Thymosin Fraction Research Articles

Quantitative analysis of mitogen-stimulated cellular proliferation: Effect of concanavalin A and thymosin interaction

Studies of mitogen stimulated lymphocyte proliferation have shown that considerable variation exists in the magnitude of response to specific mitogens. No standard method has been devised which would permit comparisons of the non-linear proliferation response between groups. In this study a mathematical correlation between mitogen stimulation and cellular response is defined using concanavalin A as the mitogen. A logistic equation was derived by least sum of squares regression analysis. The dose response relationship of concanavalin A stimulated peripheral blood mono-nuclear cell proliferation was natural logarithmic. A computing program was designed to accomodate any calculation related to proliferation assays. The efficacy of this program and the adequacy of the equation were confirmed after they were tested in thymosin fraction 5 treated cellular proliferation experiments. This study demonstrates a quantitative method for the comparisons of immunological proliferation response among groups.

Effects of Nutritional Recuperation on E‐Rosetting Lymphocytes and in Vitro Response to Thymosin in Malnourished Children

SummaryThe percentage of peripheral blood lymphocytes forming rosettes with sheep erythrocytes (E‐rosettes) was determined at admission to the INCAP Clinical Center in eight acutely malnourished Guatemalan children, and again after 14 and 28–30 days of nutritional therapy. While the mean percentage of E‐rosettes increased during therapy, the change (from 35.6 ± 10% to 43.3 ± 19%) did not reach statistical significance because of the variable response of different subjects. At each time period, however, in vitro incubation with the thymic factor, thymosin fraction 5, significantly increased the percentage of E‐rosetting lymphocytes. The presence of thymosin responsive cells in circulation after 1 month of optimal nutritional support indicates that immature T‐lymphocytes can persist in circulation in patients with severe malnutrition, even after clinical improvement. Thus, neither the percentage of E‐rosettes in peripheral blood nor their response to in vitro incubation with thymosin correlated with anthropometric measures of nutritional status in individual patients. This suggests that other nutritional or nonnutritional factors may be important modulating influences on T‐lymphocytes, and that prospective studies with thymic factor administration are warranted.

Immunoregulatory effects of fraction 5 thymus peptides. I. Thymosin α1 enhances while thymosin β4 suppresses the human autologous and allogeneic mixed lymphocyte reaction

Thymosin α1 and thymosin β4, two peptides isolated from preparations of calf thymus fraction 5, were tested in the human mixed lymphocyte reaction (MLR). Thymosin α1 was found capable of enhancing both the allogeneic and autologous MLR. On the contrary, thymosin β4 suppressed MLR proliferative responses. Study of the responses of the T cell subpopulations revealed that T4+ (helper/inducer) cells but not T8+ (suppressor/cytotoxic) are responsible for the enhanced, proliferative response to allo- and auto-antigens in the presence of thymosin α1. Both the autologous and the allogeneic proliferative responses of either T4+ cells or T8+ cells were not influenced by the addition of thynosin β4 in the cultures. However, when T4+ and T8+ subsets were cocultured, thymosin β4 was capable of activating T8+ cells to suppress the allogeneic and the autologous proliferative response of T4+ cells. These studies show that thymosin fraction 5 peptides exert immunoregulatory effects on the human MLR proliferative responses in vitro.

Immature circulating lymphocytes in severely malnourished Guatemalan children.

The percentage of peripheral blood lymphocytes forming rosettes with sheep erythrocytes (E-rosettes) was determined in 33 severely malnourished Guatemalan children, and in two groups of clinically well but mildly growth retarded children from the same environment. Mean E-rosettes in the acutely ill patients was lower than the value observed in the mildly malnourished children, although there was considerable overlap between groups. These data differ from previously published studies of severely malnourished children from other parts of the world in that not all patients had decreased values for E-rosettes, in contrast to the uniform depression reported by others. As all patients were clinically similar, the results suggest that there may be specific nutrient defects associated with protein-energy malnutrition that particularly affect immune function. In addition, in vitro incubation of lymphocytes from the acutely malnourished children with the thymic factor, thymosin fraction 5, increased the percentage of E-rosettes in a dose-dependent fashion. These data suggest that immature, thymosin-responsive T cells are present in circulation. It is possible that in vivo thymosin administration may be beneficial for malnourished individuals.

