A phase IB study on intravenous synthetic mRNA electroporated dendritic cell immunotherapy in pretreated advanced melanoma patients.

BACKGROUND: Autologous monocyte-derived dendritic cells (DCs) electroporated with synthetic messenger RNA (mRNA) encoding a CD40 ligand, a constitutively active Toll-like receptor 4 and CD70, together with mRNA encoding fusion proteins of a human leukocyte antigen (HLA)-class II targeting signal (DC-LAMP) and a melanoma-associated antigen (MAA); either MAGE-A3, MAGE-C2, tyrosinase or gp100) (TriMixDC-MEL) are superiorly immunogenic. PATIENTS AND METHODS: In this phase IB clinical trial, 24 million viable DCs were administered by four biweekly combined intradermal (id) and intravenous (iv) administrations, and a fifth administration on week 16. The number of iv-administered DCs was escalated in four sequentially treated cohorts. Immune responses were assessed by analysis of antigen specificity of blood-derived T-cells and skin infiltrating lymphocytes (SKILs). RESULTS: Fifteen patients with pretreated advanced melanoma tolerated administration of TriMixDC-MEL well. Two patients achieved a complete response and two patients a partial response. All objective responders are progression-free after a follow-up of, respectively, 24+, 28+, 33+, and 34+ months. Post-therapy antigen-specific SKILs were documented in 6 of 12 patients, and antigen-specific CD8(+) T-cells were detected in the blood of 4 of 5 patients. CONCLUSIONS: Cellular immunotherapy with TriMixDC-MEL is safe and immunogenic. Antitumor activity with durable disease control is observed across the investigated iv-dose levels. CLINICALTRIALSGOV IDENTIFIER: NCT01066390.

Release of superoxide anion by alveolar macrophages in pulmonary sarcoidosis.

Biological mechanisms involving nonprotease factors mediate the alterations of the alveolar structures which lead to the interstitial fibrosis of pulmonary sarcoidosis. Thus, we have investigated the production of oxidant species by BAL cells from 50 sarcoidosis patients and 18 healthy controls using a lucigenin-dependent CL method. Spontaneous and PMA-induced CL's were significantly higher in untreated patients and treated patients than in spontaneously cured patients or healthy controls (p less than .05). SOD inhibits 60 to 75% of spontaneous CL and 91 to 93% of PMA-induced CL. There was no apparent correlation between the CL of AM's and the radiological types, SACE levels, and gallium scans. In marked contrast, CL was significantly higher in patients with increased alveolar lymphocytosis (greater than or equal to 18%) than in patients with normal BAL. Since there were neither neutrophils nor eosinophils in BAL and since lymphocytes do not produce lucigenin-dependent CL, we believe that CL is produced by AM's. CL inhibition by SOD suggests that superoxide anion is involved in the production of CL. The release of both superoxide anion and related radicals may be of importance in the pathogenesis of pulmonary sarcoidosis.

Oxidative inactivation of alpha 1-proteinase inhibitor by alveolar epithelial type II cells.

The aim of this work was to evaluate the ability of guinea pig alveolar epithelial type II cells to generate significant amounts of reactive oxygen species to inactivate alpha 1-proteinase inhibitor (alpha 1-PI). Inactivation of alpha 1-PI was evaluated by its inhibitory activity against porcine pancreatic elastase and was expressed as a percentage. The same experiments were performed in parallel with alveolar macrophages (AM) obtained from the same animals and with MRC-5 fibroblasts. Both type II cells and AM released significant amounts of hydrogen peroxide and superoxide, whereas the fibroblasts did not. Unstimulated type II cells (0.5 +/- 2%), AM (1.2 +/- 1.5%), and fibroblasts (0.5 +/- 0.5%) were unable to inactivate alpha 1-PI. Addition of phorbol myristate acetate did not increase their ability to inactivate alpha 1-PI. In contrast, type II cells (79.7 +/- 7%) and AM (80.1 +/- 8%) dramatically inactivated alpha 1-PI in the presence of myeloperoxidase (25 mU/ml), whereas fibroblasts did not. Addition of catalase to the reaction significantly prevented the inactivation of alpha 1-PI. Western blot analysis of alpha 1-PI did not reveal a significant proteolysis of alpha 1-PI, which supports the hypothesis that, in the presence of neutrophil-derived myeloperoxidase, type II cells may oxidatively inactivate alpha 1-PI.

