RESUMO
The disease relevance of novel therapeutic agent T11TS, established first by the authors' group, was shown to ameliorate experimental glioma through multimodal mechanistic activities. T11TS reverses immunosuppression in glioma, causing profound effects on immune potentiation via peripheral, intracranial and hematopoietic cells. T-cell signaling in glioma is reversed by T11TS, modulating cytokine levels and favoring apoptotic killing of glioma cells. T11TS arrests the glioma cell cycle at the G1 phase via activation of p21. VEGF downregulation hypophosphorylates the Akt pathway. T11TS hinders endothelial cell progression and metastasis by arresting matrix degradation, inhibiting the Ras-Raf and Akt-PTEN pathways and initiating inflammatory changes, causing apoptosis. T11TS is effective against in vitro human glioma. Toxicity studies demonstrate that T11TS is nontoxic. The authors' study promise translational research with T11TS.
Glioma is a fatal brain tumor, and conventional treatments with surgery, radiotherapy, and chemotherapy often cause cancer recurrence. Therefore, newer strategies of treatment are being developed. In the authors' laboratory, a novel molecular approach with the biomolecule T11TS has been successfully applied in the eradication of glioma in an experimental rat model and on human samples. T11TS ameliorates glioma by various means, including immune augmentation and cytokine modulation; causes glioma cell death (apoptotic); halts the glioma cell cycle; and retards glioma blood vessel growth (antiangiogenic). T11TS is nontoxic. The author's study points to novel glioma therapy.
Assuntos
Antineoplásicos , Neoplasias Encefálicas , Glioma , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose , Neoplasias Encefálicas/tratamento farmacológico , Citocinas/uso terapêutico , Glioma/tratamento farmacológico , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator A de Crescimento do Endotélio VascularRESUMO
The novel anti-neoplastic glycopeptide T11TS retards glioma both in in-vitro clinical samples and in-vivo models. This study investigates the correlation between altering the glioma microenvironment with glioma arrest and death. Flow cytometry, immunoblotting, ELISA, and co-immunoprecipitation were employed to investigate glioma cell arrest and death. Results include a decline in phosphorylation of Akt and attenuation of p21 phosphorylation (Thr145,Ser146) and disassociation of p-Akt-Mdm2 and p-Akt-BAD facilitating death by Akt>BAD. T11TS influence phosphorylation patterns in two focal axes Akt>p21 and Akt>Mdm2>p53. The current article provides crucial insight in deciphering the mechanism of T11TS induced glioma cell arrest and death.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Antígenos CD58/farmacologia , Glioma/tratamento farmacológico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Antígenos CD58/uso terapêutico , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Feminino , Glioma/metabolismo , Glioma/patologia , Masculino , PTEN Fosfo-Hidrolase/análise , Fosforilação , Proteínas Proto-Oncogênicas c-mdm2/análise , Ratos , Ratos Wistar , Microambiente Tumoral , Proteína Supressora de Tumor p53/análise , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
Cryptococcus neoformans infects and disseminates in hosts with diminished T cell responses. The immunomodulator T11TS (T11 target structure) had profound potential in glioma as well as C. neoformans infected model for disease amelioration. It is been established by our group that T11TS potentiates Calcineurin-NFAT pathway in T cells of C. neoformans infected rats. We investigated the upstream Immunological Synapse (IS) molecules that are vital for the foundation of initial signals for downstream signaling, differentiation and proliferation in T cells. Improved RANTES level in the T11TS treated groups suggests potential recruitment of T cells. Down-regulation of TCRαß, CD3ζ, CD2, CD45 and CD28 molecules by cryptococcus were boosted after T11TS therapy. Heightened expression of inhibitory molecule CTLA-4 in cryptococcosis was dampened by T11TS. The decline of MHC I, MHC II and CD80 expression on macrophages by C. neoformans were enhanced by T11TS. The dampening of positive regulators and upsurge of negative regulators of the IS during cryptococcosis was reversed with T11TS therapy resulting in enhanced clearance of fungus from the lungs as envisaged by our histological studies. This preclinical study with T11TS opens a new prospect for potential immunotherapeutic intervention against the devastating C. neoformans infection with positive aspect for the long-term solution and a safer immunotherapeutic regimen.
