Double ELISA Research Articles

Study the Profile of Foot-and-mouth Disease Virus Protein by Electrophoresis and Identification of the Immunodominant Proteins.

Foot-and-mouth disease is an extremely infectious and occasionally fatal viral disease with a rapid onset and a short course that affects cloven-hoofed animals and results in considerable financial losses. Today, Foot-and-mouth disease is controlled by traditional inactivated vaccines. Due to the short duration of immunity, a study was conducted for proteins of the virus as well as obtaining immunodominant proteins to design more efficient vaccines against Foot-and-mouth disease virus. This research aims to study the profile of Foot-and-mouth disease virus protein by electrophoresis and identification of the immunodominant proteins. The purified Foot-and-mouth disease virus was purchased then the protein concentration of that solution was measured by Lowry method. SDS-PAGE was done to achieve the protein profiles of the virus and immunization of 5 guinea pigs was done, then blood samples were taken for obtaining serum. Finally, serology tests; double immunodiffusion, ELISA, and western blotting were used to evaluate antigen response to antibodies (antigenic immunization). The protein concentration was 3.5 mg/ml. In SDS-PAGE with 10% gel, the protein profile of the virus was observed. After immunization, by conducting double immunodiffusion tests, the sediment lines between the serum antibody and the antigen of the virus were formed. Also, The ELISA test showed that the antibodies were formed against the antigens. In the western blot test, two immunodominant proteins of the FMD virus were obtained. According to the results, the immunodominant proteins of the FMD virus were determined. These proteins can be used in immunological diagnostic methods and also novel vaccines.

Do Blood Phenotypes of Feline AB Blood Group System Affect the SARS-CoV-2 Antibody Serostatus in Cats?

Cats are susceptible to coronavirus infections, including infection by human severe acute respiratory syndrome coronavirus (SARS-CoV). In human ABO system blood groups, alloantibodies can play a direct role in resistance to infectious diseases. Individuals with the AB blood type were over-represented in the SARS-CoV-2 infection group. Blood type AB individuals lack both anti-A and anti-B antibodies, and therefore lack the protective effect against SARS-CoV-2 infection given by these antibodies. Starting from this knowledge, this pilot preliminary study evaluated a possible association between feline blood phenotypes A, B, and AB and serostatus for SARS-CoV-2 antibodies in cats. We also investigated selected risk or protective factors associated with seropositivity for this coronavirus. A feline population of 215 cats was analysed for AB group system blood phenotypes and antibodies against the nucleocapsid (N-protein) SARS-CoV-2 antigen using a double antigen ELISA. SARS-CoV-2 seropositive samples were confirmed using a surrogate virus neutralization test (sVNT). Origin (stray colony/shelter/owned cat), breed (DSH/non DSH), gender (male/female), reproductive status (neutered/intact), age class (kitten/young adult/mature adult/senior), retroviruses status (seropositive/seronegative), and blood phenotype (A, B, and AB) were evaluated as protective or risk factors for SARS-CoV-2 seropositivity. Seropositivity for antibodies against the SARS-CoV-2 N-protein was recorded in eight cats, but only four of these tested positive with sVNT. Of these four SARS-CoV-2 seropositive cats, three were blood phenotype A and one was phenotype AB. Young adult age (1-6 years), FeLV seropositivity and blood type AB were significantly associated with SARS-CoV-2 seropositivity according to a univariate analysis, but only blood type AB (p = 0.0344, OR = 15.4, 95%CI: 1.22-194.39) and FeLV seropositivity (p = 0.0444, OR = 13.2, 95%CI: 1.06-163.63) were significant associated risk factors according to a logistic regression. Blood phenotype AB might be associated with seropositivity for SARS-CoV-2 antibodies. This could be due, as in people, to the protective effect of naturally occurring alloantibodies to blood type antigens which are lacking in type AB cats. The results of this pilot study should be considered very preliminary, and we suggest the need for further research to assess this potential relationship.

Cancer-Associated Fibroblasts Hinder Lung Squamous Cell Carcinoma Oxidative Stress-Induced Apoptosis via METTL3 Mediated m6A Methylation of COL10A1.

