Studies on the Dutch rabbit for pyrogen test

A study was conducted on Dutch rabbits to examine their applicability for the pyrogen test in comparison with the Japanese white rabbit which, conventionally, has been mainly used for the test in Japan. The following results were obtained. Adult Dutch rabbits, so small as to weight only about 60% of average adult weight of the Japanese white, showed a food consumption as low as 40% of that in the latter. None in the Dutch rabbit group suffered dislocation of the hip-joint during restraint on a neck stock while it was frequently encountered among Japanese white rabbits. The body temperature during restraint was unstable in infantile Dutch rabbits (7-8 weeks old)(, but stable in the young (14 weeks old) and in the adult (20-47 weeks old). Adult Dutch rabbits exhibited greater febrile responses to intravenously injected bacterial pyrogen (LPS) than infantile and young Dutch rabbits and adult Japanese white rabbits. A good linear regression was observed between the dose of LPS (0.01-0.1 microgram/kg, iv) and the febrile response in adult Dutch rabbits as well as in adult Japanese white rabbits. From these data, it is concluded that the Dutch rabbit has an advantage over the Japanese white for the pyrogen test in respect of febrile responsiveness and cost of rearing.     

不同基础体温的家兔对热原检查结果的影响

对实验中使用的普通级家兔基础体温进行统计,观察不同基础体温家兔注射血液制品后的升温情况。按照中华人民共和国药典(2005)年版三部的热原检查规定进行测定,将实验家兔基础体温38.0℃~39.6℃分为3组:38.0℃~38.5℃为1组;38.6℃~39.0℃为2组;39.1℃~39.6℃为3组。注射制品后,家兔升温≥0.4℃记为升温家兔,统计升温≥0.4℃的家兔升温百分率并对其进行统计学处理。经卡方检验,升温家兔≥0.4℃的百分率1组与2组、1组与3组有显著性差异(p<0.05);2组与3组无显著性差异(p>0.05)。家兔对热原的敏感性随基础体温的高低而有明显的差异,基础体温偏低的家兔对热原更敏感,其升温幅度大于基础体温偏高的家兔。     

Comparison of febrile responsiveness of rats and rabbits to endogenous pyrogen

The fever responses of rats and rabbits were compared in detail using a single common source of semipurified endogenous pyrogen prepared from human monocytes. The characteristics and dynamics of the fever-response curves for each species were examined and their dose-response curves were determined and compared. The fevers displayed by rats were qualitatively similar to those of rabbits, but, typically, they developed and terminated more rapidly than those of rabbits. Rabbits were much more sensitive to the endogenous pyrogen than rats. The threshold dose of pyrogen required to elicit a fever was 5 times lower in the rabbit, and the slope of the rabbit's dose-response curve was 1.5 times steeper than that of the rat. The maximum fevers attainable in rabbits were approximately twice those attainable in rats. It was also shown that the more rapid febrile responses of the rat were not due to the 10-fold smaller mass of the rat; instead, we proposed that this difference was more likely due to a closer diffusional proximity of the pyrogen receptor sites to the circulation in rats. The lower sensitivity of the rat to endogenous pyrogen was attributed to a relative insensitivity of the pyrogen receptor sites in rats in the translation of the endogenous pyrogen stimulus into fever.     

实验兔脚皮炎与相对湿度的相关性及对热原筛选实验的影响分析

目的分析实验兔脚皮炎发病率与相对湿度的相关性及脚皮炎实验兔对热原筛选实验合格率的影响。方法将2006、2007年实验室相对湿度记录、实验兔脚皮炎记录及热原筛选实验记录进行汇总统计,并对数据进行生物学分析。结果实验室相对湿度超过70％时,可导致实验兔脚皮炎发病率的升高,二者存在显著相关性（P〈0．01）;有脚皮炎的实验兔可降低筛选实验的合格率,二者存在显著相关性（P〈0．01）;实验兔基础体温的高低,对筛选合格率有显著影响（P〈0．0001）。结论实验兔的脚皮炎发病率与环境相对湿度有显著的正相关性,当实验室相对湿度超过实验兔适宜湿度范围时,可导致其脚皮炎的发病率上升;有脚皮炎的实验兔与无脚皮炎的实验免相比,可显著降低热原筛选实验的合格率。     

Comparison of the rabbit pyrogen test and Limulus amoebocyte lysate (LAL) assay for endotoxin in hepatitis B vaccines and the effect of aluminum hydroxide.

