Biomedical subjects

S Hashizume

Publications and source records attributed to S Hashizume.

At least 55 records · Page 3Linked to original sources

PubMed1993

[A report of 4 cases from whom Chlamydia pneumoniae was isolated].

Chlamydia pneumoniae (C. pneumoniae) was isolated from respiratory tract of 4 girls. Case 1 is 5-year-old, diagnosed as pneumonia, had had a prolonged productive cough. She was treated with RKM with improvement of symptoms, however, C. pneumoniae was isolated repeatedly and was not deleted. Neither anti-mycoplasmal nor viral antibodies were positive and no significant pathogens were cultured from nasophrayngeal swabs. Case 2, the sister of case 1, is a 3-year-old girl with acute bronchitis treated with EM. C. pneumoniae was negative on the tenth day after treatment. Case 3, a 5-year-old girl, had a fever and was diagnosed as bronchopneumonia with a mild attack of bronchial asthma. She was initially treated with CFIX followed by therapy including EM. Her symptoms had disappeared after treatment and anti-mycoplasmal antibody was 1:320. Case 4 was an asymptomatic carrier of C. pneumoniae. Specimen was obtained at regular health examinations of junior high school. C. pneumoniae was isolated from a 14-year-old girl without respiratory symptoms. Clinical figures of C. pneumoniae infections varies from asymptomatic carrier to pneumonia. Pathogens other than C. pneumoniae could modify symptoms of infections. Precise examinations of these cases would establish a proper management of a C. pneumoniae infection.

Adolescent↗

PubMed1993

Cancer-specific binding of a mouse MAb vs. Candida krusei cytochrome c: an antigen recognized by a cancer-associated human MAb HB4C5.

More than 60 mouse monoclonal antibodies directed to cytochrome c from Candida krusei with different specificities were raised. Most of these monoclonal antibodies, except for three of them, did not cross-react with bovine cytochrome c. By the immunoblotting method, the monoclonal antibodies of clones HCC 5-13, 9-2, and 10-5 reacted with the Candida cytochrome c, which had been transferred onto nitrocellulose membrane, but those of clones HCC 1-22, 6-3, and 17-3 did not, although all these monoclonal antibodies strongly reacted with coated Candida cytochrome c on plastic immunoplates when examined by ELISA. On the contrary, monoclonal antibody activities of clones HCC 1-22, 6-3, and 17-3 in binding to the coated cytochrome c in ELISA were inhibited competitively by the addition of extra Candida cytochrome c, whereas those of clones HCC 5-13, 9-2, and 10-5 were not inhibited. Among these monoclonal antibodies, the antibody of clone HCC 6-3, which showed a good reactivity to added cytochrome c in inhibiting ELISA reaction but was not reactive with the transblotted cytochrome c on nitrocellulose, was found to be reactive with human lung cancer tissues specifically with no reactivity to normal tissues. The immunostaining of lung cancer tissue showed that this mouse monoclonal antibody to Candida cytochrome c reacted to the cytoplasmic fraction of the cancer cells specifically.

Animals↗

PubMed1993

Activity enhancement of a lung cancer-associated human monoclonal antibody HB4C5 by N-deglycosylation.

It has been known that the lung cancer-associated human monoclonal antibody HB4C5 comprises two lambda light chains of 30 and 32 kD and that the 30 kD species is exclusively responsible for the antibody activity. This study demonstrates that the two light chains were both N-glycosylated with glycosyl residues of different sizes, one of which was sensitive to neuraminidase and the other insensitive. Our unpublished data of DNA sequence for the light chain of this monoclonal antibody indicated that the light chain contains only one possible site for N-glycosylation, which located in the CDR-1. N-Deglycosylation of this monoclonal antibody under the denaturing condition resulted in the complete conversion of the two light chains into one identical polypeptide chain of 26 kD. Activity of this monoclonal antibody was found to be significantly enhanced by N-deglycosylation. All the facts described above consistently indicate that the activity of this monoclonal antibody is interfered with by the attachment of bulky glycosyl residues at the antigen binding site on the light chain. The N-deglycosylated antibody was stable under the storage conditions employed, suggesting that the glycosyl residues attached to the light chain do not play any important biological role except for interference with the antibody activity of binding antigen.

Adenocarcinoma↗

PubMed1992

Serodiagnosis of cancers by ELISA of anti-histone H2B antibody.

