Induction of central tolerance by intrathymic inoculation of adenoviral antigens into the host thymus permits long-term gene therapy in Gunn rats

Recombinant adenoviruses are highly efficient at transferring foreign genes in vivo. However, duration of gene expression is limited by the host antiviral immune response which precludes expression upon viral readministration. We tested the feasibility of prolonging gene expression by induction of central tolerance to adenoviral antigens in bilirubin-UDP-glucuronosyltransferase-1 (BUGT1)-deficient Gunn rats. Tolerance was induced by intraperitoneal injection of antilymphocyte serum, followed by intrathymic inoculation of one of the following: a recombinant adenovirus (Ad), adenovirus human UDP-glucuronosyltransferase (Ad-hBUGT1) carrying the hBUGT1 gene; a protein extract of the same virus; or viral infected hepatocytes. Controls received intrathymic injections of normal saline. After 12 d all groups were injected intravenously with 5 x 10(9) pfu of either Ad-hBUGT1 or adenovirus beta-galactosidase (Ad-LacZ) (expressing the Escherichia coli beta-galactosidase [LacZ] gene). In all three groups of tolerized rats, hBUGT1 was expressed in the liver after administration of Ad-hBUGT1, with glucuronidation of biliary bilirubin of above 95%. Serum bilirubin levels decreased from 7.2 to 1.8 mg/dl within 1 wk and remained low for 7 wk. Similar findings were observed following repeat injections given on days 45 and 112. In control rats serum bilirubin levels were reduced for only 4 wk, and viral readministration was ineffective. In all tolerized groups, but not in controls, there was a marked inhibition of appearance of neutralizing antibodies and cytotoxic lymphocytes against the recombinant adenovirus. Injection of wild type adenovirus-5 (Ad5) into the tolerized rats elicited a wild type-specific cytotoxic lymphocyte response. This is the first demonstration of Ad-directed long-term correction of an inherited metabolic disease following central tolerization with thymic antigen.     

Nutrition Screening Initiative Checklist may be a better awareness/educational tool than a screening one

OBJECTIVE: To measure responses of cows to supplemental Se intake in excess of nutritional requirements, but lower than recognized toxic dosages. ANIMALS: 24 healthy adult Holstein cows. PROCEDURE: Cows were allotted to 4 groups and fed sodium selenite to provide 0, 3, 20, or 50 mg of Se/cow/d for 90 days. Subsequently, the dosage for the group receiving 50 mg/cow/d was increased to 100 mg/cow/d for 28 d. Blood, liver specimens, feces, and urine were obtained at points during the trial. RESULTS: Serum and blood Se concentrations in groups receiving 20 or 50 mg/cow/d increased over time, compared with controls (P < 0.01). Increasing supplemental Se intake to 100 mg/cow/d further increased serum and blood Se concentrations (P < 0.05). Urine, fecal, and liver Se concentrations increased more markedly in response to treatment than did those of serum or blood. No effect of Se treatment was seen on blood cell counts or serum activities of hepatocellular enzymes. Likewise, neither titer response to rabies vaccination nor lymphocyte blastogenic response to nonspecific mitogens was affected by Se treatment. Objective or subjective physical signs of Se toxicosis were not observed at any Se dosage. CONCLUSIONS: Inorganic Se intakes of up to 50 mg/d for 90 days or 100 mg/d for 28 days by adult Holstein cows do not affect the variables measured. CLINICAL RELEVANCE: Intakes of Se as sodium selenite in amounts 10 to 30 times the nutritional requirements are unlikely to cause health problems in adult cows. Urine and feces are good test samples for detection of Se supplementation greater than requirements.     

The effect of AZT on dendritic cell number and provirus load in the peripheral blood of AIDS patients: a preliminary study

In this pilot study, the numbers of dendritic cells (DC) in peripheral blood of AIDS patients and the level of infection with HIV1 were determined before and after AZT treatment. Mononuclear cells were cultured overnight and DC were identified by their lack of labelling with antibodies specific for T, B and natural killer (NK) cells and monocytes and by their high level of staining with antibodies for MHC class II molecules. Although the numbers of DC identified by this method were lower than those identified morphologically in earlier studies (Macatonia et al., 1990), the numbers in three untreated AIDS patients were below the range seen in normals. There was also a marked rise in DC number in patients given AZT therapy. In two patients, there was a significant provirus load in the DCs which was decreased two to three weeks after the commencement of AZT therapy. The studies suggest that DC numbers and their infection levels may be markers of disease in HIV infection.     

