Cytokine responses to recombinant cholera toxin B subunit produced by Bacillus brevis as a mucosal adjuvant.

We attempted to clarify the mechanism of the mucosal adjuvanticity of recombinant cholera toxin B subunit (rCTB), which is inherently uncontaminated with the holotoxin produced by Bacillus brevis and has a powerful mucosal adjuvant activity, on cytokine responses compared with that of cholera toxin (CT). rCTB had no ability to stimulate cyclic AMP formation in mouse peritoneal macrophages (Mphi). Cytokine production by non-immunized Mphi cultured with rCTB or CT and by the spleen cells of mice co-immunized intranasally with ovalbumin (OVA) and rCTB or CT was examined. rCTB alone did not induce interleukin (IL)-1alpha/beta or IL-6 production by Mphi, but combination of rCTB with lipopolysaccharide (LPS) enhanced both IL-1alpha/beta production. Conversely, CT plus LPS suppressed IL-1alpha/beta production more than LPS alone. Both rCTB and CT suppressed IL-12 secretion induced by interferon gamma (IFN gamma) plus LPS. IL-2, IL-4, IL-5, and IL-10 were secreted by mouse spleen cells restimulated with OVA after intranasal co-administration of OVA together with rCTB, and in response to CT, the same cytokines were secreted. The different effect of rCTB on Mphi from that of CT may mean a difference between the mechanisms of rCTB and CT during the early stage of an immune response.

Mucosal immunization against hepatitis B virus by intranasal co-administration of recombinant hepatitis B surface antigen and recombinant cholera toxin B subunit as an adjuvant.

Recombinant cholera toxin B subunit (rCTB) produced by Bacillus brevis carrying pNU212-CTB has been previously found to be a potent mucosal adjuvant to aluminium-non-adsorbed tetanus toxoid (nTT) and diphtheria toxoid (nDT) co-administered intranasally, and the possibility of needle-free inoculation of these vaccines with rCTB has been suggested. In this paper we examined the potentiality of rCTB as a mucosal adjuvant to aluminium-non-adsorbed yeast-derived recombinant hepatitis B surface antigen (rHBs) being a particulate antigen when administered intranasally with rCTB. In-house ELISA showed that a mixture of rHBs (1 or 5 microg) and rCTB (10 microg) elevated not only systemic responses but also mucosal immune responses at the nasal cavity, the lung, the saliva, the small intestine and the vagina against rHBs, and these could be further increased with higher doses of antigen. With antibody isotypes of IgG, there were equally high levels of serum HBs-specific IgG1, IgG2a and IgG2b antibodies and induction of mixed Th1- and Th2-type responses was considered to occur in combination of rHBs and rCTB. Serum anti-HBs titres in almost all mice obtained from sandwich EIA using a commercial kit were higher than 1000 milli-international units ml(-1) (mIU ml(-1)). These results show that rCTB is also very effective as a mucosal adjuvant for a particulate antigen like rHBs, as well as soluble antigens like nTT and nDT reported previously, suggesting the possibility of intranasal immunization with rHBs plus rCTB in humans.

Efficient extracellular production of recombinant Escherichia coli heat-labile enterotoxin B subunit by using the expression/secretion system of Bacillus brevis and its mucosal immunoadjuvanticity.

A gene encoding the mature Escherichia coli heat-labile enterotoxin B subunit (LTB) was introduced in a vector pNU212 and expressed at high levels in Bacillus brevis HPD31. The maximum amount of recombinant LTB (rLTB) secreted into the modified 5PY medium containing erythromycin was about 350 mg l(-1) when cultivated at 30 degrees C for 8 days. The rLTB purified directly from the culture supernatant by using D-galactose immobilized agarose was identical to the native LTB with respect to the molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the amino terminal amino acid sequence. Western blot analysis with antiserum to cholera toxin B subunit (CTB) indicated that rLTB had cross-reactivity to native CTB and its GM1 binding ability was almost the same as that of the CTB. The rLTB predominantly showed the pentameric form when non-boiled samples were applied to SDS-PAGE. When rLTB was administered intranasally to mice with diphtheria toxoid (D(T)), it resulted in the substantial stimulation of D(T)-specific serum IgG antibody, and in the induction of moderate levels of D(T)-specific mucosal IgA antibody responses in the nasal cavities and in the lung, suggesting that purified rLTB acts as a promising immunoadjuvant on mucosal immunizations.

Safety evaluation of recombinant cholera toxin B subunit produced by Bacillus brevis as a mucosal adjuvant.

