RESUMO
Collagen VI is a key component of muscle basement membranes, and genetic variants can cause monogenic muscular dystrophies. Conversely, human genetic studies recently implicated collagen VI in central nervous system function, with variants causing the movement disorder dystonia. To elucidate the neurophysiological role of collagen VI, we generated mice with a truncation of the dystonia-related collagen α3 VI (COL6A3) C-terminal domain (CTD). These Col6a3CTT mice showed a recessive dystonia-like phenotype in both sexes. We found that COL6A3 interacts with the cannabinoid receptor 1 (CB1R) complex in a CTD-dependent manner. Col6a3CTT mice of both sexes have impaired homeostasis of excitatory input to the basal pontine nuclei (BPN), a motor control hub with dense COL6A3 expression, consistent with deficient endocannabinoid (eCB) signaling. Aberrant synaptic input in the BPN was normalized by a CB1R agonist, and motor performance in Col6a3CTT mice of both sexes was improved by CB1R agonist treatment. Our findings identify a readily therapeutically addressable synaptic mechanism for motor control.SIGNIFICANCE STATEMENT Dystonia is a movement disorder characterized by involuntary movements. We previously identified genetic variants affecting a specific domain of the COL6A3 protein as a cause of dystonia. Here, we created mice lacking the affected domain and observed an analogous movement disorder. Using a protein interaction screen, we found that the affected COL6A3 domain mediates an interaction with the cannabinoid receptor 1 (CB1R). Concordantly, our COL6A3-deficient mice showed a deficit in synaptic plasticity linked to a deficit in cannabinoid signaling. Pharmacological cannabinoid augmentation rescued the motor impairment of the mice. Thus, cannabinoid augmentation could be a promising avenue for treating dystonia, and we have identified a possible molecular mechanism mediating this.
Assuntos
Canabinoides , Colágeno Tipo VI , Distonia , Distúrbios Distônicos , Neurônios Motores , Plasticidade Neuronal , Animais , Canabinoides/metabolismo , Canabinoides/farmacologia , Colágeno Tipo VI/genética , Colágeno Tipo VI/metabolismo , Distonia/genética , Distonia/metabolismo , Distúrbios Distônicos/genética , Distúrbios Distônicos/metabolismo , Feminino , Masculino , Camundongos , Neurônios Motores/efeitos dos fármacos , Mutação , Plasticidade Neuronal/efeitos dos fármacos , Receptores de Canabinoides/genética , Receptores de Canabinoides/metabolismoRESUMO
Baculovirus-insect cell expression system has become one of the most widely used eukaryotic expression systems for heterologous protein production in many laboratories. The availability of robust insect cell lines, serum-free media, a range of vectors and commercially-packaged kits have supported the demand for maximizing the exploitation of the baculovirus-insect cell expression system. Naturally, this resulted in varied strategies adopted by different laboratories to optimize protein production. Most laboratories have preference in using either the E. coli transposition-based recombination bacmid technology (e.g. Bac-to-Bac®) or homologous recombination transfection within insect cells (e.g. flashBAC™). Limited data is presented in the literature to benchmark the protocols used for these baculovirus vectors to facilitate the selection of a system for optimal production of target proteins. Taking advantage of the Protein Production and Purification Partnership in Europe (P4EU) scientific network, a benchmarking initiative was designed to compare the diverse protocols established in thirteen individual laboratories. This benchmarking initiative compared the expression of four selected intracellular proteins (mouse Dicer-2, 204â¯kDa; human ABL1 wildtype, 126â¯kDa; human FMRP, 68â¯kDa; viral vNS1-H1, 76â¯kDa). Here, we present the expression and purification results on these proteins and highlight the significant differences in expression yields obtained using different commercially-packaged baculovirus vectors. The highest expression level for difficult-to-express intracellular protein candidates were observed with the EmBacY baculovirus vector system.
