Protection against Paracoccidioides brasiliensis infection conferred by the prophylactic administration of native and recombinant ArtinM

We determined the prophylactic effect of both the d-mannose-binding lectin ArtinM extracted from the seeds of Artocarpus integrifolia (jackfruit) and its recombinant counterpart during the course of experimental paracoccidioidomycosis induced in BALB/c mice. Four experimental protocols of prophylaxis were employed to evaluate the most protective regimen of ArtinM administration. It was demonstrated that the best effect was obtained by administration of two ArtinM doses on days 10 and 3 before the challenge with Paracoccidioides brasiliensis. By following this protocol, the lungs of mice that received native or recombinant ArtinM exhibited reduced fungal burden and granuloma incidence. In addition, the protocol augmented contents of IL-12, IFN-gamma, TNF-alpha and NO. On the other hand, the control group consisting of untreated infected mice had higher pulmonary levels of IL-4 and IL-10. In conclusion, prophylaxis with ArtinM significantly reproduces the effect of its therapeutic administration, i.e, it confers resistance to P. brasiliensis infection in mouse models by promoting IL-12 production and favours Th1-immunity.

Hyaluronan in vaginal secretions: association with recurrent vulvovaginal candidiasis

OBJECTIVE: We evaluated whether vaginal concentrations of hyaluronan were altered in women with recurrent vulvovaginal candidiasis (RVVC). STUDY DESIGN: Lavage samples from 17 women with acute RVVC, 27 women who were receiving a maintenance antifungal regimen, and 24 control women were tested for hyaluronan and interleukin (IL)-6, IL-12, and IL-23 by enzyme-linked immunosorbent assay. RESULTS: Median vaginal hyaluronan concentrations were 33.8 ng/mL (range, 21.6-66.3 ng/mL) in women with acute RVVC, 15.0 ng/mL (range, 11.2-50.6 ng/mL) in women who were receiving maintenance therapy, and 4.2 ng/mL (range, 3.6-12.0 ng/mL) in control subjects (P <= .02). The vaginal hyaluronan concentration was 27.4 ng/mL (range, 15.4-37.7 ng/mL) when Candida was detected by microscopy and 9.5 ng/mL (range, 7.7-14.6 ng/mL) in microscopy-negative cases (P = .0354). Elevated hyaluronan levels were associated with itching plus burning (40.7 ng/mL) or itching plus discharge (42.1 ng/mL), as opposed to itching only (6.2 ng/mL; P = .0152). Hyaluronan and IL-6 levels were correlated (P = .0009). CONCLUSION: Hyaluronan release is a component of the host response to a candidal infection and may contribute to symptoms.

Rheumatic Fever and Rheumatic Heart Disease: Cellular Mechanisms Leading Autoimmune Reactivity and Disease

Rheumatic fever (RF) is an autoimmune disease caused by the gram-positive bacteria Streptococcus pyogenes that follows a nontreated throat infection in susceptible children. The disease manifests as polyarthritis, carditis, chorea, erythema marginatum, and/or subcutaneous nodules. Carditis, the most serious complication, occurs in 30% to 45% of RF patients and leads to chronic rheumatic heart disease (RHD), which is characterized by progressive and permanent valvular lesions. In this review, we will focus on the genes that confer susceptibility for developing the disease, as well as the innate and adaptive immune responses against S. pyogenes during the acute rheumatic fever episode that leads to RHD autoimmune reactions. The disease is genetically determined, and some human leukocyte antigen class II alleles are involved with susceptibility. Other single nucleotide polymorphisms for TNF-alpha and mannan-binding lectin genes were reported as associated with RF/RHD. T cells play an important role in RHD heart lesions. Several autoantigens were already identified, including cardiac myosin epitopes, vimentin, and other intracellular proteins. In the heart tissue, antigen-driven oligoclonal T cell expansions were probably the effectors of the rheumatic heart lesions. These cells are CD4(+) and produced inflammatory cytokines (TNF alpha and IFN gamma). Molecular mimicry is the mechanism that mediated the cross-reactions between streptococcal antigens and human proteins. The elucidation of chemokines and their receptors involved with the recruitment of Th1, Th2, and Th17 cells, as well as the function of T regulatory cells in situ will certainly contribute to the delineation of the real picture of the heart lesion process that leads to RHD.

