Active targeting of cancer cells by CD44 binding peptide-functionalized oil core-based nanocapsules.

Selectivity in tumor targeting is one of the major issues in cancer treatment. Therefore, surface functionalization of drug delivery systems with active moieties, able to selectively target tumors, has become a worldwide-recognized strategy. The CD44 receptor is largely used as a biomarker, being overexpressed in several tumors, and consequently as a target thanks to the identification of the CD44 binding peptide. Here we implemented the CD44 binding peptide logic onto an oil core-polymer multilayer shell, taking into account and optimizing all relevant features of drug delivery systems, such as small size (down to 100 nm), narrow size distribution, drug loading capability, antifouling and biodegradability. Besides promoting active targeting, the oil core-based system enables the delivery of natural and synthetic therapeutic compounds. Biological tests, using curcumin as a bioactive compound and fluorescent tag, demonstrated that CD44 binding peptide-functionalized nanocapsules selectively accumulate and internalize in cancer cells, compared to the control, thanks to ligand-receptor binding.

Tunable stability of monodisperse secondary O/W nano-emulsions.

Stable and biodegradable oil in water (O/W) nano-emulsions can have a huge impact on a wide range of bio-applications, from food to cosmetics and pharmaceuticals. Emulsions, however, are immiscible systems unstable over time; polymer coatings are known to be helpful, but an effective procedure to stabilize monodisperse and biodegradable O/W nano-emulsions is yet to be designed. Here, we coat biodegradable O/W nano-emulsions with a molecular layer of biodegradable polyelectrolytes such as polysaccharides--like chitosan--and polypeptides--like polylysine--and effectively re-disperse and densify the polymer coating at high pressure, thus obtaining monodisperse and stable systems. In particular, focusing on chitosan, our tests show that it is possible to obtain unprecedented ultra-stable O/W secondary nano-emulsions (diameter sizes tunable from ∼ 80 to 160 nm and polydispersion indices below 0.1) by combining this process with high concentrations of polymers. Depending on the polymer concentration, it is possible to control the level of coating that results in a tunable stability ranging from a few weeks to several months. The above range of concentrations has been investigated using a fluorescence-based approach with new insights into the coating evolution.

Herbal hepatotoxicity: a case of difficult interpretation.

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.

Multi Tbit/in(2) storage densities with thermomechanical probes.

Exploiting the spatial resolution of scanning probes presents an attractive approach for novel data storage technologies in particular for large-scale data repositories because of their inherent potential for high storage density. We show that multi-Tbit/in(2) density can be achieved by means of thermomechanically embossing the information as indentation marks into a polymer film. The data density is determined by the nonlinear interaction between closely spaced indents and the fundamental scaling relations governing the shape and size of the indents. We find that cooperative effects in polymers give rise to a minimum indentation radius on the order of the correlation length of the cooperatively rearranged region even if formed by an infinitely sharp indenter. Thus, cooperativity coupled to alpha-transitions in polymers is evinced in a real space geometrical experiment. Furthermore, we predict that indentation marks cannot be made smaller than 5 nm in diameter, which limits the feature resolution for embossing technologies in general.

Steatohepatitis is associated with diabetes and fibrosis in genotype 1b HCV-related chronic liver disease.

Liver steatosis, diabetes mellitus and hepatitis C virus (HCV) genotype have been implicated in liver fibrosis in HCV-related chronic active hepatitis (CAH). The aim of this study was to evaluate whether steatosis and diabetes were associated with more severe liver fibrosis in patients with genotype 1b HCV-related CAH. One-hundred and eighty patients (98 men, 82 women; age range 17-68 years; median 51) infected with genotype 1b HCV underwent ultrasound examination and liver biopsy because of elevated levels of serum alanine transaminase. Based on liver histology, patients were divided into three steatosis classes: 1 (involving <33% of hepatocytes), 2 (34-66%) and 3 (>66%). Fibrosis was graded with the Ishak score (range: 0-6). Virological and epidemiologic characteristics, biochemical data, body mass index, and apparent duration of disease were recorded. Diabetes was identified according to American Diabetes Association criteria. The median fibrosis grade was 2 (23 patients had liver cirrhosis) in the three steatosis classes, with no significant differences between classes. At multivariate analysis, fibrosis was significantly related to age, alanine transaminase, diabetes, hepatitis B core antibody, steatohepatitis and grading. At binary logistic regression analysis, only diabetes and fibrosis stage were significantly associated with steatohepatitis. Steatosis was not an independent risk factor for liver disease severity in our CAH/genotype 1b HCV-infected patients. Steatohepatitis was associated as well as diabetes and affected the severity of liver fibrosis.

