RESUMO
Alemtuzumab is a humanized monoclonal antibody specific for the CD52 protein present at high levels on the surface of B and T lymphocytes. In clinical trials, alemtuzumab has shown a clinical benefit superior to that of interferon-ß in relapsing-remitting multiple sclerosis patients. Treatment with alemtuzumab leads to the depletion of circulating lymphocytes followed by a repopulation process characterized by alterations in the number, proportions and properties of lymphocyte subsets. Of particular interest, an increase in the percentage of T cells with a regulatory phenotype (Treg cells) has been observed in multiple sclerosis patients after alemtuzumab. Since Treg cells play an important role in the control of autoimmune responses, the effect of alemtuzumab on Treg cells was further studied in vitro. Alemtuzumab effectively mediated complement-dependent cytolysis of human T lymphocytes and the remaining population was enriched in T cells with a regulatory phenotype. The alemtuzumab-exposed T cells displayed functional regulatory characteristics including anergy to stimulation with allogeneic dendritic cells and ability to suppress the allogeneic response of autologous T cells. Consistent with the observed increase in Treg cell frequency, the CD25(hi) T-cell population was necessary for the suppressive activity of alemtuzumab-exposed T cells. The mechanism of this suppression was found to be dependent on both cell-cell contact and interleukin-2 consumption. These findings suggest that an alemtuzumab-mediated increase in the proportion of Treg cells may play a role in promoting the long-term efficacy of alemtuzumab in patients with multiple sclerosis.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Esclerose Múltipla/imunologia , Linfócitos T Reguladores/imunologia , Alemtuzumab , Antígenos CD/imunologia , Antígenos de Neoplasias/imunologia , Antígeno CD52 , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/imunologia , Sobrevivência Celular , Células Dendríticas/imunologia , Células Dendríticas/patologia , Feminino , Glicoproteínas/imunologia , Humanos , Interleucina-2/imunologia , Subunidade alfa de Receptor de Interleucina-2/imunologia , Masculino , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/patologia , Linfócitos T Reguladores/patologiaRESUMO
TAR DNA-binding protein 43 (TDP-43) and Fused in Sarcoma/Translocated in Sarcoma (FUS) are ribonucleoproteins associated with pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Under physiological conditions, TDP-43 and FUS are predominantly localized in the nucleus, where they participate in transcriptional regulation, RNA splicing and metabolism. In disease, however, they are typically mislocalized to the cytoplasm where they form aggregated inclusions. A number of shared cellular pathways have been identified that contribute to TDP-43 and FUS toxicity in neurodegeneration. In the present study, we report a novel pathogenic mechanism shared by these two proteins. We found that pathological FUS co-aggregates with a ribosomal protein, the Receptor for Activated C-Kinase 1 (RACK1), in the cytoplasm of spinal cord motor neurons of ALS, as previously reported for pathological TDP-43. In HEK293T cells transiently transfected with TDP-43 or FUS mutant lacking a functional nuclear localization signal (NLS; TDP-43ΔNLS and FUSΔNLS), cytoplasmic TDP-43 and FUS induced co-aggregation with endogenous RACK1. These co-aggregates sequestered the translational machinery through interaction with the polyribosome, accompanied by a significant reduction of global protein translation. RACK1 knockdown decreased cytoplasmic aggregation of TDP-43ΔNLS or FUSΔNLS and alleviated associated global translational suppression. Surprisingly, RACK1 knockdown also led to partial nuclear localization of TDP-43ΔNLS and FUSΔNLS in some transfected cells, despite the absence of NLS. In vivo, RACK1 knockdown alleviated retinal neuronal degeneration in transgenic Drosophila melanogaster expressing hTDP-43WT or hTDP-43Q331K and improved motor function of hTDP-43WT flies, with no observed adverse effects on neuronal health in control knockdown flies. In conclusion, our results revealed a novel shared mechanism of pathogenesis for misfolded aggregates of TDP-43 and FUS mediated by interference with protein translation in a RACK1-dependent manner. We provide proof-of-concept evidence for targeting RACK1 as a potential therapeutic approach for TDP-43 or FUS proteinopathy associated with ALS and FTLD.
Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Degeneração Lobar Frontotemporal , Sarcoma , Animais , Humanos , Esclerose Lateral Amiotrófica/patologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células HEK293 , Neurônios Motores/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Demência Frontotemporal/patologia , Degeneração Lobar Frontotemporal/patologia , Biossíntese de Proteínas , Sarcoma/metabolismo , Sarcoma/patologia , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo , Receptores de Quinase C Ativada/genética , Receptores de Quinase C Ativada/metabolismo , Proteínas de Neoplasias/genéticaRESUMO
Lipid rafts reportedly have a role in coalescing key signaling molecules into the immunological synapse during T cell activation, thereby modulating T cell receptor (TCR) signaling activity. Recent findings suggest that a correlation may exist between increased levels of glycosphingolipids (GSLs) in the lipid rafts of T cells and a heightened response of those T cells toward activation. Here, we show that lowering the levels of GSLs in CD4(+) T cells using a potent inhibitor of glucosylceramide synthase (Genz-122346) led to a moderation of the T cell response toward activation. TCR proximal signaling events, such as phosphorylation of Lck, Zap70 and LAT, as well as early Ca(2+) mobilization, were attenuated by treatment with Genz-122346. Concomitant with these events were significant reductions in IL-2 production and T cell proliferation. Similar findings were obtained with CD4(+) T cells isolated from transgenic mice genetically deficient in GM3 synthase activity. Interestingly, lowering the GSL levels in CD4(+) T cells by either pharmacological inhibition or disruption of the gene for GM3 synthase also specifically inhibited the differentiation of T cells to the Th(17) lineage but not to other Th subsets in vitro. Taken together with the recently reported effects of Raftlin deficiency on Th(17) differentiation, these results strongly suggest that altering the GSL composition of lipid rafts modulates TCR signaling activity and affects Th(17) differentiation.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Glicoesfingolipídeos/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Células Th17/citologia , Animais , Diferenciação Celular , Linhagem da Célula , Citocinas/biossíntese , Sinapses Imunológicas , Microdomínios da Membrana , Camundongos , Camundongos TransgênicosRESUMO
Misfolded toxic forms of alpha-synuclein (α-Syn) have been implicated in the pathogenesis of synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). The α-Syn oligomers and soluble fibrils have been shown to mediate neurotoxicity and cell-to-cell propagation of pathology. To generate antibodies capable of selectively targeting pathogenic forms of α-Syn, computational modeling was used to predict conformational epitopes likely to become exposed on oligomers and small soluble fibrils, but not on monomers or fully formed insoluble fibrils. Cyclic peptide scaffolds reproducing these conformational epitopes exhibited neurotoxicity and seeding activity, indicating their biological relevance. Immunization with the conformational epitopes gave rise to monoclonal antibodies (mAbs) with the desired binding profile showing selectivity for toxic α-Syn oligomers and soluble fibrils, with little or no reactivity with monomers, physiologic tetramers, or Lewy bodies. Recognition of naturally occurring soluble α-Syn aggregates in brain extracts from DLB and MSA patients was confirmed by surface plasmon resonance (SPR). In addition, the mAbs inhibited the seeding activity of sonicated pre-formed fibrils (PFFs) in a thioflavin-T fluorescence-based aggregation assay. In neuronal cultures, the mAbs protected primary rat neurons from toxic α-Syn oligomers, reduced the uptake of PFFs, and inhibited the induction of pathogenic phosphorylated aggregates of endogenous α-Syn. Protective antibodies selective for pathogenic species of α-Syn, as opposed to pan α-Syn reactivity, are expected to provide enhanced safety and therapeutic potency by preserving normal α-Syn function and minimizing the diversion of active antibody from the target by the more abundant non-toxic forms of α-Syn in the circulation and central nervous system.
