Trace Elements Zinc and Selenium: Their Significance in the Conditions of the Covid-19 Pandemic

Within the conditions of the ongoing COVID-19 pandemic, when many questions regarding prevention and treatment strategies remain unsolved and the search for the best antiviral agents is underway, attention should be paid to the role of trace elements zinc and selenium in increasing the body's resistance to viral infections and their direct antiviral activity against SARS-CoV-2. Experimental data show that trace elements zinc and selenium not only actthrough regulating the immune response at all levels of humoral and cellular immunity, but also can play a significant role in adjuvant therapy for viral diseases. This is especially relevant in the case of COVID-19. Studies of the direct antiviral effect of these micro-elements testify to its 3 main ways to SARS-Cov-2: I - counteraction to virus replication and its transcription through: (i) their covalent binding to the SH-group of the cysteine of the main protease M(Pro) of the virus;(ii) inhibition of its RNA polymerase activity by zinc;II - preventing the penetration of the virus into cells due to blocking SH-groups of protein disulfide isomerase (RDI) of the protein of its spikes (peplomers);III - decreasing the adsorption capacity of the virus due to the blocking of the electrostatic interaction of SARS-CoV-2 peplomers and angiotensin-converting enzyme (ACE-2) in ultra-low, uncharacteristic oxidation states (Zn+1and Se-2). The intensity of the antiviral action of these trace elements may depend on their chemical form. It was found that zinc citrate (a five-membered complex of zinc with citric acid) and monoselenium citric acid obtained with the help of nanotechnology have a greater intensity of action and higher chemical purity. Taking into account the immunostimulating and direct antiviral effect of zinc and selenium, their use in the form of pharmaceuticals and dietary supplements should be considered as adjunctive therapy for SARS-CoV-2 in patients, or as a preventive strategy for uninfected people from risk groups during the spread of COVID-19.Copyright © Publisher PH <<Akademperiodyka>> of the NAS of Ukraine, 2023.

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.

The sense of taste: Development, regeneration, and dysfunction.

Gustation or the sense of taste is a primary sense, which functions as a gatekeeper for substances that enter the body. Animals, including humans, ingest foods that contain appetitive taste stimuli, including those that have sweet, moderately salty and umami (glutamate) components, and tend to avoid bitter-tasting items, as many bitter compounds are toxic. Taste is mediated by clusters of heterogeneous taste receptors cells (TRCs) organized as taste buds on the tongue, and these convey taste information from the oral cavity to higher order brain centers via the gustatory sensory neurons of the seventh and ninth cranial ganglia. One remarkable aspect of taste is that taste perception is mostly uninterrupted throughout life yet TRCs within buds are constantly renewed; every 1-2 months all taste cells have been steadily replaced. In the past decades we have learned a substantial amount about the cellular and molecular regulation of taste bud cell renewal, and how taste buds are initially established during embryogenesis. Here I review more recent findings pertaining to taste development and regeneration, as well as discuss potential mechanisms underlying taste dysfunction that often occurs with disease or its treatment. This article is categorized under: Infectious Diseases > Stem Cells and Development Cancer > Stem Cells and Development Neurological Diseases > Stem Cells and Development.

Robust, efficient workflow to establish, culture, and functionally assess primary-isolated airway epithelial cells

