Human Peripheral Blood Lymphocytes Research Articles

Effects of 660 nm red light and 850 nm near infrared light on human blood lymphocytes

Abstract The applications of light emitting diodes (LED) as a red light therapy (RLT) source for the management of various conditions such as wound treatment, control of inflammation and pain have been growing continually. Despite its extensive medical application, the effects of RLT on living cells are still highly contested. This study is conducted to test the effects of RLT using combined LED source with 660 nm (red) and 850 nm (near infrared) light on cultured human lymphocytes in vitro. To analyze the effect of RLT on human peripheral blood lymphocyte’s proliferation, the mitotic index (cytostatic effect) is monitored under different irradiation exposure time parameters and conditions. This value indicates how a particular treatment affected cell division, either proliferatively, inhibitory, or had no effect at all. The interpretation of the results of the mitotic index is done in relation to the mitotic index of the untreated (unirradiated) control cells. Our data shows higher lymphocyte proliferation for all of the irradiated samples, and is particularly enhanced by multiple exposures to red light. The effectiveness of RLT on cell activity is of importance in determination of suitable treatment for diseases related to the immune system. To better understand the molecular and metabolic mechanisms involved in red LED-induced photobiomodulation, the study will be extended to investigate the RLT effect on cell protein synthesis.

New insights into the oxidative and cytogenotoxic effects of Tetraglyme on human peripheral blood cells

The present study investigated the oxidative and cytogenotoxic potential of Tetraethylene glycol dimethyl ether (known as Tetraglyme) on healthy human peripheral blood lymphocytes, widely used as an in vitro model for assessing the human health risk posed by different chemical compounds. In a first step, Nuclear Magnetic Resonance (1H NMR) spectroscopy, and Ultra-High Performance Liquid Chromatography-Mass Spectrometry (UHPLC-MS) were employed to estimate Tetraglyme's stability under a wide range of pH values (4–12), and thus to identify potential by-products. Thereafter, isolated lymphocytes were treated with different concentrations of Tetraglyme (0.02–20 mg L−1) for assessing its oxidative (using the DCFH-DA staining), and cytogenotoxic potential (using the trypan blue exclusion test for estimating cell viability, Comet assay, as well as the cytokinesis-block micronucleus assay, with or without the addition of S9 metabolic activation system). According to the results, Tetraglyme remains stable at pH 4, but two additional derivatives (i.e. 1-[2-(2-ethoxyethoxy)ethoxy]-2-methoxyethane [C9H20O4] and 1-ethoxy-2-(2-ethoxyethoxy)ethane (Diethylene glycol diethyl ether) [C8H18O3]) were found in traces, under alkaline conditions (pH ≥7). Moreover, although Tetraglyme (and/or its derivatives) showed negligible alterations of cell viability (>92 %) in all cases, the pronounced ROS formation, DNA damage, cell proliferation arrest, and MN frequencies in challenged cells are indicative of its oxidative and cytogenotoxic potential. The significant alterations of Cytokinesis-Block Proliferation Index (CBPI) and Micronucleus (MN) frequencies in S9 challenged cells give further evidence for the potential involvement of Tetraglyme's metabolites in the observed cytogenotoxic mode of action. Although not conclusive, the present findings give rise to further research, utilizing different cell types and biological models, for elucidating Tetraglyme's toxic mode of action, as well as its environmental and human risk.

Characterizing Human Peripheral Blood Lymphocyte Phenotypes and Their Correlations with Body Composition in Normal-Weight, Overweight, and Obese Healthy Young Adults.

