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Thymulin, A Thymic Peptide, Prevents the Overproduction of Pro-Inflammatory Cytokines and Heat Shock Protein Hsp70 in Inflammation-Bearing Mice

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Abstract

The effects of synthetic analogue of peptide hormone thymulin, which is normally produced by thymic epithelial cells, on immune cells activity and blood cytokine profile had been studied in male NMRI mice with acute inflammation induced by injection of lipopolysaccharide from gram-negative bacteria (LPS, 250 microg/100 g of body weight). Inflammation induced by LPS resulted in accumulation of several plasma pro-inflammatory cytokines, IL-1 beta, IL-2, IL-6, TNF-alpha, interferon-gamma, and also IL-10, anti-inflammatory cytokine. Thymulin previously injected in dose of 15 microg/100 g body weight, prevented the accumulation of proinflammatory cytokines in plasma. Thymulin also prevented LPS-induced up-regulation of production of several cytokines by spleen lymphocytes and peritoneal macrophages. Added in vitro, thymulin decreased the peak of TNF-alpha production in macrophages cultivated with LPS. In addition, thymulin lowered the peak of Hsp70 production induced by LPS treatment. The results indicate that thymulin having significant anti-inflammatory effect may be promising in clinical application.

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... In the present study, we tested the effects of the thymic peptide thymulin, which has been shown to exert an anti-inflammatory influence during acute inflammation [31]. In addition, we compared the efficacy of free thymulin with that of thymulin bound to polybutylcyanoacrylate (PBCA) nanoparticles. ...
... In the present work, we aimed to study the effects of thymulin on immune cell activity in a mouse model of chronic inflammation induced by daily treatment with increasing doses of LPS. Based on our earlier investigations, in which thymulin was found to have anti-inflammatory properties in vitro [21] and in acute inflammation in vivo [31], we concluded that it might exert a protective effect against the consequences of sepsis. It is important to emphasize that when applied to healthy mice, thymulin does not affect plasma levels of proinflammatory cytokines, synthesis of stress proteins (Hsp70 and Hsp90), or cellular production of nitric oxide [31]. ...
... Based on our earlier investigations, in which thymulin was found to have anti-inflammatory properties in vitro [21] and in acute inflammation in vivo [31], we concluded that it might exert a protective effect against the consequences of sepsis. It is important to emphasize that when applied to healthy mice, thymulin does not affect plasma levels of proinflammatory cytokines, synthesis of stress proteins (Hsp70 and Hsp90), or cellular production of nitric oxide [31]. Thus, thymulin has no effect on the inflammatory status of healthy animals, but does correct immune imbalances in chronic inflammation. of blots show a single representative experiment using one animal. ...
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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.
... An increased RelA/p65 (Ser276) phosphorylation, commonly attributed to PKA, corresponded with the production of IF-γ, Hsp72, and the initial phase of IL-17 production associated with the early phase of the disease, whereas increased RelA/p65 (Ser536) phosphorylation was correlated not only with the activation of IKK, SAPK/JNK, and p53, but also with the late phase of IL-17 secretion and the pathology progression. Lunin and coworkers [69,70] experienced that the thymic peptide thymulin, a metallopeptide consisting of a nonapeptide (Table 1) in complex with a zinc ion, in addition to producing effects on the neuroendocrine stress response system [69][70][71], shows analgesic activity in inflammatory conditions [72] and in severe and mild progressive forms of EAE [73,74]. Recently [75], the thymulin was bound to polybutylcyanoacrylate (PBCA) nanoparticles for enhancing the bioavailability of the free peptide [76], and the properties of both forms of the peptide were also evaluated on relapse and remitting EAE. ...
... An increased RelA/p65 (Ser276) phosphorylation, commonly attributed to PKA, corresponded with the production of IF-γ, Hsp72, and the initial phase of IL-17 production associated with the early phase of the disease, whereas increased RelA/p65 (Ser536) phosphorylation was correlated not only with the activation of IKK, SAPK/JNK, and p53, but also with the late phase of IL-17 secretion and the pathology progression. Lunin and coworkers [69,70] experienced that the thymic peptide thymulin, a metallopeptide consisting of a nonapeptide (Table 1) in complex with a zinc ion, in addition to producing effects on the neuroendocrine stress response system [69][70][71], shows analgesic activity in inflammatory conditions [72] and in severe and mild progressive forms of EAE [73,74]. Recently [75], the thymulin was bound to polybutylcyanoacrylate (PBCA) nanoparticles for enhancing the bioavailability of the free peptide [76], and the properties of both forms of the peptide were also evaluated on relapse and remitting EAE. ...
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Multiple sclerosis (MS) belongs to demyelinating diseases, which are progressive and highly debilitating pathologies that imply a high burden both on individual patients and on society. Currently, several treatment strategies differ in the route of administration, adverse events, and possible risks. Side effects associated with multiple sclerosis medications range from mild symptoms, such as flu-like or irritation at the injection site, to serious ones, such as progressive multifocal leukoencephalopathy and other life-threatening events. Moreover, the agents so far available have proved incapable of fully preventing disease progression, mostly during the phases that consist of continuous, accumulating disability. Thus, new treatment strategies, able to halt or even reverse disease progression and specific for targeting solely the pathways that contribute to the disease pathogenesis, are highly desirable. Here, we provide an overview of the recent literature about peptide-based systems tested on experimental autoimmune encephalitis (EAE) models. Since peptides are considered a unique therapeutic niche and important elements in the pharmaceutical landscape, they could open up new therapeutic opportunities for the treatment of MS.
... We found that thymulin gene therapy abrogated the M2-biased macrophage phenotype in the OVA-challenged asthmatic lungs, while the difference in the macrophage count between the induction of disease and therapeutic intervention was not significant. It is well established that lymphocytes function in coordination with other immune cells, including macrophages (50). Thus, thymulin-mediated phenotypic deviation of macrophages from the M2 phenotype may be attributed to a reduction in M2-inducing T H 2 cytokine production and release by lymphocytes. ...
... Thus, thymulin-mediated phenotypic deviation of macrophages from the M2 phenotype may be attributed to a reduction in M2-inducing T H 2 cytokine production and release by lymphocytes. However, thymulin may have directly acted on macrophages, given that macrophages have previously been shown to respond differently to thymulin depending on the pathologic state in vitro (50,51). In summary, we demonstrate here that inhaled thymulin gene therapy by mucus-penetrating particles therapeutically reverses key biochemical, histologic, and functional disease phenotypes of allergic asthma via direct and/or indirect modulation of various immune cells, including eosinophils, neutrophils, lymphocytes, and macrophages. ...
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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.
... One potential therapy that shows little or no adverse effects is the thymic peptide thymulin, a metallopeptide consisting of a nonapeptide (Glu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asp) in complex with a zinc ion, which has been reported not only to regulate maturation of lymphocytes in the thymus, but also to modulate the immune and neuroendocrine systems' activities [3]. Numerous data showed that thymulin exerts mostly inhibitory effects on the inflammatory immune response both in vitro and in vivo [4][5][6]. Thymulin also affects the neuroendocrine stress response system including its major pathway, the hypothalamic-pituitary-adrenal axis [3], and produces analgesic effects in inflammatory conditions [7]. Despite its systemic anti-inflammatory effects, thymulin is non-toxic at very large doses [8], and it has no marked effect on animal physiological status. ...
... In contrast to SAPK/JNK activation, the expression of Hsp72 was increased at the earliest stage only, decreasing to control levels at later stages. Both thymulin and PBCA-thymulin completely blocked the rEAE-induced increase in Hsp72 expression ( Figure 5C), which corresponds to our previous data showing that thymulin blocks inflammatory Hsp72 induction [4]. ...
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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.
... Other authors believe that the mechanisms of the anti-inflammatory effects of thymic peptides, including thymulin, may involve NF-B and p38 signalling cascades, which play crucial roles in inflammation [10,11]. Recently we have demonstrated that thymulin in vitro and in vivo affected the NF-B cascade in tandem with the production of proinflammatory cytokines, nitric oxide, and heat shock proteins in immune cells [12,13]. We hypothesised that the antioxidants and/or thymulin could act as adjuvants, strengthening the anti-inflammatory effect of IKK Inhibitor XII. ...
... Thymic peptide thymulin, which is a metallopeptide consisting of a nonapeptide (Glu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asp) that couples with zinc ions, mostly has inhibitory effects on immune responses [12,31] and also stimulates endocrine systems, indicating its role in the recovery from inflammatory conditions [32]. Using thymulin as an immune-modulator in the second experimental line, we tested a repertoire of cytokines, including IL-6 and IL-17. ...
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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/β kinases (IKK Inhibitor XII); with thymulin; with dietary coenzyme Q9, α-tocopherol, and β-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.
