Avaliação da absorção colostral em neonatos ovinos da raça Bergamácia / Evaluation of colostral absorption in neonates of Bergamacia breed

Objetivou-se determinar o período de absorção das macromoléculas colostrais e a transferência de imunidade passiva em cordeiros da raça Bergamácia. Avaliou-se o proteinograma sérico dos cordeiros antes da ingestão de colostro até 48 horas de vida e das frações colostrais ao nascimento e 12 horas pós-parto. Foi avaliada a concentração de proteína total no soro por refratometria e sua relação com a densidade e quantidade de gamaglobulinas presentes no colostro. A concentração sérica de gamaglobulina nos cordeiros variou de 0,111±0,07g/dL antes da ingestão de colostro a 1,609±0,72g/dL às 48 horas. Nas amostras de colostro, a concentração variou de 3,125±1,27g/dL, imediatamente após o parto, para 1,378±0,82g/dL, 12 horas após. A concentração de proteína sérica total teve acréscimo de 4,46±0,58g/dL para 5,61±0,75g/dL entre o nascimento e após 48 horas, apresentando correlação positiva com a densidade e a proteína total colostral. A absorção colostral pelo cordeiro foi ascendente até 24 horas subsequentes ao parto, quando, então, iniciou-se sua estabilização. A quantificação da proteína sérica, com uso de refratômetro nos cordeiros, pode ser usada como método para avaliar a transferência de imunidade passiva, pois está diretamente relacionada com a absorção de gamaglobulina colostral...

The period of absorption of colostrum macromolecules and the passive immunity transfer of Bergamacia lambs was determined. The serum proteinogram of lambs before the intake of colostrum up 48 hours of life and colostrum fractions between delivery and twelve hours after birth were measured. The total protein concentration in serum was evaluated by refractometry and also its relationship with the density and amount in colostrum. The serum concentration of gamma globulin in lambs from 0.111±0.07g/dL before the intake of colostrum was 1.609±0.72g/dL at 48 hours. In the colostrum samples, the concentration was 3.125±1.27g/dL immediately after delivery and 1.378±0.82g/dL twelve hours after birth. Serum total protein concentration increased from 4.46±0.58g/dL to 5.61±0.75g/dL between birth and after 48 hours, there was positive correlation with the density and total protein colostrum. The lamb had ascendant colostrum absorption subsequent to delivery, for twelve hours and then began its stabilization. The quantification of serum protein with the use of the refractometer in lambs can be used as a method to evaluate the transfer of passive immunity because it is directly related to the absorption of colostral gammaglobulin...

Durable pharmacological responses from the peptide ShK-186, a specific Kv1.3 channel inhibitor that suppresses T cell mediators of autoimmune disease.

The Kv1.3 channel is a recognized target for pharmaceutical development to treat autoimmune diseases and organ rejection. ShK-186, a specific peptide inhibitor of Kv1.3, has shown promise in animal models of multiple sclerosis and rheumatoid arthritis. Here, we describe the pharmacokinetic-pharmacodynamic relationship for ShK-186 in rats and monkeys. The pharmacokinetic profile of ShK-186 was evaluated with a validated high-performance liquid chromatography-tandem mass spectrometry method to measure the peptide's concentration in plasma. These results were compared with single-photon emission computed tomography/computed tomography data collected with an ¹¹¹In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-conjugate of ShK-186 to assess whole-blood pharmacokinetic parameters as well as the peptide's absorption, distribution, and excretion. Analysis of these data support a model wherein ShK-186 is absorbed slowly from the injection site, resulting in blood concentrations above the Kv1.3 channel-blocking IC50 value for up to 7 days in monkeys. Pharmacodynamic studies on human peripheral blood mononuclear cells showed that brief exposure to ShK-186 resulted in sustained suppression of cytokine responses and may contribute to prolonged drug effects. In delayed-type hypersensitivity, chronic relapsing-remitting experimental autoimmune encephalomyelitis, and pristane-induced arthritis rat models, a single dose of ShK-186 every 2 to 5 days was as effective as daily administration. ShK-186's slow distribution from the injection site and its long residence time on the Kv1.3 channel contribute to the prolonged therapeutic effect of ShK-186 in animal models of autoimmune disease.

