Proliferation, apoptosis and thymic involution.

The thymus reaches its maximal size at the age of 1 month in ICR mice and thereafter, the thymic cortex undergoes an exponential decline. This study was designed to compare the proliferation and apoptosis of thymocytes in different parts of the thymus of ICR female mice at the beginning and after the rapid phase of decline of the thymic cortical cellularity. The pattern of proliferation and apoptosis of the thymus was studied in situ in 1-month-old ICR female mice (10 mice) compared to mice at 7 months of age (10 mice). Staining for argyrophylic nucleolar organizer region by histochemistry was used to determine the proportion of type 2 thymocytes, which are considered as cells at S phase of the cell cycle. The mean number of type 2 cells in four random samples of 50 cells in each part of the thymus was defined as the proliferation index of this part of the thymus. In situ detection of apoptosis of thymocytes was carried out using the Apoptag kit, which can detect a single cell apoptosis. The mean number of apoptotic cells in five randomly selected fields of each part of the thymus was defined as the apoptotic index of this part of the thymus. The proliferation index of the peripheral cortex of the 1-month-old mice was 3.6 times higher than the proliferation index of the deep cortex and 5.8 times higher than the proliferation index of the medulla (P < 0.0001). The proliferation index of the peripheral cortex of the 7-month-old mice was reduced by 45% compared to the 1-month-old mice (P < 0.005). The apoptotic index of the corticomedullary junction of the 1-month-old mice was six times higher than the apoptotic index of the cortex and 18 times higher than the apoptotic index of the medulla. The apoptotic index of the thymic cortex was elevated by 66% in the 7-month-old mice compared to the 1-month-old mice (P < 0.0001). We conclude that there is a reduction of the proliferation index and an elevation of the apoptotic index of the thymic cortex in adult mice compared to young mice. These changes might account for the reduction of thymic cortical cellularity during thymic involution.     

Failure of phenobarbitone to prevent febrile convulsions.

One-hundred-sixty-five children without known neurological disorder who presented with their first febrile convulsion between the ages of six months and three years were assigned to daily phenobarbitone treatment or to a control group and followed up at a special clinic for six months. One-hundred-and-sixty-one-one children completed the trial, and of the 88 children assigned to phenobarbitone treatment 10 had further convulsions during this period compared with 14 of the 73 control children. Only 49 of those assigned to phenobarbitone took the drug regularly throughout the trial, and four of these had further febrile convulsions, a proportion not significantly different from that in the controls. All four had mean plasma phenobarbitone concentrations over 69 mumol/l (16 mug/ml) during the trial and in three the plasma concentration was at or over this figure within eight hours over 69 mumol/l (16 mug/ml) during the trial and in three the plasma concentration was at or over this figure within eight hours of the repeat convulsion. Regular phenobarbitone does not seem to prevent febrile convulsions. Attention should instead be directed to organising emergency services to allow early termination of fevrile convulsions, whether first or subsequent, to prevent irreversible brain damage.     

Directed microspore-specific recombination of transgenic alleles to prevent pollen-mediated transmission of transgenes

A major challenge for future genetically modified (GM) crops is to prevent undesired gene flow of transgenes to plant material intended for another use. Recombinase-mediated auto excision of transgenes directed by a tightly controlled microspore-specific promoter allows efficient removal of either the selectable marker gene or of all introduced transgenes during microsporogenesis. This way, transgene removal becomes an integral part of the biology of pollen maturation, not requiring any external stimulus such as chemical induction by spraying. We here show the feasibility of engineering transgenic plants to produce pollen devoid of any transgene. Highly efficient excision of transgenes from tobacco pollen was achieved with a potential failure rate of at most two out of 16 800 seeds (0.024%). No evidence for either premature activation or absence of activation of the recombinase system was observed under stress conditions in the laboratory. This approach can prevent adventitious presence of transgenes in non-GM crops or related wild species by gene flow. Such biological containment may help the deployment and management of coexistence practices to support consumer choice and will promote clean molecular farming for the production of high-value compounds in plants.     

Failure to prevent degeneration of insect muscles with pepstatin.

Minimal O₂ consumption (MOC), was elevated during pregnancy, lactation and after cold-acclimation. Since the MOC remained elevated in pregnant rats after removal of the gravid reproductive organs and fetuses, it was concluded that maternal tissues were hypermetabolic. Results of experiments using endocrine ablation (thyroidectomy) or replacement (thyroid hormones) techniques could not resolved the question of whether the thyroid was required to sustain this hypermetabolism.In order to determine whether the increased MOC during lactation was secondary to the calorigenic requirements of milk biosynthesis, the MOC was measured after removal of mammary tissue of the lactating rat. Despite the absence of lactating mammary tissue, the MOC remained elevated.Thyroidectomized rats were successfully acclimated to cold but their MOC did not change. In contrast, intact rats acclimated to cold in the same manner became better cold-acclimated, as judged by survival at colder temperatures, and their MOC was elevated. Cold-acclimation appears to consist of two components, only one of which depends on the presence of an intact thyroid gland. The presence of both components confers maximal tolerance to cold.     

