Unique subset of thymic epithelial cells defined by the expression of a novel neuroendocrine antigen

Murine monoclonal antibodies (MoAbs) were produced against a rat medullary thyroid carcinoma to identify neuroendocrine differentiation antigens. One of these antibodies (1D4) identified a novel 62- to 69-kDa antigen expressed by subsets of immune system epithelial and neuroendocrine cells. This antigen is expressed by distinct subsets of thymic epithelial and splenic reticular cells and is shared by discrete subsets of anterior pituitary and thyroid neuroendocrine cells. In the thymus, 1D4 expression identified a unique subset of stellate-shaped Ia+ medullary epithelial cells which did not react with thymosin alpha-1 antisera nor with the MoAb A2B5 specific for a GQ ganglioside expressed by thymic hormone-producing cells. The availability of the 1D4 MoAb should facilitate further characterization of 1D4+ immune system epithelial cells and may advance our understanding of neuroendocrine-immune system interactions.     

Structural requirements for thymosin beta4 in its contact with actin. An NMR-analysis of thymosin beta4 mutants in solution and correlation with their biological activity.

We examined the conformational preferences of mutants of thymosin beta4, an actin monomer sequestering protein by NMR spectroscopy in 60% (v/v) trifluoroethanol. Under these conditions, the wild-type thymosin beta4 conformation consists of an alpha-helix (helix I) extending from residues 5-16 with a more stable fragment from lysine 11 to lysine 16 and a second alpha-helix (helix II) encompassing residues 31-39. The point mutations studied here are located in helix I or in the LKKTET segment (residues 17-22) that form the two main entities of interaction with the actin molecule. The alpha-1H conformational shifts allow us to investigate the helicity of the polypeptides at the residue level and to correlate these structures with their biological activity. We determine that an extension of helix I at its C-terminal end over the LKK-segment results in loss of activity. The correct termination of this helix is connected to a specific orientation of the polypeptide essential for a cooperative action of the thymosin beta4 binding entities required for full activity.     

Cellular distribution of prothymosin alpha and parathymosin in rat thymus and spleen

By means of immunohistochemical methods, we have investigated the cellular distribution of prothymosin alpha and parathymosin in rat thymus and spleen, using specific antibodies raised against thymosin alpha-1 and against parathymosin. We observed prothymosin alpha immunoreactivity in lymphoid cells both in thymus and spleen. In the thymus, prothymosin alpha staining was more marked in cortex than in medulla. In the spleen, prothymosin alpha was found in lymphocytes of the periarteriolar lymphatic sheaths and was especially prominent in the germinal centers. Parathymosin immunoreactivity in the thymus was mainly localized in the medulla; positive cells were reticuloepithelial cells from the thymic reticulum and the blood barrier. Thymocytes were negative. In spleen, parathymosin was found in reticular cells arranged in a ring between the periarteriolar lymphatic sheath and the marginal zone. Our results do not support an exclusive role for these peptides as immune system hormones or cytokines.     

Evidence for the monomeric nature of thymosins

According to gel-filtration experiments, alpha- and beta-thymosins appear to form oligomers, which are 4-5-fold larger than the corresponding polypeptides. However, on analysis by sedimentation equilibrium ultracentrifugation, prothymosin alpha and thymosin beta 4 showed relative molecular masses of 12,800 and 4600, which are close to the values calculated from their amino acid sequences, confirming their existence in solution as discrete monomeric entities.     

The thymus AIDS connection: Thymosin in the diagnosis and treatment of individuals at risk for AIDS

The thymus gland, which plays a key role in the maturation and functioning of the lymphoid system, is implicated in the acquired immune deficiency syndrome (AIDS). The observation that the thymic hormone, thymosin α₁, is elevated in individuals at risk for AIDS (as opposed to being depressed in other immunodeficient states) has provided the first direct evidence that the thymus is malfunctioning early in the course of this deadly disease. These observations have been valuable in screening for the syndrome with a rapid radioimmunoassay and in the initiation of the first clinical trials with thymosin in high risk homosexuals and hemophiliacs. If the progressive immune paralysis in AIDS is due to a dying thymus, the early identification of asymptomatic carriers of AIDS or individuals with modest AIDS-related dysfunction may lead to therapy with thymosin or other thymomimetic agents that can restore immune function and prevent the onset of frank AIDS.     