[Studies on the activities of peptidases from human leucocytes, and human and guinea pig alveolar macrophages (author's transl)]. / Etude des activités de peptidases des leucocytes humains et des macrophages alvéolaires de l'homme et du cobaye

Peptidases activities were compared in human leucocytes, guinea pig and human alveolar macrophages. Seversl endo- and exopeptides were characterized; some of them were active at acid pH and others at neutral and alkaline pH. Leucocytes and alveolar macrophages had proteolygic activity for hemoglobin, fibrinogen, collagen and elastin. Using synthetic substrates, several enzymes were characterized: arylamidase, aminopeptidase, carboxypeptidases A and B and cathepsins A and C. The enzymatic activities were much higher in alveolar macrophages than in leucocytes.

[Comparative studies of the acid hydrolases of human leucocytes and human and guinea pig alveolar macrophages. I. Study of the activities of glycosidases, arylsulfatase and acid phosphatase (author's transl)]. / Etude comparative des hydrolases acides des leucocytes humains et des macrophages alvéolaires de l'homme et du cobaye. I. Etude des activités de glycosidases, d'arylsulfatase et de phosphatase acide

Acid hydrolase activities were compared in human leucocytes, guinea pig and human alveolar macrophages. Several enzymes were characterized: N-acetyl-beta-D-glucosaminidase, N-acetyl-alpha- and beta-D-galactosaminidase, alpha and beta-D-galactosidase, alpha-D-mannosidase, alpha-L-fucosidase, beta-D-glucuronidase, neuraminidase, acid phosphatase and arylsulfatase. The enzymatic activities were lower in leucocytes than in alveolar macrophages, higher in human macrophages than in guinea pig macrophages, except for beta-D-glucuronidase, acid phosphatase and arylsulfatase activities.

Use of alveolar macrophages in antianoxic drug studies.

Alveolar macrophages are able to adapt their energy metabolism to very difficult survival conditions. Gaseous phase culture is adaptable to alveolar macrophages because it reproduces in vitro conditions very similar to in vivo conditions. It is easy to modify the incubation gas composition for hypoxia and anaerobiosis. Metabolic changes and cell injury were evaluated in three studies carried out after 24 hr of gaseous phase culture in normoxia and in anaerobiosis with a possible treatment with 0.01 microgram/ml vincamine: 1) ATP content assay by bioluminescence, the witness of cell vitality which decreases significantly in anaerobiosis; 2) Lactate assay which shows the metabolism derivation towards the anaerobic pathways; and 3) Tritiated deoxyglucose (DOG) incorporation, which shows glucose requirements after hypoxic incubation, maintaining or recovering a certain level of energetic activity. This incorporation greatly increases after anaerobic culture. Vincamine has no activity in normoxia. The three parameters are not significantly different from control, but in anaerobiosis, vincamine reveals an interesting protective effect. ATP content decreases under treatment and DOG incorporation increases. This demonstrates that vincamine is able to maintain cell metabolic activity for a longer period of time after the beginning of hypoxic trial. Cells can better use their energy storage and the metabolic pathways which enable them to restore themselves, thanks to vincamine treatment. It has been shown that cell membrane integrity was preserved by tests using cytochalasin B. DOG was not incorporated by cells treated with cytochalasin B after 24 hr of anaerobic culture and normally incorporated by control cells.(ABSTRACT TRUNCATED AT 250 WORDS)

[The in vivo penetration of erythromycin into alveolar macrophages]. / Etude de la pénétration in vivo de l'érythromycine dans les macrophages alvéolaires.

