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ABSTRACT: Lolium multiflorum (Lm) grass pollen is the major cause of pollinosis in Southern Brazil. The objectives of this study were to investigate immunodominant components of Lm pollen allergens and the cross-reactivity of IgE with commercial grass pollen allergen extracts. Thirty-eight serum samples from patients with seasonal allergic rhinitis (SAR), 35 serum samples from patients with perennial allergic rhinitis (PAR) and 30 serum samples from non-atopic subjects were analyzed. Allergen sensitization was evaluated using skin prick test and serum IgE levels against Lm pollen extract were determined by ELISA. Inhibition ELISA and immunoblot were used to evaluate the cross-reactivity of IgE between allergens from Lm and commercial grass pollen extracts, including L. perenne (Lp), grass mix I (GI) and II (GII) extracts. IgE antibodies against Lm were detected in 100% of SAR patients and 8.6% of PAR patients. Inhibition ELISA demonstrated IgE cross-reactivity between homologous (Lm) and heterologous (Lp or GII) grass pollen extracts, but not for the GI extract. Fifteen IgE-binding Lm components were detected and immunoblot bands of 26, 28-30, and 32-35 kDa showed >90% recognition. Lm, Lp and GII extracts significantly inhibited IgE binding to the most immunodominant Lm components, particularly the 55 kDa band. The 26 kDa and 90-114 kDa bands presented the lowest amount of heterologous inhibition. We demonstrated that Lm extract contains both Lm-specific and cross-reactive IgE-binding components and therefore it is suitable for measuring quantitative IgE levels for diagnostic and therapeutic purposes in patients with pollinosis sensitized to Lm grass pollen rather than other phylogenetically related grass pollen extracts.
Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas / Sociedade Brasileira de Biofisica ... [et al.] 02/2010; 43(2):166-75. · 1.08 Impact Factor
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ABSTRACT: Indoor allergens are major causative agents in allergic disease development. Besides homes, public transport vehicles have been considered important mite and pet allergen reservoirs. Our recent studies on allergen exposure in automobiles showed that different allergen levels are found in private cars versus taxis. We quantified group 1 Dermatophagoides spp. (Der 1), Felis domesticus (Fel d 1), and Canis familiaris (Can f 1) allergen levels by ELISA in dust samples from 60 taxi and 60 private car upholstered seats. Mean levels of Der 1 and Fel d 1 were significantly higher in taxis than private cars. A significantly higher percentage of taxis (42%) harboring sensitizing levels of Der 1 compared to private cars (5%) was also found. In spite of the low mean Fel d 1 levels, comparison of the percentage of vehicles with moderate Fel d 1 levels showed a significant difference between taxis and private cars (43% vs. 20%). On the other hand, mean Can f 1 levels were significantly higher in private cars compared to taxis concomitant with a significantly higher percentage of private cars containing moderate Can f 1 levels than taxis (53% vs. 28%). We conclude that upholstered seats from Brazilian taxis but not private cars constitute an important mite allergen reservoir. Thus, additional effective measures for the reduction of allergen exposure in vehicles within the global allergen avoidance strategy should also be routinely accomplished to minimize the induction of sensitization and symptoms in allergic patients.
Journal of investigational allergology & clinical immunology: official organ of the International Association of Asthmology (INTERASMA) and Sociedad Latinoamericana de Alergia e Inmunología 02/2006; 16(1):34-6. · 2.27 Impact Factor
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ABSTRACT: A comparative study of the indirect haemagglutination (IHA), immunofluorescence (IFAT) and immunoenzymatic (ELISA) tests was carried out to determine the prevalence of Toxoplasma gondii antibodies in goats. One hundred seventy-four serum samples were obtained from four goat herds from the region of Uberlândia, State of Minas Gerais. The distribution of the animals, according to their origin, was as follow: 71 from herd I; 39 from herd II; 37 from herd III; and 27 from herd IV. Serum samples were analyzed by IHA, IFAT and ELISA, considering the reactivity of the serum samples at dilution > or = 1:64 as cut off titer for the three tests. A global seroprevalence of 18.4% was observed, with significantly higher positivity rate in the herd II (66.7%) and older animals (> 36 months). A high and significant positive correlation was found between the titers obtained by the IHA versus IFAT, IHA versus ELISA, and ELISA versus IFAT. Therefore, it can be concluded that the three analyzed tests have shown to be highly concordant and appropriate for epidemiological surveys of Toxoplasma infection in goats. Although the seroprevalence of T. gondii infection in goats is relatively low in this region as compared to other regions of the country, adequate management might be useful and essential to control the infection in the goat herds.
