Biomedical subjects

Thomas Wagner

Publications and source records attributed to Thomas Wagner.

49 records · Page 3Linked to original sources

PubMed2003

REM sleep enhancement induced by different procedures improves memory retention in rats.

Growing evidence supports the idea that sleep following learning is critically involved in memory formation. Recent studies suggest that information acquired during waking is reactivated and possibly consolidated during subsequent sleep, especially during rapid-eye movement (REM) or paradoxical sleep (PS). Critical reviews, however, have questioned PS and memory relationships, particularly because of shortcomings of the PS deprivation paradigm applied in many studies. Therefore, in the present study we used an opposite strategy, i.e. we investigated the effects of PS enhancement on memory retention. In three experiments, we found that selective PS enhancement, induced by different procedures after discrimination training in rats, results in increased retention tested 24 h later. Moreover, calculated in all animals (n = 61), there was a highly significant correlation between post-training PS values and retention scores. Our results suggest that an experimentally induced increase of PS after learning facilitates memory consolidation.

Adrenocorticotropic Hormone↗

PubMed2003

Intra-arterial instillation of microencapsulated, Ifosfamide-activating cells in the pig pancreas for chemotherapeutic targeting.

BACKGROUND: The therapeutic efficacy of intratumoral instillation of genetically engineered, CYP2B1-expressing, microencapsulated cells in combination with ifosfamide had been previously demonstrated in xenografted human pancreatic ductal carcinomas [Gene Ther 1998;5:1070-1078]. Prior to a clinical study, the feasibility of an intra-arterial application of microencapsulated cells to the pancreas and its consequences to the organ had to be evaluated. MATERIAL AND METHODS: Microencapsulated, CYP2B1-producing cells were instilled both in vivo (transfemoral angiographical access) and in vitro (perfusion model) in the splenic lobe of the pig pancreas. In vivo, animals were monitored clinically for 7 days, then treated with ifosfamide and sacrificed. In vitro, ifosfamide was administered intra-arterially. RESULTS: In all animals, 100 microcapsules could be instilled safely via the femoral route without clinical, biochemical or histological signs of pancreatitis. Histological examination revealed partial obstruction of small arteries by the capsules, without causing any parenchymal damage. In vitro, instillation reduced blood flow by half. Ifosfamide, also in combination with the capsules, did not add any damage to the pancreas. CONCLUSION: Intra-arterial instillation of microencapsulated cells to the pig pancreas is feasible and safe. Neither pancreatitis, foreign body reactions nor circulatory disturbances were observed. Clinical application of this genetically enhanced chemotherapeutic method seems possible.

Angiography↗

PubMed2003

Inhibition of endogenous thioredoxin in the heart increases oxidative stress and cardiac hypertrophy.

Thioredoxin 1 (Trx1) has redox-sensitive cysteine residues and acts as an antioxidant in cells. However, the extent of Trx1 contribution to overall antioxidant mechanisms is unknown in any organs. We generated transgenic mice with cardiac-specific overexpression of a dominant negative (DN) mutant (C32S/C35S) of Trx1 (Tg-DN-Trx1 mice), in which the activity of endogenous Trx was diminished. Markers of oxidative stress were significantly increased in hearts from Tg-DN-Trx1 mice compared with those from nontransgenic (NTg) mice. Tg-DN-Trx1 mice exhibited cardiac hypertrophy with maintained cardiac function at baseline. Intraperitoneal injection of N-2-mercaptopropionyl glycine, an antioxidant, normalized cardiac hypertrophy in Tg-DN-Trx1 mice. Thoracic aortic banding caused greater increases in myocardial oxidative stress and enhanced hypertrophy in Tg-DN-Trx1 compared with NTg mice. In contrast, transgenic mice with cardiac-specific overexpression of wild-type Trx1 did not show cardiac hypertrophy at baseline but exhibited reduced levels of hypertrophy and oxidative stress in response to pressure overload. These results demonstrate that endogenous Trx1 is an essential component of the cellular antioxidant mechanisms and plays a critical role in regulating oxidative stress in the heart in vivo. Furthermore, inhibition of endogenous Trx1 in the heart primarily stimulates hypertrophy, both under basal conditions and in response to pressure overload through redox-sensitive mechanisms.

Animals↗

PubMed2003

A matrix metalloproteinase 2 cleavable melittin/avidin conjugate specifically targets tumor cells in vitro and in vivo.

