Enzyme-linked Immune Sorbent Assay Research Articles

Vascular Endothelial Growth Factor Levels in the Gingival Crevicular Fluid of Type II Diabetes Mellitus Patients with and without Chronic Periodontitis – A Clinico-Biochemical Study

Background: Vascular endothelial growth factor (VEGF) is a multifunctional angiogenic cytokine that plays a central role in inflammation and wound healing. The role of this angiogenic factor in periodontal destruction could be significant, and VEGF could act as a potent marker of periodontal disease progression. Its role in diabetes mellitus-related complications has been evaluated, and diabetes mellitus may be a potent modulator of VEGF in periodontal disease. Objective: The objective of this study is to evaluate and compare gingival crevicular fluid (GCF) VEGF levels of systemically healthy, chronic periodontitis, and Type II diabetes mellitus participants with and without chronic periodontitis and to investigate the role of VEGF in periodontal disease progression. Materials and Methods: Eighty participants were divided into four groups based on the gingival index, plaque index, probing pocket depth, and clinical attachment level: (Group 1) healthy, (Group 2) chronic periodontitis, (Group 3) Type II diabetes mellitus without chronic periodontitis, and (Group 4) Type II diabetes mellitus with chronic periodontitis. GCF samples collected from each subject were quantified for VEGF levels using an enzyme-linked immune sorbent assay. Further, the correlation between VEGF levels within groups and with the clinical parameters was analyzed in all groups. The statistical analysis was performed using the SPSS software. Results: The mean concentration of VEGF in GCF was the highest in Group 4 (2179.24 pg/ml) followed by Group 3 (1948.32 pg/ml), Group 2 (1776.83 pg/ml), and the least in Group 1 (1266.80 pg/ml). Further, GCF VEGF levels showed a positive correlation with all of the clinical parameters. Conclusions: VEGF concentrations increased from health to disease. Type II diabetes mellitus may affect VEGF concentrations in periodontal disease. These data indicate that VEGF plays a key role in periodontal disease progression and can be considered a biomarker of periodontal disease progression.

AuNP Coupled Rapid Flow-Through Dot-Blot Immuno-Assay for Enhanced Detection of SARS-CoV-2 Specific Nucleocapsid and Receptor Binding Domain IgG.

BackgroundSerological tests detecting severe acute respiratory syndrome coronavirus−2 (SARS-CoV-2) are widely used in seroprevalence studies and evaluating the efficacy of the vaccination program. Some of the widely used serological testing techniques are enzyme-linked immune-sorbent assay (ELISA), chemiluminescence immunoassay (CLIA), and lateral flow immunoassay (LFIA). However, these tests are plagued with low sensitivity or specificity, time-consuming, labor-intensive, and expensive. We developed a serological test implementing flow-through dot-blot assay (FT-DBA) for SARS-CoV-2 specific IgG detection, which provides enhanced sensitivity and specificity while being quick to perform and easy to use.MethodsSARS-CoV-2 antigens were immobilized on nitrocellulose membrane to capture human IgG, which was then detected with anti-human IgG conjugated gold nanoparticle (hIgG-AuNP). A total of 181 samples were analyzed in-house. Within which 35 were further evaluated in US FDA-approved CLIA Elecsys SARS-CoV-2 assay. The positive panel consisted of RT-qPCR positive samples from patients with both <14 days and >14 days from the onset of clinical symptoms. The negative panel contained samples collected from the pre-pandemic era dengue patients and healthy donors during the pandemic. Moreover, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of FT-DBA were evaluated against RT-qPCR positive sera. However, the overall efficacies were assessed with sera that seroconverted against either nucleocapsid (NCP) or receptor-binding domain (RBD).ResultsIn-house ELISA selected a total of 81 true seropositive and 100 seronegative samples. The sensitivity of samples with <14 days using FT-DBA was 94.7%, increasing to 100% for samples >14 days. The overall detection sensitivity and specificity were 98.8% and 98%, respectively, whereas the overall PPV and NPV were 99.6% and 99%. Moreover, comparative analysis between in-house ELISA assays and FT-DBA revealed clinical agreement of Cohen’s Kappa value of 0.944. The FT-DBA showed sensitivity and specificity of 100% when compared with commercial CLIA kits.ConclusionThe assay can confirm past SARS-CoV-2 infection with high accuracy within 2 minutes compared to commercial CLIA or in-house ELISA. It can help track SARS-CoV-2 disease progression, population screening, and vaccination response. The ease of use of the assay without requiring any instruments while being semi-quantitative provides the avenue of its implementation in remote areas around the globe, where conventional serodiagnosis is not feasible.

