Shear‐induced unfolding activates von Willebrand factor A2 domain for proteolysis

Summary. Background: To avoid pathological platelet aggregation by von Willebrand factor (VWF), VWF multimers are regulated in size and reactivity for adhesion by ADAMTS13‐mediated proteolysis in a shear flow dependent manner. Objective and methods: We examined whether tensile stress in VWF under shear flow activates the VWF A2 domain for cleavage by ADAMTS13 using molecular dynamics simulations. We generated a full length mutant VWF featuring a homologous disulfide bond in A2 (N1493C and C1670S), in an attempt to lock A2 against unfolding. Results: We indeed observed stepwise unfolding of A2 and exposure of its deeply buried ADAMTS13 cleavage site. Interestingly, disulfide bonds in the adjacent and highly homologous VWF A1 and A3 domains obstruct their mechanical unfolding. We find this mutant A2 (N1493C and C1670S) to feature ADAMTS13‐resistant behavior in vitro. Conclusions: Our results yield molecular‐detail evidence for the force‐sensing function of VWF A2, by revealing how tension in VWF due to shear flow selectively exposes the A2 proteolysis site to ADAMTS13 for cleavage while keeping the folded remainder of A2 intact and functional. We find the unconventional ‘knotted’ Rossmann fold of A2 to be the key to this mechanical response, tailored for regulating VWF size and activity. Based on our model we discuss the pathomechanism of some natural mutations in the VWF A2 domain that significantly increase the cleavage by ADAMTS13 without shearing or chemical denaturation, and provide with the cleavage‐activated A2 conformation a structural basis for the design of inhibitors for VWF type 2 diseases.     

Treatment of compulsive behaviour in eating disorders with intermittent ketamine infusions

We have previously shown that eating disorders are a compulsive behaviour disease, characterized by frequent recall of anorexic thoughts. Evidence suggests that memory is a neocortical neuronal network, excitation of which involves the hippocampus, with recall occurring by re-excitement of the same specific network. Excitement of the hippocampus by glutamate-NMDA receptors, leading to long-term potentiation (LTP), can be blocked by ketamine. Continuous block of LTP prevents new memory formation but does not affect previous memories. Opioid antagonists prevent loss of consciousness with ketamine but do not prevent the block of LTP. We used infusions of 20 mg per hour ketamine for 10 h with 20 mg twice daily nalmefene as opioid antagonist to treat 15 patients with a long history of eating disorder, all of whom were chronic and resistant to several other forms of treatment. Nine (responders) showed prolonged remission when treated with two to nine ketamine infusions at intervals of 5 days to 3 weeks. Clinical response was associated with a significant decrease in Compulsion score: before ketamine, mean +/- SE was 44.0 +/- 2.5; after ketamine, 27.0 +/- 3.5 (t test, p = 0.0016). In six patients (non-responders) the score was: before ketamine, 42.8 +/- 3.7; after ketamine, 44.8 +/- 3.1. There was no significant response to at least five ketamine treatments, perhaps because the compulsive drive was re-established too soon after the infusion, or because the dose of opioid antagonist, nalmefene, was too low.      

Acute cytomegalovirus infection in liver transplant recipients: An independent risk for venous thromboembolism

Acute cytomegalovirus (CMV) infection is a commonly encountered complication in the post liver transplant setting. We present a case of a 71-year-old male with acute CMV infection, initially presenting with a gastrointestinal bleed due to acute CMV gastritis and later on complicated by acute venous thromboembolism occurring as an unprovoked event in the post liver transplant period. Traditional risk factors for venous thromboembolism have been well described in the medical literature. Sporadic cases of thromboembolism due to CMV infection in the immune compromised patients have been described, especially in the post kidney transplant patients. Liver transplant recipients are equally prone to CMV infection particularly in the first year after successful transplantation. Venous thromboembolism in this special population is particularly challenging due to the fact that these patients may have persistent thrombocytopenia and anticoagulation may be a challenge for the treating physician. Since liver transplantation is severely and universally limited by the availability of donor organs, we feel that this case report will provide valuable knowledge in the day to day management of these patients, whose clinical needs are complex and require a multidisciplinary approach in their care and management. Evidence and pathophysiology linking both the conditions is presented along with a brief discussion on the management, common scenarios encountered and potential impact in this special group of patients.     

