AKR1A1 Variant Associated With Schizophrenia Causes Exon Skipping, Leading to Loss of Enzymatic Activity

Schizophrenia is a heterogeneous psychiatric disorder characterized by positive symptoms such as hallucinations and delusions, negative symptoms such as anhedonia and flat affect, and cognitive impairment. Recently, glucuronate (GlucA) levels were reported to be significantly higher in serum of patients with schizophrenia than those in healthy controls. The accumulation of GlucA is known to be related to treatment-resistant schizophrenia, since GlucA is known to promote drug excretion by forming conjugates with drugs. However, the cause of GlucA accumulation remains unclear. Aldo-keto reductase family one member A1 (AKR1A1) is an oxidoreductase that catalyzes the reduction of GlucA. Genetic loss of AKR1A1 function is known to result in the accumulation of GlucA in rodents. Here, we aimed to explore genetic defects in AKR1A1 in patients with schizophrenia, which may result in the accumulation of GlucA. We identified 28 variants of AKR1A1 in patients with schizophrenia and control subjects. In particular, we identified a silent c.753G > A (rs745484618, p. Arg251Arg) variant located at the first position of exon 8 to be associated with schizophrenia. Using a minigene assay, we found that the c.753G > A variant induced exon 8 skipping in AKR1A1, resulting in a frameshift mutation, which in turn led to truncation of the AKR1A1 protein. Using the recombinant protein, we demonstrated that the truncated AKR1A1 completely lost its activity. Furthermore, we showed that AKR1A1 mRNA expression in the whole blood cells of individuals with the c.753G > A variant tended to be lower than that in those without the variants, leading to lower AKR activity. Our findings suggest that AKR1A1 carrying the c.753G > A variant induces exon skipping, leading to a loss of gene expression and enzymatic activity. Thus, GlucA patients with schizophrenia with the c.753G > A variant may show higher GlucA levels, leading to drug-resistant schizophrenia, since drug excretion by GlucA is enhanced.

Dysregulated lncRNAs are Involved in the Progress of Sepsis by Constructing Regulatory Networks in Whole Blood Cells

Sepsis is a highly heterogeneous syndrome that is caused by an unbalanced host response to an infection. Long noncoding RNAs (lncRNAs) have been reported to exert regulatory roles in a variety of biological processes, and became potential biomarkers and therapeutic targets for diverse diseases. However, current understanding on the roles of lncRNAs in sepsis is extremely limited. Herein, to decipher the underlying functions of lncRNAs, we reexplored the 83 transcriptome datasets from specimens with sepsis, no_sepsis by final diagnosis, and control. The results of differentially expressed genes (DEGs), differentially expressed lncRNA (DElncRNA) analysis, and co-expression analysis of lncRNA–mRNA pairs were obtained. We found that the expression pattern of lncRNAs was significantly activated in sepsis specimens, which was clearly distinguished in sepsis from no_sepsis and control specimens. By performing co-expression analysis, we found DElncRNAs were closely related to T-cell activation and immune response–related terms in sepsis by regulating mRNA expression in the trans manner. The lncRNA–mRNA network and the qRT-PCR test revealed that lncRNAs LINC00861, RP11-284N8.3, and CTB-61M7.2 were significantly correlated with the pathogenesis of sepsis. In addition, weighted gene co-expression analysis (WGCNA) and cis-regulation analysis also revealed sepsis-specific lncRNAs were highly associated with important biological processes correlated with sepsis. In summary, the systematic dysregulation of lncRNAs is tightly involved in the remodeling of gene expression regulatory network in sepsis, and the lncRNA–mRNA expression network may be used to refine biomarker predictions for developing novel therapeutic approaches in sepsis.

Astragalus Saponins, Astragaloside VII and Newly Synthesized Derivatives, Induce Dendritic Cell Maturation and T Cell Activation Through IL-1β Production

