Long-term liver allograft survival induced by combined treatment with rAAV-hCTLA4Ig gene transfer and low-dose FK506

BACKGROUND: Recombinant adeno-associated virus vector (rAAV) is a promising vehicle for gene delivery, but few reports have documented its application in solid organ transplantation. In a rat orthotopic liver transplantation model, we investigated the efficacy of rAAV-mediated human cytotoxic T-lymphocyte-associated antigen 4 and immunoglobulin G (hCTLA4Ig) gene transfer to induce long-term allograft survival. METHODS: Dark Agouti and Lewis rats were used as donors and recipients, respectively, in six experimental groups: (a) syngeneic control, (b) no treatment, (c) rAAV-green fluorescent protein, (d) rAAV-hCTLA4Ig, (e) low-dose FK506 for 7 days, and (f) rAAV-hCTLA4Ig and low-dose FK506 for 7 days. RESULTS: The liver allografts were rejected within 10 days when no treatment was given or rAAV-green fluorescent protein was delivered. rAAV-hCTLA4Ig transduction slightly prolonged the survival time to 11 days. Long-term survival was achieved using the combined treatment of rAAV-hCTLA4Ig and low-dose FK506, whereas grafts were rejected on day 33 in the low-dose FK506 group. A sustained hCTLA4 level in plasma was detected in the combined treatment group from day 5 to day 180. On postoperative day 5, combined treatment significantly decreased the interleukin-2 and interferon-gamma protein levels in the grafts and the number of infiltrating B, T, CD25+, CD4+, CD8+, and NK cells. CONCLUSIONS: This study shows that rAAV-hCTLA4Ig gene transfer combined with low-dose FK506 can achieve long-term liver allograft survival.     

Radiation therapy depletes extrachromosomally amplified drug resistance genes and oncogenes from tumor cells via micronuclear capture of episomes and double minute chromosomes

PURPOSE: To determine if clinically relevant doses of ionizing radiation are capable of inducing extrachromosomal DNA loss in transformed human cell lines. MATERIALS AND METHODS: The multidrug-resistant (MDR) human epidermoid KB-C1 cell line and the human neuroendocrine colon carcinoma line COLO320, which contain extrachromosomally amplified MDR1 drug resistance genes and MYCC oncogenes, were irradiated with 2 Gy fractions up to a total dose of 28 Gy. To track the fate of extrachromosomally amplified genes, cells surviving radiation therapy and unirradiated control cells were analyzed by fluorescent in situ hybridization of chromosomes using MDR1 and MYCC-specific cosmid DNA probes. In addition, total DNA and protein isolated from irradiated and control cells was subjected to Southern and Western blotting procedures, respectively, to determine amplified gene copy number and protein expression levels. Dose-response assays to follow loss of function of the MDR1 gene from KB-C1 cells were also performed. RESULTS: A significant reduction in extrachromosomal DNA, amplified gene copy number, and expression was detected in surviving cells after relatively low doses of radiation. Entrapment of extrachromosomal DNA into micronuclei was a consistent feature of irradiated cells. CONCLUSIONS: Clinically relevant doses of radiation can deplete extrachromosomal DNA in viable human malignant cells and alter their phenotype. Depletion of extrachromosomally amplified genes from tumor cells occurs via entrapment in radiation-induced micronuclei.     

Convolution method to predict drug concentration profiles of 2,3,5,6-tetramethylpyrazine following transdermal application

The objective of this work is to predict the systemic drug concentration of 2,3,5,6-tetramethylpyrazine (TMP) following transdermal application in rabbits from the in vitro skin permeation data. The in vitro skin permeation was studied in Franz diffusion cells. Pharmacokinetic evaluation of TMP following transdermal application and bolus intravenous administration were carried out in New Zealand White (NZW) rabbits. Drug concentration-time curve following transdermal application was predicted via the convolution procedure using an in vitro skin permeation data as a weighting function, and the intravenous data as an unit impulse response. The results showed that the predicted drug concentration following transdermal application by convolution method was in good agreement with the observed drug absorption profiles. These findings indicated that in vitro skin permeation tests could be useful to predict in vivo drug absorption profiles following transdermal application.     