Specific antibody modulates absorptive transport and metabolic activation of benzo[a]pyrene across Caco-2 monolayers.

It has been shown that oral anticarcinogen antibodies can decrease intestinal absorption of procarcinogens. So far, no attempts have been made to understand the potential modulatory effect of such antibodies on metabolic activation at mucosal surfaces. Moreover, the influence of naturally induced serosal-specific antibodies on absorptive transport of carcinogens remains unknown. In this study, the prototype food carcinogen benzo[a]pyrene (B[a]P) and a specific monoclonal antibody were used to address these questions in a bicompartmental model of polarized intestinal cells (Caco-2). Apical (i.e., luminal) administration of a 30-fold molar excess antibodies increased about 25-fold recovery of unmetabolized B[a]P, concomitantly with a decrease of 80% in both absorptive transport and formation of phenol metabolites. Interestingly, when metabolism was slowed down by antibodies, cross-reactive antibodies also increased at least 5-fold the extracellular levels of the 7,8-diol-B[a]P, interrupting subsequent activation steps. On the other hand, basolateral antibodies changed by 8-fold the rate of carcinogen appearance in the basolateral compartment, albeit without interfering with the apical cellular uptake or sequestration of either B[a]P or 7,8-diol-B[a]P by apical antibodies. Furthermore, basolateral antibodies reduced exposure of Caco-2 monolayers to B[a]P as demonstrated by a 50% decrease in apical efflux of 3-OH-B[a]P. These data show for the first time that both luminal and serosal antibodies may reduce the carcinogenic process by preventing high local concentrations, which would overload DNA repair mechanisms. This study also sheds light on the relevance of both naturally induced and immunoprophylactic antibodies against polycyclic aromatic hydrocarbon carcinogens.

Determinacion de los niveles de contaminacion por Mercurio en la especie Salmo Gairdnerie Irideus y el cuerpo de agua de la Laguna Suches adyacentes al Lago Titicaca del Departamento de La Paz / Determination of the levels of contamination by mercury in the species Salmo Gairdnerie Irideus and the water body of the adjacent Suches lagoon to the titicaca lake of the department of La Paz

En el presente estudio se determino la presencia de mercurio en la especie Salmo gairdnerie irideuis (trucha) y en aguas de la Laguna Suches afluente al lago Titicaca, la cual esta situada en la reserva Ulla-Ulla. La evaluacion se realizo mediante el metodo de espectrofotometria de Absorcion Atomica por generacion de vapor frio utilizando las tecnicas de oxido, reduccion para la preparacion de muestras. En 30 muestras de peces se determina que el 100 porciento tiene de contaminacion y en 25 muestras de agua de la Laguna Suches se determina que el 100 porciento de las muestras tambien presentan contaminacion. El tamaño muestral esta limitado por la poca accesibilidad de la zona esta circundada por minas pertenecientes al sector privado. La media de contaminacion por mercurio en peces es de 0.104 ppm, en agua corresponde a 0.398 ppb. Estos valores son menores a los valores permisibles que señala la Organizacion Mundial de la Salud que son de 2 ppm. Sin embargo consideramos que los mas importante de esta investigacion no son las concentraciones que se encuentran por debajo del limite permisible, sino el haber encontrado que el equilibrio ecologico de la zona esta en un inicio de contaminacion por un metal altamente toxico como es el mercurio

Binding of a putative and a known chaperone protein revealed by immunogold labeling transmission electron microscopy: A suggested use of chaperones as probes for the distribution of their target proteins.

