Serum Concentrations of TIM-3, LAG-3, and PD-1 in Patients with Hemorrhagic Fever with Renal Syndrome.

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.

Evaluation of the Neutralizing Antibody STE90-C11 against SARS-CoV-2 Delta Infection and Its Recognition of Other Variants of Concerns.

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.

Is Better Standardization of Therapeutic Antibody Quality in Emerging Diseases Epidemics Possible?

During the ongoing COVID-19 epidemic many efforts have gone into the investigation of the SARS-CoV-2-specific antibodies as possible therapeutics. Currently, conclusions cannot be drawn due to the lack of standardization in antibody assessments. Here we describe an approach of establishing antibody characterisation in emergent times which would, if followed, enable comparison of results from different studies. The key component is a reliable and reproducible assay of wild-type SARS-CoV-2 neutralisation based on a banking system of its biological components - a challenge virus, cells and an anti-SARS-CoV-2 antibody in-house standard, calibrated to the First WHO International Standard immediately upon its availability. Consequently, all collected serological data were retrospectively expressed in an internationally comparable way. The neutralising antibodies (NAbs) among convalescents ranged from 4 to 2869 IU mL-1 in a significant positive correlation to the disease severity. Their decline in convalescents was on average 1.4-fold in a one-month period. Heat-inactivation resulted in 2.3-fold decrease of NAb titres in comparison to the native sera, implying significant complement activating properties of SARS-CoV-2 specific antibodies. The monitoring of NAb titres in the sera of immunocompromised COVID-19 patients that lacked their own antibodies evidenced the successful transfusion of antibodies by the COVID-19 convalescent plasma units with NAb titres of 35 IU mL-1 or higher.

Collection of Monoclonal Antibodies Targeting SARS-CoV-2 Proteins.

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.

Non-toxic fluorescent phosphonium probes to detect mitochondrial potential.

We evaluated our phosphonium-based fluorescent probes for selective staining of mitochondria. Currently used probes for monitoring mitochondrial membrane potential show varying degrees of interference with cell metabolism, photo-induced damage and probe binding. Here presented probes are characterised by highly efficient cellular uptake and specific accumulation in mitochondria. Fluorescent detection of the probes was accomplished using flow cytometry and confocal microscopy imaging of yeast and mammalian cells. Toxicity analysis (impedimetry-xCELLigence for the cellular proliferation and Seahorse technology for respiratory properties) confirms that these dyes exhibit no-toxicity on mitochondrial or cellular functioning even for long time incubation. The excellent chemical and photophysical stability of the dyes makes them promising leads toward improved fluorescent probes. Therefore, the probes described here offer to circumvent the problems associated with existing-probe's limitations.

Diminished Resistance to Hyperoxia in Brains of Reproductively Senescent Female CBA/H Mice.

BACKGROUND We have explored sex differences in ability to maintain redox balance during acute oxidative stress in brains of mice. We aimed to determine if there were differences in oxidative/antioxidative status upon hyperoxia in brains of reproductively senescent CBA/H mice in order to elucidate some of the possible mechanisms of lifespan regulation. MATERIAL AND METHODS The brains of 12-month-old male and female CBA/H mice (n=9 per sex and treatment) subjected to 18-h hyperoxia were evaluated for lipid peroxidation (LPO), antioxidative enzyme expression and activity - superoxide dismutase 1 and 2 (Sod-1, Sod-2), catalase (Cat), glutathione peroxidase 1 (Gpx-1), heme-oxygenase 1 (Ho-1), nad NF-E2-related factor 2 (Nrf2), and for 2-deoxy-2-[18F] fluoro-D-glucose (18FDG) uptake. RESULTS No increase in LPO was observed after hyperoxia, regardless of sex. Expression of Nrf-2 showed significant downregulation in hyperoxia-treated males (p=0.001), and upregulation in hyperoxia-treated females (p=0.023). Also, in females hyperoxia upregulated Sod-1 (p=0.046), and Ho-1 (p=0.014) genes. SOD1 protein was upregulated in both sexes after hyperoxia (p=0.009 for males and p=0.011 for females). SOD2 protein was upregulated only in females (p=0.008) while CAT (p=0.026) and HO-1 (p=0.042) proteins were increased after hyperoxia only in males. Uptake of 18FDG was decreased after hyperoxia in the back brain of females. CONCLUSIONS We found that females at their reproductive senescence are more susceptible to hyperoxia, compared to males. We propose this model of hyperoxia as a useful tool to assess sex differences in adaptive response to acute stress conditions, which may be partially responsible for observed sex differences in longevity of CBA/H mice.

