Lymphocyte Apoptosis Research Articles

Magnetic resonance imaging-based approaches for detecting the efficacy of combining therapy following VEGFR-2 and PD-1 blockade in a colon cancer model

BackgroundAngiogenesis inhibitors have been identified to improve the efficacy of immunotherapy in recent studies. However, the delayed therapeutic effect of immunotherapy poses challenges in treatment planning. Therefore, this study aims to explore the potential of non-invasive imaging techniques, specifically intravoxel-incoherent-motion diffusion-weighted imaging (IVIM-DWI) and blood oxygenation level-dependent magnetic resonance imaging (BOLD-MRI), in detecting the anti-tumor response to the combination therapy involving immune checkpoint blockade therapy and anti-angiogenesis therapy in a tumor-bearing animal model.MethodsThe C57BL/6 mice were implanted with murine MC-38 cells to establish colon cancer xenograft model, and randomly divided into the control group, anti-PD-1 therapy group, and combination therapy group (VEGFR-2 inhibitor combined with anti-PD-1 antibody treatment). All mice were imaged before and, on the 3rd, 6th, 9th, and 12th day after administration, and pathological examinations were conducted at the same time points.ResultsThe combination therapy group effectively suppressed tumor growth, exhibiting a significantly higher tumor inhibition rate of 69.96% compared to the anti-PD-1 group (56.71%). The f value and D* value of IVIM-DWI exhibit advantages in reflecting tumor angiogenesis. The D* value showed the highest correlation with CD31 (r = 0.702, P = 0.001), and the f value demonstrated the closest correlation with vessel maturity (r = 0.693, P = 0.001). While the BOLD-MRI parameter, R2* value, shows the highest correlation with Hif-1α(r = 0.778, P < 0.001), indicating the capability of BOLD-MRI to evaluate tumor hypoxia. In addition, the D value of IVIM-DWI is closely related to tumor cell proliferation, apoptosis, and infiltration of lymphocytes. The D value was highly correlated with Ki-67 (r = − 0.792, P < 0.001), TUNEL (r = 0.910, P < 0.001) and CD8a (r = 0.918, P < 0.001).ConclusionsThe combination of VEGFR-2 inhibitors with PD-1 immunotherapy shows a synergistic anti-tumor effect on the mouse colon cancer model. IVIM-DWI and BOLD-MRI are expected to be used as non-invasive approaches to provide imaging-based evidence for tumor response detection and efficacy evaluation.

Immune cell early activation, apoptotic kinetic, and T-cell functional impairment in domestic pigs after ASFV CADC_HN09 strain infection.

African swine fever (ASF) caused by the African swine fever virus (ASFV) is a fatal and highly contagious disease of domestic pigs characterized by rapid disease progression and death within 2 weeks. How the immune cells respond to acute ASFV infection and contribute to the immunopathogenesis of ASFV has not been completely understood. In this study, we examined the activation, apoptosis, and functional changes of distinct immune cells in domestic pigs following acute infection with the ASFV CADC_HN09 strain using multicolor flow cytometry. We found that ASFV infection induced broad apoptosis of DCs, monocytes, neutrophils, and lymphocytes in the peripheral blood of pigs over time. The expression of MHC class II molecule (SLA-DR/DQ) on monocytes and conventional DCs as well as CD21 expression on B cells were downregulated after ASFV infection, implying a potential impairment of antigen presentation and humoral response. Further examination of CD69 and ex vivo expression of IFN-γ on immune cells showed that T cells were transiently activated and expressed IFN-γ as early as 5 days post-infection. However, the capability of T cells to produce cytokines was significantly impaired in the infected pigs when stimulated with mitogen. These results suggest that the adaptive cellular immunity to ASFV might be initiated but later overridden by ASFV-induced immunosuppression. Our study clarified the cell types that were affected by ASFV infection and contributed to lymphopenia, improving our understanding of the immunopathogenesis of ASFV.

The gut microbiota contributes to the infection of bovine viral diarrhea virus in mice.

