Retroviral transduction and expression of the human alkyltransferase cDNA provides nitrosourea resistance to hematopoietic cells

Myelosuppression is the dose-limiting toxicity for nitrosourea chemotherapy. This toxicity predominantly involves modification of the O6 position of guanine with an alkyl moiety. The enzyme responsible for repair of O6-alkylguanine adducts, O6-alkylguanine-DNA alkyltransferase (alkyltransferase), is expressed at low levels in bone marrow (BM) cells. High alkyltransferase expression prevents the cytotoxicity and carcinogenicity of nitrosoureas in several transgenic and in vitro gene transfer models. We used gene transfer using a novel myeloproliferative sarcoma virus (MPSV) based retrovirus (vM5MGMT) to express the human alkyltransferase cDNA (MGMT) in human and murine hematopoietic cells. Transduced K562 cells had very high levels of alkyltransferase expression and significantly increased resistance to 1,3-bis (2- chloroethyl) nitrosourea (BCNU) as compared with untransduced K562 cells. Primary murine BM progenitors showed a high transduction efficiency with vM5MGMT and have increased BCNU resistance in vitro. After BM transplantation with vM5MGMT-transduced BM cells and BCNU treatment of these mice, BM, spleen and thymus had a 10- to 40-fold increase in alkyltransferase expression that persisted for at least 23 weeks posttransplantation. Progenitor cells procured from mice expressing high levels of alkyltransferase also had increased resistance to BCNU. Thus, an MPSV-based retroviral vector transduces mouse and human hematopoietic cells at high efficiency and results in high levels of gene expression both in vitro and in vivo. Overexpression of the alkyltransferase protein may protect hematopoietic progenitors from nitrosourea-induced myelosuppression.     

大肠癌免疫组化表达与临床病理的关系

目的:探讨大肠癌CEA、P53、nm23、Ki-67、MRP免疫组化表达特点和相互关系,及其与临床病理的关系．方法:回顾性分析2003-01／2006-07我院收治的73例大肠癌患者的临床病理及随访资料,并对其石蜡标本采用免疫组化SP染色法检测CEA、P53、nm23、Ki-67、MRP,分析其免疫组化特点及其与临床病理之间的关系．结果:CEA、P53、nm23、Ki-67、MRP在大肠癌中的阳性表达率依次为82．2％、68．5％、75.3％、84．9％和64．4％．CEA、MRP与大肠癌患者的各因素无统计学差异．P53、Ki-67和nm23与肿瘤的Dukes分期和淋巴结转移有关, P53、Ki-67在Dukes C、D期的阳性表达率(依次为82．8％和100％1明显高于Dukes A、B期者(59．1％和75．0％)(P<0．05),而nm23在Dukes C、D期的阳性表达率(58．6％)明显低于Dukes A、B期者(86．4％)(P<0．05)．CEA与nm23的表达呈明显的负相关(r=-0．296,P=0．011),而P53和Ki-67表达之间呈现明显的正相关(r= 0．308,P=0．008),其他各指标间的表达无相关性．nm23、P53和Ki-67与预后因素关系明显,nm23在生存期≥3 a患者的阳性表达率(92．9%)高于生存期<3 a者(71．2％)(P<0．05),而P53和Ki-67在生存期≥3 a患者的阳性表达率(依次为42．9％和64.3％)明显低于生存期<3 a者(74．6％和89．8％)(P<0．05)．结论:P53、Ki-67和nm23的表达与大肠癌的侵袭转移和预后密切相关．CEA可能是大肠癌的侵袭转移的促进因素．MRP所引起的耐药机制是一个相对独立的机制．CEA、P53、nm23、Ki-67可作为判断大肠癌恶性程度、侵袭转移以及预后的指标．     

Mutations of conserved arginines in the membrane domain of erythroid band 3 lead to a decrease in membrane-associated band 3 and to the phenotype of hereditary spherocytosis

