Enzyme-linked Immunosorbent Assay Results Research Articles

Rhein Suppresses Lung Inflammatory Injury Induced by Human Respiratory Syncytial Virus Through Inhibiting NLRP3 Inflammasome Activation via NF-κB Pathway in Mice.

Rhein is one of active anthraquinone components in traditional Chinese herbal medicine Rheum palmatum L., possessing anti-inflammatory, antioxidant, antitumor, antiviral, and hepatoprotective activities. Human respiratory syncytial virus (RSV), a common virus, is able to result in pneumonia and bronchitis, which usually can be seen in infants. However, so far the effects of Rhein on RSV-induced pneumonia are still unknown. As the NLRP3 inflammasome is activated excessively, it is able to lead to inflammatory response and tissue injury in most viral infection process (including RSV infection) of respiratory tract. Therefore, we designed experiments to reveal whether Rhein can treat RSV-induced pneumonia by inhibiting NLRP3 inflammasome activation. In present research, we established the pneumonia model of BALB/C mice caused by RSV. First of all, the pathology of lung tissue and the weight of mice were evaluated, and the corresponding lung index was calculated. Additionally, the expression of pro-inflammatory mediators in serum and lung tissues, and related proteins (NLRP3, ASC and Caspase-1) of NLRP3 inflammasome and NF-κB pathway were detected by Enzyme-linked immunosorbent assay (ELISA), Real-time PCR (RT-PCR), Immunohistochemistry (IHC), and Western blot (WB), respectively. The determination of lung index and lung tissue pathological evaluation revealed that Rhein was able to alleviate lung infection and injury caused by RSV. The results of ELISA showed that Rhein was able to reduce the release of pro-inflammatory cytokines in the serum and lung tissues of RSV-induced BALB/c mice, including IL-1β, IL-6, TNF-α, IL-18, and IL-33. Additionally, it was revealed that Rhein inhibited the immune inflammatory response of RSV-infected mice, which was likely to be associated with the inhibition the NLRP3 inflammasome activation via NF-κB pathway. To sum up, our results indicated that Rhein may inhibit RSV-induced pulmonary inflammatory response effectively; meanwhile, it is emphasized that Rhein therapy is likely to be a promising treatment on the RSV-infected lung inflammation and avoidance of lung tissue damage.

Development and Potential Application of Ras Domain Containing Protein from Haemonchus contortus for Diagnosis of Goat Infection.

Simple SummaryThe early diagnosis of Haemonchosis is important for the control and effective treatment of the disease. Early stage detection of Haemonchus contortus infection has not been attempted in goats, even though the immature worms are blood-sucking. This study was carried out to detect H. contortus infection during the early and late stages in goats. The results of this study assessed that recombinant H. contortus Ras domain-containing protein (rHcRas) could detect the antibody in H. contortus-infected goat sera during the prepatent and postpatent periods with high sensitivity and specificity using immunodiagnostic techniques. Our findings illustrated that indirect ELISA based on rHcRas also had the potential to detect H. contortus infection from field samples.Haemonchus contortus is an important gastrointestinal nematode of small ruminants that causes significant mortality in goats worldwide. Diagnosis of this infection mainly depends on the evaluation of clinical signs and fecal examination. However, limitations often occur in early or mild infections. For this purpose, serological diagnosis seems to be more accurate and reliable. Ras domain-containing protein (Ras) is one of H. contortus’s excretory and secretory products (ESPs) that can be isolated from different larval stages of the nematode. In this study, the recombinant H. contortus Ras domain-containing protein (rHcRas) was expressed and purified and its diagnostic potential was evaluated. Reactions between rHcRas and goat sera were tested using Western blotting (WB). The results showed that rHcRas could be recognized by sera as early as 14 days post infection (DPI), and antibodies against rHcRas in infected goats could be maintained for over 89 days. No reaction was found between rHcRas and antibodies against Trichinella spiralis, Fasciola hepatica, or Toxoplasma gondii. An indirect enzyme-linked immunosorbent assay (ELISA) was produced based on rHcRas. The optimal coating antigen (157 ng of rHcRas/well) and serum dilutions (1:50) were determined via checkerboard titration. Indirect ELISA based on rHcRas showed 87.5% sensitivity and 90.6% specificity. The cut-off values for this experiment were determined to be 0.324 (positive) and 0.273 (negative), respectively, and the variation coefficient (CV) was less than 15%. The results of the indirect ELISA in-field examination showed that 17.6% (9/51) of the goats were infected with H. contortus, higher than the fecal examination results (15.7%, 8/51). When compared the results of the indirect ELISA and necropsy testing, 98.0% (50/51) consistency was found. These results indicated that rHcRas was a potential antigen for the diagnosis of H. contortus infection in goats.

Epidemiological investigation of porcine pseudorabies virus and its coinfection rate in Shandong Province in China from 2015 to 2018.

