Herbal traditional Chinese medicine and its evidence base in gastrointestinal disorders

Herbal traditional Chinese medicine(TCM) is used to treat several ailments, but its efficiency is poorly documented and hence debated, as opposed to modern medicine commonly providinge ffective therapies. The aim of this review article is to present a practical reference guide on the role of herbal TCM in managing gastrointestinal disorders, supported by systematic reviews and evidence based trials. A literature search using herbal TCM combined with terms for gastrointestinal disorders in Pub Med and the Cochrane database identified publications of herbal TCM trials. Results were analyzed for study type, inclusion criteria, and outcome parameters. Quality of placebo controlled, randomized, double-blind clinical trials was poor, mostly neglecting stringent evidence based diagnostic and therapeutic criteria. Accordingly, appropriate Cochrane reviews and meta-analyses were limited and failed to support valid, clinically relevant evidence based efficiency of herbal TCM in gastrointestinal diseases, including gastroesophageal reflux disease, gastric or duodenal ulcer, dyspepsia, irritable bowel syndrome, ulcerative colitis, and Crohn's disease. In conclusion, the use of herbal TCM to treat various diseases has an interesting philosophical background with a long history, but it received increasing skepticism due to the lack of evidence based efficiency as shown by high quality trials; this has now been summarized for gastrointestinal disorders, with TCM not recommended for most gastrointestinal diseases. Future studies should focus on placebo controlled, randomized, double-blind clinical trials, herbal product quality and standard criteria for diagnosis, treatment, outcome, and assessment of adverse herb reactions. This approach will provide figures of risk/benefit profiles that hopefully are positive for at least some treatment modalities of herbal TCM. Proponents of modern herbal TCM best face these promising challenges of pragmaticmodern medicine by bridging the gap between the two medicinal cultures.

Herbal Traditional Chinese Medicine and suspected liver injury: A prospective study

AIM To analyze liver tests before and following treatment with herbal Traditional Chinese Medicine(TCM) in order to evaluate the frequency of newly detected liver injury.METHODS Patients with normal values of alanine aminotransferase(ALT) as a diagnostic marker for ruling out pre-existing liver disease were enrolled in a prospective study of a safety program carried out at the First German Hospital of TCM from 1994 to 2015. All patients received herbal products, and their ALT values were reassessed 1-3 d prior to discharge. To verify or exclude causality for suspected TCM herbs, the Roussel Uclaf Causality Assessment Method(RUCAM) was used.RESULTS This report presents for the first time liver injury data derived from a prospective, hospital-based and largescale study of 21470 patients who had no liver disease prior to treatment with herbal TCM. Among these, ALT ranged from 1 × to < 5 × upper limit normal(ULN) in 844 patients(3.93%) and suggested mild or moderate liver adaptive abnormalities. However, 26 patients(0.12%) experienced higher ALT values of ≥ 5 × ULN(300.0 ± 172.9 U/L, mean ± SD). Causality for TCM herbs was RUCAM-based probable in 8/26 patients, possible in 16/26, and excluded in 2/26 cases. Bupleuri radix and Scutellariae radix were the two TCM herbs most commonly implicated. CONCLUSION In 26(0.12%) of 21470 patients treated with herbal TCM, liver injury with ALT values of ≥ 5 × ULN was found, which normalized shortly following treatment cessation, also substantiating causality.

Drug and herb induced liver injury: Council for International Organizations of Medical Sciences scale for causality assessment