Myocardial deformation abnormalities in patients with aortic regurgitation: a strain rate imaging study

Aims: Early left ventricular (LV) dysfunction in asymptomatic patientswith severe aortic regurgitation (AR) may go undetected dueto the lack of a sufficiently sensitive diagnostic tool. Ultrasonicstrain/strain rate (S/SR) imaging should now provide such sensitivityin detecting early dysfunction in regional LV systolic deformation.The aim of this study was to understand and define the changesin LV regional systolic deformation based on S/SR imaging inpatients with asymptomatic or minimally symptomatic AR. Methods and results: Eighty-one individuals were studied: 59 asymptomatic patientswith isolated non-ischaemic AR who were divided into three sub-groupssuch as mild, moderate, and severe AR and 22 age-matched healthysubjects. All patients underwent standard echocardiographicexaminations including a tissue Doppler imaging study. For LVradial deformation, the posterior wall (LVPW) was examined.To assess LV longitudinal deformation, S and SR data were acquiredfrom the LV lateral wall and septum. Radial as well as longitudinalpeak systolic SRs were significantly decreased in patients withboth moderate AR (LVPW, P = 0.0009; septum, P = 0.03; LV lateralwall, P = 0.0009) and severe AR (P < 0.0001) compared withhealthy subjects. Changes in regional LV deformation correlatedinversely both with LV end-diastolic volume and with end-systolicvolume. Conclusions: Strain rate imaging is a sensitive tool in detecting the spectrumof changes in radial and longitudinal deformation in asymptomaticor minimally symptomatic patients with AR. The index where volumewas corrected by deformation should form the basis for predictingsubclinical LV dysfunction in patients with increasing LV dilatation.     

The "common stem cell" hypothesis reevaluated: human fetal bone marrow contains separate populations of hematopoietic and stromal progenitors

There is a long-standing controversy as to whether a single bone marrow (BM)-derived cell can differentiate along both hematopoietic and stromal lineages. Both primitive hematopoietic and stromal progenitor cells in human BM express the CD34 antigen but lack expression of other surface markers, such as CD38. In this study we examined the CD34+, CD38- fraction of human fetal BM by multiparameter fluorescence- activated cell sorting (FACS) analysis and single-cell sorting. CD34+, C38- cells could be divided into HLA-DR+ and HLA-DR- fractions. After single-cell sorting, 59% of the HLA-DR+ cells formed hematopoietic colonies. In contrast, the CD34+, CD38-, HLA-DR- cells were much more heterogeneous with respect to their light scatter properties, expression of other hematopoietic markers (CD10, CD36, CD43, CD49b, CD49d, CD49e, CD50, CD62E, CD90w, CD105, and CD106), and growth properties. Single CD34+, CD38-, HLA-DR- cells sorted into individual culture wells formed either hematopoietic or stromal colonies. The presence or absence of CD50 (ICAM-3) expression distinguished hematopoietic from stromal progenitors within the CD34+, CD38-, HLA-DR- population. The CD50+ fraction had light scatter characteristics and growth properties of hematopoietic progenitor cells. In contrast, the CD50- fraction lacked hematopoietic progenitor activity but contained clonogenic stromal progenitors at a mean frequency of 5%. We tested the hypothesis that cultures derived from single cells with the CD34+, CD38- , HLA-DR- phenotype could differentiate along both a hematopoietic and stromal lineage. The cultures contained a variety of mesenchymal cell types and mononuclear cells that had the morphologic appearance of histiocytes. Immunophenotyping of cells from these cultures indicated a stromal rather than a hematopoietic origin. In addition, the growth of the histiocytic cells was independent of the presence or the absence of hematopoietic growth factors. Based on sorting more than 30,000 single cells with the CD34+, CD38-, HLA-DR- phenotype into individual culture wells, and an analysis of 864 stromal cultures initiated by single CD34+ BM cells, this study does not support the hypothesis of a single common progenitor for both hematopoietic and stromal lineages within human fetal BM.     

Clinical features and outcome of T-cell acute lymphoblastic leukemia in childhood with respect to alterations at the TAL1 locus: a Pediatric Oncology Group study

Alteration of the TAL1 locus is the most common nonrandom genetic defect in childhood T-cell acute lymphoblastic leukemia (T-ALL). To determine if rearrangements of the TAL1 proto-oncogene confer a distinct leukemic phenotype, we studied leukemic peripheral blood or bone marrow samples from 182 children with newly diagnosed T-ALL enrolled on Pediatric Oncology Group treatment protocols. Forty-eight (26%) of the samples had a local rearrangement of the TAL1 locus. Demographic and clinical features were compared for patient subgroups with and without TAL1 rearrangements. The only clinical correlates that were significantly associated with TAL1 gene rearrangements were higher white blood cell count (P = .017) and higher hemoglobin (P = .007) at diagnosis. Immunophenotypically, samples with altered TAL1 were more likely to be CD2+ (P = .001) and lack CD10 (cALLa) expression (P = .007) than those without the rearrangement. There was a trend toward improved event-free survival (EFS) in patients with TAL1 rearrangements (4-year EFS was 44% +/- 7% for patients without the rearrangements v 59% +/- 11% for those with rearrangements), but the difference was not significant (P = .34). The role of TAL1 in leukemogenesis has yet to be clearly defined, and the prognostic significance of TAL1 gene rearrangements in T-ALL deserves further study.     

普罗布考和抗氧化维生素不影响体外实验条件下内皮细胞的白细胞粘附

为探讨预防动脉粥样硬化的药物普罗布考,维生素C和维生素E是否抑制内皮细胞表面粘附分子表达和白细胞一内皮细胞的粘附,以及这种抑制是否通过影响核因子－kB的活性来实现的,在液体流动小室中进行细胞粘附实验。用ELISA方法测定内皮细胞粘附分子E－选择素的表达;用电泳迁移率分析测定内皮细胞核因子－kB的活性,经肿瘤坏死因子α刺激的内皮细胞核因子－B活性增加,粘附分子E－选择素的表达上调（是基础水平的3．5倍）,其表面HL60细胞的粘附增加（是基础水平的4－26倍）,而抗氧化剂PDTC使所有这些变化都受到抑制。PDTC浓度为18umol/L时对粘附分子E－选择素的表达呈最大半抑制;PDTC浓度为52umol/L时对内皮细胞表面HL60细胞的粘附呈最大半抑制,普罗布考,维生素C和维生素E对肿瘤坏死因子α诱导的粘附分子表达和HL60细胞与内皮细胞的粘附没有作用,对核因子－kB的活性没有影响,临床上常用的这三种抗氧化剂并未影响作为动脉粥样硬化始动机制之一的E－选择素介导的白细胞－内皮细胞粘附水平。     

The systemic inflammatory response syndrome as a predictor of bacteraemia and outcome from sepsis

Criteria defining the <it>systemic inflammatory response syndrome</it> (SIRS) were used to assess prospectively 270 clinical episodes in which blood cultures were taken from patients in general medicine. SIRS, severe sepsis and septic shock occurred in 149 (55%), 13 (5%) and 9 (3%) episodes, respectively. However, evidence of organ hypoperfusion indicating severe sepsis was recorded as sought in only 26% of episodes of SIRS. Crude mortality at 28 days increased sequentially as more SIRS criteria were met, rising from 12% in non-SIRS blood culture episodes, to 36% when all four criteria were met. Mortality from severe sepsis and septic shock was 38% and 56%, respectively. In 61/64 (95%) episodes of clinically important bacteraemia, patients fulfilled SIRS criteria when the blood culture was taken. However, the positive predictive value of SIRS for predicting bacteraemia was only 7%. Patients who did not fulfill SIRS criteria when blood cultures were taken were at low risk of bacteraemia and comprised 45% (121/270) of the study population. Three patients in this low-risk group had bacteraemia. Mortality in bacteraemic patients with severe sepsis or septic shock who were initially treated with ineffective antibiotics for up to 48 h was 80%, compared to 42% in those always treated appropriately.