Strain difference in the radiosensitivity of immunocompetent cells and its influence on the residual host-vs-graft reaction in lethally irradiated mice grafted with semiallogeneic bone marrow

A striking difference in radiosensitivity was noted between C3H/He (H-2k) and C57BL/6J (H-2b) strain mice when assessed by primary anti-SRBC PFC response of intact animals and primary cell-mediated lympholysis (CML) response of spleen cells to allogeneic cells in vitro, the C3H strain being more radioresistant. On the other hand, when C3H and B6 mice were exposed to 6.62 to 10.40 grays (Gy) of x-rays and then were transplanted with 2 X 10(6) bone marrow cells from B6C3F1 (H-2b/k) donor mice within 3 hr or at 24 hr after radiation exposure, the early mortality caused by residual host-vs-graft (HVG) reaction was much higher when C3H mice were used as recipients. Furthermore, the proportion of surviving animals manifesting host-type lymphohemopoiesis, i.e., host-type revertants, was much higher in B6C3F1 to C3H than in B6C3F1 to B6 combination. Spleen cells from such host-type revertants manifested strong anti-donor reactivity when assessed by mixed lymphocyte reaction (MLR) and/or CML in vitro. Increase of radiation doses to the recipients to 10.40 Gy resulted in 100% survival and 100% donor-type lymphohemopoiesis in both groups of chimeras. These results indicate strongly that a genetic difference in radiosensitivity of immune system of the recipients can greatly influence the magnitude of residual HVG reactions observed in hybrid to parental strain bone marrow transplantation in mice.     

Stereoselective biliary elimination of disopyramide and mono-N-desisopropyldisopyramide in humans.

The results of a previous pharmacokinetic study of disopyramide (DP) enantiomers in humans suggested that DP and/or mono-N-desisopropyldisopyramide (MND) may show stereoselective extrarenal elimination. Thus, the present study investigates the biliary elimination of DP and MND enantiomers in three patients who had undergone cholecystectomy for cholelithiasis. DP and MND enantiomers displayed biliary elimination. In both subjects, this elimination pathway showed the same characteristics: (1) biliary elimination of DP and MND was stereoselective, (2) the stereoselectivity was opposite to that observed for the metabolic and renal elimination pathways, i.e., the elimination of the (-)-(R)-enantiomer was higher than that of the (+)-(S)-enantiomer, and (3) biliary elimination of MND was higher than that of DP, for both enantiomers. Estimates of the relative contribution of the biliary clearance in the total clearance of DP and MND indicated that this elimination pathway was secondary, especially for DP. The biliary clearance (expressed as % of total clearance) was 1.9 to 4.0% for (-)-(R)-DP, 1.2 to 1.7% for (+)-(S)-DP, 7.8 to 22.9% for (-)-(R)-MND, and 5.2 to 10.5% for (+)-(S)-MND.     

Immediate and long-term effects of radiation on the immune system of specific-pathogen-free mice

Studies on the immediate and long-term effects of radiation on the immune system of specific-pathogen-free mice are summarized in this paper. There was a striking difference in the radiation response of lymphocyte subsets; B cells consist of a fairly radiosensitive homogeneous population, whereas T cells consist of a large percentage (greater than 90 per cent) of radiosensitive and a small percentage (less than 10 per cent) of extremely radioresistant subpopulations. Ly 1+ and Ly 2+ lymphocytes appear equally radiosensitive, although the percentage of radioresistant cells was slightly larger for the former (approximately 5.5 per cent) than the latter (approximately 2.5 per cent). There was a significant strain difference in the radiosensitivity of immune-response potential in mice; immunocompetent cells of C3H mice were more radioresistant than those of BALB/c, C57BL/6, and B10.BR mice. Studies on the long-term effect of radiation on immune system in mice indicated no evidence for accelerated ageing of the immunologic functions when radiation exposure was given to young adults. Preliminary results on the enhancing effect of low dose radiation on cytotoxic T cell response in vitro are also discussed.     

