Association of polymorphic variants in human genome with the COVID-2019 severity and post-COVID syndrome development

Background/Objectives: Despite intensive research of the novel coronavirus SARS-CoV-2 and COVID-2019 caused by it, factors affecting the severity of the disease remains poorly understood. Clinical manifestations of COVID-2019 may vary from asymptomatic form to pneumonia, acute respiratory distress syndrome (ARDS) and multiorgan failure. Features of individual genetic landscape of patients can play an important role in development of the pathological process of COVID-19. In this regard the purpose of this study was to investigate the influence of polymorphic variants in genes (ADD1, CAT, IL17F, IL23R, NOS3, IFNL3, IL6, F2, F13A1, ITGB3, HIF1A, MMP12, VEGFA), associated with cardiovascular, respiratory and autoimmune pathologies, on the severity of COVID-19 and post-COVID syndrome in patients from Russia. Method(s): The study included 200 patients recovered from COVID-19. Two groups of patients were formed in accordance with clinical manifestations: with mild and moderate forms of the disease. The polymorphic variants were analysed with real-time PCR using commercial kits (Syntol). Result(s): 13 SNPs (rs4961;rs1001179;rs612242;rs11209026;rs2070744;rs8099917;rs1800795;rs1799963;rs5985;rs5918;rs11549465;rs652438;rs699947) were genotyped and comparative analysis of allele frequency distribution was carried out in two groups of patients recovered from COVID-2019. Conclusion(s): Identification of polymorphic variants in genome associated with severity of pathological processes in patients infected with SARS-CoV-2 can contribute to the identification of individuals with an increased risk of severe infection process and can also serve as a basis for developing personalized therapeutic approaches to the treatment of post-COVID syndrome.

Reduced susceptibility to COVID-19 associated with ABO blood group and pre-existing anti-A and anti-B antibodies.

BACKGROUND: Susceptibility to severe acute respiratory syndrome coronavirus 2 shows individual variability in un-vaccinated and previously un-exposed individuals. We investigated the impact of ABO blood group, titers of anti-A and anti-B, other blood group antigens, and the extracellular deposition of ABH antigens as controlled by secretor fucosyltransferase 2 (FUT2) status. STUDY DESIGN AND METHODS: We studied incidents in three different hospitals between April to September 2020, where un-diagnosed coronavirus disease 2019 (COVID-19) patients were cared for by health care workers without use of personal protection and with close contact while delivering therapy. We recruited 108 exposed staff, of whom 34 were diagnosed with COVID-19. ABO blood type, titer of anti-A and -B, blood group specific alleles, and secretor status were determined. RESULTS: Blood group O was associated with lower risk of COVID-19 (OR 0.39, 95 %CI (0.16-0.92), p = 0.03) compared to non-O, i.e., blood groups A, B and AB. High titer anti-A immunoglobulin G (IgG) compared to low titer was associated with lower risk of COVID-19 (OR 0.24 95 %CI (0.07-0.78), p = 0.017). High titer of anti-B immunoglobulin M (IgM) compared to no anti-B (IgM) was associated with lower risk of COVID-19 (OR 0.16, 95 %CI (0.039-0.608), p = 0.006) and the same applies to low titer anti-B (IgM) compared to no titer (OR 0.23, 95 %CI (0.07-0.72), p = 0.012). The 33Pro variant in Integrin beta-3, that is part of human platelet antigen 1b (HPA-1b), was associated with lower risk of COVID-19 (OR 0.23, 95 %CI (0.034-0.86), p = 0.028). CONCLUSION: Our data showed that blood group O, anti-A (IgG) titer, anti-B (IgM) titer as well as HPA-1b are associated with lower risk for COVID-19.

Human Adenovirus Type 26 Infection Mediated by αvß3 Integrin Is Caveolin-1-Dependent.

