Fluorocytometric analysis of induced sputum cells in an asthmatic population.

BACKGROUND: Bronchial mucosal inflammation is the major pathogenic process in asthma. In the latest years, induced sputum (IS) examination has become an important non-invasive method of assessing airway inflammation. Flow cytometry has been recently applied to the study of IS though it is not exempt of methodological difficulties. The aim of the present study was to further study if the fluorocytometric analysis of IS could represent a reliable tool to assess the presence of bronchial activated lymphocytes in stable mild asthmatic patients. METHODS: Induced sputa from controls and asthmatic patients were processed in isotonic 3mM dithiothreitol (DTT), a mucolytic agent required for cell dispersion. The individualized cells were then stained with monoclonal antibodies for three-colour flow-cytometric analysis. Total IgE and ECP were measured in serum and in the sputum fluid phase. RESULTS: The cellularity of asthmatic sputa is enriched in eosinophils (mean, 26.63%) with respect to controls, but not in lymphocytes. However, lymphocytes from asthmatics show increased surface expression of activation markers (CD25 in T cells, CD23 in B cells). Surprisingly, no differences were observed in the detected levels of CD54 on IS lymphocytes and eosinophils between asthmatics and non-asthmatics. Furthermore, there was a significantly higher concentration of ECP and total IgE in the sputum from the asthmatic group. CONCLUSION: Fluorocytometric analysis of induced sputum is a reliable non-invasive method for the study of bronchial immune cells. It could provide complementary information on activated cells in the bronchial mucosa even in non-smokers, mild and stable asthmatics and it is reasonable to speculate that it will be useful in monitoring the effect of the treatment in these patients.

An ultrasensitive method for the detection of oligoclonal IgG bands.

We have developed an ultrasensitive isoelectrofocusing (IEF) method for the detection of oligoclonal (OGC) IgG bands in cerebrospinal fluid (CSF) and sera. In this procedure, double antibody and peroxidase immunodetection have been substituted by a single antibody labelled with alkaline phosphatase. Alkaline phosphatase immunodetection not only improves tenfold the sensitivity of the assay but also gives a sharper pattern resolution making the interpretation easier and increases the specificity of this technique. This preliminary report should be validated in a larger number of patients with and without multiple sclerosis (MS).

[T gamma-delta lymphocytes and their role in hypersensitivity processes in the digestive and respiratory mucosa]. / Los linfocitos T gammadelta y su papel en procesos de hipersensibilidad en la mucosa digestiva y respiratoria.

Specific antigenic recognition by the immune system relies on receptors that T and B lymphocytes display on their plasmatic membranes. Most T cells express an antigen receptor integrated by an a chain and a b chain, the TcR-alphabeta. There is also a second population of T cells, minority in blood, spleen and lymph nodes, which expresses a different type of TcR, the TcR-gammadelta. These cells, termed gammadelta T cells, are probably implicated in the innate immunity, and are preferentially located in epithelia, where they are known as "gammadelta intraepithelial lymphocytes" (gammadelta IEL). The intestinal gammadelta IEL population (gammadelta i-IEL) is particularly abundant and it might have, at least in part, an extrathymic origin. An increase in the gammadelta i-IEL population has been described in intestinal hypersensitivity processes like Alimentary Allergy and, characteristically, Celiac Disease, but its significance is not well known. The second best known gammadelta IEL subset is the one located in the respiratory mucosa. Recent data suggest that these gammadelta IEL might play a role in protecting the normal airway function while being also implicated in allergic airway diseases, like Allergic Rhinitis and Asthma, due to their pro-inflammatory functions. This review focuses on the general characteristics of this yet poorly known and intriguing T cell population, whose relevance is increasingly acknowledged.

