Amplification of genes encoding human myeloid membrane antigens after DNA-mediated gene transfer

Spontaneous amplification of genes encoding two different human myeloid surface antigens was observed after DNA-mediated gene transfer of cellular DNA from the human myeloid cell line HL-60 into NIH-3T3 mouse fibroblasts. Transformed recipient cells with highly amplified expression of either of two donor membrane polypeptides, gp150 or p67, were isolated with a fluorescence-activated cell sorter (FACS), using monoclonal antibodies specific for human myeloid cells. Immunoprecipitation of enzymatically radioiodinated polypeptides from the surface of transformed NIH-3T3 cells confirmed that expression of these proteins was amplified tenfold to 20-fold in comparison to their expression on human myeloid cell lines. The cellular DNA of cloned secondary and tertiary transformants expressing high levels of gp150 and p67 contained amplified sets of DNA restriction fragments that hybridized with human repetitive DNA sequences. Cytogenetic analysis of subclones overexpressing gp150 revealed extrachromosomal double minutes (DMs), whose presence correlated with the unstable expression of the membrane polypeptide. Human sequences in gp150-positive clones did not localize to chromosomes, consistent with their association with extrachromosomal DMs. By contrast, p67-positive subclones stably expressed the antigen, and in situ hybridization to metaphase spreads demonstrated that amplified human DNA sequences were integrated into a specific marker chromosome. Cytogenetic analysis of the parental NIH- 3T3 subclone used in these studies disclosed DMs in a low percentage of metaphases, suggesting that the recipient cells have a propensity for amplifying donor DNA.     

A 3-base pair deletion,c.9711_9713del,in DMD results in intellectual disability without muscular dystrophy

We have identified a deletion of 3 base pairs in the dystrophin gene (DMD), c.9711_9713del, in a family with nonspecific X-linked intellectual disability (ID) by sequencing of the exons of 86 known X-linked ID genes. This in-frame deletion results in the deletion of a single-amino-acid residue, Leu3238, in the brain-specific isoform Dp71 of dystrophin. Linkage analysis supported causality as the mutation was present in the 7.6 cM linkage interval on Xp22.11–Xp21.1 with a maximum positive LOD score of 2.41 (MRX85 locus). Molecular modeling predicts that the p.(Leu3238del) deletion results in the destabilization of the C-terminal domain of dystrophin and hence reduces the ability to interact with β-dystroglycan. Correspondingly, Dp71 protein levels in lymphoblastoid cells from the index patient are 6.7-fold lower than those in control cell lines (P=0.08). Subsequent determination of the creatine kinase levels in blood of the index patient showed a mild but significant elevation in serum creatine kinase, which is in line with impaired dystrophin function. In conclusion, we have identified the first DMD mutation in Dp71 that results in ID without muscular dystrophy.     

Squamous cell carcinoma after radiofrequency ablation for Barrett’s dysplasia

Barrett’s oesophagus(BO)is a usually indolent condition that occasionally requires endoscopic therapy.Radiofrequency ablation(RFA)is an effective endoscopic treatment for high grade dysplasia(HGD)and intramucosal cancer in BO.It has a good efficacy,durability and safety profile although complications can occur.Here we describe a case of RFA in a patient with high grade dysplasia.Although the response to treatment was initially very good with the development of neosquamous epithelium,the patient very rapidly developed a squamous cell cancer of the oesophagus confirmed on radiology,histology and immunohistochemistry.Sanger sequencing confirmed that the original HGD and the squamous cell cancer(SCC)were derived from separate clonal origins.The report highlights the fact that SCC of the oesophagus has been noted after endoscopic ablation for BO previously and suggest that ablation of BO may encourage the clonal expansion of cells carrying carcinogenic mutations once a dominant clonal population has been eradicated.     

Serum and intraperitoneal levels of amphotericin B and flucytosine during intravenous treatment of critically ill patients with Candida peritonitis

OBJECTIVES: To study the relation between serum and peritoneal levels of amphotericin B and flucytosine during intravenous treatment in patients with abdominal sepsis due to a perforated gut. PATIENTS AND METHODS: Included were consecutive patients with abdominal sepsis due to a perforated gut, who were treated intravenously with amphotericin B and/or flucytosine after surgery if an abdominal drain was present. Amphotericin B and flucytosine were measured from simultaneously collected serum and abdominal fluid samples. RESULTS: Twenty-one consecutive patients were included. Five repeated samples were taken from three patients. The time interval between the start of the medication and the first sampling was median 4.0 days (range 2-7 days). The correlation coefficient (r(2)) between serum and peritoneal levels of amphotericin B was 0.79. In nine patients (43%) with a maximum serum level of 0.28 mg/L, amphotericin B in the peritoneal fluid was undetectable. The lowest serum level that was present with a detectable peritoneal level was 0.16 mg/L. A short duration of treatment (2 days) was associated with low serum and undetectable peritoneal levels. In seven patients, flucytosine levels were measured. Peritoneal flucytosine levels did not differ significantly from serum levels. Serum and peritoneal flucytosine levels correlated well with r(2)=0.88. Peritoneal amphotericin B level was inversely correlated with C-reactive protein level on the same day (r(2)=0.30). CONCLUSIONS: It is shown, during continuous infusion, that peritoneal levels of amphotericin B are lower than serum levels. The amphotericin B serum levels should exceed 0.5 mg/L to obtain peritoneal levels above MIC values. Flucytosine levels in the abdominal fluid are comparable to serum levels and within MIC ranges.     

