Recombinant C fragment of botulinum neurotoxin B serotype (rBoNTB (HC)) immune response and protection in the rhesus monkey

Botulinum neurotoxin B (BoNTB) is a distinct protein subtype of a family of neurotoxins with the potential for use in biological warfare or terrorist attacks. This study is one in a series evaluating the immunogenicity and protective effects of recombinant vaccines against the different subtypes of botulinum toxin. The recombinant subunit vaccines encoding the C fragment portion (50 kDa) of the toxins are produced in the yeast, Pichia pastoris. In this study, groups of rhesus monkeys were vaccinated with three doses (1 and 5 μg per dose) of rBoNTB(H_c) vaccine. Total and neutralizing antibody titers were determined at various times during and postvaccination. Two groups of vaccinated monkeys plus non-vaccinated controls were actively challenged with B toxin by aerosol exposure. All monkeys receiving vaccine were protected from the toxin and no clinical signs of disease were observed, while controls displaying classic signs of botulism succumbed to the toxin challenge. Two additional groups of monkeys receiving the same vaccine regiment as the first two groups had significant levels of circulating neutralizing antibody titers up to 24 months postvaccination. This non-human primate study demonstrated the short- and long-term immunity afforded by the rBoNTB(H_c) vaccine.     

Vaccines against the category B toxins: Staphylococcal enterotoxin B, epsilon toxin and ricin

The threat of bioterrorism worldwide has accelerated the demand for the development of therapies and vaccines against the Category B toxins: staphylococcal enterotoxin B (SEB), epsilon toxin (ETX) produced by Clostridium perfringens types B and D, and ricin, a natural product of the castor bean. The diverse and unique nature of these toxins poses a challenge to vaccinologists. While formalin-inactivated toxins can successfully induce antibody-mediated protection in animals, their usefulness in humans is limited because of potential safety concerns. For this reason, research is now aimed at developing recombinant, attenuated vaccines based on a detailed understanding of the molecular mechanisms by which these toxins function. Vaccine development is further complicated by the fact that as bioterrorism agents, SEB, ETX and ricin would most likely be disseminated as aerosols or in food/water supplies. Our understanding of the mechanisms by which these toxins cross mucosal surfaces, and importance of mucosal immunity in preventing toxin uptake is only rudimentary.     

由上呼吸道感染诱发的急性点滴型银屑病皮损生态微生物研究

目的 探讨由上呼吸道感染诱发的急性点滴型银屑病皮损表面微生物群落的特点，及其与健康对照者之间的物种差异。方法 收集具有上呼吸道感染史的急性点滴型银屑病患者（银屑病组）与无其他皮肤病皮损的健康对照（健康组）各11例，应用16SrDNA测序技术进行研究。用无菌棉拭子取材，后行DNA抽提、质检，PCR，文库构建等微生物基因提取、纯化、回收过程，随后上机测序基因并行物种注释，最后对数据行α、β多样性分析寻找组内微生物种差异及组间微生物群落结构的差异，采用LEfSe分析找出差异显著的物种，由秩和检验验证差异结果。结果 2组间α多样性分析差异无统计学意义，β多样性分析发现2组存在差异的趋势。经LEfSe分析（LED值为4）与秩和检验均发现不动杆菌属物种差异具有统计学意义（P＜0.05），且在银屑病组皮损起主要作用。结论 初发或复发的具有上呼吸道感染史的急性点滴型银屑病患者皮损微生物菌群正由一个稳态向另一稳态发展，不动杆菌属在该变化中可能起到关键作用，然而整体微生物菌群的变化并不显著。     

Advances in respiratory syncytial virus vaccine development

Respiratory syncytial virus (RSV) is the most important causative agent of respiratory tract infections. Young children with chronic lung or congenital heart disease are at increased risk for severe disease. Intensive research into a candidate vaccine has yielded live attenuated vaccines and subunit vaccines, which have been studied in humans. Although immunogenic, occurrence of upper respiratory tract infection symptoms with live attenuated vaccine prohibits evaluation in young infants. Subunit vaccines include purified F protein (PFP-1 and -2) and BBG2Na. PFP vaccines are effective in seropositive children, but also induce upper respiratory symptoms. BBG2Na was being investigated in phase III clinical trials, however, further development has now been discontinued. This review discusses recent advances in RSV vaccine development.     