Background and Objectives: Obesity-associated chronic low-grade inflammation supports various systemic alterations. In this descriptive study, 122 apparently healthy adults aged 20 to 35 years were voluntarily included and classified based on body mass index (BMI) as normal-weight (NW), overweight (OW), and obese (OB). This study aims to characterize peripheral blood (PB) lymphocyte (Ly) phenotypes and investigate their correlations with body composition indices (BCIs) in healthy young adults. Materials and Methods: The following BCIs were measured: waist circumference, hip circumference, height, waist-to-hip ratio, waist-to-height ratio, total body fat mass, visceral fat level, weight, and BMI. White blood cell count (WBC), Ly absolute count, serum TNF-α, and IFN-γ were quantified. Ly subpopulations were analyzed as follows: total TLy (TTLy-CD45+CD3+), early activated TLy (EATLy-CD45+3+69+), total NKLy (TNKLy-CD45+CD3-CD56+CD16+), NKdim (low expression of CD56+), NKbright (high expression of CD56+), BLy (CD45+CD3-CD19+), T helper Ly (ThLy-CD45+CD3+CD4+), and T cytotoxic Ly (TcLy-CD45+CD3+CD8+). Results: Higher BMI has significantly higher WBC and BLy (p < 0.0001; p = 0.0085). EATLy significantly decreased from NW to OB (3.10-NW, 1.10-OW, 0.85-OB, p < 0.0001). Only EATLy exhibited significant negative correlations with all the BCIs. A significantly higher TNF-α was observed in the OW and OB groups compared to the NW group. IFN-γ increased linearly but nonsignificantly with BMI. TTLy showed a nonsignificant positive correlation with both IFN-γ and TNF-α, while EATLy showed a negative correlation, significant only for IFN-γ. NKLy subpopulations exhibited a consistent negative correlation with TNF-α, significant only for NKdim (p = 0.0423), and a nonsignificant consistent positive correlation with IFN-γ. A nonsignificant negative correlation between age and both TNKLy (r = -0.0927) and NKdim (r = -0.0893) cells was found, while a positive correlation was found with NKbright (r = 0.0583). Conclusions: In conclusion, the baseline immunological profile of PB is influenced by excessive adipose tissue in healthy young adults.

Effects of Rubus idaeus extracts on some Cancerous Cell Lines in vitro Study Using MTT Assay

Our study investigated the cytotoxicity of Rubus idaeus alcoholic and aqueous extracts prepared from ripe blackberries were collected from the farmlands north of Baghdad, the extracts prepared at the same starting concentration, and tested on two cell line, the result show high toxic effects increase with the increase of concentration. The three extracted components from Rubus idaeus (purified flavonoid, polysaccharides, and carotenes) showed cytotoxic effect towards both: the primary cell culture of normal hepatic cells (HepG2 and L20B cell), and cancer hepatic cell lines (HepG2 and L20B cell) at 100μg/ml concentration for 24 hours treatment. Purified flavonoid exerted the potent effect on both cell lines among the other two extracted components. The cytotoxicity assay was held only for the purified flavonoid to investigate the mechanism by which the purified flavonoid affected living cells toward apoptosis. The most significant reduction (p≤0.05) in cell viable count was at the concentration 100μg/ml which appear to cause the induction of cell death via mitochondrial pathway for HepG2 and L20B cell line after 24 hours exposure. The purified flavonoid had no effect on the HepG-2 cell cycle. The Immunomodulation effect for the purified flavonoid and the extracted polysaccharides on normal human peripheral blood lymphocytes showed that the purified flavonoid suppress lymphocytes proliferation, while the later increased lymphocytes proliferation significantly. The immune stimulating effect of the polysaccharides caused alteration in IL-17 and level estimated by ELISA technique towards IL-2 elevation for the normal human blood lymphocytes against IL-17 level after 4 hours exposure at concentrations (250 and 500 μg/ml), while toxicity results were shown after 4 hours.

Prospects and Methods for Studying the Proliferative Capacity of the Human Peripheral Blood Lymphocyte Subpopulations in Radiation Medicine

Prospects and Methods for Studying the Proliferative Capacity of the Human Peripheral Blood Lymphocyte Subpopulations in Radiation Medicine

Sex differences in constructing dose-response calibration curves for micronuclei using cytokinesis-blocked micronucleus assay for radiation biological dosimetry in the Iranian population

Biological dosimetry, using chromosome damage biomarkers, can be considered as a key measure for radiation overexposure assessment. Therefore, for accurate dose estimation through biodosimetry, it's imperative for each biological dosimetry laboratory to establish its own specific dose-response calibration curve. In this research, the cytokinesis-blocked micronucleus (CBMN) assay was utilized to determine the frequencies of micronuclei (MN) per binucleated cell in human peripheral blood lymphocytes exposed to x-ray radiation using a LINAC (6 MV) at doses up to 4 Gy. The aim was to establish an in vitro dose-response calibration curve in our laboratory for the Iranian demographic by analyzing blood samples of ten participants (5 males and 5 females) through CABAS and Dose Estimate software. Our findings indicate an over-dispersion of the Poisson distribution in the pooled data across both sexes, coupled with a linear-quadratic increase in MN yield with dose which was particularly pronounced in the female group. The constructed dose-response curves for micronuclei yield are represented by equations Y= (0.0102 ± 0.0016) +(0.0296 ± 0.0054) D+(0.0232 ± 0.0017) D2 and Y= (0.0084 ± 0.0010) +(0.0212 ± 0.0034) D+(0.0160 ± 0.0011) D2 for the female groups, respectively. The alpha and beta coefficients, derived from the Dose Estimate software for each male and female group, were closely comparable with those obtained from the CABAS program and previous studies. The analysis of statistical parameters such as the Weighted Chi Squared (χ2) and p-value suggests that using distinct curves for each sex, rather than a unified biodosimetry formula for pooled data, ensures more accurate radiation dose estimates. Consequently, the findings of this study provide us with the assurance to utilize the derived calibration curve of MN for upcoming biological dosimetry needs in Iran. However, to enhance the reliability of results, it's essential to use biodosimetry with diverse assays and consider all clinical and physical parameters, given the limitations of cytogenetic assays.

