Hyperinflammatory State Research Articles

COVID-19-associated pulmonary aspergillosis

Abstract Invasive pulmonary aspergillosis (IPA) is now been recognized as a possible secondary infection affecting COVID-19 recovery and increasing mortality. It can be explained due to factors such as direct epithelial injury, hypercoagulable and hyperinflammatory states, and the use of immunosuppressive treatment. We managed five such cases in a post-COVID intensive care unit facility at a tertiary care institute in North India. All patients had high oxygen demand, positive serum galactomannan (GM) index, and significant chest X-ray changes. All patients showed an initial improvement after receiving intravenous antifungal treatment with voriconazole. Systemic markers were suboptimal for the diagnosis of COVID-19-associated pulmonary aspergillosis (CAPA) with a sensitivity of less than 50%. GM assay has been validated in BAL and serum in numerous studies. Serum GM is a sensitive marker for IPA in patients with neutropenia; however, in nonneutropenic critically ill patients, serum GM exhibited a sensitivity of 25%, although a negative serum result cannot exclude the diagnosis of CAPA. The diagnosis of CAPA is challenging, and in patients with severe COVID-19 pneumonia may be associated with a worse prognosis. GM levels can be used for the diagnostic purpose of probable CAPA cases and administration of prophylactic treatment with voriconazole is still under debate.

Analysis of C-Reactive Protein, Neutrophil-to-Lymphocyte Ratio, PaO2/FiO2 Ratio on the Success of High Flow Nasal Cannula Usage in Hospitalized COVID-19 Patients

Background: Several studies had shown High Flow Nasal Cannula (HFNC) is effective in treating hypoxemic COVID-19 patients. The C-Reactive Protein and Neutrophil-to-Lymphocyte Ratio is an inflammatory marker that could predict the severity of COVID-19, where the P/F ratio infers oxygenation status. Since COVID-19-related ARDS is closely related to a hyper-inflammatory state and HFNC becomes widely utilized for hypoxemic patients, it has become important to discover reliable inflammatory biomarkers related to therapeutic HFNC success. This study aims to assess the factors that influence the success of HNFC therapy, in terms of demographic and laboratory profiles of CRP, NLR, and P/F ratio. Method: A retrospective, single-center cohort study was conducted in a tertiary care hospital in Malang, East Java from January to March 2022. Subjects of 31 PCR-confirmed, hospitalized COVID-19 patients who were treated with HFNC were included. Results: This study involved 2 groups comprised of 19 subjects with successful HFNC and 12 patients who failed. Significant demographic factors affecting successful HFNC were female gender (OR=1.46 95% CI=1.08-1.99; P=0.037) and occupation type (P=0.023). Whereas, biomarkers of CRP (8.90±6.8 mg/L vs 12.39±11.7 mg/L; P=0.656), NLR (7.24±4.66 vs 12.85±12.9; P=0.243) and P/F ratio (171.40±54 vs 148.00±40; P=0.219) were found to be non-significant between successful and failed HFNC cohorts, respectively. Conclusion: HFNC could provide a specific positive end-expiratory pressure in COVID-19 patients with contributing factors of successful HFNC being female and occupational type. However, CRP, NLR, and P/F did not contribute significantly to HFNC's success.

Circulating Reelin promotes inflammation and modulates disease activity in acute and long COVID-19 cases.

Thromboembolic complications and excessive inflammation are frequent in severe COVID-19, potentially leading to long COVID. In non-COVID studies, we and others demonstrated that circulating Reelin promotes leukocyte infiltration and thrombosis. Thus, we hypothesized that Reelin participates in endothelial dysfunction and hyperinflammation during COVID-19. We showed that Reelin was increased in COVID-19 patients and correlated with the disease activity. In the severe COVID-19 group, we observed a hyperinflammatory state, as judged by increased concentration of cytokines (IL-1α, IL-4, IL-6, IL-10 and IL-17A), chemokines (IP-10 and MIP-1β), and adhesion markers (E-selectin and ICAM-1). Reelin level was correlated with IL-1α, IL-4, IP-10, MIP-1β, and ICAM-1, suggesting a specific role for Reelin in COVID-19 progression. Furthermore, Reelin and all of the inflammatory markers aforementioned returned to normal in a long COVID cohort, showing that the hyperinflammatory state was resolved. Finally, we tested Reelin inhibition with the anti-Reelin antibody CR-50 in hACE2 transgenic mice infected with SARS-CoV-2. CR-50 prophylactic treatment decreased mortality and disease severity in this model. These results demonstrate a direct proinflammatory function for Reelin in COVID-19 and identify it as a drug target. This work opens translational clinical applications in severe SARS-CoV-2 infection and beyond in auto-inflammatory diseases.

