Assuntos
Anticorpos Monoclonais/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Paraproteinemias/diagnóstico , Paraproteinemias/imunologia , Proteinose Alveolar Pulmonar/tratamento farmacológico , Anticorpos Monoclonais/sangue , Autoanticorpos/sangue , Autoanticorpos/imunologia , Biópsia , Medula Óssea/patologia , Cromatografia Líquida , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Cadeias gama de Imunoglobulina/sangue , Cadeias gama de Imunoglobulina/imunologia , Masculino , Gamopatia Monoclonal de Significância Indeterminada/sangue , Gamopatia Monoclonal de Significância Indeterminada/diagnóstico , Gamopatia Monoclonal de Significância Indeterminada/imunologia , Paraproteinemias/sangue , Proteinose Alveolar Pulmonar/diagnóstico , Proteinose Alveolar Pulmonar/etiologia , Espectrometria de Massas em Tandem , Tomografia Computadorizada por Raios XRESUMO
BACKGROUND: Humanized immune system immunodeficient mice have been extremely useful for the in vivo analyses of immune responses in a variety of models, including organ transplantation and graft versus host disease (GVHD) but they have limitations. Rat models are interesting complementary alternatives presenting advantages over mice, such as their size and their active complement compartment. Immunodeficient rats have been generated but human immune responses have not yet been described. METHODS: We generated immunodeficient Rat Rag-/- Gamma chain-/- human signal regulatory protein alpha-positive (RRGS) rats combining Rag1 and Il2rg deficiency with the expression of human signal regulatory protein alpha, a negative regulator of macrophage phagocytosis allowing repression of rat macrophages by human CD47-positive cells. We then immune humanized RRGS animals with human peripheral blood mononuclear cells (hPBMCs) to set up a human acute GVHD model. Treatment of GVHD was done with a new porcine antihuman lymphocyte serum active through complement-dependent cytotoxicity. We also established a tumor xenograft rejection model in these hPBMCs immune system RRGS animals by subcutaneous implantation of a human tumor cell line. RESULTS: RRGS animals receiving hPBMCs showed robust and reproducible reconstitution, mainly by T and B cells. A dose-dependent acute GVHD process was observed with progressive weight loss, tissue damage, and death censoring. Antihuman lymphocyte serum (L1S1) antibody completely prevented acute GVHD. In the human tumor xenograft model, detectable tumors were rejected upon hPBMCs injection. CONCLUSIONS: hPBMC can be implanted in RRGS animals and elicit acute GVHD or rejection of human tumor cells and these are useful models to test new immunotherapies.
Assuntos
Antígenos de Diferenciação/imunologia , Proteínas de Homeodomínio/imunologia , Hospedeiro Imunocomprometido , Cadeias gama de Imunoglobulina/imunologia , Síndromes de Imunodeficiência/imunologia , Leucócitos Mononucleares/transplante , Receptores Imunológicos/imunologia , Animais , Antígenos de Diferenciação/genética , Soro Antilinfocitário/farmacologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/imunologia , Xenoenxertos , Proteínas de Homeodomínio/genética , Humanos , Cadeias gama de Imunoglobulina/genética , Síndromes de Imunodeficiência/genética , Leucócitos Mononucleares/imunologia , Ratos Sprague-Dawley , Ratos Transgênicos , Receptores Imunológicos/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cutaneous sclerosis is one of the most common clinical manifestations of chronic graft-versus-host disease (cGVHD). Donor CD4(+) T and B cells play important roles in cGVHD pathogenesis, but the role of antibodies from donor B cells remains unclear. In the current studies, we generated immunoglobulin (Ig)H(µÎ³1) DBA/2 mice whose B cells have normal antigen-presentation and regulatory functions but cannot secrete antibodies. With a murine cGVHD model using DBA/2 donors and BALB/c recipients, we have shown that wild-type (WT) grafts induce persistent cGVHD with damage in the thymus, peripheral lymphoid organs, and skin, as well as cutaneous T helper 17 cell (Th17) infiltration. In contrast, IgH(µÎ³1) grafts induced only transient cGVHD with little damage in the thymus or peripheral lymph organs or with little cutaneous Th17 infiltration. Injections of IgG-containing sera from cGVHD recipients given WT grafts but not IgG-deficient sera from recipients given IgH(µÎ³1) grafts led to deposition of IgG in the thymus and skin, with resulting damage in the thymus and peripheral lymph organs, cutaneous Th17 infiltration, and perpetuation of cGVHD in recipients given IgH(µÎ³1) grafts. These results indicate that donor B-cell antibodies augment cutaneous cGVHD in part by damaging the thymus and increasing tissue infiltration of pathogenic Th17 cells.
