Synchronization isolation method for multiple types of cells from mouse liver / 中华肝脏病杂志

Objective: To explore a simple and feasible method for the isolation and purification of hepatocytes, hepatic stellate cells (HSC), and lymphocytes from mice. Methods: The cell suspension was obtained from male C57bl/6 mice by hepatic perfusion through the portal vein digestion method and then isolated and purified by discontinuous Percoll gradient centrifugation. Trypan blue exclusion was used to determine cell viability. Glycogen staining, cytokeratin 18, and transmission electron microscopy were used to identify hepatic cells. Immunofluorescence was used to detect α-smooth muscle actin combined with desmin in HSCs. Flow cytometry was used to analyze lymphocyte subsets in the liver. Results: After isolation and purification, about 2.7×10(7) hepatocytes, 5.7×10(5) HSCS, and 4.6×106 hepatic mononuclear cells were obtained from the liver of mice with a body weight of about 22g. The cell survival rate in each group was > 95%. Hepatocytes were apparent in glycogen deposited purple-red granules and cytokeratin 18. Electron microscopy showed that there were abundant organelles in hepatocytes and tight junctions between cells. HSC had expressed α-smooth muscle actin and desmin. Flow cytometry showed hepatic mononuclear cells, including lymphocyte subsets such as CD4, CD8, NKs, and NKTs. Conclusion: The hepatic perfusion through the portal vein digestion method can isolate multiple primary cells from the liver of mice at once and has the features of simplicity and efficiency.

Hepatocyte apoptosis fragment product cytokeratin-18 M30 level and non-alcoholic steatohepatitis risk diagnosis: an international registry study / 中华医学杂志(英文版)

BACKGROUND@#Liver biopsy for the diagnosis of non-alcoholic steatohepatitis (NASH) is limited by its inherent invasiveness and possible sampling errors. Some studies have shown that cytokeratin-18 (CK-18) concentrations may be useful in diagnosing NASH, but results across studies have been inconsistent. We aimed to identify the utility of CK-18 M30 concentrations as an alternative to liver biopsy for non-invasive identification of NASH.@*METHODS@#Individual data were collected from 14 registry centers on patients with biopsy-proven non-alcoholic fatty liver disease (NAFLD), and in all patients, circulating CK-18 M30 levels were measured. Individuals with a NAFLD activity score (NAS) ≥5 with a score of ≥1 for each of steatosis, ballooning, and lobular inflammation were diagnosed as having definite NASH; individuals with a NAS ≤2 and no fibrosis were diagnosed as having non-alcoholic fatty liver (NAFL).@*RESULTS@#A total of 2571 participants were screened, and 1008 (153 with NAFL and 855 with NASH) were finally enrolled. Median CK-18 M30 levels were higher in patients with NASH than in those with NAFL (mean difference 177 U/L; standardized mean difference [SMD]: 0.87 [0.69-1.04]). There was an interaction between CK-18 M30 levels and serum alanine aminotransferase, body mass index (BMI), and hypertension ( P  < 0.001, P  = 0.026 and P  = 0.049, respectively). CK-18 M30 levels were positively associated with histological NAS in most centers. The area under the receiver operating characteristics (AUROC) for NASH was 0.750 (95% confidence intervals: 0.714-0.787), and CK-18 M30 at Youden's index maximum was 275.7 U/L. Both sensitivity (55% [52%-59%]) and positive predictive value (59%) were not ideal.@*CONCLUSION@#This large multicenter registry study shows that CK-18 M30 measurement in isolation is of limited value for non-invasively diagnosing NASH.

Interaction between PSF and cytokeratin 18 mediates PSF relocation to cell membrane and maintains chemosensitivity of myeloid leukemia / 北京大学学报(医学版)

