Enzyme-linked Immunoassay Assay Research Articles

Seroprevalence and molecular detection of Toxoplasma gondii and Neospora caninum in beef cattle and goats in Hunan province, China

BackgroundToxoplasma gondii and Neospora caninum are closely related protozoan parasites that are considered important causes of abortion in livestock, causing huge economic losses. Hunan Province ranks 12th in the production of beef and mutton in China. However, limited data are available on the seroprevalence, risk factors and molecular characterization of T. gondii and N. caninum in beef cattle and goats in Hunan province, China.MethodsSera of 985 beef cattle and 1147 goats were examined for the presence of specific antibodies against T. gondii using indirect hemagglutination test (IHAT) and anti-N. caninum IgG using competitive-inhibition enzyme-linked immunoassay assay (cELISA). Statistical analysis of possible risk factors was performed using PASW Statistics. Muscle samples of 160 beef cattle and 160 goats were examined for the presence of T. gondii DNA (B1 gene) and N. caninum DNA (Nc-5 gene) by nested PCR. The B1 gene-positive samples were genotyped at 10 genetic markers using the multilocus nested PCR-RFLP (Mn-PCR-RFLP).ResultsSpecific IgG against T. gondii were detected in 8.3% (82/985) and 13.3% (153/1147) and against N. caninum in 2.1% (21/985) and 2.0% (23/1147) of the beef cattle and goats, respectively. Based on statistical analysis, the presence of cats, semi-intensive management mode and gender were identified as significant risk factors for T. gondii infection in beef cattle. Age was a significant risk factor for T. gondii infection in goats (P < 0.05), and age > 3 years was a significant risk factor for N. caninum infection in beef cattle (P < 0.05). PCR positivity for T. gondii was observed in three beef samples (1.9%; 3/160) and seven chevon samples (4.4%; 7/160). Genotyping of PCR positive samples identified one to be ToxoDB#10. The N. caninum DNA was observed in one beef sample (0.6%; 1/160) but was negative in all chevon samples.ConclusionsTo our knowledge, this is the first large-scale serological and molecular investigation of T. gondii and N. caninum and assessment of related risk factors in beef cattle and goats in Hunan Province, China. The findings provide baseline data for executing prevention and control of these two important parasites in beef cattle and goats in China.Graphical

Helicobacter pylori infection and risk of multiple sclerosis: an updated meta-analysis.

Numerous studies have proposed that Helicobacter pylori infection may possess a protective effect in terms of future risk of multiple sclerosis (MS), however is poorly evidenced. We performed a systematic review and meta-analysis to obtain the pooled results regarding the prevalence of H. pylori infection in persons with multiple sclerosis (pwMS) and healthy controls. A comprehensive database search was performed in PubMed, Embase, and medRxiv for all relevant literature published from the inception of the databases until the August 1, 2022. The retrieved articles were first screened by title and abstract, followed by full-text screening based on the pre-established eligibility criteria. The risk of bias was assessed using the ROBINS-I tool. Data on the seroprevalence of H. pylori in pwMS and healthy controls was extracted, and a meta-analysis was performed in Review Manager Version 5.4.1. Sub-group analysis was performed in accordance with the geographical distribution (Eastern and Western countries) and the method of detection of H. pylori infection enzyme-linked-immunoassay (ELISA), Immunofluorescence, Immunochromatography). Furthermore, sensitivity analyses and publication bias were determined. The preliminary database search retrieved a total of 822 studies. Seventeen case-control studies with a total of 2721 pwMS and 2245 controls were included as a final sample size for the meta-analysis. The overall risk of bias was moderate. Overall, the rate of H. pylori infection in pwMS was not significantly different than in healthy controls (OR: 0.79 (95% CI = 0.58-1.08); I2 = 79%, p = 0.14). Subgroup analysis revealed that the rate of H. pylori infection among PwMS was not significant in both Eastern and Western countries (OR: 0.75 (95% CI = 0.52-1.08); I2 = 81%, p = 0.12). In contrast, data revealed that the prevalence of H. pylori infection in pwMS was significantly lower than that of control based on studies utilizing ELISA assays detection (OR: 0.71 (95% CI = 0.50-1.00); I2 = 81%, p = 0.05), while no significant difference was seen on studies using other assays than ELISA (OR: 1.19 (95% CI = 0.81-1.77); I2 = 0%, p = 0.38). Our findings of statistically indifferent prevalence of H. pylori infection as compared between pwMS and healthy controls suggested the absence of protective effect for risk of MS following H. pylori infection.

Streptococcal pyrogenic exotoxin B is a superantigen that induces murine splenocyte proliferation and secretion of IL-2 and IFN-γ ex vivo.

