Label-free 3D characterization of cardiac fibrosis in muscular dystrophy using SHG imaging of cleared tissue.

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.

Evaluation of the dystrophin carboxy-terminal domain for micro-dystrophin gene therapy in cardiac and skeletal muscles in the DMDmdx rat model.

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.

TRPC3, but not TRPC1, as a good therapeutic target for standalone or complementary treatment of DMD.

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.

Skeletal Muscle Regenerative Potential of Human MuStem Cells following Transplantation into Injured Mice Muscle.

After intra-arterial delivery in the dystrophic dog, allogeneic muscle-derived stem cells, termed MuStem cells, contribute to long-term stabilization of the clinical status and preservation of the muscle regenerative process. However, it remains unknown whether the human counterpart could be identified, considering recent demonstrations of divergent features between species for several somatic stem cells. Here, we report that MuStem cells reside in human skeletal muscle and display a long-term ability to proliferate, allowing generation of a clinically relevant amount of cells. Cultured human MuStem (hMuStem) cells do not express hematopoietic, endothelial, or myo-endothelial cell markers and reproducibly correspond to a population of early myogenic-committed progenitors with a perivascular/mesenchymal phenotypic signature, revealing a blood vessel wall origin. Importantly, they exhibit both myogenesis in vitro and skeletal muscle regeneration after intramuscular delivery into immunodeficient host mice. Together, our findings provide new insights supporting the notion that hMuStem cells could represent an interesting therapeutic candidate for dystrophic patients.

Major contribution of the RNA-binding domain of NS1 in the pathogenicity and replication potential of an avian H7N1 influenza virus in chickens.

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.

Quantitative proteome profiling of dystrophic dog skeletal muscle reveals a stabilized muscular architecture and protection against oxidative stress after systemic delivery of MuStem cells.

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).

Identification in GRMD dog muscle of critical miRNAs involved in pathophysiology and effects associated with MuStem cell transplantation.

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.

Shortening the unstructured, interdomain region of the non-structural protein NS1 of an avian H1N1 influenza virus increases its replication and pathogenicity in chickens.

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.

Novel chemical tyrosine functionalization of adeno-associated virus improves gene transfer efficiency in liver and retina.

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.

Deletion of the C-terminal ESEV domain of NS1 does not affect the replication of a low-pathogenic avian influenza virus H7N1 in ducks and chickens.

Highly pathogenic avian influenza (HPAI) H7N1 viruses caused a series of epizootics in Italy between 1999 and 2001. The emergence of these HPAI viruses coincided with the deletion of the six amino acids R(225)VESEV(230) at the C terminus of NS1. In order to assess how the truncation of NS1 affected virus replication, we used reverse genetics to generate a wild-type low-pathogenic avian influenza (LPAI) H7N1 virus with a 230aa NS1 (H7N1(230)) and a mutant virus with a truncated NS1 (H7N1(224)). The 6aa truncation had no impact on virus replication in duck or chicken cells in vitro. The H7N1(230) and H7N1(224) viruses also replicated to similar levels and induced similar immune responses in ducks or chickens. No significant histological lesions were detected in infected ducks, regardless of the virus inoculated. However, in chickens, the H7N1(230) virus induced a more severe interstitial pneumonia than did the H7N1(224) virus. These findings indicate that the C-terminal extremity of NS1, including the PDZ-binding motif ESEV, is dispensable for efficient replication of an LPAI virus in ducks and chickens, even though it may increase virulence in chickens, as revealed by the intensity of the histological lesions.

Specific and label-free endogenous signature of dystrophic muscle by Synchrotron deep ultraviolet radiation.

Dystrophic muscle is characterized by necrosis/regeneration cycles, inflammation, and fibro-adipogenic development. Conventional histological stainings provide essential topographical data of this remodeling but may be limited to discriminate closely related pathophysiological contexts. They fail to mention microarchitecture changes linked to the nature and spatial distribution of tissue compartment components. We investigated whether label-free tissue autofluorescence revealed by Synchrotron deep ultraviolet (DUV) radiation could serve as an additional tool for monitoring dystrophic muscle remodeling. Using widefield microscopy with specific emission fluorescence filters and microspectroscopy defined by high spectral resolution, we analyzed samples from healthy dogs and two groups of dystrophic dogs: naïve (severely affected) and MuStem cell-transplanted (clinically stabilized) animals. Multivariate statistical analysis and machine learning approaches demonstrated that autofluorescence emitted at 420-480 nm by the Biceps femoris muscle effectively discriminates between healthy, dystrophic, and transplanted dog samples. Microspectroscopy showed that dystrophic dog muscle displays higher and lower autofluorescence due to collagen cross-linking and NADH respectively than that of healthy and transplanted dogs, defining biomarkers to evaluate the impact of cell transplantation. Our findings demonstrate that DUV radiation is a sensitive, label-free method to assess the histopathological status of dystrophic muscle using small amounts of tissue, with potential applications in regenerative medicine.

