FDA/PI flow cytometry assay of complement-mediated cytotoxicity of antibodies generated during xenotransplantation.

BACKGROUND: A flow cytometry complement-mediated cytotoxicity assay (FCCA) using fluorescein diacetate (FDA) and propidium iodide (PI) to measure antibody-dependent toxicity is useful to determine the success of xenotransplant organs. We evaluated the validity of different mathematical models as a measure of cytotoxicity in FCCA. METHODS: Sera from untreated baboons (n = 7) and from immunosuppressed animals (n = 5) undergoing different xenotransplantation protocols with pig organs were tested by endogenous FCCA and a similar assay also using exogenous complement, and the results were compared with those of a complement-dependent hemolytic assay to detect anti-pig antibodies (APHA). The influence of PI/FDA staining and the use of several mathematical models were analyzed. RESULTS: For both groups of animals, we observed high correlations between the endogenous and exogenous FCCA pathways and between calculations based on PI and FDA staining. Of the four mathematical models tested--the Von Krogh equation, two exponential models, and area under the curve--the Von Krogh equation was the most appropriate in terms of goodness of fit and concordance with APHA. CONCLUSIONS: FDA/PI FCCA is useful to measure endogenous and exogenous complement-mediated cytotoxicities, and it has advantages related to identification of potential new xenoantibodies. Although all four mathematical models produced acceptable solutions, the Von Krogh equation was the best option.     

XENOTRANSPLANTATION, CONSENT AND INTERNATIONAL JUSTICE

The risk posed to the community by possible xenozoonosis after xenotransplantation suggests that some form of 'community consent' is required before whole organ animal-to-human xenotransplantation should take place. I argue that this requirement places greater obstacles in the path of ethical xenotransplantation than has previously been recognised. The relevant community is global and there are no existing institutions with democratic credentials sufficient to establish this consent. The distribution of the risks and benefits from xenotransplantation also means that consent is unlikely to be forthcoming. Proceeding on the basis of hypothetical consent to a package of global health measures that includes xenotransplantation, as Rothblatt has recently advocated, is more problematic than she acknowledges. Given that it may place the lives of citizens of poor nations at risk to benefit the citizens of wealthy nations, xenotransplantation raises significant questions of international justice.     

In pursuit of xenoreactive antibodies: where has it gotten us?

Numerous studies have aimed to overcome the barrier to xenotransplantation posed by xenoreactive antibodies and the antigens they recognize. Whether this work will eventually lead to the widespread clinical application of xenotransplantation remains unknown. However, the benefits of this research are already substantial, with research leading to dramatic new developments in fields other than xenotransplantation. Our understanding of natural immunity, particularly the nature and function of natural antibodies, has taken quantum leaps forward, with far-reaching implications. Our improved understanding of the immune response to xenografts has proven invaluable in the characterization of the human immune reaction to commonly used biological therapeutics of xenogeneic origin. Our understanding of cell surface carbohydrates and our ability to modify these carbohydrates in living animals has advanced substantially, with implications for diseases such as cancer and autoimmunity. With this in mind, it is argued that continued work in xenotransplantation is of great value, not only because of the great potential benefits of xenotransplantation, but also because of the more certain benefits that arise from setting our sights on a difficult challenge.     

Staining with Fluorescein Diacetate Correlates with Hepatocyte Function

To establish the importance of fluorescein diacetate (FDA) as a viability stain for cultured hepatocytes. we hypothesized that FDA staining would correlate positively with hepatocyte viability and function. Mixtures of live and dead cells were stained with FDA and scanned by flow cytometry. A close correlation was observed between the live cell fraction and percent viability as determined by FDA staining (R² = 0.962). Hepatocytes were also sorted into low fluorescence and high fluorescence groups. Both albumin production and lidocaine metabolism (P-450 activity) were significantly increased in the high fluorescence group compared to the low fluorescence group. An automated, fluorescence-activated assay was useful for rapid assessment of hepatocyte viability. In addition. the intensity of green fluorescence following staining with FDA correlated well with two specific measures of hepatocyte function.     