Streptococcus pyogenes is a significant human pathogen, producing a range of virulence factors, including streptococcal pyrogenic exotoxin B (SpeB) that is associated with foodborne outbreaks. It was only known that this cysteine protease mediates cleavage of transmembrane proteins to permit bacterial penetration and is found in 25% of clinical isolates from streptococcal toxic shock syndrome patients with extreme inflammation. Its interaction with host and streptococcal proteins has been well characterized, but doubt remains about whether it constitutes a superantigen. In this study, for the first time it is shown that SpeB acts as a superantigen, similarly to other known superantigens such as staphylococcal enterotoxin A or streptococcal pyrogenic exotoxin type C, by inducing proliferation of murine splenocytes and cytokine secretion, primarily of interleukin-2 (IL-2), as shown by cytometric bead array analysis. IL-2 secretion was confirmed by enzyme-linked immunosorbent assay (ELISA) as well as secretion of interferon-γ. ELISA showed a dose-dependent relationship between SpeB concentration in splenocyte cells and IL-2 secretion levels, and it was shown that SpeB retains activity in milk pasteurized for 30 min at 63°C.

HAGLR, stabilized by m6A modification, triggers PTEN-Akt signaling cascade-mediated RPE cell pyroptosis via sponging miR-106b-5p.

Consistent hyperglycaemia on retinal microvascular tissues is recognized as a vital inducer of diabetic retinopathy (DR) pathogenesis. In view of the essential functionality of long noncoding RNAs (lncRNAs) in multiple human diseases, we aim to figure out the exact role and underlying mechanisms of lncRNA HOXD Cluster Antisense RNA 1 (HAGLR) in DR pathogenesis. Serum specimens from patients with proliferative DR and healthy volunteers were collected for measuring HAGLR levels. Human primary retinal pigment epithelium (HRPE) cells kept in high glucose (HG) condition were applied to simulating hyperglycaemia of DR pathology in vitro. Cell proliferation, apoptosis, either pyroptosis was assess using Cell Counting Kit-8 TUNEL, flow cytometry, and enzyme-linked immunoassay assays. Bioinformatics analysis was subjected to examine the interaction between HAGLR and N6-methyladenosine (m6A)-bind protein IGF2BP2, as determined using RNA immunoprecipitation and RNA pull-down. Luciferase reporter assay was performed to assess the HAGLR-miR-106b-5p-PTEN axis. Levels of pyroptosis-associated biomarkers were detected using western blotting. Aberrantly overexpressed HAGLR was uncovered in the serum samples of DR patients and HG-induced HRPE cells, of which knockdown attenuated HG-induced cytotoxic impacts on cell apoptosis and pyroptosis. Whereas, reinforced HAGLR further aggravated these effects. IGF2BP2 positively regulated HAGLR in a m6A-dependent manner. HAGLR served as a sponge for miR-106b-5p to upregulate PTEN, thereby activating Akt signaling cascade. Rescue assays demonstrated that PTEN overexpression abolished the inhibition of silenced HAGLR on pyroptosis in HRPE cells. HAGLR, epigenetically modified by IGF2BP2 in an m6A-dependent manner, functioned as a sponge for miR-106b-5p, thereby activating PTEN/Akt signaling cascade to accelerate DR pathology.

Detection of the Enzymes and Toxins Produced by the Pathogenic Fungi that Cause Chias Pollen Disease on Different Date Palm Cultivars in Iraq

Mohamed, Kh.A. and M.A. Alfahd. 2023. Detection of the Enzymes and Toxins Produced by the Pathogenic Fungi that Cause Chias Pollen Disease on Different Date Palm Cultivars in Iraq. Arab Journal of Plant Protection, 41(1): 65-70. https://doi.org/10.22268/AJPP-41.1.065070 This study aimed to determine the exo-enzyme activity of some inflorescence-rot causing fungi that were isolated from different date palm cultivars such as Mauginiella scaettae, Fusarium solani, Fusarium oxysporium and Alternaria radicina and their ability to produce protease and cellulase enzymes on Czapek dox (CD) liquid media supplemented with carboxy methyl cellulose (CMC), as well as their ability to produce Fumonisin B1 toxin using the enzyme linked immunoassay assay (ELISA). The results obtained showed a discrepancy in the amount of enzymes and toxins produced by these pathogens. The fungus F. oxysporum produced the highest cellulase enzyme activity, which amounted to 1.1933 units/ml, whereas Alternaria radicina produced the highest protease enzyme activity which amounted to 50,964 units/ml. The pathogen M. scaettae produced the highest toxin concentration of 38.465 µg/kg, whereas the pathogen Fusarium solani did not produce this toxin. Keywords: Mauginiella. Scaettae, enzymes, fumonisin B1, toxins.