ROLE OF INTERLEUKIN-2, INTERLEUKIN-4 AND CLUSTER OF DIFFERENTIATION-22 AS AN IMMUNE MARKERS IN INDIVIDUALS INFECTED WITH Helicobacter pylori

Helicobacter pylori is a gram-negative, intracellular, microaerophilic bacteria which causing Peptic ulcer. This bacterium can change its shape which helps the bacteria to survive in the host gastric microenvironment. The Peptic ulcer caused by this bacterium stimulates the humoral and cellular immune response in individuals. The current study was carried out to access the role of interleukin-2, interleukin-4, and cluster differentiation-22 as immune markers in the identification of H. pylori infection. The presence of H. pylori has been diagnosed by feces test (antigen rapid test). In this study, the presence of three immunological markers viz., IL-2, IL-4, and CD22 were measured in the serum of 60 individuals infected with H. pylori and 30 healthy individuals by the Enzyme-Linked Immune-sorbent Assay method. Results of this study indicated a significant increase (P-value=0.0307*) in the concentration of IL-2 (294.27ng/ml), IL-4(151.28ng/ml), and CD22 (492.73ng/ml) in the serum of individuals infected with H. pylori while these concentrations were reported 235.98ng/ml, 116.14ng/ml and 369.33ng/ml respectively in the healthy individuals. Results of the study can be concluded that H.pylori infection stimulates the Cellular and humoral immune response which resulted in the increased production of IL-2, IL-4, and CD22.

Estimation of Serum Vitamin D2, Growth Hormone, Alkaline Phosphatase and Calcium Phosphate Product in Patients with End Stage Renal Disease

INTRODUCTION&#x0D; The chronic kidney disease (CKD) patient's calcium phosphate product, alkaline phosphatase (ALP), vitamin-D2 and human growth hormone (hGH) are altered under haemodialysis. This study aimed to evaluate these biochemical variables in conjunction with haemoglobin and blood pressure to find out their association in End Stage Renal Disease (ESRD) patients.&#x0D; MATERIAL AND METHODS&#x0D; This cross-sectional study comprised of 104 patients with ESRD undergoing haemodialysis. The estimated glomerular filtration rate (eGFR) was calculated by Cockcroft-Gault (CG) equation and calcium, phosphorus, ALP were measured by fully automated analyzer whereas vitamin-D2 and hGH were measured by sandwich and competitive enzyme linked immune sorbent assay (ELISA) techniques.&#x0D; RESULTS&#x0D; The mean age of patients was 53.12 ±16.37 years comprising 68% male. The hypovitaminosis D was 57.7% deficiency and 23.1% insufficiency states whereas hGH insufficiency was 22.1%. The calcium phosphate product was found to be increased in only 39.9% cases. The increased ALP level was observed in 64.4% cases. There was statistically significant association between hGH and Hb status (p=0.03). The significant difference in mean sodium and Ca×P of ESRD cases was observed with hypertension status (p=0.03 and p=0.01) respectively. Moreover, the significant difference in mean eGFR and hGH was observed with haemoglobin status (p=0.0001and p=0.01) respectively.&#x0D; CONCLUSION&#x0D; Increased level of ALP and hypovitaminosis-D was very common in ESRD patients undergoing dialysis with less prevalence of hGH insufficiency and calcium phosphate product increment. The anaemia and hypertension status can be pre-existing condition with ESRD which are cumbersome to control if not monitor in these patients.

Augmented secretion of IL-1α from mouse oral squamous cell carcinoma (OSCC) vcells caused by serum deprivation and hypoxia promotes immune suppressive activity of mesenchymal stromal cells

ObjectivesWe have previously reported that mouse oral squamous carcinoma (OSCC), Sq-1979-1, produces interleukin (IL)-1α, which specifically enhances the immunosuppressive activity of co-cultured mesenchymal stromal 10T1/2 cells. This study assessed the conditions promoting the production of IL-1α in Sq-1979-1 cells, which could further enhance the immunosuppressive function of 10T1/2 cells, and evaluated its expression in OSCC tissues. MethodsThe expression of IL-1α was examined by RT-PCR, western blotting, and enzyme-linked immune sorbent assay (ELISA). The interferon (IFN)- γ-producing capability of anti-CD3 antibody-stimulated mouse spleen cells co-cultured with 10T1/2 cells and conditioned medium (CM) from Sq-1979-1 cells was examined by ELISA. The function of IL-1α was examined using an anti-IL1α antibody. Immunohistochemical analysis of the OSCC tissues was performed. ResultsThe production of IL-1α from Sq-1979-1 cells was synergistically enhanced in lower serum (0.5% or 1.0% FBS) at the transcriptional level, and under hypoxia (1.0% oxygen) at the release level compared to that in the control medium supplemented with 10% FBS under normoxia. The IFN-γ-producing capability of stimulated spleen cells co-cultured with 10T1/2 cells was significantly reduced in the CMs prepared with the lower serum or under hypoxia. These functions of CMs were completely abolished by the anti-IL-1α antibody. The expression of IL-1α in OSCC tissues was prominent in the midst of a carcinomatous cellular lesion or a nearby necrotic lesion, where a supply deficiency could occur. Conclusions: IL-1α production by Sq-1979-1 cells was synergistically augmented under low serum and hypoxic conditions, which could promote the immunosuppressive activity of mesenchymal cells.

