网织红百分数法测定rHuEPO体内生物学活性的研究

本文通过对网织红百分数法测定rHuEPO体内生物学活性进行研究,找出网织红细胞百分数与rHuEPO浓度线性相关的范围。从而确定了用计数网织红细胞百分数的方法测定rHuEPO体内生物学活性。提出在rHuEPO体内生物学活性测定中选择合适的稀释液至关重要。对(3,3)法测定rHuEPO体内生物学活性进行了统计,批间CV、批内CV均接近10%,单次实验FL%平均数为3126%,并提出鼠间差异是造成FL%较大的主要原因;对(3,3)法与(2,2)法测定rHuEPO体内生物学活性进行比较,发现(2,2)法较(3,3)法更适合用于rHuEPO生产过程中rHuEPO体内生物学活性的测定。     

In vivo biological activities of recombinant human erythropoietin analogues produced by CHO cells, BHK cells and C127 cells.

The in vivo biological activity of four pharmaceutical preparations of recombinant human erythropoietin was compared. Two of the erythropoietins were produced by Chinese hamster ovary cells, CHO-K1, and the others were produced by mouse mammary cells, C127, and baby hamster kidney cells, BHK-21. The activities of the analogues were estimated by a simple cell counting method with conventional automated microcell counters. The amounts of these analogues gave straight logarithmic dose-response curves when plotted against the count of particles resistant to hemolysing reagent, which particles were mostly immature reticulocytes. The lines from the four analogues were parallel to each other. The relative activities of these analogues were 1.02, 1.19 and 1.21 when one of the analogues was arbitrarily used as the standard. These differences in the extent of the activity were not significant. Thus, the four recombinant human erythropoietin analogues, produced by four different mammalian cell lines, expressed the same biological potencies in vivo corresponding to their units, and the units used up to now by the manufacturers are equivalent. These results also draw the conclusion that the new simple in vivo bioassay can replace the existing accepted assay methods.     

A peculiar pattern of expression of the transferrin receptor (CD71) by reticulocytes in patients given recombinant human erythropoietin (rHuEPO): a novel marker for abuse in sport?

To overcome the limitation of the currently adopted direct method to detect recombinant Human Erythropoietin (rHuEpo) abuse in sport, indirect analysis of blood parameters are increasingly used as part of the anti-doping strategies. The aim of the present work is to identify whether immunophenotype modifications on erythroid cells may be indicative of previous rHuEPO administration. The study was conducted on dialyzed patients under treatment with rHuEPO (DPT). Dialyzed patients without rHuEPO therapy (DP) and volunteer donors (H) were used as controls. The analysis of erythroid cells immunophenotype, performed using a multiparametric flow cytometry technique, showed a peculiar pattern of CD71 expression following rHuEPO treatment. In particular CD71 showed an increased expression in mature and intermediate reticulocytes and a surprisingly decreased expression in immature reticulocytes. In conclusion, the analysis of reticulocyte maturation stages with TO/CD71 double staining may be considered as a valid alternative indirect method for the detection of rHuEPO abuse.     

Development of an in vitro Bioassay for Recombinant Human Erythropoietin (rHuEPO) Based on Proliferative Stimulation of an Erythroid Cell Line and Analysis of Sialic Acid Dependent Microheterogeneity: UT-7 Cell Bioassay

Determination of biological activity and its comparison with clinical behavior is important in the quality assessment of therapeutic glycoproteins. In vivo studies are usually employed for evaluating bioactivity of these glycomolecules. However, alternative methods are required to simplify the bioassay and avoid ethical issues associated with in vivo studies. Negatively charged sialic acid residues are known to be critical for in vivo bioactivity of rHuEPO. To address this need, we employed the human acute myeloid leukemia cell line UT-7 for the determination of proliferative stimulation induced by rHuEPO. Relative potencies of various intact and sugar-trimmed rHuEPO preparations were estimated using the International Standard for Human r-DNA derived EPO (87/684) as a reference for bioactivity. The cellular response was measured with a multi-channel photometer using a colorimetric microassay, based on the metabolism of the Resazurin sodium by cell viability. For a resourceful probing of physiological features of rHuEPO with significance, we obtained partly or completely desialylated rHuEPO digested by the neuraminidase enzyme without degradation of carbohydrates. Two-fold higher specific activity was shown by asialoerythropoietin in in vitro analysis compared with the sialoerythropoietin. Further, computational studies were also carried out to construct the 3D model of the erythropoietin (EPO) protein structure using standard comparative modeling methods. The quality of the model was validated using Procheck and protein structure analysis (ProSA) server tools. N–glycan units were constructed; moreover, EPO protein was glycosylated at potential glycosylation amino acid residue sites. The method described should be suitable for potency assessments of pharmaceutical formulations of rHuEPO (European Pharmacopeia, 2016).     