Modulatory effect of the fruit rind extract of Garcinia indica Choisy against gamma radiation induced damage in human peripheral blood lymphocytes: a preliminary study

Purpose Nowadays people are exposed to radiation due to various reasons, including natural, diagnostic, occupational or accidental exposure. High level of exposure to ionizing radiation can be fatal to human body. Synthetic drugs used to prevent radiation-induced damage are toxic in nature. Recently, Herbal drugs are being screened as an alternative due to their mechanism of action. Garcinia indica (G. indica) is one of the traditional medicinal plant which contains phytochemicals having several medicinal properties. Materials and methods In this study, G. indica extract was observed for its modulatory effect against 3 Gray (Gy) gamma radiation-induced damages in human peripheral blood lymphocytes. Various concentrations of G. indica extract ranging from 1 to 25 µg/mL was added to the blood post irradiation at 0 hr. Chromosomal aberration (CA) and Cytochalasin B blocked Micronuclei Cytome (CBMN) Assay were performed as per standard procedure. Results Radiomodulatory effect of Garcinia indica fruit rind extract (GIFRE) on CA and MN formation was observed in this study. Treatment of GIFRE did not affect the mitotic index. Positive inhibition percentages for dicentrics, total chromosomal aberrations and micronuclei were observed except for one instance. Conclusion Owing to the various properties of Garcinia extracts, it makes it a potential candidate to be tested for its radiomodulatory effect. Based on the results observed in this preliminary study, it could act as a radiomodulatory agent. Radiomodulatory effect of GIFRE could possibly serve it as a potential herbal medicinal alternative to current drugs. However, results of this study need to be validated on larger sample size.

Oxidative damage and cell cycle delay induced by vanadium(III) in human peripheral blood cells

Vanadium (V) is a metal that can enter the environment through natural routes or anthropogenic activity. In the atmosphere, V is present as V oxides, among which vanadium(III) oxide (V2O3) stands out. Cytogenetic studies have shown that V2O3 is genotoxic and cytostatic and induces DNA damage; however, the molecular mechanisms leading to these effects have not been fully explored. Therefore, we treated human peripheral blood lymphocytes in vitro, evaluated the effects of V2O3 on the phases of the cell cycle and the expression of molecules that control the cell cycle and examined DNA damage and the induction of oxidative stress. The results revealed that V2O3 did not affect cell viability at the different concentrations (2, 4, 8 or 16 μg/mL) or exposure times (24 h) used. However, V2O3 affected the percentage of G1- and S-phase cells in the cell cycle, decreased the expression of mRNAs encoding related proteins (cyclin D, cyclin E, CDK2 and CDK4) and increased the expression of γH2AX and the levels of reactive oxygen species. The ability of V2O3 to cause a cell cycle delay in G1-S phase may be associated with a decrease in the mRNA and protein expression of the cyclins/CDKs and with intracellular oxidative stress, which may cause DNA double-strand damage and H2AX phosphorylation.

Efficacy of MAGE-A4 long peptide as a universal immunoprevention cancer vaccine

BackgroundThe clinical application of peptide vaccines in tumor immunotherapy holds significant promise. Peptide-based tumor vaccines are currently subject to certain limitations in clinical trials, including the challenge of inducing a sustained response from CD4+ T helper cells and cytotoxic T lymphocytes (CTL), as well as human leukocyte antigen (HLA) restrictions.MethodsThrough the utilization of biological information methodology, a screening process was conducted to identify three potential long peptides that are specifically targeted by the MAGE-A4 antigen. The candidate long peptides were subjected to in vitro testing using human peripheral blood lymphocytes as samples to evaluate their immunogenicity and immune function. The antitumor properties and preliminary mechanism of the long peptide vaccine were investigated through the use of a mouse model designed for the prevention of triple negative breast cancer (TNBC).ResultsThree predicted multi-epitope long peptides targeting MAGE-A4 have shown to have a strong immunogenicity, with a total positive rate of 72% across different HLA subtypes in Chinese populations. they can also increase the levels of the costimulatory factor CD137 and tumor necrosis factor-alpha (TNF-α), activate T cells, and boost the cytotoxic activity. Results from an animal study have revealed that the long-peptide vaccine, both on its own and in combination with R848, has displayed impressive anti-tumor and target-specific capabilities. Moreover, it has the ability to increase the expression of effector memory T cells and central memory T cells.ConclusionsThis study was the first to screen three multi-epitope long peptides targeting MAGE-A4 and assess their immunogenicity, immune function, and potential as adjuvant peptides. The results showed that the MAGE-A4 long peptide vaccine can be used as a novel immunoprophylaxis method to prevent TNBC. Moreover, the proposed development model is capable of screening multiple target antigens, which lead to its clinical application.

