Comparative in vitro and in vivo assessment of genotoxic effects of etoposide and chlorothalonil by the comet assay.

The alkaline single cell gel electrophoresis (comet) assay was used to assess in vitro and in vivo genotoxicity of etoposide, a topoisomerase II inhibitor known to induce DNA strand breaks, and chlorothalonil, a fungicide widely used in agriculture. For in vivo studies, rats were sacrificed at various times after treatment and the induction of DNA strand breaks was assessed in whole blood, bone marrow, thymus, liver, kidney cortex and in the distal part of the intestine. One hour after injection, etoposide induced DNA damage in all organs studied except kidney, especially in bone marrow, thymus (presence of HDC) and whole blood. As observed during in vitro comet assay on Chinese hamster ovary (CHO) cells, dose- and time-dependent DNA effects occurred in vivo with a complete disappearance of damage 24 h after administration. Even though apoptotic cells were detected in vitro 48 h after cell exposure to etoposide, such a result was not found in vivo. After chlorothalonil treatment, no DNA strand breaks were observed in rat organs whereas a clear dose-related DNA damage was observed in vitro. The discrepancy between in vivo and in vitro models could be explained by metabolic and mechanistic reasons. Our results show that the in vivo comet assay is able to detect the target organs of etoposide and suggest that chlorothalonil is devoid of appreciable in vivo genotoxic activity under the protocol used.     

The role of the thymus in the ontogeny of the immune system

The proper development of the organs of the immune system is dependent on at least three factors: (1) the development of anlagen with the capacity to trap antigens and support the proliferation of lymphoid and plasma cell precursors; (2) the production by the bone marrow of lymphoid and plasma cell precursors which seed in the lymphoid organs; and (3) the thymus, which seeds reactive cells to the lymphoid organs and produces a humoral factor stimulating antigen-triggered proliferation of primitive lymphoid and plasma cells. Studies on cell population changes in the lymph nodes following thymectomy in mice confirm earlier evidence that most cells produced in the thymus do not seed to the lymphoid organs, but die locally in the thymus.     

T cell differentiation in lethally irradiated and reconstituted mice: stem cell influx demonstrated with immunoperoxidase technique.

Using both an anti-stem cell serum and an anti-T cell serum the influx of stem cells in mouse thymus and spleen after lethal irradiation and reconstitution was determined by immunoperoxidase staining. In both organs a rapid influx was observed reaching a maximum on Day 5 after irradiation and cell transfer. Thereafter a decline of stem cells took place while the number of T cells in the thymus increased gradually, reaching a maximum on Day 12. T cells could only be detected in the spleen after 3 weeks.     

Administration of IL-7 to normal mice stimulates B-lymphopoiesis and peripheral lymphadenopathy.

Normal mice were injected with IL-7 (500 ng, twice daily) for various periods of time up to 6 days and the cellularity and phenotypic composition of the thymus, spleen, lymph node, and bone marrow was assessed. After 6 days of treatment, significant increases in the cellularity of the spleen, lymph node, and bone marrow were observed which returned to the normal range within 6 days after cessation of treatment. After 3 days of IL-7 treatment, increased numbers of B220+/surface(s) IgM- bone marrow cells were observed. After 6 days of treatment, these numbers were still further increased and a significant population of B220+/sIgM- cells were observed in the spleen. The numbers of c mu+/sIgM- cells were also increased in the IL-7-treated mice. Analysis of the expression of B220 and BP-1 on the sIgM- bone marrow cells revealed that the B220+/BP-1+ population was dramatically increased after IL-7 treatment and the size of the B220+/BP-1- population did not differ from control mice. The pre-B cell numbers declined rapidly after the cessation of IL-7 treatment. After 6 days of IL-7 treatment, a twofold increase in the number of B cells in the spleen and lymph node was observed. The B cell numbers declined to normal values within 6 days after the cessation of IL-7 administration. In the spleens of the IL-7-treated mice, there was a significant increase in the number of B cells with an immature phenotype (e.g., sIgMhi/sIgDlo, decreased levels of Ia and FcR expression). The numbers of CD8+ and CD4+ T cells were also increased in the lymph node and spleen of the IL-7-treated mice. These numbers declined to normal levels after the cessation of IL-7 treatment.     

