Immunotherapy of intracranial G422 glioblastoma with dendritic cells pulsed with tumor extract or RNA

Objective: To investigate the anti-tumor efficacy of dendritic cell (DC)-based vaccines pulsed with tumor extracts or RNA in a mouse model of intracranial G422 glioblastoma. Methods: Bone marrow-derived DCs were pulsed ex vivo with tumor extracts or RNA. Ninety female mice harboring 4-day-old intracranial G422 glioblastomas and 126 normal mice were treated with three spaced one week apart subcutaneous injections either with PBS, unpulsed DCs, G422 tumor extracts, RNA, DCs pulsed with G422 tumor extracts (DC/extract) or with RNA (DC/RNA). Seven days after the third immunization of normal mice, the spleens of 36 of them were harvested for cytotoxic T lyphocyte (CTL) assays and the others were challenged in the brain with G422 tumor cells. All the treated mice were followed for survival. Some mice brains were removed and examined pathologically when they died. Results: Immunization using DC/extract or DC/RNA significantly induced G422-specific CTL responses compared with control groups (P<0.01). Vaccinatio     

Phenotypic and functional characteristics of dendritic cells derived from human peripheral blood monocytes

INTRODUCTION Dendritic cells(DCs)are professional anti-gen-presenting cells(APC)that are responsible for the activation of undifferentiated T cells and the generation of primary T-cell responses(Cella et al.,1997).The specific role of DCs is to capture,process and present antigens to T cells.Immunogenic and inflammatory signals are responsible for the migra-tion of DCs from tissues to lymphoid organs where they initiate an immune response.These processes induce the maturation of DCs…     

Lymphotactin enhances the in-vitro immune efficacy of dendritoma formed by dendritic cells and mouse hepatocellular carcinoma cells

Objective: To investigate the in-vitro antitumor immune responses of dendritoma formed by mouse hepatocellular carcinoma (HCC) cells and lymphotactin (Lptn) gene modified dendritic cells (DCs). Method: DCs prepared from mouse bone marrow were genetically modified by lymphotactin adenovirus, and fused with H22 cells by polyethylene glycol (PEG). RT-PCR and ELISA were employed to identify lymphotactin expression at mRNA and protein level. Cell phenotypes and fusion efficiency was detected by FACS. The stimulatory effect of DC on T cells was detected by mixed lymphocyte reaction. The cytotoxicity activity against H22 cells was assayed by LDH method. Results: Lymphotactin could be efficiently expressed by DCLptn/H22 hybridoma. DCLptn/H22 cells could induce potent T cell proliferation effect and generate strong cytotoxic T lymphocyte (CTL) reaction against allogenic H22 cells. Conclusion: Lymphotactin genetic modification could enhance the in vitro immune activity of the dendritoma.     

Successful immunological treatment of gallbladder cancer in India Case report

Gallbladder cancer has a poor outcome because of its anatomy and location. Often, the diagnosis is made very late due to its silent course. Post-operated cases do respond to chemotherapy but the survival is counted in months and the quality of life is further hampered due to toxicity of drugs. Immunotherapy holds good promise in non-responding cancers treated by conventional chemotherapeutic agents. Among various therapies, dendritic cell therapy is growing at rapid pace due to its acceptable rationale. It has been utilized in treating successfully resected stage Ill （T2, N1, M0） gallbladder cancer in one of our patients. A 48 years old lady treated with this therapy is free of metastasis with ten doses of autologous dendritic cell vaccine constructed by utilizing resected tumor lysate antigen. She has received ten doses of therapy in 14 months of her treatment. This therapy has proven to be safe and without apparent side effects. The positive clinical response obtained supports that autologous dendritic cell-based immunotherapy is a promising therapeutic approach for refractory gallbladder cancers.     

