Complete amino acid sequence analysis of a peptide isolated from the thymus that enhances release of growth hormone and prolactin.

We have previously reported that thymosin fraction 5 (TF5), a partially purified calf thymus preparation, contains a peptide(s) that can enhance the production of GH and/or PRL from rat anterior pituitary cells in vitro. Using reverse phase HPLC, we have now isolated and chemically characterized from TF5 a peptide possessing this activity. This peptide, termed MB-35, is a highly charged basic molecule of 35 amino acid residues and a mol wt of 3756. A computer-assisted search of published protein sequences has revealed that this peptide has a 100% homology with a region of the histone H2A. Biological studies using rat pituitary cells have revealed that MB-35 is active alone or in combination with GH-releasing factor (GRF) or TRH and can increase the production of GH and/or PRL beyond that achievable with GH-releasing factor and TRH alone. The observation that histone H2A, the parent molecule, is without activity is of keen interest, since it suggests that nucleoproteins may have heretofore unknown physiological activities, perhaps related to cell cycle and/or other events associated with DNA activation events.

Thymosin fraction 5 inhibits the proliferation of the rat neuroendocrine MMQ pituitary adenoma and C6 glioma cell lines in vitro.

Cytokines such as interleukin-1 (IL-1) and IL-6 stimulate the hypothalamic-pituitary-adrenal (HPA) axis. In addition, these proteins affect pituitary cell proliferation in vitro. Thymosin fraction 5 (TF5) is a partially purified preparation of the bovine thymus that enhances immune system functioning. Because TF5 similarly stimulates the HPA axis, we examined the effects of this preparation on neuroendocrine tumor cell proliferation. Cells of the PRL-secreting rat anterior pituitary adenoma, MMQ (5-50 x 10(3) cells/well), were exposed to vehicle (RPMI-1640 containing 2.5% FCS, 7.5% horse serum, and antibiotics) or TF5 (100-500 microg/ml) for up to 96 h and the proliferation of MMQ cells monitored using the MTT assay (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). TF5-mediated inhibition of cell proliferation was dependent on both TF5 concentration and the initial MMQ cell number. Minimal reductions in optical densities resulted from exposure to 100 microg/ml TF5, whereas the highest concentration of this preparation (i.e. 500 microg/ml) completely blocked MMQ cell division. The concentration-dependent effects of TF5 were particularly striking at initial plating densities of 25 and 50 x 10(3) MMQ cells/well; in contrast, all concentrations of TF5 completely inhibited MMQ cell growth at 5 and 10 x 10(3) cells/well. The antiproliferative actions of TF5 on MMQ cells were demonstrable within 24 h and remained for up to 96 h as determined by the MTT assay and actual cell counts. Because the highest densities of MMQ cells were partially refractive to the antiproliferative effects of TF5, we examined the effects of PRL (1-1000 nM) and MMQ cell conditioned medium (50%) on TF5 inhibition of MMQ adenoma proliferation. The TF5 concentration-dependent inhibition of MMQ cell growth was largely reversed by the 50% conditioned medium, whereas PRL slightly potentiated the antiproliferative actions of TF5. The proliferation of the rat C6 glioma cell line (10-30 x 10(3) cells/well) demonstrated greater sensitivity to TF5: concentrations as low as 10 microg/ml TF5 inhibited C6 cell proliferation (P < 0.01), and near-maximal inhibition was noted at 200 microg/ml TF5. Significant reductions in MMQ and C6 cell viabilities accompanied decreases in cell number and morphological analysis indicated these cells were dying by apoptosis. The peptides thymosin alpha1 (T alpha1), thymosin beta4 (T beta4), MB35, and MB40 had no effect on either MMQ or C6 cell proliferation, indicating that these TF5 components are not the principle active peptides. Therefore, TF5 was further separated into 60 fractions by preparative reverse phase HPLC. HPLC fractions 17, 25, 26, and 27 significantly suppressed MMQ cell proliferation (P < 0.01) to the same extent as TF5; other HPLC fractions had no effect. These data demonstrate a new biological property of TF5: the inhibition of cell proliferation and the induction of apoptosis in neuroendocrine tumor cells. The proliferation effects were time and concentration dependent and could be partially reversed by an activity present in the MMQ cell conditioned medium. Thus, TF5 and cytokines have opposite effects on adenoma cells because IL-2 and IL-6 stimulate GH3 cell proliferation. We propose that circulating thymic peptides may act to prevent pituitary adenoma and glioma tumor formation, an action opposed by autocrine growth factors secreted by these tumors.

