mRNA Expression In Peripheral Blood Mononuclear Cells Research Articles

Down-regulation of A20 mRNA expression in peripheral blood mononuclear cells from MDS patients

ABSTRACT Myelodysplastic syndrome (MDS) is characterized by activated inflammatory signaling and affects prognosis. Targeting inflammatory signaling may provide a way to treat the disease. We were curious whether there were changes in A20 in peripheral blood mononuclear cells (PBMC) of MDS patients. Therefore, we conducted a study with 60 clinical samples, including 30 MDS patients and 30 healthy controls. All patients with MDS were diagnosed and classified according to the criteria of the 2016 World Health Organization. The study was performed in accordance with the guidelines of the Declaration of Helsinki. Using Quantitative Real-Time RT–PCR, we discovered that A20 mRNA expression in PBMC of the MDS group was significantly lower than that in the control group (P < 0.001). Additionally, using Luminex Liquid Suspension Chip, we observed elevated plasma levels of pro-inflammatory IL-8 and TNF-α in the MDS group compared to the healthy control group (P < 0.001). We did not find a significant correlation between A20 mRNA and clinical characteristics (age, sex, concentration of hemoglobin, neutrophils count, platelets count, percent of blasts, and WHO classification) of the patients, nor between A20 mRNA and plasma cytokines (data not shown). Our study found down-regulated of A20 and increased levels of pro-inflammatory cytokines in the peripheral blood of MDS patients, providing further evidence for the activation of inflammatory signals in MDS.

Significance of triggering receptor expressed on myeloid cells-2 prognostic evaluation in hepatitis B virus-related acute-on-chronic liver failure

Objective: To explore the significance of triggering receptor expressed on myeloid cells-2 (TREM-2) prognostic evaluation so as to provide novel biological markers in clinical practice for patients with hepatitis B virus-related acute-on-chronic liver failure ( HBV-ACLF). Methods: The research subjects of this study were divided into an experimental group and a control group. Fifty HBV-ACLF cases admitted to the Department of Infectious Diseases of the First Affiliated Hospital of Nanchang University from January 1, 2019 to December 31, 2019 were selected as the experimental group. Patients were divided into survival and death groups according to the actual prognosis at discharge (self-discharge and dead patients were considered death groups, and all enrolled patients were hospitalized for more than 28 days). Twenty-five healthy subjects were chosen as the control group. Peripheral venous blood was collected from the experimental group and the control group. Plasma and peripheral blood mononuclear cells (PBMC) were isolated. The concentrations of TREM-2, interleukin (IL)-6, and IL-8 were detected in the plasma. TREM-2 mRNA expression was detected in PBMC. A single blood sample was collected from the control group, whereas five blood samples were dynamically collected from the experimental group on the day of admittance and at 7, 14, 21, and 28 days after treatment commenced. Simultaneously, upon admission, the relevant clinical indicators of HBV-ACLF patients were monitored, including the liver function test: alanine aminotransferase, aspartate aminotransferase, total bilirubin, albumin, coagulation function test: international normalized ratio, prothrombin time, and other indicators. Measurement data were expressed as mean±standard deviation (x±s). Count data were compared and analyzed using the χ(2) test. The intra-group factor mean was compared using a repeated measures ANOVA. The means were analyzed by t-tests between the two groups. Bivariate correlation analysis was used to analyze the correlation between the two variables. The value of TREM-2 as a diagnostic marker was analyzed using the receiver operating characteristic (ROC) curve. Results: The mRNA expression of TREM-2 in the PBMC of HBV-ACLF patients showed a gradually increasing trend at various time points and was significantly higher in the survival group than that of the control group at 28 days (P < 0.01), while the death group showed a gradually weakening trend at various time points and was significantly lower than the control group at 28 days (P < 0.01). (1) The levels of TREM-2 in the plasma of HBV-ACLF patients generally showed a gradually increasing trend at various time points in the survival group. The levels on the day of admission and 7, 14, 21, and 28 days after the initiation of treatment were (1.49±0.85), (1.62±0.58), (1.95±0.69), (2.33±0.71), and (2.00±0.67) ng/ml, respectively. The expression of TREM-2 in the death group showed a gradually weakening trend at various time points. The levels on the day of admission and 7, 14, 21, and 28 days after initiation of treatment were (1.40±0.73), (1.59±0.79), (1.56±0.80), (1.05±0.49), and (0.81±0.21) ng/ml, respectively. The survival group's various detection time points were higher than those of the death group, and the difference was statistically significant. The plasma level of TREM-2 in the healthy control group was (1.25±0.35) ng/ml. (2) The concentrations of IL-6 and IL-8 in the plasma of HBV-ACLF patients showed a gradually decreasing trend at various time points in the survival group. The levels on the day of admission and 7, 14, 21, and 28 days after initiation of treatment were (46.70±26.31), (33.98±20.28), (19.07±10.24), (14.76±7.84), (9.12±7.65) and (108.29±47.07), (93.85±26.53), (79.27±34.63), (56.72 ±18.30), (37.81±13.88) pg/ml, respectively. However, its concentration in the death group fluctuated within a relatively high range. The levels on the day of admission and 7, 14, 21, and 28 days after the initiation of treatment were (41.94±24.19), (36.99±19.78), (34.30±20.62), (34.14±14.52), (36.64±23.61) and (104.65±50.16), (112.98±45.03), (118.43±45.00), (111.67±40.44), (109.55±27.54) pg/ml, respectively. (3) Bivariate correlation analysis results indicated that the plasma TREM-2 content was negatively correlated with the plasma levels of pro-inflammatory cytokines IL-6 and IL-8 (r = -0.224, P = 0.025; r = - 0.223, P = 0.026). ROC curve analysis showed that the mRNA levels of TREM-2 in PBMCs at various time points for prognostic evaluation of HBV-ACLF patients were 1d=0.667, 7d=0.757, 14d=0.979, 21d=0.986, and 28d= 0.993. The areas under the ROC curve of the TREM-2 content in the plasma at various time points were 1d=0.522, 7d=0.571, 14d=0.658, 21d=0.927, and 28d=0.994. Conclusion: TREM-2 mRNA expression in PBMC and TREM-2 content in plasma have a significant relationship to the prognosis of HBV-ACLF patients and may inhibit the liver inflammatory response by regulating the secretion of pro-inflammatory cytokines IL-6 and IL-8. Dynamic monitoring of TREM-2 expression in peripheral blood is favorable for evaluating the prognostic condition of HBV-ACLF patients.

