[Surgical treatment for stage IV lung cancer].

OBJECTIVE: To find out the optimal surgical indication in stage IV lung cancer patients, we evaluated them retrospectively. METHODS & RESULTS: From 1975 to 2005, 62 patients without multiple metastases were operated at our hospital. The most common histological type was adenocarcinoma (67.7%). The metastatic lesions were lung (33.9%), brain (24.2%), liver, bone, adrenal gland and so on. The overall survival rate of stage IV lung cancer was 10.4% at 5-year. Five-year survival for patients with lung or brain metastasis who had no lymph node metastasis were significantly more superior than those with lymph node metastasis (p=0.0389, 0.0021). Four of 62 patients had 5-year survival. Two were lung and the others were brain and adrenal gland metastasis without lymph node metastasis. CONCLUSION: Stage IV lung cancer with lung or brain or adrenal gland metastasis without lymph node metastasis should be resected.

TRPV1 activation and induction of nociceptive response by a non-pungent capsaicin-like compound, capsiate.

Capsiate is a capsaicin-like ingredient of a non-pungent cultivar of red pepper, CH-19 sweet. To elucidate the mechanisms underlying the non-pungency of capsiate, we investigated whether capsiate activates the cloned capsaicin receptor, TRPV1 (VR1). In patch-clamp experiments, capsiate was found to activate TRPV1 expressed transiently in HEK293 cells with a similar potency as capsaicin. Capsiate induced nociceptive responses in mice when injected subcutaneously into their hindpaws with a similar dose dependency as capsaicin. These data indicate that the non-pungent capsiate is an agonist for TRPV1 and could excite peripheral nociceptors. In contrast to this, capsiate did not induce any significant responses when applied to the skin surface, eye or oral cavity of mice, suggesting that capsiate requires direct access to nerve endings to exhibit its effects. Capsiate was proved to have high lipophilicity and to be easily broken down in normal aqueous conditions, leading to less accessibility to nociceptors. Another highly lipophilic capsaicin analogue, olvanil, was similar to capsiate in that it did not produce irritant responses when applied to the skin surface, although it could activate TRPV1. Taken together, high lipophilicity and instability might be critical determinants for pungency and so help in understanding the effects of capsaicin-related compounds.

Release of IL-4 and IL-5 from human peripheral blood mononuclear cells co-cultured with Japanese cedar pollen antigen in vitro.

The Japanese cedar pollen (JCP) is a major allergen with respect to pollinosis in Japan. It is believed that interleukin-4 (IL-4) and interleukin-5 (IL-5) derived from lymphocytes and other cells play a pivotal role in allergic reactions. We investigated whether the JCP antigen stimulates the release of these cytokines by peripheral blood mononuclear cells (PBMCs). PBMCs from eight adults (five adults with JCP pollinosis and three adults without JCP pollinosis) were co-incubated with purified JCP antigens. IL-4 was released in response to JCP antigens in six of the eight subjects at 24 h and in three subjects at 48 h. IL-4 release at 24 h occurred in all five subjects with JCP pollinosis but in only one of the three subjects without pollinosis. IL-5 was released in response to the JCP antigen in five of the eight subjects at 24 h and 48 h, including four of the five subjects with JCP pollinosis and one of the three subjects without pollinosis. These results suggest that PBMCs were more likely to release IL-4 and IL-5 in the presence of JCP pollinosis.

Analysis of the accumulation of mutants in Sabin attenuated polio vaccine viruses passaged in Vero cells.

To confirm the safety of oral poliomyelitis vaccine (OPV) cultured in Vero cells, the genetic stability of cultured polio vaccine viruses was analysed by MAPREC (mutant analysis by PCR and restriction enzyme cleavage). The rates of mutant accumulation of the viruses passaged in Vero cells under a low multiplicity of infection (MOI) condition (approximately 10(-3.5)CCID50/cell; the same as under usual OPV production conditions) were higher than those passaged in secondary cultured monkey kidney cells. However, the rates of mutant accumulation were restrained when the viruses were cultured under a high MOI condition (approximately 10(-1.5)CCID50/cell) in Vero cells. Furthermore, neurovirulence of the passaged viruses in pollovirus susceptible transgenic mice PVR-Tg21 was shown to correlate highly with the results of MAPREC. It is expected that our results will contribute to the large scale preparation of safe and effective OPV using Vero cells.

Availability of oncogene activated production system for mass production of light chain of human antibody in CHO cells.