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.

Thymosin alpha 1 antagonizes dexamethasone and CD3-induced apoptosis of CD4+ CD8+ thymocytes through the activation of cAMP and protein kinase C dependent second messenger pathways.

It is well established that glucocorticoid hormones and anti-CD3 monoclonal antibodies induce apoptosis in immature developing thymocytes. This process can be modulated by soluble factors, anti-oxidants and adhesion receptors. Previously we have demonstrated that thymosin alpha 1 (T alpha 1), a 28-amino acid thymic peptide hormone, is a dose and time dependent antagonist of dexamethasone (DEX) and CD# induced DNA fragmentation of murine thymocytes in vitro. To further investigate the mechanism of T alpha 1 action we determined a T alpha 1 sensitive thymocyte population and examined some of the molecular events associated with T alpha 1 anti-apoptotic activity. Phenotypic analysis of the sub-populations of thymocytes, based on CD4 and CD8 expression, revealed that T alpha 1 exerts its effect on CD4+ CD8+ immature thymocytes. T alpha 1 treatment of thymocytes delays the production of free radicals and the subsequent consumption of glutathione, that is observed during both DEX and CD3 induced apoptosis. We further demonstrate that T alpha 1 stimulates the production of cAMP and activates PKC in thymocytes. These data suggest that T alpha 1 exerts an influence on the development of a population of immature T-cells in the thymus by effecting the sensitivity of thymocytes to apoptosis during the pre-selection stages of thymic development. Our studies also suggest that the mechanism of T alpha 1 action involves the induction of both cAMP and PKC dependent second messenger pathways.

Thymosinalpha1 is chemopreventive for lung adenoma formation in A/J mice.

The effects of thymosin (THN) alpha1 were investigated using the urethane injection carcinogenesis A/J mouse model. Lung adenomas were observed 2.5, 3, and 4 months after urethane injection (400 mg/kg i.p.) into female A/J mice. Daily administration of THNalpha1 (0.4 mg/kg, s.c.) reduced lung adenoma multiplicity significantly, by approximately 45, 40, and 17%, respectively, 2.5, 3, and 4 months after urethane injection. Animals treated with THNalpha1 had a significantly greater white cell density than control A/J mice. Endogenous THNalpha1-like peptides were detected in the mouse lung. By radioimmunoassay and by Western blot, prothymosin alpha was detected in the mouse lung. By immunocytochemistry, THNalpha1-like peptides were detected in all lung compartments including the bronchus, adenoma, bronchioles, and alveoli. These results indicate that exogenous THNalpha1 prevents lung carcinogenesis in A/J mice.

Immunological, isoelectric, hydrophobic and molecular weight differences between soluble and ionically membrane-bound fractions of choline-o-acetyltransferase prepared from mouse and rat brain.

Choline-O-acetyltransferase (EC 2.3.1.6; ChAT) was prepared from synaptosomal fractions (P(2)) of mouse and rat brain in the presence of proteolytic inhibitors by the method of Gray and Whittaker (1962) as modified by (Salehmoghaddam and Collier, 1976). The P(2) fraction was hypo-osmotically shocked with glass distilled water and centrifuged to separate the cytoplasmic (S(3)) and vesicle-bound (P(3)) fractions. Fraction S(3) was saved for ChAT assay and compared with the ChAT fraction eluted from the P(3) by salt at a pH 7.4 or by detergent (Benishin and Carroll, 1983). These three fractions of ChAT were then compared by molecular weights, isoelectric points, immunoblotting with monoclonal or polyclonal antibodies and hydrophobicity. The results show that the S(3) fraction of ChAT has a molecular weight of 66 K(d), whereas the ionically-bound fraction of ChAT has a molecular weight of 73-78 K(d). SDS-PAGE of these two ChAT fractions followed by immunoblotting revealed the presence of two immunoreactive bands at 28-29 K(d) and 50-51 K(d) for the ionically bound ChAT fraction. Conversely, none of these antibodies immunostained any protein bands for the S(3) ChAT fraction even though one monoclonal antibody had been prepared against this ChAT fraction and the S(3) ChAT fraction had a similar specific activity prior to SDS-PAGE as did the salt solubilized ChAT fraction. However, anti-ChAT monoclonal antibody MB16 binds the native S(3) ChAT fraction in the co-precipitation assay. The S(3) fraction of ChAT had only one isoelectric point at pH 7.8, whereas the ionically bound and detergent soluble ChAT fractions had two isoelectric points at pH 8.1-8.15 and 7.45-7.5. The S(3) ChAT fraction also differed in hydrophobicity from the other two ChAT fractions. These differences between the S(3) and salt soluble ChAT fractions were not obviated by addition of Triton X-100 and thus could not be attributed to the association of lipids with either of the fractions. We conclude that the water soluble fraction of ChAT in central nerve terminals differs in its physical properties and its subcellular location from that which ionically binds to membranes.