Memórias do Instituto Oswaldo Cruz 08/2001; 96(5):687-92. · 2.15 Impact Factor
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ABSTRACT: A total of 163 dogs with neuromuscular, respiratory and/or gastrointestinal disorders, was admitted at the Veterinary Hospital, Federal University of Uberlândia, Brazil, and submitted to serology for Toxoplasma gondii and Neospora caninum. Assays for T. gondii included indirect haemagglutination (IHA), indirect fluorescent antibody (IFAT-Tg), immunoenzymatic (ELISA), and immunoblotting (IB-Tg). Assays for N. caninum included IFAT-Nc and immunoprecipitation (IP-Nc). Based on concordant results by three serological tests (IHA, IFAT-Tg and ELISA) for T. gondii, and divergent results further confirmed by IB-Tg for reactivity to TgSAG1, the 163 sera were divided into two groups: 59 (36%) Tg-seropositive samples and 104 (64%) Tg-seronegative samples. Antibodies to Neospora were detected in 11 (6.7%) out of 163 analyzed dog sera, with 5 (3.1%) samples reactive to both parasites (Tg+/Nc+), and 6 (3.7%) reactive only to Neospora (Tg-/Nc+). Antibodies only to T. gondii were found in 54 (33%) samples. Among the 11 Neospora-positive sera analyzed by IB-Tg, the five sera Tg+/Nc+ showed strong reactivity to Toxoplasma antigens, especially to TgSAG1 (p30). No reactivity was observed to TgSAG1 in the six samples Tg-/Nc+. By IP-Nc, two highly immunodominant antigens (29 and 35kDa proteins) were recognized by all 11 IFAT-Nc positive sera. Our results suggest that the infection by N. caninum can be concomitantly present in dogs from this area, although less common, and therefore should be considered in the differential clinical diagnosis with T. gondii in dogs presenting neuromuscular, respiratory and/or gastrointestinal disorders.
Veterinary Parasitology 08/2001; 98(4):239-45. · 2.58 Impact Factor
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ABSTRACT: Epidemiologic studies have shown that the presence of IgE antibodies to house dust mite and other indoor allergens is an important risk factor for asthma.
The aim of this study was to develop a reverse ELISA (rELISA) for measuring specific IgE to Der p 2, a major Dermatophagoides pteronyssinus (Dpt) allergen, as a potential tool for followup of allergen immunotherapy.
Recombinant Der p 2 allergen or a monoclonal antibody to Der p 2 was used to coat plates in conventional ELISA (cELISA) and rELISA, respectively. Sera from 48 asthmatic patients with positive skin prick test (SPT+) to D. pteronyssinus extract were analyzed for total IgE and specific IgE to Der p 2, and the results were compared with a group of 41 SPT asthmatic and 30 SPT- control subjects.
The sensitivity of the two assays for Der p 2-specific IgE was 3.9 EU/mL and their specificities were confirmed by inhibition tests, in a dose-dependent manner. There was a significant positive correlation between cELISA and rELISA (r = 0.74; P < 0.0001). However, rELISA was more sensitive than was cELISA, regarding both the positive sera percentage (70.8% vs 52.1%) and the Der p 2-specific IgE levels (28.4 vs 4.5 EU/mL) in SPT+ asthmatic patients.
rELISA has shown to be a sensitive and alternative method for measuring Der p 2-specific IgE without using radioactive techniques. Detection of specific IgE to major allergens and relevant peptides, and identification of B cell epitopes in allergens will provide valuable information for the design of allergen analogs and peptides for immunotherapy.
