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S C Kennedy

Publications and source records attributed to S C Kennedy.

At least 19 recordsLinked to original sources
PubMed2003

Molecular phylogenetics and evolution of the endemic Hawaiian genus Adenophorus (Grammitidaceae).

Recent studies of the phylogeny of several groups of native Hawaiian vascular plants have led to significant insights into the origin and evolution of important elements of the Hawaiian flora. No groups of Hawaiian pteridophytes have been subjected previously to rigorous phylogenetic analysis. We conducted a molecular phylogenetic analysis of the endemic Hawaiian fern genus Adenophorus employing DNA sequence variation from three cpDNA fragments: rbcL, atpbeta, and the trnL-trnF intergenic spacer (IGS). In the phylogenetic analyses we employed maximum parsimony and Bayesian inference. Bayesian phylogenetic inference often provided stronger support for hypothetical relationships than did nonparametric bootstrap analyses. Although phylogenetic analyses of individual DNA fragments resulted in different patterns of relationships among species and varying levels of support for various clades, a combined analysis of all three sets of sequences produced one, strongly supported phylogenetic hypothesis. The primary features of that hypothesis are: (1) Adenophorus is monophyletic; (2) subgenus Oligadenus is paraphyletic; (3) the enigmatic endemic Hawaiian species Grammitis tenella is strongly supported as the sister taxon to Adenophorus; (4) highly divided leaf blades are evolutionarily derived in the group and simple leaves are ancestral; and, (5) the biogeographical origin of the common ancestor of the Adenophorus-G. tenella clade remains unresolved, although a neotropical origin seems most likely.

Base Sequence↗
PubMed2000

Lovastatin decreases mortality and improves liver functions in fulminant hepatic failure from 90% partial hepatectomy in rats.

BACKGROUND/AIMS: Liver insufficiency occurs when the liver cannot perform critical functions such as ammonia metabolism, gluconeogenesis, or production of coagulation factors The hypothesis of this study was that decreased function of existing hepatocytes may contribute to hepatic failure, and that the function of these cells might be increased pharmacologically. Lovastatin is a 3-hydroxy-3-methylglutaryl CoA reductase inhibitor that inhibits cholesterol biosynthesis and affects the activity of some signal transduction pathways and liver transcription factors. Changes in hepatic transcription factors during liver regeneration might result in decreased liver functions, and lovastatin might prevent these changes METHODS: Rats received 90% partial hepatectomy (90% PH), and either lovastatin or vehicle alone daily. Survival and liver functions were assessed. RESULTS: Lovastatin increased survival to 58% (vs. 6% in controls that received 90% PH without drug), decreased the peak ammonia level to 427 microM (vs. 846 microM in controls), increased the nadir of glucose to 88 mg/dl (vs. 57 mg/dl in controls), decreased the peak prothrombin time to 23 s (vs 29 s in controls), and decreased the peak activated partial thromboplastin time to 29 s (vs. 39 s in controls). The full survival and metabolic benefits were observed when lovastatin was started at 30 min after 90% PH, but lovastatin was less efficacious when started at later times. CONCLUSIONS: Lovastatin increases the function of existing hepatocytes and might be used to improve liver function after extensive hepatic resection.

Ammonia↗
PubMed1999

Myocardial kinetics of 99m technetium-Q agents: studies in isolated cardiac myocyte, isolated perfused rat heart, and canine regional myocardial ischemia models.

OBJECTIVE: Based on reports of high cellular uptake and low plasma binding of nonreducible mixed ligand Tc(III) cations (Q complexes) and high linear uptake versus blood flow of 99mTc-Q3 in canine hearts, the authors hypothesized that the two Q complexes, 99mTc-Q63 and 99mTc-Q64, would have high cell uptake and better differentiation between ischemic and nonischemic myocardium compared with other 99mTc-based compounds. METHODS: Uptake and retention kinetics of 99mTc-Q63 and 99mTc-Q64 were measured in isolated rat cardiac myocytes, isolated perfused rat hearts, and intact canines and compared with previously reported Q-based compounds, a clinically available 99mTc perfusion agent (sestamibi), and 201Tl. RESULTS: Uptake of Q63, Q64, and sestamibi by isolated cardiac myocytes was similar. Maximum extraction (Emax) of Q64 by isolated perfused rat hearts was greatest among the 99mTc agents (P < 0.02), but net extraction (Enet) of Q64 was not different from Q63 or sestamibi 3 minutes after tracer injection. By 15 minutes, 201Tl Enet was lower than Q63, Q64, and sestamibi (P < 0.05). Among 99mTc agents, the uptake versus flow of Q3, Q63, and Q64 by canine heart was superior to Q12 and sestamibi (P < 0.05). CONCLUSIONS: The activity of Q63 and Q64 in the myocardium is related to actual myocardial blood flow over a broad, clinically relevant range of flows. The ischemic-to-normal zone activity distributions of Q63 and Q64 approximate actual flow in a manner more like that of 201Tl than sestamibi or Q12. These results provide a rational foundation in support of further evaluation of Q63 and Q64 in humans.