Human adenovirus type 26 (HAdV26) has been recognized as a promising platform for vaccine vector development, and very recently vaccine against COVID-19 based on HAdV26 was authorized for emergency use. Nevertheless, basic biology of this virus, namely, pathway which HAdV26 uses to enter the cell, is still insufficiently known. We have shown here that HAdV26 infection of human epithelial cells expressing low amount of αvß3 integrin involves clathrin and is caveolin-1-independent, while HAdV26 infection of cells with high amount of αvß3 integrin does not involve clathrin but is caveolin-1-dependent. Thus, this study demonstrates that caveolin-1 is limiting factor in αvß3 integrin-mediated HAdV26 infection. Regardless of αvß3 integrin expression, HAdV26 infection involves dynamin-2. Our data provide for the first-time description of HAdV26 cell entry pathway, hence increase our knowledge of HAdV26 infection. Knowing that functionality of adenovirus vector is influenced by its cell entry pathway and intracellular trafficking, our results will contribute to better understanding of HAdV26 immunogenicity and antigen presentation when used as vaccine vector. IMPORTANCE In order to fulfill its role as a vector, adenovirus needs to successfully deliver its DNA genome to the host nucleus, a process highly influenced by adenovirus intracellular translocation. Thus, cell entry pathway and intracellular trafficking determine functionality of human adenovirus-based vectors. Endocytosis of HAdV26, currently extensively studied as a vaccine vector, has not been described so far. We present here that HAdV26 infection of human epithelial cells with high expression of αvß3 integrin, one of the putative HAdV26 receptors, is caveolin-1- and partially dynamin-2-dependent. Since caveolin containing domains provide a unique environment for specific signaling events and participate in inflammatory signaling one can imagine that directing HAdV26 cell entry toward caveolin-1-mediate pathway might play role in immunogenicity of this virus. Therefore, our results contribute to better understanding of HAdV26 infection pathway, hence, can be helpful in explaining induction of immune response and antigen presentation by HAdV26-based vaccine vector.

Changes in the proteomics of exhaled breath condensate under the influence of inhaled hydrogen in patients with post-COVID syndrome.

Aim. To study the effect of inhalation therapy with an active hydrogen (AH) on the protein composition of exhaled breath condensate (EBC) in patients with post-COVID syndrome (PCS). Material and methods. This randomized controlled parallel prospective study included 60 patients after coronavirus disease 2019 (COVID-19) with PCS during the recovery period and clinical manifestations of chronic fatigue syndrome who received standard therapy according to the protocol for managing patients with chronic fatigue syndrome (CFS). The patients were divided into 2 groups: group 1 (main) - 30 people who received standard therapy and AH inhalations (SUISONIA, Japan) for 10 days, and group 2 (control) - 30 medical workers who received only standard therapy. Patients in both groups were comparable in sex and mean age. All participants in the study were sampled with EBC on days 1 and 10. Samples were subjected to tryptic digestion and high-performance liquid chromatography combined with tandem mass spectrometry analysis using a nanoflow chromatograph (Dionex 3000) in tandem with a high-resolution time-of-flight mass spectrometer (timsTOF Pro). Results. A total of 478 proteins and 1350 peptides were identified using high resolution mass spectrometry. The number of proteins in samples after AH therapy, on average, is 12% more than before treatment. An analysis of the distribution of proteins in different groups of patients showed that only half of these proteins (112) are common for all groups of samples and are detected in EBC before, after, and regardless of hydrogen therapy. In addition to the qualitative difference in the EBC protein compositions in different groups, quantitative changes in the concentration of 36 proteins (mainly structural and protective) were also revealed, which together made it possible to reliably distinguish between subgroups before and after treatment. It is worth noting that among these proteins there are participants of blood coagulation (alpha-1-antitrypsin), chemokine- and cytokine-mediated inflammation, and a number of signaling pathways (cytoplasmic actin 2), response to oxidative stress (thioredoxin), glycolysis (glyceraldehyde-3- phosphate dehydrogenase), etc. Conclusion. The use of hydrogen therapy can contribute to the switching of a number of physiological processes, which may affect the success of recovery in PCS patients. In particular, the obtained results indicate the activation of aerobic synthesis of adenosine triphosphate in mitochondria by hydrogen therapy, which correlates well with the decrease in the blood lactate level detected by laboratory studies. At the same time, this therapy can inhibit pro-inflammatory activity, negatively affecting the coagulation and signaling pathways of integrins and apoptosis, and, in addition, activate protective pathways, tricarboxylic acid cycle, FAS signaling, and purine metabolism, which may be essential for effective recovery after COVID-19.Copyright © 2023 Vserossiiskoe Obshchestvo Kardiologov. All rights reserved.

