Peripheral Blood Leukocytes Research Articles

Transcriptomic responses of peripheral blood leukocytes to cardiac surgery after acute inflammation, and three months recovery

Traumatic perioperative conditions may trigger early systemic responses, activate leukocytes and reprogram the immune system. We hypothesize that leukocyte activation may not revert to pre-surgical states, and that protracted activation may emerge with increased risks of comorbidities. We tested this concept by examining the transcriptomes of monocytes and T cells in a representative observational cohort of patients (n = 13) admitted for elective cardiac surgery. Transcriptomes in T cells and monocytes were compared from before surgery (t0), and monocytes were analyzed longitudinally after acute (t24hr), and convalescent (t3m) time points. Monocytes and T cells expressed distinct transcriptomes, reflected by statistically significant differential expression of 558 T cell related genes. Monocytes expressed genes related to protein degradation and presented atypical activation of surface markers and cytoplasmic functions over time. Additionally, monocytes exhibited limited transcriptomic heterogeneity prior to surgery, and long-term patterns of gene expression associated with atherosclerosis showed three temporally distinct signatures. These data establish that post-cardiac surgery transcriptomes of monocytes differ even at three months compared to baselines, which may reflect latent (‘smoldering’) inflammation and persistent progression of tissue degenerative processes that should inform clinical care.

Flow Cytometry as the Tool to Define Peripheral Blood Leukocyte Signatures in Acute EBV Infection.

Primary Epstein-Barr virus (EBV) infection which can manifest as infectious mononucleosis (IM) is commonly acquired during childhood. EBV primarily invades B cells leading to a lytic reaction; the control of the infection is handled by natural killer and T cells in immunocompetent individuals. The infection has a wide spectrum of clinical findings and can lead to serious complications in patients with certain underlying immunological dysfunctions. We retrospectively investigated peripheral white blood cell populations' surface marker characteristics in IM using a comprehensive flow cytometry marker panel. Twenty-one cases of IM and seventeen EBV-seropositive cases without IM serving as controls were included. We observed novel alterations in lymphocyte, neutrophil, and monocyte populations. In addition to increased activated cytotoxic T cells and low B cells, we demonstrated high T-large granular lymphocyte (T-LGL) populations in IM cases. Furthermore, despite T cells' increased HLA-DR expression, another activation marker, CD11b, was lower in T-LGL populations. Monocytes showed increased CD16 expression; CD64 was higher in neutrophils. Our findings point to monocyte and neutrophil activation which may account for acute clinical features and may contribute to the understanding of IM immunobiology. Furthermore, they may serve as a useful tool in investigating inherited and post-transplant conditions characterized by deficiencies in controlling EBV infection.

The diagnostic value of selected immune parameters in peripheral blood of dogs with malignant mammary tumours - a preliminary study.

The main adaptive immune cells are T and B lymphocytes and they play key roles in the induction of immune responses against canine mammary tumours. Investigating these cell subpopulations may lead to more precise diagnosis of these malignancies. The percentages of CD3+, CD4+ and CD8+ T cells and of CD21+ B cells in the peripheral blood of bitches with malignant mammary tumours were compared with those in the blood of healthy animals. The phenotypic features of peripheral blood leukocytes were evaluated by flow cytometry. There was a significant difference in the mean percentages of CD3+ lymphocytes between healthy (66.7%) and metastatic dogs (46.1%), and between tumour-bearing non-metastatic (66.6%) and metastatic dogs. There was also a significant difference in CD4+ T helper cell percentages between healthy dogs (40.4%) and dogs with metastases (23.2%), and between the latter and dogs without them (35.5%). In the case of CD21+ lymphocyte subsets, a significant difference was noted between healthy animals (10.9%) and those with metastases (20.1%), and between the latter and patients without metastases (8.5%). There were also significant differences in CD3+/CD21+ ratios between the group with metastases (3.0), the healthy group (7.8), and the group without metastases (8.5). Similarly, a significant difference was noted in CD4+/CD8+ ratios between animals with metastases (1.4), bitches in the control group (2.2), and dogs without metastases (1.9). Peripheral blood leukocyte phenotypic characteristics are putative novel biomarkers. These findings may be useful in future studies improving mammary tumour diagnostic procedures, especially in metastasis detection.

