Genes In Peripheral Blood Mononuclear Cells Research Articles

Antiretroviral Therapy Suppresses RNA N6-Methyladenosine Modification in Peripheral Blood Mononuclear Cells from HIV-1-Infected Individuals.

RNA N6-methyladenosine (m6A) modification is important for regulating gene expression and innate immune responses to viral infection. HIV-1 in vitro infection induces a significant increase in m6A modification of cellular RNA; however, whether m6A levels of cellular RNA are affected by HIV-1 replication or by antiretroviral therapy (ART) in infected individuals remains unknown. Using dot blot or enzyme-linked immunosorbent assay, we measured RNA m6A levels of peripheral blood mononuclear cells (PBMCs) from healthy donors or HIV-1-infected individuals with or without ART. Using a reverse transcription-quantitative polymerase chain reaction array, we quantified expression levels of 84 type-I interferon (IFN-I)-responsive genes in PBMCs from some individuals of these three groups. RNA m6A levels in PBMCs from HIV-1 viremic patients (n = 10) were significantly higher (p ≤ .0001) compared with ART-treated individuals (n = 22) or 1.5-fold higher compared with healthy donors (n = 14). However, the increase in RNA m6A levels did not correlate with changes in the expression of 10 m6A-regulatory genes. We found significant upregulation and downregulation in the expression of several IFN-I-responsive genes from HIV-1 viremic patients (n = 4) and ART-treated patients (n = 6) compared with healthy donors (n = 5), respectively. Our results suggest that post-transcriptional m6A modification may contribute to the regulation of IFN-I-responsive gene expression during HIV-1 infection and ART.

Peripheral Blood Mononuclear Cell Gene Expression Associated with Pulmonary Microvascular Perfusion: The Multi-Ethnic Study of Atherosclerosis Chronic Obstructive Pulmonary Disease Study.

Rationale Chronic obstructive pulmonary disease (COPD) and emphysema are associated with endothelial damage and altered pulmonary microvascular perfusion. Molecular mechanisms underlying these changes are poorly understood in patients due, in part, to the inaccessibility of the pulmonary vasculature. Peripheral blood mononuclear cells (PBMC) interact with the pulmonary endothelium. Objective To test the association between gene expression in PBMCs and pulmonary microvascular perfusion in COPD. Methods The Multi-Ethnic Study of Atherosclerosis (MESA) COPD Study recruited two independent samples of COPD cases and controls with 10 or more pack-years. In both samples, pulmonary microvascular blood flow, pulmonary microvascular blood volume (PMBV), and mean transit time were assessed on contrast-enhanced MRI, and PBMC gene expression was assessed by microarray. Additional replication was performed in a third sample with PMBV measures on contrast-enhanced, dual-energy CT. Differential expression analyses were adjusted for age, gender, race-ethnicity, educational attainment, height, weight, smoking status, and pack-years. Results The 79 participants in the discovery sample had mean age of 69±6 years, 44% were female, 25% were non-white, 34% were current smokers and 66% had COPD. There were large PBMC gene expression signatures associated with pulmonary microvascular perfusion traits, with several replicated in the replication sets with MRI (n=47) or dual-energy CT scan (n=157) measures. Many of the identified genes are involved in inflammatory processes, including NF-κB and chemokine signaling pathways. Conclusions PBMC gene expression in NF-κB, inflammatory and chemokine signaling pathways was associated pulmonary microvascular perfusion in COPD, potentially offering new targetable candidates for novel therapies.

Evaluation of MMP-9, IL-6, TNF-α levels and peripheral blood mononuclear cells genes expression of MMP-9 and TIMP-1 in Iranian patients with coronary artery disease.

Coronary artery disease (CAD) is the main cause of death and is characterized by atherosclerosis in coronary arteries. Inflammation plays a crucial role in the progression and development of atherosclerosis. The present study consisted of 132 Iranian individuals who underwent coronary angiography, 65 patients with CAD, and 67 controls. The matrix metalloproteinase-9 (MMP-9), TNF-α, IL-6, and vitamin D serum levels were measured by the ELISA technique. The gene expression of MMP-9 and tissue inhibitors of metalloproteinase (TIMP-1) was estimated by real-time PCR assay. A considerable increase in levels and PBMC gene expression of MMP-9 and serum levels of IL-6 and TNF-α were found in CAD patients compared with controls. A significant decrease was detected in vitamin D levels of CAD patients in comparison with controls. A considerable direct correlation was found between MMP-9 levels and MMP-9 and TIMP1 gene expression in CAD patients. MMP-9 levels positively correlated with LDL-C in CAD patients. The correlation between TIMP1 gene expression and IL-6 levels was also negatively significant. There were positive correlations between MMP-9 levels with IL-6 and TNF-α serum levels in CAD patients. This study showed that the interaction between MMPs, TIMP1, and cytokines could play a role in the pathogenesis of atherosclerosis. The present study suggested that high levels of TNF-α and IL-6 and vitamin D deficiency in our studied patients could disturb the MMP-9/TIMP-1 balance and lipid metabolism, leading to plaque formation/ rupture in predisposed CAD patients.