RNAi-mediated gene knockdown and in vivo diuresis assay in adult female Aedes aegypti mosquitoes

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis. The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.     

Development of an enzyme immunoassay for the detection of Clostridium novyi type B alpha toxin

Clostridium novyi type B alpha toxin was purified to homogeneity and shown to have a molecular weight of 200 kD by SDS polyacrylamide gel electrophoresis. The toxin was toxoided and used to produce a pair of non-interfering monoclonal antibodies. Their specificity was confirmed by immunoblotting and bioassay. The monoclonal antibodies were used to develop an enzyme immunoassay which was more sensitive than bioassay, and permitted less than 1 ng/ml toxin to be detected in a rapid 10 min assay format. Use of the assay can eliminate the requirement for in vivo testing of novyi toxin and toxoid, provided measurements of biological activity are not required. Because of its speed and sensitivity, the assay can be used to monitor toxin production during fermentation and as an alternative to bioassay to measure antigen content during toxoiding and vaccine formulation.     

In vivo erythrocyte micronucleus assay III. Validation and regulatory acceptance of automated scoring and the use of rat peripheral blood reticulocytes, with discussion of non-hematopoietic target cells and a single dose-level limit test

The in vivo micronucleus assay working group of the International Workshop on Genotoxicity Testing (IWGT) discussed new aspects in the in vivo micronucleus (MN) test, including the regulatory acceptance of data derived from automated scoring, especially with regard to the use of flow cytometry, the suitability of rat peripheral blood reticulocytes to serve as the principal cell population for analysis, the establishment of in vivo MN assays in tissues other than bone marrow and blood (for example liver, skin, colon, germ cells), and the biological relevance of the single-dose-level test. Our group members agreed that flow cytometric systems to detect induction of micronucleated immature erythrocytes have advantages based on the presented data, e.g., they give good reproducibility compared to manual scoring, are rapid, and require only small quantities of peripheral blood. Flow cytometric analysis of peripheral blood reticulocytes has the potential to allow monitoring of chromosome damage in rodents and also other species as part of routine toxicology studies. It appears that it will be applicable to humans as well, although in this case the possible confounding effects of splenic activity will need to be considered closely. Also, the consensus of the group was that any system that meets the validation criteria recommended by the IWGT (2000) should be acceptable. A number of different flow cytometric-based micronucleus assays have been developed, but at the present time the validation data are most extensive for the flow cytometric method using anti-CD71 fluorescent staining especially in terms of inter-laboratory collaborative data. Whichever method is chosen, it is desirable that each laboratory should determine the minimum sample size required to ensure that scoring error is maintained below the level of animal-to-animal variation. In the second IWGT, the potential to use rat peripheral blood reticulocytes as target cells for the micronucleus assay was discussed, but a consensus regarding acceptability for regulatory purposes could not be reached at that time. Subsequent validation efforts, combined with accumulated published data, demonstrate that blood-derived reticulocytes from rats as well as mice are acceptable when young reticulocytes are analyzed under proper assay protocol and sample size. The working group reviewed the results of micronucleus assays using target cells/tissues other than hematopoietic cells. We also discussed the relevance of the liver micronucleus assay using young rats, and the importance of understanding the maturation of enzyme systems involved in the processes of metabolic activation in the liver of young rats. Although the consensus of the group was that the more information with regard to the metabolic capabilities of young rats would be useful, the published literature shows that young rats have sufficient metabolic capacity for the purposes of this assay. The use of young rats as a model for detecting MN induction in the liver offers a good alternative methodology to the use of partial hepatectomy or mitogenic stimulation. Additional data obtained from colon and skin MN models have been integrated into the data bases, enhancing confidence in the utility of these models. A fourth topic discussed by the working group was the regulatory acceptance of the single-dose-level assay. There was no consensus regarding the acceptability of a single dose level protocol when dose-limiting toxicity occurs. The use of a single dose level can lead to problems in data interpretation or to the loss of animals due to unexpected toxicity, making it necessary to repeat the study with additional doses. A limit test at a single dose level is currently accepted when toxicity is not dose-limiting.     

