Peripheral Blood Leukocytes Research Articles

TLR7 agonist, DSP-0509, with radiation combination therapy enhances anti-tumor activity and modulates T cell dependent immune activation

BackgroundTLR7 is a key player in the antiviral immunity. TLR7 signaling activates antigen-presenting cells including DCs and macrophages. This activation results in the adaptive immunity including T cells and B cells. Therefore, TLR7 is an important molecule of the immune system. Based on these observations, TLR7 agonists considered to become a therapy weaponize the immune system against cancer. Radiation therapy (RT) is one of the standard cancer therapies and is reported to modulate the tumor immune response. In this study, we aimed to investigate the anti-tumor activity in combination of TLR7 agonist, DSP-0509, with RT and underlying mechanism.ResultWe showed that anti-tumor activity is enhanced by combining RT with the TLR7 agonist DSP-0509 in the CT26, LM8, and 4T1 inoculated mice models. We found that once- weekly (q1w) dosing of DSP-0509 rather than biweekly (q2w) dosing is needed to achieve superior anti-tumor activities in CT26 model. Spleen cells from the mice in RT/DSP-0509 combination treatment group showed increased tumor lytic activity, inversely correlated with tumor volume, as measured by the chromium-release cytotoxicity assay. We also found the level of cytotoxic T lymphocytes (CTLs) increased in the spleens of completely cured mice. When the mice completely cured by combination therapy were re-challenged with CT26 cells, all mice rejected CT26 cells but accepted Renca cells. This rejection was not observed with CD8 depletion. Furthermore, levels of splenic effector memory CD8 T cells were increased in the combination therapy group. To explore the factors responsible for complete cure by combination therapy, we analyzed peripheral blood leukocytes (PBLs) mRNA from completely cured mice. We found that Havcr2low, Cd274low, Cd80high, and Il6low were a predictive signature for the complete response to combination therapy. An analysis of tumor-derived mRNA showed that combination of RT and DSP-0509 strongly increased the expression of anti-tumor effector molecules including Gzmb and Il12.ConclusionThese data suggest that TLR7 agonist, DSP-0509, can be a promising concomitant when used in combination with RT by upregulating CTLs activity and gene expression of effector molecules. This combination can be an expecting new radio-immunotherapeutic strategy in clinical trials.

Telomerase Reverse Transcriptase Regulates Intracellular Ca2+ Homeostasis and Mitochondrial Function via the p53/PGC-1α Pathway in HL-1 Cells.

Telomere shortening is strongly associated with cardiovascular aging and disease, and patients with shorter telomeres in peripheral blood leukocytes are at higher risk of cardiovascular diseases such as heart failure and atrial fibrillation (AF). Telomerase reverse transcriptase (TERT) maintains telomere length, and overexpression of TERT has been shown to reduce cardiomyocyte apoptosis and myocardial infarct size, and extend the lifespan of aged mice. However, the specific impact of TERT on the electrophysiology of cardiomyocytes remains to be elucidated. The aims of this study were to evaluate the role of TERT in Ca2+ homeostasis and mitochondrial function in atrial myocytes as well as the underlying mechanisms. TERT overexpressed and silenced HL-1 cells were constructed with lentiviruses, and the respective empty lentiviral vectors were used as negative controls. Then the patch clamp technique was used to record the electrophysiological characteristics such as cell action potential duration (APD) and L-type Ca2+ currents (ICa,L), flow cytometry was used to detect intracellular Ca2+ concentration and mitochondrial membrane potential (MMP), and the Seahorse assay was used to measure the oxygen consumption rate (OCR). TERT silencing led to intracellular Ca2+ overload, shortened APD, decreased ICa,L current density, altered Ca2+ gating mechanism, decreased MMP and OCR, and increased reactive oxygen species (ROS), whereas TERT overexpression led to the reverse effects. Additionally, TERT silencing resulted in intracellular Ca2+ overload with decreased expression of the SERCA2a, CaV1.2, and NCX1.1, whereas TERT overexpression had opposing effects. Furthermore, we discovered that TERT could regulate the expression of p53 and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). The expression of PGC-1α was downregulated by the p53 agonist Tenovin-6 but upregulated by the p53 inhibitor PFTα. The effects of the PGC-1α inhibitor SR-18292 on intracellular Ca2+ and cell electrophysiology were similar to those of silencing TERT, whereas the PGC-1α agonist ZLN005 produced comparable outcomes to TERT overexpression. TERT silencing-induced Ca2+ overload and mitochondrial dysfunction may be one mechanism of age-related AF. Overexpression of TERT reduced the basis for arrhythmia formation such as AF, suggesting a favorable safety profile for TERT therapy. TERT regulated intracellular Ca2+ homeostasis and mitochondrial function through the p53/PGC-1α pathway. In addition, PGC-1α might be a novel target for AF, suggesting that intervention for AF should be not limited to abnormal cation handling.

Thymic microenvironment's impact on immunosenescence.

