Upskilling haematological services for WGS and germline assessments through a regional skin biopsy training programme

NHS England Genomics introduced whole genome sequencing (WGS) with standard-of- care (SoC) genetic testing for haemato-oncology patients who meet eligibility criteria, including patients with acute leukaemia across all ages, and exhausted SoC testing. Alongside, the role of germline mutations in haematological cancers is becoming increasingly recognised. DNA samples are required from the malignant cells (somatic sample) via a bone marrow aspirate, and from non-malignant cells (germline sample) for comparator analysis. Skin biopsy is considered the gold-standard tissue to provide a source of fibroblast DNA for germline analysis. Performing skin punch biopsies is not within the traditional skillset for haematology teams and upskilling is necessary to deliver WGS/germline testing safely, independently and sustainably. A teaching programme was designed and piloted by the dermatology and haematology teams in Sheffield and delivered throughout the NHS trusts in North East & Yorkshire Genomic Laboratory Hub. The training programme consisted of a 90-min session, slides, video and practical biopsy on pork belly or synthetic skin, designed to teach up to six students at one time. To disseminate best practice, the standard operating procedure and patient information used routinely in Sheffield were shared, to be adapted for local service delivery. From January 2021 to December 2022, 136 haematology staff from 11 hospitals, including 34 consultants, 41 registrars, 34 nurses and 8 physician associates, across the NEY GLH region completed the skin biopsy training programme. Feedback from the course was outstanding, with consistently high scores in all categories. Practical components of the course were especially valued;98.6% (71/72) trainees scored the practical element of the programme a top score of 5 out of 5, highlighting that despite the challenges of delivering face-to- face teaching due to COVID-19, teaching of practical skills was highly valued;training in this way could not have been replicated virtually. Costs of the programme have been approximately 16 000, including consultant input and teaching/educational materials. Recent support has been provided by a separately funded Genomic Nurse Practitioner (GNP), with succession planning for the GNP to take over leadership from the consultant dermatologist. Plans are in place to use the remaining budget to disseminate the programme nationally. Our training programme has shown that skin biopsy can be formally embedded into training for haematology consultants, trainees, nursing team, and physician associates. Delivery of training can be effective and affordable across regional GLHs with appropriate leadership and inter-speciality coordination, and ultimately sustainable with specialist nursing staff, including GNPs.

Inherited deficiency of the OAS-RNase L pathway in children with MIS-C

Multisystem inflammatory syndrome in children (MIS-C) is a rare and severe condition that follows benign COVID-19. We report autosomal recessive deficiencies of OAS1, OAS2, or RNASEL in five unrelated children with MIS-C. The cytosolic dsRNA-sensing OAS1 and OAS2 generate 2'-5'-linked oligoadenylates (2-5A) that activate the ssRNA-degrading RNase L. Consistent with the absence of pneumonia in these patients, epithelial cells and fibroblasts defective for this pathway restricted SARS-CoV-2 normally. This contrasted with IFNAR1-deficient cells from patients prone to hypoxemic pneumonia without MIS-C. Monocytic cell lines and primary myeloid cells with OAS1, OAS2, or RNASEL deficiencies produce excessive amounts of inflammatory cytokines upon dsRNA or SARS-CoV- 2 stimulation. Exogenous 2-5A suppresses cytokine production in OAS1-but not RNase L- deficient cells. Cytokine production in RNase L-deficient cells is impaired by MDA5 or RIG-I deficiency and abolished by MAVS deficiency. Recessive OAS-RNase L deficiencies in these patients unleash the production of SARS-CoV-2-triggered, MAVS-mediated inflammatory cytokines by mononuclear phagocytes, thereby underlying MIS-C.Copyright © 2023 Elsevier Inc.

