Human papillomavirus DNA in sera of cervical cancer patients as tumor marker

Persistent infection with human papillomavirus (HPV) has been widely recognized to induce carcinoma of the uterine cervix. Viral DNA has been documented to occur as tumor DNA in the circulation of patients with primary tumors caused by viral infection. The aim of this study was to evaluate the utility of serum HPV DNA as tumor marker in cervical cancer patients. Sera taken from 94 cervical cancer patients at the date of diagnosis and follow-up serum samples from 24 patients were examined for HPV DNA using PCR enzyme immunoassay. Serum samples taken at the date of diagnosis were HPV DNA positive in 45% of all investigated samples. Sera which were HPV DNA positive at the time of diagnosis became and also remained negative after primary treatment in patients without recurrence or persistent disease. HPV DNA was positive up to 423 days before the time of clinical diagnosis of recurrence in 10 out of 13 cases (median 72 days, range 0-423 days). Serum HPV DNA seems to reflect biological activity of the tumor. Our results indicate that serum HPV DNA might be a useful additional marker for early detection of recurrence in cervical cancer patients.     

Hospital discharges and 30-day case fatality for drug poisoning: a Danish population-based study from 1979 to 2002 with special emphasis on paracetamol

Objective To examine the number of hospital discharges and 30-day case fatalities due to drug poisoning based on data from a Danish County Hospital Discharge Registry from 1979 to 2002.Methods All patients with a hospital discharge diagnosis of drug poisoning were identified and separated into groups taking: (1) opioid analgesics; (2) non-opioid analgesics; (3) anxiolytics; (4) antidepressants; (5) antipsychotics; or (6) non-specified. Paracetamol and salicylate were analysed separately. From 1994 to 2001, the total amount of drugs sold in the county was identified from a national drug database.Results A total of 13,432 patients with a median age 41.5 years at discharge of whom 59% were females accounted for 20,249 discharges for drug poisoning. The overall number of discharges remained essentially stable around 170 discharges per 100,000 inhabitants per year. From the mid-1990s, paracetamol became the most frequently used drug in poisoning with the largest increase in female teenagers. Thirty-day case fatality in poisoning with opioids was 3.6% compared with around 1% in other drug categories. For most drug categories, a sale of around 80,000 defined daily doses was associated with one hospital discharge due to drug poisoning.Conclusion The overall number of hospital discharges remained stable and seems primarily related to amount of drugs available. With almost 10 years delay, the easier access to paracetamol was followed by an increase in hospitalisation due to poisoning with paracetamol. However, although the majority of hospitalisations were found in the younger age group, the highest mortality was seen among the elderly.     

A new, rapid, fully automated method for determination of fluconazole in serum by column-switching liquid chromatography

A sensitive and rapid HPLC assay for the determination of fluconazole in serum is described. HPLC-integrated sample preparation allows direct injection of serum samples without any pretreatment. The in-line extraction technique is carried out by automatically switching from the extraction column (Lichrospher ADS C8) to the analytic column (Nucleosil C18). After 6 minutes the matrix passes the extraction column, and the retained analyte is quantitatively transferred to the analytic column, where separation by isocratic HPLC is performed. The extraction eluent is sodium dihydrogen phosphate buffer, pH 5.0 (50 mM), and the analytic eluent is acetonitrile/sodium dihydrogen phosphate buffer, pH 5.0 (50 mM) (26.8/73.2, vol/vol). Fluconazole is detected according to its absorption maximum at 210 nm. The lower limit of quantification (LLOQ) is 0.65 microg/mL, the limit of detection (LOD) is 0.2 microg/mL, and the quantification range is 0.65-23.3 microg/mL. The assay was precise with a between-run coefficient of variation of < or = 5.59%. The within-run accuracy was 99.8% and 103.4%, and the between-run accuracy was 99.2% and 99.7%, respectively, for the concentrations 23.3 microg/mL and 1.3 microg/mL. The recovery was 78%. The described procedure allows sample cleanup and determination within 20 minutes, thereby facilitating drug monitoring in clinical routine. The method was applied successfully.     

An autocrine loop for vascular endothelial growth factor is established in prostate cancer cells generated after prolonged treatment with interleukin 6

Concentrations of interleukin 6 (IL-6) and its receptor are increased in human prostate cancer. Prostate cancer LNCaP-IL-6+ cells, established after prolonged treatment with IL-6, have been found to acquire a growth advantage. Vascular endothelial growth factor (VEGF) may accelerate the growth of various tumours by stimulation of VEGF receptor 2 (VEGFR-2). To understand better the regulation of proliferation of LNCaP-IL-6+ cells, the expression of VEGF and VEGFR-2 was here investigated in the LNCaP-IL-6+ subline. VEGF was measured in cellular supernatants by enzyme-linked immunoassay. The expression of VEGFR-2 was assessed by Western blot. LNCaP-IL-6+ and control LNCaP-IL-6- cells were treated with a neutralising antibody against VEGFR-2. VEGF concentrations were 20-fold higher in LNCaP-IL-6+ than in LNCaP-IL-6- cells. The stimulatory effect of IL-6 on VEGF production was abolished by an inhibitor of the signalling pathway for phosphoinositol 3 kinase in LNCaP-IL-6+ and LNCaP-IL-6- cells. Exogenous VEGF did not stimulate proliferation in either LNCaP-IL-6+ cells or controls. VEGFR-2 was detected only in LNCaP-IL-6+ cells, in which the neutralising antibody caused a partial inhibition of cell proliferation. It was concluded that a VEGF autocrine loop is established in prostate cancer cells generated after chronic treatment with IL-6. Because of the upregulation of IL-6 in patients with prostate cancer, these findings might be clinically relevant.     

Myocytes die by multiple mechanisms in failing human hearts

We tested the hypothesis that myocyte loss in failing human hearts occurs by different mechanisms: apoptosis, oncosis, and autophagic cell death. Explanted hearts from 19 patients with idiopathic dilated cardiomyopathy (EF< or =20%) and 7 control hearts were analyzed. Myocyte apoptosis revealed by caspase-3 activation and TUNEL staining occurred at a rate of 0.002+/-0.0005% (P<0.05 versus control) and oncosis assessed by complement 9 labeling at 0.06+/-0.001% (P<0.05). Cellular degeneration including appearance of ubiquitin containing autophagic vacuoles and nuclear disintegration was present at the ultrastructural level. Nuclear and cytosolic ubiquitin/protein accumulations occurred at 0.08+/-0.004% (P<0.05). The ubiquitin-activating enzyme E1 and the ligase E3 were not different from control. In contrast, ubiquitin mRNA levels were 1.8-fold (P<0.02) elevated, and the conjugating enzyme E2 was 2.3-fold upregulated (P<0.005). The most important finding, however, is the 2.3-fold downregulation of the deubiquitination enzyme isopeptidase-T and the 1.5-fold reduction of the ubiquitin-fusion degradation system-1, which in conjunction with unchanged proteasomal subunit levels and proteasomal activity results in massive storage of ubiquitin/protein complexes and in autophagic cell death. A 2-fold decrease of cathepsin D might be an additional factor responsible for the accumulation of ubiquitin/protein conjugates. It is concluded that in human failing hearts apoptosis, oncosis, and autophagy act in parallel to varying degrees. A disturbed balance between a high rate of ubiquitination and inadequate degradation of ubiquitin/protein conjugates may contribute to autophagic cell death. Together, these different types of cell death play a significant role for myocyte disappearance and the development of contractile dysfunction in failing hearts.