Enzyme-linked Immunoadsorbent Assay Research Articles

Quantitation of hexahydrocannabinol (HHC) and metabolites in blood from DUID cases.

Hexahydrocannabinol (HHC) was first reported in the EU in May 2022. HHC has three chiral carbon atoms, but only (6aR,9R,10aR)-HHC (9R-HHC) and (6aR,9S,10aR)-HHC (9S-HHC) have been encountered in HHC products. The aim of this study was to develop and validate a method for the quantitative analysis of 9R-HHC, 9S-HHC, 11-OH-9R-HHC, 9R-HHC-COOH, 9S-HHC-COOH and 8-OH-9R-HHC. In addition, an objective was to investigate the immunochemical cross-reactivity. Blood samples from driving under the influence of drugs (DUID) cases screened positive for cannabis using enzyme-linked immunoadsorbent assay (ELISA) and confirmed negative for tetrahydrocannabinol (THC), 11-hydroxy-THC and THC-COOH were reanalyzed with a newly validated HHC method to investigate the presence of HHC and metabolites. The LC-MS-MS method was validated for matrix effects, lower limit of quantification (LLOQ), calibration model, precision, bias and autosampler stability. Cross-reactivity on an ELISA method was investigated separately for 9R-HHC-COOH and 9S-HHC-COOH at a concentration range between 5 and 200 ng/mL. The cross-reactivity was found to be 120% for 9R-HHC-COOH and 48% for 9S-HHC-COOH. In the LC-MS-MS method, 9R-HHC-COOH, 9S-HHC-COOH and 11-OH-9R-HHC showed matrix effects <25% at both concentrations, while 8-OH-9R-HHC, 9R-HHC and 9S-HHC matrix effects exceeded 25% at both concentrations but showed good precision (<10% for both inter and intra day) and low bias (<6%) in the further validation. The LLOQ was investigated and established at 0.2 ng/mL for all analytes except the carboxylated metabolites that had an LLOQ of 2.0 ng/mL. The upper LOQ was 20 and 200 ng/mL, respectively. Reanalysis of cases (n = 145) confirmed HHC and metabolites in 32 cases (22%). It was determined that the major metabolite in blood after administration of HHC was 9R-HHC-COOH followed by 11-OH-9R-HHC and that presumptive positive cases are caught by the routine ELISA screening for cannabis.

Association of serum PCSK9 levels with platelet function in patients with acute coronary syndromes.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates low-density lipoprotein (LDL) homeostasis and plays a key role in acute coronary syndrome (ACS). The cardioprotective effect of PCSK9 inhibition extends beyond LDL cholesterol reduction, involving regulation of platelet function by not yet unraveled mechanisms. Oxidized-LDL (ox-LDL) is increased during ACS and induces platelet activation via binding to platelet surface. We will evaluate serum PCSK9 and its correlation with platelet reactivity and platelet-ox-LDL binding in Chinese ACS patients. In this pilot cross-sectional study, we will enroll 115 Chinese participants aged 30 to 75 years with ACS. Blood sample will be obtained after the first maintenance dose of aspirin and clopidogrel during morning time. Serum PCSK9 will be measured by an enzyme-linked immunoadsorbent assay. Platelet reactivity will be assessed by; Platelet activation (P-selectin and GPIIbIIIa expression using flow cytometry) and; Platelet aggregation using light transmission aggregometry in response to various stimuli. On-treatment platelet reactivity is measured by adenosine diphosphate-induced platelet aggregation. Binding of ox-LDL to platelet will be evaluated by flow cytometry. Spearman correlations will be used to determine association of serum PCSK9 with platelet functional parameters and platelet-ox-LDL binding. Additionally, continuous PCSK9 levels will be categorized into tertiles of equal size to investigate its association with on-treatment platelet reactivity. This study will reveal possible relationship between serum PCSK9 and platelet reactivity in the setting of ACS which may shed light on therapeutic potential in platelet inhibition by targeting PCSK9. The study will also explore the association of serum PCSK9 and platelet-ox-LDL binding, an important mechanism for platelet-LDL interplay, to provide mechanistic insight into PCSK9-mediated regulation of platelet reactivity.

Association of Toll-Like Receptor 2 (TLR2) Expression in Placenta and Intrauterine Exposure to Hepatitis B Virus.

