Increases in expression of carbohydrate sulfotransferases CHST11 and CHST15 and decline in N-acetylgalactosamine-4-sulfatase (Arylsulfatase B, ARSB) require phospho-p38-MAPK following exposure to SARS-CoV-2 spike protein receptor binding domain in human a

Objective: Immunohistochemistry of post-mortem lung tissue from Covid-19 patients with diffuse alveolar damage demonstrated marked increases in chondroitin sulfate and CHST15 and decline in N-acetylgalactosamine-4-sulfatase. Studies were undertaken to identify the mechanisms involved in these effects. Method(s): Human primary small airway epithelial cells (PCS 301-010;ATCC) were cultured and exposed to the SARSCoV- 2 spike protein receptor binding domain (SPRBD;AA: Lys310-Leu560;Amsbio). Expression of the spike protein receptor, angiotensin converting enzyme 2 (ACE2), was enhanced by treatment with Interferon-beta. Promoter activation, DNA-binding, RNA silencing, QPCR, Western blots, ELISAs, and specific enzyme inhibitors were used to elucidate the underlying molecular mechanisms. Result(s): Treatment of the cultured cells by the SPRBD led to increased CHST15 and CHST11 expression and decline in ARSB expression. Sulfotransferase activity, total chondroitin sulfate, and sulfated glycosaminoglycan (GAG) content were increased. Phospho-T180/T182-p38-MAPK and phospho- S423/S425-Smad3 were required for the activation of the CHST15 and CHST11 promoters. Inhibition by SB203580, a phospho-p38 MAPK inhibitor, and by SIS3, a Smad3 inhibitor, blocked the CHST15 and CHST11 promoter activation. SB203580 reversed the SPRBD-induced decline in ARSB expression, but SIS3 had no effect on ARSB expression or promoter activation. Phospho-p38 MAPK was shown to reduce retinoblastoma protein (RB) S807/S811 phosphorylation and increase RB S249/T252 phosphorylation. E2F-DNA binding declined following exposure to SPRBD, and SB203580 reversed this effect. This indicates a mechanism by which SPRBD, phospho-p38 MAPK, E2F, and RB can regulate ARSB expression and thereby impact on chondroitin 4-sulfate and dermatan sulfate and molecules that bind to these sulfated GAGs, including Interleukin-8, bone morphogenetic protein-4, galectin-3 and SHP-2 (Src homology region 2-containing protein tyrosine phosphatase 2). Conclusion(s): The enzyme ARSB is required for the degradation of chondroitin 4-sulfate and dermatan sulfate, and accumulation of these sulfated GAGs can contribute to lung pathophysiology, as evident in Covid-19. Some effects of the SPRBD may be attributable to unopposed Angiotensin II, when Ang1-7 counter effects are diminished due to binding of ACE2 with the SARS-CoV-2 spike protein and reduced production of Ang1-7. Aberrant cell signaling and activation of the phospho-p38 MAPK and Smad3 pathways increase CHST15 and CHST11 production, which can contribute to increased chondroitin sulfate in infected cells. Decline in ARSB may occur as a consequence of effects of phospho-p38 MAPK on RB phosphorylation and E2F1 availability. Decline in ARSB and the resulting impaired degradation of sulfated GAGs have profound consequences on cellular metabolic, signaling, and transcriptional events. Funding is VA Merit Award.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Mechanism of interferon induction by cannabidiol (CBD) and analogues in lung cancer cells

CBD, an FDA approved drug for epilepsy, may have therapeutic potential for other diseases and is currently being tested for efficacy in cancer-related clinical trials. As the literature about CBD, especially in vitro reports, is often contradictory, increasing our understanding of its specific action on a molecular level will allow to determine whether CBD can become a useful therapy or exacerbates specific cancers in a context-dependent manner. Due to its relative lipophilicity, CBD is challenging to dispense at therapeutic concentrations;therefore, one goal is to identify cannabinoid congeners with greater efficacy and reduced drug delivery challenges. We recently showed that CBD activates interferons as a mechanism of inhibiting SARS-CoV-2 replication in lung carcinoma cells. As factors produced by the innate immune system, interferons have been implicated in both pro-survival and growth arrest and apoptosis signaling in cancer. Here we show that CBD induces interferon production and interferon stimulated genes (ISGs) through a mechanism involving NRF2 and MAVS in lung carcinoma cells. We also show that CBDV, which differs from CBD by 2 fewer aliphatic tail carbons, has limited potency, suggesting that CBD specifically interacts with one or more cellular proteins rather than having a non-specific effect. We also identified other CBD-related cannabinoids that are more effective at inducing ISGs. Taken together, these results characterize a novel mechanism by which CBD activates the innate immune system in lung cancer cells and identify related cannabinoids that have possible therapeutic potential in cancer treatment.

