Electrophoretic analysis (sds-page) of canine urinary proteins according to the stage of chronic kidney disease / [Análise eletroforética (SDS-PAGE) de proteínas urinárias de cães de acordo com o estágio da doença renal crônica]

Glomerular proteinuria is characterized by the loss of high-molecular-weight proteins (HMWPs), while tubulointerstitial proteinuria is characterized by the loss of low-molecular-weight proteins (LMWPs). The objective was to assess the molecular weight of urinary proteins (MWUP) in dogs with naturally acquired CKD and determine the proportion of HMWPs and LMWPs according to CKD stage. Twenty-eight dogs with CKD were recruited and divided into 4 groups based on serum creatinine (Cr) levels (group1: Cr<1,4, n=8; group2: 1,45,0, n=5). The control group consisted of 5 healthy dogs. The MWUP was determined by SDS-PAGE. The urinary protein-to-creatinine ratio (UP/C) was used to quantitatively assess proteinuria. The electrophoresis pattern revealed a proportionally greater loss of HMWPthan of LMWP in all groups with CKD and an increased loss of LMWP in group 4 (P<0.05). These results suggest a predominance of glomerular injuries throughout all stages of CKD in these dogs and an increase in tubulointerstitial injury towards the end-stage of the disease. The results of the present study support the recommendation of SDS-PAGE as an effective technique for the qualitative assessment of proteinuria, as well as a method for assessing the severity and location of renal injury.(AU)

A proteinúria glomerular é caracterizada pela perda de proteínas de alto peso molecular (PAPM), enquanto a proteinúria tubulointersticial se caracteriza pela perda de proteínas de baixo peso molecular (PBPM). O objetivo do trabalho foi determinar o peso molecular das proteínas urinárias (PMPU) de cães com DRC naturalmente adquirida e a proporção de PAPM e PBPM de acordo com o estágio da DRC. Foram utilizados 28 cães com DRC, divididos em quatro grupos, de acordo com o nível sérico de creatinina (cr) (grupo 1: cr<1,4, n=8; grupo 2: 1,45,0, n=5). O grupo controle era composto por cinco cães saudáveis. O PMPU foi determinado por SDS-PAGE. A relação proteína:creatinina urinária (RPCU) foi utilizada como um método quantitativo de proteinúria. A eletroforese revelou uma perda proporcionalmente maior de PAPM, quando comparada às PBPM, em todos os grupos de DRC, bem como uma perda crescente de PBPM no grupo 4 (P<0,05). Esses resultados sugerem uma predominância de lesão glomerular em todos os estágios de DRC nesses cães e uma progressão crescente na lesão túbulo-intersticial no estágio terminal da doença. Os resultados deste estudo reafirmam a recomendação de que a eletroforese de proteínas urinárias é uma técnica qualitativa efetiva de avaliação da proteinúria, bem como um método que permite avaliar a extensão e a localização da lesão renal.(AU)

Epicutaneous sensitization with nematode antigens of fish parasites results in the production of specific IgG and IgE.

Fish consumption plays an important role in the human diet. Hoplias malabaricus, trahira, is a freshwater fish widely appreciated in several Brazilian states and it is frequently infected by Contracaecum multipapillatum third-instar larvae (L3). The aim of the present study was to evaluate the allergenic potential of the C. multipapillatum L3 crude extract (CECM). BALB/c mice were immunized intraperitoneally (ip) with 10 or 50 µg CECM associated with 2 mg of aluminium hydroxide on days 0, 14 and 48. The determination of specific IgG and IgE antibody levels was done after immunization, and the late immunity was evaluated by the intradermal reaction in the ear pavilion. Epicutaneous sensitization was performed in the dorsal region, with antigenic exposure via a Finn-type chamber, containing 100 µg of chicken ovum albumin (OVA) or 100 µg CECM. After the exposures, the specific antibody levels were determined. In the ip immunization, there was a gradual increase in IgG antibody levels, independent of CECM concentration. In relation to IgE production, it was transitory, and immunization with 10 µg was more efficient than that of 50 µg. The same result was observed in the cellular hypersensitivity reaction. In the case of antigen exposure by the epicutaneous route, it was verified that only CECM was able to induce detectable levels of specific IgG and IgE antibodies. In the present study it was demonstrated that both intraperitoneal immunization and epicutaneous contact with C. multipapillatum larval antigens are potentially capable of inducing allergic sensitization in mice.