Affective reactivity to daily stressors and immune cell gene expression in the MIDUS study

Affective reactivity to stress is a person-level measurement of how well an individual copes with daily stressors. A common method of measuring affective reactivity entails the estimation of within-person differences of either positive or negative affect on days with and without stressors present. Individuals more reactive to common stressors, as evidenced by affective reactivity measurements, have been shown to have increased levels of circulating pro-inflammatory markers. While affective reactivity has previously been associated with inflammatory markers, the upstream mechanistic links underlying these associations are unknown. Using data from the Midlife in the United States (MIDUS) Refresher study (N = 195; 52% female; 84% white), we quantified daily stress processes over 10 days and determined individuals’ positive and negative affective reactivities to stressors. We then examined affective reactivity association with peripheral blood mononuclear cell (PBMC) gene expression of the immune-related conserved transcriptional response to adversity. Results indicated that individuals with a greater decrease in positive affect to daily stressors exhibited heightened PBMC JUNB expression after Bonferroni corrections (p-adjusted < 0.05). JUNB encodes a protein that acts as a transcription factor which regulates many aspects of the immune response, including inflammation and cell proliferation. Due to its critical role in the activation of macrophages and maintenance of CD4+ T-cells during inflammation, JUNB may serve as a potential upstream mechanistic target for future studies of the connection between affective reactivity and inflammatory processes. Overall, our findings provide evidence that affective reactivity to stress is associated with levels of immune cell gene expression.

An atlas of genome-wide gene expression and metabolite associations and possible mediation effects towards body mass index.

Investigating the cross talk of different omics layers is crucial to understand molecular pathomechanisms of metabolic diseases like obesity. Here, we present a large-scale association meta-analysis of genome-wide whole blood and peripheral blood mononuclear cell (PBMC) gene expressions profiled with Illumina HT12v4 microarrays and metabolite measurements from dried blood spots (DBS) characterized by targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) in three large German cohort studies with up to 7706 samples. We found 37,295 associations comprising 72 amino acids (AA) and acylcarnitine (AC) metabolites (including ratios) and 8579 transcripts. We applied this catalogue of associations to investigate the impact of associating transcript-metabolite pairs on body mass index (BMI) as an example metabolic trait. This is achieved by conducting a comprehensive mediation analysis considering metabolites as mediators of gene expression effects and vice versa. We discovered large mediation networks comprising 27,023 potential mediation effects within 20,507 transcript-metabolite pairs. Resulting networks of highly connected (hub) transcripts and metabolites were leveraged to gain mechanistic insights into metabolic signaling pathways. In conclusion, here, we present the largest available multi-omics integration of genome-wide transcriptome data and metabolite data of amino acid and fatty acid metabolism and further leverage these findings to characterize potential mediation effects towards BMI proposing candidate mechanisms of obesity and related metabolic diseases. KEY MESSAGES: Thousands of associations of 72 amino acid and acylcarnitine metabolites and 8579 genes expand the knowledge of metabolome-transcriptome associations. A mediation analysis of effects on body mass index revealed large mediation networks of thousands of obesity-related gene-metabolite pairs. Highly connected, potentially mediating hub genes and metabolites enabled insight into obesity and related metabolic disease pathomechanisms.

The role of SOD2 and NOS2 genes in the molecular aspect of bladder cancer pathophysiology