PCR detection of pirimicarb resistance in Australian field isolates of Aphis gossypii Glover (Aphididae: Hemiptera)

Aphis gossypii Glover (cotton aphid) is a major secondary pest of Australian cotton that readily develops resistance to the carbamate insecticide pirimicarb (Pirimor®) and to organophosphates generally. To test the pirimicarb resistance status of Australian strains of A . gossypii , a polymerase chain reaction (PCR) assay followed by restriction enzyme assay (REA) was designed to identify the Ace I polymorphism S431F known to be responsible for resistance. The method was tested against reference and 33 field strains collected over two consecutive seasons. Both methods confirmed pirimicarb resistance in two field strains, one from each cotton season, giving credence to the molecular technique described. The PCR assay proved specific for the Ace I gene. This PCR REA assay has the potential to replace bioassay for the routine pirimicarb resistance monitoring in A . gossypii. With the molecular assay providing results in 48 h, compared with 4–8 weeks for bioassay, such an assay could be used before insecticide control.     

Silica nanoparticles administered at the maximum tolerated dose induce genotoxic effects through an inflammatory reaction while gold nanoparticles do not

While the collection of genotoxicity data and insights into potential mechanisms of action for nano-sized particulate materials (NPs) are steadily increasing, there is great uncertainty whether current standard assays are suitable to appropriately characterize potential risks. We investigated the effects of NPs in an in vivo Comet/micronucleus (MN) combination assay and in an in vitro MN assay performed with human blood. We also incorporated additional endpoints into the in vivo study in an effort to delineate primary from secondary mechanisms. Amorphous silica NPs (15 and 55 nm) were chosen for their known reactivity, while gold nano/microparticles (2, 20, and 200 nm) were selected for their wide size range and lower reactivity. DNA damage in liver, lung and blood cells and micronuclei in circulating reticulocytes were measured after 3 consecutive intravenous injections to male Wistar rats at 48, 24 and 4h before sacrifice. Gold nano/microparticles were negative for MN induction in vitro and in vivo, and for the induction of DNA damage in all tissues. Silica particles, however, caused a small but reproducible increase in DNA damage and micronucleated reticulocytes when tested at their maximum tolerated dose (MTD). No genotoxic effects were observed at lower doses, and the in vitro MN assay was also negative. We hypothesize that silica NPs initiate secondary genotoxic effects through release of inflammatory cell-derived oxidants, similar to that described for crystalline silica (quartz). Such a mechanism is supported by the occurrence of increased neutrophilic infiltration, necrosis, and apoptotic cells in the liver, and induction of inflammatory markers TNF-α and IL-6 in plasma at the MTDs. These results were fairly consistent between silica NPs and the quartz control, thereby strengthening the argument that silica NPs may act in a similar, thresholded manner. The observed profile is supportive of a secondary genotoxicity mechanism that is driven by inflammation.     

Micronucleus evaluation in peripheral blood reticulocytes of mice treated with procarbazine hydrochloride or mitomycin C.

