A Case of a Relapsing Remitting Macrophage Activating Syndrome After Covid-19 Vaccine In a Teenager with UNC13D Heterozygous Variant of Uncertain Significance

Introduction: Macrophage activation syndrome (MAS) is a severe hyper inflammatory condition caused by the over-activation and proliferation of T cells, NK cells and macrophages. It is often associated with complications of rheumatic/immune diseases. We present a case of a 15-year-old female who experiences recurrent episodes of MAS without any known definitive underlying etiology. Case Presentation: A 15-year-old previously healthy female developed fatigue, fevers, myalgia, chest pain, splenomegaly and lymphadenopathy 10 days after receiving her first Pfizer COVID-19 vaccine. Her symptoms recurred 10 days after receiving the second dose. Her myocarditis, MIS-C, and infectious work up was negative except for positive EBV IgG. Laboratory studies revealed anemia, hypertriglyceridemia, hypofibrinogenemia, and hyperferritinemia. She initially responded to decadron;however, her symptoms recurred with steroid taper. Bone marrow biopsy revealed hemophagocytosis. Whole exome sequencing (WES) revealed a heterozygous variant of uncertain significance in UNC13D c.962C>A (p.Thr321Asn). She had multiple re-admissions with significantly elevated inflammatory markers, including extremely high IL2-R, IL-18 and CXCL9. Each episode was complicated by an acute viral infection. She responds to high dose steroids, anti-IL-1, and JAK inhibitors. Nonetheless, it has been difficult to wean decadron without triggering a flare. She continues to require increasing doses of baricitinib. Discussion(s): MAS may be seen as a complication of rheumatic diseases, as well as inborn errors of immunity. However, none of these conditions have been diagnosed in this patient despite extensive testing, including WES. The degree of her immune dysregulation has been very severe making her disease process unpredictable and extremely difficult to control. She has frequent flares precipitated by viral infections or attempts at adjusting her immunomodulators. Weaning her medications has been challenging as she continues to require increasing doses of baricitinib and corticosteroids. The UNC13D gene is associated with autosomal recessive familial hemophagocytic lymphohistiocytosis type 3 (FHL3). Our patient is heterozygous for an UNC13D variant of uncertain significance. Additional genetic inquiries with whole genome sequencing to help elucidate the underlying etiology of her severe condition is being conducted. We hypothesize she developed MAS due to a combination of genetic predisposition, prior EBV infection, and immune stress associated with the COVID-19 vaccine. [Formula presented] [Formula presented] [Formula presented]Copyright © 2023 Elsevier Inc.

Development of warm autoimmune hemolytic anemia after initiation of Ruxolitinib in a patient with STAT1 gain-of-function mutation

Introduction: STAT1 gain-of-function (GOF) disease is associated with chronic mucocutaneous candidiasis (CMC) and a broad spectrum of infectious, inflammatory, and vascular manifestations. The Janus Kinase inhibitor ruxolitinib has been used successfully for CMC and autoimmune phenomena. We describe a case of warm autoimmune hemolytic anemia (WAIHA) in a patient with STAT1 GOF disease after initiating ruxolitinib. Case report: A 36-year-old man with STAT1 c.850G>A (p.Glu284Lys) mutation presented with CMC as well as recurrent viral and bacterial infections, lymphadenopathy, enteritis, nodular regenerative hyperplasia (NRH) and splenomegaly. Immune workup confirmed a combined immunodeficiency with hypogammaglobulinemia and T-cell lymphopenia. Ruxolitinib was initiated at 5 mg twice daily (due to pre-existing thrombocytopenia) with up titration over 3 months to 20 mg twice daily. He improved with weight gain, increased energy, resolution of chronic anemia, and improved lymphadenopathy and splenomegaly on imaging. Serum CXCL9 only minimally decreased from 4660 pg/ml to 3990 pg/ml. Soon after reaching ruxolitinib 20 mg twice daily, he developed JC viremia, prompting dose reduction to 15 mg BID. Within two weeks, he developed a non-COVID upper respiratory tract infection followed by fatigue, shortness of breath with ambulation, and dark urine. Emergency evaluation revealed warm antibody positive hemolytic anemia with a hemoglobin of 5 g/dL, and worsened thrombocytopenia. He was treated with blood transfusions, pulse steroids, and high-dose IVIG with stabilization but continued hemolysis. Due to the JC viremia, there was concern to give rituximab with increased PML risk. Bone marrow showed trilineage hematopoiesis, a mild increase in megakaryocytes and RBC precursors, and a loss of B-cell progenitors with retention of mature B cells. His B and T lymphocyte numbers had increased since prior to ruxolitinib, with a predominance of Tfh1-cells (58.7% of total Tfh-cells). He was started on sirolimus with a slow taper of prednisone with continued stable hemoglobin and platelets, and resolution of hemolysis after 3 months. Conclusion(s): To our knowledge, this is the first case of a STAT1 GOF patient developing WAIHA while receiving ruxolitinib therapy. Treatment choices were complicated by the risks of PML. Sirolimus combined with ruxolitinib allowed wean of corticosteroid and subsequent resolution of hemolysis.Copyright © 2023 Elsevier Inc.

Cytokine Profile in Different Post-Covid-19 Condition Phenotypes

Background: At least 10% of SARS-CoV-2 infected patients suffer from persistent symptoms for >12 weeks, known as post-COVID-19 condition (PCC) or Long Covid. Reported symptomatology is diverse with >200 physical and neurological debilitating symptoms. Here, we analyzed pro-inflammatory cytokine levels as a potential mechanism underlying persistent symptomatology. Method(s): Clinical data and samples used belong to the KING cohort extension, which includes clinically well characterized PCC (N=358, 59 persistent symptoms evaluated), COVID-19 recovered and uninfected subjects. We used Gower distances to calculate symptom's similarity between PCC and Ward's hierarchical clustering method to identify different symptom patterns among PCC patients. Cytokine levels of randomly selected PCC, recovered and uninfected subjects (N=193) were measured on plasma samples collected >6 months after acute infection using the 30-Plex Panel for Luminex. Mann- Whitney t-test was used to compare PCC vs recovered groups and Kruskal-Wallis t-test for >2 groups comparisons (PCC vs recovered vs Uninfected and within PCC clusters). FDR correction was applied for statistical significance (p-adj). Result(s): Hierarchical clustering identified 5 different PCC clusters according to their symptomatology, where PCC3 and PCC5 clusters showed higher prevalence of women ( >80%) and more persistent symptoms, while acute COVID-19 was mild in >80% of the patients. We selected 91 PCC (belonging to each cluster), 57 recovered and 45 uninfected subjects for cytokine profiling (Table 1). 13 soluble markers were significantly elevated (IL-1beta, Eotaxin, MIP-1beta, MCP-1, IL-15, IL-5, HGF, IFN-alpha, IL-1RA, IL-7, MIG, IL-4 and IL-8) in PCC and recovered groups compared to uninfected subjects (all p-adj< 0.04). In addition, PCC subjects tended towards higher levels of IL-1RA compared to recovered group (padj= 0.071). Within PCC clusters, FGF-basic and RANTES were elevated while IL-2 and MIG were decreased in PCC3 and PCC5 compared to the other PCC clusters (all p-adj< 0.04). TNF-alpha, IP-10, G-CSF and MIP-1alpha were decreased in PCC3 and PCC5 not reaching statistical significance (all p-adj=0.07). Conclusion(s): Some cytokines remained altered in all SARS-CoV-2 infected subjects independently of persistent symptoms after 6 months from acute infection. Differences between PCC and recovered individuals are limited after correction. Importantly, PCC cytokine profiles showed differences between clusters, which suggests different PCC subsyndromes with distinct etiology. Subjects Characteristics (Table Presented).

