Objective To evaluate the effects of pyruvate Ringer' s solution on erythrocyte deformation function in the patients undergoing cardiopulmonary bypass (CPB) in vitro.Methods Thirteen patients of both sexes,aged 25-64 yr,of ASA physical status Ⅱ or Ⅲ (NAHY Ⅱ or Ⅲ),scheduled for elective cardiac valve replacement under CPB were enrolled in the study.At 10 and 60 min of CPB (T1,2) and 60 min after termination of CPB (T3),blood samples 18 ml were drawn from the radial artery and each patient's blood sample was equally divided into 6 parts (3 ml per part).The blood samples were divided into 3 groups (n =13 each):control group (group C),lactated Ringer's solution group (group R),and pyruvate Ringer's solution group (group P).In C,R and P groups,normal saline 1 ml,lactated Ringer's solution 1 ml,and pyruvate Ringer's solution 1 ml were added,respectively,and then the erythrocytes were incubated for 60 min at 37 ℃.At 30 and 60 min of incubation,the erythrocyte rigidity index (ERI) and erythrocyte deformation index (EDI) were detected.Results Compared with C and R groups,the ERI was significantly decreased at 30 and 60 min of incubation at T1,the EDI was decreased at 30 min of incubation at T1,2,and no significant change was found in ERI at T2,3 or in EDI at T3 in group P.There was no significant difference in ERI and EDI at different time points of incubation within groups.Conclusion Pyruvate Ringer's solution can enhance erythrocyte deformation function in the patients undergoing CPB.

ObjectiveTo evaluate the diagnostic value of ultrasound or CT guided percutaneous Trucut needle biopsy on the diagnosis of pancreatic tumors.Methods One hundred and twenty-four patients clinically diagnosed as pancreatic cancer without pathological diagnosis underwent percutaneous pancreatic biopsy by using Trucut needle under ultrasound or CT guidance.ResultsOne hundred and nine procedures of ultrasound-guided biopsy and 15 procedures of CT-guided biopsy were performed,and one patient received 2.3times of punctures.Tissue samples were obtained in all 124 patients,the diagnostic accuracy was 95.2％,among them 115 were adenocarcinoma,5 were cystadenoma,2 were metastasis cancer,1 was cancer of unknown origin and 1 was normal.The sensitivity,specificity,and accuracy were 99.2％ 100％,and 99.2％,respectively.Transient serum amylase increase was observed in 3 patients; 5 patients' abdominal pain aggravated,but all recovered with conservative management.One patient was found to have tumor seeding on the spot of insertion after 34 days.No other major complications occurred.ConclusionsUltrasound or CTguided percutaneous pancreatic 16 ～ 18G Trucut needle biopsy is a safe and simple procedure with excellent diagnostic value for pancreatic cancer.

Objective:To study the effect of metformin on proliferation,cell cycle and apoptosis of U937 cells.Methods: U937 cells were treated with different concentrations of metformin,collected cells in 24,48 and 72 hours.Subsequently,cell proliferation was assessed by CCK-8 assay,and the cell cycle and apoptosis were analyzed by flow cytometry (FCM).The expression of Bcl-2,Bax,p-AMPK,p53 were determined by Western blot.Results: The proliferation of U937 cells was inhibited by metformin in a time-and dose-dependent manner.Metformin-treated cells were arrested at G0/G1 phase,the cell frequency at G0/G1 phase was increased in a time-and dose-dependent manner.Metformin also induced cell apoptosis in a dose-dependent manner.It showed that 20 mmol/L metformin induced cell apoptosis in a time-dependent manner.The expression of p-AMPK,p53,Bax was up-regulated while Bcl-2 expression was down-regulated after metformin treatment.Conclusion: Metformin could inhibit the U937 cell proliferation,block the cell cycle at G0/G1 phase,and induce cell apoptosis,which may partially be attribute to the up-regulation of Bax,down-regulation of Bcl-2,activation of AMPK/p53 signaling.

OBJECTIVETo investigate the effects of mannan-binding lectin (MBL) on the functions of human polymorphonuclear cells (PMNs).

METHODSELISA and Dot blot were performed to examine the binding between MBL and the microorganisms. Flow cytometry and fluorescence microscopy were employed to analyze the phagocytosis of FITC-labeled microorganisms by the PMNs. Real-time quantitative PCR was used to detect the expression levels of IL-1β, TNF-α and CD11b mRNA in the PMNs, and ELISA used to detect the levels of TNF-α and IL-6 in the supernatants of PMN culture. Nitro-blue tetrazolium reduction assay was used to estimate the levels of superoxide production.

RESULTSMBL bound to the microorganisms in a dose-dependent manner. MBL had no significant effect on phagocytosis of C. albicans and E.coli by the PMNs in the absence of human serum, but in presence of mixed MBL-deficient human sera, MBL promoted the phagocytosis of C. albicans, which could be blocked by mannan. Mannan treatment increased the expressions of IL-1β, TNF-α, IL-6 and CD11b and enhanced superoxide production in the PMNs.

