Proteomic study of Tianxiangdan intervention in rats with myocardial ischemia.

The present study aimed to screen out the proteins with significantly differential expression through the proteomics study of Tianxiangdan intervention in rats with myocardial ischemia as well as elucidate the mechanism of the intervention. In total, 54 Wistar rats (male, 6-8 weeks old) were randomly divided into the blank group, sham operation group, and model group, with 6 rats in each, together with the model + low dose group, model + medium-dose group, and model + high dose group, with 12 rats in each. Upon successful construction of the ischemic model, low, medium, and high doses, respectively, of Tianxiangdan were administered in the groups. The rat model of coronary heart disease (CHD) with myocardial ischemia was prepared by ligating the coronary artery. The tandem mass tag-labeled quantitative proteomics technology was adopted to observe the differentially expressed proteins in the myocardium of the model rats under the action of Tianxiangdan to find the target proteins for the treatment of myocardial ischemia in CHD. A total of 3122 proteins were identified. Combined with the references, tropomyosin alpha-3 chain (TPM3), protein kinase C delta (PRKCD), myosin heavy chain 10 (MYH10), MYH6, G protein subunit alpha i2 (GNAI2), and other proteins were screened out. Western blotting was adopted for the proteomics validation, and it was found that compared with the sham operation group, the expression levels of the GNAI2, TPM3, and MYH10 proteins were upregulated in the myocardial ischemia model group but downregulated after the administration of Tianxiangdan; the differences were statistically significant (p<0.05). We conclude that Tianxiangdan could improve myocardial ischemia by downregulating the proteins, including GNAI2, TPM3, and MYH10, which might be potential targets of Tianxiangdan in the treatment of myocardial infarction.

[Development and functional verification of CAR-T cells targeting CLL-1].

Objective: To explore the development of a CAR-T cells targeting CLL-1 and verify its function. Methods: The expression levels of CLL-1 targets in cell lines and primary cells were detected by flow cytometry. A CLL-1 CAR vector was constructed, and the corresponding lentivirus was prepared. After infection and activation of T cells, CAR-T cells targeting CLL-1 were produced and their function was verified in vitro and in vivo. Results: CLL-1 was expressed in acute myeloid leukemia (AML) cell lines and primary AML cells. The transduction rate of the prepared CAR T cells was 77.82%. In AML cell lines and AML primary cells, CLL-1-targeting CAR-T cells significantly and specifically killed CLL-1-expressing cells. Compared to untransduced T cells, CAR-T cells killed target cells and secreted inflammatory cytokines, such as interleukin-6 and interferon-γ, at significantly higher levels (P<0.001) . In an in vivo human xenograft mouse model of AML, CLL-1 CAR-T cells also exhibited potent antileukemic activity and induced prolonged mouse survival compared with untransduced T cells [not reached vs 22 days (95%CI 19-24 days) , P=0.002]. Conclusion: CAR-T cells targeting CLL-1 have been successfully produced and have excellent functions.

[Analysis on poor efficacy factors in the treatment of recurrent/refractory B-cell lymphoma with CD19 CAR-T cells].

