The present work aimed at purifying the intracellular fungal metabolites, such as 16-methylheptadecanoic acid methyl ester (HDA) and 9,12-octadecadienoic acid (ODA) from marine Trichoderma, Hypocrea lixii TSK8, Hypocrea rufa SKS2 respectively, and investigating their anticancer and antioxidant effects. The two fungal metabolites were tested against two human cancer cell lines, namely oral cancer (KB) and skin carcinoma (A431) by using MTT assay. The inhibitory concentrations (IC50) against KB oral cancer cells were found to be 18.75 ± 0.12 μg/mL for HDA and 75.50 ± 0.42 μg/mL for ODA. Whereas IC50 values of HDA and ODA against A431 were found 37.5 ± 0.42 μg/mL and 72.89 ± 0.15 μg/mL, respectively. In addition, the down-regulation of heat shock protein 90 kDa (HSP90) was confirmed by using SDS-PAGE and Western blot analysis. The effect of HDA induced apoptosis via ROS-dependent internucleosomal DNA fragmentation was confirmed by AGE analysis. We further evaluated the in vivo anti-skin cancer activity of HDA in Swiss albino mice induced with skin cancer by 7,12-dimethylbenz(a)anthracene (DMBA) and croton oil (CO). The in vivo hematological, biochemical and histopathological results revealed that the fungal metabolite HDA was a highly potent anticancer compound against the skin cancer.

• Klíčová slova
• methyl ester 16-methylheptadekanové kyseliny (HDA) a 9, 12-oktodekanové kyseliny (ODA),
• MeSH
• antioxidancia izolace a purifikace   terapeutické užití   MeSH
• antitumorózní látky * MeSH
• apoptóza genetika   imunologie   účinky léků   MeSH
• fragmentace DNA MeSH
• houby cytologie   izolace a purifikace   metabolismus   MeSH
• kyseliny dekanové * izolace a purifikace   metabolismus   MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• myši MeSH
• nádorové buněčné linie cytologie   metabolismus   účinky léků   MeSH
• nádory kůže farmakoterapie   MeSH
• oxidační stres genetika   imunologie   účinky léků   MeSH
• proteiny tepelného šoku HSP90 imunologie   izolace a purifikace   metabolismus   MeSH
• statistika jako téma MeSH
• Trichoderma * genetika   izolace a purifikace   účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH

PURPOSE: The aim of this work was to demonstrate an immunostimulatory and adjuvant effect of new apyrogenic lipophilic derivatives of norAbuMDP and norAbuGMDP formulated in nanoliposomes. METHODS: Nanoliposomes and metallochelating nanoliposomes were prepared by lipid film hydration and extrusion methods. The structure of the liposomal formulation was studied by electron microscopy, AF microscopy, and dynamic light scattering. Sublethal and lethal γ-irradiation mice models were used to demonstrate stimulation of innate immune system. Recombinant Hsp90 antigen (Candida albicans) bound onto metallochelating nanoliposomes was used for immunisation of mice to demonstrate adjuvant activities of tested compounds. RESULTS: Safety and stimulation of innate and adaptive immunity were demonstrated on rabbits and mice. The liposomal formulation of norAbuMDP/GMDP was apyrogenic in rabbit test and lacking any side effect in vivo. Recovery of bone marrow after sublethal γ-irradiation as well as increased survival of mice after lethal irradiation was demonstrated. Enhancement of specific immune response was demonstrated for some derivatives incorporated in metallochelating nanoliposomes with recombinant Hsp90 protein antigen. CONCLUSIONS: Liposomal formulations of new lipophilic derivatives of norAbuMDP/GMDP proved themselves as promising adjuvants for recombinant vaccines as well as immunomodulators for stimulation of innate immunity and bone-marrow recovery after chemo/radio therapy of cancer.

