Anti-inflammatory effect of stem cells in the treatment of ischemic stroke

doi:10.3969/j.issn.2095-4344.2017.25.025

Abstract

Abstract:

BACKGROUND: A large number of preclinical experimental data have shown that stem cells can regulate the immune function, and serve the function of cell replacement.
OBJECTIVE: To summarize the anti-inflammatory effects of stem cells in the treatment of ischemic stroke, based on which, we further discuss the specific mechanisms.
METHODS: We conducted a systematic and comprehensive search in PubMed, Elsevier, Springer, Wiley, Ovid, EBSCO databases. The range of retrieval time was from 2012 to 2017. The keywords were “stem cells, stroke, inflammation, immune”. Totally 110 articles were retrieved initially, and 47 articles were included in result analysis.
RESULTS AND CONCLUSION: By reading extensive literature, we analyzed and summarized the research status quo of the anti-inflammatory effects of neural stem cells, mesenchymal stem cells and endothelial cells in the treatment of ischemic stroke. The mechanisms mainly include reducing focal inflammation, immune regulation, promoting the secretion of various neurotrophic factors, reducing secondary cell death, protecting neurons and promoting cell function recovery and further promoting the recovery of nerve function. The mechanisms underlying local immune regulation and anti-inflammatory effects of stem cells are mainly described as the shift from M1 to M2 macrophages under the intervention of stem cell factors, to intervene secreted immune cytokine profiles and exert effects on inhibition and polarization of glial cells. Further investigation is required on the anti-inflammatory effects and immune regulation of stem cell therapy for stroke.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Stem Cells, Stroke, Tissue Engineering

CLC Number:

R318

Gao Feng-juan, Gao Shan-e, Chen Xu, Sun Jian, Wang Jun-yi. Anti-inflammatory effect of stem cells in the treatment of ischemic stroke[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(25): 4088-4093.

