Dynamics of the Trypanosoma cruzi infection in adipose tissue: Assessing gene expression of PNPLA2, FASN, and ACAT1 under Benzonidazole treatment and indirect mononuclear immune cells interaction.

Trypanosoma cruzi is a parasite with a high capacity to adapt to the host. Animal models have already demonstrated that the tropism of this parasite occurs not only in cardiac/digestive tissues but also in adipose tissue (AT). That said, the consequences ofT. cruziinfection for AT and the implications of treatment with Benzonidazole in this tissue are under discussion. Here, we tested the hypothesis that T. cruzi infection in adipose tissue upon treatment with Benzonidazole (Bz) and the interaction of mononuclear immune cells (PBMC) influences the relative expression of ACAT1, FASN, and PNPLA2 genes. Thus, stem cells derived from adipose tissue (ADSC) after adipogenic differentiation were indirectly cultivated with PBMC after infection with the T. cruzi Y strain and treatment with Bz. We use the TcSAT-IAM system and RT-qPCR to evaluate the parasite load and the relative quantification (ΔCt) of the ACAT1, FASN, and PNPLA2 genes. Our results demonstrate that treatment with Bz did not reduce adipocyte infection in the presence (p-value: 0.5796) or absence (p-value: 0.1854) of cultivation with PBMC. In addition, even though there is no statistical difference when compared to the control group (AT), T. cruzi induces the FASN expression (Rq: 14.00). However, treatment with Bz in AT suggests the increases of PNPLA2 expression levels (Rq: 12.58), even in the absence of T. cruzi infection. During indirect cultivation with PBMC, T. cruzi smooths the expression of PNPLA2 (Rq: 0.824) and instigates the expression of ACAT1 (Rq: 1.632) and FASN (Rq: 1.394). Furthermore, the treatment with Bz during infection induces PNPLA2 expression (Rq: 1.871), maintaining FASN expression levels (Rq: 1.334). Given this, our results indicate that treatment with Benzonidazole did not decrease T. cruzi infection in adipose tissue. However, treating the adipocyte cells with Bz during the interaction with PBMC cells influences the lipid pathways scenario, inducing lipolytic metabolism through the expression of PNPLA2.

Treating leishmaniasis in Amazonia, part 2: Multi-target evaluation of widely used plants to understand medicinal practices.

ETHNOPHARMACOLOGICAL RELEVANCE: Leishmaniasis are widely distributed among tropical and subtropical countries, and remains a crucial health issue in Amazonia. Indigenous groups across Amazonia have developed abundant knowledge about medicinal plants related to this pathology. AIM OF THE STUDY: We intent to explore the weight of different pharmacological activities driving taxa selection for medicinal use in Amazonian communities. Our hypothesis is that specific activity against Leishmania parasites is only one factor along other (anti-inflammatory, wound healing, immunomodulating, antimicrobial) activities. MATERIALS AND METHODS: The twelve most widespread plant species used against leishmaniasis in Amazonia, according to their cultural and biogeographical importance determined through a wide bibliographical survey (475 use reports), were selected for this study. Plant extracts were prepared to mimic their traditional preparations. Antiparasitic activity was evaluated against promastigotes of reference and clinical New-World strains of Leishmania (L. guyanensis, L. braziliensis and L. amazonensis) and L. amazonensis intracellular amastigotes. We concurrently assessed the extracts immunomodulatory properties on PHA-stimulated human PBMCs and RAW264.7 cells, and on L. guyanensis antigens-stimulated PBMCs obtained from Leishmania-infected patients, as well as antifungal activity and wound healing properties (human keratinocyte migration assay) of the selected extracts. The cytotoxicity of the extracts against various cell lines (HFF1, THP-1, HepG2, PBMCs, RAW264.7 and HaCaT cells) was also considered. The biological activity pattern of the extracts was represented through PCA analysis, and a correlation matrix was calculated. RESULTS: Spondias mombin L. bark and Anacardium occidentale L. stem and leaves extracts displayed high anti-promatigotes activity, with IC50 ≤ 32 µg/mL against L. guyanensis promastigotes for S. mombin and IC50 of 67 and 47 µg/mL against L. braziliensis and L. guyanensis promastigotes, respectively, for A. occidentale. In addition to the antiparasitic effect, antifungal activity measured against C. albicans and T. rubrum (MIC in the 16-64 µg/mL range) was observed. However, in the case of Leishmania amastigotes, the most active species were Bixa orellana L. (seeds), Chelonantus alatus (Aubl.) Pulle (leaves), Jacaranda copaia (Aubl.) D. Don. (leaves) and Plantago major L. (leaves) with IC50 < 20 µg/mL and infection rates of 14-25% compared to the control. Concerning immunomodulatory activity, P. major and B. orellana were highlighted as the most potent species for the wider range of cytokines in all tested conditions despite overall contrasting results depending on the model. Most of the species led to moderate to low cytotoxic extracts except for C. alatus, which exhibited strong cytotoxic activity in almost all models. None of the tested extracts displayed wound healing properties. CONCLUSIONS: We highlighted pharmacologically active extracts either on the parasite or on associated pathophysiological aspects, thus supporting the hypothesis that antiparasitic activities are not the only biological factor useful for antileishmanial evaluation. This result should however be supplemented by in vivo studies, and attracts once again the attention on the importance of the choice of biological models for an ethnophamacologically consistent study. Moreover, plant cultural importance, ecological status and availability were discussed in relation with biological results, thus contributing to link ethnobotany, medical anthropology and biology.

