Inclusion of a universal tetanus toxoid CD4 T cell epitope P2 significantly enhanced the immunogenicity of recombinant rotavirus ΔVP8* subunit parenteral vaccines

Currently available live oral rotavirus vaccines, Rotarix^® and RotaTeq^®, are highly efficacious in developed countries. However, the immunogenicity and efficacy of such vaccines in some developing countries are low. We reported previously that bacterially-expressed rotavirus ΔVP8* subunit vaccine candidates with P[8], P[4] or P[6] specificity elicited high-titer virus neutralizing antibodies in animals immunized intramuscularly. Of note was the finding that antibodies induced with the P[8]ΔVP8* vaccine neutralized both homotypic P[8] and heterotypic P[4] rotavirus strains to high titer. To further improve its vaccine potential, a tetanus toxoid universal CD4⁺ T cell epitope P2 was introduced into P[8] or P[6]ΔVP8* construct. The resulting recombinant fusion proteins expressed in Escherichia coli were of high solubility and were produced with high yield. Two doses (10 or 20 μg/dose) of the P2-P[8]ΔVP8* vaccine or P2-P[6]ΔVP8* vaccine with aluminum phosphate adjuvant elicited significantly higher geometric mean homologous neutralizing antibody titers than the vaccines without P2 in intramuscularly immunized guinea pigs. Interestingly, high levels of neutralizing antibody responses induced in guinea pigs with 3 doses of the P2-P[8]ΔVP8* vaccine persisted for at least 6 months. Furthermore, in the gnotobiotic piglet challenge study, three intramuscular doses (50 μg/dose) of the P2-P[8]ΔVP8* vaccine with aluminum phosphate adjuvant significantly delayed the onset of diarrhea and significantly reduced the duration of diarrhea and the cumulative diarrhea score after oral challenge with virulent human rotavirus Wa (G1P[8]) strain. The P2-P[8]ΔVP8* vaccine induced serum virus neutralizing antibody and VP4-specific IgG antibody production prechallenge, and primed the pigs for higher antibody and intestinal and systemic virus-specific IFN-γ producing CD4⁺ T cell responses postchallenge. These two subunit vaccines could be used at a minimum singly or preferably in bivalent formulation to provide antigenic coverage of most of the G types of global importance.     

复方苯佐卡因凝胶的局部毒性研究

目的评价复方苯佐卡因凝胶的局部刺激性和致敏性。方法依照国家食品药品监督管理局《化学药物刺激性、过敏性和溶血性研究技术指导原则》(以下简称指导原则),采用大鼠口腔黏膜刺激性实验法、兔眼刺激性实验法以及豚鼠皮肤致敏实验法。结果复方苯佐卡因凝胶给大鼠口腔正常黏膜连续涂药3次,相当于3 g.kg-1,均无刺激和毒性反应;一次性按每只0.1 g给兔眼结膜涂药,亦未出现刺激性反应;给每只豚鼠皮肤0.2 g致敏接触和激发接触,均无过敏反应发生。结论复方苯佐卡因凝胶对口腔黏膜无刺激性,对皮肤也没有致敏性。     

Radioactive choline metabolism in guinea pig gallbladder

Acetylcholine may be released from gallbladder intrinsic nerves in response to cholecystokinin stimulation. This study characterized metabolites of [¹⁴C]choline produced in the gallbladder and released during incubation, with or without cholecystokinin-octapeptide. Radiolabeled [¹⁴C]choline was applied to the mucosal or muscle surface of intact guinea pig gallbladders in an organ bath. After radiolabeling, gallbladders were incubated with or without the contractile agonist cholecystokinin-octapeptide. Metabolites of [¹⁴C]choline were identified in gallbladder tissue and incubation buffers using HPLC and thin-layer chromatography. The major metabolites of [¹⁴C]choline were betaine and phosphocholine. [¹⁴C]Phosphocholine was incorporated slowly into [¹⁴C]phosphatidylcholine. [¹⁴C]Choline was released into buffers during incubation. [¹⁴C]Acetylcholine constituted less than 1% of radiolabel in the gallbladder. There was no identifiable [¹⁴C]acetylcholine released in buffers. Cholecystokinin-octapeptide did not affect choline metabolism. These studies showed that choline in the gallbladder is metabolized along pathways similar to those in the liver. Gallbladders released mostly choline, rather than acetylcholine, even during hormonally induced contraction.This project was supported by the Research and Development Office of the Department of Veterans Affairs.     