Circulating cellular clusters are associated with thrombotic complications and clinical outcomes in COVID-19

We sought to study the role of circulating cellular clusters (CCC)-such as circulating leukocyte clusters (CLCs), platelet-leukocyte aggregates (PLA), and platelet-erythrocyte aggregates (PEA)- in the immunothrombotic state induced by COVID-19. Forty-six blood samples from 37 COVID-19 patients and 12 samples from healthy controls were analyzed with imaging flow cytometry. Patients with COVID-19 had significantly higher levels of PEAs (p value<0.001) and PLAs (p value= 0.015) compared to healthy controls. Among COVID-19 patients, CLCs were correlated with thrombotic complications (p value= 0.016), vasopressor need (p value= 0.033), acute kidney injury (p value= 0.027), and pneumonia (p value= 0.036), whereas PEAs were associated with positive bacterial cultures (p value= 0.033). In predictive in silico simulations, CLCs were more likely to result in microcirculatory obstruction at low flow velocities (≤1mm/s) and at higher branching angles. Further studies on the cellular component of hyperinflammatory prothrombotic states may lead to the identification of novel biomarkers and drug targets for inflammation-related thrombosis.

P-370 Impaired endometrial decidualization with a hyperinflammation environment is involved in poor reproductive outcomes in adenomyosis patients

Abstract Study question Do patients with adenomyosis have an impaired inflammatory state of the endometrium that could affect implantation and pregnancy? Summary answer Adenomyosis patients show increased proinflammatory cytokines expression and deregulated decidualization markers expression in eutopic endometrium which could lead to altered endometrial receptivity and impaired implantation. What is known already Adenomyosis is an estrogen-dependent chronic inflammatory condition, characterized by the presence of endometrial glands and stroma within the myometrium. Adenomyosis patients present altered decidualization and defective embryo-endometrium communication, resulting in implantation failure, miscarriage, and other fertility-related disorders. Although the underlying mechanisms of this infertility remain unknown, it is known that a favorable immune and inflammatory uterine environment is necessary for developmental pregnancy. It has been proposed that a uterine hyperinflammatory state could be involved in adenomyosis-related infertility. For this reason, we aim to evaluate the inflammatory and endometrial receptivity status during implantation in eutopic endometrium from adenomyosis patients. Study design, size, duration Sixteen endometrial samples were collected from infertile patients with and without adenomyosis undergoing hormonal replacement therapy before in vitro fertilization (IVF) at IVIRMA Valencia between January to December 2022. Participants/materials, setting, methods Eutopic endometrial samples were obtained at secretory phase (LH + 7) from patients diagnosed with adenomyosis (n = 8) by ultrasound or hysteroscopy and patients without gynecological diseases (control, n = 8). Total RNA extraction was performed to later determine the gene expression of the decidualization-related genes Prolactin (PRL), SPP1 and PAEP by qRT-PCR. In addition, proteins were extracted from eutopic endometrium using a lysis buffer and the relative expression levels of human cytokines were measured by Human Cytokine Array (Raybiotech). Main results and the role of chance Gene expression evaluation in endometrium from adenomyosis patients compared to control showed a significant downregulation of the key marker of decidualization PRL (fold change [fc] = 0.56, p = 0.0047) and a significant upregulation of SPP1 (fc = 1.75, p = 0.009) and PAEP (fc = 1.56, p = 0.0192), both involved in endometrial receptivity and in immune regulation. Regarding cytokines expression, array results showed upregulation of different interleukins (IL) involved in the mediation of the immune and inflammatory response previously described in adenomyosis. Specifically, IL1ß (12±19.63 vs. 1.37±2.77, p = 0.009) that regulates several inflammatory responses, including cell proliferation, differentiation, and apoptosis; and IL6 (584.7±121.8 vs. 488.4±57.44, p = 0.06) that acts on the inflammation and maturation of B cells, contributing to the development of autoimmune diseases. Similarly, cytokines related to the promotion of inflammation and cellular proliferation in endometriosis, IL17a (173.6±44.75 vs. 102.10±71.85, p = 0.04) and TNFβ (430.9±139.6 vs. 315.3±37.09, p = 0.04), were also upregulated in adenomyosis compared to control. There were also other cytokines upregulated with diverse functions like an anti-inflammation response, growth factors or immune modulation: IL5 (308.1±88.77 vs. 202.6±45.72, p = 0.01), IL2 (341.4±60.38 vs. 270.8±35.13, p = 0.014), TGFα (215.3±27.04 vs. 117.8±36.67, p = 0.002), IL31 (77.74±40.57 vs. 14±15.96, p = 0.0012), IL7 (739.1±147.1 vs. 511.8±103.7, p = 0.003) and IL15 (301.9±95.73 vs. 209.8±50.62, p = 0.03). Limitations, reasons for caution Our findings are limited by the relatively small sample size and inherent biological variability of human samples. Wider implications of the findings Adenomyosis patients showed impaired decidualization and a hyperinflammatory endometrial environment that could be affecting the endometrial receptivity and consequently, the embryo implantation. These findings open insight to further investigations to study the mechanism by which exaggerated inflammation affects fertility in the context of adenomyosis to define new management approaches. Trial registration number Not applicable

Multisystem Inflammatory Syndrome in Children (MIS-C): Reduced Exercise Duration and Capacity at Six Month Follow-Up.