Essential hypertension and chronic viral hepatitis.

OBJECTIVE: Both arterial hypertension and chronic hepatitis are common disorders. The relationship between arterial pressure and liver cirrhosis has been extensively studied, but no studies are available in chronic hepatitis (CH). Recently, a few studies have reported that treatment with angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin-receptor blockers (ARBs), commonly used in arterial hypertension, reduce hepatic fibrosis in patients with viral CH and in nonalcoholic steatohepatitis. This study was aimed at comparing the evolution of post-viral CH in patients with/without concomitant essential hypertension. METHODS: Two sets of observations were carried out: (a) a cross-sectional cohort study of 95 patients with viral CH, to compare the severity of histological and biochemical data at diagnosis, in relation to pharmacologically treated essential hypertension, and (b) a retrospective study with the observation of 254 patients with CH of viral etiology, followed up from 2 to 20 years, to establish the natural history of viral CH in relation to treated essential hypertension. RESULTS: In the cross-sectional analysis, patients with treated hypertension had a significantly older age at diagnosis of CH (51.4 +/- 8.4 years vs. 46.2 +/- 12.2 in normotensive; P < 0.001) and histological evidence of less severe necro-inflammatory liver damage. ALT levels were also lower (109.8 +/- 62.5 U/L vs. 166.0+/-169.5 in normotensive; P < 0.001) as were endothelin-1 levels (0.74 +/- 0.97 vs. 1.77 +/- 1.51 fmol/mL; P < 0.001). The retrospective study confirmed an older age at diagnosis in patients with treated hypertension (48.7 +/- 9.8 vs. 41.9 +/- 11.8 years; P < 0.001) and lower death rates (2.2% vs. 11%; P < 0.05). CONCLUSIONS: The evolution of post-viral CH seems to be less severe in subjects with essential hypertension, possibly in relation to treatment with antihypertensive drugs.

Serum concentrations of the tissue polypeptide specific antigen in patients suffering from non-alcoholic steatohepatitis.

BACKGROUND: Liver histology is the gold standard for diagnosis of non-alcoholic fatty liver disease. Ethical considerations and patient choice often preclude performing a liver biopsy, especially considering the rare but potential risk. Searching for a good serological marker substitute for the invasive procedure was the aim of our study. Keratins, mainly 8 and 18, play not only a mere structural role providing mechanical stability to hepatocytes, but also represent a target via toxic stress ultimately inducing apoptosis/necrosis. Tissue polypeptide-specific antigen (TPS), a serological mirror of keratin 18, is widely used as a marker for various cancers. This antigen was assessed in three different groups who were overweight or obese. MATERIALS AND METHODS: In this cross-sectional case-control study, 48 cancer-free patients with non-alcoholic steatohepatitis (NASH, Group 1), 48 patients with pure fatty liver (FL, Group 2), and 47 volunteers (Group 3) were studied. All of them were referred to our metabolic unit for routine evaluation. RESULTS: The median (range) TPS levels were 123 (56-286) ng mL(-1) in NASH patients. FL patients and volunteers had significantly lower TPS levels, 76 (38-98) ng mL(-1) and 64 (28-87) ng mL(-1), respectively (P = 0.0001). A value of 88 ng mL(-1) in patients with underlying bright liver was associated with a high probability of NASH (sensitivity and specificity = 92% and 96%, respectively). One patient (2.1%) with FL had a TPS value > 88 ng mL(-1), but in the same group, 29 FL patients (60.4%) had an alanine aminotransferase value > 40 U L(-1). Based on a recent classification of liver fibrosis, the median (range) TPS values were significantly different among the stages: F1 (n = 23) = 100 (76-264) ng mL(-1); F2 (n = 21) = 134 (56-276) ng mL(-1); and F3 (n = 4) = 199.5 (123-286) ng mL(-1), respectively (P = 0.014). CONCLUSIONS: Our study shows that TPS is a better marker than alanine aminotransferase activity, ultrasonography or the combination of both parameters in differentiating NASH from FL.

The role of bright liver echo pattern on ultrasound B-mode examination in the diagnosis of liver steatosis.

