Application of Laser Scanning Confocal Microscopy for the Visualization of M. tuberculosis in Lung Tissue Samples with Weak Ziehl-Neelsen Staining.

One of the key requirements for the diagnosis of pulmonary tuberculosis is the identification of M. tuberculosis in tissue. In this paper, we present the advantages of specific fluorescent antibody labelling, combined with laser scanning confocal microscopy (LSCM), for the detection of M. tuberculosis in histological specimens of lung tissues. We demonstrate that the application of LSCM allows: (i) The automatic acquisition of images of the whole slice and, hence, the determination of regions for subsequent analysis; (ii) the acquisition of images of thick (20-40 µm) slices at high resolution; (iii) single bacteria identification; and (iv) 3D reconstruction, in order to obtain additional information about the distribution, size, and morphology of solitary M. tuberculosis; as well as their aggregates and colonies, in various regions of tuberculosis inflammation. LSCM allows for the discrimination of the non-specific fluorescence of bacteria-like particles and their aggregates presented in histological lung samples, from the specific fluorescence of labelled M. tuberculosis, using spectrum emission analysis. The applied method was effective in the identification of M. tuberculosis in lung histological samples with weak Ziehl-Neelsen staining. Altogether, combining immunofluorescent labelling with the application of LSCM visualization significantly increases the effectiveness of M. tuberculosis detection.

B-lymphocytes forming follicle-like structures in the lung tissue of tuberculosis-infected mice: Dynamics, phenotypes and functional activity.

During tuberculosis (TB) infection, B cells form follicles in close vicinity of lung granuloma. We assessed the dynamics of follicle formation, surface phenotypes and functional activity of lung B cells during TB course in genetically susceptible mice. The follicles appeared early post infection and peaked at weeks 7-8. Lung B cells resembled classical B2 cells (CD19+IgMloIgDhiCD1d-CD21/35intCD5-CD11b-CD43-), but differed from them by the absence of B2 marker CD23. Lung B-cells constitutively expressed MHC II molecules, presented mycobacterial antigens to immune CD4+ T-cells and produced high amounts of IL-6 and IL-11, but no classical type 1 (TNF-α, IFN-γ), or anti-inflammatory (IL-10, TGF-ß) cytokines. The total antibody response in tuberculous lung showed almost no specificity to mycobacteria. A panel of monoclonal antibodies obtained from lung B cells contained only few clones with reactivity to mycobacteria. Our results suggest that anti-TB B cell response in the lung has clear pathological and doubtful protective role.