CD8+ T Cell-Mediated Therapeutic Antitumor Effect of an Herbal Mixture Containing Ganoderma lucidum.

Although Kampo-a traditional Japanese herbal medicine-contributes in the control of tumor growth in vivo in experimental animals, most of the antitumor effects are prophylactic and not therapeutic. In this study, we determined whether oral administration of an herbal mixture containing Ganoderma lucidum (WTMCGEP; Wisteria floribunda, Trapae fructus, Myristica fragrans, Coicis semen, Ganoderma lucidum, Elfvingia applanata, and Punica granatum), anecdotally used in Japan for the palliative care of patients with cancer, exhibits a therapeutic effect on tumor growth in vivo in a hypodermic murine CT26 colorectal tumor model. An in vitro tumor assay revealed that WTMCGEP extract has some direct influence over suppression of tumor growth. In wild-type BALB/c mice, WTMCGEP did not show any antitumor effect in vivo. However, in BALB-CD1d-/- mice with partly mitigated immunosuppression by reason of them being devoid of both antitumoral type I and immunosuppressive type II natural killer T (NKT) cells, WTMCGEP therapeutically suppressed tumor growth. CD8+ T cell depletion significantly accelerated tumor growth in WTMCGEP mice; therefore, its antitumor activity was primarily in a CD8+ T cell-dependent manner. Regarding immunosuppressive cells in tumor-bearing CD1d-/- mice, WTMCGEP did not influence the abundance of tumor-infiltrating CD4+ and Forkhead box protein 3+ regulatory T cells. However, it reduced both intratumoral and splenic Ly6G+ Ly6Clo polymorphonuclear myeloid-derived suppressor cells, which were most likely involved in tumor growth inhibition related to higher frequency of intratumoral CD107a+ CD8+ T cells in these mice. Overall, these data illustrate that the deficiency of NKT cells urges WTMCGEP to exert a therapeutic antitumor effect mainly through CD8+ T cells. Our efforts are the first to scientifically demonstrate the WTMCGEP's contribution to tumor immunity.

Five-year trends in bacterial contamination of dialysis equipment.

Bedside dialysis monitoring equipment for hemodialysis are located in the bioburden section upstream of the endotoxin-retentive filter for dialysis fluid sterilization. We observed 26 equipment at our institution for bacterial contamination at least once every 4 weeks for 5 years with another ultrafiltration membrane upstream to prevent bacterial contamination. Bacterial contamination levels were highest and most diverse at the time of the first flush. During subsequent initial cleanng, the contamination level decreased, and bacterial species converged almost exclusively to one genus, namely Methylobacterium spp. During clinical use, the equipment were cleaned and disinfected daily after dialysis, and daily operations and maintenance were performed using aseptic techniques. Although the frequency of bacterial detection decreased annually, the same bacterial genotypes observed at the first flush were isolated even after long time periods and were thought to persist in the equipment possibly by forming biofilm. Pseudomonas aeruginosa was newly detected after the replacement of parts during breakdown maintenance, indicating the need to sterilize replacement parts. Thus, the bioburden should be assessed regularly as part of the management of in-house-produced dialysis fluid.

Induction of apoptosis-resistant and TGF-ß-insensitive murine CD8(+) cytotoxic T lymphocytes specific for HIV-1 gp160.

Although TGF-ß and IL-6 would turn CD8(+) T cells to differentiate into non-cytotoxic state, these treated cells were converted to cytolytic phenotypes after re-exposure to their antigenic epitope in vitro. Here, using spleen cells from TCR transgenic mice expressing TCRαß genes of clone RT1 recognizing an epitope peptide (P18-I10: RGPGRAFVTI) of HIV-1 gp160, we generated CD8(+) cytotoxic T lymphocytes (CTLs) activated by re-exposure to P18-I10 after primarily cultured with TGF-ß and IL-6 in vitro to examine their effector function. The CTLs, having strong cytotoxic activity in vitro, were not only resistant to Fas-FasL mediated apoptosis, but also insensitive to the suppression of their cytotoxicity by re-exposure to TGF-ß in vitro. Moreover, adoptive transfer experiments indicated that the CTLs are capable of eliminating recombinant vaccinia virus expressing HIV-1 gp160 in vivo. Taken together, our data suggest that TGF-ß and IL-6 may play pivotal roles in inducing apoptosis-resistant and TGF-ß-insensitive CTLs in vitro.

