Identification of Schistosoma mansoni candidate antigens for diagnosis of schistosomiasis

The development of a more sensitive diagnostic test for schistosomiasis is needed to overcome the limitations of the use of stool examination in low endemic areas. Using parasite antigens in enzyme linked immunosorbent assay is a promising strategy, however a more rational selection of parasite antigens is necessary. In this study we performed in silico analysis of the Schistosoma mansoni genome, using SchistoDB database and bioinformatic tools for screening immunogenic antigens. Based on evidence of expression in all parasite life stage within the definitive host, extracellular or plasmatic membrane localization, low similarity to human and other helminthic proteins and presence of predicted B cell epitopes, six candidates were selected: a glycosylphosphatidylinositol-anchored 200 kDa protein, two putative cytochrome oxidase subunits, two expressed proteins and one hypothetical protein. The recognition in unidimensional and bidimensional Western blot of protein with similar molecular weight and isoelectric point to the selected antigens by sera from S. mansoni infected mice indicate a good correlation between these two approaches in selecting immunogenic proteins.

Schistosoma mansoni antigens alter the cytokine response in vitro during cutaneous leishmaniasis

Schistosoma mansoni infection or associated products are able to down-modulate the type 1 CD4+ T cell inflammatory response characteristic of autoimmune diseases. In this study, we evaluated how S. mansoni antigens altered the immune response that was induced by the soluble Leishmania antigen (SLA) from cutaneous leishmaniasis (CL) patients. Cytokines were measured from the supernatants of peripheral blood mononuclear cell cultures stimulated with SLA. This was performed using the sandwich enzyme linked immunosorbent assay technique in the presence or absence of S. mansoni recombinant antigens Sm29, SmTSP-2 and PIII. The addition of S. mansoni antigens to the cultures resulted in the reduction of interferon gamma (IFN-γ) levels in 37-50% of patients. Although to a lesser extent, the antigens were also able to decrease the production of tumour necrosis factor-alpha (TNF-α). We compared patients that either had or did not have reduction in IFN-γ and TNF-α production in cultures stimulated with SLA in the presence of S. mansoni antigens. We found that there was no significant difference in the levels of interleukin (IL)-10 and IL-5 in response to S. mansoni antigens between the groups. The antigens used in this study down-modulated the in vitro proinflammatory response induced by SLA in a group of CL patients through a currently undefined mechanism.

Immunodetection of Helicobacter sp. and the associated expression of ABO blood group antigens in the gastric mucosa of captive and free-living New World primates in the Amazon region

The histo-blood group ABH antigens were first described in humans. These antigens are only present on erythrocytes from great apes and humans, while in more primitive animals they are found in tissues and body fluids. The ABH antigens are mainly distributed in tissues exposed to the external environment and potentially serve as ligands for pathogens or inhibitors of tissue connections. The objective of this paper was two-fold: (i) to determine the presence of Helicobacter sp. in the gastric mucosa of 16 captive and 24 free-living New World monkeys and (ii) to evaluate the presence of histopathological alterations related to bacterial infection and the associated expression of ABH antigens in the tissue. Stomach tissues from 13 species of monkey were assessed using haematoxylin-eosin and modified Gram staining (Hucker) methods. An immunohistochemical analysis of the tissue revealed the presence of infectious bacteria that were characteristic of the genus Helicobacter sp. The results demonstrate that various species of monkey might be naturally infected with the Helicobacter sp. and that there is an increased susceptibility to infection. This study serves as a comparative analysis of infection between human and non-human primates and indicates the presence of a new species of Helicobacter.

Profile of total IgG, IgG1, IgG2, IgG3 and IgG4 levels in sera of patients with paracoccidioidomycosis: treatment follow-up using Mexo and rPb27 as antigens in an ELISA

The levels of total of IgG, IgG1, IgG2, IgG3 and IgG4 were evaluated in 54 patients with chronic paracoccidioidomycosis (PCM) before, during and after treatment using an enzyme-linked immunosorbent assay with Mexo and recombinant Pb27 (rPb27) as the antigens. Mexo was effective in distinguishing PCM patients from individuals in the negative control group (NC) based on total IgG and rPb27 performed worse than Mexo when these two groups were compared. IgG1, IgG2, IgG3 and IgG4 could not be used to clearly distinguish PCM patients from those in the NC group using either antigen. There was no clear relationship between antibody levels and the period of treatment. The majority of patients presented with decreased antibody levels during treatment, with no statistically significant differences among the different periods of treatment. Only IgG4 presented a negative correlation between its levels and clinical improvement during treatment. In total, 65% of untreated PCM patients showed reactivity against IgG4 when the Mexo antigen was used and this reactivity decreased over the course of treatment. There was a tendency towards decreasing antibody levels during treatment, but these antibody levels did not necessarily clear after the treatment was stopped. Mexo was useful for PCM diagnosis using total IgG; however, more studies are necessary before this antigen can be used in measuring the levels of total IgG and its subclasses for monitoring patients during treatment.