RESUMO
Asthma is a chronic inflammatory disorder of the airways, increasing in prevalence worldwide. Reduced T cell apoptosis may interfere with the down-regulation of an immune response resulting in T cell accumulation contributing to the chronic inflammation of asthma. Most studies focused so far on apoptosis of eosinophils but the detailed role of T lymphocytes apoptosis in allergic diseases is unclear yet. The present experimental study was designed to discern the modulation of various apoptotic proteins of splenic T lymphocytes in a previously established rat model of Alstonia scholaris pollen induced airway allergy. Flowcytometry, immunoblotting, and immunofluorescence imaging techniques were employed for the present investigation. Annexin-V studies registered early apoptotic rate of lymphocytes with allergen sensitization and challenge which was corrected following mucosal immunotherapy. The study demonstrates that allergen sensitization and challenge reduced apoptosis of splenic T-lymphocytes via Fas mediated extrinsic pathway, Bax/Bcl2 regulated intrinsic pathway and also perforin/granzyme mediated pathway which were normalized following allergen specific intranasal immunotherapy. Inadequate T cell apoptosis in asthma appears to interfere with normal T cell elimination, resulting in T cell accumulation, which contributes to chronic inflammation and may be the major underlying cause for tissue damage which can be modulated by intranasal immunotherapy. Thus the apoptosis inducing effect of allergen immunotherapy necessitates more studies to elaborate on its effects on various effector cells of airway inflammation.
Assuntos
Apoptose/imunologia , Dessensibilização Imunológica , Hipersensibilidade/imunologia , Hipersensibilidade/terapia , Pólen/imunologia , Linfócitos T/imunologia , Animais , Apoptose/genética , Biomarcadores , Caspase 8 , Dessensibilização Imunológica/métodos , Modelos Animais de Doenças , Expressão Gênica , Hipersensibilidade/genética , Hipersensibilidade/metabolismo , Imunofenotipagem , Ativação Linfocitária/imunologia , Ratos , Baço/imunologia , Baço/metabolismo , Linfócitos T/metabolismoRESUMO
Cryptococcus neoformans, the encapsulated yeast acquired through inhalation, remains localized in lungs, but harbours the CNS in immunocompromised individuals. Several treatment regimes have failed combating this disease totally, but long-term usage of drugs leads to organ damage. As T11-target structure (T11TS) has documented profound immune potentiation, we aimed to investigate the role of microglia, pivotal immune cells of brain in ameliorating cryptococcosis, with T11TS immunotherapy. Murine model with C neoformans infection was prepared by intraperitoneal injection and the brains of rats examined 7 days post-infections for histopathology by PAS and Alcian blue staining corroborated with organ fungal burden evidencing restorative T11TS action on Cryptococcal meningitis. Immunotherapy with three doses of T11TS, a CD2 ligand, in C neoformans infected rats, upregulates toll-like receptors 2, -4 and -9 of microglia, indicating increased phagocytosis of the fungus. Flowcytometric analysis revealed increased numbers of T11TS treated brain infiltrating CD4+ and CD8+ T-lymphocytes along with increased MHC I and MHC II on microglia, activating the infiltrating lymphocytes aiding the killing mechanism. Present study also indicated that T11TS increased production of Th1 inflammatory cytokines conducive to fungal elimination while the inhibitory Th2 cytokines were dampened. This preclinical study is first of its kind to show that T11TS effected profound immune stimulation of microglial activity of C neoformans infected rats eradicating residual fungal burden from the brain and can be a useful therapeutic strategy in fighting against this deadly disease.