• MeSH
• acetylmuramyl-alanyl-isoglutamin aplikace a dávkování   analogy a deriváty   chemie   farmakologie   terapeutické užití   MeSH
• adaptivní imunita účinky léků   MeSH
• adjuvancia imunologická aplikace a dávkování   chemie   farmakologie   terapeutické užití   MeSH
• analýza přežití MeSH
• antigeny fungální imunologie   MeSH
• experimentální radiační poranění imunologie   prevence a kontrola   MeSH
• králíci MeSH
• liposomy MeSH
• mikroskopie atomárních sil MeSH
• mikroskopie elektronová rastrovací MeSH
• molekulární struktura MeSH
• myši inbrední ICR MeSH
• myši MeSH
• nanočástice MeSH
• nosiče léků chemie   MeSH
• přirozená imunita účinky léků   MeSH
• proteiny tepelného šoku HSP90 imunologie   MeSH
• protilátky fungální krev   MeSH
• rekombinantní proteiny imunologie   MeSH
• transmisní elektronová mikroskopie MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Recent studies have identified molecular events characteristic of immunogenic cell death (ICD), including surface exposure of calreticulin (CRT), the heat shock proteins HSP70 and HSP90, the release of high-mobility group box protein 1 (HMGB1) and the release of ATP from dying cells. We investigated the potential of high hydrostatic pressure (HHP) to induce ICD in human tumor cells. HHP induced the rapid expression of HSP70, HSP90 and CRT on the cell surface. HHP also induced the release of HMGB1 and ATP. The interaction of dendritic cells (DCs) with HHP-treated tumor cells led to a more rapid rate of DC phagocytosis, upregulation of CD83, CD86 and HLA-DR and the release of interleukin IL-6, IL-12p70 and TNF-α. DCs pulsed with tumor cells killed by HHP induced high numbers of tumor-specific T cells. DCs pulsed with HHP-treated tumor cells also induced the lowest number of regulatory T cells. In addition, we found that the key features of the endoplasmic reticulum stress-mediated apoptotic pathway, such as reactive oxygen species production, phosphorylation of the translation initiation factor eIF2α and activation of caspase-8, were activated by HHP treatment. Therefore, HHP acts as a reliable and potent inducer of ICD in human tumor cells.

• MeSH
• adenosintrifosfát sekrece   MeSH
• aktivace enzymů imunologie   MeSH
• antigeny CD86 biosyntéza   MeSH
• apoptóza imunologie   MeSH
• CD antigeny biosyntéza   MeSH
• dendritické buňky imunologie   MeSH
• eukaryotický iniciační faktor 2 metabolismus   MeSH
• fagocytóza imunologie   MeSH
• fosforylace MeSH
• HLA-DR antigeny biosyntéza   MeSH
• hydrostatický tlak MeSH
• imunoglobuliny biosyntéza   MeSH
• interleukin-12 sekrece   MeSH
• interleukin-6 sekrece   MeSH
• kalretikulin biosyntéza   imunologie   MeSH
• kaspasa 8 metabolismus   MeSH
• lidé MeSH
• membránové glykoproteiny biosyntéza   MeSH
• membránové proteiny biosyntéza   MeSH
• nádorové buněčné linie MeSH
• nádory imunologie   MeSH
• protein HMGB1 imunologie   sekrece   MeSH
• proteiny tepelného šoku HSP70 biosyntéza   imunologie   MeSH
• proteiny tepelného šoku HSP90 biosyntéza   imunologie   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• regulační T-lymfocyty imunologie   MeSH
• stres endoplazmatického retikula imunologie   MeSH
• TNF-alfa sekrece   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Immunogenic cell death is characterized by the early surface exposure of chaperones including calreticulin and HSPs, which affect dendritic cell (DC) maturation and the uptake and presentation of tumor antigens. It has also been shown that it is characterized by the late release of high mobility group box 1 (HMGB1), which acts through Toll-like receptor 4 (TLR4) and augments the presentation of antigens from dying tumor cells to DCs. Most of the data on immunogenic tumor cell death were obtained using mouse models. In this study, we investigated the capacity of clinically used chemotherapeutics to induce immunogenic cell death in human tumor cell lines and primary tumor cells. We found that only anthracyclines induced a rapid translocation of calreticulin, HSP70, and HSP90 to the cell surface and the release of HMGB1 12 hours after the treatment. The interaction of immature DCs with immunogenic tumor cells led to an increased tumor cell uptake and induces moderate phenotypic maturation of DCs. Killed tumor cell-loaded DCs efficiently stimulated tumor-specific IFN-γ-producing T cells. DCs pulsed with killed immunogenic tumor cells also induced significantly lower numbers of regulatory T cells than those pulsed with nonimmunogenic tumor cells. These data indicate that human prostate cancer, ovarian cancer, and acute lymphoblastic leukemia cells share the key features of immunogenic cell death with mice tumor cells. These data also identify anthracyclines as anticancer drugs capable of inducing immunogenic cell death in sensitive human tumor cells.

