RESUMO
The main problem related to the studies focusing on group-specific component protein-derived macrophage-activating factor (GcMAF) is the lack of clarity about changes occurring in different types of macrophages and related changes in their properties under the effect of GcMAF in various clinical conditions. We analyzed the antitumor therapeutic properties of GcMAF in a Lewis carcinoma model in two clinical conditions: untreated tumor lesion and tumor resorption after exposure to Karanahan therapy. GcMAF is formed during site-specific deglycosylation of vitamin D3 binding protein (DBP). DBP was obtained from the blood of healthy donors using affinity chromatography on a column with covalently bound actin. GcMAF-related factor (GcMAF-RF) was converted in a mixture with induced lymphocytes through the cellular enzymatic pathway. The obtained GcMAF-RF activates murine peritoneal macrophages (p < 0.05), induces functional properties of dendritic cells (p < 0.05) and promotes in vitro polarization of human M0 macrophages to M1 macrophages (p < 0.01). Treatment of whole blood cells with GcMAF-RF results in active production of both pro- and anti-inflammatory cytokines. It is shown that macrophage activation by GcMAF-RF is inhibited by tumor-secreted factors. In order to identify the specific antitumor effect of GcMAF-RF-activated macrophages, an approach to primary reduction of humoral suppressor activity of the tumor using the Karanahan therapy followed by macrophage activation in the tumor-associated stroma (TAS) was proposed. A prominent additive effect of GcMAF-RF, which enhances the primary immune response activation by the Karanahan therapy, was shown in the model of murine Lewis carcinoma. Inhibition of the suppressive effect of TAS is the main condition required for the manifestation of the antitumor effect of GcMAF-RF. When properly applied in combination with any chemotherapy, significantly reducing the humoral immune response at the advanced tumor site, GcMAF-RF is a promising antitumor therapeutic agent that additively destroys the pro-tumor properties of macrophages of the tumor stroma.
Assuntos
Carcinoma , Fatores Ativadores de Macrófagos , Proteína de Ligação a Vitamina D , Animais , Proteínas Sanguíneas/metabolismo , Carcinoma/tratamento farmacológico , Humanos , Ativação de Macrófagos , Fatores Ativadores de Macrófagos/metabolismo , Camundongos , Proteína de Ligação a Vitamina D/metabolismoRESUMO
Besides an antigen-presenting function and ability to induce antitumor immune responses, dendritic cells (DCs) possess a direct tumoricidal activity. We previously reported that monocyte-derived IFNα-induced DCs (IFN-DCs) of glioblastoma multiforme patients express low levels of membrane TNFα molecule (mTNFα) and have impaired TNFα/TNF-R1-mediated cytotoxicity against immortalized tumor cell line HEp-2. However, whether the observed defect could affect killer activity of glioma patient DCs against autologous tumor cells remained unclear. Here, we show that donor IFN-DCs possess cytotoxic activity against glioblastoma cell lines derived from a primary tumor culture. Granule-mediated and TNFα/TNF-R1-dependent pathways were established as the main mechanisms underlying cytotoxic activity of IFN-DCs. Glioblastoma patient IFN-DCs showed lower cytotoxicity against autologous glioblastoma cells sensitive to TNFα/TNFR1-mediated lysis, which was associated with low TNFα mRNA expression and high TACE/ADAM-17 enzyme activity. Recombinant IL-2 (rIL-2) and human double-stranded DNA (dsDNA) increased 1.5-fold cytotoxic activity of patient IFN-DCs against autologous glioblastoma cells. dsDNA, but not rIL-2, enhanced the expression of TNFα mRNA and decreased expression and activity of TACE/ADAM-17 enzyme. In addition, dsDNA and rIL-2 stimulated the expression of perforin and granzyme B (in the presence of dsDNA), suggesting the possibility of enhancing DC cytotoxicity against autologous glioblastoma cells via various mechanisms.
