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1.
Gut ; 67(1): 128-137, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28108468

RESUMO

OBJECTIVE: Current non-invasive diagnostic tests can distinguish between pancreatic cancer (pancreatic ductal adenocarcinoma (PDAC)) and chronic pancreatitis (CP) in only about two thirds of patients. We have searched for blood-derived metabolite biomarkers for this diagnostic purpose. DESIGN: For a case-control study in three tertiary referral centres, 914 subjects were prospectively recruited with PDAC (n=271), CP (n=282), liver cirrhosis (n=100) or healthy as well as non-pancreatic disease controls (n=261) in three consecutive studies. Metabolomic profiles of plasma and serum samples were generated from 477 metabolites identified by gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry. RESULTS: A biomarker signature (nine metabolites and additionally CA19-9) was identified for the differential diagnosis between PDAC and CP. The biomarker signature distinguished PDAC from CP in the training set with an area under the curve (AUC) of 0.96 (95% CI 0.93-0.98). The biomarker signature cut-off of 0.384 at 85% fixed specificity showed a sensitivity of 94.9% (95% CI 87.0%-97.0%). In the test set, an AUC of 0.94 (95% CI 0.91-0.97) and, using the same cut-off, a sensitivity of 89.9% (95% CI 81.0%-95.5%) and a specificity of 91.3% (95% CI 82.8%-96.4%) were achieved, successfully validating the biomarker signature. CONCLUSIONS: In patients with CP with an increased risk for pancreatic cancer (cumulative incidence 1.95%), the performance of this biomarker signature results in a negative predictive value of 99.9% (95% CI 99.7%-99.9%) (training set) and 99.8% (95% CI 99.6%-99.9%) (test set). In one third of our patients, the clinical use of this biomarker signature would have improved diagnosis and treatment stratification in comparison to CA19-9.


Assuntos
Biomarcadores Tumorais/sangue , Carcinoma Ductal Pancreático/diagnóstico , Detecção Precoce de Câncer/métodos , Neoplasias Pancreáticas/diagnóstico , Pancreatite Crônica/diagnóstico , Adulto , Idoso , Carcinoma Ductal Pancreático/patologia , Estudos de Casos e Controles , Diagnóstico Diferencial , Estudos de Viabilidade , Feminino , Humanos , Masculino , Metabolômica/métodos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Pancreáticas/patologia , Sensibilidade e Especificidade
2.
ESC Heart Fail ; 4(2): 178-189, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28451455

RESUMO

AIMS: Identification of metabolic signatures in heart failure (HF) patients and evaluation of their diagnostic potential to discriminate HF patients from healthy controls during baseline and exercise conditions. METHODS: Plasma samples were collected from 22 male HF patients with non-ischemic idiopathic cardiomyopathy and left ventricular systolic dysfunction and 19 healthy controls before (t0), at peak (t1) and 1 h after (t2) symptom-limited cardiopulmonary exercise testing. Two hundred fifty-two metabolites were quantified by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography (LC)-MS/MS-based metabolite profiling. RESULTS: Plasma metabolite profiles clearly differed between HF patients and controls at t0 (P < 0.05). The metabolic signature of HF was characterized by decreased levels of complex lipids and fatty acids, notably phosphatidylcholines, cholesterol, and sphingolipids. Moreover, reduced glutamine and increased glutamate plasma levels, significantly increased purine degradation products, as well as signs of impaired glucose metabolism were observed. The metabolic differences increased strongly according to New York Heart Association functional class and the addition of three metabolites further improved prediction of exercise capacity (Q2 = 0.24 to 0.35). Despite a high number of metabolites changing significantly with exercise (30.2% at t1/t0), the number of significant alterations between HF and controls was almost unchanged at t1 and t2 (30.7 and 29.0% vs. 31.3% at t0) with a similar predictive group separation (Q2 = 0.50 for t0, 0.52 for t1, and 0.56 for t2, respectively). CONCLUSIONS: Our study identified a metabolic signature of non-ischemic HF with prominent changes in complex lipids including phosphatidylcholines, cholesterol, and sphingolipids. The metabolic changes were already evident at rest and largely preserved under exercise.

