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Biomedical subjects

Roland Kaufmann

Publications and source records attributed to Roland Kaufmann.

At least 19 recordsLinked to original sources
PubMed2007

Identification of specific protein markers in microdissected hepatocellular carcinoma.

At present, the molecular mechanisms of hepatocellular carcinogenesis are not well-understood, and hepatocellular carcinoma (HCC) stays one of the most frequent and high-risk metastatic visceral neoplasms worldwide. For the identification of tumor-relevant proteins, we analyzed microdissected cells from nontumorous liver tissue (n = 28) and tissue derived from hepatic tumor center (n = 25), as well as tumor margin (n = 23). We unequivocally identified 53 proteins from hepatic tumor tissues by peptide fingerprint mapping and SELDI mass spectrometry that were separated using two-dimensional gel electrophoresis. Among a number of signals that were detected as significantly different in the protein profiling analysis, we identified for the first time ferritin light subunit (FLS) and adenylate kinase 3 alpha-like 1 (AK3), showing decreased expressions in hepatic tumor, as well as biliverdin reductase B (BVRB) that was upregulated in HCC. The use of ProteinChip technology in combination with tissue microdissection gives insight of the complex changes occurring at the protein level in hepatocellular cancer associated with tumor development and progression and resulted in three new potential diagnostically useful markers.

Biomarkers↗
PubMed2006

Long-term lowering of tumour interstitial fluid pressure reduces Ki-67 expression.

High tumour interstitial fluid pressure (TIFP) is a characteristic of most solid tumours. Recent data give first evidence that mechanical stretch induced by TIFP triggers proliferation in solid tumours. In the present study we compared two protocols of TIFP reduction on the expression of the tumour proliferation marker Ki-67: (a) short-term lowering of TIFP by a singular puncture and (b) long-term lowering of TIFP by catheterization. Utilizing two experimental tumours (A431, A549) it was found that the TIFP broke down rapidly after a singular puncture but recovered within 6h. In case of permanent catheterization no TIFP recovery was observed. After 24h tumours were excised and stained against the proliferation marker Ki-67. While a singular puncture had no effect catheterized tumours showed a significant decrease in Ki-67 expression. Our data suggest that long-term lowering of TIFP is required to reduce tumour proliferation.

Animals↗
PubMed2006

Oligonucleotides suppress IL-8 in skin keratinocytes in vitro and offer anti-inflammatory properties in vivo.

DNA codes for genetic information. Furthermore, recent findings suggest that DNA offers additional function, particularly in the recognition of microorganisms. In this study, we investigated two classes of oligodeoxynucleotides (ODN) in skin keratinocytes; namely, an ODN comprising two cytidine-phosphate-guanosine (CpG) motifs (CpG-1-phosphorothioate (PTO)) and a poly-cytidine (Non-CpG-5-PTO) as control. Both fluorescence-tagged ODN were rapidly taken up by cells and accumulated already after 5 minutes in perinuclear compartments. In order to test whether ODN convey immunological effects in keratinocytes, secretion of IL-8 was measured. Interestingly, both CpG-1-PTO and Non-CpG-5-PTO suppressed basal and tumor necrosis factor alpha-induced IL-8 levels measured in cell culture supernatants. Experiments using deletion mutant revealed a critical length of approximately 16 nucleotides conveying IL-8 suppression. Studies regarding the ODN backbone offered that PTO bondings are critical for significant IL-8 suppression. In order to substantiate the anti-inflammatory response, a contact hypersensitivity mouse model was utilized. Topical application of Non-CpG-5-PTO-containing ointments reduced ear thickness in sensitized mice. Taken together, these findings suggest an anti-inflammatory effect of ODN in epithelial cells in vitro and in vivo, indicating that DNA molecules offer distinct biological activities restricted to the physiological compartment applied. This effect seems to be independent from Toll-like receptor 9.

Administration, Topical↗
PubMed2006

Restoration of E-cadherin sensitizes human melanoma cells for apoptosis.