Elucidating Genes and Transcription Factors of Human Peripheral Blood Lymphocytes Involved in the Cellular Response upon Exposure to Ionizing Radiation for Biodosimetry and Triage: An In Silico Approach

Abstract Background Gene expression–based biodosimetry is a promising method for estimating radiation dose following exposure. A panel of highly radio-responsive genes in human peripheral blood was used in the current investigation to create and evaluate a unique gene expression–based radiological biodosimetry method. Methodology In human cellular research, we reviewed the literature on genes and proteins correlating to radiation response in vivo and in vitro. We looked at two publicly accessible independent radiation response gene expression profiles (GSE1977 and GSE1725) and identified the differentially expressed genes (DEGs). Results The obtained data exhibited 42 genes with substantial differential expression, 25 of which were upregulated and 17 of which were downregulated in ionizing radiation exposure groups compared with control groups. The gene ontology enrichment analysis revealed that the hub genes are significantly involved in the regulation of the mitotic cell cycle phase transition, regulation of the mitotic cell cycle, and mitotic cell cycle checkpoint signaling. Out of the 42 DEGs, four top genes (CDK1, CCNB1, UBC, and UBB) were obtained through network centrality features. However, the multicomponent filtering procedure for radiation response genes resulted in cyclin-dependent kinase 1 (CDK1) as a critical gene in the dataset curated. Conclusion Our findings suggest the possibility of discovering novel gene connections involved in the cellular response of human peripheral blood lymphocytes upon exposure to ionizing radiation.

Modulation of Ionizing Radiation-Induced Apoptosis by Taurine in Human Peripheral Blood Lymphocytes: Flow Cytometry-based Quantification.

Radiotherapy, a highly effective method of radiation-based treating cancers, can reduce the size of tumors and affect healthy tissues. Radiation-induced lymphopenia as a side effect of radiation therapy can reduce the effectiveness of the treatment. This study aimed to examine how taurine can protect peripheral blood lymphocytes from radiation-based apoptosis. In this experimental study, the effects of the taurine on lymphocytes were studied, and blood samples were divided into three groups: a negative control group that was not treated, a positive control group that was treated with cysteine (100 μg/ml), and a group that was treated with taurine (100 µg. mL-1) in three different doses (4, 8 & 12 Gy) before irradiation. The percentage of apoptotic and necrotic lymphocytes was measured using flow cytometry 48 and 72 hours after the irradiation, respectively. According to the groups treated with taurine, the number of lymphocytes undergoing apoptosis was lower and higher compared to the negative and positive control groups, respectively. The decrease in this value was more pronounced 48 hours after radiation compared to 72 hours. Furthermore, there was a slight increase in the number of apoptotic lymphocytes with increasing radiation dose. Taurine effectively protects human peripheral blood lymphocytes from radiation-based apoptosis.

Effect of combinatorial immune checkpoint blockade on myocardial expression of NLRP-3 and secretion of H-FABP, NT-Pro-BNP, interleukin-1β, and interleukin-6: Biochemical implications in cardio-immuno-oncology.