3.
Oncotarget ; 7(2): 1421-38, 2016 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-26623558

RESUMO

Integrated analysis of metabolomics, transcriptomics and immunohistochemistry can contribute to a deeper understanding of biological processes altered in cancer and possibly enable improved diagnostic or prognostic tests. In this study, a set of 254 metabolites was determined by gas-chromatography/liquid chromatography-mass spectrometry in matched malignant and non-malignant prostatectomy samples of 106 prostate cancer (PCa) patients. Transcription analysis of matched samples was performed on a set of 15 PCa patients using Affymetrix U133 Plus 2.0 arrays. Expression of several proteins was immunohistochemically determined in 41 matched patient samples and the association with clinico-pathological parameters was analyzed by an integrated data analysis. These results further outline the highly deregulated metabolism of fatty acids, sphingolipids and polyamines in PCa. For the first time, the impact of the ERG translocation on the metabolome was demonstrated, highlighting an altered fatty acid oxidation in TMPRSS2-ERG translocation positive PCa specimens. Furthermore, alterations in cholesterol metabolism were found preferentially in high grade tumors, enabling the cells to create energy storage. With this integrated analysis we could not only confirm several findings from previous metabolomic studies, but also contradict others and finally expand our concepts of deregulated biological pathways in PCa.


Assuntos
Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Metabolismo Energético , Perfilação da Expressão Gênica , Imuno-Histoquímica , Metabolômica , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Integração de Sistemas , Idoso , Colesterol/metabolismo , Bases de Dados Genéticas , Ácidos Graxos/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Modelos Lineares , Masculino , Metabolômica/métodos , Pessoa de Meia-Idade , Gradação de Tumores , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Fusão Oncogênica/genética , Oxirredução , Valor Preditivo dos Testes , Modelos de Riscos Proporcionais , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapia , Regulador Transcricional ERG/genética , Translocação Genética , Resultado do Tratamento
4.
Int J Cancer ; 133(12): 2914-24, 2013 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-23737455

RESUMO

Metabolomic research offers a deeper insight into biochemical changes in cancer metabolism and is a promising tool for identifying novel biomarkers. We aimed to evaluate the diagnostic and prognostic potential of metabolites in prostate cancer (PCa) tissue after radical prostatectomy. In matched malignant and nonmalignant prostatectomy samples from 95 PCa patients, aminoadipic acid, cerebronic acid, gluconic acid, glycerophosphoethanolamine, 2-hydroxybehenic acid, isopentenyl pyrophosphate, maltotriose, 7-methylguanine and tricosanoic acid were determined within a global metabolite profiling study using gas chromatography/liquid chromatography-mass spectrometry. The data were related to clinicopathological variables like prostate volume, tumor stage, Gleason score, preoperative prostate-specific antigen and disease recurrence in the follow-up. All nine metabolites showed higher concentrations in malignant than in nonmalignant samples except for gluconic acid and maltotriose, which had lower levels in tumors. Receiver -operating characteristics analysis demonstrated a significant discrimination for all metabolites between malignant and nonmalignant tissue with a maximal area under the curve of 0.86 for tricosanoic acid, whereas no correlation was observed between the metabolite levels and the Gleason score or tumor stage except for gluconic acid. Univariate Cox regression and Kaplan-Meier analyses showed that levels of aminoadipic acid, gluconic acid and maltotriose were associated with the biochemical tumor recurrence (prostate-specific antigen > 0.2 ng/mL). In multivariate Cox regression analyses, aminoadipic acid together with tumor stage and Gleason score remained in a model as independent marker for prediction of biochemical recurrence. This study proved that metabolites in PCa tissue can be used, in combination with traditional clinicopathological factors, as promising diagnostic and prognostic tools.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Próstata/metabolismo , Idoso , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Estadiamento de Neoplasias , Prognóstico , Modelos de Riscos Proporcionais , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/patologia
5.
Health Care Manage Rev ; 33(2): 103-12, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18360161

RESUMO

BACKGROUND: Clinical studies play a pivotal role in the development of new pharmaceutical drugs. Before newly developed active substances can be put on the market, the law requires that they be tested in a large number of clinical trials. The initiators of these trials are usually study groups (publicly funded trials) or the pharmaceutical industry, which often supplies the drugs for clinical research in hospitals. There is reason to believe that hospital administrators could make cost savings on drugs. PURPOSE: The purpose of this article is to quantify drug cost savings in hospitals related to clinical trials and to examine the relationship between researchers and hospital administrators with respect to clinical trials. METHODOLOGY/APPROACH: We analyzed 88 clinical trials in oncology including 29 researchers in 11 hospitals in Germany from 2002 through 2005. We also interviewed researchers and hospital administrators concerning their attitude toward these clinical trials. We propose that hospital administrators tend to focus on the economics of conducting clinical trials. FINDINGS: The results showed a drug cost saving potential of US $6.7 million (euro5.1 million) in 11 hospitals from 2002 through 2005 and an actual cost saving of US $2.0 million (euro1.5 million). The hospital administrators underestimated the difficulties that researchers experienced because of lack of personnel resources when conducting clinical trials. PRACTICE IMPLICATIONS: The hospital administrator has a financial incentive to provide internal conditions in the hospital that facilitate incentives for researchers to become involved in clinical trials. We suggest supporting researchers actively in research with additional human resources (study nurses, etc.). We propose that this would result in a higher number of patients taking part and in more clinical trials. Consequently, higher drug cost savings could also be realized.