BackgroundPseudorabies, also known as Aujeszky's disease, is caused by the pseudorabies virus (PRV) and has been recognized as a critical disease affecting the pig industry and a wide range of animals around the world, resulting in great economic losses each year. Shandong province, one of the most vital food animal-breeding regions in China, has a very dense pig population, within which pseudorabies infections were detected in recent years. The data, however, on PRV epidemiology and coinfection rates of PRV with other major swine diseases is sparse.ObjectivesThis study aimed to investigate the PRV epidemiology in Shandong and analyze the current control measures.MethodsIn this study, a total number of 16,457 serum samples and 1,638 tissue samples, which were collected from 362 intensive pig farms (≥ 300 sows/farm) covered all cities in Shandong, were tested by performing enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR).ResultsOverall, 52.7% and 91.5% of the serum samples were positive for PRV-gE and -gB, respectively, based on ELISA results. In addition, 15.7% of the tissue samples were PCR positive for PRV. The coinfection rates of PRV with porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus, and classical swine fever virus were measured; coinfection with PCV2 was 35.0%, higher than those of the other two viruses. Macroscopic and microscopic lesions were observed in various tissues during histopathological examination.ConclusionsThe results demonstrate the PRV prevalence and its coinfection rates in Shandong province and indicate that pseudorabies is endemic in pig farms in this region. This study provides epidemiological data that can be useful in the prevention and control of pseudorabies in Shandong, China.

Paeoniflorin improves the permeability of cardiac microvascular endothelial cells by regulating Src/vascular endothelial-cadherin pathway

To investigate the effect and mechanism of paeoniflorin on the permeability of cardiac microvascular endothelial cells (CMECs) in sepsis. Primary rat CMECs were isolated and cultured in vitro, and the cells in the logarithmic growth phase were used for experiments. Tetramethylazozolium colorimetry (MTT) was used to screen the safe and effective concentrations of paeoniflorin at 10, 20, and 40 μmol/L. The cells were divided into blank control group, lipopolysaccharide (LPS) group and low, medium and high concentration paeoniflorin pretreatment group. The cells in the blank control group were cultured in complete medium; the cells in the LPS group were challenged with LPS (1 mg/L) in complete medium; and the cells in the paeoniflorin pretreatment groups were pretreated with 10, 20, and 40 μmol/L paeoniflorin at 4 hours before LPS stimulation. The cells in each group were further cultured for 24 hours after LPS stimulation. The horseradish peroxidase (HRP) method was used to detect the permeability of rat CMECs. The enzyme-linked immunosorbent assay (ELISA) was used to detect the CXC chemokine ligand (CXCL1, CXCL2) levels in the cell supernatant. The real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the mRNA expressions of CXCL1 and CXCL2 in the cells. Western Blot was used to detect phosphorylated Src (p-Src), vascular endothelial-cadherin (VE-cadherin) and phosphorylated mitogen activated protein kinase (p-MAPK). Compared with the blank control group, the permeability of rat CMECs in the LPS group was significantly increased. The cell permeability was improved to some extent after paeoniflorin pretreatment at different concentrations, and the improvement was most obvious in the 40 μmol/L paeoniflorin group, with statistically significant difference as compared with the LPS group (A value: 1.61±0.07 vs. 2.13±0.06, P < 0.01). ELISA results showed that there were moderate amounts of CXCL1 and CXCL2 in the cell supernatant of rat CMECs in the blank control group. However, the secretion of CXCL1 and CXCL2 in the cell supernatant was increased significantly under the induction of LPS. After pretreatment with paeoniflorin at different concentrations, the secretion of CXCL1 and CXCL2 in the cell supernatant was significantly reduced. The most obvious inhibitory effect on CXCL1 was 40 μmol/L paeoniflorin, and the most obvious inhibition on CXCL2 was 20 μmol/L paeoniflorin, the differences were statistically significant as compared with the LPS group [CXCL1 (ng/L): 337.51±68.04 vs. 829.86±65.06, CXCL2 (ng/L): 4.48±0.11 vs. 9.41±0.70, both P < 0.01]. RT-qPCR results showed that the mRNA expressions of CXCL1 and CXCL2 in the rat CMECs were consistent with the ELISA results. LPS could increase mRNA expressions of CXCL1 and CXCL2 in the rat CMECs, and pretreatment with different concentrations of paeoniflorin could significantly reduce the mRNA expressions of CXCL1 and CXCL2. The 40 μmol/L paeoniflorin had the best inhibitory effect on CXCL1 mRNA expression, and the 20 μmol/L paeoniflorin had the best inhibitory effect on CXCL2 mRNA expression, the differences were statistically significant as compared with the LPS group [CXCL1 mRNA (2-ΔΔCt): 0.543±0.004 vs. 0.812±0.089, CXCL2 mRNA (2-ΔΔCt): 10.52±0.71 vs. 17.68±1.09, both P < 0.01]. Western Blot results showed that moderate amounts of p-Src, VE-cadherin and p-MAPK proteins were expressed in the rat CMECs in the blank control group. After LPS stimulation, the expressions of p-Src and p-MAPK proteins were increased significantly, while the expression of VE-cadherin protein was decreased significantly. After pretreatment with different concentrations of paeoniflorin, the expressions of p-Src and p-MAPK proteins in the cells were decreased to varying degrees, while the expression of VE-cadherin protein was increased, and 40 μmol/L paeoniflorin had the most obvious effect, the differences were statistically significant as compared with the LPS group [p-Src protein (p-Src/GAPDH): 1.02±0.09 vs. 1.29±0.05, p-MAPK proteins (p-MAPK/GAPDH): 0.24±0.02 vs. 0.62±0.02, VE-cadherin protein (VE-cadherin/GAPDH): 0.64±0.03 vs. 0.31±0.02, all P < 0.01]. Paeoniflorin can regulate the Src/VE-cadherin pathway in CMECs, inhibit the expression and secretion of inflammation-related proteins and chemokines, and improve the cell permeability of CMECs induced by LPS.