RESUMO
Alemtuzumab is a humanized monoclonal antibody against CD52, an antigen found on the surface of normal and malignant lymphocytes. It is approved for the treatment of B-cell chronic lymphocytic leukaemia and is undergoing Phase III clinical trials for the treatment of multiple sclerosis. The exact mechanism by which alemtuzumab mediates its biological effects in vivo is not clearly defined and mechanism of action studies have been hampered by the lack of cross-reactivity between human and mouse CD52. To address this issue, a transgenic mouse expressing human CD52 (hCD52) was created. Transgenic mice did not display any phenotypic abnormalities and were able to mount normal immune responses. The tissue distribution of hCD52 and the level of expression by various immune cell populations were comparable to those seen in humans. Treatment with alemtuzumab replicated the transient increase in serum cytokines and depletion of peripheral blood lymphocytes observed in humans. Lymphocyte depletion was not as profound in lymphoid organs, providing a possible explanation for the relatively low incidence of infection in alemtuzumab-treated patients. Interestingly, both lymphocyte depletion and cytokine induction by alemtuzumab were largely independent of complement and appeared to be mediated by neutrophils and natural killer cells because removal of these populations with antibodies to Gr-1 or asialo-GM-1, respectively, strongly inhibited the activity of alemtuzumab whereas removal of complement by treatment with cobra venom factor had no impact. The hCD52 transgenic mouse appears to be a useful model and has provided evidence for the previously uncharacterized involvement of neutrophils in the activity of alemtuzumab.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antineoplásicos/imunologia , Antígenos CD/metabolismo , Antígenos de Neoplasias/metabolismo , Antineoplásicos/imunologia , Glicoproteínas/metabolismo , Depleção Linfocítica/métodos , Modelos Animais , Adenoviridae/imunologia , Alemtuzumab , Animais , Anticorpos Monoclonais Humanizados , Anticorpos Antivirais/biossíntese , Antígeno CD52 , Citocinas/biossíntese , Citocinas/sangue , Relação Dose-Resposta Imunológica , Humanos , Tecido Linfoide/imunologia , Camundongos , Camundongos Transgênicos , Neutrófilos/imunologia , Baço/imunologia , Distribuição TecidualRESUMO
Advances in the understanding of Alzheimer's disease (AD) suggest that pathogenesis is not directly related to plaque burden, but rather to soluble toxic amyloid-beta oligomers (AßO). Therapeutic antibodies targeting Aß monomers and/or plaque have shown limited efficacy and dose-limiting adverse events in clinical trials. These findings suggest that antibodies capable of selectively neutralizing toxic AßO may achieve improved efficacy and safety. To this end, we generated monoclonal antibodies against a conformational Aß epitope predicted by computational modeling to be presented on toxic AßO but not monomers or fibrils. The resulting lead antibody, PMN310, showed the desired AßO-selective binding profile. In vitro, PMN310 inhibited AßO propagation and toxicity. In vivo, PMN310 prevented AßO-induced loss of memory formation and reduced synaptic loss and inflammation. A humanized version (huPMN310) compared favorably to other Aß-directed antibodies showing a lack of adverse event-associated binding to Aß deposits in AD brains, and greater selective binding to AßO-enriched AD brain fractions that contain synaptotoxic Aß species. Systemic administration of huPMN310 in mice resulted in brain exposure and kinetics comparable to those of other therapeutic human monoclonal antibodies. Greater selectivity for AßO and the potential to safely administer high doses of huPMN310 are expected to result in enhanced safety and therapeutic potency.
Assuntos
Doença de Alzheimer/imunologia , Peptídeos beta-Amiloides/imunologia , Anticorpos Monoclonais/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/fisiopatologia , Animais , Encéfalo/imunologia , Criança , Cognição/fisiologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Placa Amiloide/imunologia , Adulto JovemRESUMO
The efficacy and mechanism of action of therapeutic antibodies that target cancer cells have typically been evaluated using in vitro assays and long-term in vivo tumor models. To allow for a more efficient assessment of the function of candidate therapeutic antibodies, we have developed a flow cytometric-based method that rapidly and directly quantifies antibody-mediated killing in a short term in vivo assay. Target cells that express human CD52, including huCD52(+) splenocytes from huCD52 transgenic mice and Ramos cells, a CD52(+) human B cell lymphoma line, and CD52(-) reference cells were differentially labeled by using two fluorescent dyes to distinguish target and reference cell populations. Labeled cells were injected into mice with or without Campath-1H (Alemtuzumab) and then recovered for flow cytometric analysis 5 h later. We found that huCD52(+) transgenic splenocytes and Ramos cells were selectively depleted in Campath-treated animals but not in animals treated with a negative control antibody. Furthermore, it is likely that the cells were depleted in vivo by a complement-dependent mechanism since target cell depletion was significantly reversed after complement inactivation using cobra venom factor. This report demonstrates the feasibility and utility of a powerful method for the rapid evaluation in vivo of therapeutic antibody candidates for cancer.