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Purpose Palliation of bone pain can be achieved in men with androgen-independent prostate cancer treated with docetaxel and estramustine (DE) or mitoxantrone and prednisone (MP). While Southwest Oncology Group trial 99-16 demonstrated a survival improvement of DE over MP, the study also was designed to compare the palliation of disease-related symptoms. Methods Pain palliation and global quality of life (QOL) were the two primary patient-reported outcomes. Pain was measured with the Present Pain Intensity scale of the McGill Pain Questionnaire-Short Form. The European Organisation for Research and Treatment of Cancer Core Quality of Life Questionnaire C30 (QLQ-C30) and its Prostate Cancer Module (PR25) measured QOL and symptom status. Pain and analgesic use were measured at random assignment, every cycle for eight cycles, and 1 year from random assignment; the QLQ-C30 and the PR25 were administered at random assignment, before cycle four (week 10) and cycle eight (month 6) and at 1 year. In addition to the primary intent-to-treat, missing at random analysis, sensitivity analyses were performed to assess robustness of global QOL conclusions under alternative informative missing data assumptions. Results Six hundred seventy four eligible patients received DE (n = 338) or MP (n = 336). In an intention-to-treat analysis, median overall survival was 17.5 months for the DE arm and 15.6 months for the MP arm (P = .02). There were no statistically significant differences in pain palliation between the treatment arms. The sensitivity analyses showed a consistent lack of statistically significant global QOL differences for the two arms. Conclusion DE had superior clinical efficacy (overall survival, time-to-progression, and prostate-specific antigen declines) with similar global QOL and pain palliation in the MP arm.

The Ca(2+)- and calmodulin-dependent protein phosphatase calcineurin is inhibited by the immunosuppressant drug cyclosporin A in the presence of cyclophilin A or B. Of the two isoforms, cyclophilin B is more potent by a factor of 2-5 when either the phosphoprotein [32P]casein or the [32P]phosphoserine [Ser(32P)] form of the 19-residue bovine cardiac cAMP-dependent protein kinase regulatory subunit peptide RII, [Ser(32P)15]RII, is used as substrate. With [Ser(32P15]RII as substrate, the concentrations of the cyclosporin A.cyclophilin A and cyclosporin A.cyclophilin B complexes, which cause 50% inhibition of calcineurin activity, are 120 and 50 nM, respectively. Lowering the concentration of calcineurin 80% with [32P]casein as substrate lowered the apparent inhibition constant for each complex even further; 50% inhibition of calcineurin was observed at 40 nM for cyclosporin A.cyclophilin A, whereas it was less than 10 nM for cyclosporin A.cyclophilin B. In all inhibition assays with [32P]casein or [Ser(32P)15]RII, the concentration of calcineurin required for measurable phosphatase activity is such that these complexes behave as tight-binding inhibitors of calcineurin, and steady-state kinetics cannot be used to assess inhibition patterns or Ki values. Limited trypsinization of calcineurin produces a fragment that is still inhibited, indicating that the interaction of cyclosporin.cyclophilin with calcineurin does not require either calmodulin or Ca2+.

SUMMARY Real-time PCR has revolutionized the way clinical microbiology laboratories diagnose many human microbial infections. This testing method combines PCR chemistry with fluorescent probe detection of amplified product in the same reaction vessel. In general, both PCR and amplified product detection are completed in an hour or less, which is considerably faster than conventional PCR detection methods. Real-time PCR assays provide sensitivity and specificity equivalent to that of conventional PCR combined with Southern blot analysis, and since amplification and detection steps are performed in the same closed vessel, the risk of releasing amplified nucleic acids into the environment is negligible. The combination of excellent sensitivity and specificity, low contamination risk, and speed has made real-time PCR technology an appealing alternative to culture- or immunoassay-based testing methods for diagnosing many infectious diseases. This review focuses on the application of real-time PCR in the clinical microbiology laboratory.

ABSTRACT A real-time PCR method using the LightCycler (Roche Applied Science, Indianapolis, IN) was compared to a conventional PCR assay for the detection of Tropheryma whipplei in 321 clinical specimens. The LightCycler method had sensitivity and specificity comparable to the conventional method but required considerably less labor and time (3 h versus 3 to 5 days).

Purpose On the basis of reported activity of methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC) or topotecan plus cisplatin in advanced cervix cancer, we undertook a randomized trial comparing these combinations versus cisplatin alone, to determine whether survival is improved with either combination compared with cisplatin alone, and to compare toxicities and quality of life (QOL) among the regimens. Patients and Methods Eligible patients were randomly allocated to receive cisplatin 50 mg/m2 every 3 weeks (CPT); cisplatin 50 mg/m2 day 1 plus topotecan 0.75 mg/m2 days 1 to 3 every 3 weeks (CT); or methotrexate 30 mg/m2 days 1, 15, and 22, vinblastine 3 mg/m2 days 2, 15, and 22, doxorubicin 30 mg/m2 day 2, and cisplatin 70 mg/m2 day 2 every 4 weeks (MVAC). Survival was the primary end point; response rate and progression-free survival (PFS) were secondary end points. QOL data are reported separately. Results The MVAC arm was closed by the Data Safety Monitoring Board after four treatment-related deaths occurred among 63 patients, and is not included in this analysis. Two hundred ninety-four patients enrolled onto the remaining regimens: 146 to CPT and 147 to CT. Grade 3 to 4 hematologic toxicity was more common with CT. Patients receiving CT had statistically superior outcomes to those receiving CPT, with median overall survival of 9.4 and 6.5 months (P = .017), median PFS of 4.6 and 2.9 months (P = .014), and response rates of 27% and 13%, respectively. Conclusion This is the first randomized phase III trial to demonstrate a survival advantage for combination chemotherapy over cisplatin alone in advanced cervix cancer.