Lack of galectin-3 alters the balance of innate immune cytokines and confers resistance to Rhodococcus equi infection

Galectin-3 is a p-galactoside-binding lectin implicated in the fine-tuning of innate immunity. Rhodococcus equi, a facultative intracellular bacterium of macrophages, causes severe granulomatous bronchopneumonia in young horses and immunocompromised humans. The aim of this study is to investigate the role of galectin-3 in the innate resistance mechanism against R. equi infection. The bacterial challenge of galectin-3-deficient mice (gal3(-/-)) and their wild-type counterpart (gal3(+/+)) revealed that the LD50 for the gal3(-/-) mice was about seven times higher than that for the gal3(+/+) mice. When challenged with a sublethal dose, gal3(-/-) mice showed lower bacteria counts and higher production of IL-12 and IFN-gamma production, besides exhibiting a delayed although increased inflammatory reaction. Gal3(-/-) macrophages exhibited a decreased frequency of bacterial replication and survival, and higher transcript levels of IL-1 beta, IL-6, IL-10, TLR2 and MyD88. R. equi-infected gal3(+/+) macrophages showed decreased expression of TLR2, whereas R. equi-infected gal3(-/-) macrophages showed enhanced expression of this receptor. Furthermore, galectin-3 deficiency in macrophages may be responsible for the higher IL-1 beta serum levels detected in infected gal3(-/-) mice. Therefore galectin-3 may exert a regulatory role in innate immunity by diminishing IL-1 beta production and thus affecting resistance to R. equi infection.

Polymorphisms in innate immunity genes and patients response to dendritic cell-based HIV immuno-treatment

Dendritic cells (DCs)-based vaccine was demonstrated to increase HIV specific cellular immune response; however, in some HIV-infected patients, the response to the vaccine resulted to be not effective. In order to understand if the outcome of the vaccination may be influenced by the host`s genome and natural immunity, we studied the innate immune genome of HIV-infected patients previously vaccinated with DCs. We identified 15 SNPs potentially associated with the response to the immuno-treatment and two SNPs significantly associated with the modulation of the response to the DC vaccine: MBL2 rs10824792 and NOS1 rs693534. These two SNPs were also studied in different ethnic groups (Brazilians, African and Caucasian) of HIV-infected, exposed uninfected and unexposed uninfected subjects. The HIV positive Caucasian patients were also characterized by different disease progressions. Our findings suggest that, independently and/or in addition to other variables. the host`s genome could significantly contribute to the modulation of the response to the DC vaccine. (C) 2009 Elsevier Ltd. All rights reserved.

Neutrophil activation induced by ArtinM: Release of inflammatory mediators and enhancement of effector functions

The D-mannose binding lectin ArtinM from Artocarpus integrifolia, previously known as KM+ and artocarpin. is considered a stimulant of Th1-type immunity, which is able to confer resistance to some intracellular pathogens. In addition, ArtinM induces neutrophil migration by haptotaxis through simultaneous interactions of its carbohydrate recognition domains (CRDs) with glycans expressed on the extracellular matrix and the neutrophil surface. In the present study, we have expanded the characterization of ArtinM as a neutrophil activator. Exposure of neutrophils to ArtinM for 15 min resulted in tyrosine phosphorylation of intracellular proteins, a process that was selectively inhibited by D-mannose or mannotriose. Shortly after stimulation, neutrophils secreted high levels of LTB(4) and underwent shedding of L-selectin from their surface. Exposure to ArtinM enhanced neutrophil functions, such as respiratory burst and zymozan and Listeria monocytogenes phagocytosis. In addition, ArtinM-stimulated neutrophils displayed increased CXCL-8 secretion and TLR2 gene transcription. These results demonstrate that ArtinM is able to induce potent neutrophil activation, a feature that should be strongly considered in the assessment of the lectin capacity to confer resistance against infections. (C) 2009 Elsevier B.V. All rights reserved.

Assessment of Galectin-3 Polymorphism in Subjects with Chronic Chagas Disease

AbstractBackground:Galectin-3, a β-galactoside binding lectin, has been described as a mediator of cardiac fibrosis in experimental studies and as a risk factor associated with cardiovascular events in subjects with heart failure. Previous studies have evaluated the genetic susceptibility to Chagas disease in humans, including the polymorphisms of cytokine genes, demonstrating correlations between the genetic polymorphism and cardiomyopathy development in the chronic phase. However, the relationship between the galectin-3 single nucleotide polymorphism (SNP) and phenotypic variations in Chagas disease has not been evaluated.Objective:The present study aimed to determine whether genetic polymorphisms of galectin-3 may predispose to the development of cardiac forms of Chagas disease.Methods:Fifty-five subjects with Chagas disease were enrolled in this observational study. Real-time polymerase chain reaction (PCR) was used for genotyping the variants rs4644 and rs4652 of the galectin-3 gene.Results:For the SNP rs4644, the relative risk for the cardiac form was not associated with the genotypes AA (OR = 0.79, p = 0.759), AC (OR = 4.38, p = 0.058), or CC (OR = 0.39, p = 0.127). Similarly, for the SNP rs4652, no association was found between the genotypes AA (OR = 0.64, p = 0.571), AC (OR = 2.85, p = 0.105), or CC (OR = 0.49, p = 0.227) and the cardiac form of the disease.Conclusion:Our results showed no association between the different genotypes for both SNPs of the galectin-3 gene and the cardiac form of Chagas disease. (Arq Bras Cardiol. 2015; [online].ahead print, PP.0-0)

The complement inhibitor low molecular weight dextran sulfate prevents TLR4-induced phenotypic and functional maturation of human dendritic cells.

