Cellular composition and ultrastructure of the thymus of the newborn mouse after immunization of pregnant females with homologous brain antigens

Immunization is performed with 20% of water-saline extract of medulla oblongata on the 6th, 8th, 10th days of pregnancy (1 g per 200 g body mass). In the thymus cortical substance of newborn rats no statistically significant difference in content of small lympocytes, lymphoblasts and large lymphocytes, mitotically deviding cells is revealed as compared to the normal. The part of middle lymphocytes decreases up to 5.7 +/- 0.7% (control--10.0 +/- 1.7%). The content of distroying cells and fagocytic macrophages is increasing. In cytoplasm of one macrophage several fagocyted degenerating cells with pyknotic nuclei and destructively altered organells are often present. In the interlobular connective tissue an increased amount of degenerating forms of mast cells is noted. In the thymus medullary substance small lymphocytes are growing in number. Certain changes in vessels of the microcirculatory bed are revealed.     

A thymic factor increasing the blood sugar level.

The thymus extract prepared by using isotonic NaC1 solution causes hyperglycaemy and the thymectomy, too, produces the same effect. Consequently, the active substance of the thymus is produced elsewhere in the organism and being stored within the thymus, gets into the extract by homogenizing its cells. Both the thymectomy and the thymic factor increase the effect of alloxan and this sensitizing effect can be prevented by pretreatment with prednisolone.     

Erythrocyte lysis in isotonic solution of ammonium chloride: theoretical modeling and experimental verification

A mathematical model of erythrocyte lysis in isotonic solution of ammonium chloride is presented in frames of a statistical approach. The model is used to evaluate several parameters of mature erythrocytes (volume, surface area, hemoglobin concentration, number of anionic exchangers on membrane, elasticity and critical tension of membrane) through their sphering and lysis measured by a scanning flow cytometer (SFC). SFC allows measuring the light-scattering pattern (indicatrix) of an individual cell over the angular range from 10° to 60°. Comparison of the experimentally measured and theoretically calculated light scattering patterns allows discrimination of spherical from non-spherical erythrocytes and evaluation of volume and hemoglobin concentration for individual spherical cells. Three different processes were applied for erythrocytes sphering: (1) colloid osmotic lysis in isotonic solution of ammonium chloride, (2) isovolumetric sphering in the presence of sodium dodecyl sulphate and albumin in neutrally buffered isotonic saline, and (3) osmotic fragility test in hypotonic media. For the hemolysis in ammonium chloride, the evolution of distributions of sphered erythrocytes on volume and hemoglobin content was monitored in real-time experiments. The analysis of experimental data was performed in the context of a statistical approach, taking into account that parameters of erythrocytes vary from cell to cell.     

Spleen and thymus lymphoid tissue in hypoxia (a biometric study)

The spleen and thymus have been studied macro- and microscopically in rats (180-200 g body mass) on the 1st, 3d, 5th, 7th, 14th and 28th days of adaptation to a decreased atmospheric pressure in the altitude chamber corresponding to lifting to 5,000 and 7,500 m (after a preliminary gradual acclimatization) and on the 14th, 28th, 42d, 56th days of readaptation. A relative mass of the organs, the white pulp section area--the transversal section area of the spleen ratio, the summation section area of its lymph nodules have been estimated. In the thymus the cortico-medullary index (CMI) has been defined. A relative mass of the spleen increases during the first week of hypoxia, and during adaptation period it somewhat decreases and stabilizes, remaining higher than in the control. At the altitude of 5,000 m the cross section area of the lymph nodules decreases by 17% on the 28th adaptation day and at the altitude of 7,500 m--by 27% beginning from the 14th up to the 28th adaptation days. In the thymus the CMI, after some decrease during the first days of hypoxia at the altitude of 5,000 m, increases and normalizes on the 28th adaptation day, and at the altitude of 7,500 m stabilizes on the 14th - 28th days of hypoxia. When the rats are at the altitudes of 5,000 and 7,500 m the thymus lymphoid tissue reacts more quickly to the hypoxia effect and much sooner normalizes during the readaptation period than does the white pulp of the spleen. The main changes in the lymphoid tissue of the spleen and thymus take place on the 7th - 28th days of hypoxia.     