Objective: To investigate the factors influencing the efficacy of CD19 chimeric antigen receptor T (CAR-T) cells in the treatment of patients with relapsed refractory B cell lymphoma and to provide evidence for further improvement of CAR-T efficacy. Methods: A total of 34 patients with relapsed and refractory B-cell lymphoma were recruited from the Department of Hematology of Tianjin First Central Hospital from February 2017 to January 2019. All patients received CD19 CAR-T cell therapy. These patients were evaluated for efficacy, factors with poor efficacyand adverse effects. Results: The overall response rate was 58.8% (20/34) and the complete remission rate was 41.2% (14/34) after infusion of CD19 CAR-T cells in 34 patients with relapsed refractory B cell lymphoma. According to the efficacy of CAR-T cells, patients were divided into two groups, 20 in the effective group and 14 in the poorly effective group. The median am ount of CD19 CAR-T cell infusions in these two groups were 8.6 (5.0-12.7)×10(6)/kg and 9.7 (5.8-15.0) × 10(6)/kg, respectively, and the difference was not statistically significant (P=0.654). The percentage of CD19 CAR-T cells in the effective group and the poorly treated group was 10.28% (3.92%-44.16%) and 4.05% (0.92%-28.63%), respectively.The effective group had a higher proportion of CAR-T cells than the poorly treated group, but the difference was not statistically significant (P=0.371).The presence of massive mass was an unfavorable factor affecting the efficacy of CD19 CAR-T cells and the difference was statistically significant (P=0.001). Logistic regression multivariate analysis showed that the characteristics of massive tumors were still independent prognostic factors for poor efficacy of CD19 CAR-T cells (P=0.005, OR=0.039). Of all 34 patients, there were 70.6% (24/34) who showed varying degrees of adverse reactions after the infusion of CD19 CAR-T cells, mainly cytokines release syndrome (CRS). The median time of occurrence of fever was on the third day after infusion (0-11th) day. 16 patients were with grade 1 CRS, 7 with grade 2, and 1 with grade 3. After glucocorticoids and support treatment, they all showed improvements. Conclusions: CD19 CAR-T cell therapy has achieved a certain effect in CD19(+)B cell lymphoma, but has poor efficacy on some patients. Large mass tumors may be an adverse factors to CAR-T cell treatment.

[The value of rapid on-site evaluation (ROSE) of bronchoscopy in the diagnosis of pulmonary complications after allogeneic hematopoietic stem cell transplantation].

Objective: To investigate the value of rapid on-site evaluation (ROSE) of bronchoscopy in the diagnosis of pulmonary complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods: A retrospective analysis was conducted on the diagnosis and treatment process before and after ROSE examination of 12 patients with pulmonary complications after allo-HSCT who were admitted to the Department of hematology, Tianjin First Central Hospital from January 2017 to June 2019. The diagnostic accuracy of the ROSE was evaluated by comparing the initial diagnosis of ROSE with the final clinical diagnosis. At the same time, the safety of ROSE examination was evaluated and two typical cases were shared. Results: In the 12 transbronchial lung biopsies, there were 5 cases of organizing pneumonia, 3 cases of bronchiolitis obliterans with organizing pneumonia, 1 case of pulmonary fibrosis, 1 case of acute fibrinous and organizing pneumonia, 1 case of pseudomembranous tracheobronchial aspergillosis and 1 case of uncertain diagnosis evaluated by ROSE. Compared with the final clinical diagnosis, there were 10 cases of accurate diagnosis made by ROSE (10/12). All patients were well tolerant to the operation of bronchoscopy. There was only a small amount of bleeding observed during the operation, without pneumothorax and hemoptysis. And no complications related to ROSE occurred. According to the initial diagnosis of ROSE, 10 cases of non-infectious pulmonary complications were treated with methylprednisolone or other immunosuppressive agents and 1 case of Aspergillus infection was given antifungal therapy. Seven patients with non-infectious pulmonary complications improved after treatment. One patient obtained uncertain diagnosis by ROSE was later diagnosed with virus infection by next generation sequencing technology and improved after treatment with foscarnet sodium and immunoglobulin. As of June 30, 2019, 7 patients improved and 5 died. Conclusion: ROSE has the advantages of diagnostic accuracy and rapidity, and is very suitable for patients with critical illness after hematopoietic stem cell transplantation, who are in urgent need of definite diagnosis and prompt treatment, which is beneficial to improve the prognosis of patients.

[Mucormycosis in patients with hematological diseases: seven cases reports and literature review].

