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2.
J Crohns Colitis ; 12(12): 1459-1474, 2018 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-30137286

RESUMO

BACKGROUND AND AIMS: Epithelial expression of the insulin receptor in the colon has previously been reported to correlate with extent of colonic inflammation. However, the impact of insulin signalling in the intestinal mucosa is still unknown. Here, we investigated the effects of inactivating the epithelial insulin receptor in the intestinal tract, in an experimental model of inflammation-induced colorectal cancer. METHODS: The mice were generated by utilizing the intestinal- and epithelial-specific villin promoter and the Cre-Lox technology. All mice included in the cohorts were generated by crossing [vil-Cre-INSR+/-] × [INSRfl/fl] to obtain [vil-Cre-INSR-/-], and their floxed littermates [INSRfl/fl] served as the control group. For the intervention study, phosphate-buffered saline with or without insulin was instilled rectally in anaesthetized wild-type mice with chemically induced colitis. RESULTS: We found higher endoscopic colitis scores together with potentiated colonic tumorigenesis in the knockout mice. Furthermore, we showed that topically administered insulin in inflamed colons of wild-type mice reduced inflammation-induced weight loss and improved remission in a dose-dependent manner. Mice receiving rectal insulin enemas exhibited lower colitis endoscopic scores and reduced cyclooxygenase 2 mRNA expression, and developed significantly fewer and smaller tumours compared with the control group receiving phosphate-buffered saline only. CONCLUSIONS: Rectal insulin therapy could potentially be a novel treatment, targeting the epithelial layer to enhance mucosal healing in ulcerated areas. Our findings open up new possibilities for combination treatments to synergize with the existing anti-inflammatory therapies.


Assuntos
Colite , Neoplasias Colorretais , Inflamação , Insulina/administração & dosagem , Mucosa Intestinal , Administração Retal , Animais , Colite/tratamento farmacológico , Colite/etiologia , Colite/imunologia , Colite/patologia , Neoplasias Colorretais/etiologia , Neoplasias Colorretais/patologia , Neoplasias Colorretais/prevenção & controle , Troca Genética , Modelos Animais de Doenças , Endoscopia/métodos , Hipoglicemiantes/administração & dosagem , Inflamação/tratamento farmacológico , Inflamação/patologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Camundongos , Proteínas dos Microfilamentos/genética , Receptor de Insulina/imunologia
3.
Inflamm Bowel Dis ; 23(5): 739-752, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28426456

RESUMO

BACKGROUND: microRNAs (miRNAs) are small noncoding RNAs that guide degradation of mRNA and regulate protein expression. miRNA based diagnostic biomarkers for ulcerative colitis (UC) and Crohn's disease (CD) are emerging but information about the cellular localization of many miRNAs is limited and more detailed histologic evaluation of miRNA expression patterns is needed to understand their immunobiological function. METHODS: Formalin-fixed paraffin-embedded colon biopsies from 10 patients with UC and 8 patients with CD together with 9 controls were examined by RT-qPCR and quantitative in situ hybridization (ISH). The cellular expression of miR-21 positive cells was further characterized using immunohistochemical cellular markers. RESULTS: Increased levels of miR-21 and miR-126 were found in UC compared with controls and increased levels of miR-21 were observed in UC compared with CD by both RT-qPCR and quantitative in situ hybridization. miR-126 was localized to endothelial cells and miR-21 to cells in the lamina propria. Multiplex immunohistochemical staining showed miR-21 expression in subsets of CD68 macrophages and CD3 T cells in UC, however, far the majority of the miR-21 positive cells could not be categorized among CD68, CD3, and CD19 cells. CONCLUSIONS: This study shows that miR-126 levels are increased in UC and expressed in endothelial cells. miR-21 is expressed in subsets of monocytes/macrophages and T cells and may work as a potential biomarker to distinguish UC from CD. Quantitative in situ hybridization may be a powerful tool for such analysis as it combines overall expression with validation of cellular origin. Studies in larger cohorts may confirm this for clinical diagnostics.


