Cytotoxicity of oleanolic and ursolic acid derivatives toward hepatocellular carcinoma and evaluation of NF-κB involvement

Oleanolic and ursolic acids are two ubiquitous isomeric triterpene phytochemicals known for their anticancer activity. A set of derivatives of the two compounds with a modified oxidation state and lipophylicity at C-3 and C-28 positions, were prepared and tested as anticancer agents versus the lines HepG2, Hep3B and HA22T/VGH of hepatocarcinoma, a strongly aggressive tumor that is not responsive toward the standard therapies. New derivatives containing a three carbons side chain on the C-3 position were synthetized in both stereoisomeric forms by the Barbier-Grignard procedure and three of them were found to be active toward all of the three targets. The implication of the transcriptional nuclear factor NF−κB in the mechanism of action was assessed for the more active compounds in the set, as hepatocellular carcinoma (HCC) cyto-types are known to overexpress NF−κB

Intolerance of cow's milk and chronic constipation in children

Background Chronic diarrhea is the most common gastrointestinal symptom of intolerance of cow's milk among children. On the basis of a prior open study, we hypothesized that intolerance of cow's milk can also cause severe perianal lesions with pain on defecation and consequent constipation in young children. Methods We performed a double-blind, crossover study comparing cow's milk with soy milk in 65 children (age range, 11 to 72 months) with chronic constipation (defined as having one bowel movement every 3 to 15 days). All had been referred to a pediatric gastroenterology clinic and had previously been treated with laxatives without success; 49 had anal fissures and perianal erythema or edema. After 15 days of observation, the patients received cow's milk or soy milk for 2 weeks. After a one-week washout period, the feedings were reversed. A response was defined as eight or more bowel movements during a treatment period. Results Forty-four of the 65 children (68 percent) had a response while receiving soy milk. Anal fissures and pain with defecation resolved. None of the children who received cow's milk had a response. In all 44 children with a response, the response was confirmed with a double-blind challenge with cow's milk. Children with a response had a higher frequency of coexistent rhinitis, dermatitis, or bronchospasm than those with no response (11 of 44 children vs. than those with no response (11 of 44 children vs. 1 of 21, P=0.05); they were also more likely to have anal fissures and erythema or edema at base line (40 of 44 vs. 9 of 21, P<0.001), evidence of inflammation of the rectal mucosa on biopsy (26 of 44 vs. 5 of 21, P=0.008), and signs of hypersensitivity, such as specific IgE antibodies to cow's-milk antigens (31 of 44 vs. 4 of 21, P<0.001). Conclusions In young children, chronic constipation can be a manifestation of intolerance of cow's milk

Accumulation of apoE-enriched triglyceride-rich lipoproteins in patients with coronary artery disease

Triglycerides (TGs) are vehicled by multiple particles with different abilities to promote atherosclerosis. Among plasma TG-rich lipoproteins (TRLs), subspecies may or may not contain apolipoprotein E (apoE) molecules: in this study, we evaluated the relative contribution of apoE-rich and apoE-poor TRLs to coronary atherosclerosis. We selected a group of males with premature coronary artery disease (CAD) without any of the classical nonlipid risk factors and/or high plasma lipid levels and evaluated the plasma concentration of TRL subspecies in comparison with healthy controls. Patients with CAD and controls had total cholesterol and TG levels within the normal range (despite slightly, even if significantly, higher TG levels in patients with CAD) and low-density lipoprotein cholesterol levels near optimal values. Nevertheless, patients with CAD had significantly lower high-density lipoprotein cholesterol, smaller low-density lipoprotein peak particle size, and a reduced HDL2b subfraction than controls. In addition, we observed higher concentrations of total TRL in patients with CAD together with a selective increase in apoE-rich particles. All these data were confirmed after correction for TG levels. We also investigated which parameters were associated with the spread of coronary atherosclerosis. Subjects with a single-vessel disease had selectively lower levels of apoE-rich fractions than patients with a multivessel disease. This was confirmed by multivariate analysis. Patients with a premature CAD free of nonlipid conventional risk factors, despite not having elevated lipid levels, show several lipoprotein abnormalities. Besides known atherogenic alterations, the accumulation of apoE-rich TRL subfractions may represent an additive factor that can potentially promote and initiate the atherosclerotic process.Triglycerides (TGs) are vehicled by multiple particles with different abilities to promote atherosclerosis. Among plasma TG-rich lipoproteins (TRLs), subspecies may or may not contain apolipoprotein E (apoE) molecules: in this study, we evaluated the relative contribution of apoE-rich and apoE-poor TRLs to coronary atherosclerosis. We selected a group of males with premature coronary artery disease (CAD) without any of the classical nonlipid risk factors and/or high plasma lipid levels and evaluated the plasma concentration of TRL subspecies in comparison with healthy controls. Patients with CAD and controls had total cholesterol and TG levels within the normal range (despite slightly, even if significantly, higher TG levels in patients with CAD) and low-density lipoprotein cholesterol levels near optimal values. Nevertheless, patients with CAD had significantly lower high-density lipoprotein cholesterol, smaller low-density lipoprotein peak particle size, and a reduced HDL2b subfraction than controls. In addition, we observed higher concentrations of total TRL in patients with CAD together with a selective increase in apoE-rich particles. All these data were confirmed after correction for TG levels. We also investigated which parameters were associated with the spread of coronary atherosclerosis. Subjects with a single-vessel disease had selectively lower levels of apoE-rich fractions than patients with a multivessel disease. This was confirmed by multivariate analysis. Patients with a premature CAD free of nonlipid conventional risk factors, despite not having elevated lipid levels, show several lipoprotein abnormalities. Besides known atherogenic alterations, the accumulation of apoE-rich TRL subfractions may represent an additive factor that can potentially promote and initiate the atherosclerotic process

