Protective effect of exogenous peroxiredoxin 6 and thymic peptide thymulin on BBB conditions in an experimental model of multiple sclerosis.

This study aimed to assess the effects of the immunomodulator thymulin, a thymic peptide with anti-inflammatory effects, and peroxiredoxin 6 (Prdx6), an antioxidant enzyme with dual peroxidase and phospholipase A2 activities, on the blood‒brain barrier (BBB) condition and general health status of animals with relapsing-remitting experimental autoimmune encephalomyelitis (EAE), which is a model of multiple sclerosis in humans. Both thymulin and Prdx6 significantly improved the condition of the BBB, which was impaired by EAE induction, as measured by Evans blue dye accumulation, tight-junction protein loss in brain tissue, and lymphocyte infiltration through the BBB. The effect was associated with significant amelioration of EAE symptoms. Thymulin treatment was accompanied by a decrease in immune cell activation as judged by interleukin-6, -17, and interferon-gamma cytokine levels in serum and NF-kappaB cascade activation in splenocytes of mice with EAE. Prdx6 did not induce significant immunomodulatory effects but abruptly decreased EAE-induced NOX1 and NOX4 gene expression in brain tissue, which may be one of the possible mechanisms of its beneficial effects on BBB conditions and health status. The simultaneous administration of thymulin and Prdx6 resulted in complete symptomatic restoration of mice with EAE. The results demonstrate prospective strategies for multiple sclerosis treatment.

Thymulin and peroxiredoxin 6 have protective effects against streptozotocin-induced type 1 diabetes in mice.

Protective effects of peroxiredoxin 6 (PRDX6) in RIN-m5F ß-cells and of thymulin in mice with alloxan-induced diabetes were recently reported. The present work was aimed at studying the efficiency of thymulin and PRDX6 in a type 1 diabetes mellitus model induced by streptozotocin in mice. Effects of prolonged treatment with PRDX6 or thymic peptide thymulin on diabetes development were evaluated. We assessed the effects of the drugs on the physiological status of diabetic mice by measuring blood glucose, body weight, and cell counts in several organs, as well as effects of thymulin and PRDX6 on the immune status of diabetic mice measuring concentrations of pro-inflammatory cytokines in blood plasma (TNF-α, interleukin-5 and 17, and interferon-γ), activity of NF-κB and JNK pathways, and Hsp90α expression in immune cells. Both thymulin and PRDX6 reduced the physiological impairments in diabetic mice at various levels. Thymulin and PRDX6 provide beneficial effects in the model of diabetes via very different mechanisms. Taken together, the results of our study indicated that the thymic peptide and the antioxidant enzyme have anti-inflammatory functions. As increasing evidences show diabetes mellitus as a distinct comorbidity leading to acute respiratory distress syndrome and increased mortality in patients with COVID-19 having cytokine storm, thymulin, and PRDX6 might serve as a supporting anti-inflammatory treatment in the therapy of COVID 19 in diabetic patients.

Nanoparticle-based thymulin gene therapy therapeutically reverses key pathology of experimental allergic asthma.

Despite long-standing efforts to enhance care for chronic asthma, symptomatic treatments remain the only option to manage this highly prevalent and debilitating disease. We demonstrate that key pathology of allergic asthma can be almost completely resolved in a therapeutic manner by inhaled gene therapy. After the disease was fully and stably established, we treated mice intratracheally with a single dose of thymulin-expressing plasmids delivered via nanoparticles engineered to have a unique ability to penetrate the airway mucus barrier. Twenty days after the treatment, we found that all key pathologic features found in the asthmatic lung, including chronic inflammation, pulmonary fibrosis, and mechanical dysregulation, were normalized. We conducted tissue- and cell-based analyses to confirm that the therapeutic intervention was mediated comprehensively by anti-inflammatory and antifibrotic effects of the therapy. We believe that our findings open a new avenue for clinical development of therapeutically effective gene therapy for chronic asthma.

Protective Effect of PBCA Nanoparticles Loaded with Thymulin Against the Relapsing-Remitting Form of Experimental Autoimmune Encephalomyelitis in Mice.

