Immunization with PIII, a fraction of Schistosoma mansoni soluble adult worm antigenic preparation, affects nitric oxide production by murine spleen cells

Nitric oxide (NO) is an important effector molecule involved in immune regulation and defense. NO produced by cytokine-activated macrophages was reported to be cytotoxic against the helminth Schistosoma mansoni. Identification and characterization of S. mansoni antigens that can provide protective immunity is crucial for understanding the complex immunoregulatory events that modulate the immune response in schistosomiasis. It is, then, essential to have available defined, purified parasite antigens. Previous work by our laboratory identified a fraction of S. mansoni soluble adult worm antigenic preparation (SWAP), named PIII, able to elicit significant in vitro cell proliferation and at the same time lower in vitro and in vivo granuloma formation when compared either to SEA (soluble egg antigen) or to SWAP. In the present work we report the effect of different in vivo trials with mice on their spleen cells ability to produce NO. We demonstrate that PIII-immunization is able to significantly increase NO production by spleen cells after in vitro stimulation with LPS. These data suggest a possible role for NO on the protective immunity induced by PIII.

Role of defensins and cathelicidin LL37 in auto-immune and auto-inflammatory diseases.

Defensins and cathelicidins are anti-microbial peptides (AMPs) that act as natural antibiotics and are part of the innate immune defence in many species. We consider human defensins and LL37, the only human member of the cathelicidin family. In particular, we refer to the human alpha-defensins called human neutrophil peptides (HNP1 through 4), which are produced by neutrophils, HD5 and HD6, mainly expressed in Paneth cells of intestine, the human beta-defensins HBD1, HBD2 and HBD3, synthesized by epithelial cells and LL37, which is located in granulocytes, but is also produced by epithelial cells of the skin, lungs, and gut. In the last years, the study of AMPs activity and regulation has allowed to understand the important role of these peptides not only in the innate defence mechanisms against bacteria, viruses, fungi, but also in the regulation of immune cell activation and migration. Complementary studies have disclosed a role for AMPs in modulating many physiological processes that involve non-immune cells, such as activation of wound healing, angiogenesis, cartilage remodeling. Due to the pleiotropic tasks of these peptides, many of them are now being discovered to contribute to immune pathology of chronic diseases that affect skin, gut, joints; this is supported by many examples of immune-mediated pathologies in which their expression is disregulated. In this article we review the current literature that suggests a role for human defensins and LL37 in pathogenic mechanisms of several chronic diseases that are considered of auto-immune or auto-inflammatory origin.

Partial Protection of Mice against Trypanosoma cruzi after Immunizing with the TcY 72 Antigenic Preparation

A 72 kDa Trypanosoma cruzi glycoprotein recognized by the 164C11 monoclonal antibody (IgM isotype) was purified by preparative electrophoresis. The antigenic preparation obtained, named TcY 72, was used to immunize C57Bl/10 mice. The following results were observed after immunization: (1) induction of higher titres of IgG than IgM antibodies, as evaluated by indirect immunofluorescence; (2) significant DTH after injection of epimastigotes in mice footpads; (3) peak parasitemia in immunized mice was significantly reduced and animals were negative by 13 days post-infection, although the mice still succumb to infection; (4) the phenotypic analysis of spleen cell populations showed a decrease in the CD4/CD8 ratio in immunized mice. Taken as a whole, these findings indicate that TcY 72 is immunogenic and potentially important for protective immunity.

Immunopathology of Chagas disease

The main clinical forms of Chagas disease (acute, indeterminate and chronic cardiac) present strong evidences for the participation of the immune system on pathogenesis. Although parasite multiplication is evident during acute infection, the intense acute myocarditis of this phase exhibits clear ultrastructural signs of cell-mediated immune damage, inflicted to parasitized and non-parasitized myocardiocytes and to the endothelium of myocardial capillaries (microangiopathy). Inflammation subsides almost completely when immunity decreases parasite load and suppressor factors modulate host reaction, but inflammation does not disappear when the disease enters the indeterminate phase. Inflammation becomes mild and focal and undergoes cyclic changes leading to complete resolution. However, the process is maintained because the disappearance of old focal lesions is balanced by the upsurge of new ones. This equilibrium allows for prolonged host survival in the absence of symptoms or signs of disease. The chronic cardiac form is represented by a delayed-type, cell-mediated diffuse myocarditis, that probably ensues when the suppressive mechanisms, operative during the indeterminate phase, become defaulted. The mechanism responsible for the transition from the indeterminate to the cardiac form, is poorly understood.

