树突状细胞调节和细胞因子诱导的杀伤细胞的抗肿瘤机制及其在结直肠癌治疗中的应用前景

利用细胞因子诱导树突状细胞（dendritic cell，DC）和细胞因子诱导的杀伤细胞（cytokine induced killer，CIK细胞），以肿瘤抗原冲击DC，将经过抗原冲击的DC和CIK细胞按一定比例混合进行共培养，得到一种新的免疫效应细胞群，即DC调节和细胞因子诱导的杀伤细胞（DCIK细胞）。DCIK细胞具有识别和特异性杀伤恶性细胞的作用，打破了放化疗“敌我不分”的状况。DCIK细胞不但存活率高，增殖能力强．而且对肿瘤细胞具有特异性杀伤能力，为肿瘤免疫治疗开创了一条新的途径。     

骨髓瘤独特型抗原负载树突细胞介导的抗肿瘤效应研究

目的:研究骨髓瘤独特型抗原(Idiotype,Id)负载树突细胞(DC)对同源细胞因子诱导的杀伤细胞(CIK)体外抗瘤活性的影响。方法:采集健康供者外周血单个核细胞(PBMNC)用常规方法诱导DC和CIK细胞,将骨髓瘤OPM-2细胞培养上清提取的Id冲击或未冲击的DC与CIK细胞共培养(CIK、DC加CIK、Id-DC加CIK),用流式细胞术分析细胞表型,MTT法检测体外效应细胞杀伤活性。结果:在(5～20):1效靶比范围内, CIK细胞对OPM-2和K562细胞的杀伤率分别为(24．47±3．00)％～(40．64±1．62)％和(23．36±1．51)％～(42．52±2．06)％。DC加CIK及Id—DC加CIK对OPM-2和K562细胞的杀伤活性均高于CIK组,差异有统计学意义(P<0．05);而在相同效靶比之下,Id-DC加CIK对OPM-2细胞的杀伤活性最强,差异有统计学意义(P <0．05)。结论:CIK细胞对骨髓瘤细胞有强的杀伤活性,经Id负载的DC与CIK细胞共培养能进一步增强其特异性杀伤活性,对骨髓瘤可能有免疫治疗作用。     

树突细胞-细胞因子诱导的杀伤细胞在抗肿瘤生长免疫治疗中的应用

目的研究树突细胞(DC)与细胞因子诱导的杀伤细胞(CIK)在抗肿瘤生长免疫治疗中的应用情况。方法采集26例恶性肿瘤患者外周血进行细胞培养与回输,随机分为观察组(PC、CIK回输)和对照组(CIK回输)。采用流式细胞仪检测细胞免疫表型、采用酶联免疫吸附试验(ELISA)双抗体夹心法检测干扰素(IFN)-γ、白细胞介素(IL)-12、肿瘤坏死因子(TNF)-α细胞因子水平,记录患者不良反应与疗效。结果观察组CD3~+/CD8~+细胞、CD3~+/CD56~+细胞双阳性细胞比例显著高于对照组(P<0.05);CIK细胞的IFN-γ、IL-12、TNF-α细胞因子均显著低于DC-CIK细胞(P<0.05);观察组不良反应率显著低于对照组、治疗效果显著优于对照组(P<0.05)。结论肿瘤患者免疫结构受损,DC-CIK免疫功能优于单纯CIK;DC-CIK生成IFN-γ、IL-12、TNF-α细胞因子能力强、杀伤肿瘤细胞效果优;DC-CIK免疫治疗可有效缓解治疗中发热、发量减少、白细胞数量降低等副作用。     

