Influence of antigen density and immunosuppressive factors on tumor-targeted costimulation with antibody-fusion proteins and bispecific antibody-mediated T cell response

Target expression heterogeneity and the presence of an immunosuppressive microenvironment can hamper severely the efficiency of immunotherapeutic approaches. We have analyzed the potential to encounter and overcome such conditions by a combinatory two-target approach involving a bispecific antibody retargeting T cells to tumor cells and tumor-directed antibody-fusion proteins with costimulatory members of the B7 and TNF superfamily. Targeting the tumor-associated antigens EpCAM and EGFR with the bispecific antibody and costimulatory fusion proteins, respectively, we analyzed the impact of target expression and the influence of the immunosuppressive factors IDO, IL-10, TGF-β, PD-1 and CTLA-4 on the targeting-mediated stimulation of T cells. Here, suboptimal activity of the bispecific antibody at diverse EpCAM expression levels could be effectively enhanced by targeting-mediated costimulation by B7.1, 4-1BBL and OX40L in a broad range of EGFR expression levels. Furthermore, the benefit of combined costimulation by B7.1/4-1BBL and 4-1BBL/OX40L was demonstrated. In addition, the expression of immunosuppressive factors was shown in all co-culture settings, where blocking of prominent factors led to synergistic effects with combined costimulation. Thus, targeting-mediated costimulation showed general promise for a broad application covering diverse target expression levels, with the option for further selective enhancement by the identification and blockade of main immunosuppressive factors of the particular tumor environment.Deutsche KrebshilfeProjekt DEA

Influence of antigen density and immunosuppressive factors on tumor-targeted costimulation with antibody-fusion proteins and bispecific antibody-mediated T cell response

Target expression heterogeneity and the presence of an immunosuppressive microenvironment can hamper severely the efficiency of immunotherapeutic approaches. We have analyzed the potential to encounter and overcome such conditions by a combinatory two-target approach involving a bispecific antibody retargeting T cells to tumor cells and tumor-directed antibody-fusion proteins with costimulatory members of the B7 and TNF superfamily. Targeting the tumor-associated antigens EpCAM and EGFR with the bispecific antibody and costimulatory fusion proteins, respectively, we analyzed the impact of target expression and the influence of the immunosuppressive factors IDO, IL-10, TGF-β, PD-1 and CTLA-4 on the targeting-mediated stimulation of T cells. Here, suboptimal activity of the bispecific antibody at diverse EpCAM expression levels could be effectively enhanced by targeting-mediated costimulation by B7.1, 4-1BBL and OX40L in a broad range of EGFR expression levels. Furthermore, the benefit of combined costimulation by B7.1/4-1BBL and 4-1BBL/OX40L was demonstrated. In addition, the expression of immunosuppressive factors was shown in all co-culture settings, where blocking of prominent factors led to synergistic effects with combined costimulation. Thus, targeting-mediated costimulation showed general promise for a broad application covering diverse target expression levels, with the option for further selective enhancement by the identification and blockade of main immunosuppressive factors of the particular tumor environment

Intossicazione cronica da glicole etilenico in un cane: "risolutivo" l'esame istologico

