Fluorescence-based immunosensor using three-dimensional CNT network structure for sensitive and reproducible detection of oral squamous cell carcinoma biomarker.

A hierarchical three-dimensional network of carbon nanotubes on Si pillar substrate (3DN-CNTs) was developed for the accurate detection of oral squamous cell carcinoma (OSCC) in clinical saliva samples. The 3DN-CNTs were uniformly coated with a layer of aluminum oxides to enhance structural stability during biomarker detection. Cytokeratin-19 antigen (Cyfra 21-1) was utilized as a model biomarker of OSCC for fluorescence-based immunosensor using 3DN-CNTs (3DN-CNTs sensor). The 3DN-CNTs sensor enhances the sensitivity of Cyfra 21-1 detection by increasing the density of immobilized antibody through high surface area of 3DN-CNTs and enhancing the accessibility of biomolecules through the ordered pathway of hierarchical structure. The reliable detection limit for sensing of Cyfra 21-1 was estimated as in the level of 0.5 ng/mL and the quantitative estimation of Cyfra 21-1 was analyzed by 4-parameter logistic (4-PL) model for curve-fitting analysis. Clinical applicability of 3DN-CNTs sensor was evaluated through correlation with the commercially available electrochemiluminescence (ECL) detection system in the hospital. The assay results of the two systems for clinical saliva samples showed a good linear correlation. The 3DN-CNTs sensor offers great potential for accurate diagnosis of OSCC using Cyfra 21-1 biomarker in clinical fluids.

Efficacy of computed tomography-guided implantation of 125I seeds in the treatment of refractory malignant tumors accompanied with cancer pain and its influence on tumor markers in the serum.

OBJECTIVE: This study intended to explore the efficacy of computed tomography (CT)-guided implantation of iodine-125 (125I) seeds in the treatment of refractory malignant tumors with cancer pain and its influence on tumor markers in the serum. PATIENTS AND METHODS: 76 patients with refractory malignant tumors accompanied by cancer pain that received treatments in LongHua Hospital Shanghai University of Traditional Chinese Medicine from September 2013 to August 2014 were selected. They were divided into control group and observation group using a random number table (38 patients in each group). Patients in the control group received simple chemotherapy, while those in the observation group undergone CT-guided implantation of 125I seeds in combination with chemotherapy. Recent efficacy and 1-3-year survival rate were compared between the two groups of patients. The degree of pain relief after treatment was also compared between the two groups of patients. Electrochemiluminescence method was used to detect the concentrations of carcinoembryonic antigen (CEA), sugar chain antigen 199 (CA 199), sugar chain antigen 125 (CA 125), neuron-specific enolase (NSE) and cytokeratin-19-fragment (CYFRA21-1) in the two groups of patients before treatment, and 3 days, 7 days and 30 days after treatment. RESULTS: Recent disease control rate of the patients in the observation group was higher than that of the patients in the control group (p<0.05). The 1-3-year survival rate after surgery in the observation group was significantly higher than that in the control group (p<0.05). The total efficiency of pain control in the observation group was significantly higher than that in the control group (p<0.05). The levels of tumor markers in the two groups of patients were significantly decreased after treatment, while the reduction in the observation group was more evident than that in the control group (p<0.05). CONCLUSIONS: Our results showed that CT-guided implantation of 125I seeds is effective for the treatment of patients with refractory malignant tumors accompanied by cancer pain. It can reduce the levels of tumor markers, improve the survival rate and prolong the survival time of the patients.

Epithelial expression of cytokeratins 15 and 19 in vitiligo.

BACKGROUND: Cytokeratins (CK) belong to the family of intermediate filament proteins, and among them specific epithelial keratins are considered markers for stem cells activation. OBJECTIVES: This study aims to investigate the expression of CK15 and CK19 as possible stem cell markers in vitiligo during phototherapy. METHODS: The study was conducted on vitiligo patients receiving narrow-band ultraviolet therapy. Immunohistochemical staining for CK15 and CK19 was carried out, and clinical follow-up continued for 4 weeks. RESULTS: Of 28 patients, CK15 expression was demonstrated in 17 cases (61%) while CK19 expression was demonstrated in 11 cases (39%). Cells expressing positive staining were demonstrated in follicular and interfollicular epithelium. Expression was clearly demonstrated in patients younger than 20 years old, with shorter disease duration, with disease stability, and with normally pigmented hairs. Expression of cytokeratins was significantly correlated to improvement of vitiligo lesions. CONCLUSION: CK15 and CK19 are expressed in vitiligo during UV repigmentation in the follicular and interfollicular epithelium. This expression of cytokeratins was significantly correlated to improvement and can be considered valuable tool to monitor stem cells stimulation for the sake of the repigmentation process in vitiligo.

Robust ultrasensitive tunneling-FET biosensor for point-of-care diagnostics.

