Leishmania-Specific Surface Antigens Show Sub-Genus Sequence Variation and Immune Recognition

Background: A family of hydrophilic acylated surface (HASP) proteins, containing extensive and variant amino acid repeats, is expressed at the plasma membrane in infective extracellular (metacyclic) and intracellular (amastigote) stages of Old World Leishmania species. While HASPs are antigenic in the host and can induce protective immune responses, the biological functions of these Leishmania-specific proteins remain unresolved. Previous genome analysis has suggested that parasites of the sub-genus Leishmania (Viannia) have lost HASP genes from their genomes. Methods/Principal Findings: We have used molecular and cellular methods to analyse HASP expression in New World Leishmania mexicana complex species and show that, unlike in L. major, these proteins are expressed predominantly following differentiation into amastigotes within macrophages. Further genome analysis has revealed that the L. (Viannia) species, L. (V.) braziliensis, does express HASP-like proteins of low amino acid similarity but with similar biochemical characteristics, from genes present on a region of chromosome 23 that is syntenic with the HASP/SHERP locus in Old World Leishmania species and the L. (L.) mexicana complex. A related gene is also present in Leptomonas seymouri and this may represent the ancestral copy of these Leishmania-genus specific sequences. The L. braziliensis HASP-like proteins (named the orthologous (o) HASPs) are predominantly expressed on the plasma membrane in amastigotes and are recognised by immune sera taken from 4 out of 6 leishmaniasis patients tested in an endemic region of Brazil. Analysis of the repetitive domains of the oHASPs has shown considerable genetic variation in parasite isolates taken from the same patients, suggesting that antigenic change may play a role in immune recognition of this protein family. Conclusions/Significance: These findings confirm that antigenic hydrophilic acylated proteins are expressed from genes in the same chromosomal region in species across the genus Leishmania. These proteins are surface-exposed on amastigotes (although L. (L.) major parasites also express HASPB on the metacyclic plasma membrane). The central repetitive domains of the HASPs are highly variant in their amino acid sequences, both within and between species, consistent with a role in immune recognition in the host.

Cutting Edge: Nucleotide-Binding Oligomerization Domain 1-Dependent Responses Account for Murine Resistance against Trypanosoma cruzi Infection

An effective innate immune recognition of the intracellular protozoan parasite Trypanosoma cruzi is critical for host resistance against Chagas disease, a severe and chronic illness that affects millions of people in Latin America. In this study, we evaluated the participation of nucleotide-binding oligomerization domain (Nod)like receptor proteins in host response to T cruzi infection and found that Nod1-dependent, but not Nod2-dependent, responses are required for host resistance against infection. Bone marrow-derived macrophages from Nod1(-/-) mice showed an impaired induction of NF-kappa B-dependent products in response to infection and failed to restrict T cruzi infection in presence of IFN-gamma. Despite normal cytokine production in the sera, Nod1(-/-) mice were highly susceptible to T cruzi infection, in a similar manner to MyD88(-/-) and NO synthase 2(-/-) mice. These studies indicate that Nod1-dependent responses account for host resistance against T cruzi infection by mechanisms independent of cytokine production. The Journal of Immunology, 2010, 184: 1148-1152.

A Novel Pathway for Inducible Nitric-oxide Synthase Activation through Inflammasomes

Innate immune recognition of flagellin is shared by transmembrane TLR5 and cytosolic Nlrc4 (NOD-like receptor family CARD (caspase activation recruitment domain) domain containing 4)/Naip5 (neuronal apoptosis inhibitory protein 5). TLR5 activates inflammatory genes through MYD88 pathway, whereas Nlrc4 and Naip5 assemble multiprotein complexes called inflammasomes, culminating in caspase-1 activation, IL-1 beta/IL-18 secretion, and pyroptosis. Although both TLR5 and Naip5/Nlrc4 pathways cooperate to clear infections, little is known about the relative anti-pathogen effector mechanisms operating through each of them. Here we show that the cytosolic flagellin (FLA-BSDot) was able to activate iNOS, an enzyme previously associated with TLR5 pathway. Using Nlrc4- or Naip5-deficient macrophages, we found that both receptors are involved in iNOS activation by FLA-BSDot. Moreover, distinct from extracellular flagellin (FLA-BS), iNOS activation by intracellular flagellin is completely abrogated in the absence of caspase-1. Interestingly, IL-1 beta and IL-18 do not seem to be important for FLA-BSDot-mediated iNOS production. Together, our data defined an additional anti-pathogen effector mechanism operated through Naip5 and Nlrc4 inflammasomes and illustrated a novel signaling transduction pathway that activates iNOS.

