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Publications and source records attributed to R Ueda.
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A human-mouse chimeric antibody constructed in the present study was specific for a human tumor-associated antigen, common acute lymphocytic leukemia antigen. The antibody consisted of human heavy and light chain constant domains (gamma 1 and kappa type) and mouse heavy and light chain variable domains, which were derived from human plasma cell leukemia line (ARH77) and mouse hybridoma cells (NL-1) specific for common acute lymphocytic leukemia antigen, respectively. The artificially fused immunoglobulin molecules were produced in mouse myeloma cells, X63Ag8.653 which were transformed with the chimeric heavy and light chain genes formed by joining the corresponding gene segments in vitro at the J-C introns. The human heavy chain enhancer element was ligated tot he chimeric heavy and light chain genes, and this enhancer appeared to be obligatory for the efficient production of the chimeric antibody molecules. The stably transformed cells secreted the chimeric antibody, which specifically bound a common acute lymphocytic leukemia antigen expressing cell line. The amount of the chimeric antibody produced (10-30 micrograms/ml in the serum-free medium) was comparable to that made by murine hybridoma line, NL-1. The molecular weight of the chimeric heavy chain molecules was reduced from 54,000 to 50,000 upon treatment with tunicamycin, suggesting that the peptide was normally glycosylated in the transformants. The chimeric antibody exhibited complement-dependent cytotoxicity, in which glycosylation is thought to be indispensable. The antibody also mediated antibody-dependent cell-mediated cytotoxicity to the human target cells. The antibody-dependent cell-mediated cytotoxicity activity of the chimeric antibody was twice that of the murine NL-1 monoclonal antibody when human peripheral blood mononuclear cells were used as effectors.
Five hybrids reactive with monoclonal antibodies against human leukocyte common antigen (T-200) and/or lymphocyte function-associated antigen 1 (LFA-1) beta subunit were obtained from the fusion of human blood lymphocytes or T-cell chronic lymphocytic leukemia cells with BW5147 mouse T-cell leukemia cells. Chromosome analyses of 20 clones showed concordance between the presence of human chromosomes 1 and 21 and the expression of T-200 and LFA-1 beta subunit, respectively. Confirmation of human chromosomes in the hybrids was made by the electrophoretic analyses of phosphoglucomutase for chromosome 1 and superoxide dismutase for chromosome 21. The results suggested that the presence of human chromosomes 1 and 21 was essential for the expression of T-200 and LFA-1 beta subunit, respectively.
New cell lines, designated as ML-DmD1-10, were established from dissociated imaginal discs of Drosophila melanogaster. The culture medium was prepared by mixing in a 1:1 ratio Cross and Sang's M3(BF) medium, supplemented with 10% heat inactivated fetal bovine serum (FBS), with the supernatant of a primary embryonic cell culture made in the M3(BF) medium and supplementing this mixture with insulin. One cell line was established in the medium containing larval hemolymph instead of the primary culture supernatant, and another was established in fresh M3(BF) medium supplemented with insulin and FBS. In these mediums, imaginal disc cells first formed aggregates and cellular vesicles within a few weeks followed by the proliferation of thin-layered cells around them after about 1 mo. Ten cell lines have so far been established from two kinds of imaginal discs and disc mixtures. The ploidy of these cell lines was predominantly diploid. Population doubling time was about 50 to 70 h at 3 to 10 mo. after initiation of the culture. When the cell aggregates formed in vitro were implanted in metamorphosing larvae, they differentiated at high frequency into adult cuticular structures in the early phase of the primary culture. This differentiation of aggregates was also observed, though at low frequency, in a culture maintained by dilution-transfer for 6 to 15 mo. in vitro.
