Nasal administration of recombinant Neospora caninum secreting IL-15/IL-15Rα inhibits metastatic melanoma development in lung.

BACKGROUND: Metastases are the leading cause of mortality in many cancer types and lungs are one of the most common sites of metastasis alongside the liver, brain, and bones. In melanoma, 85% of late-stage patients harbor lung metastases. A local administration could enhance the targeting of metastases while limiting the systemic cytotoxicity. Therefore, intranasal administration of immunotherapeutic agents seems to be a promising approach to preferentially target lung metastases and decrease their burden on cancer mortality. From observations that certain microorganisms induce an acute infection of the tumor microenvironment leading to a local reactivating immune response, microbial-mediated immunotherapy is a next-generation field of investigation in which immunotherapies are engineered to overcome immune surveillance and escape from microenvironmental cancer defenses. METHODS: The goal of our study is to evaluate the potential of the intranasal administration of Neospora caninum in a syngeneic C57BL6 mouse model of B16F10 melanoma lung metastases. It also compares the antitumoral properties of a wild-type N. caninum versus N. caninum secreting human interleukin (IL)-15 fused to the sushi domain of the IL-15 receptor α chain, a potent activator of cellular immune responses. RESULTS: The treatment of murine lung metastases by intranasal administration of an N. caninum engineered to secrete human IL-15 impairs lung metastases from further progression with only 0,08% of lung surface harboring metastases versus 4,4% in wild-type N. caninum treated mice and 36% in untreated mice. The control of tumor development is associated with a strong increase in numbers, within the lung, of natural killer cells, CD8+ T cells and macrophages, up to twofold, fivefold and sixfold, respectively. Analysis of expression levels of CD86 and CD206 on macrophages surface revealed a polarization of these macrophages towards an antitumoral M1 phenotype. CONCLUSION: Administration of IL-15/IL-15Rα-secreting N. caninum through intranasal administration, a non-invasive route, lend further support to N. caninum-demonstrated clear potential as an effective and safe immunotherapeutic approach for the treatment of metastatic solid cancers, whose existing therapeutic options are scarce. Combination of this armed protozoa with an intranasal route could reinforce the existing therapeutic arsenal against cancer and narrow the spectrum of incurable cancers.

Vaccination of squirrel monkeys (Saimiri spp.) with nanoparticle-based Toxoplasma gondii antigens: new hope for captive susceptible species.

Squirrel monkeys (Saimiri spp.), new world primates from South America, are very susceptible to toxoplasmosis. Numerous outbreaks of fatal toxoplasmosis in zoos have been identified around the world, resulting in acute respiratory distress and sudden death. To date, preventive hygiene measures or available treatments are not able to significantly reduce this mortality in zoos. Therefore, vaccination seems to be the best long-term solution to control acute toxoplasmosis. Recently, we developed a nasal vaccine composed of total extract of soluble proteins of Toxoplasma gondii associated with muco-adhesive maltodextrin-nanoparticles. The vaccine, which generated specific cellular immune responses, demonstrated efficacy against toxoplasmosis in murine and ovine experimental models. In collaboration with six French zoos, our vaccine was used as a last resort in 48 squirrel monkeys to prevent toxoplasmosis. The full protocol of vaccination includes two intranasal sprays followed by combined intranasal and s.c. administration. No local or systemic side-effects were observed irrespective of the route of administration. Blood samples were collected to study systemic humoral and cellular immune responses up to 1 year after the last vaccination. Vaccination induced a strong and lasting systemic cellular immune response mediated by specific IFN-γ secretion by peripheral blood mononuclear cells. Since the introduction of vaccination, no deaths of squirrel monkeys due to T. gondii has been observed for more than 4 years suggesting the promising usage of our vaccine. Moreover, to explain the high susceptibility of naive squirrel monkeys to toxoplasmosis, their innate immune sensors were investigated. It was observed that Toll-like and Nod-like receptors appear to be functional following T. gondii recognition suggesting that the extreme susceptibility to toxoplasmosis may not be linked to innate detection of the parasite.