Association of interleukin 33 gene polymorphisms with susceptibility and regulation of inflammatory mediators in Systemic lupus erythematosus patients.

Studies have indicated the involvement of interleukin (IL)-33 in the pathogenesis of Systemic lupus erythematosus (SLE). This research intended to evaluate the association of IL33 gene rs1929992 and rs7044343 Single nucleotide polymorphisms (SNPs) with risk of SLE. In addition, the association between these SNPs and inflammatory cytokines was determined. In this study, 200 SLE cases and 200 healthy subjects were recruited. Using allelic discrimination Real-time PCR, IL33 gene rs1929992 and rs7044343 SNPs were genotyped. The mRNA expression levels of IL-1β, IL-6, IL-33, TNF-α were determined in the peripheral blood mononuclear cells (PBMCs). The serum levels of cytokines were also measured. The G allele (OR = 1.57, CI: 1.18-2.08, P = 0.0017), GG genotype (OR = 2.52, CI: 1.33-4.77, P = 0.0043), and GA genotype (OR = 2.12, CI: 1.34-3.34, P = 0.0011) of rs1929992 SNP was significantly associated with an increased SLE risk. The C allele (OR = 1.44, CI: 1.08-1.90; P = 0.0105), CC genotype (OR = 2.07, CI: 1.15-3.71; P = 0.0146), and CT genotype (OR = 1.61, CI: 1.02-2.53, P = 0.0395) of rs7044343 was significantly associated with increased SLE risk. The PBMC mRNA expression and serum levels of IL-1β, IL-6, IL-33, TNF-α were significantly increased in the SLE patients compared to controls. However, there was no significant difference in the mRNA expression and serum levels of IL-1β, IL-6, IL-33, and TNF-α among the SLE patients with three genotypes for both rs1929992 and rs7044343 polymorphisms. IL33 gene rs1929992 and rs7044343 SNPs are involved in SLE pathogenesis but they might not influence on the inflammatory pathway.

Prenatal exposure of diabetes and progestin-mediated autistic biomarker in peripheral blood mononuclear cells.

Despite the importance of early diagnosis and intervention, the diagnosis of autism spectrum disorders (ASDs) remains delayed as it is mostly based on clinical symptoms and abnormal behaviours appearing after 2years of age. Identification of autistic markers remains a top priority in achieving an early and effective ASD diagnosis. We have previously reported that prenatal exposure of hormones or diabetes triggers epigenetic changes and oxidative stress, resulting in gene suppression with autism-like behaviours in offspring. Here, a potential biomarker for ASD diagnosis was established through gene analysis in peripheral blood mononuclear cells (PBMCs). The study from in vivo mouse showed that prenatal hormone exposure or maternal diabetes suppresses mRNA expression of estrogen-related receptor α (ERRα), superoxide dismutase 2 (SOD2), G protein-coupled estrogen receptor (GPER) and retinoic acid-related orphan receptor α (RORA) in the brain as well as oxidative stress and mitochondrial dysfunction, subsequently triggering autism-like behaviour in mouse offspring. Also, similar gene suppression was found in hematopoietic stem cells (HSCs) and PBMC, with inherited epigenetic changes being identified on the related promoters. The human case-control study found that mRNA levels of ERRα, SOD2, GPER and RORA were significantly reduced in PBMC from ASD subjects (n= 132) compared with typically developing (n= 135) group. The receiver operating characteristic curve showed a .869 ± .021 of area under the curve for ASD subjects with 95% confidence interval of .829-.909, together with 1.000 of sensitivity and .856 of specificity. In conclusion, the combined mRNA expression in PBMC based on prenatal factor exposure-mediated gene suppression could be a potential biomarker for ASD diagnosis.