Esculin alleviates LPS-induced acute lung injury via inhibiting neutrophil recruitment and migration.

OBJECTIVES: Acute lung injury (ALI) poses a serious threat to human health globally, particularly with the Coronavirus 2019 (COVID-19) pandemic. Excessive recruitment and infiltration of neutrophils is the major etiopathogenesis of ALI. Esculin, also known as 6,7-dihydroxycoumarin, is a remarkable compound derived from traditional Chinese medicine Cortex fraxini. Accumulated evidence indicates that esculin has potent anti-inflammatory effects, but its pharmaceutical effect against ALI and potential mechanisms are still unclear. METHODS: This study evaluated the protective effect of esculin against ALI by histopathological observation and biochemical analysis of lung tissues and bronchoalveolar lavage fluid (BALF) in lipopolysaccharide (LPS)-challenged ALI mice in vivo. The effects of esculin on N-formyl-met-leu-phe (fMLP)-induced neutrophil migration and chemotaxis were quantitatively assessed using a Transwell assay and an automated cell imaging system equipped with a Zigmond chamber, respectively. The drug affinity responsive target stability (DARTS) assay, in vitro protein binding assay and molecular docking were performed to identify the potential therapeutic target of esculin and the potential binding sites and pattern. RESULTS: Esculin significantly attenuated LPS-induced lung pathological injury, reduced the levels of pro-inflammatory cytokines in both BALF and lung, and suppressed the activation of NF-κB signaling. Esculin also significantly reduced the number of total cells and neutrophils as well as myeloperoxidase (MPO) activity in the BALF. Esculin impaired neutrophil migration and chemotaxis as evidenced by the reduced migration distance and velocity. Furthermore, esculin remarkably inhibited Vav1 phosphorylation, suppressed Rac1 activation and the PAK1/LIMK1/cofilin signaling axis. Mechanistically, esculin could interact with ß2 integrin and then diminish its ligand affinity with intercellular adhesion molecule-1 (ICAM-1). CONCLUSIONS: Esculin inhibits ß2 integrin-dependent neutrophil migration and chemotaxis, blocks the cytoskeletal remodeling process required for neutrophil recruitment, thereby contributing to its protective effect against ALI. This study demonstrates the new therapeutic potential of esculin as a novel lead compound.

Integrin ß1 is a key determinant of the expression of angiotensin-converting enzyme 2 (ACE2) in the kidney epithelial cells.

The expression of the angiotensin-converting enzyme 2 (ACE2) is altered in multiple chronic kidney diseases like hypertension and renal fibrosis, where the signaling from the basal membrane proteins is critical for the development and progression of the various pathologies. Integrins are heterodimeric cell surface receptors that have important roles in the progression of these chronic kidney diseases by altering various cell signaling pathways in response to changes in the basement membrane proteins. It is unclear whether integrin or integrin-mediated signaling affects the ACE2 expression in the kidney. The current study tests the hypothesis that integrin ß1 regulates the expression of ACE2 in kidney epithelial cells. The role of integrin ß1 in ACE2 expression in renal epithelial cells was investigated by shRNA-mediated knockdown and pharmacological inhibition. In vivo studies were carried out using epithelial cell-specific deletion of integrin ß1 in the kidneys. Deletion of integrin ß1 from the mouse renal epithelial cells reduced the expression of ACE2 in the kidney. Furthermore, the downregulation of integrin ß1 using shRNA decreased ACE2 expression in human renal epithelial cells. ACE2 expression levels were also decreased in renal epithelial cells and cancer cells when treated with an integrin α2ß1 antagonist, BTT 3033. SARS-CoV-2 viral entry to human renal epithelial cells and cancer cells was also inhibited by BTT 3033. This study demonstrates that integrin ß1 positively regulates the expression of ACE2, which is required for the entry of SARS-CoV-2 into kidney cells.

What had the greatest impact on the quality of life of IBD patients on intravenous biological therapy in Montenegro during COVID-19?

