Claudia Scotti

Zidovudine-induced experimental myopathy: dual mechanism of mitochondrial damage.

Myopathy often complicates Zidovudine (AZT) treatment in patients with acquired immunodeficiency syndrome (AIDS). The pathogenesis of the myopathy is controversial, since clinical phenomena intrinsic to AIDS may interfere per se with the onset of the myopathy. In the present work we investigated the in vivo effect of AZT in an animal model species (rat) not susceptible to HIV infection. Histochemical and electron microscopic analyses demonstrated that, under the experimental conditions used, the in vivo treatment with AZT does not cause in skeletal muscle true dystrophic lesions, but rather mitochondrial alterations confined to the fast fibers. In the same animal models, the biochemical analysis confirmed that mitochondria are the target of AZT toxicity in muscles. The effects of AZT on mitochondria energy transducing mechanisms were investigated in isolated mitochondria both in vivo and in vitro. Membrane potential abnormalities, due to a partial impairment of the respiratory chain capability observed in muscle mitochondria from AZT-treated rats, closely resemble those of control mitochondria in the presence of externally added AZT. mtDNA deletion analysis by PCR amplification and Southern blot analysis did not show any relevant deletion, while mtDNA depletion analysis demonstrated a significant decrease in mtDNA in AZT-treated rats. The present findings show that AZT causes damage to mitochondria by two mechanisms: a short-term mechanism that affects directly the respiratory chain, and a long-term mechanism that alters the mitochondrial DNA thus impairing the mitochondrial protein synthesis. In addition, the ultrastructural observations indicate that the fiber types are differently affected upon AZT treatment, which poses a number of questions as to the pathogenesis of this myopathy.

Helicobacter pyloril-asparaginase: A promising chemotherapeutic agent

Bacterial L-asparaginases are amidohydrolases that catalyse the conversion of L-asparagine to L-aspartate and ammonia and are used as anti-cancer drugs. The current members of this class of drugs have several toxic side effects mainly due to their associated glutaminase activity. In the present study, we report the molecular cloning, biochemical characterisation and in vitro cytotoxicity of a novel L-asparaginase from the pathogenic strain Helicobacter pylori CCUG 17874. The recombinant enzyme showed a strong preference for L-asparagine over L-glutamine and, in contrast to most L-asparaginases, it exhibited a sigmoidal behaviour towards L-glutamine. The enzyme preserved full activity after 2 h incubation at 45 degrees C. In vitro cytotoxicity assays revealed that different cell lines displayed a variable sensitivity towards the enzyme, AGS and MKN28 gastric epithelial cells being the most affected. These findings may be relevant both for the interpretation of the mechanisms underlying H. pylori associated diseases and for biomedical applications.

Targeted Drug Delivery Using Immunoconjugates: Principles and Applications

Antibody-drug conjugates (also known as “immunoconjugates”) have only recently entered the arsenal of anticancer drugs, but the number of undergoing clinical trials including them is ever increasing and most therapeutic antibodies are now patented including their potential immunoconjugate derivatives. They typically consist of three components: antibody, linker, and cytotoxin. An antibody or antibody fragment targeted to a tumor-associated antigen acts as a carrier for drug delivery and can be conjugated by cleavable or uncleavable linkers to a variety of effector molecules, either a drug, toxin, radioisotope, enzyme (the latter also used in Antibody-Directed Enzyme Prodrug Therapy), or to drug-containing liposomes or nanoparticles. In this review, we propose a general outline of the field, starting from the diagnostic and clinical applications of this class of molecules. Special attention will be devoted to the principles and issues in molecular design (choice of tumor-associated antigen, critical milestones in antibody development, available alternatives for linkers and effector molecule, and strategies for fusion proteins building) to the importance of antibody affinity modulation to optimize therapeutic effect and the potential of emerging alternative scaffolds. Most of the power of these molecules is to reach high concentrations in the tumor, relatively unaffecting normal cells, although one drawback lies in their short half-life. In this respect, modifications of immunoconjugates, which have shown to strongly influence pharmacokinetics, like glycosylation and PEGylation, will be discussed. Undergoing clinical trials and active patents will be analyzed and problems present in clinical use will be reported.