Veratridine-induced breakdown of cytosolic acetylcholine in rat hippocampal minces: an intraterminal form of acetylcholinesterase or choline O-acetyltransferase?

Rat hippocampal minces were loaded with N-methyl-[3H]acetylcholine ([3H]ACh) in the presence of the 'poorly penetrating' acetylcholinesterase (EC 3.1.1.7, AChE) inhibitor echothiophate and the effect of the depolarizing agent veratridine determined on the subcellular storage and release of [3H]ACh and [3H]choline. Results indicated that veratridine stimulated the release of [3H]ACh from a crude vesicular fraction (P3) by a Ca2+-dependent process, while simultaneously accelerating the breakdown of cytosolic (S3) [3H]ACh. A portion of the [3H]choline derived from the hydrolyzed S3 [3H]ACh was donated to the P3 fraction for [3H]ACh formation and release. When the identical experiment was done using hippocampal minces from septal lesioned rats, veratridine did not stimulate either the Ca2+-dependent release of [3H]ACh or the hydrolysis of cytosolic [3H]ACh. Incubation of control hippocampal minces with paraoxon, an AChE inhibitor which can penetrate cholinergic nerve terminals more rapidly than echothiophate, prevented veratridine from stimulating the Ca2+-dependent release of [3H]ACh from the P3 fraction. Instead, it then stimulated the Ca2+-independent release of [3H]ACh from the S3 fraction. When minces were incubated with the choline O-acetyltransferase (EC 2.3.1.6, ChAT) inhibitor 4-(1-naphthyl)vinyl pyridine (NVP), veratridine was no longer able to stimulate the Ca2+-dependent release of labelled ACh either. Instead, veratridine stimulated the Ca2+-independent release of labelled ACh from the S3 fraction. NVP also abolished the veratridine-induced, Ca2+-dependent release of total ACh. Both paraoxon and NVP inhibited the reversible reaction of ionically bound ChAT prepared from rat brain when tested in vitro, yet paraoxon was much less potent than NVP, and was unable to inhibit this reaction at the low concentration which prevented the veratridine induced breakdown of S3 [3H]ACh during mince incubation. Veratridine depolarization of hippocampal minces stimulated the activity of a membrane-bound fraction of ChAT associated with the P3 fraction, but this fraction of ChAT did not become more sensitive to inhibition by paraoxon during tissue incubation. Veratridine depolarization of minces also increased the activity of membrane-bound AChE, but this enzyme was not inhibited by the low NVP concentration which prevented the veratridine-induced breakdown of S3 [3H]ACh. The veratridine-induced increase in membrane-bound ChAT activity was dependent on the presence of extracellular Ca2+ in the incubation medium.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of choline O-acetyltransferase (ChAT) in the BALB/C mouse spleen.