Background: Air-liquid interface (ALI) and organoid culture are key techniques for differentiating human airway epithelial cells (HAECs). The efficiency and robustness of these assays often depends on the quality of primary-isolated cells, but primary cell isolation workflows, with which the user controls the choice of isolation method, cell culture medium, and culture format, may reduce reproducibility. Therefore, an optimized, standardized workflow can enhance and support isolation of epithelial cells from diseased donors with potentially rare cystic fibrosis (CF) mutations or particularly sensitive cell populations. We have developed a standardized workflow for isolation and culture of freshly derived airway epithelial cells. Method(s): Briefly, HAECs isolated from primary tissue were expanded in PneumaCult-Ex Plus Medium for 1 week and then seeded into Corning Transwell inserts and expanded until confluency. The cells were then differentiated in PneumaCult-ALI Medium for at least 4 weeks. To assess differentiation efficiency in ALI culture, the cells were immunostained to detect Muc5AC, acetylated tubulin, and ZO-1 to identify goblet cells, ciliated cells, and apical tight junctions, respectively, aswell as SARS-CoV-2 cell entry targets angiotensin-converting enzyme 2 and transmembrane serine protease 2. Ion transport and barrier function of the ALI culturesand response to CF transmembrane conductance regulator (CFTR) correctors were also measured. In addition, freshly derived HAECs were seeded into Corning Matrigel domes in the presence of PneumaCult Airway Organoid Seeding Medium. Oneweek later, the mediumwas changed to PneumaCult Airway Organoid Differentiation Medium and maintained for an additional 3 weeks to promote cell differentiation. These airway organoids were then treated with CFTR corrector VX-809 for 24 hours, followed by 6-hour treatment with amiloride, forskolin, and genistein to induce organoid swelling. Result(s): Our results demonstrate that ALI cultures derived from CF donors displayed partial rescue of CFTR across multiple passages after treatment with VX-809. Airway organoids were found to express functional CFTR, as evidenced by forskolin treatment, which induced a 64 +/- 14% (n = 1 donor) greater organoid area than in vehicle control-treated airway organoids. Airway organoids derived from CF donors displayed a loss of forskolininduced swelling, which could be partially re-established with VX-809 treatment (29 +/- 9%, n = 3). Conclusion(s): In summary, the PneumaCult workflow supports robust, efficient culture of primary-airway epithelial cells that can be used as physiologically relevant models suitable for CF research, CFTR corrector screening, and studying airway biology.Copyright © 2022, European Cystic Fibrosis Society. All rights reserved

Ayurvedic Herbs Advised for COVID-19 Management: Therapeutic Potential and Clinical Relevance

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide. There is no effective medication for COVID-19 as of now, so it would be good to take preventive measures that not only boost our immunity but also fight against infections. The use of traditional Chinese medicine in China to treat COVID-19 patients sets the prototype demonstrating that traditional medicines can contribute to prevention and treatment successfully. In India, the Ministry of AYUSH (Ayurveda, Yoga, Unani, Siddha, Homeop-athy) released a self-care advisory during the COVID-19 crisis as a preventive aspect. This review article discusses the therapeutic potential and clinical relevance of some herbs [(Tulsi (Ocimum sanctum), Haridra (Curcuma longa), Tvaka (Cinnamon), Maricha (Piper longum), Shunthi (Zingi-ber officinale), Munakka (Dried grapes), Lavang (Syzigiumaromaticum), Pudina (Mentha arvensis), and Ajwain (Trachyspermum ammi)] advised by AUYSH to take during COVID-19 infection. They are effective in COVID-19 management, therefore, authors have discussed their detailed traditional uses as therapeutics and spotted scientific insight and clinical significance of the herbs mentioned above along with their mechanistic viewpoint, adequately, on a single platform. Provided information could be a treasure to open up a new research arena on natural products to manage human health crises effectively, caused not only by COVID-19 but also by other infectious diseases.Copyright © 2023 Bentham Science Publishers.

Immunohistochemical profile of cell cycle control, proliferation and differentiation proteins of the pulmonary atypical alveolar epithelium with COVID-19-induced diffuse alveolar damage as compared to pulmonary [lepidic-growth] adenocarcinoma.

We aim aim to compare immunophenotypic charac-teritics of atypical epithelium (AE) with COVID-19-induced diffuse alveolar damage (DAD) and pulmonary lepidic-growth adenocarcinoma, accounting for cell cycle control, proliferation and differentiation]. Methods. We examined pulmonary tissue specimens from twenty-four fatal cases of CO VID-19-induced acute respiratory damage syndrome confirmed by autopsy (Group 1) and four cases of pulmonary lepidic-growth adenocarcinoma (Group 2). Perpendicular dimensions of 10 nuclei were measured on the H&E slides, means of their sums of products (SPNM) were calculated. We have used p53, Ki67, pi6, p63 antibodies for immunohistochemical staining in each case. We evaluate colour intensity, rate of stained cells of AE and the product of these parameters. We evaluated separately Nuclear and cyto-plasmic staining (couple) and only cytoplasmic staining (cyt) for pi6 expression. We measured proliferative index only at KI-67 stained slides. U-test and Spearman rank correlation test were used for statistical analysis. Results. Expression of p63 was higher in group 1 (p=0.001), while pi6 was more frequently expressed in group 2 (p=0.002). We have found no statistically significant differences (p>0.1) in the p53 and Ki67 expression. Group 1 showed There was negative correlation between the number of days from onset of symptoms and the following variables: Ki67 (r=M).587, p=0.003);SPNM (r 0.406, p=0.049). Conclusion. The present study has shown heterogeneity in levels of cell cycle control expression, proliferation and differentiation of atypical epithelium in the pulmonary lep-idic-growth adenocarcinoma and CO VID-19-induced diffuse alveolar damage.Copyright © 2022 Izdatel'stvo Meditsina. All rights reserved.

