Gene Expression Levels Of Cytokines Research Articles

Features of Brain Damage after Neutron Irradiation of the Head and Modification of the Damage by Lactoferrin.

: Mouse heads were irradiated in a beam of neutrons and gamma rays from the IR-8 nuclear reactor. The brain cells of control and irradiated mice were isolated using Percoll. Neurons and resting and activated microglial cells were analyzed using the fluorescently labeled antibodies and flow cytometry. The level of DNA double-strand breaks in neurons was determined by γH2AX histone content. Cytokine gene expression in the hippocampus was studied by RT-PCR. Behavior and cognitive functions were studied using the open field, Morris water maze, and novel object recognition tests. LF was isolated from female colostrum by preparative ion-exchange chromatography and purified by affinity chromatography on heparin-Sepharose. : γ,n-Irradiation of the mouse head at a dose of 1.5 Gy led to an increase in the level of DNA double-strand breaks in neurons. Twenty-four hours after irradiation the total number of cells and the number of neurons in the isolated fraction of brain cells decreased, but the number of microglial cells remained unchanged. The number of resting and activated microglia did not change within 3-72 h after γ,n-irradiation. The expression level of the TNFα, IL-1β, and IL-6 genes increased 2 months after γ,n-irradiation of the mouse head at a dose of 1.5 Gy, indicating the development of neuroinflammation. At this time, irradiated mice demonstrated the anxiety-like behavior and impaired spatial and episodic memory. A single i.p. administration of human LF to mice immediately after γ,n-irradiation of the head did not affect the observed radiation-induced disturbances, but decreased the gene expression levels of TNFα, IL-1β, and IL-6 pro-inflammatory cytokines and increased the gene expression level of TGFβ anti-inflammatory cytokine in the hippocampus 2 months after radiation exposure. The obtained results indicate a partial decrease in the level of hippocampal neuroinflammation of irradiated animals treated with LF. . γ,n-Irradiation of the mouse head at a dose of 1.5 Gy leads to DNA damage of neurons and the decrease in the number of neurons. Microglia cells are more resistant to such radiation exposure. Late after head-only γ,n-irradiation, mice develop neuroinflammation, which is detected by an increase in the pro-inflammatory cytokine gene expression in the hippocampus and also by anxiety-like behavior and impaired cognitive functions. A single LF administration leads to a partial decrease in the neuroinflammation level, but does not affect the other studied parameters. The optimal dosing regimen of LF remains to be determined to preserve cognitive functions after γ,n-irradiation of the brain.

Glyburide Suppresses Inflammation-Related Colorectal Tumorigenesis Through Inhibition of NLRP3 Inflammasome.

Colorectal cancer represents one of the most serious complications of inflammatory bowel disease. The NLRP3 inflammasome plays a pivotal role in the onset and progression of inflammatory bowel disease and is also implicated in colorectal cancer. This study aimed to investigate whether NLRP3 deficiency or glyburide, a sulfonylurea used for diabetes management and known as an NLRP3 inhibitor, could suppress colitis and its related colorectal tumorigenesis. Mice were divided into three groups: a control group, a glyburide group, and an NLRP3-deficient group. We investigated acute colitis and inflammation-related tumor models using azoxymethane and dextran sodium sulfate. In the colitis model, the colonic inflammation grade was significantly increased in NLRP3-deficient mice but not in mice administered glyburide. In the colorectal carcinogenesis model, fewer colorectal tumors were observed in both NLRP3-deficient and glyburide-treated groups. Additionally, a reduction in the expression levels of inflammatory cytokine genes was detected in the colonic mucosa of the mice of these groups. These findings suggest that NLRP3 deficiency may exacerbate acute colitis, while pharmacological inhibition, as well as deficiency of NLRP3, suppresses colitis-related tumorigenesis, presumably due to the attenuation of chronic inflammation in the colorectum. Glyburide holds promise as a potential chemopreventive agent for colitis-related colorectal cancer.

Myeloid-derived growth factor promotes M2 macrophage polarization and attenuates Sjögren's syndrome via suppression of the CX3CL1/CX3CR1 axis.

Primary Sjögren syndrome (pSS) is a systemic autoimmune disease that is characterized by the infiltration of immune cells into the salivary glands. The re-establishment of salivary glands (SGs) function in pSS remains a clinical challenge. Myeloid-derived growth factor (MYDGF) has anti-inflammatory, immunomodulatory, and tissue-functional restorative abilities. However, its potential to restore SGs function during pSS has not yet been investigated. Nonobese diabetic (NOD)/LtJ mice (pSS model) were intravenously administered with adeno-associated viruses carrying MYDGF at 11 weeks of age. Salivary flow rates were determined before and after treatment. Mice were killed 5 weeks after MYDGF treatment, and submandibular glands were collected for analyses of histological disease scores, inflammatory cell infiltration, PCR determination of genes, and Western blotting of functional proteins. Furthermore, mRNA sequencing and bioinformatics were used to predict the mechanism underlying the therapeutic effect of MYDGF. Treatment of NOD/LtJ mice with MYDGF alleviated pSS, as indicated by increased salivary flow rate, reduced lymphocyte infiltration, attenuated glandular inflammation, and enhanced AQP5 and NKCC1 expression. The gene expression levels of cytokines and chemokines, including Ccl12, Ccl3, Il1r1, Ccr2, Cx3cr1, Il7, Mmp2, Mmp14, Il1b, and Il7, significantly decreased after treatment with MYDGF, as determined by RNA sequencing. Meanwhile, MYDGF inhibits infiltration of macrophages (Mϕ) in SGs, induces polarization of M2ϕ, and suppresses C-X3C motif ligand 1 (CX3CL1)/C-X3C motif receptor 1 (CX3CR1) axis. Our findings showed that MYDGF could revitalize the SGs function of pSS, inhibit infiltration of Mϕ, and promote M2ϕ polarization via suppression of the CX3CL1/CX3CR1 axis, which has implications for potential therapy for pSS.