Assuntos
Colite Ulcerativa/metabolismo , Doença de Crohn/metabolismo , Mucosa Intestinal/metabolismo , MicroRNAs/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Biópsia , Estudos de Casos e Controles , Colite Ulcerativa/patologia , Doença de Crohn/patologia , Feminino , Humanos , Hibridização In Situ , Mucosa Intestinal/patologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real
4.
Inflamm Bowel Dis ; 20(6): 1004-14, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24788222

RESUMO

BACKGROUND: Treatment with the TRPV1 agonist, capsaicin, was previously shown to protect against experimental colitis in the severe combined immunodeficiency (SCID) T-cell transfer model. Here, we investigate trpv1 gene expression in lymphoid organs and cells from SCID and BALB/c mice to identify a potential target for the anti-inflammatory effect of capsaicin. METHODS: The trpv1 expression was studied by real-time PCR in lymphoid tissues and gut of untreated and capsaicin-treated colitic SCID mice. Effects of capsaicin and a TRPV1 antagonist on T cells were studied in vitro. RESULTS: In contrast to BALB/c mice, spleen, lymph nodes, and rectum of colitic and noncolitic SCID mice express trpv1 mRNA. Capsaicin treatment in vivo attenuated T-cell transfer colitis and capsaicin in vitro also attenuated T-cell proliferation induced by enteroantigen, mitogen, and anti-CD3/CD28 beads in BALB/c, C57BL/6 mice, and B6.129X1-trpv1tm1Jul/J trpv1 knockout mice. Proliferation and cytokine secretion were fully comparable in mice with and without trpv1 expression. Likewise, enteroantigen- and mitogen-stimulated T cells from wild-type and trpv1 knockout mice were equally inhibited by capsaicin. Surprisingly, the TRPV1 antagonist BCTC also inhibited enteroantigen- and mitogen-induced T-cell proliferation. CONCLUSIONS: The trpv1 mRNA expression in lymphoid organs and the rectum of SCID mice suggests that the TRPV1 signaling in these organs could play a role in capsaicin-mediated attenuation of colitis. In addition, capsaicin-induced inhibition of T-cell proliferation of wild-type T cells lacking trpv1 expression suggests that capsaicin inhibits colitogenic T cells in a TRPV1 receptor-independent way, which might be linked to its anti-inflammatory effect.


Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Capsaicina/farmacologia , Colite/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Canais de Cátion TRPV/agonistas , Animais , Antígenos/metabolismo , Antipruriginosos/farmacologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/transplante , Proliferação de Células/efeitos dos fármacos , Colite/imunologia , Colite/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Ligantes , Tecido Linfoide/efeitos dos fármacos , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Pirazinas/farmacologia , Piridinas/farmacologia , Transdução de Sinais/imunologia , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Microglobulina beta-2/genética
5.
Oncoimmunology ; 2(4): e23659, 2013 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-23734320

RESUMO

Aberrantly glycosylated tumor antigens represent promising targets for the development of anti-cancer vaccines, yet how glycans influence immune responses is poorly understood. Recent studies have demonstrated that GalNAc-glycosylation enhances antigen uptake by dendritic cells as well as CD4+ T-cell and humoral responses, but prevents CD8+ T-cell activation. Here, we briefly discuss the relevance of glycans as candidate targets for anti-cancer vaccines.

6.
Glycoconj J ; 30(3): 227-36, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22878593

RESUMO

Protein glycosylation often changes during cancer development, resulting in the expression of cancer-associated carbohydrate antigens. In particular mucins such as MUC1 are subject to these changes. We previously identified an immunodominant Tn-MUC1 (GalNAc-α-MUC1) cancer-specific epitope not covered by immunological tolerance in MUC1 humanized mice and man. The objective of this study was to determine if mouse antibodies to this Tn-MUC1 epitope induce antibody-dependent cellular cytotoxicity (ADCC) pivotal for their potential use in cancer immunotherapy. Binding affinity of mAb 5E5 directed to Tn-MUC1 was investigated using BiaCore. The availability of Tn-MUC1 on the surface of breast cancer cells was evaluated by immunohistochemistry, confocal microscopy, and flow cytometry, followed by in vitro assessment of antibody-dependent cellular cytotoxicity by mAb 5E5. Biacore analysis demonstrated high affinity binding (KD = 1.7 nM) of mAb 5E5 to its target, Tn-MUC1. Immunolabelling with mAb 5E5 revealed surface expression of the Tn-MUC1 epitope in breast cancer tissue and cell lines, and mAb 5E5 induced ADCC in two human breast cancer cell lines, MCF7 and T47D. Aberrantly glycosylated MUC1 is expressed on the surface of breast cancer cells and a target for antibody-dependent cell-mediated cytotoxicity suggesting that antibodies targeting glycopeptide epitopes on mucins are strong candidates for cancer-specific immunotherapies.