Effect of transplantation of thymus, bone marrow and spleen cells on the regenerative processes in pathologically changed liver]

In tests on CBAXC57BL mice with experimental hepatitis, induced by carbon tetrachloride, the transplantation of cells from the lymphoid organs of healthy donors intensified the repairing process in the recipient pathologically changed liver. The most pronounced normalization was seen in the liver of the animals given thymocytes which suggests a deficit of these cells in experimental toxic hepatitis and indicates a definite role of the thymus in the repairing processes of the damaged liver.     

Tolerance to cyclosporin A-induced autologous graft-versus-host disease is mediated by a CD4+CD25+ subset of recent thymic emigrants

Our previous studies revealed that both the autoeffector and immunoregulatory T cells in cyclosporin A (CSA)-induced autologous graft-vs-host disease are recent thymic emigrants (RTEs). The autoeffector cells appear in and are released from the thymus during the first week of CSA treatment, whereas the immunoregulatory thymocytes appear during the second week but are not released until several days after cessation of CSA treatment. In the present study, the antigenic phenotypes of these functional T cell subsets were determined by immunomagnetic separation and flow immunocytometric analysis. During CSA wk 1, the autoeffector T cells in both the thymus and lymph node (LN) expressed a CD4+8+ double-positive (DP) phenotype, after which those in the LN became CD8 single positive (SP). Timed thymectomy experiments confirmed that the CD8-SP autoeffector T cells in LN originated from these DP RTEs. During CSA wk 2, the immunoregulatory thymocytes also displayed a DP phenotype. However, they were not exported to the periphery until several days after CSA treatment had been interrupted and they had acquired a CD4-SP phenotype. In LN, these immunoregulatory RTEs expressed the CD25+ marker characteristic of anergic/suppressor T cells. Cell separation and mixing experiments demonstrated that the autoeffector T cells persist in LN after cessation of CSA treatment, but their activity is not detectable in the presence of recently exported CD4+ T cells. Hence, the results indicate that tolerance to CSA-induced autologous graft-vs-host disease is actively mediated by CD25+CD4+ RTEs that suppress the function of CD8 autoeffector T cells.     

On the Role of the Reticular-epithelial Complex in Transplantation of the Thymus

Transplantation of the neonatal thymus, into young, adult hosts, resulted in massive cell death of graft cortical lymphoid tissue with apparent selective survival of the reticular-epithelial cells. The central area of the graft was progressively cleared of cell debris and the characteristic thymic architecture restored within fourteen days of grafting. Evidence obtained from the regeneration of different-sized transplants suggested that the size and shape attained by the regenerated graft was closely related to the size and shape of the donor tissue.
When donor rat thymuses were transplanted in Millipore chambers, the lymphocyte population did not reappear and after seven days only reticular-epithelial cells remained, retaining their normal appearance. However, when these thymic remnants were removed from the chambers and transplanted into secondary hosts, the thymus regenerated normally, suggesting that the lymphocytes in the regenerated gland were derived from the host. Thymic remnants after cortisol treatment of donors also formed distinct organs after grafting despite the fact that they contained few donor lymphocytes. From the differential effects of cortisol on host and transplanted thymus and the different growth characteristics of transplants it appears that transplants differ in their growth/involution control system from the host thymus.     

In vivo effect of progesteron and estrogen on thymus mass and T-cell functions in female mice

Progesteron and estradiol, administered in doses equivalent to those used for therapy caused a marked transient reduction of the thymus mass, but did not affect the cellularity of other lymphoid organs. Humoral and cellular immune response of the hormone-treated mice was normal at the time of thymus involution. The same was true for the stem-cell differentiation capacity. The remaining thymus cells after hormone treatment showed increased DNA-synthesis.     

鸡胸腺APUD细胞的存在

为了确定鸟类免疫器官内有无内分泌细胞的存在,该文应用Ｇｒｉｍｅｌｉｕｓ嗜银染色法,ＭａｓｓｏｎＦａｎｔａｎａ亲银染色法,铅苏木精法和５－ＨＴ免疫组织化学方法,对鸟胸腺,腔上囊和脾脏进行了观察。结果表明：鸡和虎斑颈槽蛇相同,仅胸腺含有少量嗜银,亲银,铅染和（或）５－免疫反应阳性的细胞,具有一定的内分泌功能,腔上囊虽与胸腺同为中枢免疫器官,但没有此类细胞,这说明二者在内分泌调节等方面且一定差异。     

Neonatal tolerance induction in the thymus to MHC-class II-associated antigens. III. Significance of hemopoietic stem cells for induction and maintenance of Mls tolerance by continuous supply of tolerance-inducing nonlymphocytes

