Thymosin Fraction Research Articles

Aspirin and thymosin increase interleukin-2 and interferon-γ production by human peripheral blood lymphocytes

Interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) have recently been added to the arsenal of synthetic biological response modifiers with important immunomodulatory activities. In this paper we have assessed the effects of acetylsalicylic acid (aspirin), thymosin alpha and thymosin fraction 5 (TF5), a partially purified calf thymic preparation, on production of IFN-gamma in vitro. Stimulation by oral aspirin of IL-2 and IFN-gamma production by peripheral blood lymphocytes (PBLs) was also studied in healthy human volunteers. Aspirin, thymosin alpha 1 and TF5 were all observed to enhance phytohemagglutinin (PHA)-stimulated production of IFN-gamma. Peak IFN-gamma production by PHA-stimulated PBLs was observed after 24 h of incubation with TF5 and after 72 h with aspirin. Stimulation by aspirin and TF5 required the presence of macrophages, and was additive and dose-dependent. The additive effects of aspirin and TF5 suggest that these agents act by different mechanisms. Oral administration of aspirin in normal volunteers significantly enhanced production of both IFN-gamma and IL-2. PHA-stimulated IFN-gamma production was greatest 24 h after aspirin ingestion; in contrast, IL-2 production was optimal 10 h after aspirin ingestion. These observations suggest that oral aspirin is an effective biological response modifier in humans and raise the possibility of a novel combination approach to immunomodulation involving cyclooxygenase inhibitors and thymosins.

Rapid Micro Isolation of Thymosin α1from Thymosin Fraction 5 by Reversed-Phase High Performance Liquid Chromatography

Abstract We have developed a rapid, efficient, and reproducible method for the purification of thymosin α1 (Tα1) from thymosin fraction 5 (TF5). This procedure can serve as a model for isolation of other biologically active peptides from TF5 in sufficient quantity for characterization. The purification procedure is based on the use of high-performance preparative/semi-preparative and analytical reversed-phase (C18 Delta-Pak) chromatographic columns. The HPLC retention time, pI, RIA, SDS-PAGE, and amino acid composition analysis have shown that natural, purified Tα1 is identical to synthetic α1.

Characterization of the immunoregulatory properties of thymosin alpha 1 on interleukin-2 production and interleukin-2 receptor expression in normal human lymphocytes

Thymosin alpha 1 (T α 1) and thymosin fraction 5 (TF5) have been shown to induce lymphocyte maturation and differentiation as well as to modulate mature immune responses to antigens and mitogens. The present study focused on the characterization of the mechanisms involved in Tα 1 and TF5 enhancement of phytohemagglutinin (PHA)-induced interleukin-2 (IL-2) secretion and interleukin-2 receptor (IL-2R) expression in human mononuclear cells. We provide evidence that TF5 and Tα 1 modulate an early event(s) during lymphocyte activation by mitogens. A short preincubation period (30 min) of non-adherent cells with thymosins, followed by extensive washing and subsequent exposure to PHA, was sufficient to enhance the production of IL-2 and the expression of IL-2R induced by the mitogen. Furthermore, the concomitant addition of PHA and thymosin during the preincubation period is not necessary for the enhancing effects to occur. We have also studied the role of macrophages on thymosin modulation of these responses. Results presented here indicate that macrophages are not essential for the interaction of thymosins with T-cells. However, macrophages are an absolute requirement during the exposure to the mitogen after preincubation with thymosins for the manifestation of TF5- and Tα 1-mediated enhancing effects on IL-2 production and IL-2R expression. Human recombinant interleukin-1 beta (rIL-1β) was able to replace this macrophage requirement, indicating that production of IL-1 by these cells is a critical event in thymosin modulation of the IL-2 system. Two-color flow cytometric analysis and experiments involving the use of highly purified helper/ inducer (Th, CD4 +) and cytotoxic/suppressor (Tc, CD8 +) T-cell populations indicated that both, Th and Tc cell populations are targets of thymosin activity. These studies provide additional evidence that thymosins play an important role in the modulation of the normal immune response and begin to define the mechanisms underlying Tα 1 immunoregulatory properties.