Pericytes may facilitate SARS-CoV-2 entry into the nervous system

Objective: Although the neurological and olfactory symptoms of coronavirus disease 2019 have been identified, the neurotropic properties of the causative virus, severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2), remain unknown. We sought to identify the susceptible cell types and potential routes of SARS-CoV-2 entry into the central nervous system, olfactory system, and respiratory system. Method(s): We collected single-cell RNA data from normal brain and nasal epithelium specimens, along with bronchial, tracheal, and lung specimens in public datasets. The susceptible cell types that express SARS-CoV-2 entry genes were identified using single-cell RNA sequencing and the expression of the key genes at protein levels was verified by immunohistochemistry. We compared the coexpression patterns of the entry receptor angiotensin-converting enzyme 2 (ACE2) and the spike protein priming enzyme transmembrane serine protease (TMPRSS)/cathepsin L among the specimens. Result(s): The SARS-CoV-2 entry receptor ACE2 and the spike protein priming enzyme TMPRSS/cathepsin L were coexpressed by pericytes in brain tissue;this coexpression was confirmed by immunohistochemistry. In the nasal epithelium, ciliated cells and sustentacular cells exhibited strong coexpression of ACE2 and TMPRSS. Neurons and glia in the brain and nasal epithelium did not exhibit coexpression of ACE2 and TMPRSS. However, coexpression was present in ciliated cells, vascular smooth muscle cells, and fibroblasts in tracheal tissue;ciliated cells and goblet cells in bronchial tissue;and alveolar epithelium type 1 cells, AT2 cells, and ciliated cells in lung tissue. Conclusion(s): Neurological symptoms in patients with coronavirus disease 2019 could be associated with SARS-CoV-2 invasion across the blood-brain barrier via pericytes. Additionally, SARS-CoV-2-induced olfactory disorders could be the result of localized cell damage in the nasal epithelium.Copyright © Wolters Kluwer Health, Inc. All rights reserved.

Exosomes/EVs: BIOXOME: A NATURAL LIPOSOME MIMETIC AS DRUG DELIVERY SYSTEM

Background & Aim: Liposomes are spherical-shaped vesicles composed of one or more lipid bilayers. The ability of liposomes to encapsulate hydro- or lipophilic drugs allowed these vesicles to become a useful drug delivery system. Natural cell membranes, such as Bioxome, have newly emerged as new source of materials for molecular delivery systems. Bioxome are biocompatible and GMP-compliant liposome-like membrane that can be produced from more than 200 cell types. Bioxome self-assemble, with in-process self-loading capacity and can be loaded with a variety of therapeutic compounds. Once close to the target tissue, Bioxome naturally fuse with the cell membrane and release the inner compound. Orgenesis is interested in evaluating the potential of Bioxome as new drug delivery system for treatment of several diseases, including skin repair, local tumour or COVID19. Methods, Results & Conclusion(s): Bioxome were obtained from adipose- derived Mesenchymal Stem Cells, with a process of organic- solvent lipid extraction, followed by lyophilization and sonication assemblage. During the sonication process, Bioxome were charged or not with several cargos. Size distribution of empty Bioxome was detected by Particle Size Analyzer (NanoSight). Electron Microscopy (EM) was performed to assess Bioxome morphology. Lipid content was evaluated by electrospray ionization system. Dose response in vitro test on human lung fibroblasts treated or not with Bioxome encapsulating a specific cargo (API) against COVID19 were performed. NanoSight analysis showed that nanoparticle size in Bioxome samples ranged between 170+/-50 nm, with a concentration ranging between 109-1010+/-106 particles/mL. EM clearly showed the double phospholipid layers that composes the Bioxome. Stability study demonstrated that Bioxome are stable in size and concentration up to 90 days at +4Cdegree or even at RT. No change in size between encapsulated Bioxome with small size (~340 Da) cargo vs empty Bioxome was observed up to 30 days storage. Lipidomic analysis approach revealed that the yield of lipids and their composition are satisfactory for a therapeutic product using Bioxome. Lastly, in the in vitro model of COVID19, Bioxome encapsulating API effectively saved cells from death (20x vs untreated cells) and at lower doses of API than these of non-encapsulated cargo (0.005 microM vs 0.1 microM). Bioxome seems to be an excellent candidate for liposome mimetic tool as drug delivery system for targeting specific organs and diseases treatment.Copyright © 2023 International Society for Cell & Gene Therapy

The relationship between salivary Fibroblast Growth Factor-2 and cortisol reactivity and psychological outcomes prior to and during the COVID-19 pandemic.

