AIM: To investigate the expression pattern of epithelial cell adhesion molecule (Ep-CAM) on normal and malignant colon tissues to evaluate its diagnostic and therapeutic significance.METHODS: cDNA encoding Ep-CAv extr...AIM: To investigate the expression pattern of epithelial cell adhesion molecule (Ep-CAM) on normal and malignant colon tissues to evaluate its diagnostic and therapeutic significance.METHODS: cDNA encoding Ep-CAv extracellular domain was cloned by reverse transcription-polymerase chain reaction (RT-PCR) from excised malignant colon tissues and inserted into a glutathione S-transferase (GST)-tagged vector. EpCAM-GST fusion protein was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and purified with glutathionesepharose. The Ep-CAM-GST fusion protein was mixed with Freund's adjuvant and Balb/c mice were immunized with it. Sp2/0 myeloma cells were fused with the spleen cells of the immunized mice. After having selected by indirect ELISA, the anti-Ep-CAM monoclonal antibodies (NAbs) were generated and the corresponding ascites were obtained.Finally, the human colon carcinoma tissue array prepared from seventy individual patients was stained with the antiEp-CAM NAbs.RESULTS: The isolated Ep-CAM cDNA sequence was identical to the data in GenBank. The expressed fusion protein was almost soluble and had a molecular weight (NW) of 53 ku.Four NAbs against Ep-CAM were obtained and designated as FMU-Ep1, FMU-Ep2, FMU-Ep3 and FMU-Ep4 respectively.Among them, FMU-Ep4 could recognize the natural EpCAM on Colo205 and SW480 cells, and all of them could be used for immunohistochemical staining of tissue sections.It was found that Ep-CAM was distributed differently in normal and various malignant colon tissues, including squamous cell carcinoma, signet-ring cell carcinoma and adenocarcinoma.In normal colon gland epithelia, Ep-CAM antigen was mainly distributed on the basolateral membrane and in the region between the basolateral membrane and the cytoplastic part near the nuclei, whereas the expression pattern of colon malignancies was mainly on the whole surface of epithelia and the expression was much higher than the normal colon tissues. The staining pattern of tissue array showed in adenocarcinoma and papillary adenocarcinoma, and the expression of Ep-CAM was increased from grade Ⅰ to grade Ⅲ.CONCLUSION: NAbs against Ep-CAM might be useful for research on the structure and function of Ep-CAM and may have diagnostic and therapeutic value to various colon carcinomas.展开更多
AIM: DPC4 is a tumor suppressor gene on chromosome 18q21.1 that has high mutant frequencies in pancreatic carcinogenesis. The purpose of this study was to investigate the role of DPC4 alterations in tumorigenesis and ...AIM: DPC4 is a tumor suppressor gene on chromosome 18q21.1 that has high mutant frequencies in pancreatic carcinogenesis. The purpose of this study was to investigate the role of DPC4 alterations in tumorigenesis and progression of pancreatic carcinomas. METHODS: We studied the immunohistochemical markers of DPC4 in 34 adenocarcinomas and 16 nonmalignant specimens from the pancreas. The 16 nonmalignant specimens from the pancreas included 8 non-neoplastic cysts and 8 normal pancreatic tissues. The relationship between DPC4 alterations and various clinicopathological parameters was evaluated by chi-square test or Fisher's exact test. Survivals were calculated using Kaplan-Meier method (by a log-rank test). RESULTS: All the 16 nonmalignant cases of the pancreas showed expression of DPC4 gene. Loss of DPC4 expression was seen in 8 of 34(23.5 %) pancreatic adenocardnomas. The frequency of loss of DPC4 expression was higher in poorly differentiated adenocarcinoma (G3) than in well and moderately differentiated adenocarcinoma (G1 and G2) histologically (P--0.037). Loss of DPC4 expression of the patients at TNM stage IV was also higher than that of the patients at TNM stages I, II and III (60.0 % at stage IV, versus14.3 % atstage I, 18.2 % at stage II, and 18.2 % at stage III) (P=0.223). The mean and median survival in patients with DPC4 expression was longer than those in patients with loss of DPC4 expression. Kaplan-Meier survival analysis demonstrated patients with DPC4 expression had a higher survival rate than patients with loss of DPC4 expression, but the difference did not reach statistical significance (P=0.879). CONCLUSION: This study suggests that DPC4 is involved in the development of pancreatic carcinoma and is a late event in pancreatic carcinogenesis, DPC4 expression may be a molecular prognostic marker for pancreatic carcinoma.展开更多
AIM: To investigate the possible association between hepatitis B virus (HBV) infection and angiographically proven coronary artery disease (CAD) in a population with relatively high prevalence of HBV.METHODS: Sera fro...AIM: To investigate the possible association between hepatitis B virus (HBV) infection and angiographically proven coronary artery disease (CAD) in a population with relatively high prevalence of HBV.METHODS: Sera from 434 patients who underwent coronary angiography were tested for HBV antigens (HBsAg, HBeAg) and antibodies (Anti-HBs, Anti-HBc and Anti-HBe) by ELISA.RESULTS: Seventy-seven percent (224/291) of the patients with CAD and 73.4% (105/143) of the patients without angiographic evidence of atherosclerosis were seropositive for HBV (P＞0.05). However, C-reactive protein (CRP) levels were significantly higher in patients with CAD (P = 0.008), while lower in HBV seropositive population (P= 0.043 and P = 0.021 after adjustment for conventional risk factors).CONCLUSION: Our results suggested HBV infection negatively correlates with CRP levels, but seems not to be associated with coronary atherosclerosis.展开更多
AIM: To probe into the genetic susceptibility of HLA-DRB1 alleles to esophageal carcinoma in Han Chinese in Hubei Province.METHODS: HLA-DRB1 allele polymorphisms were typed by polymerase chain reaction with sequence-s...AIM: To probe into the genetic susceptibility of HLA-DRB1 alleles to esophageal carcinoma in Han Chinese in Hubei Province.METHODS: HLA-DRB1 allele polymorphisms were typed by polymerase chain reaction with sequence-specific primers (PCR-SSP) in 42 unrelated patients with esophageal cancer and 136 unrelated normal control subjects and the associated HLA-DRB1 allele was measured by nucleotide sequence analysis with PCR.SAS software was used in statistics.RESULTS: Allele frequency (AF) of HLA-DRB1·0901 was significantly higher in esophageal carcinoma patients than that in the normal controls (0.2500 vs0.1397, P=0.028, the odds ratio 2.053, etiologic fraction 0.1282). After analyzed the allele nucleotide sequence of HLA-DRB1·0901 which approachs to the corresponded exon 2 sequence of the allele in genebank. There was no association between patients and controls in the rested HLA-DRB1 alleles.CONCLUSION: HLA-DRB1·0901 allele is more common in the patients with esophageal carcinoma than in the healthy controls, which is positively associated with the patients of Hubei Han Chinese. Individuals carrying HLA-DRB1·0901may be susceptible to esophageal carcinoma.展开更多
AIM:To observe the effects of low molecular weight heparin(LMWH) on platelet surface P-selectin expression and serum interleukin-8 production in rats with trinitrobenzene sulphonic acid (TNBS) induced colitis.METHODS:...AIM:To observe the effects of low molecular weight heparin(LMWH) on platelet surface P-selectin expression and serum interleukin-8 production in rats with trinitrobenzene sulphonic acid (TNBS) induced colitis.METHODS:Colitis was induced in female Sprauge-Dawley rats by colonic administration of 2, 4, 6-TNBS. LMWH, a dalteparin (150U/kg, 300U/kg) was subcutaneously administrated one hour before induction of colitis and went on once a day for 6 days. Then a half dose was given for the next 7 days. Control animals received the same volume of normal saline once a day for 14 days after treated by TNBS.Animals were sacrificed at 24h, days 7 and 14 after induction of colitis. The colon was excised for the evaluation of macroscopic and histological findings and TNF-α immunohistochemical assay. Platelet surface P-selectin expression was determined by radioimmunoassay and serum IL-8 production was assayed by ELISA method.RESULTS:LMWH treatment in a dose of 300U/kg for 14 days significantly improved colonic inflammation by histological examination. Serum IL-8 production in the 300U/kg treatment group was more significantly decreased at day 14 than that at 24h (P<0.05). However, platelet surface P-selectin expression and TNF-α staining in colonic tissue were not significantly different among the three groups.CONCLUSION:LMWH has an anti-infiammatory effect on TNBS induced colitis in rats. The effect is possibly related to inhibition of proinflammatory cytokine IL-8, but not involved platelet surface P-selectin expression.展开更多
