Targeting SIRT2 Sensitizes Melanoma Cells to Cisplatin via an EGFR-Dependent Mechanism.

Melanoma cells are resistant to most anticancer chemotherapeutics. Despite poor response rates and short-term efficacy, chemotherapy remains the main approach to treating this cancer. The underlying mechanisms of the intrinsic chemoresistance of melanoma remain unclear, but elucidating these mechanisms is important to improve the efficacy of chemotherapy regimens. Increasing evidence suggests that sirtuin 2 (SIRT2) plays a key role in the response of melanoma cells to chemotherapeutics; thus, in the present study, we evaluated the impact of shRNA-mediated and pharmacological inhibition of SIRT2 on the sensitivity of melanoma cells to cisplatin, which is used in several regimens to treat melanoma patients. We found that cells with SIRT2 inhibition revealed increased sensitivity to cisplatin and exhibited increased accumulation of γ-H2AX and reduced EGFR-AKT-RAF-ERK1/2 (epidermal growth factor receptor-protein B kinase-RAF kinase-extracellular signal-regulated kinase 1/2) pathway signaling compared to control cells. Thus, our results show that sirtuin 2 inhibition increased the in vitro efficacy of cisplatin against melanoma cells.

Complete genome sequences of Aeromonas and Pseudomonas phages as a supportive tool for development of antibacterial treatment in aquaculture.

BACKGROUND: Aquaculture is the fastest growing sector of food production worldwide. However, one of the major reasons limiting its effectiveness are infectious diseases among aquatic organisms resulting in vast economic losses. Fighting such infections with chemotherapy is normally used as a rapid and effective treatment. The rise of antibiotic resistance, however, is limiting the efficacy of antibiotics and creates environmental and human safety concerns due to their massive application in the aquatic environment. Bacteriophages are an alternative solution that could be considered in order to protect fish against pathogens while minimizing the side-effects for the environment and humans. Bacteriophages kill bacteria via different mechanisms than antibiotics, and so fit nicely into the 'novel mode of action' concept desired for all new antibacterial agents. METHODS: The bacteriophages were isolated from sewage water and characterized by RFLP, spectrum of specificity, transmission electron microscopy (TEM) and sequencing (WGS). Bioinformatics analysis of genomic data enables an in-depth characterization of phages and the choice of phages. This allows an optimised choice of phage for therapy, excluding those with toxin genes, virulence factor genes, and genes responsible for lysogeny. RESULTS: In this study, we isolated eleven new bacteriophages: seven infecting Aeromonas and four infecting Pseudomonas, which significantly increases the genomic information of Aeromonas and Pseudomonas phages. Bioinformatics analysis of genomic data, assessing the likelihood of these phages to enter the lysogenic cycle with experimental data on their specificity towards large number of bacterial field isolates representing different locations. CONCLUSIONS: From 11 newly isolated bacteriophages only 6 (25AhydR2PP, 50AhydR13PP, 60AhydR15PP, 22PfluR64PP, 67PfluR64PP, 71PfluR64PP) have a potential to be used in phage therapy due to confirmed lytic lifestyle and absence of virulence or resistance genes.

Influence of bacteriophages cocktail on European eel (Anguilla anguilla) immunity and survival after experimental challenge.

Inland fishery belongs to those branches of animal production that use very large amounts of chemotherapeutics, in particular antibiotics. The accumulation of chemotherapeutic agents in bottom sediments is a direct threat to the aquatic environment and directly affects the condition and health of the fish. Finding a preparation that could be used both prophylactically to increase the resistance of fish and therapeutically in case of infection with pathogenic bacteria, without side effects for fish and aquatic environment could be a great solution to this problem. Our aim was to determine influence of BAFADOR® the new bacteriophage-based preparation on European eel immunity and survival after experimental challenge. Application of BAFADOR® increased total protein level, immunoglobulin level, lysozyme activity and ceruloplasmin level in European eel serum. Potential killing activity and metabolic activity of spleen phagocytes as well as pronephros lymphocyte proliferation of was higher compared to control. The preparation also reduced mortality after experimental infections with the pathogenic bacteria Aeromonas hydrophila and Pseudomonas fluorescens. Our results showed that preparation BAFADOR® is well tolerated by the fish organism causing stimulation of cellular and humoral immunity parameters and reduces the mortality of the European eel after experimental challenge.

