Zbornik - Društvo genetičara Srbije

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184 ZBORNIK ABSTRAKATA III KONGRESA GENETIÈARA SRBIJE V-Usm-1 Subotica, 30. novembar - 4. decembar 2004. ANALIZA KLONALNOSTI T-ÆELIJA U TUMORSKIM UZORCIMA BOLESNICA SA KARCINOMOM DOJKE Bojana Cikota 1 , Mirjana Brankoviæ-Magiæ 2 i Z. Magiæ 1 1 Institut za medicinska istraivanja, Vojnomedicinska akademija, Beograd 2 Institut za onkologiju i radiologiju <strong>Srbije</strong>, Beograd Rezultati brojnih istraivanja su pokazali da razlièiti tumori, ukljuèujuæi i tumore dojke, mogu stimulisati specifièni T-æelijski antitumorski odgovor. Prepoznavanje tumor-specifiènog antigena (kojeg tumorska æelija eksprimira endogeno ili egzogeno kao peptid prezentovan putem antigen-prezentujuæih æelija) rezultuje klonalnom ekspanzijom i diferencijacijom naivnih CD8 + T-æelija u aktivirane efektorske æelije koje uèestvuju u ubijanju tumorskih æelija. U ovom istraivanju je analizirana klonalnost T-æelija u tumorskim uzorcima 54 bolesnice sa karcinomom dojke. Na osnovu prisustva/odsustva metastaza u regionalnim limfnim èvorovima bolesnice su klasifikovane u grupu N+ (prisutne metastaze; n=25) ili N0 (bez metastaza; n=16). Analiza klonalnosti T-æelija se zasnivala na PCR amplifikaciji dela rearaniranog gena za lanac TCR-a i PAGE. Prisustvo monoklonalne/oligoklonalne (M/O) populacije T-æelija je detektovano u 9 bolesnica iz N+ i 6 bolesnica iz N0 grupe. U svim analiziranim grupama (N + +N0, N+, N0) nije uoèena statistièki znaèajna razlika u uèestalosti relapsa izmeðu bolesnica sa M/O u odnosu na bolesnice sa poliklonskom T-æelijskom populacijom. Poreðenjem duine perioda bez bolesti (DFI) tokom prve dve godine u bolesnica sa M/O T-æelijama u odnosu na bolesnice sa poliklonskom populacijom je dobijena statistièki znaèajna razlika graniène vrednosti samo u N+ grupi. Bolesnice sa M/O T-æelijama su imale kraæi DFI od bolesnica sa poliklonskom populacijom. Ova razlika nije uoèena kada se analiza zasnivala na periodu praæenja duem od 2 godine. Dobijeni rezultati ukazuju da su populacije tumor infiltrirajuæih limfocita uglavnom poliklonalne. ANALYSIS OF T-CELL CLONALITY PATTERN IN TUMOR SAMPLES OF BREAST CANCER PATIENTS Numerous experimental evidences confirm that different tumors, including breast carcinomas, can stimulate specific T-cell mediated immune response. Recognition of tumor-associated antigen (expressed endogenously by tumor cells or exogenously as peptide presented by antigen-presenting cells) results in the clonal expansion and differentiation of naive CD8+ T-cells into activated effector cells that mediate tumor cell killing. In this study we have analyzed pattern of T-cell clonality in tumor samples of 54 breast cancer patients classified as lymph node-negative – N0 (n=16) or lymph node-positive – N+ (n=25). Analysis of T-cell clonality was based on PCR amplification of rearranged TCR chain gene followed by PAGE. Monoclonal/oligoclonal (M/O) T-cell populations were found in 9 of N + and6ofN0 breast cancer patients, respectively. In all analyzed groups (N + +N0, N+, N0), incidence of relapse was not significantly different between patients with M/O T-cells vs patients with polyclonal T-cells. Comparison of disease free interval (DFI) between the patients divided according to the presence of TCRã monoclonality/oligoclonality showed board-line significant difference only in the group of N+ patients within first 24 months of follow-up. Patients with M/O T-cell population had shorter DFI than patients with polyclonal T-cell population. Concerning the complete follow-up in the same group of patients, this difference was not observed. Our results imply that tumor infiltrating T-cells are usually polyclonal.