体外培养的树突状细胞与细胞因子诱导的杀伤细胞相互作用的研究

目的 观察同源的树突状细胞（DC）与细胞因子诱导的杀伤（CIK）细胞于体外同一培养体系共同培养时的相互影响,为临床联合应用DC和CIK细胞进行肿瘤生物治疗提供依据.方法 用无血清培养基进行DC和CIK细胞的体外培养制作,共同培养1 w后,检测CIK细胞免疫表型、杀瘤活性的变化以及DC分泌IL-12的变化.结果 DC与CIK细胞的共培养会增加CIK细胞的CD3CD56双阳性细胞的比例和非特异性增强对K562细胞的杀伤活性;同时增强DC分泌IL-12的能力.结论 体外共培养DC与CIK细胞可相互增强抗肿瘤免疫活性.     

nTreg对DC和CIK细胞疫苗治疗黑色素细胞瘤小鼠的影响

目的探讨自然调节性细胞(nTreg)对基于树突状细胞(DC)和细胞因子诱导的杀伤细胞(CIK)细胞疫苗治疗黑色素细胞瘤小鼠疗效的影响。方法制备出小鼠的黑色素瘤动物模型40只。制备删除nTreg的CIK细胞、未删除nTreg的CIK细胞及输入DC和CIK细胞后再去除nTreg的细胞,并将各组细胞输入动物模型内,观察各组细胞体外杀伤活性及动物模型生存情况。结果第1组制备的DC和CIK疫苗杀伤黑色素瘤细胞活性最强,平均杀伤活性为(98.32±6.24)%,显著高于第2组(45.53±9.13)%和第3组(51.27±8.24)%(P<0.05)。对照组小鼠肿瘤体积显著升高,治疗B和C组在第8、12天肿瘤生长被抑制,治疗A组在第6天肿瘤被抑制,第8、12天肿瘤大小显著降低。对照组小鼠在30 d内全部死亡,治疗A组仅在第3、6天分别死亡1只,治疗B组30 d内有5只存活,治疗C组30 d有3只存活,均显著高于对照组(均P<0.05)。结论 nTreg会抑制DC和CIK疫苗杀伤肿瘤细胞活性以及治疗肿瘤疗效。     

树突状细胞与细胞因子诱导的杀伤细胞共培养对白血病耐药细胞杀伤作用的实验研究

目的探讨负载P-糖蛋白(P-gp)高表达的多药耐药(MDR)白血病K562/A02细胞冻融抗原的树突状细胞(DC)与同源细胞因子诱导的杀伤细胞(CIK)共培养对MDRK562/A02杀伤作用的影响。方法提取健康人骨髓单个核细胞,常规诱导出DC及CIK,将K562/A02细胞冻融物作为抗原冲击的DC,与CIK共培养作为实验组,抗原不冲击的DC与CIK共培养作为对照组,以CIK及DC单独培养分别作为空白对照组1和空白对照组2。光镜下观察细胞形态,流式细胞术分析细胞表型,MTT法检测杀伤活性。结果实验组、对照组细胞增殖活性均大于CIK组(P<0.05)。实验组对K562/A02、K562的杀伤活性在效靶比5∶1、10∶1、20∶1时分别为(42.90±0.67)%、(49.85±0.28)%、(63.36±0.46)%和(23.56±0.43)%、(26.11±0.34)%、(34.46±0.35)%,均高于对照组及空白对照组1(P<0.05);实验组对K562/A02的杀伤活性高于K562和MCF7(P<0.05)。结论DC与CIK共培养物是一种增殖活性和细胞毒活性高于CIK的免疫活性细胞,而经冻融抗原冲击的DC与CIK共培养能明显提高对MDRK562/A02的杀伤活性。     

树突状细胞联合细胞因子诱导的杀伤细胞对胃癌细胞杀伤作用

目的探讨树突状细胞联合细胞因子诱导的杀伤细胞对胃癌细胞的杀伤作用。方法采用胃癌患者自身血液中单个核细胞(peripheral blood mononuclear cells,PBMC),经体外诱导分别扩增出DC和CIK细胞,二者共同培养后,利用MTT法检测DC细胞联合CIK细胞体外杀伤人胃癌细胞株(MNK-45、MNK-28、SG-7901)的活性。结果DC与CIK细胞共培养后得到的细胞群高表达CD3 CD56 ,平均值达到(56.74±7.63)%。通过彼此相互作用诱导出的细胞群体对胃癌细胞株MNK-45、MNK-28、SG-7901有杀伤作用,且杀伤活性随着效靶比的增加而增强。结论DC与CIK细胞共培养后有很强的增殖能力,对胃癌细胞具有杀伤活性,且其杀伤作用与胃癌细胞类型无相关性。     