Causality assessment of suspected drug induced liver injury(DILI) and herb induced liver injury(HILI) is hampered by the lack of a standardized approach to be used by attending physicians and at various subsequent evaluating levels. The aim of this review was to analyze the suitability of the liver specific Council for International Organizations of Medical Sciences(CIOMS) scale as a standard tool for causality assessment in DILI and HILI cases. PubMed database was searched for the following terms: drug induced liver injury; herb induced liver injury; DILI causality assessment; and HILI causality assessment. The strength of the CIOMS lies in its potential as a standardized scale for DILI and HILI causality assessment. Other advantages include its liver specificity and its validation for hepatotoxicity with excellent sensitivity, specificity and predictive validity, based on cases with a positive reexposure test. This scale allows prospective collection of all relevant data required for a valid causality assessment. It does not require expert knowledge in hepatotoxicity and its results may subsequently be refined. Weaknesses of the CIOMS scale include the limited exclusion of alternative causes and qualitatively graded risk factors. In conclusion, CIOMS appears to be suitable as a standard scale for attending physicians, regulatory agencies, expert panels and other scientists to provide a standardized, reproducible causality assessment in suspected DILI and HILI cases, applicable primarily at all assessing levels involved. 2014 Baishideng Publishing Group Co., Limited. All

Superior mesenteric artery syndrome:Diagnosis and management

Superior mesenteric artery(SMA)syndrome(also known as Wilkie's syndrome,cast syndrome,or aorto-mesenteric compass syndrome)is an obstruction of the duodenum caused by extrinsic compression between the SMA and the aorta.The median age of patients is 23 years old(range 0-91 years old)and predominant in females over males with a ratio of 3:2.The symptoms are variable,consisting of postprandial abdominal pain,nausea and vomiting,early satiety,anorexia,and weight loss and can mimic anorexia nervosa or functional dyspepsia.Because recurrent vomiting leads to aspiration pneumonia or respiratory depression via metabolic alkalosis,early diagnosis is required.The useful diagnostic modalities are computed tomography as a standard tool and ultrasonography,which has advantages in safety and capability of real-time assessments of SMA mobility and duodenum passage.The initial treatment is usually conservative,including postural change,gastroduodenal decompression,and nutrient management(success rates:70%-80%).If conservative therapy fails,surgical treatment(i.e.,laparoscopic duodenojejunostomy)is recommended(success rates:80%-100%).

Acute-on-chronic liver failure in patients with severe acute respiratory syndrome coronavirus 2 infection

The coronavirus disease 2019(COVID-19)pandemic caused by the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has had a significant impact on the lives of millions of people,especially those with other concomitant diseases,such as chronic liver diseases.To date,seven coronaviruses have been identified to infect humans.The main site of pathological action of these viruses is lung tissue.However,a substantial number of studies have proven that SARSCoV-2 shows affinity towards several organs,including the gastrointestinal tract and the liver.The current state of evidence points to several proposed mechanisms of liver injury in patients with COVID-19 and their combination.Liver impairment is considered to be the result of the direct effect of the virus on the hepatic tissue cells,a systemic reaction consisting of inflammation,hypoxia and cytokine storm,drug-induced liver injury,with the possible contribution of a perturbed gut-liver axis.Reactivation of chronic hepatic disease could be another factor for liver impairment in patients with SARS-CoV-2 infection.Acute-onchronic liver failure(ACLF)is a relatively new syndrome that occurs in 10%–30%of all hospitalized patients with chronic liver disease.It is crucial to recognize high-risk patients due to the increased morbidity and mortality in these cases.Several published studies have reported virus infection as a trigger factor for ACLF.However,to date,there are few relevant studies describing the presence of ACLF in patients with acute SARS-CoV-2 infection.In this minireview we summarize the current state of knowledge regarding the relation between ACLF and acute SARS-CoV-2 infection.

Contrast-enhanced endoscopic ultrasound in discrimination between focal pancreatitis and pancreatic cancer

