Comparison of Thymulin Activity with Other Measures of Marginal Zinc Deficiency.

Activity of the immunoregulatory peptide thymulin reflects differences in zinc status. This study compared thymulin activity with four other zinc status measures in rats fed zinc at either 5 or 25 ppm. Rats fed the lower zinc showed the following results compared with rats with adequate zinc intake: serum thymulin activity 61% lower, serum zinc 31% lower, serum extracellular superoxide dismutase 18% lower, serum 5'-nucleotidase activity 26% lower, and liver metallothionein 28% lower. Thus, thymulin activities showed more sensitivity to restricted zinc intake than did four other parameters.

Sphingosine-1-Phosphate Receptor 1 Is Involved in Non-Obese Diabetic Mouse Thymocyte Migration Disorders.

NOD (non-obese diabetic) mice spontaneously develop type 1 diabetes following T cell-dependent destruction of pancreatic β cells. Several alterations are observed in the NOD thymus, including the presence of giant perivascular spaces (PVS) filled with single-positive (SP) CD4⁺ and CD8⁺ T cells that accumulate in the organ. These cells have a decreased expression of membrane CD49e (the α5 integrin chain of the fibronectin receptor VLA-5 (very late antigen-5). Herein, we observed lower sphingosine-1-phosphate receptor 1 (S1P1) expression in NOD mouse thymocytes when compared with controls, mainly in the mature SP CD4⁺CD62Lhi and CD8⁺CD62Lhi subpopulations bearing the CD49e− phenotype. In contrast, differences in S1P1 expression were not observed in mature CD49e⁺ thymocytes. Functionally, NOD CD49e− thymocytes had reduced S1P-driven migratory response, whereas CD49e⁺ cells were more responsive to S1P. We further noticed a decreased expression of the sphingosine-1-phosphate lyase (SGPL1) in NOD SP thymocytes, which can lead to a higher sphingosine-1-phosphate (S1P) expression around PVS and S1P1 internalization. In summary, our results indicate that the modulation of S1P1 expression and S1P/S1P1 interactions in NOD mouse thymocytes are part of the T-cell migratory disorder observed during the pathogenesis of type 1 diabetes.

A new adenovector system for implementing thymulin gene therapy for inflammatory disorders.

Thymulin is a thymic peptide possessing anti-inflammatory effects. In order to manipulate thymulin expression in gene therapy studies, we built a bidirectional regulatable two-vector Tet-Off system and the corresponding control system. The experimental two-vector system, ETV, consists of a recombinant adenovector (RAd) harboring an expression cassette centered on a Tet-Off bidirectional promoter flanked by a synthetic gene for thymulin and the gene for humanized Green Fluorescent Protein (hGFP). The second adenovector of this system, RAd-tTA, constitutively expresses the regulatory protein tTA. When cells are co-transduced by the two adenovector components, tTA activates the bidirectional promoter and both transgenes are expressed. In the presence of the antibiotic doxycycline (DOX) transgene expression is deactivated. The control two-vector system, termed CTV, is similar to ETV but only expresses hGFP. In CHO-K1, BHK, and C2C12 cells, ETV and CTV induced a dose-dependent hGFP expression. In CHO-K1 cells, transgene expression was almost completely inhibited by DOX (1mg/ml). After intracerebroventricular injection of ETV in rats, thymulin levels increased significantly in the cerebrospinal fluid and there was high hGFP expression in the ependymal cell layer. When injected intramuscularly the ETV system induced a progressive increase in serum thymulin levels, which were inhibited when DOX was added to the drinking water. We conclude that our regulatable two-adenovector system is an effective molecular tool for implementing short and long-term anti-inflammatory thymulin gene therapy in animal models of acute or chronic inflammation.

Hormonal control of T-cell development in health and disease.

