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1.
Sci Rep ; 11(1): 17296, 2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34453089

RESUMO

Hypertrophic scars represent a common complication in burn patients. In addition to cosmetic defects, they may cause serious sensory abnormalities such as pain and itching, severe dysfunction depending on the site, and emotional disorders such as anxiety and depression. The present study aimed to identify the molecular mechanisms underlying the use of extracorporeal shock wave therapy in keratinocytes. Keratinocytes derived from hypertrophic scar tissue were cultured and expression of proliferation markers (keratin 5 and 14), activation markers (keratin 6 and 17), differentiation markers (keratin 1, 10, and involucrin), apoptosis factors (Bax, Bcl2, and Caspase 14), and proliferation/differentiation regulators (p21 and p27) was investigated to compared with that of those in keratinocytes derived from normal skin tissue. Scar-derived keratinocytes were treated with extracorporeal shock waves under 1000 impulses at 0.1, 0.2, and 0.3 mJ/mm2. Shock waves altered the molecular pattern of proliferation, activation, differentiation, and apoptosis, as well as proliferation/ differentiation regulators, including Bax, Bcl2, ASK1, p21, p27, and Notch1. In summary, we show that extracorporeal shock wave therapy regulates the proliferation and differentiation of keratinocytes derived from hypertrophic scar to maintain normal epidermal integrity.


Assuntos
Cicatriz Hipertrófica/terapia , Tratamento por Ondas de Choque Extracorpóreas/métodos , Queratinócitos/citologia , Biomarcadores/metabolismo , Caspase 14/metabolismo , Diferenciação Celular , Humanos , Queratina-14/metabolismo , Queratina-5/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pele , Resultado do Tratamento , Proteína X Associada a bcl-2/metabolismo
2.
Exp Eye Res ; 210: 108696, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34228968

RESUMO

Studies have reported that the incidence of ocular discomfort in people who often wear makeup is higher than that in the normal population. The incidence of ocular discomfort of these people may be also related to the daily ocular exposure to chemical surfactants during cleaning. The objectives of this study were to explore morphological and pathological changes in the murine ocular surface after low-dose repeated exposure to disodium cocoamphodiacetate (DC), a kind of chemical surfactant widely used in personal cleaning products, and to investigate the possible mechanisms. DC was administered in low dose (0.1%) to the ocular surface of C56BL/6 once daily for two weeks. We found that there were an increase of sodium fluorescein staining on the cornea, a significant thinning of corneal epithelial thickness, and increased TUNEL-positive cells in corneal epithelium in vivo. DC treatment also modulated the distribution of K14+ and P63+ epithelia from the limbal to the center on the cornea. In cultured murine corneal epithelial progenitor cell line (TKE2), DC treatment induced cell detachment and decreased the activation of Ak strain transforming protein (AKT), and extracellular signal-regulated kinase (ERK). And DC increased TUNEL-positive cells in vitro with increased expression of cleaved Caspase3 and B-cell lymphoma-2 associated X protein (Bax). Our results indicated that repeated low-dose DC exposure on ocular surface caused significant impairment on the structure and viability of the corneal epithelium by inhibiting epithelial proliferation and inducing apoptosis. It provides the foundations to understand the harmful effects of cleaning products daily exposure on the ocular surface.


Assuntos
Acetatos/efeitos adversos , Doenças da Córnea/induzido quimicamente , Epitélio Corneano/efeitos dos fármacos , Glicina/análogos & derivados , Limbo da Córnea/efeitos dos fármacos , Tensoativos/efeitos adversos , Acetatos/administração & dosagem , Administração Oftálmica , Animais , Apoptose , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Doenças da Córnea/metabolismo , Doenças da Córnea/patologia , Epitélio Corneano/metabolismo , Epitélio Corneano/patologia , Feminino , Fluoresceína/metabolismo , Glicina/administração & dosagem , Glicina/efeitos adversos , Queratina-14/metabolismo , Limbo da Córnea/metabolismo , Limbo da Córnea/patologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Soluções Oftálmicas , Microscopia com Lâmpada de Fenda , Coloração e Rotulagem , Tensoativos/administração & dosagem , Transativadores/metabolismo
3.
Virchows Arch ; 479(3): 515-521, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34218288