AIM: To investigate whether P28 derived from C3d can enhance the immune response to HBV-preS2/S induced by directly injection of naked plasmids containing variable repeats of P28 and HBV-preS2/S in fusion form.METHODS...AIM: To investigate whether P28 derived from C3d can enhance the immune response to HBV-preS2/S induced by directly injection of naked plasmids containing variable repeats of P28 and HBV-preS2/S in fusion form.METHODS: One to four copies of C3d-P28 coding gene,amplified by PCR and modified by restriction endonuclease sdigestion, were subcloned into a eukaryotic expression vector pVAON33 to construct pVAON33-P28, pVAON33-P28.2, pVAON33-P28.3 and pVAON33-P28.4 (pVAON33-P28.[1-4]). HBV-preS2/S coding sequence was then introduced into the pVAON33-P28.[1-4] and identified by both PCR and DNA sequencing. BALB/c mice were primed by intramuscular gene immunization with 100 μg different recombinant plasmids on day 0 and were boosted by subcutaneous inoculation with HBsAg protein (1 μg) 12 wk post-priming. The levels and avidity of specific IgG in sera collected at the indicated times from each group were determined by ELISA and NaSCN-displacement ELISA,respectively.RESULTS: HBsAg specific antibody response was elicited in groups primed with plasmids pVAON33-S2/S-P28.[1-4] and pVAON33-S2/S. However, the response against HBsAg in the groups primed with pVAON33-S2/S-P28.[1-4] was significantly higher than that in pVAON33-S2/S group, the highest level of the specific antibody response was observed in the groups primed with pVAON33-S2/S-P28.4 (P＜0.01).After secondary immunization with specific antigen, the acceleration of antibody levels was significantly higher and faster in the mice primed with DNA expressing preS2/S-P28 fusions than that with DNA expressing preS2/S only (P＜0.05).Interestingly, mice primed with DNA expressing preS2/S-P28.4 fusions maintained the highest levels of anti-HBs antibodies in all animals. The avidity assay showed that the avidity index (AI) collected at 18 wk from mice primed with pVAON33-S2/S-P28.3 and pVAON33-S2/S-P28.4 were significantly higher than that from preS2/S-DNA vaccinated mice (P＜0.01).CONCLUSION: Different repeats of C3d-P28 can enhance both humoral immune response and avidity maturation of specific antibodies induced by gene immunization, in which four copies of C3d-P28 may be necessary to achieve the most modest antibody response.展开更多
AIM:To develop a tumor DNA vaccine encoding a fusion protein of murine AFP and CTLA4,and to study its ability to induce specific CTL response and its protective effect against AFP-producing tumor.METHODS:Murine α-fet...AIM:To develop a tumor DNA vaccine encoding a fusion protein of murine AFP and CTLA4,and to study its ability to induce specific CTL response and its protective effect against AFP-producing tumor.METHODS:Murine α-fetoprotein (mAFP) gene was cloned from total RNA of Hepal-6 cells by RT-PCR.A DNA vaccine was constructed by fusion murine α-fetoprotein gene and extramembrane domain of murine CTLA4 gene.The DNA vaccine was identified by restriction enzyme analysis,sequencing and expression.EL-4 (mAFP) was developed by stable transfection of EL-4 cells with pmAFP.The frequency of cells producing IFN-γ in splenocytes harvested from the immunized mice was measured by ELISPOT.Mice immunized with DNA vaccine were inoculated with EL-4(mAFP) cells in back to observe the protective effect of immunization on tumor. On the other hand,blood samples were collected from the immunized mice to check the functions of liver and kidney.RESULTS:1.8kb mAFP cDNA was cloned from total RNA of Hepal-6 cells by RT-PCR.The DNA vaccine encoding a fusion protein of mAFP-CTLA4 was constructed and confirmed by restriction enzyme analysis,sequencing and expression.The expression of mAFP mRNA in EL-4 (mAFP) was confirmed by RT-PCR.The ELISPOT results showedthat the number of IFN-γ-producing cells in pmAFP-CTLA4 group was significantly higher than that in pmAFP,pcDNA3.1 and PBS group.The tumor volume in pmAFP-CTLA4 group was significantly smaller than that in pmAFP,pcDNA3.1 and PBS group, respectively. The hepatic and kidney functions in each group were not altered.CONCLUSION: AFP-CTLA4 DNA vaccine can stimulate potent specific CTL responses and has distinctive antitumor effect on AFP-producing tumor.The vaccine has no impact on the function of mouse liver and kidney.展开更多
The relationship between bone mineral density (BMD) and Zn, Cu, Ca levels in the meal and hair of urban and rural elderly people were studied. 470 subjects above 60 years old (urban 205 and rural 265), 178 males with ...The relationship between bone mineral density (BMD) and Zn, Cu, Ca levels in the meal and hair of urban and rural elderly people were studied. 470 subjects above 60 years old (urban 205 and rural 265), 178 males with an average age of 65.70±3.48 and 292 females with an average age of 65.90±4.02, were inquired. The BMD and Zn, Cu, Ca levels in the meal and hair were measured. The detected BMD in urban and rural female old people was significantly lower than that of the males; The contents of Ca and Zn in the meal of the urban females were significantly lower than those of the urban males; The Ca, Zn in the meal and Zn in the hair of the rural females were significantly lower than those of rural males (P< 0.05 or 0.01). The BMD, Ca intakes, Ca and Zn in the hair of the rural old people were significantly lower than those of the urban old people (P< 0 05 or 0.01). There was a correlation between BMD with the Ca, Zn of the hair and dietary Ca, Zn, Cu or between dietary Zn with Ca, Zn in the hair and Ca, Cu intakes. The Zn, Cu and Ca levels in the meal nutrients were correlated with BMD to some degrees. Lack of Ca and Zn in the meal can cause the reduction of BMD.展开更多
H-Ras is well known as one of the essential components of Ras/Raf/MEK/ERK cascade, which is a critical prosurvival signaling mechanism in most eukaryotic cells. Ras targets Raf/MEK/ERK cascade by integrating and trans...H-Ras is well known as one of the essential components of Ras/Raf/MEK/ERK cascade, which is a critical prosurvival signaling mechanism in most eukaryotic cells. Ras targets Raf/MEK/ERK cascade by integrating and transmitting extracellular signals from growth factor receptors to Raf, leading to the propagation of signals to modulate a serious of cellular survival events. Apoptosis signal-regulating kinasel (ASK1) serves as a general mediator of cell death because it is responsive to a variety of death signals. In this study, we found that H-Ras interacted with ASK1 to cause the inhibition of both ASK1 activity and ASK1-induced apoptosis in vivo, which was reversed only partially by addition of RafS621A, an antagonist of Raf, whereas MEK inhibitor, PD98059, and PI3K inhibitor, LY294002, did not disturb the inhibitory effect of H-Ras on ASK-1-induced apoptosis. Furthermore, by means of immunoprecipitate and kinase assays, we demonstrated that the interaction between H-Ras and ASK1 as well as the inhibition of ASK1 activity were dependent on the binding activity of H-Ras. These results suggest that a novel mechanism may be involved in H-Rasmediated cell survival in addition to the well established MEK/ERK and PI3K/Akt kinase-dependent enhancement of cell survival.展开更多