Comparison of two rapid tests for anti-phenolic glycolipid-I serology in Brazil and Nepal

The diagnosis of leprosy continues to be based on clinical symptoms and early diagnosis and treatment are critical to preventing disability and transmission. Sensitive and specific laboratory tests are not available for diagnosing leprosy. Despite the limited applicability of anti-phenolic glycolipid-I (PGL-I) serology for diagnosis, it has been suggested as an additional tool to classify leprosy patients (LPs) for treatment purposes. Two formats of rapid tests to detect anti-PGL-I antibodies [ML immunochromatography assay (ICA) and ML Flow] were compared in different groups, multibacillary patients, paucibacillary patients, household contacts and healthy controls in Brazil and Nepal. High ML Flow intra-test concordance was observed and low to moderate agreement between the results of ML ICA and ML Flow tests on the serum of LPs was observed. LPs were "seroclassified" according to the results of these tests and the seroclassification was compared to other currently used classification systems: the World Health Organization operational classification, the bacilloscopic index and the Ridley-Jopling classification. When analysing the usefulness of these tests in the operational classification of PB and MB leprosy for treatment and follow-up purposes, the ML Flow test was the best point-of-care test for subjects in Nepal and despite the need for sample dilution, the ML ICA test yielded better performance among Brazilian subjects. Our results identified possible ways to improve the performance of both tests.

Prevalence of antibody to hantaviruses in humans and rodents in the Caribbean region of Colombia determined using Araraquara and Maciel virus antigens

We tested sera from 286 agricultural workers and 322 rodents in the department of Córdoba, northeastern Colombia, for antibodies against two hantaviruses. The sera were analysed by indirect ELISA using the lysate of Vero E6 cells infected with Maciel virus (MACV) or the N protein of Araraquara virus (ARAV) as antigens for the detection of antibodies against hantaviruses. Twenty-four human sera were IgG positive using one or both antigens. We detected anti-MACV IgG antibodies in 10 sera (3.5%) and anti-ARAV antibodies in 21 sera (7.34%). Of the 10 samples that were positive for MACV, seven (70%) were cross-reactive with ARAV; seven of the 21 ARAV-positive samples were cross-reactive with MACV. Using an ARAV IgM ELISA, two of the 24 human sera (8.4%) were positive. We captured 322 rodents, including 210 Cricetidae (181 Zygodontomys brevicauda, 28 Oligoryzomys fulvescens and 1 Oecomys trinitatis), six Heteromys anomalus (Heteromyidae), one Proechimys sp. (Echimyidae) and 105 Muridae (34 Rattus rattus and 71 Mus musculus). All rodent sera were negative for both antigens. The 8.4% detection rate of hantavirus antibodies in humans is much higher than previously found in serosurveys in North America, suggesting that rural agricultural workers in northeastern Colombia are frequently exposed to hantaviruses. Our results also indicate that tests conducted with South American hantavirus antigens could have predictive value and could represent a useful alternative for the diagnosis of hantavirus infection in Colombia.

A retrospective search for bovine respiratory syncytial virus (BRSV) antigens in histological specimens by immunofluorescence and immunohistochemistry

Bovine respiratory syncytial virus (BRSV) has been only sporadically identified as a causative agent of respiratory disease in Brazil. This contrasts with frequent reports of clinical and histopathological findings suggestive of BRSV-associated disease. In order to examine a possible involvement of BRSV in cases of calf pneumonia, a retrospective search was performed for BRSV antigens in histological specimens submitted to veterinary diagnostic services from the states of Rio Grande do Sul and Minas Gerais. Ten out of 41 cases examined (24.4%) were positive for BRSV antigens by immunohistochemistry (IPX). Eight of these cases (19.5%) were also positive by indirect immunofluorescence (IFA), and 31 cases (75.6%) were negative in both assays. In the lungs, BRSV antigens were predominantly observed in epithelial cells of bronchioles and less frequently found in alveoli. In one case, antigens were detected only in the epithelium of the alveolar septae. The presence of antigen-positive cells was largely restricted to epithelial cells of these airways. In two cases, positive staining was also observed in cells and cellular debris in the exudate within the pulmonary airways. The clinical cases positive for BRSV antigens were observed mainly in young animals (2 to 12 month-old) from dairy herds. The main microscopic changes included bronchointerstitial pneumonia characterized by thickening of alveolar septae adjacent to airways by mononuclear cell infiltrates, and the presence of alveolar syncytial giant cells. In summary, the results demonstrate the suitability of the immunodetection of viral antigens in routinely fixed tissue specimens as a diagnostic tool for BRSV infection. Moreover, the findings provide further evidence of the importance of BRSV as a respiratory pathogen of young cattle in southeastern and southern Brazil.

