Biomedical subjects

Ilona Kovalszky

Publications and source records attributed to Ilona Kovalszky.

At least 19 recordsLinked to original sources

PubMed2006

Agrin, a novel basement membrane component in human and rat liver, accumulates in cirrhosis and hepatocellular carcinoma.

Agrin is a multifunctional heparan sulfate proteoglycan originally discovered in the neuromuscular junctions and later observed in numerous other localizations. The presence of agrin in the liver, either healthy or diseased, has formerly not been reported. We detected agrin in minor amounts in the basement membranes of blood vessels and bile ducts in the healthy liver. The proliferation of bile ductules and the formation of new septal blood vessels in liver cirrhosis, as well as neoangiogenesis in the hepatocellular carcinoma (HCC) result in a dramatic increase in the quantity of agrin. Vascular and peribiliary basement membranes were strongly immunopositive for agrin in 29/29 human liver specimens with cirrhosis and HCC. However, sinusoidal walls of regenerative nodules in the cirrhotic liver consistently remained negative. Given the selectivity of agrin for tumor microvessels, agrin immunohistochemistry may prove helpful in recognizing malignant transformation in cirrhotic livers. Similar immunohistochemical observations were made on the liver of rats exposed to a combined cirrhosis/HCC induction treatment. In both human and rats, agrin probably originates from activated myofibroblasts, vascular smooth muscle cells and biliary epithelial cells. Increased agrin expression in human specimens, in the liver of 4/4 treated rats, as well as in isolated rat liver mesenchymal cells was verified by quantitative RT-PCR. Considering that agrin binds various growth factors, and it directly interacts with cell membrane receptors such as alphav-integrins, we hypothesize a stimulatory role for agrin in neoangiogenic processes such as tumor vascularization, and a supportive role in bile ductule proliferation.

Agrin↗

PubMed2006

[Role of syndecan-1 proteoglycan in the invasiveness of HT-1080 fibrosarcoma].

Syndecan-1 is a transmembrane heparan sulfate proteoglycan which plays pivotal role in cell-cell and cell-extracellular matrix interactions. However, its implication in the establishment of malignant phenotype is still controversial. Its expression indicates differentiated phenotype in certain tumors, while it confers invasive nature for others. For the better understanding of the role of syndecan-1 in cancer we transfected HT-1080 fibrosarcoma cell line with the full and a truncated construct of syndecan-1 and established stable cell lines with them. We studied the in vitro and in vivo growth capacity and metastatic potential of the transfectants in comparison with the cell line bearing only the EGFP expression vector. Our results showed that the growth rate of syndecan transfectants increased and they developed more lung metastases than the control cells. As local growth of the full transfectant was faster than that of the 78sig we presume that the full protein and maybe the shedding is needed for the local development of the tumor, but the intracellular and transmembrane domain is sufficient to promote metastasis formation.

Animals↗

PubMed2006

Regulatory role of kinases and phosphatases on the internalisation of caveolae in HepG2 cells.

The caveolar cycle is thought to be regulated by synchronised function of kinases and phosphatases. Using ocadaic acid--a serine/threonine protein phosphatase inhibitor--and an inhibitor of tyrosine phosphatase (sodium orthovanadate) we have followed the internalisation of caveolae. Since albumin binding to its receptor (gp60) can induce pinching off of caveolae from the plasma membrane, we also used this physiological ligand to induce the internalisation. Our confocal microscopic results show that both ocadaic acid and vanadate treatments have significantly decreased caveolin (caveolin-1 and -2) labelling on the cell surface, while the cytoplasmic labelling became much stronger. Quite often large, strongly labelled "granules" appear at the perinuclear region. Very strong caveolin labelling was detected along the actin-cytoskeleton suggesting that caveolae might move along these filaments. Our electron microscopic results also show an intensive caveolae pinching off from the plasma membrane. After ocadaic acid and vanadate treatments the number of surface connected vesicles (caveolae) decreases. At the same time, large multivesicular bodies (termed caveosomes) appear in the perinuclear area of the cytoplasm. By immunoprecipitation and Western blot analysis we detect an increased tyrosine phosphorylation of a approximately 29kDa protein in ocadaic acid and vanadate treated samples. This protein was identified as caveolin-2. No significant change in the tyrosine phosphorylation of caveolin-1 was found. From these data we can conclude that caveolae internalisation is regulated by phosphorylation of caveolin-2.