Delayed mortality and attenuated thrombocytopenia associated with severe malaria in urokinase- and urokinase receptor-deficient mice.

We explored the role of urokinase and tissue-type plasminogen activators (uPA and tPA), as well as the uPA receptor (uPAR; CD87) in mouse severe malaria (SM), using genetically deficient (-/-) mice. The mortality resulting from Plasmodium berghei ANKA infection was delayed in uPA(-/-) and uPAR(-/-) mice but was similar to that of the wild type (+/+) in tPA(-/-) mice. Parasitemia levels were similar in uPA(-/-), uPAR(-/-), and +/+ mice. Production of tumor necrosis factor, as judged from the plasma level and the mRNA levels in brain and lung, was markedly increased by infection in both +/+ and uPAR(-/-) mice. Breakdown of the blood-brain barrier, as evidenced by the leakage of Evans Blue, was similar in +/+ and uPAR(-/-) mice. SM was associated with a profound thrombocytopenia, which was attenuated in uPA(-/-) and uPAR(-/-) mice. Administration of aprotinin, a plasmin antagonist, also delayed mortality and attenuated thrombocytopenia. Platelet trapping in cerebral venules or alveolar capillaries was evident in +/+ mice but absent in uPAR(-/-) mice. In contrast, macrophage sequestration in cerebral venules or alveolar capillaries was evident in both +/+ and uPAR(-/-) mice. Polymorphonuclear leukocyte sequestration in alveolar capillaries was similar in +/+ and uPAR(-/-) mice. These results demonstrate that the uPAR deficiency attenuates the severity of SM, probably by its important role in platelet kinetics and trapping. These results therefore suggest that platelet sequestration contributes to the pathogenesis of SM.

NLRP3 Suppresses NK Cell-Mediated Responses to Carcinogen-Induced Tumors and Metastases.

The NLRP3 inflammasome acts as a danger signal sensor that triggers and coordinates the inflammatory response upon infectious insults or tissue injury and damage. However, the role of the NLRP3 inflammasome in natural killer (NK) cell-mediated control of tumor immunity is poorly understood. Here, we show in a model of chemical-induced carcinogenesis and a series of experimental and spontaneous metastases models that mice lacking NLRP3 display significantly reduced tumor burden than control wild-type (WT) mice. The suppression of spontaneous and experimental tumor metastases and methylcholanthrene (MCA)-induced sarcomas in mice deficient for NLRP3 was NK cell and IFN-γ-dependent. Focusing on the amenable B16F10 experimental lung metastases model, we determined that expression of NLRP3 in bone marrow-derived cells was necessary for optimal tumor metastasis. Tumor-driven expansion of CD11b(+)Gr-1(intermediate) (Gr-1(int)) myeloid cells within the lung tumor microenvironment of NLRP3(-/-) mice was coincident with increased lung infiltrating activated NK cells and an enhanced antimetastatic response. The CD11b(+)Gr-1(int) myeloid cells displayed a unique cell surface phenotype and were characterized by their elevated production of CCL5 and CXCL9 chemokines. Adoptive transfer of this population into WT mice enhanced NK cell numbers in, and suppression of, B16F10 lung metastases. Together, these data suggested that NLRP3 is an important suppressor of NK cell-mediated control of carcinogenesis and metastases and identify CD11b(+)Gr-1(int) myeloid cells that promote NK cell antimetastatic function. Cancer Res; 72(22); 5721-32. ©2012 AACR.

Molecular definition of inflammasomes

Inflammasomes are crucial actors of the innate immunity. They consist of cytoplasmic multiprotein complexes controlling the biological activities of the inflammatory cytokines IL-1ß and IL-18. Inflammasome assembly depends on protein domain interaction. In this chapter, we focus on the biochemical aspects characterizing the components of the inflammasomes and their assembly into a high moelcuar weight protelytic complex.

