Thymulin

Are zinc-bound metallothionein isoforms (I+II and III) involved in impaired thymulin production and thymic involution during ageing?

Eugenio Mocchegiani, Robertina Giacconi, Catia Cipriano +3 more

BACKGROUND: With advancing age, thymic efficiency shows progressive decline due to thymic involution allowing impaired cell-mediated immunity and the appearance of age-related diseases. The intrinsic cause of thymic involution is still undefined. Chronic inflammation and high glucocorticoids (GCs) may be involved. However, transgenic mice, with increased GC sensitivity and over expression of GC receptors, display delayed age-associated thymic involution. This fact suggests that other substances may affect thymic involution. Among them, both isoforms of metallothioneins (MTs) I+II and III are the major candidates because their increments leads to organ atrophy in constant stress and are induced by IL-6, which increases in ageing. Enhanced MTs in ageing allows constant sequester of zinc ions and no subsequent zinc release leading to low zinc ion bioavailability for thymic efficiency. This sequester is very limited in very old age. Thus, we have investigated the MTmRNA (I+II and III) in the thymus from young, old and very old mice. METHODS: MTmRNA and IL-6mRNA (RT-PCR) in the thymus from different donors were tested. Concomitantly, TECs proliferation, zinc ion bioavailability (ratio total thymulin/active thymulin), thymulin activity and corticosterone were tested from different donors. RESULTS: Both isoforms of MTmRNA and IL-6mRNA increase in old thymus coupled with low zinc ion bioavailability, reduced TECs proliferation, impaired thymulin activity and enhanced plasma corticosterone in comparison with young. Conversely, although the thymus is involuted in very old mice because of no changes in thymus weight in comparison to old mice, reduced MTmRNA, especially MT-I+II isoforms, and low IL6mRNA occur. Concomitantly, good zinc ion bioavailability, maintained TECs proliferation, satisfactory thymulin activity and reduced corticosterone are observed in very old mice. CONCLUSIONS: The concomitant increments by high IL-6 of both MT isoforms in the thymus from old mice may be involved in thymic involution because provoking low zinc ion bioavailability, which is relevant for thymic efficiency. By contrast, the limited increments of MTs by low IL-6 induce good zinc ion bioavailability and satisfactory thymic efficiency in very old mice. Therefore, abnormal increased MTs may provoke complete thymic involution during ageing and the possible appearance of age-related diseases. If their increments are instead limited by low inflammation, healthy ageing and longevity may be reached.

The thymus-neuroendocrine axis: physiology, molecular biology, and therapeutic potential of the thymic peptide thymulin.

Paula C Reggiani, Gustavo R Morel, Gloria M Cónsole +7 more

Thymulin is a thymic hormone exclusively produced by the thymic epithelial cells. It consists of a nonapeptide component coupled to the ion zinc, which confers biological activity to the molecule. After its discovery in the early 1970s, thymulin was characterized as a thymic hormone involved in several aspects of intrathymic and extrathymic T cell differentiation. Subsequently, 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 hypophysotropic peptide. In recent years, interest has arisen in the potential use of thymulin as a therapeutic agent. Thymulin was shown to possess anti-inflammatory and analgesic properties in the brain. Furthermore, an adenoviral vector harboring a synthetic gene for thymulin, stereotaxically injected in the rat brain, achieved a much longer expression than the adenovirally mediated expression in the brain of other genes, thus suggesting that an anti-inflammatory activity of thymulin prevents the immune system from destroying virus-transduced brain cells. Other studies suggest that thymulin gene therapy may also be a suitable therapeutic strategy to prevent some of the endocrine and metabolic alterations that typically appear in thymus-deficient animal models. The present article briefly reviews the literature on the physiology, molecular biology, and therapeutic potential of thymulin.

In vitro and in vivo effects of mercuric chloride on thymic endocrine activity, NK and NKT cell cytotoxicity, cytokine profiles (IL-2, IFN-gamma, IL-6): role of the nitric oxide-L-arginine pathway.

Lory Santarelli, Massimo Bracci, Eugenio Mocchegiani

Mercury (Hg2+) affects cell-mediated immunity, including thymulin production. Thymulin, a zinc-dependent thymic hormone synthesized by thymic epithelial cells (TECs), is involved in NK cell cytotoxicity and Th1 cytokine production (IL-2 and IFN-gamma), which in turn affect both NKT and classic NK spleen cell cytotoxicity. High doses of Hg2+ induce an inflammatory status, increased production of IL-6 and consequent Th1/Th2 imbalance as well as cell-mediated immune depression. The mechanisms by which Hg+ affects the cell-mediated immune response are still unclear. The nitric oxide (NO) pathway may be implicated. The aim of this work was to further explore its noxious role in innate and adaptive immunity and to study the possible role played by the NO pathway. Young Balb/c mice treated in vivo for 1 month with 1.0 mg HgCl2/kg b.w. showed low thymulin activity, depressed NO production (as measured by nitrite and nitrate plasma levels), impaired classic NK spleen cell cytotoxicity, decreased Th1 (IL-2 and IFN-gamma) cytokine profiles, and increased IL-6 production. In vitro, 10(-6) M of HgCl2 inhibited active thymulin kinetics, TEC proliferation, NKT cell cytotoxicity and Th1 cytokine production, whereas IL-6 increased. L-arginine restored thymulin activity, TEC proliferation, NKT cytotoxicity, cytokine profiles and nitrite and nitrate plasma levels both in vivo and in vitro. Since L-arginine is the substrate for NO production, it may compensate for the cell-mediated immune defect induced by HgCl2, via the arginine-NO-pathway. L-arginine is also able to reduce glomerular kidney IgG antibodies deposits induced by higher dose of HgCl2 administration.