We previously established a ras-oncogene amplified Chinesehamster ovary (CHO) cell line, named ras clone I, as anuniversal host cell line for oncogene activated production(OAP) system to mass-produce recombinant protein by activationof the cytomegalovirus immediate early (CMV) promoter with ras protein. The lambda light chain(C5lambda) of human monoclonal antibody HB4C5 is expected tobe potentially useful for lung cancer targeting. We generated aC5lambda hyper-producing cell line by transfecting ras cloneI with the C5lambda gene expression plasmid regulated by theCMV promoter, of which productivity was 5.3 times greater thanthe hyper productive CHO cell line generated by using conventional CHO cells. Introduction of the adenovirus E1A geneinto the hyper-producing cell line derived from ras clone I resulted in further 9.5 times enhancement of the productivity,suggesting the synergistic effect of E1A and ras oncogenes on the recombinant protein production driven by the CMV promoter. In addition, intracellular accumulation of C5lambda andupregulation of BiP was found in hyper-producing cell lineswhich were introduced E1A and ras oncogene. This resultsuggests that excessive intracellular accumulation ofC5lambda protein, which might be caused by that the amount of produced C5lambda in ER is beyond the ability of CHO cells to secrete, might signal the BiP promoter. Our data imply that ras clone I is available as a general host cell for establishing the recombinant protein hyper-producing CHOcells by the OAP system, and suggest that further mass production of recombinant proteins in the OAP system can be possible by clarifying the accurate role of upregulated BiP protein.

Molecular analysis of cross-reactive human monoclonal antibody AE6F4 generated by in vitro immunization: Epitope mapping of AE6F4 antibody on 14-3-3 family proteins and cytokeratin 8.

We reported previously that adenocarcinoma-reactive human monoclonal antibody AE6F4, which had been generated by in vitro immunization method, recognizes both 14-3-3protein and cytokeratin 8 (CK8). In this study, to analyze the cross-reactivity of AE6F4 antibody, epitopes of AE6F4 antibody on 14-3-3 proteins and CK8 were studied by using synthetic linear peptide scanning technology. To determine the locations of B cell epitope, 48 and 95 of decapeptides covering the entire 14-3-3 proteins and CK8, respectively,were synthesized and binding to AE6F4 antibody was examined by ELISA. The AE6F4 antibody was strongly reactive to peptides containing amino acid sequences TLWTSDTQGD in 14-3-3 proteins and INFLRQLYEE in CK8. These results indicate that AE6F4 antibody can recognize the different peptide sequences in 14-3-3 proteins and CK8.

Establishment of hybridomas producing cancer specific human antibodies from B cell line derived from PBL of a patient with adult T cell leukemia.

Adult T cell leukemia (ATL) is a malignant disease characterized by tumorous proliferation of CD4(+) T cells infected with retrovirus human T cell leukemia virus Type-I (HTLV-I) and concurs with an autoimmune disease and cancer due to attenuated immune response. In this study, we established ATL patient derived B-cell line TM-1 producing cancer-specific IgM antibodies, and further characterized its antigen specificity by establishing hybridomas fused with human-mouse origin hetero-myeloma cell line RF-S1. We established three hybridoma cell lines termed 2E12, 3E9, and 3E10, which continuously secreted human IgM antibodies. Immunohistochemical staining of formalin-fixed tissue section using antibodies secreted from these hybridomas showed that these antibodies specifically recognized tumor sites of human colon adenocarcinomas. Antibody produced from hybridoma 3E9 bound to some of leukemic cell lines, but not to normal human PBL, which was evidenced by the flow cytometric analysis, indicating that antibody produced from 3E9 recognizes cell surface antigen specifically expressed in the leukemic cells.

Progress with inactivated poliovirus vaccines derived from the Sabin strains.

To produce a safe and effective inactivated poliovirus vaccine (IPV), we have developed S-IPV using Vero cells infected with the Sabin strains in a semi-production scale. All production steps including virus culture on microcarrier beads were highly reproducible. Mean recovery percents of infectious viruses or D-antigens during all processes for concentration, purification and inactivation were 30-50% in the three types. The S-IPV potency was adjusted for D-antigen content as determined by in-house ELISA and was comparable to WHO reference IPV derived from the virulent strains in immunogenicity tests in rats. Antibody development in more than 30 seronegative infant volunteers after two shots of S-IPV at four-week interval were 100% without notable adverse reactions. The mean antibody titres (log2) to Sabin 1, 2 and 3 viruses were 11.1, 8.3 and 8.9, respectively. The antibodies neutralized the Mahoney, MEF-1, and Saukett virulent strains with slightly inferior titres to those of the Sabin strains. D-antigens for each type of S-IPV were stable at 4 degrees C without any significant decrease over more than two years.

