RESUMO
Thrombopoietin (Tpo) is the main hematopoietic growth factor for platelet production. Plasma Tpo levels in autoimmune thrombocytopenic patients are normal or slightly elevated. Although thrombocytopenia exists, Tpo levels are not increased because the produced megakaryocytes and platelets can bind circulating Tpo, thereby normalizing Tpo levels. In this study, plasma samples from fetuses and neonates with neonatal alloimmune thrombocytopenia (NAIT), a different form of immune thrombocytopenia, were measured. Umbilical cord samples from 50 fetuses before treatment because of severe thrombocytopenia and 51 fetuses after treatment, and peripheral blood samples of 21 untreated newborns with NAIT were analyzed. As controls, plasma Tpo levels were determined in 21 umbilical cord samples of 14 nonthrombocytopenic fetuses with hemolytic disease resulting from red blood cell alloimmunization and in umbilical cord samples of 51 healthy newborns. The values were also compared with the plasma Tpo levels in 193 healthy adults. Mean Tpo levels from the groups of fetuses and neonates, including both NAIT and control plasma, were slightly but significantly elevated compared with levels in healthy adults. Tpo levels in NAIT samples were not significantly different from the levels in hemolytic disease samples or in samples from healthy newborns. Thus, as in autoimmune thrombocytopenic patients, normal Tpo levels are present in NAIT patients.
Assuntos
Feto/citologia , Doenças do Recém-Nascido/sangue , Púrpura Trombocitopênica Idiopática/sangue , Trombopoetina/sangue , Humanos , Recém-Nascido , Contagem de Plaquetas , Cordão UmbilicalRESUMO
Congenital amegakaryocytic thrombocytopenia (CAMT) is an uncommon cause of thrombocytopenia in children. CAMT is characterised by an isolated thrombocytopenia and the near absence of megakaryocytes in the bone marrow. The gene involved in the development of CAMT has recently been described. In a number of CAMT patients, mutations in the thrombopoietin (Tpo) receptor gene, c-mpl, were found to be the likely cause of the thrombocytopenia and the complete bone marrow failure that most patients develop. Measurement of Tpo plasma levels and a study of the megakaryocytopoiesis in vitro, may add to the diagnosis. At present the only curative treatment is allogeneic bone marrow transplantation.
Assuntos
Proteínas de Neoplasias , Proteínas Proto-Oncogênicas/genética , Receptores de Citocinas , Trombocitopenia/congênito , Transplante de Medula Óssea , Criança , Humanos , Megacariócitos/citologia , Mutação , Receptores de Trombopoetina , Trombocitopenia/genética , Trombocitopenia/terapia , Trombopoetina/metabolismoAssuntos
Isoantígenos/genética , Glicoproteínas de Membrana/genética , Neutrófilos/imunologia , Clonagem Molecular , Proteínas Ligadas por GPI , Humanos , Isoantígenos/química , Isoantígenos/imunologia , Isoantígenos/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Receptores de Superfície CelularRESUMO
Thrombopoietin (Tpo), the main regulator of thrombocytopoiesis, is a probable candidate to play a role in the increase in platelet counts that is frequently seen after surgery. In the current study, serial blood samples of patients that underwent major surgery were analysed with respect to Tpo kinetics, platelet turnover and inflammatory cytokines. Platelet Tpo content and plasma Tpo levels rose before platelet counts increased, suggesting that Tpo was indeed responsible for the elevation in platelet counts. In addition, an increase in interleukin 6 (IL-6) levels, but not in IL-11 and tumour necrosis factor alpha levels, was seen before the rise in Tpo concentration. In vitro, IL-6 was shown to enhance Tpo production by the HepG2 liver cell line. Thus, increased Tpo levels after surgery, possibly resulting from enhanced Tpo production under the influence of IL-6 or other inflammatory cytokines, are involved in an enhanced thrombocytopoiesis.
