RESUMO
In vivo bioluminescence imaging facilitates the non-invasive visualization of biological processes in living animals. This system has been used to track virus infections mostly in mice and ferrets; however, until now this approach has not been applied to pathogens in avian species. To visualize the infection of an important avian pathogen, we generated Marek's disease virus (MDV) recombinants expressing firefly luciferase during lytic replication. Upon characterization of the recombinant viruses in vitro, chickens were infected and the infection visualized in live animals over the course of 14 days. The luminescence signal was consistent with the known spatiotemporal kinetics of infection and the life cycle of MDV, and correlated well with the viral load measured by qPCR. Intriguingly, this in vivo bioimaging approach revealed two novel sites of MDV replication, the beak and the skin of the feet covered in scales. Feet skin infection was confirmed using a complementary fluorescence bioimaging approach with MDV recombinants expressing mRFP or GFP. Infection was detected in the intermediate epidermal layers of the feet skin that was also shown to produce infectious virus, regardless of the animals' age at and the route of infection. Taken together, this study highlights the value of in vivo whole body bioimaging in avian species by identifying previously overlooked sites of replication and shedding of MDV in the chicken host.
Assuntos
Herpesviridae , Herpesvirus Galináceo 2 , Doença de Marek , Animais , Galinhas , Furões , CamundongosRESUMO
Tricyclo-DNA (tcDNA) is a conformationally constrained oligonucleotide analog that has demonstrated great therapeutic potential as antisense oligonucleotide (ASO) for several diseases. Like most ASOs in clinical development, tcDNA were modified with phosphorothioate (PS) backbone for therapeutic purposes in order to improve their biodistribution by enhancing association with plasma and cell protein. Despite the advantageous protein binding properties, systemic delivery of PS-ASO remains limited and PS modifications can result in dose limiting toxicities in the clinic. Improving extra-hepatic delivery of ASO is highly desirable for the treatment of a variety of diseases including neuromuscular disorders such as Duchenne muscular dystrophy. We hypothesized that conjugation of palmitic acid to tcDNA could facilitate the delivery of the ASO from the bloodstream to the interstitium of the muscle tissues. We demonstrate here that palmitic acid conjugation enhances the potency of tcDNA-ASO in skeletal and cardiac muscles, leading to functional improvement in dystrophic mice with significantly reduced dose of administered ASO. Interestingly, palmitic acid-conjugated tcDNA with a full phosphodiester backbone proved effective with a particularly encouraging safety profile, offering new perspectives for the clinical development of PS-free tcDNA-ASO for neuromuscular diseases.
Assuntos
Distrofia Muscular de Duchenne/terapia , Oligonucleotídeos Antissenso/química , Ácido Palmítico/química , Animais , Terapia Genética/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Oligonucleotídeos Antissenso/efeitos adversos , Oligonucleotídeos Antissenso/farmacocinética , Distribuição TecidualRESUMO
BACKGROUND INFORMATION: Duchenne muscular dystrophy (DMD) is a neuromuscular disease caused by mutations in the gene encoding dystrophin. It leads to repeated cycles of muscle fiber necrosis and regeneration and progressive replacement of fibers by fibrotic and adipose tissue, with consequent muscle weakness and premature death. Fibrosis and, in particular, collagen accumulation are important pathological features of dystrophic muscle. A better understanding of the development of fibrosis is crucial to enable better management of DMD. Three-dimensional (3D) characterization of collagen organization by second harmonic generation (SHG) microscopy has already proven a highly informative means of studying the fibrotic network in tissue. RESULTS: Here, we combine for the first-time tissue clearing with SHG microscopy to characterize in depth the 3D cardiac fibrosis network from DMDmdx rat model. Heart sections (1-mm-thick) from 1-year-old wild-type (WT) and DMDmdx rats were cleared using the CUBIC protocol. SHG microscopy revealed significantly greater collagen deposition in DMDmdx versus WT sections. Analyses revealed a specific pattern of SHG+ segmented objects in DMDmdx cardiac muscle, characterized by a less elongated shape and increased density. Compared with the observed alignment of SHG+ collagen fibers in WT rats, profound fiber disorganization was observed in DMDmdx rats, in which we observed two distinct SHG+ collagen fiber profiles, which may reflect two distinct stages of the fibrotic process in DMD. CONCLUSION AND SIGNIFICANCE: The current work highlights the interest to combine multiphoton SHG microscopy and tissue clearing for 3D fibrosis network characterization in label free organ. It could be a relevant tool to characterize the fibrotic tissue remodeling in relation to the disease progression and/or to evaluate the efficacy of therapeutic strategies in preclinical studies in DMD model or others fibrosis-related cardiomyopathies diseases.
