RESUMEN
The platelet receptors, glycoprotein VI (GPVI) and integrin α2ß1 jointly control collagen-dependent thrombus formation via protein tyrosine kinases. It is unresolved to which extent the ITIM (immunoreceptor tyrosine-based inhibitory motif) receptor PECAM1 and its downstream acting protein tyrosine phosphatase PTPN11 interfere in this process. Here, we hypothesized that integrin α2ß1 has a co-regulatory role in the PECAM1- and PTPN11-dependent restraint of thrombus formation. We investigated platelet activation under flow on collagens with a different GPVI dependency and using integrin α2ß1 blockage. Blood was obtained from healthy subjects and from patients with Noonan syndrome with a gain-of-function mutation of PTPN11 and variable bleeding phenotype. On collagens with decreasing GPVI activity (types I, III, IV), the surface-dependent inhibition of PECAM1 did not alter thrombus parameters using control blood. Blockage of α2ß1 generally reduced thrombus parameters, most effectively on collagen IV. Strikingly, simultaneous inhibition of PECAM1 and α2ß1 led to a restoration of thrombus formation, indicating that the suppressing signaling effect of PECAM1 is masked by the platelet-adhesive receptor α2ß1. Blood from 4 out of 6 Noonan patients showed subnormal thrombus formation on collagen IV. In these patients, effects of α2ß1 blockage were counterbalanced by PECAM1 inhibition to a normal phenotype. In summary, we conclude that the suppression of GPVI-dependent thrombus formation by either PECAM1 or a gain-of-function of PTPN11 can be overruled by α2ß1 engagement.
Asunto(s)
Integrina alfa2beta1 , Trombosis , Humanos , Integrina alfa2beta1/genética , Plaquetas , Glicoproteínas , Colágeno , Trombosis/genéticaRESUMEN
INTRODUCTION: The protein tyrosine phosphatase SHP2 (PTPN11) is a negative regulator of glycoprotein VI (GPVI)-induced platelet signal under certain conditions. Clinical trials with derivatives of the allosteric drug SHP099, inhibiting SHP2, are ongoing as potential therapy for solid cancers. Gain-of-function mutations of the PTPN11 gene are observed in part of the patients with the Noonan syndrome, associated with a mild bleeding disorder. Assessment of the effects of SHP2 inhibition in platelets from controls and Noonan syndrome patients. MATERIALS AND METHODS: Washed human platelets were incubated with SHP099 and stimulated with collagen-related peptide (CRP) for stirred aggregation and flow cytometric measurements. Whole-blood microfluidics assays using a dosed collagen and tissue factor coating were performed to assess shear-dependent thrombus and fibrin formation. Effects on clot formation were evaluated by thromboelastometry. RESULTS: Pharmacological inhibition of SHP2 did not alter GPVI-dependent platelet aggregation under stirring, but it enhanced integrin αIIbß3 activation in response to CRP. Using whole-blood microfluidics, SHP099 increased the thrombus buildup on collagen surfaces. In the presence of tissue factor and coagulation, SHP099 increased thrombus size and reduced time to fibrin formation. Blood from PTPN11-mutated Noonan syndrome patients, with low platelet responsiveness, after ex vivo treatment with SHP099 showed a normalized platelet function. In thromboelastometry, SHP2 inhibition tended to increase tissue factor-induced blood clotting profiles with tranexamic acid, preventing fibrinolysis. CONCLUSION: Pharmacological inhibition of SHP2 by the allosteric drug SHP099 enhances GPVI-induced platelet activation under shear conditions with a potential to improve platelet functions of Noonan syndrome patients.
Asunto(s)
Síndrome de Noonan , Trombosis , Humanos , Plaquetas/metabolismo , Síndrome de Noonan/tratamiento farmacológico , Síndrome de Noonan/genética , Síndrome de Noonan/metabolismo , Tromboplastina/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Colágeno/metabolismo , Fibrina/metabolismo , Glicoproteínas de Membrana Plaquetaria , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismoRESUMEN
The central lymphatic system consists of the thoracic duct, cisterna chyli and the retroperitoneal lymphatics through which lymph and chyle flows. Disorders of the central lymphatic system can for instance lead to leakage (chylothorax), accumulation of fluid (lymphedema) and retrograde flow (protein losing enteropathy). Abnormalities in the central lymphatic system were overlooked for years, followed by lack of diagnostic and therapeutic options. This has changed, as the technique of intranodal contrast injection in inguinal lymph nodes brought renewed interest in the central lymphatic system. In this article, the importance of intranodal contrast injection in diagnosis and treatment of disorders of the central lymphatic system will be presented through 3 clinical cases.