The synthesizing enzyme, Choline-O-acetyl transferase (ChAT) (EC 2.3.1.6) and the degradation enzyme, acetylcholinesterase (EC 3.1.1.7) for the neurotransmitter acetylcholine, have been anatomically and biochemically characterized in the thymus of the BALB/C mouse. In the present study we continue to analyze the possibility of cholinergic immunomodulation of immune tissues by determining if ChAT is present in the BALB/C mouse spleen. Our enzymatic evaluation of ChAT activity in splenic extracts revealed .05 nmoles/min/mg protein as compared to .1 nmoles/min/mg of protein activity in controls prepared from whole brain extracts. No detectable levels of ChAT activity were observed in the serum. Immunoblotting and immunoprecipitating using the anti ChAT monoclonal antibody, MB16, demonstrated two bands in the brain and one band in the spleen. Membrane bound ChAT in the brain was composed of two subunits with apparent molecular weights of 28 and 50 kDa. The spleen demonstrated only one form of ChAT with an apparent molecular weight of 28 kDa. Immunoprecipitation of the enzyme from both the brain and spleen resulted in a recovery of 59% and 60% of the activity respectively.

Molecular weight determinations of soluble and membrane-bound fractions of choline O-acetyltransferase in rat brain.

Three fractions of choline O-acetyltransferase (ChAT) (EC 2.3.1.6) were solubilized from a nerve ending fraction of rat forebrain using three sequential washes of an increasingly chaotrophic nature (100 mM sodium phosphate, pH 7.4; 500 mM NaCl; 2% Triton DN-65) as previously described (Benishin, C.G., and P.T. Carroll (1983) J. Neurochem. 41: 1030-1039). The molecular weights of the soluble (NaP) and membrane-bound fractions (NaCl and 2% Triton DN-65) of ChAT, following partial purification, were determined using either gel filtration on Sephadex G-200, G-100 Superfine, or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by "Western blotting" and immunochemical visualization of ChAT with four different anti-ChAT monoclonal antibodies (Ab8, Ab9, 4D7, and 1E6). Results obtained with gel filtration indicated that the NaP- and Triton DN-65-solubilized fractions of ChAT had molecular weights in the range of 73,000 to 78,000, whereas the NaCl-solubilized fraction of ChAT had a molecular weight in the range of 230,000 to 240,000. Results obtained with SDS-PAGE and Western blotting indicated that all three fractions of ChAT were composed of the same nonidentical subunits.

Thymosin alpha1 is a time and dose-dependent antagonist of dexamethasone-induced apoptosis of murine thymocytes in vitro.

It is well established that glucocorticoid hormones induce apoptosis in immature developing thymocytes. Thymocyte apoptosis can be modulated by growth factors, anti-oxidants and adhesion receptors. We have previously demonstrated that thymosin alpha1 (Talpha1) antagonizes dexamethasone-induced apoptosis of CD4+CD8+ thymocytes. In the present study, we further characterize the dose and time dependence of Talpha1's antagonism of dexamethasone-induced thymocyte apoptosis. Talpha1 is effective at concentrations ranging from 2 to 100 microg/10(6) thymocytes. Talpha1 pre-treatment is necessary to achieve its anti-apoptotic activity. Talpha1 provides temporary protection to thymocytes by slowing dexamethasone's apoptotic activity up to 12 h post dexamethasone treatment. Additionally, Talpha1's activity is not sensitive to cycloheximide treatment, suggesting Talpha1's activity is independent of protein synthesis. Finally, Talpha1 is unable to antagonize apoptosis induced by the reactive oxygen species, H2O2, suggesting Talpha1's antagonism of dexamethasone occurs at the early stages of dexamethasone-induced apoptosis, prior to the production of reactive oxygen species. This evidence suggests that Talpha1 may provide a mechanism to transiently extend the life of a thymocyte during thymic selection.

Thymosin stimulates interleukin-6 production from rat spleen cells in vitro.