Properties of ras-amplified recombinant BHK-21 cells in protein-free culture.

We compared serum and protein-free cultures ofa ras-amplified recombinant BHK-21 cell line(ras-rBHK-IgG), which hyperproduces a lungcancer specific recombinant human monoclonal antibody. Ras-rBHK-IgG cells were shown to grow well, evenin protein-free medium and to be morphologicallysimilar to cells cultured in serum containing medium. However, the growth rate of ras-rBHK-IgG cellswas considerably slower in protein-free medium, whichresults in a longer maintenance period compared with cells cultured in serum containing medium. In addition, it was found that antibody production in protein-free culture had a ten times higher maximum than cells cultured in serum containing medium. On theother hand, in high density culture, using the hollowfiber bioreactor system, ras-rBHK-IgG cellscould be maintained for a month in protein-freeculture in contrast with serum culture, which onlylasted for half a month. However, the markedincrease of antibody production was not observed. A total amount of about 15 mg of the recombinantantibody, obtained in protein-free culture, was abouttwo times of that obtained in serum culture, and wasshown to be reactive to lung cancer cells in tissue. From these properties in protein-free medium, it isconcluded that protein-free culture of ras-rBHK-IgG cells is suitable for middle scaleproduction of recombinant human monoclonal antibody.

Enhanced antibody production of human-human hybridomas by retinoic acid.

The enhancement of human monoclonal antibody production by retinoic acid (RA) was evaluated usingthe human-human hybridoma cell line BD9 underserum-free culture condition. The amount of humanIgG secreted by BD9 hybriodmas was enhanced abouteight-fold by treatment with 10(-7) M of RA for 4days. Northern blot analysis showed that both mRNAlevels of the IgG light and heavy chains were markedlyincreased by RA when compared with control without RAtreatment. On the other hand, it was found thatcontinuous treatment of cells with RA was not alwaysrequired to exhibit the enhancing effect, suggestingthat RA may act as a trigger for IgG gene expression. The comparison between extra- and intracellular IgGamounts by immunoblot analysis suggests that thesecretion rate of IgG may be accelerated by RAtreatment. These results suggest that RA may be aneffective culture additive for efficient production ofhuman monoclonal antibody using human-humanhybridomas.

[Antibacterial effects of cacao mass on enterohemorrhagic Escherichia coli O157:H7].

The antimicrobial activities of aqueous cacao mass extract against enterohemorrhagic Escherichia coli (EHEC) O157:H7 006 strain were studied. Hot water extract of cacao mass (cocoa extract) was shown to inhibit the growth of EHEC O157:H7 006 strain in PBS or CAYE medium. In addition, the production of verotoxins (types 1 and 2) of EHEC O157:H7 006 strain was significantly inhibited by 8.0% cocoa extract. The cocoa extract did not neutralize the cytotoxity of verotoxins, but had inhibitory effect on adhesion of verotoxins to the target Vero cells. These results demonstrate that cacao mass has antimicrobial effects on EHEC O157:H7.

Recombinant light chain of human monoclonal antibody HB4C5 as a potentially useful lung cancer-targeting vehicle.

