Filippo Oliveri

Skeletal Muscle-Specific Ablation of raptor, but Not of rictor, Causes Metabolic Changes and Results in Muscle Dystrophy

Mammalian target of rapamycin (mTOR) is a central controller of cell growth. mTOR assembles into two distinct multiprotein complexes called mTOR complex 1 (mTORC1) and mTORC2. Here we show that the mTORC1 component raptor is critical for muscle function and prolonged survival. In contrast, muscles lacking the mTORC2 component rictor are indistinguishable from wild-type controls. Raptor-deficient muscles become progressively dystrophic, are impaired in their oxidative capacity, and contain increased glycogen stores, but they express structural components indicative of oxidative muscle fibers. Biochemical analysis indicates that these changes are probably due to loss of activation of direct downstream targets of mTORC1, downregulation of genes involved in mitochondrial biogenesis, including PGC1alpha, and hyperactivation of PKB/Akt. Finally, we show that activation of PKB/Akt does not require mTORC2. Together, these results demonstrate that muscle mTORC1 has an unexpected role in the regulation of the metabolic properties and that its function is essential for life.

Outcome of anti-HBe positive chronic hepatitis B in alpha-interferon treated and untreated patients: a long term cohort study.

BACKGROUND/AIMS We studied the influence of biochemical and virologic patterns and interferon on the outcome of anti-HBe positive chronic hepatitis B in 164 (103 treated) consecutive patients, followed-up prospectively for a mean of 6 years (21 months-12 years). METHODS Histology, biochemical and virologic profiles were characterized by monthly monitoring during the first 12 months of follow-up. Thereafter patients underwent blood and clinical controls every 4 and 6 months, respectively. Cirrhosis at follow-up histology or end stage complications of cirrhosis served as end points for the analysis of factors influencing disease progression in patients with baseline chronic hepatitis or cirrhosis, respectively. RESULTS Disease progression was associated with older age (P<0.001), absence of previous HBeAg history (P=0.017) and higher serum HBV-DNA levels (P=0.009) (more frequently observed in unremitting disease profile, P=0.012) at multivariate analysis. Fluctuations of IgM anti-HBc levels (associated with disease exacerbations, P=0.045) correlated with end stage complications in cirrhotics (P=0.011). Disease improved in 14.6 and 1.6% of treated and untreated patients, respectively (P=0.015): interferon slowed disease progression (P<0.001). CONCLUSIONS The outcome of anti-HBe positive chronic hepatitis B is worsened by older age and persistent viral replication or hepatitis exacerbations in chronic hepatitis or in cirrhotic patients, respectively. Interferon reduces by 2.5-folds disease progression.

Hepatitis B surface antigen serum levels help to distinguish active from inactive hepatitis B virus genotype D carriers.

BACKGROUND & AIMS The accurate identification of inactive (serum HBV-DNA persistently <or=2000 IU/mL) hepatitis B virus (HBV) carriers (IC) is difficult because of wide and frequent HBV-DNA fluctuations. We studied whether hepatitis B surface antigen (HBsAg) serum levels (HBsAgsl) quantification may contribute to diagnosis of HBV phases in untreated hepatitis B e antigen-negative genotype D asymptomatic carriers. METHODS HBsAgsl were measured at baseline and end of follow-up and correlated with virologic and biochemical profiles of 209 consecutive carriers followed-up prospectively (median, 29; range, 12-110 months). HBV phases were defined after 1-year monthly monitoring of HBV-DNA and transaminases. RESULTS HBsAgsl were significantly lower in 56 inactive carriers (IC) than 153 active carriers (AC): median, 62.12 (range, 0.1-4068) vs median, 3029 (range, 0.5-82,480) IU/mL; P<.001. Among AC, HBsAgsl were lower in 31 AC whose viremia remained persistently <20,000 IU/mL (AC1) than in 122 AC with fluctuations>or=20,000 IU/mL (AC2): 883 (0.5-7838) vs 4233 (164-82,480) IU/mL, P=.002. HBV infection was less productive in IC and AC1 than AC2 (log10 HBV-DNA/HBsAgsl ratios 0.25 and 0.49 vs 2.06, respectively, P<.001) and in chronic hepatitis than cirrhosis (1.97 vs 2.34, respectively; P=.023). The combined single point quantification of HBsAg (<1000 IU/mL) and HBV-DNA (<or=2000 IU/mL) identified IC with 94.3% diagnostic accuracy, 91.1% sensitivity, 95.4% specificity, 87.9% positive predictive value, 96.7% negative predictive value. During follow-up, HBsAgsl were stable in AC but declined in IC (yearly median decline, -0.0120 vs -0.0768 log10 IU/mL, respectively, P<.001), 10 of whom cleared HBsAg. CONCLUSIONS HBsAgsl vary during chronic hepatitis B e antigen-negative genotype D infection and are significantly lower in IC. Single-point combined HBsAg and HBV-DNA quantification provides the most accurate identification of IC, comparable with that of long-term tight monitoring.

