Determination of the optimal therapeutic protocols in cancer immunotherapy

Cancer immunotherapy aims at eliciting an immune system response against the tumor. However, it is often characterized by toxic side-effects. Limiting the tumor growth and, concurrently, avoiding the toxicity of a drug, is the problem of protocol design. We formulate this question as an optimization problem and derive an algorithm for its solution. Unlike the standard optimal control approach, the algorithm simulates impulse-like drug administrations. It relies on an exact computation of the gradient of the cost function with respect to any protocol by means of the variational equations, that can be solved in parallel with the system. In comparison with previous versions of this method [F. Castiglione, B. Piccoli, Optimal control in a model of dendritic cell transfection cancer immunotherapy, Bull. Math. Biol. 68 (2006) 255-274; B. Piccoli, F. Castiglione, Optimal vaccine scheduling in cancer immunotherapy, Physica A. 370 (2) (2007) 672-680], we optimize both the timing and the dosage of each administration and introduce a penalty term to avoid clustering of subsequent injections, a requirement consistent with the clinical practice. In addition, we implement the optimization scheme to simulate the case of multi-therapies. The procedure works for any ODE system describing the pharmacokinetics and pharmacodynamics of an arbitrary number of therapeutic agents. In this work, it was tested for a well known model of the tumor-immune system interaction [D. Kirschner, J.C. Panetta, Modeling immunotherapy of tumor-immune interaction, J. Math. Biol. 37 (1998) 235-252]. Exploring three immunotherapeutic scenarios (CTL therapy, IL-2 therapy and combined therapy), we display the stability and efficacy of the optimization method, obtaining protocols that are successful compromises between various clinical requirements.     

Photosynthesis responses to ozone in young trees of three species with different sensitivities, in a 2-year open-top chamber experiment (Curno, Italy)

This paper reports the findings of an open-top chamber experiment carried out in northern Italy (Forest nursery at Curno), during the 2004 and 2005 growth seasons, on Fagus sylvatica and Quercus robur seedlings and on Populus nigra cuttings, in order to test their photosynthesis response to ambient ozone. The experimental protocols were non-filtered air (NF), charcoal-filtered air (CF) and open air (OA). Tests performed included morphological features of leaves; development of foliar symptoms; chlorophyll content, determined by non-destructive means; chlorophyll fluorescence (direct fluorescence and JIP test) and gas exchanges and net photosynthesis (P_N). Main findings were as follows: (1) symptoms occurred early and were extensive in P. nigra , and they occurred later in F. sylvatica , whereas early degeneration of chlorophyll occurred in late summer in Q. robur ; (2) in conditions of ozone exposure, the three species all presented a decline in photosynthesis efficiency and a decrease in P_N, regardless of the symptomatology they displayed; (3) leaf traits are predictors of species-specific sensitivity to ozone—the high density of Q. robur foliar tissues prevents this species from developing visible symptoms and reduces the extent of physiological responses and (4) physiological responses varied from year to year in the same species—responses were lower in the second year of the experiment, when plants had become better acclimatized to plot conditions.     

PeroxiBase: the peroxidase database

Peroxidases (EC 1.11.1.x), which are encoded by small or large multigenic families, are involved in several important physiological and developmental processes. Analyzing their evolution and their distribution among various phyla could certainly help to elucidate the mystery of their extremely widespread and diversified presence in almost all living organisms. PeroxiBase was originally created for the exhaustive collection of class III peroxidase sequences from plants (Bakalovic, N., Passardi, F., et al., 2006. PeroxiBase: a class III plant peroxidase database. Phytochemistry 67, 534-539). The extension of the class III peroxidase database to all proteins capable to reduce peroxide molecules appears as a necessity. Our database contains haem and non-haem peroxidase sequences originated from annotated or not correctly annotated sequences deposited in the main repositories such as GenBank or UniProt KnowledgeBase. This new database will allow obtaining a global overview of the evolution the protein families and superfamilies capable of peroxidase reaction. In this rapidly growing field, there is a need for continual updates and corrections of the peroxidase protein sequences. Following the lack of unified nomenclature, we also introduced a unique abbreviation for each different family of peroxidases. This paper thus aims to report the evolution of the PeroxiBase database, which is freely accessible through a web server (http://peroxibase.isb-sib.ch). In addition to new categories of peroxidases, new specific tools have been created to facilitate query, classification and submission of peroxidase sequences.     

