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.     

Long-term results after reoperation for failed antireflux procedures

From January 1960 to June 1995, 185 patients underwent reoperation without esophageal resection for symptoms of recurrent gastroesophageal reflux disease. There were 102 men and 83 women. Median age was 58 years (range 20 to 84 years). A single previous antireflux operation had been performed in 147 patients, two in 33, and three in 5. The median interval between the reoperation and the previous operation was 36 months (range 1 to 291 months). Indications for reoperation were symptoms in 184 patients and a large paraesophageal hernia in one patients. The surgical approach was by means of a thoracotomy in 133 patients (71.9%), laparotomy in 27 (14.6%), and a thoracoabdominal incision in 25 (13.5%). A Nissen fundoplication was performed in 107 patients (57.8%), Belsey fundoplication in 47 (25.4%), truncal vagotomy and antrectomy with Roux-en-Y reconstruction in 17 (9.2%), anatomic hernia repair in 12 (6.5%), and Hill gastropexy in 2 (1.1%). A Collis gastroplasty was added to the fundoplication in 116 patients (62.7%), and a pyloroplasty was performed in 17 (9.2%). There was one operative death (0.5%). Complications occurred in 47 patients (25.4%). Median postoperative hospitalization was 9 days (range 5 to 58 days). Follow-up was complete in 156 patients (84.3%) and ranged from 3 to 283 months (median 44 months). Improvement occurred in 137 patients (87.8%). Functional results were classified as excellent in 65 patients (41.6%), good in 29 (18.6%), fair in 43 (27.6%), and poor in 19 (12.2%). No single operative approach or procedure proved to be functionally superior. We conclude that reoperation with esophageal preservation after a failed antireflux procedure will result in significant functional benefit and can be performed with low mortality and acceptable morbidity. The type of repair should be tailored to the individual patient.     

Humoral immunity and regulation of intrapulmonary growth of Legionella pneumophila in the immunocompetent host

The potential role of humoral immunity in regulating intrapulmonary growth of Legionella pneumophila in the immunocompetent host was investigated using a murine model of Legionnaires' disease. Intratracheal inoculation of A/J mice with a virulent strain of L. pneumophila (10(6) bacteria per mouse) resulted in the recruitment of B lymphocytes into the lung and the development of anti-L. pneumophila Ab. Opsonization of L. pneumophila in vitro with anti-L. pneumophila-specific mAb resulted in a significant decrease in intrapulmonary growth of the bacteria at 24 to 72 h postinfection. Transmission electron microscopic analysis of lung tissue from L. pneumophila- infected mice demonstrated that while there was no significant difference between phagocytosis of the unopsonized and opsonized L. pneumophila by alveolar macrophages at 24 h postinfection, phagocytosis of opsonized bacteria by alveolar mononuclear phagocytic cells was significantly enhanced at 48 h postinfection. Depletion of A/J mice of complement before intratracheal inoculation of opsonized L. pneumophila (10(6) bacteria per mouse) did not significantly alter intrapulmonary growth of L. pneumophila. These results suggest that anti-L. pneumophila Ab, produced during replicative L. pneumophila lung infections, may regulate intrapulmonary growth of L. pneumophila in the immunocompetent host by decreasing the viability of extracellular L. pneumophila and by enhancing phagocytosis of the bacteria by alveolar mononuclear phagocytic cells by a complement-independent mechanism.     

400 cycles with assisted fertilization in severe teratozoospermia

From May 1991 to July 1993, assisted fertilisation by micromanipulation (PZD, SUZI and ICSI) was performed in 397 cycles with severe teratozoospermia. In the second series of 152 cycles with SUZI, the pregnancy rate per cycle was 5% (10% per transfer). In the third series, the method was changed from SUZI to ICSI.     

Loss of parathyroid hormone-stimulated 1,25-dihydroxyvitamin D3 production in aging does not involve protein kinase A or C pathways

Intestinal calcium absorption declines with aging as a result of decreased renal 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] biosynthesis. At least part of the decline in 1,25-(OH)2D3 may be due to acquired resistance to parathyroid hormone (PTH) stimulation of renal 25-hydroxyvitamin D1-hydroxylase (1-OHase) activity. To test whether aging rats can increase 1,25-(OH)2D3 production in response to PTH, male rats of the same litter were fed a normal Ca diet and were sacrificed at 175-225 g (young rats) or 3 months later at 350-425 g (aging rats). At sacrifice, basal serum 1,25-(OH)2D3 levels (88 +/- 16 versus 49 +/- 8 pg/ml, P < 0.05) and in vitro renal proximal tubule 1-OHase activity (178 +/- 15 versus 77 +/- 5 pmol/mg protein/5 minutes, n = 6, P < 0.001) were lower in aging animals. rPTH-(1-34) (10(-11) or 10(-7) M) increased in vitro 1,25-(OH)2D3 secretion by perifused renal proximal tubules from young but not aging rats. For young and aging rats, rPTH-(1-34) (10(-7) M) increased proximal tubule cAMP-dependent protein kinase (PKA) activity, and lower concentrations (10(-11) M) stimulated translocation of protein kinase C (PKC) activity from cytosolic to soluble membrane proximal tubule cell fractions. The results of this study show that PTH activation of 1,25-(OH)2D3 production may involve both signaling pathways, with the PKC pathway responsive to lower concentrations of the hormone. The acquired resistance to PTH stimulation of 1,25-(OH)2D3 production in aging appears not to involve the hormonal activation of PKA or PKC.     

