Search

In nonalcoholic steatohepatitis animal models, an increased lipid droplet size in hepatocytes is associated with fibrogenesis. Hepatocytes with large droplet (Ld-MaS) or small droplet (Sd-MaS) macrovesicular steatosis may coexist in the human liver, but the factors associated with the predominance of one type over the other, including hepatic fibrogenic capacity, are unknown. In pre-ischemic liver biopsies from 225 consecutive liver transplant donors, we retrospectively counted hepatocytes with Ld-MaS and Sd-MaS and defined the predominant type of steatosis as involving ≥50% of steatotic hepatocytes. We analyzed a donor Patatin-like phospholipase domain-containing protein 3 (PNPLA3) rs738409 polymorphism, hepatic expression of proteins involved in lipid metabolism by RT-PCR, hepatic stellate cell (HSC) activation by α-SMA immunohistochemistry and, one year after transplantation, histological progression of fibrosis due to Hepatitis C Virus (HCV) recurrence. Seventy-four livers had no steatosis, and there were 98 and 53 with predominant Ld-MaS and Sd-MaS, respectively. In linear regression models, adjusted for many donor variables, the percentage of steatotic hepatocytes affected by Ld-MaS was inversely associated with hepatic expression of Insulin Induced Gene 1 (INSIG-1) and Niemann-Pick C1-Like 1 gene (NPC1L1) and directly with donor PNPLA3 variant M, HSC activation and progression of post-transplant fibrosis. In humans, Ld-MaS formation by hepatocytes is associated with abnormal PNPLA3-mediated lipolysis, downregulation of both the intracellular cholesterol sensor and cholesterol reabsorption from bile and increased hepatic fibrogenesis.

Cholangiocarcinomas (CCAs) comprise a mucin-secreting form, intrahepatic or perihilar, and a mixed form located peripherally. We characterized cancer stem cells (CSCs) in CCA subtypes and evaluated their cancerogenic potential. CSC markers were investigated in 25 human CCAs in primary cultures and established cell lines. Tumorigenic potential was evaluated in vitro or in xenografted mice after s.c. or intrahepatic injection in normal and cirrhotic (carbon tetrachloride-induced) mice. CSCs comprised more than 30% of the tumor mass. Although the CSC profile was similar between mucin-intrahepatic and mucin-perihilar subtypes, CD13 + CSCs characterized mixed-intrahepatic, whereas LGR5 + characterized mucin-CCA subtypes. Many neoplastic cells expressed epithelial-mesenchymal transition markers and coexpressed mesenchymal and epithelial markers. In primary cultures, epithelial-mesenchymal transition markers, mesenchymal markers (vimentin, CD90), and CD13 largely predominated over epithelial markers (CD133, EpCAM, and LGR5). In vitro , CSCs expressing epithelial markers formed a higher number of spheroids than CD13 + or CD90 + CSCs. In s.c. tumor xenografts, tumors dominated by stromal markers were formed primarily by CD90 + and CD13 + cells. By contrast, in intrahepatic xenografts in cirrhotic livers, tumors were dominated by epithelial traits reproducing the original human CCAs. In conclusion, CSCs were rich in human CCAs, implicating CCAs as stem cell–based diseases. CSC subpopulations generate different types of cancers depending on the microenvironment. Remarkably, CSCs reproduce the original human CCAs when injected into cirrhotic livers.

Cholangiocarcinoma (CCA) and its subtypes (mucin- and mixed-CCA) arise from the neoplastic transformation of cholangiocytes, the epithelial cells lining the biliary tree. CCA has a high mortality rate owing to its aggressiveness, late diagnosis and high resistance to radiotherapy and chemotherapeutics. We have demonstrated that CCA is enriched for cancer stem cells which express epithelial to mesenchymal transition (EMT) traits, with these features being associated with aggressiveness and drug resistance. TGF-β signaling is upregu-lated in CCA and involved in EMT. We have recently established primary cell cultures from human mucin- and mixed-intrahepatic CCA. In human CCA primary cultures with different levels of EMT trait expression, we evaluated the anticancer effects of: (i) CX-4945, a casein kinase-2 (CK2) inhibitor that blocks TGF-β1-induced EMT; and (ii) LY2157299, a TGF-β receptor I kinase inhibitor. We tested primary cell lines expressing EMT trait markers (vimentin, N-cadherin and nuclear catenin) but negative for epithelial markers, and cell lines expressing epithelial markers (CK19-positive) in association with EMT traits. Cell viability was evaluated by MTS assays, apoptosis by Annexin V FITC and cell migration by wound-healing assay. Results: at a dose of 10 μM, CX4945 significantly decreased cell viability of primary human cell cultures from both mucin and mixed CCA, whereas in CK19-positive cell cultures, the effect of CX4945 on cell viability required higher concentrations (>30μM). At the same concentrations, CX4945 also induced apoptosis (3- fold increase vs controls) which correlated with the expression level of CK2 in the different CCA cell lines (mucin- and mixed-CCA). Indeed, no apoptotic effects were observed in CK19-positive cells expressing lower CK2 levels. The effects of CX4945 on viability and apoptosis were associated with an increased number of γ-H2ax (biomarker for DNA double-strand breaks) foci, suggesting the active role of CK2 as a repair mechanism in CCAs. LY2157299 failed to influence cell proliferation or apoptosis but significantly inhibited cell migration. At a 50 μM concentration, in fact, LY2157299 significantly impaired (at 24, 48 and 120 hrs) the wound-healing of primary cell cultures from both mucin-and mixed-CCA. In conclusion, we demonstrated that CX4945 and LY2157299 exert relevant but distinct anticancer effects against human CCA cells, with CX4945 acting on cell viability and apoptosis, and LY2157299 impairing cell migration. These results suggest that targeting the TGF-β signaling with a combination of CX-4945 and LY2157299 could have potential benefits in the treatment of human CCA.

