Towards a framework for the evolutionary genomics of Kinetoplastids: what kind of data and how much?

The current status of kinetoplastids phylogeny and evolution is discussed in view of the recent progresses on genomics. Some ideas on a potential framework for the evolutionary genomics of kinetoplastids are presented.     

The role of intravascular ultrasound imaging in vascular brachytherapy

Intracoronary brachytherapy has recently emerged as a new therapy to prevent restenosis. Initial experimental work was achieved in animal models and the results were assessed by histomorphometry. Initial clinical trials used angiography to guide dosimetry and to assess efficacy. Intravascular ultrasound (IVUS) permits tomographic examination of the vessel wall, elucidating the true morphology of the lumen and transmural components, which cannot be investigated on the lumenogram obtained by angiography. This paper reviews the use of IVUS in the clinical studies of brachytherapy conducted to date. IVUS allows clinicians to make a thorough assessment of the remodeling of the vessel and appears to have a major role to play in facilitating understanding of the underlying mechanisms of action in this emerging field. The authors propose that state-of-the-art IVUS techniques should be employed to further knowledge of the mechanisms of action of brachytherapy in atherosclerotic human coronary arteries.     

Cutting edge: vitamin D-mediated human antimicrobial activity against Mycobacterium tuberculosis is dependent on the induction of cathelicidin

Host defense against intracellular pathogens depends upon innate and adaptive antimicrobial effector pathways. TLR2/1-activation of monocytes leads to the vitamin D-dependent production of cathelicidin and, at the same time, an antimicrobial activity against intracellular Mycobacterium tuberculosis. To determine whether induction of cathelicidin was required for the vitamin D-triggered antimicrobial activity, the human monocytic cell line THP-1 was infected with M. tuberculosis H37Ra and then activated with the active vitamin D hormone 1,25-dihydroxyvitamin D(3) (1,25D(3)). 1,25D(3) stimulation resulted in antimicrobial activity against intracellular M. tuberculosis and expression of cathelicidin mRNA and protein. Using small interfering RNA (siRNA) specific for cathelicidin, 1,25D(3)-induced cathelicidin mRNA and protein expressions were efficiently knocked down, whereas a nonspecific siRNA control had little effect. Finally, 1,25D(3)-induced antimicrobial activity was completely inhibited in the presence of siRNA against cathelicidin, instead leading to enhanced intracellular growth of mycobacteria. These data demonstrate that cathelicidin is required for the 1,25D(3)-triggered antimicrobial activity against intracellular M. tuberculosis.     

Improving model construction of profile HMMs for remote homology detection through structural alignment

Background  

Remote homology detection is a challenging problem in Bioinformatics. Arguably, profile Hidden Markov Models (pHMMs) are one of the most successful approaches in addressing this important problem. pHMM packages present a relatively small computational cost, and perform particularly well at recognizing remote homologies. This raises the question of whether structural alignments could impact the performance of pHMMs trained from proteins in the Twilight Zone, as structural alignments are often more accurate than sequence alignments at identifying motifs and functional residues. Next, we assess the impact of using structural alignments in pHMM performance.     

Identification of an altered peptide ligand based on the endogenously presented, rheumatoid arthritis-associated, human cartilage glycoprotein-39(263-275) epitope: an MHC anchor variant peptide for immune modulation

We sought to identify an altered peptide ligand (APL) based on the endogenously expressed synovial auto-epitope of human cartilage glycoprotein-39 (HC gp-39) for modulation of cognate, HLA-DR4-restricted T cells. For this purpose we employed a panel of well-characterized T cell hybridomas generated from HC gp-39-immunized HLA-DR4 transgenic mice. The hybridomas all respond to the HC gp-39(263-275) epitope when bound to HLA-DR4(B1*0401) but differ in their fine specificities. First, the major histocompatibility complex (MHC) and T-cell receptor (TCR) contact residues were identified by analysis of single site substituted analogue peptides for HLA-DR4 binding and cognate T cell recognition using both T hybridomas and polyclonal T cells from peptide-immunized HLA-DR4 transgenic mice. Analysis of single site substituted APL by cognate T cells led to identification of Phe265 as the dominant MHC anchor. The amino acids Ala268, Ser269, Glu271 and Thr272 constituted the major TCR contact residues, as substitution at these positions did not affect HLA-DR4(B1*0401) binding but abrogated T cell responses. A structural model for visualisation of TCR recognition was derived. Second, a set of non-classical APLs, modified at the MHC key anchor position but with unaltered TCR contacts, was developed. When these APLs were analysed, a partial TCR agonist was identified and found to modulate the HC gp-39(263-275)-specific, pro-inflammatory response in HLA-DR4 transgenic mice. We identified a non-classical APL by modification of the p1 MHC anchor in a synovial auto-epitope. This APL may qualify for rheumatoid arthritis immunotherapy.     