Gene recognition via spliced sequence alignment

Gene recognition is one of the most important problems in computational molecular biology. Previous attempts to solve this problem were based on statistics, and applications of combinatorial methods for gene recognition were almost unexplored. Recent advances in large-scale cDNA sequencing open a way toward a new approach to gene recognition that uses previously sequenced genes as a clue for recognition of newly sequenced genes. This paper describes a spliced alignment algorithm and software tool that explores all possible exon assemblies in polynomial time and finds the multiexon structure with the best fit to a related protein. Unlike other existing methods, the algorithm successfully recognizes genes even in the case of short exons or exons with unusual codon usage; we also report correct assemblies for genes with more than 10 exons. On a test sample of human genes with known mammalian relatives, the average correlation between the predicted and actual proteins was 99%. The algorithm correctly reconstructed 87% of genes and the rare discrepancies between the predicted and real exon-intron structures were caused either by short (less than 5 amino acids) initial/terminal exons or by alternative splicing. Moreover, the algorithm predicts human genes reasonably well when the homologous protein is nonvertebrate or even prokaryotic. The surprisingly good performance of the method was confirmed by extensive simulations: in particular, with target proteins at 160 accepted point mutations (PAM) (25% similarity), the correlation between the predicted and actual genes was still as high as 95%.     

CYP2D6 allelic frequencies in young-onset Parkinson's disease

Parkinson's disease (PD) is thought to develop as a result of interactions between genetic susceptibility factors and environmental exposures. One candidate gene is CYP2D6, which codes for the debrisoquine 4-hydroxylase cytochrome P450. Impairment of debrisoquine 4-hydroxylase activity has been associated with an increased risk of PD in patients with younger age at disease onset. Genotyping studies in patients with an older age at onset have reported modest increases in risk associated with the CYP2D6 B and A alleles; however, the risk for young-onset PD has not been adequately evaluated. We designed a case-control study to investigate the role of nonfunctional CYP2D6 allelic risk factors for young-onset PD in a sizable patient population and compared the distributions of CYP2D6 genotypes between young-onset ( < or = 51 years) PD patients (n = 108) and controls (n = 236). In contrast with the results from genotyping studies conducted among patients with an older age at onset, there were no significant differences in CYP2D6 allelic frequencies between young-onset PD cases and controls. The frequency of the B allele was slightly lower in the young-onset PD cases than in the controls (0.14 versus 0.20) (X2 = 2.66, p = 0.10). The presence of one or more B alleles was not associated with an increased risk of young-onset PD (odds ratio 0.58; 95% CI 0.33 to 1.00), nor was the presence of one or more nonfunctional alleles (i.e., A, B, D, and D2) (odds ratio 0.68; 95% CI 0.41 to 1.13). This study suggests that the young-onset PD population may differ from the older-onset population with respect to risk factors.     

Effects of hypothermia or anesthetics on hippocampal glutamate and glycine concentrations after repeated transient global cerebral ischemia

The reovirus group C temperature-sensitive mutant tsC447, whose defect maps to the S2 gene, which encodes the major core protein sigma 2, fails to assemble core particles at the nonpermissive temperature. To identify other proteins that may interact with sigma 2 during assembly, we generated and examined 10 independent revertants of the mutant. To determine which gene(s) carried a compensatory suppressor mutation(s), we generated intertypic reassortants between wild-type reovirus serotype 1 Lang and each revertant and determined the temperature sensitivities of the reassortants by efficiency-of-plating assays. Results of the efficiency-of-plating analyses indicated that reversion of the tsC447 defect was an intragenic process in all revertants. To identify the region(s) of sigma 2 that had reverted, we determined the nucleotide sequences of the S2 genes. In all revertant sequences examined, the G at nucleotide position 1166 in tsC447 had reverted to the A present in the wild-type sequence. This reversion leads to the restoration of a wild-type asparagine (in place of a mutant aspartic acid) at amino acid 383 in the sigma 2 sequence. These results collectively indicate that the functional lesion in tsC447 is Asp-383 and that this lesion cannot be corrected by alterations in other core proteins. These observations suggest that this region of sigma 2, which may be important in mediating assembly of the core particle, does not interact significantly with other reovirus proteins.