Production of recombinant antigens in plants for animal and human immunization - a review

Plants present a cost effective production system for high value proteins. There is an increasing world demand for cheap vaccines that can be readily administered to the population, especially in economically less developed regions. A promising concept is the production of vaccines in plants that could be grown locally. Expression of antigenic peptides in the palatable parts of plants can lead to the production of edible active vaccines. Two major strategies are: i) to express antigens in transgenic plants, and ii) to produce antigenic peptides on the surface of plant viruses that could be used to infect host plants. This review considers the experimental data and early results for both strategies, and discusses the potential and problems of this new technology

Glycosphingolipid antigens from Leishmania (L.) amazonensis amastigotes: Binding of anti-glycosphingolipid monoclonal antibodies in vitro and in vivo

Specific glycosphingolipid antigens of Leishmania (L.) amazonensis amastigotes reactive with the monoclonal antibodies (MoAbs) ST-3, ST-4 and ST-5 were isolated, and their structure was partially elucidated by negative ion fast atom bombardment mass spectrometry. The glycan moieties of five antigens presented linear sequences of hexoses and N-acetylhexosamines ranging from four to six sugar residues, and the ceramide moieties were found to be composed by a sphingosine d18:1 and fatty acids 24:1 or 16:0. Affinities of the three monoclonal antibodies to amastigote glycosphingolipid antigens were also analyzed by ELISA. MoAb ST-3 reacted equally well with all glycosphingolipid antigens tested, whereas ST-4 and ST-5 presented higher affinities to glycosphingolipids with longer carbohydrate chains, with five or more sugar units (slow migrating bands on HPTLC). Macrophages isolated from footpad lesions of BALB/c mice infected with Leishmania (L.) amazonensis were incubated with MoAb ST-3 and, by indirect immunofluorescence, labeling was only detected on the parasite, whereas no fluorescence was observed on the surface of the infected macrophages, indicating that these glycosphingolipid antigens are not acquired from the host cell but synthesized by the amastigote. Intravenous administration of 125I-labeled ST-3 antibody to infected BALB/c mice showed that MoAb ST-3 accumulated significantly in the footpad lesions in comparison to blood and other tissues

Idiopathic focal segmental glomerulosclerosis and HLA antigens

The objective of the present study was to investigate a possible association between HLA class II antigens and idiopathic focal segmental glomerulosclerosis (FSGS). HLA-A, -B, -DR and -DQ antigens were determined in 19 Brazilian patients (16 white subjects and three subjects of Japanese origin) with biopsy-proven FSGS. Comparison of the HLA antigen frequencies between white patients and white local controls showed a significant increase in HLA-DR4 frequency among FSGS patients (37.7 vs 17.2%, P<0.05). In addition, the three patients of Japanese extraction, not included in the statistical analysis, also presented HLA-DR4. In conclusion, our data confirm the association of FSGS with HLA-DR4 previously reported by others, thus providing further evidence for a role of genes of the HLA complex in the susceptibility to this disease

Regulation of T cell response to leishmania antigens by determinants of histocompatibility leukocyte class I and II molecules

It has been shown that HLA class I molecules play a significant role in the regulation of the proliferation of T cells activated by mitogens and antigens. We evaluated the ability of mAb to a framework determinant of HLA class I molecules to regulate T cell proliferation and interferon gamma (IFN-g) production against leishmania, PPD, C. albicans and tetanus toxoid antigens in patients with tegumentary leishmaniasis and healthy subjects. The anti-major histocompatibility complex (MHC) mAb (W6/32) suppressed lymphocyte proliferation by 90% in cultures stimulated with aCD3, but the suppression was variable in cultures stimulated with leishmania antigen. This suppression ranged from 30-67% and was observed only in 5 of 11 patients. IFN-g production against leishmania antigen was also suppressed by anti-HLA class I mAb. In 3 patients IFN-g levels were suppressed by more than 60%, while in the other 2 cultures IFN-g levels were 36 and 10% lower than controls. The suppression by HLA class I mAb to the proliferative response in leishmaniasis patients and in healthy controls varied with the antigens and the patients or donors tested. To determine whether the suppression is directed at antigen presenting cells (APCs) or at the responding T cells, experiments with antigen-primed non-adherent cells, separately incubated with W6/32, were performed. Suppression of proliferation was only observed when the W6/32 mAb was added in the presence of T cells. These data provide evidence that a mAb directed at HLA class I framework determinants can suppress proliferation and cytokine secretion in response to several antigens.