Thymosin fraction 5 (TF5) is a partially purified preparation of bovine thymus that affects the differentiation and function of T-cells in vitro. Interleukin-6 (IL-6) is a pleiotropic cytokine that induces terminal maturation of B-cells and T-cell activation and differentiation. Although TF5 had previously been shown to stimulate the production of a number of lymphokines, its effects on IL-6 were not known. In this study we determined the effect of TF5 on IL-6 production from rat spleen cells in vitro. TF5 (100 micrograms/ml) stimulated IL-6 production from splenocytes (0.75-3.0 x 10(5) cells/well) in the presence of 0.008-0.2 micrograms/well of the T-cell mitogen concanavalin-A (con-A) by 10-20 fold during a 72 h incubation period. Dose-response studies demonstrated that 10 micrograms/ml of TF5 was the lowest concentration capable of enhancing IL-6 production. The ability of TF5 to stimulate IL-6 production in the presence of con-A could be demonstrated within 24 h of incubation; longer incubation periods (48-72 h) correlated with further enhancements of IL-6 production. Partial purification of the IL-6-inducing activity from TF5 resulted in three subfractions possessing activity in the presence of con-A (MB2, MB3, MB7) and one in the absence of con-A (MB2). The previously characterized thymosin peptides T alpha 1 and T beta 4 had no effect on IL-6 production in the absence or presence of mitogen. This study reports a new biological activity for TF5 and suggests that a novel constituent of TF5 may enhance the production of IL-6 from spleen cells.

Rapid isolation of thymosin beta 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 beta 4 (T beta 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 C18) chromatographic columns. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid compositional analysis have shown that natural, purified T beta 4 is identical to synthetic T beta 4. This procedure can be used to isolate other biologically active peptides from TF5 in sufficient quantity for characterization.

Thymosin enhances the production of IL-1 alpha by human peripheral blood monocytes.

Incubation of human peripheral blood monocytes (PBM) with a partially purified thymic preparation, thymosin fraction (TF5), results in a dose dependent production of an interleukin-1 (IL-1)-like factor. The biological activity of this factor can be blocked by anti-IL-1 alpha, but not by anti-IL-1 beta which neutralizes bacterial induced IL-1 activity. Studies with further purified TF5 fractions show that this activity is not due to the well-characterized peptide, thymosin alpha 1 (T alpha 1), but rather a new thymosin peptide(s) isolated from a more basic fraction. Intraperitoneal injection of TF5 also induces the expression of a membrane-bound IL-1 (mIL-1) on mouse peritoneal cells. This study provides the first evidence that TF5 can influence macrophage activity directly by enhancing IL-1 production. This observation may help explain the mechanism by which TF5 modulates immune responses. These results also point to a more selective role for thymic hormones, growth factors and cytokines which may trigger macrophages to secrete different forms of IL-1, which can then regulate either immune and/or inflammatory processes.

Biodistribution of synthetic thymosin beta 4 in the serum, urine, and major organs of mice.

Thymosin beta 4 (T beta 4) is a peptide of 43 amino acids that was first isolated from the thymus gland and subsequently found to be ubiquitous in nature. T beta 4 functions mainly as an actin-sequestering molecule in nonmuscle cells, where its primary role is to maintain the large pool of unpolymerized G-actin in the cell. Studies on the pharmacokinetics of T beta 4 in human and other mammals have not been reported so far. In the present study, we have measured T beta 4 concentrations in serum, urine, and 10 major organs of female Swiss-Webster mice following intraperitoneal administration of 400 micrograms synthetic T beta 4. Using a modified enzymatic immunoassay, our data show a significant increase of T beta 4 in serum starting 2 min after injection and lasting for 40 min (average: 2.34 +/- 0.54 micrograms/ml). High concentrations were found in urine (59.3 +/- 7.54 micrograms/ml) at three different points after injection (20 min, 40 min, and 2 h). Of the 400 micrograms T beta 4 administered to mice 83% was recovered at the end of the study, 44.6% of which corresponded to urine, 1.4% to serum, and 37.5% to the organs. In 50% of the tested organs, the wet weight concentrations of T beta 4 increased significantly from the first 40 min to 2 h after injection in comparison to their baseline wet weight concentrations. These organs were: the brain (72 micrograms/g), heart (80 micrograms/g), liver (15 micrograms/g vs 9 micrograms/g), kidneys (65 micrograms/g vs 28 micrograms/g), and peritoneal fat (47 micrograms/g vs 13 micrograms/g). Wet weight concentrations increased in the thymus (196 micrograms/g vs 147 micrograms/g) and muscle (45 micrograms/g vs 0 micrograms/g) after 6 h of injection. The spleen showed an increase in wet weight concentrations at the 2 min timepoint (267 micrograms/g vs 161 micrograms/g). Ovaries had a biphasic increase at 40 min (72 micrograms/g vs 62 micrograms/g) and 24 h (92 micrograms/g vs 62 micrograms/g) after T beta 4 administration. In lungs, the highest wet weight increase after injection (149 micrograms/g at timepoint 6 h) was not higher than its basal wet weight concentration (153 micrograms/g). These pharmacokinetic studies of T beta 4 in mice have established that high levels of T beta 4 are found in blood following I.P. administration and the kidney rapidly removes the peptide from the circulation. The kinetics of this response should help define the proper scheduling of administration of T beta 4 during clinical trials in disorders, such as the acute respiratory distress syndrome (ARDS), associated with actin toxicity.