The role of 17ß-estradiol in the regulation of antioxidant enzymes via the Nrf2-Keap1 pathway in the livers of CBA/H mice.

AIMS: We aimed to explore the impact of surgical 17ß-estradiol (E2) deprivation/administration on the expression of antioxidant enzymes with an emphasis on the alteration of the NF-E2-related factor 2/Kelch-like ECH-associated protein 1 (Nrf2/Keap1) pathway under physiological conditions in the livers of CBA/H mice of both sexes. MAIN METHODS: Hepatic oxidative stress markers were determined by measuring lipid peroxidation and DNA damage using the comet assay. The expression and activities of two isoforms of superoxide dismutase (Sod-1, Sod-2) and catalase (Cat) were studied using real-time PCR, Western blot and spectrophotometrical analyses. The effect of E2 on Nrf2/Keap1 protein levels and localization was assessed using cytosolic and nuclear fractions. KEY FINDINGS: We demonstrate the E2-mediated repression of the antioxidant enzymes Sod-1, Sod-2 and Cat in the livers of ovariectomized mice treated with E2 and its association with a decreased level of Nrf2/Keap1 proteins in the nucleus. We observed beneficial effects of long-term E2 administration on lipid peroxidation but not on DNA damage in the livers of ovariectomized mice. SIGNIFICANCE: The results of this study may additionally confirm the protective ability of E2 in prolonging the onset of age-related disease in females that ultimately contributes to their longer lifespan.

Antitumor effect of Croatian propolis as a consequence of diverse sex-related dihydropyrimidine dehydrogenase (DPD) protein expression.

The aim of this study was to detect the antitumor properties of Croatian propolis in BALB/c male and female mice injected with 4T1 mammary carcinoma. Furthermore, the gender-dependence of this effect and the possible involvement of combined effect of propolis and 5-Fluorouracil (5FU) on dihydropyrimidine dehydrogenase (DPD) transcriptional and translational level, were determined. In combination with 5FU propolis treatment induced gender-related effects. The results of the study revealed that pretreatment of mice with propolis combined with 5FU treatment prolonged the suppressive effect of 5FU on tumor growth and reduced the number of metastasis only in male mice. Only males pretreated with propolis prior to 5FU administration had decreased DPD protein level indicating higher sensitivity to 5FU. Thus, benefitial effects of propolis in male tumor-bearing mice treated with 5FU might be explained by increased sensitivity to 5FU as the result of translationally downregulated DPD.

Superoxide dismutase and cytochrome P450 isoenzymes might be associated with higher risk of renal cell carcinoma in male patients.

Literature data support the hypothesis that oxidative stress and the accompanying antioxidant defense might play an important role in renal cell carcinoma (RCC) growth and progression. It is also known that the incidence of renal tumors is two times higher in men than in women. Thus, the aim of this study was to determine whether the oxidant/antioxidant profile of renal cell carcinoma tissue, adjacent to tumor tissue and nontumor tissue was different in male and female patients. Significantly higher lipid peroxidation (LPO) in renal cell carcinoma tissue compared to nontumor tissue was demonstrated only in male patients. Besides, gender-related difference in copper zinc superoxide dismutase (CuZnSOD) and manganese superoxide dismutase (MnSOD) in nontumor and renal cell carcinoma tissue was obtained at the level of transcription, translation and activity of these antioxidant isoenzymes. Morever, we demonstrated that the gene expression of 3 CYPs out of 7 was altered; CYP2D6 mRNA was decreased in both sexes while gender-related suppression of mRNA for CYP2E1 (women) and CYP2C19 (men) was observed. Taken together, these parameters might be potentially responsible for higher risk of renal cell carcinoma in men than in women.