RESUMO

Urothelial carcinoma is subdivided into luminal (L), basal (B), and p53-wild-type (WT) molecular subtypes, with basal and p53-WT groups showing more aggressive course and poor treatment response, respectively. The literature on molecular subtypes of UC includes a mixture of different stages. We investigated the molecular profile and outcome of pure cohort of muscle invasive bladder carcinoma (MIBC) considering two distinct patterns of muscularis propria (MP) invasion. Forty-three cystectomies harboring stage pT2 were retrospectively identified in 18 years. MP invasion was subclassified into patterns 1 (tumor encasing intact detrusor muscle bundles) and 2 (tumor dissecting/replacing detrusor muscle). Using IHC, B/L phenotypes, p53, and Ki67 were assessed, and survival data was collected. Pattern 1 invasion was noted in 16 (37%) and pattern 2 in 27 (63%), with mean age of pattern 1 being 10 years younger. B/L phenotypes were successfully determined in 83.7%; 48.8% and 34.8% revealed L and B phenotypes, respectively (indeterminate phenotype in 16.4%). Pattern 1 was associated with L phenotype (GATA3 and HER-2 expressions: p = 0.02 & p = 0.04, respectively). Ki67 ≥ 5/10HPF was noted in pattern 2 and B phenotype (p = 0.03). B phenotype showed association with p53-WT (p = 0.007). In median follow-up of 60.7 months, 63.6% of pattern 1 cases were alive without disease compared to 32% of pattern 2 (not significant). A panel of CK20 and GATA3 for luminal and CK5/6 and CK14 for basal subtypes can provide reliable molecular classification in UC. Also, morphology of MIBC can predict the molecular phenotype and the behavior of the UC.


Assuntos
Biomarcadores Tumorais/análise , Carcinoma/química , Neoplasias da Bexiga Urinária/química , Urotélio/química , Idoso , Carcinoma/classificação , Carcinoma/patologia , Carcinoma/cirurgia , Cistectomia , Bases de Dados Factuais , Feminino , Fator de Transcrição GATA3/análise , Humanos , Imuno-Histoquímica , Queratina-14/análise , Queratina-20/análise , Queratina-5/análise , Queratinas Específicas do Cabelo/análise , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Fenótipo , Valor Preditivo dos Testes , Estudos Retrospectivos , Resultado do Tratamento , Neoplasias da Bexiga Urinária/classificação , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/cirurgia , Urotélio/patologia , Urotélio/cirurgia
4.
Hua Xi Kou Qiang Yi Xue Za Zhi ; 39(3): 274-278, 2021 Jun 01.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-34041875

RESUMO

OBJECTIVES: The effect of Vps4b gene mutation on the expressions of cytokeratin 14 (CK14) and proliferating cell nuclear antigen (PCNA) in the Hertwig's epithelial root sheath (HERS) is investigated. METHODS: The bilateral mandibular tissues of mouse on postnatal days 5, 9, 11, 15, and 19 were removed. The mandibular first molar tissue sections were obtained after paraffin embedding. The CK14 and PCNA expressions in the epithelial root sheath of the normal mouse and Vps4b knockout mouse were compared through immunohistochemistry. RESULTS: On postnatal day 5, the normal mouse began to form HERS and had a strong positive PCNA expression in the HERS cells; on postnatal day 9, the HERS structure was continuous, and PCNA was positive in the HERS cells; on postnatal day 11, a small portion of HERS began to break, and PCNA was weakly positive in the HERS cells; on postnatal day 15, HERS continued to fracture; PCNA was weakly and positively expressed in the HERS cells on the root surface; on postnatal day 19, the tooth root reached normal physiological length, and PCNA was positively expressed in the HERS cells of the terminal part. Similar to the normal mouse, the gene knockout mouse also formed a HERS structure on postnatal day 5. However, HERS began to break on postnatal day 9. On postnatal day 19, only a few fragments of HERS were found on the root surface, and the root development was immature. Moreover, the expression intensity of PCNA in the gene knockout mouse was decreased. CONCLUSIONS: The Vps4b gene mutation may change the CK14 and PCNA expressions, leading to abnormal root development.