Analysis by indirect immunoperoxidase staining of tissues demonstrated that monoclonal antibody (mAb) K2.7 derived from a mouse immunized with a renal cancer cell line OS-RC-2 was reactive with the tissues of all 36 normal kidneys examined, including two fetal kidneys, 22 of 25 renal cell carcinomas (RCCs) and one of 15 gastric cancers, but not with 23 normal tissues from 11 different organs or 41 malignant tissues from 10 different organs. mAb K2.7 was reactive with the kidneys of several animal species. Positive staining was seen on tubular epithelial cells of the kidney. Thus, the antigen recognized by mAb K2.7 appeared to be an interspecies kidney specific antigen. Results obtained by protein A (PA) assays with a cultured cell panel were consistent with those of staining and confirmed the restricted expression of the antigen. Immunochemical analysis revealed that mAb K2.7 reacted with molecules of 63,000, 60,000 and 43,000 daltons.
To detect the proliferating cells in situ, a monoclonal antibody against human DNA polymerase alpha (pol alpha) was employed because this enzyme is known to be present in the nucleus of the cells in G1, S, and G2 phases. In addition, the surface phenotype of pol alpha-positive proliferating lymphocytes in diseased lymph nodes was determined by double staining consisting of immunoperoxidase and immunoalkaline phosphatase methods with various monoclonal antibodies against lymphocyte membrane antigens. In the paracortical area of lymph nodes with reactive changes, proliferating cells were 17% or less, and most of them were helper T-cells, although suppressor T-cells and B-cells also proliferate to a certain extent. In contrast, the proliferating cell population in malignant lymphomas was generally more than 40%, and it showed a single surface phenotype, indicating monoclonal proliferation. In addition, an unusual T-cell antigen phenotype of proliferating cells was observed in some cases of peripheral T-cell lymphomas. Thus, this double staining provided the authors with valuable information regarding the proportion, localization, and surface phenotype of proliferating cells, which should be useful for diagnosis of the diseases of lymphoid system.
Several lines of evidence suggest that subsets of resting lymphocytes naturally express interleukin-2 receptors (IL-2.R). Recombinant IL-2 (rIL-2) induced the enhancement of natural killer (NK) activity, the generation of activated killer (AK) cells, the proliferation of resting lymphocytes, and the production of interferon-gamma (IFN-gamma) in lymphocyte cultures. The subsets of lymphocytes mediating these responses appeared to be heterogeneous, but reside predominantly in nylon wool-passed non-T, non-B cells ("null cells" or T3- cells); in response to rIL-2, only Leu 11+T3- cells showed enhanced NK activity, and both Leu 11+T3- and Leu11-T3- cells showed predominantly AK activity, proliferation and production of IFN-gamma. These findings suggest that the T3- fraction (null cell fraction) contains predominantly cells expressing IL-2.R at the resting state. Unlike the case with activated T cells, however, none of these responses was blocked by any of three monoclonal antibodies to IL-2.R, including anti-Tac antibody at any dilution. These results indicate that IL-2.R on the resting T3- cells possess unique biological features compared to those on activated T or B cells. A most likely explanation is that T3- cells possess higher affinity IL-2.R than activated T or B cells. Other possibilities are also discussed.
Immunological phenotypes of leukemic blasts from 50 children with acute lymphoblastic leukemia (ALL) have been examined with a panel of monoclonal antibodies to evaluate their prognostic significance. Thirty-seven of them were common-ALL positive for CD10 "common-ALL antigen (CALLA)" (NL-1), CD19(B4) and HLA-DR. One was pre-B ALL negative for CALLA and another null-ALL which expressed HLA-DR alone. Six of the remaining 11 cases were traditional T-ALL positive for CD2(9.6), and the other five tentative pre-T ALL positive for CD7(Tp40) but negative for CD2. Twenty-one out of 39 patients with non-T ALL were treated with the standard regimen. The 18 children with non-T ALL having poor prognostic factors, five with pre-T ALL and six with T-ALL were treated with the more intensive regimen. The median follow-up period was 36 (range 4 to 74) months. Their disease-free survival probabilities were compared. It was found that the disease-free survival of non-T ALL patients with poor prognostic factors was comparable to that of the patients without such factors as a result of the more intensive chemotherapy. Among the patients with poor prognostic factors, those with pre-T ALL as well as those with T-ALL, which were positive for CD7 antigen, were found to have significantly short disease-free survival times (P less than 0.03). CD7 antibody is most useful for detecting ALL patients with poor prognoses.
