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S Hashizume

Publications and source records attributed to S Hashizume.

At least 37 records · Page 2Linked to original sources
PubMed1997

Cytokeratin 8 and 19 as antigens recognized by adenocarcinoma-reactive human monoclonal antibody AE6F4.

The human monoclonal antibody (MAb) AE6F4 is secreted by a human-human hybridoma line established from the in vitro immunization of normal human peripheral blood lymphocytes with the human lung adenocarcinoma cell line, A549. This MAb is strongly reactive to lung cancer tissues. In the previous study, the antigens recognized by the MAb AE6F4 were purified from A549 cells and identified as 14-3-3 protein and 31 kDa cytosolic phospholipase A2 (cPLA2). The MAb AE6F4 also binds two kinds of antigens (53 kDa and 40 kDa), which are not related to 14-3-3 protein or 31 kDa cPLA2, in the human breast adenocarcinoma cell line, MCF-7. We purified a 38 kDa antigen, which is a degradation product of 53 kDa antigen from breast adenocarcinoma MCF-7 cells using ion-exchange and hydroxyapatite column chromatography. Two partial amino acid sequences of the purified 38 kDa antigen showed 95-100% homology to human cytokeratin 8 (CK8). Two-dimensional gel electrophoresis and immunoblot analysis of intermediate filament fraction separated from MCF-7 cells demonstrated that the 53 kDa and 40 kDa antigens were CK8 and CK19, respectively. Antigenic determinants on CK8 and CK19 recognized by the MAb AE6F4 were resistant to sodium periodate treatment, although antigenic determinant on 31 kDa antigen (14-3-3 protein and(or) cPLA2) was sensitive to this treatment. These results suggest that the MAb AE6F4 reacts with both carbohydrate and peptide antigenic determinants.

Adenocarcinoma↗
PubMed1996

The use of antipoliovirus monoclonal antibodies as an improved safety test for oral live poliomyelitis vaccine.

Almost all the individual preparations of poliovirus type 3 specific monoclonal antibodies showed high neutralizing antibody titres against low-titre Sabin type 3 virus with a titer of 10(2) CCID 50/25 microliters. However, the preparations failed to neutralize high-titre virus at a titre of about 10(7) CCID50/25 microliters (undiluted virus fluid). The use of pooled monoclonal antibodies, comprising two or more individual antibodies, however, showed a high neutralizing activity for the high-titre virus suspension. The results indicate that the neutralization of high-titre Sabin type 3 virus can be achieved by the use of pooled monoclonal antibodies when appropriate antibodies are present. It seems likely that neutralization by these antibodies is achieved through the recognition of different neutralizing epitopes by specific antibodies. One of the safety tests used during the production of oral poliomyelitis vaccine (OPV) is that which employs type-specific antipoliovirus rabbit sera to detect adventitious viruses in monovalent poliovirus fluid. It has, however, proved difficult to prepare sera of this nature which can completely neutralize high-titre poliovirus having no cross-reactivity against other types of poliovirus. In our studies, pooled monoclonal antibodies showed a neutralizing activity against high-titre Sabin type 3 virus about 100-fold greater than that shown by rabbit antisera. Moreover, these antibodies appear to have two further important advantage for use in testing the safety of OPV: they showed no cross-neutralizing activity against heterotypic polioviruses; and they showed no evidence inhibiting the propagation of a number of viruses of monkey and human origin.

Animals↗
PubMed1996

Production of recombinant human monoclonal antibody using ras-amplified BHK-21 cells in a protein-free medium.

A ras oncogene-amplified recombinant BHK-21 cell line (ras-rBHK-IgG) has been established, and was shown to hyperproduce the recombinant IgG chimeric human monoclonal antibody (hMAb) AE6F4, which recognizes lung cancer cells. We found that the ras-rBHK-IgG cell could be easily cultured in a protein-free ERDF medium supplemented with iron(III) nitrate, hydroxyethyliminodiacetic acid, and non-protein synthetic attachment factor as well as in a serum-free ERDF medium supplemented with insulin, transferrin, ethanolamine, and sodium selenite. The productivity of recombinant hMAb from the cells cultured in dishes at high cell densities was higher in protein-free medium than in serum-containing medium. True high density culture of the ras-rBHK-IgG cells was done in protein-free medium using the Tecnomouse, which is a novel hollow fiber bioreactor system. After culture for 30 days in protein-free culture, a total amount of about 14 mg of the recombinant hMAb AE6F4 was obtained, and was shown to be reactive against lung cancer cells in tissues.

