RESUMO
Mouse models of active systemic anaphylaxis rely predominantly on IgG Abs forming IgG-allergen immune complexes that induce IgG receptor-expressing neutrophils and monocytes/macrophages to release potent mediators, leading to systemic effects. Whether anaphylaxis initiates locally or systemically remains unknown. In this study, we aimed at identifying the anatomical location of IgG-allergen immune complexes during anaphylaxis. Active systemic anaphylaxis was induced following immunization with BSA and i.v. challenge with fluorescently labeled BSA. Ag retention across different organs was examined using whole-body fluorescence imaging, comparing immunized and naive animals. Various mouse models and in vivo deletion strategies were employed to determine the contribution of IgG receptors, complement component C1q, myeloid cell types, and anaphylaxis mediators. We found that following challenge, Ag diffused systemically, but specifically accumulated in the lungs of mice sensitized to that Ag, where it formed large Ab-dependent aggregates in the vasculature. Ag retention in the lungs did not rely on IgG receptors, C1q, neutrophils, or macrophages. IgG2a-mediated, but neither IgG1- nor IgG2b-mediated, passive systemic anaphylaxis led to Ag retention in the lung. Neutrophils and monocytes significantly accumulated in the lungs after challenge and captured high amounts of Ag, which led to downmodulation of surface IgG receptors and triggered their activation. Thus, within minutes of systemic injection in sensitized mice, Ag formed aggregates in the lung and liver vasculature, but accumulated specifically and dose-dependently in the lung. Neutrophils and monocytes recruited to the lung captured Ag and became activated. However, Ag aggregation in the lung vasculature was not necessary for anaphylaxis induction.
Assuntos
Anafilaxia , Alérgenos , Animais , Complexo Antígeno-Anticorpo , Complemento C1q , Modelos Animais de Doenças , Imunoglobulina G , Pulmão , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Complemento , Receptores de IgGRESUMO
We describe a Kappa-on-Heavy (KoH) mouse that produces a class of highly diverse, fully human, antibody-like agents. This mouse was made by replacing the germline variable sequences of both the Ig heavy-chain (IgH) and Ig kappa (IgK) loci with the human IgK germline variable sequences, producing antibody-like molecules with an antigen binding site made up of 2 kappa variable domains. These molecules, named KoH bodies, structurally mimic naturally existing Bence-Jones light-chain dimers in their variable domains and remain wild-type in their antibody constant domains. Unlike artificially diversified, nonimmunoglobulin alternative scaffolds (e.g., DARPins), KoH bodies consist of a configuration of normal Ig scaffolds that undergo natural diversification in B cells. Monoclonal KoH bodies have properties similar to those of conventional antibodies but exhibit an enhanced ability to bind small molecules such as the endogenous cardiotonic steroid marinobufagenin (MBG) and nicotine. A comparison of crystal structures of MBG bound to a KoH Fab versus a conventional Fab showed that the KoH body has a much deeper binding pocket, allowing MBG to be held 4 Å further down into the combining site between the 2 variable domains.
