Inhibition of nuclear factor-kappaB attenuates artherosclerosis in apoE/LDLR - double knockout mice.

Nuclear factor - kappaB (NF-kappaB) is a good therapeutic target for cardiovascular disease and numerous efforts are being made to develop safe NF-kappaB inhibitors. Nowadays many authors address NF-kappaB as a major therapeutic target in atherosclerosis, especially for preventive measures, in the light of two main hypothesis of atherosclerosis: oxidation and inflammation. We hypothesized that ammonium pyrrolidinedithioocarbamate (PDTC) - a well-known inhibitor of NF-kappaB could inhibit the development of atherosclerosis in this experimental model. We used apoE/LDLR - DKO mouse model, which is considered as a one of the best models to study the anti-atherosclerotic effect of drugs. In this model PDTC inhibited atherogenesis, measured both by "en face" method (25,15+/-2,9% vs. 15,63+/-0,6%) and "cross-section" method (565867+/-39764 microm2 vs. 291695+/-30384 microm2). Moreover, PDTC did not change the profile of cholesterol and triglycerides in blood. To our knowledge, this is the first report that shows the effect of PDTC on atherogenesis in gene-targeted apoE/LDLR - double knockout mice.

Effect of curcumin on atherosclerosis in apoE/LDLR-double knockout mice.

It is widely appreciated that inflammation and oxidant stress contribute to atherogenesis. Curcumin, a polyphenolic natural compound has been reported to possess anti-inflammatory and anti-oxidant actions. We hypothesized that curcumin could inhibit the development of atherosclerosis in the apoE/LDLR-double knockout mice fed with Western diet (21% fat, 0.15% cholesterol w/w, without cholic acid). Curcumin (purity>or=98%), premixed with diet, was given for 4 months at a dose of 0.3 mg/ per day/ per mouse. In this model curcumin inhibited atherogenesis, measured both by "en face" method (25,15+/-2,9% vs. 19,2+/-0,6%, p<0,05) and "cross-section" method (565867+/-39764 microm2 vs. 299201+/-20373 microm2, p<0,05). Importantly, curcumin influenced neither the concentrations of cholesterol and triglycerides in blood nor animal body weight. To our knowledge, this is the first report that shows the anti-atherogenic effect of low dose of curcumin in fine model of atherosclerosis: gene-targeted apoE/LDLR-double knockout mice.

GADD153 is an independent prognostic factor in melanoma: immunohistochemical and molecular genetic analysis.

The main role of growth arrest and DNA damage-inducible (GADD) genes is to block proliferation at G1 and G2 checkpoints in response to DNA damage. The goal of this study was to examine the expression of GADD genes in primary melanomas with respect to prognosis. GADD34 was found in 73% of the primary melanomas investigated. GADD45 and GADD153 were positive in 60% and 80% of primary melanomas, respectively. Cox regression demonstrated that only GADD153 had any independent prognostic impact. We therefore decided to establish a PCR assay for detection of GADD153 in paraffin-embedded tissue. GADD153 deletion was found in 3/26 melanomas. None of the 3 cases with GADD153 deletion showed any expression of GADD153. Sequencing analysis detected polymorphism T-C at amino acid position 10 in 6/23 melanomas. In 6 cases with GADD153 polymorphism, GADD153 expression was found in 2 melanomas with a maximum GADD153 index of 10%. We postulate that the GADD gene family plays an important role in melanoma progression.

Interleukin 16 expression and phenotype of interleukin 16 producing cells in Crohn's disease.

BACKGROUND: The mechanisms involved in the initiation and maintenance of Crohn's disease are poorly understood. Previous studies have demonstrated an increased number of infiltrating CD4+ T cells within the inflammatory affected bowel wall in Crohn's disease. Novel therapy approaches using anti-CD4 antibodies are thought to be effective in Crohn's disease. AIMS: Interleukin 16 (IL-16) has been characterised as a chemokine with selective chemoattraction for CD4+ inflammatory T cells. In this study, cellular expression of IL-16 in Crohn's disease and ulcerative colitis was investigated. METHODS: Expression of IL-16 was analysed in tissue samples of Crohn's disease, ulcerative colitis, and normal controls by applying reverse transcription-polymerase chain reaction, non-radioactive in situ hybridisation, and immunohistochemistry. Double staining methods were used to characterise cells expressing IL-16. The amount of infiltrating CD4+ cells was determined by immunohistochemistry and correlated with the corresponding IL-16+ cell number by step sections. RESULTS: An increased number of IL-16+ cells in Crohn's disease in comparison with ulcerative colitis and control probes was demonstrated. IL-16 was expressed by CD4 and CD8 positive T cells. In addition, in active Crohn's disease there was a substantial number of IL-16 positive mast cells. The increased number of CD4+ lymphocytes correlated positively with the increased number of IL-16 positive cells in Crohn's disease. CONCLUSION: Our results demonstrate that increased expression of IL-16 in T cells and mast cells in active Crohn's disease is associated with increased numbers of CD4+ lymphocytes. Local expression of IL-16 seems to play a significant role in the initiation and persistence of the inflammatory process in Crohn's disease, presumably by IL-16 mediated recruitment of CD4+ cells, mostly lymphocytes, into the bowel wall.