Resposta imunológica a antígenos de Hysterothylacium deardorffoverstreetorum de peixes teleósteos / Imunne response against Hysterothylacium deardorffoverestreetorum from teleost fish

Anisaquidose é uma doença provocada por parasitos da família Anisakidae e se caracteriza por manifestações gastrointestinais e alérgicas. O Anisakis simplex é o parasito mais patogênico ao homem e altamente alergênico. Porém, outros anisaquídeos também são danosos aos humanos, mas é desconhecida a imunogenicidade dessas larvas. O objetivo deste trabalho foi avaliar o potencial imunogênico do parasito Hysterothylacium deardorffoverestreetorum (HD) em modelo murino. Camundongos da linhagem BALB/c foram divididos em três grupos experimentais e receberam as preparações antigênicas obtidas de larvas de HD: extrato bruto de larvas (EBH), extrato secretado/ excretado de larvas (ESH) e extrato bruto de larvas após excreção/secreção (EEH). Amostras séricas foram obtidas em diferentes dias após imunização para determinação dos níveis de anticorpos específicos pelo ensaio imunoenzimático (ELISA). Os resultados demonstram aumento na produção de imunoglobulina (Ig) G após a segunda imunização, com aumento progressivo após a terceira imunização. Já em relação à IgE, a reatividade foi mais tardia, demonstrando aumento progressivo após a terceira imunização. Foi avaliada a imunidade celular por meio da intradermorreação, como resultado estatisticamente significativo em relação ao controle utilizado. Este experimento é a primeira descrição da potencialidade patogênica desse parasito em mamíferos e representa um avanço no diagnóstico da anisaquidose humana.(AU)

Anisaquidosis is a disease caused by parasites of Anisakidae family and is characterized by gastrointestinal and allergic reactions. The Anisakis simplex is a more pathogenic Anisakidae to humans and is highly allergenic. However, other species of this family also have characteristics that are harmful to humans, but little is known about the immunogenicity this parasites. The objective of this study was to experimentally assess the immunogenic potential of the parasite Hysterothylacium deardorffoverestreetorum (H.D) in mice. Mice of inbred BALB/c strain were divided into three groups and received three immunizations of the following antigenic preparations obtained from L3 larvae H.D: Crude larval extract of H.D (CEH) Extract secreted / excreted larvae H.D. (ESH) and crude extract of larvae after excretion / secretion (EEH). Serum samples were obtained on different days after immunization to determine the levels of circulating specific antibodies by enzyme-linked immunosorbent assay (ELISA). The results show increased production of immunoglobulin (Ig) G after the second immunization with a gradual increase after the third immunization. Regarding IgE reactivity, this occurred later, demonstrating a progressive increase only after the third immunization. Cellular immunity was evaluated by intradermal, and showed statistically significant result compared to the control used. This experiment is the first description of the pathogenic potential of this parasite in mammals and represents a breakthrough in the diagnosis of human Anisakidosis.(AU)

Immunogenic activity of the fish tapeworm Pterobothrium heteracanthum (Trypanorhyncha: Pterobothriidae) in BALB/c mice.

The aim of this study was to verify the immunogenicity of Pterobothrium heteracanthum (Cestoda: Trypanorhyncha) crude protein extract (PH-CPE) in BALB/c mice. The parasites were obtained from Micropogonias furnieri (Osteichthyes: Sciaenidae). Groups of six mice were each immunized with 10, 50 or 100 µg of PH-CPE, on days 0 and 35. Both specific IgG and IgE responses were developed after immunization. The immunoblot assay revealed that specific IgG recognizes PH-CPE proteins with two molecular weight ranges, 60-75 and 30-40 kDa, and that IgE recognizes larger proteins over 120 kDa. This appears to be the first report on the immunogenicity of metacestodes within the Pterobothriidae and that PH-CPE is a potential inducer of a specific IgE response.