Glutathione S-transferase P1-1 induction influences the cellular proteostasis and apoptotic response to uremic proteins in peripheral blood leukocytes.

Glutathione S-transferase P1-1 induction influences the cellular proteostasis and apoptotic response to uremic proteins in peripheral blood leukocytes.

Interaction between RNF4 and SART3 is associated with the risk of schizophrenia

The pathogenesis of schizophrenia (SCZ) is heavily influenced by genetic factors. Ring finger protein 4 (RNF4) and squamous cell carcinoma antigen recognized by T cells 3 (SART3) are thought to be involved in nervous system growth and development via oxidative stress pathways. Moreover, they have previously been linked to SCZ. Yet the role of RNF4 and SART3 in SCZ remains unclear. Here, we investigated how these two genes are involved in SCZ by studying their variants observed in patients. We first observed significantly elevated mRNA levels of RNF4 and SART3 in the peripheral blood in both first-episode (n = 30) and chronic (n = 30) SCZ patients compared to controls (n = 60). Next, we targeted-sequenced three single nucleotide polymorphisms (SNPs) in SART3 and six SNPs in RNF4 for association with SCZ using the genomic DNA extracted from peripheral blood leukocytes from SCZ participants (n = 392) and controls (n = 572). We observed a combination of SNPs that included rs1203860, rs2282765 (both in RNF4), and rs2287550 (in SART3) was associated with increased risk of SCZ, suggesting common pathogenic mechanisms between these two genes. We then conducted experiments in HEK293T cells to better understand the interaction between RNF4 and SART3. We observed that SART3 lowered the expression of RNF4 through ubiquitination and downregulated the expression of nuclear factor E2-related factor 2 (NRF2), a downstream factor of RNF4, implicating the existence of a possible shared regulatory mechanism for RNF4 and SART3. In conclusion, our study provides evidence that the interaction between RNF4 and SART3 contributes to the risk of SCZ. The findings shed light on the underlying molecular mechanisms of SCZ and may lead to the development of new therapies and interventions for this disorder.

The impact of grape pomace extract rich in natural complex of polyphenols on morpho-functional state of leukocytes under experimental diabetes mellitus

Background. Diabetes mellitus is one of the most common diseases in the world. Under this pathology all organs and systems of an organism are damaged, inclu­ding the immune system. Peripheral blood leukocytes are an important element of this system that suffer damage under diabetes mellitus due to the influence of reactive oxygen species and reactive nitrogen species, the number of which increases fast and leads to the development of oxidative-nitrative stress. Thus, the discovery of new diabetes-correcting drugs that possess hypoglycemic, antioxidant and immunomodulatory proper­ties is one of the principal tasks. Such properties are inherent in polyphenolic compounds, a large amount of which is contained in the grape pomace. That is why the study of grape pomace extract, rich in a natural complex of polyphenols, is important to evaluate the possibility of further use of these substances as a basis for drugs that can be used in the complex therapy of diabetes mellitus. Materials and Methods. The research used peripheral blood leukocytes of the control rats, the control animals that were treated with grape pomace extract for 14 days, animals with streptozotocin-induced diabetes mellitus and rats with experimental diabetes mellitus that were treated with grape pomace extract for 14 days. To evaluate the corrective effect of the grape pomace extract rich in a natural complex of polyphenols on the state of the antioxidant defense and the L-arginine/NO systems, the activity of antioxidant enzymes, the level of oxidative modification products of proteins and lipids, the activity of NO-synthase, the content of nitrites and nitrates, 3ʹ-nitrotyrosine-modified proteins were studied. The total number of leukocytes, the white blood cell differential and the phagocytic activity, and the average cytochemical coefficients of cationic proteins and NADPH-oxidase activity were indicated to study the effect of the grape pomace extract on the functional state of leukocytes. Results and Discussion. The study revealed normalization of the total number of leukocytes and the white blood cell differential, the activity of NADPH-oxidase, superoxide dismutase, catalase and glutathione peroxidase, and the levels of the oxidative modification products of proteins and lipids, nitrites, nitrates and 3ʹ-nitrotyrosine-modified proteins, as well as an increase in the average cytochemical coefficient of cationic proteins, and a decrease of the inducible NO-synthase activity after grape pomace extract administration for 14 days to animals with streptozotocin-induced diabetes mellitus. Conclusion. The obtained results confirm the antioxidant and immunomodulatory effects of the studied extract and justify the feasibility of using grape pomace complex of polyphenolic compounds as a basis for new drugs that will be used in the complex therapy of diabetes mellitus.

