Substance P-like immunoreactivity in capsaicin-sensitive structures of the rat thymus

Unlike in mouse and hamster, the thymus of rats or guinea pigs contains measurable amounts of substance P-like immunoreactivity (SP-LI), which, in a HPLC system, eluted as authentic SP or SP sulfoxide. Ontogenetic study showed that in rats the SP-LI content of the thymus increased up to 60 days from birth, and decreased thereafter. Capsaicin, but not 6-hydroxydopamine (6-OHDA) pretreatment completely depleted thymic SP-LI content in both newborn and adult rats. Animals treated with capsaicin as newborns, but not as adults, showed lower thymus weights as compared to controls. Rats pretreated with capsaicin as adults underwent partial time-dependent recovery of thymic SP-LI content. Somatostatin-like immunoreactivity (SST-LI) of rat thymus, eluting in part as authentic SST, was unaffected both by capsaicin or 6-OHDA pretreatment. Taken together, these findings demonstrate the existence of capsaicin-sensitive structures containing SP in the rat thymus. The possible function(s) that capsaicin-sensitive structures could exert in the thymus, among which a trophic action, mediated by the efferent function of sensory neurons, remain(s) to be established.     

Studies on the termination of immunological tolerance in the mouse thymus cell population after irradiation

Immunological tolerance in the mouse thymus cell population induced by the intravenous injection of deaggregated bovine gamma globulin was terminated by whole body irradiation. After irradiation, the weight of the thymus recovered biphasically, and the termination of tolerance occurred as early as in the first phase. Both Thy-1 antigen expression and helper activity of the thymus cell population in irradiated mice recovered in parallel with the recovery of the thymus weight. Sensitivity of the regenerating thymus cell to the tolerogen was not different from that of the normal thymus cell. The first phase of thymus regeneration may be caused by the proliferation and differentiation of relatively radioresistant and tolerogen insensitive precursors residing in the thymus. Tolerogen and/or immunogen reactive thymus cells may originate from the precursor.     

The recovery of spleen-seeking and lymph-node-seeking thymus subpopulations following cortisol administration

The administration of cortisol acetate depleted the population of spleen-seeking thymus cells to a greater extent than the population of lymph node-seeking thymus cells. While the proportion of lymph node-seeking thymus cells was increased following cortisol administration to about five times that found in the normal thymus, the number of lymph node-seeking cells in the atrophied thymus was reduced to about one-fifth of the number present in the thymus of untreated mice. Cortisol administration completely eliminated the small proportion of TL-positive cells present among the lymph node-seeking population, and drastically reduced the proportion of TL-positive spleen-seeking cells. The rate of recovery of the number of spleen-seeking thymus cells was much more rapid than that of the lymph node-seeking cells. The number of spleen-seeking cells increased significantly within 12 days after the administration of cortisol, and full recovery was evident by day 20. In contrast, the number of lymph node-seeking cells started to increase only 18 days after cortisol administration, and reached the level found in the normal thymus about 8 days later. The recovery of TL-positive lymph node-seeking cells paralleled the recovery of the TL-negative lymph node-seeking population rather than that of the TL-positive spleen-seeking population. Possible developmental interrelations between the spleen-seeking and lymph node-seeking populations of thymus cells are discussed.     

Restoration of T cell depression and suppression of blood pressure in spontaneously hypertensive rats (SHR) by thymus grafts or thymus extracts

Spontaneously hypertensive rats (SHR) that develop hypertension and arterial lesions resembling human periarteritis nodosa were found to possess a selective depression of T cell functions with an appearance of natural thymocytotoxic autoantibody (NTA). The relationship between T cell depression and hypertension in these animals was investigated. The immune responsiveness of T cell-depressed SHR was completely recovered by histocompatible thymus grafts and was partially restored by histoincompatible allogeneic or xenogeneic thymus grafts or by injection of thymus extracts. Transplantation of compatible thymus tissues into neonatal SHR produced long-lasting recovery of immune functions. When complete immunologic restoration was achieved, significant suppression of high blood pressure was obtained. The SHR that showed high blood pressure were always accompanied with high NTA titers and arterial lesions. Thymus grafts or thymus extracts significantly decreased the titers of NTA. The development and dissemination of arterial lesions, which may cause increased blood flow resistance, were completely prevented by compatible thymus grafts into neonatal SHR. These results suggest that thymus grafts and thymus extracts may suppress the development of hypertension by preventing or curing the periarteritis nodosa in SHR.     

