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Biomedical subjects

T G Jensen

Publications and source records attributed to T G Jensen.

At least 19 recordsLinked to original sources
PubMed2006

Phi c31 integrase induces chromosomal aberrations in primary human fibroblasts.

Phi c31 integrase is investigated as a novel tool for nonviral gene therapy as the enzyme can direct site-specific integration into a host chromosome. In order to investigate effects of phi c31 integrase expression in normal human cells, we have generated stably transfected primary human fibroblasts expressing the enzyme. All control cells were cytogenetically normal, but in cells expressing phi c31 integrase, numerous chromosomal abnormalities including various translocations were found, suggesting that the enzyme itself acts as a mutagen.

Attachment Sites, Microbiological↗
PubMed2005

Tissue distribution and engraftment of human mesenchymal stem cells immortalized by human telomerase reverse transcriptase gene.

Engraftment of mesenchymal stem cells (MSC) in peripheral tissues for replenishing of local stem cell function has been proposed as a therapeutic approach to degenerative diseases. We have previously reported the development of an immortalized human telomerase reverse transcriptase transduced MSC line (hMSC-TERT). In the present study, we co-transduced hMSC-TERT with enhanced green fluorescent protein gene, and studied tissue distribution, engraftment, and cell survival after intracardiac and intravenous injections in immunodeficient mice. The pattern of organ distribution suggested that infused cells were efficiently arrested in microvasculature during first-pass, but only for a fraction of the infused cells was arrest followed by vascular emigration and tissue engraftment. Few engrafted cells in lungs, heart, and kidney glomeruli remained after 4 weeks. These observations are consistent with several reports on limited systemic transplantability of primary MSC. HMSC-TERT may constitute a valuable tool for mechanistic studies on how to control MSC homing and engraftment.

Animals↗
PubMed2004

Fusarium fungaemia in immunocompromised patients.

Fusarium spp. cause infections only rarely in immunologically competent hosts, but disseminated infection may occur in severely immunocompromised patients. Symptoms of disseminated infection are persistent fever, despite broad-spectrum antibacterial and antifungal treatment, associated with skin lesions, most commonly on the extremities, in 60-80% of patients. A mortality rate of 50-75% has been reported for patients with disseminated fusariosis. Despite treatment failures, amphotericin B remains the preferred drug, in part because of lack of alternatives. Voriconazole is a promising new agent, but more clinical experience is required.

Aged↗
PubMed2001

Tail-vein injection of mannan-binding lectin DNA leads to high expression levels of multimeric protein in liver.

The human plasma protein mannan-binding lectin (MBL) is an essential part of the innate immune defense system. Low levels of MBL are associated with recurrent infections and other clinically significant signs of a compromised immune defense. Previous studies have addressed the possibility of reconstitution therapy by the use of recombinant or plasma-derived protein. Natural MBL is a multimeric protein, which consists of up to 18 identical polypeptide chains. Synthesis by in vitro methods of MBL with the proper multimeric structure is difficult. We here report that mice obtain MBL levels comparable to those found in normal human plasma when injected with an MBL expression construct as naked plasmid DNA contained in a large volume of physiologic salt solution. The expression was confined to the liver and high MBL expression levels were obtained with less than 5% of the liver cells transfected. The multimeric structure of the MBL found in plasma of injected mice was similar to that of natural MBL. Thus, liver expression following injection of naked DNA is an alternative to reconstitution therapy with a protein having a complex quaternary structure.

Animals↗
PubMed2001

Cutaneous gene therapy--an update.

In this paper we review various strategies for gene transfer into the skin, their applications and show some of our own examples. The skin is the most accessible somatic tissue. Inherited skin diseases, such as epidermolysis bullosa and ichthyosis, and various systemic metabolic disorders are under investigation as potential candidate diseases for cutaneous gene transfer. Research is directed against genetic therapy of wounds and malignancies as well. So far, cutaneous gene transfer only has been used experimentally, but several clinical trials are under preparation.