Relapsing-remitting experimental autoimmune encephalomyelitis (rEAE) in mice is a model that closely resembles relapsing-remitting multiple sclerosis in humans. This study aims to investigate a new approach to modulation of the inflammatory response in rEAE mice using a thymic peptide thymulin bound to polybutylcyanoacrylate (PBCA) nanoparticles. PBCA nanoparticles were used to prolong the presence of thymulin in the blood. Cytokine levels in blood were measured by ELISA; NF-κB and SAPK/JNK cascade activation, as well as Hsp72 and p53 protein expression, were measured by Western blotting. Animal health statuses were estimated using severity scores. Results showed that the cytokine response in rEAE was multi-staged: an early phase was accompanied by an increase in plasma interferon-γ, while the interleukin (IL)-17 response was markedly increased at a later stage. The stages were attributed to rEAE induction and maintenance phases. Thymulin significantly alleviated symptoms of rEAE and lowered plasma cytokine levels both in early and later stages of rEAE, and decreased NF-κB and SAPK/JNK cascade activation. Thymulin modulated NF-kappaB pathway activity via site-specific phosphorylation of RelA/p65 protein (at Ser276 and Ser536). The effect of nanoparticle-bound thymulin was more pronounced than the effect of free thymulin. Therefore, PBCA-thymulin can be considered a prospective treatment for this pathology.

Thymulin, free or bound to PBCA nanoparticles, protects mice against chronic septic inflammation.

In the present work, we aimed to study the effects of free and polybutylcyanoacrylate nanoparticle-bound thymulin on immune cell activity in mice with chronic inflammation. NF-κB, MAPK, and PKC-θ signaling pathway activity was assessed, alongside Hsp72, Hsp90-α, and TLR4 expression and levels of apoptosis. In addition, plasma cytokines and blood and brain melatonin and serotonin levels were measured. In mice treated with gradually raised doses of lipopolysaccharide, significant increases in the activity of the signaling pathways tested, heat-shock protein and TLR4 expression, lymphocyte apoptosis, and plasma proinflammatory cytokine levels were noted. Moreover, we observed significantly heightened serotonin concentrations in the plasma and especially the brains of mice with inflammation. In contrast, melatonin levels were reduced in the tissues examined, particularly so in the brain. Treatment of these mice with thymulin alleviated fever, reduced apoptosis, increased splenic cell number, and decreased cytokine production, Hsp72, Hsp90, and TLR4 expression, and the activity of the signaling pathways examined. In addition, thymulin partially restored brain and blood serotonin and melatonin levels. Thus, thymulin suppressed the proinflammatory response in LPS-treated mice, indicating the potential of thymulin co-therapy in the treatment of sepsis. Nanoparticle-bound thymulin was more effective in several respects.

Signaling, stress response and apoptosis in pre-diabetes and diabetes: restoring immune balance in mice with alloxan-induced type 1 diabetes mellitus.

The aim of this study was to compare immune imbalances in "pre-diabetic" and diabetic mice and to evaluate the efficacy of several agents in improving the immunity of mice with type 1 diabetes. Pre-diabetic and diabetic models generated by a single or double alloxan injection were monitored for plasma glucose and pancreas immunohistochemistry. To study the immunity in pre-diabetic and diabetic Balb/C male mice; the levels of cytokines; synthesis of inducible heat shock proteins HSP72 and HSP90α; activity of the NF-κB, IFR3, SAPK/JNK, and TLR4 pathways; and apoptosis levels in thymuses were measured. Pre-diabetes resulted in a decrease in IL-4, IL-5 and IL-10 in plasma; in diabetic mice, plasma IFN-gamma, IL-6, TNF-alpha, and IL-10 were decreased. The NF-κB alternative pathway activity and TLR4 expression were significantly increased only in pre-diabetic mice, whereas SAPK/JNK activation was observed at both stages of diabetes. Other measured parameters also showed distinct altered patterns in the immunity of pre-diabetic and diabetic mice. Treatment with an inhibitor of NF-κB, thymulin, or a diet with an antioxidant improved or normalized the immune balance in diabetic mice and also notably decreased pancreatic cell damage in pre-diabetic mice.

[The influence of the thymus peptides on analgesia caused by acute and chronic immobilization].

OBJECTIVE: Our aim was to investigate the influence of thymic polypeptides on pain sensitivity and to analyze a possible role of the opioid system in the implementation of the analgesia caused by immobilization stress. METHODS: The study was performed on male Wistar rats at the Moscow state University named after M. V. Lomonosov. We studied effects of thymus peptides: thymuline (0.15 mg/kg), fraction 5 thymosin (0.25 microgram/kg) and cattle thymus extracted product (CTEP) (0.5 mg/kg) on pain sensitivity in rats using test "tail flick" without stress, with acute (3 h) and sub acute (12 h) immobilization stress. The comparison groups were animals treated with saline and spleen polypeptides. RESULTS: It is shown that preparations of thymus increase the threshold of pain sensitivity in the intact animals. Immobilization stress duration 3 and 12 h in thymus peptides treated rats caused a less pronounced increase in pain threshold than in the control groups (immobilization stress 3 h: CTEP--p = 0.025, thymuline--p = 0.022, fraction 5 thymosin--p = 0.033; immobilization stress 12 h: CTEP--p = 0.034, thymuline--p = 0.027, fraction 5 thymosin--p = 0.036). The opioid receptor blocker naloxone (1 mg/kg) did not completely block the stress-induced analgesia, indicating the presence of both opioid and non -opioid components in this state. In thymus peptides treated rats, opioid component was less pronounced than in the control groups (CTEP--p = 0.031, thymuline--p = 0.026, fraction 5 thymosin--p = 0.029). CONCLUSION: Pre-activation of the opioid system by the thymus polypeptides leads to an increase in the share of non-opioid component of the stress-induced analgesia and prevents the depletion of the opioid system in immobilization stress.

