RESUMEN
X-linked agammaglobulinemia, a B cell immunodeficiency, is caused by mutations in the Bruton's tyrosine kinase (Btk) gene. The absence of a functional Btk protein leads to a failure of B cell differentiation and antibody production. B cell receptor stimulation leads to the phosphorylation of the Btk protein and it is, therefore, likely that Btk is involved in B cell receptor signaling. As a nonreceptor tyrosine kinase, Btk is likely to interact with several proteins within the context of a signal transduction pathway. To understand such interactions, we have generated glutathione S-transferase fusion proteins corresponding to different domains of the human Btk protein. We have identified a 120-kD protein present in human B cells as being bound by the SH3 domain of Btk and which, after B cell receptor stimulation, is one of the major substrates of tyrosine phosphorylation. We have shown that this 120-kD protein is the protein product of c-cbl, a protooncogene, which is known to be phosphorylated in response to T cell receptor stimulation and to interact with several other tyrosine kinases. Association of the SH3 domain of Btk with p120cbl provides evidence for an analogous role for p120cbl in B cell signaling pathways. The p120cbl protein is the first identified ligand of the Btk SH3 domain.
Asunto(s)
Linfocitos B/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores de Antígenos de Linfocitos B/fisiología , Ubiquitina-Proteína Ligasas , Agammaglobulinemia Tirosina Quinasa , Secuencia de Bases , Línea Celular , Cartilla de ADN/química , Humanos , Datos de Secuencia Molecular , Fosfoproteínas/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-cbl , Transducción de SeñalRESUMEN
The gene which causes X-linked agammaglobulinemia, btk, has recently been identified as a cytoplasmic tyrosine kinase expressed almost exclusively in B cells, and at all stages of B-cell differentiation. To assess the possibility of involvement of this gene in childhood B-cell malignancies, cells from 23 pediatric patients with B-cell acute lymphoblastic leukemia were examined for expression and alteration of the Btk protein and also for mutations in the btk gene. Btk proteins, similar in both molecular weight and quantity to those seen in unaffected individuals, were detected in whole cell lysates from the blasts of 12/12 patients indicating that no abnormal protein was present. cDNAs from the leukemic blasts of all 23 patients were screened with specific primers covering the coding region of the btk cDNA for mutations using single strand conformation polymorphism (SSCP) analysis. No mutations were found but a nucleotide polymorphism was identified in 4/23 patients at the 3' end of btk. Although the sample size in this study was relatively small, these data suggest that btk does not appear to play a critical role in childhood B-cell leukemias.
Asunto(s)
Agammaglobulinemia/genética , Linfoma de Burkitt/genética , Ligamiento Genético , Mutación/genética , Proteínas Tirosina Quinasas/genética , Cromosoma X , Adolescente , Agammaglobulinemia Tirosina Quinasa , Western Blotting , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Proteínas Tirosina Quinasas/análisisRESUMEN
We investigated whether rhesus monkey CD34+CD11b- hematopoietic stem cells can be transduced with recombinant retroviruses carrying the human adenosine deaminase (hADA) gene by co-cultivation with a virus-producing cell line. Following autologous transplantation, polymerase chain reaction (PCR) analysis on peripheral blood mononuclear cells and granulocytes showed that the hADA-retrovirus was present in approximately 0.1% of the cells for at least 400 days post transplantation in 2 monkeys. Bone marrow that was harvested 16 months after transplantation carried ADA-overexpressing myeloid progenitor cells capable of in vitro colony formation. In addition, hADA activity could be demonstrated in T lymphocytes that were harvested 9 months post transplantation. Thus, in vitro transduction of CD34+CD11b- cells led to long-term repopulation of the hematopoietic system with transduced cells of lymphoid and myeloid lineages expressing the hADA gene. To investigate whether infusion of virus-producing cells into a rhesus monkey undergoing autologous bone marrow transplantation could lead to in vivo transfer of the recombinant retrovirus, 1 monkey was infused with CD34+CD11b- bone marrow cells (BMC) and a large quantity of virus-producing cells. Few provirus-carrying cells could temporarily be detected in this animal. This shows that in vivo gene transfer into a regenerating hemopoietic system can occur, albeit at very low efficiency.