The arrangements of the T cell receptor (TCR) beta genes were studied in lymph node specimens with the histological characteristics of angioimmunoblastic lymphadenopathy with dysproteinemia (AILD), AILD-like T cell lymphoma (AILD-T), T-zone lymphoma, or Lennert's lymphoma. Eight of 11 cases with AILD or AILD-T showed clonal rearrangements of TCR beta genes: all three AILD cases showed clonal rearrangements of TCR beta genes, while five of 8 AILD-T cases exhibited clonal rearrangement patterns. Malignant lymphoma evolved in one AILD case showed the same rearrangement band of TCR beta genes as its primary AILD lesion. The rearrangement of TCR beta genes was also observed in 2 out of 3 cases with T-zone lymphoma and 2 out of 2 cases with Lennert's lymphoma. None of the cases studied, except one AILD-T case, exhibited clonal rearrangements of immunoglobulin heavy chain genes. The results suggested that a significant proportion of AILD, AILD-T, T-zone lymphoma and Lennert's lymphoma cases are malignancies of peripheral T cell origin.
A double blind study was carried out to compare the efficacy and safety of cefixime (CFIX), a new oral cephem with cefaclor (CCL) in the treatment of 245 children weighing 10 approximately 30 kg, with acute suppurative otitis media. The daily dosages of CFIX and CCL were 3 approximately 6 mg/kg in 2 divided portions, and 20 approximately 40 mg/kg in 3 divided portions, respectively, and the drugs were administered for 7 days. The results obtained in this study are summarized as follows. Analyzed subjects were 211 patients (CFIX group: 108 patients, CCL group: 103 patients) for clinical efficacy. Efficacy rates judged by the doctor in charge were 88.9% and 83.5% in CFIX and CCL group, respectively, without significant difference between the 2 groups. Similar results were also obtained by the committee. When clinical effects were classified by clinical isolates, the efficacy rates against monomicrobial infections with Gram-negative bacteria were judged by the doctor in charge to be 100% in the CFIX group and 84.6% in the CCL group. Thus CFIX was judged to be significantly superior to CCL (P less than 0.05). The overall eradication rates of bacteria were 97.1% in the CFIX group and 90.3% in the CCL group. The eradication rate of CFIX was significantly superior to that of CCL (P less than 0.05). When improvements of individual symptoms were evaluated, regarding redness of the tympanic membrane and the tympanic cavity on the 3rd day of dosing, CFIX group (improvement in 84.1% of the cases) was significantly superior (P less than 0.05) to the CCL group (67.6%). Regarding purulent secretion on the 7th day of dosing, the CFIX group (improvement in 98.1% of the cases) was also significantly superior (P less than 0.05) to the CCL group (91.3%). Two hundred thirty eight patients were analyzed for side effects (CFIX group: 120 patients, CCL group: 118 patients). The incidence rates of side effects were 0.8% (1/120) in the CFIX group and 1.7% (2/118) in the CCL group, and there was no significant difference between the 2 drugs. From the above results, it is concluded that CFIX is a useful oral antibiotic in the treatment of acute suppurative otitis media in children. Furthermore, CFIX is expected to be equal or superior to CCL in clinical effects.
The clinical efficacy and safety of cefixime (CFIX), a new oral cephalosporin, were compared with those of cefroxadine (CXD) in patients suffering from acute lacunar tonsillitis in a double blind study. Two hundred and fifty two patients were given each orally 100 mg of CFIX b.i.d. or 250 mg of CXD t.i.d. for, in principle, 7 days. Number of patients evaluated for clinical efficacy was 202 (103 treated with CFIX and 99 treated with CXD). As for the backgrounds of patients, more severe cases were found in the CFIX group than in the CXD group (P less than 0.01). Efficacy rates evaluated by individual doctors were 88.3% in the CFIX group and 91.9% in the CXD group. There was no significant difference between the 2 groups. Efficacy rates on the third day after the initiation of treatment evaluated by the committee were 40.8% in the CFIX group and 47.9% in the CXD group with no significant difference. Efficacy rate on the 7th day, however, was 79.8% in the CFIX group and 93.4% in the CXD group, showing a significant difference (P less than 0.05). Bacteriological effectiveness were satisfactory for both groups with eradication rates of 93.4% for the CFIX and 96.9% for the CXD group. Number of patients evaluated for safety was 226 (110 treated with CFIX and 116 treated with CXD). No significant difference was observed between the 2 drug groups in incidences of side effects; gastrointestinal disturbances or rashes were noted in 6 patients (5.5%) of the CFIX group and in 5 patients (4.3%) of the CXD group. As for the abnormal laboratory findings, elevation of GOT & GPT was observed in 1 patients of the CFIX group. From these results, it was concluded that 100 mg b.i.d. of CFIX was as useful as 250 mg t.i.d. of CXD in the treatment of acute lacunar tonsillitis.