Association of IL-9, IL-10, and IL-17 Cytokines With Hepatic Fibrosis in Human Schistosoma mansoni Infection.

This is a case series study to evaluate immunological markers associated with schistosomiasis advanced fibrosis, including 69 patients from an endemic area from the State of Sergipe and from the Hepatology Service of the University Hospital in Sergipe, Brazil. Hepatic fibrosis was classified based on Niamey protocol for ultrasonography (US). Immune response to Schistosoma mansoni antigens was evaluated by stimulating peripheral blood mononuclear cells (PBMCs) from these patients with either adult worm (SWAP-10 µg/ml) or egg (SEA-10 µg/ml) antigens or purified protein derivative of turberculin (PPD-10 µg/ml) or phytohemagglutinin (PHA-1 µg/ml) for 72 h. The levels of IFN-γ, TNF-α, IL-5, IL-10, and IL-17 were measured in these supernatants by ELISA and IL-9 by Luminex. Single nucleotide polymorphisms in IL-17, IL10, and CD209 genes were genotyped using TaqMan probe by qPCR. Higher levels of IL-9, IL-10, and IL-17 were found in PBMC supernatants of patients with advanced hepatic fibrosis. Direct correlations were detected between IL-9 and IL-17 levels with US spleen sizes, portal vein diameters, and periportal thickening. The CD209 rs2287886 AG polymorphism patients produce higher IL-17 levels. Together, these data suggest a role of these cytokines in the immunopathogenesis of advanced fibrosis in human schistosomiasis.

Timing of Transcriptomic Peripheral Blood Mononuclear Cell Responses of Sheep to Fasciola hepatica Infection Differs From Those of Cattle, Reflecting Different Disease Phenotypes.

Infection with the zoonotic trematode Fasciola hepatica, common in many regions with a temperate climate, leads to delayed growth and loss of productivity in cattle, while infection in sheep can have more severe effects, potentially leading to death. Previous transcriptomic analyses revealed upregulation of TGFB1, cell death and Toll-like receptor signalling, T-cell activation, and inhibition of nitric oxide production in macrophages in response to infection. However, the differences between ovine and bovine responses have not yet been explored. The objective of this study was to further investigate the transcriptomic response of ovine peripheral blood mononuclear cells (PBMC) to F. hepatica infection, and to elucidate the differences between ovine and bovine PBMC responses. Sixteen male Merino sheep were randomly assigned to infected or control groups (n = 8 per group) and orally infected with 120 F. hepatica metacercariae. Transcriptomic data was generated from PBMC at 0, 2 and 16 weeks post-infection (wpi), and analysed for differentially expressed (DE) genes between infected and control animals at each time point (analysis 1), and for each group relative to time 0 (analysis 2). Analysis 2 was then compared to a similar study performed previously on bovine PBMC. A total of 453 DE genes were found at 2 wpi, and 2 DE genes at 16 wpi (FDR < 0.1, analysis 1). Significantly overrepresented biological pathways at 2 wpi included role of PKR in interferon induction and anti-viral response, death receptor signalling and RIG-I-like receptor signalling, which suggested that an activation of innate response to intracellular nucleic acids and inhibition of cellular apoptosis were taking place. Comparison of analysis 2 with the previous bovine transcriptomic study revealed that anti-inflammatory response pathways which were significantly overrepresented in the acute phase in cattle, including IL-10 signalling, Th2 pathway, and Th1 and Th2 activation were upregulated only in the chronic phase in sheep. We propose that the earlier activation of anti-inflammatory responses in cattle, as compared with sheep, may be related to the general absence of acute clinical signs in cattle. These findings offer scope for "smart vaccination" strategies for this important livestock parasite.

