and 120 age-matched and sexmatched controls were included in present study. Oxidative stress markers Malondialdehyde (MDA), Catalase, Superoxide Dismutase (SOD), vitamin C and vitamin E, zinc and iron indices {Iron, Total Iron Binding Capacity (TIBC) and serum ferritin} were analysed. Data was analysed using descriptive and inferential statistics using Analysis of Variance (ANOVA). Results: The mean age of SS patients was 19.01±11.43, AS was 22.01±11.78 years, control was 22.63±12.17 years, and IDA was 18.50±11.46. There was an equal distribution of both males and females in all groups. The mean of haemoglobin in SS was 7.40 g/dL, AS was 10.31 g/dL, IDA was 8.63 g/dL, and control was 14.81 g/dL. The mean value of mean corpuscular volume for SS was 65.71 fL, AS was 65.64 fL, IDA was 72.38 fL, and control was 87.68 fL. Zinc for SS was 82.65 μg/dL, AS was 82.13 μg/dL, IDA was 43.33 μg/dL, and control was 103.99 μg/dL. Conclusion: There is impairment in the oxidant and antioxidant status among patients with sickle cell anaemia with and without IDA.

Salivary gland ultrasonography in patients with connective tissue diseases: a multi-centre observational study.

US of salivary glands (SGUS) is a non-invasive tool that allows for diagnosing primary SS (pSS) or secondary SS (sSS). However, little is known about the prevalence of US findings of SS in other CTDs. The aim of this multi-centre observational study was to evaluate, in CTD patients with or without SS, the prevalence of abnormal SGUS findings and the possible association of the findings with clinical or biological phenotypes. B-Mode SGUS was performed by one operator blinded to clinical data. Each SG was semi-quantitatively rated on a scale from 0 to 4 according to the Jousse-Joulin score; a score ≥2 was considered pathological. Data for 194 patients were analysed (pSS, n = 30; sSS, n = 39; other CTDs, n = 77; controls, n = 48). SGUS findings were abnormal in 80%, 67%, 25% and 2% of patients, respectively. Independent of the underlying disease, age and sex, abnormal SGUS findings were significantly associated with presence of anti-SSA antibodies (P < 0.001), pSS (P < 0.001) and sSS (P < 0.01). Among SS patients, abnormal SGUS findings were associated with the presence of hypergammaglobulinemia, anti-SSA antibodies, objective eye dryness and increased anti-nuclear antibody level, with no difference in EULAR SS Disease Activity Index. Abnormal SGUS findings were associated with anti-SSA antibody positivity independent of the underlying disease. In SS patients, abnormal findings were associated with immunologic features and mouth involvement. Among CTD patients, SGUS changes may be associated with a particular immune profile.

Abstract 9515: Heterogeneity of Benefit on Quality of Life With an Invasive versus Conservative Strategy for Chronic Coronary Disease. Results From the ISCHEMIA Trial

Background: The ISCHEMIA trial recently showed that, on average, patients with chronic coronary disease (CCD) and at least moderate ischemia randomized to an invasive strategy (INV) reported less angina and better quality of life (QOL) than those treated conservatively (CON)—an effect sustained through follow-up. To better support shared decision-making for individual patients, we explored the heterogeneity of treatment benefit of the INV vs CON strategy within the ISCHEMIA trial. Methods: Disease-specific QOL was assessed with the Seattle Angina Questionnaire (SAQ; scores range 0-100, higher=less angina/better QOL). An ordinal (proportional odds) model for 1-year SAQ summary score (SAQ SS) produced estimates of the odds of better QOL with randomization to INV vs CON, with potential heterogeneity of treatment benefit explored via interactions between patient factors and treatment. The final model included both significant interactions (accounting for multiple testing) and main effects. Results: Among 4617 patients with CCD from 37 countries randomized to INV vs CON treatment (mean age 64 ± 10 y, 23% women, 40% diabetes), mean SAQ SS was 74.1 ± 18.9 at baseline and 85.7 ± 15.6 at 1 year (p&lt;0.001). Patients with lower baseline SAQ SS (i.e., more angina) and younger patients derived greater symptomatic treatment benefit from INV management (model-based interaction p=0.005 and p=0.004, respectively; Figure). No other patient factor was associated with a differential treatment effect. In the final model, female sex and diabetes were also associated with worse 1-year QOL, independent of treatment. Conclusion: Using data from a large multinational randomized trial, we found that younger patients and those with more angina at baseline derived greater QOL benefit from INV management. As the primary benefit of revascularization in CCD is symptom improvement, this model could be used to better inform individual patient’s decision-making for initial treatment.

