Antitumor activities of human autologous cytokineinduced killer(CIK)cells against hepatocellular carcinoma cells in vitro and in vivo

AIM: To characterize the anticancer function of cytokine-induced killer cells (CIK) and develop an adoptive immunotherapy for the patients with primary hepatocellular carcinoma (HCC), we evaluated the proliferation rate, phenotype and the antitumor activity of human CIK cells from healthy donors and HCC patients in vitro and in vivo. METHODS: Peripheral blood mononuclear cells (PBMC) from healthy donors and patients with primary HCC were incubated in vitro and induced into CIK cells in the presence of various cytokines such as interferon-gamma (IFN-gamma), interleukin-1 (IL-1), IL-2 and monoclonal antibody (mAb) against CD3. The phenotype and characterization of CIK cells were identified by flow cytometric analysis. The cytotoxicity of CIK cells was determined by (51)Cr release assay. RESULTS: The CIK cells were shown to be a heterogeneous population with different cellular phenotypes. The percentage of CD3+/CD56+ positive cells, the dominant effector cells, in total CIK cells from healthy donors and HCC patients, significantly increased from 0.1-0.13% at day 0 to 19.0-20.5% at day 21 incubation, which suggested that the CD3+ CD56+ positive cells proliferated faster than other cell populations of CIK cells in the protocol used in this study. After 28 day in vitro incubation, the CIK cells from patients with HCC and healthy donors increased by more than 300-fold and 500-fold in proliferation cell number, respectively. CIK cells originated from HCC patients possessed a higher in vitro antitumor cytotoxic activity on autologous HCC cells than the autologous lymphokine-activated killer (LAK) cells and PBMC cells. In in vivo animal experiment, CIK cells had stronger effects on the inhibition of tumor growth in Balb/c nude mice bearing BEL-7402-producing tumor than LAK cells (mean inhibitory rate, 84.7% vs 52.8%, P【0.05) or PBMC (mean inhibitory rate, 84.7% vs 37.1%, P【0.01). CONCLUSION: Autologous CIK cells are of highly efficient cytotoxic effector cells against primary hepatocellular carcinoma cells and might serve as an alternative adoptive therapeutic strategy for HCC patients.

Serum-free culture of dendritic cells from patients with chronic myeloid leukemia in vitro and estimation of their cytotoxicity

OBJECTIVE: To establish a serum-free culture system of dendritic cells (DCs) from chronic myeloid leukemia (CML) cells so that DCs vaccine may be applied to the adoptive immunotherapy of CML in the near future. METHODS: Fetal calf serum, serum-free medium and autologous serum were used for culture of DCs. The usage of cytokines was classified into two groups: group A (stem cell factor, granulocyte/macrophage colony-stimulating-factor, tumor necrosis factor-alpha and interleukin-4) and group B (granulocyte/macrophage colony-stimulating-factor, tumor necrosis factor-alpha and interleukin-4). The phenotypes of DCs were analyzed by using indirect immunofluorescence and flow cytometry. Mixed leukocyte responses were performed by methyl thiazolyl tetrazolium (MTT) assay. Chromosome analysis of DCs can be achieved by displaying G banding. T cells from CML patients were stimulated with autologous DCs and T-cell cytotoxicity was measured by (MTT) assay. RESULTS: CD34(+) cells or mononuclear cells were obtained from peripheral blood or bone marrow samples of eight patients of chronic-phase CML. Group A of serum-free medium was better than group B in expansion of total cell numbers and the rate of DCs. These results of serum-free medium were not significantly different from those of fetal calf serum medium, but the results of autologous serum medium were inferior to two groups above. The expression of major histocompatibility complex class II antigen on the surface of DCs was notable (> 50%), but the expression of CD83 and the costimulatory molecules CD86 was not noticeable (10% - 50%). Although CD1a(+)/CD14(-) DCs were potent stimulators of allogeneic lymphocytes, expansion of T cells from normal volunteers were not significant (average 27.2 fold at DCs: T cells ratio of 1:10). At day 12, CD1a(+) cells from three patients were studied by displaying G banding and Ph(+) cells in these populations were 100%, 98% and 60%, respectively. At an effector: target ratio of 40:1, 32% to 45% cytotoxicity was noted with DC-stimulated T cells against autologous leukemia cells. CONCLUSIONS: A stable serum-free culture system of CML-DCs was established. The expression of CD83 and CD86 on the surface of CML-DCs and DCs' potent stimulation of allogeneic lymphocytes were not notable. DCs in CML patients can be derived from the malignant clone and these malignant DCs could induce anti-leukemic reactivity in autologous T lymphocytes without the necessity for additional exogenous antigens.

Investigation of the cytotoxicity,antioxidative and immune-modulatory effects of Ligusticum porteri(Osha) root extract on human peripheral blood lymphocytes