Impairment of effector molecules response in diabetes induces susceptibility to Leishmania amazonensis infection.

Type 2 Diabetes is a chronic disease resulting from insulin dysfunction that triggers a low-grade inflammatory state and immune impairment. Leishmaniasis is an infectious disease characterized by chronic inflammation resulted from the parasite's immunomodulation ability. Thus, due to the delicate immune balance required in the combat and resistance to Leishmania infection and the chronic deregulation of the inflammatory response observed in type 2 diabetes, we evaluated the response of PBMC from diabetic patients to in vitro Leishmania amazonensis infection. For that, peripheral blood was collected from 25 diabetic patients and 25 healthy controls matched for age for cells extraction and subsequent experimental infection for 2 or 24 h and analyzed for phagocytic and leishmanicidal capacity by optical microscopy, oxidative stress by GSSG generation, labeling of intracellular mediators by enzyme-Linked immunosorbent assay, and cytokines measurement with cytometric beads array technique. We found that the diabetic group had a higher percentage of infected cells and a greater number of amastigotes per cell. Also, even inducing NF-kB phosphorylation and increasing TNF production after infection, cells from diabetic patients were unable to downregulate NRF2 and generate oxidative stress, which may be associated with the exacerbated levels of IL-6 observed. PBMC of diabetic individuals are more susceptible to infection by L. amazonensis and fail to control the infection over time due to the inability to generate effector microbicidal molecules.

IL-10 receptor blockade controls the in vitro infectivity of Leishmania infantum and promotes a Th1 activation in PBMC of dogs with visceral leishmaniasis.

An important strategy to reduce the risk of visceral leishmaniasis (VL) in humans is to control the infection and disease progression in dogs, the domestic reservoir of Leishmania infantum parasites. Certain therapeutic strategies that modulate the host immune response show great potential for the treatment of experimental VL, restoring the impaired effector functions or decreasing host excessive responses. It is known that the overproduction of interleukin-10 (IL-10) promotes parasite replication and disease progression in human VL as well as in canine visceral leishmaniasis (CVL). Thus, in the present study we investigated the potential of the anti-canine IL-10 receptor-blocking monoclonal antibody (Bloq IL-10R) to control and reduce in vitro infectivity of L. infantum and improve the ability of PBMC isolated from VL dogs to alter the lymphoproliferative response and intracytoplasmic cytokines. Overall, GFP+Leishmania showed lower capacity of in vitro infectivity in the presence of Bloq IL-10R. Moreover, addition of Bloq IL-10R in cultured PBMC enhanced T-CD4 and CD8 proliferative response and altered the intracytoplasmic cytokine synthesis, reducing CD4+IL-4+ cells and increasing CD8+IFN-γ+ cells after specific antigen stimulation in PBMC of dogs. Furthermore, we observed an increase of TNF-α levels in supernatant of cultured PBMC under IL-10R neutralizing conditions. Together, our findings are encouraging and reaffirm an important factor that could influence the effectiveness of immune modulation in dogs with VL and suggest that blocking IL-10R activity has the potential to be a useful approach to CVL treatment.

Lack of detectable short-term effects of a single dose of ivermectin on the human immune system.

BACKGROUND: Ivermectin is widely used in human and animal medicine to treat and prevent parasite nematode infections. It has been suggested that its mode of action requires the host immune system, as it is difficult to reproduce its clinical efficacy in vitro. We therefore studied the effects of a single dose of ivermectin (Stromectol®-0.15 mg/kg) on cytokine levels and immune cell gene expression in human volunteers. This dose reduces bloodstream microfilariae rapidly and for several months when given in mass drug administration programmes. METHODS: Healthy volunteers with no travel history to endemic regions were given 3-4 tablets, depending on their weight, of either ivermectin or a placebo. Blood samples were drawn immediately prior to administration, 4 h and 24 h afterwards, and complete blood counts performed. Serum levels of 41 cytokines and chemokines were measured using Luminex® and expression levels of 770 myeloid-cell-related genes determined using the NanoString nCounter®. Cytokine levels at 4 h and 24 h post-treatment were compared to the levels pre-treatment using simple t tests to determine if any individual results required further investigation, taking p = < 0.05 as the level of significance. NanoString data were analysed on the proprietary software, nSolver™. RESULTS: No significant differences were observed in complete blood counts or cytokine levels at either time point between people given ivermectin versus placebo. Only three genes showed a significant change in expression in peripheral blood mononuclear cells 4 h after ivermectin was given; there were no significant changes 24 h after drug administration or in polymorphonuclear cells at either time point. Leukocytes isolated from those participants given ivermectin showed no difference in their ability to kill Brugia malayi microfilariae in vitro. CONCLUSIONS: Overall, our data do not support a direct effect of ivermectin, when given at the dose used in current filarial elimination programmes, on the human immune system. Trial registration ClinicalTrials.gov NCT03459794 Registered 9th March 2018, Retrospectively registered https://clinicaltrials.gov/ct2/show/NCT03459794?term=NCT03459794&draw=2&rank=1 .

