Enterocytozoon bieneusi Infection after Hematopoietic Stem Cell Transplant in Child, Argentina.

We report a case of Enterocytozoon bieneusi infection in a pediatric hematopoietic stem cell transplant recipient in Argentina. Spores were visualized in feces using Calcofluor White and modified trichrome stainings. PCR and sequencing identified E. bieneusi genotype D in fecal samples and liver samples, confirming extraintestinal dissemination of the parasite.

Pulmonary Conventional Type 1 Langerin-Expressing Dendritic Cells Play a Role in Impairing Early Protective Immune Response against Cryptococcus neoformans Infection in Mice.

Lung dendritic cells (DC) are powerful antigen-presenting cells constituted by various subpopulations that differ in terms of their function and origin and differentially regulate cell-mediated antifungal immunity. The lung is the primary target organ of Cryptococcus neoformans and C. gattii infections, which makes it essential in the establishment of the first line of anti-cryptococcal defense. However, the lung-specific dynamics and function of DC subsets are poorly understood in cryptococcosis. In this study, we provide evidence for the in vivo function of a conventional langerin-expressing DC1 dendritic cell (LangDC1) population during the first week of intratracheal C. neoformans infection in mice. By using conditional depletion of LangDC1 after diphtheria toxin treatment of LangDTREGFP mice, we demonstrate that these animals better control the fungal infection and produce type 1 and 17 cytokines in the context of a type 2 immune response, favoring a predominance of iNOS over arginase-1 expression by pulmonary cells. Our results suggest that LangDC1 cells play a role in impairing immune response for the clearance of C. neoformans in the early stage of pulmonary infection.

Skin Immunity to Dermatophytes: From Experimental Infection Models to Human Disease.

Dermatophytoses (ringworms) are among the most frequent skin infections and are a highly prevalent cause of human disease worldwide. Despite the incidence of these superficial mycoses in healthy people and the compelling evidence on chronic and deep infections in immunocompromised individuals, the mechanisms controlling dermatophyte invasion in the skin are scarcely known. In the last years, the association between certain primary immunodeficiencies and the susceptibility to severe dermatophytosis as well as the evidence provided by novel experimental models mimicking human disease have significantly contributed to deciphering the basic immunological mechanisms against dermatophytes. In this review, we outline the current knowledge on fungal virulence factors involved in the pathogenesis of dermatophytoses and recent evidence from human infections and experimental models that shed light on the cells and molecules involved in the antifungal cutaneous immune response. The latest highlights emphasize the contribution of C-type lectin receptors signaling and the cellular immune response mediated by IL-17 and IFN-γ in the anti-dermatophytic defense and skin inflammation control.

Immunomodulatory Effect of Fasciola hepatica Excretory-Secretory Products on Macrophages.

The liver fluke, Fasciola hepatica, infects a wide range of mammals including humans and leads to chronic disease. Like other helminths, F. hepatica migrates and survives in the host tissues after penetrating the intestinal wall to enter the peritoneal cavity, and then migrates through the liver before finally inhabiting the bile ducts. To avoid the antihelminthic immune response during migration, F. hepatica releases excretory-secretory products (FhESP) that exert various immunomodulatory effects, such as alternative macrophage activation or programmed cell death induction. Here, we describe the currently available techniques for studying macrophage activation and apoptotic cell death triggered by purified FhESP originating from the adult F. hepatica fluke.

Study of Eosinophil Apoptosis Induced by Fasciola hepatica Excretory-Secretory Products.

The excretory-secretory products released by the liver fluke Fasciola hepatica (FhESP) are in close contact with the immune system and have different immunomodulatory effects associated with the parasite virulence. The control of the early immune response is crucial for the establishment of the fluke in the host. Related to this, eosinophils (Eo) are implicated as effector cells in helminthic infections, and the induction of Eo apoptosis has been demonstrated to be a remarkable immunoevasion mechanism induced by the parasite. In this chapter, we describe different techniques to assay Eo apoptosis triggered by FhESP as well as the mechanisms involved in this phenomenon.

