A tünetmentes pikkelysömörös bőr pathológiás elváltozásainak szerepe a betegség fenotípusának megjelenésében = The contribution of inherent abnormalities of non-lesional skin to disease phenotype in psoriasis

Az elvégzett kutatás alapkutatás, amely jelentős része klinikai jellegű, a pikkelysömör bőrbetegség pathomechanizmusának megértését célozza, ezáltal terápiás célpontokat jelöl ki a betegség gyógyításában. Közvetlen hasznosítás az elmúlt 4 évben nem történt, de ez nem is volt kitűzött cél, azonban a betegség kialakulásával kapcsolatban számos új adatot szolgáltattunk, melyeknek egy része szakmánk vezető folyóirataiban publikációra is került. Az utóbbi év eredményei új távlatokat nyitottak további kutatásaink célkitűzéseihez. A pályázatban vázolt munkáktól, csak annyiban tértünk el, amennyiben bizonyos eredmények más területen való hasznosítása értelemszerűen kívánkozott, valamint amennyiben kutatásunk eredményei új, fontosnak ítélhető megválaszolandó kérdéseket tettek fel. A munka eddig megjelent dolgozatai mellett 2 cikk közvetlen benyújtás előtt, kettő pedig az írás fázisában van. 1 hallgató a pályázat ideje alatt ebből a munkából írt és szerzett PhD fokozatot, jelenleg 2 hallgató PhD-ja készül részben az itt elvégzett munkákat is tartalmazva. | Within the project we carried out basic research toward the understanding of psoriasis pathomechanisms. Our research carries the possibility of new therapies in this common skin disease. We have made considerable progress in understanding key mechanisms of keratinocyte hyperproliferation, the major cutaneous characteristic of this skin disease. Our research work went according to the proposed plan, minor changes were made as required by experimental data. Some of the results have already been published in leading journals of our field, we are about to submit two papers and two is in the process of writing. One PhD student who worked on the project has successfully defended her thesis and two are presently working on their theses

Detection of a rare CDKN2A intronic mutation in a Hungarian melanoma-prone family and its role in splicing regulation

The genetic predisposition to melanoma is quite heterogeneous. The major locus for melanoma predisposition is the cell cycle regulatory CDKN2A gene on chromosome 9p21 though alterations in it have been detected in only 20 40% of melanoma prone families. However, with regard to the frequency of melanoma-prone families linked to 9p21, the frequency of germline coding mutations of the CDKN2A gene is lower than expected. We set out to investigate whether the rare IVS1+37 G/C intronic mutation of the CDKN2A gene recently identified in a Hungarian melanoma prone family, influences mRNA splicing regulation. To this end, CDKN2A minigenes containing the wild type and the mutant intronic sequence were created and transfected into HeLa cells with the aim of study of the mRNA transcripts. The results revealed the emergence of a differential splicing pattern from the wild type and the mutant minigene, suggesting that this mutation may alter the splicing of CDKN2A primary mRNA and therefore might have a pathogenetic role in familial melanoma. We believe that these results confirm the importance of the identification and characterization of CDKN2A intronic mutations with a view to improvement of our understanding of the pathogenesis and the explanation of why the frequency of germline coding mutations of the CDKN2A gene is lower than expected in melanoma-prone families linked to chromosome 9p21

Melanoma-Derived BRAF(V600E) Mutation in Peritumoral Stromal Cells: Implications for in Vivo Cell Fusion

Melanoma often recurs in patients after the removal of the primary tumor, suggesting the presence of recurrent tumor-initiating cells that are undetectable using standard diagnostic methods. As cell fusion has been implicated to facilitate the alteration of a cell's phenotype, we hypothesized that cells in the peritumoral stroma having a stromal phenotype that initiate recurrent tumors might originate from the fusion of tumor and stromal cells. Here, we show that in patients with BRAF(V600E) melanoma, melanoma antigen recognized by T-cells (MART1)-negative peritumoral stromal cells express BRAF(V600E) protein. To confirm the presence of the oncogene at the genetic level, peritumoral stromal cells were microdissected and screened for the presence of BRAF(V600E) with a mutation-specific polymerase chain reaction. Interestingly, cells carrying the BRAF(V600E) mutation were not only found among cells surrounding the primary tumor but were also present in the stroma of melanoma metastases as well as in a histologically tumor-free re-excision sample from a patient who subsequently developed a local recurrence. We did not detect any BRAF(V600E) mutation or protein in the peritumoral stroma of BRAF(WT) melanoma. Therefore, our results suggest that peritumoral stromal cells contain melanoma-derived oncogenic information, potentially as a result of cell fusion. These hybrid cells display the phenotype of stromal cells and are therefore undetectable using routine histological assessments. Our results highlight the importance of genetic analyses and the application of mutation-specific antibodies in the identification of potentially recurrent-tumor-initiating cells, which may help better predict patient survival and disease outcome