Asunto(s)
Quilotórax , Vasos Linfáticos , Linfedema , Quilotórax/diagnóstico , Quilotórax/etiología , Quilotórax/terapia , Humanos , Sistema Linfático/patología , Vasos Linfáticos/diagnóstico por imagen , Vasos Linfáticos/patología , Conducto Torácico/patologíaRESUMEN
Extensive lymphatic malformations (LMs) of the head and neck region may require tracheostomy to secure the airway. Treatment of these life-threatening LMs is usually multimodal and includes sclerotherapy and surgery, among others. Recently, systemic therapy with sirolimus has been introduced as an effective treatment for venous and lymphatic malformations; its efficacy and safety profile in patients with extensive LM requiring tracheostomy are, however, as yet not fully known. We performed a retrospective, multicenter review and identified 13 patients with an extensive LM of the head and neck region, who previously underwent placement of tracheostomy and subsequently received sirolimus treatment with the aim to improve the local respiratory situation and remove the tracheostomy. Under sirolimus therapy, tracheostomy could be reversed in 8/13 (62%) patients, a further 2/13 (15%) patients improved markedly, and removal of the tracheostomy was planned at the time of writing, while 3/13 (23%) patients showed insufficient or absent response to sirolimus, rendering tracheostomy reversal not feasible. The median duration of sirolimus treatment until removal of tracheostomy was 18 months (range, 8 months to 5.6 years). Adverse events of sirolimus therapy were common [10/13 (77%) patients], yet the majority of these were mild [9/10 (90%) patients] and only one severe adverse event was recorded, with ulceration and necrosis at a catheter insertion site. In conclusion, sirolimus can be considered an effective and safe salvage treatment in patients with extensive LM even after placement of a tracheostomy, as closure of the latter was possible in the majority of patients (62%) of our retrospective cohort. A better understanding of when to start sirolimus therapy, of the duration of treatment, and of factors allowing the prediction of treatment response will require further investigation.
RESUMEN
Ototoxicity and nephrotoxicity are potentially irreversible side effects of chemoradiotherapy with cisplatin in locally advanced head and neck cancer (LAHNC) patients. Several predictive genetic variants have been described, but as yet none in LAHNC patients. The aim of this study is to investigate genetic variants as predictors for ototoxicity and nephrotoxicity in LAHNC patients treated with cisplatin-containing chemoradiotherapy. Our prospective cohort of 92 patients was genotyped for 10 genetic variants and evaluated for their association with cisplatin-induced ototoxicity (ACYP2, COMT, TPMT and WFS1) and nephrotoxicity (OCT2, MATE and XPD). Ototoxicity was determined by patient-reported complaints as well as tone audiometrical assessments. Nephrotoxicity was defined as a decrease of ≥25% in creatinine clearance during treatment compared to baseline. A significant association was observed between carriership of the A allele for rs1872328 in the ACYP2 gene and cisplatin-induced clinically determined ototoxicity (p = 0.019), and not for ototoxicity measured by tone audiometrical assessments (p = 0.449). Carriership of a T allele for rs316019 in the OCT2 gene was significantly associated with nephrotoxicity at any time during chemoradiotherapy (p = 0.022), but not with nephrotoxicity at the end of the chemoradiotherapy. In conclusion, we showed prospectively that in LAHNC patients genetic variants in ACYP2 are significantly associated with clinically determined ototoxicity. Validation studies are necessary to prove the added value for individualized treatments plans in these patients.