AIM: To explore the virulence and the infectivity of coccoid Helicobacter ppylori(H.pylori) transformed from spiral form by exposure to sterile tap water.METHODS: Three strains of H.pylori, isolated from gastric biops...AIM: To explore the virulence and the infectivity of coccoid Helicobacter ppylori(H.pylori) transformed from spiral form by exposure to sterile tap water.METHODS: Three strains of H.pylori, isolated from gastric biopsy specimens of confirmed peptic ulcer, were converted from spiral into coccoid form by exposure to sterile tap water.Both spiral and coccoid forms of H.pylori were tested for the urease activity, and the adherence to Hep-2 cells. The presence of flagella was examined under electron microscopy. In the experimental animal infection, the spiral and coccoid forms of H.pylori originated from the same strain F49 were inoculated intragastrically into BALB/c mice respectively four times at a 3-day interval. Half of the mice from each group were sacrificed at Day 21 and Day 28 after the last inoculation. Histology and H.pylori colonization were detected by urease test of gastric mucosa, cultures of H. pylori,and electron microscopy and so on.RESULTS: The urease activity and the ability of adherence to Hep-2 cells were found to be lower in coccoid H.pylori than that in its spiral form. For example, the transformation in strain F44 led to a significant decrease of the adherence rate and adherence index from 70.0±5.3 % to 30.2±3.5 %(P＜0.01), and from 2.6±0.4 to 0.86±0.3 (P＜0.01),respectively. The flagella of coccoid H.pylori were observed under electron microscope. In the experimental infection in mice, the positive rate of gastric mucosa urease test was 93.8 % (15/16) in the group infected by spiral H.pylori and 50 % (8/16) in the group infected by coccoid H. pylori,and the estimated coccoid H.pylori colony number was 1.75 vs0,56. The positive rates of H. pyloriculture were 87.5 %(14/16) in spiral H. pylori group and 68.8 % (11/16) in coccoid H.pylorigroup. There was no significant difference in either urease test or bacterial culture rate between the groups examined at Day 21 and Day 28 after inoculation. Electron microscopic examination of the samples taken from both groups showed the adherence of H. pylori in spiral,bacillary and coccoid shapes to the epithelial cells of gastric wall. Histological examination showed the occurrence of gastric mucosal injury as indicated by various degrees of erosion, ulcer, and inflammatory cell infiltration. Mucosal injury was slighter in the mice infected by coccoid H. pylori.No positive result was obtained in the control group that received intragastrical administration of sterile tap water.展开更多
AIM: To analyze the expression levels of soluble form of CD95, CD95 ligand (sCD95 and sCD95L, respectively) in plasma and CD95 expression on CD3+ cells in livertransplanted recipients with acute rejection (AR).METHODS...AIM: To analyze the expression levels of soluble form of CD95, CD95 ligand (sCD95 and sCD95L, respectively) in plasma and CD95 expression on CD3+ cells in livertransplanted recipients with acute rejection (AR).METHODS: Peripheral blood mononuclear cells (PBMCs)were isolated from 30 clinically liver transplanted recipients.CD95 expression on CD3+ cells was quantitatively measured by two-color fluorescence activated cell sorter (FACS)analysis. Lymphocyte surface phenotypes of CD4, CD8,CD16 and CD55 were determined by flow cytometry.Plasma levels of sCD95 and sCD95L were detected byEnzyme Linked-Immuno-Sorbent Assay (ELISA). The results were compared with that from normal healthy volunteers (n = 15 individuals).RESULTS: FACS analysis showed that CD95 expression on CD3+ T cells was significantly increased in liver transplanted recipients with AR compared to that in stable recipients without rejection and infection or healthy individuals who did not undergo transplantation (18 676.93±11 588.34/molecule,6 848.20±1712.96/molecule, 6 418.01±2 001.95/molecule,respectively, P＜0.01). Whereas no significant difference was seen between liver-transplanted stable recipients and healthy individuals. Furthermore, no significant differences were detected between each group with CD4/CD8 ratio or the percentage of CD16+56+ cells. Plasma levels of sCD95were significantly higher in transplanted recipients with AR compered to that in stable recipients or healthy individuals (391.88±196.00, 201.37±30.30, 148.83±58.25 pg/mL,respectively, P＜0.01). In contrast, the plasma levels ofsCD95L in liver- transplanted recipients were not significantlydifferent from that in healthy individuals.CONCLUSION: The present results indicate that the increased CD95 expression on CD3+ cells and the increased levels of sCD95 in plasma may modify the immunological situation of the recipients after transplantation or represent the ongoing graft rejection.展开更多
AIM: To investigate the role of Peyer's patch lymphocytes in the regulation of enteric epithelial barrier and ion transport function in homeostasis and host defense. METHODS: Mouse Peyer's patch lymphocytes we...AIM: To investigate the role of Peyer's patch lymphocytes in the regulation of enteric epithelial barrier and ion transport function in homeostasis and host defense. METHODS: Mouse Peyer's patch lymphocytes were co-cultured with human intestinal epithelial cell line Caco-2 either in the mixed or separated (isolated but permeable compartments) culture configuration. Barrier and transport functions of the Caco-2 epithelial monolayers were measured with short-circuit current (Isc) technique. Release of cytokines was measured by enzyme-linked immunosorbent assay (ELISA) and cytokine mRNA expression was analyzed by semi-quantitative RT-PCR. Barrier and iontransport functions of both culture conditions following exposure to Shigella lipopolysaccharide (LPS) were also examined. RESULTS: The transepithelial resistance (TER) of the epithelial monolayers co-cultured with Peyer's patch lymphocytes was maintained whereas that of the Caco-2 monolayers alone significantly decreased after eight days in culture. The forskolin-induced anion secretion, in either absence or presence of LPS, was significantly suppressed in the both co-cultures as compared with the Caco-2 cells alone. Furthermore, only the mixed co-culture condition induced the expression and release of mIL-6 from Peyer's patch lymphocytes, which could be further enhanced by LPS. However, both co-culture conditions suppressed expression and release of epithelial hIL-8 under the unstimulated conditions, while the treatment with LPS stimulated their hIL-8 expression and release. CONCLUSION: Peyer's patch lymphocytes may modulate intestinal epithelial barrier and ion transport function in homeostasis and host defense via cell-cell contact and cytokine signaling.展开更多
AIM: To identify the scFv antibody fragments specific for hepatocellular carcinoma by biopanning from a large human naive scFv phage display library.METHODS: A large human naive scFv phage library was used to search f...AIM: To identify the scFv antibody fragments specific for hepatocellular carcinoma by biopanning from a large human naive scFv phage display library.METHODS: A large human naive scFv phage library was used to search for the specific targets by biopanning with the hepatocellular carcinoma cell line HepG2 for the positiveselecting and the normal liver cell line L02 for the counterselecting. After three rounds of biopanning, individual scFv phages binding selectively to HepG2 cells were picked out. PCR was carried out for identification of the clones containing scFv gene sequence. The specific scFv phages were selected by ELISA and flow cytometry. DNA sequences of positive clones were analyzed by using Applied Biosystem Automated DNA sequencers 3 730. The expression proteins of the specific scFv antibody fragments in E. coli HB2151were purified by the affinity chromatography and detected by SDS-PAGE, Western blot and ELISA. The biological effect of the soluble antibody fragments on the HepG2 cells was investigated by observing the cell proliferation.RESULTS: Two different positive clones were obtained and the functional variable sequences were identified.Their DNA sequences of the scFv antibody fragments were submitted to GenBank (accession nos: AY686498 and AY686499). The soluble scFv antibody fragments were successfully expressed in E. coli HB2151. The relative molecular mass of the expression products was about 36 ku,according to its predicted Mr value. The two soluble scFv antibody fragments also had specific binding activity and obvious growth inhibition properties to HepG2 cells.CONCLUSION: The phage library biopanning permits identification of specific antibody fragments for hepatocellular carcinoma and affords experiment evidence for its immunotherapy study.展开更多