Cell-cell adhesion is considered to be important in the development and maintenance of organ tissue. The spatial association between melanocytes and keratinocytes within human epidermis is achieved by homophilic interaction of E-cadherin molecules located on adjacent cells. In contrast, downregulation of E-cadherin expression in melanoma cells is considered as a key event in metastasis. Besides the adhesive properties, E-cadherin serves as a signal receptor linking to the cadherin-catenin signaling complex. As cadherins act as negative regulators of beta-catenin, a contribution to tumor formation seems likely. In the present study, it was tested whether ectopic expression of E-cadherin triggers apoptosis in human melanoma cell lines (G-361, JPC-298, SK-Mel-13). It was found that restoration of E-cadherin caused sensitization against drug-induced apoptosis. Particularly, the release of mitochondrial cytochrome c was increased in response to staurosporine. Moreover, activation of caspase-3 and caspase-8 was elevated. Similarly, DNA fragmentation, serving as a marker for advanced apoptosis, was amplified in cells transduced with E-cadherin. Interestingly, transduction with an E-cadherin construct lacking the extracellular domain showed no modified apoptosis. In conclusion, our findings suggest therapeutic strategies that enable expression of E-cadherin in order to sensitize human melanoma cells towards apoptosis.

Apoptosis↗
PubMed2006

Mechanical stretch induces clustering of beta1-integrins and facilitates adhesion.

Human epithelial cells are permanently stimulated by external mechanical forces. The present in vitro study suggests that keratinocytes respond to mechanical strain by a coordinated spatial and functional utilization of beta1-integrins and the epidermal growth factor receptor (EGFR) with impact to the adhesion properties. It was found that a single mechanical stretch applied to HaCaT keratinocytes elevates the substrate adhesion, in particular to fibronectin and collagen type IV but not to laminin indicating the relevance of beta1-integrins in this process. This was confirmed using a functional blocking antibody directed against beta1-integrins which reversed the stretch-induced adhesion. Furthermore, mechanical stretch gives rise to a rapid redistribution of beta1-integrins in clusters on the basal cell membrane, without changing the overall amount of this particular integrin subset. Concomitantly, the EGFR co-localizes with beta1-integrin suggesting a functional cooperation of both membrane proteins in mechano-signaling. This is corroborated by data showing that stretch-induced activation of the EGFR and the downstream element extracellular regulated kinase 1/2 (ERK1/2) is reversed by preincubation with beta1-integrin antibodies. Vice versa, blocking the EGFR using a specific inhibitor abrogates stretch-induced ERK1/2 activation. In summary, these results show a functional cooperation of beta1-integrins and EGFR in the adhesion complex supporting the transmission of stretch-induced signals.

Cell Adhesion↗
PubMed2006

Eukaryotic expression of the broad-spectrum chemokine receptor antagonist vMIP-II and its effects on T-cell function in vitro and in vivo.

Pro-inflammatory chemokines and their receptors exhibit elementary functions in cell migration and in Th1-driven inflammatory conditions. One therapeutic strategy to prevent accumulation of pro-inflammatory immune cells is the use of specific chemokine receptor antagonists. An interesting and promising candidate in this context is the viral antagonist MIP-II (vMIP-II) that acts on a broad spectrum of chemokine receptors. To study the in vitro and in vivo effects of vMIP-II on pro-inflammatory chemokine receptor function, we further characterized an ovalbumin-specific murine central memory Th1IF12 clone by using RT-PCR, cDNA array and cytometry. Using in vitro chemotaxis assays we show that eukaryotically generated vMIP-II strongly inhibited migration of CCL2- or CCL5-stimulated Th1 IF12 cells. Using intravital microscopy, we observed that CCL5 induced rolling of Th1 cells in the ear vasculature of C57Bl/6 mice. Pre-treatment with vMIP-II significantly reduced CCL5-induced rolling of Th1 cells to basal levels, indicating, that vMIP-II is also active in vivo (proportion of rolling cells: 19.4 +/- 3.8%, 39.8 +/- 2.9% and 26.1 +/- 3.2%). In addition, investigating the anti-inflammatory action of vMIP-II in adoptive transfer of immunity and dinitrofluorobenzene-induced cutaneous hypersensitivity reaction using C57Bl/6 mice, we show a direct inhibitory effect of vMIP-II on the sensitization phase [Delta ear swelling 62 and 37 cm x 10(-3) for controls and vMIP-II treated mice (2.5 mg/kg), respectively] and effector phase (Delta ear swelling 14.8 and 3.6 cm x 10(-3) for controls and vMIP-II treated mice (2.5 mg/kg), respectively) of cutaneous hypersensitivity. These data indicate that vMIP-II is a promising agent to interfere with chronic inflammatory (skin) diseases.

