Tumor vaccine composed of C-class CpG oligodeoxynucleotides and irradiated tumor cells induces long-term antitumor immunity

<p>Abstract</p> <p>Background</p> <p>An ideal tumor vaccine should activate both effector and memory immune response against tumor-specific antigens. Beside the CD8+ T cells that play a central role in the generation of a protective immune response and of long-term memory, dendritic cells (DCs) are important for the induction, coordination and regulation of the adaptive immune response. The DCs can conduct all of the elements of the immune orchestra and are therefore a fundamental target and tool for vaccination. The present study was aimed at assessing the ability of tumor vaccine composed of C-class CpG ODNs and irradiated melanoma tumor cells B16F1 followed by two additional injections of CpG ODNs to induce the generation of a functional long-term memory response in experimental tumor model in mice (i.p. B16F1).</p> <p>Results</p> <p>It has been shown that the functional memory response in vaccinated mice persists for at least 60 days after the last vaccination. Repeated vaccination also improves the survival of experimental animals compared to single vaccination, whereas the proportion of animals totally protected from the development of aggressive i.p. B16F1 tumors after vaccination repeated three times varies between 88.9%-100.0%. Additionally, the long-term immune memory and tumor protection is maintained over a prolonged period of time of at least 8 months. Finally, it has been demonstrated that following the vaccination the tumor-specific memory cells predominantly reside in bone marrow and peritoneal tissue and are in a more active state than their splenic counterparts.</p> <p>Conclusions</p> <p>In this study we demonstrated that tumor vaccine composed of C-class CpG ODNs and irradiated tumor cells followed by two additional injections of CpG ODNs induces a long-term immunity against aggressive B16F1 tumors.</p

Detection of V600E Mutation in BRAF Gene

Mutacija V600E predstavlja več kot 90 % vseh opisanih mutacij v genu BRAF pri različnih vrstah tumorjev. Protein, ki nastane kot produkt onkogena BRAF z mutacijo V600E (BrafV600E), sproža nenehno signaliziranje prek signalne poti RAS-RAF-MAPK, kar povzroči številnejše delitve celice in njeno maligno transformacijo. Zato ima onkogen BrafV600E pomembno vlogo pri indukciji in napredovanju tumorja ter verjetno predstavlja zgodnji dogodek v procesu maligne transformacije. Po podatkih iz literature je čezmerno izražen v različnih vrstah solidnih tumorjev, kot so melanom, metastatski rak debelega črevesa in danke, papilarni rak ščitnice, rak ledvic (RCC), hepatocelularni karcinom (HCC), velikocelični rak pljuč (NSCLC) in serozni rak jajčnikov. Na Oddelku za molekularno diagnostiko smo uvedli metodo za določanje mutacije V600E v genu BRAF. Metoda temelji na PCR-pomnoževanju in uporabi specifičnih sond. Izkazala se je kot primerna za rutinsko diagnostiko. V primerjavi z neposrednim sekveniranjem, ki velja za zlati standard, sta bili njeni občutljivost in specifičnost 100-odstotni. Zaradi velike specifičnosti je zanesljiva za ločevanje med normalnim in mutiranim genotipom BRAF in je primerna za hitro rutinsko diagnostiko.The most common BRAF mutation, which accounts for more than 90% of all BRAF mutations described in different malignancies, is a glutamic acid for valin substitution at position 600 (V600E). BRAF gene carrying V600E mutation is termed BrafV600E oncogene. The product of BrafV600E is a protein which induces constitutive signaling in cells through hyperactivation of the RAS-RAF-MAPK pathway leading finally to increased cellular proliferation and malignant transformation. BrafV600E oncogene thus plays an important role in cancer induction and progression and is probably mutated early in the process of malignant transformation. According to the literature, BrafV600E oncogene is over-expressed in diverse human solid tumors: melanoma, colorectal carcinoma, papillary thyroid carcinoma, renal cell carcinoma (RCC), hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC) and serous ovarian cancer. At the Department of Molecular Diagnostics, we introduced a method for the detection of V600E mutation in BRAF gene based on real-time PCR and on application of specific probes. The method is robust and convenient for routine diagnostics. Its sensitivity and specificity when determined in comparison to the results of direct sequencing are as high as 100%. Due to the high specificity, the method allows the discrimination between normal and mutated BRAF genotypes and is therefore suitable for fast and accurate routine diagnostics

