Tokuichiro Utsunomiya

Glycyrrhizin (20β-carboxy-11-oxo-30-norolean-12-en-3β-yl-2-O-β-d-glucopyranuronosyl-α-d-glucopyranosiduronic acid) improves the resistance of thermally injured mice to opportunistic infection of herpes simplex virus type 1

Abstract The effect of glycyrrhizin (GR) on the resistance of thermally injured mice to opportunistic herpes simplex virus type 1 (HSV) infection was investigated. We have previously reported that the susceptibility of thermally injured mice or normal mice inoculated with T6S cells (a clone of burn-associated CD8+ CD11+ TCR γ δ + type-2 suppressor T cells), to HSV infection was about 100 times greater than it was in normal mice. When thermally injured mice were treated i.p. with a 10 mg/kg dose of GR 2 and 4 days after infection of HSV, the resistance of these mice to HSV was improved to levels observed in normal mice. The adoptive transfer of splenic mononuclear cells (MNC) from normal mice treated with GR (GR-MNC) to thermally injured mice (recipients) resulted in the improved resistance of recipients to HSV infection. Normal mice inoculated with T6S cells and exposed to HSV had an 80% mortality rate, when given GR-MNC they had a 95% survival rate. The suppressor cell activity of T6S cells was clearly counteracted by GR-MNC in vitro in a mixed lymphocyte-tumor cell reaction. The type of cells responsible for anti-suppressor cells in GR-MNC was shown to be a CD4+ CD28+ TCR α β + Vicia villosa lectin-adherent T cell. These results suggest that GR may reverse the increased susceptibility of thermally injured mice to HSV infection through the induction of CD4+ contrasuppressor T cells.

Bacterial sepsis and chemokines.

Bacterial sepsis causes a high mortality rate when it occurs in patients with compromised host defenses. Severely burned patients, typical immunocompromised hosts, are extremely susceptible to infections from various pathogens, and a local wound infection frequently escalates into sepsis. In these patients, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa are familiar pathogens that cause opportunistic infections. Also, polymicrobial sepsis frequently occurs in these patients. In this review, therefore, the roles of chemokines in thermally injured patients infected with these 3 pathogens and polymicrobial sepsis will be discussed. These infections in thermally injured patients may be controlled immunologically, because immunocompetent hosts are resistant to infections with these pathogens. Classically activated macrophages (M1Mphi) are major effector cells for host innate immune responses against these infections. However, M1Mphi are not generated in thermally injured patients whose alternatively activated macrophages (M2Mphi) predominate. M2Mphi appear in patients early after severe burn injuries. M2Mphi inhibit M1Mphi generation through the secretion of CCL17 and IL-10. As a modulator of Mphi, two different subsets of neutrophils (PMN-I, PMN-II) are described. PMN-I direct the polarization of resident Mphi into M1Mphi through the production of CCL3. M2Mphi are induced from resident Mphi by CCL2 released from PMN-II. Therefore, as an inhibitor of CCL2, glycyrrhizin protects individuals infected with S. aureus. Sepsis stemming from P. aeruginosa wound infection is also influenced by CCL2 released from immature myeloid cells. A large number of immature myeloid cells appear in association with burn injuries. Host resistance to S. aureus, E. faecalis, P. aeruginosa or polymicrobial infections may be improved in thermally injured patients through the induction of M1Mphi, elimination of CCL2 and/or depletion of M2Mphi induced by CCL2.

Inhibition of burn-associated suppressor cell generation by glycyrrhizin through the induction of contrasuppressor T cells