Assuntos
Distrofia Muscular de Duchenne , Animais , Modelos Animais de Doenças , Matriz Extracelular , Fibrose , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , RatosRESUMO
Duchenne muscular dystrophy (DMD) is a muscle wasting disorder caused by mutations in the gene encoding dystrophin. Gene therapy using micro-dystrophin (MD) transgenes and recombinant adeno-associated virus (rAAV) vectors hold great promise. To overcome the limited packaging capacity of rAAV vectors, most MD do not include dystrophin carboxy-terminal (CT) domain. Yet, the CT domain is known to recruit α1- and ß1-syntrophins and α-dystrobrevin, a part of the dystrophin-associated protein complex (DAPC), which is a signaling and structural mediator of muscle cells. In this study, we explored the impact of inclusion of the dystrophin CT domain on ΔR4-23/ΔCT MD (MD1), in DMDmdx rats, which allows for relevant evaluations at muscular and cardiac levels. We showed by LC-MS/MS that MD1 expression is sufficient to restore the interactions at a physiological level of most DAPC partners in skeletal and cardiac muscles, and that inclusion of the CT domain increases the recruitment of some DAPC partners at supra-physiological levels. In parallel, we demonstrated that inclusion of the CT domain does not improve MD1 therapeutic efficacy on DMD muscle and cardiac pathologies. Our work highlights new evidences of the therapeutic potential of MD1 and strengthens the relevance of this candidate for gene therapy of DMD.
Assuntos
Distrofina , Distrofia Muscular de Duchenne , Animais , Cromatografia Líquida , Distrofina/genética , Distrofina/metabolismo , Complexo de Proteínas Associadas Distrofina/metabolismo , Terapia Genética , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Ratos , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked inherited disease caused by mutations in the gene encoding dystrophin that leads to a severe and ultimately life limiting muscle-wasting condition. Recombinant adeno-associated vector (rAAV)-based gene therapy is promising, but the size of the full-length dystrophin cDNA exceeds the packaging capacity of a rAAV. Alternative or complementary strategies that could treat DMD patients are thus needed. Intracellular calcium overload due to a sarcolemma permeability to calcium (SPCa) increase is an early and critical step of the DMD pathogenesis. We assessed herein whether TRPC1 and TRPC3 calcium channels may be involved in skeletal muscle SPCa alterations and could represent therapeutic targets to treat DMD. METHODS: All experiments were conducted in the DMDmdx rat, an animal model that closely reproduces the human DMD disease. We measured the cytosolic calcium concentration ([Ca2+]c) and SPCa in EDL (Extensor Digitorum Longus) muscle fibers from age-matched WT and DMDmdx rats of 1.5 to 7 months old. TRPC1 and TRPC3 expressions were measured in the EDL muscles at both the mRNA and protein levels, by RT-qPCR, western blot and immunocytofluorescence analysis. RESULTS: As expected from the malignant hyperthermia like episodes observed in several DMDmdx rats, calcium homeostasis alterations were confirmed by measurements of early increases in [Ca2+]c and SPCa in muscle fibers. TRPC3 and TRPC1 protein levels were increased in DMDmdx rats. This was observed as soon as 1.5 months of age for TRPC3 but only at 7 months of age for TRPC1. A slight but reliable shift of the TRPC3 apparent molecular weight was observed in DMDmdx rat muscles. Intracellular localization of both channels was not altered. We thus focused our attention on TRPC3. Application of Pyr10, a specific inhibitor of TRPC3, abolished the differences between SPCa values measured in WT and DMDmdx. Finally, we showed that a rAAV-microdystrophin based treatment induced a high microdystrophin expression but only partial prevention of calcium homeostasis alterations, skeletal muscle force and TRPC3 protein increase. CONCLUSIONS: All together our results show that correcting TRPC3 channel expression and/or activity appear to be a promising approach as a single or as a rAAV-based complementary therapy to treat DMD.