Increasing the immune activity of exosomes: the effect of miRNA-depleted exosome proteins on activating dendritic cell/cytokine-induced killer cells against pancreatic cancer

Background

Tumor-derived exosomes were considered to be potential candidates for tumor vaccines because they are abundant in immune-regulating proteins, whereas tumor exosomal miRNAs may induce immune tolerance, thereby having an opposite immune function.

Objective

This study was designed to separate exosomal protein and depleted exosomal microRNAs (miRNAs), increasing the immune activity of exosomes for activating dendritic cell/cytokine-induced killer cells (DC/CIKs) against pancreatic cancer (PC).

Methods

PC-derived exosomes (PEs) were extracted from cultured PANC-1 cell supernatants and then ruptured; this was followed by ultrafiltered exosome lysates (UELs). DCs were stimulated with lipopolysaccharide (LPS), PE, and UEL, followed by co-culture with CIKs. The anti-tumor effects of DC/CIKs against PC were evaluated by proliferation and killing rates, tumor necrosis factor-α (TNF-α) and perforin secretion. Exosomal miRNAs were depleted after lysis and ultrafiltration, while 128 proteins were retained, including several immune-activating proteins.

Results

UEL-stimulated DC/CIKs showed a higher killing rate than LPS- and PE-stimulated DC/CIKs.

Conclusions

miRNA-depleted exosome proteins may be promising agonists for specifically activating DC/CIKs against PC.

     

抑癌基因PTEN载体构建及在LOVO细胞中的表达

目的:构建抑癌基因PTEN的表达载体并在LOVO细胞中表达。方法:从人外周血中提取总RNA,通过RT-PCR扩增出PTEN基因片段,将PTEN基因连入pLenti6/V5载体。测序鉴定后,经脂质体转染入LOVO细胞,对细胞株进行RT-PCR和Western blot检测。结果:DNA测序证实pLenti6/V5-PTEN表达载体为阳性克隆;该表达载体转染LOVO细胞后上调了PTEN mRNA和蛋白的表达。结论:成功构建了pLen-ti6/V5-PTEN表达载体,为进一步研究PTEN在LOVO细胞中的功能奠定了基础。     

Parthenolide inhibits proliferation of vascular smooth muscle cells through induction of G0/G1 phase cell cycle arrest

Objective: This study is to determine the effect of the natural product parthenolide, a sesquiterpene lactone isolated from extracts of the herb Tanacetum parthenium, on the proliferation of vascular smooth muscle cells (VSMCs). Methods: Rat aortic VSMCs were isolated and cultured in vitro, and treated with different concentrations of parthenolide (10, 20 and 30 μmol/L).[3H]thymidine incorporation was used as an index of cell proliferation. Cell cycle progression and distribution were determined by flow cytometric analysis. Furthermore, the expression of several regulatory proteins relevant to VSMC proliferation including IκBα, cyclooxygenase-2 (Cox-2), p21, and p27 was examined to investigate the potential molecular mechanism. Results:Treatment with parthenolide significantly decreased the [3H]thymidine incorporation into DNA by 30%～56% relative to control values in a dose-dependent manner (P＜0.05). Addition of parthenolide also increased cell population at G0/G1 phase by 19.2%～65.7% (P＜0.05) and decreased cell population at S phase by 50.7%～84.8% (P＜0.05), which is consistent with its stimulatory effects on p21 and p27. In addition, parthenolide also increased IκBα expression and reduced Cox-2 expression in a time-dependent manner. Conclusion: Our results show that parthenolide significantly inhibits the VSMC proliferation by inducing G0/G1 cell cycle arrest. IκBα and Cox-2 are likely involved in such inhibitory effect of parthenolide on VSMC proliferation. These findings warrant further investigation on potential therapeutic implications of parthenolide on VSMC proliferation in vivo.     