Immature Circulating Lymphocytes In Severely Malnourished Guatemalan Children

SummaryThe percentage of peripheral blood lymphocytes forming rosettes with sheep erythrocytes (E‐rosettes) was determined in 33 severely malnourished Guatemalan children, and in two groups of clinically well but mildly growth retarded children from the same environment. Mean E‐rosettes in the acutely ill patients was lower than the value observed in the mildly malnourished children, although there was considerable overlap between groups. These data differ from previously published studies of severely malnourished children from other parts of the world in that not all patients had decreased values for E‐rosettes, in contrast to the uniform depression reported by others. As all patients were clinically similar, the results suggest that there may be specific nutrient defects associated with protein‐energy malnutrition that particularly affect immune function. In addition, in vitro incubation of lymphocytes from the acutely malnourished children with the thymic factor, thymosin fraction 5, increased the percentage of E‐rosettes in a dose‐dependent fashion. These data suggest that immature, thymosin‐responsive T cells are present in circulation. It is possible that in vivo thymosin administration may be beneficial for malnourished individuals.

Localization of Thymosin α1Production to Thymus Medullary Epithelial Cells by Use of Monoclonal Antibodies

The thymus is an endocrine organ which modulates T-cell immunity through the production of protein like peptides such as the thymosins. Thymosin alpha 1 was the first biologically active peptide isolated and sequenced from the partially purified thymic preparation, thymosin fraction 5, and has been extensively studied. Using synthetic Thymosin alpha 1, a heterologous rabbit antiserum has been raised and a radioimmunoassay has been developed. Although thymosin alpha 1 antibodies have been used in several histological studies, their use is limited by potential nonspecific cross-reactivities, unpredictable heterogenicity, variable affinities, and a limited unstandardized supply. In the studies, reported here, eight anti-thymosin alpha 1 monoclonal antibodies (MAbs) were produced by somatic cell fusion between spleen cells from immunized BALB/c mice and P3x64 Ag8.653 myeloma cells. The MAbs were screened for anti-thymosin alpha 1 specificity in a solid phase ELISA and a liquid phase RIA. Only those clones which secreted specific antibody as detected by both procedures were characterized for their heavy chain class and epitope specificity. The anti-thymosin alpha 1 monoclonal antibodies were then used for indirect immune fluorescence studies of perfused rat thymus. Thymosin alpha 1 containing cells were found primarily in the thymic medulla, confirming previous studies using the heterologous antisera. These studies demonstrated the specificity of the anti-thymosin alpha 1 monoclonal antibodies for immunochemical studies of intra- and extra-thymic localization of thymosin alpha 1. They also provide an important reagent for biological studies of the role of thymosin alpha 1, in vitro and in vivo.

Reduction in Responsiveness of Thymocytes to Phytohemagglutinin During Bladder Tumor Induction and Restorative Effects of Thymosin Fraction 5 in Rats

Urinary bladder tumors were induced in female Wistar rats by oral administration of N-butyl-N-(4-hydroxybutyl) nitrosamine [(BBN) CAS: 3817-11-6]. Hypertrophy or vascular formation was observed in the bladder membrane 8 weeks after BBN administration, and tumors became visible at the 15th week. At the 20th week, approximately 95% of these rats had bladder tumors. The response of thymus lymphocytes to phytohemagglutinin (PHA) in these BBN-treated rats began to decrease from the 10th week and decreased significantly from the 18th week as compared with that in untreated rats. This reduced response to PHA was slower in comparison with the reduced response to concanavalin A. When thymus lymphocytes were preincubated with thymosin fraction 5 in vitro, the response to PHA improved between the 8th week and the 20th week. Furthermore, when the thymus small lymphocyte population was separated into 3 subpopulations by discontinuous density gradient centrifugation and adhesion column, the restorative effect of the thymus hormonal product was greater in the intermediate and heavier subpopulations.

Thymosin fraction 5 stimulates secretion of immunoreactive beta-endorphin in mouse corticotropic tumor cells.

