Satomi Haga

Summary Report on the ISOBM TD-4 Workshop: Analysis of 56 Monoclonal Antibodies against the MUC1 Mucin

Sixteen research groups participated in the ISOBM TD-4 Workshop in which the reactivity and specificity of 56 monoclonal antibodies against the MUC1 mucin was investigated using a diverse panel of target antigens and MUC1 mucin- related synthetic peptides and glycopeptides. The majority of antibodies (34/56) defined epitopes located within the 20-amino acid tandem repeat sequence of the MUC1 mucin protein core. Of the remaining 22 antibodies, there was evidence for the involvement of carbohydrate residues in the epitopes for 16 antibodies. There was no obvious relationship between the type of immunogen and the specificity of each antibody. Synthetic peptides and glycopeptides were analyzed for their reactivity with each antibody either by assay of direct binding (e.g. by ELISA or BiaCore) or by determining the capacity of synthetic ligands to inhibit antibody binding interactions. There was good concordance between the research groups in identifying antibodies reactive with peptide epitopes within the MUC1 protein core. Epitope mapping tests were performed using the Pepscan analysis for antibody reactivity against overlapping synthetic peptides, and results were largely consistent between research groups. The dominant feature of epitopes within the MUC1 protein core was the presence, in full or part, of the hydrophilic sequence of PDTRPAP. Carbohydrate epitopes were less easily characterized and the most useful reagents in this respect were defined oligosaccharides, rather than purified mucin preparations enriched in particular carbohydrate moieties. It was evident that carbohydrate residues were involved in many epitopes, by regulating epitope accessibility or masking determinants, or by stabilizing preferred conformations of peptide epitopes within the MUC1 protein core. Overall, the studies highlight concordance between groups rather than exposing inconsistencies which gives added confidence to the results of analyses of the specificity of anti-mucin monoclonal antibodies.

Characterization of cells with proliferative activity after a brain injury.

The cellular responses to a brain injury are important steps in restoring the integrity and function of the brain. Proliferating cells, such as reactive astrocytes, oligodendrocyte precursor cells and microglia remodel the injured tissue. To spatially and temporally characterize the initial cellular responses in vivo, proliferating cells were pulse-labeled with BrdU soon after (the 2nd day) a cortical cryo-injury, and their fate was investigated by double labeling with an anti-BrdU antibody and antibodies to various cellular markers. Three days after the cryo-injury, a significant proportion of BrdU-positive cells were positive for NG2-proteoglycan, suggesting that oligodendrocyte progenitors (OPCs) were induced in response to injury. One-two weeks after the cryo-injury, the number of OPC was reduced and GFAP/BrdU double-positive cells, in turn, became dominant, while cells with mature oligodendrocyte markers did not increase significantly. Neuronal markers were rarely co-localized with BrdU immunoreactivity throughout the period studied. These findings imply that OPCs are prone to differentiate to astrocytes in the lesioned site. In this cryo-injury model, treatment with thyroid hormone (T4) altered cell fate; the increase in the number of GFAP/BrdU-positive cells was significantly diminished, and there was an increased number of mature oligodendrocytes (CNPase, PLP-positive) exhibiting BrdU immunoreactivity. These findings suggest that modification of proliferating progenitors in injured brain by hormonal or chemical treatment might benefit functional regeneration.

Genetic dissection of susceptibility to radiation-induced apoptosis of thymocytes and mapping of Rapop1, a novel susceptibility gene

Genetic dissection of susceptibility to radiation-induced apoptosis of thymocytes was performed by counting dead cells in histologically processed thymuses after 0.5 Gy of whole-body X-irradiation, using recombinant congenic (CcS/Dem) strains derived from inbred mouse strains BALB/cHeA (susceptible) and STS/A (resistant). A high (8/20) number of strains with lower dead cell scores than BALB/cHeA among CcS/Dem recombinant congenic strains (RCS), which contain 12.5% of STS/A genome in the genetic background of BALB/cHeA strain, indicates that the difference between BALB/cHeA and STS/A is caused by several genes and that susceptibility probably requires BALB/cHeA alleles at more than one locus. Similar results were obtained with CXS/Hg recombinant inbred (CXS/Hg) strains. Analysis of F2 hybrids between BALB/cHeA and CcS-7, one of the CcS/Dem strains that showed lower dead cell scores than BALB/cHeA, demonstrated that a novel gene (Rapop1, radiation-induced apoptosis 1) controlling susceptibility to radiation-induced apoptosis in the thymus is located in the proximal region of mouse chromosome 16.