Relationships between plasma cytokine balance and cardiac magnetic resonance imaging in long-term post-COVID follow-up: A cross-sectional preliminary study

Background: COVID-19 infection can lead to a constellation of longlasting post-infectious sequelae, including myocardial dysfunction, whose outcome is strongly affected by a fine-tuned balance between pro-and anti-inflammatory systemic immune responses. Plasma cytokines are key mediators of this immunological balance. In this preliminary study we evaluated the cross-sectional association between the circulating levels of the main pro-and anti-inflammatory cytokines and cardiac magnetic resonance (CMR) abnormalities. Method(s): 71 subjects (59% female, mean age 52+/-14) with previous diagnosis of COVID-19 infection were enrolled at our institution for MULTICOVID protocol, comprehensive of CMR and biomarkers assessment performed >3 months and <1 year following the first negative swab test. CMR protocols consisted of conventional sequences (cine, T2-weighted imaging, and late gadolinium enhancement [LGE]) and quantitative mapping sequences (T1, T2, and extracellular volume [ECV] mapping). Plasma levels of cytokines TNF-alpha, IL-1beta, IL-1alpha, IFN-alpha2, IL-6, IL-8, IL-13, IL-10, IL-17A, IL-18, IP-10, MIG and MCP-1 were quantified by Multiplex Immunoassays on the Luminex technology platform. Soluble cardiologic and biochemical biomarkers were measured by routine laboratory analysis. Result(s): After a median of 9 (IQR 6-11) months following negative swab, CMR was normal in 48 subjects, while in 23 (32%) it revealed tissue characterization abnormalities (myocardial late enhancement and/or edema). By multivariate regression analysis (adjusted for age, sex, vaccination, severity degrees of the initial COVID disease, presence of comorbidities, smoke, time interval between COVID diagnosis and CMR assessment) the cytokine ratio TNF-alpha/(IL-10+IL-13) was independently associated (OR=2.89, 95% CI 1.19-7.04, p=0.02) with CMR abnormalities. Interestingly, the cumulative pro-/anti-inflammatory cytokine ratio (IL-1beta+TNF-alpha+IFN-alpha2+IL-6+IL-17A+IL-8)/(IL-10+IL-13) showed a positive (OR=1.70, 95% CI: 1.04-2.75) and significant (p=0.03) association with CMR imaging aspects. Also, the ratio IFN-alpha2/(IL-10+IL-13), although without achieving a complete statistical significance (p=0.09), was associated positively with CMR findings. Conclusion(s): The preliminary results of this cross-sectional study suggest that the systemic inflammatory environment, long-lasting unbalanced towards a prevalent cytokine-driven pro-inflammatory condition following COVID infection, could affect the development of CMR-detectable myocardial edema and fibrosis in long-term post-COVID subjects.

Correlation between lung inflammatory cells, viral load and cytokines in fatal COVID-19

SARS-CoV2 infection induces a complex interaction between virus and host immune system, activating multiple inflammatory pathways and leading to hyperinflammation, diffuse alveolar damage (DAD), ARDS, and multiorgan failure. We aimed to correlate the quantification of viral load, inflammatory cells and cytokines in lung tissue of fatal COVID-19. We assessed inflammatory cells by multiplex immunohistochemistry, cytokines by Luminex xMAP Assay and viral load by real time PCR in autopsy lung tissue of 18 COVID-19 patients. Correlations were considered statistically significant if p<0.05. Macrophages correlated with IL-1beta (r=0.54), IL-10 (r=0.5), IFN-alpha2 (r=0.72), IFN-gamma (r=0.6), CCL20 (r=0.5), TGF-beta1 (r=0.6), TGF-beta2 (r=0.6). CD4+T cells correlated with CCL20 (r=0.6), MDC/CCL2 (r=0.53), CCL17 (r=0.5), IP-10 (r=0.6), CXCL9 (r=0.6). CD8+T cells correlated with IL-1beta (r=0.54), IL-4 (r=0.63), IL-6 (r=0.7), IL-8 (r=0.63), IL-10 (r=0.6), TNF-alpha (r=0.6), IFN-gamma (r=0.74), CCL20 (r=0.7), TGF-beta1 (r=0.7), TGF-beta2 (r=0.56), TGF-beta3 (r=0.54), MDC/CCL2 (r=0.7), CCL17 (r=0.64). Langerin dendritic cells (DC) correlated with symptom onset to death interval (r=0.6), hospitalization length (r=0.65), mechanical ventilation (MV) length (r=0.6), ICU stay (r=0.6), exudative DAD (r=-0.5), viral load (r=-0.6). Myeloid DC correlated with symptom onset to death interval (r=0.8), hospitalization length (r=0.8), MV length (r=0.8), ICU stay (r=0.8), exudative DAD (r=-0.5), viral load (r=-0.7). Viral load correlated with symptom onset to death interval (r=-0.7), hospitalization length (r=-0.8), MV length (r=-0.7), ICU stay (r=-0.8), exudative DAD (r=0.6). There is a complex temporal inflammatory modulation in severe COVID-19.