In addition to reconstituting immune competence, the thymus gland preparation, thymosin fraction 5 (TSN-5), has recently been shown to stimulate secretion of hormones from the hypothalamic-pituitary adrenal axis in vivo and from pituitary corticotropes in vitro. The purpose of the present study was to investigate the effects of TSN-5 on secretion of immunoreactive beta-endorphin (i beta-E) by mouse corticotropic tumor cells. The release of i beta-E by AtT-20 pituitary tumor cells was increased in a dose-dependent manner by concentrations of 30-600 micrograms/ml of TSN-5, whereas concentrations greater than 1,000 micrograms/ml were increasingly less effective in stimulating secretion. TSN-5 (600 micrograms/ml) significantly stimulated i beta-E release within 7 min; maximal secretory responses (up to 275% of control release) occurred by 4 hr. The secretory response of AtT-20 cells to 600 micrograms/ml TSN-5 (37.9 +/- 2.0 vs. 16.1 +/- 1.0 ng i beta-E/ml/4 hr, mean +/- SE) was similar in magnitude to release evoked by 0.1 microM corticotropin-releasing factor (CRF). Combining TSN-5 and CRF treatments increased secretion of i beta-E to nearly 600% of control levels, an effect greater than an additive influence of the two independent treatments. Whereas CRF treatment reduced the levels of i beta-E in AtT-20 cell extracts after 24-hr treatment by 45% (231.8 +/- 24.7 vs. 417.2 +/- 17.8 ng i beta-E/mg protein, CRF vs. vehicle treatments, respectively), TSN-5 did not significantly alter cellular hormone content. Neither TSN-alpha 1 nor TSN-beta 4, two of the component peptides of TSN-5, affected basal or CRF-stimulated release of i beta-E, indicating that an unidentified constituent(s) is corticotropic.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro immunomodulatory effects of interleukin-2 and thymosin fraction V in acquired immune deficiency syndrome

A monoclonal antibody (anti-Tac) that appears to bind the receptor for interleukin-2 (IL-2) was used to quantitate lymphocytes that express IL-2 receptors (IL-2R) in response to phytohemagglutinin (PHA) stimulation. 62 ± 4% of the cells expressed IL-R2 in response to PHA in twelve normal subjects compared to 22 ± 4% in fourteen patients with acquired deficiency syndrome (AIDS) ( P<0.001) and 50 ± 6% in six patients with AIDS-related complex ( P<0.1). There was no effect on IL-2R expression, when lymphocytes from seven controls were incubated with IL-2 (20u/ml) or thymosin fraction V (10u/ml) for 72 h. However, when the lymphocytes from seven patients with AIDS were incubated with IL-2, the IL-2R rose from 18 ± 3% to 31 ± 3% ( P<0.005) and with a fraction V to 29% ± 3% ( p< 0.0001). In addition, IL-2 augmented the PHA-induced proliferative responses in patients with AIDS-related complex and AIDS and normal controls, whereas thymosin fraction V had no significant effect. Thymosin fraction V also enhanced the IL-2 production of PHA-stimulated mononuclear cells obtained from six pateints with AIDS and six normal controls. These results suggest that both IL-2 and thymosin fraction V can modulate in vitro T-cell function in patients with AIDS.

Thymosin fraction 5 stimulates the release of prolactin from cultured GH 3 cells

Thymosin fraction 5, a bovine thymus preparation, has recently been implicated in the regulation of neuroendocrine function. The purpose of the present study was to investigate the effects of thymosin fraction 5 treatment upon the GH 3 rat pituitary cell line. Thymosin fraction 5 stimulated prolactin (PRL) release from these cells in a dose and time dependent manner. These results suggest that a product of the endocrine thymus may regulate the release of PRL.

Effect of two thymosin fraction 5 polypeptides on human peripheral blood lymphocytes.

Thymosin fraction 5 polypeptides beta 4 and alpha 1 were tested for their ability to affect certain immunological parameters of human peripheral blood lymphocytes (PBL). PBL were cultured with various concentrations of the peptides for 24 hours. Thymosin beta 4 was found to induce a significant decrease in the expression of the Fc alpha receptors of PBL, as well as in their ability to express antibody dependent cellular cytotoxic (ADCC) activity. In addition, this peptide had the ability to increase the percentage of T4 lymphocytes in normal and immunosuppressed donors and to decrease the percentage of T8 positive cells in normal donors. Finally, beta 4 peptide caused a small increase in the capacity of peripheral blood lymphocytes to form sheep red blood cell (SRBC) rosettes (ER). In parallel experiments thymosin alpha 1 was found inactive. The results presented here indicate that thymosin beta 4 may be used as an immunoregulatory molecule in patients with immunodeficiencies.