Blotting, Western↗

PubMed2006

The presence of human papillomavirus 16 in neural structures and vascular endothelial cells.

Human papillomavirus (HPV) is known as a strictly epitheliotropic pathogen. Our results raised the possibility that HPV 16 is present in neural cells and in the vascular endothelium. By in situ hybridization, we have detected HPV 16 E6 ORF sequence in small blood vessels and peripheral nerves adjacent to oral and cervical cancers. The same structures have clearly shown immunohistochemical reactivity for the E6 oncoprotein. These results were verified by PCR applied to E6 and L1 ORFs following microscopic laser dissection of the immunohistochemically positive nerves and vessels. These observations suggest that HPV 16 DNA and protein are present in neurons and endothelial cells in the vicinity of HPV-associated tumors. The HPV 16 genome presumably exists in a non-replicating form in the neurons and constitutively produces high levels of E6 and E7 proteins with an unknown neuropathological outcome.

Base Sequence↗

PubMed2006

Claudin-4 differentiates biliary tract cancers from hepatocellular carcinomas.

The recently identified claudins are dominant components of tight junctions, responsible for cell adhesion, polarity and paracellular permeability. Certain claudins have been shown to have relevance in tumor development, with some of them, especially claudin-4, even suggested as future therapeutic target. The aim of the present study was to analyze the expression of claudin-4 in the biliary tree, biliary tract cancers and hepatocellular carcinomas. A total of 107 cases were studied: 53 biliary tract cancers, 50 hepatocellular carcinomas, 10 normal liver and 10 normal extrahepatic biliary duct samples. Immunohistochemical analysis was performed on conventional specimens and on tissue microarrays as well. Claudin-4 was further investigated by Western blot analysis and real-time RT-PCR. Intense membranous immunolabeling was found for claudin-4 in all biliary tract cancers unrelated to the primary site of origin, namely intrahepatic, extrahepatic or gallbladder cancers. Normal biliary epithelium showed weak positivity for claudin-4. In contrast, normal hepatocytes and tumor cells of hepatocellular carcinomas did not express claudin-4. The results of Western immunoblot analysis and real-time RT-PCR were in correlation with the immunohistochemical findings. Cytokeratins, as CK7 (92%) and CK19 (83%) were mostly positive in biliary tract cancers, however, one-third of hepatocellular carcinomas also expressed CK7 (34%). HSA antibody (HepPar1) reacted with the majority of hepatocellular carcinomas (86%), while being positive in a low percentage of the biliary tract cancers (8%). In conclusion, this is the first report of a significantly increased claudin-4 expression in biliary tract cancers, which represents a novel feature of tumors of biliary tract origin. Claudin-4 expression seems to be a useful marker in differentiating biliary tract cancers from hepatocellular carcinomas and could well become a potential diagnostic tool.

Adenocarcinoma↗

PubMed2005

Stromal syndecan-1 expression is an adverse prognostic factor in oral carcinomas.

Syndecan-1, a transmembrane proteoglycan, may exert anti-proliferative effects and promote cell growth by binding various growth factors. Malignant epithelial cells often down-regulate their own syndecan-1 production, and are capable of inducing an aberrant syndecan-1 expression in stromal fibroid cells. Decreased tumor cell syndecan-1 levels in human head and neck squamous cell carcinomas indicate poor prognosis, however, no correlation between stromal syndecan-1 expression and clinical parameters has previously been established. By means of immunohistochemistry, we observed a decrease in tumor cell syndecan-1 reactivity in 33/39 oral carcinoma cases, the degree of which, however, correlated only weakly with the clinical outcome (p = 0.097). Conversely, stromal syndecan-1 positivity proved to be a significant risk factor of recurrence (Cox analysis, p = 0.03) and tumor-specific death (p = 0.023) within a 24-month period after operation. Taken together, stromal expression of syndecan-1 is a reliable factor of adverse prognosis in oral carcinomas.

