17β-estradiol (E₂) has well-established cardioprotective, antioxidant and neuroprotective role, and exerts a vast range of biological effects in both sexes. Dipeptidyl peptidase III (DPP III) is protease involved as activator in Keap1–Nrf2 signalling pathway, which is important in cellular defense to oxidative and electrophilic stress. It is generally accepted that oxidative stress is crucial in promoting liver diseases. To examine the effect of E₂ on the expression of DPP III and haeme oxygenase 1 (HO-1) in liver of adult CBA/H mice of both sexes. Gene and protein expressions of studied enzymes were determined by quantitative real-time PCR and Western blot analysis. Immunohistochemistry was performed to analyse the localization of both proteins in different liver cell types. Ovariectomy diminished expression of DPP III and HO-1 proteins. E₂ administration abolished this effect, and even increased these proteins above the control. A significant enhancement in DPP III protein was found in E₂-treated males, as well. A decrease in the expression of HO-1, but not of the DPP III gene, was detected in the liver of ovariectomized females. HO-1 protein was found localized in the pericentral areas of hepatic lobules (Kupffer cells and hepatocytes), whilst DPP III showed a uniform distribution within hepatic tissue. We demonstrate for the first time that E₂ influences the protein level of DPP III in vivo, and confirm earlier finding on HO-1 gene upregulation by 17β-estradiol. These results additionally confer new insights into complexity of protective action of E₂.

As of now, the COVID-19 pandemic has spread to over 770 million confirmed cases and caused approximately 7 million deaths. While several vaccines and monoclonal antibodies (mAb) have been developed and deployed, natural selection against immune recognition of viral antigens by antibodies has fueled the evolution of new emerging variants and limited the immune protection by vaccines and mAb. To optimize the efficiency of mAb, it is imperative to understand how they neutralize the variants of concern (VoCs) and to investigate the mutations responsible for immune escape. In this study, we show the in vitro neutralizing effects of a previously described monoclonal antibody (STE90-C11) against the SARS-CoV-2 Delta variant (B.1.617.2) and its in vivo effects in therapeutic and prophylactic settings. We also show that the Omicron variant avoids recognition by this mAb. To define which mutations are responsible for the escape in the Omicron variant, we used a library of pseudovirus mutants carrying each of the mutations present in the Omicron VoC individually. We show that either 501Y or 417K point mutations were sufficient for the escape of Omicron recognition by STE90-C11. To test how escape mutations act against a combination of antibodies, we tested the same library against bispecific antibodies, recognizing two discrete regions of the spike antigen. While Omicron escaped the control by the bispecific antibodies, the same antibodies controlled all mutants with individual mutations.

In early 2020, the COVID-19 pandemic sparked a global crisis that continues to pose a serious threat to human health and the economy. Further advancement in research is necessary and requires the availability of quality molecular tools, including monoclonal antibodies. Here, we present the development and characterization of a collection of over 40 new monoclonal antibodies directed against different SARS-CoV-2 proteins. Recombinant SARS-CoV-2 proteins were expressed, purified, and used as immunogens. Upon development of specific hybridomas, the obtained monoclonal antibody (mAb) clones were tested for binding to recombinant proteins and infected cells. We generated mAbs against structural proteins, the Spike and Nucleocapsid protein, several non-structural proteins (nsp1, nsp7, nsp8, nsp9, nsp10, nsp16) and accessory factors (ORF3a, ORF9b) applicable in flow cytometry, immunofluorescence, or Western blot. Our collection of mAbs provides a set of novel, highly specific tools that will allow a comprehensive analysis of the viral proteome, which will allow further understanding of SARS-CoV-2 pathogenesis and the design of therapeutic strategies.