Myocarditis is common in Multisystem Inflammatory Syndrome in Children (MIS-C), and the mechanism may differ from idiopathic/viral myocarditis as MIS-C involves a hyper-inflammatory state weeks after COVID-19. We sought to evaluate exercise stress testing (EST) in these patients as EST may help guide return-to-play recommendations. Retrospective cohort study evaluating ESTs (standard Bruce treadmill protocol) from MIS-C patients from 2020 to 2022, compared to myocarditis patients and age, sex, and weight matched controls from 2005 to 2019. ESTs included 22 MIS-C patients (mean age 11.9 years) with 14 cardiopulmonary and 8 cardiovascular tests, 33 myocarditis (15.5 years), and 44 controls (12.0 years). Percent-predicted peak VO2 was abnormal (< 80% predicted) in 11/14 (79%) MIS-C patients, 13/33 (39%) myocarditis, and 17/44 (39%) controls (p = 0.04). Exercise duration was shorter in MIS-C than myocarditis or control cohorts (p = 0.01). Isolated atrial or ventricular ectopy was seen in 8/22 (36%) MIS-C, 9/33 (27%) myocarditis, and 5/44 (11%) controls (p = 0.049). No arrhythmias/complex ectopy or evidence of ischemia were noted, though non-specific ST/T wave abnormalities occurred in 4/22 (18%) MIS-C, 5/33 (15%) myocarditis, and 3/44 (7%) controls. Exercise duration and percent-predicted peak VO2 were significantly reduced in MIS-C at mean 6-month follow-up compared to pre-COVID era idiopathic/viral myocarditis and control cohorts. This may be secondary to deconditioning during the pandemic and/or chronic cardiopulmonary or autonomic effects of COVID/MIS-C. Although there were no exercise-induced arrhythmias in our MIS-C patients, larger cohort studies are warranted. EST in MIS-C follow-up may help evaluate safety and timing of return to play and potentially mitigate further deconditioning.

Increased Expression of Anaphylatoxin C5a-Receptor-1 in Neutrophils and Natural Killer Cells of Preterm Infants.

Preterm infants are susceptible to infection and their defense against pathogens relies largely on innate immunity. The role of the complement system for the immunological vulnerability of preterm infants is less understood. Anaphylatoxin C5a and its receptors C5aR1 and -2 are known to be involved in sepsis pathogenesis, with C5aR1 mainly exerting pro-inflammatory effects. Our explorative study aimed to determine age-dependent changes in the expression of C5aR1 and C5aR2 in neonatal immune cell subsets. Via flow cytometry, we analyzed the expression pattern of C5a receptors on immune cells isolated from peripheral blood of preterm infants (n = 32) compared to those of their mothers (n = 25). Term infants and healthy adults served as controls. Preterm infants had a higher intracellular expression of C5aR1 on neutrophils than control individuals. We also found a higher expression of C5aR1 on NK cells, particularly on the cytotoxic CD56dim subset and the CD56- subset. Immune phenotyping of other leukocyte subpopulations revealed no gestational-age-related differences for the expression of and C5aR2. Elevated expression of C5aR1 on neutrophils and NK cells in preterm infants may contribute to the phenomenon of "immunoparalysis" caused by complement activation or to sustained hyper-inflammatory states. Further functional analyses are needed to elucidate the underlying mechanisms.

#6437 INTRABDOMINAL ABSCESS: AN UNEXPECTED CAUSE OF POSTPARTUM SEPSIS IN A VAGINALLY DELIVERED WOMAN WITH PREGNANCY RELATED ACUTE KIDNEY INJURY

Abstract Pregnancy related-Acute kidney injury (PR-AKI) is a life-threatening complication with substantial fetal and maternal mortality and morbidity. AKI is associated with increased risk of infection and/or sepsis. Intra-abdominal infections account for 11.9% of infections that complicate AKI among critically ill patients. The pathophysiology is not fully understood, several theories have been proposed; of which, AKI associated hypervolemia leads to tissue edema and bacterial translocation. Moreover, AKI induces a hyper-inflammatory state and suppresses the immune system, this may present a greater predisposition for infection. Here we report a rare case of intra-abdominal abscess presented in a woman with recovering PR-AKI three months following normal vaginal delivery. Case presentation A 20-year-old, previously healthy female patient, was admitted due to severe postpartum hemorrhage (PPH) complicating a full-term vaginal delivery. On admission, blood pressure was 170/70, heart rate was 100 beats/min, and respiratory rate was 22 breaths/min. The patient developed oliguria, generalized edema, and fever (38.50c). The blood tests are revealed in Table I. The patient had elevated liver enzymes (SGOT 197 IU/L, and SGPT 117 IU/L). The patient was started on supportive treatment with packed RBC, platelet transfusion, and fresh frozen plasma. Empiric antibiotic was administrated. The patient subsequently had general improvement and was discharged 17 days after delivery with partial recovery of kidney function. Three months later, the patient presented with throbbing pain in the lower abdomen and fever. Abdominal examination revealed pelvi-abdominal fullness along with mild tenderness with no guarding or rigidity. No obvious palpable lumps were detected. No abnormities were detected in per vaginal and per rectal examinations. CT scan was performed and revealed a large sized collection in the lower abdomen sized 6.5x5x9 cm. Ultrasound guided aspiration of 60 ml pus confirmed the diagnosis of intraabdominal abscess. Intravenous antibiotics were started with percutaneous drainage of the abscess. Discussion Intraabdominal abscess is a serious ailment, and it is associated with high mortality and morbidity if left untreated. An intra-abdominal abscess can cause symptoms such as prolonged ileus, anorexia, fever, and abdominal pain. Septic shock may eventually develop in case of delayed treatment. However rare, there should be a causative factor. Intraabdominal abscess following a normal vaginal delivery is very rare and not reported with PR-AKI. CT scan is still the most useful technique for diagnosis and treatment. Additionally, it can help guide percutaneous drainage by locating the abscess in relation to the viscera of the abdomen. The use of adequate antimicrobial drug therapy in combination is a fundamental approach. It is therefore of great importance to consider symptoms such as atypical abdominal distension or pain in women in the postpartum period and to provide thorough comprehensive evaluation. Early diagnostic consideration greatly lessens patients’ morbidity and mortality.