The rabbit pyrogen test and Limulus amoebocyte lysate (LAL) assay have been used to detect endotoxins in vaccines, but interactions between the endotoxins and proteins or aluminum hydroxide can interfere with the results. Currently, the rabbit pyrogen test is used to detect endotoxin in hepatitis B (HB) vaccines even though the HB surface protein, the active ingredient, is over-expressed in and purified from eukaryotic cells which lack endotoxin. Therefore, we examined the possibility of replacing the animal tests with the more efficient LAL test. To this end, we determined whether the aluminum hydroxide in the HB vaccines affects the rabbit pyrogen test and the LAL assay. HB vaccines and HB protein solutions spiked with lipopolysaccharide (LPS) produced almost the same dose-dependent temperature rise in rabbits, indicating that the aluminum hydroxide in the HB vaccine does not interfere with the pyrogenic response in rabbit. In contrast, a spike recovery study showed that aluminum hydroxide interfered with the LAL clot and kinetic assays; however, the LAL clot assay was effective at detecting endotoxin without loss of LAL activity after serial dilution of the samples. Furthermore, there was good correlation in the LAL clot assay between the amount of LPS added and the amount recovered. However, both turbidimetric and chromogenic kinetic assays displayed no correlation between the LPS amount added and recovered. Our results suggest that the LAL clot assay is sensitive and reliable when samples are properly prepared, and can be used to replace the rabbit pyrogen test for the detection of endotoxin in HB vaccines.     

A quantitative in vitro assay method for detecting biological activity of endotoxin using prostaglandin E2 induction in rabbit peripheral blood

The pyrogen test and the endotoxin test (the LAL test) have been playing crucial roles in detecting endotoxin in parenteral drugs. The current test methods, however, have disadvantages such as requiring a large number of animals or an inadequacy in evaluation of in vivo endotoxin activity. We attempted to establish a new assay method that can overcome the shortcomings of the current methods. We standardized the in vitro assay method by the use of prostaglandin E2 (PGE2) induction from peripheral blood of rabbits for detecting endotoxin activity. A linear dose-response regression was attained from approximately 0.15 to 5 endotoxin units/ml of Japanese national reference standard endotoxin by the in vitro assay. The assay showed a fine correlation with the pyrogen test but not with the LAL test, when endotoxins from various bacterial sources were tested. The in vitro assay was also shown to have the capability of detecting a synergistic effect of endotoxin and parenteral drugs. The in vitro PGE2 induction test using rabbit blood was, therefore, suggested to be the appropriate test method for guaranteeing the same level of safety of parenteral drugs as the pyrogen test does.     

rhBMP-2诱导异位软骨修复重建兔气管缺损的实验研究

目的：探讨重组人骨形成蛋白-2（rhBMP-2）作为激活物诱导异位软骨修复并重建免气管缺损的可行性。方法：取24只新西兰大白兔,制备气管前壁软骨1／3缺损模型。随机分为A、B组,每组12只,A组为实验组,在气管缺损处前壁颈前肌肉修补,多点注射rhBMP-2;B组于气管软骨缺损部位直接颈前肌群修补。术后观察动物一般情况,于4、8、12周取材进行大体观察、HE染色观察重建区域情况。结果：术后A组动物均存活至实验完成,B组因气道感染及气道分泌物堵管潴留致使实验兔死亡,其余动物出现皮下气肿,呼吸不畅等情况。组织学观察A组有明显的新生软骨细胞及少量软骨样组织,可见结缔组织包绕,周围肌肉组织完整,排列整齐,未见明显坏死组织,有少量淋巴细胞浸润。B组未见软骨组织生成,可见大量肉芽组织增生,结缔组织排列紊乱,伴少量坏死组织,大量淋巴浸润。结论：rhBMP-2可通过注射到颈前肌肉修补肌群中诱导软骨细胞和软骨样组织生成,减轻炎症反应,联合颈前肌瓣修复重建气管缺损能充分维持修复重建后的气道形态,具有减少术后皮下气肿、气管狭窄的作用,有望用于临床修复重建气管纽织缺损。     

Construction of a sensitive pyrogen-testing cell model by site-specific knock-in of multiple genes

A pyrogen test is crucial for evaluating the safety of drugs and medical equipment, especially those involved in injections. As existing pyrogen tests, including the rabbit pyrogen test, the limulus amoebocyte lysate (LAL) test and the monocyte activation test have limitations, development of new models for pyrogen testing is necessary. Here we develop a sensitive cell model for pyrogen test based on the lipopolysaccharides (LPS) signal pathway. TLR4, MD2, and CD14 play key roles in the LPS-mediated pyrogen reaction. We established a new TLR4/MD2/CD14-specific overexpressing knock-in cell model using the CRISPR/CAS9 technology and homologous recombination to detect LPS. Stimulation of our TLR4/CD14/MD2 knock-in cell line model with LPS leads to the release of the cytokines IL-6 and TNF-alpha, with a detection limit of 0.005 EU/ml, which is greatly lower than the lower limit of 0.015 EU/ml detected by the Tachypleus amebocyte lysate (TAL) assay.     

