Microbiology and Immunology

Because Helicobacter pylori (H. pylori) infection is a major cause of gastroduodenal diseases in humans, the eradication of H. pylori using antibiotics is very effective for the treatment of gastroduodenal diseases. However, it has recently been reported that resistance to these antibiotics is developing. In the present study, the antibacterial effect of a Kampo (traditional Japanese medicine) herbal formulation, Hochu‐ekki‐to (HET; Formula repletionis animalis et supletionis medii), against H. pylori was examined in vitro and in vivo. HET inhibited the growth of antibiotic‐resistant strains of H. pylori as well as antibiotic‐sensitive strains at a dose of 2.5 mg/ml in vitro. When 1,000 mg/kg of HET was administered orally to C57BL/6 mice for 7 days before or after inoculation with H. pylori, H. pylori in the stomach was significantly reduced in the HET‐pre‐treatment group compared with the control group. Furthermore, HET in combination with antibiotics completely eradicated the bacteria in mice. The expression of interferon (IFN)‐γ was induced in the gastric mucosa of the mice pre‐treated with HET. There were no significant differences between the colonization of H. pylori in the control and HET treatment groups in IFN‐γ gene‐deficient mice. These results suggest that the antibacterial effect of HET may be partly due to IFN‐γ induction, and that HET may be clinically useful for treatment of H. pylori infection.

The nucleic acid fraction from cells of 6 species of bacterium and 2 kinds of vertebrate, calf and salmon, was extracted and purified by the same procedures as described previously. When the spleen cells from BALB/c mice were incubated with the nucleic acid fraction from either of the bacteria, natural killer (NK) activity of the cells was remarkably elevated and the cells produced factors to activate macrophages and to inhibit viral growth. It was shown that the factor to activate macrophages was interferon (IFN)‐gamma and that to inhibit viral growth was IFN‐alpha/beta. On the other hand, the nucleic acid fraction from either of the vertebrate cells did not show such activities. Pretreatment of the bacterial nucleic acid fraction with DNase, but not with RNase, abrogated completely the biological activities. The activities of the bacterial nucleic acid were not influenced by the presence of polymyxin B, an inhibitor of lipopolysaccharide (LPS), and the spleen cells from not only BALB/c mice but also LPS‐insensitive C3H/HeJ mice were activated, indicating that the activities of the fraction were not ascribed to LPS contaminated possibly into the fraction, but to DNA itself. Intralesional injection with the bacterial DNA fraction caused regression of mouse IMC tumors, but the injection with the vertebrate DNA fraction did not. These findings prompted us to examine the biological activities of DNA samples from a variety of animals and plants, which were provided from other laboratories or purchased from manufacturers. All of the DNA samples from cells of 5 kinds of bacterium, 2 of virus and 4 of invertebrate augmented NK activity and induced IFN, more or less, in mouse spleen calls, while the DNA from 10 kinds of vertebrate, including 3 of fish and 5 of mammal, showed no such activities. The DNA from 2 species of plants, were also inactive. Possible mechanisms to explain the different biological activities of DNA from different cell sources were discussed based on our previous finding that the particular palindromic sequences with a G‐C motif (s) are required for induction of IFNs and activation of NK cells with synthetic 30‐mer oligonucleotides.

Interleukin (IL)‐17 is a proinflammatory cytokine which induces differentiation and migration of neutrophils through induction of cytokines and chemokines including granulocyte‐colony stimulating factor and CXCL8/IL‐8. IL‐17‐producing CD4⁺ T cells (Th17) have pivotal role in pathogenesis of autoimmune diseases. IL‐17 is also involved in protective immunity against various infections. IL‐17 has important role in induction of neutrophil‐mediated protective immune response against extracellular bacterial or fungal pathogens such as Klebsiella pneumoniae and Candida albicans. Importance of IL‐17 in protection against intracellular pathogens including Mycobacterium has also been reported. Interestingly, not only CD4⁺ T cells but atypical CD4^–CD8^– T cells expressing T cell receptor (TCR) γδ produce IL‐17, and IL‐17 producing cells participate in both innate and acquired immune response to infections. Furthermore, neutrophil induction may not be the only mechanism of IL‐17‐mediated protective immunity. IL‐17 seems to participate in host defense through regulation of cell‐mediated immunity or induction of antimicrobial peptides such as β‐defensins. In this review, we summarize recent progress on the role of IL‐17 in immune response against infections, and discuss possible application of IL‐17 in prevention and treatment of infectious diseases.

Virulence of hantavirus strain of SR‐11 Seoul virus and Hantaan 76–118 (HTN) of Hantaan virus were compared. Infections of both strains were lethal in newborn mice. However, inoculum required to cause lethal infection was about 4,000 times higher for strain HTN (1.65 × 10³ PFU/mouse/LD₅₀) than for strain SR‐11 (0.36 PFU). Thus, both strains were considered pathogenic to newborn mice but they possessed different levels of virulence. The assay system used for these strains in newborn mice proved to be useful in the study of hantavirus vilurence. Growth curves of the two strains in CV‐7 cell cultures were compared. Strain SR‐11 was shown to have higher activity of virus replication and virus release into the culture fluids than strain HTN. The possibility of a relationship between replication activity and high levels of virulence in mice was suggested.