Background: COVID-19 has affected the quality of life (QoL) of patients with chronic diseases, including patients with IBD. The aim of this study is to evaluate the QoL of patients with IBD on intravenous biological therapy (IvBT), through the Short Inflammatory Bowel Disease Questionnaire (sIBDQ), and to correlate the results with sociodemographic data of the patients. Method(s): This study was comprised of patients older than 18 years of age, with a pathohistologically confirmed diagnosis of IBD (Ulcerative Colitis (UC), and Crohn's Disease (CD)). The study was conducted on September and October 2020, during one of the highest incidences period of COVID-19 in our country. Patients completed the sIBDQ, and DASS-21 (Depression, Anxiety and Stress Score-21) questionnaire too, assessing their level of depression, anxiety and stress. For significant symptoms (DASS-21), we used at least moderate DASS-21 subscale score: DASS-21 Depression (>= 14), DASS-21 Anxiety (>= 10) and DASS-21 Stress (>=19). The Simple Clinical Colitis Activity Index was used to assess the disease activity of UC;for CD, the Harvey-Bradshaw Index was used. Patients who scored below 50 on the sIBDQ were those labeled with worse QoL. We also examined demographic data, data on IBD characteristics and COVID-19 data and their impact on quality of life. Result(s): Of the total number of patients (94), there were 40 (42.5%) females, 42 (44.6%) with CD. All patients have been receiving IvBTh (anti TNFalpha: Infliximab-originator and biosimilar (59 patients) and anti-integrins: Vedolizumab (35 patients)) for at least 6 months prior. The results indicated worse QoL in 25 (27%) patients. Multivariate analysis showed that the greatest impact on poor QoL during the COVD-19 pandemic were: active disease (p= 0.002), significant symptoms on the DASS21 score (p= 0.013), patients who did not regularly go to work or were not employed (p=0.017), if they had a patient with COVID-19 in their immediate environment (p=0.024) and those who had a higher degree of health concerns about coming to regular admission to biological therapy at the IBD unit. (p=0.046). Conclusion(s): 27% of IBD patients on IvBT had worse QoL during the COVID 19 pandemic. In addition to disease activity and significant psychological disturbances, other reasons for lower QoL were identified during the pandemic and are directly related to it. (Figure Presented).

Ulcerative Colitis: Rapid Evidence Review

Ulcerative colitis is a relapsing and remitting inflammatory bowel disease of the large intestine. Risk factors include recent Salmonella or Campylobacter infection and a family history of ulcerative colitis. Diagnosis is suspected based on symptoms of urgency, tenesmus, and hematochezia and is confirmed with endoscopic findings of continuous inflammation from the rectum to more proximal colon, depending on the extent of disease. Fecal calprotectin may be used to assess disease activity and relapse. Medications available to treat the inflammation include 5-aminosalicylic acid, corticosteroids, tumor necrosis factor-alpha antibodies, anti-integrin antibodies, anti-interleukin-12 and -23 antibodies, and Janus kinase inhibitors. Choice of medication and method of delivery depend on the location and severity of mucosal inflammation. Other treatments such as fecal microbiota transplantation are considered experimental, and complementary therapies such as probiotics and curcumin have mixed data. Surgical treatment may be needed for fulminant or refractory disease. Increased risk of colorectal cancer and use of immunosuppressive therapies affect the preventive care needs for these patients. (Am Fam Physician. 2022;105(4):406-411. Copyright © 2022 American Academy of Family Physicians.)Copyright © 2022 American Academy of Family Physicians. All rights reserved.

Impaired antibody seroconversion after COVID-19 vaccination and negative impact of immunosuppressive treatment in Inflammatory Bowel Disease. Results from a multicentre, prospective study of GETECCU (VACOVEII)