Asunto(s)
Antígenos CD , Técnicas de Transferencia de Gen , Células Madre Hematopoyéticas/citología , Adenosina Desaminasa/genética , Animales , Antígenos CD34 , Antígenos CD11 , Diferenciación Celular , Línea Celular , Replicación del ADN , Trasplante de Células Madre Hematopoyéticas , Humanos , Macaca mulatta , Linfocitos T/citologíaRESUMEN
Ocular gene transfer may provide a means for arresting the retinal degeneration characteristic of many inherited causes of blindness, including retinitis pigmentosa (RP). Previously, we have shown in immunodeficient animals that recombinant adeno-associated virus (rAAV) mediates transduction of photoreceptors as well as the retinal pigment epithelium (RPE) following subretinal injection. In this study we extend these observations and show that highly purified recombinant AAV vectors encoding the reporter gene LacZ transduce photoreceptors in an immunocompetent mouse strain following subretinal injection and efficiently transduce ganglion cells after intravitreal injection. Levels of transduction increase over time. Sublethal gamma-irradiation is shown to facilitate this process.
Asunto(s)
Dependovirus/genética , Técnicas de Transferencia de Gen , Degeneración Retiniana/genética , Animales , Rayos gamma , Vectores Genéticos/genética , Operón Lac/genética , Ratones , Ratones Endogámicos BALB C , Retina/patología , Retina/efectos de la radiaciónRESUMEN
We have investigated the minimal time required for efficient transduction of human hematopoietic repopulating cells using a surrogate nonobese diabetic (NOD)/severe combined immunodeficient (SCID) xenoengraftment assay. Cord blood CD34+ cells were transduced to high levels over 24-48 hr in the presence of Flt-3 ligand, stem cell factor, interleukin 3, and interleukin 6. Under these conditions, high levels of NOD/SCID repopulating activity were preserved, but the levels of gene marking in engrafting cell populations measured by expression of a reporter transgene were low. Extension of the transduction period by 24 hr (total culture period, 72 hr) under the same cytokine conditions resulted in high levels of gene marking, but on closer analysis expression was limited predominantly to the myeloid population. Efficient transduction of both lymphoid and myeloid lineages could be achieved only if the transduction protocol was extended by a further 24 hr (total culture period, 96 hr), suggesting that myeloid lineage-committed precursors are capable of repopulation, and that over shorter time periods transduction is largely restricted to this population. This adds to the emerging evidence of heterogeneity within the SRC compartment, and has important implications for the interpretation of this assay in stem cell transplantation and gene transfer studies.
Asunto(s)
Marcadores Genéticos , Vectores Genéticos , Virus de la Leucemia del Gibón/genética , Inmunodeficiencia Combinada Grave/inmunología , Animales , Antígenos CD34/genética , Citocinas/uso terapéutico , Citometría de Flujo , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/terapiaRESUMEN
Production of high-titer rAAV is essential for in vivo clinical application. One limiting factor may be the failure of existing systems to replicate the packaging genome in such a way that expression of Rep and Cap proteins is coordinately amplified. DISC-HSV (disabled single-cycle virus) is a genetically modified herpes simplex virus (HSV) that by deletion of glycoprotein H (gH) is infectious only if propagated in a complementing cell line. In this study, we have used DISC-HSV as a helper for rAAV replication, and have simulated to some extent the amplication of the rep and cap genomes seen in wtAAV infection by incorporating both these and vector sequences in HSV amplicons. Facilitated production of AAV Rep and Cap proteins translates into a considerably improved recovery of rAAV, which transduces cells of the neuroretina in vivo with high efficiency. The potential for contamination with infectious herpes particles is eliminated by the use of noncomplementing (gH-) cell lines to propagate the virus, and by standard purification methods. The use of DISC-HSV and herpes-derived amplicons for production of rAAV may be a useful strategy for future in vivo studies and for clinical application.
