The SARS-CoV-2 Antibody and Drug Research Based on the Structure of the Spike Protein and the Mechanism of Cell Entry.

COVID-19 is a severe acute respiratory syndrome caused by a novel coronavirus, SARS-CoV- 2.COVID-19 is now a pandemic, and is not yet fully under control.As the surface spike protein (S) mediates the recognition between the virus and cell membrane and the process of cell entry, it plays an important role in the course of disease transmission.The study on the S protein not only elucidates the structure and function of virus-related proteins and explains their cellular entry mechanism, but also provides valuable information for the prevention, diagnosis and treatment of COVII)-19.Concentrated on the S protein of SARS-CoV-2, this review covers four aspects: (1 ) The structure of the S protein and its binding with angiotensin converting enzyme II (ACE2) , the specific receptor of SARS-CoV-2, is introduced in detail.Compared with SARS-CoV, the receptor binding domain (RBD) of the SARS-CoV- 2 S protein has a higher affinity with ACE2, while the affinity of the entire S protein is on the contrary.(2) Currently, the cell entry mechanism of SARS-CoV-2 meditated by the S protein is proposed to include endosomal and non-endosomal pathways.With the recognition and binding between the S protein and ACE2 or after cell entry, transmembrane protease serine 2(TMPRSS2) , lysosomal cathepsin or the furin enzyme can cleave S protein at S1/S2 cleavage site, facilitating the fusion between the virus and target membrane.(3) For the progress in SARS-CoV-2 S protein antibodies, a collection of significant antibodies are introduced and compared in the fields of the target, source and type.(4) Mechanisms of therapeutic treatments for SARS-CoV-2 varied.Though the antibody and medicine treatments related to the SARS-CoV-2 S protein are of high specificity and great efficacy, the mechanism, safety, applicability and stability of some agents are still unclear and need further assessment.Therefore, to curb the pandemic, researchers in all fields need more cooperation in the development of SARS-CoV-2 antibodies and medicines to face the great challenge.Copyright © Palaeogeography (Chinese Edition).All right reserved.

SARS-CoV-2 infection disrupts trophoblast function and placental syncytial barrier integrity via its accessory protein, ORF3a

Problem: Although it is rare for a SARS-CoV-2 infection to transmit vertically to the fetus during pregnancy, there is a significantly increased risk of adverse pregnancy outcomes due to maternalCOVID- 19. However, there is a poor understanding of such risks because mechanistic studies on how SARS-CoV-2 infection disrupts placental homeostasis are significantly lacking. The SARS-CoV-2 proteome includes multiple structural and non-structural proteins, including the non-structural accessory proteinORF3a. The roles of these proteins in mediating placental infection remain undefined. We and others have shown that autophagy activity in placental syncytium is essential for barrier function and integrity. Here, we have used clinical samples and cultured trophoblast cells to evaluate syncytial integrity of placenta exposed to SARS-CoV-2. The objective of our study was to investigate potential mechanisms through which SARS-CoV-2 impairs placental homeostasis and causes adverse pregnancy outcomes. We tested the central hypothesis that an essential SARS-CoV-2 non-structural and accessory protein, ORF3a, uniquely (amongst multiple viral proteins tested) with a novel three-dimensional structure andwith no homology to any other proteins is a key modulator of placental trophoblast cell dynamics via autophagy and intracellular trafficking of a tight junction protein (TJP), ZO-1. Method(s): We used clinical samples and cultured trophoblast cells to evaluate syncytial integrity of placentas exposed to SARS-CoV- 2. Autophagic flux was measured in placental villous biopsies from SARS-CoV-2-exposed and unexposed pregnant women by quantifying the expression of autophagy markers, LC3 and P62. Trophoblast cells (JEG-3, Forskolin-treated JEG-3, HTR8/SVneo, or primary human trophoblasts (PHTs)) were transfected with expression plasmids encoding SARS-CoV-2 proteins including ORF3a. Using western blotting, multi-label immunofluorescence, and confocal imaging, we analyzed the effect of ORF3a on the autophagy, differentiation, invasion, and intracellular trafficking of ZO-1 in trophoblasts. Using coimmunoprecipitation assays, we tested ORF3a interactions with host proteins. t-tests and one-way analyses of variance (ANOVAs) with post hoc tests were used to assess the data, with significance set at P < .05. Result(s): We discovered :1) increased activation of autophagy, but incomplete processing of autophagosome-lysosomal degradation;2) accumulation of protein aggregates in placentas exposed to SARS-CoV- 2. Mechanistically, we showed that the SARS-CoV-2 ORF3a protein, uniquely 3) blocks the autophagy-lysosomal degradation process;4) inhibits maturation of cytotrophoblasts into syncytiotrophoblasts (STBs);5) reduces production ofHCG-beta, a key pregnancy hormone that is also essential for STB maturation;and 6) inhibits trophoblast invasive capacity. Furthermore, ORF3a harbors an intrinsically disordered C-terminus withPDZ-bindingmotifs.We show for the first time that, 7) ORF3a binds to and co-localizes with the PDZ domain of ZO-1, a junctional protein that is essential for STB maturation and the integrity of the placental barrier. Conclusion(s): Our work outlines a new molecular and cellular mechanism involving the SARS-CoV-2 accessory protein ORF3a that may drive the virus's ability to infect the placenta and damage placental syncytial integrity. This implies that the mechanisms facilitating viral maturation, such as the interaction of ORF3a with host factors, can be investigated for additional functionality and even targeted for developing new intervention strategies for treatment or prevention of SARS-CoV-2 infection at the maternal-fetal interface.