Assuntos
Ensaios Clínicos como Assunto/economia , Redução de Custos , Custos de Medicamentos , Administradores Hospitalares/psicologia , Pesquisadores/psicologia , Atitude , Alemanha , Humanos , Relações Interprofissionais , Entrevistas como Assunto , Serviço Hospitalar de Oncologia
6.
J Neurooncol ; 74(3): 305-9, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16132524

RESUMO

Human malignant brain tumors have a poor prognosis in spite of surgery and radiation therapy. Cubic phases consist of curved biocontinuous lipid bilayers, separating two congruent networks of water channels. Used as a host for cytotoxic drugs, the gel-like matrix can easily be applied to the walls of a surgical resection cavity. For human glioblastoma recurrences, the feasibility, safety, and short-term effects of a surgical intracavitary application of paclitaxel and carboplatin encapsulated by liquid crystalline cubic phases are examined in a pilot study. A total of 12 patients with a recurrence of a glioblastoma multiforme underwent re-resection and received an intracavitary application of paclitaxel and carboplatin cubic phases in different dosages. Six of the patients received more than 15 mg paclitaxel and suffered from moderate to severe brain edema, while the remaining patients received only a total of 15 mg paclitaxel. In the latter group, brain edema was markedly reduced and dealt medically. Intracavitary chemotherapy in recurrent glioblastoma using cubic phases is feasible and safe, yet the clinical benefit remains to be examined in a clinical phase II study.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Glioblastoma/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/líquido cefalorraquidiano , Edema Encefálico/induzido quimicamente , Carboplatina/administração & dosagem , Carboplatina/efeitos adversos , Carboplatina/líquido cefalorraquidiano , Relação Dose-Resposta a Droga , Vias de Administração de Medicamentos , Portadores de Fármacos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Paclitaxel/administração & dosagem , Paclitaxel/efeitos adversos , Paclitaxel/líquido cefalorraquidiano , Projetos Piloto , Análise de Sobrevida
7.
Radiology ; 236(2): 572-8, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16040915

RESUMO

PURPOSE: To evaluate the feasibility of gene delivery mediated with diagnostic ultrasound and plasmid DNA (pDNA) encapsulated in gas-filled microparticles (GFMP) in rodent tumor models. MATERIALS AND METHODS: This study was performed according to a protocol approved by the regional animal research committee. The model plasmid UT651 (pUT651) that contained the Escherichia coli LacZ gene for beta-galactosidase was used to demonstrate the feasibility of ultrasound-mediated gene delivery in CC531 liver tumors in rats. In preliminary experiments, a single injection of pUT651-containing GFMP was administered intraarterially (n=4) or intravenously (n=6) with simultaneous sonication (color Doppler mode, maximum mechanical index) of the GFMP passing through the capillaries of the tumors. All animals were sacrificed 2-5 days later, and liver tumors were examined for beta-galactosidase expression histochemically. Subsequently, potential medical usefulness of this delivery system was tested in nude mice bearing Capan-1 tumors (adenocarcinoma of the human pancreas) by using the plasmid RC/CMV-p16 (pRC/CMV-p16), which contains tumor suppressor gene p16. The tumor suppressor gene p16 is deleted in Capan-1 cells. Twenty-five tumor-bearing mice were classified into five groups (four to six mice per group, one treatment group, four control groups) at random. All mice were treated once weekly for 5 weeks with intravenous infusion of p16-containing GFMP or control substances with simultaneous tumor sonication with color Doppler mode ultrasound and maximum mechanical index or without ultrasound treatment. The therapeutic effect of p16 was measured as an increase in tumor volume doubling time. Data were analyzed with analysis of variance. Results were considered significant at the 5% critical level (P < .05). RESULTS: A clear expression of pDNA was found in tumors in rats treated with a combination of pUT651-containing GFMP and ultrasound; relevant controls showed a significantly lower expression of marker gene. The controlled ultrasound-triggered release of pRC/CMV-p16 from GFMP leads to a strong tumor growth inhibition, which is significant (P < .002), compared with that in controls. CONCLUSION: A combination of GFMP and ultrasound provides an effective approach for nonviral gene therapy-based cancer treatment.