Drugs that target early stages of Onchocerca volvulus: A revisited means to facilitate the elimination goals for onchocerciasis.

Several issues have been identified with the current programs for the elimination of onchocerciasis that target only transmission by using mass drug administration (MDA) of the drug ivermectin. Alternative and/or complementary treatment regimens as part of a more comprehensive strategy to eliminate onchocerciasis are needed. We posit that the addition of "prophylactic" drugs or therapeutic drugs that can be utilized in a prophylactic strategy to the toolbox of present microfilaricidal drugs and/or future macrofilaricidal treatment regimens will not only improve the chances of meeting the elimination goals but may hasten the time to elimination and also will support achieving a sustained elimination of onchocerciasis. These "prophylactic" drugs will target the infective third- (L3) and fourth-stage (L4) larvae of Onchocerca volvulus and consequently prevent the establishment of new infections not only in uninfected individuals but also in already infected individuals and thus reduce the overall adult worm burden and transmission. Importantly, an effective prophylactic treatment regimen can utilize drugs that are already part of the onchocerciasis elimination program (ivermectin), those being considered for MDA (moxidectin), and/or the potential macrofilaricidal drugs (oxfendazole and emodepside) currently under clinical development. Prophylaxis of onchocerciasis is not a new concept. We present new data showing that these drugs can inhibit L3 molting and/or inhibit motility of L4 at IC50 and IC90 that are covered by the concentration of these drugs in plasma based on the corresponding pharmacological profiles obtained in human clinical trials when these drugs were tested using various doses for the therapeutic treatments of various helminth infections.

Galectins from Onchocerca ochengi and O. volvulus and their immune recognition by Wistar rats, Gudali zebu cattle and human hosts.

BACKGROUND: During the last two decades research on animal filarial parasites, especially Onchocerca ochengi, infecting cattle in savanna areas of Africa revealed that O. ochengi as an animal model has biological features that are similar to those of O. volvulus, the aetiological agent of human onchocerciasis. There is, however, a paucity of biochemical, immunological and pathological data for O. ochengi. Galectins can be generated by parasites and their hosts. They are multifunctional molecules affecting the interaction between filarial parasites and their mammalian hosts including immune responses. This study characterized O. ochengi galectin, verified its immunologenicity and established its immune reactivity and that of Onchocerca volvulus galectin. RESULTS: The phylogenetic analysis showed the high degree of identity between the identified O. ochengi and the O. volvulus galectin-1 (ß-galactoside-binding protein-1) consisting only in one exchange of alanine for serine. O. ochengi galectin induced IgG antibodies during 28 days after immunization of Wistar rats. IgG from O. ochengi-infected cattle and O. volvulus-infected humans cross-reacted with the corresponding galectins. Under the applied experimental conditions in a cell proliferation test, O. ochengi galectin failed to significantly stimulate peripheral blood mononuclear cells (PBMCs) from O. ochengi-infected cattle, regardless of their parasite load. CONCLUSION: An O. ochengi galectin gene was identified and the recombinantly expressed protein was immunogenic. IgG from Onchocerca-infected humans and cattle showed similar cross-reaction with both respective galectins. The present findings reflect the phylogenetic relationship between the two parasites and endorse the appropriateness of the cattle O. ochengi model for O. volvulus infection research.

Benznidazole: Hero or villain of cellular immune response in chronic Chagas disease patients?