The conformation of peptide thymosin α1 in solution and in a membrane-like environment by circular dichroism and NMR spectroscopy. a possible model for its interaction with the lymphocyte membrane

The 28-residue peptide thymosin α1 was studied by circular dichroism and two-dimensional NMR. Circular dichroism indicates that thymosin α1 in water solution does not assume a preferred conformation, while in the presence of small unilamellar vesicles of dimiristoylphosphatidylcholine and dimiristoylphosphatidic acid (10:1) and in sodium dodecyl sulphate, it assumes a partly structured conformation. Presence of zinc ions produces similar effects. In a more hydrophobic environment like a solution of a mixed solvent water-2,2,2 trifluoroethanol, it adopts a structured conformation. NMR spectra indicated that in this mixture as solvent, thymosin α1 has a structure characterized by two regions. A β-turn is present between residue 5 and residue 8, while the region between residues 17 and 24 shows an α helix conformation. These changes of conformation in different environments may be considered structural requirements in the steps of its interaction with the lymphocyte membrane. In fact, these conformational changes may correspond to the first event of the mechanism of lymphocyte activation in the immune response modulation by thymosin α1.     

Effect of thymosin peptides on the chick chorioallantoic membrane angiogenesis model.

The effect of alpha- and beta-thymosin peptides, namely prothymosin alpha (ProT(alpha)), thymosin alpha(1) (T(alpha)1), parathymosin alpha (ParaT(alpha)), thymosin beta(4) (Tbeta4), thymosin beta(10) (Tbeta10), and thymosin beta(9) (Tbeta9), on the angiogenesis process was investigated using the chick chorioallantoic membrane as an in vivo angiogenesis model. The thymosin peptides tested were applied in 10 microl aliquots containing 0.01-4 nmoles of Tbeta4, Tbeta10 or Tbeta9, 0.016-6.66 nmoles of T(alpha)1, 4.1 pmoles-1.66 nmoles of ProT(alpha), and 4.4 pmoles-1.76 nmoles of ParaT(alpha). Phorbol 12-myristate 13-acetate and hydrocortisone were also used as positive and negative control, respectively. Tbeta4, ProT(alpha) and T(alpha)1 were found to enhance angiogenesis, while Tbeta10, Tbeta9 and ParaT(alpha) exhibited an inhibitory effect on the angiogenesis process. When mixtures of Tbeta4 and Tbeta10 containing active amounts of the two peptides at different proportions were applied, the promoting effect of Tbeta4 on angiogenesis was reversed in the presence of increasing concentrations of Tbeta10 and vice versa. The effect of Tbeta10, Tbeta9, ProT(alpha) and ParaT(alpha), in parallel with Tbeta4 and T(alpha)1, on the angiogenesis process was investigated for the first time as far as we know and the results of this study offer more insight into the biological regulatory roles of thymosin peptides, and provide helpful information about their therapeutic potential. Whether these agents could be used either as inhibitors of angiogenesis in disease states where uncontrolled angiogenesis is involved, e.g. in carcinogenesis, or as angiogenesis promoters that could be useful in wound healing, fracture repair, peptic ulcers etc., remains to be further studied.     

Developmental changes of serum thymosin alpha 1 and beta 4 in male and male castrated pigs: modulation by testosterone and human chorionic gonadotropin.

Thymic secretory peptides thymosin beta 4 and alpha 1 have possible endocrine roles in both immune and reproductive systems; thus, they should respond to endocrine feedback control mechanisms consistent with gonadal function. In an initial experiment, male pigs (boars; n = 90; 10/time) were bled at 1, 3, 6, 12, 18, 24, 30, 36, and 96 wk of age before and 24 h after hCG stimulation. Thymosin beta 4 concentrations were significantly depressed 24 h after hCG challenge. Testosterone concentrations increased with age up to 36 wk and were further increased with hCG stimulation (p less than 0.01). In a subsequent experiment, boars (n = 12) and barrows (males castrated shortly after birth; n = 12) were blood-sampled, administered hCG, and sampled again 24 h later at 1, 3, 6, 12, 18, and 24 wk of age. Barrows (n = 12) were administered testosterone with the same protocol. Testosterone concentrations increased in boars with maturity and were further increased from the hCG stimulation (p less than 0.01). Thymosin beta 4 concentrations decreased with age in boars and barrows (p less than 0.01), and hCG challenge depressed thymosin alpha 1 and beta 4 concentrations in boars and thymosin beta 4 in barrows (p less than 0.01). Testosterone treatment of barrows also depressed thymosin beta 4 and alpha 1 in barrows (p less than 0.01). The depression of thymosins by hCG treatment points to a role for gonadotropins in altering circulating thymosin concentrations independent of, but in conjunction with, the effect of gonadal steroids.     

