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Biomedical subjects

S Hosoi

Publications and source records attributed to S Hosoi.

At least 55 records · Page 3Linked to original sources
PubMed1991

Optimization of cell culture conditions for production of biologically active proteins.

We investigated the basic technology of cell culture conditions for production of useful substances such as cytokines, and related proteins produced by Namalwa cells. Namalwa cells (Klein, 1972), human B lymphoblastoid cells, were used for large scale production of alpha-interferon (Klein, 1979). Namalwa KJM-1, a subline of Namalwa cells, adapted to serum- and albumin-free medium, can grow at a high density above 1 x 10(7) cells/ml in suspension mode by the use of a perfusion culture system, Biofermenter, containing a cone-type cell-sedimentation column as cell separator (Sato, 1983). Several kinds of cytokine cDNA can be introduced and expressed in Namalwa KJM-1 cells (Miyaji, 1990a,b,c). Some of these were produced in large quantities by use of a gene amplification method with dhfr (Miyaji, 1990c), even though the Namalwa KJM-1 cells contained endogenous dhfr genes. For stable production of the target protein, Namalwa KJM-1 cells are very useful host cells, because they have no effective endogenous protease activity in the conditioned medium. Using Biofermenter with micro-silicone fibers and a dialysis system, the specific productivity of the target proteins was not depressed at a high cell density.

Amino Acid Sequence↗
PubMed1991

[Production of useful recombinant proteins using Namalwa KJM-1 cells adapted to serum-free medium].

We investigated the basic technology of cell culture conditions for production of useful substances such as cytokines, and related proteins produced by Namalwa cells. Namalwa cells, human B lymphoblastoid cells, were used for large scale production of alpha-interferon. Namalwa KJM-1, a subline of Namalwa cells, adapted to serum- and albumin-free medium, can grow at a high density above 1 x 10(7) cells/ml in suspension manner by the use of a perfusion culture system, Biofermenter, containing a cone-type cell-sedimentation column as cell separator. Several kinds of cytokine cDNA can be introduced and expressed in Namalwa KJM-1 cells. Some of these were produced in large quantities by use of a gene amplification method with dhfr, even through the Namalwa KJM-1 cells contained endogenous dhfr genes. For stable production of the target protein, Namalwa KJM-1 cells are very useful host cells, because they have no effective endogenous protease activity in the conditioned medium. Using Biofermenter with micro-silicone fibers and a dialysis system, the specific productivity of the target proteins was not depressed at a high cell density.

Adaptation, Biological↗
PubMed1991

Dermatophagoides pteronyssinus specific IgM antibody and its complement activation in children with bronchial asthma.

Western blotting analysis and ELISA were used to examine Dp specific IgM abs in the sera and sputa of bronchial asthmatic and non-atopic children. With Western blotting Dp specific IgM abs were detected binding diffusely to high molecular weight (greater than 65 kD) components of Dp extracts and from the intensities of the staining of the sera the asthmatic children were divided into two groups: an intensive staining and a faint staining group. The non-atopic children showed still fainter staining than the latter group. ELISA titers of Dp specific IgM abs in the intensive staining group were significantly higher than in the faint staining group and the non-atopic children (p less than 0.01). Dp specific IgM abs in sputa were also detected more intensively in asthma than in non-atopy by Western blotting. The hemolytic complement activities of the fresh sera were consumed when they were incubated with Dp extracts at 37 degrees C. The degree of consumption was significantly higher in the intensive staining group than in the faint staining group and the non-atopic children (p less than 0.05). High molecular weight fractions of Dp extracts were responsible for this complement inactivation. From these results we suspected that Dp specific IgM abs might react with high molecular weight components of Dp to induce complement activation by the classical pathway and might cause chronic inflammatory changes in the respiratory mucosa. This chronic inflammation might emphasize IgE mediated asthmatic reactions to cause late phase reactions or chronic asthma.

