Cyclic dinucleotides are second messengers in the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, which plays an important role in recognizing tumor cells and viral or bacterial infections. They bind to the STING adaptor protein and trigger expression of cytokines via TANK binding kinase 1 (TBK1)/interferon regulatory factor 3 (IRF3) and inhibitor of nuclear factor-κB (IκB) kinase (IKK)/nuclear factor-κB (NFκB) signaling cascades. In this work, we describe an enzymatic preparation of 2'-5',3'-5'-cyclic dinucleotides (2'3'CDNs) with use of cyclic GMP-AMP synthases (cGAS) from human, mouse, and chicken. We profile substrate specificity of these enzymes by employing a small library of nucleotide-5'-triphosphate (NTP) analogues and use them to prepare 33 2'3'CDNs. We also determine affinity of these CDNs to five different STING haplotypes in cell-based and biochemical assays and describe properties needed for their optimal activity toward all STING haplotypes. Next, we study their effect on cytokine and chemokine induction by human peripheral blood mononuclear cells (PBMCs) and evaluate their cytotoxic effect on monocytes. Additionally, we report X-ray crystal structures of two new CDNs bound to STING protein and discuss structure-activity relationship by using quantum and molecular mechanical (QM/MM) computational modeling.

• MeSH
• biotest MeSH
• cytokiny metabolismus   MeSH
• HEK293 buňky MeSH
• konformace proteinů MeSH
• leukocyty mononukleární účinky léků   MeSH
• lidé MeSH
• membránové proteiny chemie   metabolismus   MeSH
• nukleotidy cyklické chemická syntéza   farmakologie   MeSH
• počítačová simulace MeSH
• regulace genové exprese účinky léků   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The exceptionally high cellular uptake of gold nanorods (GNRs) bearing cationic surfactants makes them a promising tool for biomedical applications. Given the known specific toxic and stress effects of some preparations of cationic nanoparticles, the purpose of this study was to evaluate, in an in vitro and in vivo in mouse, the potential harmful effects of GNRs coated with (16-mercaptohexadecyl)trimethylammonium bromide (MTABGNRs). Interestingly, even after cellular accumulation of high amounts of MTABGNRs sufficient for induction of photothermal effect, no genotoxicity (even after longer-term accumulation), induction of autophagy, destabilization of lysosomes (dominant organelles of their cellular destination), alterations of actin cytoskeleton, or in cell migration could be detected in vitro. In vivo, after intravenous administration, the majority of GNRs accumulated in mouse spleen followed by lungs and liver. Microscopic examination of the blood and spleen showed that GNRs interacted with white blood cells (mononuclear and polymorphonuclear leukocytes) and thrombocytes, and were delivered to the spleen red pulp mainly as GNR-thrombocyte complexes. Importantly, no acute toxic effects of MTABGNRs administered as 10 or 50 μg of gold per mice, as well as no pathological changes after their high accumulation in the spleen were observed, indicating good tolerance of MTABGNRs by living systems.