Introduzione. L\u2019intossicazione da Glicole Etilenico (GE) e la conseguente insufficienza renale acuta si verificano di frequente nel cane. La particolarit\ue0 di questo caso \ue8 da ricercarsi nella malattia renale cronica (CKD) che potrebbe essersi instaurata a seguito dell\u2019assunzione del tossico in dosi subletali e da una fonte diversa dal liquido antigelo. Il soggetto inoltre presentava agenesia renale monolaterale. Descrizione del caso. Un Doberman Pinscher, maschio intero di 5 anni, \ue8 stato riferito per insufficienza renale associata a sintomatologia gastro-enterica acuta, con poliuria/polidpsia da qualche mese e lieve dimagrimento. Il cane presentava grave depressione del sensorio, soffusioni emorragiche disseminate ed ipotermia (36,3\ub0C). La pressione arteriosa sistolica era di 190-200 mm Hg e l\u2019output urinario 3 ml/Kg/h, il peso specifico urinario di 1010, con 30 mg/dl di proteine al dipstick urinario. Il sedimento urinario non presentava cristalli. Il rapporto proteine urinarie-creatinina urinaria era di 1,6 (I.R. < 0,4) ed il rapporto albumina urinaria- creatinina urinaria era di 0,25 (I.R. < 0,025). Erano presenti inoltre grave iperazotemia e iperfosfatemia, lieve panipoprotidemia, lieve incremento degli enzimi epatici e normocalcemia, grave piastrinopenia, un lieve aumento dei tempi di coagulazione e riduzione dell\u2019attivit\ue0 plasmatica dell\u2019antitrombina. La Proteina C Reattiva aveva concentrazioni di 5,57 mg/dl (I.R. <1 mg/dl). L\u2019ecografia addominale rilevava agenesia del rene destro e nefropatia sinistra con presenza di spot iperecogeni diffusi alla corticale, iperecogenicit\ue0 della corticale e della midollare con riduzione della distinzione corticomidollare. Dopo 96 ore di terapia intensiva con scarsi miglioramenti, \ue8 stata richiesta l\u2019eutanasia da parte del proprietario. L\u2019esame necroscopico ha consentito di rilevare l\u2019agenesia totale del rene destro e la relativa ipertrofia compensatoria del rene sinistro, il quale presentava la capsula leggermente ispessita e biancastra, la corticale di colore diffusamente giallastro e nella midollare numerose aree di mineralizzazione puntiformi. Di particolare rilevanza era la presenza di una pancreatite cronica fibrosa ed emorragica e la presenza nel miocardio di numerosissimi focolai di necrosi. Erano inoltre presenti altre lesioni riconducibili alla CKD: stomatite ulcerativa e necrotica, gastrite e colite emorragica, mineralizzazioni multifocali nella pleura parietale intercostale, delle valvole semilunari aortiche e nella trachea, e multifocali emorragie da vasculite. L\u2019esame istopatologico, su sezioni incluse in paraffina e fissate in formalina, ha evidenziato sia a livello della corticale sia della midollare renale una sofferenza tubulare, caratterizzata da degenerazione ed appiattimento dell\u2019epitelio dovuto alla presenza di cristalli birifrangenti di colore giallo chiaro. La natura dei cristalli \ue8 stata svelata mediante l\u2019utilizzo di colorazioni speciali: Oil Red O, Alizarin Red S, Von Kossa e Pizzolato. \uc8 stata ottenuta una negativit\ue0 per Oil Red O ed Alizarin Red S, mentre una positivit\ue0 per Von Kossa e per Pizzolato, reattivit\ue0 paradigmatica del contenuto dei cristalli di ossalato di calcio. In aggiunta, utilizzando il microscopio a luce polarizzata \ue8 stato possibile rendere meglio evidenti i numerosi cristalli birifrangenti piramidali disposti in rosette o fasci. In base a questi risultati \ue8 stata effettuata un\u2019ulteriore indagine anamnestica, nella quale \ue8 stata esclusa dal proprietario l\u2019ingestione di liquido anti-gelo e da cui \ue8 emerso che il cane viveva in casa, ma accompagnava il proprietario sul luogo di lavoro dove era a contatto con solventi e vernici. Conclusioni. I rischi connessi alla professione del proprietario, risultano essere particolarmente significativi in questo caso clinico. Il GE, contenuto principalmente nel liquido antigelo con concentrazioni del 95%, \ue8 anche un componente, seppur in concentrazioni minori di sg..