Animals↗
PubMed1990

Expression of human beta-interferon in Namalwa KJM-1 which was adapted to serum-free medium.

A Namalwa cell line, KJM-1, which was adapted to serum-free medium is thought to be a very useful host cell line for recombinant DNA technology. Thus, the utility of Namalwa KJM-1 for expression of foreign genes was examined. As a model system human beta-interferon (beta-IFN) gene was engineered for expression in this cell line. For construction of the beta-IFN expression vector pSE1 beta 1-4, the expression vector pAGE107 was constructed and used. It contains simian virus 40 (SV40) early promoter, the rabbit beta-globin RNA processing signals for splicing and polyadenylation, and SV40 early RNA processing signal for polyadenylation. In addition to the above transcription unit, pAGE107 contains the ampicillin-resistance gene and G418-resistance gene. They can confer ampicillin resistance to Escherichia coli (E. coli) and G418 resistance to animal cells. To introduce plasmid DNA into cells, electroporation is a useful method (Wong, 1982; Potter, 1984). We have established conditions for DNA-mediated transfection of Namalwa KJM-1 cell line by electroporation. Among pSE1 beta 1-4-introduced cells, clone 1-3 was further examined for the expression of beta-IFN in serum-free medium. The production level of beta-IFN was elevated with the increase of the cell density. The results indicated that the Namalwa KJM-1 cell line is useful for production of foreign gene products.

Base Sequence↗
PubMed1990

Chinese hamster ovary cells continuously secrete a cysteine endopeptidase.

The protease activity in serum-free conditioned medium of chinese hamster ovary (CHO) cells was measured using peptidyl (or aminoacyl)-4-methylcoumaryl-7-amides (MCAs) as the substrates. Aminopeptidase increased in level as amounts of nonviable cells increased during cultivation in serum-free medium, indicating that the activity seems to be originated from intracellular proteases. The activity toward Boc-Leu-Arg-Arg-MCA, which was strongly inhibited by p-chloromercuribenzonate and N-ethylmaleimide, was the strongest among those toward peptidyl-MCAs in the conditioned medium within 48 h-cultivation in serum-free medium. In contrast to the case of aminopeptidase activity, the endopeptidase activity decreased in level after 48 h-cultivation although amounts of nonviable cells increases. Thus, CHO cells continuously secrete the cysteine proteases.

Aminopeptidases↗
PubMed1990

Heat-labile, complement-like factor(s) of animal sera prevent(s) HIV-1 infectivity in vitro.

We studied inactivation of HIV-1 by fresh sera of animals. We found that while fresh sera of humans and chimpanzees (among others) did not have antiviral activity, fresh sera of several other mammals, especially those of rodents and felines, showed a dose-dependent viral-inactivating property against cell-free HIV-1; these sera were also capable of inactivating virus preadsorbed to cells, similar to neutralizing antibody. The activity was destroyed by heating to 56 degrees C, required Ca2+ but no antiviral antibody, and therefore apparently involves the classical complement pathway. The activity could not be ascribed to any single fraction of sera separated on a size exclusion HPLC column. Mouse serum (the only one tested) also inactivated HIV-2. The data are consistent with classical C-mediated pathway inactivation of HIV-1 and HIV-2 by animal sera and is the first report of any "complement-like" antilentiviral serum factor. Elucidation of this mechanism may aid in understanding the lack of activity of human serum against HIV-1 and may prove useful in combined interventive strategies against HIV-1.

Animals↗
PubMed1989

DiGeorge syndrome with hypogammaglobulinaemia: a patient with excess suppressor T cell activity treated with fetal thymus transplantation.