Assuntos
Terapia Genética/métodos , Neoplasias/diagnóstico por imagem , Neoplasias/terapia , Animais , Modelos Animais de Doenças , Estudos de Viabilidade , Gases , Masculino , Ratos , Ultrassonografia
8.
J Neurooncol ; 72(3): 209-15, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15937642

RESUMO

Implanted drug carrier systems for retarded chemotherapy against gliomas are mainly based upon polymers containing nitrosoureas. The authors have developed an intracavitary carrier system of biodegradable liquid crystalline cubic phases encapsulating carboplatin and paclitaxel and studied it for release kinetics, antitumor activity, and survival prolongation. A total of 61 Fisher rats with F98 tumors were divided into six treatment groups at day 12 post-inoculation, receiving either no treatment, surgery with partial tumor resection, or partial resection with implantation of cubic phases containing either paclitaxel and carboplatin, paclitaxel alone, carboplatin alone, or no drug. Animals were killed for tumor size analysis at day 21 post-inoculation (n=28) or were included in survival studies (n=33). Additional 12 animals received a paclitaxel/carboplatin application and were killed at different time intervals (6 h, 24 h, 48 h, 5 d, 7 d, 10 d post-agent application) for in vivo diffusion studies. Animals from the paclitaxel/carboplatin group showed a significantly smaller tumor (mean 3.25 mm2+/-SD 1.79 mm2) than animals from the control group (15.30+/-5.86 mm2; P=0.0031), animals having received the empty matrix (11.62+/-6.66 mm2; P=0.0241), and animals after tumor resection without implantation (20.87+/-3.56 mm2; P

Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/uso terapêutico , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Carboplatina/administração & dosagem , Carboplatina/uso terapêutico , Glioblastoma/tratamento farmacológico , Paclitaxel/administração & dosagem , Paclitaxel/uso terapêutico , Animais , Antineoplásicos/líquido cefalorraquidiano , Antineoplásicos Fitogênicos/líquido cefalorraquidiano , Encéfalo/metabolismo , Neoplasias Encefálicas/patologia , Carboplatina/líquido cefalorraquidiano , Cristalização , Difusão , Portadores de Fármacos , Composição de Medicamentos , Sistemas de Liberação de Medicamentos , Glioblastoma/patologia , Masculino , Transplante de Neoplasias , Paclitaxel/líquido cefalorraquidiano , Ratos , Ratos Endogâmicos F344 , Sobrevida
9.
Methods Enzymol ; 391: 200-8, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15721383

RESUMO

The LIPO-HSV-1-tk gene transfer system was developed for a 3-day pump application in a first prospective Phase I?II clinical study. Eight patients suffering from recurrent glioblastoma multiforme were treated intratumorally on the basis of convection-enhanced delivery using the nonviral vector system. It was possible to identify the target tissue together with assessment of vector distribution and gene product expression, as well as the metabolic effect of ganciclovir treatment, noninvasively, by the combination of magnetic resonance imaging and positron emission tomography as a multimodal molecular imaging system. The therapy was well tolerated without major side effects. In two of eight patients, we observed a greater than 50% reduction of tumor volume and in six of eight patients focal treatment effects. The noninvasive visualization of therapeutic effects on tumor metabolism and documentation of gene expression will be important for the further successful development and implementation of patient individual gene therapy.


Assuntos
Neoplasias Encefálicas/terapia , Genes Transgênicos Suicidas , Terapia Genética/métodos , Lipossomos , Animais , Efeito Espectador , Ganciclovir/metabolismo , Ganciclovir/uso terapêutico , Herpesvirus Humano 1/enzimologia , Herpesvirus Humano 1/genética , Humanos , Lipossomos/química , Pró-Fármacos/metabolismo , Ratos , Timidina Quinase/genética , Timidina Quinase/metabolismo
10.
Exp Eye Res ; 80(1): 93-101, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15652530

