Cytokeratin Protein Research Articles

First Case Report of Primary Testicular Diffuse Large B-Cell Lymphoma from the Western Region of Saudi Arabia

Primary testicular lymphoma (PTL) represents 1-2% of all types of non-Hodgkin lymphomas (NHLs) and 1-10% of testicular neoplasms. Up to the best of my knowledge, this is the first case of PTL of the diffuse large B-cell lymphoma (DLBCL) in a 60-year-old man presented with a painless mass in the left testis as revealed by physical examination in a tertiary care hospital in Al-Madinah Al-Munwarah in the western region of the Kingdom of Saudi Arabia (KSA). Radiological examination revealed a large well-defined heterogeneous predominantly hypo-echoic mass with increased vascularity in the upper portion of the testis. On the other hand, histopathological examination revealed a tumor involving the whole left testis, which was large (measuring 6 3.5 3.3 cm), solid and dark red with focal areas of hemorrhage and epididymal infiltration. Immunohistochemistry showed positivity of leucocyte common antigen (LCA), pan B-cell marker (CD20) and negativity of pan T-cell marker (CD3). Other immunohistochemical markers such as CD10, placental alkaline phosphatase (PLAP), cytokeratin, vimentin, desmin and S100 protein were also negative. However, there was a marked expression of Ki67 and Bcl2 markers. Accordingly, the diagnosis of DLBCL was established. The tumor was classified as stage I according to the Ann Arbor system. The case was treated by orchiectomy followed by prophylactic anthracycline-based chemotherapy and irradiation of the contralateral testis and central nervous system.

Mechanism of nanobacteria promoting apoptosis of human gallbladder epithelial cells

Objective To observe the interaction between nanobacteria (NB) and human gallbladder epithelial cells, and explore the possible mechanism of porcelain gallbladder formation. Methods Absorbing bile from porcelain gallbladder and normal gallbladder to culture nanobacteria, the morphology of NB was observed under an electron microscope. Primary cultured human gallbladder epithelial cells were obtained by cutting out some normal gallbladders, and the expression of Cytokeratin-19 (CK19) was detected by immunofluorescence. NB of 1.0 Macfarland (MCF) was induced into human gallbladder epithelial cells at 0, 12, 24, 48 h. Cell damage and apoptosis rate were detected by Cell Counting Kit-8 (CCK-8), lactate dehydrogenase (LDH) kit and flow cytometry. Protein expression of B-lymphoma-2 (bcl-2), bcl-2 related X protein (bax), cytochrome C (Cyt-C), and cleaved Caspsae-9 were detected by Western blotting. Results The positive rate of NB in porcelain gallbladder and normal gallbladder bile was 88.9% and 15.0%. The difference was statistically significant (χ2=14.39, P<0.01). Under transmission electron microscopy, NB is round and unequal in size and has a diameter of about 80 nm. Immunofl-uorescence showed that CK19 protein was positive in primary cultured human gallbladder epithelial cells. NB induced human gallbladder epithelial cells at 0, 12, 24, 48 h, cell survival rate decreased significantly at 24, 48 h, the difference was statistically significant (F=305.501, P<0.01), the release of LDH began to increase significantly at 12 h, and the difference was statistically significant (F=5351.109, P<0.05). The apoptosis rate increased significantly at 24 h and 48 h, and the difference was statistically significant (F=597.457, P<0.01). The expression of bcl-2/bax was significantly decreased by Western blotting at 24 h and 48 h, and the difference was statistically significant (F=1 336.833, P<0.01). The relative expression of Cty-c was significantly increased at 12, 24, 48 h, and the difference was statistically significant (F=1 422.101, P<0.01). Cleved Caspsae-9 increased significantly at 24 h and 48 h, and the difference was statistically significant (F=2 348.061, P<0.01). Conclusion NB promotes apoptosis of human gallbladder epithelial cells through the mitochondrial pathway, which may be closely related to the formation of porcelain gallbladder. Key words: Nanobacteria; Human gallbladder epithelial cells; Apoptosis; Porcelain gallbladder

Abstract 772: MicroRNA regulatory networks: Concomitant in situ detection of microRNA, mRNA and protein in cancer samples

