Characterization of a neurotoxigenic Clostridium butyricum strain isolated from the food implicated in an outbreak of food-borne type E botulism.

Neurotoxigenic Clostridium butyricum was isolated from the food implicated in an outbreak of clinically diagnosed type E botulism in China. PCR assay showed that the isolate (LCL 155) contained the type E botulinum toxin gene. This appears to be the first report of neurotoxigenic C. butyricum causing food-borne botulism.     

Characterization of an organism that produces type E botulinal toxin but which resembles Clostridium butyricum from the feces of an infant with type E botulism.

The apparent causative organism from the only reported case of type E infant botulism was isolated and characterized. Except for its ability to produce type E botulinal toxin, this organism (strain 5262) would be unquestionably identified as Clostridium butyricum. This is the second time an organism resembling a defined Clostridium species other than a member of the C. botulinum group has been implicated in infant botulism.     

Comparison of toxins of Clostridium butyricum and Clostridium botulinum type E.

The toxin of Clostridium butyricum strains isolated from two infants with botulism is neutralized by antitoxin for type E botulinum toxin. This toxin and that of a C. botulinum type E strain were purified by the same protocol. Both toxins were Mr 145,000 proteins which, when activated with trypsin, were composed of an H subunit of Mr 105,000 and an L subunit of Mr 50,000. The activated specific toxicity of purified butyricum toxin based on an intravenous assay was 2 X 10(8) mouse 50% lethal doses (LD50s)/mg of protein, but that based on an intraperitoneal assay was 7 X 10(7) LD50s/mg, compared with 6 X 10(7) LD50s/mg for type E toxin as determined by both methods. Immunodiffusion tests with antitoxin raised with type E toxin indicated that the two toxins were serologically very similar except for a spur formed by type E toxin. The close similarities of the two toxins suggest that toxigenic C. butyricum could arise when a wild-type strain, which is normally nontoxigenic, acquires the toxin gene of a C. botulinum type E strain.     

Gene probes for identification of the botulinal neurotoxin gene and specific identification of neurotoxin types B, E, and F.

A polymerase chain reaction method was developed for the specific detection of the botulinum neurotoxin (BoNT) gene of Clostridium botulinum. Degenerate oligonucleotide primers, designed from the nucleotide sequence of the heavy chain of the BoNT gene, amplified a specific fragment of approximately 1.1 kb from strains of C. botulinum toxin types A, B, E, F, and G and neurotoxin-producing strains of Clostridium barati and Clostridium butyricum, but no fragment was obtained from nontoxigenic strains. The fragments amplified from several strains of C. botulinum types B, E, and F were cloned in Escherichia coli and their nucleotide sequences were determined. Sequences within this region were used to design oligonucleotide probes specific for BoNT type B (BoNT/B), BoNT/E, and BoNT/F genes. An additional probe was designed for the detection of the BoNT/F gene of C. barati, which differed in sequence from BoNT/F genes of both proteolytic and nonproteolytic strains of C. botulinum.     

Characterization of the antibody response to the receptor binding domain of botulinum neurotoxin serotypes A and E

Clostridium botulinum neurotoxins (BoNTs) are the most toxic proteins for humans. The current clostridial-derived vaccines against BoNT intoxication have limitations including production and accessibility. Conditions were established to express the soluble receptor binding domain (heavy-chain receptor [HCR]) of BoNT serotypes A and E in Escherichia coli. Sera isolated from mice and rabbits immunized with recombinant HCR/A1 (rHCR/A1) from the classical type A-Hall strain (ATCC 3502) (BoNT/A1) and rHCR/E from BoNT serotype E Beluga (BoNT/E(B)) neutralized the homologous serotype of BoNT but displayed differences in cross-recognition and cross-protection. Enzyme-linked immunosorbent assay and Western blotting showed that alpha-rHCR/A1 recognized epitopes within the C terminus of the HCR/A and HCR/E, while alpha-rHCR/E recognized epitopes within the N terminus or interface between the N and C termini of the HCR proteins. alpha-rHCR/E(B) sera possessed detectable neutralizing capacity for BoNT/A1, while alpha-rHCR/A1 did not neutralize BoNT/E. rHCR/A was an effective immunogen against BoNT/A1 and the Kyoto F infant strain (BoNT/A2), but not BoNT serotype E Alaska (BoNT/E(A)), while rHCR/E(B) neutralized BoNT/E(A), and under hyperimmunization conditions protected against BoNT/A1 and BoNT/A2. The protection elicited by rHCR/A1 to BoNT/A1 and BoNT/A2 and by rHCR/E(B) to BoNT/E(A) indicate that immunization with receptor binding domains elicit protection within sub-serotypes of BoNT. The protection elicited by hyperimmunization with rHCR/E against BoNT/A suggests the presence of common neutralizing epitopes between the serotypes E and A. These results show that a receptor binding domain subunit vaccine protects against serotype variants of BoNTs.     