膀胱癌誘発剤投与ラットの胸腺リンパ球に関する研究

実験的膀胱癌誘発剤であるN-butyl-N- (4-hydroxybutyl) nitrosamine (以下BBN) の0.025%水溶液をラットに経口投与し, 以後, 発癌に至るまでの胸腺リンパ球群の one-way mixed lymphocyte reaction (以下MLR) 系における応答能の変化を経時的に検討するとともに, 胸腺リンパ球の分化・成熟に関与していると考えられる胸腺ホルモン様物質 (thymosin fraction 5, 以下TF5) を仔牛胸腺より抽出し, その実験系に対する影響について検討した. また, 浸潤圧とpHを生理的に調整したアラビアゴム溶液を分離液とする不連続性密度勾配遠心分離法と粘着カラム法を併用して, 実験群および対照群 (BBN非投与群) のラット胸腺から3種のリンパ球サブセット〔lighter small lymhocytes (LSL), intermediate small lymphocytes (ISL), heavier small lymphocytes (HSL)〕を分離し, 各サブセットについて同様に経時的変化及びTF5の影響を検討した.1) 全胸腺小リンパ球 (whole small lymphocytes, 以下WSL) のMLR応答能は, 膀胱粘膜に肥厚や血管増生などの肉眼的変化を認めるBBM投与10週ごろから低下しはじめ, 95%以上に腫瘍を観察する20週ごろより著明に低下した.2) 胸腺小リンパ球中のMLRに応答する細胞は, HSLサブセットにより多く分離され, BBN投与に伴うMLR応答能の経時的な低下傾向は, HSLサブセットにおいて顕著であった.3) TF5でWSLを前処置すると, BBN投与10週～20週において低下したMLR応答能の修復が認められたが, 95%以上に腫瘍を観察する20週以後では, その効果は著明には認められなかった.4) BBN投与により低下したMLR応答能のTF5よる修復は, HSLサブセットでもっとも顕著であった.5) 以上の結果より, BBN投与ラット胸腺リンパ球の分化・成熟機構の乱れは, 胸腺皮質リンパ球のみならず, 胸腺髄質リンパ球においても生じているようであり, さらにMLR応答能賦与に関する胸腺ホルモン様物質の分泌異常が想定された.

Suppressive effect of thymosin fraction 5 on proliferation of cultured human T lymphocytes

The effects of thymosin fraction 5 (F5), an extract of bovine thymus containing multiple polypeptides, on the proliferation of cultured T cells (CTC), a continuously proliferating subpopulation of peripheral blood T lymphocytes, stimulated by either phytohemagglutinin (PHA) or delectinated interleukin 2 (IL-2) were studied. Addition of F5 to cultures significantly and consistently inhibited CTC responsiveness to PHA, with the degree of inhibition being greater using a suboptimal concentration of mitogen. F5 did not significantly or consistently inhibit CTC proliferation induced by IL-2. These studies suggest that the suppressive effect of F5 may be primarily mediated by decreased IL-2 production instead of effects on IL-2 activity or efficiency in stimulating CTC proliferation. Since prostaglandins inhibit the proliferation of CTC in response to PHA or IL-2 (R.D. Maca (1983) Immunopharmacology 6:267), studies were undertaken to determine if the observed inhibition was mediated by effects of F5 on prostaglandin E2 (PGE2). The inhibitory effect of F5 on PHA responsiveness of CTC was not affected by the addition of indomethacin indicating that suppression by F5 is not mediated by stimulating the production or release of cyclooxygenase-derived prostaglandins, such as PGE2. Furthermore, PGE2 could not be detected in supernatants of F5-treated CTC stimulated by PHA. When PGE2 was added to F5-treated CTC cultures, the PHA response was inhibited indicating that the suppressive effects of F5 and PGE2 were additive and that F5 did not modulate the sensitivity of CTC to PGE2. These studies further demonstrate that CTC, being free of monocytes and B lymphocytes, provide a useful assay system for evaluation of the effects of F5 and other immune modulators on the function of T lymphocytes or T lymphocyte subpopulations.

