RESUMO
Effective therapies are urgently needed to safely target TDP-43 pathology as it is closely associated with the onset and development of devastating diseases such as frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP) and amyotrophic lateral sclerosis (ALS). In addition, TDP-43 pathology is present as a co-pathology in other neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Our approach is to develop a TDP-43-specific immunotherapy that exploits Fc gamma-mediated removal mechanisms to limit neuronal damage while maintaining physiological TDP-43 function. Thus, using both in vitro mechanistic studies in conjunction with the rNLS8 and CamKIIa inoculation mouse models of TDP-43 proteinopathy, we identified the key targeting domain in TDP-43 to accomplish these therapeutic objectives. Targeting the C-terminal domain of TDP-43 but not the RNA recognition motifs (RRM) reduces TDP-43 pathology and avoids neuronal loss in vivo. We demonstrate that this rescue is dependent on Fc receptor-mediated immune complex uptake by microglia. Furthermore, monoclonal antibody (mAb) treatment enhances phagocytic capacity of ALS patient-derived microglia, providing a mechanism to restore the compromised phagocytic function in ALS and FTD patients. Importantly, these beneficial effects are achieved while preserving physiological TDP-43 activity. Our findings demonstrate that a mAb targeting the C-terminal domain of TDP-43 limits pathology and neurotoxicity, enabling clearance of misfolded TDP-43 through microglia engagement, and supporting the clinical strategy to target TDP-43 by immunotherapy. SIGNIFICANCE STATEMENT: TDP-43 pathology is associated with various devastating neurodegenerative disorders with high unmet medical needs such as frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Thus, safely and effectively targeting pathological TDP-43 represents a key paradigm for biotechnical research as currently there is little in clinical development. After years of research, we have determined that targeting the C-terminal domain of TDP-43 rescues multiple patho-mechanisms involved in disease progression in two animal models of FTD/ALS. In parallel, importantly, our studies establish that this approach does not alter the physiological functions of this ubiquitously expressed and indispensable protein. Together, our findings substantially contribute to the understanding of TDP-43 pathobiology and support the prioritization for clinical testing of immunotherapy approaches targeting TDP-43.
Assuntos
Doença de Alzheimer , Esclerose Lateral Amiotrófica , Demência Frontotemporal , Doença de Pick , Camundongos , Animais , Esclerose Lateral Amiotrófica/genética , Demência Frontotemporal/genética , Doença de Alzheimer/genética , Neuroproteção , Proteínas de Ligação a DNA/metabolismo , ImunoterapiaRESUMO
Fungi cause serious life-threatening infections in immunocompromised individuals and current treatments are now complicated by toxicity issues and the emergence of drug resistant strains. Consequently, there is a need for development of new antifungal drugs. Inosine monophosphate dehydrogenase (IMPDH), a key component of the de novo purine biosynthetic pathway, is essential for growth and virulence of fungi and is a potential drug target. In this study, a high-throughput screen of 114,000 drug-like compounds against Cryptococcus neoformans IMPDH was performed. We identified three 3-((5-substituted)-1,3,4-oxadiazol-2-yl)thio benzo[b]thiophene 1,1-dioxides that inhibited Cryptococcus IMPDH and also possessed whole cell antifungal activity. Analogs were synthesized to explore the SAR of these hits. Modification of the fifth substituent on the 1,3,4-oxadiazole ring yielded compounds with nanomolar in vitro activity, but with associated cytotoxicity. In contrast, two analogs generated by substituting the 1,3,4-oxadiazole ring with imidazole and 1,2,4-triazole gave reduced IMPDH inhibition in vitro, but were not cytotoxic. During enzyme kinetic studies in the presence of DTT, nucleophilic attack of a free thiol occurred with the benzo[b]thiophene 1,1-dioxide. Two representative compounds with substitution at the 5 position of the 1,3,4-oxadiazole ring, showed mixed inhibition in the absence of DTT. Incubation of these compounds with Cryptococcus IMPDH followed by mass spectrometry analysis showed non-specific and covalent binding with IMPDH at multiple cysteine residues. These results support recent reports that the benzo[b]thiophene 1,1-dioxides moiety as PAINS (pan-assay interference compounds) contributor.