Animals↗
PubMed2006

[Dystrophic calcinosis cutis after subcutaneous administration of para-aminosalicylic acid for treatment of pulmonary tuberculosis].

An 81-year-old women presented with monstrous subcutaneous mobile masses on the ventral and ventrolateral surfaces of her thighs. Radiography as well as computer tomography showed extensive soft tissue calcifications. We interpreted these alterations as dystrophic calcinosis cutis, most likely resulting from repeated subcutaneous PAS infusions for the treatment of pulmonary tuberculosis.

Aged, 80 and over↗
PubMed2006

Calciphylaxis: no therapeutic concepts for a poorly understood syndrome?

Calciphylaxis is a very uncommon and severe disease which mainly appears in patients with chronic renal insufficiency. It presents with ischemia and necrosis of the skin, subcutaneous adipose tissue, muscles and rarely viscera. The pathogenetic mechanisms inducing calciphylaxis are for the most part unknown. The mortality rate of 80% in the first year is very high. Patients experience marked pain, recurrent infections and the constant risk of secondary sepsis. Even multidisciplinary therapeutic strategies are limited, although there are recent case reports providing promising new therapeutic options including sodium thiosulfate and cinacalcet. This review summarizes the important aspects of diagnosis, pathogenesis, prevention and the possible therapeutic strategies of this intriguing, rare and often fatal disease.

Age Factors↗
PubMed2006

The ability of different forms of heparins to suppress P-selectin function in vitro correlates to their inhibitory capacity on bloodborne metastasis in vivo.

Ample evidence suggests that many of the in vivo anti-metastatic effects by heparins reflect their actions on P-selectin-mediated binding. We hypothesized that the ability of widely used heparins and derivatives to interfere with P-selectin-dependent tumour cell interactions under flow in vitro could be used to identify anticoagulants with advanced inhibitory functions on experimental blood-borne metastasis in vivo. To test this assumption, the impact of unfractionated heparin, the low-molecular-weight heparins (LMWH) nadroparin and enoxaparin, and the synthetic pentasaccharide fondaparinux on P-selectin-dependent tumour interactions in vitro and metastasis formation in vivo were evaluated. Our data revealed that these commonly used anticoagulants widely differ in their potential to interfere with P-selectinmediated cell binding. Importantly, the superior inhibitory capacity on P-selectin function of unfractionated heparin and LMWH nadroparin as opposed to LMWH enoxaparin and synthetic heparin pentasaccharide fondaparinux strongly correlated to the inhibitory potency of each in inhibiting experimental lung metastasis in vivo. Hence, P-selectin inhibition may constitute a valuable feature to identify anticoagulants that are suitable for anticancer therapy.

Animals↗
PubMed2006

Lowering of tumor interstitial fluid pressure reduces tumor cell proliferation in a xenograft tumor model.