Introduction The placenta is a specialized organ that only performs during pregnancy and serves as an immunological barrier in preventing pathogens to reach the fetus. It has been known that toll-like receptors (TLRs) on the placenta respond to antigens, such as zymosan, lipopolysaccharide, and other viral infections. This study analyzes the distribution of TLR2 protein and hepatitis B DNA virus (HBV DNA) virus identification to gain an understanding of hepatitis B viral transmission from the mother to child. Methods We performed enzyme-linked immunoadsorbent assay of HBeAg, HBsAg titer, Anti-HBs, and Anti-HBc to 59 HBsAg-positive pregnant women and identified HBV DNA using nested PCR in their cord blood during delivery for evidence of exposure to HBV DNA. The expression of TLR2 protein in the placenta was performed using immunohistochemistry analysis. Results Intrauterine exposure to the hepatitis B virus occurred in 69.5% of all pregnant women who were HbsAg positive. TLR2 expression was predominantly identified in syncytiotrophoblast and cytotrophoblast cells with the highest score in mothers aged 20–35 years (75%), multigravida (58.3%), and mothers with term pregnancies (70.8%). Statistical analysis results showed that placental TLR2 expression did not indicate any association with hepatitis B virus DNA identified in cord blood with a p value of 0.730 and an OR of 0.650 (95% CI 0.173–2.440). Conclusion TLR2 expression is not associated with intrauterine exposure of hepatitis B virus.

Biochemical and Molecular Characterization of Pichia pastoris Cells Expressing Multiple TMOF Genes (tmfA) for Mosquito Larval Control.

Trypsin modulating oostatic factor (TMOF), a decapeptide hormone synthesized by female mosquito ovaries, ganglia and the central nervous system of Aedes aegypti, terminates trypsin biosynthesis in larvae, and blood-fed female mosquitoes. Earlier, TMOF was cloned and expressed as a single copy in Chlorella dessicata and in Saccharomyces cerevisiae cells as a potential larvicide. Here we report the use of a methylotrophic yeast cells, Pichia pastoris, that efficiently express multi copies of heterologous proteins, that are readily ingested by mosquito larvae. P. pastoris was engineered using pPICZB (Invitrogen, CA, United States), and 2 genes: gfp-tmfA and tmfA inserted between KpnI and XbaI in the multiple cloning site. The plasmid carries a strong AOXI promoter and P. pastoris KM71 and KM71H cells were transformed by homologous recombination. The synthesis of GFP-TMOF was followed using UV and clones were analyzed using southern and Northern blot analyses. Cloning tmfA into KM71H and selection on high Zeocin concentration (2.0 mg/mL) identified a clone that carried 10 copies of tmfA. A comparison between a single and high copy (10 genes) insertions using Northern blot analyses showed that a tmfA transcript was highly expressed even after 120 h. SDS-PAGE analysis of KM71 cells transformed with gfp-tmfA identified a protein band that ran at the expected Mr of 31 kDa. Enzyme Linked Immunoadsorbant Assay (ELISA) analysis of the recombinant cells showed that 1.65 × 108 and 8.27 × 107 cells produce 229 and 114 μM of TMOF, respectively, and caused 100% larval mortality when fed to groups of 5 larvae in 25 mL water. These results indicate that the recombinant P. pastoris cells could be used in the future in the marsh to control mosquito populations.

Tumor characteristics associated with engraftment of patient-derived non-small cell lung cancer xenografts in immunocompromised mice.

Patient-derived xenograft (PDX) models increasingly are used in translational research. However, the engraftment rates of patient tumor samples in immunodeficient mice to PDX models vary greatly. Tumor tissue samples from 308 patients with non-small cell lung cancer were implanted in immunodeficient mice. The patients were followed for 1.5 to approximately 6years. The authors performed histological analysis of PDXs and some residual tumor tissues in mice with failed PDX growth at 1year after implantation. Quantitative polymerase chain reaction and enzyme-linked immunoadsorbent assay were performed to measure the levels of Epstein-Barr virus genes and human immunoglobulin G in PDX samples. Patient characteristics were compared for PDX growth and overall survival as outcomes using Cox regression analyses. Disease staging was based on the 7th TNM staging system. The overall engraftment rate for PDXs from patients with non-small cell lung cancer was 34%. Squamous cell carcinomas had a higher engraftment rate (53%) compared with adenocarcinomas. Tumor samples from patients with stage II and stage III disease and from larger tumors were found to have relatively high engraftment rates. Patients whose tumors successfully engrafted had worse overall survival, particularly those individuals with adenocarcinoma, stage III or stage IV disease, and moderately differentiated tumors. Lymphoma formation was one of the factors associated with engraftment failure. Human CD8-positive and CD20-positive cells were detected in residual samples of tumor tissue that failed to generate a PDX at 1year after implantation. Human immunoglobulin G was detected in the plasma of mice that did not have PDX growth at 14months after implantation. The results of the current study indicate that the characteristics of cancer cells and the tumor immune microenvironment in primary tumors both can affect engraftment of a primary tumor sample.