Altered counts and mitochondrial mass of peripheral blood leucocytes in patients with chronic hepatitis B virus infection.

Hepatitis B virus (HBV) damages liver cells through abnormal immune responses. Mitochondrial metabolism is necessary for effector functions of white blood cells (WBCs). The aim was to investigate the altered counts and mitochondrial mass (MM) of WBCs by two novel indicators of mitochondrial mass, MM and percentage of low mitochondrial membrane potential, MMPlow%, due to chronic HBV infection. The counts of lymphocytes, neutrophils and monocytes in the HBV infection group were in decline, especially for lymphocyte (p = 0.034) and monocyte counts (p = 0.003). The degraded MM (p = 0.003) and MMPlow% (p = 0.002) of lymphocytes and MM (p = 0.005) of monocytes suggested mitochondrial dysfunction of WBCs. HBV DNA within WBCs showed an extensive effect on mitochondria metabolic potential of lymphocytes, neutrophils and monocytes indicated by MM; hepatitis B e antigen was associated with instant mitochondrial energy supply indicated by MMPlow% of neutrophils; hepatitis B surface antigen, antiviral therapy by nucleos(t)ide analogues and prolonged infection were also vital factors contributing to WBC alterations. Moreover, degraded neutrophils and monocytes could be used to monitor immune responses reflecting chronic liver fibrosis and inflammatory damage. In conclusion, MM combined with cell counts of WBCs could profoundly reflect WBC alterations for monitoring chronic HBV infection. Moreover, HBV DNA within WBCs may be a vital factor in injuring mitochondria metabolic potential.

Annual rhythm in immune functions of blood leucocytes in an ophidian, Natrix piscator

Annual variations in animal’s physiological functions are an essential strategy to deal with seasonal challenges which also vary according to the time of year. Information regarding annual adaptations in the immune-competence to cope with seasonal stressors in reptiles is scarce. The present research plan was designed to analyze the presence of circannual immune rhythms in defense responses of the leucocytes in an ophidian, Natrix piscator. Peripheral blood leucocytes were obtained, counted, and superoxide anion production, neutrophil phagocytosis, and nitrite release were tested to assess the innate immune functions. Peripheral blood lymphocytes were separated by centrifugation (utilizing density gradient) and the cell proliferation was measured. The Cosinor rhythmometry disclosed the presence of significant annual rhythms in the number of leucocytes, superoxide anion production, nitric oxide production, and proliferation of stimulated lymphocytes. The authors found that respiratory burst activity and proliferative responses of lymphocytes were crucial immune responses that showed the annual rhythm. It was summarized that the immune function of the N. piscator is a labile attribute that makes the animal competent to cope with the seasonal stressor by adjustment in the potency of response.

