Stabilization of double-stranded DNA molecule by nonhistone peptidic effector from calf thymus

A peptidic effector from calf thymus causes a strong stabilization of DNA doublestranded molecule in vitro. The active factor was isolated from aqueous ultrafiltered thymus extracts and purified by means of chromatography on DEAE-cellulose and then on Dowex 50 WX2. The purified thymic factor was characterized as a peptide of low molecular weight (<5000). The biological activity of the thymic factor cannot be attributed to a histone fragment. Melting data of the control DNA and of the DNA-active factor complex in various conditions of ionic strength and dielectric constant of the solution medium are recorded.     

In vitro allogeneic response of human lymphocytes dependent upon dialyzable plasma components and a thymic hormone, THF.

The in vitro response to allogeneic stimulation of human lymphocytes obtained from umbilical cord blood and from adult peripheral blood is dependent upon the presence of dialyzable plasma components. We observed here a reduced allogeneic response of human lymphocytes in the presence of a dialyzed human plasma as compared to their reactivity in the presence of whole human plasma. This impaired mixed lymphocyte culture response was restored by the addition of thymus humoral factor (THF), a calf thymus extract. It is suggested that dialysis depletes the plasma of its natural thymic hormone(s) content, this being replaced by the addition of THF. Restoration of the in vitro allogeneic response of human lymphocytes by THF was shown to be specific, since such effect was not observed when calf spleen extract or bovine serum albumin were tested.     

Effect of various thymic and nonthymic factors on in vitro antibody formation by spleen cells from nude mice

Thymosin and thymic humoral factor (THF), both prepared from whole thymus tissue, increased the in vitro anti-SRBC response of spleen cells from nu/nu mice. Lymphoid and nonlymphoid control preparations did not influence the response. Standard isolation procedures for thymosin and THF were employed to prepare factors from thymus lymphocytes (TL) separated from calf thymus and from thymus tissue enriched in thymus epithelium (TE) by preirradiation of the calves. The activity of the TE preparations was greater than that of the total thymus (TT) preparations. The TL preparations were marginally effective. However, the activity of the TE preparations could not be attributed exclusively to epithelium-derived factors, since spleen and lymph node extracts from the irradiated calves contained stimulatory material of an unknown origin which may also be present in the TE preparations.     

Maturational impairment of thymic lymphocytes in streptozotocin-induced diabetes in rats

Streptozotocin (STZ)-induced diabetes in rats was associated with marked decreases in thymus weight and the number of thymic lymphocytes. Histologically, the cortical lymphocytes which were present near the cortico-medullary junction in the thymus seemed to be reduced selectively in the STZ-induced diabetes. Rosette-forming cells, which bind to guinea pig erythrocytes in the presence of fetal calf serum, were also significantly decreased. Insulin treatment allayed these intrathymic changes. Preincubation of thymic lymphocytes from diabetic rats with thymosin fraction 5 significantly enhanced the percentage of rosette-forming cells to near the control level. These results suggest that a maturational impairment of thymus cortical lymphocytes may be caused in STZ-induced diabetes with hypoinsulinemia and it may be intimately related to reductions in thymus weight and the number of thymic lymphocytes.     

Thymostimulin: an immunohistochemical and immunological evaluation

The immunohistochemical and immunological properties of a rabbit serum raised against the calf thymic hormonal factor "Thymostimulin" were studied on mammalian and human thymuses, discussing the problem of species-specificity. The serum was also injected in chick embryos to study the possible interrelationship between avian thymus and Bursa of Fabricius.     

Low molecular weight peptides controlling transcription are present in the calf thymus chromatin structure

A calf thymus peptide fraction controlling DNA and chromatin template has been purified by DNA-cellulose and Dowex 50 WX2 chromatography and its amino acid composition determined. The active peptide fraction can be extracted in high pH buffer from calf thymus native chromatin previously deproteinized by chloroform-isamyl alcohol and phenol. These data demonstrate that the thymic peptide(s) is (are) a chromatin protein constituent strongly linked to DNA. The specificity in association of the peptide(s) to DNA has also been considered.     

Role of thymic hormone (THF) and of a thymic plasma recirculating factor (TPRF) in the modulation of human lymphocyte response to PHA and Con A.

