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Despite all the scientific progress in recent decades to unravel the immune processes and the way the parasite bypasses the immune system, Chagas disease is still a major public health problem, affecting an estimated 3.5 million people. Among the components that may participate in the response against the parasite, testosterone has been gaining more and more visibility. Studies indicate that the parasite itself seems to carry out steroidogenesis, in which, in co-culture with androgen precursors, T. cruzi has been shown to produce TS, but the purpose of the TS synthesized by the parasite and how this can influence its invasion glycoproteins is still unclear unknown. The aim of this study was to evaluate the influence of testosterone in Trypanosoma cruzi infection on the immune response of bone marrow-derived macrophages. Bone marrow from male rats was extracted and cultured with RMPI medium containing 30% L929 cell supernatant for macrophage differentiation. The cells were incubated for 10 days and, after this period, they were seeded in 96 wells in the amount of 1 x 10 5  cells per well. TS was added at different concentrations of 20 μM, 10 μM, 5 μM and 1 μM and then infected with the Y strain of T. cruzi, at a rate of 10 parasites per cell, with the culture remaining for six, 12 and 24 h. The supernatant was collected and the production of nitric oxide (NO), tumor necrosis factor (TNF) and the number of cell parasites was assessed by staining with 4'-6'-diamino-2-phenylindole (DAPI) and ranked by high Content Screening (HSC). The parasite was then cultured with the addition of TS, at the mentioned concentrations, leaving it for six and 12 h and then performing the RT-PCR of the mucins. DAPI staining revealed a significant increase in the number of parasites in cells containing TS. The exception was observed when 1 μM of hormone/well was used. A reduction in TNF production was found with 20 and 10 μM of TS for 6 h stimulation, although increased levels were observed with 5 and 1 μM, similar to the infected control. However, there was an increase in TNF production and not after 12 h. The relative expression of parasite glycoprotein 82 was increased with the presence of TS in the medium, regardless of time. Our data suggest that TS may contribute to cellular immunosuppression, increasing parasite infection in the cell, as well as inflammatory mediators that lead to cell and tissue damage in infected individuals, as well as the possible use of TS to allow their invasion into the cell hosts., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Importance: While prior research has established that Plasmodium gametocytes sequester in the bone marrow and can influence resident stem cells, the question of why they would choose this compartment and these cells remained a mystery. This study, for the first time, shows that being in the presence of mesenchymal stem cells (MSCs) alters the biology of the P. falciparum parasite and makes it more infectious to mosquitoes, hinting at novel mechanisms in its life cycle. This method also facilitates mosquito infections with field isolated parasites, affording research teams new infection models with parasites, which are challenging to infect into mosquitos using conventional culture methods. Finally, our findings that MSC-conditioned medium can also raise infectivity open avenues of investigation into mechanisms involved but can also serve as a practical tool for researchers hoping to increase oocyst yields., Competing Interests: The authors declare no conflict of interest.

A 5-y-old male Poodle mix was presented with intermittent vomiting, anorexia, and weight loss. Physical examination revealed emaciation, lethargy, dehydration, hypothermia, respiratory distress, and splenomegaly. Based on clinicopathologic, serologic, and parasitologic findings, diagnoses of severe leishmaniosis and dirofilariasis were made. Extracellular, intraneutrophilic, and intramonocytic Leishmania amastigotes were observed on blood smear and buffy coat smear examination. In blood smears, 0.2% of neutrophils were observed to be infected; in buffy coat smears, 0.5% of neutrophils and 0.1% of monocytes were found to be infected. Leishmania amastigotes were also found engulfed by eosinophils and neutrophil precursors in bone marrow aspiration cytology. The detection of Leishmania amastigotes in blood smears is rare, and the clinical significance is uncertain. In circulating blood, Leishmania amastigotes are primarily found phagocytized by neutrophils. Although debatable, there is growing evidence that neutrophils are used as carriers enabling the "silent entry" of the protozoa into macrophages ("Trojan horse" theory). To date, cytologic screening of blood smears for the diagnosis of canine leishmaniosis is not a routine practice. Clinical pathologists and practitioners should be aware that Leishmania amastigotes may be present in neutrophils and less frequently monocytes during blood smear evaluation; neutrophil precursors and eosinophils may also be parasitized in bone marrow specimens.

Species of the genus Leishmania are the causal agents of leishmaniasis, a disease with diametrically different clinical manifestations that have been attributed to the species and host immune response. Some Leishmania species, including Leishmania mexicana , are capable of causing both localized cutaneous leishmaniasis (LCL) and diffuse cutaneous leishmaniasis (DCL). Therefore, it is possible that intraspecific differences may exist that contribute to the development of distinct clinical forms. Dendritic cells (DC) are important host cells of Leishmania spp. parasites, and cytokine production and phagocytosis upon infection with the parasite are significant for the outcome of the disease. In the present study we analyzed the production of IL-12, TNF-α, and IL-10 by DC infected with L. mexicana amastigotes isolated from a patient with LCL (amastigote = Lac) and from a patient with DCL (amastigote = Diact) by murine DC. Furthermore, we compared the frequency of phagocytosis of L. mexicana amastigotes of each isolate by fluorescence and optical microscopy and by flow cytometry. We show that the infection of DC with Diact amastigotes elicited the secretion of IL-10, TNF-α, and IL-12 by DC to a major extent as compared to the infection with Lac amastigotes. On the other hand, Lac and Diact amastigotes were similarly phagocytosed by DC, but interestingly there were more vacuoles in DC infected with Diact amastigotes. Our results suggest that isolates from a same species of Leishmania , such as L. mexicana , with different degrees of virulence according to the clinical manifestation they cause, differ in their capacity to elicit cytokine production and form vacuoles in DC.

Helminths and their products are strong candidates for the treatment of autoimmunological disorders and allergies. Being a key population of antigen-presenting cells, dendritic cells play a crucial role in the therapeutic potential of worms. The study compares the effects of live pre-male and pre-female L4 stage Heligmosomoides polygyrus administration on the maturation and activation of the JAWS II line of immature dendritic cells. On stimulation with L4 stage H. polygyrus, JAWS II cells acquire semi-mature status and induce Th2 and regulatory responses in vitro. The strongest immunosuppressive effect on JAWS II cells was observed following stimulation with both sexes of nematodes together; this was manifested as immature dendritic cell morphology, proliferation inhibition, cell cycle change, decreased translocation of NF-κB into the nucleus, and lower expression of surface cellular costimulatory molecules CD80, CD86 and MHC I. However, greater production of proinflammatory (IL-12p70, TNF-α, IL-6) and Th2 response-promoting cytokines (IL-4) was observed by JAWS II following exposure to both sexes compared to male or female larvae alone. Sex had no influence on the viability, apoptosis process or endocytosis abilities of the JAWS II cell line. The findings indicate that the presence of only a single sex of the parasite influences a developed response, resulting in reduced proinflammatory and an antiparasitic reaction., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Murine bone marrow-derived macrophages (BMMs) can be differentiated within 10 days from ex vivo bone marrow progenitor cells by supplementing the cell growth medium with colony stimulating factor-1 (CSF-1). Mature macrophages express specific myeloid markers which can be labeled and detected by flow cytometry (FACS).BMMs are a valuable tool to investigate the interactions between the Leishmania parasites and their host cell as well as to screen anti-Leishmania components. Options for the readout of in vitro infection experiments are diverse and may range from simple counting of intracellular parasites to the determination of metabolic changes of the intracellular parasite or the infected cell, thus providing the investigator with valuable results.

