核黄素光化学法灭活Sindbis病毒的研究

目的探究核黄素光化学反应对Sindbis病毒的灭活效果。方法将10 mmol/L的核黄素0.050 mL加入到4.95 mL的Sindbis病毒(XJ-160株)悬液中,经440 nm波段的可见光(40 J/cm2)双侧照射后,接种至幼仓鼠肾细胞(BHK-21)中培养,观察细胞病变情况(CPE),检测病毒滴度,用PCR检测病毒核酸的变化,并在透射电镜下观察病毒形态。结果经终浓度为100μmol/L核黄素结合440 nm可见光作用,可将滴度为6.5 log TCID50Sindbis病毒灭活至≤0.5 logTCID50;PCR法未能扩增出病毒核酸片段;透射电镜显示病毒颗粒塌陷。结论核黄素光化学法能有效灭活Sindbis病毒,其主要作用靶点为核酸。     

利用动物模型评价核黄素光化学法灭活红细胞病毒的效果

本研究旨在评估核黄素光化学法(核黄素终浓度为150μmol/L,光照时间为20分钟,光照强度为40 000Lux)灭活红细胞中病毒的有效性。将HCMV作为指示病毒加入红细胞中。30只BALA/c小鼠设为实验组(n=10)、病毒对照组(n=10)、可见光对照组(n=5)和红细胞对照组(n=5);实验组小鼠注射经核黄素光化学法灭活处理的红细胞;病毒对照组小鼠注射未经灭活处理的红细胞;可见光对照组小鼠注射经可见光照射的红细胞;红细胞对照组小鼠注射正常红细胞。取各组小鼠进行体外病毒分离,PCR检测HCMV UL83基因,间接免疫荧光鉴定PP65抗原。结果表明:病毒对照组和可见光对照组病毒分离、PCR及间接免疫荧光检测均为阳性,而实验组和红细胞对照组所有结果均为阴性。结论:核黄素光化学法灭活红细胞病毒是有效的。     

核黄素光化学方法抑制淋巴细胞增殖及细胞因子分泌活性的实验研究

目的研究核黄素光化学方法对淋巴细胞的增殖活性及细胞因子分泌活性的抑制程度,观察该方法对淋巴细胞增殖周期的影响。方法实验分为实验组:随机选择捐献的全血将从中收集的淋巴细胞悬浮于核黄素终浓度为50μmol/L的1640缓冲溶液中,注入PVC透光转移袋内(440nm处透光率为80%),用400—500nm的可见光照射,照射量为8.8J/ml;对照组:为相同来源、悬浮于不含核黄素的1640缓冲溶液中、未接受光照处理的淋巴细胞。以植物血凝素同步刺激2个组的淋巴细胞,用MTS非放射性细胞增殖试验检测淋巴细胞的增殖活性,计算核黄素光化学处理对淋巴细胞的增殖抑制率;用酶标方法检测细胞因子;用流式细胞术观察经植物血凝素(PHA)刺激后的淋巴细胞周期变化(RPT-lymphocytes)。结果与对照组比较,实验组经过核黄素光化学法处理后的淋巴细胞(RPT-lymphocytes)对PHA刺激的增殖抑制率为(99.21±1.06)%,淋巴细胞IL-1β、-2、-6、-8及TNF-α、IFN-γ细胞因子的分泌量比对照组细胞分别降低了(95.09±2.60)%、(98.20±1.64)%、(98.77±0.97)%、(92.30±11.04)%及(98.82±1.42)%、100%;实验组S期细胞占0.73%,对照组S期细胞则占32.5%。结论可见光激发的核黄素光化学方法可有效抑制淋巴细胞的增殖活性和细胞因子分泌活性,阻止淋巴细胞进入细胞增殖周期,提示该方法可能是预防TA-GVHD的一种有效并且可行的方法。     