Figures/Tables 1

干细胞移植治疗使脑卒中损伤得到最大程度的修复，是一种科学的医学治疗方法，但其机制尚待更加深入的研究，其中干细胞的抗炎效应和免疫调节功能亟需深入研究，如将其机制解析清晰，对于干细胞用于临床治疗脑卒中会有重要的理论和现实意义。脑卒中后原位免疫细胞如小胶质细胞的活化及外周血液免疫细胞的浸润会引起炎症反应[6]。干细胞的局部免疫调节和抑炎作用主要体现在其分泌因子作用于巨噬细胞，使其发生M1到M2型转化，干预免疫分泌的细胞因子图谱，对胶质细胞进行抑制和极化等[7-9]。M1型小胶质细胞或巨噬细胞分泌破坏性的炎性介导因子，而M2型小胶质细胞或巨噬细胞则能够清除损伤的细胞或细胞碎片并分泌营养因子，对组织起到保护作用并且促进预后[10-11]。 2.1 神经干细胞短暂性脑缺血或者再灌注的急性期引发炎症反应，这种炎症伴随着血脑屏障的损害和外周白细胞浸润[12]。缺血和再灌注可引起促炎细胞因子和趋化因子的上调[13]。在鼠脑卒中模型中，Huang等[14]证明了人神经干细胞移植不仅能够通过调节免疫反应降低继发性炎性脑损害，而且还能缓解血脑屏障的渗漏，促进血脑屏障的重建。此机制更加支持其抗炎效应及促进血脑屏障修复和促进神经营养因子的分泌作用，例如主要通过降低炎性因子(如肿瘤坏死因子α、白细胞介素6和白细胞介素1β等)及细胞黏附分子(如细胞间黏附分子1和血管细胞黏附分子1等)的表达，并显著抑制小胶质细胞的活化。同时脑内的炎症因子信号会促进移植的人神经干细胞迁移到缺血性梗死损伤区。人神经干细胞除具有潜在的细胞替代作用，还可以表达趋化因子基质细胞衍生因子1，趋化因子基质细胞衍生因子1可被其趋化因子受体CXCR4吸引到脑损伤区[15]。 Ourednik等[16]将鼠源神经干细胞移植到小鼠一侧中脑后，发现神经干细胞除了具有细胞替代作用之外，还能起到保护受损但未凋亡的多巴胺能神经元的作用。对于直接来源于人胚胎干细胞或流产胎儿的神经干细胞因取材有限，不能实现同体移植，且后续的临床应用会有免疫排斥和细胞不纯等原因带来的生物安全隐患问题。人诱导多潜能干细胞来源的神经干细胞克服了以上几点，为后续临床移植提供了可能性。Eckert等[17]采用RT-PCR技术研究了人诱导多潜能干细胞来源神经干细胞移植治疗鼠脑卒中模型后炎症细胞因子的效应发现在缺血性卒中后48 h，健侧大脑半球炎性细胞因子肿瘤坏死因子a、白细胞介素6 和白细胞介素1b表达增加，相反，人诱导多潜能干细胞来源神经干细胞移植组表达减少，同时，人诱导多潜能干细胞来源神经干细胞移植组与鼠脑卒中模型组比较，细胞间黏附分子1和血管细胞黏附分子1基因表达减少，趋化因子表征小胶质细胞/巨噬细胞活化表达显著下调。因此，移植人诱导多潜能干细胞来源神经干细胞可减少炎症和小胶质细胞/巨噬细胞活化相关基因的表达。 2.2 间充质干细胞间充质干细胞的种类较多，其中研究较多的有骨髓间充质干细胞、脂肪间充质干细胞、脐带间充质干细胞等[18-24]。已有数据表明间充质干细胞不仅能够有效抑制炎性因子如肿瘤坏死因子α、白细胞介素6等的水平，而且还能缓解内质网的压力[25]。Ren等[26]也发现干扰素γ与肿瘤坏死因子a、白细胞介素1a、白细胞介素1b中的任何一种炎性细胞因子组合，可诱导间充质干细胞的免疫抑制功能。这些细胞因子刺激间充质干细胞高水平表达趋化因子和诱导型一氧化氮合酶，这两种物质又可以促进T细胞迁移聚集到间充质干细胞附近。Huang等[27]利用氧糖剥夺应激模型发现间充质干细胞通过分泌白细胞介素6和血管内皮生长因子促进神经元的恢复。Choi等[28]采用免疫组织化学的方法发现血管内注射间充质干细胞，实验组几乎没有Iba-1+细胞，而对照组含有大量的Iba-1+细胞，这些结果表明间充质干细胞可能通过减少卒中后活化的免疫细胞来减轻炎症反应。间充质干细胞的免疫正调节或者抑炎作用主要体现在抑制炎症因子白细胞介素10、肿瘤坏死因子α刺激因子6、白细胞介素6的分泌及白血病抑制因子、前列腺素E2等抑制免疫细胞进一步活化的因子的分泌，同时促进免疫细胞分泌干扰素γ、肿瘤坏死因子α、白细胞介素α/β刺激干细胞分泌正向调控因子。其中前列腺素E2能够进一步诱导巨噬细胞分泌更多的白细胞介素10，以抑制树状细胞的成熟，从而改变Th1和Th2的平衡[29-30]。间充质干细胞的这些特性促进卒中后功能的恢复。 Gu等[31]利用RT-PCR、免疫印记、免疫组织化学等方法分析发现，注射间充质干细胞后促炎因子肿瘤坏死因子a、白细胞介素1b mRNA和P-IkB-a、人磷酸化κB抑制蛋白激酶、p53蛋白等表达大量减少，相反，抗炎因子抑制性卡巴蛋白α、Bcl-2(细胞凋亡相关基因)蛋白表达显著增加，同时也发现注射间充质干细胞组，梗死面积显著减少，因此认为，间充质干细胞通过抑制核因子κB活性发挥抗炎和抗凋亡作用进而起到神经保护作用。人骨髓间充质干细胞对免疫细胞具有免疫调节功能，不仅抑制异体效应T淋巴细胞的功能，还增加调节性T淋巴细胞的功能和数量，同时促炎细胞因子干扰素γ，增强人骨髓间充质干细胞的免疫抑制特性。人骨髓间充质干细胞还可改变树突状细胞、幼稚型和效应T细胞(TH1和TH2)及自然杀伤细胞的细胞因子分泌，并且可将这些类型的细胞诱导成为一种更抗炎或更耐受的亚型，即人骨髓间充质干细胞通过增加髓源性免疫抑制细胞数量、调节单核细胞免疫抑制表型增强先天性免疫系统的调节能力[32-33]。