B cells participate in thymic negative selection of murine auto-reactive CD4+ T cells

It is well documented that thymic epithelial cells participate in the process of negative selection in the thymus. In recent years it was reported that also dendritic cells enter the thymus and contribute to this process, thus allowing for the depletion of thymocytes that are specific to peripherally expressed self-antigens. Here we report that also B cells may take part in the elimination of auto-reactive thymocytes. Using a unique mouse model we show that B cells induce negative selection of self-reactive thymocytes in a process that leads to the deletion of these cells whereas regulatory T cells are spared. These findings have direct implication in autoimmunity, as expression of a myelin antigen by B cells in the thymus renders the mice resistant to autoimmune inflammation of the CNS.     

Evaluation of the radioprotective action of mexamine based on the cellularity index of the rat thymus

Tie administration of mexamine leads to emigration of thymus cells levelling the radioprotective effect of the compound as determined by total cellularity of the organ. Processes of thymus cell depletion were additive after the effect of mexamine and ionizing radiation. It was found possible to estimate the radioprotective efficiency of mexamine with regard to thymus tissue cellularity diminution after the administration of the preparation.     

Impaired T-cell differentiation in the thymus at the early stages of acute pathogenic chimeric simian-human immunodeficiency virus (SHIV) infection in contrast to less pathogenic SHIV infection

One of the mechanisms by which HIV infection induces the depletion of CD4+ T cells has been suggested to be impairment of T-cell development in the thymus, although there is no direct evidence that this occurs. To examine this possibility, we compared T-cell maturation in the intrathymic progenitors between macaques infected with an acute pathogenic chimeric simian-human immunodeficiency virus (SHIV), which causes profound and irreversible CD4+ T-cell depletion, and macaques infected with a less pathogenic SHIV, which causes only a transient CD4+ T-cell decline. Within 27 days post-inoculation (dpi), the two virus infections caused similar increases in plasma viral loads and similar decreases in CD4+ T-cell counts. However, in the thymus, the acute pathogenic SHIV resulted in increased thymic involution, atrophy and the depletion of immature T cells including CD4(+)CD8(+) double-positive (DP) cells, whereas the less pathogenic SHIV did not have these effects. Ex vivo differentiation of CD3(-)CD4(-)CD8(-) triple-negative (TN) intrathymic progenitors to DP cells was assessed by a monkey-mouse xenogenic fetal thymus organ culture system. Differentiation was impaired in the TN intrathymic progenitors of the acute pathogenic SHIV-infected monkeys, while differentiation was not impaired in the TN intrathymic progenitors of the less pathogenic SHIV-infected monkeys. These differences suggest that dysfunction of thymic maturation makes an important contribution to the irreversible depletion of circulating CD4+ T cells in vivo.     

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.     

Absence of mitochondrial uncoupling protein 3: effect on thymus and spleen in the fed and fasted mice

Mitochondrial uncoupling protein 3 (UCP3) is constitutively expressed in mitochondria from thymus and spleen of mice, and confocal microscopy has been used to visualize UCP3 in situ in mouse thymocytes. UCP3 is present in mitochondria of thymus and spleen up to at least 16 weeks after birth, but levels decrease by a half in thymus and a fifth in spleen after three weeks, probably reflecting the suckling to weaning transition. UCP3 protein levels increase approximately 3-fold in thymus on starvation, but expression levels in spleen were unaffected by starvation. Lack of UCP3 had little effect on thymus mass or thymocyte number. However, lack of UCP3 affected spleen mass and splenocyte number (in the fasted state) and results in reduced CD4+ single positive cell numbers and reduced double negative cells in the thymus, but as a 2-fold increase in the proportion of CD4(+), CD8(+) and DP cells in spleen. Starvation attenuates these proportionate differences in the spleen. A lack of UCP3 had no apparent effect on basal oxygen consumption of thymocytes or splenocytes or on oxygen consumption due to mitochondrial proton leak. Splenocytes from UCP3 knock-out mice are also more resistant to apoptosis than those from wild-type mice. Overall we can conclude that UCP3 affects thymocyte and spleen cell profiles in the fed and fasted states.     

