RESUMO
We have determined in mice the minimum composition required for forming a vaccine adjuvant that stimulates a regulatory T (Treg) cell response to immunization, and we named the adjuvant "complete tolerogenic adjuvant." This new kind of adjuvant may let us use the well-proven "Ag with adjuvant" form of immunization for inducing Treg cell-mediated Ag-specific immunosuppression. The minimum composition consists of dexamethasone, rapamycin, and monophosphoryl lipid A at a mass ratio of 8:20:3. By dissecting the respective role of each of these components during immunization, we have further shown why immunosuppressive and immunogenic agents are both needed for forming true adjuvants for Treg cells. This finding may guide the design of additional, and potentially more potent, complete tolerogenic adjuvants with which we may form numerous novel vaccines for treating immune diseases.
Assuntos
Linfócitos T Reguladores , Vacinas , Camundongos , Animais , Imunização , Adjuvantes Imunológicos/farmacologia , ImunossupressoresRESUMO
Extracellular high-mobility group box 1 (HMGB1) is a prototypic damage-associated molecular pattern. Although a homeostatic level of extracellular HMGB1 may be beneficial for immune defense, tissue repair, and tissue regeneration, excessive HMGB1 is linked to inflammatory diseases. This prompts an intriguing question: how does a healthy body control the level of extracellular HMGB1? In this study, in the plasma of both healthy humans and healthy mice, we have identified an anti-HMGB1 IgM autoantibody that neutralizes extracellular HMGB1 via binding specifically to a 100% conserved epitope, namely HMW4 (HMGB198-112). In mice, this anti-HMW4 IgM is produced by peritoneal B-1 cells, and concomitant triggering of their BCR and TLR4 by extracellular HMGB1 stimulates the production of anti-HMW4 IgM. The ability of extracellular HMGB1 to induce its own neutralizing Ab suggests a feedback loop limiting the level of this damage-associated molecular pattern in a healthy body.
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Anticorpos Neutralizantes/sangue , Autoanticorpos/sangue , Subpopulações de Linfócitos B/imunologia , Epitopos/imunologia , Proteína HMGB1/imunologia , Imunoglobulina M/sangue , Adulto , Animais , Apolipoproteínas E/genética , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos B/metabolismo , Receptor 4 Toll-Like/metabolismo , Adulto JovemRESUMO
Maize (Zea mays L.) is one of the most important crops worldwide. In September 2020, five maize plants and soil samples were received from a farmer. The samples were collected from a maize field in Fugu County (N 39.02805, E 111.06723), Shaanxi Province, China. These maize plants had the symptoms of stunting or thinning. Soil nematodes were extracted from soils with modified Baermann funnel method for 48 h (Barker 1985). Trichotylenchus sp. were detected with populations ranging from 12 to 45 (in five soil samples) nematodes per 100 gram of fresh soil. Maize were planted in pots with soil samples containing Trichotylenchus sp. to propagate this plant-parasitic nematode at 20-28 °C in a greenhouse. After plants had grown for 60 days, active Trichotylenchus sp. were observed and picked under an anatomical lens (SZX16, Olympus, Tokyo, Japan) for an inoculation experiment. Freshly germinated maize (Limin 33) seeds were planted into paper pots with a sterilized mixture of soil and sand (4:1). Four plants were inoculated with approximately 2800 mixed-stage nematodes. The maize plants were allowed to grow under room conditions (15-20 °C) for two weeks and transferred to plastic pots (12 cm diameter × 10 cm deep) with the same soil mixture. After 30 days, all inoculated plants displayed the disease symptoms such as hypoplasia of fibrous roots and a hole at the base of the maize stem, but the Trichotylenchus sp. was not detected in maize roots and stems. The control plants did not exhibit any disease symptoms. The nematodes from the inoculated pots were identified by both morphological and molecular methods. The bodies of female and male were usually C-shaped when killed by hot water. The stylet was well developed with basal knobs. The posterior part of esophagus basal bulb did not overlap dorsally with the intestine. The female had a subcylindrical tail, but the male had a conical tail. The spicule of male was ventrally curved. The gubernaculum was well developed. Morphometric data based on females and males