Targeting myeloid leukemia with a DT390-mIL-3 fusion immunotoxin: ex vivo and in vivo studies in mice

The IL-3 receptor was expressed on a high frequency of myeloidleukemia cells and also on hematopoietic and vascular cells.We previously showed that a recombinant IL-3 fusion immunotoxin(DT₃₉₀IL-3) expressed by splicing the murine IL-3 gene to atruncated diphtheria toxin (DT₃₉₀) gene selectively killed IL-3R⁺expressing cells and was not uniformly toxic to uncommited BMprogenitor cells (Chan,C.-H., Blazar,B.R., Greenfield,L., Kreitman,R.J.and Vallera,D.A., 1996, Blood, 88, 1445–1456). Thus, weexplored the feasability of using DT₃₉₀IL-3 as an anti-leukemiaagent. DT₃₉₀IL-3 was toxic when administered to mice at dosesas low as 0.1 µg/day. The dose limiting toxicity appearedto be related to platelet and bleeding effects of the fusiontoxin. Because of these effects, DT₃₉₀IL-3 was studied ex vivoas a means of purging contaminating leukemia cells from BM graftsin a murine autologous BM transplantation. In this setting,as few as 1000 IL-3R-expressing, bcr/abl transformed myeloid32Dp210 leukemia cells were lethal. An optimal purging intervalof 10 nM/l for 8 h eliminated leukemia cells from 32Dp210/BMmixtures given to lethally irradiated (8 Gy) C3H/HeJ syngeneicmice. Mice given treated grafts containing BM and a lethal doseof 32Dp210 cells survived over 100 days while mice given untreatedgrafts did not survive (P < 0.00001). DT₃₉₀IL-3 may provehighly useful for ex vivo purging of lethal malignant leukemiacells from autologous BM grafts.     

A fold-back single-chain diabody format enhances the bioactivity of an anti-monkey CD3 recombinant diphtheria toxin-based immunotoxin

T-cell depleting anti-CD3 immunotoxins have utility in non-human primate models of transplantation tolerance and autoimmune disease therapy. We recently reported that an affinity matured single-chain (scFv) anti-monkey CD3 antibody, C207, had increased binding to T-cells and increased bioactivity in a diphtheria toxin (DT)-based biscFv immunotoxin compared with the parental antibody, FN18. However, FN18 scFvs and their mutant derivatives such as C207 did not exhibit robust bivalent character in the biscFv format. We now report that C207 in a diabody format exhibits a 7-fold increase in binding to T-cells over scFv (C207) indicating considerable divalent character for the diabody. This construct was formed by reducing the V(L)/V(H) linker to five residues and was secreted from Pichia pastoris as the non-covalent dimer. An immunotoxin based on this diabody format was secreted as a non-covalent dimer but was devoid of bioactivity and failed to bind T-cells, suggesting steric hindrance from the two large closely positioned truncated DT moieties. We constructed a single-chain diabody immunotoxin by fusing to the truncated DT C-terminus L1-VL-L1-VH-L2-VL-L1-VH where L1 is a five-residue linker and L2 is the longer (G4S)3 linker permitting interactions between the distal and proximal VL/VH domains. This 'fold-back' immunotoxin was secreted predominantly as the monomer and exhibited a 5- to 7-fold increase in bioactivity over DT390biscFv(C207) and depleted monkey T-cells in vivo.     

重组DT/bFGF融合蛋白基因表达载体的构建及表达产物的纯化

目的构建重组DT/bFGF融合蛋白表达载体,制备高纯度的DLF融合毒素。方法PCR扩增DT389和bFGF的编码DNA序列,经酶切和连接,克隆至表达载体pET-30a,转化E.coliBL21(DE3),鉴定阳性克隆菌落,经IPTG诱导表达融合蛋白DLF,镍离子螯合层析纯化,用Westernblot分析其抗原性。结果测序表明,插入片段可编码含545个氨基酸残基的融合蛋白DLF。SDS-PAGE分析表明,该融合蛋白可在大肠杆菌中表达,其表达量约占菌体总蛋白的20%,表达形式主要为包涵体。纯化后纯度达90%以上。Westernblot证实,该融合蛋白同时具有DT和bFGF两种抗原性。结论已成功构建DT/bF-GF表达载体,并获得较纯的DLF融合毒素。     

