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Biomedical subjects

F D Ledley

Publications and source records attributed to F D Ledley.

At least 19 recordsLinked to original sources
PubMed1993

Academic careers: choice and activity of graduates of a pediatric residency program 1974-1986.

In summary, our data suggest that the playing field for academic medicine is changing. It is more patient care oriented, more multifaceted and supported more by clinical dollars than in the past. Greater flexibility in what constitutes "academic success" is necessary to assure a supportive environment in which tomorrow's academic faculty can develop and flourish. To accomplish these goals promotion systems that reward not only research but also teaching and clinical care accomplishments will be necessary. Clinicians will need to be compared with clinicians, teachers with teachers, clinical investigators with clinical investigators and basic investigators with basic investigators. Sources of support will need to be more clearly targeted along activity lines with clinical dollars supporting the clinician, medical education dollars supporting the teacher-educators and federal and foundation dollars supporting research. In our department, time and effort for research (45%) approximates dollar support for this activity (44%), while clinical dollars (43%) fund to a greater degree time and effort committed to clinical care (34%), and administration and teaching dollars (13%) under fund time and effort committed to these activities (21%). This suggests the need to identify increased funding to support teaching and education. Promotion expectations for women will need to be more flexible and adjusted to family responsibilities and demands. Most of all, however, we academic faculty must support enthusiastically the importance and joy of our work. We must be encouraging to our colleagues and our students and continue to recognize that for all of the difficulties and challenges, academic life is a rewarding and fulfilling enterprise.

Academic Medical Centers↗
PubMed1992

Transduction of primary human hepatocytes with amphotropic and xenotropic retroviral vectors.

Experiments in animal models suggest that it is feasible to consider hepatic gene therapy using a strategy in which hepatocytes would be isolated by partial hepatectomy, transduced with recombinant retroviral vectors containing genes of therapeutic importance, and then transplanted back into the patient by autologous hepatocellular transplantation. The application of this strategy in clinical trials will require adapting these methods to human cells. We describe the transduction of primary human hepatocytes with two forms of retroviral vectors: amphotropic vectors, which have been used previously in clinical trials, and xenotropic vectors, which have a different host range. Human hepatocytes were harvested from organs preserved in Belzer's solution and were cultivated in a serum-free, tyrosine-free, hormonally defined medium. These cells proliferated for 3-5 days in culture, exhibited characteristic hepatocyte morphology, and expressed liver-specific functions, including phenylalanine hydroxylase, alpha 1-antitrypsin, and glutamine synthase. Transduction with an amphotropic LNL6 retroviral vector resulted in stable incorporation of the provirus into 1% of the cells as estimated by semiquantitative PCR. Consistently higher transduction efficiencies (as much as 10% of the cells) were observed with a xenotropic N2 vector. These data support the feasibility of using LNL6 as a marker gene in clinical trials of hepatocellular transplantation. These data also suggest that the efficiency of transducing hepatocytes with amphotropic vectors in animal models may not accurately reflect the utility of these vectors for human applications. Consideration should be given to the use of xenotropic vectors for optimizing the efficiency of transduction for human applications.

Animals↗
PubMed1992

Cobalamin deficiency associated with methylmalonic acidemia in a cat.

A 9-month-old sexually intact male longhair cat was examined because of lethargy, anorexia, cold intolerance, and failure to thrive since acquisition at an early age. Clinical signs of disease were less pronounced when the cat was fed a low-protein diet. Anemia, hypoglycemia, low total CO2 content, and hyperammonemia were detected. The cat was euthanatized. Urine obtained immediately before euthanasia contained a large amount of methylmalonic acid. Total serum cobalamin concentration was low. Hepatic methylmalonic-CoA mutase activity, with and without the addition of coenzyme adenosylcobalamin, was consistent with a cobalamin deficiency. Methylmalonic acidemia secondary to a putative defect in cobalamin absorption was diagnosed.

Absorption↗
PubMed1992

Phenotype of disease in three patients with identical mutations in methylmalonyl CoA mutase.

We have previously identified a mutation in the gene for methylmalonyl CoA mutase in a patient with the mut- phenotype of methylmalonic aciduria. This mutation (G717V) interferes with the binding of the deoxyadenosylcobalamin cofactor to the apoenzyme producing a mutant holoenzyme that is defective, but not completely inactive, in vitro. This report describes the clinical phenotype associated with this mutation in the original patient and two additional patients who are homozygous for this allele. All three patients presented in the first years of life with multiple episodes of life-threatening organic acidosis and hyperammonemia. None had evidence of disease in the perinatal period, and all three have low-normal intelligence. These three children exhibit a distinctive phenotype of disease that is intermediate between the fulminant and benign forms of methylmalonic aciduria. These data suggest that this phenotype is the specific consequence of the G717V mutation, and that the degree of residual enzyme activity associated with the G717V mutation is close to the threshold required in vivo for maintaining metabolic homeostasis.