Assuntos
Interleucina-6/análise , Complicações Pós-Operatórias/sangue , Trombocitose/sangue , Trombopoetina/sangue , Idoso , Artroplastia de Quadril , Artroplastia do Joelho , Plaquetas/química , Linhagem Celular , Células Cultivadas , Ponte de Artéria Coronária , Feminino , Humanos , Interleucina-6/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Pessoa de Meia-Idade , Contagem de Plaquetas , Trombopoetina/análiseRESUMO
In multiple myeloma (MM), suppression of haematopoiesis occurs as a result of expansion of malignant cells in the bone marrow. Thrombopoietin (Tpo) levels in patients with impaired platelet production are generally found to be highly elevated. To examine the circulating Tpo levels in patients with MM, Tpo levels were measured in 50 serum samples from 34 patients. Tpo levels were subsequently related to disease stage, and cell numbers and markers, i.e. platelet count, leukocyte count and haemoglobin (Hb) concentration. Elevated Tpo levels were found in association with decreased platelet counts (n=8), but also in patients with normal platelet counts (n=14). The latter group included patients without and with signs of impaired haematopoiesis, i.e. with decreased Hb concentration and decreased leukocyte count. These results show that neither platelet counts nor Tpo levels are reliable parameters to judge bone-marrow failure in patients with MM. Furthermore, in patients with MM, increased Tpo levels may play a role in the maintenance of thrombocytopoiesis. The origin of the increased Tpo levels remains to be determined.
Assuntos
Plaquetas , Hematopoese/fisiologia , Mieloma Múltiplo/sangue , Trombopoetina/sangue , Idoso , Idoso de 80 Anos ou mais , Feminino , Hemoglobinas/análise , Humanos , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Contagem de Plaquetas , Estudos RetrospectivosRESUMO
Examining peripheral blood smears provides valuable information in the diagnosis of anaemia despite large inter- and intraobserver variation. The classification of anaemia is usually based on the average erythrocyte size, referred to as the mean corpuscular volume (MCV). Microcytosis indicates a reduced haemoglobin synthesis caused by either an iron deficiency or haemoglobinopathy, a congenital disorder. Macrocytosis is the result of a disruption to the division and maturing of proerythroblasts in the bone marrow, due, for example, to vitamin B12 (folic acid) deficiency or excessive alcohol use. Furthermore, a high number of reticulocytes in the blood indicates an increased production of erythrocytes whereas a low total indicates an inadequate production level. In addition to the case history and the physical examination, the MCV and number of reticulocytes can provide guidance with respect to further diagnostic investigation.
Assuntos
Anemia/diagnóstico , Contagem de Eritrócitos/métodos , Algoritmos , Anemia/classificação , Diagnóstico Diferencial , Índices de Eritrócitos , Humanos , Internet , Contagem de ReticulócitosRESUMO
Oral mucositis is a frequent side effect of myeloablative chemo- and radiotherapy preceding stem cell transplantation. It causes pain, poor food intake, and is a port of entry for infection. We studied whether GM-CSF applied topically in the oral cavity can prevent or ameliorate this mucositis. In 36 consecutive patients undergoing a stem cell transplantation, we performed a double-blind placebo-controlled study of 300 micrograms GM-CSF in a 2% methylcellulose gel daily versus a 2% methylcellulose gel alone. Both were locally applied in the oral cavity. The primary end-point was mucositis as measured by the WHO toxicity scale for mucositis, oral assessment scale, and a subjective pain scale, all scored daily. The secondary end-points were need to give parenteral nutrition and morphine, incidence of fever and infections, and duration of neutropenia and hospitalization. No differences were found in the median subjective pain scores, WHO scores, and oral assessment scores between the placebo and the GM-CSF groups. In both groups, nine patients required morphine for pain control. Ten patients in the placebo group and 11 in the GM-CSF group received parenteral nutrition. Documented infections, use of broad-spectrum antibiotics, and number of days with fever were similar in the placebo and the GM-CSF groups. The duration of neutropenia below 0.5 x 10(9)/l (median 14.5 days in the placebo group versus 17 days in the GM-CSF group) and the duration of hospitalization (28.5 versus 29 days) was also not significantly different. We found no beneficial effect of 300 micrograms GM-CSF dissolved in a 2% methylcellulose gel applied locally for chemo- and radiotherapy-induced mucositis in patients undergoing a stem cell transplantation.