Japanese Kampo Medicine Juzentaihoto Improves Antiviral Cellular Immunity in Tumour-Bearing Hosts.

Global and antigen-independent immunosuppression by growing tumours can cause life-threating damage when concurrent with an infection in tumour-bearing hosts. In the present study, we investigated whether the oral administration of the Japanese traditional herbal (Kampo) medicine, juzentaihoto (JTT), plays a role in the improvement of antiviral cellular immunity in tumour-bearing hosts. Female BALB/c mice subcutaneously injected with murine colorectal cancer CT26 cells fed a control or JTT diet were inoculated with recombinant vaccinia virus expressing human immunodeficiency virus-1 glycoprotein 160 (vSC25). At 7 days postinfection, anti-vSC25 cellular immunity was evaluated by measuring the abundance of splenic virus-specific CD8+ T cells. JTT had no impact on CT26 tumour growth in vivo. Surprisingly, JTT augmented anti-vSC25 cellular immunity in CT26-bearing mice. Depletion of either CD25+ regulatory T (Treg) cells or myeloid-derived suppressor cells (MDSCs) also enhanced anti-vSC25 cellular immunity in tumour-bearing mice but had no therapeutic benefit against tumour growth. However, JTT had no impact on the abundance of these immunosuppressive cells. Overall, our data indicates that JTT contributes to the improvement of anti-vSC25 cellular immunity in tumour-bearing hosts possibly via a mechanism independent of CD25+ Treg cells and MDSCs, suggesting that this Kampo medicine can act as a promising antiviral adjuvant in an immunosuppressive state caused by tumours.

A nonclassical non-Valpha14Jalpha18 CD1d-restricted (type II) NKT cell is sufficient for down-regulation of tumor immunosurveillance.

The importance of immunoregulatory T cells has become increasingly apparent. Both CD4+CD25+ T cells and CD1d-restricted NKT cells have been reported to down-regulate tumor immunity in mouse tumor models. However, the relative roles of both T cell populations have rarely been clearly distinguished in the same tumor models. In addition, CD1d-restricted NKT cells have been reported to play a critical role not only in the down-regulation of tumor immunity but also in the promotion of the immunity. However, the explanation for these apparently opposite roles in different tumor models remains unclear. We show that in four mouse tumor models in which CD1d-restricted NKT cells play a role in suppression of tumor immunity, depletion of CD4+CD25+ T cells did not induce enhancement of immunosurveillance. Surprisingly, among the two subpopulations of CD1d-restricted NKT cells, Valpha14Jalpha18+ (type I) and Valpha14Jalpha18- (type II) NKT cells, type I NKT cells were not necessary for the immune suppression. These unexpected results may now resolve the paradox in the role of CD1d-restricted NKT cells in the regulation of tumor immunity, in that type II NKT cells may be sufficient for negative regulation, whereas protection has been found to be mediated by alpha-galactosylceramide-responsive type I NKT cells.

Sodium Hypochlorite is Effective against Biofilms in Dialysis Equipment.

To test the efficacy of chemical disinfectants against bacterial biofilms in hemodialysis equipment, a Center for Disease Control and Prevention (CDC)-Biofilm Reactor was used to create biofilms. Methylobacterium radiotolerance was isolated from the hemodialysis fluid and used as the test organism. We examined the efficacy of sodium hypochlorite (NaOCl) in elimination of planktonic cells compared to that in the case of biofilms. Planktonic bacteria were completely eliminated at 50 parts per million (ppm) of NaOCl, which is the lowest concentration for clinical use. The viable cell count in the biofilm reached its minimum value around a logarithmic reduction value (LRV) of 6, when the concentration was raised to 1000 ppm and the reaction time was extended by 1 hour or more. Furthermore, at 200 ppm, the LRV was elevated depending on the time. And the LRV while maintaining static conditions for 6 hours at 200 ppm was similar to that of short time at 1000 ppm. These results suggest that NaOCl has sufficient bactericidal activity even for biofilms at a practical concentration and reaction time, and that the CDC-Biofilm Reactor is an effective tool for finding useful disinfection conditions.