Immune response against toxoplasmosis-some recent updates RH: Toxoplasma gondii immune response.

AIMS: Cytokines, soluble mediators of immunity, are key factors of the innate and adaptive immune system. They are secreted from and interact with various types of immune cells to manipulate host body's immune cell physiology for a counter-attack on the foreign body. A study was designed to explore the mechanism of Toxoplasma gondii (T. gondii) resistance from host immune response. METHODS AND RESULTS: The published data on aspect of host (murine and human) immune response against T. gondii was taken from Google scholar and PubMed. Most relevant literature was included in this study. The basic mechanism of immune response starts from the interactions of antigens with host immune cells to trigger the production of cytokines (pro-inflammatory and anti-inflammatory) which then act by forming a cytokinome (network of cytokine). Their secretory equilibrium is essential for endowing resistance to the host against infectious diseases, particularly toxoplasmosis. A narrow balance lying between Th1, Th2, and Th17 cytokines (as demonstrated until now) is essential for the development of resistance against T. gondii as well as for the survival of host. Excessive production of pro-inflammatory cytokines leads to tissue damage resulting in the production of anti-inflammatory cytokines which enhances the proliferation of Toxoplasma. Stress and other infectious diseases (human immunodeficiency virus (HIV)) that weaken the host immunity particularly the cellular component, make the host susceptible to toxoplasmosis especially in pregnant women. CONCLUSION: The current review findings state that in vitro harvesting of IL12 from DCs, Np and MΦ upon exposure with T. gondii might be a source for therapeutic use in toxoplasmosis. Current review also suggests that therapeutic interventions leading to up-regulation/supplementation of SOCS-3, IL12, and IFNγ to the infected host could be a solution to sterile immunity against T. gondii infection. This would be of interest particularly in patients passing through immunosuppression owing to any reason like the ones receiving anti-cancer therapy, the ones undergoing immunosuppressive therapy for graft/transplantation, the ones suffering from immunodeficiency virus (HIV) or having AIDS. Another imortant suggestion is to launch the efforts for a vaccine based on GRA6Nt or other similar antigens of T. gondii as a probable tool to destroy tissue cysts.

Co-delivery of PLGA nanoparticles loaded with rSAG1 antigen and TLR ligands: An efficient vaccine against chronic toxoplasmosis.

Although vaccination is a promising approach for the control of toxoplasmosis, there is currently no commercially available human vaccine. Adjuvants such as delivery vehicles and immunomodulators are critical components of vaccine formulations. In this study, Poly (D, l-lactide-co-glycolide) (PLGA) nanoparticles were applied to serve as delivery system for both surface antigen-1 (SAG1), a candidate vaccine against toxoplasmosis and two TLR ligands, monophosphoryl lipid A (MPL) and imiquimod (IMQ), respectively. Compared to rSAG1 alone, CBA/J mice immunized with rSAG1-PLGA produced higher anti-SAG1 IgG antibodies titers. This response was increased by the co-administration of IMQ-PLGA (p < 0.01). Compared to IMQ-PLGA co-administration, MPL-PLGA co-administration further increased the humoral response (p < 0.01) and potentiated the Th1 humoral response. Compared to rSAG1 alone, rSAG1-PLGA, or rSAG1-PLGA mixed with IMQ-PLGA or MPL-PLGA similarly enhanced the cellular response characterized by the production of IFN-γ, IL-2, TNF-α and low levels of IL-5, indicating a Th1-biased immunity. The induced immune responses, led to significant brain cyst reductions (p < 0.01) after oral challenge with T. gondii cysts in mice immunized with either rSAG1-PLGA, rSAG1-PLGA + IMQ-PLGA, rSAG1-PLGA + MPL-PLGA formulations. Taken together the results indicated that PLGA nanoparticles could serve as a platform for dual-delivery of antigens and immunomodulators to provide efficacious vaccines against toxoplasmosis.