Background & Aims Human biliary tree stem/progenitor cells (hBTSCs) are multipotent epithelial stem cells, easily obtained from the biliary tree, with the potential for regenerative medicine in liver, biliary tree, and pancreas diseases. Recent reports indicate that human mesenchymal stem cells are able to modulate the T cell immune response. However, no information exists on the capabilities of hBTSCs to control the allogeneic response. The aims of this study were to evaluate FasL expression in hBTSCs, to study the in vitro interaction between hBTSCs and human lymphocytes, and the role of Fas/FasL modulation in inducing T cell apoptosis in hBTSCs/T cell co-cultures. Methods Fas and FasL expression were evaluated in situ and in vitro by immunofluorescence and western blotting. Co-cultures of hBTSCs with human leukocytes were used to analyze the influence of hBTSCs on lymphocytes activation and apoptosis. Results hBTSCs expressed HLA antigens and FasL in situ and in vitro . Western blot data demonstrated that hBTSCs constitutively expressed high levels of FasL that increased after co-culture with T cells. Confocal microscopy demonstrated that FasL expression was restricted to EpCAM + /LGR5 + cells. FACS analysis of T cells co-cultured with hBTSCs indicated that hBTSCs were able to induce apoptosis in activated CD4 + and CD8 + T cell populations. Moreover, the Fas receptor appears to be more expressed in T cells co-cultured with hBTSCs than in resting T cells. Conclusions Our data suggest that hBTSCs could modulate the T cell response through the production of FasL, which influences the lymphocyte Fas/FasL pathway by inducing "premature" apoptosis in CD4 + and CD8 + T cells.

Background & Aims We analysed for the first time whether recipient perioperative serum total cholesterol (sTC) concentration is associated with liver transplantation outcome. Methods We studied noncholestatic cirrhotics submitted to primary deceased-donor liver transplantation in a prospective group (n = 140) from Rome and in a validation retrospective cohort (n = 157) from Udine, Italy. Pre-ischaemia and post-reperfusion cholesterol metabolism gene mRNA was measured by RT-PCR in 74 grafts of the study group. Results At Cox regression analysis, independently from confounders including recipient MELD score, the recipient pre-operative sTC pooled quintiles 2–5, compared with the lowest quintile showed HR (95% CI) and significances for overall graft loss (GL) of 0.215 (0.104–0.444) P

Prostate apoptosis response-4 (Par-4) is a tumor suppressor protein that sensitizes cells to apoptosis; therefore, Par-4 modulation has therapeutic potential. No data currently exist on Par-4 expression in cholangiocarcinoma (CCA). We evaluated the expression of Par-4 in normal and neoplastic cholangiocytes and the effects of its pharmacological or genetic modulation. The study was performed in human and rat liver, CCA patient biopsies, and two CCA cell lines. PAR-4 was expressed in normal rat and human cholangiocytes, but its expression levels decreased in both human CCA and CCA cell lines. In both intrahepatic and extrahepatic CCA, Par-4 expression (as shown by immunohistochemistry) was inversely correlated with markers of proliferation (eg, proliferating cellular nuclear antigen) and directly correlated with apoptotic markers (eg, Bax and Bax/BCL2 ratio). Par-4 expression was decreased during CCA cell proliferation but was enhanced after apoptosis induction. Pharmacological induction of Par-4 expression in CCA cell lines by diindolymethane or withaferin A promoted activation of apoptosis and inhibition of proliferation. In contrast, specific Par-4 silencing by small-interfering RNA determined activation of CCA cell line proliferation. Par-4 is expressed in rat and human cholangiocytes and is down-regulated in both human CCA and CCA cell lines. Par-4 protein levels decrease during cell proliferation but increase during apoptosis. Pharmacological or genetic induction of Par-4 determines apoptosis of CCA cells, suggesting Par-4 targeting as a CCA treatment strategy.

Oxidized LDL (ox-LDL) have been involved in the pathogenesis of several human diseases including dermatological pathologies. Oxidative modification of low-density lipoproteins (LDL) is accompanied by both extensive degradation of its polyunsaturated fatty acids and production of lipoperoxides. These highly reactive products induce an intracellular oxidative stress with a variety of cytotoxic effects. In order to evaluate cellular damage induced by oxidative stress in epidermal cells, a human epidermoid carcinoma cell line in culture (A 431) was used as experimental model. Cell treatment with UV-oxidized LDL resulted in cytostatic and cytotoxic effects characterized by morphological and functional alterations: inhibition of cell proliferation, modifications of cytoskeleton network, microtubular derangement, loss of cell–cell and cell–substrate contacts, cell detachment and cell death by apoptosis. The ox-LDL-induced alterations were almost completely prevented by pre-incubating cells with α-tocopherol. The results presented here could be of relevance for a better comprehension of the pathogenic mechanisms of several human diseases, including dermatological pathologies, and could indicate that antioxidants such as α-tocopherol could represent an important therapeutic challenge in the maintenance of cell and tissue homeostasis in the long run.

Background/Aims We evaluated the role and mechanisms by which the GH/IGF1 axis modulates cholangiocyte proliferation. Methods GH-receptors (GH-R), IGF1, IGFBP3 (binding protein 3), IGF1-R and receptor substrates (IRS) were evaluated in cholangiocytes of normal or bile duct-ligated (BDL) rat livers. The effects of GH and IGF1 on proliferation of normal quiescent cholangiocytes and the transduction pathways involved were investigated. Results IGF1, GH-R, IGF1-R, IRS-1/2 were expressed in normal cholangiocytes and overexpressed in cholangiocytes proliferating after BDL which also secrete IGF1 in a higher amount than normal cells. IGFBP3, which may counter-regulate IGF1 effects, was decreased in BDL cholangiocytes. IGF1 promoted cholangiocyte proliferation in association with overexpression of p-IGF1R, IRS1, IRS-2, p-ERK1/2 and p-AKT. GH induced IGF1 expression and release in isolated cholangiocytes, and reproduced the effects of IGF1 but GH effects were abolished by IGF1-R blocking antibody, suggesting IGF1 as a mediator of GH. Finally, IGF1 and 17β-estradiol reciprocally potentiated their proliferative effects on cholangiocytes, and by interacting at both receptor and post-receptor levels. Conclusions Cholangiocytes respond to GH with production and release of IGF1 that modulates cell proliferation by transduction pathways involving IGF1-R, IRS1/2 and both ERK and PI3-kinase pathways. The biliary epithelium is a target of GH/IGF1 liver axis.