Assuntos
Subpopulações de Linfócitos B/imunologia , Doença Enxerto-Hospedeiro/imunologia , Isoanticorpos/imunologia , Animais , Subpopulações de Linfócitos B/metabolismo , Subpopulações de Linfócitos B/transplante , Quimiocina CCL20/metabolismo , Doença Crônica , Células Dendríticas/metabolismo , Doença Enxerto-Hospedeiro/patologia , Imunoglobulina G/análise , Cadeias Pesadas de Imunoglobulinas , Cadeias gama de Imunoglobulina/genética , Cadeias gama de Imunoglobulina/imunologia , Cadeias mu de Imunoglobulina/genética , Cadeias mu de Imunoglobulina/imunologia , Interleucina-23/metabolismo , Tecido Linfoide/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Quimera por Radiação , Pele/patologia , Organismos Livres de Patógenos Específicos , Células Th17/imunologia , Timo/patologiaAssuntos
Crioglobulinemia/imunologia , Síndrome de Guillain-Barré/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Cadeias gama de Imunoglobulina/imunologia , Cadeias kappa de Imunoglobulina/imunologia , Vasculite Leucocitoclástica Cutânea/imunologia , Vitiligo/imunologia , Biópsia , Crioglobulinemia/complicações , Crioglobulinemia/diagnóstico , Crioglobulinemia/tratamento farmacológico , Glucocorticoides/uso terapêutico , Síndrome de Guillain-Barré/diagnóstico , Síndrome de Guillain-Barré/tratamento farmacológico , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Cadeias gama de Imunoglobulina/sangue , Cadeias kappa de Imunoglobulina/sangue , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento , Vasculite Leucocitoclástica Cutânea/diagnóstico , Vasculite Leucocitoclástica Cutânea/tratamento farmacológico , Vitiligo/diagnóstico , Vitiligo/tratamento farmacológicoRESUMO
IgG molecules exert both pro- and antiinflammatory effector functions based on the composition of the fragment crystallizable (Fc) domain glycan. Sialylated IgG Fc domains have antiinflammatory properties that are attributed to their ability to increase the activation threshold of innate effector cells to immune complexes by stimulating the upregulation of the inhibitory Fcγ receptor IIB (FcγRIIB). Here, we report that IgG Fc sialylation of human monoclonal IgG1 molecules impairs their efficacy to induce complement-mediated cytotoxicity (CDC). Fc sialylation of a CD20-targeting antibody had no impact on antibody-dependent cellular cytotoxicity and did not change the affinity of the antibody for activating Fcγ receptors. In contrast, the presence of sialic acid abrogated the increased binding of C1q to Fc-galactosylated IgG1 and resulted in decreased levels of C3b deposition on the cell surface. Similar to monoclonal antibodies, sialic acid inhibited the increased C1q binding to galactosylated Fc fragments in human polyclonal IgG. In sera derived from patients with chronic inflammatory demyelinating polyneuropathy, an autoimmune disease of the peripheral nervous system in which humoral immune responses mediate tissue damage, induction of IgG Fc sialylation was associated with clinical disease remission. Thus, impairment of CDC represents an FcγR-independent mechanism by which Fc-sialylated glycovariants might limit proinflammatory IgG effector functions.
Assuntos
Linfócitos B/imunologia , Via Clássica do Complemento , Proteínas do Sistema Complemento/imunologia , Imunoglobulina G/química , Cadeias gama de Imunoglobulina/química , Ácido N-Acetilneuramínico/química , Rituximab/química , Animais , Citotoxicidade Celular Dependente de Anticorpos , Antígenos CD20/imunologia , Linfoma de Burkitt/patologia , Linhagem Celular Tumoral , Complemento C1q/imunologia , Complemento C1q/metabolismo , Citotoxicidade Imunológica , Glicosilação , Humanos , Imunoglobulina G/imunologia , Cadeias gama de Imunoglobulina/imunologia , Imunoglobulinas Intravenosas/uso terapêutico , Células Matadoras Naturais/imunologia , Depleção Linfocítica , Camundongos , Glicoproteína Mielina-Oligodendrócito/imunologia , Polirradiculoneuropatia Desmielinizante Inflamatória Crônica/imunologia , Polirradiculoneuropatia Desmielinizante Inflamatória Crônica/terapia , Processamento de Proteína Pós-Traducional , Receptores de IgG/imunologia , Rituximab/imunologiaRESUMO
The CD80/CD86-CD28 axis is a critical pathway for immuno-corrective therapy, and the cytotoxic T lymphocyte antigen 4 (CTLA4) is a promising immunosuppressor targeting the CD80/CD86-CD28 axis; however, its use for asthma therapy needs further optimization. A human CTLA4 fused with the IgCγ Fc (CTLA4Ig) and mouse CC chemokine receptor type7 (CCR7) coding sequences were inserted into a recombinant adenovirus (rAdV) vector to generate rAdV-CTLA4Ig and rAdV-CCR7. The naive dendritic cells (DCs) were infected with these rAdVs to ensure CCR7 and CTLA4Ig expression. The therapeutic effects of modified DCs were evaluated. rAdV-CTLA4Ig and rAdV-CCR7 infected DCs improved all asthma symptoms. Inflammatory cell infiltration and cytokine analysis showed that rAdV-CTLA4Ig and rAdV-CCR7-modified DC therapy reduced the number of eosinophils and lymphocyte and neutrophil infiltration in the lung. Interestingly, assessment of the humoral immunity showed that the IL-4 and IFNγ levels of the rAdV-CTLA4Ig and rAdV-CCR7-modified DC-treated mice decreased significantly and did not reverse the Th1/Th2 balance. DCs expressing CCR7 displayed guidance ability for DC migration, primarily for DCs in the inflammatory lung. Additionally, the rAdVs caused an inflammatory response by inducing DC differentiation, inflammatory cell infiltration and changes in cytokines; however, mice transplanted with rAdV-green fluorescent protein (GFP)-infected DCs displayed no asthma manifestations. In conclusion, CTLA4Ig-modified DCs exhibited a therapeutic effect on asthma, and CCR7 may guide DC homing. The combination of these two molecules may be a model for precision-guided immunotherapy.
