New insights into Vogt-Koyanagi-Harada disease

Vogt-Koyanagi-Harada disease (VKH), a well-established multiorgan disorder affecting pigmented structures, is an autoimmune disorder of melanocyte proteins in genetically susceptible individuals. Several clinical and experimental data point to the importance of the effector role of CD4+ T cells and Th1 cytokines, the relevance of searching a target protein in the melanocyte, and the relevance of the HLA-DRB1*0405 in the pathogenesis of the disease. Vogt-Koyanagi-Harada disease has a benign course when early diagnosed and adequatey treated. Full-blown recurrences are rare after the acute stage of Vogt-Koyanagi-Harada disease is over. On the other hand, clinical findings, such as progressive tissue depigmentation (including sunset glow fundus) and uveitis recurrence, indicate that ocular inflammation may persist after the acute phase. Additionally, indocyanine green angiography findings suggest the presence of choroidal inflammation in eyes without clinically detectable inflammation. The aim of this paper is to review the latest research results on Vogt-Koyanagi-Harada disease pathogenesis and chronic/convalescent stages, which may help to better understand this potentially blinding disease and to improve its treatment.

Use of L-Proline and ATP Production by Trypanosoma cruzi Metacyclic Forms as Requirements for Host Cell Invasion

The process of host cell invasion by Trypanosoma cruzi depends on parasite energy. What source of energy is used for that event is not known. To address this and other questions related to T. cruzi energy requirements and cell invasion, we analyzed metacyclic trypomastigote forms of the phylogenetically distant CL and G strains. For both strains, the nutritional stress experienced by cells starved for 24, 36, or 48 h in phosphate-buffered saline reduced the ATP content and the ability of the parasite to invade HeLa cells proportionally to the starvation time. Inhibition of ATP production by treating parasites with rotenone plus antimycin A also diminished the infectivity. Nutrient depletion did not alter the expression of gp82, the surface molecule that mediates CL strain internalization, but increased the expression of gp90, the negative regulator of cell invasion, in the G strain. When L-proline was given to metacyclic forms starved for 36 h, the ATP levels were restored to those of nonstarved controls for both strains. Glucose had no such effect, although this carbohydrate and L-proline were transported in similar fashions. Recovery of infectivity promoted by L-proline treatment of starved parasites was restricted to the CL strain. The profile of restoration of ATP content and gp82-mediated invasion capacity by L-proline treatment of starved Y-strain parasites was similar to that of the CL strain, whereas the Dm28 and Dm30 strains, whose infectivity is downregulated by gp90, behaved like the G strain. L-Proline was also found to increase the ability of the CL strain to traverse a gastric mucin layer, a property important for the establishment of T. cruzi infection by the oral route. Efficient translocation of parasites through gastric mucin toward the target epithelial cells in the stomach mucosa is an essential requirement for subsequent cell invasion. By relying on these closely associated ATP-driven processes, the metacyclic trypomastigotes effectively accomplish their internalization.

Evaluation of Leptospiral Recombinant Antigens MPL17 and MPL21 for Serological Diagnosis of Leptospirosis by Enzyme-Linked Immunosorbent Assays

Leptospirosis is a zoonosis of multisystem involvement caused by pathogenic strains of the genus Leptospira. In the last few years, intensive studies aimed at the development of a vaccine have provided important knowledge about the nature of the immunological mechanisms of the host. The purpose of this study was to analyze the immune responses to two recombinant proteins, MPL17 and MPL21 (encoded by the genes LIC10765 and LIC13131, respectively) of Leptospira interrogans serovar Copenhageni in individuals during infection. The recombinant proteins were expressed in Escherichia coli as six-His tag fusion proteins and were purified from the soluble bacterial fraction by affinity chromatography with Ni2+ -charged resin. The recombinant proteins were used to evaluate their ability to bind to immunoglobulin G (IgG) (and IgG subclass) or IgM antibodies in serum samples from patients in the early and convalescent phases of leptospirosis (n = 52) by enzyme-linked immunosorbent assays. The prevalences of total IgG antibodies against MPL17 and MPL21 were 38.5% and 21.2%, respectively. The titers achieved with MPL17 were statistically significantly higher than those obtained by the reference microscopic agglutination test. The specificity of the assay was estimated to be 95.5% for MPL17 and 80.6% for MPL21 when serum samples from individuals with unrelated febrile diseases and control healthy donors were tested. The proteins are conserved among Leptospira strains that cause human and animal diseases. MPL17 and MPL21 are most likely new surface proteins of leptospires, as revealed by liquid-phase immunofluorescence assays with living organisms. Our results demonstrate that these recombinant proteins are highly immunogenic and, when they are used together, might be useful as a means of diagnosing leptospirosis.