V-Usm-2 ZBORNIK ABSTRAKATA III KONGRESA GENETIÈARA SRBIJE 185 Subotica, 30. novembar - 4. decembar 2004. MULTIPLEKS PCR KAO BRZ, SENZITIVAN I SPECIFIÈAN METOD ZA DETEKCIJU PATOGENIH MIKROORGANIZAMA Nataša Goliæ, L. Ranin, M. Kojiæ, Ivana Strahiniæ, Ð. Fira, Maja Vukašinoviæ, Amarela Terziæ-Vidojeviæ, Jelena Begoviæ, Jelena Lozo, Katarina Krstiæ, B. Jovèiæ, Maja Tolinaèki, Milica Nikoliæ i Lj. Topisiroviæ Institut za molekularnu genetiku i genetièko inenjerstvo, Beograd U poslednjih deset godina su razvijene razlièite metode za identifikaciju i tipizaciju prokariotskih i eukariotskih organizama na DNK nivou. PCR metod omoguæava brzu i specifiènu detekciju širokog spektra bakterijskih vrsta. Multipleks PCR je brz, senzitivan i specifièan metod za detekciju patogenih mikroorganizama u jednoj PCR reakciji. Metod je zasnovan na PCR reakciji u kojoj se koristi nekoliko parova prajmera. Jedan set prajmera je najèešæe specifièan za vrstu i potièe iz regiona 16S rDNK, te se na osnovu dobijenog produkta nedvosmisleno detektuje prisustvo specifiènog mikroorganizma. Drugi set prajmera je obièno specifièan za gene koji su prisutni samo kod patogenih serotipova. PCR metod je izuzetno senzitivan i moe da detektuje vrlo mali broj patogenih æelija u uzorku (oko 10 æelija). Štaviše, ovaj metod je brz i rezultati se dobijaju u toku jednog radnog dana. Dobijeni rezultati su nedvosmilseni. Prednost multipleks PCR metoda je da omoguæuje detekciju specifiènih mikroorganizama odmah nakon infekcije, pre pojave prvih simptoma i stvaranja antitela, kao i nakon infekcije kada je titar antitela blizu normalnih vrednosti (ELISA test). U ovom radu predstavljamo multipleks PCR, kao metod za detekciju Listeria monocytogenes, Yersinia enterocolitica, Helicobacter pylori, Mycoplasma pneumoniae, Chlamidia trachomatis, Chlamidia pneumonie, Campilobacter jejuni, Borrelia burgdoferi, i Brucella sp. iz klinièkih uzoraka. MULTIPLEX PCR AS A RAPID, SENSITIVE AND SPECIFIC METHOD FOR DETECTION OF PATHOGEN MICROORGANISMS In the past decade, various methods have been developed for the identification and typing of prokaryotic and eukaryotic organisms at the DNA level. The PCR approach allows rapid and specific detection of a wide range of bacterial species. Multiplex PCR is rapid, sensitive and specific method for detection of pathogen microorganisms in one PCR reaction. The method is based on PCR reaction in which several sets of primers are used. One set of primers is usually species specific, originated from 16S rDNA, that unumbiguos detect the presence of a specific microorganism. The other set of primers is commonly specific for the genes that are predominantly present in pathogen serotypes. PCR method is extremely sensitive and very low amount of pathogen cells in the sample could be detected (aproximatelly 10 cells). Moreover, this method is fast and the analysis could be completed in one working day. The results obtained by this method are unambiguos. The most important is that by using the multiplex PCR method it becomes possible to detect the presence of the specific microorganism immediately after infection, before the apearence of the first symptoms and the antibody generation, as well as after the infection when the titer of antibodies is close to referent values (ELISA test). In this work we describe the multiplex PCRs that are used for detection of Listeria monocytogenes, Yersinia enterocolitica, Helicobacter pylori, Mycoplasma pneumoniae, Chlamidia trachomatis, Chlamidia pneumonie, Campilobacter jejuni, Borrelia burgdoferi, and Brucella sp. from the clinical specimens.
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Zbornik - Društvo genetičara Srbije

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