[Reduced thymulin production during occupational exposure to lead].

L Santarelli, L Di Lorenzo, M Valentino +8 more

Thymulin is a thymic hormone that being activated by binding a zinc ion promotes differentiation and several functions of T lymphocytes. It has been demonstrated only in experimental animals that metallic lead (Pb) is able to cause adverse effects on thymocyte number and function. The objective of this study is to evaluate the plasmatic level of active thymulin of 58 male workers being exposed for more than one year to low lead doses with respect to 59 male never exposed workers. All these were subjected to anamnesis collection, medical examination and determination of blood lead (PbB), plasmatic lead (PbPl), plasmatic thymulin, urinary lead (PbU) and urinary zinc (ZnU) levels. The mean plasma concentration of active thymulin was significantly lower in lead exposed than in non exposed workers. Active thymulin was also significantly and negatively correlated to PbB, PbPl and PbU level and resulted to be significantly and negatively influenced by PbB. Lead exposed workers had slightly higher zinc concentration in urine than non exposed workers, increasing ZnU levels by class of PbB. It is the first time that a toxic effect of lead on plasmatic active thymulin levels is demonstrated in humans, particularly in occupationally exposed workers. This study opens perspectives for further research that would both confirm the results and verify the mechanisms of action of lead on thymulin either direct or indirect and the possible role of zinc.

Thymulin and the neuroendocrine system.

Rodolfo G Goya, Oscar A Brown, Jean-Marie Pléau +1 more

Thymulin is a thymic hormone exclusively produced by the thymic epithelial cells. It consists of a nonapeptide component coupled to the ion zinc, which confers biological activity to this molecule. After its discovery in the early 1970, thymulin was characterized as a thymic hormone involved in several aspects of intra- and extrathymic T-cell differentiation. Subsequently, it was demonstrated that thymulin production and secretion is strongly influenced by the neuroendocrine system. Conversely, an emerging core of information points to thymulin as a hypophysotropic peptide. Here we review the evidence supporting the hypothesis that thymulin is an important player in the hypophyso-thymic axis.

Primary thymic endocrine failure in HIV-1-infected children.

R Consolini, A Legitimo, M Milani

Thymulin, an essential hormone for the T lymphocyte differentiation process and function, was evaluated to asses thymic endocrine function in a cohort of 17 HIV-1-infected children aged between 2 months and 14 years, 18 seroreverted subjects and 47 normal controls. The rosette inhibition assay by Dardenne and Bach (1975) is the only method available to evaluate the biologically active form of this hormone (thymulin or Zn-facteur thymique sérique, Zn-FTS), as immunoassays cannot discriminate between thymulin and the inactive form of the hormone not containing Zn (FTS). HIV-1 patients presented undetectable or significantly lowered plasma levels of thymulin. Plasma zinc levels were significantly reduced in patients although inactive, zinc-unbound thymulin molecules were not demonstrated. The investigation of inhibitory anti-thymulin molecules performed in all patients was negative. Thymulin titers did not correlate with CD4+ lymphocyte count at the different disease stages. This study suggests that a primary thymic endocrine deficiency is present in HIV children. The critical importance of these results in assessing disease progression and a potential therapeutic approach are discussed.

Immunological evaluation of patients with beta-thalassemia major.

R Consolini, A Calleri, A Legitimo +1 more

Abnormalities in the immune system and zinc homeostasis in patients with beta-thalassemia major (TM) have been reported. Since zinc ion is essential for the efficiency of the immune system and is required to induce biological activity to thymulin (Zn-FTS), a biochemically defined thymic hormone, we investigated the plasma levels of zinc and both active thymulin (Zn-FTS) and total zinc saturable thymulin (Zn-FTS+FTS) in 18 patients with TM aged between 2 and 31 years and 22 normal controls of the same age. Inhibitory molecules anti-thymulin and the distribution of lymphocyte subsets were also analyzed. Patients with TM presented significantly lowered plasma zinc and thymulin levels when compared to normal subjects. The significant enhancement of the active form of the hormone after zinc addition in vitro suggests that low thymulin values found in TM are due not to a thymic failure in synthesizing and secreting the thymic hormone, but a defect in zinc saturation of the hormone. An impairment of cell subset distribution was also demonstrated. This study shows that zinc and thymulin deficiency contribute to the complex mechanisms underlying immune dysfunction in TM.

Different age-related effects of thymectomy in myasthenia gravis: role of thymoma, zinc, thymulin, IL-2 and IL-6.

E Mocchegiani, R Giacconi, M Muzzioli +4 more

Different age-related immune pathogenetic mechanisms in myasthenia gravis (MG) have been suggested because of restoration after thymectomy (Tx) of altered zinc, thymulin (TH) and T-cell subsets exclusively in early-onset patients (younger <50 years), not in late-onset patients (older >50 years). In this context interleukin-2 (IL-2), interleukin-6 (IL-6) and thymoma are crucial because both involved in MG pathogenesis and correlated with acetylcholine receptors (AchRs) Ab production. Moreover, IL-2 and IL-6 are zinc-dependent, are altered in aging and related with zinc and TH age-dependent declines. Moreover, zinc is relevant for immune efficiency. In order to confirm these different age-related pathogenetic mechanisms further, the role of thymoma, zinc, TH, IL-2 and IL-6 is studied in MG patients with generalized MG with and without thymoma before and 1 month and 1 year after Tx. The high IL-2, IL-6, zinc, and AChR Ab levels observed before Tx are significantly correlated each other in younger MG patients (<50 years) independently by thymoma and in older MG patients (>50 years) with thymoma. No correlations exist in older MG patients without thymoma. Thymulin is not correlated with other parameters considered to be both in younger and older MG patients independently by the thymoma. Thymectomy restores zinc; immune parameters and AChR Ab are exclusively in the younger group, not in the older one. These findings suggest that IL-2 and IL-6, via zinc, rather than TH, may be involved in different age-related pathogenetic mechanisms mainly in early-onset MG. By contrast, thymoma may be involved in MG etiology in late-onset representing, as such, a useful discriminant tool for MG etiology between early and late-onset MG patients. Because autoimmune phenomena may rise in aging, a parallelism with altered immune functions during aging is discussed.