核黄素光化学法灭活单采血小板中巨细胞病毒的效果评价

目的探讨核黄素光化学法灭活单采血小板中巨细胞病毒的效果及血小板一部分生化和生理学指标的改变。方法将14ml浓度为500μmol／L核黄素加到125ml的单采血小板悬液中,再将一定滴度约6log TCID50／ml的人类巨细胞病毒标准株（HCMV AD169）分别注入上述混悬液中,经265～370nm广谱紫外光（6．2J／m1）照射8～10min后,分别加至人胚肺成纤维细胞（HELF）单层中培养,与对照细胞比较观察细胞病变情况（CPE）,测定其滴度,并观察血小板一部分体外参数的变化情况。结果经浓度50μmol／L的核黄素结合强度为6．2J／ml紫外光照射8～10min,可将滴度为6logTCID50／ml模型病毒的组织培养半数感染量（TCID50）下降至FDA规定的灭活效果标准的下限值（〈0．43logTCID50／ml）。结论核黄素结合紫外光照射可以有效灭活人类巨细胞病毒,而单采血小板的各项生化、生理学指标和阴性对照比较差异均无统计学意义。     

不同浓度叠氮溴化丙啶和照射协同灭活大肠杆菌8099 DNA效果评价

目的评价不同浓度叠氮溴化丙啶(PMA)在不同照射时间对大肠杆菌DNA灭活的效果,确定在实际使用中的最佳浓度和照射时间。方法选择相当于107cfu大肠杆菌(8099)的DNA作为灭活对象,加入终浓度为50μmol/ml、100μmol/ml、150μmol/ml和200μmol/ml的PMA,使用波长为460 nm的LED灯,分别照射5 min、10 min和15 min,然后进行Real Time PCR扩增。结果当PMA的浓度达到150μmol/ml,照射5 min时,大肠杆菌Real Time PCR扩增结果为阴性。结论在实际使用中,要完全灭活107cfu的大肠杆菌(8099)的DNA,需要≥150μmol/ml的PMA,照射时间≥5 min较合适。     

噻唑橙光化学法对红细胞中病毒及细菌灭活作用的研究

目的探讨噻唑橙(TO)光化学法对红细胞中病毒及细菌灭活效果。方法以伪狂犬病毒(PRV)和辛德毕斯(Sindbis)病毒为指示病毒,分别加入红细胞比积为20%的红细胞悬液中,TO孵育1 h后做476 nm光照射处理;研究病毒灭活动力学特征;做TO浓度(C)、光照强度(I)、光照时间(T)三因素三水平正交试验确定最优灭活条件;最优条件下检测裸照、密闭血袋中、密闭血袋充氧后的病毒灭活效果;最优条件下检测血液污染常见细菌小肠结肠炎耶尔森氏菌、金黄色葡萄球菌及表皮葡萄球菌灭活效果。结果病毒灭活动力学研究显示:在60μmol/LTO、1.06E-01 w·m-2·nm-1光强条件下,照射5 min可将大部分PRV和Sindbis病毒灭活,20 min灭活作用达峰值,分别为5.88和5.12 LogTCID50;正交试验显示:裸照时PRV及Sindbis病毒灭活最优条件均为I=1.33E-01 w·m-2·nm-1,T=20 min,C=80μmol/L;在此条件下,可灭活红细胞中PRV≥6.13LogTCID50(裸照,n=4)、(4.75±0.62)LogTCID50(密闭血袋,n=4)、(6.06±0.16)LogTCID50(密闭血袋充氧,n=4);可灭活红细胞中Sindbis病毒(5.41±0.12)LogTCID50(裸照,n=4)、(3.72±0.77)LogTCID50(密闭血袋,n=4)、(5.76±0.25)LogTCID50(密闭血袋充氧,n=4)。在此条件下细菌灭活效果:小肠结肠炎耶尔森氏菌(5.91±0.13)Log10、金黄色葡萄球菌6.8Log10、表皮葡萄球菌6.0 Log10。结论 TO光化学法可有效灭活红细胞中病毒,有氧情况下(裸照或充氧)病毒灭活效果好于密闭无氧。TO光化学法可有效灭活红细胞中细菌。     

