Supercritical assisted atomization: A novel technology for microparticles preparation of an asthma-controlling drug

The objective of this study was to produce microparticles of a new asthma-controlling drug by supercritical assisted atomization (SAA), proposed as an alternative to conventional jet-milling process. SAA is based on the solubilization of supercritical carbon dioxide in a liquid solution containing the drug; the ternary mixture is then sprayed through a nozzle, and microparticles are formed as a consequence of the enhanced atomization. SAA process parameters studied were precipitator temperature, nozzle diameter, and drug concentration in the liquid solution. Their influence was evaluated on morphology and size of precipitated particles. Spherical particles with mean particle size ranging from 1 to 3 μm of the new anti-asthma drug were produced by SAA. The mass median aerodynamic diameter (MMAD) of the SAA micronized particles and of the conventional jet-milled drug was used to compare, the results obtainable using the 2 techniques. Particularly, MMADs from 1.6 to 4.0 μm were obtained by SAA at the optimum operating conditions and by varying the concentration of the solution injected. MMAD of 6.0 μm was calculated for the jet-milled drug. SAA samples also exhibited narrower particle size distribution (PSD). A good control of particle size and distribution together with no drug degradation was obtained by SAA process. Published: October 22, 2005     

Visualization through magnetic resonance imaging of DNA internalized following "in vivo" electroporation

The ability to visualize plasmid DNA entrapment in muscle cells undergoing an "in vivo" electroporation treatment was investigated on BALB/c mice using a 7-T magnetic resonance imaging (MRI) scanner using the paramagnetic Gd-DOTA-spd complex as imaging reporter. Gd-DOTA-spd bears a tripositively charged spermidine residue that yields a strong binding affinity toward the negatively charged DNA chain (6.4 kb, K(a) = 2.2 x 10(3) M(-1) for approximately 2500 +/- 500 binding sites). Cellular colocalization of Gd-DOTA-spd and plasmid DNA has been validated by histological analysis of excised treated muscle. In vivo MRI visualization of Gd-DOTA-spd distribution provides an excellent route to access the cellular entrapment of plasmid DNA upon applying an electroporation pulse.     

Mitochondrial deoxynucleotide pools in quiescent fibroblasts: a possible model for mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)

Mitochondrial (mt) DNA depletion syndromes can arise from genetic deficiencies for enzymes of dNTP metabolism, operating either inside or outside mitochondria. MNGIE is caused by the deficiency of cytosolic thymidine phosphorylase that degrades thymidine and deoxyuridine. The extracellular fluid of the patients contains 10-20 microM deoxynucleosides leading to changes in dTTP that may disturb mtDNA replication. In earlier work, we suggested that mt dTTP originates from two distinct pathways: (i) the reduction of ribonucleotides in the cytosol (in cycling cells) and (ii) intra-mt salvage of thymidine (in quiescent cells). In MNGIE and most other mtDNA depletion syndromes, quiescent cells are affected. Here, we demonstrate in quiescent fibroblasts (i) the existence of small mt dNTP pools, each usually 3-4% of the corresponding cytosolic pool; (ii) the rapid metabolic equilibrium between mt and cytosolic pools; and (iii) the intra-mt synthesis and rapid turnover of dTTP in the absence of DNA replication. Between 0.1 and 10 microM extracellular thymidine, intracellular thymidine rapidly approaches the extracellular concentration. We mimic the conditions of MNGIE by maintaining quiescent fibroblasts in 10-40 microM thymidine and/or deoxyuridine. Despite a large increase in intracellular thymidine concentration, cytosolic and mt dTTP increase at most 4-fold, maintaining their concentration for 41 days. Other dNTPs are marginally affected. Deoxyuridine does not increase the normal dNTP pools but gives rise to a small dUTP and a large dUMP pool, both turning over rapidly. We discuss these results in relation to MNGIE.     

