Cross-reactivity between human and canine helper and suppressor T cell antigens using monoclonal antibodies RPA-T4 and HuLy-m8

The murine monoclonal antibodies RPA-T4 and HuLy-m8, specific for a framework determinant of human helper/inducer and suppressor/cytotoxic T cell antigens, cross-reacted with canine cell membrane molecules recognizing a biomolecular complex (50,000 to 55,000 daltons) similar to that described in humans. We investigated the distribution of these helper and suppressor T cell-like antigens on canine peripheral blood lymphocytes. With complement-mediated lymphocytotoxicity, 34% and 35% of the canine lymphocytes expressed the helper T cell-like antigens and the suppressor-like T cell antigens, respectively. When the lymphocytes were treated with RPA-T4 and HuLy-m8, the respective helper and suppressor function was significantly inhibited.     

Splenic outer periarterial lymphoid sheath (PALS): An immunoproliferative microenvironment constituted by antigen-laden marginal metallophils and ED2-positive macrophages in the rat

Summary In an attempt to reveal the role of antigen-laden marginal metallophil (MM) and other macrophages in the intrasplenic immune response of a specific B-cell lineage to a thymus-independent type-2 antigen (Ficoll conjugated with fluorescein isothiocyanate), simultaneous immuno-histological observations of the involved cells were performed in the rat. By newly established methods of double or triple immunostainings, time-kinetics of the following parameters were studied and compared: (1) the antigen, (2) the specific antibody-forming cells (AFC) directed to the fluorescein-isothiocyanate determinant, (3) proliferating cells labeled with 5-bromo-2-deoxyuridine (BrdU), and (4) macrophage subpopulations recognized by monoclonal antibodies (ED2 and ED3). The antigen localized stably not only in the marginal-zone macrophages but also in the MM except around the follicular area. The increase of BrdU-positive cells was observed from day 2 up to day 4 after antigen injection mostly in the periphery of the periarterial lymphoid sheath (outer PALS), which indicated antigen-induced proliferation. As a novel finding, the majority of AFC, both BrdU-positive and -negative, were either closely associated with the antigen-laden MM, or forming cell clusters with ED2-positive macrophages in the outer PALS. In contrast, there were very few AFC in juxtaposition to antigen-free MM in the follicular area or the antigen-laden marginal zone macrophages. The results led to the proposal of a hypothesis that the antigen-laden MM together with ED2-positive macrophages constitute an immunoproliferative microenvironment for the plasmacellular reaction by accumulating the antigen-specific B-cell lineage and promoting these cells to differentiate into the AFC and to proliferate in the outer PALS.Abbreviations AFC specific antibody-forming cells - BrdU 5-bromo-2-deoxyuridine - Fic-F FITC-conjugated Ficoll - FITC fluorescein isothiocyanate - HRP horseradish peroxidase - MM marginal metallophils - MZ marginal zone - PALS periarterial lymphoid sheath - PBS phosphate-buffered saline - TI2 thymus-independent type-2     

Study on placental transmission of Pneumocystis carinii in mice using immunodeficient SCID mice as a new animal model.

Placental transmission of Pneumocystis carinii in mice was examined in 39 animals obtained by caesarean section from 17 pregnant SCID females experimentally infected with P. carinii. When examined with toluidine blue O, DAPI and immunofluorescent antibody stains, P. carinii was detected in the lungs of infected mothers but not in the lungs of caesarean section-derived neonates even after the neonates were treated with dexamethasone for 8 weeks. However, 13 neonates born to five infected females developed P. carinii pneumonia. These results indicate that P. carinii cannot be transmitted transplacentally in mice.     

Characterization of murine erythropoietin receptor genes

We have isolated and characterized the murine genomic and complementary DNAs encoding erythropoietin (Epo) receptor from Epo-responsive and unresponsive mouse erythroleukemia cells. Two classes of Epo receptor cDNAs were isolated from Epo-responsive cells. One is a 55,000 Mr membrane-bound Epo receptor, and the other is a 29,000 Mr soluble Epo receptor lacking the transmembrane and cytoplasmic domains. As a result of alternative splicing, two insert sequences containing termination codons are produced, and the encoded polypeptide diverges four amino acids upstream from the transmembrane domain, adding 20 new amino acids before terminating. Amino acid sequence of the Epo receptor cDNA isolated from Epo-responsive cells was identical with that of Epo-unresponsive cells, indicating that Epo-responsiveness does not depend upon the primary structure of the Epo receptor (binding) protein. Analysis of 6.6 x 10(3) base-pairs (kb) genomic DNA segments covering complete Epo receptor gene and promoter regions revealed that potential regulatory elements (NF-E1, GF-1 or Eryf 1) for erythroid-specific and differentiation stage-specific gene expression are located in the promoter and 3' noncoding regions.     

