Biomedical subjects

Michael Gregor

Publications and source records attributed to Michael Gregor.

30 records · Page 2Linked to original sources

PubMed2003

Negative-strand RNA viral vectors: intravenous application of Sendai virus vectors for the systemic delivery of therapeutic genes.

Treatment by gene replacement is critical in the field of gene therapy. Suitable vectors for the delivery of therapeutic genes have to be generated and tested in preclinical settings. Recently, extraordinary features for a local gene delivery by Sendai virus vectors (SeVV) have been reported for different tissues. Here we show that direct intravenous application of SeVV in mice is not only feasible and safe, but it results in the secretion of therapeutic proteins to the circulation, for example, human clotting Factor IX (hFIX). In vitro characterization of first-generation SeVV demonstrated that secreted amounts of hFIX were at least comparable to published results for retroviral or adeno-associated viral vectors. Furthermore, as a consideration for application in humans, SeVV transduction led to efficient hFIX synthesis in primary human hepatocytes, and SeVV-encoded hFIX proteins could be shown to be functionally active in the human clotting cascade. In conclusion, our investigations demonstrate for the first time that intravenous administration of negative-strand RNA viral vectors may become a useful tool for the wide area of gene replacement requirements.

3T3 Cells↗

PubMed2003

Association between the C3435T MDR1 gene polymorphism and susceptibility for ulcerative colitis.

BACKGROUND & AIMS: The human multidrug resistance 1 (MDR1) gene product P-glycoprotein is highly expressed in intestinal epithelial cells, where it constitutes a barrier against xenobiotics. The finding that mdr1a knockout mice develop a form of colitis that is similar to ulcerative colitis, which can be prevented by antibiotics, indicates a barrier function for P-glycoprotein against the invasion of bacteria or toxins. Because the MDR1 single nucleotide polymorphism C3435T is associated with lower intestinal P-glycoprotein expression, we tested whether this polymorphism predisposes to development of ulcerative colitis. METHODS: Allele frequencies and genotype distributions of the C3435T single nucleotide polymorphism were investigated in 149 patients with ulcerative colitis, 126 patients with Crohn's disease, and sex-matched healthy controls. RESULTS: Significantly increased frequencies of the 3435T allele and the 3435TT genotype were observed in patients with ulcerative colitis compared with controls (3435T: P = 0.049; odds ratio, 1.4; 95% confidence interval, 1.02-1.94; 3435TT: P = 0.045; odds ratio, 2.03; 95% confidence interval, 1.04-3.95). In contrast, frequencies of the T allele and the TT genotype were the same in patients with Crohn's disease as in controls (P = 0.66 and P = 0.59, respectively). In comparison to 998 non-sex-matched controls, the effect for the TT genotype in ulcerative colitis patients was more pronounced (P = 0.0055; odds ratio, 2.1). CONCLUSIONS: The higher frequency of the 3435TT genotype in patients with ulcerative colitis corroborates the findings from the mdr1a knockout mice. The results support the notion that P-glycoprotein plays a major role in the defense against intestinal bacteria or toxins. Impairment of barrier function in 3435TT subjects could render this genotype more susceptible to the development of ulcerative colitis.

Adolescent↗

PubMed2003

Spontaneous, self-limited, non-atherosclerotic dissection of the superior mesenteric artery.

A case of spontaneous, non-atherosclerotic dissection of the superior mesenteric artery is described. The patient presented with severe abdominal pain. An initial computed tomography scan did not disclose any pathology, namely the major mesenteric arteries were perfused. The patient's initial symptoms resolved during the first 48 h under conservative treatment. Sagittal reconstruction from a follow-up computed tomography scan disclosed a 7 cm long dissection of the proximal superior mesenteric artery with thrombosis of the false lumen and residual blood flow through the true lumen. There were no signs of transmural infarction of the bowel. Anticoagulation with heparin and later with phenprocoumon was initiated and the patient is well after 6 months. Together with several other cases reported recently, and in contrast to previous experience, this case adds evidence that conservative treatment of spontaneous, non-atherosclerotic dissection of the superior mesenteric artery may be an alternative to surgery if residual blood flow is maintained.

Aortic Dissection↗

PubMed2003

Promoter methylation of INK4a/ARF as detected in bile-significance for the differential diagnosis in biliary disease.