Characterization of obesity-related diseases and inflammation using single cell immunophenotyping in two different diet-induced obesity models

BackgroundObesity is a growing problem worldwide and a major risk factor for many chronic diseases. The accumulation of adipose tissue leads to the release of significant amounts of pro-inflammatory cytokines and adipokines, resulting in a low-grade systemic inflammation. However, the mechanisms behind the development of obesity-related diseases are not fully understood. Therefore, our study aimed to investigate the pathological changes and inflammatory processes at systemic level and in individual organs in two different diet-induced mouse obesity models.MethodsMale C57BL6/J mice were fed by high-fat diet (HFD), high-fat/high-fructose diet (HFD + FR) or normal chow for 21 weeks starting at 3 months of age (n = 15 animals/group). Insulin resistance was tested by oral glucose tolerance test. Pathological changes were investigated on hematoxylin–eosin-stained liver and brown adipose tissue sections. The gene expression levels of adipokines and cytokines were analyzed by qPCR in adipose tissues, whereas serum protein concentrations were determined by multiplex immunoassays. Immunophenotyping of isolated blood, bone marrow and spleen cells was performed by single-cell mass cytometry.ResultsWeight gain, glucose intolerance and hepatic steatosis were more severe in the HFD + FR group than in the control and HFD groups. This was accompanied by a higher level of systemic inflammation, as indicated by increased expression of pro-inflammatory genes in visceral white adipose tissue and by a higher serum TNFα level. In addition, immunophenotyping revealed the increase of the surface expressions of CD44 and CD69 on various cell types, such as CD8+ and CD4 + T-cells, B-cells and macrophages, in animals with obesity.ConclusionsThe combination of HFD with fructose supplementation promotes more properly the symptoms of metabolic syndrome. Therefore, the combined high-fat/high-fructose nutrition can be a more suitable model of the Western diet. However, despite these differences, both models showed immunophenotypic changes that may be associated with increased risk of obesity-related cancer.

Transcriptomic Expression of T2-Inflammation Genes in Peripheral Blood Mononuclear Cells and Longitudinal Clinical Outcomes in Asthma: Insights from the COREA Study.

Gene expression can provide distinct information compared to clinical biomarkers in the context of longitudinal clinical outcomes in asthma patients. This study examined the association between the gene expression levels of upstream (IL-25, IL-33, and TSLP) and downstream cytokines (IL-5, IL-4, and IL-13) in the T2 inflammatory pathway with a 12-month follow-up of exacerbation, lung function, and steroid use. Transcriptomic sequencing analysis was performed on peripheral blood mononuclear cells from 279 adult asthmatics. Survival analysis and linear mixed-effect models were used to investigate potential differences between the high-level and low-level gene expression groups and the clinical outcomes. Analysis was performed separately for the upstream, downstream, and all 6 cytokines. In general, T2 inflammatory cytokine gene expression showed a weak correlation with blood eosinophil counts (all r < 0.1) and clinical outcomes. Among moderate-to-severe eosinophilic asthma (MSEA) patients, individuals with elevated levels of downstream cytokines were at increased risk of time-to-first exacerbation (p = 0.044) and a greater increase of inhaled corticosteroid use over time (p = 0.002) compared to those with lower gene expression. There was no association between baseline T2 inflammatory cytokine gene expression and the longitudinal changes in lung function over time among MSEA patients. These findings suggest that, among MSEA patients, the gene expression levels of downstream cytokines in the T2 inflammatory pathway may serve as indicators for endotyping asthma.

Mapping and Analysis of Protein and Gene Profile Identification of the Important Role of Transforming Growth Factor Beta in Synovial Invasion in Patients With Pigmented Villonodular Synovitis.