Biodistribution of synthetic thymosin alpha1 in the serum, urine and major organs of mice.

Thymosin fraction 5 (TF5), a thymic preparation, has been shown to be an immune-potentiating agent consisting of biologically active polypeptide components with hormone-like activities. Thymosin alpha1 (T alpha1) was the first biologically active polypeptide to be purified from TF5 and completely characterized. It is an acidic peptide with an isoelectric point of 4.2 and a molecular weight of 3108. T alpha1 is considered a biological response modifier which amplifies T-cell immunity. In the present study, we have studied some pharmacokinetic properties of T alpha1 by measuring its concentrations in serum, urine and ten major organs of female Swiss-Webster mice following administration of 500 microg T alpha1 intraperitoneally. Using a modified enzymatic immunoassay, our data show a significant increase of T alpha1 in serum 2 min after injection and lasting for 2 h (average: 1.55 +/- 0.27 microg/ml). In urine, at four different time points after injection (20 min, 40 min, 2 h, 6 h), increased concentrations of T alpha1 were found between 24.2 and 25.4 microg/ml (average: 25 +/- 0.47 microg/ml). Of the 500 microg T alpha1 administered to mice, 8.97% was recovered at the end of the study, of which 2% corresponded to urine, 1.25% to serum (2 ml of serum per mouse), and 5.72% to organs. Since the urine/day volume and the serum volume of any Swiss Webster mouse is ca 2 ml, additional extrapolation of the above mentioned values could show percentages of recovery close to 40% for urine and 2.5% for serum. In most of the organs, the wet weight concentrations of T alpha1 increased significantly during the first 40 min after injection in comparison to their baseline wet weight concentrations. These organs consisted of the following: thymus (33.1 +/- 3.5 microg/g vs 18 microg/g baseline); lungs (7.7 +/- 1.1 microg/g vs 1.9 microg/g baseline); spleen (15.6 +/- 0.7 microg/g vs 5.6 microg/g); kidneys (6.2 +/- 1.1 microg/g vs 3.9 microg/g); ovaries (9.2 +/- 1.4 microg/g vs 0 microg/g); and peritoneal fat (4 +/- 1 microg/g vs 0 microg/g). No significant increases were observed in the liver (1.7 +/- 0.1 microg/g vs 1.4 microg/g) and heart (0.7 +/- 0.5 microg/g vs 0 microg/g). Increased concentrations of T alpha1 were not detected in the brain and skeletal muscle tissues. These pharmacokinetic studies of T alpha1 in mice indicate that rapid renal excretion of T alpha1 represents a major source of humoral loss following I.P. administration. Recent preliminary studies in humans confirm that the kidney rapidly releases high levels of T alpha1 in urine in a time frame consistent with that observed in mice.

Thymic hormone modulation of age-induced changes in the induction of graft-versus-host disease by DBA/2J lymphocytes.