RESUMEN
BACKGROUND: Children with acute myeloid leukemia (AML) have a 70% survival rate with treatment regimens containing high doses of cytarabine and anthracyclines and, in some, hematopoietic stem cell transplantation (allo-HSCT). PROCEDURE: In this multicenter Dutch-Belgian protocol (DB AML-01), 112 children with de novo AML were included. Treatment was stratified according to day 15 bone marrow response after the first induction course. Poor responders received a second course without delay while good responders awaited hematological recovery. Patients achieving CR after two induction courses continued with three consolidation courses without HSCT in CR1. RESULTS: The overall remission rate was 93.5%. After a median follow-up of 4.1 years, three-year event-free survival (EFS) was 52.6% (95% CI, 42.9%-61.3%), three-year cumulative incidence of relapse 39.7% (95% CI, 30.1%-49.0%), and three-year overall survival (OS) 74.0% (95% CI, 64.8%-81.2%). Significantly more events occurred in patients with high WBC at diagnosis or FLT3-ITD/NPM1-WT, whereas core binding factor (CBF) leukemia had a significantly better EFS. KMT2A rearrangements and age > 10 years negatively impacted OS. CONCLUSIONS: DB AML-01 response-guided therapy results in a favorable OS, particularly for children with CBF leukemia, children younger than 10 years or with initial WBC counts below 100 × 109 /L. Outcome of patients with FLT3-ITD/NPM1-WT remains poor and warrants alternative treatment strategies.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Adolescente , Antraciclinas/administración & dosificación , Niño , Preescolar , Citarabina/administración & dosificación , Femenino , Estudios de Seguimiento , Humanos , Lactante , Recién Nacido , Leucemia Mieloide Aguda/patología , Masculino , Nucleofosmina , Pronóstico , Estudios Prospectivos , Inducción de Remisión , Tasa de SupervivenciaRESUMEN
BACKGROUND: The efficacy and safety of recombinant Escherichia coli-asparaginase (rASNase) was compared to native E.coli asparaginase (Asparaginase medac). METHODS: One hundred and ninety-nine children with newly diagnosed acute lymphoblastic leukemia were randomized to receive one of both agents at a dose of 5,000 U/m² during induction (eight doses) and 10,000 U/m² during the postinduction phase (only high-risk patients; standard- and medium-risk patients received pegaspargase). RESULTS: Median trough serum asparaginase activity levels were comparable between both groups; they ranged from 143 to 182 U/l during induction and were above the target value of 100 U/l. Complete asparagine depletion in serum was achieved in 97.9% of patients, with no significant differences between both groups. On day 33 (end of induction), only two (2%) evaluable patients in each group had measurable asparagine serum levels, and complete asparagine depletion in the cerebrospinal fluid was achieved in 98.8% and 93.6% of the patients with rASNase and Asparaginase medac, respectively. During induction, 2.1% and 5% of patients developed an allergic reaction to rASNase or Asparaginase medac, respectively. Approximately 41% of the patients in both groups had a clinical allergy or enzyme inactivation to the first dose of any asparaginase preparation in postinduction. A comparable proportion of patients in both groups developed anti-asparaginase antibodies (57%) during repeated administration of asparaginase. Minimal residual disease levels at the end of induction, 5-year event-free survival, and 5-year cumulative incidence of relapse did not differ between both groups. CONCLUSION: The efficacy, safety, and immunogenicity of both asparaginase preparations are comparable. This trial was registered at www.clinicaltrials.gov as #NCT00784017; EudraCT number 2006-003180-31.
Asunto(s)
Antineoplásicos/uso terapéutico , Asparaginasa/uso terapéutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Adolescente , Niño , Preescolar , Escherichia coli , Femenino , Humanos , Lactante , Masculino , Proteínas Recombinantes/uso terapéutico , Resultado del TratamientoRESUMEN
INTRODUCTION: Diamond-Blackfan anemia (DBA) is characterized by hypoplastic anemia, congenital anomalies, and a predisposition for malignancies. Most of our understanding of this disorder stems from molecular studies combined with extensive data input from international patient registries. OBJECTIVES: To create an overview of the pediatric DBA population in the Netherlands. METHODS: Forty-three patients diagnosed with DBA from all Dutch university pediatric hospitals were included in this study, and their clinical and genetic characteristics were collected from patient records. RESULTS: Congenital malformations were present in 24 of 43 patients (55.8%). An underlying genetic defect was identified in 26 of 43 patients (60.5%), the majority of which were found in the RPS19 gene (12 of 43, 27.9%) with 1 patient carrying a mutation in a novel DBA candidate gene, RPL9. In 31 of 35 (88.6%) patients, an initial response to glucocorticoid treatment was observed. Six patients (14.0%) underwent hematopoietic stem cell transplantation, and eleven patients (11 of 43, 25.6%) became treatment-independent spontaneously. CONCLUSION: In agreement with previous reports, the Dutch pediatric DBA population is both clinically and genetically heterogeneous. National and international registries, together with more extensive genetic testing, are crucial to increase our understanding of genotype and phenotype correlations of this intriguing disorder.