Assuntos
Antifúngicos/química , Antifúngicos/farmacologia , Cryptococcus neoformans/efeitos dos fármacos , Proteínas Fúngicas/antagonistas & inibidores , IMP Desidrogenase/antagonistas & inibidores , Tiofenos/química , Tiofenos/farmacologia , Criptococose/tratamento farmacológico , Criptococose/metabolismo , Criptococose/microbiologia , Cryptococcus neoformans/enzimologia , Proteínas Fúngicas/metabolismo , Células HEK293 , Células Hep G2 , Humanos , IMP Desidrogenase/metabolismo , Modelos Moleculares , Oxidiazóis/química , Oxidiazóis/farmacologiaRESUMO
The exocrine pancreas exhibits a distinctive capacity for tissue regeneration and renewal following injury. This regenerative ability has important implications for a variety of disorders, including pancreatitis and pancreatic cancer, diseases associated with high morbidity and mortality. Thus, understanding its underlying mechanisms may help in developing therapeutic interventions. Serotonin has been recognized as a potent mitogen for a variety of cells and tissues. Here we investigated whether serotonin exerts a mitogenic effect in pancreatic acinar cells in three regenerative models, inflammatory tissue injury following pancreatitis, tissue loss following partial pancreatectomy, and thyroid hormone-stimulated acinar proliferation. Genetic and pharmacological techniques were used to modulate serotonin levels in vivo. Acinar dedifferentiation and cell cycle progression during the regenerative phase were investigated over the course of 2 weeks. By comparing acinar proliferation in the different murine models of regeneration, we found that serotonin did not affect the clonal regeneration of mature acinar cells. Serotonin was, however, required for acinar dedifferentiation following inflammation-mediated tissue injury. Specifically, lack of serotonin resulted in delayed up-regulation of progenitor genes and delayed the formation of acinar-to-ductal metaplasia and defective acinar cell proliferation. We identified serotonin-dependent acinar secretion as a key step in progenitor-based regeneration, as it promoted acinar cell dedifferentiation and the recruitment of type 2 macrophages. Finally, we identified a regulatory Hes1-Ptfa axis in the uninjured adult pancreas, activated by zymogen secretion. Our findings indicated that serotonin plays a critical role in the regeneration of the adult pancreas following pancreatitis by promoting the dedifferentiation of acinar cells.
Assuntos
Células Acinares/citologia , Desdiferenciação Celular/fisiologia , Pâncreas Exócrino/fisiologia , Serotonina/metabolismo , Envelhecimento , Animais , Modelos Animais de Doenças , Immunoblotting , Imuno-Histoquímica , Metaplasia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Pancreatite/patologia , RegeneraçãoRESUMO
Immunoglobulin class switching from IgM to IgG in response to peptides is generally T cell-dependent and vaccination in T cell-deficient individuals is inefficient. We show that a vaccine consisting of a dense array of peptides on liposomes induced peptide-specific IgG responses totally independent of T-cell help. Independency was confirmed in mice lacking T cells and in mice deficient for MHC class II, CD40L, and CD28. The IgG titers were high, long-lived, and comparable with titers obtained in wild-type animals, and the antibody response was associated with germinal center formation, expression of activation-induced cytidine deaminase, and affinity maturation. The T cell-independent (TI) IgG response was strictly dependent on ligation of TLR4 receptors on B cells, and concomitant TLR4 and cognate B-cell receptor stimulation was required on a single-cell level. Surprisingly, the IgG class switch was mediated by TIR-domain-containing adapter inducing interferon-ß (TRIF), but not by MyD88. This study demonstrates that peptides can induce TI isotype switching when antigen and TLR ligand are assembled and appropriately presented directly to B lymphocytes. A TI vaccine could enable efficient prophylactic and therapeutic vaccination of patients with T-cell deficiencies and find application in diseases where induction of T-cell responses contraindicates vaccination, for example, in Alzheimer disease.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Peptídeos beta-Amiloides/imunologia , Linfócitos B/imunologia , Switching de Imunoglobulina/imunologia , Fragmentos de Peptídeos/imunologia , Receptor 4 Toll-Like/fisiologia , Vacinas de Subunidades Antigênicas/imunologia , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Proteínas Adaptadoras de Transporte Vesicular/genética , Transferência Adotiva , Sequência de Aminoácidos , Peptídeos beta-Amiloides/administração & dosagem , Animais , Apresentação de Antígeno , Linfócitos B/metabolismo , Antígenos CD28/deficiência , Antígenos CD28/imunologia , Ligante de CD40/deficiência , Ligante de CD40/imunologia , Centro Germinativo/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina M/biossíntese , Receptores de Lipopolissacarídeos/imunologia , Lipossomos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Dados de Sequência Molecular , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Fragmentos de Peptídeos/administração & dosagem , Receptores de Antígenos de Linfócitos B/imunologia , Linfócitos T/imunologia , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genética , Vacinação , Vacinas de Subunidades Antigênicas/administração & dosagemRESUMO