Atm-heterozygous deficiency enhances development of mammary carcinomas in p53-heterozygous knockout mice

Introduction Ataxia-telangiectasia is an autosomal-recessive disease that affects neuro-immunological functions, associated with increased susceptibility to malignancy, chromosomal instability and hypersensitivity to ionizing radiation. Although ataxia-telangiectasia mutated (ATM) heterozygous deficiency has been proposed to increase susceptibility to breast cancer, some studies have not found excess risk. In experimental animals, increased susceptibility to breast cancer is not observed in the Atm heterozygous deficient mice (Atm+/-) carrying a knockout null allele. In order to determine the effect of Atm heterozygous deficiency on mammary tumourigenesis, we generated a series of Atm+/- mice on the p53+/- background with a certain predisposition to spontaneous development of mammary carcinomas, and we examined the development of the tumours after X-irradiation.

Atm heterozygous deficiency enhances development of mammary carcinomas in p53 heterozygous knockout mice

IntroductionAtaxia-telangiectasia is an autosomal-recessive disease that affects neuro-immunological functions, associated with increased susceptibility to malignancy, chromosomal instability and hypersensitivity to ionizing radiation. Although ataxia-telangiectasia mutated (ATM) heterozygous deficiency has been proposed to increase susceptibility to breast cancer, some studies have not found excess risk. In experimental animals, increased susceptibility to breast cancer is not observed in the Atm heterozygous deficient mice (Atm+/-) carrying a knockout null allele. In order to determine the effect of Atm heterozygous deficiency on mammary tumourigenesis, we generated a series of Atm+/- mice on the p53+/- background with a certain predisposition to spontaneous development of mammary carcinomas, and we examined the development of the tumours after X-irradiation.MethodsBALB/cHeA-p53+/- mice were crossed with MSM/Ms-Atm+/- mice, and females of the F1 progeny ([BALB/cHeA × MSM/Ms]F1) with four genotypes were used in the experiments. The mice were exposed to X-rays (5 Gy; 0.5 Gy/min) at age 5 weeks.ResultsWe tested the effect of haploinsufficiency of the Atm gene on mammary tumourigenesis after X-irradiation in the p53+/- mice of the BALB/cHeA × MSM/Ms background. The singly heterozygous p53+/- mice subjected to X-irradiation developed mammary carcinomas at around 25 weeks of age, and the final incidence of mammary carcinomas at 39 weeks was 31% (19 out of 61). The introduction of the heterozygous Atm knockout alleles into the background of the p53+/- genotype significantly increased the incidence of mammary carcinoma to 58% (32 out of 55) and increased the average number of mammary carcinomas per mouse. However, introduction of Atm alleles did not change the latency of development of mammary carcinoma.ConclusionOur results indicate a strong enhancement in mammary carcinogenesis by Atm heterozygous deficiency in p53+/- mice. Thus, doubly heterozygous mice represent a useful model system with which to analyze the interaction of heterozygous genotypes for p53, Atm and other genes, and their effects on mammary carcinogenesis.

Progenitor endothelial cells on vascular grafts: An ultrastructural study

The morphology of progenitor endothelial cells on vascular graft surfaces is addressed in this report. Such cells were seen to attach to intima-expressed CD34 and Flk-1 antigen and showed positive 5-bromo-2-deoxyuridine (BrdU) uptake. We examined CD34 and Flk-1 antigen-expressing endothelial progenitor cells three-dimensionally using confocal laser scanning microscopy (CLSM). Under detailed CLSM observation, through an ameboid-form cell, these progenitor endothelial cells changed from a globular to a flattened form. We also investigated these morphological changes using scanning electron microscopy. From these results, progenitor endothelial cells were observed not only near the advancing edge of endothelium, but also around the developing intimal site. Their form also changed from globular to flattened as observed in the CLSM results. These morphological changes were seen more frequently near the advancing edge and around the developing intimal site. They attached directly to vascular prosthesis fibers and likewise covered the graft luminal surface. Progenitor endothelial cells in any form had a common surface structure. We conclude from our results that progenitor endothelial cells can attach to graft fibers directly without clotting and directly cover the graft luminal surfaces.

Complete Nucleotide Sequence of Mouse Mammary Tumor Virus from JYG Chinese Wild Mice: Absence of Bacterial Insertion Sequences in the Cloned Viral gag Gene.