Background Cancer-associated fibroblasts (CAFs) within the tumor microenvironment are key players in tumorigenesis and tumor development. Nevertheless, the regulatory mechanisms of CAFs on lung squamous cell carcinoma- (LUSC-) associated remain poorly elucidated. Methods The microarray dataset GSE22874, containing 30 specimens of primary culture of normal fibroblasts (NFs) and 8 specimens of cancer-associated fibroblasts (CAFs) samples derived from LUSC, was retrieved from the Gene Expression Omnibus (GEO) database and then calculated by using the R language (limma package) to identify differentially expressed genes (DEGs). CAF-conditioned medium (CAF-CM) was collected and used to culture LUSC cells, followed by assessment of cell proliferation, apoptosis, and oxidative stress levels by using CCK-8, annexin V-FITC/PI double staining and ELISA assays. Subsequently, COL10A1 was knocked down in CAFs to assess the role of COL10A1 in CAF regulation of LUSC behavior. Bioinformatics online analysis and MeRIP were applied to predict and test the m6A modification of COL10A1 mRNA and the regulatory relationship with METTL3. Rescue experiments were next performed to explore the effects of METTL3 and COL10A1 in CAFs on LUSC cell proliferation, apoptosis, and oxidative stress. LUSC tumor cells with or without (COL10A1-silenced) CAFs were subcutaneously inoculated in nude mice to evaluate the effect of COL10A1 in CAFs on LUSC tumor growth. Results Elevated expression of COL10A1 was found in LUSC-derived CAFs by GSE22874 dataset analysis. We discovered that COL10A1 and METTL3 was expressed in both LUSC cells and matched CAFs, while COL10A1 expression was prominently higher in CAFs than in LUSC cells. CAF-CM memorably encouraged LUSC cell proliferation and suppressed apoptosis-induced oxidative stress, which was reversed by interfering with COL10A1 expression in CAFs, suggesting that COL10A1 might be secreted by CAFs into the culture medium to exert its effects inside LUSC cells. Global m6A modification was decreased in METTL3 knocked down CAFs. M6A modification, expression levels, and stability of COL10A1 mRNA were impaired upon METTL3 knockdown in CAFs. Overexpression of COL10A1 in CAFs partially reversed the effect of METTL3 knockdown on the malignant behavior of LUSC cells. In vivo studies confirmed that CAFs accelerated LUSC tumor growth, and this effect was counteracted by COL10A1 silencing. Conclusions COL10A1 secreted by CAFs could facilitate LUSC cell proliferation and repress apoptosis-induced oxidative stress, and the mechanism was due to elevated expression mediated by METTL3 promoting its mRNA m6A modification, thereby accelerating tumor growth.

Naltrexone-Induced Cardiac Function Improvement is Associated With an Attenuated Inflammatory Response and Lipid Perioxidation in Volume Overloaded Rats.

In previous studies, upregulation of myocardial opioid receptors as well as the precursors of their endogenous ligands were detected in the failing heart due to chronic volume overload. Moreover, opioid receptor blockade by naltrexone improved left ventricular function. In parallel, inflammatory processes through cytokines have been confirmed to play an important role in the pathogenesis of different forms of heart failure. Thus, the present study examined the systemic and myocardial inflammatory response to chronic volume overload and its modulation by chronic naltrexone therapy. Chronic volume overload was induced in male Wistar rats by applying an infrarenal aortocaval fistula (ACF) for 28 days during which the selective opioid receptor antagonist naltrexone (n = 6) or vehicle (n = 6) were administered via a subcutaneously implanted Alzet minipump. The ultrastructural, morphometric and hemodynamic characterization of ACF animals were performed using an intraventricular conductance catheter in vivo and electron microscopy in vitro. Co-localization of mu-, delta- and kappa-opioid receptor subtypes (MOR, DOR, and KOR respectively) with the voltage gated L-type Ca2+ channel (Cav1.2), the ryanodine receptor (RyR), and mitochondria in cardiomyocytes as well as IL-6, IL-12, TNF-alpha, and Malondialdehyde (MDA) were determined using double immunofluorescence confocal microscopy, RT-PCR and ELISA, respectively. In rat left ventricular myocardium, three opioid receptor subtypes MOR, DOR, and KOR colocalized with Cav1.2, RyR and mitochondria suggesting a modulatory role of the excitation-contraction coupling. In rats with ACF-induced volume overload, signs of heart failure and myocardial ultrastructural damage, chronic naltrexone therapy improved cardiac function and reversed the systemic and myocardial inflammatory cytokine expression as well as lipid peroxidation. In conclusion, antagonism of the cardiodepressive effects of the myocardial opioid system does not only improve left ventricular function but also blunts the inflammatory response and lipid peroxidation.

