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1.
Vet Dermatol ; 32(2): 179-e44, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33165993

RESUMO

BACKGROUND: Keratinocyte organoids can be used as a tool to evaluate epidermal structure, function and dysfunction. OBJECTIVES: To optimize the canine keratinocyte organoid system and produce organoids that are structurally equivalent to in vivo canine epidermis, in order to enable studies that focus on epidermal diseases and diseases resulting from an impaired epidermal barrier. ANIMALS: Skin biopsies were obtained from five recently euthanized dogs of different breeds with no skin abnormalities. METHODS AND MATERIALS: Cells derived from microdissected interfollicular epidermis were seeded in basement membrane extract and epidermal organoids were grown under different media conditions. Organoids were characterized to assess cell morphology and architecture in haematoxylin and eosin-stained slides and expression of selected epidermal markers (keratin 5, keratin 10, loricrin and filaggrin) by immunohistochemical analysis and quantitative reverse transcription PCR. RESULTS: The selected epidermal markers were expressed in the same epidermal layers in the organoids cultured in expansion medium and differentiation medium as in normal interfollicular epidermis, yet restriction to the distinct layers was best achieved with expansion medium. Comparison of the mRNA expression levels of these markers revealed that relative expression is similar in organoids cultured in expansion medium and normal canine epidermis, while it differs in organoids cultured in differentiation medium. CONCLUSION AND CLINICAL IMPORTANCE: Organoids cultured in expansion medium have an equivalent structure to the interfollicular epidermis and express key marker proteins in similar proportions. Epidermal organoids are therefore a promising in vitro model to study epidermal structure, function and dysfunction.


Assuntos
Epiderme , Organoides , Animais , Diferenciação Celular , Células Cultivadas , Cães , Células Epidérmicas , Queratinócitos , Reação em Cadeia da Polimerase em Tempo Real/veterinária
2.
Vet Dermatol ; 31(3): 244-e54, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31908106

RESUMO

BACKGROUND: Genetic skin diseases in cattle are rare. CLINICAL SUMMARY: A 7-week-old female Holstein calf was presented with epidermal lesions and alopecia in the caudal region of the ears and on the neck, as well as deep bilateral ulcerative lesions on the palmar aspect of the metacarpi and dorsal aspect of the right metacarpus. Clinical, pathological and histopathological examination of the calf was suggestive of a subepidermal vesicular dermatosis. Genetic analysis identified a de novo non-sense variant affecting the aspartate dehydrogenase domain containing (ASPDH) gene, which might be associated with the formation of subepidermal vesicles in this case. CONCLUSION AND CLINICAL IMPORTANCE: The observed phenotype in the calf may represent a novel form of a vesicular skin disorder. Haploinsufficiency of the ASPDH gene might be considered as a possible cause.


Assuntos
Aminoácido Oxirredutases/genética , Doenças dos Bovinos/genética , Variação Genética , Úlcera Cutânea/genética , Úlcera Cutânea/veterinária , Alopecia/diagnóstico , Animais , Biópsia , Bovinos , Doenças dos Bovinos/diagnóstico , Códon sem Sentido , Epiderme/patologia , Feminino , Técnicas Histológicas , Pele/patologia , Úlcera Cutânea/etiologia
3.
Genes (Basel) ; 13(4)2022 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-35456456