Protective effects of three kinds of borneol on different brain regions in acute cerebral ischemia/reperfusion model rats

This study compared the ameliorating effects of L-borneol, natural borneol, and synthetic borneol on the injury of different brain regions in the rat model of acute phase of cerebral ischemia/reperfusion(I/R) for the first time, which provides a reference for guiding the rational application of borneol in the early treatment of ischemic stroke and has important academic and application values. Healthy specific pathogen-free(SPF)-grade SD male rats were randomly assigned into 13 groups: a sham-operation group, a model group, a Tween model group, a positive drug(nimodipine) group, and high-, medium-, and low-dose(0.2, 0.1, and 0.05 g·kg~(-1), respectively) groups of L-borneol, natural borneol, and synthetic borneol according to body weight. After 3 days of pre-administration, the rat model of I/R was established by suture-occluded method and confirmed by laser speckle imaging. The corresponding agents in different groups were then administered for 1 day. The body temperature was monitored regularly before pre-administration, days 1, 2, and 3 of pre-administration, 2 h after model awakening, and 1 d after model establishment. Neurological function was evaluated based on Zea-Longa score and modified neurological severity score(mNSS) 2 h and next day after awakening. The rats were anesthetized 30 min after the last administration, and blood was collected from the abdominal aorta. Enzyme-linked immunoassay assay(ELISA) was employed to determine the serum levels of tumor necrosis factor-alpha(TNF-α), interleukin-6(IL-6), IL-4, and transforming growth factor-beta1(TGF-β1). The brain tissues were stained with triphenyltetrazolium chloride(TTC) for the calculation of cerebral infarction rate, and hematoxylin-eosin(HE) staining was used for observing and semi-quantitatively evaluating the pathological damage in different brain regions. Immunohistochemistry was employed to detect the expression of ionized calcium binding adapter molecule 1(IBA1) in microglia. q-PCR was carried out to determine the mRNA levels of iNOS and arginase 1(Arg1), markers of polarization phenotype M1 and M2 in microglia. Compared with the sham-operation group, the model group and the Tween model group showed significantly elevated body temperature, Zea-Longa score, mNSS, and cerebral infarction rate, severely damaged cortex, hippocampus, and striatum, increased serum levels of IL-6 and TNF-α, and decreased serum levels of IL-4 and TGF-β1. The three borneol products had a tendency to reduce the body temperature of rats 1 day after modeling. Synthetic borneol at the doses of 0.2 and 0.05 g·kg~(-1), as well as L-borneol of 0.1 g·kg~(-1), significantly reduced Zea-Longa score and mNSS. The three borneol products at the dose of 0.2 g·kg~(-1) significantly reduced the cerebral infarction rate. L-borneol at the doses of 0.2 and 0.1 g·kg~(-1) and natural borneol at the dose of 0.1 g·kg~(-1) significantly reduced the pathological damage of the cortex. L-borneol and natural borneol at the dose of 0.1 g·kg~(-1) attenuated the pathological damage of hippocampus, and 0.2 g·kg~(-1) L-borneol attenuated the damage of striatum. The 0.2 g·kg~(-1) L-borneol and the three doses of natural borneol and synthetic borneol significantly reduced the serum level of TNF-α, and the 0.1 g·kg~(-1) synthetic borneol reduced the level of IL-6. L-borneol and synthetic borneol at the dose of 0.2 g·kg~(-1) significantly inhibited the activation of cortical microglia, and 0.2 g·kg~(-1) L-borneol up-regulated the expression of Arg1 and down-regulated the expression level of iNOS. In conclusion, the three borneol products may alleviate inflammation to ameliorate the pathological damage of brain regions of rats in the acute phase of I/R by inhibiting the activation of microglia and promoting the polarization of microglia from M1 type to M2 type. The protective effect on brain followed a trend of L-borneol &gt; synthetic borneol &gt; natural borneol. We suggest L-borneol the first choice for the treatment of I/R in the acute phase.

Comparison of Chemiluminescence Enzyme Immunoassay (Cl-ELISA) with Colorimetric Enzyme Immunoassay (Co-ELISA) for Imidacloprid Detection in Vegetables

Imidacloprid is one of the most commonly used insecticides for managing pests, thus, improving the quality and yield of vegetables. The abuse/misuse of imidacloprid contaminates the environment and threatens human health. To reduce the risk, a colorimetric enzyme-linked immunoassay assay (Co-ELISA) and chemiluminescence enzyme-linked immunoassay assay (Cl-ELISA) were established to detect imidacloprid residues in vegetables. The linear range of Co-ELISA ranged between 1.56 μg/L and 200 μg/L with a limit of detection (LOD) of 1.56 μg/L. The values for Cl-ELISA were 0.19 μg/L to 25 μg/L with an LOD of 0.19 μg/L, which are lower than those of Co-ELISA. Fortifying Chinese cabbage, cucumber, and zucchini with imidacloprid at 10, 50, and 100 μg/L yielded recoveries between 81.7 and 117.6% for Co-ELISA and at 5, 10, and 20 µg/L yielded recoveries range from 69.7 to 120.6% for Cl-ELISA. These results indicate that Cl-ELISA has a high sensitivity and a rapid detection time, saving cost (antigen and antibody concentrations) and serving as a more efficient model for the rapid detection of imidacloprid residue.