Study on the expression of TRIM7 in peripheral blood mononuclear cells of patients with sepsis and its early diagnostic value

BackgroundThe early diagnosis of sepsis is beneficial to put forward a reasonable clinical treatment plan as soon as possible. This study was to explore the expression of Tripartite Motif 7 (TRIM7) in peripheral blood mononuclear cells (PBMCs) of patients with sepsis and its diagnostic value.MethodsThis is a cross-sectional study. A total of 69 patients with infectious diseases were enrolled in the emergency room. They were divided into the sepsis group (34 cases) and the non-sepsis infection group (35 cases). There were 25 healthy subjects who were selected as the control group. The expression of TRIM7 in PBMCs was observed by immunofluorescence staining. The correlation between the expression of TRIM7 mRNA and acute physiology and chronic health evaluation II (APACHE II) score, sequential organ failure assessment (SOFA) score, white blood cell (WBC), C-reactive protein (CRP), procalcitonin (PCT), tumor necrosis factor (TNF)-α and interleukin (IL)-6 was discussed. The receiver operating characteristic (ROC) curve was utilized for evaluating the value of TRIM7 expression for the early diagnosis of sepsis.ResultsThe fluorescence intensity representing the expression level of TRIM7 in PBMCs of patients in the sepsis group was the lowest among three groups. The TRIM7 mRNA expression in PBMCs of the sepsis group was greatly decreased in comparison with that of the non-sepsis infection group and control group (P < 0.05). Spearman correlation analysis indicated that TRIM7 mRNA expression was negatively correlated with APACHE II score, SOFA score, WBC, CRP, PCT, TNF-α and IL-6. ROC curve analysis revealed that the area under curve (AUC) of TRIM7 mRNA expression in PBMCs for the diagnosis of sepsis was 0.798, with a 95% confidence interval of 0.691- 0.905, a sensitivity of 73.5%, and a specificity of 77.1%.ConclusionThe expression of TRIM7 in PBMCs of patients with sepsis is significantly down-regulated, which has certain clinical value for early diagnosis of sepsis.

The relationships between cholesterol crystals, NLRP3 inflammasome, and coronary atherosclerotic plaque vulnerability in acute coronary syndrome: An optical coherence tomography study.

Cholesterol crystals (CCs) in lesions are the hallmark of advanced atherosclerotic plaque. Previous studies have demonstrated that CCs could activate NLRP3 inflammasome, which played an important role in atherosclerotic lesion progression. However, the relationship between CCs, NLRP3 inflammasome pathway, and plaque vulnerability in patients with ACS is still not elucidated. Two hundred sixty-nine consecutive acute coronary syndrome (ACS) patients with 269 culprit lesions were included in this study. CCs and other plaque characteristics within the culprit lesion segment were evaluated by optical coherence tomography (OCT) before percutaneous coronary intervention (PCI). The NLRP3 mRNA expression in peripheral blood mononuclear cells (PBMCs) and the serum levels of interleukin (IL)-1β, IL-18, and other biological indices were measured. Cholesterol crystals were observed in 105 (39%) patients with 105 culprit lesions. There were no significant differences in baseline clinical characteristics between the patients with CCs (CCs group, n = 105) and the patients without CCs (non-CCs group, n = 164) within the culprit lesion segment except for lipoprotein(a) [Lp(a)]. The CCs group had a higher level of NLRP3 mRNA expression in PBMCs and higher levels of serum cytokine IL-1β and IL-18. OCT showed that the CCs group had longer lesion length, more severe diameter stenosis, and less minimum luminal area (MLA) than the non-CCs group (all p < 0.05). The frequency of thin-cap fibroatheroma (TCFA), thrombus, accumulation of macrophages, plaque rupture, micro-channel, calcification, spotty calcification, and layered plaque was higher in the CCs group than in the non-CCs groups (all p < 0.05). Multivariate logistic analysis revealed that the level of NLRP3 expression (OR = 10.204), IL-1β levels (OR = 3.523), IL-18 levels (OR = 1.006), TCFA (OR = 3.593), layered plaque (OR = 5.287), MLA (OR = 1.475), macrophage accumulation (OR = 2.881), and micro-channel (OR = 3.185) were independently associated with CCs. Acute coronary syndrome patients with CCs in culprit lesions had a higher expression of NLRP3, IL-1β, and IL-18, and had more vulnerable plaque characteristics than patients without CCs. CCs might have interacted with NLRP3 inflammasome activation in patients with ACS, which could contribute to plaque vulnerability in culprit lesions.