A practice-based intervention to enhance quality of care in the first 3 years of life: the Healthy Steps for Young Children Program

Context  There is growing concern regarding the quality of health care available in the United States for young children, and specific limitations have been noted in developmental and behavioral services provided for children in the first 3 years of life. Objective  To determine the impact of the Healthy Steps for Young Children Program on quality of early childhood health care and parenting practices. Design, Setting, and Participants  Prospective controlled clinical trial enrolling participants between September 1996 and November 1998 at 6 randomization and 9 quasi-experimental sites across the United States. Participants were 5565 children enrolled at birth and followed up through age 3 years. Intervention  Incorporation of developmental specialists and enhanced developmental services into pediatric care in participants' first 3 years of life. Main Outcome Measures  Quality of care was operationalized across 4 domains: effectiveness (eg, families received =" BORDER="0">4 Healthy Steps–related services or discussed >6 anticipatory guidance topics), patient-centeredness (eg, families were satisfied with care provided), timeliness (eg, children received timely well-child visits and vaccinations), and efficiency (eg, families remained at the practice for =" BORDER="0">20 months). Parenting outcomes included response to child misbehavior (eg, use of severe discipline) and practices to promote child development and safety (eg, mothers at risk for depression discussed their sadness with someone at the practice). Results  Of the 5565 enrolled families, 3737 (67.2%) responded to an interview at 30 to 33 months (usual care, 1716 families; Healthy Steps, 2021 families). Families who participated in the Healthy Steps Program had greater odds of receiving 4 or more Healthy Steps–related services (for randomization and quasi-experimental sites, respectively: odds ratio [OR], 16.90 [95% confidence interval {CI}, 12.78 to 22.34] and OR, 23.05 [95% CI, 17.38 to 30.58]), of discussing more than 6 anticipatory guidance topics (OR, 8.56 [95% CI, 6.47 to 11.32] and OR, 12.31 [95% CI, 9.35 to 16.19]), of being highly satisfied with care provided (eg, someone in the practice went out of the way for them) (OR, 2.06 [95% CI, 1.64 to 2.58] and OR, 2.11 [95% CI, 1.72 to 2.59]), of receiving timely well-child visits and vaccinations (eg, age-appropriate 1-month visit) (OR, 1.98 [95% CI, 1.08 to 3.62] and OR, 2.11 [95% CI, 1.16 to 3.85]), and of remaining at the practice for 20 months or longer (OR, 2.02 [95% CI, 1.61 to 2.55] and OR, 1.75 [95% CI, 1.43 to 2.15]). They also had reduced odds of using severe discipline (eg, slapping in face or spanking with object) (OR, 0.82 [95% CI, 0.54 to 1.26] and OR, 0.67 [95% CI, 0.46 to 0.97]). Among mothers considered at risk for depression, those who participated in the Healthy Steps Program had greater odds of discussing their sadness with someone at the practice (OR, 0.95 [95% CI, 0.56 to 1.63] and OR, 2.82 [95% CI, 1.57 to 5.08]). Conclusion  Universal, practice-based interventions can enhance quality of care for families of young children and can improve selected parenting practices.      

Characterization of radioiodinated ligand binding to amyloid beta plaques

Several novel series of iodinated compounds based on the thioflavin backbone structure have been developed and characterized. These iodinated compounds showed high specific binding to amyloid beta (Abeta) aggregates with subnanomolar to nanomolar affinities. Probes like IMPY and MIPA display high brain uptakes and fast washout in normal mice, resulting in low background signals (presumably no amyloid plaques present in normal mouse brain), whereas TZDM shows long brain retention in normal mice suggesting high nonspecific in vivo binding. It is likely that tracers, that is, IMPY or MIPA, with desirable in vivo properties, will provide the highest target to non-target ratio; therefore, they are most likely to be successful as imaging agents targeting Abeta plaques in the brain.     

Alpha 2 adrenergic receptors on GABAergic,putative sleep-promoting basal forebrain neurons

The basal forebrain plays an important role in the modulation of cortical activity and sleep-wake states. Yet its role must be multivalent as lesions reportedly diminish cortical fast activity and also cortical slow activity along with slow wave sleep (SWS). Basal forebrain cholinergic vs. GABAergic cell groups could differentially influence these processes. By labelling recorded neurons with Neurobiotin (Nb) using the juxtacellular technique and identifying them by immunostaining, we previously found that whereas all cholinergic cells increased their firing, the majority of GABAergic neurons decreased their firing in association with evoked cortical activation in urethane-anaesthetized rats. Here, we examined the possibility that such GABAergic, cortical activation 'off' cells might bear alpha 2 adrenergic receptors (alpha2AR) through which noradrenaline (NA) could inhibit them during cortical activation. First using simple dual-immunostaining for glutamic acid decarboxylase (GAD) and the alpha2AAR, we found that the majority (approximately 60%) of GAD-immunopositive (GAD+) neurons through the magnocellular preoptic nucleus (MCPO) and substantia innominata (SI) were labelled for the alpha2AAR. Second, in urethane-anaesthetized rats, we examined whether Nb-labelled, GAD+ cortical activation 'off' neurons that discharged maximally in association with cortical slow wave activity, were immunopositive for alpha2AAR. We found that all the Nb+/GAD+'off' cells were labelled for the alpha2AAR. Such cells could be inhibited in association with cortical activation and waking when noradrenergic locus coeruleus (LC) neurons discharge and be disinhibited with cortical slow waves and SWS when these neurons become inactive. We thus propose that alpha2AR-bearing GABAergic basal forebrain neurons constitute sleep-active and sleep-promoting neurons.     