Bovine viral diarrhea virus (BVDV) is prevalent worldwide and causes significant economic losses. Gut microbiota is a large microbial community and has a variety of biological functions. However, whether there is a correlation between gut microbiota and BVDV infection and what kind of relation between them have not been reported. Here, we found that gut microbiota composition changed in normal mice after infecting with BVDV, but mainly the low abundance microbe was affected. Interestingly, BVDV infection significantly reduced the diversity of gut microbiota and changed its composition in gut microbiota-dysbiosis mice. Furthermore, compared with normal mice of BVDV infection, there were more viral loads in the duodenum, jejunum, spleen, and liver of the gut microbiota-dysbiosis mice. However, feces microbiota transplantation (FMT) reversed these effects. The data above indicated that the dysbiosis of gut microbiota was a key factor in the high infection rate of BVDV. It is found that the IFN-I signal was involved by investigating the underlying mechanisms. The inhibition of the proliferation and increase in the apoptosis of peripheral blood lymphocytes (PBL) were also observed. However, FMT treatment reversed these changes by regulating PI3K/Akt, ERK, and Caspase-9/Caspase-3 pathways. Furthermore, the involvement of butyrate in the pathogenesis of BVDV was also further confirmed. Our results showed for the first time that gut microbiota acts as a key endogenous defense mechanism against BVDV infection; moreover, targeting regulation of gut microbiota structure and abundance may serve as a new strategy to prevent and control the disease.IMPORTANCEWhether the high infection rate of BVDV is related to gut microbiota has not been reported. In addition, most studies on BVDV focus on in vitro experiments, which limits the study of its prevention and control strategy and its pathogenic mechanism. In this study, we successfully confirmed the causal relationship between gut microbiota and BVDV infection as well as the potential molecular mechanism based on a mouse model of BVDV infection and a mouse model of gut microbiota dysbiosis. Meanwhile, a mouse model which is more susceptible to BVDV provided in this study lays an important foundation for further research on prevention and control strategy of BVDV and its pathogenesis. In addition, the antiviral effect of butyrate, the metabolites of butyrate-producing bacteria, has been further revealed. Overall, our findings provide a promising prevention and control strategy to treat this infectious disease which is distributed worldwide.

Lymphocyte apoptosis and its association with the inflammatory markers and disease severity in juvenile-onset systemic lupus erythematosus patients

BackgroundThe defective clearance of apoptotic bodies in juvenile-onset systemic lupus erythematosus (jSLE) potentially leads to the persistence of autoreactive lymphocytes and the perpetuation of the autoimmune response. These factors contribute to the disturbance in lymphocyte apoptosis and show potential as key determinants in the clinical course and severity of jSLE. This study evaluates the role of peripheral blood (PB) lymphocyte apoptosis in prognosis of jSLE and as a predictor for disease activity.MethodsThe study involved 100 jSLE patients and 50 healthy controls. Flow cytometry was used to analyze percentages of lymphocyte apoptosis in PB of all study participants. Plasma levels of pro-inflammatory cytokines were determined using ELISA.ResultsOur results showed that percentages of lymphocyte apoptosis in PB of jSLE patients are significantly higher than those of healthy controls. These percentages are significantly positively associated with disease activity of patients (SLEDAI-2 K). Furthermore, plasma cytokine levels (IL-17, IFN-γ and TNF-α) are significantly elevated in jSLE patients compared to their levels in healthy controls. Also, there are weak significant positive correlations between percentages of PB lymphocyte apoptosis and each of IL-17 and IFN-γ plasma levels in jSLE patients. Moreover, PB lymphocyte apoptosis percentages among jSLE patients are higher in the presence of some clinical and laboratory features than those in their absence.ConclusionPeripheral apoptotic lymphocytes could contribute to the prognosis of jSLE and could be used as a predictor for disease activity in jSLE patients.

X-ray irradiation effectively inactivated lymphocytes in transfusion in vivo monitored by the bioluminescence transfusion-associated graft-versus-host disease model.

Transfusion of cold-stored whole blood is the preferred resuscitation method for trauma patients but may cause transfusion-associated graft-versus-host disease (TA-GVHD). Standard clinical practice to prevent this is to irradiate blood components with gamma-rays. X-ray irradiations are also a safe and effective alternative to gamma-ray irradiation. We established a visual mouse model of TA-GVHD to compare the viability and function of lymphocytes exposed to gamma- and x-ray irradiation. A haploidentical transplantation mouse model was established to simulate TA-GVHD with Balb/c mice as donors and hybrid F1 CB6 mice (Balb/c × C57) as recipients. Spleen cells from Tg-Fluc+ Balb/c mice were isolated and irradiated with gamma-rays and x-rays. Lymphocyte activation, apoptosis and proliferation post phorbol 1 2-myristate 1 3-acetate (PMA) stimulation were evaluated. After transfusion, we monitored Fluc+ lymphocytes daily by bioluminescence imaging. Recipients were euthanized on day 21, and tissues were examined pathologically and for inflammatory cytokines. The viability of gamma- or x-ray irradiated lymphocytes decreased significantly with slight changes in proliferation in vivo after transfusion. Compared with the non-irradiated group, both the gamma- and x-ray irradiated groups showed significantly decreased clinical scoring and inflammatory cytokine levels. The fluorescence intensity of the body and target organs was reduced after irradiation. No recipients acquired TA-GVHD after lymphocyte transfusion subjected to gamma- or x-rays, showing that x-rays inactivate as well as gamma rays and are suitable for irradiating whole blood.