Thymosin, interleukins, isoprinosine and imuthiol do not reconstitute T-cells in athymic nude mice

Previous reports suggest that immunotherapy can induce T-cell development in athymic nude mice. Eight-week-old BALB/c nu/nu (athymic nude) mice were treated for 3, 6 and 12 weeks with thymosin fraction 5 (TF-5), buffy coat interleukins (BC-IL), recombinant IL-2 (rIL-2), a combination of TF-5 and BC-IL, isoprinosine or imuthiol. Control animals were treated with sterile saline or were given a syngeneic thymus graft. Spleen weight, cell yields and Thy 1.2 + T-cell markers were monitored and spleen cell proliferative responses to rIL-2, concanavalin A (Con A), Con A + rIL-2, phytohemagglutinin (PHA) and endotoxin (LPS) were assessed. Only mice transplanted with a syngeneic thymus showed progressive increases in both T-cell number and function. Minor changes in proliferative responses were noted at 3 weeks with all treatments; however, no biologically significant reconstitution of T-cell number or function was observed.

Randomized trial of combined modality therapy with and without thymosin fraction V in the treatment of small cell lung cancer : Scher H.I. Shank B. Chapman R et al. Solid Tumor, Immunology and Developmental Chemotherapy Services, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY 10021. Cancer Res 1988;48:1663-70

A randomized trial of thymosin fraction V (60 mg/m2 s.c. twice weekly) given during induction chemotherapy and radiation therapy was performed in 91 patients with small cell carcinoma of the lung. Induction chemotherapy consisted of four cycles of an alternating combination of drugs (cyclophosphamide/Adriamycin/vincristine and cisplatin/etoposide). Radiation to the primary complex was given to patients with limited disease. All patients received prophylactic cranial irradiation. There were 35 patients with limited disease (18 randomized to thymosin and 17 to no thymosin) and 56 with extensive disease (28 thymosin and 28 no thymosin). Pretreatment immunological parameters were comparable between the two groups. For limited disease patients the overall response rate was 100%, including 66% (21 of 32) complete responders. The median duration of response was 19 mo (range, 5-57 mo) and survival 21 mo (range, 4 days to 57 mo). The 3-yr survival was 32%. For ED patients the overall response rate was 95% with 29% (13 of 48) complete. The median duration of response was 10 mo and the median duration of survival 12 mo with 13% alive at 2 yr. A comparison of the thymosin-versus no thymosin-treated patients revealed no difference in response rate, response duration, or survival whether analyzed as a whole or by extent of disease. An analysis based on pretreatment immune function and total white blood cell and absolute lymphocyte count revealed no difference in the survival distributions. No differences in the pattern of toxicity were observed between the thymosin- versus no thymosin-treated patients. The addition of thymosin fraction V during induction chemotherapy and consolidation radiotherapy did not alter outcome.

In vivo effects of thymic epithelial culture supernate and thymosin fraction 5 on ADP-induced thromboembolic respiratory depression in mice

In vivo effects of thymic epithelial culture supernate and thymosin fraction 5 on ADP-induced thromboembolic respiratory depression in mice

In vitro effect of methionine-enkephalin and thymosin on murine expression of Thyl-2 antigen

The present study investigated the in vitro effect of methionine-enkephalin (Met-Enk) on Thyl-2 antigen density and the interaction between this opioid peptide and a thymic hormone (thymosin fraction V (TFV)). In vitro, it was found that Met-Enk at various concentrations (28 · 10−11, 28 · 10−10, 28 · 10−9 and 28 · 10−8 M ) had an increased positive effect on the Thyl-2 antigen density of Balb/C mouse thymocytes. This action was observed at all concentrations of Met-Enk. The addition of TFV to Met-Enk thymocyte cultures showed an additive effect of these two hormones. The physiological significance of these observations is that they indicate an interaction between immune and endocrine system functions.