Expression of Paraoxonase-2 in Different Stages of Cutaneous T Cell Lymphoma: Is It a Potential Biomarker for Aggressiveness?

Introduction: Paraoxonase 2 (PON2) is an intracellular membrane protein that belong to the PON gene family. This enzyme exerting a crucial role against production of reactive oxygen species within mithochondrial respiratory chain. Some reports provided clinical evidence for a link between PON2 and different types of malignancies and recently a growing attention has been focused on exploring the role of PON2 in cancer. Moreover, in contrast to solid tumors, a low expression levels of PON2 protein were detected in hematological malignancies like acute myeloid leukemia and B-cell lymphomas. The aim of this study was to investigate the role of PON2 in the cutaneous T cells Lymphoma (CTCL).Methods: We performed an immunohistochemistry analysis of PON2 protein expression at various stages CTCL. We analyzed PON2 protein using skin biopsies from stage I (n= 3) and stage II (n= 6) Mycosis Fungoides (MF) and stage III/IV (n= 5) erythrodermic MF/Sézary Syndrome (SS) and control (n=6) tissues. Values were expressed as percentage of PON2-positive staining cells, whereas the intensity of PON2 positivity was semi-quantitatively scored from negative to -/+/+++. Subsequent statistical analysis was carried out to explore the existence of correlations between intra-tumor enzyme level and clinical-pathological features at diagnosis.Results: In our preliminary study, results showed PON2 down-expression in SS compared to controls. Among CTCL, we found significant differences in enzyme levels between MF and erythrodermic MF/SS; in fact, PON2 expression was higher in patients with not erythrodermic MF compared to patients with erythrodermic MF/SS. (Table I) Moreover, we found no significant differences in enzyme levels between MF patients and controls.Conclusion: Our study is the first to demonstrate downregulation of PON2 intra-tumoral in SS patients while protein levels were higher in stage I and II MF patients. These findings seem to suggest that PON2 expression levels could be negatively related with tumor aggressiveness in CTCL: an interesting phenomenon where PON2 is upregulated in the early stages and downregulated in the late stages of CTCL. Further, in vitro studies are needed to clarify the mechanism related to PON2 expression. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Splenic Complications in Sickle Cell Disease: A Retrospective Cohort Review

▪Splenic dysfunction, particularly acute splenic sequestration crisis (ASSC), is a leading cause of death in the first decade of life of patients with sickle cell disease (SCD). Despite the severity of splenic disease in SCD, few large-scale studies have been performed to assess the natural progression of splenic complications, particularly in the less common sickle cell genotypes and with the onset of early use of hydroxyurea.We performed a retrospective chart review of patients with SCD born between January 2011 and December 2018 and treated at the Texas Children's Hospital Sickle Cell Center. We extracted data from our electronic medical record system to identify patients with documented splenic complications based on ICD9 and ICD10 codes. We then reviewed charts for these patients to confirm diagnoses and obtain additional information about these complications.We identified 688 patients treated for SCD at our center during the study period. Of these, 113 (16.4%) were diagnosed with one or more splenic complications. The overall prevalence of splenic complications was highest in patients with hemoglobin Sβ 0 thalassemia (Sβ 0) at 50.0%, compared to 20.7% in hemoglobin SS (SS) patients, 6.9% in hemoglobin SC (SC) patients, and 0% in hemoglobin Sβ + (Sβ +) patients and patients with other genotypes (Table 1). ASSC and splenectomy were likewise most prevalent in Sβ 0, less so in SS, and rare in SC, Sβ +, and other genotypes.To further delineate the incidence of ASSC, we performed an in-depth analysis of patients from our cohort for whom detailed information was available. Compared to patients with the SS genotype, the incidence rate ratio (IRR) for ASSC was significantly higher for Sβ 0 patients and significantly lower for SC patients (Table 2). The mean age of onset of ASSC was similar between SS, Sβ 0, and SC patients, but survival curves for time to the first event differed significantly between genotypes.We examined the association between early hydroxyurea therapy and ASSC in SS and Sβ 0 patients by comparing patients who initiated hydroxyurea therapy by the age of two years to those who initiated therapy after this age. ASSC were more frequent among patients initiated on hydroxyurea by the age of two years (Table 3, upper panel). We also compared patients on hydroxyurea to those not on this therapy and found a similar effect, with ASSC occurring more frequently among patients on hydroxyurea (Table 3, lower panel). Survival curves for time to first ASSC also diverged significantly between these groups of patients.Our data indicate that the prevalence and severity of splenic problems vary widely between different sickle cell genotypes in the first decade of life, with SC patients having relatively mild complications while Sβ 0 patients have the most severe and frequent complications. The association between hydroxyurea use and incidence of ASSC also suggests that hydroxyurea therapy may increase the risk of this complication of SCD, particularly when initiated before two years of age. [Display omitted] DisclosuresGeorge: Global Blood Therapeutics: Consultancy, Speakers Bureau; Wolters Kluwer: Patents & Royalties.