Structures of the human eIF4E homologous protein, h4EHP, in its m7GTP-bound and unliganded forms

All eukaryotic cellular mRNAs contain a 5' m(7)GpppN cap. In addition to conferring stability to the mRNA, the cap is required for pre-mRNA splicing, nuclear export and translation by providing an anchor point for protein binding. In translation, the interaction between the cap and the eukaryotic initiation factor 4E (eIF4E) is important in the recruitment of the mRNAs to the ribosome. Human 4EHP (h4EHP) is a homologue of eIF4E. Like eIF4E it is able to bind the cap but it appears to play a different cellular role, possibly being involved in the fine-tuning of protein expression levels. Here we use X-ray crystallography and isothermal titration calorimetry (ITC) to investigate further the binding of cap analogues and peptides to h4EHP. m(7)GTP binds to 4EHP 200-fold more weakly than it does to eIF4E with the guanine base sandwiched by a tyrosine and a tryptophan instead of two tryptophan residues as seen in eIF4E. The tyrosine resides on a loop that is longer in h4EHP than in eIF4E. The consequent conformational difference between the proteins allows the tyrosine to mimic the six-membered ring of the tryptophan in eIF4E and adopt an orientation that is similar to that seen for equivalent residues in other non-homologous cap-binding proteins. In the absence of ligand the binding site is incompletely formed with one of the aromatic residues being disordered and the side-chain of the other adopting a novel conformation. A peptide derived from the eIF4E inhibitory protein, 4E-BP1 binds h4EHP 100-fold less strongly than eIF4E but in a similar manner. Overall the data, combined with sequence analyses of 4EHP from evolutionary diverse species, strongly support the hypothesis that 4EHP plays a physiological role utilizing both cap-binding and protein-binding functions but which is distinct from eIF4E.     

Unexpected inhibition of peptidoglycan LD-transpeptidase from Enterococcus faecium by the beta-lactam imipenem

The beta-lactam antibiotics mimic the D-alanyl(4)-D-alanine(5) extremity of peptidoglycan precursors and act as "suicide" substrates of the DD-transpeptidases that catalyze the last cross-linking step of peptidoglycan synthesis. We have previously shown that bypass of the dd-transpeptidases by the LD-transpeptidase of Enterococcus faecium (Ldt(fm)) leads to high level resistance to ampicillin. Ldt(fm) is specific for the L-lysyl(3)-D-alanine(4) bond of peptidoglycan precursors containing a tetrapeptide stem lacking D-alanine(5). This specificity was proposed to account for resistance, because the substrate of Ldt(fm) does not mimic beta-lactams in contrast to the D-alanyl(4)-D-alanine(5) extremity of pentapeptide stems used by the DD-transpeptidases. Here, we unexpectedly show that imipenem, a beta-lactam of the carbapenem class, totally inhibited Ldt(fm) at a low drug concentration that was sufficient to inhibit growth of the bacteria. Peptidoglycan cross-linking was also inhibited, indicating that Ldt(fm) is the in vivo target of imipenem. Stoichiometric and covalent modification of Ldt(fm) by imipenem was detected by mass spectrometry. The modification was mapped into the trypsin fragment of Ldt(fm) containing the catalytic Cys residue, and the Cys to Ala substitution prevented imipenem binding. The mass increment matched the mass of imipenem, indicating that inactivation of Ldt(fm) is likely to involve rupture of the beta-lactam ring and acylation of the catalytic Cys residue. Thus, the spectrum of activity of beta-lactams is not restricted to transpeptidases of the DD-specificity, as previously thought. Combination therapy with imipenem and ampicillin could therefore be active against E. faecium strains having the dual capacity to manufacture peptidoglycan with transpeptidases of the LD- and DD-specificities.     

