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1.
Int J Mol Sci ; 22(7)2021 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-33806091

RESUMO

According to the literature, the autoantigen La is involved in Cap-independent translation. It was proposed that one prerequisite for this function is the formation of a protein dimer. However, structural analyses argue against La protein dimers. Noteworthy to mention, these structural analyses were performed under reducing conditions. Here we describe that La protein can undergo redox-dependent structural changes. The oxidized form of La protein can form dimers, oligomers and even polymers stabilized by disulfide bridges. The primary sequence of La protein contains three cysteine residues. Only after mutation of all three cysteine residues to alanine La protein becomes insensitive to oxidation, indicating that all three cysteines are involved in redox-dependent structural changes. Biophysical analyses of the secondary structure of La protein support the redox-dependent conformational changes. Moreover, we identified monoclonal anti-La antibodies (anti-La mAbs) that react with either the reduced or oxidized form of La protein. Differential reactivities to the reduced and oxidized form of La protein were also found in anti-La sera of autoimmune patients.


Assuntos
Autoantígenos/química , Oxirredução , Ribonucleoproteínas/química , Síndrome de Sjogren/imunologia , Anticorpos Antinucleares , Autoanticorpos/imunologia , Autoimunidade , Citocinas/metabolismo , Dissulfetos/química , Epitopos/química , Humanos , Lúpus Eritematoso Sistêmico/imunologia , Oxigênio/química , Polímeros/química , Multimerização Proteica , Estrutura Secundária de Proteína , RNA/química , Proteínas de Ligação a RNA/imunologia , Proteínas Recombinantes/química , Temperatura , Antígeno SS-B
2.
PLoS One ; 9(4): e95517, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24751697

RESUMO

There is currently growing interest in retargeting of effector T cells to tumor cells via bispecific antibodies (bsAbs). Usually, bsAbs are directed on the one hand to the CD3 complex of T cells and on the other hand to a molecule expressed on the surface of the target cell. A bsAb-mediated cross-linkage via CD3 leads to an activation of CD8+ T cells and consequently to killing of the target cells. In parallel, CD4+ T cells including TH1, TH2, TH17 cells and even regulatory T cells (Tregs) will be activated as well. Cytokines produced by CD4+ T cells can contribute to severe side effects e. g. life-threatening cytokine storms and, thinking of the immunosupressive function of Tregs, can even be counterproductive. Therefore, we asked whether or not it is feasible to limit retargeting to CD8+ T cells e. g. via targeting of the co-receptor CD8 instead of CD3. In order to test for proof of concept, a novel bsAb with specificity for CD8 and a tumor-associated surface antigen was constructed. Interestingly, we found that pre-activated (but not freshly isolated) CD8+ T cells can be retargeted via CD8-engaging bsAbs leading to an efficient lysis of target cells.


Assuntos
Anticorpos Biespecíficos/imunologia , Antígenos CD8/metabolismo , Linfócitos T CD8-Positivos/imunologia , Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Anticorpos de Cadeia Única/imunologia , Animais , Anticorpos Biespecíficos/isolamento & purificação , Complexo CD3/metabolismo , Linhagem Celular , Separação Celular , Reagentes de Ligações Cruzadas/metabolismo , Citocinas/metabolismo , Citotoxicidade Imunológica/imunologia , Humanos , Ativação Linfocitária/imunologia , Camundongos , Neoplasias/patologia , Ligação Proteica
3.
PLoS One ; 9(4): e93745, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24699869

RESUMO

Genetically engineered T lymphocytes are a promising option for cancer therapy. Prior to adoptive transfer they have to be expanded in vitro to reach therapeutically sufficient numbers. So far, no universal method exists for selective in vitro expansion of engineered T lymphocytes. In order to overcome this problem and for proof of concept we incorporated a novel unique peptide sequence of ten amino acids as epitope (E-Tag) into the binding domains of two novel chimeric antigen receptors (ECARs) directed against either prostate stem cell antigen (PSCA) for the treatment of prostate cancer (PCa) or CD33 for the treatment of acute myeloide leukemia (AML). The epitope tag then was utilized for expanding ECAR engrafted T cells by triggering the modified T cells via a monoclonal antibody directed against the E-Tag (Emab). Moreover, the E-Tag served as an efficient selection epitope for immunomagnetic isolation of modified T cells to high purity. ECAR engrafted T cells were fully functional and mediated profound anti-tumor effects in the respective models of PCa or AML both in vitro and in vivo. The method can be integrated straightforward into clinical protocols to improve therapeutic efficiency of tumor treatment with CAR modified T lymphocytes.