Abstract The canonical mode of action of microRNAs involves the binding to the 3'UTR of mRNAs thereby mediating repression of protein synthesis. MicroRNAs comprise an interesting new group of cancer biomarkers and may also be therapeutic targets in cancer and other diseases using antisense oligos (ASO). The visualization of potential molecular dynamics between a microRNA, its target mRNA and the effect of protein expression can be facilitated by combined detection in tissue sections. MicroRNAs, as well as ASOs, can be detected in tissue sections or cultured cells by in situ hybridization (ISH) using LNA probes. We have developed a novel ISH assay for microRNA detection with improved sensitivity and signal-to-noise that can be used with any LNA probe on both FFPE and frozen sections. The assay is based on double-labeled LNA™ probes and detection can be either fluorescence or chromogenic staining. We have combined the fluorescence assay with immunofluorescence, for double and triple staining. We also combined the microRNA ISH assay with mRNA in situ hybridization using the highly specific RNAscope probes. We show a variety of combinations, including examples of combined staining of microRNA-17, IL-1beta mRNA or TNF-alpha mRNA, and cytokeratin proteins in automated triple staining procedures of colon cancer samples. The focal expression of IL-1beta mRNA or TNF-alpha mRNA and the loss of microRNA-17 at the invasive front involving inflammatory cells and budding cancer cells is discussed. Citation Format: Boye Schnack Nielsen. MicroRNA regulatory networks: Concomitant in situ detection of microRNA, mRNA and protein in cancer samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 772.

Connexin 43 plays an important role in the transformation of cholangiocytes with Clonochis sinensis excretory-secretory protein and N-nitrosodimethylamine.

BackgroundClonorchis sinensis is a group I bio-carcinogen responsible for cholangiocarcinoma (CHCA) in humans. However, the mechanism by which C. sinensis promotes carcinogenesis is unclear.MethodologyUsing the human cholangiocyte line H69, we investigated cell proliferation and gap junction protein expression after stimulation with the hepatotoxin N-nitrosodimethylamine (NDMA) and/or excretory-secretory products (ESP) of C. sinensis, which induce inflammation. NDMA and ESP treatment increased proliferation by 146% and the proportion of cells in the G2/M phase by 37%. Moreover, the expression of the cell proliferation-related proteins E2F1, Ki-67, and cancer related protein cytokeratin 19 and Cox-2 increased in response to combined treatment with NDMA and ESP. The gap-junction proteins connexin (Cx) 43 and Cx26 increased. In contrast, Cx32 expression decreased in cells treated with NDMA and ESP. Silencing of Cx43 reduced cell proliferation and significantly suppressed Cx26 and Cox-2 expression.ConclusionsThese results suggest that Cx43 is an important factor in CHCA induced by C. sinensis ESP and NDMA and further investigations targeting this pathway may allow prevention of this deadly disease.

Mechanosensitivity response of epithelial HT29 cells on titanium dioxide nanotube array surface via CK8 protein expression

Titanium dioxide nanotube arrays (TNA) have been proposed as novel nanosurface modifications for biomaterial implants and nanomedicine applications. However, the molecular mechanisms of cell-TNA mechanosensitivity are poorly understood. Therefore, this study investigates the effect of cell-TNA mechanosensitivity activity via cytokeratin (CK) protein marker. Field emission scanning electron microscopy (FESEM) characterisation was conducted on TNA with epithelial HT29 cells. The CK protein marker was also analysed using immunofluorescent staining and immunoblotting techniques. FESEM results showed that cells grown on TNA exhibited enhanced cytoplasmic extension and spreading characteristics. Furthermore, the cell interaction with TNA nanosurface showed high CK8 expression, thereby indicating the clustering or aggregation of the cytoskeleton proteins. Findings suggested that epithelial HT29 cells on TNA nanotopography may involve cytoskeleton mechanosensitivity response for cellular adaptation activity.

Nisin, a food preservative produced by Lactococcus lactis, affects the localization pattern of intermediate filament protein in HaCaT cells

Nisin is a food preservative produced by Lactococcus lactis subsp. lactis. Previous blood biochemical research revealed that nisin has physiological effects in mammals; although the site of action has yet to be identified, keratinocytes have been proposed as a possible target. In this study, we investigated whether nisin affects keratinocytes by examining the effects on eukaryotic intermediate filaments in HaCaT human keratinocytes. Treatment with 93 μg/ml nisin for 24 h decreased the localization of the intermediate filament proteins cytokeratin (CK)5 and CK17 at the cell periphery, which were distributed in a limited area in a ring- or net-like shape. However, this was not observed upon treatment for 6 h. The results of a serial dilution assay revealed that the effect on CK17 localization depends on the nisin concentration and were observed at ≥47 μg/ml. Moreover, this effect was partially blocked by treatment with the calcium channel blocker bepridil. Thus, despite the long history of nisin as being safe for humans, it has measurable effects on the keratinocyte cytoskeleton. Our findings also indicate that CK5 and CK17 can serve as markers for evaluating the effects of nisin on keratinocytes.

Characterization of a Clival Chordoma Xenograft Model Reveals Tumor Genomic Instability

Patient-derived xenografts retain the genotype of the parent tumors more readily than tumor cells maintained in culture. The two previously reported clival chordoma xenografts were derived from recurrent tumors after radiation. To study the genetics of clival chordoma in the absence of prior radiation exposure we established a patient-derived xenograft at primary resection of a clival chordoma. Epicranial grafting of clival chordoma collected during surgery was performed. Tumor growth was established in a nonobese diabetic/severe combined immunodeficiency mouse and tumors have been passaged serially for seven generations. Physaliferous cell architecture was shown in the regenerated tumors, which stained positive for Brachyury, cytokeratin, and S100 protein. The tumors showed bone invasion. Single-nucleotide polymorphism analysis of the tumor xenograft was compared with the parental tumor. Copy number gain of the T gene (brachyury) and heterozygous loss of cyclin dependent kinase inhibitor 2A (CDKN2A) was observed. Heterozygous loss of the tumor-suppressor fragile histidine triad (FHIT) gene also was observed, although protein expression was preserved. Accumulation of copy number losses and gains as well as increased growth rate was observed over three generations. The patient-derived xenograft reproduces the phenotype of clival chordoma. This model can be used in the future to study chordoma biology and to assess novel treatments.

Abstract 5195: Increased circulating tumor cell (CTC) clusters are associated with significantly higher levels of HER2 expression and metastasis in stage III/IV breast cancer

Abstract Introduction: Molecular and genomic characterization of CTCs may help to understand breast cancer prognosis and predict treatment benefit, especially for metastatic or recurrent disease. Overexpression of HER2 protein has been associated with rapid cell division and the metastasis and prognosis of advanced breast cancer in the clinic. Herein, we report a correlation between the detection of CTC clusters and HER2 overexpression in otherwise biomarker negative disease. Methods: 146 whole blood samples (7.5ml/each) were collected from 95 patients with stage III/IV breast cancer at baseline, before and after systemic therapy. All samples were transported and stored at 15-30 °C in EDTA tubes. CTC enrichment and enumeration were performed in FDA approved semi-automated fluorescence CELLTRACKS ANALYZERII® System (Janssen Diagnostics) by using CELLSEARCH® CXC Kit (Cell Search) contains ferrofluid-based capture reagent particles with a magnetic core surrounded by a polymeric layer coated with antibodies targeting the Epithelial Cell Adhesion Molecule (EpCAM) antigen for capturing CTCs. Furthermore, it includes immunofluorescent staining reagents including Anti-CK-PE which is specific for the intracellular protein cytokeratin in epithelial cells, DAPI stains the cell nucleus, anti-CD45-APC is specific for leukocytes, and anti-HER-2/neu-FLU is specific for HER-2/neu antigen. The CTCs were classified based on morphology and correct phenotype as CK+, EpCAM+, DAPI+ and CD45-. Database of CTCs was generated and linked with clinical database. Kruskal-Wallis test was used for statistics. Results: Of the 146 samples analyzed, 80 samples contained CTCs, among which 54 cases did not form clusters (Group 1), 11 samples had 1-5 clusters (Group 2), and 15 had &amp;gt;5 clusters (Group 3). The average ratio of CTCs/ total EpCAM+ was directly correlated with the number of clusters and were reported as 15.36%, 25.32% and 43.65% for the 3 cohorts respectively (P&amp;lt;0.01). The highest expression of HER-2 was found in Group 3 (66.67% samples are positive, and 120 positive cells/each sample). This value was significantly higher than Group 2 (27.27% samples are positive, 24.33 positive cells/each sample) and Group 1 (20.37% samples are positive, 6.18 positive cells/each sample) (P&amp;lt;0.001). The results indicated that high level of CTCs clusters strongly associated with over expression of HER2 in CTCs. Conclusion: We demonstrate that patients with detectable CTCs clusters in comparison with CTCs with no or low numbers of clusters, high levels of CTCs clusters (&amp;gt; 5 clusters/7.5ml blood) is a strong indicator for more aggressive of CTCs with high level of HER2 expression, which tend to be more aggressive in metastasis and worse prognosis than HER2-negative breast cancers. Along with and cancer stage and HER2 status, CTCs clusters helps determine your treatment options in clinic. Citation Format: Qiang Zhang, Youbin Zhang, Lisa Flaum, Lorenzo Gerratana, William Gradishar, Leonidas Platanias, Massimo Cristofanilli. Increased circulating tumor cell (CTC) clusters are associated with significantly higher levels of HER2 expression and metastasis in stage III/IV breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5195.

Aneuploid CTC and CEC.

