Search PubMed⌕ Search

Biomedical subjects

T G Jensen

Publications and source records attributed to T G Jensen.

At least 55 records · Page 3Linked to original sources
PubMed1994

Gene transfer into cultured human epidermis and its transplantation onto immunodeficient mice: an experimental model for somatic gene therapy.

To try epidermis as a target for somatic gene therapy we studied transfected primary human keratinocytes grown in culture and grafted onto athymic mice. We have developed a novel technique for grafting cultured epidermal sheets onto mice. First, the graft is placed on the dorsal muscle fascia underneath the mouse skin using the latter as a bandage. Secondly, the mouse skin above the graft is removed, which exposes the grafted skin to open air and thus stimulates terminal differentiation. A novel method for the discrimination between murine and human epidermal cells is also presented, employing in situ hybridization with human Alu repeated DNA sequences. During monolayer culture the keratinocytes were lipofected with the gene for human growth hormone in an Epstein-Barr virus-based expression vector. The cells were allowed to develop a multilayered tissue for 5 d, secreting human growth hormone into the medium at a daily rate of at least 50 ng/cm2 of tissue. The transfected tissues were then grafted onto mice. We detected human growth hormone at levels of up to 2.6 ng/ml in mouse serum for 4 d, but later no human growth hormone could be found, although the transplants survived for months. To investigate the fate of the transfected cells in the transplanted tissue, we labeled them with the beta-galactosidase reporter gene. The cells staining positive for X-gal were found exclusively in the most superficial differentiated layers at 7 d after transplantation. This may be the main reason why no human growth hormone is found in the mouse circulation at this time.

Animals↗
PubMed1994

Disease-causing mutations in exon 11 of the medium-chain acyl-CoA dehydrogenase gene.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most commonly recognized defect of the mitochondrial beta-oxidation in humans. It is a potentially fatal, autosomal recessive inherited defect. Most patients with MCAD deficiency are homozygous for a single disease-causing mutation (G985), causing a change from lysine to glutamate at position 304 (K304E) in the mature MCAD. Only seven non-G985 mutations, all of which are rare, have been reported. Because the G985 mutation and three of the non-G985 mutations are located in exon 11, it has been suggested that this exon may be a mutational hot spot. Here we describe the results from sequence analysis of exon 11 and part of the flanking introns from 36 compound heterozygous patients with MCAD deficiency. We have identified four previously unknown disease-causing mutations (M301T, S311R, R324X, and E359X) and two silent mutations in exon 11. Our results show that exon 11 is not especially mutation prone. We demonstrate that two of the identified disease-causing mutations can be detected by restriction enzyme digestion of the PCR product from the assay for the G985 mutation, a discovery that makes this assay even more useful than before. On the basis of expression of wild-type and mutant MCAD protein in COS-7 cells, we show that the identified mutations abolish MCAD enzyme activity and that they therefore must be disease causing. The M301T, S311R, and K304E mutations are located in helix H, which makes up part of the dimer-dimer interface of the MCAD tetramer. On the basis of the three-dimensional structure of MCAD and the results from the COS-7 expression experiments, we speculate that the primary effect of the M301T and S311R mutations is on correct folding/tetramer assembly, as it has previously been observed for the K304E mutation.

Acyl-CoA Dehydrogenase↗
PubMed1993

Co-overexpression of bacterial GroESL chaperonins partly overcomes non-productive folding and tetramer assembly of E. coli-expressed human medium-chain acyl-CoA dehydrogenase (MCAD) carrying the prevalent disease-causing K304E mutation.