Los linfocitos T Gamma-Delta y su papel en procesos de hipersensibilidad en la mucosa digestiva y respiratoria

La mayoría de los linfocitos T, las células mejor conocidas del sistema inmune, expresan en su membrana un receptor para antígeno formado por una cadena Alfa y otra Beta, denominado TcR-Alfa Beta . Sin embargo, existe una segunda población de linfocitos T minoritaria en la sangre, bazo y ganglios linfáticos que presenta un TcR diferente, el TcR-Gamma Delta . Estos linfocitos, probablemente implicados en el sistema inmune innato, son sin embargo cuantitativamente importantes en los epitelios, donde se denominan "linfocitos intraepiteliales Gamma Delta" (LIE Gamma Delta). Los LIE Gamma Delta de la mucosa digestiva o i-LIE son especialmente abundantes y podrían tener, al menos en parte, un origen extratímico. Se ha descrito un aumento de la población i-LIE Gamma Delta en enfermedades intestinales como la alergia alimentaria o la enfermedad celíaca, aunque el significado de este aumento no es del todo conocido. La importancia los LIE Gamma Delta de la mucosa respiratoria es doble: por un lado, estarían encargados de la protección de la mucosa frente a agentes patógenos y, por otro, estarían implicados en procesos alérgicos como la rinitis alérgica crónica y el asma alérgica. En este trabajo se revisan las principales características de ésta, aún poco conocida pero interesante, estirpe linfoide, cuya relevancia está emergiendo gracias a los hallazgos que se han sucedido en los últimos años (AU)

Specific antigenic recognition by the immune system relies on receptors that T and B lymphocytes display on their plasmatic membranes. Most T cells express an antigen receptor integrated by an a chain and a b chain, the TcR-αβ. There is also a second population of T cells, minoritary in blood, spleen and lymph nodes, which expresses a different type of TcR, the TcR-γδ. These cells, termed γδ T cells, are probably implicated in the innate immunity, and are preferentially located in epithelia, where they are known as "γδ intraepithelial lymphocytes" (γδ IEL). The intestinal γδ IEL population (γδ i-IEL) is particularly abundant and it might have, at least in part, an extrathymic origin. An increase in the γδ i-IEL population has been described in intestinal hypersensitivity processes like Alimentary Allergy and, characteristically, Celiac Disease, but its significance is not well known. The second best known γδ IEL subset is the one located in the respiratory mucosa. Recent data suggest that these γδ IEL might play a role in protecting the normal airway fuction while being also implicated in allergic airway diseases, like Allergic Rhinitis and Asthma, due to their pro-inflammatory functions. This review focuses on the general caracteristics of this yet poorly known and intriguing T cell population, whose relevance is increasingly acknowledged (AU)

Intrathecal IgM synthesis predicts the onset of new relapses and a worse disease course in MS.

BACKGROUND: The authors have recently described that intrathecal IgM synthesis (ITMS) correlates with a higher disability in patients with clinically definite MS (CDMS). OBJECTIVE: To follow-up a group of patients with MS in the initial stages of the disease to evaluate if the presence of ITMS correlates with a worse evolution. METHODS: Oligoclonal IgM bands were performed in 22 patients with MS with a mean of 1.14 months of evolution. Patients were followed for a period ranging from 6 to 36 months (mean, 21.4 months). During follow-up, time to conversion to CDMS, number of relapses, and changes in Expanded Disability Status Scale (EDSS) score were evaluated. RESULTS: Patients were divided into two groups according to the presence (Group 1, 10 patients) or absence (Group 2, 12 patients) of ITMS. No clinical differences were observed between the groups at inclusion in the study. During the follow-up, the probability of conversion to CDMS was greater in Group 1 (90% of the patients had converted to CDMS after 8 months of follow-up) than in Group 2 (51% of patients had converted to CDMS after 36 months of follow-up) (p = 0.0001). Patients from Group 1 had more relapses (mean, 2.0) than those from Group 2 (mean, 0.58) (p = 0.02). At the end of the study, patients from Group 1 had higher EDSS scores (mean, 1.70) than those from Group 2 (mean, 0.79) (p = 0.02). CONCLUSION: The presence of oligoclonal IgM bands in CSF can be a prognostic marker in the early phases of MS.

