Observation of the Curative Effect of Acupuncture for Tonifying Kidney and Removing Blood Stasis Combined with Radiofrequency Surgery in Patients with NSCLC and the Diagnostic Efficacy of Combined Detection of NTx, BGP, and CYFRA21-1 in the Occurrence of Bone Metastases.

The curative effect observation of acupuncture for tonifying kidney and removing blood stasis combined with radiofrequency surgery in patients with non-small-cell lung carcinoma (NSCLC) and the diagnostic efficacy of combined detection of NTx, BGP, and CYFRA21-1 for bone metastases are investigated. 122 NSCLC patients admitted to our hospital from January 2019 to December 2021 are selected for the examination, and the two sets of patients are randomly divided into the study set and the control set using the random number table method, with 61 cases in each set. Patients in the control set are given CT-guided percutaneous radiofrequency ablation therapy, and patients in the study set are given a combination of acupuncture therapy for tonifying the kidney and removing blood stasis on the basis of the therapy of the control set. The experimental results show that for NSCLC patients, the application of kidney-tonifying and stasis-removing acupuncture therapy combined with radiofrequency surgery can notoriously enhance the clinical therapy effect and enhance the quality of life of patients, and the detection of NTx, BGP, and CYFRA21-1 indicators can effectively predict the prognosis.

Role of one-step nucleic acid amplification in colorectal cancer lymph node metastases detection.

Current histopathological staging procedures in colorectal cancer (CRC) depend on midline division of the lymph nodes (LNs) with one section of hematoxylin and eosin staining. Cancer cells outside this transection line may be missed, which could lead to understaging of Union for International Cancer Control Stage II high-risk patients. The one-step nucleic acid amplification (OSNA) assay has emerged as a rapid molecular diagnostic tool for LN metastases detection. It is a molecular technique that can analyze the entire LN tissue using a reverse-transcriptase loop-mediated isothermal amplification reaction to detect tumor-specific cytokeratin 19 mRNA. Our findings suggest that the OSNA assay has a high diagnostic accuracy in detecting metastatic LNs in CRC and a high negative predictive value. OSNA is a standardized, observer-independent technique, which may lead to more accurate staging. It has been suggested that in stage II CRC, the upstaging can reach 25% and these patients can access postoperative adjuvant chemotherapy. Moreover, intraoperative OSNA sentinel node evaluation may allow early CRC to be treated with organ-preserving surgery, while in more advanced-stage disease, a tailored lymphadenectomy can be performed considering the presence of aberrant lymphatic drainage and skip metastases.

Prognosis Analysis and Perioperative Research of Elderly Patients with Non-Muscle-Invasive Bladder Cancer under Computed Tomography Image of Three-Dimensional Reconstruction Algorithm.

To analyze the application value of computed tomography (CT) based on a three-dimensional reconstruction algorithm in perioperative nursing research and prognosis analysis of non-muscle-invasive bladder cancer (NMIBC), a retrospective study was performed on 124 patients with NMIBC who underwent surgical treatment in the hospital. All patients underwent CT examination based on the three-dimensional reconstruction algorithm before surgery, and transurethral resection of the bladder tumor was performed. The patients receiving conventional care were classified as the control group, and those receiving comprehensive care were classified as the case group, and the recovery status and recurrence of the two groups were compared. The results showed that the accuracy, specificity, and sensitivity of CT imaging information based on the three-dimensional reconstruction algorithm for NMIBC patients were 89.38, 93.77, and 84.39, respectively. The incidence of bladder spasm (9.68%), bladder flushing time (1.56 d), and retention of drainage tube time (2.68 d) in the case group were obviously lower compared with the control group (30.65%, 2.32 d, and 5.19 d) (P < 0.05). Serum BLCA-1 (3.72 ng/mL) and CYFRA21-1 (5.68 µg/mL) in the case group were significantly lower than those in the control group, with a statistically considerable difference (P < 0.05). Compared with the control group, the scores of role function (89.82 points), emotional function (84.76 points), somatic function (79.23 points), and social function (73.93 points) in the case group were observably higher (P < 0.05). In addition, one year after the operation, CT examination showed that the recurrence rate in the case group (6.45%) was significantly lower than that in the control group (22.58%) (P < 0.05). Therefore, CT detection based on the three-dimensional reconstruction algorithm was particularly important for preoperative diagnosis, prognosis, and recurrence monitoring of NMIBC patients. It could provide great clinical value for the diagnosis and prognosis monitoring of NMIBC.