were presented as means (range). For female (n= 20): L = 1142.7 (1002.3 to 1313.4) µm, St = 26.5 (23.9 to 29.4) µm, a = 32.8 (27.4 to 38.7), b = 6.8 (6.0 to 7.9), c = 16.3 (14.9 to 19.4), c' = 2.7 (2.3 to 3.1), v = 51.3 (30.3 to 54.8), T = 70.1 (59.0 to 78.9) µm. For male (n= 20): L = 1093.3 (960.6 to 1183.7) µm, St = 24.3 (22.1 to 26.1) µm, a = 34.9 (31.1 to 39.1), b = 6.5 (5.6 to 7.4), c = 15.1 (13.6 to 16.7), c' = 3.6 (2.9 to 4.3), T = 72.5 (63.8 to 81.1) µm, SL = 28.8 (22.2 to 32.5) µm, GL = 11.5 (7.2 to 16.1) µm. The characteristics were coincident with the description of Trichotylenchus changlingensis (Guo et a., 2015). Genomic DNA was extracted from 10 nematodes, and PCR amplifications of the D2/D3 region of 28S rRNA were performed using universal primers D2A/D3B (Castillo et al. 2003), and that of the internal transcribed spacer (ITS)-rRNA region were amplified using universal primers TW81/AB28 (Amiri et al. 2002). A 780 bp amplicon of the 28S rRNA (GenBank accession no. OM276857.1) had 90.6% (Query Cover = 95%) identity with that of Tylenchorhynchus mediterraneus (KJ461557.1). Three ITS-rRNA region sequences (GenBank accession nos. OM294652.1, OM294653.1 and OM294654.1) were obtained, and the comparison revealed that sequences OM294652.1 and OM294653.1 had 98.90% (Query Cover = 100%) identity with the ITS-rRNA region sequence of T. changlingensis isolate (MH545694.1) from China, and OM294654.1 showed a 99.09 % (Query Cover = 99%) similarity to T. changlingensis sequence (MH545693.1). The morphological and molecular characterizations confirmed that the observed nematode was T. changlingensis, which is distributed in Heilongjiang, Jilin, Liaoning, Hebei, Gansu provinces and Inner Mongolia of China (Guo et al. 2020). To our knowledge, this is the first report of T. changlingensis infecting maize and causing disease in Shaanxi Province.
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The immunosuppressant dexamethasone was shown to preferentially deplete CD4+ effector T cells while sparing regulatory T cells (Tregs) in vivo. In the current study, we show that it also preferentially depletes B-2 cells while sparing B-1 cells. In the ApoE(-/-) mouse model of atherosclerosis, in which both Tregs and B-1 cells are thought to play an atheroprotective role, we show that HSP60-targeted immunization in the presence of dexamethasone raises Ag-reactive Tregs and B-1 cells concomitantly and reduces the severity of atherosclerosis. These results indicate that dexamethasone is an adjuvant that potentiates both the Treg and B-1 responses to immunogens. This study shows that B-1 cells with a specificity for a disease-relevant Ag can be raised in vivo by immunization.
Assuntos
Adjuvantes Imunológicos/farmacologia , Antígenos/farmacologia , Aterosclerose/imunologia , Subpopulações de Linfócitos B/imunologia , Dexametasona/farmacologia , Imunização , Linfócitos T Reguladores/imunologia , Animais , Anti-Inflamatórios/farmacologia , Apolipoproteínas E , Aterosclerose/genética , Aterosclerose/patologia , Subpopulações de Linfócitos B/patologia , Modelos Animais de Doenças , Camundongos , Camundongos Knockout , Linfócitos T Reguladores/patologiaRESUMO
We previously showed that antigen immunization in the presence of the immunosuppressant dexamethasone (a strategy we termed "suppressed immunization") could tolerize established recall responses of T cells. However, the mechanism by which dexamethasone acts as a tolerogenic adjuvant has remained unclear. In the present study, we show that dexamethasone enriches CD11c(lo) CD40(lo) macrophages in a dose-dependent manner in the spleen and peripheral lymph nodes of mice by depleting all other CD11c(+) CD40(+) cells including dendritic cells. The enriched macrophages display a distinct MHC class II (MHC II)(lo) CD86(hi) phenotype. Upon activation by antigen in vivo, CD11c(lo) CD40(lo) macrophages upregulate IL-10, a classic marker for tolerogenic antigen-presenting cells, and elicit a serum IL-10 response. When presenting antigen in vivo, these cells do not elicit recall responses from memory T cells, but rather stimulate the expansion of antigen-specific regulatory T cells. Moreover, the depletion of CD11c(lo) CD40(lo) macrophages during suppressed immunization diminishes the tolerogenic efficacy of the treatment. These results indicate that dexamethasone acts as a tolerogenic adjuvant partly by enriching the CD11c(lo) CD40(lo) tolerogenic macrophages.