Plasmodium vivax apical membrane antigen-1: comparative recognition of different domains by antibodies induced during natural human infection

The Apical Membrane Antigen-1 (AMA-1) of Plasmodium sp. has been suggested as a vaccine candidate against malaria. This protein seems to be involved in merozoite invasion and its extra-cellular portion contains three distinct domains: DI, DII, and DIII. Previously, we described that Plasmodium vivax AMA-1 (PvAMA-1) ectodomain is highly immunogenic in natural human infections. Here, we expressed each domain, separately or in combination (DI-II or DII-III), as bacterial recombinant proteins to map immunodominant epitopes within the PvAMA-1 ectodomain. IgG recognition was assessed by ELISA using sera of P. vivax-infected individuals collected from endemic regions of Brazil or antibodies raised in immunized mice. The frequencies of responders to recombinant proteins containing the DII were higher than the others and similar to the ones observed against the PvAMA-1 ectodomain. Moreover, ELISA inhibition assays using the PvAMA-1 ectodomain as substrate revealed the presence of many common epitopes within DI-II that are recognized by human immune antibodies. Finally, immunization of mice with the PvAMA-1 ectodomain induced high levels of antibodies predominantly to DI-II. Together, our results indicate that DII is particularly immunogenic during natural human infections, thus indicating that this region could be used as part of an experimental sub-unit vaccine to prevent vivax malaria. (C) 2008 Elsevier Masson SAS. All rights reserved.

Innate immune lectins kill bacteria expressing blood group antigen

The expression of ABO(H) blood group antigens causes deletion of cells that generate self-specific antibodies to these antigens but this deletion limits adaptive immunity toward pathogens bearing cognate blood group antigens. To explore potential defense mechanisms against such pathogens, given these limitations in adaptive immunity, we screened for innate proteins that could recognize human blood group antigens. Here we report that two innate immune lectins, galectin-4 (Gal-4) and Gal-8, which are expressed in the intestinal tract, recognize and kill human blood group antigen-expressing Escherichia coli while failing to alter the viability of other E. coli strains or other Gram-negative or Gram-positive organisms both in vitro and in vivo. The killing activity of both Gal-4 and Gal-8 is mediated by their C-terminal domains, occurs rapidly and independently of complement and is accompanied by disruption of membrane integrity. These results demonstrate that innate defense lectins can provide immunity against pathogens that express blood group-like antigens on their surface.

The pattern recognition receptors Nod1 and Nod2 account for neutrophil recruitment to the lungs of mice infected with Legionella pneumophila

The intracellular bacterium Legionella pneumophila induces a severe form of pneumonia called Legionnaires diseases, which is characterized by a strong neutrophil (NE) infiltrate to the lungs of infected individuals. Although the participation of pattern recognition receptors, such as Toll-like receptors, was recently demonstrated, there is no information on the role of nod-like receptors (NLRs) for bacterial recognition in vivo and for NE recruitment to the lungs. Here, we employed a murine model of Legionnaires disease to evaluate host and bacterial factors involved in NE recruitment to the mice lungs. We found that L. pneumophila type four secretion system, known as Dot/Icm, was required for NE recruitment as dot/icm mutants fail to trigger NE recruitment in a process independent of bacterial multiplication. By using mice deficient for Nod1, Nod2, and Rip2, we found that these receptors accounted for NE recruitment to the lungs of infected mice. In addition, Rip2-dependent responses were important for cytokine production and bacterial clearance. Collectively, these studies show that Nod1, Nod2, and Rip2 account for generation of innate immune responses in vivo, which are important for NE recruitment and bacterial clearance in a murine model of Legionnaires diseases. (C) 2010 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Recognition by toll-like receptor 2 induces antigen-presenting cell activation and Th1 programming during infection by Neospora caninum