Dectin-1 on macrophages modulates the immune response to Fasciola hepatica products through the ERK signaling pathway.

Fasciolosis is a zoonotic disease of increasing importance due to its worldwide distribution and elevated economic losses. Previously, we demonstrated that Fasciola hepatica excretory-secretory products (FhESP) induce immunomodulatory effects on peritoneal macrophages in a Dectin-1 dependent manner. In this study, we observed that peritoneal macrophages from naive BALB/c mice stimulated in vitro with FhESP presented increased expression levels of phosphorylated extracellular-signal-regulated kinase (ERK), and this effect was dependent on Syk, protein kinase C (PKC) and Dectin-1. In this sense, we observed increased levels of arginase activity, IL-10 and TGF-ß in macrophages stimulated with FhESP, which were dependent on PKC and ERK. Furthermore, we observed that the increased arginase activity, as well as in TGF-ß and IL-10 levels, was partially dependent on IL-10 receptor signaling in macrophages that were pre-incubated with anti-IL10R before being stimulated with FhESP. Taken together, these results suggest the participation of Dectin-1 and Syk in FhESP interaction with peritoneal macrophages and the possible role of ERK and IL-10 in downstream signaling pathways involved in the immunomodulatory effects induced by Fasciola hepatica products.

IL-17-Mediated Immunity Controls Skin Infection and T Helper 1 Response during Experimental Microsporum canis Dermatophytosis.

Despite worldwide prevalence of superficial mycoses, the immune response in dermatophytosis has scarcely been investigated. In this study, we developed a model of superficial skin infection in C57BL/6 mice with Microsporum canis, a highly prevalent human pathogen. This model mimics mild inflammatory human dermatophytosis, characterized by neutrophil recruitment and fungal invasion limited to the epidermis and exhibits the establishment of a specific T helper type 17 immune response during infection. By using IL-17RA- or IL-17A/F-deficient mice we showed that, in the absence of a functional IL-17 pathway, M. canis extensively colonizes the epidermis and promotes an exaggerated skin inflammation and a shift to an IFN-γ-mediated (T helper type 1) response. IL-17 signaling was not involved in neutrophil influx to skin or fungal invasion to deeper tissues. Finally, this study shows that skin langerin-expressing cells contribute to the antifungal T helper type 17 response in vivo. In conclusion, these data directly show a dual function of IL-17 cytokines in dermatophytosis by controlling superficial infection and down-modulating a T helper type 1 antifungal response.

Fasciola hepatica excretory-secretory products induce CD4+T cell anergy via selective up-regulation of PD-L2 expression on macrophages in a Dectin-1 dependent way.

Fasciola hepatica excretory-secretory products (FhESP) induce immunomodulatory effects on macrophages. Previously, we demonstrated that these effects are dependent on Dectin-1. Therefore, the aim of this study was to determine how this affects the CD4 T-cells immune response. We observed that FhESP induce an increased expression of PD-L2 in macrophages via Dectin-1. Furthermore, in co-cultures with CD4 T-cell we observed a suppressive effect on proliferative response, down-modulation of IFN-γ and up-modulation of IL-10 via Dectin-1 on macrophages. These results suggest that FhESP induce T-cell anergy via selective up-regulation of PD-L2 expression on macrophages in a Dectin-1 dependent way.

Eosinophils elicit proliferation of naive and fungal-specific cells in vivo so enhancing a T helper type 1 cytokine profile in favour of a protective immune response against Cryptococcus neoformans infection.

