Staphylococcus aureus induces a muted host response in human blood that blunts the recruitment of neutrophils.

Staphylococcus aureus is an opportunistic pathogen and chief among bloodstream-infecting bacteria. S. aureus produces an array of human-specific virulence factors that may contribute to immune suppression. Here, we defined the response of primary human phagocytes following infection with S. aureus using RNA-sequencing (RNA-Seq). We found that the overall transcriptional response to S. aureus was weak both in the number of genes and in the magnitude of response. Using an ex vivo bacteremia model with fresh human blood, we uncovered that infection with S. aureus resulted in the down-regulation of genes related to innate immune response and cytokine and chemokine signaling. This muted transcriptional response was conserved across diverse S. aureus clones but absent in blood exposed to heat-killed S. aureus or blood infected with the less virulent staphylococcal species Staphylococcus epidermidis. Notably, this signature was also present in patients with S. aureus bacteremia. We identified the master regulator S. aureus exoprotein expression (SaeRS) and the SaeRS-regulated pore-forming toxins as key mediators of the transcriptional suppression. The S. aureus-mediated suppression of chemokine and cytokine transcription was reflected by circulating protein levels in the plasma. Wild-type S. aureus elicited a soluble milieu that was restrictive in the recruitment of human neutrophils compared with strains lacking saeRS. Thus, S. aureus blunts the inflammatory response resulting in impaired neutrophil recruitment, which could promote the survival of the pathogen during invasive infection.

Quantitative Analysis of Phagocytosis in Whole Blood Using Double Staining and Visualization.

Phagocytosis is an essential innate immunity function in humans and animals. A decrease in the ability to phagocytize is associated with many diseases and aging of the immune system. Assessment of phagocytosis dynamics requires quantification of bacteria inside and outside the phagocyte. Although flow cytometry is the most common method for assessing phagocytosis, it does not include visualization and direct quantification of location of bacteria. Here, we used double-labeled Escherichia coli cells to evaluate phagocytosis by flow cytometry (cell sorting) and confocal microscopy, as well as employed image cytometry to provide high-throughput quantitative and spatial recognition of the double-labeled E. coli associated with the phagocytes. Retention of pathogens on the surface of myeloid and lymphoid cells without their internalization was suggested to be an auxiliary function of innate immunity in the fight against infections. The developed method of bacterial labeling significantly increased the accuracy of spatial and quantitative measurement of phagocytosis in whole blood and can be recommended as a tool for phagocytosis assessment by image cytometry.

Study of the DNA damage and cell death in human peripheral blood mononuclear and HepG2/C3A cells exposed to the synthetic 3-(3-hydroxyphenyl)-7-hydroxycoumarin.

Hydroxycoumarins are an important source of biologically active compounds. Previous studies have shown that the number and position of the hydroxyl substituents in the scaffold play an important role for the observed biological activity. In the present study, 3-(3-hydroxyphenyl)-7-hydroxycoumarin was synthesized, and potential cytogenotoxic effects determined in human HepG2/C3A cells displaying phase 1 and phase 2 enzymes (metabolizing cell ability) and compared to human peripheral blood mononuclear cells (PBMC) without xenobiotics metabolizing capacity. Cell viability was determined with concentrations between 0.01 and 10 µg/ml of 3-(3-hydroxyphenyl)-7-hydroxycoumarin using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) and trypan blue tests. Genotoxicity was determined utilizing the comet assay, and the clastogenic/aneugenic potential employing the micronucleus (MN) test. The results of the in vitro cytotoxicity assays showed a significant decrease in cell viability of PBMC following exposure to 10 µg/ml concentration of the studied compound after 48 and 72 hr. Comet assay observations noted significant DNA damage in PBMC after 4 hr treatment. No marked cytogenotoxic effects were found in HepG2/C3A cells. No chromosomal mutations were observed in both cell lines. It is important to note that 3-(3-hydroxyphenyl)-7-hydroxycoumarin may exert beneficial pharmacological actions at the low micromolar range and with half-life less than 24 hr. Therefore, the results obtained encourage the continuation of studies on this new molecule for medicinal purposes, but its potential toxicity at higher concentrations and longer exposure times needs to be investigated in further studies.