Bladder cancer (BC) is a severe health problem of the genitourinary system and is characterised by a high risk of recurrence. According to the recent GLOBOCAN report, bladder cancer accounts for 3% of diagnosed cancers in the world, taking 10th place on the list of the most common cancers. Despite numerous studies, the full mechanism of BC development remains unknown. Nevertheless, precious results suggest a crucial role of oxidative stress in the development of BC. Therefore, this study explores whether the c. 47 C > T (rs4880)—SOD2, (c. 1823 C > T (rs2297518) and g.-1026 C > A (rs2779249)—NOS2(iNOS) polymorphisms are associated with BC occurrence and whether the bladder carcinogenesis induces changes in SOD2 and NOS2 expression and methylation status in peripheral blood mononuclear cells (PBMCs). In this aim, the TaqMan SNP genotyping assay, TaqMan Gene Expression Assay, and methylation‐sensitive high‐resolution melting techniques were used to genotype profiling and evaluate the expression of the genes and the methylation status of their promoters, respectively. Our findings confirm that heterozygote of the g.-1026 C > A SNP was associated with a decreased risk of BC. Moreover, we detected that BC development influenced the expression level and methylation status of the promoter region of investigated genes in PBMCs. Concluding, our results confirmed that oxidative stress, especially NOS2 polymorphisms and changes in the expression and methylation of the promoters of SOD2 and NOS2 are involved in the cancer transformation initiation of the cell urinary bladder.

TRIM5 Promotes Systemic Lupus Erythematosus Through CD4(+) T Cells and Macrophage.

Systemic lupus erythematosus (SLE) is a typical autoimmune disease characterized by the involvement of multiple organs and the production of antinuclear antibodies. This study aimed to investigate the molecular mechanism of SLE. We retrieved genome-wide gene expression levels from five public datasets with relatively large sample sizes from the Gene Expression Omnibus (GEO), and we compared the expression profiles of peripheral blood mononuclear cells (PBMCs) from SLE patients and healthy controls (HCs). The expression of seven target genes in PBMCs from 25 cases and 3 HCs was further validated by reverse-transcription quantitative PCR (RT‒qPCR). Flow cytometry was used for verifying the proportion of naive CD4(+) T cells and M2 macrophages in PBMCs from 5 cases and 4 HCs. We found 14 genes (TRIM5, FAM8A1, SHFL, LHFPL2, PARP14, IFIT5, PARP12, DDX60, IRF7, IF144, OAS1, OAS3, RHBDF2, and RSAD2) that were differentially expressed among all five datasets. The heterogeneity test under the fixed effect model showed no obvious heterogeneity of TRIM5, FAM8A1, and SHFL across different populations. TRIM5 was positively correlated with the remaining 13 genes. By separating patient samples into TRIM5-high and TRIM5-low groups, we found that up-regulated genes in the TRIM5-high group were mainly enriched in virus-related pathways. Immune cell proportion analysis and flow cytometry revealed that naive CD4(+) T cells were significantly decreased while M2 macrophages were increased in the SLE group. TRIM5 expression levels were negatively correlated with naive CD4(+) T cells but positively correlated with M2 macrophages. Our data indicated that TRIM5 might be a key factor that modulates SLE etiology, possibly through naive CD4(+) T cells and M2 macrophages.

Investigation of conceptus stimulated gene expression in buffalo peripheral blood mononuclear cells as potential diagnostic markers of early pregnancy

Exploration of novel strategies for early pregnancy diagnosis is pivotal in enhancing the reproductive potential and monetary gains from dairy herds. In buffalo, the trophectoderm cells of the elongating conceptus secrete interferon-tau that stimulates the transcription of various genes in peripheral blood mononuclear cells (PBMC) during the peri-implantation period. We explored the differential expression of classical (ISG15) and novel (LGALS3BP and CD9) early pregnancy markers in PBMC of buffaloes during various stages of pregnancy. Natural heat was detected in buffaloes by assessing the vaginal fluid, and artificial insemination (AI) was done. Whole blood was collected from the jugular vein in EDTA-containing vacutainers for PBMC isolation before AI (0-day) and 20, 25 and 40 d post-AI. On day 40, transrectal ultrasonography examination was performed to confirm pregnancy. The inseminated non-pregnant animals served as control. Total RNA was extracted using the TRIzol method. The temporal abundance of ISG15, LGALS3BP and CD9 genes in PBMC was compared between pregnant and non-pregnant groups (n = 9 per group) using real time-qPCR. Results showed transcripts of ISG15 and LGALS3BP were more abundant at 20 d in the pregnant group compared to the 0 d and 20 d values of the non-pregnant group. However, due to variability in expression, threshold (Ct) cycle of RT-qPCR alone could not distinguish pregnant and non-pregnant animals. In conclusion, ISG15 and LGALS3BP transcripts abundance in PBMCs are potential candidate biomarkers for early prediction of buffalo pregnancy 20-days post-AI, but further work is required to allow the development of a reliable new methodology.