Background: Fibroblast growth factor-2 (FGF2) is a biomarker that is associated with depression, anxiety and stress in rodents. In humans, we have previously demonstrated that salivary FGF2 increased following stress in a similar pattern to cortisol, and FGF2 (but not cortisol) reactivity predicted repetitive negative thinking, a transdiagnostic risk factor for mental illness. The current study assessed the relationship between FGF2, cortisol, and mental health before and during the COVID-19 pandemic. Methods: We employed a longitudinal correlational design using a convenience sample. We assessed whether FGF2 and cortisol reactivity following the Trier Social Stress Task (TSST) were associated with DASS-21 depression, anxiety and stress, measured at the time of the TSST in 2019-20 (n = 87; time 1), and then again in May 2020 during the first wave of COVID-19 in Sydney (n = 34 of the original sample; time 2). Results: FGF2 reactivity (but not absolute FGF2 levels) at time 1 predicted depression, anxiety, and stress across timepoints. Cortisol reactivity at time 1 was associated with stress over timepoints, and absolute cortisol levels were associated with depression across timepoints. Limitations: The sample was comprised of mostly healthy participants from a student population, and there was high attrition between timepoints. The outcomes need to be replicated in larger, more diverse, samples. Conclusions: FGF2 and cortisol may be uniquely predictive of mental health outcomes in healthy samples, potentially allowing for early identification of at-risk individuals.

Is It Cancer Hepatic Inflammatory Pseudotumor: An Important Hepatic Mass Differential

Introduction: Hepatic inflammatory pseudotumor (HIP), albeit rare, is an important pathology to be included in differentials for hepatic masses. The benign nature and treatment of this disease process should be considered especially in comparison to malignant hepatic processes. Case Description/Methods: A 66-year-old male with pre-existing history of compensated Hepatitis C cirrhosis status post direct-acting antivirals with sustained virologic response presented in shock after a syncopal episode. Initial work up revealed leukocytosis, thrombocytopenia, acute renal injury, elevated liver enzymes, and COVID-19 positive test. Patient underwent initial liver ultrasound revealing intrahepatic and extrahepatic biliary ductal dilation. Subsequent MRCP demonstrated diffuse thickening of intra and extra hepatic bile ducts suggestive of cholangitis and several hepatic masses concerning for abscesses versus possible metastatic cholangiocarcinoma. Patient improved symptomatically with antibiotics and supportive care. A liver biopsy was performed with pathology showing lymphoplasmacytic inflammation and fibroblastic infiltration suggestive of hepatic inflammatory pseudotumor. A repeat MRCP one week later showed interval decrease in size of liver lesions and repeat liver function tests also showed improvement. Patient was discharged on a course of ciprofloxacin and metronidazole. Patient had repeat MRCP 3 months after discharge, with further significant improvement in size of liver lesions. After multi-disciplinary discussion the plan was for further surveillance with imaging and labs in 2 months. Discussion(s): Inflammatory pseudotumors are benign and non-neoplastic lesions that can occur in any organ. They can appear as a malignant lesion when they arise in the liver and an accurate identification can allow for conservative management and prevent unnecessary invasive procedures. Hepatic inflammatory pseudotumors are often seen with concomitant infection or inflammatory processes. Liver biopsies distinguish these tumors from other malignant processes as they demonstrate a characteristic dense inflammatory infiltrate interspersed in stroma of interlacing bundles of myofibroblasts. This case highlights the importance of maintaining HIP on the differential diagnosis. (Figure Presented).

Cytokine Profile in Different Post-Covid-19 Condition Phenotypes

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

Inflammatory and Blood-Brain Barrier Markers after Covid-19 among People with Hiv

Background: COVID-19, the disease caused by SARS-CoV-2, has resulted in devastating morbidity and mortality worldwide. Alarming evidence indicates that long-term adverse outcomes of COVID-19 can affect all major systems of the body, including the immune, respiratory, cardiovascular, and neurological systems. While acute COVID-19 pathology does not appear to be markedly different by HIV status, long-term outcomes of COVID-19 in People with HIV (PWH) are unknown and require further investigation. This study evaluates the inflammatory profile longitudinally up to three months after COVID-19. In addition, markers of the blood-brain barrier (BBB) integrity and vascular dysfunction were also evaluated. Method(s): Plasma samples were collected from 15 males and 6 females with COVID-19 and HIV infection (COVID+/HIV+) and 9 males and 14 females with COVID-19 without HIV infection (COVID+/HIV-) between March 2020 and March 2021. Baseline samples were obtained approx. 10 days after COVID-19 diagnosis (T=0) and three months after (T=3). Mean age group for COVID+/HIV-was 45.4+/-17.8 years for males and 39.7+/-15.3 for females and for COVID+/HIV+ was 52.1+/-12.3 for males and 48.7+/-1 for females (N=15 and 6, respectively). 27 inflammatory molecules were measured by Bio-Plex Multiplex Immunoassay (Bio-Rad) and two markers of BBB and vascular dysfunction (soluble ICAM1 and S100beta) by ELISA. Result(s): Out of 27 inflammatory analytes, 20 had detectable signals. Eotaxin (CCL11) and G-CSF levels were differentially upregulated in the COVID+/HIV+ group as compared to the COVID+/HIV-group in both time point studied (Table 1). IFN-g showed sustained increased levels at T=3 in the COVID+/HIV+ group, whereas there was a significant reduction over time in the COVID+/HIV-group. At T3, inflammatory markers (IL-4, IL-8, IL-13, basic FGF, TNF-alpha, MIP-1alpha, and CCL2) either decreased or remained unchanged in both groups. In contrast, the markers of the BBB disruption and vascular dysfunction, such as S100beta and soluble ICAM-1 increased in the COVID+/HIV+ group, suggesting long-term progressive BBB and vascular alterations. Conclusion(s): HIV-1 may potentiate long COVID-19-induced neuropathology, with progressive BBB breakdown and sustained increase in eotaxin-1 and G-CSF. Plasma inflammatory markers in COVID-19 patients with or without HIV-1 co-infection.