血卟啉衍生物的抗菌活性研究

目的研究不同浓度血卟啉衍生物(HpD)对大肠杆菌和金黄色葡萄球菌的光动力杀伤作用。方法配制不同浓度HpD,分别加入大肠杆菌和金黄色葡萄球菌悬液中,分别用400nm波长光照强度20J/cm2激光照射后,将菌液涂布于LB固体培养基上,进行菌落计数。结果 HpD光动力作用对大肠杆菌和金黄色葡萄球菌的抑制效果与HpD浓度呈现剂量依赖关系,当光照强度为20J/cm2时,40μg/mlHpD浓度作用下,大肠杆菌的灭活率为81.4%,金黄色葡萄球菌的灭活率为93.69%。结论在400nm波长激光照射下,HpD对大肠杆菌和金黄色葡萄球菌均有抑制作用,且对金黄色葡萄球菌的抑制效果均优于比大肠杆菌。     

运用FQ-PCR技术监测亚甲蓝光化学法对血浆丙型肝炎病毒的灭活效果

目的通过荧光定量PCR(FQ-PCR)技术对血浆病毒灭活前后丙型肝炎病毒RNA (HCV-RNA)浓度的测定,判断亚甲蓝(MB)光化学法灭活血浆丙型肝炎病毒的效果,为临床判别病毒灭活效果提供直接和客观依据.方法4份经证实为HCV-RNA阳性的血浆分别加入MB,终浓度为1μmol/L,经可见光(大于30000Lux)照射不同时间(0、5、10、15和30min)后,分别用FQ-PCR测定这些血浆的HCV-RNA浓度,并与未用MB处理的血浆作比较,判断随照射时间不同,HCV被灭活的效果.结果4份HCV-RNA阳性病人的血浆未经处理时其病毒核酸浓度分别为8.0×105拷贝/ml、1.7×106拷贝/ml、1.6×106拷贝/ml和3.1×105拷贝/ml,加入MB未经照射时HCV-RNA浓度即明显下降,可见光照射5min后HCV-RNA浓度分别下降为未经任何处理时浓度的18.75%、23.36%、4.66%和1.27%,随照射时间延长,病毒核酸浓度逐渐下降,照射30min后,HCV-RNA浓度均下降为零.结论用MB加光照30min处理,可达到将血浆丙肝病毒灭活的效果;FQ-PCR可定量检测MB光化学法灭活丙肝病毒的过程.     

核黄素光化学法对血浆中革兰阳性和阴性指示菌的减除作用和对血小板P选择蛋白表达的影响

本研究验证核黄素（维生素B2）光化学减除血浆中指示菌的实际效果和对机采浓缩血小板的活化影响。以大肠杆菌为革兰阴性和以金黄色葡萄球菌为革兰阳性指示病原体，观察核黄素结合紫外线照射的协同效应和灭菌效果；采用流式细胞术评估灭活处理后的机采浓缩血小板的活化状态。结果表明：50μmoL／L核黄素结合6．2J／ml剂量紫外线辐照，T／E比值分别为1．44和1．58，降低大肠杆菌和金色葡萄球菌3．87和3．82Log数。血小板经病原体灭活处理后于22℃保存0天和5天时CD62p的表达率分别为4．92％和36．18％，与对照3．94％和32．03％相比表达率略有增加（P〈0．05）。结论：核黄素与紫外线照射协同效应可以减除大肠杆菌和金黄葡萄球菌指示菌，对血小板无明显影响，液体血小板部分活化状态表现为贮存损伤，此损伤程度在可接受的范围之内。     