Subjective Cognitive Decline May Be Associated With Post-operative Delirium in Patients Undergoing Total Hip Replacement: The PNDABLE Study.

Objective: Subjective cognitive decline (SCD) is associated with an increased risk of clinical cognitive disorders. Post-operative delirium (POD) is a common complication after total hip replacement. We aimed to investigate the relationship between SCD and POD in patients undergoing total hip replacement.Methods: Our study recruited 214 cognitively intact individuals from the Perioperative Neurocognitive Disorder And Biomarker Lifestyle (PNDABLE) study in the final analysis. SCD was diagnosed with Subjective Cognitive Decline Scale (SCDS), Mini-Mental State Examination (MMSE), and Montreal Cognitive Assessment (MoCA). The incidence of POD was evaluated by using Confusion Assessment Method (CAM), and POD severity was measured by using the Memorial Delirium Assessment Scale (MDAS). Preoperative cerebrospinal fluid (CSF) Aβ40, Aβ42, T-tau, and P-tau levels were measured by enzyme-linked immune-sorbent assay (ELISA).Results: Overall, the incidence of POD was 26.64% (57/214), including 32.43% (36/111) in the SCD group and 20.39% (21/103) in the NC group. With the increase of age, the incidence of POD in all age groups increased (P < 0.05). Logistic regression analysis showed that after adjusting for SCD, Aβ42, Aβ40, P-tau, and T-tau, SCD (OR 2.32, CI 1.18–4.55, P = 0.01) and the increased CSF level of P-tau (OR 1.04, CI 1.01–1.06, P < 0.001) were risk factors for POD, while the level of aβ42 (OR 0.99, CI 0.99–1.00, P < 0.001) was a protective factor for POD.Conclusion: SCD is one of the preoperative risk factors for POD.Clinical Trial Registration: This study was registered at China Clinical Trial Registry (Chictr200033439).

Estimation of HsCRP and Cardiac Troponin I in Ischemic Heart Disease Iraqi Patients

The present study is an attempt to investigate the changes in serum cardiac troponin (cTn) and HighsensitivityC-reactive protein (HsCRP) markers levels in Iraqi ischemic heart disease (IHD) patientsincluding stable angina (SA), unstable angina (UA) and myocardial infarction (MI) compared to the healthycontrols (HC), and to evaluate the importance of the HsCRP between these diseases combination groupsfrom the dignostic point of view, in addition to find a significant correlation between HsCRP level and someIHD risk factors. The IHD’s mortality surpass that of every major disease, so it is important that to getsuch research especially among Iraqi patients. IHD is defined as blood flow reduction to the heart muscledue to build-up of fatty strips (plaque) in the coronary arteries, it exists when the coronary blood flowbecomes incapable of both delivering sufficient oxygen (O2) to the myocardium and taking away the carbondioxide (CO2) from the cells. Collecting the blood samples from one hundred thirty IHD patients (during theperiod from October 2020 to February 2021) with ages ranged (30-80) years, of both genders, investigatedfor HsCRP by enzyme linked immune sorbent assay (ELISA) technique and cardiac Troponin (cTn I) byimmunochromography method. This study observed that the HsCRP marker is a reliable diagnostic indicatorin both MI and UA groups (but not in SA patients), and in all diseases combination groups, and that therewas significant correlations between each of the triglyceride level and the hypertension, and the HsCRPmarker level, only in the MI group, but not in SA and UA patients.

Development of a new EGFR antibody antagonist which exhibits potential biological effects against laryngeal cancer.

