Senecavirus A (SVA) is a major viral pathogen causing disease in pigs, and effective monitoring of SVA infection is critical for disease control. In this study, we aimed to develop a reliable ELISA method for rapidly detecting neutralizing antibodies against SVA. We used HEK293F cells to express an SVA-specific porcine Fab antibody and verified the biological activity of the Fab antibody by indirect ELISA, immunofluorescence assay, virus neutralization test, and Western blotting. The Fab antibody was biotinylated and used as a competitive antibody to establish a competitive ELISA (C-ELISA) for detecting neutralizing antibodies against SVA. We then evaluated the C-ELISA in terms of sensitivity, specificity, repeatability, and result agreement rate with the VNT. The results showed that we successfully prepared an SVA-specific porcine Fab antibody, which showed high affinity for SVA. We named this antibody 1M33Fab and designated it as Bio-1M33Fab after biotin labeling. The assay conditions were optimized as follows: the coating concentration of SVA particles being 1 μg/mL, the working concentration of Bio-1M33Fab being 0.5 μg/mL, the optimal serum dilution of 1:10, and the optimal dilution of enzyme-labeled avidin being 1:30 000. At a percent inhibition (PI) of 47%, the assay demonstrated the highest sensitivity (96.88%) and specificity (100%), with no cross-reactivity observed with the positive sera of major porcine viral diseases. The intra-assay coefficient of variation ranged from 1.12% to 7.34%, while the inter-assay coefficient of variation ranged from 1.10% to 8.97%, indicating good repeatability. In the detection of 224 clinical pig serum samples, C-ELISA and VNT showed a result agreement rate of 93.75%. In conclusion, we successfully develop a C-ELISA method for detecting neutralizing antibodies against SVA by using a porcine-derived Fab antibody, which lays a foundation for the development of detection kits.
Animals ; Swine ; Antibodies, Neutralizing/immunology* ; Enzyme-Linked Immunosorbent Assay/methods* ; Immunoglobulin Fab Fragments/immunology* ; Antibodies, Viral/immunology* ; Picornaviridae/immunology* ; Humans ; HEK293 Cells ; Swine Diseases/diagnosis* ; Picornaviridae Infections/diagnosis*

Objective To explore the relationship between 25-hydroxyvitamin D[25(OH)D]and insulin resistance.Methods A total of 112 diabetic patients hospitalized in Department of Endocrinology,the Fifth People's Hospital of Jinan from June 2021 to December 2022 were selected as observation group by using random number table method,and 61 individuals in endocrinology clinic with normal blood glucose levels were selected as control group.The levels of 25(OH)D,blood glucose level,homeostasis model assessment of insulin resistance(HOMA-IR),homeostasis model assessment of β-cell function(HOMA-β),thyroglobulin antibodies,and diabetes-related antibodies[insulin autoantibodies(IAA),islet cell antibody(ICA)and glutamic acid decarboxylase antibody(GAD)]etc indexs were measured in two groups,and the correlation between 25(OH)D and blood glucose,thyroglobulin antibody,diabetes-related antibody and pancreatic function was analyzed.Results The level of 25(OH)D in observation group was significantly lower than that in control group,and the difference was statistically significant(P＜0.05).Correlation analysis showed that the level of 25(OH)D was negatively correlated with thyroglobulin antibodies,glycosylated hemoglobin,IAA,GAD,ICA,and HOMA-IR etc(P＜0.01)in all subjects,and positively correlated with gender and HOMA-β(P＜0.01).In observation group,the level of 25(OH)D was negatively correlated with HOMA-IR(P＜0.01)and positively correlated with HOMA-β(P＜0.01).Conclusion Insulin resistance and immune damage may be related to insufficient the level of serum 25(OH)D.It is suggested that individuals with diabetes and those at high risk of diabetes should supplement with vitamin D appropriately in the early stage.

Objective To explore the relationship between 25-hydroxyvitamin D[25(OH)D]and insulin resistance.Methods A total of 112 diabetic patients hospitalized in Department of Endocrinology,the Fifth People's Hospital of Jinan from June 2021 to December 2022 were selected as observation group by using random number table method,and 61 individuals in endocrinology clinic with normal blood glucose levels were selected as control group.The levels of 25(OH)D,blood glucose level,homeostasis model assessment of insulin resistance(HOMA-IR),homeostasis model assessment of β-cell function(HOMA-β),thyroglobulin antibodies,and diabetes-related antibodies[insulin autoantibodies(IAA),islet cell antibody(ICA)and glutamic acid decarboxylase antibody(GAD)]etc indexs were measured in two groups,and the correlation between 25(OH)D and blood glucose,thyroglobulin antibody,diabetes-related antibody and pancreatic function was analyzed.Results The level of 25(OH)D in observation group was significantly lower than that in control group,and the difference was statistically significant(P＜0.05).Correlation analysis showed that the level of 25(OH)D was negatively correlated with thyroglobulin antibodies,glycosylated hemoglobin,IAA,GAD,ICA,and HOMA-IR etc(P＜0.01)in all subjects,and positively correlated with gender and HOMA-β(P＜0.01).In observation group,the level of 25(OH)D was negatively correlated with HOMA-IR(P＜0.01)and positively correlated with HOMA-β(P＜0.01).Conclusion Insulin resistance and immune damage may be related to insufficient the level of serum 25(OH)D.It is suggested that individuals with diabetes and those at high risk of diabetes should supplement with vitamin D appropriately in the early stage.

