bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Construction and Expression of Recombination Adenovirus Vector of G156A O6-methylguanine-DNA-methyltransferase.
Authors: Wang JiShi , Yang Yuan , Jiang XueJie , Li DongBo , Fang Qin 
Published: October 2019

Abstract
OBJECTIVE: To transfect G156A mutant MGMT gene into peripheral blood mononuclear cells(PBMCs), peripheral blood CD34+ cells, umbilical cord blood CD34+ cells and K562 cells through recombinant adenovirus vector and detect the expression of ΔMGMT in these cells, to study the protective effect of the ΔMGMT gene for bone marrow cells against alkylating agent.
METHODS: We obtained a full-length cDNA fragment encoding human O6-methylguanine-DNA-methytransferase (MGMT) gene from human liver tissue by RT-PCR. ΔMGMT cDNA was made by site-directed mutagenesis. Positive clones were identified by diagnostic restriction analysis and sequenced to confirm the presence of the desired mutation. ΔMGMT cDNA was released by digestion with XhoI and BamHI, purified and then ligated into adenovirus expression vector pAd5CMV-NpA. The recombinant adenovirus vectors pAd5CMV-ΔMGMT and pAd5CMV-EGFP were constructed and transfected into the packing cell lines HEK293 cells by liposome encapsulation method. The recombination adenvirus were propagated, purified, and titrated to 1x1012pfu/mL. PBMCs, peripheral blood CD34+ cells, umbilical cord blood CD34+ cells and K562 cells were incubated with a mixture containing adenovirus. Fluorescence microscopy was employed to test the transfection of recombinant adenovirus vectors.The expression of ΔMGMT gene was detected by Real-time PCR and Western Blot and contrasted to the cells which were transfected into the recombinant adenovirus including EGFP. 3-(4,5-dimethyhhiazol-2-yl)-2,5-dipheyhetrazolium bromide (MTT) and CFU-assays were used to detect the drug-resistance of the ΔMGMT transfected cells.
RESULTS: MGMT gene was cloned successfully and the mutant G156A MGMT genes were obtained. High titer recombinant adenovirus had been made. Real time-PCR showed that mutant drug resistance gene expressed strongly. Western-Blot verified that cells infected with Adv-EGFP displayed EGFP immunoreactivity and basal levels of MGMT immunoreactivity; in contrast, cells infected with Adv-ΔMGMT showed a strong ΔMGMT immunoreactivity. Fluorescence microscopy showed EGFP positive in control group. The mutant drug resistance gene target cells conferred 12.4 ∼ 18.6-fold stronger resistances to BCNU and O6-benzylguanine (BG) in MTT test than control group. CFU-assays showed the colony-forming units was more detected in transfected group compared with vacant vector group (P<0.05).
CONCLUSIONS: The results suggested that transduction of the mutant G156A MGMT into hematopoietic progenitor cells mediated by recombinant adenovirus vectors may lead to their selective resistance to the combined use of O6-benzylguanine and alkylating agents, and this study provided an experimental foundation for improving combination chemotherapy tolerance in clinical practice.
Prevention of Ph+ Acute Lymphoblastic Leukemia by Nanoparticulate Delivery of a DNA Vaccine in Syngeneic Mice.
Authors: Koechling Joachim , Rott Yvonne , Arndt Stefanie , Green Jordan , Anderson Daniel , Langer Robert , Stripecke Renata , Wittig Burghardt , Schmidt Manuel 
Published: October 2019