Assuntos
Asma/terapia , Antígeno CTLA-4/genética , Células Dendríticas/metabolismo , Imunoterapia , Receptores CCR7/metabolismo , Adenoviridae/genética , Animais , Antígeno CTLA-4/metabolismo , Movimento Celular , Células Dendríticas/imunologia , Células Dendríticas/fisiologia , Células Dendríticas/transplante , Feminino , Vetores Genéticos/genética , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Cadeias gama de Imunoglobulina/genética , Cadeias gama de Imunoglobulina/imunologia , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-4/genética , Interleucina-4/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Receptores CCR7/genéticaRESUMO
The TLR4 ligand LPS causes mouse B cells to undergo IgE and IgG1 isotype switching in the presence of IL-4. TLR4 activates two signaling pathways mediated by the adaptor molecules MyD88 and Toll/IL-IR domain-containing adapter-inducing IFN-ß (TRIF)-related adaptor molecule (TRAM), which recruits TRIF. Following stimulation with LPS plus IL-4, Tram(-/-) and Trif(-/-) B cells completely failed to express Cε germline transcripts (GLT) and secrete IgE. In contrast, Myd88(-/-) B cells had normal expression of Cε GLT but reduced IgE secretion in response to LPS plus IL-4. Following LPS plus IL-4 stimulation, Cγ1 GLT expression was modestly reduced in Tram(-/-) and Trif(-/-) B cells, whereas Aicda expression and IgG1 secretion were reduced in Tram(-/-), Trif(-/-), and Myd88(-/-) B cells. B cells from all strains secreted normal amounts of IgE and IgG1 in response to anti-CD40 plus IL-4. Following stimulation with LPS plus IL-4, Trif(-/-) B cells failed to sustain NF-κB p65 nuclear translocation beyond 3 h and had reduced binding of p65 to the Iε promoter. Addition of the NF-κB inhibitor, JSH-23, to wild-type B cells 15 h after LPS plus IL-4 stimulation selectively blocked Cε GLT expression and IgE secretion but had little effect on Cγ1 GLT expression and IgG secretion. These results indicate that sustained activation of NF-κB driven by TRIF is essential for LPS plus IL-4-driven activation of the Cε locus and class switching to IgE.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/imunologia , Switching de Imunoglobulina/imunologia , Imunoglobulina E/imunologia , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/imunologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Citidina Desaminase/genética , Citidina Desaminase/imunologia , Citidina Desaminase/metabolismo , Immunoblotting , Switching de Imunoglobulina/efeitos dos fármacos , Imunoglobulina E/genética , Imunoglobulina E/metabolismo , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Cadeias épsilon de Imunoglobulina/genética , Cadeias épsilon de Imunoglobulina/imunologia , Cadeias épsilon de Imunoglobulina/metabolismo , Cadeias gama de Imunoglobulina/genética , Cadeias gama de Imunoglobulina/imunologia , Cadeias gama de Imunoglobulina/metabolismo , Interleucina-4/imunologia , Interleucina-4/farmacologia , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Fenilenodiaminas/imunologia , Fenilenodiaminas/farmacologia , Receptores de Interleucina/genética , Receptores de Interleucina/imunologia , Receptores de Interleucina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/metabolismo , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/imunologia , Fator de Transcrição RelA/metabolismoRESUMO
In this study, the light chain (κ) and heavy chain (γ) sequences of the monoclonal antibody against vascular endothelial growth factor (VEGF) were sub-cloned into the eukaryotic pcDNA3.1 (+) (Hygro) and the pcDNA3.1 (+) (Neo) expression vectors using the traditional and homologous recombination methods. To express the antibody, the recombinant plasmids were transfected into the Chinese hamster ovary (CHO) and the K562 cell lines. The recombinant antibody was then purified using the protein A affinity chromatography. Furthermore, in order to demonstrate the inhibition of VEGF-induced mitogenesis of the recombinant antibody, the bovine aorta endothelial like cells were employed. The results showed specialization and conjunction of the recombinant antibody to the VEGF. It was also indicated that the antibody expression in the K562 cell lines was higher than the CHO cell lines. Furthermore, the in vitro VEGF inhabitation of the recombinant antibodies which were produced from the K562 cell line, and the CHO cell line, were similar. This proved that the K562 cell line is a good substitute for the CHO cell line in the production of the recombinant antibodies.
