Expression of cIAP-1 correlates with nodal metastasis in squamous cell carcinoma of the tongue.

Cellular inhibitor-of-apoptosis protein 1 (cIAP-1) is a member of the inhibitor-of- apoptosis protein family, which predominantly regulates apoptosis. It has been suggested that expression of cIAP-1 correlates with certain clinicopathological features. The possible significance of cIAP-1 expression in cervical lymph node metastasis of tongue squamous cell carcinoma (SCC) is investigated. Seventy-five tongue SCCs were analyzed by immunohistochemistry. cIAP-1 immunoreactivity patterns were nuclear in 38 (51%), cytoplasmic in 47 (63%), and concurrent in 37 (49%) cases. Nuclear, cytoplasmic and concurrent cIAP-1 immunoreactions were significantly correlated with lymph node metastasis in tongue SCCs (P=0.0011, 0.0012, and 0.0006, respectively). The cleaved caspase-3, which is a marker of tumor apoptosis, and Ki-67 index, which is a marker of tumor proliferation, were immunohistochemically examined in 21 tongue SCCs with concurrent nuclear and cytoplasmic cIAP-1 expression and with metastasis, and in 23 tongue SCCs without concurrent nuclear and cytoplasmic cIAP-1 expression and without metastasis. Concurrent cIAP-1 expression was inversely correlated with caspase-3 (P=0.0066), but was positively correlated with Ki-67 expression (P=0.0028). The mode of invasion was associated with lymph node metastasis (P=0.014) and differentiation (P=0.013), but was not correlated with cIAP-1 expression. There was no statistically significant correlation between nuclear or cytoplasmic cIAP-1 expression and the clinicopathological factors of gender, age, clinical stage or differentiation. These results suggest that both patterns of cIAP-1 are useful markers for predicting cervical lymph node metastasis in tongue SCC.

Cyclin B1 is useful to predict occult cervical lymph node metastases in tongue carcinoma.

Cyclin B1 plays an important role in regulating the G2-M transition of the cell cycle. In this study, we evaluated the immunohistochemical expression of cyclin B1 in a series of 40 oral tongue squamous cell carcinomas and analyzed the findings in relation to both clinicopathological variables and the Ki-67 labeling index. In normal squamous epithelium, cyclin B1 was localized in the nucleus and was expressed only in several cells of the basal and parabasal layers. By contrast, in dysplastic epithelium, cyclin B1 was localized in the cytoplasm and nuclei, and the numbers of cyclin B1 positive cells increased compared with normal epithelium. In tumor tissues, cyclin B1 expression was observed mainly in the cytoplasm, and the cyclin B1 labeling index ranged from 2 to 21%, with a mean of 11%. Cyclin B1 overexpression was positively correlated with occult cervical lymph node metastases and the number of mitotic cells. In addition, there was a positive relationship between labeling indices of cyclin B1 and Ki-67. These findings indicate that cyclin B1 could be a useful prognostic marker of occult cervical lymph node metastases in tongue carcinoma.

Comparison of primary structures and substrate specificities of two pullulan-hydrolyzing alpha-amylases, TVA I and TVA II, from Thermoactinomyces vulgaris R-47.

Thermoactinomyces vulgaris R-47 produces two alpha-amylases, TVA I, an extracellular enzyme, and TVA II, an intracellular enzyme. Both enzymes hydrolyze pullulan to produce panose, and also hydrolyze cyclodextrins. We cloned and sequenced the TVA I gene. The TVA I gene consisted of 1833 base pairs, and the deduced primary structure was composed of 611 amino-acid residues, including an N-terminal signal sequence consisting of 29 amino-acid residues. The similarity between the amino-acid sequence of mature TVA I with those of other pullulan/cyclodextrin-hydrolyzing enzymes, such as TVA II and Bacillus stearothermophilus neopullulanase, was only 30%, although that of TVA II with neopullulanase was 48%. TVA II prefers specific small oligosaccharides and alpha- and beta-cyclodextrins. Whereas kcat/Km values of TVA I for pullulan were larger than that of TVA II, and TVA II could not hydrolyze starch completely. TVA II was inhibited by maltose, the hydrolysate of starch, which seems to be the reason for inefficient hydrolysis of starch. These kinetic properties indicate that TVA I and TVA II have differential physiological roles in sugar metabolism extracellularly and intracellularly, respectively.