Results of enzyme-linked immunoassay of the blood serum in patients with infectious and inflammatory diseases of the anterior segment of the eye

To evaluate the clinical significance of the data obtained during enzyme-linked immunosorbent assay (ELISA) of the blood serum of patients with inflammatory diseases of the anterior eye segment compared to a group of healthy volunteers. A retrospective analysis of the results of serum ELISA of 200 patients with chronic keratoconjunctivitis and keratouveitis was performed using the solid-phase IFA method in order to detect the presence of antibodies to herpes simplex viruses of 1 and 2 types (HSV 1, 2), cytomegalovirus (CMV), Chlamydia trachomatis and Toxoplasma gondii. The control group consisted of 34 healthy volunteers with no signs of inflammation of the eye tissue. There were no significant differences in the frequency of detection of both «acute-phase» IgM and «chronic» IgG to HSV and CMV between patients of the main group and healthy volunteers (ρ≥0.05). The levels of IgG to Toxoplasma gondii in the study group were three times higher than in the control group (ρ≤0.05). A comparative analysis of the frequency of detection of antibodies to Chlamydia trachomatis showed statistically significant differences between the groups and more frequent detection of immunoglobulins classes A, G and M in patients with inflammatory eye diseases (ρ≤0.05). The absence of statistical differences with the control group in the frequency of detection of active HSV and CMV infections in patients with inflammatory diseases of the anterior eye segment when using ELISA allows to recommend the use of additional methods of examination such as polymerase chain reaction (PCR) and real-time PCR (RT-PCR) with identification of pathogen genetic material in the available biological secretions. The increase in IgG titers to toxoplasma in patients of the main group apparently indicates a cross-stimulation of antibody synthesis against the background of a chronic inflammatory process. Significant differences in the frequency of detection of all types of antibodies to chlamydiae suggests a significant etiological role of this pathogen in the development and maintenance of chronic inflammation in the anterior eye segment.

Role of the erythropoietin receptor in Lung Cancer cells: erythropoietin exhibits angiogenic potential.

Background: Recombinant human erythropoietin (rHuEPO), a hormone regulating the proliferation and differentiation of erythroid cells, is one of the prescription drugs used to treat cancer-associated anemia. However, administration of rHuEPO to cancer patients has been reported to be associated with decreased survival, and the mechanism by which it acts remains controversial. The present study aimed to investigate the expression of the EPO-receptor in lung cancer cell lines and whether rHuEPO treatment affected its growth and migration. Moreover, the angiogenic effects of rHuEPO were also explored in vivo.Methods: Expression of the EPO-receptor in lung cancer cell lines was measured by Western blotting and enzyme linked immunosorbent assays (ELISAs). Proliferation of the lung cancer cells was monitored in the presence of rHuEPO. Human umbilical vein endothelial cells (HUVECs) were used for tube formation assays in vitro, and transwell migration assays were performed to detect migration under rHuEPO treatment. Matrigel plug technology was employed to observe the angiogenic effects in both nude mice and Matrigel-containing lung cancer cell lines H838 or H1975. Microvessel density (MVD) was measured using CD31 Immunohistochemistry (IHC) staining.Results: EPO-receptor (EPO-R) was only detected in the cell lines H838 and H1339 by ELISA. However, the EPO-R protein was detected in all cell lines by Western blotting, which is in contradiction to the ELISA results. Proliferation and migration were not affected by rHuEPO treatment. However, rHuEPO promoted HUVEC tube formation in vitro and significantly induced the formation of new blood vessels in vivo. Furthermore, rHuEPO did not antagonize the inhibitory effects of Afatinib (epidermal growth factor receptor-tyrosine kinase inhibitor; EGFR-TKI) in simultaneous treatment with rHuEPO. In a 3D cell co-culture model, rHuEPO did not enhance the secretion of vascular endothelial growth factor (VEGF) in lung cancer cells or human lung fibroblast cell line MRC-5.Conclusions: We have shown that the role of EPO goes beyond erythropoiesis, also playing a strong role in angiogenesis by participating in new blood vessel formation in lung cancer models. Thus, rHuEPO may raise the risk of thrombosis and metastasis in vivo. Additionally, our results suggest that studies using commercially available EPO-R antibodies should be reexamined; some of these antibodies may not in fact recognize EPO-R.

IL-9 blockade attenuates inflammation in a murine model of methicillin-resistant Staphylococcus aureus pneumonia.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important etiology of pneumonia. Interleukin (IL)-9 is a T helper 9 (Th9) cytokine and participates in the pathogenesis of infectious diseases. Here, we investigated the role of IL-9 by using an MRSA pneumonia animal model. The BALB/c mice underwent nasal inhalation with an ST239 MRSA strain to establish the mouse model of MRSA pneumonia, and a subset of mice were intravenously injected with IL-9 neutralizing antibody or immunoglobulin (Ig) G. At 3 and 8days postinfection, the peripheral blood, bronchioalveolar lavage fluid (BALF), and lung tissues were collected. The frequencies of Th9 cells and the levels of cytokines in peripheral blood, BALF, and lung tissues were determined by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively. The colony counts of MRSA in BALF and lung tissue were detected. The lung pathological changes were examined using hematoxylin and eosin staining. Data from flow cytometry, qRT-PCR, and ELISA showed that MRSA-infected mice exhibited higher frequency of Th9 cells and higher IL-9 mRNA and protein levels in the peripheral blood, BALF, and lung tissues of mice. In contrast, the neutralization of IL-9 abrogated MRSA inoculation-induced Th9 cell generation and IL-9 production in BALF and lung tissues. Furthermore, bacterial counting and histological examination showed that the numbers of bacteria in BALF and lungs and the lung pathological scores induced by MRSA inoculation were attenuated by the neutralization of IL-9. Moreover, cell counting and ELISA results demonstrated that IL-9 neutralization diminished the MRSA inoculation-induced count of neutrophils and macrophages and levels of pro-inflammatory cytokines in BALF. Collectively, IL-9 neutralization attenuated inflammation of MRSA pneumonia by regulating Th9/IL-9 expression.

The Combination of IgA and IgG autoantibodies against Transcriptional Intermediary Factor-1γ contributes to the early diagnosis of Lung Cancer.

Objective: The aberrant expression of tumor-associated antigens (TAAs) is responsible for the release of large amounts of autoantibodies in sera, and serum autoantibody detection has been demonstrated to contribute to the early diagnosis of malignancies. Recent studies showed the closely correlation of transcriptional intermediary factor-1γ (TIF1γ) with some malignancies. In our pilot study, we found aberrantly high expression of TIF1γ protein existed in cancer tissues other than matched paracancerous tissues of patients with lung cancer (LC) at early stage by immunohistochemistry (IHC) staining. As a result, this study aims to detect the expression of autoantibodies against TIF1γ in sera of patients with LC at early stage by using enzyme-linked immunosorbent assay (ELISA) and investigate its potential value for the early diagnosis of LC.Methods: The expressions of TIF1γ protein in 60 pairs of LC tissues and matched paracancerous tissues were detected by IHC staining. The levels of anti-TIF1γ-IgA, IgG, IgM, and IgE in the sera of 248 patients with LC at early stage, 200 patients with lung benign lesions (LBL), and 218 healthy controls (HC) were detected by ELISA, respectively. Western blot was used to validate the ELISA results of serum autoantibodies against TIF1γ.Results: The positive rate of TIF1γ protein expression in LC tissues was 83.33%, which was significantly higher than 25.00% in paracancerous tissues (P<0.01). The levels and positive rates of serum anti-TIF1γ-IgM and anti-TIF1γ-IgE in early LC group had no significant difference from that in LBL group and HC group (P>0.05), while the levels and positive rates of anti-TIF1γ-IgA and anti-TIF1γ-IgG were significantly higher than that in LBL group and HC group (P<0.01), of which anti-TIF1γ-IgA showed the area under the receiver operating characteristic curve (AUC) of 0.704 for the patients with LC at early stage, with 28.20% sensitivity at 95.93% specificity, and anti-TIF1γ-IgG showed the AUC of 0.622 for the patients with LC at early stage, with 18.54% sensitivity at 94.25% specificity. The results of anti-TIF1γ-IgA and anti-TIF1γ-IgG in western blot were consistent with that in ELISA. Additionally, the combination of anti-TIF1γ-IgA and anti-TIF1γ-IgG improved the AUC to 0.734, with 38.31% sensitivity at 92.34% specificity.Conclusions: There is a strong humoral immune response to autologous TIF1γ existing in patients with early LC. Both serum anti-TIF1γ-IgA and anti-TIF1γ-IgG show the diagnostic value for the patients with LC at early stage, of which anti-TIF1γ-IgA is donstrated to be a preferable biomarker, and the combined detection of anti-TIF1γ-IgA and anti-TIF1γ-IgG might contribute to the further improvement of early diagnosis for LC.

Intermittent hypoxia improves cognition and reduces anxiety-related behavior in APP/PS1 mice.

IntroductionAlthough hypoxia can exacerbate symptoms of various neurological disorders, accumulating evidence has indicated that intermittent hypoxia (IH) may exert protective effects against brain diseases. In the present study, we aimed to determine whether exposure to IH exerts beneficial effects in a transgenic murine model of Alzheimer's disease (AD). Because comorbid anxiety is prevalent among patients with AD, we explored the effects of IH on anxiety‐like behaviors and associated factors in APP/PS1 mice.MethodsAPP/PS1 mice were subjected to IH for two weeks. We assessed cognitive performance and anxiety‐related behavior using standard behavioral assessments. Amyloid beta (Aβ) levels in the hippocampus were assessed using immunofluorescence and enzyme‐linked immunosorbent assays (ELISA). We also assessed cell morphology and brain‐derived neurotrophic factor (BDNF) expression in the hippocampus.ResultsExposure to IH significantly increased cognitive performance and decreased anxiety‐related behaviors in APP/PS1 mice. Immunofluorescence and ELISA results revealed that IH pretreatment significantly lowered Aβ levels in the cortex and hippocampus. Morphological studies validated the neuroprotective effect of IH exposure on hippocampal neurogenesis. Molecular studies revealed IH‐enhanced BDNF expression and inhibition of apoptosis‐related protein expression in the hippocampus of APP/PS1 mice.ConclusionsOur study demonstrates that IH improves cognition and reduces anxiety in a murine model of AD. Thus, further studies are required to determine whether IH can be used as a preventive/adjuvant therapy in patients with AD.