Diminution of STING Expression in the Blood of Patients with Severe or Critical COVID-19 Pneumonia

Introduction: Cytokine storm and critical COVID-19 pneumonia are caused in at least 10% of patients by inborn errors of or auto-Abs to type I IFNs. The pathogenesis of life-threatening COVID-19 pneumonia in other patients remains unknown. Methods: This study was conducted at Masih Daneshvari Hospital, Tehran, Iran. In the period of study, 75 confirmed cases of COVID-19 with presentations ranging from mild upper respiratory tract infection to lower respiratory tract infection, including moderate, severe, and critical disease, were recruited. Expression of STING mRNA was measured in peripheral blood mononuclear cells (PBMCs) and compared between patients with different severity and outcome. Results: There was a significant negative correlation between age and STING expression level (p value = 0.010). Patients with “severe to critical” illness had a 20-fold lower STING expression level compared to the “mild to moderate” group (p value = 0.001). Also, the results showed lower expressions of STING in the patients admitted to the ICU (p value = 0.015). Patients who finally died had lower expression of STING at the time of sampling (p value = 0.041). Conclusion: STING mRNA expression in PBMCs was significantly lower in older COVID-19 cases, the patients with more severe illness, who needed intensive care, and who eventually died.

Effect of ultramarathon running on iron metabolism.

Iron is a vital trace element for energy production and oxygen transportation; importantly, it is essential to athletic performance. Maintaining iron balance is tightly controlled at systemic and cellular levels. This study aimed to determine serum iron tests, hepcidin levels, and cellular iron import and export activities in peripheral blood mononuclear cells (PBMCs) in ultramarathon runners to elucidate the association of systemic inflammation response and iron metabolism. Sixteen amateur runners were enrolled. Blood samples were taken 1 week before, immediately, and 24 h after the run. Plasma hepcidin levels were measured by enzyme-linked immunosorbent assay. The expression levels of divalent metal iron transporter 1 (DMT1), ZRT/IRT-like protein 14 (ZIP14), transferrin receptor 1 (TfR1), and ferroportin (FPN) in PBMCs were measured using real-time quantitative reverse transcription-polymerase chain reaction. Serum iron concentrations and transferrin saturation significantly decreased immediately after the race and dramatically recovered 24 h post-race. Serum ferritin levels had a statistically significant rise immediately after the race and remained high 24 h after the completion of the race. Ultramarathons were associated with increased plasma interleukin-6 concentrations corresponding to the state of severe systemic inflammation and therefore boosted plasma hepcidin levels. The expression levels of DMT1 and FPN mRNA were markedly decreased immediately and 24 h after the race. The ZIP14 and TfR1 mRNA expression in PBMCs significantly decreased immediately after the race and returned to the baseline level at 24 h post-race. Positive significant correlations were observed between plasma hepcidin and ferritin levels. Iron homeostasis and systemic inflammatory response are closely interconnected. Cellular iron import and export mRNA activities in PBMCs were acutely inhibited during an ultramarathon.

Integrated genomic, transcriptomic and metabolomic analysis reveals MDH2 mutation-induced metabolic disorder in recurrent focal segmental glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) has an over 30% risk of recurrence after kidney transplantation (Ktx) and is associated with an extremely high risk of graft loss. However, mechanisms remain largely unclear. Thus, this study identifies novel genes related to the recurrence of FSGS (rFSGS). Whole genome-wide sequencing and next-generation RNA sequencing were used to identify the candidate mutant genes associated with rFSGS in peripheral blood mononuclear cells (PBMCs) from patients with biopsy-confirmed rFSGS after KTx. To confirm the functional role of the identified gene with the MDH2 c.26C >T mutation, a homozygous MDH2 c.26C >T mutation in HMy2.CIR cell line was induced by CRISPR/Cas9 and co-cultured with podocytes, mesangial cells, or HK2 cells, respectively, to detect the potential pathogenicity of the c.26C >T variant in MDH2. A total of 32 nonsynonymous single nucleotide polymorphisms (SNPs) and 610 differentially expressed genes (DEGs) related to rFSGS were identified. DEGs are mainly enriched in the immune and metabolomic-related pathways. A variant in MDH2, c.26C >T, was found in all patients with rFSGS, which was also accompanied by lower levels of mRNA expression in PBMCs from relapsed patients compared with patients with remission after KTx. Functionally, co-cultures of HMy2.CIR cells overexpressing the mutant MDH2 significantly inhibited the expression of synaptopodin, podocin, and F-actin by podocytes compared with those co-cultured with WT HMy2.CIR cells or podocytes alone. We identified that MDH2 is a novel rFSGS susceptibility gene in patients with recurrence of FSGS after KTx. Mutation of the MDH2 c.26C >T variant may contribute to progressive podocyte injury in rFSGS patients.

