Biomedical subjects
Publications and source records attributed to R M Adams.
Four tandem subtilisin-like protease genes were found on a 6,854-bp DNA fragment cloned from the alkalophilic Bacillus sp. strain LG12. The two downstream genes (sprC and sprD) appear to be transcribed independently, while the two upstream genes (sprA and sprB) seem to be part of the same transcript.
Retrovirus infection is normally limited to cells within a specific host range which express a cognate receptor that is recognized by the product of the env gene. We describe retrovirus infection of cells outside of their normal host range when the infection is performed in the presence of a replication-defective adenovirus (dl312). In the presence of adenovirus, several different ecotropic vectors are shown to infect human cell lines (HeLa and PLC/PRF), and a xenotropic vector is shown to infect murine cells (NIH 3T3). Infectivity is demonstrated by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining, selection with G418 for neomycin resistance, and PCR identification of the provirus in infected cells. Infectivity is quantitatively dependent upon both the concentration of adenovirus (10(6) to 10(8) PFU/ml) and the concentration of retrovirus. Infection requires the simultaneous presence of adenovirus in the retrovirus infection medium and is not stimulated by preincubation and removal of adenovirus from the cells before retrovirus infection. The presence of adenovirus is shown to enhance the uptake of fluorescently labeled retrovirus particles into cells outside of their normal host range, demonstrating that the adenovirus enhances viral entry into cells in the absence of the recognized cognate receptor. This observation suggests new opportunities for developing safe retroviral vectors for gene therapy and new mechanisms for the pathogenesis of retroviral disease.
Methoxylamine mutagenesis of the beta-galactosidase gene from Lactobacillus delbrückii subsp. bulgaricus was used to generate cold-sensitive variants. Two variants, P429S and L317F, were characterized kinetically in order to determine the enzymatic consequences of these mutations. The kinetic parameters Km and Vmax on the synthetic substrate o-nitrophenyl-beta-D-galactopyranoside have been determined over a temperature range of 11-45 degrees C. Only the Vmax of the two variants was significantly different than the wild-type enzyme over the temperature range studied. The Vmax of the L317F variant is reduced proportionately at all temperatures compared to the wild-type enzyme while the value of Vmax for the P429S mutant deviates from wild-type only at lower temperatures (in 2 mM Mg2+). This temperature-dependent effect on the Vmax of P429S can be suppressed by increasing the Mg2+ concentration. The results suggest that the binding of this essential metal ion is altered in the P429S variant such that its dissociation is increased by lowering the temperature.
A method is described for generating and screening variants of the beta-galactosidase from Lactobacillus delbrueckii subsp. bulgaricus sensitive to several environmental stresses, with potential application in the food industry. Chemical mutagenesis with hydroxylamine or methoxylamine was performed on the beta-galactosidase gene carried on an Escherichia coli expression vector. Mutants sensitive to cold, heat, low pH, low magnesium concentration, and the presence of urea were isolated by screening for reduced color development on beta-galactosidase indicator plates. The mutations responsible for three variant beta-galactosidases were localized, and the base substitutions were determined by DNA sequencing. The amino acid alterations associated with one low-pH-sensitive (pHs) and two urea-sensitive (Us) variants correspond to P584L (pHs1), G400S/R479Q (Us26), and G167E/E168K/E363K/V492M (Us17), respectively. Mutant pHs1 is also heat, cold, low magnesium, and urea sensitive; Us26 is also cold sensitive; and Us17 is also low-pH sensitive.
The authors established a means of effective gene transfer into human thyroid follicular cells via retroviral-mediated mechanisms. Using specific harvest and culture techniques, we investigated the selection of human thyroid cells in serum-free media. Normal adult human thyroid tissue was obtained after thyroidectomy from fresh specimens sent for frozen-section analysis. Follicular cells were harvested and grown in hormonally defined, serum-free media to prevent fibroblast growth with selection for differentiated function assessed by immunohistochemical staining for thyroglobulin. The efficiency of gene transfer into human thyroid cells was compared between the zen-beta-gal and LNL6 retroviral vectors. The zen-beta-gal retrovirus encodes the product beta-galactosidase, and gene expression was demonstrated by histochemical staining in 0.1% to 1% of the cells. An improved efficiency of 2% to 3% transduction was demonstrated using the LNL6 vector which carries the gene for neomycin resistance (NEO-R). Polymerase chain reaction (PCR) identification of the integrated proviral sequence (NEO-R gene) with Southern blot confirmation was used to quantitate LNL6 transductions and compare confluent versus actively dividing cell cultures. Follicular cell gene therapy has significant potential for treating congenital or acquired diseases of the thyroid as well as disorders of circulating proteins such as diabetes, hypopituitarism, and hemophilia. The ability to culture human follicular cells and perform effective gene transfer is paramount in the eventual realization of thyroid gene therapy.
Workers' compensation law is poorly understood by many physicians, including dermatologists. The tenets of this law throughout the country are basic and fairly straight forward, and the most important of them are nearly the same for all of the states. By understanding how the law operates and what is required of physicians, helping patients with their work-related dermatoses can be a gratifying experience.