HSPA5 Promotes Attachment and Internalization of Porcine Epidemic Diarrhea Virus through Interaction with the Spike Protein and the Endo-/Lysosomal Pathway.

Porcine epidemic diarrhea virus (PEDV) has caused huge economic losses to the global pig industry. The swine enteric coronavirus spike (S) protein recognizes various cell surface molecules to regulate viral infection. In this study, we identified 211 host membrane proteins related to the S1 protein by pulldown combined with liquid-chromatography tandem mass spectrometry (LC-MS/MS) analysis. Among these, heat shock protein family A member 5 (HSPA5) was identified through screening as having a specific interaction with the PEDV S protein, and positive regulation of PEDV infection was validated by knockdown and overexpression tests. Further studies verified the role of HSPA5 in viral attachment and internalization. In addition, we found that HSPA5 interacts with S proteins through its nucleotide-binding structural domain (NBD) and that polyclonal antibodies can block viral infection. In detail, HSPA5 was found to be involved in viral trafficking via the endo-/lysosomal pathway. Inhibition of HSPA5 activity during internalization would reduce the subcellular colocalization of PEDV with lysosomes in the endo-/lysosomal pathway. Together, these findings show that HSPA5 is a novel PEDV potential target for the creation of therapeutic drugs. IMPORTANCE PEDV infection causes severe piglet mortality and threatens the global pig industry. However, the complex invasion mechanism of PEDV makes its prevention and control difficult. Here, we determined that HSPA5 is a novel target for PEDV which interacts with its S protein and is involved in viral attachment and internalization, influencing its transport via the endo-/lysosomal pathway. Our work extends knowledge about the relationship between the PEDV S and host proteins and provides a new therapeutic target against PEDV infection.

THE SPIKE OF SARS-CoV-2 VARIANTS ALLOWS FOR MORE EFFICIENT COUNTERACTION OF BST2

Background: BST2/Tetherin is an interferon-stimulated gene with antiviral activity against enveloped viruses. Particularly, BST2 tethers virions at their site of assembly, preventing their release and spread. In addition to this primary role, BST2 is as an important bridge between the innate and adaptive immune system, since (i) BST2 routes tethered particles to lysosomes, which generates viral breakdown products that engage pattern recognition receptors;and (ii) trapped virions facilitate antibody-dependent cell-mediated cytotoxicity (ADCC). In turn, viruses have evolved mechanisms to bypass BST2, either by targeting BST2 for proteasomal/lysosomal degradation or by removing BST2 from sites of virion assembly. However, the role of BST2 in SARS-CoV-2 replication, spread, evolution, and pathogenesis remains largely unknown. Method(s): The antiviral potential of BST2 against SARS-CoV-2 was investigated by infecting different SARS-CoV-2 isolates (Hong Kong, Alpha, Beta, Delta, and Omicron) in BST2+ and BST2- cells. Culture supernatants were collected to assess virion production by ELISA and infectivity by TCID50. Infected cells were analyzed by western blot and flow cytometry to examine viral and cellular protein levels, including BST2. Transfection of individual SARS-CoV-2 ORFs and mutagenesis studies allowed us to identify the genes that the virus uses to downregulate BST2. Immunoprecipitation assays revealed protein-protein interactions and changes in ubiquitination patterns. Experiments with proteasomal and lysosomal inhibitors furthered our mechanistic understanding of how SARS-CoV-2 counteracts BST2. Finally, fluorescence microscopy studies uncovered changes in the subcellular distribution of BST2 in SARS-CoV-2 infected cells. Result(s): While BST2 reduces virion release, SARS-CoV-2 has evolved to counteract this effect. Specifically, SARS-CoV-2 uses the Spike to interact with BST2, sequester the protein at perinuclear locations, and ultimately route it for lysosomal degradation. By surveying different SARS-CoV-2 variants of concern (Alpha-Omicron), we found that each variant is more efficient than the previously circulating strain at downregulating BST2 and facilitating virion production, and that mutations in the Spike account for their enhanced BST2 antagonism. Conclusion(s): As part of its adaptation to humans, SARS-CoV-2 is improving its capacity to counteract BST2, highlighting that BST2 antagonism is important for SARS-CoV-2 infectivity and transmission.

