Targeting inflammatory components in neuropathic pain: The analgesic effect of thymulin related peptide.

Neuropathic pain is considered to be pathological in nature and has been shown to involve, at least partially, dysregulated inflammatory processes. It is a severe chronic disease that can develop following lesions to the central nervous system or to peripheral nerves. The peripheral nerve damage can be caused by either diseases such as diabetes, or by trauma. A common underlying mechanism of neuropathic pain is the presence of inflammation at the site of the damaged or affected nerve(s). This inflammatory response, especially when unresolved, initiates and maintains a cascade of events resulting in the activation of innate immune cells at the site of tissue injury. The release of inflammatory mediators such as cytokines, neurotrophic factors, and chemokines initiates local actions and can result in a more generalized immune response. The resultant neuroinflammatory environment can cause activation of glial cells, which can release, in an uncontrolled manner, more of these mediators and exasperate the situation, thus having a prominent role in nociception. The neuropathic pain pathophysiology is complex and includes peripheral and central neuronal alterations as well as neuro-immune interactions, which become more prominent during inflammatory reactions. This report focuses on how targeting inflammatory mediators may result in novel therapeutic approaches to neuropathic pain management.

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.

[Thymus and adaptive changes of bone marrow cell composition in animals of different age].

The influence of thymus removal or administration of thymalin, thymic peptide factor, on number of cells-precursors for colonies of fibroblasts (CFC-F) and for granulocyte-macrophageal colonies (CFC-GM), CD(4+)-cells in bone marrow in CBA mice of different age in some seasons was investigated. It was shown that in adult mice the orientation of influence thymic activity modulation on amount of these types cells is dependent from seasons. It was shown the season possibility of direct effect of thymic stroma supernatant, in which was thymic serum factor (FTS), or the synthetic FTS on the amount of CFC-F and CFC-GM in bone marrow of adult mice; in old mice thymic supernatant increased in vitro the amount of CD(4+)-cells. FTS level in old mice increased after administration of thymalin, the rhythm of CD(4+)-cells number in bone marrow and of corticosterone serum content restored.

Thymus hormones as prospective anti-inflammatory agents.

IMPORTANCE OF THE FIELD: Inflammatory diseases are characterized by severe immune imbalances, leading to excessive or inappropriate release of mediators, which, in turn, result in massive damage to organs and systems. Effective means to control inappropriate immune reactions are often life-critical needs. Available data on the role of thymus-derived hormones in inflammation show their great potential. AREAS COVERED IN THIS REVIEW: The review aims to systematize information for the last two decades on immune system regulation by thymic peptide hormones, with a primary focus on the role of these hormones in the systemic inflammatory response and inflammatory diseases. Anti-inflammatory potential of three thymic hormones - thymulin, thymosin-alpha, and thymopoietin - is discussed, reviewing recently published clinical and experimental studies. WHAT THE READER WILL GAIN: Our analysis revealed the regulation of inflammatory processes via thymic hormones that could be prospective for therapeutic application. This regulation may be mediated through thymic hormone effects on peripheral immune cell activities and bidirectional coupling between thymic hormones and the hypothalamic-pituitary-adrenal axis. TAKE-HOME MESSAGE: In view of the role of thymic hormones in immune and neuroendocrine systems, they could be suitable as therapeutic agents for inflammation.

Immunomodulatory role of thymulin in lung diseases.

IMPORTANCE OF THE FIELD: Inflammation is a hallmark of lung diseases. The available treatment options are unsatisfactory because they are not efficacious or induce major side effects. Alternative approaches need to be developed. Thymulin is a peptide exclusively produced in the thymus with several anti-inflammatory properties. AREAS COVERED IN THIS REVIEW: The physiological features of thymulin and data that support its potential as an anti-inflammatory treatment for lung diseases are reviewed. WHAT THE READER WILL GAIN: Thymulin has consistent beneficial effects in experimental models of lung diseases. It has a broad inhibitory effect on pro-inflammatory cytokines, suppresses p38 (a MAPK family member) and inhibits the activation of the NF-kappaB signal pathway. It is an attractive peptide for lung gene therapy because has no toxicity even at high doses and when expressed by adenoviral vectors reduces immune response against viral proteins. TAKE HOME MESSAGE: Thymulin has a selective immunomodulatory effect, enhancing anti-inflammatory and inhibiting pro-inflammatory cytokines. It suppresses p38 (implicated in glucocorticoid-resistance) and inhibits NF-kappaB activation, which has an important pathogenic role in several lung diseases. The broad spectrum of anti-inflammatory effects of this peptide in several animal models of lung disease makes thymulin a good candidate for future clinical trials.