Vitamin D and estrogen steroid hormones and their immunogenetic roles in Infectious respiratory (TB and COVID-19) diseases

The role of steroid hormones against infectious diseases has been extensively studied. From immunomodulatory action to direct inhibition of microorganism growth, hormones D3 (VD3) and 17beta-estradiol (E2), and the genetic pathways modulated by them, are key targets for a better understanding pathogenesis of infectious respiratory diseases (IRD) such as tuberculosis (TB) and the coronavirus disease-19 (COVID-19). Currently, the world faces two major public health problems, the outbreak of COVID-19, accounting for more than 6 million so far, and TB, more than 1 million deaths per year. Both, although resulting from different pathogens, the Mtb and the SARS-CoV-2, respectively, are considered serious and epidemic. TB and COVID-19 present similar infection rates between men and women, however the number of complications and deaths resulting from the two infections is higher in men when compared to women in childbearing age, which may indicate a role of the sex hormone E2 in the context of these diseases. E2 and VD3 act upon key gene pathways as important immunomodulatory players and supporting molecules in IRDs. This review summarizes the main roles of these hormones (VD3 and E2) in modulating immune and inflammatory responses and their relationship with TB and COVID-19.Copyright © Sociedade Brasileira de Genetica.

Bioavailability of iron and its potential to improve the immune system and ward off COVID-19: a review

Iron is a mineral that plays an important role, especially to prevent anaemia through the production of red blood cells. Iron also plays a role in physiological processes, such as the activation of enzymes and hormones, as well as increasing the immune system in warding off various viral infections. Therefore, iron bioavailability needs to be considered to take the greatest benefit of iron. This review discussed the factors that can affect the bioavailability of iron, various technologies to increase the bioavailability, and its potential in enhancing the immune system. Iron bioavailability can be increased by fortification, fermentation, the addition of vitamin C, and iron encapsulation. Under conditions of adequate iron intake, iron plays an important role in enhancing the immune system through controlling lymphocytes and T cell proliferation. However, excess iron consumption can be at risk of weakening the host's immune response to viruses. Therefore, the appropriate level of iron intake must be maintained accurately to be used optimally and has the potential to ward off viral infections, including the Sars-CoV-2 virus as the cause of COVID-19.Copyright © 2023, Rynnye Lyan Resources. All rights reserved.

Distinct dynamics of antigen-specific induction and differentiation of different CD11c+Tbet+ B-cell subsets.

BACKGROUND: CD11c+Tbet+ B cells are enriched in autoimmunity and chronic infections and also expand on immune challenge in healthy individuals. CD11c+Tbet+ B cells remain an enigmatic B-cell population because of their intrinsic heterogeneity. OBJECTIVES: We investigated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen-specific development and differentiation properties of 3 separate CD11c+ B-cell subsets-age-associated B cells (ABCs), double-negative 2 (DN2) B cells, and activated naive B cells-and compared them to their canonical CD11c- counterparts. METHODS: Dynamics of the response of the 3 CD11c+ B-cell subsets were assessed at SARS-CoV-2 vaccination in healthy donors by spectral flow cytometry. Distinct CD11c+ B-cell subsets were functionally characterized by optimized in vitro cultures. RESULTS: In contrast to a durable expansion of antigen-specific CD11c- memory B cells over time, both ABCs and DN2 cells were strongly expanded shortly after second vaccination and subsequently contracted. Functional characterization of antibody-secreting cell differentiation dynamics revealed that CD11c+Tbet+ B cells were primed for antibody-secreting cell differentiation compared to relevant canonical CD11c- counterparts. CONCLUSION: Overall, CD11c+Tbet+ B cells encompass heterogeneous subpopulations, of which primarily ABCs as well as DN2 B cells respond early to immune challenge and display a pre-antibody-secreting cell phenotype.