BackgroundLaryngeal cancer is a common malignant tumor of the head and neck. Clinical treatment methods mainly include radiotherapy and chemotherapy, but the toxicity and side effects of these treatments seriously affect the quality of life of patients. Currently, there are no specific anti-laryngeal cancer drugs available. Therefore, it is necessary to develop new targeted drugs for laryngeal cancer.MethodsWe established a cell model of laryngeal cancer in vitro and a TU686 xenograft model in vivo. We then carried out the related research through a series of experiments [including laser confocal microscopy, enzyme linked immune sorbent assay (ELISA) and Western blot].ResultsThe results showed that the epidermal growth factor receptor (EGFR) antibody antagonist 6E-C could not only specifically bind to EGFR, but also specifically inhibit the binding of EGF to EGFR. Further analysis indicated that 6E-C could inhibit the EGFR-mediated intracellular signaling pathway. Furthermore, 6E-C inhibited xenograft tumor growth in vivo.ConclusionsIn summary, we have successfully prepared a new anti-EGFR antibody antagonist, which exhibited anti-laryngeal cancer effects in vitro and in vivo. The current research demonstrates that the EGFR antibody antagonist 6E-C shows potential as an effective anti-laryngeal cancer agent, with potential clinical application value. This study therefore provides a solid foundation for related research in the future.

"Adjusting Internal Organs and Dredging Channel" Electroacupuncture Ameliorates Insulin Resistance in Type 2 Diabetes Mellitus by Regulating the Intestinal Flora and Inhibiting Inflammation.

IntroductionTraditional Chinese acupuncture has been demonstrated to be beneficial for treatment of type 2 diabetes mellitus (T2DM). The mechanism of acupuncture on T2DM is crucial for their biological activity as well as their usefulness as tools in biology and medicine. However, its mechanism is poorly understood.MethodsIn an effort to explore the mechanism, eight db/db mice (a type of spontaneous T2DM mouse) were treated with adjusting internal organs and dredging channel electroacupuncture (AODCEA) for 2 weeks. Another eight db/db mice were fed as T2DM group (T2DMG), and eight db/m mice were set as normal control group (NCG). Lipopolysaccharide (LPS), interleukin-6 (IL-6), and diabetes-related indicators, such as fasting blood glucose (FBG) and triglyceride (TG), were detected by enzyme-linked immune sorbent assay (ELISA). The V4 region of 16S rRNA gene was analyzed by Illumina sequencing to evaluate the effect of AODCEA on intestinal flora. The amount of short-chain fatty acids (SCFAs) in the feces were determined by gas chromatography-mass spectrometry (GC-MS).ResultsOur results indicate that AODCEA treatment can reduce diabetes-related indicators. We observed the increased probiotics such as Blautia and Lactobacillus and decreased opportunist pathogens (Alistipes, Helicobacter, Prevotella) by AODCEA interventions. Importantly, the total amount of SCFAs in the feces of T2DM mice was promoted by AODCEA. Finally, obviously alleviated systemic inflammation was exhibited through AODCEA treatment by detection of lipopolysaccharide (LPS) and interleukin-6 (IL-6) in serum.ConclusionAODCEA can reshape the structure of intestinal flora, which can increase intestinal SCFAs, affect the circulating LPS level, and reduce the inflammatory response.

Study of Glypican-3 in Patients with Hepatitis C Virus-Related Hepatocellular Carcinoma

Abstract Background: Early diagnosis and treatment of HCC can greatly improve the efficiency of treatment and extend patient life. Aim of Study: To investigate the expression of serum Glypican-3 (GPC3) in patients with Hepatocellular Carcinoma (HCC) and determine its efficacy as a screening test in early detection of HCC. Patients and Methods: This case control study involved 30 HCC patients, 30 liver cirrhotic patients and 20 healthy controls. This study had been approved by local institutional research board in Menoufia Faculty of Medicine. All subjects participated in the study voluntarily and written informed consent was obtained from each participant. Clinical exami-nation, abdominal ultrasonography and triphasic Computed Tomography (CT) for focal lesion were performed. Liver function tests were performed using clinical auto-analyzer, serum a-Fetoprotein (AFP) was measured using Enzyme-linked Immune-Sorbent Assay (ELISA) method and GPC3 was determined by ELISA kit for GPC3. Data were collected and statistically analyzed. Results: GPC3 was highly significant higher in HCC group than cirrhotic and control groups. There was highly positive significant correlation between GPC3 and child score, size of focal lesions and number of focal lesions. The sensitivity of GPC3 in diagnosis of HCC was (68.5%) and the specificity was (83.3%) at cut off point (58.2ng/ml) that elicited from the Receiver Operator Characteristic (ROC) curve with very good Area Under Curve (AUC) (0.814), whereas that the sensitivity of AFP was (66.7%) and the specificity was (66.7%) at cut off point (380ng/ml) that elicited from the ROC curve with very good AUC (0.679). Conclusion: GPC3 is highly associated with HCC and is more sensitive than AFP for early detection of HCC.