Astragaloside VII (AST VII), a plant triterpenoid saponin isolated from Astragalus species, shows promise as vaccine adjuvant, as it supports a balanced Th1/Th2 immune response. However, the underlying mechanisms of its adjuvant activity have not been defined. Here we investigated the impact of AST VII and its newly synthesized semi-synthetic analogs on human whole blood cells, as well as on mouse bone marrow-derived dendritic cells (BMDCs). Cells were stimulated with AST VII and its derivatives in the presence or absence of LPS or PMA/ionomycin and the secretion of cytokines and the expression of activation markers were analyzed by ELISA and flow cytometry, respectively. AST VII and its analogs increased the production of IL-1b; in PMA/ionomycin stimulated human whole blood cells. In LPS-treated mouse BMDCs, AST VII increased the production of IL-1b; and IL-12, and the expression of MHC II, CD86, and CD80. The strength of the IL-1b; boost correlated directly with the hydrophobicity of the AST VII compounds. In mixed leukocyte reaction, AST VII and derivatives increased the expression of the activation marker CD44 on mouse CD4+ and CD8+ T cells. In conclusion, AST VII and its derivatives strengthen pro-inflammatory responses, support dendritic cell maturation, and T cell activation in vitro. Our results provide insights into the mechanisms of the adjuvant activities of AST VII and its analogs, which will be instrumental to improve their utility as vaccine adjuvant.

Elevated methylation of the vault RNA2-1 promoter in maternal blood is associated with preterm birth

Background Preterm birth, defined as parturition before 37 completed weeks of gestation, is associated with an increased risk of neonatal complications and death, as well as poor health and disease later in life. Epigenetics could contribute to the mechanism underlying preterm birth. Results Genome-wide DNA methylation analysis of whole blood cells from 10 women (5 term and 5 preterm deliveries) was performed using an Illumina Infinium HumanMethylation450 BeadChips array. We identified 1,581 differentially methylated CpG sites in promoter regions between term and preterm birth. Although the differences were not significant after correcting for multiple tests, seven CpGs on the genomically imprinted vault RNA2-1 (VTRNA2-1; also known as non-coding RNA, nc886 or miR-886) showed the largest differences (range: 26–39 %). Pyrosequencing verification was performed with blood samples from pregnant women recruited additionally (39 term and 43 preterm deliveries). In total, 28 (34.1 %) samples showed hypomethylation of the VTRNA2-1 promoter (< 13 % methylation), while 54 (65.9 %) samples showed elevated methylation levels between 30 and 60 %. Elevated methylation of VTRNA2-1 promoter was associated with an increased risk of preterm birth after adjusting for maternal age, season of delivery, parity and white blood cell count. The mRNA expression of VTRNA2-1 was 0.51-fold lower in women with preterm deliveries (n = 20) compared with women with term deliveries (n = 20). Conclusions VTRNA2-1 is a noncoding transcript to environmentally responsive epialleles. Our results suggest that elevated methylation of the VTRNA2-1 promoter may result in increased risk of PTB caused by the pro-inflammatory cytokines. Further studies are needed to confirm the association of VTRNA2-1 methylation with preterm birth in a large population, and to elucidate the underlying mechanism.

Long-Term Hypermethylation of FcγR2B in Leukocytes of Patients with Kawasaki Disease

The Fc gamma receptor family contains several activating receptors and the only inhibitory receptor, FcγR2B. In this study, we investigated the dynamic methylation change of FcγR2B in different stages of Kawasaki disease (KD). We enrolled a total of 116 participants, which included patients with febrile diseases as controls and KD patients. Whole blood cells of KD patients were collected prior to intravenous immunoglobulin (IVIG) treatment (KD1), three to seven days after IVIG (KD2), three weeks after IVIG treatment (KD3), six months after IVIG (KD4), and one year after IVIG treatment (KD5). In total, 76 KD patients provided samples in every stage. Leukocytes of controls were also recruited. We performed DNA extraction and pyrosequencing. FcγR2B methylation levels were higher in KD3 compared to both the controls and KD1. A significantly higher methylation of FcγR2B was found in KD5 when compared with KD1. FcγR2B methylation levels in the IVIG-resistant group were lower than those in the IVIG-responsive group at KD1-3 (p = 0.004, 0.004, 0.005 respectively). This study is the first to report the dynamic change of FcγR2B methylation and to demonstrate long-term hypermethylation one year after disease onset. Hypomethylation of FcγR2B is associated with IVIG resistance.