Assuntos
Anticorpos Monoclonais/genética , Cadeias gama de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Fator A de Crescimento do Endotélio Vascular/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Aorta/efeitos dos fármacos , Aorta/crescimento & desenvolvimento , Aorta/imunologia , Apoptose/genética , Células CHO , Bovinos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Clonagem Molecular , Cricetinae , Cricetulus , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Humanos , Cadeias gama de Imunoglobulina/imunologia , Cadeias gama de Imunoglobulina/isolamento & purificação , Cadeias kappa de Imunoglobulina/imunologia , Cadeias kappa de Imunoglobulina/isolamento & purificação , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
GM (γ marker) allotypes, genetic variants of immunoglobulin γ chains, have been reported to be associated strongly with susceptibility to lung cancer, but the mechanism(s) underlying this association is not known. One mechanism could involve their contribution to humoral immunity to lung tumour-associated antigens. In this study, we aimed to determine whether particular GM and KM (κ marker) allotypes were associated with antibody responsiveness to XAGE-1b, a highly immunogenic lung tumour-associated cancer-testis antigen. Sera from 89 patients with non-small cell lung cancer (NSCLC) were allotyped for eight GM and two KM determinants and characterized for antibodies to a synthetic XAGE-1b protein. The distribution of various GM phenotypes was significantly different between XAGE-1b antibody-positive and -negative patients (P = 0·023), as well as in the subgroup of XAGE-1b antigen-positive advanced NSCLC (P = 0·007). None of the patients with the GM 1,17 21 phenotype was positive for the XAGE-1b antibody. In patients with antigen-positive advanced disease, the prevalence of GM 1,2,17 21 was significantly higher in the antibody-positive group than in those who lacked the XAGE-1b antibody (P = 0·026). This phenotype also interacted with a particular KM phenotype: subjects with GM 1,2,17 21 and KM 3,3 phenotypes were almost four times (odds ratio = 3·8) as likely to be positive for the XAGE-1b antibody as the subjects who lacked these phenotypes. This is the first report presenting evidence for the involvement of immunoglobulin allotypes in immunity to a cancer-testis antigen, which has important implications for XAGE-1b-based immunotherapeutic interventions in lung adenocarcinoma.
Assuntos
Antígenos de Neoplasias/imunologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Imunidade Humoral/imunologia , Cadeias gama de Imunoglobulina/imunologia , Cadeias kappa de Imunoglobulina/imunologia , Neoplasias Pulmonares/imunologia , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Feminino , Frequência do Gene , Haplótipos , Humanos , Imunidade Humoral/genética , Alótipos Gm de Imunoglobulina/genética , Alótipos Gm de Imunoglobulina/imunologia , Cadeias gama de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Fenótipo , Testículo/imunologia , Testículo/metabolismoRESUMO
A 45-year-old man presented with moderate proteinuria and hematuria. A renal biopsy showed mesangial/endocapillary proliferative glomerulonephritis, linear deposition of monoclonal immunoglobulin γ1 C(H) 2 heavy chain along glomerular and tubular basement membranes (GBMs and TBMs), granular deposition of κ light chain within the mesangial area, and continuous linear deposits of finely granular electron-dense materials along GBMs and TBMs. Dual immunostaining showed essentially discrete glomerular localization of γ1 C(H) 2 heavy chain and κ light chain. Monoclonal protein was not detected in urine and serum. A bone marrow aspiration showed no abnormalities. Steroid therapy led to the improvement of proteinuria and hematuria. We would classify this case as a new variant of monoclonal immunoglobulin deposition disease, light chain/heavy chain deposition disease. In contrast with light and heavy chain deposition disease, the remarkable characteristics of this variant are separate deposition of monoclonal heavy chain and light chain, deposition of largely deleted γ heavy chain lacking the C(H) 1 domain, and good response to steroid therapy.