Coronavirus disease (COVID-19) exhibits a wide spectrum of clinical severity and has been associated with specific biomarkers linked to disease progression and outcomes. This retrospective study analyzed sera from 1222 adult COVID-19 patients hospitalized at the University Hospital for Infectious Diseases in Croatia. We examined the association between several laboratory biomarker levels measured at patient admission and disease severity, fatal outcomes, viral variants and clinical parameters. Deceased patients and surviving patients with severe COVID-19 exhibited significantly elevated levels of several biomarkers on admission, including hs-troponin T, N-terminal pro-brain natriuretic peptide, creatine kinase, C-reactive protein, procalcitonin, interleukin-6, lactate dehydrogenase, lactate, urea and creatinine. Random forest models identified lymphocyte percentage, D-dimers, and hs-troponin T as the most important biomarkers for fatal outcome prediction, achieving 84.1% accuracy. Patients infected with the Delta SARS-CoV-2 variant exhibited significantly higher levels of proinflammatory, cardiac and renal biomarkers. Vaccination correlated with reduced proinflammatory parameters and higher lymphocyte proportions. Hypertension, chronic renal disease and diabetes were associated with increased cardiac, renal and metabolic biomarker levels, respectively. These findings highlight the association of several laboratory biomarkers with COVID-19 severity, viral variants, vaccination status and comorbidities, potentially offering prognostic insights into COVID-19 outcomes.

Hemorrhagic fever with renal syndrome (HFRS) is a rodent-borne disease widespread in Europe and Asia. HFRS is caused by negative-sensed single-stranded RNA orthohantaviruses transmitted to humans through inhaling aerosolized excreta of infected rodents. Symptoms of HFRS include acute kidney injury, thrombocytopenia, hemorrhages, and hypotension. The immune response raised against viral antigens plays an important role in the pathogenesis of HFRS. Inhibitory co-receptors are essential in regulating immune responses, mitigating immunopathogenesis, and reducing tissue damage. Our research showed an increased soluble form of inhibitory co-receptors TIM-3, LAG-3, and PD-1 in HFRS patients associated with disease severity. Our study aimed to investigate the impact of HFRS on the concentrations of soluble forms of inhibitory receptors TIM-3, LAG-3, and PD-1 in the patient’s serum and the potential correlation with key clinical parameters. Our study aimed to investigate the impact of HFRS on the concentrations of soluble forms of inhibitory receptors TIM-3, LAG-3, and PD-1 in the patient’s serum and their possible association with relevant clinical parameters. Using multiplex immunoassay, we found elevated levels of TIM-3, LAG-3, and PD-1 proteins in the serum of HFRS patients. Furthermore, increased levels were associated with creatinine, urea, lactate dehydrogenase concentrations, and platelet count. These findings suggest that these proteins play a role in regulating the immune response and disease progression.

At present, the novel pandemic coronavirus SARS-CoV-2 is a major global threat to human health and hence demands united research activities at different levels. Finding appropriate cell systems for drug screening and testing molecular interactions of the virus with the host cell is mandatory for drug development and understanding the mechanisms of viral entry and replication. For this, we selected human cell lines represented in the Cancer Cell Line Encyclopedia (CCLE) based on RNA-seq data determined transcript levels of ACE2 and TMPRSS2, two membrane proteins that have been identified to aid SARS-CoV-2 entry into the host cell. mRNA and protein expression of these host factors were verified via RQ-PCR and western blot. We then tested permissiveness of these cell lines towards SARS-CoV-2 infection, cytopathic effect, and viral replication finding limited correlation between receptor expression and infectability. One of the candidate cancer cell lines, the human colon cancer cell line CL-14, tested positive for SARS-CoV-2 infection. Our data argue that SARS-CoV-2 in vitro infection models need careful selection and validation since ACE2/TMPRSS2 receptor expression on its own does not guarantee permissiveness to the virus. Author summary: In the midst of the pandemic outbreak of corona-virus SARS-CoV-2 therapeutics for disease treatment are still to be tested and the virus-host-interactions are to be elucidated. Drug testing and viral studies are commonly conducted with genetically manipulated cells. In order to find a cell model system without genetic modification we screened human cell lines for two proteins known to facilitate entry of SARS-CoV-2. We confirmed and quantified permissiveness of current cell line infection models, but dismissed a number of receptor-positive cell lines that did not support viral replication. Importantly, ACE2/TMPRSS2 co-expression seems to be necessary for viral entry but is not sufficient to predict permissiveness of various cancer cell lines. Moreover, the expression of specific splice variants and the absence of missense mutations of the host factors might hint on successful infection and virus replication of the cell lines.