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Transplante de Células-Tronco Hematopoéticas , Estomatite/prevenção & controle , Administração Tópica , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Método Duplo-Cego , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Humanos , Masculino , Pessoa de Meia-Idade , Mucosa Bucal , Agonistas Mieloablativos/efeitos adversos , Estudos Prospectivos , Estomatite/induzido quimicamenteRESUMO
BACKGROUND: It has been shown in several studies that platelets play a role in the removal of TPO from the circulation. For instance, in vitro studies have shown that platelets can bind and internalize TPO, and transfusion studies have shown that the concentration of circulating TPO decreased after platelet transfusion. In the current study, the in vivo kinetics of plasma TPO levels and TPO uptake by transfused platelets is analyzed in more detail. STUDY DESIGN AND METHODS: Serial blood samples from patients who received a platelet transfusion were analyzed with respect to platelet count, plasma TPO concentration, and TPO content per platelet. In addition, the capacity of transfused platelets to bind TPO in vitro was assessed. RESULTS: Platelet counts increased immediately after transfusion, but subsequently started to decrease. Conversely, TPO levels decreased significantly but then returned to baseline level by 44 hours after transfusion. Platelet count and plasma TPO concentration were inversely correlated (r(p) = -0.9; p<0.05). The decrease in TPO concentration upon transfusion was accompanied by a significant increase in the platelet-associated TPO concentration. After transfusion, platelets isolated from the patient still displayed functional TPO receptors, as indicated by their intact capacity to bind TPO in vitro. CONCLUSION: The decrease in plasma TPO followed by the increase in platelet TPO provides evidence that platelets are responsible for the clearance of TPO in circulation. In vivo, platelets can bind and may degrade TPO upon platelet transfusion.
Assuntos
Transfusão de Plaquetas , Trombopoetina/sangue , Humanos , Cinética , Contagem de PlaquetasRESUMO
Antigen receptor gene rearrangements are applied for the PCR-based minimal residual disease (MRD) detection in acute lymphoblastic leukemia (ALL). It is known that ongoing rearrangements result in subclone formation, and that the relapsing subclone(s) can contain antigen receptor rearrangement(s) that differ from the rearrangements found in the major clone(s) at diagnosis. However, the mechanism leading to this so-called clonal evolution is not known, particularly at which time point in the disease the relapsing subclone obtains its (relative) therapy resistance. To obtain insight in clonal evolution, we followed the kinetics of several subclones in three oligoclonal ALL patients during induction therapy. Clone-specific nested PCR for immunoglobulin heavy chain or T cell receptor delta gene rearrangements were performed in limiting dilution assays on bone marrow samples taken at diagnosis, at the end of induction therapy and at possible relapse in three children with oligoclonal B-precursor ALL. We demonstrated that in all three patients the subclones were behaving differently in response to therapy. Moreover, in the two patients who relapsed, the clones that grew out during relapse showed the slowest regression or even evoluated during induction therapy and the clones that were not present at relapse showed good response to induction therapy. These results support the hypothesis that at least in some patients already at diagnosis or in the very first weeks, subclones have important differences in respect to resistance. Hence, these data give experimental evidence for the need to develop, during the first months after diagnosis, quantitative PCR assays for at least two different Ig/TCR gene rearrangement targets for every ALL patient.