After infection with Trypanosoma cruzi , the etiologic agent of Chagas disease, immunosuppression, and apoptosis of mature lymphocytes contribute to the establishment of the parasite in the host and thereby to persistence and pathology in the chronic stage of infection. In a systemic mouse model of experimental Chagas disease, we have demonstrated a strong depletion of mature B cells in the spleen during the first 2 weeks of infection. Remarkably, the decrease in this cell population commenced already in the bone marrow from infected mice and was a concomitant of an increased apoptosis in pro- and pre-B cell populations. Pro- and pre-B cells in the bone marrow showed a significant reduction accompanied by a functional disturbance of bone marrow-derived stromal cells resulting in diminished levels of IL-7, an essential factor for the development of B cell precursors. Ex vivo , stromal cells isolated from the bone marrow of infected mice had a strikingly impaired capacity to maintain the development of pro- and pre-B cells obtained from uninfected animals. Together, the reduction of an active humoral immune response during acute Chagas disease suggests to be an initial immune evasion mechanism of the parasite to establish persistent infection. Therefore, prevention of B cell depletion by rescuing the stromal cells during this early phase, could give rise to new therapeutic approaches.

Immunosuppression is a well-established risk factor for Visceral Leishmaniasis. Post-immunosuppression leishmaniasis is characterized by an increase of parasite burden, hematopoietic disorders and unusual clinical manifestations. Although there are many reports on bone marrow findings in VL, less is known about the relationship between parasite dynamics in this organ and the function of either hematopoietic stem cells and progenitor cells themselves. In the present study, we tackle these issues using a new approach of infecting human stem cells derived from bone marrow with L. infantum. Using this strategy, we show that human hematopoietic stem cells (hHSC) are able to phagocytize L. infantum promastigotes and release modulatory and pro-inflammatory cytokines, mainly TNF-α. Our results demonstrated that L. infantum infection in vitro enhances hematopoiesis, favoring the development of erythrocitic lineage through a mechanism yet unknown. Moreover, we found that L. infantum infection alters the phenotypic profile of the hematopoietic progeny; modifying the surface markers expression of differentiated cells. Thus, our study represents a rare opportunity to monitor the in vitro differentiation of human stem cells experimentally infected by L. infantum to better understand the consequences of the infection on phenotypic and functional profile of the cell progeny., (Copyright © 2017. Published by Elsevier Inc.)

Malaria infections cause several systemic and severe single- or multi-organ pathologies, killing hundreds of thousands of people annually. Considering the existing widespread resistance of malaria parasites to anti-parasitic drugs and their high propensity to develop drug resistance, alternative strategies are required to manage malaria infections. Because malaria is a host immune response-driven disease, one approach is based on gaining a detailed understanding of the molecular and cellular processes that modulate malaria-induced innate and adaptive immune responses. Here, using a mouse cerebral malaria model and small-molecule inhibitors, we demonstrate that inhibiting MEK1/2, the upstream kinases of ERK1/2 signaling, alters multifactorial components of the innate and adaptive immune responses, controls parasitemia, and blocks pathogenesis. Specifically, MEK1/2 inhibitor treatment up-regulated B1 cell expansion, IgM production, phagocytic receptor expression, and phagocytic activity, enhancing parasite clearance by macrophages and neutrophils. Further, the MEK1/2 inhibitor treatment down-regulated pathogenic pro-inflammatory and helper T cell 1 (Th1) responses and up-regulated beneficial anti-inflammatory cytokine responses and Th2 responses. These inhibitor effects resulted in reduced granzyme B expression by T cells, chemokine and intracellular cell adhesion molecule 1 (ICAM-1) expression in the brain, and chemokine receptor expression by both myeloid and T cells. These bimodal effects of the MEK1/2 inhibitor treatment on immune responses contributed to decreased parasite biomass, organ inflammation, and immune cell recruitment, preventing tissue damage and death. In summary, we have identified several previously unrecognized immune regulatory processes through which a MEK1/2 inhibitor approach controls malaria parasitemia and mitigates pathogenic effects on host organs., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Leishmaniasis is caused by protozoan parasites of the genus Leishmania In mammalians, these parasites survive and replicate in macrophages and parasite elimination by macrophages is critical for host resistance. Endosomal Toll-like receptors (TLRs) have been shown to be crucial for resistance to Leishmania major in vivo For example, mice in the resistant C57BL/6 genetic background that are triple-deficient for TLR3, -7, and -9 ( Tlr3/7/9 -/- ) are highly susceptible to L. major infection. Tlr3/7/9 -/- mice are as susceptible as mice deficient in MyD88 or UNC93B1, a chaperone required for appropriate localization of endosomal TLRs, but the mechanisms are unknown. Here we found that macrophages infected with L. major undergo autophagy, which effectively accounted for restriction of parasite replication. Signaling via endosomal TLRs was required for autophagy because macrophages deficient for TLR3, -7, and 9, UNC93B1, or MyD88 failed to undergo L. major -induced autophagy. We also confirmed that Myd88 -/- , Tlr3/7/9 -/- , and Unc93b1 -/- cells were highly permissive to L. major replication. Accordingly, shRNA-mediated suppression of Atg5, an E3 ubiquitin ligase essential for autophagosome elongation, in macrophages impaired the restriction of L. major replication in C57BL/6, but did not affect parasite replication in Myd88 -/- or Unc93b1 -/- macrophages. Rapamycin treatment reduced inflammatory lesions formed in the ears of Leishmania -infected C57BL/6 and Tlr3/7/9 -/- mice, indicating that autophagy operates downstream of TLR signaling and is relevant for disease development in vivo Collectively, our results indicate that autophagy contributes to macrophage resistance to L. major replication, and mechanistically explain the previously described endosomal TLR-mediated resistance to L. major infection., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Leishmaniasis is one of the leading globally neglected diseases, affecting millions of people worldwide. Leishmania infection depends on the ability of insect-transmitted metacyclic promastigotes to invade mammalian hosts, differentiate into amastigotes, and replicate inside macrophages. To counter the hostile oxidative environment inside macrophages, these protozoans contain anti-oxidant systems that include iron-dependent superoxide dismutases (SODs) in mitochondria and glycosomes. Increasing evidence suggests that in addition to this protective role, Leishmania mitochondrial SOD may also initiate H 2 O 2 -mediated redox signaling that regulates gene expression and metabolic changes associated with differentiation into virulent forms. To investigate this hypothesis, we examined the specific role of SODA, the mitochondrial SOD isoform in Leishmania amazonensis Our inability to generate L. amazonensis SODA null mutants and the lethal phenotype observed following RNAi-mediated silencing of the Trypanosoma brucei SODA ortholog suggests that SODA is essential for trypanosomatid survival. L. amazonensis metacyclic promastigotes lacking one SODA allele failed to replicate in macrophages and were severely attenuated in their ability to generate cutaneous lesions in mice. Reduced expression of SODA also resulted in mitochondrial oxidative damage and failure of SODA /Δ sodA promastigotes to differentiate into axenic amastigotes. SODA expression above a critical threshold was also required for the development of metacyclic promastigotes, as SODA /Δ sodA cultures were strongly depleted in this infective form and more susceptible to reactive oxygen species (ROS)-induced stress. Collectively, our data suggest that SODA promotes Leishmania virulence by protecting the parasites against mitochondrion-generated oxidative stress and by initiating ROS-mediated signaling mechanisms required for the differentiation of infective forms., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Accumulation of erythrocytic parasites in bone marrow and the spleen has been reported in cases of Plasmodium vivax malaria. If this occurs commonly, these stages represent a possible source of early, relapse-like homologous recurrences. Moreover, they might hinder the elimination of malaria from human populations. Pertinent research suggestions have been made., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Cross-presentation by MHC class I molecules allows the detection of exogenous antigens by CD8 + T lymphocytes. This process is crucial to initiate cytotoxic immune responses against many pathogens (i.e., Toxoplasma gondii) and tumors. To achieve efficient cross-presentation, dendritic cells (DCs) have specialized endocytic pathways; however, the molecular effectors involved are poorly understood. In this work, we identify the small GTPase Rab22a as a key regulator of MHC-I trafficking and antigen cross-presentation by DCs. Our results demonstrate that Rab22a is recruited to DC endosomes and phagosomes, as well as to the vacuole containing T. gondii parasites. The silencing of Rab22a expression did not affect the uptake of exogenous antigens or parasite invasion, but it drastically reduced the intracellular pool and the recycling of MHC-I molecules. The knockdown of Rab22a also hampered the cross-presentation of soluble, particulate and T. gondii-associated antigens, but not the endogenous MHC-I antigen presentation through the classical secretory pathway. Our findings provide compelling evidence that Rab22a plays a central role in the MHC-I endocytic trafficking, which is crucial for efficient cross-presentation by DCs., (© 2016 The Authors.)