The influence of co-overexpression of the bacterial chaperonins GroEL and GroES on solubility, tetramer formation and enzyme activity of three variants of heterologously-expressed human medium-chain acyl-CoA dehydrogenase (MCAD) was analysed in order to investigate the molecular mechanism underlying MCAD deficiency caused by the prevalent K304E mutation. Depending on which of the three amino acids--lysine (wild-type), glutamic acid (K304E) or glutamine (K304Q) are present at position 304 of the mature polypeptide, three different patterns were observed in our assay system: (i) solubility, tetramer formation and yield of enzyme activity of wild-type MCAD is largely independent of GroESL co-overexpression; (ii) the larger part of the K304Q mutant is insoluble without and solubility is enhanced with GroESL co-overexpression; solubility correlates with the amount of tetramer detected and the enzyme activity measured as observed for the wild-type protein. (iii) Solubility of the K304E mutant is in a similar fashion GroESL responsive as the K304Q mutant, but the amount of tetramer observed and the enzyme activity measured do not correlate with the amount of soluble K304E MCAD protein detected in Western blotting. In a first attempt to estimate the specific activity, we show that tetrameric K304E and K304Q mutant MCAD display a specific activity in the range of the wild-type enzyme. Taken together, our results strongly suggest, that the K304E mutation primarily impairs the rate of folding and subunit assembly. Based on the data presented, we propose that lysine-304 is important for the folding pathway and that an exchange of this amino acid both to glutamine or glutamic acid leads to an increased tendency to misfold/aggregate. Furthermore, exchange of lysine-304 with an amino acid with negative charge at position 304 (glutamic acid) but not with a neutral charge (glutamine) negatively affects conversion to active tetramers. A possible explanation for this latter effect--charge repulsion upon subunit docking--is discussed.

Acyl-CoA Dehydrogenase↗
PubMed1993

Correction of steroid sulfatase deficiency by gene transfer into basal cells of tissue-cultured epidermis from patients with recessive X-linked ichthyosis.

To develop an experimental model for somatic gene therapy we have tried to correct the steroid sulfatase (STS) deficiency in tissue-cultured primary epidermal keratinocytes from patients suffering from recessive X-linked ichthyosis. An efficient Epstein-Barr virus-based vector was constructed, in which full-length steroid sulfatase cDNA is located between an SV40 early promotor and processing signals. After STS gene transfer into cultured basal cells from ichthyotic skin, the cells produce large amounts of enzymatically active steroid sulfatase protein. The subpopulation of transfected cells can be made to produce approximately 100 times more STS activity than normal keratinocytes. Keratinocytes from patients suffering from recessive X-linked ichthyosis display an abnormal phenotype when developing a multilayered tissue in culture: Initially an extensive burst of keratinization is observed, followed by rapid, premature shedding and degradation of most suprabasal cell layers, leaving a culture with hyperproliferative relatively immature keratinocytes. Transfection of these immature ichthyotic cells with the functional STS construct led to an increase in the amount of retained cell material in the culture medium, indicating an increased cell maturation. It is possible to genetically label individual transfected epidermal cells with a reporter gene. Cotransfection experiments with STS and reporter gene vectors show that the cohort of transfected cells had a tendency to develop less rapidly since they became overrepresented in the smaller size classes at the same time the total population was somewhat shifted toward higher cell sizes. We interpret these results as an indication that restoration of the enzymatic activity induces a more normal maturation of the transfected keratinocytes.

Arylsulfatases↗
PubMed1993

Prevention of duodenal ulcer recurrence with penicillin. A double-blind, placebo-controlled trial.

Eradication of Helicobacter pylori is associated with a reduced recurrence of duodenal ulcer (DU). The relationship between H. pylori and DU has been interpreted as causal, but the evidence has been criticized for methodologic reasons. To ascertain whether an antibiotic with no effect on epithelial-cell integrity prevents DU recurrence, we conducted a randomized double-blind trial of phenoxymethylpenicillin (PEN), 2.4 twice daily, and placebo (PLA). Patients with an active DU and positive H. pylori culture from antral biopsy specimens were treated with 40 mg omeprazole daily for 4 weeks, but at week 2 they were allocated at random to PEN (85 patients) or PLA (85 patients) for up to 14 weeks. Those without recurrence during this treatment were followed up for another 6 months. Endoscopy and H. pylori culture were performed at the end of the treatment period and at the end of follow-up, and in between if ulcer symptoms recurred. During the treatment period the ulcer relapse rate was 5 of 58 (9%) in the PEN group and 34 of 68 (50%) in the PLA group (P < 0.0001, log-rank test), with 53% and 14%, respectively, of the patients in the two groups being H. pylori-negative. The relapse rate in the PEN group did not differ between H. pylori-negative and H. pylori-positive patients. The recurrence rate in the PEN group remained low for another 5 months but then approached the rate in the PLA group. The prevalence of H. pylori-negative patients at the end of follow-up was 20% in the PEN group and 10% in the PLA group. These data provide strong evidence that DU has a bacterial cause, with H. pylori as the likely agent.