Blockade of TGF-beta enhances tumor vaccine efficacy mediated by CD8(+) T cells.

Though TGF-beta inhibition enhances antitumor immunity mediated by CD8(+) T cells in several tumor models, it is not always sufficient for rejection of tumors. In this study, to maximize the antitumor effect of TGF-beta blockade, we tested the effect of anti-TGF-beta combined with an irradiated tumor vaccine in a subcutaneous CT26 colon carcinoma tumor model. The irradiated tumor cell vaccine alone in prophylactic setting significantly delayed tumor growth, whereas anti-TGF-beta antibodies alone did not show any antitumor effect. However, tumor growth was inhibited significantly more in vaccinated mice treated with anti-TGF-beta antibodies compared to vaccinated mice without anti-TGF-beta, suggesting that anti-TGF-beta synergistically enhanced irradiated tumor vaccine efficacy. CD8(+) T-cell depletion completely abrogated the vaccine efficacy, and so protection required CD8(+) T cells. Depletion of CD25(+) T regulatory cells led to the almost complete rejection of tumors without the vaccine, whereas anti-TGF-beta did not change the number of CD25(+) T regulatory cells in unvaccinated and vaccinated mice. Though the abrogation of CD1d-restricted NKT cells, which have been reported to induce TGF-beta production by MDSC through an IL-13-IL-4R-STAT6 pathway, partially enhanced antitumor immunity regardless of vaccination, abrogation of the NKT cell-IL-13-IL-4R-STAT-6 immunoregulatory pathway did not enhance vaccine efficacy. Taken together, these data indicated that anti-TGF-beta enhances efficacy of a prophylactic vaccine in normal individuals despite their not having the elevated TGF-beta levels found in patients with cancer and that the effect is not dependent on TGF-beta solely from CD4(+)CD25(+) T regulatory cells or the NKT cell-IL-13-IL-4R-STAT-6 immunoregulatory pathway.

Japanese Kampo Medicine Juzentaihoto Enhances Antitumor Immunity in CD1d-/- Mice Lacking NKT Cells.

Although the Japanese traditional herbal medicine (Kampo), Juzentaihoto (JTT), has been reported to have antitumor effects in several tumor models, its role in tumor immunology remains controversial. In the present study, we tested whether oral administration of JTT enhances antitumor immunity in CD1d-/- mice, in which immunosuppression was partially relieved due to the lack of NKT cells. In a subcutaneous murine syngeneic CT26 colorectal tumor model, JTT had no impact on tumor growth in wild type (WT) BALB/c mice. However, the growth rate of tumors was significantly slower in CD1d-/- mice than in WT mice. Surprisingly, JTT significantly delayed tumor growth in such CD1d-/- mice. In vivo depletion of CD8+ T cells revealed that CD8+ T cells are required for JTT's antitumor activity. Moreover, tumor-reactive cytotoxic T-lymphocytes were detected exclusively in JTT-treated mice with well-controlled tumors. JTT did not affect the number of tumor-infiltrating CD4+ regulatory T cells. On the contrary, JTT increased the degranulation marker CD107a+ CD8+ T cells and decreased Ly6G+ Ly6Clo polymorphonuclear myeloid-derived suppressor cells in tumor-infiltrating lymphocytes, most probably contributing to the suppression of tumor growth in JTT-treated mice. Nonetheless, JTT had no impact on the proportion of monocytic myeloid-derived suppressor cells. In conclusion, our results indicate that in the absence of NKT cells, JTT augments antitumor immunity by CD8+ T cells, suggesting that this Kampo medicine is a promising anticancer adjuvant when negative immune regulation is partially relieved.

Japanese Kampo medicine ninjin'yoeito synergistically enhances tumor vaccine effects mediated by CD8+ T cells.