The effects on complement component 3 of dietary variation of protein, fat and vitamin E during growth of young mice

Some studies have shown that nutrition lowers overall immunity to disease. To further elucidate the role of nutrition on immunity, we have investigated the effect on serum Complement C3 by three nutrients, protein, lipid and vitamin E, in BALB/c mice for up to 24 weeks. The group with high dietary vitamin E had slightly higher C3 for the first four months but by the 22nd week was lower than control. The groups receiving high or low protein had, respectively, high or low C3. Injection of thymosin fraction 5 increased the C3, suppressed by the low protein diet. The group on the high lipid diet had high C3 until the 14th week whereupon it declined to control level by the 22nd week. The low lipid diet resulted in low C3 during the early weeks but increased to control level by the 22nd week. We conclude that diet is important to complement mediated immunity of young mice. Protein calorie malnutrition (PCM) lowers overall immunity to disease (1, 2). The increased incidence of serious gram negative infections including sepsis, of defective inflammatory responses, and of decreased opsonizing activity in serum of PCM patients parallels the symptoms of people with congenital (3) or nutritionally (1) associated deficiency of complement. Complement C 3, a crucial intersection point where the paths of nonspecific immunity and antibody mediated immunity join on the way to destroying foreign organisms, is susceptible to: general nutritional status (4), deficiencies of calcium and magnesium (5), or high levels of lead (6). To further elucidate the role of nutrition on immunity, we have investigated the effect of three nutrients, protein, lipid and vitamin E, on serum C 3 levels in mice for up to 24 weeks. Because overnutrition can also affect immunity (7), we have studied both high and low nutrient levels. A part of protein deficiency is the damage to the thymus and thymus gland hormone production. Therefore the thymic factors on complement production during protein malnutrition is measured.

Eine neue Synthese von Thymosin α1

A New Synthesis of Thymosin α1Thymosin α1, an N‐acetylated octacosa thymus peptide, isolated first from thymosin fraction 5, is synthesized by the solid phase method using the p‐benzyloxybenzyl alcohol/polystyrene/divinylbenzene resin, Nα ‐Fmoc‐amino acids and those with tert‐butyl or Boc side chain protection. All couplings are performed with the Bop reagent. The peptide is purified by a combination of gel chromatography and preparative HPLC, its purity checked by amino acid analysis and analytical HPLC, and the biological activity tested by the E‐rosette assay.

Effect of thymosin (fraction 5) on testicular endocrine function in mice

O. P. Balezina, M. A. Pan.v, M. A. Poskonova , and A. Z. Khanazadyan, Zh. Obshch. Biol., 38, 603 (1977). B. M. Gekht and N. I. ll'ina, Neuromuscular Diseases [in Russian], 230, 613 (1973). C. C. Chang, T. Chen, and S. T. Chuang, J. Physiol. (London), 230, 6i3 (1973). A. G. Engel, E. H. Lambert, and T. Santa, Neurology (Minneapolis) , 23, 1273 (1973). D. M. Fisher, M. D. Cronnelly, R. D. Miller, and H. Sharma, Anesthesiology, 59, 220 (1983). J. McLaughlin and M. Bosman, Exp. Neurol., 52, 263 (1976). M. D. Ward, M. S. Forbes, akd T. R. Johns, Arch. Neurol. (Chicago), 32, 808 (1975).

Production of human B and T cell growth factors is enhanced by thymic hormones.