NMR structure of human thymosin alpha-1

800 MHz NMR structure of the 28-residue peptide thymosin alpha-1 in 40% TFE/60% water (v/v) has been determined. Restrained molecular dynamic simulations with an explicit solvent box containing 40% TFE/60% TIP3P water (v/v) were used, in order to get the 3D model of the NMR structure. We found that the peptide adopts a structured conformation having two stable regions: an alpha-helix region from residues 14 to 26 and two double β-turns in the N-terminal twelve residues which form a distorted helical structure.     

Thymosin alpha 1: amino acid homology with peptide T from the human immunodeficiency virus envelope

Thymosin alpha 1 has many effects on immune function and its absence in primary immunodeficiency states produce a clinical presentation similar to the one encountered in acquired immune deficiency syndrome (AIDS). Human immunodeficiency virus (HIV), the etiologic agent of AIDS, binds to T4 helper/inducer lymphocytes through specific surface receptors which include the CD4 glycoprotein. Octapeptide T, a component of the HIV envelope, mediates the binding of HIV to its receptor. In this report, we draw attention to the similarity between the amino acid sequence of thymosin alpha 1 and peptide T and its analogues. This similarity can produce a cross-reactivity between thymosin alpha 1 and HIV and may be a factor in the pathophysiology of the acquired immuno-deficiency syndrome.     

Role of helper T-lymphocytes in modulating the humoral immune response to cholera enterotoxin

The role of helper T-lymphocytes in the modulation of humoral immune response to sheep red blood cells with choleragen has been studied in vivo, the populations of cooperating marrow cells, formed in mice under the action of hydrocortisone being used as a model. In adoptive transfer marrow cells, taken from mice on day 12 after thymectomy and from mice previously treated with antithymocyte serum, have proved incapable of humoral immune response. Choleragen, similarly to theophylline, normalizes the humoral immune response of marrow cells in thymectomized mice, but inhibits this response in intact ones, while thymosin fraction 2 restores it again, thus abolishing the action of choleragen and theophylline. The opposite effects rendered by choleragen and theophylline on humoral immune response, depending on the hormonal status of the animals and the possibility of influencing these effects by means of thymosin fraction 2 indicate that the population of helper T-lymphocytes are selectively sensitive to changes in the concentration of intracellular cAMP. Their capacity for cooperative interaction in the immune process is regulated by thymic hormones and forms the basis of the mechanism permitting the modulation of humoral immune response with choleragen.     

晚期胃癌患者化疗中胸腺肽α1治疗疗效的临床研究

目的：探讨应用流式细胞仪（FCM）检测胃癌患者外周血免疫指标,以评价胸腺肽α1联合化疗对胃癌患者免疫功能的影响。方法：70例胃癌患者随机分为用药组和对照组。用药组35例采用胸腺肽α1＋化疗,对照组35例单用化疗,两组化疗方案相同。胸腺肽α1每次1.6mg,每周2次,连续4周（共8针）。28天为一疗程,共用2个疗程。采用FCM检测两组患者化疗前后外周血CD4、CD8、CD3＋CD4＋、CD3＋CD8＋免疫指标。结果：用药组CD4、CD8、CD3＋CD4＋、CD4/CD8均高于化疗前和对照组化疗后水平,均具有统计学意义（P〈0.05）。结论：胸腺肽α1配合化疗能提高患者的免疫功能,用FCM检测外周血免疫指标为临床观察肿瘤患者的免疫状态提供有效的依据。     

Acceptor specificity and tissue distribution of three human alpha-3-fucosyltransferases

