Background Sickle cell anaemia (SCA) is associated with significant morbidity from acute complications and organ dysfunction beginning in the first year of life. In the first multicenter randomised double-blinded trial in very young children with SCA, the impact of hydroxyurea (hydroxycarbamide) therapy on organ dysfunction, clinical complications, and laboratory findings, and its toxicity, were examined. Methods Eligible subjects had HbSS or Sβ0thalassaemia, were age 9–18 months at randomisation, and were not selected for clinical severity. Subjects received liquid hydroxyurea, 20 mg/kg/day, or placebo for two years. Primary study endpoints were splenic function (qualitative uptake on 99Tc spleen scan) and renal function (glomerular filtration rate by 99mTc-DTPA clearance). Additional evaluations included: blood counts, HbF, chemistry profiles, spleen function biomarkers, urine osmolality, neurodevelopment, transcranial Doppler ultrasonography, growth, and mutagenicity. Study visits occurred every two to four weeks. Findings Ninety-six subjects received hydroxyurea and 97 placebo; 86% completed the study. Significant differences were not seen for the primary endpoints, but suggestive benefit was noted in quantitative measures of spleen function. Hydroxyurea significantly decreased pain and dactylitis with trends for decreased acute chest syndrome, hospitalisation and transfusion. Hydroxyurea increased haemoglobin and HbF and decreased WBC count. Toxicity was limited to mild-moderate neutropaenia. Interpretation Although hydroxyurea treatment did not reduce splenic and renal dysfunction assessed by primary endpoint measures, it resulted in major clinical benefit because of diminished acute complications, favorable haematologic results, and a lack of unexpected toxicities. Based on the safety and efficacy data from this trial, hydroxyurea can now be considered for all very young children with SCA.

BACKGROUND Silent cerebral infarcts are the most common neurologic injury in children with sickle cell anemia and are associated with the recurrence of an infarct (stroke or silent cerebral infarct). We tested the hypothesis that the incidence of the recurrence of an infarct would be lower among children who underwent regular blood-transfusion therapy than among those who received standard care. METHODS In this randomized, single-blind clinical trial, we randomly assigned children with sickle cell anemia to receive regular blood transfusions (transfusion group) or standard care (observation group). Participants were between 5 and 15 years of age, with no history of stroke and with one or more silent cerebral infarcts on magnetic resonance imaging and a neurologic examination showing no abnormalities corresponding to these lesions. The primary end point was the recurrence of an infarct, defined as a stroke or a new or enlarged silent cerebral infarct. RESULTS A total of 196 children (mean age, 10 years) were randomly assigned to the observation or transfusion group and were followed for a median of 3 years. In the transfusion group, 6 of 99 children (6%) had an end-point event (1 had a stroke, and 5 had new or enlarged silent cerebral infarcts). In the observation group, 14 of 97 children (14%) had an end-point event (7 had strokes, and 7 had new or enlarged silent cerebral infarcts). The incidence of the primary end point in the transfusion and observation groups was 2.0 and 4.8 events, respectively, per 100 years at risk, corresponding to an incidence rate ratio of 0.41 (95% confidence interval, 0.12 to 0.99; P = 0.04). CONCLUSIONS Regular blood-transfusion therapy significantly reduced the incidence of the recurrence of cerebral infarct in children with sickle cell anemia. (Funded by the National Institute of Neurological Disorders and Stroke and others; Silent Cerebral Infarct Multi-Center Clinical Trial ClinicalTrials.gov number, NCT00072761, and Current Controlled Trials number, ISRCTN52713285.)