Pigmented villonodular synovitis (PVNS) is a rare benign proliferative disease affecting the soft-tissue lining the synovial joints and tendons. Its etiology is poorly understood, largely limiting the availability of current therapeutic options. Here, we mapped the synovial gene and protein profiles of patients with PVNS, revealed a link between synovial inflammation and invasion, and elucidated the potential molecular mechanism involved. The expression of synovial genes from 6 control individuals, 7 patients with osteoarthritis (OA), and 19 patients with PVNS was analyzed via RNA sequencing. Protein profiles from 5 control individuals, 10 patients with OA, and 32 patients with PVNS were analyzed using label-free proteomics. Microarray and reverse transcription-polymerase chain reaction analyses and immunohistochemical staining were used to evaluate inflammatory cytokine and target gene expression levels in synovial tissue, epithelial cells, and synovial fibroblasts (FLSs) derived from tissue of patients with PVNS. Various signaling pathway inhibitors, small interfering RNAs, and Western blots were used for molecular mechanism studies. Transwell migration and invasion assays were subsequently performed. In total, 522 differentially expressed proteins were identified in the tissues of patients with PVNS. By integrating RNA sequencing and microarray analyses, significant changes in the expression of epithelial-mesenchymal transition (EMT)-related genes, including transforming growth factor TGF-b induced, neural cadherin, epithelial cadherin, SNAIL, and TWIST, were confirmed in the tissue of patients with PVNS compared to the control tissue. In vitro, TGFβ induced EMT and increased epithelial cell migration and invasion. Moreover, TGFβ not only promoted interactions between epithelial cells and FLSs but also directly increased the migration and invasion abilities of FLSs by activating the classical Smad2/3 and nonclassical JNK/AKT signaling pathways. This study provides overall protein and gene profiles of PVNS and identifies the crucial role of TGFβ in synovial invasion pathology. Exploring the related molecular mechanism may also reveal a new strategy or target for PVNS therapy.

Relationship between the serum level, polymorphism and gene expression of IL-33 in samples of recurrent miscarriage Iraqi women infected with toxoplasmosis

One of the many warm-blooded hosts that toxoplasmosis-causing intracellular protozoan parasite Toxoplasma gondii can infect is humans. Cytokines are crucial to stimulate an effective immune response against T. gondii. Interleukin-33 (IL-33) is a unique anti-inflammatory cytokine that suppresses the immune response. The levels of cytokine gene expression are regulated by genetics, and the genetic polymorphisms of these cytokines play a functional role in this process. Single nucleotide polymorphisms (SNPs) are prognostic indicators of illnesses. This study aimed to determine whether toxoplasmosis interacts with serum levels of IL-33 and its SNP in miscarriage women as well as whether serum levels and IL-33 gene expression are related in toxoplasmosis-positive miscarriage women. Two hundred blood samples from patients and controls were collected from AL-Alawiya Maternity Teaching Hospital and AL-Yarmouk Teaching Hospital in Baghdad, Iraq from 2021 to 2022 in order to evaluate the serum level of IL-33 using ELISA test. For the SNP of IL-33, the allelic high-resolution approach was utilized, and real time-PCR was performed to assess gene expression. The results showed that compared to healthy and pregnant women, recurrent miscarriage with toxoplasmosis and recurrent miscarriage women had lower IL-33 concentrations. Additionally, there were significant differences among healthy women, pregnant women, and women with repeated miscarriage who experienced toxoplasmosis. Furthermore, no differences between patients and controls were revealed by gene expression data. The results revealed that recurrent miscarriage, pregnancy, and healthy women all had a slightly higher amount of the IL-33 gene fold. Additionally, the SNP of IL-33 data demonstrated that there was no significant genetic relationship between patients and controls. Recurrent miscarriage women with toxoplasmosis have showed significant differences from pregnant women in the genotypes GG and AA as well as the alleles A and G. There were notable variations between recurrent miscarriage with and without toxoplasmosis in terms of the genotypes AA and AC. The genotypes GG, AA, and allele A in recurrent miscarriage women with toxoplasmosis and recurrent miscarriage women is a protective factor. Taking together, there was a statistically significant negative correlation between toxoplasmosis and IL-33 gene expression, which calls for more quantitative investigation in order to fully comprehend the interaction of mRNA and protein.

Search in the Periphery for Potential Inflammatory Biomarkers of Dementia with Lewy Bodies and Alzheimer's Disease.

Neuroinflammation, with altered peripheral proinflammatory cytokine production, plays a major role in the pathogenesis of neurodegenerative diseases, such as Alzheimer's disease (AD), while the role of inflammation in dementia with Lewy bodies (DLB) is less known and the results of different studies are often in disagreement. The present study aimed to investigate the levels of TNFα and IL-6 in serum and supernatants, and the related DNA methylation in patients affected by DLB and AD compared to healthy controls (HCs), to clarify the role of epigenetic mechanisms of DNA promoter methylation on of pro-inflammatory cytokines overproduction. Twenty-one patients with DLB and fourteen with AD were frequency-matched for age and sex with eleven HCs. Clinical evaluation, TNFα and IL-6 gene methylation status, cytokine gene expression levels and production in serum and peripheral blood mononuclear cell (PBMC) supernatants were performed. In AD and DLB patients, higher serum levels of IL-6 and TNFα were detected than in HCs. Differences in LPS-stimulated versus spontaneous PBMCs were observed between DLB, AD, and HC in the levels of TNFα (p = 0.027) and IL-6 (p < 0.001). Higher levels were also revealed for sIL-6R in DLB (p < 0.001) and AD (p < 0.001) in comparison with HC.DNA hypomethylation in IL-6 and TNFα CpG promoter sites was detected for DLB and AD patients compared to the corresponding site in HCs. Our preliminary study documented increased levels of IL-6 and TNFα in DLB and AD patients to HCs. This overproduction can be due to epigenetic mechanisms regarding the hypomethylation of DNA promoters.