Asunto(s)
Dependovirus/genética , Amplificación de Genes , Vectores Genéticos , Simplexvirus/genética , Proteínas del Envoltorio Viral/genética , Animales , Línea Celular , Chlorocebus aethiops , Cricetinae , Dependovirus/fisiología , Eliminación de Gen , Ratones , Ratones Endogámicos BALB C , Plásmidos , Recombinación Genética , Células Vero , Replicación ViralRESUMEN
Nonviral vectors consisting of integrin-targeting peptide/DNA (ID) complexes have the potential for widespread application in gene therapy. The transfection efficiency of this vector, however, has been limited by endosomal degradation. We now report that lipofectin (L) incorporated into the ID complexes enhances integrin-mediated transfection, increasing luciferase expression by more than 100-fold. The transfection efficiency of Lipofectin/Integrin-binding peptide/DNA (LID) complexes, assessed by beta-galactosidase reporter gene expression and X-gal staining, was improved from 1% to 10% to over 50% for three different cell lines, and from 0% to approximately 25% in corneal endothelium in vitro. Transfection complexes have been optimized with respect to their transfection efficiency and we have investigated their structure, function, and mode of transfection. Both ID and LID complexes formed particles, unlike the fibrous network formed by lipofectin/DNA complexes (LD). Integrin-mediated transfection by LID complexes was demonstrated by the substantially lower transfection efficiency of LKD complexes in which the integrin-biding peptide was substituted for K16 (K). Furthermore, the transfection efficiency of complexes was shown to be dependent on the amount of integrin-targeting ligand in the complex. Finally, a 34% reduction in integrin-mediated transfection efficiency by LID complexes was achieved with a competing monoclonal antibody. The role of lipofectin in LID complexes appears, therefore, to be that of a co-factor, enhancing the efficiency of integrin-mediated transfection. The mechanism of enhancement is likely to involve a reduction in the extent of endosomal degradation of DNA.
Asunto(s)
Vectores Genéticos , Liposomas , Péptidos , Fosfatidiletanolaminas , Receptores de Fibronectina/metabolismo , Transfección/métodos , Secuencia de Aminoácidos , Animales , Unión Competitiva , Línea Celular , Córnea , Portadores de Fármacos , Humanos , Ligandos , Microscopía de Fuerza Atómica , Datos de Secuencia Molecular , Oligopéptidos/metabolismo , Péptidos/síntesis química , Péptidos/metabolismo , Compuestos de Amonio Cuaternario , Conejos , Proteínas Recombinantes de FusiónRESUMEN
Sixty adults were tested for humoral and cell-mediated immunity to varicella zoster virus (VZV), type 1 herpes simplex virus (HSV-1) and the human cytomegalovirus (CMV). Since herpesviruses share common antigens, we compared results in these individuals to assess whether our tests gave false positives due to cross-reactions. Of the 60, IgG antibody to VZV, HSV-1, CMV tested by ELISA was detected in 51 (85%), 34 (57%) and 20 (33%) respectively. All possible permutations of results were obtained and there was no evidence of cross-reactivity among the viruses. Lymphocyte transformation performed in 20 was found to be positive in 16 (80%), 13 (65%) and 8 (40%) for VZV, HSV-1 and CMV respectively. Similarly, all possible permutations of results were obtained. In addition, 8 subjects were found to have positive lymphocyte transformation responses for one or other of the viral antigens but no specific antibody was found in their sera. Thus, the antibody and lymphocyte transformation test were not influenced by possible cross-reacting antigens among the 3 herpesviruses we studied. Both tests are necessary to firmly establish whether a subject has had a prior infection.
Asunto(s)
Anticuerpos Antivirales/análisis , Citomegalovirus/inmunología , Herpesvirus Humano 3/inmunología , Activación de Linfocitos , Simplexvirus/inmunología , Adolescente , Adulto , Anticuerpos Antivirales/inmunología , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunoglobulina G/análisisRESUMEN
The majority of studies involving neutrophil integrin expression and function are performed at physiological temperatures subsequent to routine preparative procedures at 4 degrees C. We have shown that surface expression of the leukocyte integrin molecules on neutrophils is increased by cooling and subsequently re-warming of neutrophils to 37 degrees C when compared with cells held at room temperature or 37 degrees C. This increase in expression is secondary to prior cooling of the neutrophils. There is an associated increase in function of these newly expressed adhesion molecules, making the neutrophils more adherent to endothelium. Preparation of cells at 4 degrees C and subsequently warmed to 37 degrees C is stimulatory for neutrophils, probably causing translocation of intracellular stores of the leukocyte integrins to the cell surface in a manner analogous to the stimulant FMLP. Our results indicate that the cooling of neutrophils during isolation is an inappropriate method of neutrophil preparation.
