Toshiki Aji

Parasite infection and cancer: with special emphasis on Schistosoma japonicum infections (Trematoda). A review

This article contains a review of current knowledge on the association of parasite infections and cancer formation, especially that of Schistosoma japonicum (Trematoda) in man and experimental animals. The association of S. haematobium infection and bladder cancer is well known and documented. However, S. japonicum infection has also been reported to be associated with cancer, in this case hepatocellular carcinoma and/or colorectal cancer. Pathological records and analyses have shown a correlation between this infection and cancer, and pathohistological descriptions have been numerous, together with clinical case reports. Epidemiological analyses have been conducted in China and Japan and support a role of S. japonicum infection as one of the risk factors in cancer formation, along with others, such as hepatitis virus infection and alcoholic intake. Experimental results have also shown that cancer appears early and in larger numbers in experimentally infected animals given a known carcinogen. In spite of these positive end-point associations, the mechanism of schistosome-mediated enhancement of carcinogenesis is obscure. A suggestive observation is that in S. japonicum-infected mice carcinogen-metabolizing hepatic activity including P-450 was decreased so that an administered carcinogen persisted for a longer period than in uninfected mice. Further studies, both epidemiological and experimental, are needed to firmly establish the relationship between schistosome infection and cancer.

Enrichment of Foxp3+ CD4 Regulatory T Cells in Migrated T Cells to IL-6– and IL-8–Expressing Tumors through Predominant Induction of CXCR1 by IL-6

Analysis of cytokine and chemokine production by tumor cell lines including five lung cancers, a malignant mesothelioma, and a malignant melanoma recently established in our laboratory showed rather high production of IL-8 in all tumors and IL-6 in one lung cancer, the malignant mesothelioma, and the malignant melanoma. We investigated the migration of PBMCs to these tumor cells using Transwell plates and showed enrichment of Foxp3+ CD4 regulatory T cells (Tregs) in migrated T cells to both IL-6– and IL-8–producing tumors. Marked induction of CXCR1 expression on Foxp3+ CD4 Tregs by IL-6 followed by IL-8–mediated migration appeared to be responsible for enriched migration. Frequent production of IL-8 by the tumors and Treg migration to those tumors through induction of IL-8R expression by IL-6 is one of the mechanisms for tumor escape.

Evaluation of the mutagenicity and the tumor-promoting activity of parasite extracts: Schistosoma japonicum and Clonorchis sinensis.

In relation to the observed association of carcinogenesis with parasitic infections, the mutagenicity of extracts of Schistosoma japonicum and Clonorchis sinensis was examined. In the bacterial mutagenicity tests using the Ames Salmonella typhimurium strains TA98, TA100, TA97 and TA102, and Escherichia coli WP2 and WP2 uvrA pKM101 Schistosoma soluble egg antigen and a homogenate of adult Schistosoma worms showed no positive responses either in the presence or in the absence of S9 mix. Likewise, adult worm extracts of Clonorchis showed no mutagenicity. The Schistosoma soluble egg antigen showed a weak but significant activity for the induction of Epstein-Barr virus expression in viral genome-carrying human lymphoblastoid cells in culture. This phenomenon suggests that the soluble egg antigen possesses tumor-promoting activity.

Reduced levels of mutagen processing potential in the Schistosoma japonicum-infected mouse liver

Epidemiological studies have shown the presence of a positive correlation between the infection of Schistosoma japonicum and colorectal and/or liver cancers in the humans. To explore the mechanism underlying this correlation, we have investigated the mutagen-activating potentials of the liver homogenate fraction (S9) from Schistosoma japonicum infected mice and those from control mice, by use of the Ames test with 2-acetylaminofluorene, aflatoxin B1 and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) as test mutagens. Liver S9 prepared from the infected group at the 15th week after the infection showed a potential significantly lower than that from the control group. The hepatic cytochrome P-450 concentration in the infected mice was persistently low, about a half of that in the uninfected mice, during the period of 6-18 weeks after the infection. Thus, in mice bearing chronic schistosomiasis, mutagen-processing potentials are decreased.

Identification of an HLA-A24-restricted OY-TES-1 epitope recognized by cytotoxic T-cells.