In order to appreciate the in vivo penetration of erythromycin the alveolar spaces a broncho-alveolar lavage was carried out in 24 guinea pigs, 30 minutes, 1 hour 30 minutes and three hours after a single intraperitoneal injection of 50 mgms. of erythromycin. The erythromycin dose was assessed by a microbiological method in the alveolar macrophages and the supernatant of the broncho-alveolar lavage liquid. The intramacrophage concentrations of erythromycin were 3.9, 11.5 and 12 times higher than the serum concentrations at 30 minutes, 1 hour 30 and three hours respectively. The concentrations in the broncho-alveolar lavage liquid was always higher than the serum concentrations tacking account of the different dilutions estimated with relation to the glucose concentrations. At 30 minutes, 1 hour 30 minutes and three hours the alveolar macrophages contained 1.9; 7.6 and 6 times more erythromycin respectively than the lavage supernatant. From the first half hour of its administration the erythromycin was concentrated in the alveolar spaces, in particular within the macrophages. Already noted in vitro, this rapidity of erythromycin concentration in vivo in alveolar macrophages appears to be one of the reasons to explain its activity against micro-organisms developing within macrophages.

[In vivo kinetics of cotrimoxazole in alveolar macrophages]. / Cinétique in vivo du cotrimoxazole dans les macrophages alvéolaires.

The aim of this work was to study the kinetic of intramacrophage penetration of cotrimoxazole in guinea pigs which had received 100 mg/kg of sulfamethoxazole and 20 mg/kg of trimethoprim after a single intraperitoneal injection. 30 minutes, 1, 3, 6 and 24 hours after this injection an intra-cardiac blood sample was taken and pulmonary lavage was performed immediately after sacrificing the animal by cervical cord dislocation. The level of trimethoprim and sulfamethoxazole was measured in each sample by high performance liquid chromatography (HPLC). An estimation of the dilution of the supernatant was obtained by comparing the supernatant glucose with the serum glucose. The serum kinetics of trimethoprim and sulfamethoxazole progressed in a parallel fasion with time with a maximal concentration at 30 minutes (for trimethoprim: 6.7 +/- 0.9 micrograms/ml and for sulfamethoxazole 176.1 +/- 16.2 micrograms/ml). On the other hand their penetration capacity was different in the supernatant and in the alveolar macrophages: the maximal concentrations were obtained after one hour in the supernatant and after 3 hours in the cellular extract and were respectively for trimethoprim 0.43 +/- 0.07 microgram/ml and 20.9 +/- 8.06 micrograms/ml of intramacrophage water and for sulfamethoxazole 1.86 +/- 0.24 micrograms/ml and 23.8 +/- 12.7 micrograms/ml of intramacrophage water. A concentration around six times greater was noted for the trimethoprim inside the cells compared with serum and was only 0.25 time for sulfamethoxazole. On the other hand the supernatant/serum ratio showed a greater concentration for trimethoprim (4 to 10) than for sulfamethoxazole (0.6 to 1).(ABSTRACT TRUNCATED AT 250 WORDS)

Asteroseismology. Echography of young stars reveals their evolution.

We demonstrate that a seismic analysis of stars in their earliest evolutionary phases is a powerful method with which to identify young stars and distinguish their evolutionary states. The early star that is born from the gravitational collapse of a molecular cloud reaches at some point sufficient temperature, mass, and luminosity to be detected. Accretion stops, and the pre-main sequence star that emerges is nearly fully convective and chemically homogeneous. It will continue to contract gravitationally until the density and temperature in the core are high enough to start nuclear burning of hydrogen. We show that there is a relationship for a sample of young stars between detected pulsation properties and their evolutionary status, illustrating the potential of asteroseismology for the early evolutionary phases.

Kepler detected gravity-mode period spacings in a red giant star.

Stellar interiors are inaccessible through direct observations. For this reason, helioseismologists made use of the Sun's acoustic oscillation modes to tune models of its structure. The quest to detect modes that probe the solar core has been ongoing for decades. We report the detection of mixed modes penetrating all the way to the core of an evolved star from 320 days of observations with the Kepler satellite. The period spacings of these mixed modes are directly dependent on the density gradient between the core region and the convective envelope.