蓖麻蛋白诱导HeLa细胞凋亡的分子机制(英文)

目的:研究蓖麻蛋白引起的Hela细胞凋亡的形态变化及机制。方法:扫描电镜,透射电镜,Western blot,细胞周期分析、细胞毒性和细胞相对存活率测定。结果:蓖麻蛋白0.05μmol·L~(-1)引起HeLa细胞发生典型的凋亡。凋亡细胞主要表现为胞浆膜起泡,核染色质浓缩,形成新月状核或膜包裹核染色质的凋亡小体;Western blot未检测到p53、Bax和ICE的p20活性亚基,而检测到CPP32的p17活性亚基,CPP32活性升高,而ICE活性无显著改变。结论:CPP32参与了蓖麻蛋白诱导的HeLa细胞凋亡过程。     

Reductive activation of ricin and ricin A-chain immunotoxins by protein disulfide isomerase and thioredoxin reductase

Intracellular activation of ricin and of the ricin A-chain (RTA) immunotoxins requires reduction of their intersubunit disulfide(s). This crucial event is likely to be catalyzed by disulfide oxidoreductases and precedes dislocation of the toxic subunit to the cytosol. We investigated the role of protein disulfide isomerase (EC 5.3.4.1, PDI), thioredoxin (Trx), and thioredoxin reductase (EC 1.8.1.9, TrxR) in the reduction of ricin and of a ricin A-chain immunotoxin by combining enzymatic assays, SDS-PAGE separation and immunoblotting. We found that, whereas PDI, Trx, and TrxR used separately were unable to directly reduce ricin and the immunotoxin, PDI and Trx in the presence of TrxR and NADPH could reduce both ricin and immunotoxin in vitro. PDI functioned only after pre-incubation with TrxR and the reductive activation of ricin was more efficient in the presence of glutathione. Similar results were obtained with microsomal membranes or crude cell extracts. Pre-incubation with the gold(I) compound auranofin, which irreversibly inactivates TrxR, resulted in a dose-dependent inhibition of ricin and immunotoxin reduction. Reductive activation of ricin and immunotoxin decreased or was abolished in microsomes depleted of TrxR and in cell extracts depleted of both PDI and Trx. Pre-incubation of U-937, Molt-3, Jurkat, and DU145 cells with auranofin significantly decreased ricin cytotoxicity with respect to mock-treated controls (P<0.05). Conversely, auranofin failed to protect cells from the toxicity of pre-reduced ricin which does not require intracellular reduction of disulfide between the two ricin subunits. We conclude that TrxR, by activating disulfide reductase activity of PDI, can ultimately lead to reduction/activation of ricin and immunotoxin in the cell.     

Multivalent binding of ricin to bovine serum albumin-based neoglycoconjugates.

Ricin, a ribosome-inactivating protein from the plant Ricinus communis, is a heterodimeric protein. The A chain is a N-glycosidase and the B chain (RTB) is a lectin with two carbohydrate binding sites. RTB has been shown to bind asialofetuin with much greater affinity than it does galactose, supporting the hypothesis that it may exhibit multivalency. To test this, neoglycoconjugates were prepared and tested for their ability to function as ligands for ricin binding. Because the two carbohydrate binding sites on RTB are approximately 70A apart, bovine serum albumin (BSA) was used as the carbohydrate carrier. It was derivatized with either the oligosaccharide portion of asialo-GM1 or with lactose. These sugars were used because ricin was found to adhere more effectively to asialo-GM1 and LacCer immobilized on plastic than to the other glycosphingolipids tested. Results of binding studies done using surface plasmon resonance indicated that the RTB subunit of ricin exhibited a multivalent effect when it bound to the neoglycoconjugates.     