Unique biological activity of botulinum D/C mosaic neurotoxin in murine species

Clostridium botulinum types C and D cause animal botulism by the production of serotype-specific or mosaic botulinum neurotoxin (BoNT). The D/C mosaic BoNT (BoNT/DC), which is produced by the isolate from bovine botulism in Japan, exhibits the highest toxicity to mice among all BoNTs. In contrast, rats appeared to be very resistant to BoNT/DC in type C and D BoNTs and their mosaic BoNTs. We attempted to characterize the enzymatic and receptor-binding activities of BoNT/DC by comparison with those of type C and D BoNTs (BoNT/C and BoNT/D). BoNT/DC and D showed similar toxic effects on cerebellar granule cells (CGCs) derived from the mouse, but the former showed less toxicity to rat CGCs. In recombinant murine-derived vesicle-associated membrane protein (VAMP), the enzymatic activities of both BoNTs to rat isoform 1 VAMP (VAMP1) were lower than those to the other VAMP homologues. We then examined the physiological significance of gangliosides as the binding components for types C and D, and mosaic BoNTs. BoNT/DC and C were found to cleave an intracellular substrate of PC12 cells upon the exogenous addition of GM1a and GT1b gangliosides, respectively, suggesting that each BoNT recognizes a different ganglioside moiety. The effect of BoNT/DC on glutamate release from CGCs was prevented by cholera toxin B-subunit (CTB) but not by a site-directed mutant of CTB that did not bind to GM1a. Bovine adrenal chromaffin cells appeared to be more sensitive to BoNT/DC than to BoNT/C and D. These results suggest that a unique mechanism of receptor binding of BoNT/DC may differentially regulate its biological activities in animals.     

Experimental cecitis in gnotobiotic quails monoassociated with Clostridium butyricum strains isolated from patients with neonatal necrotizing enterocolitis and from healthy newborns.

Using axenic quails fed a diet containing lactose, we have investigated the potentially pathogenic roles of six Clostridium butyricum strains of human origin. Three strains (CB155-3, CB1002, and CB203-1) isolated from neonatal necrotizing enterocolitis patients and two of three strains (CB19-1 and CB25-2) isolated from healthy newborns led to cecal or crop lesions or both similar to those observed in human neonatal necrotizing enterocolitis: thickening of the cecal wall with gas cysts, hemorrhagic ulcerations, and necrotic areas. The lactose-negative strain (CB46-1) did not develop any lesions. The neuraminidase-producing strain (CB155-3) caused lesions in all monoassociated quails, whereas the other strains caused lesions in 28 to 85% of animals. Removal of dietary lactose suppressed all pathological incidence. These results show that lactose fermentation is a prerequisite in these pathological changes and stress the roles played by both the strain and the host in the expression of C. butyricum enteropathogenicity.     

Genetic confirmation of identities of neurotoxigenic Clostridium baratii and Clostridium butyricum implicated as agents of infant botulism.

Two unusual neurotoxigenic clostridia isolated from fecal specimens from patients with type F and type E infant botulism were phenotypically identical to the existing species Clostridium baratii and C. butyricum, respectively. DNA hybridization experiments confirmed that one strain was C. baratii and that the other was C. butyricum. These species therefore do contain neurotoxigenic strains and are possible causes of infant botulism.     