1909-P: The Platelet-Derived Growth Factor a—An Additional Pathophysiological Factor in Hepatic Steatosis and Fibrosis Development Both in Patients with Hepatitis C and Patients with Prediabetes

Insulin resistance (IR) represents one of the key regulatory mechanisms in the development of non - alcoholic fatty liver disease. It is a common hepatocyte injury in hepatitis C virus (HCV) infection and type 2 diabetes mellitus (T2DM). Higher plasma platelet-derived growth factor A (PDGF- A) concentration is associated with increased hepatic steatosis and fibrosis in T2DM. PDGFs are key regulators of the connective tissue formation and potent mitogens for hepatic stellate cells. We investigated whether there is an association between PDGF-A serum concentration, IR and HCV mediated hepatic steatosis and fibrosis. This cross-sectional study comprised 90 nondiabetic participants aged 47.11±1.54 years divided into 3 groups: HCV negative group with IR (HCV-/IR-, N=12), HCV positive group without IR (HCV+/IR-, N=34), HCV positive group with IR (HCV+/IR+, N=44). The fasting PDGF-A concentration was determined by enzyme-linked immunoadsorbent assay. Homeostatic Model Assessment of IR score over 1.64 was categorised as IR. Fibrosis and steatosis were evaluated by elastography. Steatosis severity accessed by Controlled Attenuation Parameter (p&amp;lt;0.001) had the mean value of 15.9 kPa in the HCV+/IR+ (p&amp;lt;0.001) revealing the highest value in HCV-/IR+ (296.5 dB/m) group. The PDGF-A concentration correlated positively with steatosis (r=0.341, p=0.007) and fibrosis (r=0.264, p=0.071). Both IR positive groups had higher PDGF-A compared to HCV+/IR- group (p=0.006). In accordance with more pronounced steatosis, the HCV-/IR+ group revealed the highest PDGF-A. Concentration of PDGF-A is associated with the severity of hepatic steatosis in HCV and non-HCV patients with IR; i.e., that PDGF-A could represent one of the key regulatory mechanisms in the pathway from hepatic steatosis towards fibrosis. These results provide better understanding of pathways that regulate fibrosis. Disclosure K. Blaslov: None. V. Kokic Males: None. S. Kokic: None. I. Kruljac: None.

Increased circulating levels of vascular endothelial growth factor C can predict outcome in resectable gastric cancer patients.

Neoangiogenesis has proven to be a relevant pathogenetic mechanism in gastric cancer (GC) and lymphatic spread represents an important well-known prognostic factor. Vascular endothelial growth factor C (VEGF-C) plays a key role in lymphangiogenesis and its blood levels in GC patients are easily measurable. This analysis aimed to investigate the prognostic role of preoperative VEGF-C blood levels. VEGF-C serum levels were determined by enzyme-linked immunoadsorbent assay (ELISA) in 186 patients observed at our institution from January 2004 until December 2009 and 82 healthy subjects. Statistical analyses were performed using SPSS 21.0. VEGF-C levels were significantly higher in GC patients (median: 287.4 pg/mL; range, 76.2-865.2 pg/mL) than in the control group (median VEGF-C: 31 pg/mL; range, 12-97 pg/mL). A significant correlation between VEGF-C levels, T, N and tumor stage has been described. The median overall survival (OS) was statistically significantly higher in pts with low serum VEGF-C levels [median: not reached (NR) vs. 26 months; P<0.0001]. Higher preoperative VEGF-C levels correlated also with earlier disease relapse and poor disease-free survival (DFS) (median NR in each subgroup, P=0.005). Furthermore, high VEGF-C levels [hazard ratio (HR) =2.7; P=0.018] and tumor grading (HR =0.44; P=0.007) were independent prognostic factors for OS at multivariate analysis. Our study showed that increased VEGF-C levels are significantly associated with advanced regional lymph node involvement and poor OS and DFS in pts with resected GC paving the way to a possible application as prognostic factor in the clinical practice.