Background & Aims Multipotent stem/progenitor cells are found in peribiliary glands throughout human biliary trees and are able to generate mature cells of hepato-biliary and pancreatic endocrine lineages. The presence of endodermal stem/progenitors in human gallbladder was explored. Methods Gallbladders were obtained from organ donors and laparoscopic surgery for symptomatic cholelithiasis. Tissues or isolated cells were characterized by immunohistochemistry and flow cytometry. EpCAM+ (Epithelial Cell Adhesion Molecule) cells were immunoselected by magnetic microbeads, plated onto plastic in self-replication conditions and subsequently transferred to distinct serum-free, hormonally defined media tailored for differentiation to specific adult fates. In vivo studies were conducted in an experimental model of liver cirrhosis. Results The gallbladder does not have peribiliary glands, but it has stem/progenitors organized instead in mucosal crypts. Most of these can be isolated by immune-selection for EpCAM. Approximately 10% of EpCAM+ cells in situ and of immunoselected EpCAM+ cells co-expressed multiple pluripotency genes and various stem cell markers; other EpCAM+ cells qualified as progenitors. Single EpCAM+ cells demonstrated clonogenic expansion ex vivo with maintenance of stemness in self-replication conditions. Freshly isolated or cultured EpCAM+ cells could be differentiated to multiple, distinct adult fates: cords of albumin-secreting hepatocytes, branching ducts of secretin receptor+ cholangiocytes, or glucose-responsive, insulin/glucagon-secreting neoislets. EpCAM+ cells transplanted in vivo in immune-compromised hosts gave rise to human albumin-producing hepatocytes and to human Cytokeratin7+ cholangiocytes occurring in higher numbers when transplanted in cirrhotic mice. Conclusions Human gallbladders contain easily isolatable cells with phenotypic and biological properties of multipotent, endodermal stem cells.

This study evaluated the applicability of microchip electrophoresis to the sizing of microsatellites suitable to genetic, clinical and forensic applications. The evaluation was performed with the D19S394 tetranucleotide (AAAG) repeat characterized by a wide variation in the repeat number (1-17) and a short recombination distance from the low-density lipoprotein (LDL)-receptor gene that makes it suitable to cosegregation analysis of familial hypercholesterolemia (FH). The study was performed with 70 carriers of two LDL-receptor mutations common in northern Italy (i.e., the 4 bp insertion in exon 10 known as FH-Savona and the D200G missense mutation in the exon 4, known as FH-Padova 1) and 100 healthy controls. The polymerase chain reaction (PCR) amplification products prepared with a cosolvent PCR protocol and an antibody-protected polymerase were directly analyzed with an apparatus for high-voltage capillary electrophoresis on microchips and laser-induced fluorescence detection equipped with chips for the analysis of 25-500 bp dsDNA fragments. The test could not be extended to dinucleotide repeats due to the resolution characteristics of the available microchip. This novel approach was able to distinguish 17 microsatellite alleles varying from 0 to 17 repeats. Many of these alleles were quite rare, but the seven more abundant accounted for over the 70% of allele distribution in control population. The standard deviation in the sizing of the most abundant alleles ranged from +0.60 to +/- 0.75 bp. This indicated that the size attribution to a conventional allele using the +/- 1 bp range around it allowed a confidence limit above the 80 %. The sizing of D19S394 obtained this way allowed the cosegregation analysis with both the FH mutations tested. Therefore, this innovative approach to microsatellite sizing was much simpler, but equally effective as traditional capillary electrophoresis, at least with tetranucleotide repeats.

The proband is a 50 year-old woman born from a consanguineous marriage. She has been suffering from angina pectoris since the age of 38 and underwent coronary bypass surgery for three-vessel disease at 48. The presence of low plasma levels of total cholesterol and high density lipoprotein (HDL) cholesterol (2.4 and 0.1 mmol/l) and apo AI (15 mg/dl), associated with corneal lesions and a mild splenomegaly suggested the diagnosis of Tangier disease. However, none of the other features of Tangier disease, including hepatomegaly, anemia and peripheral neuropathy, were present. The analysis of the dinucleotide microsatellites located in chromosome 9q31 region demonstrated that the proband was homozygous for the alleles of D9S53, D9S1784 and D9S1832. The mother and son of the proband, both with low levels of HDL cholesterol, shared one of the proband's haplotypes, whereas neither of these haplotypes was present in the normolipidemic proband's sister. The sequence of ATP-binding cassette transporter 1 (ABC1-1) cDNA obtained by reverse transcription-PCR (RT-PCR) of total RNA isolated from cultured fibroblasts showed that the proband was homozygous for a CT transition in exon 13, which caused a tryptophane for arginine substitution (R527W). This mutation was confirmed by direct sequencing of exon 13 amplified from genomic DNA. It can be easily screened, as the nucleotide change introduces a restriction site for the enzyme Afl III. R527W substitution occurs in a highly conserved region of the NH2 cytoplasmic domain of ABC1 protein. R527W co-segregates with the low HDL phenotype in the family and was not found in 200 chromosomes from normolipidemic individuals.

Both estrogen and dietary n-3 polyunsaturated fatty acids are known to be hypocholesterolemic, but appear to exert their effects by different mechanisms. In this study, the interaction between dietary fish oil (rich in n-3 polyunsaturated fatty acids) and estrogen in the regulation of hepatic cholesterol metabolism and biliary lipid secretion in rats was studied. Rats fed a low fat or a fish oil-supplemented diet for 21 days were injected with 17alpha-ethinyl estradiol (5 mg/kg body weight) or the vehicle only (control rats) once per day for 3 consecutive days. Estrogen-treatment led to a marked reduction in plasma cholesterol levels in fish oil-fed rats, which was greater than that observed with either estrogen or dietary fish oil alone. The expression of mRNA for cholesterol 7alpha-hydroxylase was decreased by estrogen in rats fed a low fat or a fish oil-supplemented diet, while the output of cholesterol (micromol/h/kg b.wt.) in the bile was unchanged in both groups. Cholesterol levels in the liver were increased by estrogen in rats given either diet, but there was a significant shift from cholesterol esterification to cholesteryl ester hydrolysis only in the fish oil-fed animals. Estrogen increased the concentration of cholesterol (micromol/ml) in the bile in rats fed the fish oil, but not the low fat diet. However, the cholesterol saturation index was unaffected. The output and concentration of total bile acid was also unaffected, but changes in the distribution of the individual bile acids were observed with estrogen treatment in both low fat and fish oil-fed groups. These results show that interaction between estrogen-treatment and dietary n-3 polyunsaturated fatty acids causes changes in hepatic cholesterol metabolism and biliary lipid secretion in rats, but does not increase the excretion of cholesterol from the body.

Polymerase chain reaction (PCR) amplification of specific alleles allowed the rapid detection of a point mutation (missense Gly528 → Asp) in exon 11 of the low density lipoprotein receptor gene which was otherwise not detectable by exon amplification and enzymatic digestion as it does not modify the normal restriction pattern. The mutant allele, designated as FH-Palermo-1 from the origin of the first carrier family identified, gave a specific PCR product of 109 bp clearly distinct from the product of 168 bp obtained from other alleles with a nonspecific couple of primers. This method allowed us to distinguish one positive sample mixed with up to 11 parts of normal DNA. Furthermore, the specific amplification product was characterized by a Bsm I restriction site not present in nonspecific products.—Cantafora, A., I. Blotta, E. Mercuri, S. Calandra, and S. Bertolini. Simple detection of a point mutation in LDL receptor gene causing familial hypercholesterolemia in southern Italy by allele-specific polymerase chain reaction. J. Lipid Res. 1998. 39: 1101–1105.