Asunto(s)
Integrinas/biosíntesis , Neutrófilos/metabolismo , Temperatura , Adulto , Fosfatasa Alcalina/metabolismo , Anticuerpos Monoclonales , Antígenos de Superficie/biosíntesis , Antígenos de Superficie/inmunología , Adhesión Celular , Separación Celular , Células Cultivadas , Endotelio Vascular/citología , Fluorescencia , Humanos , N-Formilmetionina Leucil-Fenilalanina , Neutrófilos/enzimologíaRESUMEN
A two site solid phase radioimmunoassay for detection of common food antigens is described. Bovine serum albumin, beta-lactoglobulin and ovalbumin can be detected in normal human serum at levels ranging from 0.1 to > 1000 ng/ml; sensitivity is not impaired by the presence of low levels of antibodies. Thirty min to 3 h after oral intake of milk, beta-lactoglobulin could be detected in the sera of 3 normal individuals, at a concentration of 0.1-3 ng/ml. This assay should prove useful in assessing the importance of macromolecular absorption in food allergy and in other gastrointestinal diseases.
Asunto(s)
Alérgenos/inmunología , Hipersensibilidad a los Alimentos/sangre , Alimentos , Radioinmunoensayo/métodos , Adulto , Animales , Bovinos , Hipersensibilidad a los Alimentos/metabolismo , Humanos , Absorción Intestinal , Lactoglobulinas/inmunología , Leche/efectos adversos , Ovalbúmina/inmunología , Albúmina Sérica Bovina/inmunologíaRESUMEN
A simple, objective semi-quantitative assay for yeast opsonisation by normal polymorphs has been developed. This depends on electronically counting the number of free unphagocytosed yeast particles on a Coulter counter. The method has been compared with the widely used microscope technique and shows excellent correlation. The sera of 112 unselected school-children gave a distribution curve consistent with 2 peaks; 7 gave values less than 2 S.D.s below the mean confirming the high incidence of this immunodeficiency.
Asunto(s)
Proteínas Opsoninas/metabolismo , Saccharomyces cerevisiae/inmunología , Niño , Calor , Humanos , Métodos , Fagocitosis , Valores de ReferenciaRESUMEN
An ELISA procedure to determine the distribution of human IgG subclass antibodies directed against the gram-negative bacterium Branhamella catarrhalis has been developed using commercially available monoclonal anti-IgG subclass antibodies. Using whole bacteria as coating antigen the specificity of the assay was determined and showed minimal cross-reactivity with a range of other bacteria. Estimations of IgG1, IgG2, IgG3, IgG4 and total IgG antibodies directed against this antigen were performed. All normal adult sera tested had measurable antibody levels of specific IgG1, IgG2, IgG3 and total IgG. Specific IgG4 was undetectable in the majority of adult sera. These assays will be of value for investigation of both children and adults with suspected immunodeficiency and recurrent upper respiratory tract infection.
Asunto(s)
Anticuerpos Antibacterianos/análisis , Ensayo de Inmunoadsorción Enzimática/métodos , Inmunoglobulina G/análisis , Moraxella catarrhalis/inmunología , Adulto , Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos/inmunología , Reacciones Antígeno-Anticuerpo/inmunología , Antígenos Bacterianos/inmunología , Reacciones Cruzadas/inmunología , Humanos , Inmunoglobulina G/clasificación , Reproducibilidad de los ResultadosRESUMEN
We have compared assays for products of the neutrophil respiratory burst in normal EBV-transformed B cell lines stimulated with agonists of protein kinase C. Those measuring O2- directly or its immediate product, H2O2, were successful. Of these, the most sensitive were the lucigenin- and luminol-based chemiluminescence assays for O2- and H2O2 respectively. Cell lines from CGD patients, with X-linked or autosomal recessive genetic defects in the neutrophil NADPH oxidase, did not respond in these assays, indicative of their inability to produce O2-. The defects in the lines studied encompass both proteins forming the cytochrome b-245 membrane component, and the 47 kDa cytosolic component of the NADPH oxidase. The possession of the disease associated phenotype by these cell lines provides evidence that in the normal situation both neutrophils and B cells produce O2- via the same system.