The micronucleus test with mouse peripheral blood on N-methyl-N'-nitro-N-nitrosoguanidine and mitomycin C.

The micronucleus test using mouse peripheral blood was conducted with N-methyl-N'-nitro-N-nitro-soguanidine (MNNG) and mitomycin C (MMC) as part of the 5th collaborative study supported by the Environmental Mutagen Society of Japan (CSGMT/MMS.JEMS). Male CD-1 mice were intraperitoneally injected once with 12.5-100 mg/kg of MMC. Peripheral blood was drawn at different intervals after treatment, placed on slides previously coated with acridine orange and the numbers of reticulocytes with micronuclei (MNRETs) were scored. The experiments indicated that the maximum effect of both MNNG and MMC was found about 48 h after treatment, and that the micronucleus test using peripheral blood is useful for the screening of chemicals throughout the experimental period in a single animal.     

Structural basis for the coiled-coil architecture of human CtIP

The DNA repair factor CtIP has a critical function in double-strand break (DSB) repair by homologous recombination, promoting the assembly of the repair apparatus at DNA ends and participating in DNA-end resection. However, the molecular mechanisms of CtIP function in DSB repair remain unclear. Here, we present an atomic model for the three-dimensional architecture of human CtIP, derived from a multi-disciplinary approach that includes X-ray crystallography, small-angle X-ray scattering (SAXS) and diffracted X-ray tracking (DXT). Our data show that CtIP adopts an extended dimer-of-dimers structure, in agreement with a role in bridging distant sites on chromosomal DNA during the recombinational repair. The zinc-binding motif in the CtIP N-terminus alters dynamically the coiled-coil structure, with functional implications for the long-range interactions of CtIP with DNA. Our results provide a structural basis for the three-dimensional arrangement of chains in the CtIP tetramer, a key aspect of CtIP function in DNA DSB repair.     

Point mutagenesis in mouse reveals contrasting pathogenetic effects between MEN2B- and Hirschsprung disease-associated missense mutations of the RET gene

Missense mutations of the RET gene have been identified in both multiple endocrine neoplasia (MEN) type 2A/B and Hirschsprung disease (HSCR: congenital absence of the enteric nervous system, ENS). Current consensus holds that MEN2A/B and HSCR are caused by activating and inactivating RET mutations, respectively. However, the biological significance of RET missense mutations in vivo has not been fully elucidated. In the present study, we introduced one MEN2B-associated (M918T) and two HSCR-associated (N394K and Y791F) RET missense mutations into the corresponding regions of the mouse Ret gene by genome editing (Ret^M919T, Ret^N396K and Ret^Y792F) and performed histological examinations of Ret-expressing tissues to understand the pathogenetic impact of each mutant in vivo. Ret^M919T/+ mice displayed MEN2B-related phenotypes, including C-cell hyperplasia and abnormal enlargement of the primary sympathetic ganglia. Similar sympathetic phenotype was observed in Ret^M919T/- mice, demonstrating a strong pathogenetic effect of the Ret M918T by a single-allele expression. In contrast, no abnormality was found in the ENS of mice harboring the Ret N394K or Y791F mutation. Most surprisingly, single-allele expression of RET N394K or Y791F was sufficient for normal ENS development, indicating that these RET mutants exert largely physiological function in vivo. This study reveals contrasting pathogenetic effects between MEN2B- and HSCR-associated RET missense mutations, and suggests that some of HSCR-associated RET missense mutations are by themselves neither inactivating nor pathogenetic and require involvement of other gene mutations for disease expressivity.     

Macrophage lipoprotein lipase modulates the development of atherosclerosis but not adiposity

The role of macrophage lipoprotein lipase (LpL) in the development of atherosclerosis and adiposity was examined in macrophage LpL knockout (MLpLKO) mice. MLpLKO mice were generated using cre-loxP gene targeting. Loss of LpL in macrophages did not alter plasma LpL activity or lipoprotein levels. Incubation of apolipoprotein E (ApoE)-deficient β-VLDL with peritoneal macrophages from ApoE knockout mice lacking macrophage LpL (MLpLKO/ApoEKO) led to less cholesteryl ester formation than that found with ApoEKO macrophages. MLpLKO/ApoEKO macrophages had reduced intracellular triglyceride levels, with decreased CD36 and carnitine palmitoyltransferase-1 mRNA levels compared with ApoEKO macrophages, when incubated with VLDL. Although both MLpLKO/ApoEKO and ApoEKO mice developed comparable hypercholesterolemia in response to feeding with a Western-type diet for 12 weeks, atherosclerosis was less in MLpLKO/ApoEKO mice. Epididymal fat mass and gene expression levels associated with inflammation did not differ between the two groups. In conclusion, macrophage LpL plays an important role in the development of atherosclerosis but not adiposity.