#1558 Mutational spectrum of X-linked hypophosphatemia (XLH) in Hungarian patients

Abstract Background and Aims X-linked hypophosphatemia (XLH), the most common form of hypophosphatemic rickets, is caused by genetic alterations in the PHEX gene. Prevalence of the X-linked disease is ∼1/20,000. The PHEX gene has 22 exons and encodes a 749 amino acid protein with homology to zinc metallopeptidases. The physiological protein target for PHEX cleavage is unknown. PHEX promotes mineralization by regulating the expression of FGF23. To date, more than 800 disease-causing variants in the PHEX gene have been described in XLH patients. These variants might occur across the entire gene and include missense, nonsense, frameshift, splice site, copy-number variants with a consequence of loss of PHEX protein function. Genetic confirmation of XLH has significant clinical importance from risk prediction to modern treatment options. Here we describe the genetic results of 20 mutation-positive patients from 18 unrelated families, the first such report from Hungary. Method The cohort consisted of 8 male and 12 female patients (mean age at genetic diagnosis (range): 8.25 years (3-17 years) and 12 years (2-32 years)). Genomic DNA was isolated from peripheral blood leukocytes and in the mosaic case also from mouthwash sample. PHEX sequencing was performed using next generation sequencing with a custom designed library preparation kit (Twist Biosciences) on an Illumina MiSeq instrument. For the bioinformatic analysis, NextGene software was used. All small-scale mutations were confirmed by Sanger sequencing. For copy number variation (CNV) analysis, a coverage-based pipeline was used. Detected CNVs were confirmed using multiplex ligation-dependent probe amplification (MLPA). Mutation classification was performed according to the ACMG guidelines. Results All female patients were heterozygous, while all but one male patients were hemizygous. In case of one male patient, a mosaic deletion of exon 10 was observed. Most common mutation type was nonsense (n = 7) followed by small deletions/insertions (n = 6) and splicing alterations (n = 3). One missense mutation was detected. CNVs were responsible in three cases (two large deletions and one large duplication). From the 11 most common PHEX alleles known from the literature (Hum Mutat 2022;43:143-57), three (c.2104C > T, c.2239C > T, c.871C > T) were detected in our cohort. Nine novel, previously undescribed alterations were found: c.264G > A, c.1073_1074delGA, c.1148dupA, c.1645+1G > C, c.1682G > A, c.1701-1G > C, c.2150T > G, c.2172delT, e6-15dup. Conclusion Our data shed more light on the distribution of the PHEX causative variations in the Hungarian XLH patients and opens the possibility of the state-of-the-art treatment in many of them.

Zerumbone exhibits anti-inflammatory effects by suppressing eicosanoid signaling: Evidence from LPS-induced peripheral blood leukocytes

Zerumbone, a sesquiterpene isolated from Zingiber zerumbet, has many bioactivities, exhibiting anti-inflammatory properties. However, the effect of zerumbone on the eicosanoid signaling pathway has yet to be examined. Here, we deciphered the anti-eicosanoid properties of zerumbone isolated from ginger. The molecular interaction between zerumbone and eicosanoid metabolizing enzymes (COX-2, 5-LOX, FLAP, and LTA4-hydrolase) and receptors (EP-4, BLT-1, and ICAM-1) along with NOS-2 were assessed using Auto-Dock 4.2 and visualized by chimera and Liggplot+ software. Further, the leukocytes were treated with zerumbone (1–20 μM) and activated using bacterial lipopolysaccharide (LPS-10 nM). The oxidative stress (OS) markers, antioxidant enzymes, and the eicosanoid pathway mediators such as COX-2, 5-LOX, BLT-1, and EP-4 were assessed. The molecular interaction of zerumbone with eicosanoids showed a higher binding affinity with mPGES-1, followed by NOS-2, FLAP, COX-2, LTA-4-hydrolase, and BLT-1. The concentration of 5 μM zerumbone effectively prevented the generation of reactive oxygen species (ROS) and nitric oxide (NO). Likewise, zerumbone significantly (p<0.05) inhibited COX-2, 5-LOX, NOS-2, EP-4, BLT-1, and ICAM-1 expression in LPS-induced peripheral blood leukocytes from rats. Further, the zerumbone treatment on the human PBMCs activated with LPS showed significant inhibition in the expression of ICAM1, COX-2, 5-LOX, and the generation of inflammatory cytokines compared to the control. Overall, the data presented infers that zerumbone positively modulates critical enzymes and receptors of eicosanoids in leukocytes activated with lipopolysaccharides. Thus, zerumbone can be a potential anti-eicosanoid drug in managing inflammation.