The results presented here point to the possibility that calf thymus extracts contain, in addition to the thymic hormone (THF), a second component: thymic plasma recirculating factor (TPRF). THF, which is involved in the process of T cell maturation and has been characterized as a protein of m.w. 3000 eluted in the void volume of a G-10 Sephadex column (G-10-I), caused an increased level of intracellular cAMP in umbilical cord blood lymphocytes (UCBL). This is in agreement with our previous observation that THF plays a major role in the differentiation of T cells. The second active material, TPRF, also isolated from thymic extract, is of a molecular size below 500 and was eluted in a G-10 Sephadex column at the fourth protein peak; it seems to circulate in the blood. Previously, we had observed in impaired response of UCBL to PHA and Con A stimulation in the presence of dialyzed human plasma (DHP). Our present results indicate that this impaired response is restored exclusively by TPRF. A factor with TPRF-like activity was also isolated from the plasma of normal donors; yet it was not detected in the plasma of thymectomized patients suffering from myasthenia gravis (MG). This suggests that TPRF from plasma is thymus dependent. TPRF does not affect the level of intracellular cAMP in UCBL.     

Up-regulation of VEGF expression by NGF that enhances reparative angiogenesis during thymic regeneration in adult rat

Angiogenesis is important for adult tissue regeneration as well as normal development. Vascular endothelial growth factor (VEGF) is a unique potent angiogenic factor, and plays an essential role in regulating angiogenesis during embryonic development, normal tissue growth, and tissue regeneration. Recent evidence shows that nerve growth factor (NGF) also plays a role as an angiogenic regulator as well as a well-known neurotrophic factor. The aim of this study was to investigate whether thymus regeneration accompanies reparative angiogenesis and also to evaluate whether the thymic expression of VEGF is regulated by NGF in vivo and in vitro. Here, we show that high VEGF mRNA and protein levels are concomitant with reparative angiogenesis that occurs dramatically during regeneration following acute involution induced by cyclophosphamide (CY) in the rat thymus. Fluorescent thymus angiography using FITC-dextran showed that thymic regeneration is associated with a much denser capillary network compared with normal control thymus. Furthermore, the expressions of NGF and TrkA were highly increased during thymic regeneration. We also show that NGF mediates thymic epithelial induction of VEGF expression in vitro and in vivo. Taken together, our results suggest that NGF-mediated VEGF up-regulation in thymic epithelial cells may contribute to reparative angiogenesis during thymic regeneration in adult.     

Purification and characterization of a calf thymus protein active on lipid metabolism

1. This work concerns the purification of a calf thymus protein that increases the binding of human 125I-labeled low density lipoprotein (LDL) on both human skin fibroblasts and a special line of rat liver cells, BRL 3A. 2. It was found that the thymus gland affects cholesterol metabolism via an activation of the LDL receptor pathway. 3. Moreover, the thymus protein active on the LDL receptor pathway has a different amino acid composition and molecular weight from other well-characterized thymic peptides.     

Prospects of AIDS therapy by thymic humoral factor, a thymic hormone

Research performed in our laboratory has established that thymic humoral factor (THF), a peptide hormone isolated from calf thymus, leads to clonal expansion, differentiation and maturation of T-cell lymphocytes. THF augments the response to T-cell lectins, mixed lymphocyte reactions, graft-versus-host reactivity, antibody response to SRBC, cytotoxic responses and production of interleukin-2. Clinical data based on the use of THF of natural sources in about 200 patients indicate that it regulates differentiation of T-cell precursors, leading to normalization of the ratio between helper and suppressor/cytotoxic subsets. THF reconstitutes the defective cell-mediated immunity in patients affected by various types of neoplasms and suffering from secondary immune deficiencies, as a result of chemo and/or radiotherapy. THF-gamma 2 was purified and synthesized and found to be an octapeptide which retains all the biological activity of the thymus extracts. It is presently available for extensive clinical use.     