Annals of allergy, asthma & immunology: official publication of the American College of Allergy, Asthma, & Immunology 05/2001; 86(5):545-50. · 2.83 Impact Factor
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ABSTRACT: The role of mite allergen exposure in sensitization and development of asthma has been widely recognized. Previous studies have shown that Dermatophagoides pteronyssinus and Blomia tropicalis were the most prevalent house dust mites in Brazil, while D. farinae was rarely found. The aim of this study was to measure Der f 1 and Der p 1 allergen levels in Brazilian asthmatics' and controls' homes.
Sixty-four houses (32 asthmatic, 32 control) were visited for dust sampling from five sites. Der f 1 and Der p 1 levels were measured by two-site monoclonal-antibody-based ELISAs.
The highest levels of Der f 1 and Der p 1 allergens were found in bedding samples from both asthmatics' and controls' homes. However, the geometric mean of Der f 1 levels (15.8 microgram/g of dust) was significantly higher than for Der p 1 (8.2 microgram/g of dust) in these samples. In addition, allergen levels >/=10 microgram/g of dust were found in 60-80% of the samples for Der f 1 and about 50% for Der p 1.
These results indicate that high levels of Der f 1 allergen are present in both asthmatics' and controls' homes, in contrast to previously reported data. Therefore, studies on exposure to mites should be performed in different cities, seasons and times, since the mite fauna might be subject to variations. Knowledge of the mite fauna will certainly improve the means of investigating the association between allergen exposure and sensitization, allowing to establish the inclusion of new mite extracts in inhalant skin test sets, and even to detect monosensitized patients with respiratory allergy.
International Archives of Allergy and Immunology 08/2000; 122(4):257-63. · 2.40 Impact Factor
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ABSTRACT: We characterized antigenic markers recognized by human serum samples from patients presenting with acute and chronic toxoplasmosis by the determination of immunoglobulin G (IgG) antibody avidity by a Western blot modified technique (avidity immunoblotting) that includes the dissociation of the antigen-antibody interaction with 6 or 8 M urea solutions. Human serum samples from 20 patients presenting with recent infection and from 20 patients with chronic infection were analyzed. It was observed that bands p16, p32, p38, p40, p43, p54, p60, p66, and p97 were more frequently recognized by low-avidity IgG in recent infection and by high-avidity IgG in chronic toxoplasmosis. From these antigenic bands, p38 can be characterized as an optimal antigenic marker of low avidity for recent forms of toxoplasmosis due to a significant decrease of their frequencies (from 80 to 0%) after treatment with 6 M urea solutions. The p30 antigen was not considered a good marker to distinguish acute from chronic infection since corresponding IgG antibodies were determined to have high avidity in both phases of the infection. Thus, the avidity immunoblotting assay proved to be a useful tool for determining antigenic markers of recent and chronic phases of Toxoplasma gondii infection.
Clinical and Diagnostic Laboratory Immunology 06/2000; 7(3):384-9. · 2.51 Impact Factor
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ABSTRACT: The potential of the RH strain of Toxoplasma gondii to invade trophoblast cells of the cricetid rodent Calomys callosus in a congenital infection in the initial third of pregnancy was investigated in this study using morphological and immunocytochemical approaches. The animals were intraperitoneally inoculated on the 1st day of pregnancy and the infection was observed on day 7. Various numbers of parasites could be observed inside the parasitophorous vacuoles in trophoblastic cells under light and electron microscopy. The trophoblast cells showed characteristics of healthy cells, and no alteration other than parasite vacuoles in their cytoplasm could be detected. Polyclonal or monoclonal anti-T. gondii antibodies (respectively, anti-T. gondii components and the major surface parasite antigen p30) labeled both the parasite surface and parasitophorous vacuole membranes, regardless of the number of parasites inside the compartment. In addition, p30-containing trails were detected in the extracellular matrix surrounding trophoblastic cells similar to those found with other parasites during locomotion and the invasion process. Our results show the ability of T. gondii to infect trophoblast cells during the early blastocyst-endometrial relationship and open new possibilities for more accurate study of the invasion process of this parasite and the role of the trophoblast as an embryo defense barrier.