A new hepatitis B virus strain in patients with severe anti-HBe positive chronic hepatitis B

In hepatitis B virus carriers who are anti-HBe positive despite ongoing viral replication (HBcAg in liver and HBV-DNA in serum) the natural course of hepatitis is severe and the response to interferon is low. We investigated whether a new hepatitis B virus (HBV) strain could be involved. A translational termination codon at the carboxyterminal end of the pre-C region responsible for the lack of HBeAg secretion was found in 18 of 19 HBV clones isolated from seven pedigreed patients with this clinical syndrome. The same findings were confirmed by direct sequencing. One of these patients underwent a liver transplant and HBV infection of the new liver resulted in high titered viremia and intrahepatic expression of HBcAg, without detectable HBeAg in serum. Another patient was superinfected by hepatitis delta virus (HDV) and developed high titres of total and IgM anti-HD. In spite of this, chronic hepatitis remained unchanged during 7 years of follow-up. These data strongly suggest that a viable precore minus mutant of hepatitis B virus is responsible for the lack of HBeAg in the serum of these patients. The HBV variant may explain the peculiar geographic distribution of anti-HBe positive hepatitis. The variations in the virus genome sequence may cause the more severe form of liver disease and modify the pathogenicity in the case of HDV superinfection.

Hepatitis B virus unable to secrete e antigen and response to interferon in chronic hepatitis B

BACKGROUND Anti-hepatitis e antigen-positive chronic hepatitis B is a progressive liver disease associated with precore mutant hepatitis B virus (HBV) and poor response to interferon. Therefore, precore mutant HBV may behave as an interferon-resistant virus. The relations between the prevalences of wild-type and precore mutant HBVs in baseline viremias and response to interferon were analyzed. METHODS Sera from 115 patients (59 treated and 56 untreated, followed up for 30 months) were tested using a quantitative oligonucleotide hybridization assay. RESULTS Spontaneous or interferon-induced recoveries were observed in 28.5% (6 of 21) and 47.3% (18 of 38) or in 0% (0 of 35) and 19% (4 of 21) of the patients with wild-type prevalent or mutant prevalent HBVs, respectively. Relapses occurred in 85.7% (12 of 14) and 19.4% (4 of 21) of treated patients with prevalent precore mutant and prevalent wild-type HBV, respectively (P = 0.0001). High precore mutant HBV levels (> 20% of total viremia) were associated with the lack of permanent response to interferon (P = 0.01). CONCLUSIONS Precore mutant HBV can influence the response to interferon when it reaches significant serum levels (> 20% of total viremia). Therefore, chronic hepatitis B should be treated as early as possible in its natural history before precore mutant HBV is selected as a prevalent virus.

Ablation of the mTORC2 component rictor in brain or Purkinje cells affects size and neuron morphology

The mTOR complex 2 (mTORC2) is essential in the central nervous system for normal neuronal structure and function, potentially through effects on PKC signaling and independent of the related mTOR complex 1 (mTORC1).

Doxorubicin-eluting bead-enhanced radiofrequency ablation of hepatocellular carcinoma: a pilot clinical study

BACKGROUND/AIMS Experimental studies have shown synergy between radiofrequency (RF) ablation and adjuvant chemotherapy in animal tumour models. We aimed to assess safety and efficacy of doxorubicin-eluting bead (DEB)-enhanced RF ablation in the treatment of human hepatocellular carcinoma (HCC). METHODS Twenty patients with single HCC ranging 3.3-7.0 cm (mean, 5.0 cm+/-1.4) showing evidence of residual viable tumour after standard RF ablation underwent intraarterial DEB administration (50-125 mg doxorubicin; mean, 60.2 mg+/-21.8). Follow-up period ranged 6-20 months (mean, 12 months+/-5). RESULTS No major complication occurred. No deterioration of liver function was observed. The volume of treatment-induced necrosis--as measured on imaging--increased from 48.1 cm3+/-35.7 after RF ablation to 75.5 cm3+/-52.4 after DEB administration, with an increase of 60.9%+/-39.0. The enhanced effect resulted in confirmed complete response (CR) of the target lesion in 12 (60%) of 20 patients. Incomplete response with persistence of <10% of initial tumour volume was observed in 6 (30%) of 20 patients, and local tumour progression in 2 (10%) of 20. CONCLUSIONS Intraarterial DEB administration substantially enhances the effect of RF ablation. DEB-enhanced RF ablation is safe and results in a high rate of CR in patients refractory to standard RF treatment.

Monitoring the natural course and response to therapy of chronic hepatitis B with an automated semi-quantitative assay for IgM anti-HBc.

