In this study,the carboxylated magnetic beads were coupled with bivalent nanobodies a-gainst porcine epidemic diarrhea virus(PEDV)M protein to construct immunomagnetic beads(IM-NBs-Ⅱ),The capture and enrichment function of IMNBs-Ⅱ was verified by using PEDV propaga-ted in Vero cells.A one-step RT-qPCR detection method for PEDV was established by combining the characteristics of IMNBs-Ⅱ with the detection advantages of reverse transcription fluorescence quantitative polymerase chain reaction(RT-qPCR).Specific analysis found that this method has no cross reactivity with swine fever virus,porcine reproductive and respiratory syndrome virus,por-cine parvovirus,porcine circovirus,indicating that it has good specificity.Sensitivity analysis re-vealed that the detection sensitivity of the RT-qPCR based on IMNBs-Ⅱ was increased 10 times compared to traditional RT-qPCR methods.Detection of the clinical samples confirm that the RT qPCR method based on IMNBs-Ⅱ is suitable for rapid and accurate detection of clinical feces and tissue samples.The method established in this study effectively avoids contamination issues during nucleic acid extraction,simplifies experimental procedures,and saves detection time,which pro-vides a method for efficient detection of PEDV.

In this study,the carboxylated magnetic beads were coupled with bivalent nanobodies a-gainst porcine epidemic diarrhea virus(PEDV)M protein to construct immunomagnetic beads(IM-NBs-Ⅱ),The capture and enrichment function of IMNBs-Ⅱ was verified by using PEDV propaga-ted in Vero cells.A one-step RT-qPCR detection method for PEDV was established by combining the characteristics of IMNBs-Ⅱ with the detection advantages of reverse transcription fluorescence quantitative polymerase chain reaction(RT-qPCR).Specific analysis found that this method has no cross reactivity with swine fever virus,porcine reproductive and respiratory syndrome virus,por-cine parvovirus,porcine circovirus,indicating that it has good specificity.Sensitivity analysis re-vealed that the detection sensitivity of the RT-qPCR based on IMNBs-Ⅱ was increased 10 times compared to traditional RT-qPCR methods.Detection of the clinical samples confirm that the RT qPCR method based on IMNBs-Ⅱ is suitable for rapid and accurate detection of clinical feces and tissue samples.The method established in this study effectively avoids contamination issues during nucleic acid extraction,simplifies experimental procedures,and saves detection time,which pro-vides a method for efficient detection of PEDV.

Objective:We proposed a Mingdao immune score system(MISS)to evaluate recipient's immune status after liver transplantation.Methods:From January 2017 to June 2019, retrospective analysis was conducted for 89 recipients of liver transplantation. Age/gender-matched 385 healthy controls(HC)were selected. The percentages of 30 lymphocyte subgroups of patients and HC were measured by flow cytometry. The score of each individual was calculated with our proposed MISS method. And drug concentrations and relevant clinical data were collected.Results:The normal MISS value of a healthy person was 0 score according to our criterion. In this study, the value of MISS for HC was distributed in a nearly normal fashion(-0.73±4.02). When the data from patients at different timepoints were compared, the MISS value started with -1.21±7.42 pre-operation, then declined sharply down to -8.95±8.05 at 1 month and jumped to -4.50±7.80 at 3 months. Afterward it stabilized at -4.18±7.83 between 3~12 months post-operation and finally reached -2.00±5.51 at 1 year ( P<0.05). Patients with acute rejection had higher MISS values than those without acute rejection, ( P<0.05). No significant correlation existed between blood drug concentrations and MISS values ( P>0.05). Conclusions:Our proposed MISS method may reflect the whole immune status. It is useful to manage the application of immunosuppressants in conjunctions with blood drug concentrations and liver graft function.