We investigated whether S-adenosyl-L-methionine (SAMe), dilinoleoylphosphatidylcholine (DLPC), or SAMe + DLPC influence liver lipid composition as well as acute ethanol hepatotoxicity in the isolated perfused rat liver (IPRL). SAMe (25 mg/kg intramuscularly three times a day) was administered for five consecutive days, while DLPC was administered intraperitoneally for five days. The liver was then isolated, perfused with taurocholate to stabilize bile secretion, and exposed to 0.5% ethanol for 70 min. SAMe, without changing total phospholipid (PL) content, induced an increase in the phosphatidylcholine/phosphatidylethanolamine (PC/PE) molar ratio in both liver homogenate and microsomes and a significant enrichment of 16:0-20:4 and 18:0-20:4 PC molecular species. DLPC induced a significant enrichment of PL in liver homogenate and microsomes due to a contemporary increase in PC and PE. The PC enrichment specifically involved 16:0-20:4 and 18:0-20:4 PC molecular species besides the HPLC peak containing the administered 18:2-18:2 PC species. DLPC + SAMe increased the concentration of PC in liver homogenate and microsomes due to a specific enrichment of 16:0-22:6, 16:0-20:4, and 18:0-20:4 PC molecular species, and the HPLC peak containing the administered 18:2-18:2 PC species. Ethanol acute exposure in the control IPRLs for 70 min induced a depletion of cholesterol in both liver homogenate and microsomes without significant changes in the composition of PL classes and PC molecular species. SAMe, DLPC, or SAMe + DLPC counteracted the cholesterol depletion induced by ethanol, indicating that phospholipid changes promoted by these treatments all induce a major resistance of liver membranes to the effect of ethanol. Ethanol administration in control IPRLs induced a fivefold increase of AST and LDH release in the perfusate, depletion of glutathione in homogenates and mitochondria, decreased oxygen liver consumption, and inhibition of bile flow. These effects of ethanol were significantly antagonized by SAMe. In contrast, DLPC alone only minimally attenuated enzyme release in the perfusate and the inhibitory effect of ethanol on bile flow, but it failed to influence the depletion of total and mitochondrial glutathione or the depressed oxygen consumption induced by ethanol. DLPC, administered together with SAMe, added nothing to the protective effect of SAMe against ethanol hepatotoxicity and cholestasis. In conclusion, this study demonstrates that both SAMe and DLPC induced marked modifications in the lipid composition of liver membranes with a similar enrichment of polyunsaturated PC molecular species. Only SAMe, however, significantly protected against the hepatotoxic and cholestatic effect of acute ethanol administration, an effect associated with maintained normal glutathione mitochondrial levels and oxygen liver consumption. This indicates that the protective effect of SAMe against ethanol toxicity is linked to multiple mechanisms, the maintenance of glutathione levels probably being one of the most important.

The lipolysis of chylomicrons derived from palm, olive, corn or fish oil (enriched in saturated, monounsaturated, n−6 polyunsaturated and n−3 polyunsaturated fatty acids, respectively) by rat post-heparin lipoprotein lipase in vitro was compared by measuring the release of [ 3 H ]oleate from their triacylglycerol. Chylomicrons derived from corn oil were lipolysed more rapidly than the other types in the first 20 min of the reaction, but after 120 min the total amount of triacylglycerol hydrolysed was similar with all types of chylomicrons used. The rate of lipolysis of the different types of chylomicrons also showed different dependencies on the substrate concentration. The highest Vmax values were obtained when the chylomicrons were derived from olive and corn oil and the lowest when they were derived from palm oil, while olive oil chylomicrons gave the highest Km and palm oil chylomicrons the lowest. These results indicate that differential metabolism of chylomicrons of different fatty acid composition by lipoprotein lipase may play a part in the differential rates of clearance from the blood of lipid of dietary origin demonstrated in earlier work from our laboratory.

In this study, perfused livers from Yoshida rats, either on a normal diet or on a diet with 0.3% probucol, were examined. The analysis of liver lipid content and of bile and lipoprotein secretion changes showed that probucol had a relevant effect on liver lipid biosynthesis. In particular, it reduced the production of triacylglycerols and, to a much greater extent that of cholesterol. In addition, probucol reduced plasma cholesterol concentration by decreasing esterified cholesterol in HDL1 and HDL2 fractions. Furthermore, HDL1 composition of both hepatic neosynthetized and circulating particles was strongly modified by probucol. Finally, probucol did not appear to induce significant differences in lipid bile secretion while phospholipid secretion from perfused livers was increased. These facts suggest that the hypolipidemic action of probucol is not mediated by an increase in bile steroid secretion, but rather by a direct reduction in hepatic lipoprotein cholesterol secretion. This secretion induces a modified plasma profile of HDL particles such that these variations are advantageous in terms of reverse cholesterol transport.

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.

We employed matrix-assisted laser desorption mass spectrometry (LD-MS) to detect recombinant bovine growth hormone (r-bGH) in sustained-release preparations. After preliminary extraction in phosphate buffer, LD-MS provided a precise determination of the molecular mass (Mr) of the r-bGH contained in 38 sustained-release preparations. The hormone was characterised using enzyme immunoassay, immunoblotting and amino acid sequencing. Rapid detection is essential for analysing large numbers of samples, and for monitoring the use of r-bGH in zootechnical productions and its administration as a “high-tech” drug for therapeutic purposes.

Lipoprotein and liver lipids of spontaneously hyperHpidemic Yoshida rats were compared with those of normolipidemic Wistar animals for studying their age- and strain-related differences. Both strains showed an age-related increase in the total plasma cholesterol concentration. However, the Yoshida strain had a higher content of cholesteryl esters and triglycerides than the Wistar strain in both young and adult animals (2- and 8-month-old animals, respectively). The free cholesterol content was also higher, but only in the 8-month-old animals. Both strains showed an age-related increase in the proportion of HDL1 and a symmetrical decrease in both the HDL2 and HDL3 subfractions, but the variations were more evident in the Yoshida strain. The study of strain-related differences suggested that the spontaneous hypertriglyceridemia of the Yoshida strain was not only related to the higher amount and proportion of the VLDL fraction, but also to the higher content of triglycerides in the LDL fraction. The livers of Yoshida rats accumulated more triglycerides (with an age-related progression) than those of Wistar rats. The major lipid classes in the liver of Yoshida rats contained a significantly higher proportion of monounsaturated fatty acyls. Furthermore, this proportion showed an age-related increase in all the lipid classes, but in cholesteryl esters. This suggested that liver desaturases had a relevant role in the development of hyperlipidemia, and of its age-related variations, in the Yoshida strain. In conclusion, Yoshida rats appeared a better model for the study of age-related alterations in the liver lipid metabolism since the alterations are stronger and earlier than in Wistar animals and are not complicated by the development of degenerative pathologies typical of old rats.

