ObJective To locate the cysteine-rich domains(CRD) of murine 4-1BB binding to its natural ligand. Methods A serial soluble extracellular CRDs of routine 4-1BB and 4-1BBL fusion proteins was constructed and prepared. The binding of purified 4-1BB-Igs to 4-1BBL and 4-1BB monoclonal antibody were tested using ELISA assay and Western blot analysis. Blocking experiment with 4-1BBL and 4-1BB mon-oclonal antibody was performed by ELISA assay. Results All truncated overlapped proteins containing ex-tracellular CRD Ⅱ of murine 4-1BB were able to bind to 4-1BBL by ELISA assay, excepting the CRD Ⅰ do-main alone. A 4-1BB monoclonal antibody proved to block the interaction of 4-1BB and 4-1BBI, was also able to bind to CRD Ⅱ. Conclusion Murine 4-1BBL whose specificity was mapped to CRD Ⅱ of 4-1BB ex-tracellular region with a possible conformational structure.

Objective To determine whether regulatory T cells(Tr)are increased in patients with tuberculosis and whether they are associated with its immunopathology.Meantime,to investigate the possibility of tuberculosis(TB)as a model for studying Tr functions.Methods The lymphocyte subsets were isolated from peripheral blood mononuclear cells by sorting with flow cytometry.Total cellular RNA was extracted and RT-PCR was performed to detect the Foxp3 mRNA in purified CD3+CIM+T cells,CD3+CD8+T cells and non-CD3+CD4+CD8+T cells.Using FACS analysis.we further investigated the distribution of Foxp3+ population in CD4+ CD25+T cells.Finally,we compared the percentage of CD4+CD25highFoxp3+T cells present in 51 active patients with tuberculosis and 40 uninfected healthy control subjects by FACS.The detection of Tr infiltration of Foxp3+ cells were performed with immunohistochemistry(IHC)method on tuberculosis pathological sections.Results Foxp3 was specific expressed in CD3+CD4+T cells,either in tuberculosis patients or healthy control subjects.Foxp3+ T cells took about 85%fraction of CD4+ CD25highpopulation.We used CD4+CD25high Foxp3+as a detective markers for Tr in the FACS analysis.The results showed that patients with active TB had a 4.4 fold higher percentage within the CD4+T cells in peripheral blood compared to healthy control group(modian,1.01%vs 0.23%,P<0.01).Much higher frequency of Tr were found along with T cells infiltration at the tuberculosis pathological tissues.A few individuals that we can followed indicated the expanded Tr was declined after curative treatment with operation.Conclusion Tr cells are increased in tuberculosis patients and closely correlate with its immunopathology.Tuberculosis should be a valuable model for Tr functional study.

Objectives: To evaluate the effect and safety of 14-day continuous subcutaneous insulin infusion (CSII) in type 2 diabetes patients with heart diseases.
Methods: A total of 22 consecutive type 2 diabetes patients (history ≤ 5 years) with heart diseases treated in our hospital from 2011-03 to 2013-08 were studied. There were 20 male, and the with the mean age of patieuts (48.15 ± 9.80) years, all patients without standard hypoglycemic treatment before admission. The patients received 14-day CSII for enhanced treatment and the blood glucose level, insulin function and insulin sensitivity were compared before and after the treatment.
Results: After CSII treatment, the blood glucose level was obviously decreased, fasting blood glucose (FBG) and postprandial blood glucose at 30, 60 and 120 min were improved, all P<0.001. The C peptide level was higher at 60 and 120 min alter treatweut as 2.73 (1.05-7.05)ng/ml vs 3.84 (1.22-63.39)ng/ml, P=0.004 and 3.34 (1.42-9.61)ng/ml vs 6.27 (0.93-47.39)ng/ml, P=0.004. The insulin sensitivity was improved as -1.89 ± 0.29 vs -1.70±0.31, P=0.008. With CSII treatment, there were 22.73% patients (5/22) at remission by controlling the diet and excise and 77.27% (17/22) with continuing medication. The patients were followed-up for (12.4 ± 8.5) months, there were 4/5 patients with euglycemia, 1/5 with increase blood sugar and received medication at 2 months after discharge, the rest 17 patients remained oral hypoglycemic medication.
Conclusion: CSII may quickly relieve glucotoxicity and improve insulin sensitivity in type 2 diabetes patients with heart diseases. Some patients may alleviate drug burden in clinical practice.

BACKGROUNDIt was reported that tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was a powerful pulmonary carcinogen, predominantly inducing adenocarcinoma of the lung in mouse. The aim of this study is to assay metabolites of NNK, which are 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its O-glucuronide (NNAL-Gluc), and their ratio (NNAL-Gluc/NNAL) in smokers and non-smokers' urine, and to explore the carcinogenicity of NNK among different people.