Assuntos
Dexametasona/administração & dosagem , Hipersensibilidade Tardia/tratamento farmacológico , Imunossupressores/administração & dosagem , Interleucina-10/imunologia , Macrófagos/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Animais , Antígeno B7-2/metabolismo , Antígenos CD11/metabolismo , Antígenos CD40/metabolismo , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Dexametasona/efeitos adversos , Antígenos de Histocompatibilidade Classe II/metabolismo , Hipersensibilidade Tardia/imunologia , Tolerância Imunológica , Imunossupressores/efeitos adversos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
PURPOSE AND EXPERIMENTAL DESIGN: Recombinant human IL-2 (rhIL-2) is a potent cytokine and FDA-approved anticancer drug. However, its clinical use has been limited by severe toxicity, associated primarily with systemic administration with excess protein distributing freely throughout the body. We hypothesized that rhIL-2 in alternate forms permitting more restricted localization may exert stronger antitumor efficacy and less toxicity. Here, we have tested the utility of palmitate-derivatized rhIL-2. rhIL-2 was reacted with N-hydroxysuccinimide palmitate ester. The resultant lipidated rhIL-2 (pIL-2), when mixed with cells, could spontaneously transfer from solution to cell surfaces. Next, anticancer efficacy of pIL-2 was assessed in two modalities. For adoptive T cell therapy, antitumor cytotoxic T cells (CTLs) were protein transferred ("painted") with pIL-2 and injected into mice bearing lymphoma. For in situ therapy, pIL-2 was injected intratumorally into mice bearing melanoma. Tumor growth and IL-2-associated toxicity were determined. RESULTS: In the lymphoma model, painting of the antitumor CTLs with pIL-2 markedly increased their viability and titer. In the melanoma model, intratumoral injection of pIL-2, but not rhIL-2, increased the number of activated CD8(+) T cells (IFN-γ(+)) in the spleen, reduced lung metastasis and prolonged the survival of treated mice. Moreover, while repeated intratumoral injection of rhIL-2 at an excessively high dose (10 injections of 10,000 IU/mouse) caused marked vascular leakage syndrome, the same regimen using pIL-2 caused no detectable toxicity. CONCLUSIONS: Transferring spontaneously from solution to cell surfaces, pIL-2 may bypass the current limitations of rhIL-2 and, thus, serve as a more effective and tolerable anticancer drug.
Assuntos
Imunoterapia Adotiva/métodos , Interleucina-2/administração & dosagem , Linfoma/terapia , Melanoma Experimental/terapia , Proteínas Recombinantes/administração & dosagem , Animais , Humanos , Interleucina-2/efeitos adversos , Interleucina-2/genética , Camundongos , Camundongos Transgênicos , Ácido Palmítico , Proteínas Recombinantes/efeitos adversos , Succinimidas , Linfócitos T Citotóxicos/transplanteRESUMO
Expression of receptor for advanced glycation end products (RAGE) plays a key role in the progression of prostate cancer. However, the therapeutic potential of targeting RAGE expression in prostate cancer is not yet evaluated. Therefore in this study, we have investigated the effects of silencing the expression of RAGE by RNAi approach both in vitro and in vivo. The results of this study showed that down regulation of RAGE expression by RNAi inhibited the cell proliferation of androgen-dependent (LNCaP) and androgen-independent (DU-145) prostate cancer cells. Furthermore, targeting RAGE expression resulted in apoptotic elimination of these prostate cancer cells by activation of caspase-8 and caspase-3 death signaling. Of note, the levels of prostate specific antigen (PSA) were also reduced in LNCaP cells transfected with RAGE RNAi constructs. Importantly, the RAGE RNAi constructs when administered in nude mice bearing prostate tumors, inhibited the tumor growth by targeting the expression of RAGE, and its physiological ligand, HMGB1 and by up regulating death receptors DR4 and DR5 expression. Collectively, the results of this study for the first time show that targeting RAGE by RNAi may be a promising alternative therapeutic strategy for treating prostate cancer.
Assuntos
Apoptose , Proliferação de Células , Proteína HMGB1/metabolismo , Terapia de Alvo Molecular , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapia , Animais , Caspases/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Ligantes , Masculino , Camundongos , Camundongos Nus , RNA Interferente Pequeno/genética , Receptor para Produtos Finais de Glicação Avançada/genética , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
DNA vaccines have been widely used to induce immune responses against molecular targets. In this study, we explored the possibility of using DNA vaccine combined with the immunosuppressant FK506 (tacrolimus) to antigen-specifically suppress unwanted immune responses and prevent autoimmune ovarian disease. To that end, we immunized C57BL/6 mice with a DNA vaccine encoding mouse zona pellucida 3 (ZP3) together with FK506. The immunization induced ZP3-specific CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg), which suppressed the induction of ZP3-specific delayed-type hypersensitivity in the animals. Significantly, the immunization also protected the animals from experimentally induced autoimmune ovarian disease. These results suggest that DNA vaccination in the presence of FK506 may be used to induce Treg cells and prevent AOD.
