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Introduction The effectiveness of several vaccines against coronavirus disease (COVID-19) has been reported in the real-world setting. However, it is still unknown how long antibodies persist following vaccination and whether or not the persistence of antibodies has a protective effect against COVID-19. Methods Healthcare workers who had received two doses of the BNT162b2 mRNA COVID-19 vaccine were enrolled, and a single-center study was conducted at the National Hospital Organization Hakodate National Hospital. Serum samples from all participants were collected 13–21 weeks (median: 20 weeks) after the second dose of vaccination. The antibody titers were measured using an electrochemiluminescence immunoassay (Elecsys® Anti-SARS-CoV-2 S). Data on characteristics of the participants were gathered from patient records and interview sheets. Results A total of 401 participants, among whom 70.1% were women and the median age was 42 years, were evaluated in this study. None of the participants had a definite COVID-19 history, and all participants who received complete vaccination showed positive antibody titers. The antibody titer was observed to be higher in participants with younger age (p

The rotavirus-induced diarrhea of human and animal neonates is a major public health concern worldwide. Until recently, no effective therapy is available to specifically inactivate the rotavirion particles within the gut. Passive immunotherapy by oral administration of chicken egg yolk antibody (IgY) has emerged of late as a fresh alternative strategy to control infectious diseases of the alimentary tract and has been applied in the treatment of diarrhea due to rotavirus infection. The purpose of this concise review is to evaluate evidence on the properties and performance of anti-rotavirus immunoglobulin Y (IgY) for prevention and treatment of rotavirus diarrhea in human and animal neonates. A survey of relevant anti-rotavirus IgY basic studies and clinical trials among neonatal animals (since 1994-2015) and humans (since 1982-2015) have been reviewed and briefly summarized. Our analysis of a number of rotavirus investigations involving animal and human clinical trials revealed that anti-rotavirus IgY significantly reduced the severity of clinical manifestation of diarrhea among IgY-treated subjects relative to a corresponding control or placebo group. The accumulated information as a whole depicts oral IgY to be a safe and efficacious option for treatment of rotavirus diarrhea in neonates. There is however a clear need for more randomized, placebo controlled and double-blind trials with bigger sample size to further solidify and confirm claims of efficacy and safety in controlling diarrhea caused by rotavirus infection especially among human infants with health issues such as low birth weights or compromised immunity in whom it is most needed.

In Clostridium botulinum, the characteristics of type C and D strains are quite different from other types, and they are classified as group III. They produce C2 binary toxin and C3 exoenzyme in addition to type C and D neurotoxins. Two different phages and many plasmids are identified in the organisms. The genes of neurotoxin and C3 exoenzyme are converted from toxigenic strains to non-toxigenic strains by the specific bacteriophages (phages), whereas, the C2 toxin gene is carried by large or small plasmids. Classification of type C and D strains has been in confusion because 1) antigenicity of type C and D neurotoxins is complex, 2) the cells produce two types of toxins, neurotoxin and C2 toxin, and 3) some non-toxigenic strains can be converted to produce C or D neurotoxin by the infection with phages. Until now, entire nucleotide sequences of cell chromosomes, phages, and plasmids have been determined. Since both genetic and protein-chemical analyses have been clarifying the above confusions, these data are reviewed historically.

Epidemiological studies have demonstrated a close association between infection with Helicobacter pylori (H.pylori) and the development of gastric carcinoma. Chronic H.pylori infection increases the frequency of mutation in gastric epithelial cells. However, the mechanism by which infection of H.pylori leads to mutation in gastric epithelial cells is unclear. We suspected that components in H.pylori may be related to the mutagenic response associated with DNA alkylation, and could be detected with the Ames test using a more sensitive strain for alkylating agents. Our investigation revealed that an extract of H.pylori was mutagenic in the Ames test with Salmonella typhimurium YG7108, which is deficient in the DNA repair of O(6)-methylguanine. The extract of H.pylori may contain methylating or alkylating agents, which might induce O (6)-alkylguanine in DNA. Mutagenicity of the alkylating agents N-methyl-N-nitrosourea (MNU) and N-methyl-N'-nitro-N-nitrosoguanidine in the Ames test with S.typhimurium TA1535 was enhanced significantly in the presence of the extract of H.pylori. The tested extracts of H.pylori resulted in a significant induction of micronuclei in human-derived lymphoblastoid cells. Heat instability and dialysis resistance of the extracts of H.pylori suggest that the mutagenic component in the extracts of H.pylori is a heat-unstable large molecule or a heat-labile small molecule strongly attached or adsorbed to a large molecule. Proteins in the extracts of H.pylori were subsequently fractionated using ammonium sulphate precipitation. However, all fractions expressed enhancing effects toward MNU mutagenicity. These results suggest the mutagenic component is a small molecule that is absorbed into proteins in the extract of H.pylori, which resist dialysis. Continuous and chronic exposure of gastric epithelial cells to the alkylative mutagenic component from H.pylori chronically infected in the stomach might be a causal factor in the gastric carcinogenesis associated with H.pylori.

Vibrio vulnificus is the causative agent of primary septicemia, wound infection and gastroenteritis in immunocompromised people. In this study, signature-tagged mutagenesis (STM) was applied to identify the virulence genes of V. vulnificus. Using STM, 6,480 mutants in total were constructed and divided into 81 sets (INPUT pools); each mutant in a set was assigned a different tag. Each INPUT pool was intraperitoneally injected into iron-overloaded mice, and in vivo surviving mutants were collected from blood samples from the heart (OUTPUT pools). From the genomic DNA of mixed INPUT or OUTPUT pools, digoxigenin-labeled DNA probes against the tagged region were prepared and used for dot hybridization. Thirty tentatively attenuated mutants, which were hybridized clearly with INPUT probes but barely with OUTPUT probes, were negatively selected. Lethal doses of 11 of the 30 mutants were reduced to more than 1/100; of these, the lethal doses of 2 were reduced to as low as 1/100,000. Transposon-inserted genes in the 11 attenuated mutants were those for IMP dehydrogenase, UDP-N-acetylglucosamine-2-epimerase, aspartokinase, phosphoribosylformylglycinamidine cyclo-ligase, malate Na (+) symporter and hypothetical protein. When mice were immunized with an attenuated mutant strain into which IMP dehydrogenase had been inserted with a transposon, they were protected against V. vulnificus infection. In this study, we demonstrated that the STM method can be used to search for the virulence genes of V. vulnificus.