To elucidate the molecular basis of band 3 deficiency in a recently defined subset of patients with autosomal dominant hereditary spherocytosis (HS), we screened band 3 cDNA for single-strand conformation polymorphism (SSCP). In 5 of 17 (29%) unrelated HS subjects with band 3 deficiency, we detected substitutions R760W, R760Q, R808C, and R870W that were all coinherited with the HS phenotype. The involved arginines are highly conserved throughout evolution. To examine whether or not the product of the mutant allele is inserted into the membrane, we studied one HS subject who was doubly heterozygous for the R760Q mutation and the K56E (band 3sMEMPHIS) polymorphism that results in altered electrophoretic mobility of the band 3 Memphis proteolytic fragments. We detected only the band 3MEMPHIS in the erythrocyte membrane indicating that the protein product of the mutant, R760Q, band 3 allele is absent from the red blood cell membrane. These findings suggest that the R760Q substitution, and probably the other arginine subsitutions, produce band 3 deficiency either by precluding incorporation of the mutant protein into the red blood cell membrane or by leading to loss of mutant protein from differentiating erythroid precursors.     

Detection of maternal cells in human umbilical cord blood using fluorescence in situ hybridization

Cord blood is a potential source of hematopoietic stem cells for transplantation and is being used on a growing number of patients. However, there are concerns that cord blood might be contaminated with maternal cells that could lead to graft-versus-host disease. To ascertain the extent to which maternal cell contamination of cord blood occurs, we examined 49 cord blood samples from male babies for maternal cells by fluorescence in situ hybridization using probes to the X and Y chromosomes. A minimum of 1,000 nuclei were scored from each sample, and maternal cells were found in 7 of the 49 cord bloods, at levels ranging from 0.04% to 1.0%. In addition, in 39 and 27 of the cord blood samples, respectively, we examined the CD8+ and CD34+ cell populations for maternal cells. Maternal cells were found in 5 of the 39 CD8 fractions and in 1 of the 27 CD34 fractions, at levels similar to that found in the unfractionated cord blood. In sum, maternal cells were found in either the unseparated mononuclear fraction or the CD8 or CD34 fractions in 10 of the 49 cord blood samples (20%). These results show that maternal cells are present in a substantial number of cord bloods, and that some of these maternal cells are T cells.     

Retreatment of hepatitis C patients with pegylated interferon combined with ribavirin in non-responders to interferon plus ribavirin. Is it different in real life?

Background  

More than 50% of hepatitis C viruses (HCV)-infected patients do not respond to the classical Interferon (IFN)/Ribavirin (RBV) combination therapy. The aim of this study was to evaluate the efficacy of retreatment with Peg-Interferon alpha-2b (PEG-IFN alpha-2b) plus RBV, in patients with HCV, genotypes 1 or 3, who were non-responders to the previous standard treatment with IFN/RBV.     

A Randomized‐Controlled Pilot Study Comparing ICD Implantation with and Without Intraoperative Defibrillation Testing in Patients with Heart Failure and Severe Left Ventricular Dysfunction: A Substudy of the RAFT Trial

Comparing ICD Implantation with and Without Intraoperative Defibrillation Testing. Introduction: The need to perform defibrillation testing (DT) at the time of implantable cardioverter defibrillator (ICD) insertion is controversial. In the absence of randomized trials, some regions now perform more than half of ICD implants without DT. Methods: During the last year of enrolment in the Resynchronization for Ambulatory Heart Failure Trial, a substudy randomized patients to ICD implantation with versus without DT. Results: Among 252 patients screened, 145 were enrolled; 75 randomized to DT and 70 to no DT. Patients were similar in terms of age (65.9 ± 9.3 years vs 67.9 ± 8.9 years); LVEF (24.7 ± 4.6% vs 23.6 ± 4.6%), QRS width (154.8 ± 23.5 vs 155.8 ± 23.6 ms), and history of atrial fibrillation (5% vs 6%). All 68 patients in the DT arm tested according to the protocol achieved a successful DT (≤25 J); 96% without requiring any system modification. No patient experienced perioperative stroke, myocardial infarction, heart failure (HF), intubation or unplanned ICU stay. The length of hospital stay was not prolonged in the DT group: 20.2 ± 26.3 hours versus 21.3 ± 23.0 hours, P = 0.79. One patient in the DT arm had a failed appropriate shock and no patient suffered an arrhythmic death. The composite of HF hospitalization or all‐cause mortality occurred in 10% of patients in the no‐DT arm and 19% of patients in the DT arm (HR = 0.53, 95% CI: 0.21–1.31, P = 0.14). Conclusions: In this randomized trial, perioperative complications, failed appropriate shocks, and arrhythmic death were all uncommon regardless of DT. There was a nonsignificant increase in the risk of death or HF hospitalization with DT. (J Cardiovasc Electrophysiol, Vol. 23, pp. 1313‐1316, December 2012)     