Zyxin expression levels in non-small cell lung cancer patients

Background/Aim. Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related mortality worldwide. Early detection represents one of the most promising approaches to reduce lung cancer mortality. Zyxin (ZYX) is a member of the focal adhesion protein family, recently identified as a potential early diagnostic marker for NSCLC. The aim of this study was to evaluate ZYX expression levels in NSCLC patients and compare its serum expression profiles between early and advanced clinical stages, different histological subtypes and histological grades. Methods. Blood samples were obtained from 90 patients diagnosed with NSCLC in all clinical stages and 30 patients without the clinical and radiological findings and previous history of malignancy. For the quantitative determination of human ZYX concentrations in the serum we used enzyme-linked immunoadsorbent assay (ELISA). Results. ZYX exhibited higher serum levels in NSCLC patients as compared to the control samples with exceptionally significant difference (p = 0.00). The ROC curve demonstrated a high specificity with AUC = 0.912. There were no statistically significant differences in the ZYX values between two most common NSCLC types, adenocarcinoma and squamous cell carcinoma (p = 0.758). There were no statistically significant differences in the ZYX values among different clinical stages (p = 0.518). Only 3 patients had well-differentiated tumor, and no useful data may be extracted from their samples. There were no statistically significant differences in the ZYX values between patients with moderately differentiated tumor and poorly differentiated tumor (p = 0.48). Conclusion. We found that ZYX was overexpressed in NSCLC, but its expression level was not closely correlated with the tumor size and advanced tumor, node, metastasis (TNM) stage. Our results suggest that ZYX has potential to be an early diagnostic plasma-based tumor marker for NSCLC with the same importance for both adenocarcinoma and squamous cell carcinoma.

Effect of emodin on gut microbiota of rats with acute kidney failure

The aim of this paper was to investigate the effect of emodin on gut microbiota in acute kidney injury rats( AKI). Rats were randomly divided into several groups: normal group,model group,low-dose of emodin group( 10 mg·kg~(-1)),medium-dose of emodin group( 25 mg·kg~(-1)),high-dose of emodin group( 50 mg·kg~(-1)) and control group( 5 mg·kg~(-1) of benazepril hydrochloride).The AKI model rats were established by intraperitoneal injection of small dose of gentamicin sulfate for 7 days. Two hours after intraperitoneal injection,except for the normal group and the model group,the other groups were given corresponding doses of drugs for 15 days. The serum levels of serum creatinine( SCr),urea nitrogen( BUN),plasma endotoxin level,24 h urinary protein and D-lactate in the plasma were determined by sarcosine oxidase,urease method,tal reagent method,bromo cresol chloroform method and double antibody sandwich enzyme-linked immunoadsorbent assay,respectively. Gut microbial communities were assayed by fluorescent quantitative PCR methods. HE staining was used to detect the pathological changes of the kidneys. Compared with the normal group,there were significant differences in body weight,urinary protein( UTP),bacterial endotoxin,urea nitrogen,creatinine,D-lactate in the plasma and four bacterial contents in the model group( P<0. 05). The urinary protein,urea nitrogen,D-lactate,creatinine and plasma bacterial endotoxin in control group and each emodin group were lower than those in model group,especially for high-dose of emodin( P<0. 01). Moreover,pathology resolution in high-dose emodin was better than other groups. Except for low-dose of emodin group,qRT-PCR data suggested that the amounts of Escherichia coli and Enterococcus in medication administration group were increased,while the amounts of Lactobacilli and Bifidobacterium were reduced compared with model group( P<0. 05),especially for high-dose of emodin( P<0. 01). There is a clear imbalance of gut microbiota in rats with AKI. Emodin could regulate the imbalance of gut microbiota,which might be one of the mechanisms of its effects on AKI rats.

The role of vascular endothelial growth factor, interleukin 8, and insulinlike growth factor in sustaining autophagic DIRAS3-induced dormant ovarian cancer xenografts.