Abstract
In contrast to gene therapy of solid tumors, only a few preclinical studies exist about gene therapy of acute lymphoblastic leukemia. Previously, we showed that vaccination of mice with syngeneic BCR-ABLp185 expressing leukemia cell lines modified to express costimulatory molecules and cytokines induce a systemic immunity against wild type leukemia. However, the difficulties to culture and transfect human leukemia cells limit the clinical application of leukemia cell based vaccines. Thus, we evaluated the pre-immunization of mice with DNA based vaccines subsequently challenged by the cell line BM185. In order to avoid the limitations and serious side effects associated with viral vectors we used nonviral gene delivery methods. Minimalistic immunogenically defined gene expression (MIDGE) vectors encoding a BCR-ABLp185 fusion specific peptide or GM-CSF were used as DNA vaccine and double stem-loop immunomodulators (dSLIM), containing three CpG-motifs were used as immune adjuvant. We provide biometrical and CTL data that shows specific immunization and protection of mice which received the complete vaccine BCR-ABL/GM-CSF/dSLIM by GeneGun delivery of naked DNA. Mean tumor-free survival (p=0.019) and overall survival (p=0.008) were significantly longer compared to non-vaccinated mice. Furthermore we show that BCR-ABL specific sequences are required to prevent Ph+ acute lymphoblastic leukemia by DNA vaccination. However, survival rate was moderate and only 26.7% of vaccinated mice survived and remained tumor-free. While current nonviral genetic tumor vaccine systems are less effective than viral vaccines, in particular when tumor associated antigens are weakly immunogenic, microencapsulation of tumor-specific DNA with poly (β-amino esters) might be a promising, safe and efficient nonviral delivery method for genetic vaccines. These biodegradable cationic polymers can bind and condense DNA into nanoparticles, deliver large DNA payloads and show low toxicity. Therefore, we prepared DNA-polymer complexes of MIDGE-vectors and end-modified poly (butane diol diacrylate co amino pentanol) C32-117 or C32-118 which self-assemble into particles with effective diameters of 200 nm and zeta potentials of 9.8 mV in PBS. We compared the immunization of mice receiving the vaccine BCR-ABL/GM-CSF/dSLIM by intradermal injection as naked DNA or alternatively, complexed with C32-117 or C32-118. We present data about tumor growth, survival rate, tumor free and overall survival of vaccinated and control mice.
Lymphoma and Chromosomal Instability in Artemis Deficient Mice - A Challenge for Long Term Malignancy Free Survival and Choice of Optimal Conditioning Regimen in Artemis Gene Therapy Trials.
Authors: Hauer Julia , Benjelloun Fatine , Romana Serge , Lelorc’h Marc , Fischer Alain , Villartay de Jean-Pierre , Cavazzana-Calvo Marina 
Published: October 2019

Abstract
Correction of Artemis deficiency, which causes a T−/−B−/− negative and radiosensitive type of SCID, is subject of murine gene therapy trials as an alternative treatment protocol for Hematopoietic Stem Cell Transplantation (HSCT). A conditioning regimen consisting of irradiation or busulfan in mice is indispensable for a succesfull engraftment. Gene therapy related toxicity on the basis of vector integration and conditioning regimen has been observed previously. Herein we provide evidence that Artemis deficiency harbours an additional risk factor for the development of lymphoproliferative disease. We provide in vivo evidence, that non gene corrected Artemis mice are able to develop lymphoma facilitated by irradiation and/or aging. Artemis−/− mice were irradiated at 3 Gy. Lymphoma were detected in 2 out of 10 Artemis−/− mice at the age of 4 to 6 months. Lymphoproliferative disease were exclusively of T-cell origin and arose from the thymus carrying a highly CD4+ or CD4+/CD8+ phenotype. Additionally, increased chromosomal rearrangements were detected in bone marrow cells of irradiated Artemis−/− mice compared to non irradiated control mice. Karyograms of bone marrow cells of 3 Gy-irradiated, 4 months old mice were stained with the Spectral Karyotyping (SKY) method. Analysis of 5 out of 20 metaphases revealed chromosomal translocations whereas in two out of 5 abnormal metaphases chromosome 15 was involved in the translocations. On contrary in karyograms of 4 to 6 weeks old Artemis−/− Sca1+ cells, which were used for viral transduction twenty out of twenty metaphases showed no obvious rearrangement. Even stimulation with cytokines in the course of viral transduction (SCF, FLT3L, TPO, IL6 and IL11) for five days did not increase chromosomal instability. Here we provide evidence, that Artemis−/− mice have an additional increased risk of developing hematopoietic malignancies, due to chromosomal instability which is independent of gene therapy. Human cells might not harbour the same riskfactor. However chromosomal instability in human Artemis−/− cells needs to be analysed in more detail for the choice of an optimal conditioning regimen and long term malignancy free survival in clinical trials.
Drug Resistance Gene Therapy by Simultaneous Lentiviral Overexpression of MDR1 in Combination with the MGMT P140K Mutant in Human Hematopoietic Stem Cells.
Authors: Spier Isabel , Maier Patrick , Laufs Stephanie , Veldwijk Marlon R. , Wenz Frederik , Zeller W. Jens , Fruehauf Stefan 
Published: October 2019