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Antineoplásicos/farmacologia , Anticorpos/farmacologia , Citotoxicidade Celular Dependente de Anticorpos , Citometria de Fluxo/métodos , Linfoma de Células B/imunologia , Alemtuzumab , Animais , Anticorpos/imunologia , Anticorpos Monoclonais Humanizados , Antígenos CD/imunologia , Antígenos de Neoplasias/imunologia , Antígeno CD52 , Linhagem Celular Tumoral , Corantes Fluorescentes/química , Glicoproteínas/imunologia , Humanos , Imunoterapia/métodos , Linfoma de Células B/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Camundongos Transgênicos , Organismos Livres de Patógenos EspecíficosRESUMO
Over the past decade, adenovirus (Ad)-based vectors have been used extensively in the context of cancer gene therapy. Two basic strategies have been pursued for the use of Ad vectors in cancer gene therapy: 1) approaches aimed at direct tumor cell killing through delivery of replicating oncolytic viruses or non-replicating vectors encoding tumor suppressor genes, suicide genes or anti-angiogenic genes, and 2) immunotherapeutic approaches aimed at inducing host anti-tumor immune responses that can destroy tumor cells at both primary and metastatic locations. Both strategies offer the potential of selective tumor cell destruction without damage to normal tissues. Extensive pre-clinical and clinical studies have been conducted based on these strategies. Encouraging results have been obtained but robust clinical efficacy remains elusive. Several obstacles limiting the therapeutic activity of Ad vectors have been encountered, including efficiency of tumor cell transduction and inhibition of efficacy by anti-Ad host immune responses. However, expanding knowledge in the areas of Ad biology and tumor biology continues to lead to increasingly sophisticated approaches to address these issues. A review of various Ad-based cancer gene therapy approaches and recent progress in the area are presented herein.
Assuntos
Adenovírus Humanos/genética , Terapia Genética/métodos , Vetores Genéticos , Neoplasias/terapia , Adenovírus Humanos/imunologia , Animais , Terapia Combinada , Vetores Genéticos/imunologia , Vetores Genéticos/uso terapêutico , Humanos , Imunoterapia/métodos , Modelos Biológicos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/radioterapiaRESUMO
Alemtuzumab, a monoclonal antibody directed against human CD52, is used in the treatment of MS. To characterize the impact of anti-CD52 administration, a monoclonal antibody to mouse CD52 (anti-muCD52) was generated and evaluated in EAE mouse models of MS. A single course of anti-muCD52 provided a therapeutic benefit accompanied by a reduction in the frequency of autoreactive T lymphocytes and production of pro-inflammatory cytokines. Examination of the CNS revealed a decrease in infiltrating lymphocytes, demyelination and axonal loss. Electrophysiological assessment showed preservation of axonal conductance in the spinal cord. These findings suggest that anti-CD52 therapy may help preserve CNS integrity.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígenos CD/imunologia , Antígenos de Neoplasias/imunologia , Doenças Desmielinizantes/tratamento farmacológico , Doenças Desmielinizantes/imunologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/imunologia , Glicoproteínas/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/farmacologia , Axônios/efeitos dos fármacos , Axônios/imunologia , Axônios/patologia , Antígeno CD52 , Doenças Desmielinizantes/patologia , Encefalomielite Autoimune Experimental/patologia , Glicoproteínas/antagonistas & inibidores , Humanos , Inflamação/tratamento farmacológico , Inflamação/imunologia , Inflamação/patologia , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência MolecularRESUMO
Treatment of multiple sclerosis (MS) is challenging: disease-modifying treatments (DMTs) must both limit unwanted immune responses associated with disease initiation and propagation (as T and B lymphocytes are critical cellular mediators in the pathophysiology of relapsing MS), and also have minimal adverse impact on normal protective immune responses. In this review, we summarize key preclinical and clinical data relating to the proposed mechanism of action of the recently approved DMT teriflunomide in MS. Teriflunomide selectively and reversibly inhibits dihydro-orotate dehydrogenase, a key mitochondrial enzyme in the de novo pyrimidine synthesis pathway, leading to a reduction in proliferation of activated T and B lymphocytes without causing cell death. Results from animal experiments modelling the immune activation implicated in MS demonstrate reductions in disease symptoms with teriflunomide treatment, accompanied by reduced central nervous system lymphocyte infiltration, reduced axonal loss, and preserved neurological functioning. In agreement with the results obtained in these model systems, phase 3 clinical trials of teriflunomide in patients with MS have consistently shown that teriflunomide provides a therapeutic benefit, and importantly, does not cause clinical immune suppression. Taken together, these data demonstrate how teriflunomide acts as a selective immune therapy for patients with MS.