Assuntos
Distrofia Muscular de Duchenne , Animais , Terapia Genética/métodos , Humanos , Camundongos , Camundongos Endogâmicos mdx , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/terapia , RatosRESUMO
The intrinsic necrosis of skeletal muscles in animal models of Duchenne muscular dystrophy (DMD) damages neuromuscular junctions (NMJs) with progressively altered NMJs associated with denervation and premature changes in dystrophic nerves. In the mdx mouse model of DMD, the proteins S100ß and Tau5 are significantly increased in sciatic nerves by 13 months (M) of age, far earlier (by 9 M) than in normal wildtype (WT) nerves. Since dystrophic Dmdmdx rats are reported to have a more severe dystropathology than mdx mice, we hypothesised that Dmdmdx rat nerves would show earlier neuronal changes compared with mdx nerves. We quantified levels of 8 proteins (by immunoblotting) in sciatic and radial nerves from young adult Dmdmdx rats (aged 8 M) and mdx mice (9 M), plus levels of 7 mRNAs (by qPCR) in rat nerves only. Sciatic nerves of 8 M Dmdmdx rats had more consistently increased levels of S100ß and Tau5 proteins, compared with 9 M mdx mice, supporting pronounced dystropathology in the rat model. There were no differences for mRNA levels, apart from higher gelsolin mRNA in Dmdmdx sciatic nerves. The pronounced protein changes in Dmdmdx nerves indicate a severe ongoing myonecrosis, and likely consequent myofibre denervation, for the dystrophic rat model. These data support increased neuronal proteins in dystrophic nerves as a novel pre-clinical readout of ongoing myonecrosis for DMD research. In older DMD boys, such progressive neuronal changes over many years are likely to contribute to loss of muscle function, and may complicate evaluation of late-onset clinical therapies.
Assuntos
Distrofina/genética , Distrofia Muscular de Duchenne/genética , Neurônios/patologia , Fenótipo , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Proteínas tau/metabolismo , Animais , Masculino , Camundongos , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Mutação , Neurônios/metabolismo , Ratos , Subunidade beta da Proteína Ligante de Cálcio S100/genética , Especificidade da Espécie , Proteínas tau/genéticaRESUMO
Respiratory syncytial virus (RSV) is the main cause of severe respiratory infection in young children worldwide, and no therapies have been approved for the treatment of RSV infection. Data from recent clinical trials of fusion or L polymerase inhibitors for the treatment of RSV-infected patients revealed the emergence of escape mutants, highlighting the need for the discovery of inhibitors with novel mechanisms of action. Here we describe stapled peptides derived from the N terminus of the phosphoprotein (P) that act as replication inhibitors. We demonstrate that these peptides inhibit RSV replication in vitro and in vivo by preventing the formation of the N0-P complex. The present strategy provides a novel means of targeting RSV replication with constrained macrocyclic peptides or small molecules and is broadly applicable to other viruses of the Mononegavirales order.
Assuntos
Antivirais , Peptídeos , Conformação Proteica em alfa-Hélice , Vírus Sincicial Respiratório Humano , Animais , Antivirais/farmacologia , Humanos , Camundongos , Peptídeos/farmacologia , Fosfoproteínas/farmacologia , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Replicação ViralRESUMO
Triple-negative breast cancer (TNBC) represents 10% of all breast cancers and is a very heterogeneous disease. Globally, women with TNBC have a poor prognosis, and the development of effective targeted therapies remains a real challenge. Patient-derived xenografts (PDX) are clinically relevant models that have emerged as important tools for the analysis of drug activity and predictive biomarker discovery. The purpose of this work was to analyze the molecular heterogeneity of a large panel of TNBC PDX (n = 61) in order to test targeted therapies and identify biomarkers of response. At the gene expression level, TNBC PDX represent all of the various TNBC subtypes identified by the Lehmann classification except for immunomodulatory subtype, which is underrepresented in PDX. NGS and copy number data showed a similar diversity of significantly mutated gene and somatic copy number alteration in PDX and the Cancer Genome Atlas TNBC patients. The genes most commonly altered were TP53 and oncogenes and tumor suppressors of the PI3K/AKT/mTOR and MAPK pathways. PDX showed similar morphology and immunohistochemistry markers to those of the original tumors. Efficacy experiments with PI3K and MAPK inhibitor monotherapy or combination therapy showed an antitumor activity in PDX carrying genomic mutations of PIK3CA and NRAS genes. TNBC PDX reproduce the molecular heterogeneity of TNBC patients. This large collection of PDX is a clinically relevant platform for drug testing, biomarker discovery and translational research.