The aim of this study was to determine whether the common forms of dyslipidemia could affect either the lipid composition or insulin receptor processing (down-regulation) of erythrocytes. The study included 22 patients with type IIa hypercholesterolemia, 15 patients with type IV hypertriglyceridemia and 12 patients with type IIb hyperlipidemia. Ten normolipidemic subjects were used as controls. Their erythrocyte membranes were analyzed for lipid composition and insulin receptor down-regulation. The results show that all the hyperlipidemias investigated were characterized by significant increases in the cholesterol to phospholipid molar ratio (0.56 +/- 0.08 in controls and 1.11 +/- 0.13, 1.09 +/- 0.14, 1.04 +/- 0.15, p < 0.001, in types IIa, IIb and IV, respectively). Surface insulin receptors of type IIa and IIb patients did not appear to down-regulate when compared to normal subjects, but rather up-regulated (+65.2% in controls, -1.0% and -8.7%, p < 0.001, in type IIa and IIb patients, respectively). Patients with type IV hypertriglyceridemia showed a residual capacity for insulin receptor internalization (10.7% down-regulation). Membranes of all the patients contained a higher proportion of phosphatidylethanolamine; the molar ratio of sphingomyelin to phosphatidylcholine was significantly higher in types IIb than in controls (1.22 +/- 0.11 and 1.12 +/- 0.10, p < 0.05, respectively); all the patients showed a lower content of polyunsaturated fatty acids in the major glycerophospholipid classes. However, type IV hypertriglyceridemics showed less variations, especially in the phosphatidylserine fraction. These results indicate that the alterations in lipoprotein pattern may affect both the lipid membrane equilibria and the processing ability of surface insulin receptors. Copyright 1995 S. Karger AG, Basel

Background & Aims Biliary tree, liver, and pancreas share a common embryological origin. We previously demonstrated the presence of stem/progenitor cells of endodermal origin in the adult human extrahepatic biliary tree. This study evaluated the human foetal biliary trees as sources of stem/progenitor cells of multiple endodermal-derived mature fates. Methods Human foetal intrahepatic and extrahepatic biliary tree tissues and isolated cells were tested for cytoplasmic and surface markers of stem cells and committed progenitors, as well as endodermal transcription factors requisite for a liver versus pancreatic fate. In vitro and in vivo experiments were conducted to evaluate the potential mature fates of differentiation. Results Foetal biliary tree cells proliferated clonogenically for more than 1month on plastic in a serum-free Kubota medium. After culture expansion, cells exhibited multipotency and could be restricted to certain lineages under defined microenvironments, including hepatocytes, cholangiocytes, and pancreatic islet cells. Transplantation of foetal biliary tree cells into the livers of immunodeficient mice resulted in effective engraftment and differentiation into mature hepatocytes and cholangiocytes. Conclusions Foetal biliary trees contain multipotent stem/progenitor cells comparable with those in adults. These cells can be easily expanded and induced in vitro to differentiate into liver and pancreatic mature fates, and engrafted and differentiated into mature cells when transplanted in vivo . These findings further characterise the development of these stem/progenitor cell populations from foetuses to adults, which are thought to contribute to liver and pancreas organogenesis throughout life.

As part of a study of the effects of aging on lipoprotein metabolism, the uptake and processing in vivo of cholesterol from high density lipoprotein (HDL) was compared in young (3 months of age) and mature (10-12 months of age) rats by studying the fate of HDL [3H] unesterified cholesterol or [3H] cholesteryl ester after intravenous administration. Radioactivity from [3H] unesterified cholesterol was cleared from the blood more slowly in older rats, and this difference was accounted for by decreased uptake by the liver. Uptake by other tissues were unaffected. In addition, a shift in the distribution of radioactivity across the plasma lipoprotein density range from the d = 1.125-1.250 g/ml (HDL3) to the d = 1.050-1.085 g/ml (HDL1) fraction was observed in the mature as compared to the young rat group. The secretion of radioactivity from [3H] unesterified cholesterol into bile was also decreased in the older animals, particularly in the first hour after injection of the label. In the case of HDL labeled with [3H] cholesteryl ester, clearance from the blood was similar in both age groups in the first 30 min after injection, but was significantly lower in older rats at later time points. After 180 min, less radioactivity was found in the VLDL density fraction in mature as compared to young rats, suggesting that hepatic secretion of VLDL cholesterol originating from HDL cholesteryl ester is less efficient in the older animals. The amount of radioactivity from HDL [3H] cholesteryl ester secreted in bile was less in the mature rat group at all time points measured.(ABSTRACT TRUNCATED AT 250 WORDS)

We have shown previously that the age-dependent increase in plasma cholesterol levels observed in male Wistar rats is associated with relevant changes in the lipoprotein pattern (in particular, with a much higher proportion of the HDL1 class) that are evident in animals from the age of 9 months. In this study, the possibility that a decreased catabolism of HDL1 cholesterol may cause this is evaluated by infusing this lipoprotein fraction labeled with [14C]cholesterol into both young (3.5 +/- 0.5 months) and adult (13.0 +/- 1.0 months) male Wistar rats with a permanent biliary drainage. The clearance of radioactivity from the blood compartment was slower in the older animals than in the younger ones. Conversely, the incorporation of radioactivity into plasma cholesteryl esters and the secretion of radioactivity into bile was higher in the younger animals. These results support the hypothesis that the age-related increase in HDL1 proportion is due, at least in part, to a slower liver catabolism of HDL1-cholesterol.

Plasma lipoproteins in human and rodent species show marked differences in their distribution patterns. However, more analogies are evident with hamsters than with rats. Plasma lipoproteins of golden Syrian hamster comprise significant amounts of the VLDL, LDL and HDL classes, and the HDL fraction does not appear to be divided into subclasses. HDL is the main lipoprotein class and shows an age-related reduction from 73% to 65% of total lipoproteins in young and old animals, respectively. The LDL class represents about the 12% of total lipoprotein throughout the life-span examined by us (1.5–9-month-old animals). The VLDL fraction shows an age-related increase from 15% in younger animals to 24% in older ones. We observed also that in the hamsters the distribution of cholesterol between free and esterified forms, and the distribution of phospholipid classes in the different lipoprotein fractions are more similar to those found in man than to those in the Wistar rat. This suggests that the golden Syrian hamster, particularly when older than 6 months, is a better model for experimental studies on lipoprotein metabolism than Wistar rats.