The usefulness of the acridine orange (AO) supravital staining technique for the mouse peripheral blood reticulocyte micronucleus test was investigated independently by three laboratories using the known clastogens procarbazine hydrochloride (PCZ) and mitomycin C (MMC). In all three laboratories the highest frequencies of micronucleated peripheral blood reticulocytes were observed 48 h after treatment of mice with a single dose of either MMC or PCZ. The animals responded to both chemicals in a dose-dependent manner. Although similar qualitative results were observed, mean micronucleus frequencies induced by a particular dose of a given test chemical did vary quantitatively among the three laboratories. This was most probably due to the use of slightly different scoring criteria by each examiner. This aspect needs special attention. To minimize inter-laboratory variability, therefore, we recommend establishing unequivocal criteria to distinguish the subclass of reticulocytes. These should then be used consistently by all investigators using this method. The most striking advantages of the AO supravital staining technique were the ease of slide preparation, the ease with which reticulocytes and mature erythrocytes could be distinguished by the examiners, and the occurrence of numerous scorable reticulocytes in each microscopic field, which greatly speeded up the manual counting process. The disadvantages of the staining technique were the limited scoring time due to the rapid fading of the fluorescence stain, the degradation of the cells with time, and the frequent need to search for adequate scoring areas within a microscopic field. Based on the data of this study the authors conclude that the AO supravital staining technique is highly suitable for the micronucleus assay in erythrocytic cells of mouse peripheral blood. In addition, we consider the mouse peripheral blood reticulocyte micronucleus test to be a useful tool with which to investigate the clastogenic potential of chemicals in vivo. As pretreatment of mice with Aroclor 1254 markedly increased the effect of PCZ on micronucleus induction, we suggest that the inclusion of inducers of drug metabolizing enzymes in the micronucleus test would be useful for the detection of the clastogenic potential of promutagenic chemicals.     

Sulfur-free parathyroid hormone analogues containing D-amino acids: biological properties in vitro and in vivo

Three sulfur-free analogues of bovine parathyroid hormone (bPTH) containing D-amino acids were synthesized by the solid-phase method and their biological properties compared in an in vitro bioassay (rat renal adenylate cyclase assay), a receptor assay for parathyroid hormone (PTH) (canine renal membranes), and an in vivo bioassay (chick hypercalcemia assay). The analogue [Nle8,Nle18,D-Tyr34]-bPTH-(1-34)-amide, which was found to be more than 4 times as potent in vitro as unsubstituted PTH, is the most potent analogue of PTH yet synthesized. The enhanced potency was largely attributable to increased affinity for the PTH receptor. In vivo, however, this analogue was only one-third as potent as bPTH-(1-34). Cumulative evidence suggests that the nearly 15-fold decline in the relative potency when the compound was assayed in vivo is due to the substitution of norleucine for methionine. The other analogues, [D-Val2,Nle8,D-Tyr34]bPTH-(1-34)-amide and [D-Val2,Nle8,Nle18,D=Tyr34]bPTH-(2-34)-amide, were only weakly active in vitro and in vivo, indicating that substitution with D-amino acids at the NH2 terminus of PTH causes markedly diminished receptor affinity. In fact, the placement of a D-amino acid at the NH2 terminus is more deleterious to biological activity than is omission of amino acids at positions 1 and 2.     

Nondwarf rice seedling bioassay for gibberellins

The use of nondwarf rice (Oryza sativa L.) cultivars treated with uniconazole as test plants for gibberellin (GA) bioassay instead of Tan-ginbozu dwarf rice variant was investigated. The sensitivity of six nondwarf rice cultivars to GAs was increased substantially by treatment of the seeds with uniconazole. The minimum detectable dose of a GA in the nondwarf cultivars treated with uniconazole was 1- to 1/10-fold of that in the nontreated Tanginbozu and 3- to 10-fold of that in uniconazole-treated Tanginbozu. The relative activity of several GAs on treated nondwarf rice cultivars was not largely different from that to Tan-ginbozu. Considering that seeds of nondwarf rice are available commercially, the nondwarf rice seedling assay would be useful as a simple assay for systematic analysis of GAs, and also as a routine teaching tool in high schools and universities.     