The analysis of cell surface markers with monoclonal antibodies has recently been developed and has been proved to be valuable in the diagnosis and classification of hematopoietic tumors. Occasionally, however, such serological studies have been shown to be inconclusive in identifying cell lineage and or clonal proliferation. In order to overcome these problems, two new approaches were introduced in our laboratory. First, T cell receptor and immunoglobulin gene rearrangement analysis as a means of immunomolecular marking was carried out. Second, a double immunoenzymatic staining technique for determining the surface phenotypes of proliferating lymphocytes using a monoclonal antibody against DNA polymerase alpha together with those detecting lymphocyte membrane antigens was developed. The results revealed by these techniques strongly suggested that some CD2-, CD5+, CD7+ ALL cases are of T cell origin and that AILD may be a neoplastic disease derived from either of the subsets of peripheral T cells.
Biopsy specimens of lymph nodes with the histologic characteristics of angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) were obtained from 9 cases (4 cases of AILD and 5 cases of AILD-like T lymphoma [AILD-T]) and histologically analyzed by the use of a double immunoenzymatic staining technique with the combination of a monoclonal antibody against lymphocyte membrane antigen and that against human DNA polymerase alpha (pol alpha), which is detectable in the nucleus of the cells in G1, S, and G2 phases. In all 9 cases, the pol alpha + proliferating cells had a peripheral T-cell phenotype with T11 and Leu-4 antigens, whereas proliferating B cells with B1 antigen were rarely observed. As for T-cell subset antigens, the proliferating T cells had T4+ helper/inducer phenotype in 7 cases, while T8+ suppressor/killer T cells proliferated in 2 cases, although a significant number of T4+ proliferating cells were also recognized. The study on malignant lymphomas that evolved in the 2 cases showed that the T-subset antigens on major proliferating tumor cells were the same as those found in the preceding AILD lesions, suggesting that lymphoma T cells originate from the AILD lesion. The results suggested that AILD without histologic manifestations of malignancy and AILD-T may be a neoplastic disease derived from either subset of peripheral T cells.
Hybridomas secreting monoclonal antibodies that reacted with the B6 radiation leukemia virus (RadLV)-induced leukemia B6RV2 were produced by fusion of BALB/c NS-1 myeloma cells with spleen cells from (BALB/c X B6)F1 mice immunized with B6RV2. By direct and absorption analyses with 28 B6 and BALB/c leukemias, the monoclonal antibodies NU7-4 and NU7-99 were shown to react only with B6RV2, indicating that they recognized an individually distinct antigen on B6RV2 that was identified previously with conventional (BALB/c X B6)F1 anti-B6RV2 serum. Another monoclonal antibody, NU1-132, showed relatively restricted reactivity with B6 RadLV leukemias. These three monoclonal antibodies all precipitated material of approximately 80,000 daltons, which is the same size as that precipitated by anti-xenotropic MuLV gp70 serum. Sequential immunoprecipitation analysis revealed that the molecules precipitated by NU7-4 were not removed by pretreatment of NU7-99 or NU1-132 and that the molecules precipitated by NU7-99 were not removed by NU7-4 or NU1-132. The molecules precipitated by NU1-132 were partially removed by pretreatment with NU7-4, but not with NU7-99. The molecules precipitated by these three monoclonal antibodies were removed by pretreatment with anti-xenotropic gp70. These results suggested heterogeneity of the xenotropic MuLV gp70-related molecules expressed on B6RV2 and a possible relation between serologically defined unique tumor antigens and gp70-related molecules.