• MeSH
• akutní lymfatická leukemie farmakoterapie   imunologie   patologie   MeSH
• antracykliny imunologie   farmakologie   MeSH
• buněčná smrt účinky léků   imunologie   MeSH
• dendritické buňky imunologie   patologie   MeSH
• fagocytóza účinky léků   imunologie   MeSH
• kalretikulin biosyntéza   imunologie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory prostaty farmakoterapie   imunologie   patologie   MeSH
• nádory vaječníků farmakoterapie   imunologie   patologie   MeSH
• nádory farmakoterapie   imunologie   patologie   MeSH
• protein HMGB1 imunologie   sekrece   MeSH
• proteiny tepelného šoku HSP70 biosyntéza   imunologie   MeSH
• proteiny tepelného šoku HSP90 biosyntéza   imunologie   MeSH
• T-lymfocyty účinky léků   imunologie   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Hsp90-CA is present in cell wall of Candida pseudohyphae or hyphae-typical pathogenic morphotype for both systemic and mucosal Candida infections. Heat shock protein from Candida albicans (hsp90-CA) is an important target for protective antibodies during disseminated candidiasis of experimental mice and human. His-tagged protein rHsp90 was prepared and used as the antigen for preparation of experimental recombinant liposomal vaccine. Nickel-chelating liposomes (the size around 100nm, PDI≤0.1) were prepared from the mixture of egg phosphatidyl choline and nickel-chelating lipid DOGS-NTA-Ni (molar ratio 95:5%) by hydration of lipid film and extrusion methods. New non-pyrogenic hydrophobised derivative of MDP (C18-O-6-norAbuMDP) was incorporated into liposomes as adjuvans. rHsp90 was attached onto the surface of metallochelating liposomes by metallochelating bond and the structure of these proteoliposomes was studied by dynamic light scattering, AF microscopy, TEM and GPC. The liposomes with surface-exposed C18-O-6-norAbuMDP were well recognised and phagocyted by human dendritic cells in vitro. In vivo the immune response towards this experimental vaccine applied in mice (i.d.) demonstrated both TH1 and TH2 response comparable to FCA, but without any side effects. Metallochelating liposomes with lipophilic derivatives of muramyl dipeptide represent a new biocompatible platform for construction of experimental recombinant vaccines and drug-targeting systems.

• MeSH
• antigeny fungální imunologie   metabolismus   MeSH
• biokompatibilní potahované materiály metabolismus   MeSH
• buněčná imunita MeSH
• Candida imunologie   MeSH
• chelátory chemie   metabolismus   MeSH
• dendritické buňky imunologie   metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• liposomy MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• nikl imunologie   metabolismus   MeSH
• proteiny tepelného šoku HSP90 imunologie   metabolismus   MeSH
• syntetické vakcíny imunologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