Assuntos
Membrana Celular/metabolismo , Citotoxicidade Imunológica , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Glioblastoma/etiologia , Glioblastoma/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Biomarcadores , Linhagem Celular Tumoral , Suscetibilidade a Doenças , Expressão Gênica , Glioblastoma/patologia , Humanos , Interleucina-2/metabolismo , Interleucina-2/farmacologia , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/genéticaRESUMO
BACKGROUND: Tolerogenic dendritic cells (tDCs) are considered a novel therapeutic tool in treating autoimmune diseases, allergies, and transplantation reactions. Among numerous pharmacological immune modulators, dexamethasone (Dex) is known to induce potent tolerogenicity in DCs generated from human monocytes with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), and these cells (IL-4-DCs/Dex) are being appraised as a tDC-based platform in clinical settings. Interferon-α (IFNα) represents another powerful inducer of monocyte-derived DCs, which possess higher migratory activity and stability. However, the functions of IFN-DCs/Dex have not been sufficiently analyzed and there are no comparative studies of the tolerogenicity of IFN-DCs/Dex and IL-4-DCs/Dex. This study aimed to investigate the properties of IFN-DCs/Dex in comparison with IL-4-DCs/Dex. RESULTS: DCs were obtained by cultivation of an adherent fraction of peripheral blood mononuclear cells (MNCs) in the presence of GM-CSF and IFNα or IL-4 with subsequent lipopolysaccharide-driven maturation. Dex (10-6 M) was added to the cultures at day 3. We showed that generation of IFN-DCs with Dex resulted in decrease in percentage of CD83+ and CD86+ DCs and increase in numbers of CD14+, B7-H1+, and Toll-like receptor 2 (TLR2+) DCs. Treatment with Dex downregulated pro-inflammatory cytokine production, reduced DC allostimulatory activity, and inhibited DC capacity to stimulate Th1/pro-inflammatory cytokine production, altogether evidencing the induction of a tolerogenic phenotype. As compared to IL-4-DCs/Dex, IFN-DCs/Dex were characterized by larger proportion of TLR2+ and CD14+ cells, higher production of IL-10 and lower TNFα/IL-10 ratio, more potent capacity to induce T cell anergy, and more efficiently skewed T cell cytokine balance towards Th2/anti-inflammatory profile. CONCLUSIONS: The data obtained indicate that potent tDCs could be generated by treating IFN-DCs with dexamethasone. The tolerogenic properties of IFN-DCs/Dex are better than or at least equal to those of the IL-4-DCs/Dex, as assessed by in vitro phenotypic and functional assays, suggesting these cells as a new tolerogenic vaccine platform.
Assuntos
Células Dendríticas/imunologia , Dexametasona/imunologia , Interferon-alfa/imunologia , Vacinas/imunologia , Antígenos CD/imunologia , Células Cultivadas , Citocinas/imunologia , Humanos , Inflamação/imunologia , Interleucina-10/imunologia , Leucócitos Mononucleares/imunologia , Ativação Linfocitária/imunologia , Células Th1/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
BACKGROUND: We report on the results of a phase II clinical trial of Panagen (tablet form of fragmented human DNA preparation) in breast cancer patients (placebo group n = 23, Panagen n = 57). Panagen was administered as an adjuvant leukoprotective agent in FAC and AC chemotherapy regimens. Pre-clinical studies clearly indicate that Panagen acts by activating dendritic cells and induces the development of adaptive anticancer immune response. METHODS: We analyzed 5-year disease-free survival of patients recruited into the trial. RESULTS: Five-year disease-free survival in the placebo group was 40 % (n = 15), compared with the Panagen arm - 53 % (n = 51). Among stage III patients, disease-free survival was 25 and 52 % for placebo (n = 8) and Panagen (n = 25) groups, respectively. Disease-free survival of patients with IIIB + C stage was as follows: placebo (n = 6)-17 % vs Panagen (n = 18)-50 %. CONCLUSIONS: Disease-free survival rate (17 %) of patients with IIIB + C stage breast cancer receiving standard of care therapy is within the global range. Patients who additionally received Panagen demonstrate a significantly improved disease-free survival rate of 50 %. This confirms anticancer activity of Panagen. TRIAL REGISTRATION: ClinicalTrials.gov NCT02115984 from 04/07/2014.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Quimioterapia Adjuvante/métodos , Feminino , Humanos , Estadiamento de Neoplasias , Análise de Sobrevida , Resultado do TratamentoRESUMO
BACKGROUND: This study aimed to test the hypothesis that immune dysfunction and the increased risk of spontaneous abortion in pregnant women with hyperandrogenia (HA) are caused by the reduced tolerogenic potential of dendritic cells (DCs) that results from elevated levels of dehydroepiandrosterone sulfate (DHEAS). METHODS: The phenotypic and functional properties of monocyte-derived DCs generated from blood monocytes from non-pregnant women, women with a normal pregnancy, or pregnant women with HA, as well as the in vitro effects of DHEAS on DCs in healthy pregnant women were investigated. RESULTS: In a normal pregnancy, DCs were shown to be immature and are characterized by a reduced number of CD83(+) and CD25(+) DCs, the ability to stimulate type 2 T cell responses and to induce T cell apoptosis. By contrast, DCs from pregnant women with HA had a mature phenotype, were able to stimulate both type 1 (IFN-γ) and type 2 (IL-4) T cell responses, and were characterized by lower B7-H1 expression and cytotoxic activity against CD8(+) T cells. The addition of DHEAS to cultures of DCs from healthy pregnant women induced the maturation of DCs and increased their ability to activate type 1 T cell responses. CONCLUSION: Our data demonstrated the reduction in the tolerogenic potential of DCs from pregnant women with HA, and revealed new mechanisms involved in the hormonal regulation of DCs mediated by DHEAS.