Assuntos
Anticorpos/química , Anticorpos/imunologia , Antígenos/imunologia , Cadeias Pesadas de Imunoglobulinas/química , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/imunologia , Cadeias kappa de Imunoglobulina/química , Animais , Anticorpos/genética , Anticorpos/uso terapêutico , Sequência de Bases , Sítios de Ligação de Anticorpos/genética , Bufanolídeos , Engenharia Genética , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Camundongos , Modelos Moleculares , Nicotina , Conformação ProteicaRESUMO
BACKGROUND: C3 glomerulopathy (C3G) is characterized by the alternative-pathway (AP) hyperactivation induced by nephritic factors or complement gene mutations. Mice deficient in complement factor H (CFH) are a classic C3G model, with kidney disease that requires several months to progress to renal failure. Novel C3G models can further contribute to understanding the mechanism behind this disease and developing therapeutic approaches. METHODS: A novel, rapidly progressing, severe, murine model of C3G was developed by replacing the mouse C3 gene with the human C3 homolog using VelociGene technology. Functional, histologic, molecular, and pharmacologic assays characterize the presentation of renal disease and enable useful pharmacologic interventions in the humanized C3 (C3hu/hu) mice. RESULTS: The C3hu/hu mice exhibit increased morbidity early in life and die by about 5-6 months of age. The C3hu/hu mice display elevated biomarkers of kidney dysfunction, glomerulosclerosis, C3/C5b-9 deposition, and reduced circulating C3 compared with wild-type mice. Administration of a C5-blocking mAb improved survival rate and offered functional and histopathologic benefits. Blockade of AP activation by anti-C3b or CFB mAbs also extended survival and preserved kidney function. CONCLUSIONS: The C3hu/hu mice are a useful model for C3G because they share many pathologic features consistent with the human disease. The C3G phenotype in C3hu/hu mice may originate from a dysregulated interaction of human C3 protein with multiple mouse complement proteins, leading to unregulated C3 activation via AP. The accelerated disease course in C3hu/hu mice may further enable preclinical studies to assess and validate new therapeutics for C3G.
Assuntos
Complemento C3/genética , Modelos Animais de Doenças , Glomerulonefrite Membranoproliferativa/genética , Nefropatias/genética , Animais , Complemento C3/metabolismo , Via Alternativa do Complemento/genética , Éxons , Regulação da Expressão Gênica , Glomerulonefrite Membranoproliferativa/metabolismo , Humanos , Nefropatias/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Fenótipo , Polimorfismo de Nucleotídeo Único , Insuficiência Renal/genética , Insuficiência Renal/metabolismoRESUMO
Increased activation of the contact system protein high molecular weight kininogen (HK) has been shown in plasma and cerebrospinal fluid of Alzheimer's disease (AD) patients, but its potential role in the brain has not been explored. We assessed HK levels in brain tissue from 20 AD patients and controls and modeled the effects of HK on microglia-like cells in culture. We show increased levels of HK in the hippocampus of AD patients, which colocalized with amyloid beta (Aß) deposits and activated microglia. Treatment of microglia with HK led to cell clustering and elevated levels of phagocytosed Aß. We demonstrate that microglia internalize HK and traffic it to lysosomes, which is accompanied by reduced activity of lysosomal cathepsins L and S. Our results suggest that HK accumulation in the AD hippocampus may alter microglial uptake and degradation of Aß fibrils, possibly contributing to microglial dysfunction in AD.
Assuntos
Doença de Alzheimer , Microglia , Humanos , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Catepsinas/metabolismo , Catepsinas/farmacologia , Cininogênio de Alto Peso Molecular/metabolismo , Cininogênio de Alto Peso Molecular/farmacologia , Lisossomos/metabolismo , Microglia/metabolismo , FagocitoseRESUMO
BACKGROUND AND PURPOSE: Stroke is a major cause of chronic neurological disability. There is considerable interest in understanding how acute transcriptome changes evolve into subacute and chronic patterns that facilitate or limit spontaneous recovery. Here we mapped longitudinal changes in gene expression at multiple time points after stroke in mice out to 6 months. METHODS: Adult C57BL/6 mice were subjected to transient middle cerebral artery occlusion. Longitudinal transcriptome levels were measured at 10 time points after stroke from acute to recovery phases of ischemic stroke. Localization and the number of mononuclear phagocytes were determined in the postischemic brain. Whole-mount brain imaging was performed in asplenic mice receiving GFP+ (green fluorescent protein)-tagged splenocytes. RESULTS: Sustained stroke-induced mRNA abundance changes were observed in both hemispheres with 2989 ipsilateral and 822 contralateral genes significantly perturbed. In the hemisphere ipsilateral to the infarct, genes associated with immune functions were strongly affected, including temporally overlapping innate and adaptive immunity and macrophage M1 and M2 phenotype-related genes. The strong immune gene activation was accompanied by the sustained infiltration of peripheral immune cells at acute, subacute, and recovery stages of stroke. The infiltrated immune cells were found in the infarcted area but also in remote regions at 2 months after stroke. CONCLUSIONS: The study identifies that immune components are the predominant molecular signatures and they may propagate or continuously respond to brain injury in the subacute to chronic phase after central nervous system injury. The study suggests a potential immune-based strategy to modify injury progression and tissue remodeling in ischemic stroke, even months after the initiating event.