e24016 Background: Immune checkpoint blockade in monotherapy or combinatorial regimens with chemotherapy or radiotherapy have become an integral part of oncology in recent years. Monoclonal antibodies against CTLA-4 or PD-1 or PDL-1 are the most studied ICIs in randomized clinical trials, however, more recently, an anti-LAG3 (Lymphocyte activation gene-3) antibody, Relatlimab, has been approved by FDA in combination with Nivolumab for metastatic melanoma therapy. Moreover, Atezolizumab is actually under study in association with Ipilimumab for therapy of metastatic lung cancer. Myocarditis, vasculitis and endothelitis are rarely observed in these patients on monotherapy, however new combination therapies could expose patients to more adverse cardiovascular events. Methods: human cardiomyocytes, human peripheral blood lymphocytes (hPBMCs) and combinatorial ICIs (PD-L1 and CTLA-4 or PD-1 and LAG-3 blocking agents, at 100 nM) for 48 h. After treatments, cardiac cell lysis and secretion of biomarkers of cardiotoxicity (H-FABP, troponin-T, BNP, NT-Pro-BNP), NLRP3-inflammasome and Interleukin 1 and 6 were determined through colorimetric and enzymatic assays. Mitochondrial functions were studied in cardiomyocyte cell lysates through quantification of intracellular Ca++, ATP content and NADH:ubiquinone oxidoreductase core subunit S1 (Ndufs1) levels. Histone deacetylases type 4 (HDAC-4) protein levels were also determined in cardiomyocyte cell lysates to study potential epigenetic changes induced by immunotherapy regimens. Results: Both combinations of immune checkpoint inhibitors exert more potent cardiotoxic side effects compared to monotherapies against human cardiac cells co-cultured with human lymphocytes. LDH release from cardiac cells was 43% higher in PD-L1/CTLA-4 blocking agents, and 35.7% higher in PD-1/LAG-3 blocking agents compared to monotherapies. HDAC4 and intracellular Ca++ levels were increased, instead ATP content and Ndufs1 were reduced in myocardial cell lysates (p &lt; 0.001 vs untreated cells). Troponin-T, BNP, NT-Pro-BNP and H-FABP, were also strongly increased in combination therapy compared to monotherapy regimen. NLRP3 expression, IL-6 and IL-1β levels were also increased by PDL-1/CTLA-4 and PD-1/LAG-3 combined blocking agents compared to untreated cells and monotherapies. Conclusions: Data of the present study, although in vitro, indicate that combinatorial immune checkpoint blockade, induce a pro- inflammatory phenotype, thus indicating that these therapies should be closely monitored by the multidisciplinary team consisting of oncologists, cardiologists and immunologists.

Causal Relationship Between Circulating Immune Cells and Recurrent Spontaneous Abortion: A Bidirectional Mendelian Randomization Study

ABSTRACTBackgroundRecurrent spontaneous abortion (RSA) is a serious and common complication of pregnancy caused by multiple factors. The etiology remains incompletely understood, but immunologic factors play important roles. Here, we aimed to evaluate whether circulating immune cells causally impacted RSA.MethodsIn this study, we conducted a comprehensive two‐sample Mendelian randomization (MR) study to determine the causal association between the 731 immunophenotypes of human peripheral blood lymphocytes and the number of spontaneous abortions as well as recurrent miscarriage. Sensitivity analyses were performed to assess and minimize heterogeneity and horizontal pleiotropy. Reverse MR analysis was used to assess reverse causality.ResultsAfter Bonferroni‐correction, eight immunophenotypes were significantly associated with the number of spontaneous abortions: FSC‐A on CD4+ T cell (beta = −0.051, 95% CI = [−0.085, −0.017], P‐value = 0.004), CD8 on HLA DR+ CD8+ T cell (beta = −0.040, 95% CI = [−0.067, −0.014], P‐value = 0.003), HLA DR on CD33dim HLA DR+ CD11b− (beta = −0.021, 95% CI = [−0.036, −0.005], P‐value = 0.010), HLA DR+ T cell Absolute Count (beta = 0.022, 95% CI = [0.006, 0.037], P‐value = 0.008), HLA DR+ T cell % lymphocyte (beta = 0.026, 95% CI = [0.010, 0.041], P‐value = 0.001), HLA DR+ T cell % T cell (beta = 0.023, 95% CI = [0.007, 0.039], P‐value = 0.004), HLA DR+ CD4+ T cell % lymphocyte (beta = 0.034, 95% CI = [0.007, 0.060], P‐value = 0.012), and HLA DR on B cell (beta = 0.012, 95% CI = [0.003, 0.021], P‐value = 0.010). In addition, we identified two immunophenotypes associated with recurrent miscarriage: HLA DR on B cell (OR = 0.854, 95% CI = [0.757, 0.964], P‐value = 0.011), and CD19 on naive‐mature B cell (OR = 4.595, 95% CI = [1.674, 12.617], P‐value = 0.003). There was no evidence of heterogeneity, horizontal pleiotropy and reverse causality.ConclusionsOur study demonstrated a tight link between adaptive immune cells and RSA through genetic means, thus providing potential therapeutic targets or novel diagnostic biomarkers.