Rapid identification, isolation, and evaluation on anti-neuroinflammatory activity of limonoids derivatives from the root bark of Dictamnus dasycarpus

A total of 49 limonoids derivatives were rapidly identified by UNIFI software and three new limonoids derivatives, named dasycarinone (1, DAS), isodictamdiol C (2) and dasycarinone A (3), along with nineteen known compounds, were isolated from the root bark of Dictamnus dasycarpus, named as “Baixianpi” in Chinese. Their structures were elucidated on the basis of spectroscopic data (UV, IR, HR-ESI-MS, NMR, CD spectra and OR). All the compounds were tested for anti-inflammatory activities by suppressing the nitric oxide (NO) production in lipopolysaccharide (LPS) induced BV-2 cells. DAS exhibited a strong anti-inflammatory activity with IC50 value of 1.8 μM. Nuclear Factor kappa B (NF-κB) luciferase assay and enzyme-linked immune sorbent assay indicated that DAS can suppress the release of inflammatory cytokines such as Tumor Necrosis Factor α (TNF-α), interleukin 6 (IL-6) via inactivating NF-κB signaling pathways. Moreover, we found that anti-inflammatory activities of obacunone-class are better than those of limonin-class by analyzing structure-activity relationship. Our results suggested that obacunone derivatives play an important role on anti-inflammation of Baixianpi. As a representative among them, DAS showed a strong anti-inflammatory activity via suppressing NF-κB signaling pathways.

Sitagliptin inhibits lipopolysaccharide-induced inflammatory response in human gingival fibroblasts by blocking nuclear factor-κB signaling pathway

This study was performed to clarify the effects of sitagliptin on Porphyromonas gingivalis-lipopolysaccharide (LPS)-induced inflammatory response in human gingival fibroblasts (HGFs), explore the molecular mechanism of its roles, and provide a foundation for clinical therapeutics in periodontitis. Healthy gingival samples were collected from the donors. HGFs were isolated with enzymic digestion method and identified. The effects of LPS and sitagliptin on cell viability were detected by cell-counting kit-8 (CCK8). The mRNA levels of inflammatory cytokines, namely, interleukin (IL)-6, IL-8, C-C motif ligand 2 (CCL2), and superoxide dismutase 2 (SOD2), were evaluated by quantity real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immune sorbent assay (ELISA) was used to measure the secretion protein levels of IL-6, IL-8, and CCL2. Western blot analysis was used to further investigate the activation of nuclear factor (NF)-κB signaling pathway. The effect of NF-κB pathway inhibitor BAY11-7082 on LPS-induced HGF inflammatory cytokines at the gene level was verified by qRT-PCR. Low concentrations of sitagliptin (0.1, 0.25, and 0.5 µmol·L-1) did not affect HGF growth in 24 and 48 h, whereas high concentrations of sitagliptin (5-1 000 µmol·L-1) significantly inhibited cell proliferation. Sitagliptin suppressed 5 µg·mL-1 of LPS-induced IL-6, IL-8, CCL2, and SOD2 gene expression levels in HGF in a concentration-dependent manner. Furthermore, sitagliptin significantly decreased the elevated secretion of IL-6, IL-8, and CCL2 protein induced by LPS. Western blot analysis showed that 0.5 µmol·L-1 of sitagliptin significantly inhibited LPS-induced NF-κB signaling pathway activation. Results of qRT-PCR analysis indicated that 0.5 µmol·L-1 of sitagliptin and 5 µmol·L-1 of BAY11-7082 significantly inhibited LPS-induced IL-6, IL-8, CCL2, and SOD2 gene expressions. Sitagliptin could significantly inhibit LPS-induced HGF inflammatory response by blocking the NF-κB signaling pathway activation.

Fluorescence polarization assay improves the rapid detection of human brucellosis in China

BackgroundBrucellosis is an infectious-allergic zoonotic disease caused by bacteria of the genus Brucella. Early diagnosis is the key to preventing, treating, and controlling brucellosis. Fluorescence polarization immunoassay (FPA) is a new immunoassay for relatively rapid and accurate detection of antibodies or antigens based on antigen–antibody interaction. However, there is no report on FPA-based detection of human brucellosis in China. Therefore, this study is to evaluate the value of FPA for the diagnosis of human brucellosis in China.MethodsWe recruited 320 suspected brucellosis cases who had the clinical symptoms and epidemiological risk factors between January and December, 2019. According to China Guideline for Human Brucellosis Diagnosis, the Rose Bengal test (RBT) was used for the screening test, and the serum agglutination test (SAT) was used as the confirmatory test. Brucellosis was confirmed only if the results of both tests were positive. Additionally, FPA and enzyme linked immune sorbent assay (ELISA) were compared with SAT, and their sensitivity, specificity, coincidence rate and consistency coefficient (Kappa value) as diagnostic tests were analyzed individually and in combination. The optimal cut-off value of FPA was also determined using the receiver operator characteristic (ROC) curve.ResultsThe optimum cut-off value of FPA was determined to be 88.5 millipolarization (mP) units, with a sensitivity of 94.5% and specificity of 100.0%. Additionally, the coincidence rate with the SAT test was 96.6%, and the Kappa value (0.9) showed excellent consistency. The sensitivity and specificity of FPA and ELISA combined were higher at 98.0% and 100.0% respectively.ConclusionsWhen the cut-off value of FPA test is set at 88.5 mP, it has high value for the diagnosis of brucellosis. Additionally, when FPA and ELISA are combined, the sensitivity of diagnosis is significantly improved. Thus, FPA may have potential in the future as a diagnostic method for human brucellosis in China.Graphic abstract

