Segregated Kunming mice bearing S180 sarcoma were used as tumor models and treated with rhIL-6. The tumor regressed in most of the treated mice (8/10) in which there was no tumor growth when rechallenged with the same tumor cells, whereas 9/10 of controls died as the tumor progressed. Morphologically, in the treated group, the mice had enlarged spleen (4 times larger than that of control group)with hyperplastic white pulp consisted predominantly of activated lymphocytes. Cytotoxicity of spleen cells from IL-6 treated group to autologous tumor cells was higher than that of control group (907 ?318: 387 ?144, P=0.003) and so did L929 cell line used as target (1145?164: 186?251).We also set in vitro experiment using human PBL and human melanoma and colon carcinoma cell lines (A375,LS174). These cells were treated with IL-6 respectively and the cytotoxicity was assayed. Although PBL stimulated with IL-6 killed the LS174 more efficiently,the higher cytotoxicity to LS174 is because of the increased sensitivty of LS174 to the effector cells .On the contrary,IL-6 showed no effect on the A 375 cell line. It is assumed that this difference might result from the discrepancy of the recognition molecules existed on the cell surface between these two cell lines.

Objective To construct the human interleukin-18(hIL-18) DNA plasmids vaccine and to express the eukaryotic plasmids vaccine in mammalian cell lines Cos-7 and D5.Methods Gene recombinant technique was used to construct hIL-18 eukaryotic expression vectors.Calcium phosphate method was performed to transect recombinant hIL-18 eukaryotic expression vectors into Cos-7 and D5 cells.In situ hybridization and Western Blot were implemented to verify the transient expression of recombinant hIL-18 in Cos-7 and D5.Results The eukaryotic expression plasmid pVAX1-IL 18 was constructed successfully.hIL-18 was transiently expressed in Cos-7 and D5.Conclusion The eukaryotic expression plasmid pVAX1-IL 18 was constructed.In situ hybridization and Western Blot results proved the successful transient expression of pVAX1-IL 18 in Cos-7 and D5.Therefore,the work has settled the foundation for further biological research on hIL-18,including immunogene therapy through hIL-18.

Objective To prepare the monoclonal antibody against human perforin(HP). Methods Recombinant eukaryotic expression plasmid pCDM8-HP was extracted and purified, and the BALB/C mice were immunized with the plasmid. The hybridomas producing anti-HP McAbs were established by using hybridoma technique, then the specificity of the McAbs was identified by using immunocytochemical technique and Western blot. Results Three hybridoma cell lines secreting McAbs against human perforin were established, and the three McAbs showed positive only with LAK cells containing human perforin protein, and showed negative with inactive human peripheral blood lymphocytes (PBLs). The subclasses of the three McAbs were determined as lgG2bK. Western blot results showed that the three McAbs recognized a specific band of LAK cell lysateds with molecular weight of 70.0Kd. Conclusion The three hybridoma cell lines secreting McAbs against human perforin were established and the secreted McAbs were specific.

Objective To express human B7.2 extracellular domain with prokaryote expression system and to evaluate its biological activity in vitro. Methods PCR was used to amplify the extracellular region of human B7. 2which contained both the IgV and IgC domains. The recombinant PGEX-4T-3/hB7. 2 (IgV+C) was obtained by cloning the PCR product into a prokaryote expression plasmid PGEX-4T-3 and was transformed into the host strain of DH5-α. The fusion protein consisted of GST and hB7.2(IgV+C) was identified by SDS-PAGE and Western blotting.T cell activation was observed by exposing purified T lymphocytes to the fusion protein and [3H]-TdR incorporation with the presence of the first signal imitated by anti-CD3 antibody. Results The fusion protein GST-hB7.2 (IgV+C) was produced and detected in inclusive body form from engineered bacterial cells. With the first signal existed,T lymphocytes proliferated when it was co-stimulated by the fusion protein. Conclusion These results indicated that the functional human B7.2(IgV+C) fusion protein can be produced in bacterial cells and the fusion protein displays the co-stimulatory activity in T lymphocytes activation.

Objective To analyze mRNA expressions of 7 cytoklnes which influence the immune response in lym- phocytes in pleural effusion of non-small cell lung cancer patients to evaluate the effect of local tumor microenviron- ment on anti-tumor immune response and to explore the mechanism of tumor escape. Methods Detecting the mRNA expression of IL-2,INF-γ,IL-12,IL-18,IL-10,IL-4 and TGF-β 1 in lymphocytes in pleural effusion of non-small cell lung cancer patients and tuberculotic pleurisy patients on the single cell level by using in situ hybridization. Results In the pleural effusion of non-small cell lung cancer, the mRNA expressions of IL-10,TGF-β1 and IL-4 were signifi- cantly higher than those of IL-2,IL-12,IL-18 and INF-γ,as well as these of control group. The cytokine expression levels of tuberculotic pleurisy patients were very Iow, and there were no significant differences between different cy- tokines. Conclusion Type 2 cytokines are expressed predominantly in the pleural effusion of non-small cell lung can- cer. The increased co-expression of IL-10 and TGF-β1 indicates that they might act Jointly and play a critical role in the immunosuppression of non-small cell lung cancer.