Bacteriophage-based cocktail modulates selected immunological parameters and post-challenge survival of rainbow trout (Oncorhynchus mykiss).

Recently, a rapid increase in the resistance of pathogenic bacteria to antibiotics and chemotherapeutics admitted for use in aquaculture has been observed. This happens especially often in intensive breeding. The use of drugs in closed circuits is problematic because it can damage biological filters. Therefore, in recent years, there has been a growing interest in natural methods of combating pathogens. These include bacteriophages. The aim of the study was to determine the safety of the new BAFADOR® bacteriophage-based preparation, its effect on selected immunological parameters and the effectiveness of prophylactic and therapeutic use after experimental infections with pathogenic bacteria Aeromonas hydrophila and Pseudomonas fluorescens. The use of BAFADOR® increased the activity of lysozyme, total protein level and immunoglobulin level. The level of ceruloplasmin in the rainbow trout serum remained unchanged regardless of the route of administration of the preparation. Potential killing activity and metabolic activity of spleen phagocytes and proliferation of pronephros lymphocytes were higher compared to the control group. Both therapeutic and prophylactic application of the preparation after mixed experimental infection of A. hydrophila and P. fluorescens limited the mortality of rainbow trout.

The Dichotomous Nature of AZ5104 (an EGFR Inhibitor) Towards RORγ and RORγT.

The RORC (RAR related orphan receptor C) gene produces two isoforms by alternative promoter usage: RORγ (nuclear receptor ROR-gamma isoform 1) and RORγT (nuclear receptor ROR-gamma isoform 1). Both proteins have distinct tissue distributions and are involved in several physiological processes, including glucose/lipid metabolism and the development of Th17 lymphocytes. Previously, we developed a stably transfected reporter cell line and used it to screen a library of kinase inhibitors. We found that AZ5104 acts as an RORγ agonist at low micromolar concentrations. Molecular docking analysis showed that this compound occupies the ligand binding domain of the receptor with a significant docking score. However, analysis of the biological activity of this compound in Th17 cells revealed that it downregulates RORγT expression and Th17-related cytokine production via inhibition of SRC-ERK-STAT3 (SRC proto-oncogene - extracellular regulated MAP kinase - signal transducer and activator of transcription 3). We thus identified a compound acting as an agonist of RORγ that, due to the inhibition of downstream elements of EGFR (epidermal growth factor receptor) signaling, exerts different biological activity towards a Th17-specific isoform. Additionally, our results may be relevant in the future for the design of treatments targeting signaling pathways that inhibit Th17-related inflammation in certain autoimmune disorders.

Mast cells as the strength of the inflammatory process.

The inflammatory process is a complex host defence mechanism aimed at the elimination of deleterious factors disturbing homeostasis. Inflammation consists of several interdependent stages controlled by a wide range of mediators. Those include acute phase proteins, heat shock proteins, complement components, biogenic amines, cytokines, lipid-derived mediators, reactive oxygen species, nitric oxide, proteolytic enzymes, and kinins. Due to the strategic location in the body, mast cells play a protective role in the inflammatory process, through its initiation, amplification, and resolution. Mast cells degranulate and/or newly produce, and release various mediators classified into three groups: preformed mediators, de novo synthesised lipid mediators, and newly synthesised cytokines. Those mediators have an impact on different processes occurring during inflammation, inter alia, they influence blood vessels leading to dilation, enhanced adhesion molecule expression, and increased permeability. Furthermore, mast cell mediators play a pivotal role in inflammatory cell chemotaxis, degradation of extracellular matrix proteins, impact on stationery cells and resolution of inflammation. The release of mast cell mediators and their actions constitute a highly complex and still not fully understood mechanism, which warrants further studies of the action of mast cells in inflammation. This review will focus on the current knowledge concerning the broad role of mast cells in the inflammatory process.