Predictive Value of Specific Cytokines for Lethal Covid-19 Outcome

In our study, we aimed to evaluate the significance of specific cytokines in blood plasma as predictive markers of COVID-associated mortality. Materials and methods. In plasma samples of 29 patients with PCR-confirmed COVID-19 we measured the concentrations of 47 molecules. These molecules included: interleukins and selected pro-inflammatory cytokines (IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A/CTLA8, IL-17-E/IL-25, IL-17F, IL-18, IL-22, IL-27, IFNalpha2, IFNgamma, TNFalpha, TNFbeta/Lymphotoxin-alpha(LTA));chemokines (CCL2/MCP-1, CCL3/MIP-1alpha, CCL4/MIP-1beta, CCL7/MCP-3, CCL11/Eotaxin, CCL22/MDC, CXCL1/GROalpha, CXCL8/IL-8, CXCL9/MIG, CXCL10/IP-10, CX3CL1/Fractalkine);anti-inflammatory cytokines (IL-1Ra, IL-10);growth factors (EGF, FGF-2/FGF-basic, Flt-3 Ligand, G-CSF, M-CSF, GM-CSF, PDGF-AA, PDGFAB/BB, TGFalpha, VEGF-A);and sCD40L. We used multiplex analysis based on xMAP technology (Luminex, USA) using Luminex MagPix. As controls, we used plasma samples of 20 healthy individuals. Based on the results, we applied Receiver Operating Characteristic (ROC) analysis and Area Under Curve (AUC) values to compare two different predictive tests and to choose the optimal division point for disease outcome (survivors/non-survivors). To find optimal biomarker combinations, we as used cytokines concentrations as dependent variables to grow a regression tree using JMP 16 Software.Results. Out of 47 studied cytokines/chemokines/growth factors, we picked four pro-inflammatory cytokines as having high significance in evaluation of COVID-19 outcome: IL-6, IL-8, IL-15, and IL-18. Based on the results received, we assume that the highest significance in terms of predicting the outcome of acute COVID-19 belongs to IL-6 and IL-18. Conclusion. Analyzing concentrations of IL-6 and IL-18 before administering treatment may prove valuable in terms of outcome prognosis. Copyright © Arsentieva N.A. et al., 2022.

COVID-19 AS A CONFOUNDER IN A CASE OF HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS

SESSION TITLE: Pulmonary Involvement in Critical Care Case Posters SESSION TYPE: Case Report Posters PRESENTED ON: 10/17/2022 12:15 pm - 01:15 pm INTRODUCTION: Hemophagocytic Lymphohistiocytosis (HLH) is a condition in which the body's natural ability to end an immune or inflammatory response is defective1. COVID-19 also presents with severe inflammation, and like HLH, leads to significantly elevated ferritin2. We present a case that was initially thought to be COVID-19, but the patient was diagnosed with HLH in the setting of S. aureus endocarditis. CASE PRESENTATION: A 62-year-old male with a history of atrial fibrillation, mechanical mitral valve on warfarin, type II diabetes, chronic obstructive pulmonary disease, and recently diagnosed COVID-19 presented to the hospital with progressive dyspnea. In the emergency department, he was found to be hypoxemic and in atrial fibrillation with rapid ventricular response. He had a fever of 39.3°C and his initial laboratory workup revealed hemoglobin of 11.9 g/dL, leukocytes of 5,700, platelets of 83,000, AST 35 U/L, ALT 34 U/L, CRP of 31.89 mg/dL, and ferritin of 1994 ug/L. The patient was admitted and started on dexamethasone 6 mg daily. The following day, the patient's blood work revealed a significant worsening of AST and ALT to 7280 U/L and 3319 U/L, respectively. D-dimer increased to 11861 ng/mL (DDU) and ferritin to 36,470 ug/L. On the third day of admission, his clinical status declined acutely as he became significantly bradycardic, progressing to a cardiac arrest after which he required cardiopulmonary resuscitation, intubation, and was transferred to the intensive care unit. A CT scan obtained revealed hepatomegaly of 22 cm and blood cultures were positive for S. aureus requiring vancomycin treatment. The patient was kept on dexamethasone due to concerns for HLH. Ferritin continued to worsen, reaching 50,749 ug/L. His sCD25 came back positive. Unfortunately, the patient expired on his fifth day of hospitalization after discussing with his family their goals for his care and switching his care to comfort only. DISCUSSION: HLH is a challenging condition since diagnosis is difficult and mortality is high. There are a few methods used to diagnose HLH. Usually, 5 of 8 criteria must be met, which was achieved with this patient. However, often the patient only fulfills 4 of 8 since many criteria are difficult to obtain such as bone marrow biopsy, sCD25, and CXCL9. A useful tool is the H-calculator3. Our patient scored a 180 indicating a 50-75% likelihood of HLH. Assessing the likelihood of disease is important since sCD25 and CXCL9 take time and if the patient is clinically deteriorating treatment should not be delayed. CONCLUSIONS: HLH is catastrophic and rare. Physicians should always have it as a differential diagnosis in patients with severe inflammatory states and elevated ferritins to avoid anchoring bias. If suspicion is high based on clinical evaluation and scores, treatment should not be delayed. Reference #1: Filipovich A, McClain K, Grom A. Histiocytic disorders: recent insights into pathophysiology and practical guidelines. Biol Blood Marrow Transplant. 2010;16(1 Suppl):S82-S89. doi:10.1016/j.bbmt.2009.11.014 Reference #2: Cheng L, Li H, Li L, et al. Ferritin in the coronavirus disease 2019 (COVID-19): A systematic review and meta-analysis. J Clin Lab Anal. 2020;34(10):e23618. doi:10.1002/jcla.23618 Reference #3: Fardet L, Galicier L, Lambotte O, et al. Development and validation of the HScore, a score for the diagnosis of reactive hemophagocytic syndrome. Arthritis Rheumatol. 2014;66(9):2613-2620. doi:10.1002/art.38690 DISCLOSURES: No relevant relationships by Areeka Memon No relevant relationships by Carissa Monterroso No relevant relationships by Carson Oprysko No relevant relationships by Eduardo Padrao No relevant relationships by Mouna Penmetsa

*CHILDREN WITH ACUTE SEVERE HEPATITIS OF UNDETERMINED ETIOLOGY HAVE A SYSTEMIC T CELL ACTIVATION AND INTERFERON GAMMA (IFN-gamma) ACTIVITY REMINISCENT OF HEMOPHAGOCYTIC HISTIOCYTOSIS: A POTENTIAL ROLE FOR T CELL AND IFN-gamma DIRECTED THERAPIES

