Vibrio vulnificus infection has attracted special interest because of its high mortality. A strong clinical association exists between hepatic dysfunction and increased morbidity and mortality from V. vulnificus infection. In this study, the effect of C-reactive protein (CRP), a typical hepatogenic acute phase protein, on the lethality induced by V. vulnificus lipopolysaccharide (LPS) was investigated in galactosamine-sensitized mice. The pretreatment of CRP, in a dose of at least 2mg/kg, 2hr before the challenge of LPS completely protected mice against the lethality by V. vulnificus LPS. The elevation of serum tumor necrosis factor-α (TNF-α) induced by LPS administration was not affected by CRP pretreatment. However, the LPS-or TNF-α-induced hepatotoxicity was completely prevented by CRP. These results indicate that CRP does not prevent the synthesis, but prevents the hepatotoxic action of TNF-α. The possibility that impaired production of acute phase proteins in patients with pre-existing hepatic dysfunction may predispose the higher risk of V. vulnificus infection needs to be evaluated further.

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A gene (apk) encoding the extracellular protease of Aeromonas caviae Ae6 has been cloned and sequenced. For cloning the gene, the DNA genomic library was screened using skim milk LB agar. One clone harboring plasmid pKK3 was selected for sequencing. Nucleotide sequencing of the 3.5kb region of pKK3 revealed a single open reading frame (ORF) of 1, 785bp encoding 595 amino acids. The deduced polypeptide contained a putative 16-amino acid signal peptide followed by a large propeptide. The N-terminal amino acid sequence of purified recombinant protein (APK) was consistent with the DNA sequence. This result suggested a mature protein of 412 amino acids with a molecular mass of 44kDa. However, the molecular mass of purified recombinant APK revealed 34kDa by SDS-PAGE, suggesting that further processing at the C-terminal region took place. The 2 motifs of zinc binding sites deduced are highly conserved in the APK as well as in other zinc metalloproteases including Vibrio proteolyticus neutral protease, Emp V from Vibrio vulnifcus, HA/P from Vibrio cholerae, and Pseudomonas aeruginosa elastase. Proteolytic activity was inhibited by EDTA, Zincov, 1, 10-phenanthroline and tetraethylenepentamine while unaffected by the other inhibitors tested. The protease showed maximum activity at pH 7.0 and was inactivated by heating at 80C for 15min. These results together suggest that APK belongs to the thermolysin family of metalloendopeptidases.

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Cell surface-associated materials of Actinobacillus actinomycetemcomitans were extracted by a short incubation of the cell suspension in a Tris-buffered saline in the presence and absence of a restriction enzyme, EcoRI. The supernatants (which we termed EcoRI extract and surface extract, respectively) contained a number of extracellularly released proteins. Of these proteins, four major proteins were identified by N-terminal sequencing to be the 34 and 39kDa outer membrane proteins, the GroEL-like protein, and a 47kDa protein homologous to Haemophilus influenzae enolase. Enolase activity was found in the extracts and its relative amount of activity in the EcoRI extract from a culture of the mid-exponential growth phase was estimated as 5.7% of total enzyme activity. In contrast, the relative amount of activity of another cytosolic enzyme, lactate dehydrogenase, was extremely low in the extracts and also in the culture supernatant. These results suggest the external localization of enolase in this bacterium.

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Hantaviral antibodies were detected in the sera from patients with hepatic disease of unknown etiology in Japan by several different serological diagnostic methods. A total of 105 sera from diseased patients which were negative to A-G hepatitis virus infections in the Tokyo area were tested. Among them, 3 out of 73 sera from patients with chronic hepatic disease were positive to hantaviral antibody by enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescent antibody assay (IFA) and Western blot analysis (WB). Neutralizing antibody titers of the 3 sera to Seoul virus (SEO) were 4 to 8 times higher than those to Hantaan virus (HTN). However, all of the 32 sera from patients with acute hepatitis were negative for hantaviral antibody. Among the 60 patients with chronic hepatitis in Hokkaido which were serologically negative to B and C hepatitis virus infection, one was positive for hantaviral antibody by ELISA and WB. In contrast, the sera from healthy adults in Japan, 550 from the Honshu and Kyushu regions, and 1, 000 from the Hokkaido region, were negative for hantavirus antibody. These results show that hantaviral antibodies are more frequently detected in patients with hepatic disease than in healthy adults. However, the observation that no positive sera were detected from patients with acute hepatitis implies that hantavirus might not be directly related to hepatitis.