Age-related thymic involution is characterized by the loss of T cell development and the supporting epithelial network, which are replaced by adipose tissue. We previously showed that aging functionally impairs lymphohematopoietic progenitor cells, including thymic early T cell progenitors (ETPs), contributing to thymic involution. Considering that the thymic microenvironment is essential for thymocyte incubation, we aimed to investigate its role in age-related thymic involution and the mechanisms underlying these changes. The challenge in studying these processes led us to transplant T cell-depleted fetal thymus tissue into the kidney capsule of aged mice. This model allowed us to identify the mechanisms driving age-related changes in the thymic microenvironment and to assess whether these changes could be reversed. Flow cytometry was used to detect naïve T cells (CD62L+CD44-), including CD4 CD8 double-negative, double-positive, and single-positive T cells. Real-time PCR was used to detect and quantify signal-joint T cell receptor excision circles. We rearranged δRec-ΨJα in murine peripheral blood leukocytes to evaluate the thymic output of newly developed naïve T cells in the mice and gene expression in the thymus. Age-related thymic involution decreased naïve T cells and increased memory T cells, while fetal thymus transplantation improved thymic output and T cell production and reversed the impairment of thymopoiesis due to thymic involution in aged mice. Furthermore, the expression of key cytokines was restored and ETPs in the aged mice showed normal thymic T cell development. Our study suggests that degenerative changes in the thymic microenvironment are the primary cause of thymic dysfunction, leading to immunosenescence associated with age-related thymic involution.

Biomarkers for Radiation Biodosimetry and Correlation with Hematopoietic Injury in a Humanized Mouse Model.

After a large-scale radiological or nuclear event, hundreds of thousands of people may be exposed to ionizing radiation and require subsequent medical management. Acute exposure to moderate doses (2-6 Gy) of radiation can lead to the hematopoietic acute radiation syndrome, in which the bone marrow (BM) is severely compromised, and severe hemorrhage and infection are common. Previously, we have developed a panel of intracellular protein markers (FDXR, ACTN1, DDB2, BAX, p53 and TSPYL2), designed to reconstruct absorbed radiation dose from human peripheral blood (PB) leukocyte samples in humanized mice up to 3 days after exposure. The objective of this work was to continue to use the humanized mouse model to evaluate biomarker dose-/time- kinetics in human PB leukocytes invivo, at an earlier (day 2) and later (day 7) time point, after exposure to total-body irradiation (TBI) doses of 0 to 2 Gy of X rays. In addition, to assess hematological sensitivity and radiation-induced injury, PB leukocyte cell counts, human BM hematopoietic stem cell (HSC) and progenitor cell [multipotent progenitor (MPP), common myeloid progenitor (CMP), granulocyte myeloid progenitor (GMP), megakaryocyte/erythrocyte progenitor (MEP) and multi-lymphoid progenitor (MLP)] levels were measured, and their correlation was also examined as the BM damages are difficult to assess by routine tests. Peripheral blood B-cells were significantly lower after TBI doses of 0.5 Gy on day 2 and 2 Gy on days 2 and 7; T-cells were significantly reduced only on day 2 after 2 Gy TBI. Bone marrow HSCs and MPP cells showed a dose-dependent depletion after irradiation with 0.5 Gy and 2 Gy on day 2, and after 1 Gy and 2 Gy on day 7. Circulating B cells correlated with HSCs, MPP and MLP cells on day 2, whereas T cells correlated with MPP, and myeloid cells correlated with MLP cells. On day 7, B cells correlated with MPP, CMP, GMP and MEP, while myeloid cells correlated with CMP, GMP and MEP. The intracellular leukocyte biomarkers were able to discriminate unirradiated and irradiated samples at different time points calculated by receiver operating characteristic (ROC) curve. Using machine learning algorithm methods, combining ACTN1, p53, TSPYL2 and PB-T cell and PB-B cell counts served as a strong predictor (area under the ROC >0.8) to distinguish unirradiated and irradiated samples independent of the days after TBI. The results further validated our biomarker-based triage assay and additionally evaluated the radiation sensitivity of the hematopoietic system after TBI exposures.

Multicenter Validation of lncRNA and Target mRNA Diagnostic and Prognostic Biomarkers of Acute Ischemic Stroke From Peripheral Blood Leukocytes.

