In search of a tolerance-induction strategy for cow's milk allergies: significant reduction of beta-lactoglobulin allergenicity via transglutaminase/cysteine polymerization

OBJECTIVE: To explore the use of β-lactoglobulin polymerized using microbial transglutaminase and heating to identify whether protein polymerization could reduce in vivo allergenicity and maintain in vitro and ex vivo immunoreactivity for use in tolerance-induction protocols. METHODS: Based on previous protocols applied in mice and children, we performed in vivo challenges (using a skin prick test) with native and polymerized β-lactoglobulin in adult patients with an IgE-mediated allergy to plactoglobulin. In vitro humoral immunoreactivity was analyzed using immunoblotting. Cell-mediated immunoreactivity was analyzed using ex vivo challenges with native and polymerized β-lactoglobulin and monitored by leukocyte adherence inhibition tests. RESULTS: The skin tests demonstrated that there was a significant reduction in immediate cutaneous reactivity after polymerization. Polymerization did not decrease the immunoblotting detection of s-IgE specific to β-lactoglobulin. Cell-mediated immunoreactivity, as assessed by ex vivo challenges and leukocyte adherence inhibition tests, did not exhibit significant differences between leukocytes challenged with native versus polymerized β-lactoglobulin. CONCLUSIONS: The polymerization of β-lactoglobulin decreased in vivo allergenicity and did not decrease in vitro humoral or ex vivo cell-mediated immunoreactivity. Therefore, we conclude that inducing polymerization using transglutaminase represents a promising technique to produce suitable molecules for the purpose of designing oral/ sublingual tolerance induction protocols for the treatment of allergies.

INFLUENCE OF 1-BUTANOL, 1,2-BUTANEDIOL AND 1,2,3,4-BUTANETETROL ON THE ADSORPTION OF b-LACTOGLOBULIN AT THE AIR-WATER INTERFACE / INFLUENCIA DE 1-BUTANOL, 1,2-BUTANODIOL Y 1,2,3,4-BUTANOTETROL EN LA ADSORCIÓN DE b-LACTOGLOBULINA EN LA INTERFASE AIRE-AGUA / INFLUÊNCIA DE 1-BUTANOL, 1,2-BUTANODIOL E 1,2,3,4-BUTANOTETROL NA ADSORÇÃO DE b-LACTOGLOBULINA NA INTERFACE AR-ÁGUA

In this work, a systematic study on the effect of 1-butanol, 1,2-butanediol and 1,2,3,4-butanetetrol (erythritol) on the surface tension of β-lactoglobulin in aqueous solution at pH 6.5 and 298.15 K is presented. The experimental data were used to calculate the surface pressure and were adjusted to different protein adsorption models at the liquid-air interface to explain the behavior of β-lactoglobulin in aqueous solution. The results show that the alcohols produce a significant effect on the adsorption behavior of the protein at the interface that is related to the number of hydroxyl groups.

En este trabajo se presenta un estudio sistemático del efecto de 1-butanol, 1,2-butanodiol y 1,2,3,4-butanotetrol (eritritol) sobre la tensión superficial de la β-lactoglobulina en solución acuosa a 298,15 K. Los datos experimentales fueron usados para calcular la presión superficial y se ajustaron a distintos modelos de adsorción en la interfase líquido-aire para explicar el comportamiento de la β-lactoglobulina en solución acuosa. Los resultados muestran que los alcoholes tienen un efecto significativo en el proceso de adsorción de la proteína en la interfase, relacionado con el número de grupos hidroxilo del alcohol.

Neste trabalho, um estudo sistemático sobre o efeito do 1-butanol, 1,2-butanodiol e 1,2,3,4-butanotetrol (eritritol) sobre a tensão superficial da β-lactoglobulina em soluçãoaquosa em pH 6,5 e 298,15 K é apresentado. Os dados experimentais foram utilizados para calcular a pressão de superfície e foram ajustados para diferentes modelos de adsorção de proteínas na interface líquido-ar para explicar o comportamento de β-lactoglobulina em soluçãoaquosa. Os resultados mostram que os alcoóisproduzem um efeito significativo sobre o comportamento de adsorção da proteína na interface que está relacionado com o número de grupos hidroxila.

