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.

COVID-19 patients display changes in lymphocyte subsets with a higher frequency of dysfunctional CD8lo T cells associated with disease severity.

This work examines cellular immunity against SARS-CoV-2 in patients from Córdoba, Argentina, during two major waves characterized by different circulating viral variants and different social behavior. Using flow cytometry, we evaluated the main lymphocyte populations of peripheral blood from hospitalized patients with moderate and severe COVID-19 disease. Our results show disturbances in the cellular immune compartment, as previously reported in different cohorts worldwide. We observed an increased frequency of B cells and a significant decrease in the frequency of CD3+ T cells in COVID-19 patients compared to healthy donors (HD). We also found a reduction in Tregs, which was more pronounced in severe patients. During the first wave, the frequency of GZMB, CD107a, CD39, and PD-1-expressing conventional CD4+ T (T conv) cells was significantly higher in moderate and severe patients than in HD. During the second wave, only the GZMB+ T conv cells of moderate and severe patients increased significantly. In addition, these patients showed a decreased frequency in IL-2-producing T conv cells. Interestingly, we identified two subsets of circulating CD8+ T cells with low and high CD8 surface expression in both HD and COVID-19 patients. While the percentages of CD8hi and CD8lo T cells within the CD8+ population in HD are similar, a significant increase was observed in CD8lo T cell frequency in COVID-19 patients. CD8lo T cell populations from HD as well as from SARS-CoV-2 infected patients exhibited lower frequencies of the effector cytokine-producing cells, TNF, IL-2, and IFN-γ, than CD8hi T cells. Interestingly, the frequency of CD8lo T cells increased with disease severity, suggesting that this parameter could be a potential marker for disease progression. Indeed, the CD8hi/CD8lo index helped to significantly improve the patient's clinical stratification and disease outcome prediction. Our data support the addition of, at least, a CD8hi/CD8lo index into the panel of biomarkers commonly used in clinical labs, since its determination may be a useful tool with impact on the therapeutic management of the patients.

Different cytokine and chemokine profiles in hospitalized patients with COVID-19 during the first and second outbreaks from Argentina show no association with clinical comorbidities.

Background: COVID-19 severity has been linked to an increased production of inflammatory mediators called "cytokine storm". Available data is mainly restricted to the first international outbreak and reports highly variable results. This study compares demographic and clinical features of patients with COVID-19 from Córdoba, Argentina, during the first two waves of the pandemic and analyzes association between comorbidities and disease outcome with the "cytokine storm", offering added value to the field. Methods: We investigated serum concentration of thirteen soluble mediators, including cytokines and chemokines, in hospitalized patients with moderate and severe COVID-19, without previous rheumatic and autoimmune diseases, from the central region of Argentina during the first and second infection waves. Samples from healthy controls were also assayed. Clinical and biochemical parameters were collected. Results: Comparison between the two first COVID-19 waves in Argentina highlighted that patients recruited during the second wave were younger and showed less concurrent comorbidities than those from the first outbreak. We also recognized particularities in the signatures of systemic cytokines and chemokines in patients from both infection waves. We determined that concurrent pre-existing comorbidities did not have contribution to serum concentration of systemic cytokines and chemokines in COVID-19 patients. We also identified immunological and biochemical parameters associated to inflammation which can be used as prognostic markers. Thus, IL-6 concentration, C reactive protein level and platelet count allowed to discriminate between death and discharge in patients hospitalized with severe COVID-19 only during the first but not the second wave. Conclusions: Our data provide information that deepens our understanding of COVID-19 pathogenesis linking demographic features of a COVID-19 cohort with cytokines and chemokines systemic concentration, presence of comorbidities and different disease outcomes. Altogether, our findings provide information not only at local level by delineating inflammatory/anti-inflammatory response of patients but also at international level addressing the impact of comorbidities and the infection wave in the variability of cytokine and chemokine production upon SARS-CoV-2 infection.

Antibody detection against Kunitz-type protein in Fasciola hepatica experimentally infected sheep using enzyme-linked immunosorbent assay (ELISA).

Fasciolosis is a parasitic disease considered as emerging and neglected by the WHO. Sheep are highly susceptible to this disease, and affected flocks experience decreased productivity due to increased mortality, and the reduced quality of their products, such as wool and meat. To effectively control this disease, reliable and early diagnosis is essential for making decisions regarding antiparasitic application and/or the removal of affected animals. Currently, the diagnosis of F. hepatica in sheep relies on the detection of parasite eggs in faeces, a method that becomes reliable from week 10 post-infection. Consequently, there is a need for earlier diagnostic tools based on immune response. However, obtaining antigens for antibody detection has proven to be difficult and expensive. The aim of this study was to evaluate members of the Kunitz protein family of F. hepatica expressed in the form of a fusion protein in the serological diagnosis of F. hepatica in sheep. The performance of three recombinant F. hepatica Kunitz-type inhibitors (FhKT1.1, FhKT1.3, and FhKT4) was compared with a synthetic Kunitz-type peptide (sFhKT) in sera from sheep experimentally infected with F. hepatica, using an ELISA. Of these, FhKT1.1 showed the most promising diagnostic indicators, exhibiting high precision and low cross-reactivity, and thus potential for standardized production. The results of our study demonstrated that the application of FhKT1.1 is a valuable tool for early-stage diagnosis of F. hepatica in sheep. Such an early diagnosis can aid in implementing timely interventions and effectively managing the disease in sheep populations.