Modulation of inflammatory response in mice with severe autoimmune disease by thymic peptide thymulin and an inhibitor of NF-kappaB signalling.

To investigate some cellular and molecular aspects of the autoimmune response and anti-inflammatory efficiency of potential therapeutic agents in a severe form of experimental autoimmune encephalomyelitis (sEAE), an inhibitor of NF-kappaB signalling, IKK Inhibitor XII, and/or thymic peptide thymulin, were injected intraperitoneally at 1.8 and 0.15mg/kg e.o.d, respectively, to C57BL/6 mice immunized with myelin oligodendrocyte glycoprotein and several adjuvants. The immunization induced high lethality in three weeks. The biphasic cytokine response observed in earlier and delayed phases was attributed to the activity of Th1 and Th17 cells, respectively. Phosphorylation of RelA protein from the NF-kappaB family increased during the earlier phase and decreased in the delayed phase. SAPK/JNK signalling protein and heat shock protein Hsp72 significantly increased in lymphocytes. Both the IKK Inhibitor XII and thymulin reduced disease severity, attenuated immune imbalance, and increased mouse life-span. Co-administration of the agents produced no additive effect. Both the inhibitor and thymulin reduced the Th1 response but not the Th17 response. Therefore, RelA-associated Th1 activation and RelA-independent Th17 activation occurred in sEAE. Thymulin and the inhibitor demonstrate similar patterns of activity, potentially through the RelA pathway inhibition, resulting in a partial therapeutic effect on the animals' health status.

Anti-inflammatory effects of IKK inhibitor XII, thymulin, and fat-soluble antioxidants in LPS-treated mice.

The present study was designed to compare the anti-inflammatory effects of several agents applied in vivo, namely, a synthetic inhibitor of the NF-κB cascade, fat-soluble antioxidants, and the thymic peptide thymulin. Cytokine response in LPS-treated mice was analysed in tandem with the following parameters: the synthesis of inducible forms of the heat shock proteins HSP72 and HSP90α; activity of the NF-κB and SAPK/JNK signalling pathways; and TLR4 expression. Inflammation-bearing Balb/c male mice were pretreated with an inhibitor of IKK-α/ß kinases (IKK Inhibitor XII); with thymulin; with dietary coenzyme Q9, α-tocopherol, and ß-carotene; or with combinations of the inhibitor and peptide or antioxidants. Comparable anti-inflammatory effects were observed in inflammation-bearing mice treated separately with thymulin or with dietary antioxidants administered daily for two weeks before LPS treatment. When LPS-injected mice were treated with the inhibitor and antioxidants together, neither plasma cytokines, signal proteins, nor heat shock proteins recovered more efficiently than when mice were treated with these agents separately. In contrast to antioxidant diet, the thymulin was shown to increase the effect of IKK Inhibitor XII in preventing IKK activation in LPS-treated mice.

[The impact of immunoactive drugs on passive avoidance response].

AIM: The objective of this project was to explore the influence of immunoactive drugs (tactivin, thymulin, and thymosin fraction 5) on the development of the passive avoidance conditioned reflex. MATERIALS AND METHODS: Two types of passive avoidance boxes were used--a regular two-chamber box and a modified three-chamber box, comprising a dark chamber in which rats were exposed to electrical shock, a safe dark chamber, and a light chamber in the center. RESULTS: The project has established that the memory trace persists longer under the influence of the immunoactive drugs in both models, which is consistent with the reference nootropic piracetam test results. Notably, the immunoactive drugs' mnemotropic effect was more pronounced in the modified three-chamber box than in the standard two-chamber box. Using the modified box helped to establish the influence of tactivin, thymulin, and thymosin fraction 5 on the spatial memory component. Immunotropic preparations from thymus caused the animals to select the safe chamber 24 hours later and in subsequent tests. CONCLUSION: The project's results indicate that the drugs tested do possess mnemotropic properties, so their range of clinical use can be broadened.