Acute Disease↗
PubMed1998

Therapeutic levels of human protein C in rats after retroviral vector-mediated hepatic gene therapy.

Protein C deficiency results in a thrombotic disorder that might be treated by expressing a normal human protein C (hPC) gene in patients. An amphotropic retroviral vector with a liver-specific promoter and the hPC cDNA was delivered to rat hepatocytes in vivo during liver regeneration. Expression of hPC varied from 55 to 203 ng/ml (1.3-5.0% of normal) for 2 wk after transduction. Expression increased to an average of 900 ng/ml (22% of normal) in some rats and was maintained at stable levels for 1 yr. All of these rats developed anti-hPC antibodies and exhibited a prolonged hPC half-life in vivo. The hPC was functional as determined by a chromogenic substrate assay after immunoprecipitation. We conclude that most rats achieved hPC levels that would prevent purpura fulminans, and that hepatic gene therapy might become a viable treatment for patients with severe homozygous hPC deficiency. Anti-hPC antibodies increased the hPC half-life and plasma levels in some rats, but did not interfere with its functional activity. Thus, the development of antibodies against a plasma protein does not necessarily abrogate its biological effect in gene therapy experiments.

Animals↗
PubMed1997

Therapeutic levels of functional human factor X in rats after retroviral-mediated hepatic gene therapy.

Factor X deficiency results in a rare but serious bleeding disorder that might be treated by expressing a normal factor X gene in patients. We generated an amphotropic retroviral vector with the human FX cDNA and delivered it to rat hepatocytes in vivo during liver regeneration. The human alpha1-antitrypsin promoter was chosen to direct expression because it was the most efficient of several tested in yielding expression of alpha1-antitrypsin protein from a retroviral vector in hepatocytes in vivo. We achieved expression of factor X in four rats at levels sufficient to maintain hemostasis in humans (10% to 43% of normal). The factor X was determined to be functional by using a chromogenic substrate assay after immunoprecipitation with human specific antibodies. Expression of factor X remained stable for more than 10 months in two rats. It is likely that expression will be maintained for the life of the animals, because retroviral vectors integrate into the chromosome and hepatocytes are long-lived. The high and stable levels of expression achieved using this liver-specific promoter overcomes one of the two major obstacles to successful human gene therapy for hemophilia.

3T3 Cells↗
PubMed1996

Portal branch occlusion safely facilitates in vivo retroviral vector transduction of rat liver.

Hepatic gene therapy might correct the clinical manifestations of several genetic disorders in patients. Although retroviral vectors with a strong liver-specific promoter can result in stable and therapeutic levels of expression of genes from the liver, application of these techniques in humans is limited by the need to perform one or more invasive procedures to achieve ex vivo or in vivo transduction of hepatocytes. In vivo delivery involves injection of retrovirus into the portal vein during liver regeneration. Although transduction is efficient and specific for the liver, induction of hepatocyte replication requires a 70% partial hepatectomy or administration of a liver toxin. An alternative method for inducing hepatocyte replication is to occlude branches of the portal vein. This results in apoptosis of hepatocytes in the occluded lobes and compensatory replication of the hepatocytes in the nonoccluded lobes. We demonstrate here that portal branch occlusion is nearly as effective as partial hepatectomy at facilitating retroviral vector transduction in vivo and has a lower morbidity. Portal branch occlusion could be performed in larger animals by minimally invasive techniques and has been used safely to treat human patients with liver cancer. Portal branch occlusion might ultimately be used in humans to facilitate retroviral vector transduction in vivo for the treatment of genetic diseases.

Alanine Transaminase↗
PubMed1996

Liver-directed gene therapy: a retroviral vector with a complete LTR and the ApoE enhancer-alpha 1-antitrypsin promoter dramatically increases expression of human alpha 1-antitrypsin in vivo.