DNA-PK inhibition by NU7441 sensitizes breast cancer cells to ionizing radiation and doxorubicin.

DNA-dependent protein kinase (DNA-PK) plays a key role in the repair of DNA double-strand breaks (DSBs) that are probably the most deleterious form of DNA damage. Inhibition of DNA-PK has been considered as an attractive approach to decrease resistance to therapeutically induced DNA DSBs. Ionizing radiation (IR) and doxorubicin, which induce DSBs, are used in the treatment of breast cancer. We determined the cellular concentration of DNA-PK and other DSB-activated kinases: ATM and ATR and the effect of DNA-PK inhibition by NU7441 on DNA repair, cell cycle, and survival after IR or doxorubicin treatment in three human breast cancer cell lines (MCF-7, MDA-MB-231, and T47D) representing different breast cancer subtypes. T47D cells had the highest expression of DNA-PKcs, ATM, and ATR and the most rapid rate of DNA DSB repair. IR caused a 10- to 16-fold increase in DNA-PK activity and two to threefold induction of ATM in all 3 cell lines. NU7441 inhibited IR-induced DNA-PK activity in all cell lines with IC50s in the range 0.17-0.25 µM. NU7441 retarded the repair of DSB and significantly increased the sensitivity of all cell lines to IR (4- to 12-fold) and doxorubicin (3- to 13-fold). The greatest sensitization by NU7441 was observed in MDA-MB-231 cells. NU7441 affected the cell cycle distribution in all studied cell lines; increasing accumulation of cells in G2/M phase after DNA damage. Our data indicate that DNA-PK might be an effective target for chemo- and radio-potentiation in breast cancer and suggest that further development of DNA-PK inhibitors for clinical use is warranted.

Upstream stimulating factors regulate the expression of RORγT in human lymphocytes.

Retinoic acid-related orphan receptor γT (RORγT) is the orphan nuclear receptor that regulates the development of Th17 cells and the expression of IL-17. The differentiation of Th17 cells is associated with the upregulation of RORγT mRNA, and the mechanisms regulating that process in human cells are not well understood. We investigated the transcriptional regulation of RORγT in a human lymphocytic cell line and Th17 differentiated from naive CD4+ cells from human peripheral blood. A series of experiments, including 5' deletion and in situ mutagenesis analysis of the human RORγT promoter, chromatin immunoprecipitation, and overexpression of selected transcription factors, revealed that the transcription factors upstream stimulatory factor 1 (USF-1) and USF-2 are indispensable for the transcription of RORγT in human lymphocytes. There was also upregulation of USF-1 and USF-2 during the differentiation of Th17 cells from naive CD4+ cells. In this article, we report the first analysis, to our knowledge, of the human RORγT promoter and demonstrate the role of the USF-1 and USF-2 transcription factors in regulating the expression of RORγT in human lymphocytes. Thus, USFs are important for the molecular mechanisms of Th17 differentiation, and possible changes in the expression of USFs might be of interest for inflammatory conditions with a Th17 component. Furthermore, these observations suggest a possible link between metabolic disorders in which the role of glucose-induced USF expression has already been established and autoimmune diseases in which the upregulation of RORγT is frequently detected.

Novel multiplex-PCR test for Escherichia coli detection.