树突状细胞与同源细胞因子诱导的杀伤细胞共培养细胞在肿瘤免疫治疗中的作用

目的观察树突状细胞(DC)与同源细胞因子诱导的杀伤 (CIK)细胞共培养后,共培养细胞(DC-CIK细胞)的表型、体外增殖活性和细胞毒活性的变化,并探讨CIK细胞在肿瘤治疗中的作用.方法 (1)提取3名健康献血者的外周血单个核细胞(PBMC),常规诱导出DC与CIK细胞后,将其共培养,动态观察CIK细胞和DC-CIK细胞增殖活性和表型变化;并用四甲基偶氮唑蓝(MTT)法测定其体外细胞毒活性;(2)80只BALB/c裸鼠随机分为2组,分别在前肢腋下接种A549肺腺癌细胞和BEL-7404肝癌细胞.10 d后分别选择荷A549和BEL-7404瘤大小相似的裸鼠各30只,全部一次性腹腔注射化疗药物后,每组再随机分成3组.①单独化疗组;②CIK治疗组;③DC-CIK治疗组,每组10只. 单独化疗组注射化疗药物后不再进行任何处理,另2组分别于化疗后的第2、4、6、8、10天腹腔注射CIK细胞和DC-CIK细胞进行免疫治疗.结果 (1)经DC作用后的CIK细胞CD +3CD +56双阳性细胞和CD +8细胞的数量明显升高;(2)DC-CIK细胞的增殖速度明显快于同源CIK细胞,DC-CIK细胞的细胞毒活性远高于CIK细胞;(3)在肿瘤的治疗中,CIK细胞可提高化疗的效果.化疗后25 d,CIK治疗组和DC-CIK治疗组肿瘤受抑制的程度均明显大于单独化疗组,DC-CIK组大于CIK治疗组(P<0.05).结论 DC与CIK细胞共培养细胞是一种强于CIK细胞的免疫活性细胞,在肿瘤治疗中具有更广阔的应用前景.     

DC与CIK或LAK联合对结肠癌细胞株SW480杀伤作用的研究

[目的]研究树突状细胞(DC)联合细胞因子诱导或未诱导的杀伤细胞(CIK)或淋巴因子激活的杀伤细胞(LAK)对结肠癌细胞株SW480的杀伤活性.提供DC联合CIK或LAK治疗结肠癌的实验依据.[方法]取人外周血分离出单个核细胞(PBMNC),诱导生成DC、CIK、LAK细胞;流式细胞仪检测DC经SW480肿瘤抗原冲击后的表型变化;以CIK+DC细胞、CIK细胞、LAK+DC细胞及LAK细胞作为效应细胞,SW480为靶细胞,以15∶1、30∶1、45∶1为效靶比,LDH释放法测定细胞杀伤试验活性;ELISA检测杀伤试验中干扰素γ(IFN-γ)、白细胞介素2(IL-2)、IL-12、IL-17的分泌水平.[结果]流式细胞仪检测DC经SW480肿瘤抗原冲击后,其表面分子HLA-DR、CD40、CD80和CD86表达分别平均为90.23％、73.68％、85.96％、57.55％,与未经肿瘤抗原冲击DC比较,DC成熟的表面标志分子表达明显增加(P＜0.01).相同效靶比下,CIK+DC细胞组对SW480的杀伤作用最强,明显高于其他细胞组(P＜0.01);CIK+ DC细胞组在效靶比为45∶1时,杀伤活性最强(P＜0.01);单独CIK细胞组的杀伤活性明显高于LAK+DC细胞组(P＜0.01);LAK+ DC细胞组的杀伤活性明显高于单独LAK细胞组(P＜0.01).效靶比为45∶1时,各杀伤试验细胞组上清液中IFN-γ、IL-2、IL-12、IL-17的分泌量,CIK+DC细胞组的IFN-γ、IL-12的分泌量显著高于其他细胞组(P＜0.05);LAK+DC、单独LAK细胞组IL-2的分泌量明显高于CIK+DC、单独CIK细胞组(P＜0.05);单独CIK细胞组IFN-γ的分泌量明显高于LAK+DC、单独LAK细胞组(P＜0.05).[结论]CIK+DC细胞组对SW480的杀伤活性明显强于单独CIK、LAK+ DC组、单独LAK细胞组.其机制可能是,SW480抗原致敏的DC分泌IFN-γ、IL-12等刺激、诱导CIK细胞的活化和增殖,明显增强CIK细胞杀伤SW480的活性.     

