Genome-wide transcription analysis of interaction between the human macrophage and Mycobacterium tuberculosis during concurrent drug administration by conventional and novel methods

Targeted drug delivery to alveolar macrophages harboring Mycobacterium tuberculosis (Mtb) holds promise of high efficacy against pulmonary tuberculosis (TB). It was investigated whether inhalable microparticles (MP) can rescue macrophages from ‘alternative’ activation induced by pathogenic Mtb in addition to achieving targeted drug delivery. A genome-wide transcription analysis (Affymetrix HG-U133 Plus 2.0 DNA microarray) of THP-1 cell line derived macrophages was undertaken after exposing them to infection with 10 MOI of MTB H37Rv at 0, 12 and 24 hours post infection. The Molecular markers of macrophage bactericidal activity were assayed in THP-1- and primary human peripheral blood mononuclear cell (PBMC)-derived macrophages, in the presence or absence of soluble anti-tuberculosis drugs, drug-containing MP and blank MP. About 1,500 genes were differentially upregulated and about 500 genes differentially downregulated in response to various modes of treatment. Variations were also observed in the kinetics of gene expression. Cluster analysis indicated activation of several pathways related to innate immune response (cytokines, chemokines, receptors and ligands), apoptosis, cytoskeleton and membrane remodeling, general metabolism and general housekeeping. Some of these results were validated at the functional level, by studying caspase activities, concentrations and time-courses of effector molecules , rates/extents of apoptosis and nitrite oxide induction. Production of cytokines and NO, apoptosis, and bacterial survival were studied as pharmacodynamic outcomes. Cytokine responses of THP-1 derived macrophages were estimated. MP reversed suppression of tumor necrosis factor (TNF) induced by infection, and transiently upregulated γ-interferon (IFN-γ). Drug-free MP surprisingly induced IFN-γ, but not TNF. Primary cells responded to MP, regardless of drug content, by upregulation of NO; but THP-1-derived cells did not respond to blank MP. About 19% of infected cells exposed to MP underwent apoptosis as compared to ~11% cells treated with soluble drugs or blank MP. Cell death induced by blank MP was caspase-independent. Only drug-containing MP induced apoptosis through caspase-8 and caspase-9. Bacterial survival after different treatments varied between individuals. In the best case, while untreated infection resulted in survival of 900±141 colony forming units (CFU), treatment with soluble drugs, drug-containing MP and blank MP respectively, reduced CFU counts to 8.5± 0.7, 3±1.4 and 102±138.6. The results suggest a role of the drug delivery system in macrophage activation as a component of therapeutic strategy against TB

Stress-induced detwinning in copper

Epitaxial thin films of nanotwinned fcc metals such as Cu possess unprecedented combination of high hardness and high electrical conductivity due to the unique structure of nanometer-spaced coherent twin boundaries. Recent studies using in situ nanoindentation in a TEM and atomistic modeling have provided new insights on the deformation behavior and thermomechanical stability of nanotwins that will be reviewed in this discussion. In particular, two unit processes will be highlighted. First, stress-induced migration of 3{112} incoherent twin boundary that leads to de-twinning of nanotwins. Second, a mechanism for twin 3{111} coherent twin bound dislocation multiplication at boundary

A Review on the Statistical Methods and Implementation to Homogeneity Assessment of Certified Reference Materials in Relation to Uncertainty

An importance of data analysis, methods for homogeneity test and standard uncertainty evaluation associated in any measurement for exact quantification of certified value of any product is vital to be stressed in the scientific community. Herein, we have collectively summarized the detailed discussion on the basics of statistical parameters such as mean, median, mode, standard deviation, variance, range, normal distribution, and central limit theorem. Various statistical analysis methods such asztest,ttest, Chi-squared test, and ANOVA includingFtest have also been discussed in great detail to test the homogeneity of samples for certification of the reference material. The ISO guide 35 (2006) and Guide to Uncertainty in Measurement (GUM) are primarily considered to describe the basic concept of evaluating the associated uncertainty in the light of GUM modelling approach to avoid the error in the measurement which normally occurs in many scientific reports

PLA Microparticles for Pulmonary Delivery of AntiTB drugs: Biodistribution study

A dry powder inhalable (DPI) microparticles comprising anti-tuberculosis drugs incorporated in biodegradable polymers was developed for the treatment of pulmonary tuberculosis (P. Muttil _et al_. 2007). Poly L-lactic acid (PLA) microparticles incorporating a high payload of rifabutin and isoniazid were fabricated by spray drying (Buchi 190). Microparticles were composed of PLA and the drugs (rifabutin and isoniazid) at a 2:1:1 weight ratio. Microparticles of desired high encapsulation efficiency and sustained release characteristics were produced having a diameter range of 2-10 µm (Malvern Mastersizer 2000). Differential scanning calorimetry (DSC) was carried out to study drug polymer interaction. The time course of tissue biodistribution following a single inhalation dose of microparticles was evaluated. 
Thirty-two BALB/c mice were divided into groups of four and administered the DPI using an in-house (nose only) apparatus (Kaur _et al_. 2008; Verma _et al_. 2008). A validated HPLC method was used for determination of rifabutin and isoniazid in the lungs (target organ), liver and kidneys (major sites of toxicity) at different time-points after inhalation. A comparison was made with mice receiving free drugs (intravenous) at equivalent doses. Deposition of microparticles in lungs of mice following aerosolization was also evaluated. Pharmacokinetic parameters in different organs were calculated using WinNonlin software version 5.2. Area under the concentration-time curve observed (AUC~obs~), C~max~, half-life (t~½~) and clearance (CL) in lungs following inhalation /intravenous administration were:
*Rifabutin*: AUC~obs~-96h= 1697.39 ±154.67 (187.63 ±23.93) µg/ml^-1^hr^-1^; C~max~ = 33.42±3.80 (4.17±0.31) µg.ml^-1^; t~½~= 78.08±9.42 (34.00 ±3.31) and Cl= 1.16±.22 (0.68 ±0.45) ml.h^-1^.
*Isoniazid*: AUC~obs~-24h= 566.31±123.96 (99.85 ±14.24) µg/ml^-1^hr^-1^; Cmax= 24.02±1.71 (8.16±0.93) µg.ml^-1^; t~½~= 25.88±12.16 (6.45±3.24) h; and Cl= 5.47±1.30 (0.96±0.14) ml.h^-1^.
The relative bioavailability of both drugs incorporated in microparticles was significantly higher compared with free drugs. Peak levels of isoniazid and rifabutin in lungs (target organ) were much higher than those in the liver and kidney of mice in case of inhalation as compared to intravenous administration. Inhalation of microparticles resulted in targeting both drugs to the lungs, with the effect being more pronounced in the case of rifabutin than isoniazid. High and prolonged drug concentrations and increased AUC values (~9-fold and ~6 fold increase of rifabutin and isoniazid in case of lungs) with respect to free drugs were observed. Significant decrease in drug concentration was found in the liver and kidneys. Drug levels were maintained above the minimum inhibitory concentration (MIC) in organs through out the study after administration of encapsulated drugs. Based on favorable biodistribution kinetics, these microparticles hold great potential in reducing dosing frequency and toxicity of antituberculosis drugs.
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