Objective To investigate the effect of Immutol on inducing the immune tolerance of cardiac grafts in rat models. Methods A rat model of heterotopic abdominal heart transplantation was established. The recipient rats were divided into 5 groups: blank control group (n=6); dimethyl sulfoxide (DMSO) group (n=6), in which DMSO was administered until the cardiac graft arrest; Immutol group (n=6), in which Immutol was administered until the cardiac graft arrest; ciclosporin (CsA) group (n=10), in which CsA was administered for 20 d; combined group (n=13), in which Immutol was given for 60 d combined with CsA for 20 d. The survival time and pathological changes of cardiac grafts in each group were observed. The contents of serum interleukin (IL)-10 and interferon (IFN)-γ were detected. The expression levels of indoleamine 2, 3-dioxygenase (IDO) and fibrinogen-like protein 2(Fgl2) messenger RNA(mRNA) in heart tissues of rats in each group were measured. Results In the combined group, the cardiac grafts survived for >180 d and immune tolerance was induced. The pathological score of cardiac grafts in the combined group was significantly lower than that in the CsA group at postoperative 39 d (P < 0.05). The levels of serum IL-10 and IFN-γ in the combined group were significantly higher than those in the CsA group at 9 d and 39 d after operation (both P < 0.05). The content of serum IL-10 and IFN-γ in the combined group were gradually increased over time. At postoperative 39 d, the expression levels of IDO and Fgl2 mRNA in the combined group were significantly higher than those in the CsA group (both P < 0.05). The expression level of IDO mRNA in the combined group tended to gradually elevate after operation. In the combined group, the expression level of Fgl2 mRNA at postoperative 180 d was significantly higher than those at 9 d and 39 d after operation (both P < 0.05). Conclusions Combined administration of Immutol and CsA can effectively inhibit the incidence of acute rejection, and maintain the long-term survival of the cardiac grafts and induce immune tolerance after drug withdrawal.

Objective To analyse the clinical efficacy of liver transplantation and summarize the clinical experience of perioperative management in patients with hepatic coma. Methods Clinical data of 22 patients with hepatic coma undergoing liver transplantation were retrospectively analyzed. The perioperative conditions of the recipients were observed, including operation time, warm/cold ischemia time of donor liver, intraoperative anhepatic phase of the recipients, intraoperative blood loss, intraoperative blood transfusion, early postoperative blood drug concentration and incidence of postoperative complications. The survival situation of the recipients and the influencing factors of clinical prognosis were analyzed. Results The operation time of 22 recipients was 8 (6-12) h, the warm ischemia time of donor liver was 4 (2-6) min, the cold ischemia time was 7 (5-10) h, intraoperative anhepatic phase of recipients was 80 (55-120) min, intraoperative blood loss was 1 139 (400-4 000) mL and intraoperative blood transfusion was 1 440 (0-3 600) mL.The blood concentration of tacrolimus (FK506) fluctuated between 6 and 11 ng/mL at postoperative one week. Six recipients died after liver transplantation including 1 case of primary graft liver failure, 2 cases of severe infection, 1 case of severe cerebral edema caused by cerebral hemorrhage and 2 cases of multiple organ failure. The postoperative 1 month and 1 year survival rates of hepatic coma recipients were 82% and 77%. Conclusions Liver transplantation can significantly improve the survival rate of patients with hepatic coma. Preoperative decreasing blood ammonia, controlling postoperative infection, improving renal function and formulating precise individualized immunosuppression therapy according to immune status play a pivotal role in enhancing the survival rate.