Assuntos
Adenocarcinoma/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Neoplasias da Mama/imunologia , Mucina-1/imunologia , Acetilgalactosamina/química , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Feminino , Glicosilação , Humanos , Células MCF-7 , Mucina-1/química , Mucina-1/metabolismo
7.
PLoS One ; 7(11): e50139, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23189185

RESUMO

Aberrant glycosylation of mucins and other extracellular proteins is an important event in carcinogenesis and the resulting cancer associated glycans have been suggested as targets in cancer immunotherapy. We assessed the role of O-linked GalNAc glycosylation on antigen uptake, processing, and presentation on MHC class I and II molecules. The effect of GalNAc O-glycosylation was monitored with a model system based on ovalbumin (OVA)-MUC1 fusion peptides (+/- glycosylation) loaded onto dendritic cells co-cultured with IL-2 secreting OVA peptide-specific T cell hybridomas. To evaluate the in vivo response to a cancer related tumor antigen, Balb/c or B6.Cg(CB)-Tg(HLA-A/H2-D)2Enge/J (HLA-A2 transgenic) mice were immunized with a non-glycosylated or GalNAc-glycosylated MUC1 derived peptide followed by comparison of T cell proliferation, IFN-γ release, and antibody induction. GalNAc-glycosylation promoted presentation of OVA-MUC1 fusion peptides by MHC class II molecules and the MUC1 antigen elicited specific Ab production and T cell proliferation in both Balb/c and HLA-A2 transgenic mice. In contrast, GalNAc-glycosylation inhibited the presentation of OVA-MUC1 fusion peptides by MHC class I and abolished MUC1 specific CD8+ T cell responses in HLA-A2 transgenic mice. GalNAc glycosylation of MUC1 antigen therefore facilitates uptake, MHC class II presentation, and antibody response but might block the antigen presentation to CD8+ T cells.


Assuntos
Apresentação de Antígeno/imunologia , Neoplasias/imunologia , Neoplasias/metabolismo , Acetilgalactosamina/metabolismo , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos/imunologia , Antígenos de Neoplasias/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular , Glicosilação , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Imunoglobulina G/imunologia , Ativação Linfocitária/imunologia , Camundongos , Dados de Sequência Molecular , Mucina-1/química , Mucina-1/imunologia , Mucina-1/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica/imunologia
8.
Immunology ; 133(2): 239-45, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21426340

RESUMO

Anti-CD20 monoclonal antibodies are promising for the treatment of B-cell malignancies such as chronic lymphocytic leukaemia and autoimmune diseases where auto-antibodies play an important role. Anti-CD20 such as rituximab (RTX) mediates B-cell depletion through mechanisms such as complement-mediated cytotoxicity and antibody-dependent cellular cytotoxicity. However, in haematological malignancies, such effector mechanisms can be saturated and result in release of malignant B cells with reduced levels of CD20. It has been hypothesized that this is the result of monocyte-mediated shaving of the CD20/RTX complex from the B-cell surface. Here, we confirm, that in vitro co-culture of human monocytes and RTX-labelled syngeneic B cells results in reduced expression of CD20/RTX complex on the B cell surface. This shaving mechanism was the result of active protease activity because EDTA and PMSF were able to mediate partial inhibition. Also, a series of alternative anti-CD20 antibodies representing both type I and type II antibodies were tested for their ability to induce the shaving reaction. These results demonstrate that a monocyte-mediated shaving reaction can lead to complete loss of most anti-CD20 antibodies from the surface of B cells even from healthy donors and this is an important obstacle for antibody-mediated immune therapy. The findings demonstrate the necessity of developing novel antibodies that maintain high effector functions without enabling activation of the shaving reaction.