An enzyme-linked immunosorbent assay method for serodiagnosis of cancers was developed by employing histone H2B. This method measures anti-histone H2B antibody levels in sera and includes a device for coating the plastic immunoplate with a mixture of histone H2B and diluted fetal calf serum. The coating of immunoplates with this mixture decreased apparent sensitivity of the assay compared with that in the absence of fetal calf serum, but effective reduction of nonspecific background enabled a specific assay of anti-histone H2B antibody with excellent reproducibility. By this method cancer patients were discriminated from normal healthy subjects at detection rates of 37% for lung cancer, 33% for liver cancer, 50% for pancreatic cancer, 42% for colon cancer, and 78% for cervical cancer. However, stomach and esophagus cancers showed detection rates of less than 17%, which are comparable to the values for benign diseases. It is likely that this assay method detects squamous cell carcinomas at relatively high rates.

Antibodies, Neoplasm↗

PubMed1992

Characteristics of toxin-neutralization by anti-tetanus human monoclonal antibodies directed against the three functional domains [A], [B] and [C] of the tetanus toxin molecule and a reliable method for evaluating the protective effects of monoclonal antibodies.

Five anti-tetanus human monoclonal antibodies (MAbs) produced by hybrid cell lines we established previously were characterized. Their abilities to neutralize tetanus toxin in vitro and to protect mice against challenge with toxin were studied by observing the changes in the progress of symptoms in mice. Immunostaining showed that MAbs MAb-G4 and G2 recognized the N-terminal domain, [A] and the C-terminal domain, [C] of the tetanus toxin molecule, respectively, while MAbs MAb-G1, G3 and G6 recognized its middle domain, [B]. Enzyme-linked immunosorbent assay showed that the binding affinity of MAb-G3 was 2.9 x 10(10) M-1 and those of the other MAbs were as high as approximately 10(11) M-1. In in vitro neutralization experiments, at sufficient doses all the MAbs as single reagents protected mice completely against the effect of tetanus toxin. However, at lower doses than those sufficient to rescue mice, the kinetic patterns of progress of symptoms with the individual MAbs differed with each other and, except for MAb-G4, were different from that of anti-tetanus human polyclonal antibody. They suppressed the development and/or slowed the rate of progress of symptoms for over 96 h and delayed death of the mice. We propose that the comparison of the minimum survival dose with that of human polyclonal antibody of known international units is a reliable method for estimating the actual protective activity of a MAb. Intravenous (IV) injection of doses of individual MAbs or their mixtures at over 0.03 IU per mouse protected mice from subsequent challenge with 20 MLD of tetanus toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

[The establishment of human anti-tetanus toxoid hybridomas with in vitro immunized human tonsil cells].

Human tonsil cells in vitro immunized with tetanus toxoid were fused with human-mouse heteromyeloma line RF to generate human-mouse hybridomas. Hybridoma 891112-50 was cloned and 2 subclones (891112-50-3 and -4) were obtained. The secreted antibodies from the subclones were antigen specific, since they did not cross react with three irrelevant antigens (OVA, TCS and F gamma G). The hybridomas were quite stable. After 13 passages in tissue culture flasks, they still retained their antibody secreting ability. From flow cytometry analysis the subclone 50-3 was more stable than the subclone 50-4. The human immunoglobulin contained in supernatant collected during regular passages was equivalent to 69.6 micrograms/ml.

Animals↗

PubMed1992

[Quantitative evaluation of the stability of antibody production by human-mouse hybridoma clones with flow cytometry analysis].

The paper described a method of quantitative stability analysis of antibody production by hybridoma clones with flow cytometry (FACS). Through FACS analysis the frequency of antibody-negative variants was measured. The rate of generation of antibody-negative variants/cell/generation (mu) could be calculated through Luria-Delbrück fluctuation analysis. The results showed that the value of mu was correlated to the antibody production of hybridoma clones: the larger was the mu value, the more unstable was the antibody production by the hybridoma clones. The experimental results suggested that the clone was stable if its mu value was on the magnitude of 10(-3), while the clone was not stable if its mu value was on the magnitude of 10(-2). According to requirements of single cell for FACS analysis and presence of a variety of methods for dispersing cell aggregates to single cells, the authors regard that this method is fully suitable for analysis of cells in suspension or even in the form of aggregate.

Animals↗

PubMed1991

Regulation of plaque size and host range by a vaccinia virus gene related to complement system proteins.