此外，有研究者发现，移植的异体人骨髓间充质干细胞使小胶质细胞激活减少，促炎因子(肿瘤坏死因子α、白细胞介素6、白细胞介素1b、单核细胞趋化蛋白1、巨噬细胞炎性蛋白1α)和黏附分子(细胞间黏附分子1和血管细胞黏附分子1)表达下降，以及受损的血脑屏障得到改善[34]。Aggarwal等[32]也发现机体对人骨髓间充质干细胞缺乏免疫识别和清除功能，异体移植后可显著减少移植物抗宿主病的严重程度和发生率。另有研究表明，损伤后局灶部位的促炎环境的正向调控有助于神经细胞免于进一步损伤。此外，也有研究发现脑卒中损伤后人骨髓间充质干细胞移植通过趋化信号转导通路优先转移到脾，随后来自脾脏的免疫细胞如T细胞进入全身循环系统，引起脾脏质量减少和空洞体积的增加[35-36]。人脐带间充质干细胞因其在组织中含量丰富，与人骨髓间充质干细胞相似的特性引起越来越多的重视。Cheng等[37]发现小鼠脑卒中模型移植人脐带间充质干细胞后神经功能缺损得到改善，脑水肿、脑梗死体积大量减少。人脐带间充质干细胞抑制脑卒中后外周血和梗死区炎症因子包括白细胞介素1、肿瘤坏死因子α、白细胞介素23、白细胞介素17和白细胞介素10的水平，增加转化生长因子β的水平。在脑卒中后24 h，人脐带间充质干细胞能够明显的降低Th17细胞水平，并且在脑卒中后72 h增加外周血中调节性T细胞水平。因此表明人脐带间充质干细胞通过抑制促炎性细胞因子产生和增强抗炎性细胞因子的表达来逆转炎症，减少损伤。此外，人脐带间充质干细胞还通过增加转化生长因子β水平抑制外周免疫反应，具有神经保护作用[38]。人胎盘间充质干细胞除了具有多向分化潜能还具有强大的抗炎效应。人胎盘间充质干细胞与人骨髓间充质干细胞类似，不仅抑制异体效应T淋巴细胞的功能，还增加调节性T淋巴细胞的功能和数量。同时，促炎细胞因子干扰素γ可增强人胎盘间充质干细胞的免疫抑制特性。人胎盘间充质干细胞通过增加髓源性免疫抑制细胞数量，调节单核细胞免疫抑制表型，增强先天性免疫系统地调节能力[33]。此外，人胎盘间充质干细胞上调暴露于干扰素γ的免疫调节分子人类白细胞抗原(HLA)-G的水平，并抑制介导细胞凋亡的自然杀伤细胞NK/白细胞介素2。由于具有高免疫调节和多潜能特性，人胎盘间充质干细胞对缺血性脑疾病的神经保护和神经再生作用重大。人羊膜干细胞改善脑卒中预后涉及到局部炎症的减少和免疫反应的调节，进而促进神经恢复，促进神经组织的分化，联系中断的再支配，促进有助于细胞功能修复的细胞因子，生长因子，激素或者神经递质的分泌[39]。间充质干细胞的这些特性有助于保护神经元，促进功能的恢复。由于间充质干细胞突出的免疫调节能力和神经干细胞理论上显著的细胞替代治疗作用，两者共移植有望使其各自发挥优势，获得更好的治疗效果。Walker等[40]提出移植间充质干细胞于损伤区能够激活并改变脑内微环境的原位的神经干细胞池。两者共移植既可增加神经干细胞对白细胞介素6的分泌，还可以减少神经干细胞的凋亡。 2.3 内皮祖细胞内皮祖细胞治疗缺血性脑卒中的研究已成为了新的热点。已有研究证实，虽然内皮祖细胞治疗缺血性脑卒中有很好的效果，但炎症因子的释放会影响其治疗效应。内皮祖细胞表达白细胞介素6的受体gp80，白细胞介素6依靠剂量依赖的方式促进内皮祖细胞的增值、迁移以及促进基底膜的形成，除此之外，白细胞介素6还能激活受体gp80/gp130信号通路，如下游的ERK-1/2与信号转导和STAT3 的磷酸化[41]。凝血酶作为一种有效的炎症因子，能够激活并结合于内皮祖细胞表达的具有功能的凝血酶受体PAR-1，此外，凝血酶还可以通过活化蛋白1和核因子κB路径激活白细胞介素8的合成，进而抑制内皮祖细胞的存活[41]。白细胞介素10通过激活STAT3/VEGF受体途径促进内皮祖细胞的存活。炎症因子肿瘤坏死因子α不仅可以促进内皮祖细胞的凋亡，还可通过短暂性升高细胞质Ca2+浓度使早期的内皮祖细胞表达血小板激活因子受体。血小板激活因子与血小板激活因子受体结合，促进p38的分泌，进而抑制内皮祖细胞的存活[42]。肿瘤坏死因子α还可以促进组织因子表达上调，进而促进重组因子Ⅶ(a)-依赖性的凝血酶生成活性增强，抑制内皮祖细胞的存活促进内皮祖细胞凋亡[43]。此外，肿瘤坏死因子α还通过TNF-TNFR2/ P75 信号路径抑制早期内皮祖细胞的存活，但却促进晚期的内皮祖细胞存活[44]。近年来，在心血管疾病中，因内皮祖细胞与炎症因子的相互作用以及两者之间具有千丝万缕的联系，内皮组细胞可作为炎症标记细胞。此外，临床研究发现用丁基苯酞可以动员患者体内循环系统中的内皮祖细胞，有效促进患者的预后康复[45]。 2.4 脐血干细胞脐血中含有多种细胞因子、生长因子和免疫调节因子，调节免疫细胞和成体干细胞的功能和增殖。脐血干细胞移植脑卒中动物模型后发挥抗炎效应，促进功能的恢复[46]。Vendrame 等[47]将脐血干细胞静脉注射入缺血性脑卒中动物模型后，发现移植的细胞不仅在梗死区周围发挥作用，还迁移到损伤部位发挥作用，其中还发现CD45/CD11b-和CD45/B220-阳性细胞减少的同时，伴随着促炎细胞因子的mRNA和蛋白表达减少及核因子kB的DNA结合活性减少。Yoo等[46]研究发现脐血干细胞移植1周后，在梗死区周围活化的小胶质细胞数量减少。在脑卒中细胞移植后，脑内炎症反应减弱及增加的神经保护作用促进细胞恢复。"