脐血DC与CIK共培养对白血病K562细胞杀伤活性的影响

目的观察细胞因子诱导的杀伤细胞（CIK）与同源树突状细胞（DC）共培养后DC—CIK细胞的增殖活性、表型的变化及其对白血病K562细胞杀伤活性的影响。方法采集健康产妇分娩正常足月胎儿脐血50ml,密度梯度离心法分离出脐血单个核细胞培养。收集非贴壁细胞用于诱导培养CIK,贴壁细胞诱导分化出成熟DC;将成熟DC和CIK按1：5的比例混合培养3d,用MTT法检测Dc—CIK共培养细胞对白血病K562细胞杀伤活性。结果DC与CIK共培养后,DC—CIK细胞群的增殖活性和杀伤活性明显高于单纯CIK。结论DC—CIK共培养可明显提高CIK增殖活性和细胞毒作用。     

Erythrocyte-endothelial cell adherence in sickle cell disorders

Detachment of individual sickle erythrocytes from cultured endothelial cell monolayers has been evaluated by a fluid-shearing technique in an effort to quantitate adherence at shear forces that would be anticipated in the in vivo circulation. Nonirreversibly sickled cells (non-ISC) were more adherent at normal oxygen tensions than control cells. More than 1% non-ISC remained attached to the monolayer at forces greater than physiologic shear stresses in capillary and venous circulations, and many of the most avidly attached cells, once separated, immediately reattached to adjacent endothelial cells. These data suggest that hemoglobin S-containing erythrocytes may have a higher frequency of adherence in vivo in regions of low shear stress where prolonged erythrocyte-endothelial cell contact could occur. Some of these cells detached by shear force would subsequently reattach in in vivo conditions. Plasma-enhanced attachment frequency and plasma from blood in a case of sickle crisis caused further increase. These observations further support the concept that sickle erythrocyte- endothelial cell interaction may be a significant factor in initiation of vascular occlusive events in sickle cell disease.     

Liver cell adenoma and liver cell adenomatosis

During the last three decades liver cell adenoma and liver cell adenomatosis have emerged as new clinical entities in hepato-logical practice due to the widespread use of oral contraceptives and increased imaging of the liver. On review of published series there is evidence that 10% of liver cell adenomas progress to hepatocellular carcinoma, diagnosis is best made by open or laparoscopic excision biopsy, and the preferred treatment modality is resection of the liver cell adenoma to prevent bleeding and malignant transformation. In liver cell adenomatosis, the association with oral contraceptive use is not as high as in solitary liver cell adenomas. The risk of malignant transformation is not increased compared with solitary liver cell adenomas. Treatment consists of close monitoring and imaging, resection of superficially located, large (>4 cm) or growing liver cell adenomas. Liver transplantation is the last resort in case of substantive concern about malignant transformation or for large, painful adenomas in liver cell adenomatosis after treatment attempts by liver resection.     