A vaccinia virus variant, LC16m8, and its parental Lister (Elstree) strain (LO) were employed to identify the viral gene(s) responsible for plaque size and host range: the large-plaque-forming LO strain but not the small-plaque-forming LC16m8 strain can actively proliferate in Vero (YTV) cells. Previously, we suggested that some particular gene(s) present in the HindIII D fragment of LO DNA was responsible for these biological activities. In the present experiment, the mapping of the putative gene was done by introducing various subfragments of the HindIII D fragment of LO DNA into the gene of LC16m8 strain and screening of the resultant virus variants for the capability of forming large plaques or of proliferating well in Vero cells. The results indicated that an open reading frame (called ps/hr gene) in LO HindIII D fragment was responsible for either plaque size or host range. This gene encoded a polypeptide of 317 amino acids related to the regulators of complement activation (RCA) gene family of mammals. Thus, the present genetic analysis provided direct evidence for a previously unrecognized function of RCA-related proteins encoded by the virus.

Amino Acid Sequence↗

PubMed1991

Serodiagnosis of cancer by using Candida cytochrome c recognized by human monoclonal antibody HB4C5.

Cytochrome c from various sources, such as Candida krusei, yeast, horse, and cattle, was found to be recognized by human monoclonal antibody HB4C5 specific to lung cancer. Therefore, the cytochrome c was applied to the measurement of antibody amount in patient sera with a similar reactivity to the antibody HB4C5 for serodiagnosis of cancer. The cytochrome c from Candida krusei was most valuable for the serodiagnosis of various cancers, and the yeast cytochrome c was also useful. However, horse and bovine cytochrome c did not react with antibody of the cancer patients. By using Candida cytochrome c lung, bile duct, esophagus, and liver cancers were detected at high rates of more than 50%. In the case of lung cancer, the detection rates of small-cell, squamous, large-cell and adenocarcinoma were 78%, 63%, 100%, and 34%, respectively. The rate for small-cell carcinoma was higher than that with the currently used NSE assay system, and the rate for squamous carcinoma was comparable to that with the SCC assay system, although the system using cytochrome c did not show similar reactivity to that with the SCC system. Furthermore, lung cancer was detected at early stages by using cytochrome c, and even in the case of adenocarcinoma, the rate at early stages with the cytochrome c system was higher than that with the CEA assay system. On the other hand, false positive rates of benign diseases and normal were low--8% and 2%, respectively.

Antibodies, Monoclonal↗

PubMed1991

Detection of lung cancer in clinical specimens using a human monoclonal antibody HB4C5-clone 3.

Biopsies of 18 patients with lung cancer and cellular specimens of 21 lung cancer patients were analyzed with human monoclonal antibody HB4C5-clone 3 using avidin-biotin-peroxidase techniques. Analyses with the biopsies showed that HB4C5-clone 3 reacted with 16 of 18 biopsy specimens at a high rate of about 90% and reacted with all specimens of five adenocarcinoma tissues, five of six squamous cell carcinomas, two of three large cell carcinomas, both specimens of small cell carcinomas, and both of the other types of carcinomas. In all the reactive cases, the monoclonal antibody was reactive with greater than 50% of cancer cells. With cellular specimens of lung cancer patients, HB4C5-clone 3 reacted with 7 of 13 adenocarcinoma specimens, two of six squamous cell carcinomas, and one of two small cell carcinomas. The immunoreaction with HB4C5-clone 3 was observed in sites of the cytoplasm of cancer cells. From these data, HB4C5-clone 3 is considered to be of potential use in diagnoses of tissues and cellular specimens of lung cancer.

Adenocarcinoma↗

PubMed1991

[The in vitro induction of antibody responses of human lymphocytes and its application to hybridoma production].