Validation of reference genes for the normalization of the RT-qPCR in peripheral blood mononuclear cells of septic patients

ObjectiveTo screen and validate reference genes suitable for gene mRNA expression study in peripheral blood mononuclear cells (PBMCs) between septic patients and healthy controls (HC). MethodsTotal RNA in PBMCs was extracted and RT-qPCR was used to determine the mRNA expression profiles of 9 candidate genes, including ACTB, B2M, GAPDH, GUSB, HPRT1, PGK1, RPL13A, SDHA and YWHAZ. The genes expression stabilities were assessed by both geNorm and NormFinder software. ResultsYWHAZ was the most stable gene among the 9 candidate genes evaluated by both geNorm and NormFinder in mixed and sepsis groups. The most stable gene combination in mixed group analyzed by geNorm was the combination of GAPDH, PKG1 and YWHAZ, while that in sepsis group was the combination of ACTB, PKG1 and YWHAZ. ConclusionOur first systematic analysis of the reference genes in PBMC of septic patients suggested YWHAZ was the best candidate. The combination of ACTB, PKG1 and YWHAZ could improve RT-qPCR accuracy in septic patients. Our results identified the most stable reference genes to standardize RT-qPCR of sepsis patients, which can serve as a useful tool for gene function exploration in the future.

Abstract B021: Breast cancer survivors and expression of p16INK4a, DNA damage, and the SASP prior to and following treatment

Abstract Purpose: To examine whether breast cancer therapy accelerates aging via increasing expression of cellular senescence (p16INK4a), the DNA damage response (DDR), or the proinflammatory senescence associated secretory phenotype (SASP), we measured gene expression of these pathways in a cohort of women recruited following a diagnosis of breast cancer (Stage 0-III). Methods: The RISE study is a Los Angeles-based longitudinal, observational study of biological and behavioral processes following surgery, radiation (RT) and/or chemotherapy (CT); and more persistent changes in these processes in the months after adjuvant treatment completion and initiation of endocrine therapy (6, 12, 18 month post-treatment follow-ups). Of the 270 women enrolled in the study, gene expression data was available for 184 women (aged average of 55.5 years, SD 11.2). Peripheral blood mononuclear cells (PBMC) gene expression was determined using RNA sequencing on quality-verified RNA. We determined transcript abundance for cellular senescence marker p16INK4a, genes that express proteins of the SASP, and genes responsive to DNA damage. Longitudinal data for p16INK4a, DDR and SASP were modeled using mixed models, including a quadratic term for time and its interaction with four treatment groups (1: Surgery alone [no RT or CT], RT alone, CT alone, and CT+RT), and a zero-inflated two-part model with p16INK4a. Covariates included age, BMI, race, initial surgery type, receipt of interim surgery, and endocrine therapy. Results: Analyses testing whether there was a change in p16INK4a over time by treatment group revealed that all groups experienced increases in p16INK4a expression over time. At baseline 53% of women had no detectable p16INK4a transcripts in their PBMC samples, and this proportion changed over time across all groups indicating significant increases in women expressing the marker for cellular senescence, p&amp;lt;0.0001. Women receiving CT or CT+RT had a 40% and 36% increase in percentage expressing p16INK4a (26% increase among those with surgery alone and 31% for RT alone) by 18 months post treatment compared to their pretreatment values. Analyses found a significant initial increase in DDR over time in all groups (p's&amp;lt;0.002) followed by a deceleration in all three groups except CT+RT, which continued an upward trajectory. There were also significant increases in SASP across all groups (p’s&amp;lt;0.05), with a significant quadratic effect in the RT group only (p&amp;lt;0.001). Conclusions: These results indicate that there was activation of genes associated with acceleration of biological aging across all groups after breast cancer diagnosis. Survivors had increases in p16INK4a, DNA damage response genes, and SASP from pre to 18 months post treatment for breast cancer. (NCI R01 CA237535) Citation Format: Judith E. Carroll, Catherine M. Crespi, Laura Petersen, Julie E. Bower. Breast cancer survivors and expression of p16INK4a, DNA damage, and the SASP prior to and following treatment [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr B021.

