Multiscale sensing of antibody - antigen interactions by organic transistors and single-molecule force spectroscopy

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

Humoral immune responses against the malaria vaccine candidate antigen Plasmodium vivax AMA-1 and IL-4 gene polymorphisms in individuals living in an endemic area of the Brazilian Amazon

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Expression of the classical and nonclassical HLA molecules in breast cancer

Considering that downregulation of HLA expression could represent a potential mechanism for breast carcinogenesis and metastasis, the aim of the present study was to use immunohistochemical methods to analyze the expression of HLA-Ia, HLA-DR, HLA-DQ, HLA-E, and HLA-G in invasive ductal carcinoma (IDC) of the breast and to relate this HLA profile to anatomopathological parameters. Fifty-two IDC from breast biopsies were stratified according to histological differentiation (well, moderately, and poorly differentiated) and to the presence of metastases in axillary lymph nodes. The expression of HLA molecules was assessed by immunohistochemistry, using a computer-assisted system. Overall, 31 (59.6%) out of the 52 IDC breast biopsies exhibited high expression of HLA-G, but only 14 (26.9%) showed high expression of HLA-E. A large number (41, 78.8%) of the biopsies showed low expression of HLA-Ia, while 45 (86.5%) showed high expression of HLA-DQ and 36 (69.2%) underexpressed HLA-DR. Moreover, 24 (41.2%) of 52 biopsies had both low HLA-Ia expression and high HLA-G expression, while 11 (21.2%) had low HLA-Ia expression and high HLA-E expression. These results suggest that, by different mechanisms, the downregulation of HLA-Ia, HLA-E, and HLA-DR and the upregulation of HLA-G and HLA-DQ are associated with immune response evasion and breast cancer aggressiveness.

Terapia gênica com interferon-alfa no controle do câncer colorretal

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Estudo da variabilidade da região codificadora e 3'não traduzida do gene HLA-F no Brasil

Pós-graduação em Patologia - FMB

Estudo da variabilidade da região codificadora e 3'não traduzida do gene HLA-F no Brasil

Pós-graduação em Patologia - FMB

Susceptibility to toxoplasmic retinochoroiditis is associated with HLA alleles reported to be implicated with rapid progression to AIDS

Background/aims: The frequency of HLA markers associated with rapid progression to AIDS was evaluated in Brazilian patients with AIDS exhibiting or not toxoplasmic retinochoroiditis (TRC). Methods: 98 AIDS patients (25 with TRC, 43 with anti-T. gondii antibodies but without TCR, and 30 without anti-T. gondii antibodies and without TCR) were studied. Results: The HLA-B35 was significantly increased in TRC group (p = 0.0038). Conclusion: The presence of HLA-B35 may simultaneously predispose to progression to AIDS and TRC.

Echinococcus granulosus Antigen B Structure: Subunit Composition and Oligomeric States

Background: Antigen B (AgB) is the major protein secreted by the Echinococcus granulosus metacestode and is involved in key host-parasite interactions during infection. The full comprehension of AgB functions depends on the elucidation of several structural aspects that remain unknown, such as its subunit composition and oligomeric states. Methodology/Principal Findings: The subunit composition of E. granulosus AgB oligomers from individual bovine and human cysts was assessed by mass spectrometry associated with electrophoretic analysis. AgB8/1, AgB8/2, AgB8/3 and AgB8/4 subunits were identified in all samples analyzed, and an AgB8/2 variant (AgB8/2v8) was found in one bovine sample. The exponentially modified protein abundance index (emPAI) was used to estimate the relative abundance of the AgB subunits, revealing that AgB8/1 subunit was relatively overrepresented in all samples. The abundance of AgB8/3 subunit varied between bovine and human cysts. The oligomeric states formed by E. granulosus AgB and recombinant subunits available, rAgB8/1, rAgB8/2 and rAgB8/3, were characterized by native PAGE, light scattering and microscopy. Recombinant subunits showed markedly distinct oligomerization behaviors, forming oligomers with a maximum size relation of rAgB8/3 >rAgB8/2>rAgB8/1. Moreover, the oligomeric states formed by rAgB8/3 subunit were more similar to those observed for AgB purified from hydatid fluid. Pressure-induced dissociation experiments demonstrated that the molecular assemblies formed by the more aggregative subunits, rAgB8/2 and rAgB8/3, also display higher structural stability. Conclusions/Significance: For the first time, AgB subunit composition was analyzed in samples from single hydatid cysts, revealing qualitative and quantitative differences between samples. We showed that AgB oligomers are formed by different subunits, which have distinct abundances and oligomerization properties. Overall, our findings have significantly contributed to increase the current knowledge on AgB expression and structure, highlighting issues that may help to understand the parasite adaptive response during chronic infection.