Hepatic gene therapy could improve the treatment of many inherited disorders. Although retroviral vectors result in long-term expression in hepatocytes in vivo, their low level of expression currently precludes most clinical applications. Four copies of the liver-specific apolipoprotein E (ApoE) enhancer were placed upstream of the human alpha 1-antitrypsin (hAAT) promoter in either orientation into a retroviral vector with a complete long terminal repeat (LTR) and the hAAT cDNA to generate ApoE(+)hAAT-LTR and ApoE(-)hAAT-LTR. In addition, the ApoAI promoter was placed upstream of the hAAT cDNA in a similar retroviral vector backbone. Amphotropic retroviral vectors were transferred into regenerating rat liver cells in vivo by intraportal injection. ApoE(-)hAAT-LTR and ApoE(+)hAAT-LTR led to average hAAT levels of 5 micrograms/ml (0.5% of normal levels of a very abundant protein), and 2.5 micrograms/ml, respectively, which was stable for at least 10 months after transduction. This level of serum hAAT was > 25-fold higher than what was observed from the ApoAI promoter used in this study. Serum levels of hAAT were > 15-fold higher than what was observed from retroviral vectors containing the hAAT cDNA that were analyzed previously by this lab. In some cases, improved expression was due to the promoter chosen. In other cases, the increase in expression was primarily due to the higher titers obtained by using a retroviral backbone with an intact LTR as opposed to a vector with a deletion in the LTR. The increased expression levels observed from this enhancer/promoter combination in an intact retroviral backbone may enable one to achieve therapeutic levels of clinically important genes from a retroviral vector in liver cells of animals.

Animals↗
PubMed1996

Retroviral vector sequences may interact with some internal promoters and influence expression.

Although retroviral vectors show promise for gene therapy, their expression in animals has been low. An improved understanding of how promoters function from a retroviral vector should facilitate the design of improved vectors. In this study, liver-specific promoters were cloned into a retroviral vector and expression from the retroviral long terminal repeat (LTR) and the internal promoter was analyzed. In addition, oligomerized liver-specific transcription factor binding sites were placed upstream of each promoter in an attempt to increase expression further. Additional oligomerized binding sites only increased expression slightly or inhibited expression in hepatoma cells, suggesting that this is not an effective way to increase expression from a retroviral vector. Unexpectedly, the liver-specific albumin promoter was expressed at high levels from a retroviral vector in fibroblasts, suggesting that retroviral elements functioned as an enhancer. Furthermore, the addition of HNF-4 binding sites adjacent to the albumin promoter inhibited both the LTR and albumin promoter in fibroblasts, an effect that was probably mediated by inhibitory proteins present in nonhepatic cells that can bind to HNF-4 sites. These results suggest that both positive and negative influences can be transmitted between the LTR and the albumin promoter. In contrast, the liver-specific human alpha 1-antitrypsin promoter did not appear to interact with the LTR by either of these criteria. Retroviral vectors have sequences that may inhibit expression of the LTR and some internal promoters in vivo. We hypothesize that internal promoters that do not interact with the LTR in tissue culture will be resistant to inhibitory effects of retroviral sequences in vivo.

Albumins↗
PubMed1995

Ras-transduced diethylnitrosamine-treated hepatocytes develop into cancers of mixed phenotype in vivo.

The cell of origin of hepatocellular carcinoma (HCC) is controversial. A method for marking cells of different lineages in vivo and then determining their carcinogenic potential should resolve this issue. A retroviral vector expressing activated ras and beta-gal genes (Ras-gal) was transferred into adult rat hepatocytes in vivo, and some animals were treated with diethylnitrosamine (DEN). Bile ductule cells and the putative stem cells of the liver (the oval cells) did not appear to be transduced by this method. At 1 month after transfer, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining was performed on transduced rat livers to determine the blue cluster size. Eight % of the clusters in Ras-gal-transduced, DEN-treated livers contained at least twice as many cells as the largest cluster in Ras-gal-transduced, DEN-untreated rats, demonstrating that they had acquired markedly abnormal growth properties. When the retroviral vector containing beta-gal without ras (Gal-509) was transferred into DEN-treated rats, 2.5% of the cells were present in clusters containing at least twice as many cells as the largest cluster in Gal-509-transduced, DEN-untreated animals. Thus, p21-ras may increase the percentage of cells that acquire mutations in response to DEN, or it may behave synergistically with other mutations to increase the replication rate of cells. Occasional foci in Ras-gal-transduced, DEN-treated rats had extramedullary hematopoiesis. Forty % of the Ras-gal-transduced, DEN-treated rats developed unifocal HCC, mixed HCC/cholangiocarcinoma (CC), or CC at 3-6 months after transduction, suggesting that hepatocytes can develop into HCC or CC if sufficient genetic alterations occur.

