Biomedical subjects

R Adány

Publications and source records attributed to R Adány.

At least 19 recordsLinked to original sources

PubMed2005

Identification of factor XIII-A as a marker of alternative macrophage activation.

Factor XIII subunit A of blood coagulation (FXIII-A) is known to be synthesized but not secreted by the monocyte/macrophage cell line. On the basis of its intracellular localization and substrate profile, FXIII-A is thought to be involved in certain intracellular processes. Our present study was designed to monitor the changes in FXIII-A gene expression and protein production in long-term culture of human monocytes during their differentiation into macrophages in the presence of activating agents (interleukin-4, interferon-gamma, Mycobacterium bovis BCG) inducing classical and alternative activation pathways. By using quantitative RT-PCR and fluorescent image analysis at the single-cell level we demonstrated that the expression of FXIII-A both at the mRNA as well as at the protein level is inversely regulated during the two activation programmes. Here we conclude that FXIII-A expression is an intracellular marker for alternatively activated macrophages, while its absence in monocyte-derived macrophages indicates their classically activated state.

Biomarkers↗

PubMed2003

Factor XIII subunit A as an intracellular transglutaminase.

Over the last 2 decades there has been increasing evidence that the role of factor XIII (FXIII) is not restricted to the area of hemostasis and that its subunit A functions as an intracellular enzyme in platelets and monocytes/macrophages. FXIII is already expressed during compartmentalisation of the precursors of megakaryocyte/platelet and monocyte/macrophage cell lines in the bone marrow. FXIII-A, produced by megakaryocytes, is packaged into budding platelets and is present in huge quantity in circulating ones. It seems very likely that it plays an important role in the cytoskeletal remodelling associated with the activation stages of platelets. FXIII-A can also be detected in blood monocytes and in all subsets of monocyte-derived macrophages throughout the body. FXIII-A is mainly localised in the cytoplasm, in association with cytoskeletal filaments, but at a relatively early stage of macrophage differentiation it also appears transiently in the nucleus. Cytoplasmic expression has a very close relationship with phagocytic activities. Further research is needed to understand the biological significance of its nuclear presentation.

Blood Platelets↗

PubMed2001

Chromosomal imbalances in primary and metastatic melanomas revealed by comparative genomic hybridization.

Characteristic genetic changes underlying the metastatic progression of malignant melanoma is incompletely understood. The goal of our study was to explore specific chromosomal alterations associated with the aggressive behavior of this neoplasm. Comparative genomic hybridization was performed to screen and compare genomic imbalances present in primary and metastatic melanomas. Sixteen primary and 12 metastatic specimens were analyzed. We found that the pattern of chromosomal aberrations is similar in the two subgroups; however, alterations present only in primary and/or metastatic tumors were also discovered. The mean number of genetic changes was 6.3 (range 1-14) in primary and 7.8 (range 1-16) in metastatic lesions. Frequent losses involved 9p and 10q, whereas gains most often occurred at 1q, 6p, 7q, and 8q. Distinct, high-level amplifications were mapped to 1p12-p21 and 1p22-p31 in both tumor types. Amplification of 4q12-q13.1, 7q21.3-qter and 8q23-qter were detected only in primary tumors. The 20q13-qter amplicon was present in a metastatic tumor. The number of genetic alterations were significantly higher in primary tumors which developed metastases within one year after the surgery compared to tumors without metastasis during this time period. Fluorescence in situ hybridization with centromeric and locus-specific probes was applied to validate CGH results on a subset of tumors. Comparison of FISH and CGH data gave good correlation. The aggressive behavior of melanoma is associated with accumulation of multiple genetic alterations. Chromosome regions, which differ in the primary and metastatic lesions, may represent potential targets to identify metastases-related chromosomal alterations.

Adult↗

PubMed2001

Multiplex PCR assay for screening deletions in the low density lipoprotein receptor gene.