Long noncoding RNA (lncRNA) and mRNA profiles in leukocytes have shown potential as biomarkers for acute ischemic stroke (AIS). This study aimed to identify altered lncRNA and target mRNA profiles in peripheral blood leukocytes as biomarkers and to assess the diagnostic value and association with AIS prognosis. Differentially expressed lncRNAs (DElncRNAs) and differentially expressed target mRNAs (DEmRNAs) were screened by RNA sequencing in the discovery set, which consisted of 10 patients with AIS and 20 controls. Validation sets consisted of a multicenter (311 AIS versus 303 controls) and a nested case-control study (351 AIS versus 352 controls). The discriminative value of DElncRNAs and DEmRNAs added to the traditional risk factors was estimated with the area under the curve. NAMPT-AS, FARP1-AS1, FTH1, and NAMPT were identified in the multicenter case-control study (P<0.05). LncRNA NAMPT-AS was associated with cis-target mRNA NAMPT and trans-target mRNA FTH1 in all validation sets (P<0.001). Similarly, AIS cases exhibited upregulated lncRNA FARP-AS1 and FTH1 expression (P<0.001) in the nested case-control study (P<0.001). Furthermore, lncRNA FARP1-AS1 expression was upregulated in AIS patients at discharge with an unfavorable outcome (P<0.001). Positive correlations were found between NAMPT expression level and NIHSS scores of AIS patients (P<0.05). Adding 2 lncRNAs and 2 target mRNAs to the traditional risk factor model improved area under the curve by 22.8% and 5.2% in the multicenter and the nested case-control studies, respectively. lncRNA NAMPT-AS and FARP1-AS1 have potential as diagnostic biomarkers for AIS and exhibit good performance when combined with target mRNA NAMPT and FTH1.

Elevated basophil count is associated with increased risk of endometriosis.

Immunological dysregulation plays a fundamental role in the inflammatory aspects of endometriosis. Circulating blood leukocytes, one of the most abundant immune cell populations in the human body, have been shown diagnostic significance in some diseases. Nevertheless, the association between peripheral blood leukocyte counts and endometriosis remains unexplored to date. We analysed two targeted study cohorts: a tertiary centre cohort (Endometriosis at Oxford University [ENDOX] study, 325 cases/177 controls) and a large-scale population study (UK Biobank [UKBB], 1537 cases/6331 controls). In both datasets, peripheral venous blood sample results were retrieved and counts of leukocyte subpopulations, including neutrophils, lymphocytes, monocytes, eosinophils and basophils analysed. Logistic regression models were used to investigate the association of leukocyte subtype alterations with endometriosis status, adjusting for confounding factors. We demonstrate that higher blood basophil level is associated with increased odds of endometriosis. This association was first discovered in the ENDOX cohort (basophils >0.04 x10^9/L: OR 1.65 [95%CI:1.06-2.57], P trend = 0.025) and replicated in the UKBB dataset (basophils >0.04 x10^9/L: OR 1.26 [95%CI:1.09-1.45], P trend = 0.001). Notably, women with basophil counts in the upper tercile had significantly increased odds of having stage III/IV endometriosis (ENDOX study: OR = 2.30, 95% CI [1.25 to 4.22], P trend = 0.007; UKBB study (OR = 1.40, 95% CI [1.07 to 1.85], P trend = 0.015). None of the other leukocyte subtypes showed an association. Our findings suggest an association between inflammatory responses and the pathogenesis of endometriosis; future studies are warranted to investigate whether the association is causal.

Clinical characteristics and prognosis of childhood acute lymphoblastic leukemia with CD123 expression

To investigate the expression of CD123 in children with acute lymphoblastic leukemia (ALL) and its effect on the clinical characteristics and prognosis of children with B-lineage acute lymphoblastic leukemia (B-ALL). A retrospective analysis was conducted on the clinical data of 251 children with ALL who were admitted to the Department of Hematology and Oncology, Children's Hospital of Kunming Medical University, from December 2019 to June 2022. According to the expression of CD123 at initial diagnosis, the children were divided into CD123+ group and CD123- group, and the two groups were compared in terms of clinical characteristics and treatment outcome. The factors influencing the prognosis were analyzed. Among the 251 children with ALL, there were 146 children (58.2%) in the CD123+ group. The B-ALL group had a significantly higher positive expression rate of CD123 than the acute T lymphocyte leukemia group (P<0.05). Compared with the CD123- group, the CD123+ group had significantly lower peripheral blood leukocyte count and percentage of juvenile cells and a significantly higher proportion of children with high hyperdiploid karyotype or an age of 1-10 years, with a relatively low proportion of children with E2A-PBX1 fusion gene (P<0.05). The multivariate Cox proportional-hazards regression model analysis showed that compared with the >10 years group, the 1-10 years group had a significantly higher overall survival rate (P<0.05), and compared with the high risk group, the moderate risk group had a significantly higher event-free survival rate in children with B-ALL (P<0.05). CD123 is widely expressed in children with B-ALL, and positive expression of CD123 might be an indicator for good prognosis in children with B-ALL, which is of great significance for evaluating the efficacy of remission induction therapy and survival prognosis of children with B-ALL.

