RESUMO
There is evidence that IL-22 and IL-17 participate in the pathogenesis of allergic asthma. To investigate the role of IL-22, we used IL-22 deficient mice (IL-22 KO) sensitized and challenged with ovalbumin (OVA) and compared with wild type (WT) animals exposed to OVA. IL-22 KO animals exposed to OVA showed a decreased number and frequency of eosinophils, IL-5 and IL-13 in the airways, reduced mucus production and pulmonary inflammation. In addition, IL-22 KO animals exhibited a decreased percentage and number of lung CD11c+CD11b+ cells and increased apoptosis of eosinophils. Th17 cell transfer generated from IL-22 KO to animals previously sensitized and challenged with OVA caused a reduction in eosinophil frequency and number in the airways compared to animals transferred with Th17 cells generated from WT mice. Therefore, IL-22 is deleterious with concomitant secretion of IL-17. Our findings show a pro-inflammatory role for IL-22, confirmed in a model of allergen-free and allergen-specific immunotherapy. Moreover, during the comorbidity asthma and pneumonia that induces neutrophil inflammation, IL-22 was not detrimental. Our results show that targeting IL-22 would negatively affect the survival of eosinophils, reduce the expansion or migration of CD11c+CD11b+ cells, and negatively regulate allergic asthma.
Assuntos
Asma , Pneumonia , Camundongos , Animais , Interleucina-17/genética , Asma/patologia , Pulmão/patologia , Eosinófilos , Pneumonia/patologia , Alérgenos , Comorbidade , Ovalbumina , Modelos Animais de Doenças , Líquido da Lavagem Broncoalveolar , Camundongos Endogâmicos BALB CRESUMO
Chronic pulmonary inflammation marked predominantly by CD4+IFN-γ+ cells is the hallmark of tuberculosis pathogenesis in immunocompetent adults, who are substantially affected by this disease. Moreover, CD4+Foxp3+ cell-mediated suppression contributes to infection susceptibility. We addressed the role of CD4+Foxp3+ cells in tuberculosis pathogenesis, because this aspect has not been addressed during chronic infection. We targeted CCR4, which induces the influx of CD4+Foxp3+ cells into the lungs. CCR4-/- mice exhibited a lower frequency of CD4+Foxp3+ cells at 15, 30, and 70 days of infection than their wild-type counterparts. However, only at 70 days of infection was an exacerbated IFN-γ-mediated immune response associated with apparent tuberculosis pathogenesis and susceptibility. In addition, CCR4-/- mice exhibited a decrease in the suppressor function of CD4+Foxp3+ cells. Adoptive transfer of Foxp3+ cells into infected CCR4-/- mice restored pulmonary inflammation and bacterial load to levels observed in wild-type mice. Our findings suggest that CD4+Foxp3+ cells play a time-dependent role in tuberculosis and highlight that CCR4 plays a critical role in the balance of IFN-γ-mediated inflammation by regulating the influx and function of CD4+Foxp3+ cells. Our findings are translationally relevant, as CD4+Foxp3+ cells or CCR4 could be a target for immunotherapy, considering the heterogeneity of tuberculosis in immunocompetent adults.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Interferon gama/imunologia , Mycobacterium tuberculosis/imunologia , Receptores CCR4/imunologia , Tuberculose Pulmonar/imunologia , Animais , Linfócitos T CD4-Positivos/patologia , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Interferon gama/genética , Camundongos , Camundongos Knockout , Receptores CCR4/genética , Tuberculose Pulmonar/genética , Tuberculose Pulmonar/patologiaRESUMO
M1 macrophages are more effective in the induction of the inflammatory response and clearance of Mycobacterium tuberculosis than M2 macrophages. Infected C57BL/6 mice generate a stronger cellular immune response compared with BALB/c mice. We hypothesized that infected C57BL/6 mice would exhibit a higher frequency and function of M1 macrophages than infected BALB/c mice. Our findings show a higher ratio of macrophages to M2 macrophages in the lungs of chronically infected C57BL/6 mice compared with BALB/c mice. However, there was no difference in the functional ability of M1 and M2 macrophages for the two strains in vitro. In vivo, a deleterious role for M2 macrophages was confirmed by M2 cell transfer, which rendered the infected C57BL/6, but not the BALB/c mice, more susceptible and resulted in mild lung inflammation compared with C57BL/6 mice that did not undergo cell transfer. M1 cell transfer induced a higher inflammatory response, although not protective, in infected BALB/c mice compared with their counterparts that did not undergo cell transfer. These findings demonstrate that an inflammation mediated by M1 macrophages may not induce bacterial tolerance because protection depends on the host genetic background, which drives the magnitude of the inflammatory response against M. tuberculosis in the pulmonary microenvironment. The contribution of our findings is that although M1 macrophage is an effector leucocyte with microbicidal machinery, its dominant role depends on the balance of M1 and M2 subsets, which is driven by the host genetic background.
