STAT3- and STAT5-dependent pathways competitively regulate the pan-differentiation of CD34pos cells into tumor-competent dendritic cells

The clinical outcomes of dendritic cell (DC)-based immunotherapy remain disappointing, with DCs often displaying a tenuous capacity to complete maturation and DC1 polarization in the tumor host. Surprisingly, we observed that the capacity for successful DC1 polarization, including robust IL12p70 production, could be regulated by STAT-dependent events even prior to DC differentiation. Exposure of CD34(pos) cells to single-agent granulocyte-macrophage colony-stimulating factor (GMCSF) induced multilineage, STAT5-dependent differentiation, including DCs that failed to mature in the absence of further exogenous signals. In contrast, Flt3L induced nearly global differentiation of CD34(pos) cells into spontaneously maturing DCs. IL-6 synergized with Flt3L to produce explosive, STAT3-dependent proliferation of phenotypically undifferentiated cells that nevertheless functioned as committed DC1 precursors. Such precursors not only resisted many tumor-associated immunosuppressants, but also responded to tumor contact or TGFbeta with facilitated DC maturation and IL12p70 production, and displayed a superior capacity to reverse tumor-induced T-cell tolerance. GMCSF preempted Flt3L or Flt3L plus IL-6 licensing by blocking STAT3 activation and promoting STAT5-dependent differentiation. Paradoxically, following overt DC differentiation, STAT5 enhanced whereas STAT3 inhibited DC1 polarization. Therefore, nonoverlapping, sequential activation of STAT3 and STAT5, achievable by sequenced exposure to Flt3L plus IL-6, then GMCSF, selects for multilog expansion, programming, and DC1 polarization of tumor-competent DCs from CD34(pos) cells.     

The effect of rehabilitation in a comprehensive inpatient rehabilitation unit on mobility outcome after dysvascular lower extremity amputation

Czerniecki JM, Turner AP, Williams RM, Hakimi KN, Norvell DC. The effect of rehabilitation in a comprehensive inpatient rehabilitation unit on mobility outcome after dysvascular lower extremity amputation.ObjectivesTo (1) compare the total volume of rehabilitation therapy for patients ever attending a comprehensive inpatient rehabilitation unit (CIRU) versus never during the 12 months after amputation; (2) determine whether rehabilitation in a CIRU at any time in the first year after amputation results in greater mobility success compared with other types of rehabilitation environments of care; and (3) determine for those patients treated in a CIRU, which specific patient characteristics were associated with improved mobility outcome.DesignProspective cohort study.SettingTwo Veterans Affairs medical centers.ParticipantsPatients (N=199) with peripheral vascular disease or diabetes undergoing a first unilateral major amputation were screened for participation between September 2005 and December 2008. Among these, 113 (57%) met study criteria; of these, 72 (64%) participated.InterventionEver attending a CIRU versus never attending a CIRU in first 12 months after amputation.Main Outcome MeasuresNumber of rehabilitation therapy visits, Locomotor Capability Index scores, and mobility success.ResultsThe mean number of all therapy visits for patients ever attending a CIRU was significantly greater than that for those never attending over a 12-month period (48.6 vs 22.6; P=.001). Mean total time per any rehabilitation visit was .83±.27 hours for those ever attending and .60±.20 hours for those never attending (P<.001). Patients who ever were treated in a CIRU were 17% more likely to achieve mobility success than those who were not, controlling for amputation level, major depressive episode, alcohol use, social support, total number of rehabilitation visits, and hospital site (risk difference=.17; 95% confidence interval, .09–.25; P<.001).ConclusionsRehabilitation in a CIRU resulted in improved mobility success for veterans undergoing major lower extremity amputation secondary to peripheral vascular disease or diabetes. Among those admitted to a CIRU, younger patients with greater social support, healthy weight, and without chronic obstructive pulmonary disease had the greatest probability of mobility success.     

Human target validation of phosphoinositide 3‐kinase (PI3K)β: effects on platelets and insulin sensitivity,using AZD6482 a novel PI3Kβ inhibitor

Nylander S, Kull B, Björkman JA, Ulvinge JC, Oakes N, Emanuelsson BM, Andersson M, Skärby T, Inghardt T, Fjellström O, Gustafsson D. Human target validation of phosphoinositide 3‐kinase (PI3K)β:effects on platelets and insulin sensitivity, using AZD6482 a novel PI3Kβ inhibitor. J Thromb Haemost 2012; 10: 2127–36. See also Jackson SP, Schoenwaelder SM. Antithrombotic phosphoinositide 3‐kinase β inhibitors in humans – a ‘shear’ delight! This issue, pp 2123–6. Summary. Background: Based on in vitro and animal data, PI3Kβ is given an important role in platelet adhesion and aggregation but its role in insulin signaling is unclear. Objective: To strengthen the PI3Kβ target validation using the novel, short‐acting inhibitor AZD6482. Methods and results: AZD6482 is a potent, selective and ATP competitive PI3Kβ inhibitor (IC₅₀ 0.01 μm ). A maximal anti‐platelet effect was achieved at 1 μm in the in vitro and ex vivo tests both in dog and in man. In dog, in vivo AZD6482 produced a complete anti‐thrombotic effect without an increased bleeding time or blood loss. AZD6482 was well tolerated in healthy volunteers during a 3‐h infusion. The ex vivo anti‐platelet effect and minimal bleeding time prolongation in the dog model translated well to data obtained in healthy volunteers. AZD6482 inhibited insulin‐induced human adipocyte glucose uptake in vitro (IC₅₀ of 4.4 μm ). In the euglycemic hyperinsulinemic clamp model, in rats, glucose infusion rate was not affected at 2.3 μm but reduced by about 60% at a plasma exposure of 27 μm . In man, the homeostasis model analysis (HOMA) index increased by about 10–20% at the highest plasma concentration of 5.3 μm . Conclusions: This is the first human target validation for PI3Kβ inhibition as anti‐platelet therapy showing a mild and generalized antiplatelet effect attenuating but not completely inhibiting multiple signaling pathways with an impressive separation towards primary hemostasis. AZD6482 at ‘supratherapeutic’ plasma concentrations may attenuate insulin signaling, most likely through PI3Kα inhibition.     

Reducing delirium in elderly patients with hip fracture: a multi‐factorial intervention study

Background: There is an evident need for improved management of elderly patients with trauma in order to avoid common and troublesome complications such as delirium. The aim of this study was to investigate whether an implementation of a multi‐factorial program including intensified pre‐hospital and perioperative treatment and care could reduce the incidence of delirium in elderly patients with hip fracture, cognitively intact at admission to the hospital. In addition, we explored the factors that characterize patients who developed delirium. Methods: A prospective, quasi‐experimental design was used. A total of 263 patients with hip fracture (≥65 years), cognitively intact at admission, were consecutively included between April 2003 and April 2004. On 1 October 2003, a new program was introduced. All patients were screened for cognitive impairment within 30 min after admission to the emergency department using The Short Portable Mental Status Questionnaire (SPMSQ). To screen for delirium, patients were tested within 4 h of admission and thereafter daily, using the Organic Brain Syndrome scale. Results: The number of patients who developed delirium during hospitalization was 74 (28.1%), with a decrease from 34% (45 of 132) in the control group to 22% (29 of 131) in the intervention group (P=0.031). Patients who developed delirium were statistically older, more often had >4 prescribed drugs at admission and scored less well in the SPMSQ test. Conclusion: The use of a multi‐factorial intervention program in elderly hip fracture patients, lucid at admission, reduced the incidence of delirium during hospitalization by 35%.     

Investigation of the effects of the novel anticonvulsant compound carisbamate (RWJ-333369) on rat piriform cortical neurones in vitro

Background and purpose:

Carisbamate is being developed for adjuvant treatment of partial onset epilepsy. Carisbamate produces anticonvulsant effects in primary generalized, complex partial and absence-type seizure models, and exhibits neuroprotective and antiepileptogenic properties in rodent epilepsy models. Phase IIb clinical trials of carisbamate demonstrated efficacy against partial onset seizures; however, its mechanisms of action remain unknown. Here, we report the effects of carisbamate on membrane properties, evoked and spontaneous synaptic transmission and induced epileptiform discharges in layer II-III neurones in piriform cortical brain slices.

Experimental approach:

Effects of carisbamate were investigated in rat piriform cortical neurones by using intracellular electrophysiological recordings.

Key results:

Carisbamate (50–400 µmol·L⁻¹) reversibly decreased amplitude, duration and rise-time of evoked action potentials and inhibited repetitive firing, consistent with use-dependent Na⁺ channel block; 150–400 µmol·L⁻¹ carisbamate reduced neuronal input resistance, without altering membrane potential. After microelectrode intracellular Cl⁻ loading, carisbamate depolarized cells, an effect reversed by picrotoxin. Carisbamate (100–400 µmol·L⁻¹) also selectively depressed lateral olfactory tract-afferent evoked excitatory synaptic transmission (opposed by picrotoxin), consistent with activation of a presynaptic Cl⁻ conductance. Lidocaine (40–320 µmol·L⁻¹) mimicked carisbamate, implying similar modes of action. Carisbamate (300–600 µmol·L⁻¹) had no effect on spontaneous GABA_A miniature inhibitory postsynaptic currents and at lower concentrations (50–200 µmol·L⁻¹) inhibited Mg²⁺-free or 4-aminopyridine-induced seizure-like discharges.

Conclusions and implications:

Carisbamate blocked evoked action potentials use-dependently, consistent with a primary action on Na⁺ channels and increased Cl⁻ conductances presynaptically and, under certain conditions, postsynaptically to selectively depress excitatory neurotransmission in piriform cortical layer Ia-afferent terminals.     

Does having a computerized prosthetic knee influence cognitive performance during amputee walking?

OBJECTIVE: To compare objective cognitive performance and perception of cognitive burden during walking tasks using 2 different prosthetic knees: a computerized hydraulic knee (Otto Bock C-leg) and a noncomputerized hydraulic knee (Ossur Mauch SNS). DESIGN: Two-group crossover trial, with participants randomly assigned to order of prosthesis. Participants completed assessments under 2 conditions, a self-selected speed walk and a controlled speed walk, on 2 separate occasions (precrossover, postcrossover). SETTING: Veterans Health Administration hospital. PARTICIPANTS: Eight transfemoral amputees. INTERVENTION: Computerized versus noncomputerized prosthetic knee. MAIN OUTCOME MEASURES: Objective cognitive performance measures included verbal fluency (Controlled Oral Word Association Test, Category Test), attention and working memory (serial subtraction), and walking speed during cognitive tasks. Measures of perceived cognitive burden included subjective attentional requirements of walking and cognitive tasks and subjective general cognitive burden of prosthesis. RESULTS: There were no significant differences in objective cognitive performance on any task between prostheses, nor did walking speed vary by prosthesis during the free-speed walk. Participants reported that walking required less attention while wearing the C-leg and that the C-leg was less of a cognitive burden than the noncomputerized prosthesis. CONCLUSIONS: In nondemanding walking conditions with experienced amputees, participants reported that the more costly C-leg required less cognitive attention than the noncomputerized knee. However, this subjective experience did not translate to improved performance on neuropsychologic screening instruments or on walking speed. Noncomputerized prostheses may be adequate for a majority of amputees, and further research is needed to identify particular groups of amputees (ie, new amputees, amputees with complex physical or cognitive demands) who may benefit from computerized prostheses.     

人T-bet基因的体外扩增及克隆和鉴定

目的:克隆人T-bet基因,构建其真核表达载体pEGFP-C1-T-bet。方法:实验于2005-07/2006-07在北京大学深圳医院中心实验室完成。①根据Genebank的人T-bet基因的全长cDNA序列,设计合成一对附加BglⅡ和SalⅠ两个限制性内切酶酶切位点的特异性引物。②分离人外周血单个核细胞,提取RNA,用反转录-聚合酶链反应方法将T-bet的编码序列cDNA扩增,装入pMD18-T载体并送去测序。③BglⅡ SalⅠ双酶切质粒pMD18-T-bet,经琼脂糖电泳切胶回收T-bet片段,将T-bet插入载体pEGFP-C1构建成重组真核表达载体pEGFP-C1-T-bet。④用双酶切和聚合酶链反应对插入片段进行分析和验证。结果:①反转录-聚合酶链反应产物经琼脂糖电泳结果显示在预期位置有相对分子质量为1608bp的特异性扩增带。②测序结果证实,T-bet的编码序列和Genbank中T-bet mRNA序列相同。③双酶切和聚合酶链反应结果证实插入片段序列正确。结论:实验成功扩增出人T-bet基因,构建了基因重组真核表达载体pEGFP-C1-T-bet,为探索T-bet基因对免疫细胞的调节作用和肿瘤基因治疗奠定了基础。     

大环骨架结构的倍半萜化合物——Ⅵ.马兜铃新内酯的化学结构测定

自绵毛马兜铃(Aristolochia mollissima Hance)根茎中分得九个化合物,其中已报道的七个化合物是尿囊素、马兜铃内酯、绵毛马兜铃内酯、β-谷甾醇、马兜铃酸A、9-乙氧基马兜铃内酯和9-乙氧基马兜铃内酰胺,本文报道结晶K₃的化学结构,经光谱分析(IR,¹H-NMR,¹³C-NMR,2D-NMR和MS),化学反应及X-衍射晶体分析,确证K₃为一个新骨架结构的倍半萜化合物,命名为马兜铃新内酯。