Pomegranate Peel Extract Differently Modulates Gene Expression in Gingiva-Derived Mesenchymal Stromal Cells under Physiological and Inflammatory Conditions.

Pomegranate has shown a favorable effect on gingivitis/periodontitis, but the mechanisms involved are poorly understood. The aim of this study was to test the effect of pomegranate peel extract (PoPEx) on gingiva-derived mesenchymal stromal cells (GMSCs) under physiological and inflammatory conditions. GMSC lines from healthy (H) and periodontitis (P) gingiva (n = 3 of each) were established. The lines were treated with two non-toxic concentrations of PoPEX (low-10; high-40 µg/mL), with or without additional lipopolysaccharide (LPS) stimulation. Twenty-four genes in GMSCs involved in different functions were examined using real-time polymerase chain reaction (RT-PCR). PoPEx (mostly at higher concentrations) inhibited the basal expression of IL-6, MCP-1, GRO-α, RANTES, IP-10, HIF-1α, SDF-1, and HGF but increased the expression of IL-8, TLR3, TGF-ß, TGF-ß/LAP ratio, IDO-1, and IGFB4 genes in H-GMSCs. PoPEx increased IL-6, RANTES, MMP3, and BMP2 but inhibited TLR2 and GRO-α gene expression in P-GMSCs. LPS upregulated genes for proinflammatory cytokines and chemokines, tissue regeneration/repair (MMP3, IGFBP4, HGF), and immunomodulation (IP-10, RANTES, IDO-1, TLR3, COX-2), more strongly in P-GMSCs. PoPEx also potentiated most genes' expression in LPS-stimulated P-GMSCs, including upregulation of osteoblastic genes (RUNX2, BMP2, COL1A1, and OPG), simultaneously inhibiting cell proliferation. In conclusion, the modulatory effects of PoPEx on gene expression in GMSCs are complex and dependent on applied concentrations, GMSC type, and LPS stimulation. Generally, the effect is more pronounced in inflammation-simulating conditions.

The comparison of SARS-CoV-2 antibody levels in medical personnel induced by different types of vaccines compared to the natural infection.

INTRODUCTION: During the last two and a half years, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has spread around the world. Most of the SARS-CoV-2 vaccines are designed to produce anti-SARS-CoV-2 immunoglobulin G (IgG) against the viral S-glycoprotein. The aim of this study was to measure the anti-S antibody titres among the medical personnel who had been fully vaccinated with different types of vaccines, and to compare them with those who were COVID-19 convalescents. MATERIAL AND METHODS: In this study serum was collected from 261 healthcare workers, of whom 227 were vaccinated, while 34 were recovered participants who were not immunised. Serum samples were collected 21 days after the first dose and 60 and 180 days after the second dose of the vaccines and tested with a commercial ELISA kit. RESULTS: The highest antibody level (12 AU/ml) was measured in the Pfizer-BioNTech group, followed by Sinopharm (9.3 AU/ml), Sputnik V (5.9 AU/ml), Sinovac (4.6 AU/ml) and Oxford/Astra- Zeneca vaccine (2.5 AU/ml) 60 days after the second dose of the vaccines (90 days after the first dose). The seropositivity rate for mRNA vaccine was 88.5%, for vector vaccines 86.2% and for inactivated vaccines 71.4%. When comparing these antibody levels with COVID-19 convalescents, higher antibody titres were found in vaccinated participants (5.76 AU/ml vs 7.06 AU/ml), but the difference was not significant (p = 0.08). CONCLUSIONS: Individuals vaccinated with mRNA and vector vaccines had a higher seroconversion rate compared to the group vaccinated with inactivated vaccines, or convalescents.

Immunomodulatory Activity of Punicalagin, Punicalin, and Ellagic Acid Differs from the Effect of Pomegranate Peel Extract.