Migration of peripheral T and B cells into the thymus of aging (NZB × SJL)F1 female mice

Aging NZB × SJL (NS) female mice provide a unique model of thymopathology characterized by the intrathymic accumulation of large numbers of mature T and B cells. The purpose of the present work was to examine the possibility that this phenomenon results from the invasion of the thymus by cells from the periphery. Lymphoid cells labeled with chromium-51 or indium-111 were injected into syngeneic recipients to study their patterns of in vivo migration. Lymph node (LN) or spleen cells were found to localize significantly (1–2% of injected radioactivity) into the thymus of 12-month-old NS females but not into that of young recipients or of old NS males. However, intrathymic localization of injected LN cells was observed in castrated NS males which exhibit the same thymopathology as NS females. Both radiolabeled T and B cells were found to enter the thymus of aged NS females but the latter cells about three times less efficiently than the former. Moreover, while thymocytes from young NS females were unable to recirculate to LN, those of old NS females showed increased LN-seeking capacity and part (1%) of them did migrate back into the thymus of old but not young NS females. In additional cell transfer experiments, the intrathymic migration of B cells into old NS females was further documented by using the antibody response to sheep erythrocytes as a tracer. Taken together, these observations indicate that the thymus of aging NS female mice is permeable to recirculating lymphocytes, suggesting that at least part of the mature T and B cells detected in this thymus, are migrants from the periphery.     

Long-term retention of mature NK1.1+ NKT cells in the thymus

The NKT cell pool in the thymus contains immature (NK1.1(-)) and mature (NK1.1(+)) subsets that represent distinct linear stages of a linear developmental pathway. An unexplained paradox is why immature NK1.1(-) NKT cells are mainly exported to the periphery instead of the more mature and more abundant NK1.1(+) NKT cells. In this study we have determined that mature NK1.1(+) NKT cells are retained by the thymus to form an extremely long-lived resident population capable of rapid and prolonged production of IFN-gamma and IL-4. The retention of mature NKT cells provides an explanation for why the periphery is mainly seeded by immature NK1.1(-) cells despite mature NK1.1(+) NKT cells being more abundant in the thymus. This is the first study to identify a mature T cell subset retained within the thymus and is additional evidence of the distinct developmental pathways of mainstream T cells and NKT cells.     

Evidence for Subpopulation of Mature Lymphocytes within Mouse Thymus

THERE is increasing evidence that thymus cells migrate from the thymus to the peripheral lymphoid tissues where they make up most, if not all, of the thymus-dependent population of lymphocytes^1–3. The term “thymus-derived” is thus appropriately applied to this population. Yet most thymocytes are different from peripheral lymphocytes, both in immunological competence and in surface antigenic characteristics. For example, thymocytes have more theta (θ)⁴ and less H2 antigen⁵ than do peripheral lymphocytes and in TL-positive strains of mice only thymocytes normally express the TL antigen⁶. Recently, Lance et al.⁷ found that injected thymocytes which had migrated to lymph nodes and spleen were progressively less susceptible to anti-TL and more susceptible to anti-H2 serum over the first 24 h. I report here experiments in which thymus cells injected intravenously into irradiated syngeneic mice and harvested as early as 3 h later from the peripheral lymphoid tissues can be shown to have the surface antigenic properties of peripheral thymus-derived lymphocytes rather than thymocytes. A second experiment demonstrates that at least part of the differentiation from thymocyte to thymus-derived lymphocyte seems to occur within the thymus.     

