Effect of HaCaT cells with CRISPR-Cas9-induced KRT5 mutation on co-cultured human melanocytes / 中华皮肤科杂志

Objective:To investigate the effect of KRT5 knockdown in keratinocytes on melanin content in co-cultured melanocytes, and to explain mechanisms underlying formation of hyperpigmented lesions in reticulate pigmented anomaly of the flexures (Dowling-Degos disease, DDD) .Methods:HaCaT cells with heterozygous mutations in the KRT5 gene were obtained by using clustered regularly interspaced short palindromic repeats (CRISPR) -CRISPR-associated protein 9 (Cas9) technology (experimental group) , and HaCaT cells transfected with non-targeting single guide RNA:Cas9 protein complex served as control group, both of which were in vitro co-cultured with primary human melanocyte cells (HEMn) separately. Immunofluorescence study was conducted to determine the expression of cytokeratin and melanosomes in co-cultured cells; melanin content was detected in melanocytes in different co-culture groups, which were obtained by differential trypsinization. Immunohistochemical study was performed to determine the expression of melanocyte-specific premelanosome protein 17 (Pmel17) in skin lesions in a patient with DDD carrying a KRT5 mutation and normal skin tissues in a healthy control. Results:Sanger sequencing showed a heterozygous mutation (c.1delA) at the initiation codon of exon 1 of the KRT5 gene in HaCaT cells in the experimental group, but no mutation in the KRT5 gene in the control group. Western blot analysis showed that the KRT5 protein expression was significantly lower in the experimental group (0.60 ± 0.05) than in the control group (1.00 ± 0.00, t = 32.38, P = 0.001) . Compared with the co-culture system in the control group, the number of Pmel17-labeled melanosomes markedly increased with the melanin content elevated by 52.5% ( t = -3.48, P = 0.025) in the HEMn cells co-cultured with HaCaT cells in the experimental group. Immunohistochemical study showed that the Pmel17 expression increased in the skin lesions in the DDD patient with KRT5 mutation compared with the normal skin tissues in the healthy control. Conclusion:The effect of HaCaT cells with CRISPR-Cas9-induced KRT5 mutation on the co-cultured HEMn melanocytes was verified by the successfully established in vitro co-culture system, which provides a primary cell model for further studies on interaction mechanisms between keratinocytes and melanocytes, and on pathogenesis of skin pigmentation abnormalities.

Establishment of a culture method for primary human nail matrix cells in serum-free media / 中华皮肤科杂志

Objective To establish a culture method for primary human nail matrix cells in serumfree media.Methods Nail matrix tissues were collected from 9 patients,who received nail or toe amputation and nail bed repair in Peking University Shenzhen Hospital between January 2016 and December 2016,and cultured in the serum-free DEME/F-12 media at a 37℃ incubator with an atmosphere of 5％ CO2 in air for 2-3 days.Then,primary human nail matrix cells were cultured in keratinocyte serumfree media (CnT-07),and the morphology of human nail matrix cells was observed by microscopy during the culture process.Immunofluorescence cytochemistry with anti-keratin 5 (K5) and K10 was performed to identify the acquired cells,and flow cytometry to analyze the cell purity.Results After 2 or 3 days of the culture,some cells began to crawl out from the tissue.On day 10,large cell masses were formed,some cells were morphologically similar to epithelioid cells arranged in a paving stone-like pattern,and some were flat giving a spindle-shaped or star-shaped appearance.Immunofluorescence cytochemistry showed that some cells could express both K5 and K10,which proved the existence of nail matrix cells,and 37.6％ of the cells expressed K10.Conclusion Human primary nail matrix cells could be successfully cultured by using the tissue culture method with serum-free culture media,and the nail matrix cells cultured in vitro can express both K5 and K10.