Assuntos
Encéfalo/efeitos dos fármacos , Antígenos CD58/uso terapêutico , Cryptococcus neoformans/fisiologia , Fatores Imunológicos/uso terapêutico , Imunoterapia/métodos , Meningite Criptocócica/terapia , Microglia/imunologia , Animais , Encéfalo/imunologia , Encéfalo/microbiologia , Bovinos , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Imunidade Inata/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Masculino , Meningite Criptocócica/imunologia , Microglia/patologia , Ratos , Ratos Wistar , Linfócitos T/imunologia , Receptores Toll-Like/metabolismoRESUMO
Combating gliomagenic global immunosuppression is one of the emerging key for improving prognosis in malignant glioma. Apoptosis plays a pivotal role within the adult hematopoietic system particularly in regulating the cells of immune system. Gliomagenic regulation of apoptotic mediators within bone marrow milieu has not been elucidated. We previously demonstrated that administration of membrane glycopeptides T11 target structure (T11TS) not only rejuvenate bone marrow hematopoietic stem cells (BMHSCs) from glioma mediated hibernation by inhibiting gliomagenic overexpression of Ang-1/Tie-2 but also stimulate glioma mediated diminution of expression CD34, c-kit, and Sca-1 markers. In the present study, we investigated the impact of glioma on apoptotic signaling cascades of BMHSCs and consequences following T11TS therapy. Bone marrow smear and Annexin V staining confirm gliomagenic acceleration of apoptotic fate of BMHSCs whereas T11TS treatment in glioma-bearing rats disrupted apoptosis of BMHSCs. Flowcytometry, immunoblotting, and immunofluorescence imagining results revealed multi potent T11TS not only significantly downregulates gliomagenic overexpression of Fas, Fas L, Bid, and caspase-8, the pro-apoptotic extrinsic mediators but also strongly inhibits cytosolic release of cytochrome-c, Apf-1, and Bax to deactivate gliomagenic caspase-9, 3 the key intrinsic apoptotic mediators followed by up modulation of anti-apoptotic Bcl-2 in glioma associated HSCs. T11TS is also able to diminish the perforin-granzyme B mediated apoptotic verdict of BMHSCs during gliomagenesis. The anti-apoptotic action of T11TS on glioma associated BMHSCs provide a crucial insight into how T11TS exerts its immunomodulatory action against glioma mediated immune devastation.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Células da Medula Óssea/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Glioma/tratamento farmacológico , Glicopeptídeos/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Neoplasias Experimentais/tratamento farmacológico , Evasão Tumoral/efeitos dos fármacos , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Sobrevivência Celular/efeitos dos fármacos , Feminino , Glioma/imunologia , Glioma/metabolismo , Glioma/patologia , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Masculino , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Células Tumorais Cultivadas , Microambiente TumoralRESUMO
Malignant glioma is the most fatal of astrocytic lineage tumors despite therapeutic advances. Onset and progression of gliomas is accompanied by severe debilitation of T-cell defense and T-cell survival. One of the chief contributors to T-cell survival downstream of activation is the PI3K-AKT pathway. Our prior studies showed that the novel immunotherapeutic molecule T11-target structure (T11TS) blocks T-cell apoptosis in glioma. We also showed activation of immunological synapse components and calcineurin-NFAT pathway following T11TS immunotherapy of glioma-bearing rats. This lead to investigations whether such T-cell activation upon T11TS therapy translates into activation of downstream PI3K/AKT signals which may be related to observed blockade of T-cell apoptosis. For the purpose, we assessed by flowcytometry and immunoblotting, expressions of PI3K, PDK1, AKT, p-AKT, and PTEN in splenic T-cells of normal, experimentally-induced glioma-bearing rats and glioma-bearing rats receiving first, second and third doses of T11TS. We also determined comparative nuclear translocation of NF-κB across groups. We found significant increases in T-cell expressions of PDK1, PI3K, and p-AKT in T11TS-treated animal groups compared to sharp downregulations in glioma. AKT levels remained unchanged across groups. PTEN levels declined sharply after T11TS immunotherapy. T11TS also caused enhanced NF-κB translocation to the T-cell nucleus compared to glioma group. Results showed heightened activation of the PI3K-AKT pathway in glioma-bearing rats following T11TS immunotherapy. These results illustrate the novel role of T11TS immunotherapy in ameliorating the PI3K pathway in T-cells in glioma-bearing animals to enhance T-cell survival, according greater defense against glioma. The study thus has far-reaching clinical outcomes.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Antígenos CD58/farmacologia , Glioma/tratamento farmacológico , Imunoterapia/métodos , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linfócitos T/efeitos dos fármacos , Evasão Tumoral/efeitos dos fármacos , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Antígenos CD28/imunologia , Antígenos CD28/metabolismo , Sobrevivência Celular , Etilnitrosoureia , Feminino , Glioma/enzimologia , Glioma/imunologia , Glioma/patologia , Masculino , NF-kappa B/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Fosforilação , Ratos , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/enzimologia , Linfócitos T/imunologiaRESUMO
AIM: To study the apoptosis of Foxp3+ Treg cells following Alstonia scholaris pollen sensitization-challenge and following allergen immunotherapy. MATERIALS & METHODS: Wistar rats were sensitized-challenged with Alstonia scholaris pollen and were further given intranasal immunotherapy. For the analysis of the apoptotic proteins on Treg cells by flow cytometry, multiple gating procedures were followed. RESULTS: Allergen sensitization-challenge increases Annexin-V, Fas, FasL, caspases-8, 9, 3 cytochrome-C, APAF-1, Bax, perforin-1 and granzyme-B on Treg cells which is decreased following intranasal immunotherapy. On the other hand, Bcl-2 expression is decreased in allergy and increased by immunotherapy. CONCLUSION: Apoptosis of Treg cells is increased following allergen sensitization-challenge via extrinsic, intrinsic and perforin/granzyme pathways and allergen immunotherapy decreased the sensitivity to apoptosis of Treg cells.