BACKGROUND: In different populations of the world, more than 150 genetic alterations of the LDL receptor gene have been identified; each of which can result in hypercholesterolaemia, but no hot spots in the gene were detected so far. Because of the existence of very variable genetic alterations in different ethnic communities, none of the assays developed for screening mutations/deletions in a population defined can be adapted to study the possible genetic defects. The present study was designed to develop a new, multiplex PCR-based, molecular biological method to screen the whole coding region of the LDL receptor gene. METHODS: Using primer pairs completely flanking the promoter and the entire exonal region, in the PCR reactions 83-386-bp long, DNA sequences were synthesised in seven different reaction mixtures. The reaction conditions of the multiplex PCR system were optimised in order to synthesise all exons and the promoter region of the gene using only two annealing temperatures. The products could be visualised separately by agarose gel electrophoresis/ethidium bromide staining. RESULTS: A rapid, effective test enabling the screening of DNA alterations in the entire LDL receptor gene was developed. Using this simple multiplex PCR assay, deletions affecting more than 10 bp in any part of the gene can be easily detected by a single agarose gel electrophoresis. CONCLUSIONS: The simplicity, specificity and versatility of the assay make it suitable system for routine screening of LDL receptor gene mutations in large population samples. This PCR assay can be recommended for screening of LDL-RG deletions in populations or groups at high risk for cardiovascular diseases.

Electrophoresis, Agar Gel↗

PubMed2001

Phorbol ester treatment inhibits proliferation and differentiation of cultured human skeletal muscle satellite cells by differentially acting on protein kinase C isoforms.

We have previously shown that cultured human skeletal muscle cells express five protein kinase C (PKC) isoforms (PKCalpha, -gamma, -eta, -theta, and -zeta) and that expression levels of various PKC isozymes differentially change during differentiation. In this study we investigated the effects of the PKC activator phorbol 12-myristate 13-acetate (PMA) on differentiation and on PKC isozymes of human skeletal muscle satellite cells. PMA inhibited the growth and fusion of cultured human myoblasts in a dose-dependent manner. In addition, prolonged treatment of cells with PMA suppressed the expression of the myogenic differentiation marker desmin showing similar dose-response characteristics. Furthermore, PMA also induced the intracellular translocation of PKCgamma, -eta, and -theta, whereas cellular localization of PKCalpha and -zeta were not altered. These changes in subcellular localization patterns were of great importance since only those PKC isoforms were translocated that possessed alterations in their expression levels during differentiation. Our findings, therefore, suggest that the PMA-induced inhibition of differentiation of human skeletal muscle cells is mediated by certain PKC isoforms. Moreover, these data strongly argue for differential and isozyme-specific roles of various PKC isoforms in these processes.

Biological Transport↗

PubMed2001

Analysis of genetic alterations in salivary gland tumors by comparative genomic hybridization.

In order to define and map chromosomal copy number alterations in salivary gland tumors (SGTs), a comparative genomic hybridization (CGH) technique was applied to two pleomorphic adenomas (PAs), one adenoid cystic carcinoma (ACC), and one basal cell adenocarcinoma (BCAC). The PAs exhibited regional copy number losses at 5q12.4-q14.1, 9q12-q21.13, and 16q11.2, as well as a gain at 20p12.1; among these, the losses at the 9q12-q21.11 and 16q11.2 regions were common to both PAs. The ACC showed overrepresentations of the entire regions of chromosomes 16 and 20, a regional gain at 22q12.3-q13.1, and no losses. In the BCAC, regional gains at 9p21.1-pter, 18q21.1-q22.3, and 22q11.23-q13.31 as well as losses at 2q24.2 and 4q25-q27 were seen; the gain at 22q12.3-q13.1 was common in both the ACC and the BCAC. These CGH data indicate that different genetic alterations are present in the different types of SGTs, and that the alterations involve several chromosomes. The discovery of common alterations in the same and/or different types of tumors might be important in the understanding of the development and progression of the SGTs.

Adenoma, Pleomorphic↗

PubMed2001

Factor XIII of blood coagulation as a nuclear crosslinking enzyme.