(Non-)Sense of Milk Testing in Small Ruminant Lentivirus Control Programs in Goats. Comparative Analysis of Antibody Detection and Molecular Diagnosis in Blood and Milk.

Small ruminant lentivirus (SRLV) control programs are mainly based on diagnostic tests performed on blood samples collected from sheep and goats. Since blood sampling is costly and stressful for the animals, we evaluated whether milk could be used as an inexpensive and easily collectable matrix for SRLV detection. We therefore compared SRLV detection via two commercial enzyme-linked immunosorbent assays (ELISAs) and quantitative polymerase chain reaction (qPCR) in blood and corresponding milk samples from 321 goats originating from eight different SRLV-infected farms in Flanders (Belgium). The IDscreen® ELISA had a better relative sensitivity (97% vs 93%) and specificity (100% and 97%) than the Elitest® ELISA for SRLV-specific antibody detection in milk compared to serum. The higher sensitivity correlates with a 10-fold higher analytical sensitivity of the IDscreen® test. In contrast to the overall good ELISA results, qPCR on milk cell pellets lacked sensitivity (81%) and specificity (88%), compared to molecular detection in blood leucocyte pellets. Our results show that serology is more suitable than qPCR for SRLV diagnosis, and that milk may represent an interesting matrix for a preliminary evaluation of a herd’s infection status. Serum remains however the sample of choice for control programs where it is important to identify positive animals with the highest sensitivity.

Effect of herbal cake-partitioned moxibustion on Leptin/JAK2/STAT3 in lipid-lowering pathway of hyperlipidemia rabbits

Objective: To observe the lipid-lowering effect of different transdermal absorption enhancers applied to the herbal cake-partitioned moxibustion in hyperlipidemia model rabbits, and to explore the possible mechanism. Methods: Forty New-Zealand rabbits were randomly divided into 5 groups using the random number table method, with 8 rats in each group. Rabbits in the blank group were fed routinely with normal diet; rabbits in the other groups were fed with high-fat diet for 12 weeks to establish the hyperlipidemia model. Rabbits in the blank and the model groups were not treated. After the model was prepared, rabbits in the non-transdermal absorption enhancer group received herbal cake-partitioned moxibustion without transdermal absorption enhancer; rabbits in the laurocapram group and the borneol group received herbal cake-partitioned moxibustion with laurocapram or borneol respectively. After 4 weeks of treatment, serum was collected for enzyme-linked immunosorbent assay (ELISA), and the liver tissues were isolated for immunohistochemistry, quantitative polymerase chain reaction (qPCR) and Western-blotting (WB) detection. Results: Serum ELISA results showed that leptin was significantly decreased in the model group compared with the blank group (P 0.05). The qPCR results of rabbit liver tissues showed that the mRNA expressions of leptin, Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) in the model group were significantly lower than those in the blank group (all P<0.05); compared with the model group, the mRNA expressions of leptin, leptin receptor (LR), JAK2 and STAT3 in the non-transdermal absorption enhancer, the laurocapram and the borneol groups were significantly increased (all P<0.05); compared with the non-transdermal absorption enhancer group, the mRNA expressions of leptin, LR, JAK2 and STAT3 in the laurocapram and the borneol groups were significantly increased (all P<0.05); compared with the laurocapram group, the mRNA expressions of leptin, LR, JAK2 and STAT3 in the borneol group were significantly increased (P<0.05). The trend of immunohistochemistry and WB detection results was basically consistent with the qPCR assay results. The immunohistochemistry and WB detection results of phosphorylated JAK2 (phospho-JAK2) and phosphorylated STAT3 (phospho-STAT3) were basically consistent with those of JAK2 and STAT3. Conclusion: The molecular expression of Leptin/JAK2/STAT3 pathway in the hyperlipidemia model rabbits was decreased. The molecular expression of Leptin/JAK2/STAT3 pathway was significantly increased after the herbal cake-partitioned moxibustion. The application of laurocapram and borneol, as transdermal absorption enhancers, in the herbal cake-partitioned moxibustion could more obviously up-regulate the factors of the Leptin/JAK2/STAT3 lipid-regulating pathway than the herbal cake-partitioned moxibustion alone.

Development and comparison of enzyme-linked immunosorbent assays based on recombinant trimeric full-length and truncated spike proteins for detecting antibodies against porcine epidemic diarrhea virus