Objective: To explore the clinical characteristics, diagnosis, treatment and prognosis of patients with hematological diseases that complicated by mucormycosis, and to improve the understanding and clinical diagnosis and treatment of the disease. Methods: The clinical data of 7 patients suffering from mucormycosis during September 2012 and September 2018 were retrospectively analyzed, and their clinical characteristics, treatment process and prognosis were analyzed. Results: Of 7 patients, there were 4 males and 3 females, with a median age of 36 (19-79) years old. Two patients were diagnosed as acute myeloid leukemia as the underlying disease, the other 5 patients suffered from acute B lymphoblastic leukemia, peripheral T cell lymphoma, chronic myelocytic leukemia in blastic phase, myeloproliferative neoplasm and severe aplastic anemia after transplantation, respectively. Among them, disease types of mucormycosis were pulmonary in 4 patients, rhino-orbital-cerebral in 1 patient, cutaneous in 1 patient and disseminated in 1 patient. All the cases were confirmed by biopsy histopathology. The treatment drugs were amphotericin B or liposomal amphotericin B, and posaconazole. Surgical treatment was performed in 4 patients, 3 out of 4 achieved radical debridement, and the other one had local debridement. Two patients were cured, 1 patient was improved and 4 patients died. Conclusions: The clinical manifestation and image feature of mucormycosis in patients with hematological diseases were diverse, and the mortality rate is high, diagnosis mainly depends on histopathology. Early diagnosis, control of underlying disease, improvement of immunosuppressive status, timely effective antifungal therapy and radical surgical debridement are the key points for improving the survival rate of patients with hematological diseases complicated by mucormycosis.

[Relationship between cervical lesions and the type-specific viral load of high risk HPV reflected by the Ct value of Cobas 4800 HPV system].

Objective: To explore the relationship between cervical lesions and high risk HPV (HR-HPV) viral load reflected by the cycle threshold (Ct) values of Cobas 4800 HPV (Cobas 4800) system. Methods: From August 2016 to September 2017, 7 000 women from Shenzhen, were recruited for cervical cancer screening with Cobas 4800 system and cytology co-test. Colposcope biopsies were performed on women who were positive of HPV 16, 18, and positive of HPV types other than 16,18 with cytology [≥ atypical squamous cell of undetermined signification (ASCUS)], or HPV negative but abnormal of cytology [≥ low grade squamous intraepithelial lesion (LSIL)]. The Ct values of HPV 16, 18 and all combined other types coming from Cobas 4800 system were used as an indicator of viral load to analyze the relationship between type-specific HPV load and the cervical lesions. Results: (1) Among the 7 000 screening women, 370 cases were positive for cervical cancer screening, 325 of them underwent colposcope biopsies, and coloposcopy referred rate was 87.8% (325/370). Among 325 women undergoing cervical biopsy, pathological diagnosis was 119 cases of normal cervical cervix, 151 cases of LSIL, and 55 cases of high-grade squamous intraepithelial lesion (HSIL) and above (HSIL(+); including 53 cases of HSIL, 1 case of cervical adenocarcinoma, and 1 case of cervical squamous cell carcinoma). (2) The Ct value of HPV 16 was inversely correlated with the upgrading of the lesions (r=-0.617, P=0.000), and significant different among normal cervix,LSIL and HSIL(+) (35.4±4.5 vs 31.0±6.0 vs 26.5±4.0; F=25.537, P=0.000). There was no correlation between Ct value of HPV 18 and cervical lesions (r=-0.021, P=0.902). The Ct value of other 12 HPV types was statistically difference among normal normal cervix, HSIL(+) and cervicitis (33.0±5.3 vs 29.9±7.2 vs 29.8±5.8; F=5.087, P=0.007). Among them, LSIL and HSIL(+) were significantly lower than normal cervix (P<0.05), but there was no significant difference between LSIL and HSIL(+) (P>0.05). Conclusion: The Ct value of HPV 16 detecting in Cobas 4800 system as an indicator of virus load obviously correlates with different grades of cervical lesions, therefore could be a reference of cervical lesion existence and an indicator of lesion prognosis.

[Efficacy and safety of CD19 chimeric antigen receptor T cells for the treatment of 22 patients with B-cell lymphoma].