Assuntos
Doenças Autoimunes/prevenção & controle , Proteínas do Ovo/metabolismo , Hipersensibilidade Tardia/imunologia , Glicoproteínas de Membrana/metabolismo , Doenças Ovarianas/prevenção & controle , Receptores de Superfície Celular/metabolismo , Linfócitos T Reguladores/metabolismo , Vacinas de DNA/administração & dosagem , Animais , Doenças Autoimunes/imunologia , Antígenos CD4/metabolismo , Células Cultivadas , Proteínas do Ovo/administração & dosagem , Proteínas do Ovo/genética , Proteínas do Ovo/imunologia , Feminino , Fatores de Transcrição Forkhead/metabolismo , Hipersensibilidade Tardia/induzido quimicamente , Hipersensibilidade Tardia/prevenção & controle , Tolerância Imunológica , Imunização , Imunossupressores/administração & dosagem , Imunossupressores/efeitos adversos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Glicoproteínas de Membrana/administração & dosagem , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Doenças Ovarianas/imunologia , Receptores de Superfície Celular/administração & dosagem , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Tacrolimo/administração & dosagem , Tacrolimo/efeitos adversos , Glicoproteínas da Zona PelúcidaRESUMO
Maize is one of the most important crops in the world. Heilongjiang province has the largest maize area in China. Plant-parasitic nematodes are important agricultural pests, which cause huge economic losses every year and have attracted global attention. Potato rot nematode Ditylenchus destructor is a plant-parasitic nematode with a wide range of hosts and strong survival ability in different environments, which brings risks to agricultural production. In 2020, D. destructor was detected in seven maize fields in Heilongjiang province. Morphological identification and molecular approach were used to characterize the isolated D. destructor. The observed morphological and morphometric characteristics were highly similar and consistent with the existing description. The DNA sequencing on the D2/D3 region of the ribosomal DNA 28S and the phylogenetic analysis showed that D. destructor population obtained from maize and other isolates infesting carrot, sweet potato, and potato were in subclade I supported by a 96% bootstrap value. Additionally, the phylogenetic analysis of the ITS rRNA gene sequence further indicated that this D. destructor population from maize clustered in a clade I group and belonged to ITS rRNA haplotype C. An inoculation experiment revealed that D. destructor was pathogenic on the maize seedlings in pots and caused the disease symptoms in the stem base of maize seedlings. This is the first report of D. destructor causing stem rot of maize in Heilongjiang province, and contributes additional information on disease control and safe production of maize in the region.
RESUMO
BACKGROUND: DNA vaccination is a strategy that has been developed primarily to elicit protective immunity against infection and cancer. METHODS: DNA vaccine was used, in conjunction with an immunosuppressant, to tolerize harmful autoimmunity. RESULTS: Immunization of C57BL/6 mice with MOG(35-55), a myelin oligodendrocyte glycoprotein-derived peptide, and FK506 (Tacrolimus) as a tolerogenic adjuvant stimulated regulatory dendritic cells, induced antigen-specific regulatory T cells (Treg), and protected the animals from subsequent induction of experimental autoimmune encephalomyelitis (EAE). After EAE induction, there were fewer lymphocytes, including fewer T helper 17 cells, and more Treg infiltrating the spinal cord in the immunized mice compared to in control mice. Furthermore, at the peak of the EAE manifestation, CD4 T cells in the immunized mice showed decreased expression of interferon-gamma and interleukin (IL)-17, but not IL-4, in treated mice. CONCLUSIONS: DNA vaccination, when applied with an immunosuppressant as adjuvant, can induce antigen-specific tolerance and prevent autoimmune disease.