METHODSUsing high pressure liquid chromatograph (HPLC) and gas chromatograph-mass tadom (GC-MS/MS), NNAL-Gluc and NNAL in 24h urine were detected in 8 healthy smokers, 10 lung cancer smokers and 4 healthy non-smokers.

RESULTSBoth of the two metabolites were not found in non-smokers' urine. The ratios of urine NNAL-Gluc/NNAL were greatly different among different smokers. The mean ratio of NNAL-Gluc/NNAL in healthy smokers was 4.95, and 0.5 in lung cancer smokers.

CONCLUSIONSThe results provide the first evidence for metabolite detection of tobacco-specific nitrosamine in Chinese smokers' urine . The result suggests that detoxification ability of healthy smokers is higher than that of lung cancer smokers. It may provide a detective way to screen high risk people for lung cancer in smokers.

Objective:To observe the effect of neuroendoscope-assisted drainage with lumbar cistern and large bone flap decompression in the treatment of craniocerebral injury complicated with temporal uncinate herniation.Methods:A total of 80 patients with craniocerebral trauma and temporal uncinate herniation hospitalized in Lanling County People′s Hospital from January 2017 to October 2020 were retrospectively included and divided into the observation group and the control group according to the surgical methods, with 40 patients in each group. Surgical procedures were performed by the same group of experienced neurosurgeons. The observation group was treated with neuroendoscope-assisted drainage with lumbar cistern and large bone flap decompression, while the control group was treated with large bone flap decompression only. Cephalic CT was reexamined before and 48 h after the surgery to compare the appearance rates of cisterna ambiens and suprasellar cistern. Intracranial pressure (ICP) was monitored at 3, 5 and 7 d after the surgery, and the scores of Glasgow coma scale(GCS) was recorded. Drainage time, postoperative cerebral edema and cerebral infarction complications were recorded and compared between the two groups. Six months after the surgery, the prognosis was assessed by the Glasgow prognostic scale (GPS).Results:The occurrence rates of cisterna ambiens and suprasellarcistern in the observation group were higher than those in the control group: 67.50%(27/40) vs. 45.00%(18/40), 65.00%(26/40) vs. 42.50%(17/40), χ2 = 4.11, 4.07, P<0.05. The ICP value in the observation group at 3, 5 and 7 d after the surgery were significantly lower than those in the control group, and the scores of GCS in the observation group were significantly higher than those in the control group, there were statistical differences( P<0.05). There was no statistically significant difference in drainage time between the two groups ( P>0.05). The incidence of postoperative cerebral edema in the observation group was lower than that in the control group:7.50%(3/40) vs. 25.00%(10/40), χ2 = 4.50, P<0.05. The incidence of postoperative cerebral infarction in the observation group was lower than that in the control group, and the volume of cerebral infarction was smaller than that in the control group: 5.00%(2/40) vs. 22.50%(9/40), (6.68 ± 1.75) cm 3 vs. (8.20 ± 2.15) cm 3, there were statistical differences ( P<0.05). The incidence of postoperative complications in the observation group was lower than that in the control group: 7.50%(7/40) vs. 40.00%(16/40), χ2 = 4.94, P<0.05. Six months after the surgery, the rate of good prognosis in the observation group was higher than that in the control group: 62.50%(25/40) vs. 35.00%(14/40), χ2 = 6.05, P<0.05. Conclusions:Neuroendoscope-assisted drainage with lumbar cistern and large bone flap decompression in the treatment of craniocerebral trauma and temporal uncinate herniation has good efficacy and safety.