DNA Methylation-derived biological age and long-term mortality risk in subjects with type 2 diabetes

BackgroundIndividuals with type 2 diabetes (T2D) face an increased mortality risk, not fully captured by canonical risk factors. Biological age estimation through DNA methylation (DNAm), i.e. the epigenetic clocks, is emerging as a possible tool to improve risk stratification for multiple outcomes. However, whether these tools predict mortality independently of canonical risk factors in subjects with T2D is unknown.MethodsAmong a cohort of 568 T2D patients followed for 16.8 years, we selected a subgroup of 50 subjects, 27 survived and 23 deceased at present, passing the quality check and balanced for all risk factors after propensity score matching. We analyzed DNAm from peripheral blood leukocytes using the Infinium Human MethylationEPIC BeadChip (Illumina) to evaluate biological aging through previously validated epigenetic clocks and assess the DNAm-estimated levels of selected inflammatory proteins and blood cell counts. We tested the associations of these estimates with mortality using two-stage residual-outcome regression analysis, creating a reference model on data from the group of survived patients. ResultsDeceased subjects had higher median epigenetic age expressed with DNAmPhenoAge algorithm (57.49 [54.72; 60.58] years. vs. 53.40 [49.73; 56.75] years; p = 0.012), and accelerated DunedinPoAm pace of aging (1.05 [1.02; 1.11] vs. 1.02 [0.98; 1.06]; p = 0.012). DNAm PhenoAge (HR 1.16, 95% CI 1.05–1.28; p = 0.004) and DunedinPoAm (HR 3.65, 95% CI 1.43–9.35; p = 0.007) showed an association with mortality independently of canonical risk factors. The epigenetic predictors of 3 chronic inflammation-related proteins, i.e. CXCL10, CXCL11 and enRAGE, C-reactive protein methylation risk score and DNAm-based estimates of exhausted CD8 + T cell counts were higher in deceased subjects when compared to survived.ConclusionsThese findings suggest that biological aging, as estimated through existing epigenetic tools, is associated with mortality risk in individuals with T2D, independently of common risk factors and that increased DNAm-surrogates of inflammatory protein levels characterize deceased T2D patients. Replication in larger cohorts is needed to assess the potential of this approach to refine mortality risk in T2D.

Associations of TRAF2 (rs867186), TAB2 (rs237025), IKBKB (rs13278372) Polymorphisms and TRAF2, TAB2, IKBKB Protein Levels with Clinical and Morphological Features of Pituitary Adenomas.

The aim of this study was to determine associations of TRAF2 (rs867186), TAB2 (rs237025), IKBKB (rs13278372) gene polymorphisms and TRAF2, TAB2, IKBKB protein levels with clinical and morphological features of pituitary adenomas (PAs). This case-control study included 459 individuals divided into two groups: a control group (n = 320) and a group of individuals with PAs (n = 139). DNA from peripheral blood leukocytes was isolated using salt precipitation and column method. Real-time PCR was used for TRAF2 (rs867186), TAB2 (rs237025), and IKBKB (rs13278372) SNP genotyping, and TRAF2, TAB2, IKBKB protein concentration measurements were performed by immunoenzymatic analysis tests using a commercial ELISA kit according to the manufacturer's recommendations. The labeling index Ki-67 was determined by immunohistochemical analysis using a monoclonal antibody (clone SP6; Spring Bioscience Corporation). Statistical data analysis was performed using the programs "IMB SPSS Statistics 29.0". We found significant differences in TRAF2 (rs867186) genotypes (AA, AG, GG) between groups: 79.1%, 17.3%, 3.6% vs. 55.3%, 20.9%, 23.8% (p < 0.001). The G allele was less frequent in the PA group than in controls (12.2% vs. 34.2%, p < 0.001). The AG and GG genotypes reduced PA occurrence by 1.74-fold and 9.43-fold, respectively, compared to AA (p < 0.001). In the dominant model, GG and AG genotypes reduced PA odds by 3.07-fold, while in the recessive model, the GG genotype reduced PA odds by 8.33-fold (p < 0.001). Each G allele decreased PA odds by 2.49-fold in the additive model (p < 0.001). Microadenomas had significant genotype differences compared to controls: 81.3%, 18.8%, 0.0% vs. 55.3%, 20.9%, 23.8% (p < 0.001), with the G allele being less frequent (9.4% vs. 34.2%, p < 0.001). In macroadenomas, genotype differences were 78%, 16.5%, 5.5% vs. 55.3%, 20.9%, 23.8% (p < 0.001), and the G allele was less common (13.7% vs. 34.2%, p < 0.001). The dominant model showed that GG and AG genotypes reduced microadenoma odds by 3.5-fold (p = 0.001), and each G allele reduced microadenoma odds by 3.1-fold (p < 0.001). For macroadenomas, the GG genotype reduced odds by 6.1-fold in the codominant model (p < 0.001) and by 2.9-fold in GG and AG genotypes combined compared to AA (p < 0.001). The recessive model indicated the GG genotype reduced macroadenoma odds by 5.3-fold (p < 0.001), and each G allele reduced odds by 2.2-fold in the additive model (p < 0.001). The TRAF2 (rs867186) G allele and GG genotype are significantly associated with reduced odds of pituitary adenomas, including both microadenomas and macroadenomas, compared to the AA genotype. These findings suggest a protective role of the G allele against the occurrence of these tumors.