Resolvins and cysteinyl-containing pro-resolving mediators activate resolution of infectious inflammation and tissue regeneration.

This review is a synopsis of the main points from the opening presentation by the authors in the Resolution of Inflammation session at the 8th European Workshop on Lipid Mediators held at the Karolinska Institute, Stockholm, Sweden, June 29th, 2022. Specialized pro-resolving mediators (SPM) promote tissue regeneration, control infections and resolution of inflammation. These include resolvins, protectins, maresins and the newly identified conjugates in tissue regeneration (CTRs). We reported mechanisms of CTRs in activating primordial regeneration pathways in planaria using RNA-sequencing. Also, the 4S,5S-epoxy-resolvin intermediate in the biosynthesis of resolvin D3 and resolvin D4 was prepared by total organic synthesis. Human neutrophils convert this to resolvin D3 and resolvin D4, while human M2 macrophages transformed this labile epoxide intermediate to resolvin D4 and a novel cysteinyl-resolvin that is a potent isomer of RCTR1. The novel cysteinyl-resolvin significantly accelerates tissue regeneration with planaria and inhibits human granuloma formation.

Genotoxicity and cytotoxicity potential of organoselenium compounds in human leukocytes in vitro.

In the current work, cytotoxicity and genotoxicity of different organoselenium compounds were examined using Trypan blue exclusion and alkaline comet assays with silver staining respectively. Leukocytes were subjected to a 3-hour incubation with organoselenium compounds at concentrations of 1, 5, 10, 25, 50, and 75 µM, or with the control vehicle (DMSO), at a temperature of 37 °C. The viability of the cells was evaluated using the Trypan blue exclusion method, while DNA damage was analyzed through the alkaline comet assay with silver staining. The exposure of leukocytes to different organoselenium compounds including i.e. (Z)-N-(pyridin-2-ylmethylene)-1-(2-((2-(1-((E)-pyridin-2-ylmethyleneamino)ethyl)phenyl)diselanyl)phenyl)ethanamine (C1), 2,2'(1Z,1'E)-(1,1'-(2,2'-diselanediylbis(2,1-phenylene))bis(ethane-1,1-diyl)) bis(azan-1-yl-1-ylidene)bis -methan-1-yl-1-ylidene)diphenol (C2), and dinaphthyl diselenide (NapSe)2, At concentrations ranging from 1 to 5 µM, no significant DNA damage was observed, as indicated by the absence of a noteworthy increase in the Damage Index (DI). Our results suggest that the organoselenium selenium compounds tested were not genotoxic and cytotoxic to human leukocytes in vitro at lower concentration. This study offers further insights into the genotoxicity profile of these organochalcogens in human leukocytes. Their genotoxicity and cytotoxicity effects at higher concentration are probably mediated through reactive oxygen species generation and their ability to catalyze thiol oxidation.

Role of HLA typing and chimerism analysis in post-liver transplantation GVHD in pediatrics: A systematic review.

BACKGROUND: GVHD is a well-documented complication after liver transplantation. GVHD occurs when donor immune cells mount a destructive immune response against host cells. The rarity of the GVHD complication and the nonspecific presentation of symptoms and histopathological features provide a diagnostic challenge. Therefore, diagnosis and initiation of treatment are often delayed. AIM: In this systematic review, we assessed relevant literature to better understand the utilization of HLA typing and chimerism analysis in liver transplantation. We mainly focused on their importance in diagnosing GVHD incidence after liver transplantation. RESULTS: A total of 18 articles reported 21 cases of GVHD after liver transplantation in pediatrics. Generally, there is a consensus on the advantage of HLA typing and chimerism analysis in confirming the diagnosis of GVHD after liver transplantation. However, there is an inconsistency in the timing and the application of the accurate HLA typing and chimerism analysis. CONCLUSION: Further studies are required to assess the incidence of GVHD post-LT and to determine the impact of HLA typing and chimerism analysis in assessing the risk, early determination of GVHD incidence, and improving outcomes. This systematic review highlights the gap in the field of liver transplantation and calls for revisiting the guidelines to consider HLA typing and chimerism analysis in predicting GVHD before transplantation and diagnosing GVHD incidence after liver transplantation.