Assuntos
Alérgenos/uso terapêutico , Antígenos de Plantas/uso terapêutico , Dessensibilização Imunológica/métodos , Rinite Alérgica Sazonal/terapia , Linfócitos T Reguladores/imunologia , Administração Intranasal , Alérgenos/imunologia , Alstonia/imunologia , Animais , Antígenos de Plantas/imunologia , Apoptose , Células Cultivadas , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/metabolismo , Humanos , Perforina/metabolismo , Pólen/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Wistar , Rinite Alérgica Sazonal/imunologiaRESUMO
Allergic airway diseases such as asthma and allergic rhinitis are increasing in prevalence worldwide. The theory of an altered Th1/Th2 balance in allergic diathesis has recently been termed a "procrustean paradigm" as it failed to explain many preclinical findings. Regulatory T cells (Treg) have now been shown to be critical in T-cell homeostasis and in the maintenance of peripheral tolerance to allergens. Allergen specific immunotherapy (SIT) has been shown to induce regulatory T cells in allergic patients. Among various types of SIT, intranasal immunotherapy had not been studied in detail for the treatment of allergic airway diseases. So, there was a need to study the contribution of regulatory T cells and their mechanistic pathways following intranasal immunotherapy in-vivo. It had been previously shown that intranasal allergen immunotherapy using Alstonia scholaris pollen extract abrogates allergic airway inflammation with decline in IgE and Th2 cytokine levels. The present study for the first time offers a multi-targeted approach towards attenuation of airway allergy by the generation of CD4+CD25+Foxp3+T cells and other subsets of Treg cells like Tr1 cells, Th3 cells, CTLA4+Treg cells, and also modulation of various Treg cell surface molecules like GITR, OX40, CD39 and CD73 by intranasal immunotherapy in the same animal model. This animal experiment will thus help to chart out newer molecular targets for treating allergic asthma or rhinitis.
Assuntos
Asma/terapia , Dessensibilização Imunológica/métodos , Extratos Vegetais/uso terapêutico , Rinite Alérgica/terapia , Linfócitos T Reguladores/imunologia , Células Th2/imunologia , Administração Intranasal , Alérgenos/imunologia , Alstonia/imunologia , Animais , Antígenos de Plantas/imunologia , Asma/imunologia , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/metabolismo , Humanos , Imunoglobulina E/sangue , Imunomodulação , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Extratos Vegetais/imunologia , Pólen/imunologia , Ratos , Ratos Wistar , Rinite Alérgica/imunologiaRESUMO
Compared to conventional therapy, several studies with prothrombin complex concentrate (PCC) have recently demonstrated its superior efficacy in rapidly replacing vitamin K-dependent factors for patients with life-threatening hemorrhage. We present a novel use of PCC in a patient with intracranial hypotension, who had received warfarin for treatment of cortical vein thrombosis. However, after anticoagulation, she proceeded to develop bilateral subdural hematomas with descent of cerebellar tonsils. Given the possibility of an occult dural puncture during labor analgesia, an epidural blood patch was performed after administration of PCC and normalization of coagulation parameters, with prompt improvement of the patient's headache.