Application value of cell keratin 7 ,thyroid transcription factor-1,Napsin A,cell keratin5/6,p40 and p63 immunohistochemical stains in lung adenocarcinoma and squamous cell carcinoma / 中国综合临床

Objective To study the significant immunohistochemical marker to identify lung adenocarcinoma(ADC) and squamous cell carcinoma(SCC).Methods Three hundred and twenty-nine Choose 329 cases of nonsmall-cell lung cancer (NSCLC) were chosen.Analysis of the clinical and pathological features.The expression of cell keratin 7 (CK7),thyroid transcription factor-1 (TTF-1),Napsin A,CK5/6,p40 and p63 were detected by using immunohistochemistry.Results (1) Among 329 specimens,containing 129 cases of resections,195 cases of biopsies and 5 cases of pleural effusion specimens.(2)In these cases,187 cases were classified to be ADC,142 cases were classified to be SCC.(3) CK7,TTF-1,Napsin A,CK5/6,p40,p63 sensitivity were 97.9％,87.2％,81.3％,6.4％,3.7％,18.7％ in ADC groups,and 25.4％,11.3％,0,92.3％,95.1％,98.6％ in SCC groups,and the differences of two groups were significant statistically (x2 =190.665,187.432,214.542,242.003,274.407,206.818;P＜ 0.001).(4) In the 3 IHC of ADC,CK7 had the highest sensitivity,Napsin A had the highest specificity.In the 3 IHC of SCC,p63 had the highest sensitivity,p40 had the highest specificity.Conclusion CK7,TTF-1,Napsin A,CK5/6,p40 and p63 can be a markable panel of immunohistochemistry in the differential diagnosis of NSCLC.

Weber-Cockayne Type Epidermolysis Bullosa Simplex Resulting from a Novel Mutation (c. 608T>C) in the Keratin 5 Gene

Epidermolysis bullosa simplex (EBS), an inherited genetic disorder, is most often caused by a dominant-negative mutation in either the keratin 5 (KRT5) or the keratin 14 (KRT14) gene. These keratin mutants result in a weakened cytoskeleton and cause extensive cytolysis. It is important to analyze the KRT5 or KRT14 genes of the patient and their family members by mutational analysis in order to identify genetic defects as well as the need for genetic counseling. In this study, we present a 5-year-old Korean boy who had been developing blisters and erosions on the palms of his hands and soles of his feet since infancy. In addition, while his younger sister and father showed similar clinical manifestation, his mother did not. The patient was diagnosed with EBS based on clinical manifestation, which is characterized by the presence of blisters restricted to the palms and soles, histological findings, and mutational analysis. Mutational analysis of the patient's DNA revealed a thymine-to-cytosine transition at codon 608 in the KRT-5 gene, resulting in a leucine-to-proline substitution in the keratin 5 protein. The same mutation was identified in the paternal, but not maternal, DNA. Here, we report a case of Weber-Cockayne type EBS with vesicles and bullae restricted to the palms and soles with a novel, paternally inherited mutation in KRT5 gene (exon2, c.608T>C).

Comparison of nuclear grade and immunohistochemical features in situ and invasive components of ductal carcinoma of breast / Comparação do grau nuclear e perfil imunoistoquímico nos componentes in situ e invasivo de carcinoma mamário

PURPOSE:To compare the prognostic and predictive features between in situ and invasive components of ductal breast carcinomas. METHODS:We selected 146 consecutive breast samples with ductal carcinoma in situ (DCIS) associated with adjacent invasive breast carcinoma (IBC). We evaluated nuclear grade and immunohistochemical expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), cytokeratin 5/6 (CK5/6), and epidermal growth factor receptor (EGFR) in both components, in situ and invasive, and the Ki-67 percentage of cells in the invasive part. The DCIS and IBC were classified in molecular surrogate types determined by the immunohistochemical profile as luminal (RE/PR-positive/ HER2-negative), triple-positive (RE/RP/HER2-positive), HER2-enriched (ER/PR-negative/HER2-positive), and triple-negative (RE/RP/HER2-negative). Discrimination between luminal A and luminal B was not performed due to statistical purposes. Correlations between the categories in the two groups were made using the Spearman correlation method. RESULTS:There was a significant correlation between nuclear grade (p<0.0001), expression of RE/RP (p<0.0001), overexpression of HER2 (p<0.0001), expression of EGFR (p<0.0001), and molecular profile (p<0.0001) between components in situ and IBC. CK 5/6 showed different distribution in DCIS and IBC, presenting a significant association with the triple-negative phenotype in IBC, but a negative association among DCIS. CONCLUSIONS: Our results suggest that classical prognostic and predictive features of IBC are already determined in the preinvasive stage of the disease. However the role of CK5/6 in invasive carcinoma may be different from the precursor lesions.