Gene Transfer Techniques↗
PubMed2001

Recombinant expression of human mannan-binding lectin.

Mannan-binding lectin (MBL) constitutes an important part of the innate immune defence by effecting the deposition of complement on microbial surfaces. MBL deficiency is among the most common primary immunodeficiencies and is associated with recurrent infections and symptoms of poor immune complex clearance. Plasma-derived MBL has been used in reconstitution therapy but concerns over viral contamination and production capacity point to recombinant MBL (rMBL) as a future source of this protein for clinical use. Natural human MBL is an oligomer of up to 18 identical polypeptide chains. The synthesis of rMBL has been accomplished in several mammalian cell lines, however, the recombinant protein differed structurally from natural MBL. In this, study we compare rMBL produced in myeloma cells, Chinese hamster ovary (CHO) cells, human hepatocytes, and human embryonic kidney (HEK) cells. We report that rMBL structurally and functionally similar to natural MBL can be obtained through synthesis in the human embryonic kidney cells followed by selective carbohydrate affinity chromatography.

Carrier Proteins↗
PubMed2001

LDL receptor-GFP fusion proteins: new tools for the characterisation of disease-causing mutations in the LDL receptor gene.

The function of a series of LDL receptor GFP fusion proteins with different, flexible, unstructured spacer regions was analysed. An optimised version of the fusion protein was used to analyse the effect of an LDL receptor mutation (W556S) found in FH patients and characterised as transport defective. In cultured liver cells this mutation was found to inhibit the transport of LDL receptor GFP fusion protein to the cell surface, thus leading to impaired internalisation of fluorescent labelled LDL. Co-localisation studies confirmed the retention of the mutant protein in the endoplasmic reticulum. Wild type (WT) and W556S LDL receptor GFP fusion proteins were expressed in mouse liver by means of hydrodynamic delivery of naked DNA. Two days after injection liver samples were analysed for GFP fluorescence. The WT LDL receptor GFP protein was located on the cell surface whereas the W556S LDL receptor GFP protein was retained in intracellular compartments. Thus, the GFP-tagged LDL receptor protein allows both detailed time lapse analysis and evaluations in animals for the physiological modelling of mutations. This method should be generally applicable in functional testing of gene products for aberrant processing.

Animals↗
PubMed2000

Transgene expression in human epidermal keratinocytes: cell cycle arrest of productively transfected cells.

We have analysed the consequences of liposome mediated gene transfer into human primary epidermal keratinocytes and compared non-Epstein-Barr Virus (EBV) and EBV based expression vectors that carry the genes encoding human Growth Hormone (hGH) or Enhanced Green Fluorescent Protein (EGFP). Different kinetics between the non-EBV and EBV based vectors were revealed upon subcultivation of hGH transfected keratinocytes. The keratinocytes transfected with non-EBV based vector showed a rapid reduction in hGH production. Although the EBV based vector resulted in more stable expression, this was also reduced over time. Chromatin inactivation by deacetylation was investigated by treatment with sodium butyrate and found not to be the reason for the decreasing expression. Keratinocytes divided into subpopulations enriched for either stem cells or transit amplifying cells, based on beta1-integrin expression and function, do not differ significantly with respect to susceptibility to productive transfection. However, when the keratinocytes were transfected with the EGFP gene and sorted live by FACS into EGFP negative and positive populations, only the negative cells were capable of forming significant numbers of colonies. This is consistent with the observation that the ability to incorporate BrdU was dramatically reduced in the EGFP expressing population within 24-48 h post transfection indicating an almost complete cell cycle arrest. p53 levels were unaffected by the procedures, and the keratinocyte cell line HaCat, mutated in both p53 alleles, also shows a marked reduction in clonogenic potency upon transfection. There was a slight increase of TUNEL positive apoptotic nuclei in the positive population at early time points. However, the apoptotic index was still very low. When we measured the frequency of involucrin expressing cells, we found an increase in the productively transfected population over time indicating an initiation of terminal differentiation. In contrast to the transfected cultures, keratinocytes that were transduced using a retroviral vector showed no decrease in colony forming efficiency. In conclusion we find that transgene expressing cells from transfected cultures of epidermal keratinocytes undergo cell cycle arrest and initiate terminal differentiation by mechanisms which are independent of p53 levels.