Assuntos
Fatores de Coagulação Sanguínea/administração & dosagem , Placa de Sangue Epidural/métodos , Hematoma Subdural/tratamento farmacológico , Trombose Intracraniana/tratamento farmacológico , Feminino , Hematoma Subdural/etiologia , Hemostáticos , Humanos , Trombose Intracraniana/etiologia , Resultado do Tratamento , Varfarina/efeitos adversos , Adulto JovemRESUMO
Malignant glioma continues to be a clinical challenge with an urgent need for developing curative therapeutic intervention. Apoptosis induction in tumor-associated endothelial cells represent a central mechanism that counteracts angiogenesis in glioma and other solid tumors. We previously demonstrated that intraperitoneal administration of sheep erythrocyte membrane glycopeptide T11-target structure (T11TS) in rodent glioma model inhibits PI3K/Akt pathway and Raf/MEK/ERK signaling in glioma-associated brain endothelial cells. In the present study, we investigated whether T11TS treatment influence apoptosis signaling in vivo in glioma-associated brain endothelial cells. Annexin-V/PI staining showed that T11TS treatment in glioma-induced rats increases apoptosis of glioma-associated endothelial cells within glioma milieu compared to brain endothelial cells in glioma induced and control groups. Flowcytometric JC-1 assay revealed that T11TS administration triggers loss of mitochondrial membrane potential in glioma-associated brain endothelial cells. Flowcytometry, immunoblotting, and in situ immunofluoresecnt imaging were employed to investigate the effect of T11TS on apoptotic regulatory proteins in brain endothelial cells. T11TS treatment-upmodulated expression of p53, Bax, Fas, FasL, and FADD in glioma associated endothelial cells and downregulated Bcl-2 protein. T11TS therapy induced cytochrome-c release into cytosol, activated caspase -9, 8, 3, and cleaved Bid in glioma associated brain endothelial cells. The study demonstrates that T11TS induces apoptosis in glioma-associated brain endothelial cells via p53 accumulation and activation of intrinsic as well as Fas-dependent extrinsic pathway. The pro-apoptotic action of T11TS on glioma-associated endothelial cells provides crucial insight into how T11TS exerts its anti-angiogenic function in glioma. J. Cell. Physiol. 232: 526-539, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Células Endoteliais/patologia , Glioma/patologia , Glicopeptídeos/farmacologia , Glicopeptídeos/uso terapêutico , Glicoproteínas de Membrana/farmacologia , Glicoproteínas de Membrana/uso terapêutico , Neovascularização Patológica/tratamento farmacológico , Inibidores da Angiogênese/farmacologia , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Neoplasias Encefálicas/patologia , Caspases/metabolismo , Citocromos c/metabolismo , Citosol/metabolismo , Células Endoteliais/efeitos dos fármacos , Ativação Enzimática , Proteína Ligante Fas/metabolismo , Proteína de Domínio de Morte Associada a Fas/metabolismo , Feminino , Glicopeptídeos/administração & dosagem , Masculino , Glicoproteínas de Membrana/administração & dosagem , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Modelos Biológicos , Neovascularização Patológica/patologia , Ratos , Ovinos , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/metabolismo , Receptor fas/metabolismoRESUMO
Human glioma, arising from glial cells of the central nervous system, accounts for almost 30%of all brain tumours , neoplasms with a poor prognosis and high mortality rates worldwide. In the present study we assessed tissue architectural modifications associated with macrophage lineage cells, controversial major immune effector cells within the brain, in human glioma tissue samples from eastern India. Ethically cleared post-operative human glioma samples from our collaborative neurosurgery unit with respective CT/MRI and patient history were collected from the Nodal Centre of Neurosciences in Kolkata, over 9 months. Along with conventional histopathology, samples were subjected to silver-gold staining and fluorescence tagged immunophenotyping for the detection of electron dense brain macrophage/microglia cells in glioma tissue, followed by immune-phenotyping of cells. With higher grades, CD11b+/Iba-1+ macrophage/microglia architecture with de-structured boundaries of glioma lesions indicated malfunction and invasive effector state. Present study documented a contribution of microglia to glioma progression in Eastern India.
Assuntos
Neoplasias Encefálicas/patologia , Encéfalo/patologia , Glioma/patologia , Macrófagos/patologia , Microglia/patologia , Neoplasias Encefálicas/epidemiologia , Estudos de Casos e Controles , Progressão da Doença , Seguimentos , Glioma/epidemiologia , Humanos , Incidência , Índia/epidemiologia , Estadiamento de Neoplasias , PrognósticoRESUMO
Pollen grains are well established to be an important cause of respiratory allergy. Current pharmacologic therapies for allergic asthma do not cure the disease. Allergen specific immunotherapy is the only treatment method which re-directs the immune system away from allergic response leading to a long lasting effect. The mechanism by which immunotherapy achieves this goal is an area of active research world-wide. The present experimental study was designed to develop an experimental model of allergic lung inflammation based on a relevant human allergen, Alstonia scholaris pollen, and to establish the immunological and cellular features of specific allergen immunotherapy using this same pollen extract. Our results revealed that Alstonia scholaris pollen sensitization and challenge causes eosinophilic airway inflammation with mucin hypersecretion. This is associated with increased total IgE, increased expression of FcÉRI on lung mast cells and increased levels of IL-4, IL-5 & IL-13 as confirmed by ELISA, in-situ immunofluorescence and FACS assay. Allergen specific immunotherapy reduced airway inflammation and also decreased total IgE level, FcÉRI expression, IL-4, IL-5 & IL-13 levels. It was further noted that the reduction of these levels was more by intra-nasal route than by intra-peritoneal route. Thus we present a novel animal model of Alstonia scholaris pollen allergic disease and specific allergen immunotherapy which will pave the way towards the development of better treatment modalities.