Detecting Aflatoxin B1 in Peanuts by Fourier Transform Near-Infrared Transmission and Diffuse Reflection Spectroscopy.

Aflatioxin B1 (AFB1) has been recognized by the International Agency of Research on Cancer as a group 1 carcinogen in animals and humans. A fast, batch, and real-time control and no chemical pollution method was developed for the discrimination and quantification prediction of AFB1-infected peanuts by applying Fourier transform near-infrared (FT-NIR) coupled with chemometrics. Initially, the near-infrared transmission (NIRT) and diffuse reflection (NIRR) modules were applied to collect spectra of the samples. The principal component analysis (PCA) method was employed to extract the characteristic wavelength, followed by different preprocessing methods (seven methods) to build an effective linear discriminant analysis (LDA) classification and partial least squares (PLS) quantification models. The results showed that, for both the NIRT or NIRR modules, the LDA classification models satisfactorily distinguished peanuts infected with AFB1 or from those not infected, with external validation showing a 100% correct identification rate and a 0% misjudgment rate. In addition, combined with the concentration of AFB1 in peanuts determined by enzyme-linked immunoassay assay, the best partial least squares (PLS) models were established, with a combination of the first derivative and the Norris derivative filter smoothing pretreatment (Rc2 = 0.937 and 0.984, RMSECV = 3.92% and 2.22%, RPD = 3.98 and 7.91 for NIRR and NIRT, respectively). The correlation coefficient between the predicted value and the reference value in the external verification was 0.998 and 0.917, respectively. This study highlights that both spectral acquisition modules meet the requirements of online, rapid, and accurate identification of peanut AFB1 infection in the early stages.

A copro-antigen ELISA for the detection of ascarid infections in chickens

A reliable method of diagnosing the most prevalent helminth infections in chickens is vital for developing effective control strategies. Ascaridia galli and Heterakisgallinarum are phylogenetically close nematode species that can elicit the development of cross-reactive antibodies in chickens. Therefore, an enzyme-linked immunoassay (ELISA) based on Ascaridia galli antigens in faeces of chickens to detect and quantify infections with both A. galli and H. gallinarum was developed. The ELISA utilised polyclonal antibodies that were obtained from rabbits immunised with soluble antigens isolated from A. galli. In two separate experiments, chickens were kept as uninfected controls or were orally infected with either 100 or 1000 of embryonated eggs of A. galli or H.gallinarum. Faecal samples were collected after 28–30 weeks post-infection. The ELISA was then used to quantify the concentration of soluble worm antigens in faecal samples, i.e., the amount of antigen per gram faeces, APG. The APG from infected chickens was significantly higher than non-infected groups in both experiments (P 0.001). Both 100 and 1000 infection dose groups were not significantly different (P = 0.999) in the experiment with H. gallinarum, whereas in the experiment with A. galli, APG was significantly higher in the 1000 infection group (P 0.001). A receiver operation characteristics (ROC) analysis that evaluates the qualitative performance of diagnostics tests was used to calculate the assay parameters within each mono-infection experiment. The result showed that the assay had a high diagnostics accuracy with an area-under-curve (AUC) of 0.99 in detecting infection in A. galli infected chickens and a moderate-high accuracy (AUC = 0.89) in birds infected with H. gallinarum. The diagnostic sensitivity and specificity of the assay at the optimal cut-off point equivalent to Youden index were 93% and 100% for detecting infections in A. galli experiment and 85% and 92% in H. gallinarum experiment, respectively. The correlation between faecal antigen concentration and all worm burden parameters was positive but generally low (r < 0.33), which provided less information about infection intensities. Nonetheless, these results indicate that a reliable and accurate qualitative diagnosis of the two most prevalent intestinal nematodes in chickens can be achieved using a non-invasive copro-antigen ELISA assay.