Pediatric Sickle Cell Disease and the COVID-19 Pandemic: A Year in Review at Children's National Hospital

▪Background: Over the past year, COVID-19 was declared a global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resulting in over 100 million cases and >3 million deaths worldwide according to the World Health Organization (WHO). Morbidity and mortality have been highest in adults, particularly in those with underlying conditions including hypertension, diabetes, and obesity. Children, thus far, have largely remained either asymptomatic or presented with mild symptoms. However, sickle cell disease (SCD) was classified as a risk factor for severe COVID-19 disease in both adults and pediatric patients.Objective: To describe the one-year experience of the clinical course, management, and treatment of COVID-19 in children, and young adults with SCD at Children's National Hospital (CNH).Methods: This was a single-center, observational cohort study of 55 children (age <18 years) and young adult (age ≥ 18 years) patients with SCD and PCR confirmed SARS-CoV-2 infection at CNH between March 31, 2020, and February 12, 2021 (Figure 1A).Results: Sixty-nine percent were children (N=38) and 31% were young adults (N=17). The mean age was 11.6 years with 51% females (N=28) and 49% males (N=27). Seventy-five percent of cases were Hgb SS, 15% Hgb SC, and 11% Hgb SBeta Thalassemia Zero (Table 1). Fever (45%) was the most common presenting symptom; only 9% had a loss of taste or smell (Figure 1B). Twenty-two percent were asymptomatic at presentation. Among the 40 patients who presented to the emergency department (ED) or were hospitalized, 50% (N=20) presented with vaso-occlusive pain crisis (VOC), 42% with Acute Chest Syndrome (ACS), 2% with splenic sequestration, and 2% with Venous Thromboembolism. Only 3% were admitted to the ICU (N=2 young adults, N=1 children); none of whom were on hydroxyurea. There were no differences in hospitalization rates between Hgb SS and Hgb SC patients (Table 1). Lower oxygen saturation (02 Sat)(02 Sat <95%: 62% vs 16% p=0.004), fevers (69% vs 16%, p=0.001), VOC pain crisis (50% vs 24%, p=0.047), and ACS (65% vs 3%, p<0.001) were more common in hospitalized patients vs. non-hospitalized patients.Patients with ACS experienced a longer length of stay (6 days vs. 3 days p=0.008), lower oxygen saturation (02Sat<95%: 81% vs 13% p<0.001), higher white blood cell count (13.3 vs 9.0 K/mcL, p=0.009), lower hemoglobin nadir (6.8 vs. 9.6 gm/dL, p= 0.049), and elevated D-dimers (4.1 vs. 0.8 ug/mL, p=0.002) compared to those without ACS. The types of treatment received by patients requiring hospitalization or ED visits included ceftriaxone (N=28, 70%), azithromycin (N=15, 37.5%), remdesivir (N=6,15%) convalescent plasma(N=1,2.5%). Blood transfusion was required in 29% of the 55 SCD COVID-19 cases (N=16) and 76% (N=13) of the ACS patients. Twenty-six percent (N=17) of hospitalized patients were anticoagulated with either enoxaparin or rivaroxaban according to CNH's COVID-19 anticoagulation treatment protocol. There were similar rates of healthcare utilization, SCD modifying therapies, acute COVID-19 clinical presentation, respiratory support, and laboratory findings (hematologic, inflammatory) between the children and young adults. However, young adults were more likely to be on crizanlizumab treatment (18% vs 0%, p=0.026) and have an elevated D-Dimer (4.0 vs 1.0, p=0.012) on laboratory evaluation.Conclusion: Our case series reveals that the demographics and clinical presentation between our SCD children and young adult patients with COVID-19 were similar overall. While the morbidity was high, there was no mortality. Those that were hospitalized had lower oxygen saturation levels, higher incidences of fever, and higher morbidity presenting with VOC and ACS. Patients with ACS and/or an oxygen requirement had significantly higher WBC count, lower nadir hemoglobin, and higher D-dimers in a small subset of patients supporting a pro-inflammatory and coagulopathic picture. Our study will add to a growing body of literature on SCD COVID-19 cases. [Display omitted] DisclosuresDarbari: Global Blood Therapeutics: Consultancy; Hilton Publishing Inc.: Consultancy; Novartis: Consultancy. Majumdar: Asklepion Pharma: Consultancy, Patents & Royalties: IV L-Citrulline in the use of sickle cell pain crisis. Campbell: Novartis Pharmaceuticals Corporation: Consultancy, Research Funding.