Expression and clinical significance of the m6A reader YTHDF2 in peripheral blood mononuclear cells from rheumatoid arthritis patients

As an important m6A reader, the YT521-B homology domain family 2 (YTHDF2) has been shown to regulate mRNA degradation and translation, and to be involved in inflammation. However, little is known about the role of YTHDF2 in the autoimmune-based inflammatory disease rheumatoid arthritis (RA). To begin to ascertain any role for this reader, 74 RA patients and 63 healthy controls (HC) were recruited for this study. Blood was collected from each subject and peripheral blood mononuclear cells (PBMC) isolated. Thereafter, mRNA expression of YTHDF2, interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α in the cells was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The harvested blood was also assessed for a variety of parameters, including levels of C-reactive protein (CRP), erythrocyte sedimentation rates (ESR), white blood cell counts (WBC), neutrophils counts (N)/neutrophils percentages (N%), and neutrophil:lymphocyte ratios (NLR) - each markers of inflammation during RA. The results showed that YTHDF2 mRNA expression in RA patient PBMC was decreased significantly vs that in healthy control subject cells. Further, YTHDF2 mRNA expression in RA patient PBMC negatively-correlated with ESR, CRP levels, WBC counts, as well as neutrophils counts, percentages, and NLR values. In addition, it was seen that YTHDF2 mRNA expression in RA patient PBMC was associated with host serum RF levels and treatment. Moreover, it was found that mRNA expression of IL-1β, IL-6, IL-8, and TNFα was increased in PBMC from RA patients relative to in control subject cells; however, only the increased IL-1β expression was seen to be negatively-correlated with decreased YTHDF2 mRNA expression. In conclusion, the present study illustrated that YTHDF2 expression might have some regulatory role in the underlying mechanisms associated with the autoimmune disease RA and that this m6A reader could at some point represent a potential target for regulating inflammatory responses that occur during RA.

Identification of Key Genes and Pathways in Peripheral Blood Mononuclear Cells of Type 1 Diabetes Mellitus by Integrated Bioinformatics Analysis.

Background The onset and progression of type 1 diabetes mellitus (T1DM) is closely related to autoimmunity. Effective monitoring of the immune system and developing targeted therapies are frontier fields in T1DM treatment. Currently, the most available tissue that reflects the immune system is peripheral blood mononuclear cells (PBMCs). Thus, the aim of this study was to identify key PBMC biomarkers of T1DM.Methods Common differentially expressed genes (DEGs) were screened from the Gene Expression Omnibus (GEO) datasets GSE9006, GSE72377, and GSE55098, and PBMC mRNA expression in T1DM patients was compared with that in healthy participants by GEO2R. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and protein-protein interaction (PPI) network analyses of DEGs were performed using the Cytoscape, DAVID, and STRING databases. The vital hub genes were validated by reverse transcription-polymerase chain reaction using clinical samples. The disease-gene-drug interaction network was built using the Comparative Toxicogenomics Database (CTD) and Drug Gene Interaction Database (DGIdb).Results We found that various biological functions or pathways related to the immune system and glucose metabolism changed in PBMCs from T1DM patients. In the PPI network, the DEGs of module 1 were significantly enriched in processes including inflammatory and immune responses and in pathways of proteoglycans in cancer. Moreover, we focused on four vital hub genes, namely, chitinase-3-like protein 1 (CHI3L1), C-X-C motif chemokine ligand 1 (CXCL1), matrix metallopeptidase 9 (MMP9), and granzyme B (GZMB), and confirmed them in clinical PBMC samples. Furthermore, the disease-gene-drug interaction network revealed the potential of key genes as reference markers in T1DM.Conclusion These results provide new insight into T1DM pathogenesis and novel biomarkers that could be widely representative reference indicators or potential therapeutic targets for clinical applications.

PCSK9 Induces Tissue Factor Expression by Activation of TLR4/NFkB Signaling.

Proprotein convertase subtilisin kexin 9 (PCSK9) increases LDL cholesterol (C) concentration by accelerating the hepatic degradation of the LDL receptor (R) thus promoting atherogenesis. The molecule, however, also exerts proinflammatory effects independent of circulating LDL-C by enhancing local cytokine production and activation of NFkB, a process that might involve Toll-like receptor 4 (TLR4), a crucial component of the innate immunity system. Tissue factor (TF), a glycoprotein which plays an essential role in coagulation and inflammation, is rapidly induced by circulating monocytes stimulated by proinflammatory agents through NFkB-dependent mechanisms. The aims of our study were (1) to assess whether PCSK9 may induce monocytic TF expression and (2) to evaluate whether the TLR4/NFkB signaling pathway may contribute to that effect. Experiments were carried out in peripheral blood mononuclear cells (PBMCs), THP-1 cells, and HEK293 cells transfected with plasmids encoding the human TLR4 complex. PCSK9 increased procoagulant activity (PCA), mRNA and TF protein expression in both PBMCs and THP-1 cultures. Pre-treatment with inhibitors of TLR4/NFkB signaling such as LPS-RS, CLI-095, and BAY 11-7082, downregulated PCSK9-induced TF expression. A similar effect was obtained by incubating cell cultures with anti-PCSK9 human monoclonal antibody. In TLR4-HEK293 cells, PCSK9 activated the TLR4/NFkB signaling pathway to an extent comparable to LPS, the specific agonist of TLR4s and quantitative confocal microscopy documented the colocalization of PCSK9 and TLR4s. In conclusion, PCSK9 induces TF expression through activation of TLR4/NFkB signaling.