Identification of stable reference genes in peripheral blood mononuclear cells from type 2 diabetes mellitus patients

Reference genes are obligatory for accurate normalization of mRNA transcript levels across samples and experimental conditions in Real Time-polymerase chain reaction (qRT-PCR) based quantitative gene expression assays. Selection of stably expressed reference genes is therefore crucial for ensuring reproducibility of such assays. However, there is a complete dearth of data on stability of commonly used reference genes in Peripheral Blood Mononuclear Cells (PBMCs) from Type 2 diabetes mellitus (T2DM) patients. We have evaluated the gene expression stability of 4 widely used reference genes (Beta-actin, ACTB; Peptidylprolyl Isomerase B, PPIB; Tyrosine 3 Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Zeta, YWHAZ; and Glyceraldehyde-3-Phosphate Dehydrogenase, GAPDH); in PBMCs from 39 T2DM patients and 47 normoglycemic (NGT) subjects. ACTB and YWHAZ were found to be the most stable genes in PBMCs from T2DM patients and therefore, can be recommended as suitable reference genes in similar contexts. GAPDH and PPIB expressions were not stable in PBMCs from T2DM patients. On using ACTB and YWHAZ as reference genes for measuring relative expression of GAPDH and PPIB in these subjects, relative GAPDH expression was found to be significantly lower in female T2DM patients, compared to female NGT subjects [GAPDH relative normalization unit (RNU): female T2DM (n = 19), median (Q1, Q3): 9.0 (8.1, 9.9); female NGT (n = 18): median (Q1, Q3): 10.1 (9.1, 11.0); P = 0.034]. Dysregulation of GAPDH in PBMCs from female T2DM patients could be associated with sex-specific differences in pathogenesis and outcomes of T2DM.

The immunomodulatory effects of all-trans retinoic acid and docosahexaenoic acid combination treatment on the expression of IL-2, IL-4, T-bet, and GATA3 genes in PBMCs of multiple sclerosis patients

ABSTRACT Objectives Multiple sclerosis (MS) is a potentially disabling autoimmune disease of the central nervous system. Neither the pathogenesis nor the effectiveness of treatment of MS has been fully understood. This in vitro trial evaluated the beneficial immunomodulatory effects of single and combined treatments of all-trans retinoic acid (ATRA) and docosahexaenoic acid (DHA) on the peripheral blood mononuclear cells (PBMCs) of relapsing-remitting MS (RRMS) patients who were receiving interferon beta (IFN-β). Methods The PBMCs of 15 RRMS patients were isolated, cultured, and treated with single and combined treatments of ATRA and DHA. The expressions of IL-2, IL-4, T-bet, and GATA3 genes were evaluated using real-time PCR. Results The results showed that a single treatment of ATRA could significantly suppress the gene expression of the pro-inflammatory cytokine, IL-2 (P < 0.05), and related transcription factor, T-bet (P < 0.001). The gene expression level of the anti-inflammatory cytokine, IL-4, and its transcription factor, GATA3, were not significantly changed. The expression of IL-2 and T-bet genes was significantly decreased in combination treatments of ATRA and DHA (P < 0.001). Significant suppression of IL-2 and T-bet (P < 0.001) was observed in ATRA and DHA combination therapy with half doses of their single treatment, which suggested a synergistic effect of these components. Discussion Co-administration of vitamin A and DHA, an omega-3 fatty acid derivative, may exert a synergistic effect in modulating the immune system in MS patients; however, more studies are needed to evaluate the exact effects and mechanism of their actions on the immune cells.

Evaluation of IFIT3 and ORM1 as Biomarkers for Discriminating Active Tuberculosis from Latent Infection.

Current commercially available immunological tests cannot be used for discriminating active tuberculosis (TB) from latent TB infection. To evaluate the value of biomarker candidates in the diagnosis of active TB, this study aimed to identify differentially expressed genes in peripheral blood mononuclear cells (PBMCs) between patients with active TB and individuals with latent TB infection by transcriptome sequencing. The differentially expressed genes in unstimulated PBMCs and in Mycobacterium tuberculosis (Mtb) antigen-stimulated PBMCs from patients with active TB and individuals with latent TB infection were identified by transcriptome sequencing. Selected candidate genes were evaluated in cohorts consisting of 110 patients with TB, 30 individuals with latent TB infections, and 50 healthy controls by quantitative real-time RT-PCR. Receiver operating characteristic (ROC) curve analysis was performed to calculate the diagnostic value of the biomarker candidates. Among the differentially expressed genes in PBMCs without Mtb antigen stimulation, interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) had the highest area under curve (AUC) value (0.918, 95% CI: 0.852-0.984, P<0.0001) in discriminating patients with active TB from individuals with latent TB infection, with a sensitivity of 91.86% and a specificity of 84.00%. In Mtb antigen-stimulated PBMCs, orosomucoid 1 (ORM1) had a high AUC value (0.833, 95% CI: 0.752-0.915, P<0.0001), with a sensitivity of 81.94% and a specificity of 70.00%. IFIT3 and ORM1 might be potential biomarkers for discriminating active TB from latent TB infection.