Assuntos
Linfócitos B/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Diferenciação Celular , Divisão Celular , Criança , Pré-Escolar , Humanos , Lactente , Masculino , Células-Tronco Neoplásicas/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , RecidivaRESUMO
Reinfusion of ex vivo-expanded autologous megakaryocytes together with a stem cell transplantation may be useful to prevent or reduce the period of chemotherapy-induced thrombocytopenia. In this study, we analyzed several serum-containing and serum-free media to identify the most suitable medium for megakaryocyte expansion. Moreover, two thrombopoietin (Tpo)-mimetic peptides were tested to evaluate whether they could replace Tpo in an expansion protocol. To analyze the effects of different media on megakaryocyte expansion, we used an in vitro liquid culture system. For this purpose, CD34(+) cells were isolated from peripheral blood and cultured for 8 days in the presence of Tpo and interleukin-3 (IL-3). The presence of megakaryocytes was analyzed by flow cytometric analysis after staining for CD41 expression. For our standard culture procedure, megakaryocyte medium (MK medium) supplemented with 10% AB plasma was used. Addition of 5% or 2.5% AB plasma yielded higher numbers of megakaryocytes, implying the presence of inhibitory factors in plasma. However, some plasma components are required for optimal megakaryocyte expansion because addition of less than 1% AB plasma or addition of human serum albumin instead of AB plasma resulted in the formation of lower numbers of megakaryocytes. Two commercially available serum-free media were also tested: Cellgro and Stemspan. If CD34(+) cells were cultured in Cellgro medium similar numbers of megakaryocytes were obtained as when CD34(+) cells were cultured in MK medium supplemented with 10% AB plasma. In MK medium with 2.5% AB plasma, higher numbers of megakaryocytes were cultured than in MK medium supplemented with 10% AB plasma. Therefore, Cellgro medium is not the best alternative medium. In cultures with Stemspan medium, higher numbers of megakaryocytes were obtained compared to MK medium with 10% AB plasma. Stemspan is thus a good alternative for MK medium. Two Tpo-mimetic peptides, AF13948 and PK1M, were tested for their ability to replace Tpo. In cultures with AF13948, comparable numbers of megakaryocytes were obtained as in the presence of Tpo, but in cultures with PK1M the number of megakaryocytes was lower. This study shows that high concentrations of plasma in medium inhibits megakaryocyte formation, but some plasma components are required for optimal megakaryocyte expansion. For an ex vivo expansion protocol, it is worthwhile to test several media, because the number of megakaryocytes differs widely with the medium used.
Assuntos
Meios de Cultura/farmacologia , Megacariócitos/citologia , Megacariócitos/efeitos dos fármacos , Antígenos CD34/análise , Técnicas de Cultura de Células/métodos , Divisão Celular/efeitos dos fármacos , Meios de Cultura/química , Peptídeos e Proteínas de Sinalização Intercelular , Mimetismo Molecular , Peptídeos/farmacologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/análise , Trombopoetina/farmacologiaRESUMO
To characterize fetal thrombopoiesis, we determined plasma thrombopoietin (TPO) and glycocalicin levels, platelet counts and reticulated platelets (RP) of fetuses and compared them with the respective values of their mothers. Percutaneous umbilical vein sampling in abnormal pregnancies revealed twofold higher thrombopoietin levels and 20-fold higher reticulated platelet counts, but lower levels of glycocalicin in fetuses compared with their mothers (P < 0.05). Neither the expression of platelet glycoprotein Ib and IIb on platelets nor the platelet counts were different between mothers and their fetuses. These data indicate enhanced thrombopoiesis and/or increased platelet turnover in fetuses.