Background: COVID-19 vaccination has been suggested as very effective in patients with Inflammatory Bowel Disease (IBD), but most studies assess antibody levels within a few weeks after vaccination and do not use the most recent recommendations as seroconversion cut-off. The objective of VACOVEII study is to evaluate the antibody response to vaccination at 6 months using these recommendations, the improvement after a booster dose and the effect of the immunosuppressive therapy (IST). We present the intermediate results of the study. Method(s): Spanish multicentre, prospective and case-control study. 18 years or older IBD patients fully vaccinated against COVID-19 were included. Those with previous COVID infection were not included, but not excluded for the next analyses if the infection was subsequent. Main outcomes were anti-SARS-CoV-2 spike protein antibody (anti S) concentrations and rate of seroconversion (defined above the protection threshold of 260 BAU/mL), measured 6 months after vaccination at a single centralized laboratory. The effect of IST on the main outcomes was analysed, adjusted by age, vaccine type and COVID infection. Groups of treatment considered for the analysis were: Patients without IST (without treatment or under salicylates alone), anti-TNF in combination with immunomodulators (IMM), anti-TNF in monotherapy, IMM in monotherapy, ustekinumab and anti-integrin. Result(s): We included 313 patients with IBD (46.5% ulcerative colitis and 52.3% Crohn's disease, median age 49 years) vaccinated either with non-mRNA vaccines (14%) or mRNA vaccines (86%). Baseline therapy was: 124 patients without IST, 21 with anti-TNF plus IMM, 67 with anti-TNF in monotherapy, 54 with IMM in monotherapy, 28 with ustekinumab and 19 with anti-integrin. Mean anti S concentrations were significant lower in patients with anti-TNF compared with patients without IST (Figure 1). In multivariable analysis, lower antibody concentrations were independently associated with anti-TNF treatment, non-mRNA vaccines and older age. Within the patients with no COVID infection during the follow-up, we found very low rates of seroconversion in patients with anti-TNF (14.1%), ustekinumab (30.8%) and IMM in monotherapy (34.9%), compared with patients without IST (51.5%) (Table 1). In multivariable analysis, anti-TNF treatment, non-mRNA vaccines and older age were independently associated with lower rates of seroconversion, as well as ustekinumab and IMM in monotherapy (Table 2). Conclusion(s): COVID-19 vaccine-induced antibody seroconversion in patients with IBD, measured at 6 months and according to >260 BAU as protection threshold, is clearly lower than previously reported, with a profound impact by some IST therapies, mainly anti-TNF, besides age and type of vaccine.

Longitudinal profiling of the peripheral blood transcriptome identifies risk of Major Cardiac Events after Hospitalization for COVID-19

Introduction: SARS-CoV-2 infection has profound effects on endothelial and immune cell function and coagulation, and better understanding of these events in COVID-19 would allow for targeted cardiovascular treatment and followup. Method(s): Longitudinal observational study of patients with PCR-confirmed SARS-CoV-2 infection admitted to hospital at two UK sites. Patients were enrolled within 96 hours of admission, with sampling up to day 29. RNAstabilised whole blood was processed for mRNA sequencing. Gene expression levels were compared between patients who did and did not suffer a major cardiac event (MACE) from admission to 1-year post-hospitalization. Result(s): At day 1, in acute COVID-19, no differences in gene expression were observed between those with (n=23) and without (n=140) a MACE. However, 93 significant differentially expressed genes (DEGs;adjusted pvalue<0.05;Wald test with Benjamini-Hochberg correction) were identified at day 29 between patients who suffered a MACE (n=16) or not (n=85) post-hospitalization. Neutrophil elastase (ELANE), tissue factor pathways inhibitor (TFPI) and integrin subunit alpha-2 (ITGA2B) were significantly elevated in patients who suffered a MACE. Significantly enriched pathways associated with cardiovascular events included type I interferon signalling and neutrophil chemotaxis. Conclusion(s): COVID-19 patients who experienced a MACE demonstrated significant changes in peripheral blood transcriptome 29 days after hospital admission. Significant DEGs were related to neutrophil activity, coagulation and interferon signalling, suggesting a relationship between these pathways and increased cardiovascular risk.

Myocardial Injury and Altered Gene Expression Associated With SARS-CoV-2 Infection or mRNA Vaccination.

SARS CoV-2 enters host cells via its Spike protein moiety binding to the essential cardiac enzyme angiotensin-converting enzyme (ACE) 2, followed by internalization. COVID-19 mRNA vaccines are RNA sequences that are translated into Spike protein, which follows the same ACE2-binding route as the intact virion. In model systems, isolated Spike protein can produce cell damage and altered gene expression, and myocardial injury or myocarditis can occur during COVID-19 or after mRNA vaccination. We investigated 7 COVID-19 and 6 post-mRNA vaccination patients with myocardial injury and found nearly identical alterations in gene expression that would predispose to inflammation, coagulopathy, and myocardial dysfunction.