Immunological effects of human decidual mesenchymal stem cells in spontaneous and recurrent abortions

The aim of this study was to evaluate the immunological activities of human decidual mesenchymal stem cells (MSCs) on proliferation, apoptosis and percentage of regulatory T cells (Treg) in abortions and to investigate whether these activities differ in spontaneous abortions (SA) and recurrent abortions (RA). This prospective cohort study included women who had a first-trimester abortion between 2019 and 2022. Women with uterine anomaly, endocrinological disease, known autoimmune or thrombophilic disease, and fetal chromosomal abnormality in abortion material were excluded. Decidual MSCs isolated from abortion materials were classified as spontaneous abortion-MSCs (SA-MSCs) and recurrent abortion-MSCs (RA-MSCs). Peripheral blood mononuclear cells were isolated from venous blood and co-cultured with SA-MSCs and RA-MSCs. The effects of MSCs on proliferation and apoptosis of lymphocytes, and Tregs levels were compared between SA-MSCs and RA-MSCs groups. Thirty cases (15 SA-MSCs and 15 RA-MSCs) were included in the study. The presence of MSC in co-cultures increased percentage of Treg cells while reducing proliferation and apoptosis compared to those without MSCs (p < 0.0001, p < 0.0001 and p < 0.0001). The increase in percentage of Treg cells and the reduction in apoptosis were significantly lower in the RA-MSCs group compared to the SA-MSCs group (p < 0.0001 and p < 0.001, respectively). Although the proliferation reducing effect of the presence of MSCs was lower in the RA-MSCs group compared to the SA-MSCs group, the difference was not significant (p = 0.07). MSCs contribute to maternal immunotolerance to semi-allogeneic fetus by suppressing proliferation and apoptosis, and increasing percentage of Treg cells. However, the immunoregulatory effects of MSCs are lower in RA compared to SA.

Immunosuppression in Sepsis: Biomarkers and Specialized Pro-Resolving Mediators.

Severe infection can lead to sepsis. In sepsis, the host mounts an inappropriately large inflammatory response in an attempt to clear the invading pathogen. This sustained high level of inflammation may cause tissue injury and organ failure. Later in sepsis, a paradoxical immunosuppression occurs, where the host is unable to clear the preexisting infection and is susceptible to secondary infections. A major issue with sepsis treatment is that it is difficult for physicians to ascertain which stage of sepsis the patient is in. Sepsis treatment will depend on the patient's immune status across the spectrum of the disease, and these immune statuses are nearly polar opposites in the early and late stages of sepsis. Furthermore, there is no approved treatment that can resolve inflammation without contributing to immunosuppression within the host. Here, we review the major mechanisms of sepsis-induced immunosuppression and the biomarkers of the immunosuppressive phase of sepsis. We focused on reviewing three main mechanisms of immunosuppression in sepsis. These are lymphocyte apoptosis, monocyte/macrophage exhaustion, and increased migration of myeloid-derived suppressor cells (MDSCs). The biomarkers of septic immunosuppression that we discuss include increased MDSC production/migration and IL-10 levels, decreased lymphocyte counts and HLA-DR expression, and increased GPR18 expression. We also review the literature on the use of specialized pro-resolving mediators (SPMs) in different models of infection and/or sepsis, as these compounds have been reported to resolve inflammation without being immunosuppressive. To obtain the necessary information, we searched the PubMed database using the keywords sepsis, lymphocyte apoptosis, macrophage exhaustion, MDSCs, biomarkers, and SPMs.

Evaluation of Relationship between Intrinsic Radiosensitivity (Survival Fraction at 2 Gy) and Gamma-H2AX Test and Apoptosis of Lymphocytes in Breast Cancer Patients.

Radiotherapy is one of the routine treatment strategies for breast cancer (BC) patients. Different responses of the patient to radiation due to different intrinsic radiosensitivity (RS) were induced to the researcher try to introduce a standard assay for the prediction of RS. Clonogenic assay is recognized as a gold standard method in this subject but because of some of its disadvantages, it is needed for alternative assays. In this study, two assays were evaluated for this reason in ten BC patients with different RSs. The peripheral blood of 10 volunteers with BC was obtained, and the peripheral blood mononuclear cells were extracted. After exposed with 2 Gy, survival fraction at 2 Gy (SF2) was calculated by clonogenic assay. γ-H2AX assay was performed for all patients, and apoptosis assay was evaluated for three represented categorized patients. RS of patients showed SF2 and categorized in three groups (high, medium, and low RS). Double-strand breaks (DSBs) were decreased in high radiosensitive patients, but the residual DSBs were clearly higher than other two groups. It is shown that the repair system in these patients is lower active than others. Apoptosis frequency in patient 4 is highly active which could induce the enhancement of her RS. γ-H2AX and apoptosis assays could predict the intrinsic RS, but evaluation of them separately is not sufficient for this aim. It is necessary to consider all the parameters together and consideration of the combination of assays could fit a better prediction of intrinsic RS.