To determine the relationship between the state of actin polymerization in neutrophils and the formyl-methionyl-leucyl-phenylalanine (fMLP)-induced changes in the locomotive behavior of neutrophils, the mean rate of locomotion (mROL), the percent G-actin, and the relative F-actin content of neutrophils were determined. The mROL was quantified by analysis of the locomotion of individual cells; the percentage of total actin as G-actin was measured by DNase I inhibition ; and the F-actin was determined by fluorescence-activated cell sorter (FACS) analysis of nitrobenzoxadiazol (NBD)-phallacidin-stained neutrophils .Neutrophils stimulated with fMLP exhibit a change in their mROL that is biphasic and dose dependent . The mROL of neutrophils exposed to 10-8 M fMLP, the Ko, is 11 .9 ± 2 .0 pm/min (baseline control 6 .2 ± 1 .0 /Am/min) . At 10-6 M fMLP, the mROL returns to baseline levels . Stimulation of neutrophils with fMLP also induces action polymerization . Evidence for actin polymerization includes a 26 .5% reduction in G-actin and a twofold increase in the amount of NBD-phallacidin staining of cells as determined by FACS analysis. The NBD-phallacidin staining is not due to phagocytosis, is inhibited by phalloidin, requires cell permeabilization, and is saturable at NBD-phallacidin concentrations >10-7 M. The fMLP-induced increase in NBD-phallacidin staining occurs rapidly (<2 min), is temperature dependent, and is not due to cell aggregation . Since NBD-phallacidin binds specifically to F-actin, the increase in fluorescent staining of cells likely reflects an increase in the F-actin content of fMLP-stimulated cells . FACS analysis of NBD-phallacidin-stained cells shows that the relative F-actin content of neutrophils stimulated with 10-11 -10-8 M fMLP increases twofold and remains increased at concentrations >10-8 M fMLP. Therefore, the fMLP-induced increase in F-actin content of neutrophils as determined by FACS analysis of NBD-phallacidin-stained cells coincides with a decrease in G-actin and correlates with increased mROL of neutrophils under some (10-"-i 10-8 M fMLP) but not all (>10-8 M fMLP) conditions of stimulation . Quantification of the Factin content of nonmuscle cells by FACS analysis of NBD-phallacidin-stained cells may allow rapid assessment of the state of actin polymerization and correlation of that state with the motile behavior of nonmuscle cells.Actin is a ubiquitous protein in eucaryotic cells. It exists in globular (43,000 dalton) and filamentous forms. The filamentous form (F-actin) interacts with myosin to generate the force necessary for motility in nonmuscle cells . In addition, F-actin, in conjunction with other cytoskeletal proteins, plays a major structural role within nonmuscle cells. Presumably in nonmuscle cells, as in muscle cells, actin interacts with myosin in accord with the "sliding filament hypothesis" to generate the contractile force necessary for motile behavior (1). Force generation and movement of cellular components requires the presence of myosin, ATP, and fil...

The X proteins have been reported only in association with microtubules and with ribosomes in situ, in the normal central nervous system. In addition, T has been shown to be an integral component of paired helical rdaments, the principal constituent of the neurofibriflary tangles found in brains of patients with Alzheimer disease and of most aged individuals with Down syndrome (trisomy 21). We report here the localization of the well-characterized Tau-1 monoclonal antibody to the nucleolar organizer regions of the acrocentric chromosomes and to their interphase counterpart, the fibrillar component of the nucleolus, in human neuroblastoma cells. Similar localization to the nucleolar organizer regions was also observed in other human cell lines and in one monkey kidney cell line but was not seen in non-primate species. Immunochemically, we further demonstrate the existence of the entire Tmolecule in the isolated nuclei of neuroblastoma cells. Nuclear 7 proteins, like the 7 proteins of the paired helical filaments, cannot be extracted in standard SDS-containing electrophoresis sample buffer but require pretreatment with formic acid prior to immunoblot analysis. This work indicates that 7 may function in processes not directly associated with microtubules and that highly insoluble complexes of T may also play a role in normal cellular physiology.T proteins were first identified as a family of phosphoproteins that associate with microtubules in vivo and stimulate their assembly in vitro (1, 2). Recent evidence indicates that T proteins are an integral component of the paired helical filaments, the principal constituent of the neurofibrillary tangles characteristic ofAlzheimer disease or senile dementia of the Alzheimer type (3-7). The structure of the 7 gene and of the cDNAs cloned and sequenced from the expressed mRNAs reveals a tripartite protein composed of a variable N-terminal domain, a constant central domain, and a C-terminal, tubulin-binding domain (8,9). Hence, it appears that much of the observed electrophoretic heterogeneity is generated by alternative splicing of a single RNA transcript (8-10).Initially, r was reported to be restricted to axons within the central nervous system (11). A more widespread distribution was subsequently documented, indicating the presence of T along microtubules of both the axonal and somatodendritic compartments (12). Additionally, T was observed on ribosomes in neuronal somatodendritic compartments and in glial cells (12). We report here the localization of the Tau-1 monoclonal antibody (11) to the nucleolar organizer regions (NORs) of the acrocentric chromosomes (nos. 13, 14, 15, 21, and 22), in cultured human cells. Immunolocalization is also detected in the interphase counterpart of the NORs, the fibrillar component of nucleoli. Similar localization patterns are observed in cultured monkey kidney cells but are not present in non-primate cultured cell lines. The presence of T is biochemically documented in the isolated nuclei of two human neuroblastoma cell lines. ...