Effects of Flavonoid Quercetin on Thrombo-Inflammatory Processes in Patients with Sickle Cell Disease

Introduction:Altered erythrocyte physiology in Sickle Cell Disease (SCD) results in severe vascular complications, including vaso-occlusion (VOC) along with various other manifestations, such as thrombosis and end organ damage. Moreover, platelet activation endothelial dysfunction and fibrin formation, are critical processes implicated in thrombo-inflammatory vasculopathy in SCD. Since flavonoids intake is associated with lowered adverse cardiovascular disease outcomes and cancer associated thrombosis, we are evaluating the effects of the flavonoid Isoquercetin (IQ) on thrombo-inflammatory biomarkers in patients with SCD (NCT:04514510). Here we report the mechanistic effects of Quercetin (Que), an aglycone that differs from IQ by the absence of 3-linked glucoside moiety, on markers of erythrocyte and platelet activation in patients with SCD.Methods:We studied patients with SCD (Genotype SS, mean age: 39 yrs, N=8) and ethnic matched controls (Genotype AA, mean age: 42 yrs, N=8). Erythrocyte reactive oxygen species (ROS) was measured in washed red cells by flow cytometry using the fluorescent probe 2‘, 7‘-dichlorofluorescein diacetate (DCFDA) in the presence or absence of Quercetin (Que) (100 µM). Effects of in vitro erythrocyte ROS induction by hydrogen peroxide (H 2O 2) and ROS inhibition by N-acetyl-L-cysteine (NAC) were evaluated. Baseline and agonist-induced (ADP 20 µM) platelet P-selectin expression in vitro in the presence or absence of Que (100 µM) was assessed by flow cytometry.Results:At baseline, SS erythrocytes demonstrated elevated levels of ROS reflected by greater florescent intensity with DCFDA when compared with erythrocytes from ethnic matched controls (SS mean= 1884, SD: 508.8 vs. AA mean: 1604, SD: 554.5; p value= 0.3798). Stimulation of AA erythrocytes in vitro with H 2O 2 led to accumulation of ROS that approached levels similar to those exhibited by SS erythrocytes at baseline (AA mean: 1890, SD: 748.3). Pre-treatment of SS and AA erythrocytes in vitro with the antioxidant NAC exhibited minimal effects on ROS in SS erythrocytes (SS baseline+NAC mean: 1942, SD: 526.8, p value 0.4609 and SS H 2O 2+NAC mean: 1889, SD: 437.4, p value 0.5469) but lowered ROS levels in both baseline and H 2O 2 treated AA erythrocytes (AA baseline+NAC mean: 1442, SD: 453.9, p value 0.1563 and AA H 2O 2+NAC mean: 1581, SD: 518.7, p value 0.078). Pre-treatment of SS and AA erythrocytes in vitro with Que substantially lowered erythrocyte ROS in patients and controls at the baseline (SS baseline ROS with Que mean: 1582, SD: 542.0, p value 0.0078; AA baseline ROS with Que mean: 1341, SD: 490.9, p value 0.0313; Fig. 1A), and after H 2O 2 stimulation (SS H 2O 2 induced ROS+Que mean: 1677, SD: 572.5, p value 0.0156; AA H 2O 2 induced ROS+Que mean: 1417, SD: 512, p value 0.0078). Pre-treatment of erythrocytes with a combination of Que and NAC did not reveal synergistic anti-oxidant activity (SS NAC+Que mean: 1616, SD: 464.8; AA NAC+Que mean: 1333, SD: 524.1).We simultaneously investigated the effects of Que on platelet P-selectin expression in the same subjects. As expected, baseline platelet surface P-selectin expression was higher in SS patients compared with controls (SS N=5, mean: 1.48%, Max: 4.9%, Min: 0.03%; AA N=4, mean: 0.89%, Max: 2.3%, Min: 0%; p value 0.55). Stimulation of platelet rich plasma (PRP) with ADP increased surface P-selectin expression in comparison with baseline, in both patients and controls (SS N=5, mean: 18.04%, Max: 40.4%, Min: 5.8%, p value 0.0047; AA N=4, mean: 20.60%, Max: 43.8%, Min: 5.4%; p value 0.0140). Pre-treatment of PRP with Que prior to ADP stimulation revealed a trend toward decreased platelet surface P-selectin expression in both patients and controls (SS N=5, mean: 10.49%, Max: 24.8%, Min: 3.1%; AA N=4, mean: 12.09%, Max: 21.9%, Min: 3.4%; Fig 1B) although this did not return to baseline.Conclusion:The flavonoid Quercetin appears to have a beneficial effect in lowering erythrocyte ROS and platelet surface P-selectin and warrant further evaluation as a strategy to ameliorate thrombo-inflammatory pathophysiology in sickle hemoglobinopathies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