The thymic preparations thymosin fraction 5 (TF5) and synthetic thymosin alpha 1 (T alpha 1) were examined for their ability to enhance growth factor production by human peripheral blood mononuclear cells (PBMC). The results showed that both TF5 and T alpha 1 were capable of enhancing the production of a B cell growth factor (BCGF-12kD) and T cell growth factor (TCGF; IL-2). Enhancement by T alpha 1 could be obtained at 100-200-fold lower concentrations than that seen with TF5. In contrast, no enhancement of growth factor production was obtained with control preparations of non-thymic tissue extracts at any concentrations used. It was observed that stimulation of BCGF-12kD and IL-2 was most significantly obtained when the PBMC were activated with lectin. Furthermore, no direct effect of thymic hormones on test B and T cells was observed. These observations provide the first direct evidence that production of B cell growth factors can be enhanced by thymic hormones. In addition, these studies suggest that thymic hormones may regulate B cell responses by acting on mature activated T lymphocytes.

Thymosin fraction 5 (TF5) stimulates secretion of adrenocorticotropic hormone (ACTH) from cultured rat pituitaries

In vivo administration of a partially purified thymic hormone-containing extract of the thymus gland, TF5, causes an increase in serum glucocorticoids. The lack of a direct effect of TF5 on adrenal corticosterone secretion suggests that it is mediated at the level of the pituitary. Cultured rat pituitary monolayers were used to determine if the effect is mediated by stimulation of ACTH secretion from the pituitary. Two lots of TF5, BPP100 and C114080-01, caused a dose dependent secretion of ACTH from cultured pituitary monolayers. There was a synergistic effect when the cells were treated with both TF5 and corticotropin-releasing factor (CRF). Immunoneutralization studies were done in which the cells were treated with TF5 or CRF and an antibody to CRF. The antibody completely blocked CRF induced ACTH release, but had no effect on TF5 stimulated ACTH release, suggesting that the activity is not due to a CRF-like peptide in TF5. A number of peptides isolated from TF5, and certain other peptides produced by the immune system were evaluated for their ability to stimulate ACTH secretion. These included thymosin (TSN) α 1, α 11, and β 4, prothymosin α (PT α, thymopoeitin 5 (TP5), factuer thymique serique (FTS), interferon α (INF α), INF γ, interleukin 1 (IL-1), and interleukin 2 (IL-2). None of these factors had any effect on pituitary ACTH secretion. These results demonstrate that some peptide component of TF5 causes an increase in serum corticosteroids by stimulating pituitary ACTH release.

Rapid isolation of thymosin β 4 from thymosin fraction 5 by preparative high-performance liquid chromatography

We have developed a rapid, efficient, and reproducible two-step method for the purification of thymosin β 4 (Tβ 4) from thymosin fraction 5 (TF5). This purification is based on the use of high-performance preparative/semi-preparative and analytical reversed-phase (Delta-Pak C 18) chromatographic columns. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid compositional analysis have shown that natural, purified Tβ 4 is identical to synthetic Tβ 4. This procedure can be used to isolate other biologically active peptides from TF5 in sufficient quantity for characterization.

Thymosin fraction 5 stimulates prolactin and growth hormone release from anterior pituitary cells in vitro.

Thymosin fraction 5 (TF5) is a partially purified extract of bovine thymus containing 40-60 peptides. In addition to its well documented immunopotentiating effects, TF5 reportedly modulates the secretion of some hypothalamic peptides and pituitary hormones. In this study, TF5 (10-100 micrograms/ml) stimulated PRL release from normal, MtTW15, and 7315a cells and GH release from normal and MtTW15 cells, but had no apparent effect on LH release. No changes in intracellular cAMP or cGMP levels could be correlated with these responses. Stimulation of PRL release from perifused normal anterior pituitary cells was rapid, sustained, and concentration related. Although it had no apparent effect on normal prelabeled anterior pituitary cells with respect to 45Ca2+ efflux, the calcium channel blocker D-600 inhibited TF5-mediated hormone release from these cells. Additive increases in TRH-stimulated PRL release and GRF-stimulated GH release by TF5 suggested independent mechanisms of action. Dopamine (500 nM) blocked TF5-stimulated PRL release, but somatostatin (10-100 nM) had no effect on TF5-stimulated PRL or GH release. TF5 failed to affect either basal or TRH-induced polyphosphoinositide hydrolysis. Perifused normal anterior pituitary cells prelabeled with [3H]arachidonate responded to TF5 treatment with a liberation of radioactive arachidonate and/or its metabolites. BW755c, an inhibitor of all known catabolic pathways of arachidonic acid, blocked the ability of TF5 to stimulate PRL and GH release. Reversed phase HPLC separation of TF5 into five fractions resulted in two fractions that exhibited hormone-releasing activity. These data suggest that TF5 stimulates pituitary hormone release through a mechanism different from that ascribed to TRH or GRF. The stimulus-secretion coupling mechanism involves neither polyphosphoinositide hydrolysis nor cAMP generation, but appears to be dependent on the generation of arachidonate metabolites.