Formyl-met-leu-phe (fMLP) induces actin assembly in neutrophils; the resultant increase in F-actin content correlates with an increase in the rate of cellular locomotion at fMLP concentrations _<10 -~ M (Howard, T. H., and W. H. Meyer, 1984, J. Cell Biol., 98:1265-1271. We studied the time course of change in F-actin content, F-actin distribution, and cell shape after fMLP stimulation. F-actin content was quantified by fluorescence activated cell sorter analysis of nitrobenzoxadiazole-phallacidin-stained cells (Howard, T. H., 1982, J. Cell Biol., 95(2, Pt. 2:327a). F-actin distribution and cell shape were determined by analysis of fluorescence photomicrographs of nitrobenzoxadiazole-phallacidin-stained cells. After fMLP stimulation at 25°C, there is a rapid actin polymerization that is maximal (up to 2.0 times the control level) at 45 s; subsequently, the F-actin depolymerizes to an intermediate F-actin content 5-10 rain after stimulation. The depolymerization of F-actin reflects a true decrease in F-actin content since the quantity of probe extractable from cells also decreases between 45 s and 10 min. The rate of actin polymerization (3.8 + 0.3-4.4 + 0.6% increase in F-actin/s) is the same for 10-t°-10 -6 M fMLP and the polymerization is inhibited by cytochalasin D. The initial rate of F-actin depolymerization (6.0 + 1.0-30 + 5% decrease in F-actin/min) is inversely proportional to fMLP dose. The F-actin content of stimulated cells at 45 s and 10 min is greater than control levels and varies directly with fMLP dose. F-actin distribution and cell shape also vary as a function of time after stimulation. 45 s after stimulation the cells are rounded and Factin is diffusely distributed; 10 min after stimulation the cell is polarized and F-actin is focally distributed. These results indicate that (a) actin polymerization and depolymerization follow fMLP stimulation in sequence, (b) the rate of depolymerization and the maximum and steady state F-actin content but not the rate of polymerization are fMLP dose dependent, and (c) concurrent with F-actin depolymerization, F-actin is redistributed and the cell changes shape.