The haemagglutinin (HA) complex of Clostridium botulinum type C toxin is composed of three types of subcomponents: HA33, HA17 and HA70 (also known as HA1, HA2 and HA3, respectively). Here, a 260 kDa HA17-HA70 complex was crystallized. His-tagged HA17 and maltose-binding-protein-tagged HA70 were expressed in Escherichia coli and their complex was affinity-purified using a combination of amylose resin chromatography and nickel-nitrilotriacetic acid agarose chromatography. Diffraction data were collected to 8.0 Å resolution and the crystal belonged to the tetragonal space group P4(1)2(1)2. The molecular-replacement solution indicated that one molecule of HA17 was bound to each HA70 monomer.

Clostridium botulinum produces the botulinum neurotoxin, forming a large complex as progenitor toxins in association with non-toxic non-hemagglutinin and/or several different hemagglutinin (HA) subcomponents, HA33, HA17 and HA70, which bind to carbohydrate of glycoproteins from epithelial cells in the infection process. To elucidate the carbohydrate recognition mechanism of HA70, X-ray structures of HA70 from type C toxin (HA70/C) in complexes with sialylated oligosaccharides were determined, and a binding assay by the glycoconjugate microarray was performed. These results suggested that HA70/C can recognize both α2–3- and α2–6-sialylated oligosaccharides, and that it has a higher affinity for α2–3-sialylated oligosaccharides.

Many papers report the clinical success of botulinum toxin A as a method of management of various bladder dysfunctions. The rationale was that botulinum toxin A was able to block the presynaptic release of acetylcholine from the parasympathetic efferent nerve. The efficacy might result not only from an inhibitory effect on detrusor muscle, but also some effects might be mediated by altering the afferent nerve input. This systematic literature review discusses the efficacy and safety of botulinum toxin A therapy for idiopathic detrusor overactivity, neurogenic detrusor overactivity, interstitial cystitis/painful bladder syndrome and benign prostatic hyperplasia. The information was gathered from a PubMed literature research for abstracts from recent urological meetings. Injection of botulinum toxin A appears to have a positive therapeutic effect in multiple urological conditions, such as refractory idiopathic detrusor overactivity, neurogenic detrusor overactivity, interstitial cystitis/painful bladder syndrome and benign prostatic hyperplasia. Because the United States Food and Drug Administration has approved botulinum toxin A (Botox) for injection for the treatment of urinary incontinence as a result of neurogenic detrusor overactivity (e.g. spinal cord injury, multiple sclerosis) in adults who have an inadequate response to or are intolerant of an ant cholinergic medication, the use of botulinum toxin A will spread and be a more familiar therapy in the urological arena. However, further robust evidence should be awaited. We will discuss the current use of this agent within the urological field.

A large size botulinum toxin complex (L-TC) is composed of a single neurotoxin (BoNT), a single nontoxic nonhaemagglutinin (NTNHA) and a haemagglutinin (HA) complex. The HA complex is comprised of three HA-70 molecules and three arm structures of HA-33/HA-17 that consist of two HA-33 and a single HA-17. In addition to the mature L-TC, smaller TCs are present in cultures: M-TC (BoNT/NTNHA), M-TC/HA-70 and immature L-TCs with fewer HA-33/HA-17 arms than mature L-TC. Because L-TC displays higher oral toxicity than pure BoNT, it was presumed that nontoxic proteins are critical for food poisoning. In this study, the absorption of TCs across intestinal epithelial cells was assessed by examining the cell binding and monolayer transport of serotype D toxins in the rat intestinal epithelial cell line IEC-6. All TCs, including pure BoNT, displayed binding and transport, with mature L-TC showing the greatest potency. Inhibition experiments using antibodies revealed that BoNT, HA-70 and HA-33 could be responsible for the binding and transport. The findings here indicate that all TCs can transport across the cell layer via a sialic acid-dependent process. Nonetheless, binding and transport markedly increased with number of HA-33/HA-17 arms in the TC. We therefore conclude that the HA-33/HA-17 arm is not necessarily required for, but facilitates, transport of botulinum toxin complexes.

Adamantiades-Behçet's disease (ABD) is characterized by starting with oral aphthous ulceration and developing of the systemic involvements. The pathogenesis of ABD is closely correlated with the genetic factors and the triggering factors which acquire delayed-type hypersensitivity reaction against oralstreptococcimediated by IL-12 cytokine family. HLA-B51 is associated in more than 60% of the patients and its restricted CD8+ T cell response is clearly correlated with the target tissues.Bes-1gene encoded partialS. sanguinisgenome which is highly homologous with retinal protein, and 65 kD heat shock protein (Hsp-65) released from streptococci is playing an important role with human Hsp-60 in the pathogenesis of ABD. Although Hsp-65/60 has homologies with the respective T cell epitope, it stimulates peripheral blood mononuclear cells (PBMCs) from ABD patients. On the other hand, some peptides of Hsp-65 were found to reduce IL-8 and IL-12 production from PBMCs of ABD patients in active stage.

In an attempt to prepare egg yolk immunoglobulin (IgY) to treat and prevent cholera, hens were immunized by a mixture of heat- or formalin-killed Vibrio cholerae O1 and O139 organisms, or by the recombinant cholera toxin B subunit (CTB). The IgYs were partially purified from egg yolk and orally administered to suckling mice before or after challenge with live O1 or O139 cells. The anti-O1 and O139 IgYs and the mixture of either IgY with anti-CTB IgY significantly protected the occurrence of cholera caused by both O1 and O139 infection. Since large amounts of IgY can be prepared very easily and at low cost, this seems to be a useful procedure for preventing and treating cholera.