Cell Adhesion↗
PubMed2000

Development of a skin-based metabolic sink for phenylalanine by overexpression of phenylalanine hydroxylase and GTP cyclohydrolase in primary human keratinocytes.

Phenylketonuria, PKU, is caused by deficiency of phenylalanine hydroxylase (PAH) resulting in increased levels of phenylalanine in body fluids. PAH requires the non-protein cofactor BH4 and the rate-limiting step in the synthesis of BH4 is GTP cyclohydrolase I (GTP-CH). Here we show that overexpression of the two enzymes PAH and GTP-CH in primary human keratinocytes leads to high levels of phenylalanine clearance without BH4 supplementation. Integration of multiple PAH and GTP-CH transgenes were achieved after optimized retroviral transduction. Phenylalanine clearance was measured ex vivo in primary human keratinocytes cotransduced with PAH and GTP-CH (more than 370 nmol/24 h/106 cells), a level exceeding that of a human liver cell line (HepG2 cells). Cells overexpressing either one of the enzymes alone did not clear significant amounts of phenylalanine. Transfer of the two genes into the same cell was not necessary, since cocultivation of cells transduced separately with PAH and GTP-CH also resulted in phenylalanine clearance. Thus the experiments indicate metabolic cooperation between cells overexpressing PAH and cells overexpressing GTP-CH, possibly due to intercellular transport of synthesized BH4.

Biopterins↗
PubMed2000

Antagonism between penicillin and erythromycin against Streptococcus pneumoniae in vitro and in vivo.

The combination of beta-lactam antibiotics and macrolides is often recommended for the initial empirical treatment of acute pneumonia in order to obtain activity against the most important pathogens. Theoretically, this combination may be inexpedient, as the bacteriostatic agent may antagonize the effect of the bactericidal agent. In this study, the possible interaction between penicillin and erythromycin was investigated in vitro and in vivo against four clinical isolates of Streptococcus pneumoniae with MICs of penicillin ranging from 0.016 to 0.5 mg/L and of erythromycin from 0. 25 to >128 mg/L. In vitro time-kill curves were generated with clinically relevant concentrations of penicillin (10 mg/L) and erythromycin (1 mg/L), either individually or in combination. Antagonism between penicillin and erythromycin was observed for the four isolates. In vivo interaction was investigated in the mouse peritonitis model. After intraperitoneal inoculation, penicillin and erythromycin were given either individually or in combination. For two of the four isolates, mortality was significantly higher in the groups treated with the combination of penicillin and erythromycin than in the groups treated with penicillin alone [32/36 (86%) vs. 3/12 (25%), P<0.05; and 24/36 (67%) vs. 3/12 (25%), P<0.05, respectively]. Using the mouse peritonitis model, in vivo time-kill curves showed that there was antagonism between erythromycin and penicillin for the examined isolate. The antagonism demonstrated in vitro and in vivo between penicillin and erythromycin suggests that ss-lactam antibiotics and macrolides should not be administered together unless pneumococcal infection is ruled out.

Animals↗
PubMed1999

Resistance problems in two university hospitals in Denmark.

In 1997, 80 and 89% of Staphylococcus aureus isolated from university hospitals in Odense and Hvidovre respectively were resistant to penicillin and 32.0 and 41% of Escherichia coli were resistant to ampicillin. There were low incidences of methicillin resistance in S. aureus (<1%), penicillin resistance in Streptococcus pneumoniae (3%),and gentamicin in E. coli (2%). These figures might reflect the low use of antibiotics in Denmark.