References

[1] Hao L,Zou Z,Tian H,et al.Stem cell-based therapies for ischemic stroke.BioMed Res Int.2014;2014:468748.

[2] Gervois P,Wolfs E,Ratajczak J,et al.Stem Cell-Based Therapies for Ischemic Stroke: Preclinical Results and the Potential of Imaging-Assisted Evaluation of Donor Cell Fate and Mechanisms of Brain Regeneration.Med Res Rev. 2016; 36(6):1080-1126.

[3] Mendez-Ferrer S,Michurina TV,Ferraro F,et al.Mesenchymal and haematopoietic stem cells form a unique bone marrow niche.Nature.2010;466(7308):829-834.

[4] Tobin MK,Bonds JA,Minshall RD,et al.Neurogenesis and inflammation after ischemic stroke: what is known and where we go from here.J Cereb Blood Flow Metab. 2014;34(10): 1573-1584.

[5] Lee YS,Chio CC,Chang CP,et al.Long course hyperbaric oxygen stimulates neurogenesis and attenuates inflammation after ischemic stroke.Mediators Inflamm. 2013;2013:512978.

[6] Kokaia Z.Targeting Neuroinflammation for Treatment Of Ischemic Stroke.Georgian Med News.2015;243:84-87.

[7] Omi M,Hata M,Nakamura N,et al.Transplantation of dental pulp stem cells suppressed inflammation in sciatic nerves by promoting macrophage polarization towards anti-inflammation phenotypes and ameliorated diabetic polyneuropathy.J Diabetes Investig.2016;7(4):485-496.

[8] Gliem M,Krammes K,Liaw L.Macrophage-derived osteopontin induces reactive astrocyte polarization and promotes re-establishment of the blood brain barrier after ischemic stroke.Glia.2015;63(12):2198-2207.