Parasitology Research 09/1999; 85(8-9):647-54. · 2.15 Impact Factor
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ABSTRACT: Stage conversion between bradyzoites and tachyzoites was investigated in C57BL/6 mice chronically infected with the ME-49 strain of Toxoplasma gondii. In order to promote bradyzoite-tachyzoite conversion, mice were treated in vivo with neutralizing doses of anti-gamma interferon (IFN-gamma) or anti-tumor necrosis factor alpha (TNF-alpha) antibodies. Expression of parasite-specific antigens SAG-1, SAG-2, and heat shock protein 70 (Hsp-70) was visualized in the central nervous system by immunocytochemistry and measured by photometric assay. The immunosuppressive effect of anti-IFN-gamma or anti-TNF-alpha treatment was immediate, leading to parasite stage conversion as indicated by the increased expression of tachyzoite-specific antigens (SAG-1 and SAG-2) and by rapid parasite replication. We also observed expression of high levels of Hsp-70 during a short period of conversion of bradyzoites to tachyzoites. Our data suggest that Hsp-70 may have an important role in the process of bradyzoite-tachyzoite conversion during the reactivation of chronic toxoplasmosis.
Infection and Immunity 09/1998; 66(8):3959-63. · 4.16 Impact Factor
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ABSTRACT: Serum samples from 107 cervids were examined for Toxoplasma gondii antibodies using indirect hemagglutination (IHA), indirect immunofluorescence (IFA), enzyme linked immunosorbent assay (ELISA) and Dot-ELISA. Samples were obtained from 66 marsh deer (Blastocerus dichotomus) in the State of São Paulo (Brazil) and from 41 pampas deer (Ozotocerus bezoarticus) in the State of Goiás (Brazil). Antibodies to T. gondii were found in 23 (22%) of the deer, with 18 and 5 positive samples, respectively, for B. dichotomus and O. bezoarticus. The highest prevalence of T. gondii antibodies were young adults (32%), following by adults (27%) and fawns (13%). Only one serum sample (8%) from a newborn fawn was positive in the serological tests. The convenience of the Dot-ELISA test is obvious when compared with other serological tests for both laboratory or field surveys, mainly due to its features of practicability and reagent stability.
Journal of wildlife diseases 11/1997; 33(4):896-9. · 1.08 Impact Factor
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ABSTRACT: Paired human saliva and serum samples from 60 individuals were tested for specific IgA and IgG antibodies to Toxoplasma gondii. The study in both fluids was carried out by indirect immunoenzymatic assay (ELISA). Saline antigenic extract of T. gondii was used to coat plastic surfaces upon which the samples were then incubated; monospecific conjugates of anti-IgA and anti-IgG-peroxidase were then incubated with the samples after a washing procedure to separate the unbound antibodies. The enzymatic activity was measured and the results expressed in terms of ELISA index. Toxoplasma-specific IgG antibodies were detected in 43 of the serum samples (71.7%) and in 12 of the saliva samples (20.0%) whereas Toxoplasma-specific IgA antibodies were detected in 18 of the serum samples (30.0%) and in 12 of the saliva samples (20.0%). No association was observed when the Toxoplasma-specific IgG reactive and non-reactive serum samples were compared with the reactive and non-reactive saliva samples for this class of immunoglobulin. On the other hand, a significant association was observed when the Toxoplasma-specific IgA reactive and non-reactive serum samples were compared with the reactive and non-reactive saliva for this type of antibody. In conclusion, our results show that the detection of salivary IgA reflects the serum level of this isotype but salivary IgG does not. Moreover, the isolated detection of salivary IgG may not contribute to epidemiological studies of chronic toxoplasmic infections.
