Sadaaki Sawamura

Inhibition of the Accumulation of Uremic Toxins in the Blood and Their Precursors in the Feces after Oral Administration of Lebenin®, a Lactic Acid Bacteria Preparation, to Uremic Patients Undergoing Hemodialysis

The plasma levels of phenol, p-cresol, and indican are markedly increased in uremic patients, and cannot be efficiently reduced by hemodialysis. Such uremic toxins, which are produced in the intestine as bacterial putrefactive metabolites, accumulate to a great degree in the feces of hemodialysis patients. Oral administration of Lebenin, a preparation consisting of antibiotic-resistant lactic acid bacteria, reduced the levels of fecal putrefactive metabolites to levels comparable with those of healthy subjects. Moreover, the plasma level of indican also significantly decreased in these Lebenin-treated patients. An analysis of the fecal microflora revealed that a disturbed composition of the microflora characterized by an overgrowth of aerobic bacteria is restored to normal by oral administration of Lebenin in hemodialysis patients. These results thus demonstrate that oral administration of lactic acid bacteria in uremic patients is effective in reducing the levels of uremic toxins, especially that of indican, in the blood by inhibiting bacterial production by means of correcting the intestinal microflora.

The failure of oral tolerance induction is functionally coupled to the absence of T cells in Peyer's patches under germfree conditions.

Although intestinal bacterial flora has been thought to play a role in the induction of oral tolerance, the mechanism has yet to be elucidated. We therefore examined the bacterial flora-dependent acquisition of susceptibility to oral tolerance induction using a gnotobiotic murine model. Germ-free (GF) mice exhibited a significant shortage of T cells in the PPs in comparison to SPF mice. A recovery in the number of such T cells was accomplished in the gnotobiotic mice associated with Bifidobacterium infantis or Escherichia coli but not in the gnotobiotic mice with Clostridium perfringens or Staphylococcus aureus. To examine the susceptibility to oral tolerance induction, these mice were orally given ovalbumin (OVA) as a tolerogen and then injected i.p. with the Ag. The Ag-specific IgG1 in the serum remained at a low level in both SPF and those gnotobiotic mice groups containing a sufficient number of T cells in the PPs. However, no such unresponsiveness in the Ab response was observed in GF or the other gnotobiotic mice groups containing only a few T cells in the tissues. Adoptive cell transfer analysis clearly showed that a sufficient number of T cells in the PPs is required for the induction of oral tolerance. Furthermore, the reduced expression of SLC (secondary lymphoid-tissue chemokine), which is responsible for T-cell migration to lymphoid organs, was observed in the PPs of GF mice, resulting in a shortage of T cells in the tissues. However, the reduced expression of SLC was restored even in the GF mice after conventionalization, thus suggesting that the failure of oral tolerance induction is functionally coupled to the innate absence of T cells under the GF condition.

Role of Nitric Oxide Synthase Type 2 in Acute Infection with Murine Cytomegalovirus

Whether or not NO plays a critical role in murine CMV (MCMV) infection has yet to be elucidated. In this study, we examined the role of NO in acute infection with MCMV using NO synthase type 2 (NOS2)-deficient mice. NOS2−/− mice were more susceptible to lethal infection with MCMV than NOS2+/+ mice and generated a much higher peak virus titer in the salivary gland after acute infection. A moderate increase in the MCMV titer was also observed in other organs of NOS2−/− mice such as the spleen, lung, and liver. The immune responses to MCMV infection including NK cell cytotoxicity and CTL response in NOS2−/− mice were comparable with those of NOS2+/+ mice. Moreover, the ability to produce IFN-γ is not impaired in NOS2−/− mice after MCMV infection. The peritoneal macrophages from NOS2−/− mice, however, exhibited a lower antiviral activity than those from NOS2+/+ mice, resulting in an enhanced viral replication in macrophages themselves. Treatment of these cells from NOS2+/+ mice with a selective NOS2 inhibitor decreased the antiviral activity to a level below that obtained with NOS2−/− mice. In addition, the absence of NOS2 and NOS2-mediated antiviral activity of macrophages resulted in not only an enhanced MCMV replication and a high mortality but also a consequent risk of the latency. It was thus concluded that the NOS2-mediated antiviral activity of macrophages via NO plays a protective role against MCMV infection at an early and late stage of the infection.

