Functional Reorganization and Recovery After Constraint-Induced Movement Therapy in Subacute Stroke: Case Reports

Preliminary assessments of the feasibility, safety, and effects on neuronal reorganization measured with transcranial magnetic stimulation (TMS) from Constraint-Induced Movement Therapy (CIMT) of the upper extremity were made in eight cases of subacute stroke. Within fourteen days of their stroke, patients were randomly assigned to two weeks of CIMT or traditional therapy. Baseline motor performance and cortical/subcortical representation for movement with TMS were assessed before treatment. Post-treatment assessments were made at the end of treatment and at three months after the stroke. The TMS mapping showed a larger motor representation in the lesioned hemisphere of the CIMT patients as compared to the controls at the three-month follow-up assessment. The enlarged motor representation in the lesioned hemisphere for hand movement correlated with improved motor function of the affected hand, suggesting a link between movement representation size as measured with TMS and functionality. These results suggest that TMS can be safely and effectively used to assess brain function in subacute stroke and further suggest that CIMT may enhance cortical/subcortical motor reorganization and accelerate motor recovery when started within the first two weeks after stroke.     

Uptake of antibiotics by human alveolar macrophages

To provide additional criteria for therapy of pulmonary infections caused by facultative intracellular bacteria, we studied the uptake of 12 antibiotics by alveolar macrophages (AM) obtained from healthy, young volunteers by bronchoalveolar lavage. These human AM were incubated with radiolabeled antibiotics for periods as long as 2 h. Entry of antimicrobials into the cells was determined by means of a velocity-gradient centrifugation technique. Antibiotic uptake was expressed as the ratio of the cellular to the extracellular drug concentration (C/E). Penicillin G, cefamandole, and gentamicin were taken up poorly by human AM (C/E = 0.5 to 0.8). Isoniazid achieved a cellular concentration similar to the extracellular level of the drug (C/E = 0.9). Chloramphenicol, rifampin, tetracycline, and lincomycin, drugs that are lipid-soluble, were concentrated several-fold by AM (C/E = 2 to 5). The remaining antibiotics tested, clindamycin, erythromycin, erythromycin propionate, and ethambutol, were markedly concentrated by AM (C/E = 9 to 23). Accumulation of clindamycin (C/E = 23) was a rapid, active, energy-requiring process, which appeared to be dependent upon mitochondrial oxidative metabolism. The ability of the tested antimicrobial agents to enter human AM correlates well with the efficacy of these drugs in treatment of certain intracellular pulmonary infections.     

Contrasting effects of recombinant human granulocyte-macrophage colony- stimulating factor (CSF) and granulocyte CSF treatment on the cycling of blood elements in childhood-onset cyclic neutropenia

Recombinant human granulocyte colony-stimulating factor (G-CSF) treatment has been shown to increase average neutrophil counts substantially in patients with childhood-onset cyclic neutropenia (or "cyclic hematopoiesis"), but not to eliminate the cyclic oscillations of neutrophil counts or those of other blood elements (monocytes, platelets, eosinophils, and reticulocytes) that are characteristic of this hematopoietic disorder. Indeed, oscillations of neutrophil counts are amplified during G-CSF treatment. We have compared the effects of recombinant granulocyte-macrophage-CSF (GM-CSF) with those of G-CSF in three patients with this disease (2 men and 1 woman, 17, 30, and 32 years of age). These patients were treated with GM-CSF (2.1 micrograms/kg/day, subcutaneously) for 6 weeks, preceded and followed by 6 to 13 weeks of detailed observation to document changes in the cyclic oscillations of blood neutrophils and other blood elements; two of the patients were subsequently treated with G-CSF (5.0 micrograms/kg/d, subcutaneously) and observed for comparable periods of time. Unlike G-CSF treatment, which increased average neutrophil counts more than 20-fold, GM-CSF increased neutrophil counts only modestly, from 1.6- to 3.9-fold, although eosinophilia of varying prominence was induced in each patient. However, at the same time, GM-CSF treatment dampened or eliminated the multilineage oscillations of circulating blood elements (neutrophils, monocytes, platelets, and/or reticulocytes) in each of the patients. In contrast, G-CSF treatment of the same patients markedly amplified the oscillations of neutrophil counts and caused the cycling of other blood elements (monocytes in particular) to become more distinct. These findings support the conclusion that the distinctive cycling of blood cell production in childhood-onset cyclic neutropenia results from abnormalities in the coordinate regulation of both GM-CSF-responsive, multipotential progenitor cells and G-CSF-responsive, lineage-restricted, neutrophil progenitors.     

Probing the binding specificities of human Siglecs by cell-based glycan arrays

Siglecs are a family of sialic acid–binding receptors expressed by cells of the immune system and a few other cell types capable of modulating immune cell functions upon recognition of sialoglycan ligands. While human Siglecs primarily bind to sialic acid residues on diverse types of glycoproteins and glycolipids that constitute the sialome, their fine binding specificities for elaborated complex glycan structures and the contribution of the glycoconjugate and protein context for recognition of sialoglycans at the cell surface are not fully elucidated. Here, we generated a library of isogenic human HEK293 cells with combinatorial loss/gain of individual sialyltransferase genes and the introduction of sulfotransferases for display of the human sialome and to dissect Siglec interactions in the natural context of glycoconjugates at the cell surface. We found that Siglec-4/7/15 all have distinct binding preferences for sialylated GalNAc-type O-glycans but exhibit selectivity for patterns of O-glycans as presented on distinct protein sequences. We discovered that the sulfotransferase CHST1 drives sialoglycan binding of Siglec-3/8/7/15 and that sulfation can impact the preferences for binding to O-glycan patterns. In particular, the branched Neu5Acα2–3(6-O-sulfo)Galβ1–4GlcNAc (6′-Su-SLacNAc) epitope was discovered as the binding epitope for Siglec-3 (CD33) implicated in late-onset Alzheimer’s disease. The cell-based display of the human sialome provides a versatile discovery platform that enables dissection of the genetic and biosynthetic basis for the Siglec glycan interactome and other sialic acid–binding proteins.

Immune cells are equipped with an array of glycan-binding proteins (GPBs) capable of interpreting the biological information encoded by glycans. Endogenous GBPs recognize host-derived “self” and foreign-derived “nonself” glycans and produce cues that are integrated into the signaling network of immune cells and contribute to immune homeostasis and the immune response (1). Siglecs (sialic acid–binding immunoglobulin-like lectins) serve in self-recognition and transmit immune inhibitory signals upon binding to a select repertoire of sialoglycans expressed by host cells raising the threshold for immune activation (2, 3). The human Siglec family consists of 14 functionally expressed members, and these are composed of an N-terminal V-set immunoglobulin (Ig)-like domain that mediates sialoglycan binding followed by varying numbers of C2-set Ig-like domains. Intracellularly, most Siglecs have immunoreceptor tyrosine-based inhibition motifs, and Siglec-14/15/16 carry immunoreceptor tyrosine-based activation motifs (3–7). Siglecs are broadly expressed throughout the immune system, and several Siglecs are also found outside of the immune system, such as Siglec-4 (MAG), which is expressed by oligodendrocytes and Schwann cells in the nervous system (8). Although the diverse biological functions within and outside of the immune system of Siglecs are not fully understood, Siglecs generally contribute to immune homeostasis by dampening immune activation upon recognition of sialoglycans. For example, Siglec-2 (CD22) can suppress B cell receptor activation (9), and Siglec-9 can dampen neutrophil activation (10). Cancer cells with aberrant sialoglycans and pathogens that express sialic acids can exploit Siglec signaling to modulate immune responses (11, 12). Moreover, Siglec-3 (CD33) is strongly associated with risk for Alzheimer’s disease and expressed on microglia cells (13, 14). Given the potent immune modulatory functions of Siglecs and their wide involvement in autoimmunity, infection, cancer, and neurodegeneration, Siglecs are promising therapeutic targets (7, 15). However, many of the natural ligands of Siglecs have not been fully identified, and endogenous ligands for several Siglecs including Siglec-3/CD33 remain elusive.Human cells can produce a large diversity of glycans capped with sialic acids (Sia), a family of chemically diverse sugars with N-acetylneuraminic acid (Neu5Ac) being the predominant type in humans. Sialic acids are generally found at the termini of mammalian glycans, and most types of glycoconjugates including N-glycoproteins, multiple types of O-glycoproteins, and glycolipids carry oligosaccharides capped by sialic acids (16, 17). Sialylation is one of the most complex regulated steps in glycosylation with 20 distinct Golgi-located sialyltransferase isoenzymes dedicated to catalyze transfer of sialic acids to galactose (ST3GAL1-6, ST6GAL1 and 2), N-Acetylgalactosamine (Gal-NAc) (ST6GALNAC1-6), or sialic acid (ST8SIA1-6) via α2-3, α2-6, or α2-8 linkages, respectively and with different preferences for the underlying glycan structures and types of glycoconjugate (18–20). The resulting plethora of sialic acid-containing glycans constituting the sialome of cells provides a vast catalog of ligands for Siglecs and potential for distinct instructive cues for the immune response (16). The current insight into the interactome of Siglecs is largely derived from studies with libraries of synthetic and natural glycans printed on glass arrays (21, 22). These glycan arrays have demonstrated distinct structural glycan features that drive selective binding of individual Siglecs, including the linkage type of sialic acids, the core disaccharide carrying sialic acids, and glycan modifications such as sulfation or acetylation (23–27). However, printed glycan arrays may not present glycans in the natural context of the overall glycoconjugate structure and the cell surface with spatial organization and competition dynamics limiting insight into the fine binding specificities of Siglecs and their interactions with the host cell sialome.Here, we took advantage of our recently developed cell-based glycan array strategy (28–30) and generated an expanded sialome sublibrary with the human embryonic kidney (HEK) 293 for dissection of Siglec binding properties. First, combinatorial gene knockout (KO) was used to delete distinct subsets of sialyltransferase isoenzymes or all endogenous sialylation capacity. Second, using targeted gene knock-in (KI), individual sialyltransferase isoenzymes were introduced in the absence of other isoenzymes. Finally, we introduced selected sulfotransferase isoenzymes to explore cross-talk between sialylation and sulfation. To specifically address the influence of clustered O-glycan presentation for Siglec binding, we introduced a large panel of reporter constructs designed to display human O-glycodomains derived from mucins and mucin-like O-glycoproteins with different densities and patterns of O-glycans. The cell-based sialome array reproduced previous results for binding specificities for Siglec-2 (CD22) and Siglec-9 and led to insight into the binding specificities of Siglec-4/7/15 for distinct GalNAc-type O-glycans and their presentation on O-mucin–like glycoproteins. Finally, we demonstrate that Siglec-3/7/8/15 have preferential binding to sulfated sialoglycans yet have different specificities for underlying glycoconjugate structures. We further discovered the 6′-Su-SLacNAc (Neu5Acα2-3[6-O-sulfo]Galβ1-4GlcNAc) epitope on N-glycans and glycolipids as the ligand for Siglec-3/CD33 as well as Siglec-8. In summary, the cell-based display of the human sialome enables dissection of the Siglec interactome in the natural context of a human cell and provides the biosynthetic and genetic basis for the identified ligands.     

三维重建射频损伤区域观察猪舌根射频损伤体积与射频能量及时间的关系

目的：目前临床进行隧道法舌根射频治疗时,其作用参数的设置仍缺乏统一的标准,故通过计算机三维重建射频损伤区域,分析猪舌根射频损伤体积与射频能量、时间的关系,从中得出应用舌根隧道法射频治疗的最佳作用能级和作用时间。 方法：实验于2006-06/2007-05在上海交通大学耳鼻喉科研究所完成。将36只实验用猪以射频作用能级1,2,3,4,5,6随机分成6组,每组6头猪,各个猪舌的作用时间分别设置为2,5,10,15,20,25s。用Coblation射频发生仪及Reflex55刀头进行猪舌根射频操作。射频作用后的舌根组织行连续冰冻切片,苏木精-伊红染色后,进行序列组织切片的全貌二维图像采集,对拟重建的结构进行边界提取和图像分割。将提取分割图像导入Image-Pro Solution图像处理软件,利用3D Constructor插件进行三维重建,并根据设定参数进行体积计算。用SPSS10.0统计学软件对所测数据进行统计学分析。 结果：①作用能级固定时,舌根组织射频损伤体积随时间延长而增大,符合Logarithmic回归曲线。②作用时间固定时,舌根组织射频损伤体积随能级增大而增大,符合直线回归。③射频损伤体积随能量增大而增加亦符合Logarithmic回归曲线。④Coblation射频治疗系统在能级6时,在作用10s之前,损伤体积随作用时间增加而迅速增加,其后变化趋势平缓,超过20s后损伤体积无显著增加。 结论：①舌根区域射频治疗时,舌根组织射频损伤体积与时间或能量呈Logarithmic曲线相关,与能级呈直线相关。②Coblation射频治疗系统在能级6时,最佳作用时间范围为10-20s。     

构建Loa22基因去信号肽片段原核重组表达载体

目的：构建赖型钩端螺旋体OmpA膜蛋白Loa22基因去信号肽片段的原核表达载体，并对其进行克隆表达。方法：实验于2004—12／2005—12在四川大学华西医学中心感染免疫研究室完成。以赖型钩端螺旋体017株基因组DNA为模板，PCR扩增Loa22基因去信号肽片段，亚克隆至原核表达载体pGEX-4T-1，经双酶切、PCR鉴定，筛选出阳性重组质粒克隆。经DNA测序正确后，转化大肠杆菌，利用IPTG进行诱导表达，通过SDS—PAGE鉴定表达产物。结果：PCR获得长516bp的片段。Loa22基因去信号肽片段与pGEX-4T-1的重组质粒构建成功。重组质粒经IPTG诱导后能在大肠杆菌中表达Mr45000的融合蛋白。结论：制备了Loa22基因去信号肽片段原核重组表达载体，为钩体新型疫苗的研究奠定基础。     

肝脏可溶性复合物不同剂量、不同途径移植对肿瘤细胞增殖抑制的体内外实验

目的:肝脏可溶性复合物具有保护肝脏、刺激肝组织再生等生物学活性,观察天然物质肝脏可溶性复合物对肿瘤细胞生长增殖的抑制作用.方法:实验于2006-05/2007-02在四川大学华西医院生物治疗国家重点实验室实验肿瘤研究室完成.①分离人胚胎、成年及新生小鼠肝脏组织,生理盐水清洗、剪碎、筛网过滤,用生理盐水制备混悬液,3 000 r/min离心,收集上清,制备肝脏可溶性复合物.②体外实验:用上述不同来源的肝脏可溶性复合物体外处理肿瘤细胞,四甲基偶氮唑盐比色法测定其对乳腺癌细胞EMT6增殖的影响.③体内实验:观察成年鼠肝脏可溶物质对乳腺癌细胞EMT6体内生长的抑制作用及其对荷瘤鼠生存状况的影响,包括不同给药剂量及不同给药途径两个实验,给药途径包括在接种肿瘤细胞部位的对侧腋下、同侧腋下、腹腔注射及灌胃等.结果:①体外实验显示不同来源的肝脏可溶性复合物能明显抑制肿瘤细胞EMT6增殖率,肿瘤增殖抑制率均显著高于血清白蛋白处理组(P＜0.05),并呈剂量依赖性.②成年鼠肝脏可溶物质8mg/L组抑瘤率高于2,4 mg/L组(P＜0.05),未观察到明显毒副效应.③比较不同给药途径,成年鼠肝脏可溶物质同侧注射组的抑瘤率较其他3组的抑瘤率高(P＜0.05),各成年鼠肝脏可溶物质给予组的体质量增长率比相应生理盐水对照组高(P＜0.05).④与相应生理盐水对照组比较,在同侧腋下注射成年鼠肝脏可溶物质的小鼠生存期明显延长(P＜0.05).结论:肝脏可溶性复合物具有抑制肿瘤细胞生长的作用,并且呈一定的剂量依赖性.不同的给药途径中,在接种肿瘤细胞部位的同侧腋下给药抑瘤效果最好.     

基于反向滤波器的倒谱法估计平均骨小梁间距

目的 探讨用改进的倒谱方法估计平均骨小梁间距(mean trabecular bone spacing,MTBS)的可行性.方法 提出了一种基于反向滤波器的改进的倒谱分析方法用于估计MTBS,并将该方法应用于仿真及离体牛胫骨松质骨中的实验信号.结果 改进的倒谱方法能有效减少超声换能器脉冲响应和组织散射特性对倒谱的干扰,而且实现简单,计算量小.结论 相比于传统的倒谱方法,改进的倒谱方法在估计MTBS时, 对弥散散射和噪声有更强的鲁棒性,因此估计MTBS的精度更高.