Prime/Boost Immunization with DNA and Adenoviral Vectors Protects from Hepatitis D Virus (HDV) Infection after Simultaneous Infection with HDV and Woodchuck Hepatitis Virus

Hepatitis D virus (HDV) superinfection of hepatitis B virus (HBV) carriers causes severe liver disease and a high rate of chronicity. Therefore, a vaccine protecting HBV carriers from HDV superinfection is needed. To protect from HDV infection an induction of virus-specific T cells is required, as antibodies to the two proteins of HDV, p24 and p27, do not neutralize the HBV-derived envelope of HDV. In mice, HDV-specific CD8⁺ and CD4⁺ T cell responses were induced by a DNA vaccine expressing HDV p27. In subsequent experiments, seven naive woodchucks were immunized with a DNA prime and adenoviral boost regimen prior to simultaneous woodchuck hepatitis virus (WHV) and HDV infection. Five of seven HDV-immunized woodchucks were protected against HDV infection, while acute self-limiting WHV infection occurred as expected. The two animals with the breakthrough had a shorter HDV viremia than the unvaccinated controls. The DNA prime and adenoviral vector boost vaccination protected woodchucks against HDV infection in the setting of simultaneous infection with WHV and HDV. In future experiments, the efficacy of this protocol to protect from HDV infection in the setting of HDV superinfection will need to be proven.     

Improved glioblastoma treatment with Ad5/35 fiber chimeric conditionally replicating adenoviruses

Adenovirus type 5 (Ad5)-based vectors have been used in clinical trials for glioblastoma treatment, but the capacity of Ad5 to infect human glioma cells was questioned. Seeking to improve the adenovirus transduction, we tested four Ad5-based vectors differing only in their fiber gene on permanent and short-term cultures of glioblastoma cells. A wild-type fiber Ad5 vector (Ad5.Luc) was compared to an RGD integrin-binding motif-containing fiber adenovirus (AdlucRGD) and the two fiber chimeras Ad5/3 and Ad5/35, with vector binding redirected to the Ad3 or Ad35 receptor, respectively. Compared to Ad5, the transduction of the tested short-term glioblastoma cultures with the vector Ad5/35.Luc, AdlucRGD and Ad5/3.Luc was enhanced by approximately 72%, approximately 13% and approximately 2%, respectively. To limit adenovirus spread, we aimed to develop conditionally replicative Ad5/35 vectors by targeting the expression of the essential E1 and E4 genes; in addition, some vectors had the E1Delta24 deletion. We analyzed eleven promoters for their activity in glioblastoma cells and determined the specificity of eight replicative adenovirus vectors in vitro. We evaluated the most promising vectors with E1/E4 under the control of the GFAP/Ki67 or E2F-1/COX-2 promoters, and the native Ad5 or the chimeric Ad5/35 fiber for their antineoplastic activity in a subcutaneous and intracranial glioblastoma xenograft model. Animals treated with the Ad5/35-based vectors showed significantly smaller tumors and longer survival than those treated with the homologous Ad5 vectors; no significant toxicity was observed in the intracranial model. Our data suggest that Ad5/35-based vectors are promising tools for glioblastoma treatment.     

Budgetary Impact of Treating Acute Promyelocytic Leukemia Patients with First-Line Arsenic Trioxide and Retinoic Acid from an Italian Payer Perspective

