CAMILA APARECIDA DE CARVALHO
Projetos de Pesquisa
Unidades Organizacionais
LIM/49 - Laboratório de Protozoologia, Hospital das Clínicas, Faculdade de Medicina
9 resultados
Resultados de Busca
Agora exibindo 1 - 9 de 9
- Understanding hypergammaglobulinemia in experimental or natural visceral leishmaniasis(2024) CARVALHO, Camila Aparecida de; HIRAMOTO, Roberto Mitsuyoshi; MEIRELES, Luciana Regina; ANDRADE, Heitor Franco deNonspecific hypergammaglobulinemia (HGG) occurs in symptomatic human visceral leishmaniasis (VL) caused by L. L. infantum. This study assessed this finding in experimental infection in hamsters and natural infection in dogs. The serum concentration of proteins, albumin and globulins was determined through the biuret and bromocresol green reaction, where the HGG was better expressed through the albumin/globulin (A/G) ratio. HGG was associated with a higher concentration of specific anti-glycan antibodies (BSA-G)/promastigote soluble extract (PSE) and the presence of circulating immune complexes (IC) by dissociative enzyme-linked immunoassay (ELISA). The study found monovalent IC in 37.9% (PSE) and 50% (BSA-G) of sera from infected hamsters, with increased frequency as the disease progressed. HGG was found in >60% of the samples in dogs with VL, associated with higher levels of specific immunoglobulin (Ig)A and IgM, but not IgG, determined using the PSE and BSA-G ELISA. HGG was associated with the presence of monovalent IC in 58.9% (PSE) and 63.4% (BSA-G) positive dog samples. HGG may result not only from the nonspecific activation of B cells, with greater production of specific and nonspecific antibodies, but also due to lower IgG excretion due to the presence of soluble monovalent IC. HGG correlates to the progression of VL and may be a marker for manifested disease.
- Indirect Evidence of Circulating Parasite Hapten Immune Complexes in Visceral Leishmaniasis(2019) CARVALHO, Camila Aparecida de; FERRAO, Thiago Fidelis; HIRAMOTO, Roberto Mitsuyoshi; PARTATA, Anette Kelsei; ANDRADE JUNIOR, Heitor Franco deBackground: Hypergammaglobulinemia is present in visceral leishmaniasis (VL), inducing the formation of immune complexes (ICs), which interferes in conventional serology. Parasitic haptens block antibodies, making it difficult to identify and detect them. ICs could be determined indirectly by acid dissociative ELISA (DE) seroconversion in natural and experimental VL. Methods: We determined the frequency of samples that seroconverted in DE or presented a 10% increase in DE (Delta DE) in 3 types of VLs-hamster, canine, and human samples-with larger antigen determination by direct antigen capture in experimental samples. Results: The Delta DE frequency is increased in all VL models: human (34%), canine (27%), and hamster (25%) samples. Seroconversion was present in hamster (14%), dog (1%), and human (6%) samples. During experimental infection, higher frequencies (28%) of circulating antigens were observed at the 30th day, associated with higher Delta DE (47%) and seroconversion (22%), with lower frequencies in other periods. Conclusions: The frequency of Delta ED and seroconversion samples found in natural and experimental infection suggests that specific antibodies can be blocked by low molecular weight antigens that interfere qualitatively (seroconversion) or quantitatively (Delta DE) in serology. Several antigen types may be involved, as high molecular weight proteins and low molecular weight glycoconjugates. The higher frequency of those indirect demonstrations of antibody-blocking antigen or haptens that could be acid-removed in VL has implications for the development of assays for detection of circulating or antibody-bound 1- to 3-kDa antigens, which could interfere in diagnosis and also in the immune response of the host.
- Preventive measures for "Pet Friendly" lodging facilities: association of Leishmaniasis expansion route in Sao Paulo and preventive measures for regional animals displacement(2017) CARVALHO, Camila Aparecida de; FUJITA, Dennis Minoru; NALI, Luiz Henrique da Silva; ANDRADE JUNIOR, Heitor Franco de; HIRAMOTO, Roberto Mitsuyoshi
- Scavenger receptors mediate increased uptake of irradiated T.gondii extracts by J774 macrophages(2023) COSTA, Andrea da; CARVALHO, Camila Aparecida de; NASCIMENTO, Nanci do; JR, Heitor Franco de AndradePurposeProtein extracts developed increased immunogenicity without the aid of adjuvants after gamma irradiation. Gamma irradiation of snake venom increased antivenin production by detoxification and enhanced immunity, probably due preferential uptake of irradiated venoms by macrophage scavenger receptors. We studied this uptake of irradiated soluble Toxoplasma gondii extract (STag) by the J774 macrophage cell line similar to antigen presenting cells.Material And MethodsWe labeled STag by biosynthesis in living tachyzoites with radioactive amino acids before purification and irradiation or by adding labels as biotin or fluorescein in stored STag, for quantitative studies or subcellular distribution visualization.ResultsThere was enhanced binding and uptake of irradiated STag into the cells compared to non-irradiated STag. Using fluorescein labeled antigens and morphological assays, we confirmed that cells avidly ingested both native and irradiated proteins but native STag were digested after ingestion while irradiated proteins remained in the cell, suggesting diverse intracytoplasmic pathways. Native or irradiated STag present the same in vitro sensitivity to three types of peptidases. Inhibitors of scavenger receptors (SRs) such as Dextran sulfate (SR-A1 blocker) or Probucol (SR-B blocker) affect the specific uptake of irradiated antigens, suggesting its association with enhanced immunity.ConclusionsOur data suggests that cell SRs recognize irradiated proteins, mainly SRs for oxidized proteins, leading to antigen uptake by an intracytoplasmic pathway with fewer peptidases that prolongs presentation to nascent major histocompatibility complex I or II and enhances immunity by better antigen presentation.