Assuntos
Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Transferência Adotiva , Epitopos , Humanos , Receptores de Antígenos/genética
4.
Blood ; 121(13): 2462-73, 2013 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-23365460

RESUMO

Regulated migration of hematopoietic stem cells is fundamental for hematopoiesis. The molecular mechanisms underlying stem cell trafficking are poorly defined. Based on a short hairpin RNA library and stromal cell-derived factor-1 (SDF-1) migration screening assay, we identified the histone 3 lysine 27 demethylase UTX (Kdm6a) as a novel regulator for hematopoietic cell migration. Using hematopoietic stem and progenitor cells from our conditional UTX knockout (KO) mice, we were able to confirm the regulatory function of UTX on cell migration. Moreover, adult female conditional UTX KO mice displayed myelodysplasia and splenic erythropoiesis, whereas UTX KO males showed no phenotype. During development, all UTX KO female and a portion of UTX KO male embryos developed a cardiac defect, cranioschisis, and died in utero. Therefore, UTY, the male homolog of UTX, can compensate for UTX in adults and partially during development. Additionally, we found that UTX knockdown in zebrafish significantly impairs SDF-1/CXCR4-dependent migration of primordial germ cells. Our data suggest that UTX is a critical regulator for stem cell migration and hematopoiesis.


Assuntos
Movimento Celular/genética , Hematopoese/genética , Células-Tronco Hematopoéticas/fisiologia , Histona Desmetilases/fisiologia , Animais , Células Cultivadas , Embrião não Mamífero , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Células HEK293 , Células-Tronco Hematopoéticas/metabolismo , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peixe-Zebra/embriologia , Peixe-Zebra/genética
5.
Oncoimmunology ; 2(12): e26770, 2013 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-24498554

RESUMO

We have recently described a novel modular targeting platform for T cell recruitment that not only efficiently replaces but also is superior to conventional T cell-engaging bispecific antibodies as it allows for the flexible targeting of several antigens and the delivery of co-stimulatory ligands to malignant lesions, thereby enhancing the antitumor potential of redirected T cells.

6.
J Immunol ; 189(6): 3249-59, 2012 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-22875801

RESUMO

Prostate cancer is the most common noncutaneous malignancy in men. The prostate stem cell Ag (PSCA) is a promising target for immunotherapy of advanced disease. Based on a novel mAb directed to PSCA, we established and compared a series of murine and humanized anti-CD3-anti-PSCA single-chain bispecific Abs. Their capability to redirect T cells for killing of tumor cells was analyzed. During these studies, we identified a novel bispecific humanized Ab that efficiently retargets T cells to tumor cells in a strictly Ag-dependent manner and at femtomolar concentrations. T cell activation, cytokine release, and lysis of target cells depend on a cross-linkage of redirected T cells with tumor cells, whereas binding of the anti-CD3 domain alone does not lead to an activation or cytokine release. Interestingly, both CD8+ and CD4+ T cells are activated in parallel and can efficiently mediate the lysis of tumor cells. However, the onset of killing via CD4+ T cells is delayed. Furthermore, redirecting T cells via the novel humanized bispecific Abs results in a delay of tumor growth in xenografted nude mice.


Assuntos
Anticorpos Biespecíficos/fisiologia , Anticorpos Monoclonais Humanizados/fisiologia , Antígenos de Neoplasias/biossíntese , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Proteínas de Neoplasias/biossíntese , Neoplasias da Próstata/imunologia , Células-Tronco/imunologia , Ensaio Tumoral de Célula-Tronco , Antígenos de Neoplasias/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/patologia , Morte Celular/imunologia , Epitopos de Linfócito T/imunologia , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/biossíntese , Proteínas Ligadas por GPI/imunologia , Humanos , Masculino , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/imunologia , Neoplasias da Próstata/patologia , Células-Tronco/patologia , Ensaio Tumoral de Célula-Tronco/métodos
7.
J Immunol ; 188(3): 1551-8, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22184723

RESUMO

Bispecific Abs hold great potential for immunotherapy of malignant diseases. Because the first components of this new drug class are now entering clinical trials, all aspects of their mode of action should be well understood. Several studies proved that CD8(+) and CD4(+) effector T cells can be successfully redirected and activated against tumor cells by bispecific Abs both in vitro and in vivo. To our knowledge, this study provides the first evidence that bispecific Abs can also redirect and activate regulatory T cells against a surface Ag, independently of their TCR specificity. After cross-linking, via a bispecific Ab, redirected regulatory T cells upregulate the activation markers CD69 and CD25, as well as regulatory T cell-associated markers, like CTLA-4 and FOXP3. The activated regulatory T cells secrete the immunosuppressive cytokine IL-10, but, in contrast to CD8(+) and CD4(+) effector T cells, almost no inflammatory cytokines. In addition, the redirected regulatory T cells are able to suppress effector functions of activated autologous CD4(+) T cells both in vitro and in vivo. Therefore, the potential risk for activation of regulatory T cells should be taken into consideration when bispecific Abs are applied for the treatment of malignant diseases. In contrast, an Ag/tissue-specific redirection of regulatory T cells with bispecific Abs holds great potential for the treatment of autoimmune diseases and graft rejection.


Assuntos
Anticorpos Biespecíficos/uso terapêutico , Terapia de Alvo Molecular/métodos , Linfócitos T Reguladores/efeitos dos fármacos , Anticorpos Biespecíficos/farmacologia , Antígenos de Superfície/efeitos dos fármacos , Linhagem Celular , Humanos , Interleucina-10 , Ativação Linfocitária
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