Secretory IgA, a major immunoglobulin in most bovine external secretions.

Bovine secretory IgA (SIgA), recently identified in colostrum, was shown to be homologous to human SIgA by immunologic cross-reaction. A quantitative study indicated that bovine SIgA, a minor component of colostrum, is a major immunoglobulin in most other external secretions including saliva, spermatic fluid, lacrimal, nasal and gastrointestinal secretions. SIgA was isolated from saliva. The free form of secretory component was found to be abundant in milk. A normal lactating cow produces about 1.2 g of this protein per day. Two forms of IgA were identified in serum: a normal serum IgA with no secretory antigenic determinant, and a small amount of SIgA. In vitro synthesis of SIgA by the salivary gland was studied by tissue cultures with incorporation of labeled amino acids.

Detection and coat protein gene characterization of an isolate of Grapevine virus B from corky bark-affected grapevines in Southern Brazil

An isolate of Grapevine virus B (GVB), obtained by indexing Vitis labrusca and V. vinifera grapevines on the indicator LN33, was transmitted mechanically to several Nicotiana species. The virus was partially purified from N. cavicola and the coat protein estimated at 23 kDa by SDS-PAGE. In negatively stained leaf extracts of experimentally inoculated N. cavicola and N. occidentalis, flexuous particles with cross banding were observed, predominantly measuring 750-770 x 12 nm, with a modal length of 760 nm. Decoration indicated a clear, positive reaction against AS-GVB. In DAS-ELISA, GVB was detected in N. cavicola and grapevine extracts, and Western blots showed homologous and cross reaction of GVB and GVA antisera with GVB coat protein. Using specific primers for GVB, a fragment of 594 bp, comprising the coat protein gene coding for 197 amino acids, was amplified by RT-PCR with viral RNA extracted from GVB-infected N. occidentalis. The nucleotide and the deduced amino acid sequences of the coat protein gene showed high identities with Italian and Japanese isolates of GVB.

Distinct features of Pepper yellow mosaic virus isolates from tomato and sweetpepper

Determination of virus diversity in the field is vital to support a sustainable breeding program for virus resistance of horticultural crops. The present study aimed to characterize four field potyvirus isolates found naturally infecting sweet pepper (Capsicum annuum) (Sa66 and Sa115) and tomato (Lycopersicon esculentum) (IAC3 and Sa21) plants. Their biological characteristics revealed differences among the isolates in their ability to infect distinct Capsicum spp. and tomato genotypes, and in the severity of symptoms caused by these isolates compared to the infection caused by an isolate of Pepper yellow mosaic virus (PepYMV). Absence of cross-reaction was found among the studied isolates with antiserum against Potato virus Y (PVY). However, all isolates reacted, at different intensities, with antiserum against PepYMV. All isolates showed high identity percentage (97 to 99%) of the amino acid sequence of the coat protein with PepYMV (accession AF348610) and low (69 to 80%) with other potyvirus species. The comparison of the 3' untranslated region also confirmed this finding with 97 to 98% identity with PepYMV, and of 47 to 71% with other potyviruses. The results showed that PepYMV isolates were easily differentiated from PVY by serology and that the host response of each isolate could be variable. In addition, the nucleotide sequence of the coat protein and 3' untranslated region was highly conserved among the isolates.

Decorin is one of the proteoglycans expressed in Walker 256 rat mammary carcinoma

Proteoglycan and glycosaminoglycan content was analyzed in a model of rat mammary carcinoma to study the roles of these compounds in tumorigenesis. Hyaluronic acid and proteoglycans bearing chondroitin and/or dermatan sulfate chains were detected in solid tumors obtained after subcutaneous inoculation of Walker 256 rat carcinoma cells. About 10% of sulfated glycosaminoglycan chains corresponded to heparan sulfate. The small leucine-rich proteoglycan, decorin, was identified as one of the proteoglycans, in addition to others of higher molecular weight, by cross-reaction with an antiserum raised against pig laryngeal decorin and by N-terminal amino acid sequencing. Decorin was separated from other proteoglycans by hydrophobic chromatography and its complete structure was determined. It has a molecular weight of about 85 kDa and a dermatan chain of 45 kDa with 4-sulfated disaccharides. After degradation of the glycosaminoglycan chain, three core proteins of different molecular weight (36, 46 and 56 kDa) were identified. The presence of hyaluronic acid and decorin has been reported in a variety of tumors and tumor cells. In the Walker 256 mammary carcinoma model, hyaluronic acid may play an important role in tumor progression, since it provides a more hydrated extracellular matrix. On the other hand, decorin, which is expressed by stromal cells, represents a host defense response to tumor growth.