Low molecular weight dextran sulfate (DXS) has been reported to inhibit the classical, alternative pathway as well as the mannan-binding lectin pathway of the complement system. Furthermore, it acts as an endothelial cell protectant inhibiting complement-mediated endothelial cell damage. Endothelial cells are covered with a layer of heparan sulfate (HS), which is rapidly released under conditions of inflammation and tissue injury. Soluble HS induces maturation of dendritic cells (DC) via TLR4. In this study, we show the inhibitory effect of DXS on human DC maturation. DXS significantly prevents phenotypic maturation of monocyte-derived DC and peripheral myeloid DC by inhibiting the up-regulation of CD40, CD80, CD83, CD86, ICAM-1, and HLA-DR and down-regulates DC-SIGN in response to HS or exogenous TLR ligands. DXS also inhibits the functional maturation of DC as demonstrated by reduced T cell proliferation, and strongly impairs secretion of the proinflammatory mediators IL-1beta, IL-6, IL-12p70, and TNF-alpha. Exposure to DXS leads to a reduced production of the complement component C1q and a decreased phagocytic activity, whereas C3 secretion is increased. Moreover, DXS was found to inhibit phosphorylation of IkappaB-alpha and activation of NF-kappaB. These findings suggest that DXS prevents TLR-induced maturation of human DC and may therefore be a useful reagent to impede the link between innate and adaptive immunity.

Increased titers of anti-Saccharomyces cerevisiae antibodies in Crohn's disease patients with reduced H-ficolin levels but normal MASP-2 activity.

BACKGROUND AND AIMS: Mannan-binding lectin (MBL) and ficolins are microbial pattern recognition molecules that activate the lectin pathway of complement. We previously reported the association of MBL deficiency with anti-Saccharomyces cerevisiae antibodies (ASCA) in patients with Crohn's disease (CD). However, ASCA are also frequently found in MBL-proficient CD patients. Here we addressed expression/function of ficolins and MBL-associated serine protease-2 (MASP-2) regarding potential association with ASCA. METHODS: ASCA titers and MBL, ficolin and MASP-2 concentrations were determined by ELISA in the serum of patients with CD, ulcerative colitis (UC), and in healthy controls. MASP-2 activity was determined by measuring complement C4b-fixation. Anti-MBL autoantibodies were detected by ELISA. RESULTS: In CD and UC patients, L-ficolin concentrations were significantly higher compared to healthy controls (p<0.001 and p=0.029). In contrast, H-ficolin concentrations were slightly reduced in CD and UC compared to healthy controls (p=0.037 for UC vs. hc). CD patients with high ASCA titers had significantly lower H-ficolin concentrations compared to ASCA-low/negative CD patients (p=0.009). However, MASP-2 activity was not different in ASCA-negative and ASCA-positive CD patients upon both, ficolin- or MBL-mediated MASP-2 activation. Finally, anti-MBL autoantibodies were not over-represented in MBL-proficient ASCA-positive CD patients. CONCLUSIONS: Our results suggest that low expression of H-ficolin may promote elevated ASCA titers in the ASCA-positive subgroup of CD patients. However, unlike MBL deficiency, we found no evidence for low expression of serum ficolins or reduced MASP-2 activity that may predispose to ASCA development.

The conveyor belt hypothesis for thymocyte migration: participation of adhesion and de-adhesion molecules

Thymocyte differentiation is the process by which bone marrow-derived precursors enter the thymus, proliferate, rearrange the genes and express the corresponding T cell receptors, and undergo positive and/or negative selection, ultimately yielding mature T cells that will represent the so-called T cell repertoire. This process occurs in the context of cell migration, whose cellular and molecular basis is still poorly understood. Kinetic studies favor the idea that these cells leave the organ in an ordered pattern, as if they were moving on a conveyor belt. We have recently proposed that extracellular matrix glycoproteins, such as fibronectin, laminin and type IV collagen, among others, produced by non-lymphoid cells both in the cortex and in the medulla, would constitute a macromolecular arrangement allowing differentiating thymocytes to migrate. Here we discuss the participation of both molecules with adhesive and de-adhesive properties in the intrathymic T cell migration. Functional experiments demonstrated that galectin-3, a soluble ß-galactoside-binding lectin secreted by thymic microenvironmental cells, is a likely candidate for de-adhesion proteins by decreasing thymocyte interaction with the thymic microenvironment.