Cytidine deaminase deficiency in mice enhances genetic instability but limits the number of chemically induced colon tumors.

Cytidine deaminase (CDA) catalyzes the deamination of cytidine (C) and deoxycytidine (dC) to uridine and deoxyuridine, respectively. We recently showed that CDA deficiency leads to genomic instability, a hallmark of cancers. We therefore investigated whether constitutive CDA inactivation conferred a predisposition to cancer development. We developed a novel mouse model of Cda deficiency by generating Cda-knockout mice. Cda+/+ and Cda-/- mice did not differ in lifetime phenotypic or behavioral characteristics, or in the frequency or type of spontaneous cancers. However, the frequency of chemically induced tumors in the colon was significantly lower in Cda-/- mice. An analysis of primary kidney cells from Cda-/- mice revealed an excess of C and dC associated with significantly higher frequencies of sister chromatid exchange and ultrafine anaphase bridges and lower Parp-1 activity than in Cda+/+ cells. Our results suggest that, despite inducing genetic instability, an absence of Cda limits the number of chemically induced tumors. These results raise questions about whether a decrease in basal Parp-1 activity can protect against inflammation-driven tumorigenesis; we discuss our findings in light of published data for the Parp-1-deficient mouse model.

The caecal microbiota promotes the acute inflammatory response and the loss of the intestinal barrier integrity during severe Eimeria tenella infection.

Introduction: Coccidiosis, a disease caused by intestinal apicomplexan parasites Eimeria, is a threat to poultry production. Eimeria tenella is one of the most pathogenic species, frequently causing a high prevalence of opportunistic infections. Objective: The objective of this study is to investigate the role of the microbiota in the pathogenesis of severe Eimeria tenella infection. Methods: We have previously shown that microbiota can promote parasite development. To study the effect of the microbiota on the pathogenesis of this infection, we used an experimental condition (inoculum of 10 000 oocysts E. tenella INRAE) in which the parasite load is similar between germ-free and conventional broilers at 7 days post-infection (pi). Thirteen conventional and 24 germ-free chickens were infected. Among this latter group, 12 remained germ-free and 12 received a microbiota from conventional healthy chickens at 4 days pi. Caeca and spleens were collected at 7 days pi. Results: Our results demonstrated caecal lesions and epithelium damage in conventional chickens at 7 days pi but not in germ-free infected chickens. Administration of conventional microbiota to germ-free chickens partially restored these deleterious effects. At day 7 pi, both infected conventional and germ-free chickens exhibited increased gene expression of inflammatory mediators, including IL15, IFNγ, TNFα and the anti-inflammatory mediator SOCS1, whereas the inflammatory mediators CXCLi2, CCL20, IL18, CSF1, NOS2, PTGS2, IL1ß, IL6, the receptor CCR2, and the anti-inflammatory mediators TGFß1 and IL10 were upregulated only in infected conventional chickens. Notably, the IL18, PTGS2 gene expression was significantly higher in the infected conventional group. Overall, the inflammatory response enhanced by the microbiota might be in part responsible for higher lesion scores. Epithelial tight junction protein gene expression analysis revealed a significant upregulation of CLDN1 with the infection and microbiota, indicating a potential loss of the intestinal barrier integrity. Conclusion: These observations imply that, during E. tenella infection, the caecal microbiota could trigger an acute inflammatory response, resulting in a loss of intestinal integrity. Increase in bacterial translocation can then lead to the likelihood of opportunistic infections. Hence, modulating the microbiota may offer a promising strategy for improving poultry gut health and limiting caecal coccidiosis.

Recurrence and survival in dogs with excised colorectal polyps: A retrospective study of 58 cases.

BACKGROUND: Compared to humans, colorectal polyps are relatively rare in dogs. Epidemiological and prognostic data remain accordingly sparse, although they could help veterinary clinicians in the management of these cases. OBJECTIVES: To report the epidemiological data of dogs with colorectal polyps and identify factors associated with recurrence and survival. ANIMALS: Fifty-eight client-owned dogs with colorectal polyps admitted to 7 veterinary hospitals (53 dogs from France, 5 dogs from Spain, and 4 dogs from Portugal) were included. METHODS: Retrospective multicentric cohort study. Medical records and long-term outcome of the dogs were reviewed. When available, histological samples were reassessed by 2 board-certified pathologists according to the revised Vienna classification (RVC). RESULTS: The West Highland White Terrier (WHWT) breed was significantly associated with the presence of colorectal polyps (OR: 20; 95% CI: 7.5-52; P < .001). The overall median time to recurrence was not reached after 2000 days. The overall estimated median survival time was 1640 days. WHWT breed and larger polyps were significantly associated with a shorter time of polyp recurrence after surgical removal (respectively, P = .05 and P = .01). CONCLUSIONS AND CLINICAL IMPORTANCE: The probability of recurrence of colorectal polyps in dogs is low, but increased in WHWTs and larger polyps, which might benefit from routine screening after removal. No effective predictors of polyp recurrence and survival were identified using the RVC.