LIMITED INDUCTION OF LUNG-RESIDENT MEMORY T CELLS BY SARS-CoV-2 mRNA VACCINATION

Background: Resident memory T cells (TRM) present at the respiratory tract may be essential to enhance early SARS-CoV-2 viral clearance, thus limiting viral infection and disease. While long-term antigen-specific TRM are detectable beyond 11 months in the lung of convalescent COVID-19 patients after mild and severe infection, it is unknown if mRNA vaccination encoding for the SARS-CoV-2 S-protein can induce this frontline protection. Method(s): We obtained cross-sectional paired blood and lung biopsy samples from patients (n=30) undergoing lung resection for various reasons and assigned them to one of four groups: I.) uninfected unvaccinated individuals (n=5), II.) unvaccinated long-term SARS-CoV-2 convalescent individuals (between 6.0-10.5 months post-infection;n=9), III.) uninfected and long-term vaccinated individuals (between 6.0-7.7 months after the second or third dose;n=10), and IV.) uninfected and short-term vaccinated individuals (between 1.3-1.8 months after the third or fourth dose;n=6). We determined the presence of SARS-CoV-2-specific CD4+ and CD8+ T cells in blood and lung samples after exposure of cells to M, N, and S peptide pools, followed by flow cytometry to detect TRM cells expressing interferon (IFN)gamma and/or CD107a, as a degranulation marker. Result(s): We found that the frequency of CD4+ T cells secreting IFNgamma in response to S-peptides was variable but detectable in blood and lung up to 8 months after mRNA vaccination. Moreover, the IFNgamma response of CD4+ T cells in the lung of mRNA-vaccinated patients was similar to the response found in convalescent patients. However, in mRNA-vaccinated patients, lung responses presented less frequently with a TRM phenotype compared to convalescent infected individuals and, strikingly, polyfunctional CD107a+ IFNgamma+ TRM were virtually absent in vaccinated patients. Conclusion(s): mRNA vaccines might induce memory responses within the lung parenchyma in some patients, potentially contributing to the overall disease control. However, the robust and broad TRM response established in convalescent-infected individuals may offer advantages at limiting disease if the virus is not blocked by initial mechanisms of protection, such as neutralization. Our results warrant investigation of mucosal vaccine-induced resident T cell responses in establishing superior site-specific protective immunity.

The Impact of N-butyldeoxynojirimycin and 1-deoxymannojirimycin on SARS-CoV-2 S1 Subunit and Angiotensin-Converting Enzyme 2 Interaction in Intestinal Epithelial Cells

