ObjectivesThe current method for testing new drugs against tuberculosis in vivo is the enumeration of bacteria in organs by cfu assay. Owing to the slow growth rate of Mycobacterium tuberculosis (Mtb), these assays can take months to complete. Our aim was to develop a more efficient, fluorescence-based imaging assay to test new antibiotics in a mouse model using Mtb reporter strains.MethodsA commercial IVIS Kinetic® system and a custom-built laser scanning system with fluorescence molecular tomography (FMT) capability were used to detect fluorescent Mtb in living mice and lungs ex vivo. The resulting images were analysed and the fluorescence was correlated with data from cfu assays.ResultsWe have shown that fluorescent Mtb can be visualized in the lungs of living mice at a detection limit of ∼8 × 107 cfu/lung, whilst in lungs ex vivo a detection limit of ∼2 × 105 cfu/lung was found. These numbers were comparable between the two imaging systems. Ex vivo lung fluorescence correlated to numbers of bacteria in tissue, and the effect of treatment of mice with the antibiotic moxifloxacin could be visualized and quantified after only 9 days through fluorescence measurements, and was confirmed by cfu assays.ConclusionsWe have developed a new and efficient method for anti-tuberculosis drug testing in vivo, based on fluorescent Mtb reporter strains. Using this method instead of, or together with, cfu assays will reduce the time required to assess the preclinical efficacy of new drugs in animal models and enhance the progress of these candidates into clinical trials against human tuberculosis.

The global tuberculosis epidemic is the most common cause of death after infectious disease worldwide. Increasing numbers of infections with multi- and extensively drug-resistant variants of the Mycobacterium tuberculosis complex, resistant even to newly discovered and last resort antibiotics, highlight the urgent need for an efficient vaccine. The protective efficacy to pulmonary tuberculosis in adults of the only currently available vaccine, M. bovis BCG, is unsatisfactory and geographically diverse. More importantly, recent clinical studies on new vaccine candidates did not prove to be better than BCG, yet. Here, we propose and discuss novel strategies to improve efficacy of existing anti-tuberculosis vaccines. Modulation of innate immune responses upon vaccination already provided promising results in animal models of tuberculosis. For instance, neutrophils have been shown to influence vaccine efficacy, both, positively and negatively, and stimulate specific antibody secretion. Modulating immune regulatory properties after vaccination such as induction of different types of innate immune cell death, myeloid-derived suppressor or regulatory T cells, production of anti-inflammatory cytokines such as IL-10 may have beneficial effects on protection efficacy. Incorporation of lipid antigens presented via CD1 molecules to T cells have been discussed as a way to enhance vaccine efficacy. Finally, concepts of dendritic cell-based immunotherapies or training the innate immune memory may be exploitable for future vaccination strategies against tuberculosis. In this review, we put a spotlight on host immune networks as potential targets to boost protection by old and new tuberculosis vaccines.

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Administration of lipophilic drugs is often restricted by poor aqueous solubility (especially in blood), limited membrane permeability, uncontrolled drug leakage, and aggregation of lipophilic drugs. As a new nanocarrier concept, LC@ZrO(mdp)@ZrO(HPO 4 ) core@shell nanocontainers (LC: lipophilic cavity; mdp: monododecylphosphate) are presented. As a proof of concept, the nanocontainers are used to encapsulate different types of lipophilic molecules such as the fluorescent dye lumogen red (LR), the cytostatic drug irinotecan (ITC), the insecticide cypermethrin (CM), and the tuberculosis antibiotic benzothiazinone-043 (BTZ). Synthesis strategy and material structure of the nanocontainers are discussed in detail. LR@ZrO(mdp)@ZrO(HPO 4 ), as the first example, shows intense red emission and successful incorporation of LR into the nanocontainers. As ex vivo application, CM@ZrO(mdp)@ZrO(HPO 4 ) nanocontainers can be used to repel and even kill mosquitoes or flies being in contact with the insecticide-loaded nanocontainers. The drugs ITC and BTZ-after encapsulation in ITC@ZrO(mdp)@ZrO(HPO 4 ) and BTZ@ZrO(mdp)@ ZrO(HPO 4 ) nanocontainers-show high activity at low cytotoxicity in in vitro studies against tumor cells (HeLa, SK-Mel-28, HTC116, A549, RAW264.7) and tuberculosis (Mycobacterium tuberculosis-infected macrophages). Taken together, the different lipophilic molecules (LR, CM, ITC, BTZ) point to the adaptability and performance of the novel zirconyl hydrogenphosphate nanocontainer concept. and limited membrane permeability limit the applicability of lipophilic drugs. [3d,4,5] To enable intravenous, oral, or gastrointestinal administration, appropriate carrier systems were suggested, [3c,6] especially including metal oxide nanoparticles (e.g., SiO 2 , Fe 2 O 3 , MgCO 3 , Ca 3 (PO 4 ) 2 ), [7] polymer nanoparticles (e.g., polyethylene glycol/PEG), [8] biopolymers (e.g., lipids, proteins), [3a,9] graphene, [10] as well as micelles, [6b,11] nanoemulsions, [12] and liposomes. [13] Many nanocontainer systems yet suffer from weaknesses such as uncontrolled drug leakage and aggregation of lipophilic drugs, limited cell uptake, high material complexity, unexpected toxicity and hypersensitivity, damage of cell membranes, inadequate drug loading, and poor stability in blood. [3c,5a,7b,d,8c,14] Especially, SiO 2 -based nanocarriers were described to cause toxic effects. [7b,14,15] All in all, further improvement and exploration of more robust delivery systems for lipophilic drugs are indispensable.As a proof of concept, we here present a novel nanocarrier concept, which is essentially based on lipophilic cavities loaded with drugs and an inorganic zirconyl hydrogenphosphate shell, resulting in LC@ZrO(mdp)@ZrO(HPO 4 ) core@shell nanocontainers (LC: lipophilic cavity; mdp: monododecylphosphate). These novel nanocarriers show excellent cell uptake at low toxicity. The feasibility and performance of the LC@ ZrO(mdp)@ZrO(HPO 4 ) core@shell nanocontainers are validated for the transport of different lipophilic agents, which in...