Antimalarial Activity of Extracts of Abutilon grandiflorum G. Don - A Traditional Tanzanian Medicinal Plant

The Tanzanian medicinal plant Abutilon grandiflorum G. Don was studied for its in vivo and in vitro antiplasmodial effects. The ethyl acetate extract showed prominent in vivo activity against P. vinckei vinckei in mice and in vitro against P. falciparum strains HB3 and FCB. The extract was only moderately cytotoxic if tested in vitro against the colon cell line HT29. In the in vivo study, the results were significantly influenced by the treatment schedule used, i.e. early treatment with higher doses was more successful than applying the same overall amount over a longer period. Phytochemical analysis of the extract provided no conclusive evidence for the observed parasitological effects

Tyrosine Kinase Receptor Expression in Canine Liposarcoma

The expression of tyrosine kinase receptors is attracting major interest in human and veterinary oncological pathology because of their role as targets for adjuvant therapies. Little is known about tyrosine kinase receptor (TKR) expression in canine liposarcoma (LP), a soft tissue sarcoma. The aim of this study was to evaluate the immunohistochemical expression of the TKRs fibroblast growth factor receptor 1 (FGFR1) and platelet-derived growth factor receptor-\u3b2 (PDGFR\u3b2); their ligands, fibroblast growth factor 2 (FGF2) and platelet-derived growth factor B (PDGFB); and c-kit in canine LP. Immunohistochemical labeling was categorized as high or low expression and compared with the mitotic count and MIB-1-based proliferation index. Fifty canine LPs were examined, classified, and graded. Fourteen cases were classified as well differentiated, 7 as myxoid, 25 as pleomorphic, and 4 as dedifferentiated. Seventeen cases were grade 1, 26 were grade 2, and 7 were grade 3. A high expression of FGF2, FGFR1, PDGFB, and PDGFR\u3b2 was identified in 62% (31/50), 68% (34/50), 81.6% (40/49), and 70.8% (34/48) of the cases, respectively. c-kit was expressed in 12.5% (6/48) of the cases. Mitotic count negatively correlated with FGF2 (R = -0.41; P < .01), being lower in cases with high FGF2 expression, and positively correlated with PDGFR\u3b2 (R = 0.33; P < .01), being higher in cases with high PDGFR\u3b2 expression. No other statistically significant correlations were identified. These results suggest that the PDGFR\u3b2-mediated pathway may have a role in the progression of canine LP and may thus represent a promising target for adjuvant cancer therapies

Wpływ miedzi w wodzie na katalazę w skrzelach i biochemię krwi u dorady (Sparus aurata L.)

