X-linked ichthyosis (XLI) is a recessively inherited ichthyosis. Skin barrier function of XLI patients reported in Western countries presented minimally abnormal or normal. Here, we evaluated the skin barrier properties and a skin barrier-related gene mutation in 16 Korean XLI patients who were diagnosed by fluorescence in situ hybridization and array comparative genomic hybridization analysis. Skin barrier properties were measured, cytokine expression levels in the stratum corneum (SC) were evaluated with the tape stripped specimen from skin surface, and a genetic test was done on blood. XLI patients showed significantly lower SC hydration, but normal basal trans-epidermal water loss and skin surface pH as compared to a healthy control group. Histopathology of ichthyosis epidermis showed no acanthosis, and levels of the pro-inflammatory cytokines in the corneal layer did not differ between control and lesional/non-lesional skin of XLI patients. Among the mutations in filaggrin (FLG), kallikrein 7 (KLK7), and SPINK5 genes, the prevalence of KLK7 gene mutations was significantly higher in XLI patients (50%) than in controls (0%), whereas FLG and SPINK5 prevalence was comparable. Korean XLI patients exhibited unimpaired skin barrier function and frequent association with the KLK7 gene polymorphism, which may differentiate them from Western XLI patients.
Adolescent ; Adult ; Asian Continental Ancestry Group/*genetics ; Child ; Chromosomes, Human, X ; Comparative Genomic Hybridization ; Cytokines/metabolism ; Humans ; Hydrogen-Ion Concentration ; Ichthyosis/diagnosis/*genetics/pathology ; In Situ Hybridization, Fluorescence ; Intermediate Filament Proteins/genetics ; Kallikreins/*genetics ; Male ; Polymorphism, Single Nucleotide ; Proteinase Inhibitory Proteins, Secretory/genetics ; Republic of Korea ; Skin/metabolism/*pathology ; Young Adult

X-linked ichthyosis (XLI) is a recessively inherited ichthyosis. Skin barrier function of XLI patients reported in Western countries presented minimally abnormal or normal. Here, we evaluated the skin barrier properties and a skin barrier-related gene mutation in 16 Korean XLI patients who were diagnosed by fluorescence in situ hybridization and array comparative genomic hybridization analysis. Skin barrier properties were measured, cytokine expression levels in the stratum corneum (SC) were evaluated with the tape stripped specimen from skin surface, and a genetic test was done on blood. XLI patients showed significantly lower SC hydration, but normal basal trans-epidermal water loss and skin surface pH as compared to a healthy control group. Histopathology of ichthyosis epidermis showed no acanthosis, and levels of the pro-inflammatory cytokines in the corneal layer did not differ between control and lesional/non-lesional skin of XLI patients. Among the mutations in filaggrin (FLG), kallikrein 7 (KLK7), and SPINK5 genes, the prevalence of KLK7 gene mutations was significantly higher in XLI patients (50%) than in controls (0%), whereas FLG and SPINK5 prevalence was comparable. Korean XLI patients exhibited unimpaired skin barrier function and frequent association with the KLK7 gene polymorphism, which may differentiate them from Western XLI patients.
Adolescent ; Adult ; Asian Continental Ancestry Group/*genetics ; Child ; Chromosomes, Human, X ; Comparative Genomic Hybridization ; Cytokines/metabolism ; Humans ; Hydrogen-Ion Concentration ; Ichthyosis/diagnosis/*genetics/pathology ; In Situ Hybridization, Fluorescence ; Intermediate Filament Proteins/genetics ; Kallikreins/*genetics ; Male ; Polymorphism, Single Nucleotide ; Proteinase Inhibitory Proteins, Secretory/genetics ; Republic of Korea ; Skin/metabolism/*pathology ; Young Adult