Enterohepatic Helicobacters have been shown to inhabit the intestine and hepatobiliary tracts and may be associated with a variety of diseases [1]. The enterohepatic Helicobacters were first recognized in laboratory rodents and have been considered a component of the resident microbiota, or normal flora. However, the role of enterohepatic Helicobacters in human diseases remains obscure. Helicobacter hepaticus, perhaps the most studied member of enterohepatic Helicobacter, colonizes the lower gastrointestinal tract, including the cecum, colon, and hepatobiliary system of mice [2–4]. Helicobacter hepaticus infection can cause chronic active hepatitis and typhlocolitis in immunocompetent mice and can lead to liver carcinoma in male mice of susceptible strains [5–9]. Natural and experimental infection with H. hepaticus in certain immunodeficient mice can induce inflammatory bowel disease [10,11]. Helicobacter bilis was also identified in inbred mice with chronic hepatitis [12]. Helicobacter bilis infection in immunodeficient rodents causes typhlocolitis and diarrhea [13]. These studies have prompted the increased use of murine models with H. hepaticus infection to elucidate the possible roles of Helicobacter in the development of gastrointestinal diseases in humans. Helicobacter hepaticus or H. bilis infection in humans was reported. For example, patients with chronic liver disease have been reported to have significantly higher levels of antibody to H. bilis and H. hepaticus compared to healthy subjects [14]. Although the H. pylori urease A gene (nested amplicon) has been frequently detected in bile samples, to date H. bilis-16S rRNA genes have only been detected in two cases, and H. hepaticus has not been detected [15]. These papers indicate that enterohepatic Helicobacter spp. may also infect in the human. The aim of this study was to directly detect enterohepatic Helicobacter spp. from human bile samples. We cultured bile samples taken from patients with and without hepatobiliary diseases to determine whether Helicobacter spp. were present. We used PCR and nested-PCR for detecting H. hepaticus or H. bilis. As nested-PCR for only H. hepaticus was positive, we tested the bile samples for in situ hybridization by a probe for H. hepaticus and for Western blotting by anti-H. hepaticus antibodies.

Objectives To investigate the efficacy of a newly purified neurotoxin (NTX) in male rat prostates. Several reports have suggested that intraprostatic injection of botulinum toxin type A has demonstrated efficacy against symptomatic benign prostatic hyperplasia. NTXs associate with nontoxic components and form large complexes designated "progenitor toxins." In general, progenitor toxins are used, because they are easily obtained and are more stable than NTXs. However, they have side effects for some patients in whom anti-progenitor toxins, including anti-NTX antibodies, are produced after several injections. We purified NTXs without their nontoxic components using a simple procedure. Methods Adult male Sprague-Dawley rats were injected with 5 U of NTX or saline into their prostates, which were harvested and weighed after 1 or 4 weeks. The effects of the NTX on the prostate were histologically and immunohistochemically studied using hematoxylin-eosin, synaptophysin, and terminal deoxynucleotidyl-mediated deoxyuridine triphosphate nick end labeling staining. Results In the NTX-treated rats, the prostate weight was reduced and atrophy and diffuse apoptosis were observed. Moreover, synaptophysin-positive cells in the epithelium were decreased after NTX injection. Conclusions Intraprostatic NTX injection induces prostate apoptosis and atrophic change in the rat prostate. These results suggest that NTX injection might be a promising material for treatment of patients with benign prostatic hyperplasia.

In this study, we have demonstrated that Helicobacter pylori absorbs a steroid prehormone (pregnenolone) and two androgens (dehydroepiandrosterone and epiandrosterone), glucosylates these steroids, and utilizes glucosyl-steroid hormone compounds as the membrane lipid components. The only common structure among the steroid prehormone and the two androgens is a 3beta-OH in the steroid framework. Our results indicate that the 3beta-OH in the steroid hormones is a crucial conformation required for steroid glucosylation by H. pylori. In addition, we found that H. pylori absorbs and holds estrogens possessing 3-OH (estrone and estradiol) into the membrane. The effective absorption of estrogen into the membrane appeared to be controlled by the number of hydroxyl groups modifying the steroid framework. In contrast, H. pylori induced neither membrane absorption nor glucosylation of the other steroid hormones possessing 3=O (progesterone, androstenedione and testosterone) or 3alpha-OH (androsterone). These results indicate that H. pylori selectively absorbs 3beta-OH and 3-OH steroid hormones, and utilizes only 3beta-OH steroid hormones as the materials for glucosylation.

Immune responses against heat shock protein 60 (HSP60) of pathogen-origin are thought to be defensive events which, due to molecular mimicry, misdirect to a human counterpart. Therefore, atherosclerosis may be serologically predicted by anti-HSP60 antibodies (Abs). In the present study, we analyzed the clinical prevalence of the serum IgG Abs against Helicobacter pylori (Hp)-derived HSP60 (Hp-HSP60) or its peptide fragments in patients with cardiovascular disease (CVD; n = 250), as compared to those in age- and gender-matched non-CVD patients ( n = 293). Anti-Hp cell lysate Abs frequently appeared in Hp-infected patients who were not associated with CVD. In contrast, Abs against the particular amino acid sequence Hp-HSP60 II3 (II3 region, Glu 141 –Leu 160 , in Hp-HSP60) predominantly appeared in CVD patients, as well as IgG anti-human HSP60 (Hu-HSP60 w ). Furthermore, neither titer of anti-Hp-HSP60 II3 nor anti-Hu-HSP60 w Abs was correlated with the levels of high sensitivity C-reactive protein (hsCRP). This data strongly suggested that IgG anti-Hp-HSP60 II3 Abs cross-reacted with Hu-HSP60 w were independent diagnostic markers relevant to CVD. Further, the 20 amino acid residues (Glu 141 –Leu 160 ) might be predominant CVD-associated epitopes that induce anti-Hu-HSP60 auto-Abs, whose location was predicted in the tertiary structure of Hu-HSP60.

The catabolic pathway for flavodoxin has yet to be clarified for any bacterial species. In this study, we found that the flavin mononucleotide in the flavodoxin of Helicobacter pylori is degraded to riboflavin via the phosphomonoesterase activity of class C acid phosphatase. The result is a conversion of holoflavodoxin to apoflavodoxin.