The acute toxicity, tissue distribution, and histopathology of inhaled ricin in Sprague Dawley rats and BALB/c mice

Ricin is a highly toxic ribosome-inactivating protein derived from the castor bean (Ricinus communis). Due to the relative ease of producing ricin, it is characterized as a category B priority pathogen by the Center for Disease Control and Prevention. The purpose of this study was to compare the acute toxicity, associated histopathology, as well as the regional respiratory tract deposition and clearance kinetics of inhaled ricin in rats and mice using a single pure preparation. Acute toxicity was evaluated in five groups of six animals per species exposed nose-only to ricin aerosols and followed up to 7 days post-exposure. Tissues were collected for histopathology. The calculated median lethal doses (LD??s) were 0.24 μg/kg (rats) and 0.58 μg/kg (mice). Histological changes were noted in nose, larynges, trachea, lung, thymus, and spleen of both species. Pulmonary deposition in rats inhaling 94-99?ng/L ricin for 20 min (low dose) or 40?min (high dose) were 45.9 and 96?ng/g lung, respectively. Clearance was best described by a single-component negative exponential function. Estimated lung doses were 0.38 and 1.43 μg/g·h among the low and high dose rats, respectively. In mice inhaling 94 ng/L ricin for 20 min, pulmonary deposition was 91.1?ng/g lung and the estimated tissue dose was 1.72 μg/g·h. No ricin was detected in extra-respiratory tract tissue or in excreta. Results of this study demonstrate differences exist in pulmonary deposition, clearance rates, and tissue dose and histopathological changes between rats and mice inhaling ricin.     

Inhalation Toxicity of 1,6-Hexanediamine Dihydrochloride in F344/N Rats and B6C3F1 Mice

1,6-Hexanediamine (HDA) is a high production volume chemicalwhich is used as an intermediate in the synthesis of paints,resins, inks, and textiles and as a corrosion inhibitor in lubricants.Two- and 13-week studies of the toxicity of the dihydrochloridesalt of HDA (HDDC) were conducted in male and female Fischer344/N rats and B6C3F₁ mice using whole-body inhalation exposure.Both species were evaluated for histopatho-logic and reproductiveeffects, and rats were examined for clinical chemistry and hematologicchanges. In the 2-week inhalation studies, animals were exposedto 10–800 mg HDDC/m³, 6 hr per day. All rats, all femalemice, and two of five male mice in the high-exposure group diedbefore the end of the study. Surviving mice in this group hada dose-dependent depression in body weight gain. Clinical signswere primarily related to upper respiratory tract irritationand included dyspnea and nasal discharge in both species. Treatment-relatedhistopathologic lesions included inflammation and necrosis ofthe laryngeal epithelium of both species and the tracheal epitheliumof mice, as well as focal inflammation and ulceration of therespiratory and olfactory nasal mucosa. In the 13-week inhalationstudies, animals were exposed to HDDC at concentrations of 1.6–160mg/ m³ for 6 hr per day, 5 days per week. In addition to thebase study groups, a supplemental group of rats at each exposurelevel was included to assess the effect of HDDC on reproduction.No treatment-related changes in organ weights or organ-to-body-weightratios occurred in rats, and no treatment-related clinical signsor gross lesions were seen in either species. Chemical-relatedmicroscopic lesions were limited to the upper respiratory tract(larynx and nasal passages) in the two highest exposure groupsand were similar in both species. These lesions included minimalto mild focal erosion, ulceration, inflammation, and hyperplasiaof the laryngeal epithelium, in addition to degeneration ofthe olfactory and respiratory nasal epithelium. HDDC causedno significant changes in sperm morphology or vaginal cytologyand no significant adverse effects on reproduction in rats ormice. Hematologic and clinical chemistry changes in rats wereminor and sporadic and were not accompanied by related histologicfindings. HDDC did not increase the frequency of micronucleatederythrocytes in mice. In summary, the toxicity of HDDC to ratsand mice was a result of the irritant properties of the chemical,was limited primarily to the nasal passages and upper airways,and was consistent with the effects of other irritant chemicalsadministered by inhalation.     

Translocation of ricin across polarized human bronchial epithelial cells

Due to widespread availability, toxicity, and potential for use as a bioterrorism agent, ricin is classified as a category B select agent. While ricin can be internalized by a number of routes, inhalation is particularly problematic. The resulting damage leads to irreversible pulmonary edema and death. Our study describes a model system developed to investigate the effects of ricin on respiratory epithelium. Human bronchial epithelial (HBE) cells were cultured on collagen IV-coated inserts until polarized epithelial cell monolayers developed. Ricin was added to the apical or basal medium and damage to the cell monolayer was then assessed. Within a few hours after exposure, the cell monolayer was permeable to paracellular passage of the toxin. A mouse anti-ricin antibody neutralized ricin and prevented cellular damage as long as the antibody was present before the addition of toxin. These studies suggested that effective therapeutic agents or antibodies neutralizing ricin biological activity must be present at the apical surface of epithelial cells. The in vitro system developed here provides a method by which to screen potential therapeutics for protecting lung epithelial cells against ricin intoxication.     

