Objective:To clone the yeast cytosine deaminase(YCD) gene and to elucidate the killing efficacy of yeast cytosine deaminase / 5 - fluorocytosine gene therapy system on gene- transfered cells. Methods:YCD gene was cloned and YCD expression retroviral vector was constructed,the vector was transfered into packaging cell line and high- titer virus was abtained. Then the tum origenic leukemia cell line K5 6 2 B was infected,selected and evaluated. Finally,the killing efficacy of 5 - FC on gene- transfered cell clone observed,and the 5 - FC to 5 - FU conversion of YCD- K5 6 2 B cell lysate was estim ated. Results:YCD gene was cloned and the sequence was confirm ed. A YCD expression retroviral vector,MSCV- YCD- IRES- EGFP was constructed. Transfering it into packaging cell line? NX- A ,high- titer (3.5? 10 6 CFU / ml) virus was obtained. The virus were used to infect tumorigenic leukem ia cell line,K5 6 2 B,and the infecting rate was quite high (30 % ) . A gene- transfered cell clone,YCD- K5 6 2 B,was selected,and YCD gene m RNA expression was detected in it.YCD- K5 6 2 B cell lysate demonstrated CD enzyme activity,and it could deaminase 5 - FC to 5 - FU .MTT assay showed 5 - FC could kill YCD- K5 6 2 B cell in 96 h even at lower concentration(15?mol/ L ) . Conclusion:YCD/ 5 - FC suicide gene therapy system has a significantkilling efficacy on gene- transfered cells. [

Objective To explore methylprednisolone and conventional dose prednisone treatment for hematological damage in systemic lupus erythematosus(SLE) in the near future response. Methods Hemocytopenia in 147 patients with SLE were treated by intravenous injecting methylprednisolone and conventional dose prednisone and therapy response were observed in the tenth day after treatment. Results The responses were obtained in methylprednisolone and in conventional dose prednisone increased percentage of Hb were 34.8% and 14.0%,of WBC were 76.7% and 63.0%,of Pt were 66.7% and 27.3% in two group respectively. In comparison of values of Hb,WBC,and Pt before treatment with those after treatment showed significant difference in two groups(P

Objective: To construct retroviral vector pLDAAOSN and observe the function of DAAO gene. Methods: With recombinant DNA technology, DAAO cDNA was cloned into retroviral vector (pLDAAOSN). The vector was transfected into ?XNA cells by CaPO4 method, and the DAAO cDNA was transferred by recombinant retroviral vector into leukemia cell line K562. The positive clones were obtained after G418 selection and termed K DAAO. PCR and in situ hybridization were used to identify the integration and expression of DAAO gene in K DAAO. In order to observe the function of DAAO, K DAAO was treated with 50 mm/L D-Ala. Results: Results of plasmid pLDAAOSN digested with KpnⅠ and the sequence determination confirmed pLDAAOSN contains full-length DAAO cDNA. Infectious titer generated by the packaging cells was 5.2?10 6 CFU/ml. PCR and in situ hybridization analysis showed integration of DAAO gene in K DAAO and expression of DAAO gene at mRNA level. Preliminary observation suggested that D-Ala could effectively kill K DAAO. Conclusion: Retroviral vector pLDAAOSN may be useful for futher study of gene therapy in cancer.

Objective: To investigate the effect of BSO on DAAO/D-Ala system killing K562e cells. Methods: KDFGC cell stably expressing DAAO was obtained by retrovirus transfection. The integration and expression of DAAO gene in KDFGC cells were identified by PCR and in situ hybridization. The killing effects of D-Ala on KDfGC cells treating with Immol/L BSO were observed. Results: PCR and in situ hybridization analysis confirmed integration of DAAO gene in positive clone and expression of it at mRNA level. The IC50 of KDFGC and KDFGC + BSO treating with D-Ala for 24 hours were 10.21 mmol/L and 7.92 mmol/L, respectively. The 95% confidence limits of them were different. Conclusion: BSO can enhance the killing effect of DAAO/D-Ala system on K652e cells.

Objective: To investigate the clinical treatment results of combined Tretinoin-chemotherapy protocol and kinetics of PML-RAR ? fusion gene in childhood acute promyelocytic leukemia(APL). Methods: Ten children with APL were involved in this study. Induction therapy was Tretinoin alone(6 cases),Tretinoin plus chemotherapy(3 cases) and arsenic trixode(1 case). Postremission therapy consisted of three consolidation courses with DA,MA or HA and a monthly maintenance therapy over 4-5 years. Monitoring of minimal residual disease was performed regularly by RT-PCR assay for PML-RAR ? at differential clinical stages. Results: Clinical complete remission(CR) was obtained in 9 cases (90%).After a median follow-up of 42 months(14-156 months), the estimated 5-year event-free survival was (56? 16.5)%.Four cases relapsed at 14-42 months after achieving CR and 5 cases remained continuing CR. PML-RAR ? fusion gene was positive in all cases at CR and turned negative gradually during consolidation and maintenance treatment. The duration of conversion to RT-PCR negative status varied from 6 to 42 months.Four patients who were persistent positive(2 cases) or converted to positive(2 cases) for PML-RAR ? relapsed. Conclusion: Continuous negative RT-PCR results are associated with long-term disease-free survival and may be considered as potentially curative. RT-PCR assay for detection of PML-RAR ?should be performed regularly during post-remission period. The hematological relapse could potentially be averted through treatment modification according to molecular monitoring results of PML-RAR ?.

