肿瘤热休克蛋白70联合IL-2对荷瘤小鼠的治疗作用

目的:研究肿瘤热休克蛋白70(HSP70)与IL-2联合应用对荷瘤小鼠的治疗作用。方法:用液相色谱法纯化小鼠肿瘤细胞 株中的HSP70。对纯化产物用SDS-PAGE及Western blot进行定性分析,再用毛细管电泳鉴定其纯度。通过动物实验,观察HSP70与IL-2单独应用及联合应用的抗肿瘤作用。结果:HSP70与IL-2联合应用较单独应用的治疗效果更明显,但治疗作用仍以HSP70为主。单独应用IL-2只能延长小鼠的生存期限[平均为(36.6±13.0)d],而HSP70 10μg组可使40％荷瘤小鼠的肿瘤最终全部消退,生存期最长者>90[平均生存期(>59.2±29.6)d],与对照组相比较差异显著(P<0.01)。HSP70与IL-2联合应用组可使60％荷瘤小鼠的肿瘤完全消退,平均生存期(>70.8±26.5)d,与对照组相比差异十分显著(P<0.01)。结论:合适剂量的HSP70与IL-2联合应用,对荷瘤小鼠有明显的治疗作用,可明显抑制肿瘤进展,提高生存率。以上结果对研究人类恶性肿瘤的免疫治疗具有较大的参考价值。     

微波消融联合消融灶周边注射CpG ODN诱导抗小鼠肝癌免疫效应

目的:探讨消融灶周围注射CpG ODN诱导抗肿瘤免疫反应的效能。方法:建立C57BL/6J小鼠Hepa 1-6细胞双侧皮下肝癌模型,根据右侧皮下肝癌治疗的方法分为联合治疗组(微波消融联合消融灶周边注射CpG ODN治疗)、消融治疗组、CpG ODN治疗组和对照组。观察双侧皮下肝癌体积变化、小鼠生存时间。免疫组化检测右侧肿瘤组织热休克蛋白70(HSP70)表达和左侧肿瘤组织CD4、CD8T淋巴细胞的数量。ELISA法检测各组小鼠血清IL-12、IL-10含量。LDH释放法检测各组脾源性CTL杀伤活性。另选取单侧皮下肝癌小鼠,分别给予联合治疗和消融治疗,30天后在对侧皮下再次接种同种肝癌细胞,观察再接种肿瘤的成瘤率和肿瘤体积变化。结果:双侧皮下肝癌模型小鼠联合治疗组和消融治疗组右侧肿瘤体积体积显著小于CpG ODN治疗组和对照组(P<0.05)。联合治疗组中左侧未治疗的肿瘤体积在第10天后明显小于其他各组(P<0.05)。中位生存时间分别为:联合治疗组73天,消融治疗组60天,CpG ODN治疗组55天,对照组38天。联合治疗组左侧肿瘤组织内CD8T淋巴细胞数量为(25.17±5.46)/HP,显著高于其他3组(P<0.05)。联合治疗组和消融治疗组右侧肿瘤组织HSP70标记指数分别为(39.95±9.03)%和(33.90±7.98)%,明显高于CpG ODN治疗组和对照组(P<0.05)。联合治疗组小鼠血清IL-12含量显著高于其他各组(P<0.05),IL-10含量显著低于其他各组(P<0.05)。联合治疗组中脾CTL细胞对Hepa 1-6杀伤率显著高于其他各组(P<0.05)。单侧皮下肝癌小鼠联合治疗组再接种肿瘤细胞的成瘤率为58.33%,而消融治疗组的成瘤率为83.33%,且联合治疗组的再接种肿瘤体积在第20天后明显小于消融治疗组(P<0.05)。结论:微波消融能促使肿瘤细胞HSP70表达,微波消融联合消融灶周边注射CpG ODN能激活小鼠体内的CD8 T淋巴细胞,促使小鼠体内发生Th2/Th1免疫偏移,从而产生抗肿瘤的免疫效应,抑制肿瘤生长。     