Assuntos
Pulmão/imunologia , Macrófagos/imunologia , Mycobacterium tuberculosis/imunologia , Tuberculose/imunologia , Animais , Suscetibilidade a Doenças , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/enzimologia , Óxido Nítrico Sintase Tipo II/análise , Especificidade da EspécieRESUMO
The voltage-gated potassium channel Kv1.3 is a novel target for immunomodulation of autoreactive effector memory T cells, which play a major role in the pathogenesis of autoimmune diseases. In this study, the Ts6 and Ts15 toxins isolated from Tityus serrulatus (Ts) were investigated for their immunosuppressant roles on CD4(+) cell subsets: naive, effector (TEF ), central memory (TCM) and effector memory (TEM). The electrophysiological assays confirmed that both toxins were able to block Kv1.3 channels. Interestingly, an extended Kv channel screening shows that Ts15 blocks Kv2.1 channels. Ts6 and Ts15 significantly inhibit the proliferation of TEM cells and interferon-γ production; however, Ts15 also inhibits other CD4(+) cell subsets (naive, TEF and TCM). Based on the Ts15 inhibitory effect of proliferation of all CD4(+) cell subsets, and based on its blocking effect on Kv2.1, we investigated the Kv2.1 expression in T cells. The assays showed that CD4(+) and CD8(+) cells express the Kv2.1 channels mainly extracellularly with TCM cells expressing the highest number of Kv2.1 channels. We also provide in vivo experimental evidence to the protective effect of Ts6 and Ts15 on delayed-type hypersensitivity reaction. Altogether, this study presents the immunosuppressive behaviour of Ts6 and Ts15 toxins, indicating that these toxins could be promising candidates for autoimmune disease therapy. Moreover, this is the first report illustrating the involvement of a novel K(+) channel subtype, Kv2.1, and its distribution in T-cell subsets.
Assuntos
Imunossupressores/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Venenos de Escorpião/farmacologia , Linfócitos T/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Hipersensibilidade Tardia/imunologia , Hipersensibilidade Tardia/metabolismo , Hipersensibilidade Tardia/prevenção & controle , Canal de Potássio Kv1.3/antagonistas & inibidores , Canal de Potássio Kv1.3/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Masculino , Potenciais da Membrana , Camundongos Endogâmicos BALB C , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Soroalbumina Bovina , Canais de Potássio Shab/antagonistas & inibidores , Canais de Potássio Shab/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Xenopus laevisRESUMO
The magnitude of the cellular adaptive immune response is critical for the control of Mycobacterium tuberculosis infection in the chronic phase. In addition, the genetic background is equally important for resistance or susceptibility to tuberculosis. In this study, we addressed whether lung populations of dendritic cells, obtained from genetically different hosts, would play a role in the magnitude and function of CD4(+) populations generated after M. tuberculosis infection. Thirty days post-infection, C57BL/6 mice, which generate a stronger interferon-γ (IFN-γ)-mediated immune response than BALB/c mice, exhibited a higher number and frequency of lung CD11c(+) CD11b(-) CD103(+) cells compared with BALB/c mice, which exhibited a high frequency of lung CD11c(+) CD11b(+) CD103(-) cells. CD11c(+) CD11b(-) CD103(+) cells, purified from lungs of infected C57BL/6 mice, but not from infected BALB/c mice, induced a higher frequency of IFN-γ-producing or interleukin-17 (IL-17)-producing CD4(+) cells. Moreover, CD4(+) cells also arrive at the lung of C57BL/6 mice faster than in BALB/c mice. This pattern of immune response seems to be associated with higher gene expression for CCL4, CCL19, CCL20 and CCR5 in the lungs of infected C57BL/6 mice compared with infected BALB/c mice. The results described here show that the magnitude of IFN-γ-producing or IL-17-producing CD4(+) cells is dependent on CD11c(+) CD11b(-) CD103(+) cells, and this pattern of immune response is directly associated with the host genetic background. Therefore, differences in the genetic background contribute to the identification of immunological biomarkers that can be used to design human assays to predict progression of M. tuberculosis infection.