504 UVB-induced expression of fast-responding genes is modulated by huCOP1 in keratinocytes

Ultraviolet (UV) B is the most prominent physical carcinogen in the environment leading to the development of various skin cancers. We have previously demonstrated that the human ortholog of the Arabidopsis thaliana constitutive photomorphogenesis 1 (COP1) protein, huCOP1, is expressed in keratinocytes in a UVB-regulated manner and is a negative regulator of p53 as a posttranslational modifier. However, it was not known whether huCOP1 plays a role in mediating the UVB-induced early transcriptional responses of human keratinocytes. To clarify this question we produced transgenic cell lines in which the expression of huCOP1 was stably silenced. Real-time RT-PCR array showed that the stable siRNA-mediated silencing of huCOP1 affects the UVB response of several genes within 2 h of irradiation, indicating that altered huCOP1 expression sensitizes the cells toward UVB. Pathway analysis identified a molecular network in which 13 of the 30 examined UVB-regulated genes were organized around three central proteins. Since the expression of the investigated genes was upregulated by UVB in the siCOP1 cell line, we hypothesize that huCOP1 is a repressor of the identified pathway. Several members of the network have been implicated previously in the pathogenesis of non-melanoma skin cancers; therefore, clarifying the role of huCOP1 in these skin diseases may have clinical relevance in the future

Ultraviolet B-dependent changes in the expression of fast-responding early genes is modulated by huCOP1 in keratinocytes

Ultraviolet (UV) B is the most prominent physical carcinogen in the environment leading to the development of various skin cancers. We have previously demonstrated that the human orthologue of the Arabidopsis thaliana constitutive photomorphogenesis (COP)1 protein, huCOP1, is expressed in keratinocytes in a UVB-regulated manner and is a negative regulator of p53 as a post-translational modifier. However, it was not known whether huCOP1 plays a role in mediating the UVB-induced early transcriptional responses of human keratinocytes. In this study we found that stable small-interfering (si) RNA-mediated silencing of huCOP1 affects the UVB response of several genes within 2 h of irradiation indicating that altered huCOP1 expression sensitizes the cells toward UVB. Pathway analysis identified a molecular network in which 13 out of the 30 examined UVB-regulated genes were organized around three central proteins. As the expression of the investigated genes was upregulated by UVB in the siCOP1 cell line, we hypothesize that huCOP1 is a repressor of the identified pathway. Several members of the network have been implicated previously in the pathogenesis of nonmelanoma skin cancers; therefore, clarifying the role of huCOP1 in these skin diseases may have clinical relevance in the future

Different activations of toll-like receptors and antimicrobial peptides in chronic rhinosinusitis with or without nasal polyposis

Background: Both up- and down-regulation of the Toll-like receptors (TLRs) and antimicrobial peptides (AMPs) of the sinonasal mucosa have already been associated with the pathogenesis of chronic rhinosinusitis with (CRSwNP) or without (CRSsNP) nasal polyps. Objective: Determination of the expression of all known TLR and several AMP genes and some selected proteins in association with allergy, asthma and aspirin intolerance (ASA) in CRS subgroups. Methods: Patients with CRSsNP (n=19), CRSwNP (ASA[-]:17; ASA[+]:7) and control subjects (n=12) were selected. Sinonasal samples were taken during endonasal surgery and RT-PCR was used to measure mRNA expressions of 10 TLRs, 4 defesins, lysozyme, cathelicidin and lactoferrin (LTF). In several cases immunohistochemistry was performed to evaluate protein expressions. Statistical analysis was done with the Kruskall-Wallis ANOVA, Mann-Whitney U and Student-t-test. Results: TLR2, TLR5, TLR6, TLR7, TLR8, TLR9, β-defensins 1 and 4, cathelicidin and LTF mRNA expressions were significantly (p<0.05) increased in CRSwNP, whereas only TLR2 and LTF were up-regulated in CRSsNP compared to controls. There was no statistical difference in respect of allergy, aspirin intolerance and smoking between CRSsNP, ASA(-) and ASA(+) CRSwNP patients. TLR2, TLR3, TLR4, LTF, β defensin 2 and lysozyme protein expression was found to be increased in CRSwNP stainings (p<0.05) dominantly in macrophages. Conclusions: Gene expression analysis showed markedly different expressions in CRSwNP (6 out of 10 TLR and 4 out of 7 AMP genes were up-regulated) compared to CRSsNP (1/10, 1/7). The distinct activation of the innate immunity may support the concept, that CRSsNP and CRSwNP are different endotypes of CRS. These findings were found to be independent from allergy, asthma, smoking, aspirin intolerance and systemic steroid application