The severe acute respiratory syndrome coronavirus 2 (SARSCoV- 2) targets mainly the respiratory tract. In addition to respiratory symptoms, many extrapulmonary manifestations were observed in the gastrointestinal tract and reported by SARS-CoV-2 patients, including abdominal pain, nausea, and diarrhea. SARS-CoV-2 binds initially to angiotensin-converting enzyme 2 (ACE2) on the host cell surface via its spike (S) protein before it undergoes endocytosis and fusion with the lysosomal membrane. The spike protein of SARS-CoV-2 is a heavily N- and O-glycosylated trimer. Glycosylation is an essential posttranslational modification in the life cycle of membrane and secretory proteins that affects their structural and functional characteristics as well as their trafficking and sorting patterns. This study aimed at elucidating the impact of glycosylation modulation on the trafficking of both S1 subunit and ACE2 as well as their interaction at the cell surface of intestinal epithelial cells. For this purpose, the S1 protein was expressed in COS-1 cells and its glycosylation modified using N-butyldeoxynojirimycin (NB-DNJ), an inhibitor of ER-located alpha-glucosidases I and II, and or 1-deoxymannojirimycin (dMM), an inhibitor of the Golgi-located alpha-mannosidase I. The intracellular and secreted S1 proteins were analyzed by endoglycosidase H treatment. Similarly, ACE2 trafficking to the brush border membrane of intestinal Caco-2 cells was also assessed in the presence or absence of the inhibitors. Finally, the interaction between the S1 protein and ACE2 was investigated at the surface of Caco-2 cells by co-immunoprecipitation. Our data show that NB-DNJ significantly reduced the secretion of S1 proteins in COS-1 cells, while dMM affected S1 secretion to a lesser extent. Moreover, NB-DNJ and dMM differentially affected ACE2 trafficking and sorting to the brush border membrane of intestinal Caco-2 cells. Strikingly, the interaction between S1 and ACE2 was significantly reduced when both proteins were processed by the glycosylation inhibitors, rendering glycosylation and its inhibitors potential candidates for SARS-CoV-2 treatment. This work has been supported by a grant from the German Research Foundation (DFG) grant NA331/15-1 to HYN. M.K. was supported by a scholarship from the Hannover Graduate School for Veterinary Pathobiology, Neuroinfectiology, and Translational Medicine (HGNI) and by the DFG grant NA331/15-1.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

7th Turkey in vitro Diagnostics (IVD) Symposium

The proceedings contain 75 papers. The topics discussed include: development of different product groups in the cleaning industry with synthesized nanosilver;investigation of the antimetastatic potential of thymbra spicata in human breast adenocarcinoma cells combined with standard chemotherapy;the effect of toll-interancing protein on inflammatory status of hepatocellular carcinoma cell line;surgery and inflammation: surgical inflammation;biochemistry of inflammation;its mediators and activities;pathophysiology of inflammation;classic and new biomarkers of inflammation;thymol reduces the lipopolysaccharide-induced acute kidney inflammation by modulating lysosomal stress;metabolic shift of the kynurenine pathway in inflammatory conditions;and evaluation of HBA1C level in patients with COVID-19.

A new autophagy-driven protective pathway of Haematococcus astaxanthin against chemical damage in wistar rats with gentamicin-induced acute kidney injury

Astaxanthin was established to conserve kidney function and subcellular structure through anti-oxidation and/or the free radical scavenging system, yet little research linked a new protective effect to autophagy or lysosomes. We pre-fed Wistar rats with natural astaxanthin, β-carotene, or placebo and induced acute kidney injury using gentamicin, before examining renal tissues and measuring physiological indices. Qualitative evidence from histopathological and subcellular images, along with quantitative evidence showing treatment effects on blood urea nitrogen and serum creatinine (p < 0.01), indicated that esterified Haematococcus astaxanthin surpassed β-carotene at effectively counteracting chemical damage and protecting the kidneys from injury. Proliferation of enlarged lysosomes and mediation analysis results revealing enhanced lysosomal acid phosphatase activity were consistent with the hypothesized autophagy-lysosomal pathway being up-regulated by astaxanthin intake (p < 0.05). In conclusion, the protective effect of astaxanthin against acute kidney injury exerted through the autophagy-lysosomal detoxification pathway, which totally different from the anti-oxidation and/or conventional SOD-dependent free radical scavenging system, was demonstrated with strong evidence. In light of the pandemic outbreak of novel coronavirus pneumonia associated with a virus preferentially targeting the renal tubular cells, dietary astaxanthin may help bring down incidence rate of coronavirus disease, cases of acute kidney injury secondary to the disease, and mortality rate from acute kidney injury, especially when a standard of care treatment for the infectious disease is pending. © 2023 Elsevier B.V.

Cathepsins in the extracellular space: Focusing on non-lysosomal proteolytic functions with clinical implications.

Cathepsins can be found in the extracellular space, cytoplasm, and nucleus. It was initially suspected that the primary physiological function of the cathepsins was to break down intracellular protein, and that they also had a role in pathological processes including inflammation and apoptosis. However, the many actions of cathepsins outside the cell and their complicated biological impacts have garnered much interest. Cathepsins play significant roles in a number of illnesses by regulating parenchymal cell proliferation, cell migration, viral invasion, inflammation, and immunological responses through extracellular matrix remodeling, signaling disruption, leukocyte recruitment, and cell adhesion. In this review, we outline the physiological roles of cathepsins in the extracellular space, the crucial pathological functions performed by cathepsins in illnesses, and the recent breakthroughs in the detection and therapy of specific inhibitors and fluorescent probes in associated dysfunction.