OBJECTIVE: Serotonin (5-hydroxytryptamine, 5-HT) is a potent bioactive molecule involved in a variety of physiological processes. In this study, the authors analysed whether 5-HT regulates zymogen secretion in pancreatic acinar cells and the development of pancreatic inflammation, a potentially lethal disease whose pathophysiology is not completely understood. METHODS: 5-HT regulation of zymogen secretion was analysed in pancreatic acini isolated from wild-type or tryptophan hydoxylase-1 knock-out (TPH1(-/-)) mice, which lack peripheral 5-HT, and in amylase-secreting pancreatic cell lines. Pancreatitis was induced by cerulein stimulation and biochemical and immunohistochemical methods were used to evaluate disease progression over 2 weeks. RESULTS: Absence and reduced intracellular levels of 5-HT inhibited the secretion of zymogen granules both ex vivo and in vitro and altered cytoskeleton dynamics. In addition, absence of 5-HT resulted in attenuated pro-inflammatory response after induction of pancreatitis. TPH1(-/-) mice showed limited zymogen release, reduced expression of the pro-inflammatory chemokine MCP-1 and minimal leucocyte infiltration compared with wild-type animals. Restoration of 5-HT levels in TPH1(-/-) mice recovered the blunted inflammatory processes observed during acute pancreatitis. However, cellular damage, inflammatory and fibrotic processes accelerated in TPH1(-/-) mice during disease progression. CONCLUSIONS: These results identify a 5-HT-mediated regulation of zymogen secretion in pancreatic acinar cells. In addition, they demonstrate that 5-HT is required for the onset but not for the progression of pancreatic inflammation. These findings provide novel insights into the normal physiology of pancreatic acinar cells and into the pathophysiology of pancreatitis, with potential therapeutic implications.
Assuntos
Células Acinares/patologia , Amilases/metabolismo , Precursores Enzimáticos/metabolismo , Pancreatite/enzimologia , Serotonina/fisiologia , Células Acinares/enzimologia , Citoesqueleto de Actina/patologia , Animais , Linhagem Celular , Ceruletídeo/efeitos adversos , Quimiotaxia de Leucócito , Progressão da Doença , Fibrose , Imuno-Histoquímica , Inflamação/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pancreatite/induzido quimicamente , Pancreatite/patologiaRESUMO
Synthetic peptide immunogens that mimic the conformation of a target epitope of pathological relevance offer the possibility to precisely control the immune response specificity. Here, we performed conformational analyses using a panel of peptides in order to investigate the key parameters controlling their conformation upon integration into liposomal bilayers. These revealed that the peptide lipidation pattern, the lipid anchor chain length, and the liposome surface charge all significantly alter peptide conformation. Peptide aggregation could also be modulated post-liposome assembly by the addition of distinct small molecule ß-sheet breakers. Immunization of both mice and monkeys with a model liposomal vaccine containing ß-sheet aggregated lipopeptide (Palm1-15) induced polyclonal IgG antibodies that specifically recognized ß-sheet multimers over monomer or non-pathological native protein. The rational design of liposome-bound peptide immunogens with defined conformation opens up the possibility to generate vaccines against a range of protein misfolding diseases, such as Alzheimer disease.
Assuntos
Lipossomos/química , Peptídeos/química , Deficiências na Proteostase/metabolismo , Vacinas/química , Doença de Alzheimer/metabolismo , Animais , Benzotiazóis , Dicroísmo Circular , Feminino , Humanos , Imunoglobulina G/química , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Tiazóis/químicaRESUMO
The function of lymphoid organs and immune cells is often modulated by hormones, steroids and neuropeptides produced by the neuroendocrine and immune systems. The thymus intrinsically produces these factors and a comparative analysis of the expression of neuropeptides in the thymus of different species would highlight the evolutionary importance of neuroendocrine interaction in T cell development. In this review, we highlight the evidence which describes the intrathymic expression and function of various neuropeptides and their receptors, in particular somatostatin, substance P, vasointestinal polypeptide, calcitonin gene-related peptide and neuropeptide Y, in mammals (human, rodent) and non-mammals (avian, amphibian and teleost), and conclude that neuropeptides play a conserved role in vertebrate thymocyte development.