Objective:To explore the pathogenesis of flunarizine-induced parkinsonism from gut-brain axis perspective.Methods:Thirty male C57BL/6 mice were randomly divided into control group and flunarizine group ( n=15). Mice in the control group were given 0.1 mL 50% polyethylene glycol 400+50% saline by gavage once/d for 2 weeks, while mice in the flunarizine group were given 6 mg/mL flunarizine+50% polyethylene glycol 400+50% saline by gavage at a daily dose of 30 mg/kg for 2 weeks. Body mass was recorded 1, 3, 5, 7, 10 and 14 d after drug administration, and motor function was assessed by rotarod test 14 d after drug administration; 16s RNA sequencing was performed in the feces to observe the intestinal flora; intestinal transit function was detected by Evans blue by gavage; and then, the mice were sacrificed and homogenate or frozen sections (brain and intestinal tissues) were prepared; dopamine-ergic neuron expression was detected by Western blotting; RT-qPCR was applied to detect the expressions of inflammatory factors in the substantia nigra, and immunofluorescent staining was used to detect the expressions of ZO-1 and Claudin-5 in the intestinal epithelial tissues. Results:Compared with the control group, the flunarizine group had lower body mass ratio 1, 3, 5, 7, 10 and 14 d after drug administration (ratio to body mass before drug administration). Compared with the control group, the flunarizine group had significantly shortened residence time in rod rotating and lower rotational speed when falling ( P<0.05). Compared with the control group, the flunarizine group had decreased tyrosine hydroxylase protein in the substantia nigra without significant difference ( P>0.05). Compared with the control group, the flunarizine group had significantly increased interleukin-6 and tumor necrosis factor-α in the substantia nigra (1.00±0.00 vs. 2.79±0.83; 1.00±0.00 vs. 3.39±1.37), significantly lower intestinal Evans blue propulsion rate (80.67%±4.51% vs. 50.67%±6.03%), and statistically decreased ZO-1 and Claudin-5 expressions in the colonic epithelial tissues (27.01±1.41 vs. 16.32±2.83; 37.00±2.80 vs. 24.52±2.12, P<0.05). Totally, 576 microorganisms were noted in both control group and flunarizine group, 744 in the control group alone, and 634 in the flunarizine group alone. The intestinal flora β diversity indices in the 2 groups were significantly different based on weighted Unifrac-principle coordinates analysis (PCoA, PCoA1: 39.88%; PCoA2: 30.69%). Compared with the control group, the microbial colony structure of mice in flunarizine group was dominated by phylum thick-walled bacteria and phylum warty microbacteria, and by families Muribaculaceae, Lachnospiraceae and Akkermansiaceae. Compared with the control group, the flunarizine group had significantly decreased relative abundance of Ackermannia spp. and Lactobacillus spp. in the intestinal flora ( P<0.05). Conclusion:Flunarizine may contribute to the pathogenesis of DIP by causing structural disturbances in the intestinal flora and inducing neuroinflammation based on the gut-brain axis.

Objective To establish a human lung cancer cell line that can stably express firefly luciferase (Fluc) and red fluorescent protein (RFP) gene so as to lay the foundation for the further establishment of a live-imaging lung cancer xenograft model in nude mice and therapeutic research.Methods The lentiviral vector pHBLV-FlucRFP containing luciferase and red fluorescent protein was constructed and then transfected into 293T cells for virus packaging.The complete virus was used to infect human lung cancer cell lines A549,H1975 and human B-cell lymphoma cell line K562.The stable cell lines were obtained by puromycin selection.Fluorescence microscopy and quantitative PCR were used to confirm the RFP and Fluc expression.Results The lentiviral vector pHBLV-FlucRFP was successfully constructed.Cancer cell line A549,H1975 and K562 stably expressing Fluc and RFP was obtained.The real-time quantitative PCR results showed that the relative expression of Fluc gene in the three stable infected cells was much higher than that in the corresponding wild-type cells,and the differences were statistically significant(all P＜0.05).Conclusion The human lung cancer cell line A549,H1975 and human B-cell lymphoma cell line K562 with dual expression of RFP and Fluc were obtained,which provided a new model of fluorescent cells for in vivo imaging of immunodeficient mouse models such as nude mice.

Objective:To construct an expression system of lentivirus vector encoding epidermal growth factor receptor-specific chimeric antigen receptor (EGFR-CAR) and programmed cell death ligand-1 (PD-L1) antibody.Methods:Human PD-L1-Fc protein was used to immunize BALB/c mice. Cell-fusion and subcloning were performed to screen stable hybridoma strains with high secretion of PD-L1-specific antibodies, which were identified by both ELISA and Western blot. The activity of the antibodies in blocking the binding of programmed cell death-1 (PD-1) to PD-L1 was determined by fluorescence-activated cell sorting (FACS). Antibody affinity was analyzed by Fortebio Octet96. A single-chain variable fragment (scFv) was further constructed after antibody full-length sequencing and humanization using CDR grafting method. Meanwhile, the genes encoding the light and heavy chain variable regions (VL and VH) were cloned from a hybridoma secreting antibody against human EGFR by 5′ RACE technology to construct scFv gene. The expression of scFv was confirmed using pcDNA3.1 vector. EGFR-CAR containing CD137 intracellular function domain and PD-L1-scFv was ligated using 2A gene. The synthetic single molecule was cloned into pLVX-EF1a-IRES-ZsGreen1 lentivirus expression vector, and then transfected into 293T cells using Lenti-X Packaging Single Shots (VSV-G) to prepare infectious virus. Expression of CAR on cell surface and the soluble form of PD-L1-scFv in the supernatant of transfected 293V cells were detected by FACS and ELISA.Results:A PD-L1 antibody named 11E3 with high ligand-receptor blocking performance was obtained. The humanized antibody showed a stable affinity (2.67×10 -10 mol/L) after directly grafting the mouse CDRs (CDR1, CDR2 and CDR3) to human frameworks. EGFR-scFv was effectively expressed in a form of Fc-fusion. Secretory CAR (CTZ0431-1) and membrane CAR (CTZ0431-2) expression plasmids were constructed using lentivirus vector containing EGFR-CAR and PD-L1-scFv. The infection efficiency in 293V cells was around 10%. EGFR-scFv on the cell membranes and PD-L1-scfv in the culture supernatants were detected after 293V cells were infected with CTZ0431-1. EGFR-scFv and PD-L1-scfv were expressed on the cell membranes of 293V cells infected with CTZ0431-2. The expression rate of CAR in LV-CART46407-1-transfected activated T cells was 39.3%. Conclusions:The lentivirus vectors co-expressing EGFR-CAR with moderate binding affinity and PD-L1-scFv with high binding affinity were successful constructed, which provided an essential tool for investing EGFR- and PD-L1 double targeted CAR-T cell therapy against solid tumor.