Effect of macrophage-activating factor (GcMAF-RF) upon ex vivo polarization of macrophages, activation of dendritic cells and production of cytokines by human whole blood cells

This article is the second communication in a series of articles devoted to the effects of a domestic preparation of macrophage-activating factor (GcMAF-RF) and assessment of its biological properties. The aim of this work was to study the effect of the GcMAF-RF upon M0 → M1 polarization of macrophages (Mph), and activation of the professional properties of ex vivo generated antigen-presenting dendritic cells (DC), as well as on ex vivo production of pro-inflammatory (TNFα, IL-1β, IL-6, IFNγ, IL-17, IL-18) and anti-inflammatory (TGF-β, IL-4, IL-10) cytokines, growth factors (IL-2, GM-CSF, G-CSF, VEGF) and chemokines (MCP, IL-8) by the whole blood cells from healthy donors. Mph and DC were generated from the monocytes (3 to 5×106 /ml) derived from adherent fraction of peripheral blood mononuclear cells (MNC) of healthy donors. Granulocyte/macrophage colony-stimulating factor (rhGM-CSF) was used to obtain Mph, whereas DC production was induced by GM-CSF and interferon-α. To provide M1 polarizing signals, bacterial lipopolysaccharide (LPS from E. coli 0114:B4) was used in controls. In experimental series, GcMAF-RF was added 48 h before the end of culture. The stimulating effect of the obtained Mph and DC upon cell proliferation was assessed in allogeneic mixed culture of leukocytes (alloMLC) using radiometric technique, by 3 H-thymidine incorporation. The influence index (IR) of Mph or DC upon allo-SCL was calculated as the ratio of the proliferative response of MNCs in the presence of Mph, or DC to the level of spontaneous MNC proliferation. To determine the cytokine production by human whole blood cells ex vivo, peripheral blood samples from 3 donors with two replicate GcMAF-RF preparations were used, at a total of 6 points. All variants of the study were carried out with mitogen-activated and non-activated blood cells. The cytokine content was determined by the ELISA assays. The effects of GcMAF-RF were quantified as a fold increase (FI), i.e., the ratio of cytokine production in the presence of GcMAF-RF to the level of their spontaneous production. It was shown that the GcMAF-RF preparation was as effective, as lipopolysaccharide (LPS), the standard Mph and DC activator which induces polarization of differentiated M0-macrophages into M1 cells and final maturation of DCs, manifesting by a significant increase in their allo-stimulatory activity in a mixed leukocyte culture (allo-MLC). Moreover, GcMAF-RF stimulates production of numerous cytokines and chemokines (TNFα, IL-1β, IL-6, IL-18, IL-4, IL-10, GM-CSF, G-CSF, VEGF, IL-8), by blood cells (granulocytes, lymphocytes, monocytes), thus indicating direct participation of the macrophage activator GcMAF-RF in various immune processes. The domestic GcMAF-RF drug induces polarization of macrophages M0 → M1, final maturation of DCs and allostimulating activity of Mf and DCs, and is also able to effectively stimulate circulating blood cells to synthesize cytokines/chemokines with pro-inflammatory and immunoregulatory activities.

Monocytes From Patients With Macrophage Activation Syndrome and Secondary Hemophagocytic Lymphohistiocytosis Are Hyperresponsive to Interferon Gamma

ObjectiveTo investigate the activation of the IFNγ signaling pathway in monocytes of patients with secondary hemophagocytic lymphohistiocytosis (sHLH)/macrophage activation syndrome (MAS) and to evaluate whether levels of phosphorylated STAT1 represent a biomarker for the identification of patients at early stages of the disease.MethodsFresh whole blood samples from pediatric patients with active sHLH/MAS, not receiving (n=10) and receiving glucocorticoids (n=14) at time of sampling, were prospectively collected. As disease control groups, patients with active systemic juvenile idiopathic arthritis (sJIA) without MAS, patients with sHLH/MAS in remission and patients with other rheumatic diseases were also sampled. Whole blood cells were left unstimulated or stimulated with increasing concentrations of IFNγ for 10 minutes and the intracellular Tyrosine (701)-phosphorylated STAT1 (pSTAT1) levels were evaluated in monocytes by flow cytometry.ResultsMonocytes from untreated sHLH/MAS patients showed significantly higher basal levels of pSTAT1 compared to those observed in monocytes from glucocorticoid-treated sHLH/MAS patients and from all the other disease controls. In addition, a significant increase in responsiveness to IFNγ, as assessed by increased levels of pSTAT1 following ex vivo stimulation, was observed in monocytes from untreated sHLH/MAS patients. pSTAT1 levels in monocytes distinguished patients with sHLH/MAS not treated with glucocorticoids from patients with active sJIA or with other rheumatic diseases [AUC, 0.93; 95% confidence interval 0.85-1.00, p&lt;0.001]. Statistically significant correlations between IFNG mRNA levels in whole blood cells, circulating IFNγ levels and pSTAT1 levels in sHLH/MAS monocytes were found.ConclusionOur data demonstrating higher basal levels of pSTAT1 as well as a hyperreactivity to IFNγ stimulation in monocytes from patients with sHLH/MAS point to perturbations in the activation of downstream IFNγ signaling pathway as a contributor to the hyperinflammation occurring in these patients. Finally, the observation that glucocorticoids affect pSTAT1 levels in vivo, makes it difficult to consider the measurement of pSTAT1 levels as a biomarker to identify patients at early stages of sHLH/MAS in clinical practice.