Effects of primed adipose mesenchymal stem cell‐derived exosomes on immunomodulation in Behcet uveitis

Aims/Purpose: Exosomes of interferon‐gamma (IFN‐ γ) primed (IFN + Exo) and non‐primed (IFN‐Exo) adipose‐derived mesenchymal stem cells (AdMSCs) were studied for their possible significance in the therapy of Behçet Disease (BD) uveitis (BU).Methods: Adipose tissue was used to isolate AdMSCs. Characterization as well as multipotency investigations were carried out. Exosomes of AdMSCs were also identified and characterized. Lymphocytes were isolated from the peripheral venous blood of patients with BD uveitis (n = 15) and healthy controls (n = 15). AdMSCs were preincubated for 48 h with or without IFN‐ γ. Peripheral Blood Mononuclear Cells (PBMC) of BD uveitis patients and healthy controls were cultivated with exosomes separately for 72 h. Flow cytometry was used to assess lymphocyte proliferation, viability, and apoptosis after the culture period. Real‐time polymerase chain reaction (RT‐PCR) was used to assess the expression of interleukin‐10 (IL‐10), interleukin‐17 (IL‐17), transforming growth factor‐ β (TGF‐ β), and interferon‐ γ (IFN‐ γ).Results: In patients with BD uveitis, IFN + Exo inhibited lymphocyte apoptosis. IFN + Exo reduced the viability of BD uveitis T lymphocytes. Both exosomes also raised IL‐10 level as an anti‐inflammatory cytokine and decreased IL‐17, TGF‐ β, IFN‐γ levels as a pro‐inflammatory cytokine in BD uveitis patients' lymphocytes. However, exosomes were ineffective in stimulating T lymphocyte proliferation.Conclusions: Exosomes of AdMSCs stimulated with and without IFN‐ γ decrease the abnormal inflammatory response caused by BD uveitis PBMC. This study's findings have the potential to pave the road for MSC/exosome therapy in BD uveitis.

Human papillomavirus infection affects the immune microenvironment and antigen presentation in penile cancer.

Penile squamous cell carcinoma (PSCC) is a largely neglected condition, predominantly affecting underdeveloped regions, and is associated with risk factors such as low socioeconomic status, phimosis, and human papillomavirus (HPV) infection. Unlike other urogenital cancers, its pathophysiology and therapeutic targets remain poorly understood, particularly regarding the immune response to the tumor microenvironment. This study aims to investigate immune cell infiltration profiles, dendritic cell maturation, and lymphocyte apoptosis in both HPV-positive and HPV-negative PSCC. Clinical and histopathological data, along with peripheral blood and tumor tissue samples, were collected from 30 patients (66.6% were HPV-positive and 33.3% HPV-negative), with an additional 19 healthy donors serving as controls. Tumor-infiltrating immune cells were analyzed following enzymatic digestion of tumor tissue, enabling detailed phenotypic characterization. A simulated tumor microenvironment was created using supernatants derived from primary cultures of HPV-positive PSCC tumors. Peripheral blood mononuclear cells were isolated and differentiated into dendritic cells (Mo-DCs) for further phenotyping and lymphoproliferation assays. Lymphocytes from healthy donors and patients were exposed to tumor culture supernatants to evaluate apoptosis induced by the tumor microenvironment. Results showed that HPV-positive tumors exhibited lower T lymphocyte frequencies compared to HPV-negative tumors. Additionally, patients infected with high-risk HPV demonstrated reduced maturation rates of Mo-DCs and decreased expression of co-stimulatory molecules on these cells compared to healthy donors. Furthermore, Mo-DCs from hrHPV-positive patients showed impaired lymphoproliferation capacity relative to controls, while HPV-negative patients exhibited a trend towards reduced lymphoproliferative ability. Regarding the simulated tumor microenvironment, lymphocytes from healthy donors underwent apoptosis, contrasting with patients' lymphocytes, which showed increased viability when cultured with tumor supernatants. These results underscore the impact of HPV infection on T lymphocyte infiltration, Mo-DC maturation, and lymphocyte survival in PSCC, offering critical insights for advancing our understanding of the tumor microenvironment and guiding the development of immunotherapy strategies.

Morphology of Hassall's corpuscles and their microenvironment in neonates with increased thymus weight

To study morphological features of Hassall's corpuscles (HC) and their microenvironment in newborns with increased thymus mass. The study was carried out on autopsy material of children of the first month of life. Based on the thymic index (TI), 2 groups were identified: with normal (conditional norm) and increased TI value (increased thymus weight). The standard method of histological staining and immunohistochemical methods with antibodies to Pan-CK, CK19, CD68, CD3 and p53 were used in the study. The classification proposed by A.G. Beloveshkin (2013) was used to determine the degree of maturity of HC. Nonparametric Mann-Whitney test was used to determine statistically significant differences in the groups. In the group of children with increased thymus weight, the number of HC decreased by 20%. It was found that the proportion of progressive and mature corpuscles in this group was reduced by 2.3 and 1.6 times, respectively, compared to the group of children with normal thymus weight, while the proportion of regressive corpuscles increased almost 2-fold. In the HC microenvironment, there is an increase in the total number of thymocytes, combined with a decrease in the expression of CD68, CD3 and p53 in them. A sharp decrease in CK19-expressing cells in this group is accompanied by a disruption in the formation of reticular structures characteristic of the comparison group. In the thymus with increased mass, the structural and functional organization changes: along with an increase in the total number of thymocytes in the cortical layer and a decrease in the number of macrophages, epithelial cells and HC (with a predominance of regressive corpuscles), disturbances in the processes of maturation, apoptosis and negative selection of lymphocytes occur, which can lead to development of immunogenesis disorders.