Although Japanese traditional herbal medicine (Kampo) has been widely applied to the treatment of various diseases, including cancer, their mechanisms of action have not yet been elucidated in detail, particularly regarding their role in tumor immunology. The present study investigated the antitumor effects of the Japanese Kampo medicine, ninjin'yoeito (NYT; Ren-Shen-Yang-Rong-Tang in Chinese), which was orally administered with or without an irradiated tumor cell vaccine to a subcutaneous CT26 colon carcinoma tumor model. The irradiated tumor cell vaccine in a prophylactic setting significantly delayed tumor growth in mice fed a control diet, whereas a diet containing NYT alone did not exert any antitumor effects in vivo. However, the inhibition of tumor growth was significantly greater in vaccinated mice fed the NYT diet compared with in vaccinated mice given the control diet. These results suggest that NYT synergistically enhances the effects of the antitumor vaccine. The depletion of cluster of differentiation (CD)8+ T cells abrogated these effects, indicating that antitumor activity required CD8+ T cells. Furthermore, reductions in CD4+ CD25+ and forkhead box protein 3+ T regulatory cell numbers were more apparent between vaccinated mice fed the NYT diet and non-vaccinated mice fed the control diet than between vaccinated mice and non-vaccinated mice fed the control diet, suggesting that the weaker impact of T regulatory cells contributes to the augmentation of antitumor immunity by CD8+ T cells in vaccinated mice fed with NYT. Overall, these results indicate that NYT synergistically enhances the effects of the prophylactic tumor vaccine mediated by CD8+ T cells and that this Japanese Kampo medicine has potential as a useful adjuvant agent for cancer immunotherapy.

Detection Method for Aquatic Bacteria of the Fingers, as a Potential Origin of the Aqueous Solution Contamination.

　Aquatic bacteria were isolated from the hands of working staffs by an adapted culture protocol. When the sample solution obtained by the" glove juice method" was incubated for 3 days at room temperature, viable cell counts increased up to 105-fold, and the majority of the isolated colonies were shown to be Gram-negative aquatic bacteria, which carry the risk of contaminating water. Using R2A medium, coagulase-negative staphylococci were the dominant microbes immediately after recovery from the hands. Here it was revealed that bacteria of the phylum Proteobacteria isolated from the hand can be the causative bacteria of aqueous contamination. This modification in the GJ method may be useful as an effective training protocol to demonstrate the importance of hand hygiene and clean operation for aseptic manufacturing.

Rapid detection of microbes in the dialysis solution by the microcolony fluorescence staining method (Millflex quantum).

A chemiluminescence system, Milliflex Quantum (MFQ), to detect microcolonies, has been used in the pharmaceutical field. In this study, we investigated aquatic bacteria in hemodialysis solutions sampled from bioburden areas in 4 dialysis faculties. Using MFQ, microcolonies could be detected after a short incubation period. The colony count detected with MFQ after a 48-hour incubation was 92% ± 39%, compared to that after the conventionally used 7-14-day incubation period; in addition, the results also showed a linear correlation. Moreover, MFQ-based analysis allowed the visualization of damaged cells and of the high density due to the excessive amount of bacteria. These results suggested that MFQ had adequate sensitivity to detect microbacteria in dialysis solutions, and it was useful for validating the conditions of conventional culture methods.

A case of migraine without aura that was successfully treated with an herbal medicine.

OBJECTIVE: Migraine is a common neurologic condition characterized by the disabling effects it has on the patient. Despite recent progress in drug development, better pharmacotherapies against migraine are still needed. This report describes an herbal medicine that has a strong pain-relieving effect against migraine. PATIENT: This case involved a 49-year-old woman with a 16-year history of headaches. Migraine without aura had been diagnosed at age 47 years. Despite taking antiepileptic drugs as prophylaxis, she had migraines almost three times a week, especially in the morning. As a result, 3 months before her visit to the study clinic she was prescribed sumatriptan, which she had to take for each attack. However, sumatriptan sometimes failed to work as a painkiller, and the patient did not want to continue taking it because she was worried about overuse. INTERVENTION AND OUTCOME: According to traditional Japanese (Kampo) and Chinese herbal medicine, the patient was prescribed an oral sanno-shashin-to (Xie Xin Tang) extract formula to take at night to prevent her migraines. Soon after she started taking the formula, the intensity of her morning headaches markedly decreased, to the extent that triptans were no longer needed. Furthermore, her residual headaches quickly disappeared after the additional administration of the formula in the morning. CONCLUSIONS: Sanno-shashin-to might have both prophylactic and therapeutic effects against migraine without aura. Although the underlying mechanism behind the effectiveness of this herbal medicine against migraine has not been fully elucidated, it is a possible option for the treatment of this type of headache.