Assuntos
Crotonatos/uso terapêutico , Fatores Imunológicos/uso terapêutico , Esclerose Múltipla/tratamento farmacológico , Toluidinas/uso terapêutico , Animais , Linfócitos B/imunologia , Proliferação de Células/efeitos dos fármacos , Crotonatos/farmacologia , Di-Hidro-Orotato Desidrogenase , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Hidroxibutiratos , Fatores Imunológicos/farmacologia , Esclerose Múltipla/imunologia , Esclerose Múltipla/fisiopatologia , Nitrilas , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Linfócitos T/imunologia , Toluidinas/farmacologiaRESUMO
The pathogenesis of multiple sclerosis (MS) is thought to involve peripheral activation of immune cells against central nervous system (CNS) antigens and their migration across the blood-brain barrier, leading to CNS inflammation and neurodegeneration. Alemtuzumab, a humanized anti-CD52 monoclonal antibody that rapidly depletes CD52-expressing cells from the circulation, is being investigated as a new treatment option in relapsing-remitting MS (RRMS). Clinical and radiologic results indicate robust suppression of inflammation related to the depletion of T and B lymphocytes during each treatment course of alemtuzumab. Furthermore, several lines of evidence suggest that the long-term clinical effects of alemtuzumab are attributable to qualitative changes in repopulating lymphocyte subsets potentially leading to a rebalancing of the immune system. Here, we review the contribution of data from animal models, ex vivo human studies, and clinical trials to the understanding of the mechanisms underlying the therapeutic effect of alemtuzumab in patients with RRMS.
RESUMO
Alemtuzumab is a monoclonal antibody against the CD52 antigen present at high levels on the surface of lymphocytes. While treatment of multiple sclerosis patients with alemtuzumab results in marked depletion of lymphocytes from the circulation, it has not been associated with a high incidence of serious infections. In a human CD52 transgenic mouse, alemtuzumab treatment showed minimal impact on the number and function of innate immune cells. A transient decrease in primary adaptive immune responses was observed post-alemtuzumab but there was little effect on memory responses. These results potentially help explain the level of immunocompetence observed in alemtuzumab-treated MS patients.
Assuntos
Imunidade Adaptativa/genética , Anticorpos Monoclonais Humanizados/fisiologia , Antígenos CD/imunologia , Antígenos de Neoplasias/imunologia , Glicoproteínas/imunologia , Alemtuzumab , Animais , Antígenos CD/genética , Antígenos de Neoplasias/genética , Linfócitos B/imunologia , Antígeno CD52 , Células Cultivadas , Glicoproteínas/genética , Humanos , Imunidade Inata/genética , Camundongos , Camundongos Transgênicos , Linfócitos T/imunologia , Resultado do TratamentoRESUMO
The interaction between CXCR4 on the surface of tumor cells and CXCL12 in the stroma is believed to contribute to tumor cell survival and protection against drug treatment. Inhibition of stromal survival signals by CXCR4 antagonists has been reported to render tumor cells more sensitive to chemotherapy, but little is known about potential synergy with monoclonal antibodies. In this study, administration of the small molecule CXCR4 antagonists plerixafor and GENZ-644494 was found to enhance the anti-tumor activity of the monoclonal antibodies alemtuzumab and rituximab in disseminated lymphoma models. The observed enhancement in therapeutic efficacy by CXCR4 antagonists appeared to involve several factors, including interference with the tumor-promoting signals delivered by CXCL12, disruption of the tumor/stroma interaction and mobilization of effector neutrophils capable of mediating antibody-dependent cell-mediated cytotoxicity. The involvement of neutrophils was further supported by the observed reversal in therapeutic benefit upon neutrophil depletion.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfoma/tratamento farmacológico , Receptores CXCR4/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto , Alemtuzumab , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Murinos/administração & dosagem , Anticorpos Monoclonais Murinos/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Benzilaminas , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quimiocina CXCL12/metabolismo , Quimiotaxia/efeitos dos fármacos , Ciclamos , Sinergismo Farmacológico , Citometria de Fluxo , Compostos Heterocíclicos/administração & dosagem , Compostos Heterocíclicos/farmacologia , Humanos , Linfoma/metabolismo , Linfoma/patologia , Camundongos , Camundongos SCID , Fosforilação/efeitos dos fármacos , Piridinas/administração & dosagem , Piridinas/farmacologia , Receptores CXCR4/metabolismo , Rituximab , Transdução de Sinais/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacosRESUMO
The molecular changes induced by alemtuzumab following binding of CD52 on B tumor cells were investigated. Alemtuzumab alone had no detectable impact on cell signaling but cross-linking of alemtuzumab on the surface of B tumor lines with anti-human Fc antibodies induced a transient Ca(2+) flux followed by phosphorylation of several kinases involved in stress and survival pathways, and expression of associated proteins including TNF-α. Cross-linking of alemtuzumab also induced capping and caspase-dependent apoptosis of the tumor lines. When using primary cells from B-CLL patients, alemtuzumab alone was capable of inducing protein phosphorylation and apoptosis through the cross-linking of alemtuzumab by FcγRIIb receptors on B-CLL cells. Apoptosis was prevented by blocking of FcγRIIb receptors with anti-CD32 antibody. Overall, our results indicate that cross-linking of alemtuzumab on B tumor cells can occur naturally through Fc receptor interaction and leads to the activation of specific cellular pathways and induction of apoptosis.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Apoptose/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Alemtuzumab , Anticorpos/imunologia , Anticorpos/farmacologia , Antineoplásicos/farmacologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Cálcio/metabolismo , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Immunoblotting , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de IgG/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Alemtuzumab is a monoclonal antibody that targets cell surface CD52 and is effective in depleting lymphocytes by cytolytic effects in vivo. Although the cytolytic effects of alemtuzumab are dependent on the density of CD52 antigen on cells, there is scant information regarding the expression levels of CD52 on different cell types. In this study, CD52 expression was assessed on phenotypically distinct subsets of lymphoid and myeloid cells in peripheral blood mononuclear cells (PBMCs) from normal donors. Results demonstrate that subsets of PBMCs express differing levels of CD52. Quantitative analysis showed that memory B cells and myeloid dendritic cells (mDCs) display the highest number while natural killer (NK) cells, plasmacytoid dendritic cells (pDCs) and basophils have the lowest number of CD52 molecules per cell amongst lymphoid and myeloid cell populations respectively. Results of complement dependent cytolysis (CDC) studies indicated that alemtuzumab mediated profound cytolytic effects on B and T cells with minimal effect on NK cells, basophils and pDCs, correlating with the density of CD52 on these cells. Interestingly, despite high CD52 levels, mDCs and monocytes were less susceptible to alemtuzumab-mediated CDC indicating that antigen density alone does not define susceptibility. Additional studies indicated that higher expression levels of complement inhibitory proteins (CIPs) on these cells partially contributes to their resistance to alemtuzumab mediated CDC. These results indicate that alemtuzumab is most effective in depleting cells of the adaptive immune system while leaving innate immune cells relatively intact.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antígenos CD/metabolismo , Antígenos de Neoplasias/metabolismo , Morte Celular/efeitos dos fármacos , Glicoproteínas/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Alemtuzumab , Antígenos CD/genética , Antígenos de Neoplasias/genética , Antígeno CD52 , Morte Celular/imunologia , Glicoproteínas/genética , Humanos , Leucócitos Mononucleares/imunologiaRESUMO
Overexpression of TMPRSS4, a cell surface-associated transmembrane serine protease, has been reported in pancreatic, colorectal and thyroid cancers, and has been implicated in tumor cell migration and metastasis. Few reports have investigated both TMPRSS4 gene expression levels and the protein products. In this study, quantitative RT-PCR and protein staining were used to assess TMPRSS4 expression in primary non-small cell lung carcinoma (NSCLC) tissues and in lung tumor cell lines. At the transcriptional level, TMPRSS4 message was significantly elevated in the majority of human squamous cell and adenocarcinomas compared with normal lung tissues. Staining of over 100 NSCLC primary tumor and normal specimens with rabbit polyclonal anti-TMPRSS4 antibodies confirmed expression at the protein level in both squamous cell and adenocarcinomas with little or no staining in normal lung tissues. Human lung tumor cell lines expressed