Immune response of severe malnutrition children treated according to the protocol of the World Health Organization / Respuesta inmune de niños con desnutrición grave tratada según el protocolo de la organización mundial de la salud

The aim of the study was to compare the innate immune system of severely malnourished children admitted to the Instituto de Medicina Integral Professor Fernando Figueira and treated according to the protocol of the World Health Organization (WHO) at admission and discharge. An experimental study was conducted with 20 children under two years of age. Ten of them had severe malnutrition and ten were a control group. The malnourished group consisted of hospitalized infants and it was submitted to WHO’s protocol. Children with HIV and re-admitted during the study period were excluded. A blood sample was taken at admission and at discharge. Later, an analysis of blood leukocytes, adherence index, phagocytic capacity, production of free radicals superoxide and nitric oxide was performed. Patients with severe malnutrition at hospital discharge showed improved phagocytic function, release of oxygen radicals and reduction of the number of lymphocytes when compared to the time of admission. When compared to the control group, patients at hospital discharge had lower lymphocyte values and lower production of free radicals. Thus, it can be concluded that the duration of hospitalization was insufficient to restore cell-mediated immunity and microbicide activity (AU)

El objetivo del estudio fue comparar el sistema inmune innato de niños con malnutrición grave ingresados en el Instituto de Medicina Integral Professor Fernando Figueira, tratados de acuerdo con el protocolo de la Organización Mundial de la Salud (OMS), al ingreso y al alta hospitalaria. Se llevó a cabo un estudio experimental con 20 niños menores de dos años de edad, 10 con malnutrición grave y 10 niños del grupo de control. El grupo de malnutridos se compuso de lactantes hospitalizados y sometidos al protocolo de la OMS. Se excluyeron los niños afectados por el HIV y los readmitidos durante el período del estudio. Se recogió una muestra de sangre al ingreso y otra al alta, y posterioriormente se realizó el análisis del perfil leucocitario, y el índice de adherencia, la capacidad fagocítica y la producción de los radicales libres superóxido y óxido nítrico. Los pacientes con malnutrición grave en el alta hospitalaria mostraron mejoría de la función fagocítica, la liberación de radicales oxidantes y la reducción del número de linfocitos en comparación con el ingreso hospitalario. En comparación con el grupo de control, los pacientes en el alta hospitalario presentaron valores más bajos de linfocitos y de producción de radicales libres. Por lo tanto, se puede concluir que el tiempo de hospitalización fue insuficiente para restablecer la inmunidad mediada por células, así como para restaurar la actividad microbicida (AU)

The basophil activation test in immediate drug allergy.

Diagnosis of drug allergy is not always straight forward for several reasons. First, a broad spectrum of drugs can elicit various immune-mediated diseases with distinct pathomechanism, secondly, although exact epitope identification is not mandatory for clinical diagnosis, the epitope that causes the reaction is frequently unknown, thirdly in vitro or in vivo test results might not be predictive of a clinical situation, and finally the gold standard or reference test for diagnosis, the drug challenge, is a complicated and sometimes dangerous endeavour. Upon challenge with specific allergens that cross-link membrane-bound IgE antibodies, basophils upregulate the expression of different activation markers such as CD63 and CD203c. These immunophenotypic alterations can be detected on a single-cell basis by multicolour flow cytometry using specific monoclonal antibodies in the basophil activation test (BAT). This review intends to summarise our current experience with the BAT in the diagnostic management of immediate-type allergy to drugs and related compounds that are generally (but not always) mediated by drug-specific IgE antibodies.

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

Increased leukocyte adhesiveness/aggregation in patients with inflammatory bowel disease during remission. Further evidence for subclinical inflammation.

PURPOSE: We have used a novel leukocyte adhesiveness/aggregation test (LAAT) to show that many patients with inflammatory bowel disease (IBD) in clinical remission have a subclinical low grade inflammation. METHODS: Included in the study are 500 controls, 96 patients with IBD in remission, and 106 patients in relapse. RESULTS: The percent of aggregated white blood cells detected in the peripheral blood was 5.9 +/- 3.9, 9.1 +/- 5.9, and 18.8 +/- 9.4, respectively. The difference between each group and any other was significant at P < 0.0001. Similar results were obtained when other acute phase reactants like the erythrocyte sedimentation rate, white blood cell count, differential count, and C-reactive protein level were examined. However, in a linear regression analysis, LAAT was the only significant (P < 0.0006) variable that could classify correctly each subject to the appropriate category of control and IBD in remission or relapse. CONCLUSIONS: Identification of patients with IBD in clinical remission who have ongoing inflammation may be of clinical-therapeutic relevance. The LAAT is a simple, rapid, and convenient test. The present study indicates that it is also very sensitive.