Efeito protetor das frações proteicas do soro de leite em camundongos Balb/C infectados por Escherichia coli O157: H7 / Protective effect of whey proteic fractions on Balb/C mice infected by Escherichia coli O157: H7

Avaliou-se o efeito protetor das frações proteicas do soro do leite sobre as vilosidades intestinais de camundongos Balb/C, fêmeas, infectadas por Escherichia coli O157:H7. Foram utilizados 48 animais, distribuídos aleatoriamente em oito grupos de seis fêmeas cada um. Os animais dos grupos 1 e 2 (controles) receberam dieta AIN93G padrão; os dos grupos 3 e 4, AIN93G + alfalactalbumina; os dos grupos 5 e 6, AIN93G + betalactoglobulina e os dos grupos 7 e 8, AIN93G + concentrado proteico total e água ad libitum por sete dias. No dia zero, os animais dos grupos 2, 4, 6 e 8 foram inoculados, por meio de cânula de gavagem, com 0,5mL de E. coli O157:H7, na concentração de 7 x 10(10)UFC/mL. Os animais foram acompanhados clinicamente e sacrificados, no oitavo dia experimental. Verificou-se, por meio de exames histológicos e da morfometria, que as frações betalactoglobulina e alfalactalbumina exerceram efeito protetor sobre as vilosidades intestinais do jejuno distal e do íleo (P<0,05), respectivamente. O concentrado proteico total não demonstrou efeito protetor sobre as vilosidades intestinais.

The protective effect of protein fractions of whey on intestinal villi of Balb/C female mice infected with Escherichia coli O157: H7 was evaluated. A total of 48 animals were randomly distributed into eight groups of six females each. Animals in groups 1 and 2 (controls) received AIN93G standard diet; the groups 3 and 4, AIN93G + alpha-lactalbumin; while groups 5 and 6, AIN93G + beta-lactoglobulin; and groups 7 and 8, AIN93G + total protein concentrate and water ad libitum for seven days. On day zero, animals of groups 2, 4, 6, and 8 were inoculated by gavage tube with 0.5mL of E. coli O157: H7 at a concentration of 7 x 10(10)CFU/mL. The animals were clinically followed and sacrificed on the eighth day. It was verified by histological examination and morphometry that the beta-lactoglobulin and alpha-lactalbumin exerted a protective effect on the villi of the distal jejunum and ileum (P<0.05), respectively. The total protein concentrate showed no protective effect on the villi.

EFECTO DE ALCOHOLES Y POLIOLES SOBRE LA ESTABILIDAD ESTRUCTURAL DE LA β-LACTOGLOBULLNA EN SOLUCIÓN ACUOSA / EFFECT OF ALCOHOLS AND POLYOLS ON THE STRUCTURAL STABILITY OF β-LACTOGLOBULLN IN AQUEOUS SOLUTION / EFEITO DE ALCOÓIS E POLIOLES SOB A ESTABILIDADE ESTRUTURAL DA β-LACTOGLOBULLNA EM SOLUÇÃO AQUOSA

En este trabajo se presenta un estudio sistemático sobre el efecto del incremento en el número de grupos OH de alcoholes y polioles de cuatro carbonos sobre las propiedades conformacionales de la β-lactoglobulina. Los cambios en el comportamiento de las soluciones acuosas de la proteína por la adición de 1-butanol, 1,2-butanodiol, 1,2,4-butano-triol y 1,2,3,4-butanotetrol fueron determinados por espectroscopia UV, de fluorescencia y DC en el UV lejano, y en el UV cercano a 298,15 K. Los resultados muestran que el butanol ejerce una mayor modificación en la estructura de la proteína y el efecto va disminuyendo a medida que se incrementa el número de grupos OH.

In this work we present a systematic study of the effect of the increase of OH groups in alcohols and polyols of four carbon atoms on the conformational properties of β-lactoglobulin. The changes in the behavior of the aqueous solution of the protein by the addition of 1-butanol, 1,2-butanediol, 1,2,4-butanetriol y 1,2,3,4-butano-tetrol were determined by UV, far and near UV CD spectra and fluorescence spectroscopy at 298.15K. The results show that the largest modification ofprotein structure is due to butanol and the effect decreases as the number of OH groups increase.

Neste trabalho apresenta-se um estudo sistemático sob o efeito do acréscimo do número de grupos OH de alcoóis e polióis de quatro carbonos nas propriedades conformacionais da β-lactoglobulina. As mudanças no comportamento das soluções aquosas da proteína, pela adição de 1-butanol, 1,2 butanodiol, 1,2,4 e 1,2,3,4 butanotetrol butanotriol, foram determinados por espectroscopia UV, fluorescência e DC no UV distante, e no UV próximo a 298,15 K. Os resultados mostraram que o butanol tem uma maior influencia na estrutura da proteína, e o seu efeito diminui com o aumento do número de grupos OH.

