Development of a qualitative real-time PCR for microbiological quality control testing in mammalian cell culture production.

AIMS: The aim of this study was to develop and evaluate a real-time PCR technology for microbiological control methods to examine individualized cell therapeutics, an emerging class of pharmaceutical formulations. METHODS AND RESULTS: Oligonucleotide primers and hybridization probe for bacterial detection targeting the 16SrRNA gene were adapted based on Nadkarni et al. [Microbiology148 (2002) 257]. For detection of yeast and moulds, primers and probe were designed from conserved sequences of the 18SrRNA gene in this study. The real-time PCR assays were tested on genomic DNA of Escherichia coli and Candida albicans to assess efficiency and linear dynamic range. After successful establishment of robust real-time PCRs, applicability of the assays was evaluated by extracting microbial target DNA from cell-based preparations. Different commercial DNA extraction methods were compared identifying the MagNA Pure DNA Isolation Kit III as the method of choice. Sensitivity was examined for different strains and a detection limit of 102 -103 CFU per ml in a sample containing ~106 mammalian cells per ml was achieved. CONCLUSIONS: This study reports the successful establishment of two qualitative real-time PCR assays, enabling in general the broad-range detection of microbial contaminants in a cell-based sample matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: Individualized cell therapeutics tend to have a short shelf life. Due to lengthy incubation periods, compendial testing according to current pharmacopoeial guidelines may not be applicable. We report a suitable alternative method upon which future microbiological quality control methods for such products could be based on. However, to implement valid rapid microbiological testing methods using real-time PCR technology, further challenges need to be addressed.

Evidence for a prostate cancer susceptibility locus on the X chromosome.

Over 200,000 new prostate cancer cases are diagnosed in the United States each year, accounting for more than 35% of all cancer cases affecting men, and resulting in 40,000 deaths annually. Attempts to characterize genes predisposing to prostate cancer have been hampered by a high phenocopy rate, the late age of onset of the disease and, in the absence of distinguishing clinical features, the inability to stratify patients into subgroups relative to suspected genetic locus heterogeneity. We previously performed a genome-wide search for hereditary prostate cancer (HPC) genes, finding evidence of a prostate cancer susceptibility locus on chromosome 1 (termed HPC1; ref. 2). Here we present evidence for the location of a second prostate cancer susceptibility gene, which by heterogeneity estimates accounts for approximately 16% of HPC cases. This HPC locus resides on the X chromosome (Xq27-28), a finding consistent with results of previous population-based studies suggesting an X-linked mode of HPC inheritance. Linkage to Xq27-28 was observed in a combined study population of 360 prostate cancer families collected at four independent sites in North America, Finland and Sweden. A maximum two-point lod score of 4.60 was observed at DXS1113, theta=0.26, in the combined data set. Parametric multipoint and non-parametric analyses provided results consistent with the two-point analysis. Significant evidence for genetic locus heterogeneity was observed, with similar estimates of the proportion of linked families in each separate family collection. Genetic mapping of the locus represents an important initial step in the identification of an X-linked gene implicated in the aetiology of HPC.

The missing link in rhesus monkey amniotic fluid volume regulation: intramembranous absorption.

OBJECTIVE: To investigate whether intramembranous absorption occurs in the rhesus monkey and its role in amniotic fluid (AF) volume regulation as a possible model for the human fetus. MATERIALS AND METHODS: We studied five chronically catheterized rhesus monkey fetuses (Macaca mulatta) at 126 +/- 1 (standard error) days' gestation (term approximately 165 days) with ligated esophagi and catheterized tracheae. Samples (0.5 mL each) of fetal and maternal blood and amniotic and lung fluid were collected at 0, 15, 30, 60, 120, 180, and 240 minutes after injection of 0.1 mCi (3 mL) of technetium-99m (Tc-99m) into the amniotic cavity. RESULTS: In spite of esophageal ligation, there was a rapid absorption of the Tc-99m into the fetal circulation within 15 minutes of injection. The maternal Tc-99m activity increased in parallel to fetal activity but remained lower. The fetal lung fluid Tc-99m activity increased more slowly but was equivalent to the fetal circulating level by 4 hours. CONCLUSIONS: These results suggest that intramembranous absorption occurs and may play an important role in rhesus AF volume regulation and composition. Furthermore, this animal model, which closely resembles the human, may provide valuable insight into abnormalities of human AF volume regulation.