Assuntos
Aborto Espontâneo/imunologia , Desidroepiandrosterona/metabolismo , Células Dendríticas/imunologia , Hiperandrogenismo/imunologia , Células Th1/imunologia , Células Th2/imunologia , Adulto , Diferenciação Celular , Células Cultivadas , Feminino , Humanos , Tolerância Imunológica , Interferon gama/metabolismo , Interleucina-4/metabolismo , Ativação Linfocitária , Gravidez , Risco , Equilíbrio Th1-Th2RESUMO
Dendritic cell-based vaccines are considered as a new and promising immunotherapeutic approach for cancer treatment. However, the choice of optimal protocol of dendritic cell generation in vitro represents the major challenge. Here, we compared phenotype and functional characteristics of human monocyte-derived dendritic cells (DCs) generated in the presence of IL-4/GM-CSF (IL4-DCs) and IFNα/GM-CSF (IFN-DCs). We showed that IFN-DCs displayed semi-mature phenotype and expressed higher level of CD123, TNF-related apoptosis-inducing ligand (TRAIL) and B7-H1 molecules in comparison with IL4-DCs. LPS-stimulated IFN-DCs were characterized by greater production of Th1/pro-inflammatory (IFN-γ, IL-2, IL-1ß, TNF-α, IL-17), Тh2/anti-inflammatory cytokines (IL-10, IL-5), hematopoietic growth factors (G-CSF) and chemokines (MCP-1). These data indicated more pronounced ability of IFN-DCs to induce cellular immune response as well as humoral immune response compared to IL4-DCs. LPS-stimulated IFN-DCs possessed higher direct cytotoxic activity against TRAIL-sensitive tumor cell line Jurkat and similar cytotoxicity against TRAIL-resistant tumor HEp-2 cells. Besides, IFN-DCs and IL4-DCs equally induced apoptosis of activated CD4(+) and CD8(+) T cells. These results suggest that IFN-DCs can be used as potent cell-based curative therapies for individuals with cancer.