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. (HEPATOLOGY 2011;54:2159-2172)

Both Wistar and Sprague-Dawley rat strains have been used for experimental studies on lipoprotein metabolism, although the lipoprotein characteristics of the former strain are less well known than those of the latter. We have defined more precise conditions for separating by density gradient ultracentrifugation the different lipoprotein classes from plasma of young male Wistar rats. Present results confirm that Wistar rats, like other rat strains, have negligible amounts of low density lipoproteins and levels of very low density lipoproteins much lower than high density lipoproteins. The differences between rat strains appeared in the profile of lipoprotein denser than 1.08 g/ml. In fact, we showed Wistar rats have considerable amounts of high density lipoprotein-3 subfraction with a density range between 1.17 and 1.21 g/ml, a mean diameter of 4.9 nm, and a low cholesterol content. In analogy with the Sprague-Dawley strain, Wistar rats had a small proportion of high density lipoprotein-1 subfraction (about 17% of total lipoproteins) and, at variance, in Wistar rats, we did not observe the presence of the so-called very high density lipoprotein fraction described previously in the Sprague-Dawley strain.

Insulin-induced down-regulation of erythrocyte insulin receptors is a simplified model that can provide useful information on the cell surface regulative phenomena and on role of the plasma membrane and cytoskeleton in such physiological processes. Oxidative imbalance was examined since it was shown to play an important role in numerous cellular pathologies as well as in cell aging. Specifically, the free radical inducer menadione was used in order to evaluate if this compound is able to modify (and in which manner) the down-regulation process. Biochemical, biophysical, and ultrastructural approaches were used. The results obtained seem to indicate that menadione-induced oxidative damage was able to decrease the insulin-induced down-regulation process, as measured by binding assays. This effect was accompanied by slight alterations in plasma membrane ultrastructure and insignificant variations in plasma membrane lipid composition. In addition, the decrease in membrane order, measured by electron paramagnetic resonance, which was shown to usually occur during the process of down-regulation, was not observed. In contrast, cytoskeletal protein assembly, as previously shown in other in vitro systems, appeared to be remarkably altered. Such changes in specific cytoskeletal elements could lead to the decrease of down-regulation phenomenon induced by menadione. Changes in electrophoretic pattern of some cytoskeletal proteins (e.g., spectrin) reenforce this hypothesis. Considering the importance of free radicals in cell injury, data reported here could represent a specific example of a general mechanism by which cell surface receptor expression and recycling can be modified by changes in some intracellular molecule redox status and cell ionic homeostasis.

The effects of HDL1 lipoprotein infusion on biliary lipid secretion were studied in the in vitro model of rat perfused liver. A strong increase in bile flow was observed during and after lipoprotein infusion. This caused a significant rise in cholesterol, phospholipid and bile salt secretions. However, only the percentage of cholesterol increased with respect to the other bile lipids. The changes observed in the cholesterol/phospholipid molar ratio values of liver membrane subfractions (i.e., liver plasma membrane, mitochondria plus lysosomes and microsomes) isolated from the perfused rat liver after HDL1 administration were not significant.

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 9years of age, following the fortuitous discovery of elevated serum liver enzymes in apparently healthy children. They had mixed hyperlipidemia, hepatosplenomegaly, reduced LAL activity (∼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 80year-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.

Background/Aims: Drugs with antivascular endothelial growth factor A (anti-VEGF-A) action are under clinical evaluation with encouraging results in advanced hepatocellular carcinoma (HCC). The relative VEGF-A protein expression in non-advanced HCC and in the cirrhotic non-tumoral tissue in the same patient, a variable that could be important for treatment efficacy, has been investigated with conflicting results, only using the cirrhotic tissue surrounding the neoplasm (CS). Methods: We measured, for the first time, VEGF-A expression in non-advanced HCC and in the respective CS and cirrhotic tissue at a distance from the tumour (CD), in 24 patients who underwent liver transplantation. Results: VEGF-A protein was more expressed (P

We have recently demonstrated that the delay in myoblast membrane fusion induced by cesium is accompanied by changes in isolated membrane lipids (Santini, M.T., Indovina, P.L., Cantafora, A. and Blotta, I. (1990) Biochim. Biophys. Acta 1023, 298–304). In the present study, we have investigated changes in the lipid profile of total cell homogenates and microsomal membrane fractions during myoblast membrane fusion as well as the effects that addition of cesium may have on these lipid variations in order to try to understand the production and translocation of lipids during this myogenic process. The data presented here indicate that the lipid composition of cell homogenates and microsomes varies in a different manner from isolated plasma membranes during myogenic fusion. In addition, cesium affects, in a different manner, the normally-occurring lipid production and distribution which takes place in each subcellular fraction.

Lysophosphatidylcholine is a major metabolic product in the plasma and cellular turnover of phospholipids, with well-known membrane-toxic and proinflammatory properties. Because the liver plays a key role in plasma lysophosphatidylcholine removal and biotransformation and because virtually nothing is known of these processes in a diseased organ, the hepatobiliary metabolism of lysophosphatidylcholine was investigated in rats with carbon tetrachloride-induced liver cirrhosis. Twelve adult male Wistar rats with histologically confirmed cirrhosis and 8 control animals were fitted with jugular and biliary catheters and allowed to recover. The animals were kept under constant IV infusion of taurocholate (1 mumol/min). Two microcuries of sn-1[14C]palmitoyl-lysophosphatidylcholine was administered as a single bolus. The fate of the injected radioactivity, including removal from plasma, uptake, and subcellular location in the liver and molecular and aggregative forms, was studied by combined chromatographic and radiochemical methods. Major findings were (a) that lysophosphatidylcholine has a prolonged permanence in plasma of cirrhotic rats, due both to decreased hepatic clearance and to depressed conversion into phosphatidylcholine; (b) that the rate of lysophosphatidylcholine acylation is much slower in the cirrhotic than in the normal liver, both at the microsomal and at the cytosolic level; (c) that cytosolic lysophosphatidylcholine in the cirrhotic liver, but not in the normal liver, is predominantly non-protein bound; (d) that the strict molecular selectivity of lysophosphatidylcholine acylation observed in controls is partially lost in cirrhosis; and (e) that a consistent fraction of lysophosphatidylcholine is converted into triacylglycerols in cirrhotics but not in controls. These findings show a profound derangment of lysophosphatidylcholine handling and processing in the cirrhotic liver, which is of potential pathogenetic significance.