HSP70多肽复合物修饰DCs疫苗抗胰腺癌荷瘤小鼠的实验研究

目的:探讨负载热休克蛋白70多肽复合物(HSP70-PC)抗原的树突状细胞(DCs)疫苗对胰腺癌荷瘤小鼠的免疫治疗作用.方法:采用低渗裂解、ConA-Sepharose亲和层析及ADP-Agarose亲和层析法,从小鼠胰腺癌(MPC83)肿瘤组织中纯化HSP70-PC,修饰小鼠骨髓来源的树突状细胞(DCs),制备树突状细胞HSP70多肽肿瘤疫苗,用MTT法检测混合淋巴细胞反应(MLR)中HSP70-PC致敏DC对CTL的增殖及活化效果;建立MPC83荷瘤小鼠模型,观察树突状细胞HSP70多肽肿瘤疫苗对荷瘤小鼠治疗的效果和小鼠存活期.结果:经上述方法分离、纯化获得了较高纯度的HSP70-PC蛋白质;HSP70-PC在1.5～2.0 μg/ml范围内可达到刺激树突状细胞最强效果,用HSP70-PC修饰的DCs能增强T细胞的增殖和活化能力;应用树突状细胞HSP70多肽肿瘤疫苗治疗荷瘤小鼠能显著抑制荷瘤小鼠肿瘤的生长,延长荷瘤小鼠存活期.结论:采用低压亲和层析柱可从胰腺癌瘤块中获得较高纯度HSP70多肽复合物,其修饰的DCs疫苗用于荷瘤小鼠免疫治疗有显著疗效,为临床胰腺癌生物免疫治疗奠定基础.     

IL-2与眼镜蛇毒协同抗肿瘤作用的实验研究

本文研究了白细胞介素2(IL-2)和眼镜蛇毒对小鼠移植鼠肝癌的抑制作用以及对荷移植窟小鼠NK细胞杀伤活性的影响。结果表明:IL-2或眼镜蛇毒单独使用时,前者能明显增强荷瘤鼠的NK细胞活性,可是抑瘤作用不很明显;后者虽然严重地减弱荷瘤鼠的NK活性,但抑瘤作用反而较前者为强。当两者联合使用时,其抑瘤作用及NK活性则都显著提高(P<0.005)。提示IL-2与眼镜蛇毒具有良好的协同抗小鼠移植鼠肝癌的作用。     

热休克蛋白70的抗肿瘤免疫效应研究

目的 :研究HSP70诱导的抗肿瘤免疫效应 ,为应用其治疗人类恶性肿瘤提供实验依据。方法 :应用细胞培养、液相色谱法、电泳技术、Western blot法等获得高纯度肿瘤中的HSP70 ,将其应用于同源的 61 5系小鼠 ,观察其抗肿瘤免疫效应。结果 :HSP70免疫后的小鼠能够抵抗同种肿瘤细胞的攻击 ,其中 1 0 μg免疫后的小鼠 1 0 0 %获长期生存 (>90d) ,无一死于肿瘤 ,5μg免疫后的小鼠肿瘤进展受到抑制 (平均生存 2 8 2± 4 8d) ,但无一小鼠获长期生存 ,上述两组与对照组 (平均生存 1 8 3± 3 6d)相比差异均具有显著性 (P <0 0 1 )。HSP70对荷瘤小鼠的治疗研究证明 ,5μgHSP70有一定的治疗作用 ,荷瘤小鼠平均生存 31 3± 2 9d ,而 1 0 μgHSP70治疗组的小鼠生存期 >58 8± 33 7d ,其中 40 %的小鼠所接种肿瘤完全消退 ,获长期生存(>90d) ,与其他组相比 ,差异具有显著性 (P <0 0 1 )。结论 :HSP70可诱发强大的抗肿瘤免疫效应 ,并且有良好的治疗作用 ,对于研究利用HSP70治疗人类的恶性肿瘤有重要的参考意义。     