A Method for Producing Dust-, Streak- and Hole-Free Formvar Films in Laboratories Having High Atmospheric Humidity

To establish the importance of fluorescein diacetate (FDA) as a viability stain for cultured hepatocytes. we hypothesized that FDA staining would correlate positively with hepatocyte viability and function. Mixtures of live and dead cells were stained with FDA and scanned by flow cytometry. A close correlation was observed between the live cell fraction and percent viability as determined by FDA staining (R² = 0.962). Hepatocytes were also sorted into low fluorescence and high fluorescence groups. Both albumin production and lidocaine metabolism (P-450 activity) were significantly increased in the high fluorescence group compared to the low fluorescence group. An automated, fluorescence-activated assay was useful for rapid assessment of hepatocyte viability. In addition. the intensity of green fluorescence following staining with FDA correlated well with two specific measures of hepatocyte function.     

Analysis of natural recombination in porcine endogenous retrovirus envelope genes

Human tropic Porcine Endogenous Retroviruses (PERVs) are the major concern in zoonosis for xenotransplantation because PERVs cannot be eliminated by specific pathogen-free breeding. Recently, a PERV A/C recombinant with PERV-C bearing PERV-A gp70 showed a higher infectivity (approximately 500-fold) to human cells than PERV-A. Additionally, the chance of recombination between PERVs and HERVs is frequently stated as another risk of xenografting. Overcoming zoonotic barriers in xenotransplantation is more complicated by recombination. To achieve successful xenotransplantation, studies on the recombination in PERVs are important. Here, we cloned and sequenced proviral PERV env sequences from pig gDNAs to analyze natural recombination. The envelope is the most important element in retroviruses as a pivotal determinant of host tropisms. As a result, a total of 164 PERV envelope genes were cloned from pigs (four conventional pigs and two miniature pigs). Distribution analysis and recombination analysis of PERVs were performed. Among them, five A/B recombinant clones were identified. Based on our analysis, we determined the minimum natural recombination frequency among PERVs to be 3%. Although a functional recombinant envelope clone was not found, our data evidently show that the recombination event among PERVs may occur naturally in pigs with a rather high possibility.     

在美国进行植物药开发的路径、要求及策略探讨

近年来,越来越多药企开始在美国进行植物药的临床试验申请和新药申报,美国 FDA 日益重视植物药的开发,于 2016 年 12 月 发布并实施了修订版的《行业指南:植物药》,为植物药后期开发提供更多专业建议。结合 FDA 已经批准的 2 个植物药案例,详细介绍 植物药 CMC(化学、生产、控制)、临床前研究和过往人用历史方面的基本要求,浅谈 FDA 的申报要求、审评思想和申报策略,以期 为中国中药企业的国际化开发和注册提供参考。     

Potential problems with fluorescein diacetate assays of cell viability when testing natural products for antimicrobial activity.

There are two potential problems in the use of fluorescein diacetate (FDA) as a measure of cell viability. The first is the hydrolysis of FDA to fluorescein in the absence of live cells and the second is the quenching of fluorescence by assay solutions. We show that common media components such as tryptone, peptone and yeast extract all promote hydrolysis of FDA in the absence of live cells, as do Tris-HCl and sodium phosphate buffers. As a consequence, various microbiological media promote hydrolysis of FDA in the absence of live cells. Different media were also shown to reduce the amount of visible fluorescence of fluorescein. Diluting the medium decreases the background hydrolysis of FDA as well as increases the amount of visible fluorescence. Both problems should be considered when using FDA as an indicator of cell viability when testing natural products for antimicrobial activity.     