RESUMO

The aim of this study was to optimize non-viral gene transfer conditions and investigate the effect of fibroblast growth factor-1 (FGF-1) gene transfer on human corneal endothelial cell (HCEC) proliferation. Five non-viral vectors (Lipofectin, DMRIE-C, DAC-30, Effectene, FuGene6) were used to transfect HCEC with plasmids coding for enhanced green fluorescent protein (EGFP) and FGF-1. Transfection efficiency and toxicity (n=6) were quantified and optimized using the EGFP construct by FACS-analysis. Using optimal conditions HCEC were transfected with the FGF-1 plasmid and cell proliferation as well as expression of FGF-1 were determined at days 4 and 7 by counting and western blotting, respectively. Lipofectin (17+/-2.02%) transfected HCEC more successfully than DMRIE-C (11+/-1.46%), Effectene (9+/-0.62%), FuGene (9+/-0.93%) and DAC-30 (7+/-0.59%). Toxicity of the lipids ranged from 2 to 4%. Optimal HCEC proliferation was achieved with DAC-30/FGF-1 (P<0.05), whereas all other vectors did not result in significantly increased cell proliferation. However, all of the transfected cells produced FGF-1 in different amounts as indicated by western blotting. Efficient and almost non-toxic transfer of the FGF-1 gene into HCEC can be successfully achieved by lipid-based techniques. Using optimal conditions significantly increased cell proliferation was independent on gene transfer efficiency. This may indicate that even a low transfection rate is sufficient to produce a concentration of FGF-1 that will have a stimulatory effect on HCECs.


Assuntos
Colesterol/análogos & derivados , Endotélio Corneano/fisiologia , Fator 1 de Crescimento de Fibroblastos/genética , Lipossomos , Transfecção/métodos , Western Blotting/métodos , Contagem de Células , Divisão Celular/genética , Células Cultivadas , Colesterol/genética , Células Endoteliais/fisiologia , Células Endoteliais/ultraestrutura , Endotélio Corneano/ultraestrutura , Fator 1 de Crescimento de Fibroblastos/análise , Citometria de Fluxo/métodos , Vetores Genéticos/genética , Humanos , Lipídeos/genética , Microscopia Eletrônica/métodos , Fosfatidiletanolaminas/genética , Compostos de Amônio Quaternário
11.
Anesthesiology ; 101(1): 204-11, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15220792

RESUMO

BACKGROUND: Opioid-containing leukocytes migrate to peripheral sites of inflammation. On exposure to stress, opioid peptides are released, bind to opioid receptors on peripheral sensory neurons, and induce endogenous antinociception. In later stages of Freund's complete adjuvant-induced local inflammation, monocytes/macrophages are a major opioid-containing leukocyte subpopulation, but these cells also produce proalgesic cytokines. In this study, the role of tissue monocytes/macrophages in hyperalgesia and in peripheral opioid-mediated antinociception was investigated. METHODS: After intraplantar injection of Freund's adjuvant, leukocyte subpopulations and opioid-containing leukocytes were analyzed by flow cytometry in the inflamed paw in the presence or absence of monocyte/macrophage depletion by intraplantar injection of clodronate-containing liposomes (phosphate-buffered saline and empty liposomes served as controls). Paw volume was measured with a plethysmometer. Hyperalgesia was determined by measuring heat-induced paw withdrawal latency and paw pressure threshold. Paw pressure threshold was also measured after swim stress and injection of fentanyl. RESULTS: At 48 and 96 h of inflammation, it was found that (1). monocytes/macrophages were the largest leukocyte subpopulation (> 55% of all leukocytes) and the predominant producers of opioid peptides (71-77% of all opioid-containing leukocytes in the paw), (2). clodronate-containing liposomes depleted monocytes/macrophages by 30-35% (P < 0.05), (3). hyperalgesia was unaltered by liposome injection (P > 0.05), and (4) opioid-containing leukocytes and swim stress but not fentanyl-induced antinociception were significantly decreased by clodronate-containing liposomes (P < 0.05, P > 0.05, all by t test; opioid-containing cells and swim stress-induced increase of paw pressure threshold were reduced by 35-42% and 20%, respectively). CONCLUSION: Partial depletion of tissue monocytes/macrophages impairs peripheral endogenous opioid-mediated antinociception without affecting hyperalgesia.