混合热休克蛋白/肽疫苗与IL-2联合免疫治疗肉瘤的实验研究

目的观察混合热休克蛋白/肽(mHSP/P)疫苗、环磷酰胺(Cy)及重组人IL-2(rhIL-2)联合免疫治疗小鼠肉瘤的效果。方法取小鼠S180细胞肉瘤组织,经层析分离获得混合mHSP/P疫苗,进行常规蛋白质印迹鉴定。S180细胞接种至小鼠皮下后第2天开始实验,将小鼠按mHSP/P、Cy、rhIL-2药物组合、疗程和剂量的不同依次分为B～I组,以及给予生理盐水的A组(对照组)。自肿瘤移植后至第45天,观察计算肿瘤体积;以肿瘤移植后3个月仍无瘤生长或肿瘤逐渐缩小至消失为标准,观察各组小鼠的无瘤生存情况,并进行平均生存期比较;以对照组的肿瘤体积为参照,计算第35天各组小鼠肿瘤的生长抑制率。结果蛋白质印迹分析结果显示所获目的蛋白为含有HSP110、HSP70、HSP60、Gp96的混合mHSP疫苗。3种药物联合应用的治疗效果明显优于单用mHSP/P疫苗,且多疗程较单一疗程疗效好,其中rhIL-2的最佳治疗剂量为5×104～1×105IU/只,并可使30%的小鼠肿瘤消退而长期生存(治愈),平均生存期延长22～26d,肿瘤生长抑制率为65%～76%。结论mHSP疫苗与适量rhIL-2以及小剂量Cy联合应用为临床肉瘤治疗新的免疫治疗方案提供了实验基础。     

Hsp70L1增强肿瘤细胞疫苗免疫原性的研究

目的:考察Hsp70L1对肿瘤细胞免疫原性的增强作用。方法:用RT-PCR的方法,从小鼠黑色素瘤B16细胞和C57BL/6小鼠脾脏中获得TRP2153-243及Hsp70L1基因。分别插入pcDNA3.1/V5-His真核表达载体,构建pHSP70L1、pTRP和pTRP2-Hsp3种表达载体。分别转染B16肿瘤细胞并制备坏死或凋亡瘤苗,免疫C57BL/6小鼠后移植B16肿瘤细胞,观察肿瘤生长曲线,采用流式细胞术(FCM)或微量细胞毒的方法检测荷瘤小鼠细胞因子INF-γ和CTL活性。结果:将经过HSP70L1、TRP2及TRP2-HSP基因修饰的坏死或凋亡的B16肿瘤细胞免疫正常小鼠后,观察到Hsp70L1及TRP2-Hsp基因修饰的坏死瘤苗可显著抑制荷瘤鼠肿瘤的生长,并显著促进荷瘤鼠脾脏淋巴细胞CTL活性和IFN-γ产生(P0.05,P0.01);HSP70L1免疫刺激作用在坏死瘤苗中更明显。结论:Hsp70L1可明显提高B16瘤苗的免疫原性,且对坏死细胞瘤苗的作用更显著。     

瘤内注射MIP-3α对小鼠肝癌生长抑制作用的研究

目的:探讨瘤内注射巨噬细胞炎症蛋白-3α(macrophage inflammatory protein-3α,MIP-3α)能否趋化外周树突状细胞(Dendritic cells,DCs)至肿瘤组织内,诱导特异性免疫应答。方法:成功建立小鼠皮下肝癌模型后随机分为3组,第10天起分别向MIP-3α治疗组、PBS对照组的小鼠皮下肿瘤内注射MIP-3α溶液及PBS,空白对照组小鼠不予任何处理。20天后取肿瘤组织,免疫组化法检测肿瘤内DCs、CD4+、CD8+细胞浸润情况,流式细胞术检测肿瘤内DCs浸润数量及其表型。另外,每组各10只小鼠持续观察,用于绘制肿瘤生长曲线并观察生存时间。结果:①MIP-3α治疗组的小鼠肿瘤内浸润的CD4+、CD8+细胞及DCs数量均显著高于其他两组。②MIP-3α治疗组小鼠肿瘤内浸润性DCs的CD80、CD86表达率显著高于其他两组(P<0.05)。③MIP-3α治疗组小鼠肿瘤生长速率显著低于对照组(P<0.001),生存时间较对照组明显延长(P<0.05)。结论:①瘤内注射MIP-3α可在小鼠肝癌病灶内趋化、募集外周的树突状细胞,使其摄取并提呈肿瘤抗原,有效诱导针对肝癌细胞的特异性免疫应答。②MIP-3α在小鼠皮下肝癌模型的局部微环境下可能具有促进树突状细胞成熟的作用。     