Fetal monkey surfactants after intra-amniotic or maternal administration of betamethasone and thyroid hormone.

OBJECTIVE: To compare direct intra-amniotic injection of betamethasone and thyroxine (T4) with maternal treatment and controls for accelerating pulmonary surfactant production. METHODS: Twelve pregnant monkeys (Macaca mulatta) on gestational day 125 (term 165 +/- 10 days) had surfactant protein A and B concentrations measured in amniotic fluid. In four controls, normal saline was injected into the amniotic fluid; four others (intra-amniotic) received intra-amniotic betamethasone (1 mg) and T4 (60 microg); and in four others (maternal), the dam was given betamethasone (12 mg) intramuscularly, repeated in 24 hours, plus TRH (400 microg) intravenously, repeated every 6 hours for 24 hours. Seventy-two hours after the initial amniocentesis, a hysterotomy was performed and fetal tissue and amniotic fluid harvested for determination of surfactant protein A and B concentrations and immunohistochemical staining for surfactant protein A. RESULTS: Amniotic fluid surfactant protein A was higher with intra-amniotic injection than with maternal treatment (P <.04) or controls (P =.07). Amniotic fluid surfactant protein B was higher in the intra-amniotic group than in controls (P =.06). Immunohistochemical staining for surfactant protein A in the lung tissue was increased in the intra-amniotic group compared with controls (0.145 +/- 0.01 versus 0.097 +/- 0.001, percent positive staining for surfactant protein A cells per lung tissue cells; P <.03). Birth weight was greater in the intra-amniotic group compared with the maternal group (P <.03) although not different from the controls. Finally, gut motility and the presence of formed meconium were increased in the intra-amniotic group compared with the other groups (P <.05). CONCLUSION: Intra-amniotic injection of betamethasone and T4 enhanced lung (and possibly intestinal) maturation of the preterm rhesus fetal monkey compared with maternal injections.

Sequential expression of Egr-1 and Egr-3 in hippocampal granule cells following electroconvulsive stimulation.

Previous studies examining the regulation of immediate early gene mRNAs by neuronal stimulation have revealed that two members of the Egr family of transcription factors, Egr-1 and Egr-3, display parallel response patterns. As these transcription factors compete for the same consensus sequence, we investigated how their expression and DNA binding activities are coordinated. Following electroconvulsive stimulation, which induces rapid increases in both Egr-1 and Egr-3 mRNA levels in dentate granule cells, we found that these proteins are induced sequentially. Egr-1 protein levels peak at 0.5-1 h and decay to basal levels by 4 h. In contrast, Egr-3 protein levels respond more slowly; little change is apparent at 1 h, and peak levels are not reached until 4 h following stimulation. Gel shift assays demonstrated that Egr-1 and Egr-3 DNA binding activities follow the same pattern. These findings indicate that Egr-1 and Egr-3 act in concert to mediate early and late phases, respectively, of the transcriptional response regulated by their cognate response element.

Wound complications and treatment of the infected implantable cardioverter defibrillator generator.