Dokazovanje mutacij v genu BRCA1 z analizo talitvene krivulje produktov PCR

Rak dojke je z nekaj več kot 1000 novimi bolnicami na leto na prvem mestu po številu obolelih žensk za rakastimi boleznimi v Sloveniji. številka vključuje sporadične in dedne oblike raka na dojki. Dedne oblike raka na dojki so najpogosteje povezane z mutacijami v genih BRCA1 in BRCA2, zato se številni strokovnjaki s tega področja zavzemajo za genetsko testiranje bolnikov, pri katerih obstaja sum na dedno obliko bolezni. Testiranje bolnic/-kov in njihovih sorodnikov je dokaj težavno zaradi zahtevnosti metod genetskega testiranja, stroškov testiranja ter nezadostnega poznavanja in predvidevanja vseh možnih vplivov dokazane mutacije pri nastanku raka na dojki. V tem delu predstavljamo osnovne metodološke podatke za odkrivanje petih različnih mutacij v genu BRCA1 pri bolnicah/-kih s karcinomom dojke in njihovih sorodnikih. Mutacije 1806C>T, 300T>G, 300T>A, 310G>A, 5382insC določamo s polimerazno verižno reakcijo (PCR) v realnem času in analizo talitvene krivulje. Primerjava z neposrednim sekveniranjem je pokazala, da je uporabljena metoda dovolj občutljiva in hitra za dnevno rutinsko določanje mutacij v DNA, izolirani iz periferne krvi

Screening for melanoma susceptibility gene mutations in patients with familial cutaneous melanoma

Gensko testiranje za dedno obliko malignega melanoma kože na OI Ljubljana opravljamo od leta 2005. Izbor bolnikov za testiranje poteka v okviru genetskega svetovanja. Testiramo bolnike in njihove zdrave sorodnike, če sta v družini najmanj dva obolela člana, in bolnike s primarnimi multiplimi melanomi brez obremenjujoče družinske anamneze. Na oddelku za molekularno diagnostiko z metodo sekvenčne analize iščemo in določamo neznane točkovne mutacije ter manjše delecije in insercije v eksonih genov, povezanih z nastankom malignega melanoma kože: CDKN2A, CDK4 in MC1R. Do sedaj smo testirali 70 oseb: 40 bolnikov in njihovih zdravih sorodnikov iz 28 različnih družin, obremenjenih z družinsko anamnezo, ter 30 bolnikov s primarnimi multiplimi melanomi. Sekvenčna analiza genov, povezanih z dedno obliko malignega melanoma kože, je pokazala, da so mutacije CDKN2A p16INK4a pri bolnikih z družinsko anamnezo zelo pogoste (37,5 %). Mutacij CDKN2A p14ARF in CDK4 pri slovenskih bolnikih nismo našli.At the Institute of Oncology Ljubljana, Department of Molecular Diagnostics, genetic testing for familial cutaneous melanoma (CM) was started in 2005. The patients are selected according to the guidelines of genetic counseling for CM. The screening for melanoma susceptibility genes is performed in the patients and their healthy relatives from the families with at least two affected family members and in the patients with multiple primary CM without family history. The mutation screening for melanoma susceptibility genes – CDKN2A, CDK4 and MC1R is performed by direct sequencing. Altogether 70 patients and their healthy relatives (40 patients or their relatives selected from 28 families after considering their on family history and 30 patients with multiple primary CM) have been tested so far. Our results showed high prevalence of CDKN2A p16INK4a mutations in the patients with familial CM. In CDKN2A p14ARF and CDK4, no mutations were detected in Slovenian patients

Determination of Lymphoid Proliferation Clonality – A New Method in Comparison to the in House Method

Na Oddelku za molekularno diagnostiko smo na področju diagnostike limfomov uvedli dodatno metodo za določanje klonalnosti limfoidnih proliferacij. Tudi nova metoda temelji na verižni reakciji s polimerazo (PCR) in na uporabi različnih konsenzusnih oligonukleotidnih začetnikov. Metoda je validirana in registrirana za diagnostične potrebe (oznaka CE). Z razširjenim naborom oligonukleotidnih začetnikov in uporabo večjega števila začetnikov smo, po naših izračunih, izboljšali občutljivost določanja klonalnosti limfocitov B za 23,1 %, določanje klonalnosti limfocitov T pa za 14,3 %. Teoretično lahko sedaj z obema metodama določimo monoklonalno populacijo limfocitov pri 84,6 % B- in 85,7 % T-limfomovAt the Department of Molecular Diagnostics, a new method for determining lymphoid proliferation clonality has been introduced in lymphoma diagnostics. The method is based on PCR and on the use of various consensus primers. It is validated and registered for the use in clinical diagnostics (CE-marked method). Our measurements confirmed that the use of extended number of primers improved the sensitivity of determining the lymphoid clonality of B-lymphocytes and T-lymphocytes by 23.1% and 14.3%, respectively. Both methods may, theoretically, be used in determining monoclonal population of lymphocytes in 84.6% of B- and 85.8 of T-lymphomas