Based on the capacity to transfer alpha-L-fucose onto type-1 and type-2 synthetic blood group H and sialylated acceptors, a comparison of the alpha-3-fucosyltransferase activities of different human tissues is shown. Three distinct acceptor specificity patterns are described: (I) myeloid alpha-3-fucosyltransferase pattern, in which leukocytes and brain enzymes transfer fucose actively onto H type-2 acceptor and poorly onto sialylated N-acetyllactosamine: (II) plasma alpha-3-fucosyltransferase (EC 2.4.1.152), in which plasma and hepatocyte enzymes transfer, in addition, onto the sialylated N-acetyllactosamine; (III) Lewis alpha-3 4-fucosyltransferase (EC 2.4.1.65), in which gall-bladder kidney and milk enzymes transfer, in addition, onto type-1 acceptors. The small amount (less than 10%) of alpha-3-fucosyltransferase activity found in the plasma of an alpha-3-fucosyltransferase-deficient individual had a myeloid-type acceptor pattern, suggesting that this small proportion of the plasma enzyme is derived from leukocytes. In addition to the three acceptor specificity patterns, these enzyme activities can be differentiated by their optimum pH: 8.0-8.7 for the enzymes from myeloid cells and brain. 7.2-8.0 for liver enzymes and 6.0-7.2 for gallbladder enzymes. Milk samples had two alpha-3-fucosyltransferase activities, the Lewis or alpha-3/4-fucosyltransferase under control of the Lewis gene and an alpha-3-fucosyltransferase with plasma acceptor pattern which was independent of the control of the Lewis gene. The apparent affinity for GDP-fucose of the myeloid-like enzyme was weaker than those of the plasma and Lewis-like enzymes. The apparent affinities for H type 2 and sialylated N-acetyllactosamine were stronger for exocrine secretions as compared to the plasma and myeloid enzymes. The plasma type of alpha-3-fucosyltransferase activity was more sensitive to N-ethylmaleimide and heat inactivation than the samples with myeloid-like alpha-3-fucosyltransferase activity.     

Characterization of two C-type lectin-like domain (CTLD)-containing proteins from the cDNA library of Chinese mitten crab Eriocheir sinensis

Chinese mitten crab Eriocheir sinensis is one of the most important aquaculture crustacean species in China. A cDNA library was constructed from mixed tissues of E. sinensis challenged with LPS. Eight genes involved in immune response were identified from 319 single colonies. Among them, two different C-type lectin-like domain (CTLD)-containing proteins were firstly identified in Chinese mitten crab. The full-length cDNA sequences of two C-type lectin-like domain (CTLD)-containing proteins named EsCTLDcp-1 and EsCTLDcp-2 were cloned by 5' RACE. The deduced amino acid sequences of EsCTLDcp-1 and EsCTLDcp-2 possessed several conserved features of C-type lectin subfamily. The tissue distribution of EsCTLDcp-1 and EsCTLDcp-2 was examined by Real-time PCR. In the normal Chinese mitten crab, the expression of EsCTLDcp-2 was detected in all tested tissues such as haemolymph, muscle, intestine, gill, heart, gonad and hepatopancreas, whereas in muscle, intestine, gill, heart and hepatopancreas for EsCTLDcp-1. The highest expressions of EsCTLDcp-1 and EsCTLDcp-2 were both observed in hepatopancreas. LPS significantly induced the expression of EsCTLDcp-1 and EsCTLDcp-2 in the hepatopancreas at the different time points. The induced fold change of EsCTLDcp-1 and EsCTLDcp-2 increased significantly from 2 h for EsCTLDcp-1 and 4 h for EsCTLDcp-2, and reached a maximum at 12 h, then dropped at 24 h. A differential pattern was found in Chinese mitten crab challenged with Chinese mitten crab pathogen Aeromonas hydrophila. The expression of EsCTLDcp-1 increased significantly at 2 h post-challenge crabs with A. hydrophila, then decreased at 4 h and 8 h, after that increased at 12 h and 24 h. The expression of EsCTLDcp-2 was decreased at the all time points. All these data suggest a differential role of EsCTLDcp-1 and EsCTLDcp-2 in the crab innate immune response to bacterial infection.     

