A selective inhibitor of the kinetoplastid proteasome (GNF6702) is identified that is highly efficacious in vivo, clearing the parasites that cause leishmaniasis, Chagas disease and sleeping sickness from mice, highlighting the possibility of developing a single class of drugs for these neglected diseases. Chagas disease, leishmaniasis, and sleeping sickness are caused by the kinetoplastid parasites Trypanosoma cruzi, Leishmania spp. and Trypanosoma brucei spp., respectively, and affect 20 million people worldwide. This study reports the results of a screen to find new conserved molecular targets and broad spectrum drugs that could be used to treat all three diseases. A selective inhibitor of the kinetoplastid proteasome (GNF6702) was identified as the most effective. It is highly efficacious in vivo, clearing parasites from mice in all three models of infection. GNF6702 is a non-competitive inhibitor, specific for kinetoplastid proteasome, and is well-tolerated in mice. These results highlight the possibility of developing a single class of drugs for these neglected diseases. Chagas disease, leishmaniasis and sleeping sickness affect 20 million people worldwide and lead to more than 50,000 deaths annually1. The diseases are caused by infection with the kinetoplastid parasites Trypanosoma cruzi, Leishmania spp. and Trypanosoma brucei spp., respectively. These parasites have similar biology and genomic sequence, suggesting that all three diseases could be cured with drugs that modulate the activity of a conserved parasite target2. However, no such molecular targets or broad spectrum drugs have been identified to date. Here we describe a selective inhibitor of the kinetoplastid proteasome (GNF6702) with unprecedented in vivo efficacy, which cleared parasites from mice in all three models of infection. GNF6702 inhibits the kinetoplastid proteasome through a non-competitive mechanism, does not inhibit the mammalian proteasome or growth of mammalian cells, and is well-tolerated in mice. Our data provide genetic and chemical validation of the parasite proteasome as a promising therapeutic target for treatment of kinetoplastid infections, and underscore the possibility of developing a single class of drugs for these neglected diseases.

IL-12p35^−/−p40^−/− mice are highly susceptible to Mycobacterium bovis bacillus Calmette-Guérin (BCG) or Mycobacterium tuberculosis infection. In this study IL-12p35^−/− mice, which are able to produce endogenous IL-12p40, cleared M. bovis BCG and showed reduced susceptibility to pulmonary M. tuberculosis infection, which was in striking contrast to the outcome of mycobacterial infection in IL-12p35^−/−p40^−/− mice. Resistance in wild-type and IL-12p35^−/− mice was accompanied by protective granuloma formation and Ag-specific delayed-type hypersensitivity responses, which were impaired in susceptible IL-12p35^−/− p40^−/− mice. Furthermore, IL-12p35^−/− mice, but not IL-12p35^−/−p40^−/− mice, mounted Ag-specific Th1 and cytotoxic T cell responses. In vivo therapy with rIL-12p40 homodimer restored the impaired delayed-type hypersensitivity responses in M. bovis BCG-infected IL-12p35^−/−p40^−/− mice and reverted them to a more resistant phenotype. Together, these results show evidence for a protective and agonistic role of endogenous and exogenous IL-12p40 in mycobacterial infection, which is independent of IL-12p70.

Rapid progress is being made in understanding the roles of the cerebral meninges in the maintenance of normal brain function, in immune surveillance, and as a site of disease. Most basic research on the meninges and the neural brain is now done on mice, major attractions being the availability of reporter mice with fluorescent cells, and of a huge range of antibodies useful for immunocytochemistry and the characterization of isolated cells. In addition, two-photon microscopy through the unperforated calvaria allows intravital imaging of the undisturbed meninges with sub-micron resolution. The anatomy of the dorsal meninges of the mouse (and, indeed, of all mammals) differs considerably from that shown in many published diagrams: over cortical convexities, the outer layer, the dura, is usually thicker than the inner layer, the leptomeninx, and both layers are richly vascularized and innervated, and communicate with the lymphatic system. A membrane barrier separates them and, in disease, inflammation can be localized to one layer or the other, so experimentalists must be able to identify the compartment they are studying. Here, we present current knowledge of the functional anatomy of the meninges, particularly as it appears in intravital imaging, and review their role as a gateway between the brain, blood, and lymphatics, drawing on information that is scattered among works on different pathologies.

Expressed on various cell types, the IL-4Rα is a component of both receptors for IL-4 and IL-13. Susceptibility of BALB/c mice to Leishmania major is believed to be dependent on the development of IL-4- and IL-13-producing Th2 cells, while IFN-γ secretion by Th1 cells is related to resistance. Despite a sustained development of Th2 cells, IL-4Rα-deficient BALB/c mice are able to control acute cutaneous leishmaniasis, suggesting that IL-4Rα-bearing cells other than Th2 cells contribute to susceptibility. To analyze the contribution of the IL-4Rα on macrophages, recently generated macrophage/neutrophil-specific IL-4Rα-deficient mice on a susceptible BALB/c genetic background were infected with L. major. Strikingly, macrophage/neutrophil-specific IL-4Rα-deficient mice showed a significantly delayed disease progression with normal Th2 and type 2 Ab responses but improved macrophage leishmanicidal effector functions and reduced arginase activity. Together, these results suggest that alternative macrophage activation contributes to susceptibility in cutaneous leishmaniasis.