AIM： To evaluate the contrast-enhanced endosonography as a method of differentiating inflammation from pancreatic carcinoma based on perfusion characteristics of microvessels. METHODS： In 86 patients with suspected chronic pancreatitis （age： 62± 12 years; sex： f/m 38/48）, pancreatic lesions were examined by conventional endo- scopic B-mode, power Doppler ultrasound and contrastenhanced power mode （Hitachi EUB 525, SonoVue, 2.4 mL, Bracco） using the following criteria for malignant lesions： no detectable vascularisation using conventional power Doppler scanning, irregular appearance of arterial vessels over a short distance using SonoVue contrastenhanced technique and no detectable venous vessels inside the lesion. A malignant lesion was assumed if all criteria were detectable [gold standard endoscopic ultrasound （EUS）-guided fine needle aspiration cytology, operation]. The criteria of chronic pancreatitis without neoplasia were defined as no detectable vascularisation before injection of SonoVue, regular appearance of vessels over a distance of at least 20 mm after injection of SonoVue and detection of arterial and venous vessels. RESULTS： The sensitivity and specifidty of conventional EUS were 73.2% and 83.3% respectively for pancreatic cancer. The sensitivity of contrast-enhanced EUS increased to 91.1% in 51 of 56 patients with malignant pancreatic lesion and the specificity increased to 93.3% in 28 of 30 patients with chronic inflammatory pancreatic disease.CONCLUSION： Contrast-enhanced endoscopic ultrasound improves the differentiation between chronic pancreatitis and pancreatic carcinoma.

Long-term outcome and prognostic factors of patients with hilar cholangiocarcinoma

AIM： To evaluate the long-term outcome and prognostic factors of patients with hilar cholangiocarinoma. METHODS： Ninety-six consecutive patients underwent treatment for malignant hilar bile duct tumors during 1995-2005. Of the 96 patients, 20 were initially treated with surgery （n = 2 R0 / n = 18 R1）. In non-operated patients, data analysis was performed retrospectively. RESULTS： Among the 96 patients, 76 were treated with endoscopic transpapillary （ERC, n = 45） and/or percutaneous transhepatic biliary drainage （PTBD, n = 31）. The mean survival time of these 76 patients undergoing palliative endoscopic and/or percutaneous drainage was 359 ± 296 d. The mean survival time of patients with initial bilirubin levels 〉 10 mg/dL was significantly lower （P 〈 0.001） than patients with bilirubin levels 〈 10 mg/dL. The mean survival time of patients with Bismuth stage Ⅱ （n = 8）, Ⅲ （n = 28） and Ⅳ （n = 40） was 496 =1= 300 d, 441 ± 385 d and 274 ± 218 d, respectively. Thus, patients with advanced Bismuth stage showed a reduced mean survival time, but the difference was not significant. The type of biliary drainage had no significant benefidal effect on the mean survival time （ERC vs PTBD, P = 0.806）. CONCLUSION： Initial bilirubin level is a significant prognostic factor for survival of patients. In contrast, age, tumor stage according to the Bismuth-Corlette classification, and types of intervention are not significant prognostic parameters for survival. Palliative treatment with endoscopic or percutaneous biliary drainage is still suboptimal, new diagnostic and therapeutic tools need to be evaluated.

Diagnostic approaches for cholangiocarcinoma

Cholangiocarcinomas arise from the epithelial cells of the bile ducts and are associated with poor prognosis. Despite new diagnostic approaches, the definite diagnosis of this malignancy continues to be challenging. Cholangiocarcinomas often grow longitudinally along the bile duct rather than in a radial direction. Thus, large tumor masses are frequently absent and imaging techniques, including ultrasound, CT, and MRI have only limited sensitivity. Tissue collection during endoscopic (ERCP) and/or percutaneous transhepatic (PTC) procedures are usually used to confirm a definitive diagnosis of cholangiocarcinoma. However, forceps biopsy and brush cytology provide positive results for malignancy in about only 50% of patients. Percutaneous and peroral cholangioscopy using fiber-optic techniques were therefore developed for direct visualization of the biliary tree, yielding additional information about endoscopic appearance and tumor extension, as well as a guided biopsy acquistion. Finally, endoscopic ultrasonography (EUS) complements endoscopic and percutaneous approaches and may provide a tissue diagnosis of tumors in the biliary region through fine-needle aspiration. In the future, new techniques allowing for early detection, including molecular markers, should be developed to improve the diagnostic sensitivity in this increasing tumor entity.