Spontaneous Apoptosis of Lymphocytes in Patients with Bronchial Asthma with Comorbid Pathology of Digestive Organs

Spontaneous Apoptosis of Lymphocytes in Patients with Bronchial Asthma with Comorbid Pathology of Digestive Organs

Veterinarians exposed to inhaled anesthetic present chromosome damage, apoptosis and cell cycle changes.

This cross-sectional study evaluated, for the first time, DNA damage, viability, and cell death of lymphocytes and cell cycle phases of mononuclear and polymorphonuclear cells in veterinarians exposed to the volatile anesthetic isoflurane. Veterinarians who were occupationally exposed to isoflurane (exposed group; n = 20) and matched-unexposed individuals (volunteers without occupational exposure; n = 20) were enrolled in the study. DNA damage was assessed in lymphocytes by micronucleus (MN) and phosphorylated histone gamma-H2AX (γ-H2AX). Cell viability, cytotoxicity, and the cell cycle were evaluated by flow cytometry. Isoflurane was detected in urine samples by headspace gas chromatography-mass spectrometry. Compared with unexposed subjects, veterinarians occupationally exposed to isoflurane (25.7 ± 23.7 μg/L urine) presented statistically higher MN frequencies, lymphocytic apoptosis rates, and numbers of polymorphonuclear cells in the G0/G1 stage. Additionally, the exposed group presented statistically lower proportions of viable lymphocytes and G2/M polymorphonuclear cells. Our findings indicate that veterinarians who are frequently exposed to inhaled anesthetic exhibit chromosomal and cell damage in addition to changes in peripheral blood cell proliferation.

Multi-strain probiotic combined with dietary fiber is an effective factor in the nutritional support of immunity in athletes

A priority in the prevention and correction of immune disorders in athletes is the use of products with high nutrient density, fortified with various nutrients or bioactive compounds, as well as probiotic microorganisms. Probiotics help to maintain the gut microbiota, which is actively involved in the absorption of substances and energy and increases the host immune resistance. Dietary fiber, resistant to digestion in the small intestine, is fully or partially fermented in the large intestine and acts as an essential substrate for the growth and regulation of metabolic activity of normal flora, improves peristalsis and digestion. The purpose of the study was to evaluate the impact of a multi-strain probiotic in combination with dietary fiber on the immune status of basketball athletes during the training period. Material and methods. The study was conducted with the participation of 30 male basketball athletes aged 18 to 24 years. The athletes were randomly divided into 2 groups of 15 people. Athletes in the main group received 1 capsule of multi-strain probiotic (≥1.25×1010 CFU of 10 probiotic strains of bifidobacteria and lactobacilli) and 40 g of corn bran (as a source of dietary fiber) for 23 days. Athletes in the control group received 1 placebo capsule containing maltodextrin and breadcrumbs (40 g/day). Subpopulations of peripheral blood lymphocytes were studied by flow cytometry: T lymphocytes, T helper cells, T cytotoxic lymphocytes, NK cells, NKT cells, B lymphocytes, as well as lymphocytes carrying activation markers and apoptosis marker antigen. The content of cytokines in blood serum [FGF, Eotaxin, G-CSF, GM-CSF, IFN-γ, IL-1ra, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12(p70), IL-13, IL-15, IL-17A, MCP-1, MIP-1α, MIP-1β, PDGF-BB, RANTES, TNF-α, VEGF] was determined using a multiplex immunoassay. Results. Calculation of the absolute number of lymphocytes revealed a tendency (0.05<p<0.10) to a decrease in T helper cells by the end of the observation period in athletes of the main group (497.60±27.67 vs 632.67±65.20 cells/μL), as well as a decrease (p<0.05) in the expression of the CD95/Fas apoptotic marker on peripheral blood lymphocytes of athletes of the main group compared to the beginning of the study (41.53±5.78 vs 69.53±11.79 cells/μL). At the end of the study, a significant increase in IL-9 level was found in the control group [(Me; min-max) = (0.33; 0.21-0.48) vs (0.26; 0.09-0.38) pg/ml; p<0.05; in comparison with the initial indicator]; as well as the tendency (0.05<p<0.10) towards an increase in the levels of IL-15, IL-1ra and RANTES was revealed. In the main group, at the end of the study, the level of G-CSF significantly decreased [(0.36; 0.03-0.95) vs (0.53; 0.14-1.36) pg/ml, p<0.05]. At the end of the observation period, blood serum levels of FGF, G-CSF, IL-13, IL-2 and RANTES in the athletes of the control group exceeded these indicators in the athletes of the main group whereas no significant differences in the studied cytokines were detected between the control and the main groups at the beginning of the study. Conclusion. The results of observation of basketball athletes who consumed a multi-strain probiotic in combination with corn bran (sources of arabinoxylans) in addition to the main diet for 23 days indicate a decrease in the inflammatory process activity and peripheral blood lymphocyte apoptosis, which confirms the effectiveness of probiotics and dietary fiber in sports nutrition.