Ampicillin Resistance↗
PubMed1999

How realistic is cutaneous gene therapy?

Recent progress with innovative, experimental gene therapy approaches in animals, and recent improvements in our understanding and manipulation of stem cells, gene expression and gene delivery systems, have raised plenty of hopes in essentially all branches of clinical medicine that hitherto untreatable or poorly manageable diseases will soon become amenable to treatment. Few other organ systems have received such enthusiastic reviews in recent years as to the chances and prospects of gene therapy as the skin, with its excellent accessibility and its pools of--seemingly--readily manipulated epithelial stem cells (cf. Cotsarelis et al., Exp Dermatol 1999: 8: 80-88). However, as in other sectors of clinical medicine, the actual implementation of general gene therapy strategies in clinical practice has been faced with a range of serious difficulties (cf. Smith, Lancet 1999: 354 (suppl 1): 1-4; Lattime & Gerson (eds.), Gene Therapy of Cancer, Academic Press, San Diego, 1999). Thus, it is critically important to carefully distinguish unfounded hype from justified hope in this embryonal area of dermatologic therapy, to discuss in detail what can be realistically expected from cutaneous gene therapy approaches in the next few years, and importantly, what kind of promises should not be made to our patients at this time.

Animals↗
PubMed1999

Clinical and molecular evidence of abnormal processing and trafficking of the vasopressin preprohormone in a large kindred with familial neurohypophyseal diabetes insipidus due to a signal peptide mutation.

The autosomal dominant form of familial neurohypophyseal diabetes insipidus (adFNDI) is a rare disease characterized by postnatal onset of polyuria and a deficient neurosecretion of the antidiuretic hormone, arginine vasopressin (AVP). Since 1991, adFNDI has been linked to 31 different mutations of the gene that codes for the vasopressin-neurophysin II (AVP-NPII) precursor. The aims of the present study were to relate the clinical phenotype to the specific genotype and to the molecular genetic effects of the most frequently reported adFNDI mutation located at the cleavage site of the signal peptide of AVP-NPII [Ala(-1)Thr]. Genetic analysis and clinical studies of AVP secretion, urinary AVP, and urine output were performed in 16 affected and 16 unaffected family members and 11 spouses of a Danish adFNDI kindred carrying the Ala(-1)Thr mutation. Mutant complementary DNA carrying the same mutation was expressed in a neurogenic cell line (Neuro2A), and the cellular effects were studied by Western blotting, immunocytochemistry, and AVP measurements. The clinical studies showed a severe progressive deficiency of plasma and urinary AVP that manifested during childhood. The expression studies demonstrated that the Ala(- 1)Thr mutant cells produced 8-fold less AVP than wild-type cells and accumulated excessive amounts of 23-kDa NPII protein corresponding to uncleaved prepro-AVP-NPII. Furthermore, a substantial portion of the intracellular AVP-NPII precursor appeared to be colocalized with an endoplasmic reticulum antigen (Grp78). These results provide independent confirmation that this Ala(-1)Thr mutation produces adFNDI by directing the production of a mutant preprohormone that accumulates in the endoplasmic reticulum, because it cannot be cleaved from the signal peptide and transported to neurosecretory vesicles for further processing and secretion.

Adolescent↗
PubMed1998

Rapid degradation of short-chain acyl-CoA dehydrogenase variants with temperature-sensitive folding defects occurs after import into mitochondria.