Therapeutic application of zinc in human immunodeficiency virus against opportunistic infections.

E Mocchegiani, M Muzzioli

The relevance of zinc in resistance to infections by virus, fungi and bacteria is recognized because of its pivotal role in the efficiency of the entire immune system, in particular in conferring biological activity to a thymic hormone called thymulin, which has differentiation properties on T-cell lines. In infection with human immunodeficiency virus (HIV), the zinc-bound form of thymulin (active thymulin, ZnFTS) is strongly reduced in stage IV of the disease (Centers for Disease Control and Prevention classification) with concomitant decrements in CD4(+) cell count and zincemia values. The zinc-unbound form of thymulin (inactive thymulin, FTS) is, in contrast, very high. The in vitro addition of zinc to plasma samples induces a recovery of the thymulin active form, suggesting low zinc bioavailability as the cause of impaired thymic functions with consequent CD4(+) depletion. An analysis of risk factors for the incidence of recidivism opportunistic infections shows CD4(+) depletion and zinc deficiency to have significant scores. Supplementation with zinc for 1 mo (45 mg Zn(2+)/d) associated with zidovudine (AZT) therapy in stage IV induces recovery of active zinc-bound thymulin, of zincemia, of CD4(+) cells with concomitant reduction (50%) of recidivism opportunistic infections compared with the AZT-treated group. Complete disappearance of recidivism by Candida aesophagea or Pneumocystis carinii is observed after supplementation with zinc. The relative risk factors (CD4(+) depletion and zinc-deficiency) have lower scores in the HIV-positive zinc-treated group, confirming, as such, the relevance of zinc in opportunistic infections that involve extracellular matrix. Such an assumption is indirectly confirmed with new HAART, where no opportunistic infections occur. Indeed, HIV RNA is inversely correlated with both CD4(+) and zincemia values (r = -0.73, P<0.01) in HAART-treated subjects. Lower scores for the same relative factors for the appearance of opportunistic infections are present in HAART-treated subjects compared with those treated with AZT. These findings, on the one hand, show the poor efficacy of AZT therapy compared with HAART therapy for the progression of HIV, but on the other hand, they suggest that the lack of occurrence of opportunistic infections by HAART may also result from major zinc bioavailability. This further supports the key role played by zinc against opportunistic infections in HIV with a possible independent effect by either HIV or the pathogens involved.

Homeostasis, thymic hormones and aging.

R G Goya, F Bolognani

The thymic-pituitary axis constitutes a bidirectional circuit where the ascending feedback loop is effected by thymic factors of epithelial origin. The aim of the present article is, first, to introduce the idea of an immune-neuroendocrine homeostatic network in higher animals. Next, the relevance of the thymus in this network and the possible role of this gland in the neuroendocrine imbalances associated with aging are discussed. A number of studies are next reviewed which show that the endocrine thymus produces several bioactive molecules, generally called thymic hormones, which in addition to possessing immunoregulatory properties are also active on nervous and endocrine circuits. In particular, the reported activities of thymosin fraction five, thymosin alpha 1 and thymosin beta 4 on beta-endorphin, adrenocorticotropic hormone, glucocorticoids, luteinizing hormone-releasing hormone and luteinizing hormone secretion in different animal and cell models are reviewed. The known hypophysiotropic actions of other thymic hormones like thymulin, homeostatic thymus hormone and thymus factor are also summarized, and the impact of aging on pituitary responsiveness to thymic hormones is discussed. As a conclusion, it is proposed that in addition to its central role in the regulation of the immune function, the thymus gland may extend its influence to nonimmunologic components of the body, including the neuroendocrine system. The early onset of thymus involution might, therefore, act as a triggering event which would initiate the gradual decline in homeostatic potential that characterizes the aging process.

Zinc, T-cell pathways, aging: role of metallothioneins.

E Mocchegiani, M Muzzioli, C Cipriano +1 more

Zinc is an essential trace element for many biological functions, including immune functions. Indeed zinc is required for the biological activity of a thymic hormone, called thymulin in its zinc-bound form, important for the maturation and differentiation of T-cells. With advancing age zinc, thymic functions and peripheral immune efficiency show a progressive decline. Supplementing zinc in old age restores them. Zinc is also relevant for liver extrathymic T-cell pathway, being preeminent in old age. Since zinc is also required for metallothioneins (MTs) biological functions, binding zinc with high affinity, aim of the present article is to summarize findings from our laboratory regarding the role of zinc on T-cell pathways, investigating also the possible cause of thymic involution and impaired liver extrathymic T-cell pathway in aging. Partial hepatectomy and liver regeneration are good models for this aim because of the likeness with aging for many immune functions, including thymic functions. MTs levels are increased, other than into the liver, also into the thymus during aging and in young hepatectomized (pHx) mice as compared to young sham controls. MTs may be one of the possible causes of reduced thymic efficiency and impaired liver extrathymic T-cell pathway in old age because of their higher zinc binding affinity rather than thymulin with consequent reduction of the free quota of zinc available for normal cell-mediated immunity. Following that, MTs may contribute to thymic involution and impaired peripheral immune efficiency in aging and in young pHx mice with different roles during the whole life of an organism: protective in young-adult age which may became, at least, dangerous for immune responses in aging. In order to limit or avoid this latter MTs possible role in aging, supplementing physiological zinc may be useful to improve immune responses in old age because of no interference of endogenous zinc on already high thymus MTs levels, but with caution for competition phenomena with copper, as documented in old mice and in syndrome of accelerate aging.