Parvulins are a distinct family within the peptidyl-prolyl cis-trans isomerase group of proteins that catalyse the cis-trans isomerization of proline-containing peptides. The intracellular distribution of a novel human parvulin homologue (hEPVH) has been investigated in a human kidney cell line (HEK 293) by immunogold labeling transmission electron microscopy (TEM). This showed hEPVH to be distributed throughout HEK 293 cells but in highest concentration within mitochondria. Unexpectedly, preabsorption of anti-hEPVH antiserum with recombinant hEPVH exaggerated the observed immunolabel density in a pattern that mirrored that of the endogenous hEPVH. The hEPVH protein itself was then used to label sections, and the specificity of its binding was confirmed with the use of polyclonal and monoclonal antibodies in conjunction with homologous and irrelevant protein controls. The pattern of hEPVH binding also mirrored that of endogenous hEPVH. A known chaperone protein, Pin1, was also found to bind to cells in a pattern mirroring that of the endogenous protein. This lends considerable weight to our hypothesis that hEPVH is binding to its target protein(s) within the cell, reflecting its postulated chaperone function. Finally, we suggest that chaperone proteins in general might be used as TEM probes for their target (or substrate) proteins. (J Histochem Cytochem 47:1633-1640, 1999)

Identification of a domain in human factor H and factor H-like protein-1 required for the interaction with streptococcal M proteins.

The plasma protein factor H (FH) inhibits the alternative pathway of complement activation. Previous work has shown that FH binds to group A streptococci and that the interaction does not interfere with the complement-inhibitory capacity of FH. In this work, we report a molecular analysis of this interaction. In absorption experiments with human plasma, M protein-expressing group A streptococci bound both FH and FH-like protein-1 (FHL-1), an active 42-kDa splice product of the FH-gene transcript comprising the first 7 of its 20 short consensus repeat (SCR) domains. rFHL-1 also bound to M protein-expressing streptococci, but rFH fragments containing SCR 1-5 or SCR 1-6 did not. rFHL-1 bound to purified M5 protein with an affinity that was higher than the value calculated for the interaction between FH and M5 protein. The binding of radiolabeled rFHL-1 to immobilized M5 was blocked completely by unlabeled rFHL-1, but was inhibited only partially by SCR 1-6, emphasizing the importance of SCR 7 for the interaction. In experiments with the FH-related proteins FHR-3 and FHR-4, only the former bound to M protein-expressing streptococci, again pointing to an involvement of SCR 7, since FHR-3, but not FHR-4, contains a domain that is similar to SCR 7. Finally, the interaction between rFHL-1 and purified M5 protein was inhibited by heparin, which binds FH via SCR 7. Together, these data indicate that the interaction between streptococcal M proteins and FH or FHL-1 requires SCR 7.

Immunoprecipitation of melanogenic enzyme autoantigens with vitiligo sera: evidence for cross-reactive autoantibodies to tyrosinase and tyrosinase-related protein-2 (TRP-2).

In the present study we describe the detection of TRP-2 antibodies in vitiligo patients using in vitro 35S-labelled human TRP-2 in a radioimmunoassay. Of 53 vitiligo sera examined in the assay, three (5 9%) were found to be positive for TRP-2 antibodies. In contrast, 20 control sera, sera from 10 patients with Hashimoto's thyroiditis and sera from 10 patients with Graves' disease were all negative. All three patients positive for TRP-2 antibodies (mean age 54 years, age range 50-63 years) had had vitiligo of the symmetrical type for more than 1 year and all of them also had an associated autoimmune disorder: Graves' disease in one and autoimmune hypothyroidism in two. In addition, antibodies to the melanogenic enzyme tyrosinase were present in their serum. To examine any immunological cross-reactivity between TRP-2 and tyrosinase, the three vitiligo sera positive for TRP-2 antibodies were preabsorbed with COS-7 cell extract containing either expressed TRP-2 or tyrosinase, and subsequently used in the radioimmunoassay. These absorption studies indicated that preincubation with both proteins inhibited the immunoreactivity of the positive sera in the immunoassay using in vitro translated 35S-TRP-2. This antibody cross-reactivity suggests the humoral response to the two melanogenic enzymes in these patients may not be entirely independent.

