Lymphocyte-specific reconstitution of IL-4Ra-deficient mice : characterization and infectious disease studies

Includes bibliographical references (leaves 114-142).Lymphocyte-specific reconstitution of IL-4Ra was recently established by intercrossing lymphocyte-specific human IL-R4a transgenic mice with mIL-4Ra-deficient mice. Human IL-4Ra may bind to mouse yc resulting in a chimeric receptor specific for human IL-4 but not mouse IL-4. This provides us with and inducible IL-4 system. The aim of this study was to investigate in vitro and in vivo characteristics of our novel hlL-4Ra Tg/mIL-4Ra+/- mouse model

Chapter 48 In Vivo Bioluminescence Imaging to Assess Compound Efficacy Against Trypanosoma brucei

Traditional animal models for human African trypanosomiasis rely on detecting Trypanosoma brucei brucei parasitemia in the blood. Testing the efficacy of new compounds in these models is cumbersome because it may take several months after treatment before surviving parasites become detectable in the blood. To expedite compound screening, we have used a Trypanosoma brucei brucei GVR35 strain expressing red-shifted firefly luciferase to monitor parasite distribution in infected mice through noninvasive wholebody bioluminescence imaging. This protocol describes the infection and in vivo bioluminescence imaging of mice to assess compound efficacy against T. brucei during the two characteristic stages of disease, the hemolymphatic phase (stage 1) and the encephalitic or central nervous system phase (stage 2)

A reduction in the duration of infection with Tritrichomonas foetus following vaccination in heifers and the failure to demonstrate a curative effect in infected bulls

Seven batches of 25% water-phase, oil-in-water vaccine were prepared from whole cultures of Tritrichomonas foetus. Two inoculations were given, spaced 6 weeks apart, to virgin heifers and infected bulls. A significant reduction (P < 0,01) in the duration of infection in vaccinated heifers was seen when they were challenged by being bred to a bull infected with the same isolate as that contained in the vaccine. Only 1/12 vaccinated heifers were pregnant 4,5 months after the end of the breeding season compared to 2/12 in the control group. The vaccine, therefore, has no practical advantage. Vaccine was supplied to 2 724 bulls on properties where the infection was present. From these bulls, 110 reliable results were obtained, where bulls had been infected, been inoculated and tested 1 month later. No curative effect was demonstrable with 69/110 (62,7%) bulls, remaining infected after the course of inoculations. There was also no difference between vaccine batches or between bulls of different ages. Further work on improving the vaccine is indicated. Three media suitable for the culture of T. foetus are described in detail.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. Adobe Acrobat XI Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.mn201

Where are we? : The anatomy of the murine cortical meninges revisited for intravital imaging, immunology, and clearance of waste from the brain

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

Inhibitor of serine peptidase 2 enhances Leishmania major survival in the skin through control of monocytes and monocyte-derived cells

Leishmania major is the causative agent of the neglected tropical disease, cutaneous leishmaniasis. In the mouse, protective immunity to Leishmania is associated with inflammatory responses. Here, we assess the dynamics of the inflammatory responses at the lesion site during experimental long-term, low-dose intradermal infection of the ear, employing noninvasive imaging and genetically modified L. major Significant infiltrates of neutrophils and monocytes occurred at 1-4 d and 2-4 wk, whereas dermal macrophage and dendritic cell (DC) numbers were only slightly elevated in the first days. Quantitative whole-body bioluminescence imaging of myeloperoxidase activity and the quantification of parasite loads indicated that the Leishmania virulence factor, inhibitor of serine peptidase 2 (ISP2), is required to modulate phagocyte activation and is important for parasite survival at the infection site. ISP2 played a role in the control of monocyte, monocyte-derived macrophage, and monocyte-derived DC (moDC) influx, and was required to reduce iNOS expression in monocytes, monocyte-derived cells, and dermal DCs; the expression of CD80 in moDCs; and levels of IFN-γ in situ. Our findings indicate that the increased survival of L. major in the dermis during acute infection is associated with the down-regulation of inflammatory monocytes and monocyte-derived cells via ISP2.-Goundry, A., Romano, A., Lima, A. P. C. A., Mottram, J. C., Myburgh, E. Inhibitor of serine peptidase 2 enhances Leishmania major survival in the skin through control of monocytes and monocyte-derived cells

Correction: Delayed Goblet Cell Hyperplasia, Acetylcholine Receptor Expression, and Worm Expulsion in SMC-Specific IL-4Rα–Deficient Mice