Role of ENaC in skin wound healing and phenotypic analysis of GILZ-deficient mice

In my first project, I analyzed the role of the amiloride-sensitive epithelial sodium channel ENaC) in the skin during wound healing. ENaC is present in the skin and a function in keratinocyte differentiation and barrier formation has been demonstrated. Previous findings suggested, that ENaC might be implicated in keratinocyte migration, although its role in wound healing was not analyzed yet. Using skin-specific (K14-Cre) conditional ENaC knockout and overexpressing mice, I determined the wound closure kinetic and performed morphometric measurements. The time course of wound repair was not significantly different in knockouts or transgenics when compared to control mice and the morphology of the closing wound was not altered. In my second project, I studied the glucocorticoid-induced leucine zipper (GILZ, Tsc22d3). GILZ is widely expressed and an important role has been predicted in immunity, adipogenesis and renal sodium handling. Mice were generated that constitutively lack all the functional domains of the Gilz gene. In these mice, the expression of GILZ mRNA transcripts and protein were completely abolished in all tissues tested. Surprisingly, knockout mice survived. To test whether GILZ mimicks glucocorticoid action, we studied its implication in T- and B- cell development and in a model of sepsis. We measured cytokine secretion in different inflammatory models, like in peritoneal and bone marrow-derived macrophages, in splenocytes and a model of sepsis. In all our experiments, cytokine secretion from GILZ- deficient cells was not different from controls. From 6 months onwards, knockout mice contained significantly less body fat and were lighter. Following sodium and water deprivation experiments, water and salt homeostasis was preserved. Sterility of knockout males was associated with a severe testis dysplasia, smaller seminiferous tubules, the number of Sertoli and germ cell was reduced while increased apoptosis, but not cell proliferation, was evidenced. The interstitial Leydig cell population was augmented, and higher plasma FSH and testosterone levels were found. Interestingly, the expression of the target gene Ppar2 was diminished in the testis and in the liver, but not in the skin, kidney or fat. Tsc22d1 mRNA transcript level was found to be upregulated in testis, but not in the kidney or fat tissue. In most tissue, excepted the testis, GILZ-deficient mice reveal functional redundancy amongst members of the Tsc22d family or genes involved in the same regulatory pathways. In summary, contrarily to the published in vitro data, GILZ does not play a crucial role attributed in immunology or inflammation, but we identified a novel function in spermatogenesis. -- Dans mon premier projet, j'ai analysé le rôle du canal épithélial sodique sensible à l'amiloride (ENaC) dans la cicatrisation de la peau. ENaC est présent dans la peau et il a une fonction dans la différenciation des kératinocytes et dans la formation de la barrière. Des études suggèrent qu'ENaC pourrait être impliqué dans la migration des kératinocytes, cependant, son rôle dans la cicatrisation n'a pas encore été étudié. A l'aide de souris qui surexpriment ou qui sont knockout pour ENaC, spécifiquement dans la peau (K14-Cre), j'ai analysé le temps de clôture de la cicatrice et j'ai aussi étudié la morphologie de la plaie guérissant. Chez les souris qui surexpriment ou chez les knockouts, la vitesse de fermeture et la morphologie de la cicatrice étaient identiques aux souris contrôles. Dans mon second projet, j'ai étudié le glucocorticoid-induced leucine zipper (GILZ, Tsc22d3). GILZ est largement exprimé et un rôle important a été prédit dans l'immunité, l'adipogénèse et le transport sodique rénal. Des souris ont été générées dont les domaines fonctionnels du gène Gilz sont éliminés. L'expression de GILZ en ARNm et protéine a été complètement abolie dans tous les tissus testés. Étonnamment, ces souris knockout survivent. Afin de tester si GILZ imite les effets des glucocorticoïdes, nous avons étudié son implication dans le développement des cellules T et B ainsi qu'un modèle de septicémie. Nous avons mesuré la sécrétion de cytokines à partir de différents modèles d'inflammation tels que des macrophages péritonéaux ou de moelle, de splénocytes ou encore d'un modèle de septicémie. Dans toutes nos expériences, la sécrétion de cytokines de cellules GILZ-déficientes était semblable. Dès 6 mois, les knockouts contenaient significativement moins de graisses et étaient plus légères. Suite à une privation sodique et aqueuse, l'homéostasie du sel et de l'eau était préservée. Les mâles knockouts présentaient une stérilité accompagnée d'une dysplasie testiculaire sévère, de tubules séminifères étaient plus petits et contenaient un nombre réduit de cellules de Sertoli et de cellules germinales. L'apoptose était augmentée dans ces cellules mais pas la prolifération cellulaire. Le nombre de cellules de Leydig était aussi plus élevé, ainsi que la FSH et la testostérone. L'expression du gène cible Pparγ2 était diminuée dans le testicule et le foie, mais pas dans la peau, le rein ou le tissu adipeux. L'ARNm de Tsc22d1 était plus exprimé dans le testicule, mais pas dans le rein ou le tissu adipeux. Dans la plupart des tissus, sauf le testicule, les souris knockouts révélaient une redondance fonctionnelle des autres membres de la famille Tsc22d ou de gènes impliqués dans les mêmes voies de régulation. En résumé, contrairement aux données in vitro, GILZ ne joue pas un rôle essentiel en immunologie, mais nous avons identifié une nouvelle fonction dans la spermatogénèse.