Immobilization of neural cells in three-dimensional matrices for biosensor applications

To overcome logistical difficulties with current designs of cell- or tissue-based biosensors which have individual cells or tissue slices immobilized on membranes or microelectrode arrays, we have proposed a system that uses three-dimensional cultures of neural cells immobilized in hydrogel matrices. In this design, immobilized cells would be maintained in a reservoir and then transferred to a detector platform when needed for analysis. The development of such a system relies upon a renewable supply of cells and the ability to culture cells for long periods of time in three-dimensions while maintaining their physiological function. To investigate the ability to culture neural cells in 3D matrices, embryonic rat cortical neurons and astrocytes were immobilized by matrix entrapment in a novel sugar poly(acrylate) hydrogel and collagen gels. The sugar poly(acrylate) hydrogel does not appear to support neural cell growth as a result of a lack of cell adherence, small pore size and, possibly, harshness of synthesis conditions. In contrast, collagen gels support the growth of cortical neurons, astrocytes, as well as neural progenitor cells. Evidence is also presented from immunocytochemistry and patch-clamp measurements which shows that neural progenitor cells proliferate in culture and can be induced to differentiate into neural cell types. Thus, they potentially represent a renewable cell source.     

Azurophil Granule Proteins Constitute the Major Mycobactericidal Proteins in Human Neutrophils and Enhance the Killing of Mycobacteria in Macrophages

Pathogenic mycobacteria reside in, and are in turn controlled by, macrophages. However, emerging data suggest that neutrophils also play a critical role in innate immunity to tuberculosis, presumably by their different antibacterial granule proteins. In this study, we purified neutrophil azurophil and specific granules and systematically analyzed the antimycobacterial activity of some purified azurophil and specific granule proteins against M. smegmatis, M. bovis-BCG and M. tuberculosis H37Rv. Using gel overlay and colony forming unit assays we showed that the defensin-depleted azurophil granule proteins (AZP) were more active against mycobacteria compared to other granule proteins and cytosolic proteins. The proteins showing antimycobacterial activity were identified by MALDI-TOF mass spectrometry. Electron microscopic studies demonstrate that the AZP disintegrate bacterial cell membrane resulting in killing of mycobacteria. Exogenous addition of AZP to murine macrophage RAW 264.7, THP-1 and peripheral blood monocyte-derived macrophages significantly reduced the intracellular survival of mycobacteria without exhibiting cytotoxic activity on macrophages. Immunofluorescence studies showed that macrophages actively endocytose neutrophil granular proteins. Treatment with AZP resulted in increase in co-localization of BCG containing phagosomes with lysosomes but not in increase of autophagy. These data demonstrate that neutrophil azurophil proteins may play an important role in controlling intracellular survival of mycobacteria in macrophages.     

Mycobacterial lipopeptides elicit CD4+ CTLs in Mycobacterium tuberculosis-infected humans

In searching for immunogenic molecules with the potential to induce protective immune responses against tuberculosis, we developed an ex vivo model to study frequency, phenotype, and effector functions of human T lymphocytes recognizing hydrophobic Ags of Mycobacterium tuberculosis (M.Tb). To obtain unbiased results, we characterized T lymphocytes responding to a crude cell wall extract (chloroform methanol extract of M.Tb (M.Tb-CME)) containing a broad spectrum of mycobacterial glycolipids and lipopeptides. A significant proportion of T lymphocytes recognized M.Tb-CME (290 IFN-gamma+ T cells/10(5) PBMCs) and developed to effector memory cells as determined by the expression of CD45RO and the chemokine receptors CXCR3 and CCR5. Expanded lymphocytes fulfilled all criteria required for an efficient immune response against tuberculosis: 1) release of macrophage-activating Th1 cytokines and chemokines required for the spatial organization of local immune responses, 2) cytolytic activity against Ag-pulsed macrophages, and 3) recognition of infected macrophages and killing of the intracellular bacteria. Phenotypically, M.Tb-CME-expanded cells were CD4+ and MHC class II restricted, challenging current concepts that cytotoxic and antimicrobial effector cells are restricted to the CD8+ T cell subset. Pretreatment of M.Tb-CME with protease or chemical delipidation abrogated the biological activity, suggesting that responses were directed toward mycobacterial lipopeptides. These findings suggest that lipidated peptides are presented by M.Tb-infected macrophages and elicit CD4+ cytolytic and antimicrobial T lymphocytes. Our data support an emerging concept to include hydrophobic microbial Ags in vaccines against tuberculosis.     

Induction of TNF in human alveolar macrophages as a potential evasion mechanism of virulent Mycobacterium tuberculosis.

The ability of macrophages to release cytokines is crucial to the host response to intracellular infection. In particular, macrophage-derived TNF plays an important role in the host response to infection with the intracellular pathogen Mycobacterium tuberculosis. In mice, TNF is indispensable for the formation of tuberculous granulomas, which serve to demarcate the virulent bacterium. TNF is also implicated in many of the immunopathological features of tuberculosis. To investigate the role of TNF in the local immune response, we infected human alveolar macrophages with virulent and attenuated mycobacteria. Infection with virulent strains induced the secretion of significantly higher levels of bioactive TNF than attenuated strains correlating with their ability to multiply intracellularly. Treatment of infected macrophages with neutralizing anti-TNF Abs reduced the growth rate of intracellular bacteria, whereas bacterial replication was augmented by addition of exogenous TNF. Infected and uninfected macrophages contributed to cytokine production as determined by double-staining of M. tuberculosis and intracellular TNF. The induction of TNF by human alveolar macrophages at the site of infection permits the multiplication of intracellular bacteria and may therefore present an evasion mechanism of human pathogens.