Red cell alloimmunization in sickle cell disease

Alloimmunization to red cell antigens contributes to morbidity in transfused patients. It has been recommended that blood for sickle cell patients need not be matched for antigens other than ABO and Rh(D), as there is no greater incidence of antibody production than in other multitransfused patient populations. Post transfusion alloimmunization was studied in a group of 34 sickle cell disease patients attending a U.K. haemoglobinopathy clinic. Red cell antibodies were formed in 17.6% of the transfused patients and Rhesus and Kell antibodies accounted for 66% of this total. In order to reduce alloimmunization, a policy of performing extended red cell phenotyping on the patients, and providing blood matched for Kell, and in certain circumstances the Rhesus antigens other than Rh(D), is recommended.     

Misdiagnosis of clear cell renal cell carcinoma

Clear cell renal cell carcinoma (RCC) represents the most common histological subtype of malignant kidney tumors. Based on symptoms alone, clear cell RCC is indistinguishable from other histological classes of RCC unless the tumor is present in the context of an RCC syndrome. Histopathological examination is, therefore, important to accurately identify clear cell RCC. Clear cell RCCs have characteristic morphological criteria; these tumors can be easily identified upon typical presentation, but diagnosis can be challenging when tumor cell pattern is unusual or when availability of tissue samples is limited. In this Review, the clinical, radiological and pathological characteristics of clear cell RCCs are described, as well as the potential tumors that can be confused with clear cell RCC and need to be considered in the differential diagnoses. Finally, the importance of an accurate diagnosis is highlighted in the context of the increasing use of preoperative tissue sampling and the prevalence of clear cell tumors associated with hereditary syndromes, which could have different therapeutic and prognostic implications for patients and their families.     

Red cell exchange in sickle cell disease

Red cell exchange transfusion is frequently of use in the management of patients with sickle cell disease either electively or therapeutically. Modern cell separators allow this procedure to be performed rapidly, effectively and safely. These machines have a number of advantages over manual exchange procedures. The patient remains isovolaemic, there is little loss of plasma or platelets, the procedure is relatively short and in elective circumstances can be performed on an outpatient basis. In this series 66 exchanges were performed on 21 patients with an overall increase in HbA of 70%. The COBE Spectra gave a mean increase in HbA of 77%, with the majority of patients achieving an HbA of > 90% post exchange. Automated redcell exchange was well tolerated by most patients, and adverse effects were limited to symptoms of hypocalcaemia which were easily treated, and to transfusion reactions. Cell separators can therefore be recommended for exchange transfusion in patients with sickle cell disease, who require an increase in HbA levels either prophylactically or therapeutically. They are safe, effective, easy and quick to use.     

Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines

Background

Cancer stem cells (CSCs) are regarded as the cause of tumor formation and recurrence. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs.

Methods

Human hepatoma cell lines were plated in stem cell conditioned culture system allowed for sphere forming. To evaluate the stemness characteristics of spheres, the self-renewal, proliferation, chemoresistance, tumorigenicity of the PLC/PRF/5 sphere-forming cells, and the expression levels of stem cell related proteins in the PLC/PRF/5 sphere-forming cells were assessed, comparing with the parental cells. The stem cell RT-PCR array was performed to further explore the biological properties of liver CSCs.

Results

The PLC/PRF/5, MHCC97H and HepG2 cells could form clonal nonadherent 3-D spheres and be serially passaged. The PLC/PRF/5 sphere-forming cells possessed a key criteria that define CSCs: persistent self-renewal, extensive proliferation, drug resistance, overexpression of liver CSCs related proteins (Oct3/4, OV6, EpCAM, CD133 and CD44). Even 500 sphere-forming cells were able to form tumors in NOD/SCID mice, and the tumor initiating capability was not decreased when spheres were passaged. Besides, downstream proteins DTX1 and Ep300 of the CSL (CBF1 in humans, Suppressor of hairless in Drosophila and LAG1 in C. elegans) -independent Notch signaling pathway were highly expressed in the spheres, and a gamma-secretase inhibitor MRK003 could significantly inhibit the sphere formation ability.