Cytochrome P450 gender-related differences in response to hyperoxia in young CBA mice.

Cytochrome P450 monooxygenases (CYPs) represent large class of heme-containing enzymes that catalyze the metabolism of various endogenous and exogenous substrates. Although they are found in many tissues, the function of the particular subset of their isoforms does not appear to be the same. Many CYP genes exhibit sexually dimorphic expression, while others are sex-independent. Moreover, as a source of reactive oxygen species (ROS), P450 system is believed to play the important role in various pathological conditions and diseases. The aim of this study was to observe the effect of hyperoxia on oxidant/antioxidant status in the liver of young male and female mice and to determine whether the observed effects are associated with the expression of Heme oxygenase-1 (HO-1) and CYP genes associated with stress (Cyp1a1, Cyp1a2, Cyp2a5, and Cyp2e1) or stress and gender-related responses (Cyp2b9). In this study, we demonstrated gender-related effect of hyperoxia on oxidant/antioxidant status and on expression of certain P450 enzymes. Our results suggest that females are less susceptible to hyperoxia induced oxidative stress by two major mechanisms: upregulated expression of HO-1 genes and different expression of certain P450 enzymes. Therefore, our study could provide additional data of gender-dependent responses in susceptibility to oxidative stress, chemical toxicity and drug efficiency in treatment of diseases.

Cyp4a14 overexpression induced by hyperoxia in female CBA mice as a possible contributor of increased resistance to oxidative stress.

The beneficial effects of hyperoxia have been noted in treatment of several diseases and pathological states. However, the excessive production of ROS under hyperoxic conditions can directly damage cellular macromolecules if the imbalance in antioxidant status exists. Cytochrome P450 (Cyp) 4a14 has an important role in the metabolism of lipids and as a source of ROS in oxidative stress. This study investigated the oxidant/antioxidant status as a response to hyperoxia treatment in liver of young CBA/Hr mice of both sexes and whether the observed response is mediated by Cyp4a14 via PPAR isoforms in a sex-dependent manner. The overexpression of Cyp4a14, lack of both LPO and of 4-hydroxynonenal(HNE)-protein adducts revealed by immunohistochemical analysis in hyperoxia-treated females indicates their greater resistance to hyperoxia compared to males, which is parallelled to changes in PPARbeta/delta and PPARgamma expression. These results suggest the presence of sex-dependent changes in all investigated parameters, which points out sex-related susceptibility towards oxidative stress and hyperoxia treatment of various pathological conditions and diseases.

Improved antioxidant and anti-inflammatory potential in mice consuming sour cherry juice (Prunus Cerasus cv. Maraska).

The present investigation tested the in vivo antioxidant efficacy (superoxide dismutase, SOD; catalase, CAT; glutathione peroxidase; Gpx), lipid peroxidation (LPO) and anti-inflammatory properties (cyclooxygenase-2; COX-2) of sour cherry juices obtained from an autochthonous cultivar (Prunus cerasus cv. Maraska) that is grown in coastal parts of Croatia. Antioxidant potential was tested in mouse tissue (blood, liver, and brain), LPO (liver, brain) and anti-inflammatory properties in glycogen elicited macrophages. Additionally, the concentration of cyanidin-3-glucoside, cyanidin-3-rutinoside, pelargonidin-3-glucoside, pelargonidin-3-rutinoside and total anthocyanins present in Prunus cerasus cv. Maraska cherry juice was determined. Mice were randomly divided into a control group (fed with commercial food pellets) and 2 experimental groups (fed with commercial food pellets with 10% or 50% of cherry juice added). Among the anthocyanins, the cyanidin-3-glucoside was present in the highest concentration. These results show antioxidant action of cherry juice through increased SOD (liver, blood) and Gpx (liver) activity and decreased LPO concentration. The study highlights cherry juice as a potent COX-2 inhibitor and antioxidant in the liver and blood of mice, but not in the brain. Thus, according to our study, Prunus cerasus cv. Maraska cherry juice might potentially be used as an antioxidant and anti-inflammatory product with beneficial health-promoting properties.