Experimental Cryptococcus neoformans infection in rats has been shown to have similarities with human cryptococcosis, because as in healthy humans, rats can effectively contain cryptococcal infection. Moreover, it has been shown that eosinophils are components of the immune response to C. neoformans infections. In a previous in vitro study, we demonstrated that rat peritoneal eosinophils phagocytose opsonized live yeasts of C. neoformans, thereby triggering their activation, as indicated by the up-regulation of MHC and co-stimulatory molecules and the increase in interleukin-12, tumour necrosis factor-α and interferon-γ production. Furthermore, this work demonstrated that C. neoformans-specific CD4(+) and CD8(+) T lymphocytes cultured with these activated C. neoformans-pulsed eosinophils proliferated, and produced important amounts of T helper type 1 (Th1) cytokines in the absence of Th2 cytokine synthesis. In the present in vivo study, we have shown that C. neoformans-pulsed eosinophils are also able to migrate into lymphoid organs to present C. neoformans antigens, thereby priming naive and re-stimulating infected rats to induce T-cell and B-cell responses against infection with the fungus. Furthermore, the antigen-specific immune response induced by C. neoformans-pulsed eosinophils, which is characterized by the development of a Th1 microenvironment with increased levels of NO synthesis and C. neoformans-specific immunoglobulin production, was demonstrated to be able to protect rats against subsequent infection with fungus. In summary, the present work demonstrates that eosinophils act as antigen-presenting cells for the fungal antigen, hence initiating and modulating a C. neoformans-specific immune response. Finally, we suggest that C. neoformans-loaded eosinophils might participate in the protective immune response against these fungi.

Treatment of rats with heat killed cells (HKC) of Cryptococcus neoformans var. grubii induces cellular activation in spleen and lymphatic nodes.

Our previous studies showed that the subcutaneous pretreatment of rats with heat killed cells of Cryptococcus neoformans (HKC) emulsified in complete Freund adjuvant (CFA) promotes protection against an intraperitoneal challenge with viable C. neoformans. In this model, an appropriate activation of adherent peritoneal cells after antigenic treatment is very important for the control of the infection. Here, we investigated the immune response developed in spleen and lymphatic nodes as a result of treatment with HKC-CFA, which might also contribute in the protective phenomenon of this treatment against cryptococcal infection. The results show that, compared with adjuvant alone, rats which received treatment with HKC-CFA presented a greater activation of adherent splenic cells, with up-regulation of major histocompatibility complex class II (MHC II) and CD86 expression and secretion of anticryptococcal metabolites. Furthermore, this treatment also induced an increase in the blastogenic response and the secretion of Th1 and Th2 cytokines by spleen cells in comparison with cells from CFA-phosphate-buffered saline (PBS) treated rats. On the other hand, lymph node cells from animals treated with HKC-CFA presented a rise in the expression of MHCII but not of CD86 with respect to control cells from rats treated with CFA-PBS. These cells also showed a high proliferative response and secretion of Th1-related cytokines, interleukin (IL)-12 and tumor necrosis factor (TNF). These results show that treatment of rats with HKC-CFA is able to induce an early immune response in secondary lymphoid organs, which may contribute to the protective effect induced by this treatment.

Mycetoma of the scalp due to Microsporum canis: hystopathologic, mycologic, and immunogenetic features in a 6-year-old girl.

Dermatophytic mycetoma is an extremely rare subcutaneous mycosis. Here, we report the case of a 6-year-old girl with clinical, histologic, and mycologic findings consistent with a mycetoma of the scalp caused by Microsporum canis. To our knowledge, this is the first report showing the immunologic and immunogenetic features of a patient with a recalcitrant dermatophytic mycetoma.

Rat eosinophils stimulate the expansion of Cryptococcus neoformans-specific CD4(+) and CD8(+) T cells with a T-helper 1 profile.