Seasonal and age changes in the thymus gland of the Red fox, Vulpes vulpes,

Seasonal and age changes in thymus weight and histological structure were examined in the Red fox ( Vulpes vulpes ). Growth in the fox thymus slowed down after birth compared with the last third of foetal existence, but the gland still grew rapidly to reach a peak first year weight when the cubs were 20 weeks of age. From this point the thymus in both sexes decreased markedly in weight to reach a low point by the beginning of the first breeding season. During this involution lobule structure broke down and adipose tissue and connective tissue was laid down in the gland. Recovery of the thymus towards the second year weight maximum was accompanied by the regaining of lobule structure and the gland resembled that of the juvenile again. The male thymus increased in weight from the middle of the mating season, but recovery in the female thymus was delayed until the end of lactation. Involution occurred prior to the second breeding season. Thereafter, the gland never attained the high weights seen in the first two years of life, but histological changes still occurred even in old animals. The thymus gland of animals infected with sarcoptic mange is described.     

Thymic hormones in radiation-induced immunodeficiency. I. Induction of mature interleukin 1 responsive cell in the thymus by thymosin fraction 5

The restorative effect of thymosin fraction 5 (TF5) on the thymus of gamma-irradiated mice was examined. Four different mouse strains were used in this study since earlier work determined that the degree of response to TF5 is strain dependent. The responsiveness to comitogenic effect of interleukin 1 (IL-1) was used to measure the rate of recovery of immunocompetent cells in the thymus, since only more mature PNA-, Lyt-1+-2- medullary cells respond to this monokine. Contrary to several earlier reports that radioresistant cells repopulating the thymus within the first 10 days after irradiation are mature, corticosteroid resistant, immunocompetent cells, the thymic cells from irradiated mice in all strains used had greatly reduced responses to IL-1. Daily intraperitoneal injections of TF5 increased significantly the responses of thymic cells to IL-1 in 10- to 13-weeks-old C57Bl/KsJ, C57Bl/6, C3H/HeJ, and DBA/1 mice. Older mice, 5 months or more in age, of DBA/1 strain did not respond to treatment with TF5. However, C3H/HeJ mice of the same age were highly responsive. In conclusion, (1) cells repopulating the thymus within 12 days after irradiation contain lower than normal fraction of mature IL-1 responsive cells, (2) thymic hormones increase the rate of recovery of immunocompetent cells in the thymus, and (3) the effect of thymic hormones is strain and age dependent.     

Recovery response of dividing cells in the thymus of whole-body gamma-irradiated mice.

Mice were irradiated with different doses of gamma-rays 30 min after the administration of 32P-orthophosphate. The dose-response curves determined at 72 hours after exposure showed an inflection point in the total activity present in the DNA in thymus and spleen. In the low dose-range, the dose-response curves have D0 = 55 rad (n = 2-5) for thymus and DO = 95 rad (n = 2-5) for the spleen. Thirty minutes after the administration of 32P-orthophosphate, the dividing cells from thymus were partially synchronized by the administration of 80 mg per kg body-weight hydroxyurea. At different time-intervals, the mice were irradiated with 80 rad, and the total activity of DNA was determined at 72 hours after synchronization. A significant maximum of recovery was found at 5 hours (S phase) after the administration of hydroxyurea. In similar conditions, the dose-response curves corresponding to the G1, S and M phase of the division cycle were also determined. The synchronization of dividing cells induced by hydroxyurea failed in the spleen.     