Assuntos
Citocinas/imunologia , Citocinas/metabolismo , Citotoxicidade Imunológica , Células Dendríticas/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Interferon-alfa/imunologia , Interleucina-4/imunologia , Apoptose , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular , Linhagem Celular Tumoral , Quimiocinas/imunologia , Quimiocinas/metabolismo , Células Dendríticas/metabolismo , Humanos , Lipopolissacarídeos/imunologia , Ativação Linfocitária , Fenótipo , Ligante Indutor de Apoptose Relacionado a TNF/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: We performed a multicenter, double-blind, placebo-controlled, phase II clinical trial of human dsDNA-based preparation Panagen in a tablet form. In total, 80 female patients with stage II-IV breast cancer were recruited. METHODS: Patients received three consecutive FAC (5-fluorouracil, doxorubicin and cyclophosphamide) or AC (doxorubicin and cyclophosphamide) adjuvant chemotherapies (3 weeks per course) and 6 tablets of 5 mg Panagen or placebo daily (one tablet every 2-3 hours, 30 mg/day) for 18 days during each chemotherapy course. Statistical analysis was performed using Statistica 6.0 software, and non-parametric analyses, namely Wilcoxon-Mann-Whitney and paired Wilcoxon tests. To describe the results, the following parameters were used: number of observations (n), median, interquartile range, and minimum-maximum range. RESULTS: Panagen displayed pronounced leukostimulatory and leukoprotective effects when combined with chemotherapy. In an ancillary protocol, anticancer effects of a tablet form of Panagen were analyzed. We show that Panagen helps maintain the pre-therapeutic activity level of innate antitumor immunity and induces formation of a peripheral pool of cytotoxic CD8+ perforin + T-cells. Our 3-year follow-up analysis demonstrates that 24% of patients who received Panagen relapsed or died after the therapy, as compared to 45% in the placebo cohort. CONCLUSIONS: The data collected in this trial set Panagen as a multi-faceted "all-in-one" medicine that is capable of simultaneously sustaining hematopoiesis, sparing the innate immune cells from adverse effects of three consecutive rounds of chemotherapy and boosting individual adaptive immunity. Its unique feature is that it is delivered via gastrointestinal tract and acts through the lymphoid system of intestinal mucosa. Taken together, maintenance of the initial levels of innate immunity, development of adaptive cytotoxic immune response and significantly reduced incidence of relapses 3 years after the therapy argue for the anticancer activity of Panagen. TRIAL REGISTRATION: ClinicalTrials.gov NCT02115984 from 04/07/2014.
Assuntos
Imunidade Adaptativa/efeitos dos fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , DNA/administração & dosagem , Leucopoese/efeitos dos fármacos , Imunidade Adaptativa/imunologia , Neoplasias da Mama/imunologia , DNA/química , Método Duplo-Cego , Feminino , Seguimentos , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucopoese/imunologiaRESUMO
Recent studies have revealed that besides the important role in triggering the adoptive antitumor immunity, dendritic cells (DCs) possess direct cytotoxic antitumor activity. Here, we investigated brain glioma patient monocyte-derived DCs generated in the presence of IFNα and GM-CSF (IFN-DCs). These DCs were characterized by reduced cytotoxic activity against TRAIL-resistant HEp-2 cells. The impairment of DC cytotoxic function was observed mainly in high-grade glioma patients and associated with poor survival. The dysfunction of patient DC cytotoxicity was partially restored under in vitro pretreatment of DCs with double-stranded human DNA as well as rIL-2. In contrast to healthy donors, IFN-DCs in a part of high-grade glioma patients also failed to lyse primary autologous or allogeneic glioma cells. Our findings point to possible contribution of DC impairment in tumor pathogenesis in brain glioma and justify the necessity to evaluate and correct DC cytotoxic function when exploring DCs as cancer vaccines in glioma.
Assuntos
Neoplasias Encefálicas/terapia , Células Dendríticas/imunologia , Glioma/terapia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Imunoterapia Adotiva/métodos , Interferon-alfa/imunologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Adolescente , Adulto , Idoso , Neoplasias Encefálicas/imunologia , Estudos de Casos e Controles , Linhagem Celular Tumoral , Sobrevivência Celular/imunologia , Criança , Testes Imunológicos de Citotoxicidade , Feminino , Glioma/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Humanos , Estimativa de Kaplan-Meier , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-Idade , Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Adulto JovemRESUMO
Background: Double-stranded fragmented extracellular DNA is a participant, inducer, and indicator of various processes occurring in the organism. When investigating the properties of extracellular DNA, the question regarding the specificity of exposure to DNA from different sources has always been raised. The aim of this study was to perform comparative assessment of biological properties of double-stranded DNA obtained from the human placenta, porcine placenta and salmon sperm. Methods: The intensity of leukocyte-stimulating effect of different dsDNA was assessed in mice after cyclophosphamide-induced cytoreduction. The stimulatory effect of different dsDNA on maturation and functions of human dendritic cells and the intensity of cytokine production by human whole blood cells was analyzed ex vivo. The oxidation level of the dsDNA was also compared. Results: Human placental DNA exhibited the strongest leukocyte-stimulating effect. DNA extracted from human and porcine placenta exhibited similar stimulatory action on maturation of dendritic cells, allostimulatory capacity, and ability of dendritic cells to induce generation of cytotoxic CD8+CD107a+ T cells in the mixed leukocyte reaction. DNA extracted from salmon sperm stimulated the maturation of dendritic cells, while having no effect on their allostimulatory capacity. DNA extracted from human and porcine placenta was shown to exhibit a stimulatory effect on cytokine secretion by human whole blood cells. The observed differences between the DNA preparations can be caused by the total methylation level and are not related to differences in oxidation level of DNA molecules. Conclusions: Human placental DNA exhibited the maximum combination of all biological effects.