Assuntos
Adjuvantes Imunológicos/farmacologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/prevenção & controle , Imunossupressores/farmacologia , Tacrolimo/farmacologia , Vacinas de DNA/imunologia , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas da Mielina , Glicoproteína Associada a Mielina/genética , Glicoproteína Associada a Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito , VacinaçãoRESUMO
Translationally controlled tumor protein (TCTP) is a novel anti apoptotic protein which is highly expressed in several cancer cell types including prostate cancer. However, studies investigating the role of TCTP in prostate cancer are scarce. Therefore, in this study we evaluated the effect of small interference RNA (siRNA) based knocking down of TCTP gene in prostate cancer cells. Cell proliferation and apoptosis were evaluated. Our results showed that TCTP is highly expressed in LNCaP cells compared to normal prostate epithelial cells. Transfection with TCTP siRNA specifically and drastically reduced the expression of both mRNA and protein levels of TCTP in LNCaP cells. The decreased expression of TCTP was associated with decreased viability of LNCaP cells. Further analysis of the transfected LNCaP cells showed that they undergo apoptosis via caspase-8 and caspase-3 dependent pathways. Results presented herein suggest a potential therapeutic application for prostate cancer by targeting TCTP gene using an siRNA approach.
Assuntos
Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Neoplasias da Próstata/patologia , RNA Interferente Pequeno/farmacologia , Biomarcadores Tumorais/genética , Caspase 3/metabolismo , Caspase 8/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/fisiologia , Humanos , Masculino , Neoplasias da Próstata/genética , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
One strategy for improving adoptive therapy is preconditioning the host immune environment by depleting CD4(+)CD25(+) regulatory T cells (Treg) suppressive to antitumor responses. Given that Treg increase, or selectively accumulate, within tumors and are sensitive to FasL-mediated apoptosis, we test here the hypothesis that inducing apoptosis of intratumoral Treg using FasL may improve adoptive T cell therapy. We show that FasL applied intratumorally via protein transfer decreases intratumoral Treg via inducing apoptosis in these cells. Significantly, we show that the use of FasL prior to the infusion of tumor-reactive CD8(+) T cells enhances the therapeutic efficacy of adoptive T cell transfer against established tumors, which is mediated by persistent, systemic antitumor immunity. Intratumoral FasL protein transfer also results in neutrophil infiltration of tumor. However, we show that intratumoral immunodepletion of neutrophils does not abolish the effect of FasL on adoptive transfer. Rather, the effect of FasL is completely abolished by cotransfer of Treg, isolated from the tumor-draining lymph nodes. Hence, our study shows for the first time that using FasL to predeplete intratumoral Treg provides a useful means for optimizing adoptive therapy.
Assuntos
Proteína Ligante Fas/imunologia , Imunoterapia Adotiva/métodos , Subunidade alfa de Receptor de Interleucina-2/imunologia , Linfoma/imunologia , Linfoma/terapia , Linfócitos T Reguladores/imunologia , Animais , Apoptose/imunologia , Subunidade alfa de Receptor de Interleucina-2/biossíntese , Leucemia L5178 , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Neutrófilos/imunologiaRESUMO
Non-steroidal anti-inflammatory drugs (NSAID) have shown promise as anticancer agents by inducing cell death apart from their antipyretic, anti-inflammatory and anti-thrombogenic effects. In our current study, we investigated the oxidative stress mediated cell death mechanism of a NSAID derivative NCX4040 (a nitric oxide (NO) releasing form of aspirin) in castration-resistant prostate cancer (CRPC) PC3 cell line. Our data revealed that NCX4040 is more potent than its parent compound aspirin or NO releasing compound DETA NONOate. NCX4040 significantly induced hydrogen peroxide formation with ensuing oxidative stress and mitochondrial depolarization resulting in lipid peroxidation, cell cycle arrest, inhibition of colony growth and induction of apoptosis in PC3 cells. Moreover, NCX4040 inhibited migration potential of PC3 cells by depolymerizing F-actin and promoting anoikis. Interestingly, elevated levels of NADPH oxidase 1 (NOX1), superoxide dismutase (SOD) 1 and 2 were observed upon NCX4040 treatment. However, down regulation of anti-apoptotic markers B-cell lymphoma 2 (Bcl2) and anti-oxidant thioredoxin reductase 1 (TXNRD1) expression were observed. In addition, NCX4040 down regulated cyclin D1 expression in PC3 cells further supporting the anticancer effect of NCX4040. Western blot analysis revealed that significant down regulation of key anti-apoptotic markers such as cellular inhibitor of apoptosis protein-1 (cIAP1), X-linked inhibitor of apoptosis (XIAP), survivin, and Cellular-Myc (c-Myc). On the other hand, NCX4040-treated cells showed upregulation of phosho histone H2AX (pH2AX), cleaved caspase3 and cleaved Poly [ADP-ribose] polymerase 1 (PARP1). Taken together, our data demonstrate that NCX4040 treatment enhances free radical formation which in turn induces oxidative stress leading to mitochondrial mediated cell death in metastatic PC3 cells.
