Shaping the niche in macrophages: Genetic diversity of the M. tuberculosis complex and its consequences for the infected host.

Pathogenic mycobacteria of the Mycobacterium tuberculosis complex (MTBC) have co-evolved with their individual hosts and are able to transform the hostile environment of the macrophage into a permissive cellular habitat. The impact of MTBC genetic variability has long been considered largely unimportant in TB pathogenesis. Members of the MTBC can now be distinguished into three major phylogenetic groups consisting of 7 phylogenetic lineages and more than 30 so called sub-lineages/subgroups. MTBC genetic diversity indeed influences the transmissibility and virulence of clinical MTBC isolates as well as the immune response and the clinical outcome. Here we review the genetic diversity and epidemiology of MTBC strains and describe the current knowledge about the host immune response to infection with MTBC clinical isolates using human and murine experimental model systems in vivo and in vitro. We discuss the role of innate cytokines in detail and portray two in our group recently developed approaches to characterize the intracellular niches of MTBC strains. Characterizing the niches and deciphering the strategies of MTBC strains to transform an antibacterial effector cell into a permissive cellular habitat offers the opportunity to identify strain- and lineage-specific key factors which may represent targets for novel antimicrobial or host directed therapies for tuberculosis.

Surfactant Protein A Enhances Constitutive Immune Functions of Clathrin Heavy Chain and Clathrin Adaptor Protein 2.

NF-κB transcription factors are key regulators of pulmonary inflammatory disorders and repair. Constitutive lung cell type- and microenvironment-specific NF-κB/inhibitor κBα (IκB-α) regulation, however, is poorly understood. Surfactant protein (SP)-A provides both a critical homeostatic and lung defense control, in part by immune instruction of alveolar macrophages (AMs) via clathrin-mediated endocytosis. The central endocytic proteins, clathrin heavy chain (CHC) and the clathrin adaptor protein (AP) complex AP2, have pivotal alternative roles in cellular homeostasis that are endocytosis independent. Here, we dissect endocytic from alternative functions of CHC, the α-subunit of AP2, and dynamin in basal and SP-A-modified LPS signaling of macrophages. As revealed by pharmacological inhibition and RNA interference in primary AMs and RAW264.7 macrophages, respectively, CHC and α-adaptin, but not dynamin, prevent IκB-α degradation and TNF-α release, independent of their canonical role in membrane trafficking. Kinetics studies employing confocal microscopy, Western analysis, and immunomagnetic sorting revealed that SP-A transiently enhances the basal protein expression of CHC and α-adaptin, depending on early activation of protein kinase CK2 (former casein kinase II) and Akt1 in primary AMs from rats, SP-A(+/+), and SP-A(-/-) mice, as well as in vivo when intratracheally administered to SP-A(+/+) mice. Constitutive immunomodulation by SP-A, but not SP-A-mediated inhibition of LPS-induced NF-κB activity and TNF-α release, requires CHC, α-adaptin, and dynamin. Our data demonstrate that endocytic proteins constitutively restrict NF-κB activity in macrophages and provide evidence that SP-A enhances the immune regulatory capacity of these proteins, revealing a previously unknown pathway of microenvironment-specific NF-κB regulation in the lung.

Immunomagnetic isolation of pathogen-containing phagosomes and apoptotic blebs from primary phagocytes.

Macrophages and polymorphonuclear neutrophils are professional phagocytes essential in the initial host response against intracellular pathogens such as Mycobacterium tuberculosis. Phagocytosis is the first step in phagocyte-pathogen interaction, where the pathogen is engulfed into a membrane-enclosed compartment termed a phagosome. Subsequent effector functions of phagocytes result in killing and degradation of the pathogen by promoting phagosome maturation, and, terminally, phago-lysosome fusion. Intracellular pathogenic microbes use various strategies to avoid detection and elimination by phagocytes, including induction of apoptosis to escape host cells, thereby generating apoptotic blebs as shuttles to other cells for pathogens and antigens thereof. Hence, phagosomes represent compartments where host and pathogen become quite intimate, and apoptotic blebs are carrier bags of the pathogen's legacy. In order to investigate the molecular mechanisms underlying these interactions, both phagosomes and apoptotic blebs are required as purified subcellular fractions for subsequent analysis of their biochemical properties. Here, we describe a lipid-based procedure to magnetically label surfaces of either pathogenic mycobacteria or apoptotic blebs for purification by a strong magnetic field in a novel free-flow system.