RESUMO
We investigated the influence of Panagen DNA preparations on laboratory animals and IFN-induced human dendritic cells, as well as analyzed the data from a phase II clinical trial in the therapy of breast cancer. It was shown that this treatment resulted in increased number of CD8+/perforin+ T cells in peripheral lymphoid organs of experimental animals, in mixed lymphocyte culture population and in peripheral blood of breast cancer patients. Moreover, we demonstrated that when Panagen DNA preparations are used in combination with the standard FAC-based breast cancer therapies, non-specific immune response activity remains at the same levels as observed prior to therapy, whereas in FAC-placebo patients, non-specific immunity is greatly diminished.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Citotoxicidade Imunológica/efeitos dos fármacos , DNA/farmacologia , Perforina/imunologia , Animais , Neoplasias da Mama/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Diferenciação Celular , Células Cultivadas , Ensaios Clínicos Fase II como Assunto , Células Dendríticas/citologia , Células Dendríticas/imunologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos CBA , Perforina/biossínteseRESUMO
The PD-1/B7-H1-mediated induction of T cell apoptosis/anergy as a possible mechanism of immune response failure was studied in 76 patients with pulmonary tuberculosis (TB) with normal and low-proliferative response to antigens of M. tuberculosis (purified protein derivative (PPD)). It was revealed that dendritic cells (DCs), generated in vitro from patient blood monocytes with GM-CSF + IFN-α, were characterized by increased B7-H1 expression, upproduction of IL-10, and reducing of allostimulatory activity in mixed lymphocyte culture (MLC). Moreover, DCs of patients with TB were able to enhance T cell apoptosis and to block T-cell division in MLC. It was shown that neutralizing anti-PD1 antibodies significantly decreased the proapoptogenic/tolerogenic effect of DCs. Correlation analysis revealed a direct relationship between IL-10 production and level of B7-H1 expression in the general group of investigated patients. It was demonstrated that generation of healthy donor DCs in the presence of IL-10 led to an increase in the number of DCs-expressed B7-H1 molecule, DC proapoptogenic activity, and a decrease in their allostimulatory activity. Obviously, the revealed phenomenon of the PD-1/B7-H1-mediated pro-apoptogenic activity of DCs is clinically significant since the cytotoxic/tolerogenic potential of DCs is more pronounced in patients with PPD anergy.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Imunoterapia Adotiva/métodos , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/terapia , Adolescente , Adulto , Anticorpos Neutralizantes/imunologia , Antígenos de Bactérias/imunologia , Apoptose/imunologia , Proteínas Reguladoras de Apoptose/imunologia , Antígeno B7-H1/imunologia , Citotoxicidade Imunológica , Células Dendríticas/efeitos dos fármacos , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Interferon gama/imunologia , Interferon gama/farmacologia , Interleucina-10/imunologia , Masculino , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Mycobacterium tuberculosis/imunologia , Receptor de Morte Celular Programada 1/imunologia , Adulto JovemRESUMO
To overcome immune tolerance to cancer, the immune system needs to be exposed to a multi-target action intervention. Here, we investigated the activating effect of CpG oligodeoxynucleotides (ODNs), mesyl phosphoramidate CpG ODNs, anti-OX40 antibodies, and OX40 RNA aptamers on major populations of immunocompetent cells ex vivo. Comparative analysis of the antitumor effects of in situ vaccination with CpG ODNs and anti-OX40 antibodies, as well as several other combinations, such as mesyl phosphoramidate CpG ODNs and OX40 RNA aptamers, was conducted. Antibodies against programmed death 1 (PD1) checkpoint inhibitors or their corresponding PD1 DNA aptamers were also added to vaccination regimens for analytical purposes. Four scenarios were considered: a weakly immunogenic Krebs-2 carcinoma grafted in CBA mice; a moderately immunogenic Lewis carcinoma grafted in C57Black/6 mice; and an immunogenic A20 B cell lymphoma or an Ehrlich carcinoma grafted in BALB/c mice. Adding anti-PD1 antibodies (CpG+αOX40+αPD1) to in situ vaccinations boosts the antitumor effect. When to be used instead of antibodies, aptamers also possess antitumor activity, although this effect was less pronounced. The strongest effect across all the tumors was observed in highly immunogenic A20 B cell lymphoma and Ehrlich carcinoma.