OBJECTIVE@#To identify the chaperone of polypyrimidine tractor-binding protein-associated splicing factor (PSF) in myeloid leukemia cells, and to explore the mechanism and redistributive pattern to cell surface of PSF in chemo-sensitive HL60 cells and resistant HL60/DOX cells.@*METHODS@#The eukaryotic expression vector of PSF was transfected with liposomes transiently, then flow cytometry was used to detect the expression level of PSF on the cell surface 24 h, 48 h and 72 h after vector transfections. We constructed a chimeric expression vector, streptavidin binding peptide (SBP)-PSF, meanwhile this vector was transfected and made SBP-PSF fusion protein overexpress. In addition, we used streptavidin magnetic beads to precipitate the cellular chaperonin of PSF and then identified its chaperonin by mass spectrometry (MS). Lentiviral vectors containing cytokeratin18 (K18) interference sequences were transfected into 293T cells to prepare lentivirus. HL60 and HL60/DOX cells were infected with lentivirus to obtain stable interfering K18 cell lines. Next, flow cytometry was used to test the membrane relocation level of PSF. Together, these methods confirmed the similar or different mechanisms of the PSF redistributing to membrane synergistically mediated by K18 in HL60 and HL60/DOX cells.@*RESULTS@#The expression of membrane relocated PSF was detected every day for three days (at the end of 24 h, 48 h and 72 h) after transient overexpression. The expressing rate of PSF on the cell surface was 22.4%±3.5%, 37.9%±6.0%, 58.3%±8.8%, respectively in sensitive HL60 cells, while that was 4.7%±0.5%, 3.9%±0.6%, 2.9%±0.6% , respectively in resistant HL60/DOX cells. The difference of expressing rate on each day was significant, P<0.01. We identified K18 detected by co-immunoprecipitation and mass spectrum assay which was the cellular chaperone of PSF. We found that K18 knockdown decreased the PSF expression level which redistributed on cell surface from 48.9%±5.4% to 6.2%±1.0% in sensitive HL60 cells, and from 9.11%±1.2% to 2.21%±0.51% in resistant HL60/DOX cells, respectively.@*CONCLUSION@#K18 is the intracellular chaperonin of PSF. The interaction of PSF and K18 mediates its redistribution to cell membrane in sensitive cells. While in resistant cells, PSF failed to relocate at the cell surface and accumulated in cells, which mediated resistance to chemotherapeutics.

The Use of Foxa2-Overexpressing Adipose Tissue-Derived Stem Cells in a Scaffold System Attenuates Acute Liver Injury

BACKGROUND/AIMS: For the clinical application of stem cell therapy, functional enhancement is needed to increase the survival rate and the engraftment rate. The purpose of this study was to investigate functional enhancement of the paracrine effect using stem cells and hepatocyte-like cells and to minimize stem cell homing by using a scaffold system in a liver disease model. METHODS: A microporator was used to overexpress Foxa2 in adipose tissue-derived stem cells (ADSCs), which were cultured in a poly(lactic-co-glycolic acid) (PLGA) scaffold. Later, the ADSCs were cultured in hepatic differentiation medium for 2 weeks by a 3-step method. For in vivo experiments, Foxa2-overexpressing ADSCs were loaded in the scaffold, cultured in hepatic differentiation medium and later were implanted in the dorsa of nude mice subjected to acute liver injury (thioacetamide intraperitoneal injection). RESULTS: Foxa2-overexpressing ADSCs showed greater increases in hepatocyte-specific gene markers (alpha fetoprotein [AFP], cytokeratin 18 [CK18], and albumin), cytoplasmic glycogen storage, and cytochrome P450 expression than cells that underwent the conventional differentiation method. In vivo experiments using the nude mouse model showed that 2 weeks after scaffold implantation, the mRNA expression of AFP, CK18, dipeptidyl peptidase 4 (CD26), and connexin 32 (CX32) was higher in the Foxa2-overexpressing ADSCs group than in the ADSCs group. The Foxa2-overexpressing ADSCs scaffold treatment group showed attenuated liver injury without stem cell homing in the thioacetamide-induced acute liver injury model. CONCLUSIONS: Foxa2-overexpressing ADSCs applied in a scaffold system enhanced hepatocyte-like differentiation and attenuated acute liver damage in an acute liver injury model without homing effects.