Assuntos
Anticorpos Monoclonais/imunologia , Antígenos CD20/imunologia , Linfócitos B/imunologia , Neoplasias Hematológicas/imunologia , Leucócitos Mononucleares/imunologia , Anticorpos Monoclonais Murinos/farmacologia , Citotoxicidade Celular Dependente de Anticorpos , Antineoplásicos/farmacologia , Células Cultivadas , Citometria de Fluxo , Humanos , Peptídeo Hidrolases/metabolismo , Ligação Proteica/efeitos dos fármacos , Rituximab
9.
Int J Cancer ; 128(8): 1860-71, 2011 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-21344374

RESUMO

Cancer-associated autoantibodies hold promise as sensitive biomarkers for early detection of cancer. Aberrant post-translational variants of proteins are likely to induce autoantibodies, and changes in O-linked glycosylation represent one of the most important cancer-associated post-translational modifications (PTMs). Short aberrant O-glycans on proteins may introduce novel glycopeptide epitopes that can elicit autoantibodies because of lack of tolerance. Technical barriers, however, have hampered detection of such glycopeptide-specific autoantibodies. Here, we have constructed an expanded glycopeptide array displaying a comprehensive library of glycopeptides and glycoproteins derived from a panel of human mucins (MUC1, MUC2, MUC4, MUC5AC, MUC6 and MUC7) known to have altered glycosylation and expression in cancer. Seromic profiling of patients with colorectal cancer identified cancer-associated autoantibodies to a set of aberrant glycopeptides derived from MUC1 and MUC4. The cumulative sensitivity of the array analysis was 79% with a specificity of 92%. The most prevalent of the identified autoantibody targets were validated as authentic cancer immunogens by showing expression of the epitopes in cancer using novel monoclonal antibodies. Our study provides evidence for the value of glycopeptides and other PTM-peptide arrays in diagnostic measures.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias Colorretais/sangue , Glicopeptídeos/sangue , Mucinas/sangue , Análise Serial de Proteínas , Anticorpos Monoclonais/imunologia , Autoanticorpos/imunologia , Biomarcadores Tumorais/imunologia , Estudos de Casos e Controles , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/imunologia , Ensaio de Imunoadsorção Enzimática , Epitopos/análise , Feminino , Glicopeptídeos/imunologia , Glicosilação , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Mucinas/imunologia , Prognóstico , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo
10.
Cytotherapy ; 12(6): 721-34, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20429791

RESUMO

BACKGROUND AND AIM: Dendritic cells are regarded as the most effective antigen presenting cells and coordinators of the immune response and therefore suitable as vaccine basis. Here we present results from a clinical study in which patients with malignant melanoma (MM) with verified progressive disease received vaccination with autologous monocyte-derived mature dendritic cells (DC) pulsed with p53, survivin and telomerase-derived peptides (HLA-A2+ patients) or with autologous/allogeneic tumor lysate (HLA-A2(−) patients) in combination with low-dose interleukin (IL)-2 and interferon (IFN)-alpha2b. RESULTS: Of 46 patients who initiated treatment, 10 stopped treatment within 1-4 weeks because of rapid disease progression and deterioration. After 8 weeks, 36 patients were evaluable: no patient had an objective response, 11 patients had stable disease (SD); six had continued SD after 4 months, and three patients had prolonged SD for more than 6 months. The mean overall survival time was 9 months, with a significantly longer survival (18.4 months) of patients who attained SD compared with patients with progressive disease (PD) (5 months). Induction of antigen-specific T-cell responses was analyzed by multidimensional encoding of T cells using HLA-A2 major histocompatibility complex (MHC) multimers. Immune responses against five high-affinity vaccine peptides were detectable in the peripheral blood of six out of 10 analyzed HLA-A2+ patients. There was no observed correlation between the induction of immune responses and disease stabilization. A significant lower blood level of regulatory T cells (CD25(high) CD4 T cells) was demonstrable after six vaccinations in patients with SD compared with PD. CONCLUSIONS: Vaccination was feasible and safe. Treatment-associated SD was observed in 24% of the patients. SD correlated with prolonged survival suggesting a clinical benefit. Differences in the level of regulatory T cells among SD and PD patients could indicate a significant role of these immune suppressive cells.