Rapid Determination of Conidial Viability for Entomopathogenic Hyphomycetes Using Fluorescence Microscopy Techniques

The viability of conidia from two species of deuteromycetes fungi pathogenic to insects was determined using two fluorochrome stains, fluorescein diacetate (FDA) and propidium iodide (PI). These stains were used either alone or in combination, and results were compared with standard conidial germination tests. FDA fluoresces bright green in viable conidia and PI fluoresces red in non-viable conidia, when viewed using specific fluorescence microscopic techniques. Conidia from two isolates of Paecilomyces fumosoroseus (Wize) Brown and Smith and two isolates of Beauveria bassiana (Balsamo) Vuillemin were evaluated. Conidia were suspended in deionized water and half of each suspension was treated with microwave radiation to kill all the conidia. Conidia were tested for viability in non-microwaved suspensions in a mixture (ca. 1:1) of viable and non-viable conidial suspensions, and in the microwaved suspensions that contained all non-viable conidia. No significant differences were observed for the four isolates tested between germination tests on water and agar and viability tests conducted with FDA alone or FDA in combination with PI. One isolate of B. bassiana that had been damaged in storage was also tested. Differences were observed between the actual germination and the percentage of viability determined using FDA or FDA plus PI. Damaged conidia maintained a measure of viability and fluoresced green, but did not fully germinate.     

FDA-PI荧光双色法快速评价小鼠桑椹胚活力

胚胎移植成功与否关键取决于胚胎的存活力和发育能力,但是胚胎的存活力很难预知.本实验以小鼠桑椹胚为材料,研究荧光物质二乙酰荧光素(Fluorescein diacetate,FDA)-碘化丙啶(Propidium iodide,PI)在胚胎活力检测上的应用,同时检测两种荧光物质FDA和PI对小鼠桑椹胚的毒负作用,再通过胚胎移植来最终证明荧光标记的可靠性.实验结果表明,荧光物质FDA和PI能分别标记强活力胚胎、弱活力胚胎和死胚胎,且FDA对胚胎无毒负作用,PI对透明带完整的胚胎也无毒负作用,所以FDA-PI荧光双色法是一种简捷、安全而有效的的小鼠胚胎活力评价方法.     

国家标准测定食品细菌总数培养基的改进研究

应用我国国家标准营养琼脂（GB4789 2－94简称NA）与美国食品药品管理局（FDA）标准平析以（简称SA）两种培养基对动植物食品中细菌总数进行检测对比,结果表明FDA标准平板比GＢ营养琼脂效果较好,前比后的检出率高出２３．９％,且菌落大而明显,为此,对这两种培养基进行优化筛选试验,并优选出Ｃ８培养基,扩大试验结果表明C8培养基的检出率较GB营养琼脂及FDA标准平板分别高出35．8％和9．5％。     

Modification of the fluorescein diacetate assay for screening of antifungal agents against Candida albicans: comparison with the NCCLS methods

A modified fluorescein diacetate (FDA) assay has been compared with standard NCCLS broth macrodilution and broth microdilution methods for the detection of antifungal activity. The FDA assay was performed in a medium containing bacteriological peptone, NaCl, yeast extract and glucose (0.2%, 0.1%, 0.1% and 1% w/v, respectively) and buffered with 10 mM BES buffer. The MICs of amphotericin B, fluconazole, miconazole and flucytosine (representing three major classes of antifungal agents) obtained by the three methods were compared. The results obtained with the FDA assays correlated well with the NCCLS macrodilution method for MICs of amphotericin B, miconazole and fluconazole, but not for flucytosine. However, the MIC values of flucytosine obtained with the FDA assay were well within the quality control range for the two reference strains recommended by the NCCLS. The FDA assay described is an attractive alternative to the NCCLS methods for screening for antifungal agents, with the added advantage of objectivity of fluorescence measurement.     

Production of genetically modified pigs expressing human insulin and C-peptide as a source of islets for xenotransplantation

Transgenic Research - Islet xenotransplantation is a promising treatment for type I diabetes. Numerous studies of islet xenotransplantation have used pig-to-nonhuman primate transplantation models....     

Xenotransplantation and other means of organ replacement

Exciting new technologies, such as cellular transplantation, organogenesis and xenotransplantation, are thought to be promising approaches for the treatment of human disease. The feasibility of applying these technologies, however, might be limited by biological and immunological hurdles. Here, we consider whether, and how, xenotransplantation and various other technologies might be applied in future efforts to replace or supplement the function of human organs and tissues.     