Detailed epitope mapping of SARS-CoV-2 nucleoprotein reveals specific immunoresponse in cats and dogs housed with COVID-19 patients

Since the initial emergence in December 2019, the novel Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been reported in over 200 countries, representing an unprecedented challenge related to disease control worldwide. In this context, cases of human to animal transmission have been reported, raising concern about the potential role of companion animals in the pandemic and stressing the need for reliable animal testing. In the study, a detailed epitope mapping of SARS-CoV-2 nucleoprotein, using both human and pet sera, allowed the identification of the most antigenic region in the C-terminus domain of the protein, which was used to develop an experimental double antigen-based ELISA. A panel of pre-pandemic sera and sera of animals immunized against (or naturally infected with) related coronaviruses was used to assess assay specificity at 99.5%. Positive sera belonging to animals housed with COVID-19 patients were confirmed with the experimental double-antigen ELISA using Plaque Reduction Neutralization test (PRNT) test as gold standard. The availability of a serological assay that targets a highly specific viral antigen represents a valuable tool for multispecies monitoring of Coronavirus Disease 2019 (COVID-19) infection in susceptible animals.

The Impact of Sodium Dodecyl Sulfate and 2-Mercaptoethanol on Antibody and Antigen Binding.

To evaluate the effect of sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (2-ME) on antigen-antibody binding when incubated at 100°C, which is the pretreatment temperature required for western blots. Serum that tested positive for hepatitis B surface antigen (HBsAg) plus loading buffer were mixed at a ratio of 4:1 and incubated in a water bath. We then detected HBsAg using double immunodiffusion and ELISA. The HBsAg titer was 1:512 in the control group when incubated at 37°C. Incubation with SDS at 100°C reduced the antigen titer to 1:32. The inhibitory effect on HBsAg titer reached 96.9% after incubation at 100°C with SDS and 2-ME. We detected strong inhibition of antigens in western blots via SDS and 2-ME. It is likely that false-negative results will be obtained from western blots of antigens with weak resistance to these reagents.

Fluoride exposure induces mitochondrial damage and mitophagy via activation of the IL-17A pathway in hepatocytes

As an environmental toxicant, the damage of fluoride to the body has attracted global attention. Because liver is an essential organ for fluoride accumulation and damage. Our previous studies revealed fluoride-induced hepatic injury through interleukin 17A (IL-17A) pathway, but the underlying cellular mechanism remains unclear. Hence, this research explored the mechanism of IL-17A pathway and mitophagy in fluoride-induced liver injury through the use of the mice fluorosis model, IL-17A addition fluorosis cell model, IL-17A gene knockout mice fluorosis model, flow cytometry, immunohistochemistry, fluorescence double staining, ELISA, western blotting, and other techniques. The results showed that fluoride reduced the bodyweight and liver coefficient, increased the bone fluoride content, the aspartate aminotransferase (AST), alanine aminotransferase (ALT), glutamate dehydrogenase (GDH) levels, caspase 8 and caspase 9 activities, and induced liver morphology and ultrastructure damage. Furthermore, the protein expression levels of IL-17A pathway key proteins, IL-17A, IL-17R, and Act1 were increased, but IκB was decreased after fluoride exposure. In addition, fluoride exposure elevated the mitochondrial depolarization percent, the mitochondria damage, the fluorescent spots of mitophagy, and the LC3II/LC3I protein relative expression level. To further verify the role of the IL-17A pathway in fluoride-induced hepatocyte mitochondrial damage and mitophagy disorder, the IL-17A was added and knocked out in cells of animals. The results showed that the addition of IL-17A aggravated fluoride-induced liver morphology and functional damage, activation of the IL-17A pathway, mitochondrial injury, and mitophagy, but the IL-17A knockout mitigated fluoride-induced changes. These results suggested that fluoride exposure induced mitochondrial damage and mitophagy through the IL-17A pathway in hepatocytes.