141 Sickle cell disease and physical activity: Is it safe?

Primary Subject areaHematology and Oncology - PaediatricBackgroundThough physical activity (PA) is beneficial for healthy individuals and for those with chronic disease, its impact on sickle cell disease (SCD) children remains poorly studied. Moreover, patients may refrain from PA given a perceived fear of SCD complications.Objectives1) Evaluate whether SCD children engage differently in PA compared to their peers. 2) Evaluate the cardiopulmonary impact of PA in a group of SCD patients.Design/MethodsPrior to the COVID-19 pandemic, SCD patients aged 8-17 years old consented to answer a PA questionnaire (PAQ) and have their school peers answer the same PAQ. PAQ, a well-described surrogate marker of PA, is a standardized questionnaire evaluating PA during the last 7 days. Patients and their peers were approached only during the school year. A set of HbSS patients were invited to participate in a supervised exercise test including pre- and post-effort PFT, echocardiography, EKG, measurement of troponins and NT-proBNP and a methacholine challenge to evaluate exercise-induced bronchial hyperreactivity. T-test was used as statistical analysis, using SPSS V24. The study received IRB approval.ResultsQuestionnaires from 25 SCD children (13 SS and 12 SC) were compared with 36 matched-school peers. SCD patients were overall less active (p=0.04). While their level of activity was similar at school, SCD patients were overall less active after school (p=0.028), in the evening (p=0.008) and weekends (p=0.049). While the difference was significant for both SS and SC patients, SS were less active than SC, although this difference was statistically not significant. Five HbSS patients on HU were subjected to an exercise test. There were no statistical differences in the pre- and post-PA evaluations including measurements of troponin, NT-proBNP and functional testing. None of them had exercise-induced hyperreactivity.ConclusionOverall, SCD patients are less active than their peers, mostly outside of school. The absence of cardiopulmonary modifications during PA is reassuring, although our study was limited to 5 HbSS patients on HU. Whether HU has a protective role cannot be excluded. Our results encourage us to further evaluate PA interventions outside of school in SCD patients. Interventional studies evaluating the risk-benefit ratio of PA in SCD children are further needed.

Haptoglobin Gene Polymorphism among Sickle Cell Patients in West Cameroon: Hematological and Clinical Implications.