Xenotransplantation and tolerance

The application of xenotransplantation faces daunting immunological hurdles, some of which might be overcome with the induction of tolerance. Porcine organs transplanted into primates are subject to several types of rejection responses. Hyperacute rejection mediated by naturally occurring xenoreactive antibodies and complement can be overcome without tolerance. Acute vascular rejection and cellular rejection, however, may present important opportunities for immunological tolerance, and humoral rejection might be approached by various mechanisms including (i) clonal deletion, (ii) anergy, (iii) immune deviation, (iv) induction of immunoregulatory or suppressor cells, or (v) veto cells. B-cell tolerance, useful for preventing humoral rejection, might be approached through clonal anergy. It remains to be determined, however, whether tolerance induction is required for xenotransplantation and by which means the various mechanisms of tolerance can be applied in the setting of xenotransplantation. Regardless, the study of tolerance will surely expand understanding of the physiology and pathophysiology of the immune system.     

异种移植的病毒安全性研究进展

猪-人异种移植有望解决人源器官短缺的严重问题。然而,以前病毒（provirus）形式整合入猪基因组中的猪内源性反转录病毒（porcine endogenous retrovirus,PERV）难以去除,PERV有可能通过异种移植传播给人类,甚至产生新的病毒性疾病。本文回顾了PERV与异种移植病毒安全性及我国特有小型猪中PERV的相关研究。     

应用流式细胞术检测毕赤酵母的细胞活性

选取两种细胞活性染色试剂二乙酸荧光素(fluoresceindiacetate,FDA)和碘化丙锭(propidiumiodide,PI),应用流式细胞术(flowcytometry,FCM)检测毕赤酵母细胞活性。比较FDA/PI双染色与PI单染色的FCM图谱,后者能够很好地将死活细胞区分开来并得到正确的比例。利用PI单染色检测发酵过程细胞活性的变化,甘油补料阶段几乎没有细胞死亡,进入甲醇补料阶段后,随着细胞密度的增加,细胞的活性不断降低,发酵88h时细胞活性仅为73.8%。     

The screening and identification of endogenous retrovirus free CEMPs

The endogenous retrovirus (ERV) is one kind ofretroviruses that integrated in the genome in the formof provirus and replicates with the proliferation of hostcells. The ERV may play a significant role in the evo-lution, pathology and physiology of animals[1]. Now,proviral sequences of ERV have been found in the ge-nome of many vertebrates, and the release of virionshas also been detected both in vivo and in vitro. Porcine endogenous retrovirus (PERV) embeddedin the genome of pigs belo…     

猪作为异种器官移植供体的研究进展

异种器官移植是现代和未来医学的重要研究领域之一,转基因猪有望为人类提供移植所需的器官,本对猪作为异种器官移植供体的可能性,移植引起的免疫排斥反应及病毒感染等问题进行了综述和讨论。     

The resurgent landscape of xenotransplantation of pig organs in nonhuman primates

Organ shortage is a major bottleneck in allotransplantation and causes many wait-listed patients to die or become too sick for transplantation. Genetically engineered pigs have been discussed as a potential alternative to allogeneic donor organs. Although xenotransplantation of pig-derived organs in nonhuman primates(NHPs) has shown sequential advances in recent years, there are still underlying problems that need to be completely addressed before clinical applications, including(i) acute humoral xenograft rejection;(ii) acute cellular rejection;(iii) dysregulation of coagulation and inflammation;(iv) physiological incompatibility; and(v) cross-species infection. Moreover, various genetic modifications to the pig donor need to be fully characterized, with the aim of identifying the ideal transgene combination for upcoming clinical trials. In addition, suitable pretransplant screening methods need to be confirmed for optimal donor-recipient matching, ensuring a good outcome from xenotransplantation. Herein, we summarize the understanding of organ xenotransplantation in pigs-to-NHPs and highlight the current status and recent progress in extending the survival time of pig xenografts and recipients. We also discuss practical strategies for overcoming the obstacles to xenotransplantation mentioned above to further advance transplantation of pig organs in the clinic.