Animals↗
PubMed1994

Quantitative evaluation of liver-specific promoters from retroviral vectors after in vivo transduction of hepatocytes.

Hepatic gene therapy could be used to treat a number of inherited blood diseases such as hemophilia or thrombophilia. Although liver-directed retroviral transduction can result in long-term gene expression in vivo, the low level of protein production has limited its clinical application. We reasoned that the insertion of liver-specific promoters into retroviral vectors would increase gene expression in vivo. The 347-bp human alpha 1-antitrypsin (hAAT), the 810-bp murine albumin (mAIb), the 490-bp rat phosphoenolpyruvate carboxykinase (rPECK), and the 596-bp rat liver fatty acid binding protein promoters were inserted into a Moloney murine leukemia retroviral backbone containing the hAAT reporter gene. Vectors that produced appropriately sized RNA and hAAT protein in vitro were tested in vivo by transducing regenerating rat livers. Long-term serum expression of the hAAT reporter gene was normalized to retroviral transduction efficiency as determined by using a polymerase chain reaction-based assay of genomic DNA from transduced rat livers. The hAAT, mAIb, and rPEPCK promoters were, respectively, 35-, 8-, and 0.02-fold as strong as the previously studied constitutive Pol-II promoter. We conclude that the hAAT promoter resulted in the highest expression from a retroviral vector and may result in therapeutically significant expression of other clinically significant blood proteins.

Animals↗
PubMed1994

Liver-directed gene therapy: quantitative evaluation of promoter elements by using in vivo retroviral transduction.

Liver-directed gene therapy will be applicable to many inherited diseases. Although various protocols have been devised for in vivo delivery of retrovirus, comparison of hepatocyte transduction frequencies has been difficult due to variations in retroviral titer and a paucity of DNA data. We have previously reported an in vivo rat hepatocyte transduction technique which involves 70% hepatectomy followed 24 hr later by portal vein injection of retrovirus during hepatic in-flow occlusion. In this study, we employed this method and concentrated retroviral preparations to achieve transduction of up to 15% of hepatocytes as determined by a quantitative PCR assay. As an initial step toward identifying promoters which lead to high-level long-term expression of retroviral transduced genes, we used our in vivo delivery system to compare the Moloney murine leukemia virus long terminal repeat (LTR) promoter with the promoter for the large subunit of murine RNA polymerase II (Pol-II). Human alpha 1-antitrypsin (hAAT) was used as the reporter gene to facilitate long-term analysis of expression. Serum hAAT levels were higher for the Pol-II promoter (143 ng/ml) than for the LTR promoter (50 ng/ml). This difference was consistent with the higher transduction frequency observed for the Pol-II-hAAT vector. Although serum hAAT expression was sustained for up to 1 year in six of eight Pol-II-hAAT-transduced rats and three of five LTR-hAAT-transduced rats and was proportional to hAAT mRNA level and proviral DNA frequency, in vivo expression was significantly lower than in transduced tissue culture cells. We conclude that a high frequency of in vivo transduction can be achieved by using retroviral vectors and our rapid transduction protocol, but transduced gene expression remains a serious problem. The quantitative assays described herein will facilitate in vivo comparisons of gene regulatory elements.

Animals↗
PubMed1994

Liver-directed gene therapy: evaluation of liver specific promoter elements.

Liver-directed gene therapy could dramatically alter the therapy of many inherited hematologic and metabolic diseases. We have developed a rapid, reliable, low-mortality method for the in vivo delivery in rats of retroviral vectors 24 hr after 70% hepatectomy by intraportal injection during hepatic in-flow occlusion (HIFO). Using the human alpha 1-antitrypsin (hAAT) reporter gene, we found in vivo that up to 10% of hepatocytes integrated the provirus, and serum hAAT protein levels were sustained for up to 1 year. Despite high in vivo transduction efficiencies, gene expression at the mRNA level is disappointingly low compared to in vitro transduced NIH 3T3 or Hepa A1 tissue culture cells. In this report, LNL-6-derived retroviral vectors (RV) were combined with one of two strong, liver-specific promoters, murine albumin or human alpha 1-antitrypsin, an upstream insertion of a trimer of hepatocyte nuclear factor-3 (HNF-3) binding sites, and the hAAT reporter gene. HNF-3 has been demonstrated to increase in vitro transcriptional activity [23]. Twenty-four hours after 70% hepatectomy, 10-fold concentrated (by methotrexate-resistant titer) RV-producing cell supernatant was given intraportally during a 3-min HIFO. Serum hAAT levels as quantitated with a human specific ELISA were sustained for over 40 weeks with all of the liver-specific promoter constructions. However, the hAAT protein production with the murine albumin promoter retroviral constructs decreased with time, but was sustained at levels approximately 80% of the initial serum peak levels with the constructs containing the hAAT promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

In vivo hepatocyte transduction with retrovirus during in-flow occlusion.