An improved linker for single-chain Fv with reduced aggregation and enhanced proteolytic stability

The effects of linker length on binding affinity and degreeof aggregation have been examined in the antifluorescein 4-4-20and anticarcinoma CC49 single-chain Fvs. Longer linkers in theantifluorescein sFvs have higher affinities for fluoresceinand aggregate less. A proteolytically susceptible site betweenLys8 and Ser9, in the previously reported 212 linker has beenidentified. A new linker sequence, 218 (GSTSGSGKPGSGEGSTKG)was designed in which a praline was placed at the C-terminalside of the proteolytic clip site in the 212 linker. The CC49sFv containing the 218 linker showed reduced aggregation andwas found to be more stable to proteolysis in vitro, when comparedto the CC49/212 sFv. The CC49 sFv with the longer 218 linkerhad higher affinity than CC49/212 sFv. An aggregated CC49/212sFv sample had higher affinity than CC49/218 sFv. The CC49/218and CC49/212 sFvs had similar blood clearances in mice, whilethe aggregated CC49/212 sFv remained in circulation significantlylonger. In mice bearing LS-174T human colon carcinoma xenografts,the CC49/218 sFv showed higher tumor uptake than the CC49/212sFv and lower tumor uptake than the aggregated CC49/212 sFv.The higher tumor uptake of the CC49/218 is most likely a resultof its higher resistance to proteolysis. The higher affinityand higher tumor uptake of the aggregated CC49/212 sFv are mostlikely due to the repetitive nature of the TAG-72 antigen andthe higher avidity of multivalent aggregates. When the sFvswere radiolabeled with a lutetium-chelate the CC49/218 sFv showeda lower accumulation in the liver and spleen compared to theaggregated CC49/212 sFv.     

EspA-Stx2A1融合蛋白的表达、纯化及其免疫学活性

目的在原核表达系统中表达肠出血性大肠杆菌O157∶H7(EHECO157∶H7)Ⅲ型分泌蛋白EspA与Stx2毒素A1亚单位(Stx2A1)的融合蛋白,并对表达蛋白进行纯化及免疫学活性检测。方法PCR扩增espA和stx2A1全长基因,利用重叠延伸PCR技术获得espA-stx2A1融合基因,T-A克隆后插入表达载体pET-28a(+),构建原核表达质粒pET-28a(+)-espA-stx2A1,转化大肠杆菌BL21(DE3),分别在37℃和25℃用IPTG诱导表达。以EspA单克隆抗体亲和层析柱纯化目的蛋白,免疫小鼠,检测其免疫原性及抗血清的反应原性,并以天然Stx2毒素攻击,观察保护效果。通过细胞毒试验检测免疫小鼠抗血清的体外中和作用。结果重叠延伸PCR方法扩增出1319bp的融合基因片段,重组表达质粒构建正确;目的蛋白在25℃时的表达量明显高于37℃,约占菌体总蛋白的40%。两种温度下,目的蛋白均主要以包涵体形式存在;纯化后蛋白纯度可达90%。Western blot结果证实,融合蛋白与EspA单克隆抗体和Stx2A1单克隆抗体均发生特异性反应。融合蛋白免疫小鼠制备的抗血清能分别与O157∶H7的EspA、Stx2A发生特异性免疫反应。融合蛋白免疫小鼠能够抵御致死剂量天然Stx2毒素的攻击,保护率达95%。免疫小鼠血清可以中和天然Stx2毒素对HeLa细胞的毒性作用。结论已成功表达了EspA-Stx2A1融合蛋白,纯化的蛋白显示出较好的免疫保护效果,为研制EHECO157∶H7基因工程多亚单位疫苗奠定了基础。     

A bispecific immunotoxin (DTAT13) targeting human IL-13 receptor (IL-13R) and urokinase-type plasminogen activator receptor (uPAR) in a mouse xenograft model