Platelet αIIbß3 integrin binds to SARS-CoV-2 spike protein of alpha strain but not wild type and omicron strains.

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 causes a pandemic infectious disease, Coronavirus disease 2019 (COVID-19). It causes respiratory infection. Then, it progresses into a systemic infection by involving other organs. This progression mechanism remains to be elucidated, although thrombus formation plays an important role in its progression. Platelets is involved in the thrombus formation by aggregating each other through association of activated αIIbß3 integrin with the Arg-Gly-Asp (RGD) motif-containing its ligands such as fibrinogen and von Willebrand factor. SARS-CoV-2 enters host cells through association of the spike protein (S-protein) with its receptor, angiotensin-converting enzyme 2 (ACE-2), on the host cells. While presence of ACE2 in platelets is suspicious, S-protein harbors the RGD sequences within its receptor binding domain. Therefore, it could be possible SARS-CoV-2 enter platelets through association of S-protein with αIIbß3. In this study, we found that receptor binding domain of S-protein of WT SARS-CoV-2 strain barely bound to isolated healthy human platelets. In contrast, highly toxic alpha-strain-based N501Y substitution strongly bound to platelets in a RGD dependent manner, although binding of S protein did not induce platelet aggregation or activation. This binding may serve for transferring the infection to systemic organs.

Staining of activated ß2-integrins in combination with CD137 and CD154 for sensitive identification of functional antigen-specific CD4+ and CD8+ T cells.

Common flow cytometry-based methods used for functional assessment of antigen-specific T cells rely on de novo expression of intracellular cytokines or cell surface activation induced markers. They come with some limitations such as complex experimental setting, loss of cell viability and often high unspecific background which impairs assay sensitivity. We have previously shown that staining of activated ß2-integrins either with multimers of their ligand ICAM-1 or with a monoclonal antibody can serve as a functional marker detectable on T cells after minutes (CD8+) or few hours (CD4+) of activation. Here, we present a simple method for detection of activated ß2-integrins in combination with established cell surface activation induced markers. We observed that activated ß2-integrins were still detectable after 14 hours of stimulation, allowing their detection together with CD137 and CD154. Combinatorial gating of cells expressing activated ß2-integrins and CD137 or CD154 reduced background in unstimulated samples, increasing the signal-to-noise ratio and allowing improved assessment of low-frequency T cell responses. Extracellular staining of these markers highly correlated with production of intracellular cytokines IL-2, TNF or IFNγ in CD4+ and CD8+ T cells. As an exemplary application, SARS-CoV-2 spike-specific T cell responses were assessed in individuals after COVID-19 vaccination. This method should be useful for epitope discovery projects and for the simultaneous monitoring of low-frequency antigen-specific CD4+ and CD8+ T cell responses in various physiological situations.

Receptor-binding domain of SARS-CoV-2 is a functional αv-integrin agonist.

Among the novel mutations distinguishing SARS-CoV-2 from similar coronaviruses is a K403R substitution in the receptor-binding domain (RBD) of the viral spike (S) protein within its S1 region. This amino acid substitution occurs near the angiotensin-converting enzyme 2-binding interface and gives rise to a canonical RGD adhesion motif that is often found in native extracellular matrix proteins, including fibronectin. Here, the ability of recombinant S1-RBD to bind to cell surface integrins and trigger downstream signaling pathways was assessed and compared with RGD-containing, integrin-binding fragments of fibronectin. We determined that S1-RBD supported adhesion of fibronectin-null mouse embryonic fibroblasts as well as primary human small airway epithelial cells, while RBD-coated microparticles attached to epithelial monolayers in a cation-dependent manner. Cell adhesion to S1-RBD was RGD dependent and inhibited by blocking antibodies against αv and ß3 but not α5 or ß1 integrins. Similarly, we observed direct binding of S1-RBD to recombinant human αvß3 and αvß6 integrins, but not α5ß1 integrins, using surface plasmon resonance. S1-RBD adhesion initiated cell spreading, focal adhesion formation, and actin stress fiber organization to a similar extent as fibronectin. Moreover, S1-RBD stimulated tyrosine phosphorylation of the adhesion mediators FAK, Src, and paxillin; triggered Akt activation; and supported cell proliferation. Thus, the RGD sequence of S1-RBD can function as an αv-selective integrin agonist. This study provides evidence that cell surface αv-containing integrins can respond functionally to spike protein and raises the possibility that S1-mediated dysregulation of extracellular matrix dynamics may contribute to the pathogenesis and/or post-acute sequelae of SARS-CoV-2 infection.