Recombinant lambda light chain of lung cancer-reacting human monoclonal antibody HB4C5 was expressed in Escherichia coli. Expression in bacteria ensured the generation of homogeneous light chain species devoid of activity-hampering N-linked glycosylation usually found in the light chain CDR-1 of HB4C5. Molecular engineering was also employed to eliminate the C-terminal two amino acid residues, i.e., Cys and Ser, to prevent the formation of lambda light chain dimers which are less reactive than the monomeric form. The lambda light chain was overexpressed in E. coli as inclusion bodies, which were solubilized, refolded, and treated with Aeromonas proteolytica aminopeptidase to remove the N-terminal Met with subsequent natural cyclization of the penultimate Gln residue to pyroglutamate, the same N-terminal end as that of naturally occurring lambda light chain in HB4C5. Monomeric recombinant lambda light chains, both before and after removal of the N-terminal Met residue, were 40 times more immunoreactive than the parent HB4C5. The immunostaining of lung cancer tissue sections with the recombinant lambda light chain indicated cancer-specific reactions to all specimens of adenocarcinoma, squamous cell carcinoma and large cell carcinoma histologies, but did not react with small cell carcinoma. Tumor radioimmunoimaging experiments in LC6 (lung squamous cell carcinoma line)--xenografted nude mice by the i.p. injection of 125I-labeled recombinant lambda light chain and 125I-labeled human lambda light chain control gave tumor-specific and recombinant lambda light chain-dependent images on day 5 postinjection, and images were also detectable on day 3. Biodistribution studies with 125I-labeled recombinant lambda light chain demonstrated that the lambda light chain could penetrate better into the tumor sites, both at the necrotic and solid parts of the xenograft, as compared to our previous results with 125I-labeled HB4C5 which could localize to the necrotic part only. These results suggest that the recombinant lambda light chain is potentially useful as a lung cancer-targeting vehicle, for such as radioimmunoimaging and radioimmunotherapy, with least possible adverse immunogenic effects.

Evaluation of an enzyme-linked immunosorbent assay based on binding inhibition for type-specific quantification of poliovirus neutralization-relevant antibodies.

To detect neutralization-relevant antibodies against 3 types of poliovirus (PV) without using tissue cultures and live viruses, an enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody-binding inhibition was evaluated using sera from 80 vaccinated Japanese children and 60 Pakistani poliomyelitis patients. Compared with the neutralization test, the sensitivity of the inhibition ELISA was 100% (111/111) for detection of anti-PV1 antibody, 98.3% (118/120) for anti-PV2, and 96.5% (82/85) for anti-PV3, and the specificity was 93.1% (27/29), 100% (20/20), and 92.7% (51/55), respectively. Thus, the inhibition ELISA showed excellent potential as a seroepidemiologic tool in both vaccinated and naturally-infected populations.

Detection of mutants in polio vaccine viruses using pooled antipoliovirus monoclonal antibodies.

We prepared six monoclonal antibodies (mAbs) for type 1 polioviruses, and analysed their neutralizing specificities for use in safety tests in oral poliomyelitis vaccine (OPV) production. Pools of two or more individual mAbs showed high neutralizing activity against high-titre (approximately 10(7) CCID (50)/25 microl) of Sabin type 1 virus. It was demonstrated that the pooled mAbs can be utilized effectively in detection tests of adventitious viruses, which are among the safety tests in OPV production. Moreover, some pooled mAbs were shown to be capable of detecting very small amounts of type 1 virulent viruses and mutants in high-titre Sabin type 1 virus suspensions. Neutralizing antibody titres of these pooled mAbs decreased with increasing numbers of mutants containing neurovirulent activity in high-titre Sabin type 1 viruses which were repeatedly passaged in culture. It is expected that these pooled mAbs will contribute greatly to safety tests for OPV production.

Effectiveness of vitamin A acetate for enhancing the production of lung cancer specific monoclonal antibodies.

The antibody productivity of the human-human hybridoma cell line AE6, which produces the lung cancer specific human monoclonal antibody AE6F4, was enhanced fourfold upon stimulation with 1 mug/ml of vitamin A acetate for one day. The enhancement lasted for about two weeks, and could be repeated by another stimulation with vitamin A acetate. The enhancing effect of vitamin A acetate was influenced by the cell density. Enhancement was clearly observed when the cell density was under 10(6) cells/ml. However, when the cell density was over 10(7) cells/ml, enhancement was observed weakly or not at all. Although the enhancing effect of vitamin A acetate is not unique to AE6 cells, not all human-human hybridoma cell lines show increased productivity upon VA acetate stimulation. This study suggests that the response to vitamin A acetate may be related to the properties of a particular fusion partner which the hybridoma cell inherits. The efficacy of vitamin A acetate for production of human monoclonal antibodies using human-human hybridomas is discussed.

In vitro immunization of human peripheral blood lymphocytes: establishment of B cell lines secreting IgM specific for cholera toxin B subunit from lymphocytes stimulated with IL-2 and IL-4.