Mucosal immune responses are known to play important roles in the establishment of protective immunity to microbial infections through mucosa. We examined the toxic effects of recombinant cholera toxin B subunit (rCTB) secreted by Gram-positive bacterium Bacillus brevis as a mucosal adjuvant. Incubation of guinea-pig peritoneal macrophages with cholera toxin (CT) or aluminium hydroxide gel (Al-gel) released a significantly higher activity of lactate dehydrogenase than did commercial natural CTB (CTB) or rCTB. Intraintestinal or intramuscular administration of CT, CTB or Al-gel caused severe histopathological reactions. CT also caused infiltration of neutrophils and irregular arrangement or partial loss of the respiratory epithelium. In addition, CT and CTB elicited vascular permeability-increasing effects. rCTB elicited no toxic effects to macrophages and no vascular permeability-increasing effects. Moreover, it is noticeable that no distinct local histopathological reactions were observed in the nasal cavity, the small-intestinal loop or the muscle given rCTB. These results suggest that, from a safety standpoint, rCTB is a useful candidate as mucosal vaccine adjuvant.

Induction of systemic and mucosal antibody responses in mice immunized intranasally with aluminium-non-adsorbed diphtheria toxoid together with recombinant cholera toxin B subunit as an adjuvant.

Nasal mucosal immunization is very attractive for vaccination to prevent various bacterial and viral infectious diseases because of induction of systemic and mucosal immune responses. The aim of the present study was to investigate the possibility of changing the immunization procedure of diphtheria toxoid (DT) from intramuscular or subcutaneous injection to intranasal administration. Intranasal immunization with aluminium-non-adsorbed diphtheria toxoid (nDT) together with recombinant cholera toxin B subunit (rCTB, 10 microg) induced, at a concentration of 5 Lf, high levels of serum DT-specific IgG antibody responses and high or moderate levels of the specific IgA antibody responses in all mice and only a slight level of the specific IgE antibody responses in some mice. Furthermore, sufficiently high diphtheria antitoxin titres more than 0.1 international units (IU) ml(-1) were obtained from mice which showed high levels of serum DT-specific IgG antibody responses. Under the same experimental conditions, induction of significant levels of mucosal DT-specific IgA antibody responses occurred in the nasal cavity, the lung, the saliva and vaginal secretions and the small and large intestines of all mice, although there were different titres between individual mice. Similar results were also obtained with rCTB-specific serum IgG and IgA and mucosal IgA antibody responses; serum rCTB-specific IgE antibody titres were not detected. These results show that intranasal administration of nDT with rCTB must be a very useful means for vaccination against diphtheria.

Expression of a germination-specific amidase, SleB, of Bacilli in the forespore compartment of sporulating cells and its localization on the exterior side of the cortex in dormant spores.

A germination-specific amidase of bacilli is a major spore-lytic enzyme that is synthesized with a putative signal sequence and hydrolyses spore cortex in situ. The sleB gene encoding this amidase in Bacillus subtilis and Bacillus cereus was expressed in the forespore compartment of sporulating cells under the control of sigmaG, as shown by Northern blot and primer extension analyses. The forespore-specific expression of B. subtilis sleB was further indicated by the forespore-specific accumulation of a SleB-green fluorescent protein fusion protein from which a putative secretion signal of SleB was deleted. Immunoelectron microscopy with anti-SleB antiserum and a colloidal gold-immunoglobulin G complex showed that the enzymes from both Bacillus species are located just inside the spore coat layer in the dormant spore, and in the dormant spore, the amidases appear exist in a mature form lacking a signal sequence. These results indicate that SleB is translocated across the forespore's inner membrane by a secretion signal peptide and is deposited in cortex layer synthesized between the forespore inner and outer membranes. The peripheral location of the spore-lytic enzymes in the dormant spore suggests that spore germination is initiated at the exterior of the cortex.

Intranasal or subcutaneous co-administration of recombinant cholera toxin B subunit stimulates only a slight or no level of the specific IgE response in mice to tetanus toxoid.