Extracellular matrix breakdown as well as increased expression in cancer cells and tumor microvascular endothelial cells make matrix metalloproteinase 2 (MMP2) an attractive target for cancer treatment. By taking advantage of MMP2's properties, an MMP2 cleavable melittin/avidin conjugate was designed. Melittin alone is extremely toxic to cells and induces immediate cell lysis, but becomes inactive when coupled with avidin. The incorporation of the MMP2 target sequence into the peptide was used as a means for targeting tumor cells. In vitro, the melittin/avidin conjugate showed strong cytolytic activity against cancer cells with high MMP2 activity; DU 145 prostate cancer cells and SK-OV-3 ovarian cancer cells. The conjugate exhibited very little cytolytic activity against normal L-cells that displayed low MMP2 activity. These data demonstrate the MMP2 specificity of the melittin/avidin conjugate. In vivo, the size of tumors injected with the melittin/avidin conjugate was significantly smaller as compared to untreated tumors. Therefore, due to its tumor targeting capabilities as well as its cytolytic properties in vitro and in vivo, the melittin/avidin conjugate displays the potential for use in cancer therapy.

Amino Acid Sequence↗

PubMed2002

Lung tumorigenesis associated with erb-B-2 and erb-B-3 overexpression in human erb-B-3 transgenic mice is enhanced by methylnitrosourea.

Erb-B-3 overexpression is associated with poor prognosis in non-small cell lung cancer and is often overexpressed in breast cancers. MMTVhuman-erb-B-3 transgenic mice were generated to evaluate the impact of erb-B-3 overexpression on lung and mammary gland tumorigenesis. These transgenic mice developed a high incidence of lung adenocarcinomas but not mammary gland tumors. The tumors overexpressed transgenic human [h]-erb-B-3 but also overexpressed endogenous erb-B-2, indicating that the heterodimer of h-erb-B-3-erb-B-2 was required for proliferative signal transduction to the nucleus. Lung tumor latency was shorter and the incidence higher in erb-B-3 transgenic mice treated with the methylating agent, methylnitrosourea [MNU]. In MNU treated mice, K-ras activating point mutations in codon 12, synergized with h-erb-B-3 in lung tumorogenesis. In bitransgenic MMTVrat-erb-B2/MMTV-human-erb-B-3 mice, lung tumor latency was also significantly shortened. Unlike over-expression of rat-erb-B-2, overexpression of h-erb-B-3 did not alter the incidence or latency of mammary tumors. Coupled erb-B-2 and erb-B-3 overexpression as well as K-ras activation induced signaling through mitogen-activated protein kinase (MAPK). This animal model links erb-B-3 with lung cancer, suggests that erb-B-2 and erb-B-3 heterodimerization is a necessary intermediate, and documents latency shortening by methylating agent-induced mutation of K-ras. This erb-B-3 mouse lung cancer model will help dissect genetic changes in lung tumorigenesis and may be useful for chemoprevention studies.

Animals↗

PubMed2002

H11 kinase is a novel mediator of myocardial hypertrophy in vivo.

By subtractive hybridization, we found a significant increase in H11 kinase transcript in large mammalian models of both ischemia/reperfusion (stunning) and chronic pressure overload with hypertrophy. Because this gene has not been characterized in the heart, the goal of the present study was to determine the function of H11 kinase in cardiac tissue, both in vitro and in vivo. In isolated neonatal rat cardiac myocytes, adenoviral-mediated overexpression of H11 kinase resulted in a 37% increase in protein/DNA ratio, reflecting hypertrophy. A cardiac-specific transgene driven by the alphaMHC-promoter was generated, which resulted in an average 7-fold increase in H11 kinase protein expression. Transgenic hearts were characterized by a 30% increase of the heart weight/body weight ratio, by the reexpression of a fetal gene program, and by concentric hypertrophy with preserved contractile function at echocardiography. This phenotype was accompanied by a dose-dependent activation of Akt/PKB and p70(S6) kinase, whereas the MAP kinase pathway was unaffected. Thus, H11 kinase represents a novel mediator of cardiac cell growth and hypertrophy.

Animals↗

PubMed2002

Stem cell toxicity of oxazaphosphorine metabolites in comparison to their antileukemic activity.

