IL-21 induces tumor rejection by specific CTL and IFN-gamma-dependent CXC chemokines in syngeneic mice

IL-21 is an immune-stimulatory four alpha helix cytokine produced by activated T cells. To study the in vivo antitumor activities of IL-21, TS/A murine mammary adenocarcinoma cells were genetically modified to secrete IL-21 (TS/A-IL-21). These cells developed small tumors that were subsequently rejected by 90% of s.c. injected syngeneic mice. Five days after injection, TS/A-IL-21 tumors showed numerous infiltrating granulocytes, NK cells, and to a lesser extent CD8(+) T cells, along with the expression of TNF-alpha, IFN-gamma, and endothelial adhesion molecules ICAM-1 and VCAM-1. At day 7, CD8(+) and CD4(+) T cells increased together with IFN-gamma, and the CXC chemokines IFN-gamma-inducible protein 10, monokine induced by IFN-gamma, and IFN-inducible T cell alpha-chemoattractant. The TS/A-IL-21 tumor displayed a disrupted vascular network with abortive sprouting and signs of endothelial cell damage. In vivo depletion experiments by specific Abs showed that rejection of TS/A-IL-21 cells required CD8(+) T lymphocytes and granulocytes. When injected in IFN-gamma-deficient mice, TS/A-IL-21 cells formed tumors that regressed in only 29% of animals, indicating a role for IFN-gamma in IL-21-mediated antitumor response, but also the existence of IFN-gamma-independent effects. Most immunocompetent mice rejecting TS/A-IL-21 cells developed protective immunity against TS/A-pc (75%) and against the antigenically related C26 colon carcinoma cells (61%), as indicated by rechallenge experiments. A specific CTL response against the gp70-env protein of an endogenous murine retrovirus coexpressed by TS/A and C26 cells was detected in mice rejecting TS/A-IL-21 cells. These data suggest that IL-21 represents a suitable adjuvant in inducing specific CTL responses.     

Multivesicular bodies and autophagy in erythrocyte maturation

During reticulocyte maturation, hematopoietic progenitors undergo numerous changes to reach the final functional stage which concludes with the release of reticulocytes and erythrocytes into circulation. During this process some proteins, which are not required in the mature stage, are sequestered in the internal vesicles present in multivesicular bodies (MVBs). These small vesicles are known as exosomes because they are released into the extracellular medium by fusion of the MVB with the plasma membrane. Interestingly, during this maturation process some organelles, such as mitochondria and endoplasmic reticulum, are wrapped in double membrane vacuoles and degraded via autophagy. We have demonstrated in human leukemic K562 cells a role for calcium and Rab11 in the biogenesis of MVBs and exosome release. Here we discuss evidence indicating that K562 cells present a high basal level of autophagy, and that there is an association between MVBs and autophagosomes, suggesting a role for the autophagic pathway in the maturation process of this cell type.     

The two faces of autophagy: Coxiella and Mycobacterium

In the world of pathogen-host cell interactions, the autophagic pathway has been recently described as a component of the innate immune response against intracellular microorganisms. Indeed, some bacterial survival mechanisms are hampered when this process is activated. Mycobacterium tuberculosis infection of macrophages, for example, is impaired upon autophagy induction and the bacterial phagosomes are redirected to autophagosomes. On the other hand, pathogens like Coxiella burnetii are benefited by this cellular response and subvert the autophagy process resulting in a more efficient replication. We study at the molecular level these two different faces of the autophagy process in pathogen life in order to elucidate the intricate routes modulated by the microorganisms as survival strategies.     

Determination of the novel non-peptidic HIV-protease inhibitor tipranavir by HPLC-UV after solid-phase extraction

An HPLC method previously described for the assay of amprenavir (APV), ritonavir (RTV), indinavir (IDV), saquinavir (SQV), nelfinavir (NFV), lopinavir (LPV), atazanavir (ATV), nevirapine (NVP) and efavirenz (EFV) can be also conveniently applied, with minor gradient program adjustment, for the determination of the novel non-peptidic HIV protease inhibitor tipranavir (TPV) in human plasma, by off-line solid-phase extraction (SPE) followed by HPLC coupled with UV-diode array detection (DAD). After viral inactivation by heat, the plasma is diluted with phosphate buffer (pH 7), and subjected to a SPE on a C18 cartridge. Matrix components are eliminated with a solution of 0.1% H3PO4 solution neutralised to pH 7, and TPV is eluted with MeOH. The resulting eluate is evaporated and reconstituted in 100 microl MeOH/H2O 50/50. A 40 microl volume is injected onto a Nucleosil C18 AB column and TPV is analysed by UV detection at 201 nm using a gradient elution program constituted of MeCN and phosphate buffer adjusted to pH 5.12 and containing 0.02% sodium heptanesulfonate. The calibration curves are linear up to 75 microg/ml, with a lower limit of quantification of 0.125 microg/ml. The mean absolute recovery of TPV is 77.1+/-4.0%. The method is precise with mean inter-day coefficient of variations (CVs) within 2.2-3.4%, and accurate (range of inter-day deviations from 0.7 to 1.2%). The method has been validated and is currently applied to the monitoring of TPV plasma levels in HIV patients.     