Assuntos
Alérgenos/imunologia , Dessensibilização Imunológica , Eosinófilos/imunologia , Mastócitos/imunologia , Pneumonia/terapia , Pólen/imunologia , Rinite Alérgica Sazonal/terapia , Administração Intranasal , Alstonia/imunologia , Animais , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Imunoglobulina E/sangue , Mucinoses , Pneumonia/imunologia , Ratos , Ratos Wistar , Receptores de IgE/metabolismo , Rinite Alérgica Sazonal/imunologia , Células Th2/imunologiaRESUMO
Malignant glioma is the most lethal of a wide array of CNS neoplasms. Its onset and progression are markedly associated with profound immunosupression and paralysis of T-cell survival and proliferation. Myriad immunotherapeutic strategies are presently used to target such T-cell anomalies in glioma. Our recent work has highlighted use of the novel glycopeptide, the CD2 ligand, T11 target structure (T11TS) as an immunotherapeutic agent against experimentally induced glioma in rats. We have shown that T11TS causes multi-target modulation of key components of the T-cell - antigen presenting cell (APC) immunological synapse. This consequently triggers T-cell activation so as to reverse glioma-induced changes to physiological levels. T11TS administration also causes CD2 upregulation. Earlier we also found T11TS to cause enhanced proliferation of both CD4+ and CD8+ T-cells in glioma conditions. These findings led us to believe that downstream CD2-stimulated "alternative pathway" of calcineurin-NFAT could be a possible target for modulation by T11TS. In the present paper we thus show that immunotherapy with T11TS induces a multi-targeted approach towards activation of this "alternative pathway" of T-cell signaling providing an immunotherapeutic advantage against glioma. We show here that T11TS immunotherapy causes positive modulations of the CD2 pathway-associated proteins, viz., p59fyn, protein kinase C-θ (PKC-θ), calcineurin and nuclear factor for activation of T-cells (NFAT) and hint that this may accord greater survival and proliferation advantage to T-cells of the glioma-bearing animals for augmented defence against glioma. These findings help open a molecular immunotherapeutic door - one which is directed towards clinical studies for glioma-immunotherapy using T11TS.
Assuntos
Antígenos CD2/metabolismo , Calcineurina/metabolismo , Glioma/terapia , Imunoterapia , Fatores de Transcrição NFATC/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Animais , Animais Recém-Nascidos , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/terapia , Núcleo Celular/metabolismo , Citometria de Fluxo , Imunofluorescência , Glioma/imunologia , Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Proteína Quinase C-theta , Ratos , Ovinos , Baço/citologiaRESUMO
T11 Target structure (T11TS), a membrane glycoprotein isolated from sheep erythrocytes, reverses the immune suppressed state of brain tumor induced animals by boosting the functional status of the immune cells. This study aims at aiding in the design of more efficacious brain tumor therapies with T11 target structure. We propose a mathematical model for brain tumor (glioma) and the immune system interactions, which aims in designing efficacious brain tumor therapy. The model encompasses considerations of the interactive dynamics of glioma cells, macrophages, cytotoxic T-lymphocytes (CD8(+) T-cells), TGF-ß, IFN-γ and the T11TS. The system undergoes sensitivity analysis, that determines which state variables are sensitive to the given parameters and the parameters are estimated from the published data. Computer simulations were used for model verification and validation, which highlight the importance of T11 target structure in brain tumor therapy.