Deficiency of Vitamin B12 in Type 2 Diabetic Patients Receiving Metformin Therapy at HMC Hospital Peshawar

Background: Diabetes mellitus is on the rise at an alarming rate, and it has now become a global issue. Type 2 Diabetic Mellitus is a disease characterized by disturbances in glucose, protein, and fat metabolism. Diabetes has been increasingly common in recent decades, causing significant socioeconomic burden, particularly in poorer countries.&#x0D; Objective: To investigate the prevalence of vitamin B12 deficiency in Type 2 diabetes patients taking metformin.&#x0D; Methodology: This study was conducted on T2 diabetic patients in the Diabetic Department of Diabetes and Endocrinology HMC Hospital, Peshawar, for a year (10 February 2019 to 12 March 2020). The demographic data of the recruited patients was obtained after they gave their informed written consent. Patients inquired about their metformin use history, dosage, and duration of type 2 diabetes mellitus. A 5cc venous blood sample was taken from the arm and kept at 4 degrees Celsius. Vitamin B12 levels were measured using enzyme-linked immunoassay assays after serum was withdrawn. ELISA was used to determine the Vitamin B12 level in serums.&#x0D; Results: A total of 149 people were enrolled in this trial. The patients' average age was 55 years. In this study, there were 67 (38.8%) males and 82 (55.2%) females. The individuals had a 5.12-year history of diabetes mellitus. The average HbA1c level was 8.280.32. The individuals that were included have taken metformin for at least 4.8 years. There were 25 (22.6%) patients using 1000-1500 mg metformin daily, 76 (46.4%) receiving 1500-2000 mg metformin daily, and 48 (40.7%) taking greater than 2000 mg metformin daily. There were 44 patients with a normal BMI, 80 patients who were overweight, and 25 patients who were obese. The average B12 level in the blood was 223pg/ml. Vitamin B12 deficiency was found in 96 (49.1%) of the patients. There was no vitamin B12 deficiency in 53 (45.4%) of the patients.&#x0D; Recommendations: It is strongly advised based on the findings of this study that Serum cobalamin levels should be measured in Type II diabetes patients who are taking metformin, and patients should be given R.D.A (Recommended Dietary Allowance) multivitamins to avoid sequaela of cobalamin deficiency.

Accuracy of multitarget indirect enzyme-linked immunoassay assay for detection of tuberculosis antibody.

BackgroundDiagnosis of tuberculosis (TB) is still difficult. The development of rapid and sensitive laboratory tools for the diagnosis of tuberculosis is a priority. This study aimed to develop an indirect enzyme-linked immunoassay (ELISA) assay for detection of TB antibody and explore its diagnostic value in patients with pulmonary tuberculosis (PTB) via a multi-center clinical evaluation.MethodsThe specific antigen, fusion antigen, and specific antibody peptide were obtained using molecular cloning and phage peptide library screening. An indirect ELISA assay was developed using multiple target materials. Further, the assay was validated in six institutions with clinically confirmed TB patients, non-TB patients with pulmonary disease, and healthy controls as research subjects.ResultsAn indirect ELISA assay was developed with 16 kD antigen, 11,488 (CFP10-MPT48-TB8.4) fusion antigen, and TB18 and pl2 as target antigens against TB antibody. The results of this multicenter study showed that the sensitivity, specificity, and the area under the receiver operating characteristic curve (AUC) of the assay were 48.25% [95% confidence interval (CI): 45.5–51.1%], 92.20% (95% CI: 90.7–93.5%) and 0.724 (95% CI: 0.707–0.741), respectively, and the cut-off value was 0.119. According to the meta-analysis, the combined ROC was 0.736 (95% CI: 0.692–0.779), I2=83.73%. The sensitivity of the sputum-positive PTB group (culture or smear positive) was 58.75% (95% CI: 52.96–65.00%); the sensitivity in sputum-negative group (culture or smear negative) was 37.38% (95% CI: 32.71–42.52%), respectively; the sensitivity of the sputum-positive group was significantly higher than that of sputum-negative group (OR =1.57, 95% CI: 1.29–1.92, P<0.001).ConclusionsMultitarget indirect ELISA assay based on specific-TB antigen, fusion antigen, and antibody peptide is of value for the diagnosis of PTB and can be used as an auxiliary rapid diagnostic tool to improve the sensitivity of sputum-negative TB.

Effects of Fuke Qianjin Formula on hormones and their receptors and metabonomics study in uterine fibroids model rats

