Apoptosis facilitates antigen presentation to T lymphocytes through MHC-I and CD1 in tuberculosis.

Protective immunity against Mycobacterium tuberculosis involves major histocompatibility complex class I (MHC-I)- and CD1-restricted CD8 T cells, but the mechanisms underlying antigen delivery to antigen-presenting molecules remain enigmatic. Macrophages, the primary host cells for mycobacteria, are CD1-negative. Here we show that M. tuberculosis phagosomes are secluded from the cytosolic MHC-I processing pathway and that mycobacteria-infected cells lose their antigen-presenting capacity. We also show that mycobacteria induce apoptosis in macrophages, causing the release of apoptotic vesicles that carry mycobacterial antigens to uninfected antigen-presenting cells (APCs). Inhibition of apoptosis reduced transfer of antigens to bystander cells and activation of CD8 T cells. Uninfected dendritic cells, which engulfed extracellular vesicles, were indispensable for subsequent cross-presentation of antigens, through MHC-I and CD1b, to T cells from mycobacteria-sensitized donors. This new 'detour' pathway for presentation of antigens from a phagosome-contained pathogen shows the functional significance of infection-induced apoptosis in the activation of CD8 T cells specific for both protein and glycolipid antigens in tuberculosis.

Correction of the iron overload defect in beta-2-microglobulin knockout mice by lactoferrin abolishes their increased susceptibility to tuberculosis.

As a resident of early endosomal phagosomes, Mycobacterium tuberculosis is connected to the iron uptake system of the host macrophage. beta-2-microglobulin (beta2m) knockout (KO) mice are more susceptible to tuberculosis than wild-type mice, which is generally taken as a proof for the role of major histocompatibility complex class I (MHC-I)-restricted CD8 T cells in protection against M. tuberculosis. However, beta2m associates with a number of MHC-I-like proteins, including HFE. This protein regulates transferrin receptor mediated iron uptake and mutations in its gene cause hereditary iron overload (hemochromatosis). Accordingly, beta2m-deficient mice suffer from tissue iron overload. Here, we show that modulating the extracellular iron pool in beta2m-KO mice by lactoferrin treatment significantly reduces the burden of M. tuberculosis to numbers comparable to those observed in MHC class I-KO mice. In parallel, the generation of nitric oxide impaired in beta2m-KO mice was rescued. Conversely, iron overload in the immunocompetent host exacerbated disease. Consistent with this, iron deprivation in infected resting macrophages was detrimental for intracellular mycobacteria. Our data establish: (a) defective iron metabolism explains the increased susceptibility of beta2m-KO mice over MHC-I-KO mice, and (b) iron overload represents an exacerbating cofactor for tuberculosis.

Iron Deficiency Impairs Intra-Hepatic Lymphocyte Mediated Immune Response.

Hepatic expression of iron homeostasis genes and serum iron parameters predict the success of immunosuppression withdrawal following clinical liver transplantation, a phenomenon known as spontaneous operational tolerance. In experimental animal models, spontaneous liver allograft tolerance is established through a process that requires intra-hepatic lymphocyte activation and deletion. Our aim was to determine if changes in systemic iron status regulate intra-hepatic lymphocyte responses. We used a murine model of lymphocyte-mediated acute liver inflammation induced by Concanavalin A (ConA) injection employing mice fed with an iron-deficient (IrDef) or an iron-balanced diet (IrRepl). While the mild iron deficiency induced by the IrDef diet did not significantly modify the steady state immune cell repertoire and systemic cytokine levels, it significantly dampened inflammatory liver damage after ConA challenge. These findings were associated with a marked decrease in T cell and NKT cell activation following ConA injection in IrDef mice. The decreased liver injury observed in IrDef mice was independent from changes in the gut microflora, and was replicated employing an iron specific chelator that did not modify intra-hepatic hepcidin secretion. Furthermore, low-dose iron chelation markedly impaired the activation of isolated T cells in vitro. All together, these results suggest that small changes in iron homeostasis can have a major effect in the regulation of intra-hepatic lymphocyte mediated responses.