IL-21基因治疗小鼠H22细胞皮下移植肝癌模型的效果评价

用已构建的mIL-21/pcDNA3.1重组质粒对H22细胞建立的小鼠肝癌模型进行基因治疗,观察IL-21对小鼠体内抗肿瘤免疫应答的影响及对小鼠生存的影响。采用BALB/c小鼠左腋皮下注射腹水型肝癌细胞株H22细胞建立小鼠移植肝癌模型,给荷瘤小鼠瘤体内注射mIL-21/pcDNA3.1进行基因治疗,MTT比色法检测IL-21对荷瘤小鼠T细胞增殖水平及NK细胞杀伤活性的影响,观察治疗后荷瘤小鼠生存情况及肿瘤生长情况的改变。病理检测结果显示,成功建立了小鼠移植型肝癌模型,MTT比色法显示基因治疗后小鼠T细胞增殖水平及NK细胞杀伤活性显著升高,荷瘤小鼠肿瘤生长速度减慢,生存期显著延长。IL-21基因治疗肝癌荷瘤小鼠可显著提高荷瘤小鼠体内抗肿瘤免疫应答水平,抑制肿瘤生长,延长荷瘤小鼠生存期。     

肿瘤坏死因子-α联合干扰素-γ治疗肝癌的实验研究

目的：探讨干扰素-γ体内外增强TNF-α治疗原发性肝癌（HCC）的作用。 方法： 采用结晶紫染色法测定肿瘤坏死因子-α（TNF-α）、干扰素-γ（IFN-γ）和两者联合用药体外杀灭肝癌细胞的能力。以已建立的人肝癌裸小鼠肝内移植模型为对象，采用瘤体内注射的直接给药途径，观察TNF-α、IFN-γ和联合用药体内抗肝癌的效果。 结果： 细胞毒性试验：TNF-α对肝癌细胞有较强的细胞毒性作用，呈剂量依赖性。当浓度超过107 U/L时，有明显杀伤作用(P<0.05)；联合应用IFN-γ具有协同抗瘤效果，单独应用107 U/L TNF-α和106 U/L IFN-γ时，肝癌细胞杀伤率分别为27.1％和7.9%，两者联合用药可达83.7%。单独使用IFN-γ未见移植瘤生长受抑制及荷瘤鼠生存期延长(P＞0.05)，移植瘤仅见小灶性坏死；单独应用TNF-α，表现为抑制移植瘤生长(抑制率17.2％)，移植瘤呈不完全性小片状坏死，但不能延长荷瘤鼠的生存时间(P>0.05)。两者联合用药能明显抑制肿瘤生长(抑癌率35.9%)，移植瘤呈大片状坏死，荷瘤鼠生存期延长(P<0.05)，血甲胎蛋白（AFP）浓度降低。 结论： TNF-α联合IFN-γ直接瘤体内注射有可能成为临床治疗原发性肝癌一个新的有效方法。     

体外构建的HSP70-肝癌抗原肽诱导抗原肽特异性免疫反应1

目的研究体外构建的HSP70-肝癌抗原肽复合物诱导针对肝癌的特异性免疫反应能力,为该复合物的临床应用奠定基础.方法在体外构建HSP70-肝癌抗原肽复合物,联合应用粒/巨细胞集落刺激因子(GM-CSF)及白介素-4(IL-4)直接从志愿者外周血中培养出DC;以HSP70、HSP70-肝癌抗原肽、抗原肽分别刺激DC,DC激活同源的T淋巴细胞产生细胞毒性T淋巴细胞(CTL),检测其杀伤T2细胞和肝癌细胞系的能力.结果HSP70-抗原肽、抗原肽均可诱导CD8+的抗原肽特异性CTL,而前者的诱导效果更强.结论体外构建的HSP70-抗原肽复合物具有免疫原性,HSP70可以增强抗原肽诱导特异性免疫反应的能力,HSP70-抗原肽复合物有可能作为肽疫苗用于临床肿瘤免疫治疗.     