For point-of-care (POC) applications, robust, ultrasensitive, small, rapid, low-power, and low-cost sensors are highly desirable. Here, we present a novel biosensor based on a complementary metal oxide semiconductor (CMOS)-compatible silicon nanowire tunneling field-effect transistor (SiNW-TFET). They were fabricated "top-down" with a low-cost anisotropic self-stop etching technique. Notably, the SiNW-TFET device provided strong anti-interference capacity by applying the inherent ambipolarity via both pH and CYFRA21-1 sensing. This offered a more robust and portable general protocol. The specific label-free detection of CYFRA21-1 down to 0.5 fgml(-1) or ~12.5 aM was achieved using a highly responsive SiNW-TFET device with a minimum sub-threshold slope (SS) of 37 mVdec(-1). Furthermore, real-time measurements highlighted the ability to use clinically relevant samples such as serum. The developed high performance diagnostic system is expected to provide a generic platform for numerous POC applications.

Cultured epithelial grafting using human amniotic membrane: the potential for using human amniotic epithelial cells as a cultured oral epithelium sheet.

OBJECTIVE: Human amniotic cells are a valuable source of functional cells that can be used in various fields, including regenerative medicine and tissue engineering. The aim of this study was to investigate the utility of human amniotic epithelial (hAE) cells as a new cell source for culturing stratified epithelium sheets for intraoral grafting. METHODS: Enzymatically isolated hAE cells were submerged in a serum-free, low-calcium-supplemented MCDB 153 medium without a feeder layer. The hAE cells were seeded onto a Millicell cell culture plate insert and cultured while submerged in a high-calcium medium for 4 days. Then, they were cultured at an air-liquid interface for 3 weeks. Cultures of hAE cells proliferated at the air-liquid interface. RESULTS: After 3 weeks, the hAE cells cultivated using the air-liquid interface method lead to almost 10 continuous layers of stratified epithelium without parakeratinization or keratinization. It confirmed immunohistochemically that the presence of CK10/13 and Ki-67 positive cells were spread throughout almost all the epithelial layer, and that CK19 positive cells were expressed throughout the entire epithelial layer in the cultured hAE cell sheets. Cultured hAE cells sheets showed a staining pattern similar to that of uncultured oral mucosa: ZO-1 and occludin were located in the intercellular junctions throughout all the epithelial layers. It was suggested that the hAE sheets consisted of highly-active proliferating cells and undifferentiated cells, and had a barrier function. CONCLUSIONS: These results suggested that hAE cells may be a promising cell source for the development of stratified epithelium allograft sheets using a human cell strain.

[Effects of chemotherapy combined with Chinese herbal medicine Kangliu Zengxiao decoction on tumor markers of patients with advanced non-small-cell lung cancer: a randomized, controlled trial].

BACKGROUND: Tumor markers are widely used in clinical practice and have become important indicators in assessing cancer progress. There is increasing concern that chemotherapy combined with traditional Chinese medicine has effects in decreasing the level of tumor markers. OBJECTIVE: To investigate the effects of chemotherapy combined with Kangliu Zengxiao Decoction (KLZX), a compound Chinese herbal drug, on tumor markers carbohydrate antigen 50 (CA 50), cytokeratin 19 fragment (CYFRA21-1) and carcinoembryonic antigen (CEA) in patients with advanced non-small-cell lung cancer (NSCLC) and to explore the relationships between clinical efficacy and tumor markers. DESIGN, SETTING, PARTICIPANTS AND INTERVENTIONS: Patients were included from Punan Hospital of Shanghai Pudong New District and Longhua Hospital between October 2008 and December 2009. Seventy-four subjects with advanced NSCLC were randomly assigned into treatment group (n=37) and control group (n=37). Patients in the control group were treated with chemotherapy alone while patients in the treatment group were treated with chemotherapy combined with KLZX. Chemotherapy of NP (vinorelbine + cisplatin) was given for two cycles and patients in the treatment group were administered with KLZX during chemotherapy. MAIN OUTCOME MEASURES: Levels of CA50, CYFRA21-1 and CEA before and after treatment were evaluated and the relationship between changes in levels of tumor makers and tumor size, clinical symptoms and living condition score (Karnofsky score) was analyzed. RESULTS: No patients achieved a complete remission. The disease control rates (complete remission (CR)+partial remission (PR)+no change (NC)) were 89.20% (33/37) and 70.30% (26/37) in the treatment and control group respectively (P<0.05). The levels of CA50, CYFRA21-1 and CEA were clearly decreased in the treatment group after treatment (P<0.05) while also decreased in the patients without progression of disease. There were no obvious changes of CA50, CYFRA21-1 and CEA in the control group, and there was even a trend of increase. Furthermore, the improvement rates of clinical syndrome were 51% (19/37) vs 11% (4/37) (P<0.05) in the treatment group and control group respectively. The total response rates of quality of life were 91.89% (34/37) vs 56.76% (21/37) (P<0.01) in the treatment and control group respectively. CONCLUSION: Combined chemotherapy with KLZX in treating advanced NSCLC can acquire better stabilizing tumor foci, decrease levels of tumor markers and improve the clinical symptoms and Karnofsky score.