AIM: The observation of bright liver echo pattern on ultrasound is commonly considered a sign of hepatic steatosis. However, the interference of liver fibrosis on sensitivity and specificity of bright liver echo pattern has caused many to question its effectiveness as a diagnostic tool. The objective of this study was to evaluate the sensitivity, specificity and predictive values of bright liver echo pattern for liver steatosis. PATIENTS AND METHODS: We studied 235 consecutive patients suspected of having liver disease of various aetiologies. Median age was 52 years (range, 17-72 years), and there was a male/female ratio of 1:18. All patients underwent ultrasound examination before liver biopsy and was performed by two operators. The presence or absence of bright liver echo pattern and posterior attenuation or areas with different patterns of fat infiltration were noted. Histologic evaluation was performed and graded by Ishak score. Steatosis was categorised as absent, 0-2%, 3-29% to 30-49% or >50%. RESULTS: Interobserver concordance was high. Bright liver echo pattern was found in 67% of patients with steatosis of any degree and 89% of patients with steatosis of >or=30%. Only three patients without steatosis, who had a low Ishak score, demonstrated bright liver echo pattern on ultrasonography. The sensitivity, specificity, positive predictive value and negative predictive value of bright liver echo pattern for steatosis were 64%, 97%, 96.0% and 65%, respectively. Among the subgroup of patients who had steatosis of >or=30%, the sensitivity, specificity, positive predictive value and negative predictive value of bright liver echo pattern were 91%, 93%, 89% and 94%, respectively. The sensitivity, specificity, positive predictive value and negative predictive value of posterior attenuation and/or skip areas associated with bright liver echo pattern for steatosis were 89.7%, 100%, 100% and 92.3%, respectively. Univariate analysis showed bright liver echo pattern to be associated only with steatosis and not with fibrosis. CONCLUSION: We concluded that the presence of bright liver echo pattern is a sign of liver steatosis and that liver fibrosis does not interfere with ultrasound measurements. Posterior attenuation and/or skip areas are closely related to steatosis of >or=30%.

Hepatitis C virus carriers with persistently normal ALT levels: biological peculiarities and update of the natural history of liver disease at 10 years.

Some chronic hepatitis C (CHC) patients exhibit persistently normal alanine aminotransferase (ALT) levels (PNAL). Patients with PNAL experience significantly milder disease. In order to understand the differences between CHC patients with elevated ALT levels compared with those with PNAL better, we compared epidemiological, immunological and histological findings, in particular, the value of proliferating hepatocyte activity (PCNA) between the two groups of patients. We studied 40 chronic hepatitis C virus (HCV) carriers with increased ALT who underwent liver biopsy for histological diagnosis and determination of clinical prognosis, and 24 PNAL patients under follow-up for 10 years. Immunological response to different HCV genomic epitopes was tested in both the control group and in PNAL subjects. PCNA values from liver specimens of all patients as well as liver biopsies of PNAL patients at time points 0 and 5 years were calculated according to Hall et al.Age, sex and body mass index (BMI) were not significantly different between the two groups. The median liver histology stage was significantly higher in HCV carriers vs the PNAL group (2.5, range = 2-6 vs 1.5, range = 1-2; P < 0.01). Among PNAL patients, histological stage was not statistically different at the three time points considered. Interferon (IFN)-gamma production was comparable in the two groups. PCNA was significantly higher in the group with elevated ALT levels vs the PNAL group (8%, range = 4-15%vs 5% range = 3-8%; P < 0.05) and no statistically significant differences were found in PNAL patients at time points 0, 5 and 10 years. This study confirms that progression to cirrhosis is slow or absent in PNAL patients after 10 years of follow-up. Accordingly, the hepatic proliferative activity index is low and seems to be stable over time.

Life saving cyclophosphamide treatment in a girl with giant cell hepatitis and autoimmune haemolytic anaemia: case report and up-to-date on therapeutical options.

We report the case of a girl affected by giant cell hepatitis associated with autoimmune haemolytic anaemia. Both conditions were severe with a number of life-threatening episodes of liver failure and anaemia unresponsive to several immunosuppressant drugs but cyclophosphamide. After a low-dose long-term treatment with this drug the patient is stably well without any therapy. A review of therapeutical options in this condition is also presented.

Non-alcoholic fatty liver disease in an area of southern Italy: main clinical, histological, and pathophysiological aspects.

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.

A spectrum of histopathologic findings in autoimmune liver disease.

