Citations

ABSTRACT A 300-bp plasmid fragment of the hemagglutinin gene was used as target DNA to develop a rapid real-time LightCycler (Roche Applied Science, Indianapolis, Ind.) PCR assay for laboratory detection of smallpox virus. PCR primers and probes were designed specifically for detection of smallpox virus DNA, but all viruses of the genus Orthopoxvirus tested could be detected by use of the hemagglutinin gene target sequence. Base pair mismatches in the 204-bp amplicon allowed discrimination of cowpox virus (melting temperature [ T m ], 56.40°C), monkeypox virus ( T m , 56.24°C), and vaccinia virus ( T m , 56.72°C), including the Dryvax vaccine strain, from smallpox virus ( T m , 62.45°C) by melting curve analysis. The analytical sensitivity was 5 to 10 copies of target DNA per sample. The assay was specific for members of the genus Orthopoxvirus ; the DNAs of herpes simplex virus and varicella-zoster virus were not detected by the smallpox virus LightCycler PCR.

ABSTRACT Cytomegalovirus (CMV) DNA quantitation in clinical specimens is progressively becoming a cornerstone in the diagnosis and management of CMV infection in the immunocompromised host. We evaluated two automated and reproducible PCR tests, the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.) and the COBAS AMPLICOR CMV Monitor (Roche Diagnostics, Pleasanton, Calif.), for the detection of CMV DNA in blood samples from transplant recipients with CMV infection as determined by shell vial culture. Following a log transformation analysis, the mean CMV DNA in plasma (PL), whole blood (WB), peripheral blood leukocytes (PBL), and peripheral blood mononuclear cells (PBMC) using the LightCycler was 6.79 copies per ml, 7.23 copies per ml, 6.38 copies per 2 × 10 6 cells, and 6.27 copies per 2 × 10 6 cells, respectively. This compares to 7.86 copies per ml, 8.37 copies per ml, 7.59 copies per 2 × 10 6 cells, and 7.44 copies per 2 × 10 6 cells, respectively, using COBAS AMPLICOR CMV Monitor. While higher CMV DNA levels were observed for the various blood compartments analyzed using COBAS AMPLICOR CMV Monitor, a high degree of correlation was evident between the two automated systems (jackknife correlation r = PL 0.77 [95% confidence interval (CI); 0.64, 0.90], WB 0.77 [95% CI; 0.62, 0.92], PBL 0.77 [95% CI; 0.67, 0.88], and PBMC 0.81 [95% CI; 0.72, 0.89], all P < 0.001). Therefore, we conclude that either automated diagnostic system is accurate for CMV DNA quantitation.

ABSTRACT We evaluated two automated systems, MagNA Pure (Roche Molecular Biochemicals, Indianapolis, Ind.) and BioRobot 9604 (Qiagen, Inc., Chatsworth, Calif.) as effective replacements for the manual IsoQuick method (Orca Research, Inc., Bothell, Wash.) for extraction of herpes simplex virus (HSV) DNA from dermal and genital tract specimens prior to analysis by LightCycler PCR. Of 198 specimens (152 genital, 46 dermal), 92 (46.2%) were positive for HSV DNA by LightCycler PCR after automated extraction of specimens with either the MagNA Pure or BioRobot 9604 instrument. The manual IsoQuick method yielded HSV DNA (total n = 95) from three additional specimens that were negative by the automated method ( P = 0.25, sign test). Although the mean numbers of LightCycler PCR cycles required to reach positivity differed statistically significantly among all three of the methods of extraction, the estimated means differed by no more than 1.5 cycles ( P < 0.05). Seventy (76%) of the 92 specimens that were LightCycler PCR positive by all three extraction methods were also positive by shell vial cell culture assay. HSV DNA was detected by a lower LightCycler PCR cycle number (26.1 cycles) in specimens culture positive for the virus than in culture-negative samples (33.3 cycles) ( P < 0.0001). The manual IsoQuick and automated MagNA Pure and BioRobot 9604 methods provide standardized, reproducible extraction of HSV DNA for LightCycler PCR. The decision to implement a manual versus an automated procedure depends on factors such as costs related to the number of specimens processed rather than on the minimal differences in the technical efficiency of extraction of nucleic acids among these methods.