Abstract
Background: In this study we cloned a vector based on a self-inactivating lentiviral backbone containing MDR1 (multidrug resistance 1 gene) connected by an ECMV-IRES-element with MGMT P140K (O6-BG-resistant mutant of O6-methylguanine-DNA methyltransferase). The chemoprotective potential of this HR′SIN-MDR-IRES-MGMT combination vector was compared to single vectors (HR′SIN-MDR, HR′SIN-MGMT).
Methods: HL60 and CD34+ cells were transduced with the various vectors. After chemotherapeutical treatment MTT assays were used to detect chemoresistance levels in HL60 cells, CD34+ cells were held in liquid culture under differentiation conditions and analysed by FACS for MDR1 expression.
Results: HL60 cells transduced with the combination vector showed significant chemoresistance to O6-BG/ACNU (IC50 13x higher compared to untransduced control), the IC50 of cells transduced with HR′SIN-MGMT was 35x higher. The IC50 of paclitaxel (MDR1 substrate) was 24x higher in cells transduced with HR’SIN-MDR and 25x higher with HR’SIN-MDR-IRES-MGMT compared to untransduced control. Combined exposure of cells to O6-BG/ACNU and paclitaxel showed a survival advantage of cells transduced with the combination vector (IC50 6.25x higher), for the single vectors the IC50 was 1.63x higher (MDR1) and 2.08x higher (MGMT) compared to untransduced control. Treatment of CD34+ cells with increasing concentrations of doxorubicin (up to 0.8 μM) resulted in a higher fraction of MDR1-positive cells either with HR′SIN-MDR (26.6x) or with HR′SIN-MDR-IRES-MGMT (30.6x) compared to untreated cells. After combination treatment (20μM O6-BG/16μM BCNU and 0.4μM doxorubicin) the fraction of MDR1-positive cells was higher for HR′SIN-MDR-IRES-MGMT (14x) than HR′SIN-MDR (8x) transduced cells.
Conclusion: The protective effect of the combination vector is comparable with the single vectors for monotherapy and superior for combined therapy. The combination vector presents simultaneous protective effects of two drug resistance genes, thus reducing the risk of insertional mutagenesis by one transduction process. Consequently our results might help to reduce myelotoxic side effects and increase the chemotherapeutic efficiency.
Efficient Clonal Gene Delivery into Human Embryonic Stem Cells Via Active Transgenesis Using PiggyBac Transposition.
Authors: Zhang Ting-Ting , Ulrey Clayton L. , Liu Shan-Run , Yang Rui , McConnell Sean C. , Huo Yongliang , Ryan Thomas M. 
Published: October 2019

Abstract
The regulation of any transgene integrated into patient derived ES cells (via nuclear transfer or reprogramming) needs to be studied in great detail prior to their differentiation and transplantation back into the patient. The ability to efficiently deliver transgenes and clonally isolate transduced human embryonic stem (hES) cells for further study should be a prerequisite for their general therapeutic use. In this study active transgenesis using the piggyBac (pB) transposon system has been used to efficiently deliver transgenes to hES cells. This transfection method utilizes transient expression of pB transposase (TPase) to efficiently insert transgenes flanked by pB inverted terminal repeats (ITRs) directly into the hES cell genome. In the two plasmid system, the pB TPase is produced in trans from the "Helper" plasmid and the transgene(s), inserted between pB ITRs, are provided in the "Donor" plasmid. In the single plasmid system both the transgene and the transposase are on the same DNA molecule, but only the transgene(s) is located between the ITRs. Amaxa nucleofection was used to efficiently deliver the supercoiled transposon plasmid(s) into hES cells. Here, we used the single plasmid pB system to deliver a GFP transgene to hES cells. Dozens of clonal lines of GFP transgenic human ES cells were produced in a single experiment. GFP fluorescent colonies were isolated, subcloned, and analyzed for transgene copy number. Chromosomal transgene integration sites were mapped by plasmid rescue. The pB preferred tetranucleotide target sequence, TTAA, was confirmed at each insertion site analyzed. The distribution of chromosomal insertion sites were analyzed for more than 50 mapped insertions. About 60% of the transposon insertions were within 5 kb of human RefSeq genes and 45% were located within transcription units. Additionally, we have used the two plasmid pB system to deliver a human β-globin transgene along with GFP into hES cells followed by clonal isolation. This method easily transferred more than 13 kb of exogenous DNA into the hES cells. Efficient pB-mediated active transgenesis and clonal isolation of transduced hES should enable a careful analysis of the regulation of specific transgenes during the directed differentiation of potential therapeutic hES cell subclones.
Impact of Anemia on Medical Resource Utilization and Hospitalization Cost in Women with Obstetrical Bleeding.
Authors: James Andra , Patel Snehal , Dinh Quinn 
Published: October 2019