Is there a role for autoimmunity in immune protection against malaria?

Much remains to be known about the mechanisms involved in protective immunity against malaria and the way it is acquired. This is probably the reason why, in spite of so much progress, it has not yet been possible to develop an anti-malaria vaccine able to induce parasite specific antibodies (Ab) and/or T-cells. It has been considered in the early 80s that the induction of efficient protection against the blood stage forms of Plasmodium falciparum would not be possible without simultaneously eliciting an autoimmune (AI) response against erythrocytes, even at the price of inducing an AI pathology. Despite the description of the reciprocal relationship, i.e. the protective effect of malaria on the development of AI diseases - demonstrated since 1970 - no effort has been made to verify the possible involvement of the AI response in protection against malaria. With this end in view - and in the light of the knowledge acquired in autoimmunity and the existence of the so called "natural" (not associated with pathology) autoantibodies - we propose to examine the hypothesis that the participation of the AI response (not necessarily restricted to autologous erythrocyte antigens) in the immune protection against malaria is possible or even necessary.

Cell populations in lesions of cutaneous leishmaniasis of leishmania (L.) amazonensis- infected rhesus macaques, Macaca mulatta

The cellular nature of the infiltrate in cutaneous lesion of rhesus monkeys experimentally infected with Leishmania (L.) amazonensis was characterized by immunohistochemistry. Skin biopsies from infected animals with active or healing lesions were compared to non-infected controls (three of each type) to quantitate inflammatory cell types. Inflammatory cells (composed of a mixture of T lymphocyte subpopulations, macrophages and a small number of natural killer cells and granulocytes) were more numerous in active lesions than in healing ones. T-cells accounted for 44.7 ± 13.1% of the infiltrate in active lesions (versus CD2+= 40.3 ± 5.7% in healing lesions) and T-cell ratios favor CD8+ cells in both lesion types. The percentage of cells expressing class II antigen (HLA-DR+) in active lesions (95 ± 7.1%) was significantly higher (P < 0.005) from the healing lesions (42.7 ± 12.7%). Moreover, the expression of the activation molecules CD25 (@ 16%), the receptor for interleukin-2, suggests that many T cells are primed and proliferating in active lesions. Distinct histopathological patterns were observed in lesions at biopsy, but healing lesions contained more organized epithelioid granulomas and activated macrophages, followed by fibrotic substitution. The progression and resolution of skin lesions appears to be very similar to that observed in humans, confirming the potential for this to be used as a viable model to study the immune response in human cutaneous leishmaniasis.

Troubles de la déglutition de la personne âgée [Swallowing disorders in the elderly].

Swallowing disorders are common in the elderly and may become life-threatening when they cause aspiration, inhalation pneumonia, malnutrition or dehydration. Dysphagia and malnutrition go hand in hand and lead to progressive worsening of mobility, immunity and quality of life. The aging of swallowing, so-called presbyphagia, is still not noticeable by 65. It becomes clinically relevant from 80 years onwards, especially during intercurrent and neurodegenerative disease, oncological treatments, or after trauma. This article aims to help practitioners in detecting these disorders and to implement appropriate therapeutic measures, which are often easy and may imply a multidisciplinary approach.

Lassa virus entry in antigen presenting cells and evaluation of a novel nanoparticle vaccine platform against arena viruses