Background: 30-50% of pediatric acute liver failure (PALF) is of unknown cause, or indeterminate PALF (iPALF), which frequently results in transplantation. A subset of iPALF is characterized by T-cell activation. Some children with acute severe hepatitis of unknown etiology (SH-u) can evolve to iPALF. Hemophagocytic Lymphohistiocytosis (HLH) is a well-defined hyper-inflammatory condition characterized by marked T-cell activation and frequent severe liver involvement. We postulated SH-u evolving to iPALF has hyper-inflammatory immune signatures that are identifiable before fulfilling PALF criteria, and might overlap with those seen in HLH. We compared the immune dysregulation signatures of children with HLH to children with SH-u, PALF cases with known etiologies, and healthy pediatric controls (HC). Method(s): Between 2019-2021, we prospectively enrolled 14 patients hospitalized with SH-u and 7 patients with PALF of known etiologies. Age dependent standard of care diagnostic studies were performed. SH-u was defined as ALT> 500, INR < 2, and no hepatic encephalopathy. HLH enrollees fulfilled the 2004 diagnostic criteria. High dimension T-cell immunophenotyping, cytokine and chemokine profiling (71-plex) was done for SH-u, HLH (n=5), and HC (n= 16) peripheral blood samples. T cell activation was prospectively identified by co-expression of surface activation markers HLA-DR and CD38. Based on immune studies in HC, CD8 effector memory (EM) activation of >9% distinguished patients with significant T cell activation from HC. This cutoff of >9% was therefore used to identify SH-u patients with T cell activation. Normally distributed data were compared by either a two-tailed t-test or an ordinary One-Way Anova test with Turkey's multiple comparison test. Non-normally distributed data were compared by either the Mann-Whitney test or Kruskal-Wallis test with Dunn's multiple comparisons test. P Values < 0.05 were deemed significant. Result(s): Subjects ranged in age from 4 days to 19 years old. There were no age or sex differences between the groups. One SH-u patient had prior COVID infection, but no subject met MIS-c criteria. Two SH-u patients ultimately evolved to PALF criteria with INR> 2. All patients with SH-u had higher CD8 EM T-cell activation (mean +/- SEM = 43.7+/-6.3%;range 9.2 to 81.3;p<0.0001), which was significantly higher than HC (2.9+/-0.5%) and PALF of known etiology (4.0+/-0.9%) . However, the amplitude of T-cell activation was lower in the SH-u group relative to the HLH group (90.3+/-2.7%;p<0.0001), as shown in Figure 1. A similar trend in T cell activation was noted in the CD4 compartment. Overall, the activation in the CD8 compartment was much greater than in CD4. SH-u patients had a decreased CD4/CD8 ratio compared to the PALF group. Despite higher T cell activation in patients with SH-u compared to PALF, ferritin, often used to screen for hyper-inflammation, was lower in the SH-u group when compared to PALF group (1240+/-609 vs. 39517+/-32149;p<0.05) and very significantly lower than HLH (32415 +/- 14845;p =0.002). 50% of patients with SH-u etiology had ferritin < 500 mg/L. Cytopenia (hemoglobin < 9 g/dL, ANC < 1000/mL, platelets < 100,000/mL) is characteristic of patients with HLH. Despite overlapping T cell activation with HLH, the SH-u cohort had only 2 patients with this feature: one with thrombocytopenia and one with neutropenia. Supportive of this higher T cell activation, we noted chemokines driven by IFN-gamma, CXCL9 and CXCL10, to be elevated in SH-u compared to HCs and comparable to HLH patients. As a proof of concept, 1 patient with SH-u and thrombocytopenia underwent treatment with Emapalumab (an IFN-gamma blocking antibody) along with other immune modulators both with complete liver, immune, and platelet count recovery. Conclusion(s): Our cohort of SH-u was associated with significant T-cell activation. In addition, our patients with HLH and SH-u with T cell activation had similar increased IFN-gamma activity. Despite this T cell activation, ferritin values were significantly lower in SH-u compared to PALF without T cell activation. Ferritin may not be a reliable screening test to identify SH-u patients with significant T cell activation. If validated in a larger well-defined population of SH-u, the results may suggest a role for IFN-gamma blocking agents in a subgroup of SH-u prior to PALF or before bone marrow failure development.

Salivary Cytokines in Children with and Without Severe Disease Due to sars-cov-2 Infection

Background: Most children exposed to SARS-CoV-2 virus present with mild symptoms, but some may experience severe illnesses such as Multisystem inflammatory syndrome (MISC) or respiratory failure. Currently there are no established biomarkers to predict progression to severe disease. Although specific serum cytokines have been found to be higher in adults with severe COVID-19, their role as predictors of severe disease in children remains unclear. Further, the role of salivary cytokines in COVID-19 associated inflammation is unknown. Our objective was to compare cytokine levels in saliva of children with and without severe disease due to SARS-CoV-2 infection. Methods: This prospective observational study, conducted at two tertiary children's hospitals, was supported by a grant from the National Institute of Health RADx Program. Children ≤ 18 years of age with symptoms due to SARS-CoV-2 infection (positive PCR test, serology or immunological link) were enrolled after informed consent. Severe cases were defined as the occurrence of any of the following within 30 days of testing: diagnosis of MISC or Kawasaki disease, requirement for >2L oxygen, inotropes, mechanical ventilation or ECMO, or death. A saliva sample was obtained through passive drool using MicroSAL kits (Oasis Diagnostics) and a viral transport medium (VTM-C19, Biomed). Abundance levels of six cytokines (TNFR1, IL13, IL-15, CCL7, CXCL10 and CXCL9) were measured in triplicate using microfluidic immunoassays (Ella, Protein Simple). Mean concentrations for each sample were determined against a standard curve and corrected for dilution. Levels of the six cytokines were compared between those with severe or nonsevere SARS-CoV-2 symptoms using a non-parametric t-test. The relationship between salivary levels of individual cytokines was assessed among children with severe and non-severe SARS-CoV2 using a Pearson correlation analysis Results: A total of 150 children were enrolled from 03/29/2021 to 05/30/2021 (mean age of 7.1 years ± 5.7 years, 54.6% females). Of the total, 38 (25.3%) children met criteria for severe SARS-CoV-2 infection. CXCL10 displayed significantly (fold change>2, p < 0.05) elevated levels in the saliva of children with severe SARS-CoV-2 (Figure 1). The relationship between levels of CXCL9 (MIG) and CXCL10 showed greater levels association (R2 = 0.93) in children with severe SARS-CoV-2 than in peers with non-severe SARS-CoV-2 (R2 = 0.65;Figure 2). Conclusion: In this preliminary analysis of salivary cytokines among children with SARS-CoV-2 infection, we found CXCL10 displayed differential expression with severe symptoms. These findings may provide critical information about the pathophysiology of severe SARS-CoV-2. Confirmation in further studies is necessary. Saliva concentrations of CXCL10 in children with severe SARSCoV-2 symptoms. The whisker box plots display salivary concentrations of CXCL10 in children with severe (green) and non-severe (red) SARS-CoV-2 infection as measured with next generation enzyme linked immunosorbent assay. Levels of CXCL10 (p < 0.01;fold change = 3.04) were elevated in children with severe SARS-CoV-2 symptoms on Wilcoxon testing. .