ABSTRACT An evaluation was undertaken to determine the utility of the BACTEC Peds Plus/F bottle and the BACTEC 9240 instrument (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) for the detection of clinically significant microorganisms in synovial fluid specimens. The Peds Plus/F bottle was used because in our laboratory the quantity of synovial fluid available for culture is frequently in the range of 0.5 to 3.0 ml. The culture results obtained with the Peds Plus/F bottle were compared to those obtained by a conventional agar plate method for a total of 805 synovial fluid specimens. Microbial growth was produced by 74 cultures (9.2%) from 60 patients, yielding a total of 77 microorganisms. Organisms were classified as pathogens ( n = 62), contaminants ( n = 12), or indeterminate ( n = 3) on the basis of a review of the patients' medical histories. Culture using BACTEC Peds Plus/F bottle detected statistically significantly more pathogens overall (62 versus 51 pathogens [ P = 0.001]) and statistically fewer contaminants overall (1 versus 11 contaminants [ P = 0.006]) than culture by the agar plate method. These results indicate the superior performance of the BACTEC Peds Plus/F bottle over the conventional agar plate method for the detection of clinically significant microorganisms from synovial fluid specimens.

ABSTRACTPneumocystis cariniiremains an important opportunistic fungal pathogen causing life-threatening pneumonia in patients with AIDS and malignancy. Currently, little is known about how the organism adapts to environmental stresses and maintains its cellular integrity. We recently discovered an open reading frame approximately 600 bp downstream of the region codingGSC-1, a gene mediating β-glucan cell wall synthesis inP. carinii. The predicted amino acid sequence of this new gene, termedP. carinii PHR1,exhibited 38% homology toSaccharomyces cerevisiae GAS1, a glycosylphosphatidylinositol-anchored protein essential to maintaining cell wall integrity, and 37% homology toCandida albicans PHR1/PHR2, pH-responsive genes encoding proteins recently implicated in cross-linking β-1,3- and β-1,6-glucans. In view of its homology to these related fungal genes, the pH-dependent expression ofP. carinii PHR1was examined. As inC. albicans,P. carinii PHR1expression was repressed under acidic conditions but induced at neutral and more alkaline pH.PHR1-related proteins have been implicated in glucan cell wall stability under various environmental conditions. Although difficulties withP. cariniiculture and transformation have traditionally limited assessment of gene function in the organism itself, we have successfully used heterologous expression ofP. cariniigenes in related fungi to address functional correlates ofP. carinii-encoded proteins. Therefore, the potential role ofP. carinii PHR1in cell wall integrity was examined by assessing its ability to rescue anS. cerevisiae gas1mutant with absent endogenous Phr1p-like activity. Interestingly,P. carinii PHR1DNA successfully restored proliferation ofS. cerevisiae gas1mutants under lethal conditions of cell wall stress. These results indicate thatP. carinii PHR1encodes a protein responsive to environmental pH and capable of mediating fungal cell wall integrity.