Animals↗
PubMed1996

Lung cancer-reacting human recombinant antibody AE6F4: potential usefulness in the sputum cytodiagnosis.

Human monoclonal antibody (hMAb) AE6F4 has been shown to be potentially useful for immunocytological detection of lung cancer cells in sputum. By recombinant DNA technology, IgM type hMAb AE6F4 was switched to lgG. The IgG mimic recombinant AE6F4 antibody expression plasmid was assembled using the antibody heavy chain gene, which ligated the gene encoding VH and CH1(mu) domains of hMAb AE6F4 heavy chain to the gene encoding CH2(gamma 1) and CH3(gamma 1) domains of human IgG heavy chain, and the antibody light chain gene of hMAb AE6F4. The recombinant antibody expressed by baby hamster kidney (BHK)-21 cells showed molecular size equivalence to IgG, and consisted of human mu-gamma hybrid heavy and kappa light chains. The immunological specificity of the recombinant antibody was the same as that of hMAb AE6F4 by immunoblotting analysis to the 14-3-3 protein, the putative antigen of hMAb AE6F4, and by immunohistochemical and immunocytological analyses using tissue sections and sputa of lung cancer patients. The transfected BHK-21 cells produced the recombinant antibody persistently and the productivity was greater than 20 times that by human-human hybridoma producing hMAb AE6F4.

14-3-3 Proteins↗
PubMed1995

Studies on neurovirulence in poliovirus-sensitive transgenic mice and cynomolgus monkeys for the different temperature-sensitive viruses derived from the Sabin type 3 virus.

We have studied methods for testing the neurovirulence of live poliovaccine viruses by intraspinal inoculation into mice carrying the human poliovirus receptor gene (Tg mice). A comparison of the neurovirulence of Sabin type 3 vaccine virus and related viruses using the 50% paralysis dose determined after intraspinal inoculation into the Tg mice as an index revealed a close correlation between the results of the paralysis dose in Tg mice, the neurovirulence expressed by the histopathological lesions core in monkeys, and the temperature sensitivity of the viruses. The results of experiments in the Tg mice also showed a good correlation with the number of mutations at position 472 from U to C in the 5' noncoding region in the genomes of the viruses tested. These results strongly suggest that the neurovirulence test for oral poliomyelitis vaccine using the Tg mice is an excellent method and may be used in place of the test using monkeys.

Animals↗
PubMed1995

Neurovirulence test for oral live poliovaccines using poliovirus-sensitive transgenic mice.

The transgenic mice, ICR-PVRTg21, carrying the human poliovirus receptor gene, were intraspinally inoculated with oral poliovirus vaccine viruses and the viruses that had been derived from a vaccine preparation by passagings at 38 degrees. Dose-dependent incidence of paralysis was observed in the transgenic mice inoculated with any of the viruses used. All transgenic mice showing histopathological lesions in the central nervous system appeared to display clinical signs that resembled those observed in human poliomyelitis. These suggest that relative neurovirulence levels of individual virus preparations can be estimated by values of 50% paralysis dose. Indeed, the parameter correlated well with monkey lesion scores of viruses used. Histopathological changes such as degeneration of neurons resemble those displayed by monkeys and were observed in the spinal cord, medulla oblongata, and pons. Thus, the transgenic mice, ICR-PVRTg21, may become a new animal model in place of monkeys for safety testing of oral poliovirus vaccine preparations.

Animals↗
PubMed1995

Molecular cloning of the 31 kDa cytosolic phospholipase A2, as an antigen recognized by the lung cancer-specific human monoclonal antibody, AE6F4.