Patterns of Antibodies Against Latent and Lytic Antigens of Human Herpesvirus 8 in an Endemic Population and Patients With Kaposi`s Sarcoma in Mozambique

The patterns of antibodies against latent and lytic antigens of human herpesvirus 8 (HHV-8) were assessed using immunofluorescence assays of samples from 155 persons seropositive for HHV-8 seen at public health centers and 24 patients with Kaposi`s sarcoma (KS) from Mozambique. Of the 155 persons without KS, 48(31%) had antibodies against latent antigens only, 29 (18.7%) had antibodies against lytic antigens only, and 78 (50.3%) had antibodies against both types of antigen. The HHV-8 antibody titer tended to increase with age until age 40, after which it began to decrease. High titers of antibodies against latent and lytic antigens of HHV-8 were detected mostly in persons co-infected with HIV, and these increased titers could have a predictive value. All patients with KS except four patients who were seronegative for HHV-8 had elevated titers of HHV-8 antibodies, predominantly against latent antigens. The data suggest the potential for an increase in the development of KS in this endemic area for HHV-8. J. Med. Virol. 82:1576-1581, 2010. (C) 2010 Wiley-Liss, Inc.

PDIA3, HSPA5 and viementin, proteins identified by 2-DE in the valvular tissue, are the target antigens of peripheral and heart infiltrating T cells from chronic rheumatic heart disease patients

Rheumatic fever (RF) is a post-infectious autoimmune disease due to sequel of group A streptococcus (GAS) pharyngitis. Rheumatic heart disease (RHD), the major manifestation of RF, is characterized by inflammation of heart valves and myocardium. Molecular mimicry between GAS antigens and host proteins has been shown at B and T cell level. However the identification of the autoantigens recognized by B and T cells within the inflammatory microenvironment of heart tissue in patients with RHD is still incompletely elucidated. In the present study, we used two-dimensional gel electrophoresis (2-DE) and mass spectrometry to identify valvular tissue proteins target of T cells from chronic RHD patients. We could identify three proteins recognized by heart infiltrating and peripheral T cells as protein disulfide isomerase ER-60 precursor (PDIA3), 78 kD glucose-regulated protein precursor (HSPA5) and vimentin, with coverage of 45%, 43 and 34%, respectively. These proteins were recognized in a proliferation assay by peripheral and heart infiltrating T cells from RHD patients suggesting that they may be involved in the autoimmune reactions that leads to valve damage. We also observed that several other proteins isolated by 2-DE but not identified by mass spectrometry were also recognized by T cells. The identified cardiac proteins are likely relevant antigens involved in T cell-mediated autoimmune responses in RF/RHD that may contribute to the development of RHD

Cell-Free Antigens from Paracoccidioides brasiliensis Drive IL-4 Production and Increase the Severity of Paracoccidioidomycosis

