We used WEHI clone—13 cell line as target and MTT colorimetric technique to evaluate monocyte cytotoxicity, this method is superior to the traditional~(51) Cr release technique. It is more sensitive,more rapid and less effectors needed, as well as no isotope contamination, and it is mose suitable in clinic.

Polyactin B (Pb) has been proved to have the effect of considerable tumor suppression. Recently,we used murine S_(180) Sarcoma as a model and observed the effect of Pb on the tumor—infiltrating immunocompetent cells, and also the infiltrating neutrophils and the alteration of small blood vessels within the tumor tissue. Compared with control: in Pb—treated group, there were more L_3T_4~+and Lyt_2~+ lymphocytes infiltrating in the periphery of the tumor ,and also within the tumor. In addition, the tumors had more prominant hyperemia, micro—thrombosis and neutrophil infiltration around the necrotic areas. The present findings suggest that the tumor—suppression effect of Pb might be mediated through TNF produced by immunocompetent cells.

The TNA mRNA expression in 33 samples of cervical carcinoma and 28 samples of condyloma have been observed by using ~3H and Digoxigenin labeled TNF—? cDNA probes and in situ hybridization technique, we found that the results of in situ hybrdfzation with these two kinds of labeled probes were alike, the sensitivity with ~3H labeled probe was slightly higher than that with Digoxigenin labeled probe. Nevertheless, the nonisotope probe would be used more and more in future because of its safety, rapidity and convenience in work.

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.

AIM: To elucidate the in vivo mechanisms of the proliferation of vascular smooth muscle cells (VSMCS) in injuried arteries. METHODS: A VSMCS proliferative model was constructed by injury of rabbit iliac arteries with balloon catheters and a probe designed for rabbit platelet-derived growth factor B chain (PDGF-B ) mRNA was used to detect the expression of it by intimal VSMCS on the vascular cross sections using an in situ hybridization technique at the indicated times. The relation of this expression to the proliferation of VSMCS by their expression of proliferating cell nuclear antigen (PCNA) and vascular intimal areas were estimated. RESULTS: The expression of PDGF-B mRNA of intimal VSMCS was increased when calculating the intimal PDGF-B mRNA positive cells per millimetre area at ?400 magnification with average numbers of 31.93?14.64 in 1 week group, 26 50?9 25 in 2 weeks group and 24 85?13 65 in 4 weeks group. This was in accordance with the expression of PCNA by VSMCS and the increase of intimal areas. CONCLUSION: The local production of PDGF-B by VSMCS via an autocrine mechanism is responsible for the continuous proliferation of these cells and formation of neointima after the injury. The probe designed is very useful for detecting rabbit PDGF-B mRNA.

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 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