The effect of dithiothreitol on VLA-4 detection in peripheral blood and induced sputum cells.

BACKGROUND: Induced sputum has been shown to be a reliable technique for investigating airway inflammation non-invasively. Flow cytometry could provide useful information in this area. However, the viscosity of the sample entails the use of a mucolytic agent. Dithiothreitol (DDT) is the most frequently used agent although it could affect detection of different inflammatory markers. METHODS: To measure the effect of DDT on the detection of certain adhesion molecules in eosinophils and lymphocytes, sputum was induced from seven non-smoking asthmatic and non-asthmatic subjects treated with 0.1 M DDT. The samples were analyzed by flow cytometry. Whole blood samples from the same subjects were also processed with DTT and analyzed by flow cytometry. RESULTS: Very late activated antigen-4 (VLA-4) levels on eosinophils in intracellular and surface staining were much lower than expected. VLA-4 on lymphocytes was also altered but less so than on eosinophils. VLA-4 levels were also decreased on blood cells after DTT treatment. No abnormalities were found in the detection of CD29 on eosinophils and the beta7-chain in lymphocytes. CONCLUSIONS: Flow cytometry could be used as a complementary method to induced sputum in the investigation of airway inflammation. However, DTT could interfere with the detection of some inflammatory markers, as is the case with VLA-4.

The effect of dithiothreitol on VLA-4 detection in peripheral blood and induced sputum cells

Background: Induced sputum has been shown to be a reliable technique for investigating airway inflammation non-invasively. Flow cytometry could provide useful information in this area. However, the viscosity of the sample entails the use of a mucolytic agent. Dithiothreitol (DDT) is the most frequently used agent although it could affect detection of different inflammatory markers. Methods: To measure the effect of DDT on the detection of certain adhesion molecules in eosinophils and lymphocytes, sputum was induced from seven non-smoking asthmatic and non-asthmatic subjects treated with 0.1 M DDT. The samples were analyzed by flow cytometry. Whole blood samples from the same subjects were also processed with DTT and analyzed by flow cytometry. Results: Very late activated antigen-4 (VLA-4) levels on eosinophils in intracellular and surface staining were much lower than expected. VLA-4 on lymphocytes was also altered but less so than on eosinophils. VLA-4 levels were also decreased on blood cells after DTT treatment. No abnormalities were found in the detection of CD29 on eosinophils and the β7-chain in lymphocytes. Conclusions: Flow cytometry could be used as a complementary method to induced sputum in the investigation of airway inflammation. However, DTT could interfere with the detection of some inflammatory markers, as is the case with VLA-4 (AU)

Antecedentes: El esputo inducido se ha mostrado como una técnica fiable para estudiar de un modo no invasivo, la inflamación de la vía respiratoria en el asma bronquial. La citometría de flujo puede aportar información útil en este campo. No obstante, la viscosidad de la muestra implica la utilización de una gente mucolítico, siendo el Ditiotreitol (DTT) uno de los más usados, a pesar de que puede alterar la expresión de diferentes mediadores inflamatorios. Métodos: Se indujo esputo de 7 sujetos asmáticos y no asmáticos, todos no fumadores. Se trataron las muestras con DTT 0.1M y se analizaron concitometría de flujo. Muestras de sangre de los mismos sujetos igualmente fueron procesadas con DTT 0.1M y analizadas. Resultados: Los niveles de detección de VLA-4 en la superficie y a nivel intracelular en los eosinófilos, fue más baja de lo esperado. La alteración de los niveles de VLA-4 en linfocitos fue menor que en los eosinófilos. En sangre, la detección de VLA-4 disminuyó en las muestras tratadas con DTT. No se observaron anormalidades en la detección de CD29 en eosinófilos ni en la cadena 7 en linfocitos. Conclusiones: La citometría de flujo puede ser usada en el estudio de la inflamación en la vía aérea aunque se debe tener en cuenta que el DTT altera la detección de algunos marcadores, como así se comprueba para el VLA-4 (AU)

Intrathecal IgM synthesis in neurologic diseases: relationship with disability in MS.