The Candida albicans heat shock protein 90 kDa (hsp90-CA) is an important target for protective antibodies in disseminated candidiasis of experimental mice and humans. Hsp90-CA is present in the cell wall of Candida pseudohyphae or hyphae--typical pathogenic morphotypes in both mucosal and systemic Candida infections. However, the potential protective effects of hsp90-CA-specific antibodies in vaginal candidiasis has not yet been reported. In the present study we used various vaccine formulations (recombinant hsp90-CA protein and hsp90-CA-encoding DNA vaccine) and routes of administration (intradermal, intranasal, and intravenous) to induce both hsp90-CA-specific systemic and vaginal mucosa immune responses in experimental BALB/c mice. The results showed that intradermal recombinant hsp90-CA protein priming, followed by intranasal or intradermal recombinant hsp90-CA protein boosting induced significant increases in both serum and vaginal hsp90-CA-specific IgG and IgA antibodies compared to the control group, as well as enhanced hsp90-CA-specific splenocyte responses in vitro. In the intradermally boosted group, subsequent experimental vaginal Candida infection induced additional increases in the hsp90-CA specific IgG isotype, suggesting that Candida has the ability to induce a local hsp90-specific antibody (IgG) response during vulvovaginal candidiasis. Further work is required to elucidate the importance of immunity to highly conserved antigens during infection of the human female reproductive tract where a balance between immunity to and tolerance for commonly antigens such as hsp90 is necessary for the maintenance of fertility.

• MeSH
• aplikace intranazální MeSH
• Candida albicans imunologie   MeSH
• DNA vakcíny aplikace a dávkování   imunologie   MeSH
• financování organizované MeSH
• fungální proteiny imunologie   MeSH
• fungální vakcíny aplikace a dávkování   imunologie   MeSH
• injekce intradermální MeSH
• injekce intravenózní MeSH
• kandidóza vulvovaginální imunologie   MeSH
• lymfocyty imunologie   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• proliferace buněk MeSH
• proteiny tepelného šoku HSP90 imunologie   MeSH
• protilátky fungální analýza   krev   MeSH
• sekundární imunizace MeSH
• slezina imunologie   MeSH
• syntetické vakcíny aplikace a dávkování   imunologie   MeSH
• tvorba protilátek MeSH
• vagina imunologie   mikrobiologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH

Candida albicans hsp90 protein is considered as an immunodominant potentially protective antigen suitable to induce specific T cells and specific antibodies protective against mucosal and disseminated candidiasis. Induction of autoimmunity after immunisation with hsp90 was not yet described unambiguously. Conversely they are some evidences that the anti-hsp response is protective. Consequently hsp90 vaccination is promising alternative to antifungal drug therapy.

Protein hsp90 Candida albicans patří mezi imunodominantní potenciálně protektivní antigeny schopné indukovat specifické T lymfocyty a imunoglobulíny jež mohou přispět ke kontrole mukózní ale i systémové kandidózy. Vznik autoimunity po imunizaci hsp90 nebyl jednoznačně potvrzen a spíše se ukazuje možný protektivní efekt při kontrole autoimunitních procesů. Hsp90 vakcinační strategie je tedy slibnou alternativou k současné terapii antifungálními látkami. Nalezení optimální formy hsp90 vakcinace zajišťující ochranu vakcinovaných zvířat během modelové kandidózy je prvním krokem při vývoji hsp90 vakcíny pro prevenci a terapii mukózní či systémové kandidózy u člověka.

• MeSH
• autoimunita MeSH
• Candida albicans imunologie   MeSH
• DNA vakcíny imunologie   MeSH
• fungální vakcíny MeSH
• kandidóza vulvovaginální imunologie   prevence a kontrola   terapie   MeSH
• modely nemocí na zvířatech MeSH
• myši MeSH
• proteiny tepelného šoku HSP90 imunologie   terapeutické užití   MeSH
• vakcinace MeSH
• Check Tag
• myši MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• mikrobiologie, lékařská mikrobiologie
• alergologie a imunologie
• gynekologie a porodnictví
• dermatovenerologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR