Multiple functions of thymosin ß4 in the pearl oyster Pinctada fucata suggest its multiple potential roles in artificial pearl culture.

Thymosin ß4 is a multifunctional protein in vertebrates that participates in physiological processes, such as wound healing, immune response, cell proliferation and migration. We assessed the multifarious roles of this small peptide in Pinctada fucata, an oyster commonly used in pearl culture in China. Our results showed that when P. fucata was challenged by bacterial pathogens or LPS, the relative expression level of Pfthymosin ß4 mRNA was significantly up-regulated, suggesting its involvement in immune response of the animal. Recombinant Pfthymosin ß4 (rPfthymosin ß4) was produced and showed in vitro different antibacterial activities against different pathogenic bacteria; the inhibitory effect of rPfthymosin ß4 on bacterial growth was relatively stronger in the broth culture than agar culture. The overexpression of Pfthymosin ß4 in Escherichia coli BL21(DE3) cells could improve their resistance to Cu2+, Zn2+, Cd2+, and H2O2, suggesting that Pfthymosin ß4 is likely involved with antioxidant. rPfthymosin ß4 also significantly promoted the proliferation and migration of mouse aortic vascular smooth muscle cells as indicated by MTT assay and cell scratch assay, respectively. In addition, chemically synthesized or recombinant Pfthymosin ß4 could transiently increase the circulating total hemocytes counts but down-regulated by RNAi in P. fucata. Taking together above results and previous studies suggested that Pfthymosin ß4 is potentially able to promote wound healing through enhancing antibacterial activity and antioxidant capacity, promotion of cell proliferation and migration, and increase of circulating hemocytes in P. fucata due to nucleus implantation injury. Thus, the future of recombinant Pfthymosin ß4 should be promising in the culture of pearls in P. fucata.

Thymosin participates in antimicrobial immunity in zebrafish.

Thymosin hormones, which were shown to be involved in immune system development and differentiation in previous studies, have antimicrobial functions in different animals. Zebrafish are a useful model for immunology research. Although thymosin has been reported to be involved in the embryonic development of zebrafish, it is necessary to uncover the antimicrobial function of thymosin in zebrafish. In this study, we expressed thymosin ß (Tß) in zebrafish in vitro and studied its antimicrobial function. The Tß protein consists of 45 amino acids and is conserved among its family members, especially the actin-binding motif (LKKTET). Tß was expressed in all tested tissues and was highly expressed in the brain, liver and hindgut. After Aeromonas hydrophila challenge, the Tß transcript level increased in the skin, liver, kidney, spleen, thymus, foregut, gills and midgut. Purified recombinant thymosin ß (rTß) protein was used to study the antimicrobial mechanism. rTß could inhibit the growth of Staphylococcus aureus, Aeromonas hydrophila, Vibrio anguillarum, Pseudomonas aeruginosa and Klebsiella pneumoniae. rTß also binds to and agglutinates certain bacteria. Further study showed that rTß could combine with the polysaccharides from gram-negative and gram-positive bacterial walls. All results suggested that the Tß of zebrafish plays a significant role in innate antibacterial immune responses.

Thymosins participate in antibacterial immunity of kuruma shrimp, Marsupenaeus japonicus.

Thymosins ß are actin-binding proteins that play a variety of different functions in inflammatory responses, wound healing, cell migration, angiogenesis, and stem cell recruitment and differentiation. In crayfish, thymosins participate in antiviral immunology. However, the roles of thymosin during bacterial infection in shrimp remain unclear. In the present study, four thymosins were identified from kuruma shrimp, Marsupenaeus japonicus, and named as Mjthymosin2, Mjthymosin3, Mjthymosin4, and Mjthymosin5 according the number of their thymosin beta actin-binding motifs. Mjthymosin3 was selected for further study because its expression level was the highest in hemocytes. Expression analysis showed that Mjthymosin3 was upregulated in hemocytes after challenged by Vibrio anguillarum or Staphylococcus aureus. The recombinant Mjthymosin3 protein could inhibit the growth of certain bacteria in an in vitro antibacterial test. Mjthymosins could facilitate external bacterial clearance in shrimp, and were beneficial to shrimp survival post V. anguillarum or S. aureus infection. The results suggested that Mjthymosins played important roles in the antibacterial immune response of kuruma shrimp.