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.

Microscopic and PCR-based detection of microsporidia spores in human stool samples.

Microsporidia are obligate intracellular fungi with a remarkable ability to infect a wide range of invertebrate and vertebrate hosts. Namely, Enterocytozoon bieneusi is the most frequently microsporidia reported worldwide, and mainly associated with chronic diarrhea and wasting syndrome in AIDS patients. Microscopy and PCR-based detection techniques are effective for diagnosis and identification of species and genotypes; however, these methods should be standardized in each laboratory. In this study, we performed microscopy and nested PCR techniques with PCR product sequencing to detect E. bieneusi in human stool samples. These techniques, if applied together, might prove useful for diagnosis and future epidemiological studies of intestinal microsporidiosis in Argentina.

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.

A Vaccine Based on Kunitz-Type Molecule Confers Protection Against Fasciola hepatica Challenge by Inducing IFN-γ and Antibody Immune Responses Through IL-17A Production.

Fasciola hepatica is helminth parasite found around the world that causes fasciolosis, a chronic disease affecting mainly cattle, sheep, and occasionally humans. Triclabendazole is the drug of choice to treat this parasite. However, the continuous use of this drug has led to the development of parasite resistance and, consequently, the limitation of its effectiveness. Hence, vaccination appears as an attractive option to develop. In this work, we evaluated the potential of F. hepatica Kunitz-type molecule (FhKTM) as an antigen formulated with a liquid crystal nanostructure formed by self-assembly of 6-O-ascorbyl palmitate ester (Coa-ASC16) and the synthetic oligodeoxynucleotide containing unmethylated cytosine-guanine motifs (CpG-ODN) during an experimental model of fasciolosis in mice, and we further dissected the immune response associated with host protection. Our results showed that immunization of mice with FhKTM/CpG-ODN/Coa-ASC16 induces protection against F. hepatica challenge by preventing liver damage and improving survival after F. hepatica infection. FhKTM/CpG-ODN/Coa-ASC16-immunized mice elicited potent IFN-γ and IL-17A with high levels of antigen-specific IgG1, IgG2a, and IgA serum antibodies. Strikingly, IL-17A blockade during infection decreased IgG2a and IgA antibody levels as well as IFN-γ production, leading to an increase in mortality of vaccinated mice. The present study highlights the potential of a new vaccine formulation to improve control and help the eradication of F. hepatica infection, with potential applications for natural hosts such as cattle and sheep.

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.

Cathepsin L3 From Fasciola hepatica Induces NLRP3 Inflammasome Alternative Activation in Murine Dendritic Cells.

The production of IL-1-family cytokines such as IL-1ß and IL-18 is finely regulated by inflammasome activation after the recognition of pathogens associated molecular pattern (PAMPs) and danger associated molecular patterns (DAMPs). However, little is known about the helminth-derived molecules capable of activating the inflammasome. In the case of the helminth trematode Fasciola hepatica, the secretion of different cathepsin L cysteine peptidases (FhCL) is crucial for the parasite survival. Among these enzymes, cathepsin L3 (FhCL3) is expressed mainly in the juvenile or invasive stage. The ability of FhCL3 to digest collagen has demonstrated to be critical for intestinal tissue invasion during juvenile larvae migration. However, there is no information about the interaction of FhCL3 with the immune system. It has been shown here that FhCL3 induces a non-canonical inflammasome activation in dendritic cells (DCs), leading to IL-1ß and IL-18 production without a previous microbial priming. Interestingly, this activation was depending on the cysteine protease activity of FhCL3 and the NLRP3 receptor, but independent of caspase activation. We also show that FhCL3 is internalized by DCs, promoting pro-IL-1ß cleavage to its mature and biologically active form IL-1ß, which is released to the extracellular environment. The FhCL3-induced NLRP3 inflammasome activation conditions DCs to promote a singular adaptive immune response, characterized by increased production of IFN-γ and IL-13. These data reveal an unexpected ability of FhCL3, a helminth-derived molecule, to activate the NLRP3 inflammasome, which is independent of the classical mechanism involving caspase activation.

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.

Helminth Infections: Recognition and Modulation of the Immune Response by Innate Immune Cells.

The survival of helminths in the host over long periods of time is the result of a process of adaptation or dynamic co-evolution between the host and the parasite. However, infection with helminth parasites causes damage to the host tissues producing the release of danger signals that induce the recruitment of various cells, including innate immune cells such as macrophages (Mo), dendritic cells (DCs), eosinophils, basophils, and mast cells. In this scenario, these cells are able to secrete soluble factors, which orchestrate immune effector mechanisms that depend on the different niches these parasites inhabit. Here, we focus on recent advances in the knowledge of excretory-secretory products (ESP), resulting from helminth recognition by DCs and Mo. Phagocytes and other cells types such as innate lymphocyte T cells 2 (ILC2), when activated by ESP, participate in an intricate cytokine network to generate innate and adaptive Th2 responses. In this review, we also discuss the mechanisms of innate immune cell-induced parasite killing and the tissue repair necessary to assure helminth survival over long periods of time.

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.