Frecuencias Alélicas de Beta-lactoglobulina en ganado criollo Limonero / Allelic Frequency of Beta-lactoglobulin in Limonero Creole Cattle

Con el objeto de caracterizar el gen de la beta-lactoglobulina (BLG) en la raza Criollo Limonero, se utilizó la técnica PCR-RFLP en 163 animales puros de la estación local Carrasquero (Carrasquero-estado Zulia), los genotipos fueron determinados a través de electroforesis en geles de agarosa. Las frecuencias obtenidas del locus de la BLG fueron A (0,22) y B (0,78) y las frecuencias genotípicas fueron AA (0,07 ); AB (0,29) y BB (0,64), la población estudiada se encuentra en equilibrio de Hardy-Weinberg (P<0,05), los resultados indican que la frecuencia alélica del alelo B fue más alta que la de A, siendo esto importante, ya que se han determinado los efectos de esta variante alélica de la BLG sobre la cantidad de grasa y proteínas en la leche, la selección a favor del alelo B en la población conllevará a una mejora en la calidad y rendimiento en la producción de queso, estos resultados representan un valioso aporte al conocimiento de esta raza y de su importancia, ya que, representa una alternativa para sistemas dirigidos a la producción de queso.

In order to characterize the beta-lactoglobulina gene (BLG) in the Limonero Creole cattle through PCR-RFLP technique, 163 purebreed animals were used from the Carrasquero local station (Carrasquero-Zulia State), genotypes were determined through gel electroforesis in agarosa. Gene and genotypic frequencies obtained were A (0.22) and B (0.78) and AA (0.07), AB (0.24) and BB (0.64) respectively, the population is in equilibrium of Hardy-Weinberg with (P<0.05), the results indicate that the alelic frequency of was more high B but that A, being this important one, since the effects of the variants of the BLG on the amount of fat and proteins in milk have been determined, the selection in favor of allele B in the population will entail to an improvement in the quality and yield in the cheese production, these results represent a valuable contribution the knowledge of this race and its importance, since, represents an alternative for systems directed to the cheese production.

Reproducción y fertilidad humana: aspectos biomédicos de la familia de las lipocalinas: biología, patobiología y bioclínica / Human reproduction and fertility: biomedical aspects of the lipocalin family: biology, pathobiology and bioclinic aspects

La familia de genes de las lipocalinas (LCN) está compuesta por varios miembros que comparten una estructura común y que se han duplicado en forma repetida durante la evolución expandiéndose a más de 150 genes conocidos, de ellos al menos veinte reportados en la especie humana. El grupo de proteínas de las LCN está constituido por varios elementos que comparten la propiedad común de unión de ligandos lipofílicos. Las LCN funcionan en un amplio rango de sistemas incluyendo quimiorrecepción y transporte en fisiología sensorial del gusto y olor, coloración, modulación hemato-inmune, síntesis de prostanglandina D2, neuro-fisiología, fisiología reproductiva y fertilidad, embriogénesis, proliferación y división celular, supervivencia y apoptosis celular. Es evidente su rol en patobiología y bioclínica reproductiva y de la fertilidad al observar que varias LCN tienen niveles alterados de expresión en diferentes eventos patofisiológicos. Esta revisión resume hallazgos e implicaciones

The family of the lipocalin (LCN) genes is composed by various members that share a common structure and haveduplicated repeatedly during evolution expanding to more than 150 known genes of which at least 20 have been reported in the human species. The group of lipocalin-related proteins is comprised by a number of elements which share various common properties such as binding to different lipophilic ligands. Lipocalins are involved in a broad range of systems including chemoreception and transport functions in sensory physiology of taste and smell, coloration, modulation of hemato-immune response, synthesis of prostaglandin D2, neurophysiology, physiology of reproduction and fertility, embryogenesis, cell proliferation and division, and cell survival and apoptosis. Its role is evident in the pathophysiology and bioclinical aspects of reproduction and fertility consistent with the observation of altered levels of expression of several lipocalins in different pathophysiological events. This review summarizes findings and implications on this topic.