Although the treatment of chronic Chagas disease (CCD) patients with Benznidazole (Bz) is still controversial, its use may prevent or delay the progression of the disease to the most severe forms. One of the main factors that can influence the effectiveness of the treatment is the possible cooperation between drug effect and the host immune response. Herein, we evaluated the immune response of peripheral blood mononuclear cells (PBMCs) infected with Trypanosoma cruzi and submitted to Bz treatment. Blood samples of CCD patients (n = 7) and non-infected individuals (n = 6) were drawn to obtain PBMCs. After cell culture, the supernatants were harvested and stored, and the cell analyzed by flow cytometer. The results showed that Bz positively regulated the molecular process of cell activation (CD80) and antigen presentation (HLA-DR), increased phagocytosis receptor and macrophage activation (CD64), and did not induce an exacerbated immune response. In conclusion, these results highlight the relevance of using Bz that, despite not being a true hero, it is also not a villain, as it presents a wide range of pharmacological/immunological response interactions, important for the immune balance in the clinical progression of CCD.

Endochin-like quinolone-300 and ELQ-316 inhibit Babesia bovis, B. bigemina, B. caballi and Theileria equi.

BACKGROUND: The most common apicomplexan parasites causing bovine babesiosis are Babesia bovis and B. bigemina, while B. caballi and Theileria equi are responsible for equine piroplasmosis. Treatment and control of these diseases are usually achieved using potentially toxic chemotherapeutics, such as imidocarb diproprionate, but drug-resistant parasites are emerging, and alternative effective and safer drugs are needed. The endochin-like quinolones (ELQ)-300 and ELQ-316 have been proven to be safe and efficacious against related apicomplexans, such as Plasmodium spp., with ELQ-316 also being effective against Babesia microti, without showing toxicity in mammals. METHODS: The inhibitory effects of ELQ-300 and ELQ-316 were assessed on the growth of cultured B. bovis, B. bigemina, B. caballi and T. equi. The percentage of parasitized erythrocytes was measured by flow cytometry, and the effect of the ELQ compounds on the viability of horse and bovine peripheral blood mononuclear cells (PBMC) was assessed by monitoring cell metabolic activity using a colorimetric assay. RESULTS: We calculated the half maximal inhibitory concentration (IC50) at 72 h, which ranged from 0.04 to 0.37 nM for ELQ-300, and from 0.002 to 0.1 nM for ELQ-316 among all cultured parasites tested at 72 h. None of the parasites tested were able to replicate in cultures in the presence of ELQ-300 and ELQ-316 at the maximal inhibitory concentration (IC100), which ranged from 1.3 to 5.7 nM for ELQ-300 and from 1.0 to 6.0 nM for ELQ-316 at 72 h. Neither ELQ-300 nor ELQ-316 altered the viability of equine and bovine PBMC at their IC100 in in vitro testing. CONCLUSIONS: The compounds ELQ-300 and ELQ-316 showed significant inhibitory activity on the main parasites responsible for bovine babesiosis and equine piroplasmosis at doses that are tolerable to host cells. These ELQ drugs may be viable candidates for developing alternative protocols for the treatment of bovine babesiosis and equine piroplasmosis.

Human peripheral blood mononuclear cells as an ex vivo model to study the host parasite interaction in Toxoplasma gondii.

Toxoplasma gondii is a parasite that can invade any cell in the human body. Here, we implemented and described an ex vivo model with human peripheral blood mononuclear cells (PBMCs) without using culture supplements/antibiotics and without cryopreserved cells (EXMOWS) to study the interactions between T. gondii and human cells. To establish the EXMOWS, three independent tests were carried out. Firstly, blood samples from 5 individuals were included to assess the viability and adherence of PBMCs in plate culture. In a second trial, blood samples from three seropositive and two seronegative individuals for T. gondii were used to evaluate human PBMCs cells: parasites, multiplicity of infection (MOI) 1:1, 1:3 and 1:5 at different times post infection (1 h, 6 h and 24 h). The possible immunomodulatory effect of the infection for this EXMOWS were evaluated in a third trial where HFF cells were infected with T. gondii and co-cultured with PBMCs obtained from anti-Toxoplasma IgG positive and IgG negative individuals. One hour was enough time for T. gondii infection of human PBMCs and 2 h was the minimum incubation time to guarantee adherence before carrying out any infection assay. A minimum of 1:3 MOI was necessary to guarantee efficient infection in human PBMCs with T. gondii RH-GFP. All protocols, including PBMCs isolation and stimulation, should be conducted the same day. This EXMOWS can be adapted to study the early stages of interaction with other microorganisms of human interest, without need of using cryopreservation and supplements/antibiotics.

Characterization of a phosphotyrosyl phosphatase activator homologue of the parasitic nematode Haemonchus contortus and its immunomodulatory effect on goat peripheral blood mononuclear cells in vitro.