Effects of alpha-tocopherol on noise-induced hearing loss in guinea pigs

Preventing noise-induced hearing loss (NIHL) by antioxidants is based on the hypothesis that generation of reactive oxygen species is one of the causes of NIHL. alpha-Tocopherol is a naturally occurring antioxidant with no noticeable side effects. In this study, we attempted to protect guinea pigs from developing NIHL by administering alpha-tocopherol. Pigmented male guinea pigs were exposed to a noise (4 kHz octave band, 100 dB SPL), 8 h/day for 3 days consecutively. alpha-Tocopherol (10 mg/kg or 50 mg/kg daily) was given by intraperitoneal injection from 3 days before through 3 days after the noise exposure. Auditory evoked brainstem response (ABR) thresholds at 2, 4 and 8 kHz were recorded prior to the experiment, immediately post-noise, 2 and 8 days post-noise. On day 8 post-noise, after the ABR recording, guinea pigs were decapitated and the cochleae were removed for cochlear surface preparations and scanning electron microscope (SEM) study. ABR threshold shifts of groups receiving alpha-tocopherol were significantly smaller than those of groups not receiving alpha-tocopherol at all frequencies and all time points tested except that of group 3 at 8 kHz 8 days post-noise. No hair cell loss was seen on the surface preparations, but stereocilia loss was found by SEM study. The noise-induced stereocilia loss was significantly decreased by alpha-tocopherol. These results indicate that alpha-tocopherol can attenuate the noise-induced cochlear damage. Further investigations on the preventive effect of alpha-tocopherol on NIHL in noise-exposed workers are necessary.     

Comparison of quality of life measures in heart failure

BACKGROUND: Although numerous health-related quality-of-life instruments are available to measure patients' quality of life, few studies have compared these measures directly to determine how they function in the same group of patients. OBJECTIVE: The purpose of this study was to empirically compare psychometric properties of the Chronic Heart Failure Questionnaire (CHQ), the Minnesota Living with Heart Failure Questionnaire (LHFQ), and the General Health Survey Short-form-12 (SF-12). SAMPLE: A convenience sample of 211 patients with heart failure completed baseline questionnaires; 165 patients completed the entire 26-week study. METHODS: Patients completed telephone interviews at baseline and at 4, 8, and 26 weeks after baseline. To compare mode of administration, a subset of patients (n = 173) completed face-to-face and telephone interviews. RESULTS: Patients reported low-to-moderate health-related quality-of-life overall. Reliability of the three instruments was satisfactory. Responsiveness to changing condition, as evaluated by analysis of variance, receiver operating curve characteristics, and the minimal clinically important difference method, indicated that the CHQ and LHFQ were more responsive to changing conditions than the SF-12. No major differences were noted between the scores of the face-to-face interviews and the baseline telephone interviews. The LHFQ and SF-12 were easier and took less time to administer than the CHQ. CONCLUSIONS: While all three instruments were reliable and valid, the CHQ and LHFQ were more sensitive than the SF-12 in detecting clinically important changes over time.     

Benzophenones and xanthones with isoprenoid groups from Cudrania cochinchinensis

Four new benzophenones with two isoprenoid groups, cudraphenones A-D (1-4), and three new xanthones also with two isoprenoid units, cudraxanthones P-R (5-7), were isolated from the roots of Cudraniacochinchinensis, together with 19 known phenolic compounds. The structures of the new compounds were elucidated by spectroscopic methods. Some compounds exhibited weak cytotoxicity against human oral squamous carcinoma cells (HSC-2) and normal human gingival fibroblasts (HGF). Among them, benzophenones 1-4 showed more potent cytotoxic activities against HSC-2 cells than against HGF cells. On the other hand, xanthones bearing isoprenoid groups showed much lower tumor specificity as compared with the benzophenones, except for geronthxanthone H and isoalvaxanthone. The presence of two sets of hydrophobic and hydrophilic groups in separate domains in each molecule might play a role in the mediation of tumor-specific action.     

Improving abnormal hemorheological parameters in aging guinea pigs by water-soluble extracts of Salvia miltiorrhiza Bunge

Salvia miltiorrhiza Bunge, known as Danshen in Chinese traditional medicine is effective at promoting blood circulation and removing (or decreasing) blood stasis. In the present study, we selected aging, 24-month-old guinea pigs as the animal experimental models and fed them a diet containing 75, 100 or 150 mg/(kg day) of water-soluble extract components of Salvia miltiorrhiza Bunge (WSm) for 28 days, respectively, in order to evaluate the effects of WSm on their abnormal hemorheological parameters.