Assuntos
Plaquetas/fisiologia , Sangue Fetal/química , Hematopoese , Trombopoetina/análise , Adulto , Feminino , Sangue Fetal/citologia , Humanos , Contagem de Leucócitos , Neutrófilos/citologia , Inibidores da Agregação Plaquetária/análise , Complexo Glicoproteico GPIb-IX de Plaquetas/análise , Gravidez , Segundo Trimestre da GravidezRESUMO
Several lines of evidence suggest that coagulation may induce the release of thrombopoietin (TPO) into plasma and that TPO levels are higher in disseminated intravascular coagulation. Therefore we set out to illuminate the mechanism of TPO release in the setting of experimental endotoxemia, which induces activation of coagulation and platelets. Endotoxin (lipopolysachharide [LPS], 2 ng/kg) was infused into a total of 54 healthy men in two subsequent studies. Volunteers received infusions of unfractionated heparin, low-molecular-weight heparin, lepirudin, or placebo in a randomized, placebo-controlled fashion after bolus injection of LPS. TPO levels increased on average by 27% to 38% in all groups at 6 hours (P <.05 vs baseline), although all active drugs effectively blocked coagulation. Platelet counts dropped by about 15% at 1 hour after LPS infusion, recovered after 2 days, and exceeded baseline values by 8% to 18% after 7 days (P <.001 vs baseline for all groups). Yet lepirudin blunted the LPS-induced increase in circulating P-selectin by one half (P <.005 vs placebo), whereas both heparins did not diminish the increase in this platelet or endothelial activation marker as compared with placebo. Endotoxemia enhances TPO plasma levels independent of the degree of coagulation induction, which eventually results in increased platelet numbers. Of potential clinical interest is the observation that the direct thrombin inhibitor lepirudin, in contrast to heparins, mitigated LPS-induced platelet activation.
Assuntos
Anticoagulantes/administração & dosagem , Endotoxemia/tratamento farmacológico , Endotoxemia/metabolismo , Heparina/administração & dosagem , Hirudinas/análogos & derivados , Ativação Plaquetária/efeitos dos fármacos , Trombopoetina/metabolismo , Adulto , Antitrombinas/administração & dosagem , Proteína C-Reativa/metabolismo , Método Duplo-Cego , Endotoxemia/induzido quimicamente , Hirudinas/administração & dosagem , Humanos , Interleucina-6/sangue , Lipopolissacarídeos/efeitos adversos , Masculino , Selectina-P/sangue , Fragmentos de Peptídeos/sangue , Contagem de Plaquetas , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Precursores de Proteínas/sangue , Protrombina , Proteínas Recombinantes/administração & dosagem , SolubilidadeRESUMO
Adhesion of CD34+ hematopoietic progenitor cells (HPCs) to sinusoidal endothelium probably plays a key role in homing of transplanted CD34+ HPCs to the bone marrow (BM). We have investigated the role of various adhesion molecules in the interaction of purified CD34+ HPCs derived from BM or peripheral blood (PB) and a human BM-derived endothelial cell line. Adhesion of CD34+ HPCs to endothelial cells was measured with the use of a double-color flow microfluorimetric adhesion assay. In this assay, adhesion is measured under stirring conditions, simulating blood flow in sinusoidal marrow vessels. Adhesion of PB CD34+ cells to human BM endothelial cells (HBMECs) was observed only after interleukin (IL)-1beta prestimulation of the endothelial cells. This adhesion was strongly increased after addition of phorbol-myristate acetate (PMA). Adhesion of PB CD34+ cells to IL-1beta-prestimulated HBMECs was inhibited by blocking monoclonal antibodies (mAbs) against E-selectin and by neuraminidase treatment of the PB CD34+ cells. mAbs against very late activation antigen (VLA)-4 inhibited adhesion only when the E-selectin-mediated interaction was prevented. No clear inhibiting effect was found with blocking mAbs against beta2-integrins. Stimulation with the beta1-integrin-activating mAb, 8A2, induced adhesion of CD34+ cells to endothelial cells. In conclusion, stimulation of both endothelial cells and CD34+ HPCs is necessary for adhesion of CD34+ HPCs to endothelial cells. We furthermore demonstrated that E-selectin and VLA-4 mediated this adhesion.
