Thymulin, a thymic peptide, prevents the overproduction of pro-inflammatory cytokines and heat shock protein Hsp70 in inflammation-bearing mice.

The effects of synthetic analogue of peptide hormone thymulin, which is normally produced by thymic epithelial cells, on immune cells activity and blood cytokine profile had been studied in male NMRI mice with acute inflammation induced by injection of lipopolysaccharide from gram-negative bacteria (LPS, 250 microg/100 g of body weight). Inflammation induced by LPS resulted in accumulation of several plasma pro-inflammatory cytokines, IL-1 beta, IL-2, IL-6, TNF-alpha, interferon-gamma, and also IL-10, anti-inflammatory cytokine. Thymulin previously injected in dose of 15 microg/100 g body weight, prevented the accumulation of proinflammatory cytokines in plasma. Thymulin also prevented LPS-induced up-regulation of production of several cytokines by spleen lymphocytes and peritoneal macrophages. Added in vitro, thymulin decreased the peak of TNF-alpha production in macrophages cultivated with LPS. In addition, thymulin lowered the peak of Hsp70 production induced by LPS treatment. The results indicate that thymulin having significant anti-inflammatory effect may be promising in clinical application.

The effect of synthetic thymulin on cell surface marker expression by avian T-cell precursors.

The effects of the in vitro exposure of avian bone marrow (BM) cells and thymocytes to synthetic thymulin were studied. Two T-cell differentiation markers, PNA binding site and CT-1a expression, were used to examine cell maturation. Enhanced PNA binding to both BM cells and thymocytes resulted following an in vitro thymulin exposure but cell proliferation was not affected. Scatchard analysis supported the conclusion that PNA binding affinity was significantly increased and thus responsible for the observed increase in PNA binding. Flow cytometric analysis suggested that the induced PNA+ thymocyte population may be a different population from the one exhibiting enhanced CT-1a expression following thymulin exposure. Taken together, the observations suggest that cells can express their further differentiation states without undergoing proliferation following in vitro thymulin stimulation.

Synthesis of analogs of the serum thymic nonapeptide, "facteur thymique serique" (FTS). Part II.

New analogs of FTS (Facteur Thymique Sérique), less than (Formula: see text), a circulating thymic factor, were prepared by replacing the amino acid residues in positions, 1, 3, 4, 5, 6 and 3 and 6 together. Five other analogs of C-terminal heptapeptide were prepared by replacing the amino acid residues in position 3 or 6. These peptides were synthesized using conventional synthesis in solution.

Acid-labile anchoring linkages for solid phase synthesis of C-terminal asparagine peptides using the Fmoc strategy.

Two acid-labile substituted benzylamine type anchoring linkages, 4-benzoxy-2,6-dimethoxybenzylamine and 2-benzoxy-4,6-dimethoxybenzylamine, for solid phase synthesis of peptide amides were prepared. The Na-9-fluorenylmethyloxycarbonyl (Fmoc) amino acids could be easily attached to the resins with DCC/HOBt (loading 0.5-0.6 mmol/g resin). After final removal of the Na-protecting groups, treatment with TFA (50-95%) yielded amino acid and peptide amides in high purity. As we could show for the synthesis of thymulin (FTS, pGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn), these two resins with anchoring linkages are well suited for the synthesis of C-terminal Asn peptides using protected aspartic acid derivative as starting material.

Solid-phase synthesis of peptides with C-terminal asparagine or glutamine. An effective, mild procedure based on N alpha-fluorenylmethyloxycarbonyl (Fmoc) protection and side-chain anchoring to a tris(alkoxy)benzylamide (PAL) handle.

Attempts to anchor Fmoc-asparagine or glutamine as p-alkoxybenzyl esters for solid-phase peptide synthesis are fraught with difficulties. A convenient and effective method to prepare peptides with C-terminal asparagine or glutamine involves quantitative attachment of N alpha-Fmoc-C alpha-tert.-butyl aspartate or glutamate via the free omega-carboxyl groups to a tris(alkoxy)benzylamino (PAL) support. Chain elongation proceeds normally by standard Fmoc chemistry, and treatment with acid, e.g., CF3COOH--CH2Cl2, 90 min at 25 degrees, releases the desired peptides in greater than 95% yields without side reactions at the C-terminus. Feasibility of the approach has been demonstrated by the syntheses of the C-terminal octapeptide from human proinsulin, H-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-OH, and the serum thymic factor pGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn-OH.

Synthesis of analogs of the serum thymic nonapeptide, "facteur thymique serique" (FTS). Part I.

Analogs of FTS (Facteur Thymique Sérique), less than :formula: (see text), a circulating thymic factor, were prepared by replacing the amino acid residues in positions 1, 2, 8 and 9, or by shortening the nonapeptide chain at the N- or C-terminal end. These peptides were synthesized by two different schemes using the conventional synthesis in solution.