PURPOSE: There is a need to enhance endobiliary cytotechniques by molecular marker lesions. This is of special significance for patients with primary sclerosing cholangitis, a disease predisposing for the development of cholangiocarcinoma. The INK4a/ADP ribosylation factor (ARF) locus encodes two tumor suppressor genes: p16INK4a and p14ARF. p16INK4a has been shown to be of major significance in cholangiocarcinoma. EXPERIMENTAL DESIGN: In an effort to evaluate the potential diagnostic role of p16INK4a and p14ARF promoter methylation in biliary disease, endoscopical obtained bile specimens of 71 patients were analyzed (26 choledocholithiasis, 6 with normal results, 23 bile duct carcinoma, 5 gall bladder carcinoma). Eleven patients with primary sclerosing cholangitis were enrolled. RESULTS: Merely 6% of specimens (2 of 32) obtained from patients without evidence for malignant biliary disease but 53.5% of malignancies (15 of 28) showed p16 promoter methylation (p14: 3 and 46.2%, respectively). The concordance of methylation rates detected in either bile or tissue specimens was high. In primary sclerosing cholangitis, a similar prevalence of methylation was detected as in malignant disease. CONCLUSIONS: This study demonstrates: (a) a high frequency and specificity of INK4a/ARF methylation in malignant biliary disease compared with mere cholangitis; and (b) the capability to detect these alterations reliably in endoscopically obtained bile. Thus, INK4a/ARF's promoter methylation status represents a candidate marker for the endoscopic diagnosis of biliary disease.

Adult↗

PubMed2002

The down regulated in adenoma (dra) gene product binds to the second PDZ domain of the NHE3 kinase A regulatory protein (E3KARP), potentially linking intestinal Cl-/HCO3- exchange to Na+/H+ exchange.

Intestinal electroneutral NaCl absorption is mediated by parallel operation of Na(+)/H(+) and Cl(-)/HCO(3)(-) exchange in the enterocyte apical membrane. The ion transporters involved are Na(+)/H(+) exchanger 3 (NHE3) and the down regulated in adenoma (dra) gene product. cAMP-mediated inhibition of NHE3 requires the transporter to bind to the second PDZ (PSD95, disk large, ZO1) domain of the adapter protein NHE3 kinase A regulatory protein (E3KARP). Because the C-terminal four amino acids of dra are ETKF (glutamate-threonine-lysine-phenylalanine), resembling a PDZ interaction motif, we hypothesized that dra may also bind to one of the PDZ domains of E3KARP. In vitro the ETKF motif of dra binds to the second PDZ domain of E3KARP, the affinity being comparable to that of the known ligand CFTR. The C-terminal phenylalanine, which is an unconventional residue in PDZ interaction motifs, can only be substituted by the classical residue leucine, but not by other hydrophobic residues (valine, isoleucine). Immunofluorescence colocalizes dra, NHE3, and E3KARP in the apical compartment of human proximal colon. We suggest a model in which both NHE3 and dra bind to the second PDZ domain of E3KARP and that linking of the transporters occurs through dimerization of E3KARP. In such a model, the first PDZ domain would remain available for instance for signal transduction proteins.

Adenoma↗

PubMed2002

Critical role of NF-kappaB and stress-activated protein kinases in steroid unresponsiveness.

Glucocorticoid resistance is a serious clinical problem in chronic inflammatory diseases, because many patients with rheumatoid arthritis, asthma, or Crohn's disease fail to respond to steroid treatment. The molecular mechanisms underlying this unresponsiveness, however, are completely unknown. The effects of steroids are largely mediated by the interference of the glucocorticoid receptor (GR) with proinflammatory transcription factors. In the present study, we therefore investigated the activation of the transcription factors nuclear factor-kappaB (NF-kappaB), activator protein-1 (AP-1), and the upstream kinases p38 and c-Jun N-terminal kinase (JNK) in steroid-sensitive and steroid-resistant patients with Crohn's disease. We demonstrated that steroid-sensitive and steroid-resistant patients reveal a remarkably different cellular activation pattern of these proinflammatory mediators. In steroid-sensitive patients, activation of NF-kappaB, AP-1, p38, and JNK was mainly found in lamina propria macrophages. In contrast, steroid-resistant patients revealed activation of all these mediators mostly in epithelial cells. The functional interference of the proinflammatory mediators with the glucocorticoid response was supported by reporter gene assays. Expression of NF-kappaB and, interestingly, also JNK1 and p38 inhibited the activity of the GR. Thus, our results suggest that steroid resistance is associated with increased epithelial activation of stress-activated protein kinases and NF-kappaB, which might inhibit the anti-inflammatory action of a limited number of GRs.

Caco-2 Cells↗

PubMed2002

Endothelial injury mediated by cytotoxic T lymphocytes and loss of microvessels in chronic graft versus host disease.