Genetic Characterization of Clostridium botulinum Associated with Type B Infant Botulism in Japan

The 15 proteolytic Clostridium botulinum type B strains, including 3 isolates associated with infant botulism in Japan, were genetically characterized by phylogenetic analysis of boNT/B gene sequences, genotyping, and determination of the boNT/B gene location by using pulsed-field gel electrophoresis (PFGE) for molecular epidemiological analysis of infant botulism in Japan. Strain Osaka05, isolated from a case in 2005, showed a unique boNT/B gene sequence and was considered to be a new BoNT/B subtype by phylogenetic analysis. Strain Osaka06, isolated from a case in 2006, was classified as the B2 subtype, the same as strain 111, isolated from a case in 1995. The five isolates associated with infant botulism in the United States were classified into the B1 subtype. Isolates from food samples in Japan were divided into the B1 and the B2 subtypes, although no relation with infant botulism was shown by PFGE genotyping. The results of PFGE and Southern blot hybridization with undigested DNA suggested that the boNT/B gene is located on large plasmids (approximately 150 kbp, 260 kbp, 275 kbp, or 280 kbp) in five strains belonging to three BoNT/B subtypes from various sources. The botulinum neurotoxin (BoNT) of Osaka05 was suggested to have an antigenicity different from the antigenicities of BoNT/B1 and BoNT/B2 by a sandwich enzyme-linked immunosorbent assay with the recombinant BoNT/B-C-terminal domain. We established a multiplex PCR assay for BoNT/B subtyping which will be useful for epidemiological studies of type B strains and the infectious diseases that they cause.Infant botulism is neuromuscular paralysis caused by the botulinum neurotoxin (BoNT) produced in the intestines after the germination and outgrowth of ingested spores of Clostridium botulinum, which is an anaerobic spore-forming bacterium (7, 9). On the basis of the antigenic specificity of BoNT, C. botulinum strains are divided into seven serotypes (serotypes A to G), and the species has been separated into four groups (groups I to IV) by cultural characteristics (24). BoNT is encoded by an approximately 3.8-kb gene, which is preceded by several nontoxic component genes (17, 30). BoNT is released from the bacteria as a single polypeptide chain of 150 kDa and is cleaved by endogenous or exogenous proteases into a 50-kDa light chain and a 100-kDa heavy chain. The heavy chain contains two functional domains, the N-terminal domain (H_N) and the C-terminal domain (H_C). H_C can be further divided into two distinct subdomains: the N-terminal domain (H_CN) and the C-terminal domain (H_CC) (5, 38).Recently, the subtype classification was confirmed by the diversity of the amino acid sequences within each serotype (13, 37). BoNT serotype A (BoNT/A) has been divided into four subtypes (subtypes A1, A2, A3, and A4) (2, 10). BoNT/B has been divided into three subtypes from type B group I (subtypes B1, B2, and B3), one subtype from group I bivalent strains that express another BoNT type, in addition to BoNT/B (bivalent), and one subtype from type B group II (nonproteolytic) (13). BoNT/E has been divided into four subtypes from C. botulinum type E (subtypes E1, E2, E3, and E6) and two subtypes from BoNT/E-producing C. butyricum (subtypes E4 and E5) (6).Since infant botulism was first recognized in the United States in 1976 (27, 31), it is now the most common disease caused by C. botulinum. This disease affects children up to 6 months old, but with rare exceptions it affects individuals of other ages. The symptoms are characterized by constipation, generalized weakness, and various neurological disorders (9). Cases represent a spectrum of disease, ranging from subclinical infection to the most fulminant form of the disease, which is unexpected sudden death (3). Almost all cases of infant botulism have been caused by proteolytic C. botulinum type A and B strains. Since the first occurrence of infant botulism in Japan caused by C. botulinum type A in 1986 (29), there have been 24 cases; 16 were caused by type A strains, 3 were caused by type B strains, 1 was caused by a type C strain, and 1 was caused by a C. butyricum strain producing BoNT/E. The types of toxin in the other three cases were not described (16). We previously indicated that the original BoNT/B2 produced by strain 111, which was isolated from the first case of type B infant botulism in Japan in 1995, showed antigenic and biological properties different from those of the authentic BoNT/B (B1) produced by strain Okra (15, 20, 22). Two additional cases of type B infant botulism with typical symptoms occurred in Osaka Prefecture in 2005 and 2006. We eventually isolated two proteolytic C. botulinum type B strains, designated Osaka05 and Osaka06, respectively.In the study described here, to better understand the background of type B infant botulism, we determined the genetic characteristics of proteolytic C. botulinum type B isolates by comparison of the nucleotide sequences of boNT/B and nontoxic component genes, the pulsed-field gel electrophoresis (PFGE) genotypes, the boNT/B gene location by PFGE and Southern blot hybridization, and the antigenicity of the new BoNT/B subtype. We developed multiplex PCR assays for the detection and identification of the BoNT/B subtypes.     