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A simian/human immunodeficiency virus (SHIV)-NM3n containing the human nef, but not the monkey nef, and vpr genes of SIV was inoculated into two cynomolgus monkeys, resulting in systemic infection with a minimum level of transient virus load. In order to study the nature of immune responses associated with the prevention of a pathogenic SHIV, the SIIIV-NM3n-inoculated monkeys and three naive monkeys were intravenously challenged with a pathogenic SHIV containing the envelope gene of HIV-1 89.6. After the heterologous virus challenge, all of the SHIV-NM3n-inoculated animals completely avoided the loss of CD4+ T lymphocytes in PBMC as well as lymphoid tissues compared to pathogenic SHIV-injected control animals. The inhibition of CD4+ cell depletion was associated with maintaining the proliferative response of helper T-cells against SIV p27 in the previously nonpathogenic virus-inoculated animals following the pathogenic virus challenge. Furthermore, the decline of CD28+ cells, the increase in CD95+ cells, and the enhancement of in vitro apoptosis in PBMC were inhibited in the non-pathogenic virus-inoculated animals. These results suggest that nonpathogenic SHIV-NM3n infection induces the protection of monkeys from heterologous pathogenic viruses that may be associated with blocking the change in immune responses and the cell loss induced by a pathogenic virus.

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Three major components of Malassezia globosa were isolated from 2-ME extracts of this fungus by ion-exchange column chromatography and are referred to as Malg46a, Malg46b and Malg67, respectively. IgE antibodies to these components in the sera of patients with AD were detected by immunoblots. In Western blot, IgE antibodies to Malg46b were most frequently detected in the sera of AD patients. Dot blot with the Malg46b-containing fraction immunologically reacted with 69% of the sera of the patients, and with 83% of the sera of the patients who were positive for IgE antibodies to the 2-ME extract of M. globosa in the Western blot. The intensities generated for each dot correlated well with the total intensities generated for the 2-ME extract of M. globosa in the Western blot (r=0.763). In the lectin blot, Con A reacted with both Malg46a and Malg46b but not with Malg67. The polyclonal antibody to Malg46b reacted strongly only with the 2-ME extract of M. globosa and reacted slightly with M. restricta. In conclusion, a glycoprotein, Malg46b of M. globosa, is dominantly expressed in this fungus and is a possible major antigen for IgE antibodies in patients with AD.

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Mice vaccinated with a combination of two Staphylococcus aureus antigens consisting of a recombinant collagen-binding protein (CnBP) and alpha-toxoid (α-toxoid) were significantly protected from intramammary challenge infection with S. aureus. The average number of bacteria recovered from the glands of mice vaccinated with the combination of CnBP/α-toxoid was significantly lower compared to the average number of bacteria recovered from the glands of mice vaccinated with only CnBP or α-toxoid or controls (P≤0.01). Histopathological examination of mammary glands of mice vaccinated with CnBP together with α-toxoid showed no pathological changes, whereas glands of mice vaccinated with CnBP or α-toxoid alone developed severe mastitis and showed both focal and disseminated necrosis.

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We used the reverse transcription polymerase chain reaction (RT-PCR) and culture methods to study the presence of Helicobacter pylori in the gastric and oral samples from a total of 116 gastritis and peptic ulcer patients, including 58 with oral cancer. Detection rates of H. pylori were 46.6% in stomach samples and 12.1% in oral swab samples. All of the oral cancer surface swab samples were positive for H. pylori, as were their gastric samples suggesting that oral H. pylori derived from the stomach. The culture supernatants of Streptococcus mutans and Prevotella intermedia inhibited the growth of the H. pylori strain and caused the formation of the coccal form. In cases where H. pylori was detected in the oral cavity samples, including the oral cancer surface samples, it was believed that this species had colonized the stomach and were present in the oral cavity only as a transient organism.

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The complete nucleotide sequence of the gene encoding an alkaline serine proteinase (aprP) of Bacillus pumilus TYO-67 was determined. The sequence analysis showed an open reading frame of 1, 149bp (383 amino acids) that encoded a signal peptide consisting of 29 residues and a propeptide of 79 residues. The deduced 3 amino acid residues, D³², H⁶⁴, and S²²¹, were identical with 3 essential amino acids in the catalytic center of subtilases. The sequence around these residues revealed that APRP was a new member of the true subtilisin subgroup of the subtilisin family. The highest homology was found in subtilisin NAT at 64.4% in the DNA sequence. The residue S¹⁸⁹ of APRP was different from those of other subtilases.