The multifaceted nature of IL-10: regulation, role in immunological homeostasis and its relevance to cancer, COVID-19 and post-COVID conditions.

Interleukin-10 (IL-10) is a pleiotropic cytokine that has a fundamental role in modulating inflammation and in maintaining cell homeostasis. It primarily acts as an anti-inflammatory cytokine, protecting the body from an uncontrolled immune response, mostly through the Jak1/Tyk2 and STAT3 signaling pathway. On the other hand, IL-10 can also have immunostimulating functions under certain conditions. Given the pivotal role of IL-10 in immune modulation, this cytokine could have relevant implications in pathologies characterized by hyperinflammatory state, such as cancer, or infectious diseases as in the case of COVID-19 and Post-COVID-19 syndrome. Recent evidence proposed IL-10 as a predictor of severity and mortality for patients with acute or post-acute SARS-CoV-2 infection. In this context, IL-10 can act as an endogenous danger signal, released by tissues undergoing damage in an attempt to protect the organism from harmful hyperinflammation. Pharmacological strategies aimed to potentiate or restore IL-10 immunomodulatory action may represent novel promising avenues to counteract cytokine storm arising from hyperinflammation and effectively mitigate severe complications. Natural bioactive compounds, derived from terrestrial or marine photosynthetic organisms and able to increase IL-10 expression, could represent a useful prevention strategy to curb inflammation through IL-10 elevation and will be discussed here. However, the multifaceted nature of IL-10 has to be taken into account in the attempts to modulate its levels.

Metabolic reprogramming through mitochondrial biogenesis drives adenosine anti-inflammatory effects: new mechanism controlling gingival fibroblast hyper-inflammatory state.

Fibroblasts are the dominant stromal cells in the gingival lamina propria with a well-established relevance in regulation of inflammation, and in innate immunity. This is exemplified by their hypersecretion of CXCL8, enhancing leukocyte infiltration in chronic and sustained inflammatory conditions. We have previously shown adenosine to be a key metabolic nucleoside that regulates stromal inflammation, but the underlying mechanisms linking adenosine to the metabolic status of fibroblasts and to the resultant inflammatory response are unclear. This study examined, by seahorse real-time cell metabolic analysis, the bioenergetics of the stromal fibroblast response to extracellular adenosine and IL-1β, focusing on CXCL8 secretion by primary human gingival fibroblasts (HGF). Markers of the glycolytic pathway and mitochondrial biogenesis were tracked through immunoblot. Further, the influence of adenosine on mitochondrial accumulation was measured by uptake of MitoTracker Red fluorescent probe and assessment of the role of FCCP (a mitochondrial uncoupler) in CXCL8 secretion and mitochondrial accumulation. Our results show that the anti-inflammatory response of HGF to extracellular adenosine, typified by reduced CXCL8 secretion, is mediated by mitochondrial oxidative phosphorylation, reflected in higher oxygen consumption rate (OCR). In the presence of IL-1β, adenosine-treated cells induced higher ATP production, basal respiration and proton leak compared to IL-1β without adenosine. Surprisingly, adenosine had no additional effect on the IL-1β-induced higher glycolysis rate demonstrated by the extracellular acidification rate (ECAR). In addition, the higher OCR in adenosine-stimulated cells was not due to the mitochondrial fuel dependency or capacity, but due to an increase in mitochondrial biogenesis and accumulation in the cells with concomitant decrease in mitophagy-required p-PINK1 marker. We detected the accumulation of functional mitochondria with increased activation of the AMPK/SIRT1/PGC-1α pathway. The adenosine-induced uptake of MitoTracker was abrogated by PGC-1α inhibition with SR-12898. In addition, the adenosine effects on reduced CXCL8 were ablated by treatment with FCCP, a potent uncoupler of mitochondrial oxidative phosphorylation. Our findings reveal a key role for mitochondrial bioenergetics in regulation of CXCL8-mediated inflammation by HGF through the adenosine/AMPK/SIRT1/PGC-1α axis. Therapeutically targeting this pathway in gingival fibroblasts might be a promising future strategy to modulate stromal-mediated sustained hyper-inflammatory responses.