The most common form of neurologic injury in sickle cell anemia (SCA) is silent cerebral infarction (SCI). In the Silent Cerebral Infarct Multi-Center Clinical Trial, we sought to identify risk factors associated with SCI. In this cross-sectional study, we evaluated the clinical history and baseline laboratory values and performed magnetic resonance imaging of the brain in participants with SCA (HbSS or HbS␤°thalassemia) between the ages of 5 and 15 years with no history of overt stroke or seizures. Neuroradiology and neurology committees adjudicated the presence of SCI. SCIs were diagnosed in 30.8% (251 of 814) participants who completed all evaluations and had valid data on all prespecified demographic and clinical covariates. The mean age of the participants was 9.1 years, with 413 males (50.7%). In a multivariable logistic regression analysis, lower baseline hemoglobin concentration (P < .001), higher baseline systolic blood pressure (P ‫؍‬ .018), and male sex (P ‫؍‬ .030) were statistically significantly associated with an increased risk of an SCI. Hemoglobin concentration and systolic blood pressure are risk factors for SCI in children with SCA and may be therapeutic targets for decreasing the risk of SCI. This study is registered at www.clinicaltrials.gov as #NCT00072761. IntroductionSilent cerebral infarcts (SCIs) have been recognized by neuroimaging in neurologically normal older adult populations since 1981 1 and were documented in sickle cell anemia (SCA) soon afterward. 2 As with overt stroke, SCIs represent a clinical finding that is common in older adults without SCD, but they appear during early childhood in persons with SCA. SCIs are defined as an MRI signal abnormality visible on 2 views on the T2-weighted images (axial and coronal) that must measure at least 3 mm in one dimension; further, the person deemed to have an SCI must have an absence of focal neurologic deficit compatible with the anatomic location of the brain lesion. 3 SCI is the most common form of neurologic injury among children with SCA, occurring in at least 27% before 6 years of life 4 and 37% by 14 years of life. 5 SCIs in children with SCA are associated with increased risk of future overt strokes and new or progressive SCIs. 6,7 In addition, children with SCA and SCI have been found to have poorer cognitive function The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. 3684BLOOD, 19 APRIL 2012 ⅐ VOLUME 119, NUMBER 16For personal use only. on March 28, 2019. by guest www.bloodjournal.org From than children with SCA with normal MRI of the brain 8-10 or sibling controls. 10,11 Clinical and laboratory risk factors for SCI have been evaluated only sparingly. In the most rigorous study to date, the investigators from the Cooperative Study for Sickle Cell Disease (CSSCD) described risk factors associated with SCI in 42 participants, comparing them with 188 controls with ...

abnormal spindle, a gene required for normal spindle structure and function in Drosophila melanogaster, lies immediately adjacent the gene tolloid at 96A/B. It encodes a 220-kD polypeptide with a predicted pI of 10.8. The recessive mutant allele asp1 directs the synthesis of a COOH terminally truncated or internally deleted peptide of ∼124 kD. Wild-type Asp protein copurifies with microtubules and is not released by salt concentrations known to dissociate most other microtubule-associated proteins. The bacterially expressed NH2-terminal 512-amino acid peptide, which has a number of potential phosphorylation sites for p34cdc2 and MAP kinases, strongly binds to microtubules. The central 579-amino acid segment of the molecule contains one short motif homologous to sequences in a number of actin bundling proteins and a second motif present at the calmodulin binding sites of several proteins. Immunofluorescence studies show that the wild-type Asp protein is localized to the polar regions of the spindle immediately surrounding the centrosome. These findings are discussed in relation to the known spindle abnormalities in asp mutants.

Background Silent cerebral infarct (SCI) is the most common cause of serious neurological disease in sickle cell anemia (SCA), affecting approximately 22% of children. The goal of this trial is to determine whether blood transfusion therapy will reduce further neurological morbidity in children with SCI, and if so, the magnitude of this benefit. Procedure The Silent Cerebral Infarct Transfusion (SIT) Trial includes 29 clinical sites and 3 subsites, a Clinical Coordinating Center, and a Statistical and Data Coordinating Center, to test the following hypothesis: prophylactic blood transfusion therapy in children with SCI will result in at least an 86% reduction in the rate of subsequent overt strokes or new or progressive cerebral infarcts as defined by magnetic resonance imaging (MRI) of the brain. The intervention is blood transfusion versus observation. Two hundred and four participants (102 in each treatment assignment) will ensure 85% power to detect the effect necessary to recommend transfusion therapy (86% reduction), after accounting for 10% drop out and 19% crossover rates. MRI examination of the brain is done at screening, immediately before randomization and study exit. Each randomly assigned participant receives a cognitive test battery at study entry, 12–18 months later, and study exit and an annual neurological examination. Blood is obtained from all screened participants for a biologic repository containing serum and a renewable source of DNA. Conclusion The SIT Trial could lead to a change in standard care practices for children affected with SCA and SCI, with a consequent reduction in neurological morbidity.