Study on the possible factors influencing the expression of H-2 restriction specificity and Ir phenotype of antigen-specific proliferative T cells with various types of radiation chimeras

To better understand the factors described previously as influencing the manifestation of H-2 restriction specificity and Ir phenotype of T cells from radiation bone marrow chimeras, we also examined H-2 restriction specificity (Ir phenotype) of antigen (DNP-OVA, (T, G)-A-L, (H, G)-A-L)-specific proliferative T cells generated in various types of H-2 incompatible radiation chimeras prepared under our specific-pathogen-free (SPF) condition. The results indicated the following: (a) T cells generated in F1----parent bone marrow chimeras preferentially manifested host-type H-2 restriction specificity and Ir phenotype, regardless of the radiation dose (8.70 vs 11.59 Gy); (b) T cells recovered from twice-reconstituted F1----(PA----PB) chimeras manifested primary host (PB)-type Ir phenotype; (c) T cells which were recovered from (B10.Thy-1.1 X B10.BR.Thy-1.1)F1----parent (Thy-1.2) bone marrow chimeras and treated with anti-Thy-1.2 plus complement to deplete host-derived T cells still manifested preferentially the restriction specificity for host-type H-2; (d) PA-derived T cells which had differentiated in a fully allogeneic host (PB) environment of (PA + PB)----PB chimeras manifested fully allogeneic host-type Ir phenotype; (e) T cells from F1----parent chimeras that were prepared with 13-day fetal liver cells also manifested host H-2-restricted Ir phenotype; and (f) host preference for Ir phenotype of antigen-specific proliferative T cells was observed even in the case of F1----parent bone marrow chimeras reconstituted with "intact" bone marrow cells. The data suggest that thymic APCs, surviving host T cells or the source of stem cells (adult bone marrow vs 13-day fetal liver), do not necessarily play a significant role in the manifestation of H-2 restriction specificity and Ir phenotype of T cells generated in H-2 incompatible radiation chimeras.     

Cellular events during radiation-induced thymic leukemogenesis in mice: abnormal T cell differentiation in the thymus and defect of thymocyte precursors in the bone marrow after split-dose irradiation

Cellular events during the development of thymic lymphomas in young B10.BR mice given leukemogenic split-dose irradiation were studied by examining the differentiation of functional T lymphocyte precursors in the regenerating thymus. It was found that leukemogenic radiation treatment resulted in a sustained depression of the level of thymic cytotoxic T lymphocyte precursors (CTLp) and of mixed lymphocyte reactivity of thymus cells when assessed between 1 and 4 mo after irradiation, in spite of the fact that the total number of thymocytes was restored to the normal level within 2 mo and continued to increase thereafter. In vitro mixing studies of normal thymocytes with thymus cells from split-dose irradiated mice provided no evidence for active suppression as a mechanism for this depressed activity. The ability of bone marrow cells from split-dose irradiated mice to regenerate the thymus and to differentiate into functional CTLp was examined by use of supralethally irradiated Thy-1 congenic recipients. Reconstitution of supralethally irradiated B10.BR Thy-1.2 mice with normal bone marrow from B10.BR Thy-1.1 mice resulted in the complete repopulation of host-thymus with donor-derived cells when assessed at 4 wk after reconstitution. Lymphocytes from the regenerating thymus of these animals were shown to contain high levels of CTLp which were donor-derived. On the other hand, when the recipient mice were reconstituted with bone marrow cells from donor mice which had been split-dose irradiated 1 mo earlier, regeneration of the recipient thymus was severely depressed when assessed at 4 wk to 3 mo after reconstitution. Although variable but small numbers of donor-derived Thy-1+ cells were detected, CTL activity for alloantigen could not be induced in these donor-derived cells. The results suggest that T cell precursors derived from split-dose irradiated donor mice were unable to undergo active proliferation and differentiation into functional CTLp. The significance of these findings on radiation-induced thymic leukemogenesis is discussed.     