The intravenous administration of dimethylethanolamine in the rat promotes a selective enrichment of liver membranes with polyunsaturated phosphatidylcholines. The effect of dimethylethanolamine pretreatment on cholestasis induced by estradiol 17 beta-D-glucuronide, a potent cholestatic agent, was assessed in this study. Dimethylethanolamine, dissolved in sodium-taurocholate was infused intravenously (0.3 mg/kg/min) for 15 hr. One group of control rats (estradiol 17 beta-D-glucuronide controls) received the bile salt only. An estradiol 17 beta-D-glucuronide bolus was then injected intravenously (10.4 mg/kg) into dimethylethanolamine-pretreated and estradiol 17 beta-D-control rats, and its effect on bile flow and biliary lipid secretion was compared for 3 hr. The estradiol 17 beta-D-glucuronide inhibitory effect on bile flow and biliary lipid secretion was significantly antagonized by dimethylethanolamine pretreatment. The maximum inhibition of bile flow was found 30 min after estradiol 17 beta-D-glucuronide administration, when it decreased from 3.5 +/- 0.4 microliters/min/100 gm (basal) to 0.9 +/- 0.3 microliters/min/100 gm in estradiol 17 beta-D-glucuronide controls, whereas in dimethylethanolamine-pretreated rats this decreased only from 3.2 +/- 0.4 (basal) to 2.3 +/- 0.4 microliters/min/100 gm. Bile flow and the biliary secretion of cholesterol, phosphatidylcholine and bile salts were significantly higher in the dimethylethanolamine-pretreated rats than in estradiol 17 beta-D-glucuronide controls (p less than 0.02) during the cholestatic phase. The inhibitory effect of estradiol 17 beta-D-glucuronide on bile flow was associated with a marked decrease of membrane fluidity (p less than 0.001) assessed by 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy and with a cholesterol enrichment of microsomes, sinusoidal and canalicular liver plasma membranes and inhibition of sinusoidal Na+,K(+)-ATPase activity (p less than 0.05). These membrane alterations persisted 180 min after estradiol 17 beta-D-glucuronide administration despite complete normalization of bile flow. Dimethylethanolamine pretreatment significantly counteracted the reduction of membrane fluidity (p less than 0.001), the cholesterol enrichment and the inhibition of Na+,K(+)-ATPase (p less than 0.05) promoted by estradiol 17 beta-D-glucuronide administration in all membrane subfractions 30 and 180 min after administration. In addition, dimethylethanolamine-pretreated rats had more polyunsaturated fatty acids in membrane phosphatidylcholine with respect to the control groups. Dilatation of canaliculi and loss of microvilli were evident in estradiol 17 beta-D-glucuronide controls 180 min after estradiol 17 beta-D-glucuronide administration.(ABSTRACT TRUNCATED AT 400 WORDS)

The metabolic fate of [14C]cholesterol carried in the VLDL, LDL, HDL2 and HDL3 lipoprotein fractions has been investigated in bile-fistula rats with jugular vein cannulation. After the depletion of bile salt pool, the labelled lipoprotein fraction was administered either with or without the continuous infusion of sodium taurocholate. The proportion of labelled steroids secreted in the bile within 3 h after the lipoprotein administration was generally higher when the exogenous bile salt was infused. Specifically, the HDL2 fraction induced the secretion of relatively high proportions of labelled steroids, mainly bile salts, either with or without the taurocholate infusion. The other lipoprotein fractions increased the proportion of free cholesterol in steroid bile secretion under taurocholate administration. The label associated to the LDL fraction showed a higher biliary secretion and a more steady clearance from plasma if the exogenous bile salt was not administered. In this same condition, the administration of HDL3 fraction gave the highest values of radioactivity recovered in the liver (mainly as free cholesterol) and a significant increase of cholesterol in the biliary secretion. The present results suggest that each lipoprotein fraction tested may contribute in a peculiar manner to bile salt and cholesterol biliary secretion. Both the expression of apo-E and apo B,E receptors and the levels of circulating bile salts appear to have a role in this process.

We have recently demonstrated that cesium ions delay the sharp decrease in both membrane conductivity and membrane permittivity of chick embryo myoblasts seen at fusion (Santini, M.T., Bonincontro, A., Cametti, C. and Indovina, P.L. (1988) Biochim. Biophys. Acta 945, 56–64). Analysis of the conductivity dispersion data (obtained in the radiowave frequency range) indicated that cesium delays fusion by about 30 h. We suggested that cesium is affecting both active ionic transport by blocking potassium channels as well as interfering with membrane lipid and/or protein charges. In the present study, we have investigated both the possible role of membrane lipids in myoblast fusion and the possible effects of cesium on these lipids. Our data indicate that lipid changes do occur in the isolated myoblast plasma membrane of controls during myogenic differentiation especially prior to fusion and that in cesium cultures these variations do not occur. These variations are in accordance with current membrane fusion theory. Specifically, there is a decrease in bilayer-stabilizing lipids (phosphatidylcholine) and an increase in bilayer-destabilizing ones (phosphatidylethanolamine and phosphatidic acid) and cholesterol during the fusion process. In addition, although slight, during fusion there appears to be a decrease in phosphatidylinositol which is believed to be involved in the inositol phosphate second messenger system. In cesium cultures, in which fusion is greatly delayed, the same lipid changes do not take place and those that are observed seem to reflect the fusion delay.

We investigated the effect of statins and statins plus ezetimibe in 65 FH heterozygotes carrying LDLR-defective or LDLR-negative mutations as well as the effect of ezetimibe monotherapy in 50 hypercholesterolemic (HCH) patients intolerant to statins. PCSK9 and NPC1L1 genes were analysed to assess the role of genetic variants in response to therapy. In FH patients combined therapy reduced LDL-C by 57%, irrespective of the type of LDLR mutation. The additional decrease of plasma LDL-C induced by ezetimibe showed wide inter-individual variability (from -39% to -4.7%) and was negatively correlated with percent LDL-C decrease due to statin alone (r=-0.713, P0.001). The variable response to statins was not due to PCSK9 gene variants associated with statin hyper-sensitivity. The highest response to ezetimibe was observed in a carrier of R174H substitution in NPC1L1, which had been found to be associated with high cholesterol absorption. In HCH patients, ezetimibe monotherapy induced a variable decrease of plasma LDL-C (from -47.7% to -13.4%). To investigate this variability, we sequenced NPC1L1 gene in patients with the highest and the lowest response to ezetimibe. This analysis showed a higher prevalence of the G allele of the c.816 CG polymorphism (L272L) in hyper-responders, an observation confirmed also in FH patients hyper-responders to ezetimibe. In both FH and HCH patients, the G allele carriers tended to have a higher LDL-C reduction in response to ezetimibe. These observations suggest that in FH heterozygotes LDL-C reduction following combined therapy reflects a complex interplay between hepatic synthesis and intestinal absorption of cholesterol.