The physiology of the thymus, the primary lymphoid organ in which T cells are generated, is controlled by hormones. Data from animal models indicate that several peptide and nonpeptide hormones act pleiotropically within the thymus to modulate the proliferation, differentiation, migration and death by apoptosis of developing thymocytes. For example, growth hormone and prolactin can enhance thymocyte proliferation and migration, whereas glucocorticoids lead to the apoptosis of these developing cells. The thymus undergoes progressive age-dependent atrophy with a loss of cells being generated and exported, therefore, hormone-based therapies are being developed as an alternative strategy to rejuvenate the organ, as well as to augment thymocyte proliferation and the export of mature T cells to peripheral lymphoid organs. Some hormones (such as growth hormone and progonadoliberin-1) are also being used as therapeutic agents to treat immunodeficiency disorders associated with thymic atrophy, such as HIV infection. In this Review, we discuss the accumulating data that shows the thymus gland is under complex and multifaceted hormonal control that affects the process of T-cell development in health and disease.

Semaphorin 3F and neuropilin-2 control the migration of human T-cell precursors.

Neuropilins and semaphorins are known as modulators of axon guidance, angiogenesis, and organogenesis in the developing nervous system, but have been recently evidenced as also playing a role in the immune system. Here we describe the expression and role of semaphorin 3F (SEMA3F) and its receptor neuropilin-2 (NRP2) in human T cell precursors. NRP2 and SEMA3F are expressed in the human thymus, in both lymphoid and non-lymphoid compartments. SEMA3F have a repulsive effect on thymocyte migration and inhibited CXCL12- and sphingosine-1-phosphate (S1P)-induced thymocyte migration by inhibiting cytoskeleton reorganization prior to stimuli. Moreover, NRP2 and SEMA3F are expressed in human T-cell acute lymphoblastic leukemia/lymphoma primary cells. In these tumor cells, SEMA3F also blocks their migration induced by CXCL12 and S1P. Our data show that SEMA3F and NRP2 are further regulators of human thymocyte migration in physiological and pathological conditions.

Physiology and therapeutic potential of the thymic peptide thymulin.

Thymulin is a thymic hormone exclusively produced by the epithelial cells of the thymus. After its discovery and initial characterization in the '70s, it was demonstrated that the production and secretion of thymulin are strongly influenced by the neuro-endocrine system. Conversely, a growing body of evidence, to be reviewed here, suggests that thymulin is a hypophysiotropic peptide. Additionally, a substantial body of information pointing to thymulin and a synthetic analog as anti-inflammatory and analgesic peptides in the central nervous system brain and other organs will be also reviewed. In recent years, a synthetic DNA sequence encoding a biologically active analog of thymulin, metFTS, was constructed and cloned in a number of adenovectors. These include bidirectional regulatable Tet-Off vector systems that simultaneously express metFTS and green fluorescent protein and that can be down-regulated reversibly by the addition of the antibiotic doxycycline. A number of recent studies indicate that gene therapy for thymulin may be an effective therapeutic strategy to prevent some of the hormonal and reproductive abnormalities that typically appear in congenitally athymic (nude) mice, used as a suitable model of neuroendocrine and reproductive aging. Summing up, this article briefly reviews the publications on the physiology of the thymulin-neuroendocrine axis and the anti-inflammatory properties of the molecule and its analog. The availability of novel biotechnological tools should boost basic studies on the molecular biology of thymulin and should also allow an assessment of the potential of gene therapy to restore circulating thymulin levels in thymodeficient animal models and eventually, in humans.

Mouse basophils reside in extracellular matrix-enriched bone marrow niches which control their motility.

Basophils co-express FcεRIα and CD49b, the α-2 chain of integrin-type receptor VLA-2 (α2ß1), which recognizes type-1 collagen as a major natural ligand. The physiological relevance of this integrin for interactions with extracellular bone marrow matrix remains unknown. Herein, we examined the expression of several receptors of this family by bone marrow-derived basophils sorted either ex-vivo or after culture with IL-3. Having established that both populations display CD49d, CD49e and CD49f (α-4, α-5 and α-6 integrins subunits, respectively), we addressed receptor functions by measuring migration, adhesion, proliferation and survival after interacting with matched natural ligands. Type I collagen, laminin and fibronectin promoted basophil migration/adhesion, the former being the most effective. None of these ligands affected basophil viability and expansion. Interactions between basophils and extracellular matrix are likely to play a role in situ, as supported by confocal 3D cell imaging of femoral bone marrow sections, which revealed basophils exclusively in type-1 collagen-enriched niches that contained likewise laminin and fibronectin. This is the first evidence for a structure/function relationship between basophils and extracellular matrix proteins inside the mouse bone marrow.