Absolute binding free energies of the antiviral peptide ATN-161 with protein targets of SARS-CoV-2.

The interactions of the antiviral pentapeptide ATN-161 with the closed and open conformations of the α5ß1 integrin, the SARS-CoV-2 major protease, and the omicron variant spike protein complexed with hACE2 were studied using molecular docking and molecular dynamics simulation. Molecular docking was performed to obtain ATN-161 binding poses with these studied protein targets. Subsequently, molecular dynamics simulations were performed to verify the ligand stability at the binding site of each protein target. Pulling simulations, umbrella sampling, and weighted histogram analysis method were used to obtain the potential of mean force of each system and calculate the Gibbs free energy of binding for the ATN-161 peptide in each binding site of these protein targets. The results showed that ATN-161 binds to α5ß1 integrin in its active and inactive form, binds weakly to the omicron variant spike protein complexed with hACE2, and strongly binds to the main protease target.Communicated by Ramaswamy H. Sarma.

α-Parvin Defines a Specific Integrin Adhesome to Maintain the Glomerular Filtration Barrier.

BACKGROUND: The cell-matrix adhesion between podocytes and the glomerular basement membrane is essential for the integrity of the kidney's filtration barrier. Despite increasing knowledge about the complexity of integrin adhesion complexes, an understanding of the regulation of these protein complexes in glomerular disease remains elusive. METHODS: We mapped the in vivo composition of the podocyte integrin adhesome. In addition, we analyzed conditional knockout mice targeting a gene (Parva) that encodes an actin-binding protein (α-parvin), and murine disease models. To evaluate podocytes in vivo, we used super-resolution microscopy, electron microscopy, multiplex immunofluorescence microscopy, and RNA sequencing. We performed functional analysis of CRISPR/Cas9-generated PARVA single knockout podocytes and PARVA and PARVB double knockout podocytes in three- and two-dimensional cultures using specific extracellular matrix ligands and micropatterns. RESULTS: We found that PARVA is essential to prevent podocyte foot process effacement, detachment from the glomerular basement membrane, and the development of FSGS. Through the use of in vitro and in vivo models, we identified an inherent PARVB-dependent compensatory module at podocyte integrin adhesion complexes, sustaining efficient mechanical linkage at the filtration barrier. Sequential genetic deletion of PARVA and PARVB induces a switch in structure and composition of integrin adhesion complexes. This redistribution of these complexes translates into a loss of the ventral actin cytoskeleton, decreased adhesion capacity, impaired mechanical resistance, and dysfunctional extracellular matrix assembly. CONCLUSIONS: The findings reveal adaptive mechanisms of podocyte integrin adhesion complexes, providing a conceptual framework for therapeutic strategies to prevent podocyte detachment in glomerular disease.

Phage-Derived Oncolytic Viruses with 3C from Seneca Valley Virus for Targeted Therapy of Cervical Cancer

Cancer gene therapy based on various gene delivery vectors has some potential but also has obvious disadvantages. In this study, a new M13 phage-based oncolytic virus is constructed that carried the RGD peptides to target tumor cells and the 3C gene of Seneca Valley virus (SVV) preceded by a eukaryotic initial transcriptional region (ITR) to transcribe an oncolytic protein to kill tumor cells. Recombinant virus particles of 1200 nm in length are obtained in large quantities by transfecting the recombinant M13 phage plasmid into the host BL2738 and are investigated in vitro in tumor cells and in vivo in tumor-bearing mice to evaluate their antitumor effect. The experiments using Hela cells confirm that the engineered M13 phage can target and enter Hela cells, and express the SVV 3C protein, resulting in apoptosis of target cells by upregulating the expression of caspase 3. Furthermore, the results of experiments in vivo also show that the recombinant phage significantly inhibits the enhanced tumor volume in nude mice compared to the control groups. The M13 phage may be engineered to fuse with a variety of oncolytic proteins to inhibit the growth of tumor cells in the future, providing a promising phage-based targeted oncolytic reagent.