Assuntos
Antígenos CD/imunologia , Antígeno CD11c/imunologia , Cadeias alfa de Integrinas/imunologia , Interferon gama/imunologia , Interleucina-17/imunologia , Pulmão/imunologia , Mycobacterium tuberculosis/imunologia , Células Th17/imunologia , Tuberculose Pulmonar/imunologia , Animais , Antígenos CD/metabolismo , Antígeno CD11b/imunologia , Antígeno CD11b/metabolismo , Antígeno CD11c/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/microbiologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Modelos Animais de Doenças , Feminino , Genótipo , Imunidade Celular , Cadeias alfa de Integrinas/metabolismo , Interferon gama/metabolismo , Interleucina-17/metabolismo , Pulmão/metabolismo , Pulmão/microbiologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/patogenicidade , Fenótipo , Transdução de Sinais , Especificidade da Espécie , Células Th17/metabolismo , Células Th17/microbiologia , Fatores de Tempo , Tuberculose Pulmonar/genética , Tuberculose Pulmonar/metabolismo , Tuberculose Pulmonar/microbiologiaRESUMO
In various types of snake venom, the major toxic components are proteinases and members of the phospholipase A2 family, although other enzymes also contribute to the toxicity. In this study, we evaluated the proteolytic, phospholipase, and L-Amino acid oxidase activities in the venom of five Bothrops species-Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi, and Bothrops alternatus-all of which are used in the production of commercial antivenom, prepared in horses. The enzymatic activities of each species' venom were classified as high, moderate, or low. B. moojeni venom demonstrated the highest enzymatic activity profile, followed by the venom of B. neuwiedi, B. jararacussu, B. jararaca, and B. alternatus. To our knowledge, this is the first study to compare all of these enzymes from multiple species, which is significant in view of the activity of L-amino acid oxidase across Bothrops species.
Assuntos
Bothrops , Venenos de Crotalídeos/enzimologia , Animais , Brasil , Bovinos , Venenos de Crotalídeos/química , L-Aminoácido Oxidase/química , Peptídeo Hidrolases/química , Fosfolipases/química , Proteólise , Ovinos , Especificidade da EspécieRESUMO
Approximately 20,000 snakebites are registered each year in Brazil. The classical treatment for venomous snakebite involves the administration of sera obtained from immunized horses. Moreover, the production and care of horses is costly, and the use of heterologous sera can cause hypersensitivity reactions. The production of human antibody fragments by phage display technology is seen as a means of overcoming some of these disadvantages. The studies here attempted to test human monoclonal antibodies specific to Bothrops jararacussu against other Bothrops sp. venoms, using the Griffin.1 library of human single-chain fragment-variable (scFv) phage antibodies. Using the Griffin.1 phage antibody library, this laboratory previously produced scFvs capable of inhibiting the phospholipase and myotoxic activities of Bothrops jararacussu venom. The structural and functional similarities of the various forms of phospholipase A2 (PLA2) in Bothrops venom served as the basis for the present study wherein the effectiveness of those same scFvs were evaluated against B. jararaca, B. neuwiedi, and B. moojeni venoms. Each clone was found to recognize all three Bothrops venoms, and purified scFvs partially inhibited their in vitro phospholipase activity. In vivo assays demonstrated that the scFv clone P2B7 reduced myotoxicity and increased the survival of animals that received the test venoms. The results here indicate that the scFv P2B7 is a candidate for inclusion in a mixture of specific antibodies to produce a human anti-bothropic sera. This data demonstrates that the human scFv P2B7 represents an alternative therapeutic approach to heterologous anti-bothropic sera available today.
Assuntos
Anticorpos Monoclonais/imunologia , Antivenenos/imunologia , Bothrops/imunologia , Venenos de Crotalídeos/imunologia , Anticorpos de Cadeia Única/imunologia , Animais , Anticorpos Monoclonais/química , Antivenenos/química , Técnicas de Visualização da Superfície Celular , Creatina Quinase/sangue , Venenos de Crotalídeos/química , Feminino , Técnica de Placa Hemolítica , Humanos , Camundongos , Fosfolipases A2/química , Especificidade da Espécie , Homologia Estrutural de ProteínaRESUMO
In Brazil, the species Tityus serrulatus is responsible for the most severe cases of scorpion envenomation. There is currently a need for new scorpion anti-venoms that are more effective and less harmful. This study attempted to produce human monoclonal antibodies capable of inhibiting the activity of T. serrulatus venom (TsV), using the Griffin.1 library of human single-chain fragment-variable (scFv) phage antibodies. Four rounds of phage antibody selection were performed, and the round with the highest phage antibody titer was chosen for the production of monoclonal phage antibodies and for further analysis. The scFv 2A, designated serrumab, was selected for the production and purification of soluble antibody fragments. In a murine peritoneal macrophage cell line (J774.1), in vitro assays of the cytokines interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-10 were performed. In male BALB/c mice, in vivo assays of plasma urea, creatinine, aspartate transaminase, and glucose were performed, as well as of neutrophil recruitment and leukocyte counts. It was found that serrumab inhibited the TsV-induced increases in the production of IL-6, TNFα, and IL-10 in J774.1 cells. The in vivo inhibition assay showed that serrumab also prevented TsV-induced increases in the plasma levels of urea, creatinine, aspartate transaminase, and glucose, as well as preventing the TsV-induced increase in neutrophil recruitment. The results indicate that the human monoclonal antibody serrumab is a candidate for inclusion in a mixture of specific antibodies to the various toxins present in TsV. Therefore, serrumab shows promise for use in the production of new anti-venom.
Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Antivenenos/imunologia , Proteínas de Insetos/imunologia , Venenos de Escorpião/imunologia , Escorpiões/fisiologia , Anticorpos de Cadeia Única/imunologia , Animais , Anticorpos Monoclonais Humanizados/biossíntese , Anticorpos Monoclonais Humanizados/uso terapêutico , Antivenenos/biossíntese , Antivenenos/uso terapêutico , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Proteínas de Insetos/antagonistas & inibidores , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Biblioteca de Peptídeos , Venenos de Escorpião/antagonistas & inibidores , Venenos de Escorpião/toxicidade , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/uso terapêuticoRESUMO
The hybrid created from the crossbreeding of European and African bees, known as the Africanised bee, has provided numerous advantages for current beekeeping. However, this new species exhibits undesirable behaviours, such as colony defence instinct and a propensity to attack en masse, which can result in serious accidents. To date, there is no effective treatment for cases of Africanised bee envenomation. One promising technique for developing an efficient antivenom is the use of phage display technology, which enables the production of human antibodies, thus avoiding the complications of serum therapy, such as anaphylaxis and serum sickness. The aim of this study was to produce human monoclonal single-chain Fv (scFv) antibody fragments capable of inhibiting the toxic effects of Africanised bee venom. We conducted four rounds of selection of antibodies against the venom and three rounds of selection of antibodies against purified melittin. Three clones were selected and tested by enzyme-linked immunosorbent assay to verify their specificity for melittin and phospholipase A2. Two clones (C5 and C12) were specific for melittin, and one (A7) was specific for phospholipase A2. In a kinetic haemolytic assay, these clones were evaluated individually and in pairs. The A7-C12 combination had the best synergistic effect and was chosen to be used in the assays of myotoxicity inhibition and lethality. The A7-C12 combination inhibited the in vivo myotoxic effect of the venom and increased the survival of treated animals.
Assuntos
Antivenenos/imunologia , Venenos de Abelha/toxicidade , Meliteno/imunologia , Fosfolipases A2/imunologia , Animais , Anticorpos Monoclonais/imunologia , Venenos de Abelha/imunologia , Abelhas , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Mordeduras e Picadas de Insetos/imunologia , Mordeduras e Picadas de Insetos/terapia , Camundongos , Anticorpos de Cadeia Única/imunologia , SobrevidaRESUMO
Monoclonal antibodies represent the fastest growing class of biopharmaceutical products and have a host of applications in medical research, diagnosis, therapy, and basic science. The production of recombinant monoclonal antibodies has revolutionized the generation of immunoglobulins, and their use represents a strategic breakthrough, affecting the global pharmaceutical market for therapeutic proteins. In the present work, a review of scFv, and the number of related patents, has been carried out. The results show that several countries have scFv patents, most notably the United States, China and United Kingdom. The target of these scFv antibodies was also assessed and the results demonstrate that most are directed toward cancer therapy.
Anticorpos monoclonais representam a classe de maior crescimento em produtos de biofármacos e possuem várias aplicações em pesquisa médica, diagnóstico, terapias e ciência básica. A produção de anticorpos monoclonais recombinantes revolucionou a geração de imunoglobulinas e sua utilização implica em avanço estratégico, afetando o mercado farmacêutico global de proteínas terapêuticas. No presente trabalho, uma revisão sobre scFv e a relação do seu número de patentes foi analisada. Os resultados mostram que vários países apresentam patentes de scFv com destaque para os Estados Unidos, China e Reino Unido. Os alvos desses anticorpos também foram avaliados e as análises revelaram que a maioria é destinado a terapias contra o câncer.