Assuntos
Células Epiteliais , Raiz Dentária , ATPases Associadas a Diversas Atividades Celulares , Animais , Complexos Endossomais de Distribuição Requeridos para Transporte , Queratina-14 , Camundongos , Camundongos Knockout , Antígeno Nuclear de Célula em Proliferação
5.
Elife ; 102021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34009125

RESUMO

Embryonic taste bud primordia are specified as taste placodes on the tongue surface and differentiate into the first taste receptor cells (TRCs) at birth. Throughout adult life, TRCs are continually regenerated from epithelial progenitors. Sonic hedgehog (SHH) signaling regulates TRC development and renewal, repressing taste fate embryonically, but promoting TRC differentiation in adults. Here, using mouse models, we show TRC renewal initiates at birth and coincides with onset of SHHs pro-taste function. Using transcriptional profiling to explore molecular regulators of renewal, we identified Foxa1 and Foxa2 as potential SHH target genes in lingual progenitors at birth and show that SHH overexpression in vivo alters FoxA1 and FoxA2 expression relevant to taste buds. We further bioinformatically identify genes relevant to cell adhesion and cell locomotion likely regulated by FOXA1;FOXA2 and show that expression of these candidates is also altered by forced SHH expression. We present a new model where SHH promotes TRC differentiation by regulating changes in epithelial cell adhesion and migration.


Assuntos
Diferenciação Celular , Autorrenovação Celular , Células Epiteliais/metabolismo , Proteínas Hedgehog/metabolismo , Células-Tronco/metabolismo , Papilas Gustativas/metabolismo , Animais , Animais Recém-Nascidos , Adesão Celular , Linhagem da Célula , Movimento Celular , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Fator 3-alfa Nuclear de Hepatócito/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Fator 3-beta Nuclear de Hepatócito/genética , Fator 3-beta Nuclear de Hepatócito/metabolismo , Queratina-14/genética , Queratina-14/metabolismo , Masculino , Transdução de Sinais , Paladar , Papilas Gustativas/citologia , Transcriptoma
6.
Ocul Surf ; 21: 145-159, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33930539

RESUMO

PURPOSE: Dry eye disease (DED) is characterized by loss of tear film stability that becomes self-sustaining in a vicious cycle of pathophysiological events. Currently, desiccation stress (DS) is the dominant procedure for inducing DED in mice, however its' effect on limbal epithelial stem cells (LESCs) has been overlooked. This study aimed to establish a DS model via the use of a novel hardware to investigate the impact on the ocular surface including LESCs. METHODS: A mouse transporter unit was customized to generate a dehumidified environment. C57BL/6J mice were exposed to mild DS and injected with scopolamine hydrobromide (SH) or remained untreated (UT) under standard vivarium conditions for 10 consecutive days (n = 28/group). Clinical assessments included phenol red tear-thread test, fluorescein staining and optical coherence tomography assessments. Histopathological and immunofluorescence was used to evaluate tissue architecture, goblet cell (GC) status, lacrimal gland (LG) inflammation and epithelial phenotype on the ocular surface. Whole flat-mounted corneas were immunostained for keratin-14 (K14), then imaged by confocal microscopy and analyzed computationally to investigate the effect of DS on LESCs. RESULTS: Custom modifications made to the animal transporter unit resulted in dehumidified cage relative humidity (RH) of 43.5 ± 4.79% compared to the vivarium 53.9 ± 1.8% (p = 0.0243). Under these conditions, aqueous tear production in mice was suppressed whilst corneal permeability and corneal irregularity significantly increased. H&E staining indicated stressed corneal basal epithelial cells and increased desquamation. DS-exposed mice had reduced GC density (41.0 ± 5.10 GC/mm vs 46.9 ± 3.88 GC/mm, p = 0.0482) and LGs from these mice exhibited elevated CD4+ cell infiltration compared to controls. DS elicited K14+ epithelial cell displacement, as indicated by increased fluorescence signal at a distance of 50-100 µm radially inwards from the limbus [0.63 ± 0.053% (DS) vs 0.54 ± 0.060% (UT), p = 0.0317]. CONCLUSIONS: Application of mild DS using customized hardware and SH injections generated features of DED in mice. Following DS, ocular surface epithelial cell health decreased and LESCs appeared stressed. This suggested that potential downstream effects of DS on corneal homeostasis are present, a phenomenon that is currently under-investigated. The method used to induce DED in this study enables the development of a chronic model which more closely resembles disease seen in the clinic.