ObjectiveTo investigate the effects of different fractions from Fuke Qianjin Formula (妇科千金方, FKQJF) on uterine leiomyoma (UL) to determine the best fraction. MethodsFKQJF was extracted and isolated to obtain polysaccharides (FKP), flavonoids (FKF), and grease (FKG). 140 female SPF SD rats were divided into 14 groups [model (MOD), normal control (NC), Gouliuqing (GLQ), Mifepristone (MFST), FKQJF, low, medium, and high dose of polysaccharides (l-FKP, m-FKP, and h-FKP), low, medium, and high dose of flavonoids (l-FKF, m-FKF, and h-FKF), low, medium, and high dose of grease (l-FKG, m-FKG, and h-FKG)], and uterine fibroids model rats were treated with drugs for four weeks. Serum levels of estrogen and progesterone were measured using enzyme-linked immunoassay assay (ELISA) kits. The expression of estrogen receptor (ER-α, ER-β) and progesterone receptor (PR) in the uterus was observed using immunohistochemistry (IHC). Serum metabolite profiles and FKG were analyzed using gas chromatography-mass spectrometry (GC-MS). ResultsFKQJF, h-FKF, m-FKG, and h-FKG significantly downregulated the estrogen level in the uterine fibroid model rats (P < 0.01). FKQJF, h-FKF, and h-FKG significantly reduced the level of progesterone in the uterine fibroid model rats (P < 0.01). The levels of ER-α, ER-β, and PR in uterine fibroid model rats were significantly decreased by FKQJF and h-FKG (P < 0.01). The levels of ER-α, ER-β, and PR in the fibroid model rats were decreased by m-FKG (P < 0.05). Additionally, serum metabolism results revealed that h-FKG and FKQJF could regulate related endogenous metabolites and make the pathological indices of uterine fibroids in rats close to the normal group. Forty-six components were identified in the oil, accounting for 91.97% of the total oil components. ConclusionFKQJF and h-FKG showed a significant anti-myoma activity and significantly improved the pathological state of the uterus in rats with hysteromyoma. The mechanism of action may be related to the regulation of estrogen progesterone and its receptor in uterine fibroid model animals. These findings proved the effect of FKQJF on uterine leiomyoma and provided an experimental basis for its clinical research and application.

Interleukin-10 (IL-10) 1082 promoter polymorphisms and plasma IL-10 levels in patients with bacterial sepsis.

Background. Interleukin-10 (IL-10) is a multifunctional cytokine which has been seen to play a relevant role in the pathogenesis of sepsis. We examined the association between a single nucleotide polymorphism (SNP) in IL-10-1082G/A in patients with sepsis in Cali city.Methods. A total of 100 patients with sepsis and 50 control subjects were enrolled in this study. Blood samples were collected from all patients in EDTA containing tubes. IL-10-1082G/A gene promoter polymorphism was analyzed by Sequence Specific Polymerase Chain Reaction (SS-PCR), while levels of serum IL-10 were measured by Enzyme Linked Immunoassay Assay (ELISA) in patients with sepsis and healthy controls.Results. AA homozygous genotype was found more frequently in patients (32%), compared with controls (18%). AA homozygous patients showed an increased risk of developing infection by Gram-negative bacteria (OR = 2,875; 95% CI = 1.162-7.113; p = 0.020), and significantly high plasma levels of IL-10 (OR = 4.800, 95% CI 1.652-13.944; p = 0.002). AA homozygous patients high plasma IL-10 levels have greater risk of developing sepsis (63.6%; OR = 4,894; 95% CI: 1,337-17,909; p = 0.002). In this group, Afro-Colombian individuals were overrepresented among the sepsis patients with high plasma IL-10 levels (OR = 1.661; 95% CI: 1.408-1.959; p = 0.036).Conclusion. Our study concluded that AA genotype of IL-10-1082G/A polymorphism is a risk factor for high IL-10 production and development of sepsis by Gram negative bacteria, especially in Afro-Colombian patients.

Seroprevalence of neutralizing antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among healthcare workers in Makkah, Saudi Arabia

ObjectiveThe new coronavirus disease 2019 (COVID-19) is a major health problem worldwide. The surveillance of seropositive individuals serves as an indicator to the extent of infection spread and provides an estimation of herd immunity status among population. Reports from different countries investigated this issue among healthcare workers (HCWs) who are “at risk” and “sources of risk” for COVID-19. This study aims to investigate the seroprevalence of COVID-19 among HCWs in one of the COVID-19 referral centers in Makkah, Saudi Arabia using three different serological methods. MethodsIn-house developed enzyme-linked immunoassay (ELISA), commercially available electro-chemiluminescence immunoassay (ECLIA), and microneutralization (MN) assay were utilized to determine the seroprevalence rate among the study population. 204 HCWs participated in the study. Both physicians and nurses working in the COVID-19 and non COVID-19 areas were included. Twelve out of 204 were confirmed cases of COVID-19 with variable disease severity. Samples from recovered HCWs were collected four weeks post diagnosis. ResultsThe overall seroprevalence rate was 6.3% (13 out of 204) using the in-house ELISA and MN assay and it was 5.8% (12 out of 204) using the commercial ECLIA. Among HCWs undiagnosed with COVID-19, the seroprevalence was 2% (4 out 192). Notably, neutralizing antibodies were not detected in 3 (25%) out 12 confirmed cases of COVID-19. ConclusionsOur study, similar to the recent national multi-center study, showed a low seroprevalence of SARS-Cov-2 antibodies among HCWs. Concordance of results between the commercial electro-chemiluminescence immunoassay (ECLIA), in-house ELISA and MN assay was observed. The in-house ELISA is a promising tool for the serological diagnosis of SARS-CoV-2 infection. However, seroprevalence studies may underestimate the extent of COVID-19 infection as some cases with mild disease did not have detectable antibody responses.