The thermally dimorphic fungus Paracoccidioides brasiliensis (Pb) is the causative agent of paracoccidioidomycosis (PCM), one of the most frequent systemic mycosis that affects the rural population in Latin America. PCM is characterized by a chronic inflammatory granulomatous reaction, which is consequence of a Th1-mediated adaptive immune response. In the present study we investigated the mechanisms involved in the immunoregulation triggered after a prior contact with cell-free antigens (CFA) during a murine model of PCM. The results showed that the inoculation of CFA prior to the infection resulted in disorganized granulomatous lesions and increased fungal replication in the lungs, liver and spleen, that paralleled with the higher levels of IL-4 when compared with the control group. The role of IL-4 in facilitating the fungal growth was demonstrated in IL-4-deficient- and neutralizing anti-IL-4 mAb-treated mice. The injection of CFA did not affect the fungal growth in these mice, which, in fact, exhibited a significant diminished amount of fungus in the tissues and smaller granulomas. Considering that in vivo anti-IL-4-application started one week after the CFA-inoculum, it implicates that IL-4-CFA-induced is responsible by the mediation of the observed unresponsiveness. Further, the characterization of CFA indicated that a proteic fraction is required for triggering the immunosuppressive mechanisms, while glycosylation or glycosphingolipids moieties are not. Taken together, our data suggest that the prior contact with soluble Pb antigens leads to severe PCM in an IL-4 dependent manner.

Identification and Characterization of Ixodes scapularis Antigens That Elicit Tick Immunity Using Yeast Surface Display

Repeated exposure of rabbits and other animals to ticks results in acquired resistance or immunity to subsequent tick bites and is partially elicited by antibodies directed against tick antigens. In this study we demonstrate the utility of a yeast surface display approach to identify tick salivary antigens that react with tick-immune serum. We constructed an Ixodes scapularis nymphal salivary gland yeast surface display library and screened the library with nymph-immune rabbit sera and identified five salivary antigens. Four of these proteins, designated P8, P19, P23 and P32, had a predicted signal sequence. We generated recombinant (r) P8, P19 and P23 in a Drosophila expression system for functional and immunization studies. rP8 showed anti-complement activity and rP23 demonstrated anti-coagulant activity. Ixodes scapularis feeding was significantly impaired when nymphs were fed on rabbits immunized with a cocktail of rP8, rP19 and rP23, a hall mark of tick-immunity. These studies also suggest that these antigens may serve as potential vaccine candidates to thwart tick feeding.

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.

Neospora caninum excreted/secreted antigens trigger CC-chemokine receptor 5-dependent cell migration

Neospora caninum, the causative agent of neosporosis, is an obligate intracellular parasite considered to be a major cause of abortion in cattle throughout the world. Most studies concerning N. caninum have focused on life cycle, seroepidemiology, pathology and vaccination, while data on host-parasite interaction, such as host cell migration, mechanisms of evasion and dissemination of this parasite during the early phase of infection are still poorly understood. Here we show the ability of excreted/secreted antigens from N. caninum (NcESAs) to attract monocytic cells to the site of primary infection in both in vitro and in vivo assays. Molecules from the family of cyclophilins present on the NcESAs were shown to work as chemokine-like proteins and NcESA-induced chemoattraction involved G(i) protein signaling and participation of CC-chemokine receptor 5 (CCR5). Additionally, we demonstrate the ability of NcESAs to enhance the expression of CCR5 on monocytic cells and this increase occurred in parallel with the chemotactic activity of NcESAs by increasing cell migration. These results suggest that during the first days of infection, N. caninum produces molecules capable of inducing monocytic cell migration to the sites of infection, which will consequently enhance initial parasite invasion and proliferation. Altogether, these results help to clarify some key features involved in the process of cell migration and may reveal virulence factors and therapeutic targets to control neosporosis. (C) 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Direct effect of Plasmodium vivax recombinant vaccine candidates AMA-1 and MSP-1(19) on the innate immune response