[9] Hu X,Leak RK,Shi Y,et al.Microglial and macrophage polarization-new prospects for brain repair.Nature Rev Neurol. 2015;11(1):56-64.

[10] Mosser DM,Edwards JP.Exploring the full spectrum of macrophage activation.Nat Rev Immunol.2008;8(12): 958-969.

[11] David S, Kroner A.Repertoire of microglial and macrophage responses after spinal cord injury.Nat Rev Neurosci.2011; 12(7):388-399.

[12] Jin R,Yang G,Li G.Inflammatory mechanisms in ischemic stroke: role of inflammatory cells.J Leukoc Biol.2010;87(5):779-789.

[13] Rossi B,Angiari S,Zenaro E,et al.Vascular inflammation in central nervous system diseases: adhesion receptors controlling leukocyte-endothelial interactions. J Leukoc Biol. 2011;89(4):539-556.

[14] Huang L,Wong S,Snyder EY,et al.Human neural stem cells rapidly ameliorate symptomatic inflammation in early-stage ischemic-reperfusion cerebral injury.Stem Cell Res ther. 2014;5(6):129.

[15] Imitola J,Raddassi K,Park KI,et al.Directed migration of neural stem cells to sites of CNS injury by the stromal cell-derived factor 1alpha/CXC chemokine receptor 4 pathway. Proc Natl Acad Sci U S A.2004;101(52):18117-18122.

[16] Ourednik J,Ourednik V,Lynch WP,et al.Neural stem cells display an inherent mechanism for rescuing dysfunctional neurons.Nat Biotechnol.2002;20(11):1103-1110.

[17] Eckert A,Huang L,Gonzalez R,et al.Bystander Effect Fuels Human Induced Pluripotent Stem Cell-Derived Neural Stem Cells to Quickly Attenuate Early Stage Neurological Deficits After Stroke.Stem Cells Transl Med.2015;4(7):841-851.

[18] Marti-Fabregas J,Crespo J,Delgado-Mederos R,et al. Endothelial progenitor cells in acute ischemic stroke.Brain Behav.2013;3(6):649-655.

[19] Li D,Zhang M,Zhang Q,et al.Functional recovery after acute intravenous administration of human umbilical cord mesenchymal stem cells in rats with cerebral ischemia- reperfusion injury.Intractable Rare Dis Res.2015; 4(2):98-104.

[20] Zhang X,Gao F,Yan Y,et al.Combination therapy with human umbilical cord mesenchymal stem cells and angiotensin-converting enzyme 2 is superior for the treatment of acute lung ischemia-reperfusion injury in rats.Cell Biochem Funct.2015;33(3):113-120.

[21] Zhou F,Gao S,Sun C,et al.Adipose-derived stem cells in stroke treatment: translational possibility and mechanism. Chin Med J.2014;127(20):3657-3663.

[22] Dharmasaroja P.Bone marrow-derived mesenchymal stem cells for the treatment of ischemic stroke.J Clin Neurosci. 2009;16(1):12-20.

[23] Liu N,Zhang Y,Fan L,et al.Effects of transplantation with bone marrow-derived mesenchymal stem cells modified by Survivin on experimental stroke in rats.J Transl Med.2011;9:105.

[24] Sun J,Wei ZZ,Gu X,et al.Intranasal delivery of hypoxia- preconditioned bone marrow-derived mesenchymal stem cells enhanced regenerative effects after intracerebral hemorrhagic stroke in mice.Exp Neurol.2015;272:78-87.

[25] Zhu XY,Urbieta-Caceres V,Krier JD,et al.Mesenchymal stem cells and endothelial progenitor cells decrease renal injury in experimental swine renal artery stenosis through different mechanisms.Stem Cells.2013;31(1):117-125.

[26] Ren G,Zhang L,Zhao X,et al.Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide.Cell Stem Cell.2008; 2(2):141-150.

[27] Huang P,Gebhart N,Richelson E,et al.Mechanism of mesenchymal stem cell-induced neuron recovery and anti-inflammation.Cytotherapy.2014;16(10):1336-1344.

[28] Choi C,Oh SH,Noh JE,et al.Attenuation of Postischemic Genomic Alteration by Mesenchymal Stem Cells: a Microarray Study.Mol Cells.2016;39(4):337-344.