Favorable outcomes following allogeneic transplantation in adults with hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a syndrome marked by a severe hyperinflammatory state characterized by aberrant T- and natural killer-cell activity leading to prolonged hypercytokinemia and can be rapidly fatal if not diagnosed and treated early. While upfront therapy is aimed at reducing hyperinflammation and controlling possible triggers, allogeneic hematopoietic stem cell transplantation (HSCT) is indicated for primary and relapsed/refractory cases to attain sustained remission. While this has been explored extensively in the pediatric population, there are limited data on adults undergoing HSCT for HLH. We analyzed transplant outcomes in an adult HLH population in the modern era who were transplanted at Dana-Farber Cancer Institute from 2010 onwards. Patients were uniformly transplanted on a reduced intensity platform incorporating early administration of alemtuzumab with standard infectious and graft-versus-host disease (GVHD) prophylaxis. Engraftment was documented for all patients. At 3years after transplantation, overall survival (OS) was 75% (95% confidence interval [CI], 51-89) while 3-year progression-free survival (PFS) was 71% (95% CI, 46-86). The 3-year cumulative incidence of relapse was 15% (95% CI, 3.4-33). There were no isolated HLH relapses without relapse of malignancy. The cumulative incidence of nonrelapse mortality at 3 years was 15% (95% CI, 3.5-34). Infectious complications and GVHD outcomes were comparable to standard reduced-intensity conditioning (RIC) transplantation at our institute. Mixed chimerism was common but did not correlate with transplant outcomes. Our data suggest that the immune defect in HLH can be abrogated with allogeneic transplantation using a reduced intensity regimen with early administration of alemtuzumab as preconditioning, providing a potentially curative option for this difficult disease.

Concomitant Autoimmunity in Endometriosis Impairs Endometrium-Embryo Crosstalk at the Implantation Site: A Multicenter Case-Control Study.

Endometriosis and autoimmune diseases share a hyper-inflammatory state that might negatively impact the embryo-endometrium crosstalk. Inflammatory and immune deregulatory mechanisms have been shown to impair both endometrial receptivity and embryo competence at the implantation site. The aim of this study was to investigate the potential additional impact of co-existing autoimmunity in women affected by endometriosis on the early stages of reproduction. This was a retrospective, multicenter case-control study enrolling N = 600 women with endometriosis who underwent in vitro fertilization-embryo transfer cycles between 2007 and 2021. Cases were women with endometriosis and concomitant autoimmunity matched based on age and body mass index to controls with endometriosis only in a 1:3 ratio. The primary outcome was the cumulative clinical pregnancy rate (cCPR). The study found significantly lower cleavage (p = 0.042) and implantation (p = 0.029) rates among cases. Autoimmunity (p = 0.018), age (p = 0.007), and expected poor response (p = 0.014) were significant negative predictors of cCPR, with an adjusted odds ratio of 0.54 (95% CI, 0.33-0.90) for autoimmunity. These results suggest that the presence of concomitant autoimmunity in endometriosis has a significant additive negative impact on embryo implantation. This effect might be due to several immunological and inflammatory mechanisms that interfere with both endometrial receptivity and embryo development and deserves further consideration.

CD16+ monocytes are involved in the hyper-inflammatory state of Prader-Willi Syndrome by single-cell transcriptomic analysis.

Patients with Prader-Willi syndrome (PWS) have a reduced life expectancy due to inflammation-related disease including cardiovascular disease and diabetes. Abnormal activation of peripheral immune system is postulated as a contributor. However, detailed features of the peripheral immune cells in PWS have not been fully elucidated. Serum inflammatory cytokines were measured in healthy controls (n=13) and PWS patients (n=10) using a 65- multiplex cytokine assays. Changes of the peripheral immune cells in PWS was assessed by single-cell RNA sequencing (scRNA-seq) and high-dimensional mass cytometry (CyTOF) using peripheral blood mononuclear cells (PBMCs) from PWS patients (n=6) and healthy controls (n=12). PWS patients exhibited hyper-inflammatory signatures in PBMCs and monocytes were the most pronounced. Most inflammatory serum cytokines were increased in PWS, including IL-1β, IL-2R, IL-12p70, and TNF-α. The characteristics of monocytes evaluated by scRNA-seq and CyTOF showed that CD16+ monocytes were significantly increased in PWS patients. Functional pathway analysis revealed that CD16+ monocytes upregulated pathways in PWS were closely associated with TNF/IL-1β- driven inflammation signaling. The CellChat analysis identified CD16+ monocytes transmitted chemokine and cytokine signaling to drive inflammatory process in other cell types. Finally, we explored the PWS deletion region 15q11-q13 might be responsible for elevated levels of inflammation in the peripheral immune system. The study highlights that CD16+ monocytes contributor to the hyper-inflammatory state of PWS which provides potential targets for immunotherapy in the future and expands our knowledge of peripheral immune cells in PWS at the single cell level for the first time.