Thymus involution induced by mouse hepatitis virus A59 in BALB/c mice.

Mouse hepatitis virus A59 (MHV-A59) infection of adult BALB/c mice induced a severe, transient atrophy of the thymus. The effect was maximal at 1 week after infection, and thymuses returned to normal size by 2 weeks after infection. There was no effect of glucocorticoids, since thymus atrophy was also found in adrenalectomized, infected mice. In infected thymus, immature CD4+ CD8+ lymphocytes were selectively depleted, and apoptosis of lymphocytes was increased. The MHV receptor glycoprotein MHVR was detected on thymus epithelial cells but not on T lymphocytes. In a small number of stromal epithelial cells, but in very few lymphocytes, the viral genome was detectable by in situ hybridization. These observations suggested that MHV-A59-induced thymic atrophy results not from a generalized lytic infection of T lymphocytes but rather from apoptosis of immature double-positive T cells that might be caused by infection of a small proportion of thymus epithelial cells or from inappropriate secretion of some factor, such as a cytokine.     

Loss of lymphocyte cyclic AMP dependent protein kinase activity in malignant melanoma.

Cyclic AMP dependent protein kinase activity was depressed in whole thymus and spleen as well as isolated splenic lymphocytes from B16 melanoma bearing C57B1/6J mice as compared to control animals. A similar loss of enzyme activity was observed in human peripheral blood lymphocytes from melanoma bearing patients as compared to normal subjects. An unaltered level of activity in the heart of tumor bearing mice suggested some specificity for the lymphoid system. This depressed enzyme activity was the result of a diminished Vmax for cAMP stimulated calf histone phosphorylation. The tumor bearing state in the mouse was also accompanied by a depletion of small lymphocytes from both thymus and spleen and it is hypothesized that the losses of lymphocytes and cAMP dependent protein kinase activity are related.     

Arginine Depletion by Arginine Deiminase Does Not Affect Whole Protein Metabolism or Muscle Fractional Protein Synthesis Rate in Mice

Due to the absolute need for arginine that certain cancer cells have, arginine depletion is a therapy in clinical trials to treat several types of cancers. Arginine is an amino acids utilized not only as a precursor for other important molecules, but also for protein synthesis. Because arginine depletion can potentially exacerbate the progressive loss of body weight, and especially lean body mass, in cancer patients we determined the effect of arginine depletion by pegylated arginine deiminase (ADI-PEG 20) on whole body protein synthesis and fractional protein synthesis rate in multiple tissues of mice. ADI-PEG 20 successfully depleted circulating arginine (<1 μmol/L), and increased citrulline concentration more than tenfold. Body weight and body composition, however, were not affected by ADI-PEG 20. Despite the depletion of arginine, whole body protein synthesis and breakdown were maintained in the ADI-PEG 20 treated mice. The fractional protein synthesis rate of muscle was also not affected by arginine depletion. Most tissues (liver, kidney, spleen, heart, lungs, stomach, small and large intestine, pancreas) were able to maintain their fractional protein synthesis rate; however, the fractional protein synthesis rate of brain, thymus and testicles was reduced due to the ADI-PEG 20 treatment. Furthermore, these results were confirmed by the incorporation of ureido [¹⁴C]citrulline, which indicate the local conversion into arginine, into protein. In conclusion, the intracellular recycling pathway of citrulline is able to provide enough arginine to maintain protein synthesis rate and prevent the loss of lean body mass and body weight.     

Body mass is less important than bird order in determining the molecular rate for bird mitochondrial DNA

A negative relationship between body mass and molecular evolution rates has been suggested, and recently a correlation equation has been published based on mitochondrial genomic data of 475 bird species and their body masses. Here, we re‐analysed these data and show that the bird order as a proxy of monophyletic groups was a stronger predictor of the molecular rate than the body mass. We provide order‐specific molecular substitution rates. Only three orders (Galliformes, Gruiformes, Pelecaniformes) showed a very clear negative correlation, and specific correlation equations are given for these. The molecular rates of bird orders differed strongly at similar mean body masses, and we suggest that the previously described trend across all birds may arise as smaller species also tend to have characteristic life histories, namely faster turnover of generations, higher fecundity and shorter lifespans.     