Assuntos
Dessecação , Síndromes do Olho Seco , Animais , Modelos Animais de Doenças , Queratina-14 , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco , Lágrimas
7.
Toxicol In Vitro ; 74: 105162, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33839235

RESUMO

Silymarin is a flavonoid complex isolated from the plant Silybum marianum which is well known for its antioxidant, hepatoprotective and immunomodulatory effects. Since little is known about its anti-inflammatory properties and healing effects, our study focused on whether or not silymarin components reduce inflammation and support epidermis regeneration. Lipopolysaccharides (LPS) and sodium dodecyl sulfate (SDS) were used to induce inflammation in normal human epidermal keratinocytes (NHEKs) and reconstructed epidermis (RHE), respectively. The expression of pro-inflammatory cytokines (IL-1, IL-6 and IL-8) in NHEKs and RHE was measured by enzyme - linked immunosorbent assay (ELISA). The expression of cytokeratin 14 and loricrin in RHE was detected by immunofluorescent analysis. Hematoxylin and eosin staining was used for the morphological evaluation of RHE. It was determined that 2, 3 - dehydrosilybin (DHSB) downregulated the production of selected pro-inflammatory cytokines produced by NHEKs. Although all layers of RHE displayed full thickness, when SDS was applied, cell detachment was seen in the stratum corneum and loricrin expression was diminished.


Assuntos
Anti-Inflamatórios/farmacologia , Epiderme/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Quercetina/farmacologia , Silimarina/farmacologia , Citocinas/metabolismo , Epiderme/metabolismo , Humanos , Inflamação/induzido quimicamente , Queratina-14/metabolismo , Queratinócitos/metabolismo , Lipopolissacarídeos/toxicidade , Proteínas de Membrana/metabolismo , Dodecilsulfato de Sódio/toxicidade
8.
Am J Respir Cell Mol Biol ; 65(1): 103-113, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33789072

RESUMO

Airway basal cells are crucial for regeneration of the human lung airway epithelium and are believed to be important contributors to chronic obstructive pulmonary disease (COPD) and other lung disorders. To reveal how basal cells contribute to disease and to discover novel therapeutic targets, these basal cells need to be further characterized. In this study, we optimized a flow cytometry-based cell sorting protocol for primary human airway basal cells dependent on cell size and NGFR (nerve-growth factor receptor) expression. The basal cell population was found to be molecularly and functionally heterogeneous, in contrast to cultured basal cells. In addition, significant differences were found, such as KRT14 expression exclusively existing in cultured cells. Also, colony-forming capacity was significantly increased in cultured cells showing a clonal enrichment in vitro. Next, by single-cell RNA sequencing on primary basal cells from healthy donors and patients with Global Initiative for Chronic Obstructive Lung Disease stage IV COPD, the gene expression revealed a continuum ranging from healthy basal cell signatures to diseased basal cell phenotypes. We identified several upregulated genes that may indicate COPD, such as stress response-related genes GADD45B and AHSA1, together with with genes involved in the response to hypoxia, such as CITED2 and SOD1. Taken together, the presence of healthy basal cells in stage IV COPD demonstrates the potential for regeneration through the discovery of novel therapeutic targets. In addition, we show the importance of studying primary basal cells when investigating disease mechanisms as well as for developing future cell-based therapies in the human lung.


Assuntos
Células Epiteliais/metabolismo , Pulmão/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Mucosa Respiratória/metabolismo , Antígenos de Diferenciação/metabolismo , Células Cultivadas , Células Epiteliais/patologia , Humanos , Queratina-14/metabolismo , Pulmão/patologia , Chaperonas Moleculares/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Receptores de Fator de Crescimento Neural/metabolismo , Mucosa Respiratória/patologia
10.
Pediatr Dermatol ; 38(2): 436-441, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33471381

RESUMO

BACKGROUND: Epidermolysis bullosa simplex (EBS) is a heterogeneous group of inherited disorders characterized by skin fragility due to intraepidermal separation. Most cases result from heterozygous mutations in KRT5 or KRT14; however, a minority of affected individuals carry mutations in non-keratin genes including DST encoding an epithelial isoform of dystonin. DST-associated EBS is transmitted as an autosomal recessive trait. Here, we report a series of EBS patients carrying bi-allelic DST mutations and review previously reported cases aiming to delineate phenotype-genotype correlations. METHODS: Whole-exome and direct sequencing were used for variant analysis. Review of previously reported cases was performed. RESULTS: Mutation analysis revealed DST mutations in five patients belonging to three families. Two variants have not been previously reported: c.7097dupA (p.Tyr2366X) and c.7429delC (p.Leu2477Serfs*13). We identified an additional six cases in the literature, bringing the total number of individuals affected with EBS due to DST variants to 11. Patients displayed distinctive phenotypes regardless of the causative variant. CONCLUSIONS: The current study expands the clinical and genetic spectrum of DST-associated EBS subtype.