Asunto(s)
Anemia de Diamond-Blackfan/diagnóstico , Anemia de Diamond-Blackfan/genética , Adolescente , Anemia de Diamond-Blackfan/epidemiología , Anemia de Diamond-Blackfan/terapia , Niño , Preescolar , Terapia Combinada , Anomalías Congénitas/diagnóstico , Anomalías Congénitas/genética , Femenino , Estudios de Seguimiento , Estudios de Asociación Genética , Pruebas Genéticas , Variación Genética , Genotipo , Humanos , Lactante , Recién Nacido , Masculino , Países Bajos/epidemiología , Fenotipo , Polimorfismo de Nucleótido Simple , Sistema de RegistrosRESUMEN
Erwinia asparaginase is an important component in the treatment of pediatric acute lymphoblastic leukemia. A large variability in serum concentrations has been observed after intravenous Erwinia asparaginase. Currently, Dutch Childhood Oncology Group protocols dose alterations are based on trough concentrations to ensure adequate asparaginase activity (≥100 IU/L). The aim of this study was to describe the population pharmacokinetics of intravenous Erwinia asparaginase to quantify and gather insight into inter-individual and inter-occasion variability. The starting dose was evaluated on the basis of the derived population pharmacokinetic parameters. In a multicenter prospective observational study, a total of 714 blood samples were collected from 51 children (age 1-17 years) with acute lymphoblastic leukemia. The starting dose was 20,000 IU/m2 three times a week and adjusted according to trough levels from week three onwards. A population pharmacokinetic model was developed using NONMEM® A 2-compartment linear model with allometric scaling best described the data. Inter-individual and inter-occasion variability of clearance were 33% and 13%, respectively. Clearance in the first month of treatment was 14% higher (P<0.01). Monte Carlo simulations with our pharmacokinetic model demonstrated that patients with a low weight might require higher doses to achieve similar concentrations compared to patients with high weight. The current starting dose of 20,000 IU/m2 might result in inadequate concentrations, especially for smaller, lower weight patients, hence dose adjustments based on individual clearance are recommended. The protocols were approved by the institutional review boards. (Registered at NTR 3379 Dutch Trial Register; www.trialregister.nl).
Asunto(s)
Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Asparaginasa/administración & dosificación , Asparaginasa/farmacocinética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Administración Intravenosa , Adolescente , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Modelos Estadísticos , Vigilancia de la Población , Reproducibilidad de los ResultadosRESUMEN
Treatment with cisplatin-containing chemotherapy regimens causes hearing loss in 40-60% of cancer patients. It has been suggested that genetic variants in the genes encoding thiopurine S-methyltransferase (TPMT) and catechol O-methyltransferase (COMT) can predict the development of cisplatin-induced ototoxicity and may explain interindividual variability in sensitivity to cisplatin-induced hearing loss. Two recently published studies however, sought to validate these findings and showed inconsistent results. The aim of this study was to evaluate the role of polymorphisms in the TPMT and COMT genes in cisplatin-induced ototoxicity. Therefore we investigated two independent cohorts of 110 Dutch and 38 Spanish patients with osteosarcoma and performed a meta-analysis including all previously published studies resulting in a total population of 664 patients with cancer. With this largest meta-analysis performed to date, we show that the influence of TPMT and COMT on the development of cisplatin-induced hearing loss may be less important than previously suggested.
Asunto(s)
Catecol O-Metiltransferasa/genética , Cisplatino/efectos adversos , Pérdida Auditiva/inducido químicamente , Pérdida Auditiva/genética , Metiltransferasas/genética , Adolescente , Adulto , Antineoplásicos/efectos adversos , Antineoplásicos/uso terapéutico , Neoplasias Óseas/tratamiento farmacológico , Niño , Preescolar , Cisplatino/uso terapéutico , Femenino , Genotipo , Humanos , Masculino , Países Bajos , Osteosarcoma/tratamiento farmacológico , Polimorfismo de Nucleótido Simple , España , Adulto JovenRESUMEN
BACKGROUND: UNC13D, encoding the protein munc13-4, is essential in intracellular trafficking and exocytosis of lytic granules. Mutations in this gene are associated with familial hemophagocytic lymphohistiocytosis type 3 (FHL3), a genetically heterogeneous, rare autosomal recessive immune disorder. How mutations affect function of munc13-4 is poorly understood. Since 2006 we genetically identified seven FHL patients with mutations in UNC13D. PROCEDURES: Here, we report for the first time a c.2695C>T (p.Arg899X) mutation in exon 28 of UNC13D in three young unrelated Dutch patients. The mutation causes a premature stop codon and encodes munc13-4(1-899), which lacks the C-terminal C2 domain. Genealogical research and haplotyping of the patient families demonstrated that a single ancestral founder introduced the mutation in the Netherlands. We then characterized the mutant protein phenotypically in cell biological and immunological assays. RESULTS: Munc13-4(1-899) was correctly targeted to CD63-positive secretory lysosomes, although its stability was reduced and dynamic turnover on the granule membrane became uncoupled from receptor signaling. In accord, and in contrast to wild-type munc13-4, ectopically expressed mutant failed to rescue degranulation in cells with silenced endogenous munc13-4. CONCLUSIONS: The functional and clinical data showed that this novel Dutch founder mutation leads to severe early onset of FHL3 due to misfolding and degradation of munc13-4(1-899).