CK19 Predicts Recurrence and Prognosis of HBV Positive HCC.

BACKGROUND: Cytokeratin is associated with the recurrence and metastasis of some cancers and tends to increase the malignancy of the disease. It is getting more and more attention in cancer research. Abnormal expression of cytokeratin 19 (CK19) has been reported as an important prognostic factor in cancers. CK19 is a marker of bile duct cells, liver progenitor cells (HPCs), and early hepatoblasts, and its expression is associated with poor prognosis in patients diagnosed with hepatocellular carcinoma (HCC). The purpose of this study was to evaluate the predictive value of CK19 for tumor recurrence after radical resection in patients with hepatitis B virus (HBV) positive HCC. METHODS: This study was a retrospective study conducted in two institutions. A total of 674 patients with HBV positive HCC who underwent radical HCC resection from January 2010 to May 2020 were included in this study. Chi-square test or Fisher's exact test was used to compare the classification variables and continuous variables were compared by t-test or Wilcoxon rank sum test. Cox regression model was used for univariate and multi-variable survival analyses. Based on the results of the multi-variable analyses of Cox regression, the nomogram of 2-year recurrence-free survival (RFS) was plotted. The model was validated internally in the Hangzhou cohort (training set) and then externally in the Lanzhou cohort (test set) and the effectiveness of the model was tested. RESULTS: For all 674 patients, 223 cases (33.1%) were positive and 451 cases (66.9%) were negative for CK19. The 2-year RFS rate was higher in patients with CK19 negative than in patients with CK19 positive. In the training set, correlation analysis showed that CK19 expression was correlated with preoperative potassium (P value(P) = 0.030), satellite nodules (P < 0.001) and microvascular invasion (P = 0.020). In the test set, CK19 expression was correlated with postoperative platelet (P = 0.038), satellite nodules (P = 0.003), microvascular invasion (P = 0.011), and maximum tumor size (P = 0.039). Univariate Cox regression correlation analyses showed that CK19 expression was correlated with preoperative potassium (P value(P) = 0.030), satellite nodules (P < 0.001), and microvascular invasion (P = 0.020). Training and test sets showed that postoperative platelet (> 300/L), CK19, satellite nodules in the training set, microvascular invasion, maximum tumor size, and tumor boundary were adverse factors for predicting RFS. Multi-variable analyses showed that in the training set, postoperative platelet > 300/L (hazard ratios (HR) = 2.753, 95% confidence interval (95%CI):1.234-6.142, P = 0.013), CK19 (HR = 1.410, 95%CI:1.006-1.976, P = 0.046), satellite nodule (HR = 1.476, 95%CI:1.026-2.120, P = 0.036), microvascular invasion (HR = 2.927, 95%CI:2.006-4.146, P < 0.001), incomplete tumor capsule (HR = 1.539, 95%CI:1.012-2.341, P = 0.044) were independent prognostic indicator of poor RFS. In the test set, postoperative platelet > 300/L (HR = 2.816, 95%CI:1.043-7.603, P = 0.041), CK19 (HR = 1.586, 95%CI:1.016-2.475, P = 0.042), satellite nodule (HR = 1.706, 95%CI:1.067-2.728, P = 0.026), microvascular invasion (HR = 1.611, 95%CI:1.034-2.510, P = 0.035), and tumor without capsule (HR = 1.870, 95%CI:1.120-3.120, P = 0.017) were independent prognostic indicators of poor RFS. The C-index for the nomogram was 0.698 (95%CI: 0.654-0.742) and the C-index for the test set was 0.670 (95%CI: 0.616-0.724). Both internal and external verification showed good results in identification and calibration. CONCLUSION: CK19 plays a key role in tumor malignancy through overexpression and the expression of CK19 is an independent adverse factor affecting recurrence; therefore, CK19 can be used as a potential biomarker to predict adverse prognosis after surgery and adjuvant therapy in HCC patients.

