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Biomedical subjects

U Stein

Publications and source records attributed to U Stein.

At least 19 recordsLinked to original sources
PubMed2006

Uptake, biodistribution, and time course of naked plasmid DNA trafficking after intratumoral in vivo jet injection.

Nonviral jet injection is an applicable technology for in vivo gene transfer of naked DNA. However, little is known about the biodistribution and clearance of jet-injected DNA, or about its localization within tissue and cells. Therefore, in this study we analyzed the intratumoral and systemic biodistribution of jet-injected naked DNA in human colon carcinoma-bearing NCr-nu/nu mice, which were jet-injected with the pCMVbeta plasmid DNA. Intratumoral and systemic plasmid DNA biodistribution was analyzed 5, 10, 20, and 40 min and 3, 6, 24, 48, and 72 hr after jet injection, using quantitative real-time polymerase chain reaction. In the tumors, a rapid drop in naked DNA load within 24 hr of jet injection was shown. Detailed analysis of intratumoral distribution of rhodamine-labeled DNA revealed the presence of plasmid DNA within tumor cells 5 min after jet injection and further accumulation of significant DNA amounts in the cell nuclei 30 to 60 min after jet injection. In the blood, DNA amounts rapidly dropped within 10 to 40 min of jet injection to less than 0.001 pg of plasmid per 250 ng of tissue DNA and only minimal plasmid DNA dissemination was detected in liver, lung, spleen, kidney, and ovaries, which was cleared 3 to 6 hr after jet injection. By contrast, in heart, bone marrow, and brain almost no plasmid DNA was detectable.

Animals↗
PubMed2005

Enhanced repair of articular cartilage defects in vivo by transplanted chondrocytes overexpressing insulin-like growth factor I (IGF-I).

Traumatic articular cartilage lesions have a limited capacity to heal. We tested the hypothesis that overexpression of a human insulin-like growth factor I (IGF-I) cDNA by transplanted articular chondrocytes enhances the repair of full-thickness (osteochondral) cartilage defects in vivo. Lapine articular chondrocytes were transfected with expression plasmid vectors containing the cDNA for the Escherichia coli lacZ gene or the human IGF-I gene and were encapsulated in alginate. The expression patterns of the transgenes in these implants were monitored in vitro for 36 days. Transfected allogeneic chondrocytes in alginate were transplanted into osteochondral defects in the trochlear groove of rabbits. At three and 14 weeks, the quality of articular cartilage repair was evaluated qualitatively and quantitatively. In vitro, IGF-I secretion by implants constructed from IGF-I-transfected chondrocytes and alginate was 123.2+/-22.3 ng/10(7) cells/24 h at day 4 post transfection and remained elevated at day 36, the longest time point evaluated. In vivo, transplantation of IGF-I implants improved articular cartilage repair and accelerated the formation of the subchondral bone at both time points compared to lacZ implants. The data indicate that allogeneic chondrocytes, transfected by a nonviral method and cultured in alginate, are able to secrete biologically relevant amounts of IGF-I over a prolonged period of time in vitro. The data further demonstrate that implantation of these composites into deep articular cartilage defects is sufficient to augment cartilage defect repair in vivo. These results suggest that therapeutic growth factor gene delivery using encapsulated and transplanted genetically modified chondrocytes may be applicable to sites of focal articular cartilage damage.

Alginates↗
PubMed2005

Current TNM classification of renal cell carcinoma evaluated: revising stage T3a.