Protein carbonylation, cellular dysfunction, and disease progression

Carbonylation of proteins is an irreversible oxidative damage, often leading to a loss of protein function, which is considered a widespread indicator of severe oxidative damage and disease-derived protein dysfunction. Whereas moderately carbonylated proteins are degraded by the proteasomal system, heavily carbonylated proteins tend to form high-molecular-weight aggregates that are resistant to degradation and accumulate as damaged or unfolded proteins. Such aggregates of carbonylated proteins can inhibit proteasome activity. Alarge number of neurodegenerative diseases are directly associated with the accumulation of proteolysis-resistant aggregates of carbonylated proteins in tissues. Identification of specific carbonylated protein(s) functionally impaired and development of selective carbonyl blockers should lead to the definitive assessment of the causative, correlative or consequential role of protein carbonylation in disease onset and/or progression, possibly providing new therapeutic approaches.     

H2O2 induces abnormal tail flexure in Xenopus embryos: similarities with Paraquat teratogenic effects

BACKGROUND: As previously shown, Paraquat (PQ) treatments of Xenopus developing embryos mainly induce a characteristic developmental alteration we named "abnormal tail flexure." PQ oxidative activity has been indicated as the cause of this malformation. Since PQ evokes reactive oxygen species (ROS), among which hydroxyl radicals (OH(*)), and H(2)O(2) can be converted to (OH(*)) via Fenton reaction, we compared here the lethal and teratogenic potentials of both oxidants by using the Frog Embryo Teratogenesis Assay-Xenopus (FETAX), in order to grasp eventual similarities in their teratogenic activity. METHODS: Xenopus embryos were exposed, from stage 8 to stage 47, at 368, 491, 612, and 735 microM H(2)O(2) and 0.388 microM PQ. The probit analysis of H(2)O(2) mortality and malformed larva percents gave a 598.82 microM Lethal Concentration 50% (LC(50)) and 536.04 microM Teratogenic Concentration 50% (TC(50)) from which a 1.11 Teratogenic Index (T.I.) has been calculated. This T.I. value should allow the classification of H(2)O(2) as a non-teratogenic compound. RESULTS: A comparison of H(2)O(2) mortality and malformed larva percents with those obtained from PQ exposure showed the higher embryotoxicity of PQ, but, markedly, both compounds mainly induced the "abnormal tail flexure." Histological analysis of both H(2)O(2) and PQ malformed embryo tails showed a similar distorted morphology of both somites and myocytes. Some of muscle cells were necrotic and affected by an apical enlargement as well as a detachment from the connective tissue of intersomitic boundaries. CONCLUSIONS: In our opinion, both of the tested chemicals likely weaken the mechanical bridge connecting the myocyte contractile apparatus to the extracellular matrix, therefore causing the detachment of some of tail myocytes from their connectival septum as well as their apical enlargement. This could lead to the unbalance of tail tensional forces and, in turn, to the appearance of the "abnormal tail flexure."     

Synthesis of new enantiomerically pure macrocycles containing phenanthroline subunits

New enantiomerically pure macrocycles have been prepared by combining 1,10-phenanthroline 2,9-dicarboxylic acid and two alpha-amino-acids linked through spacers. Different diamine linkers have been employed in order to modify the dimensions and the properties of the macrocycles whose structures have been studied by 1H and 13C NMR spectroscopy. The ability of the (L)-valine containing macrocycles to bind metal ions and phenolic molecules has been investigated by 1H NMR experiments and Molecular Mechanics calculations.     

The determinants of stability in the human prion protein: insights into folding and misfolding from the analysis of the change in the stabilization energy distribution in different conditions

The dynamic evolution of the PrP(C) from its NMR-derived conformation to a beta-sheet-rich, aggregation-prone conformation is studied through all-atom, explicit solvent molecular dynamics in different temperature and pH conditions. The trajectories are analyzed by means of a recently introduced energy decomposition approach aimed at identifying the key residues for the stabilization and folding of the protein. It is shown that under native conditions the stabilization energy is concentrated in regions of the helices H1 and H3, whereas under misfolding conditions (low pH, high temperature, or mutations in selected sites) it is spread out over helix H2. Misfolding appears to be a rearrangement of the chain that disrupts most of the native secondary structure of the protein, producing some beta-rich conformations with an energy distribution similar to that of the native state.     

Structure elucidation of new compounds from acidic treatment of the progestins gestodene and drospirenone

Gestodene acidic treatment afforded a single rearrangement product, namely 13-beta-ethyl-18,19-dinorpregna-4,14,16-trien-3,20-dione 3, which was originated through HCl-catalyzed Rupe rearrangement. Drospirenone acidic treatment yielded two epimeric lactones by addition of HCl to the 6beta,7beta-cyclopropane ring, namely 7beta-(chloromethyl)-15beta,16beta-methylene-3-oxo-17beta-pregn-4-ene-21,17-carbolactone 4 and 7beta-(chloromethyl)-15beta,16beta-methylene-3-oxo-17alpha-pregn-4-ene-21,17-carbolactone 5. The structure of the compounds was assessed by spectroscopic and crystallographic methods.