Journal of Oral Pathology and Medicine 05/1997; 26(4):187-91. · 1.63 Impact Factor
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ABSTRACT: We evaluated the titers of anti-T. gondii antibodies by various serological tests in 40 serum samples from dogs exhibiting clinical signs of infectious diseases. Indirect immunofluorescence (IgG-IFI), indirect haemagglutination (IHA and M-Toxo) and immunoenzymatic (ELISA and PA-ELISA) tests were carried out. Titers > or = 64 were considered as positive. Anti-Toxoplasma antibodies were found in 9 (22.5%), 14 (35%), 14 (35%) and 12 (30%) samples, respectively for IHA, IgG-IFI, ELISA and PA-ELISA. The results showed that 57% were negative in all tests and 43% of the dogs presented antibodies to T. gondii; from these, 20% were positive in all three tests with high titers of antibodies and 23% were positive in only one or two tests with low titers of antibodies and mainly related to the IFI and ELISA tests. We observed 5 (12.5%) and 1 (2.5%) reactive samples, respectively, by M-Toxo and IHA with or without 2-mercapthoethanol, in the attempt to detect specific IgM. We can conclude that serodiagnosis of toxoplasmosis in dog have to be based on the combination of serological tests (IFI and ELISA) and with emphasis at the determination of the titers and the classes of the specific antibodies.
Memórias do Instituto Oswaldo Cruz 01/1997; 92(6):785-9. · 2.15 Impact Factor
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ABSTRACT: Using a modified enzyme-linked immunosorbent assay that included dissociation of antigen antibody complexes with 6M urea solution, we analyzed the avidity of Toxoplasma-specific IgG in aqueous humor and serum samples from 24 patients with toxoplasmic chorioretinitis. As a control, we studied aqueous humor and serum samples from 14 cataract patients without history of uveitis and serum samples from 10 patients with recent primary systemic toxoplasmic infection without ocular lesions. IgG avidity was markedly lower in aqueous humor samples from patients with toxoplasmic chorioretinitis than in serum samples, despite those samples presenting higher levels of Toxoplasma-specific IgG than in serum samples. The detection of the low-avidity Toxoplasma-specific antibodies can offer a valuable aid to make a specific etiologic diagnosis and perhaps contribute to understand the pathogenic mechanisms of ocular toxoplasmosis.
Applied parasitology 03/1994; 35(1):1-7.
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ABSTRACT: An experimental model for acquired and congenital ocular toxoplasmosis as well as a model to induce experimental autoimmune uveitis (EAU) was investigated in Calomys callosus. Toxoplasma gondii, ME-49 strain, was used to infect males and pregnant- and not pregnant-females while S-antigen, a major glycoprotein of the retinal photoreceptor cell, was used to induce EAU. The ocular lesions elicited by T. gondii were characterized by the presence of cysts, free tachyzoites and inflammatory cells in the retina or related tissues. In the congenital form, 40% of the fetus presented ocular lesions, i.e., presence of cysts in the retina, vitreous, and extra-retinal tissues. In the acquired form, 75% of the females and 50% of the males presented unilateral ocular cysts both at 21 and 47 days post-infection. It was also demonstrated that S-antigen was not uveitogenic in the C. callosus model. No lesion was observed in the animals exclusively immunized with this retinal component, even when jacalin was used as additional adjuvant for polyclonal response to the retinal antigen. It can be concluded that C. callosus may constitute in a promising model for study both acquired and congenital ocular toxoplasmosis, particularly when it is important to make sure that a non autoimmune process is involved in the genesis of the ocular infection.
Memórias do Instituto Oswaldo Cruz 94(1):103-14. · 2.15 Impact Factor
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ABSTRACT: An enzyme-linked immunosorbent assay (ELISA) has been developed to detect IgG and IgM antibodies in human sera against a synthetic tripeptide derived from a hybrid peptide containing 3 specific epitopes from Trypanosoma cruzi. This assay was compared in Brazil with one using conventional antigen, the alkaline crude extract. Serum samples were divided into positive (40 samples) or negative (107 samples) for Chagas disease. Positive samples included 9 serum samples from patients with acute Chagas disease, while negative samples included 57 samples from patients suffering from viral diseases. The total percentages of IgG positive samples from patients with chronic Chagas disease for alkaline extract and synthetic tripeptide were 93.5% and 100%, respectively. All samples from patients with acute Chagas disease were confirmed positive for IgM antibodies by using both the tripeptide and the alkaline extract. However, the results for anti-T. cruzi IgM in the group of chronic Chagas disease patients demonstrated that 41.9% were positive for IgM with the alkaline extract, while the synthetic peptide showed a significantly lower number of positive samples (12.9%). The serum samples from healthy people showed similar results for both antigens. However, 40% of the serum samples from patients presenting with viral diseases were IgM positive for Chagas disease when assayed with conventional antigen; with the synthetic tripeptide as antigen, 100% of this group of samples were found to be negative. Thus, as the results of ELISA with synthetic tripeptide showed higher rates of sensitivity and specificity than ELISA with conventional antigen, the former should be included as a laboratory tool in the serodiagnosis of Chagas disease.