Objective: To investigate the efficacy and safety of CD19 chimeric antigen receptor T (CAR-T) lymphocytes for the treatment of B cell lymphoma. Methods: A total of 22 patients with B-cell lymphoma from February 1, 2017 to July 1, 2018 were reviewed to evaluate the efficacy and adverse reactions of CD19 CAR-T. Results: Of 22 patients with B-cell lymphoma received CD19 CAR-T cells, the median dose of CAR-T cells was 7.2 (2.0-12.0) ×106/kg. Nine of 12 cases of relapse refractory patients were overall response. Complete remission (CR) occurred in 2 of 12 patients, partial remission (PR) in 7 of 12 patients. The overall response in minor residual disease positive (MRD) group was 8 of 10 patients. CD19 CAR-T cells proliferated in vivo and were detectable in the blood of patients. The peak timepoints of CAR-T cells proliferated in the relapsed refractory and MRD positive groups were 12 (5-19) and 4.5 (1-12) days after treatment respectively, and among peripheral blood cells, CAR-T cells accounted for 10.10% (3.55%-24.74%) and 4.02% (2.23%-28.60%) of T lymphocytes respectively. The MRD positive patients achieved sustained remissions during a median follow-up of 8 months (rang 3-18 months) . None of all the patients relapsed during a median follow-up time of 10 months (3-18 months) . However, 7 PR responders of the relapsed refractory patients maintained a good condition for 1.5-6.0 months. One patient bridged to hematopoietic stem cell transplantation, another one sustained remission for 12 months. Cytokine-release syndrome (CRS) occurred in 14 patients with grade 1-2 CRS in MRD positive group and grade 3 CRS in relapsed refractory group. Conclusions: CAR-T cell therapy not only played a role in the rescue treatment of relapsed and refractory patients, but also produced a surprising effect in the consolidation and maintenance of B-cell lymphoma. CD19 CAR-T cells might be more effective in the treatment of MRD positive B-cell lymphoma patients than in the refractory or relapsed cases. High response rate was observed with fewer adverse reactions.

[The usage of comprehensive geriatric assessment in elderly patients with acute myeloid leukemia: a multicenter, prospective study].

Objective: To evaluate the feasibility and potential value of comprehensive geriatric assessment (CGA) in elderly (≥60 years) patients with newly diagnosed acute myeloid leukemia (AML) in China. Methods: The CGA results of 83 newly diagnosed AML (non-APL) patients from 16 hospitals in Beijing and Tianjin between March 2016 and December 2017 were prospectively collected and analyzed. The clinical data, treatment and follow-up information were also collected. Results: Of 83 newly diagnosed elderly AML patients, 81 patients (97.6%) completed all designated CGA assessment. The median number of impaired scales of the CGA assessment in the studied population was 2(0-6). Sixteen patients (19.3%) showed no impairments according to the geriatric assessment scales implem ented by this study. The distributions of impaired scales were as follows: impairment in ADL, 55.4%; IADL impairment, 42.2%; MNA-SF impairment, 48.2%; cognitive impairment, 15.7%; GDS impairment, 31.7%; HCT-CI impairment, 19.5%, respectively. In patients with "good" ECOG (n=46), the proportion of impairment for each CGA scale ranged from 6.5% to 37.0% and 32 patients (68.9%) had at least one impaired CGA scale. Survival analysis showed that the number of impaired scales of the CGA was significantly correlated with median overall survival (P=0.050). Conclusions: CGA was a tool with feasibility for the comprehensive evaluation in elderly AML patients in China. Combined with age and ECOG, CGA may be more comprehensive in assessing patients' physical condition.

[Effect of PD-1 inhibitor Nivolumab on the proliferation and cytotoxicity of anti-CD19 chimeric antigen receptor T cells].