Mucosal immunization with Lactiplantibacillus plantarum-displaying recombinant SARS-CoV-2 epitopes on the surface induces humoral and mucosal immune responses in mice

BackgroundThe use of probiotic lactic acid bacteria as a mucosal vaccine vector is considered a promising alternative compared to the use of other microorganisms because of its “Generally Regarded as Safe” status, its potential adjuvant properties, and its tolerogenicity to the host. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease (COVID-19), is highly transmissible and pathogenic. This study aimed to determine the potential of Lactiplantibacillus plantarum expressing SARS-CoV-2 epitopes as a mucosal vaccine against SARS-CoV-2.ResultsIn this study, the possible antigenic determinants of the spike (S1–1, S1–2, S1–3, and S1–4), membrane (ME1 and ME2), and envelope (E) proteins of SARS-CoV-2 were predicted, and recombinant L. plantarum strains surface-displaying these epitopes were constructed. Subsequently, the immune responses induced by these recombinant strains were compared in vitro and in vivo. Most surface-displayed epitopes induced pro-inflammatory cytokines [tumor necrosis factor alpha (TNF-α and interleukin (IL)-6] and anti-inflammatory cytokines (IL-10) in lipopolysaccharide-induced RAW 264.7, with the highest anti-inflammatory to pro-inflammatory cytokine ratio in the S1–1 and S1–2 groups, followed by that in the S1–3 group. When orally administered of recombinant L. plantarum expressing SARS-CoV-2 epitopes in mice, all epitopes most increased the expression of IL-4, along with induced levels of TNF-α, interferon-gamma, and IL-10, specifically in spike protein groups. Thus, the surface expression of epitopes from the spike S1 protein in L. plantarum showed potential immunoregulatory effects, suggesting its ability to potentially circumvent hyperinflammatory states relevant to monocyte/macrophage cell activation. At 35 days post immunization (dpi), serum IgG levels showed a marked increase in the S1–1, S1–2, and S1–3 groups. Fecal IgA levels increased significantly from 21 dpi in all the antigen groups, but the boosting effect after 35 dpi was explicitly observed in the S1–1, S1–2, and S1–3 groups. Thus, the oral administration of SARS-CoV-2 antigens into mice induced significant humoral and mucosal immune responses.ConclusionThis study suggests that L. plantarum is a potential vector that can effectively deliver SARS-CoV-2 epitopes to intestinal mucosal sites and could serve as a novel approach for SARS-CoV-2 mucosal vaccine development.

Abstract 265: Coronavirus Infection Induced Interferon Signaling Promotes Atherosclerotic Plaque Vulnerability

Atherosclerosis is a chronic inflammatory vascular disease characterized by the formation of lipid-rich plaques inside arteries. The rupture of vulnerable plaques and consequent acute complications, such as myocardial infarction and stroke, are the leading causes of morbidity and mortality worldwide. It is known that pre-existing cardiovascular comorbidities pose a risk for severe outcomes in COVID-19, though the underlying pathophysiology is not fully understood. To investigate the role of coronavirus infection in established atherosclerotic plaques, we developed an atherosclerotic model of natural murine beta coronavirus infection with mouse hepatitis virus strain-A59 (MHV-A59). We fed Ldlr-/- mice a Western diet for three months prior to MHV-A59 intranasal inoculation and assessed plaque morphology at day post infection (dpi) 7 and dpi 30. Respiratory infection in mice recapitulated many aspects of COVID-19 pathophysiology. More strikingly, virus was detected in atherosclerotic aorta and induced multiple features of vulnerable plaques, a prerequisite for plaque rupture, characterized by thinner fibrous caps, less collagen content, and larger necrotic cores. The phenotype was persistent at dpi 30 when the mice were fully recovered. To further explore the underlying mechanism, we performed single-cell RNA sequencing (scRNA-seq) on infected aortas isolated at dpi 7. Our data revealed that interferon &amp;#947; was exclusively upregulated in activated T cells and elicited various responses across multiple cell populations within infected aortas. The drastic responses that potentially contribute to plaque instability include activation of a more proinflammatory endothelial cell phenotype, downregulation of TGF- &amp;#946; mediated fibroblast to myofibroblast transition, and enhanced degradation of extracellular matrix and smooth muscle cell senescence. Moreover, macrophages in infected plaques activate inflammasomes, release IL-18, and undergo pyroptosis, thereby further contributing to the hyperinflammatory state of the atherosclerotic plaques. Taken together, our data shed light on a molecular mechanism for coronavirus infection induced plaque instability and provide a small animal model to explore future therapeutics.

The use of elevated ferritin levels in neonates and young infants as a screening lab for HLH

Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory state due to primary genetic defect in T/NK cell cytotoxicity or secondary to infection, rheumatologic process, or malignancy. Activated macrophages/cytotoxic lymphocytes lead to elevated cytokines and T-cell activation. Ferritin is a screening tool, with levels >10,000 mug/L reported to be 90% sensitive and 96% specific for HLH. Its use in neonatal/early infancy hasn’t been specifically evaluated.In our retrospective case review we included patients 3 months or younger, treated in the past 5 years at our institution with an elevated ferritin >5000 mug/L. 20 patients were evaluated, demographic/diagnostic information seen in table 1. Average ferritin was 44 515 IQR (9922–54 853) Four patients, (20%) were diagnosed with primary HLH (pHLH), with non-HLH conditions including: 4 (20%) sepsis/infection, 4 (20%) with acute liver failure/galactosemia, 3 (15%) hematologic complications, and 5 (25%) without definitive diagnosis.All patients with HLH had splenomegaly and thrombocytopenia (< 100); 2 patients with pHLH had anemia (< 9), and 3 with ANC< 1000. Of those without HLH, 1 had splenomegaly, 9 with anemia, 6 with thrombocytopenia, and 1 with neutropenia. Soluble IL-2 receptor (sIL2R), a specific marker for T-cell activation was measured in 12 patients. pHLH patients had sIL2R levels >10 000 U/ml: pHLH average 31 432 (11 000–47 474), without pHLH average 2039 (1123–3161). sIL2R is more specific than ferritin but there are limitations (turn-around-time and locations performing). Figure 1 depicts ferritin/sIL2R levels in these groups.We utilized direct flow-based detection of T-cell activation markers for identification of HLH. As a proof of concept, patient 19 had fulminant HSV and a ferritin >200 000 mug/L. HLH diagnostic criteria were fulfilled, the T-cell activation profile was unremarkable (Figure 2). HLH treatment was not initiated; an unremarkable sIL2R result followed which validated our approach.A broad spectrum of etiologies drives high ferritin in neonatal/early infancy and can cause ferritin of >5000 mug/L. Thus, HLH 2004 criteria can be fulfilled in patients without pHLH resulting in inadvertent use of immune suppression. sIL2R and/or T-cell activation flow should be obtained when evaluating and initiating treatment for HLH. Further, genetic evaluation for other conditions is warranted in this patient population. [Display omitted] [Display omitted] Table 1(abstract: 128) Patient characteristics and information: laboratory values, genetics, and underlying diagnosis included.PatientPeak Ferritin (ng/ml)Large SpleenHgb (g/dL)Platelets (thou/ul)NCFibrinogen (mg/dl)SIL2R (units/ml)CXCL9 (pg/ml)PerforinCD107aGenetic FindingFinal DiagnosisCurrent Status127436+13.45042602761123N/ADecreasedLowNo definitive diagnosisUnknownAlive217491–10.43609700224N/N/AN/AN/ANon-contributoryABO incompatibilityAlive3106635–10809110132N/N/AN/AN/ANon-contributoryUnknownAlive482270–6.511540179N/N/AN/AN/AGALT, compound heterozygoteGalactosemiaDeceased59423N/A9.64010420621665N/AN/AN/AWES without definitive diagnosisUnknownAlive6150000+11.7632143024411,0006603N/ALowSTXBP2(homozygous)pHLHDeceased724304–11.91610480185N/AN/AN/AN/ANot collectedUnknownDeceased87494–8.841960603161N/AN/AN/APendingGalactosemiaAlive911412–9.1408770912144N/AN/AN/AWES without definitive diagnosisAcute liver failureAlive1014175–7.93721575173N/AN/AN/AN/ANo definitive diagnosisFulminant liver failureDeceased1117032–10.9332106023,000N/AAbsentPresentPRF1 (homozygous)pHLHDeceased12140000–5.52211880601644N/AN/AN/ANon-contributoryVitamin-K def hemorrhageDeceased139673–9.96992190216N/AN/AN/AN/ANon-contributoryCOVID-19 infectionAlive1410171–8.52952480N/2297N/AN/AN/ANot collectedViral hepatitisAlive155906–8.6103N/1081791N/AN/AN/ANot collectedCampylobacter bacteremiaAlive1614855+7.2101806844256325N/ALowRAB27A compound heterozygotepHLH, Griscelli SyndromeAlive179615–8.2263N/N/N/AN/AN/ALowIFIH1 heterozygous, pending further evaluationUnknownAlive1818730–7.61851940165N/AN/AN/AN/ANot collectedIntrauterine hydrops, secondary to Rh alloimmunization, liver failureAlive19>200000–6.3411400872493N/AN/AN/ATNFRSF13B HeterozygousHSV2 viremiaAlive2013675+6.8404506047474N/AN/ALowUNC13D, compound heterozygotepHLHDeceased

PO-01-140 ATRIAL FIBRILLATION AND SEPTIC SHOCK: A FIVE-YEAR RETROSPECTIVE OBSERVATIONAL STUDY OF ANTICOAGULATION USAGE, BLEEDING AND STROKE RATES