The objective of this study was to estimate the net cost of arsenic trioxide (ATO) added to all-trans retinoic acid (ATRA) compared to ATRA plus chemotherapy when used in first-line acute promyelocytic leukemia (APL) treatment for low to intermediate risk patients from the perspective of the overall Italian healthcare systemA Markov model was developed with 3 health states: stable disease, disease event and death. Each month, patients could move from stable to disease event or die from either state. After a disease event, patients discontinued initial treatment and switched to the other regimen as second-line therapy. Treatment regimens, efficacy and adverse events were derived from published sources and expert opinion; unit costs were collected from standard Italian sources. Clinical outcomes and costs for pre-ATO and post-ATO scenarios were combined with population and product utilization information to calculate the total budgetary impact using a 3-year time horizon; one-way sensitivity analyses were conducted. Three-year cumulative pharmacy costs for ATO+ATRA were €46,700 per-patient versus €6,500 for ATRA+chemotherapy; however, medical costs for ATO+ATRA were €12,300 per-patient versus €30,200 for ATRA+chemotherapy. The total budgetary impact was estimated to be an additional €127,300, €312,500 and €477,800 in the first, second and third years, respectively. The model was most sensitive to changes in the cost of the ATO+ATRA regimen during the consolidation phase. Budgetary impact models are valuable to payers making formulary decisions regarding the access and affordability of new medicines. The cost of treatment analysis showed that pharmacy costs for ATO+ATRA were higher than for ATRA+chemotherapy, while all other evaluated costs were lower for ATO+ATRA treated patients. The average budgetary impact was €305,900 per year overall, representing a 3.5% increase. Further research is needed to determine the cost-effectiveness of ATO+ATRA compared to the current first-line standard of care in APL.     

Adipokines promote lipotoxicity in human skeletal muscle cells

Studies have shown the implication of specific adipokines or fatty acids (FA) in the pathogenesis of insulin resistance. However, the interplay of adipokines with FA remains poorly understood. This study aimed to investigate the combined effects of adipokines and low concentrations of palmitic acid (PA, 100 μmol/l) on skeletal muscle metabolism. Human skeletal muscle cells were incubated with adipocyte-conditioned medium (CM), PA or PA+CM, and FA transporter and FA metabolism were analysed. CM-incubation increased CD36 level (1.8 fold) and PA-uptake (1.4 fold). However, only co-application of PA+CM resulted in profound lipid accumulation (5.3 fold), 60% reduction of PA-oxidation and 3.5 fold increased diacylglycerol content. Our results support a novel role for adipokines in the pathogenesis of T2D by increasing the lipotoxic potential of PA, notably of low concentrations. This implies an increased lipotoxic risk already at an early stage of weight gain, when lipolysis has not yet contributed to increased plasma free FA levels.     

Protein O-glycosylation analysis

Abstract This review provides an overview on the methods available for analysis of O-glycosylation. Three major themes are addressed: analysis of released O-glycans including different O-glycan liberation, derivatization, and detection methods; analysis of formerly O-glycosylated peptides yielding information on O-glycan attachment sites; analysis of O-glycopeptides, representing by far the most informative but also most challenging approach for O-glycan analysis. Although there are various techniques available for the identification of O-linked oligosaccharides, the focus here is on MS fragmentation techniques such as collision-induced fragmentation, electron capture dissociation, and electron transfer dissociation. Finally, the O-glycan analytical challenges that need to be met will be discussed.     

Sphingolipids: Critical players in Alzheimer's disease

Alzheimer's disease is characterized by the progressive accumulation of extracellular deposits of the amyloid β-peptide (Aβ) and intraneuronal aggregates of the microtubule associated protein tau. Strong genetic, biochemical and cell biological evidence indicates critical roles of Aβ in the initiation of the pathogenic process, while tau might mediate its toxicity and neurodegeneration. Aβ is generated by proteolytic processing of the amyloid precursor protein (APP) by β- and γ-secretases. Alternatively, APP can also be cleaved by α-secretase within the Aβ domain, thereby precluding subsequent production of Aβ. APP and the three secretases are integral membrane proteins and follow secretory and endocytic trafficking pathways. Thus, the membrane lipid composition could play important roles in trafficking and metabolism of Alzheimer's disease related proteins. Sphingolipids and especially complex gangliosides are abundant and characteristic components of neuronal membranes. Together with cholesterol, they confer unique characteristics to membrane domains, thereby regulating subcellular trafficking and signaling pathways. Thus, sphingolipids emerged to important modulators of biological processes including cell growth, differentiation, and senescence. Defects in sphingolipid catabolism are long known to cause severe lysosomal storage disorders, often characterized by neurological phenotypes. In recent studies it became evident that impaired sphingolipid metabolism could also be involved in Alzheimer's disease.     