Assuntos
Isquemia Encefálica/diagnóstico por imagem , Isquemia Encefálica/imunologia , Movimento Celular/fisiologia , Imunidade Celular/fisiologia , Recuperação de Função Fisiológica/fisiologia , Transcrição Gênica/fisiologia , Animais , Isquemia Encefálica/genética , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Immunodeficient mice reconstituted with a human immune system represent a promising tool for translational research as they may allow modeling and therapy of human diseases in vivo. However, insufficient development and function of human natural killer (NK) cells and T cell subsets limit the applicability of humanized mice for studying cancer biology and therapy. Here, we describe a human interleukin 15 (IL15) and human signal regulatory protein alpha (SIRPA) knock-in mouse on a Rag2-/- Il2rg-/- background (SRG-15). Transplantation of human hematopoietic stem and progenitor cells into SRG-15 mice dramatically improved the development and functional maturation of circulating and tissue-resident human NK and CD8+ T cells and promoted the development of tissue-resident innate lymphoid cell (ILC) subsets. Profiling of human NK cell subsets by mass cytometry revealed a highly similar expression pattern of killer inhibitory receptors and other candidate molecules in NK cell subpopulations between SRG-15 mice and humans. In contrast to nonobese diabetic severe combined immunodeficient Il2rg-/- (NSG) mice, human NK cells in SRG-15 mice did not require preactivation but infiltrated a Burkitt's lymphoma xenograft and efficiently inhibited tumor growth following treatment with the therapeutic antibody rituximab. Our humanized mouse model may thus be useful for preclinical testing of novel human NK cell-targeted and combinatory cancer immunotherapies and for studying how they elicit human antitumor immune responses in vivo.
Assuntos
Células Matadoras Naturais/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Modelos Animais de Doenças , Humanos , Imunidade Inata/imunologia , Subunidade gama Comum de Receptores de Interleucina/imunologia , Interleucina-15/imunologia , Linfócitos/imunologia , Camundongos , Camundongos SCID , Receptores Imunológicos/imunologia , Rituximab/imunologiaRESUMO
The Adisintegrin and metalloprotease domain-containing (ADAM) family of proteins is involved in cell adhesion, migration, proteolysis, and signaling. Many ADAMs are required for reproduction; however, the role of Adam6 has remained largely unknown. In the course of humanizing the mouse immunoglobulin heavy chain (IgH) locus, we generated Adam6-deficient mice that demonstrate severe subfertility. We decided to elucidate the role of ADAM6 in fertility and explore the underlying mechanisms. Despite normal sperm development and motility, Adam6-deficient mice display diminished male fertility, have abnormal sperm adhesion, and most importantly cannot transition from uterus to oviduct. To test whether ADAM6 is required for sperm's binding to extracellular matrix (ECM) components, we used a panel of ECM components and showed that unlike normal sperm, Adam6-deficient sperm cannot bind fibronectin, laminin, and tenascin. Reintroduction of Adam6 into these deficient mice repaired sperm interaction with ECM, restored male fertility, and corrected the sperm transport deficit. Together, our data suggest that ADAM6, either alone or in complex with other proteins, aids sperm transport through the female reproductive tract by providing a temporary site of attachment of sperm to ECM components prior to ascent into the oviduct.