人肝癌组织中p53与HSP70相互作用的初步研究

目的 :探讨人肝癌中热休克蛋白 70 (HSP70 )与 p5 3的相互作用。方法 :用免疫组织化学染色法 ,从 12例肝癌组织中筛选HSP70与 p5 3均呈阳性表达的标本 ,并以免疫共沉淀法提取之。然后用SDS PAGE及Westernblot分析双阳性标本中两种蛋白的存在形式。结果 :用免疫组织化学法检测到 12例肝癌组织中有 3例为双阳性 ,用抗HSP70mAb免疫共沉淀的样品 ,可检测到 p5 3蛋白。用抗p5 3mAb免疫共沉淀的样品也可检测到HSP70蛋白。结论 :人肝癌中p5 3与HSP70以复合物的形式而存在 ,此可为肝癌的发病机制及免疫治疗的研究提供新的思路     

Message in a bottle: role of the 70-kDa heat shock protein family in anti-tumor immunity

Extracellular heat shock protein 70 (HSP70) is a potent agent for tumor immunotherapy, which can break tolerance to tumor-associated antigens and cause specific tumor cell killing by cytotoxic CD8+ T cells. The pro-immune effects of extracellular HSP70 are, to some extent, extensions of its molecular properties as an intracellular stress protein. The HSP70 are characterized by massive inducibility after stress, preventing cell death by inhibiting aggregation of cell proteins and directly antagonizing multiple cell death pathways. HSP70 family members possess a domain in the C terminus that chaperones unfolded proteins and peptides, and a N-terminal ATPase domain that controls the opening and closing of the peptide binding domain. These properties not only enable intracellular HSP70 to inhibit tumor apoptosis, but also promote formation of stable complexes with cytoplasmic tumor antigens that can then escape intact from dying cells to interact with antigen-processing cells (APC) and stimulate anti-tumor immunity. HSP70 may be released from tumors undergoing therapy at high local extracellular concentrations, and send a danger signal to the host leading to APC activation. Extracellular HSP70 bind to high-affinity receptors on APC, leading to activation of maturation and re-presentation of the peptide antigen cargo of HSP70 by the APC. The ability of HSP70-peptide complexes (HSP70-PC) to break tolerance and cause tumor regression employs these dual properties as signaling ligand and antigen transporter. HSP70-PC thus coordinately activate innate immune responses and deliver antigens for re-presentation by MHC class I and II molecules on the APC cell surface, leading to specific anti-tumor immunity.     

热休克蛋白70融合肿瘤抗原基因的DNA疫苗研究进展

在抗肿瘤免疫治疗中,已经发展了多种形式的热休克蛋白70(HSP70)相关治疗性疫苗,这些疫苗主要包括蛋白疫苗、肽疫苗、细胞疫苗及DNA疫苗等,其中HSP70融合DNA疫苗凭借其自身的优势引起人们的关注。它不但可诱导高效、持久的抗肿瘤免疫应答,而且可以维持免疫记忆。本文就HSP70在肿瘤治疗性DNA疫苗中的作用和应用作一综述。     

A 77-kilodalton protein of Cryptococcus neoformans, a member of the heat shock protein 70 family, is a major antigen detected in the sera of mice with pulmonary cryptococcosis.

Heat shock proteins (HSPs) from several pathogenic microbes have been shown to be target molecules of humoral responses as well as cellular immune responses. However, little is known about target molecules in pulmonary cryptococcosis. Western blotting analysis revealed that experimentally induced pulmonary cryptococcosis in (BALB/c x DBA/2)F1 mice was associated with the appearance of serum antibodies to a 77-kDa protein derived from Cryptococcus neoformans as well as to 18-, 22-, 25-, 36-, and 94-kDa proteins. Since the 77-kDa band also reacted with rabbit polyclonal antibodies against 70-kDa HSP (HSP70) family members, the protein was predicted to be a member of the HSP70 family. We also purified HSP70 directly from a C. neoformans cell extract by Mono Q fast protein liquid chromatography and ATP-agarose affinity column chromatography and showed that it was positive in immunoblot analysis using either serum from C. neoformans-infected mice or rabbit anti-HSP70 antibodies. N-terminal amino acid sequencing of this purified protein confirmed that the 77-kDa protein was a member of the HSP70 protein family. A 66-kDa protein, which coincidentally purified with the HSP70 protein and was identified as a member of the HSP60 family by N-terminal amino acid sequencing, was not reactive with sera from C. neoformans-infected mice. Thus, a protein associated with the HSP70 family and derived from C. neoformans was a major target molecule of the humoral response in murine pulmonary cryptococcosis.     

