Tumorspecifikus ellenanyagfragmens tisztítási és tesztelési eljárásainak kidolgozása a diagnosztikus alkalmazhatóság céljából = Development of purification and test procedures of tumorspecific immunoglobulin fragments for their further diagnostic use

A szolid tumorokban fellelhető B sejtek kiaknázása volt a cél. A tumort infiltráló B sejtek (TIL-B) tumorspecifikus antigénkötő kapacitását nemzetközileg is elsőként igazoltuk. Az eljárás diagnosztikus célú tumorfelismerő "jelölő molekulákat" eredményez. A tématervi munka sokrétű volt: 1/Új molekuláris biológiai és biotechnológiai módszerek, 2/ preparatív fehérjetisztítási munkák, 3/ összehasonlító DNS elemzések, 4/ különböző ellenanyag - antigén kötődési tesztek, 5/ immun hiszto- és citokémiai jelölési technikák történtek. Eredményeink a célkitűzéseknek megfelelőek. A medulláris emlőkarcinómából nyert egyláncú ellenanyag fragmens tisztítása sikerült. Tumorsejtkultúra lemezek és fagyasztott tumorszövet metszetek a reagensünkkel pozitív reakciót adtak immunfluoreszcencs tesztben konfokális mikroszkópos értékeléssel. Melanomák, kolorektális karcinomák és több emlőtumor típus paraffinos metszetein immunhisztokémiai eljárással is pozitív reakciót kaptunk az új GD3 gangliozid ellenanyaggal. A TIL-B immunglobulin fág megjelenítéses módszer lehetővé tette további érdekes immunglobulin variábilis régiók szelektálását invaziv ductalis emlőcarcinomákban és melanomákban. GD3 gangliozid specifikus ellenanyag fragmensünk a tumorprogresszió és invázió szempontjából fontos struktúrákat szelektíven és megfelelően jelölte a tumorszöveti mintákon. Ezen szialilált glikoszfingolipidek differenciál diagnosztikus értékét alapozta meg a munka, lehetőséget nyitva mikrometastazisok kimutatására is. | We aimed to reveal the capacity of B cells present in small amounts in solid tumors. We were internationally the first to prove the characteristic tumorspecific binding capacity of tumor infiltrating B cells (TIL-B). Our startegy helps to develop novel diagnostic tumor recognizing "labelling arrow molecules". The project work consisted of: 1/ Novel molecular biological and biotechnological techniques, 2/ preparative protein purification processes, 3/ comparative DNA analyses, 4/ different antibody ? antigen binding assays, 5/ immunohistochemical and immunocytochemical methods. The results correlated our specific aims. We could purify our medullary breast carcinoma originated antibody fragment. Tumor cell chamber slides and frozen tissue sections gave positive reactions with our reagent in immunofluorescence - confocal laser microscopy. On paraffin embedded tissue sections of melanomas, colorectal carcinomas and some other breast cancer types we got positive reaction with this unique GD3 ganglioside specific antibody. Due to our TIL-B immunglobulin phage display technique further antibody variable region genes were selected in invasive ductal breast carcinomas and melanomas. The GD3 ganglioside specific antibody labelled selectively the structures important for tumorprogression and invasion on tumor tissue specimens. The work supports the differential diagnostic value of these sialylated glycosphingolipides, opening a window towards the detection of tumor micrometastases

Differential expression of proteoglycans on the surface of human melanoma cells characterized by altered experimental metastatic potential

Heparan sulphate (HS) and chondroitin sulphate (CS) proteoglycans (PGs) frequently have opposite biologic functions in cell-matrix adhesion as well as in the regulation of cell proliferation. Data revealed that sulphated glycosaminoglycans (sGAGs) (sugar chains of PGs) are differently expressed in tumor cells characterized by different metastatic potential; the more metastatic cells contain a higher HS/CS ratio. As the proliferative capacity of tumor cells is also frequently altered in parallel with their metastatic potential, it was not clear whether observed PG alterations reflect changes in cell proliferation or metastatic potential. The cell-associated PG expression and sGAG biosynthesis was studied in tumor cells of human melanoma lines characterized by different experimental metastatic potential to the mouse liver but similar in vitro/in vivo proliferation rates. Using antibodies against PGs we found different expression of PG epitopes in melanoma lines, except from the melanoma antigen. Unlike the low CSPG (melCSPG) metastatic melanoma cells, the cell line with high metastatic capacity contained a higher proportion of positive cells for surface-HSPG without the coexpression of certain cartilage-type CSPG epitopes (recognized by MAb HSFPG 529) as well as by an increased pericellular HS/CS ratio due to intracellular accumulation/retention of CS. Immunocytochemistry of adherent cells revealed HSPGs at substrate-attached membrane areas only in cases of highly metastatic melanoma cells. These data further support our view that the absolute or relative dominance of HSPGs over CSPGs at the cell surface of metastatic tumor cells can be considered a marker of a more metastatic phenotype