Plasma Levels of K18 Fragments Do Not Correlate with Alcoholic Liver Fibrosis

BACKGROUND/AIMS: Noninvasive markers of liver fibrosis in alcoholic liver disease (ALD) are crucial to establish early intervention. Previous studies have suggested that plasma levels of cleaved keratin-18 (K18; M30) fragments can predict the severity of liver disease. The aim of this study was to correlate plasma M30 levels with stages of liver fibrosis in ALD. METHODS: Patients with ALD (n=139, 79.1% males) and liver histology were included, and plasma samples were collected to quantify plasma M30 levels. Patients were stratified into five groups by fibrosis stage (F0=14; F1=15; F2=35; F3=17; and F4=58) according to the Kleiner score. Differences between groups were evaluated using the chi-square test or analysis of variance. Trends by fibrosis stage were calculated by logistic regression analysis, and sensitivity, specificity and positive and negative predictive values were determined. RESULTS: There were no significant differences in M30 levels among fibrosis stages. The correlation between plasma M30 levels and fibrosis was poor (Pearson’s correlation coefficient=0.13, Spearman rho=0.20 [p=0.02]), and M30 levels did not correlate with alcohol-specific histological features. However, significant correlations of M30 levels with aspartate aminotransferase (Spearman rho=0.653, p 200 U/L reveal a sensitivity for predicting cirrhosis of 84.5% with a negative predictive value of 73.5%. CONCLUSIONS: Plasma M30 levels are often elevated in ALD and correlate with serum transaminases but do not reflect fibrosis. The usefulness as a prognostic marker awaits evaluation in prospective studies.

Expression of M30 and M65 in celiac disease. Analytical cross-sectional study

ABSTRACT BACKGROUND: The role of villous atrophy in apoptosis, a distinctive feature of celiac disease, is a matter of controversy. The aim of this study was to determine the apoptosis rate through immunohistochemical staining for M30 and M65 in celiac disease cases. DESIGN AND SETTING: Analytical cross-sectional study in a tertiary-level center. METHODS: Duodenal biopsies from 28 treatment-naive patients with celiac disease, 16 patients with potential celiac disease, 10 patients with a gluten-free diet and 8 controls were subjected to immunohistochemical staining for the end-apoptotic marker M30 and the total cell death marker M65. H-scores were compared. Several laboratory parameters were recorded concomitantly, and at the one-year follow-up for celiac disease and potential celiac disease patients. RESULTS: There was a significant difference in H-score for M30 expression between the celiac disease, potential celiac disease and gluten-free diet groups (P = 0.009). There was no significant difference in H-score for M65 expression. There was a positive correlation between the H-score for M30 expression and the anti-tissue transglutaminase immunoglobulin A (anti-tTgIgA) and anti-tissue transglutaminase immunoglobulin G (anti-tTgIgG) levels (R = 0.285, P = 0.036; and R = 0.307, P = 0.024, respectively); and between the H-score for M65 expression and the anti-tTgIgA and anti-tTgIgG levels (R = 0.265, P = 0.053; and R=0.314, P = 0.021, respectively). There was no difference between celiac disease and potential celiac disease patients regarding the laboratory parameters selected. CONCLUSION: The rates of apoptosis and nutritional deficiencies in patients with potential celiac disease were similar to those in patients with celiac disease.

The role of serum ck-18, mmp-9 and tipm-1 levels in predicting r0 resection in patients with gastric cancer / O papel dos níveis séricos de ck-18, mmp-9 e tipm-1 na predição da ressecção r0 em pacientes com câncer gástrico

ABSTRACT Background: Gastric cancer is the 3rd most common cause of death in men and the 5th common in women worldwide. Today, surgery is the only curative therapy. Currently available advanced imaging modalities can predict R0 resection in most patients, but it can only be detected with certainty in the perioperative period. Aim: To determine the role of serum CK18, MMP9, TIMP1 levels in predicting R0 resection in patients with gastric cancer. Methods: Fifty consecutive patients scheduled for curative surgery with gastric adenocarcinoma diagnosed between 2013-2015 were included. One ml of blood was taken from the patients to analyze CK18, MMP9 and TIMP1. Results: CK18, MMP9 and TIMP1 levels were positively correlated with pathological N and the stage (p<0,05). CK-18, MMP-9 and TIMP-1 averages in positive clinical lymph nodes and in clinical stage 3, were found to be higher than the averages of those with negative clinical lymph nodes and in clinical stage 2 (p<0,05). Conclusion: Although serum CK-18, MMP-9 and TIMP-1 preoperatively measured in patients scheduled for curative surgery did not help to evaluate gastric tumor resectability, they were usefull in predicting N3-stage.