[29] Ma S,Xie N,Li W,et al.Immunobiology of mesenchymal stem cells.Cell Death Differ.2014;21(2):216-225.

[30] Nemeth K,Mezey E.Bone marrow stromal cells as immunomodulators. A primer for dermatologists.J Dermatol Sci.2015;77(1):11-20.

[31] Gu N,Rao C,Tian Y,et al. Anti-inflammatory and antiapoptotic effects of mesenchymal stem cells transplantation in rat brain with cerebral ischemia. J Stroke Cerebrovasc Dis.2014; 23(10): 2598-2606.

[32] Aggarwal S,Pittenger MF.Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood. 2005; 105(4):1815-1822.

[33] Chen PM,Liu KJ,Hsu PJ,et al.Induction of immunomodulatory monocytes by human mesenchymal stem cell-derived hepatocyte growth factor through ERK1/2.J Leukoc Biol. 2014;96(2):295-303.

[34] Borlongan CV,Kaneko Y,Maki M,et al. Menstrual blood cells display stem cell-like phenotypic markers and exert neuroprotection following transplantation in experimental stroke.Stem Cells Dev. 2010;19(4):439-452.

[35] Murray KN,Buggey HF,Denes A,et al.Systemic immune activation shapes stroke outcome. Mol Cell Neurosci. 2013;53:14-25.

[36] Acosta SA,Tajiri N,Hoover J,et al.Intravenous Bone Marrow Stem Cell Grafts Preferentially Migrate to Spleen and Abrogate Chronic Inflammation in Stroke. Stroke. 2015; 46(9):2616-2627.

[37] Cheng Q,Zhang Z,Zhang S,et al. Human umbilical cord mesenchymal stem cells protect against ischemic brain injury in mouse by regulating peripheral immunoinflammation. Brain Res.2015;1594:293-304.

[38] Yoo SW,Chang DY,Lee HS,et al.Immune following suppression mesenchymal stem cell transplantation in the ischemic brain is mediated by TGF-beta.Neurobiol Dis. 2013;58:249-257.

[39] Broughton BR,Lim R,Arumugam TV,et al.Post-stroke inflammation and the potential efficacy of novel stem cell therapies: focus on amnion epithelial cells.Front Cell Neurosci. 2013;6:66.

[40] Walker PA,Harting MT,Jimenez F,et al.Direct intrathecal implantation of mesenchymal stromal cells leads to enhanced neuroprotection via an NFkappaB-mediated increase in interleukin-6 production.Stem Cells Dev.2010;19(6):867-876.

[41] Li YF,Ren LN,Guo G,et al.Endothelial progenitor cells in ischemic stroke: an exploration from hypothesis to therapy.J Hematol Oncol.2015;8:33.

[42] Balestrieri ML,Giovane A,Milone L,et al.Endothelial progenitor cells express PAF receptor and respond to PAF via Ca(2+)-dependent signaling.Biochim Biophys Acta. 2010; 1801(10):1123-1132.

[43] Cuccuini W,Poitevin S,Poitevin G,et al.Tissue factor up-regulation in proinflammatory conditions confers thrombin generation capacity to endothelial colony-forming cells without influencing non-coagulant properties in vitro.J Thromb Haemost.2010;8(9):2042-2052.

[44] Sasi SP,Song J,Park D,et al.TNF-TNFR2/p75 signaling inhibits early and increases delayed nontargeted effects in bone marrow-derived endothelial progenitor cells. J Biol Chem.2015;290(45):27014.

[45] Zhao H,Yun W,Zhang Q,et al.Mobilization of Circulating Endothelial Progenitor Cells by dl-3-n-Butylphthalide in Acute Ischemic Stroke Patients.J Stroke Cerebrovasc Dis.2016; 25(4):752-760.

[46] Yoo J,Kim HS,Seo JJ,et al.Therapeutic effects of umbilical cord blood plasma in a rat model of acute ischemic stroke. Oncotarget,2016;7(48):79131-79791.

[47] Vendrame M,Gemma C,de Mesquita D,et al. Anti-inflammatory effects of human cord blood cells in a rat model of stroke.Stem Cells Dev.2005;14(5):595-604.