Suppression and modulation of the host immune response to parasitic nematodes have been extensively studied. In the present study, we cloned and produced recombinant phosphotyrosyl phosphatase activator protein from Haemonchus contortus (rHCPTPA), a parasitic nematode of small ruminants, and studied the effect of this protein on modulating the immune response of goat peripheral blood mononuclear cells. Enzymatic assays revealed that rHCPTPA enhanced the p-nitrophenylphosphate phosphatase activity of bovine PP2A1. Immunohistochemical tests verified that the HCPTPA protein was localised mainly in the bowel wall and on the body surface of worms. It was also shown that serum produced by goats artificially infected with H. contortus successfully recognised rHCPTPA, which conjugated with goat peripheral blood mononuclear cells. The rHCPTPA was then co-incubated with goat peripheral blood mononuclear cells to assess its immunomodulatory effects on proliferation, apoptosis, cytokine secretion, migration and nitric oxide production. Our results showed that rHCPTPA suppressed the proliferation of goat peripheral blood mononuclear cells stimulated by concanavalin A and induced apoptosis in goat peripheral blood mononuclear cells. After rHCPTPA exposure, IFN-γ and IL-2 expression was markedly reduced, whereas secretion of IL-10 and IL-4 was significantly elevated, in goat peripheral blood mononuclear cells. Moreover, rHCPTPA down-regulated nitric oxide production and migration of goat peripheral blood mononuclear cells in a dose-dependent manner. These results illuminate the interaction between parasites and hosts at the molecular level, suggest a possible immunomodulatory target and contribute to the search for innovative proteins that might be candidate targets for drugs and vaccines.

Innate immune responses to malaria-infected erythrocytes in pregnant women: Effects of gravidity, malaria infection, and geographic location.

BACKGROUND: Malaria in pregnancy causes maternal, fetal and neonatal morbidity and mortality, and maternal innate immune responses are implicated in pathogenesis of these complications. The effects of malaria exposure and obstetric and demographic factors on the early maternal immune response are poorly understood. METHODS: Peripheral blood mononuclear cell responses to Plasmodium falciparum-infected erythrocytes and phytohemagglutinin were compared between pregnant women from Papua New Guinea (malaria-exposed) with and without current malaria infection and from Australia (unexposed). Elicited levels of inflammatory cytokines at 48 h and 24 h (interferon γ, IFN-γ only) and the cellular sources of IFN-γ were analysed. RESULTS: Among Papua New Guinean women, microscopic malaria at enrolment did not alter peripheral blood mononuclear cell responses. Compared to samples from Australia, cells from Papua New Guinean women secreted more inflammatory cytokines tumor necrosis factor-α, interleukin 1ß, interleukin 6 and IFN-γ; p<0.001 for all assays, and more natural killer cells produced IFN-γ in response to infected erythrocytes and phytohemagglutinin. In both populations, cytokine responses were not affected by gravidity, except that in the Papua New Guinean cohort multigravid women had higher IFN-γ secretion at 24 h (p = 0.029) and an increased proportion of IFN-γ+ Vδ2 γδ T cells (p = 0.003). Cytokine levels elicited by a pregnancy malaria-specific CSA binding parasite line, CS2, were broadly similar to those elicited by CD36-binding line P6A1. CONCLUSIONS: Geographic location and, to some extent, gravidity influence maternal innate immunity to malaria.

Characterization of ovine monocyte activity when cultured with Haemonchus contortus larvae in vitro.

AIMS: The objective of this study was to identify and characterize cell populations within ovine peripheral blood mononuclear cells (PBMCs) associated with Haemonchus contortus (Hc) larval morbidity and impairment in vitro. METHODS AND RESULTS: Monocytes and lymphocytes were separated from PBMC from parasite-resistant St. Croix (STC) sheep and parasite-susceptible Suffolk (SUF) sheep. Cells were cultured with Hc third stage larvae (L3) for 9 h. Larval morbidity was assessed using ATP concentration. Activation status was determined through gene expression analysis and enzyme inhibition. Enzymes arginase-1 (Arg1) and inducible nitric oxide synthase (iNOS) were inhibited using BEC (S-(2-boronoethyl)-I-cysteine) and 1400W (N-(3-(aminomethyl)benzyl)acetamidine), respectively. Larval ATP was lower when cultured with STC-derived monocytes (0.015 µmol/L ATP) compared to SUF-derived monocytes (0.067 µmol/L ATP) (P < .001), or lymphocytes from either breed (STC: 0.085 µmol/L, SUF: 0.112 µmol/L ATP) (P < .001). SUF-derived monocytes displayed higher expression of M1 genes, whereas STC-derived monocytes displayed M2 genes continuously. Inhibition of Arg1 decreased monocyte function in both breeds, whereas iNOS inhibition restored SUF-derived monocyte function. CONCLUSIONS: Together, these data indicate STC-derived monocytes favour M2 phenotype when exposed to L3, where SUF-derived monocyte function resembled M1 phenotype and described potential for improving Suffolk sheep through modulating inflammatory responses.