Growth hormone modulates migration of thymocytes and peripheral T cells.

In the context of immunoneuroendocrine cross talk, growth hormone (GH) exerts pleiotropic effects in the immune system. For example, GH-transgenic mice, as well as animals and humans treated with GH, exhibit enhanced cellularity in the thymus. GH also stimulates the thymic microenvironment, augmenting chemokine and extracellular matrix (ECM) production, with consequent increase in ECM- and chemokine-driven thymocyte migratory responses. Peripheral T cell migration triggered by laminin or fibronectin was enhanced in cells from GH-transgenic versus wild-type control adult mice, as seen for CD4(+) and CD8(+) T cells from mesenteric lymph nodes. Migration of these T lymphocytes, triggered by the chemokine CXCL12, in conjunction with laminin or fibronectin, was also enhanced compared with control counterparts. Considering that GH can be used as an adjuvant therapy in immunodeficiencies, including AIDS, the concepts defined herein, that GH enhances developing and peripheral T cell migration, provide new clues for future GH-related immune interventions.

Neonatal thymulin gene therapy prevents ovarian dysgenesis and attenuates reproductive derangements in nude female mice.

Congenitally athymic (nude) female mice show severe ovarian dysgenesis after puberty, which seems to be consequential to a number of neuroendocrine derangements described in these mutants. Thus, considerable evidence suggests that thymulin, a thymic peptide, may be involved in thymus-pituitary communication. In order to clarify the relevance of thymulin for the maturation of the female reproductive system, we assessed at hypothalamic, pituitary, ovarian, and uterine level the preventive action of neonatal thymulin gene therapy (NTGT) on the changes that typically occur after puberty in congenitally athymic female mice. We injected (im) an adenoviral vector harboring a synthetic DNA sequence encoding a biologically active analog of thymulin, methionine-serum thymic factor, in newborn nude mice (which are thymulin deficient) and killed the animals at 70-71 d of age. NTGT in the athymic mice restored the serum thymulin levels. Morphometric analysis revealed that athymic nudes have reduced numbers of brain GnRH neurons and pituitary gonadotropic cells as compared with heterozygous controls. NTGT prevented these changes and also rescued the premature ovarian failure phenotype typically observed in athymic nude mice (marked reduction in the number of antral follicles and corpora lutea, increase in atretic follicles). Serum estrogen, but not progesterone, levels were low in athymic nudes, a reduction that was partially prevented by NTGT. Little to no morphological changes were observed in the endometrium of female nudes. The delay in the age of vaginal opening that occurs in athymic nudes was significantly prevented by NTGT. Our results suggest that thymulin plays a relevant physiologic role in the thymus-hypothalamo-pituitary-gonadal axis.

Semaphorins and neuropilins: new players in the neuroendocrine control of the intrathymic T-cell migration in humans.

Cell migration is a key event for proper intrathymic T-cell differentiation, and several ligand-receptor interactions contribute to the well-co ordinated movement of developing thymocytes within the thymic lobules. Herein we summarize recent data that place semaphorin 3A (Sema3A) and its receptor neuropilin 1 (NRP1) as further players in the physiological process of cell migration in the human thymus. These molecules, as well as class A plexins (necessary for the intracellular signalling transduction triggered by Sema3A-NRP1 ligation), are constitutively expressed by both developing thymocytes and components of the thymic microenvironment, including epithelial and dendritic cells. Functionally, Sema3A decreases the adhesion of human thymocytes on thymic epithelial cell monolayers and exerts per se a dose-dependent chemorepulsive effect on human thymocytes. Moreover, Sema3A inhibits chemoattractant migratory responses induced by other ligands, including fibronectin, laminin and CXCL12 (chemokine CXC motif ligand 12). These data should be placed in the context of the concept that migration of developing T cells is a multivectorial system, in which the resulting migration vector derives from a balance of several simultaneous and/or sequential ligand-receptor pair interactions. Accordingly, semaphorins and neuropilins can be considered as further players in the system.

Inhibitory effect of semaphorin-3A, a known axon guidance molecule, in the human thymocyte migration induced by CXCL12.