PURPOSE: : Recent studies of rare cases of pT3a renal cell carcinoma extending directly into the adrenal gland showed worse survival than in other pT3a cases and recategorization as stage pT4 was suggested. We assessed the prognostic validity of a stage pT3a diagnosis based on perirenal fat infiltration. MATERIALS AND METHODS: : The records of 1,794 patients with renal cell carcinoma who underwent surgical resection between 1975 and 2000 at our institution were analyzed retrospectively. Focusing on pT3a tumors, as defined by perirenal fat infiltration, numerous clinical and histopathological parameters were investigated by univariate and multivariate statistical methods with cancer specific survival as the primary end point. RESULTS: : We identified 237 of 1,794 patients with perirenal fat infiltration, classified as having pT3a disease. In patients with pT3a tumors tumor size was a significant parameter predicting survival. The most significant cutoff value for tumor size in pT3a disease was 7 cm. Patients with distant metastasis had a worse prognosis independent of T classification. Therefore, to assess the prognostic value of the current T classification in regard to T3a tumors we excluded patients with tumor stage cM+ for further subgroup analysis. Survival comparison of pT1 pNall, cM0 (744 of 1,794 cases) and pT3a pNall, cM0 7 cm or less (100 of 237) as well as pT2 pNall, cM0 (265 of 1,794) and pT3a pNall, cM0 greater than 7 cm (93 of 237) yielded similar results. After splitting pT3a into a modified T1/T2 classification a significant difference in 5-year survival analysis for a modified T1/T2 stage was found (pT1 plus pT3a less than 7 cm 90% vs pT2 plus pT3a greater than 7 cm 73%, p <0.001). Subsequently multivariate analysis in all 1,794 patients showed that modified T stage was an independent significant predictor of cancer specific survival. CONCLUSIONS: : We suggest revising the current pT3a classification based on perirenal fat infiltration but rendering a modified pT1/pT2 classification, which resolves pT3a cases without the loss of prognostic validity. Perirenal fat infiltration should not be used to assign T category. Tumors directly infiltrating the adrenal gland should be reclassified as T4.

Carcinoma, Renal Cell↗
PubMed2004

[Interstitial cystitis. Current aspects of diagnosis and therapy].

Interstitial cystitis is a chronic, heterogeneous syndrome that is characterized by the classic symptoms of bladder pain, nycturia, and pollakiuria. Population studies have shown an increase in prevalence and incidence. Different hypotheses exist about the etiology and pathogenesis. Epithelial dysfunction and neuro-urothelial interaction are the most frequently used causal models. Utilization of the official diagnosis criteria of the NIDDK (National Institute of Diabetes, Digestive and Kidney Disease) should be flexible and individual in order not to overlook early forms of IC. Since there is no pathognomonic marker, diagnosis of IC is a combination of typical patient history and exclusion of differential diagnoses. Cystoscopy and biopsies are not specific; however, they can provide useful information on extent and aid in treatment choice. The effectiveness of conservative therapy has been increased by using electromotive drug administration (EMDA) in instillation therapy.

Causality↗
PubMed2004

Validation of the inflammatory bowel disease questionnaire IBDQ-D, German version, for patients with ileal pouch anal anastomosis for ulcerative colitis.

BACKGROUND AND AIMS: The inflammatory bowel disease questionnaire (IBDQ) is the standard instrument for assessment of health-related quality of life (HRQOL) in patients with inflammatory bowel diseases. It has not been validated for patients with ileal pouch anal anastomosis (IPAA) and ulcerative colitis (UC). METHODS: To determine acceptance (percentage of completed items), reliability (Cronbach's alpha of the IBDQ-D subscales) and convergent validity (correlations of the IBDQ subscales with the questionnaires used for validation) 61 patients with UC (age 52.7 +/- 13.9 years; 47 % female, 53 % male) and IPAA completed the German (Competence Network IBD) version of the Inflammatory Bowel Disease Questionnaire (IBDQ-D), the Short Form Health Survey (SF-36) the Hospital Anxiety and Depression Scale German Version (HADS-D) and the Giessener Symptom List (GBB 24). Face validity was assessed by a physicians' and patients' panel. All 37 patients underwent endoscopy making it possible to differentiate between patients with and without pouchitis (discriminant validity). RESULTS: With 97.7 % completed items the acceptance was high. Cronbach's alpha value for the subscales ranged from 0.71 to 0.93. Missing items covering extraintestinal manifestations of IBD were criticized by patients. The correlation coefficients with comparable subscales of other instruments ranged between 0.41 and 0.76. Patients with clinical pouchitis scored significantly lower in all subscales than patients without pouchitis (p < 0.001). CONCLUSION: The IBDQ-D has good acceptance, reliability, convergent and discriminant validity, but limited face and construct validity in patients with IPAA and UC.

Adult↗
PubMed2003

Genetic imbalances with impact on survival in colorectal cancer patients.