The role of iron in infections with intracellular bacteria.

The requirement for iron as a critical component for cellular processes has long been appreciated. During infection with intracellular bacteria, iron is required by both the host cell and the pathogen that inhabits the host cell. Macrophages require iron as a cofactor for the execution of important antimicrobial effector mechanisms, including the NADPH dependent oxidative burst and the production of nitrogen radicals catalysed by the inducible nitric oxide synthase. On the other side of the equation, intracellular bacteria such as Salmonella typhimurium and Mycobacterium tuberculosis have an obligate requirement for iron to support their growth and survival inside cells. This brief report summarises the background to our work on iron modulation in infections with these two organisms and highlights key observations on how modulation of host iron status disturbs the equilibrium between host and pathogen and can determine the outcome of infection.

Use of electronic medical records (EMR) for oncology outcomes research: assessing the comparability of EMR information to patient registry and health claims data.

UNLABELLED: Electronic medical records (EMRs) are used increasingly for research in clinical oncology, epidemiology, and comparative effectiveness research (CER). OBJECTIVE: To assess the utility of using EMR data in population-based cancer research by comparing a database of EMRs from community oncology clinics against Surveillance Epidemiology and End Results (SEER) cancer registry data and two claims databases (Medicare and commercial claims). STUDY DESIGN AND SETTING: DEMOGRAPHIC, CLINICAL, AND TREATMENT PATTERNS IN THE EMR, SEER, MEDICARE, AND COMMERCIAL CLAIMS DATA WERE COMPARED USING SIX TUMOR SITES: breast, lung/bronchus, head/neck, colorectal, prostate, and non-Hodgkin's lymphoma (NHL). We identified various challenges in data standardization and selection of appropriate statistical procedures. We describe the patient and clinic inclusion criteria, treatment definitions, and consideration of the administrative and clinical purposes of the EMR, registry, and claims data to address these challenges. RESULTS: Sex and 10-year age distributions of patient populations for each tumor site were generally similar across the data sets. We observed several differences in racial composition and treatment patterns, and modest differences in distribution of tumor site. CONCLUSION: Our experience with an oncology EMR database identified several factors that must be considered when using EMRs for research purposes or generalizing results to the US cancer population. These factors were related primarily to evaluation of treatment patterns, including evaluation of stage, geographic location, race, and specialization of the medical facilities. While many specialty EMRs may not provide the breadth of data on medical care, as found in comprehensive claims databases and EMR systems, they can provide detailed clinical data not found in claims that are extremely important in conducting epidemiologic and outcomes research.

Monitoring intracellular labile iron pools: A novel fluorescent iron(III) sensor as a potential non-invasive diagnosis tool.

The physiological and pathophysiological importance of intracellular redox active "labile" iron has created a significant need for improved noninvasive diagnostic tools to reliably monitor iron metabolism in living cells. In this context, fluorescent iron-sensitive chemosensors in combination with digital fluorescence spectroscopic methods have proven to be highly sensitive and indispensable tools to determine cellular iron homeostasis. Recently, application of fluorescent iron sensors has led to the identification of a complex sub-cellular iron compartmentation. Cell organelle-specific iron sensors will significantly contribute to enhance fundamental knowledge of cellular iron trafficking, representing a crucial prerequisite for the future development of therapeutic strategies in iron dysregulatory diseases. Here we present physicochemical characterization and functional investigation of a new 3-hydroxypyridin-4-one based fluorescent iron(III) sensor, exclusively monitoring labile iron pools in the endosomal/lysosomal compartments. In vitro studies of the fluorescein labeled probe were carried out in murine bone marrow derived macrophages. Endosomal/lysosomal accumulation of the probe was revealed by confocal microscopy. Flow cytometry analyses demonstrated high sensitivity of the probe towards exogenous alterations of intracellular iron concentrations as well as in response to the chelation potency of iron chelators, clinically approved for treatment of iron-overload related diseases.

Withholding iron as a cellular defence mechanism--friend or foe?