A bispecific immunotoxin (IT) called DTAT13 was synthesized in order to target simultaneously the urokinase-type plasminogen activator receptor (uPAR)-expressing tumor neovasculature and IL-13 receptor expressing glioblastoma cells with the goal of intratumoral administration for brain tumors. The recombinant hybrid was created using the non-internalizing N-terminal fragment (ATF) of uPA and the IL-13 molecule for binding plus the catalytic and translocation portion of diphtheria toxin (DT) for killing. The 71 kDa protein was highly selective for human glioblastoma in vitro showing no loss on binding compared with DTAT and DTIL13 controls. In vivo, DTAT13 caused the regression of small tumors when administered at 10 micro g/day given on a five-dose schedule every other day. DTAT13 was able to target both overexpressed uPAR and the vasculature, as demonstrated by its ability to kill HUVEC cells. Also, mortality studies indicated that DTAT13 was less toxic than DTAT or DTIL13. These findings indicate that bispecific IT may allow treatment of a broader subset of antigenically diverse patients while simultaneously reducing the exposure to toxin required than if two separate agents were employed.     

Effects of a Fully Humanized Type II Anti-CD20 Monoclonal Antibody on Peripheral and CNS B Cells in a Transgenic Mouse Model of Multiple Sclerosis

Successful therapy with anti-CD20 monoclonal antibodies (mAbs) has reinforced the key role of B cells in the immunopathology of multiple sclerosis (MS). This study aimed to determine the effects of a novel class of anti-CD20 mAbs on vascular and extravascular central nervous system (CNS)-infiltrating B cells in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Male hCD20xhIgR3 mice and wild-type C57BL/6 (B6) mice were immunized with human myelin oligodendrocyte glycoprotein (MOG)_1–125 to induce EAE. While hCD20xhIgR3 mice were injected intravenously with an anti-human CD20 mAb (5 mg/kg) (rituximab (a type I anti-CD20 mAb) or obinutuzumab (a type II anti-CD20 mAb), B6 mice received the anti-mouse CD20 antibody 18B12. Neither mAb affected clinical disease or serum antibody levels. Obinutuzumab and rituximab had an impact on splenic and CNS-infiltrated B cells with slightly differential depletion efficacy. Additionally, obinutuzumab had beneficial effects on spinal cord myelination. B cell depletion rates in the 18B12/B6 model were comparable with those observed in obinutuzumab-treated hCD20xhIgR3 mice. Our results demonstrate the usefulness of anti-CD20 mAbs for the modulation of B cell-driven peripheral immune response and CNS pathology, with type II antibodies potentially being superior to type I in the depletion of tissue-infiltrating B cells.     

Targeting glioblastoma multiforme with an IL-13/diphtheria toxin fusion protein in vitro and in vivo in nude mice

Fusion proteins composed of tumor binding agents and potentcatalytic toxins show promise for intracranial therapy of braincancer and an advantage over systemic therapy. Glioblastomamultiforme (GBM) is the most common form of brain cancer andoverexpresses IL-13R. Thus, we developed an interleukin-13 receptortargeting fusion protein, DT₃₉₀IL13, composed of human interleukin-13and the first 389 amino acids of diphtheria toxin. To measureits ability to inhibit GBM, DT₃₉₀IL13 was tested in vitro andfound to inhibit selectively the U373 MG GBM cell line withan IC₅₀ around 12 pmol/l. Cytotoxicity was neutralized by anti-human-interleukin-13antibody, but not by control antibodies. In vivo, small U373MG glioblastoma xenografts in nude mice completely regressedin most animals after five intratumoral injections of 1 µgof DT₃₉₀IL13 q.o.d., but not by the control fusion protein DT₃₉₀IL-2.DT₃₉₀IL13 was also tested against primary explant GBM cellsof a patient's excised tumor and the IC₅₀ was similar to thatmeasured for U373 MG. Further studies showed a therapeutic windowfor DT₃₉₀IL13 of 1–30 µg/injection and histologystudies and enzyme measurements showed that the maximum tolerateddose of DT₃₉₀IL13 had little effect on kidney, liver, spleen,lung and heart in non-tumor-bearing immunocompetent mice. Together,these data suggest that DT₃₉₀IL13 may provide an important,alternative therapy for brain cancer.     