Early virological events in various tissues of newborn monkeys after intrarectal infection with pathogenic simian human immunodeficiency virus

Children infected with human immunodeficiency virus type 1 often have higher viral loads and progress to acquired immunodeficiency syndrome more rapidly than adults. In our previous study of simian-human immunodeficiency virus (SHIV)-infected adult monkeys, immature CD4CD8 double-positive T cells in the thymus and jejunum decreased faster than mature CD4 single-positive T cells. Here, we examined the effect of virus replication on immature T cells from the same SHIV-inoculated newborn monkeys having more immature T cells than adults. The infectious viruses were more abundantly detected in the thymus than in other tissues at both 13 and 26 days post-infection (dpi). However, mature CD4(+) T cells in the thymus declined after 13 dpi and immature CD3(-) CD4 single-positive T cells remained at 26 dpi. These results suggested that many immature CD4(+) T cells in the thymus of newborns support the production of infectious viruses even after the depletion of mature CD4(+) T cells.     

Effect of substances of a polypeptide nature isolated from the thymus, cerebral cortex and substantia alba on the cellular and humoral indices of immunity in thymectomized mice]

Effects of low molecular weight polypeptides (M. W. lower 10,000) isolated from the calf thymus, cortex and white matter of the brain by extraction with acetic acid on the cellular and humoral immune responses were studied in experimental thymectomized mature CBA mice. Thymectomy reduced markedly the number of T-cells in the spleen. Accordingly, the ability to generate both Ig M and IgG antibody forming cells as well as humoral antibodies to thymus-dependent antigen, SRBC, was significantly suppressed in the animals. Subcutaneous administration of 1 micron/g (body weight) of the thymus and brain cortex polypeptides during 8 days not only completely restored T-cells population in the spleen and immune responsibility but also elevated these values 1.5-2 fold in comparison with sham controls which had been given saline solution. The preparation from the white matter of the brain lacked biological activity.     

Histopathological changes in the thymus of the white mouse after infection with Toxoplasma gondii (author's transl)]

Three days after infection of mice with a virulent strain of T. gondii first histopathological destructions of the thymus are visible. The number of lymphocytes decreases step by step till to the animals' day of death on the 7th day p.i. At this time the cortex has lost all its thymocytes. Electronmicroscopical pictures show destruction of most of the reticulum cells and lymphocytes. In those cells which are still alive multiplicaiton of Toxoplasma trophozoites is to be seen. A lot of parasites are lying extracellular in the detritus of the destroyed thymus-cells. After infection of mice with an avirulent strain of T. gondii there is a loss of lymphocytes in the cortex of the thymus too. Starting at day 5th to 7th p.i. it reaches its peak at the time of the 10th to 15th day after infection. After this period restitution of lymphocytes in the cortex is going on. At about the 30th day p.i. replacement of all the lymphocytes is finished. Sometimes the cortex seems to be enlarged, that means now there are more thymocytes in the cortex than in uninfected controls. Neither reticulum cells nor lymphocytes show destruction of their ultrastructure. Only a process of activation of the lymphocytes can be seen by electronmicroscopy. In the lymphocytes the mitochondria are enlarged and there are more than in the controls. At the same time Golgi-apparatus and endoplasmatic reticulum become prominent.     

Thymus cell migration: cells migrating from thymus to peripheral lymphoid organs have a "mature" phenotype

To gain information on the lineage relationship of cells leaving the thymus, we studied the phenotype of thymus emigrants within hours of their exit. The migrants were identified in the peripheral lymphoid organs by their fluorescence, 3 to 4 hr after intrathymic injection of a solution of fluorescein isothiocyanate, a technique that initially only labels thymocytes. Migrants identified in this way were analyzed with rhodamine-anti-Thy-1 or rhodamine peanut agglutinin (PNA). They were found to express Thy-1 antigen and PNA binding sites at levels very similar to those found on the majority of peripheral T cells or medullary thymocytes and quite different from cortical thymocytes. Taken together with our previous experiments on Lyt-1, Lyt-2, and H-2 levels, the data show that cells leaving the thymus are quite mature in phenotype and are indistinguishable from peripheral T cells by all the criteria examined.     

