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The deflection of magnetic beads in a microfluidic channel through magnetophoresis can be improved if the particles are somehow focused along the same streamline in the device. We design and fabricate a microfluidic device made of two modules, each one performing a unit operation. A suspension of magnetic beads in a viscoelastic medium is fed to the first module, which is a straight rectangular-shaped channel. Here, the magnetic particles are focused by exploiting fluid viscoelasticity. Such a channel is one inlet of the second module, which is a H-shaped channel, where a buffer stream is injected in the second inlet. A permanent magnet is used to displace the magnetic beads from the original to the buffer stream. Experiments with a Newtonian suspending fluid, where no focusing occurs, are carried out for comparison. When viscoelastic focusing and magnetophoresis are combined, magnetic particles can be deterministically separated from the original streamflow to the buffer, thus leading to a high deflection efficiency (up to ~96%) in a wide range of flow rates. The effect of the focusing length on the deflection of particles is also investigated. Finally, the proposed modular device is tested to separate magnetic and non-magnetic beads.

The stabilization of oil in water nano-emulsions by means of a polymer coating is extremely important; it prolongs the shelf life of the product and makes it suitable for a variety of applications ranging from nutraceutics to cosmetics and pharmaceutics. To date, an effective methodology to assess the best formulations in terms of thermodynamic stability has yet to be designed. Here, we perform a complete physicochemical characterization based on isothermal titration calorimetry (ITC) compared to conventional dynamic light scattering (DLS) to identify polymer concentration domains that are thermodynamically stable and to define the degree of stability through thermodynamic functions depending upon any relevant parameter affecting the stability itself, such as type of polymer coating, droplet distance, etc. For instance, the method was proven by measuring the energetics in the case of two different biopolymers, chitosan and poly-L-lysine, and for different concentrations of the emulsion coated with poly-L-lysine.

In this paper, we report on a general approach for the detection of a specific tumoural biomarker directly in serum. Such detection is made possible using a protein-binding peptide selected through an improved phage display technique and then conjugated to engineered microparticles (MPs). Protein biomarkers represent an unlimited source of information for non-invasive diagnostic and prognostic tests; MP-based assays are becoming largely used in manipulation of soluble biomarkers, but their direct use in serum is hampered by the complex biomolecular environment. Our technique overcomes the current limitations as it produces a selective MP--engineered with an antifouling layer--that 'captures' the relevant protein staying impervious to the background. Our system succeeds in fishing-out the human tumour necrosis factor alpha directly in serum with a high selectivity degree. Our method could have great impact in soluble protein manipulation and detection for a wide variety of diagnostic applications., (© 2014 The Author(s) Published by the Royal Society. All rights reserved.)

The only currently recommended treatment for nonalcoholic fatty liver disease (NAFLD) is lifestyle modification. Preliminary studies of silybin showed beneficial effects on liver function. Realsil (RA) comprises the silybin phytosome complex (silybin plus phosphatidylcholine) coformulated with vitamin E. We report on a multicenter, phase III, double-blind clinical trial to assess RA in patients with histologically documented NAFLD. Patients were randomized 1:1 to RA or placebo (P) orally twice daily for 12 months. Prespecified primary outcomes were improvement over time in clinical condition, normalization of liver enzyme plasma levels, and improvement of ultrasonographic liver steatosis, homeostatic model assessment (HOMA), and quality of life. Secondary outcomes were improvement in liver histologic score and/or decrease in NAFLD score without worsening of fibrosis and plasma changes in cytokines, ferritin, and liver fibrosis markers. We treated 179 patients with NAFLD; 36 were also HCV positive. Forty-one patients were prematurely withdrawn and 138 patients analyzed per protocol (69 per group). Baseline patient characteristics were generally well balanced between groups, except for steatosis, portal infiltration, and fibrosis. Adverse events (AEs) were generally transient and included diarrhea, dysgeusia, and pruritus; no serious AEs were recorded. Patients receiving RA but not P showed significant improvements in liver enzyme plasma levels, HOMA, and liver histology. Body mass index normalized in 15% of RA patients (2.1% with P). HCV-positive patients in the RA but not the P group showed improvements in fibrogenesis markers. This is the first study to systematically assess silybin in NAFLD patients. Treatment with RA but not P for 12 months was associated with improvement in liver enzymes, insulin resistance, and liver histology, without increases in body weight. These findings warrant further investigation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background and Aim: Refractory ascites in liver-cirrhosis is associated with a poor prognosis. We performed a prospective study to investigate whether aggressive nutritional-support could improve outcomes in cirrhotic patients., Methods: Cirrhotic patients undergoing serial large-volume paracentesis for refractory-ascites were enrolled and randomized into three groups. Group A received post-paracentesis intravenous nutritional-support in addition to a balanced oral diet and a late-evening protein snack, group B received the same oral nutritional-protocol as the first group but without parenteral support, and group C (the control group) received a low-sodium or sodium-free diet. Clinical, anthropometric and laboratory nutritional parameters and biochemical tests of liver and renal function were reported for 12 months of follow-up., Results: We enrolled 120 patients, who were randomized into three groups of equal size. Patients on the nutritional-protocol showed better preservation of clinical, anthropometric and laboratory nutritional parameters that were associated with decreased deterioration of liver function compared with patients on the low-sodium or sodium-free diet (group C). Groups A and B had lower morbidity and mortality rates than the control group (C). Mortality rates were significantly better in patients who were treated with parenteral-nutritional-support than for the other two groups. In patients who were on the nutritional-protocol, there was a reduction in the requirement of taps for the treatment of refractory ascites., Conclusions: Post-paracentesis parenteral-nutritional-support with a balanced oral diet and an evening protein snack appears to be the best care protocol for patients with liver-cirrhosis that has been complicated by refractory-ascites., (© 2011 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd.)