ADULT-ONSET STILL'S DISEASE AND MACROPHAGE ACTIVATION SYNDROME FOLLOWING COVID-19 PNEUMONIA

CASE: A 41-year-old woman with recent COVID-19 pneumonia presented to the hospital with several months of fever, polyarthralgia, and weight loss. She reported waxing and waning shoulder, elbow, wrist, hip, knee, and ankle pain without identifiable triggers. She had no pertinent medical or family history. Vital signs were only notable for fever of 40C which recurred daily. Exam revealed tenderness to palpation of multiple joints;her skin had no rash, purpura, or nodules. Hepatosplenomegaly and axillary lymphadenopathy were noted. Infectious workup was negative for bacterial, viral, fungal, mycobacterial, parasitic, and protozoal infections. Initial studies demonstrated hemoglobin 8.2 mg/dL, lymphopenia, and aspartate transaminase 58 U/L. Flow cytometry, excisional lymph node biopsy, and bone marrow biopsy were negative for lymphoproliferative disease. Rheumatologic workup revealed elevated ferritin, triglycerides, Interleukin-6, soluble Interleukin-2 receptor (sIL-2R), and “C-X-C Motif Chemokine Ligand 9” (CXCL9);extensive rheumatologic serologies were otherwise negative. Her clinical picture was consistent with Macrophage Activation Syndrome (MAS). She also met diagnostic criteria for Adult-Onset Still's Disease (AOSD) given arthralgia, fever, lymphadenopathy, splenomegaly, abnormal liver function test (LFT), and otherwise negative workup. Her presentation suggested COVID-19 triggered AOSD which triggered MAS. We administered intravenous immune globulin (IVIG) and high-dose steroids. She clinically improved and was discharged with oral steroids. She returned to the hospital two months later for fever, arthralgia, and faint, evanescent rash with elevated erythrocyte sedimentation rate, C-reactive protein, ferritin, lactate dehydrogenase, and LFT consistent with an AOSD flare. She received intravenous steroids and Anakinra. Symptoms resolved, and she was discharged with plans to continue Anakinra and oral steroids. At followup, she had resolution of all symptoms. IMPACT/DISCUSSION: COVID-19 has many chronic complications, including triggering of underlying rheumatic disease. This sequence of events suggests that COVID-19 Pneumonia triggered an underlying diagnosis of AOSD. AOSD should be considered in the differential diagnosis of patients with quotidian fever and arthralgia following COVID-19 infection. AOSD is a diagnosis of exclusion and requires ruling out infectious, malignant, and rheumatic etiologies. AOSD may trigger MAS, a dysregulated immune response to underlying inflammation, and should be considered in patients with suspected infection refractory to treatment who have fever, splenomegaly, cytopenias, and elevated ferritin, triglycerides, sIL-2R, and CXCL9. CONCLUSION: COVID-19 has many chronic complications. AOSD may manifest after COVID-19 infection and should be considered in the differential diagnosis of patients with persistent fever and arthralgia. MAS should be suspected in patients with systemic inflammation refractory to treatment. AOSD may cause MAS.

Biomarker Levels Concur with a Risk Stratification Criteria in Covid-19 Pneumonia, a Role for a Prognostic-Tool for Identification of Clinical Deteriorators

Rationale: COVID-19 patients present with a number of clinical symptoms ranging from mild, moderate to severe, while only a subgroup of patients, who requires high-dependency critical care resources, accounts for most of the COVID-19 associated health care expenditure and death. A reliable prognostic tool is therefore required to identify patients at risk of developing severe COVID-19 pneumonia. To address this unmet need, we tested a wide range of potentially important peripheral blood biomarkers in a group of clinically risk-stratified COVID-19 patients in order to identify most relevant candidate biomarker(s) predictive of disease progression. Methods: Patients and healthy controls recruited to this study are summarised in Figure 1. Biomarkers levels were analysed using ANOVA across the severity groups. Spearman-correlation coefficients against pairs of average levels from each biomarker within severity-group and healthy controls were assembled into a 76x76 matrix and agglomerative hierarchical clustering was applied to generate the final heatmaps. Linear-discriminant analysis (LDA) was carried out on a reduced optimised set of biomarkers to explore the boundaries between the clinical severity groups.Results: Degree of lymphopaenia, neutrophil levels, TNF-α, INR-levels, and pro-inflammatory cytokines;IL6, IL8, CXCL9 and D-dimers were significantly increased in COVD-19 patients compared to healthy controls (p<0.05, 95% C.I.). C3a and C5 was significantly elevated in all categories of severity compared to healthy controls (p<0.05), C5a levels were significantly different between “moderate” and “severe” categories (p<0.01). sC5b-9 was significantly elevated in the “moderate” and “severe” category of patients compared to healthy controls (p<0.001).Heatmap analysis demonstrated distinct visual differences of biomarker profiles between the clinical severity groups. LDA on the deteriorators, non-deteriorators and healthy volunteers as a combined function of the predictor variables: C3, eosinophil-counts, granulocyte colony-stimulating factor (G-CSF), fractalkine, IL10, IL27, LTB4, lymphocyte count, MIG/CXCL9, M-CSF, platelet count and sC5b-9 showed clear separation between the groups based on biomarker/blood-count levels.Conclusions: Diagnostic and clinical assessments followed by robust statistical and machine learning approaches could identify peripheral blood biomarkers for prognostic stratification of patients in COVID-19. Our results would be helpful for clinicians and supports the use of point of care devices that can quantify multiple analytes. (Lui G, et al., Pointof- care detection of cytokines in cytokine storm management and beyond: Significance and challenges. VIEW. 2021;2: 1-20.). Such would allow for more efficient management and resource allocation. 1 (Figure Presented).