Comparative study of ß-thymosin in two scallop species Argopecten irradians and Chlamys farreri.

The ß-thymosin (Tß) proteins participate in numerous biological processes, such as cell proliferation and differentiation, anti-inflammatory and antimicrobial mechanism. To date, Tß proteins have been well studied in vertebrates, especially mammals. While limited Tß or Tß-like proteins have been reported in invertebrates. Moreover, rare information of Tß or Tß-like proteins is available in scallop species yet. In the present study, two Tß homologues, AiTß and CfTß, were identified and characterized from two scallop species bay scallop Argopecten irradians and Zhikong scallop Chlamys farreri. They were both 41 amino acid peptide and contained one THY domain, a highly conserved actin-binding motif and two conserved helix forming regions. Tissue distribution and expression profiles of their mRNA transcripts were roughly similar yet different in detail, while their recombinant proteins exhibited different immunomodulation activity on the downstream immune parameters. These results collectively indicated that the function of Tß family in scallop were functionally differentiated.

Thymosin ß4 is involved in the antimicrobial immune response of Golden pompano, Trachinotus ovatus.

Thymosin beta belongs to the thymosin family, which consists of a series of highly conserved peptides involved in various biological processes. In teleosts, understanding of the immunological functions of thymosin beta is limited, particularly in vivo, which is essentially unknown. In the current study, we cloned and identified thymosin beta 4 from the teleost fish Golden pompano (Trachinotus ovatus), which we have named TroTß4. We investigated the expression patterns and functions of TroTß4 in both in vivo and in vitro assays. TroTß4 is composed of 44 amino acids and shares high sequence identities with known thymosin ß4 species in other teleosts, which contains a highly conserved actin-binding motif (LKKTET). The expression of TroTß4 was most abundant in immune organs, and was significantly up-regulated in response to infection bacterial with one of a number of bacteria (including Edwardsiella tarda, Vibrio harveyi, and Streptococcus agalactiae). Purified recombinant TroTß4 (rTroTß4) inhibited the growth of bacteria, as measured using an automatic growth curve analyzer, indicating that TroTß4 has antimicrobial functions. When administered in vivo, overexpression of TroTß4 in T. ovatus, bacterial colonization of tissues was significantly reduced. In contrast, when a DNA vector-based siRNA technology was used to knock down TroTß4 expression, bacterial dissemination and colonization of tissues increased significantly. Taken together, these results provide the first in vivo evidence to indicate that teleost thymosin beta 4 plays a significant role in innate antibacterial immune responses in addition to in vitro bacteriostatic activity. This provides valuable information regarding the biological functions of teleost thymosin beta.

Function of Thymosin Beta-4 in Ethanol-Induced Microglial Activation.

BACKGROUND/AIMS: Neuroinflammation mediated by activated microglia may play a pivotal role in a variety of central nervous system (CNS) pathologic conditions, including ethanol-induced neurotoxicity. The purpose of this study was to investigate the function of Tß4 in ethanol-induced microglia activation. METHODS: Quantitative real-time PCR was conducted to assess the expression of Tß4 and miR-339-5p. Western blot analysis was used to measure the expression of Tß4, phosphorylated p38, ERK, JNK, Akt, and NF-x03BA;B p65. The concentration of TNF-α and IL-1ß was determined using ELISA. NO concentration was measured using a nitric oxide colorimetric BioAssay Kit. Double immunofluorescence was performed to determine Tß4 expression, in order to assess microglial activation in neonatal mouse FASD model. RESULTS: Increased Tß4 expression was observed in ethanol treated microglia. Knockdown of Tß4 enhanced ethanol-induced inflammatory mediators tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) and nitric oxide (NO) in BV-2 cells was performed. Exogenous Tß4 treatment significantly inhibited expression and secretion of these inflammatory mediators. Tß4 treatment attenuated p38, ERK MAPKs, and nuclear factor-kappa B (NF-x03BA;B) pathway activation, and enhanced miR-339-5p expression induced by ethanol exposure in microglia. A neonatal mouse fetal alcohol spectrum disorders (FASD) model showed that Tß4 expression in the microglia of the hippocampus was markedly enhanced, while Tß4 treatment effectively blocked the ethanol-induced increase in inflammatory mediators, to the level expressed in vehicle-treated control animals. CONCLUSION: This study is the first to demonstrate the function of Tß4 in ethanol-induced microglia activation, thus contributing to a more robust understanding of the role of Tß4 treatment in CNS disease.

Subcellular dissemination of prothymosin alpha at normal physiology: immunohistochemical vis-a-vis western blotting perspective.

BACKGROUND: The cell type, cell status and specific localization of Prothymosin α (PTMA) within cells seemingly determine its function. PTMA undergoes 2 types of protease proteolytic modifications that are useful in elucidating its interactions with other molecules; a factor that typifies its roles. Preferably a nuclear protein, PTMA has been shown to function in the cytoplasm and extracellularly with much evidence leaning on pathognomonic status. As such, determination of its cellular distribution under normal physiological context while utilizing varied techniques is key to illuminating prospective validation of its distinct functions in different tissues. Differential distribution insights at normal physiology would also portent better basis for further clarification of its interactions and proteolytic modifications under pathological conditions like numerous cancer, ischemic stroke and immunomodulation. We therefore raised an antibody against the C terminal of PTMA to use in tandem with available antibody against the N terminal in a murine model to explicate the differences in its distribution in brain cell types and major peripheral organs through western blotting and immunohistochemical approaches. RESULTS: The newly generated antibody was applied against the N-terminal antibody to distinguish truncated versions of PTMA or deduce possible masking of the protein by other interacting molecules. Western blot analysis indicated presence of a truncated form of the protein only in the thymus, while immunohistochemical analysis showed that in brain hippocampus the full-length PTMA was stained prominently in the nucleus whereas in the stomach full-length PTMA staining was not observed in the nucleus but in the cytoplasm. CONCLUSION: Truncated PTMA could not be detected by western blotting when both antibodies were applied in all tissues examined except the thymus. However, immunohistochemistry revealed differential staining by these antibodies suggesting possible masking of epitopes by interacting molecules. The differential localization patterns observed in the context of nucleic versus cytoplasmic presence as well as punctate versus diffuse pattern in tissues and cell types, warrant further investigations as to the forms of PTMA interacting partners.

Prothymosin α and a prothymosin α-derived peptide enhance T(H)1-type immune responses against defined HER-2/neu epitopes.

BACKGROUND: Active cancer immunotherapies are beginning to yield clinical benefit, especially those using peptide-pulsed dendritic cells (DCs). Different adjuvants, including Toll-like receptor (TLR) agonists, commonly co-administered to cancer patients as part of a DC-based vaccine, are being widely tested in the clinical setting. However, endogenous DCs in tumor-bearing individuals are often dysfunctional, suggesting that ex vivo educated DCs might be superior inducers of anti-tumor immune responses. We have previously shown that prothymosin alpha (proTα) and its immunoreactive decapeptide proTα(100-109) induce the maturation of human DCs in vitro. The aim of this study was to investigate whether proTα- or proTα(100-109)-matured DCs are functionally competent and to provide preliminary evidence for the mode of action of these agents. RESULTS: Monocyte-derived DCs matured in vitro with proTα or proTα(100-109) express co-stimulatory molecules and secrete pro-inflammatory cytokines. ProTα- and proTα(100-109)-matured DCs pulsed with HER-2/neu peptides induce TH1-type immune responses, prime autologous naïve CD8-positive (+) T cells to lyse targets expressing the HER-2/neu epitopes and to express a polyfunctional profile, and stimulate CD4+ T cell proliferation in an HER-2/neu peptide-dependent manner. DC maturation induced by proTα and proTα(100-109) is likely mediated via TLR-4, as shown by assessing TLR-4 surface expression and the levels of the intracellular adaptor molecules TIRAP, MyD88 and TRIF. CONCLUSIONS: Our results suggest that proTα and proTα(100-109) induce both the maturation and the T cell stimulatory capacity of DCs. Although further studies are needed, evidence for a possible proTα and proTα(100-109) interaction with TLR-4 is provided. The initial hypothesis that proTα and the proTα-derived immunoactive decapeptide act as "alarmins", provides a rationale for their eventual use as adjuvants in DC-based anti-cancer immunotherapy.

Prothymosin-alpha inhibits HIV-1 via Toll-like receptor 4-mediated type I interferon induction.