DNA encoding individual mycobacterial antigens protects mice against tuberculosis

Over the last few years, some of our experiments in which mycobacterial antigens were presented to the immune system as if they were viral antigens have had a significant impact on our understanding of protective immunity against tuberculosis. They have also markedly enhanced the prospects for new vaccines. We now know that individual mycobacterial protein antigens can confer protection equal to that from live BCG vaccine in mice. A critical determinant of the outcome of immunization appears to be the degree to which antigen-specific cytotoxic T cells are generated by the immune response. Our most recent studies indicate that DNA vaccination is an effective way to establish long-lasting cytotoxic T cell memory and protection against tuberculosis.

The leaves of green plants as well as a cyanobacterium, a red alga, and fungi contain insulin-like antigens

We report the detection of insulin-like antigens in a large range of species utilizing a modified ELISA plate assay and Western blotting. We tested the leaves or aerial parts of species of Rhodophyta (red alga), Bryophyta (mosses), Psilophyta (whisk ferns), Lycopodophyta (club mosses), Sphenopsida (horsetails), gymnosperms, and angiosperms, including monocots and dicots. We also studied species of fungi and a cyanobacterium, Spirulina maxima. The wide distribution of insulin-like antigens, which in some cases present the same electrophoretic mobility as bovine insulin, together with results recently published by us on the amino acid sequence of an insulin isolated from the seed coat of jack bean (Canavalia ensiformis) and from the developing fruits of cowpea (Vigna unguiculata), suggests that pathways depending on this hormone have been conserved through evolution.

Analysis of HLA-A antigens and C282Y and H63D mutations of the HFE gene in Brazilian patients with hemochromatosis

The hemochromatosis gene, HFE, is located on chromosome 6 in close proximity to the HLA-A locus. Most Caucasian patients with hereditary hemochromatosis (HH) are homozygous for HLA-A3 and for the C282Y mutation of the HFE gene, while a minority are compound heterozygotes for C282Y and H63D. The prevalence of these mutations in non-Caucasian patients with HH is lower than expected. The objective of the present study was to evaluate the frequencies of HLA-A antigens and the C282Y and H63D mutations of the HFE gene in Brazilian patients with HH and to compare clinical and laboratory profiles of C282Y-positive and -negative patients with HH. The frequencies of HLA-A and C282Y and H63D mutations were determined by PCR-based methods in 15 male patients (median age 44 (20-72) years) with HH. Eight patients (53%) were homozygous and one (7%) was heterozygous for the C282Y mutation. None had compound heterozygosity for C282Y and H63D mutations. All but three C282Y homozygotes were positive for HLA-A3 and three other patients without C282Y were shown to be either heterozygous (N = 2) or homozygous (N = 1) for HLA-A3. Patients homozygous for the C282Y mutation had higher ferritin levels and lower age at onset, but the difference was not significant. The presence of C282Y homozygosity in roughly half of the Brazilian patients with HH, together with the findings of HLA-A homozygosity in C282Y-negative subjects, suggest that other mutations in the HFE gene or in other genes involved in iron homeostasis might also be linked to HH in Brazil.

Induction of interleukin-10 by HIV antigens in peripheral mononuclear cells of health care workers after occupational exposure to HIV-1-positive blood

Evaluation of HIV-induced IL-2 production by peripheral blood mononuclear cells (PBMC) and HIV-specific T helper and cytotoxic T lymphocyte (CTL) responses in health care workers (HCW) occupationally exposed to HIV reveals a high rate of response to HIV among non-seroconverters. IL-10 is also known to interfere with HIV infection in vitro. To evaluate the induction of IL-10 by HIV antigens in HCW occupationally exposed to HIV, 18 HCW with percutaneous injury were enrolled in this study, 9 of them exposed to HIV-contaminated blood, and 9 exposed to HIV-negative blood. PBMC were incubated on plates coated with HIV-1 antigens, and IL-10 was measured in supernatants by ELISA. Five of nine HCW exposed to HIV-contaminated blood presented HIV-induced IL-10. Two of nine HCW exposed to HIV-negative source patients also had detectable levels of HIV-induced IL-10, one of them in the sample obtained on the day of accidental exposure. There was a relationship between the type of device involved in injury and IL-10 production. Individuals exposed to hollow needles or scalpels presented HIV-induced IL-10, whereas those exposed to solid needles and to digital puncture did not, suggesting a relationship between infectious load and IL-10. Although occupational exposure to HIV leads to a low rate of seroconversion, these individuals can develop an antigen-specific immune response characterized in our study by induction of IL-10 in PBMC in vitro.