Endotoxin-mediated regulation of nuclear factor-kappaB nuclear translocation and activation in the hippocampus of the central nervous system: modulation by intracerebroventricular treatment with thymulin and the immunomodulatory role of the IkappaB-alpha/pIkappaB-alpha pathway.

The transcription factor nuclear factor kappaB (NF-kappaB) is one member of a ubiquitously expressed family of Rel-related transcription factors that serve as critical regulators of many proinflammatory genes and immunomodulators. Nevertheless, the immunomodulatory potential of thymulin and its effect on NF-kappaB in vivo, and particularly in the central nervous system (CNS), is not well characterized. In this study, the role of endotoxin (ET/LPS) in regulating NF-kappaB was deciphered in various compartments of the CNS. Stereotaxic localization reverberated specific intracerebroventricular (ICV) injection of ET into the CNS, with or without pretreatment with ICV thymulin. Treatment with ET (1 microg for 45 min; ICV) upregulated the expression and nuclear localization of NF-kappaB(1) (p50), NF-kappaB(2) (p52), RelA (p65), RelB (p68) and c-Rel (p75) in the hippocampus (HC), an effect abrogated, in a dose-dependent manner, by ICV pretreatment (30 min) with thymulin. Thymulin modulated the phosphorylation of IkappaB-alpha in the HC by upregulating the cytosolic accumulation of IkappaB-alpha and downregulating its phosphorylation (pIkappaB-alpha). Further analysis of the DNA-binding activity revealed an upregulated activity in the HC relative to saline-constitutive expression of the RelA (p65) subunit, the specificity of which was determined by a mutant oligonucleotide of RelA and a cold, non-specific competitor. ET did not induce the DNA-binding activity of NF-kappaB in the diencephalon (DE) or substantia nigra (SN) at various time points, when compared with baseline levels of expression. Intraperitoneal (IP) injections of ET (25 microg for 15 min) in vivo upregulated the expression of NF-kappaB subunits in the liver and reduced the cytosolic accumulation of IkappaB-alpha by inducing pIkappaB-alpha. Furthermore, IP pretreatment with thymulin followed by ICV injection of ET attenuated and reduced the DNA-binding activity of NF-kappaB in the HC. These results indicate that ICV injection of ET regulates the nuclear translocation and activation of NF-kappaB subunits within specific compartments in the brain, an effect particularly localized to the hippocampus. Additionally, thymulin attenuated the ET-induced response, with particular involvement of the transduction pathway implicating IkappaB-alpha, the major cytosolic inhibitor of NF-kappaB. The in vivo molecular regulation of thymulin via the NF-kappaB pathway is critical to understanding the alleviating anti-inflammatory role of this nonapeptide and paving the way to unraveling pathways associated with neuroimmune interactions mediating proinflammatory signals in the CNS.

Thymulin gene therapy prevents the reduction in circulating gonadotropins induced by thymulin deficiency in mice.