Re-expression of the imprinted tumor suppressor gene DIRAS family GTPase 3 (DIRAS3) (aplysia ras homology member I [ARHI]) induces autophagy and tumor dormancy in ovarian cancer xenografts, but drives autophagic cancer cell death in cell culture. The current study explored the tumor and host factors required to prevent autophagic cancer cell death in xenografts and the use of antibodies against those factors or their receptors to eliminate dormant autophagic ovarian cancer cells. Survival factors (insulinlike growth factor 1 [IGF-1], vascular endothelial growth factor [VEGF], and interleukin 8 [IL-8]) were detected with growth factor arrays and measured using enzyme-linked immunoadsorbent assay analysis. Phosphorylation of protein kinase B (AKT), phosphorylation of extracellular signal-regulated kinase (ERK), nuclear localization of translocation factor EB (TFEB) or forkhead box O3a (FOXo3a), and expression of microtubule-associated proteins 1A/1B light chain 3B (MAPLC3B; LC3B) were examined using Western blot analysis. The effect of treatment with antibodies against survival factors or their receptors was studied using DIRAS3-induced dormant xenograft models. Ovarian cancer cells grown subcutaneously in nude mice exhibited higher levels of phosphorylated ERK/AKT activity and lower levels of nuclear TFEB/FOXo3a, MAPLC3B, and autophagy compared with cells grown in culture. Induction of autophagy and dormancy with DIRAS3 was associated with decreased ERK/AKT signaling. The addition of VEGF, IGF-1, and IL-8 weakened the inhibitory effect of DIRAS3 on ERK/AKT activity and reduced DIRAS3-mediated TFEB or FOXo3a nuclear localization and MAPLC3B expression in ovarian cancer cells. Treatment with antibodies against VEGF, IL-8, and IGF receptor inhibited the growth of dormant xenografts, thereby prolonging survival from 99 to >220 days (P<.05) and curing a percentage of mice. Treatment with a combination of anti-VEGF, anti-IL-8, and anti-IGF receptor antibodies prevented the outgrowth of dormant cells and prolonged survival in a preclinical model.

Platelet-specific antibodies and differences in their expression in childhood immune thrombocytopenic purpura predicts clinical progression.

ABSTRACTImportanceImmune thrombocytopenic purpura (ITP) is the most common bleeding disorder in children. Despite the highly spontaneously remission, still almost 20% of cases progress into chronic or refractory ITP, which seriously affects children's quality of life. Currently there is no method to predict the initial stage of childhood ITP.ObjectivesTo evaluate platelet‐specific antibodies and compare differences in their expression in childhood ITP to predict clinical progression.MethodsThis is a single‐center prospective cohort study from April 2014 to October 2015. We enrolled children initially diagnosed as ITP. Anti‐GPIIb/IIIa and GPIb/IX antibodies were assayed by enzyme‐linked immunoadsorbent assay (ELISA) and patients were followed up for 1 year. We also analyzed the relationship between the expression of the platelet‐specific antibodies GPIIb/IIIa and GPIb/IX and their respective clinical prognoses.ResultsOverall, 134 cases were enrolled including 77 boys and 57 girls with a median age of 19 months (range: 1 to 159). Positive rates of anti‐platelet antibodies were 79.8%. After a 1‐year observation period, 84.3% were diagnosed as newly diagnosed ITP and 13.4% were diagnosed as chronic ITP. Patients with anti‐GPIIb/IIIa antibody had a higher risk for newly diagnosed ITP compared with patients who were anti‐GPIb/IX antibody positive only (93% vs 25%, P = 0.005; 87% vs 25%, P = 0.014, respectively). There were more anti‐GPIb/IX antibody positive only cases, diagnosed as chronic ITP, compared with anti‐GPIIb/IIIa antibody positive only cases and double GPIIb/IIIa and GPIb/IX antibody positive cases (75% vs 7%, P = 0.005; 75% vs 13%, P = 0.014, respectively). InterpretationInterpretationPatients with anti‐GPIIb/IIIa antibody (either single or double) were predicted to have a good prognosis, whereas anti‐GPIb/ IX antibody only predicted a poor prognosis. These results should be confirmed via a larger cohort multicenter study.

STUDY OF SERUM ADIPONECTIN AND HIGH SENSITIVITY C-REACTIVE PROTEIN IN DIABETIC PATIENTS

Objectives: Adiponectin, a major cytokine from adipose tissue, and high sensitivity C-reactive protein (hs-CRP), well-established markers of inflammation are known to be associated with increased risk of cardiovascular disorders (CVD). Therefore the objective of our study was to evaluate the levels of these parameters and determine their correlation with glycemia to assess the cardiovascular risks in the patients with Type 2 diabetes mellitus.Methods: This study was conducted in the Department of Biochemistry, Santosh Medical College, Ghaziabad, with 25 Type 2 diabetic patients and 25 age and sex-matched controls. Ethical clearance from the institution and informed consent from the patients were taken before the study. Adiponectin was analyzed by enzyme-linked immunoadsorbent assay, and blood sugar and CRP were estimated by the kit based method.Results: Fasting blood sugar (FBS, 158.2±37.2) and hs-CRP (3.97±1.54) were significantly high, adiponectin was significantly low in the patients with diabetes compared to controls (80.52±9.72, 1.27±0.75, and 10.78±1.69, respectively, p&lt;0.05). Adiponectin showed a negative correlation with FBS (r=−0.427) and hs-CRP (r=−0.336), but the correlation was significant only in case of FBS (p&lt;0.05). hs-CRP positively correlated with FBS (r=0.568) and was statistically significant.Conclusion: The results of our study further support the role of adiponectin and hs-CRP as a predictive biomarker of CVD risks in the patients suffering from Type 2 diabetes.

