Alfredo Cantafora

Multipotent stem/progenitor cells in human biliary tree give rise to hepatocytes, cholangiocytes, and pancreatic islets

Multipotent stem/progenitors are present in peribiliary glands of extrahepatic biliary trees from humans of all ages and in high numbers in hepato‐pancreatic common duct, cystic duct, and hilum. They express endodermal transcription factors (e.g., Sox9, SOX17, FOXA2, PDX1, HES1, NGN3, PROX1) intranuclearly, stem/progenitor surface markers (EpCAM, NCAM, CD133, CXCR4), and sometimes weakly adult liver, bile duct, and pancreatic genes (albumin, cystic fibrosis transmembrane conductance regulator [CFTR], and insulin). They clonogenically expand on plastic and in serum‐free medium, tailored for endodermal progenitors, remaining phenotypically stable as undifferentiated cells for months with a cell division initially every ≈36 hours and slowing to one every 2‐3 days. Transfer into distinct culture conditions, each comprised of a specific mix of hormones and matrix components, yields either cords of hepatocytes (express albumin, CYP3A4, and transferrin), branching ducts of cholangiocytes (expressing anion exchanger‐2‐AE2 and CFTR), or regulatable C‐peptide secreting neoislet‐like clusters (expressing glucagon, insulin) and accompanied by changes in gene expression correlating with the adult fate. Transplantation into quiescent livers of immunocompromised mice results in functional human hepatocytes and cholangiocytes, whereas if into fat pads of streptozocin‐induced diabetic mice, results in functional islets secreting glucose‐regulatable human C‐peptide. Conclusion: The phenotypes and availability from all age donors suggest that these stem/progenitors have considerable potential for regenerative therapies of liver, bile duct, and pancreatic diseases including diabetes. (HEPATOLOGY2011;)

Inherited Apolipoprotein A-V Deficiency in Severe Hypertriglyceridemia

Objective— Mutations in LPL or APOC2 genes are recognized causes of inherited forms of severe hypertriglyceridemia. However, some hypertrigliceridemic patients do not have mutations in either of these genes. Because inactivation or hyperexpression of APOA5 gene, encoding apolipoprotein A-V (apoA-V), causes a marked increase or decrease of plasma triglycerides in mice, and because some common polymorphisms of this gene affect plasma triglycerides in humans, we have hypothesized that loss of function mutations in APOA5 gene might cause hypertriglyceridemia. Methods and Results— We sequenced APOA5 gene in 10 hypertriglyceridemic patients in whom mutations in LPL and APOC2 genes had been excluded. One of them was found to be homozygous for a mutation in APOA5 gene (c.433 C>T, Q145X), predicted to generate a truncated apoA-V devoid of key functional domains. The plasma of this patient was found to activate LPL in vitro less efficiently than control plasma, thus suggesting that apoA-V might be an activator of LPL. Ten carriers of Q145X mutation were found in the patient’s family; 5 of them had mild hypertriglyceridemia. Conclusions— As predicted from animal studies, apoA-V deficiency is associated with severe hypertriglyceridemia in humans. This observation suggests that apoA-V regulates the secretion and/or catabolism of triglyceride-rich lipoproteins.

The molecular basis of lecithin:cholesterol acyltransferase deficiency syndromes: a comprehensive study of molecular and biochemical findings in 13 unrelated Italian families.

Objective—To better understand the role of lecithin:cholesterol acyltransferase (LCAT) in lipoprotein metabolism through the genetic and biochemical characterization of families carrying mutations in the LCAT gene. Methods and Results—Thirteen families carrying 17 different mutations in the LCAT gene were identified by Lipid Clinics and Departments of Nephrology throughout Italy. DNA analysis of 82 family members identified 15 carriers of 2 mutant LCAT alleles, 11 with familial LCAT deficiency (FLD) and 4 with fish-eye disease (FED). Forty-four individuals carried 1 mutant LCAT allele, and 23 had a normal genotype. Plasma unesterified cholesterol, unesterified/total cholesterol ratio, triglycerides, very-low-density lipoprotein cholesterol, and pre-&bgr; high-density lipoprotein (LDL) were elevated, and high-density lipoprotein (HDL) cholesterol, apolipoprotein A-I, apolipoprotein A-II, apolipoprotein B, LpA-I, LpA-I:A-II, cholesterol esterification rate, LCAT activity and concentration, and LDL and HDL3 particle size were reduced in a gene–dose-dependent manner in carriers of mutant LCAT alleles. No differences were found in the lipid/lipoprotein profile of FLD and FED cases, except for higher plasma unesterified cholesterol and unesterified/total cholesterol ratio in the former. Conclusion—In a large series of subjects carrying mutations in the LCAT gene, the inheritance of a mutated LCAT genotype causes a gene–dose-dependent alteration in the plasma lipid/lipoprotein profile, which is remarkably similar between subjects classified as FLD or FED.