Assuntos
Glomerulonefrite Membranoproliferativa/patologia , Cadeias gama de Imunoglobulina/imunologia , Cadeias kappa de Imunoglobulina/imunologia , Paraproteinemias/patologia , Antagonistas de Receptores de Angiotensina/uso terapêutico , Anti-Hipertensivos/uso terapêutico , Biomarcadores/metabolismo , Proliferação de Células , Membrana Basal Glomerular/metabolismo , Membrana Basal Glomerular/ultraestrutura , Glomerulonefrite Membranoproliferativa/tratamento farmacológico , Glomerulonefrite Membranoproliferativa/imunologia , Glucocorticoides/uso terapêutico , Humanos , Cadeias gama de Imunoglobulina/metabolismo , Cadeias kappa de Imunoglobulina/metabolismo , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Masculino , Pessoa de Meia-Idade , Paraproteinemias/tratamento farmacológico , Paraproteinemias/imunologia , Prednisolona/uso terapêutico , Resultado do TratamentoRESUMO
Tumor necrosis factor (TNF)-α is produced in brain in response to acute cerebral ischemia, and promotes neuronal apoptosis. Biologic TNF inhibitors (TNFIs), such as the etanercept, cannot be developed as new stroke treatments because these large molecule drugs do not cross the blood-brain barrier (BBB). A BBB-penetrating biologic TNFI was engineered by fusion of the type II human TNF receptor (TNFR) to each heavy chain of a genetically engineered chimeric monoclonal antibody (MAb) against the mouse transferrin receptor (TfR), designated as cTfRMAb-TNFR fusion protein. The cTfRMAb domain of the fusion protein acts as a molecular Trojan horse to deliver the fused TNFR across the BBB. Etanercept or the cTfRMAb-TNFR fusion protein (1 mg/kg) was administered intravenously in adult mice subjected to 1-hour reversible middle cerebral artery occlusion up to 90 minutes after the occlusion. Neuroprotection was assessed at 24 hours or 7 days after occlusion. The cTfRMAb-TNFR fusion protein treatment caused a significant 45%, 48%, 42%, and 54% reduction in hemispheric, cortical, and subcortical stroke volumes, and neural deficit, respectively. Intravenous etanercept had no therapeutic effect. Biologic TNFIs can be reengineered for BBB penetration, and the IgG-TNFR fusion protein is therapeutic after delayed intravenous administration in experimental stroke.
Assuntos
Encéfalo/patologia , Cadeias gama de Imunoglobulina/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Receptores do Fator de Necrose Tumoral/uso terapêutico , Acidente Vascular Cerebral/prevenção & controle , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Administração Intravenosa , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Encéfalo/efeitos dos fármacos , Células CHO , Cricetinae , Humanos , Cadeias gama de Imunoglobulina/administração & dosagem , Cadeias gama de Imunoglobulina/imunologia , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/prevenção & controle , Camundongos , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/imunologia , Receptores da Transferrina/imunologia , Receptores do Fator de Necrose Tumoral/administração & dosagem , Receptores do Fator de Necrose Tumoral/imunologia , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/uso terapêutico , Acidente Vascular Cerebral/patologiaRESUMO
Emerging evidence suggests that the catalytically inactive ErbB3 (HER3) protein plays a fundamental role in normal tyrosine kinase receptor signaling as well as in aberrant functioning of these signaling pathways, resulting in several forms of human cancers and some mental disorders. Here we report the generation of a specific anti-ErbB3 antibody intended for use in diagnosing disease or therapeutic application. By using the hybridoma technique, one cell line (2E(12)C(3)) stably producing anti-ErbB3 antibody was obtained. Its molecular weight was about 185 kDa and its isotype was IgG 2a and κ, respectively. The affinity constant (Kaff) of the anti-ErbB3 MAb was 5.83×10(10) M(-1). This antibody may become a useful tool for diagnostic and therapeutic targeting of ErbB3-expressing cancers or helpful in highlighting the etiology of schizophrenia.
Assuntos
Anticorpos Monoclonais Murinos/biossíntese , Anticorpos Antineoplásicos/biossíntese , Cadeias gama de Imunoglobulina/biossíntese , Receptor ErbB-3/imunologia , Animais , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Monoclonais Murinos/uso terapêutico , Anticorpos Antineoplásicos/imunologia , Anticorpos Antineoplásicos/uso terapêutico , Afinidade de Anticorpos , Linhagem Celular Tumoral , Feminino , Humanos , Hibridomas , Cadeias gama de Imunoglobulina/imunologia , Cadeias gama de Imunoglobulina/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Receptor ErbB-3/metabolismoRESUMO
BACKGROUND: Gamma heavy chain disease with underlying thyroid pathology is rare. There are 5 reported cases in the English literature, including the present case of an elderly female with γ heavy chain disease with underlying lymphoplasmacytic lymphoma of the thyroid who initially presented with long-standing goiter and chronic thyroiditis. METHODS: The protein studies and histopathologic findings in her thyroid are described. Her case is compared with reported cases of γ heavy chain disease with thyroid involvement. RESULTS: Initial impression on most cases was chronic thyroiditis; however pathology showed 3 cases with plasmacytoma and 2 with lymphoplasmacytic infiltrate. All were diagnosed and followed up using serum and urine electrophoresis. CONCLUSION: Gamma heavy chain disease has a protean manifestation; however there appears to be a more uniform pattern of the disease when it is associated with the thyroid. The inclusion of protein studies in cases diagnosed with chronic thyroiditis by FNA may aid in establishing γ heavy chain disease with underlying thyroid involvement. In this case serum and urine electrophoresis, and immunofixation studies which are simple and affordable tests facilitated the hematologic workup and follow up.