Short- and long-term effects of whole-body irradiation with fission neutrons or X rays on the thymus in CBA mice

Young adult (6 weeks old) female CBA mice were exposed to whole-body irradiation with either 2.5-Gy fast fission neutrons of 1 MeV mean energy or 6.0-Gy 300 kVp X rays at centerline dose rates of 0.1 and 0.3 Gy/min, respectively. The weight of spleen and animal and the weight, cellularity, and histological structure of the thymus were studied at different times after irradiation. Thymic recovery after whole-body irradiation showed a biphasic pattern with minima at 5 and 21 days after irradiation and peaks of regeneration at Days 14 and 42 after X irradiation or at Days 14 and 70 after neutron irradiation. After the second phase of recovery, a marked decrease in relative thymus weight and cellularity was observed, which lasted up to at least 250 days after irradiation. Splenic recovery showed a monophasic pattern with an overshoot on Day 21 after irradiation. After neutron irradiation a late decrease in relative spleen and animal weight was observed. The observed late effects on thymus and spleen weight and thymus cellularity are discussed in terms of a persistent defect in the bone marrow.     

Thymic irradiation inhibits the rapid recovery of TH1 but not TH2-like functions of CD4+ T cells after total lymphoid irradiation.

Four to six weeks after total lymphoid irradiation (TLI), there is a selective deficit in the CD4+ T cells which secrete IL-2, proliferate in the MLR, and induce GVHD (Th1-like functions). A similar deficit in CD4+ T cells which secrete IL-4 and help antibody responses (Th2-like functions) is not observed. In the present study, shielding of the thymus with lead during TLI increased the Th1-like functions of CD4+ cells. Mice without thymus shields showed a marked selective reduction in the medullary stromal cells identified with the monoclonal antibody, MD1, and the severe reduction was prevented with thymus shields. Thus, shielding the thymus prevents the depletion of thymic medullary stromal cells and allows for a rapid recovery of Th1-like functions in the mouse spleen after TLI. Th2-like functions recover rapidly after TLI whether or not the thymus is irradiated.     

Changes in functional activity of the synthetic apparatus of rat thymocytes under acute and chronic gamma-irradiation

The changes in functional activity of rat thymocyte synthetic apparatus (synthetic activity) under acute (7.5 Gy) and continuous (dose rates 14.4 and 0.43 cGy/day) gamma-irradiation were studied by the fluorescent microspectral analysis. It has been shown that after the acute irradiation the changes in synthetic activity occurred in three main stages. The stages reflect the depression and activation of synthetic processes that is due to interphase and reproductive cell death and urgent recovery of thymus cellularity and secondary repopulating. Under continuous irradiation with a dose rate 14.4 cGy/day in long-term period both the decrease of thymocyte synthetic activity (in most animals) and activation (in the animals with pronounced symptoms of radiation damage) were observed. This reflects the depression processes in immune system and augmentation of immunoreactivity due to mass antigen influence of transformed cells and infectious agents on thymocytes. Under low dose ionizing irradiation (dose rate 0.43 cGy/day) the undulating changes in synthetic processes in thymus cells were observed. This depends on the recurrence of depression and recovery processes in the blood-forming tissue.     

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.     

Morphofunctional characteristics of the thymus and muscle regenerates under laser irradiation and alloplasty of adult muscle tissue in the area of trauma

The effect of muscle tissue alloplasty and He-Ne laser radiation on the skeletal muscle regeneration and thymus function was studied. Allogenic muscle tissue was implanted from an adult rat. Without laser irradiation (series 1), initial enhancement of thymus recovery observed on day 7 after alloplasty (a characteristic stress response to operation) was followed by gradual destructive changes in the thymus tissue. On day 30 after alloplasty, connective tissue developed in the implantation area in muscle regenerates, and the muscle tissue accounted for 64 ± 2%. Implantation of unirradiated allografts into the muscles of recipient rats preirradiated with a He-Ne laser (series 2) resulted in a nearly complete destruction of the thymus and suppression of its function; the mitotic index of thymocytes was low. These changes were observed throughout the experiment starting immediately after the operation. In this case, the allogenic transplant retained the ability to develop: the 30-day repairing muscles consisted of 71 ± 2% of muscle tissue. When an allograft preirradiated with a He-Ne laser was implanted into unirradiated rats (series 3), thymus destruction at the beginning of the postoperative period was much less significant than in series 2 but more pronounced than in series 1. Then, thymus recovered more rapidly, the allogenic transplant was resorbed, and the muscle tissue in the regenerates accounted for 62 ± 3%.     