Assuntos
Vacinas Anticâncer , Células Dendríticas/metabolismo , Melanoma/terapia , Neoplasias Cutâneas/terapia , Linfócitos T/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/patologia , Células Dendríticas/transplante , Feminino , Seguimentos , Antígeno HLA-A2/metabolismo , Humanos , Interferon-alfa/administração & dosagem , Interleucina-2/administração & dosagem , Ativação Linfocitária , Masculino , Melanoma/imunologia , Melanoma/mortalidade , Melanoma/patologia , Melanoma/fisiopatologia , Pessoa de Meia-Idade , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/fisiopatologia , Análise de Sobrevida , Linfócitos T/imunologia , Linfócitos T/patologia
11.
AIDS ; 23(11): 1329-40, 2009 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-19528789

RESUMO

OBJECTIVE: To investigate the potential to induce additional cytotoxic T-lymphocyte (CTL) immunity during chronic HIV-1 infection. DESIGN: We selected infrequently targeted or subdominant but conserved HLA-A*0201-binding epitopes in Gag, Pol, Env, Vpu and Vif. These relatively immune silent epitopes were modified as anchor-optimized peptides to improve immunogenicity and delivered on autologous monocyte-derived dendritic cells (MDDCs). METHODS: Twelve treatment-naïve HLA-A*0201 HIV-1-infected Danish individuals received 1 x 10 MDDCs subcutaneously (s.c.) (weeks 0, 2, 4 and 8), pulsed with seven CD8 T-cell epitopes and three CD4 T-cell epitopes. Epitope-specific responses were evaluated by intracellular cytokine staining for interferon-gamma, tumor necrosis factor alpha and interleukin-2 and/or pentamer labeling 3 weeks prior to, 10 weeks after and 32 weeks after the first immunization. RESULTS: Previously undetected T-cell responses specific for one or more epitopes were induced in all 12 individuals. Half of the participants had sustained CD4 T-cell responses 32 weeks after immunization. No severe adverse effects were observed. No overall or sustained change in viral load or CD4 T-cell counts was observed. CONCLUSION: These data show that it is possible to generate new T-cell responses in treatment-naive HIV-1-infected individuals despite high viral loads, and thereby redirect immunity to target new multiple and rationally selected subdominant CTL epitopes. Further optimization could lead to stronger and more durable cellular responses to selected epitopes with the potential to control viral replication and prevent disease in HIV-1-infected individuals.


Assuntos
Vacinas contra a AIDS , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/uso terapêutico , Regulação Viral da Expressão Gênica , Infecções por HIV/prevenção & controle , HIV-1 , Adulto , Doença Crônica , Células Dendríticas/imunologia , Epitopos de Linfócito T/imunologia , Feminino , Infecções por HIV/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Carga Viral
12.
J Immunother ; 31(8): 771-80, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18779742

RESUMO

Therapeutic dendritic cell (DC) vaccination against cancer is a strategy aimed at activating the immune system to recognize and destroy tumor cells. In this nonrandomized phase 1/2 trial, we investigated the safety, feasibility, induction of T-cell response, and clinical response after treatment with a DC-based vaccine in patients with metastatic renal cell carcinoma. Twenty-seven patients with progressive cytokine-refractory metastatic renal cell carcinoma were vaccinated with DCs loaded with either a cocktail of survivin and telomerase peptides or tumor lysate depending on their HLA-A2 haplotype, and low-dose IL-2 was administered concomitantly. Tumor response, immune response, and serum IL-6 and YKL-40 were measured during treatment. Vaccine generation was successful in all patients and no serious adverse events were observed. None of the patients had an objective response but 13/27 patients obtained disease stabilization (SD) for more than 8 weeks. An antigen-specific immune response was demonstrated in 6/6 patients tested. Furthermore, significant alterations in serum YKL-40 and IL-6 were found during treatment. In conclusion, DC vaccination in our setting is feasible and without severe toxicity. Almost half of the patients obtained SD, and in more than 1/3 of the patients, SD persisted for more than 6 months. However, the evaluation of SD is difficult to interpret in the absence of a randomized trial and, therefore, these results should be interpreted with caution. Antigen-specific immune responses were observed in a subset of the treated patients.