OY‐TES‐1 was identified as a human homologue of the mouse, guinea pig, and pig proacrosin binding protein sp32 precursor. Differential expression levels of OY‐TES‐1 mRNA between testis and other normal tissues, and its expression in cancers indicated that OY‐TES‐1 would be classified as a cancer/testis antigen and considered to be a candidate of target antigen for cancer immunotherapy. In this study, we showed identification of HLA‐A24‐binding OY‐TES‐1 peptide, TES401–409 (KTPFVSPLL) recognized by CD8 T‐cells. Purified CD8 T‐cells from healthy donors stimulated in vitro with the peptide‐pulsed autologous DC and PBMC produced IFN7 in response to the peptide‐pulsed PBMC and showed cytotoxicity against the peptide‐pulsed autologous EBV‐B specifically. Furthermore, cytotoxicity was also observed against an OY‐TES‐1 mRNA‐expressing tumor line, LK79. The retention time of the fraction in HPLC of the acid eluate from LK79 cells that showed positive sensitization against autologous EBV‐B cells in recognition by CD8 CTL was the same as that of the fraction of the TES401–409 peptide itself, suggesting that the TES401–409 was a naturally processed peptide on LK79.

Retention of a mutagen, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), in the liver of mice infected with Schistosoma japonicum

Regarding the mechanism underlying the suspected enhancement of hepatic cancers among Schistosoma japonicum-infected humans, we hypothesized that mutagen exposures in the livers of patients may be enhanced due to the parasitic infection. To explore this possibility, we have done a model experiment using mice and a carcinogenic mutagen, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). Mice infected with Schistosoma japonicum were intravenously administered Trp-P-2, and the mutagenic activities of the mouse serum and of the liver tissue extracts, which were observable during the 6-h period after the administration, were investigated. The level of serum indirect mutagenicity, which probably reflected the amount of unmetabolized Trp-P-2, was higher in the infected animals than in uninfected control animals. Direct mutagenicity in the serum, on the other hand, was higher in the control animals than in the infected mice. Furthermore, the liver tissue extracts from infected mice showed higher indirect-mutagenicity than those from the controls. These data suggest that the infection results in a decreased metabolism and an increased retention of Trp-P-2 in the liver. Consistent with this phenomenon, pigments in the liver formed by the schistosome infection were found to be an efficient adsorbent for Trp-P-2. Thus, the possibility exists that these pigments, which contain hematin as a major constituent, may function as a reservoir for the mutagen, thereby prolonging the exposure period of the liver to the mutagen.

HLA-DRB1*0410-Restricted Recognition of XAGE-1b37-48 Peptide by CD4 T Cells

XAGE‐1b belongs to cancer/testis (CT) antigens, and has been shown to be expressed frequently in lung cancers and to elicit an antibody response in patients with XAGE‐1b‐expressing tumors. In this study, we investigated an XAGE‐1b peptide recognized by CD4 T cells. CD4 T cells were purified from PBMC of a healthy donor and stimulated with pooled 25‐mer peptides overlapped with 15 amino acids spanning the entire XAGE‐1b protein. The generation of XAGE‐1b‐specific CD4 T cells was shown by IFN7 secretion assay. A CD4 T cell clone OHD1 was obtained by limiting dilution. OHD1 recognized two overlapping peptides, XAGE1‐b33–49 and XAGE‐1b37–52, by ELISPOT assay. A peptide XAGE‐1b38–46 which was included in both XAGE‐1b33–49 and XAGE‐1b37–52 was predicted to be a DRB1*0410‐restricted 9‐mer peptide by a computer‐based program. We identified the 12‐mer peptide XAGE‐1b37–48 as a new XAGE‐1b epitope restricted to HLA‐DRB1*0410.

Cryptic CTL Epitope on a Murine Sarcoma Meth A Generated by Exon Extension as a Novel Mechanism

Using the recently developed ELISPOT cloning methodology, we obtained cDNA clone S35 coding for the Ag epitope recognized by a murine sarcoma Meth A-specific CTL clone AT-1. Analysis of truncated S35 constructs and overlapping peptides revealed that the peptide epitope was LGAEAIFRL. AT-1 CTL lysed peptide-pulsed CMS8 cells at a nanomolar concentration, and the peptide strongly stimulated IFN-γ production in AT-1 CTL. Sequence homology indicated that the S35 was derived from a mouse homologue of human retinoic acid-regulated nuclear matrix-associated protein (ramp). The ramp gene consisted of 15 exons. The majority of the ramp mRNA was the transcript normally spliced between exons 14 and 15, but a minor population of mRNA with an extended exon 14 was also present in Meth A cells. The epitope was derived from the newly created open reading frame, which resulted from extension of exon 14 after splicing of the adjacent intronic sequence.