COVID-19 Pandemic: An Overview of its Origin, Current Status, and Ongoing Clinical Trials

The COVID-19 pandemic is raging across the globe, with the total active cases increas-ing each day. Globally over 63 million COVID-19cases and more than 1.4 million deaths have been reported to WHO. Throughout the world, academicians, clinicians and scientists are working tirelessly on developing a treatment to combat this pandemic. The origin of novel SARS-CoV-2 virus still remains foggy but is believed to have originated from a bat coronavirus RaTG13 with which it shares approximately 96% sequence similarity. In the present review, the authors have pro-vided an overview of the COVID-19 pandemic, epidemiology, transmission, developments related to diagnosis, drugs and vaccines, along with the genetic diversity and lifecycle of the SARS-CoV-2 based on the current studies and information available.Copyright © 2022 Bentham Science Publishers.

Collateral damage of the COVID-19 pandemic on lysosomal storage diseases: A single unit experience: Collateral damage of COVID-19

Aim: COVID-19 epidemic caused significant problems in countries' health systems. The direct and indirect effects of this unprecedented outbreak on patients with rare diseases are not clear. The aim of this study is to identify the pandemic-related problems encountered by adults with LSDs and parents who have children with LSDs and to evaluate the socio-economic and psychological effects of the COVID-19 outbreak in the lysosomal diseases community. Material(s) and Method(s): Our study included 27 adult patients receiving ERT treatment and 19 parents of child patients who were followed up with LSD diagnosis. Sociodemographic Data Form and COVID-19 Traumatic Stress Scale were applied to patients and their parents. Result(s): We found that treatment was disrupted in 44.4% of adult patients and 36.84% of child patients due to the COVID-19 outbreak. We found that 50% of adults and 80% of parents had a fear of going to the hospital due to the COVID-19 pandemic. Economic distress was higher in the treatment-disrupted group. Also, patients who had a fear when going to the hospital for ERT had higher "fear/threat of infection" scores. Discussion(s): The pandemic brought wide-ranging changes in the treatment and follow-up of patients with rare diseases. Home therapy appears to be the most effective way to maintain access to treatment during a pandemic;however, the personnel involved should be monitored and much attention should be paid to the proper use of personal protective equipment. It is also of great importance to provide adequate social, economic and psychological support to individuals.Copyright © 2022, Derman Medical Publishing. All rights reserved.

A novel diG motif in ORF3a protein of SARS-Cov-2 for intracellular transport.

The ongoing SARS-CoV-2/COVID-19 pandemic caused a global public health crisis. Yet, everyone's response to SARS-CoV-2 infection varies, and different viral variants confer diverse pathogenicity. Thus, it is imperative to understand how viral determinants contribute to COVID-19. Viral ORF3a protein is one of those viral determinants, as its functions are linked to induction of cell and tissues damages, disease severity and cytokine storm that is a major cause of COVID-19-related death. ORF3a is a membrane-associated protein. Upon synthesis, it is transported from endoplasmic reticulum, Golgi apparatus to plasma membrane and subcellular endomembranes including endosomes and lysosomes. However, how ORF3a is transported intracellularly remains elusive. The goal of this study was to carry out a systematic mutagenesis study to determine the structural relationship of ORF3a protein with its subcellular locations. Single amino acid (aa) and deletion mutations were generated in the putative function-relevant motifs and other regions of interest. Immunofluorescence and ImageJ analyses were used to determine and quantitate subcellular locations of ORF3a mutants in comparison with wildtype ORF3a. The wildtype ORF3a localizes predominantly (Pearson's coefficients about 0.8) on the membranes of endosomes and lysosomes. Consistent with earlier findings, deletion of the YXXΦ motif, which is required for protein export, retained ORF3a in the Golgi apparatus. Interestingly, mutations in a double glycine (diG) region (aa 187-188) displayed a similar phenotype to the YXXΦ deletion, implicating a similar role of the diG motif in intracellular transport. Indeed, interrupting any one of the two glycine residues such as deletion of a single (dG188), both (dG187/dG188) or substitution (G188Y) of these residues led to ORF3a retention in the Golgi apparatus (Pearson's coefficients ≥0.8). Structural analyses further suggest that the diG motif supports a type-II ß-turn between the anti-parallel ß4 and ß5 sheets and connects to the YXXΦ motif via hydrogen bonds between two monomers. The diG- YXXΦ interaction forms a hand-in-hand configuration that could facilitate dimerization. Together, these observations suggest a functional role of the diG motif in intracellular transport of ORF3a.