The oxazaphosphorine agent cyclophosphamide (CP) is an alkylating agent with a relative low stem cell toxicity. The aim of this study was to further evaluate the stem cell toxicity of the active metabolites of CP and its structural analogue ifosfamide (IFO) in comparison to their antileukemic efficacy. Cells of different malignant hematologic disorders (HL-60, HS-Sultan and THP-1) and CD34+ stem cells were treated with cytotoxic CP-metabolite mafosfamide (MAFO) and IFO-metabolites 4-hydroxy-IFO (4-OH-IFO) and chloroacetaldehyde. The clonogenity of the cells was investigated by using a colony-forming assay. All metabolites reduced the formation of both tumor-derived colonies and stem cell-derived CFU-GMs in a concentration-dependent manner. Our data showed a relative tumor-specific, stem cell protecting action of the substances tested with a higher toxicity against tumor cells (IC(50) against HS-Sultan: MAFO 1.1 microM; 4-OH-IFO 1.3 microM; CAA 3 microM) than against stem cells (IC(50) MAFO 14.8 microM; 4-OH-IFO 16.9 microM; CAA 14 microM). However, while the cytotoxic action of 4-OH-IFO corresponded to MAFOs activity, CAAs cytotoxic effect against the hematologic tumor cells was lower. In conclusion, the results confirm the observed cytotoxicity of CAA against solid tumors for cells of malignant hematologic disorders. Although the relative cytotoxic specificity of CAA is lower than for 4-OH-IFO and MAFO, also CAA, like 4-OH-IFO and MAFO, was found to be in part a tumoricidal, stem cell protecting substance.

Acetaldehyde↗

PubMed2002

Quantitative polymerase chain reaction-based assay with fluorogenic Y-chromosome specific probes to measure bone marrow chimerism in mice.

UNLABELLED: A quantitative duplex polymerase chain reaction (PCR) method using the LightCycler detection system was developed to generate a reproducible and convenient method to quantitatively measure chimeric states in murine transplantational models using male and female BALB/c mice. MATERIALS AND METHODS: DNA mixtures isolated from male and female murine BALB/c bone marrow cells were analyzed with Y-chromosome and control autosomal GAPDH specific primers by either monoplex or duplex real-time quantitative PCR using the LightCycler detection system to determine chimeric states. RESULTS: High specific Y-chromosome and control autosomal GAPDH primers gave a detection sensitivity of approximately 0.01% using LightCycler PCR. Analysis of standard curve distribution of diluted DNA samples accurately matched the proportional dilutions. Y6 primers run on male DNA samples against a female background showed amplification slope initiations reflecting the amounts of male DNA contained in these mixtures. Mixed samples run with GAPDH primers showed amplification corresponding to 100% DNA amounts of limiting dilution runs which were used as controls. Calculated engraftment generated by monoplex PCR lacked reproducibility. Quantitation using duplex PCR using specific detection probes for both Y-chromosome Y6 and autosomal GAPDH primer amplicons allowed precise and reproducible calculations of engraftment levels. CONCLUSION: Duplex PCR using specific detection probes for Y-chromosome Y6 and autosomal GAPDH primer amplicons allows for rapid, precise and specific engraftment determination after transplantation of male BALB/c marrow cells into female BALB/c recipients.

Animals↗

PubMed2002

Ex vivo expansion of long-term culture initiating marrow cells by IL-10, SCF, and IL-3.

BACKGROUND: Ex vivo expansion of progentior cells may shorten hematopoietic regeneration after myeloablative chemoradiotherapy, increase target cells for gene therapy, and improve purging of progenitor cell components. STUDY DESIGN AND METHODS: Marrow cells were incubated for 1 week in suspension culture with and without IL-10, IL-3, and SCF. As long-term culture initiating cells (LTC-ICs) represent early hematopoietic progenitors in vitro, these cells were quantified at initiation and after a 1-week culture period in a limiting dilution assays. Additionally, immunophenotyping of cells before and after culture was performed. RESULTS: In six experiments, marrow cells cultured for 1 week with IL-10, IL-3, and SCF showed a significant increase (almost doubling) in LTC-ICs as compared with marrow cells before expansion. Additionally, an increased proliferative capacity of LTC-ICs was achieved with a sevenfold increase of committed colony-forming cells and a 10-fold proliferation of high proliferative potential colony-forming cells. Immunophenotyping revealed a sevenfold increase of CD34+ CD45 RA- cells in IL-10-, IL-3-, SCF-stimulated suspension cultures. In unstimulated cultures, no LTC-ICs were maintained after 1 week. CONCLUSION: Expansion of LTC-ICs by IL-10, IL-3, and SCF has not been shown so far. This in vitro model allows expansion of LTC-IC if compared with the input of progenitor cells without extensive progenitor cell manipulation. This should be an attractive model for in vitro purging, gene transfer, or expansion of progenitor cells to allow rapid engraftment after myeloablative chemotherapy.

Adult↗

PubMed2002

Ultrastructural changes and activation differences in platelet concentrates stored in plasma and additive solution.