Objective To investigate the correlation between serum leptin level and body mass index(BMI)in in-fants with cyanosis congenital heart disease,and the relationship between leptin and Ob gene receptor(Ob-R)and hypoxia-inducible factor 1α(HIF-1α)in myocardium.Methods A total of 52 children under 6 months of age with congenital heart disease who underwent surgical treatment in the Department of Congenital Heart Surgery,Fuwai Hospital from January 2019 to October 2020 were included in this study.According to the arterial partial pressure of oxygen(PaO2)of 90 mmHg,they were divided into cyanotic group(n=30)and acyanotic group(n=22).Their height and weight were collected to calculate BMI.The serum leptin level was measured by ELISA.The ex-pressions of HIF-1α and Ob-R in myocardial tissue were detected by RT-PCR and Western blot.In animal mod-el,SD rats were divided into normoxia group and hypoxia intervention group,which were subjected to continuous hypoxia(10％ O2)for 4 weeks.The hypoxia intervention group received intraperitoneal injection of HIF-1α in-hibitor digoxin(2 mg/kg)daily from the 14 th to 21st day of hypoxia,respectively.The body weight of rats was recorded,and the expressions of HIF-1α and Ob-R were detected by RT-qPCR and Western blot.Results Com-pared with the acyanosis group,the cyanosis group had a significantly lower BMI(P<0.05)and a lower leptin/BMI ratio(leptin/BMI)(P<0.05).Spearman correlation analysis confirmed that serum leptin in the circulatory system was positively correlated with BMI(P<0.05).In the cyanosis group,the expression of Ob-R increased with the upregulation of HIF-1α,showing a positive correlation.In animal model,with the down-regulation of HIF-1α expression in digoxin injection,the Ob-R level was significantly lower than that in the control group(P<0.05),the trend of weight loss was significantly inhibited(P<0.05).The right ventricular hypertrophy in-dex was significantly lower than that in the control group(P<0.05).Conclusions HIF-1α regulates the expres-sion of Ob-R in myocardial tissue,and the mechanism of its association with leptin and Ob-R may help to find new therapeutic target for improving the prognosis of infants with congenital heart disease.

ObjectiveTo explore the role of aryl hydrocarbon receptor/suppressor of cytokine signaling 2/nuclear transcription factor-κB (AHR/SOCS2/NF-κB) signaling in the reduction of spinal cord injury (SCI) inflammation with every-other-day fasting (EODF). MethodsA total of 36 healthy male Sprague-Dawley rats were randomly divided into sham group (n = 12), model group (n = 12) and EODF group (n = 12). The latter two groups were established C₅ spinal cord hemi-clamp model. The EODF group received EODF, namely fasting and feeding per 24 hours (water free), for two weeks after operation. They were assessed with Grooming Test before operation, and one, seven and 14 days after operationt, respectively. Pathological injury of spinal cord was observed with HE staining 14 days after operation, while the expression of AHR, SOCS2 and NF-κB proteins were detected with Western blotting, and the mRNA of AHR, SOCS2, inhibitor α of NF-κB (IκBα), NF-κB and tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) were measured with reverse transcription real-time quantitative polymerase chain reaction. ResultsCompared with the sham group, the Grooming Test score decreased in the other two groups (P < 0.05), and cavity, edema and structural disorder appeared in the spinal cord. Compared with the model group, the pathology of EODF group improved, and the Grooming Test score increased (P < 0.05). The mRNA and protein expression of AHR and SOCS2 increased (P < 0.05), the mRNA and protein expression of NF-κB decreased (P < 0.05), the mRNA expression of IκBα increased (P < 0.05), and the mRNA expression of TNF-α and IL-6 decreased (P < 0.05). ConclusionEODF may promote the recovery of motor function of forelimb in rats with SCI, and relieve damage and inflammation, which may associate with the activation of AHR/SOCS2/NF-κB signaling pathway.