Detection of drug-induced apoptosis by flow cytometry after alkaline extraction of ethanol fixed cells

A new flow cytometric method was developed to detect apoptotic cells with fragmented DNA and to determine cell cycle distribution of viable cells, in the same sample, by propidium iodide staining. Apoptosis, in HT58 human B lymphoma cells, was induced by etoposide and/or by staurosporine. Using appropriate alkaline solutions (between 1-10 mN NaOH in 150 mM saline) followed by neutralization with buffer solution, the fragmented DNA can be extracted quantitatively from ethanol fixed cells. Further, good resolution of the cell cycle distribution can be obtained in unimpaired cells without RNase treatment. Furthermore, unlike the widely used hypotonic-detergent extraction of unfixed cells, the suggested extraction method can prevent drug-induced disintegration of dead cells when karyorrhexis occurs. This work was supported by Hungarian National Research Foundation (OTKA I/352 and OTKA II/2622).     

Radical cystectomy with or without adjuvant polychemotherapy for non-organ-confined transitional cell carcinoma of the urinary bladder: Prognostic impact of lymph node involvement

Objectives

To analyze the effectiveness of adjuvant polychemotherapy after radical cystectomy for nonorgan-confined transitional cell bladder cancer (Stages pT3b, pT4a, and/or pN1 or pN2).

Methods

Of 166 consecutive patients undergoing cystectomy at two institutions from 1987 to 1993, 80 received adjuvant polychemotherapy with methotrexate, vinblastine, and cisplatin plus doxorubicin (MVAC) or epirubicin (MVEC), whereas 86 had cystectomy only. The patients were evaluated for relapse-free survival and length of progression-free interval on the basis of follow-up data obtained in 1995 and 1996.

Results

Kaplan-Meier analysis revealed a significantly higher progression-free rate for patients after adjuvant chemotherapy (P=0.0002, log-rank test). With and without adjuvant chemotherapy, prognosis declined in a stepwise manner, depending on the extent of lymph node involvement. Nevertheless, the superior prognosis of the chemotherapy group could be demonstrated at each lymph node stage. Of the 166 patients, 49 had initially entered a prospective trial comparing adjuvant with no adjuvant treatment. That study was discontinued in December 1990 after an interim analysis revealed a significant prognostic advantage in favor of the 26 patients randomized to receive chemotherapy compared with the 23 control patients. Current follow-up data continue to demonstrate a significant improvement in progression-free survival in favor of patients randomized to receive adjuvant chemotherapy (P=0.0040). The follow-up period of patients living free of disease ranges from 58 to 96 months.

Conclusions

Adjuvant chemotherapy with MVAC/MVEC leads to significant prolongation of relapse-free survival and improvement of the definitive cure rate after radical cystectomy for locally advanced transitional cell carcinoma of the urinary bladder.     

Elevated risk ofHelicobacter pylori infection in submarine crews

In a prospective study designed to elucidate the route of transmission ofHelicobacter pylori, the seroprevalence and incidence ofHelicobacter pylori infection was determined in the following branches of the armed forces presumed to be at increased risk of acquiring transmissible diseases by the fecal-oral or oral-oral route: German submarine crews (n=64, mean age 26.2 years) and regular officers of the French infantry (n=51, mean age 26.5 years) who had served for a minimum of three years. The submarine crews were compared with air force staff (n=74, mean age 23.7 years), and the French officers with French infantry recruits (n=135, mean age 20.5 years) who started their service at the beginning of the study. The frequency of IgG and IgA antibody responses to the 120, 88, 86 and 82 kDa proteins was determined by the immunoblot method. The frequency of a positive antibody response was strongly dependent on age (p<0.001). When results were controlled for age by the logistic regression analysis, the submarine crews revealed significantly increased frequencies of the IgG and IgA responses compared to air force staff. The antibody responses of French officers and recruits were not significantly different. It is concluded that submarine crews serving during their missions in an overcrowded space with extremely limited sanitary facilities must be considered a high-risk group forHelicobacter pylori infection. These results strongly suggest person-to-person transmission ofHelicobacter pylori, by either the oral-oral or the fecal-oral route.     

Tissue connections in a transplantable virus-producing sebaceous adenoma of the mouse

Summary The distribution, size, and configuration of intercellular junctions in the sebaceous tumor of mice were examined using the freeze-fracture technique. Three types of junctions were observed: desmosomes, gap, and tight junctions. Tight junctions in general consisted of short linear unbranched fibrils, and macular or complex tight junctional patterns were present only occasionally. Gap junctions ranged from small sports of 0.9×10^-3 m² to areas of approximately 0.46 m². Desmosomes were the most frequent junctional specializations, and it is concluded that they are at least partially responsible for this tumor not being metastatic. Filipin in conjunction with freeze-fracture showed filipin-sterol complexes on the plasma membrane, the nuclear envelope, and the membranes of the endoplasmic reticulum and mitochondria. The intercellular junctions were devoid of these complexes indicating that these regions are low in cholesterol.     

RA 642, a pyrimido-pyrimidine-derivative with vasodilating and hypertensive potency

Summary 2,2-[(4,8-bis(diethylamino)-pyrimido [5,4-d]-pyrimidine-2,6-diyl)di-(2-methoxyethyl)imino]diethanol), RA 642, combines hypertensive and vasodilating effects. In anaesthetized animals arterial blood pressure was increased by i.v. doses of 0.25–4 mg/kg in cats and 0.025–0.25 mg/kg in dogs. In conscious dogs, 25 mm increase of mean blood pressure was achieved with 0.2 mg/kg i.v. and 18.8 mg/kg p.o. Cerebral blood flow was enhanced and calculated cerebral vascular resistance was reduced by RA 642. Total peripheral resistance was diminished by 0.25–1.0 mg/kg i.v. A vasodilatation of femoral and coronary vessels was shown after intraarterial injection. This effect as well as a BaCl₂-antagonism in the isolation ileum is explained by a papaverine-like relaxant effect on smooth muscle. Activity on peripheral adrenergic receptors was excluded. Hypertension was abolished in spinalized cats, indicating a central mechanism of this effect.     

Early [(11)C]Flumazenil/H(2)O positron emission tomography predicts irreversible ischemic cortical damage in stroke patients receiving acute thrombolytic therapy

BACKGROUND AND PURPOSE: Central benzodiazepine receptor ligands, such as [(11)C]flumazenil (FMZ), are markers of neuronal integrity and therefore might be useful in the differentiation of functionally and morphologically damaged tissue early in ischemic stroke. We sought to assess the value of a benzodiazepine receptor ligand for the early identification of irreversible ischemic damage to cortical areas that cannot benefit from reperfusion. METHODS: Eleven patients (7 male, 4 female, aged 52 to 75 years) with acute, hemispheric ischemic stroke were treated with alteplase (recombinant tissue plasminogen activator; 0.9 mg/kg according to National Institute of Neurological Disorders and Stroke protocol) within 3 hours of onset of symptoms. At the beginning of thrombolysis, cortical cerebral blood flow ([(15)O]H(2)O) and FMZ binding were assessed by positron emission tomography (PET). Those early PET findings were related to the change in neurological deficit (National Institutes of Health Stroke Scale) and to the extent of cortical damage on MRI or CT 3 weeks after the stroke. RESULTS: Hypoperfusion was observed in all cases, and in 8 patients the values were below critical thresholds estimated at 12 mL/100 g per minute, comprising 1 to 174 cm(3) of cortical tissue. Substantial reperfusion was seen in most of these regions 24 hours after thrombolysis. In 4 cases, distinct areas of decreased FMZ binding were detected. Those patients suffered permanent lesions in cortical areas corresponding to their FMZ defects (112 versus 146, 3 versus 3, 2 versus 1, and 128 versus 136 cm(3)). In the other patients no morphological defects were detected on MRI or CT, although blood flow was critically decreased in areas ranging in size up to 78 cm(3) before thrombolysis. CONCLUSIONS: These findings suggest that imaging of benzodiazepine receptors by FMZ PET distinguishes between irreversibly damaged and viable penumbra tissue early after acute stroke.     

Once upon a time...

In this, the first of two articles discussing literature for and about children, we will be considering how writing for the young has changed, reflecting different and evolving perspectives on childhood. In the second article we will be asking whether literature can be used creatively and usefully in the training of doctors. The suggestion for the topic arose from a session we organised for paediatricians in the Communication and Management module of the MSc in Child Health at Leeds University.     

Combining capecitabine and gemcitabine in patients with advanced pancreatic carcinoma: a phase I/II trial.

PURPOSE: Preclinical studies indicate positive interactions between capecitabine, an oral fluorouracil precursor, and gemcitabine, the current standard treatment for advanced pancreatic carcinoma (APC). In this study, we investigated the addition of capecitabine to gemcitabine treatment for patients with APC. PATIENTS AND METHODS: This multicenter study included patients na?ve to chemotherapy who had histologically or cytologically confirmed, nonresectable or metastatic pancreatic carcinoma. Gemcitabine was given at a fixed dose of 1,000 mg/m(2) on days 1 and 8 of a 21-day cycle. Capecitabine was given in increasing doses orally bid for 14 days followed by a 1-week rest. The maximum-tolerated dose (MTD) was defined as one dose level below the dose causing dose-limiting toxicity (DLT) in >or= one third of a cohort of six patients. We included an additional 15 patients at the MTD. RESULTS: Thirty-six patients were included. DLT occurred at a dose of 800 mg/m(2) bid of capecitabine and consisted of myelotoxicity and mucositis. Hand-foot syndrome was not observed, and other toxic effects were mild. Thus, in this regimen, the recommended dose of capecitabine is 650 mg/m(2) bid. In 27 patients with measurable disease, we observed one complete and four partial remissions. In addition, significant drops (> 50% from baseline value) of the tumor marker CA 19-9 occurred in 14 of 24 assessable patients. CONCLUSION: The combination of capecitabine and gemcitabine is well tolerated, with apparent efficacy in patients with APC. Therefore, it is currently being compared with gemcitabine monotherapy in a phase III study.