RESUMO
Angiotensin I-converting enzyme (ACE, CD143) plays a crucial role in blood pressure regulation, vascular remodeling, and immunity. A wide spectrum of mAbs to different epitopes on the N and C domains of human ACE have been generated and used to study different aspects of ACE biology, including establishing a novel approach-conformational fingerprinting. Here we characterized a novel set of 14 mAbs, developed against human seminal fluid ACE. The epitopes for these novel mAbs were defined using recombinant ACE constructs with truncated N and C domains, species cross-reactivity, ACE mutagenesis, and competition with the previously mapped anti-ACE mAbs. Nine mAbs recognized regions on the N domain, and 5 mAbs-on the C domain of ACE. The epitopes for most of these novel mAbs partially overlap with epitopes mapped onto ACE by the previously generated mAbs, whereas mAb 8H1 recognized yet unmapped region on the C domain where three ACE mutations associated with Alzheimer's disease are localized and is a marker for ACE mutation T877M. mAb 2H4 could be considered as a specific marker for ACE in dendritic cells. This novel set of mAbs can identify even subtle changes in human ACE conformation caused by tissue-specific glycosylation of ACE or mutations, and can detect human somatic and testicular ACE in biological fluids and tissues. Furthermore, the high reactivity of these novel mAbs provides an opportunity to study changes in the pattern of ACE expression or glycosylation in different tissues, cells, and diseases, such as sarcoidosis and Alzheimer's disease.
Assuntos
Anticorpos Monoclonais , Mapeamento de Epitopos/métodos , Peptidil Dipeptidase A , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Células CHO , Cricetinae , Cricetulus , Epitopos/genética , Glicosilação , Humanos , Mutação , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/imunologia , Peptidil Dipeptidase A/metabolismo , Domínios ProteicosRESUMO
A preparation of human genomic fragmented double-stranded DNA (dsDNA) was used as maturation stimulus in cultures of human dendritic cells (DCs) generated in compliance with the interferon protocol. Culturing of the DCs in medium with 5µg/ml of the DNA preparation was associated with a decrease in the relative proportion of CD14 + cells and an increase in that of CD83 + cells. These changes are markers of DC maturation. The efficiency with which the DNA preparation was able to elicit DC maturation was commensurate with that of lypopolysaccharide from bacterial cell, the standard inducer of DC maturation. Generated ex vivo, matured in the presence of the human DNA preparation, pulsed with tumor antigens mouse DCs were used as a vaccine in biological tests for its antitumor activity. The experimental results demonstrate that reinfusion of mature pulsed with tumor antigens DCs cause a statistically significant suppression of tumor graft growth.