Transactions of the Royal Society of Tropical Medicine and Hygiene 93(6):603-6. · 2.16 Impact Factor
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ABSTRACT: Active infection by T. gondii was evaluated by immunoassay for soluble SAG-1 (p30), the major surface antigen from T. gondii, specific antibodies and immune complexes in human cerebrospinal fluid (CSF) samples. A total of 263 samples of CSF were collected from hospitalized patients presenting neurological disorders and analyzed for antibodies to HIV. Patients were divided into two groups: HIV positive (n = 96) or HIV negative (n =167). The results of the assays showed that 45% of all samples were positive for soluble SAG-1. Toxoplasma Ag/Ab immune complexes were detected in 19% of the CSF samples and 62% were positive for T. gondii- specific IgG. A combination of these assays in the presence of clinical findings consistent with active Toxoplasma infection may predict the presence of toxoplasmic encephalitis. Moreover, detection of soluble SAG-1 in the CSF of these individuals appears consistent with active infection.
Revista do Instituto de Medicina Tropical de São Paulo 41(6):329-38. · 1.00 Impact Factor
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ABSTRACT: Levels of total and specific anti-Trypanosoma cruzi immunoglobulin E (IgE) were determined by immunoenzymatic assay among 101 samples of pericardial fluid from patients who had died in one trypanosomiasis endemic area in central Brazil. These samples were divided into 6 groups. Group I, 17 samples from patients with the cardiac form of Chagas disease; group II, 11 samples from patients with the digestive form of Chagas disease, presenting megaoesophagus and/or megacolon; group III, 41 samples from patients with the indeterminate form of Chagas disease; group IV, 4 samples from patients with both cardiac and digestive forms of Chagas disease; group V, 5 samples from patients who suddenly died and were seropositive for T. cruzi antibodies; group VI, 23 samples, used as a control group, which came from patients seronegative for T. cruzi antibodies. Significantly high levels of total IgE were observed in groups I, II, III, IV and V when compared with group VI (mean concentrations 708-1157 iu/mL compared with 394 iu/mL). In groups I-V, 32 samples (41%) had specific anti-T. cruzi IgE antibodies. The individual percentage positivity rates in these groups were 64.7% (group I), 45.4% (group II), 34.1% (group III), nil (group IV), and 40.0% (group V). A significant correlation between total IgE and specific anti-T. cruzi IgE was observed only in the samples from patients with the cardiac form of Chagas disease (group I).
Transactions of the Royal Society of Tropical Medicine and Hygiene 90(5):578-81. · 2.16 Impact Factor
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ABSTRACT: Calomys callosus, Rengger 1830 (Rodentia, Cricetidae), a wild rodent found in Central Brazil, was studied to investigate its susceptibility to Toxoplasma gondii experimental infection and its humoral immune response against this protozoa. The electrophoretic profile of the serum proteins of C. callosus showed that IgG, which shows no affinity to Protein A, has higher cross reactivity with rat IgG than with IgG from other rodents. The susceptibility assay was performed by inoculation groups of animals with various suspensions of T. gondii tachyzoites from 10(2) to 10(6) parasites. All animals died between 3 and 9 days after infection and the kinetics of antibody synthesis was determined. Basically, they recognized predominantly the immunodominant antigen SAG-1 (P30). The immunohistochemistry assays revealed that the liver was the most heavily infected organ, followed by the spleen, lungs, intestine, brain and kidneys. It can be concluded that C. callosus is an excellent experimental model for acute phase of Toxoplasma infection.
Memórias do Instituto Oswaldo Cruz 93(1):103-7. · 2.15 Impact Factor