Maternal ego development and mother-infant interaction in drug-abusing women

The efficacy and safety of remifentanil and alfentanil for patients undergoing major abdominal surgery were compared. Premedicated patients received a loading dose of remifentanil (1.0 microgram.kg-1; n = 116) and a continuous infusion of 0.5 microgram.kg-1.min-1, or a loading dose of alfentanil (25 micrograms.kg-1; n = 118) and a continuous infusion of 1.0 microgram.kg-1.min-1. Propofol was administered (10 mg every 10 s) until loss of consciousness. Patients' lungs were ventilated with 66% nitrous oxide and 0.5% (end-tidal) isoflurane in oxygen. The study drug infusion rate was reduced by 50% 5 min after intubation. Alfentanil was discontinued 15 min before the end of surgery, whereas remifentanil was continued in the immediate postoperative period at a reduced dose. Responses to intubation (28%) and skin incision (17%) occurred approximately twice as often in the alfentanil group (15% and 8%; p = 0.014 and p = 0.037, respectively). More patients receiving alfentanil had one or more responses to surgery (72% vs. 57%; p = 0.016). The time to spontaneous respiration, adequate respiration, response to verbal command and time to recovery room discharge were similar. However, owing to decreased variability, the time to extubation was shorter with remifentanil than with alfentanil (p = 0.048). There was a similar overall incidence of adverse events in both groups, 82% and 75% of patients, respectively. Adverse events associated with remifentanil were rapidly controlled by dose reductions. The incidence of intra-operative hypotension and bradycardia was higher in the remifentanil group (p < or = 0.033). An initial remifentanil infusion rate of 0.1 microgram.kg-1.min-1 titrated to individual need provided postoperative pain relief in the presence of adequate respiration in 71% of patients. When using remifentanil in the immediate postoperative setting, rapid administration of bolus doses and infusion rate increases resulted in a relatively high incidence of muscle rigidity, respiratory depression and apnoea. Changing the postoperative regimen to avoid rapid changes in remifentanil blood concentration resulted in more effective analgesia and dramatically reduced the incidence of adverse events during this period. In patients undergoing major abdominal surgery, remifentanil appears to offer superior intra-operative haemodynamic stability during stressful surgical events compared with alfentanil without compromising recovery from anaesthesia. Remifentanil can be administered as a postoperative analgesic agent at a starting dose of 0.1 microgram-.kg-1.min-1; however, it should only be used in the presence of adequate supervision and monitoring of the patient. Administration of bolus doses is not recommended in this setting.     

De novo heme proteins from designed combinatorial libraries

We previously reported the design of a library of de novo amino acid sequences targeted to fold into four-helix bundles. The design of these sequences was based on a "binary code" strategy, in which the patterning of polar and nonpolar amino acids is specified explicitly, but the exact identities of the side chains is varied extensively (Kamtekar S, Schiffer JM, Xiong H, Babik JM, Hecht MH, 1993, Science 262:1680-1685). Because of this variability, the resulting collection of amino acid sequences may include de novo proteins capable of binding biologically important cofactors. To probe for such binding, the de novo sequences were screened for their ability to bind the heme cofactor. Among an initial collection of 30 binary code sequences, 15 are shown to bind heme and form bright red complexes. Characterization of several of these de novo heme proteins demonstrated that their absorption spectra and resonance Raman spectra resemble those of natural cytochromes. Because the design of these sequences is based on global features of polar/ nonpolar patterning, the finding that half of them bind heme highlights the power of the binary code strategy, and demonstrates that isolating de novo heme proteins does not require explicit design of the cofactor binding site. Because bound heme plays a key role in the functions of many natural proteins, these results suggest that binary code sequences may serve as initial prototypes for the development of large collections of functionally active de novo proteins.     

Seroquel restores sensorimotor gating in phencyclidine-treated rats

Phencylidine (PCP) is a psychotomimetic noncompetitive glutamate antagonist that has been used in studies of the neural substrates of psychosis. Both schizophrenic patients and PCP-treated rats exhibit reduced amounts of prepulse inhibition (PPI) of the startle reflex, which is the normal inhibition of startle that occurs when the starting noise is preceded 30 to 500 msec by a weak prepulse. The present study assessed the effects of seroquel (ICI 204,636), a mixed D2/5-hydroxytryptamine2 antagonist with a preclinical profile suggestive of potential antipsychotic efficacy, on the PCP-induced disruption of PPI. Clozapine, risperidone and haloperidol were also studied as comparison compounds. PCP (1.25 mg/kg) significantly reduced PPI, with prepulses that were 1 to 12 dB above background. Seroquel and clozapine significantly restored PPI in PCP-treated rats, whereas haloperidol and risperidone did not. Similar findings were obtained in studies using separate animals, a slightly lower dose of PCP (1.0 mg/kg) and a high dose of each of these antipsychotics. Separate studies verified that risperidone and haloperidol restored PPI in apomorphine-treated rats. In the present studies, seroquel exhibited a profile consistent with those exhibited by other "atypical" antipsychotics.     