Assuntos
Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/terapia , Imunoterapia , Glicoproteínas de Membrana/imunologia , Modelos Biológicos , Animais , Animais Recém-Nascidos , Neoplasias Encefálicas/patologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Glioma/imunologia , Glioma/patologia , Glioma/terapia , Humanos , Interferon gama/farmacologia , Análise dos Mínimos Quadrados , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Glicoproteínas de Membrana/administração & dosagem , Análise Numérica Assistida por Computador , Ratos , Ovinos , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Malignant gliomas represent one of the most aggressive and hypervascular primary brain tumors. Angiopoietin-1, the peptide growth factor activates endothelial Tie-2 receptor promoting vessel maturation and vascular stabilization steps of angiogenesis in glioma. Epidermal growth factor receptor (EGFR) and Tie-2 receptor on endothelial cells once activated transmits signals through downstream Raf/MEK/ERK pathway promoting endothelial cell proliferation and migration which are essential for angiogenesis induction. The in vivo effect of sheep erythrocyte membrane glycopeptide T11-target structure (T11TS) on angiopoietin-1/Tie-2 axis, EGFR signaling and Raf/MEK/ERK pathway in glioma associated endothelial cells has not been investigated previously. The present study performed with rodent glioma model aims to investigate the effect of T11TS treatment on angiopoietin-1/Tie-2 signaling, EGFR activity and Raf/MEK/ERK pathway in glioma associated endothelial cells within glioma milieu. T11TS administration in rodent glioma model inhibited angiopoietin-1 expression and attenuated Tie-2 expression and activation in glioma associated brain endothelial cells. T11TS treatment also downregulated total and phosphorylated EGFR expression in glioma associated endothelial cells. Additionally T11TS treatment inhibited Raf-1 expression, MEK-1 and ERK-1/2 expression and phosphorylation in glioma associated brain endothelial cells. Thus T11TS therapy remarkably inhibits endothelial angiopoietin-1/Tie-2 signaling associated with vessel maturation and simultaneously antagonizes endothelial cell proliferation signaling by blocking EGFR activation and components of Raf/MEK/ERK pathway. Collectively, the findings demonstrate a multi-targeted anti-angiogenic activity of T11TS which augments the potential for clinical translation of T11TS as an effective angiogenesis inhibitor for glioma treatment.
Assuntos
Angiopoietina-1/metabolismo , Neoplasias Encefálicas/metabolismo , Antígenos CD2/farmacologia , Células Endoteliais/metabolismo , Glioma/metabolismo , Neovascularização Patológica/metabolismo , Receptor TIE-2/metabolismo , Animais , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Neoplasias Encefálicas/patologia , Células Endoteliais/efeitos dos fármacos , Receptores ErbB/metabolismo , Feminino , Glioma/patologia , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases , Masculino , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neovascularização Patológica/patologia , Proteínas Proto-Oncogênicas c-raf , Ratos , OvinosRESUMO
Glioma angiogenesis is the result of the interaction between cancer cells with endothelial cells, and the surrounding inflammatory cells. This interaction plays a crucial role in directing the neo-formation of blood vessels. In the carcinogenic milieu, inflammatory cytokines secreted from inflammatory cells affect endothelial cell functions that are indispensable for tumor growth and metastatic propagation. TNF-α, referred to as the 'inflammatory switch', has shown its potential as an inflammatory agent by activation of IL-8 and IL-6 through NF-κB mediated pathway. Therefore, inhibitors of angiogenesis appear to be promising therapeutic agents for advanced gliomas. Previous studies from our lab showed that T11TS, a membrane glycoprotein, has antiangiogenic and antineoplastic activities in experimental animals and human samples. The present experimental study was designed to evaluate the effect of T11TS therapy on inflammatory cytokine expression of TNF-α, IL-8, IL-6 and their downstream associated molecule NF-κB in vivo. Our results revealed that T11TS therapy induced downregulation of TNF-α, IL-8, IL-6, and NF-κB confirmed by FACS assay and ELISA. In situ-immunofluorescence results hint that T11TS has the efficacy to stop the inflammation related to angiogenesis. Moreover, upregulation of IL-4 and IL-10 in microglia after T11TS therapy helps in complete abrogation of glioma inflammation and angiogenesis. These effects might contribute to the antineoplastic activity of T11TS.
Assuntos
Inibidores da Angiogênese/administração & dosagem , Antineoplásicos/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Antígenos CD58/administração & dosagem , Células Endoteliais/efeitos dos fármacos , Glioma/tratamento farmacológico , Microglia/efeitos dos fármacos , Neovascularização Patológica/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/imunologia , Antígenos CD58/isolamento & purificação , Citocinas/metabolismo , Células Endoteliais/imunologia , Glioma/irrigação sanguínea , Glioma/imunologia , Humanos , Mediadores da Inflamação/metabolismo , Modelos Animais , NF-kappa B/metabolismo , Neovascularização Patológica/imunologia , Ratos , Ratos Endogâmicos , Transdução de Sinais/efeitos dos fármacosRESUMO
T-cell-mediated immune responses are typically low in conditions of malignant glioma which has been known to cause marked immunesuppression and dysregulate major T-cell signaling molecules. Thus, T-cell-based immunotherapies are currently in vogue in the treatment of malignant glioma. The novel glycopeptide, T11TS/S-LFA-3/S-CD58 has previously been shown by our group to be highly efficacious in glioma abrogation in in vivo and in vitro conditions. This glycopeptide ligands to the costimulatory CD2 molecule on T-cells, causing profound immune stimulation leading to glioma abrogation, suggesting probable involvement of T11TS in modulation of the T-cell signaling pathway. The present study offers a multi-targeted approach towards repair of some of the key components of the immunological synapse at the T-cell-APC interface and is therefore the first of its kind to offer a holistic model of restoration of immunological synapse components so as to trigger T-cells towards activation against glioma. The study thus indicates that the totally dysregulated molecular events at the immunological synapse in glioma are restored back to normal levels with the administration of T11TS, which finally culminates in glioma abrogation. The present study thus delineates an important T-cell signaling approach whereby T11TS acts as an anti-neoplastic agent, thus helping to chart out newer avenues in the fight against gliomas.