Double-Blind Method↗
PubMed1993

A rare disease-associated mutation in the medium-chain acyl-CoA dehydrogenase (MCAD) gene changes a conserved arginine, previously shown to be functionally essential in short-chain acyl-CoA dehydrogenase (SCAD).

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a serious and potentially fatal inherited defect in the beta-oxidation of fatty acids. Approximately 80% of patients with MCAD deficiency are homozygous for a single disease-causing mutation (G985). The remaining patients (except for a few cases worldwide) are compound heterozygous with G985 in one allele. By sequencing of cloned PCR-amplified MCAD cDNA from a G985 compound heterozygous patient, we identified a C-to-T transition at position 157 as the only change in the entire coding sequence of the non-G985 allele. The presence of the T157 mutation was verified in genomic DNA from the patient and her mother by a PCR-based assay. The mutation changes conserved arginine at position 28 (R28C) of the mature MCAD protein. The effect of the T157 mutation on MCAD protein was investigated by expression of mutant MCAD cDNA in COS-7 cells. On the basis of knowledge about the three-dimensional structure of the MCAD protein, we suggest that the mutation destroys a salt bridge between arginine28 and glutamate86, thereby affecting the formation of enzymatically active protein. Twenty-two additional families with compound heterozygous patients were tested in the PCR-based assay. The T157 mutation was identified in one of these families, which had an MCAD-deficient child who died unexpectedly in infancy. Our results indicate that the mutation is rare. It is, however, noteworthy that a homologous mutation has previously been identified in the short-chain acyl-CoA dehydrogenase (SCAD) gene of a patient with SCAD deficiency, suggesting that the conserved arginine is crucial for formation of active enzyme in the straight-chain acyl-CoA dehydrogenases.

Acyl-CoA Dehydrogenase↗
PubMed1992

Expression of wild-type and mutant medium-chain acyl-CoA dehydrogenase (MCAD) cDNA in eucaryotic cells.

An effective EBV-based expression system for eucaryotic cells has been developed and used for the study of the mitochondrial enzyme medium-chain acyl-CoA dehydrogenase (MCAD). 1325 bp of PCR-generated MCAD cDNA, containing the entire coding region, was placed between the SV40 early promoter and polyadenylation signals in the EBV-based vector. Both wild-type MCAD cDNA and cDNA containing the prevalent disease-causing mutation A to G at position 985 of the MCAD cDNA were tested. In transfected COS-7 cells, the steady state amount of mutant MCAD protein was consistently lower than the amount of wild-type human enzyme. The enzyme activity in extracts from cells harbouring the wild-type MCAD cDNA was dramatically higher than in the controls (harbouring the vector without the MCAD gene) while only a slightly higher activity was measured with the mutant MCAD. The mutant MCAD present behaves like wild-type MCAD with respect to solubility, subcellular location, mature protein size and tetrameric structure. In immunoblot comparisons, the MCAD protein was present in normal fibroblasts, but essentially undetectable in patient fibroblasts homozygous for the prevalent mutation. We suggest that the MCAD protein carrying this mutation has an impaired ability to form correct tetramers, leading to instability and subsequent degradation of the enzyme. This finding is discussed in relation to the results from expression of human MCAD in Escherichia coli, where preliminary results show that production of mutant MCAD leads to the formation of aggregates.

Acyl-CoA Dehydrogenase↗
PubMed1991

Intracranial abscesses in Odense Hospital. Survey of bacteriology, epidemiology, and treatment with antibiotics, 1963-1989.

Fifty-nine patients were operated or punctured in 60 incidents of brain abscess from 1963-1989, twice as many in men as in women. The number of cases tripled in 1980 to an incidence of 3.6 per million inhabitants per year, supposedly due to the advent of computerized tomography. Simultaneously, the aetiology changed from staphylococci and Gram negative rods to dominance of streptococci and Haemophilus aphrophilus. Apart from temporal abscesses, there was no correlation between localisation in the brain and the bacterial species isolated. Ninety-five per cent of the specimens from untreated patients gave growth, but so did specimens from six of 18 patients treated with relevant antibiotics up to 11 days before puncture. Therefore, we recommend removal of pus by excision or puncture.