A male infant with DiGeorge syndrome had hypogammaglobulinaemia with a normal number of B cells. CD3(+) T cells were reduced and the CD4(+)/CD8(+) ratio was reversed. Proliferative responses of T cells to mitogens and to allogeneic cells were low. The pokeweed mitogen (PWM)-induced B cell differentiation assay revealed a higher than normal suppressor T cell activity. This suggests that some T cells had differentiated into functionally mature cells resulting in an imbalance of regulatory T cell functions and that excess suppressor activity might play a role in hypogammaglobulinaemia. Fetal thymus transplantation improved both cellular and humoral immunity. The patient's susceptibility to viral and bacterial infections, proliferative response of T cells and serum Ig concentration returned to normal. The excess suppressor activity seen before transplantation disappeared. Hypocalcaemia did not improve. These results show that fetal thymus transplantation was effective not only in reconstituting cellular immunity but also in normalizing the imbalance of regulatory T cell functions in this patient with DiGeorge syndrome.

Agammaglobulinemia↗
PubMed1989

Evaluation of production and characterization of monoclonal antibodies to human IgG of four subclasses.

Human IgG of four subclasses, semi-purified from pooled human serum by a series of DEAE ion exchange and protein A affinity chromatographies, were used as immunogens and initial screening antigens to produce subclass-specific and -restricted monoclonal antibodies (McAbs). These McAbs were bound to CNBr-activated Sepharose 4B and utilized in immunoaffinity chromatography to prepare four polyclonal human IgG subclasses of satisfactory purities, which were then used as final screening antigens. Subclass-specific McAbs thus chosen were further evaluated for subclass- and especially allotype-specificity using a panel of monoclonal IgG myeloma proteins with representative Gm markers for each subclass in micro enzyme-linked immunosorbent assay (ELISA). A total of 10 clones of subclass-specific McAbs (one for anti-IgG1, three anti-IgG2, two anti-IgG3, four anti-IgG4) were established. Among them, IgG2-specific clones of HG2-30F and HG2-56F, IgG3-specific HG3-7C and HG3-32C, and IgG4-specific HG4-53G McAbs were superior to the corresponding specificity standard McAbs chosen by the Human Immunoglobulins Subcommittee of the WHO/International Union of Immunological Societies (IUIS) in 1985. As allotype-specific McAbs, HG1-1E for G1m(az) and HG3-3B for G3m(b) were obtained. In micro ELISA of this study as well as all protocols of the previous WHO/IUIS collaborative study, antigens (myeloma IgG subclasses) were immobilized or fixed to a solid phase, resulting in possible variations in their epitope expressions. We developed a new assay system, micro radioimmunoassay (RIA), in which reactivities of McAbs against free IgG subclasses in solution can be evaluated. HG2-30F, having extremely high reactivities to coated IgG2 in micro ELISA, remarkably reduced its reactivities to free IgG2 in solution in micro RIA. Two other clones also showed some different reactivities in micro RIA and micro ELISA. We believe that this micro RIA is valuable for evaluation of McAbs reactivities against native human IgG subclasses in solution.

Animals↗
PubMed1989

Further evidence for dilution-dependent dissociation of C1q in human serum.

Dissociation of the C1q subcomponent in native C1 upon dilution was reexamined by using ultracentrifugation in a sucrose gradient and high performance liquid chromatography system with a size exclusion column for separating the dissociated C1q fractions. The antigenic content of C1q in each fraction was detected by ELISA and Western blotting; binding to erythrocyte antibody was also determined. The results confirmed a previous claim that C1q in native C1 dissociated as a function of dilution: up to 14.5% of C1q antigen was in the low molecular weight form (approximate S value: 4-5). Commercial preparations of purified C1q also contained C1q antigen in the low molecular weight form.

Centrifugation, Density Gradient↗
PubMed1988

Activation of human C1r: Western blot analysis reveals slow and dose-dependent activation.