Haptoglobin is a protein involved in protecting the body from the harmful effects of free hemoglobin. The haptoglobin gene exhibits a polymorphism, and the different genotypes do not have the same capacity to combat the free hemoglobin effects. The present study aimed at determining the polymorphic distribution of haptoglobin in sickle cell patients (SCPs) from West Cameroon and their impact on the hematological parameters, as well as clinical manifestations of the disease severity. Haptoglobin genotype of 102 SCPs (SS) and 115 healthy individuals (60 AA and 55 AS) was determined by allele-specific polymerase chain reaction, and the complete blood count was determined using the AutoAnalyser. Results showed that the genotype Hp2-2 was significantly (p < 0.05) represented in SS patients (54%) than in controls AA and AS (27% and 29%, respectively), while Hp2-1 was mostly found (p < 0.05) in AS (42%) and AA (38%), against 15% in SS. The allelic distribution in SS patients was Hp2: 0.613, Hp1S: 0.304, and Hp1F: 0.084. In AA and AS controls, the proportions of the Hp1 and Hp2 alleles were similar (around 0.5 each), with 0.282 for Hp1S and 0.218 for Hp1F in AS and 0.283 for Hp1S and 0.258 for Hp1F in AA. The distribution of the haptoglobin genotypes did not reveal any significant difference across hematological parameters and clinical manifestations of disease severity in SCP and controls. SCP with Hp1S-1F genotype presented the highest level of hemoglobin. Although Hp2-2 was more frequent in SS patients, it appeared not to be related to the hematological parameters and to the disease's severity. Further investigations are necessary to explore the impact of Hp polymorphism such as antioxidant, lipid profile, and functionality of some tissues in SCP in Cameroon.

Management of sagittal craniosynostosis: morphological comparison of eight surgical techniques

The aim of this study was to carry out a retrospective multicentre study comparing the morphological outcome of 8 techniques used for the management of sagittal synostosis versus a large cohort of control patients. Computed tomographic (CT) images were obtained from children CT-scanned for non-craniosynostosis related events (n = 241) and SS patients at preoperative and postoperative follow-up stages (n = 101). No significant difference in morphological outcomes was observed between the techniques considered in this study. However, the majority of techniques showed a tendency for relapse. Further, the more invasive procedures at older ages seem to lead to larger intracranial volume compared to less invasive techniques at younger ages. This study can be a first step towards future multicentre studies, comparing surgical results and offering a possibility for objective benchmarking of outcomes between methods and centres.

Tre-O1-03 - Response to brentuximab vedotin in advanced stage MF/Sézary syndrome with low CD-30 expression: “real-life” data of the German Cutaneous Lymphoma Network