Vitamin C Regulates the Profibrotic Activity of Fibroblasts in In Vitro Replica Settings of Myocardial Infarction.

Extracellular collagen remodeling is one of the central mechanisms responsible for the structural and compositional coherence of myocardium in patients undergoing myocardial infarction (MI). Activated primary cardiac fibroblasts following myocardial infarction are extensively investigated to establish anti-fibrotic therapies to improve left ventricular remodeling. To systematically assess vitamin C functions as a potential modulator involved in collagen fibrillogenesis in an in vitro model mimicking heart tissue healing after MI. Mouse primary cardiac fibroblasts were isolated from wild-type C57BL/6 mice and cultured under normal and profibrotic (hypoxic + transforming growth factor beta 1) conditions on freshly prepared coatings mimicking extracellular matrix (ECM) remodeling during healing after an MI. At 10 µg/mL, vitamin C reprogramed the respiratory mitochondrial metabolism, which is effectively associated with a more increased accumulation of intracellular reactive oxygen species (iROS) than the number of those generated by mitochondrial reactive oxygen species (mROS). The mRNA/protein expression of subtypes I, III collagen, and fibroblasts differentiations markers were upregulated over time, particularly in the presence of vitamin C. The collagen substrate potentiated the modulator role of vitamin C in reinforcing the structure of types I and III collagen synthesis by reducing collagen V expression in a timely manner, which is important in the initiation of fibrillogenesis. Altogether, our study evidenced the synergistic function of vitamin C at an optimum dose on maintaining the equilibrium functionality of radical scavenger and gene transcription, which are important in the initial phases after healing after an MI, while modulating the synthesis of de novo collagen fibrils, which is important in the final stage of tissue healing.

Contemporary Concise Review 2022: Interstitial lung disease.

Novel genetic associations for idiopathic pulmonary fibrosis (IPF) risk have been identified. Common genetic variants associated with IPF are also associated with chronic hypersensitivity pneumonitis. The characterization of underlying mechanisms, such as pathways involved in myofibroblast differentiation, may reveal targets for future treatments. Newly identified circulating biomarkers are associated with disease progression and mortality. Deep learning and machine learning may increase accuracy in the interpretation of CT scans. Novel treatments have shown benefit in phase 2 clinical trials. Hospitalization with COVID-19 is associated with residual lung abnormalities in a substantial number of patients. Inequalities exist in delivering and accessing interstitial lung disease specialist care.

Oral lesions in patients with severe COVID-19 in the National Institute of Respiratory Diseases in Mexico: Case series