Assuntos
Analgésicos Opioides/farmacologia , Fentanila/farmacologia , Hiperalgesia/induzido quimicamente , Inflamação/patologia , Macrófagos/patologia , Monócitos/patologia , Analgésicos não Narcóticos/farmacocinética , Analgésicos não Narcóticos/farmacologia , Analgésicos Opioides/administração & dosagem , Animais , Ácido Clodrônico/farmacocinética , Ácido Clodrônico/farmacologia , Fentanila/administração & dosagem , Citometria de Fluxo , Pé/patologia , Adjuvante de Freund , Temperatura Alta , Hiperalgesia/patologia , Hiperalgesia/psicologia , Inflamação/induzido quimicamente , Injeções , Lipossomos , Masculino , Medição da Dor/efeitos dos fármacos , Pressão , Ratos , Ratos Wistar
12.
Appl Environ Microbiol ; 70(2): 1040-50, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14766587

RESUMO

We analyzed the biochemical composition of the magnetosome membrane (MM) in Magnetospirillum gryphiswaldense. Isolated magnetosomes were associated with phospholipids and fatty acids which were similar to phospholipids and fatty acids from other subcellular compartments (i.e., outer and cytoplasmic membranes) but were present in different proportions. The binding characteristics of MM-associated proteins were studied by selective solubilization and limited proteolysis. The MM-associated proteins were further analyzed by various proteomic approaches, including one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Edman and mass spectrometric (electrospray ionization-mass spectrometry-mass spectrometry) sequencing, as well as capillary liquid chromatography-mass spectrometry-mass spectrometry of total tryptic digests of the MM. At least 18 proteins were found to constitute the magnetosome subproteome, and most of these proteins are novel for M. gryphiswaldense. Except for MM22 and Mms16, all bona fide MM proteins (MMPs) were encoded by open reading frames in the mamAB, mamDC, and mms6 clusters in the previously identified putative magnetosome island. Eight of the MMPs display homology to known families, and some of them occur in the MM in multiple homologues. Ten of the MMPs have no known homologues in nonmagnetic organisms and thus represent novel, magnetotactic bacterium-specific protein families. Several MMPs display repetitive or highly acidic sequence patterns, which are known from other biomineralizing systems and thus may have relevance for magnetite formation.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Compostos Férricos , Membranas Intracelulares/metabolismo , Magnetospirillum/ultraestrutura , Proteoma , Sequência de Aminoácidos , Proteínas de Bactérias/química , Compostos Férricos/química , Compostos Férricos/metabolismo , Membranas Intracelulares/química , Magnetospirillum/metabolismo , Espectrometria de Massas , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular
13.
Ann Neurol ; 54(4): 479-87, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14520660

RESUMO

In a prospective phase I/II clinical study, we treated eight patients suffering from recurrent glioblastoma multiform with stereotactically guided intratumoral convection-enhanced delivery of an HSV-1-tk gene-bearing liposomal vector and systemic ganciclovir. Noninvasive identification of target tissue together with assessment of vector-distribution volume and the effects of gene therapy were achieved using magnetic resonance imaging and positron emission tomography. The treatment was tolerated well without major side effects. In two of eight patients, we observed a greater than 50% reduction of tumor volume and in six of eight patients focal treatment effects. Intracerebral infusion of contrast medium before vector application displayed substantial inhomogeneity of tissue staining indicating the need of test infusions to monitor the mechanical distribution of vectors. Visualization of therapeutic effects on tumor metabolism and documentation of gene expression using positron emission tomography indicated that molecular imaging technology appears to be essential for the further development of biological treatment strategies.


Assuntos
Ganciclovir/administração & dosagem , Terapia Genética , Glioblastoma/terapia , Imageamento por Ressonância Magnética , Timidina Quinase/metabolismo , Tomografia Computadorizada de Emissão , Adulto , Idoso , Encéfalo/patologia , Mapeamento Encefálico , Feminino , Gadolínio DTPA/metabolismo , Ganciclovir/metabolismo , Ganciclovir/uso terapêutico , Vetores Genéticos/uso terapêutico , Herpesvirus Humano 1/enzimologia , Herpesvirus Humano 1/metabolismo , Humanos , Processamento de Imagem Assistida por Computador/métodos , Lipossomos/metabolismo , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Timidina Quinase/genética , Fatores de Tempo
14.
J Histochem Cytochem ; 51(9): 1237-40, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12923249

RESUMO

We present a simple method based on transmission electron microscopy that allows investigation of the early steps of polyplex-mediated transfection without the use of labeled DNA. The ultrastructural analysis showed internalization of 0.2-1-micro m aggregates composed of 30-50-nm subunits. In addition, new details of the internalization process were revealed, suggesting an unspecific cell entry mechanism of large DNA aggregates.