For many years it has been recognized that sex steroids have profound effects on bone metabolism. The current perception is that estrogen decreases bone resorption and androgen increases bone deposition. To investigate the potential for androgens to directly modulate bone resorption, we have examined avian osteoclast and human and mouse osteoclast-like cells for androgen responsiveness. There was a dose-dependent decrease in resorption activity in response to α-dihydrotestosterone (α-DHT), β-DHT, testosterone, or the synthetic androgen RU1881. This decrease was blocked by cotreatment with the specific androgen antagonist hydroxyflutamide. Further examination of avian osteoclasts revealed that the cells exhibited specific and saturable nuclear binding of tritiated RU1881 and that α-DHT stimulated the activity of the androgen response element as measured by using a chloramphenicol acetyltransferase reporter plasmid. In addition, avian osteoclasts responded to androgen treatment with elevated production and secretion of transforming growth factor β, a well documented response to androgen exposure in other cell systems. Treatment with either α-DHT or β-DHT for 24 hours resulted in a significant dose-dependent decrease in secretion of cathepsin B and tartrate-resistant acid phosphatase. This response to β-DHT, a stereoisomer of α-DHT that is inactive in other androgen receptor-dependent systems, supports the hypothesis that the osteoclast androgen receptor has unusual ligand-binding properties. Taken together, these results confirm the presence of functional androgen receptors in these cells and support the conclusion that osteoclasts are able to respond directly to androgens in vitro and thus are potential androgen target cells in vivo .

Cyclic ADP ribose (cADPR) has been shown to trigger Ca 2+ release from intracellular stores through ryanodine receptor/channel. In our previous study we observed that all- trans- retinoic acid stimulates cADPR synthesis by ADP ribose cyclase (ADPR cyclase) in cultured epithelial cells. We have now investigated whether cADPR may play a signaling role in action of β-estradiol (E 2 ), an archetypal steroid superfamily hormone, upon its major target organ, uterus, in vivo . Administration of E 2 to gonadectomized rats (0.2 mg/kg per day for 7 days) resulted in an ≈Δ + 300% increase of ADPR cyclase activity in extracts from uterus, but in liver, brain, or skeletal muscle ADPR cyclase was unchanged. Most of the E 2 -stimulated uterine ADPR cyclase was associated with membranes. The higher ADPR cyclase activity in response to E 2 was due to the increase of V MAX without change in K m . Simultaneous administration of estrogen antagonist tamoxifen (8 mg/kg per day) with E 2 (0.2 mg/kg per day) prevented an increase in ADPR cyclase. In uterine extracts from E 2 -treated rats, the rate of cADPR inactivation by cADPR hydrolase and the activity of NADase was increased, but to a much lesser degree than activity of ADPR cyclase. Our results indicate that E 2 , via action to its nuclear receptors in vivo , increases ADPR cyclase activity in uterus. We propose that some of the estrogen effects, and by extension the effects of other steroid superfamily hormones, upon specialized cellular functions and upon hormone-induced gene expression in target cells, are mediated by cADPR-Ca 2+ release pathway.

Nitric oxide (NO), synthesized from l -arginine by NO synthases (NOS), plays an essential role in the regulation of cerebrovascular tone. Adenoviral vectors have been widely used to transfer recombinant genes to different vascular beds. To determine whether the recombinant endothelial NOS (eNOS) gene can be delivered in vivo to the adventitia of cerebral arteries and functionally expressed, a replication-incompetent adenoviral vector encoding eNOS gene (AdCMVNOS) or β-galactosidase reporter gene (AdCMVLacZ) was injected into canine cerebrospinal fluid (CSF) via the cisterna magna (final viral titer in CSF, 10 9 pfu/ml). Adventitial transgene expression was demonstrated 24 h later by β-galactosidase histochemistry and quantification, eNOS immunohistochemistry, and Western blot analysis of recombinant eNOS. Electron microscopy immunogold labeling indicated that recombinant eNOS protein was expressed in adventitial fibroblasts. In AdCMVNOS-transduced arteries, basal cGMP production and bradykinin-induced relaxations were significantly augmented when compared with AdCMVLacZ-transduced vessels ( P < 0.05). The increased receptor-mediated relaxations and cGMP production were inhibited by eNOS inhibitors. In addition, the increase in cGMP production was reversed in the absence of calcium, suggesting that the increased NO production did not result from inducible NOS expression. The present study demonstrates the successful in vivo transfer and functional expression of recombinant eNOS gene in large cerebral arteries. It also suggests that perivascular eNOS gene delivery via the CSF is a feasible approach that does not require interruption of cerebral blood flow.