Research of the FLG mutation in various ethnic groups revealed non-overlapping mutation patterns. In addition, Japanese and Chinese atopic patients showed somewhat different mutations. These ethnic differences make the research on Korean patients mandatory; however, no systematic research on Korean atopic dermatitis (AD) patients has been performed. This study aims to investigate the genetic polymorphism of FLG in Korean atopic dermatitis patients. The study was made up of three groups including 9 Ichthyosis vulgaris (IV) patients, 50 AD patients and 55 normal controls: the ichthyosis group was incorporated due to the reported association between the FLG mutation and IV. In comparison to other sequencing methods, the overlapping long-range PCR was used. We revealed the genetic polymorphism of filaggrin in Koreans, and at the same time, we discovered nonsense mutations in p.Y1767X and p.K4022X in Korean AD patients. By using FLG sequencing techniques confirmed in this study, new mutations or genetic polymorphisms with ethnic characteristics would be detected and further larger studies of repeat number polymorphisms could be performed.
Adult ; Alleles ; Asian Continental Ancestry Group/*genetics ; Base Sequence ; Codon, Nonsense ; DNA/blood/chemistry/metabolism ; DNA Mutational Analysis ; Dermatitis, Atopic/*genetics ; Female ; Genotype ; Heterozygote ; Humans ; Ichthyosis Vulgaris/genetics ; Intermediate Filament Proteins/*genetics ; Male ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide

Alpha-internexin (INA) is a proneuronal gene-encoding neurofilament interacting protein. INA is overexpressed mostly in oligodendroglial phenotype gliomas, is related to 1p/19q codeletion, and is a favorable prognostic marker. We studied INA expression in oligodendrogliomas (ODGs) and glioblastomas (GBMs) to verify its association with several molecular phenotypes, 1p/19q codeletion, and epidermal growth-factor-receptor (EGFR) amplification. A total of 230 low- and high-grade ODG and GBM cases was analyzed for INA expression by immunohistochemical staining; and 1p/19q and EGFR gene status was examined by fluorescence in-situ hybridization. INA was positive in 80.3% of ODGs and in 34.3% of GBMs. 1p/19q codeletion was detected in 77.0% of ODGs and 5.5% of GBMs. INA and 1p/19q codeletion were strongly correlated (P < 0.001). The specificity of INA expression for 1p/19q codeletion was 70.8%, while sensitivity was 100%; positive predictive value was 72.5%, and negative predictive value was 29.2% in all 228 tumors. INA expression was correlated with better progression-free survival (PFS) and overall survival (OS) (P = 0.001). In conclusion, INA expression has high specificity and sensitivity to predict 1p/19q codeletion, and it is well correlated with PFS of both ODGs and GBMs. Therefore, INA expression could be a simple, reliable, and favorable prognostic and surrogate marker for 1p/19q codeletion and long term survival.
Adult ; Brain Neoplasms/*metabolism/mortality/pathology ; Chromosomes, Human, Pair 1 ; Chromosomes, Human, Pair 19 ; Female ; Gene Deletion ; Glioblastoma/*metabolism/mortality/pathology ; Humans ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Intermediate Filament Proteins/genetics/*metabolism ; Kaplan-Meier Estimate ; Male ; Middle Aged ; Oligodendroglioma/*metabolism/mortality/pathology ; Phenotype ; Predictive Value of Tests ; Prognosis ; Receptor, Epidermal Growth Factor/genetics/metabolism

OBJECTIVETo explore the P75NTR expression in the mouse testis and its relationship with nestin.

METHODSWe observed the location of the expressions of P75NTR and nestin in the testis of the nestin-GFP transgenic mouse on postnatal day (PND) 5, 14 and 30 using immunofluorescence, and detected the expression levels of P75NTR in the testicular tissue of mice in different age groups by real-time quantitative PCR (RTqPCR) and flow cytometry. Then we cultured the P75NTR positive cells in neural stem cell culture medium and observed their neuronal differentiation capacity by orientation differentiation.