The thymus-pituitary axis and its changes during aging.

R G Goya, O A Brown, F Bolognani

The pituitary-thymic axis constitutes a bidirectional circuit where the ascending feedback loop is effected by thymic factors of epithelial origin. The aim of the present article is to review the evidence demonstrating that aging brings about a progressive disruption in the integration of this network. In doing so, we briefly review the experimental evidence supporting the view that immune and neuroendocrine aging are interdependent processes. The advantages and limits of the nude mouse as a model of thymus-dependent accelerated aging is also discussed. Next, we review a number of studies which show that the endocrine thymus produces several bioactive molecules, generally called thymic hormones, which in addition to possessing immunoregulatory properties are also active on nervous and endocrine circuits. In particular, the reported activities of thymosin fraction 5 (TF5), thymosin alpha-1 and thymosin beta-4 on beta-endorphin, ACTH, glucocorticoids, LHRH and LH secretion in different animal and cell models are reviewed. The known hypophysiotropic actions of other thymic hormones like thymulin, homeostatic thymus hormone (HTH) and thymus factor are summarized. Aging has a significant impact on pituitary responsiveness to thymic hormones. Thus, it has been reported that TF5 and HTH have thyrotropin-inhibiting activity in young but not in old rats. Furthermore, intravenous administration of HTH was also able to reduce plasma GH and increase corticosterone levels in both young and old rats, although these responses were much weaker in the old animals. Further evidence on this topic is discussed. It is proposed that in addition to its central role in the regulation of the immune function, the thymus gland may extend its influence to nonimmunologic components of the body, including the neuroendocrine system. The early onset of thymus involution might therefore act as a triggering event which would initiate the gradual decline in homeostatic potential that characterizes the aging process.

The immune-endocrine loop during aging: role of growth hormone and insulin-like growth factor-I.

W Burgess, Q Liu, J Zhou +6 more

Why a primary lymphoid organ such as the thymus involutes during aging remains a fundamental question in immunology. Aging is associated with a decrease in plasma growth hormone (somatotropin) and IGF-I, and this somatopause of aging suggests a connection between the neuroendocrine and immune systems. Several investigators have demonstrated that treatment with either growth hormone or IGF-I restores architecture of the involuted thymus gland by reversing the loss of immature cortical thymocytes and preventing the decline in thymulin synthesis that occurs in old or GH-deficient animals and humans. The proliferation, differentiation and functions of other components of the immune system, including T and B cells, macrophages and neutrophils, also demonstrate age-associated decrements that can be restored by IGF-I. Knowledge of the mechanism by which cytokines and hormones influence hematopoietic cells is critical to improving the health of aged individuals. Our laboratory has recently demonstrated that IGF-I prevents apoptosis in promyeloid cells, which subsequently permits these cells to differentiate into neutrophils. We also demonstrated that IL-4 acts much like IGF-I to promote survival of promyeloid cells and to activate the enzyme phosphatidylinositol 3'-kinase (PI 3-kinase). However, the receptors for IGF-I and IL-4 are completely different, with the intracellular beta chains of the IGF receptor possessing intrinsic tyrosine kinase activity and the alpha and gammac subunit of the heterodimeric IL-4 receptor utilizing the Janus kinase family of nonreceptor protein kinases to tyrosine phosphorylate downstream targets. Both receptors share many of the components of the PI 3-kinase signal transduction pathway, converging at the level of insulin receptor substrate-1 or insulin receptor subtrate-2 (formally known as 4PS, or IL-4 Phosphorylated Substrate). Our investigations with IGF-I and IL-4 suggest that PI 3-kinase inhibits apoptosis by maintaining high levels of the anti-apoptotic protein Bcl-2. The sharing of common activation molecules, despite vastly different protein structures of their receptors, forms a molecular explanation for the possibility of cross talk between IL-4 and IGF-I in regulating many of the events associated with hematopoietic differentiation, proliferation and survival.

Zinc and immune function: the biological basis of altered resistance to infection.

A H Shankar, A S Prasad

Zinc is known to play a central role in the immune system, and zinc-deficient persons experience increased susceptibility to a variety of pathogens. The immunologic mechanisms whereby zinc modulates increased susceptibility to infection have been studied for several decades. It is clear that zinc affects multiple aspects of the immune system, from the barrier of the skin to gene regulation within lymphocytes. Zinc is crucial for normal development and function of cells mediating nonspecific immunity such as neutrophils and natural killer cells. Zinc deficiency also affects development of acquired immunity by preventing both the outgrowth and certain functions of T lymphocytes such as activation, Th1 cytokine production, and B lymphocyte help. Likewise, B lymphocyte development and antibody production, particularly immunoglobulin G, is compromised. The macrophage, a pivotal cell in many immunologic functions, is adversely affected by zinc deficiency, which can dysregulate intracellular killing, cytokine production, and phagocytosis. The effects of zinc on these key immunologic mediators is rooted in the myriad roles for zinc in basic cellular functions such as DNA replication, RNA transcription, cell division, and cell activation. Apoptosis is potentiated by zinc deficiency. Zinc also functions as an antioxidant and can stabilize membranes. This review explores these aspects of zinc biology of the immune system and attempts to provide a biological basis for the altered host resistance to infections observed during zinc deficiency and supplementation.