Enhancer deregulation inTET2-mutant clonal hematopoiesis is associated with increased COVID-19 severity and mortality

Background: COVID-19 causes significant morbidity and mortality, albeit with considerable heterogeneity among affected individuals. It remains unclear which host factors determine disease severity and survival. Given the propensity of clonal hematopoiesis (CH) to promote inflammation in healthy individuals, we investigated its effect on COVID-19 outcomes. Method(s): We performed a multi-omics interrogation of the genome, epigenome, transcriptome, and proteome of peripheral blood mononuclear cells from COVID-19 patients (n=227). We obtained clinical data, laboratory studies, and survival outcomes. We determined CH status and TET2-related DNA methylation. We performed single-cell proteogenomics to understand clonal composition in relation to cell phenotype. We interrogated single-cell gene expression in isolation and in conjunction with DNA accessibility. We integrated these multi-omics data to understand the effect of CH on clonal composition, gene expression, methylation of cis-regulatory elements, and lineage commitment in COVID-19 patients. We performed shRNA knockdowns to validate the effect of one candidate transcription factor in myeloid cell lines. Result(s): The presence of CH was strongly associated with COVID-19 severity and all-cause mortality, independent of age (HR 3.48, 95% CI 1.45-8.36, p=0.005). Differential methylation of promoters and enhancers was prevalent in TET2-mutant, but not DNMT3A-mutant CH. TET2- mutant CH was associated with enhanced classical/intermediate monocytosis and single-cell proteogenomics confirmed an enrichment of TET2 mutations in these cell types. We identified celltype specific gene expression changes associated with TET2 mutations in 102,072 single cells (n=34). Single-cell RNA-seq confirmed the skewing of hematopoiesis towards classical and intermediate monocytes and demonstrated the downregulation of EGR1 (a transcription factor important for monocyte differentiation) along with up-regulation of the lncRNA MALAT1 in monocytes. Combined scRNA-/scATAC-seq in 43,160 single cells (n=18) confirmed the skewing of hematopoiesis and up-regulation of MALAT1 in monocytes along with decreased accessibility of EGR1 motifs in known cis-regulatory elements. Using myeloid cell lines for functional validation, shRNA knockdowns of EGR1 confirmed the up-regulation of MALAT1 (in comparison to wildtype controls). Conclusion(s): CH is an independent prognostic factor in COVID-19 and skews hematopoiesis towards monocytosis. TET2-mutant CH is characterized by differential methylation and accessibility of enhancers binding myeloid transcriptions factors including EGR1. The ensuing loss of EGR1 expression in monocytes causes MALAT1 overexpression, a factor known to promote monocyte differentiation and inflammation. These data provide a mechanistic insight to the adverse prognostic impact of CH in COVID-19.

The Effect of FOXP3+Regulatory T Cells on Infectious and Inflammatory Diseases

CD4+CD25+FOXP3+regulatory T cells (Tregs) contribute to the maintenance of immune homeostasis and tolerance in the body. The expression levels and functional stability of FOXP3 control the function and plasticity of Tregs. Tregs critically impact infectious diseases, especially by regulating the threshold of immune responses to pathogenic microorganisms. The functional regulatory mechanism and cell-specific surface markers of Tregs in different tissues and inflammatory microenvironments have been investigated in depth, which can provide novel ideas and strategies for immunotherapies targeting infectious diseases.Copyright © 2021. All rights reserved.

NEI 2022 Abstracts

The proceedings contain 96 papers. The topics discussed include: practical pharmacotherapy for opioid use disorder in the age of fentanyl;can COVID-19 cause acute psychosis in pediatric patients? a case report;a survey of bullying experiences in a child and adolescent psychiatric clinic population;acute emergence of suicidal thoughts following Lemborexant initiation: an adverse reaction case report;assessing the unmet clinical need and opportunity for digital therapeutic intervention in schizophrenia: perspective from people with schizophrenia;rapid antidepressant effects and MADRS item improvements with AXS-05 (DEXTROMETHORPHAN-BUPROPION), an oral NMDA receptor antagonist in major depressive disorder: results from two randomized double-blind, controlled trials;targeting lncRNA NEAT1 impedes Alzheimers disease progression via MicroRNA-193a mediated CREB/BDNF and NRF2/NQO1 pathways;and impact of AXS-05 (DEXTROMETHORPHAN-BUPROPION), an Oral NMDA receptor antagonist, on Anhedonic symptoms in major depressive disorder.