Assuntos
Antígenos CD2/metabolismo , Antígenos CD58/metabolismo , Glioma/prevenção & controle , Glicopeptídeos/uso terapêutico , Sinapses Imunológicas/imunologia , Linfócitos T/imunologia , Animais , Apoptose , Neoplasias Encefálicas/induzido quimicamente , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/prevenção & controle , Antígenos CD2/imunologia , Antígenos CD58/imunologia , Etilnitrosoureia/toxicidade , Feminino , Citometria de Fluxo , Imunofluorescência , Glioma/induzido quimicamente , Glioma/imunologia , Ativação Linfocitária , Masculino , Camundongos , Mutagênicos/toxicidade , Transdução de Sinais , Linfócitos T/metabolismo , Linfócitos T/patologiaRESUMO
During glioma development, angiogenesis plays a crucial role in growth and vascularization of primary brain tumors. T11 target structure (T11TS), a bioactive molecule, has been documented as an anti-neoplastic agent in glioma-induced rats and also in human glioma in vitro. This novel molecule induces apoptosis of tumor cells by way of immune potentiation and impairs the glioma cell cycle, but its role in glioma angiogenesis has not been worked out in detail. Matrix metalloproteinases (MMPs) are enzymes promoting tumor angiogenesis by enzymatically remodeling the extracellular matrix and altering surface protein expression such as integrin αv and the matrix-bound proteins like TGF-ß1. The present study was formulated to assess the efficacy of T11TS in the modulations of MMP-2 and -9 and their endogenous inhibitors (TIMP-1 and TIMP-2) as well as modulations of integrin αv and TGF-ß1 in glioma-induced rats and also on the phenotypic markers of endothelial cells (CD31 and CD34). The parameters used were zymography, western blot, and flow cytometric analyses. It was observed that T11TS administration significantly downregulates the expression of matrix metalloproteinase-2 and -9 along with its ligand integrin αv and upregulates TIMP-1 and TIMP-2. In situ immunofluorescence and FACS results revealed that T11TS administration decreased the expression of the phenotypic markers (CD31/PECAM1, CD34), inhibiting the cell grip and also downregulating TGF-ß1 expression (ELISA) from microglia cells in the glioma microenvironment. These results suggest that T11TS suppresses the expression of positive angiogenic growth factors and potentiates the expression of negative regulators in glioma-associated endothelial cells (ECs), resulting in an anti-angiogenic effect on glioma-induced angiogenesis.
Assuntos
Inibidores da Angiogênese/farmacologia , Células Endoteliais/metabolismo , Glioma/metabolismo , Glicopeptídeos/farmacologia , Neovascularização Patológica/metabolismo , Animais , Western Blotting , Células Endoteliais/efeitos dos fármacos , Citometria de Fluxo , Imunofluorescência , Glioma/irrigação sanguínea , Integrina alfaV/metabolismo , Metaloproteinases da Matriz/metabolismo , Ratos , Inibidores Teciduais de Metaloproteinases/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
T11 target structure (T11TS), a membrane glycoprotein has been documented with anti neoplastic activity in glioma bearing animal model in our lab. In this study, we have evaluated the phagocytic potential, expression of VEGF, TNF-α in T11TS treated and untreated macrophages in all four grades of glioma. The data indicates the significant enhancement of phagocytosis in T11TS treated macrophages of grades I and II glioma. There was significant up regulation in TNF-α and significant down regulation in VEGF expression in T11TS treated macrophages in grade I and II glioma. We also attempted to know any possible apoptotic role of T11TS in tumor cells by comparing Bax and Bcl2 in treated and untreated tumor cells of all four grades. We found significant up regulation in Bax expression and down regulation in Bcl2 expression of grades I and II glioma. The outcome may help in pushing this molecule into pharmaceutical domain.