Lack of BIC and microRNA miR-155 expression in primary cases of Burkitt lymphoma

We previously demonstrated high expression of primary-microRNA BIC (pri-miR-155) in Hodgkin lymphoma (HL) and lack of expression in most non-Hodgkin lymphoma subtypes including some Burkitt lymphoma (BL) cases. Recently, high expression of BIC was reported in BL in comparison to pediatric leukemia and normal peripheral-blood samples. In this study, we extended our series of BL cases and cell lines to examine expression of BIC using RNA in situ hybridization (ISH) and quantitative RT-PCR (qRT-PCR) and of miR-155 using Northern blotting. Both BIC RNA ISH and qRT-PCR revealed no or low levels of BIC in 25 BL tissue samples [including 7 Epstein-Barr virus (EBV)-positive cases] compared to HL and normal controls. In agreement with these findings, no miR-155 was observed in BL tissues. EBV-negative and EBV latency type I BL cell lines also showed very low BIC and miR-155 expression levels as compared to HL cell lines. Higher levels of BIC and miR-155 were detected in in vitro transformed lymphoblastoid EBV latency type III BL cell lines. An association of latency type III infection and induction of BIC was supported by consistent expression of BIC in 11 and miR-155 in 2 posttransplantation lymphoproliferative disorder (PTLD) cases. In summary, we demonstrated that expression of BIC and miR-155 is not a common finding in BL. Expression of BIC and miR-155 in 3 latency type III EBV-positive BL cell lines and in all primary PTLD cases suggests a possible role for EBV latency type III specific proteins in the induction of BIC expression.     

Comparison of extracellular and intracellular potency of botulinum neurotoxins

Levels of botulinum neurotoxin (BoNT) proteolytic activity were compared using a cell-free assay and living neurons to measure extracellular and intracellular enzymatic activity. Within the cell-free reaction model, BoNT serotypes A and E (BoNT/A and BoNT/E, respectively) were reversibly inhibited by chelating Zn2+ with N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN). BoNT/E required relatively long incubation with TPEN to achieve total inhibition, whereas BoNT/A was inhibited immediately upon mixing. When na?ve Zn2+-containing BoNTs were applied to cultured neurons, the cellular action of each BoNT was rapidly inhibited by subsequent addition of TPEN, which is membrane permeable. Excess Zn2+ added to the culture medium several hours after poisoning fully restored intracellular toxin activity. Unlike TPEN, EDTA irreversibly inhibited both BoNT/A and -E within the cell-free in vitro reaction. Excess Zn2+ did not reactivate the EDTA-treated toxins. However, application of EDTA-treated BoNT/A or -E to cultured neurons demonstrated normal toxin action in terms of both blocking neurotransmission and SNAP-25 proteolysis. Different concentrations of EDTA produced toxin preparations with incrementally reduced in vitro proteolytic activities, which, when applied to living neurons showed undiminished cellular potency. This suggests that EDTA renders the BoNT proteolytic domain conformationally inactive when tested with the cell-free reaction, but this change is corrected during entry into neurons. The effect of EDTA is unrelated to Zn2+ because TPEN could be applied to living cells before or after poisoning to produce rapid and reversible inhibition of both BoNTs. Therefore, bound Zn2+ is not required for toxin entry into neurons, and removal of Zn2+ from cytosolic BoNTs does not irreversibly alter toxin structure or function. We conclude that EDTA directly alters both BoNTs in a manner that is independent of Zn2+.     

Differential contribution of the residues in C-terminal half of the heavy chain of botulinum neurotoxin type B to its binding to the ganglioside GT1b and the synaptotagmin 2/GT1b complex