Yinchenzhufu decoction protects against alpha-naphthylisothiocyanate-induced acute cholestatic liver injury in mice by ameliorating disordered bile acid homeostasis and inhibiting inflammatory responses.

ETHNOPHARMACOLOGICAL RELEVANCE: Intrahepatic cholestasis is a common condition of many liver diseases with few therapies. Yinchenzhufu decoction (YCZFD) is a representative traditional Chinese herbal formula used for treating jaundice and liver disease. AIM OF THE STUDY: To investigate the hepatoprotective effect of YCZFD against cholestatic liver injury and reveal its potential mechanism. MATERIALS AND METHODS: Mice with alpha-naphthyl isothiocyanate (ANIT)-induced intrahepatic cholestasis were orally administered YCZFD at doses of 3, 6, and 12g crude drug/kg for 2 weeks followed by subsequent analyses. A serum metabolomics study was then performed to explore the different metabolites influenced by YCZFD using ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap hybrid mass spectrometry (UPLC-LTQ-Orbitrap-MS/MS).The levels of individual bile acids in the serum, liver, and bile were determined by UPLC-MS/MS. The expression of metabolic enzymes, transporters, inflammatory factors, and cytokeratin-19 (CK-19) was determined by real-time PCR, western blotting, and immunohistochemistry. RESULTS: YCZFD administration decreased the serum biochemical indexes and ameliorated pathological damage, such as hepatic necrosis and inflammatory cell infiltration. Serum metabolomics revealed that the metabolites influenced by YCZFD were mainly associated with bile acid metabolism and inflammation. YCZFD administration effectively ameliorated the disordered bile acid homeostasis. The bile acid transporter, multidrug-resistance associated protein 2 (Mrp2), and the metabolic enzyme, cytochrome P450 2b10 (Cyp2b10), were upregulated in the YCZFD intervention group compared to those in the ANIT-induced group. YCZFD administration also significantly inhibited nuclear factor-κB (NF-κB) and its phosphorylation and decreased the expression of proinflammatory cytokines including tumor necrosis factor-α, interleukin-1ß, and intercellular adhesion molecule-1 in ANIT-induced cholestatic mice. Additionally, the level of CK-19 was lower in the YCZFD intervention group than in the ANIT-induced cholestatic mice. CONCLUSION: YCZFD administration ameliorated disordered bile acid homeostasis, inhibited NF-κB pathway-mediated inflammation, and protected the liver from bile duct injury. Therefore, YCZFD exerted a protective effect against cholestatic liver injury.

Genetic Lineage Tracing of Biliary Epithelial Cells.

Lineage tracing of liver cells is a powerful tool to understand liver embryonic development, healthy liver cell homeostasis, tissue repair, and regeneration. Lineage tracing of biliary epithelial cells (BECs) in the adult liver has been used to assess the contribution of the biliary epithelium to liver injury, regeneration, and disease. These studies have shown the contribution of BECs to the expansion of ductular reaction (DR) and liver progenitor cells (LPCs) and eventually the generation of new hepatocytes. Few genetic lineage-tracing mouse models have been proved to trace BECs. This chapter is focused on lineage tracing of BECs in mouse models of liver injury and regeneration. First, we mention different existing approaches to trace the biliary epithelium based on proteins specifically expressed by BECs such as sex-determining region Y-box 9 (SOX9), osteopontin (OPN), and cytokeratin-19 (KRT19). Second, we describe mouse models that can be used to evaluate cell fate during liver injury and regeneration (i.e., partial hepatectomy (PHx), acute liver injury models, and chronic liver damage models such as 3,5-diethoxycarbonyl-1,4-dihydro-collidin (DDC) diet, choline-deficient ethionine-supplemented (CDE) diet, or chronic carbon tetrachloride (CCl4) administration). Third, we suggest possible readouts to assess BECs fate based on immunofluorescence analysis.

Loss of trefoil factor 1 inhibits biliary regeneration but accelerates the hepatic differentiation of progenitor cells in mice.