Cu is a transition metal essential for every organism, but an excess can lead to toxicity caused by oxidative stress, which can disturb the natural antioxidant defence systems. Since catalase (CAT) is a major enzyme involved in the decomposition of oxygen peroxide, the main goal of this study was to evaluate changes in the concentration and activity of CAT by means of enzymatic and immunohistochemical methods. Modifications in the blood biochemistry profile of Cu-exposed gilthead sea bream (Sparus aurata L.) were also studied. In gills of the exposed fish, Cu significantly increased throughout the study to a maximum of 6.9±4.7 μg g–1 wet weight at the end of the treatment. Immunohistochemistry (IHC) evidenced a brown cytoplasmic stain in the epithelial cells of the primary and secondary lamellae and in pillar cells, while enzyme activity was localized in the epithelium and pillar cells of both primary and secondary lamellae and appeared as a strong cytoplasmic stain particularly at the base of the primary lamellae. IHC and enzymohistochemistry (EHC) quantitative analysis suggested that the main variations in amount and activity of the enzyme were recorded 15 days after exposure (both IHC and EHC expressed a decrease in CAT in exposed fish compared to controls, P<0.0001) and 28 days after exposure (IHC recorded increased CAT in exposed specimens compared to controls, P<0.0001; EHC evidenced a decrease in CAT in exposed subjects compared to controls, P<0.0001). The biochemical blood profile was monitored with a standard blood biochemistry panel. An increase in plasma urea was evident only in exposed fish, while – as a function of time – a decrease of glucose in both exposed and control fish was apparent. The three investigation methods evidenced that CAT was effective against Cu toxicity, and the increase in plasma urea could be considered a suitable metabolic marker of Cu exposure in fish.Miedź jest metalem przejściowym niezbędnym w każdym organizmie, lecz jej nadmiar może prowadzić do skutków toksycznych powodowanych przez stres oksydacyjny, który zakłóca naturalne systemy obrony antyoksydacyjnej. Ponieważ katalaza (CAT) jest jednym z głównych enzymów uczestniczących w rozkładzie nadtlenku tlenu, głównym celem badań była ocena zmian zawartości i aktywności CAT za pomocą metod enzymatycznych i immunohistochemicznych. Badano również zmiany w biochemicznym profilu krwi u dorady (Sparus aurata L.) poddanej działaniu Cu. Stwierdzono znacząco wyższe stężenie tego metalu w skrzelach ryb, na które oddziaływała miedź, aż do maksymalnej zawartości 6,9±4,7 μg g–1 mokrej masy pod koniec doświadczenia. Badania immunohistochemiczne (IHC) ujawniły brązową plamkę cytoplazmatyczną w komórkach nabłonka blaszek pierwszego i drugiego rzędu oraz w komórkach filarowych, podczas gdy aktywność enzymatyczna została zlokalizowana w nabłonku oraz w blaszkach pierwszego i drugiego rzędu, i ujawniła się jako silna plamka cytoplazmatyczna, szczególnie u podstawy blaszek pierwszego rzędu. Analiza ilościowa wyników badań IHC oraz enzymohistochemicznych (EHC) sugeruje, iż główna zmienność w ilości oraz aktywności enzymu nastąpiła w 15. dniu po ekspozycji (zarówno IHC, jak i EHC wykazały spadek zawartości CAT u ryb wystawionych na działanie Cu w porównaniu z kontrolą, P<0,0001) oraz w 28. dniu po ekspozycji (IHC wykazało wzrost CAT u ryb wystawionych na Cu w porównaniu z kontrolą, P<0,0001; EHC ujawniło spadek CAT u ryb doświadczalnych w porównaniu z kontrolą, P<0,0001). Biochemiczny profil krwi monitorowano za pomocą standardowego zestawu biochemicznego krwi. Wzrost zawartości mocznika we krwi ujawnił się jedynie u ryb poddanych działaniu Cu, natomiast spadek zawartości glukozy – z upływem czasu – wystąpił u ryb doświadczalnych i kontrolnych. Trzy zastosowane metody badawcze udowodniły, iż CAT skutecznie przeciwstawia się toksyczności Cu, a wzrost zawartości mocznika we krwi można uznać za odpowiedni marker metaboliczny oznaczający wystawienie ryb na działanie miedzi

Effects of waterborne copper on gills catalase and blood biochemistry in gilthead seabream (Sparus aurata L.)

Cu is a transition metal essential for every organism, but an excess can lead to toxicity caused by oxidative stress, which can disturb the natural antioxidant defence systems. Since catalase (CAT) is a major enzyme involved in the decomposition of oxygen peroxide, the main goal of this study was to evaluate changes in the concentration and activity of CAT by means of enzymatic and immunohistochemical methods. Modifications in the blood biochemistry profile of Cu-exposed gilthead sea bream (Sparus aurata L.) were also studied. In gills of the exposed fish, Cu significantly increased throughout the study to a maximum of 6.9\ub14.7 \u3bcg g\u20131 wet weight at the end of the treatment. Immunohistochemistry (IHC) evidenced a brown cytoplasmic stain in the epithelial cells of the primary and secondary lamellae and in pillar cells, while enzyme activity was localized in the epithelium and pillar cells of both primary and secondary lamellae and appeared as a strong cytoplasmic stain particularly at the base of the primary lamellae. IHC and enzymohistochemistry (EHC) quantitative analysis suggested that the main variations in amount and activity of the enzyme were recorded 15 days after exposure (both IHC and EHC expressed a decrease in CAT in exposed fish compared to controls, P<0.0001) and 28 days after exposure (IHC recorded increased CAT in exposed specimens compared to controls, P<0.0001; EHC evidenced a decrease in CAT in exposed subjects compared to controls, P<0.0001). The biochemical blood profile was monitored with a standard blood biochemistry panel. An increase in plasma urea was evident only in exposed fish, while \u2013 as a function of time \u2013 a decrease of glucose in both exposed and control fish was apparent. The three investigation methods evidenced that CAT was effective against Cu toxicity, and the increase in plasma urea could be considered a suitable metabolic marker of Cu exposure in fish