Effects of age and oligoasthenozoospermia on telomeres of sperm and blood cells

Research questionHow do age and normo- or oligoasthenozoospermia affect telomere length dynamics in spermatozoa and blood? DesignSperm and blood samples were collected from a cohort of 37 men aged 25 and under and 40 men aged 40 and over, with either normozoospermia (NZ) or oligoasthenozoospermia (OAZ). Telomere length was evaluated using quantitative fluorescence in-situ hybridization. Telomerase mRNA (TERC and TERT) and shelterin (TRF1) gene expression were analysed using quantitative real-time polymerase chain reaction. TRF1 protein immunoreactivity was also evaluated using immunofluorescence. ResultsMean sperm telomere length (STL) increased with age in the NZ group; older NZ men accumulated the longest telomeres (P < 0.001). In peripheral blood mononuclear cells (PBMC), mean telomere length decreased with age in NZ groups, although not reaching statistical significance. Interestingly, the younger OAZ group had the shortest mean telomere length (versus young NZ, P = 0.0081; versus old NZ, P = 0.0116; versus old OAZ, P = 0.0009) and accumulated the highest percentage of short telomeres compared with the other groups (overall P = 0.0017). Analysis of TERC and TERT mRNA expression in spermatozoa and PBMC did not show significant differences among groups. Statistically significant positive correlations were found between STL and seminal parameters in younger NZ men (P = 0.009 for sperm count and P = 0.007 for total progressive motility). Protein immunoreactivity of TRF1 in blood was not significantly different in all groups analysed. ConclusionsThe OAZ group did not show the increase of STL with age that is seen in NZ individuals, suggesting that telomere length elongation mechanisms fail in OAZ patients. In PBMC, younger OAZ individuals showed significantly shorter mean telomere length, suggesting that this parameter could be a good biomarker of OAZ in younger OAZ patients. Telomerase gene and TRF1 mRNA expression and TRF1 protein immunoreactivity did not differ significantly between groups, and so these factors cannot be used as OAZ biomarkers.

Dietary obesity and glycemic excursions cause a parallel increase in STEAP4 and pro-inflammatory gene expression in murine PBMCs.

The balance between pro-atherogenic and anti-atherogenic factors is very crucial in thedevelopmentof atherosclerotic lesions. Although the expression of the six-transmembrane epithelial antigen of the prostate 4 (STEAP4) in myeloid cells is known to be atheroprotective,there is not a single study reporting on the status of STEAP4 expression in circulating monocytes in the early stages of diet-induced obesity or in events of glycemic excursions. We induced glycemic spikes twice daily for a 1-week duration to rats fed on regular chow and western diet, and analyzed gene expression changes in the peripheral blood mononuclear cells (PBMCs).We also conducted experiments on RAW 264.7 cells to gain insight into some of our in vivo findings. Diet-induced obesity and glycemic excursions independently caused a significant increase in STEAP4 mRNA expression in PBMCs.This was also accompanied by an induction of a substantial number of pro-inflammatory cytokines, chemokines, and chemokine receptors.However, the combined effect of western diet and hyperglycemic spikes was subtle and non-additive.In the in vitro setting, either glucose spikes, persistent hyperglycemia,or a combination of palmitic acid and insulin resulted in a parallel increase in expression of STEAP4 and pro-inflammatory genes.This was, however, significantly abrogated with 4-octyl itaconate or attenuated by inhibitors of p38MAPK and NF-kB. STEAP4 expression in mononuclear cells is induced by increasing inflammation or oxidative stress.The observed increase in STEAP4 expression in circulating monocytes due to visceral obesity or glycemic excursions is a compensatory response. The online version contains supplementary material available at 10.1007/s13340-021-00542-1.

Selected transient receptor potential channel genes' expression in peripheral blood mononuclear cells of multiple sclerosis.

Transient receptor potential channels have responsibilities in many cellular processes such as cytokine production, cell differentiation, and cytotoxicity by affecting intracellular cation levels or intracellular signal pathways. Multiple sclerosis is a chronic autoimmune central nervous system (CNS) disease caused by environmental and genetic factors. In this study, we aim to investigate TRPV1-TRPV4, TRPM2, TRPM4, TRPM7, TRPC6, and TRPA1 mRNA expression levels, which are associated with the inflammatory process, in the peripheral blood mononuclear cells (PBMCs) of relapsing-remitting multiple sclerosis (RRMS) patients. Thirty-five healthy controls and age-gender matched thirty patients with RRMS were involved in the study. TRPC6, TRPA1, TRPM2, TRPM4, TRPM7, TRPV1, TRPV2, TRPV3, and TRPV4 PBMCs mRNA expression levels were determined by qPCR. In the present study, the TRPC6, TRPM7, TRPV1, TRPV3, and TRPV4 mRNA expressions of RRMS patients in PBMCs decreased at a significant level compared to the healthy control group (p = .000, p = .000, p = .044, p = .000, p = .004, respectively). The decreased expression of TRPC6, TRPM7, TRPV1, TRPV3, and TRPV4 in PBMCs may be associated with the pathogenesis of MS. Further studies are required to understand the mechanism of the relation between these TRP channels and MS and other autoimmune diseases.