Assuntos
Doença das Cadeias Pesadas/diagnóstico , Glândula Tireoide/imunologia , Neoplasias da Glândula Tireoide/diagnóstico , Tireoidite/diagnóstico , Macroglobulinemia de Waldenstrom/diagnóstico , Idoso , Idoso de 80 Anos ou mais , Biópsia por Agulha Fina , Eletroforese das Proteínas Sanguíneas , Doença Crônica , Feminino , Doença das Cadeias Pesadas/complicações , Doença das Cadeias Pesadas/imunologia , Humanos , Cadeias gama de Imunoglobulina/imunologia , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Plasmocitoma/diagnóstico , Plasmocitoma/imunologia , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/complicações , Neoplasias da Glândula Tireoide/imunologia , Tireoidite/complicações , Tireoidite/imunologia , Macroglobulinemia de Waldenstrom/complicações , Macroglobulinemia de Waldenstrom/imunologiaRESUMO
When interleukin-2 (IL-2) receptor γ-chain (γ(C))-sharing cytokine receptors on T cells bind their specific ligands (IL-2, -4, -7, -9, -15 or -21), they initiate a variety of cell signals that promote survival, differentiation or antiviral or antitumor cytolytic functions. Although expression of the γ(C) is constitutive across T-cell subsets, the varying expression of other receptor complex components can regulate cytokine signalling and function. Impaired γ(C) cytokine activity in HIV infection, and the role of γ(C) cytokines in CD8(+) T-cell function and homeostasis, implicates these molecules among potential contributors to the observed decline of cytolytic activity (CTL) in HIV disease. In particular, this review will be highlighting information about the IL-7 receptor (IL-7R) complex, which is composed of the γ(C) and the IL-7Rα (CD127) chains. There has been an abundance of HIV-related CD127 research and its important role in CD8(+) T-cell survival and function. The expression of CD127 undergoes dramatic changes throughout the course of T-cell responses in HIV infection. The expression of CD127 is significantly decreased in progressive HIV disease, whereas effective antiretroviral therapy results in its recovery. Observations of impaired IL-7 activity in HIV(+) individuals have suggested that CD127 has an important role in HIV immunopathogenesis. In addition, a soluble form of CD127 (sCD127) is upregulated in the plasma of HIV(+) individuals. Hence, CD127 is being increasingly considered as a marker of disease prognosis, and related information may provide insight into understanding the expression and role of other γ(C) receptors in HIV disease and contribute to the development of novel cytokine-based therapeutics.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Infecções por HIV/imunologia , Cadeias gama de Imunoglobulina/imunologia , Receptores de Interleucina-7/genética , Animais , Expressão Gênica/imunologia , Humanos , Receptores de Citocinas , Receptores de Interleucina-7/imunologiaRESUMO
In plasma cells, immunoglobulin heavy chain (IgH) secretory-specific mRNA is made in high abundance as a result of both increased promoter proximal poly(A) site choice and weak splice-site skipping. Ell2, the eleven-nineteen lysine rich leukemia gene, is a transcription elongation factor that is induced â¼6-fold in plasma cells and has been shown to drive secretory-specific mRNA production. Reducing ELL2 by siRNA, which reduced processing to the secretion-specific poly(A) site, also influenced the methylations of histone H3K4 and H3K79 on the IgH gene and impacted positive transcription factor b (pTEFb), Ser-2 carboxyl-terminal phosphorylation, and polyadenylation factor additions to RNA polymerase II. The multiple lineage leukemia gene (MLL) and Dot1L associations with the IgH gene were also impaired in the absence of ELL2. To investigate the link between histone modifications, transcription elongation, and alternative RNA processing in IgH mRNA production, we performed chromatin immunoprecipitation on cultured mouse B and plasma cells bearing the identical IgH γ2a gene. In the plasma cells, as compared with the B cells, the H3K4 and H3K79 methylations extended farther downstream, past the IgH enhancer to the end of the transcribed region. Thus the downstream H3K4 and H3K79 methylation of the IgH associated chromatin in plasma cells is associated with increased polyadenylation and exon skipping, resulting from the actions of ELL2 transcription elongation factor.