PERFIL IMUNOLÓGICO DE DOADORES DE SANGUE COM E SEM INFECÇÃO PRÉVIA POR SARS-COV-2

Objetivo: Comparar o perfil imunológico de indivíduos positivos e negativos para IgG anti-SARS-CoV-2. Métodos: Amostras de soro (n = 7.837) de doadores de sangue da Fundação Hemominas, coletadas no período de março a dezembro de 2020, foram testadas por quimioluminescência para IgG anti-SARS-CoV-2. As amostras positivas foram separadas em quatro grupos considerando-se os intervalos interquartis do index de anticorpos detectados no teste sorológico. Amostras positivas e negativas foram utilizadas na dosagem de citocinas (IL-2, IL-4, IL-6, IL-10, TNF, IFN-gama e IL-17A) e quimiocinas (CXCL8, CCL5, CXCL9, CCL2 e CXCL10). Resultados: Dos doadores testados, 441 (5,6%) foram positivos para IgG anti-SARS-CoV-2 com mediana de index de 3,65 (IQR 2,43-5,40). As concentrações séricas (ng/mL) de IL-10 (mediana, 0,51;IQR, 0,18-0,86;p < 0,0001), TNF (mediana, 0,65;IQR, 0,00-0,77;p = 0,0279) e IFN-gama (mediana, 0,36;IQR, 0,00-0,88;p < 0,0001) foram significativamente maiores em doadores de sangue positivos para IgG anti-SARS-CoV-2. As concentrações séricas (ng/mL) de CXCL8 (mediana, 13,60;IQR, 5,98-28,04;p = 0,0013), CCL5 (mediana, 4.017,00;IQR, 2.674,00-4.736,00;p < 0,0001), CXCL9 (mediana, 33,08;IQR, 17,88-54,14;p < 0,0001), CCL2 (mediana, 40,39;IQR, 23,38-61,52;p = 0,0068) e CXCL10 (mediana, 111,70;IQR, 56,98-178,00;p < 0,0001) foram significativamente maiores em doadores de sangue positivos para IgG anti-SARS-CoV-2. Análises de correlação revelaram que todas as citocinas (exceto IL-4, IL-6 e IL-17A) têm correlação negativa significativa com o index de IgG anti-SARS-CoV-2, mas com coeficiente de Spearman (r) menores que 0,5. Todas as quimiocinas testadas tiveram correlação negativa significativa, com destaque para CCL5 (r = -0,79), CXCL9 (r = -0,57) e CXCL10 (r = -0,51). Discussão: A análise do perfil imunológico de indivíduos positivos e negativos evidenciou que a produção de IgG anti-SARS-CoV-2 depende de uma resposta imune inata caracterizada pela alta concentração sérica de quimiocinas e de uma resposta pró-inflamatória potencializada por TNF e IFN-gama e regulada por IL-10. Os resultados evidenciam ainda que a produção de mais anticorpos contra o vírus depende de uma síntese controlada de citocinas e quimiocinas, indicando que menores níveis destes biomarcadores estão relacionados à maior produção de IgG anti-SARS-CoV-2. Conclusão: Os resultados deste estudo evidenciaram que um perfil imune pró-inflamatório associado a biomarcadores de resposta imune inata é importante para o desenvolvimento de anticorpos IgG anti-SARS-CoV-2. Suporte financeiro: Fundação HEMOMINAS, CNPq.

ANÁLISE DE CITOCINAS E QUIMIOCINAS NO PLASMA DE PACIENTES COM COVID-19 E DOADORES DE SANGUE ASSINTOMÁTICOS INFECTADOS COM SARS-COV-2

Objetivos: Comparar níveis de citocinas e quimiocinas entre indivíduos infectados com SARS-CoV-2 com COVID-19 grave ou com a forma assintomática da infecção, a fim de avaliar quais destes marcadores biológicos de resposta inflamatória são indicadores de gravidade da infecção viral. Métodos: Foram analisados dados clínicos de 48 pacientes com COVID-19 hospitalizados no Hospital Eduardo de Menezes (FHEMIG, MG) no período de 07 de julho a 21 de novembro de 2020, que necessitaram ou não de assistência em terapia intensiva (grupos UTI e sem UTI, respectivamente). Foi feita a quantificação dos níveis de citocinas (IL-2, IL-4, IL-6, IL-10, TNF, IFN-, IL-17A) e quimiocinas (CCL2, CCL5, CXCL8, CXCL9 e CXCL10) do plasma de 14 pacientes UTI e 17 pacientes sem UTI que tinham COVID-19 grave no momento da coleta da amostra, além de 24 doadores de sangue da Fundação Hemominas com infecção ativa pelo SARS-CoV-2 (RT-PCR positiva) que eram assintomáticos no momento da coleta da amostra. Resultados: A análise clínica dos pacientes UTI (n = 19) em comparação àqueles que não usaram UTI (n = 29) não mostrou diferença estatística quanto à frequência de comorbidades e de sinais e sintomas para COVID-19 na admissão hospitalar. A comorbidade mais comum foi hipertensão (62,5%), seguida por diabetes (37,5%) e obesidade (22,9%). Tosse, dispneia, febre, fatiga e mialgia foram os sinais e sintomas mais prevalentes de COVID-19 em ambos os grupos. Entretanto, os pacientes UTI desenvolveram doença grave ou crítica que requereu um período de hospitalização em média duas vezes mais longo que o grupo sem UTI (p < 0,001). O conjunto dos pacientes com COVID-19 mostrou níveis significativamente mais altos de IL6, IL10 e CCL5 que os doadores assintomáticos. Na comparação dos grupos dos pacientes houve diferença significativa apenas para IFN, com níveis mais elevados nos pacientes UTI. Discussão: Apesar do grupo de pacientes UTI apresentarem quadro de COVID-19 mais grave que os pacientes sem UTI, a frequência de sinais e sintomas da doença e de comorbidades não foi significativamente diferente entre os grupos. A evolução da COVID-19 de assintomática para grave e crítica tem sido associada com intensa resposta inflamatória, o que está de acordo com maiores níveis de IL6, IL10 e CCL5 observados nos pacientes em comparação aos doadores assintomáticos. Nível de IFN pode ser especial indicador de gravidade da doença. Conclusão: Marcadores biológicos, como citocinas e quimiocinas, podem ser melhores preditores de evolução da COVID-19 que sinais clínicos e sintomas. Suporte financeiro: Fundação Hemominas e SES/MG.