Previous reports have indicated thatHelicobacter pyloriheat-shock protein 60 (H. pylori-HSP60), as an immunodominant antigen, induces interleukin (IL)-8 production in human monocytes. The exact mechanism by whichH. pylori-HSP60 induces IL-8 production in monocytes has not been fully elucidated. In the present study, the downstream pathway by whichH. pylori-HSP60 induces IL-8 secretion in human monocytic cell lines was investigated. IntactH. pylori, heat-killedH. pyloriandH. pylorirecombinant HSP60 (rHpHSP60) all induced the secretion of IL-8 and the activation of mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) and p38, but not c-Jun N-terminal kinase (JNK), up to 24 h in NOMO1 cells. The specific inhibitors PD98059 and U0126 (for ERK1/2 signalling) and SB203580 (for p38 MAPK signalling) down-regulated IL-8 secretion from rHpHSP60-treated NOMO1 cells. An anti-Toll-like receptor (TLR)2 antibody or TLR2 small interfering RNA (siRNA) partially inhibited the secretion of IL-8, and anti-TLR2 antibody also suppressed activation of ERK and p38 MAPK in rHpHSP60-treated NOMO1 cells. These reactions were associated with nuclear factor-κB (NF-κB)-mediated transcriptional activation, since U0126, SB203580 and the anti-TLR2 antibody decreased NF-κB activation. Taken together, the results suggest that ERK and p38 MAPK signalling linked to the TLR2 recognition receptor in human monocytes may be an important pathway inH. pylori-HSP60-induced IL-8 secretion.

Botulinum neurotoxin produced by Clostridium botulinum type B is in the form of a complex of 12S and 16S toxins. Food-borne botulism is caused by these complex toxins which are ingested orally and absorbed from the digestive tract. Here, we show that the human milk SIgA binds to the type B16S toxin. The binding of SIgA to 16S toxin and HA was inhibited by carbohydrates such as galactose, suggesting that the interaction of carbohydrate side chain of the SIgA with the HA of the 16S toxin is important for SIgA-16S complex formation. We also demonstrate that SIgA inhibits the attachment of 16S toxin to intestinal epithelial cells. These data suggest that the interaction of antigen nonspecific SIgA with 16S toxin has a large influence on the absorption of 16S toxin from the intestinal epithelium, and that SIgA may provide insight into developing a therapeutic agent for type B food-borne botulism.

Human rotavirus samples from 54 children with acute gastroenteritis in Myanmar in 2011 were subjected to reverse transcription-PCR to determine their G and P types. On G typing, G2 (24/54; 44.4%) was found to be the most prevalent, followed by G12 (17/54; 31.5%) and G1 (1/54; 1.9%). Mixed cases with G2 and G12 were found in 12 of the 54 (22.2%) samples. On P typing, P[4] was found to be the most predominant (29/54; 53.7%), followed by P[8] (17/54; 31.5%) and P[6] (4/54; 7.4%). Mixed cases with P[4] and P[8] were detected in 4 of 54 (7.4%) samples. Thus, occurrence of G2 and unusual G12 in high proportions was characteristic of human rotaviruses in Myanmar in this study setting.

Geranylgeranylacetone (GGA) is used to treat patients suffering from peptic ulcers and gastritis. We examined the effect of GGA on Helicobacter pylori, which is a causative factor of gastrointestinal diseases. Previously, we have reported that GGA binds specifically to the molecular chaperone HSP70. In this paper, we report that GGA bounds to H. pylori HSP70 (product of the DnaK gene) with 26-times higher affinity than to human HSP70 and induced large conformational changes as observed from surface plasmon resonance and circular dichroism. Binding of GGA suppressed the activity of the H. pylori chaperone. GGA also altered several characteristics of H. pylori cells. GGA-treated cells elicited enhanced interleukin-8 production by gastric cancer cell lines and potentiated susceptibility to complement as compared to untreated cells. GGA also caused morphological alterations in H. pylori as reflected in fewer coccoid-like cells, suggesting that GGA converts H. pylori to an actively dividing, spiral state (vegetative form) from a non-growing, coccoid state. This morphological conversion by GGA resulted in accelerated growth of H. pylori. These results suggest a model in which GGA sensitizes H. pylori to antibiotic treatment by converting the cells to an actively growing state.

Vibrio vulnificus is the causative agent of primary septicemia, wound infection and gastroenteritis in immunocompromised people. In this study, signature-tagged mutagenesis (STM) was applied to identify the virulence genes of V. vulnificus. Using STM, 6,480 mutants in total were constructed and divided into 81 sets (INPUT pools); each mutant in a set was assigned a different tag. Each INPUT pool was intraperitoneally injected into iron-overloaded mice, and in vivo surviving mutants were collected from blood samples from the heart (OUTPUT pools). From the genomic DNA of mixed INPUT or OUTPUT pools, digoxigenin-labeled DNA probes against the tagged region were prepared and used for dot hybridization. Thirty tentatively attenuated mutants, which were hybridized clearly with INPUT probes but barely with OUTPUT probes, were negatively selected. Lethal doses of 11 of the 30 mutants were reduced to more than 1/100; of these, the lethal doses of 2 were reduced to as low as 1/100,000. Transposon-inserted genes in the 11 attenuated mutants were those for IMP dehydrogenase, UDP-N-acetylglucosamine-2-epimerase, aspartokinase, phosphoribosylformylglycinamidine cyclo-ligase, malate Na (+) symporter and hypothetical protein. When mice were immunized with an attenuated mutant strain into which IMP dehydrogenase had been inserted with a transposon, they were protected against V. vulnificus infection. In this study, we demonstrated that the STM method can be used to search for the virulence genes of V. vulnificus.

