In addition to direct bactericidal activities, such as phagocytosis and generation of reactive oxygen species (ROS), neutrophils can regulate the inflammatory response by undergoing apoptosis. We found that infection of human neutrophils with Mycobacterium tuberculosis (Mtb) induced rapid cell death displaying the characteristic features of apoptosis such as morphologic changes, phosphatidylserine exposure, and DNA fragmentation. Both a virulent (H37Rv) and an attenuated (H37Ra) strain of Mtb were equally effective in inducing apoptosis. Pretreatment of neutrophils with antioxidants or an inhibitor of NADPH oxidase markedly blocked Mtb-induced apoptosis but did not affect spontaneous apoptosis. Activation of caspase-3 was evident in neutrophils undergoing spontaneous apoptosis, but it was markedly augmented and accelerated during Mtb-induced apoptosis. The Mtb-induced apoptosis was associated with a speedy and transient increase in expression of Bax protein, a proapoptotic member of the Bcl-2 family, and a more prominent reduction in expression of the antiapoptotic protein Bcl-xL. Pretreatment with an inhibitor of NADPH oxidase distinctly suppressed the Mtb-stimulated activation of caspase-3 and alteration of Bax/Bcl-xL expression in neutrophils. These results indicate that infection with Mtb causes ROS-dependent alteration of Bax/Bcl-xL expression and activation of caspase-3, and thereby induces apoptosis in human neutrophils. Moreover, we found that phagocytosis of Mtb-induced apoptotic neutrophils markedly increased the production of proinflammatory cytokine TNF-α by human macrophages. Therefore, the ROS-dependent apoptosis in Mtb-stimulated neutrophils may represent an important host defense mechanism aimed at selective removal of infected cells at the inflamed site, which in turn aids the functional activities of local macrophages.

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Our findings are consistent with the belief that a mutation at position 1401 in rrs leads to resistance to capreomycin, amikacin and kanamycin. The data also show that tlyA is not a sensitive genetic marker for capreomycin resistance in clinical isolates of M. tuberculosis, as mutations in this gene are infrequent and not all mutations in tlyA lead to capreomycin resistance.

Recent studies have shown that human neutrophils play a significant protective role in mycobacteria infection. When encountered with mycobacteria, neutrophils exhibit the typical early bactericidal responses including phagocytosis and generation of reactive oxygen intermediates (ROI), but the underlying mechanisms are largely unknown. The present study shows that stimulation of neutrophils with an attenuated strain of Mycobacterium tuberculosis H37Ra (Mtb) led to a tyrosine kinase-dependent ROI production in these cells. Stimulation with Mtb induces a rapid and transient tyrosine phosphorylation of several proteins, one of which was identified as phospholipase C gamma 2 (PLC gamma 2). Several tyrosine-phosphorylated proteins were associated with the PLC gamma 2 precipitates from Mtb-stimulated neutrophils, of which pp46 was characterized as the Shc adapter protein. A role for PLC gamma 2-Shc association in the generation of ROI is supported by the observations that stimulation with Mtb causes the activation of p38 mitogen-activated protein kinase (MAPK), a downstream target of the Shc/Ras signaling cascade, and that the effect of genistein on ROI production coincided with its ability to inhibit both PLC gamma 2-Shc association and p38 MAPK activation. Moreover, pretreatment of neutrophils with a PLC inhibitor markedly suppresses the Mtb-stimulated ROI production as well as p38 MAPK activation in these cells. Taken together, these results indicate that stimulation of neutrophils with Mtb triggers the tyrosine phosphorylation of PLC gamma 2 and its association with Shc, and that such association is critical for the Mtb-stimulated ROI production through activating p38 MAPK.

The Swedish Cervical Cytology Biobank (SCCB) is the first national initiative of a prospective repository for liquid-based gynecological cell samples (LBC) from women participating in organized cervical cancer screening programs. Development and implementation of a nationally standardized method for the handling and long-term storage of cervical cell samples has been a primary goal for the Swedish hub of The Biobanking and Molecular Resource Infrastructure (BBMRI.se, www.bbmri.se). The sample handling protocol was developed through i) review of the literature on biobanking processes, ii) wide consultation within the academic community, and iii) various verification assays in collaboration with the clinical cytology laboratories. A general quality management system, covering all aspects of sample handling and storage, has been established. BBMRI.se financed the development and implementation of SCCB. The protocol established in the pilot project in Stockholm is now being implemented in other counties in Sweden, and during this year, more than 120,000 LBC samples will be processed for biobanking nationwide. SCCB is embedded in a comprehensive cytology diagnostic registry and will be linked with the national cancer registry to constitute a nearly inexhaustible resource for performance of fundamental and applied biologic research.