Efficient generation of autologous peripheral blood-derived cytotoxic T lymphocytes against poorly immunogenic human tumors using recombinant CD80-adenovirus together with interleukin 12 and interleukin 2.

To generate CTLs against poorly immunogenic human tumor cells, we transfected the human CD80 gene into the tumor cells using a replication-deficient adenovirus (Ad) vector. The successful surface expression of CD80 was obtained in both cultured tumor cell lines and primary cultured tumor cells. Transduction of CD80 alone was not sufficient to induce cytotoxicity of peripheral blood lymphocytes against allogeneic tumor cell lines except for melanoma cells. We, therefore, investigated a combined effect of CD80-Ad-infected tumor cells and interleukin 12 (IL-12). Although 7-day cultivation of autologous or allogeneic lymphocytes with CD80-Ad-infected tumor cells and IL-12 slightly enhanced cytotoxicity against some allogeneic tumor cells, no substantial cytotoxicity was observed against autologous tumor cells. When we extended the culture period to 14 days in the presence of IL-2, a prominent enhancement of cytotoxicity was observed against both allogeneic and autologous tumor cells. Cytotoxicity against autologous tumor cells, but not against allogeneic tumor cells, was efficiently inhibited by anti-CD3 monoclonal antibody. Furthermore, the selective cytotoxicity against a panel of targets indicated that the induced CTLs recognize specific antigens on autologous tumor cells. These results suggest that stimulation with a combination of IL-12- and CD80-modified tumor cells and subsequent expansion with IL-2 may efficiently generate tumor-specific CTLs from autologous peripheral blood lymphocytes. Our data imply that the combination of CD80 transduction and suitable cytokines is useful for enhancing antitumor immunity to poorly immunogenic human tumors.

Anti-malarial activity of leaf-extract of hydrangea macrophylla, a common Japanese plant.

To find a new anti-malarial medicine derived from natural resources, we examined the leaves of 13 common Japanese plants in vitro. Among them, a leaf-extract of Hydrangea macrophylla, a common Japanese flower, inhibited the parasitic growth of Plasmodium falciparum. The IC50 of Hydrangea macrophylla leaf extract to Plasmodium falciparum was 0.18 microg/ml. The IC50 to NIH 3T3-3 cells, from a normal mouse cell line, was 7.2 microg/ml. Thus, selective toxicity was 40. For the in vivo test, we inoculated Plasmodium berghei, a rodent malaria parasite, to ddY mice and administered the leaf-extract of Hydrangea macrophylla (3.6 mg/0.2 ml) orally 3 times a day for 3 days. Malaria parasites did not appear in the blood of in the treated mice, but they did appear in the control group on day 3 or 4 after inoculation with the parasites. When leaf extract was administered to 5 mice 2 times a day for 3 days, malaria parasites did not appear in 4 of the mice but did appear in 1 mouse. In addition, the leaf-extract was administered orally 3 times a day for 3 days to Plasmodium berghei infected mice with a parasitemia of 2.7%. In the latter group, malaria parasites disappeared on day 3 after initiating the treatment, but they appeared again after day 5 or 6. Although we could not cure the mice entirely, we confirmed that the Hydrangea macrophylla leaf extract did contain an anti-malarial substance that can be administered orally.

[Caregiver burden and subjective health level in primary caregivers of stroke patients].