Molecular circadian clock disruption in the leukocytes of individuals with type 2 diabetes and overweight, and its relationship with leukocyte–endothelial interactions

Aims/hypothesisAlterations in circadian rhythms increase the likelihood of developing type 2 diabetes and CVD. Circadian rhythms are controlled by several core clock genes, which are expressed in nearly every cell, including immune cells. Immune cells are key players in the pathophysiology of type 2 diabetes, and participate in the atherosclerotic process that underlies cardiovascular risk in these patients. The role of the core clock in the leukocytes of people with type 2 diabetes and the inflammatory process associated with it are unknown. We aimed to evaluate whether the molecular clock system is impaired in the leukocytes of type 2 diabetes patients and to explore the mechanism by which this alteration leads to an increased cardiovascular risk in this population.MethodsThis is an observational cross-sectional study performed in 25 participants with type 2 diabetes and 28 healthy control participants. Clinical and biochemical parameters were obtained. Peripheral blood leukocytes were isolated using magnetic bead technology. RNA and protein lysates were obtained to assess clock-related gene transcript and protein levels using real-time PCR and western blot, respectively. Luminex XMAP technology was used to assess levels of inflammatory markers. Leukocyte–endothelial interaction assays were performed by perfusing participants’ leukocytes or THP-1 cells (with/without CLK8) over a HUVEC monolayer in a parallel flow chamber using a dynamic adhesion system.ResultsParticipants with type 2 diabetes showed increased BMAL1 and NR1D1 mRNA levels and decreased protein levels of circadian locomotor output cycles kaput (CLOCK), cryptochrome 1 (CRY1), phosphorylated basic helix-loop-helix ARNT like 1 (p-BMAL1) and period circadian protein homologue 2 (PER2). Correlation studies revealed that these alterations in clock proteins were negatively associated with glucose, HbA1c, insulin and HOMA-IR levels and leukocyte cell counts. The leukocyte rolling velocity was reduced and rolling flux and adhesion were enhanced in individuals with type 2 diabetes compared with healthy participants. Interestingly, inhibition of CLOCK/BMAL1 activity in leukocytes using the CLOCK inhibitor CLK8 mimicked the effects of type 2 diabetes on leukocyte–endothelial interactions.Conclusions/interpretationOur study demonstrates alterations in the molecular clock system in leukocytes of individuals with type 2 diabetes, manifested in increased mRNA levels and decreased protein levels of the core clock machinery. These alterations correlated with the impaired metabolic and proinflammatory profile of the participants with type 2 diabetes. Our findings support a causal role for decreased CLOCK/BMAL1 activity in the increased level of leukocyte–endothelial interactions. Overall, our data suggest that alterations in core clock proteins accelerate the inflammatory process, which may ultimately precipitate the onset of CVD in patients with type 2 diabetes.Graphical

Hemopexin regulates acute-phase inflammatory response against pathogenetic infections in black rockfish (Sebastes schlegelii)

Hemopexin as an acute-phase protein binds heme with a high affinity and participates in a variety of processes of physiology and pathology. Hemopexin plays an important role in regulating inflammatory response by regulating pro-/anti-inflammatory cytokines. In this study, the hemopexin gene was identified from black rockfish. The open reading frame of the black rockfish hemopexin (SsHPX) gene was predicted and amplified. The different expression patterns of SsHPX in microbial-challenged tissues and peripheral blood leukocytes (PBLs) were analyzed by RT-qPCR. The results indicated that SsHPX showed high evolutionary conservations in sequence and structure. The expression of SsHPX was significantly induced in a time-dependent manner in black rockfish intestine after Edwardsiella piscicida infection, while the evidently down-regulations were observed in the PBLs following the stimulations of two microbial ligands. The recombinant SsHPX protein (rSsHPX) exhibited microbial binding ability. In addition, rSsHPX could reduce bacterial proliferation with effect, thereby alleviating the inflammatory responses induced by bacteria. Moreover, almost all the expressions of pro-inflammatory cytokines (tnfα, il-1β, il-8 and il-17c) and anti-inflammatory cytokine (IL-10) were inhibited in black rockfish intestine cells stimulated by the mixing solution of LPS + rSsHPX comparing with the group with LPS stimulation. Overall, this study provides a theoretical basis for investigating the key roles of an acute-phase protein hemopexin in bacteriostatic ability and inflammatory regulations in black rockfish during bacterial infection. These findings also contribute to a better understanding of the relatively stable and conserved biological function of hemopexin during species evolution.

Investigate the impact of laser dosages on the regeneration of peripheral blood leukocytes after gamma radiation exposure

This research aims to examine how treatment with lasers affects the regeneration of peripheral blood leukocytes following ionizing radiation exposure. Furthermore, how may laser treatment be administered to people who have inadvertently come into contact with ionizing radiation? After being subjected to one dose of five Gy, non-lethal (1.5 Gy/min) from gamma radiation (cobalt-60), the mice developed radiation-induced sickness, as evidenced by a drop in white blood cell count and hematological symptoms. The mice were subsequently subjected to non-ionizing radiation using a 20 W power laser (Nd: YAG 1064 nm). Within the first two weeks after the application of gamma rays for 30 min, there were a total of five laser treatments. Laser energy yielded a total of 60,000 J/cm2. The energy density in each treatment was 12,000 J/cm2. Based on the peripheral blood examination, the study establishes that the use of lasers had an important influence on the number of lymphocytes two weeks after irradiation. Furthermore, following laser treatment, the lymphocyte lineage of white blood cells recovered more quickly. In gamma-irradiated mice, the study showed that the treatment laser under examination increased the overall counts of several types of white blood cells, indicating an indefinite effect. The investigation's findings may have an impact on how radiation illness is treated for specific patients. The results of this investigation may have a substantial influence on the treatment of radiation sickness in people.