Conventional circulating tumor cell (CTC) detection technologies are restricted to large tumor cells (> white blood cells (WBCs)), or those unique carcinoma cells with double positive expression of surface epithelial cell adhesion molecule (EpCAM) for isolation, and intracellular structural protein cytokeratins (CKs) for identification. With respect to detecting the full spectrum of highly heterogeneous circulating rare cells (CRCs), including CTCs and circulating endothelial cells (CECs), it is imperative to develop a strategy systematically coordinating all tri-elements of nucleic acids, biomarker proteins, and cellular morphology, to effectively enrich and comprehensively identify CRCs. Accordingly, a novel strategy integrating subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH), independent of cell size variation and free of hypotonic damage as well as anti-EpCAM perturbing, has been demonstrated to enable in situ phenotyping multi-protein expression, karyotyping chromosome aneuploidy, and detecting cytogenetic rearrangements of the ALK gene in non-hematologic CRCs. Symbolic non-synonymous single nucleotide variants (SNVs) of both the TP53 gene (P33R) in each single aneuploid CTCs, and the cyclin-dependent kinase inhibitor 2A (CDKN2A) tumor suppressor gene in each examined aneuploid CECs, were identified for the first time across patients with diverse carcinomas. Comprehensive co-detecting observable aneuploid CTCs and CECs by SE-iFISH, along with applicable genomic and/or proteomic single cell molecular profiling, are anticipated to facilitate elucidating how those disparate categories of aneuploid CTCs and CECs cross-talk and functionally interplay with tumor angiogenesis, therapeutic drug resistance, tumor progression, and cancer metastasis.

Clinicopathologic characterisitics of pancreatic acinar cell carcinomas

Objective: To investigate clinical, pathological and immunohistochemical features of pancreatic acinar cell carcinoma. Methods: A retrospective review of surgical and pathological databases between 2011 and 2016 at PLA General Hospital was collected and 14 cases of acinar cell carcinoma (ACC) of the pancreas were identified. EnVision immunohistochemistry was used to detect the expression of Trypsin, bcl-10 and cytokeratin(CK) proteins. Results: The patients included nine cases of pure ACC, 3 cases of mixed acinar ductal carcinoma, 1 case of mixed acinar-neuroendocrine carcinoma and acinar-ductal-neuroendocrine carcinoma, respectively. Tumors involved different anatomic locations of the pancreas, including eight involving the head of pancreas, four in the body and tail, one in the uncinate process and one in a heterotopic pancreas. Two patients had lymph node and liver metastases before surgery. Microscopically, the tumor was hypercellular with less fibroblastic proliferation and tumor cells arranged in acinar or solid pattern. The well differentiated tumor cells showed eosinophilic, granular cytoplasm with single prominent nucleoli, while the poorly differentiated tumor cells tended to grow in solid sheets. Immunohistochemically, the tumor cells were positive for pan-cytokeratin (14/14), Trypsin (12/14) and bcl-10 (11/14). Stains for CK7 and CK19 were negative (11/14 and 3/4). According to the pTNM staging, there were 7 cases at stageⅠ, 3 at stage ⅡA, 3 at stage Ⅲ and 1 at stage Ⅳ. With average postoperative follow-up of 6-58 months, the median disease-free survival time was 16 months. Conclusions: Pancreatic acinar cell carcinoma is a rare and relatively indolent malignant tumor with characteristic histopathological and immunohistochemical features. Accurate pathological diagnosis plays an important role in patients' treatment and evaluation of prognosis.

The alterations of cytokeratin and vimentin protein expressions in primary esophageal spindle cell carcinoma