CONCLUSIONMBL can promote phagocytosis of microorganisms by PMNs and increase the expressions of proinflammatory cytokines from PMNs in a complement lectin pathway-dependent manner.

Candida albicans ; immunology ; Cells, Cultured ; Cytokines ; immunology ; Escherichia coli ; immunology ; Humans ; Mannose-Binding Lectin ; blood ; Neutrophils ; immunology ; Phagocytosis ; Superoxides ; immunology

OBJECTIVETo prepare the trimeric subunits of recombinant human mannan-binding lectin (MBL) with biological activities.

METHODSA prokaryotic expression vector containing human MBL N-terminal deletant (rhMBLδN) gene we previously constructed was transformed into E. coli for efficient expression of rhMBLδN fusion protein. Based on the principle that the collagen polypeptides tend to self-assembly into the tertiary structure of proteins by forming a triple helix due to the characteristic properties of the collagen proteins, rhMBLδN fusion protein was limitedly hydrolyzed with thrombin. The obtained rhMBLδN polypeptide was repeatedly dialyzed in 50 mmol/L PBS (pH7.2) and ddH(2)O, and the final product was analyzed for its bioactivities using a ligand-binding assay and a C4d deposition assay.

RESULTSrhMBLδN polypeptide with a relative molecular mass of about 20 000 was obtained by limited proteolysis of rhMBLδN fusion protein with thrombin. Repeated dialyses of rhMBLδN polypeptides in 50 mmol/L PBS and ddH(2)O resulted in the isolation of the trimeric subunit trhMBLδN (with a relative molecular mass of about 50 000), which contained a collagen-like helix. The trhMBLδN protein had a higher ligand-binding activity than rhMBLδN polypeptide, and acquired the activity to initiate the lectin pathway of complement activation, but the activities were lower than those of natural MBL.

CONCLUSIONWe have successfully obtained the bioactive trimeric subunit of rhMBL, trhMBLδN, and this structural subunit is also the functional subunit of the MBL molecule.

Complement Activation ; Escherichia coli ; metabolism ; Genetic Vectors ; Humans ; Mannose-Binding Lectin ; biosynthesis ; genetics ; isolation & purification ; Recombinant Proteins ; biosynthesis

Objective To evaluate the effect and mechanism of terminal fucosylation inhibitor 2-deoxy-D-galactose (2-D-gal) on ciclosporin (CsA)-induced renal epithelial-mesenchymal transition (EMT). Methods Fifteen male C57BL/6 mice aged 8-10 weeks were randomly and evenly divided into the control group (Ctrl group), CsA group and CsA+2-D-gal group (n=5). The expression levels of fucosyltransferase 1 (FUT1), EMT-associated proteins including E-cadherin, Vimentin, α-smooth muscle actin (α-SMA) in the kidney tissues of the Ctrl and CsA groups were detected by Western blot. The expression levels of terminal fucose in the kidney tissues of Ctrl and CsA groups were determined by immunofluorescence. The renal fibrosis of mice in each group was evaluated by Masson staining. The blood urea nitrogen and serum creatinine levels of mice in each group were detected. The in vitro EMT model of renal tubular epithelial cell HK2 was induced by CsA. HK2 cells were stimulated with 0, 2.5, 5.0 and 10.0 μmol/L CsA for 24 h, respectively. In addition, HK2 cells were divided into the Ctrl, 2-D-gal, CsA and CsA+2-D-gal groups. The morphology of HK2 cells after stimulation with different concentrations of CsA and in each group was observed. The expression levels of FUT1, E-cadherin, Vimentin and α-SMA in HK2 cells after stimulation with different concentrations of CsA and in each group were detected by Western blot. The expression level of terminal fucose in HK2 cells of the Ctrl and CsA groups was measured by immunofluorescence. Results Compared with the Ctrl group, the relative expression of E-cadherin protein was down-regulated, those of FUT1, Vimentin and α-SMA proteins were up-regulated (all P < 0.05), and that of terminal fucose in the mouse kidney tissues was up-regulated in the CsA group. Compared with the Ctrl group, the blood urea nitrogen and serum creatinine levels in the CsA and CsA+2-D-gal groups were up-regulated (all P < 0.05). Compared with the CsA group, the blood urea nitrogen and serum creatinine levels in the CsA+2-D-gal group were down-regulated (both P < 0.05). Compared with the Ctrl group, the collagen fiber deposition was increased and the relative expression of α-SMA protein was up-regulated in the mouse kidney tissues of CsA and CsA+2-D-gal groups (all P < 0.05). Compared with the CsA group, the collagen fiber deposition was decreased and the relative expression of α-SMA protein in the mouse kidney tissues was down-regulated in the CsA+2-D-gal group (both P < 0.05). With the increase of CsA concentration, the morphology of HK2 cells gradually became longer and thinner from original normal cobblestone shape, the relative expression levels of FUT1, Vimentin and α-SMA protein in HK2 cells were up-regulated, and that of E-cadherin protein was down-regulated in a concentration-dependent manner. Compared with the Ctrl group, the expression level of terminal fucose of HK2 cells was up-regulated in the CsA group. After CsA treatment combined with 2-D-gal intervention, the morphology of HK2 cells in the CsA+2-D-gal group was restored to resemble that of normal HK2 cells. Compared with the CsA group, the relative expression of E-cadherin protein in HK2 cells was up-regulated, whereas those of Vimentin and α-SMA proteins were down-regulated in the CsA+2-D-gal group (all P < 0.05). Conclusions CsA may induce EMT both in vivo and in vitro, and the terminal fucosylation is increased. 2-D-gal may inhibit CsA-induced EMT by suppressing the terminal fucosylation.