Assuntos
Proteínas ADAM/metabolismo , Infertilidade Masculina/genética , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/fisiologia , Proteínas ADAM/genética , Animais , Feminino , Masculino , Camundongos , Camundongos Knockout , Oviductos , Motilidade dos Espermatozoides/genéticaRESUMO
In the RV144 gp120 HIV vaccine trial, decreased transmission risk was correlated with Abs that reacted with a linear epitope at a lysine residue at position 169 (K169) in the HIV-1 envelope (Env) V2 region. The K169 V2 response was restricted to Abs bearing Vλ rearrangements that expressed aspartic acid/glutamic acid in CDR L2. The AE.A244 gp120 in AIDSVAX B/E also bound to the unmutated ancestor of a V2-glycan broadly neutralizing Ab, but this Ab type was not induced in the RV144 trial. In this study, we sought to determine whether immunodominance of the V2 linear epitope could be overcome in the absence of human Vλ rearrangements. We immunized IgH- and Igκ-humanized mice with the AE.A244 gp120 Env. In these mice, the V2 Ab response was focused on a linear epitope that did not include K169. V2 Abs were isolated that used the same human VH gene segment as an RV144 V2 Ab but paired with a mouse λ L chain. Structural characterization of one of these V2 Abs revealed how the linear V2 epitope could be engaged, despite the lack of aspartic acid/glutamic acid encoded in the mouse repertoire. Thus, despite the absence of the human Vλ locus in these humanized mice, the dominance of Vλ pairing with human VH for HIV-1 Env V2 recognition resulted in human VH pairing with mouse λ L chains instead of allowing otherwise subdominant V2-glycan broadly neutralizing Abs to develop.
Assuntos
Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Vacinas contra a AIDS/imunologia , Motivos de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , Epitopos , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias lambda de Imunoglobulina/imunologia , CamundongosRESUMO
BACKGROUND: Anaphylaxis can proceed through distinct IgE- or IgG-dependent pathways, which have been investigated in various mouse models. We developed a novel mouse strain in which the human low-affinity IgG receptor locus, comprising both activating (hFcγRIIA, hFcγRIIIA, and hFcγRIIIB) and inhibitory (hFcγRIIB) hFcγR genes, has been inserted into the equivalent murine locus, corresponding to a locus swap. OBJECTIVE: We sought to determine the capabilities of hFcγRs to induce systemic anaphylaxis and identify the cell types and mediators involved. METHODS: hFcγR expression on mouse and human cells was compared to validate the model. Passive systemic anaphylaxis was induced by injection of heat-aggregated human intravenous immunoglobulin and active systemic anaphylaxis after immunization and challenge. Anaphylaxis severity was evaluated based on hypothermia and mortality. The contribution of receptors, mediators, or cell types was assessed based on receptor blockade or depletion. RESULTS: The human-to-mouse low-affinity FcγR locus swap engendered hFcγRIIA/IIB/IIIA/IIIB expression in mice comparable with that seen in human subjects. Knock-in mice were susceptible to passive and active anaphylaxis, accompanied by downregulation of both activating and inhibitory hFcγR expression on specific myeloid cells. The contribution of hFcγRIIA was predominant. Depletion of neutrophils protected against hypothermia and mortality. Basophils contributed to a lesser extent. Anaphylaxis was inhibited by platelet-activating factor receptor or histamine receptor 1 blockade. CONCLUSION: Low-affinity FcγR locus-switched mice represent an unprecedented model of cognate hFcγR expression. Importantly, IgG-related anaphylaxis proceeds within a native context of activating and inhibitory hFcγRs, indicating that, despite robust hFcγRIIB expression, activating signals can dominate to initiate a severe anaphylactic reaction.