The authors studied the intrathecal IgM synthesis (ITMS) in paired sera and CSF samples from 65 patients with MS, 28 with CNS infection, 40 with other neurologic diseases and eight control subjects. ITMS was found in 30 patients with MS and in 20 with CNS infection, but not in patients with other neurologic diseases or in control subjects. In infectious samples, the ITMS is likely a primary response. In MS group, it was associated with higher Expanded Disability Status Scale index (p = 0.017).

A sensitive and reproducible method for the detection of oligoclonal IgM bands.

We have developed a sensitive and specific isoelectrofocusing (IEF) method for the detection of oligoclonal (OGC) IgM in the cerebrospinal fluid (CSF) and sera. In this procedure, ampholytes, in the range pH 5-8, were used to improve the resolution, and serum was diluted in saline to avoid IgM precipitation. Samples were treated with 50 mM dithiothreitol (DTT) at pH 9.5 to reduce IgM and improve the migration of the protein. Using an anti-IgM labelled with alkaline phosphatase, the limit of detection was found to be 0.1 ng in 5 microl of sample.

Anti-transglutaminase IgA ELISA: clinical potential and drawbacks in celiac disease diagnosis.

BACKGROUND: Since the identification of tissue transglutaminase (tTG) as the antigen for the anti-endomysial antibodies (EMA), several antigen-specific immunoassays have been reported for celiac disease (CD) screening. A first objective was to evaluate the suitability for CD screening of three different IgA tTG ELISAs, two of them based on guinea pig liver tTG (gp-tTG) (an in-house ELISA with a partially purified extract and a commercial ELISA with purified gp-tTG antigen) and a third recombinant human tTG (rh-tTG) ELISA. The results are compared with EMA and with the final clinical diagnosis. A second objective was to analyze antibody reactivities in those patients with anti-tTG and EMA discrepancies. METHODS: ELISA and EMA tests were used to measure IgA anti-tTG levels in sera from 259 patients (107 had CD and 72 had Type I diabetes mellitus). RESULTS: The purified gp-tTG ELISA was highly sensitive (97.7%) and specific (98.8%) in the detection of CD, almost equaling EMA. Rh-tTG ELISA did not improved the sensitivity of EMA, but its specificity was slightly superior. Immunoblot analysis with partially purified gp-tTG extract, the antigen most frequently used for anti-tTG detection, showed that the majority of false positives were due to IgA reactivities to contaminant proteins present in the liver antigenic extract. This low specificity was particularly problematic in diabetics. CONCLUSION: Purified tTG ELISAs, either with purified guinea pig liver or recombinant human antigens, can be used as quantitative and observer-independent alternatives to the traditional and time-consuming EMA in the screening of CD.

Interleukin-6 dimers produced by endothelial cells inhibit apoptosis of B-chronic lymphocytic leukemia cells.

Tumoral lymphocytes from patients with B-chronic lymphocytic leukemia (B-CLL) are long-lived cells in vivo, but they die rapidly by apoptosis in vitro. Here, it is reported that endothelial cells (ECs) inhibit the apoptosis of B-CLL cells, as determined by 4 different flow cytometric methods, and that this antiapoptotic effect is mediated mainly by soluble factor(s), as can be deduced from the following findings. First, EC-conditioned medium (ECCM) inhibited the apoptotic rate in B-CLL to approximately 50% of control. Second, the antiapoptotic effect mediated by EC/B-CLL cell contact was more apparent than real; using a fluorescence-based phagocytosis assay, it was demonstrated that this effect was due to the phagocytic capacity of ECs, which internalized apoptotic cells. Third, the protective effect of ECCM was associated neither with proliferation nor differentiation signals. Fourth, the survival factor was a dimeric form of IL-6 because anti-IL-6 antibodies completely neutralized the antiapoptotic effect mediated not only by the crude ECCM but also by the 45- to 55-kd active fractions obtained after gel filtration, which contained high levels of IL-6. These IL-6 dimers (IL-6(D)) were noncovalently associated. Sixth, human recombinant IL-6(D) (hrIL-6(D)) inhibited B-CLL apoptosis, whereas hrIL-6 monomers (hrIL-6(M)) did not. Binding and functional competition experiments showed not only that monomers and dimers had similar affinity for the IL-6R, but also that hrIL-6(M) inhibited the antiapoptotic activity of hrIL-6(D). These data suggest that IL-6(D) derived from ECs promote the survival of B-CLL cells.