Holistic View on the Structure of Immune Response: Petri Net Model.

The simulation of immune response is a challenging task because quantitative data are scarce. Quantitative theoretical models either focus on specific cell-cell interactions or have to make assumptions about parameters. The broad variation of, e.g., the dimensions and abundance between lymph nodes as well as between individual patients hampers conclusive quantitative modeling. No theoretical model has been established representing a consensus on the set of major cellular processes involved in the immune response. In this paper, we apply the Petri net formalism to construct a semi-quantitative mathematical model of the lymph nodes. The model covers the major cellular processes of immune response and fulfills the formal requirements of Petri net models. The intention is to develop a model taking into account the viewpoints of experienced pathologists and computer scientists in the field of systems biology. In order to verify formal requirements, we discuss invariant properties and apply the asynchronous firing rule of a place/transition net. Twenty-five transition invariants cover the model, and each is assigned to a functional mode of the immune response. In simulations, the Petri net model describes the dynamic modes of the immune response, its adaption to antigens, and its loss of memory.

ROLE OF IFNgamma IN PATHOGENESIS OF SARS-CoV-2 INFECTION.

To date, there is no consensus explaining the relationship between varying concentrations of IFNgamma and the severity of infection caused by SARS-CoV-2. The aim of this article was to analyze and formulate conclusions from the selected studies and publications, which, in sum, provide a potentially reasonable view on the role of IFNgamma in COVID-19 pathogenesis. This article highlights current data on the immunological role of IFNgamma which affects differentiation of naive T helper cells, acting as a polarizing factor. It activates the major histocompatibility complex (MHC) class I and II, by increasing the expression of MHC I/II subunits, inhibiting replication of the viral particles by initiating activation of interferon-stimulated genes followed by subsequent synthesis of antiviral proteins. Moreover, IFNgamma activates the production of cytokines by T cells, enhancing cytotoxic activity of the T killers. IFNgamma exerts immunostimulatory and immunomodulatory effects via STAT1, SOCS1 and PIAS genes, thus regulating activation of the JAK-STAT signaling pathway. A number of studies were considered where the patterns of changes in serum IFNgamma concentration were examined in viral infections and SARS-CoV-2. We performed a systemic analysis of the results of studies that showed a relationship between high concentrations of IFNgamma and COVID-19 severity. In a number of studies, the significantly high levels of IFNgamma in COVID-19 patients were often associated with a poor outcome of the disease. The median values of the IFNgamma concentration in severe COVID-19 were found to be significantly higher compared to the results obtained in the cases of moderate severity. It shows an increase, in parallel with viral load in the nasopharyngeal samples upon worsening of the clinical condition. Based on the data on the decreased IFNgamma concentrations in convalescent patients, the mechanism of antagonism between IFNgamma and IL-4 is considered, where the decreases serum concentrations of IFNgamma along with increasing level of IL-4 may be an indirect proof of normal adaptive immune response with subsequent development of antibodies to SARS-CoV-2 and gradual elimination of the virus from the body. Moreover, the evidence is discussed that the patients harboring some parasitic infections (Toxoplasma gondii, Cryptosporidium, Blastocystis hominis, Giardia duodenalis, Entamoeba histolytica) with persistently elevated level of IFNgamma are at reduced risk for severe course of COVID-19. Copyright © 2022, SPb RAACI.

Molecular mechanisms underlying regulatory T cell deficiency and potential drugs in COVID-19