核黄素光化学法杀灭血浆中细菌的实验研究

目的研究核黄素光化学法对血浆中细菌的杀灭效果。方法采用模拟试验方法,对核黄素与紫外线协同杀灭血浆中细菌的效果进行观察。结果血浆中加入300μmol/L核黄素单独作用,几乎无杀菌作用。单独紫外线照射对血浆中细菌杀灭所需时间较长。在血浆中加入300μmol/L核黄素经辐射强度1 920μW/cm2的紫外光照射10 min,对血浆中大肠埃希菌和金黄色葡萄球菌杀灭率均达100%。结论核黄素与紫外线协同作用对血浆中大肠埃希菌和金黄色葡萄球菌具有较好杀灭效果。     

核黄素光化学作用对淋巴细胞分泌细胞因子的影响研究

目的探讨用核黄素光化学法预防输血相关移植物抗宿主病(TA-GVHD)的可行性。方法随机从的全血中收集淋巴细胞,将淋巴细胞悬浮于核黄素终浓度为50μmol/L的1640培养液中,注入440 nm处透光率为80%的PVC透光塑料袋内。用400—500 nm的可见光照射,照射量为8.8 J/ml。对照组为相同来源、悬浮于不含核黄素的1640培养液中,未接受光照处理的淋巴细胞。以植物血凝素(PHA)、CD3单抗、CD28单抗等同步刺激实验组和对照组淋巴细胞,用酶标方法检测细胞因子分泌量。结果经过核黄素光化学法处理后的淋巴细胞,接受PHA刺激后,实验组淋巴细胞IL-1β、IL-2、IL-4、IL-6、IL-8、IL-10、TNF-α和INF-γ的分泌量比对照组细胞分别降低了(99.47±0.80)%、(99.81±0.46)%、(84.26±24.20)%、(99.86±1.23)%、(92.30±11.04)%、(89.50±15.66)%、(99.98±0.06)%和100%;接受CD3单抗刺激后,实验组淋巴细胞IL-1β、IL-6、IL-8、IL-10、TNF-α和INF-γ的分泌量比对照组细胞分别降低了(99.32±1.30)%、(99.37±1.16)%、(93.31±7.86)%、(93.04±14.16)%、(84.70±10.22)%和100%。接受CD3单抗和CD28单抗联合刺激后,实验组淋巴细胞IL-1β、IL-6、IL-8、TNF-α和INF-γ的分泌量比对照组细胞分别降低了(96.88±7.25)%、(98.55±3.61)%、(94.35±4.93)%、(88.69±10.42)%和100%。结论核黄素光化学法处理可使淋巴细胞失去生成GVHD相关细胞因子的能力,初步证明核黄素光化学法能在细胞水平有效阻断淋巴细胞通过接受抗原诱导表达细胞因子参与TA-GVHD病理改变的通路。     

Photochemical inactivation of viruses and bacteria in platelet concentrates by use of a novel psoralen and long-wavelength ultraviolet light

BACKGROUND: A photochemical treatment process has been developed for the inactivation of viruses and bacteria in platelet concentrates. This process is based on the photochemical reaction of a novel psoralen, S- 59, with nucleic acids upon illumination with long-wavelength ultraviolet light (UVA, 320–400 nm). STUDY DESIGN AND METHODS: High levels of pathogens were added to single-donor platelet concentrates containing 3 to 5 × 10(11) platelets in 300 mL of 35-percent autologous plasma and 65-percent platelet additive solution. After treatment with S-59 (150 microM) and UVA (0-3 J/cm2), the infectivity of each pathogen was measured with established biologic assays. In vitro platelet function after photochemical treatment was evaluated during 7 days of storage by using a panel of 14 assays. The in vivo recovery and life span of photochemically treated platelets were evaluated after 24 hours of storage in a primate transfusion model. RESULTS: The following levels of pathogen inactivation were achieved:>10(6.7) plaque-forming units (PFU) per mL of cell-free human immunodeficiency virus (HIV),>10(6.6) PFU per mL of cell-associated HIV,>10(6.8) infectious dose (ID50) per mL of duck hepatitis B virus (a model for hepatitis B virus),>10(6.5) PFU per mL of bovine viral diarrhea virus (a model for hepatitis C virus),>10(6.6) colony-forming units of Staphylococcus epidermidis, and>10(5.6) colony-forming units of Klebsiella pneumoniae. Expression of integrated HIV was inhibited by 0.1 microM S- 59 and 1 J per cm2 of UVA. In vitro and in vivo platelet function were adequately maintained after antiviral and antibacterial treatment. CONCLUSION: Photochemical treatment of platelet concentrates offers the potential for reducing transfusion-related viral and bacterial diseases.     