BACKGROUND: The aim of this study was to demonstrate how ultrastructural morphology of platelets stored in different media correlate with the appearance of particular activation markers on their cell surface. STUDY DESIGN AND METHODS: Concentrates of buffy coat-derived platelets were stored in plasma or a glucose-free citrate-acetate-NaCl platelet additive solution (PAS2, Baxter Healthcare Corp.). Activation markers on platelets were measured by flow cytometry and compared with changes in the platelet cell surface as demonstrated by electron microscopy. Levels of the vasoactive cytokines vascular endothelial growth factor (VEGF) and RANTES (regulated upon activation, normal T-cell expressed and secreted) were determined in the storage medium of the platelet concentrate. RESULTS: The activation markers CD62P and CD63 and the binding of thrombospondin measured by flow cytometry were expressed to a higher extent in the PAS2 group compared with the plasma group. The difference reached significance on Day 3 (CD62P: 66.37 +/- 2.44 vs. 37.83 +/- 2.03, p < 0.001; CD63: 42.11 +/- 3.29 vs. 34.84 +/- 2.04, p < 0.05; and thrombospondin binding: 18.84 +/- 3.9 vs. 13.98 +/- 3.87, p < 0.001, respectively). The form factor that is related to changes of the platelet shape was determined by image analysis and correlated significantly with the cell surface expression of CD62P (p < 0.001) and with CD63 (p < 0.05) and with thrombospondin binding (p < 0.05). The chemokines VEGF and RANTES were measured at higher levels in the PAS2 group. CONCLUSIONS: With exception of baseline activation probably due to necessary handling procedures, platelets remain relatively unaltered and more stable in plasma in comparison to storage in PAS2.

Acetates↗

PubMedDate not supplied

Combined HSV-TK/GCV and secondary lymphoid tissue chemokine gene therapy inhibits tumor growth and elicits potent antitumor CTL response in tumor-bearing mice.

Suicide gene therapy in combination with pro-drugs represents an attractive approach to the treatment of cancer. Unfortunately this approach is limited by difficulty in targeting all tumor cells, especially those at the distant metastases associated with the most complex tumors. For this reason, attempts to stimulate global anti-tumor immune responses at the sites of effective suicide gene/pro-drug-mediated tumor cell destruction are appealing. Here we show that, by including a gene coding for secreted secondary lymphoid tissue chemokine (SLC) along with the herpes simplex virus thymide kinase (HSV-TK) gene in a bicistronic vector for anti-tumor gene therapy in conjunction with the pro-drug ganciclovir (GCV), we are able to mediate a greatly enhanced anti-tumor effect in the murine B16 melanoma tumor model. The data presented suggests that this enhanced antitumor effect is the result of a strong induced CTL immune response resulting from the recruitment of immune cells to the site of HSV-TK/GCV-induced tumor destruction by the potent chemokine SLC.

Animals↗

PubMedDate not supplied

Efficient translocation and apoptosis induction by adenovirus encoded VP22-p53 fusion protein in human tumor cells in vitro.

BACKGROUND: p53 mutations are one of the most frequent genetic alterations in cancer. Various mechanisms of delivering p53 protein into tumor cells, such as plasmids, retroviruses and adenoviruses, have been widely used in experimental studies. Although these methods are relatively successful, the transduction rate into surrounding cells is still a limiting factor. Recent studies have shown that fusing VP22 (important for intercellular transport) to p53 and delivering this protein to cells in the form of an adenovirus is a very efficient method of getting p53 into cells. RESULTS: In the present study, the effect of adenovirus encoded VP22-p53 fusion protein on p53 negative human cancer cells (LNCaP, SK-OV-3, OVCAR-3, DU 145) was investigated. A functional VP22-p53 fusion protein was produced as verified by immunofluorescence and analysis of p21 expression. Induction of p21 expression (target gene for p53) confirmed p53 was functional and immunofluorescence staining using p53 antiserum demonstrated the intercellular trafficking ability of the VP22 portion of the fusion protein. CONCLUSION: In conclusion, VP22-p53 showed efficient translocation into tumor cells, inhibition of tumor cell proliferation and induction of apoptosis. These characteristics make the fusion protein an attractive method for introducing p53 into human cells as well as a potential candidate for gene therapy.

Adenoviridae↗

PubMedDate not supplied

[Morphology of bullet wounds when using police ammunition, type QD-P.E.P].

6 years after introduction of the police ammunition QD-P.E.P. (9 mm caliber), 9 bullet wounds are presented from forensic investigations along with the results of experimental shots at pig carcasses. The evaluation reveals the characteristic pattern of such bullet wounds: a skin defect which is almost as large as the caliber and has a sharp-edged border thus looking like it has been punched out; the contusion ring ("abrasion ring") is conspicuously narrow. These findings can be explained by the shape and design of the projectile. In regular police work immediate incapacitation has not always been achieved, depending on the entry location of the bullet. Experimental shots at pig carcasses showed that in the case of shots at extremities, the residual energy of the exiting bullet may be sufficient to generate another penetrating bullet wound.

Accidents↗