The recombinant apical membrane antigen 1 (AMA-1) and 19-kDa fragment of merozoite surface protein (MSP-1(19)) are the lead candidates for inclusion in a vaccine against blood stages of malaria due to encouraging protective studies in humans and animals. Despite the importance of an efficacious malaria vaccine, vaccine-related research has focused on identifying antigens that result in protective immunity rather than determining the nature of anti-malarial immune effector mechanisms. Moreover, emphasis has been placed on adaptive rather than innate immune responses. In this study, we investigated the effect of Plasmodium vivax vaccine candidates Pv-AMA-1 and Pv-MSP-1(19) on the immune response of malaria-naive donors. Maturation of dendritic cells is altered by Pv-AMA-1 but not Pv-MSP-1(19), as observed by differential expression of cell surface markers. In addition, Pv-AMA-1 induced an increased production of MIP-1 alpha/CCL3 and decreased production of TARC/CCL17 levels in both dendritic cells (DCs) and peripheral blood mononuclear cells (PBMCs). Finally, a significant pro-inflammatory response was elicited by Pv-AMA-1-stimulated PBMCs. These results suggest that the recombinant vaccine candidate Pv-AMA-1 may play a direct role on innate immune response and might be involved in parasite destruction. (C) 2007 Elsevier Ltd. All rights reserved.

Taenia crassiceps cysticerci: Characterization of the 14-kDa glycoprotein with homologies to antigens from Taenia solium cysticerci

Glycoproteins from the total vesicular fluid of Taenia crassiceps (VF-Tc) were prepared using three different purification methods, consisting of ConA-lectin affinity chromatography (ConA-Tc), preparative electrophoresis (SDS-PAGE) (14gp-Tc), and monoclonal antibody immunoaffinity chromatography (18/14-Tc). The complex composition represented by the VF-Tc and ConA-Tc antigens revealed peptides ranging from 101 - to 14-kDa and from 92- to 12-kDa, respectively. Immunoblotting using lectins confirmed glucose/mannose (glc/man) residues in the 18- and 14-kDa peptides, which are considered specific and immunodominant for the diagnosis of cysticercosis, and indicated that these fractions are glycoproteins. Serum antibodies from a patient with neurocysticercosis that reacted to the 14gp band from T. crassiceps (Tc) were eluted from immunoblotting membranes and showed reactivity to 14gp from Taenia solium. In order to determine the similar peptide sequence, the N-terminal amino acid was determined and analyzed with sequences available in public databases. This sequence revealed partial homology between T. crassiceps and T solium peptides. In addition, mass spectrometry along with theoretical M(r) and pI of the 14gp-Tc point suggested a close relationship to some peptides of a 150-kDa protein complex of the T solium previously described. The identification of these common immunogenic sites will contribute to future efforts to develop recombinant antigens and synthetic peptides for immunological assays. (C) 2009 Elsevier Inc. All rights reserved.

Passive immunization with monoclonal antibody against a 70-kDa putative adhesin of Sporothrix schenckii induces protection in murine sporotrichosis

Cell-mediated and innate immunity are considered the most important mechanisms of host defense against fungus infections. However, recent studies demonstrated that specific antibodies show different degrees of protection against mycosis. In a previous study, antigens secreted by Sporothrix schenckii induced a specific humoral response in infected animals, mainly against the 70-kDa molecule, indicating a possible participation of antibodies to this antigen in infection control. in the present study, an IgG1 mAb was produced against a 70-kDa glycoprotein of S. schenckii in order to better understand the effect of passive immunization of mice infected with S. schenckii. Results showed a significant reduction in the number of CFU in organs of mice when the mAb was injected before and during S. schenckii infection. Similar results were observed when T-cell-deficient mice were used. Moreover, in a second schedule treatment, the mAb was injected after infection was established, and again we observed a significant reduction in CFU associated with an increase of IFN-gamma production. Also, the 70-kDa antigen is shown to be a putative adhesin present on the surface of this fungus. In conclusion, we report for the first time the protective effect of a specific antibody against S. schenckii.

Immunology of Dermatophytosis

The immune response to infection by dermatophytes ranges from a non-specific host mechanism to a humoral and cell-mediated immune response. The currently accepted view is that a cell-mediated immune response is responsible for the control of dermatophytosis. Indeed, some individuals develop a chronic or recurrent infection mediated by the suppression of a cell-mediated immune response. The immune response to Trichophyton is unusual in that this fungus can elicit both immediate hypersensitivity (IH) and delayed-type hypersensitivity (DTH) in different individuals when they are submitted to a skin test reaction. Understanding the nature and function of the immune response to dermatophytes is an exciting challenge that might lead to novel approaches in the treatment and immunological prophylaxis of dermatophytosis.