Escherichia coli is a diverse and ubiquitous strain of both commensal and pathogenic bacteria. In this study, we propose the use of multiplex polymerase chain reaction (PCR), using amplification of three genes (cydA, lacY, and ydiV), as a method for determining the affiliation of the tested strains to the E. coli species. The novelty of the method lies in the small number of steps needed to perform the diagnosis and, consequently, in the small amount of time needed to obtain it. This method, like any other, has some limitations, but its advantage is fast, cheap, and reliable identification of the presence of E. coli. Sequences of the indicated genes from 1,171 complete E. coli genomes in the NCBI database were used to prepare the primers. The developed multiplex PCR was tested on 47,370 different Enterobacteriaceae genomes using in silico PCR. The sensitivity and specificity of the developed test were 95.76% and 99.49%, respectively. Wet laboratory analyses confirmed the high specificity, repeatability, reproducibility, and reliability of the proposed test. Because of the detection of three genes, this method is very cost and labor-effective, yet still highly accurate, specific, and sensitive in comparison to similar methods. IMPORTANCE: Detection of E. coli from environmental or clinical samples is important due to the common occurrence of this species of bacteria in all human and animal environments. As commonly known, these bacteria strains can be commensal and pathogenic, causing numerous infections of clinical importance, including infections of the digestive system, urinary, respiratory, and even meninges, particularly dangerous for newborns. The developed multiplex polymerase chain reaction test, confirming the presence of E. coli in samples, can be used in many laboratories. The test provides new opportunities for quick and cheap analyses, detecting E. coli using only three pairs of primers (analysis of the presence of three genes) responsible for metabolism and distinguishing E. coli from other pathogens from the Enterobacteriaceae family. Compared to other tests previously described in the literature, our method is characterized by high specificity and sensitivity.

A Rapid and Inexpensive PCR Test for Mastitis Diagnosis Based on NGS Data.

Mastitis is a common mammary gland disease of dairy cattle caused by a wide range of organisms including bacteria, fungi and algae. Mastitis contributes to economic losses of dairy farms due to reduced yield and poor quality of milk. Since the correct identification of pathogens responsible for the development of mastitis is crucial to the success of treatment, it is necessary to develop a quick and accurate test to distinguish the main pathogens causing this disease. In this paper, we describe the development of a test based on the multiplex polymerase chain reaction (PCR) method allowing for the identification of Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis and Staphylococcus aureus. When creating our test, we relied on the results from new generation sequencing (NGS) for accurate determination of species affiliation. The multiplex PCR test was verified on 100 strains including veterinary samples, ATCC and Polish Collection of Microorganisms (PCM) reference strains. The obtained results indicate that this test is accurate and displays high specificity. It may serve as a valuable molecular tool for the detection of major mastitis pathogens.

Epigenetic regulation of CD34 and HIF1A expression during the differentiation of human mast cells.

Mast cells differentiate from circulating pluripotent hematopoietic progenitors. During this differentiation, the progenitor cells are exposed to changes in oxygen availability. HIF1A is the major sensor of oxygen concentration in mammalian cells. We investigated the expression of HIF1A during the in vitro differentiation of peripheral blood-derived progenitors into human mast cells. In a series of experiments, we determined the changes in CD34 expression, selected mast cell markers, and HIF1A in human mast cell cultures. While the expression of CD34 dramatically decreased, the expression of mast cell-specific genes, including FCER1A, MS4A2, TPSB2, and CMA1, steadily increased. HIF1A expression similarly increased during mast cell differentiation, reaching its maximum level at five weeks of culture. The analysis of the promoter methylation status showed decreasing levels of methylation at the HIF1A promoter, increasing levels of methylation at the CD34 promoter, and no significant changes in other genes. In silico analysis of the promoter regions of these genes revealed large CpG islands in close proximity to the HIF1A and CD34 transcription initiation sites, but not in other investigated genes. In conclusion, in vitro mast cell differentiation was associated with decreased CD34 expression and increased HIF1A expression. These changes were paralleled with changes in the methylation status of the respective promoters, suggesting that DNA methylation-dependent epigenetic regulation mediates the gene expression changes involved in maintaining the phenotype of hematopoietic stem cells and mature mast cells. Therefore, the baseline expression of HIF1A is epigenetically regulated in a cell type- and differentiation stage-specific fashion.

Hypoxia modulates human mast cell adhesion to hyaluronic acid.