Studies on Bacterial Pyrogenicity: III. Specificity of the United States Pharmacopeia Rabbit Pyrogen Test

The specificity of the first or “presumptive” portion of the USP rabbit pyrogen test was investigated by use of a new absolute standard of reference. The reference standard was a 0.9% sodium chloride solution prepared to be pyrogen-free. Details of the preparation were described. The hypothesis was explored that the temperature response of rabbits after intravenous injection of the standard solution was independent of exogenous pyrogen. Reactions observed among the rabbits in our colony allowed a classification of these animals ranging from “consistently reliable” to “consistently unreliable.” Details of the experimental results and implications for pyrogen testing are discussed. The recommendation was made that all rabbit test animals be “screened” in sham and actual tests before being used for pyrogen testing.     

兔肾积水模型的建立及SPECT和CT灌注成像

目的探索建立肾盂输尿管连接部梗阻所致肾积水的动物模型的可行性;探讨CT灌注成像对积水肾脏肾功能的评估价值。方法10周龄雄性新西兰兔50只随机分组,假手术组20只,分离左侧输尿管后直接关腹。模型组30只,选用腰大肌包埋输尿管造成左侧肾盂输尿管连接部梗阻。术前两组进行单光子发射计算机体层成像（SPECT）比较左肾功能,检验无差异后在术后3月分别行左肾SPECT、CT灌注,以病理检查为佐证,观察两组参数变化,进行CT灌注各项参数和GFR的统计学相关性分析。结果模型组建模成功达70%,呈慢性肾积水病理表现,左肾皮髓质CT灌注参数BF、BV、PS均下降,与相应GFR呈高度正相关。结论腰大肌包埋输尿管的模型制作方法具有可行性。CT灌注参数可作为肾功能状态的评定指标,具有一定的临床指导意义。     

Differences in endogenous pyrogen fevers induced by iv and icv routes in rabbits

We have compared the characteristics of fevers produced by endogenous pyrogen administered by the intravenous (iv) and by the intracerebroventricular (icv) routes in conscious rabbits. Fevers induced by the intracerebroventricular route have a longer latency to onset, a less steep rise in body temperature, and a longer time to peak elevation in body temperature than do fevers induced by the intravenous route. Furthermore, a dose of indomethacin (2 mg/kg) administered intravenously, which is effective in markedly attenuating fevers produced by the intravenous route, was completely without effect on fevers induced by the intracerebroventricular route. On the other hand, when indomethacin (500 micrograms) was infused intracerebroventricularly, it markedly reduced fevers induced by the subsequent injection of endogenous pyrogen into the contralateral cerebral ventricle, but such pretreatment had little effect on fevers elicited by intravenous injections of endogenous pyrogen. It is concluded that the sites of action of endogenous pyrogen in response to intravenous injections of pyrogen are different from those responding to intracerebroventricular injections of pyrogen and that this is manifest in several distinct differences in the characteristics of the two fevers. These results indicate that the intracerebroventricular model of fever production is not appropriate for the study of the normal pathogenesis of fever.     

重组人戊型肝炎疫苗细菌内毒素检查方法的建立

确立重组人戊型肝炎疫苗原液的细菌内毒素检查方法。供试品参照《中华人民共和国药典》(2005年版三部)热原检查法进行热原检查,结果符合规定。该供试品同时参照《中华人民共和国药典》(2005年版三部)细菌内毒素检查法要求进行试验。供试品溶液在40μg/m l浓度下,确定内毒素限值为40EU/m l。供试品在该内毒素限值下干扰试验有效,且细菌内毒素检查法符合规定。该疫苗用内毒素检查法代替热原检查法,方法可行。     

A study of the variability in the febrile responses of rabbits to endogenous pyrogen