Role of interleukin-2 and interleukin-18 in newly diagnosed type 2 diabetes mellitus.

The study aimed to compare the levels of anti-inflammatory interleukin-2 (IL-2) and proinflammatory interleukin-18 (IL-18) among newly diagnosed type 2 diabetes mellitus (T2DM) and nondiabetic volunteers, to predict their roles as markers in the diagnosis of newly diagnosed T2DM. In the study, 60 subjects were enrolled (30 T2DM cases and 30 non-diabetic controls). Biochemical parameters such as fasting plasma glucose (FBS), glycated haemoglobin (HbA1c), high sensitivity C-reactive protein (hs-CRP) and lipid profile were estimated in auto-analyser. Serum IL-2 and IL-18 levels were assessed by enzyme-linked immune sorbent assay (ELISA). Significant differences were observed in the levels of interleukins among study groups. The median (95% confidence interval) of IL-2 in cases and controls were 8.55 (6.07-47.23) and 45.87 (12.81-145.4) (p=0.02). The median (95% CI) of IL-18 on the other hand in cases and controls were 691.6 (580.3-872.6) and 511.1 (452.6-557.5) (p=0.0014). Our study is the first to correlate IL-2 and IL-18 in newly diagnosed T2DM patients. Findings from this study highlight the anti-inflammatory role of IL-2 and proinflammatory role of IL-18 in T2DM. ROC analysis helped predict their role as markers in T2DM diagnosis.

Punicalagin inhibits pro-inflammatory cytokines induced by influenza A virus

IntroductionInfluenza A virus causes severe pneumonia in humans, leading to high morbidity and mortality. Studies have shown that an excessive immune response in overproduction of pro-inflammatory cytokines can exacerbate complications of influenza virus infection. Thus immune-targeted therapies are an appropriate strategy to control the disease. This study was conducted to investigate the effect of punicalagin on expression and concentration levels of tumor necrosis factor alpha (TNFα), interleukin 6 (IL-6) and interleukin1-β in Madin-Darby Canine Kidney (MDCK) cells. MethodsIn this experimental study, confluent MDCK cells were infected with influenza A virus (H1N1; PR8) and treated with punicalagin at different concentrations. After incubation, mRNA expression and concentration levels of TNFa, IL-6 and IL1-β were investigated using real-time polymerase chain reaction (PCR) and enzyme-linked immunesorbent assay (ELISA), respectively. ResultsThe results of quantitative Real-time PCR showed a significant increase in TNFa, IL-6 and IL1-β mRNA expression in control virus compared to cell control, and a significant, dose-dependent decrease in these expression levels in punicalagin-treated virus infected cells compared to control virus (P < 0.05). The ELISA results also confirmed the dose-dependent decrease in TNFa, IL-6 and IL1-β concentrations in protein levels in punicalagin treated, virus infected cells compared to control virus (P < 0.05). ConclusionsOur results demonstrated the anti-inflammatory effects of punicalagin, and therefore suggests that the compound can be used as an appropriate treatment of choice to control cytokines-induced inflammation in influenza A virus infection.

Hypoxic stimulation leads to the changes of inflammatory factors in mucosal epithelial cells of nasal polyps