Assuntos
Neuropeptídeos/metabolismo , Sistemas Neurossecretores/imunologia , Sistemas Neurossecretores/metabolismo , Timo/metabolismo , Animais , Humanos , Timócitos/imunologia , Timócitos/metabolismo , Timócitos/fisiologia , Timo/imunologia , Vertebrados/imunologiaRESUMO
The function of lymphoid organs and immune cells is often modulated by peptides and hormones produced by the neuroendocrine and immune systems. We have previously reported the intrathymic expression of neuropeptides in the thymus of different species and that neuropeptides can influence murine thymocyte development in vitro. To further explore the evolutionary nature of neuroendocrine interactions in the thymus, we identified the expression of calcitonin-gene-related peptide, neuropeptide Y, somatostatin (SOM), substance P and vasointestinal polypeptide, as well as their receptors on chicken thymic epithelial cells (TEC) and thymocytes by immunofluorescence and reverse transcription polymerase chain reaction (RT-PCR). All the studied neuropeptides and their receptors were found to be expressed in both TEC and thymocytes, suggesting that intrathymic neuroendocrine interactions may take place within the avian thymus. In order to elucidate whether such interactions play a role in avian thymocyte development, neuropeptides and their antagonists were added to embryonic thymus organ cultures and found to influence chicken thymopoiesis. In particular, an antagonist of SOM increased the proportion of double-positive thymocytes, while SOM itself appeared to inhibit the early stages of thymocyte development. Taken together, these data provide further evidence to suggest that neuropeptides play a conserved role in vertebrate thymocyte development.
Assuntos
Galinhas/imunologia , Células Epiteliais/metabolismo , Neuropeptídeos/metabolismo , Receptores de Neuropeptídeos/metabolismo , Linfócitos T/metabolismo , Timo/metabolismo , Animais , Células Cultivadas , Embrião de Galinha , Galinhas/genética , Galinhas/metabolismo , Células Epiteliais/citologia , Técnicas de Cultura de Órgãos , Timo/citologia , Timo/embriologiaRESUMO
The public health threat posed by a looming 'post-antibiotic' era necessitates new approaches to antibiotic discovery. Drug development has typically avoided exploitation of membrane-binding properties, in contrast to nature's control of biological pathways via modulation of membrane-associated proteins and membrane lipid composition. Here, we describe the rejuvenation of the glycopeptide antibiotic vancomycin via selective targeting of bacterial membranes. Peptide libraries based on positively charged electrostatic effector sequences are ligated to N-terminal lipophilic membrane-insertive elements and then conjugated to vancomycin. These modified lipoglycopeptides, the 'vancapticins', possess enhanced membrane affinity and activity against methicillin-resistant Staphylococcus aureus (MRSA) and other Gram-positive bacteria, and retain activity against glycopeptide-resistant strains. Optimised antibiotics show in vivo efficacy in multiple models of bacterial infection. This membrane-targeting strategy has potential to 'revitalise' antibiotics that have lost effectiveness against recalcitrant bacteria, or enhance the activity of other intravenous-administered drugs that target membrane-associated receptors.
Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Daptomicina/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Vancomicina/farmacologia , Animais , Antibacterianos/metabolismo , Antibacterianos/farmacocinética , Bactérias/classificação , Sobrevivência Celular/efeitos dos fármacos , Glicopeptídeos/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Masculino , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacosRESUMO
T cells are an integral part of a functional immune system with the majority being produced in the thymus. Of all the changes related to immunosenescence, regression of the thymus is considered one of the most universally recognised alterations. Despite the reduction of thymic size, there is evidence to suggest that T cell output is still present into old age, albeit much diminished; leading to the assumption that thymocyte development is normal. However, current data suggests that recent thymic emigrant from the aged thymus are functionally less responsive, giving rise to the possibility that the generation of naïve T cell may be intrinsically impaired in the elderly. In light of these findings we discuss the evidence that suggest aged T cells may be flawed even before exiting to the periphery and could contribute to the age-associated decline in immune function.