Distinct requirements for IL-7 in development of TCR gamma delta cells during fetal and adult life

TCRgammadelta-transgenic IL-7(-/-) mice were generated to determine whether T cells containing productively rearranged TCRgammadelta genes have additional requirements for IL-7 within the thymus or peripheral lymphoid tissues. Differences in developmental requirements for IL-7 by TCRgammadelta cells were noted and were linked to derivation from fetal- vs adult-type precursors in the thymus. Although TCRgammadelta cells are absent from IL-7(-/-) mice, TCRgammadelta cells were restored to the thymus and periphery by expression of TCRgammadelta transgenes. Endogenous TCRgamma chains were expressed by IL-7(+/-) but not IL-7(-/-) TCRgammadelta-transgenic mice, providing direct support for findings that IL-7 is necessary for rearrangement and expression of TCRgamma genes. The number of TCRgammadelta thymocytes was 10-fold reduced in TCRgammadelta-transgenic IL-7(-/-) embryos; however, adult TCRgammadelta-transgenic IL-7(-/-) or IL-7(+/-) mice had similar numbers of fetal thymus-derived TCRgammadelta cells in their skin. Thus, fetal TCRgammadelta cells required IL-7 for TCR rearrangement, but not for proliferation or survival in the periphery. In contrast, the numbers of TCRgammadelta cells in other tissues of TCRgammadelta-transgenic IL-7(-/-) mice were not completely restored. Moreover, coincident with the transition from the first to second wave of T cell precursors maturing in neonatal thymus, thymus cellularity of TCRgammadelta-transgenic IL-7(-/-) mice dropped significantly. These data indicated that in addition to TCRVgamma gene rearrangement, TCRgammadelta cells differentiating from late fetal liver or adult bone marrow precursors have additional requirements for IL-7. BrdU incorporation studies indicated that although IL-7 was not required for TCRgammadelta cell proliferation, it was required to prolong the life span of mature TCRgammadelta cells.     

The effect of initial tonicity on freeze/thaw injury to human red cells suspended in solutions of sodium chloride

Human red blood cells, suspended in solutions of sodium chloride, have been frozen to temperatures between -2 and -14 degrees C and thawed, and the extent of hemolysis was measured. In parallel experiments, red cells were exposed to similar cycles of change in the composition of the suspending solution, but by dialysis at 21 degrees C. The tonicity of the saline in which the cells were initially suspended was varied between 0.6x isotonic and 4x isotonic; some samples from each experimental treatment were returned to isotonic saline before hemolysis was measured. It was found that the tonicity of the saline used to suspend the cells for the main body of the experiment affected the amount of hemolysis measured: raising the tonicity from 0.6x to 1x to 2x reduced hemolysis, both in the freezing and in the dialysis experiments, whereas raising the tonicity further to 4x reversed that trend. There was little difference between the freeze/thaw and the dialysis treatments for the cells suspended in 1x or 2x saline, whether or not the cells were returned to isotonic conditions. However, the cells suspended in 0.6x saline showed greater damage from freezing and thawing than from the comparable change in the composition of the solution, whether or not they were returned to isotonic conditions. Cells that were suspended in 4x saline and exposed to changes in salt concentration by dialysis showed less hemolysis when they were assayed in the 4x solution than cells that had received the comparable freezing/thaw treatment, but when the experiment included a return to isotonicity, the two treatments gave similar results. Returning the cells to isotonic saline had a negligible affect on the cells in 0.6x and 1x saline, but caused considerable hemolysis in the 2x and 4x samples, more so after dialysis than after freezing and thawing. We conclude that cells suspended in 0.6x and 4x saline behave differently from cells suspended in 1x and 2x saline and hence that cells suspended in a range of solutions of differing initial tonicity should not be treated as a homogeneous population. We argue that an effect of the unfrozen fraction of water (U) cannot be distinguished, within the framework of these freeze/thaw experiments alone, from an effect of initial tonicity, and that the biphasic nature of the correlation between haemolysis and U makes a causal connection improbable.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ontogeny and organization of the stationary non-lymphoid cells in the human thymus