Purification of Native Fasciola hepatica Fatty Acid-Binding Protein and Induction of Alternative Activation of Human Macrophages.

This chapter presents the different techniques to purify the native forms of Fasciola hepatica fatty acid-binding protein (Fh12) using size exclusion chromatography and isoelectric focusing (IEF). Also, it presents the procedure to study the immunological effect of the purified protein Fh12 using monocyte-derived macrophages (MDM) obtained from healthy human donors. For this purpose, I present the procedure to isolate and culture peripheral blood mononuclear cells (PBMCs) to generate alternatively activated macrophages (AAMΦ) by in vitro exposure to Fh12.

Leishmania infantum amastin protein incorporated in distinct adjuvant systems induces protection against visceral leishmaniasis.

The control measures against visceral leishmaniasis (VL) include a precise diagnosis of disease, the treatment of human cases, and reservoir and vector controls. However, these are insufficient to avoid the spread of the disease in specific countries worldwide. As a consequence, prophylactic vaccination could be interesting, although no effective candidate against human disease is available. In the present study, the Leishmania infantum amastin protein was evaluated regarding its immunogenicity and protective efficacy against experimental VL. BALB/c mice immunized with subcutaneous injections of the recombinant protein with or without liposome/saponin (Lip/Sap) as an adjuvant. After immunization, half of the animals per group were euthanized and immunological evaluations were performed, while the others were challenged with L. infantum promastigotes. Forty-five days after infection, the animals were euthanized and parasitological and immunological evaluations were performed. Results showed the development of a Th1-type immune response in rAmastin-Lip and rAmastin-Sap/vaccinated mice, before and after infection, which was based on the production of protein and parasite-specific IFN-γ, IL-12, GM-CSF, and nitrite, as well as the IgG2a isotype antibody. CD4+ T cells were mainly responsible for IFN-γ production in vaccinated mice, which also presented significant reductions in parasitism in their liver, spleen, draining lymph nodes, and bone marrow. In addition, PBMC cultures of treated VL patients and healthy subjects stimulated with rAmastin showed lymphoproliferation and higher IFN-γ production. In conclusion, the present study shows the first case of an L. infantum amastin protein associated with distinct delivery systems inducing protection against L. infantum infection and demonstrates an immunogenic effect of this protein in human cells.

Combined in vitro IL-12 and IL-15 stimulation promotes cellular immune response in dogs with visceral leishmaniasis.

Domestic dogs are the main reservoir of Leishmania infantum, a causative agent of visceral leishmaniasis (VL). The number of human disease cases is associated with the rate of canine infection. Currently available drugs are not efficient at treating canine leishmaniasis (CanL) and months after the treatment most dogs show disease relapse, therefore the development of new drugs or new therapeutic strategies should be sought. In CanL, dogs lack the ability to mount a specific cellular immune response suitable for combating the parasite and manipulation of cytokine signaling pathway has the potential to form part of effective immunotherapeutic methods. In this study, recombinant canine cytokines (rcaIL-12, rcaIL-2, rcaIL-15 and rcaIL-7) and soluble receptor IL-10R1 (rcasIL-10R1), with antagonistic activity, were evaluated for the first time in combination (rcaIL-12/rcaIL-2, rcaIL-12/rcaIL-15, rcaIL-12/rcasIL-10R1, rcaIL-15/rcaIL-7) or alone (rcasIL-10R1) to evaluate their immunomodulatory capacity in peripheral blood mononuclear cells (PBMCs) from dogs with leishmaniasis. All the combinations of recombinant proteins tested were shown to improve lymphoproliferative response. Further, the combinations rcaIL-12/rcaIL-2 and rcaIL-12/rcaIL-15 promoted a decrease in programmed cell death protein 1 (PD-1) expression in lymphocytes. These same combinations of cytokines and rcaIL-12/rcasIL-10R1 induced IFN-γ and TNF-α production in PBMCs. Furthermore, the combination IL-12/IL-15 led to an increased in T-bet expression in lymphocytes. These findings are encouraging and indicate the use of rcaIL-12 and rcaIL-15 in future in vivo studies aimed at achieving polarization of cellular immune responses in dogs with leishmaniasis, which may contribute to the development of an effective treatment against CanL.