Since 1980, the automatic implantable cardioverter defibrillator (ICD) has evolved as effective therapy for prevention of sudden cardiac death following documented sustained ventricular tachycardia or fibrillation. During a 5-year period, 412 ICD devices were implanted at the University of Michigan Hospitals with a wound complication rate of 4.1%. In this group, there were 13 infections, 3 erosions of the generator pocket, and 1 wound hematoma. Of the 16 patients with infection or erosion, 12 patients were treated with a rectus abdominis muscle flap closure and 4 with ICD generator removal. In 83% (n = 12) of the muscle flap patients, the wound healed uneventfully. Preoperative chest CT scanning was found to be helpful in identifying probable infection of the epicardial leads. In these cases, all hardware had to be removed to achieve resolution of the infection. We concluded that rectus abdominis muscle flaps were helpful in salvaging infected or exposed ICD generators in the absence of infected epicardial leads.

Technetium Tc 99m rapidly crosses the ovine placenta and intramembranous pathway.

OBJECTIVE: This study examined the movement of the soluble ion technetium Tc 99m across the ovine placenta and intramembranous pathway. STUDY DESIGN: Nineteen fetal sheep at 131 +/- 1 (SE) days' gestation were studied. After a 1-hour control period technetium Tc 99m was injected into either a fetal vein (n = 7), the amniotic cavity (n = 5), or a maternal vein (n = 5). Maternal and fetal blood, fetal urine, and amniotic and allantoic fluid were sampled during the control period and for 8 hours after the injection. Fetal urine was drained externally throughout the experiment. In five animals technetium Tc 99m was injected intraamniotically after the fetus was killed with air emboli and sampled as described. RESULTS: Intrafetally injected technetium Tc 99m rapidly crossed the placenta; then it entered and was concentrated in the amniotic cavity. Intraamniotically injected technetium Tc 99m rapidly entered into the fetal circulation. The maternally injected technetium Tc 99m rapidly crossed the placenta into the fetus, suggesting a half-time for placental exchange of < 50 minutes. The technetium Tc 99m injected into the dead fetus group demonstrated significantly less maternal absorption than in the live fetus group. CONCLUSIONS: The soluble ion technetium Tc 99m demonstrated a much more rapid movement in both directions across the ovine placenta then previously demonstrated for the smaller ion sodium. Technetium Tc 99m rapidly crossed the intramembranous pathway bidirectionally, suggesting a high permeability of the intramembranous pathway. Minimal maternal absorption of technetium Tc 99m in the dead fetus group suggests little transmembranous absorption by the mother.

Changes in rates of salivary estriol increases before parturition at term.

OBJECTIVE: The aim of this study was to characterize the increases of salivary estriol concentrations before the onset of labor at term. STUDY DESIGN: Salivary estriol concentrations were measured in weekly patient-collected samples by means of a sensitive (mean +/- SD threshold, 0.025 +/- 0.001 ng/mL; coefficient of variation, 3.8%) direct enzyme immunoassay in a microtiter plate format. The salivary estriol concentrations in 16 healthy pregnant women were characterized from 30 weeks' gestation until the time of parturition and delivery. Samples were stored frozen at collection and analyzed in batches after delivery. RESULTS: The median salivary estriol concentration profile revealed a nonlinear rise beginning from 30 weeks' gestation (0.89 ng/mL) until term (2.70 ng/mL, an increase of 201%). At 35 weeks' gestation the salivary estriol concentration median value increased sharply (positive inflection point, 50%-93% increase) at a demarcation between a slower increase during early pregnancy and a more rapid increase during late pregnancy. This positive inflection point associated with a late pregnancy increase characterized subgroups of pregnancies according to the lengths of gestation as follows: early term (delivered at <38 weeks 1 day's gestation), middle term (delivered at 38 weeks 1 day-40 weeks' gestation), and late term (delivered at >40 weeks' gestation). Five weeks before delivery the mean (+/-SEM) rate of rise in salivary estriol concentration was 0.50 +/- 0.13 ng/mL per week to 0.84 +/- 0.26 ng/mL per week in the early term group. The increase in rate for the middle term group was 0.32 +/- 0.06 ng/mL per week to 0.37 +/- 0.26 ng/mL per week, whereas in the late term group the rate of salivary estriol concentration rise was 0.37 +/- 0.03 ng/mL per week to -0.03 +/- 0.25 ng/mL per week. CONCLUSION: These data demonstrate in normal pregnancies (1) that a direct, nonradiometric measure of salivary estriol concentration can be used to monitor the late pregnancy increase in estriol production, (2) that 35 weeks' gestation marks a positive inflection point of the onset of increased estriol production, and (3) that the late pregnancy rise in salivary estriol concentration shows distinct patterns that tend to be characteristic of the length of pregnancy. These data support the concept that the rate of increase of estriol production is related to the timing of the onset of labor.

