Development and evaluation of an internally controlled semiautomated PCR assay for quantification of cell-free cytomegalovirus

Quantification of circulating human cytomegalovirus (HCMV) is useful in clinical contexts such as virological surveillance of bone marrow transplant recipients and monitoring of antiviral therapy. This report describes an internally controlled, quantitative, semiautomated, HCMV genome assay that was developed primarily to measure HCMV DNA in the plasma of severely leucopaenic patients. It exhibits greater sensitivity, wider dynamic range and higher sample throughput than a number of previously described commercial and "in-house" assays. Viral DNA extraction from EDTA plasma samples was automated using a BioRobot 9604 (Qiagen). HCMV strain AD169 was used to prepare a calibration curve and murine cytomegalovirus (MCMV) strain Smith was added as internal control to all calibration standards and test samples. Amplification was performed using a set of primers based on the HCMV UL50 region, capable of amplifying both human and murine CMV. The yield of biotinylated polymerase chain reaction (PCR) products was estimated using HCMV-specific and MCMV-specific enzyme-labelled probes and automated chemiluminescence detection. Log-transformed HCMV-to-MCMV signal ratios were calculated and used for quantification of test samples against simultaneously extracted MCMV-spiked calibration standards. Evaluation of the assay sensitivity by Probit analysis demonstrated a 95% probability of detection at 100 HCMV genomes per ml of plasma; the dynamic range was shown to be > or = 4 log(10). A total of 315 samples from 61 bone marrow transplant patients were analysed by both the quantitative PCR (qPCR) and by a previously validated nested nonquantitative PCR (NQPCR). A high level of concordance (90%) was observed between the two assays, although the qPCR assay exhibited slightly greater sensitivity.     

Topical glutamine therapy in experimental inflammatory bowel disease

This study investigated the effects of glutamine and steroid enemas on disease activity in an animal model of colitis. Colitis was induced in male Wistar rats by intracolonic instillation of 30 mg trinitrobenzenesulphonic acid in 50% ethanol (TNBS/E). Controls were given an isovolumetric bolus of normal saline. After 24 h, animals were randomised to receive enemas (1 mL twice daily) of prednisolone (200 mg/L), or L-glutamine (500 g/L) or the suspending agent (placebo). On day 8, the colon was weighed and the degree of inflammation assessed using a colon macroscopic score (CMS). Thymic weight, splenic weight, percentage gain in body weight (%GBW), food intake, plasma interleukin-6 (IL6) and plasma alpha(2)-macroglobulin (alpha(2)M) were also determined. There was a significant increase in CMS, colon weight, splenic weight, IL6 and alpha(2)M in TNBS/E animals compared to controls (P< 0.01). There was also a significant decrease in %GBW, food intake and thymic weight in TNBS/E animals (P< 0.01). The therapeutic enema of prednisolone reduced colonic inflammation (CMS, colon weight), improved thymic weight, %GBW and food intake, and reduced plasma IL6 concentrations (P< 0.05). In contrast administration of glutamine enemas was associated with an exaggerated acute phase protein (alpha(2)M) response (P< 0.05) and failed to improve the colonic and systemic inflammatory response in this experimental model of colitis.     

Development of a soy food frequency questionnaire to estimate isoflavone consumption in US adults.

OBJECTIVE: To develop a dietary assessment instrument to measure soy food consumption and isoflavone intake and test it for reliability. DESIGN: A soy food frequency questionnaire, designed for use in this study, was administered twice to participants, separated by a 2-week interval. The questionnaire was tested for reproducibility of estimates of soy food consumption and isoflavone (genistein and daidzein) intake. SUBJECTS/SETTING: A convenience sample of 51 faculty, staff, and students was recruited from a naturopathic university. STATISTICAL ANALYSES PERFORMED: Intraclass correlation coefficients were used to assess reproducibility of estimates of soy food intake and genistein and daidzein intake. RESULTS: Correlation coefficients comparing mean soy food servings per month between the 2 administrations of the questionnaire ranged from 0.50 for soy yogurt to 0.89 for tempeh. Correlation coefficients for genistein and daidzein intake estimated by the 2 administrations of the questionnaire were the same: 0.89. Mean intake (+/- standard deviation) of genistein and daidzein was 7 +/- 10 and 4 +/- 6 mg/day, respectively. Fifteen soy foods contributed 95% of the total genistein and daidzein intake: tofu, soy yogurt, tempeh, soy milk, low-fat tofu, soy flour, miso, soy protein isolate, low-fat soy milk, veggie soy burger, textured vegetable protein, miso soup, cooked soybeans, soy hot dogs, and natto (fermented soy beans). APPLICATIONS: The soy food frequency questionnaire developed in this study provided highly reproducible estimates of soy food consumption and isoflavone intake. This instrument may be a useful tool in studies of the associations between isoflavone exposure and risk for chronic disease.     