Impact of inhalation exposure modality and particle size on the respiratory deposition of ricin in BALB/c mice

Ricin is a toxic lectin derived from the seed of Ricinus communis (castor plant). It is lethal in small quantities when disseminated as an aerosol. We determined the impact of using two types of exposure chambers and different particle sizes on the deposition of ricin aerosols in mice. Initially, two types of inhalation exposure chambers (whole-body [WB] or nose-only [NO]) were compared using the same size aerosol (1 micro m) to determine the potential impact upon respiratory deposition and presented dose. We then assessed the role of particle size on deposition by using aerosols with two distinctly sized particle distributions. Selected organs were collected at four time points after exposure and were analyzed by quantitative enyzme-linked immunosorbent assay (ELISA) and epifluorescence microscopy. Results of the exposure chamber comparison, using 1- micro m particles only, indicated approximately 50% of the total ricin in the 4 organs was detected in the lung tissue 1 h after exposure. The trachea and nasopharyngeal region of the animals exposed using the WB chamber contained significantly more ricin than those of animals exposed in the NO chamber. Histopathology indicated an accumulation of ricin in both the tracheobronchial and pulmonary regions with pronounced bronchiolar degradation 48 h postexposure. When particles larger than 3 micro m were used, results indicated a considerable amount of ricin initially detected in the trachea, although this finding was discounted due to the heterodispersity of the particles generated. Interestingly, no animals died as a result of exposure to the equivalent of 4 LD50s (as determined using a 1- micro m particle) when exposed to the larger size distribution of particles. This result indicates a differential lethality that is contingent upon aerosol size.     

Comparison between the mechanisms of action of plant toxins ricin and viscumin on the stage of intracellular dissociation

Pharmacological effects of mistletoe extracts are determined by the concentration of three toxic lectins: mistletoe lectin I (MLI, or viscumin), MLII, MLIII. These proteins, as well as ricin, belong to ribosome-inactivating proteins type 2 (RIP2). However, the extracts from the plant Ricinus communis, containing ricin, are highly toxic. Ricin is about 30 times more effective in cell culture than viscumin. The dissociation of subunits and the transmembrane transport of catalytic subunit into the cytoplasm are needed to obtain the cytotoxic effect of RIP2. In this paper, hybridomas producing monoclonal antibodies against catalytic subunits of ricin and viscumin are described. Monoclonal antibodies against different epitopes, including one localized in intra-subunit area of catalytic subunits of ricin and viscumin, do not inhibit the enzymatic activity of these proteins in cell-free system. These hybridomas are resistant to the cytotoxic action of native toxins. Protective effect of antibodies are about the same for both toxins, though the dissociation of the subunits of ricin is more effective. The causes of the differences in activity of plant toxins as pharmacological agents, and the importance of above mentioned epitopes for neutralizing antibodies at the clinical applications of mistletoe extracts are discussed.     

The use of anti-ricin antibodies to protect mice intoxicated with ricin

Antibodies raised in rabbits to ricin or its constituent polypeptide chains are effective at rescuing mice from ricin intoxication if given soon enough after administration of the toxin. The maximum safe period for intravenous injection of 100 micrograms antibody is 40 min after intravenous injection of 1 microgram ricin, increasing to 640 min in the case of subcutaneously injected ricin. The antibodies raised against the isolated A and B chains are as effective as those against whole ricin. The antibody is as effective when administered intracerebrally as by the intravenous route; this result, combined with the very high toxicity for ricin administered intracerebrally suggests a neurotoxic role for the lectin.     

Inhalation toxicology of ricin preparations: animal models, prophylactic and therapeutic approaches to protection

Ricin is a toxin and seed protein produced by the castor oil plant, Ricinus communis. The toxin is a dimeric protein consisting of an enzymic A chain and a B chain with lectin properties aiding the uptake of the whole molecule into cells. Ricin has been considered a possible military threat for several decades and is now also of some concern as a terrorist agent. The inhalation route is of primary concern in these situations, although previous attacks with ricin have used other approaches. Medical countermeasures against ricin are urgently required and the strategy adopted has been first to understand the nature of the problem, in this case the inhalation toxicology of ricin, followed by the preparation of vaccine antigens. Toxoided ricin and modified recombinant A chain components have been examined in terms of efficacy as potential vaccine candidates in protection of animal models against inhaled ricin, primarily in laboratories both in the United Kingdom and in the United States. One recombinant A chain vaccine has been taken through to clinical trials in the United States and should become commercially available in the next few years. Toxoided ricin has also been used as an antigen to prepare antitoxin antibodies for therapeutic treatment following poisoning. In this review, a synopsis of the inhalation toxicology of ricin and approaches to medical prophylaxis and therapy of poisoning is given, based on work conducted at our laboratory and at other research institutes.     