Modulator Effect of AT1 Receptor Knockdown on THP-1 Macrophage Proinflammatory Activity.

We evaluated the gene expression of PPAR-γ in THP-1 macrophages using PMA (60 ng/mL). For the silencing, cells were incubated with the siRNA for 72 h and telmisartan (10 µM) was added to the medium for 24 h. After that, cells were incubated during 1 and 24 h, respectively, with Ang II (1 µM). The gene expression levels of AT1R, NF-κB, and cytokines (IL-1β, TNF-α, and IL-10) were measured by RT-qPCR. We observed that silencing of the AT1 receptor causes a decrease in the expression of mRNA of proinflammatory cytokines (IL-1β and TNF-α), NF-κB, and PPAR-γ. We conclude that AT1R gene silencing is an alternative to modulating the production of proinflammatory cytokines such as TNF-α and IL-1β via NF-κB in macrophages and having high blood pressure decrease.

High potency of linalool-zinc oxide nanocomposite as a new agent for cystic echinococcosis treatment.

This current in vitro, ex vivo, and in vivo research aims to evaluate and analyze the linalool-zinc oxide nanocomposite (Lin-ZNP) for treating cystic echinococcosis. Lin-ZNP was synthesized using an ethanolic solution of polyvinyl alcohol. The protoscolicidal effects of Lin-ZNP were tested on hydatid cyst protoscoleces (PTS) in both in vitro and ex vivo by eosin exclusion test. The study also examined the impact on caspase-3 gene expression and the external structure of PTS. The in vivo effect was measured by examining hydatid cysts' quantity, dimensions, and weight in mice intraperitoneally infected with 0.5 mL of PTS solution containing 1,000 PTS. The antioxidant and inflammatory cytokine gene expression levels were examined using real-time PCR. Lin-ZNP significantly (P < 0.001) killed the PTS in both in vitro and ex vivo in a dose- and time-dependent manner. The treated PTS exhibited creases and protrusions as a result of bleb formation and upregulation in the gene expression of caspase-3. Upon treatment with Lin-ZNP, there was a significant (P < 0.001) reduction in the number, diameter, and weight of the hydatid cysts. Treatment with Lin-ZNP nanocomposite led to a significant increase in the expression of antioxidant genes and a notable decrease in oxidative stress markers, and the expression levels of IL-4 and IL-10. Lin-ZNP has the potential to act as a scolicidal agent and demonstrates promise in controlling hydatid cysts in a mouse model, attributed to its antioxidant and anti-inflammatory properties. However, additional studies in clinical trials are needed to confirm the use of Lin-ZNP for treating hydatidosis.

Differential Gene Expression in Porcine Lung Compartments after Experimental Infection with Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae (Mhyo) is the causative agent of porcine enzootic pneumonia (EP), as well as one of the main pathogens involved in the porcine respiratory disease complex. The host-pathogen interaction between Mhyo and infected pigs is complex and not completely understood; however, improving the understanding of these intricacies is essential for the development of effective control strategies of EP. In order to improve our knowledge about this interaction, laser-capture microdissection was used to collect bronchi, bronchi-associated lymphoid tissue, and lung parenchyma from animals infected with different strains of Mhyo, and mRNA expression levels of different molecules involved in Mhyo infection (ICAM1, IL-8, IL-10, IL-23, IFN-α, IFN-γ, TGF-β, and TNF-α) were analyzed by qPCR. In addition, the quantification of Mhyo load in the different lung compartments and the scoring of macroscopic and microscopic lung lesions were also performed. Strain-associated differences in virulence were observed, as well as the presence of significant differences in expression levels of cytokines among lung compartments. IL-8 and IL-10 presented the highest upregulation, with limited differences between strains and lung compartments. IFN-α was strongly downregulated in BALT, implying a relevant role for this cytokine in the immunomodulation associated with Mhyo infections. IL-23 was also upregulated in all lung compartments, suggesting the potential involvement of a Th17-mediated immune response in Mhyo infections. Our findings highlight the relevance of Th1 and Th2 immune response in cases of EP, shedding light on the gene expression levels of key cytokines in the lung of pigs at a microscopic level.

