Correlation between the expression level of coiled-coil domain-containing protein 80 and obesity / 中华预防医学杂志

<p><b>OBJECTIVE</b>To explore the relationship between expression of coiled-coil domain-containing protein 80(CCDC80) and obesity in serum and adipose tissues.</p><p><b>METHODS</b>A cross-sectional survey was conducted in a hospital in Tangshan in September 2010. 100 people including 78 healthy people and 22 with type-2 diabetes were recruited. Another 36 female patients with benign tumor of Obstetrics and Gynecology were also recruited. Demographic characteristics and serum samples were collected from all subjects, basic biochemical indicators were tested. All subjects were divided into 'Normal Weight' and 'Overweight and Obese' according to their BMI (BMI <24.0 kg/m(2); BMI≥24 kg/m(2)). Serum CCDC80 of the 100 subjects was detected by ELISA. Visceral and subcutaneous fat were derived from the other 36 female subjects, and Real-time PCR was used to detect CCDC80 mRNA expression in adipose tissues. Pearson correlation and multiple linear regression were used to analyze the correlation between serum or adipose CCDC80 expression and waist, BMI, and other biochemical indicators.</p><p><b>RESULTS</b>The age of 100 subjects was (52.9±8.4) years old. 44% of them were women (44 cases) and 56% of them were men (56 cases). After dividing them into three groups according to their BMI, covariance analysis were conducted, and age and gender were adjusted. The HDL-C level was significantly different among three groups (F = 10.73, P < 0.001): 'Overweight and obese combined with diabetes' group ((0.90±0.06) mmol/L)< 'Overweight and obese' group ((1.14±0.04) mmol/L) < 'Normal weight' group ((1.28±0.05) mmol/L). The adjusted expression of serum CCDC80 of the 100 subjects was (5.84±0.16) pg/ml, (5.81±0.98) pg/ml among men and (5.97±0.89) pg/ml among women, and there was no significant difference (t = -0.812, P = 0.419) between genders. ANOVA revealed that there was no significant differences of the expression of serum CCDC80 among three groups (F = 1.06, P = 0.351), 'Normal weight' group was (5.84±0.16) pg/ml, 'overweight and obese' group was (6.11±0.14) pg/ml, and 'Overweight and obese combined with diabetes' group was (5.84±0.19) pg/ml. The analysis showed that FBG had a negative correlation with serum CCDC80 (R(b) = -0.223, P = 0.026). Multivariate linear regression had a similar result, with 1 mmol/L increase of serum FBG, serum CCDC80 decreases for 0.24 pg/ml (β = -0.24, 95% CI: -0.21--0.02). There was also a negative correlation between serum CCDC80 and FBG in overweight and obese people (R(a) = -0.368, P = 0.013). Besides, CCDC80 mRNA was detected in both subcutaneous and visceral adipose tissue of 36 cases, the expression level was 0.06±0.02 for subcutaneous fat, was 0.05±0.04 for visceral fat, and the expression in visceral fat was lower (0.05±0.03) than that in subcutaneous fat (0.06±0.03) (t = 2.50, P = 0.025) among overweight and obese group. There was a negative correlation between waist and visceral CCDC80 mRNA expression (r = -0.472, P = 0.035).</p><p><b>CONCLUSION</b>This study suggested that CCDC80 may be involved in energy and insulin metabolism, and plays a protective role in obesity and diabetes.</p>

Study on blocking the leukemia immune escape after BMT by Fas-Fas ligand pathway / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>To investigate if bone marrow transplantation (BMT) with bone marrow mononuclear cells (BMMCs) transducted with murine soluble Fas gene (sFas) using adenovirus vector could block the immune escape of leukemia cells eliminate the residual leukemia cells and reduce their relapse.</p><p><b>METHODS</b>The recombinant adenovirus vector with murine sFas, adsFas, and the control vector adEGFP were constructed using homologous recombination between two plasmids in Escherichia coli. BMT was carried out after the BMMCs were infected with Adenoviruses. The mice models of leukemia/lymphoma were constructed by inoculating female C57BL/6 mice (H-2b) with 10(5) EL4 cells/mouse through caudal vein. Donors of bone marrow grafts were syngeneic male mice. BMMCs were infected with AdsFas or AdEGFP 24 hours before (Group D or E). The following three groups were simultaneously used: Group A, no BMMCs transplanted; Group B, transplanted with BMMCs not infected with adenoviruses; Group C, only transfusing EL4 cells, neither irradiation nor BMT. The hematopoietic reconstitution, generation of leukemia/lymphoma and the survival rate were observed in all groups after BMT.</p><p><b>RESULTS</b>The adenovirus vectors were successfully constructed. The titre of virus after purification was up to 2.5 x 10(11) pfu/ml. Spleen indices examined 11 days after BMT were not obviously different among Group B, D and E (P > 0.05), but indices in Group A were significantly lower than those in the latter three groups (P < 0.01). Counts of leukocytes and platelets on +30 day showed mice were reconstituted satisfactorily in Group B and D, but very low in Group C and E. The Y-chromosomes existed 2 months after BMT and examination of bone marrow cytology showed that Group B and D were almost normal, but Group C and E had plenty of lymphoblast-like tumor cells. Tumors were obviously observed in the mice of Group C and E by histopathological examination, but the mice in Group B and D were normal. The survival rates were 0 (0/4) in Group A, 100% in Group B (6/6) and D (16/16), 12.5% (2/16) in Group C and 6.25% (1/16) in Group E respectively. It is demonstrated that, in contrast with the control (Group EGFP), survival rate was significantly increased in the sFas Group (P < 0.01).</p><p><b>CONCLUSIONS</b>The transfer of sFas gene by adenovirus changed the prognosis state of leukemia/lymphoma mice after auto-BMT. The transduction of sFas might block the effect of the immune escape of EL4 cells through FasL. These results could thus provide a new direction to find a way to treat the leukemia and its recurrence after BMT.</p>