Gene therapy research would be facilitated by a technically simple procedure for transducing hepatocytes in vivo. Previously reported methods have employed partial hepatectomy followed 24 hr later by asanguineous perfusion of the regenerating liver with retrovirus. We have developed a simpler method of in vivo transduction in which we deliver an intraportal bolus of retrovirus to the regenerating rodent liver during a brief period of hepatic in-flow occlusion. On Day 0, adult male Sprague-Dawley rats (N = 19) underwent 70% hepatectomy to induce hepatocyte replication. On Day 1, retroviral supernatant was harvested from an amphotropic retroviral packaging cell line that packaged an LNL6-derived vector containing the cytomegalovirus promoter driving expression of the Escherichia coli beta-galactosidase (beta gal) gene. Twenty-four hours after partial hepatectomy, experimental rats (N = 17) received 6 x 10(5) colony-forming units of retrovirus by intraportal injection during a 3-min occlusion of the hepatic artery and portal vein. Control rats (N = 2) received intraportal medium (without retrovirus), also during in-flow occlusion. The procedure required 20-25 min, and the survival rate was 84%. Cryostat sections were prepared from liver biopsies obtained on Post-transduction Days 8 and 15 and stained with 5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside to detect beta gal expression. Light microscopic examination of Day 8 sections from surviving experimental rats (N = 14) revealed 0.10-1.00% blue (i.e., transduced) hepatocytes per low power field, while sections from control rats (N = 2) exhibited no blue cells. Day 15 sections from experimental rats revealed a somewhat lower frequency of hepatocytes expressing beta gal.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Coagulation, fibrinolytic, and inhibitory proteins in acute myocardial infarction and angina pectoris.

BACKGROUND AND METHOD: The role of thrombus formation in the pathogenesis of acute myocardial infarction (AMI) and unstable angina pectoris has been well established. However, comprehensive and systematic studies of the blood coagulation, fibrinolytic, and inhibitory proteins are not available in patients with these conditions. Fourteen patients with AMI, 10 patients with angina pectoris, and 32 normal volunteers were studied. Plasma antigen concentrations and/or activities of high-molecular-weight kininogen (HMWK), fibrinogen, fibronectin, plasminogen, D-dimer, tissue plasminogen activator (t-PA), alpha 2-antiplasmin, alpha 2-macroglobulin, alpha 1-antitrypsin, protein C, total and free protein S, antithrombin III (AT-III), von Willebrand factor (vWF), factors (F) XII, XI, IX, VIII, VII, X, V, II, and XIII, and plasma antiplasmin activity were measured using appropriate functional or immunologic assays. RESULTS: The AMI group showed a significant reduction in F XII activity, F XII activity-to-concentration ratio, and HMWK concentration. In addition, the AMI patients exhibited a significant elevation of plasma F XI activity, F IX concentration, and F IX activity, and vWF, fibrinogen, D-dimer, and t-PA concentrations. This was associated with significant reductions in F V, F II, and AT-III activity-to-concentration ratio. Many of the changes observed in AMI patients were also present in patients with angina pectoris. Furthermore, the latter group exhibited an elevation of F VIII activity, alpha 2-macroglobulin activity, and alpha 1-antitrypsin concentration and a significant reduction of antiplasmin activity despite a normal alpha 2-antiplasmin concentration. CONCLUSIONS: The observed reduction of the plasma F XII activity-to-antigen concentration ratio combined with a reduced HMWK concentration suggests intrinsic pathway activation, while the elevation of the D-dimer concentration indicates thrombin generation and fibrin formation and degradation in the AMI group. The latter changes were also present in patients with angina pectoris. Both AMI and angina groups showed several other abnormalities of the coagulation, fibrinolytic, and inhibitory systems. The results suggest the presence of a prothrombotic state associated with the activation of coagulation and fibrinolytic systems in patients with acute myocardial ischemia or infarction.

Adult↗
PubMed1989

Colopleural fistula due to strangulated Bochdalek hernia in an adult.

An elderly patient presented with a right sided pneumothorax due to strangulation of part of the colon through a congenital Bochdalek hernia. Congenital posterolateral diaphragmatic hernia of Bochdalek is rare in an adult and strangulation with pneumothorax has not been reported before.

Aged↗