Inhibitory effect of downregulating G protein-coupled receptor class C group 5 member A expression on lipopolysaccharide-induced inflammatory response in human gingival fibroblasts

Objective: To clarify the effect and the mechanism of G protein-coupled receptor class C group 5 member A (GPRC5A) on lipopolysaccharide (LPS)-induced inflammatory response in human gingival fibroblasts (GFs), thus to provide a foundation for delving into the role of G protein coupled receptor (GPCR) in periodontitis. Methods: Gingival tissue samples were collected from 3 individuals periodontally healthy (health group) and 3 patients with periodontitis (periodontitis group) in Shandong Stomatological Hospital from December 2022 to February 2023. The expressions of GPRC5A of the two groups were detected by immunohistochemistry staining. GFs used in this study were isolated from a portion of gingiva for the extraction of impacted teeth in School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University from December 2022 to February 2023. GFs were isolated with enzymic digestion and transfected with 30, 50 and 80 μmol/L small interfering RNA-GPRC5A (siGPRC5A) or small interfering RNA-negative control (siNC), regarded as the experimental group and the negative control one, respectively. The silencing efficiency of siGPRC5A was evaluated by real-time fluorescence quantitative PCR (RT-qPCR). Experiments were then conducted using these cells which were divided into four groups of negative control (NC), LPS, siGPRC5A+LPS and siGPRC5A. The mRNA and protein levels of GPRC5A in GFs under 1 mg/L LPS-induced GFs inflammatory state were evaluated by RT-qPCR and Western blotting analysis after GPRC5A knockdown. RT-qPCR was used to detect the gene expression levels of the inflammatory cytokines in GFs induced by LPS, namely, interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α, prostaglandin endoperoxide synthase 2 (PTGS2) after GPRC5A knockdown. Western blotting analysis and immunofluorescence staining were used to further investigate the activation of nuclear factor-kappa B (NF-κB) signaling pathway. Results: Immunohistochemistry staining showed that the expression of GPRC5A in gingival tissues of periodontitis group (0.132±0.006) increased compared with that in periodontally healthy group (0.036±0.019) (t=8.24, P=0.001). Meanwhile, RT-qPCR results showed that the gene expression levels of GPRC5A at different time point (2, 6, 12, 24 h) in LPS-induced GFs (0.026±0.002, 0.042±0.005, 0.004±0.000, 0.016±0.000) were upregulated compared with those in the NC group (0.004±0.000, 0.004±0.000, 0.002±0.000, 0.007±0.000) (all P<0.001), respectively, and peaked at 6 h. The 50 μmol/L group displayed the most significant decrease in siGPRC5A expression (31.16±3.29) compared with that of the siNC group (100.00±4.88) (F=297.98, P<0.001). The results of RT-qPCR and Western blotting analysis showed that siGPRC5A (0.27±0.03, 0.71±0.00) suppressed the expressions of GPRC5A at both gene and protein levels, while LPS (1.30±0.10, 1.43±0.03) was able to promote the expressions of GPRC5A compared with those of the NC group (1.00±0.01, 1.00±0.00)(all P<0.001). The siGPRC5A+LPS group (0.39±0.03, 1.06±0.16) also inhibited the increase of GPRC5A at both gene and protein levels induced by LPS (1.30±0.10, 1.43±0.03) (F=208.38, P<0.001; F=42.04, P<0.001). RT-qPCR results showed that the expressions of IL-8, IL-1β, IL-6, TNF-α, and PTGS2 at the gene level in LPS group were highly increased compared with those in the NC group (all P<0.001). siGPRC5A significantly suppressed LPS-induced expressions of these inflammatory cytokines in GFs (all P<0.001). Western blotting analysis showed that the levels of p65 and IκBα protein phosphorylation in the LPS group were highly increased compared with those in the NC group, and siGPRC5A could effectively suppressed LPS-induced protein phosphorylation (all P<0.01). Furthermore, immunofluorescence staining showed that NF-κB p65 in the control group was mainly concentrated in the cytoplasm, and partially translocated to the nucleus under the stimulation of LPS. siGPRC5A was able to inhibit LPS-induced intranuclear translocation of p65 to a certain extent. Conclusions: GPRC5A expression was upregulated in periodontitis, and GPRC5A knockdown inhibited LPS-induced inflammation. Moreover, GPRC5A played a role in inflammation regulation by interacting with NF-κB signaling pathway.

Significant beneficial effects of 12-weeks add-on yoga therapy on antipsychotic-stabilized schizophrenia patients through epigenetic modulation: novel findings from a randomized controlled study

