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.

Método citoquímico para evaluar adherencia en leucocitos polimorfonucleares neutrófilos / Cytochemical method for evaluating adherence in polymorphonuclear neutrophil leucocytes

Se desarrolló un método citoquímico cuantitativo y sencillo para evaluar adherencia en leucocitos polimorfonucleares neutrófilos. Las células fueron aisladas por gradiente de densidad a partir de sangre periférica de 47 donantes voluntarios que acudieron al Servicio de Transfusiones del Instituto Superior de Medicina Militar "Dr. Luis Díaz Soto". La cualidad adhesiva se amplificó mediante la determinación de proteínas totales por el método de Lowry, luego de incubar los fagocitos en placas plásticas de cultivo de 24 pozos. El mayor número de valores se encontró entre 6 y 10 mg/dL, lo que representó el 70 porciento de los sujetos evaluados. La combinación de la placa de cultivo como superficie de adhesión y la determinación de proteínas como forma de amplificar esta cualidad, constituye una novedad del método propuesto

Slow cluster formation of purified human or rhesus T cells requires protein kinasse C and LFA-1

Ab: Homotropic T cell adhesion, as generally studied, consists of a rapid, transient binding process that is measured over a 15-120 min. period. Here we report a slow type of adhesion process occurring with human or rhesus T cells, purified from peripheral blood, that manifests itself by the formation of rounded, multi-layer clusters which may contain hundreds of cells. The maximal number and size of the clusters peak 1-2 days after the addition of phorbol ester, an absolute requirement. The number of clusters formed is proportional to phorbol ester concentration up to 1.25 ng/mL. Phorbol esters such as phorbol myristate acetate (PMA), phorbol dibutyrate (PDB), and 7-octylindolactam (OIL) induced optimal cluster formation at 1-13 ng/mL, levels slightly higher than that required to induce mitogenesis of purified T cells. Phorbol itself and the alpha-form of the ester were inactive. Both cluster formation and mitogenesis (stimulated by Con A or anti-CD3) are completely inhibited by staurosporin at 12.5 ng/mL. Even at 2.5 ng/mL, 74 percent of cluster formation was inhibited, which strongly implies a crucial role for protein kinase C. In the presence of accessory cells, T cell clusters were suppressed. Monoclonal Ab such as anti-CD3, mouse anti-CD3 followed by anti-mouse IgG, anti-CD4, anti-CD4A, anti-CD2, anti-CD8, and anti-CD45 did not induce cluster formation. None were inhibitory or stimulatory in the presence of PMA, except for anti-CD3 which enhanced cluster formation by 26 percent. However, anti-LFA-1 beta-chain (mouse monoclonal) completely blocked cluster formation over the range studied (63-1000 ng/mL) for both human and rhesus cells; rat anti-LFA-1 only blocked human cell adhesion. Anti LFA-1 only partially inhibited T cell mitogenesis. These results show that slow cluster formation shares the LFA-1 and phorbol ester requirements of the rapid adhesion of T cells requiring LFA-1 and ICAM-1. However, cluster occurs at a very low phorbol ester concentration, appears more sensitive to staurosporin inhibition, and is not stimulated via the TCR receptor like the rapid adhesion process. We hypothesize that certain neuronal processes, induced by phorbol ester, and which also show a similar protein kinase C activation time course, may share mechanisms in common with cluster formation

Colorimetric leukocyte adherence inhibition assay: a new auxiliary technique for the diagnosis of Schistosomiasis

We describe a modification of the leukocyte adherence inhibition assay (LAI) in which we propose the use of 3-(4,5-dimethylthiazole-2-yl)-2-5-diphenyltetrazolium bromide (MTT) dye which is taken up and reduced by mitochondria. The method was tested by screening peripheral blood leukocytes from Schistosoma mansoni-infected patients. Peripheral blood leuckocytes from patients (N=2) but not from the blood of normal subjects (N=10) failed to adhere to glass in the presence od soluble adult worm antigenic preparation (SWAP). The non-adherence index (NAI) values for schistosomiasis patients were in the range of 11.0 to 72.3 (mean ñ SEM = 29.3 ñ 4.3), whereas the values for normal subjects were -56.0 to +2.0(-25.9 ñ 7.6) and those for treated patients -59.6 to +4.0 (-19.3 ñ 5.8). Our results show that the colorimetric LAI assay can be used as an auxiliary test for the diagnosis of schistosomiasis