BACKGROUND: Vascular endothelial cells form the interface between recipient tissues and circulating alloreactive donor T cells after allogeneic stem cell transplantation. Vascular injury has been seen in patients with acute graft versus host disease (GVHD) in the skin. We aimed to see whether vascular injury mediated by cytotoxic T lymphocytes and microvessel loss arises in patients with chronic GVHD. METHODS: We investigated eight patients with acute GVHD and ten with chronic GVHD for signs of endothelial injury and microvessel loss by measurement of von Willebrand factor (vWF) in plasma and blood vessel density in biopsy samples taken from lesional skin. Controls consisted of nine patients without GVHD who survived for longer than 100 days and nine healthy people. Inflammation and endothelial injury were assessed in selected samples by immunostaining for CD8 T cells, activated cytotoxic T lymphocytes, and vascular endothelial cells. FINDINGS: We identified more extensive loss of microvessels in the skin of patients with GVHD (median 66 capillaries/mm(2); IQR 16-98) than of healthy controls (205 capillaries/mm(2); 157-226; p=0.005). Patients with GVHD had higher concentrations of vWF (238%; 168-288) than did those without GVHD (102%; 88-118; p=0.0005). Perivascular CD8 T cell infiltrates in skin correlated with vWF plasma concentrations in patients with GVHD (p=0.01), and activated cytotoxic T lymphocytes and endothelial injury were present in these same samples. INTERPRETATION: Host endothelial cells are a target of alloreactive donor cytotoxic T lymphocytes. Substantial blood vessel loss may lead to impaired blood perfusion and tissue fibrosis, the hallmark lesion of chronic GVHD.

Adolescent↗

PubMed2002

Caspase-8 and Apaf-1-independent caspase-9 activation in Sendai virus-infected cells.

Apoptotic cell death is of central importance in the pathogenesis of viral infections. Activation of a cascade of cysteine proteases, i.e. caspases, plays a key role in the effector phase of virus-induced apoptosis. However, little is known about pathways leading to the activation of initiator caspases in virus-infected host cells. Recently, we have shown that Sendai virus (SeV) infection triggers apoptotic cell death by activation of the effector caspase-3 and initiator caspase-8. We now investigated mechanisms leading to the activation of another initiator caspase, caspase-9. Unexpectedly we found that caspase-9 cleavage is not dependent on the presence of active caspases-3 or -8. Furthermore, the presence of caspase-9 in mouse embryonic fibroblast (MEF) cells was a prerequisite for Sendai virus-induced apoptotic cell death. Caspase-9 activation occurred without the release of cytochrome c from mitochondria and was not dependent on the presence of Apaf-1 or reactive oxygen intermediates. Our results therefore suggest an alternative mechanism for caspase-9 activation in virally infected cells beside the well characterized pathways via death receptors or mitochondrial cytochrome c release.

Animals↗

PubMed2002

Epigenetic control of the E-cadherin gene (CDH1) by CpG methylation in colectomy samples of patients with ulcerative colitis.

E-cadherin belongs to the cadherin family of calcium-dependent cell-adhesion molecules. The cadherins play an essential role in biological processes such as ordering of cell sorting, migration, and differentiation, and their malfunctioning is connected with neoplasia. Neoplastic progression in patients with chronic ulcerative colitis is characterized by the development of epithelial dysplasia. Transcriptional silencing of tumor-suppressor genes by promoter methylation has been observed in different types of human cancers and dysplasia. To explore the mode of E-cadherin regulation, 156 biopsy samples from 26 patients with long-standing ulcerative colitis were screened. To detect the methylation status of our samples, a methylation-specific PCR was applied. Methylation of the E-cadherin (CDH1) promoter was detected in 93% of the patients with dysplastic biopsy samples, in contrast to only 6% of the patients without dysplasia (P < 0.001). We also examined the level of synthesis of E-cadherin protein by immunohistochemical staining in different paraffin-embedded samples of dysplastic and non-dysplastic origin in a subset of our patients. Samples with dysplasia displayed reduced levels, whereas samples without dysplasia revealed normal E-cadherin protein synthesis. These results show that the E-cadherin promoter is subjected to epigenetic control in colorectal ulceration. Obviously, this event may play an important role in the progression from chronic inflammation to colorectal cancer. For this reason, methylation of the CDH1 promoter is an attractive new biomarker for detecting ulcerative colitis patients with a high risk for developing colorectal cancers.

Aneuploidy↗

PubMed2002

Down-regulated in adenoma mediates apical Cl-/HCO3- exchange in rabbit, rat, and human duodenum.