Most disease-causing missense mutations in short-chain acyl-CoA dehydrogenase (SCAD) and medium-chain acyl-CoA dehydrogenase are thought to compromise the mitochondrial folding and/or stability of the mutant proteins. To address this question, we studied the biogenesis of SCAD proteins in COS-7 cells transfected with cDNA corresponding to two SCAD missense mutations, R22W (identified in a patient with SCAD deficiency) or R22C (homologous to a disease-associated R28C mutation in medium-chain acyl-CoA dehydrogenase deficiency). After cultivation at 37 degreesC the steady-state amounts of SCAD antigen and activity in extracts from cells transfected with mutant SCAD cDNAs were negligible compared with those of cells transfected with SCAD wild type cDNA, documenting the deleterious effect of the two mutations. Analysis of metabolically labeled and immunoprecipitated SCAD wild type and mutant proteins showed that the two mutant proteins were synthesized as the 44-kDa precursor form, imported into mitochondria and processed to the mature 41.7-kDa form in a normal fashion. However, the intramitochondrial level of matured mutant SCAD proteins decreased rapidly to very low levels, indicating a rapid degradation of the mutant proteins at 37 degreesC. A rapid initial elimination phase was also observed following cultivation at 26 degreesC; however, significantly higher amounts of metabolically labeled and immunoprecipitated mature mutant SCAD proteins remained detectable. This corresponds well with the appreciable steady-state levels of SCAD mutant enzyme activity observed at 26 degreesC. In addition, confocal laser scanning microscopy of immunostained cells showed that the SCAD mutant proteins were localized intramitochondrially. Together, these results show that newly synthesized SCAD R22W and R22C mutant proteins are imported and processed in the mitochondrial matrix, but that a fraction of the proteins is rapidly eliminated by a temperature-dependent degradation mechanism. Thermal stability profiles of wild type and mutant enzymes revealed no difference between the two mutants and the wild type protein. Furthermore, the turnover of the SCAD mutant enzymes in intact cells was comparable to that of the wild type, indicating that the rapid degradation of the mutant SCAD proteins is not due to lability of the correctly folded tetrameric structure but rather to elimination of partly folded or misfolded proteins along the folding pathway.

Acyl-CoA Dehydrogenase↗
PubMed1998

Detection of extended-spectrum beta-lactamases. The reliability of methods for susceptibility testing as used in Denmark.

The susceptibility testing methods used in Denmark were evaluated with respect to their ability to detect cephalosporin resistance with cefuroxime as indicator, especially resistance caused by extended-spectrum beta-lactamases (ESBLs). Two methods for determination of minimal inhibitory concentration (MIC), three agar diffusion methods, a disc approximation test and the ESBL-Etest were used against a panel of strains producing well-known beta-lactamases. The tablet diffusion test (Rosco Neo Sensitabs) as the most used in Denmark had the lowest detection rate of cefuroxime resistance among ESBL-producing strains. The prediffusion method, which is only used at one laboratory, was the most reliable method for such detection. The MIC methods were in good agreement, but the detection rate for resistance due to ESBLs was low and depended on the antibiotics used. The disc approximation test and the ESBL-Etest both resulted in an acceptable ESBL detection rate. The latter tests discriminated between isolates producing the frequent chromosome-mediated and the in Denmark probably very rare ESBL-mediated cephalosporin resistance. For the evaluation of susceptibility tests such strains require special attention.

Alcaligenes↗
PubMed1997

Retrovirus-mediated gene transfer of ornithine-delta-aminotransferase into keratinocytes from gyrate atrophy patients.

Gyrate atrophy is a progressive blindness associated with deficiency of ornithine aminotransferase (OAT). The strategy of using an autologous keratinocyte graft, modified to express high levels of OAT as an ornithine-catabolizing skin-based enzyme sink, is investigated. Two OAT-containing retroviral vectors were constructed with or without a resistance gene. When packaged in a retroviral vector particle generated with the gibbon ape leukemia (GALV) virus envelope (PG13), these vectors could readily transduce >50% of target keratinocytes. The transduced keratinocytes in culture expressed up to 75-fold more OAT than normal control keratinocytes and these gene-modified cells extracted [14C]ornithine more efficiently than controls. The vector prepared without neo transduced cells more efficiently and led to higher levels of OAT expression than the neo-containing vector. Ornithine catabolism was maintained at high levels when the transduced patient keratinocytes were differentiated in vitro as a multilayered cutaneous organoid.

3T3 Cells↗