OBJECTIVETo investigate which of the two immunoglobulin (Ig)-like domains, the immunoglobulin variable region homologous domain IgV (hB7.2 IgV) and the immunoglobulin constant region homologous domain IgC (hB7.2 IgC) on the human B7.2 molecule contains receptor binding sites, and to evaluate whether the B7.2 protein expressed in bacteria has biological activity in vitro.

METHODSThree fragments of hB7.2 IgV,hB7.2 IgC and the complete extracellular region of human B7.2 containing both the IgV and IgC domains,hB7.2 Ig (V+C), were amplified by PCR and subcloned into pGEM-Teasy. Three recombinants,pGEX-4T-3-hB7.2 IgV,pGEX-4T-3-hB7.2 IgC and pGEX-4T-3-hB7.2 Ig (V+C), were generated by cloning the fragments into a prokaryote expression plasmid (pGEX-4T-3) and transformed into the host strain E. coli DH5alpha. The relevant target fusion proteins consisting of GST and hB7.2 IgV,hB7.2 IgC and hB7.2 Ig (V+C), were identified by SDS-PAGE and Western blotting. With the presence of the first signal imitated by anti-CD3 antibody, T cell activation was observed by exposing purified T lymphocytes to each soluble form of the three bacterially-produced human B7.2 fusion proteins by [(3)H]-TdR incorporation.

RESULTSThree recombinant fusion proteins of human B7.2, GST-hB7.2 IgV, GST-hB7.2 IgC and GST-hB7.2 Ig (V+C) were produced and detected in inclusion body form from engineered bacteria. With the first signal present,T lymphocytes proliferated when co-stimulated by bacterially-produced either GST-hB7.2 Ig (V+C) or GST-hB7.2 IgV fusion proteins, but not by GST-hB7.2 IgC.

CONCLUSIONSFunctional human B7.2 fusion protein can be produced in bacteria. The IgV-like domain of human B7.2 is sufficient for B7.2 to interact with its counter-receptors and co-stimulate T lymphocytes.

Antigens, CD ; pharmacology ; B7-2 Antigen ; Escherichia coli ; genetics ; Humans ; Immunoglobulin Constant Regions ; pharmacology ; Immunoglobulin Variable Region ; pharmacology ; Lymphocyte Activation ; Membrane Glycoproteins ; pharmacology ; Plasmids ; Recombinant Fusion Proteins ; pharmacology ; T-Lymphocytes ; immunology

OBJECTIVETo develop an efficient expression, purification system of recombinant Escherichia coli heat-labile enterotoxin B subunit (rLTB) and study its activity against mucosal immunoadjuvant by nasal immunization.

METHODSA recombinant, pMMB68-LTB was generated by cloning the LTB cDNA fragment into an expression vector (pMMB68) and transformed it into the host strain marine vibrio VSP60. The relevant target protein was identified using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Sephacryl S-100 gel filtration chromatography was carried out for purification of rLTB in engineering bacteria VSP60. BALB/c mice received hen egg lysozyme (HEL) alone or combined with rLTB by nasal administration. After three times immunization, IgG and IgA antibody levels in serum or small intestine wash samples were determined using ELISA.

RESULTSrLTB protein was highly expressed in VSP60. After gel filtration with Sephacryl S-100, the purity of rLTB reached 98.1%, the yield rate was about 52%. After immunization, IgG and IgA antibody responses specific to HEL in system and mucosa of HEL + rLTB groups were significantly increased, compared with the HEL alone group (P < 0.001).

CONCLUSIONSA set of protocols for large-scale rLTB preparation has been established, which is simple, efficient and applicable. The rLTB protein we prepared was proved to be a powerful mucocal adjuvant, which could greatly enhance systemic and mucosal immune responses to nasally co-administered antigen.

Adjuvants, Immunologic ; administration & dosage ; pharmacology ; Administration, Intranasal ; Animals ; Bacterial Toxins ; administration & dosage ; isolation & purification ; pharmacology ; Blotting, Western ; Electrophoresis, Polyacrylamide Gel ; Enterotoxins ; administration & dosage ; isolation & purification ; pharmacology ; Escherichia coli ; Escherichia coli Proteins ; Immunization ; Mice ; Mice, Inbred BALB C ; Nasal Mucosa ; immunology ; Recombinant Proteins ; administration & dosage ; isolation & purification ; pharmacology

