Newly Isolated Animal Pathogen Corynebacterium silvaticum Is Cytotoxic to Human Epithelial Cells

Corynebacterium silvaticum is a newly identified animal pathogen of forest animals such as roe deer and wild boars. The species is closely related to the emerging human pathogen Corynebacterium ulcerans and the widely distributed animal pathogen Corynebacterium pseudotuberculosis. In this study, Corynebacterium silvaticum strain W25 was characterized with respect to its interaction with human cell lines. Microscopy, measurement of transepithelial electric resistance and cytotoxicity assays revealed detrimental effects of C. silvaticum to different human epithelial cell lines and to an invertebrate animal model, Galleria mellonella larvae, comparable to diphtheria toxin-secreting C. ulcerans. Furthermore, the results obtained may indicate a considerable zoonotic potential of this newly identified species.     

Bioactive IL7-diphtheria fusion toxin secreted by mammalian cells

A number of targeted cytotoxic agents have been developed that selectively kill malignant or otherwise pathological cells. These engineered proteins consist of a potent cytotoxic element connected to a ligand domain that binds to specific molecules on the surface of the target cell. Several of these agents have shown promise in clinical trials and one is currently administered to patients. A significant technical obstacle that has impeded the development of some of these toxins is the difficulty of preparing certain recombinant proteins in properly folded forms. These fusion proteins have generally been produced in bacteria requiring them to be denatured and renatured in vitro. For some proteins this is an efficient process whereas for others it is not. We describe here a system to produce fusion toxins rapidly and efficiently by engineering mammalian cells to secrete them as properly folded molecules which can be purified in native form from cell culture medium. We have used this system to produce highly active preparations of DAB(389)-IL7, a molecule consisting of the catalytic and transmembrane domains of diphtheria toxin fused to interleukin 7. This system is generalizable and can be used to produce and evaluate rapidly fusion toxins incorporating novel or uncharacterized ligands.     

Site-directed and deletion mutational analysis of the receptor binding domain of the interleukin-6 receptor targeted fusion toxin DAB389-IL-6

We have used site-directed and in-frame deletion mutationalanalysis in order to explore the structural features of theIL–6 portion of the diphtheria toxin-related interleukin–6(IL–6) fusion toxin DAB₃₈₉-IL–6 that are essentialfor receptorbinding and subsequent inhibition of protein synthesisin target cells. Deletion of the first 14 amino acids of theIL–6 component of the fusion toxin did not alter eitherreceptor binding affinity or cytotoxk potency. In contrast,both receptor binding and cytotoxic activity were abolishedwhen the C–terminal 30 amino acids of the fusion toxinwere deleted. In addition, we explored the relative role ofthe disulfide bridges within the IL–6 portion of DAB₃₈₉-IL–6in the stabilization of structure required for receptor-binding.The analysis of mutants in which the substitution of eitherCys⁴⁴⁰, Cys⁴⁴⁶, Cys⁴⁶⁹ or Cys⁴⁷⁹ to Ser respectively, demonstratesthat only the disulfide bridge between Cys⁴⁶⁹ and Cys⁴⁷⁹ isrequired to maintain a functional receptor binding domain. Inaddition, the internal in-frame deletion of residues 435–451,which includes Cys⁴⁴⁰ and Cys⁴⁴⁶, was found to reduce, but notabolish receptor binding affinity. These results further demonstratethat the disulfide bridge between Cys⁴⁴⁰ and Cys⁴⁴⁶ is not essentialfor receptor-binding. However, the reduced cytotoxic potencyof DAB₃₈₉-IL6(435–451) suggests that the conformationand/or receptor binding sites associated with this region ofthe fusion toxin is/are important for maintaining the wild typereceptor binding affinity and cytotoxic potency.     

Failure of Diphtheria Toxin Model to Induce Parkinson-Like Behavior in Mice

Rodent models of Parkinson’s disease are based on transgenic expression of mutant synuclein, deletion of PD genes, injections of MPTP or rotenone, or seeding of synuclein fibrils. The models show histopathologic features of PD such as Lewi bodies but mostly only subtle in vivo manifestations or systemic toxicity. The models only partly mimic a predominant loss of dopaminergic neurons in the substantia nigra. We therefore generated mice that express the transgenic diphtheria toxin receptor (DTR) specifically in DA neurons by crossing DAT-Cre mice with Rosa26 loxP-STOP-loxP DTR mice. After defining a well-tolerated DTx dose, DAT-DTR and DTR-flfl controls were subjected to non-toxic DTx treatment (5 × 100 pg/g) and subsequent histology and behavioral tests. DAT protein levels were reduced in the midbrain, and tyrosine hydroxylase-positive neurons were reduced in the substantia nigra, whereas the pan-neuronal marker NeuN was not affected. Despite the promising histologic results, there was no difference in motor function tests or open field behavior. These are tests in which double mutant Pink1^−/−SNCA^A53T Parkinson mice show behavioral abnormalities. Higher doses of DTx were toxic in both groups. The data suggest that DTx treatment in mice with Cre/loxP-driven DAT-DTR expression leads to partial ablation of DA-neurons but without PD-reminiscent behavioral correlates.     