Rat thymic epithelial cells in vitro and in situ: characterization by immunocytochemistry and morphology

A new method for the long-term culture of pure rat thymic epithelial cells was established. The cultures were characterized by immunocytochemistry, electron microscopy and proliferation assays. Non-epithelial thymic cells were eliminated with a reliable and reproducible pre-plating method, by differential trypsin treatment of the cultures and by addition of horse serum to the culture medium instead of fetal calf serum. The final cultures contained more than 95% pure epithelial cells as evidenced by immunostaining for cytokeratin. Ultrastructural studies indicated that these cells are physiologically active epithelial cells with tonofilaments, desmosomes and filopods. The subsets of the thymic epithelial cells in vitro were investigated by comparing their staining pattern with that obtained in situ using several subtype-selective antibodies. Thymic epithelial cells in vitro showed a preferential expression of subcapsular/perivascular and medullary markers. Only few cultivated cells were of cortical origin. In the first to the fourth subcultures, some cells were immunopositive for the thymus hormone/factor thymulin. The proliferation of thymic epithelial cells was stimulated by horse serum and to a lesser extend by fetal calf serum. The adenylate cyclase activators isoproterenol and forskolin, and the glucocorticoid cortisol inhibited the proliferation. Received: 12 May 1995 / Accepted: 13 October 1995     

Thymic humoral factor gamma 2: purification and amino acid sequence of an immunoregulatory peptide from calf thymus

Thymic humoral factor gamma 2 (THF-gamma 2), an octapeptide essential for immune regulation, was purified from calf thymus. The purification of THF-gamma 2, monitored in vitro and in vivo in mouse splenocyte proliferation assays, was achieved by gel filtration of low molecular weight thymus extracts followed by ion-exchange chromatography and sequential reversed-phase high-performance liquid chromatography. The process yielded 5 micrograms of THF-gamma 2/1000 kg of thymus tissue. The concentration of THF-gamma 2 required for augmentation of lymphocyte proliferation and interleukin 2 production was 5 ng/mL in vitro and 10 ng/kg per mouse in vivo. THF-gamma 2 has the amino acid sequence Leu-Glu-Asp-Gly-Pro-Lys-Phe-Leu. The proposed structure has been confirmed because a peptide was synthesized on the basis of this sequence that showed activity identical with that of the biological molecule. It shows no homology to the amino acid sequence of other thymic hormones nor is it part of any peptide or protein of known sequence. THF-gamma 2 retains essentially all of the biological activity of the thymus extract from which it is derived.     

A putative 12 transmembrane domain cotransporter expressed in thymic cortical epithelial cells

We have isolated a full-length cDNA clone (thymic stromal origin (TSO)-1C12) from a SCID thymus library using a probe from a PCR-based subtractive library enriched for sequences from fetal thymic stromal cells. TSO-1C12 mRNA is expressed mainly in the thymic cortex and is highly enriched in SCID thymus. Expression per cell is highest during fetal thymus development and decreases after day 16. Antipeptide Abs immunoprecipitated a hydrophobic, plasma membrane glycoprotein (thymic stromal cotransporter, TSCOT) whose translated sequence has weak homology to bacterial antiporters and mammalian cation cotransporters with 12 transmembrane domains. TSCOT represents a new member of this superfamily that is highly expressed in thymic cortical epithelial cells.     

Genetic control of the immune response to collagen. III. Coordinate restriction of cellular cooperation and antigen responsiveness by thymus-directed maturation

The H-2 restriction of T helper cells from thymus-reconstituted nude mice was examined. Hybrid athymic mice were bred from BALB/c.nu and C57BL/6.nu parental strains and reconstituted with fetal thymus tissue from either parental strain. T helper cells from these mice, immunized to SRBC, were restricted to cooperation with B cells of the thymic H-2 haplotype. These T helper cells were shown to have originated from the F1 host by functional sensitivity to antisera and complement. The H-2 restriction of thymus-reconstituted F1 nude mice was further investigated by examining expression of the Ir-collagen phenotype. Results showed that the level of antibody produced in response to type I calf collagen in thymus chimeras correlates with the H-2 haplotype (high responder or low responder) of the reconstituting thymus. These experiments indicate that the thymus environment of T cell maturation influences both the H-2 restriction and Ir-phenotype of a responding immune system.     