Assuntos
Anafilaxia/imunologia , Receptores de IgG/imunologia , Animais , Modelos Animais de Doenças , Proteínas Ligadas por GPI/imunologia , Técnicas de Introdução de Genes , Humanos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BLRESUMO
Two activating mouse IgG receptors (FcγRs) have the ability to bind monomeric IgG, the high-affinity mouse FcγRI and FcγRIV. Despite high circulating levels of IgG, reports using FcγRI-/- or FcγRIV-/- mice or FcγRIV-blocking antibodies implicate these receptors in IgG-induced disease severity or therapeutic Ab efficacy. From these studies, however, one cannot conclude on the effector capabilities of a given receptor, because different activating FcγRs possess redundant properties in vivo, and cooperation between FcγRs may occur, or priming phenomena. To help resolve these uncertainties, we used mice expressing only FcγRI to determine its intrinsic properties in vivo. FcγRIonly mice were sensitive to IgG-induced autoimmune thrombocytopenia and anti-CD20 and anti-tumour immunotherapy, but resistant to IgG-induced autoimmune arthritis, anaphylaxis and airway inflammation. Our results show that the in vivo roles of FcγRI are more restricted than initially reported using FcγRI-/- mice, but confirm effector capabilities for this high-affinity IgG receptor in vivo.
Assuntos
Anticorpos Bloqueadores/uso terapêutico , Linfócitos B/imunologia , Imunoterapia/métodos , Púrpura Trombocitopênica Idiopática/imunologia , Receptores de IgG/metabolismo , Animais , Afinidade de Anticorpos , Modelos Animais de Doenças , Hepatectomia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Púrpura Trombocitopênica Idiopática/terapia , Receptores de IgG/genética , EsplenectomiaRESUMO
Mice genetically engineered to be humanized for their Ig genes allow for human antibody responses within a mouse background (HumAb mice), providing a valuable platform for the generation of fully human therapeutic antibodies. Unfortunately, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which their genetic humanization was carried out. Heretofore, HumAb mice have been generated by disrupting the endogenous mouse Ig genes and simultaneously introducing human Ig transgenes at a different and random location; KO-plus-transgenic humanization. As we describe in the companion paper, we attempted to make mice that more efficiently use human variable region segments in their humoral responses by precisely replacing 6 Mb of mouse Ig heavy and kappa light variable region germ-line gene segments with their human counterparts while leaving the mouse constant regions intact, using a unique in situ humanization approach. We reasoned the introduced human variable region gene segments would function indistinguishably in their new genetic location, whereas the retained mouse constant regions would allow for optimal interactions and selection of the resulting antibodies within the mouse environment. We show that these mice, termed VelocImmune mice because they were generated using VelociGene technology, efficiently produce human:mouse hybrid antibodies (that are rapidly convertible to fully human antibodies) and have fully functional humoral immune systems indistinguishable from those of WT mice. The efficiency of the VelocImmune approach is confirmed by the rapid progression of 10 different fully human antibodies into human clinical trials.
Assuntos
Formação de Anticorpos , Genes de Imunoglobulinas , Alelos , Animais , Linfócitos B/imunologia , Citometria de Fluxo , Humanos , Camundongos , MutaçãoRESUMO
Genetic humanization, which involves replacing mouse genes with their human counterparts, can create powerful animal models for the study of human genes and diseases. One important example of genetic humanization involves mice humanized for their Ig genes, allowing for human antibody responses within a mouse background (HumAb mice) and also providing a valuable platform for the generation of fully human antibodies as therapeutics. However, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which they were genetically humanized. Heretofore, most genetic humanizations have involved disruption of the endogenous mouse gene with simultaneous introduction of a human transgene at a new and random location (so-called KO-plus-transgenic humanization). More recent efforts have attempted to replace mouse genes with their human counterparts at the same genetic location (in situ humanization), but such efforts involved laborious procedures and were limited in size and precision. We describe a general and efficient method for very large, in situ, and precise genetic humanization using large compound bacterial artificial chromosome-based targeting vectors introduced into mouse ES cells. We applied this method to genetically humanize 3-Mb segments of both the mouse heavy and κ light chain Ig loci, by far the largest genetic humanizations ever described. This paper provides a detailed description of our genetic humanization approach, and the companion paper reports that the humoral immune systems of mice bearing these genetically humanized loci function as efficiently as those of WT mice.