Endoscopic transpapillary brush cytology and forceps biopsy in patients with hilar cholangiocarcinoma

AIM: To evaluate the sensitivity of brush cytology and forceps biopsy in a homogeneous patient group with hilar cholangiocarcinoma.METHODS: Brush cytology and forceps biopsy were routinely performed in patients with suspected malignant biliary strictures. Fifty-eight consecutive patients undergoing endoscopic retrograde cholangio-pancreatography (ERCP) including forceps biopsy and brush cytology in patients with hilar cholangiocarcinoma between 1995-2005.RESULTS: Positive results for malignancy were obtained in 24/58 patients (41.4%) by brush cytology and in 31/58 patients (53.4%) by forceps biopsy. The combination of both techniques brush cytology and forceps biopsy resulted only in a minor increase in diagnostic sensitivity to 60.3% (35/58 patients). In 20/58 patients (34.5%), diagnosis were obtained by both positive cytology and positive histology, in 11/58 (19%) by positive histology (negative cytology) and only 4/58 patients (6.9%) were confirmed by positive cytology (negative histology).CONCLUSION: Brush cytology and forceps biopsy have only limited sensitivity for the diagnosis of malignant hilar tumors. In our eyes, additional diagnostic techniques should be evaluated and should become routine in patients with negative cytological and histological findings.

Reduced bone mineral density and altered bone turnover markers in patients with non-cirrhotic chronic hepatitis B or C infection

AIM: Previous studies suggest that loss of bone mineral density (BMD) frequently occurs in patients with chronic viral liver disease, presenting with histologically proven liver cirrhosis. However, little is known about the occurrence of bone disease in non-cirrhotic patients with chronic hepatitis B or C. Therefore, it was the aim of this study to evaluate this particular population for BMD and bone turnover markers. METHODS: Biochemical markers of bone turnover and BMD were measured in 43 consecutive patients with HCV (n = 30) or HBV (n = 13) infection without histological evidence for liver cirrhosis. Mean age was 49 years (range 26-77 years). BMD was measured by dual X-ray absorptiometry in the femoral neck (FN) and the lumbar spine (LS) region. In addition, bone metabolism markers were measured. RESULTS: BMD was lowered in 25 (58%) of the patients with chronic hepatitis B or C (FN; 0.76 (0.53-0.99); LS: 0.96 (0.62-1.23) g/cm2). Eight (32%) osteopenic patients were diagnosed with osteoporosis. Bone-specific alkaline phosphatase (P= 0.005) and intact parathyroid hormone (iPTH) (P = 0.001) were significantly elevated in the more advanced stages of fibrosis. Mean T-score value was lower in patients with chronic hepatitis C as compared to patients suffering from chronic hepatitis B; however, the difference was not statistically significant (P= 0.09). CONCLUSION: There was a significantly reduced BMD in non-cirrhotic patients with chronic hepatitis B or C infection. Alterations of bone metabolism already occurred in advanced liver fibrosis without cirrhosis. According to our results, these secondary effects of chronic viral hepatitis should be further investigated.

Cirrhotic cardiomyopathy:A cardiologist's perspective

Cardiac dysfunction is frequently observed in patients with cirrhosis, and has long been linked to the direct toxic effect of alcohol. Cirrhotic cardiomyopathy(CCM) has recently been identified as an entity regardless of the cirrhosis etiology. Increased cardiac output due to hyperdynamic circulation is a pathophysiological hallmark of the disease. The underlying mechanisms involved in pathogenesis of CCM are complex and involve various neurohumoral and cellular pathways, including the impaired β-receptor and calcium signaling, altered cardiomyocyte membrane physiology, elevated sympathetic nervous tone and increased activity of vasodilatory pathways predominantly through the actions of nitric oxide, carbon monoxide and endocannabinoids. The main clinical features of CCM include attenuated systolic contractility in response to physiologic or pharmacologic strain, diastolic dysfunction, electrical conductance abnormalities and chronotropic incompetence. Particularly the diastolic dysfunction with impaired ventricularrelaxation and ventricular filling is a prominent feature of CCM.The underlying mechanism of diastolic dysfunction in cirrhosis is likely due to the increased myocardial wall stiffness caused by myocardial hypertrophy,fibrosis and subendothelial edema,subsequently resulting in high filling pressures of the left ventricle and atrium.Currently,no specific treatment exists for CCM.The liver transplantation is the only established effective therapy for patients with end-stage liver disease and associated cardiac failure.Liver transplantation has been shown to reverse systolic and diastolic dysfunction and the prolonged QT interval after transplantation.Here,we review the pathophysiological basis and clinical features of cirrhotic cardiomyopathy,and discuss currently available limited therapeutic options.

