Shen Guan-xin

The change of interleukin-6 and tumor necrosis factor in patients with obstructive sleep apnea syndrome.

SummaryThe levels of lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-α) expression in culture of peripheral blood mononuclear cells (PBMC) and the plasma levels of IL-6 and TNF-α in the patients with obstructive sleep apnea syndrome (OSAS) were measured and the relationship between OSAS and IL-6 or TNF-α expression studied. Both IL-6 and TNF-α were detected by using ELISA in 22 patients with OSAS and 16 normal controls. The levels of LPS-induced IL-6 (787.82±151.97 pg/ml) and TNF-α (4165.45±1501.43 pg/ml) expression in the supernatant of the culture of PBMC and plasma level of IL-6 (50.67±4.70 pg/ml) and TNF-α (299.09±43.57 pg/ml) in the patients with OSAS were significantly higher than those in the normal controls (in the supernatant of the culture of PBMC: 562.69±197.54 pg/ml and 1596.25±403.08 pg/ml respectively; in the plasma; 12.69±2.75 pg/ml and 101.88±21.27 pg/ml respectively). There were significantly positive correlation between the levels of IL-6 and TNF-α and the percentage of time of apnea and hyponea, as well as the percentage of time spending at SaO2 below 90 % in the total sleep time. It was concluded that LPS-induced IL-6 and TNF-α levels as well as plasma IL-6 and TNF-α levels in the patients with OSAS were up-regulated, which may be associated with the pathogenesis of OSAS.

Studies on lymphocyte subpopulations and NK cell activities in epileptic patients.

SummaryThe subpopulations of T lymphocytes, B lymphocytes and macrophages of the peripheral blood in 58 cases of epilepsia were studied with McAb by IFA method. The activity of natural killer (NK) cells of penpheric blood monocyte was investigated by51Cr-releasing method. 22 healthy donors of about the same age served as controls. The results showed that the percentage of T3 and T4 lymphocytes in epileptic patients was decreased, and the ratio of T4/T8 was markedly reduced as compared to control group. The percentage of T8 lymphecytes was increased. There were no significant changes in B lymphocytes and macrophages. The NK cell activity also showed decrease. The results suggested abnormality in cellular immunity in epilepsia, which may be involved in the pathogenetic mechanism of the disease.

Studies on monoclonal anti-idiotypic antibodies to human B cell leukemia.

SummaryMurine immunocytoma cell line, NS-1, was fused with spleen cells of Balb/C mice which had been stimulated by tolerogenic disaggregated human gamma globulin and immunized by purified serum IgM from the patient with chronic B cell leukemia (B-CLL). 10 hybridoma cell lines secreting monoclonal anti-idiotype (anti-Id) antibodies to human CLL were obtained. The McAbs were subclasses belonging to IgM and of IgG mouse. Specificity and biologic characters of the monoclonal anti-Id antibodies from culture fluid or ascites were assayed by ELISA, indirect mixed ELISA sandwich, ELISA inhibition, immunofluorescence (IF) and IF inhibition. The study also proved that monoclonal anti-Id antibodies could react with homologous IgM, but not with Ig from normal donors or a panel of patients with myeloma. The results of IF and IF inhibition assay showed that monoclonal anti-Id antibodies were bound to lymphocytes of patient with B-CLL. Their reactivity was inhibited by homologous IgM, but not by lymphocytes of patients with ALL or lymphoma. Monoclonal anti-Id antibodies were heterogenous reactive patterns with cell lines in vitro.

Studies on monoclonal anti-isotypic and anti-idiotypic antibodies against leukemia and myeloma: I. preparation and characteristics of monoclonal anti-isotypic antibodies to igm from B chronic lymphocytic leukemia

SummaryFive hybridomas producing McAbs against human serum IgM-isotype were obtained by fusing the myeloma NS-1 cell and the RALB/C murine spleen cell which had been immunized by serum IgM of patient with B chronic lymphocytic leukemia (B-CLL). These McAbs only reacted with human IgM, not with other immunoglobulins in ELISA and immune double diffusion test. An approximate positive rate of peripheral blood lymphocyte (PBL) was got when these McAbs and the McAbs aginst B cell were tested by indirecl immunofluorescent assay. The positive rate was similar to that obtained by direct immunofluorescent test. Immunoblotting showed that the molecular weight of the antigen to these McAbs was 70–90 Kd, indicating that the antigen was IgM. The practical value of the McAbs against human IgM was also discussed.