Objective: To Evaluation the effect of PD-1 inhibitor Nivolumab on the proliferation and cytotoxicity of anti-CD19 chimeric antigen receptor T cells (CD19-CAR-T) in vitro. Methods: Five patients with high PD-1 expression in peripheral blood and five healthy volunteers were selected. These peripheral blood mononuclear cells were used as the source of T cells to prepare CD19-CAR-T cells. Different doses (72, 36, 18 µg/ml) of Nivolumab was added on day 8 to the culture medium. Patient T cells incubated with 72 µg/ml Nivolumab and CD19-CAR-T cells of healthy volunteers were used as controls. CCK-8, lactate dehydrogenase (LDH) cytotoxicity assay and ELASA were used to detect the proliferation capacity, the specific cytotoxicity and the inflammatory factor secretion. Results: ①T cells from patients with high expression of PD-1 as the source of CD19-CAR-T cells did not affect transfection rate compared with that of healthy volunteers [(32.80±7.22)% vs (35.10±5.84)%, t=-0.554, P=0.593]. ②Incubation of CD19-CAR-T cells with 72 µg/ml Nivolumab did not affect CD19-CAR-T cell proliferation, but its cytotoxicity was significantly higher than that of CD19-CAR-T cells alone or patients' T cells +72 µg/ml Nivolumab (all P<0.001), there was no significant difference in the killing activity between the 72 µg/ml and 36 µg/ml Nivolumab treated CD19-CAR-T cells on Pfeiffer cells (P=0.281, 0.267, respectively), and they were all higher than those of 18 µg/ml Nivolumab treated CD19-CAR-T cells (all P<0.001). ③Different doses of PD-1 inhibitor Nivolumab combined with CD19-CAR-T cells does not affect the secretion of IFN-γ and IFN-α (all P>0.05). Conclusion: Combination of 36 µg/ml PD-1 inhibitor and CD19-CAR-T cells could reduce the drug toxicity and enhance the cytotoxicity.

[The specific cytotoxicities of chimeric antigen receptor-engineered T cells on different lymphomas].

Objective: To investigate the specific cytotoxicities of the second and third generations of chimeric antigen receptor (CAR) -engineered T cells (CAR-T) on different lymphomas. Methods: CAR-Ts were prepared by lentivirus packaging and infection of T cells. CCK-8, ELISA and Lactate dehydrogenase cytotoxicity assay were applied to detect the proliferation capacity, the secretion level of inflammatory factor and the specific cytotoxicity. Flow cytometry assay showed the specific cytotoxicity and residual level of CAR-T in lymphomas of treated mice. Results: The results showed that the third generation CAR-T had greater capacity of the specific cytotoxicity and proliferation capacity than of the second generation CAR-T. But there was no prominent change of the secretion level of inflammatory factor. The specific cytotoxicity of the second generation CAR-T on highly aggressive lymphomas Raji was more prominent than in inert EHEB, but also could achieve satisfactory effect. The tumor burden in the mice injected with Raji was lower than in the mice injected with EHEB from nude mice experiment. But the residual level of CAR-T in the EHEB-injected mice was higher than in the Raji-injected ones. So the second generation CAR-T was more suitable for the treatment of indolent lymphoma. Conclusion: The second generation CD19 CAR-T could treat aggressive lymphoma in a relatively short period, while the second generation CD19 CAR-T need a longer time in vivo to achieve satisfactory curative effect on the noble lymphoma.

Protective effects of deferasirox and N-acetyl-L-cysteine on iron overload-injured bone marrow.

Using an iron overload mouse model, we explored the protective effect of deferasirox (DFX) and N-acetyl-L-cysteine (NAC) on injured bone marrow hematopoietic stem/progenitor cells (HSPC) induced by iron overload. Mice were intraperitoneally injected with 25 mg iron dextran every 3 days for 4 weeks to establish an iron overload (Fe) model. DFX or NAC were co-administered with iron dextran in two groups of mice (Fe+DFX and Fe+NAC), and the function of HSPCs was then examined. Iron overload markedly decreased the number of murine HSPCs in bone marrow. Subsequent colony-forming cell assays showed that iron overload also decreased the colony forming capacity of HSPCs, the effect of which could be reversed by DFX and NAC. The bone marrow hematopoiesis damage caused by iron overload could be alleviated by DFX and NAC.

[Establishment of macrophage model of iron overload in vitro and the injury induced by oxidative stress on macrophage with iron overload].