Subtilisin-like proteases have been grouped into six families based on a sequence of the catalytic domain. One of the six is the kexin family, of which furin is a representative protease. All members of the kexin family, except one, are from eukaryotes. The one prokaryotic protease is a serine protease of Aeromonas sorbria (ASP). Here, we examined the substrate specificity of ASP based on the cleavage of short peptides. The results showed that ASP preferentially cleaves the peptide bond following two basic residues, one of which is Lys, but not the bond following a single basic residue. This indicates that the tertiary structure around the catalytic domain of ASP resembles, but is not identical to that of furin. Prekallikrein was cleaved into four fragments by ASP, indicating that the protein must be cleaved at specific sequences.

The bacterium Clostridium botulinum type C produces a progenitor toxin (C16S toxin) that binds to O-linked sugar chains terminating with sialic acid on the surface of HT-29 cells prior to internalization [A. Nishikawa, N. Uotsu, H. Arimitsu, J.C. Lee, Y. Miura, Y. Fujinaga, H. Nakada, T. Watanabe, T. Ohyama, Y. Sakano, K. Oguma, Biochem. Biophys. Res. Commun. 319 (2004) 327–333] [21]. Based on this, it was hypothesized that the C16S toxin is internalized via clathrin-coated pits. To examine this possibility, the internalized toxin was observed with a fluorescent antibody using confocal laser-scanning microscopy. The confocal images clearly indicated that the C16S toxin was internalized mainly via clathrin-coated pits and localized in early endosomes. The toxin was colocalized with caveolin-1 which is one of the components of caveolae, however, implying the toxin was also internalized via caveolae. The confocal images also showed that the neurotoxin transported to the endosome was transferred to the Golgi apparatus. However, the non-toxic components were not merged with the Golgi marker protein, TGN38, implying the neurotoxin was dissociated from progenitor toxin in endosomes. These results suggested that the C16S toxin was separated to the neurotoxin and other proteins in endosome and the neurotoxin was further transferred to the Golgi apparatus which is the center for protein sorting.

The 650 kDa large toxin complex (L-TC) produced by Clostridium botulinum serotype D strain 4947 (D-4947) has a subunit structure composed of unnicked components, i.e. neurotoxin (NT), non-toxic non-haemagglutinin (NTNHA) and three haemagglutinin subcomponents (HA-70, HA-33 and HA-17). In this study, subunit interactions were investigated through the susceptibilities of the toxin components to limited trypsin proteolysis. Additionally, complex forms were reconstituted in vitro by various combinations of individual components. Trypsin treatment of intact D-4947 L-TC led to the formation of mature L-TC with nicks at specific sites of each component, which is usually observed in other strains of serotype D. NT, NTNHA and HA-17 were cleaved at their specific sites in either the single or complex forms, but HA-33 showed no sign of proteolysis. Unlike the other components, HA-70 was digested into random fragments as a single form, but it was cleaved into two fragments in the complex form. Based on the relative position of exposed or hidden regions of the individual components in the complex derived from their tryptic susceptibilities, an assembly model is proposed for the arrangement of individual subunits in the botulinum L-TC.

Interleukin 8 (IL8) is usually produced in both epithelial and monocytic cells during bacterial infections, causing inflammation. Helicobacter pylori induces production of IL8 from gastric epithelial cells via its cag pathogenicity island (cag PAI) system, LPS and outer-membrane protein. In some bacteria, heat-shock protein 60 (HSP60) also elicits a strong pro-inflammatory response in cells of the innate immune system. Three recombinant H. pylori HSP60 (rHSP60) proteins of different sizes were produced and one of these was used to raise two monoclonal antibodies (2E7 and 7B5). IL8 production was found to be induced in cultured monocytic cells treated with H. pylori cells or rHSP60 proteins, as measured by ELISA, and the amount of IL8 produced was dose-dependent. Pre-incubation of H. pylori cells or rHSP60 preparations with the antibody 2E7 significantly inhibited IL8 production from monocytic cells. These results indicated that HSP60 is closely associated with IL8 production in monocytic cells.

Clostridium botulinum type C 16S progenitor toxin consists of a neurotoxin (NTX), a non-toxic non-HA (NTNH), and a haemagglutinin (HA). The HA acts as an adhesin, allowing the 16S toxin to bind to intestinal epithelial cells and erythrocytes. In type C, these bindings are dependent on sialic acid. The HA consists of four distinct subcomponents designated HA1, HA2, HA3a and HA3b. To identify the binding subcomponent(s) of HA of type C 16S toxin, all of the HA-subcomponents and some of their precursor forms were produced as recombinant proteins fused to glutathione S-transferase (GST). These proteins were evaluated for their capacity to adhere to intestinal epithelial cells of guinea pig and human erythrocytes. GST-HA1, GST-HA3b and GST-HA3 (a precursor form of HA3a and HA3b) bound intestinal epithelial cells and erythrocytes, whereas GST alone, GST-HA2 and GST-HA3a did not. GST-HA3b and GST-HA3 showed neuraminidase-sensitive binding to the intestinal epithelial cells and erythrocytes, whereas GST-HA1 showed neuraminidase-insensitive binding. TLC binding assay revealed that GST-HA3b and GST-HA3 recognized sialosylparagloboside (SPG) and GM3 in the ganglioside fraction of the erythrocytes, like native type C 16S toxin [Inoue, K. et al. (1999). Microbiology 145, 2533-2542]. On the other hand, GST-HA1 recognized paragloboside (PG; an asialo- derivative of SPG) in addition to SPG and GM3. Deletion mutant analyses of GST-HA3b showed that the C-terminal region of HA3b is important for its binding activity. Based on these data, it is concluded that the HA component contains two distinct carbohydrate-binding subcomponents, HA1 and HA3b, which recognize carbohydrates in different specificities.