PspGI,a type II restriction endonuclease from the extreme thermophile Pyrococcus sp.: structural and functional studies to investigate an evolutionary relationship with several mesophilic restriction enzymes

We present here the first detailed biochemical analysis of an archaeal restriction enzyme. PspGI shows sequence similarity to SsoII, EcoRII, NgoMIV and Cfr10I, which recognize related DNA sequences. We demonstrate here that PspGI, like SsoII and unlike EcoRII or NgoMIV and Cfr10I, interacts with and cleaves DNA as a homodimer and is not stimulated by simultaneous binding to two recognition sites. PspGI and SsoII differ in their basic biochemical properties, viz. stability against chemical denaturation and proteolytic digestion, DNA binding and the pH, MgCl(2) and salt-dependence of their DNA cleavage activity. In contrast, the results of mutational analyses and cross-link experiments show that PspGI and SsoII have a very similar DNA binding site and catalytic center as NgoMIV and Cfr10I (whose crystal structures are known), and presumably also as EcoRII, in spite of the fact that these enzymes, which all recognize variants of the sequence -/CC-GG- (/ denotes the site of cleavage), are representatives of different subgroups of type II restriction endonucleases. A sequence comparison of all known restriction endonuclease sequences, furthermore, suggests that several enzymes recognizing other DNA sequences also share amino acid sequence similarities with PspGI, SsoII and EcoRII in the region of the presumptive active site. These results are discussed in an evolutionary context.     

Monitoring cytosolic pH of carboxysome-deficient cells of Synechocystis sp. PCC 6803 using fluorescence analysis

Disruption of the ccmM gene in the cyanobacterium Synechocystis sp. PCC 6803 causes a deficiency of carboxysomes and impairs growth in ambient CO2. The effect of this gene defect on cellular metabolism was investigated using electron microscopy, biochemical and fluorescence analysis. Mutant cells were devoid of the characteristic dense polyhedral bodies called carboxysomes. The photosynthetic oxygen evolution was considerably lower in mutant cells compared to wild type, while Rubisco activity in cell extracts was similar. During photosynthetic CO2-dependent oxygen evolution, Rubisco Vmax dropped from 142 micromol mg-1 chlorophyll h-1 (WT) to 77 micromol mg-1 chlorophyll h-1 in the mutant cells, and the Km for Ci (inorganic carbon) increased from 0.5 mM (WT) to 40 mM. The fluorescent indicator, acridine yellow, was used for non-invasive measurements of cytoplasmic pH changes in whole cells induced by addition of Ci, making use of the decrease in fluorescence yield that accompanies cytoplasmic acidification. The experimental results indicate that control of the cytoplasmic pH is linked to the internal carbon pool (Ci). Both wild-type and ccmM-deficient cells showed a linear response of acridine yellow fluorescence quenching and, thus, of internal acidification, with respect to externally added inorganic carbon. However, the fluorescence analysis of mutant (carboxysome-free) cells indicated slower kinetics of Ci accumulation.     

Phospholipid synthesis is decreased in neuronal tissue in a mouse model of Sandhoff disease

Sandhoff disease is a progressive neurodegenerative disorder caused by mutations in the HEXB gene which encodes for the beta-subunit of beta-hexosaminidase A and B, resulting in ganglioside GM(2) accumulation in the brain. We now demonstrate that phospholipid metabolism is altered in both cultured neurons and in brain tissue from a mouse model of Sandhoff disease, the Hexb-/- mouse. Metabolic labelling using [methyl-(14)C]choline and l-[3-(3)H]serine demonstrated reduced incorporation of [methyl-(14)C]choline into phospholipids in brain tissue but not in liver or spleen. Phospholipid mass was also reduced in brain. The activities of CTP : phosphocholine cytidylyltransferase (CCT) and phosphatidylserine synthase were also reduced in brain tissue from Hexb-/- mice, probably because of post-translational modification as no changes were observed in levels of enzyme expression. The relevance of these findings to Sandhoff disease in human patients is strengthened by observations made over 30 years ago on autopsy tissue of Tay Sachs and Sandhoff disease patients, in which reduced phospholipid levels were observed. We suggest that changes in phospholipid metabolism are not simply because of loss of neuronal tissue as a result of degeneration but rather may cause degeneration, and we discuss the possible effects that changes in phospholipid metabolism could play in the neuropathophysiology of Sandhoff disease.