Lipid-labeling facilitates a novel magnetic isolation procedure to characterize pathogen-containing phagosomes.

Here we describe a novel approach for the isolation and biochemical characterization of pathogen-containing compartments from primary cells: We developed a lipid-based procedure to magnetically label the surface of bacteria and visualized the label by scanning and transmission electron microscopy (SEM, TEM). We performed infection experiments with magnetically labeled Mycobacterium avium, M. tuberculosis and Listeria monocytogenes and isolated magnetic bacteria-containing phagosomes using a strong magnetic field in a novel free-flow system. Magnetic labeling of M. tuberculosis did not affect the virulence characteristics of the bacteria during infection experiments addressing host cell activation, phagosome maturation delay and replication in macrophages in vitro. Biochemical analyses of the magnetic phagosome-containing fractions provided evidence of an enhanced presence of bacterial antigens and a differential distribution of proteins involved in the endocytic pathway over time as well as cytokine-dependent changes in the phagosomal protein composition. The newly developed method represents a useful approach to characterize and compare pathogen-containing compartments, in order to identify microbial and host cell targets for novel anti-infective strategies.

Comparison of Lornoxicam and Rofecoxib in Patients with Activated Osteoarthritis (COLOR Study).

BACKGROUND AND OBJECTIVE: Impaired mobility and pain mean a loss of quality of life for patients with rheumatic diseases. Therefore the initial aim of therapy is rapid and efficient analgesia in order to achieve the best possible result for these patients. Lornoxicam is a strong analgesic and anti-inflammatory NSAID with balanced cyclo-oxygenase (COX)-1/COX-2 inhibition and excellent tolerability. In the course of the development of selective COX-2 inhibitors, it was maintained that COX-2 inhibitors decrease the risk of injury to the upper gastrointestinal (GI) tract with a similar efficacy to that of classic NSAIDs. However, a clinical trial comparing both substances has never been performed. In the present study we investigated the treatment of patients with osteoarthritis with lornoxicam in comparison with treatment with the selective COX-2 inhibitor rofecoxib. This multicentre clinical investigation focused on efficacy and tolerability. PATIENTS AND METHODS: A total of 2520 patients (most of them with osteoarthritis) were treated over 25 days on average. Before and after treatment patients documented their individual scores for pain on movement, at rest and during the night, and their individual duration of morning stiffness as well as the consequent grade of restriction. At the end of the study all individuals involved judged the efficacy and safety of the therapy. RESULTS: All improvements in each efficacy parameter were clinically relevant in each treatment group and significantly superior (p < 0.001) in the lornoxicam group. Pain on movement (-45.3%), at rest (-42.0%) and at night (-42.5%) was reduced by rofecoxib, whereas improvements after treatment with lornoxicam exceeded those effects significantly (-55.8%, -55.8% and -59.9%, respectively). Shortening of the duration of morning stiffness was significantly (p < 0.001) more pronounced with lornoxicam (-66.6%) than with rofecoxib (-50.2%). Nearly three times as many patients discontinued rofecoxib treatment because of lack of efficacy compared with lornoxicam treatment (8.9% versus 3.4%). Physicians judged lornoxicam to be markedly superior to rofecoxib, since excellent efficacy was observed in 40.9% of all cases versus 20.1% with rofecoxib. Serious adverse events did not occur. Adverse events were reported in 5.4% of all lornoxicam patients compared with 12.0% of the rofecoxib recipients (p < 0.001). GI symptoms showed a slight trend of being less frequent following rofecoxib therapy. CONCLUSIONS: The results of this study confirmed the efficacy and safety of both drugs. Lornoxicam and rofecoxib are effective in the treatment of patients with activated osteoarthritis; the analgesic and anti-inflammatory effects of lornoxicam are significantly superior to those of rofecoxib without inferiority in tolerability.