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Biomedical subjects

F D Ledley

Publications and source records attributed to F D Ledley.

At least 73 records · Page 4Linked to original sources
PubMed1992

The use of DiI-marked hepatocytes to demonstrate orthotopic, intrahepatic engraftment following hepatocellular transplantation.

A novel method is described for marking primary hepatocytes with the fluorescent dye DiI prior to hepatocellular transplantation and identifying these cells within the hepatic parenchyma of recipient animals by fluorescence microscopy and flow cytometry. Optimal conditions are described for marking cells with DiI in suspension or in monolayer cultures prior to transplantation. DiI is shown to be nontoxic to hepatocytes and not to be exchanged between adjacent cells in vitro. Histological analysis of transplanted tissues shows DiI staining of engrafted hepatocytes and phagocytotic cells (Kupffer cells). This analysis shows that hepatocytes engraft within the hepatic parenchyma and exhibit a histological appearance indistinguishable from normal by conventional hematoxylin and eosin staining. Many previous reports of hepatocellular transplantation have been limited by their inability to unequivocally identify transplanted cells within the liver. These data illustrate the importance of having specific markers for transplanted cells that engraft in an orthotopic location and assume a normal morphological appearance.

Animals↗
PubMed1992

Cloning and expression of a mutant methylmalonyl coenzyme A mutase with altered cobalamin affinity that causes mut- methylmalonic aciduria.

Distinct genotypic and phenotypic forms of methylmalonyl CoA mutase (MCM) apoenzyme deficiency can be delineated by biochemical analysis of mutant fibroblasts. One form, designated mut-, expresses a phenotype in which residual enzyme activity is evident in cultured cells exposed to high concentrations of hydroxycobalamin. We describe cloning of an MCM cDNA from cells exhibiting a mut- phenotype and characterization of the mutant gene product overexpressed in primary muto human fibroblasts and Saccharomyces cerevisiae. Three novel base changes were observed. Recombinant clones containing one of these base changes (G717V) express four characteristics of the mut- phenotype: failure to constitute [14C]propionate incorporation activity in fibroblasts assayed under basal cell culture conditions, constitution of [14C]propionate incorporation activity in fibroblasts stimulated with 0.1-1.0 micrograms/ml hydroxycobalamin, interallelic complementation with alleles bearing an R93H mutation, and an apparent Km (adenosylcobalamin) 1,000-fold higher than normal. These results demonstrate that the G717V mutation produces the mut- phenotype and localizes determinants for adenosylcobalamin binding near the carboxyl terminus of MCM.

Amino Acid Sequence↗
PubMed1992

DiI as a marker for cellular transplantation into solid organs.

Research in cellular transplantation is frequently compromised by an inability to identify transplanted cells engrafting into orthotopic sites if they exhibit normal morphology and no unique antigenic markers. A method is described for using the fluorescent dye DiI as a marker for cell transplantation studies. This dye is not metabolized or exchanged between cells in vitro or in vivo and enables identification of engrafted cells by fluorescence microscopy, flow cytometry or fluorescence-activated cell sorting. Applications are described in autologous hepatocellular and thyroid follicular cell transplantation.

Animals↗
PubMed1992

The quality of medical science.

Is the quality of science aimed at understanding and treating disease inferior to more basic investigations of basic biology, or does the different quality of medical science reflect a distinct nature? This duality in the meaning of quality is central to any critique of the quality of medical science. The nature of medical science, which deals with the dysfunction of integrated genetic, epigenetic, environmental, or stochastic phenomenon, is distinct from that of basic science which seeks to describe discrete biological processes. In physical sciences it is generally accepted that there are practical boundaries between disciplines such as quantum physics, thermodynamics, and chemistry even though, in theory, such investigations are related. In meteorology, the development of chaos theory establishes that there are even theoretical limits to the ability to predict large scale processes from their essential, constituent processes. So too, the quality of medical science may be distinct from that of basic biology.

Attitude↗
PubMed1992

Prospects for academically trained pediatricians in academic medicine.

There is widespread concern about the commitment and ability of physicians to enter careers in academic medicine. We studied MEDLINE citations by 150 academically trained pediatricians to determine whether they remained in academic medicine and to assess the nature of their contribution. We identified 2098 citations by 122 of the 150 individuals which reflected substantive involvement in clinical and basic research. Most individuals published their first papers within three years after residency and thereafter exhibited a stable level of productivity. Individuals who published prior to completing residency were most likely to enter academic medicine and achieve a high level of productivity. These reassuring data suggest that individuals who pursue an academic track may assume the traditional role of the physician scientist, and suggest measures which may be taken to attract more physicians to academics.

Academic Medical Centers↗
PubMed1991

Identification of a point mutation in the topoisomerase II gene from a human leukemia cell line containing an amsacrine-resistant form of topoisomerase II.