The range of body temperature increases elicited by a standard dose of endogenous pyrogen (0.5 ml/kg iv) was examined in a population of 26 male New Zealand White rabbits. Although the mean maximum increase in rectal temperature was 0.88 +/- 0.06 degree C (SE), individual responses varied from 0.4 degree to 1.5 degree C. Three representative animals that responded to the standard dose of pyrogen with small, intermediate, and large febrile responses were selected and challenged with the same dose of pyrogen on eight separate occasions, and the variability of these responses was examined. There was little variability within the characteristic responses of any particular animal to the repeated challenges. The variability of the febrile responses elicited by both intravenous and intracerebroventricular administration of the same pyrogen was examined and compared using another group of 11 rabbits. The variability in response to the intravenous route was similar to that found in the larger population, whereas the variation in response to the intracerebroventricular route was smaller, and all 11 animals had fevers that were greater than 1 degrees C. It is concluded that the variability of the febrile responses of rabbits to intravenous pyrogen was due to differences between individual sensitivities of animals to the intravenously administered pyrogen. This difference in sensitivity may be due to a difference in the amount of pyrogen that reaches the putative receptor sites, or to a difference in the density or effectiveness of receptor sites in translating the pyrogenic stimulus into a fever response.     

Control of variance in experimental studies of hyperlipidemia using the WHHL rabbit

Between-animal variability has frustrated many experimental studies in outbred animal models of human disease. Variability that arises from genetic heterozygosity can be minimized by use of experimental designs that match littermates (polyzygotic twins) across control and treatment groups. Poor breeding vigor has prevented use of this experimental design in the WHHL rabbit model of hyperlipidemia and atherosclerosis. A comparison of reproduction in WHHL and normal rabbits demonstrated that litter size is limited by functional deficits at ovulation, implantation, and gestation in WHHL females. Superovulation of females reliably produced expanded litters of WHHL rabbits. Plasma lipids were measured in expanded litters of Japanese White WHHL (JW-WW) and English Half-lop WHHL (EHL-WW) rabbits. The variance of plasma cholesterol within sibships was two- to three-fold less than that between-litters. Intraclass correlation of total cholesterol within litters of EHL-WW was 0.72 and within litters of JW-WW was 0.67. These data provide evidence of genetic modulation of hypercholesterolemia in WHHL rabbits and demonstrate that experimental designs in which littermates are paired across groups can decrease the number of animals needed or increase the sensitivity of hypothesis tests by two- to threefold.     

静脉注射用人免疫球蛋白细菌内毒素检测

探索静脉注射用人免疫球蛋白(IVIG)细菌内毒素含量的鲎试验检测方法。根据《中国药典》(2000版)细菌内毒素含量的检测方法进行,将待检品用NaOH调pH至中性,稀释至2.4倍可用标示灵敏度为0.25EU/m l的特异性鲎试剂进行细菌内毒素检测,结果与家兔法进行比较,并且在样品中加入定量内毒素0.6 EU/m l用两种方法进行对比试验。结果表明用细菌内毒素检测法(鲎试验法)检测静脉注射用人免疫球蛋白是可行的。     

Dietary cholesterol absorption, and sterol and bile acid excretion in hypercholesterolemia-resistant white rabbits

The New Zealand white (NZW) rabbit fed a 0.1% cholesterol-enriched diet (CD) typically responds (normoresponsive, NR) by quickly developing hypercholesterolemia. To study the underlying mechanisms responsible for the widespread phenomenon of inter-individual variability of response to dietary cholesterol, a unique hypercholesterolemia-resistant (RT) rabbit model was developed. These animals were utilized to investigate selected potential mechanisms that might enable the RT animal to compensate for dietary cholesterol overload. When rabbits were fed the low-cholesterol stock diet, there was no significant difference in the plasma cholesterol concentrations of the NR and the RT animals. However, a significant rise was observed in the NR rabbits within 1 month of their being placed on the cholesterol-enriched diet; the plasma cholesterol concentration of the RT animals was not affected. During consumption of the cholesterol diet the cholesterol absorption rate was somewhat greater in the NR rabbits (P less than 0.05), whereas intestinal transit times and the fecal excretion of neutral steroids were substantially the same in both groups. In contrast, the fecal bile acid excretion of the RT animals was more than twice as great (P less than 0.0001) as that of the NR animals. We conclude that the response to dietary cholesterol is a heritable trait in these rabbits and that, although less dietary cholesterol was absorbed by the RT animals, it appears that a major mechanism controlling plasma cholesterol levels involves the rate of conversion of cholesterol to bile acids and their subsequent excretion.     