We describe studies in a canine model aimed at establishing methods for ex vivo gene delivery to thyroid follicular cells. Canine follicular cells were harvested from tissue obtained by unilateral lobectomy, grown in thyrotropin-containing media, and transduced with amphotropic retroviral vectors carrying Escherichia coli beta-galactosidase or Tn7 neomycin-resistance genes. Up to 30% of cells were transduced with retroviral vectors containing the neomycin resistance gene, and transduced cells could be selected with G418. Significantly, transduced and selected cells exhibited the morphology of thyroid follicular cells and continued to express thyroglobulin. To assess the viability of cultivated and transduced cells for transplantation, cells were stained with the vital fluorescent dye DiI, recovered by trypsinization, and transplanted into the contralateral thyroid lobe of autologous animals. Engraftment was demonstrated by fluorescence microscopy and identification of proviral sequences 7-10 days after transplantation. Proviral transcripts were evident using coupled reverse transcription and the polymerase chain reaction using total RNA from transplanted glands. Thyroid follicular cells may represent an attractive target for gene therapy due to their proliferative potential, their large protein synthetic and secretory capacity, and their susceptibility to regulation. The thyroid might be a target for therapy of congenital or acquired thyroid diseases as well as disorders requiring regulated expression of proteins in the circulation. This work demonstrates the feasibility of ex vivo gene delivery to thyroid follicular cells that may be used in future investigations.
Strategies for hepatic gene therapy have been proposed that involve isolation of primary hepatocytes and introduction of recombinant genes into these cells in culture, followed by autologous hepatocellular transplantation (HCT). Consideration of clinical applications requires data suggesting that HCT can be performed safely in human subjects in addition to data indicating that recombinant gene expression can reverse a disease process. This report describes preclinical studies that underlie a clinical trial of HCT in which hepatocytes would be labeled with a marker gene to facilitate assessment of engraftment in the recipient. Human hepatocytes were harvested from liver segments preserved in Belzar's solution and transduced with an amphotropic retroviral vector carrying a recombinant marker gene (neomycin phosphotransferase II). Human hepatocytes were recovered from monolayer culture, stained with the fluorescent dye 1,1'-dioctadecyl-3,3,3,3'-tetra-methylindo-carbocyanine perchlorate (DiI) and transplanted into severe combined immunodeficient mice by splenic injection. Engrafted hepatocytes were identified in the liver and spleen of severe combined immunodeficient mice but not immunocompetent controls. Two large animal models of HCT are described. In a dog model, neomycin phosphotransferase II-containing hepatocytes were identified in the liver 7 wk after transplantation. In a baboon model, autologous HCT with DiI-stained cells demonstrated that transplanted cells assume a normal morphology and constitute up to 5% of hepatocytes. These data demonstrate transduction and transplantation of human hepatocytes and the feasibility of HCT in large animals. On the basis of these studies, the proposed clinical trial for gene transfer and transplantation in human subjects has been approved by the National Institutes of Health and the Food and Drug Administration.(ABSTRACT TRUNCATED AT 250 WORDS)
Of the large number of young persons entering the work force each year, few choose their work with more than cursory consideration of their physical ability to perform it. This is especially true of atopic persons. When the first manifestation of the disease is hand eczema triggered by the first work experience, often after the subject has undergone many months of training, the answer becomes very difficult. It often requires a job change, with accompanying economic and personal loss. When the work is responsible for aggravation or activation of the condition, the entire gamut of social services comes into play, including medical, legal, rehabilitation, and workers compensation. These lead, after months and often years, to job changes that may or may not alleviate or ameliorate the condition. For these reasons dermatologists need to have greater awareness of the position of the atopic subject in the workplace.
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.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
More than 1100 patients were tested with methylchloroisothiazolinone-methylisothiazolinone, 100 ppm, in aqueous and petrolatum-based patch test materials from 1985 to 1987 by members of the North American Contact Dermatitis Group. Thirteen reactions to the aqueous materials and 10 to the petrolatum-based materials were observed. Irritant reactions were infrequent, and about half the reactions were deemed relevant. From 1984 to 1985, patch tests with this substance at a concentration of 250 ppm in petrolatum were conducted. Thirteen persons were identified as allergic, but three others were sensitized by the patch test procedure. Sensitization as not observed in tests with aqueous or petrolatum-based substance at a concentration of 100 ppm, and this concentration appears to be the best compromise between safety and sensitive detection of allergy. Use tests are helpful but not infallible as a guide in establishing relevance with methylchloroisothiazolinone-methylisothiazolinone. Wash-off products are frequently well tolerated by patients with positive reactions to this substance.
As the first step in validating a criteria-based, self-report depression questionnaire specifically for children and adolescents and to determine the prevalence of self-reported depressive symptoms, we studied 3,294 high school students of mixed ethnic background in a large urban school district. They completed the Weinberg Screening Affective Scale. The 21-item Beck Depression Inventory was also completed to allow comparison with a previous study. The prevalence of clinically significant depressive symptoms suggesting depression by self-report ranged from 18% on the Beck Depression Inventory to 13% on the Weinberg Screening Affective Scale. Hispanic females had the highest scores, while white males had the lowest. Being behind in school, female, and nonwhite predicted more self-reported depressive symptoms.
Hypersensitivity to thimerosal in vaccines has been reported to induce persistent local reactions, urticarial and generalized exanthematic eruptions, and, in the case of the hepatitis B vaccine, urticaria with asthma. The authors describe two cases of extensive reactions, one in a patient who did not form antibodies to the principal vaccine antigen. Although not all thimerosal-sensitive patients develop adverse reactions to vaccines containing this material, there is a potential risk, and the reactions can be very long lasting.
Alstroemeria (Peruvian or Inca lily) has found particular favor because of its beauty and durability. However, it may induce a dermatitis so severe that workers have to change jobs. The dermatitis is chronic, with fissuring at the tips of the fingers bilaterally. Itching is often a less prominent symptom. Preventative measures are of little benefit, and many floral shops are vanishing the plant.