OBJECTIVETo elucidate the killing activity of yeast cytosine deaminase/5-fluorocytosine (YCD/5-FC) gene therapy system on gene-transferred tumorigenic cell line K562B in vivo.

METHODK562B cell was infected with high titer virus and a gene transferred cell clone, YCD-K562B, was selected. Twelve male SCID mice of 4 week old were divided into 2 groups at random and both YCD-K562B and K562B cells were implanted to each mice. 5-FC or saline was given i. p for 10 days after tumor developed, and relative tumor volume was measured every 3 days. At the end of experiment, animals were sacrificed and the specimens were processed for histopathological examination.

RESULTSAt the end of experiment (21 days after tumor cell implantation), the relative tumor volume of the 4 groups were: YCD-K562B + 5-FC 2.922 +/- 0.581, YCD-K562B + saline 24.434 +/- 4.790, K562B + 5-FC 22.701 +/- 2.350 and K562B + saline 24.460 +/- 1.670; t-test analysis showed that 5-FC could kill cells (YCD-K562B) in vivo (P = 0.0001), but had no effect on the growth of gene-untransferred cells (K562B) (P = 0.096). In YCD-K562B + 5-FC group, relative tumor volume reduced in 3 approximately 6 days after treatment (the minimum was 0.681). Necrosis around artery could be found in the tumor of YCD-K562B + 5-FC group.

CONCLUSIONYCD/5-FC suicide gene therapy system has a significant in vivo killing activity to gene-transferred tumorigenic YCD-K562B cell.

Animals ; Cytosine Deaminase ; Flucytosine ; metabolism ; pharmacology ; Genetic Therapy ; methods ; Humans ; K562 Cells ; Male ; Mice ; Mice, SCID ; Neoplasm Transplantation ; Neoplasms, Experimental ; genetics ; therapy ; Nucleoside Deaminases ; genetics ; metabolism ; Saccharomyces cerevisiae ; enzymology ; Transfection ; Treatment Outcome ; Xenograft Model Antitumor Assays

Objective To evaluate the diagnostic and therapeutic significance of serum free light chain (sFLC) in primary systemic(AL) amyloidosis. Methods Twenty-five patients with AL amyloidosis,including 18 men and 7 women with a mean age of 54(47-77) years old, were enrolled from October, 2005to May, 2010. sFLC was measured by immunoturbidimetric assay. The type of monoclonal light chain was judged upon sFLC κ/λ and its sensibility was compared with serum immunofixation and immunohistochemical analysis. Four patients were treated with M (T)D (melphalan/thalidomideand, dexamethasone), one with VD (velcade and dexamethasone) and four with high-dose melphalan followed by autologous stem cell support. The changes of sFLC were serially determined before and after treatment. Results Among the 25 patients with AL amyloidosis, two were κ light chains of precursor protein and 23 were λ light chains. Mean plasma cell in bone marrow was 3.5% (0-15%). Nineteen (76%) patients had abnormal elevated sFLC and abnormal κ/λ ratios, and 17(68% ) patients with immunofixation positive. The sFLC test had similar sensitivity as serum immunofixation (P = 0. 727 ). Twenty-one (84%) patients were shown to have either κor λ immunoreactive amyloid deposits on biopsied tissues. The sFLC test combined with serum immunofixation allowed the M protein to be detected in 22 (88%) patients. The positive rates of immunohistochemical analysis combined with sFLC test and/or serum immunofixation were 96%. Four patients with hematologic response showed obvious improvement in visceral organ involvement, but illness of 5 patients without hematologic response kept stable or progressed. Conclusions sFLC test is a sensitive qualitative and quantitative method to detect M protein. Preliminary data show the patients with obvious sFLC level decrease and/or κ/λ recovery to normal may have a high percentage of improved organs function. sFLC is critical index in diagnosing AL amyloidosis, which might help efficacy assessment.

OBJECTIVETo explore the feature of primary light chain amyloidosis patients treated with high-dose melphalan with auto peripheral blood stem cell transplantation (auto-PBSCT) and bortezomib plus dexamethasone (VD).

METHODSThirty-eight patients diagnosed from September 2004 to September 2012 were analyzed retrospectively, including 15 cases received auto-PBSCT, 23 cases exposed with VD.