Endo-Lysosomal Cation Channels and Infectious Diseases.

Among the infectious diseases caused by pathogenic microorganisms such as bacteria, viruses, parasites, or fungi, the most prevalent ones today are malaria, tuberculosis, influenza, HIV/AIDS, Ebola, dengue fever, and methicillin-resistant Staphylococcus aureus (MRSA) infection, and most recently Covid-19 (SARS-CoV2). Others with a rather devastating history and high fatality rates such as plague, cholera, or typhus seem less threatening today but have not been eradicated, and with a declining efficacy of current antibiotics they ought to be watched carefully. Another emerging issue in this context is health-care associated infection. About 100,000 hospitalized patients in the USA ( www.cdc.gov ) and 33,000 in Europe ( https://www.ecdc.europa.eu ) die each year as a direct consequence of an infection caused by bacteria resistant to antibiotics. Among viral infections, influenza is responsible for about 3-5 million cases of severe illness, and about 250,000 to 500,000 deaths annually ( www.who.int ). About 37 million people are currently living with HIV infection and about one million die from it each year. Coronaviruses such as MERS-CoV, SARS-CoV, but in particular the recent outbreak of Covid-19 (caused by SARS-CoV2) have resulted in large numbers of infections worldwide with an estimated several hundred thousand deaths (anticipated fatality rate: <5%). With a comparatively low mortality rate dengue virus causes between 50 and 100 million infections every year, leading to 50,000 deaths. In contrast, Ebola virus is the causative agent for one of the deadliest viral diseases. The Ebola outbreak in West Africa in 2014 is considered the largest outbreak in history with more than 11,000 deaths. Many of the deadliest pathogens such as Ebola virus, influenza virus, mycobacterium tuberculosis, dengue virus, and cholera exploit the endo-lysosomal trafficking system of host cells for penetration into the cytosol and replication. Defects in endo-lysosomal maturation, trafficking, fusion, or pH homeostasis can efficiently reduce the cytotoxicity caused by these pathogens. Most of these functions critically depend on endo-lysosomal membrane proteins such as transporters and ion channels. In particular, cation channels such as the mucolipins (TRPMLs) or the two-pore channels (TPCs) are involved in all of these aspects of endo-lysosomal integrity. In this review we will discuss the correlations between pathogen toxicity and endo-lysosomal cation channel function, and their potential as drug targets for infectious disease therapy.

Prolonged fever and inflammation in a young child in the midst of the pandemic - it's not always MIS-C

Case Report: Prolonged fever in children is a symptom that is seen in many different diseases, infections, malignancies, and autoimmune conditions. This can, at times, make the correct diagnosis challenging. A previously healthy 10-year-old male was transferred to our institution with one week history of fever, fatigue, abdominal pain, and vomiting. Laboratory studies demonstrated pancytopenia, transaminitis, electrolyte abnormalities, elevated pro-inflammatory markers & D-Dimer, and hypoalbuminemia. COVID-19 IgG was reactive. Due to the severity in presentation the patient was transferred to the ICU with a presumptive diagnosis of MIS-C. Hewas started on IVIG as well as a five-day course of high-dose methylprednisolone per protocol. Aspirin was added, but later discontinued, due to worsening thrombocytopenia. CT imaging with contrast showed small bilateral pleural effusions & periportal edema, mild splenomegaly, and echocardiogram showed diffuse dilation of the left main and left anterior descending arteries. Given the laboratory findings the differential diagnosis was expanded, Ehrlichia caffeensis serology was sent and empiric Doxycycline started. EBV Nuclear Antigen IgG antibody and EBV Viral Capsid Antigen IgM Antibody resulted as positive suggesting recent or reactivated infection. Respiratory viral PCR with COVID-19, Cytomegalovirus and Parvovirus PCR were negative. Despite initial treatment, the patient continued to have persistent fever, severe pancytopenia, and high ferritin up to 24 426 ng/mL, raising suspicion for Haemophagocytic Lymphohistiocytosis (HLH). Soluble interleukin-2 level was elevated & his presentation was then considered to be more consistent with HLH given that he met 6/8 criteria. Screening for primary HLH including CD107a, perforin and granzyme B, SAP, and XIAP resulted in the latter three being normal but CD107a was abnormal. Next generation sequencing for primary criteria was negative. E. Chaffeensis resulted positive: IgM 1:80, IgG 1:256. MIS-C and HLH have overlapping features but differ in some clinical manifestations. Timely recognition and management is paramount as the management differs. This case illustrates the importance of performing a broad search for potential causes, allowing for appropriate and timely treatment. COVID-19 serology alone should not be the basis for diagnosis of MIS-C in a patient with fever and inflammation. This is important as SARS-CoV2 becomes endemic. Infections such as EBV and Ehrlichiosis should be on the differential particularly in endemic areas and during seasons of higher prevalence for the latter, as these have been well documented to cause HLH. Copyright © 2023 Southern Society for Clinical Investigation.