Modified metabolism of a carcinogen, 3-amino-1-methy-5H-pyrido[4,3-b]indole (Trp-P-2), by liver S9 from Schistosoma japonicum-infected mice

Schistosoma japonicum infection has been associated with an increased incidence of liver and colorectal cancers in humans. To explore the mechanisms underlying this association, we investigated the carcinogen-metabolizing properties of liver S9 preparations from S. japonicum-infected mice and compared them with those of S9 from uninfected animals. When the carcinogen 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) was incubated with these S9s and the products were analyzed by high-performance liquid chromatography, we observed that the S9 from infected mice had a lower ability to convert Trp-P-2 into 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2(NHOH)), an activated form of promutagenic Trp-P-2, than the S9 from uninfected mice. We found that both of these S9 preparations have a high ability to reduce Trp-P-2(NHOH) into Trp-P-2; however, the infected-mouse S9 showed a significantly greater reducing power than the control S9. This difference appears to be responsible for the observed lower mutagen-activating potential of the infected mouse S9. These results suggest that hepatic enzyme activities of S. japonicum-infected mice are quantitatively different from those of normal mice.

Abstract 3653: Enrichment of Foxp3+ CD4 Tregs in migrated T-cells to IL-6- and IL-8-expressing tumors through predominant induction of CXCR1 by IL-6

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: Analysis of cytokine and chemokine production by tumor cell lines including 5 lung cancers, a malignant mesothelioma and a malignant melanoma recently established in our laboratory showed rather high production of IL-8 in all tumors and IL-6 in one lung cancer, the malignant mesothelioma and the malignant melanoma. We investigated migration of PBMCs to these tumor cells using Transwell plates and showed enrichment of Foxp3+ CD4 Tregs in migrated T-cells to both IL-6- and IL-8-producing tumors. Marked induction of CXCR1 expression on Foxp3+ CD4 Tregs by IL-6 followed by IL-8-mediated migration appeared to be the mechanisms for enriched migration. Frequent production of IL-8 by the tumors and Treg migration to those tumors through induction of IL-8 receptor expression by IL-6 is one of the mechanisms for tumor escape. Purpose: In this study, we investigated cytokine and chemokine production by tumor cell lines including 5 lung cancers, a malignant mesothelioma and a malignant melanoma recently established in our laboratory. We observed IL-8 production in all tumors and IL-6 production in one lung cancer, a malignant mesothelioma and a malignant melanoma. We investigated migration of Foxp3+ CD4 Tregs in PBMCs to those tumor cells using Transwell plates. Experimental Design: Cytokine and chemokine production was analyzed in tumor cell lines including 5 lung cancers, a malignant mesothelioma and a malignant melanoma recently established in our laboratory. IL-8 production was detected in all tumors and IL-6 production in 3 of them. We then investigated the role of IL-6 and IL-8 on migration of Foxp3+ CD4 Tregs to those tumor cells using Transwell plates. Induction of IL-8 receptor on Foxp3+ CD4 T-cells by IL-6 was investigated by flow cytometry and its role on their migration was examined by anti-IL-8R or anti-IL-6R blocking. Furthermore, to investigate relationships between the effect of IL-6 and STAT3 signaling in the induction of IL-8R expression, we examined whether IL-8R expression would inhibit by blocking of STAT3 signaling. Results: We showed enrichment of Foxp3+ CD4 Tregs in migrated T-cells to both IL-6 and IL-8-producing tumors. Marked induction of CXCR1 (IL-8RA) expression was observed on Foxp3+ CD4 Tregs by the treatment with IL-6. Migration of IL-6-treated Foxp3+ CD4 T-cells to the tumors was blocked by the addition of anti-CXCR1 or anti-IL-6R. Furthermore, high level of IL-6 was detected in pleural effusion in most of lung cancer patients.Conclusions: Frequent production of IL-8 by the tumors and Treg migration to those tumors through induction of IL-8 receptor expression by IL-6 is one of the mechanisms for tumor escape. The findings indicate that the blocking of Treg migration by inhibiting IL-6-mediated IL-8 receptor expression could be a new strategy for tumor immunotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3653. doi:10.1158/1538-7445.AM2011-3653