Introduction SARS-CoV-2 is a neurotropic, mucotropic, and sialotropic virus that can affect the salivary glands' function, taste sensations, smell, and oral mucosa integrity.1 The oral cavity is a perfect habitat for SARS-CoV-2 invasion due to the special affinity the virus has for cells with angiotensinconverting enzyme (ACE2) receptors, such as those from the respiratory tract, oral mucosa, tongue, and salivary glands. Aphthous lesions with necrosis and hemorrhagic crusts have been described to manifest more regularly in older adults with immunosuppression and severe COVID-19 infection;one hypothesis for the development of aphthous lesions and/or ulcers is given due to the ACE2 receptor and the SARS-CoV-2 interaction, which could alter the epithelial lining of salivary glands and keratinocytes, causing lesions in the oral cavity.4 At the same time, different etiological factors such as infections, immune system alterations, and direct trauma to the oral mucosa or epithelium,5 may be related to the stress of a prolonged hospital stay.6 Including pressure in the oral cavity conditioned by the prone position, malposition of the endotracheal tube (mainly in the corners of the lips),7 medication-related nutritional deficiencies8 such as lopinavir, and ritonavir, oseltamivir, hydroxychloroquine, among others.9-12 Thrombotic vasculopathy secondary to COVID-19 has also been described, induced by system mediators in the microvascular walls, which impairs endothelial cells, and activates coagulation factors13 and a possible hypersensitivity reaction of the mucosa to the presence of SARS-CoV-2 in the epithelium14,15;there is also the hypothesis that it could be associated with an exanthem pattern induced by the inflammatory action of the SARSCoV-2 virus,16 presented as increased levels of cytokines (including interleukin-1, tumor necrosis facto-a), and arachidonic acid metabolites (prostaglandins) secondary to the stem cell factor production and the basic fibroblast growth factor of keratinocytes from the basal layer, in relation to post-inflammatory pigmentations that could appear in areas previously affected by trauma or chronic inflammation.17 Oral manifestations in COVID-19 patients appear, on many occasions, even before respiratory symptoms, although exanthematic lesions observed in COVID-19 patients can also be observed in other viral processes. Physical examination revealed a patient in a supine position with orotracheal intubation and orogastric tube, with aphthous-type ulcers, some of them had blood crusts of different sizes on the lower lip (both skin and mucosa), dorsum, and lateral edge of the tongue, gum, and vestibular fornix (Figure 3). Initial physical examination shows the patient in a supine position supported by high-flow nasal prongs, upper and lower lips edema and ulcer-like lesions with hematic crusts on both lips (Figure 4), topical management with steroids and GELCLAIRE® Oral Gel (glycyrrhetinic acid and polyvinylpyrrolidone) is observed.

Mechanisms of Atrial Fibrillation in Covid-19

Atrial fibrillation (AF) is the most frequent form of cardiac arrhythmia in COVID-19 infected patients. The occurrence of AF paroxysms is often associated with the acute period of infection in time. At the same time, the pathophysiological mechanisms of the occurrence of AF associated with COVID-19 remain insufficiently studied. The review considers the available literature data on the influence of factors such as reduced availability of angiotensin-converting enzyme 2 receptors, interaction of the virus with the cluster of differentiation 147 and sialic acid, increased inflammatory signaling, "cytokine storm", direct viral damage to the endothelium, electrolyte and acid-alkaline balance in the acute phase of severe illness and increased sympathetic activity.Copyright © Autors 2023.

Persistence of inflammatory and vascular damage mediators 5 months after acute COVID-19

Background: In the acute phase, patients with severe COVID-19 exhibit pulmonary inflammation and vascular injury, as well as an exaggerated cytokine response. Aim(s): To describe the inflammatory cytokine and vascular injury mediator profiles in patients previously hospitalised with COVID-19, and to compare these profiles with those in healthy controls and in patients recovering from severe sepsis of other aetiology. Method(s): Plasma levels of 28 different cytokine, chemokine, angiogenic and vascular injury markers were measured by MSD V-PLEX multiplex assays in 49 post-COVID patients 5.0+/-1.9 (mean+/-SD) months after hospitalisation with COVID-19 pneumonia, 11 post-sepsis patients (5.4+/-2.9 months after hospitalised non-COVID sepsis) and 18 healthy controls. Kruskal-Wallis or ANOVA were used to compare groups;false discovery rate correction (Benjamini Hochberg) allowed for multiple testing. Result(s): In the post-COVID group, IL-6, TNFalpha, SAA, MCP1, Tie-2, Flt1, PIGF and CRP were significantly elevated, whereas IL-7 and bFGF were significantly depressed. The differences in TNFalpha, SAA, MCP1, Tie-2, Flt1, IL-7 and bFGF appeared unique to the post-COVID group, but increased IL-6, PIGF and CRP levels were also seen in postsepsis patients compared with controls. In post-COVID patients we found strong negative spearman correlations between each of IL-6 (r=-0.51) and CRP (r=-0.57) with TLCO %predicted (p<0.001) and positive correlations with post-recovery CT abnormality scores: IL-6 (r=0.28) and CRP (r=0.46), p<0.05. Conclusion(s): A unique signature of inflammatory and vascular damage markers is seen months after acute COVID19 infection. Further research is needed to determine their pathophysiological significance.