AIM: TO investigate the expression of several important molecules involved in major histocompatibility complex (MHC) dass I presentation pathway in primary hepatocellular carcinoma (HCC), and to determine whether cyto...AIM: TO investigate the expression of several important molecules involved in major histocompatibility complex (MHC) dass I presentation pathway in primary hepatocellular carcinoma (HCC), and to determine whether cytotoxic T lymphocyte (CTL) vaccine therapy was suitable for HCC.METHODS: Labeled streptavidin biotin (LSAB) method of immunohisto-chemistry was used to study 33 HCC tissue specimens.RESULTS: Most HCC tissues and adjacent histological normal hepatocytes expressed HLA-I antigens,TAP, and B7, expression of B7 was especially strong, and there was no significant difference between them (P>0.05).CONCLUSION: The MHC class I presentation pathway in primary hepatocellular carcinoma may not be abnormal or dysfunctional, and CTL could kill these tumor cells.Thus, it is suitable and practicable to design and construct CTL vaccine against HCC.展开更多
Interleukin-4 is a cytokine produced by activated T cells, mast cells, and basophils that elicits many important biological responses[1] (see Tab 1). These responses range from the regulation of helper T cell differen...Interleukin-4 is a cytokine produced by activated T cells, mast cells, and basophils that elicits many important biological responses[1] (see Tab 1). These responses range from the regulation of helper T cell differentiation[2] and the production of IgE[3] to the regulation of the adhesive properties of endothelial cells via VCAM-1[4]. In keeping with these diverse biological effects, high-affinity binding sites for IL-4 (Kd 20 to 300 pM) have been detected on many hematopoietic and non-hematopoietic cell types at levels ranging from 50 to 5000 sites per cell[5],This review will focus on the discrete signal transduction pathways activated by the IL-4 receptor and the coordination of these individual pathways in the regulation of a final biological outcome.展开更多
OBJECTIVE This article is to verify feasibility and validity of autologous cytokine-induced killer cell (Auto-CIK) treatment in solid malignancypatients.METHODS Amplification, phenotypic characteristics, cytokine secr...OBJECTIVE This article is to verify feasibility and validity of autologous cytokine-induced killer cell (Auto-CIK) treatment in solid malignancypatients.METHODS Amplification, phenotypic characteristics, cytokine secretion,antitumor cytotoxicity and clinical response to Auto-CIK derived from 65cases of solid tumor patients with different pathological types and clinicalstages were compared with LAKs in a large-scale clinical trial,RESUL'r$ We found that seriousness of disease and metastatic status hadno influence on effective components and antitumor immunological activityof Auto-ClK. Comparing cytotoxicity against various tumor cells with LAKsat various effector to target ratios, ClKs showed more effective cytotoxicityagainst NK sensitive or non-sensitive solid tumor cell lines at a low E/T ratio(6:1) which suggests indirectly that Auto-CIK had a longer effective time invivo than LAKs. These results suggest that CIKs are more suitable forimmunotherapy for those solid malignancy patients at high risk of relapse orrecurrence.CONCLUSIONS Our experimental data were consistent ~ith the reportedconclusion that the potent antitumor activity of Auto-ClK mainly rooted in theCD4- part of CIKs, including CD3~CD56~ cells and CD8 ~ CTLs. The CD4~part of ClKs seemed to have no direct tumor lytic activity. The results indicatethat the special "Thl bias" and enhanced cytotoxicity against K562 cellsoccurred in PBMCs after multicycles of Auto-ClK infusions suggesting theinduction of a "Thl shift" and rectification of "Th2 dominance" in PBMC afterAuto-CIK treatments.展开更多
AIM: To observe the localization of TRAIL/TRAIR (DR4, DR5,DcR1, DcR2) in the fetal pancreas.METHODS: Fetal pancreas of 32 weeks of pregnancy wereobtained from induced abortions, embedded in paraffin, and4-μm sections...AIM: To observe the localization of TRAIL/TRAIR (DR4, DR5,DcR1, DcR2) in the fetal pancreas.METHODS: Fetal pancreas of 32 weeks of pregnancy wereobtained from induced abortions, embedded in paraffin, and4-μm sections were prepared. The localization of TRAIL/TRAILR in fetal pancreas was investigated by fluorescenceimmunohistochemical method combined with laser scanningconfocal microscopy.RESULTS: TRAIL immunoreactive cells were mainly locatedon the periphery of the pancreas islets. There were a fewDcR1 and DcR2 positive cells whereas there were noimmunoreactive cells of DR4 and DR5 in the pancreas islets.In the acini and the ducts of the exocrine pancreas therewere no TRAIL/TRAILR immunoreactive cells.CONCLUSION: This study not only describes thedistribution of TRAIL/TRAILR in the fetal pancreas, but alsoprovides a morphological basis for deducing the functionof TRAIL/TRAILR in pancreas, suggesting that in normalpancreatic islets, the pancreatic cells are resistant towardsapoptosis too.展开更多
AIM: To observe the protective effect of rhIL-1β on pancreatic islets of alloxan-induced diabetic rats. METHODS: Protection of rhIL-1β on pancreatic islets of alloxan-induced diabetic rats (n=5) was demonstrated wit...AIM: To observe the protective effect of rhIL-1β on pancreatic islets of alloxan-induced diabetic rats. METHODS: Protection of rhIL-1β on pancreatic islets of alloxan-induced diabetic rats (n=5) was demonstrated with methods of immunohistochemistry and stereology. The concentration of serum glucose was measured by GOD method and that of serum insulin by RIA. RESULTS: The concentration of serum glucose increased but that of insulin decreased after administration of alloxan (150mg/kg), and the volume density and numerical density of the islets were zero. In rhIL-1β pretreated rats, although the concentration of serum insulin decreased (from 11.9±3.0mIU/L to 6.1±1.6mIU/L,P<0.05), that of glucose was at normal level compared with the control group. As compared with alloxan group, the concentration of serum glucose in rhIL-1β pretreated rats decreased (from 19.4±8.9mmol/L to 12.0±4.0mmol/L, P<0.05) and the volume density increased(0/L to. 1/L, P<0.05). CONCLUSION: rhIL-1β pretreatment may have protective effect on the islets of alloxan-induced diabetic rats.展开更多
AIM: To investigate the combined effect of STI571 and p27 gene clone on the regulation of proliferation, cell cycle and apoptosis of K562 cell line.METHODS: p27 gene was obtained by RT-PCR, and its sequence was approv...AIM: To investigate the combined effect of STI571 and p27 gene clone on the regulation of proliferation, cell cycle and apoptosis of K562 cell line.METHODS: p27 gene was obtained by RT-PCR, and its sequence was approved to be correct. Then p27-pcDNA3.1 vector was constructed and transfected into K562 cell line.p27-pcDNA3.1-K562 cell clone was screened by G418 after transfection, p27 protein was identified by Western blot.MTT was used to detect the survival rate of the cell. Flow cytometry was used to detect cell cycle and apoptosis index.RESULTS: The expression of p27 protein could be detected by Western blot in p27-pcDNA3.1-K562 cells. A strong inhibition of cell proliferation was observed in p27-pcDNA3.1-K562 cells as compared with that of the control (pcDNA3.1-K562 cells). The cells at G0/G1 phase were significantly increased, and cells at S phase were greatly declined.The apoptosis index was increased greatly after p27-pcDNA3.1-K562 cells were treated with STI571, and survival rate of the cell was markedly declined (0.35-0.58,P<0.05-0.048 vs STI571-K562 cell, 0.35-0.72, P<0.01-0.001 vs p27-K562 cell).CONCLUSION: p27 and STI571 have a synergistic action on inhibition of proliferation and induction of apoptosis on K562 cells.展开更多