• MeSH
• autofagie účinky léků   MeSH
• kvartérní amoniové sloučeniny metabolismus   MeSH
• lidé MeSH
• lyzozomy účinky léků   metabolismus   MeSH
• mezenchymální kmenové buňky cytologie   účinky léků   MeSH
• mikrofilamenta účinky léků   metabolismus   MeSH
• mutageny toxicita   MeSH
• myši inbrední C57BL MeSH
• nádorové buněčné linie MeSH
• nanotrubičky chemie   toxicita   ultrastruktura   MeSH
• podocyty účinky léků   metabolismus   MeSH
• pohyb buněk účinky léků   MeSH
• poškození DNA MeSH
• slezina účinky léků   patologie   MeSH
• sulfhydrylové sloučeniny metabolismus   MeSH
• tkáňová distribuce MeSH
• trombocyty účinky léků   patologie   ultrastruktura   MeSH
• zlato metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Dendritic cell (DC)-based vaccines pulsed with high hydrostatic pressure (HHP)-inactivated tumor cells have recently been shown to be a promising tool for prostate cancer chemoimmunotherapy. In this study, DC-based vaccines, both pulsed and unpulsed, were as effective as docetaxel (DTX) in reducing prostate tumors in the orthotopic transgenic adenocarcinoma of the mouse prostate (TRAMP) model. However, we did not observe any additive or synergic effects of chemoimmunotherapy on the tumor growth, while only the combination of DTX and pulsed dendritic cells resulted in significantly lower proliferation detected by Ki67 staining in histological samples. The DC-based vaccine pulsed with HHP-treated tumor cells was also combined with another type of cytostatic, cyclophosphamide, with similar results. In another clinically relevant setting, minimal residual tumor disease after surgery, administration of DC-based vaccines after the surgery of poorly immunogenic transplanted TRAMP-C2, as well as in immunogenic TC-1 tumors, reduced the growth of tumor recurrences. To identify the effector cell populations after DC vaccine application, mice were twice immunized with both pulsed and unpulsed DC vaccine, and the cytotoxicity of the spleen cells populations was tested. The effector cell subpopulations were defined as CD4+ and NK1.1+, which suggests rather unspecific therapeutic effects of the DC-based vaccines in our settings. Taken together, our data demonstrate that DC-based vaccines represent a rational tool for the treatment of human prostate cancer.

• Publikační typ
• časopisecké články MeSH

High hydrostatic pressure (HHP) has been shown to induce immunogenic cell death of cancer cells, facilitating their uptake by dendritic cells (DC) and subsequent presentation of tumor antigens. In the present study, we demonstrated immunogenicity of the HHP-treated tumor cells in mice. HHP was able to induce immunogenic cell death of both TC-1 and TRAMP-C2 tumor cells, representing murine models for human papilloma virus-associated tumors and prostate cancer, respectively. HHP-treated cells induced stronger immune responses in mice immunized with these tumor cells, documented by higher spleen cell cytotoxicity and increased IFNγ production as compared to irradiated tumor cells, accompanied by suppression of tumor growth in vivo in the case of TC-1 tumors, but not TRAMP-C2 tumors. Furthermore, HHP-treated cells were used for DC-based vaccine antigen pulsing. DC co-cultured with HHP-treated tumor cells and matured by a TLR 9 agonist exhibited higher cell surface expression of maturation markers and production of IL-12 and other cytokines, as compared to the DC pulsed with irradiated tumor cells. Immunization with DC cell-based vaccines pulsed with HHP-treated tumor cells induced high immune responses, detected by increased spleen cell cytotoxicity and elevated IFNγ production. The DC-based vaccine pulsed with HHP-treated tumor cells combined with docetaxel chemotherapy significantly inhibited growth of both TC-1 and TRAMP-C2 tumors. Our results indicate that DC-based vaccines pulsed with HHP-inactivated tumor cells can be a suitable tool for chemoimmunotherapy, particularly with regard to the findings that poorly immunogenic TRAMP-C2 tumors were susceptible to this treatment modality.

• MeSH
• antigeny nádorové metabolismus   MeSH
• antitumorózní látky aplikace a dávkování   MeSH
• cytotoxicita imunologická MeSH
• dendritické buňky cytologie   MeSH
• experimentální nádory farmakoterapie   terapie   MeSH
• hydrostatický tlak MeSH
• imunitní systém MeSH
• imunoterapie metody   MeSH
• infekce papilomavirem farmakoterapie   terapie   MeSH
• interferon gama metabolismus   MeSH
• interleukin-12 metabolismus   MeSH
• lidé MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádory prostaty farmakoterapie   metabolismus   terapie   MeSH
• protinádorové vakcíny chemie   MeSH
• slezina imunologie   MeSH
• taxoidy aplikace a dávkování   MeSH
• toll-like receptor 9 metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Standard-of-care chemo- or radio-therapy can induce, besides tumor cell death, also tumor cell senescence. While senescence is considered to be a principal barrier against tumorigenesis, senescent cells can survive in the organism for protracted periods of time and they can promote tumor development. Based on this emerging concept, we hypothesized that elimination of such potentially cancer-promoting senescent cells could offer a therapeutic benefit. To assess this possibility, here we first show that tumor growth of proliferating mouse TC-1 HPV-16-associated cancer cells in syngeneic mice becomes accelerated by co-administration of TC-1 or TRAMP-C2 prostate cancer cells made senescent by pre-treatment with the anti-cancer drug docetaxel, or lethally irradiated. Phenotypic analyses of tumor-explanted cells indicated that the observed acceleration of tumor growth was attributable to a protumorigenic environment created by the co-injected senescent and proliferating cancer cells rather than to escape of the docetaxel-treated cells from senescence. Notably, accelerated tumor growth was effectively inhibited by cell immunotherapy using irradiated TC-1 cells engineered to produce interleukin IL-12. Collectively, our data document that immunotherapy, such as the IL-12 treatment, can provide an effective strategy for elimination of the detrimental effects caused by bystander senescent tumor cells in vivo.