Anticoagulation (AC) for atrial fibrillation (AF) is a cornerstone of management, and the benefit of mitigating stroke risk is accompanied by an increased risk of bleeding. The risk and benefit of AC in septic shock remains unclear as this disruptive hyperinflammatory state leads to multi-organ dysfunction, which can include coagulopathies. To further understand the risk of bleeding and stroke in patients with AF and septic shock, we aimed to study the rates of these events in addition to AC usage for this population between 2016 and 2020. In this retrospective observational study, we used ICD-10 coding to identify patients with AF and septic shock. Initial query found 604 patients. 52 were excluded for the absence of AF and 24 were excluded for requiring AC for other reasons. Admission for septic shock and evidence of AF on electrocardiogram were confirmed through chart review. Bleeding was defined using the Bleeding Academic Research Consortium (BARC) classification system. For 528 patients, mean age was 64.8 years, 325 (61.6%) were male, and 203 (38.4%) were female. 314 died (59.5%) and 227 (43%) had new-onset AF. Mean CHA2DS2-Vasc and HAS-BLED scores were 3.2 and 2.6, respectively. AC was started for 195 patients (36.9%), not started for 289 (54.7%), and continued for 43 (8.1%). AC was stopped for 74/195 (38.3%) patients after starting AC and for 20/43 (46.5%) patients after continuing AC. 157 had bleeding episodes (29.7%): 9 BARC 1 (1.7%), 46 BARC 2 (8.7%), 63 BARC 3a (11.9%), 20 BARC 3b (3.8%), 6 BARC 3c (1.1%), 5 BARC 4 (0.9%), 3 BARC 5a (0.6%), and 5 BARC 5b (0.9%). After starting AC, 70/195 (35.9%) patients bled, and this included all fatal bleeding cases. Bleeding occurred in 11/43 (25.6%) patients after continuing AC and 76/289 (26.3%) patients after not starting AC. 8 (1.5%) patients had a stroke: 4 hemorrhagic and 4 ischemic. 3 hemorrhagic strokes occurred after starting AC and 1 occurred after not starting AC. 1 ischemic stroke occurred after starting AC and 3 after not starting AC. In patients with AF and septic shock, we found that bleeding occurred more often and with the highest severity after starting AC. The rate of bleeding when not starting AC may indicate an increased baseline risk associated with sepsis. Notably, the rate of ischemic stroke was relatively low, which may suggest that bleeding risk may outweigh the benefit of stroke prevention in this population. Further studies are needed to optimize AC strategies for these patients.

Coronavirus Disease 2019, Diabetes, and Inflammation: A Systemic Review.

People with cardiometabolic diseases [namely type 2 diabetes (T2D), obesity, or metabolic syndrome] are more susceptible to coronavirus disease 2019 (COVID-19) infection and endure more severe illness and poorer outcomes. Hyperinflammation has been suggested as a common pathway for both diseases. To examine the role of inflammatory biomarkers shared between COVID-19 and cardiometabolic diseases, we reviewed and evaluated published data using PubMed, SCOPUS, and World Health Organization COVID-19 databases for English articles from December 2019 to February 2022. Of 248 identified articles, 50 were selected and included. We found that people with diabetes or obesity have (i) increased risk of COVID-19 infection; (ii) increased risk of hospitalization (those with diabetes have a higher risk of intensive care unit admissions) and death; and (iii) heightened inflammatory and stress responses (hyperinflammation) to COVID-19, which worsen their prognosis. In addition, COVID-19-infected patients have a higher risk of developing T2D, especially if they have other comorbidities. Treatments controlling blood glucose levels and or ameliorating the inflammatory response may be valuable for improving clinical outcomes in these patient populations. In conclusion, it is critical for health care providers to clinically evaluate hyperinflammatory states to drive clinical decisions for COVID-19 patients.

Anti-Inflammatory and Antioxidant Response in COVID-19 Infection

SARS-CoV-2 virus infection starts with the internalization of the viral particle into the host cells, mainly the upper respiratory system epithelial cells which have the highest expression of the ACE2 receptor which is essential for the internalization process. The pathophysiology of severe forms of COVID-19 disease results not only from direct, cytopathic viral effect but also from immune response dysregulation of the host resulting in hyperinflammatory state and oxidative stress. The nuclear factor erythroid 2-related factor 2 (Nrf2) ability to protect cells and induce a rapid anti-inflammatory and antioxidant response primarily depends on its constitutive cellular expression, which can be affected by numerous endogenous and exogenous factors. The binding of Nrf2 to cellular receptors leads to the transcription of a large number of genes encoding various antioxidant enzymes and other cytoprotective molecules, including heme oxygenase-1(HO-1). Activation of HO-1 results in antioxidant, anti-inflammatory and anti-apoptotic effects. Based on previous studies, the Nrf2/HO-1 pathway provides protection against oxidative stress and inflammatory and immune response which is significant in COVID-19 infection, which is characterized by a strong hyperinflammatory response. This narrative review aims to describe the role of the hyperinflammatory response in the development of COVID-19 infection, with a focus on the NrF2/HO-1 pathway.

Endothelial dysfunction in COVID-19 with smoking

Endothelial dysfunction is one of the mechanisms that most likely to play a role in the occurrence of poor clinical outcomes in patients with COVID-19. The hyperinflammatory and pro-coagulation state in COVID-19 suggests an important role for the endothelium, both as an effector contributing to inflammation and thrombosis, as well as a target organ. This state of endothelial dysfunction is also believed to be exacerbated by a history of previous smoking. Tobacco can increase the severity of infectious diseases such as influenza, by increasing viral replication through suppression of antiviral mechanisms, and changes in cytokine patterns in cells also increase ACE2 expression. Smoking is associated with endothelial dysfunction because it can increase the concentration of free radicals and proinflammatory cytokines. So, it is very possible that SARS-CoV-2 will damage the endothelium of smokers who have previously been injured as a result of smoking habits. The literature that we used in this study was obtained through searching engines from the Google Scholar and PubMed databases, with the inclusion criteria being literature that used English. We managed to take 1,613 literature articles, then we selected 81 articles that were most relevant to the discussion of our study. In this study we conclude that endothelial dysfunction may play a role in the mechanism of clinical deterioration in COVID-19 and may be exacerbated by a history of previous smoking.