Whether recombinant cholera toxin B subunit (rCTB) co-administered intranasally or subcutaneously with aluminium-non-adsorbed tetanus toxoid (nTT) can induce the production of tetanus toxoid (TT)-specific IgE antibodies in mice was investigated compared with aluminium-adsorbed tetanus toxoid (aTT) administered intranasally or subcutaneously. Mice immunized intranasally or subcutaneously with nTT together with rCTB showed a high level of TT-specific serum IgG antibody response and no or a slight level of TT-specific serum IgE antibody response. On the other hand, in mice vaccinated intranasally or subcutaneously with aTT alone, higher levels of TT-specific IgG and IgE antibodies were induced in comparison with intranasal or subcutaneous inoculation of nTT together with rCTB. These results suggest that intranasal or subcutaneous co-administration of rCTB with nTT is better than intranasal or subcutaneous administration of aTT to avoid IgE-mediated allergic reactions.

Cloning of a novel gene yrbB, encoding a protein located in the spore integument of Bacillus subtilis.

A DNA fragment (2.7 kbp) containing three deduced open reading frames, orf1, orf2 and orf3 (partial sequence), was isolated from the genomic library of Bacillus subtilis using an antiserum raised against spore integument, and was sequenced. orf2 was 519 nucleotides long and encoded a protein of 172 amino acids with a predicted molecular size of 19,552, corresponding to the protein which reacted with the antiserum. Immunoelectron microscopic observation indicated that YrbB, the product of orf2, was located within the spore integument, mainly in the cortex layer with a part in the inner region of the coat layer.

Protein W, a spore-specific protein in Myxococcus xanthus, formation of a large electron-dense particle in a spore.

The gene for the major spore-specific protein, termed protein W, was cloned, and it was found that protein W is composed of 426 amino acid residues including 31% charged (133 residues) and 39% hydrophobic (166 residues) amino acids. In the protein, a motif consisting of five amino acid residues [(V/L/I)-R-E-R-(V/L/I)] is repeated 28 times, and another motif [M-M-(P/G)-Q-G] five times. Protein W is synthesized during a very late stage of development, forming a single, large electron-dense particle (200-400 nm in diameter) inside a spore. X-ray microanalysis of the particle revealed that it contains a high amount of phosphate in addition to calcium and magnesium. It is proposed that protein W consisting of highly charged repetitive sequences is a polyphosphate storage protein to store energy in spores. The disruption of the gene for protein W resulted in delayed fruiting body formation and a lower spore yield.

Systemic and mucosal immune responses of mice to aluminium-adsorbed or aluminium-non-adsorbed tetanus toxoid administered intranasally with recombinant cholera toxin B subunit.

For the purpose of changing the immunization procedure of tetanus toxoid from intramuscular or subcutaneous injection, which has been in practice for a long time, to intranasal administration, we examined systemic and mucosal immune responses of mice to aluminium-adsorbed tetanus toxoid (aTT) and aluminium-non-adsorbed tetanus toxoid (nTT) inoculated intranasally with recombinant cholera toxin B subunit (rCTB). Intranasal immunization with aTT induced, at a concentration of 0.5 Lf, high levels of TT-specific serum IgG antibody titres and moderate levels of TT-specific serum IgA antibody titres in the presence and absence of rCTB. Induction of high or moderate levels of mucosal TT-specific IgA antibody responses was observed with and without rCTB in the lung, the nasal cavity, the small and large intestines and the vagina. Generally speaking, the co-administration of aTT and rCTB showed higher mucosal TT-specific IgA antibody titres when compared with the administration of aTT alone. In case of intranasal administration of nTT, the dose of 5 Lf was necessary and stimulated, only in the presence of rCTB (10 micrograms), high levels of tetanus toxoid (TT)-specific serum IgG antibody responses in all mice examined and moderate or slight levels of TT-specific IgA antibody responses in the nasal, pulmonary and small and large intestinal lavages of a few mice. All mice intranasally immunized with aTT alone or nTT and rCTB escaped onset of tetanus. This is the first report concerned with the mucosal adjuvant activity of an aluminium compound. Judging from these results, intranasal administration of aTT with and without rCTB or nTT with rCTB appears to be a very useful means for a vaccination against tetanus with respect to ease, safety, certainty, low cost and no need for an injection needle.

A spore coat protein, CotS, of Bacillus subtilis is synthesized under the regulation of sigmaK and GerE during development and is located in the inner coat layer of spores.