The ORF7a protein of SARS-CoV-2 initiates autophagy and limits autophagosome-lysosome fusion via degradation of SNAP29 to promote virus replication.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is closely related to various cellular aspects associated with autophagy. However, how SARS-CoV-2 mediates the subversion of the macroautophagy/autophagy pathway remains largely unclear. In this study, we demonstrate that overexpression of the SARS-CoV-2 ORF7a protein activates LC3-II and leads to the accumulation of autophagosomes in multiple cell lines, while knockdown of the viral ORF7a gene via shRNAs targeting ORF7a sgRNA during SARS-CoV-2 infection decreased autophagy levels. Mechanistically, the ORF7a protein initiates autophagy via the AKT-MTOR-ULK1-mediated pathway, but ORF7a limits the progression of autophagic flux by activating CASP3 (caspase 3) to cleave the SNAP29 protein at aspartic acid residue 30 (D30), ultimately impairing complete autophagy. Importantly, SARS-CoV-2 infection-induced accumulated autophagosomes promote progeny virus production, whereby ORF7a downregulates SNAP29, ultimately resulting in failure of autophagosome fusion with lysosomes to promote viral replication. Taken together, our study reveals a mechanism by which SARS-CoV-2 utilizes the autophagic machinery to facilitate its own propagation via ORF7a.

HYDROXYCHLOROQUINE CAN BREAK YOUR HEART

SESSION TITLE: The Cardiac Intensivist 2 SESSION TYPE: Rapid Fire Case Reports PRESENTED ON: 10/18/2022 12:25 pm - 01:25 pm INTRODUCTION: Hydroxychloroquine and chloroquine are medications derived from aminoquinoline. They are disease-modifying antirheumatic drugs used in the treatment of systemic lupus erythematosus (SLE). Although well tolerated, they do have side effects such as retinopathy, vacuolar myopathy, neuropathy, and as seen in our patient, cardiotoxicity. CASE PRESENTATION: Patient is a 48 year old female with a past medical history significant for chronic kidney disease secondary to autosomal dominant polycystic kidney disease, SLE on hydroxychloroquine who presented to the emergency department complaining of weakness. On arrival the patient was found to be in cardiogenic shock. Her transthoracic echocardiogram revealed a reduced ejection fraction of 37% and a large pericardial effusion concerning for tamponade physiology. Her COVID-19 PCR test was positive. She was taken for emergent pericardiocentesis which revealed 300cc of exudative fluid. Patient’s right heart catheterization revealed mean pulmonary capillary wedge pressure of 23 mmHg, pulmonary artery pressures of 44 mmHg/24 mmHg, mean 31mmHg, cardiac index 1.1L/min/m² by thermodilution, 1.7 L/min/m² by Fick. Following right heart catheterization and intra aortic balloon pump placement, the patient was admitted to the medical intensive care unit (MICU) and placed on intravenous inotropic and vasopressor support. Shortly after arrival to the MICU, patient had an increase in vasopressor requirements. Bedside ultrasound revealed cardiac tamponade. Patient had approximately 400cc of bloody pericardial fluid removed from her pericardial drain. The decision was made for emergent venoarterial extracorporeal membrane oxygenation (ECMO) to be initiated. Endomyocardial biopsy was performed which revealed vacuolization in the cytoplasm of several myocytes as well as lymphocytes in the interstitium of the endocardium. The vacuoles found in the cardiac myocytes were PAS positive. These biopsy results are consistent with hydroxychloroquine cardiotoxicity. The patient’s hydroxychloroquine was discontinued. In addition to hemodynamic support, she also received intravenous immunoglobuluin and systemic steroids. After a prolonged hospitalization she was successfully discharged. DISCUSSION: Cardiotoxicity is a rare adverse reaction seen with hydroxychloroquine. A 2018 systematic review revealed 127 cases of cardiac toxicity associated with the use of hydroxychloroquine or chloroquine. Most patients had been treated with the medication for a prolonged period of time and the toxicity is dose dependent. The mechanism behind hydroxychloroquine and chloroquine induced cardiomyopathy is believed to be secondary to lysosomal dysfunction as a result of toxic phospholipid accumulation in cardiomyocytes. CONCLUSIONS: In patients with new onset cardiomyopathy, a detailed medication reconciliation should be conducted to evaluate for toxins such as hydroxychloroquine and chloroquine. Reference #1: Della Porta, A., Bornstein, K., Coye, A., Montrief, T., Long, B., & Parris, M. A. (2020). Acute chloroquine and hydroxychloroquine toxicity: A review for emergency clinicians. The American Journal of Emergency Medicine. Reference #2: Abbi, B., Patel, S., Kumthekar, A., Schwartz, D., & Blanco, I. (2020). A Case of Cardiomyopathy With Long-term Hydroxychloroquine Use. JCR: Journal of Clinical Rheumatology, 26(8), e300. Reference #3: Chatre, C., Roubille, F., Vernhet, H., Jorgensen, C., & Pers, Y. M. (2018). Cardiac complications attributed to chloroquine and hydroxychloroquine: a systematic review of the literature. Drug safety, 41(10), 919-931. DISCLOSURES: no disclosure on file for Joseph Adams;no disclosure on file for Suliman Alradawi;No relevant relationships by George Kalapurakal No relevant relationships by Mohammed Siddiqui