Assuntos
Vacinas Anticâncer/uso terapêutico , Carcinoma de Células Renais/terapia , Células Dendríticas/transplante , Imunoterapia Adotiva , Neoplasias Renais/terapia , Adipocinas , Adulto , Idoso , Sequência de Aminoácidos , Carcinoma de Células Renais/secundário , Linhagem Celular Tumoral , Proteína 1 Semelhante à Quitinase-3 , Células Dendríticas/imunologia , Feminino , Glicoproteínas/sangue , Humanos , Proteínas Inibidoras de Apoptose , Interleucina-2/imunologia , Interleucina-2/uso terapêutico , Interleucina-6/sangue , Estimativa de Kaplan-Meier , Neoplasias Renais/patologia , Lectinas , Masculino , Proteínas Associadas aos Microtúbulos/imunologia , Pessoa de Meia-Idade , Dados de Sequência Molecular , Peptídeos/imunologia , Survivina , Linfócitos T Citotóxicos/imunologia , Telomerase/imunologia , Vacinação
13.
Vaccine ; 26(36): 4716-24, 2008 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-18616968

RESUMO

We have previously established a cancer vaccine using autologous DCs, generated by in vitro stimulation with IL-4 and GM-CSF, and pulsed with six HLA-A*0201 binding wild-type p53 derived peptides. This vaccine was used in combination with low-dose interleukin-2 in a recently published clinical Phase II trial where 26 HLA-A2+ patients with progressive late-stage metastatic breast cancer (BC) were included. Almost 1/3rd of the patients obtained stable disease or minor regression during treatment with a positive correlation to tumour over-expression of p53. In the present study, we performed a comprehensive analysis of the effector stage of the p53-specific CD8+ T cells by the use of Dextramer Technology and multicolour FACS. Pre- and post-treatment blood samples from eight BC patients were analysed. Independent of clinical outcome p53-specific T cells were phenotypic distinctly antigen experienced (CD44high, CCR-7low and CD62Llow). Furthermore, fresh blood from 18 cancer patients included in the vaccination trial were prospectively examined for more general treatment associated quantitative and qualitative changes in T cell subpopulations. We found that the frequency of CD4+ CD25high regulatory T cells was almost doubled after only 4 weeks of weekly vaccination and low-dose IL-2. In addition, a decrease in the percentage of CD27highCCR-7high CD4/CD8 naïve T cells was measured particularly in patients with progressive disease during vaccination. Finally, prior to immunotherapy a higher percentage of both CD28 and CD27 positive CD8naïve/early effector memory T cells were present in chemotherapy-treated patients.


Assuntos
Neoplasias da Mama/terapia , Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Interleucina-2/administração & dosagem , Subpopulações de Linfócitos/imunologia , Proteína Supressora de Tumor p53/imunologia , Vacinação/métodos , Adjuvantes Imunológicos/administração & dosagem , Neoplasias da Mama/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Citometria de Fluxo , Humanos , Leucócitos Mononucleares/imunologia , Linfócitos T Reguladores/imunologia
14.
J Immune Based Ther Vaccines ; 5: 9, 2007 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-17662155

RESUMO

Dendritic cells (DCs) are potent antigen-presenting cells and critical for the priming of CD8+ T cells. Therefore the use of these cells as adjuvant cells has been tested in a large number of experimental and clinical vaccination studies, in particular cancer vaccine studies. A number of protocols are emerging that combine vaccination with CTL expanding strategies, such as e.g. blockade of CTLA-4 signalling. On the other hand, the lifespan and in vivo survival of therapeutic DCs have only been addressed in a few studies, although this is of importance for the kinetics of CTL induction during vaccination. We have previously reported that DCs loaded with specific antigens are eliminated by antigen specific CTLs in vivo and that this elimination affects the potential for in vivo CTL generation. We now show that CTLA-4 blockade increases the number of DC vaccine induced LCMV gp33 specific CTLs and the lysis of relevant in vivo targets. However, the CTLA-4 blockage dependent expansion of CTLs also affect DC survival during booster DC injections and our data suggest that during a booster DC vaccine, the largest increase in CTL levels is already obtained during the first vaccination.