Sangju Cold Granule exerts anti-viral and anti-inflammatory activities against influenza A virus in vitro and in vivo

Ethnopharmacological relevanceSangju Cold Granule (SJCG) is a classical traditional Chinese medicine (TCM) prescription described in “Item Differentiation of Warm Febrile Diseases”. Historically, SJCG was employed to treat respiratory illnesses. Despite its popular usage, the alleviating effect of SJCG on influenza A virus infection and its mechanisms have not been fully elucidated. Aim of the studyInfluenza is a severe respiratory disease that threatens human health. This study aims to assess the therapeutic potential of SJCG and the possible molecular mechanism underlying its activity against influenza A virus in vitro and in vivo. Materials and methodsUltrahigh-performance liquid chromatography (UPLC)-Q-Exactive was used to identify the components of SJCG. The 50% cytotoxic concentration of SJCG in MDCK and A549 cells were determined using the CCK-8 assay. The activity of SJCG against influenza A virus H1N1 was evaluated in vitro using plaque reduction and progeny virus titer reduction assays. RT-qPCR was performed to obtain the expression levels of inflammatory mediators and the transcriptional regulation of RIG-I and MDA5 in H1N1-infected A549 cells. Then, the mechanism of SJCG effect on viral replication and inflammation was further explored by measuring the expressions of proteins of the RIG-I/NF-kB/IFN(I/III) signaling pathway by Western blot. The impact of SJCG was explored in vivo in an intranasally H1N1-infected BALB/c mouse pneumonia model treated with varying doses of SJCG. The protective role of SJCG in this model was evaluated by survival, body weight monitoring, lung viral titers, lung index, lung histological changes, lung inflammatory mediators, and peripheral blood leukocyte count. ResultsThe main SJCG chemical constituents were flavonoids, carbohydrates and glycosides, amino acids, peptides, and derivatives, organic acids and derivatives, alkaloids, fatty acyls, and terpenes. The CC50 of SJCG were 24.43 mg/mL on MDCK cells and 20.54 mg/mL on A549 cells, respectively. In vitro, SJCG significantly inhibited H1N1 replication and reduced the production of TNF-α, IFN-β, IL-6, IL-8, IL-13, IP-10, RANTES, TRAIL, and SOCS1 in infected A549 cells. Intracellularly, SJCG reduced the expression of RIG-I, MDA5, P–NF-κB P65 (P–P65), P-IκBα, P-STAT1, P-STAT2, and IRF9. In vivo, SJCG enhanced the survival rate and decreased body weight loss in H1N1-infected mice. Mice with H1N1-induced pneumonia treated with SJCG showed a lower lung viral load and lung index than untreated mice. SJCG effectively alleviated lung damage and reduced the levels of TNF-α, IFN-β, IL-6, IP-10, RANTES, and SOCS1 in lung tissue. Moreover, SJCG significantly ameliorated H1N1-induced leukocyte changes in peripheral blood. ConclusionsSJCG significantly reduced influenza A virus and virus-mediated inflammation through inhibiting the RIG-I/NF-kB/IFN(I/III) signaling pathway. Thus, SJCG could provide an effective TCM for influenza treatment.

Effect of β-glucans on rainbow trout (Oncorhynchus mykiss) IgM+ B cells

β-glucans are carbohydrates present in the cell wall of many fungi, which are often used as immunostimulants in feeds for farmed species. Their capacity to activate innate immune responses directly acting on innate cell populations has been widely documented in fish. However, whether they can affect the functionality of adaptive immune cells has been scarcely explored. In this context, in the current work, we have determined the effects of β-glucans on rainbow trout blood IgM+ B cells in the presence or absence of 2,4,6-trinitrophenyl hapten conjugated to lipopolysaccharide (TNP-LPS), a model antigen. For this, rainbow trout peripheral blood leukocytes were incubated with different doses of β-glucans or media alone in the presence or absence of TNP-LPS for 48 h. The size, levels of expression of surface MHC II, antigen processing and phagocytic capacities and proliferation of IgM+ B cells were then studied by flow cytometry. The number of IgM-secreting cells in the cultures was also estimated by ELISpot. β-glucans significantly decreased the levels of surface MHC II expression and the antigen processing capacities of these cells, especially in the presence of TNP-LPS, while they increased their phagocytic activity. On their own, β-glucans slightly activated the proliferation of IgM+ B cells but reduced that induced by TNP-LPS. In contrast, β-glucans significantly increased the number of cells secreting IgM in the cultures. This effect of β-glucans on the IgM-secreting capacity of B cells was also confirmed through a feeding experiment, in which the IgM-secreting capacity of blood leukocytes obtained from fish fed a β-glucan-supplemented diet for one month was compared to that of leukocytes obtained from fish fed a control diet. Altogether, these findings contribute to increase our knowledge regarding the effects of β-glucans on fish adaptive responses.