One-step procedure for the determination of thymosin beta 4 in small tissue samples and its separation from other thymosin beta 4-like peptides by high-pressure liquid chromatography

Thymosin beta 4 has been determined by a simple and fast one-step procedure in different tissues of rats. The tissues (1 to 40 mg) were disintegrated and deproteinized by homogenization in perchloric acid. After neutralization by potassium hydroxide the supernatant solution was used for determining thymosin beta 4 by reverse-phase HPLC without further manipulations. Not only does this procedure avoid artificial proteolysis as effectively as extraction of tissues by guanidinium chloride or boiling buffer, but it offers two further advantages. First, no additional steps--as for example desalting--are necessary prior to HPLC and thus the risk of losing thymosin beta 4 is eliminated. Using this procedure thymosin beta 4 is recovered quantitatively. The method is linear over the range 0.04 to 1.13 nmol and thymosin beta 4 is well separated from other thymosin beta 4-like peptides known to be present in mammals; i.e., thymosin beta Ala4, thymosin beta 9, thymosin beta 10, and thymosin beta Arg10. Second, the acid-insoluble pellet of the same extract can be used to determine the DNA content of the sample. Thus it is possible to relate thymosin beta 4 to DNA, which then allows comparing cells of different tissues and cell lines to one another. This procedure is also applicable to small peptides soluble in perchloric acid.     

Isolation and characterization of thymosin beta 9 Met from pork spleen

We have identified a new thymosin beta 4-like peptide in pork spleen. The new peptide (12 mg) and thymosin beta 4 (33 mg) were isolated from 230 g of spleen by solid phase extraction, preparative isoelectric focusing, and HPLC. The new peptide was termed thymosin beta 9 Met to indicate its close relationship to thymosin beta 9 from calf. The only difference from thymosin beta 9 is the substitution of leucine by methionine at position 6. This peptide replaces thymosin beta 10 which is the minor thymosin beta 4-like peptide in most mammals, e.g., in man, rat, mouse, cat, and rabbit. The structure was determined by amino acid analysis, tryptic digestion, and carboxypeptidase digestion. Pork spleen contains 192 micrograms of thymosin beta 4 and 117 micrograms of thymosin beta 9 Met per gram of tissue.     

Effects of thymic polypeptides on the thymopoiesis of mouse embryonic stem cells

The thymus provides a unique cellular and hormonic microenvironment for the development of immunocompetent T cells. Thymic polypeptides have been widely used clinically for the treatment of tumors, infectious diseases and immune deficiency diseases. They have already shown the ability to stimulate the maturation of hematopoietic stem cells towards the CD3+CD4+ T cell lineage. However, their effects on the thymopoiesis of embryonic stem cells are still unexplored. In this paper, we compared the effects of three thymic polypeptides, thymopentin (TP5), thymosin alpha-1 (Talpha-1) and thymopeptides on the in vitro thymopoiesis of mouse embryonic stem (ES) cells. Using the embryoid body induction system, we found that both Talpha-1 and thymopeptides effectively induced ES cells to differentiate sequentially into the CD3+ and CD4+/CD8+ T cells. These T cells had T cell receptor (TCR) Vbeta gene rearrangement and most were TCRalphabeta T cells. We also found that the expression of the Notch receptor and its ligands Delta-like-1 and Delta-like-4 gradually increased during the induction. However, TP5 failed to induce the T cell differentiation of the ES cells. In summary, this is the first report to demonstrate that Talpha-1 can stimulate the T cell early stage differentiation from ES cells using the embryoid body protocol. These findings provide a powerful model for studying T cell development and may open new venues for the clinical application of Talpha-1.     

Effect of dietary cyclic nigerosylnigerose on intestinal immune functions in mice

We examined the dietary effects of cyclic nigerosylnigerose (CNN), a dietary indigestible oligosaccharide with four D-glucopyranosyl residues linked by alternating alpha-(1-->3)- and alpha-(1-->6) glucosidic linkages, on the intestinal immune function of mice, and the effects were compared with those of alpha-(1-->3)-linked oligosaccharide (nigerooligosaccharides, NOS) or alpha-(1-->6)-linked oligosaccharide (isomaltooligosaccharides, IMO). BALB/c mice were fed with 1-5% CNN, 5% IMO, or 12.5% NOS for 4 weeks, and the intestinal mucosal immune responses were determined. In the 1-5% CNN fed groups, the amounts of IgA in feces increased significantly. In addition, IgA, transforming growth factor-beta1 (TGF-beta1), and interleukin-6 (IL-6) secretion by Peyer's patch (PP) cells were enhanced in CNN fed mice. In the 5% CNN group, pH in the cecum decreased, and the amounts of lactic acid and butyric acid increased. These findings were not observed in the NOS- or IMO-fed group of mice. They suggest that CNN supplementation changes the intestinal environment of microflora and indirectly enhances the immune function in the gut.