Assuntos
Células da Medula Óssea/citologia , Selectina E/farmacologia , Endotélio Vascular/citologia , Células-Tronco Hematopoéticas/citologia , Integrinas/fisiologia , Receptores de Retorno de Linfócitos/fisiologia , Molécula 1 de Adesão de Célula Vascular , Antígenos CD34/sangue , Adesão Celular/efeitos dos fármacos , Adesão Celular/imunologia , Linhagem Celular , Células-Tronco Hematopoéticas/imunologia , Humanos , Integrina alfa4beta1 , Molécula 1 de Adesão de Célula Vascular/fisiologiaRESUMO
OBJECTIVE: Reinfusion of ex vivo expanded autologous megakaryocytes together with stem cell transplantation may be useful to prevent or reduce the period of chemotherapy-induced thrombocytopenia. We compared the megakaryocyte expansion potential of CD34(+) stem cells derived from different sources: cord blood (CB), peripheral blood (PB), bone marrow from adults (ABM), and bone marrow from children (ChBM). Three different growth factor combinations were tested to identify the best combination for each of the sources. MATERIALS AND METHODS: CD34(+) cells were isolated from CB, PB, ABM, or ChBM and cultured in an in vitro liquid culture system in the presence of thrombopoietin (Tpo), Tpo + interleukin (IL-1), or Tpo + IL-3. After 8 days, proliferation was determined and the cultured cells were identified with lineage-specific surface markers by flow cytometry. RESULTS: Cultures with ChBM-derived CD34(+) cells showed the lowest level of expansion of megakaryocytes and gave rise to more profound formation of myeloid and monocytic cells. In cultures with BM- or PB-derived cells, presence of IL-3 reduced the number of immature megakaryocytes (CD34(+)CD41(+) cells). However, in CB cultures, the number of CD34(+)CD41(+) cells was highest in cultures with Tpo + IL-3. Overall, cultures with CB CD34(+) cells yielded the highest number of megakaryocytes, but these cells showed reduced ploidization and lower level of CD41 expression, suggesting less maturation. CONCLUSIONS: Each of the different CD34(+) cell sources responded differently to cytokine stimulation. For PB and ABM, the cytokine combination Tpo + IL-1 is most suitable to obtain high numbers of both immature and mature megakaryocytes for transfusion purposes. For CB, Tpo + IL-3 is better.
Assuntos
Antígenos CD34/sangue , Células da Medula Óssea/imunologia , Sangue Fetal/citologia , Megacariócitos , Células-Tronco/citologia , Adulto , Células da Medula Óssea/fisiologia , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Criança , Pré-Escolar , Citocinas/farmacologia , Sangue Fetal/imunologia , Sangue Fetal/fisiologia , Hematopoese/efeitos dos fármacos , Humanos , Imunofenotipagem , Interleucina-1/farmacologia , Interleucina-3/farmacologia , Megacariócitos/efeitos dos fármacos , Megacariócitos/fisiologia , Ploidias , Células-Tronco/imunologia , Células-Tronco/fisiologia , Trombopoetina/farmacologiaRESUMO
Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare disorder of undefined aetiology. The disease presents with severe thrombocytopenia and absence of megakaryocytes in the bone marrow. Furthermore, CAMT patients may develop bone marrow aplasia. To obtain more insight into the mechanism underlying CAMT, five children were analysed. All patients had increased plasma thrombopoietin (Tpo) levels, indicating a platelet production defect. Bone marrow-derived CD34+ stem cells from three patients were cultured in an in vitro liquid culture system to study megakaryocytopoiesis. CD34+ cells from two of the three patients failed to differentiate into megakaryocytes. The lack of megakaryocyte formation could imply that a defect in the c-mpl gene, encoding the Tpo receptor, exists. Sequencing of c-mpl revealed mutations in four of five patients. Three patients had point mutations and/or a deletion in the coding regions of c-mpl. All point mutations led to an amino acid substitution or to a premature stop codon. In one patient, a homozygous mutation in the last base of intron 10 was found that resulted in loss of a splice site. This study showed that mutations in c-mpl could be the cause of thrombocytopenia in CAMT in the majority of patients. Furthermore, Tpo has been shown to have an anti-apoptotic effect on stem cells. Therefore, mutations in c-mpl might not only affect megakaryocyte formation but may also impair stem cell survival, which could explain the occurrence of bone marrow failure as final outcome in patients with CAMT.