Transcystic or Transductal Stone Extraction during Single-Stage Treatment of Choledochocystolithiasis: A Systematic Review

Background

Choledochocystolithiasis can be managed by endoscopic retrograde cholangiopancreaticography (ERCP) or laparoscopically by transcystic (TC) or transductal (TD) stone extraction.

Objective

The aim of this study was to systematically review safety and effectiveness of combined endoscopic/laparoscopic management versus total laparoscopic management for choledochocystolithiasis with specific emphasis on TC versus TD stone extraction.

Methods

MEDLINE/PubMed, EMBASE, the Cochrane Library, and clinicaltrials.gov were searched systematically to identify trials on combined endoscopic/laparoscopic and total laparoscopic management for choledochocystolithiasis. Laparoscopic common bile duct (CBD) exploration was divided into TD and TC approach. Primary outcomes were successful stone clearance from CBD, postoperative/procedural morbidity, and mortality.

Results

Eight randomized trials with 965 patients were included. Successful bile duct clearance varied between 52.6 and 97 % in the ERCP groups, 80.4 and 100 % in the TC groups, and 58.3 and 100 % in the TD groups. There were more bile leaks after TD stone extraction (11 %) than after ERCP (1 %) and TC stone extraction (1.7 %). Total morbidity varied between 9.1 and 38.3 % in the ERCP groups, 7 and 10.5 % in the TC groups, and 18.4 and 26.7 % in the TD groups. Methodological and statistical heterogeneity among the trials precluded a meaningful meta-analysis.

Conclusion

Stone clearance rates are comparable between the three modalities, but TD stone extraction is associated with a higher risk of bile leaks and should only be performed by highly experienced surgeons. TC stone extraction seems a more accessible technique with lower complication rates. If unsuccessful, per- or postoperative endoscopic stone extraction is a viable option.     

Cytogenetic studies in non-African Burkitt lymphoma

A particular translocation between chromosomes 8 and 14 has been found repeatedly in cytogenetic studies of Burkitt lymphoma, both of African and non-African origin. We report here our findings in cytogenetic studies of direct tumor preparations from 18 non-African Burkitt lymphoma patients, 9 of whom also had cell lines available for study. A t(8;14) was found in direct tumor material in 10 of the 18 patients. Seven of the 9 cell lines had a t(8;14). A total of 15 patients had either a t(8;14) or a 14q+ present in tumor material and/or cell lines. In addition, 8 patients had a peculiar marker chromosome 1. The t(8;14) was not found in every malignant cell and, where present, it was rarely the sole karyotypic abnormality. The relationship of the t(8;14) to the evolution of the tumor is discussed.     

Rebanding for Slippage After Gastric Banding: Should We Do It?

Background

Laparoscopic adjustable gastric banding (LAGB) is a commonly performed bariatric procedure. LAGB is frequently complicated by slippage. Possible treatment for slippage is rebanding, but long-term effects are unknown. The aim of this study was to investigate whether rebanding after gastric band slippage is associated with weight loss failure.

Methods

This was a post hoc analysis of a prospectively collected database of 627 consecutive LAGB patients. Rebanding for slippage was performed in 81 patients. The effect of rebanding on weight loss was evaluated by three analyses: (1) in 81 rebanded patients, weight loss was compared before and after rebanding, separately for patients in whom primary LAGB was successful or unsuccessful; (2) 81 rebanded patients were matched to 81 patients without slippage for prognostic variables and compared for weight loss after rebanding; (3) multivariate logistic regression was performed whether rebanding was independently associated with weight loss failure.

Results

The chance of a fair result of rebanding for patients following primary successful (n?=?34) and unsuccessful LAGB (n?=?22) was 62 and 27 % after median follow-up of 113 and 97 months, respectively. There was no difference in weight loss failure between 81 rebanded patients and 81 matched patients: 54 vs 59 % (P?=?0.43). In multivariate analysis, rebanding was not significantly associated with weight loss failure: adjusted odds ratio 1.42; 95 % confidence interval 0.85–2.38; P?=?0.18.

Conclusion

In general, rebanding after LAGB has no negative effect on weight loss. However, patients in whom LAGB was unsuccessful prior to rebanding have poor long-term weight loss results.     

The use of 7-amino actinomycin D in identifying apoptosis: simplicity of use and broad spectrum of application compared with other techniques

The detection and quantitation of apoptotic cells is becoming increasingly important in the investigation of the role of apoptosis in cellular proliferation and differentiation. The pathogenesis of hematologic disorders such as aplastic anemia and the development of neoplasia are believed to involve dysregulation of apoptosis. To quantitate accurately the proportion of apoptosis cells within different cell types of a heterogeneous cell population such as blood or bone marrow, a method is required that combines the analysis of large numbers of cells with concurrent immunophenotyping of cell surface antigens. In this study, we have evaluated such a method using the fluorescent DNA binding agent, 7-amino actinomycin D (7AAD), to stain three diverse human cell lines, induced to undergo apoptosis by three different stimuli. Flow cytometric analysis defines three populations on the basis of 7AAD fluorescence and forward light scatter. We have shown by cell sorting and subsequent morphological assessment and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling that the populations defined by 7AAD represent live, apoptotic, and late-apoptotic/dead cells. This method is quick, simple, reproducible, and cheap and will be a valuable tool in the investigation of the role of apoptosis in normal physiology and in disease states.