Immune-neuroendocrine interactions during aging: Age-dependent thyrotropin-inhibiting activity of thymosin peptides

Thymosin fraction 5 (TF-5), a partially purified thymic preparation, has been previously shown to have luteinizing hormone-releasing hormone (LH-RH)-releasing activity in perfused rat hypothalamus as well as in vivo stimulatory effect on the pituitary-adrenal axis in prepubertal monkeys. We report here the effect of TF-5 on the TSH-thyroid axis in young (3 months) and old (25 months) Sprague-Dawley male rats. Conscious free-moving animals carrying an indwelling atrial cannula received a single dose of 5 mg/kg body wt. of either bovine serum albumin (BSA) or TF-5 via the cannula. In the young rats, TF-5 induced a marked reduction of plasma thyrotropin (TSH) which was significantly greater than the normal circadian decline observed in the BSA-treated controls. The old males displayed high basal levels of TSH which showed no circadian rhythmicity, and did not respond to TF-5. Thyroxine (T 4), triiodothyronine (T 3), corticosterone, and prolactin levels were not affected by TF-5 at the dose levels tested. The old rats had significantly lower basal levels of T 4, but not T 3, than their young counterparts. The synthetic peptides thymosin α-1 and serum thymic factor, which are components of TF-5, had no effect on the above hormones when injected in doses up to 5 μg/kg body wt. Acute thymectomy in 3-month-old males induced a significant increase in basal levels of TSH without affecting plasma T 4 or T 3. These results suggest that the thymus has an inhibitory action on TSH in the rat, which is not mediated by the thyroid gland. Our results also suggest an age-related desensitization of the TSH system to thymic influence in this species.

In vitro effect of a thymic epithelial culture supernate or thymosin fraction 5 on rabbit platelet aggregation and intracellular cyclic AMP levels.

Supernates of thymic epithelial cell culture (STEC) strongly inhibit aggregation induced by addition of adenosine diphosphate (ADP: 1 microM) or thrombin (0.5 unit per ml) to washed platelet suspensions and accelerated the restoration from ADP-triggered aggregation. At the same time, STEC increased the level of platelet adenosine 3',5'-cyclic monophosphate (cyclic AMP) in a dose-dependent manner. Depending on the concentration used, thymosin fraction 5 increased the level of intracellular cyclic AMP ranging between 5 and 100 micrograms per ml, as well as inhibiting ADP-induced platelet aggregation. The activities of both STEC and thymosin fraction 5 were found to act exclusively on cyclic AMP phosphodiesterase activity in platelets. In contrast the supernates from Chang, HeLa, or HCC-M cells did not affect platelet aggregation induced by ADP, but slightly increased the cyclic AMP level (Chang, HeLa). Within 2 min after the treatment with STEC, more than 50% of the maximum inhibitory activity on platelet aggregation and increases in intracellular cyclic AMP were observed. These activities disappeared following STEC treatment with pronase E. STEC activity was found predominantly in the 1,000-50,000-dalton fractions. These activities were not altered when STEC was treated by adenosine deaminase. The level of prostaglandin E (PGE) derivatives in STEC was about two times that found in the control culture medium. These data suggest that the biological activity of STEC in the platelets might be attributed to thymosinlike polypeptides and PGE1.