Single-Cell Analysis Reveals Characteristics of Myeloid Cells in Pulmonary Pasc

Background: Although our understanding of immunopathology in the risk and severity of COVID-19 disease is evolving, a detail of immune response in long-term consequences of COVID-19 infection remains unclear. Recently, few studies have detailed the immune and cytokine profiles associated with PASC. However, dysregulation of immune system driving pulmonary PASC is still largely unknown. Method(s): To characterize the immunological features of PPASC, we performed droplet-based scRNA-sequencing using 10X genomics to study the transcriptomic profiles of peripheral blood mononuclear cells (PBMCs) from participants naive to SARS-CoV-2 (NP, n=2) and infected with SARS-CoV-2 with chronic pulmonary symptoms (PPASC, n=2). Result(s): Analysis of more than 34,000 PBMCs by integrating our dataset with previously reported control datasets generated cell distribution and identified 11 immune cell types based on canonical gene expression. The proportion of myeloid-lineage cells (CD14+monocyte, CD16+monocyte, and dendritic cells) and platelets were increased in PPASC compared with those of NP. Specifically, PPASC displayed up-regulation of VEGFA and transcription factors, such as ATF2, ELK, and SMAD in myeloid-lineage cells. Also, TGF-beta and WNT signaling pathways were up-regulated in these cell population. Cell-cell interaction analysis identified that myeloid-lineage cells in PPASC participated in regulation of fibrosis and immune response, such as VEGFA (increased) and MIF (decreased) interactions. Conclusion(s): Together, this study provides high-resolution insights into immune landscape in PPASC. Our results emphasize differences in myeloid lineage-mediated fibrosis and immunity between PPASC and NP, suggesting they could act as potential pathological drivers of PPASC. (Figure Presented).

Rising level of pulmonary dioxin might trigger CMV and HSV-1 reactivation via bHLH/PAS/AhR:ARNT transcription pathway in lungs of immunocompetent COVID-19 patients with ARDS

Study objective. It has been shown that human common viruses are new target genes for host cell dioxin receptor transcriptional (AhR-ARNT) complex initially proven to up-regulate mammalian genes containing dioxin-response elements (DRE) in the promoters [doi:10.1016/j.ijid.2012.05.265]. Initially, transactivation of HIV-1 and HBV by 2,3,7,8-tetrachlodibenzop- dioxin (TCDD) at low nanomolar range was demonstrated [doi:10.3109/00498259309057034]. Noteworthy, transactivation of human cytomegalovirus (CMV) was shown with 0.3 ppt dioxin, i.e. lower than its current background level in the general population (~3.0 ppt). Recently, reactivation of CMV infection was found to influence worse clinical outcome following SARS-CoV-2 infection (doi: 10.1186/s12979-020- 00185-x). Other findings showed that CMV and herpes simplex virus 1 (HSV-1) reactivation were observed in immunocompetent patients with COVID-19 acute respiratory distress syndrome (ARDS) (doi.org/10.1186/s13054-020-03252-3). Addressing occurrence of Herpesviridae reactivation in severe COVID-19 patients, and still unspecified real triggers of CMV and HSV-1 reactivations, we tested TCDD, which current body burden (DBB) ranges from 20 pg/g (TEQ in fat) in general population to 100 pg/g in older people. Methods. In Silico quantitation of active DRE in promoters of viral genes. Virus DNA hybridization assay. Clinical and epidemiological analyses. Results and Discussion. In this study, a computational search for DRE in CMV and HSV-1 genes was performed by SITECON, a tool recognizing potentially active transcriptional factor binding sites. In silico analysis revealed in regulatory region of CMV IE genes from 5 to 10 DRE, and from 6 to 8 DRE in regulatory region of HSV-1 IE genes.We established that a low picomolar TCDD can trigger up-regulation of CMV and HSV-1 genes via AhR:Arnt transcription factor in macrophage(doi.org/10.1016/ j.ijid.2012.05.265) and glial human cell lines (doi.org/10.1016/j. jalz.2016.06.1268), respectively. In fact, viral reactivation may be triggered in COVID-19 ARDS patients by higher pulmonary TCDD concentrations, because "lipid storm" within lungs of severe COVID-19 patients has been recently reported (doi.org/ 10.1101/2020.12.04.20242115). TCDD is known as the most potent xenobiotic, which bioaccumulates and has estimation half-life in humans of up to 10 yr. Due to hydrophobic character (Log P octanol/water: 7.05), TCDD partitions into inflammatory lipids in lung tissue thus augmenting its local concentration. Population-based epidemiological data on SARS-CoV-2 first wave of pandemic revealed high level of CMV seropositivity and cumulative mortality rate 4.5 times in Lombardi region of Italy, where after Seveso industrial accident TCDD plasma level in pre-exposed subjects is 15 times the level in rest of Italy (doi. org/10.3389/fpubh.2020.620416). Also, Arctic Native (AN) peoples consume dioxin-contaminated fat in seafood and have TCDD DBB, i.e. 7 times that in general population. To the point of this paper, their COVID-19 mortality is 2.2 times of that among non-AN Alaskans (doi: 10.15585/mmwr.mm6949a3). Conclusion(s): TCDD in the picomolar range may trigger CMV expression in lung cells and commit virus to the lytic cycle, which can be applied to reactivation of Herpesviridae infection in immunocompetent patients with COVID-19 ARDS syndrome.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