Nasopharyngeal colonization by pathogenic bacteria: effect of polymorphisms in innate immune genes of young children

Nasopharyngeal bacteria can asymptomatically colonize the nasopharynx of infants and young children but are also associated with the development of respiratory infections and diseases. Such nasopharyngeal bacteria include Streptococcus pneumoniae, Moraxella catarrhalis, Haemophilus influenzae and Staphylococcus aureus. The host defense against invading pathogens is largely relies germline-encoded pattern recognition receptors (PRR), which are expressed on the cells of innate immunity, and different cytokines. These include toll-like receptors (TLR), mannose-binding lectin (MBL) and different cytokines such as IL-17A. Single nucleotide polymorphisms (SNP) in these receptors and cytokines have been reported. The aim of this study was to investigate genetic polymorphisms in the genes for TLR2, 3 and 4, MBL as well as for IL-17A and their associations with nasopharyngeal pathogenic bacterial colonization during a two-year follow-up. The study revealed that polymorphisms in TLRs, MBL2 and IL17A are associated with the nasopharyngeal bacterial colonization in young children. Healthy young (2.6 months of age) children with variant types of MBL2, TLR2 R753Q or TLR4 D299G had an increased risk to be colonized by S. pneumonia, S. aureus or M. catarrhalis, respectively. Moreover, variant types of MBL2 in healthy children with might facilitate human rhinovirus (HRV)-induced S. pneumoniae colonization at 2.6 months of age. The polymorphism of TLR4 D299G was shown to be associated with M. catarrhalis colonization throughout the whole two-year follow-up (2.6, 13 and 24 months of age) and also with the bacterial load of this pathogen. Also, the polymorphism of IL17A G152A was shown to be associated with increased risk to be colonized by S. pneumoniae at 13 and 24 months of age. Furthermore, the results suggest that IL17A G152A has an effect on production of serum IL-17A already at young age. In conclusion, the results of this study indicate that polymorphisms in the key PRRs and IL17A seem to play an important role to colonization of S. pneumoniae, M. catarrhalis, and S. aureus in healthy young Finnish children. The nasopharyngeal colonization by these pathogenic bacteria may further promote the development of respiratory infections and may be related to development of asthma and allergy in the later life of children. These findings offer a possible explanation why some children have more respiratory infections than other children and provide a rational basis for future studies in this field.

Pertussis: infection, vaccination and gene polymorphisms

Pertussis or whooping cough is a human respiratory tract infection and a vaccine-preventable disease that is caused by Bordetella pertussis bacteria. Pertussis vaccination has been part of the Finnish national vaccine program since 1952. Despite extensive vaccinations, the incidence of pertussis has increased in many countries during the last decades. Large epidemics have been observed also in countries with high vaccine coverage. Inter-individual variation in immune responses is always encountered after vaccination. Low vaccine responses may cause vulnerability to pertussis even straight after vaccination. Reasons for low responses are not fully understood. The innate immune system is responsible for the initial recognition of pathogens and vaccine antigens. The role of innate immunity on pertussis immunity has not been thoroughly investigated. Mannose-binding lectin (MBL) and toll-like receptor 4 (TLR4) are important molecules of the innate immune system and in the recognition of pathogens. Cytokines form a signaling network that have a notable role in immune responses after infections as well as after vaccinations. Single nucleotide polymorphism (SNP) is common in genes encoding these molecules and the polymorphisms have been reported to affect vaccine response after viral and bacterial vaccines. This study investigated the gene polymorphisms of MBL2, TLR4 and interleukin (IL)-10 promoter and their association with vaccine responses after acellular pertussis (aP) vaccination in Finnish adolescents and infants. Cell-mediated immune responses were investigated ten years after the previous pertussis vaccinations in young adults. In addition, the role of MBL deficiency in pertussis infection susceptibility was evaluated. The results of this study show that subjects with TLR4 polymorphism had lower antibody production and persistence after aP vaccination compared with normal allele. A specific SNP in the TLR4 gene was associated with decreased antibody responses and persistence in adolescents after aP booster vaccination. Cell-mediated immune responses were partly detected ten years after the previous vaccination; booster vaccine clearly enhanced the responses. In addition, subjects with IL-10 polymorphism had altered cell-mediated immune responses. MBL deficiency was found to be more frequent in pertussis patients than healthy controls but the polymorphism of MBL2 was not associated with antibody responses after acellular pertussis vaccination. The novel finding of this study was that genetic variation in the innate immune system seems to play a role in altered pertussis vaccine responses as well as in pertussis infection. These new findings enlighten the mechanisms behind the low responses after pertussis vaccination and help to predict risk factors related to this phenomenon.