Assuntos
Peptídeos , Plasmídeos/ultraestrutura , Transporte Biológico , Técnicas de Transferência de Genes , Genes Reporter , Humanos , Luciferases/genética , Luciferases/metabolismo , Microscopia Eletrônica , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Transfecção , Células Tumorais Cultivadas
16.
J Mol Med (Berl) ; 81(6): 355-62, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12732930

RESUMO

Duchenne muscular dystrophy (DMD), caused by the absence of dystrophin, is associated with decreased muscle cell proliferation. An increased p21 mRNA level in DMD patients may be involved in the process. In this context we are interested to improve the proliferation of primary human skeletal muscle cells (SkMC) by a reduction in the cell cycle proteins p21 and p57 using the appropriate antisense oligonucleotides (ASO). Therefore a transfection procedure needs to be optimized in which the oligonucleotide enters the SkMC with a minimal loss of cell vitality and high efficiency. Three different formulations, Effectene, DAC40, and SuperFect, were compared. Proliferation was analyzed comparing cells transfected with p21 and/or p57 ASO vs. cells transfected with scrambled ASO using a bromodeoxyuridine assay. Under optimal conditions (a mixture of 0.25 microg ASO, 5 microl Effectene, 0.8 microl enhancer) SkMC transfected with p21 ASO reveal an average increase in cell proliferation of 32.5+/-11% after 24 h. p57 ASO shows the same effect, but concomitant transfection of p21 and p57 does not enhance it. A cell vitality of 78+/-14% after 24 h was determined by the MTT test. SkMC transfected with DAC40 reveal a maximal increase in proliferation of 38+/-7% after 48 h and show a vitality of 65+/-8%. In contrast to both these formulations, SuperFect was found to be highly toxic for SkMC, with more than 70% dead cells after 24 h. The increase in proliferation, the functional biological effect of p21 ASO, is well correlated with a decrease in p21 detected by western blot analysis of 31.6% for Effectene. Transfection efficiency was measured directly by FACS analysis using FITC-labeled ASO and data showing ASO internalization in 75.8+/-11.2% of the cell population for Effectene and 74.4+/-6.6% cells for DAC40. Taken together transient transfection of p21 or p57 ASO into primary human SkMC using Effectene significantly improves their proliferation compared to transfection with scrambled ASO without a major loss of cell vitality. This represents a basis for the transfer of this technique to dystrophin-deficient SkMC cultures and the introduction of the short interference-RNA technique which might enhance the effect on cell proliferation.


Assuntos
Ciclinas/genética , Distrofina/genética , Distrofia Muscular de Duchenne/terapia , Mioblastos Esqueléticos/fisiologia , Proteínas Nucleares/genética , Oligonucleotídeos Antissenso , Transfecção , Animais , Diferenciação Celular , Divisão Celular , Sobrevivência Celular , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p57 , Ciclinas/metabolismo , Distrofina/biossíntese , Expressão Gênica/efeitos dos fármacos , Terapia Genética , Humanos , Camundongos , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Mioblastos Esqueléticos/citologia , Proteínas Nucleares/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
17.
Ophthalmic Res ; 35(2): 117-24, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12646753

RESUMO

PURPOSE: Gene transfer of immunoregulatory cytokines could contribute to reduce rejection of corneal grafts. The aim of our study was to examine the gene transfer efficiency of liposomal vehicles compared to adenoviral vectors for transferring the Epstein-Barr-virus-derived interleukin 10 homologue (viral IL-10, vIL-10) into corneal endothelial cells and organ-cultured human corneas (HC) in vitro. METHOD: To test liposomal efficiency, 2 lipid formulations (SP-Chol/DOPE 20/80 and DDAB/DOPE 30/70 in various concentrations) were complexed with a plasmid containing the vIL-10 cDNA in an eukaryotic expression vector (pcDSRalpha-BCRF-I). The complexes were transferred to (1) subconfluent bovine corneal endothelial cells (BCEC) after 1 passage and to (2) HC stored in organ culture. In addition, BCEC and HC were transduced with the recombinant adenoviral vector encoding for vIL-10 (AdvIL-10). Secretion of vIL-10 in the supernatants from both transfected BCEC and HC was measured by specific ELISA. RESULTS: For gene transfer in BCEC, both transfection methods (liposomes and adenovirus) led to high secretion of vIL-10 [>2 ng/ml (liposomes) and <150 ng/ml (adenovirus) per 5,000 initially planted BCEC]. Expression levels in BCEC were dependent on the concentration of applied liposomes. For gene transfer in HC, only the adenoviral transduction technique achieved a high production of vIL-10, whereas liposomal transfection led only to low vIL-10 secretion (4.8 microg/ml vs. 95 pg/ml per quarter of cornea). CONCLUSION: For transfection of corneal endothelial cells in culture, liposomes can be considered as a safe and useful alternative method of gene transfer avoiding side-effects of viral vectors. However, for transfection of organ-cultured HC, adenoviral vectors are superior to liposomal vehicles.