A review of fibroblast-like synoviocytes in the pathogenesis of Rheumatoid arthritis: Their activation and the inhibition of their apoptosis

Rheumatoid Arthritis (RA) is a systemic, autoimmune, inflammatory disease characterized by synovial hyperplasia, inflammatory cell infiltration in the synovial tissues, and progressive destruction of cartilage and bones. This disease often leads to chronic disability. More recently, activation of synovial fibroblasts (SFs) has been linked to innate immune responses and several cellular signalingpathways that ultimately result in the aggressive and invasive stages of RA. SFs are the major sources of pro-inflammatory cytokines in RA synovium. They participate in maintaining the inflammatory state that leads to synovial hyperplasia and angiogenesis in the inflamed synovium. The altered apoptotic response of synovial and inflammatory cells has been connected to these alterations of inflamed synovium. RA synovial fibroblasts (RASFs) have the ability to inhibit several apoptotic proteins that cause their abnormal proliferation. This proliferation leads to synovial hyperplasia. Apoptotic pathway proteins have thus been identified as possible targets for modifying the pathophysiology of RA. This review summarizes current knowledge of SF activation and its roles in the inhibition of apoptosis in the synovium, which is involved in joint damage during the effector phase of RA development.Copyright © 2022 Biomedpress.

Developing advanced cell culture models to study COVID19-associated pulmonary fibrosis

Patients with severe COVID-19-associated pneumonia are at risk to develop pulmonary fibrosis. To study the underlying mechanisms, we aim to develop advanced cell culture models that reliably reflect COVID-19-related profibrotic microenvironment. To identify key cellular players, we performed pilot immunohistochemistry analysis on lung tissue from COVID-19 patients with fibrosis collected during autopsy. Results revealed diffuse alveolar damage with macrophage infiltration, and myofibroblast accumulation with enriched collagen deposition surrounding the damaged alveoli. To mimic SARS-CoV-2 infection in alveoli, we infected human primary type II alveolar epithelial cells (AEC2) and found enhanced signaling of profibrotic cytokine transforming growth factor beta (TGFbeta) in some donors. To recreate the early fibrotic niche, an alveolar-macrophage-fibroblast (AMF) tri-culture model was established. After infecting AEC2 with SARS-CoV-2 in this AMF model, gene expression analysis provided evidence for fibroblast-to-myofibroblast transition. Furthermore, we found that overexpression of SARS-CoV-2 papain-like protease (PLpro) can promote TGFbeta signaling in HEK293T and A549 cells. After infecting AEC2 with SARS-CoV-2 PLpro lentivirus in the AMF model, we found signs of epithelial-to-mesenchymal transition and fibroblast-to myofibroblast transition. In future studies, we will use a detailed analysis of COVID-19-associated lung fibrosis with other types of lung fibrosis, to further refine COVID-19-related fibrosis models, including lung-on-chip models.

Dynamics and Heterogeneity of the Lung Immunopathology in Severe COVID-19

Background: The key impact of SARS-CoV-2 is its ability to cause a life-threatening infection in the lung. Aim(s): Using spatially resolved multiplex imaging the present study decodes the immunopathological complexity of severe COVID-19. Method(s): Autopsy lung tissue from 18 COVID-19 patients was used to map immune and structural cells in acute/exudative, intermediate and advanced diffuse alveolar damage (DAD) through multiplex immunohistochemistry and spatial statistical analyses. Cytokine profiling, viral, bacteria and fungi detection and transcriptome analyses were also performed. Result(s): All cases displayed concomitant patterns of DAD. The spatially resolved multiplex data revealed intricate patchworks of mm -size microenvironments representing distinct immunological niches. In-depth analysis of DAD areas revealed that the temporal/spatial DAD progression is associated with expansion of adaptive immune cells, macrophages, CD8 T cells, fibroblasts, angiogenesis and lymphangiogenesis. Viral load correlated positively with acute DAD and negatively with disease/hospital length. Cytokines correlated mainly with macrophages and CD8 T cells. Pro-coagulation and acute repair markers were enriched in acute DAD whereas intermediate/advanced DAD had a molecular profile of elevated humoral and innate immune responses and extracellular matrix production. Conclusion(s): Our unraveling of the spatio-temporal immunopathology in COVID-19 cases exposes the heterogeneous dynamics of acute viral infection and subsequent responses that occur side-by-side in the lungs. This complex disease feature has important implications for disease management and development of novel immunemodulatory treatments.