Relationships between bile salts hydrophilicity and phospholipid composition in bile of various animal species

Bile salts and phospholipids from bile of chicken, dog, sheep, rat, ox, pig, guinea-pig and man were analyzed by high-performance liquid chromatography. Bile salts showed marked differences in their hydrophilic properties, owing to hydroxyl structure and type of conjugation. Phospholipids were generally similar, containing 90-95% of phosphatidylcholine which was made of molecular species containing palmitic acid in the sn-1 position. The comparative analysis of bile salts and phosphatidylcholines profile demonstrated that bile salts hydrophilicity influences the quantity of phosphatidylcholine in bile but not the quality.

High performance liquid chromatographic analysis of molecular species of phosphatidylcholine - development of quantitative assay and its application to human bile

In this paper we propose a novel, rapid and simple high-performance liquid chromatographic (HPLC) method for the identification and quantitation of individual phosphatidylcholine (PC) molecular species from natural mixtures. To overcome difficulties deriving from the lack of adequate standards and from the variability of the responses to UV spectrophotometric detectors currently used in HPLC analysis, we first fractionated and quantitated the major molecular species of a commercial egg PC by means of a preparative column. The identification of PC molecular species was confirmed by gas-liquid chromatographic analysis of fatty acids. We employed the fractions recovered from preparative HPLC to determine the detector calibration factors of the individual molecular species separated using an analytical, high-speed, reversed-phase HPLC column. The proposed method seems to be adequate for the analysis of PC from many biological sources. Its application to the analysis of human hepatic and gallbladder biliary PC is shown.

AGING AND RED BLOOD CELL MEMBRANE : A STUDY OF CENTENARIANS

Successful aging, characterized by little or no loss in physiological functions, should be the usual aging process in centenarians. It is known that well-preserved physiological functions depend on the proper functioning of cell systems. In this article we focus on cell membrane integrity and study the red blood cell membrane to evaluate the effect of physiological aging in centenarians. Fifteen healthy, self-sufficient centenarians, mean age 103 years, were examined by assessing hemocytometric values and some relevant characteristics of the erythrocyte membrane, i.e., the cholesterol/phospholipid molar ratio, the distribution of phospholipid classes and their fatty acid composition, the integral and skeletal protein profiles. The centenarians showed a significant decrease in the red blood cell count (p < 0.0002), hemoglobin (p < 0.0002), and hematocrit (p < 0.0005). The red blood cell membrane showed a significantly increased cholesterol/phospholipid molar ratio (p < 0.01), with a concomitant increase in polyunsaturated fatty acids in phosphatidylcholine (p < 0.001) and, to a lesser extent, in phosphatidylethanolamine. The electrophoretic pattern of membrane proteins was qualitatively normal compared to controls but the densitometric analysis showed a significant increase in the integral protein band 4.2 (p < 0.05) and in the skeletal protein actin (p < 0.001). Extreme longevity seems to be associated with a substantial integrity of the erythrocyte membrane. Moreover, the evident increase in polyunsaturated fatty acids and in actin are likely to improve the membrane fluidity and to strengthen the membrane structure.

Cholesteryl Ester Storage Disease (CESD) due to novel mutations in the LIPA gene.

Cholesteryl Ester Storage Disease (CESD) is a rare recessive disorder due to mutations in LIPA gene encoding the lysosomal acidic lipase (LAL). CESD patients have liver disease associated with mixed hyperlipidemia and low plasma levels of high-density lipoproteins (HDL). The aim of this study was the molecular characterization of three patients with CESD. LAL activity was measured in blood leukocytes. In two patients (twin sisters) the clinical diagnosis of CESD was made at 9 years of age, following the fortuitous discovery of elevated serum liver enzymes in apparently healthy children. They had mixed hyperlipidemia, hepatosplenomegaly, reduced LAL activity (approximately 5% of control) and heteroalleic mutations in LIPA gene coding sequence: (i) the common c.894 G>A mutation and (ii) a novel nonsense mutation c.652 C>T (p.R218X). The other patient was an 80 year-old female who for several years had been treated with simvastatin because of severe hyperlipidemia associated with low plasma HDL. In this patient the sequence of major candidate genes for monogenic hypercholesterolemia and hypoalphalipoproteinemia was negative. She was found to be a compound heterozygote for two LIPA gene mutations resulting in 5% LAL activity: (i) c.894 G>A and (ii) a novel complex insertion/deletion leading to a premature termination codon at position 82. These findings suggest that, in view of the variable severity of its phenotypic expression, CESD may sometimes be difficult to diagnose, but it should be considered in patients with severe type IIb hyperlipidemia associated with low HDL, mildly elevated serum liver enzymes and hepatomegaly.