Experimental Cryptococcus neoformans infection in rats has been shown to have similarities with human cryptococcosis, revealing a strong granulomatous response and a low susceptibility to dissemination. Moreover, it has been shown that eosinophils are components of the inflammatory response to C. neoformans infections. In this in vitro study, we demonstrated that rat peritoneal eosinophils phagocytose opsonized live yeasts of C. neoformans, and that the phenomenon involves the engagement of FcγRII and CD18. Moreover, our results showed that the phagocytosis of opsonized C. neoformans triggers eosinophil activation, as indicated by (i) the up-regulation of major histocompatibility complex (MHC) class I, MHC class II and costimulatory molecules, and (ii) an increase in interleukin (IL)-12, tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) production. However, nitric oxide (NO) and hydrogen peroxide (H(2) O(2) ) synthesis by eosinophils was down-regulated after interaction with C. neoformans. Furthermore, this work demonstrated that CD4(+) and CD8(+) T lymphocytes isolated from spleens of infected rats and cultured with C. neoformans-pulsed eosinophils proliferate in an MHC class II- and class I-dependent manner, respectively, and produce important amounts of T-helper 1 (Th1) type cytokines, such as TNF-α and IFN-γ, in the absence of T-helper 2 (Th2) cytokine synthesis. In summary, the present study demonstrates that eosinophils act as fungal antigen-presenting cells and suggests that C. neoformans-loaded eosinophils might participate in the adaptive immune response.

Differential activation of peritoneal cells by subcutaneous treatment of rats with cryptococcal antigens.

Previous studies in our laboratory have shown that the subcutaneous pretreatment of rats with heat-killed cells (HKC) of Cryptococcus neoformans emulsified in complete Freund adjuvant (CFA) promotes protective immunity against an intraperitoneal challenge with C. neoformans. In contrast, subcutaneous treatment with the capsular polysaccharide (PSC) emulsified in CFA exacerbates the cryptococcal infection. The purpose of this study was to analyze the mechanisms involved in these phenomena. Adherent peritoneal cells from rats treated with HKC-CFA showed upregulated ED2, CD80, and CD86 expression; an increase in the level of production of anticryptococcal metabolites; and the enhanced production of interleukin-12 (IL-12) in comparison with the findings for cells from rats treated with CFA-phosphate-buffered saline (PBS). Adherent peritoneal cells from rats treated with PSC-CFA, however, also presented upregulated ED2, CD80, and CD86 expression compared to the level of expression for peritoneal cells from controls, but these cells showed an increase in arginase activity and decreased levels of production of IL-12 and tumor necrosis factor (TNF) compared with the activity and levels of production by peritoneal cells from CFA-PBS-treated rats. In addition, treatment with HKC-CFA resulted in a rise in the phagocytic and anticryptococcal activities of adherent peritoneal cells compared to those for control rats. However, adherent peritoneal cells from rats treated with PSC-CFA presented a reduction in anticryptococcal activity in comparison with that for cells from animals treated with CFA-PBS. These results show the differential activation between adherent peritoneal cells from HKC-CFA- and PSC-CFA-treated rats, with this differential activation at the primary site of infection possibly being responsible, at least in part, for the phenomena of protection and exacerbation observed in our model.

Cryptococcus neoformans glucuronoxylomannan induces macrophage apoptosis mediated by nitric oxide in a caspase-independent pathway.

Glucuronoxylomannan (GXM) is the major component of Cryptococcus capsular polysaccharide, which represents an essential virulence factor for this yeast. Cryptococcus neoformans infections in immunocompetent rats are associated with inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production by macrophages. This study demonstrates in vitro and in vivo that GXM promotes iNOS expression with NO production in rat macrophages. GXM also induced macrophage apoptosis after 48 h of culture, with this phenomenon being prevented by the iNOS inhibitor, aminoguanidine. The NO-induced macrophage apoptosis triggered by GXM was dependent on interactions with CD18, Fcgamma receptor II and protein kinase C activation, without participation of tyrosine kinases or mitogen-activated protein kinases. Furthermore, this study reveals that GXM down-regulates the macrophage caspase-3 activity, induces a caspase-independent cell death and promotes depolarization of mitochondria membrane potential with increased cytosolic expression of the apoptosis-inducing factor. Taken together, this study describes the pathways and mechanisms involved in the macrophage apoptosis promoted by GXM through NO generation. These findings indicate new mechanisms of immunomodulation for the main capsular polysaccharide of C. neoformans.