Specific and dynamic detection of palytoxins by in vitro microplate assay with human neuroblastoma cells

Palytoxin is one of the most complex and biggest molecules known to show extreme acute toxicity. The dinoflagellate Ostreopsis spp., the producer organism of palytoxin, has been shown to be distributed worldwide, thus making palytoxin an emerging toxin. Rat-derived hepatocytes (Clone 9) and BE (2)-M17 human neuroblastoma cells were used to test palytoxin or palytoxin-like compounds by measuring the cell metabolic rate with Alamar Blue. The dose-dependent decrease in viability was specifically inhibited by ouabain in the case of BE (2)-M17 neuroblastoma cells. This is a functional, dynamic and simple test for palytoxins with high sensitivity (as low as 0.2 ng/ml). This method was useful for toxin detection in Ostreopsis extracts and naturally contaminated mussel samples. A comparative study testing toxic mussel extracts by LC (liquid chromatography)-MS/MS (tandem MS), MBA (mouse bioassay), haemolysis neutralization assay and a cytotoxicity test indicated that our method is suitable for the routine determination and monitoring of palytoxins and palytoxin-like compounds.     

The micronucleus assay using peripheral blood reticulocytes from mitomycin C- and cyclophosphamide-treated rats

It used to be believed that the use of rat peripheral blood for the micronucleus assay would be difficult because micronucleated erythrocytes are captured and destroyed by the spleen quickly. We have applied an acridine orange (AO) supravital staining method to rat peripheral blood using AO-coated glass slides. Normal and splenectomized SD rats were treated once with mitomycin C (i.p.) or cyclophosphamide (p.o.), and 5 μl of blood was collected at intervals from the tail vein between 0 and 72 h after treatment. For comparison, bone marrow cells were smeared conventionally 30 h after treatment. Although the frequencies of spontaneous and chemically induced micronucleated reticulocytes (MNRETs) from normal rats were lower on average in the highest dose group than those of splenectomized rats, the incidence of micronuclei among type I and II reticulocytes in normal rats at 48 h was almost identical to the incidence of RNA-containing erythrocytes with micronucleus in bone marrow. Thus, we suggest that rat peripheral reticulocytes can be used as target cells for the micronucleus assay.     

The micronucleus assay using peripheral blood reticulocytes from mitomycin C- and cyclophosphamide-treated rats.

It used to be believed that the use of rat peripheral blood for the micronucleus assay would be difficult because micronucleated erythrocytes are captured and destroyed by the spleen quickly. We have applied an acridine orange (AO) supravital staining method to rat peripheral blood using AO-coated glass slides. Normal and splenectomized SD rats were treated once with mitomycin C (i.p.) or cyclophosphamide (p.o.), and 5 microliters of blood was collected at intervals from the tail vein between 0 and 72 h after treatment. For comparison, bone marrow cells were smeared conventionally 30 h after treatment. Although the frequencies of spontaneous and chemically induced micronucleated reticulocytes (MNRETs) from normal rats were lower on average in the highest dose group than those of splenectomized rats, the incidence of micronuclei among type I and II reticulocytes in normal rats at 48 h was almost identical to the incidence of RNA-containing erythrocytes with micronucleus in bone marrow. Thus, we suggest that rat peripheral reticulocytes can be used as target cells for the micronucleus assay.     

1986 Survey of genetic toxicology testing in industry,government contract,and academic laboratories

Results of the 1986 Genetic Toxicology Association's survey of industrial, government, contract, and academic laboratories on the status of several assays in genetic toxicology are presented below. 1. The most commonly used assay was the Salmonella typhimurium/mammalian microsomal (Ames) assay, which was used by 83% of all respondents. 2. The next five (5) most commonly used assays were in vitro cytogenetics (72%), in vivo cytogenetics (59%), CHO HGPRT gene mutation (55%), the micronucleus assay (53%), and L517BY gene mutation (45%). 3. The assay showing the greatest percentage increase in routine use was the micronucleus assay which went from 14% in 1984 to 34% in 1986, an increase of 20%. 4. Other assays which increased in routine use were CHO HGPRT mutation (+18%); in vitro cytogenetics (+14%); L5178Y gene mutation (+9%), and the Ames assay (+5%). 5. Routine use of in vitro UDS assays declined by 6%; use of in vitro SCE assays declined by 12%. 6. There was no change in the rate of routine use of in vivo cytogenetics or in vivo SCE assays. 7. Assays routinely performed on contract included the Salmonella assay, CHO HGPRT gene mutation, in vitro cytogenetics, in vitro UDS, in vivo cytogenetics, the micronucleus assay, L5178Y gene mutation, and the Drosophila sex-linked recessive lethal assay. 8. Four assays were being developed by five or more laboratories. These included in vitro SCE (8); the micronucleus assay (7); in vivo SCE (6); and DNA adduct formation (5). 9. A total of 17 assays had been abandoned by one or more laboratories. However, since no assay had been given up by more than three laboratories no conclusions can be drawn about the overall robustness of any of the assays on the survey form.     