Induction of type I interferons (IFN) is a central feature of innate immune responses to microbial pathogens and is mediated via Toll-like receptor (TLR)-dependent and -independent pathways. Prothymosin-alpha (ProTalpha), a small acidic protein produced and released by CD8(+) T cells, inhibits HIV-1, although the mechanism for its antiviral activity was not known. We demonstrate that exogenous ProTalpha acts as a ligand for TLR4 and stimulates type I IFN production to potently suppress HIV-1 after entry into cells. These activities are induced by native and recombinant ProTalpha, retained by an acidic peptide derived from ProTalpha, and lost in the absence of TLR4. Furthermore, we demonstrate that ProTalpha accounts for some of the soluble postintegration HIV-1 inhibitory activity long ascribed to CD8(+) cells. Thus, a protein produced by CD8(+) T cells of the adaptive immune system can exert potent viral suppressive activity through an innate immune response. Understanding the mechanism of IFN induction by ProTalpha may provide therapeutic leads for IFN-sensitive viruses.

Prothymosin alpha: a ubiquitous polypeptide with potential use in cancer diagnosis and therapy.

The thymus is a central lymphoid organ with crucial role in generating T cells and maintaining homeostasis of the immune system. More than 30 peptides, initially referred to as "thymic hormones," are produced by this gland. Although the majority of them have not been proven to be thymus-specific, thymic peptides comprise an effective group of regulators, mediating important immune functions. Thymosin fraction five (TFV) was the first thymic extract shown to stimulate lymphocyte proliferation and differentiation. Subsequent fractionation of TFV led to the isolation and characterization of a series of immunoactive peptides/polypeptides, members of the thymosin family. Extensive research on prothymosin α (proTα) and thymosin α1 (Tα1) showed that they are of clinical significance and potential medical use. They may serve as molecular markers for cancer prognosis and/or as therapeutic agents for treating immunodeficiencies, autoimmune diseases and malignancies. Although the molecular mechanisms underlying their effect are yet not fully elucidated, proTα and Tα1 could be considered as candidates for cancer immunotherapy. In this review, we will focus in principle on the eventual clinical utility of proTα, both as a tumor biomarker and in triggering anticancer immune responses. Considering the experience acquired via the use of Tα1 to treat cancer patients, we will also discuss potential approaches for the future introduction of proTα into the clinical setting.

Regional distribution and cell type-specific subcellular localization of Prothymosin alpha in brain.

Prothymosin alpha (ProTα) is an acidic nuclear protein implicated in several cellular functions including cell survival. ProTα is found in the central nervous system, but the regional and cell type-specific expression patterns are not known. In this study, our immunohistochemical analysis demonstrated that ProTα is expressed ubiquitously throughout adult brain with difference in the intensity of region-specific protein reactivity. Interestingly, the highest ProTα signals were observed in the brain regions relevant to neurogenesis, such as sub-ventricular zone, granular cell layer of dentate gyrus, as well as granule cell layer of olfactory bulb. Strong immunoreactivity was also found in habenula, ependymal cells lining the dorsal third and fourth ventricle, and in neurons in the Purkinje cell layer of cerebellum. We showed that ProTα was strictly localized in the nuclei of neurons, while it was found in the cytosolic space of astroglial and microglial processes and cell body in the adult brain. To clarify the phenomenon underlying cytosolic localization of ProTα in non-neuronal cells, ZVAD-fmk, a caspase-3 inhibitor, was delivered intracerebroventricularly in the brain. At the follow-up 24 h after ZVAD-fmk injection, we found that nuclear intensity of ProTα was significantly increased in astrocytes, whereas the ProTα expression was not affected in microglia. The present study would contribute toward better understanding of physiological and pathophysiological roles of ProTα in the brain.

Phenotypic characterization and ontogeny of mesodermal-derived and endocrine epithelial components of the human thymic microenvironment.