mRNA expression and delivery efficacy of lipid nanoparticles in the cells breast tumor microenvironment: in vitro and in vivo evaluation

During the COVID-19 pandemics we have all witnessed the clinical importance of mRNA as current vaccines and future therapeutics. mRNA therapies have a potential to revolutionize cancer treatment. Delivery of mRNA requires lipid nanoparticles (LNP) to protect the cargo from degradation. mRNA has a negative charge and depends on positively charged lipids to be encapsulated in LNP. These lipids can be either ionizable at certain pH or constantly cationic. Even though previous studies had evaluated the formulation properties of ionizable and cationic LNP systems, there is the need to understand their specificity in terms of mRNA delivery and protein expression in breast cancer tumor microenvironment. The objective of this work was to assess the kinetics of LNP cellular uptake and mRNA expression inv breast cancer (BC) cells and fibroblasts, the most frequent cell type in the tumor microenvironment cells, while studying the mechanisms involved in differential behaviors of LNP formulated with cationic and ionizable lipids. To achieve this goal mRNA-LNP containing ionizable lipids (LNP-A) and cationic lipids (LNP-B) were designed and formulated using Nanoassemblr Benchtop microfluidics mixer (Precision NanoSystems). mRNA-LNP were characterized for size, zeta potential using dynamic light scattering (DLS) and mRNA encapsulation efficiency using RiboGreen assay. LNP were tagged with rhodamine lipid to investigate the uptake kinetic and a reporter GFP mRNA to evaluate mRNA expression in murine 4T1 and human MCF7, MDA-231, SUM-159 and T47D breast cancer cells and BJ fibroblasts. Live fluorescence microscopy imaging, IncuCyte S3, was used to determine the LNP uptake and GFP mRNA expression. In vitro biocompatibility was assessed with WST-1 assay. Additionally, expression of mRNA delivered from LNP in tumor microenvironment was evaluated in vivo in a syngeneic 4T1 breast cancer model using mRNA luciferase and IVIS imaging. mRNA-LNPs possessed an average diameter of 77 - 107 nm, narrow size distribution, neutral zeta potential and high mRNA encapsulation efficiency (>94%). Our results demonstrated that mRNA expression was higher in breast cancer cells when delivered from LNP-A formulation and in BJ fibroblasts when delivered from LNP-B. LNP-A, the ionizable LNP, was tested in the breast cancer cells to confirm the efficacy of the delivery. The highest transfection efficacy, from high to low, T-47D, MCF7, SUM-159, 4T1 and MDA-231.We have further investigated the cellular uptake mechanisms of LNP using uptake pathway inhibitors for caveolae endocytosis, clathrin endocytosis, and phagocytosis. Our data confirm that there are differences in mechanisms that govern the uptake of mRNA LNP in breast cancer cells and fibroblasts. Clathrin-mediated endocytosis was active in 4T1 breast cancer cells for ionizable and cationic LNP. Interestingly, despite in vitro differences in uptake and mRNA expression, in vivo results show that both formulations efficiently delivered luciferasemRNA in the tumor microenvironment. Histology results demonstrated similar luciferase expression for both LNP in tumors. Additionally, we were able to confirm the prominent presence of fibroblast and similar distribution in the 4T1 subcutaneous model which could explain the similar efficacy of cationic and ionizable LNP. Understanding uptake and mRNA expression of different LNP formulations in the tumor microenvironment can help in achieving the necessary protein expression for breast cancer therapies. Furthermore, determining the most efficient carrier in early stages may reduce the time required for clinical translation. Acknowledgement: This research was supported in part by CPRIT Core for RNA Therapeutics and Research.

Broncho-alveolar expression of metalloproteinases and tissue growth factors as predictors of clinical severity in Sars-Cov-2 pneumonia