Assuntos
Diferenciação Celular/efeitos dos fármacos , DNA/farmacologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Animais , Antígenos CD/metabolismo , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Carcinoma de Ehrlich/imunologia , Carcinoma de Ehrlich/prevenção & controle , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Feminino , Humanos , Imunoglobulinas/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos CBA , Fator de Necrose Tumoral alfa/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Antígeno CD83RESUMO
The immunosuppressive properties of vascular endothelial growth factors (VEGFs) suggest a new role of angiogenic factors in T cell modulation in cancer and pregnancy. Most of VEGF effects on T cells are mediated through the VEGF receptor type 2 (VEGFR-2). This study aims to investigate the role of placental growth factor (PlGF) as a selective VEGFR-1 ligand in the modulation of human T cells functions. For this, PBMCs from healthy donors were stimulated with anti-CD3 mAbs (a-CD3) or Concanavalin A (ConA) in the absence or presence of PlGF and assessed for T cell proliferation, IL-10 production, programmed cell death, and the expression of inhibitory receptors (PD-1, CTLA-4, TIM-3) using radiometric (3 H-thymidine incorporation) and FACS analysis. We showed that most T cells in freshly isolated PBMCs lacked VEGFR-1. However, activation with a-CD3 or ConA strongly increased the percentages of VEGFR-1 expressing CD4+ and CD8+ T cells. PlGF in a wide dose range suppressed PBMC cell proliferation, inhibiting both CD4+ and CD8+ T cells. Blockade of VEGFR-1, but not VEGFR-2 with neutralizing Abs completely abolished the suppressive effect of PlGF. Furthermore, we found that treatment with PlGF up-regulated IL-10 production in CD4+ and CD8+ T cells, promoted CD8+ T cells apoptosis and enhanced the expression of inhibitory receptors (PD-1 and TIM-3) on activated T cells. Our in vitro findings suggest the involvement of PlGF/VEGFR-1 signaling in the modulation of T cell responses in a-CD3-stimulated PBMCs.
Assuntos
Apoptose/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Fator de Crescimento Placentário/imunologia , Transdução de Sinais/imunologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/imunologia , Receptor Celular 2 do Vírus da Hepatite A/imunologia , Humanos , Interleucina-10/imunologia , Receptor de Morte Celular Programada 1/imunologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/imunologiaRESUMO
The key role of T cells in hepatitis C virus (HCV) elimination and the ability of dendritic cells (DCs) to induce antiviral T cell responses suggest that DC vaccines could be a promising approach in the treatment of chronic HCV infection. The aim of our study was to evaluate, whether immunotherapy with DCs is safe and elicits anti-HCV T cell responses. Ten patients with HCV (genotype 1) were vaccinated with monocyte-derived DCs, generated in the presence of IFN-α (IFN-DCs) and pulsed with recombinant HCV Core and NS3 proteins. Treatment schedule included four subcutaneous vaccinations with 1 week interval and six vaccinations with month interval. No serious adverse events or an increase in hepatitis C biochemical activity were registered after vaccination. Using ex vivo assays for the detection of proliferative responses, IFN-γ production and CD8+ degranulation have shown that immunotherapy elicited antigen-specific responses in all patients although individual heterogeneity existed within their types, magnitude, and timing. Core/NS3-specific proliferative response and CD8+ T cell degranulation have already been registered after the first course of vaccination. Of note, Core-specific responses had higher magnitude. The appearance of antigen-specific IFN-γ responses was registered after the second vaccination course. Vaccination did not cause Th2 response and expansion of the CD4+CD25+CD127- regulatory T cells. Generated immune responses failed to provide virus elimination. Nevertheless, there were inverse correlations between viral load and NS3-specific proliferation (R S = 0.62; p = 0.05) and IFN-γ secretion (R S = - 0.82; p = 0.001) at 6-month post-treatment period. Immunotherapy with IFN-DCs was safe and elicited HCV-specific T cell responses which were insufficient to eliminate viruses but could be implicated in the restriction of viral replication.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Hepacivirus/fisiologia , Hepatite C Crônica/terapia , Imunoterapia Adotiva/métodos , Adulto , Células Cultivadas , Células Dendríticas/transplante , Feminino , Seguimentos , Hepatite C Crônica/imunologia , Humanos , Interferon-alfa/imunologia , Interferon gama/metabolismo , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Vacinação , Proteínas do Core Viral/imunologia , Carga Viral , Proteínas não Estruturais Virais/imunologiaRESUMO
Besides initiation of tumor-specific T cell immunity, dendritic cells (DCs) are endowed with tumoricidal activity. Previously, we showed that monocyte-derived DCs of high-grade glioma patients generated in the presence of interferon alpha (IFNα) (IFN-DCs) have impaired cytotoxic activity against tumor necrosis factor alpha (TNFα)-sensitive HEp-2 tumor cells. Herein, we demonstrate that decreased transmembrane TNFα (tmTNFα) expression, but not soluble TNFα (sTNFα) production by high-grade glioma patient IFN-DCs, determines the defective tumoricidal activity against TNFα-sensitive HEp-2 cells. Blocking TNFα-converting enzyme or stimulation of patient IFN-DCs with rIL-2 or dsDNA enhances tmTNFα expression on IFN-DCs and significantly increases their cytotoxicity. Decreased tmTNFα expression on patient IFN-DCs is not caused by downregulation of pNFκB. Neither rIL-2 nor dsDNA upregulates tmTNFα expression on patient IFN-DCs via an increase of pNFκB. The current study shows an important role of tmTNFα as mediator of IFN-DC tumoricidal activity and as molecular target for the restoration of defective DC killer activity in high-grade glioma patients.