Immunological memory for HIV-1 induced in rabbits by immune poly(A)+ RNA

New Zealand rabbits were used to demonstrate the in vitro and in vivo transfer of reactivity, including immunological memory, to a synthetic peptide corresponding to residues 586-606 of the gp-160 protein of human immunodeficiency virus (HIV-1). The transfers were mediated by immune poly (A)+ RNA from lymphoid organs(spleen and mesenteric nodules) harvested after immunization of a sheep with the peptide (8subcutaneous injections plus glucan and complete Freund's adjuvant using a total of 1750 *g peptide). Immunological reactivity was detected by the leukocyte adherence inhibition (LAI) test for cellular immunity. A dose of 150 *g poly (A) RNA ml-1 10 *******7 leukocytes -1 or 2.0 *g poly(A)+ RNA ml-1 10*******7 leukocytes-1 was used for in vitro transfer. For in vivo transfer the recipient rabbits received 3,000 *g poly(A)- RNA or 20*g poly(A)+ RNA. The mean non-adherence index(NAI) obtained in vitro was 10ñ7 for leukocytes treated with poly (A)- RNA and 60ñ10 leukocytes treated with poly(A)+ RNA. The poly(A)+ RNA fravction induced a primary-like response and memory cells in vivo. The poly (A)- RNA fraction had no effect. Since sheep are refractory to, and rabbits are sensitive to HIV-1, we suggest the use of this animal model for testing the immunomodulating effect of anti-HIV-1 immune poly(A)+ RNA

In vivo and in vitro measurement of cell mediated immunity in Marek's disease: role of reticuloendothelial system.

Dinitrofluorobenzene (DNFB) contact-sensitivity test and leucocyte adherence inhibition (LAI) test were performed in this study as in vivo and in vitro tests to measure the cell-mediated immunity (CMI) in chickens subjected to stimulation of reticuloendothelial (RE) system, depletion of RE system and other experimental groups after being challenged with Marek's disease (MD) virus. It was found that CMI was lower in the birds with depleted RE system and infected control birds, whereas CMI was higher in the birds with activated RE system and vaccinated birds as revealed by DNFB contact-sensitivity test. In cases of LAI test, the number of LAI-positive birds were highest in the chicks with depleted RE system particularly in 3rd and 4th month of age, when the incidence of MD was also maximum.

Imunidade especifica em pacientes com carcinoma do pancreas. Estudo preliminar. / Specific immunity in patients with carcinoma of the pancreas.Preliminary report

No presente estudo, investigamos a imunidade especifica mediada por celulas em pacientes portadores de carcinoma do pancreas. Para tanto utilizamos o teste de inibicao da aderencia de leucocitos. Em nosso estudo o ensaio para inibicao da aderencia leucocitaria mostrou-se especifico para pacientes portadores de cancer de pancreas sendo negativo quando se utilizou antigeno derivado de tumor nao-pancreatico. A aplicacao clinica deste ensaio bem como a relativa facilidade de seus aspectos tecnicos permitem afirmar que a inibicao da aderencia de leucocitos venha a se tornar um ensaio util para avaliacao da imunidade tumoral, especialmente importante para neoplasias onde o diagnostico precoce e essencial, tais como o carcinoma do pancreas

Immunological study of typhoid fever in man. II Cell-mediated immune response.

The development of cell-mediated immune response to lipopolysaccharide and Barber protein from Salmonella typhi was investigated in patients suffering from typhoid fever. The cell-mediated immunity as measured by the leukocyte adherence inhibition test, was demonstrable in 77% of patients with typhoid fever but only in 5.6% of healthy controls. It was found that cell-mediated immune response appeared after the first week of illness and persisted for at least 4 weeks. The time course development of cell-mediated immune response and humoral immune response was correlated but the magnitude of each response was independent on one another.