[Study of immunologic reactivity of patients with cancer of the stomach based on the data of the adhesive capacity of lymphocytes]. / Izuchenie immunologicheskoi reaktivnosti u bol'nykh rakom zheludka po dannym opredeleniia adgezivnoi sposobnosti limfotsitov.

In patients with gastric cancer, the increase in adhesive lymphocytic capacity, as compared to a control, was revealed. It is indicative of the reduction in production of lymphokines, causing inhibition of adhesion. The highest value of the index was noted in IV stage gastric cancer. The results of study of the adhesive properties of the peripheric blood lymphocytes allow to judge about the state of cellular immunity, and in the complex with the other clinico-laboratory findings, are of a prognostic value.

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

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 leukocytes from patients (N = 21) but not from the blood of normal subjects (N = 10) failed to adhere to glass in the presence of 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 +/- SEM = 29.3 +/- 4.3), whereas the values for normal subjects were -56.0 to +2.0 (-25.9 +/- 7.6) and those for treated patients -59.6 to +4.0 (-19.3 +/- 5.8). Our results show that the colorimetric LAI assay can be used as an auxiliary test for the diagnosis of schistosomiasis.

Twenty-four hour leukocyte adherence inhibition test in thyroid cancer patients.

The effect of TG cell depletion taking place during 24 hour incubation on the leukocyte adherence inhibition phenomenon was investigated in patients suffering from cancer of the thyroid and healthy volunteers. The depletion coincided with the increase of the adherence inhibition if PPD was used as antigen in both groups, but the reaction to the tumour associated antigen could not be brought into relation with the depletion of TG cells.

New micro-glass-tube leukocyte adherence inhibition assay assessing cell adherence of mononuclear cell subpopulations defined by monoclonal antibodies.

A new micro-glass-tube leukocyte adherence inhibition (LAI) assay which is appropriate for detecting delayed type hypersensitivity in vitro has been developed for human leukocytes. Enumeration of adherent cells is replaced by a cellular radioimmunoassay determining antibody binding of the monoclonal reagents, OKT4, OKT8 and OKM1, to glass-adherent cells, fixed by glutaraldehyde or formaldehyde. An LAI reactivity to purified protein derivative of tuberculin (PPD) was detectable in donors giving a positive PPD skin test with OKT4 reagent, but not with the other two reagents.

Schistosoma mansoni: cell-mediated immunity evaluated by antigen-induced leukocyte adherence inhibition assay.

Peripheral blood leukocytes (PBL) from schistosomiasis patients fail to adhere to glass in the presence of soluble egg antigens (SEA) or soluble worm adult antigenic preparation (SWAP). These leukocytes are non-reactive with S. mansoni unrelated antigens (C. albicans and bovine albumin). Supernatants obtained from cultures of mononuclear cells of patients with antigens were able to inhibit granulocyte adherence to glass. The inhibition of antigen-induced adherence (LAI assay) was not observed when PBL or supernatants were obtained from normal subjects or from schistosomiasis patients after chemotherapy. These results show that under the conditions tested, leukocytes appear to react directly with SEA or SWAP thus losing their property of adherence to glass.

[Mechanisms, kinetics and specificity of the leukocyte adherence inhibition test in breast cancer]. / Izuchenie mekhanizmov, kinetiki i spetsifichnosti testa ingibitsii adgezii leikotsitov pri rake molochnoi zhelezy.

The simplified micromodification of the leukocyte adhesion inhibition (LAI) test has shown that the LAI tests both for lymphocytes and for leukocytes may be equally used as additional methods of diagnostics of the early stages of breast cancer. The detailed study of the kinetics and cellular mechanisms of the method has shown that the LAI reaction belongs to the earliest reactions of T-cellular immunity and is mediated by T-cell lymphokine. The study of sensitivity and specificity of the method revealed that the suggested micromodification of the LAI test may be used in experimental and clinical research for the study of the subpopulation of T-cells-producers of the LAI factor as well as for monitoring of the T-cell immunity in the cancer patients during treatment.