Intrathymic T cell differentiation takes place within the thymic lobules and depends on interactions between developing thymocytes and cells of the thymic microenvironment. Along with differentiation, thymocytes migrate in an oriented progression, which is tightly regulated by a number of interactions, including one mediated by the chemokine CXCL12. It has been shown recently that SEMA-3A, a soluble member of the semaphorin family, is also involved in this human thymocyte migration and can have a chemorepulsive and de-adhesive role. Herein, we study the role of SEMA-3A on the CXCL12-driven migration of human thymocytes. We have shown that SEMA-3A is able to inhibit the chemotaxis triggered by CXCL12. Such an inhibition was seen in respect to immature and mature CD4/CD8-defined thymocyte subsets and can be reverted specifically by neutralizing anti-SEMA-3A mAb. We have also shown that SEMA-3A consistently down-regulates CXCR4 membrane expression in all CD4/CD8-defined thymocyte subsets, and this down-regulation is accompanied by a decrease in the phosphorylation of FAK and ZAP-70 protein kinases. Taken together, these results demonstrate the involvement of SEMA-3A in the regulation of CXCL12-driven human thymocyte migration, where it acts as a physiological antagonist.

Growth hormone is a modulator of lymphocyte migration.

Cell migration is crucial for intrathymic T cell differentiation and export of mature T lymphocytes to the peripheral lymphoid organs. The intrinsic regulation of T cell migration, mediated by adhesion molecules and chemokines, can be influenced by a number of endogenous factors, such as hormones, as for instance growth hormone (GH). Laminin deposition was enhanced in GH-treated mice and in GH-transgenic animals, compared with corresponding controls, and thymocyte adhesion to laminin was increased by in vivo GH treatment. An enhancing effect was also observed ex vivo in relation to the number of migrating cells in laminin-coated transwell chambers. Additionally, we found that the chemokine CXCL12, in conjunction with laminin, further enhanced the migration of thymocytes previously exposed to high concentrations of GH in vivo. Moreover, an increase in CXCL12 production has been detected in the thymus of GH-transgenic mice as well as in primary thymic epithelial cell cultures derived from these animals, as compared to age-matched wild-type counterparts. In keeping with these data, in vivo experiments showed that GH favors the trafficking of naive CD4+CD8- recent thymic emigrants to the peripheral lymph nodes. In addition, we found that migration of lymphocytes from mesenteric lymph nodes of GH-transgenic mice, triggered by the chemokine CXCL12, in conjunction with laminin or fibronectin, was enhanced, when compared to lymphocytes from control mice. Since GH-based therapy has been used in human and experimental infectious diseases, this hormone can be envisioned as an additional therapeutic tool in situations in which increasing lymphocyte numbers and migration are required for correcting a given pathological state.

Thymulin-based gene therapy and pituitary function in animal models of aging.

Thymulin is a thymic hormone exclusively produced by the thymic epithelial cells. After its discovery and initial characterization in the 1970s, it was demonstrated that thymulin production and secretion is strongly influenced by the neuroendocrine system. Conversely, a growing core of information, to be reviewed here, points to thymulin as a hypophysiotropic peptide. Additionally, thymulin was shown to possess anti-inflammatory and analgesic properties in the brain. In recent years, a synthetic DNA sequence coding for a biologically active analog of thymulin, metFTS, was constructed and cloned in different adenoviral vectors. These include bidirectional regulatable Tet-Off vector systems that simultaneously express metFTS and green fluorescent protein and that can be downregulated reversibly by the addition of the antibiotic doxycycline. A number of recent studies suggest that thymulin gene therapy may be a suitable therapeutic strategy to prevent some of the endocrine and reproductive alterations that typically appear in congenitally athymic (nude) mice, taken as a suitable model of neuroendocrine and reproductive aging. The present article briefly reviews the literature on the physiology of the thymulin-pituitary axis as well as on the new molecular tools available to exploit the therapeutic potential of thymulin.

Thymulin related peptide attenuates inflammation in the brain induced by intracerebroventricular endotoxin injection.