The human monoclonal antibody AE6F4 specifically reacts with human lung cancer tissues but does not with normal tissues. This monoclonal antibody recognizes a cytosolic 31 kDa antigen in the cancer cells. In a previous study, we elucidated that the 31 kDa antigen belonged to a family of proteins collectively designated as 14-3-3 proteins, which were known as protein kinase-dependent activators of tyrosine/trytophan hydroxylases, or protein kinase C inhibitor proteins. Here we report molecular cloning of the 31 kDa antigen from the human lung adenocarcinoma cell line, A549. Sequencing analysis indicates that the cloned cDNA is identical to that of previously reported human placental cytosolic phospholipase A2 (cPLA2), which is also a member of the 14-3-3 protein family. Western analysis demonstrated that a 31 kDa recombinant cPLA2 expressed in monkey COS cells was recognized by the AE6F4 monoclonal antibody. Binding of the monoclonal antibody to the recombinant cPLA2 was abolished when treated with sodium periodate, suggesting that not only are carbohydrate chains associated with the cPLA2, but they also play a crucial role in antigen recognition by the monoclonal antibody.

14-3-3 Proteins↗
PubMed1995

Molecular cloning of yeast cytochrome c-like polypeptide expressed in human lung carcinoma: an antigen recognizable by lung cancer-specific human monoclonal antibody.

We previously determined the amino acid sequence to the epitope (ATLFKTR) of cytochrome c from Candida krusei, which is cross-reactive to the lung cancer-specific human monoclonal antibody HB4C5. Here we report that an antigen messenger RNA, which codes for a structure similar to the cytochrome c epitope, is expressed in the human lung adenocarcinoma A549. Sequencing analysis has revealed that this messenger RNA encodes a novel 190 amino acid polypeptide of 21-kDa containing an amino acid sequence (ALLFFT) similar to the cytochrome c epitope, although the total messenger RNA sequence is apparently different from the cytochrome c messenger RNA. Western analysis indicated that an antibody-recognizable 21-kDa antigen which has the same molecular weight as the predicted polypeptide is expressed in the A549 adenocarcinoma. The in vitro translated product of the antigen messenger RNA and synthesized ALLFFT peptide were both shown to be reactive with the monoclonal antibody, indicating that this protein contains the epitope which enables A549 cells to specifically react with the antibody. The antigen mRNA was not expressed in non-transformed fibroblasts, suggesting that the antigen mRNA expression was associated with cellular transformation. Also in part of the antigen nucleotide sequence, there was a segment that had about 90% homology to the long terminal repeat sequence (no. 297-475) of the human endogenous retrovirus HERV-K10, which was related to the mouse mammary tumor virus.

Adenocarcinoma↗
PubMed1995

Immunocytochemical detection of lung cancer cells with monoclonal antibodies to 14-3-3 proteins.

Murine monoclonal antibodies were raised against 14-3-3 proteins, the antigen of human monoclonal antibody AE6F4 which had been shown potentially useful for the immunochemical diagnosis of lung cancer via sputum cytology. Enzyme-linked immunosorbent assays of the murine anti-14-3-3 monoclonal antibodies with isolated bovine brain 14-3-3 isoforms showed that the antibodies were classified into four different profiles of isoform reactivity. The comparison of 14-3-3 isoform and lung cancer tissue on the reactivity with murine monoclonal antibodies indicated that beta isoform can be responsible for cancer recognition, whereas human monoclonal antibody AE6F4 showed preferential binding to zeta isoform. No murine monoclonal antibody of the same isoform specificity as human monoclonal antibody AE6F4 was obtained. Since murine monoclonal antibodies with different isoform specificities could immunostain lung cancer cells in sputum successfully, the combination use of murine monoclonal anti-14-3-3 antibodies with human monoclonal antibody AE6F4 is potentially useful for facilitating the sputum cytodiagnosis of lung cancer.

14-3-3 Proteins↗
PubMed1995

A human monoclonal antibody to carbohydrate moiety of carcinoembryonic antigen.