A Novel Calcium-Dependent Protein Kinase 1 Inhibitor Potently Prevents Toxoplasma gondii Transmission to Foetuses in Mouse.

Treatments currently used to prevent congenital toxoplasmosis are non-specific of Toxoplasma gondii and have grievous side effects. To develop a more specific and less toxic drug, we have designed SP230, an imidazo[1,2-b]pyridazine salt targeting the Toxoplasma gondii calcium-dependent protein kinase 1 (TgCDPK1) and active against acute toxoplasmosis in mice. Efficiency of SP230 to inhibit foetal transmission of the parasite was evaluated in a mouse model of congenital toxoplasmosis. Swiss mice were infected at mid-pregnancy with tachyzoites or cysts of the ME49 strain of T. gondii by intraperitoneal and oral route, respectively, and treated with SP230 at 50 mg/kg for 5 days by the same routes. Parasite burden in organs of dams and in foetuses was measured by quantitative PCR. Intraperitoneal administration of SP230 drastically reduced the number of parasites (more than 97% of reduction) in the brain and lungs of dams, and led to a reduction of 66% of parasite burden in foetuses. Oral administration of SP230 was particularly efficient with 97% of reduction of parasite burdens in foetuses. SP230 did not impact number and weight of offspring in our conditions. This inhibitor of TgCDPK1 is a promising candidate for the development of alternative therapeutics to treat infected pregnant women.

Neospora caninum glycosylphosphatidylinositols used as adjuvants modulate cellular immune responses induced in vitro by a nanoparticle-based vaccine.

Neospora caninum causes abortion in ruminants, leading to important economic losses and no efficient treatment or vaccine against neosporosis is available. Considering the complexity of the strategies developed by intracellular apicomplexan parasites to escape immune system, future vaccine formulations should associate the largest panel of antigens and adjuvants able to better stimulate immune responses than natural infection. A mucosal vaccine, constituted of di-palmitoyl phosphatidyl glycerol-loaded nanoparticles (DGNP) and total extract (TE) of soluble antigens of Toxoplasma gondii, has demonstrated its efficacy, decreasing drastically the parasite burden. Here, DGNP were loaded with N. caninum TE and glycosylphosphatidylinositol (GPI) of N. caninum as Toll-like receptor (TLR) adjuvant able to induce specific cellular and humoral immune responses. Activation of TLR2 and TLR4 signalling pathway in HEK reporter cells induced by GPI was abrogated after its incorporation into DGNP. However, in murine bone marrow-derived dendritic cells, an adjuvant effect of GPI was observed with higher levels of interleukin (IL)-1ß, reduced levels of IL-6, IL-12p40 and IL-10, and decreased expression of major histocompatibility complex (MHC) molecules. GPI also modulated the responses of bovine peripheral blood mononuclear cells, by increasing the production of IFN-γ and by decreasing the expression of MHC molecules. Altogether, these results suggest that GPI delivered by the DGNP might modulate cell responses through the activation of an intracellular pathway of signalisation in a TLR-independent manner. In vivo experiments are needed to confirm the potent adjuvant properties of N. caninum GPI in a vaccine strategy against neosporosis.

Engineering and Functional Evaluation of Neutralizing Antibody Fragments Against Congenital Toxoplasmosis.

Maternal-fetal transmission of Toxoplasma gondii tachyzoites acquired during pregnancy has potentially dramatic consequences for the fetus. Current reference-standard treatments are not specific to the parasite and can induce severe side effects. In order to provide treatments with a higher specificity against toxoplasmosis, we developed antibody fragments-single-chain fragment variable (scFv) and scFv fused with mouse immunoglobulin G2a crystallizable fragment (scFv-Fc)-directed against the major surface protein SAG1. After validating their capacity to inhibit T. gondii proliferation in vitro, the antibody fragments' biological activity was assessed in vivo using a congenital toxoplasmosis mouse model. Dams were treated by systemic administration of antibody fragments and with prevention of maternal-fetal transmission being used as the parameter of efficacy. We observed that both antibody fragments prevented T. gondii dissemination and protected neonates, with the scFv-Fc format having better efficacy. These data provide a proof of concept for the use of antibody fragments as effective and specific treatment against congenital toxoplasmosis and provide promising leads.