We retrospectively studied 42 liver biopsy specimens from 39 patients who met serologic and histologic criteria of autoimmune liver diseases. We found 10 cases of overlap syndrome (OLS), 10 autoimmune cholangitis (AIC), 10 primary biliary cirrhosis (PBC), and 9 autoimmune hepatitis (AIH) type 1. The following results were obtained: (1) Granulomas and biliary duct lesions were more prominent in PBC and AIC than in OLS and AIH. (2) Bile duct loss was not observed in AIH cases. (3) Features of hepatocellular damage such as piecemeal necrosis, spotty lobular necrosis, and confluent necrosis, were much more prevalent in OLS and AIH than in PBC and AIC. (4) HLA-DR antigen expression by hepatocytes was more frequent in AIH and OLS, whereas the expression of the same antigen by the bile duct epithelium was more frequent in PBC and AIC. We conclude there is a morphologic spectrum in autoimmune liver diseases, in which PBC forms one end of the spectrum, AIH the other, OLS the middle but closer clinically and histologically to AIH than to PBC, and AIC, which seems to be an antimitochondrial antibody-negative subtype of PBC.

Marked genetic similarities between hepatitis B virus-positive and hepatitis C virus-positive hepatocellular carcinomas.

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.

Chronic cryptogenic hepatitis in childhood is unrelated to hepatitis G virus.

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.

Laparoscopy-guided biopsy in diagnosis of liver disorders in children.

To determine the safety and advantages of laparoscopic liver biopsy in pediatric liver disorders, we reviewed the medical records of 80 children affected by liver disease of various etiologies who underwent this procedure from 1986 to 1996. The main indicators for laparoscopic biopsy were increased risk of bleeding (i.e., mild to moderate coagulation abnormalities in patients probably affected by cirrhosis) and/or previous poorly informative blind needle liver biopsy (65 cases), and the need for a large amount of liver tissue for biochemical assays (10 cases). After inspection of the liver surface, at least two core biopsies were performed using a Tru-cut needle. We encountered difficulties with the biopsy in only four cases, due to a hard consistency of the liver. Bleeding time from the liver orifice was greatly reduced by positioning a fibrin plug (50-120 s vs 5-10 s, on average). In 15 patients, a large excisional biopsy was also successfully performed. Our results confirm an important role for laparoscopy in the diagnosis of cirrhosis (30% of bioptic false negative diagnoses in this series) and show that in selected cases laparoscopy-guided needle or excisional biopsy is an easy, useful and safe alternative to percutaneous blind liver biopsy.

Prolonged and high dose recombinant interferon alpha-2b alone or after prednisone priming accelerates termination of active viral replication in children with chronic hepatitis B infection.

BACKGROUND: There is no generally accepted treatment for chronic hepatitis B (HB) infection in children. OBJECTIVES: To evaluate the efficacy of a prolonged course of high dose interferon alone or after prednisone priming in children with chronic HB infection. METHODS: The outcome of 31 children with HB e antigen (HBeAg)-positive chronic hepatitis who randomly received either no treatment (n = 9) or 10 million units of interferon alpha-2b/m2, alone (n = 13) or after prednisone priming (n = 9), three times weekly for 1 year was studied. RESULTS: One patient withdrew from treatment. By the end of the first year treatment induced a loss of HB virus DNA and HBeAg from serum in 10 of 21 patients (48%), and a loss of HB surface antigen (HBsAg) in 4 (19%). Alanine aminotransferase values became normal in one patient (4.8%). Response rates in the two groups of treated patients were similar. In controls only one patient lost HBeAg and HBV DNA (11%; P = 0.05), and none lost HBsAg or showed alanine aminotransferase normalization (P = 0.21 and 0.70, respectively). After a posttreatment 2-year follow-up there were still no differences in the response rates of the two treatments; of the 21 pooled treated patients, 61% lost HBeAg and DNA and 67% normalized alanine aminotransferase (vs. 33 and 44% of controls, respectively; P = 0.32 and 0.40). Reversion to HBeAg and HBV DNA negativity in treated patients occurred significantly earlier (P = 0.02 and 0.006, respectively) than in controls. No further patient lost HBsAg, but one reacquired HBsAg. Treated patients had posttreatment histologic scores better than controls (P = 0.03). CONCLUSIONS: Our medium term follow-up results indicate that a prolonged course of high dose interferon in children with chronic HB infection, regardless of prednisone priming, poorly affects response rates but significantly speeds termination of active viral replication.