Zinc, thymic endocrine activity and mitogen responsiveness (PHA) in piglets exposed to maternal aflatoxicosis B1 and G1.

E Mocchegiani, A Corradi, L Santarelli +6 more

Growth retardation, thymic involution and impaired peripheral immune efficiency are constant events in piglets exposed to maternal aflatoxicosis. Zinc may play a key role because of its requirement for good immune responses, including thymic endocrine activity. Zinc is required to activate a thymic hormone, i.e. thymulin (ZnFTS), which is responsible for cell-mediated immunity. Zinc deficiency and decreased thymic endocrine activity are present in piglets fed from sows exposed to aflatoxins (AF) B1 and G1 as compared with healthy control piglets. In particular, active ZnFTS is decreased while concentrations of inactive thymulin (FTS) are high. The in vitro addition of zinc up to the plasma samples induces a reduction of inactive thymulin. The lymphocytes mitogen responsiveness (PHA) is decreased and a thymic cortical lymphocyte depletion is also present. These data suggest that the thymic defect, followed by impaired peripheral immune efficiency, may largely depend by the low peripheral zinc bioavailability to saturate all thymulin molecules produced.

Thymic endocrinology.

J W Hadden

The thymus involutes relatively early in life; cellular immune deficiencies of aging correspond to decline in function of the hypothalamic-pituitary-endocrine axis. Recent studies point to important roles for the pituitary, the pineal, and the autonomic nervous system as well as the thyroid, gonads and adrenals in the thymus integrity and function. Thymic function at the local level requires complex cellular interactions among thymic stromal cells and developing thymocytes involving paracrine and autocrine mediators including interleukins (ILs) 1, 2, 6, 7, 8, colony-stimulating factors (CSFs), interferon-gamma, thymosin alpha 1, and zinc-thymulin. An important endocrine function of the thymus is to package zinc in zinc-thymulin for delivery to the periphery. Thymic involution has been treated with interleukins, thymic hormones, growth hormone, prolactin, melatonin, zinc, and others. Our work to reverse thymic involution in hydrocortisone-treated, aged mice with interleukins, thymosin alpha 1, and zinc will be reviewed. Recent efforts to treat successfully immune deficiency in aged and cancer-bearing humans will be presented.

Zinc and metallothioneins on cellular immune effectiveness during liver regeneration in young and old mice.

E Mocchegiani, D Verbanac, L Santarelli +4 more

Partial hepatectomy in young mice (pHx) induces thymic atrophy, disregulation of thymocytes subsets and a strong accumulation of zinc in thymic tissue after 1-2 days of liver regeneration. Zinc is relevant for good immune functioning. Restoration of zinc into both the thymus and thymocytes subsets in the late period of liver regeneration is observed in young pHx mice. These findings have suggested a link between the thymus and the liver influencing T-cell functions and involving zinc. This kind of link could be relevant in aging because thymic involution, negative crude zinc balance and crippled immune functions are constant events. The preminence of a liver extrathymic T-cell pathway after pHx or during aging has been suggested. Thus the study of pHx in young and old mice may offer a good model to better understand the role played both by thymic involution and by liver extrathymic T-cell pathway and the role of zinc in these physiological processes during aging. Young pHx mice after 1-2 days of liver regeneration show: reduced thymic endocrine activity, increment of double negative (DN) thymocytes subsets, impairment of peripheral immune efficiency (PHA, NK activity and IL-2) and negative crude zinc balance, which are all restored in the late period of liver regeneration. By contrast the thymic and peripheral immune defects and the negative crude zinc balance, already present in old sham mice, are not modified during liver regeneration in old pHx mice. Circulating leukocytes and lymphocytes are not significantly modified both in young and old pHx mice as compared to respective sham controls. Zinc may also be crucial for extrathymic T-cells pathway, being preminent in aging, rather than in young age, due to its metallothioneins (MT) binding capacity. MT are significantly increased in young pHx and in aging inducing a low zinc-free quota for thymic and peripheral immune efficiency in young pHx mice, and for extrathymic T-cell pathway, in old age. Thus low zinc bioavailability, due to MT, may play a pivotal role, not only for thymocytes but also for liver extrathymic T-cell pathway.

Zinc-controlled Th1/Th2 switch significantly determines development of diseases.

J E Sprietsma

Functional, excessive-possibly temporary-deficiencies of the trace element zinc can change immune functions prematurely from predominantly cellular Th1 responses to humoral Th2 responses. T helper (Th1) cells produce cytokines such as interleukin-2 (IL-2) and interferon gamma, thereby controlling viral infections and other intracellular pathogens more effectively than Th2 responses through cytokines such as IL-4, IL-5, IL-6 and IL-10. The accelerated shift from the production of extra Th1 cells during these cellular immune activities to more Th2 cells with their predominantly humoral immune functions, caused by such a zinc deficiency, adversely influences the course of diseases such as leprosy, schistosomiasis, leishmaniasis and AIDS, and can result in allergies. It is noteworthy that AIDS viruses (HIVs) do not replicate in Th1 cells, which probably contain more zinc, but preferentially in the Th0 and Th2 cells; all the more so, because zinc and copper ions are known to inhibit intracellular HIV replication. Considering the above Th1/Th2 switch, real prospects seem to be offered of vaccination against such parasites as Leishmania and against HIVs.