Hypoxia is an inherent factor in the inflammatory process and is important in the regulation of some immune cell functions, including the expression of mast cell pro- and anti-inflammatory mediators. Hypoxia also influences cell adhesion to the extracellular matrix (ECM). Hyaluronic acid is one of the major components of the ECM that is involved in inflammatory and tissue regeneration processes in which mast cells play a prominent role. This prompted us to investigate the effects of hypoxia on the expression of hyaluronic acid receptors in mast cells and mast cell adhesion to this ECM component. We found that human LAD2 mast cells spontaneously adhered to hyaluronic acid in a CD44-dependent manner and that reduced oxygen concentrations inhibited or even completely abolished this adhesion process. The mechanism of hypoxia downregulation of mast cell adhesion to hyaluronic acid did not involve a decrease in CD44 expression and hyaluronidase-mediated degradation of adhesion substrates but rather conformational changes in the avidity of CD44 to hyaluronic acid. Hypoxia-mediated regulation of mast cell adhesion to extracellular matrix components might be involved in the pathogenic accumulation of mast cells observed in the course of certain diseases including rheumatoid arthritis and cancer.

RT-qPCR-based tests for SARS-CoV-2 detection in pooled saliva samples for massive population screening to monitor epidemics.

Swab, RT-qPCR tests remain the gold standard of diagnostics of SARS-CoV-2 infections. These tests are costly and have limited throughput. We developed a 3-gene, seminested RT-qPCR test with SYBR green-based detection designed to be oversensitive rather than overspecific for high-throughput diagnostics of populations. This two-tier approach depends on decentralized self-collection of saliva samples, pooling, 1st-tier testing with highly sensitive screening test and subsequent 2nd-tier testing of individual samples from positive pools with the IVD test. The screening test was able to detect five copies of the viral genome in 10 µl of isolated RNA with 50% probability and 18.8 copies with 95% probability and reached Ct values that were highly linearly RNA concentration-dependent. In the side-by-side comparison, the screening test attained slightly better results than the commercially available IVD-certified RT-qPCR diagnostic test DiaPlexQ (100% specificity and 89.8% sensitivity vs. 100% and 73.5%, respectively). Testing of 1475 individual clinical samples pooled in 374 pools of four revealed 0.8% false positive pools and no false negative pools. In weekly prophylactic testing of 113 people within 6 months, a two-tier testing approach enabled the detection of 18 infected individuals, including several asymptomatic individuals, with substantially lower cost than individual RT-PCR testing.

Procyanidins from Japanese quince (Chaenomeles japonica) fruit induce apoptosis in human colon cancer Caco-2 cells in a degree of polymerization-dependent manner.

Plant proanthocyanidins, including procyanidins, display various biological activities. Here we report an inhibition of human colon cancer Caco-2 cell growth by the extract from Japanese quince fruit and the procyanidin-rich fractions of the extract. We observed that the amount of apoptotic Caco-2 cells increased by 52.1% vs. control after 72-h incubation with 50 µg extract/mL, as assessed by flow cytometry and image cytometry. Under the same experimental conditions the corresponding values for human colon cancer HT-29 cells and for rat normal intestinal IEC-6 cells were 5.0% and 8.1%, respectively. The extract fractions enriched with higher oligomers exhibited the highest proapoptotic activity. In conclusion, the Japanese quince procyanidins exhibited proapoptotic activity in Caco-2 cells within a submilimolar concentration range.

Nucleocapsid and Spike Proteins of the Coronavirus SARS-CoV-2 Induce IL6 in Monocytes and Macrophages-Potential Implications for Cytokine Storm Syndrome.

The pandemic of the new coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) has led to the deaths of more than 1.5 million people worldwide. SARS-CoV-2 causes COVID-19, which exhibits wide variation in the course of disease in different people, ranging from asymptomatic and mild courses to very severe courses that can result in respiratory failure and death. Despite the rapid progression of knowledge, we still do not know how individual cells of the immune system interact with the virus or its components, or how immune homeostasis becomes disrupted, leading to the rapid deterioration of a patient's condition. In the present work, we show that SARS-CoV-2 proteins induce the expression and secretion of IL-6 by human monocytes and macrophages, the first line cells of antiviral immune responses. IL-6 may play a negative role in the course of COVID-19 by inhibiting Th1-dependent immunity and stimulating Th17 lymphocytes, thus leading to an increased probability of a cytokine storm.