The molecular targets of ivermectin and lotilaner in the human louse Pediculus humanus humanus: New prospects for the treatment of pediculosis.

Control of infestation by cosmopolitan lice (Pediculus humanus) is increasingly difficult due to the transmission of parasites resistant to pediculicides. However, since the targets for pediculicides have no been identified in human lice so far, their mechanisms of action remain largely unknown. The macrocyclic lactone ivermectin is active against a broad range of insects including human lice. Isoxazolines are a new chemical class exhibiting a strong insecticidal potential. They preferentially act on the γ-aminobutyric acid (GABA) receptor made of the resistant to dieldrin (RDL) subunit and, to a lesser extent on glutamate-gated chloride channels (GluCls) in some species. Here, we addressed the pediculicidal potential of isoxazolines and deciphered the molecular targets of ivermectin and the ectoparasiticide lotilaner in the human body louse species Pediculus humanus humanus. Using toxicity bioassays, we showed that fipronil, ivermectin and lotilaner are efficient pediculicides on adult lice. The RDL (Phh-RDL) and GluCl (Phh-GluCl) subunits were cloned and characterized by two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes. Phh-RDL and Phh-GluCl formed functional homomeric receptors respectively gated by GABA and L-glutamate with EC50 values of 16.0 µM and 9.3 µM. Importantly, ivermectin displayed a super agonist action on Phh-GluCl, whereas Phh-RDL receptors were weakly affected. Reversally, lotilaner strongly inhibited the GABA-evoked currents in Phh-RDL with an IC50 value of 40.7 nM, whereas it had no effect on Phh-GluCl. We report here for the first time the insecticidal activity of isoxazolines on human ectoparasites and reveal the mode of action of ivermectin and lotilaner on GluCl and RDL channels from human lice. These results emphasize an expected extension of the use of the isoxazoline drug class as new pediculicidal agents to tackle resistant-louse infestations in humans.

[Antitumoral microorganisms: The Swiss army knife of immunotherapy]. / Micro-organismes anti-cancéreux et armement - Le couteau suisse de l'immunothérapie.

Research on viruses, bacteria and protozoa-based immunotherapy has been on the rise for several years. The antitumoral efficacy of these microorganisms relies on three main mechanisms: Destruction of tumor cells, stimulation of the immune response and reprogramming of the tumor microenvironment. In order to optimize their immunotherapeutic action, these microorganisms can be genetically engineered to enhance their tumor-targeting efficacy or to vectorize immunostimulating molecules and/or antibodies. To this aim, molecular engineering allows the design of new antibody formats optimizing their functions. From whole antibodies to tandem single-chain variable fragments, various antibody formats can be vectorized by microorganisms to target receptors such as immune checkpoints or recruit immune effector cells within the tumor. Such possibilities broaden the arsenal of immunotherapeutic cancer treatment. This review focuses on these innovations and their advantages for immunotherapy.

TITLE: Micro-organismes anti-cancéreux et armement - Le couteau suisse de l'immunothérapie. ABSTRACT: Depuis plusieurs années, la recherche sur les micro-organismes pour une utilisation à des fins d'immunothérapie antitumorale est en plein essor. L'efficacité antitumorale de ces micro-organismes repose sur trois mécanismes principaux : la destruction des cellules tumorales, la stimulation du système immunitaire et la reprogrammation du microenvironnement tumoral. Afin d'optimiser leur action immunothérapeutique, ces micro-organismes peuvent être génétiquement modifiés pour les rendre capables de vectoriser des molécules immunostimulantes ou des anticorps. Par ingénierie moléculaire, il est désormais possible de diversifier les formats et fonctions de ces anticorps afin d'inhiber les points de contrôle immunitaire ou encore de recruter les cellules immunitaires effectrices au site de la tumeur. Cette Synthèse s'intéresse particulièrement à ces innovations et à leurs avantages en immunothérapie.