Altered thymic endocrine activity along with impairments of peripheral zinc metabolism and T-lymphocyte populations are associated with myasthenia gravis: a follow-up study.

F Licastro, E Mocchegiani, N Fabris +6 more

Thymic endocrine activity was assessed by a bioassay to determine the basal activity of thymulin (TH), a zinc dependent hormone, and its in vitro reactivation in two different age groups of patients with myasthenia gravis (MG). Before thymectomy, basal TH plasma levels were increased in patients over the age of 50 years. Plasma zinc levels were increased in all patients, this increment being very high in old patients. One year after thymectomy both TH and zinc plasma levels decreased. While zinc plasma levels were within the normal ranges for their respective ages, TH levels were lower in young and higher in old patients than in age comparable controls. Young patients with MG showed increased CD3,DR positive peripheral T-cells as well as lymphocytes with the CD16,CD56 phenotype. An increment of CD3 positive cells along with CD4 and CD16,CD56 positive cells were found in older patients. Thymectomy partially affected blood lymphocyte representation only in young patients, since CD3,DR T-cells decreased one year after surgery. No significant variations in T-cell representation were found in old patients after thymectomy. Immunosuppression in thymectomized patients did not significantly affected TH and zinc plasma levels. Very high levels of TH and the presence of additional alterations in T-lymphocyte subsets in old patients suggested that differential age related pathogenetic immunological mechanisms might be associated with the disease.

Reversibility of the thymic involution and of age-related peripheral immune dysfunctions by zinc supplementation in old mice.

E Mocchegiani, L Santarelli, M Muzzioli +1 more

With advanced ageing the zinc pool undergoes progressive reduction as shown by the low zinc plasma levels and the negative crude zinc balance, both in humans and in rodents. It has been suggested that such zinc deficiency might be involved in many age-related immunological dysfunctions, including thymic failure. The relevance of zinc for good functioning of the entire immune system is, at present, well documented. In particular, zinc is required to confer biological activity to one of the best-known thymic peptides, thymulin, which is responsible for cell-mediated immunity. In deep zinc deficiencies, in humans and other animals, the low thymulin levels are due not to a primary failure of the thymus, but to a reduced peripheral saturation of thymic hormones by zinc ions. In aged mice both a reduced peripheral saturation of the hormone and a decreased production by the thymus were present. Oral zinc supplementation in old mice (22 months old) for 1 month induced a complete recovery of crude zinc balance from negative (-1.82) to positive values (+1.47), similar to those of young animals (+1.67). A full recovery of thymic functions with a regrowth of the organ and a partial restoration of the peripheral immune efficiency, as measured by mitogen responsiveness (PHA and ConA) and natural killer cell (NK) activity, were observed after zinc supplementation. These findings clearly pin-point for relevance of zinc for immune efficiency and suggest that the age-related thymic involution and peripheral immunological dysfunctions are not intrinsic and irreversible events but are largely dependent on the altered zinc pool.

The treatment of zinc deficiency is an immunotherapy.

J W Hadden

Role of the low zinc bioavailability on cellular immune effectiveness in cystic fibrosis.

E Mocchegiani, M Provinciali, G Di Stefano +5 more

An altered cellular immune response as a secondary phenomenon has been suggested to be probably involved in the bronchopulmonary infections by Pseudomonas aeruginosa in cystic fibrosis (CF). The difficulty to eradicate with modern anti-pseudomonal antibiotics the bronchopulmonary infections has led us to further investigate the possible existence of other cellular immune defects and their cause. Alterations in zinc turnover are present in CF. Zinc is relevant for good immune functioning. In particular, zinc is required to confer biological activity to thymulin (ZnFTS), a biochemically defined thymic hormone with a modulating action on cell-mediated immunity. The zinc-unbound form (FTS) is inactive and it can be unmasked by in vitro zinc addition to the plasma samples revealing the total amount of circulating thymulin (active + inactive). Marginal zinc deficiencies may prevent peripheral biological activation of active thymulin. Total zinc-saturable thymulin fractions in CF are similar to those observed in normal subjects, whereas the active quota is strongly reduced associated with concomitant high plasma levels of inactive thymulin compared to the values of healthy children (P < 0.01). A strict correlation exists between zinc and thymic hormone-saturable fraction (r = 0.87, P < 0.01) in CF. These findings suggest that the defect is not due to a thymic failure but to a reduced peripheral saturation of thymulin by zinc ions. This defect might depend on augmented plasma concentration of alpha 2-macroglobulin, which has a higher binding affinity for zinc than thymulin. T cell subsets are normal in CF. Reduced NK cell number and activity are present. Also, plasma IL-2 levels are reduced. The existence of positive correlations between zinc and IL-2 (r = 0.79, P < 0.01) and between zinc or active thymulin and NK activity (r = 0.70, P < 0.01 and r = 0.88, P < 0.01, respectively) suggest a close link among zinc failure, impaired IL-2 activity, low thymulin level, and reduced NK activity in CF patients with both normal and growth retardation. Although the role of NK cells is unknown in CF, a zinc supplementation, in order to induce a complete saturation of thymulin molecules, to correct some cellular immune defects and to improve the growth, may be suggested.

A trial of zinc supplementation in young rural Gambian children.