The expression of diacylglycerol kinase isoforms α and ζ correlates with the progression of experimental autoimmune encephalomyelitis in rats

Multiple sclerosis (MS) is characterized by neuroinflammation and neurodegeneration, whose precise processes are not fully understood. Diacylglycerol kinase (DGK) isozymes of α, β, γ and ζ expressed abundantly in the brain and/or the immune system, may be regulatory targets for MS. In this study, we analyzed the four DGK isozymes along the induction, peak and recovery phases in an experimental autoimmune encephalomyelitis (EAE) rat model of MS. The expression of these DGK isozymes and the diacylglycerol (DAG) pathway in the EAE rat brainstems were analyzed by qRT-PCR, immunohistochemistry, immunofluorescence double staining, western blotting and ELISA. Our results showed that the mRNA content of the four DGK isozymes decreased significantly, and their immunoreactivity in myelin sheathes (DGKα, β) and neurons (DGKγ, ζ) became weaker at the beginning of the induction phase. With the progressive increase in clinical signs, DGKα, DGKγ and DGKζ mRNA increased and DGKβ mRNA decreased, and microglia were involved in the formation of perivascular cuffing. In the peak phase, both DGKα and DGKζ were expressed in neurons and inflammatory cells, and DGKζ was also positive in microglia. During the recovery phase, the mRNA content and immunoreactivity of these DGK isozymes generally reached normal levels. Moreover, our results revealed that changes in DAG accumulation and PKCδ phosphorylation were almost the same as those of DGKα and DGKζ mRNA. In summary, the four DGK isozymes are involved in the EAE process. The predominant and broad presence of DGKα and DGKζ suggests that they may regulate the pathological process by attenuating DAG/PKCδ pathway signaling during EAE evolution.

Antithrombin III level in children with nephrotic syndrome, its correlation to thromboembolic complications, and serum albumin level.

Nephrotic syndrome (NS) is one of the most common pediatric diseases with many complications. Thromboembolic complication is the most serious complication. The aim of this study was to predict the possible risk of thromboembolic complication development in children with NS due to antithrombin III deficiency. This study was conducted in the Outpatient Nephrology Clinic of Children's Hospital in Fayoum University Hospital. It included 27 children with NS and 27 healthy children as a control group in an analytic study with cross-sectional comparative design. Laboratory investigations were done in the form of complete blood picture, serum levels of albumin, total protein, creatinine, urea, cholesterol, triglycerides, urine analysis, albumin/creatinine ratio, prothrombin time, and INR. The serum antithrombin III level was measured by double ELISA technique. Data analysis was performed using the Statistical Package for the Social Sciences software version 18. Student's t-test was used to compare measures of two independent groups of quantitative data. One-way ANOVA test was used to compare more than two independent groups of quantitative data. Kruskal-Wallis test was used in comparing more than two independent nonparametric groups. Bivariate Pearson correlation test was used to test the association between variables. The level P ≤0.05 was considered significant. There were significant decreases in antithrombin III, albumin, and total protein levels in the study group during relapse and improved after steroid. There were no thromboembolic complications detected among the study group. NS causes heavy proteinuria with loss of many important proteins as antithrombin III. Serum antithrombin III level is significantly decreased in children with NS, and it correlated with serum albumin. Although patients in the study have thrombocytosis, hypercholesterolemia, and decreased serum level of antithrombin III, none of the children in the the study showed thrombotic complication, so we conclude that, thromboembolism is uncommon in children with NS may be due to early diagnosis and proper treatment.

Sero Prevalence and Risk factors forSheep Pox and Lumpy Skin Disease and Their Comparison to Capri Pox Double Antigen Multispecies ELISA in Khartoum and Kordofan States in Sudan

A cross-sectional survey was performed in the Kordofan region, from March to September 2011 was compared to Capripox Double Ag ELISA for multispecies. The estimated overall sero-prevalence of sheep pox in Kordofan region was 73.4% determined by virus neutralization and was prevalent in both South and North Kordofan states at 85% and 63.6% respectively. However, Seroprevalence for lumpy skin A cross-sectional survey was performed in the Kordofan region, from March to September 2011 was compared to Capripox Double Ag ELISA for multispecies. The estimated overall sero-Prevalence of sheep pox in Kordofan region was 73.4% determined by virus neutralization and was prevalent in both South and North Kordofan states at 85% and 63.6% respectively. However, Sero-Prevalence for lumpy skin disease was 5% and 62% for sheep pox by using Capripox Double Ag ELISA. The serological information was used to identify potential risk factors associated with sheep pox outbreaks. The risk factors identified were the breed, age, sex, species, movement patterns, herd size and geographic region. In addition, a questionnaire explored producer’s knowledge about the disease in the Sudan. The results of the questionnaire were that both nomadic as well as fixed farmers were generally aware of sheep pox as a disease, but most did not have full knowledge about the disease. Greater than half of producers experienced the disease in the past 2 years and did not have their sheep vaccinated.disease was 5% and 62% for sheep pox by using Capripox Double Ag ELISA. The serological information was used to identify potential risk factors associated with sheep pox outbreaks. The risk factors identified were the breed, age, sex, species, movement patterns, herd size and geographic region. In addition, a questionnaire explored producer’s knowledge about the disease in the Sudan. The results of the questionnaire were that both nomadic as well as fixed farmers were generally aware of sheep pox as a disease, but most did not have full knowledge about the disease. Greater than half of producers experienced the disease in the past 2 years and did not have their sheep vaccinated.