OBJECTIVE： Ligusticum ported is a traditional Native American herb. The roots of L. ported are traditionally used in the treatment of many diseases, however, its cytotoxicity, antioxidative and immune- modulatory effects need to be investigated. In this study, we evaluated the effects of the root extract at different doses on human peripheral blood lymphocytes （PBLs）. METHODS： The lymphocytes were incubated with different concentrations of the root extracts （0, 50, 100, 200, and 400 μg/mL） and harvested every 6 h for 2 d （P〈0.05）. The protective effect of the herb against oxidative damage was determined by inducing oxidative stress with the administration of 50 μmol/L of hydrogen peroxide （H202）. RESULTS： Treatments with L. ported at 200 and 400 pg/mL increased the viability of PBLs. The deleterious effect of H2O2 was ameliorated by 400μg/mL L. ported treatment. Addition of 400 μg/mL L. ported reduced lipid peroxidation in stressed PBLs by 94% （P〈0.05）. Treatment with 400 μg/mL of L. ported resulted in a 26.4% increase of reduced glutathione levels. Activities of superoxide dismutase and catalase increased by 17.5% and 55.2% respectively, when stressed PBLs were treated with 400 μg/mL L. ported for 2 d （P〈0.05）. Treatment with 400 μg/mL L. ported increased interferon-γand interleukin-2 expressions in H2O2-challenged PBLs （P〈0.05）, however, the root extract did not cause a significant difference in interleukin-10 levels compared to the control （P〉0.05）. CONCLUSION： The findings suggest that L involving protective effects against oxidative ported might be a potential immune-modulating agent damage.

Evaluation of antileishmanial,antibacterial and brine shrimp cytotoxic potential of crude methanolic extract of Herb Ocimum basilicum(Lamiacea)

OBJECTIVE:To collect and screen for ethnopharmacological properties(antileishmanial,antibacterial and brine lethality assays) of medicinal plant Ocimum basilicum from Peshawar region(34.008 latitude and 71.57 altitudes).METHODS:In the present study a general antileishmanial activity against Leishmania tropica strain was carried out.The antibacterial potential of the plant was performed against 06 gram positive and 06 gram negative bacteria.Brine shrimp cytotoxicity assay at different concentrations were investigated.RESULTS:The anti-promastigotes profile of the plant showed good antileishmanial activity exhibited LC_(50) value 21.67 μg/mL.The result for gram positive antibacterial activity revealed that the O.basilicum leaves extract possesses significant inhibitory activity at highest two concentrations ranging from20.66 ± 0.31 to 31.86 ± 0.80 for Clostridium perfringens type C and Bacillus subtitilis,respectively,as compared to the gentamycin(27.36 ± 0.55 and21.80 ± 0.72,respectively).For gram negative bacteria good activity was observed.A highest zone of inhibition was recorded for Pseudomonas aeroginosa(28.83 ± 0.28) atthe highest concentration(10 mg/mL).The LC_(50) value obtained for brine shrimp lethality assay was 91.56 μg/mL.CONCLUSION:The herb basil possesses effective cidal activities which make this plant a good candidate for the isolation of antiprotozoal and antibacterial compounds which may lead to the development of novel drug.

Influence on the immune function of the human peripheral blood mononuclear cells transfected by retrovirus-mediated HSV-tk gene

Graft-versus-host disease （GVHD） is a severe complication and a major source of morbidity and mortality after allogeneic hematopoietic stem cell transplantation （HSCT）. Although T cell depletion of the allogeneic HSCT can efficiently prevent GVHD, it is associated with increased graft rejection and relapse of the malignant disease. To preserve the beneficial effects of donor T cells and avoid their GVHD effects, some approaches have been explored. One of them is to transfer a special suicide gene into the donor T lymphocytes so that they become more sensitive to a specific drug that is ordinarily not toxic. The most commonly used suicide gene is the thymidine kinase-encoding gene of herpes simplex virus （HSV-tk）.

Effects of anti-HPV16E6-ribozyme on phenotype and gene expression of a cervical cancer cell line

OBJECTIVE: To investigate the effects of anti-HPV16E6-ribozyme (HRz) on phenotype and gene expression of a cervical cancer cell line. METHODS: HRz was designed by computer programs. HRz's activity was identified by cleavage experiments in vitro. HRz and empty eukaryotic plasmids were transfected into CaSKi cells with lipofectin, then renamed CaSKi-R and CaSKi-P, respectively. The expression of ribozyme in transfected cells was observed by RNA dot blot. The amounts of E6 mRNA in three kinds of cells lines were detected by Northern blot. Cell growth curves and soft agar forming ability were studied. The ability of each cell line to form tumors was assessed in nude mice. Apoptosis rates and expression of c-myc, bcl-2, p53 and Fas were detected by flow cytometry (FCM). Antigens of tumor cells, HLA-1, HLA-2, B7-1 and B7-2 were also detected. NK, LAK, and CD(3)AK cells were induced. Their cytotoxicities were detected in CaSKi-R, CaSKi-P, and CaSKi cells. RESULTS: In vitro cleavage reaction demonstrated that HRz could cleave HPV16E6 mRNA in a site-specific manner. HRz could be expressed stably in transfected CaSKi cells. Northern blot analysis showed that E6 mRNA levels were lower in CaSKi-R than in CaSKi. The growth rate of CaSKi-R was slower than those of CaSKi and CaSKi-P. The soft agar-forming rate of CaSKi-R was lower compared with those of CaSKi and CaSKi-P cells. The ability of CaSKi-R to form tumors in nude mice was also poor. The apoptosis rate of CaSKi-R cells was much higher than those of CaSKi and CaSKi-P. HRz could reduce the expression of E6, c-myc and bcl-2 proteins, and increase the expression of p53 as well. HRz could increase the expression of HLA-2, B7-1 and B7-2 antigens. The cytotoxicity of NK, LAK and CD(3)AK cells was much higher in CaSKi-R than in CaSKi-P and CaSKi cells. CONCLUSION: HRz not only reverses the malignant phenotype of CaSKi cells partially, but also induces apoptosis in the cells, and increases sensitivity of CaSKi cells to immune cells.