BackgroundThe accumulated evidence has indicated the diagnostic role of cytokeratin (CK) and vimentin protein immunoassay in primary esophageal spindle cell carcinoma (PESC), which is a rare malignant tumor with epithelial and spindle components. However, it is largely unknown for the expression of CK and vimentin in pathological changes and prognosis of PESC.MethodsEighty-two PESC patients were identified from the esophageal and gastric cardia cancer database established by Henan Key Laboratory for Esophageal Cancer Research of Zhengzhou University. We retrospectively evaluated CK and vimentin protein expressions in PESC. Clinicopathological features were examined by means of univariate and multivariate survival analyses. Furthermore, the co-expression value of cytokeratin and vimentin was analyzed by receiver operating characteristic (ROC) curve.ResultsThe positive pan-cytokeratins AE1/AE3 (AE1/AE3 for short) staining was chiefly observed in cytoplasm of epithelial component tumor cells, with a positive detection rate of 85.4% (70/82). Interestingly, 19 cases showed AE1/AE3 positive staining both in epithelial and spindle components (23.2%). However, AE1/AE3 expression was not observed with any significant association with age, gender, tumor location, gross appearance, lymph node metastasis and TNM stage. Furthermore, AE1/AE3 protein expression does not show any effect on survival. Similar results were observed for vimentin immunoassay. However, in comparison with a single protein, the predictive power of AE1/AE3 and vimentin proteins signature was increased apparently than with single signature [0.75 (95% CI = 0.68–0.82) with single protein v.s. 0.89 (95% CI = 0.85–0.94) with AE1/AE3 and vimentin proteins]. The 1-, 3-, 5- and 7-year survival rates for PESC patients in this study were 79.3%, 46.3%, 28.0% and 15.9%, respectively. Multivariate analysis demonstrated age and TNM stage were independent prognostic factors for overall survival (P = 0.036 and 0.003, respectively). It is noteworthy that only 17.1% patients had a PESC accurate diagnosis by biopsy pathology before surgery (14/82). 72.4% PESC patients with biopsy pathology before surgery had been diagnosed as squamous cell carcinoma.ConclusionThe present study demonstrates that cytokeratin and vimentin protein immunoassay is a useful biomarker for PESC accurate diagnosis, but not prognosis. The co-expression of cytokeratin and vimentin in both epithelial and spindle components suggest the possibility of single clone origination for PESC.

Technical note: Method for isolation of the bovine sweat gland and conditions for in vitro culture

Apocrine sweat glands in bovine skin are involved in thermoregulation. Human, horse, and sheep sweat gland epithelial cells have been isolated and grown in vitro. The present study was conducted to identify a method to isolate bovine sweat glands and culture apocrine bovine sweat gland epithelial cells in vitro. Mechanical shearing, collagenase digestion, centrifugation, and neutral red staining were used to identify and isolate the apocrine glands from skin. Bovine sweat glands in situ and after isolation comprised 2 major cell types consisting of a single layer of cuboidal epithelial cells resting on a layer of myoepithelial cells. In situ, the glands were embedded in a collagen matrix primarily comprising fibroblasts, and some of these cells were also present in the isolated material. The isolated material was transferred to complete medium (keratinocyte serum-free medium, bovine pituitary extract, and human recombinant epidermal growth factor + 2.5% fetal bovine serum) in a T 25 flask (Falcon, Franklin Lakes, NJ) with media film and then incubated at 37°C for 24 h. After sweat glands adhered to the bottom of the flask, an additional 2 mL of complete medium was added and the medium was changed every 3 d. Isolated apocrine sweat glands and bovine sweat gland epithelial cells were immunostained for cytokeratin and fibroblast specific protein, indicating fibroblast-free cultures.

Abstract P1-01-14: The hemidesmosome protein collagen 17A1 is required for collective invasion and growth of mammary tumor organoids

Abstract Background: Invasion is a fundamental step in metastasis, the leading cause of breast cancer-related death. We have shown previously that primary breast tumors invade collectively as clusters of cancer cells (Cheung et al. Cell 2013). These clusters are led by a small subpopulation of cancer cells that highly express the basal epithelial protein cytokeratin 14 (K14) and other stem cell genes (Cheung et al. PNAS 2016). K14+ leader cells metastasize as clusters, reverting to a proliferative growth state upon colonization of a secondary site (Cheung and Ewald. Science 2016). The mechanisms regulating their transitions between collective invasion and growth states remains poorly understood. Understanding these mechanisms could produce new insights into tumor cell collectivity and uncover new ways to treat disseminated cell clusters that have already metastasized. Results: RNA-seq analysis of K14+ and K14- tumor cells revealed that K14+ tumor cells have increased expression of genes encoding the hemidesmosome, a major adhesion complex that plays an essential role in anchoring epithelial cells to the basement membrane. The highest upregulated hemidesmosome gene was collagen 17A1 (COL17A1), a transmembrane protein and structural component of the hemidesmosome that is involved in cell migration during wound healing and tissue regeneration. Consistent with these RNA-seq data, we observed by IF staining in vivo that K14 and COL17A1 are typically co-expressed in collective invasive strands at the invasive front of MMTV-PyMT mammary carcinomas. We next applied ex-vivo 3D culture assays to determine the function of COL17A1 in K14+ cell invasion and proliferation. In mammary tumor organoids we observed that COL17A1 is expressed in basal K14+ tumor cells. However, while the COL17A1 endodomain is uniformly expressed, the ectodomain is expressed in patches, indicating active ectodomain cleavage, a process that reduces cellular motility (Franzke et al. The EMBO Journal 2002). Surprisingly, loss of total COL17A1 expression by shRNA-knockdown significantly impairs tumor organoid growth ˜2 fold and prevents invasion in basement membrane rich matrigel. Further studies are underway to investigate the role of COL17A1 during collective invasion in other extracellular matrix environments. Conclusions: Taken together, our data shows that invasive leader cells express COL17A1 in 3D culture and in vivo and that this protein is required for tumor organoid invasion and growth. Therefore, therapeutically targeting COL17A1, a cell surface protein, could prevent both the migratory and proliferative phases of breast cancer metastasis. We are currently investigating how COL17A1 and its shed COL17A1 ectodomain enable leader cells to toggle between invasive and proliferative states. Citation Format: Greenwood E, Cane Gasull P, Moore B, Cheung K. The hemidesmosome protein collagen 17A1 is required for collective invasion and growth of mammary tumor organoids [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-01-14.

