RESUMEN
The mechanisms that mediate acute exacerbations in chronic inflammatory diseases are not understood. As IL-8 is a potent chemoattractant for neutrophils in acute inflammatory lesions, we investigated the role of fibroblast-mast cell interactions in short-term IL-8 release. Human gingival fibroblasts were co-cultured with human mast cells (HMC). The concentration of IL-8 in co-culture medium was measured by ELISA. HMC-fibroblast co-cultures showed >8-fold higher IL-8 secretion than fibroblasts or HMC alone. Increased IL-8 secretion required HMC-fibroblast intercellular contact, was enhanced by serum and was blocked by the gap junction inhibitor ß-glycyrrhetinic. Calcein-dye transfer showed intercellular, gap junction communication between HMC and fibroblasts that was dependent in part on hyaluronic acid on the cell surface of fibroblasts. IL-8 secretion by fibroblasts was strongly promoted by hyaluronic acid. Pre-treatment of HMC with thapsigargin provoked 15-fold higher IL-8 production by fibroblasts in co-cultures. Chemotaxis of mouse neutrophils was enhanced 2-fold in response to conditioned medium from HMC-fibroblast co-cultures. We conclude that mast cells adhere to fibroblasts and promote IL-8 secretion, which enhances neutrophil chemotaxis and the inflammatory response.
Asunto(s)
Fibroblastos/metabolismo , Mediadores de Inflamación/metabolismo , Mastocitos/metabolismo , Transducción de Señal , Adolescente , Animales , Células Cultivadas , Quimiotaxis , Niño , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/metabolismo , Expresión Génica , Ácido Glicirretínico/farmacología , Humanos , Ácido Hialurónico/metabolismo , Inflamación/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Ratones , Neutrófilos/fisiología , Vías SecretorasRESUMEN
BACKGROUND AND OBJECTIVE: Human subjects affected by inflammatory diseases, such as periodontitis, may be at increased risk for the development of cardiovascular diseases and calcification of atheromas; however, the potential mechanisms have not been defined. Alpha-2-Heremans Schmid glycoprotein (fetuin A) is an abundant serum glycoprotein of ~49 kDa that inhibits ectopic arterial calcification. We examined whether matrix metalloproteinases (MMPs), which are increased in inflammatory diseases, including periodontitis, bind and degrade fetuin and alter its ability to inhibit calcification in vitro. MATERIAL AND METHODS: Binding and cleavage of fetuin by MMPs were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in-silico analyses and mass spectrometry. The effects of intact and MMP-degraded human fetuin on mineralization were measured in a cell-free assay. RESULTS: From in-silico analyses and literature review, we found that only MMP-3 (stromelysin) and MMP-7 (matrilysin) were predicted to cleave human fetuin at levels that were physiologically relevant. In-vitro assays showed that MMP-7, and, to a lesser extent, MMP-3, degraded human fetuin in a time- and dose-dependent manner. Fetuin peptides generated by MMP-7 cleavage were identified and sequenced by mass spectrometry; novel cleavage sites were found. Hydroxyapatite mineralization in vitro was strongly inhibited by fetuin (> 1 µm), as was MMP-3-cleaved fetuin, while MMP-7-cleaved fetuin was threefold less effective in blocking mineralization. CONCLUSION: MMP-7 and, to a lesser extent, MMP-3, affect the ability of fetuin to inhibit the formation of hydroxyapatite in vitro. These data suggest that the MMPs increased in inflammatory diseases, such as periodontitis, could affect regulation of mineralization and potentially enhance the risk of calcified atheroma formation.