The study of salivary pepsin content and laryngopharyngeal reflux scale in 91 asymptomatic volunteers

To investigate the content of pepsin in salivary, and to assess the laryngophargeal lesions based on the reflux founding score (RFS) scale in asymptomatic volunteers, in order to provide a reference for the diagnosis of laryngopharyngeal reflux. A total of 91 asymptomatic subjects were recruited in this study. Participants provided a fasting saliva specimen for pepsin measurement using enzyme-linked immunoadsorbent assay, completed the reflux symptom index (RSI) assessment and underwent laryngostroboscopic examination using a rigid endoscope. Their RFS were graded according to the laryngeal findings. The median concentration of pepsin in 91 asymptomatic volunteers was 55.5 μg/L (range 3.53-191.64 μg/L). The mean individuals RSI was 2.24±2.34, and the mean individuals RFS was 5.78±1.74. Our data demonstrate that certain concentration of pepsin was detected and showed a higher RFS score in asymptomatic volunteers.

Mechanisms of Antiulcer Effect of an Active Ingredient Group of Modified Xiao Chaihu Decoction.

The present study aimed to investigate the antiulcer activities and mechanisms of action of an active ingredient group (AIG) of Modified Xiao Chaihu Decoction (MXCD). The gastroprotective action of the AIG was studied in ethanol-induced, pylorus ligature-induced, and acetic acid-induced in vivo gastric ulcer models. The enzyme-linked immunoadsorbent assay (tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2), and epidermal growth factor (EGF)), nitrate reductase assay (nitric oxide (NO)), western blot analysis (Bax, Bcl-2, cleaved-caspase-3, and cleaved-PARP (poly (ADP-Ribose) polymerase)), histological analysis (HE), and immunohistochemical analysis (HSP-70, p-AKT, and PCNA) were used to evaluate the anti-inflammatory, antiapoptotic, and healing properties of AIG. Numerous mechanisms are involved in the antiulcer activity of AIG, including the increase of PGE2, NO, and EGF content and a reduction in TNF-α levels. The upregulation of HSP-70, p-AKT, and PCNA seems to be directly linked to the healing effect of AIG. Bax, Bcl-2, cleaved-caspase-3, and cleaved-PARP also play a key role in this process. The AIG exerted gastroprotective effects by reducing antisecretory, anti-inflammatory, and antiapoptotic mechanisms. In addition, it promotes cell proliferation. Therefore, activation of the PI3K/AKT signaling pathway may play an important role in cell proliferation.

Changes and clinical significance of CD4+ CD25+ Foxp3+ regulatory T cell and relative cell factors in peripheral blood of steroid-resistant allergic rhinitis patients

Objective To investigate the changes of CD4+ CD25+ Foxp3+ regulatory T cell (Treg) and relative cell factors in peripheral blood of steroid-resistant allergic rhinitis (AR) patients, and to study their functions in occurrence of steroid-resistant AR. Methods The CD4+ CD25+ Foxp3+ Treg cell in 30 cases general physical examination (The control group), 30 cases steroid-sensitive AR patients (steroid-sensitive group) and 30 cases steroid-resistant AR patients (steroid-resistant group) were detected by flow cytometry. The levels of serum interleukin 10 (IL-10) and transforming growth factor β1 (TGF-β1) in the three groups were detected by enzyme-linked immunoadsorbent assay (ELISA). Results Compare to the control group, proportion of CD4+ CD25+ Foxp3+ Treg cell and the levels of serum IL-10, TGF-β1 in steroid-sensitive group and steroid-resistant group were significantly reduced (P<0.01). The proportion of CD4+ CD25+ Foxp3+ Treg cell and the levels of serum IL-10, TGF-β1 in steroid-resistant group were decreased significantly than steroid-sensitive group (P<0.01). The proportion of CD4+ CD25+ Foxp3+ Treg cell were positively correlated with the serum IL-10, and TGF-β1(P<0.01). Conclusions The reduced proportion of CD4+ CD25+ Foxp3+ Treg cell and lower serum IL-10, TGF-β1 play an important role in the occurrence of steroid-resistant AR patients. Key words: Rhinitis, allergic, perennial; CD4-positive T-lymphocytes; T-lymphocytes, regulatory; Interleukin-10/ME; Transforming growth factor beta/ME