Assuntos
Genes de Imunoglobulinas , Animais , Cromossomos Artificiais Bacterianos , Células-Tronco Embrionárias/imunologia , Recombinação Homóloga , Humanos , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase , TransgenesRESUMO
BACKGROUND: Our aim was to explore previously unknown long-term outcomes of self-directed personal care services for young adults with intellectual disabilities and limitations in activities of daily living. MATERIALS AND METHODS: The present authors utilized participatory action research and qualitative content analysis in interviewing 11 unpaid familial programme representatives of young adults with intellectual disabilities, ages 23-34, who were eligible for income-based Medicaid and enrolled five or more years in a Cash and Counseling-based programme of self-direction in the United States. RESULTS: Young adults are represented as receiving services and supports in a supportive and stable environment, with previously identified short-term programme benefits evident over the long-term. Young adults are also transitioning to adulthood at home with their families as primary social support and caregivers, bridging a service gap. CONCLUSIONS: Our results show that self-direction helps meet these young adults' personal care and community engagement needs over time.
Assuntos
Atividades Cotidianas , Aconselhamento , Deficiência Intelectual , Pessoas com Deficiência Mental/psicologia , Adulto , Cuidadores/psicologia , Feminino , Pesquisa sobre Serviços de Saúde , Serviços de Assistência Domiciliar , Humanos , Masculino , Medicaid , Estados Unidos , Adulto JovemRESUMO
Numerous studies have demonstrated the short-term effectiveness of the Cash and Counseling model option of participant-directed home and community-based personal care service programs for Medicaideligible recipients with disabilities requiring long-term care. However, long-term experiences with participant-directed services have yet to be examined for these individuals. We addressed this gap in the literature through participatory action research and qualitative content analysis. Working together as coresearchers with members of the National Participant Network, a peer organization for people interested in or enrolled in participant-directed services, we interviewed 17 adults enrolled in one state's Cash and Counseling-based program. Participants' ages ranged from 40 to 83 years, had been enrolled for at least 5 years, and acted as their own representative within the program. Our major findings show (a) the program's flexibility allowed for adaptation to meet participants' changing needs over time and (b) that program attendants helped connect participants with community in multiple ways. In this article, we provide important policy and practice implications for participant-directed programs for people with disabilities.
Assuntos
Administração de Caso/organização & administração , Pessoas com Deficiência/reabilitação , Serviços de Assistência Domiciliar/organização & administração , Assistência de Longa Duração/organização & administração , Medicaid/organização & administração , Adulto , Idoso , Idoso de 80 Anos ou mais , Pesquisa Participativa Baseada na Comunidade , Feminino , Pesquisa sobre Serviços de Saúde , Humanos , Entrevistas como Assunto , Masculino , Pessoa de Meia-Idade , Pesquisa Qualitativa , Qualidade da Assistência à Saúde , Estados Unidos , United States Dept. of Health and Human ServicesRESUMO
mAb therapy for experimental metastatic melanoma relies on activating receptors for the Fc portion of IgG (FcγR). Opposing results on the respective contribution of mouse FcγRI, FcγRIII, and FcγRIV have been reported using the gp75-expressing B16 melanoma and the protective anti-gp75 mAb TA99. We analyzed the contribution of FcγRs to this therapy model using bioluminescent measurement of lung metastases loads, novel mouse strains, and anti-FcγR blocking mAbs. We found that the TA99 mAb-mediated effects in a combination therapy using cyclophosphamide relied on activating FcγRs. The combination therapy, however, was not more efficient than mAb therapy alone. We demonstrate that FcγRI and, unexpectedly, FcγRIII contributed to TA99 mAb therapeutic effects, whereas FcγRIV did not. Therefore, FcγRIII and FcγRI are, together, responsible for anti-gp75 mAb therapy of B16 lung metastases. Our finding that mouse FcγRIII contributes to Ab-induced tumor reduction correlates with clinical data on its human functional equivalent human FcγRIIIA (CD16A).