Objective : To analyze the epidemiological characteristics and pathogen spectrum of hand，foot mouth disease (HFMD) in Anhui province from 2015 to 2022，and to provide scientific evidence for prevention and control measures of HFMD． Methods : The surveillance data of hand，foot and mouth disease in Anhui province from 2015 to 2022 were analyzed by descriptive epidemiology. Real-time PCR was used to detect and classify HFMD samples. Results : A total of 650 590 HFMD cases were reported in Anhui province from 2015 to 2022，including 1 406 se- vere cases and 17 deaths．The annual reported incidence was 131. 45 /100 000．The epidemic features of“low incidence in odd years and high incidence in even years”were presented from 2015 to 2019．The incidence showed a continuous decline from 2020 to 2022．The monthly distribution showed the characteristics of bimodal epidemic，and the main peak was not obvious in 2020．Hefei，Fuyang，Bozhou，Chuzhou and Suzhou ranked the top five cities in terms of cumulative incidence．The age of onset was mainly distributed in children aged 5 years and below，accounting for 89. 26% of the total cases．The male to female ratio was 1. 48 ∶ 1．A total of 28 657 laboratory-confirmed cases had been reported from 2015 to 2022．EV71 cases accounted for 10. 57% ，Cox A16 cases accounted for 24. 90% ，and other enterovirus cases accounted for 64. 53%．The dominant pathogens showed dynamic changes in different years．Since 2018，the proportion of EV71 decreased significantly，and the proportion of other enteroviruses gradually increased to become the dominant pathogens．Among other enteroviruses，Cox A6 strain was dominant (80. 48% ) ． Conclusion This study suggests that the prevention and control of HFMD in Anhui province should be paid more attention from April to July and from October to December．The focus areas are the cities in northern Anhui and Hefei where the floating population is large．The focus of prevention and control is on children aged 5 years and below．Other enteroviruses have become the dominant pathogens of hand-foot-mouth disease in Anhui province，Cox A6 strain is dominant.

Nowadays, more attentions have been paid on brown adipose tissue (BAT) because BAT plays a great role in individuals with obesity and metabolic diseases. 18F-fluorodeoxyglucose (FDG) PET/CT is noninvasive and sensitive to detect the location, volume, and metabolic activity of BAT. 18F-FDG PET/CT for BAT provides new insight for the prevention and clinical management of obesity and diabetes. The review summarizes the methods to activate BAT, the preparation of patients, 18F-FDG PET/CT imaging techniques and clinical application.

Objective:To determine the effects of Foxp3-overexpressing lung cancer cells on activated CD4+T lymphocyte.Methods: Stable Foxp3-overexpressing lung cancer cells NCIH-1299,NCIH-hFoxp3,was generated by transfection of NCIH-1299 cells with plasmid pcDNA3-hFoxp3 mediated by Lipofectamine 2000 and by selection with G418,and validated by quantitative PCR and Western blot.The expression levels of IL-8 and IL-10 secreted by NCIH-hFoxp3 and NCIH-control were measured by ELISA.IL-2 secrection by activated human CD4+T lymphocyte which was tested after stimulation with 20% conditioned medium of NCIH-hFoxp3 and NCIH-control cells.The proliferation of activated human CD4+ T lymphocytes was assessed by MTT after coculture with NCIH-hFoxp3 cells.The adhesive ability of activated human CD4+ T lymphocytes was probed with NCIH-hFoxp3 cells by immunocytochemistry.Results: Compared with NCIH-control cells,NCIH-hFoxp3 secreted high level of IL-10 and low level of IL-8.NCIH-hFoxp3 with Foxp3 overexpression significantly suppressed the proliferation,adhesive potential and IL-2 expression by activated CD4+ T cells.Conclusion: Suppression of immune activities of activated CD4+ T cells by Foxp3 overexpression in lung cancer cells may correlate with cytokine IL-8 and IL-10,which can contribute lung cancer progression.