Upon activation, naive T-helper cells can differentiate into two major distinct subsets, T helper 1 (Th1) and T helper 2 (Th2), as defined by their effector functions and cytokine secretion patterns. Cytokine milieu a...Upon activation, naive T-helper cells can differentiate into two major distinct subsets, T helper 1 (Th1) and T helper 2 (Th2), as defined by their effector functions and cytokine secretion patterns. Cytokine milieu and costimulatory molecules have been shown to play an essential role in determining T helper differentiation. However, it is still unclear how the effects of signals of co-stimulatory molecules and cytokines are exerted during T helper differentiation. We show evidence suggesting that while cytokine signals initiate differentiation program, the selective action of death effectors determines the endpoint balance of differenti-展开更多
基金Supported by the National Key Basic Research Special Funds (NKBRSF), No. 2001CB510004
文摘AIM: To investigate the expression pattern of epithelial cell adhesion molecule (Ep-CAM) on normal and malignant colon tissues to evaluate its diagnostic and therapeutic significance.METHODS: cDNA encoding Ep-CAv extracellular domain was cloned by reverse transcription-polymerase chain reaction (RT-PCR) from excised malignant colon tissues and inserted into a glutathione S-transferase (GST)-tagged vector. EpCAM-GST fusion protein was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and purified with glutathionesepharose. The Ep-CAM-GST fusion protein was mixed with Freund's adjuvant and Balb/c mice were immunized with it. Sp2/0 myeloma cells were fused with the spleen cells of the immunized mice. After having selected by indirect ELISA, the anti-Ep-CAM monoclonal antibodies (NAbs) were generated and the corresponding ascites were obtained.Finally, the human colon carcinoma tissue array prepared from seventy individual patients was stained with the antiEp-CAM NAbs.RESULTS: The isolated Ep-CAM cDNA sequence was identical to the data in GenBank. The expressed fusion protein was almost soluble and had a molecular weight (NW) of 53 ku.Four NAbs against Ep-CAM were obtained and designated as FMU-Ep1, FMU-Ep2, FMU-Ep3 and FMU-Ep4 respectively.Among them, FMU-Ep4 could recognize the natural EpCAM on Colo205 and SW480 cells, and all of them could be used for immunohistochemical staining of tissue sections.It was found that Ep-CAM was distributed differently in normal and various malignant colon tissues, including squamous cell carcinoma, signet-ring cell carcinoma and adenocarcinoma.In normal colon gland epithelia, Ep-CAM antigen was mainly distributed on the basolateral membrane and in the region between the basolateral membrane and the cytoplastic part near the nuclei, whereas the expression pattern of colon malignancies was mainly on the whole surface of epithelia and the expression was much higher than the normal colon tissues. The staining pattern of tissue array showed in adenocarcinoma and papillary adenocarcinoma, and the expression of Ep-CAM was increased from grade Ⅰ to grade Ⅲ.CONCLUSION: NAbs against Ep-CAM might be useful for research on the structure and function of Ep-CAM and may have diagnostic and therapeutic value to various colon carcinomas.
文摘AIM: DPC4 is a tumor suppressor gene on chromosome 18q21.1 that has high mutant frequencies in pancreatic carcinogenesis. The purpose of this study was to investigate the role of DPC4 alterations in tumorigenesis and progression of pancreatic carcinomas. METHODS: We studied the immunohistochemical markers of DPC4 in 34 adenocarcinomas and 16 nonmalignant specimens from the pancreas. The 16 nonmalignant specimens from the pancreas included 8 non-neoplastic cysts and 8 normal pancreatic tissues. The relationship between DPC4 alterations and various clinicopathological parameters was evaluated by chi-square test or Fisher's exact test. Survivals were calculated using Kaplan-Meier method (by a log-rank test). RESULTS: All the 16 nonmalignant cases of the pancreas showed expression of DPC4 gene. Loss of DPC4 expression was seen in 8 of 34(23.5 %) pancreatic adenocardnomas. The frequency of loss of DPC4 expression was higher in poorly differentiated adenocarcinoma (G3) than in well and moderately differentiated adenocarcinoma (G1 and G2) histologically (P--0.037). Loss of DPC4 expression of the patients at TNM stage IV was also higher than that of the patients at TNM stages I, II and III (60.0 % at stage IV, versus14.3 % atstage I, 18.2 % at stage II, and 18.2 % at stage III) (P=0.223). The mean and median survival in patients with DPC4 expression was longer than those in patients with loss of DPC4 expression. Kaplan-Meier survival analysis demonstrated patients with DPC4 expression had a higher survival rate than patients with loss of DPC4 expression, but the difference did not reach statistical significance (P=0.879). CONCLUSION: This study suggests that DPC4 is involved in the development of pancreatic carcinoma and is a late event in pancreatic carcinogenesis, DPC4 expression may be a molecular prognostic marker for pancreatic carcinoma.
基金Supported by Major State Basic Research Development Program of China, No. G2000056903the National High Technology Research and Development Program of China, No. 2004AA215242
文摘AIM: To investigate the possible association between hepatitis B virus (HBV) infection and angiographically proven coronary artery disease (CAD) in a population with relatively high prevalence of HBV.METHODS: Sera from 434 patients who underwent coronary angiography were tested for HBV antigens (HBsAg, HBeAg) and antibodies (Anti-HBs, Anti-HBc and Anti-HBe) by ELISA.RESULTS: Seventy-seven percent (224/291) of the patients with CAD and 73.4% (105/143) of the patients without angiographic evidence of atherosclerosis were seropositive for HBV (P＞0.05). However, C-reactive protein (CRP) levels were significantly higher in patients with CAD (P = 0.008), while lower in HBV seropositive population (P= 0.043 and P = 0.021 after adjustment for conventional risk factors).CONCLUSION: Our results suggested HBV infection negatively correlates with CRP levels, but seems not to be associated with coronary atherosclerosis.
文摘AIM: To probe into the genetic susceptibility of HLA-DRB1 alleles to esophageal carcinoma in Han Chinese in Hubei Province.METHODS: HLA-DRB1 allele polymorphisms were typed by polymerase chain reaction with sequence-specific primers (PCR-SSP) in 42 unrelated patients with esophageal cancer and 136 unrelated normal control subjects and the associated HLA-DRB1 allele was measured by nucleotide sequence analysis with PCR.SAS software was used in statistics.RESULTS: Allele frequency (AF) of HLA-DRB1·0901 was significantly higher in esophageal carcinoma patients than that in the normal controls (0.2500 vs0.1397, P=0.028, the odds ratio 2.053, etiologic fraction 0.1282). After analyzed the allele nucleotide sequence of HLA-DRB1·0901 which approachs to the corresponded exon 2 sequence of the allele in genebank. There was no association between patients and controls in the rested HLA-DRB1 alleles.CONCLUSION: HLA-DRB1·0901 allele is more common in the patients with esophageal carcinoma than in the healthy controls, which is positively associated with the patients of Hubei Han Chinese. Individuals carrying HLA-DRB1·0901may be susceptible to esophageal carcinoma.