Skeletal involution by age-associated oxidative stress and its acceleration by loss of sex steroids

Both aging and loss of sex steroids have adverse effects on skeletal homeostasis, but whether and how they may influence each others negative impact on bone remains unknown. We report herein that both female and male C57BL/6 mice progressively lost strength (as determined by load-to-failure measurements) and bone mineral density in the spine and femur between the ages of 4 and 31 months. These changes were temporally associated with decreased rate of remodeling as evidenced by decreased osteoblast and osteoclast numbers and decreased bone formation rate; as well as increased osteoblast and osteocyte apoptosis, increased reactive oxygen species levels, and decreased glutathione reductase activity and a corresponding increase in the phosphorylation of p53 and p66(shc), two key components of a signaling cascade that are activated by reactive oxygen species and influences apoptosis and lifespan. Exactly the same changes in oxidative stress were acutely reproduced by gonadectomy in 5-month-old females or males and reversed by estrogens or androgens in vivo as well as in vitro.We conclude that the oxidative stress that underlies physiologic organismal aging in mice may be a pivotal pathogenetic mechanism of the age-related bone loss and strength. Loss of estrogens or androgens accelerates the effects of aging on bone by decreasing defense against oxidative stress.     

Limiting TCR expression leads to quantitative but not qualitative changes in thymic selection

Thymic selection is controlled in part by the avidity of the interaction between thymocytes and APCs. In agreement, the selective outcome can be modulated by altering the expression levels of selecting ligands on APCs. Here we test the converse proposition, i. e., whether changing TCR levels on thymocytes can alter the selective outcome. To this end, we have generated mice in which all thymocytes express two transgenic TCRs simultaneously (dual TCR-expressing (DTE) mice), the class I-restricted HY TCR and the class II-restricted AND TCR. Due to mutual dilution, surface expression levels of the two individual transgenic TCRs are diminished in DTE relative to single TCR-expressing mice. We find that thymic selection is highly sensitive to these reductions in TCR surface expression. Positive selection mediated by the AND and HY TCRs is severely impaired or abolished, respectively. Negative selection of the HY TCR in male DTE mice is also partly blocked, leading to the appearance of significant numbers of double positive thymocytes. Also, in the periphery of male, but not female, DTE mice, substantial numbers of single positive CD8bright cells accumulate, which are positively selected in the thymus but by a highly inefficient hemopoietic cell-dependent process. Overall our results favor the interpretation that the outcome of thymic selection is not determined solely by avidity and the resulting signal intensity, but is also constrained by other factors such as the nature of the ligand and/or its presentation by different subsets of APCs.     

Mechanism of thymic involution (analysis of intrinsic and extrinsic aspects)

The mechanisms of thymic involution with age are analyzed in terms of both intrinsic and extrinsic aspects. The intrinsic factors include: decrease in the number of thymocyte-precursors (pro-T cells) in the bone marrow, in emigration of pro-T cells into the thymus, in proliferation of thymic lymphocytes and decrease in extrathymic factors promoting proliferation of thymic lymphocytes. The extrinsic factors capable to exert modulating effects on the thymic function are humoral factors of the hypothalamic-pituitary origin. It seems much easier to manipulate the extrinsic factors by controlling neuro-endocrine stimulation, and to restore the thymic involution. This is quite encouraging, because it permits restoring the impaired immune functions of the aged people.     

Failure of histamine antagonists to prevent hypoxic pulmonary vasoconstriction in dogs.

The role of histamine as a mediator of hypoxic pulmonary vasoconstriction was examined in intact anesthetized dogs. Antagonism of histamine vasoconstrictor (H1) receptors with a classic antihistaminic drug (chlorpheniramine) failed to prevent or modify the pulmonary vascular responses to hypoxia (10% O2). Blockade of histamine vasodilator (H2) receptors with a newly synthesized blocking agent (metiamide) potentiated the vasoconstriction induced by hypoxia and prevented the normal increase in heart rate. Combined H1- and H2-receptor blockade also did not prevent or reduce the hypoxic pulmonary pressor response, although it did effectively abolish the cardiovascular actions of infused histamine. In other dogs, histamine infused (3.6 mug/kg per min) during hypoxia attenuated the pulmonary vasoconstriction induced by hypoxia. The results imply that, in the dog, histamine does not mediate hypoxic pulmonary vasoconstriction. However, histamine does appear to be released during hypoxia, and it may play a role in modulating the pulmonary vascular responses to hypoxia by opposing the hypoxia induced vasoconstriction. The results also imply that histamine may be responsible for the increase in heart rate during hypoxia.     