Genetic and structure of novel coronavirus COVID-19 and molecular mechanisms in the pathogenicity of coronaviruses

The recently identified 2019 novel coronaviruses (2019-nCoV) has caused extra-human infections. 2019-nCoV identified a global threat that is causing an outbreak of unusual viral pneumonia in patients with severe acute respiratory syndrome (SARS)-coronaviruses 2 (SARS-CoV-2). Considering the relatively high identity of the receptor-binding domain (RBD) in 2019-nCoV and SARS-CoV, it is urgent to assess the cross-reactivity of anti-SARS-CoV antibodies with 2019-nCoV spike protein, which could have important implications for rapid development of vaccines and therapeutic antibodies against 2019-nCoV. The zinc metallopeptidase angiotensin-converting enzyme 2 (ACE2) is the only known human homolog of the key regulator of blood pressure ACE. ACE2 also serves as the cellular entry point for the SARS virus, therefore, a prime target for pharmacological intervention. SARS-CoV-2 uses the SARS-CoV receptor for entry and the serine protease transmembrane protease serine 2 for spike (S) protein priming. That it is still necessary to develop novel mAbs that could bind specifically to 2019-nCoV RBD. Cell entry of coronaviruses depends on the binding of the viral S proteins to cellular receptors and S protein priming by host cell proteases. A transmembrane protease serine 2 inhibitor approved for clinical use blocked entry and might constitute a treatment option. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention. This review will help understand the biology and potential risk of CoVs that exist in richness in wildlife such as bats. We provide a brief introduction to the pathogenesis of SARS-CoV and Middle East respiratory syndrome-CoV and interaction between the RBD of coronavirus spike protein and ACE2.

Children with COVID-19 Demonstrate Distinct Serum Cytokines Profiles According to Clinical Presentations

Background. Almost 4 million children have tested positive for Coronavirus Disease 2019 (COVID-19) as of June 3 2021, representing 14% of all cases in USA. Children present with diverse clinical findings including the multisystem inflammatory syndrome in children (MIS-C). In this study, we measured serum cytokine concentrations in children with COVID-19 to identify differences in immune profiles according to clinical presentations. Methods. A total of 133 children 0-21 years of age with COVID-19 were enrolled at Nationwide Children's Hospital, in Columbus, Ohio. Nasopharyngeal swab RT-PCR testing was used for SARS-CoV-2 detection and quantification. Clinical and laboratory information were obtained, and blood samples were collected for measurement of cytokines with a 92-plex inflammation assay (Olink). Normalized cytokine expression levels in patients were compared with serum samples from 66 pre-pandemic agematched healthy controls. Results. COVID-19 children included: 1) those identified by universal screening (n=47);2) moderate disease (ward;n=48);3) severe disease (PICU;n=20);4) MIS-C (n=18). Children identified by universal screening were hospitalized for trauma, appendicitis or new onset diabetes among others. Children with symptomatic COVID-19 had significantly higher SARS-CoV-2 viral loads than children with MIS-C or those identified via universal screening. Concentrations of interferon (IFN) related cytokines (IFNg, CXCL9, CXCL10, CXCL11), interleukins (IL6, IL8, IL10, IL17A, IL18, IL24) and other inflammatory cytokines (TGF, TNF, VEGF, MCP, CD40) were significantly increased in children with acute COVID-19 and MIS-C compared with children identified by universal screening and healthy controls. These cytokines were positively correlated with C-reactive protein, D-dimer and disease severity in COVID-19, but negatively correlated with viral loads (Fig 1). MIS-C showed stronger inflammatory response than acute COVID-19 (Fig 2). Correlation of Age-adjusted cytokine expression values with viral load, disease severity, CRP and D-dimer. Pearson correlation coefficient is shown for each pair. Red: positive correlation;blue: negative correlation Cytokines that differentiate MIS-C from acute COVID-19 Heatmap shows the differential expressed cytokines between MIS-C and acute severe COVID-19 (padj<0.05, FC>2). The age-adjusted expression values are normalized the median of healthy controls. Red: up-regulation, blue: down-regulation. Conclusion. We identified three cytokine clusters in children with COVID-19 according to clinical presentations. Correlations of serum cytokines with clinical/laboratory parameters could be used to identify potential biomarkers associated with disease severity in COVID-19.

Rapid and Sustained Decline in CXCL-10 (IP-10) Annotates Clinical Outcomes Following TNF-α Antagonist Therapy in Hospitalized Patients with Severe and Critical COVID-19 Respiratory Failure

Background. TNFα and IFN-γ may synergize to induce cytokine-driven lethal hyperinflammation and immune exhaustion in COVID-19 illness. Methods. To assess TNFα-antagonist therapy, 18 hospitalized adults with hypoxic respiratory failure and COVID-19 pneumonia received single-dose infliximab-abda therapy 5mg/kg intravenously between April and December 2020. The primary endpoint was time to increase in oxygen saturation to fraction of inspired oxygen ratio (SpO2/FiO2) by ≥ 50 compared to baseline and sustained for 48 hours. Secondary endpoints included 28-day mortality, dynamic cytokine profiles (Human Cytokine 48-Plex Discovery Assay), secondary infections, duration of supplemental oxygen support and hospitalization. Hospitalized patients with SARS-COV2 infection and pneumonia that were referred to the infliximab-abda study team for evaluation. Results. Patients were predominantly in critical respiratory failure (15/18, 83%), male (14/18, 78%), above 60 years (median 63 yrs, range 31-80), race-ethnic minorities (13/18, 72%), lymphopenic (13/18, 72%), steroid-treated (17/18, 94%), with a median ferritin of 1953ng/ml. Sixteen patients (89%) met the primary endpoint within a median of 4 days, 15/18 (83%) recovered from respiratory failure, and 14/18 (78%) were discharged in a median of 8 days and were alive at 28-day follow-up. Deaths among three patients ≥ 65 years age with pre-existing lung disease or multiple comorbidities were attributed to secondary lung infections. Mean plasma IP-10 levels declined sharply from 9183 pg/ml to 483 pg/ml at Day 3 and 146 pg/ml at Day 14/discharge. Significant declines in IFN-γ, TNFα, IL-27, IL-6 (baseline above 10pg/ml), CRP and ferritin were specifically observed at Day 3 whereas other cytokines were unaffected. Among 13 lymphopenic patients, six (46%) had resolution of lymphopenia by day 3, and 11 by day 14. CXCR3-ligand (IP-10 and CXCL-9) declines were strongly correlated among patients with lymphopenia reversal (Day 3, Pearson r: 0.98, p-value: 0.0006). following treatment with infliximab-abda. The status of the patient at last follow-up (discharged, alive or dead) is indicated. ECMO: extracorporeal membrane oxygenation Control of inflammatory markers and cytokines following infliximab therapy Values from individuals are connected with solid lines, with deceased individuals indicated in red. Statistics: n=18, paired ratio t-test compared to baseline;∗: P<0.05, ∗∗: P<0.01, ∗∗∗: P<0.001, ∗∗∗∗: P<0.0001, n.s.: not significant. Conclusion. Consistent with a central role of TNFα, the clinical and cytokine data indicate that infliximab-abda may rapidly abrogate pathological inflammatory signaling to facilitate clinical recovery in severe and critical COVID-19. Randomized studies are formally evaluating infliximab therapy in this context. Funding: National Center for Advancing Translational Sciences.