The inhibitory effect of glycyrrhizin (GR), an anti‐inflammatory Chinese herbal drug extracted from licorice roots, on the generation of suppressor T cells in thermally injured mice (TI‐mice) was investigated. The burn‐associated suppressor T cell (BTs cell) activity was demonstrated in splenic mononuclear cells (SMNC) from mice 2 to 8 days after thermal injury when suppressor cell activity was assayed in a one‐way mixed lymphocyte reaction. However, when TI‐mice were treated with GR, SMNC harvested 6 days after thermal injury showed no suppressor cell activity. This activity of GR demonstrated a dose‐response effect, with a dose of 10 mg/kg exhibiting peak levels of the activity. Since GR had no direct inactivating activities against BTs cells in vitro, the inhibitory effect of SMNC, derived from TI‐mice treated with GR, on the activity of BTs cells was examined in the same mixed lymphocyte reaction system, and the results showed that the SMNC from GR‐treated mice 6 days after thermal injury counteracted the activity of BTs cells. The type of cell responsible for this inhibition of BTs cell activities was a CD3+, L3T4+, Vicia villosa lectin‐adherent T cell with the same phenotypic properties previously exhibited by contrasuppressor cells. These results suggest that GR may regulate the generation of BTs cells through the induction of contrasuppressor cells. Since there are many reports describing septic infections due to the appearance of BTs cells in postburn patients, it may be possible to apply GR or blood preparations containing contrasuppressor cell populations induced by GR in healthy volunteers into immunosuppressed burn patients to avoid infections.

Antiviral effect of gingyo-san, a traditional chinese herbal medicine, on influenza A2 virus infection in mice

Gingyo-san is a crude drug containing extracts from 7 medicinal plants and fermented soybeans in a specific ratio. It has been used clinically in China as a therapeutic agent for the common cold. In the present study, we examined the antiviral effect of this agent on influenza virus infection in mice. Gingyo-san and its components were administered orally to mice 1 day before, then 1 and 4 days after the inhalation of a mouse-adopted strain of influenza A2 (H2N2) virus. After infection with a 10 LD50 of the virus, 100% of mice treated with 10 mg/kg of the agent survived as compared with a 0% survival of control mice treated with saline. Also, the mean survival days were increased and consolidation scores were decreased in treated mice as compared with those of control mice. Two components contained in the agent, extracts from Glycyrrhizae radix and Arctii fructus, expressed antiviral activities in mice infected with influenza virus. However, in vitro growth of influenza virus in MDCK cells or viability of the virus was not affected by these extracts or Gingyo-san. From these results Gingyo-san was shown to be an antiviral agent in mice infected with a lethal amount of a mouse-adopted strain of influenza A2 virus.

In vitro induction of anti-type 2 T cells by glycyrrhizin

Glycyrrhizin (GR), an active component of licorice roots, has been previously shown to induce the generation of anti-type 2 T cells (anti-BI2T cells), which are able to counteract the activity of burn-induced CD8+ type 2 T cells (BI2T cells), in thermally injured and normal mice. In the present study, anti-BI2T cells were generated in vitro in cultures of spleen cells stimulated with GR. Anti-BI2T cells were induced in vitro when splenic mononuclear cells (SMNC) were stimulated in vitro for 24 to 72 h with 0.1-10 micrograms/ml of GR. Anti-BI2T cell activity was detected when suppressor macrophages (M phi) were depleted from SMNC after stimulation with GR. However, the GR-stimulated generation of anti-BI2T cells required the participation of M phi or their sonicated fractions. Anti-BI2T cells induced in vitro by GR were identified as Vicia villosa lectin adherent IFN7 producing-CD4+ T cells lacking the ability to produce IL-2, IL-4 or IL-10. These results indicate that anti-BI2T cells are generated by GR in vitro. M phi are not only necessary for the generation of anti-BI2T cells induced by GR but also inhibit their activity.

A relationship between the generation of burn-associated type 2 T cells and their antagonistic cells in thermally injured mice

Anti-type 2 T cells, generated in the spleens of thermally injured mice following the appearance of burn-associated CD8+, CD11b+, TCR gamma/delta + type 2 T cells (BA2T cells), have previously been shown to improve the resistance of thermally injured mice to Herpes virus infections. Anti-type 2 T cells, CD4+, CD28+, TCR alpha/beta +, IFN-gamma-producing T cell, are able to counteract the activity of BA2T cells which have been shown to be key cells in the increased susceptibility of thermally injured mice to these infections. In the present study, part of the generation mechanisms of anti-type 2T cells was examined in mice. Anti-type 2 T cells were detected in the spleens of unburned mice 3-7 days after i.v. inoculation (2 x 10(6) cells/mouse) of BA2T cells or a BA2T cell clone, defined as T6S cells. The suppressor cell activity of T6S cells was completely inhibited when they were co-cultured with anti-type 2 T cells from mice inoculated with T6S cells. Similar phenotypic and biological profiles of anti-type 2 T cells acquired from burned mice were expressed by T6S cell-induced anti-type 2 T cells. These results indicated that anti-type 2 cells may be generated in response to the appearance of BA2T cells in thermally injured mice. Immunoregulatory circuits may be involved in the generation of anti-type 2 T cells.