Cell-Cycle Inhibition by Helicobacter pylori L-Asparaginase

Helicobacter pylori (H. pylori) is a major human pathogen causing chronic gastritis, peptic ulcer, gastric cancer, and mucosa-associated lymphoid tissue lymphoma. One of the mechanisms whereby it induces damage depends on its interference with proliferation of host tissues. We here describe the discovery of a novel bacterial factor able to inhibit the cell-cycle of exposed cells, both of gastric and non-gastric origin. An integrated approach was adopted to isolate and characterise the molecule from the bacterial culture filtrate produced in a protein-free medium: size-exclusion chromatography, non-reducing gel electrophoresis, mass spectrometry, mutant analysis, recombinant protein expression and enzymatic assays. L-asparaginase was identified as the factor responsible for cell-cycle inhibition of fibroblasts and gastric cell lines. Its effect on cell-cycle was confirmed by inhibitors, a knockout strain and the action of recombinant L-asparaginase on cell lines. Interference with cell-cycle in vitro depended on cell genotype and was related to the expression levels of the concurrent enzyme asparagine synthetase. Bacterial subcellular distribution of L-asparaginase was also analysed along with its immunogenicity. H. pylori L-asparaginase is a novel antigen that functions as a cell-cycle inhibitor of fibroblasts and gastric cell lines. We give evidence supporting a role in the pathogenesis of H. pylori-related diseases and discuss its potential diagnostic application.

Glutaminase activity determines cytotoxicity of l-asparaginases on most leukemia cell lines

L-Asparaginase (ASNase) is a front-line chemotherapy for acute lymphoblastic leukemia (ALL), which acts by deaminating asparagine and glutamine. To evaluate the importance of glutaminase activity, we exploited a recently developed mutant of Helicobacter pylori ASNase (dm HpA), with amino acid substitutions M121C/T169M. The mutant form has the same asparaginase activity as wild-type but lacks glutaminase activity. Wild-type and dm HpA were compared with the clinically used ASNases from Escherichia coli (l-ASP) and Erwinia chrysanthemi (ERWase). Asparaginase activity was similar for all isoforms, while glutaminase activity followed the rank order: ERWase>l-ASP>wild-type HpA>dm HpA. Cytotoxic efficacy of ASNases was tested on 11 human leukemia cell lines and two patient-derived ALL samples. Two cell lines which we had previously shown to be asparagine-dependent were equally sensitive to the asparaginase isoforms. The other nine lines and the two patient-derived samples were more sensitive to isoforms with higher glutaminase activities. ERWase was overall the most effective ASNase on all cell lines tested whereas dm HpA, having the lowest glutaminase activity, was the least effective. These data demonstrate that asparaginase activity alone may not be sufficient for ASNase cytotoxicity, and that glutaminase activity may be required for full anti-leukemic efficacy.

Genotype-phenotype correlation in Pompe disease, a step forward

BackgroundPompe’s disease is a progressive myopathy caused by mutations in the lysosomal enzyme acid alphaglucosidase gene (GAA). A wide clinical variability occurs also in patients sharing the same GAA mutations, even within the same family.MethodsFor a large series of GSDII patients we collected some clinical data as age of onset of the disease, presence or absence of muscular pain, Walton score, 6-Minute Walking Test, Vital Capacity, and Creatine Kinase. DNA was extracted and tested for GAA mutations and some genetic polymorphisms able to influence muscle properties (ACE, ACTN3, AGT and PPARα genes).We compared the polymorphisms analyzed in groups of patients with Pompe disease clustered for their homogeneous genotype.ResultsWe have been able to identify four subgroups of patients completely homogeneous for their genotype, and two groups homogeneous as far as the second mutation is defined “very severe” or “potentially less severe”. When disease free life was studied we observed a high significant difference between groups. The DD genotype in the ACE gene and the XX genotype in the ACTN3 gene were significantly associated to an earlier age of onset of the disease. The ACE DD genotype was also associated to the presence of muscle pain.ConclusionsWe demonstrate that ACE and ACTN3 polymorphisms are genetic factors able to modulate the clinical phenotype of patients affected with Pompe disease.

Bioinformatic analysis of pathogenic missense mutations of activin receptor like kinase 1 ectodomain

Activin A receptor, type II-like kinase 1 (also called ALK1), is a serine-threonine kinase predominantly expressed on endothelial cells surface. Mutations in its ACVRL1 encoding gene (12q11-14) cause type 2 Hereditary Haemorrhagic Telangiectasia (HHT2), an autosomal dominant multisystem vascular dysplasia. The study of the structural effects of mutations is crucial to understand their pathogenic mechanism. However, while an X-ray structure of ALK1 intracellular domain has recently become available (PDB ID: 3MY0), structure determination of ALK1 ectodomain (ALK1EC) has been elusive so far. We here describe the building of a homology model for ALK1EC, followed by an extensive bioinformatic analysis, based on a set of 38 methods, of the effect of missense mutations at the sequence and structural level. ALK1EC potential interaction mode with its ligand BMP9 was then predicted combining modelling and docking data. The calculated model of the ALK1EC allowed mapping and a preliminary characterization of HHT2 associated mutations. Major structural changes and loss of stability of the protein were predicted for several mutations, while others were found to interfere mainly with binding to BMP9 or other interactors, like Endoglin (CD105), whose encoding ENG gene (9q34) mutations are known to cause type 1 HHT. This study gives a preliminary insight into the potential structure of ALK1EC and into the structural effects of HHT2 associated mutations, which can be useful to predict the potential effect of each single mutation, to devise new biological experiments and to interpret the biological significance of new mutations, private mutations, or non-synonymous polymorphisms.