RESUMO Racional: Câncer gástrico é a terceira causa mais comum de morte em homens e a quinta em mulheres em todo o mundo. Atualmente a cirurgia é a única terapia curativa. As modalidades de imagem avançadas atualmente disponíveis podem prever a ressecção R0 na maioria dos pacientes, mas ela só pode ser detectada durante o perioperatório. Objetivo: Determinar o papel dos níveis séricos de CK18, MMP9 e TIMP1 na predição da ressecção R0 em pacientes com câncer gástrico. Métodos: Foram incluídos no estudo pacientes consecutivos agendados para operação curativa entre 2013-2015. Foi retirado 1 ml de sangue dos pacientes incluídos para estudar CK18, MMP9 e TIMP1. Resultados: Os níveis de CK18, MMP9 e TIMP1 foram positivamente correlacionados com o N patológico e o estadiamento (p<0,05). As médias CK-18, MMP-9 e TIMP-1 das pessoas com linfonodos positivos e aqueles em estágio clínico 3 foram superiores às médias das pessoas com linfonodos negativos e estágio clínico 2 (p<0,05). Conclusão: Embora as dosagens séricas de CK-18, MMP-9 e TIMP-1 em pacientes agendados para operação curativa por adenocarcinoma gástrico não ajudem a ter ideia de ressecabilidade tumoral, ela foi útil na predição de estadiamento N3.

Differentiation Potential of Breast Milk-Derived Mesenchymal Stem Cells into Hepatocyte-Like Cells

Human breast milk stem cells (hBSCs) contain a population of cells with the ability to differentiate into various cell lineages for cell therapy applications. The current study examined the differentiation potential of hBSCs into hepatocytes- like cells. The cells were isolated from the breast milk and were treated with hepatogenic medium containing hepatocyte growth factor, insulin-like growth factor and dexamethasone for 7 days subsequently; Oncostatin M was added to the culture media. RT-PCR and immunocytochemistry were performed to detect the hepatogenic markers. The glycogen storage and the ability of the cells to absorb and release indocynanin green were also tested. The data showed that most of the differentiated cells formed cell aggregates after the 30th day, with more cells accumulated to form spheroids. RT-PCR revealed the expression of the hepatic nuclear factor, albumin, cytokeratin 18 and 19, cytochrome P2B6, glucose-6-phospahtase and claudin. The functional assays also showed glycogen storage and omission of indicynine green. Our study demonstrated hBSCs are novel population that can differentiate into hepatocyte-like cells.

Heparin/Collagen 3D Scaffold Accelerates Hepatocyte Differentiation of Wharton's Jelly-Derived Mesenchymal Stem Cells

Both mature and stem cell-derived hepatocytes lost their phenotype and functionality under conventional culture conditions. However, the 3D scaffolds containing the main extracellular matrix constitutions, such as heparin, may provide appropriate microenvironment for hepatocytes to be functional. The current study aimed to investigate the efficacy of the differentiation capability of hepatocytes derived from human Wharton's jelly mesenchymal stem cells (WJ-MSCs) in 3D heparinized scaffold. In this case, the human WJ-MSCs were cultured on the heparinized and non-heparinized 2D collagen gels or within 3D scaffolds in the presence of hepatogenic medium. Immunostaining was performed for anti-alpha fetoprotein, cytokeratin-18 and -19 antibodies. RT-PCR was performed for detection of hepatic nuclear factor-4 (HNF-4), albumin, cytokeratin-18 and -19, glucose-6-phosphatase (G6P), c-met and Cyp2B. The results indicated that hepatogenic media induced the cells to express early liver-specific markers including HNF4, albumin, cytokeratin-18 and 19 in all conditions. The cells cultured on both heparinized culture conditions expressed late liver-specific markers such as G6P and Cyp2B as well. Besides, the hepatocytes differentiated in 3D heparinized scaffolds stored more glycogen that indicated they were more functional. Non-heparinized 2D gel was the superior condition for cholangiocyte differentiation as indicated by higher levels of cytokeratin 19 expression. In conclusion, the heparinized 3D scaffolds provided a microenvironment to mimic Disse space. Therefore, 3D heparinized collagen scaffold can be suggested as a good vehicle for hepatocyte differentiation.