Characterization of fertilization-blocking monoclonal antibody 1G12 with human sperm-immobilizing activity.

A mouse hybridoma (1G12) producing sperm-immobilizing MoAb to human sperm was established and characterized in order to study the antigens relevant to sperm immobilization by antibodies. MoAb 1G12 had strong sperm-immobilizing and agglutinating activities and also showed a fertilization-blocking activity on in vitro fertilization tests. The antibody absorption experiments showed that MoAb 1G12 reacted not only to ejaculated sperm but also human seminal plasma, suggesting that the corresponding antigen might be a sperm coating antigen. The MoAb also reacted with peripheral blood lymphocytes. In histochemical studies, the epithelia of corpus epididymis were most strongly stained. Ejaculated sperm were stained with a granular pattern for their entire surface by immunofluorescence. MoAb 1G12 recognized polymorphic glycoproteins of 15-25 kD in the ejaculated sperm extract in Western blot analysis. After deglycosilation of the sperm extract, only a single staining band of under 15 kD was detected by MoAb 1G12. This suggests that the antigen epitope recognized by MoAb 1G12 might be a peptide of the core portion of the glycoprotein. MoAb 1G12 might be a useful tool for studying the mechanism of egg-sperm interaction, and also be applied to identifying the corresponding antigen by using gene technology.

Human cytotrophoblastic cells absorb the NK blocking activity of monoclonal BA11.

PROBLEM: R80K is a polymorphic alloantigeneic protein present on human placental trophoblast and on paternal B lymphocytes and monocytes. This protein, unlike the former candidate TLX antigen, stimulates a protective maternal immune response in vivo. A murine monoclonal BA11 antibody, directed against R80K, prevents abortion in three murine pregnancy-failure models and inhibits human and murine NK activity. We attempted to define the target of BA11 in the human NK assay system. METHODS: A CELISA method was used to detect R80K antigen on the surface of different cells using the BA11 antibody. The effect, on human peripheral blood NK activity against K562, by BA11 before and after absorption by different cells, including the K562 target, was determined. RESULTS: R80K was detected on term placental syncytio and cytotrophoblast and on BeWo cells, by CELISA. BA11 suppressed NK lysis of K562 cell sin a dose-dependent manner. Absorption of the BA11 by BeWo and by cytotrophoblastic cells significantly decreased the NK-inhibitory activity. There was minimal absorption by K562 and BA11-pretreateed K562 cells remained susceptible to NK lysis. By contrast, BA11-pretreated peripheral blood cells lost all NK activity. CONCLUSIONS: The inhibition of NK killing of K562 cells by BA11 is more complex than simple masking of a trophoblast cell-associated molecule in K562 necessary for recognition in NK cells.

PeptideYY and insulin coexist in beta-granules in B cells of the Madagascan lizard, Zonosaurus laticaudatus.

PYY, a 36-amino-acid peptide belonging to the PP family, is often colocalized with glucagon in A cells in the gastroenteropancreatic system of vertebrates. However, both immunocytochemical and immunohistochemical methods reveal this peptide in insulin-containing beta-granules in the lizard Zonosaurus laticaudatus. Absorption tests of PYY antiserum with insulin did not abolish the immunostaining of beta-granules with PYY antiserum, ruling out a cross-reaction between the PYY antiserum and insulin. This feature may be a reflection of the persistence of an ontogenetic character or a result of an adaptative mechanism that selectively activates the PP family of genes in the cellular line of B rather than of A cells.

Penner serotyping of Campylobacter isolates from poultry, with absorbed pooled antisera.