Rationale for evaluation of PLN-74809 treatment in participants with primary sclerosing cholangitis in Phase 2a study INTEGRIS-PSC

Background and aims: Transforming growth factor beta (TGF-beta) signalling is a key driver of liver fibrosis. In primary sclerosing cholangitis (PSC), integrins over-expressed on injured cholangiocytes (alpha-v/beta-6) and myofibroblasts (alpha-v/beta-1) regulate TGFbeta activity. PLN-74809 is an oral, once-daily, dual-selective inhibitor of integrins alpha-v/beta-6 and alpha-v/beta-1 in development for the treatment of PSC and idiopathic pulmonary fibrosis. It has shown favourable tolerability in over 280 healthy participants, reduced TGF-beta signalling and achieved high target engagement in human lungs. Pre-clinical evaluation of antifibrotic activity resulting from dual integrin inhibition was performed to support clinical evaluation. Method: PLN-74809 was administered orally for 6 weeks in BALBc. Mdr2-/- mice with established fibrosis. A tool alpha-v/beta-6 and alpha-v/beta-1 inhibitor compound, PLN-75068, was tested therapeutically in a diet-induced mouse model of biliary fibrosis using 3, 5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC). Hepatic collagen was quantified by hydroxyproline (OHP) and collagen proportionate area (CPA) and TGF-beta signalling by phosphorylated SMAD3 (pSMAD3) levels. An ex vivo study evaluated the effects of 2-day treatment with PLN-74809 on the expression of profibrotic genes, COL1A1 and COL1A2, in precision-cut liver slices (PCLivS) from tissue explants of participants with biliary fibrosis (n = 2 PSC;n = 2 primary biliary cholangitis [PBC]). A review of available blinded safety data from the enrolling Phase 2a study in participants with PSC was performed (NCT04480840). Results: PLN-74809 dose-dependently reduced OHP (up to ∼30%, p < 0.05), CPA (up to ∼50%, p < 0.05) and pSMAD3 (up to ∼40%, p < 0.001) in the BALBc.Mdr2-/- mouse model, as well as COL1A1 and COL1A2 gene expression (up to ∼30%, p = 0.0789) in PCLivS from tissue explants of participants with PSC and PBC. PLN-75068 reduced OHP (up to ∼20%, p < 0.05) in DDC-injured mice in a dose-dependent manner. PLN-74809waswell tolerated in participants with PSC. Most adverse events (AEs)were mild;nonewere severe. The most common AE was mild headache. One participant experienced serious AEs at least 20 days after the last dose of study drug, deemed not related by the investigator. One participant prematurely discontinued due to COVID-19. PLN-74809 pharmacokinetics in participants with PSC were consistent with those of healthy participants. Conclusion: Pharmacological inhibition of integrins alpha-v/beta-6 and alpha-v/beta-1 demonstrated antifibrotic activity in two models of biliary fibrosis and in PCLivS from participants with PSC or PBC. Available safety findings from participants with PSC enrolled in the ongoing Phase 2a INTEGRIS-PSC study, continue to support the favourable tolerability profile of PLN-74809.

A case of COVID-19 treated with mycotherapy

A COVID-19 patient at the limit of hospitalization (SpO2 at 93%), treated at home, found benefit in the use of antiviral medicinal mushrooms. The main agent would be Cordyceps sinensis, used in traditional Chinese medicine from 620 BC. This fungus has been studied by numerous scientists who have demonstrated its antiviral action against HIV-1, the syncytial respiratory virus, the coxsackie B3 virus, the A and “H1N1” influenza viruses, the Epstein Barr Virus. This patient treated at a time when treatments with antiretrovirals, enoxaparin or hydroxychloroquine were not yet permitted at home showed an immediate reversal of symptoms after taking Cordyceps sinensis and beta-glucans extracted from other mushrooms. The antiviral properties of the components of the drug used in this clinical case are described through the literature (mini review). The strong point, which would explain the rapid action (if it is not placebo), is the known affinity of the beta-glucans of the medicinal mushroom wall, towards the integrin-based receptors, usually present on neutrophil leukocytes, but also on the “thorns” of the CoV-2. This strong peculiarity that CoV-2 has become a weak point, if we get to inhibit it before it attacks the cells of the respiratory system.