Relationship between DNA ploidy-related parameters and the deletions in mismatch repair genes MLH1 and MSH2 in lentigo maligna and malignant melanomas.

Defects in DNA mismatch repair genes MLH1 and MSH2, first described in hereditary nonpolyposis colon cancer (HNPCC), have been postulated to be responsible for malignant transformation in several tumours. To date there are no data on cutaneous tumours. Using a PCR assay it was possible to identify deletions in MSH2 (exonic regions 12 and 13) in 16 of 47 lentigos maligna and in 10 of 36 malignant melanomas. Deletions in MLH1 (exonic regions 15 and 16) were found in 11 of 47 lentigos and in 15 of 36 melanomas. Comparison of DNA ploidy-related parameters between lentigos with and without exonic deletions in MSH2 and MLH1 did not show any significant differences. In contrast, melanomas positive and negative for exons 12 and 13 (MSH2) (26/36 and 10/36, respectively) differed significantly with respect to the percentages of diploid cells (P = 0.0179) and tetraploid cells (P = 0.0042). Comparison of melanomas positive and negative for exons 15 and 16 (MLH1) (21/36 and 15/36, respectively) showed significant differences in the percentage of aneuploid cells between 2c and 4c (P = 0.0141) and tetraploid cells (P = 0.0404). In summary, deletions in DNA mismatch repair proteins MSH2 and MLH1 were present both in lentigo maligna and in melanomas and correlated with DNA ploidy-related parameters in malignant melanomas.

Relation between DNA ploidy status and the expression of the DNA-mismatch repair genes MLH1 and MSH2 in cytological specimens of melanoma lymph node and liver metastases.

DNA-mismatch repair is essential for preventing genetic instability, and its important protective role has been demonstrated in several tumors. The main aim of this study was to investigate the expression of MLH1 and MSH2 (on the RNA level) in melanoma liver and lymph node metastases, and to define the relation between DNA ploidy status and mismatch repair gene expression. MLH1 was found in 29/33 melanoma lymph node and in 5/17 melanoma liver metastases. MSH2 was present in 26/33 lymph node and 5/17 liver metastases. A comparison of MLH1 and MSH2 positive and negative melanoma metastases showed that there were highly significant differences in the percentages of diploid cells, aneuploid cells between 4c and 8c, octaploid cells, and 5c exceeding rate. This fact confirms the strong relation between the loss of DNA-mismatch repair gene expression and advanced DNA aneuploidy status in melanoma metastases.

Downregulation of the retinoblastoma gene expression in the progression of malignant melanoma.

The retinoblastoma gene is a cell cycle regulator preventing cells from entering into S-phase. An altered expression of the retinoblastoma gene has been reported in the majority of human malignancies. The main aim of this study was to investigate retinoblastoma gene expression in the full spectrum of melanoma progression from naevus to melanoma metastases by applying immunohistochemistry and RT-PCR. All naevi with and without dysplasia showed high expression of the retinoblastoma gene. In primary melanomas, Rb-positive cells were found in 82 out of 106. Loss of expression correlated with an increase in Clark level and shorter survival rates. An independent prognostic role of the retinoblastoma gene was confirmed by Cox multivariate analyses (p < 0.01). In melanoma metastases, retinoblastoma gene expression (at the RNA level) was found in 18 out of 26 melanoma lymphatic metastases, and in 2 out of 5 liver metastases. Our results indicate a downregulation of the retinoblastoma gene in the progression of melanocytic tumours.

Analysis of the DNA content in the progression of recurrent and metastatic melanomas.

Ploidy status and ploidy related parameters of 18 primary melanomas, 32 recurrences and 18 lymphatic metastases were investigated applying CAS200 image analyzer. All the tumours investigated were either suspicious for aneuploidy (Auer III) or clearly aneuploid (Auer IV). Primary melanomas differed from recurrent tumours concerning the percentage of aneuploid cells between 4c and 8c and 5c ER. Comparison of cutaneous tumours with lymphatic metastases showed a significant difference concerning the percentage of aneuploid cells between 2c and 4c. An already high aneuploidy rate in primary tumours suggests that recurrent and metastatic clones of cells are present in early stages and that aneuploidy status in primary melanomas could be regarded as one of the risk factors of recurrences and metastases.