Adult↗

PubMed2005

Prognostic significance of high-risk HPV status in advanced cervical cancers and pelvic lymph nodes.

OBJECTIVE: In this study, we investigated the presence of high-risk (HR) HPV types most prevalent in the Hungarian population in surgically removed cervical cancers and pelvic lymph nodes. The aim of our work was to determine the prognostic significance of HPV status in the lymph nodes draining the tumor. METHODS: Primary tumor specimens from 150 patients and 900 lymph node samples (six per case) were studied. Fifty-six/150 were early (FIGO IA-IB) stage, while 94/150 were advanced (FIGO IIA-IIIB) stage cancers. Beside histopathological evaluation, DNA extracted from the tissue samples was subjected to nested PCR to detect characteristic type-specific sequences of HPVs 16, 18 and 33. Moreover, clinicopathological data were collected for an average 48-month postoperative follow-up period for the purposes of statistical analysis. RESULTS: The presence of HR-HPV types in the lymph nodes shows no correlation with disease-free survival, whereas the presence of lymph node metastases significantly decreases life expectancy (P = 0.002). Lymph nodes with metastases more frequently carry HR-HPV than nodes with no evidence of tumorous infiltration (65% versus 36%, P < 0.001); however, a high number of metastases surrounding HR-HPV-positive tumors were found negative for the viruses (42/120). CONCLUSIONS: HR-HPV status of pelvic lymph nodes draining cervical cancers has no noticeable influence on the life expectancy of the patients. HR-HPV-positive tumor cells do not necessarily have a selective advantage in forming metastases. Presumably, a number of alterations in cellular genes rather than the presence of papillomavirus DNA may have a decisive role in the progression of cervical cancers.

Adult↗

PubMed2005

Invasive growth and topoisomerase-switch induced by tumorous extracellular matrix in osteosarcoma cell culture.

Osteosarcoma cells are capable of extracellular matrix (ECM) synthesis. The ability of ECM to trigger the proliferation of a novel osteosarcoma cell line (OSCORT) was tested in this study in relation to a known tumor ECM, isolated from Engelbreth-Holm-Swarm (EHS) sarcoma (EHS-ECM). OSCORT was grown in monolayer, in EHS-ECM and in ECM deposited by the cells (OSCORT-ECM). Both EHS-ECM and OSCORT-ECM increased the proliferation and migration of OSCORT cells. Among the ECM biopolymers, heparan sulfate proteoglycan (HSPG) and fibronectin enhanced invasive growth, collagen type IV reduced it, while laminin had no effect. Among the ECM components HSPG and collagen IV increased both the synthesis and activation of collagenase type IV, and all the ECM components substantially increased beta1 integrin levels in the cells. The majority of ECM biopolymers decreased the level of topoisomerase I (except laminin) and elevated topoisomerase II (except fibronectin) in OSCORT. The switch in the ratio between the activities of topoisomerases I and II was mainly due to HSPG. The HSPG synthesized by OSCORT cells is described as agrin, which is a novel finding. The present study showed that HSPG (agrin) showed the most remarkable stimulatory action on the growth and migration of OSCORT cells. HSPG-induced topoisomerase II-induction deserves further experimentation, to discover its relevance to tumor progression.

Adolescent↗

PubMed2005

Changes of cell adhesion and extracellular matrix (ECM) components in cervical intraepithelial neoplasia.