A Literature Review on the Antiviral Mechanism of Luteolin

Background: Viral infections pose some of the most serious human health concerns worldwide. The infections caused by several viruses, including coronavirus, hepatitis virus, and human immunodeficiency virus, are difficult to treat. Method(s): This review details the findings of a literature search performed on the antiviral properties of luteolin. The keywords engaged in the search are "virus" along with "luteolin." Results: Luteolin possesses antiviral properties, which is the basis for the current review. It is an important natural flavonoid with numerous important biological properties, including anti-inflammatory, immune regulatory, and antitumor effects, and is found in vegetables, fruits, and several medicinal plants. Recent studies have revealed that many traditional Chinese medicines that contain luteolin inhibit the replication of coronaviruses. Conclusion(s): Luteolin effectively inhibits the replication of coronavirus, influenza virus, enterovirus, rotavirus, herpes virus, and respiratory syncytial virus, among others. In particular, it prevents viral infection by improving the body's nonspecific immunity and antioxidation capacity and inhibiting many pathways related to virus infection and replication, such as MAPK, PI3K-AKT, TLR4/8, NF-kappaB, Nrf-2/hemeoxygenase-1, and others. It also regulates the expression of some receptors and factors, including hepatocyte nuclear factor 4alpha, p53, NLRP3, TNF-alpha, and interleukins, thereby interfering with the replication of viruses in cells. Luteolin also promotes the repair of damaged cells induced by proinflammatory factors by regulating the expression of inflammatory molecules. The overall effect of these processes is the reduction in viral replication and, consequently, the viral load. This review summarizes the antiviral effect of luteolin and the mechanism underlying this property.Copyright © The Author(s) 2023.

CO-SPROUT: A PILOT DOUBLE-BLINDED RANDOMISED CONTROL TRIAL of BROCCOLI SPROUT for PREGNANT WOMEN with COVID-19: STUDY PROTOCOL

Background: COVID-19 is caused by SARS-CoV-2 and has is responsible for over 619 million infections and over 6.5 million deaths globally since identification in 2019. Infection during pregnancy is associated with increased adversity including increased risks of admission to intensive care, increased ventilatory support, preeclampsia, preterm birth and maternal death. Vaccination remains the best protection against severe disease. The majority of trials for novel or repurposed COVID-19 therapies including mRNA vaccinations have excluded pregnant or lactating women despite being an at-risk population. Broccoli sprout extract contains a naturally occurring phytonutrient sulforaphane which upregulates the Nrf2 transcription factor resulting in expression of antioxidant proteins, anti-inflammatory effects and has demonstrated anti-viral effects in-vitro . Severe COVID-19 results in excessive cytokine production resulting in a proinflammatory state with significant oxidative stress and multi-organ dysfunction with evidence of placental abnormalities in almost half of infected mothers. Method(s): CO-Sprout is a pilot, double blinded, placebo controlled randomised trial that is recruiting pregnant women ( n = 60) between 20 and 36 weeks completed gestation with COVID-19 diagnosed within 5 days. Participants are randomised to either broccoli sprout capsules (containing 21 mg sulforaphane) or identical placebo (microcrystalline cellulose) twice daily for 14 days. The primary outcome will be duration (days) of COVID-19 related symptoms and other exploratory outcomes including unplanned hospital admissions, birth outcomes, inflammatory markers, microbiome and placental changes. Patients are recruited through maternity departments at Monash Health and Jessie McPherson Private Hospital. Result(s): Trial in progress. Conclusion(s): Trial results to be published after trial completion.