AIMS: To investigate the prognostic significance of chromosomal alterations in colorectal cancer patients. Histopathological tumour classification is still considered to be the gold standard for the characterization of solid tumours. However, it is well known that such established parameters do not satisfactorily predict the clinical outcome in individual cases. Markers that reliably predict survival are needed. These markers should guide the clinical treatment of neoplastic disease. METHODS AND RESULTS: Chromosomal imbalances in 61 colorectal carcinoma specimens in 37 patients determined by comparative genomic hybridization were correlated with patient survival using custom-made computer software which enabled the assessment of individual chromosomal loci. Kaplan-Meier analysis revealed that over-representations of 2p14-15, 6q23-6q24, 15q22-15q23, 22q11.2 and deletions of 1p36.1-36.2, 4q31.3, 4q35, 8q12-q21, 8p11.2 and 9p22 were significantly associated with shorter disease-specific survival, whereas over-expression of 20q13.3 and deletion of 18q11.2 were significantly associated with longer disease-specific survival in this collection of colorectal cancers. Multivariate Cox proportional hazards regression models consistently identified gains of 2p14-15, 15q22-23, 22q11.2 and losses of 1p36.1-36.2 and 4q35 as independent markers of shorter patient survival carrying greater significance than the classical clinicopathological parameters of nodal status and tumour grade. CONCLUSIONS: These five markers allow a molecular categorization of patients into high and low clinical risk groups. Thus, the genomic data have refined the histopathological classification highlighting the necessity for a supplementary genetically based stratification of colorectal cancer.

Adenocarcinoma↗
PubMed2002

Pentoxifylline influences drug transport activity of P-glycoprotein and decreases mdrl gene expression in multidrug resistant mouse leukemic L1210/VCR cells.

The effects of pentoxifylline (PTX) on intracellular accumulation of doxorobicin (DOX), DOX cytotoxicity and expression of Pgp in multidrug resistant L1210/VCR cell line were investigated. PTX (100 mg/l) was able to enhance the DOX accumulation in resistant cells. The maximum intracellular levels of DOX were reached after treatment with PTX for 24 hours (total duration of PTX-treatment was 72 hours). The levels of mdrl mRNA (measured by RT-PCR method) were decreased 2-fold in the presence of 100 mg/l PTX (minimum reached within 48 hours) in comparison to control cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Hyperthermia-induced nuclear translocation of transcription factor YB-1 leads to enhanced expression of multidrug resistance-related ABC transporters.

Genotoxic stress leads to nuclear translocation of the Y-box transcription factor YB-1 and enhanced expression of the multidrug resistance gene MDR1. Because hyperthermia is used for the treatment of colon cancer in combination with chemoradiotherapy, we investigated the influence of hyperthermia on YB-1 activity and the expression of multidrug resistance-related genes. Here we report that hyperthermia causes YB-1 translocation from the cytoplasm into the nucleus of human colon carcinoma cells HCT15 and HCT116. Nuclear translocation of YB-1 was associated with increased MDR1 and MRP1 gene activity, which is reflected in strong efflux pump activity. However, a combination of hyperthermia and drug treatment effectively reduced cell survival of the HCT15 and HCT116 cells. These results demonstrate that activation of MDR1 and MRP1 gene expression and increased efflux pump activity after hyperthermia were insufficient to cause an increase in drug resistance in colon cancer cell lines. The ability of hyperthermia to abrogate drug resistance in the presence of an increase in functional MDR proteins may provide an explanation for the efficacious results seen in the clinic in colon cancer patients treated with a combination of hyperthermia and chemotherapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Nutmeg (myristicin) poisoning--report on a fatal case and a series of cases recorded by a poison information centre.

In literature, cases of nutmeg abuse have been described repeatedly, but only one fatal case of poisoning was reported [1]. In the present case, myristicin (4 microg/ml) was detected for the first time in the postmortal serum of a 55-year-old woman. Identification was achieved with the aid of UV-VIS spectroscopy and TLC; for quantification, HPLC was used. Because also flunitrazepam (0.072 microg/ml) was found, death had probably been due to the combined toxic effect of both substances. From 1996 to 1998, in a series of cases, seven poisonings with nutmeg were recorded by the Erfurt Poison Information Centre. Even where higher doses (20-80 g of powder) had been ingested, a life-threatening situation was never observed. In one of these cases, a myristicin blood level of 2 microg/ml was measured 8h after ingestion of two to three tablespoonful of nutmeg powder (approx. 14-21 g, or 280-420 mg/kg).