Molecular and Functional Characterization of GABA Receptor Subunits GRD and LCCH3 from Human Louse Pediculus Humanus Humanus.

Human louse Pediculus humanus is a cosmopolitan obligatory blood-feeding ectoparasite causing pediculosis and transmitting many bacterial pathogens. Control of infestation is difficult due to the developed resistance to insecticides that mainly target GABA (γ-aminobutyric acid) receptors. Previous work showed that Pediculus humanus humanus (Phh) GABA receptor subunit resistance to dieldrin (RDL) is the target of lotilaner, a synthetic molecule of the isoxazoline chemical class. To enhance our understanding of how insecticides act on GABA receptors, two other GABA receptor subunits were cloned and characterized: three variants of Phh-grd (glycine-like receptor of Drosophila) and one variant of Phh-lcch3 (ligand-gated chloride channel homolog 3). Relative mRNA expression levels of Phh-rdl, Phh-grd, and Phh-lcch3 revealed that they were expressed throughout the developmental stages (eggs, larvae, adults) and in the different parts of adult lice (head, thorax, and abdomen). When expressed individually in the Xenopus oocyte heterologous expression system, Phh-GRD1, Phh-GRD2, Phh-GRD3, and Phh-LCCH3 were unable to reconstitute functional channels, whereas the subunit combinations Phh-GRD1/Phh-LCCH3, Phh-GRD1/Phh-RDL, and Phh-LCCH3/Phh-RDL responded to GABA in a concentration-dependent manner. The three heteromeric receptors were similarly sensitive to the antagonistic effect of picrotoxin and fipronil, whereas Phh-GRD1/Phh-RDL and Phh-LCCH3/Phh-RDL were respectively about 2.5-fold and 5-fold more sensitive to ivermectin than Phh-GRD1/Phh-LCCH3. Moreover, the heteropentameric receptor constituted by Phh-GRD1/Phh-LCCH3 was found to be permeable and highly sensitive to the extracellular sodium concentration. These findings provided valuable additions to our knowledge of the complex nature of GABA receptors in human louse that could help in understanding the resistance pattern to commonly used pediculicides. SIGNIFICANCE STATEMENT: Human louse is an ectoparasite that causes pediculosis and transmits several bacterial pathogens. Emerging strains developed resistance to the commonly used insecticides, especially those targeting GABA receptors. To understand the molecular mechanisms underlying this resistance, two subunits of GABA receptors were cloned and described: Phh-grd and Phh-lcch3. The heteromeric receptor reconstituted with the two subunits was functional in Xenopus oocytes and sensitive to commercially available insecticides. Moreover, both subunits were transcribed throughout the parasite lifecycle.

Key Limitations and New Insights Into the Toxoplasma gondii Parasite Stage Switching for Future Vaccine Development in Human, Livestock, and Cats.