Introduction and objectives: Advanced stage mycosis fungoides/Sézary syndrome (MF/SS) are difficult to treat and associated with an inferior prognosis. Brentuximab vedotin (BV) is a frequently used agent in higher stages, particularly in cases with a high expression of CD30. The ALCANZA trial, which included MF patients with a CD30 expression >10%, demonstrated high response rates and a good tolerability. However, its effectiveness in advanced stage MF/SS syndrome with low CD30-expression (<10%) still has to be systematically evaluated. Therefore, the question is whether an equally good response with BV is achievable in these CTCL cases. Material and methods: Between 2018 - 2020, 32 patients in 11 centres with predominantly advanced MF/SS and low CD-30 expression (<10%) were treated with BV in the recommended dosage, retrospective evaluation of therapy response was done according to the EORTC-ISCL criteria. Results: In all patients (n=32) at least one other systemic therapy preceded BV treatment. The median number of previous treatments was 3. Thirty patients suffering from MF (IB: 2; IIA: 1; IIB: n=18; III: 1; IV: 9; total ≥IIB: 27) and 2 SS patients were analyzed. As a result, 17 of 31 patients (OR: 54.8%) responded to therapy (CR n=4/31 12.9%, PR n=13/31 41.9%) after 6 cycles. The median progression-free interval among patients with OR was 6.4 months (range: 0.5–15.5 months, mean ± SD: 6.4±3.8, n=16). In 37.5% (n=12) the CD-30 Expression was 5-9% and in 62.5% (n=20) CD-30-Expression was <5%. Conclusions: In conclusion, with 54.8% OR, BV in MF/SS with low CD-30 expression (<10%) shows a comparable response rate compared to CTCL with CD-30 expression ≥10% (56-73%) and CR (12.9% vs. 3–35%) but with a lower PFS (6.4 vs. 11 months). In summary, based on this data the treatment of MF/SS with low CD-30 expression with BV seems to be an effective therapeutical option and justifies its use in challenging cases independent of the degree of CD30 positivity. Advanced stage mycosis fungoides/Sézary syndrome (MF/SS) are difficult to treat and associated with an inferior prognosis. Brentuximab vedotin (BV) is a frequently used agent in higher stages, particularly in cases with a high expression of CD30. The ALCANZA trial, which included MF patients with a CD30 expression >10%, demonstrated high response rates and a good tolerability. However, its effectiveness in advanced stage MF/SS syndrome with low CD30-expression (<10%) still has to be systematically evaluated. Therefore, the question is whether an equally good response with BV is achievable in these CTCL cases. Between 2018 - 2020, 32 patients in 11 centres with predominantly advanced MF/SS and low CD-30 expression (<10%) were treated with BV in the recommended dosage, retrospective evaluation of therapy response was done according to the EORTC-ISCL criteria. In all patients (n=32) at least one other systemic therapy preceded BV treatment. The median number of previous treatments was 3. Thirty patients suffering from MF (IB: 2; IIA: 1; IIB: n=18; III: 1; IV: 9; total ≥IIB: 27) and 2 SS patients were analyzed. As a result, 17 of 31 patients (OR: 54.8%) responded to therapy (CR n=4/31 12.9%, PR n=13/31 41.9%) after 6 cycles. The median progression-free interval among patients with OR was 6.4 months (range: 0.5–15.5 months, mean ± SD: 6.4±3.8, n=16). In 37.5% (n=12) the CD-30 Expression was 5-9% and in 62.5% (n=20) CD-30-Expression was <5%. In conclusion, with 54.8% OR, BV in MF/SS with low CD-30 expression (<10%) shows a comparable response rate compared to CTCL with CD-30 expression ≥10% (56-73%) and CR (12.9% vs. 3–35%) but with a lower PFS (6.4 vs. 11 months). In summary, based on this data the treatment of MF/SS with low CD-30 expression with BV seems to be an effective therapeutical option and justifies its use in challenging cases independent of the degree of CD30 positivity.

Tre-O2-04 - Mogamulizumab induces long term immune restoration and reshapes tumoral heterogeneity in Sézary syndrome

Mogamulizumab, an anti-CCR4 monoclonal antibody, has shown to increase progression-free survival in cutaneous T-cell lymphoma. We hypothesized that, besides the targeted depletion of Sézary cells (SCs), mogamulizumab may contribute to immunological changes, improving clinical response. We also explored potential phenotypic changes of SCs during treatment, potentially resulting in resistance/tumor escape to therapy. We have prospectively followed 26 SS patients with B2 stage before mogamulizumab initiation. We performed extended phenotypic characterization of malignant and benign compartments, using KIR3DL2, TCRVb and/or CD26 markers, every month, up to one year. Prior to mogamulizumab, the non-malignant (benign) subset of CD4+ T-cells displayed exhausted phenotypes. When comparing SCs, benign and controls CD4+ T cells, our data showed a gradient of PD1, TIGIT, DNAM, CD27, CD28 and CD70 expression. All patients presented SCs with heterogeneous surface phenotypes and differential expression of individual markers were found within distinct malignant subsets. An early complete blood response was observed in 17/26 patients and was associated to a higher baseline CCR4 expression on SCs. A drastic decrease in benign T-cells and activated Treg absolute counts was observed during the first 4 weeks. Long-term follow-up revealed the emergence of an immune restoration involving CD8+ and naive and TSCM CD4+ subsets, concomitant with almost complete disappearance of exhausted T lymphocytes. Finally, the development of resistance/tumor escape to mogamulizumab therapy allowed us to longitudinally follow phenotypic characteristics of SCs and revealed the emergence of CCR4–SCs displaying significant changes in their heterogeneity patterns. In conclusion, mogamulizumab is not only aimed at eradicating malignant-cells but is likely contributing to the restoration of an efficient immunity. This is of importance as a competent immune status is required for effective anti-infectious and anti-tumoral responses as well as for the efficacy of further immunotherapies.