RNA-sequencing unravels unique host-response to infection with SARS-CoV-2

Introduction/Aim: As the causative agent of COVID-19, SARS-CoV-2 remains a global cause for concern. Compared to other highly pathogenic coronaviruses (SARS-CoV and MERS-CoV), SARS-CoV-2 exhibits stronger transmissibility but less lethality, indicating that SARS-CoV-2 displays unique characteristics, despite the partial genomic proximity. Thus, we aim to employ RNA sequencing to define transcriptional differences in epithelial responses following infection with SARS-CoV-2 compared to pathogenic SARS-CoV and MERS-CoV, and low pathogenic HCoV-229E. Method(s): Primary human bronchial epithelial cells (PBEC) were differentiated for 6 weeks at the air-liquid interface (ALI) before parallel infection by the 4 different coronaviruses (n = 4). After infection following apical application of coronaviruses at low dose (MOI 0.1), cells were harvested for bulk RNA sequencing. Gene were considered significant with a fold change (FC) > 2 and false discovery rate of FDR < 0.05. Inhibitor experiments were conducted on CALU-3 cells using DIM-C-pPhOH 10 muM (NR4A1 antagonist), Sp600125 10 muM (JNK inhibitor), T-5224 10 muM (AP-1 transcription factor inhibitor) and Cytosporone B (CsB 5 muM;NR4A1 agonist) preincubated for 1 h with these compounds and subsequently infected with SARS-CoV-2 or MERS-CoV (MOI of 1). Samples were collect 24 h later for PCR. Result(s): PCR and RNA-Seq demonstrated that all tested coronaviruses efficiently infected ALI-PBEC and replicated over 72 h (p < 0.05). RNA sequencing analysis revealed that infection with SARS-CoV, MERS-CoV and HCoV-229E resulted in largely similar transcriptional responses by the epithelial cells. However, whereas infection with these viruses was accompanied by an increased expression of genes associated with JNK/AP-1 signalling, including FOS, FOSB and NR4A1 (FC > 1, FDR < 0.05), no such increase was observed following SARS-CoV-2 infection. Further, we found that an NR4A1 antagonist reduced viral replication of MERS and SARs-CoV-2 100-fold in Calu-3 cells. Conclusion(s): In conclusion, these data suggest that SARS-CoV-2-infected ALI-PBEC exhibit a unique transcriptional response compared to other coronaviruses, which might relate to the pathogenicity of the virus.

Extracellular vesicles as delivery vectors to enhance killing of bacteria and viruses by airway-recruited human neutrophils

Background: Polymorphonuclear neutrophils (PMNs) recruited to the airway lumen in cystic fibrosis (CF) undergo a rapid transcriptional program, resulting in exocytosis of granules and inhibition of bacterial killing. As a result, chronic infection, feed-forward inflammation, and structural tissue damage occur. Because CF airway PMNs are also highly pinocytic, we hypothesized that we could deliver protein- and ribonucleic acid (RNA)-based therapies to modulate their function to benefit patients. We elected to use extracellular vesicles (EVs) as a delivery vector because they are highly customizable, and airway PMNs have previously been shown by our group to process and use their cargo efficiently [1]. Furthermore, our prior work on CF airway PMNs [2] led to identification of the long noncoding RNA MALAT1, the transcription factor Ehf, and the histone deacetylase/long-chain fatty deacylase HDAC11 as potential targets to modulate CF airway PMN dysfunction. Method(s): H441 human club epithelial cells were chosen for EV production because they efficiently communicate with lung-recruited primary human PMNs [1]. Relevant constructs were cloned into an expression plasmid downstream of a constitutive cytomegalovirus or U6 promoter with an additional puromycin selection cassette. EVs were generated in serumdepleted media and purified by differential centrifugation. Quality and concentration of EVs was determined by electron microscopy and nanoparticle tracking analysis and cargo content by western blot (protein) or qualitative reverse transcription polymerase chain reaction (RNA). Enhanced green fluorescent protein and messenger ribonucleic acid (mRNA) were used as controls. To test delivery to primary human PMNs, generated EVs were applied in the apical fluid of an airway transmigration model [2]. PMN activation was assessed by flow cytometry, and bacterial (PA01 and Staphylococcus aureus 8325-4) killing and viral (influenza Avirus [IAV] H1N1/PR/8/34;SARS-CoV-2/Washington) clearance assays were conducted. Result(s): To package protein, we used EV-loading motifs such as the tetraspanin CD63, Basp1 amino acids 1-9, and the palmitoylation signal of Lyn kinase. To load mRNA, a C'D box motif recognized by the RNA-binding protein L7Ae was included in the 3' untranslated region of the expressed RNA, and CD63-L7Ae was co-expressed. Airway-recruited PMNs treated with EVs containing small interfering RNAs against MALAT1 or HDAC11 showed greater ability to clear bacteria. Conversely, PMNs treated with constructs encasing MALAT1 or HDAC11 efficiently cleared IAV and SARSCoV- 2. PMNs expressing Ehf showed greater clearance of bacteria and viruses. Conclusion(s): Our findings suggest mutually exclusive roles of MALAT-1 and HDAC11 in regulating bacterial and viral clearance by airway-recruited PMNs. Expression of Ehf in airway PMNs may be a pathogen-agnostic approach to enhancing clearance by airway-recruited PMNs. Overall, our study brings proof-of-concept data for therapeutic RNA/protein transfer to airway-recruited PMNs in CF and other lung diseases and for use of EVs as a promising method for cargo delivery to these cells. It is our expectation that, by treating the immune compartment of CF airway disease, pathogentherapies, such as antibiotics will be more effective, and epithelial-targeted therapies, such as CFTR modulators, will have greater penetrance into the cell types of interest.Copyright © 2022, European Cystic Fibrosis Society. All rights reserved

Post-Transplantation Cyclophosphamide for the Prevention of Graft-Versus-Host Disease Should Not Limit Transplantation for Acute Lymphoblastic Leukemia in People with Trisomy 21