Background/Purpose: The 2019 outbreak of coronavirus disease COVID-19 causes immune system disruption. Recent studies reported that the decrease or depletion of regulatory T cell (Treg) may be responsible for overstimulation of the immune system and lung damage in patients with severe COVID-19. This study aims to find the molecular mechanisms and genetic biomarkers associated with Tregs in COVID-19, providing new ideas for the treatment of COVID-19. Method(s): RNA sequencing data of peripheral blood mononuclear cells (PBMC) from 252 COVID-19 infected patients and 69 healthy controls (HC) were obtained from the GEO database. The Tregs composition of COVID-19 samples was quantified using the CIBERSORT deconvolution method. The differential genes (DEGs) were identified by the limma R package. Gene co-expression network analysis (WGCNA) was used to identify the gene. Differentially expressed Tregs-related genes (DETregRGs) were obtained by intersecting DEGs with the highly related modular genes obtained in the previous step. The potential biological functions and pathways of DETregRGs were then explored. Protein-protein interaction (PPI) networks were subsequently constructed to identify hub genes. In addition, the prediction of small molecule drugs for the potential treatment of COVID-19 was made using the CMap database. Result(s): After the weighted gene co-expression network analysis (WGCNA), the turquoise module was highly correlated with Treg expression and a total of 134 DEGs was identified as DETregRGs. These genes were mainly involved in GO biological processes, such as the inflammatory response, and T cell differentiation of thymus. Then, 11 hub genes (including RPS12, RPL21, RPS3A, CD8B, CD3D, TRAT1, RPS6, CD3E, CD28, RPL3, and CD4) were ranked based on Molecular Complex Detection (MCODE) analysis. The TregRG score of COVID-19 patients showed significantly lower than HC, calculated by the 'singscore' algorithms. After the signature query of the CMap database, the KU-0063794, an mTOR inhibitor ranked second in the negative enrichment score, may restore immune system dysregulation caused by increased Th17 differentiation and decreased Treg differentiation during SARS-CoV- 2 infection. Conclusion(s): Our study examined in detail the molecular mechanisms underlying the inadequacy of Tregs in patients with COVID-19 infection. mTOR inhibitors may improve COVID-19 symptoms by expanding Tregs which may be one of the potential therapeutic methods that need further investigation. (Figure Presented).

GATA2 deficiency presenting as fever and rash

Case Report: Our patient is an 8-year-old Caucasian female with a history of choanal atresia, first degree heart block, recurrent urinary tract infections, and recent COVID-19 infection, who initially presented with an episode of syncope and vomiting. By history, she had two weeks of daily fever and an intermittent nonspecific rash. She was diagnosed with a UTI 5 days prior to presentation but had not defervesced despite treatment. Shewas initially found to be in shock with tachycardia and poor perfusion and was treated with fluid resuscitation, antipyretics, and empiric antibiotics. Labs were significant for leukopenia, elevated inflammatory markers, lactic acidosis, coagulopathy, and mildly elevated troponin. Chest x-ray showed abnormal but non-specific widespread infiltrates. She was initially treated with IVIG and pulse steroids for a working diagnosis of MIS-C, however she did not improve and a more extensive infectious, oncologic, and rheumatologic work-up was performed. Her workup revealed a disseminated Mycobacterium abscessus infection. Bone marrow biopsy revealed myelodysplasia with monosomy 7. Her buccal swab testing revealed a heterozygous germline mutation in the GATA2 gene, a variant that is predicted to cause loss of normal protein function. She is presently on multidrug regimen for her mycobacterial infection. Her myelodysplasia evolved into an acute leukemia, and she is undergoing chemotherapy for that at this time. Discussion(s): GATA2 deficiency, first identified in 2011, is a rare immune disorder resulting in a wide variety of clinical presentations. It is caused by a germline mutation of the GATA2 gene that disrupts blood cell differentiation, resulting in decreased or absent monocytes, B cells, NK cells, and dendritic cells1. This case presented multiple challenges due to the broad range of differential diagnoses. This patient was ultimately diagnosed with myelodysplastic syndrome associated with monosomy 7 and GATA2 deficiency, confirmed by FISH testing. Due to the presentation and lab derangements this patient had, there was a delay in targeted treatment while managing her cytopenias and presumed pulmonary infection. GATA2 deficiency carries a high risk of progression from myelodysplastic syndrome to acute myelogenous leukemia. The best long-term treatment for GATA2 deficiency is hematopoietic stem cell transplant, which is the ultimate goal for our patient. Copyright © 2023 Southern Society for Clinical Investigation.