Total and individual free fatty acid concentrations in liver cirrhosis

The finding of high plasma free fatty acid (FFA) levels in cirrhotic patients has been attributed either to decreased hepatic clearance or to enhanced fat mobilization. To better clarify these hypotheses, total and individual FFA and glycerol levels were determined in 21 cirrhotic patients with different degrees of hepatocellular damage (evaluated by liver function tests), portal hypertension (evaluated by endoscopy and clinical signs), and nutritional status (evaluated by anthropometric and biohumoral parameters) and in 10 age- and sex-matched healthy subjects. Glucose tolerance and insulin and glucagon levels were determined in all individuals. Well-nourished and malnourished patients were identified within the cirrhotic group. Plasma FFA and glycerol concentrations were well correlated (r = 0.47, P less than 0.05), levels being significantly higher in cirrhotic individuals than in controls (746.6 +/- 46.29 SE v 359.22 +/- 40.82 mumol/L, P less than 0.001 for plasma FFA; 150.1 +/- 3.12 v 82.5 +/- 9.2 mumol/L, P less than 0.01 for glycerol). Plasma FFA and glycerol showed no correlation with the liver function test results or portal hypertension parameters. Interestingly, plasma levels of FFA and glycerol were influenced by the nutritional status, significantly higher FFA levels being observed in the well-nourished than in the malnourished patients (842.5 +/- 47.5 v 563.4 +/- 78 mumol/L, P less than 0.005). Furthermore, a positive correlation was found between plasma glycerol level and percentage of triceps skinfold (r = 0.45, P less than 0.05). No correlation was found between plasma levels of FFA or glycerol and glucose tolerance, insulin and glucagon.(ABSTRACT TRUNCATED AT 250 WORDS)

Impaired human gallbladder lipid absorption in cholesterol gallstone disease and its effect on cholesterol solubility in bile

BACKGROUND & AIMS The role of the gallbladder in gallstone pathogenesis is still unclear. We examined the effects of gallbladder mucosal lipid absorption on lipid composition and cholesterol crystallization in bile. METHODS The in vitro-isolated, intra-arterially perfused gallbladder model was used (1) to compare the absorption rates of lipids from standard bile by gallbladders obtained from 7 patients with cholesterol gallstones and 6 controls; and (2) to measure the microscopic cholesterol crystal detection time in cholesterol-enriched pig bile before and after lipid absorption by the pig gallbladder. RESULTS Control gallbladders, but not cholesterol gallstone gallbladders, significantly reduced cholesterol (P < 0.02) and phospholipid (P < 0.01) and increased bile salt (P < 0.01) molar percentages in bile over a 5-hour period by efficient and selective cholesterol and phospholipid absorption. A histomorphometric study of the epithelial cells showed significantly higher values for nuclear density (P < 0.01) and nuclear (P < 0.05) and cytoplasmic (P < 0.05) areas in the cholesterol gallstone than the control group. Sequential microscopy of cholesterol-enriched pig bile showed significantly shorter cholesterol filament (P < 0.01) and typical cholesterol plate (P < 0. 02) detection times before than after exposure of bile to the gallbladder lipid absorption. CONCLUSIONS In cholesterol gallstone disease, the human gallbladder epithelium loses its capacity to selectively and efficiently absorb cholesterol and phospholipids from bile, even if it is hyperplastic and hypertrophic. This epithelial dysfunction eliminates the positive effect that the normal gallbladder exerts on cholesterol solubility in bile and might be a pathogenetic cofactor for cholesterol gallstone formation.

Comparison of the hepatic uptake and processing of cholesterol from chylomicrons of different fatty acid composition in the rat in vivo

The effect of the fatty acid composition of chylomicrons on the uptake and processing of the cholesterol they carry was investigated in the rat in vivo. Rats kept on a standard low fat pellet diet and tube fed a single dose of palm, olive, corn or fish oil (rich in saturated, n-9 monounsaturated, n-6 polyunsaturated and n-3 polyunsaturated fatty acids, respectively) were used to prepare [3H]cholesterol-labelled chylomicrons of different fatty acid composition. These were then injected intravenously into rats (kept on the standard diet), and the clearance of radioactivity from the blood, distribution in the plasma lipoprotein density fractions, uptake by the liver and appearance in the bile were studied. [3H]Cholesterol from fish and corn oil chylomicrons was cleared from the blood more rapidly than that from palm and olive oil chylomicrons. After 180 min the proportion of the radioactivity present in the plasma in high density lipoprotein (HDL) was less when the chylomicrons were derived from palm oil as compared to any of the other oils. Approx. 40% of the administered label was recovered in the liver after 180 min in all experiments. The percentage of the injected radioactivity secreted into bile during 180 min was significantly higher with corn and fish oil chylomicrons than with palm oil chylomicrons, with chylomicrons from olive oil in an intermediate position, and these differences were most pronounced between 60 and 120 min after administration of the label. These studies clearly demonstrate that the fatty acid composition of chylomicrons has important effects on the hepatic uptake and processing of the cholesterol they carry, with enrichment with polyunsaturated fatty acids leading to an increased rate of uptake and more rapid removal from the body via the bile as compared to enrichment with saturated or monounsaturated fatty acids.