Unique human tumor antigens: immunobiology and use in clinical trials

The individual, unique tumor Ags, which characterize each single tumor, were described 50 years ago in rodents but their molecular characterization was limited to few of them and obtained during the last 20 years. Here we summarize the evidence for the existence and the biological role of such Ags in human tumors, although such evidence was provided only during the last 10 years and by a limited number of studies, a fact leading to a misrepresentation of unique Ags in human tumor immunology. This was also due to the increasing knowledge on the shared, self-human tumor Ags, which have been extensively used as cancer vaccines. In this review, we highlight the biological and clinical importance of unique Ags and suggest how they could be used in clinical studies aimed at assessing their immunogenic and clinical potential both in active and adoptive immunotherapy of human tumors.     

Synaptophysin I selectively specifies the exocytic pathway of synaptobrevin 2/VAMP2

Biogenesis and recycling of synaptic vesicles are accompanied by sorting processes that preserve the molecular composition of the compartments involved. In the present study, we have addressed the targeting of synaptobrevin 2/VAMP2 (vesicle-associated membrane protein 2), a critical component of the synaptic vesicle--fusion machinery, in a heterotypic context where its sorting is not confounded by the presence of other neuron-specific molecules. Ectopically expressed synaptophysin I interacts with VAMP2 and alters its default surface targeting to a prominent vesicular distribution, with no effect on the targeting of other membrane proteins. Protein-protein interaction is not sufficient for the control of VAMP2 sorting, which is mediated by the C-terminal domain of synaptophysin I. Synaptophysin I directs the sorting of VAMP2 to vesicles before surface delivery, without influencing VAMP2 endocytosis. Consistent with this, dynamin and alpha-SNAP (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein) mutants which block trafficking at the plasma membrane do not abrogate the effect of synaptophysin I on VAMP2 sorting. These results indicate that the sorting determinants of synaptic vesicle proteins can operate independently of a neuronal context and implicate the association of VAMP2 with synaptophysin I in the specification of the pathway of synaptic vesicle biogenesis.     

Proteomic analysis of spinal cord of presymptomatic amyotrophic lateral sclerosis G93A SOD1 mouse

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease, whose primary mechanisms or causes are still not defined and for which no effective treatment is available. We have recently reported that before disease onset the level of tyrosine nitrated proteins is increased in the G93A SOD1 transgenic mouse model of ALS. In the present investigation, we carried out a proteomic analysis of spinal cord extracts from G93A SOD1 mice at the presymptomatic stage of the disease to further unravel primary events in the pathogenesis and tentatively screen for potential pharmacological targets. Using a robust two-dimensional gel electrophoresis-based proteomic approach, we detected a number of proteins differentially represented in presymptomatic mice in comparison with controls. Alterations of these proteins correlate with mitochondrial dysfunction, aggregation, and stress response. Moreover, we found a variation in the isoform pattern of cyclophilin A, a molecular chaperone that protects cells from the oxidative stress.     

Long-term study on symptomless human metapneumovirus infection in hematopoietic stem cell transplant recipients

From October 2004 through October 2006 a study was performed to evaluate the prevalence of human Metapneumovirus (hMPV) infection in adult hematopoietic stem cell transplant (HSCT) recipients. Sequential nasopharyngeal aspirates (NPA) were collected independently from respiratory symptoms and evaluated for hMPV-RNA by polymerase chain reaction (PCR) and sequence analysis. Results indicate epidemiological and molecular differences between the 2004-2005 and 2005-2006 periods and that hMPV seems not to symptomatically affect HSCT patients or cause late respiratory sequelae. In addition, data collected suggest a hospital origin of hMPV infection in most HSCT patients during the 2004-2005 period.