Assuntos
Histonas/metabolismo , Cadeias gama de Imunoglobulina/biossíntese , Plasmócitos/metabolismo , Sinais de Poliadenilação na Ponta 3' do RNA/fisiologia , Transcrição Gênica/fisiologia , Fatores de Elongação da Transcrição/metabolismo , Animais , Linhagem Celular , Cromatina/genética , Cromatina/imunologia , Cromatina/metabolismo , Éxons/fisiologia , Histona-Lisina N-Metiltransferase , Histonas/genética , Histonas/imunologia , Cadeias gama de Imunoglobulina/genética , Cadeias gama de Imunoglobulina/imunologia , Metilação , Camundongos , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/imunologia , Proteína de Leucina Linfoide-Mieloide/metabolismo , Plasmócitos/citologia , Plasmócitos/imunologia , Poliadenilação/fisiologia , RNA Polimerase II/genética , RNA Polimerase II/imunologia , RNA Polimerase II/metabolismo , RNA Interferente Pequeno/genética , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/imunologiaRESUMO
Several molecules, involved in the intracellular communication network, have been identified as the cause of primary immunodeficiencies. In most cases, these molecules are exclusively expressed in hematopoietic cells, being involved in cell development and/or functionality of terminal differentiated cells of immune system. In the case of γc, the abundance of the protein suggests a potential pleiotropic effect of the molecule. Immune and endocrine systems participate to an integrated network of soluble mediators that communicate and coordinate responsive cells to achieve effector functions in an appropriate fashion. It has been demonstrated a novel dependence of GH signaling on the common cytokines receptor γc in certain cell types, supporting the hypothesis of an interplay between endocrine and immune system. The evidence that different receptors share a few molecules may certainly lead to a better knowledge on the mechanism of coordination and integration of several pathways implicated in the control of cell growth and proliferation under physiological or pathogenic conditions. This review focuses on the γc as a common transducing element shared between several cytokines and growth hormone receptors, indicating a further functional link between endocrine and immune system.
Assuntos
Sistema Endócrino/imunologia , Sistema Imunitário , Cadeias gama de Imunoglobulina/genética , Cadeias gama de Imunoglobulina/imunologia , Transdução de Sinais , Animais , Sequência Conservada , HumanosRESUMO
The innate immune system is crucial for host defense and immunosurveillance against pathogens and tumor cells. IL-15 is a pleiotropic cytokine with important effects on cells of the innate and adaptive immune systems. The NK cell- and CD8(+) T cell-mediated functions of IL-15 against tumor cells have been well documented. However, it has not been established whether IL-15 has innate anti-tumor functions independent of these cells. Here, we explored the innate anti-tumor potential of IL-15 using a B16F10 melanoma tumor model. IL-15tg mice exhibited significantly more resistance to tumor growth and metastasis compared to B6 mice, and to IL-15(-/-) mice, which exhibited increased susceptibility to B16F10 challenge. In vivo depletion of NK cells and CD8(+) T cells abrogated the innate resistance to B16F10 cells in B6 but not in IL-15tg mice. In addition, lung macrophages from IL-15tg mice produced significantly higher levels of NO and IL-12 compared with macrophages from B6 or IL-15(-/-) mice. To examine whether IL-15 has innate anti-tumor activity independent of NK cells and CD8(+) T cells, we developed Ad-Op-hIL-15; this resulted in significantly higher levels of biologically active hIL-15. Delivery of Ad-Op-hIL-15 into RAG-2(-/-)/gamma(c)(-/-) mice significantly suppressed tumor burden in the lungs compared with the control adenovirus vector. Our results show that IL-15 can have innate anti-tumor activity independent of NK cells and CD8(+) T cells and the common gamma(c)R.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Imunidade Inata/imunologia , Interleucina-15/imunologia , Células Matadoras Naturais/imunologia , Melanoma/imunologia , Imunidade Adaptativa/imunologia , Adenoviridae , Animais , Linfócitos T CD8-Positivos/patologia , Contagem de Células , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Humanos , Cadeias gama de Imunoglobulina/imunologia , Interleucina-12/imunologia , Células Matadoras Naturais/patologia , Pulmão/imunologia , Pulmão/patologia , Macrófagos/imunologia , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/biossíntese , Fenótipo , Transdução de Sinais/imunologiaRESUMO
Decoy receptors, such as the human tumor necrosis factor receptor (TNFR), are potential new therapies for brain disorders. However, decoy receptors are large molecule drugs that are not transported across the blood-brain barrier (BBB). To enable BBB transport of a TNFR decoy receptor, the human TNFR-II extracellular domain was re-engineered as a fusion protein with a chimeric monoclonal antibody (MAb) against the human insulin receptor (HIR). The HIRMAb acts as a molecular Trojan horse to ferry the TNFR therapeutic decoy receptor across the BBB. The HIRMAb-TNFR fusion protein was expressed in stably transfected CHO cells, and was analyzed with electrophoresis, Western blotting, size exclusion chromatography, and binding assays for the HIR and TNFalpha. The HIRMAb-TNFR fusion protein was radio-labeled by trititation, in parallel with the radio-iodination of recombinant TNFR:Fc fusion protein, and the proteins were co-injected in the adult Rhesus monkey. The TNFR:Fc fusion protein did not cross the primate BBB in vivo, but the uptake of the HIRMAb-TNFR fusion protein was high and 3% of the injected dose was taken up by the primate brain. The TNFR was selectively targeted to brain, relative to peripheral organs, following fusion to the HIRMAb. This study demonstrates that decoy receptors may be re-engineered as IgG fusion proteins with a BBB molecular Trojan horse that selectively targets the brain, and enables penetration of the BBB in vivo. IgG-decoy receptor fusion proteins represent a new class of human neurotherapeutics.