Clinical Features and Pattern of Antiphospholipid Antibodies in Patients of Antiphospholipid Syndrome (APS) with Systemic Lupus Erythematosus (SLE)

Background: Evaluation of antiphospholipid antibodies (aPL) profile in Systemic lupus erythematosus (SLE) patients with obstetric and thrombotic complications and to see the association of different anti phospholipid antibodies in different clinical features of Antiphospholipid syndrome (APS) in SLE patients. Materials and methods: Cross sectional prospective interdisciplinary study was conducted between July 2019 to January 2021 at the Section of Chemical Pathology and Department of Rheumatology, Fatima Memorial Hospital Lahore. After taking IRB approval, diagnosed cases of APS with SLE presenting with thrombosis and pregnancy complication were studied. After informed consent, performa was filled by patient including age, gender and clinical symptoms. Blood sample were taken for Anti-beta-2-glycoprotein 1(anti-b2GPI), anticardiolipin (aCL) analyze on Alegria based on ELISA. Results: Total of 60 diagnosed patients of SLE, presenting with thrombosis or pregnancy complications were included in the study. Mean age of patients was 31.3 ± 5.3 years. There were 56 (93.3%) female and 4 (6.7%) male patients. Non-thrombotic lupus manifestations in decreasing orders were; cutaneous features in 40 (66.7%), oral ulcers in 33 (58.3%), arthritis and nephritis each in 23 (38.3%), neuropsychiatric manifestation in 7 (11.6%), and serositis in 4 (6.7%) patients. Among thrombotic manifestations; pregnancy loss was reported in 30 (50%) with 22 (36%) in first trimester and 8 (14%) in 2nd trimester, deep venous thrombosis of extremities was noted in 14 (23.3%), thromboembolic stroke in 11 (18.3%), arterial thrombosis in 5 (8.3%), and pre-eclampsia was reported in 4 (7.1%) patients. Out of 60 patients with SLE, 18 (30%) were positive with single APLS antibody, 28 (46.7%) were positive with double –antibodies and 14 (23.3%) were positive with triple positive APLS. Person who had five thrombotic events was triple ELISA positive as compared to person who had 1 or two thrombotic events were double ELISA positive. Conclusion: aPLS antibodies of any type and number leads to cumulative obstetric and thrombotic complication in SLE, which is higher in high risk antibody profiles.

MiR-149-5p mitigates tumor necrosis factor-α-induced chondrocyte apoptosis by inhibiting TRADD.

Chondrocyte apoptosis as a prominent characteristic is usually accompanied by cartilage degeneration in osteoarthritis (OA). Herein, we aimed to determine the roles of miR-149-5p in tumor necrosis factor-α (TNF-α)-induced chondrocyte apoptosis. Human chondrocytes were cultured with TNF-α to establish an apoptosis cell model in vitro. After transfection with miR-149-5p mimics or co-expression with TRADD in chondrocytes, cell viability, apoptosis, inflammatory cytokines, mRNA and protein expression were measured using CCK8, Annexin V-FITC double staining, ELISA assays, RT-qPCR and western blotting, respectively. TNF-α-induced chondrocyte apoptosis occurred in association with the inhibition of cell proliferation, the elevation of inflammatory cytokine levels and the activation of TRADD and caspase-3/8 signaling. The post-transcriptional regulatory mechanism suggested that TRADD was a direct target of miR-149-5p, and overexpression of miR-149-5p resulted in the down-regulation of TRADD protein expression in chondrocytes. In addition, miR-149-5p mimics had the ability to attenuate TNF-α-induced inflammation and apoptosis, while transfection with TRADD vector neutralized the protective effects of miR-149-5p on TNF-α-induced chondrocyte dysfunction. miR-149-5p inversely regulated TNF-α-mediated chondrocyte damage by inhibiting TRADD-modulated caspases signaling. The miR-149-5p/TRADD signaling pathway might be a promising therapeutic target for the treatment of OA.