Assuntos
Baculoviridae/genética , Vetores Genéticos/genética , Proteínas Recombinantes/metabolismo , Animais , Linhagem Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-abl/genética , Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteínas Recombinantes/genética , Ribonuclease III/genética , Ribonuclease III/metabolismo , Células Sf9RESUMO
Antibody-mediated complement-dependent cytotoxicity (CDC) on malignant cells is regulated by several complement control proteins, including the inhibitory complement factor H (fH). fH consists of 20 short consensus repeat elements (SCRs) with specific functional domains. Previous research revealed that the fH-derived SCRs 19-20 (SCR1920) can displace full-length fH on the surface of chronic lymphocytic leukemia (CLL) cells, which sensitizes CLL cells for e.g. CD20-targeting therapeutic monoclonal antibody (mAb) induced CDC. Therefore, we constructed lentiviral vectors for the generation of cell lines that stably produce mAb-SCR-fusion variants starting from the clinically approved parental mAbs rituximab, obinutuzumab and ofatumumab, respectively. Flow-cytometry revealed that the modification of the mAbs by the SCRs does not impair the binding to CD20. Increased in vitro lysis potency compared to their parental mAbs was corroborated by showing specific and dose dependent target cell elimination by CDC when compared to their parental mAbs. Lysis of CLL cells was not affected by the depletion of NK cells, suggesting that antibody-dependent cellular cytotoxicity plays a minor role in this context. Overall, this study emphasizes the crucial role of CDC in the elimination of CLL cells by mAbs and introduces a novel approach for enhancing CDC by directly fusing fH SCR1920 with mAbs.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Antígenos CD20 , Fator H do Complemento , Leucemia Linfocítica Crônica de Células B , Rituximab , Humanos , Antígenos CD20/imunologia , Antígenos CD20/genética , Fator H do Complemento/imunologia , Fator H do Complemento/metabolismo , Fator H do Complemento/genética , Leucemia Linfocítica Crônica de Células B/imunologia , Rituximab/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Linhagem Celular TumoralRESUMO
The antitumor activity of monoclonal antibodies in the treatment of chronic lymphocytic leukemia is mediated mainly by antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Unfortunately, the efficacy of complement-dependent cytotoxicity is strongly restricted due to the expression and acquisition of regulators of complement activation by lymphocytic leukemia cells. Whereas the role of membrane regulators of complement activation, such as CD55 and CD59, has been investigated in detail in chronic lymphocytic leukemia, the involvement of soluble regulators of complement activation, such as complement factor H, has not yet been reported. Propidium iodide staining was performed to investigate the efficacy of ofatumumab and factor H-derived short-consensus-repeat 18-20 in the induction of complement-dependent cytotoxicity on primary chronic lymphocytic leukemia cells from 20 patients. Deposition of complement C3 fragments was monitored by western blot analysis. Expression of CD20, CD55 or CD59 was determined by FACS analysis. Replacement of factor H with short consensus repeat 18-20 significantly increased the susceptibility of primary chronic lymphocytic leukemia cells to ofatumumab-induced complement-dependent cytotoxicity. More importantly, addition of short-consensus-repeat 18-20 was able to overcome complement- resistance occurring during treatment with ofatumumab alone. Use of short consensus repeat 18-20 is likely to prolong the turnover time of active C3b fragments generated on the target cells following ofatumumab-induced complement activation, thereby improving specific killing of chronic lymphocytic leukemia cells by complement-dependent cytotoxicity. The relative contribution of factor H to the protection of chronic lymphocytic leukemia cells against complement-dependent cytotoxicity was comparable to that of CD55. Our data suggest that, by abrogating factor H function, short consensus repeat 18-20 may provide a novel approach that improves the complement-dependent efficacy of therapeutic monoclonal antibodies.
Assuntos
Anticorpos Monoclonais/farmacologia , Fator H do Complemento/farmacologia , Sequência Consenso/efeitos dos fármacos , Citotoxinas/farmacologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Células Cultivadas , Fator H do Complemento/genética , Sequência Consenso/genética , Citotoxinas/uso terapêutico , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Humanos , Leucemia Linfocítica Crônica de Células B/patologiaRESUMO
B cells are one of the targets of Friend virus (FV) infection, a well-established mouse model often used to study retroviral infections in vivo. Although B cells may be effective in stimulating cytotoxic T lymphocyte responses, studies involving their role in FV infection have mainly focused on neutralizing antibody production. Here we show that polyclonal activation of B cells promotes their infection with FV both in vitro and in vivo. Furthermore, we demonstrate that complement opsonization of Friend murine leukemia virus (F-MuLV) enhances infection of B cells, which correlates with increased potency of B cells to activate FV-specific CD8(+) T cells.