Assuntos
Distonina/genética , Epidermólise Bolhosa Simples/genética , Humanos , Queratina-14/genética , Queratina-5/genética , Mutação , Fenótipo
11.
Mol Cell Endocrinol ; 520: 111081, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33181234

RESUMO

During puberty, the mammary gland undergoes an intense growth, dependent on the interplay between the Epidermal Growth Factor Receptor (EGFR) in the stroma and different mammary epithelial receptors. We hypothesize that EGFR expressed in the mammary epithelium also has a role in puberty and the epithelial cells can self-sustain by EGFR-mediated autocrine signaling. We adopted mammary cell lines from different species, as in vitro model for the epithelium, and we observed that EGFR-signaling positively affects their survival and proliferation. Once deprived of external growth factors, mammary cells still showed strong Erk 1/2 phosphorylation, abolished upon EGFR inhibition, coupled with a further reduction in survival and proliferation. Based on gene expression analysis, three EGFR-ligands (AREG, EREG and HBEGF) are likely to mediate this autocrine signaling. In conclusion, internal EGFR-activating signals sustain mammary epithelial cell proliferation and survival in vitro.


Assuntos
Células Epiteliais/citologia , Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Humanas/citologia , Transdução de Sinais , Animais , Comunicação Autócrina , Bovinos , Ciclo Celular , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Receptores ErbB/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Queratina-14/metabolismo , Queratina-18/metabolismo , Ligantes , Camundongos , Receptor ErbB-2/metabolismo , Especificidade da Espécie
12.
J Immunoassay Immunochem ; 42(3): 236-251, 2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-33213275

RESUMO

Molecular subtyping of urothelial carcinoma (UC) is similar to that of breast cancer and is based on the developmental biology approach. The aim of the present study is to assess the prognostic impact of CK5, CK14, and CK20 expression in urinary bladder cancer (UBC) with the potential to stratify them into different subtypes. The current study examined the immunohistochemical expression of CK5, CK14, and CK20 in 90 specimens of UBC. CK5 was expressed in 81.1% of the cases and was significantly associated with old age, muscle invasion, presence of bilharziasis, and tendency for poor overall survival. CK20 was expressed in 47.8% of the cases and was associated with nonmuscle invasion and pure UC while 50% of the cases expressed CK14 that were associated with muscle invasion and perineural invasion. Most squamous cell carcinoma and those associated with bilharziasis were belonged to Ck5+/CK20- subgroup while pure UC and those lacked bilharziasis were located in the Ck5+/CK20+ subgroup. The basal group (Ck5+/CK14+/CK20-) showed high proliferative features compared to the intermediate group (Ck5+/CK14-/CK20-). Generally, presence of CK5 is associated with adverse features especially in the group lacking CK20; however, basal and intermediate subgroups share CK5 expression but they show different proliferative capacities, so their distinction by CK14 is helpful.


Assuntos
Biomarcadores Tumorais/biossíntese , Queratina-14/biossíntese , Queratina-20/biossíntese , Queratina-5/biossíntese , Neoplasias da Bexiga Urinária/imunologia , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/imunologia , Feminino , Humanos , Imuno-Histoquímica , Queratina-14/imunologia , Queratina-20/imunologia , Queratina-5/imunologia , Masculino , Pessoa de Meia-Idade , Neoplasias da Bexiga Urinária/diagnóstico
13.
Arch Toxicol ; 95(2): 715-726, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33211169