Introduction: Use of hematopoietic cell transplantation (HCT) in patients with trisomy 21 (+21) is infrequent given concerns about increased toxicity with cytotoxic chemotherapy.1 Due to increasing evidence of benefit from post-HCT cyclophosphamide (PTCy) for graft-vs.-host disease (GVHD) prophylaxis and lack of prior descriptions in patients with +21,2-4 we report on 2 patients with +21 and acute lymphoblastic leukemia (ALL) who underwent HCT with PTCy. Method(s): Retrospective data were collected from 2 patients with ALL and +21 who underwent allogeneic HCT with PTCybased GVHD prophylaxis from 2019 to 2021. Data collected included age, disease risk, HCT-CI, GVHD incidence, and survival. Result(s): Patient 1 is a 22-year-old male and patient 2 a 25-year-old female. Both had Ph-negative, B-cell ALL. Patient 1 had ETV6/RUNX1 rearrangement, del 12p, gain of X, and he had recurrence of measurable residual disease (MRD) after initial MRD-negative CR with two lines of therapy pre-HCT. Patient 2 had normal cytogenetics and relapsed disease with 4 prior lines of therapy. Both achieved MRD-negativity pre-HCT. Both received fludarabine and melphalan conditioning, and patient 1 also received thiotepa 2.5 mg/kg. PTCy was given on days +3 and 4 at 50 mg/kg with sirolimus and tacrolimus for GVHD prophylaxis. Patient 1 had a haploidentical donor and received one dose of rabbit ATG (1 mg/kg) on day +5. Patient 2 had a matched unrelated donor. There was no significant delay in engraftment of ANC (day 16-19) or platelets (day 15-16). Patient 2 developed acute GVHD at day 30 (stage I skin, stage II GI) that resolved with steroids which were tapered off by day 96 without recurrence. Sirolimus stopped at day 79 (pt 1) and 103 (pt 2) and tacrolimus was stopped at day 274 (pt 1) and 469 (pt 2). Patient 1 developed a sirolimus-induced pericardial effusion at day 84 which did not recur after sirolimus discontinuation. Patient 2 developed moyamoya 8 months post-HCT during tacrolimus taper without other GVHD symptoms. Response to steroids was noted, so tacrolimus was restarted for residual neurological deficit. Neither patient developed chronic GVHD or left ventricular ejection fraction decline, and neither patient had disease relapse at follow-up of 30 and 16 months respectively. Patient 2 developed COVID pneumonia 16 months post-HCT and died while in CR. Patient 1 remains alive, in CR, and off immunosuppression nearly 3 years post HCT. Conclusion(s): Allogeneic HCT with PTCy at standard doses did not appear prohibitively toxic in patients with +21 when administered after reduced-intensity conditioning. In this case series, GVHD rates seemed consistent with larger series in patients without +21. Moyamoya development is associated with autoimmunity in patients with +21 and hence may have been GVHD-related5. Trisomy 21 should not be a barrier to patients otherwise eligible for HCT, even with PTCy prophylaxis.Copyright © 2023 American Society for Transplantation and Cellular Therapy

Immunologic Profile of Severe COVID-19 Patients in Alborz Province, Iran

Background: The coronavirus disease 2019 (COVID-19) pandemic has prompted researchers to look for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenicity in depth. Immune system dysregulation was one of the major mechanisms in its pathogenesis. The evidence regarding the levels of interferons (IFNs) and pro-and anti-inflammatory cytokines in COVID-19 patients is not well-established. Objective(s): Therefore, this study evaluated the expression level of type-I, II, III IFNs, along with interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-10 (IL-10), and FOXP3 genes in patients with severe COVID-19 to provide additional insights regarding the regulation of these cytokines during COVID-19 infection. Method(s): Peripheral blood mononuclear cells were isolated from two groups, including severe COVID-19 patients and healthy con-trols. Ribonucleic acid was extracted to evaluate the expression level of IFN-a, IFN-b, IFN-g, IFN-la, IL-1, IL-6, IL-10, and FOXP3 genes using real-time polymerase chain reaction. The correlations between the expression levels of these genes were also assessed. Result(s): A total of 40 samples were divided into two groups, with each group consisting of 20 samples. When comparing the severe COVID-19 group to the controls, the expression levels of IFN-g, tumor necrosis factor-alpha (TNF-alpha), IL-6, and IL-10 genes were sig-nificantly higher in the severe COVID-19 group. The two groups had no significant differences in IFN-a, IFN-b, IFN-la, IL-1, and FOXP3 expression. The correlation analysis revealed a negative correlation between type I and type III IFNs (i.e., IFN-a and IFN-la) and pro-inflammatory cytokines (i.e., IL-1 and IL-10). Conclusion(s): This study suggests the possible upregulation of IFN-g, IL-6, IL-10, and TNF-alpha during SARS-CoV-2 pathogenicity. The pre-liminary findings of this study and those reported previously show that the levels of IFNs and pro-and anti-inflammatory cytokines are not uniformly expressed among all COVID-19 patients and might differ as the disease progresses to the severe stage.Copyright © 2023, Author(s).