Acquired immunodeficiency syndrome-associated non-Hodgkin's lymphomas and other malignancies in patients with hemophilia

Non-Hodgkin's lymphoma (NHL) is the most common human immunodeficiency virus (HIV)-associated malignancy in hemophiliacs. We studied the incidence and clinicopathologic features of NHL in 3,041 hemophiliacs followed at 18 US Hemophilia Centers between 1978 and 1989. Of the 1,295 (56.6%) who were HIV(+), 253 (19.5%) developed acquired immunodeficiency syndrome (AIDS), of whom 14 (5.5%) developed NHL. Three NHL occurred in HIV(-) hemophiliacs, for a 36.5-fold greater risk in HIV(+) than HIV(-) hemophiliacs (P < .001). The NHL incidence rate was 29-fold greater than in the US population by Surveillance, Epidemiology, and End Results (SEER) estimates (P < .001). Between 0 and 4 lymphomas have been observed per year between 1978 and 1989. At presentation 13 (92.9%) of the HIV(+) NHL were extranodal. Ten were stage IV, 1 stage II, and 3 stage IE. Ten (71.4%) were high-grade, 3 (21.4%) intermediate-grade, and 1 (7.1%) was a low-grade B-cell lymphoma. Epstein-Barr virus (EBV) DNA was detected in 36% by in situ hybridization, including one central nervous system (CNS) lymphoma. The mean CD4 cell count at NHL diagnosis was 64/mm3, the mean latency from initial HIV infection was estimated to be 59 months, and the median survival was 7 months. The incidence of basal cell carcinoma in HIV(+) hemophiliacs was 18.3-fold greater than in HIV(-) hemophiliacs (P < .001) and 11.4-fold greater than in the US population (P < .001). In conclusion, incidence rates of NHL and basal cell carcinoma in HIV(+) hemophiliacs are significantly increased over rates in HIV(-) hemophiliacs and over rates in the US population. Clinicopathologic presentation of NHL in HIV(+) hemophiliacs is similar to that in HIV(+) homosexual men.     

Poikilocytic hereditary elliptocytosis associated with spectrin Alexandria: an alpha I/50b Kd variant that is caused by a single amino acid deletion

Hereditary elliptocytosis (HE) is a heterogeneous disorder of red blood cells frequently associated with abnormal limited tryptic digestion of the alpha I domain of spectrin and impaired spectrin dimer self- association. We studied two related individuals with poikilocytic hereditary elliptocytosis (HE) of different severity. Limited tryptic digestion of spectrin from these individuals showed the presence of a variant alpha I/50b Kd peptide at the expense of the normal alpha I/80 Kd peptide. Amino acid sequence analysis of the abnormal peptide showed that the proteolytic cleavage occurred after the arginine at position 470 of the alpha spectrin chain. Spectrin from these patients had an impaired ability to undergo self-association, as evidenced by increased amounts of spectrin dimers in 4 degrees C extracts of erythrocyte membrane from affected individuals. The polymerase chain reaction was used to study the DNA sequence of the alpha spectrin gene encoding the region of the alpha spectrin chain surrounding the abnormal proteolytic cleavage site. We detected the in-frame deletion of the trinucleotide CAT, encoding histidine 469, two amino acid residues to the N-terminal side of the abnormal proteolytic cleavage site between residues 470 and 471. Similar to many other defects of spectrin associated with HE, this deletion occurs in helix three of repeat 5 of the proposed triple helical model of spectrin repeats.     

高效液相色谱法测定消痰咳片的含量

目的：采用反相色谱法同时测定消痰咳片中两种主要成分甲氧苄啶和磺胺体的含量。方法：以乙腈：0.1%H3PO4(15:85)为流动相，检测波长254nm，HPLC法测定含量。结果：试验表明，甲氧苄啶和磺胺林在0.8-8ug范围内呈良好的线性关系，回归方程分别为Y＝－1127.1＋110.2x(r=0.9994),Y=-1852.3 256.2x(r=0.9996),相对标准偏差分别为2.1%和0.8%。结论：该方法简便、准确、可靠。