Assuntos
Anticorpos Monoclonais/metabolismo , Antígenos CD/imunologia , Encéfalo/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Receptor de Insulina/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Encéfalo/efeitos dos fármacos , Química Encefálica , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Feminino , Vetores Genéticos , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Cadeias gama de Imunoglobulina/imunologia , Cadeias gama de Imunoglobulina/metabolismo , Macaca mulatta , Camundongos , Engenharia de Proteínas/métodos , Receptores do Fator de Necrose Tumoral/imunologia , Receptores do Fator de Necrose Tumoral/metabolismo , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacocinéticaRESUMO
Endoglin has been identified as a promising cell surface antigen for vascular targeting approaches in cancer therapy, e.g. employing antibody molecules as targeting moieties. However, in vivo analysis of such strategies in mouse models requires antibodies recognizing endoglin on mouse endothelial cells. Here we describe the isolation of single-chain Fv fragments (scFvs) from phage display libraries, which bind to the extracellular region of mouse endoglin. One of these clones, scFv mE12, showed strong (K(d)=11 nM) and selective binding to purified endoglin and also to the endoglin-expressing mouse endothelioma cell line eEnd.2. This antibody recognized a linear epitope located in the N-terminal region (aa 27-361) of endoglin. Cell binding was further increased by generating a bivalent scFv-Fc fusion protein composed of scFv mE12 and the human gamma1 Fc part. Moreover, scFv mE12 was endowed with an additional cysteine residue in the linker region and applied for the generation of anti-endoglin scFv immunoliposomes capable of selectively binding to endoglin-expressing cells. Thus, anti-mouse endoglin scFv mE12 should be useful to analyze vascular targeting strategies in mice.
Assuntos
Sistemas de Liberação de Medicamentos , Epitopos/imunologia , Regiões Constantes de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Proteínas Recombinantes de Fusão/imunologia , Animais , Especificidade de Anticorpos/imunologia , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos/métodos , Endoglina , Epitopos/genética , Humanos , Regiões Constantes de Imunoglobulina/genética , Regiões Constantes de Imunoglobulina/uso terapêutico , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/uso terapêutico , Cadeias gama de Imunoglobulina/genética , Cadeias gama de Imunoglobulina/imunologia , Cadeias gama de Imunoglobulina/uso terapêutico , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/uso terapêuticoRESUMO
PURPOSE: We previously generated a mouse monoclonal antibody (mAb) specific for the tumor-associated GD2 ganglioside antigen. Here, we describe the development of a chimeric anti-GD2 mAb for more effective tumor immunotherapy. EXPERIMENTAL DESIGN: We cloned the cDNA encoding the immunoglobulin light and heavy chains of the 60C3 anti-GD2 mAb, and constructed chimeric genes by linking the cDNA fragments of the variable regions of the murine light and heavy chains to cDNA fragments of the human kappa and gamma1 constant regions, respectively. RESULTS: The resultant chimeric anti-GD2 mAb, c.60C3, showed identical binding affinity and specificity to that of its murine counterpart. Both c.60C3 and 60C3 were rapidly internalized by tumor cells at 37 degrees C. When human serum and human natural killer cells were used as effectors in complement-mediated cytotoxicity and antibody-dependent cell cytotoxicity, respectively, c.60C3 was more effective in killing GD2-expressing tumor cells. However, c.60C3 was ineffective at inducing cell death by apoptosis, although binding of 60C3 induced apoptotic death in vitro. In an in vivo, GD2-expressing, syngeneic tumor model, i.v. injection of c.60C3, but not of 60C3, significantly suppressed tumor growth in mice (P<0.0005). CONCLUSION: Immune effector functions mediated by this antibody and its potentially reduced immunogenicity make chimeric c.60C3 a promising therapeutic agent against neuroectodermic tumors.