BACKGROUND: Our recent study has shown that pomegranate peel extract (PEx) showed significant immunomodulatory activity, which might be caused by ellagitannins. The aim of this work was to test the hypothesis that ellagitannin components act synergistically in the modulation of cytokine production. METHODS: Human peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated with phytohemagglutinin and treated with different concentrations of PEx or punicalagin (PG), punicalin (PN), and ellagic acid (EA), alone or with their combinations. Cytotoxicity, cell proliferation, and cytokine production were determined. RESULTS: Non-cytotoxic concentrations of all compounds significantly inhibited cell proliferation. IC50 values (µg/mL) were: EA (7.56), PG (38.52), PEx (49.05), and PN (69.95). PEx and all ellagitannins inhibited the levels of TNF-α, IL-6, and IL-8, dose-dependently, and their combinations acted synergistically. PEx and all ellagitannins inhibited Th1 and Th17 responses, whereas the lower concentrations of PEx stimulated the production of IL-10, a Treg cytokine, as did lower concentrations of EA. However, neither component of ellagitannins increased Th2 response, as was observed with PEx. CONCLUSIONS: The combination of PG, PN, and EA potentiated the anti-inflammatory response without any significant synergistic down-modulatory effect on T-cell cytokines. The increased production of IL-10 observed with PEx could be attributable to EA, but the examined ellagitannins are not associated with the stimulatory effect of PEx on Th2 response.

Immunomodulatory Properties of Pomegranate Peel Extract in a Model of Human Peripheral Blood Mononuclear Cell Culture.

Pomegranate peel extract (PoPEx) has been shown to have antioxidant and anti-inflammatory properties, but its effect on the adaptive immune system has not been sufficiently investigated. In this study, the treatment of human peripheral blood mononuclear cells (PBMC) with PoPEx (range 6.25-400 µg/mL) resulted in cytotoxicity at concentrations of 100 µg/mL and higher, due to the induction of apoptosis and oxidative stress, whereas autophagy was reduced. At non-cytotoxic concentrations, the opposite effect on these processes was observed simultaneously with the inhibition of PHA-induced PBMC proliferation and a significant decrease in the expression of CD4. PoPEx differently modulated the expression of activation markers (CD69, CD25, ICOS) and PD1 (inhibitory marker), depending on the dose and T-cell subsets. PoPEx (starting from 12.5 µg/mL) suppressed the production of Th1 (IFN-γ), Th17 (IL-17A, IL-17F, and IL-22), Th9 (IL-9), and proinflammatory cytokines (TNF-α and IL-6) in culture supernatants. Lower concentrations upregulated Th2 (IL-5 and IL-13) and Treg (IL-10) responses as well as CD4+CD25hiFoxp3+ cell frequency. Higher concentrations of PoPEx increased the frequency of IL-10- and TGF-ß-producing T-cells (much higher in the CD4+ subset). In conclusion, our study suggested for the first time complex immunoregulatory effects of PoPEx on T cells, which could assist in the suppression of chronic inflammatory and autoimmune diseases.

Seroprevalence of SARS-CoV-2 antibodies among primary healthcare workers in the Republic of Srpska, Bosnia & Herzegovina: A cross-sectional study.

Healthcare workers (HCW) in primary healthcare centres in the Republic of Srpska, Bosnia and Herzegovina, are on the first combat line with COVID-19. This study aimed to assess the seroprevalence of SARS-CoV-2 among HCW at the primary healthcare centres and to analyse the risk exposure to COVID-19, clinical signs and vaccination status. A cross-sectional study was conducted among HCW at the selected primary healthcare centres between 19 March and 30 April 2021. Antibodies against the SARS-CoV-2 virus were detected by enzyme-linked immunosorbent assay (ELISA). A total of 1,023 HCW (mean age 45 years; 71% female) were included in the study. The anti-SARS-CoV-2 antibodies were detected in 69.5% of all participants. There was a significant difference in seropositivity among primary healthcare centres from different geographical regions. As many as 432 (42%) of all participants had confirmed COVID-19 symptoms before the study and, 84.8% of them were seropositive. This study showed that 702 primary HCW were vaccinated with any of these vaccines: Sputnik V, Sinopharm, Pfizer/Biontech. High titre of SARS-CoV-2 antibodies was found amongst those who received one (92.6%) or both (97.2%) doses of vaccines. In this study, we report high prevalence of SARS-CoV-2 antibody among HCW in primary healthcare in the Republic of Srpska, Bosnia and Herzegovina during the third pandemic wave.