Sources of immunized lymphocytes constitute one of the main obstacles in the production of human monoclonal antibodies. We tried to get them through in vitro immunization. Cells from excised tonsils or trauma spleens were used for the induction of antibody responses in vitro. Antibodies to different antigens including sheep red blood cells, ovalbumin, tetanus toxoid, and hepatitis B surface antigens were induced in 7-14 days' cultures. Taking tetanus toxoid as antigen, we analysed the various factors required for antibody induction with statistics analysis, which included cell separation method, T cell conditioned medium, antigen dosage, serum content, and concentration of mitogen PWM and LPS. The results showed: (1) The cell separation method influenced the antibody production significantly in comparison with other factors. It signified that immune cells' combination was the most influential factor. (2) Serum also constituted quite important influencing factor especially in the later period of culture. However, it did not make much differences if it attended 10% or so. The antigens and mitogens tended to be used at low concentration. (3) Due to the significant variation among individuals and among different antigens, it is suggested to set up the culture system with some flexibility so as to adapt to the variation in cells and antigens from different sources. The present culture system we use includes nylon wall column separation of cells, suitable range of antigens (three doses instead of one), and either 10% T cell conditioned medium or a mixture of 1 microgram PWM/ ml + 0.1 microgram LPS/ml. The human B lymphocytes stimulated in vitro with tetanus toxoid were used for the construction of human hybridomas.

Animals↗

PubMed1991

Serodiagnosis of cancer using porcine carboxypeptidase A as an animal antigen recognized by human monoclonal antibody HB4C5.

Protein from hog which is recognized by human monoclonal antibody (HB4C5), generated from a patient with large cell lung carcinoma, was identified as carboxypeptidase A by comparison of the protein with carboxypeptidase A in enzymatic activity, immunologic reactivity, and amino acid sequence. Carboxypeptidase A activity was also found in human cancer tissue, and purified antigen from cancer tissue recognized by the antibody HB4C5 was reacted with rabbit anti-carboxypeptidase A serum, indicating that carboxypeptidase A is an antigen of HB4C5. Since large amounts of carboxypeptidase A can be obtained from porcine sources, a simple method for its purification was established. The fraction which was most reactive with HB4C5 was obtained from acetone powder of porcine pancreas by successive applications of water extraction, ammonium sulfate precipitation, trypsin treatment, and Mono Q column chromatography. Its apparent molecular weight was 40,000, according to SDS polyacrylamide gel electrophoresis. When the reactivity of IgG in sera with the purified carboxypeptidase A was measured, the detection rates for lung, ovary, larynx, uterus, and liver cancer were more than 50%, while the rates for stomach and breast cancer were around 30%, and pancreatic cancer, benign diseases, and normal controls were minimally detected.

Amino Acid Sequence↗

PubMed1991

Histone H2B as an antigen recognized by lung cancer-specific human monoclonal antibody HB4C5.

Histone H2B was demonstrated to be an immunoreactive material recognized by the human monoclonal antibody HB4C5, which had been already established to be specific for lung cancers. The inhibitory effect of histone H2B on the activity of HB4C5 antibody to immunostain the cytoplasmic antigen in lung adenocarcinoma tissue indicated that histone H2B at least had antigenic determinant comparable to the cytoplasmic antigen. A mouse anti-histone H2B monoclonal antibody could immunostain the cytoplasm of lung adenocarcinoma cells in sliced tissue sections in the same manner as the human monoclonal antibody HB4C5. Enzyme-linked immunosorbent assay of HB4C5 antibody on plastic immunoplates coated with histone H2B also showed specific reactivity of this antibody with histone H2B, and the reaction was effectively inhibited when extra histone H2B or mouse anti-histone H2B monoclonal antibody was added to the reaction mixture. These results consistently lead us to a conclusion that histone H2B possesses antigenicity to the human monoclonal antibody HB4C5.

Adenocarcinoma↗

PubMed1991

Characterization of a lung cancer-associated human monoclonal antibody HB4C5.

A human monoclonal antibody HB4C5, which is known to be associated with lung cancers, was characterized. Two kinds of light chains with 30-kD and 32-kD molecular mass were found to comprise the molecule of this IgM antibody. Various combinations of the two light chains could be contained in this IgM antibody molecule, which might have resulted in the heterogeneity of this antibody with different reactivities. The presence of the 30-kD light chain was indispensable for the antibody activity of cancer recognition. Monomeric 180-kD units of the antibody, having 32-kD light chains exclusively, which showed marginal activity of cancer recognition, could be removed by immunosorbent column chromatography with histone H2B-Sepharose 4B. F(ab')2 of the HB4C5 antibody containing the active 30-kD component as the dominant light-chain constituent could be purified by hydroxyapatite chromatography by HPLC, facilitating clinical applications of this lung cancer-associated monoclonal antibody of human origin.

Antibodies, Monoclonal↗

PubMed1990

Development and evaluation of the TD97 measles virus vaccine.