RESUMO

Non-inflammatory alopecia is a frequent skin problem in dogs, causing damaged coat integrity and compromised appearance of affected individuals. In this study, we examined the Cesky Fousek breed, which displays atypical recurrent flank alopecia (aRFA) at a high frequency. This type of alopecia can be quite severe and is characterized by seasonal episodes of well demarcated alopecic areas without hyperpigmentation. The genetic component responsible for aRFA remains unknown. Thus, here we aimed to identify variants involved in aRFA using a combination of histological, genomic, and transcriptomic data. We showed that aRFA is histologically similar to recurrent flank alopecia, characterized by a lack of anagen hair follicles and the presence of severely shortened telogen or kenogen hair follicles. We performed a genome-wide association study (GWAS) using 216 dogs phenotyped for aRFA and identified associations on chromosomes 19, 8, 30, 36, and 21, highlighting 144 candidate genes, which suggests a polygenic basis for aRFA. By comparing the skin cell transcription pattern of six aRFA and five control dogs, we identified 236 strongly differentially expressed genes (DEGs). We showed that the GWAS genes associated with aRFA are often predicted to interact with DEGs, suggesting their joint contribution to the development of the disease. Together, these genes affect four major metabolic pathways connected to aRFA: collagen formation, muscle structure/contraction, lipid metabolism, and the immune system.


Assuntos
Estudo de Associação Genômica Ampla , Transcriptoma , Alopecia/genética , Alopecia/patologia , Alopecia/veterinária , Animais , Cães , Folículo Piloso , Pele/patologia , Transcriptoma/genética
4.
Genes (Basel) ; 12(2)2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33504055

RESUMO

Canine cutaneous epitheliotropic T-cell lymphoma (CETL) and immune-mediated T-cell predominant dermatoses (IMD) share several clinical and histopathological features, but differ substantially in prognosis. The discrimination of ambiguous cases may be challenging, as diagnostic tests are limited and may prove equivocal. This study aimed to investigate transcriptional differences between CETL and IMD, as a basis for further research on discriminating diagnostic biomarkers. We performed 100bp single-end sequencing on RNA extracted from formalin-fixed and paraffin-embedded skin biopsies from dogs with CETL and IMD, respectively. DESeq2 was used for principal component analysis (PCA) and differential gene expression analysis. Genes with significantly different expression were analyzed for enriched pathways using two different tools. The expression of selected genes and their proteins was validated by RT-qPCR and immunohistochemistry. PCA demonstrated the distinct gene expression profiles of CETL and IMD. In total, 503 genes were upregulated, while 4986 were downregulated in CETL compared to IMD. RT-qPCR confirmed the sequencing results for 5/6 selected genes tested, while the protein expression detected by immunohistochemistry was not entirely consistent. Our study revealed transcriptional differences between canine CETL and IMD, with similarities to human cutaneous lymphoma. Differentially expressed genes are potential discriminatory markers, but require further validation on larger sample collections.


Assuntos
Suscetibilidade a Doenças , Doenças do Cão/etiologia , Regulação Neoplásica da Expressão Gênica , Linfoma Cutâneo de Células T/veterinária , Dermatopatias/veterinária , Animais , Biópsia , Suscetibilidade a Doenças/imunologia , Doenças do Cão/diagnóstico , Cães , Imuno-Histoquímica , Transcriptoma
5.
Genes (Basel) ; 11(8)2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32759649

RESUMO

The transcriptome profile and differential gene expression in telogen and late anagen microdissected hair follicles and the interfollicular epidermis of healthy dogs was investigated by using RNAseq. The genes with the highest expression levels in each group were identified and genes known from studies in other species to be associated with structure and function of hair follicles and epidermis were evaluated. Transcriptome profiling revealed that late anagen follicles expressed mainly keratins and telogen follicles expressed GSN and KRT15. The interfollicular epidermis expressed predominately genes encoding for proteins associated with differentiation. All sample groups express genes encoding for proteins involved in cellular growth and signal transduction. The expression pattern of skin-associated genes in dogs is similar to humans. Differences in expression compared to mice and humans include BMP2 expression mainly in telogen and high KRT17 expression in the interfollicular epidermis of dogs. Our data provide the basis for the investigation of the structure and function of canine skin or skin disease and support the use of dogs as a model for human cutaneous disease by assigning gene expression to specific tissue states.