Chromosomal damage, gene expression and alternative transcription in human lymphocytes exposed to mixed ionizing radiation as encountered in space

Astronauts travelling in space will be exposed to mixed beams of particle radiation and photons. Exposure limits that correspond to defined cancer risk are calculated by multiplying absorbed doses by a radiation-type specific quality factor that reflects the biological effectiveness of the particle without considering possible interaction with photons. We have shown previously that alpha radiation and X-rays may interact resulting in synergistic DNA damage responses in human peripheral blood lymphocytes but the level of intra-individual variability was high. In order to assess the variability and validate the synergism, blood from two male donors was drawn at 9 time points during 3 seasons of the year and exposed to 0–2 Gy of X-rays, alpha particles or 1:1 mixture of both (half the dose each). DNA damage response was quantified by chromosomal aberrations and by mRNA levels of 3 radiation-responsive genes FDXR, CDKN1A and MDM2 measured 24 h post exposure. The quality of response in terms of differential expression of alternative transcripts was assessed by using two primer pairs per gene. A consistently higher than expected effect of mixed beams was found in both donors for chromosomal aberrations and gene expression with some seasonal variability for the latter. No synergy was detected for alternative transcription.

COMBINATORIAL IMMUNE CHECKPOINT BLOCKADE INCREASES MYOCARDIAL SECRETION OF H–FABP, NT–PRO–BNP, NLRP–3 INFLAMMASOME, INTERLEUKIN–1Β AND INTERLEUKIN–6: BIOCHEMICAL IMPLICATIONS IN CARDIO–IMMUNO–ONCOLOGY

Abstract Background Immune checkpoint blockade have become an integral part of oncology in recent years. Monoclonal antibodies against CTLA–4 or PD–1 or PDL–1 are the most studied ICIs in randomized clinical trials, however, more recently, an anti–LAG3 monoclonal antibody, Relatlimab, has been approved by FDA for combinatorial treatment with Nivolumab for metastatic melanoma. Moreover, anti PD–L1 blocking agent Atezolizumab is actually under study in association with Ipilimumab as innovative thearpy for metastatic lung cancer. Myocarditis, vasculitis and endothelitis are rarely observed in these patients on monotherapy, however new combination therapies and the use of new targets in immunotherapy could expose patients to more adverse cardiovascular events. Purpose In this study, we highlighted the cardiotoxic properties of new Combinatorial immune checkpoint blockade therapies. Methods Human cardiomyocytes co–cultured with human peripheral blood lymphocytes (hPBMCs) were exposed to monotherapy and combinatorial ICIs (PD–L1 and CTLA–4 or PD–1 and LAG–3 blocking agents, at 100 nM) for 48 h. After treatments, cardiac cell lysis and secretion of biomarkers of cardiotoxicity (H–FABP, NT–Pro–BNP), NLRP3–inflammasome and Interleukin 1 and 6 were determined through colorimetric and enzymatic assays. Results Both combinations of immune checkpoint inhibitors exert more potent cardiotoxic side effects compared to monotherapies against human cardiac cells co–cultured with human lymphocytes. LDH release from cardiac cells was 43% higher in PD–L1/CTLA–4 blocking agents, and 35.7% higher in PD–1/LAG–3 blocking agents compared to monotherapies. NT–Pro–BNP and H–FABP, were also strongly increased in combination therapy with respect to monotherapies. NLRP3, IL–6 and IL–1β levels were also increased by PDL–1/CTLA–4 and PD–1/LAG–3 combined blocking agents compared to untreated cells and monotherapies. Conclusion Data of the present study, although in vitro, indicate that combinatorial immune checkpoint blockade, induce a pro– inflammatory phenotype, thus indicating that these therapies should be closely monitored by the multidisciplinary team consisting of oncologists, cardiologists and immunologists.

Labilization of the DNA structure in peripheral blood lymphocytes of COVID-19 patients