Group A rotaviruses (RVA) are a major cause of acute infantile gastroenteritis. The viral genome comprises 11 double‐stranded RNA segments and the respective gene segments are classified into more than eight genotypes, according to the nucleotide sequence similarities. So far, it has been difficult to amplify full‐length sequences of long RNA segments of rotaviruses by one‐time only RT‐PCR (especially in the genes for the viral proteins VP1, VP2, VP3 and VP4). In this study, a set of universal primers to amplify all 11 segments of RVA was designed by aligning the nucleotide sequences of the typical rotavirus strains. Using these primers and a high‐fidelity and rapid DNA polymerase in a one‐step reverse transcription polymerase chain reaction, almost the entire length of all 11 segments of the seven rotavirus strains Wa, DS‐1, Hochi, 69M, WI61, M37 and SA11‐S1 were accurately and rapidly amplified. In addition, all 11 segments of rotavirus obtained from a fecal specimen were successfully amplified. In conclusion, the method described here will be useful as an RVA detection system and protocol for complete analysis of the 11 genome sequences.

Shiga toxin (Stx)‐producing Escherichia coli (STEC) strains isolated from wild deer in Japan were examined. A total of 43 fecal samples were collected 4 times from 4 different sites around Obihiro City, Hokkaido, Japan, in June and July 1997. Seven STEC strains were isolated by PCR screening, all of them were confirmed by ELISA and Vero cell cytotoxicity assay to be producing only active Stx type 2 (Stx2). Moreover, they seemed to carry the hemolysin and eaeA genes of STEC O157:H7, and some isolates harbored large plasmids which were similar to the 90‐kilobase virulence plasmid of STEC O157:H7. Based on their plasmid profiles, antibiotic resistance patterns, PCR‐based DNA fingerprinting data obtained by using random amplified polymorphic DNA (RAPD) and the stx2 gene sequences, all isolates were divergent from each other except for 3 isolates from the first and second samplings. A DNA sequence analysis of representative isolates revealed that deer originating STEC strains were closely related to each other, but not to the Stx2‐producing STEC strains isolated from a mass outbreak in Obihiro at the same time. A phylogenic analysis of the deduced Stx2 amino acid sequences demonstrated that three distinct clusters existed in the deer originating STEC strains and that the Stx of deer originating STEC was closely associated with that originating from humans, but not those of STEC originating from other animals. These results suggest that STEC contamination of deer carcasses should be considered as a potential source of human infection and adequate sanitary inspection of meat for human consumption is also essential for wild animals.

RD3–0028, a compound with a benzodithiin structure, was found to be a potent inhibitor of respiratory syncytial virus (RSV) replication. Its action is specific; no activity is seen against influenza A virus, measles virus, herpes simplex virus type 1 or 2, or human cytomegalovirus. A time‐dependent drug addition experiment indicated that the antiviral activity occurs in the late stage of the RSV replication cycle, since this compound completely inhibited syncytium formation even when added up to 16 hr after the infection of cell monolayers at an MOI of 3. RD3–0028 had no direct virucidal effect on RSV. Western blotting analysis showed that RD3–0028 significantly decreased the amount of RSV proteins released into the cell culture medium. Moreover, five independent isolates of the RSV long strain were selected for growth in RD3–0028 (5–20 μg/ml). These resistant viruses were more than 80‐fold less sensitive to RD3–0028 than the long strain. The F gene segment of each of these viruses was sequenced and in each case the mutant RNA segment contained at least one sequence alteration, converting asparagine 276 to tyrosine (F1 protein). These results suggest that RD3–0028 inhibits RSV replication by interfering with intracellular processing of the RSV fusion protein, or a step immediately thereafter, leading to loss of infectivity.

Infection of primary cultures of total splenic and thymic cells from BALB/c and C3H/HeN mice with CVB4 E2 and JVB strains has been investigated. The presence of positive‐strand viral RNA within cells was determined by semi‐nested RT‐PCR, and viral replication was attested by detection of intracellular negative‐strand viral RNA and by release of infectious particles in culture supernatants. Viral replication occurred with both CVB4 strains to an extent dependent on the genetic background of the host. No interferon‐α production was detected in the supernatants of CVB4‐infected cultures using biological titration. Together these results suggest that infection of splenic and thymic cells can play a role in virus dissemination, and therefore in the pathophysiology of CVB4 infections.

Recently we discovered a bacterial strain (MS‐02–063) that produces large amounts of red pigment from coastal area of Nagasaki Prefecture, Japan. Comparative 16S rDNA gene sequencing analysis revealed that strain MS‐02–063 was phylogenetically closely related to γ‐proteobacterium Hahella sp. MBIC 3957 that produces prodigiosin. However, some physiological and biochemical differences between strain MS‐02–063 and Hahella sp. MBIC 3957 were observed. The red pigment (RP‐063) produced by this isolate was highly purified from the culture supernatant. It was speculated that RP‐063 might be prodigiosin‐like pigment in physical properties and biological activities such as antibacterial and cytotoxic activity. Antibacterial activity of RP‐063 was examined by an agar dilution method. The results indicated that RP‐063 showed antibacterial activity for specific for pathogenic gram‐positive bacteria such as Staphylococcus aureus. The potency of antibacterial activity against S. aureus was nearly equal to those of tetracycline. Moreover, RP‐063 showed inhibition of the superoxide generation by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐stimulated mouse macrophage RAW 264.7 cell line. Prodigiosin members have a wide variety of biological properties, including anticancer and antimalarial, etc. Especially, potent immunosuppressive properties have been reported for prodigiosin members with the mechanism of action different from that of the other well known immunosuppressors in atopic dermatitis therapy such as cyclosporin A, FK506 and rapamycin. It is suggested that RP‐063 may be able to arrest the inflammation caused by superantigens secreted from S. aureus, which colonized skin on atopic dermatitis as well as suppression of activated lymphocyte proliferation and superoxide generation from leucocytes.