Relationship between liver injury and serum cytokeratin 18 levels in asymptomatic hepatitis B virus carriers and in patients with chronic hepatitis B infection

Background and study aims: Apoptosis represents a well-known mechanism of cell death involved in most chronic liver injuries. Our aim was to investigate the serum fragment level of cytokeratin 18 [CK18], M30, in asymptomatic hepatitis B virus [HBV] carriers and patients with chronic hepatitis B [CHB] and to evaluate the relationship between serum M30 levels and the severity of hepatic injury

Patients and methods: Asymptomatic HBV carriers [n = 169], patients with CHB [n = 100], and healthy control subjects [n = 43] were enrolled in the study. Serum CK18 [M30] levels were analysed in all subjects. Liver biopsy for histopathological assessment was performed in asymptomatic HBV carriers and in patients with CHB infection

Results: Serum CK18 [M30] levels were significantly higher in asymptomatic HBV carriers [198.77 +/- 77.62 U/L] than in healthy control subjects [146.92 +/- 40.18 U/L]. Patients with CHB [283.02 +/- 147.45 U/L] had significantly higher CK18 [M30] levels than asymptomatic HBV carriers [p = 0.001]. The diagnostic efficacy of CK18 [M30] levels in distinguishing patients with HBeAg-negative CHB from asymptomatic HBV carriers was found to be moderate [c-statistics: 0.695], and the diagnostic cut-off value of CK18 [M30] was 262 U/L [specificity: 85%, sensitivity: 48%, positive likelihood ratio: 3.35, and negative likelihood ratio: 0.60]. There was a positive correlation between serum CK18 [M30] levels and histological activity index scores in asymptomatic HBV carriers and patients with CHB

Conclusions: Serum CK18 [M30] levels may be a valuable indicator in distinguishing asymptomatic HBV carriers from patients with HBeAg-negative CHB when considered together with ALT and HBV-DNA levels

Induced differentiation of rat kidney stem cells into renal tubular epithelial cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the differentiation capability of kidney stem cells (KSCs) into renal tubular epithelial cells (RTECs).</p><p><b>METHODS</b>KSCs isolated from the renal papilla of 4-week-old SD rats were co-cultured with hypoxia-exposed RTEC in induced medium (containing activin A, BMP-7, and retinoic acid) and renal epithelial cell growth medium (REGM) alternately. The KSCs cultured in MSC medium served as the control. The KSC differentiation rates in both groups were determined using flow cytometry, immunofluorescence assay and qRT-PCR.</p><p><b>RESULTS</b>Flow cytometry showed a CK-18 positive rate of 6.5Percnt; in the control KSC group and of 44.2% in the induced group. Immunofluorescence assay detected the positivity for mature epithelial cell markers CK-18, E-cadherin, and ZO-1 in the induced cells. The results of qRT-PCR showed significantly increased expression of E-cadherin and AQP-1 mRNAs in the induced cells compared with the control cells (P<0.01).</p><p><b>CONCLUSION</b>Rat KSCs can be induced to differentiate into RTECs in vitro.</p>

Detection of cytokeratin18 and cytokeratin19 gene expression in blood and tumor tissue of nasopharyngeal carcinoma patients by RT-PCR / 临床耳鼻咽喉头颈外科杂志

UNLABELLED@#Abstract@*OBJECTIVE@#To measure the expression of CK18 and CK19 in the cells from peripheral blood and tumor tissue of the nasopharyngeal carcinoma patients,to test whether CK 18 and CK 19 could be biomarkers of nasopharyngeal carcinoma fordiagnosis.@*METHOD@#The mRNA was extracted from the blood and carcinoma tissue of nasopharyngeal carcinoma and was reversed transcription to cDNA. The 3 pairs primers were designed for RT-PCR and the fold value was calculated to evaluated expression by ΔCT.@*RESULT@#There are no statistical differences between the CK18 and CK19 gene expression and the gender, age and metastasis in tumor tissue of 45 nasopharyngeal carcinoma patients (P>0. 05). There are significant differences among 3 pathological stages and 2 genes expressed increase as the grade malignancy (P<0. 05). The detecting of the 2 genes expression from blood cells shows that CK18 and CK19 had a high positive ratio 64% and 75% respectively. Meanwhile this method showed a same detection characteristic in tumor and blood, the positive.rate of CK18 and CK19 genes in metastasis is higher than non-metastasis. The results showed CK18 has a high specificity and CK19 has a high sensitivity for prognosis and all relapsed cases are associated with the expression of CK18 and CK19.@*CONCLUSION@#CK18 and CK19 may be used as biomarkers of nasopharyngeal carcinoma for diagnosis.