Synthesis of an immunogenic conjugate and of two 125I-labelled derivatives of the "facteur thymique sérique".

[4-Aminobenzoyl-Gln1]FTS was synthesized from a suitably-protected derivative of the "facteur thymique sérique" (FTS). It was diazotized and coupled to bovine serum albumin, yielding an immunogenic conjugate in which all the functional groups of the peptide remained intact. [4-Aminobenzoyl-Gln1]FTS and another synthetic compound, [3-(4-hydroxyphenyl)propionyl-Gln1]FTS, were 125I-labelled. The binding of these radioactive peptides of the anti-FTS antibodies obtained with our immunogenic conjugate was inhibited by unlabelled FTS.

Synthesis and biological activity of eight thymulin analogues.

Eight analogues of thymulin, a thymic nonapeptide involved in several aspects of T-cell differentiation, were synthesized by the conventional method in solution. Four were modified in residue 7 (Ala, D-Ala, D-Leu or Sar instead of Gly) and two in residue 8 (D-Ser or Thr instead of Ser); in the others, the Gly6-Gly7 sequence was replaced either by a single glycyl residue or by a triglycyl sequence. The biological activity of the analogues was determined in the rosette assay: five exhibited a prolonged activity in vivo with respect to thymulin. All the analogues inhibited the binding of tritiated thymulin to thymulin receptors on three human lymphoblastoid T-cell lines (HSB2, 1301 and CEM) with the same order of magnitude as non-labelled thymulin.

Suppression of acute experimental allergic encephalomyelitis by synthetic serum thymic factor. Clinical, histopathological, and immunohistochemical studies.

Acute experimental allergic encephalomyelitis (EAE) was induced in Hartley guinea pigs and Lewis rats, which were then treated with synthetic serum thymic factor (FTS). When a dose of 30 micrograms/100 g body weight of FTS was subcutaneously administered to the animals on days--1 (before inoculation), 4, 9 and 15 intermittently, clinical symptoms of acute EAE were suppressed. Histopathological evaluation showed that the severity of EAE in FTS-treated guinea pigs was less than in untreated guinea pigs. Immunohistochemical examination showed that the numbers of OX6+, W3/25+, W3/13+ and OX19+ cells in FTS-treated rats were less than in untreated rats and that the number of OX8+ cells in FTS-treated rats was greater than in untreated rats. These findings suggest that FTS induced OX8+ cells in inflammatory lesions and suppressed inflammation in acute EAE.

A microparticle enhanced nephelometric immunoassay (Nephelia) applied to thymulin measurement.

This article describes a microparticle enhanced nephelometric immunoassay (Nephelia) applied to the quantification of the thymic peptide hormone thymulin. Nephelia uses antibody recognition by the antithymulin antiserum in a competitive reaction between free thymulin and thymulin bound to the microspheres. The binding between microsphere and thymulin is achieved with the aid of a protein carrier. The sensitivity of the competitive reaction varied with the protein carrier and the antiserum sample. The most efficient reaction was obtained with the thymulin-metallothionein-microsphere conjugates; as little as 5 pg/ml of thymulin could be detected. In adult human serum or in its ultra-filtrate, spiked synthetic thymulin was totally recovered. Measurement of thymulin in serum or ultra-filtrate samples demonstrated the presence of interference from molecules in the serum. Nephelia led to the same conclusions as those reported with other methods and performs as well, and is more simple to use than radio- or enzymo-immunoassays for thymulin measurement.

A pioneer synthesis of conjugates of a thymic peptide hormone and polyamines.

To synthesize peptidic derivatives of polyamines, sequential reactions were designed for the polyfunctional peptides and polyfunctional amines which utilize specific protection and deprotection. Utilizing these reactions, a thymic nonapeptide, a lymphocyte-differentiating hormone, was coupled with spermidine and spermine to yield, respectively: Glp-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn-NH(CH2)3NH(CH2)4NH2; Glp-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn-NH(CH2)3NH-(CH2)4NH(CH2) 3NH2. Both conjugates inhibited rather than stimulated incorporation of [3H]-thymidine into DNA at levels 20-fold that of spermine. This activity is less than that based on the percent of the spermine moeity (ca. 20%) in the conjugate.

Biological activity of synthetic analogs of serum thymic factor.

A series of analogs of serum thymic factor (Facteur Thymique Sérique; FTS) devoid of C- and N-terminal few residues were synthesized and their ability to induce Thy-l (theta) antigen in vitro on mouse T lymphocyte precursors was examined. The pentapeptide moiety (Lys-Ser-Gln-Gly-Gly) seems to be important for the expression of agonistic activity. Some of the short chain peptides showed antagonistic properties. Synergistic activation by two inactive analogs was observed.