Accumulating evidences have assigned a central role to parasite-derived proteins in immunomodulation. Here, we report on the proteomic identification and characterization of immunomodulatory excretory-secretory (ES) products from the metacestode larva (tetrathyridium) of the tapeworm Mesocestoides corti (syn. M. vogae). We demonstrate that ES products but not larval homogenates inhibit the stimuli-driven release of the pro-inflammatory, Th1-inducing cytokine IL-12p70 by murine bone marrow-derived dendritic cells (BMDCs). Within the ES fraction, we biochemically narrowed down the immunosuppressive activity to glycoproteins since active components were lipid-free, but sensitive to heat- and carbohydrate-treatment. Finally, using bioassay-guided chromatographic analyses assisted by comparative proteomics of active and inactive fractions of the ES products, we defined a comprehensive list of candidate proteins released by M. corti tetrathyridia as potential suppressors of DC functions. Our study provides a comprehensive library of somatic and ES products and highlight some candidate parasite factors that might drive the subversion of DC functions to facilitate the persistence of M. corti tetrathyridia in their hosts., Competing Interests: The authors have declared that no competing interests exist.

Apoptosis is one of the mechanisms used by host cells to remove unwanted intracellular organisms, and often found to be subverted by pathogens through use of host anti-apoptotic proteins. In the present study, with the help of in vitro and in vivo approaches, we documented that the macrophage anti-apoptotic protein myeloid cell leukemia 1 (MCL-1) is exploited by the intra-macrophage parasite Leishmania donovani to protect their "home" from actinomycin D-induced mitochondria-dependent apoptosis. Among all the anti-apoptotic BCL-2 family members, infection preferentially up-regulated expression of MCL-1 at both the mRNA and protein levels and compared with infected control, MCL-1-silenced infected macrophages documented enhanced caspase activity and increased apoptosis when subjected to actinomycin D treatment. Phosphorylation kinetics and ChIP assay demonstrated that infection-induced MCL-1 expression was regulated by transcription factor CREB (cAMP-response element-binding protein) and silencing of CREB resulted in reduced expression of MCL-1 and increased apoptosis. During infection, MCL-1 was found to be localized in mitochondria and this was significantly reduced in Tom70-silenced macrophages, suggesting the active role of TOM70 in MCL-1 transport. In the mitochondria, MCL-1 interacts with the major pro-apoptotic protein BAK and prevents BAK-BAK homo-oligomer formation thereby preventing cytochrome c release-mediated mitochondrial dysfunction. Silencing of MCL-1 in the spleen of infected mice showed decreased parasite burden and increased induction of splenocyte apoptosis. Collectively our results showed that L. donovani exploited the macrophage anti-apoptotic protein MCL-1 to prevent BAK-mediated mitochondria-dependent apoptosis thereby protecting its niche, which is essential for disease progression., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

We previously showed that LIGHT and its receptor herpes virus entry mediator (HVEM) are important for development of optimal CD4(+) Th1 cell immunity and resistance to primary Leishmania major infection in mice. In this study, we further characterized the contributions of this molecule in dendritic cell (DC) maturation, initiation, and maintenance of primary immunity and secondary anti-Leishmania immunity. Flow-cytometric studies showed that CD8α(+) DC subset was mostly affected by HVEM-Ig and lymphotoxin β receptor-Ig treatment. LIGHT signaling is required at both the priming and the maintenance stages of primary anti-Leishmania immunity but is completely dispensable during secondary immunity in wild type mice. However, LIGHT blockade led to impaired IL-12 and IFN-γ responses and loss of resistance in healed CD40-deficient mice after L. major challenge. The protective effect of LIGHT was mediated primarily via its interaction with lymphotoxin β receptor on CD8α(+) DCs. Collectively, our results show that although LIGHT is critical for maintenance of primary Th1 response, it is dispensable during secondary anti-Leishmania immunity in the presence of functional CD40 signaling as seen in wild type mice., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Tissue-infiltrating Ly6C(hi) monocytes play diverse roles in immunity, ranging from pathogen killing to immune regulation. How and where this diversity of function is imposed remains poorly understood. Here we show that during acute gastrointestinal infection, priming of monocytes for regulatory function preceded systemic inflammation and was initiated prior to bone marrow egress. Notably, natural killer (NK) cell-derived IFN-γ promoted a regulatory program in monocyte progenitors during development. Early bone marrow NK cell activation was controlled by systemic interleukin-12 (IL-12) produced by Batf3-dependent dendritic cells (DCs) in the mucosal-associated lymphoid tissue (MALT). This work challenges the paradigm that monocyte function is dominantly imposed by local signals after tissue recruitment, and instead proposes a sequential model of differentiation in which monocytes are pre-emptively educated during development in the bone marrow to promote their tissue-specific function., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Group 2 innate lymphoid cells (ILC2s) are often found associated with mucosal surfaces where they contribute to protective immunity, inappropriate allergic responses, and tissue repair. Although we know they develop from a common lymphoid progenitor in the bone marrow (BM), the specific lineage path and transcriptional regulators that are involved are only starting to emerge. After ILC2 gene expression analysis we investigated the role of Bcl11b, a factor previously linked to T cell commitment, in ILC2 development. Using combined Bcl11b-tom and Id2-gfp reporter mice, we show that Bcl11b is expressed in ILC2 precursors in the BM and maintained in mature ILC2s. In vivo deletion of Bcl11b, by conditional tamoxifen-induced depletion or by Bcl11b(-/-) fetal liver chimera reconstitution, demonstrates that ILC2s are wholly dependent on Bcl11b for their development. Notably, in the absence of Bcl11b there is a concomitant expansion of the RORγt(+) ILC3 population, suggesting that Bcl11b may negatively regulate this lineage. Using Nippostrongylus brasiliensis infection, we reveal that the absence of Bcl11b leads to impaired worm expulsion, caused by a deficit in ILC2s, whereas Citrobacter rodentium infection is cleared efficiently. These data clearly establish Bcl11b as a new factor in the differentiation of ILC2s., (© 2015 Walker et al.)