Transcriptome in Human Mycoses

Mycoses are infectious diseases caused by fungi, which incidence has increased in recent decades due to the increasing number of immunocompromised patients and improved diagnostic tests. As eukaryotes, fungi share many similarities with human cells, making it difficult to design drugs without side effects. Commercially available drugs act on a limited number of targets and have been reported fungal resistance to commonly used antifungal drugs. Therefore, elucidating the pathogenesis of fungal infections, the fungal strategies to overcome the hostile environment of the host, and the action of antifungal drugs is essential for developing new therapeutic approaches and diagnostic tests. Large-scale transcriptional analyses using microarrays and RNA sequencing (RNA-seq), combined with improvements in molecular biology techniques, have improved the study of fungal pathogenicity. Such techniques have provided insights into the infective process by identifying molecular strategies used by the host and pathogen during the course of human mycoses. This chapter will explore the latest discoveries regarding the transcriptome of major human fungal pathogens. Further we will highlight genes essential for host–pathogen interactions, immune response, invasion, infection, antifungal drug response, and resistance. Finally, we will discuss their importance to the discovery of new molecular targets for antifungal drugs. © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2014, 2022.

The possibilities of using the effects of ozone therapy in neurology

OBJECTIVES: The beneficial effects of ozone therapy consist mainly of the promotion of blood circulation: peripheral and central ischemia, immunomodulatory effect, energy boost, regenerative and reparative properties, and correction of chronic oxidative stress. Ozone therapy increases interest in new neuroprotective strategies that may represent therapeutic targets for minimizing the effects of oxidative stress. METHOD(S): The overview examines the latest literature in neurological pathologies treated with ozone therapy as well as our own experience with ozone therapy. The effectiveness of treatments is connected to the ability of ozone therapy to reactivate the antioxidant system to address oxidative stress for chronic neurodegenerative diseases, strokes, and other pathologies. Application options include large and small autohemotherapy, intramuscular application, intra-articular, intradiscal, paravertebral and epidural, non-invasive rectal, transdermal, mucosal, or ozonated oils and ointments. The combination of different types of ozone therapy stimulates the benefits of the effects of ozone. RESULT(S): Clinical studies on O2-O3 therapy have been shown to be efficient in the treatment of neurological degenerative disorders, multiple sclerosis, cardiovascular, peripheral vascular, orthopedic, gastrointestinal and genitourinary pathologies, fibromyalgia, skin diseases/wound healing, diabetes/ulcers, infectious diseases, and lung diseases, including the pandemic disease caused by the COVID-19 coronavirus. CONCLUSION(S): Ozone therapy is a relatively fast administration of ozone gas. When the correct dose is administered, no side effects occur. Further clinical and experimental studies will be needed to determine the optimal administration schedule and to evaluate the combination of ozone therapy with other therapies to increase the effectiveness of treatment.Copyright © 2021 Neuroendocrinology Letters.

Functional Role of Natural Antioxidants in Controlling Oxidative Stress Associated with SARS-CoV-2 Infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a pathogenic coronavirus that emerged in late 2019, resulting in coronavirus disease (COVID-19). COVID-19 can be potentially fatal among a certain group of patients. Older age and underlying medical illness are the major risk factors for COVID-19-related fatal respiratory dysfunction. The reason for the pathogenicity of COVID-19 in the older age group remains unclear. Factors, such as coagulopathy, cytokine storm, metabolic disrup-tion, and impaired T cell function, may worsen the symptoms of the disease. Recent literature has indicat-ed that viral infections are particularly associated with a high degree of oxidative stress and an imbalance of antioxidant response. Although pharmacological management has taken its place in reducing the severity of COVID-19, the antioxidants can serve as an adjunct therapy to protect an individual from oxidative damage triggered by SARS-CoV-2 infection. In general, antioxidant enzymes counteract free radicals and prevent their formation. The exact functional role of antioxidant supplements in reducing disease symptoms of SARS-CoV-2 infection remains mostly unknown. In this review, the functional role of natural antioxidants in SARS-CoV-2 infection management is discussed in brief.Copyright © 2022 Bentham Science Publishers.