Rôles physiopathologiques du complément dans le syndrome coronarien aigu et implications thérapeutiques

Les efforts investis pour diminuer les risques de développer un infarctus du myocarde sont nombreux. Aujourd’hui les médecins prennent connaissance des divers facteurs de risque connus prédisposant aux syndromes coronariens aigus (SCA) dans le but de prendre en charge les patients «à risque» [1]. Bien que le suivi rigoureux et le contrôle de certains facteurs de risque modifiables aient permis une meilleure gestion des cas de SCA, les cas d’infarctus persistent de manière encore trop fréquente dans le monde. Puisque d’importantes études ont démontré que les SCA pouvaient survenir sans même la présence des facteurs de risque conventionnels [2, 3], les chercheurs se sont penchés sur un autre mécanisme potentiellement responsable de l’avènement des SCA : l’inflammation. L’inflammation joue un rôle prépondérant dans l’initiation, la progression et les complications de l’athérosclérose [4, 5] mais aussi dans les situations post-infarctus [6, 7]. Au cours des dernières années, le contrôle du processus inflammatoire est devenu une cible de choix dans la prévention et le traitement des SCA. Cependant, malgré les efforts investis, aucun de ces traitements ne s’est avéré pleinement efficace dans l’atteinte du but ultime visé par une diminution de l’inflammation : la diminution de la mortalité. Le complément est un système complexe reconnu principalement pour son rôle primordial dans l’immunité [2]. Cependant, lorsqu’il est activé de manière inappropriée ou excessive, il peut être à l’origine de nombreux dommages cellulaires caractéristiques de plusieurs pathologies inflammatoires dont font partie les complications de l’athérosclérose et des événements post-infarctus. Le travail effectué dans le cadre de mon doctorat vise à établir les rôles physiopathologiques du complément dans les interactions de l’axe thrombose-inflammation caractéristiques des SCA dans le but ultime d’identifier des cibles thérapeutiques permettant le développement de nouvelles approches pour la prévention et le traitement de ces pathologies. Les principaux résultats obtenus durant mon cursus suggèrent d’abord que la voie alterne du complément peut représenter une cible thérapeutique de choix dans les maladies coronariennes aiguës puisque l’activation terminale du complément semble y être principalement causée par l’activation du cette voie. De faibles niveaux sériques de MBL (mannan-binding lectin) et une activation terminale négligeable du complément caractérisent plutôt la maladie coronarienne stable. En comparant l’activité relative de chacune des voies du complément chez des cohortes de patients traités ou non par un anticorps spécifique à la protéine C5 du complément (pexelizumab), un second volet démontre quant à lui qu’une inhibition de l’activation du C5 n’a pas d’effet bénéfique majeur sur l’inhibition de la formation du complexe sC5b-9 ou sur les événements cliniques subséquents. Par conséquent, nous avons exploré, à l’aide d’un modèle in vitro, les raisons de l’inefficacité du traitement. Les résultats révèlent que le blocage du C5 avec le pexelizumab inhibe la production de l’anaphylatoxine pro-inflammatoire C5a et du complexe terminal du complément sans toutefois avoir d’effet sur l’apoptose des cellules endothéliales produites induite par le sérum des patients atteints de STEMI. Finalement, une autre section stipule que l’atorvastatine diminue l’activation du complément induite par les plaquettes sanguines chez des patients hypercholestérolémiques, mettant en évidence l’importance du rôle de cette statine dans la réduction des effets délétères de l’activation du système du complément médié par les plaquettes. Ensemble, l’étude du rôle spécifique des différentes voies d’activation du complément dans des contextes pathologiques variés, l’analyse des effets d’une inhibition spécifique de la protéine C5 du complément dans la progression des SCA et la mise en évidence des interactions entre l’activation du complément et les plaquettes activées ont contribué au développement d’une meilleure connaissance des rôles physiopathologiques du complément dans la progression de la maladie coronarienne.

Jacalin interaction with human immunoglobulin A1 and bovine immunoglobulin G1: Affinity constant determined by piezoelectric biosensoring

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Real-time monitoring and kinetic parameter estimation of the affinity interaction of jArtinM and rArtinM with peroxidase glycoprotein by the electrogravimetric technique

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)