Integrity of the thymus during perinatal life is necessary for a proper maturation of the pituitary-gonadal axis in mice and other mammalian species. Thus congenitally athymic (nude) female mice show significantly reduced levels of circulating gonadotropins, a fact that seems to be causally related to a number of reproductive derangements described in these mutants. Interestingly, a number of in vitro studies suggest that the thymic peptide thymulin may be involved in thymus-pituitary communication. To determine the consequences of low serum thymulin in otherwise normal animals, we induced short (8 days)- and long (33 days)-term thymulin deficiency in C57BL/6 mice by neonatally injecting (intraperitoneally) an anti-thymulin serum and assessed their circulating gonadotropin levels at puberty and thereafter. Control mice received an irrelevant antiserum. Gonadotropins were measured by radioimmunoassay and thymulin by bioassay. Both long- and short-term serum thymulin immunoneutralization resulted in a significant reduction in the serum levels of gonadotropins at 33 and 45 days of age. Subsequently, we injected (intramuscularly) an adenoviral vector harboring a synthetic DNA sequence (5'-ATGCAAGCCAAATCTCAAGGTGGATCCAACTAGTAG-3') encoding a biologically active analog of thymulin, methionine-FTS, in newborn nude mice (which are thymulin deficient) and measured circulating gonadotropin levels when the animals reached 52 days of age. It was observed that neonatal thymulin gene therapy in the athymic mice restored their serum thymulin levels and prevented the reduction in circulating gonadotropin levels that typically emerges in these mutants after puberty. Our results indicate that thymulin plays a relevant physiological role in the thymus-pituitary-gonadal axis.

Partial prevention of hepatic lipid alterations in nude mice by neonatal thymulin gene therapy.

During adult life athymic (nude) male mice display not only a severe T-cell-related immunodeficiency but also endocrine imbalances and a moderate hyperglycemia. We studied the impact of congenital athymia on hepatic lipid composition and also assessed the ability of neonatal thymulin gene therapy to prevent the effects of athymia. We constructed a recombinant adenoviral vector, RAd-metFTS, expressing a synthetic DNA sequence encoding met-FTS, an analog of the thymic peptide facteur thymique sérique (FTS), whose Zn-bound biologically active form is known as thymulin. On postnatal day 1-2 homozygous (nu/nu) nude and heterozygous (nu/+) mice were injected with 10(8) pfu of RAd-metFTS or RAd-betagal (control vector) intramuscularly. The animals were processed at 52 d of age. Serum thymulin, glycemia, hepatic phospholipid FA composition and free and esterified cholesterol were determined. Adult homozygous male nudes were significantly (P < 0.01) hyperglycemic when compared with their heterozygous counterparts (2.04 vs. 1.40 g/L, respectively). The relative percentage of 16:0, 18:1 n-9, and 18:1n-7 FA was lower, whereas that of 18:0, 20:4n-6, and 22:6n-3 FA was higher, in hepatic phospholipid (PL) of nu/nu animals as compared with their nu/+ counterparts. Some of these alterations, such as that in the relative content of 22:6n-3 in liver PL and the unsaturation index, were completely or partially prevented by neonatal thymulin gene therapy. We conclude that the thymus influences lipid metabolism and that thymulin is involved in this modulatory activity.

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.

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.

[Biologically active thymic factors and melatonin-producing pineal function in mice in aging].

The aging peculiarities of thymic factors influence on pineal gland function in CBA mice were investigated. It was shown that in serum of the adult mice (3-4 m) melatonin level increased significantly in 3 h after one injection of thymic preparation "thymaline". The activating influence ofthymaline depended on season and linked with increased level of thymic serum factor (FTS). Thymic stroma supernatant of adult mice with high FTS level increased melatonin-producing pineal function in vitro. There was no activating influence of thymic factors on pineal function of old mice (23-24 mo) in vivo and in vitro as well as the pineal seasonal reaction on the administration of thymaline. The importance of thymic factors influence on melatonine level for the immune system rhythmic functioning was discussed.

Effects of infantile thymectomy on ovarian functions and gonadotrophin-induced ovulation in prepubertal mice: role of thymulin.