Synergistic enhancement of CD8+ T cell-mediated tumor vaccine efficacy by an anti-transforming growth factor-beta monoclonal antibody.

PURPOSE: Transforming growth factor-beta (TGF-beta) is an immunosuppressive cytokine, having direct suppressive activity against conventional CD4(+) and CD8(+)T cells and natural killer cells, thereby inhibiting tumor immunosurveillance. Here, we investigated possible synergy between anti-TGF-beta (1D11) and a peptide vaccine on induction of antitumor immunity, and the mechanisms accounting for synergistic efficacy. EXPERIMENTAL DESIGN: The effect of combination treatment with a peptide vaccine and anti-TGF-beta was examined in a subcutaneous TC1 tumor model, as well as the mechanisms of protection induced by this treatment. RESULTS: Anti-TGF-beta significantly and synergistically improved vaccine efficacy as measured by reduction in primary tumor growth, although anti-TGF-beta alone had no impact. The number of tumor antigen-specific CTL with high functional avidity as measured by IFN-gamma production and lytic activity was significantly increased in vaccinated mice by TGF-beta neutralization. Although TGF-beta is known to play a critical role in CD4(+)Foxp3(+) Treg cells, Treg depletion/suppression by an anti-CD25 monoclonal antibody (PC61) before tumor challenge did not enhance vaccine efficacy, and adding anti-TGF-beta did not affect Treg numbers in lymph nodes or tumors or their function. Also, TGF-beta neutralization had no effect on interleukin-17-producing T cells, which are induced by TGF-beta and interleukin-6. Absence of type II NKT cells, which induce myeloid cells to produce TGF-beta, was not sufficient to eliminate all sources of suppressive TGF-beta. Finally, the synergistic protection induced by anti-TGF-beta vaccine augmentation was mediated by CD8(+) T cells since anti-CD8 treatment completely abrogated the effect. CONCLUSIONS: These results suggest that TGF-beta blockade may be useful for enhancing cancer vaccine efficacy.

Cross-regulation between type I and type II NKT cells in regulating tumor immunity: a new immunoregulatory axis.

Negative immunoregulation is a major barrier to successful cancer immunotherapy. The NKT cell is known to be one such regulator. In this study we explored the roles of and interaction between the classical type I NKT cell and the poorly understood type II NKT cell in the regulation of tumor immunity. Selective stimulation of type II NKT cells suppressed immunosurveillance, whereas stimulation of type I NKT cells protected against tumor growth even when responses were relatively skewed toward Th2 cytokines. When both were stimulated simultaneously, type II NKT cells appeared to suppress the activation in vitro and protective effect in vivo of type I NKT cells. In the absence of type I, suppression by type II NKT cells increased, suggesting that type I cells reduce the suppressive effect of type II NKT cells. Thus, in tumor immunity type I and type II NKT cells have opposite and counteractive roles and define a new immunoregulatory axis. Alteration of the balance between the protective type I and the suppressive type II NKT cell may be exploited for therapeutic intervention in cancer.

Induction of hepatic injury by hepatitis C virus-specific CD8+ murine cytotoxic T lymphocytes in transgenic mice expressing the viral structural genes.

In the present study, we generated killer cells specific for hepatitis C virus (HCV) structural protein by re-stimulation of immune spleen cells from H-2(d) haplotype transgenic (Tg) mice, expressing the core, E1, E2, and NS2 genes of HCV regulated by the Cre/loxP switching system. The generated killer cells were conventional CD8(+)L(d) class-I MHC molecule-restricted cytotoxic T lymphocytes (CTLs) and specific for the HCV E1 structural protein. Because the CTLs could also kill hepatocytes from the Tg mice expressing HCV structural proteins in vitro, we attempted to transfer those CTLs intravenously into interferon regulatory factor-1 (IRF-1) negative, CD8-deficient Tg mice representing the HCV structural genes on hepatocytes to examine whether the inoculated CD8(+) CTLs can eliminate hepatocytes expressing the HCV genes in vivo. We observed an elevation of serum ALT level as well as damage of the liver tissue histologically. To our knowledge, this is the first demonstration to show that HCV-specific CD8(+) CTLs specifically attack hepatocytes expressing the HCV structural proteins both in vitro and in vivo.