基金Supported by the Hubei Provincial Natural Science Foundation,No.2000J047
文摘AIM:To observe the effects of low molecular weight heparin(LMWH) on platelet surface P-selectin expression and serum interleukin-8 production in rats with trinitrobenzene sulphonic acid (TNBS) induced colitis.METHODS:Colitis was induced in female Sprauge-Dawley rats by colonic administration of 2, 4, 6-TNBS. LMWH, a dalteparin (150U/kg, 300U/kg) was subcutaneously administrated one hour before induction of colitis and went on once a day for 6 days. Then a half dose was given for the next 7 days. Control animals received the same volume of normal saline once a day for 14 days after treated by TNBS.Animals were sacrificed at 24h, days 7 and 14 after induction of colitis. The colon was excised for the evaluation of macroscopic and histological findings and TNF-α immunohistochemical assay. Platelet surface P-selectin expression was determined by radioimmunoassay and serum IL-8 production was assayed by ELISA method.RESULTS:LMWH treatment in a dose of 300U/kg for 14 days significantly improved colonic inflammation by histological examination. Serum IL-8 production in the 300U/kg treatment group was more significantly decreased at day 14 than that at 24h (P<0.05). However, platelet surface P-selectin expression and TNF-α staining in colonic tissue were not significantly different among the three groups.CONCLUSION:LMWH has an anti-infiammatory effect on TNBS induced colitis in rats. The effect is possibly related to inhibition of proinflammatory cytokine IL-8, but not involved platelet surface P-selectin expression.
基金Supported by the Major State Basic Research Development Program of China,No.2001CB510006 National Science Found for Distinguished Young Scholars from NSFC,No.39925031 and Key Research Program of STCSM, No.03DZ19229
文摘AIM: To investigate whether P28 derived from C3d can enhance the immune response to HBV-preS2/S induced by directly injection of naked plasmids containing variable repeats of P28 and HBV-preS2/S in fusion form.METHODS: One to four copies of C3d-P28 coding gene,amplified by PCR and modified by restriction endonuclease sdigestion, were subcloned into a eukaryotic expression vector pVAON33 to construct pVAON33-P28, pVAON33-P28.2, pVAON33-P28.3 and pVAON33-P28.4 (pVAON33-P28.[1-4]). HBV-preS2/S coding sequence was then introduced into the pVAON33-P28.[1-4] and identified by both PCR and DNA sequencing. BALB/c mice were primed by intramuscular gene immunization with 100 μg different recombinant plasmids on day 0 and were boosted by subcutaneous inoculation with HBsAg protein (1 μg) 12 wk post-priming. The levels and avidity of specific IgG in sera collected at the indicated times from each group were determined by ELISA and NaSCN-displacement ELISA,respectively.RESULTS: HBsAg specific antibody response was elicited in groups primed with plasmids pVAON33-S2/S-P28.[1-4] and pVAON33-S2/S. However, the response against HBsAg in the groups primed with pVAON33-S2/S-P28.[1-4] was significantly higher than that in pVAON33-S2/S group, the highest level of the specific antibody response was observed in the groups primed with pVAON33-S2/S-P28.4 (P＜0.01).After secondary immunization with specific antigen, the acceleration of antibody levels was significantly higher and faster in the mice primed with DNA expressing preS2/S-P28 fusions than that with DNA expressing preS2/S only (P＜0.05).Interestingly, mice primed with DNA expressing preS2/S-P28.4 fusions maintained the highest levels of anti-HBs antibodies in all animals. The avidity assay showed that the avidity index (AI) collected at 18 wk from mice primed with pVAON33-S2/S-P28.3 and pVAON33-S2/S-P28.4 were significantly higher than that from preS2/S-DNA vaccinated mice (P＜0.01).CONCLUSION: Different repeats of C3d-P28 can enhance both humoral immune response and avidity maturation of specific antibodies induced by gene immunization, in which four copies of C3d-P28 may be necessary to achieve the most modest antibody response.
文摘AIM:To develop a tumor DNA vaccine encoding a fusion protein of murine AFP and CTLA4,and to study its ability to induce specific CTL response and its protective effect against AFP-producing tumor.METHODS:Murine α-fetoprotein (mAFP) gene was cloned from total RNA of Hepal-6 cells by RT-PCR.A DNA vaccine was constructed by fusion murine α-fetoprotein gene and extramembrane domain of murine CTLA4 gene.The DNA vaccine was identified by restriction enzyme analysis,sequencing and expression.EL-4 (mAFP) was developed by stable transfection of EL-4 cells with pmAFP.The frequency of cells producing IFN-γ in splenocytes harvested from the immunized mice was measured by ELISPOT.Mice immunized with DNA vaccine were inoculated with EL-4(mAFP) cells in back to observe the protective effect of immunization on tumor. On the other hand,blood samples were collected from the immunized mice to check the functions of liver and kidney.RESULTS:1.8kb mAFP cDNA was cloned from total RNA of Hepal-6 cells by RT-PCR.The DNA vaccine encoding a fusion protein of mAFP-CTLA4 was constructed and confirmed by restriction enzyme analysis,sequencing and expression.The expression of mAFP mRNA in EL-4 (mAFP) was confirmed by RT-PCR.The ELISPOT results showedthat the number of IFN-γ-producing cells in pmAFP-CTLA4 group was significantly higher than that in pmAFP,pcDNA3.1 and PBS group.The tumor volume in pmAFP-CTLA4 group was significantly smaller than that in pmAFP,pcDNA3.1 and PBS group, respectively. The hepatic and kidney functions in each group were not altered.CONCLUSION: AFP-CTLA4 DNA vaccine can stimulate potent specific CTL responses and has distinctive antitumor effect on AFP-producing tumor.The vaccine has no impact on the function of mouse liver and kidney.
基金This project was supported by a grant from natural sci ences foundation of Henan province (964021500).
文摘The relationship between bone mineral density (BMD) and Zn, Cu, Ca levels in the meal and hair of urban and rural elderly people were studied. 470 subjects above 60 years old (urban 205 and rural 265), 178 males with an average age of 65.70±3.48 and 292 females with an average age of 65.90±4.02, were inquired. The BMD and Zn, Cu, Ca levels in the meal and hair were measured. The detected BMD in urban and rural female old people was significantly lower than that of the males; The contents of Ca and Zn in the meal of the urban females were significantly lower than those of the urban males; The Ca, Zn in the meal and Zn in the hair of the rural females were significantly lower than those of rural males (P< 0.05 or 0.01). The BMD, Ca intakes, Ca and Zn in the hair of the rural old people were significantly lower than those of the urban old people (P< 0 05 or 0.01). There was a correlation between BMD with the Ca, Zn of the hair and dietary Ca, Zn, Cu or between dietary Zn with Ca, Zn in the hair and Ca, Cu intakes. The Zn, Cu and Ca levels in the meal nutrients were correlated with BMD to some degrees. Lack of Ca and Zn in the meal can cause the reduction of BMD.