Hybridoma BSRF-S-97, secreting a human monoclonal antibody of IgG1 subclass reactive to the carcinoembryonic antigen, was generated by fusing the regional lymph node lymphocytes from a cervical cancer patient with RF-S1 human-mouse heteromyeloma fusion line. This monoclonal antibody was found specifically reactive to carinoembryonic antigen-producing cell lines, including those of cervical cancer (SKG-II), mucinous type ovarian cancer (RMUG-L), stomach cancer (MKN-45), and lung cancer (PC-10). The monoclonal antibody reactivity with pepsin- and periodate-treated carcinoembryonic antigen demonstrated that this monoclonal antibody recognizes the carbohydrate moiety of carcinoembryonic antigen specifically. Possibilities of the monoclonal antibody reaction with mucin and blood-group antigens were excluded by the comparative studies with a placental mucin-containing protein which reacted with carcinoembryonic antigen-specific rabbit polyclonal antibody. The monoclonal antibody conjugated with Pseudomonas exotoxin showed potent regression effects on the growth of the MKN-45 cell line in both the dish culture and xenografted nude mice, indicating potential usefulness of this human monoclonal antibody as a promising tumor targeting vehicle.

Animals↗
PubMed1994

Human cell clones, RSa and UVr-1, differing in their capability for UV-induced virus reactivation and phenotypic mutation.

UVr-1 is a human cell clone established as a variant with increased resistance to cell killing by ultraviolet light (UV, principally 254 nm wavelength) from a UV-sensitive cell clone, RSa. Both cells have been characterized to have much the same capacity of UV-induced DNA repair synthesis in whole cells, and the parent RSa cells were recently found to be hypermutable. In the present study UVr-1 cells were characterized in comparison RSa cells with respect to UV-induced virus reactivation and phenotypic mutation. Survival levels of UV-irradiated vaccinia virus and herpes simplex virus type 1 (HSV-1) were much the same in logarithmically proliferating UVr-1 and RSa cells. Correlated with these host cell reactivation levels, the same extent of UV-induced DNA repair replication synthesis was observed in isolated nuclei of the two cell clones. Enhancement of survival levels of UV-irradiated HSV-1 was detected when proliferating RSa cells were irradiated with UV prior to the virus infection. In contrast, this enhanced virus reactivation (EVR) was not detected in similarly irradiated and infected UVr-1 cells. As for phenotypic mutation frequencies assessed by the cloning efficiency of cells with increased resistance to ouabain cell killing (OuaR), OuaR mutants were not obtained from UVr-1 cells either with or without UV irradiation. When the proliferation of cells was synchronized, both EVR and OuaR mutations were detected in RSa cells irradiated with UV at any cell cycle phase, being greatest in the later half of the G1 phase. However, there was no detectable EVR or mutation in any phase of synchronous UVr-1 cells. The hypomutability of UVr-1 cells and hypermutability of RSa cells in a G1 cell cycle phase was also found even if 4-nitroquinoline 1-oxide was used as a mutagen or mutant cells with increased resistance to 6-thioguanine cell killing were estimated.

Cell Cycle↗
PubMed1994

Specific reactivity of human monoclonal antibody AE6F4 against cancer cells in tissues and sputa from lung cancer patients.

In vitro immunization of normal human peripheral lymphocytes by the co-cultivation with A549 cells (a human lung adenocarcinoma cell line), followed by the hybridization with the partner cell line A4H12, resulted in the efficient generation of hybridomas which produce human monoclonal antibodies of IgG or IgM isotype. Immunohistochemical and immunocytological studies showed that one of those monoclonal antibodies, termed AE6F4, was specifically reactive to lung cancer tissues as well as cancer cells in sputa.

Antibodies, Monoclonal↗
PubMed1994

The 14-3-3 protein as the antigen for lung cancer-associated human monoclonal antibody AE6F4.