Clostridium botulinum type B neurotoxin was effectively bound to synaptotagmin 2 (Stg2) associated with ganglioside GT1b, however, the molecular interaction between the neurotoxin and the Stg2/GT1b complex has not been identified. Previously, we found that infant botulism-related strain 111 generated a low activity of the neurotoxin (111/NT), which differed in some amino acid residues, especially in the carboxyl terminal half of the heavy chain (H(C)), from the original neurotoxin of strain Okra (Okra/NT) associated with a food-borne botulism. In this study, we evaluated the binding capabilities of site-directed mutants of Okra/H(C) to the Stg2/GT1b complex and to GT1b alone, and investigated the relationship between the toxic action and receptor binding. Replacement of K1187 and E1190 with glutamic acid and lysine, respectively, which substituted for the 111/NT residues, caused a reduction of binding affinity to the Stg2/GT1b complex, suggesting that both these residues contribute to the different binding affinity between Okra/NT and 111/NT. Substitution of four residues, H1240, S1259, W1261 and Y1262, which form a ganglioside pocket, drastically decreased the binding of H(C) to the Stg2/GT1b complex and to GT1b. Mutation in the residues, K1186, E1189, K1191 and K1260 reduced the binding of H(C) to GT1b alone, but not to the Stg2/GT1b complex. Analyses of effects of mutant toxins on toxicity of BoNT/B to cerebellar granule cells suggest the association of cell toxicity with binding to Stg2/GT1b complex but not that to GT1b alone.     

Escape-avoidance learning in two strains of inbred mice when two types of response can be alternatively effective

Three experiments demonstrated that escape-avoidance learning is closely related to the response type dominantly occurred in mice of an inbred strain. Inescapable shock elicited locomotion (L-typed response) in C57BL/6 mice, and rearing or jumping (R-typed response) in C3H/He (Experiment 1). In the shuttle-box situation where both of these responses can be effective to terminate CS tone and electric shock, C57BL established L-typed avoidance mainly, while C3H tended to learn avoidance by R-typed response (Experiment 2). The two strains learned the escape in the shuttle-box by the same types of response as they showed in avoidance learning (Experiment 3). These findings lead us to conclude that different inbred strains of mice innately react to electric shock by their specific types of response. Strain differences in escape-avoidance learning could thus be attributed to the compatibility of the specific response type with requirement of the task situation.     

天花粉蛋白免疫抑制作用的功能性肽段筛选及其作用机制

在人工合成天花粉蛋白(Tk)多个重叠肽段的基础上,采用前期工作中筛选出的天花粉蛋白高敏感(C57BL/6)和低敏感(C3H/He)小鼠近交系,建立OVA特异的体外二次应答增殖系统,检测各肽段抑制能力,并通过细胞剔除及过继试验确认发挥作用的细胞,采用ELISA方法检测细胞因子分泌格局的改变。结果筛选到5条具有明显免疫抑制功能的Tk衍生肽,其中PE和PS对两个品系皆显示抑制活性,而PQ、PB及PJ仅在C57BL/6小鼠中诱导抑制。Tk衍生肽段改变了OVA特异性增殖系统中细胞因子分泌的格局,使得以Th1/Tc1型细胞因子为主状态向Th2/Tc2偏移,表现为IL-4和IL-10分泌增加,而IFN-γ分泌减少。剔除CD8+T细胞明显解除Tk及其肽段的抑制作用。表明Tk的某些肽段与全蛋白一样具有免疫抑制功能,其机制可能与诱导T细胞向CD8+Tc2方向偏移有关。     

The lpf gene cluster for long polar fimbriae is not involved in adherence of enteropathogenic Escherichia coli or virulence of Citrobacter rodentium

Using the enteropathogenic Escherichia coli (EPEC) genome sequence, we found that EPEC E2348/69 has an lpfABCDE gene cluster homologous (about 60% identical at the protein level) to the Salmonella long polar fimbria (LPF) operon. To determine whether this operon is essential for adherence, the lpfABCD(E2)(3) genes were deleted from EPEC strain E2348/69 by allelic exchange. Analysis of the resulting EPECDeltalpfABCD(E23) strain showed no change in adherence to HeLa cells or to human intestinal biopsy cells in the in vitro organ culture (IVOC) system compared to the wild type. Sera from volunteers experimentally infected with E2348/69 showed no antibody response to the major subunit protein, LpfA. These results suggested that the lpf(E23) gene cluster is not necessary for EPEC adherence and attaching/effacing (A/E) lesion formation on human biopsy samples and is not expressed during human infection. We also identified an lpf gene cluster in Citrobacter rodentium strain ICC168 (lpf(cr)). A DeltalpfA(cr) mutant of ICC168 retained wild-type adherence and A/E lesion-forming activity on HeLa cells. C3H/HeJ mice were infected with a wild-type C. rodentium strain and its lpfA(cr) isogenic mutant. Both strains were recovered at high levels in stools, and there were no significant differences between the groups both in terms of the number of CFU/organ (colon and cecum) and in terms of the amount of hyperplasia, as measured by weight. Similar results were observed in a second mouse strain, C57BL/6. These data suggest that in addition to playing no apparent role in EPEC pathogenesis, lpf(cr) is not required for C. rodentium virulence in either the C3H/HeJ or C57BL/6 mouse model.     