Higher CCL22+ Cell Infiltration is Associated with Poor Prognosis in Cervical Cancer Patients.

The chemokine CCL22 recruits regulatory T (T-reg) cells into tumor tissues and is expressed in many human tumors. However, the prognostic role of CCL22 in cervical cancer (CC) has not been determined. This study retrospectively analyzed the clinical significance of the expression of CCL22 and FOXP3 in 230 cervical cancer patients. Immunohistochemical staining analyses of CCL22 and FOXP3 were performed with a tissue microarray. Double immunofluorescence staining, cell coculture, and ELISA were used to determine CCL22 expressing cells and mechanisms. The higher number of infiltrating CCL22+ cells (CCL22high) group was associated with lymph node metastasis (p = 0.004), Fédération Internationale de Gynécologie et d’Obstétrique (FIGO) stages (p = 0.010), therapeutic strategies (p = 0.007), and survival status (p = 0.002). The number of infiltrating CCL22+ cells was positively correlated with that of infiltrating FOXP3+ cells (r = 0.210, p = 0.001). The CCL22high group had a lower overall survival rate (OS), compared to the CCL22low group (p = 0.001). However, no significant differences in progression free survival (PFS) were noted between the two groups. CCL22high was an independent predictor of shorter OS (HR, 4.985; p = 0.0001). The OS of the combination group CCL22highFOXP3high was significantly lower than that of the combination group CCL22lowFOXP3low regardless of the FIGO stage and disease subtype. CCL22highFOXP3high was an independent indictor of shorter OS (HR, 5.284; p = 0.009). The PFS of group CCL22highFOXP3high was significantly lower than that of group CCL22lowFOXP3low in cervical adenocarcinoma, but CCL22highFOXP3high was not an independent indicator (HR, 3.018; p = 0.068). CCL22 was primarily expressed in M2-like macrophages in CC and induced by cervical cancer cells. The findings of our study indicate that cervical cancer patients with elevated CCL22+ infiltrating cells require more aggressive treatment. Moreover, the results provide a basis for subsequent, comprehensive studies to advance the design of immunotherapy for cervical cancer.

Gastrodin relieves inflammation injury induced by lipopolysaccharides in MRC-5 cells by up-regulation of miR-103.

The beneficial function of gastrodin towards many inflammatory diseases has been identified. This study designed to see the influence of gastrodin in a cell model of chronic obstructive pulmonary disease (COPD). MRC‐5 cells were treated by LPS, before which gastrodin was administrated. The effects of gastrodin were evaluated by conducting CCK‐8, FITC‐PI double staining, Western blot, qRT‐PCR and ELISA. Besides this, the downstream effector and signalling were studied to decode how gastrodin exerted its function. And dual‐luciferase assay was used to detect the targeting link between miR‐103 and lipoprotein receptor‐related protein 1 (LRP1). LPS induced apoptosis and the release of MCP‐1, IL‐6 and TNF‐α in MRC‐5 cells. Pre‐treating MRC‐5 cells with gastrodin attenuated LPS‐induced cell damage. Meanwhile, p38/JNK and NF‐κB pathways induced by LPS were repressed by gastrodin. miR‐103 expression was elevated by gastrodin. Further, the protective functions of gastrodin were attenuated by miR‐103 silencing. And LRP1 was a target of miR‐103 and negatively regulated by miR‐103. The in vitro data illustrated the protective function of gastrodin in LPS‐injured MRC‐5 cells. Gastrodin exerted its function possibly by up‐regulating miR‐103 and modulating p38/JNK and NF‐κB pathways.

PO-289 Dual inhibition of WNT/β-catenin signalling and histone deacetylation as a new strategy to eliminate breast cancer stem cells by augmentation of apoptosis