Asunto(s)
Metaloproteinasa 7 de la Matriz/metabolismo , Unión Proteica/efectos de los fármacos , Calcificación Vascular/inducido químicamente , alfa-2-Glicoproteína-HS/efectos adversos , alfa-2-Glicoproteína-HS/antagonistas & inhibidores , Sistema Libre de Células , Durapatita/metabolismo , Electroforesis en Gel de Poliacrilamida , Humanos , Espectrometría de Masas , Metaloproteinasa 3 de la Matriz/metabolismo , Estadísticas no Paramétricas , Calcificación Vascular/metabolismo , alfa-2-Glicoproteína-HS/metabolismoRESUMEN
The formation of myofibroblasts in valve interstitial cell (VIC) populations contributes to fibrotic valvular disease. We examined myofibroblast differentiation in VICs from porcine aortic valves. In normal valves, cells immunostained for alpha-smooth muscle actin (alpha-SMA, a myofibroblast marker) were rare (0.69 +/- 0.48%), but in sclerotic valves of animals fed an atherogenic diet, myofibroblasts were spatially clustered and abundant (31.2 +/- 6.3%). In cultured VIC populations from normal valves, SMA-positive myofibroblasts were also spatially clustered, abundant (21% positive cells after 1 passage), and stained for collagen type I and vimentin but not desmin. For an analysis of stem cells, two-color flow cytometry of isolated cells stained with Hoechst 33342 demonstrated that 0.5% of VICs were side population cells; none stained for SMA. Upon culture, sorted side population cells generated approximately 85% SMA-positive cells, indicating that some myofibroblasts originate from a rare population with stem cell characteristics. Plating cells on rigid collagen substrates enabled the formation of myofibroblasts after 5 days in culture, which was completely blocked by culture of cells on compliant collagen substrates. Exogenous tensile force also significantly increased SMA expression in VICs. Isotope-coded affinity tags and mass spectrometry were used to identify differentially expressed proteins in myofibroblast differentiation of VICs. Of the nine proteins that were identified, cofilin expression and phospho-cofilin were strongly increased by conditions favoring myofibroblast differentiation. Knockdown of cofilin with small-interfering RNA inhibited collagen gel contraction and reduced myofibroblast differentiation as assessed by the SMA incorporation into stress fibers. When compared with normal valves, diseased valves showed strong immunostaining for cofilin that colocalized with SMA in clustered cells. We conclude that in VICs, cofilin is a marker for myofibroblasts in vivo and in vitro that arise from a rare population of stem cells and require a rigid matrix for formation.
Asunto(s)
Factores Despolimerizantes de la Actina/metabolismo , Válvula Aórtica/metabolismo , Diferenciación Celular , Fibroblastos/metabolismo , Enfermedades de las Válvulas Cardíacas/metabolismo , Hipercolesterolemia/complicaciones , Células Madre/metabolismo , Factores Despolimerizantes de la Actina/genética , Actinas/metabolismo , Animales , Válvula Aórtica/patología , Técnicas de Cultivo de Célula , Proliferación Celular , Células Cultivadas , Colágeno/metabolismo , Grasas de la Dieta/efectos adversos , Modelos Animales de Enfermedad , Fibroblastos/patología , Fibrosis , Citometría de Flujo , Geles , Enfermedades de las Válvulas Cardíacas/etiología , Enfermedades de las Válvulas Cardíacas/patología , Hipercolesterolemia/etiología , Hipercolesterolemia/metabolismo , Hipercolesterolemia/patología , Marcaje Isotópico , Espectrometría de Masas , Fosforilación , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Esclerosis , Células Madre/patología , Fibras de Estrés/metabolismo , Estrés Mecánico , Porcinos , Factores de TiempoRESUMEN
Phosphoinositides regulate several actin-binding proteins but their role at intercellular adhesions has not been defined. We found that phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) was generated at sites of N-cadherin-mediated intercellular adhesion and was a critical regulator of intercellular adhesion strength. Immunostaining for PI(4,5)P2 or transfection with GFP-PH-PLCdelta showed that PI(4,5)P2 was enriched at sites of N-cadherin adhesions and this enrichment required activated Rac1. Isoform-specific immunostaining for type I phosphatidylinositol 4-phosphate 5 kinase (PIP5KI) showed that PIP5KIgamma was spatially associated with N-cadherin-Fc beads. Association of PIP5KIgamma with N-cadherin adhesions was in part dependent on the activation of RhoA. Transfection with catalytically inactive PIP5KIgamma blocked the enrichment of PI(4,5)P2 around beads. Catalytically inactive PIP5KIgamma or a cell-permeant peptide that mimics and competes for the PI(4,5)P2-binding region of the actin-binding protein gelsolin inhibited incorporation of actin monomers in response to N-cadherin ligation and reduced intercellular adhesion strength by more than twofold. Gelsolin null fibroblasts transfected with a gelsolin severing mutant containing an intact PI(4,5)P2 binding region, demonstrated intercellular adhesion strength similar to wild-type transfected controls. We conclude that PIP5KIgamma-mediated generation of PI(4,5)P2 at sites of N-cadherin contacts regulates intercellular adhesion strength, an effect due in part to PI(4,5)P2-mediated regulation of gelsolin.