Liver organogenesis and cancerogenesis share common mechanisms. HOX genes control normal development, primary cellular processes and are characterized by a unique genomic network organization. Less is known about the involvement of HOX genes with liver cancerogenesis. The comparison of the HOX gene network expression between nontumorous livers and hepatocellular carcinomas (HCCs) highlights significant differences in the locus A HOX genes, located on chromosome 7, with a consistent overexpression of HOXA13 mRNA thus validating this gene deregulation as a feature of HCC. HOXA13 is a determinant of gut primordia and posterior body structures. Transcriptome analysis of HCC/nontumorous liver mRNAs, selected on the basis of HOXA13 overexpression, recognizes a set of deregulated genes. The matching of these genes with previously reported HCC transcriptome analysis identifies cell-cycle and nuclear pore-related HCC phenotype displaying poor prognosis. HOXA13 and HOXA7 homeoproteins share a consensus sequence that physically links eIF4E nuclear bodies acting on the export of specific mRNAs (c-myc, FGF-2, vascular endothelial growth factor (VEGF), ornithine decarboxylase (ODC) and cyclin D1). We report the protein-protein interaction between HOXA13 and eIF4E in liver cancer cells and the deregulation of eIF4E mRNA and protein in cell cycle/nuclear pore HCC group phenotype and in T4 stage HCCs, respectively. Thus, transcriptional and post-transcriptional HOXA13 deregulation is involved in HCC possibly through the mRNA nuclear export of eIF4E-dependent transcripts., (Copyright © 2011 UICC.)

Background and Aim: We aimed to validate the non-invasive criteria for the characterization of portal vein thrombosis (PVT) in patients with cirrhosis and hepatocellular carcinoma (HCC). In a prospective study, we examined the impact of arterial hypervascularity, as established by the European Association for the Study of the Liver and the American Association for the Study of Liver Diseases recommendations for the non-invasive diagnosis of HCC, as a criterion for characterizing macroscopic PVT (EASL/AASLD extension criteria)., Methods: A total of 96 cases of PVT detected using ultrasonography in patients with cirrhosis and HCC were included in the study. When coincidental arterial hypervascularity was detected by contrast perfusional ultrasonography and helical computed tomography, the thrombus was considered malignant according to our EASL/AASLD extension criteria. In all cases, an ultrasound-guided biopsy examination of the thrombus was performed., Results: Coincidental hypervascularity was found in 54 of 96 nodules (56.2%), and all were malignant upon biopsy (100% positive predictive value). Twenty-four (25%) had negative results with both techniques (non-vascular thrombus). Biopsies showed HCC in five non-vascular thrombi (5.3% of all thrombi) and in 13 of 18 thrombi with a hypervascularity result from only one technique., Conclusions: The EASL/AASLD extension criteria for non-invasive diagnosis of malignant thrombosis were satisfied in 75.2% of malignant thrombi; thus, a biopsy is frequently required in this setting. However, in the presence of coincidental hypervascularity of a thrombus with both techniques, a biopsy is not required (absolute positive predictive value for malignancy). Relying on imaging techniques in thrombi could miss the diagnosis of malignant portal invasion in up to 24.9% of cases., (© 2011 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd.)

Herbal medicines are widely used in the world and are generally considered effective and safe, although many studies have demonstrated their potential toxic effects, particularly for the liver. We present a case of a woman, who developed a mixed cholestatic/hepatocellular liver injury due to herbal products. Firstly, she was admitted to Division of Surgery for right upper abdominal pain and jaundice and, for the suspect of biliary obstruction, she underwent to cholecystectomy. For persistence of liver enzymes elevation, she was admitted to our Gastroenterology Unit. We excluded every etiologies of hepatitis and, after an intensive dialogue with the patient, we obtained a history of herbal medicines use. Then, we performed a liver biopsy which was compatible with hepatotoxic injury. Therapy with ursodeoxycholic acid (UDCA) was started. Liver function tests returned to normal in two months. We describe this clinical case to encourage the communication doctor/patient in phytotherapy area and physician knowledge about efficacy and side effects of herbal medicine to avoid delayed diagnosis.