Neuroendocrine regulation of thymus hormones: Hypothalamic dependence of “facteur thymique serique” level

The influence of the hypothalamus extract (HE) on the blood level of the “facteur thymique serique” (FTS) was studied. Hypothalami collected from young mice were pooled, homogenated in saline, and centrifuged; finally, the supernatant injected in old or thymectomized mice with no detectable levels of FTS. In the old mice the treatment results in a reappearance of this circulating thymic hormone; in contrast, in adult thymectomized animals, the HE injection was not able to induce FTS activity. When HE donors were pretreated with thymosin fraction 3, known to contain FTS among others active peptides, the capacity of such a hypothalamus preparation to induce reappearance of FTS in old animals is greatly diminished. The data presented here suggest that the capacity of the thymus to secrete FTS depends on a hypothalamic factor, and therefore the absence of this thymic hormone in the aged reflects a failure of the thymus linked to its impaired neurologic control. On the other hand it seems evident that a feedback system operates in order to regulate the release of this hypothalamus stimulatory factor.

Circulating thymulin and thymosin-α 1 activity in pediatric acquired immune deficiency syndrome: In vivo and in vitro studies

Twenty-five children with acquired immune deficiency syndrome (AIDS) or AIDS-related complex had a characteristic pattern of T cell deficiency. Abnormally low plasma thymulin levels preceded the development of peripheral blood T cell abnormalities. In contrast to patients with congenital T cell deficiencies, our patients had elevated serum levels of thymosin-alpha 1. Treatment with thymosin fraction 5 in three children with AIDS resulted in only transient clinical and immunologic improvement.

Modulation of interleukin 2 receptor expression on normal human lymphocytes by thymic hormones.

The expression of interleukin 2 receptors (IL-2R) is a critical step leading to normal lymphocyte proliferation. Since thymosin fraction 5 (TF5), a thymic hormone preparation, enhances lymphoproliferative responses of human cells, we examined the effects of TF5 on the expression of IL-2R on mitogen-stimulated human lymphocytes. TF5 significantly increased the percentage and antigen density of cells expressing IL-2R after stimulation with an optimal concentration of phytohemagglutinin (PHA) when the cells from the same donor exhibited suboptimal responses to PHA alone. The same effect was observed with a suboptimal PHA concentration and with OKT3 monoclonal antibody stimulation. Thymosin alpha 1, a synthetic polypeptide originally isolated in its native form from TF5, was also able to increase IL-2R expression in response to PHA, suggesting that it is the active species in TF5. The enhancement of IL-2R expression was paralleled by increased proliferative responses. Increased IL-2R expression appears to be the direct effect of thymic hormones, since abrogation of interleukin 2 production by cyclosporin A did not affect TF5-mediated enhancement of PHA-induced IL-2R expression. These results point to a physiological role of thymic hormones in the maintenance of normal levels of IL-2R expression. This immunoregulatory activity of thymic hormones might be relevant in the treatment of conditions where there is decreased IL-2R expression, such as the acquired immune-deficiency syndrome, or in the restoration of normal IL-2R expression to lymphocytes from aged individuals.

Marked reduction in percentage of rosette-forming thymocytes of rats during induction of bladder tumors and restorative effect of thymosin in vitro.

Urinary bladder carcinoma was induced in W rats by oral administration of N-butyl-N-(4-hydroxybutyl) nitrosamine [(BBN) CAS: 3817-11-6], and rosette formation of the thymus lymphocytes with Hartley strain guinea pig erythrocytes was studied until the 20th week after BBN administration. Percentage of rosette-forming cells (RFC's) began to decrease about the 8th week (stage of hypertrophy or vascular formation in the bladder wall) and remarkably declined around the 12th week (before the incidence of carcinoma) after the administration. However, no significant reduction of RFC percentage was noted in the control (without BBN) group during the observation period. Pretreatment of thymic lymphocytes from BBN-treated rats with thymosin fraction 5 (TF5), an extract from calf thymus, significantly enhanced the percentage of RFC near to that of the control level. Two (#9 and 13) subfractions separated from TF5 by high-performance liquid chromatography were found to be more effective in recovering the percentage of RFC. These results show that a maturational impairment of thymus lymphocytes may be caused from precancerous stage in BBN-administered rats and that this impairment would be restored by the thymus products in vitro.