Objective To investigate the relationship between immune tolerance and the changes of helper T cell (Th), regulatory T cell (Treg) cytokines, related signaling pathway proteins during immune tolerance process in rat models of liver transplantation. Methods The orthotopic liver transplantation rat models were established by double-cuff technique. All rats were divided into 3 groups. In the operative control group (n=6), sham operation was performed without liver transplantation. In the short-term group (n=10), the rats survived for 10 d after liver transplantation. In the immune tolerance group (n=10), the rats survived for 100 d after operation and the function of the transplanted liver was restored to normal. The expression levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), Th1 cytokines [interferon (IFN)-γ, interleukin (IL)-2 and tumor necrosis factor (TNF)-α], Th2 cytokines (IL-4, IL-5, IL-6 and IL-13), Th17 cytokines [granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-17A], Treg cytokines [IL-10, transforming growth factor (TGF)-β and IL-12p] were quantitatively measured. The serum sample of rats in each group was detected by protein chip analysis. Results Compared with the operative control group, the AST level in the short-term group was significantly down-regulated, whereas the ALT level was significantly up-regulated (both P < 0.05). However, the AST and ALT levels did not significantly differ between the immune tolerance group and operative control group (both P > 0.05). In the liver tissues of rats in each group, the expression levels of Th1 cytokines IFN-γ and IL-2 in the short-term group were significantly higher than those in the operative control group (both P < 0.05). The expression level of Th2 cytokine IL-4 in the immune tolerance group was significantly lower than that in the operative control group (P < 0.05). The expression levels of Th2 cytokines IL-5, IL-6 and IL-13 in the short-term group were significantly lower than those in the operative control group (all P < 0.05). The expression level of IL-17A in the immune tolerance group was significantly higher than that in the operative control group (P < 0.05). In the immune tolerance group, the expression levels of IL-10and IL-12p were significantly higher than those in the operative control group (both P < 0.05). The expression level of TGF-β in the short-term group was significantly higher than that in the operative control group (P < 0.05). Compared with the operative control group, the expression levels of intercellular adhesion molecule (ICAM)-1, pro-platelet basic protein (Ppbp), Neuropilin-2, Notch-2 protein in the short-term group were significantly up-regulated (all P < 0.05). The expression levels of CXC chemokine ligand 17 (CXCL17), ICAM-1 and Neuroleptin-2 protein were markedly up-regulated (all P < 0.05), whereas that of B7-1 protein was significantly down-regulated (P < 0.05) in the immune tolerance group. Conclusions Treg cytokines (IL-10, TGF-β and IL-12p), IL-6, IL-17 and trans-membrane signaling pathway molecules (ICAM-1, Neuropilin-2, B7-1 proteins) play a pivotal role in the natural immune tolerance process of rat models of liver transplantation.

Abdomen, Acute ; diagnosis ; Adult ; Humans ; Male ; Pancreatic Neoplasms ; diagnosis ; Shock, Hemorrhagic ; diagnosis ; Young Adult

Objective To study whether citrate lock is superior to heparin lock in the prevention of catheter related infections,bleeding complications and catheter malfunctions among hemodialysis patients with tunneled catheters.Methods By searching in Pubmed,the Cochrane Library,EMBASE,Ovid,WanFang,VIP,CNKI and CBM databases as well as related journals,qualified randomized controlled trials were included in a Meta-analysis using Revman 5.0 and STATA 10.0 software.The endpoints included catheter related infection,bleeding complication,thrombolytic treatment,catheter removal for malfunction,catheter thrombosis and all-cause death.Results Fifteen randomized controlled trials were included with 1621 patients involved.Eight studies compared citrate alone with heparin lock,while 7 trials focused on citrate in combination with other antimicrobials.Pooled analysis demonstrated that incidence of catheter related infections in patients receiving citrate lock decreased by 47％ compared with those on heparin (RR=0.53,95％CI 0.36-0.77,P ＜ 0.01).Subgroup analysis by types of citrate lock indicated that all combined lock solutions of citrate and other antimicrobials (citrate + gentamicin,citrate + taurolidine,citrate + methylene blue +methylparaben + propylparaben) were superior to heparin lock in preventing catheter-related infections (P =0.01,0.04,0.01,respectively); citrate alone seemed to reduce catheter-related infection risk (RR =0.68),but no statistically significant difference was observed (95％CI 0.38-1.21,P=0.19).There were fewer patients with bleeding complications in citrate group (RR =0.53,95％ CI 0.34-0.S4,P ＜ 0.01),while citrate showed no advantage over heparin lock in terms of thrombolytic treatment (P =0.93),catheter removal for malfunction (P =0.35),catheter thrombosis (P =0.64) and all-cause death (P =0.35).Conclusions For hemodialysis patients with tunneled catheters,combined lock solutions of citrate and other antimicrobials,rather than citrate alone,are superior to heparin in preventing catheter related infections.Citrate locks are associated with less bleeding complications,and are comparable to heparin in the maintenance of catheter patency.