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Apoptose/efeitos dos fármacos , Aspirina/análogos & derivados , Peróxido de Hidrogênio/farmacologia , Nitrocompostos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Neoplasias da Próstata/patologia , Aspirina/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Masculino , Oxidantes/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Células Tumorais Cultivadas , CicatrizaçãoRESUMO
PURPOSE: The modification of therapeutic dendritic cells (DC) with various immunostimulatory molecules represents a useful means for improving the antitumor efficacy of DC transfer-based immunotherapy. We have evaluated the feasibility of modifying therapeutic DCs with multiple immunostimulatory molecules using a time-efficient, protein transfer (or protein "painting")-based method. EXPERIMENTAL DESIGN: Bone marrow-derived DCs were painted with either control protein human IgG (hIgG) or three immunostimulatory molecules, SLC, 4-1BBL, and TRANCE (the triad protein). Painted DCs were injected intratumorally into mice bearing established tumors. Subsequently, the capacities of painted DCs to migrate to the draining lymph nodes, recruit the host T cells, promote Th1 cytokine responses, and elicit therapeutic antitumor responses were evaluated. RESULTS: The triad protein transfer yields a uniform population of DCs that coexpress all three of the proteins. Compared with the hIgG-painted DCs, the triad protein-painted DCs migrate more efficiently to the draining lymph nodes and show enhanced capabilities to induce T cell infiltration of tumors and to promote Th1 cytokine responses in vivo. Furthermore, in both the EG.7 and TRAMP-C2 tumor models, compared with the DCs painted with hIgG or only one of the three proteins, the triad protein-painted DCs, upon adoptive transfer, elicit stronger therapeutic responses against established tumors. Importantly, the antitumor responses of the triad protein-painted DCs are mediated by systemic antitumor immunity. CONCLUSIONS: This study establishes, for the first time, the feasibility of optimizing DC transfer-based immunotherapy via combinatorial protein transfer of therapeutic DCs with an array of immunostimulatory molecules.
Assuntos
Antineoplásicos/farmacologia , Células Dendríticas/metabolismo , Imunoterapia Adotiva/instrumentação , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Vacinas Anticâncer , Transplante de Células , Feminino , Fluoresceínas/farmacologia , Humanos , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Imunoterapia Adotiva/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Succinimidas/farmacologia , Células Th1/metabolismo , Fatores de TempoRESUMO
T cells ectopically expressing costimulators are pathogenic and contribute to autoimmunity against self-antigens. Given that tumor antigens are often self-antigen or mutated self-antigens, we hypothesize that neoexpressing a costimulator on tumor-reactive T cells may likewise enhance their reactivity to tumor. To test this hypothesis, we have expressed B7-1 on OT-1 CD8+ T-cell receptor transgenic T cells via protein transfer (or protein "painting"). Naïve OT-1 T cells, after being painted with B7-1, can self-costimulate themselves, elicit enhanced proliferative and CTL responses to E.G7-ovalbumin tumor cells (expressing a cognate antigen), and become resistant to CD4+CD25+ regulatory T-cell-mediated suppression. Importantly, these T cells, when coimplanted with E.G7-ovalbumin tumor cells into a syngeneic host, are three to nine times more potent than are control T cells (mock painted with human IgG) in inhibiting tumor growth. Further, on transfer into mice bearing established E.G7-ovalbumin tumors, B7-1-painted ex vivo-amplified OT-1 T cells induced complete tumor regression in 65% of treated mice, whereas the control T cells did so in only 28% of treated mice. Finally, on transfer into mice bearing less immunogenic 4T1 breast tumors, B7-1-painted tumor-reactive CD8+ T cells improved the survival of treated mice to a greater extent than did the control T cells. Hence, this study establishes that arming tumor-reactive T cells with a costimulator can enhance their antitumor efficacy.