Asunto(s)
Actinas/metabolismo , Cadherinas/metabolismo , Gelsolina/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Unión Competitiva , Adhesión Celular , Pollos , Activación Enzimática , Fibroblastos/citología , Fibroblastos/ultraestructura , Humanos , Isoenzimas/metabolismo , Ratones , Células 3T3 NIH , Péptidos/metabolismo , Unión Proteica , Ratas , Transducción de Señal , Proteínas de Unión al GTP rho/metabolismoRESUMEN
BACKGROUND: Increased levels of active neutrophil collagenase (MMP-8) in the gingival crevicular fluid (GCF) are associated with progressive periodontitis. The measurement of this enzyme in GCF could facilitate diagnosis. However, assays with sufficient sensitivity to detect collagenase in whole-mouth GCF currently use radiolabeled substrates and require several days to complete. To provide more rapid analyses of collagenase activity that are better adapted to clinical studies, we developed and validated a novel assay (soluble biotinylated-collagen assay: SBA) based on chemiluminescent detection of biotinylated collagen digestion products. METHODS: The concordance of the novel SBA assay with a radioactive collagen substrate assay was assessed by parallel analyses of enzyme from 35 neutrophil preparations and from 41 samples of GCF from periodontitis patients, followed by Pearson correlation analysis. To test whether the assay appropriately measured MMP-8 activity, enzyme activity was assessed after incubation with specific collagenase blockers. We examined the diagnostic utility of the SBA in cross-sectional and longitudinal analyses of 125 patients with adult periodontitis, 5 patients with early-onset periodontitis, 1 edentulous patient, and in 32 control patients without periodontitis. RESULTS: The assay detected <56 pg collagen degraded/hour/microl sample, which is comparable to the most sensitive radioactive assay. The total assay time was 22 hours and reproducibility on replicate measurements was high (r = 0.96). In direct comparisons of MMP-8 activity in GCF with enzyme from peripheral blood neutrophils using the SBA and radioactive assays, there was a high correlation (r = 0.97). As expected, EDTA and TIMP-1 and -2, known inhibitors of MMP-8, completely blocked enzyme activity with this assay. Cross-sectional and longitudinal analyses of GCF showed that MMP-8 activity was >18-fold higher in severe periodontitis than in stable periodontitis and decreased to <25% of pretreatment levels following therapy. Based on measurements of collagenase activity in different disease groups, we estimated a value of 80 nano units as a threshold for severe periodontitis. CONCLUSIONS: These results indicate that active MMP-8 is detected in GCF by a novel assay that is specific, simple, rapid, and reproducible and which may facilitate diagnostic discrimination between stable and progressive lesions.
Asunto(s)
Pruebas Enzimáticas Clínicas , Líquido del Surco Gingival/enzimología , Metaloproteinasa 8 de la Matriz/análisis , Periodontitis/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Análisis de Varianza , Animales , Western Blotting , Estudios de Casos y Controles , Progresión de la Enfermedad , Femenino , Humanos , Modelos Lineales , Estudios Longitudinales , Masculino , Inhibidores de la Metaloproteinasa de la Matriz , Persona de Mediana Edad , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Estadísticas no Paramétricas , PorcinosRESUMEN
Neutrophil collagenase (matrix metalloproteinase 8 [MMP-8]) is an important mediator of tissue destruction in inflammatory diseases. Studies of anaerobic periodontal infections have shown that active MMP-8 in gingival crevicular fluid is associated with the degradation of periodontal tissues in progressive periodontitis whereas the latent enzyme is predominant in gingivitis. Since the activation of MMP-8 appears to be a crucial step in periodontitis, we have examined the activation of MMP-8 in gingival crevicular fluid samples by using a soluble biotinylated collagen substrate. Analysis of gingival crevicular fluid in periodontitis, gingivitis, and controls revealed sixfold (P < 0.001)-higher levels