Interleukin 4 receptor alpha (IL-4Ralpha) 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-4Ralpha pathway inhibits this response. How this response manifests itself in vivo is unknown. In this study, smooth muscle cell IL-4Ralpha-deficient mice (SM-MHC(Cre)IL-4Ralpha(-/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-4Ralpha was absent from alpha-actin-positive smooth muscle cells, while other cell types showed normal IL-4Ralpha expression, thus demonstrating efficient cell-type-specific deletion of the IL-4Ralpha gene. N. brasiliensis-infected SM-MHC(Cre)IL-4Ralpha(-/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-4Ralpha-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

Anti-Trypanosomal Proteasome Inhibitors Cure Hemolymphatic and Meningoencephalic Murine Infection Models of African Trypanosomiasis

Current anti-trypanosomal therapies suffer from problems of longer treatment duration, toxicity and inadequate efficacy, hence there is a need for safer, more efficacious and 'easy to use' oral drugs. Previously, we reported the discovery of the triazolopyrimidine (TP) class as selective kinetoplastid proteasome inhibitors with in vivo efficacy in mouse models of leishmaniasis, Chagas Disease and African trypanosomiasis (HAT). For the treatment of HAT, development compounds need to have excellent penetration to the brain to cure the meningoencephalic stage of the disease. Here we describe detailed biological and pharmacological characterization of triazolopyrimidine compounds in HAT specific assays. The TP class of compounds showed single digit nanomolar potency against Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense strains. These compounds are trypanocidal with concentration-time dependent kill and achieved relapse-free cure in vitro. Two compounds, GNF6702 and a new analog NITD689, showed favorable in vivo pharmacokinetics and significant brain penetration, which enabled oral dosing. They also achieved complete cure in both hemolymphatic (blood) and meningoencephalic (brain) infection of human African trypanosomiasis mouse models. Mode of action studies on this series confirmed the 20S proteasome as the target in T. brucei. These proteasome inhibitors have the potential for further development into promising new treatment for human African trypanosomiasis

In vivo imaging of trypanosome-brain interactions and development of a rapid screening test for drugs against CNS stage trypanosomiasis.

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

TLR2 signaling in skin non-hematopoietic cells induces early neutrophil recruitment in response to Leishmania major infection

Neutrophils are rapidly recruited to the mammalian skin in response to infection with the cutaneous Leishmania pathogen. The parasites use neutrophils to establish the disease, however, the signals driving early neutrophil recruitment are poorly known. Here, we identified the functional importance of TLR2 signaling in this process. Using bone-marrow chimeras and immunohistology we identified the TLR2-expressing cells involved in this early neutrophil recruitment to be of non-hematopoietic origin. Keratinocytes are damaged and briefly in contact with the parasites during infection. We show that TLR2 triggering by L. major is required for their secretion of neutrophil-attracting chemokines. Furthermore, TLR2 triggering by L. major phosphoglycans is critical for neutrophil recruitment impacting negatively on disease development, as shown by better control of lesion size and parasite load in Tlr2-/- compared to wild type infected mice. Conversely, restoring early neutrophil presence in Tlr2-/- mice through injection of wild type neutrophils or CXCL1 at the onset of infection resulted in delayed disease resolution comparable to that observed in wild type mice. Taken together, our data demonstrate a new role for TLR2-expressing non-hematopoietic skin cells in the recruitment of the first wave of neutrophils following L. major infection, a process delaying disease control

Attempts to Image the Early Inflammatory Response during Infection with the Lymphatic Filarial Nematode Brugia pahangi in a Mouse Model

Helminth parasites remain a major constraint upon human health and well-being in many parts of the world. Treatment of these infections relies upon a very small number of therapeutics, most of which were originally developed for use in animal health. A lack of high throughput screening systems, together with limitations of available animal models, has restricted the development of novel chemotherapeutics. This is particularly so for filarial nematodes, which are long-lived parasites with a complex cycle of development. In this paper, we describe attempts to visualise the immune response elicited by filarial parasites in infected mice using a non-invasive bioluminescence imaging reagent, luminol, our aim being to determine whether such a model could be developed to discriminate between live and dead worms for in vivo compound screening. We show that while imaging can detect the immune response elicited by early stages of infection with L3, it was unable to detect the presence of adult worms or, indeed, later stages of infection with L3, despite the presence of worms within the lymphatic system of infected animals. In the future, more specific reagents that detect secreted products of adult worms may be required for developing screens based upon live imaging of infected animals