Prevalence of variations in melanoma susceptibility genes among Slovenian melanoma families

<p>Abstract</p> <p>Background</p> <p>Two high-risk genes have been implicated in the development of CM (cutaneous melanoma). Germline mutations of the CDKN2A gene are found in < 25% of melanoma-prone families and there are only seven families with mutation of the <it>CDK4 </it>gene reported to date. Beside those high penetrance genes, certain allelic variants of the <it>MC1R </it>gene modify the risk of developing the disease.</p> <p>The aims of our study were: to determine the prevalence of germline <it>CDKN2A </it>mutations and variants in members of families with familial CM and in patients with multiple primary CM; to search for possible <it>CDK4 </it>mutations, and to determine the frequency of variations in the <it>MC1R </it>gene.</p> <p>Methods</p> <p>From January 2001 until January 2007, 64 individuals were included in the study. The group included 28 patients and 7 healthy relatives belonging to 25 families, 26 patients with multiple primary tumors and 3 children with CM. Additionally 54 healthy individuals were included as a control group. Mutations and variants of the melanoma susceptibility genes were identified by direct sequencing.</p> <p>Results</p> <p>Seven families with CDKN2A mutations were discovered (7/25 or 28.0%). The L94Q mutation found in one family had not been previously reported in other populations. The D84N variant, with possible biological impact, was discovered in the case of patient without family history but with multiple primary CM. Only one mutation carrier was found in the control group. Further analysis revealed that c.540C>T heterozygous carriers were more common in the group of CM patients and their healthy relatives (11/64 vs. 2/54). One p14ARF variant was discovered in the control group and no mutations of the <it>CDK4 </it>gene were found.</p> <p>Most frequently found variants of the <it>MC1R </it>gene were T314T, V60L, V92M, R151C, R160W and R163Q with frequencies slightly higher in the group of patients and their relatives than in the group of controls, but the difference was statistically insignificant.</p> <p>Conclusion</p> <p>The present study has shown high prevalence of p16INK4A mutations in Slovenian population of familial melanoma patients (37%) and an absence of p14ARF or <it>CDK4 </it>mutations.</p

Causes and Measures to Reduce Food Waste and Surplus Food Donation in Educational Institutions in Slovenia