IntroductionComplementary and alternative therapy, especially yoga, is emerging as an important treatment modality for various complex disorders. Yoga therapy has reportedly been demonstrated to exhibit clinical benefits in schizophrenia. However, the modulatory effects of yoga therapy on the pathobiological pathways of schizophrenia are inadequately explored. Immune dysregulation is a widely recognized etiopathological construct of schizophrenia. It is not precisely known whether yoga therapy can modulate the expression of immune molecules by regulating gene expression and epigenetic processes in schizophrenia.ObjectivesTo understand the impact of 12-weeks add-on yoga therapy on the immune-inflammatory pathway in schizophrenia by examining plasma levels and gene expression levels of cytokines and complement proteins as well as by profiling promoter DNA methylation pattern of genes coding for cytokines and complement proteins.MethodsFifty-seven schizophrenia patients fulfilling DSM-V criteria were recruited into the study and randomized into Yoga therapy (n=28) and waitlist control (n=29) groups. Plasma levels of IL-1β, IL-6, IL-10, IL-17, C1q, C2, C3, C4, C5, C5a, Factor B and Factor H by Multiplex Suspension Assay, quantification of gene expression of Il1b, Il6, Il10, IL17, C3, C4 and C5 genes by quantitative PCR and promoter DNA methylation of Il1b, Il6, Il10, Il17, C3, C4 and C5 genes by pyrosequencing were carried out in all the study participants.ResultsPlasma levels of IL-1β (Z score= 2.42, p=0.02) dropped significantly and C2 (Z score= 2.24, p=0.03) levels increased after 12-weeks of yoga therapy. The expression of Il1b (Z score=2.45, p=0.01) and Il6 (Z score=2.07, p=0.04) genes were significantly downregulated, while the levels of C4 (Z score=2.23, p=0.03) gene was upregulated in schizophrenia patients of yoga therapy group. Two CpG sites in the promoter region of Il1b (all p≤0.05) and Il6 (all p≤0.05) genes and three CpG sites in the promoter region of C4 (all p&lt;0.05) gene were hypermethylated, while two CpG sites in the gene body of Il6 (all p≤0.05) gene and two CpG sites in the promoter region of Il10 (all p ≤0.05) gene were hypomethylated after 12-weeks of yoga therapy in schizophrenia patients.ConclusionsOur findings provide important insights into the mode of action of yoga therapy in schizophrenia. This study for the first time reports the epigenetic effects of yoga therapy on immune-inflammatory pathway in schizophrenia.Disclosure of InterestNone Declared

Porphyromonas gingivalis Strain W83 Infection Induces Liver Injury in Experimental Alcohol-Associated Liver Disease (ALD) in Mice

The liver plays a vital role in the defense against infections. Porphyromonas gingivalis (P. gingivalis), a dominant etiologic oral bacterium implicated in periodontal disease (PD), has been associated with various systemic diseases. This study aimed to investigate the influence of P. gingivalis on alcohol-associated liver diseases (ALD). Mice were fed a Lieber–DeCarli liquid diet containing 5% ethanol for 10 days after an initial adaptation period on a diet with lower ethanol content for 7 days. Two days before tissue sample collection, the mice were administered P. gingivalis strain W83 (Pg) through intraperitoneal injection (IP). Pair-fed mice with Pg infection (PF+Pg) exhibited an activated immune response to combat infections. However, alcohol-fed mice with Pg infection (AF+Pg) showed liver injury with noticeable abscess lesions and elevated serum alanine aminotransferase (ALT) levels. Additionally, these mice displayed liver infiltration of inflammatory monocytes and significant downregulation of proinflammatory cytokine gene expression levels; and AF+Pg mice also demonstrated increased intrahepatic neutrophil infiltration, as confirmed by chloroacetate esterase (CAE) staining, along with elevated gene expression levels of neutrophil cytosol factor 1 (Ncf1), neutrophilic inflammation driver lipocalin 2 (Lcn2), and complement component C5a receptor 1 (C5ar1), which are associated with neutrophilic inflammation. Interestingly, compared to PF+Pg mice, the livers of AF+Pg mice exhibited downregulation of gene expression levels of NADPH oxidase 2 (Cybb), the leukocyte adhesion molecule Cd18, and the Toll-like receptor adaptor Myd88. Consequently, impaired clearance of P. gingivalis and other bacteria in the liver, increased susceptibility to infections, and inflammation-associated hepatic necrotic cell death were observed in AF+Pg mice, which is likely to have facilitated immune cell infiltration and contributed to liver injury. Furthermore, in addition to the Srebf1/Fasn pathway induced by alcohol feeding, Pg infection also activated carbohydrate response element-binding protein (ChREBP) in AF+Pg mice. In summary, this study demonstrates that P. gingivalis infection, acting as a “second hit”, induces dysfunction of immune response and impairs the clearance of bacteria and infections in alcohol-sensitized livers. This process drives the development of liver injury.

Expression Dynamics Of Cytokine Genes Is Related To The Apremilast Treatment Effectiveness In Patients With Severe Psoriasis