正常豚鼠胆囊平滑肌M受体亚型mRNA表达的研究

目的 研究M受体亚型mRNA在正常豚鼠胆囊平滑肌的表达.方法 10周龄白化豚鼠10只,RT-PCR检测胆囊平滑肌M受体亚型mRNA的表达.结果 豚鼠胆囊平滑肌有M受体4种亚型mRNA的表达,各亚型mRNA表达从强到弱顺序依次为M3、M2、M4及M1,无M5受体mRNA的表达.M3与M2受体mRNA的表达量差异无显著性(P＞0.05),M3、M2受体mRNA的表达量分别与M4和M1受体mRNA的表达量相比,差异均有显著性(P＜0.05).结论 胆囊平滑肌有4种M受体亚型mRNA表达即M1～M4,提示这4种亚型可能均参与迷走神经递质乙酰胆碱激发的胆囊收缩.     

慢病毒载体介导IL-4基因修饰BMSCs内耳局部应用治疗免疫性感音神经性聋的实验研究

目的:探讨白细胞介素4(IL-4)基因修饰骨髓间充质干细胞(BMSCs)内耳局部植入对免疫性感音神经性聋动物内耳病理损伤和生理功能障碍的调节与治疗作用.方法:采用钥孔嘁血蓝蛋白(KLH)抗原在已致敏的豚鼠圆窗龛局部免疫,造成免疫性感音神经性聋动物模型33只.分为IL-4基因修饰BMSCs组(A组)、空载慢病毒感染的BMSCs对照组(B组,即BMSCs空载对照组)和模拟手术对照组(C组),均将BMSCs细胞悬液经鼓阶开窗植入内耳. 提取豚鼠BMSCs,重组IL-4基因的慢病毒载体体外转染BMSCs,成功后鼓阶开窗植入内耳.采用免疫荧光组织化学法和免疫酶组织化学法 分别观察导入内耳的BMSCs 及IL-4基因产物在内耳组织结构中的分布和表达情况.应用酶联免疫吸附试验(ELISA)和听性脑干诱发电位(ABR)测试观察血清抗KLH特异性抗体水平和听觉功能变化,并行内耳石蜡切片光镜观察.结果:局部KLH免疫后与内耳局部BMSCs植入后2周,特异性抗KLH抗体水平差异无统计学意义;A组和B组ABR Ⅲ波阈值不同程度降低,但前者阈值降低更为明显,两组间比较差异有统计学意义(P＜0.05).免疫组织化学结果显示BMSCs(荧光反应阳性细胞)主要分布在鼓阶、前庭阶,酶反应阳性(IL-4基因产物)部位主要集中于螺旋神经节、骨螺旋板唇部、Corti器、血管纹、耳蜗骨壁以及蜗管中的BMSCs的细胞和其周围.内耳光镜观察结果显示:A组和B组仅在鼓阶内有絮状物,注射部位有少量红细胞和白细胞;C组可见螺旋神经节和蜗轴小血管周围有单个核细胞浸润,部分听损耳还可见螺旋神经节细胞数目减少、不同程度的膜迷路积水、蜗管内有絮状物和漂浮细胞. 结论:重组IL-4基因的慢病毒载体植入内耳后,可在内耳迁移并产生基因产物IL-4.经IL-4基因修饰的BMSCs和空载在体外成功地转染BMSCs.经鼓阶途径BMSCs内耳移植均可明显减轻免疫性感音 神经性聋动物的内耳免疫炎症反应和听觉功能损伤,前者作用更为显著.从而提示BMSCs(包括经IL-4基因修饰的BMSCs)局部应用可对免疫性内耳病的免疫炎症损伤产生一定的调节和治疗作用,并有向病变部位迁移、聚集的倾向.     

Aseptic knee effusion associated with calcified guinea worms

Reported is a case of aseptic knee effusion associated with the presence of a calcified guinea worm, Dracunculus medinensis, in close proximity to the joint. The patient, a native of Nigeria, presented with chronic right knee pain and swelling. He did well with symptomatic treatment including non-steroidal antiinflammatory drugs, rest, ice, and elevation of the leg. Dracunculiasis is prevalent in parts of Asia and Africa, but has been described only rarely in the United States.     

Prophylaxis with human serum butyrylcholinesterase protects guinea pigs exposed to multiple lethal doses of soman or VX

Human serum butyrylcholinesterase (Hu BChE) is currently under advanced development as a bioscavenger for the prophylaxis of organophosphorus (OP) nerve agent toxicity in humans. It is estimated that a dose of 200 mg will be required to protect a human against 2 × LD₅₀ of soman. To provide data for initiating an investigational new drug application for the use of this enzyme as a bioscavenger in humans, we purified enzyme from Cohn fraction IV-4 paste and initiated safety and efficacy evaluations in mice, guinea pigs, and non-human primates. In mice, we demonstrated that a single dose of enzyme that is 30 times the therapeutic dose circulated in blood for at least four days and did not cause any clinical pathology in these animals. In this study, we report the results of safety and efficacy evaluations conducted in guinea pigs. Various doses of Hu BChE delivered by i.m. injections peaked at ∼24 h and had a mean residence time of 78–103 h. Hu BChE did not exhibit any toxicity in guinea pigs as measured by general observation, serum chemistry, hematology, and gross and histological tissue changes. Efficacy evaluations showed that Hu BChE protected guinea pigs from an exposure of 5.5 × LD₅₀ of soman or 8 × LD₅₀ of VX. These results provide convincing data for the development of Hu BChE as a bioscavenger that can protect humans against all OP nerve agents.     