Transplantation tolerance: a look at the nonhuman primate literature in the light of modern tolerance theories

Ever since the beginning of clinical transplantation, investigators have searched for a way to transplant tissues from one person to another without chronic immunosuppression. That goal, known as allograft tolerance, has remained clinically elusive. In the past decade, however, many of the fundamental principles of tolerance have been redefined, and biological agents capable of exploiting them in vivo have been developed. Accordingly, experimental methods for tolerance induction have rapidly evolved in concert with a growing understanding of physiological tolerance to self and the development of novel immunoreactive reagents. In general, old world monkeys have become the pre-clinical testing ground for methods that have shown reasonable promise for clinical application, particularly antibodies or other biological agents with limited cross-species reactivity. As such, a survey of the nonhuman primate experience in transplantation is representative of all reasonably successful experimental attempts to develop clinically applicable tolerance regimens. This article summarizes many of the concepts currently unfolding in the tolerance literature. It also reviews the techniques for tolerance induction that have been and are currently being investigated in nonhuman primates. The validity of these models is summarized, and the older literature is reinterpreted in light of recent changes in our understanding of tolerance.     

Severe micrognathia, cleft palate, absent olfactory tract, and abnormal rib development: cerebro-costo-mandibular syndrome or a new syndrome?

We report on a family in which two sibs had apparently absent ribs and severe micrognathia on prenatal ultrasonography. The pregnancies were terminated at 19 and 12 weeks of gestation, respectively. Autopsy findings in the first fetus (19 weeks of gestation) included severe micrognathia, a U-shaped defect of the soft palate, marked postnuchal edema, absent olfactory bulbs, and cribriform plate and rib abnormalities. The ribs consisted of cartilage anteriorly, with only a small amount of fibrous tissue present laterally and posteriorly. The second fetus (12 weeks gestation) had agnathia, with a large U-shaped defect in the soft palate. There was moderate postnuchal edema. The ribs were unossified and there were gaps in the cartilage where primitive mesenchyme was present posteriorly and laterally. These findings are consistent with a severe form of cerebro-costo-mandibular syndrome. The early fetal histopathology of both cases suggests a possible mechanism by which the characteristic "rib gaps" of cerebro-costo-mandibular syndrome may develop, with evidence for abnormal function of a gene or genes involved in regulation of rib chondrogenesis.     

Sleep-disordered breathing in patients with myelomeningocele: the missed diagnosis

Moderate to severe sleep-disordered breathing (SDB) was identified in 20% (17 of 83) of children with spina bifida/myelomeningocele (SB/MM) at the Montreal Children's Hospital. The prevalence of SDB in patients with SB/MM elsewhere has not been determined. To establish current practices for identifying SDB in patients with SB/MM, questionnaires were sent to the coordinators of the 212 spina-bifida clinics in Canada and in the United States. Eighty-six (41%) questionnaires were returned, representing data on 13 349 patients. Although 67% of the responding centers reported availability of cardiorespiratory sleep studies, only 996 (7.5%) patients with SB/MM had been tested and only 418 (3.1%) patients had been diagnosed with SDB. Across clinics, the prevalence of SDB was directly related to the frequency of testing. Of 380 deaths over the past 10 years, SDB and sudden unexplained death during sleep were identified as the cause of death in 49 (12.8%) and 34 (8.9%) patients, respectively. Moderate to severe SDB may not have been identified in a significant number of patients with SB/MM because they have not been tested.     

Axonal regeneration in the central nervous system of aplysia californica determined by anterograde transport of biocytin

Rhythmic biting, a component of consummatory feeding behavior in the sea hare Aplysia californica, is eliminated following bilateral cerebral-buccal connective (CBC) crushes and recovers within 14 days postlesion. To assess axonal regeneration after CBC lesions, we used biocytin backfills of CBCs followed by fluorescence labeling with streptavidin-lissamine rhodamine. Anterograde transport of biocytin showed up to 1 mm of outgrowth by regenerating axons at 3 days postlesion. At 7 days postlesion, the regenerated axons approached or had entered the ipsilateral buccal neuropil and exhibited numerous varicosities; the average rate of axonal growth was 326 microm/day for the longest, most rapidly growing axons labeled in the CBC. The number of varicosities on labeled axons, suggestive of intercellular interactions, was increased dramatically at all times postlesion. At 14 and 20 days postlesion, regenerated axons branched extensively in the ipsilateral buccal neuropil, entered the contralateral buccal neuropil, and entered peripheral nerves on both sides of the midline. At these later times postlesion, some labeled axons encircled unlabeled buccal cell bodies and exhibited branches containing numerous varicosities, indicative of axosomatic contacts. Some regenerating axons were observed in the sheath of the CBC, but the vast majority of labeled axons remained confined to the connective core, as in control preparations. The bilateral projections within the buccal ganglia of labeled cerebral-to-buccal axons and the large number of varicosities present on these processes are indicative of regenerating axons and synapses that likely contribute to the functional recovery of rhythmic biting.