Enhancers regulate genes linked to severe and mild childhood asthma

BackgroundChildren with severe asthma suffer from recurrent symptoms and impaired quality of life despite advanced treatment. Underlying causes of severe asthma are not completely understood, although genetic mechanisms are known to be important. ObjectiveThe aim of this study was to identify gene regulatory enhancers in leukocytes, to describe the role of these enhancers in regulating genes related to severe and mild asthma in children, and to identify known asthma-related SNPs situated in proximity to enhancers. MethodsGene enhancers were identified and expression of enhancers and genes were measured by Cap Analysis Gene Expression (CAGE) data from peripheral blood leukocytes from children with severe asthma (n = 13), mild asthma (n = 15), and age-matched controls (n = 9). ResultsFrom a comprehensive set of 8,289 identified enhancers, we further defined a robust sub-set of the high-confidence and most highly expressed 4,738 enhancers. Known single nucleotide polymorphisms, SNPs, related to asthma coincided with enhancers in general as well as with specific enhancer-gene interactions. Blocks of enhancer clusters were associated with genes including TGF-beta, PPAR and IL-11 signaling as well as genes related to vitamin A and D metabolism. A signature of 91 enhancers distinguished between children with severe and mild asthma as well as controls. ConclusionsGene regulatory enhancers were identified in leukocytes with potential roles related to severe and mild asthma in children. Enhancers hosting known SNPs give the opportunity to formulate mechanistic hypotheses about the functions of these SNPs.

Expression and Significance of PKM1 in Acute Myeloid Leukaemia.

To investigate the expression level of pyruvate kinase M1 (PKM1) in patients with acute myeloid leukaemia (AML) as well as its clinical significance. A case-control study. Place and Duration of the Study: Department of Haematology, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, China, from January 2013 to 2023. The expression levels of PKM1 and pyruvate kinase m2 (PKM2) in the bone marrow of 65 AML patients (excluding M3) and 31 healthy volunteers were determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), a method that measures fluorescence in real-time. The associations between PKM1, PKM2 expressions, clinical parameters, and the survival and prognosis of AML patients were analysed. AML patients showed higher PKM1 expression compared to controls. The area under the curve (AUC) of the receiver operating characteristics (ROC) was 0.65 (p = 0.017). PKM1 expression was correlated with peripheral blood leukocyte count (r = -0.276, p = 0.026), CCAAT enhancer-binding protein alpha CEBPA mutation (r = -0.306, p = 0.014), and chemotherapy-induced response (r = -0.292, p = 0.018). Patients with high PKM1 expression had a lower remission rate (p = 0.019) and long-term survival rate (p = 0.034) than those with low PKM1 expression. Patients with AML showed a rise in PKM2 levels; however, the variation was not statistically significant (p >0.05). PKM1 expression is upregulated in AML and patients with high PKM1 expression have a lower survival rate. PKM1, Acute myeloid leukaemia, Clinical prognosis.

Telomere length as a predictor of therapy response and survival in patients diagnosed with ovarian carcinoma

Impaired telomere length (TL) maintenance in ovarian tissue may play a pivotal role in the onset of epithelial ovarian cancer (OvC). TL in either target or surrogate tissue (blood) is currently being investigated for use as a predictor in anti-OvC therapy or as a biomarker of the disease progression, respectively. There is currently an urgent need for an appropriate approach to chemotherapy response prediction.We performed a monochrome multiplex qPCR measurement of TL in peripheral blood leukocytes (PBL) and tumor tissues of 209 OvC patients. The methylation status and gene expression of the shelterin complex and telomerase catalytic subunit (hTERT) were determined within tumor tissues by High-Throughput DNA methylation profiling and RNA sequencing (RNA-Seq) analysis, respectively. The patients sensitive to cancer treatment (n = 46) had shorter telomeres in PBL compared to treatment-resistant patients (n = 93; P = 0.037). In the patients with a different therapy response, transcriptomic analysis showed alterations in the peroxisome proliferator-activated receptor (PPAR) signaling pathway (q = 0.001). Moreover, tumor TL shorter than the median corresponded to better overall survival (OS) (P = 0.006). TPP1 gene expression was positively associated with TL in tumor tissue (P = 0.026).TL measured in PBL could serve as a marker of platinum therapy response in OvC patients. Additionally, TL determined in tumor tissue provides information on OvC patients' OS.