Intracellular localization and distribution of Factor XIII subunit A (FXIIIA) was investigated in association with monocyte-macrophage differentiation in a long term culture of human monocytes by light- and electron microscopical as well as biochemical and immunobiochemical techniques. To allow the detection of FXIIIA in cells with well-preserved ultrustructure, immunosera against glutaraldehyde-derivatized recombinant FXIIIA were developed in rabbits, then characterized and used in this study. In the early phase of macrophage differentiation intranuclear accumulation of FXIIIA was detected as a transient phenomenon in cells of the 2nd day culture by optical sectioning with 0,7 microm steps in laser scanning confocal microscopy and immunoblotting technique. FXIIIA could be detected by immunoelectron microscopic postembedding staining over electrodense DNA-containing areas. Fluoresceinated monodansylcadaverine incorporation assay was used to demonstrate that FXIIIA is not only present in the nuclei, but also expresses its transglutaminase activity. Our finding of the nuclear accumulation of FXIIIA in differentiating human macrophages is also unique in that a blood clotting factor has, for the first time, been localized in nuclei and has been shown to be an intracellular crosslinking enzyme. The possible role of nuclear FXIIIA in association with cellular processes involving chromatin structure remodeling, such as cell death, cell differentiation or cellular proliferation requires further in-depth investigation.

Cell Culture Techniques↗

PubMed2000

[Fluorescence in situ hybridization in the diagnosis and follow-up of chronic myelogenous leukemia].

Chronic myelogenous leukaemia is a clonal myeloproliferative stem cell disease. Its cytogenetical hallmark is the Philadelphia chromosome (Ph) or the BCR/ABL fusion gene. Their identification is important both in the diagnosis and the follow-up of the disease. In our department we have investigated the BCR/ABL gene arrangement in 21 patients with fluorescence in situ hybridization. The aim of the analysis in freshly suspected patients without any previous therapy was to confirm diagnosis and mapping the ratio of Philadelphia positive cells. In contrast to the 95-100% Ph-positivity of mononuclear cells by classical cytogenetical examinations we found BCR/ABL gene arrangement only in various but always lower proportions. Therefore the latter examination gives a better representation of residual normal hemopoesis. Out of 9 patients who had received interferon treatment for at least 6 months, 4 gave a major, 4 a minor cytogenetical answer and in 1 case there was no cytogenetical response. Seven patients reached a complete and 2 a partial hematological remission. Among 5 other patients receiving interferon treatment, in 2 cases with double Ph-positivity we found a rapid progression. The data of 3 patients had to be excluded from the evaluation due to the so far short following time.

Diagnosis, Differential↗

PubMed2000

Effect of tumor-conditioned medium on intercellular communication and proliferation of Balb/c 3T3 cells.

The possible role of tumor cell-derived factors in the regulation of gap junctional intercellular communication and proliferation of fibroblasts was studied in a model system of Balb/c 3T3 cells growing in tumor conditioned medium by Lucifer Yellow CH dye-transfer and BrdU incorporation assays. Six to 24 h incubation of Balb/c 3T3 cells in a medium conditioned by WiDr adenocarcinoma cells enhanced the gap junctional communication between the cells by 25-40% as revealed by intercellular transfer of a fluorescent dye Lucifer Yellow CH. Simultaneously the cell proliferation rates were examined and found to be reduced by 23% at 24 h treatment. Since adenocarcinoma cells are known to secrete different growth factor-like polypeptides into their conditioned medium, we suppose that tumors that produce these molecules might alter their host environment through the enhancement of cell-cell communication thereby facilitating the exchange of modulatory factors.

3T3 Cells↗

PubMed2000

[Trends of premature mortality from cardiovascular diseases in Hungary and the European Union, 1970-1997].