Cancer genesis: stem tumour cells as an MHC-null/HSP70 - very high 'primordial self' escaping both MHC-restricted and MHC-non- restricted immunesurveillance

I have previously assumed that in tumours there are stem cells, that owing to the morphophysiologic properties shared with embryonal cells I have defined as 'para-embryonal' cells (PECs). Owing to a blocking mutation, PECs might be able to express only the genic program upstream from the block, but not that downstream. As a consequence, PECs might lack in genic differentiated products, such as MHC molecules, and might be very rich in primitive genic products, such as HSP70 molecules. Like embryonal cells, PECs might carry out induction on adjoining hyperplastic cells, thus transforming them, only phenotypically, into 'differentiated para-embryonal cells' (DPECs), endowed with both MHC and HSP70 molecules. In such a way, nuclei of MHC-non-expressing/HSP70-high expressing stem tumour cells might be surrounded by layers of MHC-expressing/HSP70-expressing non-stem tumour cells. Such a structural tumour organization, actually found by C. Cordon Cardo et al. with regard to the MHC molecule expression, might be responsible for interference phenomena versus the MHC-non-restricted immune cells, such as macrophages and NK cells. So, these cells, the only potentially able to recognize and eliminate MHC-non-expressing stem tumour cells (PECs), might spare them, thus rendering cancer a biological process without any natural immunological solution. Now, I would like to theorically demonstrate that cancer might be a process without immunological solution from the very beginning: the first stem tumour cell might be tolerated as a sort of 'primordial self' because of its MHC-null/HSP70-very high phenotype, recognizable by neither the MHC-restricted nor the MHC-non-restricted immunesurveillance systems of the host. Possible biological roles of HSP70 molecules might account for the immunesurveillance escape of stem tumour cells. Existence of these cells appears to be confirmed by the recent experiments of immunotherapy with autologous tumour-specific HSPs carried out by P. K. Srivastava; moreover, their 'self' nature appears to be confirmed by the most recent experiments of compatible bone marrow allograft carried out by A. M. Carella. On this ground, the main steps for a resolutive antitumour immunotherapy are proposed.     

Response to hepatocarcinoma Hca-F of mice immunized with heat shock protein 70 from elemene combo tumor cell vaccine

To analyze immune response to murine hepatocarcinoma Hca-F of mice immunized with heat shock protein 70 （HSP70） derived from elemene combo tumor cell vaccine （EC-TCV） of Hca-F, HSP70 was isolated from EC-TCV by ADP affinity chromatography. Mice were immunized with HSP70 intraperitoneally three times and spleen calls were sampled. For cells, their proliferation and cytotoxicity against Hca-F were measured with MTT assay and their phenotypes were analyzed with flow cytometry. Spleen cells of immunized mice with HSP70 exhibited more potent cytotoxicity against Hca-F and proliferation than that of normal control mice, but less potent than that of mice immunized with EC-TCV. Among three groups, the percent of T6 T lymphocytes in the mice immunized with HSP70 （35.5%） was the highest compared with 6.25% in normal mice, and 28.4% in the mice immunized with EC-TCV. Immunization of HSP70 derived from EC-TCV could elicit potent immune response to Hca-F. HSP70 is one of elements inducing anti-tumor immune responses against Hca-F.     

HSP60, HSP70 and HSP90 from Trichinella spiralis as targets of humoral immune response in rats

This study identifies three heat shock proteins (HSPs) using purified preparations from Trichinella spiralis larvae. The proteins: HSP60, HSP70 and HSP90 were found to be targets of the humoral immune response in rats. Three approaches were adopted to obtain T. spiralis HSP-enriched material and/or to purify HSPs to homogeneity. The former product was prepared using affinity chromatography on gelatin sepharose and elution with ATP. Pure 90 kDa-protein was isolated from parasite extract by sequential DEAE (A50) column chromatograpy and preparative electrophoresis. Immunoblot analysis using monoclonal antibodies to HSP60, HSP70 and HSP90 detected the HSP60 and HSP70 in the affinity-purified product and HSP90 in the product obtained by sequential anionic chromatography and preparative electrophoresis. Finally, the reactivy of preimmune, T. spiralis immune and irrelevant immune rat sera on immunoblots were also examined. Only sera taken from infected rats at time-points after day 7 following the first infection exhibited activity against 60, 70 and 90 kDa proteins on blots. The fact that the serum antibodies were anti-HSP was established by immunoadsorption of HSPs to microtiter plates coated with anti-HSP60, anti-HSP70, or anti-HSP90 and using rat sera, positive on blots, to also give positive scores by continued enzyme-linked immunosorbent assay.