Assuntos
Dosagem de Genes , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias de Mama Triplo Negativas/genética , Animais , Classe I de Fosfatidilinositol 3-Quinases/genética , Feminino , GTP Fosfo-Hidrolases/genética , Regulação Neoplásica da Expressão Gênica , Heterogeneidade Genética , Humanos , Proteínas de Membrana/genética , Camundongos , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Transplante de Neoplasias , Medicina de Precisão , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Proteína Supressora de Tumor p53/genéticaRESUMO
BACKGROUND: Non-structural protein NS1 of influenza A viruses harbours several determinants of pathogenicity and host-range. However it is still unclear to what extent each of its two structured domains (i.e. RNA-binding domain, RBD, and effector domain, ED) contribute to its various activities. METHODS: To evaluate the respective contributions of the two domains, we genetically engineered two variants of an H7N1 low pathogenicity avian influenza virus harbouring amino-acid substitutions that impair the functionality of either domain. The RBD- and ED-mutant viruses were compared to their wt- counterpart in vivo and in vitro, notably in chicken infection and avian cell culture models. RESULTS: The double substitution R38A-K41A in the RBD dramatically reduced the pathogenicity and replication potential of the virus, whereas the substitution A149V that was considered to abrogate the IFN-antagonistic activity of the effector domain entailed much less effects. While all three viruses initiated the viral life cycle in avian cells, replication of the R38A-K41A virus was severely impaired. This defect was associated with a delayed synthesis of nucleoprotein NP and a reduced accumulation of NS1, which was found to reach a concentration of about 30 micromol.L- 1 in wt-infected cells at 8 h post-infection. When overexpressed in avian lung epithelial cells, both the wt-NS1 and 3841AA-NS1, but not the A149V-NS1, reduced the poly(I:C)-induced activation of the IFN-sensitive chicken Mx promoter. Unexpectedly, the R38A-K41A substitution in the recombinant RBD did not alter its in vitro affinity for a model dsRNA. When overexpressed in avian cells, both the wt- and A149V-NS1s, as well as the individually expressed wt-RBD to a lesser extent, enhanced the activity of the reconstituted viral RNA-polymerase in a minireplicon assay. CONCLUSIONS: Collectively, our data emphasized the critical importance and essential role of the RNA-binding domain in essential steps of the virus replication cycle, notably expression and translation of viral mRNAs.
Assuntos
Vírus da Influenza A Subtipo H7N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H7N1/patogenicidade , Influenza Aviária/virologia , Motivos de Ligação ao RNA/fisiologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/fisiologia , Substituição de Aminoácidos , Animais , Linhagem Celular , Embrião de Galinha , Galinhas , Modelos Animais de Doenças , Cães , Expressão Gênica , Regulação Viral da Expressão Gênica , Vírus da Influenza A Subtipo H7N1/genética , Células Madin Darby de Rim Canino , Motivos de Ligação ao RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas Virais/biossíntese , Virulência/genéticaRESUMO
Marek's disease is a multi-faceted highly contagious disease affecting chickens caused by the Marek's disease alphaherpesvirus (MDV). MDV early infection induces a transient immunosuppression, which is associated with thymus and bursa of Fabricius atrophy. Little is known about the cellular processes involved in primary lymphoid organ atrophy. Here, by in situ TUNEL assay, we demonstrate that MDV infection results in a high level of apoptosis in the thymus and bursa of Fabricius, which is concomitant to the MDV lytic cycle. Interestingly, we observed that in the thymus most of the MDV infected cells at 6 days post-infection (dpi) were apoptotic, whereas in the bursa of Fabricius most of the apoptotic cells were uninfected suggesting that MDV triggers apoptosis by two different modes in these two primary lymphoid organs. In addition, a high decrease of cell proliferation was observed from 6 to 14 dpi in the bursa of Fabricius follicles, and not in the thymus. Finally, with an adapted absolute blood lymphocyte count, we demonstrate a major B-lymphopenia during the two 1st weeks of infection, and propose this method as a potent non-invasive tool to diagnose MDV bursa of Fabricius infection and atrophy. Our results demonstrate that the thymus and bursa of Fabricius atrophies are related to different cell mechanisms, with different temporalities, that affect infected and uninfected cells.