• MeSH
• antitumorózní látky farmakologie   MeSH
• bystander efekt účinky léků   MeSH
• časové faktory MeSH
• cytokiny genetika   metabolismus   MeSH
• experimentální nádory genetika   metabolismus   terapie   MeSH
• imunoterapie adoptivní metody   MeSH
• interleukin-12 biosyntéza   farmakologie   MeSH
• kombinovaná terapie MeSH
• myši inbrední C57BL MeSH
• nádorové buněčné linie MeSH
• stárnutí buněk účinky léků   MeSH
• taxoidy farmakologie   MeSH
• tumor burden účinky léků   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Downregulation of MHC class I expression on tumour cells, a common mechanism by which tumour cells can escape from specific immune responses, can be associated with coordinated silencing of antigen-presenting machinery genes. The expression of these genes can be restored by IFNγ. In this study we documented association of DNA demethylation of selected antigen-presenting machinery genes located in the MHC genomic locus (TAP-1, TAP-2, LMP-2, LMP-7) upon IFNγ treatment with MHC class I upregulation on tumour cells in several MHC class I-deficient murine tumour cell lines (TC-1/A9, TRAMP-C2, MK16 and MC15). Our data also documented higher methylation levels in these genes in TC-1/A9 cells, as compared to their parental MHC class I-positive TC-1 cells. IFNγ-mediated DNA demethylation was relatively fast in comparison with demethylation induced by DNA methyltransferase inhibitor 5-azacytidine, and associated with increased histone H3 acetylation in the promoter regions of APM genes. Comparative transcriptome analysis in distinct MHC class I-deficient cell lines upon their treatment with either IFNγ or epigenetic agents revealed that a set of genes, significantly enriched for the antigen presentation pathway, was regulated in the same manner. Our data demonstrate that IFNγ acts as an epigenetic modifier when upregulating the expression of antigen-presenting machinery genes.

• MeSH
• down regulace MeSH
• epigeneze genetická MeSH
• fibrosarkom genetika   imunologie   metabolismus   MeSH
• geny MHC třídy I * MeSH
• interferon gama genetika   imunologie   metabolismus   MeSH
• metylace DNA * MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorové buňky kultivované MeSH
• prezentace antigenu genetika   MeSH
• regulace genové exprese u nádorů MeSH
• signální transdukce MeSH
• transfekce MeSH
• upregulace MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

CD4(+)CD25(+)Foxp3(+) T regulatory cells (Tregs) and CD1d-restricted invariant natural killer T (iNKT) cells are two cell types that are known to regulate immune reactions. Depletion or inactivation of Tregs using specific anti-CD25 antibodies in combination with immunostimulation is an attractive modality especially in anti-tumour immunotherapy. However, CD25 is not expressed exclusively on Tregs but also on subpopulations of activated lymphocytes. Therefore, the modulatory effects of the specific anti-CD25 antibodies can also be partially attributed to their interactions with the effector cells. Here, the effector functions of iNKT cells were analysed in combination with anti-CD25 mAb PC61. Upon PC61 administration, α-galactosylceramide (α-GalCer)-mediated activation of iNKT cells resulted in decreased IFN-γ but not IL-4 production. In order to determine whether mutual interactions between Tregs and iNKT cells take place, we compared IFNγ production after α-GalCer administration in anti-CD25-treated and "depletion of regulatory T cell" (DEREG) mice. Since no profound effects on IFNγ induction were observed in DEREG mice, deficient in FoxP3(+) Tregs, our results indicate that the anti-CD25 antibody acts directly on CD25(+) effector cells. In vivo experiments demonstrated that although both α-GalCer and PC61 administration inhibited TC-1 tumour growth in mice, no additive/synergic effects were observed when these substances were used in combination therapy.