Transforming growth factor-β1 induces intestinal myofibroblast differentiation and modulates their migration

AIM： To investigate the effects of transforming growth factor β1 （TGF-β1） on the differentiation of colonic lamina propria fibroblasts （CLPF） into myofibroblasts in vitro.METHODS： Primary CLPF cultures were incubated with TGF-β1 and analyzed for production of m-smooth muscle actin （α-SMA）, fibronectin （FN） and FN isoforms. Migration assays were performed in a modified 48-well Boyden chamber. Levels of total and phosphorylated focal adhesion kinase （FAK） in CLPF were analyzed after induction of migration.did not change α-SMA levels, while TGF-β1 treatment for 6 d significantly increased α-SIVlA production. Short term incubation （6 h） with TGF-β1 enhanced CLPF migration, while long term treatment （6 d） of CLPF with TGF-β1 reduced migration to 15%-37% compared to untreated cells. FN and FN isoform mRNA expression were increased after short term incubation with TGF-β1 （2 d） in contrast to long term incubation with TGF-β1 for 6 d. After induction of migration, TGF-β1-preincubated CLPF showed higher amounts of FN and its isoforms and lower levels of total and phosphorylated FAK than untreated cells.CONCLUSION： Long term incubation of CLPF with TGF-β1 induced differentiation into myofibroblasts with enhanced α-SMA, reduced migratory potential and FAK phosphorylation, and increased FN production. In contrast, short term contact （6 h） of fibroblasts with TGF-β1 induced a dose-dependent increase of cell migration and FAK phosphorylation without induction of α-SMA production.

Case of clear-cell hepatocellular carcinoma that developed in the normal liver of a middle-aged woman

A 36-year-old woman was admitted to our department for close examination of a liver tumor that was found during a medical checkup. Abdominal US, CT and MRI showed a tumor in segment 7 (S7) of the liver. Although imaging suggested hepatocellular carcinoma, laboratory tests showed no abnormality in liver function, hepatitis virus markers were negative, and tumor markers including protein induced by vitamin K absence or antagonist Ⅱ (PIVKA-Ⅱ), α-fetoprotein (AFP), carbohydrate antigen 19-9 (CA19-9), and carcinoembryonic antigen (CEA) were all within normal ranges. Upon aspiration biopsy of the liver, the histopathological diagnosis was moderately differentiated hepatocellular carcinoma. Therefore, right hepatectomy was performed. Although a part of the tumor was necrotic, about 60% of the viable part showed a clear-cell variant. Consequently, it was diagnosed as clear-cell hepatocellular carcinoma. It was noted that the background liver tissue was normal. This case is worthy of reporting because development of clear-cell hepatocellular carcinoma in the normal liver of a middle-aged woman is rarely seen.