It is a well known fact that in Hungary--as in the European Countries, in general--the trends of premature mortality are mainly determined by the trends of mortality caused by cardiovascular diseases. The timeliness of our present study on changes in trends of early cardiovascular mortality in the period of 1970-1997 in Hungary in comparison with trends of EU countries is underlined by the upcoming access of Hungary to the European Union. The evaluation is based on WHO data, the relative risk of premature mortality due to different forms of cardiovascular diseases for different sex and age groups of the Hungarian population is expressed as a ratio between standardized mortality rates of Hungarian groups and that of EU-average and of countries specified. Detailed data demonstrate that the risk of early death caused by cardiovascular--especially ischaemic heart and cerebrovascular--diseases, in contrast with EU countries, is significantly increased in Hungary, most significantly in age groups of 35-44 and 45-64 years for both sexes. The authors' results draw the attention to the possible shortcomings of health care systems and the lack of comprehensive health promotion (including prevention) programs in Hungary.

Adolescent↗

PubMed2000

Mutation of the RB1 gene caused unilateral retinoblastoma in early Age.

Fluorescence in situ hybridization (FISH) was applied for the detection of the retinoblastoma tumor-suppressor gene deletion on retinoblastoma tumor cells obtained from the unilateral tumor of a 3-month-old boy. Both retinoblastoma tumor cells and peripheral lymphocytes of the patient showed one hybridization signal per cell at the retinoblastoma-1 locus, indicating that one copy of the gene was deleted. Peripheral blood lymphocytes obtained from the patient's parents had two copies per cell for the gene. Retinoblastoma nuclear phosphoprotein expression could not be detected in the tumor tissue. No copy number alterations were detected with ten different centromeric DNA probes in the tumor cells. The deletion at the RB1 locus detected by FISH suggested that this gene alteration was heritable. The parental peripheral blood lymphocytes did not show the loss of the gene; thus the first deletion may have taken place in either of the parental germ cells. The second somatic mutation of the RB1 gene was probably under the detection limit of FISH. The second allelic alterations were detected by using the polymerase chain reaction for all exons of the retinoblastoma gene.

Genes, Retinoblastoma↗

PubMed2000

Stage-related superoxide anion production of granulocytes of gynecologic cancer patients.

OBJECTIVE: To measure superoxide anion production of polymorphonuclear leukocytes (PMNLs) in 58 gynecologic cancer patients and compare to that of healthy controls. METHODS: PMNLs were separated from peripheral blood samples by Ficoll and subsequent Percoll gradient sedimentation. Baseline and phorbol-dibutyrate (100 nmol/l) stimulated superoxide anion production was measured spectrophotometrically as superoxide dismutase inhibitable reduction of ferricytochrome c (50 micromol/l) absorbance. Differences between the mean superoxide anion production of different patient groups and the control group were assessed by Student's t-test. RESULTS: The mean superoxide anion production of PMNLs of healthy controls was 1.855 nM/min/3 x 10(5) cells (SD=0.211 nM/min/3 x 10(5) cells). Superoxide anion production of gynecologic cancer patients and healthy controls varied in a wide range. PMNLs of patients had lower baseline and stimulated activity than those of healthy volunteers. The frequency of a mean superoxide production at least 2 x SD below the control showed a parallel increase with advancing stage. CONCLUSION: Granulocytes of gynecologic cancer patients have reduced capacity and inducibility of superoxide anion production already at an early stage of disease.

Anions↗

PubMed2000

Liver metastatic ability of human melanoma cell line is associated with losses of chromosomes 4, 9p21-pter and 10p.

Genetic changes underlying the aggressive progression of human cutaneous melanoma are not completely understood. In order to characterise genetic alterations associated with the metastatic behaviour of this neoplasm we used comparative genomic hybridisation (CGH) in combination with fluorescence in situ hybridisation (FISH) on an experimental metastatic model of three related human melanoma cell lines. Tumour lines were selected based on their various metastatic capacity to liver in immunosuppressed mice. The parental cell line (A2058) was a human amelanotic melanoma cell line, adaptation of this line to in vivo growth as xenograft the HT168 tumour and its cell line was established. After intrasplenic transplantation of HT168 cells into immunosuppressed mice, a highly metastatic variant (HT168-M1) was selected. Several chromosomal aberrations common to all three lines indicating common clonal origin, as well as additional non-shared chromosomal changes were found. The original cell line (A2058) exhibited the highest number of genetic changes. Chromosomal alterations present only in the highly metastatic line (HT168-M1) involved losses on chromosome 4, 9p21.3-pter and 10p. Chromosome copy number patterns and the nature of chromosome 4 loss were further investigated by FISH using different centromeric probes and a chromosome 4 painting probe. According to our CGH and FISH results we assume that alterations present only in the aggressive metastatic subline are associated with the increased - metastatic potential. Our observations further support the hypothesis, based on some recently published data, that certain (so far unidentified) suppressor genes having an important role in tumour progression are located on these chromosomes.