Phe496 and Leu497 are essential for receptor binding and cytotoxic action of the murine interleukin-4 receptor targeted fusion toxin DAB389-mIL-4

DAB₃₈₉-mIL-4 is a murine interleukin-4 (mIL-4) diphtheria toxin-relatedfusion protein which has been shown to be selectively toxicto cells expressing the mIL-4 receptor. In this report, we haveused site-directed and in-frame deletion mutagenesis to studythe role of the putative C-terminal -helix (helix E) of themIL-4 component of DAB₃₈₉-mIL-4 in the intoxication process.We demonstrate that deletion of the C-terminal 15 amino acidsof the fusion toxin leads to loss of cytotoxicity. The substitutionof Phe496 with either Pro, Ala or Tyr, results in a > 20-folddecrease in cytotoxic activity of the respective mutant fusiontoxins. In addition, substitution of Leu497 with either Alaor Glu results in a similar loss of cytotoxic activity. Allof these mutant forms of the mIL-4 fusion toxin demonstratea significant decrease in binding affinity (K_i) to the mIL-4receptor in a competitive radioligand binding assay. In markedcontrast, however, the substitution of Asp495 with Asn resultsin a 4-fold increase in cytotoxic potency and binding affinityto mIL-4 receptor bearing cells in vitro.     

Diphtheria toxin receptor-binding domain substitution with interleukin 6: genetic construction and interleukin 6 receptor-specific action of a diphtheria toxin-related interleukin 6 fusion protein

We have genetically replaced that portion of the diphtheriatoxin structural gene which encodes the native receptor-bindingdomain with a synthetic gene encoding the cytokine interleukin6 (IL-6/IFN-ß2/BSF-2). The resulting gene fusion encodesthe chimeric toxin DAB₃₈₉-IL-6. Following expression and purification,we demonstrate that DAB₃₈₉-IL-6 is selectively cytotoxic foreukaryotic cells bearing the interleukin 6 receptor. In addition,the cytotoxic action of DAB₃₈₉-IL-6 is shown to require bindingto the IL-6 receptor, internalization by receptor-mediated endocytosisand passage through an acidic compartment. Following the deliveryof the catalytically active fragment A to the cytosol of targetcells, cellular protein synthesis is inhibited by the ADP-ribosylationof elongation factor 2. While eukaryotic cells which are devoidof the IL-6 receptor are uniformly resistant to the action ofthis fusion toxin, the data presented suggest that a minimalnumber of IL-6 receptors may be necessary to mediate the internalizationof sufficient levels of DAB₃₈₉-IL-6 to result in the intoxicationof target cells.     

精制白喉标准毒素及精制锡克试验毒素的制备与检定

采用纯度为2000Lf/mgPN以上的精制白喉毒素,加到甘油明胶缓冲液中,制成约1000MLD/ml的精制白喉标准毒素。放置4℃保存10年,其MLD/Lf仅下降13.6％,证明稳定性良好;而原制白喉标准毒素在同样条件下MLD/Lf竟下降94.6％,稳定性很差,其放置的前3年,MLD/Lf呈急剧直线下降,后7年则呈缓慢下降。用精制白喉标准毒素共生产精制锡克试验毒素10批,经检定,MRD/ml和MLD/ml皆符合我国规程要求;结合力,7批符合英国药典规定,另3批经20％稀释亦能达到上述要求。原制锡克试验毒素的结合力一般为精制的2～5倍。     

精制白喉毒素的完全类毒化

精制白喉毒素加0.0125mol 赖氨酸,再加甲醛经适当的时间解毒,即可转化为完全类毒化的无毒性逆转的精制白喉类毒素。此等精白类的抗原性、效力稳定性、配成吸附制剂的吸附度以及解毒过程的 Lf/ml 损失,皆明显地优于单独用甲醛解毒生产的精白类。~H-赖氨酸结合试验表明,类毒化后,赖氨酸已结合到白喉类毒素分子中。     

用无细胞百日咳抗原配制的吸附精制百白破制剂的特性和人群反应及血清学效果

用50L发酵罐培养百日咳菌,经硫酸铵盐析法提取保护性抗原,戊二醛解毒后,与白喉、破伤风类毒素及Al(OH)_3配合制成的吸附精制百白破制剂(APDT),质量符合规程要求。给3～5月龄婴幼儿接种后全身和局部副反应很低,与现行吸附百白破制剂(WPDT)有非常显著性差异。免后血清中抗-PT和抗-FHA抗体均有显著增长,与WPDT制剂组相比,APDT的抗-PT和抗-FHA抗体增长更为显著。APDT免后抗白喉、抗破伤风抗体滴度均超过保护水平。本制剂的质量及免疫效果均达到了国外同类产品水平。     

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.