Assuntos
Anticorpos Monoclonais/uso terapêutico , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Receptores de IgG/uso terapêutico , Proteínas Virais/imunologia , Animais , Anticorpos Bloqueadores/uso terapêutico , Arbovírus/imunologia , Hibridomas , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/terapia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de IgG/deficiência , Receptores de IgG/genéticaRESUMO
The vasculature of the CNS is structurally and functionally distinct from that of other organ systems and is particularly prone to developmental abnormalities and hemorrhage. Although other embryonic tissues undergo primary vascularization, the developing nervous system is unique in that it is secondarily vascularized by sprouting angiogenesis from a surrounding perineural plexus. This sprouting angiogenesis requires the TGF-ß and Wnt pathways because ablation of these pathways results in aberrant sprouting and hemorrhage. We have genetically deleted Gpr124, a member of the large family of long N-terminal group B G protein-coupled receptors, few members of which have identified ligands or well-defined biologic functions in mammals. We show that, in the developing CNS, Gpr124 is specifically expressed in the vasculature and is absolutely required for proper angiogenic sprouting into the developing neural tube. Embryos lacking Gpr124 exhibit vascular defects characterized by delayed vascular penetration, formation of pathological glomeruloid tufts within the CNS, and hemorrhage. In addition, they display defects in palate and lung development, two processes in which TGF-ß and/or Wnt pathways also play important roles. We also show that TGF-ß stimulates Gpr124 expression, and ablation of Gpr124 results in perturbed TGF-ß pathway activation, suggesting roles for Gpr124 in modulating TGF-ß signaling. These results represent a unique function attributed to a long N-terminal group B-type G protein-coupled receptor in a mammalian system.
Assuntos
Sistema Nervoso Central/irrigação sanguínea , Sistema Nervoso Central/embriologia , Neovascularização Fisiológica/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Animais , Embrião de Mamíferos , Engenharia Genética , Técnicas Histológicas , Imuno-Histoquímica , Hibridização In Situ , Pulmão/embriologia , Pulmão/metabolismo , Camundongos , Análise em Microsséries , Palato/embriologia , Palato/metabolismo , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Proteínas Wnt/metabolismoRESUMO
Clostridioides difficile is the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis in adults. Various C. difficile strains circulate currently, associated with different outcomes and antibiotic resistance profiles. However, most studies still focus on the reference strain 630 that does not circulate anymore, partly due to the lack of immunological tools to study current clinically important C. difficile PCR ribotypes. The goal of this study was to generate monoclonal antibodies recognizing various epidemic ribotypes of C. difficile. To do so, we immunized mice expressing human variable antibody genes with the Low Molecular Weight (LMW) subunit of the surface layer protein SlpA from various C. difficile strains. Monoclonal antibodies purified from hybridomas bound LMW with high-affinity and whole bacteria from current C. difficile ribotypes with different cross-specificities. This first collection of anti-C. difficile mAbs represent valuable tools for basic and clinical research.
RESUMO
Clostridioides difficile (C. difficile), a gram-positive anaerobic and spore-forming bacterium, is the leading cause of nosocomial antibiotic-associated diarrhea in adults which is characterized by high levels of recurrence and mortality. Surface (S)-layer Protein A (SlpA), the most abundantly expressed protein on the bacterial surface, plays a crucial role in the early stages of infection although the nature of its involvement in C. difficile physiology is yet to be fully understood. Anti-S-layer antibodies have been identified in the sera of convalescent patients and have been correlated with improved outcomes of C. difficile infection (CDI). However, the precise mechanisms by which anti-S-layer antibodies confer protection to the host remain unknown. In this study, we report the first monoclonal antibodies (mAbs) targeting the S-layer of reference strain 630. Characterization of these mAbs unraveled important roles for the S-layer protein in growth, toxin secretion, and biofilm formation by C. difficile, with differential and even opposite effects of various anti-SlpA mAbs on these functions. Moreover, one anti-SlpA mAb impaired C. difficile growth and conferred sensitivity to lysozyme-induced lysis. The results of this study show that anti-S-layer antibody responses can be beneficial or harmful for the course of CDI and provide important insights for the development of adequate S-layer-targeting therapeutics.