A proteomics analysis of cell signaling pathways in colon cancer.

608 Background: Colorectal cancer is the second most common cancer in women and third most common cancer in men. Major signaling pathways are known to be deregulated in cancer, however, there are only a few studies addressing activation status of more than one or two pathways in colorectal cancer in comparison with the normal tissue. The present study aims to compare the expression pattern of proteins associated with cell signaling in paired tumor and non-tumor samples of patients with colon cancer, as well as to define the cluster of proteins to differentiate patients with non-metastatic (Dukes’ grade B) and metastatic (Dukes’ grade C&amp;D) colon cancer. Methods: Frozen tumor and non-tumor samples were collected after tumor resection from 19 patients with colon cancer. The "Panorama" (Sigma-Aldrich) Antibody Microarray-Cell Signaling kits were used for the analyses. The expression ratios of paired tumor/non-tumor samples were calculated for the each protein. We employed R packages ‘samr’, ‘gplots’, ‘supclust’ ( pelora, wilma algorithms), ‘glmnet’ for the differential expression analysis, supervised clustering and penalized logistic regression, respectively. Results: Significance analysis of microarrays revealed 9 up-regulated proteins, including protein kinase C gamma, c-Myc, MDM2, bCOP, p14ARF, p57kip2, GRB2, APP, pan cytokeratin, and 1 down-regulated protein (GAP1) in tumoral mucosa. Pan cytokeratin and amyloid precursor protein were up-regulated in tumor vs. non-tumor tissue, and were selected in the predictive cluster to discriminate colon cancer type. Higher levels of S-100b and phospho-Tau-pSer199/202 were confirmed as the predictors of non-metastatic colon cancer by all of employed regression/clustering methods. Conclusions: Deregulated proteins in colon cancer are involved in oncogenic signal transduction, cell cycle control, and regulation of cytoskeleton formation/transport. Further studies are needed to validate potential protein markers of colon cancer development and metastatic progression.

The Prognostic Value of Cytokeratin and Sal-Like Protein 4 Expression in Hepatocellular Carcinoma and Intra-Hepatic Cholangiocarcinoma in Taiwan.

Background: We previously reported that modulation of cytokeratin18 induces pleomorphism of liver cells, higher cell motility, and higher drug sensitivity to sorafenib treatment of hepatoma cells. These relationships were established by in vitro experiments. The aim of this study was to determine the in vivo association between cytokeratin expression and tumor behavior, as well as cancer stem cells of hepatocellular carcinoma and intra-hepatic cholangiocarcinoma in Taiwan.Methods: Cytokeratins and sal-like protein 4 expression was determined in 83 hepatocellular carcinoma and 30 intra-hepatic cholangiocarcinoma specimens by immunohistochemistry. The relationship between cytokeratins and sal-like protein 4 expression with hepatitis virus infection, clinicopathologic factors, and survival was analyzed. Further, the correlation among cytokeratins and sal-like protein 4 expression was studied.Results: In addition to cytokeratin8/18, the expression of cytokeratin7/19 and sal-like protein 4 was noted in hepatocellular carcinoma; however, only cytokeratin19 expression had a significant correlation with poor overall survival and poor disease-free survival. The expression of cytokeratins and sal-like protein 4 was not correlated with hepatitis virus infection. The expression of cytokeratin19, but not 7, 8, and 18, was correlated with sal-like protein 4 expression in hepatocellular carcinoma. Cytokeratin7 expression was decreased and the sal-like protein 4 expression was absent in all 30 intra-hepatic cholangiocarcinoma cases. The expression of cytokeratins had not statistically significant correlation with overall and disease-free survival in patients with intra-hepatic cholangiocarcinoma.Conclusions: The expression of cytokeratin19 was associated with sal-like protein 4 expression, as well as poor overall and disease-free survival in hepatocellular carcinoma patients in Taiwan.