OBJECTIVETo clone human CD45 gene PTPRC and establish Hela cells overexpressing recombinant human CD45 protein.

METHODSThe intact cDNA encoding human CD45 amplified using RT-PCR from the total RNA extracted from peripheral blood mononuclear cells (PBMCs) of a healthy donor was cloned into pMD-18T vector. The CD45 cDNA fragment amplified from the pMD-18T-CD45 by PCR was inserted to the coding region of the PcDNA3.1-3xflag vector, and the resultant recombinant expression vector PcDNA3.1-3xflag-CD45 was transfected into Hela cells. The expression of CD45 in Hela cells was detected by flow cytometry and Western blotting, and the phosphastase activity of CD45 was quantified using an alkaline phosphatase assay kit.

RESULTSThe cDNA fragment of about 3 900 bp was amplified from human PBMCs and cloned into pMD-18T vector. The recombinant expression vector PcDNA3.1-3xflag-CD45 was constructed, whose restriction maps and sequence were consistent with those expected. The expression of CD45 in transfected Hela cells was detected by flow cytometry and Western blotting, and the expressed recombinant CD45 protein in Hela cells showed a phosphastase activity.

CONCLUSIONThe cDNA of human CD45 was successfully cloned and effectively expressed in Hela cells, which provides a basis for further exploration of the functions of CD45.

Blotting, Western ; Cloning, Molecular ; DNA, Complementary ; Genetic Vectors ; HeLa Cells ; Humans ; Leukocyte Common Antigens ; genetics ; Leukocytes, Mononuclear ; Polymerase Chain Reaction ; Recombinant Proteins ; genetics ; Transfection

OBJECTIVETo explore the significance of plasma levels of mannan-binding lectin (MBL)-associated serine protease 2 (MASP2) in children with upper respiratory tract infection (URTI).

METHODSA total of 103 children with URTI and 35 healthy children were examined for plasma levels of MASP2 and C-reactive protein (CRP). According to CRP levels, white blood cell count (WBC), stage of infection, and administration of treatments, the children with URTI were divided into the elevated CRP group (n=48) and the normal CRP group (n=54), elevated WBC group (n=61) and normal WBC group (n=40), the early stage of infection without treatment group (n=68) and mid-late stage of infection with treatment group (n=35).

RESULTSPlasma MASP2 levels was significantly higher in URTI group than in the healthy control group (P<0.001) and showed a close correlation with age (r=0.302, P<0.01). Plasma MASP2 level was significantly correlated with CRP level in elevated CRP group (r=0.310, P<0.05) but not in normal CRP group (P>0.05), correlated with WBC in elevated WBC group (r=0.392, P<0.01) but not in normal WBC group (P>0.05), and was significantly higher in early stage infection without treatment group than in mid-late stage of infection with treatment group (P<0.01). MASP2, MBL2 and CRP genes had a common binding site for the transcription factor HNF-4α.

CONCLUSIONSMASP2 may be an acute-phase protein, and its plasma level might serve as a new reference index in the diagnosis of URTI in children.

C-Reactive Protein ; metabolism ; Case-Control Studies ; Child ; Humans ; Leukocyte Count ; Mannose-Binding Protein-Associated Serine Proteases ; metabolism ; Respiratory Tract Infections ; blood

The twenty-first century has already recorded more than ten major epidemics or pandemics of viral disease, including the devastating COVID-19. Novel effective antivirals with broad-spectrum coverage are urgently needed. Herein, we reported a novel broad-spectrum antiviral compound PAC5. Oral administration of PAC5 eliminated HBV cccDNA and reduced the large antigen load in distinct mouse models of HBV infection. Strikingly, oral administration of PAC5 in a hamster model of SARS-CoV-2 omicron (BA.1) infection significantly decreases viral loads and attenuates lung inflammation. Mechanistically, PAC5 binds to a pocket near Asp49 in the RNA recognition motif of hnRNPA2B1. PAC5-bound hnRNPA2B1 is extensively activated and translocated to the cytoplasm where it initiates the TBK1-IRF3 pathway, leading to the production of type I IFNs with antiviral activity. Our results indicate that PAC5 is a novel small-molecule agonist of hnRNPA2B1, which may have a role in dealing with emerging infectious diseases now and in the future.
Animals ; Mice ; Antiviral Agents/pharmacology* ; COVID-19 ; Hepatitis B virus ; Interferon Type I/metabolism* ; SARS-CoV-2/drug effects* ; Heterogeneous-Nuclear Ribonucleoprotein Group A-B/antagonists & inhibitors*