Proteomic and bioinformatic analyses of possible target-related proteins of gambogic acid in human breast carcinoma MDA-MB-231 cells / 中国天然药物

Gambogic acid (GA) is an anticancer agent in phase ‖b clinical trial in China but its mechanism of action has not been fully clarified. The present study was designed to search the possible target-related proteins of GA in cancer cells using proteomic method and establish possible network using bioinformatic analysis. Cytotoxicity and anti-migration effects of GA in MDA-MB-231 cells were checked using MTT assay, flow cytometry, wound migration assay, and chamber migration assay. Possible target-related proteins of GA at early (3 h) and late stage (24 h) of treatment were searched using a proteomic technology, two-dimensional electrophoresis (2-DE). The possible network of GA was established using bioinformatic analysis. The intracellular expression levels of vimentin, keratin 18, and calumenin were determined using Western blotting. GA inhibited cell proliferation and induced cell cycle arrest at G2/M phase and apoptosis in MDA-MB-231 cells. Additionally, GA exhibited anti-migration effects at non-toxic doses. In 2-DE analysis, totally 23 possible GA targeted proteins were found, including those with functions in cytoskeleton and transport, regulation of redox state, metabolism, ubiquitin-proteasome system, transcription and translation, protein transport and modification, and cytokine. Network analysis of these proteins suggested that cytoskeleton-related proteins might play important roles in the effects of GA. Results of Western blotting confirmed the cleavage of vimentin, increase in keratin 18, and decrease in calumenin levels in GA-treated cells. In summary, GA is a multi-target compound and its anti-cancer effects may be based on several target-related proteins such as cytoskeleton-related proteins.

Propofol or sevoflurane anaesthesia does not affect hepatic integrity as assessed by the M30 & M65 cell death markers & liver enzymes.

Background & objectives: General anaesthetics may induce apoptosis. The pro-apoptotic/necrotic markers M30 (caspase-cleaved cytokeratin-18) and M65 (intact cytokeratin-18) have been used to identify early apoptosis in liver disease. The aim of this study was to detect the effect of propofol and sevoflurane anaesthesia on these markers and blood transaminase levels in female patients undergoing elective surgery. Methods: Sixty-seven women undergoing mastectomy or thyroidectomy under general anaesthesia were randomly allocated to the propofol or sevoflurane groups. Venous blood samples for measuring the apoptotic and necrotic markers M30 and M65 as well as for measuring the alanine aminotransferase (ALT) and the aspartate aminotransferase (AST) liver enzymes were collected before induction of anaesthesia, immediately after completion of surgery, and 24 and 48 h postoperatively. Results. The M30 values preoperatively and 0, 24 and 48 h postoperatively were 280±229, 300±244, 267±198 and 254±189 U/l in the propofol group and 237±95, 242±109, 231±94 and 234±127 U/l in the sevoflurane group, respectively. The M30 values did not differ within or between the groups. The M65 levels at the same time intervals were 470±262, 478±271, 456±339 and 485±273 in the propofol group and 427±226, 481±227, 389±158 and 404±144 U/l in the sevoflurane group, respectively. No significant changes were found in the M65 either within or between the propofol and the sevoflurane groups. The ALT and AST levels did not change at these time intervals. Interpretation & conclusions: Under the present study design propofol or sevoflurane anaesthesia did not induce apoptosis or affected the liver function as assessed by the M30, M65 markers and liver enzymes in patients undergoing mastectomy or thyroidectomy under general anaesthesia.

Clinical Correlation between Serum Cytokeratin-18 and Metabolic Parameters in Patients with Sonographic Non-alcoholic Fatty Liver Disease / 대한소화기학회지

BACKGROUND/AIMS: The serum cytokeratin-18 (CK-18) has been suggested to be a surrogate marker of non-alcoholic fatty liver disease (NAFLD). The aim of this study was to investigate the correlation between CK-18 and metabolic parameter in NAFLD patients. Correlation between CK-18 and macronutrient composition was also assessed. METHODS: A total of 212 subjects were recruited. Blood chemistry including fasting glucose, cholesterol level, AST, ALT, and CK-18 were compared. Data on calorie intake and carbohydrate consumption were acquired by five-day-diet diary using 24 hour recall method. RESULTS: Plasma CK-18 were markedly increased in patient with NAFLD compared with control group (420.4+/-282.3 vs. 313.6+/-179, p<0.001). Plasma CK-18 were positively correlated with systolic blood pressure (r=0.130), ALT (r=0.503) and negatively correlated with HDL cholesterol (r=-0.246). NAFLD patients with metabolic syndrome had higher CK-18 level than those without metabolic syndrome (484.0 vs. 372.1 U/L, p=0.021). When NAFLD patients were subdivided into two groups with CK-18 cut-off value of 400 U/L, patients with CK-18 level over 400 U/L showed higher body mass index (28.0+/-4.5 vs. 25.5+/-4.3), subcutaneous abdominal fat (283.5+/-172.2 vs. 195.7+/-147.8), AST (52.7+/-26.3 vs. 40.7+/-23.5) and ALT (102.0+/-52.6 vs. 61.2+/-32.2). Calorie intake (r=0.301) and carbohydrate intake (r=0.305) also showed positive correlation with CK-18. CONCLUSIONS: Plasma CK-18 showed positive correlation with metabolic parameters as well as calorie and carbohydrate intake when its cut-off value of greater than 400 U/L was used.