Based on significant amount of evidence, it is now generally believed, that one underlying cause for neurodegenerative diseases, could be dysregulation in inflammatory processes. The actual mechanisms involved are not yet well understood. Several studies have demonstrated the potent analgesic and anti-inflammatory actions of thymulin related peptide (PAT), in different animal pain models. In this study, we investigated the efficacy of PAT in a recently developed model of neuroinflammation, in conscious rats, caused by intracerbroventricular (ICV) injection of endotoxin (ET). Our results indicate that ICV injection of PAT alone did not elicit significant alteration of nociceptive thresholds, while ET injections produced significant thermal hyperalgesia and cold allodynia. Pretreatment with PAT resulted in significant alleviation of ET-induced hyperalgesia and increased body temperature. In other sets of experiments, ICV injection of ET resulted in a significant elevation in the concentration of pro-inflammatory mediators measured in different areas of the brain; this elevation was significantly following pretreatment with PAT. Taken together these results provide evidence in support of our hypothesis that as a potent anti-inflammatory and analgesic peptide, PAT might have potential therapeutic use for the treatment of neurodegenerative conditions induced by silent or overt inflammation.

Nutritional imbalances and infections affect the thymus: consequences on T-cell-mediated immune responses.

The thymus gland, where T lymphocyte development occurs, is targeted in malnutrition secondary to protein energy deficiency. There is a severe thymic atrophy, resulting from massive thymocyte apoptosis (particularly affecting the immature CD4+CD8+ cell subset) and decrease in cell proliferation. The thymic microenvironment (the non-lymphoid compartment that drives intrathymic T-cell development) is also affected in malnutrition: morphological changes in thymic epithelial cells were found, together with a decrease of thymic hormone production, as well as an increase of intrathymic contents of extracellular proteins. Profound changes in the thymus can also be seen in deficiencies of vitamins and trace elements. Taking Zn deficiency as an example, there is a substantial thymic atrophy. Importantly, marginal Zn deficiency in AIDS subjects, children with diarrhoea and elderly persons, significantly impairs the host's immunity, resulting in an increased risk of opportunistic infections and mortality; effects that are reversed by Zn supplementation. Thymic changes also occur in acute infectious diseases, including a severe thymic atrophy, mainly due to the depletion of CD4+CD8+ thymocytes, decrease in thymocyte proliferation, in parallel to densification of the epithelial network and increase in the extracellular matrix contents, with consequent disturbances in thymocyte migration and export. In conclusion, the thymus is targeted in several conditions of malnutrition as well as in acute infections. These changes are related to the impaired peripheral immune response seen in malnourished and infected individuals. Thus, strategies inducing thymus replenishment should be considered as adjuvant therapeutics to improve immunity in malnutrition and/or acute infectious diseases.

Pleiotropic modulation of thymic functions by growth hormone: from physiology to therapy.

In the context of the cross-talk between the neuroendocrine and immune systems, it is well known that growth hormone (GH) exerts physiological effects in central as well as peripheral compartments of the immune system. GH modulates a variety of thymic functions, including proliferation of thymocytes and thymic epithelial cells (TEC). Accordingly, GH-transgenic mice, as well as animals and humans treated with exogenous GH, exhibit an enhanced cellularity in the organ. GH also stimulates the secretion of cytokines and chemokines by the thymic microenvironment, as well as the production of extracellular matrix proteins. These effects lead to an increase in thymocyte migratory responses and intrathymic traffic of developing T cells, including the export of thymocytes from the organ, as ascertained by experimental studies with intrathymic injection of GH in normal mice and with GH-transgenic animals. Most likely, GH effects in the thymus are mediated by an IGF-1/IGF-1 receptor circuitry, which physiologically operates in nonstimulated conditions in both thymocytes and TECs. Since GH enhances thymus replenishment and increases intrathymic T-cell traffic, ultimately modulating thymocyte exit, it should be placed as a potential adjuvant therapeutic agent in the treatment of immunodeficiencies associated with thymic atrophy, and examples recently appeared in the literature are promising and strongly indicate that GH can be beneficial for individuals suffering severe immunodeficiency.

Combined role of extracellular matrix and chemokines on peripheral lymphocyte migration in growth hormone transgenic mice.