Using murine monoclonal antibodies TE-4 and TE-7 raised against human thymic stroma, we identified two distinct and mutually exclusive thymic microenvironment components: the thymic endocrine epithelium (TE-4+) and mesodermal-derived fibrous stroma (TE-7+). TE-4-reactive epithelium did not react with antibody TE-7, contained thymosin alpha 1 and keratin, and expressed other known markers of thymic endocrine epithelium (A2B5 and p19). Moreover, TE-4+ thymic epithelial cells strongly expressed class I (HLA-A, -B and -C) and class II (Ia-like) major histocompatibility complex (MHC) antigens. In contrast, TE-7+ thymic fibrous stroma did not react with antibody TE-4, did not contain thymosin alpha 1 nor keratin, and did not express the thymic endocrine epithelium markers A2B5 and p19. TE-7+ thymic stromal cells weakly expressed class I and did not express class II MHC antigens. Both TE-4+ and TE-7+ thymic microenvironment compartments were identifiable in thymus from 7 wk gestation through adult life. At 7 wk fetal gestation, TE-7+ stroma surrounded a cylindrical TE-4+, A2B5+ thymic epithelial rudiment. Between 10 and 15 wk fetal gestation, TE-7+ thymic stroma surrounded early thymic lobules. By 15 wk fetal gestation, antibody TE-4 defined subcapsular cortical and medullary zones of endocrine thymic epithelium, while antibody TE-7 bound to interlobular fibrous septae, vessels, and thymic fibrous capsule. While otherwise specific for endocrine thymic epithelium, antibody TE-4 reacted with the basal layer of squamous epithelium in skin, tonsil, conjunctiva, and upper esophagus.

Prothymosin alpha immunoactive carboxyl-terminal peptide TKKQKTDEDD stimulates lymphocyte reactions, induces dendritic cell maturation and adopts a beta-sheet conformation in a sequence-specific manner.

Prothymosin alpha (ProTalpha) is a small acidic polypeptide with important immunostimulatory properties, which we have previously shown to be exerted by its carboxyl (C)-terminus. It exerts immunoenhancing effects through stimulation of monocytes via toll-like receptor (TLR) triggering. Here, we assayed the activity of synthetic peptides homologous to ProTalpha's C-terminus to stimulate lymphocyte functions, in particular natural killer cell cytotoxicity of peripheral blood mononuclear cells isolated from healthy donors. A synthetic decapeptide TKKQKTDEDD was identified as the most potent lymphocyte stimulator. The activity of this peptide was sequence-specific and comparable to that of the intact molecule, suggesting that ProTalpha's immunoactive segment encompasses the nuclear localization signal sequence of the polypeptide. Because ProTalpha stimulates immune responses in a monocyte-dependent manner, we further investigated whether the entire molecule and its peptide TKKQKTDEDD specifically act on monocytes and show that both can promote maturation of monocyte-derived dendritic cells (DC). Finally, knowing that, under specific conditions, ProTalpha forms amyloid fibrils, we studied the amyloidogenic properties of its C-terminal peptide segments, utilizing ATR FT-IR spectroscopy and transmission electron microscopy (negative staining). Although the peptide TKKQKTDEDD adopts an antiparallel beta-sheet conformation under various conditions, it does not form amyloid fibrils; rather it aggregates in globular particles. These data, in conjunction with reports showing that the peptide TKKQKTDEDD is generated in vivo upon caspase-cleavage of ProTalpha during apoptosis, strengthen our hypothesis that immune response stimulation by ProTalpha is in principle exerted via its bioactive C-terminal decapaptide, which can acquire a sequence-specific beta-sheet conformation and induce DC maturation.

In Vitro Immunodetection of Prothymosin Alpha in Normal and Pathological Conditions.

Prothymosin alpha (ProTα) is a highly acidic polypeptide, ubiquitously expressed in almost all mammalian cells and tissues and consisting of 109 amino acids in humans. ProTα is known to act both, intracellularly, as an anti-apoptotic and proliferation mediator, and extracellularly, as a biologic response modifier mediating immune responses similar to molecules termed as "alarmins". Antibodies and immunochemical techniques for ProTα have played a leading role in the investigation of the biological role of ProTα, several aspects of which still remain unknown and contributed to unraveling the diagnostic and therapeutic potential of the polypeptide. This review deals with the so far reported antibodies along with the related immunodetection methodology for ProTα (immunoassays as well as immunohistochemical, immunocytological, immunoblotting, and immunoprecipitation techniques) and its application to biological samples of interest (tissue extracts and sections, cells, cell lysates and cell culture supernatants, body fluids), in health and disease states. In this context, literature information is critically discussed, and some concluding remarks are presented.

Thymosin-ß12 characteristics and function in Urechis unicinctus.