Adult T cell leukemia associated antibody (ATLA-Ab) positive persons were screened by indirect immunofluorescence (IF) testing. Their lymphocytes were collected from concentrated red blood cells (CRC), and cultured in vitro with and without phytohemagglutinin (PHA) for 10 days. The expression of ATL virus (ATLV) positive lymphocytes during the in vitro culture was then analyzed by IF assay using mouse monoclonal antibody ATL-19 reactive to p19 core protein of ATLV. 97% of ATLA-Ab positive CRC (36 cases) demonstrated ATLV positive lymphocytes after being cultured for more than 10 days with PHA, whereas, none of ATLA-Ab negative CRC (22 cases) demonstrated ATLV positive lymphocytes. All of the 10 ATLA-Ab positive CRC that were stored for 2, 4, and 7 days contained lymphocytes which expressed ATLV after in vitro culture, while 7 of 10 CRC stored for 14 days and only 1 of 10 CRCs stored for 20 days, expressed ATLV positive lymphocytes. This data indicates that almost all of the ATLA-Ab positive blood contained ATLV positive lymphocytes, and that the in vitro appearance of these ATLV positive lymphocytes was reduced by storing the CRC for more than 14 days.
Three IgM mouse monoclonal antibodies, NL-9, Thy-22, and HL-5, which were produced primarily against human hematopoietic cells, were tested for their reactivity with various mouse cell lines and were found to react predominantly with mouse embryonal carcinoma cells. Thy-22 reacted with 2-cell-stage mouse embryos, whereas the other two antibodies were not reactive at this stage. All three antibodies, however, reacted with 8-cell-stage embryos. At the blastocyst stage, Thy-22 reacted with the entire surface of the trophectoderm cells, whereas the reactivity of NL-9 and HL-5 was weaker and was polarized on the mural trophectoderm. Immunohistological examination of 6th-day mouse embryos using anti-complement immunofluorescence demonstrated that the embryonic ectoderm was positive for all three antibodies: the reaction of NL-9 and Thy-22 was uniformly distributed over these cells, whereas HL-5 predominantly stained the luminal aspects of the cells lining the proamniotic cavity. Visceral-endoderm cells and trophoblastic cells were positive with all three monoclonal antibodies, whereas the parietal endoderm, extraembryonic ectoderm, and ectoplacental cone were negative. In 19th-day fetuses and adult tissues, certain epithelial cells were stained by these three antibodies. The biochemical nature of the antigens detected was also investigated. Farr's assay showed that both NL-9 and Thy-22 precipitated approximately 10% of the high-molecular-mass glycopeptides isolated from F9 cells, while HL-5 reacted with about 5% of these glycopeptides. The reactivity of the three antibodies against the glycopeptides was completely inhibited by the presence of X-hapten-conjugated silica.(ABSTRACT TRUNCATED AT 250 WORDS)
Representative works in the field of 1) serological typing of hematopoietic tumor, 2) serum diagnosis of solid tumor, 3) tumor imaging and 4) antibody against oncogene products were introduced in this article to cover recent progress in diagnosis of cancer with mouse monoclonal antibody. In addition, points to be considered when to start producing antibodies were mentioned.
A cloned cDNA segment of 442 bp which contains the region coding the N-terminal sequence of rat DNA polymerase beta polypeptide was used as a probe in Southern hybridization analysis to identify the gene for this enzyme. This probe hybridized with DNA from mouse and human in addition to rat DNA, and the BamHI fragment containing DNA polymerase beta gene sequence was unique in size in each species: 15.8 kb for rat DNA, 8.7 kb for mouse DNA and 10.1 kb for human DNA. DNA extracted from a panel of 12 independent human-mouse somatic cell hybrids was analyzed by Southern blot hybridization with the cloned cDNA probe. The results indicate that the gene for the human DNA polymerase beta sequence (POLB) is located on chromosome 8.