First-trimester hemoglobin, haptoglobin genotype, and risk of gestational diabetes mellitus in a retrospective study among Chinese pregnant women

BackgroundThis study aimed to assess whether the Haptoglobin (Hp) genotype influences the relationship between hemoglobin (Hb) levels and the development of gestational diabetes mellitus (GDM). Additionally, it sought to evaluate the interaction and joint association of Hb levels and Hp genotype with GDM risk.MethodsThis retrospective study involved 358 women with GDM and 1324 women with normal glucose tolerance (NGT). Peripheral blood leukocytes were collected from 360 individuals at 14–16 weeks’ gestation for Hp genotyping. GDM was diagnosed between 24–28 weeks’ gestation. Interactive moderating effect, joint analysis, and mediation analysis were performed to evaluate the crosslink of Hb levels and Hp genotype with GDM risk.ResultsWomen who developed GDM had significantly higher Hb levels throughout pregnancy compared to those with NGT. Increase first-trimester Hb concentration was associated with a progressive rise in GDM incidence, glucose levels, glycosylated hemoglobin levels, Homeostasis Model Assessment for Insulin Resistance (HOMA-IR) values, cesarean delivery rates, and composite neonatal outcomes. Spline regression showed a significant linear association of GDM incidence with continuous first-trimester Hb level when the latter exceeded 122 g/L. Increased first-trimester Hb concentration was an independent risk factor for GDM development after adjusting for potential confounding factors in both the overall population and a matched case-control group. The Hp2–2 genotype was more prevalent among pregnant women with GDM when first-trimester Hb exceeded 122 g/L. Significant multiplicative and additive interactions were identified between Hb levels and Hp genotype for GDM risk, adjusted for age and pre-pregnancy BMI. The odds ratio (OR) for GDM development increased incrementally when stratified by Hb levels and Hp genotype. Moreover, first-trimester Hb level partially mediated the association between Hp genotype and GDM risk.ConclusionIncreased first-trimester Hb levels were closely associated with the development of GDM and adverse pregnancy outcomes, with this association moderated by the Hp2–2 genotype.

Peripheral blood leukocyte Telomere length and endometriosis: A Mendelian randomization study

BackgroundThe link between peripheral blood leukocyte telomere length (LTL) and endometriosis has remained uncertain. In order to investigate this association, a two-sample Mendelian randomization(MR) analysis was performed. MethodsWe extracted Single-nucleotide polymorphisms (SNPs) associated with LTL from a published genome-wide association study (GWAS) comprising 472,174 individuals. Data on endometriosis, including its seven subtypes, were sourced from the iue open gwas project. Four methods were employed for MR: Inverse-variance weighted analysis (IVW), Mendelian randomization-Egger regression (MR Egger), weighted-median analysis, and Weighted Mode. ResultsGenetically determined LTL was identified as a factor that can promote the occurrence of endometriosis. With every 1-SD increase in LTL, the risk of endometriosis increased by 26 % (OR = 1.260, 95 % CI = 1.073 to 1.479; P = 0.005). Genetically determined LTL also contributed to endometriosis subtypes: intestine (OR = 3.584, 95 % CI = 1.597 to 8.041; P = 0.002), ovary (OR = 1.308, 95 % CI = 1.033 to 1.655; P = 0.026), rectovaginal septum and vagina (OR = 1.360, 95 % CI = 1.000 to 1.851; P = 0.049). There was no observed causal relationship between LTL and the other four subtypes. ConclusionThis study, utilizing genetic data, offers evidence that longer LTL may cause increased risks of endometriosis, specifically endometriosis of the intestine, ovary, rectovaginal septum and vagina. These findings not only suggest that LTL may serve as a predictive factor for assessing the prevalence of three endometriosis subtypes but also provide new insights into the study of endometriosis pathogenesis.

Longitudinal biomarker evaluation in Fabry disease patients receiving lentivirus-mediated gene therapy

Aim: In 2016, a team of Canadian researchers initiated the world’s first gene therapy clinical trial for Fabry disease. The study, aiming to determine the safety and toxicity of lentivirus α-galactosidase A transduced autologous CD34+ cells in adult males with Fabry disease (n = 5), was conducted at three Canadian centers. The objective of the present work was to evaluate a profile of Fabry disease-related biomarkers in five participants during a 5-year surveillance period, as part of the evaluation of this novel therapy. Methods: Sixteen globotriaosylceramide (Gb3) isoforms and eight globotriaosylsphingosine (lyso-Gb3) analogues were measured by LC-MS/MS in plasma and urine at numerous time points before and after gene therapy. Plasma and peripheral blood leukocyte α-galactosidase A activity were also measured. Results: Levels of urine and plasma lyso-Gb3 and analogues, and urine Gb3 were lower after gene therapy and while treated with enzyme replacement therapy (ERT) compared to baseline (before gene therapy) in participants treated with ERT only. Three participants chose to cease ERT treatment at some point after gene therapy. Two of these patients had lower levels of urine and plasma lyso-Gb3 and analogues compared to baseline, whereas one participant had higher levels of these biomarkers compared to baseline. An increase in urine Gb3 was observed when ERT treatment ceased after gene therapy (P &lt; 0.05) in all three participants. Conclusion: The evaluation of this complete glycosphingolipid biomarker profile was useful in monitoring the biochemical response to gene therapy in this cohort of five patients with Fabry disease.