Identification of Tumor Antigens Among the HLA Peptidomes of Glioblastoma Tumors and Plasma.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor with poor prognosis to most patients. Immunotherapy of GBM is a potentially beneficial treatment option, whose optimal implementation may depend on familiarity with tumor specific antigens, presented as HLA peptides by the GBM cells. Further, early detection of GBM, such as by a routine blood test, may improve survival, even with the current treatment modalities. This study includes large-scale analyses of the HLA peptidome (immunopeptidome) of the plasma-soluble HLA molecules (sHLA) of 142 plasma samples, and the membranal HLA of GBM tumors of 10 of these patients' tumor samples. Tumor samples were fresh-frozen immediately after surgery and the plasma samples were collected before, and at multiple visits after surgery. In total, this HLA peptidome analysis involved 52 different HLA allotypes and resulted in the identification of more than 35,000 different HLA peptides. Strong correlations were observed in the signal intensities and in the repertoires of identified peptides between the tumors and plasma-soluble HLA peptidomes of the individual patients, whereas low correlations were observed between these HLA peptidomes and the tumors' proteomes. HLA peptides derived from Cancer/Testis Antigens (CTAs) were selected based on their presence among the HLA peptidomes of the patients and absence of expression of their source genes from any healthy and essential human tissues, except from immune-privileged sites. Additionally, peptides were selected as potential biomarkers if their levels in the plasma-sHLA peptidome were significantly reduced after the removal of tumor mass. The CTAs identified among the analyzed HLA peptidomes provide new opportunities for personalized immunotherapy and for early diagnosis of GBM.

Polybia occidentalis and Polybia fastidiosa venom: a cytogenotoxic approach of effects on human and vegetal cells.

The venoms of wasps are a complex mixture of biologically active compounds, such as low molecular mass compounds, peptides, and proteins. The aim of the study was to evaluate the action of wasp venoms, Polybia occidentalis and Polybia fastidiosa, on the DNA of human leukocytes and on the cell cycle and genetic material of the plant model Lactuca sativa L. (lettuce). The cultured leukocytes were treated with the venoms and then evaluated by the comet assay. On another assay, seeds were exposed to a venom solution; the emitted roots were collected and the occurrence of cell cycle alterations (CCAs) and DNA fragmentation were evaluated by agarose gel electrophoresis and TUNEL assay. The results demonstrated that the venom of both wasps induces several CCAs and reduces the mitotic index (MI) on treated cells. They induced damage on human leukocytes DNA. High frequencies of fragments were observed in cells exposed to P. occidentalis venom, while those exposed to P. fastidiosa showed a high frequency of non-oriented chromosome. Both venoms induced the occurrence of various condensed nuclei (CN). This alteration is an excellent cytological mark to cell death (CD). Additionally, CD was evidenced by positive signals in TUNEL assay, by DNA fragmentation in agarose gel electrophoresis with vegetal cells, and by DNA fragmentation of the human leukocytes evaluated. Furthermore, human leukocytes exposed to the venom of P. fastidiosa had high rate of damage. The data demonstrate that both vegetal and human cells are adequate to evaluate the genotoxicity induced by venoms.

Identification of Tumor Antigens Among the HLA Peptidomes of Glioblastoma Tumors and Plasma.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor with poor prognosis to most patients. Immunotherapy of GBM is a potentially beneficial treatment option, whose optimal implementation may depend on familiarity with tumor specific antigens, presented as HLA peptides by the GBM cells. Furthermore, early detection of GBM, such as by a routine blood test, may improve survival, even with the current treatment modalities. This study includes large-scale analyses of the HLA peptidome (immunopeptidome) of the plasma-soluble HLA molecules (sHLA) of 142 plasma samples, and the membranal HLA of GBM tumors of 10 of these patients' tumor samples. Tumor samples were fresh-frozen immediately after surgery and the plasma samples were collected before, and at multiple visits after surgery. In total, this HLA peptidome analysis involved 52 different HLA allotypes and resulted in the identification of more than 35,000 different HLA peptides. Strong correlations were observed in the signal intensities and in the repertoires of identified peptides between the tumors and plasma-soluble HLA peptidomes of the individual patients, whereas low correlations were observed between these HLA peptidomes and the tumors' proteomes. HLA peptides derived from Cancer/Testis Antigens (CTAs) were selected based on their presence among the HLA peptidomes of the patients and absence of expression of their source genes from any healthy and essential human tissues, except from immune-privileged sites. Additionally, peptides were selected as potential biomarkers if their levels in the plasma-sHLA peptidome were significantly reduced after the removal of tumor mass. The CTAs identified among the analyzed HLA peptidomes provide new opportunities for personalized immunotherapy and for early diagnosis of GBM.