Acidosis↗
PubMed1992

Propionate metabolism in cultured human cells after overexpression of recombinant methylmalonyl CoA mutase: implications for somatic gene therapy.

Strategies for somatic gene therapy must consider the metabolic consequences of expressing the recombinant gene product in addition to methods for gene transfer and expression. We describe studies of propionate metabolism in cultured cells transfected with methylmalonyl CoA mutase (MCM), the enzyme deficient in mut methylmalonic acidemia. Transfection of MCM into mut fibroblasts restores propionate metabolism to normal levels in a dose-dependent manner. Overexpression of MCM, or the addition of excess propionate, carnitine, or cobalamin, does not increase propionate metabolism in normal human fibroblasts, lymphoblasts, or hepatoma cells, although hepatic cells exhibit > 10-fold higher levels of propionate metabolism. Significantly, the restoration of propionate metabolism in mut fibroblasts is disproportionately greater than the efficiency of transfection, suggesting the presence of a cooperative phenomenon between cells. Intercellular participation in propionate metabolism is evident in cocultures of MCM-deficient and propionyl CoA carboxylase-deficient cells. We conclude that the liver is the preferred target for gene therapy of MCM deficiency because of its greater capacity for propionate metabolism and that cooperation between cells could enhance the biological effect of a subpopulation of cells transformed with recombinant MCM.

Amino Acid Metabolism, Inborn Errors↗
PubMed1992

Correction of methylmalonyl-CoA mutase deficiency in Mut0 fibroblasts and constitution of gene expression in primary human hepatocytes by retroviral-mediated gene transfer.

Methylmalonic acidemia is an often fatal inborn error of organic acid metabolism due to deficiency of methylmalonyl-CoA mutase. The cloning of genes encoding this enzyme and the advent of technologies for gene transfer have introduced the possibility of somatic gene therapy for this disorder. Gene therapy may require replacement of the defective enzyme in hepatocytes, which have a greater capacity for propionate metabolism than other somatic cells and represent the principle physiological site of propionate metabolism. We describe construction of an amphotropic retroviral vector containing the human methylmalonyl-CoA mutase cDNA. This vector is shown to transduce primary MCM-deficient fibroblasts and restore levels of [14C]propionate metabolism by cultures of nonselected cells to normal. This vector will transduce primary human hepatocytes and direct transcription of recombinant human MCM from the integrated provirus. This work demonstrates the feasibility of retroviral-mediated gene transfer of methylmalonyl-CoA mutase into primary human cells, including hepatocytes which represent a difficult, but potentially necessary, target for gene therapy of methylmalonic acidemia.

3T3 Cells↗
PubMed1992

The challenge of follow-up for clinical trials of somatic gene therapy.

Although extensive efforts have been made to optimize the safety of vectors for somatic gene transfer and somatic gene therapy, the safety of these agents must ultimately be assessed in clinical trials. A statistically significant assessment of safety will be complicated by the relatively small number of patients who will be enrolled in initial clinical trials, the need for long-term longitudinal follow-up of patients and perhaps their progeny, and the traditionally poor participation in long-term follow-up by many patients. This article reviews the potential risks of retroviral-mediated gene transfer, the statistical power required to assess the true incidence of potential complications, the number of patients who may participate in clinical trials involving retroviral vectors, and the factors that make thorough follow-up and uniform data ascertainment difficult. The role of the FDA in assessing the safety of retroviral vectors and the potential role of registries for patient tracking and data ascertainment are discussed.

Clinical Trials as Topic↗
PubMed1992

Somatic gene therapy in gastroenterology: approaches and applications.

Somatic gene therapy represents a new approach for treating a variety of genetic and acquired diseases and has many potential applications in gastroenterology. Somatic gene therapy is based on the ability to transfer recombinant genes efficiently into somatic tissues, such as the liver or intestine, and achieve expression of the recombinant gene product. Gene transfer could be used to replace genetically defective gene functions or prevent the progression of acquired or multifactorial diseases. The therapeutic application of gene transfer technologies requires development of methods for achieving efficient transfer and expression of recombinant gene products, research directed at developing strategies for altering the course of disease, and clinical research aimed at assessing both the technical feasibility and therapeutic efficacy of this approach.

Ethics, Medical↗
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↗