Objective To examine whether Tim-3 plays a protective role in paraquat poisoning induced excessive immune response and tissue damage based on the critical roles of Tim-3 controlling inflammatory response.Methods A paraquat poisoning model was established in wild type and in Tim-3 transgenic C57BL/6 mice by intraperitoneal injection of paraquat (40 mg/kg) .In addition, C57BL/6 mice with paraquat poisoning were injected with Tim-3 soluble protein( sTim-3) or control protein to see the effect of Tim-3 blocking on the progression of paraquat poisoning.Samples were collected at 6 and 24 h after paraquat injection respectively and were examined for tissue damage, cytokine expression and paraquat metabolism.Results After paraquat poisoning, there was significantly attenuated tissue damage in the lungs and kidneys and decreased TNF-α,IL-6 and IL-1 beta expression in the PBMCs or in the serum from Tim-3 transgenic mice compared to wild type mice.The serum concentration of paraquat in Tim-3 transgenic mice was also significantly decreased.However, in sTim-3 treated paraquat poisoning mice, there was significantly increased cytokine expression and tissue damage compared to control protein treated mice.The in vitro data showed that Tim-3 signaling negatively regulated macrophages mediated inflammatory response.Conclusion Tim-3 plays a critical role in maintaining the homeostasis after paraquat poisoning. Further investigation on the regulatory roles of Tim-3 in inflammation will shed new light on the pathogenesis of paraquat poisoning and provide new therapeutic strategies.

Objective:To determine the effects of enforced expression of Foxp3 in lung cancer cell with regards to proliferation and tumorgeneity. Methods: A stable subline NCIH-hFoxp3 was established by liopfectamin-mediated pcDNA plasmid transfection carrying exogenous hFoxp3. The growth curve and secrection of IL-8 and IL-10 of NCIH-hFoxp3 were evaluated using MTT and ELISA, respectively. The in vivo tumorigeneity was assessed as well by inoculation of NCIH-hFoxp3 subcutaneously in nude mice. Results:Lung cancer cell NCIH-hFoxp3 with enforced expression of Foxp3 proliferated slowly but exihited increased in vivo tumorgeneity compared with corresponding control subline. In addition,increased expression of hFoxp3 in NCIH-hFoxp3 augmented secretion and at-tenuated secretion of IL-8 and IL-10,respectively. Conclusion:Increased expression of Foxp3 may promote progression of lung cancer cell by change of cellular microenvironment and evasion of immune surveillance.

Objective:To express and purify the fusion protein of GST-FILIP-1L and prepare its polyclonal antibody. Methods:The constructed recombinant expression vectors pGEX-4T3-FILIP-1L were transformed into Escherichia Coli BL21. FILIP-1L fusion protein was induced by IPTG and purified by Glutathion Sepharse 4B . The rabbit was immunized by the purified fusion protein,and pro-duced serum with anti-FILIP-1L antibody. The titer of polyclonal antibody was detected by ELISA, the anti-FILIP-1L polyclonal antibody was purified by Active and its combining specificity with FILIP-1L protein was further identified by Western blot. Results:The GST-FILIP-1L fusion protein was highly expressed in E. coli, and its specific polyclonal antibody was obtained after the immunization. The polyclonal antibody purified by Active Ester Agrose was able to combine specially with FILIP-1L protein and transformed FILIP-1L protein in 293 cells and FILIP-1L protein of liver cancer cells, respectively. Conclusion: The GST-FILIP-1L fusion protein was expressed successfully,the anti-GST-FILIP-1L polyclonal antibodies with high titer and specificity are successfully prepared,these antibodies provide an useful experimental tool to research the biological function of the FILIP-1L protein.

Objective To clone and express the alkaline protease AprA , one important virulence factor secreted by Pseudomonas aeruginosa(PAE)in Escherichia coli, to clone and express the inhibitor of AprA (AprI) and its substrate flagellin , and to detect the function of AprA and the inhibitory function of AprI .Methods The genes encoding AprA ,AprI and flagellin gene were amplified respectively by PCR using PAE PAO 1 genome DNA as the template .The expression vec-tors (pET-28a-AprA, pET-28a-AprI and pET-28a-Flagellin) were constructed and transformed into E.coli BL21(DE3) respectively.The recombinant AprA protein was expressed by IPTG induction and purified via denaturing and renaturation. The recombinant AprI and flagellin were expressed and purified by Ni 2+affinity chromatography .The cleavage activities of AprA on flagellin were detected by SDS-PAGE.Results Recombinant AprA , AprI and flagellin protein were expressed and purified .It was demonstrated that AprA cleaved flagellin , which was blocked by AprI .Conclusion Recombinant AprA could cleave its substrates as an alkaline protease , and its inhibitor AprI inhibits the activities of AprA .This study will contribute to further investigations on the role of AprA in the pathogenesis of PAE .