Background: Factors responsible for the progression of primary biliary cirrhosis (PBC) are still poorly understood. In the present study, we investigated the associations between the stage of PBC and the immune reaction triggered by oxidative stress; the presence of obesity, steatosis,steatohepatitis; and other toxic, metabolic, or steatogenic factors., Methods: We studied clinical, laboratory, and histological data for 274 untreated patients with serum antimitochondrial antibody (AMA)-positive PBC. Circulating IgG against human serum albumin adducted with malondialdeyde, the major product of lipid peroxidation, was measured in these patients and in a group of 98 sex-, age and body mass index (BMI)-matched controls., Results: Steatosis was present in 40.5% of all patients. Steatohepatitis was present in 14.9% of all patients. There was a significant association between the frequencies of steatosis and steatohepatitis and the worsening of PBC. Circulating IgG against lipid peroxidation products was significantly higher in the PBC patients than in the controls. Titers of lipid peroxidation-related antibodies were significantly increased in patients with steatosis and inpatients at more advanced stages. Bivariate analysis revealed a significant association between indirect evidence of oxidative stress, steatosis, steatohepatitis, age, BMI, frequency of diabetes, alcohol intake, iron grade after Perl's stain, and PBC stage. Logistic regression analysis confirmed that the titers of antibodies against lipid peroxidation products (odds ratio 4.5, p< .001, 95% confidence interval 3.9–14.4), the presence of steatosis (odds ratio 4.1, 95% confidence interval 2.5–15.4, p< .001), higher BMI (odds ratio 3.9, p< .021, 95%confidence interval 1.4–9.5), and alcohol intake (males ≥ 30 g/day, females ≥ 20 g/day, odds ratio 4.5,95% confidence interval 1.3–19.8, p< .029) were independently associated with more advanced stages of the disease., Conclusions: The immune reactions triggered by oxidative stress, steatosis, obesity, and alcohol intake are independent predictors of PBC stage progression.

Objectives: Few published studies have examined the results obtained from repeat liver biopsies in obese patients with nonalcoholic fatty liver disease (NAFLD). The progressive form of this disease may be largely limited to a subgroup of NAFLD patients with nonalcoholic steatohepatitis (NASH). The presence of intralobular fibronectin (Fn) and other variables was investigated in relation to subsequent fibrosis progression., Methods: In this prospective study, 271 obese patients admitted to the hospital with NAFLD and abnormal liver enzymes were scheduled to undergo a repeat liver biopsy at least 5 years after the initial biopsy. After excluding cirrhotic patients, basal biopsy specimens obtained from patients who underwent a second liver biopsy were stained with antibodies against Fn. The progression of fibrosis in the follow-up sample was correlated with the amount of Fn and other clinicopathological variables., Results: We obtained a second liver biopsy from 149 patients after a median time of 6.4 years. Of these, 132 showed suitable Fn staining for semi-quantitative assessments. In all, 44 out of 83 patients (53%) with basal NASH showed fibrosis progression by at least one stage in the second liver biopsy. The amount of Fn (odds ratio=14.1; P<0.001), a diagnosis of hypertension (odds ratio=4.8; P=0.028), and homeostasis model assessment parameter of insulin resistance (HOMA-IR) scores (>8, odds ratio=1.9; P=0.004) were independent predictive factors of worsening fibrosis., Conclusions: A semi-quantitative assessment of the amount of parenchymal Fn present at an early stage in obese patients with NASH is valuable for predicting the progression of fibrosis. Similarly, lobular Fn deposition may be a sensitive and early indicator of active fibrogenetic processes in the liver. Hypertension and higher HOMA-IR scores are other clinical independent risk factors that predict the progression of fibrosis.

Background: Differently from chronic hepatitis C, factors associated with hepatic steatosis in children with chronic hepatitis B are not clearly elucidated., Objective: Aim of this study was to investigate prevalence of steatosis at liver biopsy in HBV-infected children., Study Design: A retrospective study including 56 children with chronic hepatitis B undergoing liver biopsy at median age of 8.1 years. In all patients demographic, anthropometric, clinical and laboratory data were evaluated at the time of liver biopsy., Results: Steatosis was present in 2 (4%) children. BMI was significantly higher in 2 patients with steatosis compared with those without steatosis. Demographic, biochemical and virological parameters did not differ between children with and those without steatosis., Conclusions: Liver steatosis in HBV-infected children seems to be related to obesity and metabolic factors rather than to viral factors. Detection of steatosis in non-obese children with HBV infection requires a careful investigation to rule out other causes of fatty liver.