Modulation of human natural killer cell cytotoxic activity, lymphokine production, and interleukin 2 receptor expression by thymic hormones.

Thymic hormone preparations have been shown to modulate natural killer (NK) activity in vivo in mice. We have investigated the effects of thymosin fraction 5 (TF5) on the in vitro NK cell activity of highly purified human large granular lymphocytes (LGL). The results indicate that TF5 but not kidney fraction 5 (a preparation used as control) is able to enhance the spontaneous NK activity of normal LGL. In addition, TF5 exhibited additive effects with recombinant interferon-alpha in enhancing NK activity in vitro. TF5 also enhanced interleukin 2 production and interleukin 2 receptor expression as well as interferon-gamma production in mitogen-stimulated LGL. Thymosin-alpha 1, a synthetic polypeptide originally isolated in its native form from TF5, also exhibited enhancing effects on LGL activities, suggesting that it is the active species in TF5. These results indicate that thymic hormones might regulate NK activity through the induction of lymphokine production and receptor expression by LGL.

In vitro effects of thymosin and lithium on lymphoproliferative responses of normal donors and HIV seropositive male homosexuals with AIDS-related complex

The in vitro effects of thymosin fraction 5 (TF5) and lithium chloride (LiCl) on the ability of peripheral blood mononuclear cells (PBMC) obtained from 37 normal male donors and 33 male patients with AIDS-related complex (ARC) to respond to alloantigenic stimulation (mixed leukocyte reaction, MLR) and to produce interleukin 2 (IL-2) in response to mitogens were studied. TF5 significantly increased MLR responses in normal donors ( P < 0.01) and in a group of 33 ARC patients with depressed cellular immunity ( P < 0.05). Similar effects were observed when LiCl was added to the MLR assays in both the normal and the ARC patient groups. Furthermore, TF5 and LiCl exhibited additive immunoenhancing properties. In 10 normal donors TF5 enhanced phytohemaggutinin (PHA)-induced IL-2 production as well as IL-2 production in response to pokeweed mitogen (PWM) ( P < 0.02). TF5-mediated enhancement of IL-2 production by PBMC obtained from ARC patients was observed in response to both mitogens, i.e., PHA and PWM. Additionally, LiCl increased PHA-induced IL-2 production in both normal subjects and ARC patients. LiCl and TF5 together had an additive effect in the enhancement of IL-2 production in both groups of subjects. Our data extend previous observations regarding the immunoregulatory activities of TF5 and LiCl and provide evidence that PBMC obtained from ARC patients have the potential to respond in vitro to these agents. The significance of these findings is discussed.

Thymic extracts and lymphokine-containing supernatant fluids stimulate the pituitary-adrenal axis, but not cerebral catecholamine or indoleamine metabolism

Injection of thymosin fraction 5 (TF5) or the supernatant fluid from concanavalin Astimulated rat spleen cells into mice significantly increased plasma concentrations of corticosterone at 1 or 3 h. However, measurement of the concentrations of the catecholamines, norepinephrine and dopamine, the indoleamine, serotonin, and their major metabolites, in prefrontal cortex, parietal cortex, nucleus accumbens, septum, caudate-putamen, hypothalamus, and brain stem did not indicate any statistically significant changes. Nor was there any alteration in splenic norepinephrine content. These data suggest that TF5 and lymphokines do not cause a generalized stress response, but rather a selective activation of the pituitary-adrenal axis, probably by causing release of adrenocorticophic hormone from the pituitary. There was no evidence that the corticotropin-releasing activity of TF5 was related to an effect on hypothalamic biogenic amines. These data are discussed in the light of previous results obtained with lymphokine-containing supernatant fluids.

Effects of nutritional recuperation on E-rosetting lymphocytes and in vitro response to thymosin in malnourished children.

The 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.