Adolescent↗
PubMed1990

High-frequency transfection of cultured human epidermal basal cells that differentiate to form a multilayered tissue.

Primary cultures of human keratinocytes form a multilayered tissue. By incubating the tissue cultures in Ca2(+)-free medium the differentiated cell layers can be stripped off leaving a basal cell monolayer. We have developed a method for high-frequency transfection of these epidermal basal cells with genes inserted into Epstein-Barr virus-based expression vectors. Using the Escherichia coli lac z gene as a marker gene, the transient and long-term expression and the fate of the transfected cells were studied. During regeneration of the multilayered tissue most of the transfected basal cells enlarge and undergo differentiation, but a minor population remains as basal cells. Incubation with the tumor promotor 12-O-tetradecanoylphorbol-13-acetate results in an increase in the proportion of transfected keratinocytes that are small, suggesting a relative expansion of the immature cell pool.

Adult↗
PubMed1990

Measles incidence, vaccine efficacy, and mortality in two urban African areas with high vaccination coverage.

Measles incidence, vaccine efficacy, and mortality were examined prospectively in two districts in Bissau where vaccine coverage for children aged 12-23 months was 81% (Bandim 1) and 61% (Bandim 2). There was little difference in cumulative measles incidence before 9 months of age (6.1% and 7.6%, respectively). Between 9 months and 2 years of age, however, 6.1% contracted measles in Bandim 1 and 13.7% in Bandim 2. Even adjusting for vaccination status, incidence was significantly higher in Bandim 2 (relative risk 1.6, P = .04). Even though 95% of the children had measles antibodies after vaccination, vaccine efficacy was not more than 68% (95% confidence interval [CI] 39%-84%) and was unrelated to age at vaccination. Unvaccinated children had a mortality hazard ratio of 3.0 compared with vaccinated children (P = .002), indicating a protective efficacy against death of 66% (CI 32%-83%) of measles vaccination. These data suggest that it will be necessary to vaccinate before age 9 months to control measles in hyperendemic urban African areas.

Age Factors↗
PubMed1988

Trial of high-dose Edmonston-Zagreb measles vaccine in Guinea-Bissau: protective efficacy.

In a randomised study of 558 children in an urban African community, the protective effect of the Edmonston-Zagreb (EZ) measles vaccine given in a dose of 40,000 plaque forming units from the age of 4 months was compared with the effects of a standard dose (6000 tissue culture infectious units) of Schwarz measles vaccine given from the age of 9 months. During two years of follow-up, all 14 clinical cases of measles occurred in the Schwarz group; 10 of the children contracted measles before vaccination and 4 after measles vaccination. Thus the EZ vaccine provided significant protection against measles both before and after the usual age of vaccination. Among the children who were exposed to measles at home, those given EZ vaccine were better protected than either unvaccinated children or those given the Schwarz vaccine.

Age Factors↗
PubMed1985

Nutritional assessment indications of postburn complications.

This study evaluates the use of nutritional assessment indexes measured on the fifth day after injury to predict subsequent wound infections, episodes of septicemia, and other infectious complications in burned patients. Nutritional assessment data collected included anthropometric measurements (weight, height, triceps skinfold, and upper-arm circumference); biochemical testing (serum albumin concentration, serum transferrin concentration, total lymphocyte count, creatinine/height index, and nitrogen balance); and recall skin antigen testing. Seventy-four adult patients (mean age of 35 years) who had burn injuries ranging from 10% to 96% total body surface were studied. Indexes predictive of imminent wound infection include serum transferrin concentrations less than 150 mg/dl (p = .0006) and anergy to the skin test battery (p = .01). Those indexes were also prognostic of the development of septicemia (p = .0002 and p = .0001, respectively). Although statistically insignificant, total lymphocyte count also showed a similar trend toward predicting complications. Serum albumin concentration (less than 3.0 gm/dl), creatinine/height index (less than 60% standard), percent ideal body weight (less than 80%), percent weight change, and nitrogen balance did not contribute to group discrimination. Severe depletion of the visceral protein compartment was documented in a large percentage of patients on the fifth postburn day. Serum transferrin concentration and recall skin antigen testing can be helpful in identifying thermally injured patients who are at high risk of infectious complications.