Aktivált T-limfociták, dendritikus sejtek és immunreguláló mechanizmusok kimutatása melanómás és fej-nyaki daganatos betegek őrszemnyirokcsomóiban - klinikai jelentőség és immunterápiai vonatkozások = Prevalence of activated T lymphocytes and dendritic cells as well as immune regulatory mechanisms in sentinel lymph nodes of melanoma and head and neck cancer patients - clinical impact and immunotherapeutic approaches

Bár a szentinel nyirokcsomók (SLN) a tumorellenes immunválasz kialakulásának fontos színterei, immunológiai sajátosságaikat kevesen tanulmányozták. Vizsgálatainkban melanómás betegek SLN-mintáin meghatároztuk az OX40+ aktivált T-limfociták, FOXP3+ regulátor T-sejtek, DC-LAMP+ érett dendritikus sejtek (DC) és CD123+ plazmacitoid DC-k mennyiségét, és összevetettük nem-szentinel csomókban (NSLN) talált értékekkel, valamint elemeztük a beteg- és tumorjellemzők, illetve a betegség kimenetele függvényében. A markerek megjelenése az SLN-ekben magasabb szintű volt az NSLN-ekhez viszonyítva, ami az őrszemnyirokcsomók immunológiai kompetenciájára utal. Pozitív SLN-státus esetén a FOXP3+ sejtek magas denzitása a betegség progressziójával és rövidebb túléléssel járt. A többi marker esetén nem találtunk szignifikáns összefüggést a betegség kimenetelével. A primer melanómákat infiltráló immunsejtek közül (a korábbi vizsgálataink szerint prognosztikus értékű aktivált T-sejt- és DC-szám mellett) a B-sejtek magas denzitása önmagában, illetve az aktivált T-sejtek nagy mennyiségével együttesen kedvező prognózisra utalt, míg a FOXP3+ limfociták mennyisége nem mutatott összefüggést a túléléssel. Eredményeink arra utalnak, hogy az immunaktivációra jellemző markerek megjelenése a primer tumorban erősebben befolyásolja a betegség kimenetelét, mint az SLN-ekben mért prevalenciájuk. Az őrszemnyirokcsomók magas FOXP3+ T-sejtdenzitása ugyanakkor az SLN-pozitív esetekben rossz prognózissal járt együtt. | Sentinel lymph nodes (SLNs) are critical sites of the development of antitumor immune response, still only few studies have aimed at investigating their immunological status. We determined the prevalence of OX40+ activated T cells, FOXP3+ regulatory T cells, DC-LAMP+ mature dendritic cells (DCs) and CD123+ plasmacytoid DCs in melanoma SLNs. Cell density values were compared to those in non-sentinel nodes (NSLNs), and analyzed with regard to associations with clinicopathological parameters and disease outcome. SLNs contained higher amount of all cell types compared to NSLNs, indicating the functional competence of sentinel nodes. High density of FOXP3+ T lymphocytes showed association with disease progression and shorter survival in SLN-positive patients, while the other cell types studied did not prove of prognostic importance. Of immune cells infiltrating primary melanomas, high amount of B lymphocytes, alone or in combination with high activated T-cell density, correlated with favorable outcome, similarly to DCs and activated T cells found of prognostic importance in our previous studies. Infiltration of FOXP3+ cells in primary melanomas, on the other hand, showed no association with survival. Our results suggest that immune activation-associated markers in the primary tumor may have a higher impact than those in SLNs on the prognosis of melanoma patients. On the other hand, FOXP3+ cell density in sentinel nodes of SLN-positive cases was found predictive of disease outcome

Immune cell profile of sentinel lymph nodes in patients with malignant melanoma - FOXP3+ cell density in cases with positive sentinel node status is associated with unfavorable clinical outcome