The Intensity of Blood Lymphocyte Apoptosis in Chronically Exposed Individuals with Obligate Forms of Precancerous Conditions.

We studied the apoptotic death of peripheral blood lymphocytes in individuals chronically exposed to radiation with a history of obligate forms of precancerous conditions. The study included 242 subjects chronically exposed to low-intensity radiation in the range of low and medium doses (mean dose of irradiation of the red bone marrow 555±39 mGy). Of these, 121 subjects had obligate forms of precancerous conditions. In irradiated individuals with obligate forms of precancers, a statistically significant increase in the frequency of apoptosis of peripheral blood lymphocytes at an early and late stages was revealed. Correlation analysis revealed no dependence of the apoptotic death of peripheral blood lymphocytes on the dose of red bone marrow irradiation.

Inhibiting Endothelial Cell-Mediated T Lymphocyte Apoptosis with Integrin-Targeting Peptide-Drug Conjugate Filaments for Chemoimmunotherapy of Triple-Negative Breast Cancer.

Tumor-associated endothelial cells (TECs) limit antitumor immunity via inducing apoptosis of infiltrating T lymphocytes through a Fas ligand (FasL) mediated mechanism. Herein, this work creates a peptide-drug conjugate (PDC) by linking 7-ethyl-10-hydroxycamptothecin (SN38) to hydrophilic segments with either RGDR or HKD motif at their C-terminus through a glutathione-responsive linker. The PDCs spontaneously assemble into filaments in aqueous solution. The PDC filaments containing 1% of SN38-RGDR (SN38-HKD/RGDR) effectively target triple-negative breast cancer (TNBC) cells and TECs with upregulated expression of integrin, and induce immunogenic cell death (ICD) of tumor cells and FasL downregulation of TECs. SN38-HKD/RGDR increases infiltration, activity, and viability of CD8+ T cells, and thus inhibits the growth of primary tumors and pulmonary metastasis. This study highlights the synergistic modulation of cancerous cells and TECs with integrin-targeting PDC filaments as a promising strategy for TNBC chemoimmunotherapy.

The signal pathway of melatonin mediates the monochromatic light-induced T-lymphocyte apoptosis in chicken thymus

Our previous study revealed that under monochromatic red light (RL), the melatonin nuclear receptor reduces the proliferation activity of broiler thymic lymphocytes through the P65 signaling pathway. The main objective of this study was to investigate the signal mechanism by which RL decreases thymic lymphocyte proliferation. Initially, broilers were purchased and randomly assigned to be fed under white light (WL), red light (RL), green light (GL), and blue light (BL). Pinealectomy was performed 3 d later, and the broilers were euthanized after 14 d. The results showed that the expression of the antiapoptotic proteins Bcl-2/Bcl-xl decreased under RL, while the expression of the pro-apoptotic factor Bax/caspase-3 and the pro-inflammatory factors INF-γ/TNF-α/IL-6 increased. After pinealectomy, the expression of Bax/TNF-α/IL-6 increased in conjunction with the decrease in Bcl-2 expression. In vitro experiments demonstrated that exogenous melatonin decreased the expression of Bax/TNF-α/IL-6 in thymic lymphocytes of chicks reared under RL. This melatonin-induced effect was enhanced by SR1078 (RORα/RORγ agonist) but attenuated by SR3335 (RORα antagonist) and BAY (P65 antagonist). These findings revealed that the melatonin nuclear receptor RORα/RORγ promotes the expression of the pro-apoptotic factor Bax/caspase-3 and the pro-inflammatory factors INF-γ/TNF-α/IL-6, while inhibiting the expression of the antiapoptotic factor Bcl-2/Bcl-xl. Our research suggested the signaling pathway of monochromatic red light impacts the apoptosis of thymus lymphocytes in broiler.

Transcriptomic Analyses Indicate That Pre-Leukemic B Cells from People with Monoclonal B Lymphocytosis Are Surprisingly Much Less Active Than Normal B Cells from People with Monoclonal B Lymphocytosis and from Healthy Donors