Mink cell focus-forming murine leukemia virus infection induces apoptosis of thymic lymphocytes

In a previous study we identified the subpopulations of thymus cells that were infected by the lymphomagenic MCF13 murine leukemia virus (MLV) (F. K. Yoshimura, T. Wang, and M. Cankovic, J. Virol. 73:4890-4898, 1999) and observed an effect on thymus size by virus infection. In this report we describe our results which demonstrate that MCF13 MLV infection of thymuses reduced the number of T lymphocytes in this organ. Histological examination showed diffuse lymphocyte depletion, which was most striking in the CD4(+) CD8(+) lymphocyte-enriched cortical zone. Consistent with this, flow cytometric analysis showed that the lymphocytes which were depleted were predominantly the immature CD3(-) CD4(+) CD8(+) and CD3(+) CD4(+) CD8(+) cells. A comparison of the percentages of live, apoptotic, and dead cells of the gp70(+) and gp70(-) thymic lymphocytes suggested that this effect on thymus cellularity is a result of virus infection. Studies of the survival of thymic T lymphocytes in culture showed that cells from MCF13 MLV-inoculated mice underwent greater apoptosis and death than cells from control animals. Assays for apoptosis included 7-amino-actinomycin D staining, DNA fragmentation, and cleavage of caspase-3 and poly(ADP-ribose) polymerase proenzymes. Our results suggest that apoptosis of thymic lymphocytes by virus infection is an important step in the early stages of MCF13 MLV tumorigenesis.     

PP2A phosphatase suppresses function of the mesenchymal invasion regulator NEDD9

The mesenchymal mode of cancer cell invasion characterized by active adhesion turnover and a polarized actin cytoskeleton, is critically regulated by the adaptor protein NEDD9/HEF1/Cas-L. While it is known that NEDD9 is subject to extensive phosphorylation modification, the molecules that determine NEDD9 phosphorylation to stimulate adhesion turnover and mesenchymal cell morphologies are currently unknown. Earlier studies have suggested that the serine/threonine phosphatase PP2A regulates interconversion between a low molecular mass NEDD9 phosphoform and higher molecular mass phosphoforms. However, previous studies have used chemical inhibitors to block PP2A activity. In the present study we therefore aimed to specifically inhibit PP2A activity via siRNA and dominant negative approaches to investigate the effect of PP2A on interconversion between 115 kDa and 105 kDa NEDD9 and determine the functional consequence of PP2A activity for NEDD9 function. Strikingly, we find that while the phosphatase inhibitor Calyculin A indeed abrogates detachment-induced dephosphorylation of the 115 kDa NEDD9 phosphoform, PP2A depletion does not inhibit 115 kDa to 105 kDa interconversion. Our data suggest instead that PP2A targets discrete NEDD9 phosphorylation modifications separate to the events that mediate interconversion between the two forms. Functionally, PP2A depletion increases NEDD9 mediated cell spreading and mutation of S369 in the serine-rich region of NEDD9 to aspartate mimics this effect. Importantly, mutation of S369 to alanine abrogates the ability of dominant negative PP2A to increase NEDD9-mediated cell spreading. Collectively, our data reveal that the tumour suppressor PP2A may act via S369 to regulated NEDD9-mediated cell spreading.     

Reasons for the quantitative differences in the formation of chromatin breakdown products following exposure to neutrons and gamma radiation

Relative content of salt-soluble chromatin fragments in rat thymus increased after the effect of neutrons, as compared to gamma-radiation, while cell depletion of the organ was similar with both types of radiation. This was not associated with the rate and extent of chromatin degradation in thymocytes and was an indication of death of a larger number of thymocytes after the effect of neutrons.     