Abstract
Objective: Anemia during pregnancy has been associated with adverse maternal and fetal outcomes. Although women with obstetrical bleeding are at increased risk for developing anemia, little is known about the prevalence and burden associated with anemia in hospitalized women with this condition. This study was conducted to estimate the prevalence, demographic characteristics, medical resource utilization, and hospitalization cost associated with anemia in hospitalized women with obstetrical bleeding in the U.S.
Methods: The Healthcare Cost and Utilization Project Nationwide Inpatient Sample (2003) was queried using ICD-9-CM codes to identify all pregnancy-related discharges as well as discharges with diagnosis codes for conditions associated with obstetrical bleeding. Descriptive statistics were used to evaluate demographic characteristics, medical resource utilization components, and hospitalization cost for two groups: cases with anemia and cases without anemia.
Results: Of the estimated 4,525,714 pregnancy-related discharges in the U.S. in 2003, more than 250,000 recorded diagnosis codes associated with obstetrical bleeding. Nearly 1 in 5 of these women had an anemia diagnosis. A diagnosis of anemia in hospitalized women with obstetrical bleeding was associated with a 9-fold increase in blood transfusion (p<.0001), 33% longer average length of stay (p<.0001), and 50% higher mean cost per hospitalization (p<.0001).
Conclusions: Anemia and blood transfusion are frequently observed in hospitalized women with obstetrical bleeding. To improve outcomes in these patients and alleviate the adverse impact of anemia on postpartum health status, greater provider awareness of the prevalence and burden of illness associated with anemia in hospitalized women with obstetrical bleeding is warranted.
Trend and Geographic Variations in the Incidence of Waldenstrom’s Macroglobulinemia in the United States over 17 Years from 1988 to 2004.
Authors: Wang Michael , Zhou Yuhong , Wang Haijun , Yi Qing , Zhang Yanxia , Alexanian Raymond , Kwak Larry , Du Xin 
Published: October 2019

Abstract
Background: Waldenstrom’s Macroglobulinemia (WM) is an uncommon subtype of B cell malignancy. The updated trend of its incidence in the United States has not been reported.
Objective: To better understand the incidence and epidemiological features of WM in the United States.
Methods: We conducted a population-based incidence study with 1463 patients (pts) with WM identified from the Surveillance, Epidemiology, and End Results (SEER) tumor registries over 17 years. From the SEER data, 78,558 pts of all ages diagnosed with non-Hodgkin’s lymphoma (NHL) were identified between 1988 and 2004 in 9 SEER areas. Of these pts, 1463 had pathologically confirmed WM, accounting for 2% of NHL. The incidence with 95% confidence intervals were generated from SEER*Stat Software and was age-adjusted to the U.S. year 2000 standard population. Annual Percent Change (APC)for the incidence was calculated and considered to be statistically significant if p value was less than 0.05. The crude 1-year survival rate was calculated as the ratio of the number of pts who survived over 1 year and the number of pts diagnosed with WM.
Results: Of the1463 pts with WM, median age at diagnosis was 73. Overall incidence rate was 0.37/100,000/year, which increased with age from 0.03 in pts aged <50 to 1.03 in pts aged 60–69 and 2.76 in pts aged 80 or older. The age-adjusted incidence did not change significantly from 0.34 in 1988 compared with 0.36 in 2004. Therefore was no significant Annual Percent Change (0.97%, p>0.05). Incidence of WM was higher in men (0.52) than in women (0.26) (P<0.001). There were substantial geographic variations (p<0.001) in the incidence of WM. The age-adjusted incidence was highest in Seattle (0.49) and lowest in Atlanta (0.19). The incidence was higher in Caucasians (0.40) than in African-Americans (0.17) and other races (0.20). Annual Percentage Change was 1.3% in Caucasians (p<0.05).
Conclusion: The overall incidence of Waldenstrom’s Macroglobulinemia did not change significantly from 1988 to 2004, and was significantly higher in male Caucasians. There were substantial geographical variations in the incidence of Waldenstrom’s Macroglobulinemia in the United States.
Thalidomide-Dexamethasone vs Interferon-α-Dexamethasone as Maintenance Treatment after ThaDD Induction for Multiple Myeloma (MM): Final Analysis of a Prospective, Randomized Study.
Authors: Offidani Massimo , Polloni Claudia , Corvatta Laura , Piersantelli Maria-Novella , Galieni Piero , Visani Giuseppe , Alesiani Francesco , Catarini Massimo , Brunori Marino , Burattini Maurizio , Centurioni Riccardo , Ferranti Mario , Giuliodori Luciano , Candela Marco , Mele Anna , Marconi Monica , Leoni Pietro 
Published: October 2019