Development of a microassay technique for neutrophil adherence.

The development of a microassay for neutrophil adherence is described. The nylon fibre column consisted of 10 mg of fibre inserted into a micropipette tip. A vacuum harvester was constructed to aspirate the neutrophil-containing fluid in the columns into disposable test-tubes for quantitation of neutrophil concentrations. In this way, the neutrophil requirement was reduced from 10(7) cells to 10(5) cells. A complete assay can be performed efficiently and rapidly using less than 1 ml of blood.

[Effect of tumor antigens on leukocyte adherence in the blood in stomach cancer]. / Vliianie antigenov opukholei na prilipaemost' leikotsitov krovi pri rake zheludka.

The reaction of changes in leukocyte adherence was compared in 79 cases of stomach cancer, 44 cases of other gastric diseases and healthy subjects. Tumor antigens were shown to inhibit the adherence of lymphocytes and granulocytes in 61 out of 79 cancer patients (77,2%); adherence inhibition was more frequently observed in patients with stage I-III tumors than in cases of stage IV tumors. The removal of the antigens of normal mucosa from tumor tissue extracts did not affect the frequency of positive reactions in cancer patients significantly. However, the frequency dropped from 22-34 to 2.3-13.6% in the group of non-tumor patients.

Role of T-cells in the mechanism of reactivity of the microplate leukocyte adherence inhibition assay.

To characterize the role of mononuclear cells in the microplate leukocyte adherence inhibition assay, enriched populations of T-cells, B-cells, macrophages, and a nylon wool-adherent fraction consisting of B-cells and macrophages were prepared by a two-stage adherence procedure from spleen cells of normal C57BL/6J mice and mice bearing progressively growing murine colon adenocarcinoma 38 (MCA-38) tumors. These studies indicate that the reactive cell undergoing specific antigen-induced adherence inhibition was present in the macrophage fraction. This cell was programmed to undergo specific antigen-induced adherence inhibition by an MCA-38-sensitized B-cell. An enriched population of MCA-38-sensitized T-cells but not normal T-cells abolished the in vitro leukocyte adherence inhibition response of MCA-38-reactive cells. Pretreatment of the MCA-38-sensitized T-cell fraction with anti-Thy 1:2 serum and complement but not anti-immunoglobulin and complement or carbonyl iron to selectively deplete macrophages abolished the regulatory effect of the MCA-38-sensitized T-cell fraction. MCA-38-sensitized T-cells could prevent MCA-38-sensitized B-cells from programming normal macrophages in vitro but could not abolish the leukocyte adherence inhibition response of presensitized macrophages. Temporal studies of suppressor T-cell activity were performed throughout different phases of progressive tumor growth. Suppressor T-cell could be detected as early as 4 days post-MCA-38 tumor cell inoculation and persisted throughout MCA-38 progressive tumor growth. Titration studies of suppressor T-cells revealed the need for 4 times the number of MCA-38-sensitized T-cells from small tumor bearers (Day 4) in comparison to large tumor bearers (Day 20) to achieve the same degree of suppression. Thus, in the MCA-38 system, sensitized T-cells programming of normal macrophages. This negative regulatory influence increased in magnitude with the progressive growth of the MCA-38 tumor.

Humoral antitumor immune responses in patients with breast cancer measured with the leukocyte adherence inhibition technique.

A modification of the hemacytometer leukocyte adherence inhibition (LAI) test was described. In this modification, 0.25% serum from patients with breast cancer was added with the relevant antigen to the assay system with the use of trypsinized leukocytes from control persons as indicator cells. The modified assay measured a humoral immune response. In studies of patients with untreated breast cancer (stages I and II) with the use of a KCl extract from a breast carcinoma or from MCF-7 cells as antigens, the modified LAI test was found to be at least as sensitive as was the ordinary test. In a blind study on sera collected from patients with breast cancer 0.5-2 years before the LAI measurements and stored at -20 degrees C, 15 of 18 (83%) patients had a positive response. Whereas the ordinary LAI test is limited to the use of fresh blood, the present test can be performed with small amounts of serum that can be frozen and stored.