of active collagenase in periodontitis (n = 12) samples compared to gingivitis (n = 17) samples, which exhibited low levels of activity, while controls (n = 25) showed no activity. After gingival crevicular fluid was collected, no further activation of latent collagenase occurred in vitro. Although both MMP-1 and MMP-8, but not MMP-13, could be detected by immunoblots, blocking antibodies to MMP-1 showed that collagenase activity was largely contributed by MMP-8, which was localized to the matrix of diseased tissues. The MMP-8 in gingival crevicular fluid migrated primarily as a 60-kDa form with smaller amounts of a 78-kDa species, whereas MMP-8 isolated from peripheral neutrophils migrated at 70 and 89 kDa, corresponding to active and latent forms of the enzyme, respectively. Most of the MMP-8 in the 60- and 70-kDa bands selectively bound to tissue inhibitor of metalloproteinase 2 and collagen, indicating that most, but not all, of the enzyme in these bands was in an activated form. However, the amounts of the 78- and 60-kDa forms from gingival crevicular fluid in different samples did not correlate (r2 = 0.028) with the latent and active enzyme measured by collagenase assay. Collectively, these studies have identified distinct forms of latent and active MMP-8 in gingival crevicular fluid that appear to result from a unique activation mechanism that occurs in periodontitis. The complexity of MMP-8 activation is further indicated by the presence of latent, activated, and superactivated forms of MMP-8 in the 60- and 70-kDa bands obtained from gingival crevicular fluid and neutrophil samples, respectively.
Asunto(s)
Colagenasas/metabolismo , Neutrófilos/enzimología , Periodontitis/enzimología , Estudios de Casos y Controles , Colágeno/metabolismo , Colagenasas/química , Activación Enzimática , Estabilidad de Enzimas , Líquido del Surco Gingival/enzimología , Gingivitis/enzimología , Humanos , Inmunohistoquímica , Técnicas In Vitro , Metaloproteinasa 1 de la Matriz , Metaloproteinasa 13 de la Matriz , Metaloproteinasa 8 de la Matriz , Peso Molecular , Periodontitis/etiología , Inhibidor Tisular de Metaloproteinasa-2RESUMEN
Fetal rat calvaria cells (RC cells) grown in long term culture in the presence of ascorbic acid and organic phosphate proliferate and differentiate to form mineralized nodules of bone. Since transforming growth factor beta (TGF-beta), interleukin 1-alpha (IL-1 alpha) and epidermal growth factor (EGF) affect both bone resorption and bone formation, we have studied the ability of these growth factors to affect plasminogen activators and plasminogen activator inhibitors release by RC cells at different times throughout this proliferation/differentiation sequence. Cultures in log phase growth (day 4), when first multilayering (day 7) and when bone nodules were forming (day 13) were exposed to either TGF-beta, IL-1 alpha, EGF or vehicle. Conditioned medium was collected after 6 and 24 h and plasminogen activators and plasminogen activator inhibitors were analysed by fibrin autography and reverse fibrin autography. TGF-beta-mediated changes in plasminogen activator were apparent at day 4. By day 7 two molecular weight species of plasminogen activator were noted; a 65 kDa species, prominent at 24 h exposure was blocked by anti-tPA antibody, and a 38 kDa plasminogen activator, prominent after 6 h of stimulation was not blocked by anti-tPA antibody. Plasminogen activator-plasminogen activator inhibitor complexes are also increased. IL-1 alpha caused similar increases in plasminogen activator and plasminogen activator inhibitor with maximal activity measured at day 13, coincident with the time when bone nodules were forming. EGF-mediated changes were less by comparison. TGF-beta significantly decreased bone nodule formation after both a 6 and 24 h serum-free exposure, whereas IL-1 alpha and EGF decreased nodule number only after the 24 h exposure. The data suggest that the three factors influence the expression of plasminogen activator and plasminogen activator inhibitor by RC cells and their effect is different at different times of culture.