Parthenolide inhibits proliferation of vascular smooth muscle cells through induction of G0/G1 phase cell cycle arrest

Objective  This study is to determine the effect of the natural product parthenolide, a sesquiterpene lactone isolated from extracts of the herb Tanacetum parthenium, on the proliferation of vascular smooth muscle cells (VSMCs). Methods  Rat aortic VSMCs were isolated and cultured in vitro, and treated with different concentrations of parthenolide (10, 20 and 30 μmol/L). [³H]thymidine incorporation was used as an index of cell proliferation. Cell cycle progression and distribution were determined by flow cytometric analysis. Furthermore, the expression of several regulatory proteins relevant to VSMC proliferation including IκBα, cyclooxygenase-2 (Cox-2), p21, and p27 was examined to investigate the potential molecular mechanism. Results  Treatment with parthenolide significantly decreased the [³H]thymidine incorporation into DNA by 30%∼56% relative to control values in a dose-dependent manner (P<0.05). Addition of parthenolide also increased cell population at G₀/G₁ phase by 19.2%∼65.7% (P<0.05) and decreased cell population at S phase by 50.7%∼84.8% (P<0.05), which is consistent with its stimulatory effects on p21 and p27. In addition, parthenolide also increased IκBα expression and reduced Cox-2 expression in a time-dependent manner. Conclusion  Our results show that parthenolide significantly inhibits the VSMC proliferation by inducing G₀/G₁ cell cycle arrest. IκBα and Cox-2 are likely involved in such inhibitory effect of parthenolide on VSMC proliferation. These findings warrant further investigation on potential therapeutic implications of parthenolide on VSMC proliferation in vivo. Project (No. 491020-W50315) supported by the Foundation of the Health Bureau of Zhejiang, China     

Immortalization of human umbilical vein endothelial cells with telomerase reverse transcriptase and simian virus 40 large T antigen

Objective: To establish normally conditionally-immortalized human umbilical vein endothelial cells (HUVECs) by ectopic expression of the human telomerase catalytic enzyme (hTERT) and simian virus 40 large T (SV40 LT) antigen. Methods: Primary HUVECs were transfected with recombinant retrovirus containing hTERT or SV40 LT respectively. Subsequently drug resistant cell clones were screened and expanded for further studies. Endothelial cell biomarkers were confirmed by examination. Results: The morphological phenotype of the transfected cells was similar to the non-transfected cells. Von Willebrand factor, hTERT and SV40 LT could be detected in transfected HUVECs. Moreover, higher telomerase activity in transfected cells was maintained for over 50 population doublings compared with only low level of endogenous telomerase transiently at early population doublings in primary HUVECs. When exposed to TNF-α(tumor necrosis factor-α), the expression of E-selectin in transfected cells was significantly up-regulated, but no alteration of endothelial lipase was found. Conclusion: Ectopic coexpression of hTERT and SV40 LT can effectively immortalize HUVECs without tumorigenicity in vitro. Immortalized HUVECs may be an ideal target of further molecular function studies.     

Gastric carcinoma with osteoclast-like giant cells: a case report and review of the literature

Gastric carcinoma with osteoclast-like giant cells (OGCs) is an extremely rare tumor. So far, only six cases have been reported in the literature. Here we report an additional case of this tumor in a Chinese 78-year-old man presented with abdominal pain, vomiting, and hematemesis. Physical examination and gastroscopy revealed a tumor in the gastric antrum. The biopsy and pathological findings indicated a gastric adenocarcinoma with OGCs, which were present in both the tumor and the metastatic lymph nodes. Further immunohistochemical staining indicated that OGCs were reactive with CD68, CD45, and vimentin protein, but not with pancytokeratin, carcinoembryonic antigen, or epithelial membrane antigen, suggesting the monocytic/histiocytic derivation of these OGCs. In situ hybridization for Epstein-Barr virus showed no nuclear positivity in either adenoearcinoma or OGCs. Postoperative follow-up showed that the patient had survived for at least 6 months without recurrence. Further investiga-tion is warranted to clearly define the prognostic significance of OGCs in gastric carcinoma.