Effect of flavonoid-rich meals and low-flavonoid meals based on the dietary reference intakes for Japanese, using basic foodstuffs on the gene expression of inflammatory cytokines in the whole blood cells from adult men of normal or light overweight

Introduction: Flavonoids have a variety of functions, such as antioxidant activity, and are expected to have a disease prevention effect. In order to verify the disease risk reduction effect of flavonoids, we carried out a crossover trial in seven adult men of normal or light overweight who ingested flavonoid-rich meals, with a diverse combination of basic foodstuffs, and low-flavonoid meals and compared blood disease-related inflammatory markers.Methods: On the first two days of the study, seven male volunteers were provided with low-flavonoid meals (flavonoid content below the detection limit of HPLC: less than 0.24 mg/meal) three times a day as a washout. For the next seven days, they were fed flavonoid-rich meals (46.9 ± 8.1 mg/meal) or low-flavonoid meals. Blood samples were collected from all the volunteers before breakfast on the third day, after the washout and before breakfast on the tenth day. The test was consisted of one cycle from the first day to the tenth day, and the participants carried out two cycles. Flavonoid concentrations in plasma and gene expression of inflammatory cytokine (interleukin 1 beta, interleukin 6, interleukin 18, and tumor necrosis factor-α) in whole blood cells were compared before and after the intervention. Gene expression in whole blood cells was measured using real time RT-PCR.Results: We found a significant increase in plasma flavonoid concentration (quercetin, kaempferol, daidzein, and genistein) upon intervention with flavonoid-rich meals (p < 0.05). In addition, the inflammatory cytokine gene expression was reduced in the subjects with a body mass index of more than, but not less than, 25 kg/m2 compared with that observed after the intake of low-flavonoid meals.Conclusion: These results suggest that flavonoid-rich meals have an anti-inflammatory effect in obese persons who are likely to have chronic inflammation.Keywords: Flavonoids, inflammatory cytokines, flavonoid-rich meal, human study

The Changes of Whole Blood Cells and Plasma Proteins in Donors After Plateletpheresis: 42 Donors With a Interval of 14-16 Days and More Than 20 Times

Background To study the changes of whole blood cells and plasma proteins in donors after plateletpheresis with multiple donations. Materials and Methods From October 2015 to September 2019, 42 donors with a plateletpheresis interval of 14-16 days and more than 20 times were selected as the research subjects. The venous blood samples were collected from the first and the last screening before plateletpheresis. The result of last screening before plateletpheresis as the observation group, and the first as the control group. Then, the venous blood samples was detected. Results The whole blood cells and plasma proteins in donors after plateletpheresis changes within a normal range in the two groups. The PLT counts in the the observation group was 220.1±40.4 x109/L, which was no statistically significant compared with the change of 216.6±44.5 x109/L in the control group(P>0.05). The HGB in the the observation group was 142.8±10.2 g/L, which was no statistically significant compared with the change of 142.1±8.3g/L in the control group(P>0.05). The HCT in the the observation group was 43.50±3.2%, which was no statistically significant compared with the change of 44.1±2.8% in the control group(P>0.05). The serum TP levels in the the observation group was 70.4±4.7g/L, which was no statistically significant compared with the change of 69.0±4.8g/L in the control group(P>0.05). The serum ALB levels in the observation group was 46.3±2.3g/L, which was no statistically significant compared with the change of 45.8±2.3g/L in the control group(P>0.05). Conclusion There have no effect on the whole blood cells and plasma proteins in donors after plateletpheresis with multiple donations.