Conclusions

Nonadherent tumor spheres from hepatoma cell lines cultured in stem cell conditioned medium possess liver CSC properties, and the CSL-independent Notch signaling pathway may play a role in liver CSCs.     

Actin filament debranching regulates cell polarity during cell migration and asymmetric cell division

The formation of the branched actin networks is essential for cell polarity, but it remains unclear how the debranching activity of actin filaments contributes to this process. Here, we showed that an evolutionarily conserved coronin family protein, the Caenorhabditis elegans POD-1, debranched the Arp2/3-nucleated actin filaments in vitro. By fluorescence live imaging analysis of the endogenous POD-1 protein, we found that POD-1 colocalized with Arp2/3 at the leading edge of the migrating C. elegans neuroblasts. Conditional mutations of POD-1 in neuroblasts caused aberrant actin assembly, disrupted cell polarity, and impaired cell migration. In C. elegans one-cell−stage embryos, POD-1 and Arp2/3, moved together during cell polarity establishment, and inhibition of POD-1 blocked Arp2/3 motility and affected the polarized cortical flow, leading to symmetric segregation of cell fate determinants. Together, these results indicate that F-actin debranching organizes actin network and cell polarity in migrating neuroblasts and asymmetrically dividing embryos.

Cell polarity is a fundamental feature of virtually all eukaryotic cells and plays crucial roles in a wide range of cellular processes, including cell motility, asymmetric cell division, and cell signaling (1). The establishment of cell polarity involves the asymmetric assembly of distinct cellular components to perform specialized functions. The actin-related protein (Arp) 2/3 complex-dependent branched actin networks and the pushing force they produce provide the principal means for cells to remodel the plasma membrane during cellular polarization (2). For example, in the leading edge of a migrating cell, the local Arp2/3-nucleated actin polymerization powers asymmetric projections of the plasma membrane (3). During asymmetric cell division of the Caenorhabditis elegans zygote, an actomyosin flow is central to the transport of the polarity PAR proteins into defined subcellular domains (4).Actin filaments'' continuous assembly must be balanced by actin depolymerization to ensure a constant supply of actin monomers for new growth. The Arp2/3 complex potency in actin nucleation empowers this complex as an essential regulator to organize the actin cytoskeleton. While Arp2/3 by itself is biochemically inactive, interactions with nucleation-promoting factors (NPFs) such as the Wiskott Aldrich syndrome protein (WASP)/WASP family verproline-homologous (WASP/WAVE) family proteins shift the Arp2/3 complex from its open, inactive conformation to a closed, active conformation (5, 6). The conformationally activated Arp2/3 complex then binds to the side of preexisting actin filaments to nucleate a branch from the mother filament (7–12). Conversely, nucleation by Arp2/3 can be inhibited by several binding partners, including glia maturation factor (GMF), Gadkin, Arpin, and Coronin, whose activities replenish available pools of actin monomers and Arp2/3 complexes for sustained actin assembly (13–18).The coronin family proteins are conserved actin regulators (19). The phylogenetic analysis grouped coronin genes into three types (19, 20). The best-characterized coronin is the Type I coronin (e.g., Coronin 1B) that binds to actin filaments through the β-propeller structure and to the Arp2/3 complex via its N terminus. These interactions block the docking of Arp2/3 onto actin filaments or facilitate debranching the existing actin network (20). Coronin 1B simultaneously interacts with the Slingshot phosphatase to dephosphorylate and activate ADF/Cofilin proteins that sever actin filaments, thereby promoting the actin network disassembly (13). Despite significant progress on Type I coronin, the activity and function of other coronins remain unclear. In particular, Type III coronins, known as POD-1 in C. elegans and Drosophila or Coronin7 in Dictyostelium and humans, contain two tandem coronin repeats, making them distinct from other coronins (19–21). POD-1 was biochemically isolated from C. elegans oocytes (22), and its mutations disrupted the polarity and architecture in early C. elegans embryos and impaired midlife touch sensitivity of the nematode (21, 23). However, it remains unclear how the Type III coronin functions. The Drosophila homolog of POD-1 is required for correct axon guidance, and the purified Dpod-1 cross-links the actin and microtubule cytoskeletons (24), whereas the mammalian Coronin7 was implicated in the Golgi morphology and function (25, 26), demonstrating the functional divergence of this family of coronin. Here, we show that the C. elegans POD-1 debranches Arp2/3-nucleated actin filaments in vitro and that POD-1 regulates cell polarity by remodeling the actin cytoskeleton during cell migration and asymmetric cell division.     

