Immunocytochemical characterization of viruses and antigenic macromolecules in viral vaccines

Gold immunolabeling combined with negative staining (GINS) provides a valuable immunocytochemical approach that allows a direct ultrastructural definition of all viral vaccine constituents that share common antigenic features with pathogenic viral particles. These results have implications for the development of viral vaccines since it has been demonstrated that incomplete viral particles such as natural empty capsides and Rotavirus-like particles lacking the infective genome are potential candidates for the production of neutralizing antibodies. Furthermore comparative results of the application of GINS to either inactivated vaccines or unfixed samples provide direct evidence that even after inactivation specific antigenic sites are still available for gold immunolabeling.     

Identification of 4-hydroxynonenal as a cytotoxic product originating from the peroxidation of liver microsomal lipids

During the NADPH-Fe induced peroxidation of liver microsomal lipids, products are formed which show various cytopathological effects including inhibition of microsomal glucose-6-phosphatase. The major cytotoxic substance has been isolated and identified as 4-hydroxy-2,3-trans-nonenal. The structure was ascertained by means of ultraviolet, infrared and mass spectrometry and high-pressure liquid chromatographic analysis. Moreover, 4-hydroxynonenal, prepared by chemical synthesis, was found to reproduce the biological effects brought about by the biogenic aldehyde. Preliminary investigations suggest that as compared to 4-hydroxynonenal very low amounts of other 4-hydroxyalkenals, namely 4-hydroxyoctenal, 4-hydroxydecenal and 4-hydroxyundecenal are also formed by actively peroxidizing liver microsomes. In the absence of NADPH-Fe liver microsomes produced only minute amounts of 4-hydroxyalkenals. The biochemical and biological effects of synthetic 4-hydroxyalkenals have been studied in great detail in the past. The results of these investigations together with the finding that 4-hydroxyalkenals, in particular 4-hydroxynonenal, are formed during NADPH-Fe stimulated peroxidation of liver microsomal lipids, may help to elucidate the mechanism by which lipid peroxidation causes deleterious effects on cells and cell constituents.     

Hypoxia modulation of peroxisome proliferator-activated receptors (PPARs) in human glioblastoma stem cells. Implications for therapy

Gliobastoma (GB), the most common adult brain tumor, infiltrates normal brain area rendering impossible the complete surgical resection, resulting in a poor median survival (14–15 months), despite the aggressive multimodality treatments post‐surgery, such as radiation and chemo‐therapy. GB is characterized by hypoxic and necrotic regions due to a poorly organized tumor vascularization, leading to inadequate blood supply and consequently to hypoxic and necrotic areas. We have previously shown that, under hypoxia GB primary cells increased the expression of stemness markers as well as the expression of the nuclear receptor peroxisome proliferator‐activated receptor α (PPARα) and also the crucial role played by PPARs in mouse neural stem cells maintenance and differentiation. Due to the importance of lipid signaling in cell proliferation and differentiation, in this work, we analyzed the expression of PPARs in GB neurospheres both in normoxic and hypoxic conditions. The results obtained suggest a differential regulation of the three PPARs by hypoxia, thus indicating a possible therapeutic strategy to counteract GB recurrencies. J. Cell. Biochem. 113: 3342–3352, 2012. © 2012 Wiley Periodicals, Inc.     

Distinct cellular responses induced by saporin and a transferrin-saporin conjugate in two different human glioblastoma cell lines

Glioblastoma multiforme (GBM) is the most common primary brain tumour in adults, with a median survival of ~12-18 months post-diagnosis. GBM usually recurs within 12 months post-resection, with poor prognosis. Thus, novel therapeutic strategies to target and kill GBM cells are urgently needed. The marked difference of tumour cells with respect to normal brain cells renders glioblastoma a good candidate for selective targeted therapies. Recent experimental strategies focus on over expressed cell surface receptors. Targeted toxins represent a new class of selective molecules composed by a potent protein toxin and a carrier ligand. Targeted toxins approaches against glioblastoma were under investigation in phase I and II clinical trials with several immunotoxins (IT)/ligand toxins such as IL4-Pseudomonas aeruginosa exotoxin A (IL4-PE, NBI-3001), tumour growth factor fused to PE38, a shorter PE variant, (TGF)alpha-TP-38, IL13-PE38, and a transferrin-C diphtheriae toxin mutant (Tf-CRM107). In this work, we studied the effects of the plant ribosome-inactivating saporin and of its chimera transferrin-saporin against two different GBM cell lines. The data obtained here indicate that cell proliferation is affected by the toxin treatments but that different mechanisms are used, directly linked to the presence of an active or inactive p53. A model is proposed for these alternative intracellular pathways.     