Although the regeneration of the adult liver depends on hepatic progenitor cells (HPCs), many uncertainties regarding hepatic regeneration in the injured liver remain. Trefoil factor family 1 (TFF1), a secretory protein predominantly expressed in the gastrointestinal tract, is responsible for mucosal restitution. Here, we investigated the role of TFF1 in liver regeneration using a mouse model of hepatic injury (choline-deficient ethionine-supplemented diet and carbon tetrachloride administration) and genetically engineered mice (TFF1 knockout (TFF1-/-)). Immunohistochemistry analysis of human liver samples revealed TFF1 expression in the hepatocytes close to ductular reaction and the regenerating biliary epithelium in injured liver. The number of cytokeratin 19 (CK19)-positive bile ducts was significantly decreased in the TFF1-/- mice after liver injury. Notch pathway in the TFF1-/- mice was also downregulated. HPCs in the control mice differentiated into biliary cells (CK19+/SRY HMG box 9 (SOX9)+) more frequently. In contrast, HPCs in the TFF1-/- mice more frequently differentiated into a hepatic lineage (alpha fetoprotein+/SOX9+) after acute liver damage. Hepatocyte proliferation was upregulated, and the liver weight was increased in TFF1-/- mice in response to chronic liver damage. Thus, TFF1 is responsible for liver regeneration after liver injury by promoting HPC differentiation into a biliary lineage and inhibiting HPC differentiation into a hepatic lineage.

A feasibility study of the toxic responses of human induced pluripotent stem cell-derived hepatocytes to phytochemicals.

Herbal medicines have been increasingly used in the last three decades. Despite their popularity, safety issues with herbal products need to be addressed. We performed a feasibility study of the toxic responses of human induced pluripotent stem cell-derived hepatocytes (iHep cells) to phytochemicals in comparison with hepatoblasoma-derived HepG2 cells and long-term human hepatocytes (LTHHs). The iHep cells expressed typical hepatocyte markers cytochrome P450 3A4 (CYP3A4), hepatocyte nuclear factor 4α, and albumin despite the expression of immature markers α-fetoprotein and cytokeratin 19. We studied the responses of iHep cells to phytochemicals saikosaponin D, triptolide, deoxycalyciphylline B, and monocrotaline with different mode of toxicity employing MTS and lactate dehydrogenase (LDH) assays. Saikosaponin D and triptolide caused dose-dependent cytotoxicity in the iHep cells, which were more sensitive than LTHHs and HepG2 cells. Saikosaponin D-induced cytotoxicity tightly correlated with increased LDH leakage in the iHep cells. Although deoxycalyciphylline B did not exhibit toxic effect on the iHep and HepG2 cells when compared with LTHHs, it decreased CYP3A7 expression in the iHep cells and increased CYP1A2 expression in HepG2 cells. We hereby show the feasibility of using iHep cells to detect toxic effects of phytochemicals.

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.

Baicalein inhibits acinar-to-ductal metaplasia of pancreatic acinal cell AR42J via improving the inflammatory microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers. Recent research has demonstrated that chronic pancreatitis (CP) is associated with an increased risk of PDAC, partly due to acinar-to-ductal metaplasia (ADM). Baicalein has been shown to exert anti-inflammatory and anti-tumor effects for CP or PDAC, respectively. The aim of our study was to investigate the effect of baicalein, and the putative underlying mechanism, on inflammatory cytokines-induced ADM of rat pancreatic acinar cell line AR42J. To investigate ADM and baicalein effects in vitro, AR42J were treated with recombinant rat Tumor Necrosis Factor alpha (rTNFα) with or without baicalein for 5 days. Results showed that rTNFα-induced AR42J cells switched their phenotype from dominantly amylase-positive acinar cells to dominantly cytokeratin 19-positive ductal cells. Moreover, expression of the transcripts for TNFα or Hes-1, a Notch target, was up-regulated in these cells. Interestingly, baicalein reduced the population of ADM as well as cytokines gene expression but not Hes-1. Baicalein inhibited NF-κB activation induced by rTNFα in AR42J, but no effect on Notch 1activation. Moreover, baicalein suppressed the secretion of TNFα and Nitric Oxide (NO) in macrophages stimulated with LPS and further inhibited ADM of conditional medium-treated AR42J cells. Baicalein also suppressed the inflammatory response of LPS-activated macrophages, thereby inhibited ADM of AR42J by altering their microenvironment. Taken together, our study indicates that baicalein reduces rTNFα-induced ADM of AR42J cells by inhibiting NF-κB activation. It also sheds new light on Chinese material medica therapy of pancreatitis and thereby prevention of PDAC.