Objective: To investigate the roles of hypoxic stimulation in the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP) by comparing the variation and differences of inflammatory factors secreted from epithelial cells of nasal polyps and normal nasal mucosa under hypoxic stimulation. Methods: Sixty-eight patients who were diagnosed with CRSwNP from June 2015 to January 2018 at China-Japan Union Hospital of Jilin University were analyzed, including 36 males and 32 females, aged (45.2±12.5) years. Nasal polyps mucosa was included in CRS-NP group and inferior turbinate mucosa was included in CRS-IT group. According to the degree of eosinophil infiltration in histopathologic results, each of these two groups was further divided into eosinophil infiltration and non-eosinophil infiltration as Eos-NP group (n=34), Non-Eos-NP group (n=34), Eos-IT group (n=20) and Non-Eos-IT group (n=20). The inferior turbinate mucosa of twenty-five patients who were diagnosed with cyst of paranasal sinus or deviation of nasal septum was classified as control group (n=25), including 14 males and 11 females, aged (42.8±10.2) years. The expression of interleukin 17A (IL-17A), interferon γ (IFN-γ), tumor necrosis factor α (TNF-α) and hypoxia-inducible factor 1α (HIF-1α) in each group was analyzed by immunohistochemical staining. After 0, 24 and 48 h hypoxic stimulation, the secretion of IL-17A, IFN-γ, TNF-α in primary nasal mucosa epithelial cells of each group was tested by enzyme-linked immune sorbent assay (ELISA) experiment; the expression of HIF-1α was tested by immunofluorescent staining and imaging and Western blot. SPSS 17.0 software and two-way ANOVA were used for statistical analysis. Results: Immunohistochemical staining showed that the expression of IL-17A and TNF-α was much higher in control group (optical density (OD) value was 0.37±0.03, 0.53±0.02, respectively) and the expression of IFN-γ and HIF-1α was much higher in Eos-IT group (OD value was 0.47±0.03, 0.39±0.02, respectively). The secretion of IL-17A and TNF-α was much lower in control group than that in other groups under normal condition. After 48 h hypoxic stimulation, the secretion of IL-17A and TNF-α was much higher in control group compared with other groups. The secretion of IFN-γ in Eos-NP group was much higher than that in control group under normal condition ((13.7±1.3) pg/ml vs (11.1±1.6) pg/ml, P<0.05). After 48 h hypoxic stimulation, there was no difference of IFN-γ between control group and Eos-NP group. The expression of HIF-1α decreased in Eos-NP group and Non-Eos-NP group while increased in CRS-IT group and control group upon prolonged exposure to hypoxia. HIF-1α was mostly located at cytoplasm of epithelial cells in control and CRS-IT group while mainly located at nucleus of epithelial cells in CRS-NP group. Conclusions: The secretion of IL-17A, TNF-α, IFN-γ and the expression of HIF-1α show significant difference between normal nasal mucosa, polyps and inferior turbinate of CRSwNP under hypoxic stimulation, presenting different subcellular localization. This illustrates the proteins above are involved in transcription and regulation of the gene responsible for the pathogenesis of CRSwNP.

MicroRNA-138-5p Regulates Hippocampal Neuroinflammation and Cognitive Impairment by NLRP3/Caspase-1 Signaling Pathway in Rats.

PurposeNeuroinflammation is an essential causative factor in the pathogenesis and progression of cognitive impairment. The present study aims to evaluate the critical role of microRNA-138-5p (miR-138-5p) in hippocampal neuroinflammation and cognitive impairment through the NLRP3/caspase-1 signaling pathway in rats.Material and MethodsWe established the cognitive impairment rat model and RM (Rat microglia) microglial cellular inflammation model by intracerebroventricular (icv) injection or stimulation of lipopolysaccharide (LPS). Morris water maze (MWM) and Y-maze tests were performed to assess the cognitive behaviors. Quantitative real-time polymerase chain reaction (qRT-PCR), Enzyme-linked immune-sorbent assay (ELISA) and Western blot analysis were utilized to evaluate mRNA or protein expression. Bioinformatic analysis and dual-luciferase reporter gene assay were performed to verify the targeting relationship between NLRP3 and miR-138-5p. Besides, Hematoxylin and eosin (H&E) staining and immunohistochemistry were applied to observe the neuronal morphology and detect the positive cells of the hippocampus, respectively.ResultsCompared to the control groups, LPS-treated rats exhibited significantly impaired learning and memory in MWM and Y-maze tests. The expression of NLRP3, caspase-1 and pro-inflammation cytokines (IL-1β and IL-18) were upregulated, while miR-138-5p was downregulated both in rat hippocampus and RM cells treated with LPS. MiR-138-5p is downregulated in microarray data of cognitive impairment animals and could directly target the 3ʹ-UTR of NLRP3. Furthermore, upregulation of miR-138-5p improved impaired cognitive functions, while inhibited hippocampal neuroinflammation demonstrated by decreased expression of NLRP3/caspase-1 axis, pro-inflammation cytokines and microglial activation. This study demonstrates for the first time that miR-138-5p suppresses the hippocampal NLRP3/caspase-1 signaling pathway activation in cognition impaired rats.ConclusionThe low expression of miR-138-5p after LPS administration may contribute to the activation of the NLRP3/caspase-1 pathway, leading to hippocampal neuroinflammation and cognitive impairment in rat models. These findings indicate a promising therapeutic avenue for cognitive disorders.