Toxoplasmosis is a parasitic disease affecting human, livestock and cat. Prophylactic strategies would be ideal to prevent infection. In a One Health vaccination approach, the objectives would be the prevention of congenital disease in both women and livestock, prevention/reduction of T. gondii tissue cysts in food-producing animals; and oocyst shedding in cats. Over the last few years, an explosion of strategies for vaccine development, especially due to the development of genetic-engineering technologies has emerged. The field of vaccinology has been exploring safer vaccines by the generation of recombinant immunogenic proteins, naked DNA vaccines, and viral/bacterial recombinants vectors. These strategies based on single- or few antigens, are less efficacious than recombinant live-attenuated, mostly tachyzoite T. gondii vaccine candidates. Reflections on the development of an anti-Toxoplasma vaccine must focus not only on the appropriate route of administration, capable of inducing efficient immune response, but also on the choice of the antigen (s) of interest and the associated delivery systems. To answer these questions, the choice of the animal model is essential. If mice helped in understanding the protection mechanisms, the data obtained cannot be directly transposed to humans, livestock and cats. Moreover, effectiveness vaccines should elicit strong and protective humoral and cellular immune responses at both local and systemic levels against the different stages of the parasite. Finally, challenge protocols should use the oral route, major natural route of infection, either by feeding tissue cysts or oocysts from different T. gondii strains. Effective Toxoplasma vaccines depend on our understanding of the (1) protective host immune response during T. gondii invasion and infection in the different hosts, (2) manipulation and modulation of host immune response to ensure survival of the parasites able to evade and subvert host immunity, (3) molecular mechanisms that define specific stage development. This review presents an overview of the key limitations for the development of an effective vaccine and highlights the contributions made by recent studies on the mechanisms behind stage switching to offer interesting perspectives for vaccine development.

Neospora caninum: a new class of biopharmaceuticals in the therapeutic arsenal against cancer.

BACKGROUND: Microorganisms that can be used for their lytic activity against tumor cells as well as inducing or reactivating antitumor immune responses are a relevant part of the available immunotherapy strategies. Viruses, bacteria and even protozoa have been largely explored with success as effective human antitumor agents. To date, only one oncolytic virus-T-VEC-has been approved by the US Food and Drug Administration for use in biological cancer therapy in clinical trials. The goal of our study is to evaluate the potential of a livestock pathogen, the protozoan Neospora caninum, non-pathogenic in humans, as an effective and safe antitumorous agent. METHODS/RESULTS: We demonstrated that the treatment of murine thymoma EG7 by subcutaneous injection of N. caninum tachyzoites either in or remotely from the tumor strongly inhibits tumor development, and often causes their complete eradication. Analysis of immune responses showed that N. caninum had the ability to 1) lyze infected cancer cells, 2) reactivate the immunosuppressed immune cells and 3) activate the systemic immune system by generating a protective antitumor response dependent on natural killer cells, CD8-T cells and associated with a strong interferon (IFN)-γ secretion in the tumor microenvironment. Most importantly, we observed a total clearance of the injected agent in the treated animals: N. caninum exhibited strong anticancer effects without persisting in the organism of treated mice. We also established in vitro and an in vivo non-obese diabetic/severe combined immunodeficiency mouse model that N. caninum infected and induced a strong regression of human Merkel cell carcinoma. Finally, we engineered a N. caninum strain to secrete human interleukin (IL)-15, associated with the alpha-subunit of the IL-15 receptor thus strengthening the immuno-stimulatory properties of N. caninum. Indeed, this NC1-IL15hRec strain induced both proliferation of and IFN-γ secretion by human peripheral blood mononuclear cells, as well as improved efficacy in vivo in the EG7 tumor model. CONCLUSION: These results highlight N. caninum as a potential, extremely effective and non-toxic anticancer agent, capable of being engineered to either express at its surface or to secrete biodrugs. Our work has identified the broad clinical possibilities of using N. caninum as an oncolytic protozoan in human medicine.

Corrigendum: Blockade of IL-33R/ST2 Signaling Attenuates Toxoplasma gondii Ileitis Depending on IL-22 Expression.

[This corrects the article DOI: 10.3389/fimmu.2019.00702.].

Effective Nanoparticle-Based Nasal Vaccine Against Latent and Congenital Toxoplasmosis in Sheep.