The Effect of Astragapolysaccharide on the Lymphocyte Proliferation and Airway Inflammation in Sensitized Mice

SummaryIn order to investigate the regulating role of Astragapolysaccharide (APS) in the mice model of asthmatic airway inflammation, the airway eosinophil number, spleen T lymphocyte proliferation, level of IL-2 production and their relationships were studied in sensitized mice and sensitized mice treated with different concentrations of APS. The results showed that the number of eosinophils as well as lymphocytes in the airway of the sensitized animals were significantly increased, and a marked positive correlation between the inflammation cells and spleen T lymphocyte proliferation was found. Moreover, there was a positive correlation between inflammation cells and the level of IL-2 production. The APS of given dosage could significantly reduce the number of eosinophils in the airway of the sensitized animals. At the same time the level of IL-2 secreted by spleen T lymphocytes stimulated with ConA was also significantly decreased and there was a marked positive correlation between them. Our results suggested that APS of given dosage could prevent antigen-induced the number of eosinophils infiltrating into the airway of sensitized mice and inhibit the proliferation and activation of lymphocyte and IL- 2 production. Through its immuno-regulating effect, APS can be helpful in the treatment of asthma.

Determination of IL-2 and cytotoxicity of killer cells in MTT colorimetry

SummaryThe activity of interleukine 2 (IL-2) in culture supernatants of lymphokine-activated killer (LAK) cells and tumor infiltrating lymphocytes (TIL) as well as cytotoxicity of LAK cells on cultured leukemic cells were determined by MTT colorimetry. The results showed that higher activity of IL-2 in culture supernatant of LAK and TIL cells was found it could be used to support the culture of IL-2 dependent cell lines. The significant cytotoxicity of LAK cells on leukemic cell lines could be found in vitro, and it was consistent with the ratio of effector cells to target cells. The number of living leukemic cells is consistently related with the concentration of formazan metabolite of MTT. It suggested that the numbers of living cells and cytotoxicity of LAK cells could be estimated by determination of formazan metabolite OD value.

Expression of anti-CD4 human/murine chimeric antibody and their killer tumor activity

SummaryFrom the mouse hybridoma cell line secreting an anti-CD4 monoclonal antibody (McAb), total RNA was prepared. The VH and VL genes were amplified by RT-PCR with family specific primer pairs. The PCR products were cloned into pGEM-T vectors, then tranfected into JM109. The VH and VL genes were analyzed by automatic DNA sequencer. According to Kabat classification, the VH and VL genes belong to the mouse Ig heavy subgroup I (A) and k chain subgroup I, respectively. The VH and VL genes were subcloned into pγ1-Expr and pk-Expr respectively, then transfected into XL2-Blue. The VH-pγ1 and VL-pk were transfected by electroporation into mouse myeloma cell X63Ag8. 653. The transfectoma cells were selected by G418 screening, and then supernatant of cultured transfectoma were analyzed by ELISA and immunofluorescence techniques. We have acquired transfectoma cells secreting anti-CD4 chimeric antibodies. These chimeric antibodies are able to kill tumor cells specificallyin vitro.From the mouse hybridoma cell line secreting an anti-CD4 monoclonal antibody (McAb), total RNA was prepared. The VH and VL genes were amplified by RT-PCR with family specific primer pairs. The PCR products were cloned into pGEM-T vectors, then tranfected into JM109. The VH and VL genes were analyzed by automatic DNA sequencer. According to Kabat classification, the VH and VL genes belong to the mouse Ig heavy subgroup II (A) and kappa chain subgroup III, respectively. The VH and VL genes were subcloned into p gamma 1-Expr and p kappa-Expr respectively, then transfected into XL2-Blue. The VH- p gamma 1 and VL- p kappa were transfected by electroporation into mouse myeloma cell X63Ag8. 653. The transfectoma cells were selected by G418 screening, and then supernatant of cultured transfectoma were analyzed by ELISA and immunofluorescence techniques. We have acquired transfectoma cells secreting anti-CD4 chimeric antibodies. These chimeric antibodies are able to kill tumor cells specifically in vitro.