Toll-like receptor 1 as a possible target in non-alcoholic fatty liver disease

Toll-like receptors (TLRs) in the liver compartment have repeatedly been attributed to the development of non-alcoholic fatty liver disease (NAFLD). Knowledge on TLR expression in blood cells and their relation to intestinal microbiota and NAFLD development is limited. Here, we determined TLR expression patterns in peripheral blood mononuclear cells (PBMCs) of NAFLD patients and controls, their relation to intestinal microbiota and the impact of TLRs found altered in NAFLD development. Markers of intestinal permeability in blood and TLR mRNA expression in PBMCs were determined in 37 NAFLD patients and 15 age-matched healthy controls. Fecal microbiota composition was evaluated in 21 NAFLD patients and 9 controls using 16S rRNA gene amplicon sequencing. Furthermore, TLR1−/− and C57BL/6 mice (n = 5–6/group) were pair-fed a liquid control or a fat-, fructose- and cholesterol-rich diet. Intestinal microbiota composition and markers of intestinal permeability like zonulin and bacterial endotoxin differed significantly between groups with the latter markers being significantly higher in NAFLD patients. Expression of TLR1-8 and 10 mRNA was detectable in PBMCs; however, only TLR1 expression, being higher in NAFLD patients, were significantly positively correlated with the prevalence of Holdemanella genus while negative correlations were found with Gemmiger and Ruminococcus genera. TLR1−/− mice were significantly protected from the development of diet-induced NAFLD when compared to wild-type mice. While intestinal microbiota composition and permeability differed significantly between NAFLD patients and healthy subjects, in PBMCs, only TLR1 expression differed between groups. Still, targeting these alterations might be a beneficial approach in the treatment of NAFLD in some patients.

Abstract P139: Diet-induced Shifts In HDL Profiles Are Associated With Changes In Immune Parameters

Introduction: Preclinical and population-based studies have demonstrated immunomodulatory and anti-inflammatory properties of HDL; however, it is unclear whether diet-induced changes in HDL profiles can directly impact markers of immune inflammation. Hypothesis: We hypothesized that daily consumption of different egg-based diets, which have been shown to differentially impact serum lipids and lipoprotein profiles, would induce changes in HDL parameters that correspond to shifts in clinical and molecular immune markers. Methods: Healthy men and women (18-35y, BMI &lt; 30kg/m 2 or &lt; 30% body fat for men and &lt; 40% body fat for women, n = 26) participated in a 16-week randomized, crossover intervention trial (NCT03577223), in which they consumed an egg-free diet for 4 weeks, followed by a 4-week diet containing either 3 whole eggs or 3 egg whites per day. Participants then followed a 4-week egg-free diet washout period, before switching to the alternative whole egg or egg white diet treatment. Fasting lipoprotein profiles, complete blood cell counts, and gene expression in peripheral blood mononuclear cells (PBMCs) was measured at the end of each diet period. Results: While no changes in total HDL particles, average HDL particle diameter, or the concentration of small (7.4-8.0 nm) or medium (8.1-9.5 nm) HDL particles was observed, the concentration of large HDL particles (10.8nm) was increased by whole egg intake as compared to intake of egg whites. A trend toward increased apolipoprotein A1 during the whole egg diet period as compared to the egg-free diet period was additionally observed. In line with our hypothesis, changes in large HDL concentrations between whole egg vs. egg white periods were inversely correlated with total white blood cell counts and absolute lymphocyte counts. CD8A mRNA expression in PBMCs was inversely correlated with concentrations of total and large HDL particles. Importantly, while serum choline and betaine were increased by whole egg intake compared to the other diet periods, no differences in pro-atherogenic and inflammatory trimethylamine-N-oxide (TMAO) between diet periods was observed. Conclusion: These findings suggest that diet-induced changes in HDL profiles are directly associated with changes in immune parameters.

Effects of Vitamin D and Dexamethasone on Lymphocyte Proportions and Their Associations With Serum Concentrations of 25-Hydroxyvitamin D3In Vitro in Patients With Multiple Sclerosis or Neuromyelitis Optica Spectrum Disorder.