Engineering of Helicobacter pylori L-asparaginase: characterization of two functionally distinct groups of mutants.

Bacterial L-asparaginases have been used as anti-cancer drugs for over 4 decades though presenting, along with their therapeutic efficacy, several side effects due to their bacterial origin and, seemingly, to their secondary glutaminase activity. Helicobacter pylori type II L-asparaginase possesses interesting features, among which a reduced catalytic efficiency for L-GLN, compared to the drugs presently used in therapy. In the present study, we describe some enzyme variants with catalytic and in vitro cytotoxic activities different from the wild type enzyme. Particularly, replacements on catalytic threonines (T16D and T95E) deplete the enzyme of both its catalytic activities, once more underlining the essential role of such residues. One serendipitous mutant, M121C/T169M, had a preserved efficiency vs L-asparagine but was completely unable to carry out L-glutamine hydrolysis. Interestingly, this variant did not exert any cytotoxic effect on HL-60 cells. The M121C and T169M single mutants had reduced catalytic activities (nearly 2.5- to 4-fold vs wild type enzyme, respectively). Mutant Q63E, endowed with a similar catalytic efficiency versus asparagine and halved glutaminase efficiency with respect to the wild type enzyme, was able to exert a cytotoxic effect comparable to, or higher than, the one of the wild type enzyme when similar asparaginase units were used. These findings may be relevant to determine the role of glutaminase activity of L-asparaginase in the anti-proliferative effect of the drug and to shed light on how to engineer the best asparaginase/glutaminase combination for an ever improved, patients-tailored therapy.

Molecular Alterations in Fibroblasts Exposed to Helicobacter pylori Broth Culture Filtrate: A Potential Trigger of Autoimmunity?

Dear Editor, Gastric epithelial cells are the immediate and major target of Helicobacter pylori in vivo, yet even normal human fibroblasts are sensitive to H. pylori broth culture filtrate [1]. In the latter system, gene profiling evidenced a significant upregulation of genes involved both in the cell-cycle and structural machineries (Table 1, in white), but, beyond these modifications, which by themselves were not unexpected, an unusual gene set, typically expressed in neuronal cells, was also upregulated (Table 1, in grey). Among these genes, at least peripheral myelin protein 22 (PMP22), myelin associated glycorpotein (MAG), and glutamate decarboxylase 67-kDa isoform (GAD67), are known to act as self-antigens in autoimmune diseases [2,3] and we demonstrated by cytometry a significant increase in

Activin Receptor-like kinase 1: a novel anti-angiogenesis target from TGF-β family.

Anti-angiogenic therapy represents a very promising approach in cancer treatment, as most tumors needs to be supplied by a functional vascular network in order to grow beyond the local boundaries and metastatize. The accessibility of vessels to drug delivery and the broad spectrum of cancers treatable with the same compound have arisen interest in research of suitable molecules, with several, especially targeting the VEGF pathway, entered in clinical trials and approved by the Food and Drug Administration. Despite good results, the major hurdle resides in the limited duration of an effective clinical response before tumors start to grow again. Thus, researchers are looking for different alternative targets for a combined and parallel multi-targeting of angiogenic signaling circuits. Activin Receptor-like kinase 1 (ALK1) is a TGF-β type I receptor with high affinity for the BMP9 member of Bone Morphogenic Proteins superfamily: it is expressed mainly, even if not exclusively, on endothelial cells and seems to be involved in the regulatory phase of angiogenesis. Despite a non-completely elucidated mechanism, the targeting of this pathway, both by a soluble ALK1-Fc receptor developed by Acceleron Pharma and by a fully human monoclonal antibody developed by Pfizer, has achieved encouraging results. After having briefly summarized the state of the art of anti-angiogenic therapy, we will first review existing evidence about the molecular mechanisms of ALK1 signaling and we will then analyse in detail the pre-clinical and clinical data available about these two drugs.