Leishmania infantum heat shock protein 83 for the serodiagnosis of tegumentary leishmaniasis

The serologic assay is an important tool in the diagnosis of leishmaniasis. One of the most commonly used tests is enzyme-linked immunosorbent assay (ELISA). Since total Leishmania promastigotes are used as antigen in the routine assay, false-positive reactions are frequent due to cross-reaction with sera from other diseases, mainly Chagas' disease. Therefore, an antigen that determines less cross-reactivity has been pursued for the serodiagnosis of leishmaniasis. In the present study we analyzed the use of recombinant Leishmania infantum heat shock protein (Hsp) 83 in ELISA for the serodiagnosis of cutaneous (N = 12) and mucocutaneous leishmaniasis (N = 14) and we observed the presence of anti-L. infantum Hsp 83 antibodies in all samples as well as anti-Leishmania total antigen antibodies. When cross-reactivity was tested, chronic Chagas' disease patients (N = 10) did not show any reactivity. Therefore, we consider this L. infantum Hsp 83 to be a good antigen for routine use for serodiagnosis of tegumentary leishmaniasis.

Autoimmunity and molecular mimicry in tropical spastic paraparesis/human T-lymphotropic virus-associated myelopathy

Viruses share antigenic sites with normal host cell components, a phenomenon known as molecular mimicry. It has long been suggested that viral infections might trigger an autoimmune response by several mechanisms including molecular mimicry. More than 600 antiviral monoclonal antibodies generated against 11 different viruses have been reported to react with 3.5% of cells specific for uninfected mouse organs. The main pathological feature of tropical spastic paraparesis/human T-lymphotropic virus type I (HTLV-I)-associated myelopathy (TSP/HAM) is a chronic inflammation of the spinal cord characterized by perivascular cuffing of mononuclear cells accompanied by parenchymal lymphocytic infiltration. We detected the presence of autoantibodies against a 98- to 100-kDa protein of in vitro cultured human astrocytes and a 33- to 35-kDa protein from normal human brain in the serum of HTLV-I-seropositive individuals. The two cell proteins exhibited molecular mimicry with HTLV-I gag and tax proteins in TSP/HAM patients, respectively. Furthermore, the location of 33- to 35-kDa protein cross-reaction correlated with the anatomical spinal cord areas (in the rat model) in which axonal damage has been reported in several cases of TSP/HAM patients. Our experimental evidence strongly suggests that the demyelinating process occurring in TSP/HAM may be mediated by molecular mimicry between domains of some viral proteins and normal cellular targets of the spinal cord sections involved in the neurodegeneration.

Molecular tools to investigate Rhizoctonia solani distribution in soil

Real-time PCR protocols were developed to detect and discriminate 11 anastomosis groups (AGs) of Rhizoctonia solani using ribosomal internal transcribed spacer (ITS) regions (AG-1-IA, AG-1-IC, AG-2-1, AG-2-2, AG-4HGI+II, AG-4HGIII, AG-8) or beta-tubulin (AG-3, AG-4HGII, AG-5 and AG-9) sequences. All real-time assays were target group specific, except AG-2-2, which showed a weak cross-reaction with AG-2tabac. In addition, methods were developed for the high throughput extraction of DNA from soil and compost samples. The DNA extraction method was used with the AG-2-1 assay and shown to be quantitative with a detection threshold of 10-7 g of R. solani per g of soil. A similar DNA extraction efficiency was observed for samples from three contrasting soil types. The developed methods were then used to investigate the spatial distribution of R. solani AG-2-1 in field soils. Soil from shallow depths of a field planted with Brassica oleracea tested positive for R. solani AG-2-1 more frequently than soil collected from greater depths. Quantification of R. solani inoculum in field samples proved challenging due to low levels of inoculum in naturally occurring soils. The potential uses of real-time PCR and DNA extraction protocols to investigate the epidemiology of R. solani are discussed.

Quantitation of apolipoprotein B-48 in triacylglycerol-rich lipoproteins by a specific enzyme-linked immunosorbent assay

This paper describes the use of an antiserum, specific for apolipoprotein (apo) B-48, in a competitive, enzyme-linked immunosorbent assay (ELISA) for apo B-48 in triacylglycerol-rich lipoprotein (TRL) fractions prepared from fasting and post-prandial plasma samples. Previously we showed the antiserum to act as an effective immunoblotting agent following sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Its use in this ELISA indicates that the antiserum recognises the C-terminal region of the protein on the surface of lipoprotein particles. The ELISA had a sensitivity of less than 37 ng/ml and intra- and inter-assay coefficients of variation of 3.8% and 8.6%, respectively. There was no cross-reaction in the ELISA against serum albumin, ovalbumin, thyroglobulin, or apo B-100 (purified by immunoaffinity chromatography), and high lipid concentrations (as Intralipid) did not interfere. A low density lipoprotein fraction reacted in the ELISA but SDS-PAGE-Western blot analysis confirmed the presence, in the fraction, of a small amount of apo B-48, indicating the existence of low density dietary-derived lipoprotein particles. ELISA and SDS-PAGE-Western blot analysis were used to measure apo B-48 in 12 series of postprandial samples collected from 4 diabetic and 8 normal subjects, following test meals of varying fat content. The mean correlation between the two methods was r = 0.74. The mean fasting concentration of apo B-48 in the TRL fractions from 15 healthy men was 0.46 μg/ml of plasma.