Erythropoietin-Mediated Neuroprotection in Insects Suggests a Prevertebrate Evolution of Erythropoietin-Like Signaling.

The cytokine erythropoietin (Epo) mediates protective and regenerative functions in mammalian nervous systems via activation of poorly characterized receptors that differ from the "classical" homodimeric Epo receptor expressed on erythroid progenitor cells. Epo genes have been identified in vertebrate species ranging from human to fish, suggesting that Epo signaling evolved earlier than the vertebrate lineage. Studies on insects (Locusta migratoria, Chorthippus biguttulus, Tribolium castaneum) revealed Epo-mediated neuroprotection and neuroregeneration. Recombinant human Epo (rhEpo) prevents apoptosis by binding to a janus kinase-associated receptor, stimulation of STAT transcription factors, and generation of factors that prevent the activation of proapoptotic caspases. Insect neurons were also protected by a neuroprotective but nonerythropoietic Epo splice variant, suggesting similarity with mammalian neuroprotective but not with homodimeric "classical" Epo receptors. Additionally, rhEpo promotes the regeneration of neurites in primary cultured insect brain neurons and after nerve crush in an in vivo preparation. In contrast to neuroprotective and regenerative effects shared with mammalian species, no evidence for a role of Epo signaling in the regulation of neuro- or gliogenesis was found in insects. Similar structural and functional characteristics of the Epo binding receptors, partly shared transduction pathways that prevent apoptosis and the functional implication in neuroprotective and neuroregenerative processes in both mammalian and insect species, suggest that Epo-like signaling was already established in their last common ancestor. Originally functioning as a tissue-protective response to unfavorable physiological situations, cell injury, and pathogen invasion, Epo was later adapted as a humoral regulator of erythropoiesis in the vertebrate lineage.

Neuroprotection and endocytosis: erythropoietin receptors in insect nervous systems.

Erythropoietin (Epo) plays a dual role as an erythropoiesis-stimulating hormone and a locally produced cytoprotectant in various vertebrate tissues. Splice variants and engineered derivatives of Epo that mediate neuroprotection but do not stimulate erythropoiesis suggest that alternative receptors, different from the 'classical' homodimeric receptor involved in haematopoiesis, mediate neuroprotective Epo functions. Previous studies on grasshoppers demonstrated neuroprotective and neuroregenerative effects of Epo that involved similar transduction pathways as in mammals. To advance the characterization of yet unidentified neuroprotective Epo receptors, we studied the neuroprotective potency of the human non-erythropoietic Epo splice variant EV-3 in primary cultured locust brain neurons. We demonstrate that EV-3, like Epo, protects locust neurons from hypoxia-induced apoptotic death through activation of the Janus kinase/signal transducer and activator of transcription transduction pathway. Using the fluorescent dye FM1-43 to quantify endocytotic activity we show that both Epo and EV-3 increase the number of fluorescently labelled endocytotic vesicles. This reveals that binding of Epo to its neuroprotective receptor induces endocytosis, as it has been described for the mammalian homodimeric Epo-receptor expressed by erythroid progenitors. Reduction in Epo-stimulated endocytotic activity following pre-exposure to EV-3 indicated that both Epo and its splice variant bind to the same receptor on locust neurons. The shared neuroprotective potency of Epo and EV-3 in insect and mammalian neurons, in the absence of erythropoietic effects of EV-3 in mammals, suggests a greater similarity of the unidentified nervous Epo receptors (or receptor complexes) across phyla than between mammalian haematopoietic and neuroprotective receptors. Insects may serve as suitable models to evaluate the specific protective mechanisms mediated by Epo and its variants in non-erythropoietic mammalian tissues.