A new yeast display vector permitting free scFv amino termini can augment ligand binding affinities

Yeast surface display and sorting by flow cytometry are now widely used to direct the evolution of protein binding such as single-chain antibodies or scFvs. The available commercial yeast display vector pYD1 (Invitrogen) displays the protein of interest flanked on the N-terminus by Aga2, the disulfide of which binds the myristylated surface membrane protein Aga1. We have noted that two anti-CD3epsilon scFvs expressed as fusion proteins suffer a 30- to 100-fold loss of affinity when placed NH(2) terminal to either truncated toxins or human serum albumin. In the course of affinity maturing one of these scFv (FN18) using pYD1 we noted that the affinity towards the ectodomain of monkey CD3epsilongamma was too low to measure. Consequently we rebuilt pYD1 tethering the scFv off the NH(2) terminus of Aga2. This display vector, pYD5, now gave a positive signal displaying FN18 scFv with its ligand, monkey CD3epsilongamma. The apparent equilibrium association constant of the higher affinity scFv directed at human CD3epsilongamma increased approximately 3-fold when displayed on pYD5 compared with pYD1. These data show that for certain yeast-displayed scFvs a carboxy-tethered scFv can result in increased ligand-scFv equilibrium association constants and thereby extend the low range of affinity maturation measurements.     

Direct T-2 Toxicity on Human Skin—Fibroblast Hs68 Cell Line—In Vitro Study

T-2 toxin is produced by different Fusarium species, and it can infect crops such as wheat, barley, and corn. It is known that the T-2 toxin induces various forms of toxicity such as hepatotoxicity, nephrotoxicity, immunotoxicity, and neurotoxicity. In addition, T-2 toxin possesses a strong dermal irritation effect and can be absorbed even through intact skin. As a dermal irritant agent, it is estimated to be 400 times more toxic than sulfur mustard. Toxic effects can include redness, blistering, and necrosis, but the molecular mechanism of these effects still remains unknown. This in vitro study focused on the direct toxicity of T-2 toxin on human skin—fibroblast Hs68 cell line. As a result, the level of toxicity of T-2 toxin and its cytotoxic mechanism of action was determined. In cytotoxicity assays, the dose and time-dependent cytotoxic effect of T-2 on a cell line was observed. Bioluminometry results showed that relative levels of ATP in treated cells were decreased. Further analysis of the toxin’s impact on the induction of apoptosis and necrosis processes showed the significant predominance of PI-stained cells, lack of caspase 3/7 activity, and increased concentration of released Human Cytokeratin 18 in treated cells, which indicates the necrosis process. In conclusion, the results of an in vitro human skin fibroblast model revealed for the first time that the T-2 toxin induces necrosis as a toxicity effect. These results provide new insight into the toxic T-2 mechanism on the skin.     

The result of feeding palmitoyl glycerol on lymph and plasma lipids

Palmitoyl glycerol is toxic when fed to mice, but the toxicity is alleviated by supplementing the toxic diet with 2–4% oleate or linoleate at the expense of sucrose. Lipid and fatty acid composition of lymph and plasma were studied in mice fed chow and palmitoyl glycerol diets to help explain the toxicity mechanism. When mice were fed chow, intestinal lymph contained a high proportion of saturated fatty acids; when they were given palmitoyl glycerol, the proportion approached 90% saturated fatty acids. The cholesteryl ester fraction was higher in lymph from mice fed a toxic diet than when the diet was fortified with supplemental safflower oil. However, there were no differences between diets in lipid composition of blood plasma. Similarly, except for plasma cholesterol esters, there were no differences in fatty acid composition between mice fed palmitoyl glycerol as the only fat or supplemented with a protective unsaturated fat. In the plasma, cholesteryl palmitate was elevated and cholesteryl oleate and cholesteryl linoleate were depressed when mice were given a toxic diet. Although a monoacylglycerol was toxic when fed, the percentages of monoacylglycerols in lymph or plasma were not materially elevated. The findings indicate that neither the total proportion of saturated fatty acids nor the amount of circulating monoacylglycerols was directly involved in the toxicity of palmitoyl glycerol.     