Asunto(s)
Linfocitos B/metabolismo , Enfermedad Granulomatosa Crónica/metabolismo , Superóxidos/metabolismo , Acridinas/química , Línea Celular , Grupo Citocromo c/metabolismo , Fluoresceínas/química , Humanos , Peróxido de Hidrógeno/metabolismo , Técnicas In Vitro , Mediciones Luminiscentes , Luminol/química , Nitroazul de Tetrazolio/química , Oxidación-ReducciónRESUMEN
We report the development of a relatively quick and simple method for the assessment of X inactivation status for carrier determination in families affected by X-linked agammaglobulinemia (XLA). This method utilises an immunomagnetic separation technique for B cell purification and a polymerase chain reaction (PCR) based assay for the determination of methylation status at the androgen receptor (AR) gene locus to assess whether X inactivation is random or non-random at this locus. We report the results we have obtained using this assay to investigate females known to be carriers of various X-linked immunodeficiency disorders. In addition, we investigated four females from different families affected by XLA, two of whom were of unknown carrier status, and we discuss the results obtained with this and other X-inactivation assays. A similar assay has recently been described by Allen et al. (1992) and applied to members of one family affected by XLA.
Asunto(s)
Agammaglobulinemia/genética , Compensación de Dosificación (Genética) , Tamización de Portadores Genéticos/métodos , Agammaglobulinemia/diagnóstico , Linfocitos B/metabolismo , Secuencia de Bases , Separación Celular , ADN/química , ADN/genética , Cartilla de ADN/genética , Femenino , Ligamiento Genético , Humanos , Metilación , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Receptores Androgénicos/genética , Cromosoma XRESUMEN
Five infants with a triad of symptoms comprising generalized erythroderma, failure to thrive, and diarrhea are presented. All of these children demonstrated significant immunologic abnormalities. This is a recognizable clinical syndrome that reflects immunodeficiency; however, the responsible immunodeficiencies do not appear to be the same in every case. Early investigation and appropriate therapy may considerably reduce morbidity, and mortality in children with this syndrome.
Asunto(s)
Dermatitis Exfoliativa , Diarrea Infantil , Insuficiencia de Crecimiento , Síndromes de Inmunodeficiencia , Preescolar , Dermatitis Exfoliativa/congénito , Dermatitis Exfoliativa/inmunología , Dermatitis Exfoliativa/terapia , Diarrea Infantil/inmunología , Diarrea Infantil/terapia , Insuficiencia de Crecimiento/inmunología , Insuficiencia de Crecimiento/terapia , Femenino , Humanos , Inmunoglobulinas/análisis , Síndromes de Inmunodeficiencia/inmunología , Lactante , MasculinoRESUMEN
We used three different preparations of varicella zoster virus (VZV) to sensitise mononuclear cells obtained from VZV immune donors. These were autologous infected fibroblasts, live cell free virus and heat inactivated cell free virus. After 14 days of in vitro sensitisation and expansion with interleukin-2, the mononuclear cells which had been exposed to autologous infected fibroblasts had generated mainly cells of the cytotoxic/suppressor phenotype (CD8) while those stimulated with cell free virus (live or heat inactivated) had generated cells of the helper/inducer phenotype (CD4). Functional assays showed that the effector cells generated after exposure to autologous infected fibroblasts lysed autologous virus infected target cells but not uninfected cells. Effector cells generated in the same way but lacking HLA identity with the virus infected target cells failed to demonstrate cytotoxicity. None of these effector cells showed any significant natural killer cell activity. No specific cytotoxicity was obtained by effector cells generated after exposure to cell-free virus. We conclude that the way in which VZV antigen is presented to the mononuclear cells influences the cell type responding in tissue culture. These findings would be useful in the generation of T cell clones of different cell surface phenotype and function.
Asunto(s)
Varicela/inmunología , Herpesvirus Humano 3/inmunología , Linfocitos T/inmunología , Médula Ósea/inmunología , Células Cultivadas , Citotoxicidad Inmunológica , Fibroblastos/inmunología , Antígenos HLA/análisis , Humanos , Inmunidad , Fenotipo , Piel/inmunología , Linfocitos T/clasificación , Linfocitos T/microbiologíaRESUMEN
A common opsonic defect occurring in 7% of the Caucasian population is associated with low serum levels of the lectin mannose binding protein (MBP). This study sought to determine whether the deficiency was also present in a Chinese population using sera obtained from 100 healthy Chinese children (age range 6 weeks-16 years). The distribution profiles of MBP levels and C3b/C3bi fragments binding to mannan coated plates were both bimodal and similar to the corresponding Caucasian profiles. Serum MBP levels were low in 9% of the Chinese children and all of these sera generated low levels of C3b/C3bi fragments. Overall there was a high significant correlation between MBP levels and C3b opsonin generation (r = 0.77; P less than 0.001). By analogy with similar findings in a Caucasian population we believe this correlation to be a reflection of antibody independent complement activation by MBP. In a pilot study of DNA obtained from three adult Chinese with low MBP levels the point mutation causing MBP deficiency in Caucasians was identified in all three cases.