RESULTSImmunofluorescence showed the expressions of P75NTR and nestin in the Leydig cells of the mouse testis. RTqPCR and flow cytometry exhibited the peak of the P75NTR expression on PND 14. The positive rates of P75NTR were (2.88 +/- 0.52), (9.54 +/- 1.81) and (2.63 +/- 0.43)% on PND 5, 14 and 30, respectively. The P75NTR positive cells obtained also expressed nestin and P75NTR and had the capacity of neuronal differentiation.

CONCLUSIONP75NTR and nestin are co-expressed in the Leydig cells of the mouse testis, and the P75NTR positive cells have the ability of neural differentiation, which is presumably attributed to neural crest cells.

Animals ; Intermediate Filament Proteins ; genetics ; metabolism ; Leydig Cells ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Nerve Tissue Proteins ; genetics ; metabolism ; Nestin ; Receptor, Nerve Growth Factor ; genetics ; metabolism ; Testis ; cytology ; metabolism

OBJECTIVEDuring the process of tissue remodeling in human tumor transplantation models, the roles of the inoculated tumor cells and host tissue in tumor progression is still largely unknown. The aim of this study was to investigate the relationships and interactions between these two sides using GFP-RFP double fluorescence tracing technique.

METHODSRed fluorescence protein (RFP) gene was stably transfected into glioma stem cell line SU3, then SU3-RFP cells were transplanted into the brain of athymic nude mice with green fluorescence protein (GFP) expression. After the intracerebral tumors were formed, the relationship and interaction between GFP cells and RFP cells were analyzed. Highly proliferative GFP cells were screened out, and monocloned with micro-pipetting. DNA content assay, chromosome banding and carcinogenicity test of the GFP cells were performed to observe the GFP cells' cancerous phenotype in nude mice.

RESULTSIn the transplantable tumor tissue, besides a great quantity of RFP cells, there were still a proportion of GFP cells and GFP/RFP fusion cells. The proportion of RFP cells, GFP cells and GFP/RFP cells were (88.99 ± 1.46)%, (5.59 ± 1.00)%, and (4.11 ± 1.020)%, respectively. Two monoclonal host GFP cells (H1 and H9) were cloned, which demonstrated the properties of immortality, loss of contact inhibition, and ultra-tetraploid when cultured in vitro. Both H1 and H9 cells expressed CNP, a specific marker of oligodendrocytes. The GFP cells also demonstrated 100% tumorigenic rate and high invasive properties in vivo.

CONCLUSIONSIn this glioma transplantation model, the transplanted tumor tissues contained not only transplanted glioma stem cells but also cancerous host GFP cells. Our findings offer important clues to further research on the relationships among different members in the tumor microenvironment.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase ; metabolism ; Animals ; Brain ; cytology ; metabolism ; Cell Communication ; Cell Line, Tumor ; Cell Transformation, Neoplastic ; Glioma ; metabolism ; pathology ; Green Fluorescent Proteins ; metabolism ; Humans ; Intermediate Filament Proteins ; metabolism ; Luminescent Proteins ; genetics ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Nude ; Neoplasm Transplantation ; Neoplastic Stem Cells ; cytology ; metabolism ; Nerve Tissue Proteins ; metabolism ; Nestin ; Neuroglia ; cytology ; metabolism ; Transfection ; Tumor Microenvironment

OBJECTIVETo study the effect of lentivirus-mediated RNA interference (RNAi) of nestin on the metastatic potential of human melanoma cell line UACC903.

METHODSA lentiviral vector for RNAi targeting the coding region of human nestin mRNA (nestin-RNAi-LV) and another control vector containing a nonsense sequence were constructed. The vectors were transfected into UACC903 cells, and nestin expression in the cells was detected by RT-PCR, immunofluorence assay and Western blotting. The invasive ability and migration of the transfected UACC903 cells was evaluated using Transwell and scrape assay, respectively. Fluorescence assay was used to examine the expressions of E-cadherin, N-cadherin and β-catenin in the cells.