Differential expression of carcinoembryonic antigen-related cell adhesion molecule-5 (CEACAM5) and dipeptidyl peptidase‐4 (DPP4) with detection of Middle East respiratory syndrome-coronavirus in peripheral blood

BackgroundMiddle East respiratory syndrome-coronavirus (MERS-CoV) utilizes CD26 (dipeptidyl peptidase‐4) and CD66e or CEACAM5 (carcinoembryonic antigen-related cell adhesion molecule 5) receptors for cell infection. Peripheral blood mononuclear cells (PBMCs) play a critical role in mounting adaptive immune response against the virus. This study was performed to assess the expression of CD26 and CD66e on PBMCs and their susceptibility to MERS-CoV infection. MethodsSurface expression of CD26 and CD66e receptors on PBMCs from MERS-CoV patients (n = 20) and healthy controls (n = 20) was assessed by flow cytometry and the soluble forms were determined by enzyme-linked immunosorbent assay (ELISA). MERS-CoV UpE and Orf1a genes in PBMCs were detected by using Altona diagnostics reverse transcription polymerase chain reaction (RT-PCR) kit. ResultsMean fluorescent intensity (MFI) of CD66e was significantly higher on CD4 + lymphocytes (462.4 ± 64.35 vs 325.1 ± 19.69; p < 0.05) and CD8 + lymphocytes (533.8 ± 55.32 vs 392.4 ± 37.73; p < 0.04) from patients with MERS-CoV infection compared to the normal controls. No difference in MFI for CD66e was observed on monocytes (381.8 ± 40.34 vs 266.8 ± 20.6; p = 0.3) between the patients and controls. Soluble form of CD66e among MERS-CoV patients was also higher than the normal controls (mean= 338.7 ± 58.75 vs 160.7 ± 29.49 ng/mL; p < 0.01). Surface expression of CD26 on PBMCs and its soluble form were no different between the groups. MERS-CoV was detected by RT-PCR in 16/20 (80%) patients from whole blood, among them 8 patients were tested in PBMCs, 4/8 (50%) patients were positive. ConclusionIncreased expression levels of CD66e (CEACAM5) may contribute to increased susceptibility of PBMCs to MERS-CoV infection and disease progression.

Fatty acids homeostasis during fasting predicts protection from chemotherapy toxicity

Fasting exerts beneficial effects in mice and humans, including protection from chemotherapy toxicity. To explore the involved mechanisms, we collect blood from humans and mice before and after 36 or 24 hours of fasting, respectively, and measure lipid composition of erythrocyte membranes, circulating micro RNAs (miRNAs), and RNA expression at peripheral blood mononuclear cells (PBMCs). Fasting coordinately affects the proportion of polyunsaturated versus saturated and monounsaturated fatty acids at the erythrocyte membrane; and reduces the expression of insulin signaling-related genes in PBMCs. When fasted for 24 hours before and 24 hours after administration of oxaliplatin or doxorubicin, mice show a strong protection from toxicity in several tissues. Erythrocyte membrane lipids and PBMC gene expression define two separate groups of individuals that accurately predict a differential protection from chemotherapy toxicity, with important clinical implications. Our results reveal a mechanism of fasting associated with lipid homeostasis, and provide biomarkers of fasting to predict fasting-mediated protection from chemotherapy toxicity.

Agomelatine Changed the Expression and Methylation Status of Inflammatory Genes in Blood and Brain Structures of Male Wistar Rats after Chronic Mild Stress Procedure.

The preclinical research conducted so far suggest that depression development may be influenced by the inflammatory pathways both at the periphery and within the central nervous system. Furthermore, inflammation is considered to be strongly connected with antidepressant treatment resistance. Thus, this study explores whether the chronic mild stress (CMS) procedure and agomelatine treatment induce changes in TGFA, TGFB, IRF1, PTGS2 and IKBKB expression and methylation status in peripheral blood mononuclear cells (PBMCs) and in the brain structures of rats. Adult male Wistar rats were subjected to the CMS and further divided into matched subgroups to receive vehicle or agomelatine. TaqMan gene expression assay and methylation-sensitive high-resolution melting (MS-HRM) were used to evaluate the expression of the genes and the methylation status of their promoters, respectively. Our findings confirm that both CMS and antidepressant agomelatine treatment influenced the expression level and methylation status of the promoter region of investigated genes in PBMCs and the brain. What is more, the present study showed that response to either stress stimuli or agomelatine differed between brain structures. Concluding, our results indicate that TGFA, TGFB, PTGS2, IRF1 and IKBKB could be associated with depression and its treatment.