The Penner serotyping system, based on detection of heat-stable antigens with a passive haemagglutination technique, was used in studies on Campylobacter epidemiology in poultry. Preparation of specific antisera by absorption allowed the use of pooled antisera. Over 80% of the Campylobacter isolates were typable with this modified Penner serotyping system. Typability of strains was clearly affected by storage of the strains before actual typing. Extracted antigens appeared to be stable for at least 6 months at 4 degrees C. Therefore, it is advisable to store extracted antigens from freshly isolated Campylobacter strains instead of reculturing frozen-stored strains, when actual typing cannot be performed directly after primary isolation. Untypability of isolates may partly be explained by the detection of Campylobacter serovars not yet represented in the serotyping system. Experiments on repeated serotyping of several Campylobacter strains did not suggest any serovar instability within the strains.

Anticuerpos contra una estructura vascular y endocardio en la trypanosomiasis americana / Antibodies against a vascular structure and endocardium american trypanosomiasis

En este trabajo se estudió un grupo de enfermos provenientes de una zona endémica con una miocardiopatía compatible clinicamente con enfermedad de Chagas crónica. El criterio adoptado para el diagnóstico demostró ser útil y puede ser aplicado en trabajos de investigación clínica. Se señala el riesgo del empleo de la serología como elemento diagnóstico de enfermedad. Se describe un nuevo anticuerpo (FAC) que reacciona con endocardio, sustancia conectiva interfibrilar y estructuras vasculares, sin especificidad de especie y con capacidad de fijar complemento. Se demostró que puede pertenecer tanto a la IgM como a la IgG. Algunos hechos sugieren su relación con la infección T. cruzi. Por estudios de absorción de sueros que contenían estos anticuerpos y otros con actividad antitrypanosoma surgen evidencias en favor de la existencia de 2 sistemas inmunológicos: FAC representaría uno de estos sistemas y su especificidad inmunológica estaría vinculado a antígenos presentes solo en el T. cruzi. El FAC tiene una alta prevalencia en la zona endémica estudiada, y no fue encontrado en los controles sanos o enfermos de la ciudad de Buenos Aires. Además dentro de la zona endémica su prevalencia fue significativamente mayor en los enfermos con cardiopatía crónica compatible clinicamente con enfermedad de Chagas que en los controles asintomáticos. Esta asociación sugiere la posibilidad de utilizar este elemento FAC como complemento para el diagnóstico de enfermedad de Chagas y como diagnóstico diferencial con otras miocardiopatías crónicas. Su posible significado patogénico es discutido. (AU)

Anticuerpos contra una estructura vascular y endocardio en la trypanosomiasis americana / Antibodies against a vascular structure and endocardium american trypanosomiasis

En este trabajo se estudió un grupo de enfermos provenientes de una zona endémica con una miocardiopatía compatible clinicamente con enfermedad de Chagas crónica. El criterio adoptado para el diagnóstico demostró ser útil y puede ser aplicado en trabajos de investigación clínica. Se señala el riesgo del empleo de la serología como elemento diagnóstico de enfermedad. Se describe un nuevo anticuerpo (FAC) que reacciona con endocardio, sustancia conectiva interfibrilar y estructuras vasculares, sin especificidad de especie y con capacidad de fijar complemento. Se demostró que puede pertenecer tanto a la IgM como a la IgG. Algunos hechos sugieren su relación con la infección T. cruzi. Por estudios de absorción de sueros que contenían estos anticuerpos y otros con actividad antitrypanosoma surgen evidencias en favor de la existencia de 2 sistemas inmunológicos: FAC representaría uno de estos sistemas y su especificidad inmunológica estaría vinculado a antígenos presentes solo en el T. cruzi. El FAC tiene una alta prevalencia en la zona endémica estudiada, y no fue encontrado en los controles sanos o enfermos de la ciudad de Buenos Aires. Además dentro de la zona endémica su prevalencia fue significativamente mayor en los enfermos con cardiopatía crónica compatible clinicamente con enfermedad de Chagas que en los controles asintomáticos. Esta asociación sugiere la posibilidad de utilizar este elemento FAC como complemento para el diagnóstico de enfermedad de Chagas y como diagnóstico diferencial con otras miocardiopatías crónicas. Su posible significado patogénico es discutido.