Asunto(s)
Proteínas Portadoras/genética , Adolescente , Pueblo Asiatico/genética , Proteínas Portadoras/análisis , Niño , Preescolar , Complemento C3b/análisis , Humanos , Lactante , Recién Nacido , Lectinas de Unión a Manosa , Mutagénesis Sitio-Dirigida , Proteínas Opsoninas/sangre , Población Blanca/genéticaRESUMEN
A reciprocal relationship between erythrocyte ATP and deoxy-ATP levels has been noted in an immunodeficient child with adenosine deaminase (ADA) deficiency during therapy with red cell transfusions. The sum of red cell ATP plus deoxy-ATP equalled the normal complement of ATP prior to any form of therapy. dATP, dADP and dAMP levels were found in the same ratio (10:1:0.1) as the adenine nucleotides ATP, ADP and AMP. Red cell ATP levels were low, not high or normal as found by others in ADA deficiency, but no deoxyadenosine nucleotides could be found in peripheral blood mononuclear cells. Erythrocyte ATP depletion has recently been identified as a serious consequence of anti-leukaemic therapy with ADA inhibitors; it may thus be an important but hitherto unrecognised contributing factor in the clinical expression of inherited ADA deficiency.
Asunto(s)
Adenosina Desaminasa/deficiencia , Adenosina Trifosfato/sangre , Nucleótidos de Desoxiadenina/sangre , Eritrocitos/análisis , Nucleósido Desaminasas/deficiencia , Adenosina/metabolismo , Humanos , Lactante , MasculinoRESUMEN
S21 (DXS17) and pXG12 (DXS94), two probes linked to the locus of X-linked agammaglobulinaemia (XLA), were used for genetic prediction in 13 such families. A method of allowing for nonallelic genetic heterogeneity was demonstrated in the calculation of the genetic risks, specifying a certain proportion of unlinked families. We further estimated the impact due to the uncertainty of the proportion of unlinked families on the final genetic risks in each family and this can be taken into account during genetic counselling.
Asunto(s)
Agammaglobulinemia/genética , Cromosoma X , Alelos , ADN/genética , ADN/aislamiento & purificación , Femenino , Tamización de Portadores Genéticos , Ligamiento Genético , Humanos , Masculino , Modelos Genéticos , Linaje , Factores de RiesgoRESUMEN
Capillary leak after cardiopulmonary bypass operations for correction of congenital heart defects is universally seen in children and often causes significant morbidity and mortality. Since neutrophil-mediated endothelial injury has been implicated as a pathogenetic mechanism, a prospective controlled descriptive study was performed to investigate possible activation pathways during and after the bypass procedure. Eighteen children undergoing operations, nine with cardiopulmonary bypass and nine neurosurgical craniotomy (i.e., operations without bypass), had samples of arterial blood collected at intervals before, during, and after operations. In six of nine cardiac patients circulating interleukin-8 concentrations rose from less than 30 pg/ml to very high concentrations (> 500 pg/ml); in the remaining three patients small rises (peak 57 to 81 pg/ml) were also seen. In all nine, the rise commenced at the time of rewarming, toward the end of bypass, and peaked 1 to 3 hours thereafter. Interleukin-8 release correlated significantly with length of bypass. Interleukin-1 alpha and interleukin-1 beta were not found, and traces of tumor necrosis factor-alpha were detected in one patient only. Circulating elastase alpha 1-antitrypsin concentrations rose simultaneously and correlated significantly with interleukin-8 (p < 0.001) in patients with cardiac disease, as did absolute neutrophil counts (p < 0.001). In contrast, only one of nine patients with neurosurgical disease (undergoing an unusually long operation and exchange transfusion) had a rise in circulating interleukin-8 to levels greater than 500 pg/ml (p < 0.01). The two samples from this patient with elevated interleukin-8 were the only neurosurgical samples with elevated elastase. This study demonstrates the release of interleukin-8 into the circulation after pediatric hypothermic cardiopulmonary bypass and supports the suggestion that this cytokine plays a role in the pathophysiology of capillary leak through neutrophil degranulation.