varying levels of TMPRSS4 mRNA in vitro. Interestingly, tumor cell lines with high levels of TMPRSS4 mRNA failed to show detectable TMPRSS4 protein by either immunoblotting or flow cytometry. However, protein levels were increased under hypoxic culture conditions suggesting that hypoxia within the tumor microenvironment may upregulate TMPRSS4 protein expression in vivo. This was supported by the observation of TMPRSS4 protein in xenograft tumors derived from the cell lines. In addition, staining of human squamous cell carcinoma samples for carbonic anhydrase IX (CAIX), a hypoxia marker, showed TMPRSS4 positive cells adjacent to CAIX positive cells. Overall, these results indicate that the cancer-associated TMPRSS4 protein is overexpressed in NSCLC and may represent a potential therapeutic target.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Hipóxia Celular , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/biossíntese , Serina Endopeptidases/biossíntese , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animais , Antígenos de Neoplasias/análise , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Anidrase Carbônica IX , Anidrases Carbônicas/análise , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Pulmão/metabolismo , Neoplasias Pulmonares/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Nus , RNA Mensageiro/biossíntese , Serina Endopeptidases/genética , Transcrição Gênica , Microambiente Tumoral , Regulação para CimaRESUMO
Mesenchymal stem cells (MSCs) were discovered as a rare population of non-hematopoietic stem cells that reside in the bone marrow and interact closely with hematopoietic stem cells to support their growth and differentiation. MSCs are multipotent cells that have the ability to differentiate into cells of the mesenchymal lineage including adipocytes, osteocytes and chondrocytes and they have been reported to home to areas of tissue injury and participate in tissue repair. More recently, MSCs have also been described to possess anti-inflammatory and immunomodulatory properties that can affect multiple arms of the immune system. MSCs have been shown to inhibit T and B cell proliferation, downregulate the lytic activity of cytotoxic T lymphocytes and NK cells, inhibit the maturation and antigen-presenting function of dendritic cells and modulate macrophage function through both contact-dependent and contact-independent mechanisms. The administration of MSCs in models of autoimmune disease such as collagen-induced arthritis, EAE and autoimmune diabetes has provided additional evidence for an immunoregulatory role of MSCs supporting their use in controlling autoimmunity. The administration of allogeneic MSCs as immunosuppressive agents represents a viable approach as they appear to be largely non-immunogenic and clinical trials with allogeneic MSCs are currently underway in graftversus- host disease, Crohn's disease and type I diabetes indications. The immunomodulatory properties, mechanism of action and potential clinical utility of MSCs are reviewed herein.
Assuntos
Imunidade Celular , Células-Tronco Mesenquimais/imunologia , Transplante de Células-Tronco , Animais , Células Apresentadoras de Antígenos/imunologia , Doenças Autoimunes/terapia , Diferenciação Celular , Ensaios Clínicos como Assunto , Citocinas/imunologia , Modelos Animais de Doenças , Humanos , Imunidade Humoral , Células Matadoras Naturais/imunologia , Células-Tronco Mesenquimais/citologia , CamundongosRESUMO
Autoimmune uveitis is an inflammatory disorder of the eye that can lead to pain and vision loss. Steroids and immunosuppressive drugs are currently the only therapeutics for uveitis and have serious ocular and systemic toxicities. Therefore, safer alternative therapeutics are desired. Alpha-melanocyte stimulating hormone (α-MSH) is a neuropeptide that suppresses effector T cell functions, induces regulatory T cells and has beneficial effects in certain autoimmune and transplant models. A novel d-amino acid peptide analog of native α-MSH (dRI-α-MSH) was produced that was protected from protease digestion and had increased selectivity for the melanocortin-1 receptor. Systemic delivery of the dRI-α-MSH analog dramatically suppressed disease progression and retained retinal architecture in the experimental autoimmune uveitis (EAU) model. Local delivery by periorbital injection was equally effective. Importantly, treatment with the novel dRI-α-MSH analog suppressed uveitis with a similar magnitude to the corticosteroid, dexamethasone. Data indicate that the novel dRI-α-MSH analogs show anti-inflammatory activities and have potential therapeutic use in uveitis and other autoimmune diseases.