Neospora caninum is an apicomplexan parasite responsible for major economic losses due to abortions in cattle. Toll-like receptors (TLRs) sense specific microbial products and direct downstream signaling pathways in immune cells, linking innate, and adaptive immunity. Here, we analyze the role of TLR2 on innate and adaptive immune responses during N. caninum infection. Inflammatory peritoneal macrophages and bone marrow-derived dendritic cells exposed to N. caninum-soluble antigens presented an upregulated expression of TLR2. Increased receptor expression was correlated to TLR2/MyD88-dependent antigen-presenting cell maturation and pro-inflammatory cytokine production after stimulation by antigens. Impaired innate responses observed after infection of mice genetically deficient for TLR2((-/-)) was followed by downregulation of adaptive T helper 1 (Th1) immunity, represented by diminished parasite-specific CD4(+) and CD8(+) T-cell proliferation, IFN-gamma:interleukin (IL)-10 ratio, and IgG subclass synthesis. In parallel, TLR2(-/-) mice presented higher parasite burden than wild-type (WT) mice at acute and chronic stages of infection. These results show that initial recognition of N. caninum by TLR2 participates in the generation of effector immune responses against N. caninum and imply that the receptor may be a target for future prophylactic strategies against neosporosis. Immunology and Cell Biology (2010) 88, 825-833; doi:10.1038/icb.2010.52; published online 20 April 2010

The Role of Toll-Like Receptor 2 in the Recognition of Aggregatibacter actinomycetemcomitans

Background: Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) is a Gram-negative bacterium present in the oral cavity and is usually associated with localized aggressive periodontitis. Isolated antigens from A. actinomycetemcomitans can activate innate immune cells through Toll-like receptors (TLRs), which are molecules that recognize structural components conserved among microorganisms. In this study, we evaluate the role of TLR2 in the recognition of A. actinomycetemcomitans. Methods: Macrophages and neutrophils from knockout mice with targeted disruption of TLR2 (TLR2(-/-) mice) and wild-type mice were collected and used for the subsequent assays. The production of cytokines and chemokines was evaluated by enzyme-linked immunosorbent assay (ELISA), and the presence of apoptotic cells was determined by flow cytometry. In addition, the mechanisms that modulate the outcome of A. actinomycetemcomitans-induced periodontal disease in TLR2(-/-) mice were examined. Results: The results show that TLR2-deficient mice developed more severe periodontitis after A. actinomycetemcomitans infection, characterized by significantly higher bone loss and inflammatory cell migration to periodontal tissues. The inflammatory cell influx into the peritoneal cavities of TLR2(-/-) mice was three-fold lower than that observed for the littermate controls. A significantly diminished production of the cytokines tumor necrosis factor-alpha and interleukin-1 beta as well as the chemokine CC-ligand-5 in the peritoneal cavities of TLR2(-/-) mice was observed. In addition, a high frequency of apoptotic cells in the inflammatory exudates from TLR2(-/-) mice was observed. Phagocytosis and nitric oxide production was diminished in cells from TLR2(-/-) mice, facilitating the dissemination of the pathogen to the spleen. Conclusion: The results of this study highlight the involvement of TLR2 in recognizing A. actinomycetemcomitans and its essential role in controlling A. actinomycetemcomitans infection. J Periodontot 2009,80:2070-2019.

Mycoplasmal lipid-associated membrane proteins and Mycoplasma arthritidis mitogen recognition by serum antibodies from patients with rheumatoid arthritis

Mycoplasmal lipid-associated membrane proteins (LAMPs) and Mycoplasma arthritidis mitogen (MAM superantigen) are potent stimulators of the immune system. The objective of this work was to detect antibodies to MAM and LAMPs of Mycoplasma hominis and M. fermentans in the sera of patients affected by rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) to identify mycoplasmal products that can be involved in the etiopathogenesis of these autoimmune diseases. Serum samples from female RA and SLE patients and controls, recombinant MAM, and LAMPs of M. hominis PG21 and M. fermentans PG18 were used in Western blot assays. A similar frequency of sera from patients and controls reactive to MAM was detected. A larger number of M. hominis and M. fermentans LAMPs were recognized by sera from RA patients than controls, but no differences were detected between sera from SLE patients and controls. Among the LAMPs recognized by IgG antibodies from RA patients, proteins of molecular masses in a range of < 49 and a parts per thousand yen20 KDa (M. hominis) and < 102 and a parts per thousand yen58 KDa (M. fermentans) were the most reactive. These preliminary results demonstrate the strong reactivity of antibodies of RA patients with some M. hominis and M. fermentans LAMPs. These LAMPs could be investigated as mycoplasmal antigens that can take part in the induction or amplification of human autoimmune responses.