15.
Cancer Immunol Immunother ; 56(9): 1485-99, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17285289

RESUMO

p53 Mutations are found in up to 30% of breast cancers and peptides derived from over-expressed p53 protein are presented by class I HLA molecules and may act as tumor-associated epitopes in cancer vaccines. A dendritic cell (DC) based p53 targeting vaccine was analyzed in HLA-A2+ patients with progressive advanced breast cancer. DCs were loaded with 3 wild-type and 3 P2 anchor modified HLA-A2 binding p53 peptides. Patients received up to 10 sc vaccinations with 5 x 10(6) p53-peptide loaded DC with 1-2 weeks interval. Concomitantly, 6 MIU/m(2) interleukine-2 was administered sc. Results from a phase II trial including 26 patients with verified progressive breast cancer are presented. Seven patients discontinued treatment after only 2-3 vaccination weeks due to rapid disease progression or death. Nineteen patients were available for first evaluation after 6 vaccinations; 8/19 evaluable patients attained stable disease (SD) or minor regression while 11/19 patients had progressive disease (PD), indicating an effect of p53-specific immune therapy. This was supported by: (1) a positive correlation between p53 expression of tumor and observed SD, (2) therapy induced p53 specific T cells in 4/7 patients with SD but only in 2/9 patients with PD, and (3) significant response associated changes in serum YKL-40 and IL-6 levels identifying these biomarkers as possible candidates for monitoring of response in connection with DC based cancer immunotherapy. In conclusion, a significant fraction of breast cancer patients obtained SD during p53-targeting DC therapy. Data encourage initiation of a randomized trial in p53 positive patients evaluating the impact on progression free survival.


Assuntos
Neoplasias da Mama/terapia , Vacinas Anticâncer/uso terapêutico , Células Dendríticas/imunologia , Glicoproteínas/sangue , Interleucina-6/sangue , Fragmentos de Peptídeos/imunologia , Proteína Supressora de Tumor p53/imunologia , Adipocinas , Adulto , Idoso , Biomarcadores Tumorais/sangue , Neoplasias da Mama/imunologia , Proteína 1 Semelhante à Quitinase-3 , Células Dendríticas/transplante , Feminino , Humanos , Lectinas , Pessoa de Meia-Idade , Vacinação
16.
Vaccine ; 25(15): 2823-31, 2007 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-17254671

RESUMO

The purpose of the present study is to perform a global screening for new immunogenic HLA class I (HLA-I) restricted cytotoxic T cell (CTL) epitopes of potential utility as candidates of influenza A-virus diagnostics and vaccines. We used predictions of antigen processing and presentation, the latter encompassing 12 different HLA class I supertypes with >99% population coverage, and searched for conserved epitopes from available influenza A viral protein sequences. Peptides corresponding to 167 predicted peptide-HLA-I interactions were synthesized, tested for peptide-HLA-I interactions in a biochemical assay and for influenza-specific, HLA-I-restricted CTL responses in an IFN-gamma ELISPOT assay. Eighty-nine peptides could be confirmed as HLA-I binders, and 13 could be confirmed as CTL targets. The 13 epitopes, are highly conserved among human influenza A pathogens, and all of these epitopes are present in the emerging bird flu isolates. Our study demonstrates that present technology enables a fast global screening for T cell immune epitopes of potential diagnostics and vaccine interest. This technology includes immuno-bioinformatics predictors with the capacity to perform fast genome-, pathogen-, and HLA-wide searches for immune targets. To exploit this new potential, a coordinated international effort to analyze the precious source of information represented by rare patients, such as the current victims of bird flu, would be essential.


Assuntos
Epitopos de Linfócito T/imunologia , Antígenos HLA-A/imunologia , Antígenos HLA-B/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Linfócitos T Citotóxicos/imunologia , Adulto , Idoso , Animais , Aves , Feminino , Testes Genéticos , Genoma Viral , Humanos , Virus da Influenza A Subtipo H5N1/genética , Vírus da Influenza A/genética , Influenza Aviária/virologia , Influenza Humana/sangue , Influenza Humana/virologia , Masculino , Proteínas de Membrana Transportadoras/imunologia , Proteínas de Membrana Transportadoras/metabolismo , Pessoa de Meia-Idade , Complexo de Endopeptidases do Proteassoma/metabolismo , Linfócitos T Citotóxicos/virologia
17.
Cancer Lett ; 231(2): 247-56, 2006 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-16399226