Human monoclonal antibody (MAb) AE6F4, which had been shown potentially useful for the immunocytological detection of lung cancer cells in sputum, was characterized for its antigen(s). Of the three MAb-reacting materials found in A549 cells by the immunoblotting analysis, the cytoplasmic 31-kDa protein extractable with phosphate-buffered saline was evidenced as the most plausible antigen by its highest content and outstanding affinity to the MAb AE6F4-derivatized Sepharose 4B column. This 31-kDa protein was identified by the amino acid sequence analysis of the CNBr-cleaved fragment as the 14-3-3 family of proteins, the members of which are known to play important physiological roles such as in the regulation of neurotransmitter levels and intracellular signal transduction. The purified 14-3-3 protein from bovine brain showed a comparable MAb-reacting activity to that of the 31-kDa protein from A549 cells in the enzyme-linked immunosorbent assay (ELISA). The significant reactivity of bovine 14-3-3 protein by MAb AE6F4, shown by the cross inhibition of antibody binding to the coated 31-kDa antigen in ELISA as well as by the inhibition of immunostaining with lung cancer tissues, consistently demonstrated that the antigen(s) recognized by the MAb was involved in the 14-3-3 protein family. It was found that the expression of the 14-3-3 protein was significantly enhanced in lung cancer tissues compared with the neighboring normal part of the lung as examined by the immunoblotting method. These results implicated that some member(s) of the 14-3-3 protein family can be the tumor marker(s), providing a rational basis for the immunocytological diagnosis of lung cancer with this human MAb.

14-3-3 Proteins↗
PubMed1994

Accumulation enhancement of human monoclonal antibody HB4C5 to lung tumor xenografts by N-deglycosylation.

UNLABELLED: The fractional uptake of intact monoclonal antibodies by tumors is relatively low. Various methods to alter the molecular structure have been used to augment tumor uptake. These chemical manipulations, however, may alter the specificity of antibody binding. METHODS: Comparative studies of biodistribution, radioimmunoimaging and macroautoradiography in LC-6 xenografted mice were conducted with the 125I-labeled intact and N-terminal deglycosylated monoclonal antibodies to evaluate the effect on deglycosylation on antibody binding. RESULTS: The removal of N-glycosyl residues from this monoclonal antibody significantly enhanced specific localization of the radioactivity to the tumor, especially to its necrotic fraction. Nonspecific accumulation of radioactivity to the necrotic fraction of the tumor was excluded by biodistribution studies demonstrating selective accumulation of 125I-labeled monoclonal antibody after coadministration of 125I-monoclonal antibody (intact or N-deglycosylated) with 131I-labeled control IgM. CONCLUSION: The lung cancer-associated human monoclonal antibody HB4C5, which recognizes histone H2B as the antigen, accumulates specifically to the necrotic fraction of tumor. The uptake is enhanced by removal of N-terminal glycosyl residues from the antigen-binding site of the light chain.

Animals↗
PubMed1993

Enhanced production of human monoclonal antibodies by the use of fructose in serum-free hybridoma culture media.

It was found that the production of human monoclonal antibodies (MoAbs) by human-human hybridomas can be significantly enhanced by replacing glucose with fructose in the dish culture medium. Optimization of initial concentrations of fructose and glutamine, another influencing factor for MoAb production, enabled an enhanced production of human MoAb 2.1 times higher than that obtained using the conventional culture media employing glucose. It was shown by kinetic analysis that enhanced MoAb production at the optimum fructose concentration can be attributed to the retention of high specific antibody production rates and diminished time lag during the course of culture. These dish culture results with fructose-containing medium were successfully applied to the continuous perfusion culture with a slight modification, where 2.9- and 1.9-fold enhancements in specific antibody production rate and MoAb concentration, respectively, were attained as compared with the conventional glucose-containing medium. An inverse relationship was observed between the secreted concentrations of lactic acid and MoAb when the hybridoma was cultured in the media containing varying concentrations of fructose, i.e., the lower the lactic acid concentration, the higher the MoAb production and vice versa, suggesting that fructose at appropriate concentrations in the medium can serve as an alternative sugar for the efficient production of human MoAbs, with reduced pH shifts, for the serum-free culture of human-human hybridomas.

Antibodies, Monoclonal↗
PubMed1993

Assessment of Chlamydia trachomatis-specific IgA and IgG serum antibodies in genitourinary Chlamydia trachomatis infection--comparative study between HITAZYME and IPAzyme.