OP-1 GUT METATRANSCRIPTOMICS AND METABOLOMICS REVEAL ASSOCIATION OF CYSTEINE AND PURINE METABOLISMS WITH METABOLIC ASSOCIATED FATTY LIVER DISEASE (MAFLD)

The gut microbiome represents a niche for biomarkers discovery to risk-stratify MAFLD patients. However, each population may have unique microbiome signatures and studies are needed in Latin America where MAFLD prevalence and severity are high. To identify gut metatranscriptome and metabolome signatures associated with MAFLD and steatohepatitis (SH) in Argentina. Stool samples, diet, demographic and clinical data were obtained from 33 biopsy-proven patients (12 simple steatosis -SS- and 21 SH) and 19 healthy volunteers (HV). PNPLA3 rs738409 SNP was genotyped. HPLC, flow injection analysis with MS/MS in tandem and MetaboAnalyst-v4.0 were used for metabolomics. RNA-seq was performed in NovaSeq6000®. bioBakery-v1.8 and Maaslin2-v1.2.0 were used for data analysis. BMI was higher in MAFLD patients, particularly in SH (q=4.49e-06). After adjusting for BMI, 89 and 53 gene family clusters were differentially expressed between HV and MAFLD and between SS and SH, respectively (q<0.1). Pathways related to sulfur oxidation, short-chain fatty acid metabolism, purine metabolism and lipopolysaccharide synthesis were enriched in MAFLD patients when compared with HV and in SH when compared with SS, whereas folate synthesis was enriched in SS patients (q<0.1). Gene expression associated with Desulfobacteraceae bacteria harbored most of the functional features of MAFLD patients when compared with HV, and of SH patients within the case group (Figure). The PNPLA3 GG genotype was related to decreased hydrolysis of glycerolipids, high expression of Clostridium cadaveris and low expression of Desulfobacteraceae bacteria associated genes (q<0.1). Higher concentrations of xanthine, implicated in purine metabolism, and of the sulfur amino acid cysteine were detected in the stool of MAFLD patients when compared with HV, and of SH patients within the case group (BMI-adjusted q<0.1). Cysteine and purine metabolisms are strongly related to MAFLD and SH in Argentinian patients. Cysteine and xanthine could be useful as potential biomarkers.

Labial gland-derived mesenchymal stem cells and their exosomes ameliorate murine Sj\xf6gren's syndrome by modulating the balance of Treg and Th17 cells

BackgroundSjögren's syndrome (SS) is a chronic, systemic autoimmune disorder characterized by sicca syndrome and/or systemic manifestations. The disease severely affects the health and life of patients, and the treatment of SS has always been a clinical challenge and essentially palliative. Mesenchymal stem cells (MSCs) have been reported to exert immunomodulatory effects and as a potential novel therapeutic strategy for SS. Labial gland-derived MSCs (LGMSCs) are a population of resident stem cells in the labial gland, first isolated by our group. Exosomes released by MSCs contain a large variety of bioactive molecules and considered to function as an extension of MSCs.MethodsLGMSCs were isolated from patients who were needed surgery to remove the lip mucocele and LGMSCs derived exosomes (LGMSC-Exos) were isolated by ultracentrifugation. The non-obese diabetic (NOD) mice were treated with LGMSCs or LGMSC-Exos by tail vein injection. The saliva flow rate of mice was determined and salivary glands were dissected and stained with hematoxylin and eosin. In vitro, peripheral blood mononuclear cells (PBMCs) from SS patients were cocultured with LGMSCs or LGMSC-Exos. Percentage of T helper 17 (Th17) cells and regulatory T (Treg) cells were determined by flow cytometry. The serum levels of cytokines in NOD mice and in the supernatant of the co-culture system by ELISA.ResultsTreatment with LGMSCs or LGMSC-Exos reduced inflammatory infiltration in the salivary glands, and restored salivary gland secretory function in NOD mice. Importantly, LGMSCs or LGMSC-Exos were demonstrated to inhibit the differentiation of Th17 cells but promote the induction of Treg cells in NOD mice and PBMCs from SS patients in vitro, accompanied by reduced interleukin 17 (IL-17), interferon gamma, and IL-6 levels and enhanced transforming growth factor beta and IL-10 secretion by T cells.ConclusionsLGMSCs are potential candidates for MSCs-based therapy and LGMSC-Exos might be utilized for establishing a new cell-free therapy against SS.