Cell-cell and cell-extracellular matrix interaction is crucial in tumor progression. Tight junction (TJ) proteins as occludin and claudins (CLDNs) play important role in this process together with several extracellular matrix components, as syndecan. Our previous work suggested significant changes in the expression of claudins even in the early stages of cervical carcinogenesis. The aim of our present work was to study the expression of occludin and syndecan-1, as compared to CLDNs, in early phases of cervical carcinogenesis. Paraffin sections of 50 samples were studied by immunohistochemistry, including cervical intraepithelial neoplasias (CINI-II-III), in situ carcinomas (CIS) and normal cervical samples. Occludin and CLDN-2 were found colocalized in the basal layer, while syndecan-1 and CLDN-1, -4 and -7 were coexpressed in the parabasal and intermedier layers in normal epithelia. Intensity of occludin staining decreased in CIN/CIS lesions, although it was more extended towards the upper epithelial layers with inverse relation with grades, as seen in the case of CLDN-2 expression. CLDN-1, -2, -4 and -7 were detected in the entire epithelium in CIN, showing decrease in CIS. The progression of CIN was associated with reduced syndecan-1 expression, in contrast to CLDN-1, -4 and -7 which increased toward CIS. The obtained data suggest that significant changes occur in the composition of cell adhesion complexes even in early stages of cervical carcinogenesis. The pattern of expression is characteristic for the alteration, the changes in the different components, however, are not parallel with each other.

Biomarkers, Tumor↗

PubMed2004

[Proteoglycans in the liver].

Proteoglycans are macromolecules formed by a protein core to which sugar chains are covalently attached. They are present on the cell surface and in the ECM of living things. In normal liver syndecan-1 is the dominant transmembrane proteoglycan, trace amounts of ECM proteoglycans are in the stromal components. The amounts of proteoglycans we studied increase in liver cirrhosis. In liver cancer abnormal localization of syndecan-1 and stroma rich in agrin was characteristic. The core proteins as well as the sugar chains of proteoglycans interact with and modulate the effect of regulatory factors. This implies that structural alterations of proteoglycans contribute to the development of malignant phenotype. Heparan sulfate chains of liver cancer are undersulfated with decreased or altered biological activity. Their binding capacity for transcription factor decreases, and they do not inhibit topoisomerase I enzyme. Truncated form of syndecan-1 lacking the extracellular domain of the molecule induces differentiation of hepatoma cell line and inhibits the shedding of syndecan-1. This phenomenon calls attention to the importance of syndecan-1 shedding in the regulation of cell behavior.

Animals↗

PubMed2004

Phospholipid barrier to fibrinolysis: role for the anionic polar head charge and the gel phase crystalline structure.

The massive presence of phospholipids is demonstrated in frozen sections of human arterial thrombi. Purified platelet phospholipids and synthetic phospholipids retard in vitro tissue-type plasminogen activator (tPA)-induced fibrinolysis through effects on plasminogen activation and plasmin function. The inhibition of plasminogen activation on the surface of fibrin correlates with the fraction of anionic phospholipid. The phospholipids decrease the amount of tPA penetrating into the clot by 75% and the depth of the reactive surface layer occupied by the activator by up to 30%, whereas for plasmin both of these parameters decrease by approximately 50%. The phospholipids are not only a diffusion barrier, they also bind the components of the fibrinolytic system. Isothermal titration calorimetry shows binding characterized with dissociation constants in the range 0.35-7.64 microm for plasmin and tPA (lower values with more negative phospholipids). The interactions are endothermic and thermodynamically driven by an increase in entropy, probably caused by the rearrangements in the ordered gel structure of the phospholipids (in line with the stronger inhibition at gel phase temperatures compared with liquid crystalline phase temperatures). These findings show a phospholipid barrier, which should be overcome during lysis of arterial thrombi.

Anions↗

PubMed2004

Endoscopic diagnosis of cytomegalovirus infection of upper gastrointestinal tract in solid organ transplant recipients: Hungarian single-center experience.