The micronucleus assay with mouse peripheral blood reticulocytes using acridine orange-coated slides with triethylenemelamine.

A micronucleus assay using mouse peripheral blood and supravital staining with acridine orange (AO) was validated by two laboratories on triethylenemelamine-treated mice. Dose- and time-dependent increases in micronucleated peripheral reticulocytes were observed. This new method can be used as an alternative to the conventional bone marrow micronucleus assay.     

Improved specificity in gentamicin bioassay

An optimized bioassay for determination of gentamicin concentrations in serum of patients is presented. It was possible to enhance the exactness and representibility of the bioassay by means of a standardized methodical process, a suitable arrangement of the assay and reading at fifteen-fold enlargement. A systematic arrangement of the assay with randomized blocks is a safeguard against larger errors during the assay. The standard deviations in 5 culture plates for each serum sample amounted to about 0.3 mg/l envolving thus variation coefficients under 10%. The specificity of the bioassay was determined by means of the analytical procedure. The determination of gentamicin in blood serum, as it is generally known, may be masked in depencence on the concentration by heparin, vitamins, sodium chloride and sodium phosphate. Our model assays have revealed that for recognition of trouble factors in determination of gentamicin the evaluation by means of the parallel-line-assay according to the four point method should be performed. Inactivation of gentamicin for example by influence of phosphate cannot be recognized when evaluation is determined by linear regression. The bioassay, as a simple and economic method for control of treatment and pharmacokinetic investigations is available for any clinical and bacteriological laboratory.     

Effects of phenylhydrazine or recombinant human erythropoietin on deformability and activity of dehydrogenase glucose-6-phosphate and acetylcholinesterase in Wistar rats blood enriched in reticulocytes

Deformability and activity of the enzymes: acetylcholinesterase (AChE) and dehydrogenase glucose-6-phosphate (G-6-PD), were assayed for RBC enriched in immature reticulocytes. Reticulocytosis was evoked by administration of two different drugs: recombinant human erythropoietin (rHuEPO) and phenylhydrazine (PHZ) to two groups of Wistar rats. After treatment with the former compound, a group of animals exhibited 17.33% reticulocytes in blood whereas a group of rats treated with the latter drug reached 57.66% of these cells in blood. A marked decrease in RBC deformability was found in both groups of animals. AChE did not significantly change activity neither in PHZ-treated nor in rHuEPO-treated rats, whereas G-6-PD activity was significantly decreased in the PHZ-treated group.     

Enhancement of therapeutic protein in vivo activities through glycoengineering

Delivery of protein therapeutics often requires frequent injections because of low activity or rapid clearance, thereby placing a burden on patients and caregivers. Using glycoengineering, we have increased and prolonged the activity of proteins, thus allowing reduced frequency of administration. Glycosylation analogs with new N-linked glycosylation consensus sequences introduced into the protein were screened for the presence of additional N-linked carbohydrates and retention of in vitro activity. Suitable consensus sequences were combined in one molecule, resulting in glycosylation analogs of rHuEPO, leptin, and Mpl ligand. All three molecules had substantially increased in vivo activity and prolonged duration of action. Because these proteins were of three different classes (rHuEPO is an N-linked glycoprotein, Mpl ligand an O-linked glycoprotein, and leptin contains no carbohydrate), glycoengineering may be generally applicable as a strategy for increasing the in vivo activity and duration of action of proteins. This strategy has been validated clinically for glycoengineered rHuEPO (darbopoetin alfa).