BACKGROUND: Besides being a preferential site of early metastasis, the sentinel lymph node (SLN) is also a privileged site of T-cell priming, and may thus be an appropriate target for investigating cell types involved in antitumor immune reactions. METHODS: In this retrospective study we determined the prevalence of OX40+ activated T lymphocytes, FOXP3+ (forkhead box P3) regulatory T cells, DC-LAMP+ (dendritic cell-lysosomal associated membrane protein) mature dendritic cells (DCs) and CD123+ plasmacytoid DCs by immunohistochemistry in 100 SLNs from 60 melanoma patients. Density values of each cell type in SLNs were compared to those in non-sentinel nodes obtained from block dissections (n = 37), and analyzed with regard to associations with clinicopathological parameters and disease outcome. RESULTS: Sentinel nodes showed elevated amount of all cell types studied in comparison to non-sentinel nodes. Metastatic SLNs had higher density of OX40+ lymphocytes compared to tumor-negative nodes, while no significant difference was observed in the case of the other cell types studied. In patients with positive sentinel node status, high amount of FOXP3+ cells in SLNs was associated with shorter progression-free (P = 0.0011) and overall survival (P = 0.0014), while no significant correlation was found in the case of sentinel-negative patients. The density of OX40+, CD123+ or DC-LAMP+ cells did not show significant association with the outcome of the disease. CONCLUSIONS: Taken together, our results are compatible with the hypothesis of functional competence of sentinel lymph nodes based on the prevalence of the studied immune cells. The density of FOXP3+ lymphocytes showed association with progression and survival in patients with positive SLN status, while the other immune markers studied did not prove of prognostic importance. These results, together with our previous findings on the prognostic value of activated T cells and mature DCs infiltrating primary melanomas, suggest that immune activation-associated markers in the primary tumor may have a higher impact than those in SLNs on the prognosis of the patients. On the other hand, FOXP3+ cell density in SLNs, but not in the primary tumor, was found predictive of disease outcome in melanoma patients

The impact of extreme weather conditions and municipal sewage disposal on vegetation using sentinel images, SE Hungary

Land application of municipal sewage can enhance the productivity of biomass production systems and can contribute to a better resistance to extremities in water supply. Understanding the impacts requires the examination of changes in soil characteristics and plant productivity. In this study (paralelly to the soil analyises) Sentinel 2B vegetation index series are assessed to monitor the biomass anomalies related to different water supply conditions and the sewage disposal in the period of 2016-2018 in a study area in SE Hungary (Újkígyós). In the humid year of 2016 there were no spatial differences in biomass production of vegetation stands, but as a result of the dry summer in 2017, biomass production deficiencies were observed on the areas of the ancient river meanders, characterised by different soil properties compared to the surrounding areas. In general, the direct impact of municipal sewage disposal could be detected on biomass by increased productivity, the response is probably related to the nutrient status of the site

Új immunterápias lehetősegek az emlőrák kezelésében

The prognostic value of tumor infiltrating lymphocytes in breast cancer has long been recognized by histopathologists. These observations were reaffirmed by recent immunohistochemistry and gene expression profiling studies that also revealed an association between greater chemotherapy sensitivity and extensive lymphocytic infiltration in early stage breast cancers treated with neoadjuvant chemotherapy. These results suggest that local anti-tumor immune response can at least partially control cancer growth and may mediate the antitumor effects of chemotherapy. However, until recently, there was no direct clinical evidence to demonstrate that enhancing anti-tumor immune response could lead to clinical benefit in breast cancer patients. The recent development of clinically effective immune checkpoint inhibitors made it possible to test the therapeutic impact of augmenting the local anti-tumor immune response. Two Phase I clinical trials using single agent anti-PD-1 (MK-3475, pembrolizumab) and anti-PD-L1 (MPDL3280A, atezolizumab) antibodies demonstrated close to 20% tumor response rates in heavily pretreated, metastatic, triple negative breast cancers. The most remarkable feature of the responses was their long duration. Several patients had disease control close to a year, or longer, which has not previously been seen with chemotherapy regimens in this patient population. A large number of clinical trials are currently underway with these and similar drugs in the neoadjuvant, adjuvant and metastatic settings to define the role of this new treatment modality in breast cancer

Impact of Low-Dose Municipal Sewage Sludge Compost Treatments on the Nutrient and the Heavy Metal Contents in a Chernozem Topsoil Near Újkígyós, Hungary: A 5-Year Comparison

Agriculture is one of the major fields, where sewage sludge can be used. Its high nutrient content can contribute to the improvement of important soil properties, such as nutrient content, water balance and soil structure. However, sewage sludge may contain hazardous components, such as pathogens and pollutants. Therefore, it is important to monitor the effects of its field application. In this paper, we assessed the impacts of two low-dose (2.5 m3/ha) municipal sewage sludge compost applications (in 2013 and in 2017) in a 5.6 ha arable land in southeast Hungary (near Újkígyós), located in the Hungarian Great Plain. The nutrient and the heavy metal contents in the upper soil layer (0-30 cm) of the studied Chernozem soils were compared between two sampling campaigns in 2013 (before the compost applications) and in 2018 (after the compost applications). Basic soil properties (pH, salinity, humus content, carbonate content, Arany yarn number) complemented with nutrient content (K2O, P2O5, NO2+ NO3) and heavy metal content (Cd, Co, Cr, Cu, Ni, Pb and Zn) analyses were performed. The results show that no significant change can be noticed in the baseline parameters over the 5-year period. The slight increase in the P2O5, NO2+ NO3 content is closely related to the beneficial effects of the sewage sludge deposition. The soil-bound heavy metal load did not increase significantly as a result of the compost treatments, only nickel showed a slight increase in the topsoil. In all cases the heavy metal concentrations did not reach the contamination thresholds set by Hungarian standards. The results provided positive evidences proving that low dose municipal sewage sludge compost disposal on agricultural land is safe, and can be considered as a sustainable soil amendment for agriculture in compliance with legal requirements