BackgroundSince 2010, outbreaks of genotype 2 (G2) porcine epidemic diarrhea virus (PEDV) have caused high mortality in neonatal piglets and have had devastating impacts on the swine industry in many countries. A reliable serological assay for evaluating the PEDV-specific humoral and mucosal immune response is important for disease survey, monitoring the efficacy of immunization, and designing strategies for the prevention and control of PED. Two PEDV spike (S) glycoprotein-based indirect enzyme-linked immunosorbent assays (ELISAs) were developed using G2b PEDV-Pintung 52 (PEDV-PT) trimeric full-length S and truncated S1–501 proteins derived from the human embryonic kidney (HEK)-293 cell expression system. The truncated S1–501 protein was selected from a superior expressed stable cell line. The sensitivity and specificity of these two ELISAs were compared to immunostaining of G2b PEDV-PT infected cells and to a commercial nucleocapsid (N)-based indirect ELISA kit using a panel of PEDV negative and hyperimmune sera.ResultsThe commercial N-based ELISA exhibited a sensitivity of 37%, a specificity of 100%, and a fair agreement (kappa = 0.37) with the immunostaining result. In comparison, the full-length S-based ELISA showed a sensitivity of 97.8%, a specificity of 94%, and an almost perfect agreement (kappa = 0.90) with the immunostaining result. Interestingly, the S1–501-based ELISA had even higher sensitivity of 98.9% and specificity of 99.1%, and an almost perfect agreement (kappa = 0.97) with the immunostaining result. A fair agreement (kappa< 0.4) was seen between the commercial N-based ELISA and either of our S-based ELISAs. However, the results of the full-length S-based ELISA shared an almost perfect agreement (kappa = 0.92) with that of S1–501-based ELISA.ConclusionsBoth full-length S-based and S1–501-based ELISAs exhibit high sensitivity and high specificity for detecting antibodies against PEDVs. Considering the high protein yield and cost-effectiveness, the S1–501-based ELISA could be used as a reliable, sensitive, specific, and economic serological test for PEDV.

Human cystic echinococcosis: Serological diagnosis by indirect hemagglutination test, enzyme-linked immunosorbent assay, immunoelectrophoresis, and immunoblotting in surgically confirmed patients versus cases diagnosed by imaging techniques

IntroductionHuman cystic echinococcosis (CE) diagnosis is based on imaging findings and serology. Serology is the first-line test followed by imaging tests. Materials and methodsA total of 268 serum samples from CE patients were included in this study. The serodiagnosis was made simultaneously by indirect hemagglutination assay (IHA), enzyme-linked immunosorbent assay (ELISA), immunoblotting test (IB), and immunoelectrophoresis (IEP). ResultsIn serum samples belonging to surgically confirmed CE patients, we observed a percentage of positivity of 83.7% [77.2%–89.0%] and 80.7% [73.9%–86.4%] for IHA and IgG-ELISA, respectively. IgG-IB was associated with a higher positivity rate than IEP with 81.3% [74.6%–86.9%] and 62.0% [54.2%–69.5%], respectively. IHA and ELISA results were analyzed using cut-off generated by receiver operating curves. The best diagnostic performances were achieved by IHA (cut-off ≥1/128) and ELISA (>1.16 index). Sensitivities reported in patients with suggestive imaging findings and positive Echinococcus IB were 86.2% [78.0%–92.2%], 72.5% [62.8%–80.9%], 49.0% [39.0%–59.1%] for IHA, IgG-ELISA, and IEP, respectively. All tests gave false negative results in the confirmed CE group. Overall, 18.6% of negative results were obtained by IgG-IB test. Cross-reactivities with non-hydatid serum samples were observed in all tests. Only one patient carrying Taenia saginata serum cross-reacted with 8/12 kDa band by IB. We observed specificity at 73% [63.2%–81.4%], 87% [78.8%–92.9%], 99% [94.6%–100.0%], and 99% [94.6%–100.0%] with IHA, IgG-ELISA, IEP, and IgG-IB, respectively. Serology was less sensitive (74%) in lung cysts. Sensitivity was better in liver cysts, especially by IgG-IB (96%).

Development of a human scFv antibody targeting the lethal Iranian cobra (Naja oxiana) snake venom

Snakebite is one of the health concerns worldwide. Naja oxiana is one of the venomous snakes with a high mortality rate. Anti-serum therapy is the only treatment of the victims. However, in some cases, antiserum injection leads to some side effects in host like serum sickness and anaphylactic shock. It is crucial to develop a neutralizing agent with low side effects. The human antibody library (non-immunized library) was used to isolate specific antibodies against N.oxiana venom components. Four rounds of biopanning were performed to enrich scFv-displaying phages against the venom of N. oxiana. Enrichment of scFv-displaying phages against N. oxiana venom was analyzed by polyclonal Enzyme-Linked Immunosorbent Assay (ELISA). Specific antibody fragments against N. oxiana venom were selected through monoclonal ELISA, and were expressed in E. coli bacterial cells. Purification of the selected clones was performed by using nickel affinity chromatography. Neutralization and protective capacity of specific antibody fragments were analyzed in C57BL/6 mice (i.v. injection). Results of biopanning and polyclonal ELISA demonstrate a successful enrichment process. Five specific antibody fragments with the highest signal in monoclonal ELISA were selected, expressed, and purified. The purity of expressed antibody fragments was monitored by SDS-PAGE and Western blot. The selected antibody fragments were able to neutralize two LD50 of N. oxiana venom and protected all mice when injected 15 min post-envenomation. The data indicate that such selected antibodies are promising tools for further studies and in the development of novel protective agents against N. oxiana venom.

CENP-U function on TNBC.