TGF-β1-Mediated Activation of SERPINE1 is Involved in Hemin-Induced Apoptotic and Inflammatory Injury in HT22 Cells.

BackgroundIntracerebral hemorrhage (ICH) is a severe subtype of stroke with high mortality and morbidity. Serpin Family E Member 1 (SERPINE1) has been documented to be upregulated following ICH, however, the participation of SERPINE1 in the development of ICH has never been studied.MethodsHemin was utilized to develop an in vitro model of ICH. Gene levels were evaluated by the use of quantitative reverse transcription polymerase chain reaction, Western blot, as well as enzyme-linked immunoassay assay. The activity of caspase-3 was determined using a commercial kit. Cell viability and apoptosis were assessed using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and Terminal deoxynucleotidyl transferase (TdT) d UTP Nick-End Labeling assay.ResultsSERPINE1 was upregulated in hemin-treated HT22 cells. Silencing of SERPINE1 attenuated hemin-induced inhibition of cell viability. Moreover, knockdown of SERPINE1 repressed hemin-induced apoptosis in HT22 cells, as evidenced by the decrease in the number of TUNEL positive cells, caspase-3 activity, and Bax expression, and the increase in Bcl-2 expression. Meanwhile, knockdown of SERPINE1 repressed hemin-induced inflammation in HT22 cells, as indicated by reduced levels of tumor necrosis factor-α, interleukin-6 (IL-6), IL-1β, and inducible nitric oxide synthase. We also found that transforming growth factor-beta 1 (TGF-β1) induced SERPINE1 expression in a dose-dependent manner. Besides, SERPINE1 knockdown attenuated the effects of TGF-β1 on hemin-induced neuronal damage.ConclusionTGF-β1-induced SERPINE1 activation exacerbated hemin-induced apoptosis and inflammation in HT22 cells, manifesting a novel mechanism for ICH progression.

Electroacupuncture Pretreatment Alleviates LPS-Induced Acute Respiratory Distress Syndrome via Regulating the PPAR Gamma/NF-Kappa B Signaling Pathway.

Electroacupuncture (EA) is reported to possess anti-inflammatory properties and has beneficial effects on acute respiratory distress syndrome (ARDS). However, the underlying mechanisms of the effects of EA on ARDS remain unclear. This study aims to investigate the protective effect of EA on LPS-induced ARDS. In this study, Sprague-Dawley male rats were treated with EA at Hegu (LI4) for 45 minutes before LPS instillation (0.4 mg/kg, 100 ul). H&E staining, wet-to-dry weight (W/D) ratio, PaO2, and protein content in BALF were employed to determine the function of lung tissues. Inflammatory cytokines in serum and BALF were detected by enzyme-linked immunoassay assay (ELISA). The levels of oxidative stress markers were detected to determine the oxidative stress status. Cell apoptosis was observed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining and western blot. Here, we found that EA pretreatment effectively alleviated lung pathological damage. Moreover, EA suppressed the oxidative stress damage by upregulating glutathione and superoxide dismutase and downregulating malondialdehyde. EA pretreatment also regulated apoptosis-related proteins, such as Bax and Bcl-2. We found that peroxisome proliferators-activated receptors γ (PPARγ) play a critical role during ARDS, EA up-regulated the expression of PPARγ, which inhibited the activation of nuclear factor-kappa B (NF-κB) and decreased the inflammatory cytokines (interleukin-1β, interleukin-6, and tumor necrosis factor-α). When rats were treated with GW9662, a selective PPARγ antagonist, these effects of EA were reversed. Our study demonstrated that EA pretreatment had a beneficial effect on LPS-induced ARDS in rats by anti-inflammatory, antioxidative, and antiapoptotic properties which was regulated via PPARγ/NF-κB signaling pathway.

MYC Participates in Lipopolysaccharide-Induced Sepsis via Promoting Cell Proliferation and Inhibiting Apoptosis.