Involvement of the α7-nicotinic acetylcholine receptors in the anti-inflammatory action of the thymulin-related peptide (PAT).

BACKGROUND AND PURPOSE: Peptide analog of thymulin (PAT) has been shown to have anti-hyperalgesic and anti-inflammatory properties in animal models of inflammation. Recent reports suggest that the peripheral cholinergic system has an anti-inflammatory role mediated by α7-nicotinic acetylcholine receptor (α7-nAChR). Our aim is to investigate whether the action of PAT is mediated, via the cholinergic pathway. EXPERIMENTAL APPROACH: The anti-hyperalgesic and anti-inflammatory action of PAT was assessed in rat models of inflammatory nociceptive hyperactivity (carrageenan and endotoxin) and in a mice air-pouch model for localized inflammation, respectively; the possible attenuation of PAT's effects by pretreatment with the α7-nAchR specific antagonist methyllycaconitine citrate (MLA) was also investigated. In another series of experiments, using two electrode recordings, the effect of PAT on the α7-nAChRs, expressed in Xenopus Oocytes, was also determined. KEY RESULTS: Administration of PAT reversed inflammatory nociceptive hyperactivity and cold and tactile hyperactivity in rats. This effect was partially or totally prevented by MLA, as assessed by different behavioral pain tests. Treatment with PAT also reduced the alteration of cytokines and NGF levels by carrageenan injection in the mouse air pouch model; this effect was partially antagonized by MLA. Electrophysiological recording demonstrated that PAT significantly potentiated the α7-nAchR expressed in Xenopus Oocytes. These effects were not observed when a control peptide, with a reverse sequence (rPAT), was utilized. CONCLUSIONS AND IMPLICATIONS: The behavioral and electrophysiological observations described in this report demonstrate that PAT mediates, at least partially, its anti-inflammatory action by potentiating the α7-nAChR. These results indicate that PAT has a potential for new therapeutic applications as anti-inflammatory and analgesic agent.

Development of broiler chickens after treatment with thymulin 5cH: a zoo technical approach.

Modulation of immune response due to thymulin 5cH has been previously observed. The aim of the present study is to evaluate the development of broiler chickens treated with thymulin 5cH by conventional zoo technical indices, phytohemaglutinin induced inflammation test and histomorphometric analysis of lymphoid organs (thymus, Fabricius bursa and spleen). Animals were divided in two groups: (a) test: birds with free access to thymulin 5cH diluted into the drink water and (b) control: birds with free access to water only, from the 1st to the 42nd day of life. All experimental procedures were done in blind. The results show that thymulin 5cH treated group had increased productivity index compared to control (391.45 versus 261.93) associated with higher viability in the 7th week (p = 0.013), and a possible shunt to B lymphocyte activity. The data suggest that thymulin 5cH could be a viable method to improve productivity in poultry production due to its immune modulation properties.

Thymus peptides regulate activity of RAW 264.7 macrophage cells: inhibitory analysis and a role of signal cascades.

OBJECTIVES: The aim of this study was to reveal T-lymphocyte-independent mechanisms of thymic peptide-mediated immunomodulation. METHODS: The effects of two thymic peptides- thymulin and thymopentin were studied in cultured RAW 264.7 macrophages (lipopolysaccharide-stimulated or unstimulated) by measuring cytokine production and signal protein levels. RESULTS: Both peptides increased proinflammatory cytokine secretion by unstimulated RAW 264.7 macrophages and these effects were blocked by the NF-κB cascade inhibitor, stress-activated protein kinase (SAPK)/JNK cascade inhibitor and, to a lesser extent, Toll-like 4 receptor activity inhibitor. In macrophages stimulated by bacterial lipopolysaccharide, peptides alone did not affect cytokine secretion, but significantly enhanced effects of each of the inhibitors. Thymopentin increased activation of both NF-κB and SAPK/JNK cascades in unstimulated macrophages, while thymulin significantly decreased activation of the SAPK/JNK but not NF-κB cascade in LPS-stimulated macrophages. Thymulin and thymopentin increased production of the heat shock protein HSP72 both in LPS-stimulated and unstimulated cells. CONCLUSIONS: Thymulin and thymopentin are effective anti-inflammatory modulators with direct actions on innate immune cells; the effects involve multiple signal cascades, including NF-κB and SAPK/JNK pathways. Since signaling cascades are now considered to be targets for new therapies, thymic peptides may be prospective modulators of signaling cascades, acting alone or in combination with other agents.

Neonatal thymulin gene therapy in nude mice: Effects on the morphology of the pituitary corticotrope population.