Molecular characterization of murine humoral immune response to botulinum neurotoxin type A binding domain as assessed by using phage antibody libraries.

To produce antibodies capable of neutralizing botulinum neurotoxin type A (BoNT/A), the murine humoral immune response to BoNT/A binding domain (H(C)) was characterized at the molecular level by using phage antibody libraries. Mice were immunized with BoNT/A H(C), the spleens were harvested, and single-chain Fv (scFv) phage antibody libraries were constructed from the immunoglobulin heavy and light chain variable region genes. Phage expressing BoNT/A binding scFv were isolated by selection on immobilized BoNT/A and BoNT/A H(C). Twenty-eight unique BoNT/A H(C) binding scFv were identified by enzyme-linked immunosorbent assay and DNA sequencing. Epitope mapping using surface plasmon resonance in a BIAcore revealed that the 28 scFv bound to only 4 nonoverlapping epitopes with equilibrium constants (Kd) ranging from 7.3 x 10(-8) to 1.1 x 10(-9) M. In a mouse hemidiaphragm assay, scFv binding epitopes 1 and 2 significantly prolonged the time to neuroparalysis, 1.5- and 2.7-fold, respectively, compared to toxin control. scFv binding to epitopes 3 and 4 showed no protection against neuroparalysis. A combination of scFv binding epitopes 1 and 2 had an additive effect on time to neuroparalysis, which increased to 3.9-fold compared to the control. The results suggest that there are two "productive" receptor binding sites on H(C) which lead to toxin internalization and toxicity. Blockade of these two epitopes with monoclonal antibodies may provide effective immunoprophylaxis or therapy against BoNT/A intoxication.     

Molecular immune recognition of botulinum neurotoxin B. The light chain regions that bind human blocking antibodies from toxin-treated cervical dystonia patients. Antigenic structure of the entire BoNT/B molecule

We recently mapped the regions on the heavy (H) chain of botulinum neurotoxin, type B (BoNT/B) recognized by blocking antibodies (Abs) from cervical dystonia (CD) patients who develop immunoresistance during toxin treatment. Since blocking could also be effected by Abs directed against regions on the light (L) chain, we have mapped here the L chain, using the same 30 CD antisera. We synthesized, purified and characterized 32 19-residue L chain peptides that overlapped successively by 5 residues (peptide L32 overlapped with peptide N1 of the H chain by 12 residues). In a given patient, Abs against the L chain seemed less intense than those against H chain. Most sera recognized a limited set of L chain peptides. The levels of Abs against a given region varied with the patient, consistent with immune responses to each epitope being under separate MHC control. The peptides most frequently recognized were: L13, by 30 of 30 antisera (100%); L22, by 23 of 30 (76.67%); L19, by 15 of 30 (50.00%); L26, by 11 of 30 (36.70%); and L14, by 12 of 30 (40.00%). The activity of L14 probably derives from its overlap with L13. The levels of Ab binding decreased in the following order: L13 (residues 169-187), L22 (295-313), L19 (253-271), and L26 (351-369). Peptides L12 (155-173), L18 (239-257), L15 (197-215), L1 (1-19) and L23 (309-327) exhibited very low Ab binding. The remaining peptides had little or no Ab-binding activity. The antigenic regions are analyzed in terms of their three-dimensional locations and the enzyme active site. With the previous localization of the antigenic regions on the BoNT/B H chain, the human Ab recognition of the entire BoNT/B molecule is presented and compared to the recognition of BoNT/A by human blocking Abs.     