The role of hemopoietic stem cells and other cell types in the induction and maintenance of immunologic tolerance in the thymus was investigated by intravenous injection of Mls-semi-allogeneic cells into newborn mice less than 24 hr after birth. Mls-specific tolerance was induced by inoculation of peritoneal cells and thymus cells, and the tolerant state was compared with that induced by bone marrow cells which had hemopoietic stem cell activity and were able to create a stable chimera in both central and peripheral lymphoid organs. When peritoneal or thymus cells were injected, the level of tolerance attained was proportional to the number of cells injected, though peritoneal cells were 20 times as effective as thymus cells. In vivo functions of tolerance-inducing cells and their immediate precursors were radiosensitive and belonged to a Thy-1-, nylon-wool-nonadherent (probably non-B), weakly Sephadex G-10-adherent cell population. Tolerance induced by peritoneal cell injections was transient, starting to terminate within the first 2 weeks of life, while tolerance caused by bone marrow cell injections persisted through more than 6 weeks. Such transient tolerance induced by the former became long-lasting when followed by an additional injection of bone marrow cells, which did not cause thymic lymphocyte chimerism. All data indicated that bone marrow stem cells were engaged in tolerance induction and maintenance by continuously supplying tolerance-inducing nonlymphocytes.     

Short time observations of morphological changes and cholinesterase distribution in lymphatic organs of the mouse after corticosteroid and x-ray treatment

Summary The changes in thymus, spleen and lymph nodes of the mouse after a single cortisone application or a single whole body x-irradiation were investigated morphologically and histochemically. During 24 h the alterations following the cortisone application are at all stages examined inditinguishable from those following the x-irradiation.The first signs of lymphocyte destruction can be observed already in the first two hours after treatment. Almost at the same time macrophages accumulate at the sites of cell death in the lymphatic organs studied. The eosinophils display a different behaviour. While they accompany the macrophages in the thymus already at the first stages, they appear in the spleen and lymph nodes with a latency of 6 and 8 h, respectively. The highest amount of necrotic cells is found ten hours after both treatments. At the same time the accumulation of macrophages and eosinophils reaches its maximum.The cholinesterase in the lymphatic organs is largely the true cholinesterase. The enzyme-activity increases in the cortex of the thymus gradually 6 h after treatment, showing the highest deposit of reaction product at 10 h. In spleen and lymph nodes the cholinesterase shows only slight variations.The possible role of the cholinesterase-activity in these non-cholinergic tissues is discussed.     

Reciprocal T cell responses in the liver and thymus of mice injected with syngeneic tumor cells.

We investigated the T cell responses in various tissues, especially in the liver and thymus, of mice injected with syngeneic tumors. This study was undertaken since recent evidence indicated that the liver is one of the important immune organs for T cell proliferation. When C3H/He mice were intraperitoneally injected with mitomycin-treated syngeneic MH134 tumors (1 x 10(7)/mouse), a transient increase of liver mononuclear cells (MNC) was induced, showing a peak at Day 4 after injection. Histological study of such liver showed a sinusoidal dilatation and an accumulation of MNC in the sinusoids. The most predominant MNC induced were double negative (CD4-8-) alpha beta T cells and gamma delta T cells. These gamma delta T cells varied, showing unique time-kinetics. Despite a continuous increase of whole liver MNC and alpha beta T cells, the proportion of gamma delta T cells in the liver decreased beginning 4 days after injection. In contrast with the response in the liver, a striking decrease in the cell number of thymocytes was induced after tumor injection, showing a basal level at Day 6. This hypocellularity in the thymus appears to be an inverted response of the lymphocytosis in the liver. At this time, a corresponding decrease in the proportion of double positive (CD4+8+) T cells was always seen in the thymus. Analysis of cell proliferative response showed that the increase of liver MNC after tumor injection was accompanied by augmented proliferation, whereas the decrease of thymocytes was accompanied by depressed proliferation. The present results indicate that there exists a unique, reciprocal response of T lymphocytes between the liver and thymus, and that the presence of tumor appears to stimulate T cell response in the liver but alternatively inactivates such response in the thymus.     

Short time observations of morphological changes and cholinesterase distribution in lymphatic organs of the mouse after corticosteroid and X-ray treatment.