Animals↗

PubMed2000

Characteristic distribution patterns of tenascin in laryngeal and hypopharyngeal cancers.

OBJECTIVES: Progression of malignant neoplasias is accompanied by alteration of the extracellular matrix (ECM) composition. Tenascin is known as a member of the adhesion-modulating family of ECM macromolecules; thus its expression and distribution may have significant influence on tumor cell proliferation and invasiveness. STUDY DESIGN: The present study was carried out to determine the distribution pattern of tenascin in laryngeal and hypopharyngeal cancer samples. METHODS: In double and triple immunofluorescent staining reactions the detection of tenascin was combined with labelings for cytokeratin (marker protein of epithelial cells), for CD-34 (endothelial cell surface glycoprotein), and for a reaction with Ki-67 monoclonal antibody (nuclear antigen in proliferating cells). RESULTS: In laryngeal cancers, in early stages of tumor growth a markedly enhanced production of tenascin at the tumor host interphase was observed. In the later stages of tumor progression, a high number of blood vessels located in the tumorous tissues were also strongly labeled for tenascin. Around these vessels a significant number of proliferating tumor cells could be detected. In contrast, in hypopharyngeal cancers this vasculature-associated staining pattern could be observed from the very early stage of tumor development. In laryngeal and in hypopharyngeal cancers, tenascin upregulation strongly correlated with metastasis formation, early tumor recurrence, and lethal outcome of the disease. CONCLUSIONS: Clinical and immunohistologic data indicate that the accumulation of tenascin in the tumor blood vessels is an unfavorable prognostic indicator in laryngeal and hypopharyngeal cancers.

Adult↗

PubMed1999

Involvement of chromosome losses in the progression and metastasis formation of a human malignant melanoma.

To characterize the possible cytogenetic link between a primary tumor and its metastasis, interphase cytogenetic analysis was performed on tumor cells and cutaneous metastasis from a male patient with malignant melanoma by using fluorescence in situ hybridization. The numbers of distinct hybridization domains specific for ten different pericentromeric sequences were used as indicators of copy numbers of these chromosomes. In the primary tumor, the majority of cells had two copies of these chromosomes, but significant numbers of nuclei also were present with one and three copies. In addition, in almost all cells, both sex chromosomes were abnormal; nullisomy of the Y chromosome was associated with X disomy. The corresponding metastatic tumor cells were predominantly monosomic; only the distribution of chromosomes 11 and 7 was similar to the primary tumor. In the metastatic tumor, the sex chromosomes had a normal copy number; that is, one Y and one X were detected. These data indicate that both the initiation and the progression of this melanoma are associated with chromosome losses.

Aged↗

PubMed1998

Single-cell measurement of superoxide anion and hydrogen peroxide production by human neutrophils with digital imaging fluorescence microscopy.