Hemophagocytic lymphohistiocytosis sans triggers

Case Report Hemophagocytic Lymphohistiocytosis (HLH) is an hyperinflammatory state due to hyperactivation of macrophages and T-cells which rarely affects adults. It can be familial or sporadic. Triggers are infections, auto-immune diseases, malignancies, and immune checkpoint inhibitors. HLH diagnostic criteria are fever, splenomegaly, bicytopenia, hypertriglyceridemia, hemophagocytosis, low/absent NK-cellactivity, elevated ferritin, and high-soluble interleukin-2-receptor (IL-2R). Five out of eight criteria are required for diagnosis. A 54-year-old female was noted to have leukopenia during a routine visit with her family physician. Follow up labs revealed worsening leukopenia, anemia and a normal platelet count. She received Amoxicillin/Clavulanic acid for a presumed upper respiratory infection and developed nausea, diarrhea and decreased appetite. She was referred to Hematology Oncology for leukopenia. During workup she developed fatigue, night sweats and high fevers. Workup revealed WBC 2400 mcL, microcytic anemia, transaminitis with lactate dehydrogenase of 1725 U/L and ferritin of >15000 ng/ mL . Peripheral blood smear showed leukopenia without immature cells or blasts and mild microcytic erythrocytes. Further tests detected CXCL-9 of 125050 pg/mL, D-dimer of >5000 ng/mL and interleukin-2-receptor of 20604 pg/ mL. EBV, CMV, HSV, HHV-6, parvovirus, bartonella, leishmaniasis, bacteria and COVID-19 were negative. Computed tomography of the chest, abdomen and pelvis did not reveal lymphadenopathy. Brain imaging showed no abnormalities. Cerebrospinal fluid cytology was unremarkable. Bone marrow biopsy (BMBX) showed prominent histiocytic phagocytosis of erythroid precursors and platelets. HLH-94 treatment protocol including weekly steroid and etoposide initiated. Patient's fever, night sweats and leukopenia resolved during hospitalization, with subsequent down trending of ferritin to 103 ng/ml, CXCL-2 to 2663 pg/mL and interleukin-2-receptor to 2,265 pg/mL. Repeat BMBX revealed significant improvement. HLH is a rare life-threatening diagnosis. This patient with nonspecific symptoms was diagnosed with HLH (fever, bicytopenia, elevated ferritin, high-soluble IL-2R and hemophagocytic lymphohistiocytosis on BMBX). Several HLH gene mutations were tested including PRF1, UNC13D, STXBP2, although none was mutated. No infectious, rheumatologic or oncologic triggers were detected. Early diagnosis and treatment are critical. Without treatment, survival is measured in months due to multiorgan failure. This syndrome rarely presents in the absence of triggers which may cause delay in diagnosis and successful treatment. 5-year overall survival with HLH 94 protocol is 54% as opposed to 0% prior to the advent of this protocol. Etoposide and steroids are the mainstay of HLH-94. Cyclosporine can be added in the maintenance phase and hematopoietic stem cell transplant is reserved for familial or relapsed HLH.

COVID-19 pandemic - related chilblains: clinical and immunological characterization of an Italian cohort

Introduction: During COVID-19 pandemic, acute acral chilblain-like lesions (ACBLL), reminiscent of lupus pernio, were observed during both first and second COVID-19 peak among patients with highly suspected (but mostly unconfirmed) infection with SARS-CoV-2.The aetiology of this phenomenon has not been elucidated yet and pathogenetic mechanism remains unknown. Several studies have investigated cytokine and chemokine profile in patients with COVID-19 but an accurate characterization of ACBLL patients is lacking. Objectives: We aimed to describe the clinical, laboratory and immunological features of children presenting with ACBLL referred to our Institute during the COVID-19 pandemic spread. Methods: We prospectively collected data of children referred to our Institute from April 1st 2020 to February 28th 2021. We investigate the presence of SARS-CoV2 infection through RT-PCR from nasopharingeal swabs and three different serologic kit. All patients underwent a laboratory work-up including coagulation, viral serology and autoantibodies panel. Finally, we analysed peripheral blood IFN signature, a panel of inflammatory biomarkers in serum/plasma by a flow cytometry bead array (CXCL10, CXCL9, IL-6, IL-1β,TNFα) and the presence of SARS-CoV2 T specific lymphocytes. Results: We examined 36 children during the first peak, and 11 children during the second COVID-19 peak (F: 28 median age 12 y), at a median delay of 26 days after symptoms onset (2-73 days). Fifteen patients (31%) presented non-specific systemic symptoms preceding ACBLL onset. Nine patients (19%) reported a possible contagion from a close contact. All patients presented stereotypical features resembling classical chilblains with acral erythematousedematous violaceous plaques and nodules localized on the toes (n= 35, 74%), the fingers (n=5, 10%) or on both sites (n=7, 15%). SARSCoV- 2 RNA detection resulted negative except for 2 patients. Furthermore, ten patients observed during the first wave showed a recurrence during the second (F:6), which developed 1-4 weeks after the second COVID-19 peak the clinical features were comparable to those of the previous episode. Five of them (50%) reported nonspecific systemic symptoms before onset and/or close contact with SARS-CoV2 positive subject. Repeated SARS-CoV-2 specific IgG/IgA tests were negative for all patients except for three cases (two of them with positive swabs). Neither common virus serology nor coagulation studies revealed significative results. Two patients presented positive ANA and anti β2 glycoprotein, respectively. A positive IFN signature was detected in 12/ 33 patients (36%).Among the 35 patients tested, the cytokine array showed high levels of IP10 (n= 35, range 12.4-739 pg/ml, n.v. 0.0-0.2 pg/ml) and a mild increase of IL-6 (n=21, range 2.4-401 pg/ml, n.v. 0.5-2.2pg/ml), without alterations of CXCL9, IL-1β and TNFa. The detection of SARS-CoV2 specific lymphocytes showed the presence of SARS-CoV2 specific lymphocytes in 9/17 (52%) patients tested (validated with positive and negative controls), only one of them with a positive serological test. Conclusion: Albeit the pathogenetic mechanism of ACBLL remains to be elucidated, our preliminary results showed a significant increase in serum IP10 levels, not frankly associated with a peripheral blood IFN signature, which is instead a characteristic of pernio-related chilblains. We also proved the presence of a T-specific memory against in 50% of the tested patients, despite the negativity of coltures and serological tests, strengthening the link between SARS-CoV2 infection and this peculiar clinical manifestation.