Quantitation of cytidine-5'-monophospho-N-acetylneuraminic acid in human leukocytes using LC-MS/MS: method development and validation.

The biosynthesis of sialic acid (Neu5Ac) leads to the intracellular production of cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac), the active sialic acid donor to nascent glycans (glycoproteins and glycolipids) in the Golgi. UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase myopathy is a rare autosomal recessive muscular disease characterized by progressive muscle weakness and atrophy. To quantify the intracellular levels of CMP-Neu5Ac as well as N-acetylmannosamine (ManNAc) and Neu5Ac in human leukocytes, we developed and validated robust liquid chromatography-tandem mass spectrometry methods. A fit-for-purpose approach was implemented for method validation. Hydrophilic interaction chromatography was used to retain three hydrophilic analytes. The human leukocyte pellets were lysed and extracted in a methanol-water mixture and the leukocyte extract was used for LC-MS/MS analysis. The lower limits of quantitation for ManNAc, Neu5Ac and CMP-Neu5Ac were 25.0, 25.0 and 10.0 ng/ml, respectively. These validated methods were applied to a clinical study.

Anti-ß2GPI/ß2GPI induces human neutrophils to generate NETs by relying on ROS.

Neutrophils participate in the regulation of pathogens by phagocytosis as well as by generating neutrophil extracellular traps (NETs). Antiphospholipid antibodies, particularly those targeting beta-2-glycoprotein I (ß2GPI), stimulate monocytes, platelets, and endothelial cells with prothrombotic participation. This study aimed to explore NET generation in response to anti-ß2GPI/ß2GPI. A series of experiments involving the separation of primary human leukocytes, NETosis quantification using propidium iodide, exploration of NETosis by fluorescence microscopy, western blotting, examination of free Zn2+ using FluoZin-3, and reactive oxygen species (ROS) examination with dihydrorhodamine 123 were performed in this study. We found that anti-ß2GPI/ß2GPI triggered NETosis, resembling phorbol 12-myristate 13-acetate (PMA)-induced NETosis in magnitude and morphology. The anti-ß2 GPI/ß2 GPI complex in isolation stimulated NETs without relying on p38, protein kinase B (AKT), extracellular signal-related kinase (ERK) 1/2, and zinc signals. NET generation was unaffected by the NADPH oxidase suppressor DP1. The anti-ß2 GPI/ß2 GPI complex stimulated ROS generation without relying on NADPH oxidase, which may participate in NET generation triggered via the anti-ß2 GPI/ß2 GPI complex. In summary, our results indicate that the anti-ß2 GPI/ß2 GPI complex reinforced NET generation by relying on ROS. THE SIGNIFICANCE OF THE PAPER IN THE CONTEXT OF CURRENT KNOWLEDGE: Neutrophils as one of the first lines of defence and essential in the response to pathogen invasion. They eradicate bacteria via phagocytosis or by releasing antimicrobial proteins in degranulation. In this study, we explored the capability of anti-ß2 GPI/ß2 GPI to stimulate NETosis, demonstrating that anti-ß2 GPI/ß2 GPI is a promising method for triggering NET. Anti-ß2 GPI/ß2 GPI induced ROS generation without relying on NADPH oxidase, which contributes to NETosis independently of ERK1/2, Zn2+ , or AKT. Our results showed that anti-ß2GPI/ß2GPI triggered NETosis, resembling PMA-induced NETosis in magnitude as well as morphology. The anti-ß2 GPI/ß2 GPI complex in isolation stimulated NETs without relying on p38, AKT, ERK1/2, or zinc signals. The anti-ß2 GPI/ß2 GPI complex stimulated ROS generation without relying on NADPH oxidase, which may participate in NET generation triggered via the anti-ß2 GPI/ß2 GPI complex.