IntroductionEpigenetic changes play a critical role in the regulation of cancer stem cell (CSC) properties and the development of drug resistance. Modulation of histone acetylation program is closely related to differentiation and apoptosis process. CSCs, a subset of tumour cells, are responsible for disease relapse because of an acquired resistance to apoptosis and the Wnt signalling which is associated with cell survival/self-renewal and differentiation, is re-activated in these cells. Therefore, in the present study, we focused on a possible cytotoxic/apoptotic effect of the combination of niclosamide (Wnt/β-catenin pathway inhibitor) and Valproic acid (VPA, histone deacetylase inhibitor) on breast CSCs.Material and methodsThe effect of niclosamide (1 µM, 24 hour pre-treatment) and VPA (0.63–5 mM) combination on the viability of MCF-7s cells (CSCs-enriched population) were demonstrated by the ATP assay. Acetylated histone H3 levels at selected doses for the combination were assessed by ELISA. Protein levels associated with the Wnt/β-catenin signalling pathway, EMT, and histone modifications were shown by western blotting. Cell death mode was investigated via Hoechst 33342/PI double staining, M30 ELISA, real-time PCR (gene levels associated with apoptosis and autophagy) and western blotting (protein levels associated with apoptosis, autophagy and ER stress).Results and discussionsWe found that combination therapy exhibited a marked decrease in cell viability by inducing extrinsic apoptosis along with the stronger Wnt inhibition and increased histone H3 acetylation in MCF-7s cells. Furthermore, it was found that mesenchymal markers (fibronectin, N-cadherin, and ZEB1) were decreased at 72 hour and cytokeratin 18 as an epithelial marker was re-expressed in which H3K9ac and H3K4me3 were also increased. In addition, ER stress and blockade of autophagic flux have also been shown to be involved in this process.ConclusionIn conclusion, the future success of this combination approach in targeting CSCs and converted CSCs to non-CSCs may hold significant promise for successful treatment of breast cancer.

Evaluation of SD BIOLINE H. pylori Ag rapid test against double ELISA with SD H. pylori Ag ELISA and EZ-STEP H. pylori Ag ELISA tests

BackgroundHelicobacter pylori antibody titters fall very slowly even after successful treatment. Therefore, tests detecting H. pylori antibody lack specificity and sensitivity. On the other hand, H. pylori stool antigen tests are reported as an alternative assay because of their reliability and simplicity. However, the comparative performance of H. pylori stool antigen tests for detecting the presence of the bacterium in clinical specimens in the study area is not assessed. Therefore, in this study we evaluated the performance of SD BIOLINE H. pylori Ag rapid test with reference to the commercially available EZ- STEP ELISA and SD BIOLINE H. pylori Ag ELISA tests.MethodsStool samples were collected to analyse the diagnostic performance of SD BIOLINE H. pylori Ag rapid test kit using SD H. pylori Ag ELISA kit and EZ- STEP ELISA tests as a gold standard. Serum samples were also collected from each patient to test for the presence of H. pylori antibodies using dBest H. pylori Test Disk. Sensitivity, specificity, predictive values and kappa value are assessed. P values < 0.05 were taken statistically significant.ResultsStool and serum samples were collected from 201 dyspeptic patients and analysed. The sensitivity, specificity, positive and negative predictive values of the SD BIOLINE H. pylori Ag rapid test were: 95.6% (95% CI, 88.8–98.8), 92.5% (95%CI, 89–94.1%), 86.7% (95% CI, 80.5–89.6), and 97.6% (95% CI, 993.9–99.3) respectively.ConclusionThe performance of SD BIOLINE H. pylori Ag rapid test was better than the currently available antibody test in study area. Therefore, the SD BIOLINE Ag rapid stool test could replace and be used to diagnose active H. pylori infection before the commencement of therapy among dyspeptic patients.

Study of prevalence of dermatophytes among human immunodeficiency virus/AIDS patients in Shadan Institute of Medical Sciences and Teaching Hospital and Research Centre, Hyderabad, Telangana, India.

Introduction:The objective of the study was to evaluate the prevalence of dermatophytoses in human immunodeficiency virus (HIV) patients as well as to analyze the influence of CD4+ T-cell level in the Dermatology and STD Outpatient Department of Shadan Institute of Medical Sciences Teaching Hospital and Research Centre, Himayat sagar road, Hyderabad (Telangana state). The patients were tested for dermatophytic infections, as well as for the CD4+ T-cell counts. A total of 120 HIV-seropositive patients were included in this study, among which 38 were diagnosed of dermatophytosis. A majority of patients were in the 21–30 years’ age group. Tinea cruris was seen in majority of the cases, with Trichophyton rubrum being the most common culprit.Background:Cutaneous fungal infections have been reported worldwide as being one of the most common human infectious diseases in clinical practice. Dermatophytoses in individuals with HIV infection seem to manifest with atypical, multiple, or extensive lesions more frequently.Aims:The aim of this study was to determine the prevalence of dermatophytic infections among HIV-seropositive patients and their relation with CD4 count.Materials and Methods:This single-center prospective study was conducted in all HIV-seropositive patients (by double ELISA methods) who attended the Dermatology and STD Outpatient Department of Shadan Institute of Medical Sciences Teaching Hospital and Research Centre, Himayat sagar road, Hyderabad (Telangana state), from March 2015 to September 2016. They were screened for cutaneous fungal infections and those who tested positive were recruited for this study.Results:A total of 120 HIV-seropositive patients were included in this study, among which 38 were diagnosed of dermatophytosis. Most patients were in the 21–30 years’ age group. Tinea cruris was the most common variant, and T. rubrum was the most common offending pathogen. It was also found that the CD4+ T-cell count does not influence the occurrence of dermatophytoses.Conclusion:Superficial fungal infections are a common yet significant problem in HIV infection. They are characterized by the diversity of clinical aspects; the lesions are mostly caused by T. rubrum. It is essential that optimum treatment should be administered.