Specific immunodiagnosis of hepatoma by tube leukocyte adherence inhibition assay and a modified method of repeated tube leukocyte adherence inhibition assay.

Tube leukocyte adherence inhibition (LAI) assays were performed with normal liver extract as nonspecific antigen and with hepatoma extract as specific antigen in patients with hepatoma. Titration experiments revealed that the optimal extract concentration was 400 micrograms/ml when expressing the results in terms of a nonadherence index. The results of tube LAI assays were positive in 26 of 40 cases (65%) of hepatoma. The results were negative in all cases of other liver diseases and other cancers. The tube LAI assay was repeated after discarding the non-adherent cells in the initial tube LAI assay with normal liver extract. The nonadherence index of the repeated tube LAI assay we devised was significantly higher than that of the original tube LAI assay (p less than 0.001) in patients with hepatoma. Ten of 12 patients with hepatoma in whom the results of the original tube LAI assay were negative showed positive results in the repeated tube LAI assay. The present study suggests that the problem of false-negative results in tube LAI assay can be solved by repeating the tube LAI assay.

A computerized tube leukocyte adherence inhibition assay to detect antitumor immunity in early human cancer: a review of two years' experience.

A 2-year experience with a computerized in vitro assay of leukocyte adherence inhibition (LAI) indicated that patients with cancer of either the colorectum, stomach, pancreas, breast, or lung expressed antitumor immunity to an organ-type specific neoantigen. About 1% of the 1,299 control subjects with benign or malignant disease had a positive LAI assay and 2.5% of patients with inflammatory disease of the colorectum, stomach, pancreas, or lung had a positive LAI assay when tested against tumor extracts of the same organ. Of the 351 patients with cancer of either the colorectum (111), stomach (40), pancreas (28), lung (57), or breast (116) who harbored a microfocus of cancer, 80% or more were LAI positive. As the stage of the cancer advanced, fewer patients were LAI positive. Nine of 45 patients (20%) with colon adenomas had a positive LAI response to colon cancer antigen and 17 of 204 patients (8%) with benign breast disease reacted to the breast cancer antigen. Our results suggested that the leukocytes of some of the LAI-positive patients with either colon adenomas or benign breast disease were responding to an organ-specific neoantigen. Whether the acquisition of a cell surface tumor antigen by the colon adenomas and the dysplastic lesions of the breast implies an irretrievable step to ultimate malignancy is unknown. Most patients exhibit a vigorous antitumor immune response when the cancer exists as a microfocus, and some even before the neoplasm has acquired the capacity to invade.

Automation of the leukocyte adherence inhibition assay. Counting live mononuclear cells with an automated light microscope system.

Adaptation of an automated light microscope system to the leukocyte adherence inhibition assay provides a rapid automated assay of cell mediated immunity. Comparison between the numbers of live mononuclear cells counted by eye and by machine yields no statistical differences in the per cent adherence or in the standard errors when performing either 10 or 20 replicate counts per antigen-cell mixture. Using the cell counter in a semi-automated mode, the counting is performed 10 times as fast in comparison to the manual method. A semi-automated system is described. The procedure requires from 4-6 X 10(5) mononuclear cells and from 2-3 micrograms crude KCl extract using a standard hemocytometer. The value of the technique lies in its availability as a rapid assay for both research applications and immunologic monitoring in the clinical laboratory.

Arming of normal leukocytes with sera from patients with adenocarcinoma of the prostate.

A tumor-associated antigen-induced leukocyte adherence inhibition assay was used to evaluate the effect of serum from patients with adenocarcinoma of the prostate on the antitumor reactivity of normal leukocytes. Peripheral blood leukocytes from 53 normal (control) subjects were armed with serum from 22 patients with localized (Stage A) and metastatic (Stage D) prostatic cancer and reacted with allogenic extract of malignant prostate as specific tumor-associated antigen. Leukocytes pre-treated with serum from patients with Stage A cancer show significantly stronger responses to malignant prostate than do those pretreated with serum from patients with Stage D cancer, which induced little or no response. This may be attributed to an "arming factor" present in the sera of patients with an initial stage of prostatic cancer which appears to be capable of sensitizing normal leukocytes and making them specifically reactive to tumor extract. The specificity of arming with individual and pooled patient's sera was delineated by the use of extracts from other genitourinary tumors.