Aging induces a number of changes in the immune system, including the involution of the thymus which results in the loss of thymic hormone production and alteration in T cell function. One age-dependent change in immune response is the increasing risk of developing acute or chronic form of graft-versus-host disease (GVHD) following bone marrow transplantation as the age of the recipient increases. A murine model of GVHD that has been extensively studied is one in which injection of C57BL/6 spleen cells into unirradiated B6D2F1 mice results in an acute form of GVHD characterized by cytolytic T lymphocytes (CTL), suppressor cells, runting, and occasionally death. In contrast, injection of DBA/2J spleen cells results in a chronic form of GVHD characterized by a lack of CTL and hyperproduction of immunoglobulin and autoantibodies. This study shows that the GVHD response of DBA/2J spleen cells is dependent on the age of the donor DBA/2J mice. If spleen cells from DBA/2J mice older than 3 months are injected into B6D2F1 recipients, CTL and lack of immunoglobulin production indicative of acute GVHD result. Administration of thymosin fraction 5, a collection of thymic hormones, to DBA/2J mice older than 3 months caused spleen cells from these treated mice to give a GVHD response characteristic of the chronic form of GVHD in B6D2F1 recipients. Thus, thymic hormones were able to modulate the changes in GVHD responses of DBA/2 lymphocytes that occur as the mice age. Preliminary fractionation of TF5 has indicated that there are at least two active thymic peptides present in TF5.

Effects of synthetic thymosin-alpha 1 and its analogs on fertilizability of human sperm: search for a biologically active, stable epitope.

Thymosin-alpha 1 (T alpha 1) and six T alpha 1 analogs were synthesized to study structure-function relationships and to search for the biologically active and stable epitope(s) that would have clinical application in the treatment of male infertility. Four of these analogs were prepared by modification/substitution of N- and C-terminal amino acids of T alpha 1 peptide, and the other two analogs were fragments having only N-16 amino acids (N-terminal) or C-14 amino acids (C-terminal), respectively, of the T alpha 1 peptide. T alpha 1 and these six analogs were tested for their effects on human sperm penetration rates in the sperm penetration assay (SPA). T alpha 1 significantly (p < .0001) increased the penetration rates in SPA, with the strongest enhancing effect at 0.5 microgram/100 microL concentration. Of the six analogs tested only two, T alpha 1-Gly-NH2 and T alpha 1-C14, retained the enhancing effects in SPA. None of the analogs decreased the penetration rates or affected sperm motility compared to control. The enhancing activity resides primarily in an epitope, the C-terminal 14 amino acids of T alpha 1. However, for maximal effect both N- and C-terminal amino acids (serine and asparagine, respectively) have to be intact and unmodified. The T alpha 1-Gly-NH2 analog that had its C-terminal protected was as potent as the intact T alpha 1 peptide. T alpha 1 and this analog may have clinical applications in treatment of male-factor-mediated infertility.

A novel thymosin peptide stimulates interleukin-6 release from rat C6 glioma cells in vitro.

Thymosin fraction 5 (TF5) is a partially purified preparation of the bovine thymus possessing immunopotentiating properties. TF5 also stimulates the hypothalamic-pituitary-adrenal axis in vivo and anterior pituitary hormone release in vitro. Interleukin-6 (IL-6) is an inflammatory, pyrogenic cytokine that also stimulates hypothalamic and anterior pituitary hormone release. We hypothesized that TF5 may activate the neuroendocrine system in part via the stimulation of central cytokine production. Therefore, we determined the effects of TF5 on IL-6 release from rat C6 glioma cells in vitro. Glioma cells (25-100 x 10(3)) were exposed to vehicle (RPMI-1640) or TF5 (10-1,000 micrograms/ml) in 96-well plates (200 microliters incubation volume) for 4-24 h to determine optimal cell number and incubation period conditions. TF5 (1,000 micrograms/ml) stimulated IL-6 release from 100 x 10(3) C6 cells/well by 9-fold following a 24-hour incubation (p < 0.01). Reducing the number of cultured C6 cells to either 50 or 25 x 10(3) cells/well resulted in diminished IL-6 responses to TF5. TF5 stimulated C6 cell IL-6 release in a time-dependent manner (4-24 h) at all concentrations tested. A 24-hour incubation period provided the largest TF5-stimulated increases in IL-6 release compared with shorter time intervals (i.e., 4-8 h). Pretreatment of C6 glioma cells with 1 microM phorbol myristate acetate (PMA) for 24 h completely blocked the subsequent stimulation of IL-6 release by PMA (20-250 nM) and partially blocked by 50% the TF5 stimulation of this cytokine. Peptides previously purified from TF5 had no effect on IL-6 release at 50-1,000 nM [i.e., thymosin alpha 1 (T alpha 1), thymosin beta 4 (T beta 4), MB35, MB40]. Therefore, TF5 was further fractionated into 7 pools by preparative reverse phase high performance liquid chromatography (HPLC). HPLC pools P1 (fractions 1-8) and P2 (fractions 9-12) significantly increased C6 cell IL-6 release (p < 0.01) to the same extent as 250 micrograms/ml TF5. Other HPLC pooled fractions (P3-P7) had no effect on IL-6 release from C6 glioma cells. P1 and P2 stimulated a 50- and 10-fold increase in IL-6 release, respectively, at a protein concentration of 1.0 microgram/ml. Therefore, P1 was more potent and displayed a greater efficacy for the stimulation of IL-6 release compared to P2. Analysis of individual fractions of P1 and P2 revealed that 1 microgram/ml of fraction 6 was as efficacious as 250 micrograms/ml TF5 for the stimulation of IL-6 release. These data indicate that one or more peptide components of TF5 enhance glial cell production of IL-6. In addition, the thymosin-stimulated production of extracellular IL-6 is mediated partially by one or more isoforms of protein kinase C. We hypothesize that a peptide product of the thymus transported across the CNS blood-brain barrier may stimulate the glial cell production of IL-6 and affect neuronal, neuroendocrine and/or inflammatory processes.