Asunto(s)
Codón de Terminación , Linfohistiocitosis Hemofagocítica , Proteínas de la Membrana , Mutación Puntual , Pliegue de Proteína , Proteínas , Proteolisis , Animales , Degranulación de la Célula/genética , Línea Celular Tumoral , Humanos , Linfohistiocitosis Hemofagocítica/genética , Linfohistiocitosis Hemofagocítica/metabolismo , Lisosomas/genética , Lisosomas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Países Bajos , Estructura Terciaria de Proteína , Proteínas/genética , Proteínas/metabolismo , Ratas , Tetraspanina 30/genética , Tetraspanina 30/metabolismoRESUMEN
The typical form of hemolytic uremic syndrome (D+HUS) is a thrombotic microangiopathy that causes acute renal failure in children. The etiology of this disease is a toxin called Shiga-like toxin (Stx), present in certain strains of gram-negative bacteria. Vascular endothelial cell (EC) injury appears to be central in the pathogenesis of D+HUS. Thrombomodulin (TM) is a glycoprotein present in EC with anti-thrombogenic properties. The objective of this study was to investigate the effects of Stx on the surface expression of TM in EC using an in vitro culture of human glomerular microvascular endothelial cells. We also evaluated other inflammatory mediators [tumor necrosis factor-alpha (TNF-alpha) and lipopolysaccharide], which are known to increase Stx receptor expression and are potentially involved in the pathogenesis of D+HUS. Stx2 induced a significant decrease of TM expression in this cell type after pre-incubation with TNF-alpha. This decrease could not be attributed to the inhibition of protein synthesis only, as cycloheximide, another inhibitor of protein synthesis, did not affect TM surface expression. These results suggest that the Stx2-induced decrease of TM expression in glomerular EC might contribute to the local procoagulant state present in D+HUS.
Asunto(s)
Endotelio Vascular/metabolismo , Mesangio Glomerular/irrigación sanguínea , Síndrome Hemolítico-Urémico/etiología , Síndrome Hemolítico-Urémico/metabolismo , Trombomodulina/metabolismo , Células Cultivadas , Células Endoteliales/citología , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Humanos , Mediadores de Inflamación/metabolismo , Leucina/farmacocinética , Microcirculación , Toxina Shiga II/farmacología , TritioRESUMEN
BACKGROUND: Plasma hemopexin has been shown to induce proteinuria after intrarenal infusion in rats, as well as glomerular alterations identical to those seen in corticosteroid-responsive nephrotic syndrome (CRNS). The question emerged whether also renal cells are potentially able to release hemopexin. METHODS: Normal human mesangial cells (HMC) were incubated overnight in serum-free medium with or without tumor necrosis factor-alpha (TNF-alpha) (10 ng/mL). Parallel cultures were supplemented with prednisolone (10-3 mol/L). Concentrated supernatants were analyzed by Western blotting, using antihemopexin immunoglobulin G (IgG). Antitransferrin IgG served as control antibody. In addition, cytospins were stained using polyclonal or monoclonal antihemopexin IgG. A part of the cells was used for RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR), to study hemopexin mRNA. RESULTS: Eighty five kD bands were exclusively detected by antihemopexin IgG in the Western blots in supernatants from TNF-alpha-stimulated cultures and to a lesser extent in prednisolone-treated cultures. Cells from TNF-alpha-stimulated cultures stain positive for hemopexin in contrast to those from prednisolone-treated or nonstimulated cultures. RT-PCR data suggest that mRNA for hemopexin is up-regulated in TNF-alpha-treated versus prednisolone-treated HMC. CONCLUSION: Stimulated HMC are able to release hemopexin in vitro in a corticosteroid-dependent manner. As preliminary data indicate that mesangial hemopexin is able to affect glomerular anionic sites, it is conceivable that stimulated mesangium may contribute to enhanced glomerular permeability in CRNS through local hemopexin release.