The empowerment of translational research: lessons from laminopathies

The need for a collaborative approach to complex inherited diseases collectively referred to as laminopathies, encouraged Italian researchers, geneticists, physicians and patients to join in the Italian Network for Laminopathies, in 2009. Here, we highlight the advantages and added value of such a multidisciplinary effort to understand pathogenesis, clinical aspects and try to find a cure for Emery-Dreifuss muscular dystrophy, Mandibuloacral dysplasia, Hutchinson-Gilford Progeria and forms of lamin-linked cardiomyopathy, neuropathy and lipodystrophy.     

Maintenance of luminal NADPH in the endoplasmic reticulum promotes the survival of human neutrophil granulocytes

The present study demonstrates the expression of hexose-6-phosphate dehydrogenase and 11 beta-hydroxysteroid dehydrogenase type 1 in human neutrophils, and the presence and activity of these enzymes in the microsomal fraction of the cells. Their concerted action together with the previously described glucose-6-phosphate transporter is responsible for cortisone-cortisol interconversion detected in human neutrophils. Furthermore, the results suggest that luminal NADPH generation by the cortisol dehydrogenase activity of 11 beta-hydroxysteroid dehydrogenase type 1 prevents neutrophil apoptosis provoked by the inhibition of the glucose-6-phosphate transporter. In conclusion, the maintenance of the luminal NADPH pool is an important antiapoptotic factor in neutrophil granulocytes.     

Ac10c: a medium-ring, cycloaliphatic Calpha,alpha-disubstituted glycine. Incorporation into model peptides and preferred conformation.

Two complete series of N-protected oligopeptide esters to the pentamer level from 1-amino-cyclodecane-1-carboxylic acid (Ac10c), an alpha-amino acid conformationally constrained through a medium-ring Calphai <--> Calphai cyclization, and either the L-Ala or Aib residue, along with the N-protected Ac10c monomer and homo-dimer alkylamides, were synthesized using solution methods and fully characterized. The preferred conformation of these model peptides was assessed in deuterochloroform solution using FT-IR absorption and 1H NMR techniques. Furthermore, the molecular structures of two derivatives (Z-Ac10c-OH and Fmoc-Ac10c-OH) and two peptides (the dipeptide ester Z-Ac10c-L-Phe-OMe and the tripeptide ester Z-Aib-Ac10c-Aib-OtBu) were determined in the crystal state using X-ray diffraction. The experimental results support the view that beta-bends and 3(10)-helices are preferentially adopted by peptides rich in Ac10c, the third largest cycloaliphatic C(alpha,alpha)-disubstituted glycine known. This investigation allowed us to complete a detailed conformational analysis of the whole 1-amino-cycloalkane-1-carboxylic acid (Ac(n)c, with n = 3-12) series, which represents the prerequisite for our recent proposal of the 'Ac(n)c scan' concept.     

A one-year, randomised, multicentre trial comparing insulin glargine with NPH insulin in combination with oral agents in patients with type 2 diabetes.

AIMS: The aim of the trial was to compare the efficacy and safety of the new, long-acting basal insulin, insulin glargine (LANTUS(R)), with NPH human insulin, each administered in a combination regimen with oral antidiabetic drugs in patients with Type 2 diabetes. METHODS: In a multicentre, open, randomised study, 570 patients with Type 2 diabetes, aged 34 - 80 years, were treated for 52 weeks with insulin glargine or NPH insulin given once daily at bedtime. Previous oral antidiabetic therapy was continued throughout the study. RESULTS: There was a clinically relevant decrease in glycosylated haemoglobin (GHb) values from baseline to endpoint with both drugs (insulin glargine: - 0.46 %; NPH insulin: - 0.38 %; p = 0.415); also, this difference was statistically significant in the subgroup of overweight patients with BMI > 28 kg/m 2 (insulin glargine: - 0.42 %, NPH insulin: - 0.11 %; p = 0.0237). Over the entire treatment period, NPH insulin-treated patients (41 %) and insulin glargine-treated patients (35 %) experienced a similar level of symptomatic hypoglycaemia. A statistically significant difference was observed in the number of patients treated with NPH insulin who reported at least one episode of nocturnal hypoglycaemia compared with those treated with insulin glargine in the overall population and in the overweight subgroup (overall: 24 % vs. 12 %, p = 0.002; overweight: 22.2 % vs. 9.5 %, p = 0.0006), using the Cochran-Mantel-Haenszel test. These differences were most pronounced in insulin-na?ve and overweight (BMI > 28 kg/m 2) sub-groups. The incidence of adverse events was similar for the two treatments. CONCLUSIONS: This study demonstrated that insulin glargine is as effective as NPH insulin in achieving glycaemic control in patients with Type 2 diabetes, and is associated with fewer episodes of symptomatic hypoglycaemia, particularly nocturnal episodes.