Conformation similarities of ricin A-chain and trichosanthin

The conformation of ricin A-chain from castor bean was studied by circular dichroism at pH 4.7, 7 and 9 and compared with that of trichosanthin from the Chinese herb Tianhuafen. The CD spectra of ricin A-chain and trichosanthin were nearly identical at each of the three pHs. Analysis of the data indicated that, like trichosanthin, ricin A-chain had about 29%α-helix and 42%β-sheet but no β-turn. However, there was a subtle difference in the CD spectra in 20 mm sodium dodecyl sulfate, the addition of which at pH 7 slightly increased the helicity and decreased the content of β-sheet of ricin A-chain in contrast to a larger increase in helicity at the expense of β-sheet for trichosanthin, thus indicating a different stability against the surfactant. Native ricin A-chain and trichosanthin had about the same amount of secondary structure, which supports the belief that a high degree of sequence homology of the two proteins [Zhang & Wang (1986) Nature 321, 477–478] may lead to a conformational similarity between them, even though the two proteins are not taxonomically related.     

重组人干扰素α2b注射液治疗儿童急性上呼吸道感染的临床效果及药理机制

目的 探究重组人干扰素α2b注射液治疗儿童急性上呼吸道感染的临床效果及药理机制.方法 66例急性上呼吸道感染患者,随机分为治疗组和对照组,各33例.两组患儿均给予常规综合性治疗,治疗组在常规综合性基础上给予重组人干扰素α2b注射液.比较两组治疗效果及临床症状消失时间.结果 治疗组治疗总有效率93.9％高于对照组的75....     

Transfer of ricin toxicity by spleen cells

The ability of spleen cells to internalise ricin and release it in a form capable of killing an untreated cell population has been studied. Potential donor cells were incubated with a range of ricin concentrations and the amount of ricin subsequently released was found to be related to the ricin concentration of the primary incubation. Ricin release was detected either by using radiolabelled toxin or by measuring its cytotoxic effects. Cells which, after incubation with ricin, were washed with lactose or anti-ricin IgG had a slightly reduced ability to transfer the toxin to other cells, suggesting that much of the ricin had been internalised by the donor cells. The presence of 100 mM lactose in the incubation medium was unable to inhibit the uptake of ricin and did not prevent the release of competent toxin. Cells carrying the toxic material were lethal to animals and in vitro the released toxin was as susceptible to inactivation by anti-ricin IgG as was free ricin.     

Quantification of ricinine in rat and human urine: a biomarker for ricin exposure

Ricin is a toxalbumin derived from the castor bean plant, Ricinus communis. Ricinine is an alkaloid (3-cyano-4-methoxy-N-methyl-2-pyridone) that shares a common plant source with ricin, and its presence in urine infers ricin exposure. A new quantification method for ricinine was developed that uses solid-phase extraction to prepare 1-mL urine samples (81% recovery) for a 5-min, isocratic high-performance liquid chromatography method, followed by electrospray ionization tandem mass spectrometry. Protonated molecular ions were selected in the multiple reaction monitoring mode and quantified by isotope dilution with (13)C(6)-labelled ricinine as the internal reference. Urine pools enriched with ricinine at two concentrations were characterized as quality control materials and then used to validate the method. The method limit of quantification was 0.083 ng/mL, even with a confirmation ion of low relative abundance. Ricinine was stable in human urine when heated at 90 degrees C for 1 h, and during storage at 25 degrees C and 5 degrees C for 3 weeks. The method was applied to an animal exposure study, a crude ricin preparation scheme, and a forensic analysis. These studies show that ricinine can be measured in rat urine at least 48 h after exposure. Ricinine is present in crude preparations of ricin, and it can be found in human urine after a lethal exposure to ricin.