Inducible Nitric Oxide Synthase Deficiency Does Not Affect the Susceptibility of Mice to Atherosclerosis but Increases Collagen Content in Lesions

Background —Although endothelial nitric oxide synthase (NOS) is antiatherogenic, the role of inducible NOS (iNOS) in the development of atherosclerosis is not established. Methods and Results —We compared the susceptibility of iNOS knockout (iNOS−/−) and wild-type (iNOS+/+) mice to the development of atherosclerosis induced by feeding an atherogenic diet for 15 weeks. Plasma lipid level, atherosclerotic lesion size, and cellular density in the lesions were all similar in the 2 strains (lesion size: iNOS+/+ 285±73×103 &mgr;m2, iNOS−/− 293±82×103 &mgr;m2, n=10). iNOS mRNA was detected in the lesions of iNOS+/+ but not iNOS−/− mice through RT-PCR. Immunohistochemically, iNOS+/+ mice showed iNOS staining in macrophages and medial smooth muscle cells in the lesions. Nitrotyrosine staining showed a similar distribution, whereas it was absent in iNOS−/− mice. There was no apparent difference in the intensity or distribution of vascular cell adhesion molecule-1 staining in the lesions of the 2 strains. However, the lesions of iNOS+/+ mice showed a markedly decreased extracellular collagen content compared with those of iNOS−/− mice Conclusions —iNOS induction does not affect the development of atherosclerosis in mice fed an atherogenic diet, but the resulting lesions show decreased levels of extracellular collagen and may be more fragile.

Growth inhibition of Clostridium difficile by intestinal flora of infant faeces in continuous flow culture

Growth of Clostridium difficile was inhibited more strongly in continuous flow (CF) culture with C. difficile-negative faeces of infants than with C. difficile-positive faeces. Culture of faecal flora of infants yielded a greater variety of bacterial species in C. difficile-negative than in C. difficile-positive faeces. In the mixed CF culture of C. difficile with Enterococcus avium, Bacteroides distasonis, Eubacterium lentum, C. ramosum, C. perfringens and either Escherichia coli or Klebsiella pneumoniae isolated from C. difficile-negative faeces, inhibition of growth of C. difficile was demonstrated when the pH of the culture medium was decreased. Amino-acid analysis of CF cultures showed considerable utilisation of aspartic acid, serine, threonine, arginine and asparagine. A marked increase in concentrations of citrulline and ornithine was found in the culture that inhibited growth of C. difficile. The addition of citrulline and ornithine into a Gifu anaerobic medium (GAM) broth produced no inhibition of growth of C. difficile. The addition of the mixture of the depleted amino acids (aspartic acid, serine, threonine, arginine and asparagine) to the culture filtrate or adjustment of the pH of the culture filtrate induced considerable growth of C. difficile. These results suggest that the inhibition of growth of C. difficile may be due to consumption of amino acids by intestinal flora, and not to the presence of inhibitors produced by the intestinal flora.

Role of the Indigenous Microbiota in Maintaining the Virus-Specific CD8 Memory T Cells in the Lung of Mice Infected with Murine Cytomegalovirus

The potent role of indigenous microbiota in maintaining murine CMV (MCMV)-specific memory T cells, which were measured by multimer staining, was investigated using germfree (GF) mice. When the BALB/c mice bred under specific pathogen-free (SPF) conditions were i.p. infected with 0.2 LD50 of MCMV, high frequencies of CD69+/CD44+ MCMV-specific CD8 T cells were noted in the lungs even at 6–12 mo after infection (11.1 ± 3.2 and 9.8 ± 0.9%, respectively). In contrast, even though the viral load and expression levels of mRNA of such cytokines as IL-2, IL-7, IL-15, and IFN-γ in the lungs of MCMV-infected GF mice were comparable to those of infected SPF mice, the frequencies of MCMV-specific CD8 T cells in the lungs of infected GF mice were kept lower than 1% at 6–12 mo after infection. In addition, the reconstitution of microbiota of MCMV-infected GF mice by orally administering a fecal suspension prepared from SPF mice restored the frequencies of both CD8+/multimer+ and CD8+/multimer− T cells to levels similar to those found in SPF mice. These results suggested the indigenous microbiota to play a crucial role in the expansion and maintenance of viral-specific CD8 memory T cells, probably by cross-reactivity between the antigenic epitope of the MCMV-specific memory T cells and the variety of peptides derived from the members of the microbiota. Such cross-reactivity may thus be a major feature of those cells.