Spontaneous activation of C1r in the presence of EDTA was examined by a Western blot. Partially purified native C1r was prepared by ultracentrifugation of fresh serum in 10 to 30% sucrose gradient; final concentration of C1r was one-sixth of the original serum. C1(-)-INH was not detectable by a single radial immunodiffusion (less than 0.5% of serum). The results demonstrated that 1) the rate of spontaneous activation of C1r was slow (less than 10% in 30 min); 2) it was concentration-dependent; 3) it was enhanced by activated C1r; and 4) it was almost completely suppressed by serine protease inhibitors up to 1 h. These results were inconsistent with an intramolecular autoactivation model of C1r in the fluid phase and suggested intermolecular activation by contaminating protease or activated C1r.

Adult↗
PubMed1988

[Induction of LAK cells by high density dialyzing culture device and its cytotoxicity].

Lymphokine activated killer (LAK) cells are generated by culture of lymphocytes with interleukin 2 (IL-2) in short term culture (3 to 5 days) and are used for adoptive immunotherapy for advanced cancer patients. The culture condition hitherto reported are essentially based on the rotating culture system, in which the maximum cell density was at 2 X 10(6) cell/ml and the cell recovery was usually less than 100%. The inability to induce LAK cells efficiently in vitro made the culturing of cells for therapy rather difficult and costly work because the mean infusion dose of LAK cells of one patient requires more than 1 X 10(10)/ml. We have therefore attempted to culture lymphocytes in 10 times higher concentration comparing with conventional methods. By using a new dialyzing culture system under continuous regulation of the amount of infused IL-2, nutrition medium, and pO2 and pCO2, we could culture cells at 2 X 10(7)/ml for more than 21 days and the resulted LAK cells showed a 100 times increase of activity on a per cell basis. By limiting dilution procedure, these killer cells mostly express T cell markers such as CD3 and CD8 but dose not express CD16.

Cell Division↗
PubMed1987

Activation of human C1: analysis with Western blotting reveals slow self-activation.

The first component of human complement was separated from C1-INH by sucrose linear gradient ultracentrifugation. Activation of C1 was studied in the absence and presence of immune complexes; activation was monitored by SDS-PAGE and Western blot. When the partially purified native C1 preparation was incubated at 37 degrees C without immune complexes, activated C1s appeared after 30 min in the case of eightfold dilution with respect to the original serum, and after 45 min with 32-fold dilution. Kinetics of appearance of activated C1r was the same as that of activated C1s. From the following results, we concluded that spontaneous activation may be partially due to proteolytic enzymes contaminating the preparation: 1) a nonspecific protease inhibitor, PMSF, completely inhibited spontaneous activation but did not inhibit the activation of C1 by immune complexes; 2) alpha 2-macroglobulin partially inhibited spontaneous activation, and 3) although spontaneous activation in the absence of PMSF was relatively slow, activated C1 accelerated spontaneous activation that was completely blocked by C1-INH. In contrast to spontaneous activation, the partially purified native C1 was rapidly activated by immune complexes: within 5 min almost all C1 was activated by rabbit IgG anti-human IgM-human IgM complexes. These results support conclusions derived from activation studies when using native C1 and hemolytic assays, and do not support those derived from the activation studies with reconstituted C1 and SDS-PAGE analysis. We suggest that the contradictions can be resolved if one assumes that C1 activation can be both an intra- and intermolecular process; which process dominates is determined by the state of C1 and by experimental conditions.

Antigen-Antibody Complex↗
PubMed1987

A quantitative enzyme-linked immunosorbent assay for Dermatophagoides pteronyssinus-specific immunoglobulin G.

A monoclonal mouse anti-human IgG was used to develop an enzyme-linked immunosorbent assay (ELISA) for the measurement of Dermatophagoides pteronyssinus (DP)-specific IgG in human sera. This monoclonal antibody (HG2-25) binds to all subclasses of IgG but not to IgA, IgM, or IgE. For the assay, the DP antigen is coated firmly on polystyrene beads through physical adsorption and any leakable antigen is washed off. The assay gives satisfactory reproducibility and parallelism of the dilution curves. Using 0.1% human serum albumin as a substitute for the DP-specific IgG preabsorbed diluent gave extremely low backgrounds and high sensitivity. Horseradish peroxidase-labeled HG2-25 prepared with the optimum degree of conjugation and free of polymerized conjugates gave responses fairly proportional to the doses. This ELISA gives a satisfactory recovery and is not affected by nonspecific IgG levels in human sera.