• MeSH
• antigeny CD1d imunologie   metabolismus   MeSH
• ELISA MeSH
• experimentální nádory farmakoterapie   imunologie   patologie   MeSH
• exprese genu účinky léků   imunologie   MeSH
• forkhead transkripční faktory imunologie   metabolismus   MeSH
• galaktosylceramidy aplikace a dávkování   imunologie   farmakologie   MeSH
• interferon gama genetika   imunologie   metabolismus   MeSH
• interleukin-4 genetika   imunologie   metabolismus   MeSH
• Kaplanův-Meierův odhad MeSH
• mezibuněčné signální peptidy a proteiny genetika   imunologie   metabolismus   MeSH
• monoklonální protilátky aplikace a dávkování   imunologie   farmakologie   MeSH
• myši inbrední C57BL MeSH
• myši transgenní MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• NKT buňky účinky léků   imunologie   metabolismus   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• protokoly antitumorózní kombinované chemoterapie terapeutické užití   MeSH
• průtoková cytometrie MeSH
• receptor interleukinu-2 - alfa-podjednotka imunologie   metabolismus   MeSH
• regulační T-lymfocyty účinky léků   imunologie   metabolismus   MeSH
• tumor burden účinky léků   imunologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Downregulation of MHC class I expression on the cell surface is a common mechanism by which tumour cells, including cervical carcinoma, can escape the T cell-mediated anti-tumour immunity. This downregulation represents an obstacle for the efficacy of anti-tumour vaccines. In this study, we investigated the efficacy of prophylactic peptide and peptide-pulsed dendritic cell-based vaccines in a murine model of experimental MHC class I-deficient tumours (TC-1/A9), expressing E6/E7 oncogenes derived from HPV16, and compared the efficacy of particular vaccination settings to anti-tumour protection against parental MHC class I-positive TC-1 tumours. Peptide vaccine based on the 'short' peptide E749-57 harbouring solely the CTL epitope and co-administered to the C57BL/6 mice with CpG oligodeoxynucleotide (CpG ODN) 1826 was effective against MHC class I-positive but not -deficient tumours, while the 'longer' peptide E744-62 (peptide 8Q, harbouring CTL and Th epitopes)-based vaccines were also effective against MHC class I-deficient tumours. We have compared the adjuvant efficacies of two CpG ODN, CpG ODN 1826 and CpG ODN 1585. The 8Q peptide immunisation combined with CpG ODN 1585 inhibited growth of the TC-1/A9 tumours more effectively as compared to CpG ODN 1826. Further, we investigated the efficacy of cellular vaccines based on ex vivo cultured dendritic cells pulsed with either E749-57 or E744-62 peptides and matured with CpG ODN 1826. Unlike in the peptide immunisation setting, treatment with dendritic cells pulsed with a 'short' peptide resulted in the tumour growth inhibition, albeit weaker as compared to the immunisation with the longer peptide. Our data demonstrate that peptide and dendritic cell-based vaccines can be designed to elicit protective immunity against MHC class I-deficient tumours.

• MeSH
• adjuvantní radioterapie metody   MeSH
• CpG ostrůvky MeSH
• dendritické buňky cytologie   MeSH
• epitopy chemie   MeSH
• geny MHC třídy I MeSH
• lidé MeSH
• myši MeSH
• oligonukleotidy genetika   MeSH
• Papillomavirus E7 - proteiny chemie   MeSH
• peptidy chemie   MeSH
• protinádorové vakcíny chemie   MeSH
• průtoková cytometrie MeSH
• regulace genové exprese MeSH
• subjednotkové vakcíny genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH