Fractionated carbon dioxide (CO2) laser treatment contributes to trans-nail penetration of rhodamine B and changes of cytokine microenvironment

Lasers in Medical Science - This study is to determine the role of the fractional CO2 laser in topical drug delivery and the impact of local immune responses. Experimental rabbit nails were treated...     

Evidence for a role of anti-ADAMTS13 autoantibodies despite normal ADAMTS13 activity in recurrent thrombotic thrombocytopenic purpura

Background

Severe ADAMTS13 deficiency is a critical component of the pathogenesis of idiopathic thrombotic thrombocytopenic purpura but is found only in about 60% of patients clinically diagnosed with this disease.

Design and Methods

Over a period of 8 years and six episodes of thrombotic thrombocytopenic purpura we studied the evolution of the anti-ADAMTS13 antibody response in a patient using different ADAMTS13 assays and epitope mapping.

Results

Anti-ADAMTS13 autoantibodies were found in all episodes but were inhibitory only in the last two episodes. In a flow-based assay, normal ADAMTS13 activity was found only during the first disease episode, while ADAMTS13 activity was normal using a static assay in episodes 1 and 3, and severely deficient in the last two episodes. Fluorescence evolution in a modified fluorescence resonance energy transfer assay using a von Willebrand factor A2 domain peptide substrate was linear in episodes 1, 5 and 6, but increased exponentially in episodes 3 and 4. Despite the variable functional characteristics of the anti-ADAMTS13 autoantibodies, their principal epitope was the ADAMTS13 spacer domain in all episodes.

Conclusions

The patient is unique as he displayed features of maturation or shaping of the anti-ADAMTS13 autoantibody response during the course of multiple episodes of thrombotic thrombocytopenic purpura. Anti-ADAMTS13 autoantibodies may be important in vivo despite normal ADAMTS13 activity in routine assays. Consequently, treatment decisions should not be based solely on activity assay results.     

Site-directed mutagenesis of platelet glycoprotein Ib alpha demonstrating residues involved in the sulfation of tyrosines 276, 278, and 279

The interaction between platelet glycoprotein (GP) Ib alpha and von Willebrand factor (VWF) is essential for initiation of hemostasis. The sulfation of the 3 tyrosine residues 276, 278, and 279 in GPIb alpha is an important posttranslational modification that seems to promote the interaction with VWF. The environment where sulfation of tyrosines occurs has been proposed to contain highly acidic residues. This investigation has examined the highly acidic region from Asp249 to Asp287 in the mature GPIb alpha protein. Changes to most of the carboxylic acids in this region resulted in decreased reactivity to VWF. Only 3 mutants (Glu270Gln, Asp283Asn, Asp283Asn/Glu285Gln/Asp287Asn) resulted in the abolition of sulfation. Two novel mutations were also created. First, a deletion of the 7 amino acids from Tyr276 to Glu282 led to a loss of sulfation and totally abolished VWF binding in the presence of botrocetin. This confirms that it is these 3 tyrosines that undergo sulfation and that this region is crucial for botrocetin-mediated VWF binding. The second mutation involves changing the lysine residues at 253, 258, and 262 to alanine. This also led to distinct changes in VWF binding and abolition of sulfation.     

Biodegradable nanoparticles mimicking platelet binding as a targeted and controlled drug delivery system

This research aims to develop targeted nanoparticles as drug carriers to the injured arterial wall under fluid shear stress by mimicking the natural binding ability of platelets via interactions of glycoprotein Ib-alpha (GPIbα) of platelets with P-selectin of damaged endothelial cells (ECs) and/or with von Willebrand factor (vWF) of the subendothelium. Drug-loaded poly(d,l-lactic-co-glycolic acid) (PLGA) nanoparticles were formulated using a standard emulsion method and conjugated with glycocalicin, the external fraction of platelet GPIbα, via carbodiimide chemistry. Surface-coated and cellular uptake studies in ECs showed that conjugation of PLGA nanoparticles, with GPIb, significantly increased nanoparticle adhesion to P-selectin- and vWF-coated surfaces as well as nanoparticle uptake by activated ECs under fluid shear stresses. In addition, effects of nanoparticle size and shear stress on adhesion efficiency were characterized through parallel flow chamber studies. The observed decrease in bound nanoparticle density with increased particle sizes and shear stresses is also explained through a computational model. Our results demonstrate that the GPIb-conjugated PLGA nanoparticles can be used as a targeted and controlled drug delivery system under flow conditions at the site of vascular injury.     

Mobilization of circulating endothelial progenitor cells after endovascular therapy for ruptured cerebral aneurysms

Emerging evidence shows that circulating endothelial progenitor cells (EPCs) promote regeneration of the endothelium at sites of vessel injury. This study was designed to test the hypothesis that EPCs are mobilized in patients who had ruptured cerebral aneurysm (CA) and underwent endovascular therapy. Fourteen patients with ruptured CAs were recruited and blood samples were analyzed after coil embolization surgery. Blood samples were also obtained from 18 healthy control subjects who had no evidence of CAs and did not undergo endovascular surgery. We measured the numbers of circulating EPCs, levels of plasma vascular endothelial growth factor (VEGF) and platelet counts. EPCs significantly increased in patients with ruptured CAs and underwent surgical coil embolization, reaching a peak level on day 14 post operation. The levels of plasma VEGF and platelet counts also significantly increased in parallel with the increase in EPCs, leading to significant positive correlations of circulating EPCs with VEGF in plasma (r = 0.636, P < 0.01) and platelet counts (r = 0.721, P < 0.001), respectively. The finding suggests that EPCs are mobilized upon surgery and may play a critical role in repairing injured vascular endothelium. Levels of EPCs in peripheral blood could also serve as a prognostic marker for the outcomes of ruptured cerebral aneurysms after surgical repair.     

Changes in circulating human endothelial progenitor cells after brain injury

Endothelial progenitor cells (EPCs) are mobilized from the bone marrow to blood circulation in response to tramatic or inflammatory stimulations. Once released, they actively seek and home to the sites of vascular injury to promote vascular repair. We monitored changes of EPC counts in peripheral blood of 29 patients with traumatic brain injury for up to 21 days. We showed that the levels of circulating EPCs within the first 48 h of injury were lower than control subjects, but increased over time-reaching plateau around 7 days post-injury at a level that was significantly higher than controls. The initial EPC reduction, which was severe in patients with severe injury Glasgow Coma Scale [GCS] < 12), differs from the acute increase in EPC counts found in patients with cardiovascular injury. The subsequent increase in circulating EPCs is primarily through bone marrow mobilization because the cells were stained predominantly for CD133, which labels immature EPCs, but not CD34 (which stains cell of endothelial lineage). The increase appeared earlier in male patients and was greater in those younger than 50 years of age. Changes in circulating EPCs during follow-up periods correlated with platelet, but not leukocyte counts. These results suggest that EPC mobilization following traumatic brain injury may take a different course compared to that associated with body or vascular injuries.     

TLR3-1377C/T基因多态性及表达水平与儿童肠道病毒71型脑炎易感性的探讨

目的 研究Toll样受体(TLRs)基因TLR3-1377C/T位点基因多态性及TLR3表达水平与儿童肠道病毒71型(EV71)脑炎易感性之间的关系。方法 收集EV71感染患儿187例(脑炎组59例和无脑炎组128例)与同期健康体检儿童232例进行病例对照研究。采用聚合酶链反应限制性片段长度多态性(PCR-RFLP)方法对TLR3-1377C/T基因多态性进行检测,采用ELISA检测血清TLR3水平。结果 与EV71感染无脑炎组相比,脑炎组TLR3-1377C/T位点基因型分布及等位基因频率的差异无统计学意义(P0.05)。与对照组相比,EV71感染脑炎组、无脑炎组的血清TLR3水平均显著增高(P0.05),以无脑炎组最高(P0.05)。脑炎组的EV71病毒载量高于无脑炎组(P0.01)。1岁或≥1岁的EV71感染脑炎组、无脑炎组患儿的血清TLR3水平均较相应对照组增高(P0.05),其中无脑炎组TLR3水平高于相应年龄的脑炎组(P0.05)。脑炎组≥1岁患儿的TLR3浓度高于1岁者(P0.05);无脑炎组及对照组的TLR3浓度在1岁或≥1岁患儿之间的差异无统计学意义(P0.05)。EV71感染脑炎组,1岁患儿所占比例高于≥1岁者(P0.05)。结论 TLR3-1377C/T位点的基因多态性与EV71脑炎的发生无明显相关性。TLR3的低表达可能导致对病毒复制的抑制作用减弱,促进了EV71脑炎的发生。婴儿EV71感染后血清TLR3的表达不足可能是其合并脑炎的重要因素。     

血清胱抑素C浓度测定对病毒性脑炎患儿肾功能损害的诊断价值

目的：探讨血清胱抑素C(Cyst C)浓度在评估病毒性脑炎患儿肾小球滤过功能中的价值。方法：92例病毒性脑炎患儿为病例组,50例健康儿童为对照组,又根据肾小球滤过率（GFR）将病例组分成肾功能正常组、代偿组、失代偿组和衰竭及终末期组。检测各组血清Cyst C、尿素氮(BUN)、肌酐(Cr)浓度。结果：① 与对照组比较,病例组BUN、Cr及Cyst C均显著增高(P0.05）,在其余各病例亚组间差异有统计学意义（P<0.01）。③Cyst C与BUN、Cr呈显著正相关,与GFR呈显著负相关。结论：病毒性脑炎患儿存在不同程度的肾功能损害;Cyst C是一种比BUN和Cr更好地反映肾小球滤过功能的指标,检测血清Cyst C对病毒性脑炎患儿肾脏功能的监测有重要的临床应用价值。     

Platelet activation patterns in platelet size sub-populations: differential responses to aspirin in vitro

Abstract  

Circulating platelets are heterogeneous in size and structure. Whether this translates into differences in platelet function and efficacy of antiplatelet therapy is unclear. Hence, we decided to investigate the activation patterns among different platelet populations differentiated by size, and to compare the inhibitory effects of aspirin in these populations. Circulating platelets from 9 healthy volunteers were separated by size and stratified into the largest and smallest quintiles. Platelets were stimulated with 75 μM arachidonic acid (AA), 10 μM ADP or 25 μM TRAP. Alpha-granule protein secretion and expression (P-selectin, VWF, fibrinogen), surface-protein activation (activated integrin αIIbβ3) were assessed. Platelet thromboxane B₂ (TxB₂) synthesis following AA stimulation was measured in vitro before and after incubation with 265 μM aspirin. Reticulated (juvenile) platelets were assessed using thiazole orange staining. A greater number of large platelets in the largest quintile were reticulated compared with the smallest quintile (6.1 ± 2.8% vs. 1.2 ± 1.5% respectively, p < 0.001). Larger platelets also synthesized more TxB₂ than small platelets both before (1348 ± 276 pg/mL vs. 1023 ± 214 pg/mL, respectively, p = 0.01) and after aspirin (1029 ± 190 pg/mL vs. 851 ± 159 pg/mL, respectively, p = 0.03). After stimulation with each agonist, a greater proportion of large platelets bound fibrinogen, VWF, P-selectin and activated integrin αIIbβ3 than small platelets both in the presence and in the absence of in vitro aspirin. In an in vitro setting, large platelets appear to be more active than small platelets and continue to be more active even after in vitro aspirin.