Development of an immunoenzyme test system for diagnosis if rabies

Rabies remains one of the most serious and pressing problems for veterinary science and medicine. According to the data of Rosselkhoznadzor Information and analytical center, the Russian Federation has been an area of natural focal endemicity for many years [13]. An important stage in the control and eradication of rabies in its laboratory diagnosis by detection of virus antigen in the pathological material. The aim of the study was to design an enzyme-linked immunosorbent assay (ELISA) test kit intended for the laboratory diagnosis of rabies in agricultural, domestic, and wild carnivores. Obtaining specific components for the kit, the GNKI “Sheep” master seed strain of rabies virus, the standard CVS strain of rabies virus, and white mice of the BALB line were used.In order to obtain high-activity of specific immunoglobulins (Igs ), the processes of antirabies serum production, immunoglobulin isolation, and purification have been improved as well as the conditions for immunoglobulin conjugation with peroxidase. It was found that the binding ratio should not to be lower than 0.45–0.6.A method of inactivation of rabies virus antigen has been developed, which eliminates the risk of being infected for laboratory workers and shows high specific activity in serological tests. The optimal concentrations of the reactants (immunoglobulin, antigen, conjugated) were determined; basic conditions for conducting the reaction were identified (pH of the carbonate/bicarbonate coating buffer, the optimum temperature, and duration of incubation) that ensure the required level of accuracy and sensitivity of the method.As a result of the conducted studies, a "Kit of preparations for the laboratory diagnosis of animal rabies in animals by ELISA method" was developed. The correlation between results obtained by the immunofluorescence (IF) method and the enzyme-linked immunoadsorbent assay (ELISA) methods was determined; the compliance coefficient constituted 98.8%, the sensitivity threshold of ELISA method being 3.3 Ig LD50 / ml. The sensitivity, specificity, and reproducibility of the developed test system were determined through laboratory and production tests.Â

Vitamin D and hypertension: Prospective study and meta-analysis.

ObjectivesThe study sought to determine the link between vitamin D concentrations and incident hypertension in prospective study and meta-analysis.MethodsThe study was embedded in the Kailuan Study, a population-based cohort of adults that contains underground miners. In 2012, we studied 2,456 men and women free of prevalent hypertension, age 21 to 67 at baseline. Serum 25-hydroxyvitamin D was measured from previously frozen baseline samples using ELISA (Enzyme-Linked ImmunoadSorbent Assay). We use the logistic regression analysis to estimate the odd radio (ORs) 95% confidence intervals (CIs) for 25-hydroxyvitamin D [25(OH)D] concentrations with incident hypertension. To help place our new data in context, we conducted a systemic review and meta-analysis of previous prospective reports of vitamin D and hypertension.ResultsDuring a median follow-up of 2 years, 42.6% of the cohort (n = 1047) developed hypertension. Compared with the 25-hydroxyvitamin D >30ng/ml, 25-hydroxyvitamin D <20 ng/ml was associated with a greater hypertension risk (OR: 1.225 [95% CI: 1.010 to 1.485] p = 0.04), although the association was attenuated and not statistically significant after adjusting for potential confounders (OR: 1.092 [95% CI: 0.866 to 1.377] p = 0.456). This meta-analysis included seven prospective studies for 53,375 participants using adjusted HR founded a significant association between vitamin D deficiencies and incident hypertension (HRs = 1.235 (95% CI: 1.083 to 1.409, p = 0.002)).ConclusionLower serum 25-hydroxyvitamin D concentrations were not associated with a greater risk of incident hypertension. More research is needed to further determine the role of 25-hydroxyvitamin D in hypertension prevention and therapy.