Assuntos
Mutação Puntual/genética , Trombocitopenia/genética , Trombopoetina/genética , Substituição de Aminoácidos , Antígenos CD34 , Divisão Celular , Células Cultivadas , Pré-Escolar , Progressão da Doença , Feminino , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Lactente , Recém-Nascido , Masculino , Megacariócitos/citologia , Trombopoetina/sangueRESUMO
A 52-year-old previously healthy man was admitted to the hospital with haematuria, painful micturition and fever. Laboratory investigation showed the presence of a haemolytic uraemic syndrome (HUS), characterized by haemolysis, renal insufficiency and mental disturbances. A urinary tract infection caused by a verotoxin-producing E. coli other than O157:H7 was diagnosed. Treatment of this infection resulted in his complete recovery from the illness. Both the search for a focus outside the gastrointestinal tract and the search for verotoxin genes by specific polymerase chain reaction can be crucial in a patient with HUS without preceding diarrhoea.
Assuntos
Infecções por Escherichia coli/diagnóstico , Escherichia coli , Síndrome Hemolítico-Urêmica/microbiologia , Anti-Infecciosos/uso terapêutico , Ciprofloxacina/uso terapêutico , Escherichia coli/classificação , Escherichia coli/isolamento & purificação , Síndrome Hemolítico-Urêmica/tratamento farmacológico , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Infecções Urinárias/tratamento farmacológico , Infecções Urinárias/microbiologiaRESUMO
Thrombopoietin is produced at a constant rate by the liver and kidney and is removed from the circulation upon binding and subsequent uptake via the Tpo receptor, c-Mpl, expressed by platelets and mega-karyocytes. Apart from uptake, this study shows that platelets can also function as a storage pool for Tpo. Upon stimulation with various platelet agonists, full-length biologically active Tpo was released by platelets. Platelet fractionation experiments indicated that this Tpo most likely is contained in the granules. When platelets were preincubated with Tpo-peptide mimetic or truncated Tpo prior to maximal activation, a three- to fivefold increment in Tpo release was seen. whereas, the release of other granule proteins such as vWF-propeptide or serotonin remained unchanged. Therefore, the Mpl agonists might compete with Mpl-bound Tpo, thereby releasing Tpo into the platelet supernatant. Intravascular release of Tpo by platelets might occur in patients with massive platelet activation, as occurs in patients with disseminated intravascular coagulation. The Tpo concentration in these patients is elevated (p <0.01) and correlates with markers for thrombin generation, TAT complexes and F1+2(r(p)= 0.8 and 0.9; p <0.01). This suggests that the increment in Tpo concentration was attributed to Tpo release by activated platelets in vivo, which might be instrumental in subsequent stimulation of thrombocytopoiesis.