TLR7 in channel catfish (Ictalurus punctatus) is expressed in the endolysosome and is stimulated by synthetic ssRNA analogs, imiquimod, and resiquimod.

Toll-like receptors (TLRs) are pivotal pattern recognition receptors (PRRs) and key mediators of innate immunity. Despite the significance of channel catfish (Ictalurus punctatus) in comparative immunology and aquaculture, its 20 TLR genes remain largely functionally uncharacterized. In this study, our aim was to determine the catfish TLR7 agonists, signaling potential, and cellular localization. Using a mammalian reporter system, we identified imiquimod and resiquimod, typical ssRNA analogs, as potent catfish TLR7 agonists. Notably, unlike grass carp TLR7, catfish TLR7 lacks the ability to respond to poly (I:C). Confocal microscopy revealed predominant catfish TLR7 expression in lysosomes, co-localizing with the endosomal chaperone protein, UNC93B1. Furthermore, imiquimod stimulation elicited robust IFNb transcription in peripheral blood leukocytes isolated from adult catfish. These findings underscore the conservation of TLR7 signaling in catfish, reminiscent of mammalian TLR7 responses. Our study sheds light on the functional aspects of catfish TLR7 and contributes to a better understanding of its role in immune defense mechanisms.

Novel insights into the ontogeny of basophils.

Basophils are the least common granulocytes, accounting for <1% of peripheral blood leukocytes. In the last 20 years, analytical tools for mouse basophils have been developed, and we now recognize that basophils play critical roles in various immune reactions, including the development of allergic inflammation and protective immunity against parasites. Moreover, the combined use of flow cytometric analyses and knockout mice has uncovered several progenitor cells committed to basophils in mice. Recently, advancements in single-cell RNA sequencing (scRNA-seq) technologies have challenged the classical view of the differentiation of various hematopoietic cell lineages. This is also true for basophil differentiation, and studies using scRNA-seq analysis have provided novel insights into basophil differentiation, including the association of basophil differentiation with that of erythrocyte/megakaryocyte and the discovery of novel basophil progenitor cells in the mouse bone marrow. In this review, we summarize the recent findings of basophil ontogeny in both mice and humans, mainly focusing on studies using scRNA-seq analyses.

Fetal Clinical and Paraclinical Outcomes in HIV-Positive Pregnant Women.

Background Adverse pregnancy outcomes in women with human immunodeficiency virus (HIV) infection remain significantly increased. Untreated maternal infection primarily leads to fetal complications, such as intrauterine growth restriction, stillbirth, or preterm birth. Concerning both maternal and fetal complications that can appear in pregnancy associated with HIV infection, the purpose of the study was to determine fetal and maternal demographic characteristics and the correlation between blood count parameters and poor fetal prognosis. Methods We conducted a quantitative study utilizing document review as the data collection method. This study encompassed a cohort of nine HIV-positive pregnant women who delivered at the Obstetrics and Gynecology Department of the University Emergency Hospital in Bucharest from January 1, 2021, to December 31, 2023. A comparative cohort of nine healthy pregnant women who delivered during the same period in the same facility was selected using stratified random sampling. We examined maternal and fetal demographic parameters and neonatal outcomes, reporting them to paraclinical laboratory data. Results The incidence of pregnancy-related HIV infections was 0.16%. The mean age of patients in the selected group was 29.88 ± 5.53. There was no statistically significant correlation between maternal clinical and paraclinical parameters in the HIV-positive and HIV-negative groups. Although there was a slightly negative difference in the fetal weight at birth, the 1-min APGAR (appearance, pulse, grimace, activity, and respiration) score, and the intrauterine growth restriction between the two groups, there was a statistically significant association between admission to the neonatal intensive care unit (NICU) and the neonates from HIV-positive pregnancies. In our study, we observed preterm deliveries in 22.22% of cases, and we did not record any stillbirths. The 1-min APGAR score was correlated with the value of leukocytes in peripheral blood. Vertical transmission was established to be 11.11% independent of maternal blood count parameters. Conclusion HIV infection during pregnancy leads to a higher risk of admission to the NICU. Fetal leukocytosis is indicative of a lower 1-min APGAR score.The primary emphasis of therapeutic intervention during pregnancy should center on vigilant monitoring of maternal viral load and the timely administration of antiretroviral therapy to enhance fetal outcomes.

