Increased intracellular growth of Mycobacterium avium in HIV-1 exposed monocyte-derived dendritic cells

Intracellular growth of Mycobacterium avium subspecies and global transcriptional responses in human macrophages after infection

“…Bacterial inocula for the gene expression assay were prepared from seven days subcultures grown on plates of Middlebrook 7H10 (BD Diagnostics) with 10% OADC (BD Diagnostics) at 37°C. To adjust the number of bacteria in the inoculum, hsp 65 real-time PCR [13] was performed on serial dilutions in HBSS (Invitrogen, Oslo, Norway) of a suspension adjusted to McFarland standard 2.0, homogenised through a 23 G needle to minimise clumping.…”

“…Enumeration of mycobacteria and cells was achieved by performing a duplex real time PCR of a 103 bp long segment of the mycobacterial hsp65 gene and a 150 bp long segment of the human β-globin gene on the lysates, as described by Salte et al [13]. Briefly, the segment of the mycobacterial gene hsp65 ( GroEl2 ) was amplified using the primers MycoFP1 (5′CGAGGCGATGGACAAGGT-3′) and TB 12 (5′CTTGTCGAACCGCATACCCT-3′), and the VIC tagged TaqMan-MGB probe MycoPr1 (5′-AACGAGGGCGTCACCGTCG-3′) [14].…”

“…Briefly, the segment of the mycobacterial gene hsp65 ( GroEl2 ) was amplified using the primers MycoFP1 (5′CGAGGCGATGGACAAGGT-3′) and TB 12 (5′CTTGTCGAACCGCATACCCT-3′), and the VIC tagged TaqMan-MGB probe MycoPr1 (5′-AACGAGGGCGTCACCGTCG-3′) [14]. The segment of the human β-globin gene was amplified by using the primers BG-F (5′-TGCCTATCAGAAAGTGGTGGCT-3′) and BG-R (5′-GCTCAAGGCCCTTCATAATATCC-3′), and the FAM tagged TaqMan-MGB probe BG-TAQ (5′-TGGCTAATGCCCTGGCCCACAA-3′) [13,15]. Dilutions of known concentrations of commercially available mycobacterial DNA (ATCC-19015D-5, LGC Standards, Middlesex, UK) and human DNA (Applied Biosystems, Foster City, CA, USA) were run together with the samples and used to create standard curves.…”

“…Dilutions of known concentrations of commercially available mycobacterial DNA (ATCC-19015D-5, LGC Standards, Middlesex, UK) and human DNA (Applied Biosystems, Foster City, CA, USA) were run together with the samples and used to create standard curves. The conversion of the known amount of standard DNA to number of bacterial and human genomes were described by Salte et al [13], as well as the validation of the number of mycobacterial targets against CFU counts. Total volume of each reaction mixture was 20 μl, consisting of 8 μl lysate, 10 μl Quantitect® mastermix (Quiagen, West Sussex, UK) and 1 μl of a primer/probe mix for each target, with a stock concentration of 8 μM for each primer and 4 μM for the probe, resulting in a final primer- and probe concentration of 0.4 μM and 0.2 μM, respectively.…”

Antimycobacterial Activity Some Different Lamiaceae Plant Extracts Containing Flavonoids and Other Phenolic Compounds

“…In addition, infection with Human immunodeficiency virus type 1 (HIV-1) disrupts immunological control of Mycobacterium infections due to the loss of CD4+ T cells. Salte et al (2011) reported that Mycobacterium avium is one of the most common opportunistic infections among AIDS patients. Snider et al (1985) examined the transmission of MDR-TB strains from adult to child contacts and confirmed the progression of the disease by DNA fingerprint studies.…”