In vitro immunization (IVI) techniques have a great potential in the production of human monoclonal antibodies (MAbs) against various antigens. An IVI method of human peripheral blood lymphocytes (PBL) has been developed with a human lung adenocarcinoma cell line in our laboratory. Although several cancer specific human MAbs were successfully generated by using this IVI method, it was not available for soluble antigens, which prompted us to improve the method for generation of human MAbs against soluble antigens. IVI with soluble antigens was effectively caused by the addition of muramyl dipeptides, interleukin-2 and interleukin-4. It was found that the difference of sensitivity of lymphocytes depending upon donors could be overcome by finding the optimal concentrations of IL-2 and IL-4. IVI of human PBL was performed with cholera toxin B subunit (CTB) and the immunized B cells were transformed by Epstein-Barr virus. Anti-CTB antibody was detected using an indirect ELISA. B cells producing anti-CTB antibodies were directly cloned by a soft agar cloning method.

A new internal ribosomal entry site 5' boundary is required for poliovirus translation initiation in a mouse system.

Four mutants of the virulent Mahoney strain of poliovirus were generated by introducing mutations in nucleotides (nt) 128 to 134 of the genome, a region that contains a part of the stem-loop II (SLII) structure located within the internal ribosomal entry site (IRES; nt 120 to 590) (K. Shiroki, T. Ishii, T. Aoki, Y. Ota, W.-X. Yang, T. Komatsu, Y. Ami, M. Arita, S. Abe, S. Hashizume, and A. Nomoto, J. Virol. 71:1-8, 1997). These mutants (SLII mutants) replicated well in human HeLa cells but not in mouse TgSVA cells that had been established from the kidney of a poliovirus-sensitive transgenic mouse. Their neurovirulence in mice was also greatly attenuated compared to that of the parental virus. The poor replication activity of the SLII mutants in TgSVA cells appeared to be attributable to reduced activity of the IRES. Two and three naturally occurring revertants that replicated well in TgSVA cells were isolated from mutants SLII-1 and SLII-5, respectively. The revertants recovered IRES activity in a cell-free translation system from TgSVA cells and returned to a neurovirulent phenotype like that of the Mahoney strain in mice. Two of the revertant sites that affected the phenotype were identified as being at nt 107 and within a region from nt 120 to 161. A mutation at nt 107, specifically a change from uridine to adenine, was observed in all the revertant genomes and exerted a significant effect on the revertant phenotype. Exhibition of the full revertant phenotype required mutations in both regions. These results suggested that nt 107 of poliovirus RNA is involved in structures required for the IRES activity in mouse cells.

Estimation of the neurovirulence of poliovirus by non-radioisotope molecular analysis to quantify genomic changes.

Mutant analysis by polymerase chain reaction and restriction enzyme cleavage (MAPREC) has been developed for poliovirus to determine quantitatively for the presence of genomic changes in particular nucleotide sequences correlate with the characteristic of neurovirulence for monkeys. Currently the MAPREC is scheduled to be used as a routine safety test for oral poliomyelitis vaccine (OPV). Radioisotopes (RI) are used in MAPREC for quantitative determinations, a circumstance likely to limit its use. We investigated the possibility of developing a modified MAPREC, which did not require the use of radioisotopes, and developed a procedure designated NON-RI MAPREC. Conventional MAPREC and NON-RI MAPREC were then used in a series of studies in which analyses were performed on Sabin type 1 and Sabin type 3 attenuated vaccine polioviruses prepared under various conditions. Under the experimental conditions used, the stability of the genome of type 1 virus was shown to be markedly greater than that of the type 3 virus, and the frequency of mutants was observed to vary in relation to both the virus strain and the virus inoculum used. The results of the studies relating to the two analytical procedures used indicated that the reproducibility of both methods was of a similarly high order, but that MAPREC had a somewhat broader range of sensitivity than NON-RI MAPREC. As the quantity of genomic changes in OPV relating to neurovirulent properties are within the range of detection by NON-RI MAPREC, this procedure can be used as a quality control test for OPV.

[A case of hepatic arterial infusion chemotherapy for multiple liver metastasis from breast cancer].

A 52-year-old woman underwent Auchinclass' operation for breast cancer. The histological type was papillotubular carcinoma. One year and 5 months after operation, multiple liver tumors were found on the CT scan and multiple bone metastasis on MRI. The former were treated by hepatic artery infusion chemotherapy with epirubicin and 5-FU using a subcutaneous implanted pump and the latter by 50 Gy irradiation. The patient began to complain of abdominal pain and discomfort after hepatic artery infusion, so all treatment was discontinued. Six months later the patient died of respiratory failure due to pleural dissemination. No liver mass was detected, and bone metastasis was not changed in section tissues. This suggested that the therapy for a breast cancer patient with distant metastasis must be considered according to the region of recurrence.