Assuntos
Atrofia/veterinária , Galinhas , Herpesvirus Galináceo 2/fisiologia , Tecido Linfoide/patologia , Doença de Marek/fisiopatologia , Doenças das Aves Domésticas/fisiopatologia , Animais , Apoptose , Atrofia/patologia , Atrofia/fisiopatologia , Atrofia/virologia , Proliferação de Células , Tecido Linfoide/fisiopatologia , Linfopenia , Doença de Marek/patologia , Doença de Marek/virologia , Doenças das Aves Domésticas/patologia , Doenças das Aves Domésticas/virologiaRESUMO
Synthetic peptides derived from the heptad repeat (HR) of fusion (F) proteins can be used as dominant negative inhibitors to inhibit the fusion mechanism of class I viral F proteins. Here, we have performed a stapled-peptide scan across the HR2 domain of the respiratory syncytial virus (RSV) F protein with the aim to identify a minimal domain capable of disrupting the formation of the postfusion six-helix bundle required for viral cell entry. Constraining the peptides with a single staple was not sufficient to inhibit RSV infection. However, the insertion of double staples led to the identification of novel short stapled peptides that display nanomolar potency in HEp-2 cells and are exceptionally robust to proteolytic degradation. By replacing each amino acid of the peptides by an alanine, we found that the substitution of residues 506 to 509, located in a patch of polar contacts between HR2 and HR1, severely affected inhibition. Finally, we show that intranasal delivery of the most potent peptide to BALB/c mice significantly decreased RSV infection in upper and lower respiratory tracts. The discovery of this minimal HR2 sequence as a means for inhibition of RSV infection provides the basis for further medicinal chemistry efforts toward developing RSV fusion antivirals.
Assuntos
Antivirais/farmacologia , Peptídeos/farmacologia , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Proteínas Virais de Fusão/química , Internalização do Vírus/efeitos dos fármacos , Administração Intranasal , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Antivirais/síntese química , Sítios de Ligação , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/síntese química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteólise , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/química , Vírus Sincicial Respiratório Humano/crescimento & desenvolvimento , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Replicação Viral/efeitos dos fármacosRESUMO
Proteomic profiling plays a decisive role in the elucidation of molecular signatures representative of a specific clinical context. MuStem cell based therapy represents a promising approach for clinical applications to cure Duchenne muscular dystrophy (DMD). To expand our previous studies collected in the clinically relevant DMD animal model, we decided to investigate the skeletal muscle proteome 4 months after systemic delivery of allogenic MuStem cells. Quantitative proteomics with isotope-coded protein labeling was used to compile quantitative changes in the protein expression profiles of muscle in transplanted Golden Retriever muscular dystrophy (GRMD) dogs as compared to Golden Retriever muscular dystrophy dogs. A total of 492 proteins were quantified, including 25 that were overrepresented and 46 that were underrepresented after MuStem cell transplantation. Interestingly, this study demonstrates that somatic stem cell therapy impacts on the structural integrity of the muscle fascicle by acting on fibers and its connections with the extracellular matrix. We also show that cell infusion promotes protective mechanisms against oxidative stress and favors the initial phase of muscle repair. This study allows us to identify putative candidates for tissue markers that might be of great value in objectively exploring the clinical benefits resulting from our cell-based therapy for DMD. All MS data have been deposited in the ProteomeXchange with identifier PXD001768 (http://proteomecentral.proteomexchange.org/dataset/PXD001768).
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Musculares/transplante , Distrofia Muscular Animal/terapia , Proteoma/genética , Transplante de Células-Tronco , Células-Tronco/metabolismo , Animais , Cães , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Internet , Anotação de Sequência Molecular , Células Musculares/citologia , Células Musculares/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patologia , Estresse Oxidativo , Proteoma/metabolismo , Proteômica/métodos , Software , Células-Tronco/citologia , Resultado do TratamentoRESUMO
BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked muscle disease that leads to fibre necrosis and progressive paralysis. At present, DMD remains a lethal disease without any effective treatment, requiring a better understanding of the pathophysiological processes and comprehensive assessment of the newly identified therapeutic strategies. MicroRNAs including members of the muscle-specific myomiR family have been identified as being deregulated in muscle of DMD patients and in mdx mice used as a model for DMD. In recent years, the Golden Retriever muscular dystrophy (GRMD) dog has appeared as the crucial animal model for objectively assessing the potential of new innovative approaches. Here, we first aim at establishing the muscle expression pattern of five selected miRNAs in this clinically relevant model to determine if they are similarly affected compared with other DMD contexts. Second, we attempt to show whether these miRNAs could be impacted by the systemic delivery of a promising stem cell candidate (referred to as MuStem cells) to implement our knowledge on its mode of action and/or identify markers associated with cell therapy efficacy. METHODS: A comparative study of miRNAs expression levels and cellular localization was performed on 9-month-old healthy dogs, as well as on three sub-sets of GRMD dog (without immunosuppression or cell transplantation, with continuous immunosuppressive regimen and with MuStem cell transplantation under immunosuppression), using RT-qPCR and in situ hybridization. RESULTS: We find that miR-222 expression is markedly up-regulated in GRMD dog muscle compared to healthy dog, while miR-486 tends to be down-expressed. Intriguingly, the expression of miR-1, miR-133a and miR-206 does not change. In situ hybridization exploration reveals, for the first time, that miR-486 and miR-206 are mainly localized in newly regenerated fibres in GRMD dog muscle. In addition, we show that cyclosporine-based immunosuppression, classically used in allogeneic cell transplantation, exclusively impacts the miR-206 expression. Finally, we demonstrate that intra-arterial administration of MuStem cells results in up-regulation of miR-133a and miR-222 concomitantly with a down-expression of two sarcomeric proteins corresponding to miR-222 targets. CONCLUSION: We point out a differential muscle expression of miR-222 and miR-486 associated with the pathophysiology of the clinically relevant GRMD dog model with a tissue localization focused on regenerated fibres. We also establish a modified expression of miR-133a and miR-222 subsequent to MuStem cell infusion.