Assuntos
Antígeno B7-1/imunologia , Linfócitos T CD8-Positivos/imunologia , Imunoterapia Adotiva/métodos , Neoplasias Experimentais/terapia , Animais , Feminino , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias Experimentais/imunologia , Engenharia de Proteínas/métodosRESUMO
Epidemiological studies have demonstrated a relationship between cancer incidence and dietary habits. Especially intake of certain essential nutrients like vitamins has been shown to be beneficial in experimental studies and some clinical trials. Vitamin K (VK) is an essential nutrient involved in the blood clotting cascade, and there are considerable experimental data demonstrating its potential anticancer activity in several cancer types including prostate cancer. Previous in vitro and in vivo studies have focused mainly on anti-oxidative effects as the underlying anticancer mechanism of VK. However, recent studies reveal that VK inhibits the growth of cancer cells through other mechanisms, including apoptosis, cell cycle arrest, autophagy, and modulation of various transcription factors such as Myc and Fos. In the present review, we focus on the anticancer effect of dietary VK and its analogs on prostate cancer, with an emphasis on the signaling pathways that are activated following exposure to these compounds. This review also highlights the potential of VK and its derivatives as an adjuvant treatment in combination with other vitamins or with chemotherapeutic drugs. Based on our recent results and a review of the existing literature, we present evidence that VK and its derivatives can potentially be explored as cancer therapy, especially for prostate cancer.
RESUMO
Antibodies are widely available and cost-effective research tools in life science, and antibody conjugates are now extensively used for targeted therapy, immunohistochemical staining, or in vivo diagnostic imaging of cancer. Significant advances in site-specific antibody labeling technologies have enabled the production of highly characterized and homogenous conjugates for biomedical purposes, and some recent studies have utilized site-specific labeling to synthesize bifunctional antibody conjugates with both imaging and drug delivery properties. While these advances are important for the clinical safety and efficacy of such biologics, these techniques can also be difficult, expensive, and time-consuming. Furthermore, antibody-drug conjugates (ADCs) used for tumor treatment generally remain distinct from conjugates used for diagnosis. Thus, there exists a need to develop simple dual-labeling methods for efficient therapeutic and diagnostic evaluation of antibody conjugates in pre-clinical model systems. Here, we present a rapid and simple method utilizing commercially available reagents for synthesizing a dual-labeled fluorescent ADC. Further, we demonstrate the fluorescent ADC's utility for simultaneous targeted therapy and molecular imaging of cancer both in vitro and in vivo. Employing non-site-specific, amine-reactive chemistry, our novel biopharmaceutical theranostic is a monoclonal antibody specific for a carcinoembryonic antigen (CEA) biomarker conjugated to both paclitaxel and a near-infrared (NIR), polyethylene glycol modified (PEGylated) fluorophore (DyLight™ 680-4xPEG). Using in vitro systems, we demonstrate that this fluorescent ADC selectively binds a CEA-positive pancreatic cancer cell line (BxPC-3) in immunofluorescent staining and flow cytometry, exhibits efficient internalization kinetics, and is cytotoxic. Model studies using a xenograft of BxPC-3 cells in athymic mice also show the fluorescent ADC's efficacy in detecting tumors in vivo and inhibiting tumor growth more effectively than equimolar amounts of unconjugated drug. Overall, our results demonstrate that non-selective, amine-targeting chemistry is an effective dual-labeling method for synthesizing and evaluating a bifunctional fluorescent antibody-drug conjugate, allowing concurrent detection, monitoring and treatment of cancer.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/administração & dosagem , Sistemas de Liberação de Medicamentos , Corantes Fluorescentes/administração & dosagem , Imunoconjugados/uso terapêutico , Paclitaxel/administração & dosagem , Neoplasias Pancreáticas/tratamento farmacológico , Animais , Anticorpos Monoclonais/imunologia , Antineoplásicos/uso terapêutico , Antígeno Carcinoembrionário/imunologia , Linhagem Celular Tumoral , Corantes Fluorescentes/uso terapêutico , Humanos , Masculino , Camundongos , Camundongos Nus , Paclitaxel/uso terapêutico , Neoplasias Pancreáticas/diagnóstico por imagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Hepatoma-derived growth factor (HDGF) is a heparin-binding growth factor, which has previously been shown to be expressed in a variety of cancers. HDGF overexpression has also previously been correlated with a poor prognosis in several cancers. The significance of HDGF in prostate cancer, however, has not been investigated. Here, we show that HDGF is overexpressed in both androgen-sensitive LNCaP cells and androgen-insensitive DU145, 22RV1, and PC-3 cells. Forced overexpression enhanced cell viability of RWPE-1 cells, whereas HDGF knockdown reduced cell proliferation in human prostate cancer cells. We also show that HDGF may serve as a survival-related protein as ectopic overexpression of HDGF in RWPE cells up-regulated the expression of antiapoptosis proteins cyclin E and BCL-2, whereas simultaneously down-regulating proapoptotic protein BAX. Western blot analysis also showed that HDGF overexpression modulated the activity of phospho-AKT as well as NF-kB, and these results correlated with in vitro migration and invasion assays. We next assessed the therapeutic potential of HDGF inhibition with a HDGF monoclonal antibody and vitamin k2, showing reduced cell proliferation as well as inhibition of NF-kB expression in HDGF overexpressed RWPE cells treated with a HDGF monoclonal antibody and vitamin K2. Collectively, our results suggest that HDGF is a relevant protein in prostate oncogenesis and may serve as a potential therapeutic target in prostate cancer.