The objectives of this study were to determine the effectiveness of a novel, trypsinized density gradient treatment designed to remove viruses from semen and to evaluate sperm viability after treatment. Exp. 1: Cryopreserved human semen (n = 6 donors) was layered on 2-mL columns of 45% Isolate (Irvine Scientific, Santa Ana, CA, USA) with or without 0.25% trypsin (trypsin-exposed and control, respectively), which overlaid 2-mL columns of 90% Isolate with or without 10 µg/mL soy-based trypsin inactivator (Sigma, St. Louis, MO, USA) and centrifuged (700g for 30 min). The layering of multiple density gradients is facilitated by a novel polypropylene tube insert, which also prevents contamination when extracting treated sperm (USA and international patents pending). Pellets were washed and then incubated at room temperature. Sperm were examined (motility and supravital staining) at 0, 2, 24, and 48 h post-treatment and the results evaluated using Wilcoxon Signed Rank and Rank Sum tests. Exp. 2: A cytopathic cell (MT-2) assay was conducted (6 replicates) to determine the effect of trypsin (1-min exposure) on HIV-1 RNA infectivity. Viral replication was assessed by syncytium formation and p24 antigen production. Exp. 3: Two pools of fresh human semen (N1 = 3 and N2 = 8 donors) were inoculated (1:1) with 1 × 108 copies/mL of cultured HIV-1 RNA, and one pool (N2) was inoculated (1:1) with plasma collected from patients infected with either Hepatitis B DNA (HBV) or C (HCV) RNA viruses; spiked and non-spiked aliquots were processed as in Exp. 1. Treated sperm pellets were analyzed for HIV-1 or HBV and HCV concentrations by the Bayer Versant branched DNA (bDNA; version 3.0) and/or the Roche Amplicor quantitative RT-PCR (1.5 ultrasensitive) assays at Toga Laboratories (Pty), Ltd. (Edenvale, South Africa). As a result of Exp. 1, there was significantly (P < 0.05) lower motility (but not supravital staining) between trypsin-treated and control sperm at 0 h (58.0 vs. 69.3%) and 2 h (54.7 vs. 62.9%) post-washing; however, no differences were noted after 24 h (P > 0.05). In Exp. 2, trypsin exposure affected HIV-1 RNA infectivity negatively as compared to controls in terms of MT-2 cell syncytium formation and p24 antigen production. Results of the bDNA and/or RT-PCR assays in Exp. 3 indicated that the procedure effectively reduced HIV-1, HBV, and HCV viral copies in the spiked semen samples to undetectable levels or levels below clinical relevance. In conclusion, the novel trypsin density gradient procedure was effective for removing HIV-1, HBV and HCV from spiked semen without markedly affecting sperm survival. Extrapolation of these results to natural infections may be unfounded for viruses (e.g. HBV) that are thought to integrate into sperm chromatin.

ABSTRACT We compared the performance characteristics of a real-time PCR method, the LightCycler vanA / vanB detection assay (Roche Diagnostics Corporation, Indianapolis, Ind.) to that of Enterococcosel agar (BBL, Sparks, Md.) for direct detection of vancomycin-resistant enterococci (VRE) from 894 perianal stool swabs. For 421 of 894 swabs, the result for LightCycler PCR was compared to an Enterococcosel plate containing vancomycin at 6 μg/ml; for the remaining 473 swabs, the result for LightCycler PCR was compared to an Enterococcosel plate containing 8 μg/ml vancomycin. The LightCycler method produced considerably more positive results than either the Enterococcosel plate containing vancomycin at 6 μg/ml ( n = 25 versus n = 11; sensitivity, 100%; specificity, 97%; positive predictive value [PPV], 42%; negative predictive value [NPV], 100%) or the Enterococcosel plate containing vancomycin at 8 μg/ml ( n = 31 versus n = 10; sensitivity, 100%; specificity, 95%; PPV, 32%; NPV, 100%). When possible, additional testing, including culture, LightCycler PCR, and/or a conventional PCR method (PCR-restriction fragment length polymorphism assay), were performed on either the original specimens or original cultures or subsequent specimens for cases in which the original specimen was positive by LightCycler PCR but the Enterococcosel plate was negative. This additional testing demonstrated positive results for 7 of 14 (50%) evaluable discordant specimens which initially tested as LightCycler PCR positive but culture negative using the Enterococcosel plate containing vancomycin at 6 μg/ml and 12 of 17 (71%) evaluable discordant specimens which initially tested as LightCycler positive but culture negative using the Enterococcosel plate containing vancomycin at (8 μg/ml). These results demonstrate that the LightCycler VRE detection assay is considerably more sensitive than the standard culture method for detecting VRE directly from perianal swab specimens. The LightCycler assay also provides results much faster than culture (∼3.5 versus ≥72 h). The use of this test could have important implications for the effective control and prevention of nosocomial outbreaks of VRE.