ß-thymosin family comprise a series of heat-stable multifunctional polypeptides involved in actin regulation, anti-inflammation, wound healing, cell migration, angiogenesis, cardiac protection, antimicrobial processes and antiviral immunity. The roles of Tß12 (thymosin-ß12) in marine invertebrates is still largely unknown, especially in terms of antibacterial immunity. In this study, we cloned the Tß12 gene with an ORF of 126 bp coding 41 amino acids from Urechis unicinctus. Tissue distribution analysis by qRT-PCR used TBP as reference gene showed that Tß12 was widely expressed in all tissues, and the transcript levels were the highest in the body wall, followed by the coelomic fluid, and the lowest in the intestines and anal sacs. After LPS (lipopolysaccharides) injection, Tß12 expression in the body was first elevated significantly at 3 h (p < 0.05), indicating that the body wall was the first defense line of the innate immune system; in the coelomic fluid, the Tß12 mRNA levels increased after LPS injection, with a significant increase occurring at 6 h, showing that coelomic fluid functioned as the second defense line of the innate immune system. In the midgut and anal sacs, a significant increase in the Tß12 level occurred at 24 h, suggesting that the midgut and anal sacs may act as accessory organs for the innate immune system. Moreover, U. unicinctus Tß12 recombinants can effectively inhibit the growth of both gram-negative and gram-positive bacteria. These results indicate that U. unicinctus Tß12 plays important roles in innate antibacterial immune responses, which can deepen our understanding of Tß12 in marine invertebrates.

Neutralization of HTLV-III/LAV replication by antiserum to thymosin alpha 1.

An antiserum prepared against thymosin alpha 1, a hormone secreted by the thymus gland, effectively neutralized the AIDS-associated virus [HTLV-III/LAV (clone BH-10)] and blocked its replication in H9 cells. Reverse transcriptase activity and expression of the HTLV-III/LAV antigens p15 and p24 were inhibited by purified immunoglobulin G preparations of antisera to thymosin alpha 1. The antiviral activity of the antiserum was found to be due to a region of homology between thymosin alpha 1 and p17, a product of the gag gene of HTLV-III/LAV. Comparison of the primary sequences of thymosin alpha 1 and the gag protein revealed a 44% to 50% homology in an 18-amino acid region, between positions 11 and 28 on thymosin alpha 1 and 92 and 109 on the gag protein. The effectiveness of the thymosin alpha 1 antiserum and of immunoglobulin G-enriched preparations in blocking replication of HTLV-III(BH-10) in H9 cells suggests a novel approach to the development of an AIDS vaccine. A vaccine directed against the gag protein might overcome the problem of genetic drift in the envelope region of the virus and be useful against all genetic variants of HTLV-III/LAV.

Molecular cloning and expression analysis of a beta-thymosin homologue from a gastropod abalone, Haliotis diversicolor supertexta.

The beta-thymosins are a family of highly conserved small peptides with multiple functions. In this study, we isolated the full-length cDNA of a beta-thymosin homologue from a gastropod abalone Haliotis diversicolor supertexta which we named ab-TMSB. The full-length cDNA of ab-TMSB consists of 499 bp with an ORF encoding a 43 amino acids protein. The deduced amino acid sequence of ab-TMSB shows 61-76% identity to other beta-thymosins and shares a conserved actin-binding domain. The phylogenetic analysis revealed that ab-TMSB was branched with Sycon raphanus beta-thymosin and clustered with Strongylocentrotus purpuratus beta-thymosin. Quantitative real-time PCR showed that ab-TMSB was ubiquitously expressed in abalone and highly expressed in hemocyte. Moreover, the expression level of ab-TMSB in hemocyte was upregulated after LPS challenge. Taken together, these results indicate that ab-TMSB is a beta-thymosin homologue and may be involved in the immune response of abalone H. diversicolor supertexta.

Immunocytological and preliminary immunohistochemical studies of prothymosin alpha, a human cancer-associated polypeptide, with a well-characterized polyclonal antibody.