The integrity of the thymus during early life is necessary for a proper maturation of the neuroendocrine system, including the adrenal axis. The thymic metallopeptide thymulin seems to be a central physiologic mediator of thymus-pituitary communication. Furthermore, neonatal thymulin gene therapy has been shown to prevent the typical alterations of gonadotrophic cell number and morphology and serum gonadotropin levels in nude female mice. In the present study we assessed the impact of athymia and the effect of neonatal thymulin gene therapy on the corticotropic cell population in nude mice. The effect of thymulin administration to adult nudes on their hypothalamic content of corticotropin-releasing hormone (CRH) and the adrenal content of corticosterone was also determined. We used an adenoviral vector expressing a synthetic gene for the thymic peptide thymulin (metFTS) termed RAd-FTS. On postnatal day 1 or 2, heterozygous (nu/+) and homozygous (nu/nu) pups of both sexes received a single bilateral i.m. injection of RAd-FTS or RAd-GFP, a control vector. On postnatal day 71, mice were bled and sacrificed, and their pituitaries were immediately dissected, fixed and immunostained for corticotropin. Morphometry was performed by means of an image-analysis system. The following parameters were calculated: volume density (VD: Σ cell area/reference area), cell density (CD: number of cells/reference area), and cell surface (CS: expressed in µm²). Serum thymulin levels were measured by a bioassay, and CRH as well as corticosterone were determined by IRMA and RIA, respectively. Neonatal thymulin gene therapy in the athymic mice restored their serum thymulin levels and increased corticotrope CD, VD and CS in both control and athymic mice. Athymic mice showed only a marginal reduction in corticotrope CD, VD and CS. In these mutants hypothalamic CRH content was slightly increased, whereas adrenal corticosterone tended to be lower. Thymulin administration to adult mice tended to reverse these changes. Our results suggest a possible modulating effect of thymulin on the corticotrope population and the adrenal gland, confirming the existence of a bidirectional thymus-pituitary-adrenal axis.

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.

Thymulin and zinc (Zn2+)-mediated inhibition of endotoxin-induced production of proinflammatory cytokines and NF-kappaB nuclear translocation and activation in the alveolar epithelium: unraveling the molecular immunomodulatory, anti-inflammatory effect of thymulin/Zn2+ in vitro.

The immunomodulatory potential of thymulin and zinc (Zn(2+)) in the perinatal alveolar epithelium is not well characterized. In an in vitro model of fetal alveolar type II epithelial cells (FATEII), we have investigated the exhibition of an anti-inflammatory activity of this peptide hormone. Thymulin selectively ameliorated, in a dose-dependent manner, the endotoxin (ET/LPS [lipopolysaccharide])-induced release of IL-1beta, but not IL-6 or TNF-alpha. Furthermore, Zn(2+), an anti-inflammatory antioxidant, which is required for the biological activity of thymulin, independently reduced the secretion of IL-1beta, TNF-alpha and, to a lesser extent, at a supraphysiologic dose (1 mM), IL-6. The underlying cellular and molecular pathways associated with the anti-inflammatory effect of thymulin and Zn(2+) in the alveolar epithelium are not well established. Further in this study, the role of cyclic AMP (cAMP) in the anti-inflammatory effect of thymulin was investigated, in addition to unraveling the possible involvement of the NF-kappaB pathway. Interestingly, thymulin upregulated, in a dose- and time-dependent manner, the release of the nucleotide cAMP. To understand whether the inhibitory effect of thymulin on cytokine release is cAMP-dependent, Forskolin, a labdane diterpene known to elevate intracellular cAMP, was shown to reduce the secretion of IL-1beta and TNF-alpha, but not IL-6, an effect mimicked by dibutyryl-cAMP (dbcAMP), an analog of cAMP. Alveolar epithelial cells treated with thymulin markedly showed a downregulation of the nuclear translocation of RelA (p65), the major transactivating member of the NF-kappaB family, in addition to NF-kappaB(1) (p50) and c-Rel (p75), an effect mildly substantiated with Zn(2+). Furthermore, thymulin/Zn(2+) reduced, in a dose-dependent manner, the DNA-binding activity of NF-kappaB (RelA/p65). These results indicate that the anti-inflammatory effect of thymulin, which is mediated by cAMP, is NF-kappaB-dependent and involves the downregulation of the release of proinflammatory cytokines, particularly IL-1beta, an effect synergistically amplified, at least in part, by Zn(2+). The molecular regulation of thymulin via a NF-kappaB-dependent pathway is critical to understanding the anti-inflammatory alleviating role of this nonapeptide in regulating proinflammatory signals.

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.