The clinical significance of a semi-quantitative microparticle enzyme immunoassay (IMx Core-M, Abbott) was evaluated for detection of IgM-class antibodies against the hepatitis B core antigen (IgM anti-HBc) in 136 hepatitis B surface antigen (HBsAg) positive individuals (96 chronic HBV carriers, 20 patients with chronic HBV-HDV infections and 20 patients with acute hepatitis B) and 50 HBV-negative controls. Baseline and follow-up sera (4-11 samples) were analysed from 79 carriers with chronic hepatitis B, 44 of whom were treated with interferon. IMx indexes above 3,000 were found in 95% of the acute hepatitis B patients and above 0.300 in 91.5% of patients with ongoing chronic hepatitis B. IMx indexes between 0.200 and 0.300 were observed in (a) patients with recent HBeAg to anti-HBe seronconversion (6-12 months) and normal serum ALT levels, (b) patients immuno-tolerant to HBV infection and without liver disease despite high levels of viremia, and (c) patients with anti-HBe-positive chronic hepatitis B during 7-13-month intervals of asymptomatic carriage between episodes of disease reactivation. IMx indexes below 0.200 were detected in all HBV-negative individuals and healthy HBV carriers, in 14 (70%) of 20 chronic hepatitis D patients and in all but 1 of 22 interferon-treated patients with histological remission of liver disease, 5-12 months after clearance of viremia and normalization of serum ALT levels. In contrast, IMx indexes remained above 0.200 in all patients with hepatitis B reactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Long term response to therapy of chronic anti-HBe-positive hepatitis B is poor independent of type and schedule of interferon.

Objective:The response rate to alpha interferon (IFN) of chronic anti-HBe–positive hepatitis B is variable. We studied whether type, dose, and schedule of IFN, and type and frequency of posttreatment monitoring, influence the response rate.Methods:Seventy-two consecutive anti-HBe–positive chronic hepatitis B patients (59 male and 13 female, median age 41 yr) stratified by sex and histology were randomly allocated to three treatment arms. Twenty-seven patients (A) received 10 million units alpha-N1 IFN i.m. t.w. for 24 wk (total dose: 720 million units); 21 (B) received 9 million units alpha-2a IFN i.m. t.w. for 4 wk, followed by 18 million units for 12 wk and 9 million units for 8 wk (972 million units); 24 (C) received 2 alpha-2a IFN courses (9 million units i.m. t.w. for 16 and 12 wk separated by a 6-month interval [756 million units]). Primary response was defined by normal ALT and serum HBV-DNA levels below 10 pg/ml at the end of therapy and sustained response by normal ALT (tested monthly), undetectable HBV-DNA and IgM anti-HBc (<7 I.U. Paul Ehrlich Institute) (tested every 3 months) during the posttreatment follow-up.Results:At the end of treatment, 12, 8, and 13 patients from groups A, B, and C, respectively, were responders. At the 18-month follow-up, two patients in group A and only one in groups B and C maintained the response. Overall, after 34 months (median posttreatment follow-up), three patients were long term responders, whereas three showed a sustained remission after relapse.Conclusions:The rate of long term response to interferon of anti-HBe–positive chronic hepatitis B is poor, independent of IFN type, dose, or schedule; the more stringent the monitoring, the higher the relapse rate.

Muscle-selective synaptic disassembly and reorganization in MuSK antibody positive MG mice

MuSK antibody seropositive (MuSK+) Myasthenia Gravis (MG) patients present a distinct selective fatigue, and sometimes atrophy, of bulbar, facial and neck muscles. Here, we study the mechanism underlying the focal muscle involvement in mice with MuSK+ experimental autoimmune MG (EAMG). 8 week-old female wildtype C57BL6 mice and transgenic mice, which express yellow fluorescence protein (YFP) in their motor neurons, were immunized with the extracellular domain of rat MuSK and compared with control mice. The soleus, EDL, sternomastoid, omohyoid, thoracic paraspinal and masseter muscles were examined for pre- and postsynaptic changes with whole mount immunostaining and confocal microscopy. Neuromuscular junction derangement was quantified and compared between muscles and correlated with transcript levels of MuSK and other postsynaptic genes. Correlating with the EAMG disease grade, the postsynaptic acetylcholine receptor (AChR) clusters were severely fragmented with a subsequent reduction also of the presynaptic nerve terminal area. Among the muscles analyzed, the thoracic paraspinal, sternomastoid and masseter muscles were more affected than the leg muscles. The masseter muscle was the most affected, leading to denervation and atrophy and this severity correlated with the lowest levels of MuSK mRNA. On the contrary, the soleus with high MuSK mRNA levels had less postsynaptic perturbation and more terminal nerve sprouting. We propose that low muscle-intrinsic MuSK levels render some muscles, such as the masseter, more vulnerable to the postsynaptic perturbation of MuSK antibodies with subsequent denervation and atrophy. These findings augment our understanding of the sometimes severe, facio-bulbar phenotype of MuSK+ MG.