Prothymosin alpha (ProTalpha) is a nuclear polypeptide of great biological and, possibly clinical, importance, because its expression levels have been associated with early diagnosis/prognosis of human cancer. It is therefore interesting to raise easily available and cost-effective antibodies that would be applied to develop reliable ProTalpha immunodiagnostics. In this study, New Zealand white rabbits and laying hens were parallel immunized against intact ProTalpha or the synthetic fragments ProTalpha[1-28], ProTalpha[87-109], and ProTalpha[101-109], all conjugated to keyhole limpet hemocyanin (KLH). The corresponding antibodies G and Y were immunochemically evaluated in parallel with ELISA and Western blot systems and applied to fluorescence immunocytology experiments using various cancer cell lines and normal cells. The antibody G raised against ProTalpha[101-109]/KLH had excellent functional characteristics in the Western blot and immunocytology experiments, where the fluorescent signal was almost exclusively shown in the cell nucleus independently of the cells assayed. The above antibody has been applied to preliminary IHC staining of human cancer prostate tissues, leading to a high percentage of clearly and intensively stained nuclei in the adenocarcinoma tissue; this antibody can be further used in cancer tissue immunostaining and in research concerning the role of ProTalpha in tumorigenesis.

Thymosin-alpha1 modulates dendritic cell differentiation and functional maturation from human peripheral blood CD14+ monocytes.

Although thymosins have been demonstrated to have immunomodulatory effects, it is still not clear whether they could affect dendritic cells (DCs), the most professional antigen-presenting cells. The objective of this study was to determine the effect and potential mechanisms of thymosin-alpha1 (Talpha1) on DC differentiation and functional maturation. Human peripheral blood CD14(+) monocytes were purified by using a magnetic separation column and cultured with GM-CSF and IL-4 to differentiate into immature DCs (iDCs). In the presence of Talpha1, iDC surface markers CD40, CD80, MHC class I and class II molecules were significantly upregulated as measured by flow cytemotry analysis. However, Tbeta4 or Tbeta10 did not show these effects on iDCs. There was an approximately 30% reduction in antigen (FITC-conjugated dextran)-uptake by Talpha1-treated iDCs as compared with non-Talpha1-treated iDCs. In addition, Talpha1-treated matured DCs (mDCs) showed an increased stimulation of allogeneic CD3(+) T-cell proliferation as measured by a mixed-lymphocyte reaction assay. Talpha1-treated mDCs also increased the production of several Th1- and Th2-type cytokines as measured by a Bio-Plex cytokine assay. Furthermore, rapid activation of p38 MAPK and NFkappaB was seen in Talpha1-treated iDCs as measured by a Bio-Plex phosphoprotein assay. Thus, Talpha1 significantly enhances DC differentiation, activation, and functions from human peripheral blood CD14(+) monocytes possibly through a mechanism of the activation of p38 MAPK and NFkappaB pathways. This study provides a basis to further evaluate Talpha1 as a possible adjuvant for a DC-directed vaccine or therapy.

Thymosin alpha1: an endogenous regulator of inflammation, immunity, and tolerance.

Thymosin alpha1 (Talpha1), first described and characterized by Allan Goldstein in 1972, is used worldwide for the treatment of some immunodeficiencies, malignancies, and infections. Although Talpha1 has shown a variety of effects on cells and pathways of the immune system, its central role in modulating dendritic cell (DC) function has only recently been appreciated. As DCs have the ability to sense infection and tissue stress and to translate collectively this information into an appropriate immune response, an action on DCs would predict a central role for Talpha1 in inducing different forms of immunity and tolerance. Recent results have shown that Talpha1: (a) primed DCs for antifungal Th1 resistance through Toll-like receptor (TLR)/MyD88-dependent signaling and this translated in vivo in protection against aspergillosis; (b) activated plasmacytoid DCs (pDC) via the TLR9/MyD88-dependent viral recognition, thus leading to the activation of interferon regulatory factor 7 and the promotion of the IFN-alpha/IFN-gamma-dependent effector pathway, which resulted in vivo in protection against primary murine cytomegalovirus infection; (c) induced indoleamine 2,3-dioxygenase activity in DCs, thus affecting tolerization toward self as well as microbial non-self-antigens, and this resulted in vivo in transplantation tolerance and protection from inflammatory allergy. Talpha1 is produced in vivo by cleavage of prothymosin alpha in diverse mammalian tissues. Our data qualify Talpha1 as an endogenous regulator of immune homeostasis and suggest that instructive immunotherapy with Talpha1, via DCs and tryptophan catabolism, could be at work to control inflammation, immunity, and tolerance in a variety of clinical settings.