Background — Psoriasis is an immune-mediated genetic skin disease with a deregulated immune response governed by a proinflammatory cytokine network. Apremilast has demonstrated high safety and tolerability both in clinical trials and in clinical practice. The effectiveness of the apremilast use in clinical practice may differ from major clinical trials. Our study assessed changes in the levels of immune gene expression in patients suffering from severe psoriasis in the course of apremilast treatment in order to investigate the predictors of its effectiveness. Methods — We assessed the expression levels of IFNγ, IRF3, GLIS1, HR, STAT1, STAT3, VEGFA, ICAM1, TNF, IL1α, IL1β, IL4, IL6, IL10, IL11, IL12B, IL17A, IL17F, IL18, IL20, IL21, IL22, IL23A, IL25, IL31, IL33 genes in both lesional and nonlesional skin before the treatment, as well the expression at lesional skin after the treatment. RNA expression was assessed in skin biopsy samples by RT-PCR using TaqMan probes with StepOne5 equipment and normalized with endogenous control. The study included 16 patients diagnosed with a moderate-to-severe or severe psoriasis using clinical examination by a dermatologist. The clinical outcome after 26 weeks of apremilast treatment was assessed with delta PASI, resulting in a patient group with high effectiveness of treatment (delta PASI&gt;75%) and a group including all other patients. Results — We confirmed elevated levels of expression in STAT1, IFNγ, IL1β, IL12B, IL17A, IL17F, IL20, IL21, IL22, and IL23A genes in lesional vs. nonlesional psoriatic skin samples, while GLIS1 gene expression was reduced. The expression levels of cytokine genes after apremilast treatment decreased considerably in cytokines IFNγ, IL1β, IL20, IL21, and IL22; and to a lesser extent in STAT1, IL6, IL17F, IL22 and IL31. In the group of those who effectively responded to treatment with apremilast, a five-to-eleven-fold reduction in the expression level of the IL1B, IL6, and IL17F genes was observed, as compared with other patients. Conclusion — The increased expression of cytokine genes in lesional vs. nonlesional skin was reduced after apremilast treatment of psoriasis. We established that fold changes in the expression of the IL1β, IL6 and IL17F genes during treatment with apremilast were different in groups of patients with different therapy outcomes. Hence, we propose that they are the predictors of the effectiveness of apremilast treatment for severe psoriasis.

Effect of 900 MHz radiofrequency electromagnetic radiation emitted from mobile phone on testicular immunity and the associated risk of testicular germ cell tumor

The emission of radiofrequency electromagnetic radiation (RF-EMR) from mobile phones has been implicated in causing inflammatory changes in the testis. Nevertheless, the direct association of these changes with the development of testicular germ cell tumors (TGCT) remains unclear. Therefore, we purposed to investigate the effect of RF-EMR exposure on inflammatory changes in the testis, cytokine gene expression levels, and the incidence of TGCT. Twenty male Wistar albino rats were randomly assigned to either the study or control groups. The study group was exposed to RF-EMR at 900 MHz, 26 V/m, and a specific absorption rate (SAR) of 0.14 W/kg for 4 h/day over 8 weeks. Histopathological analysis, vitality analysis using Annexin V, and real-time polymerase chain reaction for the analysis of interleukin 1 (IL-1), IL-4, IL-10, tumor necrosis factor-alpha, and interferon-gamma (IFN-&amp;gamma;) cytokine gene expressions were performed on the testicular tissue. The median testis weight (163.0 g [133.0 &amp;ndash; 183.0] vs. 179.0 g [134.0 &amp;ndash; 195.0], P = 0.012) and volume (0.95 cm3 [0.800 &amp;ndash; 1.400] vs. 1.100 cm3 [1.050 &amp;ndash; 1.500], P = 0.031) of the study group were significantly lower compared to the control group. The seminiferous tubule damage (P &lt; 0.001) and interstitial edema (P = 0.042) were significantly higher in the study group. The tunica albuginea thickness was significantly reduced in the study group (P &lt; 0.001). The fold changes in the expression levels of IL-4 and IFN-&amp;gamma; increased significantly in the study group. Our findings indicate that RF-EMR exposure causes structural, histopathological, and inflammatory toxic effects on the testis. The observed elevation in gene expression levels of IL-4 and IFN-&amp;gamma; cytokines following RF-EMR exposure suggests their potential role as regulators of TGCT initiation, thereby offering a viable potential therapeutic target in combination with current treatments. Nonetheless, future well-designed studies are necessary to validate our findings.

Organotypic lung tissue culture as a preclinical model to study host- influenza A viral infection: A case for repurposing of nafamostat mesylate

Reliable and effective models for recapitulation of host-pathogen interactions are imperative for the discovery of potential therapeutics. Ex vivo models can fulfill these requirements as the multicellular native environment in the tissue is preserved and be utilized for toxicology, vaccine, infection and drug efficacy studies due to the presence of immune cells. Drug repurposing involves the identification of new applications for already approved drugs that are not related to the prime medical indication and emerged as a strategy to cope with slow pace of drug discovery due to high costs and necessary phases to reach the patients. Within the scope of the study, broad-spectrum serine protease inhibitor nafamostat mesylate was repurposed to inhibit influenza A infection and evaluated by a translational ex vivo organotypic model, in which human organ-level responses can be achieved in preclinical safety studies of potential antiviral agents, along with in in vitro lung airway culture. The safe doses were determined as 10 µM for in vitro, whereas 22 µM for ex vivo to be applied for evaluation of host-pathogen interactions, which reduced virus infectivity, increased cell/tissue viability, and protected total protein content by reducing cell death with the inflammatory response. When the gene expression levels of specific pro-inflammatory, anti-inflammatory and cell surface markers involved in antiviral responses were examined, the significant inflammatory response represented by highly elevated mRNA gene expression levels of cytokines and chemokines combined with CDH5 downregulated by 5.1-fold supported the antiviral efficacy of NM and usability of ex vivo model as a preclinical infection model.