31 Background: Centromere protein (CENP-U) gene, as an important constitutive kinetochorecomponent, plays significant roles in cell caryomitosis. Our previous research found that about 25% of transgenic mice with CENP-U amplified had suffered breast cancer in body surface. Also, in highly aggressive breast cancer cell lines, CENP-U presented the highest expression. Furthermore, the CENP-U protein expression obviously increased in human invasive breast carcinoma compared with the normal gland and intra-ductal tissue.Here we aimed to investigate the biological functions and potential molecular mechanism of CENP-U in breast cancer tumorigenesis. Methods: Gene knockdown was accomplished by transient transfection. In addition, cell proliferation was analyzed with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and 5-Ethynyl-2'-deoxyuridine (EdU) assay while cell apoptosis was measured by flow cytometry using an annexin V-FITC apoptosis detection kit. Then, VEGF secretion in the supernatant was detected with an enzyme-linked immunosorbent assay (ELISA). Furthermore,Vascular endothelial markers of nude mouse were discovered by immunohistochemistry. And the signaling activation in the breast cancer cells was accessed by western blot using specific antibodies. Results: We observed higher CENP-U expression in breast cancer compared to adjacent normal breast tissue, especially in TNBC.The MTT and EdU assay showed that CENP-U promoted the proliferation of the triple negative breast cancer (TNBC) cells while CENP-U downregulated promoted cell apoptosis. Furthermore, the ELISA results revealed that CENP-U significantly promoted the production and secretion of VEGF in TNBC cells. In addition, CENP-U downregulated inhibits tumor growth and angiogenesis in vivo. Importantly, CENP-U targeted HIF-1α,VEGFA and COX-2, which were shown by the specific antibodies in the western blot. Conclusions: The results showed that CENP-U influence the proliferation, apoptosis and angiogenesis especially of TNBC cells. But the affected signaling pathways require further study.

Postorthodontic Relapse Prevention by Administration of Grape Seed (Vitis vinifera) Extract Containing Cyanidine in Rats.

Objective The aim of this study was to analyze the effect of grape seed extract containing cyanidin on osteoclastogenesis (by means of receptor activator of nuclear factor-κ B ligand [RANKL] and osteoprotegerin [OPG] levels) and the number of osteoclasts during orthodontic relapse in Wistar rats. Materials and Methods This study is an in vivo quasi experimental research. A total of 32 male Wistar rats were used in the study, which were randomly split equally into two groups, grape seed (GS) and control group (CG). All rats were given an orthodontic force of 35 cN using a stainless steel 3-spin coil spring that was activated for 7 days and then conditioned to be passive. During this phase, the GS group was administered grape seed extract containing cyanidin once per day. Orthodontic appliances were removed from both groups afterward, and then the alveolar bone tissue was isolated consecutively according to observation days (days 1, 3, 7, and 14), while OPG and RANKL levels were analyzed in their gingival crevicular fluid using a specific enzyme-linked immunosorbent assay (ELISA). Tissues were then stained with hematoxylin–eosin (H&E) and observed under a light microscope to count the number of osteoclast cells. Data were analyzed statistically using an independent t -test ( p < 0.05). Results The number of osteoclasts in the GS group was significantly lower than that in the CG group on all experiment days ( p = 0.021; p = 0.001; p = 0.024; p = 0.001; p < 0.05). ELISA results showed that the RANKL level of the GS group was significantly lower on days 3 and 7 ( p = 0.025; p = 0.039; p < 0.05), while the OPG level was significantly higher on days 1 and 3 in the GS group than in the CG group ( p = 0.039; p = 0.021; p < 0.05). Conclusion Grape seed extract can prevent postorthodontic relapse movement by inhibiting osteoclastogenesis and reducing the number of osteoclasts in Wistar rats.

Paraoxonase 1 Was Negatively Associated With Intervertebral Disc Degeneration.

An in vivo and in vitro study of the correlation between Paraoxonase 1 (PON1) and intervertebral disc degeneration (IVDD). The aim of this study is to clarify the expression and role of PON1 on the process of IVDD. IVDD is responsible for most of the spinal degenerative diseases. Inflammation and oxidative stress can deteriorate the living environment of nucleus pulposus (NP) cells, leading to IVDD. PON1 is an enzyme reported to have anti-inflammatory and anti-oxidative effects. There is no study about the correlation of PON1 expression with IVDD. Immunohistochemical (IHC), hematoxylin and eosin (H&E) staining, and Western blot examined the expression of PON1 in 88 human disc samples (male: female 43: 45) and rat models (n = 5 each group). The level of PON1 is measured in the tumor necrosis factor (TNF)-α and oxidative stress (H2O2)-induced degenerative NP cell models using Western blot and reverse transcription-polymerase chain reaction (RT-qPCR). The TNF-α, interleukin (IL)-1β, Mito superoxide (SOX), aggrecan, and collagen II are detected in nucleus pulposus (NP) cells transfected with si-RNA of PON1 using Enzyme-Linked Immunosorbent Assay (ELISA), mitoSOX staining Western blot, and RT-qPCR. The expression of PON1 is significantly suppressed in human and rat degenerative intervertebral discs. The level of PON1 is significantly decreased in TNF-α and oxidative stress (H2O2)-induced degenerative NP cell models. ELISA results show that the level of TNF-α and IL-1β obviously increased; Mito SOX staining indicates that the Mito SOX fluorescence significantly increased, and the expression of aggrecan and collagen reduced in NP cells transfected with si-RNA of PON1. Our study indicates that low PON1 expression is predictive of severe IVDD; PON1 plays an important role of keeping the homeostatic balance of intervertebral discs, and therapeutic approach regarding PON1 may be helpful to alleviate IVDD in the future. N/A.