Introduction. Chronic lymphocytic leukemia (CLL) follows a stepwise developmental process, beginning in a specific group of normal B lymphocytes that undergo clonal expansion. These clonal expansions, known as monoclonal B lymphocytosis (MBL), are considered to be essential precursors of CLL based on immunologic, genetic, and epidemiologic studies. Since MBL represents the overabundance of a single CD5 + B cell, we tested how different the transcriptomes of MBL cells were from normal B cells (NBC) in people with MBL (NBC-MBL) and NBC in healthy donors (NBC-HD), expecting to identify activations pathways in the MBL cells that would explain clonal expansion. Methods. We collected and sorted specific cell populations from 13 patients with monoclonal B lymphocytosis (MBL): CD5 +CD19 +CD20 dimIgL-restricted MBL cells and CD5 -CD19 +CD20 + NBC. Additionally, we obtained CD5 -CD19 +CD20 + NBC from 13 healthy donors (NBC-HD). RNA was extracted and sequenced using SMART-Seq v4 Ultra Low Input and HiSeq platform. DESeq2 was used to analyze RNA-seq data. To identify genes with differential expression between these populations, the transcriptomes of MBL cells were compared to NBC from HD, employing a cutoff of P&amp;lt;0.05 and Log2fc ratio &amp;gt;± 1.5. We also used paired sample t-tests to compare 13 MBL cells to NBC from the same MBL patients. Significantly differentially expressed genes (DEG) resulting from these analyses were then subjected to Ingenuity Pathway Analysis (IPA). Results. When comparing the gene profiles of 13 MBL B cells to 13 NBC-HD cells, we identified 2695 DEG. Next, we compared gene profiles between MBL B cells and NBC-MBL cells obtained from 13 MBL patients, identifying 4821 genes. Using IPA, down-regulated B Cell Receptor Signaling, Cytokine Signaling, and Tumor Microenvironment Pathway were identified in both analyses. Upon overlapping the genes from these two analyses, we found that 803 genes passed the cutoff in both analyses. IPA analysis showed that 14 signaling pathways, including IL-33, IL-17, and HIF1a, were significantly downregulated in MBL cells. Moreover, the negative signaling pathway (Role of p14/p19ARF in Tumor Suppression) was upregulated in MBL cells. PDCD1 expression was significant higher in MBL (Log2Fc 6.8, P&amp;lt;0.0001) and 11 PDCD1 target genes were significantly regulated in MBL cells. IL-6 (Log2Fc -5.4, P&amp;lt;0.0001) expression was significantly lower in MBL and 47 IL-6 target genes were significantly regulated in MBL cells. Surprisingly, genes related to cell activation, proliferation and movement were largely downregulated in MBL cells. There were 2052 genes unique to the analysis MBL vs NBC-HD, IPA analysis showed several T-cell related signaling pathways e.g., T cell Exhaustion Signaling Pathway and T Lymphocyte Apoptosis were upregulated. In the 4018 genes unique in the analysis MBL vs NB-MBL. IPA results showed all the signaling pathways were downregulated in MBL. Discussion. Surprisingly, our transcriptomic analyses indicate that MBL cells are much less active than NBC from people with MBL and from HD. Specifically, we found that many cytokine signaling pathways and genes related with cell activation, proliferation and movement are downregulated in MBL cells. Also, PDCD1 is highly expressed in MBL cells, and PDCD1 target genes are also significantly regulated in MBL cells. This is consistent with the immunosuppressed features of MBL cells since interaction between PD-1 on B cells and its ligands in the microenvironment negatively influence the survival, proliferation, and immune evasion of CLL and MBL cells. Collectively, although functional studies are needed, these transcriptomic data strongly suggest that MBL cells are not in an activated state and are less active than even the NBC from the same person with MBL.

Light Chain Amyloidosis Plasma Cells Show Specific Chromatin Accessibility and Transcriptome