RESUMO

Dendritic cells (DCs) were pulsed with the H-2K(b) binding OVA(257-264)-peptide (SIINFEKL), and used as one single-injection vaccine in combination with anti-CTLA-4 monoclonal antibody (mAb) to treat mice inoculated 3 days previously with 3x10(5) E.G7-OVA lymphoma cells. Neither DC vaccination nor CTLA-4 blockage alone prevented tumor growth in tumor challenged mice. In contrast, the combination of one vaccination and injection of anti-CTLA-4 mAb lead to rejection or retarded tumor growth in more than 60% of the mice. The OVA-transgene or the SIINFEKL-epitope was not lost in the progressing tumors of vaccinated mice, however, the highest degree of anti-SIINFEKL reactivity of host CTLs in an IFN-gamma ELISPOT assay was found only in mice showing complete tumor rejection. Vaccinated mice having rejected E.G7-OVA tumors were capable of rejecting subsequent challenges with 1x10(6) E.G7-OVA tumor cells, and later on these mice even rejected wild-type EL-4 tumor cells indicating that tumor epitope spreading takes place during the process of vaccination-induced E.G7-OVA rejection. In agreement with these observations, mice having rejected E.G7-OVA tumors showed long lasting CTL memory in spleen and bone marrow towards both the SIINFEKL-peptide and other EL-4-derived tumor rejecting epitopes.


Assuntos
Antígenos de Diferenciação/imunologia , Vacinas Anticâncer/uso terapêutico , Células Dendríticas/imunologia , Linfoma/tratamento farmacológico , Fragmentos de Peptídeos/uso terapêutico , Timoma/tratamento farmacológico , Animais , Anticorpos Monoclonais/farmacologia , Antígenos CD , Medula Óssea/imunologia , Antígeno CTLA-4 , Quimioterapia Combinada , Proteínas do Ovo/imunologia , Epitopos/imunologia , Feminino , Antígenos H-2/metabolismo , Linfoma/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Fragmentos de Peptídeos/imunologia , Transdução de Sinais , Baço/imunologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Timoma/imunologia , Transgenes/fisiologia , Vacinação
18.
Cancer Immunol Immunother ; 53(7): 633-41, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-14985857

RESUMO

Peptides derived from over-expressed p53 protein are presented by class I MHC molecules and may act as tumour-associated epitopes. Due to the diversity of p53 mutations, immunogenic peptides representing wild-type sequences are preferable as a basis for a broad-spectrum p53-targeting cancer vaccine. Our preclinical studies have shown that wild-type p53-derived HLA-A2-binding peptides are able to activate human T cells and that the generated effector T cells are cytotoxic to human HLA-A2+, p53+ tumour cells. In this phase I pilot study, the toxicity and efficacy of autologous dendritic cells (DCs) loaded with a cocktail of three wild-type and three modified p53 peptides are being analysed in six HLA-A2+ patients with progressive advanced breast cancer. Vaccinations were well tolerated and no toxicity was observed. Disease stabilisation was seen in two of six patients, one patient had a transient regression of a single lymph node and one had a mixed response. ELISpot analyses showed that the p53-peptide-loaded DCs were able to induce specific T-cell responses against modified and unmodified p53 peptides in three patients, including two of the patients with a possible clinical benefit from the treatment. In conclusion, the strategy for p53-DC vaccination seems safe and without toxicity. Furthermore, indications of both immunologic and clinical effect were found in heavily pretreated patients with advanced breast cancer. An independent clinical effect of repeated administration of DCs and IL-2 can not of course be excluded; further studies are necessary to answer these questions.


Assuntos
Neoplasias da Mama/terapia , Vacinas Anticâncer/uso terapêutico , Células Dendríticas/imunologia , Fragmentos de Peptídeos/imunologia , Proteína Supressora de Tumor p53/imunologia , Vacinação , Adulto , Idoso , Neoplasias da Mama/imunologia , Epitopos de Linfócito T/imunologia , Feminino , Antígeno HLA-A2/imunologia , Humanos , Interferon gama/metabolismo , Metástase Linfática , Pessoa de Meia-Idade , Projetos Piloto
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