Objective: The purpose of our study was to investigate the predictive abilities of clinical and computed tomography (CT)features for outcome prediction in patients with coronavirus disease (COVID-19). Materials and Methods: The clinical and CT data of 238 patients with laboratory-confirmed COVID-19 in our two hospitalswere retrospectively analyzed. One hundred sixty-six patients (103 males; age 43.8 ± 12.3 years) were allocated in thetraining cohort and 72 patients (38 males; age 45.1 ± 15.8 years) from another independent hospital were assigned in thevalidation cohort. The primary composite endpoint was admission to an intensive care unit, use of mechanical ventilation, ordeath. Univariate and multivariate Cox proportional hazard analyses were performed to identify independent predictors. Anomogram was constructed based on the combination of clinical and CT features, and its prognostic performance wasexternally tested in the validation group. The predictive value of the combined model was compared with models built on theclinical and radiological attributes alone. Results: Overall, 35 infected patients (21.1%) in the training cohort and 10 patients (13.9%) in the validation cohortexperienced adverse outcomes. Underlying comorbidity (hazard ratio [HR], 3.35; 95% confidence interval [CI], 1.67–6.71;p < 0.001), lymphocyte count (HR, 0.12; 95% CI, 0.04–0.38; p < 0.001) and crazy-paving sign (HR, 2.15; 95% CI, 1.03–4.48;p = 0.042) were the independent factors. The nomogram displayed a concordance index (C-index) of 0.82 (95% CI, 0.76–0.88),and its prognostic value was confirmed in the validation cohort with a C-index of 0.89 (95% CI, 0.82–0.96). The combinedmodel provided the best performance over the clinical or radiological model (p < 0.050). Conclusion Underlying comorbidity, lymphocyte count and crazy-paving sign were independent predictors of adverseoutcomes. The prognostic nomogram based on the combination of clinical and CT features could be a useful tool for predictingadverse outcomes of patients with COVID-19.

Objective: To investigate the clinical, biochemical and genetic features of four carnitine-acylcarnitine translocase deficiency cases. Methods: Four cases diagnosed with carnitine-acylcarnitine translocase deficiency from Guangxi Maternal and Child Health Hospital were studied. DNA was extracted from dry blood filter for gene analysis. SLC25A20 gene analysis was performed in 1 case and the whole exon sequence analysis was performed in 3 cases. Results: Retrospective study on unrelated carnitine-acylcarnitine translocase deficiency patients, the age of onset was 1-28 d, the age of death were 1.5-30 d, main clinical features were hypoglycemia (4 cases), arrhythmia (2 cases), sudden death (2 cases). Biochemical test showed hypoglycemia (1.2-2.0 mmol/L) , elevated creatine kinase (955-8 361 U/L) and creatine kinase isozyme(199-360 U/L), normal or decreased free carnitine level (3.70-27.07 μmol/L) , elevated long-chain acylcarnitine (palmityl carnitine 1.85-14.84 μmol/L). The gene tests showed that all 4 cases carried SLC25A20 gene c.199-10T> G homozygous mutation, inherited from their parents. By analyzing the haplotype, we found that the mutation loci of C. 199-10T> G were all in the same haplotype. Conclusion The c.199-10T> G mutation is an important molecular cause of carnitine-acylcarnitine translocase deficiency, which has relatively high frequency in Guangxi population, and is related to the founder effect.

The classical prescription Yangweitang， derived from Zhengzhi Zhunsheng， is specialized in treating syndromes of chill and fever due to exogenous pathogens， inner-cooling， and malaria， and it has been included in the Catalogue of Ancient Classical Formulas （the First Batch） published by the National Administration of Traditional Chinese Medicine （TCM） in 2018. Through bibliographical research， the relevant ancient books and modern documents were systematically sorted out， and it was found that there were many prescriptions related to the Yangweitang from Zhengzhi Zhunsheng. They were interwoven with Yangweitang from Zhengzhi Zhunsheng and widely used in clinical practice. In order to clarify their history and evolution， this paper combed the historical origin of Yangweitang and its related prescriptions and conducted textual analysis on key information such as semantic composition， herb origin， processing method， and efficacy. A total of 896 pieces of data on Yangweitang from Zhengzhi Zhunsheng were collected. 26 pieces of effective data were included after the screening， involving 17 ancient TCM books. Then， a total of 28 pieces of data on prescriptions related to the Yangweitang from Zhengzhi Zhunsheng were included， involving 23 ancient TCM books for reference. The textual analysis showed that Yangweitang originated from the Renshen Yangweitang recorded in Taiping Huimin Heji Jufang in the Song dynasty. Based on the original formula， medical experts from later generations have modified it into many different versions. A comparative analysis showed that Yangweitang from different generations had similar compositions， and the herb origin and processing method were basically clear. The recommended prescriptions are as follows： 37.3 g of Pinelliae Rhizoma Praeparatum Cum Alumine， Magnoliae Officinalis Cortex（fried with ginger juice）， and frying with rice water Atractlodis Rhizoma， 27.98 g of Citri Exocarpium Rubrum， 18.65 g of Pogostemon cablin leaf， Tsaoko Fructus， Poria， and Ginseng Radix et Rhizoma， and 9.33 g of Glycyrrhizae Radix et Rhizoma. They could be ground into a coarse powder， with 14.92 g for every dose， and they could be orally taken after being decocted with 450 mL of water， 7 g of fresh ginger， and 2 g of Mume Fructus to 270 mL in warm conditions. Yangweitang from Zhengzhi Zhunsheng has the effect of warming the middle and releasing the external， and it can treat many syndromes including spleen and stomach disharmony caused by chill and fever due to exogenous pathogens and inner-cooling， as well as all kinds of malaria. Modern clinical applications mainly focus on chronic atrophic gastritis and other digestive system diseases.