Gypenosides Ameliorate Carbon Tetrachloride-Induced Liver Fibrosis by Inhibiting the Differentiation of Hepatic Progenitor Cells into Myofibroblasts.

Gypenosides (GPs), the predominant components of Gynostemma pentaphyllum, exert antifibrotic effects; however, the mechanisms underlying their ability to ameliorate liver fibrosis are unclear. Liver fibrosis was induced in C57BL/6 mice via subcutaneous injection of 10% carbon tetrachloride (CCl[Formula: see text] three times a week for two weeks. Then, CCl4 was administered in conjunction with intragastric GPs for another three weeks. For in vitro analyses, WB-F344, hepatatic progenitor cells (HPCs) were treated with transforming growth factor beta 1 (TGF-[Formula: see text]1) with or without GPs for 48[Formula: see text]h. The results showed that alanine aminotransferase (ALT) and aspartate transaminase (AST) activity, deposition of collagen, hydroxyproline content, and expression of alpha-smooth muscle actin ([Formula: see text]-SMA) and collagen type I (Col I) were significantly decreased after treatment with GPs ([Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text]). In the 5M CCl4 group, the expression of HPC markers, Sox9 and cytokeratin 19 (CK19), was significantly increased compared with the normal or GPs-treated group ([Formula: see text], [Formula: see text]). Immunostaining showed that the number of Sox9 and [Formula: see text]-SMA double-positive cells was higher in the 5M CCl4 group than in the normal group, but the addition of GPs caused this cell number to decrease. In WB-F344 cells, the expression of [Formula: see text]-SMA and Col I was significantly increased after treatment with TGF-[Formula: see text], whereas in the GPs treatment group, expression was markedly decreased ([Formula: see text]). The levels of TGF-[Formula: see text] and TGF-[Formula: see text]R1 were markedly reduced after GPs treatment both in vivo and in vitro. In conclusion, GPs ameliorated CCl4-induced liver fibrosis via the inhibition of TGF-[Formula: see text] signaling, consequently inhibiting the differentiation of HPCs into myofibroblasts.

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.

Loss of keratin 19 favours the development of cholestatic liver disease through decreased ductular reaction.

Keratins (K) are cytoprotective proteins and keratin mutations predispose to the development of multiple human diseases. K19 represents the most widely used marker of biliary and hepatic progenitor cells as well as a marker of ductular reaction that constitutes the basic regenerative response to chronic liver injury. In the present study, we investigated the role of K19 in biliary and hepatic progenitor cells and its importance for ductular reaction. K19 wild-type (WT) and knockout (KO) mice were fed: (a) 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC); (b) cholic acid (CA); (c) a choline-deficient, ethionine-supplemented (CDE) diet; or (d) were subjected to common bile duct ligation (CBDL). The bile composition, liver damage, bile duct proliferation, oval cell content and biliary fibrosis were analysed. In untreated animals, loss of K19 led to redistribution of the K network in biliary epithelial cells (BECs) but to no obvious biliary phenotype. After DDC feeding, K19 KO mice exhibited (compared to WTs): (a) increased cholestasis; (b) less pronounced ductular reaction with reduced ductular proliferation and fewer oval cells; (c) impaired Notch 2 signalling in BECs; (d) lower biliary fibrosis score and biliary bicarbonate concentration. An attenuated oval cell proliferation in K19 KOs was also found after feeding with the CDE diet. K19 KOs subjected to CBDL displayed lower BEC proliferation, oval cell content and less prominent Notch 2 signal. K19 deficiency did not change the extent of CA- or CBDL-induced liver injury and fibrosis. Our results demonstrate that K19 plays an important role in the ductular reaction and might be of importance in multiple chronic liver disorders that frequently display a ductular reaction.

Coexistence of malignant struma ovarii and cervical papillary thyroid carcinoma.