The diagnosis of familial hypercholesterolemia (FH) in unselected children is difficult due to the frequent overlap of cholesterol values in affected and non-affected and the paucity of physical signs. Nevertheless, detection and treatment of FH in childhood has been advocated to prevent atherosclerosis in these patients. Here, we report the results of a screening program in a cohort of 157 unrelated, hypercholesterolemic (HC) children (age range 2-15 years; mean 8.3+/-3.4 years) carried out by a combination of family study and molecular analysis of the LDLR gene. On the basis of the familial phenotype, 27 (17.2%) were classified as probable FH and 49 (31.2%) as affected by FCHL. Among probable FH children, 14 (51.8%) carried mutant LDLR alleles, giving an overall 8.9% prevalence of FH. Most of LDLR variants were already reported, but three new mutations G266C, T368M, and D451Y were identified. Beside increased TC and LDL-C (p0.001), FH children showed decreased HDL-C (p0.05) and higher prevalence of family history of CAD when compared to non-FH children. None presented tendon xanthomas. We estimated that LDL-C3.9 mmol/L was the best cut off value for diagnosing FH in these children, showing 79% sensitivity and 71.0% specificity. We propose the use of a LDL-C cut off level associated with a family study to identify FH among HC children.

Autosomal recessive hypercholesterolemia (ARH) is a rare disorder, due to complete loss of function of an adaptor protein (ARH protein) required for receptor-mediated hepatic uptake of LDL. ARH is a phenocopy of homozygous familial hypercholesterolemia (HoFH) due to mutations in LDL receptor (LDLR) gene; however, previous studies suggested that ARH phenotype is less severe than that of HoFH. To test this hypothesis we compared 42 HoFH and 42 ARH patients. LDLR and ARH genes were analysed by Southern blotting and sequencing. LDLR activity was measured in cultured fibroblasts. In ARH plasma LDL cholestrol (LDL-C) level (14.25+/-2.29 mmol/L) was lower than in receptor-negative HoFH (21.38+/-3.56 mmol/L) but similar to that found in receptor-defective HoFH (15.52+/-2.39 mmol/L). The risk of coronary artery disease (CAD) was 9-fold lower in ARH patients. No ARH patients

We describe a quantitative assay of dsDNA based on real-time PCR measurement of fluorescence due to the interaction of PicoGreen dye with dsDNA. An aliquot of 1 to 5 ml of the sample is mixed with 45 ml of diluted PicoGreen reagent within an optical PCR tube. This is placed into the real-time apparatus set to read SYBR Green I dye at the end of three cycles of 94 degrees C for 30 s and 65 degrees C for 30 s. The averaged fluorescence value is converted into DNA amount using a calibration curve prepared with lambda-DNA standard. The calibration curve has a dynamic linear range from 0.20 to 50 ng and a standard deviation variability below 5.0%. In conclusion, this method allows reliable determinations on minimal amounts of DNA from biological samples and PCR products in clinical applications of molecular biology.

Patients homozygous or Compound heterozygous for LDLR mutations or double heterozygous for LDLR and apo B R3500Q mutation have higher LDL-C levels. more extensive xanthomatosis and more severe premature coronary disease (pCAD) than simple heterozygotes for mutations in either these genes or for missense mutations in PCSK9 gene. It is not known whether combined mutations in LDLR and PKCS9 are associated with such a severe phenotype. We sequenced Apo B and PCSK9 genes in two patients with the clinical diagnosis of homozygous FH who were heterozygous for LDLR gene mutations. Proband Z.P. (LDL-C 13.39 mmol/L and pCAD) was heterozygous for an LDLR mutation (p.E228K) inherited from her father (LDL-C 8.07 mmol/L) and a PCSK9 mutation (p.R496W) from her mother (LDL-C 5.58 mmol/L). Proband L.R. and her sister (LDL-C 11.51 and 10.47 mmol/L. xanthomatosis and carotid atherosclerosis) were heterozygous for all LDLR mutation (p.Y419X) inherited from their mother (LDL-C 6.54 mmol/L) and a PCSK9 mutation (p.N425S) probably from their deceased father. The LDL-C levels in double heterozygotes of these two families were 56 and 44% higher than those found in simple heterozygotes for the two LDLR mutations, respectively. The two PCSK9 Mutations are novel and were not found in I 10 controls and 80 patients with co-dominant hypercholesterolemia. These observations indicate that Fare missense Mutations of PCSK9 may worsen the clinical phenotype of patients carrying LDLR mutations.

We studied a three generation family with co-dominant monogenic hypercholesterolemia and hypoalphalipoproteinemia. The proband, a 48 year-old male, was found to be heterozygous for a previously reported mutation in LDL receptor (LDL-R) gene (IVS15-3 ca) and a novel mutation in exon 6 of lecithin cholesterol acyltransferase (LCAT) gene (c.803 GA) causing a non-synonymous amino acid substitution (p.R244H). These mutations segregated independently in the family. The LDL-R mutation was associated with high levels of LDL-C (6.20-9.85 mmol/L) and apo B (170-255 mg/dL), comparable to those previously reported in carriers of the same mutation. The LCAT mutation was associated with low levels of HDL-C (0.67-0.80 mmol/L) and apo A-I (96-110 mg/dL). The proband had reduced LCAT function, as measured by cholesterol esterification rate (29 nmol/(mL/h) versus 30-60 nmol/(mL/h)), LCAT activity (10 nmol/(mL/h) versus 20-55 nmol/(mL/h)) and LCAT mass (2.87 microg/mL versus 3.1-6.7 microg/mL). Carriers of LCAT mutation had lower LCAT activity and a tendency to reduced cholesterol esterification rate (CER) and LCAT mass as compared to non-carrier family members. The LCAT mutation was not found in 80 control subjects and 60 patients with primary hypoalphalipoproteinemia. Despite the unfavourable lipoprotein profile, the proband had only mild clinical signs of atherosclerosis. This unexpected finding is probably due to the intensive lipid lowering treatment the patient has been on over the last decade.