Bioassay for thymic extracts: guinea pig spleen lymphocytes-rabbit red blood cells rosette method

An assay is described for assessing the potency of thymic extracts by the capacity of guinea pig spleen lymphocytes to form rosettes with rabbit red blood cells. Its sensitivity and reproducibility have been evaluated by statistical analysis of the data obtained testing different batches of the calf thymus extract TP-1.     

Purification of core-binding factor, a protein that binds the conserved core site in murine leukemia virus enhancers.

The Moloney murine leukemia virus causes thymic leukemias when injected into newborn mice. A major genetic determinant of the thymic disease specificity of the Moloney virus genetically maps to two protein binding sites in the Moloney virus enhancer, the leukemia virus factor b site and the adjacent core site. Point mutations introduced into either of these sites significantly shifts the disease specificity of the Moloney virus from thymic leukemia to erythroleukemia (N. A. Speck, B. Renjifo, E. Golemis, T. Frederickson, J. Hartley, and N. Hopkins, Genes Dev. 4:233-242, 1990). We have purified several polypeptides that bind to the core site in the Moloney virus enhancer. These proteins were purified from calf thymus nuclear extracts by selective pH denaturation, followed by chromatography on heparin-Sepharose, nonspecific double-stranded DNA-cellulose, and core oligonucleotide-coupled affinity columns. We have achieved greater than 13,000-fold purification of the core-binding factors (CBFs), with an overall yield of approximately 19%. Analysis of purified protein fractions by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis reveals more than 10 polypeptides. Each of the polypeptides was recovered from an SDS-polyacrylamide gel, and those in the molecular size range of 19 to 35 kDa were demonstrated to have core-binding activity. The purified CBFs were shown by DNase I footprint analyses to bind the core site in the Moloney virus enhancer specifically, and also to core motifs in the enhancers from a simian immunodeficiency virus, the immunoglobulin mu chain, and T-cell receptor gamma-chain genes.     

Localization of thymostimulin in mouse and rat thymuses

Anti-bovine thymostimulin serum reacts with reticulo-epithelial cells in the cortex and in the medulla of rat and mouse thymuses. The immuno-reaction is present in a smaller number of cells which have more pallid color than those found in calf thymus. Comparative examination as to the distribution of other thymic factors and as to species-specificity was conducted.     

Amplification of the proliferative response to alloantigen by a factor present in an extract of syngeneic thymic lymphoid cells: demonstration of optimal conditions and target cell for factor activity.

A factor extracted from syngeneic thymic lymphoid cells (thymocytes) is shown to amplify the proliferative (MLC) response of syngeneic lymphoid cells to alloantigen in vitro. The optimal conditions for an effect of the thymus factor are quantitatively defined by kinetic and dose-response studies. Other variables that could potentially influence the activity of the thymus factor, such as the presence of 2-mercaptoethanol and the source of alloantigen, are identified. Factor activity can be recovered from semi-allogeneic thymocytes, as well as syngeneic thymocytes. The factor appears to predominantly effect the proliferative response of T cells localized in peripheral lymphoid organs. As such, this factor appears to be distinct from the variety of previously described factors derived from thymic reticuloepithelial elements that are thought to primarily induce the differentiation of T cell precursors found predominantly in bone marrow. Several possible mechanisms of action of this thymocyte-derived factor are considered.     

Identification and in situ localization of the "thymic nurse cell" in man

The observation of the "thymic nurse cell" (TNC), a reticuloepithelial cell with intracytoplasmic lymphocytes, in suspension of murine thymic tissue prompted us to investigate the existence of this cell in cell suspension, as well as in tissue sections of the human thymus. TNC-like cells were enriched in suspension by enzymatic disintegration of thymic tissue and 1 X G sedimentation over 50% fetal calf serum gradients. TNC-like cells were negative for lysosomal enzymes: in this respect, as well as in light microscopic morphology, the cells were different from tissue macrophages with intracytoplasmic lymphocytes. In electron microscopy, TNC-like cells showed reticuloepithelial characteristics. In 1-micron tissue sections, clusters of lymphocytes with a possible reticuloepithelial nucleus were observed close to blood capillaries in the cortical area. Ultrastructural analysis confirmed the epithelial nature of this cell, as well as its location adjacent to blood capillaries. We concluded that there is in situ existence of TNC in man. This observation enables studies on the role of TNC in intrathymic T cell maturation.