A distinctive form of soluble CD8 is secreted by stimulated CD8+ cells in HIV-1-infected and high-risk individuals.

The aim of the present study was to investigate the biochemical structure and pathogenic significance of the soluble CD8 (sCD8) present in the serum of HIV-1-infected individuals. In a longitudinal study of a cohort of HIV-infected homosexuals and the amount of sCD8 detected in the plasma was correlated with changes in lymphocyte subsets and with the clinical course of HIV infection. The level of sCD8 in the plasma, the percentage, and the absolute number of CD8+CD38+ cells were increased in HIV-seronegative, high-risk homosexuals and in seropositive HIV+ individuals. The plasma concentration of serum sCD8 showed a significant correlation with the absolute number of CD8+ and CD8+CD38+ cells in HIV+ homosexuals. In addition to a molecule with a molecular weight (m.w.) of 30 kDa, sCD8 isolated from the plasma of HIV-1-infected individuals and of healthy controls was found to consist of two molecules, one with a m.w. of 57 to 62 kDa and another with a m.w. of 66 to 70 kDa. The former was the predominant molecule in normal individuals, while the latter was the predominant molecule in HIV-negative high-risk homosexuals and in HIV-infected individuals. The latter molecule, secreted by chronically stimulated CD8+ cells, seems to be present in the circulation as a dimer. While it was previously shown that CD8 can be shed from the cell membrane in vitro, the present study indicates that in vivo-stimulated CD8+ cells release a distinctive form of soluble CD8.

Human immunodeficiency virus contains an epitope immunoreactive with thymosin alpha 1 and the 30-amino acid synthetic p17 group-specific antigen peptide HGP-30.

We have reported that an antiserum prepared against thymosin alpha 1 [which shares a region of homology with the p17 protein of the acquired immunodeficiency syndrome (AIDS)-associated human immunodeficiency virus] effectively neutralized the AIDS virus and prevented its replication in H9 cells. Using HPLC and immunoblot analysis, we have identified from a clone B, type III human T-lymphotropic virus (HTLV-IIIB) extract a protein with a molecular weight of 17,000 that is immunoreactive with thymosin alpha 1. In contrast, no immunoreactivity was found in retroviral extracts from a number of nonhuman species including feline, bovine, simian, gibbon, and murine retroviruses. Heterologous antiserum prepared against a 30-amino acid synthetic peptide analogue (HGP-30) does not cross-react with thymosin alpha 1 but does react specifically with the p17 protein of the AIDS virus in a manner identical to that seen with an HTLV-IIIB p17-specific monoclonal antibody. The demonstration that this synthetic analogue is immunogenic and that antibodies to HGP-30 cross-react not only with the synthetic peptide but also with the HTLV-IIIB p17 viral protein provides an additional, and potentially more specific, candidate for development of a synthetic peptide vaccine for AIDS. In addition, the p17 synthetic peptide (HGP-30) may prove to be useful in a diagnostic assay for the detection of AIDS virus infection in seronegative individuals.