Toxoplasma gondii is a parasitic protozoan of worldwide distribution, able to infect all warm-blooded animals, but particularly sheep. Primary infection in pregnant sheep leads to millions of abortions and significant economic losses for the livestock industry. Moreover, infected animals constitute the main parasitic reservoir for humans. Therefore, the development of a One-health vaccine seems the best prevention strategy. Following earlier work, a vaccine constituted of total extract of Toxoplasma gondii proteins (TE) associated with maltodextrin nanoparticles (DGNP) was developed in rodents. In this study we evaluated the ability of this vaccine candidate to protect against latent and congenital toxoplasmosis in sheep. After two immunizations by either intranasal or intradermal route, DGNP/TE vaccine generated specific Th1-cellular immune response, mediated by APC-secretion of IFN-γ and IL-12. Secretion of IL-10 appeared to regulate this Th1 response for intradermally vaccinated sheep but was absent in intranasally-vaccinated animals. Finally, protection against latent toxoplasmosis and transplacental transmission were explored. Intranasal vaccination led to a marked decrease of brain cysts compared with the non-vaccinated group. This DGNP/TE vaccine administered intranasally conferred a high level of protection against latent toxoplasmosis and its transplacental transmission in sheep, highlighting the potential for development of such a vaccine for studies in other species.

Recent Advances in the Roles of Neutrophils in Toxoplasmosis.

Neutrophils are now recognized as major components of the response to Toxoplasma gondii by their contribution to parasite elimination by a number of mechanisms. This article focuses on recent advances in the understanding of the mechanisms of migration, cytokine release, and formation of extracellular traps by neutrophils during toxoplasmosis.

Exploration and Modulation of Antibody Fragment Biophysical Properties by Replacing the Framework Region Sequences.

In order to increase the successful development of recombinant antibodies and fragments, it seems fundamental to enhance their expression and/or biophysical properties, such as the thermal, chemical, and pH stabilities. In this study, we employed a method bases on replacing the antibody framework region sequences, in order to promote more particularly single-chain Fragment variable (scFv) product quality. We provide evidence that mutations of the VH- C-C' loop might significantly improve the prokaryote production of well-folded and functional fragments with a production yield multiplied by 27 times. Additional mutations are accountable for an increase in the thermal (+19.6 °C) and chemical (+1.9 M) stabilities have also been identified. Furthermore, the hereby-produced fragments have shown to remain stable at a pH of 2.0, which avoids molecule functional and structural impairments during the purification process. Lastly, this study provides relevant information to the understanding of the relationship between the antibodies amino acid sequences and their respective biophysical properties.

Babesia divergens glycosylphosphatidylinositols modulate blood coagulation and induce Th2-biased cytokine profiles in antigen presenting cells.

Glycosylphosphatidylinositols (GPIs) are glycolipids described as toxins of protozoan parasites due to their inflammatory properties in mammalian hosts characterized by the production of interleukin (IL)-1, IL-12 and tumor necrosis factor (TNF)-α. In the present work, we studied the cytokines produced by antigen presenting cells in response to ten different GPI species extracted from Babesia divergens, responsible for babesiosis. Interestingly, B. divergens GPIs induced the production of anti-inflammatory cytokines (IL-2, IL-5) and of the regulatory cytokine IL-10 by macrophages and dendritic cells. In contrast to all protozoan GPIs studied until now, GPIs from B. divergens did not stimulate the production of TNF-α and IL-12, leading to a unique Th1/Th2 profile. Analysis of the carbohydrate composition of the B. divergens GPIs indicated that the di-mannose structure was different from the evolutionary conserved tri-mannose structure, which might explain the particular cytokine profile they induce. Expression of major histocompatibility complex (MHC) molecules on dendritic cells and apoptosis of mouse peritoneal cells were also analysed. B. divergens GPIs did not change expression of MHC class I, but decreased expression of MHC class II at the cell surface, while GPIs slightly increased the percentages of apoptotic cells. During pathogenesis of babesiosis, the inflammation-coagulation auto-amplification loop can lead to thrombosis and the effect of GPIs on coagulation parameters was investigated. Incubation of B. divergens GPIs with rat plasma ex vivo led to increase of fibrinogen levels and to prolonged activated partial thromboplastin time, suggesting a direct modulation of the extrinsic coagulation pathway by GPIs.