Investigating the Relationship between Telomere-Related Gene Variants and Leukocyte Telomere Length in Optic Neuritis Patients.

Optic neuritis (ON) is a condition marked by optic nerve inflammation due to various potential triggers. Research indicates a link between telomeres and inflammation, as studies demonstrate that inflammation can lead to increased telomere shortening. Aim: We aimed to determine the associations of telomere-related telomeric repeat binding factor 1 (TERF1) rs1545827, rs10107605, and telomeric repeat binding factor 2 (TERF2) rs251796 polymorphisms and relative leukocyte telomere length (LTL) with the occurrence of ON. Methods: In this research, a total of 73 individuals diagnosed with optic neuritis (ON) were studied and the control group included 170 individuals without any health issues. The DNA samples were obtained from peripheral blood leukocytes, which were purified using the DNA salting-out technique. Real-time polymerase chain reaction (RT-PCR) assessed single-nucleotide polymorphisms (SNPs) and relative leukocyte telomere lengths (LTL). The data obtained were processed and analyzed using the "IBM SPSS Statistics 29.0" program. Results: Our study revealed the following results: in the male group, TERF2 rs251796 (AA, AG, and TT) statistically significantly differed between the long and short telomere group, with frequencies of 65.7%, 22.9%, and 2.0% in long telomeres, compared to 35.1%, 56.8%, and 8.1% in the short telomere group (p = 0.013). The TERF2 rs251796 CT genotype, compared to CC, under the codominant genetic model, was associated with 4.7-fold decreased odds of telomere shortening (p = 0.005). Meanwhile, CT+TT genotypes, compared to CC under the dominant genetic model, were associated with 3.5-fold decreased odds of telomere shortening (p = 0.011). Also, the CT genotype, compared to CC+TT, under the overdominant genetic model, was associated with 4.4-fold decreased odds of telomere shortening (p = 0.004). Conclusions: The current evidence may suggest a protective role of TERF2 rs251796 in the occurrence of ON in men.

Effect of Immunity to SARS-CoV-2 Virus on Blood Cellular Composition

Relevance. The new coronavirus infection (COVID-19) is still a public health problem and a threat to socio-economic well-being. Most studies have focused predominantly on humoral immunity, and there are no data on the cellular composition of blood in dynamics. Aim. To study the dynamics of changes in blood cellular composition depending on the type of immunity formed (natural, hybrid, breakthrough, postvaccinal) to SARS-CoV-2 virus. Materials and Methods. A total of 130 volunteers participated in the study. Immunophenotyping of peripheral blood leukocytes using flow cytometry was performed. The presence of specific IgG antibodies to N-protein SARS-CoV-2, total IgA and cytokines (IL-4, IL-10, IFN-γ, TNF-α) was assessed in serum by ELISA. Results and Discussion. A statistically significant increase in BL was recorded in volunteers with hybrid immunity 1 month (14,0% (12,3–16,4%)) after vaccination compared to healthy volunteers (9,1% (6,4–10,2%), p = 0,0007) and people with primary COVID-19 infection (10,2% (8,3–12,1%), p = 0,0134). In volunteers with natural and hybrid immunity, as well as in revaccinated people, an increase in B1-cells (CD3-CD19+CD5+CD27-) was observed during 3–9 months of observation. It is shown that the increase of B-lymphocytes with «switched» class of synthesized antibodies was detected in people with breakthrough immunity. Increased levels of T-lymphocytes expressing HLA-DR were recorded in all individuals during 6–9 months of follow-up. Volunteers with breakthrough immunity showed a significant increase in the positivity index when assessing the presence of specific IgG class antibodies to the coronavirus N-protein compared with volunteers with natural and hybrid immunity. Conclusions. Vaccination promotes protective immunity sufficient for timely activation of memory T- and B-cells in breakthrough immunity and maintenance of immunologic efficacy in hybrid immunity against COVID-19. The results help to assess the strain of innate and adaptive immunity in novel coronavirus infection and to fill gaps in the understanding of immunopathogenesis in COVID-19.