Assuntos
Doenças Autoimunes/tratamento farmacológico , Imunossupressores/uso terapêutico , Uveíte/tratamento farmacológico , alfa-MSH/análogos & derivados , alfa-MSH/uso terapêutico , Sequência de Aminoácidos , Animais , Doenças Autoimunes/imunologia , Feminino , Melanoma Experimental/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fragmentos de Peptídeos/uso terapêutico , Uveíte/imunologia , alfa-MSH/biossínteseRESUMO
PURPOSE: Placental growth factor (PlGF) is an angiogenic protein. Upregulation of PlGF has been observed in the clinic following antiangiogenic regimens targeting the VEGF pathway. PlGF has been proposed as a therapeutic target for oncology. sFLT01 is a novel fusion protein that neutralizes mouse and human PlGF (mPlGF, hPlGF) and mouse and human VEGF-A (mVEGF-A, hVEGF-A). It was tested in syngeneic and xenograft tumor models to evaluate the effects of simultaneously neutralizing PlGF and VEGF-A and to investigate changes observed in the clinic in preclinical models. EXPERIMENTAL DESIGN: Production of PlGF and VEGF-A by B16F10 and A673 cancer cells in vitro was assessed. Mice with subcutaneous B16F10 melanoma or A673 sarcoma tumors were treated with sFLT01. Tumor volumes and microvessel density (MVD) were measured to assess efficacy. Serum levels of hVEGF-A, hPlGF, and mPlGF at early and late time points were determined by ELISA. RESULTS: Exposure of cancer cell lines to sFLT01 caused a decrease in VEGF secretion. sFLT01 inhibited tumor growth, prolonged survival, and decreased MVD. Analysis of serum collected from treated mice showed that sFLT01 administration caused a marked increase in circulating mPlGF but not hPlGF or hVEGF. sFLT01 treatment also increased circulating mPlGF levels in non-tumor-bearing mice. CONCLUSION: With the tumor cell lines and mouse models we used, antiangiogenic therapies that target both PlGF and VEGF may elicit a host response rather than, or in addition to, a malignant cell response that contribute to therapeutic resistance and tumor escape as suggested by others.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Melanoma Experimental/tratamento farmacológico , Proteínas da Gravidez/sangue , Sarcoma de Ewing/tratamento farmacológico , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/uso terapêutico , Animais , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Humanos , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Fator de Crescimento Placentário , Proteínas da Gravidez/antagonistas & inibidores , Proteínas da Gravidez/genética , Proteínas da Gravidez/metabolismo , Proteínas Recombinantes de Fusão/uso terapêutico , Sarcoma de Ewing/metabolismo , Transdução de Sinais , Microambiente Tumoral , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/sangue , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
sFLT01 is a novel fusion protein that consists of the VEGF/PlGF (placental growth factor) binding domain of human VEGFR1/Flt-1 (hVEGFR1) fused to the Fc portion of human IgG(1) through a polyglycine linker. It binds to both human VEGF (hVEGF) and human PlGF (hPlGF) and to mouse VEGF (mVEGF) and mouse PlGF (mPlGF). In vitro, sFLT01 inhibited the proliferation of human umbilical vein endothelial cells and pericytes stimulated by either hVEGF or hPlGF. In vivo, sFLT01 had robust and significant antitumor activity in numerous preclinical subcutaneous tumor models including H460 non-small cell lung carcinoma, HT29 colon carcinoma, Karpas 299 lymphoma, MOLM-13 AML (acute myeloid leukemia), 786-O, and RENCA renal cell carcinoma (RCC). sFLT01 also increased median survival in the orthotopic RENCA RCC model. sFLT01 had strong antiangiogenic activity and altered intratumoral microvessel density, blood vessel lumen size and perimeter, and vascular and vessel areas in RCC models. sFLT01 treatment resulted in fewer endothelial cells and pericytes within the tumor microenvironment. sFLT01 in combination with cyclophosphamide resulted in greater inhibition of tumor growth than either agent used alone as a monotherapy in the A673 Ewing's sarcoma model. Gene expression profiling indicated that the molecular changes in the A673 sarcoma tumors are similar to changes observed under hypoxic conditions. sFLT01 is an innovative fusion protein that possessed robust antitumor and antiangiogenic activities in preclinical cancer models. It is a dual targeting agent that neutralizes both VEGF and PlGF and, therefore, has potential as a next generation antiangiogenic therapeutic for oncology.