Herbal hepatotoxicity:Challenges and pitfalls of causality assessment methods

The diagnosis of herbal hepatotoxicity or herb induced liver injury(HILI) represents a particular clinical and regulatory challenge with major pitfalls for the causality evaluation.At the day HILI is suspected in a patient,physicians should start assessing the quality of the used herbal product,optimizing the clinical data for completeness,and applying the Council for International Organizations of Medical Sciences(CIOMS) scale for initial causality assessment.This scale is structured,quantitative,liver specific,and validated for hepatotoxicity cases.Its items provide individual scores,which together yield causality levels of highly probable,probable,possible,unlikely,and excluded.After completion by additional information including raw data,this scale with all items should be reported to regulatory agencies and manufacturers for further evaluation.The CIOMS scale is preferred as tool for assessing causality in hepatotoxicity cases,compared to numerous other causality assessment methods,which are inferior on various grounds.Among these disputed methods are the Maria and Victorino scale,an insufficiently qualified,shortened version of the CIOMS scale,as well as various liver unspecific methods such as thead hoc causality approach,the Naranjo scale,the World Health Organization(WHO) method,and the Karch and Lasagna method.An expert panel is required for the Drug Induced Liver Injury Network method,the WHO method,and other approaches based on expert opinion,which provide retrospective analyses with a long delay and thereby prevent a timely assessment of the illness in question by the physician.In conclusion,HILI causality assessment is challenging and is best achieved by the liver specific CIOMS scale,avoiding pitfalls commonly observed with other approaches.

Assessment of metastatic liver disease in patients with primary extrahepatic tumors by contrast-enhanced sonography versus CT and MRI

AIM： To evaluate contrast-enhanced ultrasonography （CEUS） using SonoVue in the detection of liver metastases in patients with known extrahepatic primary tumors versus the combined gold standard comprising CT, MRI and clinical/histological data. METHODS： It is an international multicenter study, and there were 12 centres and 125 patients （64 males, 61 females, aged 59 ± 11 years） involved, with 102 patients per protocol. Primary tumors were colorectal in 35%, breast in 27%, pancreatic in 17% and others in 21%. CEUS using SonoVue was employed with a Iow-mechanical-index technique and contrast-specific software using Siemens Elegra, Philips HDI 5000 and Acuson Sequoia; continuous scanning for at least five minutes. RESULTS： CEUS with SonoVue increased significantly the number of focal liver lesions detected versus unenhanced sonography. In 31.4% of the patients, more lesions were found after contrast enhancement. The total numbers of lesions detected were comparable with CEUS （55）, triple-phase spiral CT （61） and HRI with a liverspecific contrast agent （53）. Accuracy of detection of metastatic disease （i.e. at least one metastatic lesion） was significantly higher for CEUS （91.2%） than for unenhanced sonography （81.4%） and was similar to that of triple-phase spiral CT （89.2 %）. In 53 patients whose CEUS examination was negative, a follow-up examination 3-6 months later confirmed the absence of metastatic lesions in 50 patients （94.4%）. CONCLUSION： CEUS is proved to be reliable in the detection of liver metastases in patients with known extrahepatic primary tumors and suspected liver lesions.

Expression of c-kit receptor in human cholangiocarcinoma and in vivo treatment with imatinib mesilate in chimeric mice