Respiratory Function of Mitochondria in Patients with Coronary Heart Disease with Implantable Cardioverter-Defibrillator.

We studied the respiratory activity of mitochondria in peripheral blood leukocytes from 36 patients with coronary heart disease (CHD) and a history of ventricular tachyarrhythmias required cardioverter-defibrillator implantation. The measurements were carried out in incubation buffers with different oxidation substrates (succinate and pyruvate-malate mixture). In pyruvate-malate incubation buffer, oxygen consumption rate and respiratory control coefficients in patients with triggered device did not differ significantly from those in patients without cardioverter-defibrillator triggering. At the same time, respiratory control coefficients were below the reference values. In succinate buffer, values of mitochondrial parameters were significantly lower in patients with triggered devices. Our findings indicate that mitochondria of patients with non-triggered cardioverters-defibrillators have better functional and metabolic plasticity. It was concluded that activity of respiratory processes in mitochondria could be an indicator that should be taken into the account when assessing the risk of developing ventricular tachyarrhythmias.

Comparative Analysis of PD-1, PD-L1 and CTLA-4 Expression in Infiltrating Leukocytes in Tumor Tissue and Peripheral Blood in Patients with Renal Cell Carcinoma.

Abstract Clear cell renal cell carcinoma (ccRCC) is the most prevalent subtype of kidney cancer. Despite the availability of treatment options, the overall prognosis for ccRCC remains poor, mainly for the acquired resistance to conventional therapies. Current evidence indicates that the immune system is indirectly involved in tumor progression, where T cells play a critical role in antitumor immunity. In addition, the functional state of infiltrating leukocytes predicts response to therapy in most types of cancer. In the present study, we describe the nature of infiltrating leukocytes in tumor tissue and the exhaustion-associated markers PD-1, PD-L1 and CTLA-4 in blood and renal tissue obtained from patients diagnosed with ccRCC. Our results indicated that within the tumor microenvironment, the main source of inhibitory molecule PD-L1 are the infiltrating monocytes whereas the most expressed inhibitory marker in lymphoid cells are PD-1 in CD8+ cells, but CTLA-4 in CD4+ cells. More importantly, the phenotype remains consistent across central and peripheral tissues, suggesting that exhaustion-associated markers could be measured in blood, which is a less invasive alternative to conventional histopathologic evaluation.

Evaluation of the STAT1 mRNA expression in peripheral blood cells as a predictive marker of early pregnancy in Awassi ewes