Two proteins representing the heavy-chain subunits of botulinum neurotoxin types C and D were expressed in Escherichia coli , and their vaccine potential was evaluated. Mice were vaccinated with doses ranging from 0.5 to 10 μg and were challenged with 10 to 10 5 50% lethal doses of toxin. For the type C subunit protein, C50, two doses of 2 μg were required for full protection, while, for type D subunit protein, D50, two 1-μg doses were required. A bivalent vaccine consisting of a mixture of these two proteins also provided protection against both botulinum neurotoxin type C and type D challenge. Antibody levels in serum were determined by both enzyme-linked immunosorbent assays and serum neutralization assays

最も繁用されている化粧品防腐剤であるパラベンは, 近年次々とその有害性が報告されている。そこでわれわれはピオニンと1,2-pentanediolをパラベンのように化粧品防腐剤として使用できるかを細菌学的見地から評価した。市販化粧品にピオニンを添加して抗菌活性を検討し, ピオニンはさまざまな化粧品製剤において強力な抗菌活性を発揮することを確認した。また, ローション製剤においてピオニンと1,2-pentanediolの併用添加は, パラベン添加よりも抗菌活性が上回ることを見出した。さらにパラベン無添加で行ったピオニンと1,2-pentanediol含有クリーム製剤中での抗菌試験では, 局方における防腐剤の条件を十分満たすことが明らかとなった。この製剤を健常ヒト皮膚へ7日間連続塗布し検討したところ, 皮膚常在菌数の日常変動に対して有意な影響を及ぼさなかった。これらよりピオニンと1,2-pentanediolの併用は, 化粧品製剤の新しい防腐処方となることが示唆された。

G12 rotaviruses are emerging rotavirus strains causing severe diarrhea in infants and young children worldwide. However, the whole genomes of only a few G12 strains have been fully sequenced and analyzed. In this study, we sequenced and characterized the complete genomes of six G12 strains (RVA/Human-tc/MMR/A14/2011/G12P[8], RVA/Human-tc/MMR/A23/2011/G12P[6], RVA/Human-tc/MMR/A25/2011/G12P[8], RVA/Human-tc/MMR/P02/2011/G12P[8], RVA/Human-tc/MMR/P39/2011/G12P[8], and RVA/Human-tc/MMR/P43/2011/G12P[8]) detected in six stool samples from children with acute gastroenteritis in Myanmar. On whole genomic analysis, all six Myanmarese G12 strains were found to have a Wa-like genetic backbone: G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 for strains A14, A25, P02, P39, and P43, and G12-P[6]-I1-R1-C1-M1-A1-N1-T1-E1-H1 for strain A23. Phylogenetic analysis showed that most genes of the six strains examined in this study were genetically related to globally circulating human G1, G3, G9, and G12 strains. Of note is that the NSP4 gene of strain A23 exhibited the closest relationship with the cognate genes of human-like bovine strains as well as human strains, suggesting the occurrence of reassortment between human and bovine strains. Furthermore, strains A14, A25, P02, P39, and P43 were very closely related to one another in all the 11 gene segments, indicating derivation of the five strains from a common origin. On the other hand, strain A23 consistently formed distinct clusters as to all the 11 gene segments, indicating a distinct origin of strain A23 from that of strains A14, A25, P02, P39, and P43. To our knowledge, this is the first report on whole genome-based characterization of G12 strains that have emerged in Myanmar. Our observations will provide important insights into the evolutionary dynamics of spreading G12 rotaviruses in Asia.

Clostridium botulinum types C and D produce a 16 S (500 kDa) toxin that is formed by conjugation of neurotoxin with a non-toxic component (nonTox). The amino acid sequences of type C and D nonTox components are almost identical. In a previous report it was proposed that nonTox is necessary for the effective absorption of the toxin from the small intestine. This suggested the hypothesis that mucosal immunity against nonTox in the small intestine might prevent the absorption of both C- and D-16 S toxins. The nonTox was purified from a mutant strain, (C)-N71, that does not produce neurotoxin. This nonTox or detoxified C-16 S toxin were mixed with adjuvant (a mutant form of heat-labile toxin of Escherichia coli), and inoculated into mice via the nasal or oral route, or both. The mice inoculated nasally four times with nonTox or toxoid produced high levels of antibodies (including IgA) against the immunogens, both in intestinal fluids and sera. When these nonTox-immunised mice were challenged orally with 2 and 20 oral minimum lethal doses (MLD) of C- or D-16 S toxins, the same results were obtained with both C and D; the mice survived after challenge with 2 MLD of either C or D but were killed by 20 MLD of either toxin although the time to death was significantly longer than in the control non-immunised mice. These results indicate that the local anti-nonTox antibodies reduce absorption of both C- and D-16 S toxins from the small intestine. The C-16 S toxoid-immunised mice showed similar behaviour with type D toxin challenge, probably due to the same mechanism, but were protected against 20 MLD of C-16 S toxin.

A molecular chaperonin in mammals, heat shock protein 60 (HSP60) is constitutively expressed in the mitochondria at a low level and is rapidly up-regulated under stress. However, the role of HSP60 in the lymphoid tissues has not been well clarified. In the present study, expression of HSP60 was examined by flow cytometry and immunohistochemistry in normal peripheral blood mononuclear cells, reactive lymphoid tissues, and malignant lymphomas. HSP60 was found to be present constitutively at low levels in a fraction of resting T cells and most monocytes. The blastic change upon mitogen stimulation induced HSP60 at much higher levels in more T, B and NK cells. In normal lymphoid tissues, HSP60 was expressed preferentially in the cytoplasm of large-sized lymphoid cells and macrophages in the germinal centers and the interfollicular area.In non-Hodgkin lymphomas strong expression of HSP60 was detected in most cases of diffuse large B-cell lymphoma, follicular lymphoma, and grade 3 and NK/T cell lymphoma. No immunostaining was observed in low grade B-cell lymphomas, including follicular lymphoma, grade 1 and B-lymphoblastic lymphomas. HSP60 immunoreactivity was variable in T-cell lymphomas. Intense expression of HSP60 was observed in Reed-Sternberg cells in all cases of Hodgkin lymphoma.

Using SDS-PAGE, we found that one subcomponent, hemagglutinin (HA-33), from the Clostridium botulinum progenitor toxin of type D strain 1873 and type C strain Yoichi had slightly smaller molecular sizes than those of type C and D reference strains, but other components did not. Based on N- and C-terminal sequence analyses of HA-33, a deletion of 31 amino acid residues from the C-terminus at a specific site was observed in the HA-33 proteins of both strains. The progenitor toxins from both strains showed poor hemagglutination activities, titers of 2(1) or less, which were much lower than titers from the reference strains (2(6)), and did not bind to erythrocytes. These results suggest strongly that the short C-terminal region of the HA-33 plays an essential role in the hemagglutination activity of the botulinum progenitor toxin. Additionally, a sequence motif search predicted that the C-terminal region of HA-33 has a carbohydrate-recognition subdomain.