Galectin Domain Containing Protein from Haemonchus contortus Modulates the Immune Functions of Goat PBMCs and Regulates CD4+ T-Helper Cells In Vitro.

Galectins are glycan-binding proteins that are widely expressed and distributed in mammalian tissues as well as cells of innate and adaptive immune responses. CD4+ T-helper cells differentiate into effector subsets in response to cytokines. T helper 9 cells are one of the recently described subsets of effector T cells that are relatively new and less studied. In this study, galectin domain containing protein from Haemonchus contortus (Hc-GDC) was cloned, expressed in pET32a, and immunoblotting was performed. Localization of recombinant (r)Hc-GDC on outer and inner surface of H. contortus worm and binding with goat Peripheral Blood Mononuclear cells (PBMCs) were performed using immunofluorescence assay. Moreover, effects of rHc-GDC on proliferation, apoptosis, cell migration, and the nitric oxide production in goat PBMCs were evaluated. Furthermore, modulatory effects of rHc-GDC on production of Th1, Th2, and Th9 cells were evaluated by flowcytometry and on interferon gamma, interleukin (IL)-4 and IL-9 were evaluated by quantitative real-time polymerase chain reaction. The results demonstrated that rHc-GDC was successfully cloned, expressed in expression vector as well as in the gut surface of adult H. contortus worm and successful binding with PBMCs surface were observed. Immunoblotting results revealed that rHc-GDC is an important active protein of H. contortus excretory and secretory products. Moreover, the interaction of rHc-GDC with host cells increased the production of Th2, Th9 cells, IL4, IL-9, PBMC proliferation, nitric oxide, and cell migration. No effects of rHc-GDC were observed on PMBC apoptosis, production of Th1 cells, and secretions of IFN- and IL-10 cytokines. These findings indicate that recombinant GDC protein from H. contortus modulates the immune functions of goat PBMCs and has the potential to enhance protective immunity by inducing T helper-9-derived IL-9 in vitro.

Biosafety Evaluation of Equine Umbilical Cord-Derived Mesenchymal Stromal Cells by Systematic Pathogen Screening in Peripheral Maternal Blood and Paired UC-MSCs.

Background: The growing interest in mesenchymal stromal cells (MSCs) in equine medicine, together with the development of MSC biobanking for allogeneic use, raises concerns about biosafety of such products. MSCs derived from umbilical cord (UC) carry an inherent risk of contamination by environmental conditions and vertical transmission of pathogens from broodmares. There is yet no report in the scientific literature about horses being contaminated by infected MSC products, and no consensus about systematic infectious screening of umbilical cord-derived mesenchymal stromal cells (UC-MSCs) to ensure microbiological safety of therapeutic products. Objectives: To develop a standard protocol to ensure UC-MSC microbiological safety and to assess the risk of vertical transmission of common intracellular pathogens from broodmares to paired UC-MSCs. Study Design and Methods: Eighty-four UC and paired peripheral maternal blood (PMB) samples were collected between 2014 and 2016. Sterility was monitored by microbiological control tests. Maternal contamination was tested by systematical PMB PCR screening for 14 pathogens and a Coggins test. In case of a PCR-positive result regarding one or several pathogen(s) in PMB, a PCR analysis for the detected pathogen(s) was then conducted on the associated UC-MSCs. Results: Ten out of 84 UC samples were contaminated upon extraction and 6/84 remained positive in primo culture. The remaining 78/84 paired PMB & UC-MSC samples were evaluated for vertical transmission; 37/78 PMB samples were PCR positive for Equid herpesvirus (EHV)-1, EHV-2, EHV-5, Theileria equi, Babesia caballi, and/or Mycoplasma spp. Hepacivirus was detected in 2/27 cases and Theiler Diseases Associated Virus in 0/27 cases (not performed on all samples due to late addition). All paired UC-MSC samples tested for the specific pathogen(s) detected in PMB were negative (37/37). Main Limitations: More data are needed regarding MSC susceptibility to most pathogens detected in PMB. Conclusions: In-process microbiological controls combined with PMB PCR screening provide a comprehensive assessment of UC-MSC exposure to infectious risk, vertical transmission risk appearing inherently low.