Long-term social recognition memory in adult male rats: factor analysis of the social and non-social behaviors

A modified version of the intruder-resident paradigm was used to investigate if social recognition memory lasts at least 24 h. One hundred and forty-six adult male Wistar rats were used. Independent groups of rats were exposed to an intruder for 0.083, 0.5, 2, 24, or 168 h and tested 24 h after the first encounter with the familiar or a different conspecific. Factor analysis was employed to identify associations between behaviors and treatments. Resident rats exhibited a 24-h social recognition memory, as indicated by a 3- to 5-fold decrease in social behaviors in the second encounter with the same conspecific compared to those observed for a different conspecific, when the duration of the first encounter was 2 h or longer. It was possible to distinguish between two different categories of social behaviors and their expression depended on the duration of the first encounter. Sniffing the anogenital area (49.9% of the social behaviors), sniffing the body (17.9%), sniffing the head (3%), and following the conspecific (3.1%), exhibited mostly by resident rats, characterized social investigation and revealed long-term social recognition memory. However, dominance (23.8%) and mild aggression (2.3%), exhibited by both resident and intruders, characterized social agonistic behaviors and were not affected by memory. Differently, sniffing the environment (76.8% of the non-social behaviors) and rearing (14.3%), both exhibited mostly by adult intruder rats, characterized non-social behaviors. Together, these results show that social recognition memory in rats may last at least 24 h after a 2-h or longer exposure to the conspecific.

Immune cells and oxidative stress in the endotoxin tolerance mouse model

Sepsis is a systemic inflammatory response that can lead to tissue damage and death. In order to increase our understanding of sepsis, experimental models are needed that produce relevant immune and inflammatory responses during a septic event. We describe a lipopolysaccharide tolerance mouse model to characterize the cellular and molecular alterations of immune cells during sepsis. The model presents a typical lipopolysaccharide tolerance pattern in which tolerance is related to decreased production and secretion of cytokines after a subsequent exposure to a lethal dose of lipopolysaccharide. The initial lipopolysaccharide exposure also altered the expression patterns of cytokines and was followed by an 8- and a 1.5-fold increase in the T helper 1 and 2 cell subpopulations. Behavioral data indicate a decrease in spontaneous activity and an increase in body temperature following exposure to lipopolysaccharide. In contrast, tolerant animals maintained production of reactive oxygen species and nitric oxide when terminally challenged by cecal ligation and puncture (CLP). Survival study after CLP showed protection in tolerant compared to naive animals. Spleen mass increased in tolerant animals followed by increases of B lymphocytes and subpopulation Th1 cells. An increase in the number of stem cells was found in spleen and bone marrow. We also showed that administration of spleen or bone marrow cells from tolerant to naive animals transfers the acquired resistance status. In conclusion, lipopolysaccharide tolerance is a natural reprogramming of the immune system that increases the number of immune cells, particularly T helper 1 cells, and does not reduce oxidative stress.

The effects of nitric oxide on the immune response during giardiasis

Nitric oxide (NO) is a free radical synthesized from L-arginine by different isoforms NO-synthases. NO possesses multiple and complex biological functions. NO is an important mediator of homeostasis, and changes in its generation or actions can contribute or not to pathological states. The knowledge of effects of NO has been not only important to our understanding of immune response, but also to new tools for research and treatment of various diseases. Knowing the importance of NO as inflammatory mediator in diverse infectious diseases, we decided to develop a revision that shows the participation/effect of this mediator in immune response induced against Giardia spp. Several studies already demonstrated the participation of NO with microbicidal and microbiostatic activity in giardiasis. On the other hand, some works report that Giardia spp. inhibit NO production by consuming the intermediate metabolite arginine. In fact, studies in vitro showed that G. lamblia infection of human intestinal epithelial cells had reduced NO production. This occurs due to limited offer of the crucial substrate arginine (essential aminoacid for NO production), consequently reducing NO production. Therefore, the balance between giardial arginine consumption and epithelial NO production could contribute to the variability of the duration and severity of infections by this ubiquitous parasite.