Study on blocking the tumor immune escape by Fas ligand pathway / 中国实验血液学杂志

The expression of Fas ligand (FasL) on the membrane of many kinds of leukemia or solid tumor cells played an important role in the immune escape of tumor cells. This study was aimed to know if the soluble Fas (sFas), expressed by adenovirus, could block the immune escape of tumor cells by FasL pathway. The two recombinant adenoviral vectors, AdsFas with murine soluble Fas gene and AdEGFP with enhanced GFP protein gene, were constructed by homologous recombination between two plasmids in Escherichia coli with the AdEasy adenovirus vector system. The viruses were propagated and purified by two times ultracentrifugation. Their titres were detected by plaque assays. The expressed protein was evaluated by Western blot analysis. Then the tumor EL4 cells were infected with AdsFas and AdEGFP respectively. The apoptosis ratio of the target cells-YAC-1 cells induced by EL4 cells was respectively detected by (3)H-thymidine ((3)H-TdR) labeling. The results showed that the recombinant adenoviral vectors AdsFas and AdEGFP were successfully obtained. The titres of viruses purified by two times ultracentrifugation were up to 10(11) pfu/ml by plaque assays. The sFas protein was highly expressed in the target cells by Western blot analysis. After the EL4 cells were transfected with the adenoviruses AdsFas, the apoptosis rate of YAC-1 cells in the sFas transfection group (respectively 6%, 7% and 9% when the effector:target (E:T) was 3:1, 10:1 and 30:1) was obviously lower than that in the control group (respectively 28%, 37% and 45%), P < 0.01. But when the EL4 cells were transfected with AdEGFP, the apoptosis rate of YAC-1 cells (respectively 30%, 36% and 48%) was similar to the control group, P > 0.05. In conclusion, the transfer of sFas by adenovirus could inhibit the apoptosis of Fas(+) cells-YAC-1 cells induced by tumor EL4 cells. It showed that the transduction of sFas could block the effect of the immune escape of EL4 cells through FasL in vitro. These results thus provide a new direction to find a way to treat tumors.

Retroviral vector-mediated in vitro expression of human soluble fas / 中国实验血液学杂志

Leukemic cells from patients expressed high level FasL cause apoptosis of autologous activated T cells via the Fas/FasL pathway. To investigate the role of soluble Fas (sFas) in reversing this process, a retroviral-mediated expression vector pLXIN-sFas was established. A retroviral-mediated expression system of human sFas was established in vitro and the biological activity of the expression product sFas was observed. To obtain the soluble Fas cDNA, the specific part of the full-length Fas cDNA was deleted by multiple PCR. After pLXIN-sFas packaged by PA317 cells, it was transferred into the target cell COS-7. The quantity of the sFas was (2.2 +/- 0.7) micro g/ml in supernatant of cultured COS-7 cells, and it could greatly inhibit apoptosis of Jurket cells induced by anti-Fas antibody. Our results suggested that the recombinant is able to express the target proteins in vitro and it has the perfect biological activity.

Study on expression and function of fas ligand in human myeloid leukemia cells / 中国实验血液学杂志

To determine whether the Fas receptor-Fas ligand (FasR-FasL) system, which triggers apoptosis in sensitive cells, is an important mechanism of cytotoxicity in myeloid leukemia. FasL expression was investigated in myeloid leukemia cells and its upregulation by a combination of IL-2 and INF-gamma, +/- as well as its function of inducing Jurkat cells to apoptosis mainly by flow cytometry (FCM). Results showed that leukemia cells expressed more FasL (3.59 +/- 1.05)% than that expressed in the healthy individuals (0.36% +/- 0.16)%, P < 0.001 and the FasL was upregulated (7.78 +/- 3.40)%, P < 0.01 when treated with IL-2 and IFN-gamma. Leukemia cells were co-cultivated with Jurkat cells for 24 hours. Then Jurkat cells were labeled with FITC-annexin V and PE-CD3 to assess apoptosis by FCM. The leukemia cells, which had been incubated with IL-2 and IFN-gamma, induced more Jurkat cells to apoptosis than the ones that freshly isolated from the peripheral blood mononuclear cells, which raised the figure from (8.28 +/- 1.61)% to (10.73 +/- 2.16%). And the supernatant derived from the former killed more Jurkat cells than the latter. It was concluded that human myeloid leukemia cells expressed high levels of functional FasL that can kill Jurkat T-cells by apoptosis. FasR-FasL sys tem could play a role in the "immune escape" and relapse of the leukemia. The induction of apoptosis through the Fas pathway might be a novel and effective approach for leukemia immunotherapy.