Evaluation of patients with bladder outlet obstruction and mild international prostate symptom score followed up by watchful waiting

OBJECTIVES: To examine the variability of bladder outlet obstruction and mild lower urinary tract symptoms in patients with benign prostatic hyperplasia (BPH) followed up by watchful waiting. METHODS: The International Prostate Symptom Score (IPSS) has four questions related to voiding symptoms and three related to filling symptoms. Scores of 0 to 7, 8 to 19, and 20 to 35 represent mild, moderate, and severe symptoms, respectively. Over a period of 36 months the IPSS questionnaire was administered to 479 patients 50 to 81 years old (mean age 63) with BPH. A pressure-flow study was used to determine the presence of bladder outlet obstruction. On the basis of their scores, the patients were classified into 50 with mild, 227 with moderate, and 202 with severe symptoms. In the present study only patients with a mild score were analyzed. RESULTS: Of 50 patients with mild symptoms, 16 (32%) had bladder outlet obstruction. After a period of 9 to 22 months (mean 17) of watchful waiting, these 16 patients were reviewed. Twelve (75%) of the 16 had bladder outlet obstruction reconfirmed by pressure-flow studies, and 3 (18.8%) of 16 had increased symptoms (moderate symptomatic) and underwent treatment (1 began pharmacologic treatment, and 2 chose transurethral resection). A total of 4 (25%) of 16 patients still had mild voiding disturbances and refused the second urodynamic evaluation. The remaining 34 patients with no obstruction had annual routine follow-up and had persistent mild symptom scores and normal uroflowmetric results. These patients did not undergo another pressure-flow evaluation. CONCLUSIONS: A pressure-flow study is routinely avoided in patients with a mild IPSS. From symptoms alone it was not possible to diagnose bladder outlet obstruction in these patients. Pressure-flow studies and symptom profiles measure different aspects of the clinical condition. After a mean follow-up of 17 months of watchful waiting, 13 (81.2%) of 1 6 patients were clinically stable. Because the need for therapy is dictated by quality of life, it is difficult to propose treatment for patients with minimal symptoms, even in the presence of bladder outlet obstruction.     

Assessing various rehabilitation strategies for coarse coal washery reject dumps in the Hunter Valley,Australia. I. Chemical characteristics

At several coal mines in the Hunter Valley (New South Wales, Australia), the use of coarse coal washery reject (chitter) as a top-dressing material has resulted in successful establishment rates of tree and shrub species by direct seeding. However, concerns have arisen about the potential to downgrade the rural capability of the land for grazing, poor understanding of the material's long-term benefits, and spontaneous combustion issues. To ascertain the suitability of chitter as a final landform surface, a revegetation research trial was initiated at United Collieries coal mine in the Hunter Valley of New South Wales. Experimental plots were established in autumn (May 1998) and spring (November 1998) on a freshly shaped stockpile of chitter. The revegetation trials incorporated four substrate treatments, namely a 1?m and 20?cm capping of overburden, a 20?cm topsoil dressing and a treatment of only chitter. Each of the four substrate treatments incorporated five revegetation treatments, namely pasture species and mulch (straw), pasture species and no mulch, native seed (shrub and tree) and mulch, native seed and no mulch, and planted tubestock trees. Chemical characteristics (pH, EC, total N, nitrate-N, available-P, total P, total S, Ca, Na, Mg, K, organic C and soil moisture) across the substrate and vegetation treatments were assessed at 0, 3, 6 and 12 months after establishment.

Laboratory analyses of soil samples from the autumn and spring substrate sowings concluded that the chitter treatment contained a significantly higher concentration of organic carbon (23 – 41%), Na (2.8 – 13 me/100g), total N (2600 – 5300?mg/kg) and S (900 – 4000?mg/kg), and pH was elevated (7.5 – 9.8), relative to the other substrates. The topsoil substrate had significantly greater moisture content and more nitrate-N in comparison to the overburden and chitter materials, and exhibited a significantly lower pH over time. The two overburden treatments showed similar chemical properties and contained significantly higher levels of available P relative to topsoil and chitter. In comparison to recommended levels, all substrates recorded adequate levels of Ca, Mg and K across the autumn and spring assessments and were generally deficient in nitrate-N. In the overburden materials, levels of organic carbon, electrical conductivity, exchangeable bases, total P and available P were generally adequate. However, the pH (8.1 – 8.9) and S (200 – 700?mg/kg) content of overburden was consistently high, and deficiencies in total and available nitrogen were commonly detected. Assessment of substrate samples across vegetation treatments showed few differences with only K, available P and nitrate-N varying significantly between treatments. In both the autumn and spring trials, the concentration of available P in pasture plots was significantly higher in comparison to the other vegetation treatments, while K levels were significantly higher in the pasture and mulch treatment relative to tubestock plots. This probably resulted from greater uptake of these nutrients by trees and shrubs compared to pasture species. In the autumn trials, seeded native tree plots contained significantly more nitrate-N than the other treatments, probably due to N-fixation by Acacia spp. Across both establishment seasons, topsoil provided the best chemical substrate for the establishment of vegetation and chitter was deemed the least hospitable substrate, while the different vegetation treatments appeared to have little effect on the chemical characteristics of the substrates. Vegetation characteristics across the treatments are presented in an accompanying paper (Charnock and Grant, Assessing various rehabilitation strategies for coarse coal washery reject dumps in the Hunter Valley, Australia. II. Vegetation characteristics. International Journal of Surface Mining, Reclamation and Environment, 2005 (in review)).