The obligate intracellular parasite, Toxoplasma gondii, disseminates through its host inside infected immune cells. We hypothesize that parasite nutrient requirements lead to manipulation of migratory properties of the immune cell. We demonstrate that 1) T. gondii relies on glutamine for optimal infection, replication and viability, and 2) T. gondii-infected bone marrow-derived dendritic cells (DCs) display both "hypermotility" and "enhanced migration" to an elevated glutamine gradient in vitro. We show that glutamine uptake by the sodium-dependent neutral amino acid transporter 2 (SNAT2) is required for this enhanced migration. SNAT2 transport of glutamine is also a significant factor in the induction of migration by the small cytokine stromal cell-derived factor-1 (SDF-1) in uninfected DCs. Blocking both SNAT2 and C-X-C chemokine receptor 4 (CXCR4; the unique receptor for SDF-1) blocks hypermotility and the enhanced migration in T. gondii-infected DCs. Changes in host cell protein expression following T. gondii infection may explain the altered migratory phenotype; we observed an increase of CD80 and unchanged protein level of CXCR4 in both T. gondii-infected and lipopolysaccharide (LPS)-stimulated DCs. However, unlike activated DCs, SNAT2 expression in the cytosol of infected cells was also unchanged. Thus, our results suggest an important role of glutamine transport via SNAT2 in immune cell migration and a possible interaction between SNAT2 and CXCR4, by which T. gondii manipulates host cell motility.

Previously, we showed that treating macrophages with ATP impairs the intracellular growth of Leishmania amazonensis, and that the P2X7 purinergic receptor is overexpressed during leishmaniasis. In the present study, we directly evaluated the effect of periodate-oxidized ATP (oATP) on parasite control in Leishmania-infected macrophages. We found that oATP impaired the attachment/entrance of L. amazonensis promastigotes to C57BL/6 mouse macrophages in a P2X7 receptor-independent manner, as macrophages from P2X7(-/-) mice were similarly affected. Although oATP directly inhibited the growth of axenic promastigotes in culture, promoted rapid ultrastructural alterations, and impaired Leishmania internalization by macrophages, it did not affect intracellular parasite multiplication. Upon infection, phagosomal acidification was diminished in oATP-treated macrophages, accompanied by reduced endosomal proteolysis. Likewise, MHC class II molecules expression and ectoATPase activity was decreased by oATP added to macrophages at the time of parasite infection. These inhibitory effects were not due to a cytotoxic effect, as no additional release of lactate dehydrogenase was detected in culture supernatants. Moreover, the capacity of macrophages to produce nitric oxide and reactive oxygen species was not affected by the presence of oATP during infection. We conclude that oATP directly affects extracellular parasite integrity and macrophage functioning., (© 2014 International Society for Advancement of Cytometry.)

Dendritic cells (DCs) are the antigen-presenting cells capable of activating naïve T cells. Although CD4+ T cells are crucial for Cryptosporidium parvum clearance, little is known about the role of DCs in the immune response to this parasite. In this study, the interaction between mouse DCs and C. parvum was investigated both in vitro and in vivo. For in vitro experiments, mouse bone marrow-derived dendritic cells (BMDCs) derived from wild-type C57B1/6 or MyD88-/- or C3H/HeJ mice and DC cell line DC2.4 were pulsed with C. parvum. Active invasion of parasites was demonstrated by parasite colocalization with host cell membranes and actin-plaque formation at the site of attachment. DC activation induced by the parasite invasion was demonstrated by upregulation of costimulatory molecules CD40, CD80, and CD86, as well as inflammatory cytokines IL-12, TNF-α, and IL-6. BMDCs derived from MyD88-/- and C3H/HeJ mice failed to produce IL-12 in response to C. parvum, suggesting the importance of TLR-dependent signaling pathway specially presence of a functional TLR4 pathway, for C. parvum-induced cytokine production. In vivo experiments showed that both parasite antigens and live parasites were transported to mice mesenteric lymph nodes. All together, these data suggest that DCs play a key role in host immune responses to C. parvum and pathogenesis of the disease., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

CD4⁺ T cell differentiation is regulated by specialized antigen-presenting cells. Dendritic cells (DCs) produce cytokines that promote naive CD4⁺ T cell differentiation into T helper 1 (Th1), Th17, and inducible T regulatory (iTreg) cells. However, the initiation of Th2 cell responses remains poorly understood, although it is likely that more than one mechanism might be involved. Here we have defined a specific DC subset that is involved in Th2 cell differentiation in vivo in response to a protease allergen, as well as infection with Nippostrongylus brasiliensis. We have demonstrated that this subset is controlled by the transcription factor interferon regulatory factor 4 (IRF4), which is required for their differentiation and Th2 cell-inducing function. IRF4 is known to control Th2 cell differentiation and Th2 cell-associated suppressing function in Treg cells. Our finding suggests that IRF4 also plays a role in DCs where it controls the initiation of Th2 cell responses., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Osteopontin (OPN) is a multifunctional protein participating in the regulation of different Th cell lineages and critically involved in the initiation of immune responses to diverse pathogens. Our study goal was to verify whether OPN helps modulate the protective Th1 and Th17 cytokine responses in C57BL/6 mice infected with Trypanosoma cruzi, the etiological agent of Chagas disease. Parasite infection induced OPN release from murine macrophages in vitro and acute Chagas mice displayed enhanced serum levels of this cytokine at the peak of parasitemia. Upon administration of a neutralizing anti-OPN antibody, recently infected mice presented lower Th1 and Th17 responses, increased parasitemia and succumbed earlier and at higher rates to infection than non-immune IgG-receiving controls. The anti-OPN therapy also resulted in reduced circulating levels of IL-12 p70, IFN-γ, IL-17A and specific IgG(2a) antibodies. Furthermore, antibody-mediated blockade of OPN activity abrogated the ex vivo production of IL-12 p70, IFN-γ and IL-17A, while promoting IL-10 secretion, by spleen macrophages and CD4(+) T cells from T. cruzi-infected mice. Th1 and Th17 cytokine release induced by OPN preferentially involved the α(v)β(3) integrin OPN receptor, whereas concomitant down-modulation of IL-10 production would mostly depend on OPN interaction with CD44. Our findings suggest that, in resistant C57BL/6 mice, elicitation of protective Th1 and Th17 cytokine responses to T. cruzi infection is likely to be regulated by endogenous OPN., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The protozoan parasite Plasmodium falciparum, responsible for the most severe form of malaria, is able to sequester from peripheral circulation during infection. The asexual stage parasites sequester by binding to endothelial cell receptors in the microvasculature of various organs. P. falciparum gametocytes, the developmental stages responsible for parasite transmission from humans to Anopheles mosquitoes, also spend the almost ten days necessary for their maturation sequestered away from the peripheral circulation before they are released in blood mainstream. In contrast to those of asexual parasites, the mechanisms and cellular interactions responsible for immature gametocyte sequestration are largely unexplored, and controversial evidence has been produced so far on this matter. Here we present a systematic comparison of cell binding properties of asexual stages and immature and mature gametocytes from the reference P. falciparum clone 3D7 and from a patient parasite isolate on a panel of human endothelial cells from different tissues. This analysis includes assays on human bone marrow derived endothelial cell lines (HBMEC), as this tissue has been proposed as a major site of gametocyte maturation. Our results clearly demonstrate that cell adhesion of asexual stage parasites is consistently more efficient than that, virtually undetectable of immature gametocytes, irrespectively of the endothelial cell lines used and of parasite genotypes. Importantly, immature gametocytes of both lines tested here do not show a higher binding efficiency compared to asexual stages on bone marrow derived endothelial cells, unlike previously reported in the only study on this issue. This indicates that gametocyte-host interactions in this tissue are unlikely to be mediated by the same adhesion processes to specific endothelial receptors as seen with asexual forms.