Anticorpos anti-trypanosoma cruzi como preditivos de infecção por leishmania infantum

Leishmania infantum and Trypanosoma cruzi are trypanosomatids of medical importance and are, respectively, the etiologic agents of visceral leishmaniasis (VL) and Chagas disease (CD) in Brazil. People infected with L. infantum or T. cruzi may develop asymptomatically, enabling the transmission of pathogens through blood transfusion and / or organs. The assessment of the infection by T. cruzi is included among the tests performed for screening blood donors in Brazil, however, there is no availability of tests for Leishmania. Serological tests for T. cruzi are very sensitive, but not specific, and may have cross-reactions with other microorganisms. Thus, the aim of this study was to determine the prevalence of Leishmania infection in blood donors and assess whether the serological test for T. cruzi detect L. infantum. Among the 300 blood samples from donors, discarded in 2011, 61 were T. cruzi positive, 203 were from donors with other infections and 36 were from handbags with low blood volume, but without infection. We also assessed 144 samples from donors without infections and able to donate blood, totaling 444 subjects. DNA was extracted from blood samples of all to perform quantitative PCR (qPCR) to detect Leishmania DNA. The buffy coat obtained from all samples was grown in Schneider medium supplemented and NNN. All samples were evaluated for the presence of anti-Leishmania antibody. The serological results indicate a percentage of 22% of Leishmania infection in blood samples obtained from discarded bags. A total of 60% of samples positive in ELISA for T. cruzi were negative by IFI, used as confirmatory test, ie 60% false positive for Chagas. Among these samples false positive for Chagas, 72% were positive by ELISA for Leishmania characterizing the occurrence of cross reaction between serologic assays. Of the 300 cultures performed, 18 grew parasites that were typed by qPCR and specific isoenzymes, found the species Leishmania infantum crops. Among the 18 cultures, 4 were purged from scholarships for low volume and all negative serology blood bank, thus demonstrating that there is a real risk of Leishmania transmission via transfusion. It is concluded that in an area endemic for leishmaniasis in Brazil, serological diagnosis performed to detect infection by T. cruzi among blood donors can identify infection by L. infantum and although cause false positive for Chagas, this cross-reactivity reduces the risk of Leishmania infection via blood transfusion, since tests are not applied specific detection of the parasite. In this way, there remains the need to discuss the implementation of a specific serological screening test for Leishmania in endemic countries such as Brazil

Isolation and immunophenotypic characterization of mesenchymal stem cells derived from equine species adipose tissue

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

Visual detection of turkey coronavirus RNA in tissues and feces by reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with hydroxynaphthol blue dye

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

Use of crude, FML and rK39 antigens in ELISA to detect anti-Leishmania spp. antibodies in Felis catus

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

Structural and functional characterization of an acidic platelet aggregation inhibitor and hypotensive phospholipase A(2) from Bothrops jararacussu snake venom

An acidic (pI similar to 4.5) phospholipase A(2) (BthA-I-PLA(2)) was isolated from Bothrops jararacussu snake venom by ion-exchange chromatography on a CM-Sepharose column followed by reverse phase chromatography on an RP-HPLC C-18 column. It is an similar to13.7 kDa single chain Asp49 PLA(2) with approximately 122 amino acid residues, 7 disulfide bridges, and the following N-terminal sequence: 'SLWQFGKMINYVMJGESGVLQYLSYGCYCGLGGQGQPTDATDRCCFVHDCC(51). Crystals of this acidic protein diffracted beyond 2.0 Angstrom resolution. These crystals are monoclinic and have unit cell dimensions of a = 33.9, b = 63.8, c = 49.1 Angstrom, and beta = 104.0degrees. Although not myotoxic, cytotoxic, or lethal, the protein was catalytically 3-4 tithes more active than BthTX-II, a basic D49 myotoxic PLA(2) from the same venom and other Bothrops venoms. Although it showed no toxic activity, it was able to induce time-independent edema, this activity being inhibited by EDTA. In addition, BthA-I-PLA(2) caused a hypotensive response in the rat and inhibited platelet aggregation, Catalytic, antiplatelet and other activities were abolished by chemical modification with 4-bromophenacyl bromide, which is known to covalently bind to His48 of the catalytic site. Antibodies raised against crude B. jararacussu venom recognized this acidic PLA(2), while anti-Asp49-BthTX-II recognized it weakly and anti-Lys49-BthTX-I showed the least cross-reaction. These data confirm that myotoxicity does not necessarily correlate with catalytic activity in native PLA(2) homologues and that either of these two activities may exist alone. BthA-I-PLA(2), in addition to representing a relevant molecular model of catalytic activity, is also a promising hypotensive agent and platelet aggregation inhibitor for further studies. (C) 2002 Elsevier B.V. All rights reserved.

Interference of vaccinal antibodies on serological diagnosis of leptospirosis in vaccinated buffalo using two types of commercial vaccines

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

Variations in the sensitivity of different primers for detecting Wolbachia in Anastrepha (diptera: tephritidae)

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