Impact of the NRF2 activator, stabilised synthetic sulforaphane, on systemic inflammation in COVID19: results from the STAR-COVID19 trial

Introduction: Dysregulated immune responses are implicated in the pathogenesis of severe COVID19 and may be modulated by the transcription factor Nrf2. Hypothesis: Treatment with stabilised, synthetic sulforaphane (S-SFN)-an Nrf2 inducer-improves clinical status in hospitalised patients with suspected COVID19 pneumonia by curbing the inflammatory response. Method(s): Double-blind RCT of S-SFN (300mg, once daily, 14 days;EudraCT 2020-003486-19) in patients hospitalised with confirmed or suspected COVID19, in Dundee, UK. The primary outcome was the 7 point WHO Clinical Status scale at day 15. Blood samples were taken on days 1, 8 and 15 for measurement of 45 serum cytokines using the Olink Target48 panel. Key neutrophil functions were assessed including migration, phagocytosis and bacterial killing. Result(s): 133 participants were randomized (placebo n=68, S-SFN n=65) from Nov 2020 to May 2021. S-SFN treatment did not improve clinical status at day 15 (adjusted OR 0.87 95%CI 0.41-1.83). In serum, Nrf2 target TGFalpha was significantly increased at day 15 in those receiving S-SFN treatment compared with placebo (p=0.004;linear mixed effects model). Other targets implicated in cytokine storm, including IL6, IL1beta and TNFalpha, were unchanged. Patients receiving Tocilizumab (n=20) were excluded from exploratory analyses due to a strong impact upon IL6 levels, leading to significant increases at day 8 across the study population (p=0.015). S-SFN treatment did not significantly affect neutrophil function. Conclusion(s): S-SFN treatment modulated select Nrf2 targets but did not modulate key cytokines. Further analyses to delineate drug activity are ongoing.

Differentially expressed FOXO1, NFE2L2 and NFKB1 mRNAs are associated with differentially regulated corona- and influenza viral receptor genes and Toll-like receptor pathway genes in human bronchial epithelial cells under hyperbaric oxygen exposure

As hyperbaric oxygen (HBO) has been shown to mitigate the COVID-19 symptoms, we were interested in studying whether HBO exposure affects expression of viral entry genes and innate immune genes in the air-liquid interface (ALI)-cultured human bronchial epithelial cells (HBECs) derived from normal individuals (NHBECs) and age-matched COPD patients (DHBECs), which were cultured under normoxia or daily exposure of 40-min hyperbaric oxygen (HBO) with 100% O2 at 2.5 ATA for 28 days in total. We found for the first time that HBO exposure differentially regulated mucociliary differentiation of HBECs by respectively decreasing and increasing expression of CGRP, MUC5AC, FOXJ1, NOTCH3 and HEYL in NHBECs and DHBECs, and respectively decreased and increased FOXO1 expression whereas increased and decreased NFE2L2 and NFKB1 expression in NHBECs and DHBECs, in association with respectively decreased and increased expression the SARS-CoV-2 entry genes ACE2 and 2 TMPRSS2 and the tight junction protein genes TJP1 and TJP2, and in association with respectively increased and decreased expression of the cell growth and inflammatory transcription factors SRF, c-FOS and TP63, as well as the TLR pathway genes TLR3, AKT1, IL-1B, IL-5, IL-6, IL-33, IRAK4 and NFKBIA in NHBECs and DHBECs. (Figure Presented).

Small airway function after Covid-19 infection

Introduction: Remaining respiratory symptoms is common after covid-19. The pathophysiology behind this is unclear, with spirometry often within the normal range. We hypothesised that impairment in the small airways can be an explanation. Aim(s): To investigate if the function of the small airways is impaired after covid-19. Method(s): 28 (18 females) post covid-19 subjects aged 27-63 yrs performed spirometry, DLCO, nitrogen (N ) multiple breath washout (MBW) and impulse oscillometry (IOS). Three out of 28 were hospitalized due to covid-19. Median (IQR) days between first symptom and participation was 216 (108;372). Results were compared to findings in 10 (7 females) non-covid-19 subjects, aged 29-63 yrs, and reported in z-score (z) related to GLI reference equations for spirometry and DLCO, while two healthy cohorts (n=400 and n=158) provided local reference values for N MBW and IOS, respectively. Result(s): IOS derived frequency dependence of resistance (FDR) and MBW derived Sacin were the only outcomes significantly different in post covid-19 subjects, median (IQR), 0.01 kPa/L/s (0.00;0.02) vs 0.04 kPa/L/s (0.01;0.06), p=0.028 for FDR and 0.070 (0.046;0.076) vs 0.009 (0.075;0.121), p=0.017 for Sacin. FDR was >1.96 z in 2/28 whileS >1.96 z was found in 14/28. In post covid-19 subjects with S >1.96 z, dyspnea were more common than in those with normal Sacin, (8/14 vs 2/14, p=0.049). FEV1 and DLCO were > -1.96 z in all but one subject. Conclusion(s): Ventilation heterogeneity at the entrance to the acinar airways were common in this small sample of post covid-19 subjects, as assessed by Sacin, and may explain the experience of dyspnea in these subjects, despite normal spirometry and DLCO. A larger study is ongoing.