Dendritic cell (DC)-derived IL-1α/β plays a critical role in the induction of T helper type 1 (Th1)-dependent immunity against Leishmania. DCs from susceptible BALB/c mice produce less IL-1α/β when compared with resistant C57BL/6 mice, contributing to aberrant Th2 development and ultimate death of infected mice. We have extended our studies of the role of IL-1 in leishmaniasis using IL-1RA(-/-) BALB/c mice that are characterized by upregulated IL-1 receptor signaling. Unexpectedly, infection of IL-1RA(-/-) mice led to significantly worsened disease outcome with larger lesions, dramatically higher parasite burdens, and decreased IFN-γ production by antigen-specific T cells. We determined that IL-1RA(-/-) DCs were more mature already in the steady state, exhibited less phagocytotic capacity, and IL-12 production in response to various stimuli was impaired. Our data suggest that in addition to effects on Th education, IL-1α/β signaling also modulates DC homeostasis with increased signaling, leading to downmodulation of IL-12 synthesis and worsened disease outcome after infection with Leishmania major. Thus, the complex regulation of various members of the IL-1 cytokine family mediated through effects on both DCs and T cells critically contributes to disease outcome against this important human pathogen.

Background: The bone marrow is considered to be an important storage of parasites in Leishmania-infected dogs, although little is known about cellular genesis in this organ during canine visceral leishmaniasis (CVL)., Methodology/principal Findings: The aim of the present study was to evaluate changes in erythropoiesis and leucopoiesis in bone marrow aspirates from dogs naturally infected with Leishmania chagasi and presenting different clinical statuses and bone marrow parasite densities. The evolution of CVL from asymptomatic to symptomatic status was accompanied by increasing parasite density in the bone marrow. The impact of bone marrow parasite density on cellularity was similar in dogs at different clinical stages, with animals in the high parasite density group. Erythroid and eosinophilic hypoplasia, proliferation of neutrophilic precursor cells and significant increases in lymphocytes and plasma cell numbers were the major alterations observed. Differential bone marrow cell counts revealed increases in the myeloid:erythroid ratio associated to increased numbers of granulopoietic cells in the different clinical groups compared with non-infected dogs., Conclusions: Analysis of the data obtained indicated that the assessment of bone marrow constitutes an additional and useful tool by which to elaborate a prognosis for CVL.

There are >2 million new cases of leishmaniasis annually, and no effective vaccine has been developed to prevent infection. In murine infection, Leishmania mexicana, which lives intracellularly in host macrophages, has developed pathways to hijack host IgG to induce a suppressive IL-10 response through FcγRs, the cell-surface receptors for IgG. To guide vaccine development away from detrimental Ab responses, which can accompany attempts to induce cell-mediated immunity, it is crucial to know which isotypes of IgG are pathogenic in this infection. We found that IgG1 and IgG2a/c induce IL-10 from macrophages in vitro equally well but through different FcγR subtypes: IgG1 through FcγRIII and IgG2a/c through FcγRI primarily, but also through FcγRIII. In sharp contrast, mice lacking IgG1 develop earlier and stronger IgG2a/c, IgG3, and IgM responses to L. mexicana infection and yet are more resistant to the infection. Thus, IgG1, but not IgG2a/c or IgG3, is pathogenic in vivo, in agreement with prior studies indicating that FcγRIII is required for chronic disease. This calls into question the assumption that macrophages, which should secrete IL-10 in response to IgG1 and IgG2a/c immune complexes, are the most important source of IL-10 generated by IgG-FcγR engagement in L. mexicana infection. Further investigations are required to better determine the cell type responsible for this immunosuppressive FcγRIII-induced IL-10 pathway and whether IgG2a/c is protective.

In mammalian hosts, Leishmania parasites are obligatory intracellular organisms that invade macrophages (M phi) and dendritic cells (DC). In M phi, the production of nitric oxide (NO) catalyzed by the inducible nitric oxide synthase (iNOS) has been implicated as a major defense against Leishmania infection. The modulation of this microbicidal mechanism by different species of Leishmania has been well studied in M phi. Although DC are permissive for infection with Leishmania both in vivo and in vitro, the effect of this parasite in the expression of iNOS and NO production in these cells has not been established. To address this issue, we analyzed the regulation of iNOS by Leishmania mexicana amastigotes in murine bone marrow-derived dendritic cells (BMDC) stimulated with LPS and IFN-gamma. We show that the infection of BMDC with amastigotes down regulated NO production and diminished iNOS protein levels in cells stimulated with LPS alone or in combination with IFN-gamma. The reduction in iNOS protein levels and NO production did not correlate with a decrease in iNOS mRNA expression, suggesting that the parasite affects post-transcriptional events of NO synthesis. Although amastigotes were able to reduce NO production in BMDC, the interference with this cytotoxic mechanism was not sufficient to permit the survival of L. mexicana. At 48 h post-infection, BMDC stimulated with LPS+IFN-gamma were able to eliminate the parasites. These results are the first to identify the regulation of iNOS by L. mexicana amastigotes in DC., ((c) 2010 Elsevier Inc. All rights reserved.)