NEGATIVE MATERNAL EFFECT REVISITED: A TEST ON TWO POPULATIONS OF ORCHESELLA CINCTA L. (COLLEMBOLA: ENTOMOBRYIDAE)

A Dutch population of Orchesella cincta had been demonstrated to exhibit a negative maternal effect on age at first reproduction, which caused alternation of short and long generations. The adaptive significance of such a mechanism was assumed to be associated with the bivoltine life cycle of Dutch O. cincta. We expected that it would be absent in a non bivoltine population sampled in Siena, Italy. To test this hypothesis we performed a parent-offspring regression experiment with both populations simultaneously. The experiment showed that there was no negative maternal effect in both populations. We leave open the question of the cause of the discrepancy between the previous result with the Dutch population and the present result. The results of our experiment were also used to determine heritabilities of the traits age, mass and number of molts at first reproduction, and size of the first clutch. The estimates of heritabilities were often not significantly different from zero, especially in the Italian population which had only one significant heritability.     

Gangliosides as markers of cortisone-sensitive and cortisone-resistant rabbit thymocytes: characterization of thymus-specific gangliosides and preferential changes of particular gangliosides in the thymus of cortisone-treated rabbits

Neutral glycosphingolipids and gangliosides in rabbit thymus, spleen, bone marrow, and erythrocyte ghosts were analyzed by conventional chemical and enzymatic procedures and negative ion fast atom bombardment mass spectrometry (FABMS). Thymus gangliosides showed a characteristic composition. Major gangliosides comprising 75% of the total thymus gangliosides were sialosyl lacto-N-neo-tetraosyl- and sialosyl lacto-N-nor-hexaosylceramides containing NeuGc and palmitic acid. These major thymus gangliosides were not detected in spleen, bone marrow, or erythrocytes, whereas GD1a, which was not present in the thymus even in a trace amount, was present in spleen and bone marrow. In addition, the major gangliosides in rabbit thymus were preferentially reduced when an animal was given an intraperitoneal injection of cortisone acetate, as found on analysis 48 h later. The decrease was accompanied by a concomitant increase in NeuAc-containing GM3 with longer chain fatty acids.     

The effect of hydrocortisone on the alkaline proteinase activity in the target organs of rats]

The effect of hydrocortisone on the thymus and liver proteinases activity in the alkaline pH range was studied on rats. It is shown that the alkaline proteinases are activated in the thymus and inhibited in the liver of hormone-treated animals. Possible relationship between the effect of glucocorticoids on protein metabolism and alkaline proteinases activity is suggested.     

Opposing reactivities of subpopulations of T lymphocytes toward syngeneic tumor cells: separation of early thymocytes.

The present communication is a continuation of earlier studies which indicated that interaction between syngeneic tumors and those lymphocytes in the early stages of thymic processing can result in enhanced tumor growth in vivo. The thymocytes involved in this tumor enhancement were found previously in the rapidly dividing subpopulation of subcapsular cortical thymocytes, both in the untreated thymus and in the thymus undergoing repopulation after cortisone depletion. In the present experiments we have isolated this small subpopulation of early thymocytes. After cortisone injection such cells could be separated from the medullary cortisone-resistant thymocytes since the latter cells exhibit a high level of surface H-2 antigens and were thus lysed preferentially by anti-H-2 serum and complement. The repopulating subcapsular early thymocytes, which were resistant to this treatment, were incapable of responding to PHA while their basal proliferation rate was undiminished, and the majority of the cells were found to be dividing. When such low H-2 early thymocytes were injected together with three different tumors into syngeneic mice their tumor-enhancing activity was evident. It is clear that such early thymocytes are not devoid of biologic reactivity and their release from the thymus could have decisive results.     