RESULTSThe lentiviral vector nestin-RNAi-LV was constructed successfully. Compared with the control vector, nestin-RNAi-LV resulted in obviously reduced expression of nestin mRNA and protein, lowered migration ability of UACC903 cells, and reduced cell adhesion and invasiveness (P<0.05).

CONCLUSIONLentivirus-mediated nestin RNAi can specifically inhibit nestin expression to cause decreased cell migration and invasiveness of human melanoma cell line UACC903.

Cell Line, Tumor ; Cell Movement ; genetics ; Genetic Vectors ; Humans ; Intermediate Filament Proteins ; genetics ; metabolism ; Lentivirus ; genetics ; Melanoma ; pathology ; Neoplasm Metastasis ; genetics ; Nerve Tissue Proteins ; genetics ; metabolism ; Nestin ; RNA Interference ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics

To construct the recombinant adenovirus vector expressing specific siRNA for rat retinoic acid receptor-beta (RARbeta) gene, and to detect its effect on RARbeta expression and neuronal differentiation of all-trans retinoic acid (ATRA) treated mesenchymal stem cells (MSCs). First, we designed four pairs of siRNA sequence for rat RARbeta gene and annealed complementary oligonucleotides in vitro, then cloned double-stranded DNA in pSES-HUS vector containing U6/H1 double-promoter and recombinated with the backbone vector to construct pAd-siRARbeta plasmid. We infected MSCs by using adenovirus Ad-siRARbeta which was packaged in HEK293 cell line, then performed Real-time, Western blotting and immunoflourencence to detect the expression of RARbeta. We used combination of ATRA and MNM to induce MSCs into neural-like cells, then performed Real-time PCR and immunoflourencence to detect neuronal specific markers of induced neural cells. By using PCR, endonuclease cutting and gene sequencing, we confirmed that the target genes were correctly cloned in adenovirus vector. We could observe more than 60% RFP-positive MSCs at 24 h after adenovirus infection. The expression of RARbeta was significantly increased to 16.5 +/- 2.34 fold in ATRA treated MSCs (P < 0.05) and located in nucleus. Three of four pairs siRNA could effectively inhibit the expression of RARbeta with inhibition efficacy of (66.26 +/- 9.12)%, (48.70 +/- 5.78)%, (64.09 +/- 0.53)% (P < 0.05), especially siRNA-pool group with inhibition efficacy of (78.09 +/- 4.24)% (P < 0.01). Combination of ATRA and MNM induced MSCs into neural-like cells which expressed neuronal specific markers, Nestin, NSE, MAP-2, and Tau. Immunoflourencence result showed that about 50-88 present of cells were positive for Nestin, NSE, Tjul, however, adenovirus medicated expression of siRARbeta could effectively inhibit the expression level of neural specific proteins and the ratio of positive stained cells (P < 0.05). Therefore, we successfully constructed the recombinant adenovirus vector containing siRNA for rat RARP gene, adenovirus could effectively infect MSCs and inhibit the expression of induced RARbeta in ATRA treated MSCs, then inhibit neuronal differentiation of MSCs.
Adenoviridae ; genetics ; metabolism ; Animals ; Cell Differentiation ; genetics ; Down-Regulation ; HEK293 Cells ; Humans ; Intermediate Filament Proteins ; metabolism ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Nerve Tissue Proteins ; metabolism ; Nestin ; Neurons ; cytology ; RNA, Small Interfering ; genetics ; Rats ; Receptors, Retinoic Acid ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection ; Tretinoin ; pharmacology