Neospora caninum is an intracellular protozoan pathogen that causes abortion in cattle. This parasite elicits a typical type 1 immune response in host animals, and it is widely believed that the strong type 1 immune response during pregnancy may result in fetal death. Pro-inflammatory and/or inflammatory cytokines produced during either primary or secondary pathogen exposure are supposed to be the mediators of abortion. The present study defined cytokine production by murine naïve dendritic cells and spleen cells in response to whole Neospora tachyzoites (live, heat-killed, freeze-killed) or whole-cell tachyzoite lysate in the form of total (NcAg), soluble (sNcAg), or insoluble antigen (isNcAg). All tachyzoite and antigen preparations at high doses stimulated high levels of interleukin (IL) -12, interferon (IFN) -gamma, and tumor necrosis factor (TNF) -alpha, except for heat-killed tachyzoites and sNcAg, which induced moderate level of IL-12 and very low levels of IFN-gamma and TNF-alpha. In general, whole N. caninum tachyzoites were more effective in inducing IL-12, IFN-gamma, and TNF-alpha than the lysate antigen preparations. It appears that the heat-killed N. caninum tachyzoites were less potent in eliciting IFN-gamma or IL-10, but more effective in inducing IL-4. Thus, heat-inactivated tachyzoites or sNcAg alone may not be powerful enough to elicit strong type 1 immune responses against the disease. The present study comprehensively studied the production of critical cytokine by the murine dendritic cells and spleen cells in response to N. caninum; these results may facilitate a better understanding of antigen priming and aid in the design of vaccines/adjuvants against neosporosis.

Dendritic cell (DC)-expressed CD40 is shown to play crucial roles in eliciting effector T cell responses, primarily the proinflammatory CD4(+) Th subsets and cytotoxic CD8(+) T cells that eliminate various infections and tumors, respectively. In contrast, DCs are also implied in the generation of regulatory T cells (Tregs) that counteract the functions of the proinflammatory Th subsets and exacerbate infections. However, the role of DC-expressed CD40 in the generation of Tregs is unknown. In this study, we generated bone marrow-derived DCs from mice (on a BALB/c background) expressing different levels of CD40 and tested their relative efficiency in generating Tregs. We observed that low levels of CD40 expression were required for efficient Treg generation. DCs expressing low levels of CD40 induced Tregs, whereas DCs expressing high levels of CD40 induced effector T cells, possibly CD8(+)CD40(+) T cells with a contraregulatory activity; the adoptive transfer of the former DC exacerbated whereas the latter significantly reduced Leishmania donovani infection in BALB/c mice. Similarly, priming of mice with leishmanial Ag-pulsed DCs expressing high levels of CD40 induced host protection against L. donovani challenge infection. In contrast, priming with the low CD40-expressing DC resulted in aggravated infection as compared with the control mice. The results establish that CD40 can play differential roles in Treg differentiation and determine the course of infection. We demonstrate that the knowledge can be efficiently used in adoptive cell transfer therapy against an infectious disease.

Autophagy is a homeostatic process promoting cell survival in periods of stress. The induction of the autophagic machinery has also been implicated in both innate and adaptive immunity. Leishmania donovani, which is the causative pathogen of visceral leishmaniasis, is an intracellular parasite that invades and multiplies in bone marrow macrophages. We describe the induction of host cell autophagic machinery during acute natural bone marrow infection by L. donovani complex, detected by LC3B immunoblot. The successful treatment with liposomal amphotericin B resulted in the resolution of this phenomenon. Even though the role of autophagy in parasite biology has been previously studied, our findings show for the first time the in vivo host cell LC3B conversion as a marker of the induction of the autophagic machinery during infection with Leishmania parasite in real time conditions.

Infection of macrophages with the protozoan parasite Toxoplasma gondii results in inhibition of a large panel of LPS-responsive cytokines, including TNF-alpha, while leaving others such as IL-10 intact. Recent studies provide evidence that the parasite interferes with chromatin remodeling at the TNF-alpha promoter that is normally associated with LPS stimulation, but that is not required for TLR4 induction of IL-10. Here, we examined the effect of Toxoplasma on IL-10 induced by simultaneous signaling through TLR4 and FcgammaR, a combined stimulus that triggers histone H3 covalent modification at the IL-10 promoter resulting in high level IL-10 cytokine production. We show that the parasite inhibits high level IL-10 production and prevents histone H3 Ser(10) phosphorylation and Lys(9/14) acetylation at the IL-10 promoter. These results provide compelling evidence that T. gondii targets the host cell chromatin remodeling machinery to down-regulate cytokine responses in infected macrophages.

Severe malarial anemia causes considerable mortality and morbidity in endemic areas. Possible mechanisms underlying the anemia include lysis of parasitized and nonparasitized red cells as well as parasite product-mediated effects on erythropoiesis. The latter include suppression of erythropoiesis, dyserythropoiesis, and ineffective erythropoiesis. Present transmission electron microscope data in two cases of Pasmodium vivax malaria show a hitherto undescribed mechanism contributing to malarial anemia, namely, infection of erythroblasts by parasites and their subsequent degradation. No parasites were detected in the peripheral blood but parasites were found in the bone marrow. These findings emphasise the value of bone marrow examination in the diagnosis and eradication of malaria.

Proinflammatory responses induced by Plasmodium falciparum glycosylphosphatidylinositols (GPIs) are thought to be involved in malaria pathogenesis. In this study, we investigated the role of MAPK-activated protein kinase 2 (MK2) in the regulation of tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-12, two of the major inflammatory cytokines produced by macrophages stimulated with GPIs. We show that MK2 differentially regulates the GPI-induced production of TNF-alpha and IL-12. Although TNF-alpha production was markedly decreased, IL-12 expression was increased by 2-3-fold in GPI-stimulated MK2(-/-) macrophages compared with wild type (WT) cells. MK2(-/-) macrophages produced markedly decreased levels of TNF-alpha than WT macrophages mainly because of lower mRNA stability and translation. In the case of IL-12, mRNA was substantially higher in MK2(-/-) macrophages than WT. This enhanced production is due to increased NF-kappaB binding to the gene promoter, a markedly lower level expression of the transcriptional repressor factor c-Maf, and a decreased binding of GAP-12 to the gene promoter in MK2(-/-) macrophages. Thus, our data demonstrate for the first time the role of MK2 in the transcriptional regulation of IL-12. Using the protein kinase inhibitors SB203580 and U0126, we also show that the ERK and p38 pathways regulate TNF-alpha and IL-12 production, and that both inhibitors can reduce phosphorylation of MK2 in response to GPIs and other toll-like receptor ligands. These results may have important implications for developing therapeutics for malaria and other infectious diseases.

The onset of T(h)1 immunity is in part regulated by genetic background. To elucidate the cell type carrying critical factors determining the T(h)1 response, we employed Rag-2(-/-) mice on Leishmania major-susceptible BALB/c and -resistant B10.D2 backgrounds. By using bone marrow (BM) chimeras generated by the transplantation of B10.D2 BM cells into BALB/c-Rag-2(-/-) mice, and vice versa, it was shown that hematopoietic cells carry factors determining the disease outcome and T(h)1 response against L. major infection. B10.D2-Rag-2(-/-) mice reconstituted with BALB/c CD4(+) T cells exhibited a T(h)1 response and controlled L. major infection. Wild-type BALB/c mice inoculated with L. major-parasitized B10.D2-Rag-2(-/-) splenocytes also exhibited a T(h)1 response and a mild disease outcome, whereas such a T(h)1 response was not induced when CD11c(+) dendritic cells (DCs) were depleted from parasitized B10.D2-Rag-2(-/-) splenocytes. T(h)1 response was reconstituted by the addition of L. major-parasitized B10.D2 DCs but not L. major-parasitized BALB/c DCs to DC-depleted parasitized B10.D2-Rag-2(-/-) splenocytes. These results indicate that DCs determine the outcome of the disease upon L. major infection.