Adolescent↗
PubMed1984

Determination of nutritional status in critical care.

The application of the standard battery of nutritional assessment tests, measurements, and calculations is of limited value in the immediate nutritional diagnosis of the critically ill patient. Many of these tests are affected by stress, making it impossible to distinguish the etiology of abnormalities during the early postinjury period. Altered test results because of stress or injury include: increased excretion of creatinine; increased white blood cell (WBC) counts; increased anergy to skin tests; and decreased serum albumin, transferrin, thyroxin-binding prealbumin, and retinol-binding protein concentrations. Therefore, in assessing the nutritional status of the critically ill patient, it is important to focus on indexes that are realistic relative to the patient's metabolic status. Timing of data collection is of keen importance; data collected after fluid resuscitation and the peak metabolic response to injury on postinjury days 5 through 10 are usually more meaningful than those collected immediately after hospital admission. Admission assessment is necessary only to identify the high-risk patient in need of immediate nutrition intervention, to estimate energy/nutrition and fluid requirements, and to provide guidelines for planning nutrition care. This article presents recommendations for initial assessment, postcatabolism assessment, and serial assessment of the critically ill patient. Guidelines for estimating energy/nutrient requirements and timing data collection are also presented.

Anthropometry↗
PubMed1983

Delayed hypersensitivity skin testing: response rates in a surgical population.

The results of this study indicate that skin test responses should be measured at both 24 and 48 hours post-injection and considered reactive or positive if induration of greater than or equal to 5 mm. is noted at either time. The practice of measuring responses at 48 hours only is neither appropriate nor recommended as it may produce a large percentage of false negative results. Erythema without induration was noted in a small percentage of patients. Future studies should evaluate the efficacy of erythema greater than or equal to 15 mm. in the absence of induration as a reactive response to mumps antigen. The validity of data interpretation must ultimately be based on correlations with morbidity and mortality. Streptokinase-streptodornase, mumps, and trichophyton were the three antigens with highest response rates, and this battery detected 94% of the reactive or positive responses to the five-antigen battery.

Adolescent↗
PubMed1982

Nutrition in orthopaedic surgery.

One hundred and twenty-nine patients undergoing orthopaedic surgical procedures were found to have an average incidence of clinical and subclinical malnutrition of 42.4 per cent per patient. The lowest incidence of nutritional depletion was identified in patients undergoing total hip-replacement surgery (28.6 per cent), followed by the elective surgical group (35.3 per cent) and patients with multiple trauma and femoral fractures (58.6 per cent). Trauma and major surgery were shown to induce a state of malnutrition and a loss of immunocompetence. A significant (p less than 0.05) correlation between subnormal nutritional indices and the development of complications was identified. A comprehensive approach to the detection and correction of malnutrition in orthopaedic patients is described.

Adolescent↗
PubMed1981

Implementation of a multidisciplinary nutritional assessment program.

Depending on the severity of malnutrition, protein depletion is associated with general debilitation, inanition, slow healing of wounds, delayed convalescence, increased susceptibility to infection, and other pathophysiologic conditions. In an effort to decrease the incidence and severity of these complications, Hermann Hospital and the University of Texas Medical School at Houston have implemented a comprehensive nutritional assessment program to identify clinical and subclinical malnutrition and to implement aggressive, effective therapy. A multidisciplinary team approach to nutritional assessment, therapy, and research can lead to an increased awareness of the importance of nutritional support and optimal nutritional care for the hospitalized patient under all conditions at all times.

Anthropometry↗
PubMed1981

Marketing strategy: a key leverage point for dietitians.

The time is right for planned change in the profession of dietetics. The concept of the nutrition support team offers the clinical dietitian a means of increasing the visibility and effectiveness of dietetic services. Dietitians must take advantage of the renewed interest in nutrition and intervene assertively to improve nutritional care through role expansion. By focusing on a systematic marketing strategy to expand their current role, dietitians can become proactive participants in role change, rather than reactive victims to changes dictated by others. When goals, objectives, and plans are made explicit, definite progress toward desired role changes can be recognized. This case study demonstrates that this change in role is possible within the present state of health case systems and that physicians are willing and even eager to allow dietitians to assume a high degree of responsibility for nutritional care.

Dietary Services↗