We assessed the C. trachomatis antibody assay kit HITAZYME (Hitachi Chemical Co., Ltd.) using clinical specimens. This kit is based on an enzyme immunoassay (EIA) which utilized purified Chlamydia trachomatis outer membrane antigen as the solid phase antigen. Twenty-nine untreated male urethritis patients, 816 pregnant housewives, 188 cervicitis patients, and 76 pelvic inflammatory disease patients were tested. Agreement between the HITAZYME test and antigen detection in infected area was assessed, and a comparison was made with IPAzyme (a commercially available indirect immunoperoxidase assay kit). 1) Summary of HITAZYME and IPAzyme IgA: Agreement between the two assays was relatively good, i.e., 82.6% (916/1109). However, 5.5% (61/1109) were HITAZYME (-), IPAzyme (+), and 11.9% (132/1109) were HITAZYME (+), IPAzyme (-). Thus, in quite a few cases the results did not agree. IgG: Agreement between the two assays was 73.7% (817/1109). Agreement was relatively low, 24.4% (271/1109) were HITAZYME (-), IPAzyme (+). 2) In the cases of disagreement, more specific Western blot analysis was performed to check the reactivity of the anti-C. trachomatis antibody. When IgA was used, agreement between HITAZYME and Western blot analysis was 69.6% (16/23), and agreement between IPAzyme and Western blot analysis was 30.4% (7/23), whereas when IgG was used, agreement between HITAZYME and Western blot analysis was 80.0% (12/15), and agreement between IPAzyme and Western blot analysis was 20.0% (3/15). There was significantly greater agreement with HITAZYME than with IPAzyme. In other words, HITAZYME had greater specificity when reacted with C. trachomatis antigen than IPAzyme. 3) The IgA antibody-positive rate in antigen (+) cases (male urethritis: 72.7%, pregnant housewives: 65.7%, cervicitis: 70.3%, pelvic inflammatory disease: 70.0%) was significantly (p < 0.01) higher than in antigen (-) cases (male urethritis: 16.7%, pregnant housewives: 13.6%, cervicitis: 22.6%, pelvic inflammatory disease: 30.4%). Therefore, IgA antibody can serve as a suitable indicator for active infection. 4) The IgG antibody-positive rate in antigen (-) female cases was 15.5% using HITAZYME and significantly (p < 0.01) lower than with IPAzyme. HITAZYME had greater specificity than IPAzyme. In conclusion, HITAZYME has relatively good sensitivity and specificity. Moreover, since it is an EIA assay, it allows objective evaluation of results. It permits processing of a large number of specimens because it is easy to perform. Thus, HITAZYME is a superior antibody assay for C. trachomatis. It can be used when antigen tests are difficult to perform. It is strongly anticipated that HITAZYME will be able to be used clinically as a screening test.

Chlamydia Infections↗
PubMed1993

[Polypeptide profiles of Chlamydia pneumoniae strains isolated from Japan and western blot analysis using patients' sera].

Polypeptide profiles were compared among Chlamydia pneumoniae strain of AC-43, which was isolated from a Japanese child, and other strains of C. pneumoniae. There was no distinctive differences among these strains. All strains showed marked band on 39.5 KDa area, which is equivalent with the major outer membrane protein (MOMP). Patients infected with C. pneumoniae were analyzed by Western blot using AC-43. Reaction for 98, 68, 60 KDa proteins were recognized among sera which showed positive results for anti-C. pneumoniae IgG antibody. There were two family members from whom C. pneumoniae was not isolated and anti-C. pneumoniae IgG antibody was negative. Sera obtained from them also showed weak reaction on 98 KDa protein area. Strong reaction on MOMP area was demonstrated in one patient from whom C. pneumoniae was isolated. Monoclonal antibody produced from AC-43 strain showed no reaction against homologous antigen. Reaction to 98 KDa protein was detected in all of the patients infected with C. pneumoniae. However, recognition to MOMP of C. pneumoniae seems to be different among patients. This result might suggest the presence of subtype among C. pneumoniae strains.

Blotting, Western↗