Abstract
Maintenance therapy with Thalidomide in MM offer controversial results. De novo or relapsing MM patients presenting at least a minor response after ThaDD were randomized to receive Interferon 3 MU x 3/week or thalidomide 100 mg /d. Both groups were given also Dexamethasone 20 mg/d x 4 days every month. Actually, we have randomized 50 patients in both treatment arms. The two groups were matched for main prognostic factors and response. During maintenance, both the ID and TD regimens improved response obtained by induction in only 10% and 11% of patients, respectively (p=0.832). After a median 2-years maintenance follow-up for both arms, 30 ID patients (60%) relapsed vs 17 (33%) TD ones (p=0.009). Time-to-progression (TTP) was significantly higher in the TD group vs the ID one (p= 0.024). So that TTP amounted to 23% in the ID group vs the 44% in the TD one. In addition, 3-years overall survival (OS) was significantly better in the TD arm with a value of 67% vs 46% of ID (p = 0.030). Both treatment arms were overall fairly well tolerated: fever, anorexia, weight loss, fatigue, liver and heart function abnormalities and hematologic toxicities were significantly more frequent in the ID cohort whereas neurotoxicity was somewhat more frequent in the TD one. This turned into a rate of therapy dropouts rate significantly higher in the ID group than in the TD one (26% vs 8%; p=0.017). Anyway, estimated risk for treatment interruption due to side effects in a 3-years period with thalidomide was only 21% vs 44% for interferon (p =0.014). We concluded that, in MM patients responding to standard induction therapy, low-dose Thalidomide maintenance therapy is feasible even in the long run and offers a significantly longer control over residual disease when compared to standard maintenance regimen.
A Transgenic Mouse Model of Multiple Myeloma Shows Heterogeneity in Drug Response with Alterations Due to Selective Gene Modifications.
Authors: Gosse Mary A. , Van Ness Brian G. 
Published: October 2019

Abstract
Multiple myeloma (MM) is a type of cancer characterized by malignant plasma cells (PCs) in the bone marrow. One difficulty in treating patients is that there is no single genetic event or abnormality common to all patients, resulting in heterogeneity of genes expressed, tumor aggressiveness, and response to therapy. In addition, mouse models of myeloma are limited. However, deregulation of MYC and BCL-XL are both common in patients’ tumors. Our lab has previously shown that a transgenic mouse line targeting overexpression of c-Myc and Bcl-xL to PCs results in an early expansion of non-malignant PCs, which then develop into clonal malignancies in multiple tissue types. Tumors develop quickly (median 16 weeks) in all mice and these cells can be adoptively transferred to syngeneic recipient mice to form tumors in as little as 10 days. Our lab has analyzed cell surface markers and generated genetic profiles on these Bcl-xL/Myc tumor cells, which show genetic heterogeneity in B-cell, PC, and myeloma markers, similar to human MM. Additionally, these tumor cells can be grown in vitro. The purpose of this study was to evaluate different drug sensitivities of our Bcl-xL/Myc primary tumor lines, and to determine if the sensitivities of these Bcl-xL/Myc tumors are dependant on the deregulated expression of specific genes. After treating cells with varying concentrations of dexamethasone, melphalan, and doxorubicin for a number of days, it was discovered that different tumor lines from the Bcl-xL/Myc transgenic mouse line have a range of sensitivity, from highly resistant to extremely sensitive. Three of these cell lines show a dose-response to varying concentrations of dexamethasone. Additionally, one cell line shows evidence of IL-6 protection against dexamethasone-induced apoptosis. Next, Bcl-xL/Myc primary cell lines were treated with BH3I-2′, a Bcl-xL small molecule inhibitor, at varying concentrations. Cell lines overexpressing Bcl-xL were more resistant to BH3I-2′, compared to S194 control cells, which express Bcl-xL at normal levels. One of the Bcl-xL/Myc cell lines did not show an increase in apoptosis or decrease in cell viability, but maintained the same number of cells over four days, whereas in other cell lines, viability started to decrease after 12 hours. Bcl-xL/Myc cell lines were also treated with siRNA targeted against Ezh2 and Cyclin D1, D2 and D3 transcripts, which have been shown to be deregulated in human myeloma as well as our own cell lines. These tumor lines are being treated with dexamethasone, melphalan and doxorubicin to evaluate the cell lines’ new sensitivities to chemotherapy. In total, our results show the Bcl-xL/Myc transgenic mouse tumor model demonstrates genetic heterogeneity that may be associated with heterogeneity in tumor responses. This serves as a useful model for human myeloma progression and allows us to examine the role of selective gene regulation in therapeutic response.