Antigenic similarity between stimulators of immunogenesis of a polypeptide nature from the thymus and cerebral cortex

Rabbit antisera against low molecular weight polypeptides from the thymus (thymosin and thymarin), cortex (cortexin) and white matter of the brain of the calves were cross-absorbed with these polypeptides and tested in the complement fixation test with these preparations and in the complement-dependent cytotoxicity test with thymic and bone marrow cells. The results showed that thymosin, thymarin and cortexin are antigenically similar, but differ in antigenic structure from polypeptide from white matter of the brain. Biological effect of polypeptides from the thymus and brain cortex is connected with thymus-depending lymphocytes and does not depend on B-cells. Cross absorbtion revealed that antisera against polypeptides from thymus and cortex of the brain contain antibody both against common antigens and antigens specific for appropriate preparation only. Antigenic set of polypeptide from the thymus (thymarin) corresponds more closely to thymic antigen as compared to polypeptide from the brain cortex (cortexin).     

Low-molecular homogeneous thymus fraction stimulating immunogenesis

The influence of purified acetic acid thymus extract (thymarin) and its three fractions isolated by ion-exchange chromatography upon immune response to SRBC in mice was studied. Each fraction was identified as homogeneous individual material of polypeptide nature. Crude thymus extract and one of its fractions with an approximate molecular weight of 5000 exerted a strong stimulating effect on the thymus-dependent response: formation of IgM- and IgG-antibody producing cells and on the circulating antibody level.     

Induction and long-term maintenance of Thy-1 positive T lymphocytes: Derivation from continuous bone marrow cultures

In the present study we investigated the presence of T-lymphocyte progenitors in the long-term murine bone marrow culture system described by Dexter: mature Thy-1 antigen-bearing T lymphocytes are lost in these cultures after a few days. By culturing nonadherent cells from such cultures in the presence of a supernatant of concanavalin A-stimulated spleen cells, a source of T-cell growth factor, we found that Thy-1 positive blast cells proliferated together with a second population of Thy-1 negative cells. These two populations of cells have been maintained in long-term in vitro cultures by passaging the cells in fresh conditioned medium at regular intervals. Moreover, we have been able to establish pure cultures of the Thy-1-bearing blast cells after separating them from the non-T cells using their adherence property to plastic surfaces. Long-term cultures of T lymphocytes can thus be established from long-term marrow cultures as well as from the spleen, thymus or fresh bone marrow.     

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.     

Endogenous thymic factors regulating cell proliferation and analysis of their mechanism of action.

Endogenous factors inhibiting the proliferation of T-lymphocytes were investigated which may function as modulators of T-lymphocyte production within the thymus. An extract from calf thymus (T4) enriched in lymphocyte chalone arrests rat thymocytes at the G1 leads to S boundary and in the S phase of the cell cycle in short-term cultures. It also inhibits the proliferative response of human peripheral blood lymphocytes to PHA-P in a time-dependent manner, as well as the spontaneous proliferation of in vitro cultured human chronic leukaemic lymphoblasts. This crude extract contains two active moities which can be isolated by molecular filtration on Sephadex G-75 column. A species non-specific, cell line selectivity inhibitory effect is characteristic of the high molecular weight fraction (mol. wt. greater than 40,000). This activity is resistant to moderate heat treatment and trypsin but is sensitive to mild alkaline hydrolysis and to RNase A digestion. About ten protein components and a toluidine blue positive substance can be detected by analytical polyacrylamide gel electrophoresis. The active inhibitor, a proposed protein-RNA complex, might be identical with the chalone. The low molecular weight, non-dialysable factor (T4-4) inhibits [3H]thymidine incorporation into acid insoluble DNA in a cell non-specific manner. A possible relationship between the two activities is discussed.     

Pineal control of immune status and hematological changes in blood and bone marrow of male squirrels (Funambulus pennanti) during their reproductively active phase

In addition to pineal control of reproduction in seasonal breeders, melatonin is also known to influence various immune parameters. In the present experiment, we assessed the effect of exogenous melatonin treatment on different hematological parameters of peripheral blood and bone marrow cells, together with histological observations of spleen and thymus blastogenic response and stimulation ratio, and hormonal assays (melatonin and testosterone) of Indian palm squirrel (Funambulus pennanti) during their reproductively active phase when endogenous melatonin levels are low. Daily subcutaneous injection of melatonin (25 microg/100 g body mass.) at 17.30-18.00 h to adult male squirrels for 60 consecutive days during May-June significantly increased the lymphocyte count of blood and bone marrow and the blastogenic response/percent stimulation ratio of spleen and thymus. Histological observation showed densely packed thymocytes and splenocytes. During this period, peripheral testosterone level was high and melatonin was low establishing an inverse relationship as noted earlier for this squirrel. In pinealectomized squirrels, decreased total leukocyte count and percent lymphocyte count in peripheral blood and bone marrow, along with a decreased cell density in spleen and thymus was observed histologically. Further, melatonin treatment of pinealectomized squirrels resulted in restoration of the immune parameters in line with a normal control level. We suggest that during the reproductively active period of male Indian palm squirrels the lymphoid organs were sensitive to melatonin; hence, the exogenous melatonin treatment had an immuno-enhancing effect.     