Immunoglobin light chain amyloidosis (AL) is a monoclonal gammopathy characterized by the accumulation of malignant plasma cells (PC) in the bone marrow. These PCs secrete misfolded immunoglobulin light chains fibrils, which deposit in various organs, leading to organ dysfunction. Unlike other monoclonal gammopathies such as Multiple Myeloma, both its rarity and the lower BM PC burden has limited the deep study of the tumor population. The aim of this study was to analyze the transcriptome and epigenome of the PC from patients with AL amyloidosis to identify the role of chromatin modifications in the pathogenesis of the disease. Highly purified clonal PCs were obtained from bone marrow aspirates samples of 58 AL amyloidosis patients at diagnosis, 19 monoclonal gammopathy of undetermined significance (MGUS), 149 MM and 17 healthy donors. RNA was obtained from PCs and Illumina Stranded Total RNA-seq protocol was performed according to manufacturer's specifications. Accessible chromatin mapping was performed using FAST-ATACseq (Corces et al, 2016) with minor modifications. The data obtained were aligned with the GRCh38 version of the human genome and custom pipelines were used for computational analyses. Expression of immunoglobulin genes was removed for RNAseq analysis. Unsupervised principal component analysis of the RNAseq and ATACseq data showed a clear distinction between PCs from AL patients, MM and HD. To obtain a reliable measure of the magnitude of epigenomic and transcriptome changes in AL, we compared the RNAseq and ATACseq data of AL with MM or HD. The RNAseq analysis showed some clear transcriptional programs (TPs) related to AL and different from MM and HD (Figure 1): TP1 showed 89 genes upregulated and associated with positive regulation of protein kinases and TP2 showed 274 genes downregulated and correlated with immunoregulatory interactions and mitotic regulation. On the other hand, TP3 included 560 downregulated genes associated with glutamate pathway and glycosylation of proteins and TP4 revealed 362 upregulated genes linked with B cell activation. Both patterns were identified in AL patients and HD in comparison with MM patients. Regarding chromatin accessibility, we detected a similar number of ATAC peaks between the AL and MGUS samples and with a significant difference compared to the MM samples (p=0.00014), suggesting that AL and PCs from MGUS have a lower chromatin accessibility (Figure 2). These results provide evidence that the biological perturbations in the chromatin accessibility and transcriptome of PCs from AL patients are highly specific. Although the results depicted in Figure 1 demonstrate that AL patients clustered together by relatively homogenous chromatin accessibility and transcriptome, compared with MM patients, we further investigated whether there were any discernible differences among AL patients themselves. To address this, we conducted an analysis of chromatin accessibility and transcriptome in relation to various factors, including age, gender, organ affected, light chain isotype, percentage of tumoral PCs, presence of t(11,14) or amp1q. Remarkably, we identified differences in the transcriptome between AL patients with and without t(11;14), identifying, 46 upregulated genes linked with T cell and lymphocyte apoptosis and inflammatory cytokines production and 257 downregulated genes linked with FGFR family ligand binding and activation and protein ubiquitination and phosphorylation, in t(11,14) positive AL patients. These results suggest that t(11;14) may exert a significant modulation on the transcriptome of PCs of AL patients. Finally, scRNAseq was performed to address the heterogeneity of PCs. Preliminary analysis has shown a remarkable homogeneity in malignant PCs from AL patients with very limited transcriptional differences between PCs clusters. In summary, in this study we have characterized the PCs of AL patients revealing a homogeneous and specific chromatin accessibility and transcriptome that could contribute to explain the differences between AL and other gammopathies, such as MM. In addition, the identification of these epigenetic and transcriptomic alterations in AL will allow us to better understand the aberrant biology driving this disease, such as those AL with t(11;14). Moreover, these findings open new avenues for the development of innovative therapeutic strategies to improve the outcomes for AL patients.

Association of &lt;i&gt;NR3C1 Bcl1&lt;/i&gt; gene polymorphism with impaired programmed cell death of lymphocytes in patients with atopic bronchial asthma

Atopic asthma is a chronic disease characterized by airway obstruction, bronchial hyperresponsiveness, and inflammation. Patients show increased activation of immune cells in the airways, especially T-lymphocytes, leading to chronic inflammation. The lymphocytes of asthma patients are known to have an impairment of the type 1 and 2 programmed cell death, i.e., apoptosis and autophagy, thus contributing to prolongation and intensification of inflammatory process. As compared to apoptosis, autophagy may also contribute to cell survival under stress conditions. Its disruption and hyperactivation leads to exacerbation of allergic responses. Glucocorticoids are the main drugs for the treatment of atopic bronchial asthma by activating the glucocorticoid receptor, thus triggering anti-inflammatory response and apoptosis of the cells. However, some patients exhibit resistance to therapy due to various factors, including single nucleotide polymorphisms of NR3C1 glucocorticoid receptor gene. The highest association between asthma severity and resistance to therapy was found for the GG variant of the NR3C1 Bcl1 polymorphism. Common molecular pathways for glucocorticoid receptor activation and programmed cell death and mediating molecules suggest a significant role for the polymorphic receptor variant in cell death. The aim of our study was to evaluate the effect of a single nucleotide polymorphism (G allele, i.e., Bcl1 polymorphism of NR3C1 gene) of glucocorticoid receptor on expression levels of genes that regulate apoptosis (BCL2, CASP3) and autophagy (BECN1, LC3) in lymphocytes of patients with moderate and severe atopic bronchial asthma. The study was performed with peripheral blood samples of 24 patients aged 20 to 45 years with an established diagnosis of moderate to severe atopic bronchial asthma. Using PCR technique with restriction fragment length polymorphism (RFLP) assay, the patients were distributed according to the genotypes of the BclI polymorphism of the NR3C1 gene: 12 patients with CC genotype, 8 persons with GC genotype, and 4 cases with GG genotype. The lymphocytes were isolated in Ficoll density gradient and cultivated with dexamethasone under the conditions of nutrient depletion. The level of gene expression was determined by real-time PCR. When studying associations between various genotypes of Bcl1 polymorphism and expression of cell death marker genes, the anti-apoptotic reactions were detected in lymphocytes of patients with GG polymorphism under the influence of dexamethasone thus being a potential mechanism for development of resistance to glucocorticosteroid therapy in asthma. Impaired activation of BECN1 gene expression in patients with the GG genotype may suggest deregulation of the autophagy in this group of patients, as a mode of programmed cell death. Moreover, in patients with GC genotype during long-term cultivation, exposure to dexamethasone increases the expression of the LC3 gene, indicating a more pronounced activation of autophagy. Hence, this work demonstrates differences in response of lymphocytes to synthetic glucocorticoid therapy, and probable effect of G allele (Bcl1 polymorphism) on dysregulation of programmed cell death under the influence of dexamethasone.