RESUMO

We previously demonstrated that immunohistochemistry for γ-H2AX, a biomarker of DNA damage, is useful for early detection of urinary bladder carcinogens in rats. In a 28-day repeated-dose study, γ-H2AX was shown to have high sensitivity for detection of bladder carcinogens. However, no reports have evaluated whether a combination of multiple biomarkers may further improve sensitivity. Accordingly, in this study, we aimed to evaluate the applicability of bladder tissue and cancer stem cell markers, including cytokeratin 14 (KRT14), aldehyde dehydrogenase 1A1 (ALDH1A1), and cluster of differentiation 44 (CD44), as complementary markers for early detection of bladder carcinogens. Bladder samples obtained from male F344 rats orally treated with 14 bladder carcinogens and five nonbladder carcinogens for 28 days were used for immunohistochemical analysis of stem cell markers. In the bladder carcinogen-treated rats, increases in KRT14, ALDH1A1, and CD44 expression were observed in 9, 10, and 10 out of 14 groups, respectively, whereas the five nonbladder carcinogens did not cause upregulation of these markers. Although most epithelial cells with KRT14 or ALDH1A1 expression were also positive for CD44, KRT14 and ALDH1A1 expression were mutually exclusive. Twelve bladder carcinogens showed increases in at least one of the three markers, indicating that the combined evaluation showed higher sensitivity than the use of individual markers alone. Importantly, two of three bladder carcinogens that did not induce γ-H2AX immunostaining showed stem cell marker expression. Our results demonstrated that these stem cell markers may be useful as complementary markers for γ-H2AX in evaluation of bladder carcinogens.


Assuntos
Aldeído Desidrogenase/metabolismo , Receptores de Hialuronatos/metabolismo , Queratina-14/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Bexiga Urinária/metabolismo , Família Aldeído Desidrogenase 1/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Carcinogênese/metabolismo , Carcinógenos/toxicidade , Detecção Precoce de Câncer/métodos , Histonas/metabolismo , Imuno-Histoquímica , Masculino , Células-Tronco Neoplásicas/efeitos dos fármacos , Fosfoproteínas/metabolismo , Ratos , Ratos Endogâmicos F344 , Retinal Desidrogenase/metabolismo , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Bexiga Urinária/patologia
14.
Cell Transplant ; 29: 963689720964381, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33040596

RESUMO

The human amniotic membrane is a highly abundant and readily available tissue that may be useful for regenerative medicine and cell therapy. The amniotic membrane stem cells can differentiate into multiple cell lineages; they have low immunogenicity and anti-inflammatory functions. This research aims to examine the protocols for the isolation of human amniotic membrane stem cells, including their phenotypic characterization and in vitro potential for differentiation toward keratinocytes. Human placentas were obtained from selected cesarean-sectioned births. We isolated amniotic stem cells by trypsin and collagenase B digestion and centrifuged with Percoll. After monolayer expansion of adherent cells, the cells were characterized by immunocytology with octamer-binding transcription factor 4 and differentiated into keratinocytes by treating the cells with insulin, hydrocortisone, BMP-4, and vitamin C. Protocol for isolation of stem cells from amniotic membrane has high efficiency. Differentiation markers of stem cells into keratinocytes, such as vimentin, cytokeratin (CK) 14, and CK19, were determined by reverse transcription-polymerase chain reaction increase over time in culture. Stem cells isolated from the amniotic membrane can differentiate into keratinocytes. It has opened the prospect of using stem cells to regenerate skin and clinical applications.


Assuntos
Âmnio/citologia , Diferenciação Celular , Queratinócitos/citologia , Células-Tronco/citologia , Diferenciação Celular/genética , Proliferação de Células/genética , Separação Celular , Células Cultivadas , Humanos , Queratina-14/genética , Queratina-14/metabolismo , Queratina-19/genética , Queratina-19/metabolismo , Queratinócitos/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células-Tronco/metabolismo
15.
Development ; 147(19)2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32994165

RESUMO

Salivary glands exert exocrine secretory function to provide saliva for lubrication and protection of the oral cavity. Its epithelium consists of several differentiated cell types, including acinar, ductal and myoepithelial cells, that are maintained in a lineage-restricted manner during homeostasis or after mild injuries. Glandular regeneration following a near complete loss of secretory cells, however, may involve cellular plasticity, although the mechanism and extent of such plasticity remain unclear. Here, by combining lineage-tracing experiments with a model of severe glandular injury in the mouse submandibular gland, we show that de novo formation of acini involves induction of cellular plasticity in multiple non-acinar cell populations. Fate-mapping analysis revealed that, although ductal stem cells marked by cytokeratin K14 and Axin2 undergo a multipotency switch, they do not make a significant contribution to acinar regeneration. Intriguingly, more than 80% of regenerated acini derive from differentiated cells, including myoepithelial and ductal cells, that appear to dedifferentiate to a progenitor-like state before re-differentiation into acinar cells. The potential of diverse cell populations serving as a reserve source for acini widens the therapeutic options for hyposalivation.