The primary objective of this study was to measure the expression profile of the signal transducer activator transcription 1 (STAT1) in the circulatory leukocytes of non-pregnant/pregnant ewes and test the possibility that it could be useful for detecting early pregnancy. Whole blood was collected from 20 ewes (control) and another 5 samples were taken on days (15, 25, 35, and 50). Also, blood was collected from 30 ewes on day 15 post insemination (PI) and another 10 samples were taken on days (25, 35, and 50) PI. Total RNA was isolated from peripheral blood leukocytes (PBLs), and STAT1 expression levels were determined by quantitative real-time PCR (qRT-PCR). Gene expression for STAT1 mRNA was 5.8-fold and 5.1 times higher in gravid as opposed to non-gravid ewes. The reliable cut-off point &gt; 1.45 was established using the receiver operating characteristic (ROC) curve, and the accuracy (Acc) for detecting pregnancy was 82%, 93%, 70% and 60%, and the sensitivity (Se) was 83%, 80%, 60% and 33% and Specificity (Sp) 95% on days 15, 25, 35 and 50, respectively. The mRNA expression was lower in ewes that control non-pregnant than pregnant ewes on days 15 and 25. So The best period to detect pregnancy was 15-25 days PI.

Xiaoyao Pills, a Chinese patent medicine, treats mild and moderate depression: A randomized clinical trial combined with DNA methylation analysis

BackgroundXiaoyao pills (XYP) is a commercial Chinese patent medicine used in the treatment of depression. However, the mechanisms underlying its therapeutic effects, as well as the patients who can benefit from XYP, have not been evaluated so far. ObjectivesTo this end, we conducted a double-blinded, random, and placebo-controlled clinical trial of orally administered XYP in patients with depression. MethodsThe 17-item Hamilton Depression Rating Scale (HAMD-17) scores were recorded at baseline, and every 2 weeks after the start of treatment. To further elucidate the epigenetic mechanism of XYP, we performed mRNA sequencing and genome-wide DNA methylation sequencing using peripheral blood leukocytes of patients and healthy. ResultsXYP effectively alleviated the symptoms in patients with mild or moderate depressive disorders, particularly that of psychomotor retardation. XYP restored aberrant gene expression and DNA methylation patterns associated with depression, and the normalization of DNA methylation correlated with downregulation of several genes. In addition, altered DNA methylation levels in the XYP-treated samples were attributed to increased expression of the DNA methyltransferase DNMT1. ConclusionsOur study provides new insights into the epigenetic mechanism underlying depression and the therapeutic effects of XYP, along with an experimental basis for using XYP in the treatment of depression. Trial Registration InformationThe name of the registry and number: U.S. Clinical Trials Registry. The link to the registration: ClinicalTrials.gov ISRCTN12746343 (https://www.isrctn.com/ISRCTN12746343). The name of the trial register is “Efficacy and safety of the Xiaoyao pill for improving the clinical symptoms of stagnation of liver qi (chi) and spleen deficiency”. The clinical trial registration number is ISRCTN12746343.

Comprehensive analysis of circRNA expression profiles in postmenopausal women differing in bone mineral density.

Postmenopausal osteoporosis (PMOP) seriously endangers the bone health of older women. Although there are currently indicators to diagnose PMOP, early diagnostic biomarkers are lacking. Circular ribonucleic acid (circRNA) has a stable structure, regulates gene expression, participates in the pathological process of disease, and has the potential to become a biomarker. The purpose of this study was to investigate circRNAs that could be used to predict patients with early PMOP. Ribonucleic acid (RNA) sequencing was performed on peripheral blood leukocytes from 15 female patients to identify differential circRNAs between different groups. Using bioinformatics analysis, enrichment analysis was performed to discover relevant functions and pathways. CircRNA-micro ribonucleic acid (miRNA) interaction analysis and messenger ribonucleic acid (mRNA) prediction and network construction help us to understand the relationship between circRNA, miRNA, and mRNA. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to validate the gene expression of candidate circRNAs. We screened out 2 co-expressed differential circRNAs, namely hsa_circ_0060849 and hsa_circ_0001394. By analyzing the regulatory network, a total of 54 miRNAs and 57 osteoporosis-related mRNAs were identified, which, as potential downstream target genes of hsa_circ_0060849 and hsa_circ_0001394, may play a key role in the occurrence and development of PMOP. The occurrence and development of PMOP is regulated by circRNAs, and hsa_circ_0060849 and hsa_circ_0001394 can be used as new diagnostic markers and therapeutic targets for early PMOP.