Regions recognized on the light chain of botulinum neurotoxin type A by T lymphocytes of SJL and BALB/c mice primed with inactivated toxin

Lymph node cells (LNC) from SJL (H-2^s) and BALB/c (H-2^d) mice primed once with inactivated botulinum neurotoxin type A (BoNT/A) were examined for their T-cell responses to each of 32 synthetic overlapping peptides (19 residues each, L1–L32) that encompass the entire L chain (residues 1–448) of BoNT/A. LNC of SJL gave strong responses to 6 regions on, L2 (residues 15–23), L10/11/12 (127–173), L19 (253–271) and L21 (281–299), and moderate to weak responses to L9 (113–131), L14/15 (183–215) and L27 (365–383). In BALB/c, LNC gave a substantial T-cell response only against peptide L12 (residues 155–173), and responded very weakly to 9 other peptides. The results were compared with the recognition profiles determined previously in these two strains after multiple BoNT/A injections. Overall responses to the L-chain peptides of T cells in later profiles were found to be somewhat weakened in SJL and stayed essentially at a similar level in BALB/c, although responses to BoNT/A increased. In SJL, response to L10 (127–145) remained the highest in the later profile. Strong responses against L12 (155–173) observed in both strains at early stage were reduced to an insignificant level. Cross-reactivity to tetanus neurotoxin by BoNT/A-specific T cells was observed in SJL but not in BALB/c. Design of an effective synthetic peptide vaccine will require incorporation of both T cell- and Ab-recognition elements of the BoNT molecule. Significance and possible implications of these results on BoNT/A-specific T-cell responses of BoNT-treated patients are discussed.     

Ganglioside GT1b as a complementary receptor component forClostridium botulinumneurotoxins

Clostridium botulinumtype B neurotoxin (BoNT/B) recognizes a complex of synaptotagmin II and ganglioside GT1b or GD1a as the high-affinity toxin binding site. Recombinant deletion mutants of synaptotagmin II allowed us to demonstrate that the N-terminal domain including the transmembrane region retains BoNT/B binding activity while the C-terminal domain is not involved in constituting the BoNT/B receptor. BoNT/B binding to reconstituted lipid vesicles containing synaptotagmin II and gangliosides showed that GT1b and GD1a confer the difference in the maximum binding capacity but not in the dissociation constant. The direct binding of GT1b to the deletion mutants revealed that the transmembrane region is required to bind GT1b, suggesting that synaptotagmin II binds to the ceramide portion of gangliosides within the plasma membrane. A monoclonal antibody against GT1b effectively inhibited not only BoNT/B binding to the reconstituted lipid vesicles and brain synaptosomes but also type A BoNT (BoNT/A) binding to brain synaptosomes. In addition, the monoclonal antibody antagonized the action of both BoNT/A and BoNT/B on synaptic transmission of rat superior cervical ganglion neurons. These results suggest that GT1b functions as a component of the receptor complex.     

Failure of Epstein-Barr virus-specific cytotoxic T lymphocytes to lyse B cells transformed with the B95-8 strain is mapped to an epitope that associates with the HLA-B8 antigen.

There are two types, A and B, of Epstein-Barr virus (EBV) and B95-8 represents the common type A laboratory strain. Herein, we show in a family study that paternal EBV-specific cytotoxic T lymphocytes (CTL) generated in short-term cultures following stimulation with the autologous B95-8-transformed lymphoblastoid cell line (LCL) or B cells freshly infected with the B95-8 isolate did not lyse haploidentical B95-8 LCL expressing the HLA-A1, -B8, -DR3 paternal haplotype. In contrast, the haploidentical B95-8 LCL expressing the HLA-A11, -B51, -DR7 paternal haplotype was strongly lysed. Moreover, paternal CTL generated in response to stimulation with the B95-8 LCL expressing the haploidentical HLA-A1, -B8, -DR3 paternal haplotype included an allogeneic response against the maternal haplotype but no EBV-specific response as shown by the poor lysis of the autologous LCL target cells. However, stimulation with the haploidentical HLA-A11, -B51, -DR7 paternal haplotype resulted in the generation of both an allogeneic and an EBV-specific response. CTL clones were generated from two HLA-B8+ donors in response to stimulation with the autologous type A LCL transformed with wildtype EBV. The clones were cross-reactive for an immunodominant B95-8-associated peptide epitope that interacted with the HLA-B8 allele but failed to lyse B95-8-transformed LCL targets unless the targets were pre-coated with the exogenous peptide. A CTL clone that was initially stimulated with the autologous BL74 LCL lysed the spontaneous autologous LCL and spontaneous LCL from an HLA-B8+ donor, but failed to lyse the B95-8 LCL from that donor. The observed haplotype preference can be explained in terms of sequence variation between the B95-8 and the corresponding wildtype epitope. Our findings may help to clarify the role of EBV in the pathogenesis of primary Sjögren''s syndrome which is closely associated with HLA-B8.