Adolescent↗
PubMed2001

Nonviral in vivo gene delivery into tumors using a novel low volume jet-injection technology.

The jet-injection technology has developed as an applicable alternative to viral or liposomal gene delivery systems. In this study a novel, low-volume, 'high-speed jet injector' hand-held system was used for the direct gene transfer of naked DNA into tumors. Lewis-lung carcinoma bearing mice were jet-injected with the beta-galactosidase (LacZ), the green fluorescence (GFP) or the human tumor necrosis factor alpha (TNF-alpha) gene carrying vector plasmids. The animals received five jet injections into the tumor at a pressure of 3.0 bar, delivering 3--5 microl plasmid DNA (1 microg DNA/microl in water) per single jet injection. The jet injection of DNA leads to a widespread expression pattern within tumor tissues with penetration depths of 5--10 mm. Analysis of tumor cryosections revealed moderate LacZ or GFP expression at 48 h and strong reporter gene expression 72 h and 96 h after jet injection. The simultaneous jet injection of the TNF-alpha and LacZ carrying vectors demonstrated efficient expression and secretion of both the cytokine, as well as LacZ expression within the tumor 24 h, 48 h, 72 h, 96 h and 120 h after jet injection. These studies demonstrate the applicability of jet injection for the efficient in vivo gene transfer into tumors for nonviral gene therapy of cancer using minimal amounts of naked DNA.

Animals↗
PubMed2001

Magnesium deficiency-induced anorexia in hyperphagic obese Zucker rats.

In clinical practice diabetes mellitus is the most significant cause of hypomagnesemia and Mg depletion. The obese Zucker rat approaches non-insulin-dependent type II diabetes; lean Zucker rats being suitable controls. Using this disease model the influence of dietary Mg deficiency was studied: animals received a diet providing only approximately 25 per cent of the Mg requirement; controls received drinking water fortified with Mg (16 mmol/L). During 125 days ad libitum feeding, Mg-deficient obese rats consumed nearly 50 per cent less feed pellets and gained 50 per cent less body weight than their obese counterparts. This effect was not fully reversible indicating Mg depletion. Blood glucose reflected food consumption, no glucosuria was detectable using test strips. In the heart muscle Mg was decreased and Ca increased in Mg-deficient rats indicating increased cardiac risk. When the rats were pair fed with lean Mg-deficient controls the development of obesity was prevented. Despite pronounced Mg deficiency blood glucose remained unaffected and no glucosuria was detectable. In future experiments the production of marginal Mg deficiency not inducing anorexia should be applied to study the pathogenetic role of Mg depletion in obese Zucker rats.

Animals↗
PubMed2000

Cloning and initial analysis of the human multidrug resistance-related MVP/LRP gene promoter.

The lung resistance-related protein (LRP) was identified as the human major vault protein (MVP), and is overexpressed in various multidrug-resistant cancer cell lines and clinical samples. We characterized DNA sequences upstream to the transcription initiation site of the MVP gene in the human non-small cell lung cancer cell line SW-1573. A 1.9-kb and a shortened 0.7-kb fragment of the 5'-upstream genomic region show strong promoter activity in chloramphenicol acetyltransferase (CAT) reporter assays. The promoter is TATA-less and contains an inverted CCAAT-box and a Sp1 site located near to a p53 binding motif. An alternative 3'-splice site of intron 1 results in a splicing variant within the 5'-untranslated region of MVP mRNA.

5' Untranslated Regions↗
PubMed2000

Mdr1 promoter-driven tumor necrosis factor-alpha expression for a chemotherapy-controllable combined in vivo gene therapy and chemotherapy of tumors.

Cancer gene therapy approaches are often designed as single-agent treatments; however, greater therapeutic effect might be obtained if combined with an established conventional treatment regimen such as chemotherapy. In this context, conditional promoters are useful tools, because they may be induced by therapeutic modalities. The human multidrug resistance gene (mdr1) promoter is inducible by cytostatic drugs and can be employed for the chemotherapy-regulated expression of therapeutic genes. In this in vivo study, the human mdr1 promoter fragment (-207 to +158) was used for drug-inducible expression of human tumor necrosis factor-alpha (TNF-alpha) in the vector construct pM3mdr-p-hTNF. The single doxorubicin and vincristine treatment of nude mice xenografted with pM3mdr-p-hTNF-transduced MCF-7 mammary tumors resulted in drug-induced and time-dependent elevation of intratumoral TNF-alpha expression at the mRNA and protein level. The highest drug induction was achieved at 2 days after drug application, as reflected by a maximum 25-fold increase in TNF-alpha secretion in the tumor. This drug-induced TNF-alpha expression is more effective in inhibiting tumor growth compared with the growth of tumors transduced with constitutively TNF-alpha-expressing vectors in combination with chemotherapy.