Human monocytes stimulated by Shiga toxin 1a via globotriaosylceramide release proinflammatory molecules associated with hemolytic uremic syndrome.

The life-threatening sequela of hemorrhagic colitis induced by Shiga toxins (Stx)-producing Escherichia coli (STEC) infections in humans is hemolytic uremic syndrome (HUS), the main cause of acute renal failure in early childhood. The key step in the pathogenesis of HUS is the appearance of Stx in the blood of infected patients because these powerful virulence factors are capable of inducing severe microangiopathic lesions in the kidney. During precocious toxemia, which occurs in patients before the onset of HUS during the intestinal phase, Stx bind to several different circulating cells. An early response of these cells might include the release of proinflammatory mediators associated with the development of HUS. Here, we show that primary human monocytes stimulated with Shiga toxin 1a (Stx1a) through the glycolipid receptor globotriaosylceramide released larger amounts of proinflammatory molecules (IL-1ß, TNFα, IL-6, G-CSF, CXCL8, CCL2, CCL4) than Stx1a-treated neutrophils. The mediators (except IL-1ß) are among the top six proinflammatory mediators found in the sera from patients with HUS in different studies. The molecules appear to be involved in different pathogenetic steps of HUS, i.e. sensitization of renal endothelial cells to the toxin actions (IL-1ß, TNFα), activation of circulating monocytes and neutrophils (CXCL8, CCL2, CCL4) and increase in neutrophil counts in patients with poor prognosis (G-CSF). Hence, a role of circulating monocytes in the very early phases of the pathogenetic process culminating with HUS can be envisaged. Impairment of the events of precocious toxemia would prevent or reduce the risk of HUS in STEC-infected children.

The novel lipid mediator PD1n-3 DPA: An overview of the structural elucidation, synthesis, biosynthesis and bioactions.

Resolvins, protectins and maresins are individual families of specialized pro-resolving mediators biosynthesized from the dietary n-3 polyunsaturated fatty acids eicosapentaenoic acid and docosahexaenoic acid. These enzymatically oxygenated polyunsaturated lipid mediators were first elucidated during the resolution phase of acute inflammation in animal models of self-limited inflammation. Specialized pro-resolving mediators display potent bioactions when administrated in vivo. Biosynthetic pathway studies have revealed that individual lipoxygenases and cyclooxygenase-2 converts eicosapentaenoic acid and docosahexaenoic acid into distinct families of the resolvins, protectins and maresins. Recently n-3 docosapentaenoic acid was found to be a substrate for the biosynthesis of several novel families of specialized pro-resolving mediators. One example is PD1n-3 DPA. During the 6th European Workshop on Lipid Mediators, Frankfurt, Germany, the structural elucidation, total organic synthesis, studies on the biosynthetic pathway, as well as the potent anti-inflammatory and pro-resolving properties of PD1n-3 DPA were presented. Herein, we provide an overview of these topics for the new member PD1n-3 DPA of the super-family of pro-resolving mediators.

Murine RAW 264.7 cell line as an immune target: are we missing something?

The popular murine macrophage cell line, RAW 264.7, is often used to initially screen natural products for bioactivity and to predict their potential effect in vivo or on primary cells. The cell line response is considered to reflect the potential human de novo response, and is used to evaluate the effective bioactivity of the product. Here, we compared the cytokine response of RAW 264.7 cells to shark cartilage (SC) with that of human leukocytes to determine whether the cell line response was a reliable predictor of the cytokine response one can expect from similarly stimulated human primary cells. Results not only revealed significant differences in the nature and level of TNFα produced by cells in vitro, but also showed that while the primary cell response included an upregulation in the production of IL-1ß such a response was absent in RAW 264.7 cells. This suggests that had we relied on RAW 264.7 cells alone to assess the cytokine-inducing capacity of SC, the comprehensive Th1 response (shown in an earlier study) induced by SC in primary cells, consisting of release of several proinflammatory cytokines and chemokines, would not have been revealed. We conclude, therefore, that assays using only RAW 264.7 cells to initially screen for and assess immune reactivity of test products will not necessarily provide a comprehensive picture of the immunomodulatory properties of the substance under investigation, and can in fact be misleading with regard to the overall bioactive potential of the substance on an initial screen.