Aislamiento y caracterización de antígenos principales en Anisakis simplex / Isolation and characterization of major antigens from Anisakis simplex

La anisakiasis o anisakidosis es una enfermedad causada por la larva de Anisakis simplex tras la ingesta de pescado crudo o poco cocinado. Se han descrito cuadros gastrointestinales, gastroalérgicos y un tercer grupo que correspondería con la llamada hipersensibilidad de Anisakis. Caracterizar los antígenos implicados en esta reacción alérgica fue la meta del estudio. Métodos: Tras la obtención de las larvas del parásito se realizó un marcaje metabólico con Leu-C14 y posterior homogeneizado de éstas y de las restantes larvas sin marcar. Se identificaron las distintas proteínas presentes en el parásito y en el huésped por PAGE-SDS y posterior autorradiografía, diferenciando así las proteínas que sintetiza el nematodo. Se fraccionó un extracto de Anisakis por filtración en gel (Sephacryl S-200), y se ensayaron las fracciones obtenidas por inmunodetección. Resultados: Se aisló una proteína de 22 KDa que estaba presente en el grupo de aquellas que sintetizaba el parásito. La fracción que contenía esta proteína fue reconocida en todos los casos en los sueros de pacientes hipersensibilizados a Anisakis simplex. Posteriormente se analizó su punto isoeléctrico, presentando un pl de aproximadamente 5.5. Conclusiones: Se ha logrado caracterizar una proteína de 22kDa y pl 5.5 que podría estar implicada en las reacciones de hipersensibilidad de algunos pacientes frente a Anisakis simplex. (AU)

Intestinal intraepithelial lymphocytes contain a CD3- CD7+ subset expressing natural killer markers and a singular pattern of adhesion molecules.

Intestinal intraepithelial lymphocytes (i-IEL) represent one of the largest, non-organized lymphoid population in the body. They are located outside the epithelial basement membrane among the mucosal epithelial cells. We, and previously other groups, have reported the presence of a CD7+CD3-IEL subset in the epithelium of human small intestine. This subset is drastically reduced in coeliac disease (CD) patients. In the present work we accomplish a better phenotypic characterization of this CD3-IEL subset and demonstrate the expression of typical natural killer (NK) cell markers. Most, if not all, CD3-CD7+ cells express NKPR1 (CD161)[98% +/- 2] and CD122[92% +/- 6]. In addition, a variable percentage express CD2[55% +/- 16], CD94[24% +/- 18], CD56[44% +/- 21] and CD16[12% +/- 4], however, no CD57 expression was observed. Moreover, these cells contain perforin granules[75% +/- 5], supporting a potential cytolytic ability. Regarding adhesion molecules, CD18 and CD44 expression is absent, which is consistent with a limited capacity of migration. Altogether, these data suggest the presence of intraepithelial NK cells in human intestinal epithelium, a compartment where cytotoxic effectors have not been clearly defined.

Lack of crossreaction with Bet v 1 in patients sensitized to Dau c 1, a carrot allergen.