High tumor interstitial fluid pressure (TIFP) is a characteristic of most solid tumors. TIFP may hamper adequate uptake of macromolecular therapeutics in tumor tissue. In addition, TIFP generates mechanical forces affecting the tumor cortex, which might influence the growth parameters of tumor cells. This seems likely as, in other tissues (namely, blood vessels or the skin), mechanical stretch is known to trigger proliferation. Therefore, we hypothesize that TIFP-induced stretch modulates proliferation-associated parameters. Solid epithelial tumors (A431 and A549) were grown in Naval Medical Research Institute nude mice, generating a TIFP of about 10 mm Hg (A431) or 5 mm Hg (A549). Tumor drainage of the central cystic area led to a rapid decline of TIFP, together with visible relaxation of the tumor cortex. It was found by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis that TIFP lowering yields a decreased phosphorylation of proliferation-associated p44/42 mitogen-activated protein kinase and tumor relaxation. In confirmation, immunohistochemical staining showed a decrease of tumor-associated proliferation marker Ki-67 after TIFP lowering. These data suggest that the mechanical stretch induced by TIFP is a positive modulator of tumor proliferation.

Animals↗
PubMed2006

Different expression of calgizzarin (S100A11) in normal colonic epithelium, adenoma and colorectal carcinoma.

The aim of the study was to detect proteomic markers usable to distinguish colorectal carcinoma from colon adenoma for a better understanding of the molecular mechanisms in the process of tumourigenesis. Therefore, we microdissected colon carcinoma tissue, epithelial colon adenoma tissue as well as normal adjacent colon epithelium and determined protein profiles by SELDI-TOF MS. A multitude of significantly different signals was detected. For their identification colon biopsis were lysed and subjected to a two-dimensional gel electrophoresis for separation. Subsequently, we identified nearly 100 proteins by tryptic digestion, peptide fingerprint mapping and database search. Calgizzarin (S100A11; S100C) identified by peptide fingerprint mapping correlated very well with a significantly differentially expressed signal found in prior protein profiling. Using an immunodepletion assay we confirmed the identity of this signal as calgizzarin. To localise calgizzarin in tissues we performed immunohistochemistry. For further confirmation of the identity of calgizzarin we re-analysed IHC-positive as well as IHC-negative tissue sections on ProteinChip arrays. This work demonstrates that biomarkers in colorectal cancer can be detected, identified and assessed by a proteomic approach comprising tissue-microdissection, protein profiling and immunological techniques.

Adenoma↗
PubMed2006

PAR-type thrombin receptors in renal carcinoma cells: PAR1-mediated EGFR activation promotes cell migration.

Cross-talk between G-protein-coupled receptor (GPCR) and epidermal growth factor receptor (EGFR) signaling systems is established in a wide variety of normal and neoplastic cell types. Here, we show that proteinase-activated receptor 1 (PAR1) mediates the tyrosine phosphorylation of EGFR in human renal carcinoma cells expressing PAR1 and PAR3 endogeneously. This GPCR-EGFR signal transduction pathway cross-talk requires matrix metalloproteinase activity and is involved in the regulation of renal carcinoma cell migration across a collagen barrier as shown using a Boyden chamber type assay. Our data therefore document a regulatory role of PAR1-mediated EGFR transactivation in cancer cell chemotactic migration. Further, our results underline the importance of PAR1-mediated pathways in kidney cancer cells and suggest that the thrombin/PAR1 system mediating EGFR transactivation may play a role in the progression of this tumor entity.

Cell Line, Tumor↗
PubMed2005

Temozolomide in combination with interferon-alfa versus temozolomide alone in patients with advanced metastatic melanoma: a randomized, phase III, multicenter study from the Dermatologic Cooperative Oncology Group.