Assuntos
Plaquetas/metabolismo , Proteínas de Neoplasias , Ativação Plaquetária/fisiologia , Receptores de Citocinas , Trombopoetina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Coagulação Sanguínea/fisiologia , Plaquetas/citologia , Coagulação Intravascular Disseminada/sangue , Feminino , Hematopoese/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Peso Molecular , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Trombopoetina , Proteínas Recombinantes/química , Sepse/sangue , Frações Subcelulares/química , Trombina/biossíntese , Trombopoetina/farmacologia , Células Tumorais CultivadasRESUMO
BACKGROUND: Adhesion of haematopoietic progenitor cells (HPC) to human bone marrow endothelial cells (HBMEC) plays a key role in homing of HPC to bone marrow. Here we describe four new HBMEC cell lines that can be used to study the (specific) adhesion of HPC to HBMEC. DESIGN: HBMEC were immortalised with a retroviral construct containing the human papilloma virus 16 E6/E7 genes. Four cell lines were characterised. RESULTS: The cell lines showed their endothelial nature by the expression of von Willebrand Factor and VE-cadherin (CD144). Electron microscopic analysis revealed normal endothelial-cell characteristics, including the presence of Weibel-Palade bodies and intercellular junction structures. An extensive phenotypic analysis of the cell-lines was performed, they were found to resemble primary HBMEC. The only difference found was the absence of expression of E-selectin (CD62e) and VCAM-1 (CD106) on resting HBMEC cell lines. Upon stimulation with IL-1beta the expression of E-selectin, VCAM-1 and ICAM-1 (CD54) was upregulated. All resting cell lines bound CD34+ HPC. Adhesion was increased by addition of the phorbol ester PMA. Two cell lines showed increased binding upon IL-1beta prestimulation. Highest adhesion was observed after the combination of IL-1beta prestimulation of the endothelial cells and addition of PMA. Binding of CD34+ HPC to HBMEC was compared with the binding to human umbilical vein endothelial cell lines and to a human dermal microvascular endothelial cell line (HMEC-1). So far, we have only found relatively less binding of HPC to IL-1beta prestimulated HMEC-1 cells, which could be explained by a reduced induction of E-selectin and VCAM-1 upon IL-1beta stimulation of these cells. CONCLUSION: The immortalised HBMEC cell lines have maintained their normal phenotype for the majority of characteristics examined. The expression of E-selectin and VCAM-1, which are not constitutively expressed on the cell lines, can be induced by stimulation of the endothelial cells with IL-1beta. The cell lines have furthermore maintained their capability to bind HPC. They will therefore be useful to investigate the interactions between HPC and HBMEC involved in homing of HPC.
Assuntos
Células da Medula Óssea/ultraestrutura , Técnicas de Cultura de Células/métodos , Endotélio Vascular/ultraestrutura , Antígenos CD34/análise , Células da Medula Óssea/química , Células da Medula Óssea/metabolismo , Linhagem Celular Transformada/química , Linhagem Celular Transformada/metabolismo , Linhagem Celular Transformada/ultraestrutura , DNA/análise , Selectina E/metabolismo , Endotélio Vascular/química , Endotélio Vascular/metabolismo , Citometria de Fluxo , Células-Tronco Hematopoéticas/química , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/ultraestrutura , Humanos , Imunofenotipagem , Molécula 1 de Adesão Intercelular/metabolismo , Microscopia Eletrônica , Veias Umbilicais/citologia , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
The applicability of different PCR-based assays for fetal RHD and K1 genotyping using DNA isolated from uncultured amniotic fluid cells has been tested prospectively: cord blood serotyping served as a control. For RHD genotyping, DNA was amplified with PCRs specific for RHD exon 7, the 3'-non-coding region and intron 4, using standard conditions. The results of these three separate assays were compared to those of a newly-developed multiplex PCR, simultaneously amplifying six regions of RHD. The PCRs analysing the 3'-non-coding region or intron 4 often yielded false-negative results or no results at all. Results of the exon 7 PCR and of the multiplex PCR always corresponded with postnatal serotyping, the multiplex PCR having the advantage of analysing six RHD-specific exons simultaneously. For K1 genotyping, two different PCR-based assays, both analysing the presence of T578C in the KEL gene, were applied. With the first method, a consensus 740-bp product of the KEL gene was amplified and subsequently specifically digested. As we were not able to obtain any PCR product from amniotic fluid DNA, we developed a new K1-specific PCR, amplifying a fragment of 91 bp only in cases of K1-positivity. With this PCR, all K1 genotyping results (n=30) correctly predicted the phenotypes. We conclude that fetal RHD and K1 genotyping can be performed reliably with DNA from uncultured amniotic fluid cells.