Objective This study aimed to explore the potential mechanism of MYC proto-oncogene, BHLH Transcription Factor (MYC) gene, on sepsis. Materials and Methods In this experimental study, rat-derived H9C2 cardiomyocyte cells were cultured in vitro, followed by lipopolysaccharide (LPS) treatment with different concentration gradients. The cholecystokinin octapeptide (CCK-8) assay, enzyme-linked immunoassay (ELISA) assay, quantitative reverse transcription polymerase chain reaction (qRT-PCR), cell transfection, Western blot and flow cytometry were used to observe the cellular apoptosis and proliferation of cells in both treated LPS groups and normal control group. Results The result of CCK-8 assay showed that silencing MYC inhibited cellular proliferation of sepsis in absence or presence of LPS treatment. ELISA assay showed that the expressions of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were decreased in MYC silenced group, but they were increased after LPS treatment. Moreover, Flow cytometry assay showed that MYC silencing contributed to the apoptosis of sepsis cells. Furthermore, the expression of inflammatory factors showed that MYC silencing elevated the expression of inflammation factors. ConclusionMYC might take part in the process of LPS induced sepsis through suppressing apoptosis and inducing cell proliferation. Moreover, MYC might reduce inflammation during the progression of LPS induced sepsis.

First report of Neospora caninum infection in pigs in China.

Neospora caninum is a protozoan parasite which can infect many mammals and birds with a worldwide distribution. However, no molecular data are available about the occurrence of N.caninum in pigs. In this study, the serological and molecular prevalence of N.caninum infection in farmed pigs were investigated in Hunan province, China, between January 2017 and December 2018. A total of 1,500 serum samples collected from 10 herds in Hunan province were evaluated using a competitive-inhibition enzyme-linked immunoassay assay (cELISA). The overall seroprevalence of N.caninum in the examined pigs was 1.9%. The seroprevalence of N.caninum ranged from 0.3% to 4.6% among different regions in Hunan province of China (p<.05). DNA was extracted from brain samples, and the Nc-5 gene and ITS-1 region were amplified and then sequenced. Three (0.5%) of the examined 600 brain tissues were found to contain N.caninum DNA. Our phylogenetic analyses indicated that N.caninum samples were classified into two distinct groups. Although the prevalence is low within the pig groups investigated, our results revealed the emergence of N.caninum infection in pigs in China. The finding of the present study provides molecular evidence that the pigs are the natural intermediate host of N.caninum and may have major epidemiological importance.

ASSESSING NUCLEIC ACID TESTING VERSUS ELIZA FOR BLOOD VIRUSES DETECTION IN SOME BLOOD BANKS

INTRODUCTION: The goal of any transfusion service is to provide adequate and safe blood and blood products that meet the needs of patients. Transfusion-transmissible viral infections, such as hepatitis C virus (HCV), hepatitis B virus (HBV), and human immunodeficiency virus (HIV), remain a major public health problem in developing countries. Nucleic acid testing (NAT) is a molecular technique for screening blood donations to reduce the risk of transfusion transmitted infections (TTIs) in the recipients, thus providing an additional layer of blood safety. AIM OF THE WORK: To assess the importance of implementing NAT assay to detect donors during window period which are not detected with Enzyme-linked immunoassay (ELISA). SUBJECTS AND METHODS: This cross sectional study was conducted at the Egyptian Abbassia regional Blood Transfusion center (ARBTC) at Abbassia, Cairo. Blood donation collected from 10000 voluntary donors from January 2016 to June 2016 and tested with both ID NAT and ELISA assays for HBV, HCV and HIV. RESULTS: NAT testing has the potential to detect viral nucleic acids of HIV 1-2, HBV, and HCV earlier than current screening methods. CONCLUSION: The implementation of NAT screening for three viruses has improved blood safety and reduced transfusion-transmitted infections.

The association of IL-18 gene promoter polymorphisms and the levels of serum IL-18 on the risk of multiple sclerosis

ObjectivesMultiple sclerosis is an inflammatory condition of the central nervous system whose etiology is influenced by immunologic, genetic, and environmental factors. Aim of the present study was to determine if any relation exists between IL-18 −137C/G and −607C/A gene promoter polymorphisms on the individual susceptibility of multiple sclerosis and also to investigate the possible effect of IL-18 activity regarding this kind of polymorphism and MS. Patients and methods113 patients with clinically definite MS and 135 ethnically-matched controls were participated in this study. IL-18 −137C/G and −607C/A gene promoter polymorphisms were analyzed by Sequence Specific Polymerase Chain Reaction (SS-PCR), while levels of serum IL-18 were measured by Enzyme Linked Immunoassay Assay (ELISA) in patients with MS and healthy controls. ResultsOur results showed that the IL-18 −607AA genotype indicated 6 times higher risk in the development of MS (OR=6.883; 3.17–14.96; p<0.001). According to our findings, smoking seems to be an important confounding factor in MS patients with carrying IL-18 −607 AA and CA+AA genotypes. However, no meaningful association was found with IL-18 −137C/G gene promoter polymorphism. ConclusionIn conclusion, we suggest that IL-18 −607C/A gene promoter polymorphism is a major genetic factor for determining individual susceptibility to MS, where smoking status also increases the risk of MS.