CFNAs of RBCs affect the release of inflammatory factors through the expression of CaMKIV in macrophages

BackgroundBlood transfusions reportedly modulate the recipient’s immune system. Transfusion-related immunomodulation has been suggested as a mechanism of some adverse clinical outcomes. Extracellular nucleic acids circulate in plasma and activate relevant immune responses, but little is known about their mechanism of action in transfusion-related immunomodulation (TRIM). The aim of this study was to investigate the effects of cell-free nucleic acids (CFNAs) produced by red blood cells (RBCs) on innate immunity, especially peripheral blood mononuclear cells (PBMCs) and macrophages, and to investigate the mechanism of action. MethodsDifferentially expressed genes (DEGs) between PBMCs exposed to RBC-produced CFNA and normal PBMCs were analyzed by gene expression data combined with bioinformatics. KEGG and GO enrichment analyses were performed for the DEGs, and in vitro experiments were performed for the effects of key genes on the release of inflammatory factors from macrophages. ResultsAnalysis of microarray data showed that exposure of monocytes to RBC-produced CFNAs increased the expression of genes involved in the innate immune response, including chemokines, chemokine receptors, and innate response receptors, and that calcium channel activity was highly regulated, with a key gene being CaMKIV. CaMKIV played a critical role in LPS-induced inflammatory factor release from macrophages, which was exacerbated by overexpression of the CaMKIV gene. ConclusionRBCs regulate the release of inflammatory factors during blood transfusion by releasing CFNAs and affecting expression of the CaMKIV gene in PBMCs or macrophages, which is a potential regulatory mechanism of blood transfusion–related immune regulation and related adverse reactions.

Derailed peripheral circadian genes in polycystic ovary syndrome patients alters peripheral conversion of androgens synthesis.

Do circadian genes exhibit an altered profile in peripheral blood mononuclear cells (PBMCs) of polycystic ovary syndrome (PCOS) patients and do they have a potential role in androgen excess? Our findings revealed that an impaired circadian clock could hamper the regulation of peripheral steroid metabolism in PCOS women. PCOS patients exhibit features of metabolic syndrome. Circadian rhythm disruption is involved in the development of metabolic diseases and subfertility. An association between shift work and the incidence of PCOS in females was recently reported. This is a retrospective case-referent study in which peripheral blood samples were obtained from 101 control and 101 PCOS subjects. PCOS diagnoses were based on Rotterdam Consensus criteria. This study comprised 101 women with PCOS and 101 control volunteers, as well as Swiss albino mice treated with dehydroepiandrosterone (DHEA) to induce PCOS development. Gene expression analyses of circadian and steroidogenesis genes in human PBMC and mice ovaries and blood were executed by quantitative real-time PCR. We observed aberrant expression of peripheral circadian clock genes in PCOS, with a significant reduction in the core clock genes, circadian locomotor output cycles kaput (CLOCK) (P ≤ 0.00001), brain and muscle ARNT-like 1 (BMAL1) (P ≤ 0.00001) and NPAS2 (P ≤ 0.001), and upregulation of their negative feedback loop genes, CRY1 (P ≤ 0.00003), CRY2 (P ≤ 0.00006), PER1 (P ≤ 0.003), PER2 (P ≤ 0.002), DEC1 (P ≤ 0.0001) and DEC2 (P ≤ 0.00005). Transcript levels of an additional feedback loop regulating BMAL1 showed varied expression, with reduced RORA (P ≤ 0.008) and increased NR1D1 (P ≤ 0.02) in PCOS patients in comparison with the control group. We also demonstrated the expression pattern of clock genes in PBMCs of PCOS women at three different time points. PCOS patients also exhibited increased mRNA levels of steroidogenic enzymes like StAR (P ≤ 0.0005), CYP17A1 (P ≤ 0.005), SRD5A1 (P ≤ 0.00006) and SRD5A2 (P ≤ 0.009). Knockdown of CLOCK/BMAL1 in PBMCs resulted in a significant reduction in estradiol production, by reducing CYP19A1 and a significant increase in dihydrotestosterone production, by upregulating SRD5A1 and SRD5A2 in PBMCs. Our data also showed that CYP17A1 as a direct CLOCK-BMAL1 target in PBMCs. Phenotypic classification of PCOS subgroups showed a higher variation in expression of clock genes and steroidogenesis genes with phenotype A of PCOS. In alignment with the above results, altered expression of ovarian core clock genes (Clock, Bmal1 and Per2) was found in DHEA-treated PCOS mice. The expression of peripheral blood core clock genes in DHEA-induced PCOS mice was less robust and showed a loss of periodicity in comparison with that of control mice. N/A. We could not evaluate the circadian oscillation of clock genes and clock-controlled genes over a 24-h period in the peripheral blood of control versus PCOS subjects. Additionally, circadian genes in the ovaries of PCOS women could not be evaluated due to limitations in sample availability, hence we employed the androgen excess mouse model of PCOS for ovarian circadian assessment. Clock genes were assessed in the whole ovary of the androgen excess mouse model of PCOS rather than in granulosa cells, which is another limitation of the present work. Our observations suggest that the biological clock is one of the contributing factors in androgen excess in PCOS, owing to its potential role in modulating peripheral androgen metabolism. Considering the increasing prevalence of PCOS and the rising frequency of delayed circadian rhythms and insufficient sleep among women, our study emphasizes the potential in modulating circadian rhythm as an important strategy in PCOS management, and further research on this aspect is highly warranted. This work was supported by the RGCB-DBT Core Funds and a grant (#BT/PR29996/MED/97/472/2020) from the Department of Biotechnology (DBT), India, to M.L. B.S.J. was supported by a DST/INSPIRE Fellowship/2015/IF150361 and M.B.K. was supported by the Research Fellowship from Council of Scientific & Industrial Research (CSIR) (10.2(5)/2007(ii).E.U.II). The authors declare no competing interests. N/A.