The TD97 strain vaccine virus was prepared from the Tanabe strain measles virus by low-temperature passages in primary cell cultures and ultraviolet (UV) mutagenesis. The TD97 strain exhibited the following characteristics: highly temperature sensitive, neither multiplying nor forming any plaques at 40 degrees C in Vero cells; genetically stable, maintaining high temperature sensitivity after ten successive passages in CE cells at 30 degrees C or 35 degrees C; and M proteins of this virus about 1 KD slower in mobility in SDS-PAGE than that of the Tanabe strain. The TD97 strain was further confirmed to be attenuated by an inoculation test into primate brain. In field trials, 752 healthy children were inoculated with a live virus vaccine prepared with this strain, and the following results were obtained: the seroconversion rate was 97% (517/533), and the average HI antibody titer was 2(5.2). An antibody-increasing effect was also observed in children who were initially seropositive. In children who seroconverted, the rates of fever were 15.7% (55/351) for 37.5 degrees C or higher and 4.0% (14/351) for 39 degrees C or higher. The rash rate was 7.7% (27/351), and the incidence of local reaction was 5.4% (19/351). The TD97 strain is thus considered to be suitable in use for an attenuated measles vaccine.

Animals↗

PubMed1990

Stimulation of proliferation and immunoglobulin M production by lactoferrin in human-human and mouse-mouse hybridomas cultures in serum-free conditions.

The effects of growth factors, such as insulin, transferrin, lactoferrin, ethanolamine, and selenium, on proliferation and IgM production of human-human hybridomas HB4C5 cells in a serum-free enriched RDF (eRDF) medium were studied. Among them, lactoferrin markedly stimulated proliferation and IgM production of the cells. Another iron-binding protein, transferrin, stimulated proliferation of HB4C5 cells as well as lactoferrin, but its stimulatory effect on IgM production was negligible. The proliferation and IgM production of HB4C5 cells gave some detectable delays in conventional ITES-eRDF medium at low cell densities, but the delays were avoided by the addition of lactoferrin. However, eRDF medium supplemented with lactoferrin could not support proliferation and IgM production of the cells at high cell densities. For proliferation and IgM production of HB4C5 cells, eRDF medium supplemented with 25 micrograms/ml lactoferrin, 10 microM ethanolamine, 35 micrograms/ml transferrin, and 2.5 nM selenium (LETS-eRDF) gave the best result. Lactoferrin stimulated proliferation of human-human and mouse-mouse hybridomas producing IgG or IgM, but stimulation of Ig production was detected only in IgM producers. These results suggest that cell proliferation, IgM production, and IgG production of hybridomas are regulated by different mechanisms.

Animals↗

PubMed1990

Establishment of stable mouse/human-human hybrid cell lines producing large amounts of anti-tetanus human monoclonal antibodies with high neutralizing activity.

To establish stable hybrid cell lines producing human anti-tetanus antibody with high toxin-neutralizing activity, peripheral lymphocytes from humans hyperimmunized with tetanus toxoid were, after in vitro antigen stimulation, fused with a mouse/human heteromyeloma or human lymphoblastoid cell line and cloned. Unlike the IgM secretors (six clones), the IgG secretors we obtained (six clones) produced anti-tetanus human monoclonal antibodies with high neutralizing activity (the highest one, cell line G2, 4.3 IU/100 micrograms IgG). Appropriate combinations of three or four kinds of monoclonal antibodies of the IgG type resulted in markedly increased neutralizing activity comparable with that of anti-tetanus human polyclonal immunoglobulin preparations currently used clinically on the basis of toxin-specific IgG content. Five of these cell lines produced 10-20 micrograms of antibody per ml for more than 3 months. The cell line G2 produced 6 mg of antibody per day in serum-free medium in a 500-ml bioreactor in perfusion culture and 13-104 mg in a nude mouse. These cell lines satisfied, for the first time, the minimal requirements for applying human monoclonal antibodies to clinical use.

Animals↗

PubMed1990

Intrauterine Chlamydia trachomatis infection in a premature infant.

Intrauterine Chlamydia trachomatis infection was strongly suspected in a premature infant born in the 32nd week of gestation. The membranes were artificially ruptured at the time of delivery. This infant showed a high titer of specific IgM antibody to Chlamydia trachomatis at one hour after birth. He showed mild respiratory distress and was treated with oral erythromycin for three weeks. He was discharged home at the age of 46 days.

Chlamydia Infections↗