The Significance of Caspase-Cleaved Cytokeratin 18 in Pleural Effusion / 결핵및호흡기질환

BACKGROUND: Apoptosis plays a role in the development of pleural effusion. Caspase-cleaved cytokeratin 18, a marker for epithelial cell apoptosis, was evaluated in pleural effusion. METHODS: A total of 79 patients with pleural effusion were enrolled. The underlying causes were lung cancer (n=24), parapneumonic effusion (n=15), tuberculous effusion (n=28), and transudates (n=12). The levels of M30, an epitope of caspase-cleaved cytokeratin 18, were measured in blood and pleural fluids using enzyme-linked immunosorbent assay along with routine cellular and biochemical parameters. The expression of M30 was evaluated in the pleural tissues using immunohistochemistry for M30. RESULTS: The M30 levels in pleural fluid were significantly higher in patients with tuberculosis (2,632.1+/-1,467.3 U/mL) than in patients with lung cancer (956.5+/-618.5 U/mL), parapneumonic effusion (689.9+/-413.6 U/mL), and transudates (273.6+/-144.5 U/mL; all p<0.01). The serum levels were not significantly different among the disease groups. Based on receiver operating characteristics analysis, the area under the curve of M30 for differentiating tuberculous pleural effusion from all other effusions was 0.93. In the immunohistochemical analysis of M30, all pathologic types of cancer cells showed moderate to high expression, and the epithelioid cells in granulomas showed high expression in tuberculous pleural tissues. CONCLUSION: Caspase-cleaved cytokeratin 18 was most prominently observed in tuberculous pleural effusion and showed utility as a clinical marker. The main source of M30 was found to be the epithelioid cells of granulomas in tuberculous pleural tissues.

Clinicopathologic features of papillary tumors of the pineal region / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the clinicopathologic features of papillary tumor of the pineal region (PTPR).</p><p><b>METHOD</b>Three hundred and eighty six cases of pineal region and posterior third ventricle tumors, two newborn and two adult pineal glands were analyzed by HE, PAS and immunohistochemistry of 16 antibodies (EnVision method).</p><p><b>RESULTS</b>Five cases of PTPR were diagnosed with mixed papillary features and densely cellular areas, and included one recurrent case. In the papillary areas, the vessels were lined by one or several layers of cuboidal/columnar cells; the vessel wall was hyalinized. In the densely cellular areas, sheets or nests of tumor cells were seen. The tumor cells of these five cases were immunoreactive to CK, CK8/18, synaptophysin, MAP2, nestin, S-100, and vimentin. Four cases were immunoreactive to NSE and CgA; and 2 cases were immunoreactive to NF. All five cases were negative for EMA, CK5/6, CEA, and NeuN. Ki-67 labeling index ranged from 1% to 6%.Three patients were alive, and the recurrent one died.</p><p><b>CONCLUSIONS</b>PTPR occurs in patients with over a wide age range, from children to adults, and is more commonly found in male than female. PTPR is composed of both papillary and solid areas, characterized by epithelial cytology, and needs to be differentiated from ependymoma. PTPR may originate from the specialized ependymocytes of the subcommissural organ. The prognostic factors are early diagnosis, complete surgical resection and radiotherapy.</p>

Noninvasive predictors of nonalcoholic steatohepatitis in Korean patients with histologically proven nonalcoholic fatty liver disease