Introduction. Available data indicate the SARS-CoV-2 coronavirus to be potent of impairing DNA repair processes and cause oxidative stress, which can lead to the accumulation of DNA damage in human cells. However, the DNA-damaging effect of the virus has not yet been sufficiently studied. The purpose of the research was to study the ability of SARS-CoV-2 to cause DNA damage in human peripheral blood lymphocytes. Materials and methods. One hundred forty COVID-19 patients and 24 donors of the control group are included in the study. The level of DNA fragmentation in lymphocytes was determined by alkaline DNA-comet assay. Statistical differences between the mean medians of the «%DNA in the comet tail» (tail DNA%) were assessed using Student’s t-test. The Jeffers test was used to compare the proportions of cells with different levels of DNA-damage. Statistical differences between groups were assessed using the Mann-Whitney test. Results. In the COVID-19 patients, an increase in the level of breaks and alkali-labile sites in DNA was revealed when compared to controls (p = 0.025). In the group of patients infected with SARS-CoV-2, the proportion of comets with DNA damage of up to 5% decreased (p = 0.009), while the proportion of comets containing more than 10% DNA tail increased (p = 0.000). The number of atypical comets compared to the control increased by 3.7 and 5.9 times with mild and moderate severity of the disease, respectively (r = 0.993; p = 0.001). In the association with diseases – coronary heart disease (CHD) and diabetes mellitus type II (DM type 2), the level of DNA fragmentation in lymphocytes statistically significantly increased compared to the group of patients without these diseases. Limitations. A limitation is the lack of data on DNA-structure damage in severe COVID-19 disease. Conclusion. SARS-CoV-2 infection leads to labilization of the DNA structure in human peripheral blood lymphocytes. The level of DNA damage depends on the severity of COVID-19 and the presence of comorbid diseases: CHD and DM type 2. The results of the study are important for understanding the mechanisms of action of the virus on human immunocompetent cells.

Abstract 2506: Novel biomarkers that predict response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer

Abstract Background: Neoadjuvant chemoradiotherapy (nCRT) followed by surgery is standard of care for locally advanced rectal cancer (LARC). About 20% patients receiving nCRT alone experience a pathologic complete response (pCR), while up to 25% patients exhibit a poor response. Currently, there is no biomarker to predict response to nCRT. We hypothesized that inherited ability to recognize and repair DNA damage, especially lethal double-strand breaks (DSBs), manifest in lymphocytes, would yield a reproducible test predicting therapeutic response to nCRT. Methods: Expression of DNA damage response (DDR) proteins was studied using peripheral blood lymphocytes (PBLs) from LARC patients and healthy controls. PBLs from LARC patients collected either before nCRT or after a standard course of ~5.5 weeks of nCRT were assessed by immunofluorescence (IF) for phospho-γH2AXSer139 and by Luminex multi-analyte platform (xMAP) approaches. The xMAP assay was modified from a qualitative to a semi-quantitative assay that measures the following DDR-associated proteins- total ATR, phospho-γH2AXSer139, total MDM2, phospho-Chk1S345, phospho-Chk2T68, phospho-p53S15, total p21. The analytical performance of the assay was assessed with vendor-provided reference controls and banked PBLs from healthy controls (n=50). LARC patients were segregated by neoadjuvant rectal (NAR) score to determine if tested biomarkers correlated with nCRT response. We used two independent sets of LARC biospecimens for xMAP analysis (set 1, n=48; set 2, n=44). Results: IF-based assessment: Using PBLs from LARC patients, we found that poor responders (PoR; NAR score&amp;gt;14; n=21) had significantly lower γH2AX foci than complete responders (CR; NAR score&amp;lt;1; n=21) (P&amp;lt;0.0001, logistic regression). No significant difference was observed in positive γH2AX foci from PBLs drawn pre-nCRT versus post-nCRT from the same patient (p=0.4961, n=11, Wilcoxon test). xMAP assay performance: The performance assessment showed linear sample curves, precision with acceptable inter- and intra-assay coefficients of variability, and high reproducibility with ~1% outliers identified in replicates. Clinical associations using xMAP assay: levels of six proteins (ATR, p-γH2AXSer139, MDM2, p-Chk1S345, p-Chk2T68, p-p53S15) significantly differentiated CRs from PoRs (p-values and False Discovery Rate ⇐ 1e-5). The strongest associations with nCRT response were observed for: ATR, MDM2 and γH2AX. Conclusion: 1) we standardized an xMAP assay to assess DDRs in human PBLs, and 2) found novel biomarkers that predict response to nCRT in LARC patients using minimally invasive PBL biospecimen. In ongoing work, we are using a larger biospecimen set to establish clinical validity parameters. Citation Format: Adria Hasan, Elena V. Demidova, Philip Czyzewicz, Karthik Devarajan, Margret B. Einarson, Donald Baldwin, Erica A. Golemis, Joshua E. Meyer, Sanjeevani Arora. Novel biomarkers that predict response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2506.

Evaluation of acute, 28-day, 13-week repeated dose oral toxicity and genotoxicity of a herbal extract (HemoHIM G) from Angelica sinensis, Ligusticum chuanxiong, and Peaonia lactiflora.