Assuntos
Cães/genética , Folículo Piloso/metabolismo , Transcriptoma , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Folículo Piloso/crescimento & desenvolvimento , Queratinas/genética , Queratinas/metabolismo
6.
PLoS One ; 14(2): e0212645, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30794648

RESUMO

Bald thigh syndrome is a common hair loss disorder in sighthounds. Numerous possible causes, including environmental conditions, trauma, stress, endocrinopathies and genetic components have been proposed, but only endocrinopathies have been ruled out scientifically. The overall goal of our study was to identify the cause of bald thigh syndrome and the pathological changes associated with it. We approached this aim by comparing skin biopsies and hair shafts of affected and control dogs microscopically as well as by applying high-throughput technologies such as genomics, transcriptomics and proteomics. While the histology is rather unspecific in most cases, trichogram analysis and scanning electron microscopy revealed severe structural abnormalities in hair shafts of affected dogs. This finding is supported by the results of the transcriptomic and proteomic profiling where genes and proteins important for differentiation of the inner root sheath and the assembly of a proper hair shaft were downregulated. Transcriptome profiling revealed a downregulation of genes encoding 23 hair shaft keratins and 51 keratin associated proteins, as well as desmosomal cadherins and several actors of the BMP signaling pathway which is important for hair shaft differentiation. The lower expression of keratin 71 and desmocollin 2 on the mRNA level in skin biopsies corresponded with a decreased protein expression in the hair shafts of affected dogs. The genetic analysis revealed a missense variant in the IGFBP5 gene homozygous in all available Greyhounds and other sighthounds. Further research is required to clarify whether the IGFBP5 variant represents a predisposing genetic risk factor. We conclude from our results that structural defects in the hair shafts are the cause for this well-known disease and these defects are associated with a downregulation of genes and proteins essential for hair shaft formation. Our data add important knowledge to further understand the molecular mechanisms of HF morphogenesis and alopecia in dogs.


Assuntos
Alopecia , Doenças do Cão , Cabelo , Pele , Alopecia/genética , Alopecia/metabolismo , Alopecia/patologia , Alopecia/veterinária , Animais , Doenças do Cão/genética , Doenças do Cão/metabolismo , Doenças do Cão/patologia , Cães , Feminino , Regulação da Expressão Gênica , Cabelo/metabolismo , Cabelo/patologia , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/biossíntese , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Queratinas/biossíntese , Queratinas/genética , Masculino , Pele/metabolismo , Pele/patologia
7.
PLoS One ; 12(10): e0186469, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29065140

RESUMO

Alopecia X is a hair cycle arrest disorder in Pomeranians. Histologically, kenogen and telogen hair follicles predominate, whereas anagen follicles are sparse. The induction of anagen relies on the activation of hair follicle stem cells and their subsequent proliferation and differentiation. Stem cell function depends on finely tuned interactions of signaling molecules and transcription factors, which are not well defined in dogs. We performed transcriptome profiling on skin biopsies to analyze altered molecular pathways in alopecia X. Biopsies from five affected and four non-affected Pomeranians were investigated. Differential gene expression revealed a downregulation of key regulator genes of the Wnt (CTNNB1, LEF1, TCF3, WNT10B) and Shh (SHH, GLI1, SMO, PTCH2) pathways. In mice it has been shown that Wnt and Shh signaling results in stem cell activation and differentiation Thus our findings are in line with the lack of anagen hair follicles in dogs with Alopecia X. We also observed a significant downregulation of the stem cell markers SOX9, LHX2, LGR5, TCF7L1 and GLI1 whereas NFATc1, a quiescence marker, was upregulated in alopecia X. Moreover, genes coding for enzymes directly involved in the sex hormone metabolism (CYP1A1, CYP1B1, HSD17B14) were differentially regulated in alopecia X. These findings are in agreement with the so far proposed but not yet proven deregulation of the sex hormone metabolism in this disease.


Assuntos
Alopecia/veterinária , Cabelo , Alopecia/genética , Animais , Biomarcadores/metabolismo , Cães , Feminino , Masculino , Receptores de Calcitriol/metabolismo , Células-Tronco/metabolismo
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