PURPOSE: Temozolomide (TMZ) has shown efficacy in metastatic melanoma equal to that of dacarbazine (DTIC), the standard chemotherapeutic agent for melanoma. As the combination with interferon-alfa (IFN-alpha) appears superior to single-agent DTIC regarding response rates, the purpose of this study was to compare TMZ alone and TMZ plus IFN-alpha in terms of objective response (OR), overall survival, and safety in a prospective, randomized, multicenter trial. PATIENTS AND METHODS: Two hundred ninety-four patients with untreated stage IV metastatic melanoma (American Joint Committee on Cancer staging system) were randomly assigned to receive either oral TMZ alone (200 mg/m2/day; days 1 through 5 every 28 days) or in combination with subcutaneous IFN-alpha (5 MU/m2; days 1, 3, and 5 every week). RESULTS: Two hundred eighty-two patients were eligible for an intent-to-treat analysis, 271 patients were treated per protocol. In the TMZ + IFN-alpha arm, 33 (24.1%) of 137 patients responded to therapy (partial or complete remission) whereas in the monotherapy arm, in 18 (13.4%) of 134 patients, a response was evident. Thus, the response rate was significantly higher in the combination arm (P = .036). Median survival time was 8.4 months for patients treated with TMZ (95% CI, 7.07 to 9.27) and 9.7 months for those treated with the combination (95% CI, 8.26 to 11.18; P = .16). Dose modifications and interval prolongations due to hematologic toxicity were significantly more frequent in the TMZ + IFN-alpha arm (P < .001). CONCLUSION: In metastatic melanoma treatment with TMZ + IFN-alpha leads to a significantly superior OR rate compared to treatment with TMZ alone, which did not translate into prolonged survival in our study population.

Adult↗
PubMed2005

Detection of human beta defensin-1 and -2 by RT-competitive multiplex PCR.

Although, the human epithelium is constantly challenged by a broad spectrum of microorganisms, invasive infections are rather rare. Recent findings suggest the expression of antimicrobial peptides by skin cells in order to provide an innate defensive barrier. In particular, peptides of the beta-defensin family offer antimicrobial activity against different pathogens including bacteria and fungi. Within this peptide family, hBD-1 is rather constitutively expressed while hBD-2 and hBD-3 expression depends on environmental conditions. The present paper introduces RT-competitive multiplex PCR as a precise tool to detect hBD-1 and hBD-2 expression on the transcriptional level. The method makes use of co-amplification of synthetic competitors along with referring wildtype targets. Competitor- and wildtype-derived products differ in size allowing signal discrimination using agarose gel electrophoresis. Regulation of gene transcripts is evaluated by comparison of competitor and corresponding wildtype signals. It was found that primary human keratinocytes stimulated with Escherichia coli cells for 8 h offered an upregulation of hBD-2 to about 2,000 fold, while hBD-1 was only marginally regulated. RT-competitive multiplex PCR is a simple and accurate method that enables new insights into defensin regulation under physiological and pathophysiological conditions.

DNA Primers↗
PubMed2005

Proteinase-activated receptors (PARs)--the PAR3 Neo-N-terminal peptide TFRGAP interacts with PAR1.

Thrombin activates proteinase-activated receptor (PAR)1, PAR3 and PAR4 by a unique mechanism that involves cleavage of the receptor and exposure of a new N-terminal domain acting as a tethered ligand. Synthetic peptides based on the proteolytically revealed receptor sequence can selectively activate PAR1 or PAR4 independently of receptor cleavage. However, corresponding peptides for PAR3 have not been identified thus far. Here, we demonstrate that the synthetic peptide TFRGAP representing the 1st six residues of the new amino terminus of PAR3 induced ERK activation in human A-498 carcinoma cells endogeneously expressing PAR1 and PAR3. This effect was completely abolished by single alanine substitution at positions 3, 4 and 6 in the peptide. Since the specific PAR1 antagonist RWJ 56110 completely abolished TFRGAP-induced ERK activation in A-498 cells we speculate that TFRGAP does signal MAPK via interaction with PAR1. This was underlined by experiments on PAR1-/- mouse lung fibroblasts (KOLF cells) that stably overexpress human PAR1 and PAR3, respectively. While TFRGAP was without effect on ERK activation in PAR3+ KOLF cells, it induced MAPK activation in KOLF cells transfected with PAR1. These studies provide evidence that analogues of the PAR3 tethered ligand can mediate cell signaling by interaction with PAR1-type thrombin receptors.

Alanine↗