Review: mRNA delivery systems in vaccinations and COVID-19

mRNA vaccines open promising avenues for overcoming a variety of diseases due to their high therapeutic utilities, rapid growth capacities, and safe administration potentials. With the emergence of COVID-19, the use of mRNA vaccines has become even more widespread and far-reaching. However, for mRNA to be delivered to target cells and tissues, several obstacles must be overcome. For instance, naked mRNAs get easily and hastily degraded by ribonucleases in tissues and the bloodstream, and since mRNAs are large and polyanionic molecules, they cannot passively diffuse across cell membranes. Even though mRNAs are internalized by APCs, they must be able to reach the cytoplasm and escape endo-lysosomal traffic. Therefore, distinctive transport systems for efficient encapsulation of mRNAs using nanocarrier systems are required to ensure their delivery to cells’ cytoplasm. At this point, non-viral gene delivery systems such as polymers and lipids come to the fore, in which they can overcome the biological barriers and provide efficient delivery of mRNAs. Recently, mRNA vaccines have been used as a powerful weapon against COVID-19 pandemic which has affected the whole world since December 2019. This was clear by the emergence of Pfizer-BioNTech and Moderna vaccines, which offered mRNA vaccines with auspicious treatment abilities. A variety of carrying candidates have been utilized in such vaccines as polymers, metal nanoparticles, as well as LNPs, which each comes with its cons and pros in delivering mRNA. All of these mentioned points will be clarified and discussed in detail in this review paper.

Multi-Centre Study to Assess the Safety of Alglucosidasi and of Laronidasi in Home Infusion Setting

Background - During Coronavirus Disease-19 (COVID- 19) pandemic, the temporary and exceptional authorization 341/2020 0f the Italian Drug Agency (AIFA), allowed to guarantee the adherence to treatment for patients with lysosomal storage disorders (LSDs) through home therapy. Stable patients affected with Pompe Disease and Mucopolysaccharidosis type I (MPS I) could then receive regular Enzyme Replacement Therapy (ERT) infusions at home. Indeed, a data collection seemed to us a good opportunity to assess the safety of home infusions and thus fill the existing information gap. Methods - This is an Italian, multicenter, non-interventional, double cohort study sponsored by SanofiGenzyme with both retrospective and prospective data collection to obtain safety information on ERT treatment of Pompe Disease and Mucopolysaccharidosis type I (MPS I) patients in a home-care setting. The study will enroll 60 patients at 15 sites. The retrospective observation will start from the first ERT infusions in a homecare setting and the prospective observation will last after 12 months from the enrollment. During the control visits, investigators will administer the questionnaires and will record any documented clinical data occurred during the home infusions. Objectives - This study aims at obtaining safety information on patients with Pompe Disease treated with Myozyme® (alglucosidase alfa) and of patients with MPS I treated with Aldurazyme® (laronidase) in a home-care setting, as well as evaluating personal satisfaction of both cohorts of patients and documenting infusion compliance. Conclusions - The outcomes will mirror real-life management of patients in home-care infusion setting, including safety profile, treatment compliance and quality of life.