Circulating endothelial cells, bone marrow-derived endothelial progenitor cells and proangiogenic haematopoietic cells in cancer: From biology to therapy

Vascularization, a hallmark of tumorigenesis, is classically thought to occur exclusively through angiogenesis (i.e. endothelial sprouting). However, there is a growing body of evidence that endothelial progenitor cells (EPCs) and proangiogenic hematopoietic cells (HCs) are able to support the vascularization of tumors and may therefore play a synergistic role with angiogenesis. An additional cell type being studied in the field of tumor vascularization is the circulating endothelial cell (CEC), whose presence in elevated numbers reflects vascular injury. Levels of EPCs and CECs are reported to correlate with tumor stage and have been evaluated as biomarkers of the efficacy of anticancer/antiangiogenic treatments. Furthermore, because EPCs and subtypes of proangiogenic HCs are actively participating in capillary growth, these cells are attractive potential vehicles for delivering therapeutic molecules. The current paper provides an update on the biology of CECs, EPCs and proangiogenic HCs, and explores the utility of these cell populations for clinical oncology

Szennyvíziszap-komposzttal történő kezelés hatása a talaj CO2-respirációjára, tápanyag- és nehézfém-tartalmára csernozjom talajokon

A szennyvíziszap olyan szerves anyagokat, mikro- és makrotápanyagokat tartalmaz, amelyek mezőgazdasági felhasználás esetén javítják a talaj termékenységét. Ráadásul a szerves anyagok és a mikrobiológiai aktivitás növekedésével a talaj CO2-respirációja javulhat, és többlet CO2 kötődhet meg a talajban, ami hosszú távon csökkenti a légköri széndioxid-koncentrációt. Ugyanakkor a kihelyezésnek lehetnek káros következményei is, ha a szennyvíziszap túl sok szerves anyagot, nitrogént vagy nehézfémet tartalmaz. Kutatásunkban csernozjomtalajokon vizsgáltuk a növények által felvehető tápanyagoknak és nehézfémeknek, valamint a szerves anyagnak a változását alacsony dózisú települési szennyvíziszap-komposzt kihelyezése esetén (2,5-35 m3/ha/év). A talaj CO2-forgalmának becslésére terepi respirációs méréseket végeztünk. A terepi mintavételezések és mérések 2018-ban és 2019-ben zajlottak Újkígyós és Kardos települések közelében (Békés megye), melyek során a kísérleti parcellákról átlagtalajmintákat (0-30 cm és 30-60 cm mélységből) és talajvízmintákat gyűjtöttünk be a tápanyag- és nehézfém-koncentrációk változásának nyomon követésére (Újkígyóson), illetve öt alkalommal mértük a talaj CO2-respirációját mind Újkígyóson, mind Kardoson. A talajmintákon talajtani alapvizsgálatokat (pH, szervesanyag-tartalom, fizikai féleség, karbonát- és sótartalom) végeztünk, illetve standard extrakciós eljárásokkal meghatároztuk a tápanyagok (K2O, P2O5, N-formák, szerves anyagok) és egyes nehézfémek koncentrációit. A laboratóriumi vizsgálatok eredményei azt mutatták, hogy a szennyvíziszap-komposzttal kezelt területeken szignifikánsan megnövekedett a talaj K2O, P2O5 és NO2- + NO3- tartalma, ugyanakkor sem a szervesanyag-, sem a nehézfém-tartalomban nem következett be jelentős változás a kontrollhoz képest. Hasonlóképpen nem tudtuk igazolni a szennyvíziszap-kezelések utáni intenzívebb CO2-respirációt az egyik vizsgálati területen sem. Összességében eredményeink meggyőzően bizonyították, hogy a csak kommunális eredetű, iparból származóval nem terhelt alacsony dózisú települési szennyvíziszap-komposzttal kezelt területeken ez fenntartható trágyázási gyakorlat lehet, mely magas N-, P- és K-tartalmával olyan formában szolgálja a növénytermesztést, hogy közben nem szennyezi a talajvizet. A talaj CO2-forgalmának markáns módosításához vélhetően alacsonyak voltak a kihelyezett dózisok