C J Bates, P H Evans, M Dardenne +7 more

The present study tested the hypothesis that inadequate Zn intake might be responsible for failure to thrive and impaired catch-up growth in young rural Gambian children, and that Zn supplements might be beneficial. Gambian children might be deprived of Zn because of its poor availability from their predominantly plant-based diet. Rural Gambian children (110; fifty boys, sixty girls) aged between 0.57 and 2.30 years were divided into two matched groups, one to receive 70 mg Zn twice weekly for 1.25 years, and the other a placebo. Growth and mid-upper-arm circumference were measured at weekly intervals throughout the study and illnesses were monitored. Capillary blood and urine samples were collected at 0, 2 and 8 weeks. Body weights and arm circumferences showed a linear increase, plus a seasonal effect (rainy season faltering). For body weight there was no significant overall effect of the supplement. For arm circumference, a very small (2%) but significant (P < 0.01) difference favoured the supplemented group. Plasma thymulin was much lower at the first clinic than at the second and third clinics, and in vitro Zn stimulation was greater at the first clinic. There was, however, no effect of Zn in vivo. Likewise, Zn did not significantly benefit T-cell numbers or ratios, secretory IgA in urine, circulating hormone levels or biochemical indices of Zn status. One index of intestinal permeability, i.e. lactulose: creatinine, was improved (P < 0.02) by the supplement, but the lactulose: mannitol value was not; this requires further investigation. Dietary Zn deficiency is, thus, unlikely to be of major overall importance for rural Gambian children's ability to thrive, and blanket Zn supplementation is not justified. There may, however, be vulnerable sub-groups who would benefit from Zn supplements.

Thymic involution in aging. Prospects for correction.

J W Hadden, P H Malec, J Coto +1 more

The thymus produces several putative thymic hormones: thymosin alpha 1, thymulin, and thymopoietin, which have been reported to circulate and to act on both prothymocytes and mature T cells in the periphery, thus maintaining their commitment to the T cell system. These endocrine influences decline with age and are associated with "thymic menopause" and cellular immune senescence, which contribute to the development of diseases in the aged. Thymus endocrinology is characterized by the action of many hormones and hormone-like substances on the cellular components of the thymus, including thymocytes, thymic epithelial cells, and thymic stromal cells. The intrathymic environment is characterized by a complex network of paracrine, autocrine, and endocrine signals involving both interleukins and thymic peptides, which can be envisioned to operate in a synergistic network to carry the evolving T cell through its stepwise development to a mature T cell. Extrathymic influences regulating the secretory function of thymic epithelial cells and the stepwise evolution of T cells can be ascribed to circulating interleukins, mainly IL1 and IL2, derived from activation and secretion of leukocytes in the periphery. These interleukins act in a synergistic fashion at all levels of T cell development by the induction of high-affinity IL2 receptors and the resultant IL2-dependent proliferative responses. To determine whether exogenous administration of interleukins would induce T lymphocyte development in aged mice, we chemically thymectomized aged mice with a steroid hormone and treated them with mixed interleukins or thymic hormones such as thymosin. We found that mixed interleukins, but not thymosin, restored thymic weight and cellularity and enhanced thymocyte responses to interleukins and mitogen. Thymosin potentiated the effect.

Thymic dysfunction in childhood T-acute lymphoblastic leukemia: a possible linkage with a primary thymus involvement.

R Consolini, A Legitimo, A Giorgianni +1 more

Experimental models, clinical and histopathological observations suggest a thymic origin of childhood T acute lymphoblastic leukemia (T-ALL). We studied thymic epithelial function in childhood T-ALL as compared to normal controls in order to improve our understanding of the cellular immunodeficiency mechanisms operating in a thymus-linked malignant process. The levels of Facteur Thymique Sérique (FTS) were measured in 9 patients at diagnosis, according to the rosette inhibition assay of Dardenne & Bach (1975). This method is based on the capacity of human serum containing FTS activity to confer on rosette-forming cells (RFC) from adult thymectomized mice a sensitivity to azathioprine identical to that of normal mouse RFC. All patients presented low age-corrected titres of FTS. No zinc deficiency was found, suggesting that low FTS levels are not related to unexpressed FTS biological activity. Plasma from all the children studied contained factors capable of inhibiting the biological activity of FTS in vitro. However, the nature of this inhibitor has not yet been elucidated. Our study shows the presence of a thymic dysfunction in childhood T-ALL, which could partially explain the immunodeficiency described in these patients. The linkage of the leukemic process with a primitive thymic involvement is discussed.

Interleukin 1 regulates secretion of zinc-thymulin by human thymic epithelial cells and its action on T-lymphocyte proliferation and nuclear protein kinase C.

J A Coto, E M Hadden, M Sauro +2 more

Thymic epithelial cells (TEC) are known to secrete thymic hormones that influence maturation of T lymphocytes. One of these peptides, thymulin, requires zinc in an equimolar ratio for biological activity. A previous study [Cousins, R. J. & Leinart, A. S. (1988) FASEB J. 2, 2884-2890] showed that interleukin 1 (IL-1) in vivo stimulates zinc uptake by the thymus. Both the alpha and beta forms of IL-1, which stimulate proliferation of human TEC, also stimulate their uptake of zinc in vitro, and this latter stimulation is both dependent and independent of proliferation. Zinc induces zinc accumulation without proliferation. Two other stimulants of proliferation, bovine pituitary extract and epidermal growth factor, stimulate zinc uptake by TEC, but only in a manner dependent on proliferation. Utilizing in situ hybridization, we show that the IL-1 alpha and beta forms and zinc induce metallothionein mRNA expression TEC. Metallothionein is thought to be involved in the transfer of zinc to thymulin. IL-1 was shown to stimulate the secretion of thymulin as measured both by its ability to stimulate induction of IL-2 receptor-positive lymphocytes from human peripheral blood lymphocytes and by the azathioprine-sensitive rosette assay. In addition, the zinc-thymulin complex in the presence, but not absence, of IL-1 stimulates nuclear protein kinase C in isolated lymphocyte nuclei. IL-1 apparently regulates the synthesis or secretion and delivery of zinc-thymulin complex to the T-lymphocyte system.