Myoepithelioma of the Palate: A Case Report

Myoepithelioma, a benign tumor, occurs with a frequency of 1.5% among all salivary gland tumors, and it most commonly affects the parotid gland. Nearly the entire epithelium is composed of neoplastic myoepithelial cells exhibiting spindle, plasmacytic, epithelial-like, and clear cells. Case Report : A 52-year-old woman with myoepithelioma of the palate. The patient was referred to our hospital with a chief complaint of swelling on the right side of the palate. Following clinical and intraoral examinations, the tumor was removed under general anesthesia. Viewed microscopically, the tumor was seen to be surrounded by a fbrous membrane, primarily composed of a solid growth of neoplastic myoepithelial cells, virtually surrounding a hyaline-like eosinophilic substance. Immunohistochemical examination revealed strongly-positive reactions by the tumor cells for widespectrum cytokeratin (CK wide), vimentin, and S-100 proteins. CK 7, smooth muscle actin (SMA), p63, and glial fibrillary acidic protein (GFAP) gave weakly-positive reactions. Conclusion: Based on the fndings, a pathological diagnosis of myoepithelioma was reached.

Differentiation of human umbilical cord Wharton\u2019s jelly-derived mesenchymal stem cells into endometrial cells

BackgroundWharton’s jelly-derived mesenchymal stem cells (WJ-MSCs) are a novel and promising strategy for tissue engineering because of their ability to differentiate into many cell types. We characterized the differentiation of WJ-MSCs into endometrial epithelial cell (EEC)-like and endometrial stromal cell (ESC)-like cells and assessed the effect of 17β-estradiol and 8-Br-cAMP on the differentiation system.MethodsWJ-MSCs were treated in two ways to differentiate into EEC-like and ESC-like cells respectively: cocultured with ESCs in control/differentiation medium (17β-estradiol, growth factors); and cultured in control/differentiation medium (8-Br-cAMP alone or 8-Br-cAMP plus 17β-estrogen and growth factors). Three signaling pathway inhibitors (SB203580, PD98059, H89) were used to investigate the mechanism of WJ-MSC differentiation into ESC-like cells. Immunofluorescence, western blot and flow cytometry analyses were used to analyze expression of epithelial markers and stromal cell markers. Enzyme-linked immunosorbent assays were used to test the production of secretory proteins associated with the differentiation of ESC-like cells.Results17β-estradiol at 1 μM downregulated vimentin and CD13 and upregulated cytokeratin and CD9 proteins, promoting the differentiation of WJ-MSCs into EEC-like cells in the coculture system. 8-Br-cAMP at 0.5 mM upregulated vimentin and CD13 and downregulated CK and CD9, promoting the differentiation of WJ-MSCs into ESC-like cells. Prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1) were upregulated and the protein kinase A (PKA) signaling pathway was activated, whereas extracellular signal-regulated (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) were not affected.Conclusions17β-estradiol at 1 μM is a good inducer for facilitating the differentiation of WJ-MSCs into EEC-like cells. 8-Br-cAMP plus estrogen and growth factors can induce the differentiation of WJ-MSCs into ESC-like cells. During the differentiation of WJ-MSCs into ESC-like cells, PRL and IGFBP1 were upregulated by the treatment and the PKA signaling pathway was activated, whereas ERK1/2 and p38 MAPK were not affected. These findings suggest a promising approach to the treatment of endometrial damage and other endometrial diseases and suggest new applications for WJ-MSCs in clinical practice.

Development and characterization of cholangioids from normal and diseased human cholangiocytes as an in vitro model to study primary sclerosing cholangitis

Primary sclerosing cholangitis (PSC) is an incurable, fibroinflammatory biliary disease for which there is no effective pharmacotherapy. We recently reported cholangiocyte senescence as an important phenotype in PSC while others showed that portal macrophages accumulate in PSC. Unfortunately, our ability to explore cholangiocyte senescence and macrophage accumulation has been hampered by limited in vitro models. Thus, our aim was to develop and characterize a three-dimensional (3D) model of normal and diseased bile ducts (cholangioids) starting with normal human cholangiocytes (NHC), senescent NHC (NHC-sen), and cholangiocytes from PSC patients. In 3D culture, NHCs formed spheroids of ~5000 cells with a central lumen of ~150 μm. By confocal microscopy and western blot, cholangioids retained expression of cholangiocyte proteins (cytokeratin 7/19) and markers of epithelial polarity (secretin receptor and GM130). Cholangioids are functionally active, and upon secretin stimulation, luminal size increased by ~80%. Cholangioids exposed to hydrogen peroxide exhibited cellular senescence and the senescence-associated secretory phenotype (SASP; increased IL-6, p21, SA‐β-Gal, yH2A.x and p16 expression). Furthermore, cholangioids derived from NHC-sen or PSC patients were smaller and had slower growth than the controls. When co-cultured with THP-1 macrophages, the number of macrophages associated with NHC-sen or PSC cholangioids was five- to seven-fold greater compared to co-culture with non-senescent NHC. We observed that NHC-sen and PSC cholangioids release greater number of extracellular vesicles (EVs) compared to controls. Moreover, conditioned media from NHC-sen cholangioids resulted in an ~2-fold increase in macrophage migration. In summary, we developed a method to generate normal and diseased cholangioids, characterized them morphologically and functionally, showed that they can be induced to senescence and SASP, and demonstrated both EV release and macrophage attraction. This novel model mimics several features of PSC, and thus will be useful for studying the pathogenesis of PSC and potentially identifying new therapeutic targets.