Assuntos
Clostridioides difficile , Microbioma Gastrointestinal , Adulto , Humanos , Anticorpos Monoclonais/uso terapêutico , Morte CelularRESUMO
Ig and T-cell receptor (TCR) variable-region gene exons are assembled from component variable (V), diversity (D) and joining (J) gene segments during early B and T cell development. The RAG1/2 endonuclease initiates V(D)J recombination by introducing DNA double-strand breaks at borders of the germ-line segments. In mice, the Ig heavy-chain (IgH) locus contains, from 5' to 3', several hundred V(H) gene segments, 13 D segments, and 4 J(H) segments within a several megabase region. In developing B cells, IgH variable-region exon assembly is ordered with D to J(H) rearrangement occurring on both alleles before appendage of a V(H) segment. Also, IgH V(H) to DJ(H) rearrangement does not occur in T cells, even though DJ(H) rearrangements occur at low levels. In these contexts, V(D)J recombination is controlled by modulating substrate gene segment accessibility to RAG1/2 activity. To elucidate control elements, we deleted the 100-kb intergenic region that separates the V(H) and D clusters (generating ΔV(H)-D alleles). In both B and T cells, ΔV(H)-D alleles initiated high-level antisense and, at lower levels, sense transcription from within the downstream D cluster, with antisense transcripts extending into proximal V(H) segments. In developing T lymphocytes, activated germ-line antisense transcription was accompanied by markedly increased IgH D-to-J(H) rearrangement and substantial V(H) to DJ(H) rearrangement of proximal IgH V(H) segments. Thus, the V(H)-D intergenic region, and likely elements within it, can influence silencing of sense and antisense germ-line transcription from the IgH D cluster and thereby influence targeting of V(D)J recombination.
Assuntos
Linfócitos B/metabolismo , Rearranjo Gênico de Cadeia Pesada de Linfócito B/fisiologia , Cadeias Pesadas de Imunoglobulinas/biossíntese , Região Variável de Imunoglobulina/biossíntese , RNA Antissenso/biossíntese , Transcrição Gênica/fisiologia , Alelos , Animais , DNA Intergênico/genética , DNA Intergênico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Loci Gênicos/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Camundongos , Camundongos Mutantes , RNA Antissenso/genética , Linfócitos T/metabolismoRESUMO
The granularity and structure of the International Council for Harmonisation's (ICH) Medical Dictionary for Regulatory Activities (MedDRA) are useful for precise coding of adverse events (AEs) for data analysis. In product labeling for healthcare practitioners, however, the granularity of MedDRA Preferred Terms (PTs) can obscure the communication of adverse reactions (ARs). Driven by a focus on patient safety, business needs, and regulatory guidance, many sponsors and regulators have begun to develop institution-specific approaches to clustering similar AR terms in medical product prescribing information on a product-by-product basis. However, there are no agreed upon conventions that describe which AR terms may be appropriate to group together. In order to improve safety communication to patients and healthcare providers, there is an urgent need for a harmonized international approach to the creation and use of groups of MedDRA PTs which we refer to as "MedDRA Labeling Groupings (MLGs)" in medical product prescribing information. Given its long-standing contributions towards the design of Standardised MedDRA Queries (SMQs), the Council for International Organizations of Medical Sciences (CIOMS) convened an Expert Working Group (EWG) with involvement of multiple major stakeholders to produce a consensus document on principles and points to consider in the development of MLGs. The CIOMS MLG EWG identified variations in grouping of MedDRA PTs in product labels, and in the current document, proposes a strategy for improving the communication of drug safety labeling. It is envisaged that the use of these consensus recommendations would be voluntary and applied to product labels in a manner that is consistent with existing regulatory frameworks.