Aim: To clarify which method has accuracy: 2nd generation contrast-enhanced ultrasound or biopsy of portal vein thrombus in the differential diagnosis of portal vein thrombosis., Methods: One hundred and eighty-six patients with hepatocellular carcinoma and portal vein thrombosis underwent in blinded fashion a 2nd generation contrast-enhanced ultrasound and biopsy of portal vein thrombus; both results were examined on the basis of the follow-up of patients compared to reference-standard., Results: One hundred and eight patients completed the study. Benign thrombosis on 2nd generation contrast-enhanced ultrasound was characterised by progressive hypoenhancing of the thrombus; in malignant portal vein thrombosis there was a precocious homogeneous enhancement of the thrombus. On follow-up there were 50 of 108 patients with benign thrombosis: all were correctly diagnosed by both methods. There were 58 of 108 patients with malignant thrombosis: amongst these, 52 were correctly diagnosed by both methods, the remainder did not present malignant cells on portal vein thrombus biopsy and showed on 2nd generation contrast-enhanced ultrasound an inhomogeneous enhancement pattern. A new biopsy during the follow-up, guided to the area of thrombus that showed up on 2nd generation contrast-enhanced ultrasound, demonstrated an enhancing pattern indicating malignant cells., Conclusion: In patients with hepatocellular carcinoma complicated by portal vein thrombosis, 2nd generation contrast-enhanced ultrasound of portal vein thrombus is very useful in assessing the benign or malignant nature of the thrombus. Puncture biopsy of thrombus is usually accurate but presents some sampling errors, so, when pathological results are required, 2nd generation contrast-enhanced ultrasound could guide the sampling needle to the correct area of the thrombus.

Background/aims: Liver iron deposits are frequent in patients with non-alcoholic steato-hepatitis (NAFLD), but their role is not well defined. To investigate the effect of liver iron excess on the prevalence of hepatocellular carcinoma (HCC) in patients with NASH-related cirrhosis., Methods: Hepatic iron was measured retrospectively with a semiquantitative method in liver biopsies of 153 patients with NASH-related cirrhosis: 51 with HCC and 102 controls without HCC, matched for age, sex and stage of liver disease. The corrected total iron score (0-60) was the sum of three scores: the hepatocytic iron score (0-36), sinusoidal iron score (0-12), and portal iron score (0-12), multiplied by 3/3, 2/3, or 1/3 depending on the localisation of the iron in the nodules., Results: Conditional logistic regression analysis showed that iron deposits (corrected total iron score>0) were more frequent in HCC patients than in controls. The median corrected total iron score was significantly higher in HCC patients than in controls. The liver iron overload was sinusoidal., Conclusions: Iron deposition in the liver was more frequent in patients with NASH-related cirrhosis with HCC than in HCC-free controls. Liver iron overload may be associated with development of HCC in patients with NASH-related cirrhosis.

Lathosterolosis (LS) is a defect of cholesterol biosynthesis due to the deficiency of the enzyme sterol-C5-desaturase. Only two patients have been described to date, both presenting with multiple malformations, mental retardation, and liver involvement. In addition in one of them pathological examination revealed mucolipidosis-like inclusions on optic microscopy analysis, and peculiar lysosomal lamellar bodies on electron microscopy analysis. This study is focused on a better characterization of the clinical phenotype of LS. We describe a further case in a fetus, sibling of the first patient reported, presenting with neural tube defect, craniofacial and limb anomalies, and prenatal liver involvement. The fetal phenotype suggests the possible occurrence of significant intrafamilial variability in LS, and expands the phenotypic spectrum of the disease. Histological examination of autopsy samples from the fetus and skin fibroblasts from the living sibling suggested that the mucolipidosis-like picture previously reported is not a constant feature of LS, being possibly associated with the most severe biochemical defects, but confirmed the ultrastructural finding of lamellar inclusions. The LS phenotype appears to be characterized by the distinctive association of a recognizable pattern of congenital anomalies, involving axial and appendicular skeleton, liver, central nervous and urogenital systems, and lysosomal storage. This condition partially overlaps with other defects of sterol metabolism, suggesting intriguing pathogenic links among these conditions., (Copyright (c) 2007 Wiley-Liss, Inc.)

Unlabelled: Hepatocellular nodules in cirrhosis include regenerative (large regenerative, LRN) and dysplastic (low and high grade, LGDN and HGDN) nodules, early and grade 1 HCC (eHCC-G1), and overt HCC. The differential diagnosis may be particularly difficult when lesions such as HGDN and eHCC-G1 are involved. We investigated the diagnostic yield of a panel of 3 putative markers of hepatocellular malignancy such as HSP70, glypican 3 (GPC3), and glutamine synthetase (GS). We selected 52 surgically removed nonmalignant nodules (15 LRNs, 15 LGDNs, 22 HGDNs) and 53 HCCs (10 early, 22 grade 1, and 21 grade 2-3) and immunostained them for HSP70, GPC3, and GS. The sensitivity and specificity of the individual markers for the detection of eHCC-G1 were 59% and 86% for GS, 69% and 91% for GPC3, and 78% and 95% for HSP70. We identified 2 main phenotypes: (1) all negative, seen in 100% LRN and LGDN, 73% HGDN and 3% eHCC-G1; (2) all positive, a feature detected in less than half the eHCC-G1. Using a 3-marker panel, when at least 2 of them, regardless which, were positive, the sensitivity and specificity for the detection of eHCC-G1 were respectively 72% and 100%; the most sensitive combination was HSP70+/GPC3+ (59%) when a 2-marker panel was used., Conclusion: The adopted panel of 3 markers is very helpful in distinguishing eHCC-G1 from dysplastic nodules arising in cirrhosis.