The changes in thymus, spleen and lymph nodes of the mouse after a single cortisone application or a single whole body x-irradiation were investigated morphologically and histochemically. During 24 h the alterations following the cortisone application are at all stages examined indistinguishable from those following the x-irradiation. The first signs of lymphocyte destruction can be observed already in the first two hours after treatment. Almost at the same time macrophages accumulate at the sites of cell death in the lymphatic organs studied. The eosinophils display a different behaviour. While they accompany the macrophages in the thymus already at the first stages, they appear in the spleen and lymph nodes with a latency of 6 and 8 h, respectively. The highest amount of necrotic cells is found ten hours after both treatments. At the same time the accumulation of macrophages and eosinophils reaches its maximum. The cholinesterase in the lymphatic organs is largely the true cholinesterase. The enzyme-activity increases in the cortex of the thymus gradually 6 h after treatment, showing the highest deposit of reaction product at 10 h. In spleen and lymph nodes the cholinesterase shows only slight variations. The possible role of the cholinesterase-activity in these non-cholinergic tissues is discussed.     

Cyclosporin A-induced autologous graft-versus-host disease: a prototypical model of autoimmunity and active (dominant) tolerance coordinately induced by recent thymic emigrants.

Cyclosporin A (CSA)-induced autologous graft-vs-host disease (autoGVHD) is an autoimmune syndrome initiated by autoeffector T cells presumed to be exported from the thymus during CSA treatment. The appearance of noncytotoxic immunoregulatory T cell activity after cessation of CSA treatment is also thymus dependent. In the present study, we have tested the hypothesis that both autoeffector and immunoregulatory T cells in CSA-treated rats are recent thymic emigrants (RTEs). Local syngeneic graft-vs-host reaction (synGVHR) and timed thymectomy (Tx) assays revealed that autoeffector T cells appear initially in the thymus and are promptly exported to lymph nodes (LN) during the first week of CSA treatment. In contrast, immunoregulatory thymocytes are first detectable by local synGVHR inhibition assays during the second week of CSA treatment but are not exported to LN until approximately 4 days post-CSA. Both the autoeffector and immunoregulatory T cells in LN express Thy-1, a selective marker for RTEs in the rat. However, the autoeffector RTEs have a CD4+8+ phenotype, whereas the immunoregulatory RTEs have a CD4+8- phenotype. Thus, the coordinate formation in and release from the thymus cortex and medulla of autoeffector and immunoregulatory T cells in CSA-treated rats directly demonstrates that centrally induced, nondeletional tolerance can serve as a fail-safe mechanism by which clones of autoeffector T cells that have escaped intrathymic negative selection for self-MHC class II Ag can be suppressed postthymically.     

MNNG对细胞DNA合成及其分布的影响

本文用流式细胞光度术(FCM)等方法研究了MNNG,ENNG和DMS对HeLa细胞DNA含量分布的影响。经MNNG(6.8μmol/L)处理后,细胞分裂减少,DNA合成速率下降,S期细胞的比例随处理时间的延长而增加。DMS显示有类似的现象而ENNG的效应则较小。     

Changes in the rat lymphoid organs in acute hypoxia

Changes occurring in the rat thymus, spleen, mesenteric lymph nodes have been studied by means of some histological and cytofluorimetrical methods under the effect of an acute hypoxia that imitates the rise to 7,000 m above the sea level for 1 h and to 6,500 m for 6 h. Under the effect of hypoxia, migration of differentiated lymphocytes out of the lymphoid organs is increasing, certain essential shifts in temporal parameters take place in the mitotic cycle of the lymphocytes, contents of nucleic acids in the lymphoid cells change. The phenomena mentioned demonstrate that under the acute hypoxic stress, intensified differentiation processes of the lymphocytes in the thymus, spleen and the lymph node take place and the lymphoid tissue functional activity increases.     