Asunto(s)
Huesos/embriología , Factor de Crecimiento Epidérmico/farmacología , Interleucina-1/farmacología , Activadores Plasminogénicos/metabolismo , Inactivadores Plasminogénicos/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Fosfatasa Alcalina/metabolismo , Animales , Huesos/metabolismo , Diferenciación Celular , División Celular , Células Cultivadas , Ratas , Ratas Endogámicas , Factores de TiempoRESUMEN
We have investigated whether lymphatic endothelial cells in culture produce plasminogen activators (PAs) and their inhibitors (PAIs) and if these activities can be modulated by the inflammatory cytokine Tumor Necrosis Factor alpha (TNF-alpha). Examination by reverse fibrin autography of the conditioned medium from these cells revealed a PAI of Mr 50 kDa. Also evident by fibrin autography were two species of PAs, of Mr 110 kDa and Mr 60 kDa. The 110 kDa protein co-migrated with the PA-PAI complexes and the 60 kDa protein co-migrated with tissue Plasminogen Activator (tPA). Functional and immunological assays indicated the human TNF-alpha increased the type 1 plasminogen activator inhibitor (PAI-1) in a time dependent manner. Treatment of the cells with recombinant human TNF-alpha for 24 hours resulted in a 3 to 7 fold increase in the amount of PAI released into the conditioned media. Immunoblot analysis identified the PAI in the TNF-alpha treated cell conditioned media, as PAI-1. Deposition of PAI-1 in the extracellular matrix then became apparent. TNF-alpha increased 4 fold the amount of tPA-PAI-1 complexes (Mr 110 kDa) detected in the conditioned media. Free tPA (Mr 60 kDa) decreased to 1/5 of control. Net fibrinolytic activity, as determined by a chromogenic substrate assay, decreased after TNF-alpha treatment. No urokinase type Plasminogen Activator (uPA) activity was detected in control or treated cells. This fibrinolytic activity may be important in maintaining free fluid movement in the interstitium and lymphatic vessels and in inflammatory states this potential may be decreased by the increase in PAI-1.
Asunto(s)
Endotelio Linfático/metabolismo , Activadores Plasminogénicos/biosíntesis , Inactivadores Plasminogénicos/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Bovinos , Células Cultivadas , Endotelio Linfático/efectos de los fármacos , Fibrinólisis/efectos de los fármacosRESUMEN
During a 6-year period (1977 to 1982) blood samples from 152 Canadian patients were referred to the national reference laboratory of the Canadian Red Cross Society because the referring hospitals had not been able to determine the cause of the patients' severe nonhemolytic transfusion reactions. Twenty-one patients were found to be IgA deficient, and 12 of them had strong class-specific anti-IgA antibodies, which were presumed to have been responsible for the reactions. The spectrum of symptoms that accompanied these violent reactions was documented for 10 of the patients. As a probable minimum, the incidence of anti-IgA-mediated reactions averaged 1.3 per million units of blood or blood products transfused during this period.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Inmunoglobulina A/inmunología , Reacción a la Transfusión , Canadá , Antígenos HLA/inmunología , Humanos , Deficiencia de IgA , Inmunoglobulina A/análisisRESUMEN
This study was undertaken to document the incidence of immediate, nonhemolytic transfusion reactions and to identify a technique or set of techniques that would best identify the different causes of these reactions. A variety of tests were employed to detect lymphocyte, granulocyte, platelet and anti-IgA antibodies. During this study 26,318 units of blood components were transfused on 5,030 occasions. 191 immediate, nonhemolytic reactions were experienced giving an incidence per unit of 0.73%. Blood specimens from 101 of these patients were investigated along with serum from 57 patients who showed no reaction to transfusion as controls. We show that standard B cell lymphocytotoxicity testing is the technique with which most antibodies can be detected (64% of reactors positive vs. 30% of controls, p less than 0.001). Additional tests did not significantly increase the level of antibody detection.
Asunto(s)
Reacción a la Transfusión , Anticuerpos Antiidiotipos/aislamiento & purificación , Plaquetas/inmunología , Pruebas Inmunológicas de Citotoxicidad , Femenino , Granulocitos/inmunología , Antígenos HLA/inmunología , Humanos , Inmunoglobulina A/inmunología , Isoanticuerpos/aislamiento & purificación , Linfocitos/inmunología , MasculinoRESUMEN
Monitoring of donors with selective IgA deficiency, i.e., with less than 0.5 mg IgA/l, led to the observation that serum IgA levels wer not constant. Small but significant fluctuations in IgA levels were noted (coefficient of variance: 143%) which were greater than the variability inherent in the testing methodology (coefficient of variance: 10%). These fluctuations created difficulties in terms of defining IgA-deficient blood products and had implications with respect to the mechanisms involved in IgA deficiency. With respect to supplying IgA-deficient blood products, in our experience a cutoff level of 0.5 mg/l should be the maximum permissible IgA concentration in order to ensure that no adverse reactions occur in individuals with class-specific anti-IgA.