Percutaneous liver biopsy: A safe outpatient procedure?

Following the discovery of hepatitis C virus, more liver biopsies (LB) than before are being performed to assess the severity of liver disease. In this study, following the recommendations for outpatient LB made by the Patient Care Committee of the American Gastroenterological Association, we assessed the feasibility and benefits of LB performed as an outpatient versus inpatient procedure over the last 7 years in our centre. The study included 1,581 patients consecutively examined in our institute; all LBs were performed by a single operator with a 16-gauge needle using the Menghini technique, and in all cases the puncture site was determined using prebiopsy ultrasound. Liver lesions were classified using grading and staging scores. Ultrasound-guided LB of focal lesions were excluded from this study. LB was performed on 1,318 outpatients and 263 hospitalized patients. The mean age of the hospitalized patients was higher than that of the outpatients (p < 0.0001). As major side effects, one death and one haemoperitoneum requiring blood transfusion were recorded in the hospitalized patients. As minor side effects, one haemorrhage occurred in the hospitalized patients, whereas a case of haemobilia and 2 cases of subcapsular haematoma were recorded in the outpatients. In both groups pain at the puncture site was the most frequent minor complication which easily resolved after non-steroid drug administration. Severe histological diagnoses, both in terms of grading and staging, were significantly associated with hospitalized patients. In conclusion, by carefully selecting patients and using prebiopsy ultrasound to assess the puncture site, outpatient LB can be safely performed in most cases; this procedure should be more widely used, because it has met with the favour of patients who are able to return home the same day and reduces public health care service costs. Copyright © 2001 S. Karger AG, Basel

Low-density lipoproteins generated during an oral fat load in mild hypertriglyceridemic and healthy subjects are smaller, denser, and have an increased low-density lipoprotein receptor binding affinity.

Triglyceride-rich lipoproteins generated during the postprandial phase are atherogenic. Large very low-density lipoproteins (LDLs) or chylomicrons (CMs) are not as atherogenic as their remnants (Rem). Small and dense LDLs are associated with cardiovascular disease. Low-density lipoprotein size is partly under genetic control and is considered as a relatively stable LDL feature. In this article, we present data on retinyl palmitate kinetics correlated with the modification of LDL features in terms of size, density, and in vitro receptor binding affinity after an oral fat load. Six nondiabetic, hypertriglyceridemic (HTG) patients and 6 healthy controls were examined. Low-density lipoprotein size was assessed by gradient gel electrophoresis, and LDL density by density gradient ultracentrifugation. Low-density lipoprotein binding affinity was tested by in vitro competition binding assay on normal human skin fibroblasts (HSFs) and hepatoma cells (HepG2). Kinetic parameters were estimated in CM and Rem fractions by compartmental modeling. Hypertriglyceridemic patients showed significantly higher triglyceride area and a slower CM fractional catabolic rate. Postprandial LDL density increased both in HTG patients and in the control group with a significant difference between groups at 6 hours. Fasting LDL size was lower in HTG patients vs controls but decreased similarly in the postprandial phase. Low-density lipoprotein size and density postprandial modifications were not correlated with any investigated parameter. Postprandial LDLs were internalized more efficiently by HSF than baseline LDL only in the HTG group. In conclusion, postprandial LDLs are smaller and denser compared with fasting LDLs after an oral fat load. Postprandial LDLs also slightly increased their affinity to the HSF cell receptors