Previous evidence indicated that growth hormone (GH) modulates cell migration in the thymus, and that extracellular matrix and chemokines are involved. Herein, we studied migration of peripheral lymphocytes derived from spleen and lymph nodes of GH-transgenic (GH-Tg) mice. We initially found that the relative cell numbers (normalized per gram of body weight) in lymph nodes and spleens from GH-Tg were higher at all ages tested (2-3, 7 and 12 months), as compared to wild type age-matched controls. Functionally, we found that lymphocyte migration triggered by laminin or fibronectin was enhanced in cells from GH-Tg versus control mice, independent of the organ from which the cells were derived (as ascertained in young adult animals). However, such an enhancement in migration was statistically significant only for CD4+ and CD8+ T cells from mesenteric lymph nodes. Migration of lymphocytes from mesenteric lymph nodes of GH-Tg mice, triggered by the chemokine CXCL12, in conjunction with laminin or fibronectin, was enhanced compared to lymphocytes from control mice. Rather surprisingly, the membrane levels of the corresponding extracellular matrix or chemokine receptors in peripheral lymphoid organs of GH-Tg mice did not necessarily correlate with the changes seen in migratory responses. In conclusion, our data show for the first time that GH alters lymphocyte migration in the periphery of the immune system. Considering that GH is used as an adjuvant therapeutic agent in immunodeficiencies, including AIDS, the concepts defined herein provide relevant background knowledge for future GH-related immune interventions.

RNA interference-mediated knockdown of CD49e (α5 integrin chain) in human thymic epithelial cells modulates the expression of multiple genes and decreases thymocyte adhesion.

BACKGROUND: The thymus is a central lymphoid organ, in which bone marrow-derived T cell precursors undergo a complex process of maturation. Developing thymocytes interact with thymic microenvironment in a defined spatial order. A component of thymic microenvironment, the thymic epithelial cells, is crucial for the maturation of T-lymphocytes through cell-cell contact, cell matrix interactions and secretory of cytokines/chemokines. There is evidence that extracellular matrix molecules play a fundamental role in guiding differentiating thymocytes in both cortical and medullary regions of the thymic lobules. The interaction between the integrin α5ß1 (CD49e/CD29; VLA-5) and fibronectin is relevant for thymocyte adhesion and migration within the thymic tissue. Our previous results have shown that adhesion of thymocytes to cultured TEC line is enhanced in the presence of fibronectin, and can be blocked with anti-VLA-5 antibody. RESULTS: Herein, we studied the role of CD49e expressed by the human thymic epithelium. For this purpose we knocked down the CD49e by means of RNA interference. This procedure resulted in the modulation of more than 100 genes, some of them coding for other proteins also involved in adhesion of thymocytes; others related to signaling pathways triggered after integrin activation, or even involved in the control of F-actin stress fiber formation. Functionally, we demonstrated that disruption of VLA-5 in human TEC by CD49e-siRNA-induced gene knockdown decreased the ability of TEC to promote thymocyte adhesion. Such a decrease comprised all CD4/CD8-defined thymocyte subsets. CONCLUSION: Conceptually, our findings unravel the complexity of gene regulation, as regards key genes involved in the heterocellular cell adhesion between developing thymocytes and the major component of the thymic microenvironment, an interaction that is a mandatory event for proper intrathymic T cell differentiation.

Growth hormone modulates migration of developing T cells.

In the context of the cross-talk between the neuroendocrine and immune systems, it is well known that growth hormone (GH) exerts physiological effects in central as well as peripheral compartments of the immune system. GH modulates a variety of thymic functions. For example, GH upregulates proliferation of thymocytes and thymic epithelial cells. Accordingly, GH-transgenic mice, as well as animals and humans treated with exogenous GH, exhibit an enhanced cellularity in the thymus organ. GH also stimulates the secretion of thymic hormones, cytokines, and chemokines by the thymic microenvironment as well as the production of extracellular matrix proteins. These effects lead to an increase in thymocyte migratory responses and intrathymic traffic of developing T cells, including the export of thymocytes from the thymus organ, as ascertained by experimental studies with intrathymic injection of GH in normal mice and with GH-transgenic animals. Because GH promotes a replenishment of the thymus and an increase of thymocyte export, it has been applied as a potential adjuvant therapeutic agent in the treatment of immunodeficiencies associated with thymic atrophy.