Studies on monoclonal anti-isotypic and anti-idiotypic antibodies against leukemia and myeloma: I. study on monoclonal anti-idiotypic antibodies against λ chain protein of myeloma

SummaryWe used A chain extracted from urine of patient with myeloma (IgD λ) as antigen for immunizing BALB/C mice, and 86 hybridoma cell clones secreting monoclonal antibodies (McAb) were obtained after fusing twice and cloning 3–4 times. 15 of these clones secreted monoclonal anti-idiotypic antibodies (anti-Id McAb). The results showed that 12 of 15 anti-Id antibodies reacted only with homologous λ chain and IgD, not with A chain, κ chain, IgG, IgA, IgM, IgD, IgE, albumin and paraglobulin from normal subjects. Indirect immunofluorescent assay demonstrated that positive reaction rate between anti-Id McAbs and peripheral blood lymphocytes or bone marrow cells of the patient with myeloma was up to 23 %. No reaction was observed between anti-Id McAb and peripheral blood lymphocytes or bone marrow cells from normal subjects. Some of these McAbs presented positive reaction with plasmacytoma cell lines cultured in our laboratory.

Immunomodulation of musk-moxa-string therapy in patients with scrofula.

SummaryThe effects of musk-moxa-string therapy on the immune system in man were investigated in 39 patients with scrofula. Before treatment, the numbers of peripheral blood (PB) CD3+ and DC4+ cells and the ratio CD4+/ CD8+ were found to be lower in patients with scrofula than in normal subjects, while those of B cells and DR+ cells were higher. Response of peripheral blood mononuclear cells (PBMC) to phytohemagglutinin (PHA) diminished in patients with scrofula. At month 2–6 of musk-moxa-string therapy the number of PB CD8+ cells showed slight diminution along with significant increaes in CD3+ and CD4+ cells and CD4+/CD8+ ratio in total lymphocytes (P<0.001). In vitro a marked increased blastogenic response to mitogen stimulation with PHA was observed in PBMC of patients with scrofula after treatment (P<0.001). In contrast, B lymphocytes, monocytes, DR+ cells and blastogenic response to concanavalin A and pokeweed mitogen were not influenced by musk-moxa-string therapy.

Preparation of AHTG-DNR conjugates and their antitumor effect in vitro.

Summary100%, 75%, 50%, 25% and 12.5% oxidized dextran T10 (Dex T1o) were used as intermediate carriers for conjugating drug daunorubicin (DNR) and antibody anti-human thymocitic globulin (AHTG), to form different immunoconjugates, AHTG:Dex:DNR. It was demonstrated that the conjugate with 25% oxidized Dex T10 as intermediate carrier linked more DNR molecules than the others. The degree of its substitution was 10–11 moles of DNR per mole of AHTG. Moreover, because the amount to reducing agent sodium borohydride (NaBH4), required for the reduction reaction, was relatively small, its damaging effect on AHTG and DNR was lessened accordingly.The antitumor effect of AHTG: Dex: DNR in vitro was tested by using 24-h cytotoxicity assay, with CEM as target cell. Cytotoxic effect of the conjugate was proven and the LD56 was 10.68 μg/ml. However, it showed only slight cytotoxic effect on non-target cell K562. When 10 min cytotoxicity assay was performed to show the specific tumor-killing effect of the conjugate, it revealed an obvious cytotoxic activity toward CEM, with the LD50 being 14.79 μg/ml, but hardly toward K562. These results suggest that AHTG:Dex:DNR possesses specific cytotoxic effect.