Alteration of T Cell Responses against Sarscov- 2 Omicron Variant after Vaccination with Mrna Booster in Lung Cancer Patients

Background The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant has demonstrated high transmissibility and possesses several spike protein mutations that allow for evasion of previously established immunity.1 mRNA vaccines against the spike protein of the ancestral strain of the virus have been reported to induce robust T cell immunity against the omicron variant when examined in healthy individuals. 2 However, the effectiveness of the booster vaccine doses in late-stage lung cancer patients undergoing active anti-PD-1/ PD-L1 agent immunotherapy has yet to be investigated.3 Methods To address this question, we assessed both CD8+ and CD4+ T cell responses using a modified activationinduced marker (AIM) assay that was performed on peripheral blood mononuclear cells (PBMCs), which was coupled with high dimension spectral flow cytometry analyses. The PBMCs were obtained using cryopreserved blood samples collected from The COVID-19 Vaccine Study of Infections and Immune REspoNse (SIIREN) trial, and a total of 51 patient samples (20 non-cancer patients and 31 lung cancer patients) were assessed. Results Our observations included that booster vaccines induced CD8+ T cell response in both non-cancer subjects and lung cancer patients against ancestral strain and omicron variant, while only marginal induction or trend was detected for CD4+ T cells in normal subjects. Pertinent results also consisted of identification of distinct subpopulation dynamics involving varying degrees of differentiation of antigen-specific CD8+ and CD4+ T cells in lung cancer patients compared to non-cancer subjects, thus demonstrating evidence of dysfunction. Another noteworthy finding included the observation of sex biased T cell responses with female lung cancer patients demonstrating more efficient antigen-specific T cell responses compared to males. Conclusions We conclude that lung cancer patients in our study cohort have substantial qualitative deviation in their T cell response to mRNA vaccine from the normal individuals. This altered response may be a consequence of altered T cell differentiation states, resulting in the high degree of heterogeneity of AIM+ T cells identified in booster vaccinated individuals. Moreover, the dampened T cell response to omicron in cancer patients could implicate that less protection was established by vaccination for lung cancer patients, especially given that humoral response is also reduced in cancer patients.4 This further highlights the need for heightened protective measures for cancer patients to minimize the risk of breakthrough infection with the omicron and other future variants of SARS-CoV-2.5.

Vitamin D, the immune system, and its relationship with diseases

Background: Vitamin D is classified as an immunomodulatory hormone that is synthesized because of skin exposure to sunlight. It is known to come into play during the regulation of hormone secretion, immune functions, cell proliferation, and differentiation. Its deficiency can cause many diseases and their associated pleiotropic effects. In addition, in relation to its eminent function as regards adaptive immune response and innate immune response, vitamin D level is associated with immune tolerance. Method(s): Literature search prior to May 2021 was conducted through selected websites, including the MEDLINE, Embase, Web of Science, Cochrane Central, www.ClinicalTrials.gov, PubMed, Science Direct, Google Scholar, and EFSA. Result(s): Vitamin D is found effective for the regulation of hormone secretion, immune functions, and cell proliferation along with differentiation. Its role as an immune modulator is based on the presence of receptors on many immune cells and the synthesis of its active metabolite from these cells. Vitamin D, an immune system modulator, inhibits cell proliferation and stimulates cell differentiation. A fair number of immune system diseases, encompassing autoimmune disorders alongside infectious diseases, can occur because of low serum vitamin D levels. Supplementation of vitamin D has positive effects in lessening the severity nature of disease activity;there exists no consensus on the dose to be used. Conclusion(s): It is figured out that a higher number of randomized controlled trials are essential to evaluate efficacy pertaining to clinical cases, treatment duration, type, and dose of supplementation and pathophysiology of diseases, immune system functioning, and the effect of vitamin D to be administered. Copyright © 2022, The Author(s).

Protocol for SARS-CoV-2 infection of kidney organoids derived from human pluripotent stem cells.

This protocol presents the use of SARS-CoV-2 isolates to infect human kidney organoids, enabling exploration of the impact of SARS-CoV-2 infection in a human multicellular in vitro system. We detail steps to generate kidney organoids from human pluripotent stem cells (hPSCs) and emulate a diabetic milieu via organoids exposure to diabetogenic-like cell culture conditions. We further describe preparation and titration steps of SARS-CoV-2 virus stocks, their subsequent use to infect the kidney organoids, and assessment of the infection via immunofluorescence. For complete details on the use and execution of this protocol, please refer to Garreta et al. (2022).1.