Comparative Effects of Deferiprone and Salinomycin on Lead-Induced Disturbance in the Homeostasis of Intrarenal Essential Elements in Mice

Lead (Pb) exposure induces severe nephrotoxic effects in humans and animals. Herein, we compare the effects of two chelating agents, salinomycin and deferiprone, on Pb-induced renal alterations in mice and in the homeostasis of essential elements. Adult male mice (Institute of Cancer Research (ICR)) were randomized into four groups: control (Ctrl)—untreated mice administered distilled water for 28 days; Pb-exposed group (Pb)—mice administered orally an average daily dose of 80 mg/kg body weight (BW) lead (II) nitrate (Pb(NO₃)₂) during the first two weeks of the experimental protocol followed by the administration of distilled water for another two weeks; salinomycin-treated (Pb + Sal) group—Pb-exposed mice, administered an average daily dose of 16 mg/kg BW salinomycin for two weeks; deferiprone-treated (Pb + Def) group—Pb-exposed mice, administered an average daily dose of 20 mg/kg BW deferiprone for 14 days. The exposure of mice to Pb induced significant accumulation of the toxic metal in the kidneys and elicited inflammation with leukocyte infiltrations near the glomerulus. Biochemical analysis of the sera revealed that Pb significantly altered the renal function markers. Pb-induced renal toxicity was accompanied by a significant decrease in the endogenous renal concentrations of phosphorous (P), calcium (Ca), copper (Cu) and selenium (Se). In contrast to deferiprone, salinomycin significantly improved renal morphology in Pb-treated mice and decreased the Pb content by 13.62% compared to the Pb-exposed group. There was also a mild decrease in the renal endogenous concentration of magnesium (Mg) and elevation of the renal concentration of iron (Fe) in the salinomycin-treated group compared to controls. Overall, the results demonstrated that salinomycin is a more effective chelating agent for the treatment of Pb-induced alterations in renal morphology compared to deferiprone.     

Acute and chronic nephrotoxicity of platinum nanoparticles in mice

Platinum nanoparticles are being utilized in various industrial applications, including in catalysis, cosmetics, and dietary supplements. Although reducing the size of the nanoparticles improves the physicochemical properties and provides useful performance characteristics, the safety of the material remains a major concern. The aim of the present study was to evaluate the biological effects of platinum particles less than 1 nm in size (snPt1). In mice administered with a single intravenous dose of snPt1, histological analysis revealed necrosis of tubular epithelial cells and urinary casts in the kidney, without obvious toxic effects in the lung, spleen, and heart. These mice exhibited dose-dependent elevation of blood urea nitrogen, an indicator of kidney damage. Direct application of snPt1 to in vitro cultures of renal cells induced significant cytotoxicity. In mice administered for 4 weeks with twice-weekly intraperitoneal snPt1, histological analysis of the kidney revealed urinary casts, tubular atrophy, and inflammatory cell accumulation. Notably, these toxic effects were not observed in mice injected with 8-nm platinum particles, either by single- or multiple-dose administration. Our findings suggest that exposure to platinum particles of less than 1 nm in size may induce nephrotoxicity and disrupt some kidney functions. However, this toxicity may be reduced by increasing the nanoparticle size.     

Single-chain Fv with manifold N-glycans as bifunctional scaffolds for immunomolecules

Unlike natural antibodies, single-chain Fv (sFv) proteins normally lack asparagine-linked glycosylation. Many designed immunoconjugates and other therapeutics currently employ the advantageous conjugation chemistry or targeting properties provided by the glycoprotein oligosaccharide domain. sFv proteins with engineered N-glycan designs were evaluated in Pichia pastoris for glycosylation efficiency, expression level, oligosaccharide chain length and composition, and affinity. In contrast to nearly all natural glycoproteins, the engineered attachment of N-glycans conveniently near the polypeptide C- terminus was found to produce the optimal results. Furthermore, the percentage modification and chain length of the attached mannose chains were controllable by the use of tandem and overlapping Asn-X-Thr tripeptide sites. The glycosylated sFv mannose chains could be effectively conjugated to polyethylene glycol and the resulting conjugate displayed a 10-fold increased circulating life in mice. The potential to control polymer:sFv or drug:sFv molar ratios by site- specific conjugation may substantially improve the therapeutic efficacy of these minimal antigen-binding molecules.      