Production and characterization of a bovine T cell-specific monoclonal antibody identifying a mature differentiation antigen.

A monoclonal antibody (MAb), BLT-1, with specificity for bovine mature T cells was prepared by somatic cell hybridization of myeloma NS-1 and spleen cells from BALB/c mice hyperimmunized with bovine T lymphocytes. The MAb reacted with over 92% of nylon wool-nonadherent lymphocytes (T cells) but not with nylon wool-adherent EAC-positive lymphocytes (B cells) in the indirect immunofluorescence assay. It is an IgM, with kappa-light chains, which fixed complement well and killed over 95% of mature T cells in complement-mediated cytotoxicity assays. It reacted with the same proportions of peripheral lymphoid cells (peripheral blood, lymph nodes, and spleen) as the polyclonal goat anti-bovine thymocyte serum (GABTS), but only with 25% of GABTS-positive thymocytes. Immunoperoxidase staining of frozen tissue sections showed that the BLT-1-positive cells were located in the medulla of the thymus and in the T lymphocyte areas of lymph nodes. Western immunoblotting assays showed that the BLT-1-reactive membrane antigen is a 22,000 m.w. protein which was inducible in bovine thymocytes with bovine thymic hormones, thymosin fraction 5, thymosin alpha 1, and thymopentin ORF-18150, indicating that it is a mature T lymphocyte differentiation antigen. The thymosin alpha 1 and thymopentin were found to show additive effects on mature T cell antigen expression by bovine thymocytes.

Thymosin fraction 5: Effects on T cell functions in mice immunosuppressed by severe dietary protein deficiency

The present studies were performed to determine the effects of severe protein deficiency and subsequent injection of thymosin fraction 5 (TF5) on T and B cell functions. BALB/c mice, 4 weeks old, were fed a normal protein (21%), a low protein (4%) or a protein free (0%) diet and then injected with TF5 or buffer (PBS). A significant increase was observed in the PHA (phytohemagglutinin) and LPS (lipopolysaccharide) induced mitogenesis with increasing age of the well-nourished, PBS injected animals. The severely protein malnourished mice, PBS injected and the well nourished mice, injected with TF5 had smaller increases in both B and T cell mitogenesis with increasing age. TF5 injection of the malnourished mice increased PHA and LPS mitogenesis nearly to the levels of the well-nourished mice. The protein malnourished mice consistently had higher serum corticosteroid levels than controls. No changes in serum corticosteroids were observed with TF5 injection of controls, but there was a significant decrease in the corticosteroid levels of the severely malnourished with TF5 injection. Cytoxicity assays of T cell function, antibody dependent cellular cytoxicity and cytoxicity to mouse thymona tumor cells, in mice fed moderately protein deficient diets showed suppression compared to controls fed 20% protein. TF5 injection partially and temporarily increased these functions in the malnourished mice.

Thymosin effects on immunoglobulin synthesis and suppressor T cell activity in normal subjects and patients with primary biliary cirrhosis

Peripheral blood lymphocytes from patients with primary biliary cirrhosis previously have been reported to demonstrate reduced pokeweed mitogen-stimulated immunoglobulin synthesis and diminished function of suppressor T cells. To determine whether thymic hormone preparations reverse these immunologic defects in vitro, the effects of thymosin fraction 5 and thymosin α 1 on immunoglobulin synthesis and concanavalin A-induced suppression of immunoglobulin synthesis were investigated in normal subjects and patients with primary biliary cirrhosis. In normal subjects, no effects of thymosin were observed on unstimulated and pokeweed mitogen-stimulated immunoglobulin synthesis, nor on Con A-induced suppressor cell activity. Lymphocytes from patients with PBC synthesized less IgG and IgM than normals when stimulated by pokeweed mitogen, and this difference was enhanced by both thymosin fraction 5 and thymosin α 1. Con A suppression of immunoglobulin synthesis was abnormal in only one PBC subject so that thymosin effects on impaired suppressor T cell activity could not be tested.