English

Contagious caprine pleuropneumonia (CCPP) is an economically important and potentially fatal disease of small ruminants caused by Mycoplasma capricolum subsp. capripneumoniae (Mccp). We designed this cross-sectional study to investigate the seroepidemiology of CCPP in the small ruminants of Oman. For this purpose, we sampled a total of 4015 small ruminants (2119 goats and 1896 sheep) from 510 flocks belonging to different governorates of Oman. A commercial competitive enzyme-linked immune-sorbent assay (cELISA) was used to test the samples. Prevalence (%) along with 95% confidence intervals (CI) was calculated, and a univariable analysis was conducted to screen different risk factors. Furthermore, a binary logistic regression model was built at the animal and flocklevel (Table 3). In total, 147 (28.8%, CI 24.9, 33.0) flocks tested positive for the CCPP and the seroprevalence ranged from 10.0 to 53.8% in various governorates (p = 0.001). The prevalence was significantly (P&lt;0.001) high in goats (28%, CI 23.8, 32.5) as compared to sheep (13.1%, CI 24.9, 33.0). At flock level; the open herds (OR 2.08, 1.33, 3.27), having a location in the coastal regions (OR 1.70, 1.14, 2.53) and flock size of more than 100 animals (OR 2.45, 1.54, 3.91) were the significant risk factors for CCPP in Oman. At the animal level; goats (OR 2.87, CI 2.17, 3.81), and small ruminants above the age of one year (OR 2.23, CI 1.38-3.59) were found more likely to acquire CCPP. We suggest that a control program based on the changes in the management system to minimize the risk factors and a possible mass vaccination should be devised to check CCPP in Oman.

Mac-2 binding protein glycan isomer as noninvasive tool to assess liver fibrosis in children with chronic liver disease.

This study is aimed to measure the value of serum Mac-2 binding protein glycan isomer (M2BPGI) in children with chronic liver diseases in comparison with liver biopsy and serum biomarkers. Comparative cross-sectional study included 100 children with chronic liver diseases and 50 healthy age/sex-matched control group. All subjects were evaluated via medical history, clinical, radiological and laboratory examinations. Liver biopsy was performed for studied patients and serum M2BPGI level was measured by Enzyme Linked Immune Sorbent Assay (ELISA) in all studied subjects. Serum M2BPGI level increased more significantly in chronic liver disease patients (6.04±2.72ng/ml) than in healthy controls (1.12±0.83ng/ml) (P<0.001). M2BPGI level was significantly elevated with progressive fibrosis (P<0.001), and differed significantly between high and low Child-Pugh score, pediatric end-stage liver disease score and model for end-stage liver disease score score. Serum M2BPGI was correlated with serum biomarkers and degree of fibrosis in patients. M2BPGI could be used as one of noninvasive tools for detecting and staging of hepatic fibrosis in Egyptian children with chronic liver disease.

Polystyrene microplastics lead to pyroptosis and apoptosis of ovarian granulosa cells via NLRP3/Caspase-1 signaling pathway in rats

Microplastics (MPs) considered as a new persistent environmental pollutant could enter into the circulatory system and result in decrease of sperm quantity and quality in mice. However, the effects of Polystyrene MPs (PS MPs) on the ovary and its mechanism in rats remained unclear. In this present study, thirty-two healthy female Wistar rats were exposed to different concentrations of 0.5 µm PS MPs dispersed in deionized water for 90 days. Using hematoxylin-eosin (HE) staining, the number of growing follicles was decreased compared to the control group. In addition, the activity of glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD) were decreased while the expression level of malondialdehyde (MDA) was increased in ovary tissue. Confirmed by immunohistochemistry, the integrated optical density of NLRP3 and Cleaved-Caspase-1 had been elevated by 13.9 and 14 in granulosa cells in the 1.5 mg/kg/d group. Furthermore, compared to the control group, the level of AMH had been decreased by 23.3 pg/ml while IL-1β and IL-18 had been increased by 32 and 18.5 pg/ml in the 1.5 mg/kg/d group using the enzyme-linked immune sorbent assay (ELISA). Besides, the apoptosis of granulosa cells was elevated measured by terminal deoxyribonucleotide transferase-mediated nick end labeling (TUNEL) staining and flow cytometry. Moreover, western blot assays showed that the expressions of NLRP3/Caspase-1 signaling pathway related factors and Cleaved-Caspase-3 were increased. These results demonstrated that PS MPs could induce pyroptosis and apoptosis of ovarian granulosa cells via the NLRP3/Caspase-1 signaling pathway maybe triggered by oxidative stress. The present study suggested that exposure to microplastics had adverse effects on ovary and could be a potential risk factor for female infertility, which provided new insights into the toxicity of MPs on female reproduction.