During infection one critical host defence strategy is an attempt to withhold iron from invading pathogens. This is achieved by by proinflammatory cytokines such as IL-6 inducing hepcidin. The net result of this is the removal of iron from the circulation and its sequestration within cells, including cells of the immune system such as macrophages. As macrophages are central cells for controlling infections with intracellular bacteria such as Salmonella and Mycobacteria, modulation of iron by hepcidin can lead to the provision of an ideal cellular iron source for these pathogens. Here we discuss how activation of macrophages with IFN-gamma not only up-regulates antimicrobial effector mechanisms but also modulates iron regulatory proteins such as ferroportin to reduce intracellular iron availability.

Systemic administration of tolerogenic dendritic cells ameliorates murine inflammatory arthritis.

The expression of various cell surface molecules and the production of certain cytokines are important mechanisms by which dendritic cells (DC) are able to bias immune responses. This paper describes the effects of the inflammatory cytokine tumor necrosis factor (TNF)-α on DC phenotype and function. TNF-α treatment resulted in upregulation of MHC class II and CD86 in the absence of increased cell surface CD40 and CD80 or the production of IL-12. Additionally TNF-α treated cells were able to bias T cell responses towards an anti-inflammatory profile. On a note of caution this tolerogenic phenotype of the DC was not stable upon subsequent TLR-4 ligation as a 4 hour pulse of the TNF-α treated DC with lipopolysaccharide (LPS) resulted in the restoration of IL-12 production and an enhancement of their T cell stimulatory capacity which resulted in an increased IFN-γ production. However, TNF-α treated DC, when administered in vivo, were shown to ameliorate disease in collagen induced arthritis, an experimental model of inflammatory joint disease. Mice receiving TNF-α treated DC but not LPS matured DC had a delayed onset, and significantly reduced severity, of arthritis. Disease suppression was associated with reduced levels of collagen specific IgG2a and decreased inflammatory cell infiltration into affected joints. In summary the treatment of DC with TNF-α generates an antigen presenting cell with a phenotype that can reduce the pro-inflammatory response and direct the immune system towards a disease modifying, anti-inflammatory state.

Targeting the lysosome: fluorescent iron(III) chelators to selectively monitor endosomal/lysosomal labile iron pools.

Iron-sensitive fluorescent chemosensors in combination with digital fluorescence spectroscopy have led to the identification of a distinct subcellular compartmentation of intracellular redox-active "labile" iron. To investigate the distribution of labile iron, our research has been focused on the development of fluorescent iron sensors targeting the endosomal/lysosomal system. Following the recent introduction of a series of 3-hydroxypyridin-4-one (HPO) based fluorescent probes we present here two novel HPO sensors capable of accumulating and monitoring iron exclusively in endosomal/lysosomal compartments. Flow cytometric and confocal microscopy studies in murine macrophages revealed endosomal/lysosomal sequestration of the probes and high responsiveness toward alterations of vesicular labile iron concentrations. This allowed assessment of cellular iron status with high sensitivity in response to the clinically applied medications desferrioxamine, deferiprone, and deferasirox. The probes represent a powerful class of sensors for quantitative iron detection and clinical real-time monitoring of subcellular labile iron levels in health and disease.

Iron chelation via deferoxamine exacerbates experimental salmonellosis via inhibition of the nicotinamide adenine dinucleotide phosphate oxidase-dependent respiratory burst.

Competition for cellular iron (Fe) is a vital component of the interaction between host and intracellular pathogen. The host cell requires Fe for the execution of antimicrobial effector mechanisms, whereas most bacteria have an obligate requirement for Fe to sustain growth and intracellular survival. In this study, we show that chelation of host Fe in vivo exacerbates murine salmonellosis, resulting in increased bacterial load and decreased survival times. We further demonstrate that host Fe deprivation results in an inability to induce the NADPH oxidase-dependent production of reactive oxygen, an essential host defense mechanism for the early control of Salmonella typhimurium infection. Thus, altering the equilibrium of intracellular Fe influences the course of infection to the benefit of the pathogen.