RESULTSThe median follow-up duration for the patients was 34 months (range, 1-112 months), including auto-PBSCT group of 38 months (range, 5-112 months) and VD group of 31 months (range, 1-108 months). The organ response rate in all the patients was 39.5% (15/38), and the organ response rate between these two groups has no significant difference [33.3% (5/15) vs 43.5% (10/23), P=0.532]. However, the median time of organ response was significant difference [6 (3-10) months vs 3 (1-6) months, respectively (P=0.032)]. The 3-year overall survival (OS) rates in the two groups were 72.0% and 66.9%, and their average survival were 84.7 months and 75.9 months, respectively (P=0.683). In the patients with auto-PBSCT, the occurrence of III-IV grade of bone marrow suppression (P<0.001), fever (P<0.001), nausea and infection (P=0.006) were obviously higher than those with VD, but there was no statistically significant difference in pulmonary infection (P=0.069) and bloodstream infection (P=0.059).

CONCLUSIONSThe preliminary results have presented that primary light chain amyloidosis patients treated with auto-PBSCT or VD had similar organ response rate and survival. However, more adverse events occurred in the group of auto-PBSCT.

Amyloidosis ; therapy ; Bortezomib ; therapeutic use ; Dexamethasone ; therapeutic use ; Humans ; Immunoglobulin Light-chain Amyloidosis ; Melphalan ; therapeutic use ; Myeloablative Agonists ; therapeutic use ; Peripheral Blood Stem Cell Transplantation ; Retrospective Studies

OBJECTIVETo investigate the in vitro killing efficiency of D-amino acid oxidase (DAAO)/D-alanine (D-Ala) system on K562e cells.

METHODSK(DfGC) cell line stably expressing DAAO was obtained by retrovirus transfection technique. The integration and expression of DAAO gene were identified by PCR and in situ hybridization. The killing activities of D-Ala to DAAO(+) cells alone or the mixtures of DAAO(+) and DAAO(-) cells in different ratios were observed. H(2)O(2) production by K(DfGC) cell was measured by phenol red oxidation assay.

RESULTSPCR and in situ hybridization analysis confirmed the integration of DAAO gene in positive clone and its mRNA expression. There was no significant difference in cell proliferation between the two kinds of K562 cells. Ninety percent of K(DfGC) cells was killed by 12.5 mmol/L D-Ala after 24 hour-treatment and the H(2)O(2) levels were in accord with the killing activities of D-Ala. When K(DfGC) was mixed with K562e at different ratio, no significant bystander effect could be found after treating with 15.0 mmol/L D-Ala for 24 hours.

CONCLUSIONThe leukemia cell line K562e was sensitive to DAAO/D-Ala system and there was no significant bystander effects observed within this cells.

Alanine ; metabolism ; pharmacology ; Cell Survival ; drug effects ; D-Amino-Acid Oxidase ; genetics ; metabolism ; Gene Expression ; Humans ; Hydrogen Peroxide ; metabolism ; K562 Cells ; cytology ; drug effects ; metabolism ; Plasmids ; genetics ; RNA, Messenger ; genetics ; metabolism ; Transfection

Objective:To identify clinical features,possible risk factors and treatment related to rituximab-associated interstitial pneumonia(RTX-IP). Methods:The clinicopathological characteristics,immune phenotype and treatment of six patients with diffuse large B-cell lymphoma(DLBCL)develped after receiving R-CDOP treatment were retrospectively analyzed. Results:Six patients had agranulocytosis or granulocytopenia within 1 week before RTX-IP diagnosis,and the median interval time was 3 courses of treatment.All six patients had double-expressor lymphoma(DEL)or triple-expressor lymphoma(TEL).Of the six patients,four had germinal-center B-cell-like lymphoma(GCB),and two had non-GCB.The expression of Ki-67 was＞70％,except for one patient with transformed lymphoma(TL).After treatment with methylprednisolone for about 1 week,all patients'chest CT showed inflammatory absorption.However,one patient developed pneumocystis carinii pneumonia during the process of hormone reduction,and recovered after 27 days of comprehensive treatment with hormones and anti-pneumocystosis therapy.All patients received CDOP regimen(a total of 8 courses)for the treatment of the primary disease,and the process was smooth. Conclusion:R-CDOP regimen may lead to a high incidence of RTX-IP in DLBCL patients(30.0％).The DLBCL patients with DEL or TEL,GCB subtype,TL and high Ki-67 expression were more liable to develop RTX-IP,and the recovery of agranulocytosis may be related to the pathogenesis of RTX-IP.High-resolution CT scan can provide valuable evidence for early diagnosis of RTX-IP.Metagenomic next-generation sequencing(mNGS)helps to distinguish IP from pathogen infections.High dose of glucocorticoids is effective treatment strategy.At the same time,it is necessary to strengthen the prevention and treatment of infection in the process of glucocorticoids application.