Neural tube defects (NTD) are morphogenetic alterations due to a defective closure of neural tube. Hepatocyte growth factor (HGF)/c-met system plays a role in morphogenesis of nervous system, lung, and kidney. HGF/c-met morphogenetic effects are mediated by signal transducers and activators of transcription (STAT)3 and both HGF and c-met genes are regulated from p53. The aim of our study was to analyze mRNA and protein expressions of p53, HGF, c-met, and STAT3 in fetuses with NTD. By reverse transcriptase-polymerase chain reaction and immunohistochemistry, we analyzed neural tissues from four NTD fetuses and the corresponding non-malformed lungs, kidneys and placentas. We found a reduced mRNA expression of HGF/c-met/STAT3 pathway, in the malformed nervous systems and placentas. The reduced expression of this pathway correlated with the absence of p53 in all these samples. On the contrary, detectable expression levels of p53, HGF, c-met, and STAT3 were observed in non-malformed lungs and kidneys obtained from the same fetuses. Comparable results were obtained by immunohistochemistry, with the exception of p53, which was undetected in all fetal tissues. In conclusion, in NTD fetuses, both the defective neural tube tissue and the placenta have a reduction in all components of the p53/HGF/c-met/STAT3 cascade. This raises the possibility of using the suppression of these genes for early diagnosis of NTD especially on chorionic villus sampling.

Background/aims: Prevalence and significance of steatosis in children with chronic hepatitis C are not well defined. We analysed the prevalence of steatosis in children with chronic hepatitis C and its relationship with clinical, laboratory features and response to interferon., Methods: Sixty-four consecutive children with CHC undergoing liver biopsy were retrospectively evaluated., Results: Twenty-five percent of children showed mild to moderate steatosis. Only one child was infected by genotype 3. Body mass index did not significantly differ between children with and without steatosis. Although no significant difference in necroinflammatory and fibrosis scores between children with and without steatosis was found, 3 (18.7%) of 16 patients with steatosis and only one (2.1%) of 48 patients without steatosis had a fibrosis score >2 (P<0.05). Forty-seven children (13 with steatosis) received interferon after liver biopsy. A sustained response was observed in 3 (23%) children with steatosis and in 18 (53%) without steatosis., Conclusions: Histological evidence of steatosis is detectable in a quarter of children with CHC. Differently from adults, genotypes other than 3 may be associated with steatosis independently from classical metabolic risk factors. Children with steatosis seem to have more severe fibrosis and lower rates of sustained response to interferon therapy compared to children without steatosis.

Homeobox-containing genes play a crucial role in odontogenesis. After the detection of Dlx and Msx genes in overlapping domains along maxillary and mandibular processes, a homeobox odontogenic code has been proposed to explain the interaction between different homeobox genes during dental lamina patterning. No role has so far been assigned to the Hox gene network in the homeobox odontogenic code due to studies on specific Hox genes and evolutionary considerations. Despite its involvement in early patterning during embryonal development, the HOX gene network, the most repeat-poor regions of the human genome, controls the phenotype identity of adult eukaryotic cells. Here, according to our results, the HOX gene network appears to be active in human tooth germs between 18 and 24 weeks of development. The immunohistochemical localization of specific HOX proteins mostly concerns the epithelial tooth germ compartment. Furthermore, only a few genes of the network are active in embryonal retromolar tissues, as well as in ectomesenchymal dental pulp cells (DPC) grown in vitro from adult human molar. Exposure of DPCs to cAMP induces the expression of from three to nine total HOX genes of the network in parallel with phenotype modifications with traits of neuronal differentiation. Our observations suggest that: (i) by combining its component genes, the HOX gene network determines the phenotype identity of epithelial and ectomesenchymal cells interacting in the generation of human tooth germ; (ii) cAMP treatment activates the HOX network and induces, in parallel, a neuronal-like phenotype in human primary ectomesenchymal dental pulp cells., (2005 Wiley-Liss, Inc.)

We investigated the metabolic defect(s) of four children who presented with isolated cryptogenic chronic liver disease, coagulopathy, and abnormalities of several unrelated serum glycoproteins. Analysis of the patients' serum glycoproteins and fibroblasts suggest they have a novel congenital disorder of glycosylation (CDG). All had abnormal transferrin (Tf) isoelectric focusing (IEF) profiles. More detailed analysis of Tf by electrospray ionization mass spectrometry (ESI-MS) showed a plethora of abnormal glycosylations that included loss of 1-2 sialic acids and 1-2 galactose units, typical of Group II defects. Tf from two patients also lacked 1-2 entire oligosaccharide chains, typical of Group One disorders. Total serum N-glycans were analyzed by HPLC and matrix-assisted laser desorption/ionization mass spectrometry and also showed increased proportion of neutral glycan chains lacking sialic acids and galactose units. Analysis of patient fibroblasts eliminated CDG-Ia, through CDG-Ih, -IL and CDG-IId. Our results suggest that a subset of children with clinically asymptomatic, cryptogenic hypertransaminasemia and/or liver steato-fibrosis may represent a novel type of CDG-X with an unknown defect(s). Clinicians are encouraged to test such patients for abnormal Tf glycosylation by ESI-MS.