Spectrum of Transfusion Transmitted Infections Among Apparently Healthy Blood Donors in Blood Bank RIMS Ranchi From 2008 to 2014

Context: Apparently healthy blood donors might carry out transfusion transmitted infections which might be fatal for the donor himself as well as the recipient later on.Aim: To assess the seroprevalence of transfusion-transmitted infections (TTI) among apparently healthy blood donors in Ranchi, India.Methods and Materials: All blood samples were screened for HIV-I and II, HBsAg, HCV (by using third generation enzyme-linked immunoadsorbent assay technique), Syphilis (by Rapid Plasma Reagin Kit), and Malaria antigen both for plasmodium falciparum and plasmodium vivax (by One step, rapid, immunochromatographic test).Result: On screening of 106,306 blood units for TTIs, 1,462 (1.37 %) donors were found positive for one of the TTIs. Highest prevalence was for HBV (970 donors - 0.91%) ranging from 0.73% to 1.19%. This was followed by Malaria (272 donors - 0.25%) ranging from 0.08% to 0.48%, HCV (127 donors - 0.11%) ranging from 0.04% to 0.25%, HIV (72 donors - 0.06%) ranging from 0.03% to 0.10%, and syphilis (21 donors 0.01%) ranging from 0.00% to 0.09%.Conclusions: Apparently healthy blood donors are not omitted from the danger of transfusion-transmitted infection.

Association of Soluble CD40 Ligand WithDuration of Atrial Fibrillation andWith Intensity of Spontaneous Echocardiographic Contrast.

The authors tested the hypothesis that the inflammatory response of nonvalvular atrial fibrillation (NVAF) is associated with elevated soluble CD40 ligand (sCD40L). NVAF is generally believed to be an inflammatory disease process. sCD40L represents a sensitive invivo indicator of platelet activation and may serve as an "inflammatory and thrombotic thermometer." Plasma sCD40L was measured using enzyme-linked immunoadsorbent assay in 109 NVAF cases (60.9 ± 15 years of age; 26% women) and 48normal sinus rhythm (NSR) controls (62.3 ± 15 years of age; 44% women). Patients were separated by arrhythmia duration of<1 month (n= 21), 1 to 12 months (n= 18), and >12 months (n= 70). Median sCD40L level was significantly higher in NVAF cases than in NSR controls (321 pg/ml vs. 238 pg/ml, respectively; p= 0.029). This difference was driven by higher levels in patients with NVAF duration for<1 month (552pg/ml) and 1 to 12 months (328 pg/ml). NVAF patients with arrhythmia duration for over 1 year had sCD40L levels not significantly different from those of NSR controls. An sCD40L concentration of 552 pg/ml distinguished NVAF patients with dysrhythmia duration of<1 month (area under the curve [AUC] of 0.72; p= 0.0010) or duration for ≤12months (AUC: 0.69; p= 0.0003) from NSR controls. Circulating sCD40L levels were also significantly higher among patients with mild spontaneous echocardiogram contrast (SEC) (p= 0.0378) and those with moderate SEC (p= 0.007) compared with NSR controls. sCD40L levels are significantly higher in NVAF patients than in NSR controls but only for up to 1 year after development of dysrhythmia. An sCD40L concentration of 552 pg/ml can help to assess development or recurrence of asymptomatic NVAF.

Serum levels of RIPK3 and troponin I as potential biomarkers for predicting impaired left ventricular function in patients with myocardial infarction with ST segment elevation and normal troponin I levels prior percutaneous coronary intervention.

The current study examined the serum levels of receptor-interacting protein kinase 3 (RIPK3) in 51 patients with New York Heart Association (NYHA) class III-IV heart failure, 53 patients with myocardial infarction with ST elevation (STEMI), and 19 healthy subjects serving as a control group. An enzyme-linked immunoadsorbent assay (ELISA) was used to measure the levels of RIPK3 expression in serum. The area under the receiver operating characteristic curve (AUC) was then used to evaluate the predictive performance of RIPK3 and troponin I in patients with STEMI. In patients with normal levels of troponin I prior to percutaneous coronary intervention (PCI), serum levels of RIPK3 and troponin I after PCI were sufficient to differentiate patients with a preserved left ventricular ejection fraction (LVEF) from those with impaired left ventricular function after PCI (AUC = 0.780 (95% CI: 0.565-0.995, p = 0.043) with a sensitivity of 76.9% and a specificity of 71.4% vs. AUC = 0.735 (95% CI: 0.530-0.941, p = 0.038) with a sensitivity of 88.2% and a specificity of 63.6% at the optimal cutoff values, respectively). Moreover, elevated levels of troponin I after PCI were associated with an increased risk of an LVEF < 50% prior to discharge (odds ratio, 1.014; 95 % CI, 1.001 to 1.027; p = 0.03), while elevated levels of RIPK3 were not associated with such a risk. The current findings suggest that in patients with normal levels of troponin I prior to PCI, serum levels of RIPK3 and troponin I can serve as a potential marker to identify patients with a decreased LVEF, thus possibly allowing an early shift to more intensive therapy.