Expression of multiple stem cell markers in dental pulp cells cultured in serum-free media.

INTRODUCTION: Stem cell lines are usually grown in medium containing animal products. Fetal bovine serum (FBS) is an important additive for cell growth; however, the allergenic potential and the possibility of contamination when we use a medium containing serum would be a barrier to transplantation and consequently to the introduction of cell therapy methods into clinical applications. METHODS: Dental mesenchymal cells were isolated and expanded in vitro and maintained in 4 different serum-free media (SFMs): SFM#1 (ITS-X, embryotrophic factor [ETF]); SFM#2 (ITS-X); SFM#3 (ETF); and SFM#4 (ETF, sodium pyruvate, ascorbic acid, fibroblast growth factor [FGF-a], acidic). Viability, proliferative, and immunocytochemical tests for the cells were performed by using 4 stem cell markers (CD44H, CK19, nestin, and P63) for ectoderm, mesoderm, and endoderm. RESULTS: Viability tests showed a significant difference between the control and SFMs in both deciduous tooth pulp cells (DTPCs) and wisdom tooth pulp cells (WTPCs). However, all SFMs demonstrated 84%-90% viability, whereas the control showed 90%-93%. In both DTPCs and WTPCs, SFM#1 had the highest proliferation rate among the 4 SFMs. Immunocytochemistry stained positive stem cell markers most intensely in cells cultured with SFM#1. Furthermore, all stem cell markers for ectoderm, mesoderm, and endoderm were expressed in the cells cultured with SFM#1. CONCLUSIONS: SFM#1 showed an acceptable survival rate, the highest proliferation rate, and the strongest expression of all the stem cell markers. SFM#1 proved to be a suitable medium for the culture of human dental pulp stem cells and to preserve pluripotency in differentiation.

Isolation, culture and immortalisation of hepatic oval cells from adult mice fed a choline-deficient, ethionine-supplemented diet.

Oval cells have great potential for use in cell therapy to treat liver disease, however this cannot be achieved until the factors which govern their proliferation and differentiation are better understood. We describe a method to establish primary cultures of murine oval cells, and the derivation of two novel lines from these. Primary cultures from the livers of wildtype or TAT-GRE lacZ transgenic mice subjected to a choline-deficient, ethionine-supplemented diet comprised up to 80% oval cells at day 7 based on A6 or CK19 staining. Cell lines were clonally derived, which underwent spontaneous immortalisation following prolonged maintenance in culture. Immunostaining and RT-PCR demonstrated they express hepatocytic and biliary markers and they were therefore termed "bipotential murine oval liver" (BMOL) cells. Under proliferating culture conditions, BMOL or BMOL-TAT cells abundantly expressed oval cell and biliary markers, whereas mature hepatocytic markers were upregulated when the growth conditions were changed to facilitate differentiation. Hepatic differentiation of BMOL-TAT cells could be traced by measuring the expression of their lacZ transgene, which is driven by a promoter element from tyrosine aminotransferase (TAT), a marker of adult hepatocytes. Interestingly, haematopoietic markers were upregulated in superconfluent cultures, indicating a possible multipotentiality. None of the cell lines grew in semi-solid agar, nor did they form tumours in nude mice, suggesting they are non-tumourigenic. These novel murine oval cell lines, together with a reliable method for isolation and culture of primary oval cells, will provide a useful tool for investigating the contribution of oval cells to liver regeneration.

[A 42-month disease free survival case of combined hepatocellular-cholangiocarcinoma with lymph node metastases treated with multimodal therapy].

Combined hepatocellular and cholangiocarcinoma (HCC-CC) is a rare type of liver cancer. We herein report a case of HCC-CC with lymph node metastases treated by multimodality therapy. The patient has been alive for more than 42 months. A 52-year-old man with a 9 cm diameter mass lesion in the liver was admitted to our hospital. The tumor was diagnosed as peripheral type of cholangiocarcinoma. Preoperative transhepatic arterial chemoenbolization (TACE) was performed. An accumulation pattern of lipiodol after TACE and an increase of serum alpha-fetoprotein led us to diagnosis of combined HCC-CC. A three segmentectomies of the liver and dissection of the local lymph nodes were performed. A histological examination of the resected specimen showed combined HCC-CC with lymph node metastases. Alpha fetoprotein, cytokeratins 7 and 19 were partially positive with immunohistochemical staining. The final diagnosis was a mixed type of combined HCC-CC. To improve a poor prognosis of combined HCC-CC, adjuvant chemotherapy with CDDP, 5 FU and radiation therapy were achieved. Fortunately, the patient is alive without any recurrence for 42 months after the operation.