Participation of the cyclase system in the molecular mechanisms of differentiation control of immunocompetent cells

The effects of polypeptide thymic and bone marrow factors on the cyclase system of rat thymus and spleen lymphocytes were studied. It was shown that the induction of maturation of rat T-lymphocyte precursors into immunocompetent T-cells under effects of the thymic factor is accompanied by the activation of the adenylate cyclase system and the elevation of the cAMP/cGMP ratio. The observed increase in the cGMP level in splenic lymphocytes suggests the presence of cAMP- and cGMP-dependent steps of in the reaction of T-lymphocyte maturation. The bone marrow factor causes an elevation of the cAMP level only in splenic lymphocytes, which points to differences in the lymphocyte subpopulations that are sensitive to thymus and bone marrow factors. Impairments in T-lymphocyte maturation in patients with immunodeficiencies are concomitant with shifts in the isoenzyme spectrum of lactate dehydrogenase in peripheral blood lymphocytes as well as with changes in the sensitivity of the cAMP system to the thymic factor and isoproterenol. These disturbances are relieved by administration of the thymic factor. The roles of the cyclase system and polypeptide thymic and bone marrow factors in the molecular mechanisms of immunocompetent cell maturation are discussed.     

Role of protein kinase C in cyclic AMP-mediated suppression of T-lymphocyte activation following burn injury.

Major burn injury induces T-lymphocyte dysfunction. Previous studies suggest that prostaglandin (PG) E2, which is elevated post-burn, is the causative factor via a cyclic AMP-dependent process. The present study was conducted to elucidate the mechanism by which cAMP induces T-lymphocyte dysfunction following burn injury. Splenocytes were isolated from mice 7 days after receiving a scald burn covering 25% of their total body surface or sham procedure. ConA-induced proliferation by splenocytes from burned mice was significantly suppressed. Macrophage depletion of the splenocyte cultures abrogated the suppression. Concanavalin A-stimulated proliferation by macrophage-depleted splenocytes was suppressed by PGE2 and dibutyryl cAMP in both groups. The IC50 of these cAMP-elevating agents, however, was approximately 100-fold lower for cells from burned mice, indicating an increased sensitivity to cAMP. PGE2 did not suppress PMA/Ca2+ ionophore-induced T-lymphocyte activation. Addition of PMA to ConA-stimulated cultures prevented the suppression of proliferative responses by PGE2, whereas Ca2+ ionophore had no effect. Thus, the suppression of T-lymphocyte activation following burn injury is macrophage-dependent, related to an increased sensitivity to cAMP and due to an uncoupling of cell surface receptors from protein kinase C activation.     

Endogenous Thymic Factors Regulating Cell Proliferation and Analysis of Their Mechanism of Action

Endogenous factors inhibiting the proliferation of T-lymphocytes were investigated which may function as modulators of T-lymphocyte production within the thymus. an extract from calf thymus (T4) enriched in lymphocyte chalone arrests rat thymocytes at the G₁ S boundary and in the S phase of the cell cycle in short-term cultures. It also inhibits the proliferative response of human peripheral blood lymphocytes to PHA-P in a time-dependent manner, as well as the spontaneous proliferation of in vitro cultured human chronic leukaemic lymphoblasts. This crude extract contains two active moities which can be isolated by molecular filtration on Sephadex G-75 column. A species non-specific, cell line selectivity inhibitory effect is characteristic of the high molecular weight fraction (mol. wt. > 40,000). This activity is resistant to moderate heat treatment and trypsin but is sensitive to mild alkaline hydrolysis and to RNase A digestion. About ten protein components and a toluidine blue positive substance can be detected by analytical polyacrylamide gel electrophoresis. the active inhibitor, a proposed protein-RNA complex, might be identical with the chalone. The low molecular weight, non-dialysable factor (T4–4) inhibits [³H]thymidine incorporation into acid insoluble DNA in a cell non-specific manner. A possible relationship between the two activities is discussed.     