Influence of Ceramic Debris on Osteoblast Behaviors: AnIn VivoStudy

ObjectiveWear‐induced aseptic loosening has been accepted as one of the main reasons for failure of total hip arthroplasty. Ceramic wear debris is generated following prosthesis implantation and plays an important part in the upregulation of inflammatory factors in total hip arthroplasty. The present study investigates the influence of ceramic debris on osteoblasts and inflammatory factors.MethodsCeramic debris was prepared by mechanical grinding of an aluminum femoral head and added to cultures of MC3T3‐E subclone 14 cells at different concentrations (i.e. 0, 5, 10, and 15 μg/mL). Cell proliferation was evaluated using a Cell Counting Kit (CCK‐8), and cell differentiation was assessed by mRNA expression of alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). In addition, cell bio‐mineralization was evaluated through alizarin red S staining, and release of tumor necrosis factor alpha (TNF‐α), interleukin‐1 beta (IL‐1β), and interleukin‐6 (IL‐6) was measured through enzyme‐linked immunosorbent assays (ELISA). Furthermore, mRNA expression of Smad1, Smad4, and Smad5 and protein expression of phosphorylated Smad1, Smad4, and Smad5 were measured by reverse transcriptase polymerase chain reaction (RT‐PCR) and western blotting.ResultsThe ceramic debris had irregular shapes and sizes, and analysis of the size distribution using a particle size analyzer indicated that approximately 90% of the ceramic debris was smaller than 3.2 μm (2.0 ± 0.4 μm), which is considered clinically relevant. The results for mRNA expression of ALP, OCN, and OPN and alizarin red S staining indicated that cell differentiation and bio‐mineralization were significantly inhibited by the presence of ceramic debris at all tested concentrations (P < 0.05, and the values decreased gradually with the increase of ceramic debris concentration), but the results of the CCK‐8 assay showed that cell proliferation was not significantly affected (P > 0.05; there was no significant difference between the groups at 1, 3, and 5 days). In addition, the results of ELISA, RT‐PCR, and western blotting demonstrated that ceramic debris significantly promoted the release of inflammatory factors, including TNF‐α, IL‐β, and IL‐6 (P < 0.05, and the values increased gradually with the increase of ceramic debris concentration), and also greatly reduced the mRNA expression of Smad1, Smad4, and Smad5 (the values decreased gradually with the increase of ceramic debris concentration) as well as protein expression of phosphorylated Smad1, Smad4, and Smad5.ConclusionCeramic debris may affect differentiation and bio‐mineralization of MC3T3‐E subclone 14 cells through the bone morphogenetic protein/Smad signaling pathway.

Enhance the affinity of nanobodies to CD47 antigen by fusion expression of COMP48 self-folding peptide

Objective To modify CD47 nanobody with the self-folding peptide human cartilage oligomeric matrix protein (COMP48) so as to enhance its affinity to CD47 antigen. Methods The fusion sequences of COMP48 and CD47 nanobody (VHHB1) were designed and synthesized, and the recombinant plasmid pET22b-VHHB1-COMP48 was constructed and transformed into E. coli BL21 (DE3) to induce expression of the fusion protein. The binding specificity and affinity of the fusion protein and the antigen CD47 were detected by Western Blot, indirect enzyme-linked immunosorbent assay (ELISA) and non-competitive ELISA. Results The recombinant VHHB1-COMP48 was expressed in BL21(DE3) by inducing with 1 mmol/L IPTG and purified at 90% homogenous in IMAC. Western Blot results showed that the recombinant protein VHHB1-COMP48 specifically binds to antigen CD47 but not to unrelated protein. The indirect ELISA and non-competitive ELISA results showed that the affinity of the conjugated recombinant protein VHHB1-COMP48 was enhanced compared to that of the non-conjugated nanobody, and the difference was statistically significant (P<0.01). Through non-competitive ELISA, the constants of affinity and dissociation constants were 6.97×107 L/mol and 1.434×10-8 mol/L, respectively. Conclusions The affinity of the nanobody for the antigen can be improved by conjugating a human cartilage matrix protein (COMP48) after the nanobody. Key words: Nanobody; Human cartilage oligomeric matrix protein; Affinity maturation; Self-folding peptide; CD47

Monitoring and risk assessment of tetracycline residues in foods of animal origin.

A total of 450 samples consisting of red meat, poultry meat, aquatic product and raw milk were collected during winter 2016 and summer 2017. 22.2% (100/450) of collected meat and raw milk samples were found to be contaminated with antibiotic residues in the initial screening using Premi®test. According to the enzyme linked immunosorbent assay (ELISA) results, the mean tetracyclines (TCs) concentration of meat samples determined as follows: chicken (155.41µg/kg) > turkey (138.68µg/kg) > quail (130.7µg/kg) > cow (108.92µg/kg) > calf (105.18µg/kg) > goat (99.4µg/kg) > sheep (95.22µg/kg) > rainbow trout (35.62µg/kg) > shrimp (31.80µg/kg). The content of TCs in cow, goat and sheep milk samples were found to be ranged 45.6-163.5µg/L, 72.4-101.1µg/L and 65.5-98.9µg/L, respectively. 5.7% (26/450) of samples had TCs confirmed the ELISA results using high performance liquid chromatography coupled with ultra-violet detection, although the concentration of TCs residues in samples was higher than that of ELISA.