Lymphocyte transformation assay for C neoformans antigen is not reliable for detecting cellular impairment in patients with Neurocryptococcosis

Background: Cryptococcus neoformans causes meningitis and disseminated infection in healthy individuals, but more commonly in hosts with defective immune responses. Cell-mediated immunity is an important component of the immune response to a great variety of infections, including yeast infections. We aimed to evaluate a specific lymphocyte transformation assay to Cryptococcus neoformans in order to identify immunodeficiency associated to neurocryptococcosis (NCC) as primary cause of the mycosis. Methods: Healthy volunteers, poultry growers, and HIV-seronegative patients with neurocryptococcosis were tested for cellular immune response. Cryptococcal meningitis was diagnosed by India ink staining of cerebrospinal fluid and cryptococcal antigen test (Immunomycol-Inc, SP, Brazil). Isolated peripheral blood mononuclear cells were stimulated with C. neoformans antigen, C. albicans antigen, and pokeweed mitogen. The amount of H-3-thymidine incorporated was assessed, and the results were expressed as stimulation index (SI) and log SI, sensitivity, specificity, and cut-off value (receiver operating characteristics curve). We applied unpaired Student t tests to compare data and considered significant differences for p<0.05. Results: The lymphotoxin alpha showed a low capacity with all the stimuli for classifying patients as responders and non-responders. Lymphotoxin alpha stimulated by heated-killed antigen from patients with neurocryptococcosis was not affected by TCD4+ cell count, and the intensity of response did not correlate with the clinical evolution of neurocryptococcosis. Conclusion: Response to lymphocyte transformation assay should be analyzed based on a normal range and using more than one stimulator. The use of a cut-off value to classify patients with neurocryptococcosis is inadequate. Statistical analysis should be based on the log transformation of SI. A more purified antigen for evaluating specific response to C. neoformans is needed.

Identification of Leishmania infantum chagasi proteins in urine of patients with visceral leishmaniasis: a promising antigen discovery approach of vaccine candidates

Visceral leishmaniasis (VL) is a serious lethal parasitic disease caused by Leishmania donovani in Asia and by Leishmania infantum chagasi in southern Europe and South America. VL is endemic in 47 countries with an annual incidence estimated to be 500 000 cases. This high incidence is due in part to the lack of an efficacious vaccine. Here, we introduce an innovative approach to directly identify parasite vaccine candidate antigens that are abundantly produced in vivo in humans with VL. We combined RP-HPLC and mass spectrometry and categorized three L. infantum chagasi proteins, presumably produced in spleen, liver and bone marrow lesions and excreted in the patients urine. Specifically, these proteins were the following: Li-isd1 (XP_001467866.1), Li-txn1 (XP_001466642.1) and Li-ntf2 (XP_001463738.1). Initial vaccine validation studies were performed with the rLi-ntf2 protein produced in Escherichia coli mixed with the adjuvant BpMPLA-SE. This formulation stimulated potent Th1 response in BALB/c mice. Compared to control animals, mice immunized with Li-ntf2+ BpMPLA-SE had a marked parasite burden reduction in spleens at 40 days post-challenge with virulent L. infantum chagasi. These results strongly support the proposed antigen discovery strategy of vaccine candidates to VL and opens novel possibilities for vaccine development to other serious infectious diseases.

Pre- and Posttransplant Monitoring of Alloantibodies by Complement-Dependent Cytotoxicity and Luminex Methodologies in Liver Transplantation

Background. This study evaluated the influence of circulating anti-HLA antibodies on outcomes of 97 liver allografts from deceased donors. Methods. Human leukocyte antigen (HLA) antibody screening was performed by both complement-dependent cytotoxicity (CDC) and multiparameter Luminex microsphere-based assays (Luminex assay). Results. The agreements between T- and B- cell CDC and Luminex assays were 67% and 77% for pre- and posttransplant specimens, respectively. Graft dysfunction was not associated with either positive pretransplant CDC or Luminex panel-reactive antibody (PRA) values. Likewise, positive posttransplant T- or B- cell CDC PRA values were not associated with graft dysfunction. In contrast, posttransplant Luminex PRA values were significantly higher among patients with graft dysfunction compared with subjects with good outcomes (P = .017). Conclusion. Posttransplant monitoring of HLA antibodies with Luminex methodology allowed identification of patients at high-risk for poor graft outcomes.