A flow cytometric approach for studying alterations in the cytoplasmic concentration of calcium ions in immune cells following stimulation with thymic peptides.

[Ca(2+)]i alterations are vital in signaling pathways of cell activation. We tried to detect such changes, in intracellular signaling pathways downstream TLR4 in immune cells, following stimulation with prothymosin alpha (proTα) and its decapeptide proTα(100-109). Human leukocytes were activated with LPS, proTα or proTα(100-109), directly or after 24h stimulation, while neutrophils were directly challenged. Cells were loaded with Fluo-4 and cytoplasmic Ca(2+) alterations were recorded by flow cytometry. Direct challenge with 20 µg/mL LPS induced a measurable [Ca(2+)]i increase in macrophages and neutrophils. Monocytes and macrophages incubated for 24h with LPS, proTα or proTα(100-109) and challenged with LPS, displayed a robust response. Lymphocytes and iDCs exhibited no alterations. Conclusively, we assessed a flow cytometry-based method for monitoring Ca(2+) ion influx changes in immune cells. Their stimulation with proTα or proTα(100-109) generates an activating background, similar to LPS, allowing for the detection of [Ca(2+)]i alterations induced upon subsequent challenge.

The ACAT2 expression of human leukocytes is responsible for the excretion of lipoproteins containing cholesteryl/steryl esters.

Acyl-coenzymeA:cholesterol acyltransferase 2 (ACAT2) is abundantly expressed in intestine and fetal liver of healthy human. Our previous studies have shown that in monocytic cells the low-level expression of human ACAT2 gene with specific CpG-hypomethylated promoter is regulated by the CCAAT/enhancer binding protein (C/EBP) transcription factors. In this study, we further report that the ACAT2 gene expression is attributable to the C/EBPs in the human leukocytes and correlated with the excretion of fluorescent lipoproteins containing the ACAT2-catalyzed NBD22-steryl esters. Moreover, this lipoprotein excretion can be inhibited by the ACAT2 isoform-selective inhibitor pyripyropene A (PPPA) in a dose-dependent manner, and employed to determine the half maximum inhibitory concentration (IC50) values of PPPA. Significantly, it is found that the differentiation-inducing factor all-trans retinoic acid, but not the proinflammatory cytokine tumor necrosis factor-α, enhances this ACAT2-dependent lipoprotein excretion. These data demonstrate that the ACAT2 expression of human leukocytes is responsible for the excretion of lipoproteins containing cholesteryl/steryl esters (CE/SE), and suggest that the excretion of lipoproteins containing the ACAT2-catalyzed CS/SE may avoid cytotoxicity through decreasing the excess intracellular cholesterols/sterols (especially various oxysterols), which is essential for the action of the human leukocytes.

Daily variations in the expression of miR-16 and miR-181a in human leukocytes.

Circadian rhythms are controlled by a molecular mechanism that is organized in transcriptional and translational feedback loops of gene expression. Recent studies have been demonstrating the involvement of microRNAs (miRs) in post-transcriptional/translational control of circadian rhythms. In the present study we aimed to analyze the daily variations of miR-16 and miR-181a expression in human leukocytes. These miRs were independently associated with hematopoiesis and circadian rhythms in previous studies using experimental models. Peripheral blood from 6 subjects was sampled in a 24 hour period for expression analysis using quantitative real-time PCR (RT-qPCR). Initially, we evaluated the expression stability of RNU6-2, RNU1A-1, RNU5A-1, SNORD-25, SCARNA-17 and SNORA-73A as candidate genes for normalization of RT-qPCR data. The combination of the four most stable genes (SNORA-73A/SCARNA-17/SNORD-25/RNU6-2) was indicated to provide a better normalization of miRs expressions. The results show a daily variation of miR-181a and miR-16 expression in human leukocytes, suggesting a potential participation of these genes in the modulation of the circadian rhythms present in blood cells.

Exploring the Impact of French Raw-Milk Cheeses on Oxidative Process Using Caenorhabditis elegans and Human Leukocyte Models.