Altered expression of cytokeratin 7 and CD117 in transitional mucosa adjacent to human colorectal cancer

The multi-step progression of colorectal cancer through precancerous lesions (adenoma and dysplasia) is associated with cumulative molecular alterations, a number of which have also been demonstrated to be present in morphologically normal transitional mucosa adjacent to colorectal cancer. The cytoskeletal protein cytokeratin 7 (CK7) and the receptor tyrosine kinase, KIT proto-oncogene receptor tyrosine kinase (CD117), encoded by the proto-oncogene c-Kit, are lacking in normal colorectal crypt epithelium and are aberrantly expressed in a subset of colorectal cancer. The aim of the present study was to evaluate the expression of CK7 and CD117 in morphologically normal transitional mucosa adjacent to colorectal cancer. Immunohistochemical staining for CK7 and CD117 was performed in the mucosa adjacent to five groups of surgically resected colorectal tumors [low-grade adenoma, high-grade adenoma, mucosal adenocarcinoma, small-sized invasive adenocarcinoma (≤2 cm) and large-sized invasive adenocarcinoma (>2 cm)]. CK7 was expressed in the mucosa adjacent to a subset of colorectal tumors, and the positivity ratio increased according to tumor grade from low-grade adenoma up to small-sized invasive adenocarcinoma (61.2%). However, the positivity ratio of CK7 in the mucosa adjacent to the large-sized invasive adenocarcinoma (25.0%) was significantly lower compared with that of the next lower grade. CD117 was also expressed in the mucosa adjacent to a subset of colorectal tumors. In contrast to CK7, the positivity ratio of CD117 increased according to tumor grade from low-grade adenoma all the way through to the large-sized invasive adenocarcinoma (45.0%). Based on these results, the mechanism of CK7 and CD117 expression in the transitional mucosa adjacent to colorectal cancer may be different, and analysis of their individual expression may provide novel insights into the development and progression of colorectal cancer.

Expression of protein components desmosomal apparatus and cytoskeleton of the epidermis in disease Hailey-Hailey

Hailey-Hailey disease (benign familial chronic pemphigus Guzhero-Hailey-Hailey) - hereditary vesiculo-bullous dermatosis, where mutation of ATP2C1 gene results in morphological changes, which cause an abnormal location of molecules in intercellular adhesion. It is believed that the pathology may involve both the inner surface of the plasma membrane proteins of keratinocytes and desmosomal and adhesion molecules in epidermal intercellular connections. This present research provides the results of immunohistochemical study on the distribution of desmosomal cadherin (desmogleins 1, 2, 3), desmosomal proteins (plakoglobin, desmoplakin I, periplakin), intercellular junctions molecules (E-cadherin, cadherin complex) in desmosomal apparatus and cytoskeletal proteins (cytokeratins 5, 14, suprabasal layers of epidermis) in clinically intact and affected skin of patients with Hailey-Hailey disease. The research also detected violation in localization of the protein components in the system of intercellular junctions and the cytoskeleton from severe immunohistochemical reaction to disappearance and appearance in places not typical for its localization. Thus, the most severe expression of protein molecules was observed in the areas of acantholysis and the formation of intra-epidermal blisters with the penetration of specific material into the cytoplasm of keratinocytes and some acantholytic cells. Besides, in the clinically intact skin areas of patients the most intense damage (almost complete absence of expression or its “masking”) was identified as a damage with the cadherin adhesion molecules. The paradoxical phenomenon - intranuclear and/or perinuclear luminescence was marked by cytokeratin 5, desmogleins and cadherins. The complex mechanism of Hailey-Hailey disease involving various adhesion molecules and protein components of intercellular connections system requires ongoing detailed study of the expression of multiple adhesion molecules associated with the system of intercellular junctions. The acquired knowledge will bring understanding of the sequence of events in pathology (acantholysis) development and the identification of a number of additional differential diagnostic signs of Hailey-Hailey disease.