Systemic delivery of allogenic muscle stem cells induces long-term muscle repair and clinical efficacy in duchenne muscular dystrophy dogs.

Duchenne muscular dystrophy (DMD) is a genetic progressive muscle disease resulting from the lack of dystrophin and without effective treatment. Adult stem cell populations have given new impetus to cell-based therapy of neuromuscular diseases. One of them, muscle-derived stem cells, isolated based on delayed adhesion properties, contributes to injured muscle repair. However, these data were collected in dystrophic mice that exhibit a relatively mild tissue phenotype and clinical features of DMD patients. Here, we characterized canine delayed adherent stem cells and investigated the efficacy of their systemic delivery in the clinically relevant DMD animal model to assess potential therapeutic application in humans. Delayed adherent stem cells, named MuStem cells (muscle stem cells), were isolated from healthy dog muscle using a preplating technique. In vitro, MuStem cells displayed a large expansion capacity, an ability to proliferate in suspension, and a multilineage differentiation potential. Phenotypically, they corresponded to early myogenic progenitors and uncommitted cells. When injected in immunosuppressed dystrophic dogs, they contributed to myofiber regeneration, satellite cell replenishment, and dystrophin expression. Importantly, their systemic delivery resulted in long-term dystrophin expression, muscle damage course limitation with an increased regeneration activity and an interstitial expansion restriction, and persisting stabilization of the dog's clinical status. These results demonstrate that MuStem cells could provide an attractive therapeutic avenue for DMD patients.

Myopathy with oval inclusions in a domestic shorthair cat.

Case summary: An 18-month-old castrated male domestic shorthair cat was presented with a 2-month history of collapse and severe weakness, particularly affecting the pelvic limbs. A biceps femoris muscle biopsy revealed excessive variability in myofibre size, mild necrosis, minimal centronucleation and scattered 10 µm intracytoplasmic oval inclusions. The inclusions appeared amphophilic with haematoxylin and eosin, blue with Gomori trichrome and unstained with nicotinamide adenine dinucleotide dehydrogenase tetrazolium reductase staining. ATPase staining revealed a normal mosaic pattern and atrophy of both type 1 and 2 myofibres. The pathological diagnosis was a myopathy with inclusions. In contrast to previous feline myofibre inclusions previously reported in the literature, inclusions were not identified after immunohistochemistry using anti-desmin, tubulin, spectrin, laminin, LAMP and LC3 antibodies. After supportive care and corticosteroid treatment, clinical improvement was noted and the cat was discharged 10 days after initial presentation. Clinical and neurological re-examinations were performed at 1, 3, 6 and 9 months after discharge. Owner contact at both 10 and 30 months post-discharge confirmed that persistent muscular weakness was present. Relevance and novel information: This case report describes a novel and slowly progressive feline myopathy associated with oval amphophilic inclusions unreactive to immunostaining, which have not been previously reported in feline myopathies.

Establishment of a culture model for the prolonged maintenance of chicken feather follicles structure in vitro.

Protocols allowing the in vitro culture of human hair follicles in a serum free-medium up to 9 days were developed 30 years ago. By using similar protocols, we achieved the prolonged maintenance in vitro of juvenile feather follicles (FF) microdissected from young chickens. Histology showed a preservation of the FF up to 7 days as well as feather morphology compatible with growth and/or differentiation. The integrity of the FF wall epithelium was confirmed by transmission electron microscopy at Day 5 and 7 of culture. A slight elongation of the feathers was detected up to 5 days for 75% of the examined feathers. By immunochemistry, we demonstrated the maintenance of expression and localization of two structural proteins: scaffoldin and fibronectin. Gene expression (assessed by qRT-PCR) of NCAM, LCAM, Wnt6, Notch1, and BMP4 was not altered. In contrast, Shh and HBS1 expression collapsed, DKK3 increased, and KRT14 transiently increased upon cultivation. This indicates that cultivation modifies the mRNA expression of a few genes, possibly due to reduced growth or cell differentiation in the feather, notably in the barb ridges. In conclusion, we have developed the first method that allows the culture and maintenance of chicken FF in vitro that preserves the structure and biology of the FF close to its in vivo state, despite transcriptional modifications of a few genes involved in feather development. This new culture model may serve to study feather interactions with pathogens or toxics and constitutes a way to reduce animal experimentation.

The Internal Conduit System of the Swine Inverted Lymph Node.