Summary Ontogenetic differentiation of the human thymus was investigated in 50 embryos by means of light and electron microscopic methods in an attempt to clarify the morphogenesis of the complicated microecology of thymic tissue. At the 8th gestational week (g.w.), the primordium of the thymus contains almost exclusively undifferentiated epithelial cells. At the 10th g.w., the epithelial cells in the central part are spindle-shaped. During the subsequent weeks the cortical region of the thymus becomes separated into lobes by mesenchymal septa containing hemopoietic precursor cells and large electronlucent cells with irregularly shaped nuclei. The latter cells are also found in the deeper presumptive medullary regions of the thymus; they differentiate into interdigitating reticulum cells (IDC). The permeation of the medulla of the thymus by non-epithelial IDC occurs concurrently with the formation of cortical and medullary epithelial cells. Between the 12th and 14th g.w. the cortical and medullary differentiation is completed. At this time-stage cortical small lymphocytes differ in morphological shape from medullary lymphocytes, the latter acquiring the appearance of immunocompetent T cells and establishing intimate contact with the IDC.These findings indicate that the thymic cortex and medulla contain different epithelial cells. In addition, the thymic medulla displays cells characterized by the morphology of typical interdigitating reticulum cells of peripheral lymphoid tissue. The structural pattern of the thymus is correlated to morphologically differing lymphoid cell populations in the cortical and medullary regions.This investigation was supported by grants from the Deutsche Forschungsgemeinschaft and by the Sonderforschungsbereich 111The authors dedicate this paper to Professor Helmut Leonhardt on the occasion of his 60th birthday. The authors also appreciate the excellent technical assistance of Mrs. I. Knauer, Mrs. H. Waluk and Mrs. H. Siebke     

IgG Fc receptor-bearing cells during early lymphoid cell development in the chicken

Cells from intraembryonic mesenchyme, yolk sac, bursa of Fabricius, and thymus from chicken embryos at different stages of development were studied for the presence of IgG Fc receptors by EA-rosette formation and binding of heat-aggregated chicken IgG (agg IgG). Cells with Fc receptors were found in high frequency in the intraembryonic mesenchyme as early as on the third day of incubation, in the yolk sac on the 7th day, in the bursa on the 10th day, and in the thymus on the 16th day of embryonic development. In the bursa the number of agg IgG binding cells increased with the age of the embryo and remained high after hatching, whereas in the thymus the peak value (76%) was observed on the 16th embryonic day, and after hatching only about 10% of the cells expressed the agg IgG receptors. The results also suggest that the appearance of IgG Fc receptors precedes the expression of B-L (Ia-like) antigens and of cytoplasmic and surface immunoglobulins on early lymphoid cells of the chicken embryo.     

Characteristics of the binding of the anticancer agents mitoxantrone and ametantrone and related structures to deoxyribonucleic acids

The binding constants for interaction of the anticancer agents mitoxantrone and ametantrone and several congeners with calf thymus DNA and the effects of ionic strength changes have been determined spectrophotometrically. The agents show a preference for certain sequences, particularly those with GC base pairs, and the magnitude of the specificity depends on the specific substituents on the anthraquinone ring system. The binding constant for mitoxantrone with calf thymus DNA in 0.1 M Na+, pH 7, is approximately 6 X 10(6) M-1, and the rate constant for the sodium dodecyl sulfate driven dissociation of mitoxantrone from its calf thymus DNA complex under the same solution conditions and 20 degrees C was determined to be 1.3 s-1. The unwinding angle of mitoxantrone determined independently by viscosity measurements and by a novel assay employing calf thymus topoisomerase shows excellent agreement for a value of 17.5 degrees. The viscosity increase of sonicated calf thymus DNA varies considerably with the substituent on the anthraquinone ring system. Binding studies employing T4 and phi w-14 DNAs in which the major groove is occluded and the reverse experiment with anthramycin-treated calf thymus DNA indicate at least part of the mitoxantrone molecule may lie in the minor groove.     