Antineoplastic Agents↗
PubMed2000

Malignant odontogenic myxoma of the maxilla: case with cytogenetic confirmation.

An odontogenic myxoma of the maxilla with an aggressive clinical course is presented. The tumour arose in a 53-year-old patient, recurred two times after extended maxillectomy and ultimately caused the patient's death by uncontrollable local disease with infiltration of the cranial cavity. Microscopically, the tumour showed histological features of a low grade malignant myxosarcoma with cellular areas, enhanced mitotic activity and nuclear pleomorphism. Cytogenetic analysis revealed an unexpectedly aberrant hypertetraploid chromosome complement, that was considered as incompatible with the usual karyotypic patterns of benign tumours.

Humans↗
PubMed2000

High-copy cDNA amplification of minimal total RNA quantities for gene expression analyses.

This protocol describes a PCR-based cDNA amplification technique of small total RNA quantities, optimized for determination and verification of gene expression variations in cells or tissue specimen. A proportional amplification of rare and abundant transcripts is thereby achieved by initial random hexamer-primed reverse transcription of total RNA. Compared to established oligo(dT)-primed techniques, this approach generates shorter than full length copies of long RNAs which leads to a normalized cDNA pool for a more adequate PCR-amplification. Subsequent double oligo(dA) tailing of the synthesized total cDNA strands and the utilization of heteropolymeric primers allow a highly specific, up to 500-fold PCR-amplification of the total cellular RNA amount. Thus, obstacles in availability of RNA from limited sources, such as human biopsies or microdissected histological sections, can be overcome. The amplified total cDNA (atcDNA) is shown to be applicable for confirmation of differential gene expression, as demonstrated in this protocol by expression analysis of the multidrug resistance-associated genes mdr1, mrp1 and lrp, using human cell lines as well as microdissected human tissue sections.

Base Sequence↗
PubMed2000

Viral vectors for gene transfer: a review of their use in the treatment of human diseases.

The efficient delivery of therapeutic genes and appropriate gene expression are the crucial issues for clinically relevant gene therapy. Viruses are naturally evolved vehicles which efficiently transfer their genes into host cells. This ability made them desirable for engineering virus vector systems for the delivery of therapeutic genes. The viral vectors recently in laboratory and clinical use are based on RNA and DNA viruses processing very different genomic structures and host ranges. Particular viruses have been selected as gene delivery vehicles because of their capacities to carry foreign genes and their ability to efficiently deliver these genes associated with efficient gene expression. These are the major reasons why viral vectors derived from retroviruses, adenovirus, adeno-associated virus, herpesvirus and poxvirus are employed in more than 70% of clinical gene therapy trials worldwide. Among these vector systems, retrovirus vectors represent the most prominent delivery system, since these vectors have high gene transfer efficiency and mediate high expression of therapeutic genes. Members of the DNA virus family such as adenovirus-, adeno-associated virus or herpesvirus have also become attractive for efficient gene delivery as reflected by the fast growing number of clinical trials using these vectors. The first clinical trials were designed to test the feasibility and safety of viral vectors. Numerous viral vector systems have been developed for ex vivo and in vivo applications. More recently, increasing efforts have been made to improve infectivity, viral targeting, cell type specific expression and the duration of expression. These features are essential for higher efficacy and safety of RNA- and DNA-virus vectors. From the beginning of development and utilisation of viral vectors it was apparent that they harbour risks such as toxicities, immunoresponses towards viral antigens or potential viral recombination, which limit their clinical use. However, many achievements have been made in vector safety, the retargeting of virus vectors and improving the expression properties by refining vector design and virus production. This review addresses important issues of the current status of viral vector design and discusses their key features as delivery systems in gene therapy of human inherited and acquired diseases at the level of laboratory developments and of clinical applications.

Adenoviridae↗