破伤风毒素及其AB片段对金鱼的毒力

作者研究了破伤风毒素及其 AB 片段对金鱼和小鼠的毒力,发现16ng 的毒素即可使金鱼麻痹和死亡;26μg 的 AB 片段即可使小鼠麻痹和死亡,而对金鱼却无作用。当抗 C 或 AB 片段的抗体与毒素结合后,可阻断毒素对金鱼的致麻痹作用。上述结果表明,破伤风毒素可作用于金鱼的外周神经系统,而 AB 片段则不能。毒素与其受体结合是毒素对金鱼作用所必需的。     

破伤风毒素的提纯及形成离子通道的特性

应用硫酸铵分段沉淀和 Ultrogei AcA_(34)凝胶过滤对破伤风毒进行了提纯,并对形成的离子通道在中毒作用中的功能进行了讨论。提纯的毒素与抗粗制毒素血清形成一条沉淀线,于聚丙烯酰胺凝胶电泳中形成单一的蛋白带,分子量为15万,等电点为5.95,毒力为1～3×10~4MLD/μg 蛋白。给小鼠注射低剂量毒素引起迟发性强直性痉挛;注射高剂量毒素引起快速的类肉毒毒素的松弛性麻痹,直至死亡。应用 Patch clamp 技术,破伤风毒素于人工磷脂膜上形成离子通道。只有当膜两侧存有 pH 梯度时毒素才形成离子通道,因此是 pH 依赖的。通道的电导系数为9 PS。     

Effects of metallic coating on the combustion toxicity of engineering plastics

Experiments were conducted on 34 plastic materials having a variety of metallic coatings to determine the toxicity of their thermal decomposition products. Mice were exposed for 30 min in a dome exposure chamber to the products obtained by ramp-heating the samples from 200°C to 800°C. An LC₅₀ value was obtained for each material. Postmortem examinations were conducted on all dead mice, and on survivors after 14 days, to determine the gross pathological effects of exposure; particular attention was devoted to pulmonary pathology. The exposure protocol chosen has been extensively criticized, but it is very useful to study the effects of stress on mice, which was the most important part of this work. Experiments were made involving unrestrained mice in groups of four, restrained mice in groups of four and unrestrained single mice. The LC₅₀ values for single unrestrained mice were greater, by factors of 2–3, than those for four restrained mice, with the differences being shown to be statistically significant. This suggests that stress on the test animals will tend to reduce the LC₅₀ values in bench-scale smoke toxicity tests. The LC₅₀ values for all of the materials tested were equal to or higher than the value of 8 mg1^?1 representative of the contribution of carbon monoxide to post-flashover fires. Moreover, no ‘supertoxicants’ were found in the smoke of any of the materials tested. Finally, the coatings did not adversely affect the smoke toxicity of the substrate materials by a factor higher than 2–3 in any of the cases investigated. Uncoated polyethylene was the most toxic substrate material tested (LC₅₀ = 16 mgl^?1) and uncoated NORYL® resin was the least toxic (LC₅₀ = 91mgl^?1). Metallic coatings involving Cu, Ni, graphite, and Zn typically had no statistically significant effect on the smoke toxicity of the substrate materials, although Ni coatings increased the smoke toxicity of ABS I and of white polycarbonate structural foam, by factors of 2–3. Overall smoke toxicities were well correlated with production of carbon monoxide (r=0.84) and carbon dioxide (r=0.82); oxygen levels and chamber temperature did not vary beyond acceptable limits. The materials tested generating the more toxic smokes (including polyethlene, polystyrene, and several polycarbonates) produced severe lung damage at low concentrations. The LC₅₀ of these materials was also typically greater than predicted on the basis of CO production. Other materials (including several coating on NORYL® resin and Lexan® polycarbonate) produced pulmonary damage at higher concentrations amd had LC₅₀ values more closely correlated with CO production. None of the polyurethane materials tested produced severe lung damage at the concentrations employed.     