The purpose of this study was to provide information on the burden and subjective health level experienced by caregivers of stroke patients. We assessed 32 patient/caregiver units attending a day care center, and investigated the characteristics and burden of caregivers. Subjective health level of caregivers was assessed using the Todai Health Index (THI). The functional level of patients was measured according to the Functional Independence Measure (FIM). Of the caregivers, 40.6% expressed a perception of being burdened. The burden of caregivers was associated with economic status, concern for future ability to provide care, and functional levels (ability of toileting, transfer, locomotion) of patients. Mean scale scores of THI did not differ significantly from scores for community women, and was not related to the perception of burden of caregivers. The situation of care for the subjects in this study can be considered to be relatively good. However, for the general caregiver of stroke patients, perception of burden and subjective health level may be affected to a greater extent than seen in this study.

[Studies on generation of cytotoxic T lymphocytes against oral squamous carcinoma by gene-transduction of a co-stimulatory molecule, CD80].

The effect of gene-transduction of a co-stimulatory molecule, CD80, on generation of cytotoxic T lymphocytes (CTL) against oral squamous cell carcinoma (SCC) was investigated. Long-term or primarily-established short-term cultured oral SCC cell lines were transfected with the human CD80 gene using a replication-deficient recombinant adenovirus (Ad). High levels of CD80 expression were obtained in most tumor cell lines examined. Allogeneic peripheral blood mononuclear cells (PBMC) co-cultured with CD80-transduced tumor cells for 7 days elicited high cytotoxicity against melanoma (526 mel) and neuroblastoma (IMR32) cells, but not against oral SCCs (HSC3 and Ca9-22). Addition of either IL-2 or IL-12 failed to induce specific cytotoxicity against oral SCC cell lines. The combined effect of CD80-transduced tumor cells and cytokines, IL-2 and IL-12, on generation of CTL in allogeneic and autologous system was investigated. PBMC from three SCC patients elicited MHC-restricted and TCR-dependent cytotoxicity against autologous SCC, although the level of induced cytotoxicity varied. In contrast, allogeneic PBMC obtained from healthy donors exhibited non-specific cytotoxicity alone. These results suggested that CD80-transduction may be effective in oral SCC for generating tumor specific CTL in an autologous system.

Tumour rejection by gene transfer of 4-1BB ligand into a CD80(+) murine squamous cell carcinoma and the requirements of co-stimulatory molecules on tumour and host cells.

NRS1 is a murine squamous cell carcinoma that constitutively expresses the co-stimulatory molecule CD80 at a high level yet grows as a tumour in syngeneic C3H mice. We examined the effect of gene transfer of the 4-1BB ligand (4-1BBL) into NRS1 cells. Introduction of the 4-1BBL gene efficiently elicited anti-tumour immune responses in syngeneic mice which acquired specific immunity against wild-type tumour. T-cell depletion studies showed that CD8(+), but not CD4(+) T cells were essential for tumour eradication. Our results suggest that the transduced 4-1BBL is more effective than the spontaneously expressed CD80 for generation of primary anti-tumour CD8(+) T-cell responses. In addition to CD80 and CD86, the host-derived 4-1BBL is also involved in the secondary anti-tumour responses. This study indicates the complicated contribution of 4-1BBL, CD80 and CD86 on tumour and host cells in anti-tumour immune responses and a possible therapeutic application of 4-1BBL for human tumour vaccination and gene therapy.

A neopullulanase-type alpha-amylase gene from Thermoactinomyces vulgaris R-47.

Shimizu et al. and ourselves have reported some enzymatic properties of an alpha-amylase from Thermoactinomyces vulgaris R-47 that hydrolyzed pullulan to produce panose [M. Shimizu et al., Agric. Biol. Chem., 42, 1681-1688 (1978); Y. Sakano et al., Agric. Biol. Chem., 46, 1121-1129 (1982)]. In this study, we cloned a gene for an alpha-amylase, which was different from the one mentioned above but also hydrolyzed pullulan to produce panose, from T. vulgaris R-47, and analyzed the entire primary structure of the gene. We designated the previously reported enzyme as T. vulgaris alpha-amylase I (TVA I), and this novel enzyme as T. vulgaris alpha-amylase II (TVA II). The nucleotide sequence had an open reading frame of 1755 base pairs corresponding to a protein of 585 amino acid residues. Although this novel alpha-amylase, TVA II, hydrolyzed both pullulan and starch, the ratio of pullulan-hydrolyzing activity to starch-hydrolyzing activity of the enzyme was higher than that of TVA I, and the primary structure of the enzyme resembled neopullulanase, which scarcely hydrolyzed starch, rather than that of TVA I.