Atopic allergy is characterized by an increase in IgE antibodies that signal through the high-affinity Fcepsilon receptor (FcepsilonRI) to induce the release of inflammatory mediators from mast cells. For unknown reasons, the prevalence of allergic diseases has recently increased steeply in the developed world. However, this increase has not been mirrored in developing countries, even though IgE concentrations are often greatly elevated in individuals from these countries, owing to nonspecific IgE induction by universally present parasitic worms. Here we offer one explanation for this paradox based on the properties of ES-62, a molecule secreted by filarial nematodes. We found that highly purified, endotoxin-free ES-62 directly inhibits the FcepsilonRI-induced release of allergy mediators from human mast cells by selectively blocking key signal transduction events, including phospholipase D-coupled, sphingosine kinase-mediated calcium mobilization and nuclear factor-kappaB activation. ES-62 mediates these effects by forming a complex with Toll-like receptor 4, which results in the sequestration of protein kinase C-alpha (PKC-alpha). This causes caveolae/lipid raft-mediated, proteasome-independent degradation of PKC-alpha, a molecule important for the coupling of FcepsilonRI to phospholipase D and mast cell activation. We also show that ES-62 is able to protect mice from mast cell-dependent hypersensitivity in the skin and lungs, indicating that it has potential as a novel therapeutic for allergy.

Leishmania are intracellular parasites that multiply as the amastigote form in the macrophages of their vertebrate hosts. Since vaccines against leishmaniases are still under development, the control of these diseases relies on prompt diagnosis and chemotherapy in infected humans as well as in dogs, which are the main reservoir of Leishmania infantum, in Mediterranean countries. To establish the macrophage type to be used as an in vitro model for antileishmanial chemotherapeutic studies, we analysed the susceptibility of human peripheral blood derived macrophages, macrophages derived from mouse bone marrow, mouse peritoneal macrophages and macrophages differentiated from cell lines U-937 and DH82 to infection by two L. infantum strains, one obtained from a human leishmanial infection and other from a canine infection. Both strains displayed comparable behaviour in their capacity of infecting the different macrophage types. Human peripheral blood macrophages and DH82 cells were less infectable by both strains. U-937, mouse peritoneal macrophages and mouse bone marrow derived macrophages are the most active cells to phagocytose the parasites. However, U-937 cell line appears to be the most useful as Leishmania infection model providing an unlimited source of homogeneous host cells with reproducibility of the results, is less time consuming, less expensive and tolerate high doses of first line drugs for human and canine visceral leishmaniasis treatment.

The ectoparasitic astigmatid mite Psoroptes ovis causes sheep scab, a highly contagious, severe allergic dermatitis associated with damage to the fleece and hide, loss of condition and occasional mortality. The scab lesion is characterized by a massive infiltration of eosinophils that begins very rapidly after infection. This paper reports the finding that mite-derived factors directly enhance the migration of ovine eosinophils in vitro. Significant (p < 0.01) and dose-dependent (r = 0.972 +/- 0.018 (SD)) activity was initially identified in whole mite extracts, by comparison with medium controls in an assay based on modified Boyden chambers and ovine bone marrow target cells. Similar pro-migratory activity (p < 0.005; r = 0.928 +/- 0.069 (SD)) was detected in washes containing mite excretory/secretory material. By direct comparison with migration ratios (n = 3) for defined chemotactic (rmeotaxin = 3.430 +/- 0.360 (SD)) and chemokinetic (rminterleukin-5 = 0.982 +/- 0.112 (SD)) stimuli it was determined that the activity in both mite extracts (0.992 +/- 0.038 (SD)) and mite washes (0.969 +/- 0.071 (SD)) was chemokinetic. Subsequent experiments (n = 3) in which live mites were incorporated directly into the in vitro assay system indicated that they produced factors that significantly (p < 0.001) enhanced eosinophil migration to a degree directly related to mite numbers (r = 0.993 +/- 0.005 (SD)). The identity of the factor(s) responsible is uncertain, but their presence suggests that mites may be capable of directly activating eosinophils in vivo, and raises the possibility that mites could directly influence, perhaps even initiate, the rapid early tissue eosinophilic response observed in experimental sheep scab infections.

In Brazil, sophisticated techniques currently employed for diagnosis of visceral leishmaniasis, such as polymerase chain reaction-based assays, are only available in major research centers, whereas conventional methods are still used in many areas where the disease occurs. In the state of Mato Grosso do Sul, in the country's Center-West Region, visceral leishmaniasis has recently emerged in many cities, and duration of the disease, from the onset of symptoms to diagnosis, has been short. Considering that results of diagnostic tests may depend on the phase of the disease, we compared direct examination of bone marrow aspirates (BMAs), BMA culture, and serology by Indirect Immunofluorescence Antibody Test (IFAT) for diagnosis in children, according to time of evolution (< or = 30 days or > 30 days) and to spleen size (< or = 5 cm or > 5 cm) at admission. Duration of the illness did not interfere with test positivity: direct smear examination and IFAT were positive in more than 80% of patients, as was culture in around 60%. Results of positive microscopy, however, where predominant in patients with larger spleens. Thanks to the association of traditional techniques, only a few patients had to begin a treatment trial without confirming the diagnosis. Conventional methods for diagnosis of visceral leishmaniasis are still indispensable in our region, and training professionals in basic techniques should be incremented. The highest sensitivity in laboratory diagnosis among the cases investigated was that obtained with a combination of BMA direct examination and IFAT, nearing 100%.

Trypanosoma cruzi causes Chagas' disease, a systemic infection that affects cells of meso-, endo-, and ectodermic origin. However, as far as we know, the presence of T. cruzi stages in bone has not been reported previously, and it has scarcely been investigated in cartilage. We inoculated 7- and 20-day-old (8 and 15 g) NMRI albino mice i.p. with metacyclic trypomastigotes from Rhodnius prolixus used for xenodiagnosis of mice previously infected with mammalian, human, and triatomines isolates, characterized by randomly amplified polymorphic DNA as zymodeme 1 (equivalent to T. cruzi I). Tissular parasitism (quantified according to the number of pseudocysts/50 fields 400x) showed amastigotes, intermediate forms, or trypomastigotes in sternum chondroblasts, osteoblasts, macrophages, and fibroblasts; chondrocyte and osteocyte invasion was rare. All isolates parasitized bone marrow macrophages, with few amastigotes. We observed marked associated myotropism, with or without inflammatory infiltration; there were small numbers of intensely parasitized mononuclear cells in perichondrium and periosteum. We discuss the results in relation to the marked differences of the T. cruzi tropism toward the different types of sternum cells, and, additionally, we outline the possibility of transmitting parasitized bone marrow through transplants. The fact of finding parasite stages in sternum bone and cartilage may be considered important due to the studies on Chagas' disease paleoparasitology that are based on histological and molecular analysis.