Protective effect of Shigyaku-to, a traditional Chinese herbal medicine, on the infection of herpes simplex virus type 1 (HSV-1) in mice

The antiviral activity of Shigyaku-to (TJS-109), a traditional Chinese herbal medicine, was investigated in mice infected with herpes simplex virus type 1 (HSV-1). TJS-109 is a combination of the medicinal plant extracts fromZingiberis siccatum rhizoma,Aconiti tuber andGlycyrrhizae radix in a specific proportion. Mice infected with a 10 LD50 dose of HSV-1 were treated with TJS-109 orally at doses of 1.25 to 20 mg/kg 2 days before, and 1 and 4 days after the infection. The treated groups had 80% (1.25 mg/kg), 40% (5 mg/kg) and 23% (20 mg/kg) mortality rates 25 days after the infection as compared with a 100% mortality rate in control mice treated with saline. When HSV-1 infected mice (recipients) received CD8+T cell fractions derived from spleens of mice treated with TJS-109 (donors), 70% of recipients survived, as compared with 0% survivors in the groups of mice treated with saline, B cell fractions, CD4+ T cell fractions or macrophage-enriched fractions prepared from the same donors. TJS-109 did not show any virucidal activities against HSV-1 or any virostatic activities on the growth of HSV-1 in Vero cells. These results suggest that TJS-109 protected mice exposed to lethal amounts of HSV-1 through the activation of CD8+ T cells.

Effect of a traditional Chinese herbal medicine, Kanzo-bushi-to, on the production of interleukin-4 from a clone of burn-associated CD8+ suppressor T cells

Effect of Kanzo-bushi-to (TJS-038), one of the Japanese Kampo medicines originating from traditional Chinese herbal medicine, on the activity of a clone of burn-associated CD8+ suppressor T cells (T6S cells) was investigated. The suppressor cell activity of T6S cells in the mixed lymphocyte-tumor cell reaction (MLTR) was not changed when various concentrations of TJS-038 were added to the MLTR. The suppressor cell activity of T6S cells was greatly inhibited by CD3+ CD4+ CD28+ TCR alpha/beta+ Vicia villosa lectin-adherent cells (TJS cells) purified from spleen cells of mice treated with TJS-038 at an oral dose of 50 mg/kg. The suppressor cell activity of T6S cells was not demonstrated in the MLTR assay when anti-IL-4 monoclonal antibody was added. Also the production of IL-4 from T6S cells was not detected, when they were co-cultured with TJS cells. These results suggest that TJS-038 stimulates the generation of contrasuppressor T cells (TJS cells) that inhibit the suppressor cell activity of T6S cells and the production of IL-4 from T6S cells.

The antiviral effect of Keishi-ni-eppi-ichi-to, a tranditional Chinese herbal medicine, on influenza A2(H2N2) virus infection in mice

The antiviral effect of Keishi-ni-eppi-ichi-to (TJS-064), a traditional Chinese herbal medicine, was investigation in mice infected with influenza A2(H2N2) virus. When mice exposed to 5 LD50 dose of the virus were treated orally with a 70 mg/kg dose of TJS-064 1 day before and 1 day and 4 days after the infection, 100% survived over a 25-day experimental period. At the end of this period all the control mice, treated with saline alone, had died; their mean survival time in days (MSD) was 11.2 days. When mice infected with a 10 LD50 dose of the virus were treated with TJS-064, the MSD was >17.4 days and there was a 50% survival rate, while the control group had a MSD of 8.7 days and 0% survival rate. No significant antiviral effect TJS-064 was observed when the agent was administered orally to mice infected with a 100 LD50 or large dose of influenza virus. Pulmonary consolidation, virus titers in lung tissues and HAI titers in sera of infected mice treated with TJS-064 were all significantly lower than those of infected mice treated with saline. Interferon activities were detected in sera of mice treated with the agent at a dose of 100 mg/kg orally. Since viricidal and viristatic activities of the agent against influenza virus were not demonstrated, the antiviral effects of TJS-064 may be expressed through the hosts antiviral functions including interferon production.