HL-60/AMSA is a human leukemia cell line that is 50- to 100-fold more resistant to the cytotoxic actions of the topoisomerase II-reactive intercalator amsacrine than is its drug-sensitive HL-60 parent line. Previously, we have shown that the topoisomerase II from HL-60/AMSA is also resistant to inhibition by amsacrine and other intercalating agents. We therefore sought the molecular basis for the resistance of the topoisomerase II of HL-60/AMSA and, by inference, of the HL-60/AMSA line itself. We report the cloning and sequencing of the topoisomerase II genes from both the sensitive and resistant leukemia cell lines using polymerase chain reaction technology. We have identified a single base change associated with the drug-resistant form of topoisomerase II. This mutation is present in both cloned HL-60/AMSA complementary DNA and extracted HL-60/AMSA genomic DNA. A rapid assay for this mutation in clinical samples has been developed and applied to the DNA of cells from both normal volunteers and leukemia patients. Thus far, the HL-60/AMSA genotype has not been identified in the cells from any individual, suggesting that this genotype is indeed a mutation and not an allelic form of topoisomerase II. The novel assay developed will allow a rapid search for the prevalence of this mutation in clinical samples from patients with leukemia who have relapsed following intercalator therapy.

Amino Acid Sequence↗
PubMed1991

Is there methylmalonyl CoA mutase in Aspergillus nidulans?

In most animal species and many prokaryotes, methylmalonyl CoA mutase catalyzes isomerization between methylmalonyl CoA and succinyl CoA using adenosylcobalamin as a cofactor. We describe the absence of this enzyme in Aspergillus nidulans based on the absence of enzyme activity in vitro and the failure to metabolize methylmalonate or grow in media containing this organic acid as the sole carbon source. These data contrast previous assumptions that propionate may be metabolized through propionyl CoA and methylmalonyl CoA to the TCA cycle in this organism. This is consistent with the separate evolution of these pathways in animals and lower eukaryotes due to the distinct endosymbiotic origin of their mitochondria.

Aspergillus nidulans↗
PubMed1991

Mouse hepatocytes migrate to liver parenchyma and function indefinitely after intrasplenic transplantation.

One approach to gene therapy for hepatic diseases is to remove hepatocytes from an affected individual, genetically alter them in vitro, and reimplant them into a receptive locus. Although returning hepatocytes to the liver itself would be advantageous, the feasibility of this approach has never been evaluated due to the inability to distinguish donor from host hepatocytes. To unambiguously identify transplanted hepatocytes after transplantation, and to better quantitate their number and degree of liver function, two transgenic mouse lines were generated in a C57BL/6 background. The first expresses the Escherichia coli beta-galactosidase gene from the relatively liver-specific human alpha 1-antitrypsin (hAAT) promoter and allows transgenic hepatocytes to be readily identified after 5-bromo-4-chloro-3-indolyl beta-D-galactoside staining; the second produces the hAAT protein under control of the same promoter, which enables hepatocyte survival and maintenance of liver function to be quantitated by measuring the serum levels of hAAT. Hepatocytes isolated from transgenic donors were transplanted into nontransgenic C57BL/6 recipients by intrasplenic injection. Surprisingly, a large fraction of these cells were identified within the liver parenchyma but not the spleen at 2 months after transplantation. The high levels of serum hAAT detected in transplant recipients were stable for greater than 6 months, suggesting that established cells will survive indefinitely. These results have important implications for liver organogenesis and hepatic gene therapy.

Animals↗
PubMed1991

Clinical application of genotypic diagnosis for phenylketonuria: theoretical considerations.

Identification of mutations within the phenylalanine hydroxylase gene which cause phenylketonuria has introduced the possibility of diagnosing phenylketonuria by direct analysis of the genome. Genotypic analysis could be used for identifying homozygotes in the newborn period, for prenatal diagnosis, or for heterozygote detection in general populations. Establishing the clinical utility of genotypic diagnosis, however, will require characterization of the cohort of patients identified by genotypic diagnosis, correlation of mutant genotypes with specific biochemical and developmental phenotypes, and consideration of how genotypic diagnosis might contribute to improving the clinical outcome in individuals at risk for mental retardation due to hyperphenylalaninemia.

Genotype↗
PubMed1991

Clinical considerations in the design of protocols for somatic gene therapy.

Despite two decades of investigating the potential for somatic gene therapy in curing human disease, few clinical trials are being proposed. This delay is due in part to limitations of existing methods for gene transfer and to the recognized need to proceed judiciously into this controversial arena. Delay is also caused by a disjunction between the traditional precedents and principles of clinical investigation and the procedures instituted to regulate somatic gene therapy. The premise of this report is twofold: first, that more extensive clinical investigation of gene transfer technologies would be beneficial to patients, medicine, and basic science and second, that clinical investigations could be expedited by appealing to the established experience in clinical investigation without compromising the scientific excellence and discipline essential for this highly public process. This report develops a clinical perspective on potential applications of existing gene transfer technologies and the issues involved in developing experimental protocols. The initiation of clinical trials should be a primary goal of gene therapy research programs.

Animals↗
PubMed1991

Hepatocellular transplantation in acute hepatic failure and targeting genetic markers to hepatic cells.