Prognostic significance of newly defined ploidy related parameters in melanoma.

5c exceeding rate is the parameter, most frequently showing prognostic impact. The CAS200 image analyzer makes possible the measurement of additional parameters defining single subfractions of cells, as for example the ratios of diploid, aneuploid, tetraploid, octaploid and 16-ploid cells. The main objective of this study was to define the prognostic significance of these new parameters in 106 primary melanomas with known survival time. 29 out of 106 melanomas were euploid. 77 out of 106 showed an aneuploid histogram. Multivariate analysis with Cox regression demonstrated that the percentage of aneuploid cells between 2c and 4c and the percentage of aneuploid cells between 4c and 8c, but not 5c exceeding rate, were able to influence survival time.

Proliferative activity in the progression of pigmented skin lesions, diagnostic and prognostic significance.

Proliferative compartments of a tumour can be determined cytophotometrically, by in situ hybridisation or by immunohistochemical detection of Ki67 antigen. The main objective of this study was to analyse the proliferative activity during the progression of pigmented skin lesions with respect to differential diagnostic and prognostic applications. The material investigated consisted of 209 pigmented skin lesions (31 naevi, 30 dysplastic naevi, 106 primary melanomas, 20 lymphatic and 22 organ melanoma metastases). Comparison of the ratios of cells in the S-phase gained by two different methods (cytometry, in situ hybridisation) did not show any significant differences. The correlations between Ki67 and S-phase indices in every diagnostic group were highly significant. The results of forward and backward Cox regression were identical and only Ki67 showed an independent prognostic influence (p < 0.001, coefficient in regression 0.02) with change in risk 2% and confidence limit ranging between 1.1% and 2.9%.

Comparative study of the expression of DNA mismatch repair genes, the adenomatous polyposis coli gene and growth arrest DNA damage genes in melanoma recurrences and metastases.

The main goal of this study was to examine the expression of DNA mismatch repair genes (MLH1, MSH2, PMS1 and PMS2), the adenomatous polyposis coli (APC) gene and growth arrest DNA damage inducible (GADD) genes (GADD34, GADD45 and GADD153) in the different stages of melanoma recurrences and metastases, and to identify any mutual consistencies in their expression pattern. All the cases of primary melanoma examined showed a reduced expression of DNA repair genes. These results demonstrate that disturbances of DNA repair begin in the early stages of melanoma. No significant differences were found in the expression of these markers between cutaneous melanomas and their recurrences and metastases (P> 0.05). Eighteen significant correlations between markers were found in the primary melanomas, and 10 significant correlations were observed in the first recurrences of melanoma. In contrast, 27 statistically significant relationships were demonstrated in metastatic lymph nodes. The different correlations found in primary and metastatic tumours confirmed the hypothetical difference in marker interaction in the diagnostic groups investigated. Our results suggest that DNA repair genes may play an important role in the recurrence and metastasis of melanomas.

Analysis of the DNA mismatch repair proteins expression in malignant melanomas.

The importance of properly functioning DNA mismatch repair has been shown in several tumour types both hereditary and sporadic, but not yet in malignant melanomas. The aim of this study was to examine the expression of DNA mismatch repair genes (MLH1, MSH2, PMS1 and PMS2) in primary melanomas and to define their possible prognostic impact in 106 primary melanomas. MLH1 was found in 64 and MSH2 in 61 out of 106 melanomas. PMS1 and PMS2 proteins were present in 69 and 67 tumours, respectively. Loss of the expression of DNA mismatch repair proteins correlated with the increase of Clark levels. Cox regression analysis demonstrated some prognostic significance for PMS1 (forward p = 0.0018 and backward selections p = 0.0277), MLH1 (only forward selection p = 0.0081) and MSH2 (only backward selection p = 0.0115).

MAGE-1, GAGE-1/-2 gene expression in FNAB of classic variant of papillary thyroid carcinoma and papillary hyperplasia in nodular goiter.

Differentiation of the papillary variant of papillary thyroid carcinoma (PTC) from papillary hyperplasia in nodular goiter may be difficult in fine-needle aspiration biopsy (FNAB) by means of morphology alone. To improve cytodiagnostic accuracy the occurrence of MAGE-1, GAGE-1/-2 gene expression was analyzed by means of RT-PCR. The genes investigated are recognized by autologous T lymphocytes and are expressed in carcinomas of various sites e.g. lung, ovary, colon but not in non-neoplastic tissue except testis. Routinely obtained smears with cytologic diagnosis of PTC confirmed by histology (n=20) and diagnosis of nodular goiter (n=10) were investigated. The MAGE-1, GAGE-1/-2 PCR products were found in 6/20 of the carcinomas but in none of the benign lesions. To identify PCR products automatic gene-sequencing in all positive cases was performed. The data indicate that MAGE-1, GAGE-1/-2 gene expression may give additional information to delineate PTC from papillary hyperplasia in FNAB.