OBJECTIVE: To establish macrophage iron overload model in vitro by co-culture macrophages with iron, and to explore the effect of iron overload on cell reactive oxygen species (ROS) and the impact of ROS on macrophages. METHOD: Iron overload group were treated with different concentrations (0, 5, 10, 20, 40, 80 µmol/L respectively) of ferric ammonium citrate (FAC). The control group was the group of macrophages without FAC treatment. We detected the number and state of cells, metabolic activity, the change of phagocytosis, the levels of ROS and reactive nitrogen, and changes of related oxidative stress signaling pathways in different groups. Changes in the above indexes were detected after application of deferasirox (DFX) to remove iron and the antioxidant N -acetylcysteine (NAC) to clear excess oxidative stress. RESULTS: (1)The levels of labile iron pool (LIP) in macrophages co-cultivated with iron was increased with the increase of iron concentration in a dose-dependent manner. The LIP levels was the highest in the macrophages treated with 80 µmol/L. (2)The increase of FAC concentration, the metabolic activity of macrophages in the 5 FAC-treated groups decreased to 51.58%, 40.98%, 16.23%, 3.46%, and 0.05% of the activity level of the control group (all P< 0.05). The group with the metabolic activity decreased to 16.23% (20 µmol/L) was selected as the iron overload group for the following experiments. (3)Compared with the control group, the number of macrophages in the iron overload group reduced to 32.80% (P<0.05), and the state of cells changed from adherence to partial suspension. The phagocytosis of macrophages in the iron overload group reduced to 20.40% of the control group (P<0.05). (4)Our further experiment showed that the levels of ROS and the activity nitrogen in the iron overload group increased by 7.71-and 1.45-fold compared with the control group (both P<0.05). The RT-PCR showed up-regulated mRNA expression of genes related with ROS production, i. e. nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX 4) gene related with ROS production and inducible nitric oxide synthase (iNOS) gene related with reactive nitrogen production, down-regulated mRNA expression of glutathione peroxidase 1 (GPX1) gene which participated in ROS clearance. Moreover, mRNA expression of phosphatidylinositol-3-kinase (PI3K) gene involved in oxidative stress signaling pathway in the iron overload group was up-regulated, while fork head protein O3 (FOXO3) which regulated oxidative stress through negative feedback showed a down-regulation level of mRNA expression compared with the control group. (5)After iron chelation and antioxidant treatment, the above-mentioned damage in the iron overload group were partially reversed. CONCLUSIONS: The damages of iron overload on macrophages may be mediated by inducing oxidative stress and activating oxidative stress signaling pathways. Our established model provides a method to explore the mechanism of iron overload on macrophage, and may shed some new light on possible therapeutic target in treating iron overload patients.

Pemetrexed versus gefitinib as a second-line treatment in advanced nonsquamous nonsmall-cell lung cancer patients harboring wild-type EGFR (CTONG0806): a multicenter randomized trial.