Assuntos
Endotélio Corneano/fisiologia , Técnicas de Transferência de Genes , Interleucina-10/genética , Adenoviridae/genética , Animais , Bovinos , Córnea/citologia , Técnicas de Cultura , Citocinas/genética , Expressão Gênica , Vetores Genéticos , Humanos , Interleucina-10/biossíntese , Lipossomos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Tempo
18.
Biomaterials ; 24(8): 1409-18, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12527282

RESUMO

Serum protein adsorption to the surface of particulate synthetic drug carrier systems has a major influence on their uptake by phagocytes. The influence of alpha2-human serum glycoprotein (alpha2GP) on the phagocytosis of various surface modified microparticles was studied in dendritic cells (DC) and was compared with a potent opsonin, IgG, and a dysopsonin, human serum albumin (HSA). The microparticles were administered to DC before and after the incubation with alpha2GP, IgG and HSA in single, binary or ternary protein systems and in whole blood serum. Phagocytosis of microparticles was vastly affected by the surface character of the microparticles themselves and by the adsorption of the proteins. Poly-L-lysine (PLL)-modified microparticles were under all conditions internalized with highest efficiency which is suggested to be mediated by their positive surface charge. The adsorption of commonly phagocytosis promoting proteins reduced the uptake of PLL-modified particles and is explained by compensation of the positive surface charge by the adsorbed negatively charged proteins. In all other particle types tested, freshly adsorbed alpha2GP was found to exhibit a strong phagocytosis promoting activity which was comparable to that of adsorbed IgG. Interestingly, this opsonic activity was lost already 2 h after adsorption to the particle surface. Protein adsorption from binary and ternary protein systems and from whole blood serum occurred in a competitive manner. Significant inhibition of phagocytosis was observed, even when HSA was combined with strong opsonins such as alpha2GP or IgG or in mixtures of all three proteins, indicating the importance of studying the influence of protein adsorption in protein mixtures.


Assuntos
Proteínas Sanguíneas/metabolismo , Células Dendríticas/fisiologia , Adsorção , Ligação Competitiva , Materiais Biocompatíveis , Células Cultivadas , Sistemas de Liberação de Medicamentos , Humanos , Imunoglobulina G/metabolismo , Teste de Materiais , Microesferas , Fagocitose , Polilisina , Ligação Proteica , Albumina Sérica/metabolismo , alfa-Macroglobulinas/metabolismo
19.
Pharm Res ; 19(3): 250-7, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11934230

RESUMO

PURPOSE: To produce and characterize a nonviral ultrasound-controlled release system of plasmid DNA (pDNA) encapsulated in gas-filled poly(D,L-lactide-co-glycolide) microparticles (PLGA-MPs). METHODS: Different cationic polymers were used to form pDNA/polymer complexes to enhance the stability of pDNA during microparticle preparation. The physico-acoustical properties of the microparticles, particle size, pDNA integrity, encapsulation efficiency and pDNA release behavior were studied in vitro. RESULTS: The microparticles had an average particle size of around 5 microm. More than 50% of all microparticles contained a gas core, and when exposed to pulsed ultrasound as used for color Doppler imaging create a signal that yields typical color patterns (stimulated acoustic emission) as a result of the ultrasound-induced destruction of the microparticles. Thirty percent of the pDNA used was successfully encapsulated and approximately 10% of the encapsulated pDNA was released by ultrasound within 10 min. CONCLUSIONS: Plasmid DNA can be encapsulated in biodegradable gas-filled PLGA-MPs without hints for a structural disintegration. A pDNA release by ultrasound-induced microparticle-destruction could be shown in vitro.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Gases/administração & dosagem , Terapia Genética/métodos , Ácido Láctico/administração & dosagem , Plasmídeos/administração & dosagem , Ácido Poliglicólico/administração & dosagem , Polímeros/administração & dosagem , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Microesferas , Tamanho da Partícula , Copolímero de Ácido Poliláctico e Ácido Poliglicólico
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