Immunocytochemical Study for Detection of Lactoglobulin in Alveolar Macrophage of Cow Milk Aspirated Mouse / 소아알레르기및호흡기학회지

PURPOSE: It is difficult to detect small amount of aspiration into the lungs due to the lack of safe, sensitive and specific diagnostic tool. Recently, in animal studies, it has been reported that immunocytochemistry for lactoglobulin can be used to detect the minimal aspiration of cow milk. So, we tried to determine the difference between immunocytochemistry for lactoglobulin and Oil Red O stain of alveolar macrophages in cow milk aspirated mice. METHODS: Fifty seven mice with 6-8 weeks old and 30-40 g weighing were used. Mice received either single or multiple intranasal instillation of 0.05 ml cow milk for study and saline for control under the anesthesia with ketamine and xylazine. The trachea of mouse was cannulated with 20G Jelco needle and then, mouse lungs were lavaged 3 times with 0.5 ml of phosphate buffer solution at 4 hours, 12 hours, 24 hours after the last milk or saline instillation. Cells in bronchoalveolar lavage fluid(BALF) were stained with Oil Red O and immunocytochemistry for beta-lactoglobulin. RESULTS: After single aspiration of milk, no cellular difference was found in bronchoalveolar lavage fluid(BALF) when compared with saline aspirated group at 4 hours. But after repeated aspiration of milk, significant change was observed in the number of alveolar macrophage, neutrophil, lymphocyte and eosinophil. Immunocytochemical reactivity was not observed in alveolar macrophages of saline aspirated group. Lipid-laden alveolar macrophages were recovered rarely in Oil Red O staining. Immunocytochemical staining displayed stain-positive alveolar macrophages for beta-lactoglobulin at 4 hours after milk aspiration, it had a peak at 12 hours and decreased markedly at 24 hours. Immunocytochemical stain positive alveolar macrophages appeared similarly in number between single and repeated aspiration group. CONCLUSION: These observations suggested that alveolar macrophages could be detected more easily on immunocytochemistry for lactoglobulin than Oil Red O stain and immunocytochemistry could be used as a sensitive & specific diagnostic method for the detection of milk aspiration.

Análise de polimorfismos do gene da beta-lactoglobulina em vacas da raça Nelore e efeitos sobre o peso à desmama de suas progênies

Weaning weights from a Nelore herd were used after adjustment of means for 205 days of age, sex, age of dam, sire and weaning month, and resulted into two groups of cows that differed by the weaning weight of their calves. The least square means (LSM) and standard error (SE) were for heavy group 163.21± 2.18kg and for light group 134.44± 2.18kg, with 41 animals in each group. These animals were genotyped by DNA polymorphisms of beta -lactoglobulin gene, using PCR-RFLP. After amplification and digestion of a beta-lactoglobulin gene fragment between II and III exon, genotypes 1AA, 24AB and 56BB were identified, with 0.16 and 0.84 frequencies for A and B alleles, respectively. The 24AB and 56BB cows showed calves with LSM± SE of 149.50± 4.17kg and 148.44± 2.73kg respectively, for weaning weight. No difference (P>0.05) was found and the heavy and light groups were similar for the allelic frequencies for this gene. The dam’s genotype did not affect the weaning weight of the calves. This suggests of having other factors, genetic or non-genetic, with major magnitude that affect the weaning weight.

Informações sobre peso à desmama de bezerros Nelore foram utilizadas após ajuste para idade padrão aos 205 dias, sexo, idade da mãe, touro e mês de desmama, para separar as reprodutrizes em dois grupos, segundo o peso de suas crias. As médias de peso dos bezerros ajustadas pelo método dos quadrados mínimos e erros-padrão (LSM± SE) foram para os grupos pesados (P) e leves (L) 163,21± 2,18kg e 134,44± 2,18kg, respectivamente, com 41 animais em cada grupo. Essas reprodutrizes foram submetidas a coleta de sangue para estudo de polimorfismos do gene da beta-lactoglobulina, por meio da técnica de PCR-RFLP. A amplificação e a digestão de um fragmento do gene da beta-lactoglobulina entre o éxon II e III identificou os genótipos 1AA, 24AB e 56BB, com as freqüências de 0,16 e 0,84 para os alelos A e B, respectivamente. Os 24 animais com genótipo AB apresentaram LSM± SE de peso de seus produtos de 149,50± 4,17kg, e os 56 animais de genótipo BB tiveram média de 148,44± 2,73kg. O teste do qui-quadrado não apresentou significância (P>0,05), isto é, os grupos P e L não diferiram entre si quanto às freqüências alélicas apresentadas para esse gene. O genótipo das reprodutrizes não afetou o peso à desmama de suas crias, o que sugere haver outros fatores genéticos e não genéticos de maior magnitude que afetam o peso à desmama.

A study on the immune reaction to the milk protein antigens in the postpartum mother, newborn baby, and children

No abstract available.