Inborn defects of cholesterol biosynthesis are a group of metabolic disorders presenting with mental retardation and multiple congenital anomalies (MCA/MR syndromes). Functional and structural liver involvement has been reported as a rare (2.5-6%) complication of the Smith-Lemli-Opitz syndrome (SLOS) and it has not been fully characterized. Here, we report on a long-term follow-up study of four patients with SLOS, and one case with lathosterolosis who presented with liver disease and underwent an extensive diagnostic work-up. Reports of liver involvement in cholesterol biosynthesis defects are reviewed. Two main different patterns of liver involvement emerged: progressive cholestasis, and stable isolated hypertransaminasemia. In our series, the first pattern was found in two patients with SLOS and one with lathosterolosis, and the second in two SLOS cases. Cholestasis was associated with early lethality and normal serum gamma-glutamyl-transferase (GGT) levels in SLOS, while possible prolonged survival and high GGT levels were seen in lathosterolosis. Hepatic fibrosis was present in both conditions. Liver biopsy performed in one of our SLOS patients with isolated hypertransaminasemia, showed only mild hydropic degeneration of the hepatocytes. The presence of liver involvement in 16% of the SLOS patients diagnosed at our Center suggests that this complication might have been underestimated in previously reported cases, possibly overshadowed by the severity of multiple malformations. Fetal hepatopathy, cholestasis, and isolated hypertransaminasemia can occur also in other disorders of cholesterol biosynthesis, such as mevalonic aciduria, desmosterolosis, Conradi-Hunermann syndrome, Greenberg dysplasia, and Pelger-Huet homozygosity syndrome. This group of inherited disorders should be considered in the differential diagnosis of patients presenting with liver disease associated with developmental delay and/or multiple malformations. Periodic liver function evaluations are recommended in these patients.

Background/aims: We studied the prevalence of non-alcoholic fatty liver disease and non-alcoholic-steatohepatitis in patients with metabolic-syndrome but normal liver enzymes. The histological findings of patients with normal liver enzymes and non-alcoholic-steatohepatitis were compared with those of a control-group with persistently abnormal liver enzymes., Methods: Patients presenting with normal liver enzymes were enrolled in the study and underwent liver biopsy. Prevalence of non-alcoholic-steatohepatitis and risk factors for fibrosis and cirrhosis were evaluated. Data from a control-group with non-alcoholic-steatohepatitis and abnormal liver enzymes were used to compare the histological findings., Results: Fifty-eight of the 80 patients enrolled had varying degrees of non-alcoholic steatohepatitis, of these 26 had fibrosis and 8 silent cirrhosis. The association of metabolic-syndrome, female-sex, a long-history of obesity and body mass index>45 were considered to be independent risk-factors for fibrosis. Comparing the histological findings of cases and controls we found a similar severity of steatosis and fibrosis, with a greater prevalence of ballooning degeneration and glycogenated-nuclei rather than lobular-inflammation., Conclusions: In the subjects selected according to our criteria, liver enzyme levels could not be used as surrogate markers of non-alcoholic-steatohepatitis. Histological hallmarks of patients with metabolic-syndrome, normal liver enzymes and non-alcoholic-steatohepatitis consist to a lesser degree of lobular-inflammation and a more severe ballooning and glycogenated-nuclei.

Hepatoid adenocarcinoma (HAC) is a special type of extrahepatic adenocarcinoma, which has a striking morphologic similarity to hepatocellular carcinoma. Seven HACs arising in the stomach and one in the lung, all with liver metastasis, were studied. They shared clinical features, such as old age, high serum alpha-fetoprotein level, aggressive behavior, and hepatic tumor in absence of risk factors for hepatocellular carcinoma (HCC). Morphologically, tumors were characterized by an admixture of tubulo-and/or papillary adenocarcinoma with hepatoid foci. In six cases, liver metastases showed an exclusive hepatoid differentiation, virtually indistinguishable from HCC with solid growth pattern. As HAC and HCC cannot be differentiated on the basis of morphology alone, differences in immunohistochemical reaction patterns would be of considerable diagnostic help. Immunostaining for CK7, CK8, CK18, CK19, CK20, alpha-fetoprotein, p-CEA, and HepPar1 revealed that hepatoid areas of both primary and metastatic HAC have a specific immunoprofile, distinctive of this entity. On the one hand, positivity of virtually all HACs for alpha-fetoprotein, CK8, CK18, and the membranous, canalicular staining for polyclonal carcinoembryonic antigen underline its hepatoid nature. On the other hand, positive staining for CK19 and CK20 and frequent negativity for HepPar1 in both primary tumors and their metastases were distinctive features of HAC. Furthermore, HAC differs from combined hepatocellular cholangiocarcinoma, being negative for CK7. In addition, for comparison of immunohistochemical results, we stained with the same antibody panel a tissue microarray of 121 HCCs. Comparative genomic hybridization study of three HAC supports their hepatoid differentiation as aberrations found in HAC are common in HCC (4q-, 8p-), and hepatoblastoma (Xq+), respectively.