Assuntos
Envelhecimento/fisiologia , Linfócitos T/fisiologia , Timo/citologia , Animais , Ciclo Celular , Células Cultivadas , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Humanos , Linfopoese/fisiologia , Camundongos , Linfócitos T/efeitos dos fármacosRESUMO
T-cell development is characterised by a complex series of events in the thymus, which results in the development of self-restricted immunocompetent lymphocytes. We have previously reported the expression of neuropeptides in the thymus of various species, highlighting the evolutionary importance of neuroendocrine interactions in thymocyte development. Despite the many physiological and functional similarities in their immune systems, no study has addressed the importance of neuropeptides and thymic hormones in T-cell development in Xenopus. Immunohistochemical analysis revealed that the neuropeptides substance P, neuropeptide Y, somatostatin, calcitonin gene related peptide, and vasoactive intestinal polypeptide and the thymic hormones thymosin alpha1, thymosin beta4, and thymopoietin are found in the Xenopus thymus. This was further corroborated by RT-PCR. Furthermore, double staining revealed that neuropeptides and thymic hormones are coexpressed within the epithelial cell component of the thymus. These results show that neuropeptides and thymic hormones are expressed in the thymus of Xenopus, and suggest that they are likely to play a role in T-cell development.
Assuntos
Neuropeptídeos/metabolismo , Timopoietinas/metabolismo , Timosina/metabolismo , Timo/metabolismo , Xenopus/metabolismo , AnimaisRESUMO
Age-associated thymic involution is one of the most dramatic and ubiquitous changes in the immune system, although the precise mechanisms involved still remain obscured. Several hypotheses have been proposed incorporating extrinsic and intrinsic factors, however, changes in the thymic microenvironment itself is one of the least investigated. We therefore decided to undertake a detailed histological examination of the aging thymus in order to elucidate possible mechanisms of thymic atrophy. This investigation provides insight into the changes within the murine thymus with age, demonstrating a new approach to quantify protein expressional differences while preserving the thymic architecture. There is a decline in expression of thymic epithelial cell-specific makers and an increase in fibroblast content in the aging mouse thymus. This is concurrent with a disorganization of the thymic compartments, a morphological transformation within the epithelial cells and alterations of their archetypal staining patterns. Furthermore, this is linked to a rise in apoptotic cells and the novel finding of increased senescence in the thymus of older mice that appears to be colocalized in the epithelial compartment. These changes within the thymic epithelial cells may be in part accountable for thymic atrophy and responsible for the decline in T-cell output.
Assuntos
Envelhecimento/fisiologia , Timo/citologia , Fatores Etários , Animais , Apoptose/fisiologia , Atrofia/patologia , Biomarcadores , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Timo/anatomia & histologia , Timo/fisiopatologia , Sobrevivência de TecidosRESUMO
It is now becoming apparent that the immune system undergoes age-associated alterations, which accumulate to produce a progressive deterioration in the ability to respond to infections and to develop immunity after vaccination, both of which are associated with a higher mortality rate in the elderly. Immunosenescence, defined as the changes in the immune system associated with age, has been gathering interest in the scientific and health-care sectors alike. The rise in its recognition is both pertinent and timely given the increasing average age and the corresponding failure to increase healthy life expectancy. This review attempts to highlight the age-dependent defects in the innate and adaptive immune systems. While discussing the mechanisms that contribute to immunosenescence, with emphasis on the extrinsic factors, particular attention will be focused on thymic involution. Finally, we illuminate potential therapies that could be employed to help us live a longer, fuller and healthier life.
Assuntos
Envelhecimento/imunologia , Sistema Imunitário/fisiologia , Idoso , Células-Tronco Hematopoéticas/fisiologia , Humanos , Tolerância Imunológica , Imunidade Celular/fisiologia , Imunidade Inata/fisiologiaRESUMO
Evidence suggests that the immune and neuroendocrine systems cross talk by sharing ligands and receptors. Hormones and neuropeptides produced by the neuroendocrine system often modulate the function of lymphoid organs and immune cells. We have previously reported the intrathymic expression of somatostatin (SOM) in the mouse and that several neuropeptides, most notably calcitonin-gene-related peptide (CGRP), neuropeptide Y (NPY), SOM and substance P (SP), can modulate thymocyte development. However, little is known about the intrathymic expression of these neuropeptides either in the mouse or in other species. Moreover, a comparative analysis of the expression of these molecules would highlight the evolutionary importance of intrathymic neuroendocrine interactions in T-cell development. We have studied the expression of different neuropeptides in the thymus of zebrafish, Xenopus, avians, rodent, porcine, equine and human by immunohistochemistry and reverse transcription-polymerase chain reaction. We found that CGRP, NPY, SOM, SP and vasointestinal polypeptide (VIP) are expressed in the thymus of all species investigated. The thymic location of many of these neuropeptides was conserved and appears to be within the stromal compartments. Interestingly, in the avian thymus the expression of CGRP, SOM and SP appears to change depending on the age of the tissue. These findings suggest that neuropeptides may play an important role in T-cell development and provide further evidence of cross talk between the immune and neuroendocrine systems.