祖师麻止痛喷雾剂对豚鼠的长期毒性研究

目的:观察祖师麻止痛喷雾剂对豚鼠的长期毒性,为临床安全用药提供依据。方法:将50只豚鼠随机分为空白对照组、高剂量完整皮肤组、高剂量破损皮肤组、低剂量完整皮肤组、低剂量破损皮肤组,连续给药4周。随时观察记录其一般情况,用药后做血液学及血液生化指标检查,计算脏器系数并进行组织病理学检查。结果:祖师麻止痛喷雾剂各剂量组未见毒性反应,与空白对照组比较差异均无统计学意义(P>0.05),停药后也未见药物延迟性毒性反应。结论:长期给予豚鼠祖师麻止痛喷雾剂无明显毒性,推论临床拟用剂量安全。     

高频听力损失对豚鼠低频区时间分辨率的影响

目的 构建豚鼠高频听力损失模型,通过听觉惊跳反射前抑制方法观察高频听力损失后,低频区时间分辨率的变化.方法 采用简单随机法将豚鼠分为实验组(6只)和对照组(4只),实验组暴露于声压级110 dB的12 kHz纯音下30 h,建立8 kHz以上听力损失模型,对照组未作处理.于噪声暴露前、暴露后2周、4周、6周及8周等五个时间点进行听觉惊跳反射前抑制试验和听觉脑干反应(ABR)测试.在听觉惊跳反射前抑制试验中,选用0.5～2 kHz,0.5～4 kHz和0.5～8 kHz三种背景噪音频率(均在听敏度正常的低频段)来观察低频区时间分辨率的变化.结果 噪声暴露后2周,实验组动物呈现8 kHz以上频率陡降型高频听力损失,在16 kHz、32 kHz和48 kHz频率均有平均55 dB的阈移,与声暴露前相比差异均有统计学意义(P值均＜0.05),而1 kHz、2 kHz、4 kHz和8 kHz在噪音暴露前后差异均无统计学意义(P值均＞0.05).在8周的实验期内,实验组高频听力损失仍然存在.对照组豚鼠所有频率的听敏度在实验期内各个时间点差异均无统计学意义(P值均＞0.05).实验组在8 kHz的背景噪音下,声暴露后第2周抑制能力减弱,与暴露前相比差异具有统计学意义(P=0.036),其余时间点和暴露前相比差异无统计学意义(P值均＞0.05);在4 kHz的背景噪音下,声暴露后所有时间点的抑制能力与暴露前相比差异均无统计学意义(P值均＞0.05);在2 kHz的背景噪音下,声暴露后第6周、第8周抑制能力降低,与暴露前相比差异具有统计学意义(P值均＜0.05).结论 高频听力损失可降低正常低频区的时间分辨率.

Abstract：

Objective To constitute the animal model of high frequency hearing loss and observer the temporal processing abilities of low frequency regions using prepulse inhibition of auditory startle response (gap-PPI).Methods Ten guinea pigs were randomly grouped into two groups: the high frequency hearing loss group with six guinea pigs and the control group with four guinea pigs.The former group was exposed to 12 kHz tone at 110 dB SPL for 30 hours to establish the high frequency hearing loss above 8 kHz and the latter group received no stimuations.Before and two,four,six and eight weeks after noise exposure,gapPPI and auditory brainstem response (ABR) were recorded in both groups.In the gap-PPI experiment,three different background noises as 0.5-2 kHz,0.5-4 kHz and 0.5-8 kHz were applied to test the temporal gap.Results High frequency hearing loss above 8 kHz was shown two weeks after noise exposure.The averaged ABR thresholds of 16 kHz,32 kHz and 48 kHz were elevated about 55 dB and shown statistical significance compared to those before exposure (P ＜ 0.05＝.No significant difference of ABR thresholds were shown between 1 kHz,2 kHz,4 kHz and 8kHz before and after noise exposure(P ＞0.05).In the control group,the ABR thresholds remained stable during experiment.In the gap-PPI test,two weeks after noise exposure of 8 kHz,the experiment group showed attenuated inhibition ability and recovered gradually four weeks after noise exposure.No statistical differences of inhibition ability at time points of two,four,six and eight weeks after noise exposure of 4 kHz were detected when compared with that of pre-exposure.Under the background noise of 2 kHz,the inhibition ability attenuated and reached statistical significance at 6-8 weeks after noise exposure.Conclusion The high frequency hearing loss might induce an impairment of the temporal processing in the low frequency region.