BACKGROUND: CTONG0806 assessed the efficacy of pemetrexed versus gefitinib as second-line treatment in advanced nonsquamous nonsmall-cell lung cancer (NSCLC) harboring wild-type epidermal growth factor receptor (EGFR). PATIENTS AND METHODS: Patients with locally advanced or metastatic nonsquamous NSCLC harboring wild-type EGFR, detected by direct sequencing, and previously treated with platinum-based chemotherapy were randomized to receive gefitinib (250 mg/day) orally or pemetrexed (500 mg/m(2)) i.v. on day 1 of a 21-day cycle until disease progression or unacceptable toxicity. The primary end point was progression-free survival (PFS). The Independent Review Committee (IRC) evaluated all pictorial data. RESULTS: From February 2009 to August 2012, 161 patients were enrolled, and 157 were assessable (81 in the gefitinib arm, 76 in the pemetrexed arm). Baseline characteristics were balanced between the two arms. The median PFSs were 4.8 versus 1.6 months in the pemetrexed and gefitinib arms, respectively [hazard ratio (HR) 0.54, 95% confidence interval (CI) 0.40-0.75, P < 0.001] as confirmed by IRC evaluation (5.6versus 1.7 months, HR 0.53, 95% CI 0.38-0.75, P < 0.001). The median overall survival (OS) showed a trend of superiority in the pemetrexed arm (12.4 versus 9.6 months, HR 0.72, 95% CI 0.49-1.04, P = 0.077). Quality-of-life assessment showed no marked difference between the arms. No unexpected adverse events were found. Of 108 patients with sufficient DNA samples, EGFR mutation status was re-tested by Scorpion amplification refractory mutation system (ARMS); 32 (29.6%) tested positive (19 in the pemetrexed arm, 13 in the gefitinib arm; median PFS: 8.1 versus 7.0 months, HR 0.94, 95% CI 0.43-2.08, P = 0.877). CONCLUSIONS: CTONG0806 is the first trial to show significant improvement in PFS and an improved OS trend with pemetrexed compared with gefitinib as second-line setting treatment of EGFR wild-type advanced nonsquamous NSCLC. ARMS is superior to direct sequencing in excluding false-negative patients. CLINICALTRIALSGOV IDENTIFIER: NCT00891579.

Gastric cancer exosomes promote tumour cell proliferation through PI3K/Akt and MAPK/ERK activation.

BACKGROUND: Exosomes are nanometer-sized vesicles that are released by normal and neoplastic cells. Previous studies have focused on the interaction between tumour-derived exosomes and the immune system, as a consequence of immune suppression or enhancement. However, the effects of tumour-derived exosomes on tumour cells themselves have not been well studied. AIMS: To investigate the effects of gastric cancer exosomes on tumour cell proliferation and the possible mechanisms. METHODS: By serial centrifugation and sucrose gradient ultracentrifugation, we isolated and purified the exosomes from gastric cancer SGC7901 cells, then viewed them by electron microscopy. Cell proliferation was measured by 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide assay. Protein expression was assayed by Western blotting. RESULTS: SGC7901-cell-derived exosomes promoted the proliferation of SGC7901 and BGC823 cells. The increase in proliferation induced by exosomes was accompanied by activation of Akt and extracellular-regulated protein kinase, and phosphoinositide 3-kinase or extracellular-regulated protein kinase inhibitor partially reversed the proliferative effect of exosomes. Moreover, the exosome-induced increase in activity of Akt and extracellular-regulated protein kinase coincided with decreased expression of the Casitas B-lineage lymphoma family of ubiquitin ligases. CONCLUSION: Gastric cancer exosomes promoted tumour cell proliferation, at least in part, by activation of PI3K/Akt and mitogen-activated protein kinase/extracellular-regulated protein kinase pathways. The decreased expression of Casitas B-lineage lymphoma proteins might have contributed to the activation of Akt and extracellular-regulated protein kinase.

A new potyvirus from tuberose ( Polianthes tuberosa) in China.

Tuberose plants with mild mottle symptoms, growing in a glasshouse in Hangzhou, China, contained virions and inclusion bodies typical of a potyvirus. The virus was mechanically transmitted to tuberose but not to 14 other test plant species. A fragment of 4607 nucleotides, corresponding to the 3'-half of a typical potyvirus was amplified by RT-PCR using degenerate primers and sequenced. The most similar sequence in the databases was that of Tuberose mild mosaic virus (TuMMV) from Taiwan and this was the only virus significantly related to it in phylogenetic analyses. The new sequence had 71.1% nt and 76.6% aa identity to TuMMV in the coat protein. Western blot analyses using antisera raised to expressed coat protein showed that the two viruses were serologically related. Although there are no substantial biological data to distinguish the Hangzhou isolate from TuMMV, the molecular difference between the two virus isolates is similar to, or slightly greater than, that between several pairs of well-established potyvirus species. These results therefore suggest that the Hangzhou isolate should be regarded as a new member of the genus Potyvirus, and we have tentatively named it Tuberose mild mottle virus.