Therapeutic DNA Vaccine Encoding Peptide P10 against Experimental Paracoccidioidomycosis

Paracoccidioidomycosis (PCM), caused by Paracoccidioides brasiliensis, is the most prevalent invasive fungal disease in South America. Systemic mycoses are the 10th most common cause of death among infectious diseases in Brazil and PCM is responsible for more than 50% of deaths due to fungal infections. PCM is typically treated with sulfonamides, amphotericin B or azoles, although complete eradication of the fungus may not occur and relapsing disease is frequently reported. A 15-mer peptide from the major diagnostic antigen gp43, named P10, can induce a strong T-CD4+ helper-1 immune response in mice. The TEPITOPE algorithm and experimental data have confirmed that most HLA-DR molecules can present P10, which suggests that P10 is a candidate antigen for a PCM vaccine. In the current work, the therapeutic efficacy of plasmid immunization with P10 and/or IL-12 inserts was tested in murine models of PCM. When given prior to or after infection with P. brasiliensis virulent Pb 18 isolate, plasmid-vaccination with P10 and/or IL-12 inserts successfully reduced the fungal burden in lungs of infected mice. In fact, intramuscular administration of a combination of plasmids expressing P10 and IL-12 given weekly for one month, followed by single injections every month for 3 months restored normal lung architecture and eradicated the fungus in mice that were infected one month prior to treatment. The data indicate that immunization with these plasmids is a powerful procedure for prevention and treatment of experimental PCM, with the perspective of being also effective in human patients.

Cutting Edge: Brazilian Pemphigus Foliaceus Anti-Desmoglein 1 Autoantibodies Cross-React with Sand Fly Salivary LJM11 Antigen

The environmental factors that contribute to the development of autoimmune diseases are largely unknown. Endemic pemphigus foliaceus in humans, known as Fogo Selvagem (FS) in Brazil, is mediated by pathogenic IgG4 autoantibodies against desmoglein 1 (Dsg1). Clusters of FS overlap with those of leishmaniasis, a disease transmitted by sand fly (Lutzomyia longipalpis) bites. In this study, we show that salivary Ags from the sand fly, and specifically the LJM11 salivary protein, are recognized by FS Abs. Anti-Dsg1 monoclonal autoantibodies derived from FS patients also cross-react with LJM11. Mice immunized with LJM11 generate anti-Dsg1 Abs. Thus, insect bites may deliver salivary Ags that initiate a cross-reactive IgG4 Ab response in genetically susceptible individuals and lead to subsequent FS. Our findings establish a clear relationship between an environmental, noninfectious Ag and the development of potentially pathogenic autoantibodies in an autoimmune disease. The Journal of Immunology, 2012, 189: 1535-1539.

Involvement of TSSA (trypomastigote small surface antigen) in Trypanosoma cruzi invasion of mammalian cells

TSSA (trypomastigote small surface antigen) is a polymorphic mucin-like molecule displayed on the surface of Trypanosoma cruzi trypomastigote forms. To evaluate its functional properties, we undertook comparative biochemical and genetic approaches on isoforms present in parasite stocks from extant evolutionary lineages (CL Brener and Sylvio X-10). We show that CL Brener TSSA, but not the Sylvio X-10 counterpart, exhibits dose-dependent and saturable binding towards non-macrophagic cell lines. This binding triggers Ca2+-based signalling responses in the target cell while providing an anchor for the invading parasite. Accordingly, exogenous addition of either TSSA-derived peptides or specific antibodies significantly inhibits invasion of CL Brener, but not Sylvio X-10, trypomastigotes. Non-infective epimastigote forms, which do not express detectable levels of TSSA, were stably transfected with TSSA cDNA from either parasite stock. Although both transfectants produced a surface-associated mucin-like TSSA product, epimastigotes expressing CL Brener TSSA showed a similar to 2-fold increase in their attachment to mammalian cells. Overall, these findings indicate that CL Brener TSSA functions as a parasite adhesin, engaging surface receptor(s) and inducing signalling pathways on the host cell as a prerequisite for parasite internalization. More importantly, the contrasting functional features of TSSA isoforms provide one appealing mechanism underlying the differential infectivity of T. cruzi stocks.

A novel HLA-E allele, E*01:03:05, identified in two Brazilian individuals

Brazilian National Research Council (CNPq/Brazil)