Study of IL-17 and Intercellular Adhesion Molecule-1 in Conjunctivochalasis Using Correlation Analysis.

The aim of this study was to observe the expression of interleukin (IL)-17 and intercellular adhesion molecule (ICAM)-1 in conjunctivochalasis (CCH) and to analyze the correlations between cytokines and the severity of CCH. Serum samples were collected from 22 patients with CCH and 18 normal controls (NCs). The Ocular Surface Disease Index, tear film break-up time, Schirmer I test, and corneal fluorescein staining were used to evaluate the ocular surface signs and symptoms. The concentrations of IL-17, IL-23, and ICAM-1 in serum and cellular supernatants were measured by enzyme-linked immunosorbent assays, and the gene expression levels of cytokines were measured by a quantitative real-time polymerase chain reaction. The relationships between serum concentrations of IL-17, IL-23, and ICAM-1 with clinical ocular surface parameters in CCH were analyzed using the Spearman correlation analysis. The concentrations of IL-17 and ICAM-1 in serum and cellular supernatants of CCH were significantly higher than those of NCs (all P < 0.001). The concentrations of IL-23 in serum and cellular supernatants of CCH showed no significant difference from those of NCs ( P > 0.05). The mRNA expression levels of IL-17 and ICAM-1 in conjunctival fibroblasts of CCH were significantly higher than those of NCs (all P < 0.001). The mRNA expression of IL-23 in conjunctival fibroblasts of CCH was higher than that of NCs, without a significant difference ( P > 0.05). Furthermore, the serum concentrations of IL-17 and ICAM-1 were positively correlated with Ocular Surface Disease Index and fluorescein staining (all P < 0.05), and negatively correlated with break-up time and Schirmer I test of CCH (all P < 0.05). The expression levels of IL-17 and ICAM-1 were significantly increased in CCH serum and associated with the disease severity. We postulate that IL-17 and ICAM-1 may play a role in the pathogenesis of CCH. IL-17 and ICAM-1 antagonists may be a potential treatment option for CCH in the future.

T Cell-Induced Colitis Is Exacerbated by Prolonged Stress: A Comparison in Male and Female Mice.

Psychological stress exposure is well recognized to exacerbate inflammatory bowel disease (IBD) but the mechanisms involved remain poorly understood. In this study, chronic T cell-mediated colitis was induced by adoptively transferring CD4+CD45RBhigh splenic T cells from C57BL/6 WT donor mice into Rag1tm1Mom mice. Two weeks after T cell transfer, mice were exposed to a prolonged restraint stressor (RST) for 8 h per day for 6 consecutive days. The colitis phenotype was assessed via histopathology and semi-quantitative rt-PCR at humane endpoints or 10 weeks post-T-cell transfer. Mice that received the T cell transplant developed chronic colitis marked by increases in colonic histopathology and inflammatory cytokines. Colonic histopathology was greater in males than females regardless of RST exposure but RST exposure increased histopathology scores in females such that they reached scores observed in the males. This pattern was consistent with cytokine gene expression and protein levels in the colon (especially for IFN-γ, IL-17A, and TNF-α). Serum cytokine levels were not strongly affected by exposure to the stressor. Using a murine model of chronic T cell-mediated colitis, this study demonstrates that biological sex strongly influences colonic inflammation and exposure to chronic stress has a more pronounced effect in females than in males.

Investigation of apoptotic and inflammatory markers in hypoxia induced human cornea epithelial cells co‐cultured with limbal mesenchymal stem cells

Aims/Purpose: Here we aim to investigate the effect of hypoxic conditions (5% O2) on human cornea epithelial cells (HCECs) and also by creating a system in which limbal mesenchymal stem cells (LMSCs) are co‐cultured with HCECs, the effect of LMSC is examined on this subject via paracrine mechanism.Methods: HCECs are either mono‐cultured or co‐cultured with LMSC under hypoxic and normoxic (%21 O2) conditions. HCECs of after 6, 24, and 48 h of incubation were harvested for comprehension of changes in RNA and protein expression levels. Bcl2, Bax, Erk1/2 protein levels are checked through western blot analysis, whereas expression levels of cytokines, which are IL‐6, IL‐8, IL‐10 and TGF‐β1, are investigated via RT‐qPCR.Results: Both anti‐inflammatory and pro‐inflammatory cytokine gene expression levels in co‐cultured model increased upon hypoxia and normoxia conditions compared to cytokines in mono cultured HCECs. In addition to the increase in cytokine levels in the co‐culture condition, this increase is less in the hypoxia exposed co‐culture in comparison to the normoxic co‐culture model. Furthermore, anti‐ and pro‐apoptotic protein expression levels have significantly changed upon co‐cultured both in normoxia and hypoxia conditions.Conclusions: In conclusion, it is observed that hypoxia with co‐cultured model decreased inflammation and acted as crucial role in cell death mechanism. These results were further confirmed with apoptotic protein ratio in hypoxic co‐cultured cells. The underlying mechanism will be examined in future experiments.