Assuntos
MicroRNAs/metabolismo , Células Musculares/transplante , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/terapia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Transplante de Células-Tronco/métodos , Animais , Biomarcadores/metabolismo , Ciclosporina/farmacologia , Ciclosporina/uso terapêutico , Modelos Animais de Doenças , Cães , Regulação para Baixo , Imunofluorescência , Humanos , Terapia de Imunossupressão/métodos , Imunossupressores/farmacologia , Imunossupressores/uso terapêutico , Hibridização In Situ , Injeções Intra-Arteriais , Camundongos , Camundongos Endogâmicos mdx , MicroRNAs/efeitos dos fármacos , Células Musculares/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular Animal/patologia , Distrofia Muscular de Duchenne/patologia , Cadeias Pesadas de Miosina/metabolismo , Células-Tronco/metabolismo , Regulação para CimaRESUMO
UNLABELLED: Chikungunya virus (CHIKV) is a member of a globally distributed group of arthritogenic alphaviruses that cause weeks to months of debilitating polyarthritis/arthralgia, which is often poorly managed with current treatments. Arthritic disease is usually characterized by high levels of the chemokine CCL2 and a prodigious monocyte/macrophage infiltrate. Several inhibitors of CCL2 and its receptor CCR2 are in development and may find application for treatment of certain inflammatory conditions, including autoimmune and viral arthritides. Here we used CCR2(-/-) mice to determine the effect of CCR2 deficiency on CHIKV infection and arthritis. Although there were no significant changes in viral load or RNA persistence and only marginal changes in antiviral immunity, arthritic disease was substantially increased and prolonged in CCR2(-/-) mice compared to wild-type mice. The monocyte/macrophage infiltrate was replaced in CCR2(-/-) mice by a severe neutrophil (followed by an eosinophil) infiltrate and was associated with changes in the expression levels of multiple inflammatory mediators (including CXCL1, CXCL2, granulocyte colony-stimulating factor [G-CSF], interleukin-1ß [IL-1ß], and IL-10). The loss of anti-inflammatory macrophages and their activities (e.g., efferocytosis) was also implicated in exacerbated inflammation. Clear evidence of cartilage damage was also seen in CHIKV-infected CCR2(-/-) mice, a feature not normally associated with alphaviral arthritides. Although recruitment of CCR2(+) monocytes/macrophages can contribute to inflammation, it also appears to be critical for preventing excessive pathology and resolving inflammation following alphavirus infection. Caution might thus be warranted when considering therapeutic targeting of CCR2/CCL2 for the treatment of alphaviral arthritides. IMPORTANCE: Here we describe the first analysis of viral arthritis in mice deficient for the chemokine receptor CCR2. CCR2 is thought to be central to the monocyte/macrophage-dominated inflammatory arthritic infiltrates seen after infection with arthritogenic alphaviruses such as chikungunya virus. Surprisingly, the viral arthritis caused by chikungunya virus in CCR2-deficient mice was more severe, prolonged, and erosive and was neutrophil dominated, with viral replication and persistence not being significantly affected. Monocytes/macrophages recruited by CCL2 thus also appear to be important for both preventing even worse pathology mediated by neutrophils and promoting resolution of inflammation. Caution might thus be warranted when considering the use of therapeutic agents that target CCR2/CCL2 or inflammatory monocytes/macrophages for the treatment of alphaviral (and perhaps other viral) arthritides. Individuals with diminished CCR2 responses (due to drug treatment or other reasons) may also be at risk of exacerbated arthritic disease following alphaviral infection.