BACKGROUND/AIMS: The aims of this study were (1) to identify the useful clinical parameters of noninvasive approach for distinguishing nonalcoholic steatohepatitis (NASH) from nonalcoholic fatty liver disease (NAFLD), and (2) to determine whether the levels of the identified parameters are correlated with the severity of liver injury in patients with NASH. METHODS: One hundred and eight consecutive patients with biopsy-proven NAFLD (age, 39.8+/-13.5 years, mean+/-SD; males, 67.6%) were prospectively enrolled from 10 participating centers across Korea. RESULTS: According to the original criteria for NAFLD subtypes, 67 patients (62.0%) had NASH (defined as steatosis with hepatocellular ballooning and/or Mallory-Denk bodies or fibrosis > or =2). Among those with NAFLD subtype 3 or 4, none had an NAFLD histologic activity score (NAS) below 3 points, 40.3% had a score of 3 or 4 points, and 59.7% had a score >4 points. Fragmented cytokeratin-18 (CK-18) levels were positively correlated with NAS (r=0.401), as well as NAS components such as lobular inflammation (r=0.387) and ballooning (r=0.231). Fragmented CK-18 was also correlated with aspartate aminotransferase (r=0.609), alanine aminotransferase (r=0.588), serum ferritin (r=0.432), and the fibrosis stage (r=0.314). A fragmented CK-18 cutoff level of 235.5 U/L yielded sensitivity, specificity, and positive and negative predictive values of 69.0%, 64.9%, 75.5% (95% CI 62.4-85.1), and 57.1% (95% CI 42.2-70.9), respectively, for the diagnosis of NASH. CONCLUSIONS: Serum fragmented CK-18 levels can be used to distinguish between NASH and NAFL. Further evaluation is required to determine whether the combined measurement of serum CK-18 and ferritin levels improves the diagnostic performance of this distinction.

Epithelioid Trophoblastic Tumor: Clinicopathologic and Immunohistochemical Analysis of Three Cases

Epithelioid trophoblastic tumor is an unusual type of trophoblastic tumor. Here we report on the clinicopathologic and immunohistochemical features of three cases of epithelioid trophoblastic tumor. All three patients were of reproductive age and presented with vaginal bleeding and mild elevation of human chorionic gonadotropin (hCG). All patients underwent a hysterectomy. The tumors consisted of epithelioid intermediate trophoblastic cells that were mononucleated and eosinophilic, or showed clear cytoplasm on microscopic examination. One case presented with a focal choriocarcinoma component. Immunohistochemically, the tumors displayed diffuse positivity for cytokeratin 18, E-cadherin, epidermal growth factor receptor, and p53 and focal positivity for p63 and hCG. However, expression of alpha-inhibin and placental alkaline phosphatase was almost negative. Tests for human placental lactogen and epithelial membrane antigen were also negative in all cases.

Evaluation of the diagnostic value of serum and tissue apoptotoic cytokeratin- 18 in patients with chronic hepatitis C

Hepatitis C virus [HCV] is considered the most common aetiology of chronic liver disease [CLD] in Egypt. The disease severity ranges from mild illness to cirrhosis and hepatocellular carcinoma. A role for apoptosis in liver damage caused by HCV chronic infection has been suggested. Cytokeratin 18 [CK-18] is the major intermediate filament protein in the liver and is a known caspase substrate in hepatocyte apoptosis. Therefore, we analysed the serum and tissue levels of CK-18 in patients with chronic HCV infection to evaluate its role in hepatocyte apoptosis. We also correlated CK-18 expression with the severity of hepatic pathology. This study examined 80 Egyptian patients with liver disease. There were 69 patients with chronic hepatitis C and 11 patients with hepatitis C-induced cirrhotic changes. Fifteen healthy controls were also included in the study. The levels of CK-18 fragment were quantified in paired serum and liver biopsy samples. The serum and tissue CK-18 levels were reduced in chronic HCV patients compared to early cirrhosis patients. This result indicates that serum levels of CK-18 and the hepatic expression of CK-18 might play an important role in disease progression. The serum and tissue levels of CK-18 were significantly increased and directly correlated with inflammation severity, stage of fibrosis, and ALT levels in the chronic HCV group and the cirrhotic liver group. There was no significant difference in viral load between patient cohorts. The serum level and the hepatic expression of CK-18 are related to disease activity and are directly correlated with METAVIR scoring. This result suggests that serum CK-18 levels may be useful for monitoring disease activity in chronic HCV and liver cirrhosis patients