Assuntos
Linfócitos B/virologia , Linfócitos T CD8-Positivos/imunologia , Proteínas do Sistema Complemento/imunologia , Vírus da Leucemia Murina de Friend/imunologia , Vírus da Leucemia Murina de Friend/patogenicidade , Animais , Células Cultivadas , CamundongosRESUMO
Hemolytic-uremic syndrome (HUS) is a life-threatening disorder characterized by hemolytic anemia, thrombocytopenia, and renal insufficiency. It is caused mainly by infections with enterohemorrhagic Escherichia coli (EHEC). Recently, Shiga toxin 2, the best-studied virulence factor of EHEC, was reported to interact with complement, implying that complement may be involved in the pathogenesis of EHEC-induced HUS. The aim of the present study was to investigate whether or not the serine protease EspP, an important virulence factor of EHEC, interacts with complement proteins. EspP did not have any effect on the integrity of factor H or factor I. However, EspP was shown to cleave purified C3/C3b and C5. Cleavage of the respective complement proteins also occurred in normal human serum (NHS) as a source of C3/C3b or C5 or when purified complement proteins were added to the supernatant of an EspP-producing wild-type strain. Edman degradation allowed unequivocal mapping of all three main C3b fragments but not of the three main C5 fragments. Complement activation was significantly downregulated in all three pathways for C5-depleted serum to which C5, preincubated with EspP, was added (whereas C5 preincubated with an EspP mutant was able to fully reconstitute complement activation). This indicates that EspP markedly destroyed the functional activity, as measured by a commercial total complement enzyme-linked immunosorbent assay (Wieslab). Downregulation of complement by EspP in vivo may influence the colonization of EHEC bacteria in the gut or the disease severity of HUS.
Assuntos
Ativação do Complemento/efeitos dos fármacos , Complemento C3/metabolismo , Complemento C3b/metabolismo , Complemento C5/metabolismo , Escherichia coli Êntero-Hemorrágica/enzimologia , Proteínas de Escherichia coli/metabolismo , Serina Endopeptidases/metabolismo , Complemento C3/química , Complemento C3b/química , Complemento C5/química , Proteínas de Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica , Síndrome Hemolítico-Urêmica/microbiologia , Humanos , Serina Endopeptidases/genéticaRESUMO
Stroke induces a multiphasic systemic immune response, but the consequences of this response on atherosclerosis-a major source of recurrent vascular events-have not been thoroughly investigated. We show that stroke exacerbates atheroprogression via alarmin-mediated propagation of vascular inflammation. The prototypic brain-released alarmin high-mobility group box 1 protein induced monocyte and endothelial activation via the receptor for advanced glycation end products (RAGE)-signaling cascade and increased plaque load and vulnerability. Recruitment of activated monocytes via the CC-chemokine ligand 2-CC-chemokine receptor type 2 pathway was critical in stroke-induced vascular inflammation. Neutralization of circulating alarmins or knockdown of RAGE attenuated atheroprogression. Blockage of ß3-adrenoreceptors attenuated the egress of myeloid monocytes after stroke, whereas neutralization of circulating alarmins was required to reduce systemic monocyte activation and aortic invasion. Our findings identify a synergistic effect of the sympathetic stress response and alarmin-driven inflammation via RAGE as a critical mechanism of exacerbated atheroprogression after stroke.
Assuntos
Alarminas/metabolismo , Aterosclerose/metabolismo , Encéfalo/metabolismo , Animais , Aterosclerose/patologia , Encéfalo/patologia , Imunidade Inata/fisiologia , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologiaRESUMO
When bound to the envelope of viruses, factor H (FH), a soluble regulator of complement activation, contributes to the protection against a potent immune defense mechanism, the complement-mediated lysis (CML). Thus, removing FH from the surface renders viruses, such as HIV, susceptible to CML. For a proof of concept, we developed a construct consisting of recombinant bifunctional single-chain variable fragment (scFv) based on a monoclonal antibody against Friend murine leukemia virus (F-MuLV) envelope protein gp70, which was coupled to specific binding domains (short consensus repeats 19-20; SCR1920) of FH. We used Pichia pastoris as expression system in common shake flasks and optimized expression in high density bench top fermentation. Specific binding of recombinant scFv was proven by flow cytometry. The recombinant scFv-SCR significantly enhanced CML of F-MuLV in vitro implying that FH binding to the viral surface was impaired by the scFv-SCR. This novel concept to enhance virolysis may provide a new approach for antiviral treatment.