Nitric oxide targets bronchiolar epithelial cells in murine cytomegalovirus-associated disease in lungs that are free of the virus

Summary. At 4 weeks after intraperitoneal (i.p.) injection of 0.2LD50 (50% lethal dose) of murine cytomegalovirus (MCMV) in adult BALB/c mice, productive virus and the viral DNA were detected only in the salivary glands, but not in the lungs. A single i.p. injection of anti-CD3 mAb to these mice provoked pulmonary lesions, which included a thickening of the interstitium and peribronchiolar areas, accompanied with a cellular infiltration in those areas. As a result, half of the mice died. In a histochemical analysis with anti-nitrotyrosine polyclonal Ab, bronchiolar epithelial cells were stained with this Ab, thus demonstrating that peroxynitrite, which was biochemically derived from nitric oxide (NO), injured those cells. Similarly, when T cells of iNOS+/+ mice, which had been infected with MCMV 4 weeks before, were activated by a single injection of anti-CD3 mAb, 37.5% of the mice died. Nitrotyrosine was also detected in the bronchiolar epithelial cells in these mice. In contrast, none of MCMV-infected iNOS−/− mice died after the anti-CD3 injection. No pathological changes were noted in the histological findings of the lungs of those mice. An intranasal injection of bacterial superantigen, staphylococcal enterotoxin B (SEB), demonstrated the same histopathological changes in the lungs and mortality in BALB/c mice as those in mice i.p. injected with anti-CD3. Therefore, T-cell responsiveness to stimulation with anti-CD3 or a superantigen was presumably modified by MCMV infection. MCMV-associated pneumonitis in the present study was thus mediated not by a direct viral attack but by iNOS-derived NO, which can be induced by the cytokines from the T cells. In addition, it was demonstrated that the NO produced by the cytokine-mediated pathway targeted bronchiolar epithelial cells.

Microbial Ecology and Tonsillar Infection

The results of quantitative and qualitative analyses of tonsillar bacterial flora in chronic tonsillitis patients and healthy subjects are reported. Based on these results, the pathogenesis of tonsillar infections is discussed from the standpoint of bacterial ecology.

The Role of Intestinal Bacterial Flora in the Tuning of the T Cell Repertoire

The role of the intestinal bacterial flora on the Vbeta repertoire was examined using the gnotobiotic murine model. The ratio of Vbeta6-positive T cells in the periphery of DBA/2 mice under SPF conditions was only 2.2% (mean, n = 4), since the cells were eliminated by the endogenous superantigen Mls(a). However, the ratio in germ-free (GF) mice was 31.7%. Similarly, the contamination of the GF Mice with the intestinal flora from SPF mice reduced the ratio of Vbeta6 in GF mice from 22.9% to 13.7%. In contrast, in BALB/c mice (Mls(b)) in which Vbeta6 cells do not react with this endogenous superantigen, the ratio of Vbeta6 cells do not react with this endogenous superantigen, the ratio of Vbeta6 of SPF mice (15.4%, mean, n = 3) was found to be comparable to that of GF mice (15.6%, n = 3). These data suggested that the absence of intestinal flora deteriorated a part of the Mls(a) determinant, which reacted with the Vbeta6 T cells and thereby eliminated them, thus resulting in an increase of these cells in GF mice. Moreover, the alloantigenicity of minor histocompatible alloantigen(s) (mHAg) in SPF mice, which was detected in H-2 identical MLR experiments and a murine graft-versus-host (GVH) model, was reduced in GF and decontaminated SPF mice, thus indicating that the intestinal flora upregulated the mHAg including a part of Mls determinant. These results therefore suggest that the intestinal flora plays a role in the upregulation of mHAg including a part of endogenous superantigen and the consequent tuning of the Vbeta repertoire.