Development of B cell subsets: effect of priming on in vitro TNP-LPS responsiveness.

Mice were injected with DNP-derivatives of thymus dependent (DNP-OA) and thymus-independent (DNP-Ficoll and TNP-LPS) antigens, and the response to TNP-LPS in vitro was studied after several priming periods. DNP-OA priming decreases the amount of cells responding to TNP-LPS in vitro. In the case of DNP-Ficoll and TNP-LPS-primed cells, there is an initial burst of responsiveness to TNP-LPS, which progressively decreases until an abolishment of TNP-LPS responsiveness is found at day 40 after immunization. The sensitivity to TNP-LPS reappears as new precursor cells differentiate into mature cells. We suggest that B cells progressively gain the capacity to respond to thymus-independent and thymus-dependent antigens (B1-B2 differentiation) and that challenge with a particle antigen increases the ratio of maturation throughout the pathway.     

Cutting edge: identification of the targets of clonal deletion in an unmanipulated thymus

Autoreactive thymocytes can be eliminated by clonal deletion during their development in the thymus. The precise developmental stage(s) at which clonal deletion occurs in a normal thymus has been difficult to assess, in large part because of the absence of a specific marker for TCR-mediated apoptosis. In this report, we reveal that Nur77 expression can be used as a specific marker of clonal deletion in an unmanipulated thymus and directly identify TCRintCD4+CD8+ and semimature CD4+CD8- thymocytes as the principal targets of deletion. These data indicate that clonal deletion normally occurs at a relatively late stage of development, as cells mature from CD4+CD8+ thymocytes to single-positive T cells.     

Viral infection of the thymus.

We have examined infection of the thymus during congenitally acquired chronic lymphocytic choriomeningitis virus (LCMV) infection of mice, a classic model of antigen-specific T-cell tolerance. Our results show that (i) infection starts at the fetal stage and is maintained throughout adulthood, and (ii) this chronic infection of the thymus can be eliminated by transfer of virus-specific cytotoxic T lymphocytes (CTL) that infiltrate the thymus and clear all viral products from both medullary and cortical regions. Elimination of virus from the thymus results in abrogation of tolerance. During the fetal stage, the predominant cell type infected is the earliest precursor of T cells with a surface phenotype of Thy1+ CD4- CD8- J11d+. In the adult thymus, infection is confined primarily to the cortisone-resistant thymocytes present in the medullary region. The infected cells are CD4+ and J11d+. The presence of J11d, a marker usually associated with immature thymocytes, on infected single positive CD4+ "mature" thymocytes is intriguing and suggests that infection by this noncytolytic virus may affect development of T cells. There is minimal infection of the CD8+ medullary thymocytes or of the double positive (CD4+ CD8+) cells present in the cortex. Infection within the cortex is confined to the stromal cells. Interestingly, there is infection of the double negative (CD4- CD8-) thymocytes in the adult thymus, showing that even during adulthood the newly developing T cells are susceptible to infection by LCMV. Virus can be eliminated from the thymuses of these carrier mice by adoptive transfer of medullary region first and then from the thymic cortex. This result clearly shows the need to reevaluate the widely held notion that mature T cells are unable to reenter the thymus. In fact, in our experiments the donor T cells made up to 20 to 30% of the total cells in the thymus at 5 to 7 days after the transfer. The number of donor T cells declined as virus was eliminated from the thymus, and at 1 month posttransfer, the donor T cells were hardly detectable. The results of this study examining the dynamics of viral infection and clearance from the thymus, the primary site of T-cell development, have implications for understanding tolerance induction in chronic viral infections.