- Activity of cycloartane-type triterpenes and sterols isolated from Musa paradisiaca fruit peel against Leishmania infantum chagasi(2014) SILVA, A. A. S.; MORAIS, S. M.; FALCAO, M. J. C.; VIEIRA, I. G. P.; RIBEIRO, L. M.; VIANA, S. M.; TEIXEIRA, M. J.; BARRETO, F. S.; CARVALHO, C. A.; CARDOSO, R. P. A.; ANDRADE-JUNIOR, H. F.The aim of the study was to evaluate in vitro the antileishmanial activity of triterpenes and sterols isolated from Musa paradisiaca (banana) fruit peel used traditionally to treat leishmaniasis. The compounds were isolated from the ethanolic extract of the peel of the banana fruit by column chromatography. The chemical structure of compounds was determined by H-1 and C-13 - nuclear magnetic resonance spectroscopy. The cytotoxicity was measured in RAW 264.7 cells and LLC-MK2. Leishmanicidal activity against L infantum chagasi promastigotes was performed by the MTT colorimetric method and activity against amastigotes was assayed in mammalian cells using in situ ELISA method. Five compounds were identified, consisting of three triterpenes: cycloeucalenone, 31-norcyclolaudenone and 24-methylene-cicloartanol and a mixture of two sterols: beta-sitosterol and stigmasterol. With the exception of cycloeucalenone, all compounds showed statistically similar activity against promastigote to peritamidine. While, acting against amastigotes, excluding 31-norcyclolaudenone, other compounds showed activity similar to amphotericin B. All compounds showed low cytotoxicity in mammalian cells. Conclusion: This study partially confirms the use of Musa paradisiaca in folk medicine against leishmaniasis. Further in vivo studies are necessary to evaluate the efficacy.
- Early high avidity specific IgG production in experimental hamster visceral leishmaniasis(2020) CARVALHO, Camila Aparecida de; FERRAO, Thiago Fidelis; CAVALCANTE, Fernanda Siqueira; FREITAS, Flavia Regina Novais de; MEIRELES, Luciana Regina; ANDRADE JUNIOR, Heitor Franco deVisceral leishmaniasis (VL) byLeishmania (Leishmania) infantumis epidemic in Brazil. Hypergammaglobulinemia appears early in patients with VL and is ineffective. Usually, high-affinity IgG B cells are selected during most infections, a critical step for an effective humoral response. The avidity of IgG antibodies in VL is unexplored due to the absence of temporal parameters in most patients, associated to low clinical significance. Experimental infection models overcome this fact, allowing the monitoring of the disease temporal evolution. In this study, the avidity of IgG antibodies was evaluated in experimental models, in infection in hamsters, and in immunization in rabbits. Specific IgG antibodies were detected by ELISA, using chaotropic solution to determine avidity, as reported for viral infections. The levels of IgG antibodies correlated with the progression of experimental infection in hamsters or antigenic stimulation in immunized rabbits. However, IgG avidity was high early in infected animals, even in early periods (> 80%), while in immunized rabbits, they had early antibodies of low avidity with progressive maturation, similar as other infections. These data suggest that the affinity maturation of the avidity of anti-LeishmaniaIgG antibodies promoted at an early stage, influencing the appropriate interaction between antigens and affecting the disease progression. This fact could be associated to monovalent immune complexes, as reported in human and experimental VL. This scenario may be related to an independent process of immune cell activation by the parasite but absent in antigen preparation used as immunogens.