Recombinant immunotoxin targeting GPC3 is cytotoxic to H446 small cell lung cancer cells.

Glypican-3 (GPC3) is a cell membrane glycoprotein that regulates cell growth and proliferation. Aberrant expression or distribution of GPC3 underlies developmental abnormalities and the development of solid tumours. The strongest evidence for the participation of GPC3 in carcinogenesis stems from studies on hepatocellular carcinoma and lung squamous cell carcinoma. To the best of our knowledge, the role of the GPC3 protein and its potential therapeutic application have never been studied in small cell lung carcinoma (SCLC), despite the known involvement of associated pathways and the high mortality caused by this disease. Therefore, the aim of the present study was to examine GPC3 targeting for SCLC immunotherapy. An immunotoxin carrying an anti-GPC3 antibody (hGC33) and Pseudomonas aeruginosa exotoxin A 38 (PE38) was generated. This hGC33-PE38 protein was overexpressed in E. coli and purified. ADP-ribosylation activity was tested in vitro against eukaryotic translation elongation factor 2. Cell internalisation ability was confirmed by confocal microscopy. Cytotoxicity was analysed by treating liver cancer (HepG2, SNU-398 and SNU-449) and lung cancer (NCI-H510A, NCI-H446, A549 and SK-MES1) cell lines with hGC33-PE38 and estimating viable cells number. A BrdU assay was employed to verify anti-proliferative activity of hGC33-PE38 on treated cells. Fluorescence-activated cell sorting was used for the detection of cell membrane-bound GPC3. The hGC33-PE38 immunotoxin displayed enzymatic activity comparable to native PE38. The protein was efficiently internalised by GPC3-positive cells. Moreover, hGC33-PE38 was cytotoxic to HepG2 cells but had no effect on known GPC3-negative cell lines. The H446 cells were sensitive to hGC33-PE38 (IC50, 70.6±4.6 ng/ml), whereas H510A cells were resistant. Cell surface-bound GPC3 was abundant on the membranes of H446 cells, but absent on H510A. Altogether, the present findings suggested that GPC3 could be considered as a potential therapeutic target for SCLC immunotherapy.

Examining the Effects of an Anti-Salmonella Bacteriophage Preparation, BAFASAL®, on Ex-Vivo Human Gut Microbiome Composition and Function Using a Multi-Omics Approach.

Salmonella infections (salmonellosis) pose serious health risks to humans, usually via food-chain contamination. This foodborne pathogen causes major food losses and human illnesses, with significant economic impacts. Overuse of antibiotics in the food industry has led to multidrug-resistant strains of bacteria, and governments are now restricting their use, leading the food industry to search for alternatives to secure food chains. Bacteriophages, viruses that infect and kill bacteria, are currently being investigated and used as replacement treatments and prophylactics due to their specificity and efficacy. They are generally regarded as safe alternatives to antibiotics, as they are natural components of the ecosystem. However, when specifically used in the industry, they can also make their way into humans through our food chain or exposure, as is the case for antibiotics. In particular, agricultural workers could be repeatedly exposed to bacteriophages supplemented to animal feeds. To our knowledge, no studies have investigated the effects of such exposure to bacteriophages on the human gut microbiome. In this study, we used a novel in-vitro assay called RapidAIM to investigate the effect of a bacteriophage mixture, BAFASAL®, used in poultry farming on five individual human gut microbiomes. Multi-omics analyses, including 16S rRNA gene sequencing and metaproteomic, revealed that ex-vivo human gut microbiota composition and function were unaffected by BAFASAL® treatment, providing an additional measure for its safety. Due to the critical role of the gut microbiome in human health and the known role of bacteriophages in regulation of microbiome composition and function, we suggest assaying the impact of bacteriophage-cocktails on the human gut microbiome as a part of their safety assessment.

DNA methylation-dependent suppression of HIF1A in an immature hematopoietic cell line HMC-1.