MEANS FOR INHIBITION OF PRODUCTION OF STAPHYLOCOCCUS ENTEROTOXINS AND THEIR ELIMINATION FROM BIOLOGICAL SUBSTRATES

Aim. Expansion of arsenal of means capable of inhibiting production of staphylococci entero-toxins (SE) and having an ability to eliminate them from biological substrates, as well as reducing the growth of staphylococci. Materials and methods. Reference strain of Staphylococcus aureus FRI 722 was used as SE producer type A (SEA), S. aureus S6 715H - as SE type В producer (SEB). Polymethylsiloxane polyhydrate (PMSPH) was used at concentrations of 1.82, 9.09 and 18.2%. Results. By using gel double diffusion method and ELISA we have established that a 18.2% solution of PMSPH (enterosgel; PMSPH - 70 g, purified water - 30 g per 100 g of the product) is an optimal concentration for inhibition of production of staphylococcus enterotoxin type A by 100 and more times, and production of staphylococci enterotoxin type В - by more than 300 times. Conclusion. PMSPH is able to eliminate staphylococci enterotoxins type A and В from biological substrates for more than 50% and significantly reduce growth of staphylococci.

8-methoxypsoralen reduces AKT phosphorylation, induces intrinsic and extrinsic apoptotic pathways, and suppresses cell growth of SK-N-AS neuroblastoma and SW620 metastatic colon cancer cells

Ethnopharmacological relevance8-methoxypsoralen (8-MOP) is a furanocoumarin and an active compound of a traditional Egyptian medicinal plant Ammi majus L, whose juice/fruit has been used for many years in folk phototherapy for the treatment of vitiligo or a hyperproliferative skin disorder, psoriasis. 8-MOP together with UVA light is also used as an anticancer drug for the treatment of cutaneous T-cell lymphoma. However, furanocoumarins exert anticancer activity even without UV irradiation. Aim of the studyEvaluation UV-independent anticancer activity of 8-MOP in human cancer cell lines and identification of the mechanisms involved in this action. Results could provide new data about a potential role of 8-MOP in prevention and growth suppression in a broad spectrum of cancers. Materials and methods8-MOP (99%, HPLC/MS assay) was isolated from A. majus fruits by chromatographic methods. The effect of 8-MOP on cell viability was evaluated by the MTT test in several human cancer cell lines. Anti-proliferative activity of 8-MOP was evaluated by the BrdU assay in neuroblastoma (SK-N-AS) and metastatic colon cancer (SW620) cells. The Hoechst/PI staining was used for morphological analysis of cell death. An annexin V-FITC/PI double labelling and Cell Death Detection ELISA kit were used to detect apoptosis. The expression of apoptosis-associated proteins and the AKT activation status were determined by Western blot analysis. Results8-MOP inhibited cell growth in several cancer cell lines. The SK-N-AS and SW620 cells were the most sensitive to the compound. 8-MOP reduced the phosphorylation of AKT308, decreased the expression of Bcl-2, increased the Bax protein level, and activated caspases −8, −9, and −3 in both cell lines. Conclusions8-MOP impairs the PI3K/AKT signalling pathway and, independently of photoactivation, can inhibit the growth of neuroblastoma and colon cancer cells by induction of apoptosis via intrinsic and extrinsic pathways.

LOW FALSE RECENT RATE OF LIMITING ANTIGEN AVIDITY ASSAY COMBINED WITH HIV-1 RNA DATA IN BOTSWANA

BackgroundCross-sectional tests for recency of HIV infection are increasing in utility for estimating HIV incidence and evaluating impact of interventions. However, they have been shown to misclassify individuals with long...