A novel polar-based human face recognition computational model

Motivated by a recently proposed biologically inspired face recognition approach, we investigated the relation between human behavior and a computational model based on Fourier-Bessel (FB) spatial patterns. We measured human recognition performance of FB filtered face images using an 8-alternative forced-choice method. Test stimuli were generated by converting the images from the spatial to the FB domain, filtering the resulting coefficients with a band-pass filter, and finally taking the inverse FB transformation of the filtered coefficients. The performance of the computational models was tested using a simulation of the psychophysical experiment. In the FB model, face images were first filtered by simulated V1- type neurons and later analyzed globally for their content of FB components. In general, there was a higher human contrast sensitivity to radially than to angularly filtered images, but both functions peaked at the 11.3-16 frequency interval. The FB-based model presented similar behavior with regard to peak position and relative sensitivity, but had a wider frequency band width and a narrower response range. The response pattern of two alternative models, based on local FB analysis and on raw luminance, strongly diverged from the human behavior patterns. These results suggest that human performance can be constrained by the type of information conveyed by polar patterns, and consequently that humans might use FB-like spatial patterns in face processing.

The Anopheles albitarsis complex with the recognition of Anopheles oryzalimnetes Wilkerson and Motoki, n. sp. and Anopheles janconnae Wilkerson and Sallum, n. sp. (Diptera: Culicidae)

The Anopheles (Nyssorhynchus) albitarsis complex includes six species: An. albitarsis, Anopheles oryzalymnetes Wilkerson and Motoki, n. sp., Anopheles marajoara, Anopheles dencorum, Anopheles janconnae Wilkerson and Sallum, n. sp., and An. albitarsis F. Except for An. deancorum, species of the complex are indistinguishable when only using morphology. The problematic distinction among species of the complex has made study of malaria transmission and ecology of An. albitarsis s.l. difficult. Consequently, involvement of species of the An. albitarsis complex in human Plasmodium transmission is not clear throughout its distribution range. With the aim of clarifying the taxonomy of the above species, with the exception of An. albitarsis F, we present comparative morphological and morphometric analyses, morphological redescriptions of three species and descriptions of two new species using individuals from populations in Brazil, Paraguay, Argentina and Venezuela. The study included characters from adult females, males, fourth-instar larvae, pupae and male genitalia of An. albitarsis, An. deaneorum and An. oryzalimnetes n. sp. For An. janconnae n. sp. only characters of the female, male and male genitalia were analysed. Fourth-instar larvae and pupae and male genitalia characteristics of all five species are illustrated. Bionomics and distribution data are given based on published literature records

Interleukin-22 forms dimers that are recognized by two interleukin-22R1 receptor chains

Interleukin-22 (IL-22) is a class 2 cytokine whose primary structure is similar to that of interleukin 10 (IL-10) and interferon-gamma (IFN-gamma). IL-22 induction during acute phase immune response indicates its involvement in mechanisms of inflammation. Structurally different from IL-10 and a number of other members of IL-10 family, which form intertwined inseparable V-shaped dimers of two identical polypeptide chains, a single polypeptide chain of IL-22 folds on itself in a relatively globular structure. Here we present evidence, based on native gel electrophoresis, glutaraldehyde cross-linking, dynamic light scattering, and small angle x-ray scattering experiments, that human IL-22 forms dimers and tetramers in solution under protein concentrations assessable by these experiments. Unexpectedly, low-resolution molecular shape of IL-22 dimers is strikingly similar to that of IL-10 and other intertwined cytokine dimeric forms. Furthermore, we determine an ab initio molecular shape of the IL-22/IL-22R1 complex which reveals the V-shaped IL-22 dimer interacting with two cognate IL-22R1 molecules. Based on this collective evidence, we argue that dimerization might be a common mechanism of all class 2 cytokines for the molecular recognition with their respective membrane receptor. We also speculate that the IL-22 tetramer formation could represent a way to store the cytokine in nonactive form at high concentrations that could be readily converted into functionally active monomers and dimers upon interaction with the cognate cellular receptors.