Expression of DNA mismatch repair genes in naevi.

The significant difference of DNA mismatch repair genes expression between naevi and melanomas was demonstrated by our research group in the previous study. The main aim of this study was to compare the expression of MLH1, MSH2, PMS1 and PMS2 in 31 naevus cell naevi, 12 fibromatous naevi and 30 dysplastic naevi. The expression of DNA mismatch repair proteins was found in all naevi investigated. However, the expression (percentage of positively stained cells) was significantly higher in naevus cell naevi than in dysplastic and fibromatous ones.

Relation between two independent DNA-repair pathways in different groups of naevi.

The relation between 2 DNA-repair systems was investigated in 3 groups of naevi (naevus cell naevi, dysplastic naevi, fibromatous naevi) using the correlation coefficient according to Spearman. In the group of naevus cell naevi, only 1 significant correlation between MSH2 and GADD34 expression was found. In the group of dysplastic naevi, 9 significant and highly significant correlations between GADD genes and mismatch repair genes were found. In the group of fibromatous naevi, MLH1 correlated significantly with GADD45 and highly significant with GADD34 expression. Different correlations in naevi groups investigated show the different functional connections between the genes of DNA repair.

Growth arrest DNA damage gene expression in naevi.

Thirty-one naevus cell naevi, 30 dysplastic naevi and 12 fibromatous naevi were stained for the presence of p53 and Growth Arrest DNA Damage genes. All naevus cell naevi and fibromatous naevi were highly positive for GADD genes and negative for p53. Dysplastic naevi had significantly lower GADD34 and GADD153 expression as well as higher p53 expression in relation to the other naevi groups. The absence or decrease of GADD genes expression in naevus may indicate a potential malignant transformation.

Bcl2 and Bax expression in naevi and melanomas and their relation to ploidy status and proliferation.

Twenty-five naevi, 53 primary melanomas, 36 melanoma metastases were stained immunohistochemically for the presence of Bcl2, Bax and Ki-67 antigen in order to define the relation between apoptosis regulators and proliferative activity. Additionally, ploidy status and S-phase were measured. Bax demonstrated a tendency to increase and Bcl2 was decreasing along with melanoma progression. In the group of euploid cases Bcl2 showed a strong significant correlation with Bax expression (p = 0.0001) and both Bcl2 and Bax correlated with Ki-67 expression and S-phase. In the group of aneuploid cases only the correlation Bcl2-Ki-67 was preserved. Others did not reach the level of statistical significance.

How can one explain aneuploidy status in fibromatous and lipomatous naevus cell naevus?

34 lightly fibromatous, 23 heavily fibromatous, 5 lipomatous and 10 naevus cell naevi were stained with Feulgen kit in order to evaluate their ploidy status with CAS 200 image analyzer. 26/34 lightly fibromatous, 18/23 heavily fibromatous, and 5/5 lipomatous naevi were either suspicious for aneuploidy (Auer III) or clearly aneuploid (Auer IV). In contrast all 10/10 naevus cell naevi were euploid. Proliferation (S-phase) was not increased in naevi fibromatously and lipomatously changed. The mechanisms leading to aneuploidy are discussed.

Loss of DNA-mismatch repair gene expression in oral melanomas.

The defect of DNA mismatch repair is postulated to be responsible for malignant transformation in many types of tumours. The main aim of this study was to evaluate the expression of DNA mismatch repair proteins in 29 cases of oral melanomas and to relate this to the ploidy status of the lesions. MLH1 expression was found in 4/29, MSH2 in 6/29, PMS2 in 2/29 and PMS1 in 0/29 cases investigated. The range of positively stained cells did not exceed 50% with MSH2, and PMS2, or 5% with MLH1. Loss of the DNA mismatch repair gene expression correlated with high aneuploidy ratio, observed in totally negative cases.

Loss of growth arrest DNA damage genes expression in oral melanomas.

29 oral melanomas were stained immunohistochemically for the GADD34, GADD45 and GADD153. GADD34 was found in 5/29 melanomas and its expression did not exceed 21%, averaging 4.1% of melanoma cells. GADD45 was observed in 8/29 oral melanomas and cell positivity averaged 2.8%. GADD153 was found in 2/29 melanomas and the percentage of positive cells ranged between 0 and 31%, averaging 1.2%. Not one significant correlation between GADD genes in oral melanomas was found. Loss of GADD gene expression and lack of correlation between them show advanced disturbances in their cooperation, leading to a high genetic instability of oral melanoma cells.