文摘H-Ras is well known as one of the essential components of Ras/Raf/MEK/ERK cascade, which is a critical prosurvival signaling mechanism in most eukaryotic cells. Ras targets Raf/MEK/ERK cascade by integrating and transmitting extracellular signals from growth factor receptors to Raf, leading to the propagation of signals to modulate a serious of cellular survival events. Apoptosis signal-regulating kinasel (ASK1) serves as a general mediator of cell death because it is responsive to a variety of death signals. In this study, we found that H-Ras interacted with ASK1 to cause the inhibition of both ASK1 activity and ASK1-induced apoptosis in vivo, which was reversed only partially by addition of RafS621A, an antagonist of Raf, whereas MEK inhibitor, PD98059, and PI3K inhibitor, LY294002, did not disturb the inhibitory effect of H-Ras on ASK-1-induced apoptosis. Furthermore, by means of immunoprecipitate and kinase assays, we demonstrated that the interaction between H-Ras and ASK1 as well as the inhibition of ASK1 activity were dependent on the binding activity of H-Ras. These results suggest that a novel mechanism may be involved in H-Rasmediated cell survival in addition to the well established MEK/ERK and PI3K/Akt kinase-dependent enhancement of cell survival.
基金the Natural Science Fundation of Fujian Province,China,No.C95031
文摘AIM: To explore the virulence and the infectivity of coccoid Helicobacter ppylori(H.pylori) transformed from spiral form by exposure to sterile tap water.METHODS: Three strains of H.pylori, isolated from gastric biopsy specimens of confirmed peptic ulcer, were converted from spiral into coccoid form by exposure to sterile tap water.Both spiral and coccoid forms of H.pylori were tested for the urease activity, and the adherence to Hep-2 cells. The presence of flagella was examined under electron microscopy. In the experimental animal infection, the spiral and coccoid forms of H.pylori originated from the same strain F49 were inoculated intragastrically into BALB/c mice respectively four times at a 3-day interval. Half of the mice from each group were sacrificed at Day 21 and Day 28 after the last inoculation. Histology and H.pylori colonization were detected by urease test of gastric mucosa, cultures of H. pylori,and electron microscopy and so on.RESULTS: The urease activity and the ability of adherence to Hep-2 cells were found to be lower in coccoid H.pylori than that in its spiral form. For example, the transformation in strain F44 led to a significant decrease of the adherence rate and adherence index from 70.0±5.3 % to 30.2±3.5 %(P＜0.01), and from 2.6±0.4 to 0.86±0.3 (P＜0.01),respectively. The flagella of coccoid H.pylori were observed under electron microscope. In the experimental infection in mice, the positive rate of gastric mucosa urease test was 93.8 % (15/16) in the group infected by spiral H.pylori and 50 % (8/16) in the group infected by coccoid H. pylori,and the estimated coccoid H.pylori colony number was 1.75 vs0,56. The positive rates of H. pyloriculture were 87.5 %(14/16) in spiral H. pylori group and 68.8 % (11/16) in coccoid H.pylorigroup. There was no significant difference in either urease test or bacterial culture rate between the groups examined at Day 21 and Day 28 after inoculation. Electron microscopic examination of the samples taken from both groups showed the adherence of H. pylori in spiral,bacillary and coccoid shapes to the epithelial cells of gastric wall. Histological examination showed the occurrence of gastric mucosal injury as indicated by various degrees of erosion, ulcer, and inflammatory cell infiltration. Mucosal injury was slighter in the mice infected by coccoid H. pylori.No positive result was obtained in the control group that received intragastrical administration of sterile tap water.
文摘AIM: To analyze the expression levels of soluble form of CD95, CD95 ligand (sCD95 and sCD95L, respectively) in plasma and CD95 expression on CD3+ cells in livertransplanted recipients with acute rejection (AR).METHODS: Peripheral blood mononuclear cells (PBMCs)were isolated from 30 clinically liver transplanted recipients.CD95 expression on CD3+ cells was quantitatively measured by two-color fluorescence activated cell sorter (FACS)analysis. Lymphocyte surface phenotypes of CD4, CD8,CD16 and CD55 were determined by flow cytometry.Plasma levels of sCD95 and sCD95L were detected byEnzyme Linked-Immuno-Sorbent Assay (ELISA). The results were compared with that from normal healthy volunteers (n = 15 individuals).RESULTS: FACS analysis showed that CD95 expression on CD3+ T cells was significantly increased in liver transplanted recipients with AR compared to that in stable recipients without rejection and infection or healthy individuals who did not undergo transplantation (18 676.93±11 588.34/molecule,6 848.20±1712.96/molecule, 6 418.01±2 001.95/molecule,respectively, P＜0.01). Whereas no significant difference was seen between liver-transplanted stable recipients and healthy individuals. Furthermore, no significant differences were detected between each group with CD4/CD8 ratio or the percentage of CD16+56+ cells. Plasma levels of sCD95were significantly higher in transplanted recipients with AR compered to that in stable recipients or healthy individuals (391.88±196.00, 201.37±30.30, 148.83±58.25 pg/mL,respectively, P＜0.01). In contrast, the plasma levels ofsCD95L in liver- transplanted recipients were not significantlydifferent from that in healthy individuals.CONCLUSION: The present results indicate that the increased CD95 expression on CD3+ cells and the increased levels of sCD95 in plasma may modify the immunological situation of the recipients after transplantation or represent the ongoing graft rejection.
基金Supported by Strategic Program of Chinese University of Hong Kong,and Distinguished Yong Investigator Fund of the National Natural Science Foundation of China,30029002
文摘AIM: To investigate the role of Peyer's patch lymphocytes in the regulation of enteric epithelial barrier and ion transport function in homeostasis and host defense. METHODS: Mouse Peyer's patch lymphocytes were co-cultured with human intestinal epithelial cell line Caco-2 either in the mixed or separated (isolated but permeable compartments) culture configuration. Barrier and transport functions of the Caco-2 epithelial monolayers were measured with short-circuit current (Isc) technique. Release of cytokines was measured by enzyme-linked immunosorbent assay (ELISA) and cytokine mRNA expression was analyzed by semi-quantitative RT-PCR. Barrier and iontransport functions of both culture conditions following exposure to Shigella lipopolysaccharide (LPS) were also examined. RESULTS: The transepithelial resistance (TER) of the epithelial monolayers co-cultured with Peyer's patch lymphocytes was maintained whereas that of the Caco-2 monolayers alone significantly decreased after eight days in culture. The forskolin-induced anion secretion, in either absence or presence of LPS, was significantly suppressed in the both co-cultures as compared with the Caco-2 cells alone. Furthermore, only the mixed co-culture condition induced the expression and release of mIL-6 from Peyer's patch lymphocytes, which could be further enhanced by LPS. However, both co-culture conditions suppressed expression and release of epithelial hIL-8 under the unstimulated conditions, while the treatment with LPS stimulated their hIL-8 expression and release. CONCLUSION: Peyer's patch lymphocytes may modulate intestinal epithelial barrier and ion transport function in homeostasis and host defense via cell-cell contact and cytokine signaling.
基金Supported by the Major State Basic Research Development Program of China, 973 Program, No. 2002CB513100
文摘AIM: To identify the scFv antibody fragments specific for hepatocellular carcinoma by biopanning from a large human naive scFv phage display library.METHODS: A large human naive scFv phage library was used to search for the specific targets by biopanning with the hepatocellular carcinoma cell line HepG2 for the positiveselecting and the normal liver cell line L02 for the counterselecting. After three rounds of biopanning, individual scFv phages binding selectively to HepG2 cells were picked out. PCR was carried out for identification of the clones containing scFv gene sequence. The specific scFv phages were selected by ELISA and flow cytometry. DNA sequences of positive clones were analyzed by using Applied Biosystem Automated DNA sequencers 3 730. The expression proteins of the specific scFv antibody fragments in E. coli HB2151were purified by the affinity chromatography and detected by SDS-PAGE, Western blot and ELISA. The biological effect of the soluble antibody fragments on the HepG2 cells was investigated by observing the cell proliferation.RESULTS: Two different positive clones were obtained and the functional variable sequences were identified.Their DNA sequences of the scFv antibody fragments were submitted to GenBank (accession nos: AY686498 and AY686499). The soluble scFv antibody fragments were successfully expressed in E. coli HB2151. The relative molecular mass of the expression products was about 36 ku,according to its predicted Mr value. The two soluble scFv antibody fragments also had specific binding activity and obvious growth inhibition properties to HepG2 cells.CONCLUSION: The phage library biopanning permits identification of specific antibody fragments for hepatocellular carcinoma and affords experiment evidence for its immunotherapy study.