Human African trypanosomiasis (HAT) manifests in two stages of disease: firstly, haemolymphatic, and secondly, an encephalitic phase involving the central nervous system (CNS). New drugs to treat the second-stage disease are urgently needed, yet testing of novel drug candidates is a slow process because the established animal model relies on detecting parasitemia in the blood as late as 180 days after treatment. To expedite compound screening, we have modified the GVR35 strain of Trypanosoma brucei brucei to express luciferase, and have monitored parasite distribution in infected mice following treatment with trypanocidal compounds using serial, non-invasive, bioluminescence imaging. Parasites were detected in the brains of infected mice following treatment with diminazene, a drug which cures stage 1 but not stage 2 disease. Intravital multi-photon microscopy revealed that trypanosomes enter the brain meninges as early as day 5 post-infection but can be killed by diminazene, whereas those that cross the blood-brain barrier and enter the parenchyma by day 21 survived treatment and later caused bloodstream recrudescence. In contrast, all bioluminescent parasites were permanently eliminated by treatment with melarsoprol and DB829, compounds known to cure stage 2 disease. We show that this use of imaging reduces by two thirds the time taken to assess drug efficacy and provides a dual-modal imaging platform for monitoring trypanosome infection in different areas of the brain. Trypanosoma brucei, a parasite transmitted by the bite of tsetse fly, is responsible for the disease human African trypanosomiasis (HAT). In advanced stages of HAT, trypanosomes invade the central nervous system (CNS), resulting in an array of neurological symptoms, and eventually death. Existing drugs for treatment of HAT are highly unsatisfactory and new safe drugs are urgently needed. Currently, potential drugs for HAT are screened in a mouse model that relies on the emergence of trypanosomes from tissues and their detection in blood. This can take up to 200 days, making selection and further development of new drugs slow and costly. Here, we employ in vivo imaging and genetically modified trypanosomes to monitor parasite distribution throughout the body in live infected mice. Our bioluminescence imaging approach provides sensitive detection of trypanosomes at sites of infection, allowing more rapid and more effective in vivo screening of candidate HAT drugs. Higher resolution intra-vital microscopy was used to investigate trypanosome dynamics in the brain and their accessibility to drugs during infection. These approaches allow more sensitive real time tracking of trypanosomes during chronic infections and will provide new insights about trypanosome pathogenesis in future experiments.

Interleukin 4 receptor α (IL-4Rα) is essential for effective clearance of gastrointestinal nematode infections. Smooth muscle cells are considered to play a role in the type 2 immune response–driven expulsion of gastrointestinal nematodes. Previous studies have shown in vitro that signal transducer and activator of transcription 6 signaling in response to parasitic nematode infection significantly increases smooth muscle cell contractility. Inhibition of the IL-4Rα pathway inhibits this response. How this response manifests itself in vivo is unknown. In this study, smooth muscle cell IL-4Rα–deficient mice (SM-MHC^CreIL-4Rα^−/lox) were generated and characterized to uncover any role for IL-4/IL-13 in this non–immune cell type in response to Nippostrongylus brasiliensis infection. IL-4Rα was absent from α-actin–positive smooth muscle cells, while other cell types showed normal IL-4Rα expression, thus demonstrating efficient cell-type–specific deletion of the IL-4Rα gene. N. brasiliensis–infected SM-MHC^CreIL-4Rα^−/lox mice showed delayed ability to resolve infection with significantly prolonged fecal egg recovery and delayed worm expulsion. The delayed expulsion was related to a delayed intestinal goblet cell hyperplasia, reduced T helper 2 cytokine production in the mesenteric lymph node, and reduced M3 muscarinic receptor expression during infection. Together, these results demonstrate that in vivo IL-4Rα–responsive smooth muscle cells are beneficial for N. brasiliensis expulsion by coordinating T helper 2 cytokine responses, goblet hyperplasia, and acetylcholine responsiveness, which drive smooth muscle cell contractions. Intestinal parasitic worm infections are a major public health concern, with more than 1 billion people infected worldwide. Symptoms associated with these infections are similar to that of other intestinal illnesses, including irritable bowel syndrome. It is likely that the immune response required to expel the worm can also, when activated inappropriately, cause the symptoms of irritable bowel syndrome. This makes understanding parasitic worm infections important in their own right and also as a model for other intestinal illnesses. In previous studies, we demonstrated the crucial importance of interleukin 4 receptor α (IL-4Rα) responsiveness for worm expulsion in global IL-4Rα–deficient mice. In this study, we specifically addressed the role of IL-4Rα responsiveness in a novel smooth muscle cell–specific IL-4Rα–deficient mouse model. These mice showed decreased ability to control the worm infection, with delayed expulsion and reduced protective immune responses. These data provide compelling evidence for smooth muscle cell IL-4Rα being an important coordinator of both the immune and physiological responses to intestinal worm infections. A proposed model is suggested with IL-4Rα responsiveness on smooth muscle cells coordinating T helper 2 cytokine responses, goblet hyperplasia, and acetylcholine-driven smooth muscle contractions for optimal worm expulsion.