Asunto(s)
Disgammaglobulinemia/inmunología , Deficiencia de IgA , Autoanticuerpos , Donantes de Sangre , Transfusión Sanguínea , Humanos , Inmunoglobulina A/inmunología , Inmunoglobulina A/metabolismoRESUMEN
Agglutinating antisera were induced in rabbits against a purified genetic variant of human albumin and against normal albumin. These antisera had different specificities, as determined by inhibition experiments with the purified variant and normal albumin. The results suggest that genetic variants of human albumin differ antigenically from the normal protein. It is conceivable that antibodies against normal human albumin could be induced following albumin administration to individuals homozygous for an albumin variant.
Asunto(s)
Epítopos/genética , Variación Genética , Albúmina Sérica/genética , Anciano , Animales , Formación de Anticuerpos , Electroforesis en Gel de Poliacrilamida , Epítopos/inmunología , Femenino , Humanos , Inmunodifusión , Inmunoelectroforesis , Sustancias Macromoleculares , Peso Molecular , Conejos , Albúmina Sérica/inmunología , Reacción a la TransfusiónRESUMEN
As determined in rabbits, the in vivo plasma survival, i.e. half-life (t 1/2), of albumin produced by the cold-ethanol procedure did not differ significantly from that of native albumin (p less than 0.5). Plasma survivals of reprocessed and heat-ethanol produced albumins were significantly less than that of the native protein (0.02 less than p less than 0.05). Chemically-modified albumin had a considerably reduced t 1/2 (p less than 0.001) and was the only albumin shown to be antigenically different from the native protein. Variations in t 1/2 s of albumin preparations could not be attributed to differences in their polymer contents.
Asunto(s)
Antígenos/inmunología , Albúmina Sérica/metabolismo , Animales , Especificidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Etanol/farmacología , Semivida , Calor , Humanos , Sueros Inmunes/inmunología , Sustancias Macromoleculares , Preservación Biológica/métodos , Conejos , Albúmina Sérica/inmunología , Albúmina Sérica/aislamiento & purificaciónRESUMEN
Covalently linked albumin dimers and polymers had plasma half-lives (t 1/2s) in mice which were not significantly different from that of native albumin. The elimination of a chemically treated albumin monomer was also not appreciably different from that of the native protein, but it was significantly faster than covalently linked albumin dimers and polymers. Essentially similar results were obtained when natural monomers, dimers and trimers were compared, although precise t 1/2 data may have been affected by the dissociation of natural polymers into appreciable amounts of monomer in vivo.
Asunto(s)
Albúmina Sérica/metabolismo , Animales , Semivida , Humanos , Ratones , Ratones Endogámicos C3H , Polímeros/metabolismoRESUMEN
To enable the detection of IgG class, anti-IgA antibodies (IgG-aIgA) and to investigate the possible occurrence of IgE class, anti-IgA antibodies (IgE-aIgA), we developed a solid phase immunoradiometric assay (IRA), which uses purified IgA coupled covalently to microcrystalline cellulose as an immunosorbent. Radiolabeled, Fc specific anti-IgG and anti-IgE antibodies were used to detect specific aIgA after incubation of test sera or controls with the immunosorbent. IgG-aIgA were detected by the IRA in 100 and 67 per cent of control sera with class specific and limited specificity aIgA. The IRA was sensitive to approximately two ng of class specific IgG-aIgA. IgG-aIgA also were detected by IRA in 7.9 per cent of sera from patients with urticarial transfusion reactions and 73 per cent of sera from patients with ataxia telangiectasia and IgA deficiency. Sera from 50 normal blood donors did not have detectable IgG-aIgA. Tests for IgE-aIgA were negative in all cases, including control sera with class specific IgG-aIgA. We conclude that the IRA is a sensitive and reproducible method for detection of class specific and limited specificity IgG-aIgA, and that IgE-aIgA do not mediate urticarial transfusion reactions.