Cellular basis of the immunohematologic defects observed in short-term semiallogeneic B6C3F1 -- C3H chimeras: evidence for host-versus-graft reaction initiated by radioresistant T cells

Lethally irradiated C3Hf mice reconstituted with a relatively low dose (2 × 10⁶) of B6C3F₁ bone marrow cells (B6C3F₁ → C3Hf chimeras) frequently manifest immunohematologic deficiencies during the first month following injection of bone marrow cells. They show slow recovery of antibody-forming potential to sheep red blood cells (SRBC) as compared to that observed in syngeneic (C3Hf → C3Hf or B6C3F₁ → B6C3F₁) chimeras. They also show a deficiency of B-cell activity as assessed by antibody response to SRBC following further reconstitution with B6C3F₁-derived thymus cells 1 week after injection of bone marrow cells. A significant fraction of B6C3F₁ → C3Hf chimeras was shown to manifest a sudden loss of cellularity of spleens during the second week following injection of bone marrow cells even though cellularity was restored to the normal level within 1 week. The splenic mononuclear cells recovered from such chimeras almost invariably showed strong cytotoxicity against target cells expressing donor-type specific H-2 antigens (H-2^b) when assessed by ⁵¹Cr-release assay in vitro. The effector cells responsible for the observed anti-donor specific cytotoxicity were shown to be residual host-derived T cells. These results indicate strongly that residual host T cells could develop anti-donor specific cytotoxicity even after exposure to a supralethal dose (1050 R) of radiation and that the immunohematologic disturbances observed in short-term F₁ to parent bone marrow chimeras (B6C3F₁ → C3Hf) were due to host-versus-graft reaction (HVGR) initiated by residual host T cells. The implication of these findings on the radiobiological nature of the residual T cells and the persistence of potentially anti-donor reactive T-cell clones in long-surviving allogeneic bone marrow chimeras was discussed.     

X-inactivation is stably maintained in mouse embryos deficient for histone methyl transferase G9a

One of the two X chromosomes becomes inactivated during early development of female mammals. Recent studies demonstrate that the inactive X chromosome is rich in histone H3 methylated at Lys-9 and Lys-27, suggesting an important role for these modifications in X-inactivation. It has been shown that in the mouse Eed is required for maintenance of X-inactivation in the extraembryonic lineages. Interestingly, Eed associates with Ezh2 to form a complex possessing histone methyltransferase activity predominantly for H3 Lys-27. We previously showed that G9a is one of the histone methyltransferases specific for H3 Lys-9 and is essential for embryonic development. Here we examined X-inactivation in mouse embryos deficient for G9a. Expression of Xist, which is crucial for the initiation of X-inactivation, was properly regulated and the inactivated X chromosome was stably maintained even in the absence of G9a. These results demonstrate that G9a is not essential for X-inactivation.     

Differential expression of cell-wall-related genes during the formation of tracheary elements in the Zinnia mesophyll cell system

Plants, animals and some fungi undergo processes of cell specialization such that specific groups of cells are adapted to carry out particular functions. One of the more remarkable examples of cellular development in higher plants is the formation of water-conducting cells that are capable of supporting a column of water from the roots to tens of metres in the air for some trees. The Zinnia mesophyll cell system is a remarkable tool with which to study this entire developmental pathway in vitro. We have recently applied an RNA fingerprinting technology, to allow the detection of DNA fragments derived from RNA using cDNA synthesis and subsequent PCR-amplified fragment length polymorphisms (cDNA-AFLP), to systematically characterize hundreds of the genes involved in the process of tracheary element formation. Building hoops of secondary wall material is the key structural event in forming functional tracheary elements and we have identified over 50 partial sequences related to cell walls out of 600 differentially expressed cDNA fragments. The Zinnia system is an engine of gene discovery which is allowing us to identify and characterize candidate genes involved in cell wall biosynthesis and assembly.