Lymph nodes (LN) are the crossroad where naïve lymphocytes, peripheral antigens and antigen presenting cells contact together in order to mount an adaptive immune response. For this purpose, LN are highly organized convergent hubs of blood and lymphatic vessels that, in the case of B lymphocytes, lead to the B cell follicles. Herein take place the selection and maturation of B cell clones producing high affinity antibodies directed against various antigens. Whereas the knowledge on the murine and human LN distribution systems have reached an exquisite precision those last years, the organization of the antigens and cells circulation into the inverted porcine LN remains poorly described. Using up to date microscopy tools, we described the complex interconnections between afferent lymphatics and blood vessels, perifollicular macrophages, follicular B cells and efferent blood vessels. We observed that afferent lymphatic sinuses presented an asymmetric Lyve-1 expression similar to the one observed in murine LN, whereas specialized perifollicular sinuses connect the main afferent lymphatic sinus to the B cell follicles. Finally, whereas it was long though that mature B cells egress from the inverted LN in the T cell zone through HEV, our observations are in agreement with mature B cells accessing the efferent blood circulation in the efferent, subcapsular area. This understanding of the inverted porcine LN circuitry will allow a more accurate exploration of swine pathogens interactions with the immune cells inside the LN structures. Moreover, the mix between similarities and differences of porcine inverted LN circuitry with mouse and human normal LN shall enable to better apprehend the functions and malfunctions of normal LN from a new perspective.

PTEN contributes to profound PI3K/Akt signaling pathway deregulation in dystrophin-deficient dog muscle.

Duchenne muscular dystrophy is the most common and severe form of muscular dystrophy, and although the genetic basis of this disease is well defined, the overall mechanisms that define its pathogenesis remain obscure. Alterations in individual signaling pathways have been described, but little information is available regarding their putative implications in Duchenne muscular dystrophy pathogenesis. Here, we studied the status of various major signaling pathways in the Golden Retriever muscular dystrophy dog that specifically reproduces the full spectrum of human pathology. Using antibody arrays, we found that Akt1, glycogen synthase kinase-3beta (GSK3beta), 70-kDa ribosomal protein S6 kinase (p70S6K), extracellular signal-regulated kinases 1/2, and p38delta and p38gamma kinases all exhibited decreased phosphorylation in muscle from a 4-month-old animal with Golden Retriever muscular dystrophy, revealing a deep alteration of the phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase pathways. Immunohistochemistry analysis revealed the presence of muscle fibers exhibiting a cytosolic accumulation of Akt1, GSK3beta, and phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase (PTEN), an enzyme counteracting PI3K-mediated Akt activation. Enzymatic assays established that these alterations in phosphorylation and expression levels were associated with decreased Akt and increased GSK3beta and PTEN activities. PTEN/GSK3beta-positive fibers were also observed in muscle sections from 3- and 36-month-old animals, indicating long-term PI3K/Akt pathway alteration. Collectively, our data suggest that increased PTEN expression and activity play a central role in PI3K/Akt/GSK3beta and p70S6K pathway modulation, which could exacerbate the consequences of dystrophin deficiency.

Characterization of brain dystrophins absence and impact in dystrophin-deficient Dmdmdx rat model.

Duchenne Muscular Dystrophy (DMD) is a severe muscle-wasting disease caused by mutations in the DMD gene encoding dystrophin, expressed mainly in muscles but also in other tissues like retina and brain. Non-progressing cognitive dysfunction occurs in 20 to 50% of DMD patients. Furthermore, loss of expression of the Dp427 dystrophin isoform in the brain of mdx mice, the most used animal model of DMD, leads to behavioral deficits thought to be linked to insufficiencies in synaptogenesis and channel clustering at synapses. Mdx mice where the locomotor phenotype is mild also display a high and maladaptive response to stress. Recently, we generated Dmdmdx rats carrying an out-of frame mutation in exon 23 of the DMD gene and exhibiting a skeletal and cardiac muscle phenotype similar to DMD patients. In order to evaluate the impact of dystrophin loss on behavior, we explored locomotion parameters as well as anhedonia, anxiety and response to stress, in Dmdmdx rats aged from 1.5 to 7 months, in comparison to wild-type (WT) littermates. Pattern of dystrophin expression in the brain of WT and Dmdmdx rats was characterized by western-blot analyses and immunohistochemistry. We showed that dystrophin-deficient Dmdmdx rats displayed motor deficits in the beam test, without association with depressive or anxiety-like phenotype. However, Dmdmdx rats exhibited a strong response to restraint-induced stress, with a large increase in freezings frequency and duration, suggesting an alteration in a functional circuit including the amygdala. In brain, large dystrophin isoform Dp427 was not expressed in mutant animals. Dmdmdx rat is therefore a good animal model for preclinical evaluations of new treatments for DMD but care must be taken with their responses to mild stress.