CONTEXT: Struma ovarii is an uncommon monodermal teratoma in which thyroid tissue is the predominant element. Malignant transformation of struma ovarii is an even rarer occurrence. CASE PRESENTATION: We describe a 42-year-old woman who underwent a total abdominal hysterectomy and bilateral salpingo-oophorectomy for a symptomatic left pelvic mass. Histology revealed malignant struma ovarii with classical papillary thyroid carcinoma expression. Ultrasonography of the cervical neck showed thyroid micronodules and a dominant 1-cm nodule in the left thyroid lobe. As the ovarian tumor was large, the patient underwent a total thyroidectomy with the intention of administering ¹³¹I therapy in an adjuvant setting. Histology of the cervical thyroid gland revealed bilateral multifocal papillary thyroid carcinoma with extrathyroidal extension and perithyroidal lymph node metastasis. METHODS: Morphological (microscopy), immunohistochemical (Hector Battifora mesothelial cell 1, cytokeratin-19, galectin-3), and molecular (BRAF V600E, RAS, RET-PTC) characteristics and clonality analysis of the cervical thyroid and ovarian tumors were explored to distinguish them as separate malignancies. RESULTS: The thyroid-type tumors from the cervical gland and ovary were discordant in terms of tissue histology and level of cytokeratin-19 expression. The clinical features and tumor profile results supported the independent existence of these two embryologically related, although topographically distinct, malignancies. CONCLUSION: Our findings provided support for synchronous, albeit distinct, primary tumors in the ovary and cervical thyroid. "Field cancerization" and early genomic instability may explain multifocality in all thyroid-type tissue. In this regard, patients with malignant struma ovarii should undergo imaging of their thyroid gland for coexisting disease and thyroidectomy recommended for suspected malignancy or in preparation for radioiodine therapy.

Alteration in apoptosis and cell cycle by celecoxib and/or fish oil in 7,12-dimethyl benzene (α) anthracene-induced mammary carcinogenesis.

Cyclooxygenase (COX)-2 inhibition by nonsteroidal anti-inflammatory drugs is a useful approach for cancer prevention but has several side effects. A novel approach combining these chemopreventive agents at low doses with dietary elements has been suggested to augment their effects and reduce side effects. Dietary fats, particularly, n-3 polyunsaturated fatty acids (PUFA) also exert cancer chemopreventive effect mediated through COX-2 inhibition. Therefore, the present study was designed to investigate the effect of combined dosage of celecoxib and n-3 PUFA-rich fish oil in experimental mammary carcinogenesis. Female Wistar rats were distributed into control and DMBA-treated groups. The groups were further subdivided based on pretreatment with celecoxib and/or fish oil. The animals were maintained for 90 days before sacrifice. To analyze the role of redox signaling, the two mediators, reactive oxygen species and calcium, and their effects on c-myc expression were evaluated. The chemopreventive effect was assessed by measurement of cell proliferation, apoptosis, and p53 in isolated mammary epithelial cells. Increased redox signaling with enhanced c-myc, p53 expression, and augmented apoptotic and proliferative rate were observed in carcinogen-treated animals. Pretreatment of carcinogen-treated animals with celecoxib and/or fish oil altered redox signaling with reduced c-myc, p53 expression, apoptosis, and proliferation. However, a combination dosage of celecoxib and fish oil had a better chemopreventive effect. The results suggest that a combination of celecoxib and fish oil is more effective in the chemoprevention of experimental mammary carcinogenesis, and this effect can be attributed to the modification of redox signaling.

Expression of specificity protein transcription factors in pancreatic cancer and their association in prognosis and therapy.

Pancreatic cancer is an aggressive malignancy with poor prognosis. Pancreatic adenocarcinoma is one of the leading causes of cancer-related deaths in the United States. Due to the aggressive nature of this malignancy, there is a serious concern for identifying effective targets, and adopting novel strategies for therapy. Members of the Specificity Protein (Sp) family of transcription factors, Sp1, Sp3, and Sp4 regulate the expression of a number of genes associated with cancer cell proliferation, differentiation, and metastasis. Sp1 levels are upregulated in pancreatic cancer cell lines, and surgically resected human pancreatic adenocarcinoma. Sp1 overexpression in tumor tissues is associated with aggressive disease, poor prognosis and inversely correlated with survival. Sp1 is also known to affect angiogenesis by regulating the expression of vascular endothelial growth factor and its receptors. Results from clinical studies suggest Sp1 as new biomarker to identify aggressive pancreatic ductal adenocarcinoma. The pharmacological inhibition of Sp1 using agents such as celecoxib, mithramycin, curcumin, and tolfenamic acid has showed promising results in pre-clinical studies and demonstrated Sp transcription factors as potential targets for pancreatic cancer therapy. This review summarizes studies showing the association of Sp proteins with this malignancy, with a special emphasis on pre-clinical studies that tested strategies to target Sp transcription factors for inhibiting human pancreatic cancer cell proliferation and tumor growth in laboratory animals. The results showed remarkable efficacy and suggest that such approaches have the potential for high success in developing clinically relevant strategies for treating pancreatic cancer.