BackgroundClear associations have been found between vitamin D deficiency and several autoimmune diseases including multiple sclerosis (MS). However, the benefits of vitamin D supplementation on disease management remain a matter of debate.Objective and MethodsPatients with MS (N=12) and neuromyelitis optica spectrum disorder (NMOSD; N=12) were enrolled along with 15 healthy controls. Changes in lymphocyte subset proportions during stimulation of their peripheral blood mononuclear cells (PBMCs) with the active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), and correlations with serum concentrations of the vitamin D precursor 25-hydroxyvitamin D3 (serum 25(OH)D3) were explored. The impact of 1,25(OH)2D3 stimulation on the expression of vitamin-D-responsive genes in immune cells was also investigated.ResultsIn both MS and NMOSD, stimulation of PBMCs with 1,25(OH)2D3 followed by steroid suppressed the proliferation of total lymphocytes and T cells. The ratio of CD19+CD27+ memory B cells (Bmem) to all B cells after stimulation with 1,25(OH)2D3 was negatively correlated with serum 25(OH)D3 in MS (Spearman’s ρ=–0.594, p=0.042), but positively correlated in NMOSD (Pearson’s r = 0.739, p=0.006). However, there was no relationship between the ratio of Bmem to CD19+CD24+CD38+ regulatory B cells and serum 25(OH)D3 in either MS or NMOSD. In addition, the level of 1,25(OH)2D3-induced CYP24A1 mRNA expression in PBMCs was significantly and negatively correlated with serum 25(OH)D3 (for ΔCT, r=0.744, p=0.014) in MS.ConclusionThese findings suggest a beneficial impact of stimulation of PBMCs with vitamin D followed by steroid on the T-cell population. The association between patient serum 25(OH)D3 and the proportion of Bmem under immune-cell stimulation differed between MS and NMOSD. Further investigations are warranted with larger patient populations.

Functional variants in the chromosome 4q21 locus contribute to allergic rhinitis risk by modulating the expression of N-acylethanolamine acid amidase.

Previous haplotype-based association studies identified chromosome 4q21 as an allergic rhinitis (AR) susceptibility locus; however, the functional role of 4q21 single nucleotide polymorphisms (SNPs) on AR risk remains unclear. To investigate the functional effect of 4q21 SNPs on AR risk by conducting cohort-based functional genomics and genetic association analyses. The associations between 4q21 SNPs and mRNA expression levels of three 4q21-associated genes (SDAD1, NAAA and CXCL9) in peripheral blood mononuclear cells (PBMCs) were assessed in a Singapore/Malaysia Chinese cohort (n=291). Exon expression levels of these genes in PBMCs were tested against the tag-SNP genotypes in a Singapore Chinese cohort (n=30). Serum protein levels of these genes were assessed with tag-SNP genotypes in a Singapore Chinese cohort (n=193). SNP functions were characterized through luciferase assay. In a Singapore Chinese cohort (n=1794), we confirmed the associations between functional SNPs and AR. Forty SNPs in 4q21 showed significant associations with NAAA (but not SDAD1 or CXCL9) mRNA expression in PBMCs, of which were tagged by two tag-SNPs, rs17001237 and rs2242470. Both tag-SNPs rs2242470 and rs12648687 (a proxy for rs17001237) were also significantly associated with the expression level of NAAA exon 1. Tag-SNP rs12648687 was correlated with serum NAAA level. A four promoter SNPs-haplotype tagged by rs17001237 influenced the NAAA promoter activity in HEK293T cells. Lastly, individuals carrying the risk allele A of rs12648687 exhibited significantly higher AR risk in the Singapore Chinese population. The rs17001237 linkage set SNPs in the 4q21 locus are associated with NAAA expression at both gene and protein levels ex vivo, have functional consequences in vitro and contribute to AR susceptibility in our study population. Our findings provided a better understanding of the genetic mechanism that contributes to AR pathogenesis.

Abnormal expression of autophagy-related proteins in immune thrombocytopenia.

Autophagy is a highly conserved protein degradation pathway that is essential for affecting some autoimmune diseases. Immune thrombocytopenia (ITP) is a common autoimmune disorder, and the complex dysregulation of cellular immunity has been observed; however, the relationship between autophagy-related proteins and immune responses in ITP remains unclear. Using real-time quantitative polymerase chain reaction (RT-PCR), the mRNA expression levels of Beclin-1, SQSTM1/p62 and LC3 were measured in the peripheral blood mononuclear cells (PBMCs) of 20 newly diagnosed patients with active ITP, 16 ITP patients in remission and 21 healthy volunteers. The stained Beclin-1 and SQSTM1/p62 proteins were also observed in the bone marrow of active ITP patients and normal controls by immunofluorescence. SQSTM1/p62 mRNA expression in PBMCs in newly diagnosed patients was significantly decreased. At the same time, Beclin-1 mRNA was increased significantly. During the remission stages, the levels of these autophagy-related proteins were comparable with those observed in healthy controls. Taken together, these results suggest that the aberrant expression of autophagy-related proteins might be involved in the pathogenesis of ITP. Further study of the autophagy pathway may provide a new strategy and direction for the treatment of ITP.