Changes in the H-alloantigen-recognizing function of mouse lymphoid cells following hydrocortisone administration]

The capacity of spleen, thymus, and bone marrow cells of intact (control) and of hydrocortisone-treated mice CBA to induce the lymph node type of graft-v-host reaction (GVHR) in hybrids F1 (CBA X c57bl) was studied. After hydrocortisone injection (2.5 mg per mouse) the donor spleen cells became more active in GVHR, considering the value of lymph node indices and immunoblast content in the regional lymph node as compared with a control group. Following transplantation of thymus cells taken from the hydrocortison-treated donors the immunoblast count was higher, although the lymph node weight remained the same as in the control group. On the contrary, following the transfer of the bone marrow cells from the hydrocortisone-treated mice the lymph nodes enlarged, while the immunoblast count remained as low as in control. Consequently, exogenously conditioned increase in the hydrocortisone level was accompanied by an enrichment of the spleen and thymus cell populations with T-lymphocytes, proliferating in response to H-alloantigens.     

An age-dependent thymic secretion modulates testicular function

The acetone extract obtained from the thymuses of 14-day-old rats contains a factor that interacts with hCG in the adult testis cells and inhibits testosterone production. Experiments were designed to investigate the possible secretion of this factor. The media from the incubation of thymuses from 14-day-old rats were processed by molecular sieve chromatography and the fractions assayed using a bioassay with testicular cells in vitro. A fraction of approx. 30 kDa was found to inhibit the hCG-stimulated production of testosterone. In addition, the influence of age on the release of the active fraction was investigated. The inhibitory effect of this thymus product was greatest in the neonatal period (1-14 days) and declined thereafter towards the onset of puberty. The age-related decline of the inhibitory activity correlated with relative thymus weight and also with the amount of protein released to the incubation media. Thymic fraction activity is, however, present in the adult gland. These results suggest that the thymus secretes active agents that are able to modulate the response of testicular cells to hCG and that their release seems to be age-related.     

Ia-positive nonlymphoid cells and T cell development in murine fetal thymus organ cultures: monoclonal anti-Ia antibodies inhibit the development of T cells

A fetal thymus organ culture system has been developed to study the differentiation of murine thymus-derived immunocompetent cells (T cells) such that cell yields can be easily monitored. This system has been used to study the effects of monoclonal anti-I-A antibodies on the growth of T cells. The addition of anti-I-A antibodies, but not anti-H2K monoclonal antibodies, to fetal thymus organ cultures resulted in a decreased yield of lymphoid cells. Anti-I-A-treated cultures did not produce cells that gave an immune response in MLC assays. Anti-I-A antibodies stained a small subpopulation of nonlymphoid cells in untreated cultures by indirect immunofluorescence that were no longer detectable in cultures that had been pretreated with anti-I-A antibody. Culture of fetal thymus lobes at low temperature (20 degrees C) for 1 wk resulted in a decrease in lymphocyte production, as well as a concomitant increase in the frequency of Ia-positive nonlymphoid cells. Co-culture of fetal liver or anti-thy-1 plus complement-treated adult bone marrow with such Ia-positive cell-enriched fetal thymus lobes at 37 degrees C resulted in the production of T cells. Anti-Thy-1.1 or -1.2 staining by indirect immunofluorescence of cells obtained from co-cultures that differed at the Thy-1 locus showed that the T cells produced were derived from the bone marrow or fetal liver. T cell production occurred in both syngeneic and allogeneic cocultures. However, if co-cultures were made by using 14-day gestation fetal thymus instead of fetal liver or bone marrow as donors of T cell precursors, T cell growth was observed only in syngeneic combinations. These results suggest that Ia-positive nonlymphoid cells play a role in the development of T cells in the fetal thymus, and that "thymus processed" T cell progenitors (but not the more immature progenitors in the fetal liver or bone marrow) are self-Ia restricted in their differentiation.     

Morphofunctional status of the thymus in guinea pigs exposed to thymus and bone marrow preparations

Study of the effect of the polypeptide thymic factor (thymalin) and the polypeptide bone marrow factor (hemalin) on the morphofunctional status of the thymus in guinea-pigs has shown that injection of thymalin into animals increases the count of small lymphocytes whereas injection of hemalin the count of medium-sized lymphocytes and lymphoblasts. Injection of thymalin magnifies the size of the nuclei of the reticuloendothelial cells.     

Capacity of thymic cells to effect target cell lysis following treatment with concanavalin A

The capacity of lymph node, thymus and bone marrow cells to mediate a cytolytic effect on target monolayers in the presence of concanavalin A was investigated. It was found that freshly prepared rat lymph node cells, but not thymus or bone marrow cells, lyse mouse fibroblasts in cultures treated with concanavalin A. A population of thymic cells did, however, manifest cytolytic capacity in the presence of concanavalin A after having been first sensitized and selected on mouse fibroblasts. Thus, sensitized thymus cells possess a lytic potency which can be activated under proper conditions.     