Analysis of SHP-1-mediated down-regulation of the TRK-T3 oncoprotein identifies Trk-fused gene (TFG) as a novel SHP-1-interacting protein

SHP-1 is a cytoplasmic SH2 domain containing protein-tyrosine phosphatase (PTP) involved in the negative regulation of multiple signaling pathways in hematopoietic, nervous, and epithelial cells. The thyroid TRK-T3 oncogene consists of the NTRK1 tyrosine kinase domain fused in-frame with sequences of the TFG (TRK-fused gene), encoding a protein of unknown function. TFG contains a coiled-coil domain responsible for TRK-T3 oligomerization. In addition, recent analysis of the sequences outside of the coiled-coil domain suggested possible interactions with other proteins. Based on the presence of a putative SHP-1 SH2-binding site within the TFG sequences, we have investigated the role of the SHP-1 phosphatase in TRK-T3 oncoprotein signaling. In this study we show that SHP-1 interacts with and down-regulates TRK-T3. We provide evidence that SHP-1 SH2 and catalytic domains, respectively, associate with the TFG- and NTRK1-derived portions of TRK-T3. Our data contribute to the definition of cellular mechanisms involved in thyroid tumorigenesis. Moreover, it reveals TFG as a novel protein able to modulate SHP-1 activity.     

Denaturing HPLC-based approach for detection of COL7A1 gene mutations causing dystrophic epidermolysis bullosa

Dystrophic epidermolysis bullosa (DEB) is a rare clinically heterogeneous genodermatosis due to genetic defects in type VII collagen gene (COL7A1). Identification of COL7A1 mutations is a challenge since this gene comprises 118 exons and more than 300 mutations scattered over the gene have been reported. Here, we describe for the first time the use of denaturing high performance liquid chromatography (DHPLC) for COL7A1 mutation detection. To validate the method, exon-specific DHPLC conditions were applied to screen DNA samples from patients carrying known COL7A1 mutations. Abnormal DHPLC profiles were obtained for all known mutations. Subsequent DHPLC analysis of 17 DEB families of unknown genotype allowed the identification of 21 distinct mutations, 9 of which were novel. The DHPLC mutation detection rate was significantly higher compared with our mutation scanning rate with conventional techniques (97% vs 86%), indicating DHPLC as the method of choice for COL7A1 molecular characterization in DEB patients.     

Phospholipase A2 hydrolysis of supported phospholipid bilayers: a neutron reflectivity and ellipsometry study

We have investigated the phospholipase A(2) catalyzed hydrolysis of supported phospholipid bilayers using neutron reflection and ellipsometry. At the hydrophilic silica-water interface, hydrolysis of phosphatidylcholine bilayers by phospholipase A(2) from Naja mossambica mossambica venom is accompanied by destruction of the bilayer at an initial rate, which is comparable for DOPC and DPPC but is doubled for POPC. The extent of bilayer destruction at 25 degrees C decreases from DOPC to POPC and is dramatically reduced for DPPC. Neutron reflectivity measurements indicate that the enzyme penetrates into the bilayers in increasing order for DOPC, POPC, and DPPC, while the amount of enzyme adsorbed at the interface is smallest for DPPC and exhibits a maximum for POPC. Penetration into the hydrophobic chain region in the bilayer is further supported by the fact that the enzyme adsorbs strongly and irreversibly to a hydrophobic monolayer of octadecyltrichlorosilane. These results are rationalized in terms of the properties of the reaction products and the effect of their accumulation in the membrane on the kinetics of enzyme catalysis.     