Atropa belladonna L. water extract: modulator of extracellular matrix formation in vitro and in vivo.

Previously, we found that treatment of cutaneous wounds with Atropa belladonna L. (AB) revealed shortened process of acute inflammation as well as increased tensile strength and collagen deposition in healing skin wounds (Gál et al. 2009). To better understand AB effect on skin wound healing male Sprague-Dawley rats were submitted to one round full thickness skin wound on the back. In two experimental groups two different concentrations of AB extract were daily applied whereas the control group remained untreated. For histological evaluation samples were removed on day 21 after surgery and stained for wide spectrum cytokeratin, collagen III, fibronectin, galectin-1, and vimentin. In addition, in the in vitro study different concentration of AB extract were used to evaluate differences in HaCaT keratinocytes proliferation and differentiation by detection of Ki67 and keratin-19 expressions. Furthermore, to assess ECM formation of human dermal fibroblasts on the in vitro level fibronectin and galectin-1 were visualized. Our study showed that AB induces fibronectin and galectin-1 rich ECM formation in vitro and in vivo. In addition, the proliferation of keratinocytes was also increased. In conclusion, AB is an effective modulator of skin wound healing. Nevertheless, further research is needed to find optimal therapeutic concentration and exact underlying mechanism of action.

Cytokeratin-19 and mammaglobin gene expression in circulating tumor cells from metastatic breast cancer patients enrolled in North Central Cancer Treatment Group trials, N0234/336/436/437.

PURPOSE: To investigate the associations between baseline and posttreatment circulating tumor cell (CTC) gene expression and outcome of patients enrolled in four North Central Cancer Treatment Group metastatic breast cancer (MBC) trials in which specimens were shipped (at 4°C) from community-based sites to a reference laboratory (Mayo Clinic, Rochester, MN). EXPERIMENTAL DESIGN: Blood was collected at treating sites from MBC patients before (baseline), during, and at the end of treatment with erlotinib + gemcitabine (N0234), sorafenib (N0336), irinotecan + cetuximab (N0436), or paclitaxel-poliglumex + capecitabine (N0437). CTCs from 10 mL of EDTA blood were enriched with CD45 depletion, 24 to 30 hours postblood collection. Reverse transcription/quantitative PCR was used to determine cytokeratin-19 (CK19) and mammaglobin (MGB1) mRNA levels in CTCs from up to 13 (N0234), 16 (N0336), 18 (N0436), and 39 (N0437) patients. The gene expressions were normalized to ß(2)-microglobulin and calibrated to healthy blood using the 2(-ΔΔCq) algorithm; positivity was defined as 2 or more. RESULTS: CK19+mRNA cells were detected in 56% to 75% and MGB1+mRNA cells in 23% to 38% of 86 patients at baseline. CK19+mRNA cells were detected in 30% to 67% and MGB1+mRNA cells in 14% to 64% of 110 postbaseline serial samples. The presence of baseline CK19+mRNA cells (P = 0.01) but not MGB1+mRNA cells (P = 0.14) was significantly associated with shorter overall survival. A decrease in MGB1+mRNA levels (baseline-week 8) seemed to be associated with clinical response (P = 0.05). CONCLUSIONS: CTC gene expression analysis conducted by a reference laboratory is feasible when blood is collected from treating sites and processed 24 to 30 hours postcollection. The presence of baseline CK19+mRNA CTCs was associated with poor prognosis; a decrease in MGB1+mRNA CTCs may help predict response to therapy of MBC patients.