AIM： To investigate the c-kit expression in biliary tract cancer cell lines and histological sections from patients with extrahepatic cholangiocarcinoma （CC） and to evaluate the efficacy of in vitro and in vitro treatment with imatinib mesilate. METHODS： The protein expression of c-kit in the human biliary tract cancer cell lines Mz-ChA-2 and EGI-1 and histological sections from 19 patients with extrahepatic CC was assessed by immunoblotting, immunocytochemistry, and immunohistochemistry. The anti-proliferative effect of imatinib mesilate on biliary tract cancer cell lines Mz-ChA-2 and EGI-1 was studied in vitro by automated cell counting. In addition, immunodeficient NMRI mice （Taconic^TM） were subcutaneously injected with 5 × 10^6 cells of cell lines MzChA-2 and EGI-1. After having reached a tumour volume of 200 mm^3, daily treatment was started intraperitoneally with imatinib mesilate at a dose of 50 mgikg or normal saline （NS）. Tumor volume was calculated with a Vernier caliper. After 14 d, mice were sacrificed with tumors excised and tumor mass determined.RESULTS： Immunoblotting revealed presence of c-kit in Mz-ChA-2 and absence in EGI-1 cells. Immunocytochemistry with c-kit antibodies displayed a cytoplasmatic and membraneous localization of receptor protein in Mz-ChA-2 cells and absence of c-kit in EGI-1 cells, c-kit was expressed in 7 of 19 （37%） extrahepatic humanCC tissue samples, 2 showed a moderate and 5 a rather weak immunostaining. Imatinib mesilate at a low concentration of 5 μmoliL caused a significant growth inhibition in the c-kit positive cell line Mz-ChA-2 （31%）, but not in the c-kit negative cell line EGI-1 （0%） （P〈0.05）. Imatinib mesilate at an intermediate concentration of 10 μmoliL inhibited cellular growth of both cell lines （51% vs 57%）. Imatinib mesilate at a higher concentration of 20 μmoliL seemed to have a general toxic effect on both cell lines. The IC50 values were 9.7 μmoliL and 11 μmoliL, respectively. After 14 d of in vitro treatment with imatinib mesilate, using the chimeric mouse model, c-kit positive Mz-ChA-2 tumors had a significantly reduced volume and mass as compared to NS treatment （P〈 0.05）. In contrast to that, treatment of mice bearing c-kit negative EGI-1 tumors did not result in any change of tumor volume and mass as compared to NS treatment. CONCLUSION： c-kit expression is detectable at a moderate to low protein level in biliary tract cancer. Imatinib mesilate exerts marked effects on tumor growth in vitro andin vitro dependent on the level of c-kit expression.

Treatment of biliary tract cancer with NVP-AEW541:Mechanisms of action and resistance

AIM:To investigate in vitro treatment with NVPAEW541,a small molecule inhibitor of insulin-like growth factor-1 receptor(IGF-1R),in biliary tract cancer (BTC),since this disease is associated with a poor prognosis due to wide resistance to chemotherapeutic agents and radiotherapy.METHODS:Cell growth inhibition by NVP-AEW541 was studied in vitro in 7 human BTC cell lines by automated cell counting.In addition,the anti-tumoral mechanism of NVP-AEW541 was studied by Western blotting,cell cycle analysis and reverse transcription-polymerase chain reaction(RT-PCR).Anti-tumoral drug effect in combination with gemcitabine,5-fluorouracil(5-FU)and Polo-like kinase 1 inhibitor BI2536 was also studied. RESULTS:In vitro treatment with NVP-AEW541 suppressed growth in all human BTC cell lines,however response was lower in gallbladder cancer.Treatment withNVP-AEW541 was associated with dephosphorylation of IGF-1R and AKT.In contrast,phosphorylation of p42/p44 and Stat3 and expression of Bcl-xL were inconsistently downregulated.In addition,treated cells showed cell cycle arrest at the G1/S-checkpoint and an increase in sub-G1 peak.Moreover,IGF-1R and its ligands IGF-1 and IGF-2 were co-expressed in RT-PCR,suggesting an autocrine loop of tumor cell activation.Combined with gemcitabine,NVP-AEW541 exerted synergistic effects, particularly at low concentrations,while effects of combination with 5-FU or BI 2536 were only additive. CONCLUSION:Our findings suggest that NVP-AEW541 is active against BTC in vitro and potentiates the efficacy of gemcitabine.

Innovative immunohistochemistry identifies MMP-9 expressing macrophages at the invasive front of murine HCC

AIM:To investigate the proteolytic contribution of tumor-associated macrophages(TAM)in tumor invasion,we analyzed whether TAM at the invasive front of small HCC in Abcb4-/--mice show an enhanced expression of MMP-9. METHODS:Liver cryosections of the hepatocellular carcinoma(HCC)invasive front from 12 mo old Abcb4-/--mice were stained for collagen typeⅠand MMP-9 using Alexa488 and Alexa568 labeled secondary antibodies.Afterwards,the Alexa568 dye was bleached and the macrophage marker F4/80 was visualized using Alexa568 labeled secondary antibodies.Finally, photographs of the invasive tumor front were digitally overlaid and analyzed. RESULTS:After complete bleaching of the primary dye,specific fluorescence staining of a third antigen, here F4/80,was successfully performed on the same histological section.With this method,we were able to identify conglomerates of matrix metalloproteinase (MMP-9)expressing macrophages within the tumor capsule of HCC. CONCLUSION:MMP-9 expressing macrophages are involved in matrix remodelling at the invasive tumor front of HCC.The described staining protocol provides a simple yet powerful extension of conventional immuno-histochemistry,facilitating visualization of at least three different antigens plus nuclei in one single histological section.