Animals↗
PubMed1987

High levels of antibodies to streptococcal cell membrane antigens specifically bound to monoclonal antibodies in acute poststreptococcal glomerulonephritis.

We produced 15 immunoglobulin G class monoclonal antibodies against antigens of the streptococcal cell membrane (SCM) of Streptococcus pyogenes (T type 12, Tanaka strain) and determined the levels in human sera of antibodies against Triton-X-extracted antigens specifically bound to each of these 15 monoclonal antibodies by enzyme-linked immunosorbent assay. Sample sera were obtained from 10 normal controls (group 1), 10 patients with streptococcal pharyngitis without sequelae (group 2), and 8 patients with acute poststreptococcal glomerulonephritis (APSGN) (group 3). Anti-streptolysin O (ASO) titers of the sera increased in the order of groups 1, 2, and 3. There was no relationship between ASO titer and the level of anti-SCM antibodies, and there was no significant difference in the level of anti-SCM antibodies determined with each of the 15 monoclonal antibodies between group 1 and group 2 sera. Group 3 sera had higher levels of antibodies to SCM antigens specifically bound to each of 14 of these 15 monoclonal antibodies than group 1 or group 2 sera did. Of these 14 monoclonal antibodies, 9 reacted with the four SCM antigens separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to a nitrocellulose sheet. These results suggest that high levels of antibodies to SCM antigens are related to the development of APSGN and that the systemic immune response to SCM antigens is involved in the pathogenesis of APSGN.

Acute Disease↗
PubMed1987

CYBEST-CDMS Model 2. Automated cell dispersion and monolayer smearing system for CYBEST.

The CYBEST automated cell dispersion and monolayer smearing system (CDMS) employed an autosyringing device and a modified centrifuge to produce specimens with an adequate number of dispersed cells per square millimeter. Since the units of the system were separate, the centrifugation chambers had to be transferred manually. The CYBEST-CDMS Model 2, described in this paper, uses a robot manipulator in a unified system combining the cell dispersion and centrifugation units. Samples in centrifugation chambers are automatically transferred from the autosyringing unit to the centrifugation unit. Using its input and output conveyers, the system can process specimens without interruption.

Automation↗
PubMed1987

CYBEST Model 4. Automated cytologic screening system for uterine cancer utilizing image analysis processing.

CYBEST (Cyto-Biologic Electronic Screening System) utilizes image analysis technology for the automated prescreening of cervical cytology specimens. CYBEST Model 3, which includes a television scan system and automatic shading control, achieved our initial goal of rapid specimen processing (no more than three minutes to achieve a final specimen assessment). This paper describes CYBEST Model 4, developed in 1981; with the minicomputer of Model 3 replaced by a microcomputer, Model 4 is considerably smaller, about the size of a business desk. A new parameter, the intranuclear configuration (chromatin pattern), was added to the four parameters used in Model 3. The five parameters now used for the assessment and ranking of cytologic abnormalities are nuclear size, nuclear-cytoplasmic ratio, nuclear optical absorption, nuclear shape and intranuclear configuration. The other features of Model 4 are almost the same as those of Model 3. As an optional function, individual parameter measurement data, assessment of atypicality grade and cell images can be displayed on the CRT monitor by pointing to a cell with a light pen system. After completion of screening of a specimen, the ten cells judged to be most abnormal can be called automatically into the microscope optical field or the CRT monitor (in order ranging from the cell with the highest atypicality rank down) along with their associated data and the system's assessment of the specimen. By connection to a small business computer, all data can be transferred to a floppy disk for later retrieval.

Cytodiagnosis↗