Factors controlling the recirculation of hematopoietic stem cells. IV. The effect of thymosin on the migration and differentiation of hematopoietic stem cells

Thymosine, a thymus hormone, restores the thymectomy induced deterioration of the routine pathways of migration and differentiation ofhemopoietic stem cells in mice. Administration of thymosine together with bone marrow cells from thymectomized mice to irradiated recipients also restores the level of migration and differentiation of hemopoietic stem cells. The inducing effect of thymosine on the maturation of T-lymphocyte precursors, which in their turn restore the usual rate of migration and differentiation of hemopoietic stem cells, has been suggested.     

Role of cyclic AMP on differentiation of T- and B-lymphocytes during the immune induction.

Spleen cell cultures from genetically thymus-deficient nude mice were restored with a T-cell replacing factor obtained from normal spleen cells of Balb/c-Ig^b mice stimulated with concanavalin A. Treatment of these cultures with an inhibitory dose of cyclic AMP did not result in reduction of the number of specific antibody-forming cells after stimulation by antigen, whereas the same treatment led to inhibition in cultures restored with normal hydrocortisone-resistant thymus lymphocytes. Further experiments lead to the conclusion that the early effect of cAMP on the immune induction seen in vitro reflects inhibition of the production or secretion of a T-cell factor which is a prerequisite for triggering B-cells with a thymus-dependent antigen.     

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.     

Suppressor T cells in thymus-reconstituted nude mice: regulation of mitogen-induced transformation

A population of glass-wool-adherent splenic cells which could suppress the response of other spleen cell populations to T-cell mitogens was isolated from thymus-reconstituted nude mice. Such adherent cells were characterized as sensitive to anti-Thy 1.2 and complement treatment. Glass-wool-adherent cells from athymic mice do not have suppressor activity to self or normal littermate NAC; however, these mice possess precursor suppressor cells as demonstrated by isolation of glass-wool-adherent T regulatory cells in thymus-grafted nude mice. Such cells are generated in either freshly obtained or in vitro cultured thymus. Evidence for suppressor T cells of host genotype was supported by their sensitivity to host-specific anti-Thy serum treatment as well as their generation in alymphoid thymus grafts. Prior anti-Thy 1.2 treatment of GAC partially removed the suppressor activity: however, macrophages and B lymphocytes were shown not to be secondary regulatory cells or suppressor mediators, thus mature T lymphocytes with low amounts of Thy 1.2 antigen may be responsible for this residual suppression. Further characterization of GAC indicates that active cell growth is required for their regulatory function, as irradiation removed the suppressor activity. This report provides evidence for the presence of a T-lymphocyte subpopulation which has a regulatory function and requires a thymus in the generation of these cells.     

Lymphokine-induced cytotoxic cell generation in thymus and spleen cell cultures

The effect of a supernatant (SN-A) obtained from PMA-stimulated IL-2 producing EL-4 cells on cytotoxic cell induction in thymocyte and splenocyte cultures was evaluated. SN-A, but not recombinant IL-2 alone, induced cytotoxic cell differentiation in thymus cell cultures, thus indicating that factors distinct from IL-2 are required for effector cell generation. INF-gamma takes part in the process, and Ia+ accessory cell presence is also strictly required in thymus cultures for lymphokine-induced cytotoxic cell generation. Cytotoxic activity in thymocyte cultures was due only to Thy 1+ Lyt 2+ cells having a broad spectrum of target cell specificity, while in spleen cell cultures an effector population with NK activity was also generated.     

Influences of clotting factors (thrombin, factor XIII) and of fibronectin on the growth of tumor cells and leukemic cells in vitro

Thrombin, factor XIII and fibronectin were incubated with cultures of mouse sarcoma cells, human cervix carcinoma cells (HeLa cells) and cells of an acute lymphoblastic leukemia. Thrombin induced a significant increase of 3H-thymidine uptake into cells with a 1,5- to 2-fold increase of cell count. The cells of an acute lymphoblastic leukemia showed a similar response to the influence of thrombin. Factor XIII in tumor cells merely induced an increase of 3H-thymidine uptake, the cell count remained constant. The cells of an acute lymphoblastic leukemia showed under the influence of factor XIII a significant increase of cell count and thymidine uptake. HeLa cell growth was optimal at low fibronectin concentrations. Fibronectin concentrations of 1 mg/ml to 3 mg/ml inhibited HeLa and mouse sarcoma cell growth.