Abrogation of tolerance to a chronic viral infection

This study documents failure of peripheral tolerance mechanisms in a chronic viral infection and shows that T cell tolerance to a viral Ag seen as self from fetal life can be broken despite the presence of this Ag in extrathymic tissues. Congenital infection of mice with lymphocytic choriomeningitis virus (LCMV) results in T cell tolerance to the virus. Such mice become carriers for life harboring virus in many tissues including the thymus and exhibit no LCMV-specific CTL responses. Our previous studies have documented the curing of this congenitally acquired chronic infection after adoptive transfer of CD8+ T cells from LCMV-immune mice and the presence of host-derived, LCMV-specific CTL in these "cured" carriers. In this study we have examined the mechanism by which these carriers acquired T cell competence and show that these CTL differentiated from the bone marrow after elimination of viral Ag from the thymus. These results demonstrate that even when a chronic infection has been established in utero, the adult thymus retains the ability to restore immunocompetence to the host and to provide protection against reinfection. Surprisingly, these LCMV specific CTL were acquired at a time when infectious virus and intracellular viral Ag, although cleared from the thymus, were readily detectable in organs such as the kidney, testes, and brain. In fact, active viral replication in peripheral tissues was ongoing when these mice acquired new virus-specific T cells. These results show that clearance of virus form the thymus was sufficient to abrogate tolerance to a congenitally acquired chronic infection and that Ag in peripheral tissues did not tolerize newly developing T cells. These findings suggest that mechanisms that operate on immature cells within the thymus to silence self-reactive T cells are effective in induction of tolerance to viruses, but mechanisms of tolerizing mature T cells are likely to breakdown. This has implications for virus-induced autoimmunity and for treatment of chronic infections.     

Localisation of Ig heavy chain mRNA positive cells in Atlantic cod (Gadus morhuaL.) tissues; identified byin situhybridisation

Localisation of immunoglobulin heavy chain (IgH) producing cells was determined in sections from head kidney, spleen, thymus, gills, gut, skin, heart and liver from the Atlantic cod. In general, IgH mRNA positive cells were detected in all organs examined and were mainly located to the connective tissue surrounding the vascular system in these organs. In the head kidney and spleen, IgH mRNA positive cells appeared as single distributed cells or as dense clusters, whereas in the thymus only single distributed positive cells were observed. The percentage of Ig heavy chain mRNA positive (plasma) cells in the head kidney, spleen and thymus was estimated at about 1% of the total cell mass. The number of IgH mRNA positive cells was lower than this in all the other organs examined.     

The functional capabilities of cells leaving the thymus

There has been a controversy for many years over the functional status of cells that leave the thymus (thymus migrants) to populate the peripheral lymphoid organs. Are they immunoincompetent like cortical thymocytes and so probably derived from them, or are they functionally mature like medullary thymocytes? Until recently the techniques used to assess putative thymus migrants have been indirect, but it is now possible to measure the function of recent thymus migrants directly. We used intrathymic injection of a solution of fluorescein isothiocyanate to label thymocytes, and used electronic cell sorting to purify the fluorescent cells that accumulate in the periphery over the following 3 to 4 hr. The migrants have been enriched from an original frequency of about 1:1000 in lymph nodes and spleen, to greater than 98% purity. These cells have been compared with normal peripheral T cells for proliferative and cytotoxic precursor activity in a high cloning efficiency, lectin-induced, limit dilution culture system and in an allospecific limit dilution system. The frequency of precursors of proliferative lymphocytes and cytotoxic lymphocytes and the size of the clones produced is the same for recent migrants and peripheral T cells. Thus by the criteria of proliferation and cytotoxic responses to mitogens and generation of allospecific CTL, thymus migrants, a few hours after their emigration from the thymus, are fully immunocompetent; we therefore see no evidence of a post-thymic precursor-type cell that requires major maturation steps after leaving the thymus.     

Organ-specific autoimmune disease induced in mice by elimination of T cell subsets. V. Neonatal administration of cyclosporin A causes autoimmune disease

Cyclosporin A (CsA), a potent immunosuppressive drug, caused organ-specific autoimmune disease, such as gastritis with anti-parietal cell autoantibodies or oophoritis with anti-oocyte autoantibodies, in BALB/c mice when the drug was administered daily for 1 wk to newborns. Administration to adult mice did not. CsA abrogated the production of L3T4+ T cells and Lyt-2+ T cells in the thymus. Consequently, these T cells were substantially depleted from the peripheral lymphoid organs, especially when the drug was administered from the day of birth. Autoimmune disease was prevented when CsA-treated newborn mice were inoculated with splenic T cells from normal syngeneic mice. However, removal of the thymus immediately after neonatal CsA treatment produced autoimmune disease with a higher incidence and in a wider spectrum of organs, i.e., thyroiditis, sialoadenitis of the salivary gland, gastritis, insulitis of the endocrine pancreas, adrenalitis, oophoritis, or orchitis. Each autoimmune disease was accompanied by the development of circulating autoantibodies specific for the corresponding organ Ag. Immunopathology of these autoimmune diseases was quite similar to that of human organ-specific autoimmune diseases.