Data from experimental hepatocarcinogenesis and recent studies in humans have suggested that the emergence of hepatocellular carcinoma (HCC) is a stepwise process. However, despite abundant experimental data, the precise molecular mechanisms and genetic alterations involved in human liver carcinogenesis are still unclear. Comparative genomic hybridization was used to analyze 26 hepatocellular nodules obtained from patients undergoing liver transplantation or surgical resection for HCC. According to the criteria proposed by the International Working Party, 16 nodules were classified as multiacinar regenerative nodules (MRN), 4 as low-grade dysplastic nodules (LG-DN), and 6 as high-grade dysplastic nodules (HG-DN). Our aim was to investigate the possible genetic differences between MRN, LG-DN, and HG-DN. The whole group of nodules showed only a few aberrations (mean 1.1/case), without any significant pattern. This finding is comparable to what happens in non-neoplastic tissue. On the contrary, in three of six HG-DN, we found deletions of 8p and gains of 1q. LG-DN and MRN did not show these chromosomal imbalances. These results confirm the important role of allelic losses on 8p as well as of gains of 1q in HCC. We conclude that the genes that are important in early stages of hepatocarcinogenesis are probably located on these chromosomal arms.

Background/aims: Studies on non-alcoholic fatty liver disease (NAFLD) have included chronic liver damage attributed to various causes. Our investigation was held to observe the main clinical, histological, and pathophysiological aspects of NAFLD in patients not exposed to any known cause of chronic liver disease., Methods: We evaluated, in 84 in-patients (male/female, 66/18; median age, 36 years), the clinical and biochemical characteristics of NAFLD, and particularly its association with diabetes, dyslipidemia, hyperinsulinemia and/or with the increase of parameters of oxidative stress (blood levels of malonyldialdehyde, 4-hydroxynonenal and total plasma antioxidant capacity)., Results: Ninety percent of patients had an increased body mass index (BMI), 35% had dyslipidemia, 40% had sub-clinical diabetes (only 3% had overt diabetes), 60% had hyperinsulinemia, and more than 90% had enhanced levels of lipid peroxidation markers. In 48 patients who had consented to liver biopsy, we found: 14 with simple steatosis, 32 with steatohepatitis, and two with cirrhosis., Conclusions: Our data indicate that in our country, NAFLD may occur in young males with an increased BMI, with or without hyperinsulinemia, dyslipidemia and diabetes, generally associated with disorders of redox status, and that it may be differentiated from steatosis to steatohepatitis or cirrhosis only with a liver biopsy.

Hepatocellular carcinoma (HCC) is one of the most common neoplasms worldwide. Well-established risk factors include infections with two very different viruses: the DNA virus causing hepatitis B (HBV) and the RNA virus inducing hepatitis C (HCV). In order to determine whether genetic differences exist between HBV- and HCV-induced HCC, 41 HCC samples of known vival status were examined by comparative genomic hybridization (CGH). The analysis revealed frequent deletions of 1p (24%), 4q (39%), 6q (41%), 8p (44%), 9p (24%), 11q (24%), 12q (22%), and 13q (39%), as well as common gains of 1q (46%), 6p+ (20%), 8q+ (41%), 11q (27%), and 17q+ (37%). There was no significant difference in the number and type of chromosomal imbalances between 25 HCV- and 16 HBV-infected tumours. This is consistent with models suggesting that HBV and HCV cause cancer through non-specific inflammatory and regenerative processes, rather than through virus-specific interactions with defined target genes. Chromosomal imbalances were also unrelated to the grade and stage of HCC. This may suggest that most gross genomic alterations occur early during HCC development and that further progression of these tumours may be associated with other types of genetic changes, not detectable by CGH. In summary, these data show that characteristic gross genomic changes occur in HCC, but these alterations at present do not appear to have diagnostic or prognostic applications., (Copyright 2000 John Wiley & Sons, Ltd.)