BACKGROUND & AIMS: Duodenal bicarbonate secretion is in part mediated by an apical Cl-/HCO3- exchanger of unknown molecular nature. The recently discovered dra (down-regulated in adenoma) gene encodes a transport protein (DRA) for SO4(2-), Cl-, and HCO3-. The aim of this study was to investigate whether DRA may be the duodenal apical Cl-/HCO3- exchanger. METHODS: DRA, Na+/H+ exchanger (NHE) isoform 3, and anion exchanger isoform (AE) 2 messenger RNA expression levels were studied in rat, rabbit, and human gastrointestinal tract by semiquantitative reverse-transcription polymerase chain reaction and in situ hybridization (DRA in human intestine). The subcellular localization of DRA was determined by Western analysis and immunohistochemistry. Using rabbit and rat duodenal brush border membrane vesicles, anion exchange characteristics were investigated. RESULTS: DRA expression was high in duodenum and colon of all species, whereas NHE3 messenger RNA expression was low in duodenum and high in colon. Western analysis and immunohistochemistry showed an apical localization for DRA. Rabbit and rat duodenal brush border membrane vesicles showed 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive Cl-/Cl-, HCO3-/Cl-, SO4(2-)/Cl-, and Cl-/SO4(2-) exchange, with evidence for one major brush border membrane Cl-/anion exchanger, an affinity for Cl- > HCO3-, and a much higher affinity for SO4(2-) in rat than rabbit. The strong predominance of DRA over NHE3 and NHE2 expression in duodenum was paralleled by much higher Cl-/HCO3- than Na+/H+ exchange rates in brush border membrane vesicles and likely explains the high duodenal HCO3- secretory rates. CONCLUSIONS: These data suggest that DRA is the major apical anion exchanger in the duodenum as well as the colon and the likely transport protein for duodenal electroneutral HCO3- secretion.

Adenoma↗

PubMed2002

Azathioprine therapy and adverse drug reactions in patients with inflammatory bowel disease: impact of thiopurine S-methyltransferase polymorphism.

The efficacy of the immunosuppressants azathioprine and 6-mercaptopurine has been well established in the therapy of inflammatory bowel diseases (IBD). However, its use has been complicated by a high incidence of serious adverse drug reactions such as hematotoxicity, hepatotoxicity, pancreatitis and gastrointestinal disturbances. Whereas azathioprine-related pancytopenia has been clearly linked to thiopurine S-methyltransferase (TPMT) polymorphism limited data are available to explain gastrointestinal side effects. In a retrospective analysis of 93 adults with IBD and azathioprine therapy both phenotyping and genotyping was used to explore systematically the relationship between TPMT and azathioprine-related adverse reactions. At time of inclusion, 69 patients were still receiving azathioprine therapy and had never experienced side effects. Azathioprine had been withdrawn in 10 patients for non-medical reasons or lack of response and 14 patients (15%) had stopped medication or were on reduced dose due to severe azathioprine-related side effects. Nine of these 14 patients had developed gastrointestinal side effects (hepatotoxicity, n = 3; pancreatitis, n = 3; others, n = 3), but their normal red blood cell TPMT activities were in accordance to TPMT wild-type. TPMT deficiency in one patient had led to pancytopenia whereas only two of the remaining four patients with hematotoxicity displayed an intermediate phenotype of TPMT. This study demonstrates that azathioprine-related gastrointestinal side effects are independent of the TPMT polymorphism. Nevertheless pharmacogenetic testing for TPMT prior to commencing thiopurine therapy should become routine practice in order to avoid severe hematotoxicity in TPMT deficient patients and lowering the incidence of hematological side effects in individuals heterozygous for TPMT.

Adult↗

PubMed2002

Validation of the EuroQol questionnaire in patients with inflammatory bowel disease.

OBJECTIVE: The EuroQol EQ-5D is a generic questionnaire for describing and valuing patients' health-related quality of life. The purpose of the study was to analyse the construct validity, criterion validity, test-retest reliability and responsiveness of the EQ-5D in patients with inflammatory bowel disease. METHODS: 152 consecutive patients with inflammatory bowel disease (123 with Crohn's disease and 29 with ulcerative colitis) completed the EQ-5D, the SF-36 and the Inflammatory Bowel Disease Questionnaire (IBDQ). Of the study group, 66 patients filled in the EQ-5D a second time after a 2-week gap, including a transition question. Disease activity was measured by the Crohn's Disease Activity Index (CDAI) and by Rachmilewitz's Clinical Activity Index (CAI). RESULTS: The EQ-5D showed a moderate ceiling effect. Correlation between the EQ-5D visual analogue scale (EQ VAS) score and CDAI/CAI was r = -0.65/r = -0.71 (both P < 0.001). Levels of responses to EQ-5D items and the EQ VAS score were significantly better for patients in remission than for patients with active disease (all P < 0.01). For the total sample, coefficients of correlation between the EQ VAS score and SF-36 and IBDQ scores ranged between 0.37 and 0.73 (all P < 0.0001). When repeated, the EQ-5D was reliable in stable patients (intraclass correlation coefficient for EQ VAS = 0.77, kappa statistic for items 0.39 to 1.00); the EQ VAS was responsive in patients who, in the transition question, indicated an improvement in health state (effect size 0.79). CONCLUSIONS: The EQ-5D is reasonably valid, reliable and responsive in patients with inflammatory bowel disease. It can be used to generate preference-based valuations of health-related quality of life in inflammatory bowel disease.

Adolescent↗