Mtb h37ra

Mtb H37Ra was obtained from ATCC (ATCC no. 25177). RPMI 1640 medium, FITC-conjugated anti-human IgG antibody, Lysotracker Red, SYTO 9 and anti-rabbit Alexa Fluor antibody were from Invitrogen. Rabbit anti-iNOS and anti-LAMP-1 antibodies were from Thermo Pierce Biotech. Anti-CD14 mAb was from Boehringer-Mannheim GmbH. Rabbit anti-sheep RBC antibody was from Rockland Immunochemicals. mAbs against Mtb-Acr (IT-4) and LAM (CS-35) were gifted by UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. Affinity purified peroxidase-conjugated anti-human and anti-mouse IgG antibodies, anti-calmodulin antibody, o-phenylenediamine (OPD), diaminobenzidine (DAB), 2 µM amine-modified green fluorescent polystyrene beads, citrate phosphate dextrose and other laboratory chemicals were from Sigma-Aldrich. BD optEIA cytokine ELISA kits and Middlebrook (MB) media and supplements were obtained from BD Biosciences. Fluorescent NO probe FL2E was purchased from Strem Chemicals, Inc. Ficoll-isopaque was obtained from GE-Biosciences. Ninety-six-well clear glass bottom black imaging plates were from Thermo Nunc.

After the BCG (5 × 10⁴ CFU/0.2 mL/subcutaneous) vaccination, 5 mice per group were subcutaneously immunized on their backs with 10 μg of Ag85B and Rv2882c emulsified with dimethyl dioctadecylammonium bromide (DDA; Sigma-Aldrich) and 25 μg monophosphoryl lipid A (MPL; Sigma-Aldrich) in a total volume of 0.2 mL two times over a 4-week interval. After 6 weeks, the vaccinated mice were infected with Mtb H37Ra (ATCC 25177). Briefly, following anesthetization with a xylazine:zoletil (9:1) mixture, 5 mice per group were intratracheally infected with 50-μL suspensions at infectious dose of 10⁶ CFU of H37Ra per mouse lung.

Strains of avirulent Mtb H37Ra or virulent Mtb H37Rv were obtained from the American Type Culture Collection (ATCC). Irradiated H37Rv (iH37Rv) was obtained through BEI Resources (NIAID, NIH: Mycobacterium tuberculosis, Strain H37Rv, Gamma-Irradiated Whole Cells, NR-14819). Mtb H37Ra and Mtb H37Rv stocks were propagated in Middlebrook 7H9 broth (Becton Dickinson), made up in low endotoxin WFI H₂O (Merck Millipore) and supplemented with ADC (albumin, dextrose, catalase) (Becton Dickinson) and 0.05% Tween 80 (Sparks Lab Supplies). Aliquots were stored at −80°C, thawed and propagated in Middlebrook 7H9 broth to log phase prior to use.

Mtb H37Ra (American Type Culture Collection 25177) was grown in Middlebrook 7H9 medium supplemented with OADC for 4 days at 37˚C. High-flux polysulfone hollow fiber cartridges (C2011, FiberCell Systems Inc.) were equilibrated for 3 days with 7H9 OADC. Initial PK data were obtained for Bio-AMS diluted in 7H9 OADC and infused with a syringe pump into the hollow fiber system. A clearance flow rate that simulated a Bio-AMS half-life of 9 to 10 hours in the extracapillary space was used. To determine the susceptibility of H37Ra to Bio-AMS under defined PK parameters, a new hollow fiber cartridge pre-equilibrated with 7H9 OADC was inoculated with 20 ml of a bacterial suspension of 10⁴ to 10⁶ CFU/ml. The culture was allowed to establish in the cartridge extracapillary space under a constant flow of 7H9 OADC (30 ml/hour) for 2 days before the first Bio-AMS infusion. To achieve a C_max of at least 9 µM in the extracapillary space of the hollow fiber system, 1.42 mg of Bio-AMS was infused for 60 min at a flow rate of 30 ml/hour. This infusion was repeated on an almost daily basis for 18 days. Bio-AMS concentrations in hollow fiber samples were quantified by mass spectrometry, except that only 1 µl volumes of extracted samples were injected. PK analysis of drug concentrations attained in both the central reservoir and extracapillary space was performed on days 0 and 14.