Herbalife hepatotoxicity: Evaluation of cases with positive reexposure tests

AIM: To analyze the validity of applied test criteria and causality assessment methods in assumed Herbalife hepatotoxicity with positive reexposure tests. METHODS: We searched the Medline database for suspected cases of Herbalife hepatotoxicity and retrieved 53 cases including eight cases with a positive unintentional reexposure and a high causality level for Herbalife. First, analysis of these eight cases focused on the data quality of the positive reexposure cases, requiring a baseline value of alanine aminotransferase(ALT) < 5 upper limit of normal (N) before reexposure, with Nas the upper limit of normal, and a doubling of the ALT value at reexposure as compared to the ALT value at baseline prior to reexposure. Second, reported methods to assess causality in the eight cases were evaluated, and then the liver specific Council for International Organizations of Medical Sciences (CIOMS) scale validated for hepatotoxicity cases was used for quantitative causality reevaluation. This scale consists of various specific elements with scores provided through the respective case data, and the sum of the scores yields a causality grading for each individual case of initially suspected hepatotoxicity. RESULTS: Details of positive reexposure test conditions and their individual results were scattered in virtually all cases, since reexposures were unintentional and allowed only retrospective rather than prospective assessments. In 1/8 cases, criteria for a positive reexposure were fulfilled, whereas in the remaining cases the reexposure test was classified as negative (n = 1), or the data were considered as uninterpretable due to missing information to comply adequately with the criteria (n = 6). In virtually all assessed cases, liver unspecific causality assessment methods were applied rather than a liver specific method such as the CIOMS scale. Using this scale, causality gradings for Herbalife in these eight cases were probable (n = 1), unlikely (n = 4), and excluded (n = 3). Confounding variables in- cluded low data quality, alternative diagnoses, poor exclusion of important other causes, and comedication by drugs and herbs in 6/8 cases. More specifically, problems were evident in some cases regarding temporal association, daily doses, exact start and end dates of product use, actual data of laboratory parameters such as ALT, and exact dechallenge characteristics. Short-comings included scattered exclusion of hepatitis A-C, cytomegalovirus and Epstein Barr virus infection with only globally presented or lacking parameters. Hepatitis Evirus infection was considered in one single patient and found positive, infections by herpes simplexvirus and varicella zoster virus were excluded in none. CONCLUSION: Only one case fulfilled positive reexposure test criteria in initially assumed Herbalife hepatotoxicity, with lower CIOMS based causality gradings for the other cases than hitherto proposed.

Cronkhite-Canada syndrome associated with myelodysplastic syndrome

We report a case of Cronkhite-Canada syndrome(CCS)associated with myelodysplastic syndrome(MDS).A 54-year-old woman,diagnosed as MDS the prior year after evaluation of anemia,visited our hospital with the chief complaint of epigastric discomfort.She also had dysgeusia,alopecia,atrophic nail change,and pigmentation of the palm,all of which began several months ago.Blood tests revealed severe hypoalbuminemia.Colonoscopy(CS)showed numerous,dense,red polyps throughout the colon and rectum.Biopsy specimens showed stromal edema,infi ltration of lymphocytes,and cystic dilatation of the crypt.Her clinical manifestations and histology were consistent with CCS.We prescribed corticosteroids,which dramatically improved her physical findings,laboratory data,and endoscopic fi ndings.This is the first report of CCS in a patient with MDS.