OBJETIVO: Comparar características prognósticas e preditivas entre os componentes in situ e invasivo de carcinomas ductais da mama. MÉTODOS: Selecionamos 146 amostras mamárias consecutivas com carcinoma ductal in situ (CDIS) associado com carcinoma invasivo (CI) adjacente. Avaliamos grau nuclear e a expressão imunoistoquímica de receptor de estrogênio (RE), receptor de progesterona (RP), receptor do fator de crescimento epidérmico humano 2 (HER2), citoqueratina 5/6 (CK5/6) e o receptor do fator de crescimento epidérmico (EGFR) em ambos componentes, in situ e invasor, e a porcentagem de células marcadas pelo Ki-67 no componente invasivo. CDIS e CI foram classificados nos tipos moleculares, determinados pelo perfil imunoistoquímico, como luminal (RE/RP-positivo/HER2-negativo), triplo-positivo (RE/RP/HER2-positivo), HER2-puro (RE/RP-negativo/HER2-positivo) e triplo-negativo (RE/RP/HER2-negativo). A discriminação entre luminal A e Luminal B não foi feita por motivos estatísticos. Correlações entre as categorias dos dois grupos foram feitas pelo método de correlação de Spearman. RESULTADOS: Houve significante associação entre grau nuclear (p<0,0001), expressão de RE/RP) (p<0,0001), superexpressão de HER2 (p<0,0001), expressão de EGFR (p<0,0001) e perfil molecular (p<0,0001) entre os componentes in situ e invasivo. CK5/6 mostrou distribuição distinta em CDIS e CI, apresentando significante associação com o fenótipo triplo-negativo em CI, mas uma associação negativa ente os CDIS. CONCLUSÕES:Nossos resultados sugerem que as características prognósticas e preditivas clássicas dos CI estão já determinadas no estágio pré-invasivo da doença. Entretanto, o papel da CK5/6 no carcinoma invasivo pode ser diferente daquele das lesões precursoras.

Doença de Dowling-Degos: apresentação clínica e histopatológica clássica / Dowling-Degos disease: classic clinical and histopathological presentation

A doença de Dowling-Degos é uma genodermatose rara que consiste numa desordem pigmentar reticulada. Caracteriza-se pela presença de máculas hiperpigmentadas nas regiões flexurais com distribuição em rede; lesões tipo comedão no dorso e na região cervical; e cicatrizes cribriformes na face, particularmente periorais. Apresentamos um caso de um paciente de 51 anos, masculino, com lesões tipo macrocomedões, cicatrizes cribriformes, cistos e máculas hipercrômicas no dorso, tórax anterior, axilas, pescoço, região genital e face. Relatava ter dois filhos, três irmãos e o pai com quadro semelhante. As biópsias de pele foram características da doença de Dowling-Degos, mostrando dilatação folicular, epiderme digitiforme, com áreas de aspecto de "chifre de veado" e focos de hiperpigmentação da camada basal.

Dowling-Degos disease (DDD) is a rare genetic disease of the skin (reticulate pigmented anomaly), clinically characterized by flexural brown pigmented reticulate macules, comedo-like papules on the back, neck and pitted perioral or facial scars. We present the case of a 51 year-old man with macrocomedo-like lesions, pitted scars, cysts, hyperpigmented macules in his back, chest, axillae, neck, groin and face. The patient reported having two children, three brothers and a father with a similar condition. The histopathology of the skin biopsies was very characteristic of Dowling-Degos disease, showing dilated follicular, fingerlike projections called rete ridges (dermal pegs), with thinning of the suprapapillary plates, resulting in an "antler-like" pattern and increased pigmentation of the basal layer.