The purpose of this study was to measure the thickness of canine epidermis at various anatomical sites according to localization of cornified envelopes (involucrin and filaggrin), keratins (keratin 10, 5), and their mRNA expression. This was done in the skin of five breeds of dogs including seven poodles, six golden retrievers, six Shih Tzus, four pugs, and four Labrador retrievers. Epidermal thickness of the stratum corneum and nucleated epidermal layer was significantly different. The greatest thickness was observed in the digital web area and the thinnest epidermis was in the axilla. Epidermal thickness was also significantly different between the breeds (p < 0.05). Immunohistochemical staining scores revealed significant decreases of involucrin, filaggrin, and keratin 10 in the ventral and weight-bearing sites, and a relative increase of keratin 5 (p < 0.05). q-PCR analysis showed that their the levels of mRNA were positively correlated with expression of the corresponding proteins in skin samples (p < 0.05). The present study is the first to report the relationship between epidermal gene expression and histologic morphology of the skin in normal dogs. Further studies will be essential to fully understand the pathogenesis of skin barrier dysfunctions in canines.
Animals ; DNA, Complementary/genetics/metabolism ; Dogs/anatomy & histology/genetics/*metabolism ; Gene Expression Regulation/*physiology ; Intermediate Filament Proteins/genetics/*metabolism ; Keratin-10/genetics/*metabolism ; Keratin-5/genetics/*metabolism ; Polymerase Chain Reaction/methods/veterinary ; Protein Precursors/genetics/*metabolism ; RNA/genetics/metabolism ; Skin/anatomy & histology/metabolism

OBJECTIVETo study the clinical and pathologic features of gastric schwannomas.

METHODSThe macroscopic and microscopic features of 9 cases of gastric schwannoma were analyzed. Immunohistochemical study for S-100 protein, CD117, CD34, neurofilament, desmin, nestin, glial fibrillary acidic protein, platelet derived growth factor-alpha (PDGFR-α) and vimentin was carried out. Mutation analysis of c-kit gene (exon 9, 11, 13 and 17) and PDGFR-α gene (exon 12 and 18) in 1 case was examined by PCR amplification and direct sequencing.

RESULTSThe patients included 5 males and 4 females. The age of patients ranged from 42 to 81 years (median = 56.5 years). The size of the tumors ranged from 2 to 9 cm in greatest diameter. Follow-up data in 8 cases (from 1 month to 65 months) showed no evidence of recurrence or metastasis. Gross examination showed that gastric schwannomas were homogeneous, firm, yellow-white and bore no true fibrous capsule. Histologically, all cases were composed of fascicles of spindle cells associated with nuclear palisading, Verocay body formation and peripheral cuff of reactive lymphoid aggregates. Some of them showed degenerative changes including cyst formation, calcification, hemorrhage, necrosis and hyalinization. Immunohistochemical study showed that the tumor cells were strongly positive for S-100 protein and vimentin. There was various degree of staining for nestin (8/9) and glial fibrillary acidic protein (6/9). They were negative for CD117, CD34, neurofilament, desmin and smooth muscle actin. One case showed focal positivity for PDGFR-α (1/9), with no mutations found.

CONCLUSIONSGastric schwannomas share similar histologic features with conventional soft tissue schwannomas, in addition to the presence a reactive lymphoid cuff. The clinical, macroscopic, histologic and immunohistochemical features of gastric schwannomas were different from those of gastrointestinal stromal tumors and leiomyomas.

Adult ; Aged ; Aged, 80 and over ; Diagnosis, Differential ; Exons ; Female ; Follow-Up Studies ; Gastrectomy ; methods ; Gastrointestinal Stromal Tumors ; metabolism ; pathology ; Glial Fibrillary Acidic Protein ; metabolism ; Humans ; Intermediate Filament Proteins ; metabolism ; Leiomyoma ; metabolism ; pathology ; Leiomyosarcoma ; metabolism ; pathology ; Male ; Middle Aged ; Mutation ; Nerve Tissue Proteins ; metabolism ; Nestin ; Neurilemmoma ; metabolism ; pathology ; surgery ; Neurofibroma ; metabolism ; pathology ; Receptor, Platelet-Derived Growth Factor alpha ; genetics ; metabolism ; S100 Proteins ; metabolism ; Stomach Neoplasms ; metabolism ; pathology ; surgery ; Vimentin ; metabolism