Orthotopic liver transplantation (OLT) represents the only therapeutic option for many patients with end-stage liver disease as well as many inborn genetic errors of hepatic metabolism. Despite dramatic progress in methods for OLT, the utilization of this procedure is limited by its considerable morbidity and mortality, by a chronic shortage of organs for transplant, and by difficulty arranging funding for many patients. Many children with fulminant hepatic failure do not receive OLT because this technology is unavailable or unaffordable. Hepatocellular transplantation (HCT), in which isolated, heterologous hepatocytes from a donor liver would be infused into the diseased organ in order to provide essential hepatic functions, could provide a much needed therapeutic alternative to OLT in the treatment of some causes of hepatic insufficiency. Experiments in animals have demonstrated that several genetic deficiencies of hepatic metabolism as well as experimental induced hepatic failure in animals can be reversed by HCT. Despite this experience, HCT has never been attempted in human subjects. This protocol represents the first proposed clinical trial of HCT. We are proposing a clinical trial in which HCT would be attempted as a therapeutic intervention in children with acute hepatic failure who have no other medical or surgical options. This proposal is intended to establish surgical methods for HCT and to evaluate the feasibility of this procedure for treating hepatic disease in humans. It is our expectation that HCT may provide short-term support for patients awaiting organ availability, a "bridge to recovery" allowing patients with fulminant hepatic failure to recover, or a long-term repopulation of the patient's liver with healthy donor cells. One of the major limitations of many animal studies in HCT is that, since the donor hepatocytes are often indistinguishable from those of the host, it has often been difficult to demonstrate a clear correlation between engraftment and the therapeutic effect. In order to verify engraftment independent of the therapeutic response, we propose to "mark" the donor hepatocytes by transducing these cells with a recombinant retroviral vector (LNL6) carrying a marker gene (NEO-R, neomycin phosphoribosyl transferase). The presence of this marker will enhance the ability to identify transplanted cells in the host using assays for the NEO-R gene or transcribed NEO-R mRNA. The LNL6 vector has been approved for human use and has been used as a marker gene for transplanted cells in human subjects without any reported adverse effects. We would like to emphasize that this is a proposal with therapeutic intent.(ABSTRACT TRUNCATED AT 400 WORDS)

Acute Disease↗
PubMed1991

Genetic characterization of a MUT locus mutation discriminating heterogeneity in mut0 and mut- methylmalonic aciduria by interallelic complementation.

Genetic complementation of fibroblasts from patients with methylmalonic aciduria (MMA) defines a unique class of allelic mutations arising from mutations at the locus encoding the methylmalonyl coenzyme A (CoA) mutase apoenzyme. Various phenotypes of MMA have been delineated including complete absence of enzyme activity (mut0) and abnormal enzyme activity with an elevated Km for adenosylcobalamin (mut-). We describe genetic studies on a cell line (WG1130) from a patient with mut0 MMA which exhibited an unusual complementation phenotype, complementing with three of nine mut0 cell lines and four of five mut- cell lines. This suggests that interallelic complementation occurs between mutant alleles in WG1130 and subsets of alleles associated with both mut0 and mut- phenotypes. The methylmalonyl CoA mutase cDNA was cloned from WG1130 and found to contain a G354----A (Arg93----His) mutation. Gene transfer of this mutant clone into primary fibroblasts from patients with MMA confirms that this mutation expresses a mut0 phenotype when transferred into a mut0 cell line with low levels of mRNA but can contribute to apoenzyme function when transferred into mut cell lines which show correction with WG1130 by somatic cell complementation. These results point to further heterogeneity within both mut0 and mut- and may enable identification of mutations affecting discrete components of apoenzyme function.

Alleles↗
PubMed1991

Differential diagnosis of mut and cbl methylmalonic aciduria by DNA-mediated gene transfer in primary fibroblasts.

Methylmalonic aciduria can be caused by mutations in the gene encoding the methylmalonyl coenzyme A mutase apoenzyme (mut) or genes required for the provision of cofactor B12 (cbl). The mut and cbl forms are classically differentiated by somatic cell complementation. We describe a novel method for differential diagnosis of mut and cbl methylmalonic aciduria using DNA-mediated gene transfer of a methylmalonyl CoA mutase cDNA clone. Gene transfer of a functional methylmalonyl CoA mutase cDNA clone into mut fibroblasts reconstitutes holoenzyme activity measured by metabolism of [14C]-propionate in culture. Identical gene transfers into cbl fibroblasts have no effect. This method is used for the differential diagnosis of mut and cbl genotypes in cells from patients with a clinical diagnosis of methylmalonic aciduria and is shown to be a facile, sensitive, and specific method for genetic diagnosis. This work establishes the principle of using DNA-mediated gene transfer to identify the genotype of diseases which can result from mutations at several different genetic loci. This type of differential genotypic diagnosis will be particularly important for establishing the applicability of somatic gene therapy in individual patients.

Amino Acid Metabolism, Inborn Errors↗