Z odpadno hrano se srečujejo vse izobraževalne institucije, ki jim ostanki hrane pomenijo tako finančno breme kot tudi možnost za izobraževanje na tem področju. V teoretičnem delu smo preučili problematiko odpadne hrane po svetu in Sloveniji ter organizacijo prehrane na različnih ravneh izobraževanja in ugotovili, da je smotrno prioritetno ukrepanje na nižjih ravneh izobraževanja. Na višjih nivojih izobraževanja se organizacija in strežba obrokov oddaljujeta od osrednje upravljavske ekipe izobraževalnega zavoda. Pregledali smo možne vzroke za kopičenje odpadne hrane in ukrepe za preprečevanje nastanka odpadne hrane ter ugotovili, da na količino odpadne hrane vplivajo število neprevzetih obrokov, prevelike količine skuhane hrane, prevelike porcije, nepravilno shranjevanje živil ter prehranske preference. Za preprečevanje kopičenja presežkov hrane lahko izobraževalne institucije uvajajo fleksibilne porcije, vključujejo učence v proces oblikovanja jedilnikov, izobražujejo na področju presežne in odpadne hrane, izvajajo preglede stanja, postavijo dodatne zahteve za ponudnike ali zamenjajo ponudnika v javnem razpisu, končni ukrep pri ravnanju z neizogibno količino odpadne hrane pa je lahko šolski kompost ali vrt. Ugotovili smo tudi, da vrtci in osnovne šole že izvajajo več projektov kot srednje šole in visokošolski zavodi. V empiričnem delu smo preverjali razlike v vzrokih za nastanek odpadne hrane med izobraževalnimi institucijami in ugotovili, da srednje šole neprevzete obroke pogosteje dojemajo kot pomemben vzrok za kopičenje presežkov hrane kot ostale izobraževalne institucije. V osnovnih in srednjih šolah so bili neprevzeti obroki označeni kot najpomembnejši vzrok, medtem ko je bil v vrtcih ta večinoma neatraktivnost obroka, med ponudniki v visokošolskih zavodih pa nepravilno shranjevanje živil. Poizvedeli smo o možnostih uvajanja različnih ukrepov ter ugotovili, da se izobraževalne institucije najpogosteje poslužujejo fleksibilnih porcij. Ta ukrep zlasti izvaja največ osnovnih šol, medtem ko vsi vrtci kot ukrep uvajajo spodbujanje otrok k jedi. Srednje šole so označile, da presežke hrane večinoma ponudijo dijakom za domov, medtem ko so zaposleni v visokošolskih zavodih in ponudniki organizirane prehrane največkrat navedli, da za študente izobešajo plakate na temo odpadne hrane. Ker je doniranje presežkov hrane lahko učinkovit neposreden način zmanjševanja presežkov hrane in posledično povzročanja odpadne hrane, nas je v raziskavi zanimalo, kako pogosto izobraževalne institucije donirajo presežno hrano. Ugotovili smo, da se doniranje redko izvaja. Vrtci, osnovne šole in srednje šole so kot najpogostejši zadržek za doniranje presežkov navedle varnost živil in s tem povezano odgovornost v postopku donacije, medtem ko za visokošolske zavode največji zadržek predstavljata logistika in organizacija doniranja.All educational institutions are confronted with the issue of food waste, that is simultaneously representing both a financial burden and an opportunity for education in this field. In the theoretical part, we examined the problem of food waste worldwide and in Slovenia, as well as the organisation of meals at different levels of education. We identified the need to take priority action in lower of education. At higher levels of education, the organization and serving of meals is detached from the management team at the educational institution. Further, we examined possible causes of food waste accumulation and measures to prevent food waste and conclude that the number of unclaimed meals, too much food cooked in advance, overportioning of meals, improper storage of food and dietary preferences (including the appearance and attractiveness of food) all have an impact on the amount of food waste. In order to prevent the accumulation of surplus food, educational institutions can introduce flexible portions, involve students in the menu design process and testing new menus, educate on surplus food and food waste, through the implementation of a diary forms, additional requirements for providers and through the educational opportunities of school gardens and composting unavoidable inedible food waste. In this respect, we have also found that kindergartens and primary schools are already implementing more projects than secondary schools and higher education institutions. In the empirical part, we examined differences in the causes of food waste between educational institutions and found that secondary schools are more likely to perceive unclaimed meals as an important cause of food surplus accumulation compared to other educational institutions. In elementary and intermediate schools, unclaimed meals were identified as the most important cause. Kindergartens however, identified the attractiveness/appearance of the meal so the most important factor. Furthermore, among the organised meal providers in higher education institutions, improper storage of ingredients was emphasized. We further queried the existing possibilities and measures for surplus food and food waste reduction, where offering flexible portions are most commonly used in educational institutions. In particular, most elementary schools are implementing this measure, while all kindergartens actively encourage children to eat. Intermediate schools indicated that they often offer surplus food to students to take home, while employees of higher education institutions and organised meal providers most frequently indicated that they put up food waste educational posters aimed to students. As donations of surplus food can be an effective direct way to reduce food surpluses and consequently food waste, our survey also inquired as to how often educational institutions donate surplus food. Respondents indicate that food surplus donation is rarely done. Kindergartens, primary schools and secondary schools referred to food safety and the corresponding liability in the process as the most common cause of reluctance to donate, while for higher education institutions the logistics and organisation of the donation were the cause of the most common reluctance

Clonality analysis of lymphoid proliferations using the BIOMED-2 clonality assays: a single institution experience

BACKGROUND: Clonality determination in patients with lymphoproliferative disorders can improve the final diagnosis. The aim of our study was to evaluate the applicative value of standardized BIOMED-2 gene clonality assay protocols for the analysis of clonality of lymphocytes in a group of different lymphoid proliferations. MATERIALS AND METHODS. With this purpose, 121 specimens from 91 patients with suspected lymphoproliferations submitted for routine diagnostics from January to December 2011 were retrospectively analyzed. According to the final diagnosis, our series comprised 32 cases of B-cell lymphomas, 38 cases of non-Hodgkin’s T-cell lymphomas and 51 cases of reactive lymphoid proliferations. Clonality testing was performed using the BIOMED-2 clonality assays. RESULTS: The determined sensitivity of the TCR assay was 91.9%, while the sensitivity of the IGH assay was 74.2%. The determined specificity of the IGH assay was 73.3% in the group of lymphomas and 87.2% in the group of reactive lesions. The determined specificity of the TCR assay was 62.5% in the group of lymphomas and 54.3% in the group of reactive lesions. CONCLUSIONS: In the present study, we confirmed the utility of standardized BIOMED-2 clonality assays for the detection of clonality in a routine diagnostical setting of non-Hodgkin’s lymphomas. Reactions for the detection of the complete IGH rearrangements and reactions for the detection of the TCR rearrangements are a good choice for clonality testing of a wide range of lymphoid proliferations and specimen types while the reactions for the detection of incomplete IGH rearrangements have not shown any additional diagnostic value