The HMC-1 cell line represents the phenotype of immature mast cells. The HIF1A gene product HIF-1alpha plays key roles in maintaining oxygen homeostasis in eukaryotic organisms and is involved in many processes, including immune response and hematopoiesis. In this study we investigated HIF1A expression in HMC-1 immature hematopoietic cells and CD34+ hematopoietic progenitors. HMC-1 cells exhibited exceptionally low levels of HIF1A expression compared to other cell lines as determined by real-time PCR, and multipotent CD34+ hematopoietic progenitors in bone marrow exhibited significantly lower levels of HIF1A mRNA compared to mature blood cells in peripheral blood. We searched for the mechanisms responsible for suppression of HIF1A expression in HMC-1 cells and obtained evidence for a DNA methylation-dependent process. In vitro methylation of the HIF1A promoter resulted in a decrease in its transcriptional activity and the level of DNA methylation in the HIF1A promoter region in analyzed cell lines was negatively correlated with HIF1A expression. Furthermore, the DNA demethylating agent 5'-azacytidine increased HIF1A expression, and MeCP2 protein was preferentially associated with the HIF1A promoter in vivo. In conclusion, we report that the HIF1A gene in HMC-1 immature hematopoietic cells is suppressed by a process dependent on DNA methylation, and we present evidence indicating downregulation of HIF1A expression in multipotent CD34+ hematopoietic progenitors.

HIF-1alpha is up-regulated in activated mast cells by a process that involves calcineurin and NFAT.

Mast cells play important roles in many pathological conditions where local hypoxia is observed, including asthma, rheumatic diseases, and certain types of cancer. Here, we investigated how expression of the hypoxia-inducible factor 1, alpha subunit gene (HIF1A), is regulated in mast cells. The product of HIF1A is hypoxia-inducible factor 1alpha (HIF-1alpha), is a major nuclear transcription factor modulating gene expression in response to hypoxic conditions. We observed that under hypoxic conditions, exposure of mast cells to ionomycin and substance P resulted in significant up-regulation of HIF1A expression as compared with resting mast cells incubated under identical conditions. The ionomycin-mediated increase in HIF-1alpha protein levels was sensitive to the transcription inhibitor actinomycin D and to inhibitors of calcineurin, cyclosporin A (CsA), and FK506. The increased HIF-1alpha protein level was paralleled by a severalfold increase in HIF-1alpha mRNA that could be also inhibited with actinomycin D and CsA. The HIF1A promoter activity was significantly increased in ionomycin-activated mast cells, and the promoter activity could be inhibited by CsA and FK506. Furthermore, in situ mutagenesis experiments showed that the ionomycin-mediated HIF1A promoter activity depends on a conservative NFAT-binding site. Thus, accumulation of HIF-1alpha in activated mast cells requires up-regulation of HIF1A gene transcription and depends on the calcineurin-NFAT signaling pathway.

SARS-CoV-2 Proteins Induce IFNG in Th1 Lymphocytes Generated from CD4+ Cells from Healthy, Unexposed Polish Donors.

The outbreak of the SARS-CoV-2 virus in December 2019 has caused the deaths of several hundred thousand people worldwide. Currently, the pathogenesis of COVID-19 is poorly understood. During the course of COVID-19 infection, many patients experience deterioration, which might be associated with systemic inflammation and cytokine storm syndrome; however, other patients have mild symptoms or are asymptomatic. There are some suggestions that impaired cellular immunity through a reduction in Th1 response and IFNG (interferon gamma) expression, as well as cross-reactivity with common cold coronaviruses, might be involved in the differential COVID-19 course. Here, we show that CD4+ cells isolated from unexposed healthy donors that were differentiated towards the Th1 lineage in the presence of SARS-CoV-2 proteins exhibited induction of IFNG. Interestingly, the same cells induced to differentiate towards a Th17 lineage did not exhibit changes in IFNG expression or Th17-related cytokines. This suggests the cellular response to SARS-CoV-2 viral proteins is primarily associated with Th1 lymphocytes and may be dependent on past infections with common cold coronaviruses or vaccinations that induce unspecific cellular responses, e.g., BCG (Bacillus Calmette-Guérin). Thus, our results might explain the high variability in the course of COVID-19 among populations of different countries.