Arenaviruses are a large and diverse family of viruses that merit significant attention as causative agents of severe hemorrhagic fevers in humans. Lassa virus (LASV) in Africa and the South American hemorrhagic fever viruses Junin (JUNV), Machupo (MACV), and Guanarito (GTOV) have emerged as important human pathogens and represent serious public health problems in their respective endemic areas. A hallmark of fatal arenaviruses hemorrhagic fevers is a marked immunosuppression of the infected patients. Antigen presenting cells (APCs) such as macrophages and in particular dendritic cells (DCs) are early and preferred targets of arenaviruses infection. Instead of being recognized and presented as foreign antigens by DCs, arenaviruses subvert the normal mechanisms of pathogen recognition, invade DCs and establish a productive infection. Viral replication perturbs the DCs' ability to present antigens and to activate T and B cells, contributing to the marked virus-induced immunosuppression observed in fatal disease. Considering their crucial role in the development of an anti-viral immune response, the mechanisms by which arenaviruses, and in particular LASV, invade DCs are of particular interest. The C-type lectin DC-specific Intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) was recently identified as a potential entry receptor for LASV. The first project of my thesis focused therefore on the investigation of the role of DC-SIGN in LASV entry into primary human DCs. My data revealed that DC-SIGN serves as an attachment factor for LASV on human DCs and can facilitate capture of free virus and subsequent cell entry. However, in contrast to other emerging viruses, of the phlebovirus family, I found that DC-SIGN does likely not function as an authentic entry receptor for LASV. Moreover, I was able to show that LASV enters DCs via an unusually slow pathway that depends on actin, but is independent of clathrin and dynamin. Considering the lack of effective treatments and the limited public health infrastructure in endemic regions, the development of protective vaccines against arenaviruses is an urgent need. To address this issue, the second project of my thesis aimed at the development of a novel recombinant arenavirus vaccine based on a nanoparticle (NPs) platform and its evaluation in a small animal model. During the first phase of the project I designed, produced, and characterized suitable vaccine antigens. In the second phase of the project, I generated antigen-conjugated NPs, developed vaccine formulations, and tested the NPs for their ability to elicit anti-viral T cell responses as well as anti-viral antibodies. I demonstrated that the NPs platform is able to activate both cellular and humoral branches of the adaptive anti-viral immunity, providing proof-of-principle. In sum, my first project will allow, in a long term perspective, a better understanding of the viral pathogenesis and contribute to the development of novel antiviral strategies. The second project will expectidly offer a new treatment option against arenaviruses.

Infections by protozoa in immunocompromised hosts

Protozoa are among the most important pathogens that can cause infection in immunocompromised patients. They infect particularly individuals with impaired cell immunity, such as those with hematologic neoplasias, those submitted to transplant of solid organs, those under high-dose corticosteroid therapy, and carriers of the human immunodeficiency virus. Among the protozoa that most commonly cause disease in immunocompromised individuals are Toxoplasma gondii, Trypanosoma cruzi, Cryptosporidium parvum, Isospora belli, Cyclospora cayetanensis and microsporidia; the former two cause severe encephalitis and myocarditis, and the others cause gastrointestinal infections. Early diagnosis and prompt institution of specific therapy for each of these organisms are basic measures to decrease morbidity and mortality associated with these infections.

Copper, selenium, and zinc status and balances after major trauma.

To investigate the trace elements (TE) losses and status after trauma, 11 severely injured patients (Injury Severity Score: 29 +/- 6), admitted to the ICU were studied from the day of injury (D0) until D25. Balance studies were started within 24 hours after injury, until D7. Serum and urine samples were collected from D1 to D7, then on D10, 15, 20, and 25. Intravenous TE supplementation was initiated upon admission. SERUM: Selenium (Se) and zinc (Zn) levels were decreased until D7 and were normal thereafter. LOSSES: TE urinary excretions were higher than reference ranges until D20 in all patients. Fluid losses through drains contained large amounts of TE. BALANCES: Balances were slightly positive for copper (Cu) and Zn, and negative for Se from D5 to D7 despite supplements. Cu status exhibited minor changes compared to those observed with the Zn and Se status: Serum levels were decreased and losses increased. Considering the importance of Se and Zn in free radical scavenging, anabolism, and immunity, current recommendations for TE supplements in severely traumatized patients ought to be revised.

Studies on the virulence and attenuation of Trypanosoma cruzi using immunodeficient animals

Tissue invasion and pathology by Trypanosoma cruzi result from an interaction between parasite virulence and host immunity. Successive in vivo generations of the parasite select populations with increasing ability to invade the host. Conversely, prolonged in vitro selection of the parasite produces attenuated sublines with low infectivity for mammals. One such subline (TCC clone) has been extensively used in our laboratory as experimental vaccine and tested in comparative experiments with its virulent ancestor (TUL). The experiments here reviewed aimed at the use of immunodeficient mice for testing the infectivity of TCC parasites. It has not been possible to obtain virulent, revertant sublines by prolonged passaged in such mice.