Effects of castration on thymocyte development in two different models of thymic involution

Age-associated thymic involution is accompanied by decreased thymic output. This adversely affects general immune competence and T cell recovery following cytoreductive treatments such as chemotherapy. A causal link between increasing sex steroids and age-related thymic atrophy is well established. Although castration has been demonstrated to regenerate the atrophied thymus, little is known about how this is initiated or the kinetics of thymocyte regeneration. The present study shows that although castration impacts globally across thymocyte development in middle-aged mice, the regenerative effects are initiated in the immature triple-negative compartment and early T lineage progenitors (ETP). Specifically, there was a reduction in number of ETP with age, which was restored following castration. There was, however, no change in ETP reconstitution potential in ETP at this age or following castration. Furthermore, in a chemotherapy-induced model of thymic involution, we demonstrate castration enhances intrathymic proliferation and promotes differentiation through the triple-negative program. Clinically, reversible sex steroid ablation is achieved hormonally, and thus presents a means of ameliorating immune inadequacies, for example, following chemotherapy for bone marrow transplantation. By improving our understanding of the kinetics of thymic recovery, this study will allow more appropriate timing of therapy to achieve maximal reconstitution, especially in the elderly.     

The role of sex steroids and gonadectomy in the control of thymic involution

A major underlying cause for aging of the immune system is the structural and functional atrophy of the thymus, and associated decline in T cell genesis. This loss of na?ve T cells reduces adaptive immunity to new stimuli and precipitates a peripheral bias to memory cells against prior antigens. Whilst multiple mechanisms may contribute to this process, the temporal alliance of thymic decline with puberty has implicated a causative role for sex steroids. Accordingly ablation of sex steroids induces profound thymic rejuvenation. Although the thymus retains some, albeit highly limited, function in healthy adults, this is insufficient for resurrecting the T cell pool following cytoablative treatments such as chemo- and radiation-therapy and AIDS. Increased risk of opportunistic infections and cancer relapse or appearance, are a direct consequence. Temporary sex steroid ablation may thus provide a clinically effective means to regenerate the thymus and immune system in immunodeficiency states.     

Analysis of the human neonatal thymus: evidence for a transient thymic involution

The neonatal period is marked by the impairment of the major components of both innate and adaptive immunity. We report a severe depletion of cortical CD4+CD8+ double-positive thymocytes in the human neonatal thymus. This drastic reduction in immature double-positive cells, largely provoked by an increased rate of cell death, could be observed as early as 1 day after birth, delaying the recovery of the normal proportion of this thymocyte subset until the end of the first month of postnatal life. Serum cortisol levels were not increased in newborn donors, indicating that the neonatal thymic involution is a physiological rather than a stress-associated pathological event occurring in the perinatal period. Newborn thymuses also showed increased proportions of both primitive CD34+CD1- precursor cells and mature TCRalphabetahighCD69-CD1-CD45RO+/RAdull and CD45ROdull/RA+ cells, which presumably correspond to recirculating T lymphocytes into the thymus. A notable reinforcement of the subcapsular epithelial cell layer as well as an increase in the intralobular extracellular matrix network accompanied modifications in the thymocyte population. Additionally neonatal thymic dendritic cells were found to be more effective than dendritic cells isolated from children's thymuses at stimulating proliferative responses in allogeneic T cells. All these findings can account for several alterations affecting the peripheral pool of T lymphocytes in the perinatal period.     

Dual immunofluorescence studies of cortisone-induced thymic involution: evidence for a major cortical component to cortisone-resistant thymocytes

Cortisone-resistant thymocytes (CRT) have been used as the experimental equivalent of medullary thymocytes for the past 15 yr. Studies with CRT have provided evidence that the medullary population is similar to mature T cells in phenotype and function and may therefore be the major source of thymus emigrants. However, we have recently demonstrated that CRT differ from medullary thymocytes in their expression of the homing receptor molecule recognized by the monoclonal antibody MEL-14. Thus, many CRT express high levels of the MEL-14-defined homing receptor, whereas medullary thymocytes are MEL-14- to MEL-14lo. In normal adult mice, only 1 to 3% of thymocytes are MEL-14hi; these cells are located exclusively in the cortex and many are phenotypically and functionally mature. In this study we have used dual immunofluorescence techniques to further characterize those thymocytes resistant to cortisone treatment. Aside from being of mature phenotype with respect to expression of peanut agglutinin binding sites and the cell surface molecules H-2K, Ly-1, Lyt-2, and L3T4, CRT can be divided into MEL-14lo and MEL-14hi subpopulations, suggesting that they may actually be derived from both the medullary and the MEL-14hi cortical thymocyte subsets.     

Expression of nerve growth factor is upregulated in the rat thymic epithelial cells during thymus regeneration following acute thymic involution

Neuroimmune networks in the thymic microenvironment are thought to be involved in the regulation of T cell development. Nerve growth factor (NGF) is increasingly recognized as a potent immunomodulator, promoting "cross-talk" between various types of immune system cells. The present study describes the expression of NGF during thymus regeneration following acute involution induced by cyclophosphamide in the rat. Immunohistochemical stain demonstrated not only the presence of NGF but also its upregulated expression mainly in the subcapsular, paraseptal, and perivascular epithelial cells, and medullary epithelial cells including Hassall's corpuscles in both the normal and regenerating thymus. Biochemical data obtained using Western blot and RT-PCR supported these results and showed that thymic extracts contain NGF protein and mRNA, at higher levels during thymus regeneration. Thus, our results suggest that NGF expressed in these thymic epithelial cells plays a role in the T lymphopoiesis associated with thymus regeneration during recovery from acute thymic involution.