The effects of thymectomy performed on 10-day-old (Tx-10) mice on spontaneous puberty and the ovulatory response induced by gonadotrophin treatment were analysed, together with the effects of thymulin replacement from 10 days of age. Infantile thymectomy induced a delay of puberty, a decrease in serum 17beta-oestradiol concentration and a reduced total number of follicles. Injection of thymulin (12 ng/g body weight) to Tx-10 mice resulted in an earlier onset of puberty, a decrease in the weights of ovaries and uterus, and an increase in serum 17beta-oestradiol concentrations. In control and Tx-10 mice, treatment with pregnant mare serum gonadotrophin (PMSG) (5 IU) at 25 days of age resulted in ovulation and the numbers of ova shed by ovulating animals were similar. When the animals were injected with 1 IU PMSG ovulation did not occur. In Tx-10 mice, both 1 and 5 IU PMSG increased the number of follicles to values similar to those observed in the controls. In Tx-10 mice the sequential injection of PMSG (1 IU) and human chorionic gonadotrophin (hCG) (3 IU) resulted in ovulation, but the number of ova shed was lower than in controls. When these animals were injected daily with thymulin, an increase in the number of ova shed and serum 17beta-oestradiol concentrations was observed. The uterine weight of Tx-10 mice was always significantly reduced in response to gonadotrophin treatment. Thymulin injection in PMSG-hCG-treated Tx-10 mice provoked a significant increase in uterine weight. The results suggest that the presence of the thymus after the neonatal period is necessary to normal ovarian development and function. The increase in gonadotrophin-induced ovarian response produced by thymulin replacement indicates that this peptide has a role in this process as one of the connecting signals between thymus and ovaries.

Studies on the gonadotropin-releasing activity of thymulin: changes with age.

We assessed the ability of thymulin, a zinc-dependent nonapeptide produced by the thymic epithelial cells, to influence the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from dispersed anterior pituitary (AP) cells from young, adult, and senescent female rats. Perifusion of young and senescent AP cells with thymulin doses of 10(-6) to 10(-5) M gave a significant stimulatory response for LH but not FSH. Gonadotropin release was always lower in the senescent cells. AP cells from both age groups incubated with 10(-8) to 10(-3) M thymulin showed a time- and dose-dependent response for both gonadotropins, with a maximal stimulation at 10(-7) M. Preincubation of thymulin with an antithymulin serum completely quenched the secretagogue activity of the hormone. Coincubation of thymulin with the secretagogue gonadotropin-releasing hormone (GnRH) revealed a synergistic effect on LH release and an additive effect on the release of FSH. The calcium chelator EGTA blocked the gonadotropin-releasing activity of thymulin in AP cells. The cAMP enhancers, caffeine, NaF, and forskolin significantly increased the thymulin-stimulated release of gonadotropins. The inositol phosphate enhancer LiCl potentiated the action of thymulin on gonadotropins. It is concluded that the gonadotropin-releasing activity documented here for thymulin is an age- and receptor-dependent effect mediated in part by calcium, cAMP, and inositol phosphates.

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

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.

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

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.

The humoral activity of the avian thymic microenvironment.

The thymic microenvironment (composed of the lymphoepithelial stroma and the secretory products of the thymic epithelium) provides the required milieu for the development of the thymus-derived lymphocytes (T cells). There is limited information characterizing or identifying the active secretory components of the avian thymus. The work discussed here has focused on examination of the presence, regulation, and activity of one of the thymic hormones (thymulin) in the chicken. A thymulin-like product has been shown to exist in chicken serum as assessed by the mammalian bioassay and an ELISA immunoassay; thymectomy removes this product from the serum. Serum thymulin activity has been shown to be directly related to the thyroid status of the chick with the functionally hypothyroid Cornell sex-linked dwarf strain having lower levels than the euthyroid K strain. Alterations in circulating thymulin concentrations produced by daily thymulin injections resulted in an altered profile of the major peripheral blood T cell subpopulations and produced significant changes in the autoimmune pathology present within the Obese strain chicken. These approaches represent preliminary attempts to study the role of thymulin in avian immune development and in immune-neuroendocrine interactions.

Thymulin and its role in immunomodulation.

Even though thymulin was isolated, sequenced and characterised some 20 years ago and later identified as a thymic hormone involved in immunomodulation, much more work is still required to further understanding of the mechanisms of action(s) of this peptide. Since the observation, by a semiquantitative bioassay, of diminished levels of thymulin in immunodeficiency and autoimmune disease, new data obtained by radioimmunoassay have not only confirmed previous observations but also demonstrated that thymulin plays a role in the interaction between the immune system and the neuro-endocrine system. In this paper we give an up to date account of recent developments in research into the role of thymulin in immunomodulation.