Immunogenicity of a Whole-Cell Pertussis Vaccine with Low Lipopolysaccharide Content in Infants

The lack of a clear correlation between the levels of antibody to pertussis antigens and protection against disease lends credence to the possibility that cell-mediated immunity provides primary protection against disease. This phase I comparative trial had the aim of comparing the in vitro cellular immune response and anti-pertussis toxin (anti-PT) immunoglobulin G (IgG) titers induced by a cellular pertussis vaccine with low lipopolysaccharide (LPS) content (wP(low) vaccine) with those induced by the conventional whole-cell pertussis (wP) vaccine. A total of 234 infants were vaccinated at 2, 4, and 6 months with the conventional wP vaccine or the wP(low) vaccine. Proliferation of CD3(+) T cells was evaluated by flow cytometry after 6 days of peripheral blood mononuclear cell culture with stimulation with heat-killed Bordetella pertussis or phytohemagglutinin (PHA). CD3(+), CD4(+), CD8(+), and T-cell receptor gamma delta-positive (gamma delta(+)) cells were identified in the gate of blast lymphocytes. Gamma interferon, tumor necrosis factor alpha, interleukin-4 (IL-4), and IL-10 levels in super-natants and serum anti-PT IgG levels were determined using enzyme-linked immunosorbent assay (ELISA). The net percentage of CD3(+) blasts in cultures with B. pertussis in the group vaccinated with wP was higher than that in the group vaccinated with the wP(low) vaccine (medians of 6.2% for the wP vaccine and 3.9% for the wP(low) vaccine; P = 0.029). The frequencies of proliferating CD4(+), CD8(+), and gamma delta(+) cells, cytokine concentrations in supernatants, and the geometric mean titers of anti-PT IgG were similar for the two vaccination groups. There was a significant difference between the T-cell subpopulations for B. pertussis and PHA cultures, with a higher percentage of gamma delta(+) cells in the B. pertussis cultures (P < 0.001). The overall data did suggest that wP vaccination resulted in modestly better specific CD3(+) cell proliferation, and gamma delta(+) cell expansions were similar with the two vaccines.

Human Airway Epithelial Cell Responses to Neisseria lactamica and Purified Porin via Toll-Like Receptor 2-Dependent Signaling

The human airway epithelium is constantly exposed to microbial products from colonizing organisms. Regulation of Toll-like receptor (TLR) expression and specific interactions with bacterial ligands is thought to mitigate exacerbation of inflammatory processes induced by the commensal flora in these cells. The genus Neisseria comprises pathogenic and commensal organisms that colonize the human nasopharynx. Neisseria lactamica is not associated with disease, but N. meningitidis occasionally invades the host, causing meningococcal disease and septicemia. Upon colonization of the airway epithelium, specific host cell receptors interact with numerous Neisseria components, including the PorB porin, at the immediate bacterial-host cell interface. This major outer membrane protein is expressed by all Neisseria strains, regardless of pathogenicity, but its amino acid sequence varies among strains, particularly in the surface-exposed regions. The interaction of Neisseria PorB with TLR2 is essential for driving TLR2/TLR1-dependent cellular responses and is thought to occur via the porin`s surface-exposed loop regions. Our studies show that N. lactamica PorB is a TLR2 ligand but its binding specificity for TLR2 is different from that of meningococcal PorB. Furthermore, N. lactamica PorB is a poor inducer of proinflammatory mediators and of TLR2 expression in human airway epithelial cells. These effects are reproduced by whole N. lactamica organisms. Since the responsiveness of human airway epithelial cells to colonizing bacteria is in part regulated via TLR2 expression and signaling, commensal organisms such as N. lactamica would benefit from expressing a product that induces low TLR2-dependent local inflammation, likely delaying or avoiding clearance by the host.