- Natural versus Recombinant Viral Antigens in SARS-CoV-2 Serology: Challenges in Optimizing Laboratory Diagnosis of COVID-19(2020) MEIRELES, Luciana Regina; SILVA, Angelica Moura Freixeira da; CARVALHO, Camila Aparecida; KESPER, Norival; GALISTEO JR., Andres Jimenez; SOARES, Camila Pereira; ARAUJO, Danielle Bastos; DURIGON, Edison Luiz; OLIVEIRA, Danielle Bruna Leal; MORGANTI, Ligia; CHURA-CHAMBI, Rosa Maria; ANDRADE JR., Heitor Franco deOBJECTIVES: COVID-19 is a public health emergency of international concern whose detection in recovered asymptomatic patients is dependent on accurate diagnosis as it enables the estimation of the susceptibility of the population to the infection. This demand has resulted in the development of several commercial assays employing recombinant proteins, but the results of these assays are not reliable as they do not involve comparison with natural viral antigens. We independently used the SARS-CoV-2 whole viral antigen (WVA) and recombinant nucleocapsid protein (rNP) to develop in-house ELISAs for IgG detection; the results of these ELISAs were then compared to obtain reliable results. METHODS: WVA and rNP ELISAs were performed on COVID-19 negative sera from patients before the pandemic in Brazil, and on RT-qPCR-positive or SARS-CoV-2-IgG against rNP and IgG against WVA-positive samples from recently infected patients in Sao Paulo, Brazil. RESULTS: Both ELISAs detected a large fraction of infected patients but exhibited certain drawbacks. Higher signals and lower numbers of false-negatives were observed in rNP ELISA; however, a higher fraction of false-ositives was observed in control groups. A high number of false-negatives was observed with WVA ELISA. Correlating the results of rNP and WVA ELISAs resulted in improved performance for COVID-19 diagnosis. CONCLUSION: The choice of antigen is an important aspect in optimizing the laboratory diagnosis of COVID-19. The use of rNP ELISA for the detection of anti-SARS-CoV-2 IgG antibodies seems promising, but comparison of the results with those of WVA ELISA is crucial for accurate test development prior to commercialization. IgG serology using several assays, and with the spectral patterns of SARS-CoV-2, resulted in confusing information that must be clarified before the establishment of diagnostic serology criteria.
- Serum antibodies blocked by glycan antigens in canine visceral leishmaniasis serology are mostly IgA immune complexes(2021) CARVALHO, Camila Aparecida de; HIRAMOTO, Roberto Mitsuyoshi; MEIRELES, Luciana Regina; ANDRADE JUNIOR, Heitor Franco deImmune complexes (ICs) are found in canine visceral leishmaniasis (CVL) and interfere with the serum detection of antibodies. Dissociation of these monovalent complexes by dissociative enzyme-linked immunosorbent assay (ELISA) removes false-negative results and allows some characterization of antibodies and antigens. We studied the serology of dogs with suspected CVL in an endemic area, testing two Leishmania (Leishmania) [L. (L.)] infantum antigens. We analysed the presence of immunoglobulin G (IgG), immunoglobulin A (IgA) and immunoglobulin M (IgM) antibodies specific to promastigote soluble extract (PSE) and low-molecular weight glycans (glycan-bovine serum albumin (BSA) complex - GBC) by conventional and dissociative ELISA. Our results showed a significant fraction of IgA ICs (46.5% for PSE and 47.6% for GBC), followed by IgG ICs (10% for PSE and 23.5% for GBC). IgM ICs were more frequent for PSE (22.7%). Hypergammaglobulinaemia in CVL would be related to the presence of IgA and IgG ICs, resulting in deficient elimination of these antibodies. Our data confirmed the presence of ICs that can generate false-negative results in conventional serology. The production of IgA antibodies and the high frequency of blockade by glycan antigens suggest the active participation of this immunoglobulin and its ICs in the immunopathology of CVL, indicating a new path for further research.
- High levels of serum glycans monovalent IgG immune complexes detected by dissociative ELISA in experimental visceral leishmaniasis(2019) CARVALHO, Camila Aparecida de; FERRAO, Thiago Fidelis; FREITAS, Flavia Regina Novais de; ANDRADE JUNIOR, Heitor Franco deVisceral leishmaniasis (VL) is epidemic in Brazil with an increasing incidence of human cases and canine reservoirs, with host hypergammaglobulinemia. Conventional enzyme-linked immunosorbent assay (cELISA) based on several parasitic antigens is the main method for diagnosis and indication of treatment. Dissociative ELISA (dELISA) uses acidic treatment to free immunoglobulin G (IgG) from immune complexes, and its use revealed a significant positive fraction of suspected cases with negative serology. Looking for small molecules or haptens that block IgG antibodies, we purified by molecular exclusion chromatography, 1000-3000 MW molecules from promastigote soluble extract, mostly oligosaccharides comprising 6-13 sugar residues using MALDI-TOF analysis. Glycan-BSA complex (GBC) was constructed by conjugating promastigote glycans to BSA molecules, allowing their use in the solid support in cELISA or dELISA. Sera from experimentally infected hamsters showed higher levels of blocked monomeric IgG during infection, mostly against GBC, which was also present in lower concentrations in the promastigote soluble extract dELISA. Those data show that most of the specific monomeric IgG in serum are blocked by haptens composed by glycans produced by the parasite, better detected in the high dilution of sera in the dELISA assays. dELISA is a useful technique for detecting blocked monomeric antibodies that could have difficult clearance from blood, which could result in hypergammaglobulinemia.