The spore coat of Bacillus subtilis has a unique morphology and consists of polypeptides of different sizes, whose synthesis and assembly are precisely regulated by a cascade of transcription factors and regulatory proteins. We examined the factors that regulate cotS gene expression and CotS assembly into the coat layer of B. subtilis by Northern blot and Western blot analysis. Transcription of cotS mRNA was not detected in sporulating cells of sigmaK and gerE mutants by Northern blot analysis. By Western blot analysis using anti-CotS antibody, CotS was first detected in protein samples solubilized from wild-type cells at 5 h after the start of sporulation. CotS was not detected in the vegetative cells and spores of a gerE mutant or in the spores of mutants deficient in sigmaE, sigmaF, sigmaG, or sigmaK. CotS was detected in the sporangium but not in the spores of a cotE mutant. The sequence of the promoter region of cotS was similar to the consensus sequences for binding of sigmaK and GerE. These results demonstrate that sigmaK and GerE are required for cotS expression and that CotE is essential for the assembly of CotS in the coat. Immunoelectron microscopic observation using anti-CotS antibody revealed that CotS is located within the spore coat, in particular in the inner coats of dormant spores.

Recombinant cholera toxin B subunit acts as an adjuvant for the mucosal and systemic responses of mice to mucosally co-administered bovine serum albumin.

We examined the mucosal adjuvant activity of recombinant cholera toxin B subunit (rCTB) produced by Bacillus brevis carrying pNU212-CTB by intranasal or oral co-administration of bovine serum albumin (BSA). Intranasal administration stimulated a high level of BSA-specific serum IgG antibody response and BSA-specific IgA antibody responses in the nasal and pulmonary lavages. Oral administration induced a moderate level of BSA-specific serum IgG antibody and a low level of BSA-specific IgA antibody in the large intestinal washes. These results show that CTB alone can act as an intranasal or oral delivery carrier; it also has strong adjuvant properties for stimulating serum IgG and mucosal IgA immune responses to unrelated, non-coupled antigens after intranasal or oral co-immunization.

Localization of germination-specific spore-lytic enzymes in Clostridium perfringens S40 spores detected by immunoelectron microscopy.

The localization of germination-specific spore-lytic enzymes, an amidase and a muramidase, in Clostridium perfringens S40 spores was examined by immunoelectron microscopy with respective antisera raised against the enzymes and a colloidal gold-immunoglobulin G complex. For both antisera, immunogold particles were visualized on the outside of the cortex of dormant spores, and they were not detected in germinated spores and decoated spores.

Expression and secretion of the S2 subunit of pertussis toxin in Bacillus brevis.

We have been trying to develop a mass production system for each of the subunits (S2, S3, S4, S5) of the pertussis toxin B oligomer (PTB) by using a Bacillus brevis-pNU212 system. In consequence a moderately efficient expression-secretion system for S2 was constructed by fusing the mature S2 gene from Bordetella pertussis Tohama with the signal-peptide coding region of pNU212 and by introducing the plasmid pNU212-S2 into B. brevis HPD31 by electroporation. The clone producing S2 secreted about 70 mg of recombinant S2 (rS2) per liter of PY-erythromycin medium after 5 days incubation at 37 degrees C. The rS2 purified by an ammonium sulfate fractionation at 30-50% saturation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was identical to the native S2 in respect to the molecular weight determined by SDS-PAGE and the amino terminal amino acid sequence. The nucleotide sequence of the S2 gene in B. pertussis Tohama inserted into pNU212 was identical with that of the S2 gene in other virulent B. pertussis strains except that an adenine at position 52 of the latter was replaced by a guanine in the former, causing an amino acid substitution (glycine in the former for serine in the latter) at position 18.

[Fragmentation and solubilization of filamentous appendages of Bacillus cereus spores].

Filamentous appendages were removed from the surfaces of Bacillus cereus spores by treatment with sodium thioglycolate and purified by filtration through a glass microfiber paper and a membrane filter. The basic structure of the isolated appendages resembled that of the native appendages on the spores. The results of electron microscopical analysis, amino acid analysis, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) attested the purity of the preparation. The purified filamentous appendages were fragmented by treatment with 2N sodium hydroxide at 30C but neither with SDS nor mercaptoethanol, and the fragmentation progressed with the prolongation of the treatment period. When observed under an electron microscope, each fragmented appendage appeared to be rod shaped with an apparent axial hole and helical or disk-like arrangement of subunits. In the agar gel immunodiffusion test, a soluble fraction of the appendages obtained by treatment with sodium hydroxide formed only a single precipitation line against the antibody to the intact appendages. The molecular mass of the subunit separated by SDS-PAGE was approximately 10kDa. The 10-kDa protein was distributed over the surface of the intact appendages in immunoelectron microscopy. The antigenicity of the appendage subunits disappeared upon treatment with 2N sodium hydroxide for longer than 24h. Neither intact appendages nor intact spores agglutinated sheep or guinea-pig erythrocytes.