BACKGROUND: Cytomegalovirus (CMV) is considered to be the major cause of upper gastrointestinal (GI) symptoms in organ transplant recipients. In the diagnosis of GI CMV infection the detection of the virus in the mucosa is essential. The aim of the study was to evaluate the significance of CMV, detected in biopsy specimens from stomach and duodenum of solid organ transplant recipients. METHODS: Data of 227 elective upper endoscopies on symptomatic organ transplant recipients were evaluated for clinical symptoms, endoscopic changes and conventional histologic alterations of mucosal biopsy samples. Qualitative PCR was performed for detection of the presence of CMV-DNA in each biopsy materials. RESULTS: CMV-DNA was detected in biopsy samples of 91 patients (40.1%) while only in 20 cases (8.8%) the signs of CMV infections were found by conventional histology (p < 0.00001). No considerable differences could be observed in symptomatic, histologic alterations between CMV-PCR positive and negative groups. There were no endoscopic changes in 25.3% of CMV-PCR positive and 5.1% of negative patients. CONCLUSIONS: Qualitative PCR is an accurate method for the detection of CMV in the mucosa of the GI tract. Further investigations are needed for determination of the exact pathological role of detected CMV.

Adult↗

PubMed2003

[The molecular diagnostics of viruses].

For many years data of cancer research indicated that viruses can cause cancer. Virus infections induce cancer by different mechanisms. To predict the significance of a viral DNA fragment in human cells we have to be aware of the changes the particular virus is able to induce there.However, no matter which mechanisms of viral carcinogenesis are utilized, generally other factors (environmental, chemical, immunodeficiency, etc.) are also needed to induce invasive cancer in human. Before the introduction of nucleic acid based detection technique virus identification was a long and cumbersome process. This has been eliminated by the invention of recombinant gene technology and polymerase chain reaction. Virus nucleic acid can be detected without amplification using Southern, Northern and in situ hybridization. Techniques for target (polymerase chain reaction)or signal (hybrid capture, tyramine) amplification improved the sensitivity of detection. In the meantime, for the successful use of the arsenal of new methods we have to consider the characteristic feature of molecular virus research. A major achievement of molecular virus detection is that it proved the pathological significance of viruses in human cancers even in those where this was not expected. Hopefully these informations will increase the effort for elimination of oncogene virus infections.

Animals↗

PubMed2003

Myosin: a noncovalent stabilizer of fibrin in the process of clot dissolution.

Myosin modulates the fibrinolytic process as a cofactor of the tissue plasminogen activator and as a substrate of plasmin. We report now that myosin is present in arterial thrombi and it forms reversible noncovalent complexes with fibrinogen and fibrin with equilibrium dissociation constants in the micromolar range (1.70 and 0.94 microM, respectively). Competition studies using a peptide inhibitor of fibrin polymerization (glycl-prolyl-arginyl-proline [GPRP]) indicate that myosin interacts with domains common in fibrinogen and fibrin and this interaction is independent of the GPRP-binding polymerization site in the fibrinogen molecule. An association rate constant of 1.81 x 10(2) M(-1) x s(-1) and a dissociation rate constant of 3.07 x 10(-4) s(-1) are determined for the fibrinogen-myosin interaction. Surface plasmon resonance studies indicate that fibrin serves as a matrix core for myosin aggregation. The fibrin clots equilibrated with myosin are stabilized against dissolution initiated by plasminogen and tissue-type plasminogen activator (tPA) or urokinase (at fibrin monomer-myosin molar ratio as high as 30) and by plasmin under static and flow conditions (at fibrin monomer-myosin molar ratio lower than 15). Myosin exerts similar effects on the tPA-induced dissolution of blood plasma clots. Covalent modification involving factor XIIIa does not contribute to this stabilizing effect; myosin is not covalently attached to the clot by the time of complete cross-linking of fibrin. Thus, our in vitro data suggest that myosin detected in arterial thrombi binds to the polymerized fibrin, in the bound form its tPA-cofactor properties are masked, and the myosin fibrin clot is relatively resistant to plasmin.

Binding Sites↗

PubMed2002

Proteoglycans and tumor progression: Janus-faced molecules with contradictory functions in cancer.