Protein quality in Mirasol pathogen reduction technology–treated,apheresis‐derived fresh‐frozen plasma

BACKGROUND: The Mirasol pathogen reduction technology (PRT) system for plasma is based on a riboflavin (vitamin B₂) and ultraviolet (UV) light treatment process resulting in pathogen inactivation due to irreversible photo‐oxidative damage of nucleic acids. The purpose of this study was to evaluate the in vitro protein quality of apheresis‐derived plasma treated with riboflavin and UV light in comparison with untreated fresh‐frozen plasma (FFP). STUDY DESIGN AND METHODS: Twenty apheresis plasma samples (270 ± 10 mL) were combined with 35 ± 5 mL of riboflavin solution (500 µM), yielding a mean 60 µM final riboflavin concentration, and then exposed to UV light (6.24 J/mL). Riboflavin and UV light–treated plasma was then flash frozen, within 8 hours of collection, generating treated FFP. Treated FFP was thawed and analyzed using standard coagulation assays, and the percent retention of protein activity was reported, relative to untreated, paired controls. RESULTS: Plasma proteins demonstrated different sensitivities to riboflavin and UV treatment. The amount of total protein remained unchanged. After treatment, fibrinogen (antigen) showed 99% retention; Factor (F)XII, FXIII, ADAMTS‐13, and von Willebrand factor (ristocetin cofactor) 96% to 100%. Fibrinogen retained 77% activity, FII 80%, FVIIIc 75%, and FV 73% after treatment. Antithrombin, protein S, plasminogen, and α₂‐antiplasmin retained between 91 and 100% activity. CONCLUSION: The results from this study demonstrate that coagulant and anticoagulant proteins in riboflavin and UV light–treated (PRT) apheresis plasma are well preserved.     

核黄素联合紫外线A照射对血浆中伪狂犬病毒的灭活效果

目的考察核黄素联合紫外线A对血浆中伪狂犬病毒的灭活效果。方法以伪狂犬病毒为模拟病毒,以Vero细胞为培养细胞,用病毒感染细胞,制备病毒增殖液;分别采用5、10、15及20J/cm2联合核黄素处理血浆,观察处理前后血浆病毒灭活的效果,筛选灭活病毒合适的紫外线剂量;将含有伪狂犬病毒血浆的样本分为实验组:采用上述筛选的紫外线强度照射联合核黄素处理;对照组1:单独采用紫外线A照射;对照组2:单独采用核黄素处理;阴性对照组:未采用紫外线照射和核黄素处理;分别在实验前后采用96孔细胞病变法,对照细胞病变效应,根据Reed-Muench公式计算病毒滴度。结果采用不同强度的紫外线联合核黄素处理血浆,紫外线强度为15及20J/cm2的灭活血浆病毒的效果明显,处理后病毒滴度分别下降4.55和4.39logs;实验组和对照组1病毒滴度分别下降4.55和4.28logs,有统计学意义(P<0.05);对照组2病毒滴度降低1.93logs,没有病毒灭活效果。结论核黄素联合紫外线可灭活血浆中伪狂犬病毒,单独紫外线照射也具有灭活血浆伪狂犬病毒的效果;而仅单独采用核黄素处理而未联合紫外线照射,对血浆中伪狂犬病毒灭活效果不明显。     