家兔发热时脑脊液和血浆中精氨酸加压素含量的变化及禁水的抗热作用

本实验观察了发热家兔脑脊液(CSF)和血浆中精氨酸加压素(AVP)含量的变化及禁水对家兔内毒素(ET)发热效应的影响。实验结果表明:1.隔区注射AVP可明显抑制家兔ET性发热效应;2.发热组家兔CSF和血浆中AVP含量较正常组明显降低;3.禁水可明显对抗家兔ET性发热效应,其抗热作用与CSF和血浆中AVP含量升高有关;4.禁水也可使正常家兔体温水平下移。上述实验结果提示,AVP可能是家兔体内一种内源性退热物质,同时在正常体温调节中也可能发挥一定的作用。     

Rabbit IL-1. Cloning, expression, biologic properties, and transcription during endotoxemia

The cloning, sequencing, expression, and biologic activities of rabbit IL-1 alpha and beta are described. A cDNA library was constructed in lambda gt10 by using polyadenylated RNA extracted from rabbit adherent splenic macrophages 4 h after stimulation with endotoxin. By using the cDNA for human IL-1 beta and IL-1 alpha as hybridization probes, cDNA for both forms of rabbit IL-1 were isolated. The cDNA for rabbit IL-1 beta encodes a precursor polypeptide of 268 amino acids with an overall homology to human IL-1 beta of 74% (81% in the mature region coding for a 17.5 kDa carboxyl-terminal protein). The similarity between the two rabbit IL-1 forms is 31% for the entire molecule and 34% for the mature protein. The mature polypeptides of both forms were expressed in Escherichia coli. The recombinant proteins were purified to homogeneity and tested in a variety of biologic assays. Both forms produced typical endogenous pyrogen fevers in rabbits and augmented murine thymocyte and Th cell proliferation. Rabbit IL-1 alpha and beta were more pyrogenic in rabbits than human rIL-1 beta, whereas human rIL-1 alpha and beta were slightly more potent lymphocyte-activating factors. The recombinant rabbit proteins induced PGE and IL-1 production from human PBMC in vitro. A RIA for human IL-1 alpha did not recognize rabbit IL-1 alpha or beta, but rabbit IL-1 beta cross-reacted (as much as 30%) in a RIA for human IL-1 beta. Rabbits were injected with endotoxin and mRNA for both forms of IL-1 were observed primarily in the spleen and liver. The mRNA reached maximal levels after 60 min, then declined rapidly over the next 3 h, but were still present after 24 h. Liver tissue removed 4 h after endotoxin infusion produced lymphocyte-activating factors which were neutralized by more than 90% with a combination of goat anti-rabbit IL-1 alpha and anti-IL-1 beta.     

Biological Parameters of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) Fed on Rabbits,Sheep, and Cattle

In order to determine the effect of various hosts on feeding performance of Rhipicephalus (Boophilus) microplus, we used 3 mammalian species as hosts, cattle (Qinchuan), sheep (T an), and rabbits (Japanese white rabbit) for infest-ing ticks. Five hundreds of R. microplus larvae were exposed to each animal (3 animals/host species). Tick recoveries were 11.0%, 0.47%, and 5.5% from cattle, sheep, and rabbits, respectively. The averages of tick feeding periods were not significantly different on cattle, sheep, and rabbits, 28.8, 25.3, and 26.7 days, respectively. The average weights of individual engorged female from cattle, sheep, and rabbits were 312.5, 219.1, and 130.2 mg, respectively and those of egg mass weights each to 85.0, 96.6, and 17.8 mg. The highest egg hatching rate was in the ticks from cattle (96.0%), fol-lowed by those from rabbits (83.0%) and sheep (19.2%). These data suggest that rabbits could be as an alternative host to cultivate R. microplus for evaluating vaccines and chemical and biological medicines against the tick in the laboratory, although the biological parameters of ticks were less than those from cattle.     

Trypsinized Human Group O Erythrocytes as an Alternative Hemagglutinating Agent for Japanese Encephalitis Virus

Trypsinized human group O erythrocytes were found to be a suitable alternative to gander cells in hemagglutination (HA) and hemagglutination inhibition (HAI) tests for Japanese encephalitis (JE) virus. In the HAI test, no cross-reactions against JE virus were observed with immune sera containing antibody to taxonomically related or unrelated viruses, with mouse brain antigen, or with nonantibody serum inhibitors; specific antibody rise could be detected in an immunized rabbit. Gander and trypsinized human group O cells gave comparable titers in the HAI test, but the latter were preferable since (i) they required less challenging HA antigen, being more sensitive to agglutination by JE virus, and (ii) all human and some animal sera investigated were devoid of natural agglutinins for these cells, thereby eliminating or reducing the need for prior adsorption with packed cells.