Haemagglutinin (HA) activity of Clostridium botulinum type A 19S and 16S toxins (HA-positive progenitor toxin; HA(+)-PTX) was characterized. HA titres against human erythrocytes of HA(+)-PTX were inhibited by the addition of lactose, D-galactose, N-acetyl-D-galactosamine and D-fucose to the reaction mixtures. A direct glycolipid binding test demonstrated that type A HA(+)-PTX strongly bound to paragloboside and some neutral glycolipids, but did not bind to gangliosides. Type A HA(+)-PTX also bound to asialoglycoproteins (asialofetuin, neuraminidase-treated transferrin), but not to sialoglycoproteins (fetuin, transferrin). Although glycopeptidase F treatment of asialofetuin abolished the binding of HA(+)-PTX, endo-alpha-N-acetylgalactosaminidase treatment did not. Thus these results can be interpreted as indicating that type A HA(+)-PTX detects and binds to Gal beta 1-4GlcNAc in paragloboside and the N-linked oligosaccharides of glycoproteins. Regardless of neuraminidase treatment, type A HA(+)-PTX bound to glycophorin A which is a major sialoglycoprotein on the surface of erythrocytes. Both native glycophorin A and neuraminidase-treated glycophorin A inhibited the binding of erythrocytes to type A HA(+)-PTX. Since the N:-linked oligosaccharide of glycophorin A is di-branched and more than 50% of this sugar chain is monosialylated, type A HA(+)-PTX probably bound to the unsialylated branch of the N-linked oligosaccharide of glycophorin A and agglutinated erythrocytes. One subcomponent of HA, designated HA1, did not agglutinate native erythrocytes, although it did bind to erythrocytes, paragloboside and asialoglycoproteins in a manner quite similar to that of HA(+)-PTX. These results indicate that type A HA(+)-PTX binds to oligosaccharides through HA1.

In order to clarify the presence and verotoxin (VT) inhibitory activity of globotriaosylceramide (Gb3) in bovine milk, we analyzed neutral glycosphingolipids (GSLs) from bovine milk and investigated the inhibitory effect of bovine milk Gb3 on the cytotoxicity of VT2. Five species of neutral GSLs, designated as N-1, N-2, N-3, N-4, and N-5, were separated on thin-layer chromatography (TLC). N-1, N-2, and N-3 showed the same mobility as glucosylceramide, lactosylceramide, and Gb3 on the TLC plate, respectively. N-4 and N-5 GSLs migrated below globoside on the TLC plate. N-3 GSL having the same TLC mobility as Gb3 from bovine milk was immunologically identified as Gb3 by monoclonal antibody against Gb3, anti-CD77 monoclonal antibody. Furthermore, the effect of bovine milk Gb3 on VT2-induced cytotoxicity was investigated. We found that treatment of VT2 with bovine milk Gb3 can reduce the cytotoxic effect of VT2.

The effect of intestinal IgA antibody against the receptor for verotoxin (VT), globotriaosylceramide (Gb3), on VT-mediated cytotoxicity was examined. Intestinal IgA antibodies against Gb3 were prepared by oral immunization of mice with Gb3 and adjuvant monophosphoryl lipid A (MPL)-containing liposomes composed of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylserine and cholesterol (1 : 1 : 2, molar ratio) (PS-liposome). Oral administration with Gb3 and MPL-containing PS-liposome induced significant IgA responses to Gb3 in the intestinal lavage fluid in all mice tested. Furthermore, anti-Gb3 IgA antibodies in the lavage fluid effectively inhibited the cytotoxicity of VT2 to Vero cells in a dose-dependent manner. These results suggest that anti-Gb3 IgA antibodies produced in the intestinal tract, upon oral immunization with Gb3-containing liposome, function as inhibitors against VT and also indicate the potential usefulness of oral PS-liposome vaccines containing MPL for the induction of a protective mucosal immune response against intestinal diseases.

Type E botulinum toxin (BoNT/E)-producing Clostridium butyricum strains isolated from botulism cases or soil specimens in Italy and China were analyzed by using nucleotide sequencing of the bont/E gene, random amplified polymorphic DNA (RAPD) assay, pulsed-field gel electrophoresis (PFGE), and Southern blot hybridization for the bont/E gene. Nucleotide sequences of the bont/E genes of 11 Chinese isolates and of the Italian strain BL 6340 were determined. The nucleotide sequences of the bont/E genes of 11 C. butyricum isolates from China were identical. The deduced amino acid sequence of BoNT/E from the Chinese isolates showed 95.0 and 96.9% identity with those of BoNT/E from C. butyricum BL 6340 and Clostridium botulinum type E, respectively. The BoNT/E-producing C. butyricum strains were divided into the following three clusters based on the results of RAPD assay, PFGE profiles of genomic DNA digested with Sma Io rXhoI, and Southern blot hybridization: strains associated with infant botulism in Italy, strains associated with food-borne botulism in China, and isolates from soil specimens of the Weishan lake area in China. A DNA probe for the bont/E gene hybridized with the nondigested chromosomal DNA of all toxigenic strains tested, indicating chromosomal localization of the bont/E gene in C. butyricum. The present results suggest that BoNT/E-producing C. butyricum is clonally distributed over a vast area. Type E botulinum toxin (BoNT/E)-producing Clostridium butyricum was first isolated from two cases of infant botulism in Italy in 1984 (1, 8). In 1997, we isolated BoNT/E-producing C. butyricum from the food implicated in food-borne botulism in China (10). Because our results indicated that type E food-borne botulism can be caused by BoNT/E-producing C. butyricum, we reexamined the cultural and biochemical properties of BoNT/E-producing organisms that had previously been isolated from type E food-borne botulism cases and found that two isolates were identifiable as C. butyricum (9). In addition, we isolated several strains of BoNT/E-producing C. butyricum from soil specimens of China (9). In 1998, an outbreak of food-borne botulism was reported in India and was strongly suggested to be caused by BoNT/E-producing C. butyricum (2). These studies indicate that soil is the principal habitat of BoNT/E-producing C. butyricum and that this organism may be widely distributed throughout the world (9). For improved surveillance of BoNT/E-producing C. butyricum, biochemical and genetic analysis of this organism is required. In this study, we performed molecular analysis of the strains isolated in Italy and China, by using nucleotide sequencing of the bont/E gene, random amplified polymorphic DNA (RAPD) assay, pulsed-field gel electrophoresis (PFGE), and Southern blot hybridization for the bont/E gene.