马蹄香对小鼠的急性毒性及对大鼠的亚急性毒性观察

目的观察马蹄香(Valeviana jatamansi Jones)对小鼠的急性毒性及对大鼠的亚急性毒性,以评价其安全性。方法给小鼠灌服5 000、7 500、10 000 mg/kg的马蹄香,对照组灌胃1%羧甲基纤维素钠(CMC),观察小鼠的中毒症状,记录小鼠的死亡数,采用Red-Munchen法计算半数致死量(LD50),并检查小鼠组织和脏器的病理变化;对大鼠分别灌服4 000、1 000和200 mg/kg的马蹄香,连续给药14 d,对照组灌胃CMC水溶液,给药后观察14 d,检测大鼠体重、脏器系数、血常规指标、血生化指标及组织病理变化情况。结果急性毒性试验结果显示,灌胃马蹄香后,小鼠短时间内出现活动减少,而后趋于正常,病理学检测未发现小鼠组织或脏器有明显异常改变,LD50>10 000 mg/kg;亚急性毒性试验结果显示,马蹄香各剂量组大鼠的增重、脏器系数、血常规指标、血生化指标及组织病理变化与对照组比较,差异均无统计学意义(P>0.05)。结论马蹄香具有较好的安全性。     

Chemistry and Toxicology of Major Bioactive Substances in Inocybe Mushrooms

Mushroom poisoning has always been a threat to human health. There are a large number of reports about ingestion of poisonous mushrooms every year around the world. It attracts the attention of researchers, especially in the aspects of toxin composition, toxic mechanism and toxin application in poisonous mushroom. Inocybe is a large genus of mushrooms and contains toxic substances including muscarine, psilocybin, psilocin, aeruginascin, lectins and baeocystin. In order to prevent and remedy mushroom poisoning, it is significant to clarify the toxic effects and mechanisms of these bioactive substances. In this review article, we summarize the chemistry, most known toxic effects and mechanisms of major toxic substances in Inocybe mushrooms, especially muscarine, psilocybin and psilocin. Their available toxicity data (different species, different administration routes) published formerly are also summarized. In addition, the treatment and medical application of these toxic substances in Inocybe mushrooms are also discussed. We hope that this review will help understanding of the chemistry and toxicology of Inocybe mushrooms as well as the potential clinical application of its bioactive substances to benefit human beings.     

Omega-3 Polyunsaturated Fatty Acid Attenuates Uremia-Induced Brain Damage in Mice

Although the cause of neurological disease in patients with chronic kidney disease (CKD) has not been completely identified yet, recent papers have identified accumulated uremic toxin as its main cause. Additionally, omega-3 polyunsaturated fatty acid (ω-3 PUFA) plays an important role in maintaining normal nerve function, but its protective effects against uremic toxin is unclear. The objective of this study was to identify brain damage caused by uremic toxicity and determine the protective effects of ω-3 PUFA against uremic toxin. We divided mice into the following groups: wild-type (wt) sham (n = 8), ω-3 PUFA sham (n = 8), Fat-1 sham (n = 8), ischemia-reperfusion (IR) (n = 20), and ω-3 PUFA+IR (n = 20) Fat-1+IR (n = 20). Brain tissue, kidney tissue, and blood were collected three days after the operation of mice (sham and IR operation). This study showed that Ki67 and neuronal nuclei (NeuN) decreased in the brain of uremic mice as compared to wt mice brain, but increased in the ω-3 PUFA-treated uremic mice and the brain of uremic Fat-1 mice as compared to the brain of uremic mice. The pro-apoptotic protein expressions were increased, whereas anti-apoptotic protein expression decreased in the brain of uremic mice as compared to wt mice brain. However, apoptotic protein expression decreased in the ω-3 PUFA-treated uremic mice and the brain of uremic Fat-1 mice as compared to the brain of uremic mice. Furthermore, the brain of ω-3 PUFA-treated uremic mice and uremic Fat-1 mice showed increased expression of p-PI3K, p-PDK1, and p-Akt as compared to the brain of uremic mice. We confirm that uremic toxin damages the brain and causes cell death. In these injuries, ω-3 PUFA plays an important role in neuroprotection through PI(3)K-Akt signaling.