Mammary tumors of a newly isolated strain of Chinese wild mouse (JYG mouse) harbor exogenous mouse mammary tumor virus (MMTV). The complete nucleotide sequence of exogenous JYG-MMTV was determined on the proviral 5′ long terminal repeat (LTR) (partial)-gag- pol- env- 3′ LTR (partial) fragment cloned into a plasmid vector and the cDNA sequence from JYG-MMTV producing cells. Similarly to the other MMTV species the LTR of JYG-MMTV contains an open reading frame (ORF). The amino acid sequence of the JYG-MMTV ORF resembles that of SW-MMTV (92% identity) and endogenous Mtv-7 (93% identity) especially at the C-terminal region. Thus, a functional similarity in T-cell receptor Vß recognition as a superantigen is implicated among these MMTV species. Analysis of the viralgag nucleotide sequence revealed that this gene is not disrupted by the bacterial insertion sequence IS1 or IS2, which have been reported to be present in the majority of the plasmids containing thegag region. Comparison of amino acid sequences of JYG-MMTV with those of BR6-MMTV showed that over 96% of the amino acids ofgag, pol, protease andenv products are identical. These results suggest the intact nature of the nucleotide sequence of the near full-length MMTV genome cloned in the plasmid.

Monoclonal antibodies against CA125-bearing antigenic molecule fragments; reactivity with mucinous ovarian tumours and lung cancers

We first established a cell strain, SHIN-3, from human ovarian serous cystadenocarcinoma, and performed antigen analysis for CA125 which appeared to be massively secreted by the SHIN-3 cells. Protein digestion analysis revealed that a low molecular weight peptide of 49 kDa showed antigen activity. In the present study, we describe a mouse monoclonal antibody against this low molecular peptide, presumed to be part of the CA125-bearing antigenic molecule. Purified antigen prepared from culture supernatant was adsorbed to nitrocellulose membranes and injected intrasplenically in mice. Of the obtained 398 clones, 10 clones were selected by screening in an ELISA test. Of the 10, two were further selected, i.e. SH-9. This hybridoma produces IgG1 monoclonal antibodies. Immunoblotting analysis revealed that SH-9 recognizes the low molecular peptide used as the immunogen. Immunohistological examination with the SH-9 MAb revealed that the antigen reacted with the bronchial epithelium, cervical glands of the uterus and other various normal tissues. Of tumorous tissues, the antibody mainly reacted with ovarian tumours, but positive reactions were also observed with pulmonary adenocarcinomas or squamous cell carcinomas. Surprisingly the positive rate was high in mucinous tumour of the ovary, while no positive reaction was observed in serous tumours. Dot-blot assay using SH-9 revealed that 17/19 (90%) sera of lung cancer patients were positive for the titre suggesting that SH-9 may be useful to set up a serum test for lung cancers.

Cardiac functions and Taurine’s actions at different extracellular calcium concentrations in forced swimming stress-loaded rats

Modulation of the sinus rate and contractile force by taurine at different extracellular Ca2+ concentrations ([Ca2+]o) was examined using rat right atria loaded with forced swimming stress. Serum concentration of corticosterone profoundly increased in stress-loaded rats as compared with native rats. The taurine level in serum also increased in stress-loaded rats, but was not changed in the different heart tissues and aorta. Heat-shock protein (HSP72) was detectable in cardiac muscles and in the lumen of cardiac blood vessels of stress-loaded rats using a monoclonal antibody. Increasing [Ca2+]o (from 0.9 to 3.6 mM) enhanced the sinus rate and contractile force in a [Ca2+]o-dependent fashion in native rats, but not in stress-loaded rats. Taurine (1–20 mM) caused a negative chronotropic and inotropic effect in a concentration-dependent manner. At 1.8 mM [Ca2+]o, the negative chronotropic effect of taurine (10–20 mM) was attenuated in stress-loaded rats as compared with native rats. These results indicate that swimming stress causes a release of taurine into the serum and reduces the sensitivity to [Ca2+]o. Taurine administration might, in part, exhibit the protective actions on acute stress-induced responses.

Cloning in a plasmid of an MMTV from a wild Chinese mouse: sequencing of the viral LTR

Plasmid subcloning by conventional techniques of full length exogenous mouse mammary viruses (MMTV) has not been realized because of the involvement of host-mediated structural changes in the viral gag gene. To circumvent this problem, an alternative subcloning method, excision of phagemid (pBluescript SK) from lambda ZAP II, was successfully used to subclone a novel exogenous MMTV (JYG-MMTV) provirus fragment containing an intact gag gene. Sequence analysis revealed that the LTR of this virus is significantly different from the LTR of C3H-MMTV in the U3 region.