Background: Systemic and pulmonary uncontrolled activation of pro-inflammatory pathways following severe acute respiratory syndrome coronavirus 2 (Sars-Cov-2) infection can lead to development of serious short- and long-term complications such as ARDS and lung fibrosis. Mounting evidence reveals a positive correlation between cytokine overexpression in bronchoalveolar lavage fluid (BALF) and the severity of respiratory involvement in Sars-Cov-2 patients. We aimed to compare levels of metalloprotease 9 (MMP9) and fibrogenic Growth factors (VEGF, a-CTGF, FGF, PDGF) in BALF of intermediate medicine ward (IMW) Sars-Cov-2 and Intensive Care Unit (ICU) mechanically ventilated patients. Method(s): Sars-Cov-2 infection was diagnosed by Reverse transcriptase-polymerase chain reaction (RT-PCR) on respiratory samples. 10 IMW and 10 ICU patients were enrolled. ELISA assay was used to quantify growth factors and MMP9 on UV rays inactivated BAL fluid samples. Result(s): BALFs collected from ICU patients showed higher levels of Connective Tissue Growth Factor (CTGF;p<0.05) and MMP-9 (p<0,05) whereas inward patients with moderate pneumonia displayed higher titles of Vascular Endothelial Growth Factor (VEGF;p<0,05). FGF values were below detection limit in 90% of samples. No statistical difference in Platelet Derived Growth Factor (PDGF) levels were found between the two groups. Conclusion(s): Analogously to what observed for pro-inflammatory cytokines, early alveolar expression of MMP9 and CTGF are associated to a more severe outcome and might play a role in determining fibrotic evolution while VEGF does not seem to play a major role.

A CASE REPORT OF 'RETROPERITONEAL FIBROSIS CAUSING RENOVASCULAR HYPERTENSION' ASSOCIATED WITH SARS-CoV2

Introduction: Retroperitoneal fibrosis (RPF) is a rare disease which can be primary (idiopathic) or secondary to drugs, tumors or infections. We are reporting the first case of RPF causing renal atrophy, renal artery stenosis and renovascular hypertension associated with SARS-CoV2. Method(s): A 37-year-old female nurse presented to her PCP with a new-onset of hypertension. She had recovered from severe SARS-CoV2 infection merely two months ago. Physical examination was remarkable for BP 170/110 mmHg, HR 88 beats/min, BMI of 31 alongside trace pitting edema. Initial lab data showed her creatinine to be 1.1mg/dl and ultrasound of her kidneys showed an atrophied right kidney with a size of 7.8 cm while the left kidney was 11.6 cm. An ultrasound KUB of that same time showed that the size of the right kidney was 10.4 cm and left 11.5 with normal renal parenchyma. She was started on amlodipine 10 mg and valsartan 160 mg per day. Two weeks later she was referred to a nephrologist when her creatinine was increased to 3.1 mg/dl. Renovascular hypertension secondary to right renal artery stenosis or thrombus was suspected. Autoimmune & hypercoagulable work up was negative. CT angiogram showed an ill-defined, poorly enhancing retroperitoneal soft tissue thickening draping the mid abdominal aorta, the origin of SMA, and bilateral renal arteries which terminated above the aortoiliac bifurcation. This, RPF, involved segment of 8.6 cm of the mid and lower abdominal aorta, causing moderate narrowing of proximal SMA, short segment narrowing of proximal left main and accessory renal artery, and diffuse long segmental narrowing of the right main renal artery. RPF encasement of right renal artery lead to poor right renal nephrogram and atrophic kidney. (Figure no A: Abdominal contrast-enhanced computed tomographic (CT) scan showing the encasement of the both renal arteries by the retroperitoneal fibrosis (RPF).Figure no B : Renal angiogram showing the renal artery stenosis on right side) Acute kidney injury (AKI) was initially thought to be due to angiotensin receptor blockade in the setting of bilateral renal artery stenosis. Valsartan was swapped for metoprolol and the serum creatinine levels decreased to 1.5 mg/dl in two weeks. Prednisone was started for RPF at a dose of 60 mg per day with a slow taper over 4 months. Over the next 8 weeks, creatinine became normal and blood pressure was controlled with amlodipine 2.5 mg/day. Subsequently at 4 months her creatinine was 1.0 mg/dl and she was off all anti-hypertensive drugs. A repeat CTA after 6 months showed that there was significant reduction in RPF. Atrophic right kidney was noted without any significant interval change. RPF, renal artery stenosis, renovascular hypertension and right renal atrophy was strongly suspected to be associated with SARS-Cov2 since none of these were identified prior to her suffering from SARS-CoV2. Result(s): [Formula presented] [Formula presented] Conclusion(s): To our knowledge, this is the first case of RPF associated with SARS-CoV-2 causing renovascular hypertension and renal atrophy. Local and systemic production of IL-6, TGF- beta and Th2 cytokines has been demonstrated in idiopathic RPF and pulmonary fibrosis due to SARS-CoV2. The presumptive pathogenesis could involve SARS-Cov2 induced release of IL-6 and other cytokines which can activate B cells and fibroblasts. No conflict of interestCopyright © 2023