PCL- And Gelatin-based Electrospun Biological Scaffolds For In Vitro Lung Tissue Engineering

Purpose/Objectives: Acute and chronic respiratory diseases constitute a substantial socioeconomic burden on a global scale, as made abundantly clear in the last two years with the rampant coronavirus disease 2019 (COVID-19) pandemic. Alas, the development of new therapies for pathological respiratory conditions has been hindered by the inadequacy of current preclinical models, which often fail to provide reliable predictions on drug safety and efficacy in humans. In particular, considerable anatomical and physiological differences between the respiratory systems of commonly used animal models and humans are one of the main issues leading to high drug attrition and clinical failure rates. Accordingly, the generation of physiologically relevant preclinical lung models for early drug development and pharmaceutical research is urgently needed. In this work, poly(ϵ-caprolactone) (PCL) and gelatin were used as raw materials to produce electrospun scaffolds for in vitro lung tissue engineering, in order to generate human biomimetic platforms for preclinical drug safety and efficacy testing. Methodology: PCL and gelatin were mixed at varying volume ratios: 1:0 (PP), 6:1 (PPG61), 4:1 (PPG41), and 2:1 (PPG21), so as to determine the optimal gelatin concentration for cell adhesion and growth. Poly(vinylpyrrolidone) (PVP) was added to every polymer mixture to facilitate the electrospinning process, and electrospun fibrous matrices were fabricated using a needleless electrospinning technique. Scaffold morphology, chemical composition, and wettability were characterized with scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and water contact angle analysis, respectively. Biocompatibility testing was performed using human bronchial (16HBE) and alveolar (A549) epithelial cell lines, consisting of cell metabolic activity, proliferation, and adhesion evaluation over two weeks of in vitro culture. Results: All polymer blends resulted in the formation of electrospun scaffolds with a nanofibrous structure. The addition of gelatin in PPG61 scaffolds improved fiber morphology compared to PP formulations, but increasing proportions of this polymer in PPG41 and PPG21 mats caused a larger number of defects, such as beading and branching. FTIR analysis confirmed the presence of PCL and PVP in PP scaffolds, as well as the addition of gelatin in all PPG blends. Moreover, as expected, all scaffolds were hydrophilic, with water contact angles below 90°, being suitable for protein adsorption and cell adhesion. Regarding 16HBE and A549 cell viability, surprisingly, no major differences were found between the different formulations over the two-week culture period, showing that all polymer blends were equally capable of promoting cell adhesion and growth. While PP scaffolds significantly outperformed PPG electrospun mats in early timepoints, no such differences were identified at the end of the experimental period. Conclusion/Significance: These results suggested that PCL, PVP, and/or gelatin blend electrospun scaffolds are conducive to lung epithelial cell adhesion and proliferation. Nevertheless, further studies investigating epithelial cell differentiation and function should be conducted to fully assess the suitability of these biomaterials as platforms for in vitro lung tissue engineering.

The Role of Endoplasmic Reticulum Stress in Differentiation of Cells of Mesenchymal Origin.

Endoplasmic reticulum (ER) is a multifunctional membrane-enclosed organelle. One of the major ER functions is cotranslational transport and processing of secretory, lysosomal, and transmembrane proteins. Impaired protein processing caused by disturbances in the ER homeostasis results in the ER stress. Restoration of normal ER functioning requires activation of an adaptive mechanism involving cell response to misfolded proteins, the so-called unfolded protein response (UPR). Besides controlling protein folding, UPR plays a key role in other physiological processes, in particular, differentiation of cells of connective, muscle, epithelial, and neural tissues. Cell differentiation is induced by the physiological levels of ER stress, while excessive ER stress suppresses differentiation and can result in cell death. So far, it remains unknown whether UPR activation induces cell differentiation or if UPR is initiated by the upregulated synthesis of secretory proteins during cell differentiation. Cell differentiation is an important stage in the development of multicellular organisms and is tightly controlled. Suppression or excessive activation of this process can lead to the development of various pathologies in an organism. In particular, impairments in the differentiation of connective tissue cells can result in the development of fibrosis, obesity, and osteoporosis. Recently, special attention has been paid to fibrosis as one of the major complications of COVID-19. Therefore, studying the role of UPR in the activation of cell differentiation is of both theoretical and practical interest, as it might result in the identification of molecular targets for selective regulation of cell differentiation stages and as well as the potential to modulate the mechanisms involved in the development of various pathological states.