Prevalence of pancreatitis in liver diseases of various etiologies: an analysis of 107,754 adult autopsies in Japan.

The frequency of acute and chronic pancreatitis is 3.3 and 2.1%, respectively, in 107,754 adult autopsies in Japan. Acute pancreatitis is highly associated with liver diseases of various etiologies such as subacute hepatitis (16.1%), fulminant hepatitis (13.5%), biliary cirrhosis (10.5%), cholangiocarcinoma (8.6%) and postnecrotic cirrhosis (7.1%). Chronic pancreatitis is also closely related to various liver diseases. It is suggested that the portal venous stasis in liver diseases may predispose the patients to develop pancreatitis regardless of the etiology of liver diseases.

Flow analysis of silicate rocks for zirconium.

A flow analysis system involving the on-line configuration of an anion-exchange column has been examined to enrich and determine trace concentrations of zirconium of several ppm to hundred ppm levels in silicate rocks and minerals. About 100 mg of sample is decomposed by fusion with a mixture of boric acid and lithium carbonate and taken up with 1M hydrochloric acid to a total of 100 ml. Depending upon the concentration of zirconium, either a 1- or 4-ml aliquot is introduced into an aqueous carrier stream, merged with sulphuric acid and passed through a small volume anion-exchange column. The enriched zirconium is then back eluted with hydrochloric acid, colour-developed with Arsenazo III, and detected spectrophotometrically at 665 nm.

Mucin-producing tumor of the pancreas.

A new pancreatic tumor, called mucin-producing tumor, has received great attention in Japan. These tumors are found inside the pancreatic duct and produce large quantities of copious mucus. The authors examined 22 cases of these tumors histologically and histochemically. In 12 malignant cases, the tumors inside the ducts consisted of cancerous lesions over small areas along with papillary or atypical hyperplasia. Tumors in ten benign cases mainly consisted of papillary hyperplasia. Except for three patients with carcinoma in situ, cancerous tumors infiltrated the pancreatic parenchyma and, in some cases, were observed invading the bile duct or duodenum. A mucous histochemical study showed evidence of sialomucin in malignant cases; neutral mucin was dominant in benign cases. Characteristics of this disease were also compared with 13 cases of mucinous cystic neoplasm. From the results, it was concluded that these two diseases can be classified into the same conceptual category.

Permeability of dormant spores of Bacillus subtilis to malachite green and crystal violet.

The permeability of dormant spores of Bacillus subtilis to malachite green (MG) and crystal violet (CV) was examined by using potassium trichloro(eta 2-ethylene)platinum(II) (KTPt) as an electron-opaque marker for the dyes. The spores were treated with the dyes and other substances at 30 degrees C for 30 min or at 80 degrees C for 5 min. When the spores were incubated in 50 mM-MG solution at 30 degrees C and in 50 mM-CV solution at 30 degrees C or 80 degrees C, many small electron-dense precipitates, which were chemical complexes of dyes and platinum, were seen, mainly around the boundary between the inner and outer coat regions. The spores treated under the above conditions were not stained. Treatment with 50 mM-MG alone or a mixture of 25 mM-oxalic acid and 50 mM-CV at 80 degrees C made the spores stainable and dye-TPt precipitates were observed mainly in the outer pericortex region. Pretreatment with 25 mM-oxalic acid and 5% (v/v) phenol at 80 degrees C followed by 50 mM-CV treatment at 30 degrees C gave the same results as above. It was considered from these results that the inner coat itself might function as the primary permeability barrier to MG and CV, and that a secondary barrier to the dyes might exist around the cortex region.

Presence of proteins derived from the vegetative cell membrane in the dormant spore coat of Bacillus subtilis.

To confirm the presence of the outer spore membrane in dormant spore coats of Bacillus subtilis, the proteins from vegetative cell membrane and dormant spore coat fractions were compared by immunoblot assay with antibodies prepared against both preparations. The spore coat fraction contained at least 11 proteins antigenically identical to those in the vegetative cell membranes. Further, the cytochemical localization of the proteins derived from vegetative cell membrane in dormant spores was examined by an immunoelectron microscopy method with a colloidal gold-immunoglobulin G complex. The colloidal gold particles were observed in the coat region and around the core region of dormant spore. These results have provided evidence that some proteins from vegetative cell membrane remain in the dormant spore coat region of B. subtilis, although it is not clear whether the outer membrane persists as an intact functional entity or not.