Assuntos
Anticorpos Biespecíficos/química , Proteínas Recombinantes/química , Anticorpos de Cadeia Única/química , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/metabolismo , Sequência de Bases , Clonagem Molecular , Fator H do Complemento/imunologia , Fator H do Complemento/metabolismo , Fermentação , Vírus da Leucemia Murina de Friend/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Dados de Sequência Molecular , Pichia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/metabolismo , Proteínas do Envelope Viral/imunologiaRESUMO
Dendritic cells (DC) represent the most potent antigen presenting cells and induce efficient cytotoxic T lymphocyte (CTL) responses against viral infections. Targeting antigens (Ag) to receptors on DCs is a promising strategy to enhance antitumor and antiviral immune responses induced by DCs. Here, we investigated the potential of CD11c-specific single-chain fragments (scFv) fused to an immunodominant peptide of Friend retrovirus for induction of virus-specific T cell responses by DCs. In vitro CD11c-specific scFv selectively targeted viral antigens to DCs and thereby significantly improved the activation of virus-specific T cells. In vaccination experiments DCs loaded with viral Ag targeted to CD11c provided improved rejection of FV-derived tumors and efficiently primed virus-specific CTL responses after virus challenge. Since the induction of strong virus-specific T cell responses is critical in viral infections, CD11c targeted protein vaccines might provide means to enhance the cellular immune response to prophylactic or therapeutic levels.
Assuntos
Antígenos Virais/imunologia , Antígeno CD11c/imunologia , Células Dendríticas/imunologia , Retroviridae/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/virologia , Sequência de Aminoácidos , Animais , Antígenos Virais/química , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Imunização , Epitopos Imunodominantes/química , Epitopos Imunodominantes/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Ovalbumina/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/imunologia , Especificidade da Espécie , Baço/citologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologiaRESUMO
PURPOSE OF REVIEW: New evidence is provided that the complement system is not only an effective component of the innate immunity, but is also involved in bridging innate and adaptive immune response to control retroviral infections. RECENT FINDINGS: The complement contributes to the control of retroviral replication by various strategies, such as complement-mediated lysis, triggering of B-cell responses by trapping the virus on follicular dendritic cells in the germinal center or enhancing of antigen presentation and thus the induction of virus-specific cytotoxic T lymphocytes. HIV has evolved mechanisms to escape from complement-meditated neutralization and counteracts these immune responses by escaping from lysis, using follicular dendritic cells as anchor to generate a latent viral reservoir and enhancing the infection of antigen-presenting cells. SUMMARY: This review will discuss the complex interactions of complement and complement receptors with retroviruses and review the escape mechanisms, which protect this virus family from complement-mediated destruction.
Assuntos
Proteínas do Sistema Complemento/imunologia , Infecções por HIV/imunologia , HIV/imunologia , Imunidade Adaptativa , HIV/patogenicidade , Humanos , Evasão da Resposta Imune , Imunidade Inata , Linfócitos T Citotóxicos/imunologiaRESUMO
Strategies for antibody-mediated cancer immunotherapy, such as active immunization with virus-like particle (VLP)-based vaccines, are gaining increasing attention. We developed chimeric hepatitis B virus core antigen (HBcAg)-VLPs that display a surface epitope of the highly selective tumor-associated cell lineage marker claudin-18 isoform 2 (CLDN18.2) flanked by a mobility-increasing linker. Auto-antibodies elicited by immunization with these chimeric HBcAg-VLPs in 2 relevant species (mouse and rabbit) bind with high precision to native CLDN18.2 at physiologic densities on the surface of living cells but not to the corresponding epitope of the CLDN18.1 splice variant that differs by merely one amino acid. The induced auto-antibodies are capable of efficiently killing CLDN18.2 expressing cells in vitro by complement-dependent and antibody-dependent cell-mediated cytotoxicity. Moreover, they provide partial protective immunity against the challenge of mice with syngeneic tumor cells stably expressing CLDN18.2. Our study provides a first proof-of-concept that immunization combining VLPs as antigen carriers with specific conformational epitopes of a highly selective differentiation antigen may elicit auto-antibodies with high cytocidal and tumoricidal potential.