Mechanism of red blood cell aging: Relationship of cell density and cell age

The human red cell has a life span of 120 days. The mechanism that determines cell removal from the circulation with such precision remains unknown. Most studies of red cell aging have been based on analysis of cells of progressively increasing age separated by density. The relationship between red cell age and density has been recently challenged, and the hypothesis has been put forward that cell death is not the result of a progressive deterioration of essential cell constituents. This theory was based on preliminary observations in transient erythroblastopenia of childhood, which could not later be confirmed. When the relationship between cell aging and increasing density is critically reviewed, it appears to be based on firm experimental evidence, confirmed by in vivo demonstration of decreasing survival of cells of increasing age. Analysis of studies using buoyant density gradients reveals that this technique can easily distinguish the single exponential slope of decline for those cell components that change progressively throughout the red cell life span from the biphasic decline of those that decrease drastically at the reticulocyte-mature red cell transition. The view that the aging of the red cell and its removal from the circulation result from a progressive series of events during the 120 days of its life span appears to be the most consistent with the available data. Density separation, validated by much experimental evidence, remains a most useful technique for the study of the mechanism of aging of the red cell. © 1993 Wiley-Liss, Inc.     

Red cell rheology in stomatocyte-echinocyte transformation: roles of cell geometry and cell shape

The influence of the shape of the red blood cell during stomatocyte- echinocyte transformation on its deformability was studied by microsieving through pores with diameters of 2.6, 4.5, and 6.9 micron. A stomatocytic transformation was produced by chlorpromazine (0.02, 0.1, and 0.5 mmol/L) and an echinocytic transformation by sodium salicylate (7.5, 30, and 120 mmol/L). For spherostomatocytes, an increase in filtration resistance through 2.6 and 4.5 micron pores was observed, whereas for spheroechinocytes, a decrease in filtration resistance through 2.6 micron pores was found. Larger pores (6.9 micron) were not sensitive to those shape changes. The changes in deformability can be explained by the fact that the surface area of (sphero)-stomatocytes decreased, whereas that of (sphero)-echinocytes increased; the cell volume remained essentially constant. Echinocytes produced by 24-hour adenosine triphosphate depletion differed from drug- induced echinocytes: they had an increased cell volume at constant surface area and consequently an increased filtration resistance through 2.6- and 4.5-micron filter pores. Shape changes with spicule formation are therefore not a homogeneous entity, and cell geometric factors (eg, surface area and volume) must be assessed with care. The viscosity of red cell suspensions at a hematocrit level of 45% was higher for drug-induced echinocytes than discocytes or stomatocytes at all shear rates tested. We conclude that the normal discocyte represents an optimum shape for the flow in vivo since a stomatocytic transformation could impair the passage through the microcirculation (decrease in cell filterability) and an echinocytic transformation could impair the flow in larger vessels (increase in blood viscosity).     

Automated red cell exchange in sickle cell disease

We used multiple optical trapping to study the mechanism of red cell (dis)aggregation. Two sets of optical 'tweezers' were used to bring two red blood cells together to form a two-cell aggregate and then to pull them apart, to study the interaction between the cells.
We found that cross-bridging occurred in normal reversible aggregation as we observed binding and the occurrence of small tethers between opposite cell membranes. Furthermore, the cells could only be parted by sliding them side by side with a maximum velocity in the order of μm/s indicating accumulation of the cross-bridges.