BACKGROUND: Pollen-related food allergies to fresh fruit and vegetables are a well-known clinical phenomenon. Allergens related to Bet v 1 are responsible for these cross-reactions. OBJECTIVE: To characterize the allergen recognized by four carrot-allergic patients. METHODS: Sera from four patients showing strong immediate systemic reactions after contact or ingestion of raw carrot were studied by immunoblotting. The 18-kD allergen, named Dau c 1, was isolated by ethanol precipitation and specific extraction after SDS-PAGE and its N-terminal amino acid sequence was determined. RESULTS: All the patients had significant levels of specific IgE to carrot, but no specific IgE to birch pollen was detected in any of them. IgE immunodetection with the sera only recognized a single band of around 18 kD in raw carrot and in celery (with weaker reaction). No reactive band was found with birch pollen. These results were confirmed using a polyclonal anti-carrot antiserum. The carrot IgE-binding protein had a pl of 4.2 and its N-terminal sequence was homologous to that of Bet v 1 and to allergens previously described in celery and other foods. The four patients studied were not sensitized to birch pollen and three of them tolerated fruit ingestion. CONCLUSION: The whole study indicated that a sensitization to Dau c 1 induces IgE antibodies that do not cross-react with birch pollen allergens.

Flow cytometry description of a novel CD3-/CD7+ intraepithelial lymphocyte subset in human duodenal biopsies: potential diagnostic value in coeliac disease.

Intraepithelial lymphocytes (IEL) represent a heterogeneous cellular compartment of unknown functions and controversial ontogeny. Previous observations in humans indicate that the majority of IEL subsets express the CD3 complex associated with either the alphabeta or the gammadelta T-cell receptor components, and describe the characteristic increase of CD3+TCRgammadelta+ IEL in coeliac disease. In the present work, we analyze the surface antigen expression of intraepithelial lymphocytes isolated from duodenal biopsies of control subjects and coeliac disease patients. We describe a CD3-CD7 + IEL subset frequently found in control subjects (41.41+/-21.8), with the following features: 1) most of these cells are CD45R0+ CD103+ and CD44- CD28- CD5-; 2) a significant percentage express CD56 (44.7%+/-21.3), CD2 (55.1%+/-16.2), and CD94 (16.2%+/-7.3). Furthermore, they are CD122+ and CD25-; 3) this CD3- IEL subset exhibit an activated phenotype expressing higher levels of CD69, CD103, and CD38 than the CD3+ subset. Interestingly, this CD3- subset is drastically reduced in CD patients (2.2+/-2.9 in active disease, 6.3+/-4.6 in treated patients versus 41.4+/-21.8 in control subjects). The imbalanced ratio "increased TCRgammadelta versus decreased CD3- CD7+" is a permanent finding in CD patients following clinical and histological remission. This parameter might provide helpful diagnostic information (easily obtained by 3-color FCM from diagnostic biopsies), and suggest a potential implication in the pathogenesis of coeliac disease.

Soluble class I antigen secretion by peripheral blood lymphocytes in multiple sclerosis.

We have quantified the soluble class I antigen (sHLA) secretion by peripheral blood lymphocytes of 26 multiple sclerosis (MS) patients. Thirteen of them were in a stable phase of the disease, 6 on relapse and 7 suffered from a progressive MS. sHLA secretion was reduced in the presence or absence of phytohemagglutinin in patients with either active or stable MS, being normal after stimulation with a monoclonal antibody anti-CD3. In MS patients, lymphocyte proliferation and immunoglobulin secretion were found to be similar to those of 29 healthy blood donors who comprised the group of controls. These results reflect systemic anomalies in the cell activation process in MS, which seem to be independent of the disease activity. Whether these alterations are specific to MS or are common to other inflammatory CNS diseases remains to be elucidated.

Intrathecal synthesis of soluble class I antigens in multiple sclerosis.

We have studied the intrathecal synthesis of soluble class I antigens (sHLA), reflected by the index IH = (CSF sHLA/serum sHLA)/(CSF albumin/serum albumin), in multiple sclerosis (MS). IH was increased in patients in relapse, but normal in patients in remission; these findings show that there is a high lymphocyte activation within the central nervous system in patients with clinically active MS.