Infection with Helicobacter pylori induces humoral immune responses against various antigens of the bacterium. Heat shock proteins (hsps) are immunodominant antigens in various diseases including H. pylori infection. In the present study, we measured the anti-hsp antibody titers in 42 patients with H. pylori -infected peptic ulcers during a bacterial eradication study. The patients were treated with a proton pump inhibitor and antimicrobial agents to eradicate the organism. Their sera were obtained at pretreatment and at 1 month and 6 months after the eradication therapy. The titers of immunoglobulin G antibodies to the H. pylori hsp, whole-cell lysate, and urease (30-kDa subunit) antigens in serum were measured by a capture enzyme-linked immunosorbent assay. The levels of H. pylori hsp60 antibodies in sera collected 1 month after treatment had declined significantly, even when changes in the titers of antibodies to whole-cell and urease antigens were not apparent. These results suggest that measurement of antibodies to H. pylori hsp60 in serum is useful for the early monitoring of the effectiveness of eradication therapy.

Helicobacter pylori is a major etiological agent in gastroduodenal disorders. The adhesion of H. pylori to gastric epithelial cells is the initial step of H. pylori infection. Inhibition of H. pylori adhesion is thus a therapeutic target in the prevention of H. pylori infection. We have reported that rebamipide and ecabet sodium, mucoprotective antiulcer agents, independently inhibit H. pylori adhesion. However, the antiadhesion activity of each antiulcer agent was incomplete. Experiments were performed to evaluate the combined effect of rebamipide and ecabet sodium on H. pylori adhesion to gastric epithelial cells. MKN-28 and MKN-45 cells, derived from human gastric carcinomas, were used as target cells. Twelve clinical isolates of H. pylori were used in this study. We evaluated the effects of rebamipide and ecabet sodium, individually and in combination, on H. pylori adhesion to target cells quantitatively using our previously established enzyme-linked immunosorbent assay. Rebamipide and ecabet sodium each partially inhibited H. pylori adhesion. In contrast, adhesion was almost completely inhibited by pretreating target cells and H. pylori with the combination of rebamipide and ecabet sodium. Our studies suggest that the synergistic antiadhesion activity of rebamipide and ecabet sodium is greater than that of each antiulcer agent alone.

The 16S toxin and one subcomponent of haemagglutinin (HA), designated HA1, were purified from a type D culture of Clostridium botulinum by a newly established procedure, and their HA activities as well as that of purified type C 16S toxin were characterized. SDS-PAGE analysis indicated that the free HA1 forms a polymer with a molecular mass of approximately 200 kDa. Type C and D 16S toxins agglutinated human erythrocytes in the same manner. Their HA titres were dramatically reduced by employing erythrocytes that had been previously treated with neuraminidase, papain or proteinase K, and were inhibited by the addition of N-acetylneuraminic acid to the reaction mixtures. In a direct-binding test to glycolipids such as SPG (NeuAc alpha2-3Gal beta1-4GlcNAc beta1-3Gal beta1-4Glc beta1-Cer) and GM3 (NeuAc alpha2-3Gal beta1-4Glc beta1-Cer), and glycoproteins such as glycophorin A and/or B prepared from the erythrocytes, both toxins bound to sialylglycolipids and sialoglycoproteins, but bound to neither neutral glycolipids nor asialoglycoproteins. On the basis of these results, it was concluded that type C and D 165 toxins bind to erythrocytes through N-acetylneuraminic acid. HA1 showed no haemagglutination activity, although it did bind to sialylglycolipids. We therefore speculate that binding to glycoproteins rather than to glycolipids may be important in causing haemagglutination by type C and D 16S toxins.

BACKGROUND—Development of gastric mucosa associated lymphoid tissue (MALT) lymphoma is thought to be closely associated with host immune reactions to Helicobacter pylori. AIM—To investigate humoral immune responses in patients with MALT lymphoma to antigens shared by H pylori and human gastric epithelial cells. METHODS—Sera were obtained from H pylori positive patients with MALT lymphoma (n = 11) or other gastroduodenal diseases (peptic ulcer, n = 40; non-ulcer dyspepsia, n = 20) and from H pylori negative healthy control subjects (n = 10). Antibodies to HGC-27 human gastric epithelial cells and human recombinant heat shock protein (Hsp) 60 were examined using an enzyme linked immunosorbent assay (ELISA) and immunoblotting. RESULTS—Antibody titres to HGC-27 cells were significantly elevated in H pylori positive patients with MALT lymphoma when compared with titres in patients with other gastroduodenal diseases and in healthy subjects. Immunoblotting of sera from patients with MALT lymphoma often detected a band with a molecular mass corresponding to Hsp60, and both ELISA and immunoblotting showed elevated antibody titres to the recombinant human Hsp60. Antigenic similarity between Hsp60 and H pylori HspB was documented by immunoblotting experiments. CONCLUSIONS—Autoantibodies reactive with host gastric epithelial cells are often increased in MALT lymphoma, and Hsp60 is a major target antigen. Immune responses induced by immunological cross reactivity between H pylori HspB and human Hsp60 in gastric epithelium may be involved in the development of MALT lymphoma. Keywords: Helicobacter pylori; mucosa associated lymphoid tissue lymphoma; heat shock protein; autoantibody