Assuntos
Infecções por Alphavirus/imunologia , Artrite/imunologia , Vírus Chikungunya/fisiologia , Neutrófilos/imunologia , Receptores CCR2/deficiência , Infecções por Alphavirus/genética , Infecções por Alphavirus/virologia , Animais , Artrite/genética , Artrite/virologia , Febre de Chikungunya , Vírus Chikungunya/genética , Fator Estimulador de Colônias de Granulócitos/imunologia , Humanos , Interleucina-10/imunologia , Interleucina-1beta/imunologia , Camundongos , Camundongos Knockout , Infiltração de Neutrófilos , Receptores CCR2/genética , Receptores CCR2/imunologiaRESUMO
Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disorder caused by mutations in the dystrophin gene, without curative treatment yet available. Our study provides, for the first time, the overall safety profile and therapeutic dose of a recombinant adeno-associated virus vector, serotype 8 (rAAV8) carrying a modified U7snRNA sequence promoting exon skipping to restore a functional in-frame dystrophin transcript, and injected by locoregional transvenous perfusion of the forelimb. Eighteen Golden Retriever Muscular Dystrophy (GRMD) dogs were exposed to increasing doses of GMP-manufactured vector. Treatment was well tolerated in all, and no acute nor delayed adverse effect, including systemic and immune toxicity was detected. There was a dose relationship for the amount of exon skipping with up to 80% of myofibers expressing dystrophin at the highest dose. Similarly, histological, nuclear magnetic resonance pathological indices and strength improvement responded in a dose-dependent manner. The systematic comparison of effects using different independent methods, allowed to define a minimum threshold of dystrophin expressing fibers (>33% for structural measures and >40% for strength) under which there was no clear-cut therapeutic effect. Altogether, these results support the concept of a phase 1/2 trial of locoregional delivery into upper limbs of nonambulatory DMD patients.
Assuntos
Dependovirus/genética , Distrofina/genética , Membro Anterior/fisiopatologia , Distrofia Muscular de Duchenne/terapia , RNA Nuclear Pequeno/genética , Animais , Estudos de Coortes , Modelos Animais de Doenças , Cães , Relação Dose-Resposta a Droga , Éxons , Terapia Genética , Vetores Genéticos/administração & dosagem , Humanos , Infusões Intravenosas , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/fisiopatologia , RNA Nuclear Pequeno/metabolismoRESUMO
Currently circulating H5N1 influenza viruses have undergone a complex evolution since the appearance of their progenitor A/Goose/Guangdong/1/96 in 1996. After the eradication of the H5N1 viruses that emerged in Hong Kong in 1997 (HK/97 viruses), new genotypes of H5N1 viruses emerged in the same region in 2000 that were more pathogenic for both chickens and mice than HK/97 viruses. These, as well as virtually all highly pathogenic H5N1 viruses since 2000, harbour a deletion of aa 80-84 in the unstructured region of the non-structural (NS) protein NS1 linking its RNA-binding domain to its effector domain. NS segments harbouring this mutation have since been found in non-H5N1 viruses and we asked whether this 5 aa deletion could have a general effect not limited to the NS1 of H5N1 viruses. We genetically engineered this deletion in the NS segment of a duck-origin avian H1N1 virus, and compared the in vivo and in vitro properties of the WT and NSdel8084 viruses. In experimentally infected chickens, the NSdel8084 virus showed both an increased replication potential and an increased pathogenicity. This in vivo phenotype was correlated with a higher replicative efficiency in vitro, both in embryonated eggs and in a chicken lung epithelial cell line. Our data demonstrated that the increased replicative potential conferred by this small deletion was a general feature not restricted to NS1 from H5N1 viruses and suggested that viruses acquiring this mutation may be selected positively in the future.
Assuntos
Vírus da Influenza A Subtipo H1N1/fisiologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Embrião de Galinha , Galinhas , Citocinas/genética , DNA Viral/genética , Vírus da Influenza A Subtipo H1N1/genética , Virus da Influenza A Subtipo H5N1/química , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/fisiologia , Influenza Aviária/imunologia , Influenza Aviária/patologia , Influenza Aviária/virologia , Interferon Tipo I/biossíntese , Pulmão/patologia , Pulmão/virologia , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Deleção de Sequência , Especificidade da Espécie , Carga Viral , Proteínas não Estruturais Virais/genética , Virulência/genética , Virulência/fisiologia , Replicação Viral/genética , Replicação Viral/fisiologiaRESUMO
Introduction: Global microplastic (MP) pollution is now well recognized, with humans and animals consuming and inhaling MPs on a daily basis, with a growing body of concern surrounding the potential impacts on human health. Methods: Using a mouse model of mild COVID-19, we describe herein the effects of azide-free 1 µm polystyrene MP beads, co-delivered into lungs with a SARS-CoV-2 omicron BA.5 inoculum. The effect of MPs on the host response to SARS-CoV-2 infection was analysed using histopathology and RNA-Seq at 2 and 6 days post-infection (dpi). Results: Although infection reduced clearance of MPs from the lung, virus titres and viral RNA levels were not significantly affected by MPs, and overt MP-associated clinical or histopathological changes were not observed. However, RNA-Seq of infected lungs revealed that MP exposure suppressed innate immune responses at 2 dpi and increased pro-inflammatory signatures at 6 dpi. The cytokine profile at 6 dpi showed a significant correlation with the 'cytokine release syndrome' signature observed in some COVID-19 patients. Discussion: The findings are consistent with the recent finding that MPs can inhibit phagocytosis of apoptotic cells via binding of Tim4. They also add to a growing body of literature suggesting that MPs can dysregulate inflammatory processes in specific disease settings.