We have demonstrated that the microculture method (MCM) enables the diagnosis of visceral leishmaniasis (VL) with samples from both the bone marrow (BM) and peripheral blood (PB). The MCM is superior to the traditional culture method (TCM) as determined by its higher sensitivity in the detection of promastigotes and the more rapid time for emergence of promastigotes. The sensitivity of MCM (100% in BMs and 77.8-100% in PB) was considerably higher than that of the TCM (37.5-100% in BMs and 0-100% in PB) according to decreasing parasite density (P < 0.05). The concentration of parasites in buffy coats has increased the sensitivity of both methods, especially that of the MCM. Detection of promastigotes by MCM requires lower amounts of culture media (25-50 microL) and shorter incubation periods (2-7 days) than TCM (2.5-3.5 mL and 15-35 days, respectively). MCM was found to be valuable with the advantages of simplicity and sensitivity, in addition to being cost-effective in the routine diagnosis for VL in Adana Turkey.

The mechanism of anemia in severe falciparum malaria is still not completely understood. The purpose of this study was to determine whether apoptosis in the erythroid lineage causes anemia in falciparum malaria. Bone marrow aspirated from 8 severe falciparum malaria patients, 3 normal volunteers and 5 retrospective normal bone marrow smears were investigated. By light microscopic study, 5 of 8 hyperparasitemic patients had hypocellular bone marrows and erythroid hypoplasia, whereas the other 3 patients had normal cellularity. The mean myeloid : erythroid ratio of these 5 patients was significantly (p < or = 0.05) higher than normal. Apoptosis of bone marrow nucleated cells (BMNC) could be determined from the exposure of phosphatidylserine (PS) on the cell membrane but not DNA fragmentation (180-250 bp) or ultrastructural morphology. The percentages of apoptotic BMNC and apoptotic erythroid cells in bone marrow from each patient and controls varied from low to high, and were not associated with parasitemia. This study suggests that destruction of erythroid lineage, particularly through apoptosis regulation, cannot solely account for anemia in falciparum malaria.

In this study, we have established conditions for generating Leishmania amazonensis recombinants stably expressing the firefly luciferase gene. These parasites produced significant bioluminescent signals for both in vitro studies and the development of an in vivo model, allowing the course of the parasitism to be readily monitored in real time in the living animals such as laboratory mice. First, a model was established, using parasite-infected mouse macrophages for rapidly determining the activity of drugs against intracellular amastigotes. Results indicated that recombinant Leishmania can be reliably and confidently used to monitor compounds acting on intracellular amastigote-harbouring macrophages. Secondly, temporal analyses were performed following inoculation of metacyclic promastigotes into the ear dermis of BALB/c mice and the bioluminescent light transmitted through the tissue was imaged externally using a charge coupled device (CCD) camera. Bioluminescent signals, measured at the inoculation site and in the draining lymph node of mice containing these parasites correlated well with the more classical quantification of parasites. These assays prove that the real-time bioluminescent assay is not only sensitive but also more rapid than culture-base techniques allowing to monitor parasite-load before any clinical signs of leishmaniasis are detectable. In short, this luciferase imaging study is useful to monitor the efficacy of anti-leishmanial drugs on live cell culture and to trace leishmanial infection in animal models.

Defocusing microscopy was used for real-time observation and quantification of membrane surface dynamics in murine bone marrow macrophages. Small random membrane fluctuations (SRMF), possibly metabolic driven, were detected uniformly over all membrane surface. Morphological and dynamical parameters of ruffles, such as shape, dimensions, and velocity of propagation, were analyzed. Optical tweezers were used to promote phagocytosis of single Leishmania amazonensis amastigotes by selected macrophages. Analysis of ruffling activity on the macrophages before and during phagocytosis of the parasites indicated that increased ruffling response near forming phagosomes, most likely induced by the parasite, accelerates phagocytosis. The effects of temperature decrease on the dynamics of membrane surface fluctuations and on the phagocytosis of parasites were used to determine the overall activation energies involved in these processes. The values obtained support the existence of strong correlation between membrane motility and phagocytic capacity.

With increasing donor age, the potential of transmitting diseases from donor to recipient reaches new dimensions. Potentially transmittable diseases from donors include infections, congenital disorders, and acquired illnesses like autoimmune diseases or malignancies of hematological or nonhematological origin. While established nonmalignant or malignant diseases might be easy to discover, early-stage hematological diseases like CML, light-chain multiple myelomas, aleukemic leukemias, occult myelodysplastic syndromes and other malignant and nonmalignant diseases might not be detectable by routine screening but only by invasive, new and/or expensive diagnostic tests. In the following article, we propose recommendations for donor work-up, taking into consideration the age of the donors. In contrast to blood transfusions, stem cells from donors with abnormal findings might still be acceptable for HCT, when no other options are available and life expectancy is limited. This issue is discussed in detail in relation to the available donor and stem cell source. Finally, the recommendations presented here aim at harmonized worldwide work-up for donors to insure high standard quality.

A 22-year-old Japanese man noticed pyrexia and diarrhea after travel to Guinea. Notable physical findings included hepatosplenomegaly. Treatment with oral quinine and minocycline was started after definitive diagnosis of falciparum malaria by blood smear. Initially, parasitemia and body temperature decreased but by the third night of therapy his temperature increased to 40 degrees C with a slight increase of parasite count. When quinine treatment was changed to atovaquone/proguanil, his temperature dropped immediately and complete plasmodial elimination was confirmed on microscopic examination. Subsequent recrudescence of the disease was not observed. It was concluded that the antimalarial treatment with atovaquone/proguanil might become invaluable in Japan.

We used the model of murine leishmaniasis to evaluate the signals enabling Ag-pulsed dendritic cells (DC) to prime a protective Th1 response in vivo. Bone marrow-derived DC (BMDC) that had been activated by TNF-alpha or CD40 ligation were not able to induce protection against leishmaniasis in susceptible BALB/c mice. In contrast, all mice vaccinated with a single dose of Leishmania major Ag-pulsed BMDC stimulated by prior in vitro exposure to CpG-containing oligodeoxynucleotides (ODN) were completely protected, had a dramatic reduction in parasite burden, and developed an Ag-specific Th1 response. Importantly, systemic administration of CpG ODN was not required. Protection mediated by ex vivo CpG ODN-activated and Ag-pulsed DC was solid, as documented by resistance to reinfection with a higher parasite dose, and long-lasting, as immunized mice were still protected against L. major challenge 16 wk after vaccination. A significantly increased level of protection could also be elicited in resistant C57BL/6 mice. Surprisingly, IL-12 expression by the immunizing BMDC was not required for induction of host resistance. In contrast, the availability of IL-12 derived from recipient cells was essential for the initial triggering of protective immunity by transferred BMDC. Together, these findings demonstrate that the type of stimulatory signal is critical for activating the potential of DC to induce a Th1 response in vivo that confers complete protection against an intracellular pathogen. Moreover, they show that the impact of activated DC on the initiation of a protective Th cell response in vivo may be independent of their ability to produce IL-12.