Assuntos
Células Epiteliais/citologia , Células Epiteliais/metabolismo , Glândulas Salivares/citologia , Glândulas Salivares/metabolismo , Animais , Proteína Axina/metabolismo , Diferenciação Celular/fisiologia , Humanos , Queratina-14/metabolismo , Camundongos , Células-Tronco/citologia , Células-Tronco/metabolismo , Glândula Submandibular/citologia , Glândula Submandibular/metabolismo
16.
Sci Rep ; 10(1): 14486, 2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32879384

RESUMO

The rules governing Medicinal Products in the European Union necessitates the production of cell-based therapy in good manufacturing practice facilities. The produced cells may need several hours in transportation to reach the application sites. In this study, we investigated four candidate solutions for transporting human keratinocytes. The solutions are (1) normal saline, (2) saline with 2.5% human serum albumin (Saline + HSA), (3) chemically defined, xeno-free keratinocyte media and (4) keratinocyte media with pituitary bovine extract (PBE-media). One million keratinocytes from three donors were suspended in each solution and kept at 4 °C for up to 24 h. Cells kept in Saline + HSA showed higher viability after 1, 3 and 24 h. Then, equal number of viable cells were seeded on collagenous matrix and cultured for 48 h. The adhesion and colonization were higher in the cells kept in PBE-media, while the keratinocyte surface marker, cytokeratin 14, was present in all studied groups. These results confirmed the suitability of Saline + HSA as a cell transportation solution for clinical use, which will be the choice for the planned clinical trial. Keratinocyte PBE-media can be an alternative for cells transported for research purpose, if the same media type is going to be used in the following experiments.


Assuntos
Queratinócitos/metabolismo , Regeneração , Albumina Sérica Humana , Pele/patologia , Animais , Biópsia , Bovinos , Adesão Celular , Sobrevivência Celular , Células Cultivadas , Ensaios Clínicos como Assunto , Meios de Cultura , Humanos , Imuno-Histoquímica , Queratina-14/metabolismo , Queratinócitos/citologia , Hipófise/metabolismo , Precursores de Proteínas/metabolismo , Transplante de Pele , Temperatura , Pesquisa Médica Translacional
17.
Anim Genet ; 51(5): 829-832, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32657488

RESUMO

Epidermolysis bullosa simplex (EBS) is a hereditary blistering disease affecting the skin and mucous membranes. It has been reported in humans, cattle, buffaloes and dogs, but so far not in cats. In humans, EBS is most frequently caused by variants in the KRT5 or KRT14 genes. Here, we report a case of feline epidermolysis bullosa simplex and describe the causative genetic variant. An 11-month-old male domestic shorthair cat presented with a history of sloughed paw pads and ulcerations in the oral cavity and inner aspect of the pinnae, starting a few weeks after birth. Clinical and histopathological findings suggested a congenital blistering disease with a split formation within the basal cell layer of the epidermis and oral mucous epithelium. The genetic investigation revealed a homozygous nonsense variant in the KRT14 gene (c.979C>T, p.Gln327*). Immunohistochemistry showed a complete absence of keratin 14 staining in all epithelia present in the biopsy. To the best of our knowledge, this is the first report of feline EBS, and the first report of a spontaneous pathogenic KRT14 variant in a non-human species. The homozygous genotype in the affected cat suggests an autosomal recessive mode of inheritance.


Assuntos
Doenças do Gato/genética , Epidermólise Bolhosa Simples/veterinária , Queratina-14/genética , Animais , Doenças do Gato/patologia , Gatos , Códon sem Sentido , Epidermólise Bolhosa Simples/genética , Epidermólise Bolhosa Simples/patologia , Queratina-14/metabolismo , Masculino
18.
Elife ; 92020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32729832

RESUMO

Keratinocytes are the most abundant cell type in the epidermis, the most superficial layer of skin. Historically, epidermal-innervating sensory neurons were thought to be the exclusive detectors and transmitters of environmental stimuli. However, recent work from our lab (Moehring et al., 2018) and others (Baumbauer et al., 2015) has demonstrated that keratinocytes are also critical for normal mechanotransduction and mechanically-evoked behavioral responses in mice. Here, we asked whether keratinocyte activity is also required for normal cold and heat sensation. Using calcium imaging, we determined that keratinocyte cold activity is conserved across mammalian species and requires the release of intracellular calcium through one or more unknown cold-sensitive proteins. Both epidermal cell optogenetic inhibition and interruption of ATP-P2X4 signaling reduced reflexive behavioral responses to cold and heat stimuli. Based on these data and our previous findings, keratinocyte purinergic signaling is a modality-conserved amplification system that is required for normal somatosensation in vivo.