Experimental anisakid infections in mice.

Anisakidosis is a human parasitic disease caused by infections with members of the Anisakidae family. Accidental infection after fish intake affects the gastrointestinal tract as a consequence of mechanical damage caused by migrating larvae. Infections can also trigger allergies, hives, severe asthma or anaphylaxis with angioedema. Although mouse models of intraperitoneal antigenic stimulation exist, enabling immunological studies, few models using gastric introduction of live larvae are available for the study of immunological and gastrointestinal damage in mice. This study was designed to characterize serum reactivity against Anisakis spp. and Contracaecum spp. in Balb/c mice following orogastric inoculation and to assess gastrointestinal damage. These anisakid species were classified at the Universidade Federal Fluminense (UFF) School of Veterinary Medicine and materials for live larval inoculation were developed at the UFF Immunobiology laboratory. Live larvae were inoculated following injection with a NaCl solution. Blood samples were collected and sera screened for immunoglobulin (Ig)E and IgG anti-larva responses to both nematodes, specific for somatic and excretory/secretory antigens, by enzyme-linked immunosorbent assay (ELISA). The means of the optical densities were analysed using analysis of variance (ANOVA) with Tukey's post-hoc test and the general linear model. This analysis identified the presence of anti-IgG seroreactivity to both somatic and excretory/secretory Anisakis antigens in inoculated animals compared with controls (P< 0.001), and no gastric or intestinal damage was observed. These experiments demonstrated that introduction of live Contracaecum spp. into the gastrointestinal tract did not elicit serum sensitization in animals.

Mapping IgE-binding epitopes of Ric c 1 and Ric c 3, allergens from Ricinus communis, by mast cell degranulation assay.

Ric c 1 and Ric c 3 are the major castor bean allergens. In order to identify continuous IgE-epitopes in Ric c 1 and Ric c 3, pools of sera from rats immunized with a pool of 2S albumin from these seeds, Ric c 1 and Ric c 3 overlapping synthetic peptides, were used to screen for IgE-binding epitopes. The allergenic properties were monitored by mast cell degranulation assays, histamine quantification and human-IgE binding. Large and small chains isolated from these proteins present allergenic properties. Four continuous epitopes were identified in Ric c 3 and two in Ric c 1. This knowledge may allow the induction of protective antibody responses to antagonize the IgE recognition.

On the hsp26 of Saccharomyces cerevisiae.

The function of the small size hsps in Saccharomyces cerevisiae has yet to be convincingly established. In this paper we present some aspects of the physiology of hsp26. Several mutant strains were analyzed with respect to the expression of the HSP26 gene using anti-hsp26 antibody for identification. The bcy1 mutant which lacks the regulating subunit of protein kinase A failed to produce full expression of HSP26 under heat shock whereas a ras2 mutation which lowers significantly the level of cAMP, produced no detectable effect. During normal growth hsp26 protein is induced during diauxie and its synthesis continues during the second exponential phase. Both BCY1 and CYR1 genes seen to be required for induction during the transition phase albeit not directly but rather interacting with some other regulatory component. The structure of hsp26 is discussed by homology with other small hsps.

A study of the adjuvant effect of nigrosin on antibody synthesis and phagocytic activity of peritoneal macrophages of inbred mice.

The dye nigrosin has an adjuvant effect on the synthesis of specific IgE and hemagglutinating antibodies in B6D2F1 mice. Phagocytosis of opsonized sheep red blood cells by peritoneal macrophages was enhanced by treatment with the dye, indicating a possible increase in the expression of Fc and C3b receptors of macrophages.