Differential recovery of intestine, bone marrow, and thymus of rats with solid tumors following 5-fluorouracil administration.

Time relationships for recovery of several host organs from toxic effects of 5-fluorouracil were determined in ACI rats bearing Morris hepatoma 3924A. A single injection of 150 mg/kg body weight 5-fluorouracil (the LD10) resulted in loss of 90% of the tibial bone marrow, 60% of the intestinal mucosa, and 90% of the thymus as measured by total DNA content of the organs. Organ DNA contents following 150 mg/kg of the drug were minimal on day 3 for intestine and on day 5 for marrow and thymus. A return to pretreatment or higher levels of DNA was observed by day 4 for intestine, day 11 for tibial marrow, and day 19 for thymus. Incorporation of 3H-deoxyuridine into host organ DNA after 150 mg/kg 5-fluorouracil was inhibited 36 hrs for intestine, 3 days for thymus, and 5 days for tibial bone marrow. Inhibition of 3H-deoxyuridine incorporation into DNA was similar for 50, 100, and 150 mg/kg doses both in tumor and in host organs, but recovery of 3H-deoxyuridine incorporation and DNA content of host organs began later with the higher doses of 5-fluorouracil. Maximal incorporation of 3H-deoxyuridine into DNA was observed on day 4 for intestine, day 8 for marrow, and day 9 for thymus after treatment with 150 mg/kg 5-fluorouracil. Animal lethality following the second of two 150 mg/kg injections of 5-fluorouracil was related to the extent of recovery of intestinal mucosa and bone marrow at the time of the second injection. Survival decreased to 0% for normal rats when the interval between injections was 3-4 days, improved at 5 days and was 100% when the interval was 10-11 days.     

Thymectomy in the neonatal rat.

A method for removal of the thymus in the newborn rat employing measures aimed at reducing the mortality incurred by the surgical procedure, cannibalism and infection is described in detail. Tranquilisation of the dam, sterile technique, magnification of the operative site for thymus aspiration, warming to 37 degrees C during the recovery phase, and application of pheromones to the offspring prior to their reunion with the dam, all proved important in increasing the survival rate.     

Generation of an active protein-tyrosine kinase from lymphocytes by proteolysis

The major NaCl-stimulated protein-tyrosine kinase activity found in soluble thymus extracts, as measured by the phosphorylation of angiotensin I, is a 40-kDa enzyme known as p40 (Zioncheck, T. F., Harrison, M. L., and Geahlen, R. L. (1986) J. Biol. Chem. 261, 15637-15643). Antibodies prepared against p40 cross-react with a 72-kDa protein-tyrosine kinase (p72) from spleen or thymus that is closely related to p40 by peptide-mapping experiments. The recovery of p40 from spleen homogenates is reduced while the recovery of p72 is enhanced by the addition of high concentrations of leupeptin or soybean trypsin inhibitor to the homogenization media. The generation of p40 in spleen homogenates occurs with a concomitant increase in protein-tyrosine kinase activity. Activated catalytic fragments of 38-43 kDa can be generated by the treatment of partially purified p72 with trypsin or papain. The p72 protein-tyrosine kinase is found at the highest levels in spleen, thymus, and lung, tissues that also have high protein-tyrosine kinase activity and generate high levels of p40 following homogenization. p72 is also found in certain T and B cell-derived cell lines and in NIH3T3 cells.     