Objectives: The aim of this study was to define the features of chronic cryptogenic hepatitis (CCH) in childhood and to investigate whether it is related to hepatitis G virus infection., Methods: Forty-six children (24 males; age range, 1.5 to 17 years) with CCH were studied. CCH was diagnosed when serum alanine aminotransferase concentrations were more than 1.5 times normal for longer than 6 months without any apparent cause of liver disease., Results: No patient had acute symptomatic onset or had received a blood transfusion. Three had undergone minor surgical procedures. All appeared to be healthy during follow-up (median, 4.2 years; range, 1 to 10 years). Hypertransaminasemia was the only aberrant liver function test. Elevated serum alanine aminotransferase values alternated with normal values in 40 children (86.9%). Five children (10.8%) had a spontaneous sustained (>12 months) remission of hypertransaminasemia. Twelve (26%) had laboratory signs of autoimmunity, but none fulfilled the criteria for autoimmune hepatitis. Of 20 children who underwent liver biopsy, 13 (65%) had minimal chronic hepatitis, 4 (20%) had mild chronic hepatitis and 3 (15%) had moderate chronic hepatitis. Serum hepatitis G virus RNA was detected in 2 girls (4%) whose risk factor was a hepatitis G virus-infected mother and a minor surgical procedure, respectively. In 12 families at least 1 other member had chronic liver disease., Conclusions: Childhood CCH seems to be a symptomless disease characterized by isolated hypertransaminasemia with onset during the first 4 years of life and mild to moderate histologic liver lesions. Although the frequency of spontaneous remissions is low, childhood CCH seems, in the short run, to be a nonprogressive disease. Hepatitis G virus does not play a major role in CCH.

The analysis of the literature on limb body wall complex reveals a varied and rather confused spectrum of cases. However, we noticed the presence of at least 2 clearly distinguishable phenotypes. The first phenotype shows craniofacial defects and amniotic bands and/or adhesion; the second--without craniofacial defects--presents urogenital anomalies, anal atresia, and abdominal placental attachment, together with a persistence of the extra-embryonic coelom. We think these 2 phenotypes are the consequence of different pathogenetic mechanisms. The pathogenesis of the first type can be related to an early vascular disruption, while the pathogenesis of the second one is attributable to an intrinsic embryonal maldevelopment. Eight cases of the second phenotype were identified and the pathological findings proving this maldevelopmental origin are described.

Six children in whom long-standing hypertransaminasemia of unknown cause led to an initial diagnosis of chronic or protracted cryptogenic hepatitis were found to have asymptomatic celiac disease. Administration of a gluten-free diet caused a prompt improvement of both hepatic and intestinal biochemical/histologic abnormalities. Hepatic damage may be another "atypical" form of celiac disease in children.

Background: Structural hemoglobinopathies usually are inherited as autosomic dominant traits; de novo mutations are uncommon. Analytical and preparative procedures for the characterization of an abnormal hemoglobin are complex and time-consuming. Mass spectrometer analysis allows a rapid identification of the amino acid substitution., Methods and Results: A cyanotic 7-year-old girl was found to have 16% methemoglobin. Laboratory data showed the presence of an abnormal hemoglobin, which was isolated by collecting the abnormal peak from DEAE and globin chains from CM52. The amino acid substitution was rapidly identified by FAB mass spectroscopic analysis, leading to the recognition of HbM Hyde Park. These data were confirmed by molecular analysis (Southern blot and DNA sequencing). Neither the parents nor a sister showed any abnormality; non-paternity was excluded by blood group serology and HLA typing., Conclusions: This is a case of HbM Hyde-Park arising as a de novo mutation. FAB mass spectroscopic analysis is a rapid and useful analytical method for identifying aminoacid substitution.

We describe the first case of renal tubular dysgenesis in a newborn female who survived 15 days. The immunohistochemical and lectin binding studies confirmed the lack of proximal tubule differentiation. Electron microscopy showed undifferentiated tubular epithelium. Widely patent cranial fontanels were present in the proposita. The father and sister showed acrocephalosyndactyly Saethre-Chotzen type. The parents were not related and there was no familial history of renal pathology.

Following previous trials about hypo- and hyper-protein diets and their negative effects on some ocular structures when administered in selected stages of rats' growth, Authors have presented a comparative study of retinal alterations between light (LM) and electron (EM) microscopic analysis. LM showed a vacuolar degeneration of retinal optic fiber and ganglion cell layers where EM, performed at the same time, showed damages of the nervous tissue consisting of microtubule diminution, optic fiber molding and glial cell proliferation.

We report a case of a 19-month-old infant with congenital hepatic fibrosis. The clinical features consisted of portal hypertension with massive gastrointestinal bleeding, recurrent cholangitis, and cystic dysplasia of the kidneys, without impairment of renal function. The dramatic course of the disease required surgical treatment. Congenital hepatic fibrosis with life-threatening gastrointestinal bleeding is extremely rare below three years of age. The purpose of the present report is to stress the need of looking for varices in all cases of congenital hepatic fibrosis, even when occurring in early infancy.

The authors studied the distribution and the composition of collagen in neoplastic tissue from mastectomy specimens of 15 patients treated for an infiltrating ductal carcinoma NOS, in comparison to normal tissue of the opposite quadrant. The study was performed by light microscopy, microspectrophotometry, and electrophoretic methods. The results demonstrate a characteristic stromal organization of breast cancer in relation to collagen type I content.