Excretory-secretory products from Fasciola hepatica induce eosinophil apoptosis by a caspase-dependent mechanism.

Eosinophils (Eo) are known to be important effector cells in the host defense against helminth parasites. Excretory-secretory products (ESP) released by helminths have shown wide immunomodulatory properties, such as the induction of cellular apoptosis. We investigated the ability of ESP from Fasciola hepatica to induce Eo apoptosis. In this work, we observed that ESP induced an early apoptosis of rat peritoneal eosinophils and that this phenomenon was time- and concentration-dependent. Furthermore, we demonstrated that activation of protein tyrosine kinases (TyrK) and caspases were necessary to mediate the Eo apoptosis induced by the ESP, and that carbohydrate components present in these antigens were involved in this effect. We have described for the first time the ability of ESP from F. hepatica to modify the viability of Eo by apoptosis induction. Besides that, we have observed Eo apoptosis in the liver of rats 21 days after F. hepatica infection. The diminution in Eo survival in early infection could be a parasite strategy in order to prevent a host immune response.

Heat killed cells of Cryptococcus neoformans var. grubii induces protective immunity in rats: immunological and histopathological parameters.

Different clinical parameters which included cell-mediated immune (CMI) response, were evaluated in a model of disseminated cryptococcosis in rats. The experimental animals were pretreated four days prior to their exposure to Cryptococcus neoformans var. grubii with either heat killed cells of this yeastlike pathogen (HKC) or capsular polysaccharide (CPS) emulsified in complete Freund adjuvant (CFA). Rats treated with HKC-CFA and intraperitoneally infected with C. neoformans var. grubii had significantly better clearance of yeasts from tissues, a lower concentration of the cryptococcal capsular polysaccharide, glucuronoxylomannan (GXM), in serum and tissues, and better histopathological parameters compared to unpretreated infected rats. In contrast, rats treated with CPS-CFA presented an exacerbation of infection with a significantly higher fungal burden in tissues, a higher concentration of GXM in serum, and worse histopathological parameters compared to similar unpretreated infected rats. In addition, HKC-CFA treatment produced a T helper 1 (Th1) profile with improvements in the spleen cell proliferative response, in the level of INFgamma production by CD4 T cells, and in the nitric oxide (NO) production by peritoneal cells. On the other hand, rats treated with CPS-CFA showed an increased level of the immunoregulatory cytokine IL10 production by CD4 T cells, but no modification in the NO production by peritoneal cells.

Immunosuppression, interleukin-10 synthesis and apoptosis are induced in rats inoculated with Cryptococcus neoformans glucuronoxylomannan.

Glucuronoxylomannan (GXM) is the major Cryptococcus neoformans capsular polysaccharide and represents the main virulence factor of this fungus. In in vitro studies we have demonstrated previously that this acidic and high-molecular-weight polysaccharide suppresses lymphoproliferation, modulates cytokine production and promotes apoptosis in spleen mononuclear (Spm) cells from rats. In this study we demonstrate that these phenomena also occur in vivo after the intracardiac inoculation of GXM into normal Wistar rats. The results of this study show suppression of the proliferative response Spm cells to concanavalin A (Con A) or heat-killed C. neoformans (HKCn) in the first 2 weeks after polysaccharide administration. In addition, increased levels of interleukin (IL)-10 were produced by Con A-stimulated Spm cells, coinciding with immunohistochemical GXM detection in the white pulp of spleen. In particular, high production of IL-10 with diminution of IL-2, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha synthesis were detected 14 days after GXM administration. In situ cell death detection by TdT-mediated biotin-dUTP nick-end labelling (TUNEL) reaction in sections of spleen, lung and liver demonstrates apoptosis in tissues with deposits of GXM. These data demonstrate the in vivo ability of GXM to modify cytokine synthesis by Spm cells and to promote host cell apoptosis.