HemoHIM G is a functional food ingredient composed of a triple herbal combination of Angelica sinensis, Ligusticum chuanxiong, and Paeonia lactiflora, to improve impaired immune function. Considering the pharmacological benefits of its constituent herbal components, HemoHIM G is anticipated to have various health benefits; however, its toxicity has not been thoroughly evaluated. Here, we conducted a comprehensive study to assess the safety of HemoHIM G in terms of acute oral toxicity, 13-week repeat-dose toxicity, and genotoxicity. In the oral acute toxicity study, Sprague-Dawley rats were orally administered a single dose of HemoHIM G at 5000mg/kg/day, the limit dose for the acute study. No abnormal findings or adverse effects were observed in this study, as confirmed by gross pathology. A 13-week repeated-dose toxicity study was conducted with HemoHIM G at doses of 1250, 2500, and 5000mg/kg/day to examine the subchronic toxicity in both male and female rats after 28days of dose-range finding study. No test substance-related clinical signs or mortality was observed at any of the tested doses. Gross pathology, hematology, blood chemistry, and histopathology were within normal ranges, further supporting the safety of HemoHIM G. Therefore, the NOAEL of HemoHIM G was considered to be at 5000mg/kg/day for both sexes of rats. Bacterial reverse mutation tests, a chromosome aberration test in human peripheral blood lymphocytes, and a mouse micronuclei test were conducted to identify the potential genotoxicity of HemoHIM G. HemoHIM G is non-mutagenic and non-clastogenic. Collectively, these findings provide valuable evidence for the safe use of HemoHIM G as a functional food ingredient.

In vitro and in vivo assessment of indomethacin-induced genotoxicity: protection by curcumin.

Indomethacin is one of the most widely used non-steroidal anti-inflammatory drugs. This study aimed to investigate the protective effects of curcumin against indomethacin-induced genotoxicity. For in vitro studies, human peripheral blood lymphocytes were obtained from a healthy volunteer and treated for 24 h as follows: vehicle control, indomethacin at 100 and 200 μΜ, indomethacin (100 μM and 200 μM) plus curcumin (27 μM). For in vivo experiments, mice received a single i.p dose of curcumin (100 mg/kg) and after 30 min genotoxicity induction was carried out by a single i.p injection of indomethacin at 10, 20, and 40 mg/kg. After 24 h, bone marrow cells were obtained from mice femurs. Genotoxicity was evaluated using a micronucleus assay. Oxidative damage was also inspected both in vitro and in vivo. In-vitro studies indicated that co-treatment with curcumin caused a significant decrease in the average micronuclei percentage and MDA level, and a significant increase in GSH concentration compared to the groups treated only with indomethacin. In-vivo findings revealed that pretreatment with curcumin induced a significant increase in the average ratio of polychromatic erythrocyte/normochromic erythrocyte, GSH concentration and caused a significant decrease in the average percentage of micronuclei and MDA level, in comparison with the group treated only with indomethacin. Curcumin attenuated indomethacin-induced genotoxicity both in vitro and in vivo. These effects might be partially exerted by decreasing oxidative stress. Further studies are required to elucidate the exact genoprotective mechanism of curcumin against indomethacin.

Testing of arctic insect hemolymph as a secondary agent in applied cryopreservation

BACKGROUND: Cold hardiness of insects from extremely cold regions is based on a principle of natural cryoprotection, which is associated with physiological mechanisms provided by cryoprotectants. OBJECTIVE: Since arctic cold-hardy insects are producers of highly effective cryoprotectants, in this study, the hemolymph of Aporia crataegi L. and Upis ceramboides L. from an extremely cold area (Yakutia) was tested as a secondary component of cryoprotective agents (CPA) for cryopreservation (−80°C) of human peripheral blood lymphocytes and skin fibroblasts. MATERIALS AND METHODS: Lymphocytes and skin fibroblasts were treated with various combinations of DMSO and hemolymph extract and step-wise cooled to −80°C. Post-cryopreservation cell viability was assessed by vital staining and morphological appearance. RESULTS: Viability was higher when cells were frozen with a mixture containing DMSO and Upis ceramboides hemolymph compared to the cells frozen in DMSO, while cells frozen with DMSO and Aporia crataegi hemolymph did not survive. The fact that hemolymph of not every cold-resistant insect can be used as a secondary agent along with DMSO indicates that only a unique combination of hemolymph components and its compatibility with cells might result in a positive effect. CONCLUSION: Although the use of insect hemolymph as a complementary agent in applied cryopreservation is a problem in terms of practical application, such studies could initiate new trends in the search for the most successful hemolymph-like cryoprotectant systems.