Immunohistochemical analysis of keratin distribution in eccrine poroma.

Although eccrine poroma has been thought of as a neoplasm of the intradermal eccrine duct, this interpretation has not been entirely confirmed. In this study, twenty-five cases of eccrine poroma were retrieved and analyzed by immunohistochemical techniques, using various kinds of monoclonal antikeratin antibodies. Comparative immunohistochemical observations of eccrine poroma and normal eccrine glands revealed that the poroma cells expressed immunophenotypes similar to those of the basal cells of dermal eccrine ducts. Sweat-ductlike structures showed similar staining patterns to those observed in the inner cells of dermal eccrine ducts. Some cystic spaces were similar to those observed in the secretory cells of eccrine glands. Eccrine poroma is, therefore, speculated to originate via the proliferation and expansion of the basal cells of eccrine ducts, although it is very difficult to prove the histogenesis. Some tumor cells may differentiate toward inner cells of the eccrine ducts, forming ductal lumina, whereas other tumor cells differentiate toward eccrine secretory regions, forming some cystic spaces.

A novel enzyme immunoassay commonly applied for ten strains of Pyricularia oryzae.

Antiserum against a strain of the rice blast fungus Pyricularia oryzae was elicited in rabbits immunized with its cell fragments emulsified with incomplete Freund's adjuvant. The fragments were also used as solid-phase antigens. A highly sensitive, competitive type enzyme-linked immunosorbent assay for P. oryzae was developed by using these two preparations as the immune reagents together with the use of beta-D-galactosidase-labeled anti-rabbit IgG as the tracer. Cross-reactivity of nine different strains of P. oryzae were measured by the assay. Sensitivity and accuracy of the assay was improved by choosing the cell fragments of the least cross-reactive strain as the solid-phase antigen. The improved method was successfully applied for sensitive and accurate assay of all ten strains of P. oryzae with the common measuring range between 1 and 100 ng per tube. Other species of microorganisms had little reactivity in this immunoassay indicating that the assay is specific to P. oryzae group microorganisms.

Rapid induction of CD95 ligand and CD4+ T cell-mediated apoptosis by CD137 (4-1BB) costimulation.

We investigated the cytolytic mechanism by CD4+ T cells in anti-CD3 mAb-induced redirected cytotoxicity against a murine Fc receptor-bearing mastocytoma (P815) transfected with either CD80 or CD137 ligand (CD137L). CD137 costimulation preferentially induced anti-CD3-induced redirected cytotoxicity within 4 h. This cytotoxicity was efficiently abrogated by the addition of anti-CD137L or anti-CD95L mAb, or by treatment with a broad caspase inhibitor, Z-VAD, suggesting that the induced cytotoxicity against CD137L-P815 is dependent on CD95L-mediated apoptosis. In contrast, the cytotoxicity against CD80-P815, but not CD137L-P815 was efficiently inhibited by an inhibitor of perforin-dependent cytotoxicity, concanamycin A. Involvement of CD95L in the CD137L-dependent cytotoxicity was confirmed by a failure of induction of cytotoxicity by CD4+ T cells from CD95L-gene mutated gid mice. A rapid and remarkable induction of CD95L transcription within 1 h was observed by CD137L costimulation. These results demonstrated that CD137L costimulation induces a rapid induction of CD95L on CD4+ T cells and leads to apoptosis of CD95-sensitive target cells. This biological function of CD137 in CD4+ T cells may play an important role for immune homeostasis.