Assuntos
COVID-19 , Modelos Animais de Doenças , Imunidade Inata , Pulmão , Microplásticos , SARS-CoV-2 , Animais , COVID-19/imunologia , COVID-19/virologia , Imunidade Inata/efeitos dos fármacos , SARS-CoV-2/imunologia , SARS-CoV-2/fisiologia , Camundongos , Pulmão/imunologia , Pulmão/virologia , Pulmão/patologia , Citocinas/metabolismo , Humanos , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Feminino , Síndrome da Liberação de Citocina/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Betacoronavirus/imunologia , PandemiasRESUMO
Getah virus (GETV) is an emerging mosquito-borne virus with economic impact on the livestock industry in East Asia. In this study, we successfully produced GETV virus-like particles (VLPs) in insect cells using the baculovirus expression vector system. We show that the GETV envelope glycoproteins were successfully expressed at the surface of the insect cell and were glycosylated. VLPs were isolated from the culture fluid as enveloped particles of 60-80 nm in diameter. Two 1 µg vaccinations with this GETV VLP vaccine, without adjuvant, generated neutralizing antibody responses and protected wild-type C57/BL6 mice against GETV viremia and arthritic disease. The GETV VLP vaccine may find application as a horse and/or pig vaccine in the future.
RESUMO
We previously reported that human muscle-derived stem cells (hMuStem cells) contribute to tissue repair after local administration into injured skeletal muscle or infarcted heart in immunodeficient rodent models. However, extrapolation of these findings to a clinical context is problematic owing to the considerable differences often seen between in vivo findings in humans versus rodents. Therefore, we investigated whether the muscle regenerative behavior of hMuStem cells is maintained in a clinically relevant transplantation context. Human MuStem cells were intramuscularly administered by high-density microinjection matrices into nonhuman primates receiving tacrolimus-based immunosuppression thereby reproducing the protocol that has so far produced the best results in clinical trials of cell therapy in myopathies. Four and 9 weeks after administration, histological analysis of cell injection sites revealed large numbers of hMuStem cell-derived nuclei in all cases. Most graft-derived nuclei were distributed in small myofiber groups in which no signs of a specific immune response were observed. Importantly, hMuStem cells contributed to simian tissue repair by fusing mainly with host myofibers, demonstrating their capacity for myofiber regeneration in this model. Together, these findings obtained in a valid preclinical model provide new insights supporting the potential of hMuStem cells in future cell therapies for muscle diseases.
Assuntos
Estudo de Prova de Conceito , Animais , Humanos , Fibras Musculares Esqueléticas/fisiologia , Transplante de Células-Tronco/métodos , Músculo Esquelético/fisiologia , Masculino , Fusão Celular , FemininoRESUMO
Decades of biological and clinical research have led to important advances in recombinant adeno-associated viruses rAAV-based gene therapy gene therapy. However, several challenges must be overcome to fully exploit the potential of rAAV vectors. Innovative approaches to modify viral genome and capsid elements have been used to overcome issues such as unwanted immune responses and off-targeting. While often successful, genetic modification of capsids can drastically reduce vector yield and often fails to produce vectors with properties that translate across different animal species, such as rodents, non-human primates, and humans. Here, we describe a chemical bioconjugation strategy to modify tyrosine residues on AAV capsids using specific ligands, thereby circumventing the need to genetically engineer the capsid sequence. Aromatic electrophilic substitution of the phenol ring of tyrosine residues on AAV capsids improved the in vivo transduction efficiency of rAAV2 vectors in both liver and retinal targets. This tyrosine bioconjugation strategy represents an innovative technology for the engineering of rAAV vectors for human gene therapy.