Asunto(s)
Antineoplásicos/farmacología , Mesangio Glomerular/citología , Mesangio Glomerular/fisiología , Hemopexina/genética , Factor de Necrosis Tumoral alfa/farmacología , Antiinflamatorios/farmacología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/fisiología , Expresión Génica/efectos de los fármacos , Humanos , Síndrome Nefrótico/fisiopatología , Prednisolona/farmacologíaRESUMEN
The diarrhea-associated form of the hemolytic uremic syndrome (D+ HUS) is characterized by a triad of symptoms, namely thrombocytopenia, hemolytic anemia, and acute renal failure. Histopathological studies of patients with D+ HUS show microthrombi in arterioles and glomeruli of the kidney. Recently, it was suggested that antiphospholipid antibodies might play a pathogenic role in D+ HUS. However, an epiphenomenon could not be excluded. In this study we investigated the relationship between antiphospholipid antibodies and clinical symptoms in 22 patients with the classical form of HUS (D+ HUS). The first sample was obtained on the day of admission. The next samples were taken on day 7 and 14. We measured anticardiolipin (aCL) antibodies (IgM, IgA, and IgG) in the samples using an ELISA. A significant increase in IgM (60%) and IgG (41%) aCL antibodies was seen in patients versus controls. No relationship between aCL antibody levels and severity of renal failure could be demonstrated. These data suggest that antiphospholipid antibodies are increased, but have not been shown to have a role in the pathogenesis of the microangiopathy seen in D+HUS.
Asunto(s)
Anticuerpos Antifosfolípidos/análisis , Cardiolipinas/inmunología , Síndrome Hemolítico-Urémico/inmunología , Enfermedad Aguda , Niño , Preescolar , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunoglobulina A/análisis , Inmunoglobulina G/análisis , Inmunoglobulina M/análisis , Lactante , Pruebas de Función Renal , Recuento de Leucocitos , Masculino , Diálisis Peritoneal , Recuento de PlaquetasRESUMEN
BACKGROUND: Shiga toxin (Stx)-producing E. coli is a causative agent of the epidemic form of hemolytic uremic syndrome (HUS), the most common cause of acute renal failure in children. Endothelial injury and leukocyte activation are instrumental to the development of microangiopathic lesions. To obtain more insight into the mechanisms favoring endothelium-leukocyte interaction, we studied (1) the effect of Stx-2 on leukocyte adhesion and transmigration in human endothelial cells under flow; (2) the effect of Stx-2 on endothelial expression of monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) and their functional role in the adhesive phenomena; and (3) the role of nuclear factor-kappaB (NF-kappaB) in endothelial chemokine expression. METHODS: For adhesion experiments, human umbilical vein endothelial cells (HUVEC) and human glomerular endothelial cells (GEC) were incubated for 24 hours with Stx-2 (25 pmol/L), with or without anti-IL-8 or MCP-1 antibodies, and then exposed to leukocyte suspension under flow (1.5 dynes/cm2). IL-8 and MCP-1 expression was evaluated in Stx-2 treated endothelial cells (6 hours) by Northern blot. NF-kappaB activity was assessed by electrophoretic mobility shift assay. The role of NF-kappaB in Stx-induced chemokines was evaluated by transfecting HUVEC with an adenovirus coding for IkappaBalpha. RESULTS: Stx-2 significantly enhanced the number of leukocytes that adhered and then migrated across the endothelium. Stx-2 increased the expression of IL-8 and MCP-1, which was preceded by NF-kappaB activation. Blocking of endothelial IL-8 and MCP-1 with corresponding antibodies significantly inhibited Stx-induced leukocyte adhesion and migration either in HUVEC or GEC. Adenovirus-mediated gene transfer of IkappaBalpha down-regulated IL-8 and MCP-1 mRNA and also inhibited the adhesion and transmigration of leukocytes in Stx-treated HUVEC. CONCLUSIONS: Stx-2 via a transcriptional activation mechanism specifically mediated by NF-kappaB up-regulates endothelial MCP-1 and IL-8. Both chemokines are important modulators of leukocyte adhesion and transmigration under flow. These findings might be relevant to understand the nature of microvascular lesions in HUS and open future perspectives for better treatment of microvascular thrombosis.