Assuntos
Adenocarcinoma/patologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Neoplasias da Próstata/patologia , Adenocarcinoma/metabolismo , Androgênios , Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular , Transformação Celular Neoplásica , Ensaios de Seleção de Medicamentos Antitumorais , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Masculino , Terapia de Alvo Molecular , NF-kappa B/metabolismo , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neoplasias Hormônio-Dependentes/metabolismo , Neoplasias Hormônio-Dependentes/patologia , Próstata/citologia , Neoplasias da Próstata/metabolismo , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes/metabolismo , Vitamina K 2/farmacologiaRESUMO
BACKGROUND AND AIMS: Anti-HMGB1 autoimmunity plays a role in systemic lupus erythematosus (SLE). Because SLE increases atherosclerosis, we asked whether the same autoimmunity might play a role in atherogenesis. METHODS: We looked for the induction of HMGB1-specific B and T cell responses by a western-type diet (WTD) in the Apoe(-/-) mouse model of atherosclerosis. We also determined whether modifying the responses modulates atherosclerosis. RESULTS: In the plasma of male Apoe(-/-) mice fed WTD, the level of anti-HMGB1 antibodies (Abs) was detected at â¼50 µg/ml, which was â¼6 times higher than that in either Apoe(-/-) mice fed a normal chow or Apoe(+/+) mice fed WTD (p ≤ 0.0005). The Abs were directed largely toward a novel, dominant epitope of HMGB1 named HMW4; accordingly, compared with chow-fed mice, WTD-fed Apoe(-/-) mice had more activated HMW4-reactive B and T cells (p = 0.005 and p = 0.01, respectively). Compared with mock-immunized mice, Apoe(-/-) mice immunized with HMW4 along with an immunogenic adjuvant showed proportional increases in anti-HMW4 IgG and IgM Abs, HMW4-reactive B-1 and B-2 cells, and HMW4-reactive Treg and Teff cells, which was associated with â¼30% increase in aortic arch lesions (p ≤ 0.01) by two methods. In contrast, Apoe(-/-) mice immunized with HMW4 using a tolerogenic adjuvant showed preferential increases in anti-HMW4 IgM (over IgG) Abs, HMW4-reactive B-1 (over B-2) cells, and HMW4-specific Treg (over Teff) cells, which was associated with â¼40% decrease in aortic arch lesions (p ≤ 0.03). CONCLUSIONS: Anti-HMGB1 autoimmunity may potentially play a role in atherogenesis.
Assuntos
Aterosclerose/genética , Autoimunidade/genética , Dieta Ocidental/efeitos adversos , Proteína HMGB1/genética , Animais , Antígenos/sangue , Aorta Torácica/patologia , Linfócitos B/imunologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Inflamação , Lipídeos/sangue , Lipídeos/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoERESUMO
Antigen-presenting cells (APC) can be refaced with "protein paints" that change the appearance of their T cell-oriented trans signal arrays. Our group has developed three categories of protein paints suitable for this kind of APC engineering: artificial glycosylphosphatidylinositol (GPI) proteins, palmitated-protein A:Fc*1 fusion protein conjugates, and trans signal converter proteins. Protein paints have been devised with either immune enhancement or suppression in mind. Costimulator * GPI and palmitated-protein A costimulator * Fcgamma1 conjugates can be used to augment the immune-activating potential of tumor cells. Alternatively, protein paints can be designed to transform APC into artificial veto cells, in essence creating Trojan horses capable of inhibiting pathogenic T cells. Trans signal converter proteins (TSCP) have been devised for this purpose. Our first paradigmatic inhibitory TSCP, CTLA-4 * Fas ligand, binds to APC, and in so doing, simultaneously blocks B7 costimulation (via CTLA-4) and sends inhibitory trans signals (via Fas ligand) to T cells with dramatic efficacy. Protein transfer offers a number of advantages over gene transfer in facilitating quantitative and combinatorial protein expression and simplifying in vivo applications; the palette of protein paints with immunotherapeutic potential will undoubtedly continue to evolve.