Effect of mode of delivery in nulliparous women on neonatal intracranial injury.

BACKGROUND: Infants delivered by vacuum extraction or other operative techniques may be more likely to sustain major injuries than those delivered spontaneously, but the extent of the risk is unknown. METHODS: From a California data base, we identified 583,340 live-born singleton infants born to nulliparous women between 1992 and 1994 and weighing between 2500 and 4000 g. One third of the infants were delivered by operative techniques. We evaluated the relation between the mode of delivery and morbidity in the infants. RESULTS: Intracranial hemorrhage occurred in 1 of 860 infants delivered by vacuum extraction, 1 of 664 delivered with the use of forceps, 1 of 907 delivered by cesarean section during labor, 1 of 2750 delivered by cesarean section with no labor, and 1 of 1900 delivered spontaneously. As compared with the infants delivered spontaneously, those delivered by vacuum extraction had a significantly higher rate of subdural or cerebral hemorrhage (odds ratio, 2.7; 95 percent confidence interval, 1.9 to 3.9), as did the infants delivered with the use of forceps (odds ratio, 3.4; 95 percent confidence interval, 1.9 to 5.9) or cesarean section during labor (odds ratio, 2.5; 95 percent confidence interval, 1.8 to 3.4), but the rate of subdural or cerebral hemorrhage associated with vacuum extraction did not differ significantly from that associated with forceps use (odds ratio for the comparison with vacuum extraction, 1.2; 95 percent confidence interval, 0.7 to 2.2) or cesarean section during labor (odds ratio, 0.9; 95 percent confidence interval, 0.6 to 1.4). CONCLUSIONS: The rate of intracranial hemorrhage is higher among infants delivered by vacuum extraction, forceps, or cesarean section during labor than among infants delivered spontaneously, but the rate among infants delivered by cesarean section before labor is not higher, suggesting that the common risk factor for hemorrhage is abnormal labor.

No evidence for a role of BRCA1 or BRCA2 mutations in Ashkenazi Jewish families with hereditary prostate cancer.

BACKGROUND: Two genes responsible for hereditary breast cancer (BRCA1 and BRCA2) have been identified, and predisposing mutations identified. Several studies have provided evidence that germline mutations in BRCA1 and BRCA2 confer an increased risk of prostate cancer. Based on these findings, one might expect to find an increased frequency of mutations in these genes in family clusters of prostate cancer. The Ashkenazi Jewish population is unique in that it has an approximate 2% incidence of specific founder BRCA1 and BRCA2 mutations (i.e., 185delAG and 5382insC in BRCA1, and 6174delT in BRCA2). METHODS: To address the question of whether or not mutations in either of these genes were overrepresented in prostate cancer families, we searched for these mutations in germline DNA samples collected from affected and unaffected members of 18 Ashkenazi Jewish families, each having at least 3 first-degree relatives affected with prostate cancer. RESULTS: No mutations were found in the BRCA1 gene in any of the 47 individuals tested. One individual possessed a BRCA2 mutation (6174delT). This individual was unaffected at the time of analysis, but had an affected paternal uncle, and an affected first cousin, neither of whom harbored the mutant gene. CONCLUSIONS: In this sample of Ashkenazi prostate cancer families, the frequency of founder BRCA1 and BRCA2 mutations was not elevated, suggesting that such mutations will account for only a small, perhaps minimal, fraction of familial prostate cancer.