I-A-positive nonlymphoid cells and T cell development in murine fetal thymus organ cultures: interleukin 1 circumvents the block in T cell differentiation induced by monoclonal anti-I-A antibodies

A fetal thymus organ culture system has been used to monitor the influence of interleukin 1 (IL 1) on the production of functional T cells as assessed by cell recoveries and MLC assays. We had shown earlier that the addition of monoclonal anti-I-A antibody inhibited the development of functional T cells as well as the expression of Ia on nonlymphoid cells recovered from fetal thymus organ cultures. The addition of purified recombinant IL 1 to anti-I-A-treated cultures reversed the inhibition of T cell growth induced by anti-I-A. IL 1 also induced the reexpression of Ia on the surfaces of nonlymphoid cells that could be recovered from the cultures. The "rescue" effect of IL 1 on anti-I-A-treated fetal thymus lobes was manifested in spite of the fact that the addition of IL 1 to untreated cultures had little effect on T cell development. To determine if IL 1 had a physiologic role in the development of the fetal thymus in organ culture, highly specific goat antibodies to IL 1 were added to organ cultures. These antibodies inhibited the development of T cells in organ cultures as determined by cell recovery and MLC reactivity. These results are consistent with the conclusion that IL 1 is an important mediator in the growth and development of functional T cells in the fetal thymus.     

Intravital imaging of the mouse thymus using 2-photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus. Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation. Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KI(gfp/gfp) mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.     

Thymus lipids in continuously irradiated rats.

Male Wistar rats were irradiated continuously with a daily dose of 0.19 Gy (120 days), 0.57 Gy (90 days) and 0.96 Gy (35 days) of gamma rays. An other group of rats was irradiated continuously with graded doses of gamma rays, up to total exposures ranging from 3.83-19.15 Gy. Depending on both the daily dose and total exposure, there was a decrease in phospholipid content in the thymus which correlated well with thymus weight changes. The decrease in triacylglycerol content was a less reliable sign of radiation damage. The phospholipid content reflecting the patterns of organ cellularity is a valuable indicator of the extent as well as recovery from radiation-induced injury to the thymus.     

Ultrastructural changes in the thymus of the turtle Mauremys caspica in relation to the seasonal cycle

Summary Changes in the ultrastructure of the thymus of the turtle Mauremys caspica, with special reference to its non-lymphoid components, were studied in relation to the seasonal cycle. The thymic cortex contains framework-forming epithelial-reticular cells and free macrophages, while the medulla includes, in addition, mature and presumptive pro-interdigitating cells. The ultrastructural features of these cells are generally similar to those described for non-lymphoid components of the mammalian thymus. The turtle thymus undergoes cortical involution in spring, with recovery periods in May–June and during autumn. A moderate involution occurs in winter. At the beginning of spring, cortical (but not medullary) epithelial-reticular cells show degenerative changes, probably related to high levels of circulating testosterone. In spring and autumn, mature interdigitating cells are absent, but macrophages, monocytes, and pro-interdigitating cells are found. During May–June, the cortical epithelial-reticular population recovers and macrophages, monocytes, and interdigitating cells are actively phagocytic. In summer, the epithelial-reticular cells in both cortex and medulla display normal ultrastructural features; mature and immature interdigitating cells are absent and some macrophages are detected occasionally. The results suggest that non-lymphoid components of the reptilian thymus can play a role in governing T-lymphocyte differentiation, and that the thymic cortex and medulla exhibit different cycles of seasonal activity.     

Repair processes of hemopoiesis after applying cyclophosphamide. II. Quantitative changes in basic proteins and DNA in the bone marrow, spleen, and thymus

The effect of cyclophosphamide on the organ weight, DNA and basic protein content in nuclei of bone marrow, thymus and spleen of rats was studied. Animals were examined 6, 12 and 24 hours and 3, 5, 10, 20 and 30 days after intraperitoneal application of cyclophosphamide in the doses of 100 and 200 mg X kg-1 of body weight. From the 5th day after application a marked decrease in weight and cellularity of all organs was observed. Amounts of DNA and basic proteins in nuclei of bone marrow and spleen were higher as compared with controls and their increase occurred prior to recovery of cellularity and organ weight, that was initiated within 5 and 10 days after cyclophosphamide treatment. Changes in the thymus persisted for a longer period of time and recovery was incomplete even at the day 30 after cyclophosphamide application. In accordance with these data no increase in DNA and basic protein amounts in the thymus nuclei was observed.