Repeated exposure to enhanced UV-B radiation in successive generations increases developmental instability (leaf fluctuating asymmetry) in a desert annual

Populations of the desert annual Dimorphotheca sinuata , derived from a common seed stock, were exposed concurrently over four successive generations to either ambient (representing no stratospheric ozone depletion) or elevated (representing 20% stratospheric ozone depletion) UV-B levels during their complete life cycle. Leaf fluctuating asymmetry (FA) was measured in populations of plants grown from seeds of selected generations which had experienced different UV-B exposure histories, and from seeds collected from a wild population of this species which grows in a naturally enhanced UV-B environment. These measured plants had been grown in a greenhouse under essentially UV-B-free conditions. Leaf FA was significantly increased by greater numbers of enhanced UV-B exposures in the parentage of the seed. There was a linear to exponential dose–response relationship between number of UV-B exposure iterations in seed parentage and leaf FA, suggesting that damage to DNA caused by UV-B exposure during plant development may not be fully repaired, and thus be inherited by offspring and accumulated over successive generations in this species. Leaf FA of plants grown from seed from the wild population was not significantly greater than that of control plants whose parentage experienced only ambient UV-B exposures, although this negative result may have been due to low sampling intensity and measurement resolution, and the relatively low UV-B enhancement experienced by the wild population. We conclude that leaf FA may constitute a relatively sensitive yet inexpensive means of quantifying UV-B damage to plants.     

In vivo administration of monoclonal antibodies to the CD3 T cell receptor complex induces cell death (apoptosis) in immature thymocytes.

Some thymocytes, upon activation via the TCR complex in vitro, undergo apoptotic cell death. In this report, we examine the cell death induced in the thymus after administration of anti-CD3 or anti-TCR antibodies. We found that shortly after antibody injection, cortical thymocytes undergo apoptosis as characterized by morphologic changes and DNA fragmentation. Anti-CD3 administration led to depletion of nearly all CD4+CD8+ thymocytes, and approximately 50% of CD4+CD8- thymocytes. This depletion predominantly affected cells bearing low levels of CD3, although some depletion also occurred among cells expressing intermediate and high levels. Administration of an anti-TCR antibody also induced apoptosis, but affected significantly fewer thymocytes than anti-CD3. This effect was probably not due to different binding affinities for the two antibodies, because both antibodies show similar dose response effects in an in vitro model of activation-induced apoptosis. This work demonstrates that findings on activation-induced apoptosis in vitro can be extended to the in vivo situation, and further, that the activation of cortical thymocytes, in situ, results in apoptosis and removal of the activated cells. The possible relationships between this activation-induced cell death in immature thymocytes and the process of negative selection of autoreactive T cells is discussed.     

Total lymphoid irradiation leads to transient depletion of the mouse thymic medulla and persistent abnormalities among medullary stromal cells

Mice given multiple doses of sublethal irradiation to both the thymus and the peripheral lymphoid tissues showed major transient, and some persistent disruptions in general thymic architecture and in thymic stromal components. At 2 wk after total lymphoid irradiation (TLI), the thymus lacked identifiable medullary regions by immunohistochemical analyses. Medullary stromal cells expression MHC Ag or a medullary epithelial cell Ag, as well as medullary macrophages, were undetectable. Instead, the processes of cortical epithelial cells were observed throughout the entire thymus. Strikingly, thymocyte subsets with mature phenotypes (CD4+CD8- and CD4-CD8+) were present in the apparent absence of a medulla. This early, gross effect was rapidly reversed such that by 1 to 2 mo after TLI, medullary areas with MHC Ag-positive cells were evident. However, abnormalities in a subset of medullary stromal cells appeared to be more persistent. Medullary epithelial cells, identified by the MD1 mAb, were greatly reduced in number and abnormally organized for at least 4 mo after TLI. In addition, macrophages containing endogenous peroxidase activity, normally abundant in medullary regions, were undetectable at all times examined after TLI. Therefore, this irradiation regimen induced both transient and long term effects in the thymus, primarily in medullary regions. These results suggest that TLI may be used as an experimental tool for studying the impact of selective depletion of medullary stromal cells on the development of specific T cell functions.