Protein delivery by porous cationic maltodextrin-based nanoparticles into nasal mucosal cells: Comparison with cationic or anionic nanoparticles.

Different types of biodegradable nanoparticles (NPs) have been studied as delivery systems for proteins into nasal mucosal cells, especially for vaccine applications. Such a nanocarrier must have the ability to be loaded with proteins and to transport this payload into mucosal cells. However, comparative data on nanoparticles' capacity for protein loading, efficiency of subsequent endocytosis and the quantity of nanocarriers used are either lacking or contradictory, making comparisons and the choice of a best candidate difficult. Here we compared 5 types of nanoparticles with different surface charge (anionic or cationic) and various inner compositions as potential vectors: the NPL (cationic maltodextrin NP with an anionic lipid core), cationic and anionic PLGA (Poly Lactic co-Glycolic Acid) NP, and cationic and anionic liposomes. We first quantified the protein association efficiency and NPL associated the largest amount of ovalbumin, used as a model protein. In vitro, the delivery of fluorescently-labeled ovalbumin into mucosal cells (airway epithelial cells, dendritic cells and macrophages) was assessed by flow cytometry and revealed that the NPL delivered protein to the greatest extent in all 3 different cell lines. Taken together, these data underlined the potential of the porous and cationic maltodextrin-based NPL as efficient protein delivery systems to mucosal cells.

Blockade of IL-33R/ST2 Signaling Attenuates Toxoplasma gondii Ileitis Depending on IL-22 Expression.

Oral T. gondii infection (30 cysts of 76K strain) induces acute lethal ileitis in sensitive C57BL/6 (B6) mice with increased expression of IL-33 and its receptor ST2 in the ileum. Here we show that IL-33 is involved in ileitis, since absence of IL-33R/ST2 attenuated neutrophilic inflammation and Th1 cytokines upon T. gondii infection with enhanced survival. Blockade of ST2 by neutralizing ST2 antibody in B6 mice conferred partial protection, while rmIL-33 aggravated ileitis. Since IL-22 expression further increased in absence of ST2, we blocked IL-22 by neutralizing antibody, which abrogated protection from acute ileitis in ST2 deficient mice. In conclusion, severe lethal ileitis induced by oral T. gondii infection is attenuated by blockade of ST2 signaling and may be mediated in part by endogenous IL-22.

In vitro cellular responses to Neospora caninum glycosylphosphatidylinositols depend on the host origin of antigen presenting cells.

Neosporosis due to Neospora caninum causes abortions in farm animals such as cattle. No treatment and vaccine exist to fight this disease, responsible for considerable economic losses. It is thus important to better understand the immune responses occurring during the pathogenesis to control them in a global strategy against the parasite. In this context, we studied the roles of N. caninum glycosylphosphatidylinositols (GPIs), glycolipids defined as toxins in the related parasite Plasmodium falciparum. We demonstrated for the first time that GPIs could be excreted in the supernatant of N. caninum culture and trigger cell signalling through the Toll-like receptors 2 and 4. In addition, antibodies specific to N. caninum GPIs were detected in the serum of infected mice. As shown for other protozoan diseases, they could play a role in neutralizing GPIs. N. caninum GPIs were able to induce the production of tumour necrosis factor-α, interleukin(IL)-1ß and IL-12 cytokines by murine macrophages and dendritic cells. Furthermore, GPIs significantly reduced expression of major histocompatibility complex (MHC) molecules of class I on murine dendritic cells. In contrast to murine cells, bovine blood mononuclear cells produced increased levels of IFN-γ and IL-10, but reduced levels of IL-12p40 in response to GPIs. On these bovine cells, GPI had the tendency to up-regulate MHC class I, but to down-regulate MHC class II. Altogether, these results suggest that N. caninum GPIs might differentially participate in the responses of antigen presenting cells induced by the whole parasite in mouse models of neosporosis and in the natural cattle host.