基金Supported by the National Natural Science Foundation of China, No. 30070855
文摘AIM: TO investigate the expression of several important molecules involved in major histocompatibility complex (MHC) dass I presentation pathway in primary hepatocellular carcinoma (HCC), and to determine whether cytotoxic T lymphocyte (CTL) vaccine therapy was suitable for HCC.METHODS: Labeled streptavidin biotin (LSAB) method of immunohisto-chemistry was used to study 33 HCC tissue specimens.RESULTS: Most HCC tissues and adjacent histological normal hepatocytes expressed HLA-I antigens,TAP, and B7, expression of B7 was especially strong, and there was no significant difference between them (P>0.05).CONCLUSION: The MHC class I presentation pathway in primary hepatocellular carcinoma may not be abnormal or dysfunctional, and CTL could kill these tumor cells.Thus, it is suitable and practicable to design and construct CTL vaccine against HCC.
文摘Interleukin-4 is a cytokine produced by activated T cells, mast cells, and basophils that elicits many important biological responses[1] (see Tab 1). These responses range from the regulation of helper T cell differentiation[2] and the production of IgE[3] to the regulation of the adhesive properties of endothelial cells via VCAM-1[4]. In keeping with these diverse biological effects, high-affinity binding sites for IL-4 (Kd 20 to 300 pM) have been detected on many hematopoietic and non-hematopoietic cell types at levels ranging from 50 to 5000 sites per cell[5],This review will focus on the discrete signal transduction pathways activated by the IL-4 receptor and the coordination of these individual pathways in the regulation of a final biological outcome.
文摘OBJECTIVE This article is to verify feasibility and validity of autologous cytokine-induced killer cell (Auto-CIK) treatment in solid malignancypatients.METHODS Amplification, phenotypic characteristics, cytokine secretion,antitumor cytotoxicity and clinical response to Auto-CIK derived from 65cases of solid tumor patients with different pathological types and clinicalstages were compared with LAKs in a large-scale clinical trial,RESUL'r$ We found that seriousness of disease and metastatic status hadno influence on effective components and antitumor immunological activityof Auto-ClK. Comparing cytotoxicity against various tumor cells with LAKsat various effector to target ratios, ClKs showed more effective cytotoxicityagainst NK sensitive or non-sensitive solid tumor cell lines at a low E/T ratio(6:1) which suggests indirectly that Auto-CIK had a longer effective time invivo than LAKs. These results suggest that CIKs are more suitable forimmunotherapy for those solid malignancy patients at high risk of relapse orrecurrence.CONCLUSIONS Our experimental data were consistent ~ith the reportedconclusion that the potent antitumor activity of Auto-ClK mainly rooted in theCD4- part of CIKs, including CD3~CD56~ cells and CD8 ~ CTLs. The CD4~part of ClKs seemed to have no direct tumor lytic activity. The results indicatethat the special "Thl bias" and enhanced cytotoxicity against K562 cellsoccurred in PBMCs after multicycles of Auto-ClK infusions suggesting theinduction of a "Thl shift" and rectification of "Th2 dominance" in PBMC afterAuto-CIK treatments.
基金the grants of the National Key Basic Research Program of China,No.2001CB510004
文摘AIM: To observe the localization of TRAIL/TRAIR (DR4, DR5,DcR1, DcR2) in the fetal pancreas.METHODS: Fetal pancreas of 32 weeks of pregnancy wereobtained from induced abortions, embedded in paraffin, and4-μm sections were prepared. The localization of TRAIL/TRAILR in fetal pancreas was investigated by fluorescenceimmunohistochemical method combined with laser scanningconfocal microscopy.RESULTS: TRAIL immunoreactive cells were mainly locatedon the periphery of the pancreas islets. There were a fewDcR1 and DcR2 positive cells whereas there were noimmunoreactive cells of DR4 and DR5 in the pancreas islets.In the acini and the ducts of the exocrine pancreas therewere no TRAIL/TRAILR immunoreactive cells.CONCLUSION: This study not only describes thedistribution of TRAIL/TRAILR in the fetal pancreas, but alsoprovides a morphological basis for deducing the functionof TRAIL/TRAILR in pancreas, suggesting that in normalpancreatic islets, the pancreatic cells are resistant towardsapoptosis too.
基金Supported by the National Natural Science Foundation of China,No.39870109
文摘AIM: To observe the protective effect of rhIL-1β on pancreatic islets of alloxan-induced diabetic rats. METHODS: Protection of rhIL-1β on pancreatic islets of alloxan-induced diabetic rats (n=5) was demonstrated with methods of immunohistochemistry and stereology. The concentration of serum glucose was measured by GOD method and that of serum insulin by RIA. RESULTS: The concentration of serum glucose increased but that of insulin decreased after administration of alloxan (150mg/kg), and the volume density and numerical density of the islets were zero. In rhIL-1β pretreated rats, although the concentration of serum insulin decreased (from 11.9±3.0mIU/L to 6.1±1.6mIU/L,P<0.05), that of glucose was at normal level compared with the control group. As compared with alloxan group, the concentration of serum glucose in rhIL-1β pretreated rats decreased (from 19.4±8.9mmol/L to 12.0±4.0mmol/L, P<0.05) and the volume density increased(0/L to. 1/L, P<0.05). CONCLUSION: rhIL-1β pretreatment may have protective effect on the islets of alloxan-induced diabetic rats.
基金Supported by the National Natural Science Foundation of China,No. 39770336
文摘AIM: To investigate the combined effect of STI571 and p27 gene clone on the regulation of proliferation, cell cycle and apoptosis of K562 cell line.METHODS: p27 gene was obtained by RT-PCR, and its sequence was approved to be correct. Then p27-pcDNA3.1 vector was constructed and transfected into K562 cell line.p27-pcDNA3.1-K562 cell clone was screened by G418 after transfection, p27 protein was identified by Western blot.MTT was used to detect the survival rate of the cell. Flow cytometry was used to detect cell cycle and apoptosis index.RESULTS: The expression of p27 protein could be detected by Western blot in p27-pcDNA3.1-K562 cells. A strong inhibition of cell proliferation was observed in p27-pcDNA3.1-K562 cells as compared with that of the control (pcDNA3.1-K562 cells). The cells at G0/G1 phase were significantly increased, and cells at S phase were greatly declined.The apoptosis index was increased greatly after p27-pcDNA3.1-K562 cells were treated with STI571, and survival rate of the cell was markedly declined (0.35-0.58,P<0.05-0.048 vs STI571-K562 cell, 0.35-0.72, P<0.01-0.001 vs p27-K562 cell).CONCLUSION: p27 and STI571 have a synergistic action on inhibition of proliferation and induction of apoptosis on K562 cells.
文摘Upon activation, naive T-helper cells can differentiate into two major distinct subsets, T helper 1 (Th1) and T helper 2 (Th2), as defined by their effector functions and cytokine secretion patterns. Cytokine milieu and costimulatory molecules have been shown to play an essential role in determining T helper differentiation. However, it is still unclear how the effects of signals of co-stimulatory molecules and cytokines are exerted during T helper differentiation. We show evidence suggesting that while cytokine signals initiate differentiation program, the selective action of death effectors determines the endpoint balance of differenti-