Egg Consumption Modulates Expression of Genes Involved in T Cell Activation and Differentiation That Correlate With HDL Profiles

ObjectivesHDL is known to be an important modulator of T cell activity. We have previously demonstrated that whole eggs modulate HDL profiles when compared to egg whites; thus, we investigated whether different egg-based diets affect expression of genes involved in T cell activation and differentiation in a manner that correlates with changes in HDL profiles.MethodsA 16-week randomized, crossover intervention trial was conducted in which healthy, young adult men and women (18–35 y, n = 26) completed a 4-week egg-free dietary period, followed by random assignment to a 4-week 3 whole egg/day or 3 egg white/day diet period. Participants then entered a 4-week egg-free washout period before switching to the alternative egg treatment for 4 weeks. Fasting HDL profiles and peripheral blood mononuclear cell (PBMC) gene expression immune arrays were measured at the conclusion of each dietary period.ResultsOur data showed that whole egg intake had the greatest impact on PBMC expression of genes related to T cell activation and differentiation. Compared to the egg-free diet, expression of CD4, CD34, and CD68 were significantly reduced following the whole egg period. We similarly observed trends toward decreased expression of HLA-DRA, CCL2, TGF-beta, and CCR4 following the whole egg diet, and increased expression of IL-12 beta, CLTA4, and PF4. When analyzing changes in gene expression between the egg white and whole egg diet periods, we identified a total of 25 and 21 genes related to immunity and inflammation that significantly correlated with changes in the number of large HDL particles and apolipoprotein A1 (apoA1) concentrations in serum, respectively. 68% and 76% of genes that correlated with large HDL and apoA1, respectively, were related to modulation of T cell activity, suggesting that egg-induced changes in HDL profiles may have stronger modulatory effects on T cells compared to other immune cell subtypes within PBMC samples.ConclusionsOur findings suggest that whole eggs promote anti-inflammatory and modulatory changes in gene expression related to T cell activation and differentiation, which directly correlate with changes in HDL profiles.Funding SourcesThis study was funded by an Agriculture and Food Research Initiative Grant from the USDA National Institute of Food and Agriculture.

Methylomic and transcriptomic characterization of postoperative systemic inflammatory dysregulation

In this study, we define and validate a state of postoperative systemic inflammatory dysregulation (PSID) based on postoperative phenotypic extremes of plasma C-reactive protein concentration following major abdominal surgery. PSID manifested clinically with significantly higher rates of sepsis, complications, longer hospital stays and poorer short, and long-term outcomes. We hypothesized that PSID will be associated with, and potentially predicted by, altered patterns of genome-wide peripheral blood mononuclear cell differential DNA methylation and gene expression. We identified altered DNA methylation and differential gene expression in specific immune and metabolic pathways during PSID. Our findings suggest that dysregulation results in, or from, dramatic changes in differential DNA methylation and highlights potential targets for early detection and treatment. The combination of altered DNA methylation and gene expression suggests that dysregulation is mediated at multiple levels within specific gene sets and hence, nonspecific anti-inflammatory treatments such as corticosteroids alone are unlikely to represent an effective therapeutic strategy.