Thymic endocrinology.

J W Hadden

Thymus endocrinology is characterized by the action of various hormones on the thymus endocrine milieu consisting of thymocytes, thymic epithelial cells and thymic stromal cells. Extrathymic hormonal influences include pituitary-derived hormones, such as prolactin and indirectly by ACTH via hydrocortisone from the adrenal, by thyroid-stimulating hormone (TSH) via thyroid hormones from the thyroid, and by LH and RH via sex steroids from gonads and adrenal. In addition, the thymus produces several putative thymic hormones: thymosin alpha 1, thymulin and thymopoietin, which have been reported to circulate and to act on both prothymocytes and mature T-cells in the periphery thus maintaining their commitment to the T-cell system and its functions. These endocrine influences decline with age and are associated with "thymic menopause" and cellular immune senescence contributing to the development of diseases in the aged. The intrathymic environment is characterized by a complex network of paracrine and autocrine endocrine signals involving both interleukins and thymic peptides. Thymic epithelial cells respond to IL-1 with proliferation and secretion of IL6 and GM-CSF. They similarly respond to cellular interactions with the production of IL1. Thymic epithelial cells also secrete thymic hormones, as exemplified by the zinc-thymulin complex, under stimulation with IL1 and other hormonal influences. Thymic stromal cells contribute, at minimum, IL1. These various interleukin and thymic hormone influences can be envisioned to operate in a synergistic interactive network to carry the evolving T-cell through its stepwise development to a mature T-cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Arginine-containing compounds and thymic endocrine activity.

N Fabris, E Mocchegiani

The frequent association of malnutrition, infectious disease and aging has stressed the role played by some nutrients on the immune efficiency and particularly on the age-dependent immunological decline. Since arginine has been proven to enhance immune efficiency as demonstrated by the observation that supplemental dietary arginine accelerates would healing and increases thymus weight, we have evaluated the influence of oral administration of arginine on the age-associated immune deficiencies and in particular on the reduced thymic endocrine activity, as measured by the circulating level of one of the best known thymic peptides, i.e. thymulin. Thirty days oral treatment with arginine at the dose of 0.03 g/Kg b.w./day in 20 month old mice induces a full recovery of thymic endocrine activity and a significant increase of PHA responsiveness by spleen cells, when compared with untreated age-matched controls. In humans, oral administration of a commercially available arginine-lysin combination (Lysargin, Baldacci, Pisa, Italia) at the dose of 4 gr. of arg. + 4 gr. of Lysine induces a significant increment of thymulin blood level both in elderly and in cancer patients and at peripheral level, an increase of CD4+ lymphocyte subpopulation. These findings confirm the immunomodulation role of arginine and suggest that on the target of arginine as the thymus and particularly its endocrine activity. Furthermore arginine and arginine-containing compounds may offer a new therapeutical approach to restore thymic immunodeficiencies associated with age or secondary to pathologies inducing thymic deterioration such as trauma, stress and cancer.

Thymulin, a zinc-dependent hormone.

J F Bach, M Dardenne

Thymulin (formerly called FTS) is a well defined nonapeptide hormone produced by thymic epithelial cells. Its biological activity and antigenicity depend upon the presence of the metal zinc in the molecule. This pharmacologically active metallopeptide induces the differentiation of T-cells and enhances several functions of the various T-cell subsets in normal or partially thymus-deficient recipients. Its effect on suppressor T-cells is, so far, the most remarkable and should be the first to find useful clinical applications. The peptide is a natural hormone, available in synthetic form. It is not toxic and one may foresee its clinical use as one of the major immunoregulatory agents in the near future.

Serum thymulin in human zinc deficiency.

A S Prasad, S Meftah, J Abdallah +4 more

The activity of thymulin (a thymic hormone) is dependent on the presence of zinc in the molecule. We assayed serum thymulin activity in three models of mildly zinc-deficient (ZD) human subjects before and after zinc supplementation: (a) two human volunteers in whom a specific and mild zinc deficiency was induced by dietary means; (b) six mildly ZD adult sickle cell anemia (SCA) subjects; and (c) six mildly ZD adult non-SCA subjects. Their plasma zinc levels were normal and they showed no overt clinical manifestations of zinc deficiency. The diagnosis of mild zinc deficiency was based on the assay of zinc in lymphocytes, granulocytes, and platelets. Serum thymulin activity was decreased as a result of mild zinc deficiency and was corrected by in vivo and in vitro zinc supplementation, suggesting that this parameter was a sensitive indicator of zinc deficiency in humans. An increase in T101-, sIg-cells, decrease in T4+/T8+ ratio, and decreased IL 2 activity were observed in the experimental human model during the zinc depletion phase, all of which were corrected after repletion with zinc. Similar changes in lymphocyte subpopulation, correctable with zinc supplementation, were also observed in mildly ZD SCA subjects. Inasmuch as thymulin is known to induce intra- and extrathymic T cell differentiation, our studies provide a possible mechanism for the role of zinc on T cell functions.