Photochemical treatment of platelet concentrates with amotosalen and long-wavelength ultraviolet light inactivates a broad spectrum of pathogenic bacteria

BACKGROUND: Bacterial contamination of platelet (PLT) concentrates can result in transfusion-transmitted sepsis. A photochemical treatment (PCT) process with amotosalen HCl and long-wavelength ultraviolet light (UVA), which cross-links nucleic acids, was developed to inactivate bacteria and other pathogens in PLT concentrates. STUDY DESIGN AND METHODS: High titers of pathogenic aerobic and anaerobic Gram-positive bacteria (10 species), aerobic Gram-negative bacteria (7 species), and spirochetes (2 species) were added to single-donor PLT concentrates containing 3.0 x 10(11) to 6.0 x 10(11) PLTs in approximately 300 mL of 35 percent plasma and 65 percent PLT additive solution (InterSol, Baxter Healthcare) or saline. After PCT with 150 micro mol per L amotosalen and 3 J per cm(2) UVA, residual bacterial levels were detected by sensitive microbiologic methods. RESULTS: The level of inactivation of viable bacteria was expressed as log reduction. Log reduction of Gram-positive bacteria for Staphylococcus epidermidis was > 6.6; for Staphylococcus aureus, 6.6; for Streptococcus pyogenes, > 6.8; for Listeria monocytogenes, > 6.3; for Corynebacterium minutissimum, > 6.3; for Bacillus cereus (vegetative), > 5.5; for Lactobacillus sp., > 6.4; for Bifidobacterium adolescentis, > 6.0; for Propionibacterium acnes, > 6.2; and for Clostridium perfringens, > 6.5. Log reduction of Gram-negative bacteria for Escherichia coli was > 6.4; for Serratia marcescens, > 6.7; for Klebsiella pneumoniae, > 5.6; for Pseudomonas aeruginosa, 4.5; for Salmonella choleraesuis, > 6.2; for Yersinia enterocolitica, > 5.9; and for Enterobacter cloacae, 5.9. Log reduction of spirochetes for Treponema pallidum was 6.8 to 7.0, and for Borrelia burgdorferi, > 6.9. CONCLUSION: PCT inactivates high levels of a broad spectrum of pathogenic bacteria. The inactivation of bacteria in PLT concentrates offers the potential to prospectively prevent PLT-transfusion-associated bacteremia.     

Leishmania inactivation in human pheresis platelets by a psoralen (amotosalen HCl) and long-wavelength ultraviolet irradiation

BACKGROUND: Leishmania spp. are protozoans that cause skin and visceral diseases. Leishmania are obligate intracellular parasites of mononuclear phagocytes and have been documented to be transmitted by blood transfusion. STUDY DESIGN AND METHODS: This study examines whether Leishmania can be inactivated in human platelet (PLT) concentrates by a photochemical treatment process that is applicable to blood bank use. Human PLT concentrates were contaminated with Leishmania mexicana metacyclic promastigotes or mouse-derived Leishmania major amastigotes and were exposed to long-wavelength ultraviolet (UV) A light (320-400 nm) plus the psoralen amotosalen HCl. RESULTS: Neither treatment with amotosalen nor UVA alone had an effect on Leishmania viability; however, treatment with 150 micromol per L amotosalen plus 3 J per cm(2) UVA inactivated both metacyclic promastigotes and amastigotes to undetectable levels, more than a 10,000-fold reduction in viability. CONCLUSIONS: This study demonstrates the effectiveness of photochemical treatment to inactivate Leishmania in PLT concentrates intended for transfusion. Both metacylic promastigotes, which represent the infectious form from the sand fly vector, and amastigotes, which represent the form that grows in mononuclear phagocytes, were extremely susceptible to photochemical inactivation by this process. Thus, the photochemical treatment of PLT concentrates inactivates both forms of Leishmania that would be expected to circulate in blood products collected from infected donors.