Synthesis, In Vitro and In Silico Analysis of New Oleanolic Acid and Lupeol Derivatives against Leukemia Cell Lines: Involvement of the NF-κB Pathway

Oleanolic acid (OA) and Lupeol (LU) belong to the class of natural triterpenes and are endowed with a wide range of biological activities, including cytotoxicity toward several cancer cell lines. In this context, we investigated a set of compounds obtained from the two natural precursors for the cytotoxicity against leukemia HL60 cells and the multidrug-resistant (MDR) variant HL60R. Six new semi-synthetic triterpenes have been synthetized, fully characterized, and were investigated together with other triterpenes compounds for their pharmacological mechanism of action. The interaction of the more cytotoxic compounds with the nuclear factor kappa B (NF-κB) pathway has been also evaluated with the aid of docking. The lupane-like compounds were more active than the precursor, while the oleane-like compounds showed more complex behavior. Both OA and LU derivatives possess a similar interaction pattern with the p65 subunit of NF-κB, justifying the similar trend in their ability to inhibit the binding of p65 to DNA. Further, some of the derivatives tested were able to increase IκB-α levels preventing the translocation of NF-κB to the nucleus. In conclusion, this study offers a deeper insight on the pharmacological action of triterpenes toward leukemia cells, and it improves the background useful for the development of new anti-cancer drugs

Guinea pig transglutaminase immunolinked assay does not predict coeliac disease in patients with chronic liver disease

Background - It has been suggested that serological screening for coeliac disease (CD) should be performed in patients with chronic unexplained hypertransaminasaemia. Aims - To evaluate the specificity for CD diagnosis of serum IgA antitissue transglutaminase (tTG) determination in consecutive patients with chronic hypertransaminasaemia using the most widely utilised ELISA based on tTG from guinea pig as the antigen. Patients and methods - We studied 98 patients with chronic hypertransaminasaemia, evaluated for the first time in a hepatology clinic. Serum anti-tTG and antiendomysial (EmA) assays were performed. Patients positive for EmA and/or anti-tTG were proposed for intestinal biopsy. Finally, all sera were reassayed for anti-tTG using an ELISA based on human recombinant tTG as the antigen. Results - A total of 94/98 hypertransaminasaemic patients were positive for hepatitis virus markers, with 82/98 (83%) positive for anti-hepatitis C virus. Liver histology showed that most patients had mild or moderate chronic hepatitis while severe fibrosis or overt liver cirrhosis was found in 20/98. CD screening showed that 15/98 (16%) hypertransaminasaemic subjects had anti-tTG values in the same range as CD patients; however, IgA EmA were positive in only 2/98 (2%). Distal duodenal biopsy, performed in nine patients, showed subtotal villous atrophy in the two EmA+/anti-tTG+ patients but was normal in 7/7 EmA-/anti-tTG+ subjects. The presence of anti-tTG+ values in EmA- patients was unrelated to particular gastrointestinal symptoms, other associated diseases, severity of liver histology, or distribution of viral hepatitis markers. There was a significantly higher frequency of positive serum autoantibodies (anti-nuclear, antimitochondrial, antismooth muscle, and anti-liver-kidney microsomal antibodies) in anti-tTG+/EmA- patients than in the other subjects (9/13 v 10/83; p<0.003). Also, a correlation was found between serum gamma globulin and anti-tTG values (p<0.01). When sera were tested with the ELISA based on human tTG as the antigen, no false positive results were observed: only the two EmA+ patients with atrophy of the intestinal mucosa were positive for anti-tTG while all others were negative, including those false positive in the ELISA based on guinea pig tTG as the antigen. Conclusions - In patients with elevated transaminases and chronic liver disease there was a high frequency of false positive anti-tTG results using the ELISA based on tTG from guinea pig as the antigen. Indeed, the presence of anti-tTG did not correlate with the presence of EmA or CD. These false positives depend on the presence of hepatic proteins in the commercial tTG obtained from guinea pig liver and disappear when human tTG is used as the antigen in the ELISA system. We suggest that the commonly used tTG ELISA based on guinea pig antigen should not be used as a screening tool for CD in patients with chronic liver disease