HD 181068: a red giant in a triply eclipsing compact hierarchical triple system.

Hierarchical triple systems comprise a close binary and a more distant component. They are important for testing theories of star formation and of stellar evolution in the presence of nearby companions. We obtained 218 days of Kepler photometry of HD 181068 (magnitude of 7.1), supplemented by ground-based spectroscopy and interferometry, which show it to be a hierarchical triple with two types of mutual eclipses. The primary is a red giant that is in a 45-day orbit with a pair of red dwarfs in a close 0.9-day orbit. The red giant shows evidence for tidally induced oscillations that are driven by the orbital motion of the close pair. HD 181068 is an ideal target for studies of dynamical evolution and testing tidal friction theories in hierarchical triple systems.

Potential of amorphous microporous silica for ibuprofen controlled release.

Amorphous microporous silica (AMS) xerogel materials were synthesized in an acid-catalyzed sol-gel process. The porosity of AMS was adapted by varying sol-gel synthesis parameters including the molar hydrolysis ratio (r-value), HCl:Si molar ratio, the type of silicon alkoxide source and the solvent. AMS particles of millimeter size were loaded with ibuprofen, by heat treatment and melt impregnation. In vitro release experiments were performed in simulated gastric and intestinal fluid. The release kinetics were critically depending on the AMS particle size distribution and the micropore diameter. The release was interpreted as configurational diffusion in the AMS micropores. The stability of unloaded and ibuprofen loaded AMS material upon storage was investigated using nitrogen physisorption, DSC analysis and in vitro release experiments. Ibuprofen loaded AMS formulations show remarkable stability, which can be attributed to the presence of ibuprofen molecules in the channels, functioning as scaffolds to support the pore structure.

Type II alveolar epithelial cell in vitro culture in aerobiosis.

A method of Type II alveolar epithelial cell culture in aerobiosis has been developed. Isolation of Type II cells was performed by digesting guinea-pig lung tissue with crude trypsin and elastase and using discontinuous Percoll density gradients. The Type II cells, as identified by light and electron microscopy, were cultured in aerobiosis for up to six days, in direct contact with the atmosphere in conditions mimicking those present in the lower respiratory tract. Significant activities of cellular superoxide dismutase (SOD), manganese dependent superoxide dismutase (Mn-SOD), catalase and glutathione peroxidase (GSH-Px) were found at the time of isolation. In contrast, cell glutathione content varied widely from one experiment to another. Changes of antioxidant enzymes were evaluated during cell culture in aerobiosis. SOD, Mn-SOD and catalase were significantly decreased after three days but were not significantly different between a three day and six day culture. Antioxidant changes did not influence the cell culture. In marked contrast, decrease in cell glutathione was associated with rapid cell death, whereas good cell survival was obtained at high levels of cell glutathione. Cell culture in aerobiosis will permit a precise evaluation of the effects of gases, particularly oxidant gases, on a primary culture of Type II alveolar epithelial cells.

Prevention of in vitro oxidant-mediated alveolar macrophage injury by cellular glutathione and precursors.

To evaluate the toxic effects of various oxidants on alveolar macrophages (O2, NO2, tobacco smoke and silica), we used an original method of cell culture in aerobiosis, which permitted direct contact between the atmosphere and the target cells. Our results demonstrated that the variations of cell sensitivity to the cytotoxic effects of oxidants were associated with various levels in cellular antioxidant equipment. A significant correlation was found between cytotoxicity and antioxidant enzymes (superoxide dismutase and catalase) and/or cellular glutathione. Addition of N-acetylcysteine, a polypeptide known to have an antioxidant activity and to be a precursor of glutathione, was responsible for a decrease of oxidant-mediated cytotoxicity. Whether this protective effect was due to an increase in glutathione cell content or to a scavenger effect of N-acetylcysteine still needs to be elucidated.