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The magnesium salt of R-form lipopolysaccharide (LPS) from Klebsiella pneumoniae strain LEN-111 (O3-:K1-) that was prepared after the removal of cationic materials by electrodialysis formed essentially the same ordered hexagonal lattice structure with a lattice constant of 14 to 15nm as the original non-electrodialyzed preparation of the R-form LPS. When the magnesium salt was suspended in 50mM glycine buffer or Tris buffer at pH 1.4 to 9.5 and kept at 4C for 24hr, its content of Mg was markedly decreased, and its hexagonal lattice structure was changed to a swollen hexagonal lattice structure with extended lattice constants at pH 1.4 and to a loose mesh-like structure at pH 3.0 or higher. In the original non-electrodialyzed preparation of the R-form LPS, the release of Mg and disintegration of the hexagonal lattice structure did not occur by suspending in buffers at pH 1.4 to 8.5 at 4C for 24hr, but occurred only at pH 9.0 or higher. The results suggest that organic cations that can be removed by electrodialysis play some part in tight binding to Mg²⁺ and in stabilizing the ordered hexagonal assembly of the R-form LPS.

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Sialyl Lewis X (SLe^x), an E-selectin ligand, was conjugated with carboxymethylpullulan (CMPul) and the disposition characteristics of this conjugate after intravenous administration were investigated using. mice with ear edema. The concentration of ³H-labeled SLe^x-CMPul in the spleen was significantly high. When CMPul was modified with a saccharide unable to bind to E-selectin, this splenic accumulation was not observed. The uptake of radiolabeled SLe^x-CMPul by the spleen was completely inhibited by a 100-fold molar of cold SLe^x-CMPul but not by a sialyl N-acetyllactosamine-CMPul conjugate (SLN-CMPul). Microautoradiography analyses revealed that SLe^x-CMPul accumulated in the marginal zone of the spleen.

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HIV-1 infection induces the expression of high level of GM2 ganglioside on infected cells and IgM antibody (Ab) against GM2 can cause complement (C)-mediated cytolysis of HIV-1-infected cells. Since GM2 is immunogenic in human, we proposed that an anti-GM2 IgM Ab may be produced by some HIV-1-infected patients and the titer of this Ab might provide some insight into the progress of the disease. On this premise, the amount of IgM Ab against GM2 was determined in 124 HIV-1-infected patients and 111 seronegative donors. As expected, the anti-GM2 IgM Ab titers of the patients was significantly higher than that of the seronegative donors while the total IgM levels remained unchanged. In addition, we determined the CD4⁺ cell count and the HIV-RNA load in the HIV-1-infected patients. The results showed a positive correlation between the anti-GM2 IgM Ab titer and CD4⁺ cell count but a negative correlation between the anti-GM2 IgM Ab titer and HIV-RNA load. These suggest that anti-GM2 IgM Ab induced and/or enhanced by HIV-1 infection causes C-mediated cytolysis of HIV-1-infected cells in vivo to a certain extent, and may help lower the plateau level of the HIV-RNA load. Therefore, the amount of IgM Ab against GM2 may be related to the prognosis of HIV-1 infected patients.

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Norwalk virus and Sapporo virus were approved as type species of the genus “Norwalk-like viruses” and the genus “Sapporo-like viruses, ” respectively, in the family Caliciviridae. A total of 116 stool specimens containing Norwalk virus (NV) or Sapporo virus (SV) were tested by RT-PCR and Southern hybridization to evaluate nine sets of PCR primers and seven internal oligonucleotide probes in the RNA dependent RNA polymerase region of NV and SV for detection and differentiation of viruses in the NV and SV. Fifty-five stool samples were collected from 11 outbreaks of NV and/or SV gastroenteritis in an infant home, where residents were infants under 2 years of age, in Sapporo, Japan. Sixty specimens were obtained in Sapporo from sporadic cases in children, mainly under 6 years of age, of acute gastroenteritis due to small round structured viruses detected by EM. There is no single primer pair to detect all NV and SV, and at least three primer pairs, G1 set, G2 set and Sapp35/Sapp36, are required to detect viruses in the NV and SV clades. Many NV and SV strains were successfully classified into one of the NV/genogroup I, NV/genogroup II and SV by single-round RT-PCR and Southern hybridization. The new detection method for SV reported in this study combined with those for NV previously reported may elucidate the importance of Norwalk virus and Sapporo virus as a cause of viral gastroenteritis in all age groups in the world.

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