Leishmania pathogenesis is primarily studied using the disease-inducing promastigote stage of Leishmania major. Despite many efforts, all attempts so far have failed to culture the disease-relevant multiplying amastigote stage of L. major. Here, we established a stably growing axenic L. major amastigote culture system that was characterized genetically, morphologically, and by stage-specific DsRed protein expression. We found parasite stage-specific disease development in resistant C57BL/6 mice. Human neutrophils, as first host cells for promastigotes, do not take up amastigotes. In human macrophages, we observed an amastigote-specific complement receptor 3-mediated, endocytotic entry mechanism, whereas promastigotes are taken up by complement receptor 1-mediated phagocytosis. Promastigote infection of macrophages induced the inflammatory mediators TNF, CCL3, and CCL4, whereas amastigote infection was silent and resulted in significantly increased parasite numbers: from 7.1 ± 1.4 (after 3 h) to 20.1 ± 7.9 parasites/cell (after 96 h). Our study identifies Leishmania stage-specific disease development, host cell preference, entry mechanism, and immune evasion. Since the amastigote stage is the disease-propagating form found in the infected mammalian host, the newly developed L. major axenic cultures will serve as an important tool in better understanding the amastigote-driven immune response in leishmaniasis.

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Administering immunoregulatory cells to patients as medicinal agents is a potentially revolutionary approach to the treatment of immunologically mediated diseases. Presently, there are no satisfactory, clinically applicable methods of tracking human cells in patients with adequate spatial resolution and target cell specificity over a sufficient period of time. Laser ablation–inductively coupled plasma mass spectrometry (LA-ICP-MS) represents a potential solution to the problem of detecting very rare cells in tissues. In this article, this exquisitely sensitive technique is applied to the tracking of gold-labeled human regulatory macrophages (Mregs) in immunodeficient mice. Optimal conditions for labeling Mregs with 50-nm gold particles were investigated by exposing Mregs in culture to variable concentrations of label: Mregs incubated with 3.5 × 109 particles/ml for 1 h incorporated an average of 3.39 × 108 Au atoms/cell without loss of cell viability. Analysis of single, gold-labeled Mregs by LA-ICP-MS registered an average of 1.9 × 105 counts/cell. Under these conditions, 100% labeling efficiency was achieved, and label was retained by Mregs for ≥36 h. Gold-labeled Mregs adhered to glass surfaces; after 24 h of culture, it was possible to colabel these cells with human-specific 154Sm-tagged anti–HLA-DR or 174Yb-tagged anti-CD45 mAbs. Following injection into immunodeficient mice, signals from gold-labeled human Mregs could be detected in mouse lung, liver, and spleen for at least 7 d by solution-based inductively coupled plasma mass spectrometry and LA-ICP-MS. These promising results indicate that LA-ICP-MS tissue imaging has great potential as an analytical technique in immunology.

Background Leishmania (L.) species are the causative agent of leishmaniasis. Due to the lack of efficient vaccine candidates, drug therapies are the only option to deal with cutaneous leishmaniasis. Unfortunately, chemotherapeutic interventions show high toxicity in addition to an increased risk of dissemination of drug-resistant parasites. An appropriate laboratory animal based model is still missing which allows testing of new drug strategies in the context of human immune cells in vivo . Methodology/Principal Findings Humanized mice were infected subcutaneously with stationary phase promastigote L. major into the footpad. The human immune response against the pathogen and the parasite host interactions were analyzed. In addition we proved the versatility of this new model to conduct drug research studies by the inclusion of orally given Miltefosine. We show that inflammatory human macrophages get infected with Leishmania parasites at the site of infection. Furthermore, a Leishmania -specific human-derived T cell response is initiated. However, the human immune system is not able to prevent systemic infection. Thus, we treated the mice with Miltefosine to reduce the parasitic load. Notably, this chemotherapy resulted in a reduction of the parasite load in distinct organs. Comparable to some Miltefosine treated patients, humanized mice developed severe side effects, which are not detectable in the classical murine model of experimental leishmaniasis. Conclusions/Significance This study describes for the first time L. major infection in humanized mice, characterizes the disease development, the induction of human adaptive and innate immune response including cytokine production and the efficiency of Miltefosine treatment in these animals. In summary, humanized mice might be beneficial for future preclinical chemotherapeutic studies in systemic (visceral) leishmaniasis allowing the investigation of human immune response, side effects of the drug due to cytokine production of activated humane immune cells and the efficiency of the treatment to eliminate also not replicating (“hiding”) parasites.

Efficient formation of early GCs depends on the close interaction between GC B cells and antigen-primed CD4 + follicular helper T cells (T FH)Eur. J. Immunol. 2014Immunol. . 44: 2955Immunol. -2967 Introduction GCs represent anatomical regions within secondary lymphoid organs in which the clonal expansion, somatic hypermutation, and affinity-based selection of B cells take place in response to a broad range of antigens. Several interactions between APCs and T cells are necessary before GC B cells can start the genetic program of differentiation into high-affinity plasma B cells [1]. It is firmly established that T-cell help is fundamental for early GC development [2]. In particular, activated follicular helper T cells (T FH ) cells express CXCR5 and migrate into the B-cell follicle in response to CXCL13 expressed by follicular DCs (FDCs) [3][4][5][6]. These T FH cells form stable conjugates with follicular B cells [7][8][9]. It has been proposed that physical conjugation between T FH and B cells is necessary for GC B-cell differentiation, which entails the expression of activation-induced cytidine deaminase (AID) and the B-cell lymphoma 6 (BCL-6) proto-oncogene [10,11]. Although GC responses and affinity maturation of GC B cells have been extensively studied, less is known about the early events that govern antigen-specific T FH -cell activation within skindraining lymph nodes (SDLNs) after cutaneous challenge with antigens. It is generally thought that APCs bearing cognate antigen are crucial for driving T FH -cell differentiation [12] and that ongoing presentation of antigens by skin-associated DCs may be necessary for licensing T FH cells to promote subsequent GC B-cell differentiation. It is assumed that any DC subset might be capable of initially activating T FH -cell precursors in response to dermal antigens [12,13]; however, the phenotype of DC responsible for T FH -cell licensing has not been analyzed within SDLNs.A previous study has shown that functionally active T FH cells form tight conjugates with B cells in SDLNs after cutaneous infection with Leishmania (L.) major parasites [7]. In addition, it is accepted that CD4 + T cells in general require signals from distinct DC subsets in order to become functionally competent in that model [14,15]. Thus, differentiation of T FH cells might be dependent on distinct DC subsets [16]. In this work, we investigated which types of DCs might be involved in T FH -dependent GC formation within SDLNs after L. major infection. Lang-hDTR mice were selectively depleted of either Langerhans cells (LCs) The results presented in this article support the view that LCs are necessary for initially activating T FH -cell precursors after cutaneous challenge with L. major parasites. However, this requirement for LCs is not universally true for all antigens, so we propose the novel concept that distinct DC subsets are responsible for early GC B-cell differentiation, depending on the kind of antigen. Understanding the role of LCs in antibody production against a broad range of...