Besides flow cytometry, fluorescence microscopy combined with computerized image analysis offers an alternative tool for assessing phagocyte oxidant generation at the single-cell level. This technique provides an opportunity for the direct visualization of cells and simultaneous measurement of cellular fluorescence intensity. Thus, we developed a simple method for the quantitative evaluation of intracellular superoxide anion and hydrogen peroxide production with image cytometry by using hydroethidine and dihydrorhodamine 123 dyes, respectively. Human neutrophils stimulated with phorbol dibutyrate and labeled by these fluorogenic substrates showed intense, well recognizable red or green fluorescence. The intensity of signals from individual granulocytes of cytospin preparations were quantitatively measured in digitized images. There was a great heterogeneity in response to the stimulus within the granulocyte population as shown by the integrated fluorescence intensity values. In agreement with the results of parallel flow cytometric experiments, this simple image analysis performed on cells of cytospin preparations was able to detect the defects in the oxidative metabolism of neutrophils from patients with cervix carcinoma. We demonstrated that even minor alterations in superoxide anion/hydrogen peroxide generation can be detected by image cytometry as efficiently as by flow cytometry. This result validates imaging microscopy as an alternative to flow cytometry in such experiments. In addition, the image cytometric technique allows the observation of the kinetics of free radical production in individual cell under adherent conditions. Therefore, we carried out image analysis of the oxidative burst of neutrophils adherent to uncoated glass and fibronectin- and type IV collagen-coated surfaces in response to stimulation with phorbol dibutyrate or N-formyl-methionyl-leucyl-phenylalanine. We elaborated a calibration technique for the quantitative measurement of the ethidium bromide generation mediated by superoxide anion within individual adherent granulocytes. The ethidium bromide production varied between 0.48 and 1.17 amol/cell/min.

Anions↗

PubMed1998

Expression of invasion markers CD44v6/v3, NM23 and MMP2 in laryngeal and hypopharyngeal carcinoma.

Twelve laryngeal squamous cell carcinoma cases (7 laryngeal and 5 hypopharyngeal cancer; 15 samples) were analysed by immunohistochemistry for the expression of invasion markers CD44v6/v3, NM23 and matrix metalloproteinase, MMP2. The laryngeal epithelium showed CD44v6+/v3+/NM23-/MMP2- phenotype. When tumors were grouped into TNM categories the phenotype of the T2 and T3 tumors was similar, characterised by decreased CD44v3+ and lack of MMP2 expressions. Meanwhile the NM23 expression was more frequent in T3 tumors. In T4 stage the frequency of NM23 and MMP2 positive cases increased (5/6 and 4/6, respectively) but there was no correlation with the appearence of lymph node metastasis. Comparison of the phenotype of laryngeal and hypopharyngeal tumors, irrespective of the TNM stages, revealed characteristic differences: T2 stage laryngeal tumors showed decreased CD44v3 and occasional NM23 and MMP2 positivity, while in T3 stage these tumors were characterised by increased frequency of NM23 positivity. The phenotype of the hypopharyngeal tumors was significantly different with a high frequency of MMP2 positive cases (5/6) and NM23+/low CD44v3+ phenotype. The sharp differences in the phenotypes of laryngeal and hypopharyngeal carcinomas were connected to the differences in their invasive capacity unlike to the size of the tumors, since the T4 stage hypopharyngeal tumors had a significantly smaller size than laryngeal ones, even at lower stages.

Antigens, Neoplasm↗

PubMed1998

Factor XIIIa as a nerve-associated transglutaminase.

Recent findings have led to changes in the traditional concept of nerve recovery, including the realization that injured nerves, like any other injured tissue, need the assistance of blood-derived cells and factors in order to heal. We show that factor XIIIa (FXIIIa, the potentially active a2subunit of factor XIII), an enzyme that participates in blood coagulation by stabilizing the fibrin clot, is also active in the nervous system where it may play a key role in the healing of injured tissue. We demonstrate that the plasma, macrophages and nerves of fish contain a 55 kDa form of transglutaminase that cross-reacts immunologically with the a-subunit of FXIII in mammals (80 kDa). The fish enzyme in the plasma, unlike its mammalian counterpart, is active, pointing to a difference in control of the coagulation pathway in the two species. Analysis of FXIIIa expression in mammalian neural tissues and their response to injury revealed high levels of the enzyme in media conditioned by peripheral nerves as compared with medium conditioned by nerves of the central nervous system. Furthermore, similarity was observed in the postinjury behavior of FXIIIa in regenerating nerve tissues (peripheral nervous system of mammals and the central nervous system of fish). We suggest that the postinjury level of factor XIIIa in the nervous system may be related to the tissue's regenerative capacity, and that FXIIIa may therefore be a link underlying a possible association between the processes of blood coagulation and nerve healing.

Animals↗