Understanding the details of the molecular mechanism of tumor dissemination revealed that several proteoglycan species are involved in the process but their role can be described as Janus-faced. One level of proteoglycan alterations is at the expression of their genes coding for the core protein. Characteristically, in progressing tumors two patterns emerged: loss or neoexpression of surface proteoglycans (PG) depending on the initial expression pattern of the cell type of origin. The situation is similarly complex concerning the changes of glycosaminoglycan (GAG) of the PG during tumor progression. This is due to the fact that the majority of PGs involved is hybrid molecule meaning that their core protein can be glycanated both with chondroitin and heparan sulfate. However, such an alteration in glycanation of PG may fundamentally change the function of the molecule, especially the one operating at the cell surface. Among the extracellular PGs, decorin emerged as inhibitor of progression while perlecan as a promoter of the process. Analysis of the available data indicate that during metastatization tumor cells must express at least one cell surface HSPG species from the syndecan-glypican-CD44v3 group. Furthermore, the HS-chain of these proteoglycan(s) carry important molecular signatures (suphution or epimerization patterns). Experimental data suggest that tumor cell surface heparan sulfate (PG) may provide a target for specific anti-metastatic interventions.

Animals↗

PubMed1997

Altered Proteoglycan Gene Expression in Human Biliary Cirrhosis.

Proteoglycans play key roles in the physiological assembly of extracellular matrices and in the modulation of growth factor activities. During liver regeneration there is a profound remodelling of the connective tissue network with a concurrent alteration in proteoglycan gene expression. In the present study we have analyzed in detail the biochemical and molecular properties of the proteoglycans associated with biliary cirrhosis. The three major proteoglycans of human liver, namely decorin, syndecan and perlecan, were markedly elevated in the cirrhotic parenchyma as compared to normal liver tissue. Particularly elevated (eight fold) was the perlecan. This proteoglycan had not only heparan sulfate but also chondroitin and dermatan sulfate. Reverse transcriptase PCR revealed a marked enhancement of decorin and syndecan expression and detectable message for perlecan was found only in the cirrhotic liver. These results indicate that significant proteoglycan alterations are associated with the development of biliary cirrhosis and provide basis for future studies aimed at the characterization of the molecular events involved in the regulation of extracellular matrix deposition in this common human disease.

Journal Article↗

PubMed1996

Marker Enzymes of Rat Chemical Hepatocarcinogenesis in Human Liver Tumors.

Reduced glucose-6-phosphatase, increased GGT activity and reduction of cytochrome P-450 content are considered to be markers of chemical hepatocarcinogenesis in rats. The significance of these changes were studied in certain human liver lesions; adenoma, focal nodular hyperplasia and hepatocellular carcinoma all developed in non-cirrhotic livers. Enzymes showed normal values in 4 out of 5 adenomas, in 2/13 FNH and in 4/18 HCC samples. The decreased cP-450 content in HCC proved to be the most consistent alteration (12/18). Only 3 HCC samples possessed changes off all enzymes. These data suggest that at least those enzymes which are used as markers in rat chemical hepatocarcinogenesis have little or no biological significance in human liver tumors, primarily due to the intertumoral heterogeneity of enzyme activity. Such heterogeneity was observed in the peritumoral "normal" liver tissue, too.

Journal Article↗

PubMed1995

Role of Proteoglycans in Tumor Progression.

Data is now starting to accumulate on the differential expression of PGs in tumor cells of various invasive/metastatic potential. This is not so surprising if one considers the key functions that PGs play in the regulation of cell proliferation, adhesion and motility. However, characterization of PG expression in individual tumor types still awaits further detailed studies. Data on melanomas clearly indicate that PG phenotype is both specific and also promiscuous in a sense that ectopic expression of certain tissue specific PGs can occur in various tumors. Expression of a metastatic phenotype-specific splice variants of CD44 provides an example for the possible marker-function of PG. This also raises the hope that some PGs could be used as diagnostic/prognostic tools in pathology or even as a therapeutic targets against tumor dissemination. On the other hand, specific glycanation inhibitors may also be used for the modulation of tumor PG exist and the invasive phenotype.

Journal Article↗