Assuntos
Queratinócitos/fisiologia , Sensação Térmica/fisiologia , Animais , Cálcio/metabolismo , Células Cultivadas , Temperatura Baixa , Feminino , Temperatura Alta , Humanos , Queratina-14/antagonistas & inibidores , Queratina-14/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos/metabolismo , Sciuridae , Células Receptoras Sensoriais/metabolismo , Transdução de Sinais , Especificidade da Espécie
19.
PLoS One ; 15(7): e0235295, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32687504

RESUMO

Spontaneous mutations in the SHANK-associated RH domain interacting protein (Sharpin) resulted in a severe autoinflammatory type of chronic proliferative dermatitis, inflammation in other organs, and lymphoid organ defects. To determine whether cell-type restricted loss of Sharpin causes similar lesions, a conditional null mutant was created. Ubiquitously expressing cre-recombinase recapitulated the phenotype seen in spontaneous mutant mice. Limiting expression to keratinocytes (using a Krt14-cre) induced a chronic eosinophilic dermatitis, but no inflammation in other organs or lymphoid organ defects. The dermatitis was associated with a markedly increased concentration of serum IgE and IL18. Crosses with S100a4-cre resulted in milder skin lesions and moderate to severe arthritis. This conditional null mutant will enable more detailed studies on the role of SHARPIN in regulating NFkB and inflammation, while the Krt14-Sharpin-/- provides a new model to study atopic dermatitis.


Assuntos
Dermatite Atópica/genética , Inflamação/genética , Queratina-14/genética , Proteínas do Tecido Nervoso/genética , Proteína A4 de Ligação a Cálcio da Família S100/genética , Animais , Apoptose/genética , Artrite/genética , Artrite/patologia , Dermatite Atópica/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Humanos , Imunoglobulina E/genética , Inflamação/patologia , Integrases/genética , Interleucina-18/genética , Queratinócitos/metabolismo , Queratinócitos/patologia , Camundongos , NF-kappa B/genética , Fenótipo , Transdução de Sinais
20.
Clin Genet ; 98(2): 179-184, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32484238

RESUMO

Epidermolysis bullosa (EB) is a heritable blistering disorder. We performed a next-generation sequencing-based multigene panel test and successfully predicted 100% of the EB types, including, 36 EB simplex (EBS), 13 junctional EB (JEB), 86 dystrophic EB (DEB), and 3 Kindler EB. Chinese JEB and recessive DEB (RDEB) patients have relatively mild phenotypes; for severe type separately accounts for 45.5% and 23.8%, respectively. We identified 96 novel and 49 recurrent pathogenic variants in 11 genes, although we failed to detect the second mutation in one JEB and five RDEB patients. We identified one novel p.E475K mosaic mutation in the clinically normal mother of one out of 13 EBS patients with KRT5 mutations, one recurrent p.G2034R mosaic mutation, and one novel p.G2043R mosaic mutation in the clinically normal relatives of two out of 19 dominant DEB patients. This study shows that next-generation technology could be an effective tool in diagnosing EB.


Assuntos
Colágeno Tipo VII/genética , Epidermólise Bolhosa Juncional/genética , Epidermólise Bolhosa/genética , Queratina-14/genética , Queratina-5/genética , China/epidemiologia , Epidermólise Bolhosa/classificação , Epidermólise Bolhosa/epidemiologia , Epidermólise Bolhosa/patologia , Epidermólise Bolhosa Juncional/classificação , Epidermólise Bolhosa Juncional/epidemiologia , Epidermólise Bolhosa Juncional/patologia , Feminino , Predisposição Genética para Doença , Genética Populacional , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Mosaicismo , Mutação/genética , Fenótipo
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