Transcriptional profiling of gamma delta T cells identifies a role for vitamin D in the immunoregulation of the V gamma 9V delta 2 response to phosphate-containing ligands

Vitamin D is a steroid hormone that, in addition to its well-characterized role in calcium/phosphate metabolism, has been found to have regulatory properties for immune system function. The nuclear vitamin D receptor is widely expressed in tissues, but has also been shown to be regulated by hormones, growth factors, and cytokines. In this study we show that activation of human Vdelta2Vgamma9 T cells by nonpeptidic monoalkyl phosphates such as isopentenyl pyrophosphate leads to the up-regulation of the vitamin D receptor via a pathway that involves the classical isoforms of protein kinase C. We further show that this receptor is active by demonstrating that the ligand 1alpha,25-dihydroxyvitamin D3 (vitD3) significantly inhibits in a dose-dependent fashion phospholigand-induced gammadelta T cell expansion, IFN-gamma production, and CD25 expression. We also show that vitD3 negatively regulates signaling via Akt and ERK and, at high concentrations, potentiates Ag-induced cell death. As such, these data provide further support for the immunoregulatory properties of vitamin D, and suggest that the ability of vitD3 to negatively regulate the proinflammatory activity of gammadelta T cells may contribute to the protection this vitamin affords against inflammatory and autoimmune disorders dependent upon Th1-type responses.     

CIN85 regulates the ligand-dependent endocytosis of the IgE receptor: a new molecular mechanism to dampen mast cell function

Ligation of the high-affinity receptor for IgE (Fc epsilonRI), constitutively expressed on mast cells and basophils, promotes cell activation and immediate release of allergic mediators. Furthermore, Fc epsilonRI up-regulation on APC from atopic donors is involved in the pathophysiology of allergic diseases. In consideration of the clinical relevance of the IgE receptor, the down-modulation of Fc epsilonRI expression in mast cells may represent a potential target for handling atopic diseases. In an effort to identify new molecular mechanisms involved in attenuating Fc epsilonRI expression and signaling, we focused our attention on CIN85, a scaffold molecule that regulates, in concert with the ubiquitin ligase Cbl, the clathrin-mediated endocytosis of several receptor tyrosine kinases. In the present study, we show that endogenous CIN85 is recruited in Cbl-containing complexes after engagement of the Fc epsilonRI on a mast cell line and drives ligand-induced receptor internalization. By confocal microscopic analysis, we provide evidence that CIN85 directs a more rapid receptor sorting in early endosomes and delivery to a lysosomal compartment. Furthermore, biochemical studies indicate that CIN85 plays a role in reducing the expression of receptor complex. Finally, we demonstrate that CIN85-overexpressing mast cells are dramatically impaired in their ability to degranulate following Ag stimulation, suggesting that the accelerated internalization of activated receptors by perturbing the propagation of Fc epsilonRI signaling may contribute to dampen the functional response. This role of CIN85 could be extended to include other multimeric immune receptors, such as the T and B cell receptors, providing a more general molecular mechanism for attenuating immune responses.     

Inhibition of NF-kappa B and oxidative pathways in human dendritic cells by antioxidative vitamins generates regulatory T cells

Dendritic cells (DCs) are central to T cell immunity, and many strategies have been used to manipulate DCs to modify immune responses. We investigated the effects of antioxidants ascorbate (vitamin C) and alpha-tocopherol (vitamin E) on DC phenotype and function. Vitamins C and E are both antioxidants, and concurrent use results in a nonadditive activity. We have demonstrated that DC treated with these antioxidants are resistant to phenotypic and functional changes following stimulation with proinflammatory cytokines. Following treatment, the levels of intracellular oxygen radical species were reduced, and the protein kinase RNA-regulated, eukaryotic translation initiation factor 2alpha, NF-kappaB, protein kinase C, and p38 MAPK pathways could not be activated following inflammatory agent stimulation. We went on to show that allogeneic T cells (including CD4(+)CD45RO, CD4(+)CD45RA, and CD4(+)CD25(-) subsets) were anergized following exposure to vitamin-treated DCs, and secreted higher levels of Th2 cytokines and IL-10 than cells incubated with control DCs. These anergic T cells act as regulatory T cells in a contact-dependent manner that is not dependent on IL-4, IL-5, IL-10, IL-13, and TGF-beta. These data indicate that vitamin C- and E-treated DC might be useful for the induction of tolerance to allo- or autoantigens.