Assuntos
DNA/imunologia , Células Dendríticas/imunologia , Glioma/imunologia , Glioma/patologia , Interleucina-2/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Proliferação de Células/efeitos dos fármacos , Criança , Células Dendríticas/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/imunologia , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/biossíntese , Adulto JovemRESUMO
Recurrent herpes simplex labialis caused predominantly with herpes simplexvirus 1(HSV-1) is a major problem, for which various treatments have minimal impact. Given the important role of the immune system in controlling virus infection, an activation of virus-specific immune responses, in particular,using dendritic cell (DCs) vaccines, seems to be a promising approach for the treatment of patients with frequent recurrences of herpes labialis. The current paper presents the results of a pilot study of the safety and efficacy of DC vaccines in 14 patients with recurrent HSV-1 infections. DCs were generated in presence of GM-CSF and IFN-alpha and were loaded with HSV-1 recombinant viral glycoprotein D (HSV1gD). DCs cells were injected subcutaneously as 2 courses of vaccination during 9 months. Immunotherapy with DCs did not induce any serious side effects and resulted in more than 2-fold reduction in the recurrence rate and significant enhancement of the inter-recurrent time during the 9 months of treatment and subsequent 6-month follow-up period. An obvious clinical improvement was accompanied with an induction of an antigen-specific response to HCV1gD and a normalization of reduced mitogenic responsiveness of mono-nuclear cells. According to long-term survey data (on average 48 months after the beginning of therapy), 87% of respondents reported the decreased incidence of recurrent infection. At this time, most patients (85.7%) responded to HCV1gD stimulation. The data obtained suggests that dendritic cell vaccines may be a promising new approach for the treatment of recurrent labial herpes.
Assuntos
Células Dendríticas/imunologia , Herpes Labial/terapia , Vacinas contra o Vírus do Herpes Simples/imunologia , Adolescente , Adulto , Feminino , Vacinas contra o Vírus do Herpes Simples/administração & dosagem , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Prevenção Secundária , Resultado do Tratamento , Adulto JovemRESUMO
In the present study 37 patients with surgical infection were investigated and a new set of diagnostic tests for detection of major syndromes of systemic inflammation - systemic inflammatory response syndrome (SIRS), compensatory anti-inflammatory response syndrome (CARS) and mixed antagonist response syndrome (MARS) - was developed. In summary, we have demonstrated that patients with surgical infection were characterized by an immunodeficiency with significant reduction of mitogen-induced proliferation and IL-2/IL-4 production in vitro combined with decrease of HLA DR(+) monocytes. Furthermore, it was revealed that the patient's serum exhibited substantially enhanced suppressive and inflammatory activities as well as the level of C-reactive protein. We have defined the negative correlation between the serum inflammatory and suppressive activities (SIA and SSA) that was most prominent at the early stage of disease. Since the changes of serum bioactivity in the course of surgical infection were prominent and coherent, we supposed that tested activities might reflect the distinctive features of systemic inflammation. In according to this assumption, the patients were divided into 3 subgroups with predominance of SIRS, CARS and MARS by using the SIA and SSA expression. It has been shown that SIRS was more frequently detected at the early stage, whereas CARS - at the late stage of disease. Patients with SIRS, CARS or MARS significantly differed by the content of CD8(+) T and CD72(+) B lymphocytes, the concentration of IgG and IgA, the production of IL-2 and IL-4. Finally, the data obtained from patients, those were studied repeatedly, showed the possibility of transformation of the major systemic inflammatory syndromes during the disease course. Our findings suggest that measurement of serum inflammatory and suppressive activities may help to differentiate patients with SIRS, CARS or MARS and to select the appropriate strategy of immunotherapy.