Fermented foods, including cheeses, have garnered increased interest in recent years for their potential health benefits. This study explores the biological properties of eight French raw-milk cheeses-goat cheese, Saint-Nectaire, Cantal, Bleu d'Auvergne, Roquefort, Comté, Brie de Meaux, and Epoisses-on oxidative processes using both in vivo (Caenorhabditis elegans) and in vitro (human leukocytes) models. A cheese fractionation protocol was adapted to study four fractions for each cheese: a freeze-dried fraction (FDC) corresponding to whole cheese, an apolar (ApE), and two polar extracts (W40 and W70). We showed that all cheese fractions significantly improved Caenorhabditis elegans (C. elegans) survival rates when exposed to oxidative conditions by up to five times compared to the control, regardless of the fractionation protocol and the cheese type. They were also all able to reduce the in vivo accumulation of reactive oxygen species (ROS) by up to 70% under oxidative conditions, thereby safeguarding C. elegans from oxidative damage. These beneficial effects were explained by a reduction in ROS production up to 50% in vitro in human leukocytes and overexpression of antioxidant factor-encoding genes (daf-16, skn-1, ctl-2, and sod-3) in C. elegans.

Relevance of Comet Assay and Phosphorylated-Hsp90α in Cancer Patients' Peripheral Blood Leukocytes as Tools to Assess Cisplatin-based Chemotherapy Clinical Response and Disease Outcome.

Cisplatin (cPt) is a commonly used treatment for solid tumors. The main target of its cytotoxicity is the DNA molecule, which makes the DNA damage response (DDR) crucial for cPt-based chemotherapy. Therefore, it is essential to identify biomarkers that can accurately predict the individual clinical response and prognosis. Our goal was to assess the usefulness of alkaline comet assay and immunocytochemical staining of phosphorylated Hsp90α (p-Hsp90α), γH2AX, and 53BP1 as predictive/prognostic markers. Pre-chemotherapy peripheral blood leukocytes were exposed to cPt in vitro and collected at 0, 24 (T24), and 48 (T48) hr post-drug removal. Healthy subjects were also included. Baseline DNA damage was elevated in cancer patients (variability between individuals was observed). After cPt, patients showed increased γH2AX foci/nucleus (T24 and T48). Both in healthy persons and patients, the nuclear p-Hsp90α and N/C (nuclear/cytoplasmic) ratio augmented (T24), decreasing at T48. Favorable clinical response was associated with high DNA damage and p-Hsp90α N/C ratio following cPt. For the first time, p-Hsp90α significance as a predictive marker is highlighted. Post-cPt-DNA damage was associated with longer disease-free survival and overall survival. Our findings indicate that comet assay and p-Hsp90α (a marker of DDR) would be promising prognostic/predictive tools in cP-treated cancer patients.

Potential radioprotective properties of arbutin against ionising radiation on human leukocytes in vitro.

Arbutin is a simple phenolic glucoside biosynthesised in many plant families. Some of the everyday foods that contain arbutin are species of the genus Origanum, peaches, cereal products, coffee and tea and Arctostaphyllos uva ursi L. leaves. Arbutin possesses various beneficial effects in the organism, and was confirmed effective in the treatment of urinary tract infections as well as in preventing skin hyperpigmentation. It shows antioxidant and anti-inflammatory properties, and antitumor activity. The aim of this study was to explore potential radioprotective properties of arbutin in concentrations of 11.4 µg/mL, 57 µg/mL, 200 µg/mL and 400 µg/mL administered as a pre-treatment for one hour before exposing human leukocytes to ionising radiation at a therapeutic dose of 2 Gy. The alkaline comet assay was used to establish the levels of primary DNA damage, and cytokinesis-block micronucleus (CBMN) cytome assay to determine the level of cytogenetic damage. None of the tested concentrations of single arbutin showed genotoxic and cytotoxic effects. Even at the lowest tested concentration, 11.4 µg/mL, arbutin demonstrated remarkable potential for radioprotection in vitro, observed both at the level of primary DNA damage, and using CBMN cytome assay. The best dose reduction compared with amifostine was observed after pre-treatment with the highest concentration of arbutin, corresponding to 400 µg/mL. Promising results obtained on the leukocyte model speak in favour of extending similar experiments on other cell and animal models.