... In this model, thymulin effect was related to the inhibition of expression of the proinflammatory cytokine IL-6 and to the suppression of p38 MAPK pathway [71]. The protective effect of thymulin against inflammation was further analysed by Lunin and coworkers in an animal model with inflammation induced by lipopolysaccharide (LPS) from gram negative bacteria [72]. These authors showed that the peptide prevented the accumulation of IL-1, IL-6, TNFand Interferon-gamma (IFN-) in plasma and the LPS-induced upregulation of production of cytokines by splenic lymphocytes and macrophages. ...
... Interestingly, this effect was accompanied by a significant decrease in the heat-shock protein HSP70 by splenic lymphocytes induced by LPS treatment. Since they also demonstrated that hyperactivation of immune cells by bacterial toxin was accompanied by increase of HSP70 production, the decrease in HSP70 production induced by thymulin suggests that this peptide reduces stress response to endotoxin, a finding confirmed by the lowering of proinflammatory cytokines in the plasma [72]. ...
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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 is 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.
... They also affect the expression of T-cell markers, such as CD90, CD3, CD4 and CD8 (Bene, Faure, & Bordigoni, 1982), as well as the extrathymic maturation of T-cells (Inagaki-Ohara, Kobayashi, & Nishimura, 1996). The activity of these hormones has also been recently reported in the bloodstream, which affects extrathymic immune cells (Lunin, Khrenov, Novoselova, Parfenyuk, & Novoselova, 2008;Lunin & Novoselova, 2010). The molecular targets of the thymic hormones have not been identified, and there is limited data on their activity mechanisms. ...
... For instance, T-cell membranes may have specific receptors that bind thymic hormones with high affinities (Gastinel et al., 1982;Pléau, Fuentes, Morgat, & Bach, 1980). In addition, we and others have shown that these hormones affect intracellular signalling cascades in conjunction with the production of pro-inflammatory and anti-inflammatory cytokines, NO and heat-shock proteins in immune cells (Lunin et al., 2008;Lunin et al., 2009). However, the primary binding target of these peptide hormones has not been identified. ...
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We propose a hypothesis that the T-cell receptor is a possible target of thymic hormones. We modelled the conformational dynamics of thymopentin and its structural variants in solution, as well as the interactions of these short peptides with the proposed molecular target. Thymopentin is a five-amino-acid fragment of the thymic hormone thymopoietin (residues 32 to 36) that reproduces the immunomodulatory activity of the complete hormone. Using molecular dynamics and flexible docking methods, we demonstrated high-affinity binding of thymopentin and its prospective mimetics with the T-cell receptor. The calculated biological activity spectra of thymopentin and its two promising modifications can be used in immunomodulatory activity screenings with live systems.
... Furthermore, addition of thymulin, in vitro, reduced the peak of TNFα production in macrophages cultivated with LPS. Additionally, thymulin lowered the production of the heat-shock protein, Hsp70, by splenic lymphocytes induced by LPS [116]. ...
Article
Introduction A large volume of data indicates that the known thymic hormones, thymulin, thymopoietin, thymosin-α, thymosin-β, and thymic humoral factor-y2, exhibit different spectra of activities. Although large in volume, available data are rather fragmented, resulting in a lack of understanding of the role played by thymic hormones in immune homeostasis. Area covered Existing data compartmentalizes the effect of thymic peptides into 2 categories: influence on immune cells, both immature and mature, and interconnection with neuroendocrine systems. The current study draws attention to a third aspect of the thymic peptide effect that has not been clarified yet, wherein ubiquitous and highly abundant intranuclear precursors of peptides referred to as “thymic peptides” play a fundamental role in all somatic cells. Expert Opinion Our analysis indicated that, under certain stress related conditions, these precursors are cleaved to form immunologically active peptides that rapidly leave the nucleus and intracellular spaces, to send “distress signals” to the immune system, thereby acting as stress sensors. We propose that these peptides may form a link between somatic cells and immune as well as neuroendocrine systems. This model may provide a better understanding of the mechanisms underlying immune homeostasis, leading thereby to the development of new therapeutic regimes utilizing the characteristics of thymic peptides.
... В экспериментах по изучению влияния тимулина на воспаление, индуцированное липополисахаридом, показано, что его защитный эффект может быть связан с предотвращением накопления провоспалительных цитокинов ИЛ-1, ИЛ-6, фактора некроза опухоли альфа (ФНОα) и интерферона гамма в плазме крови и снижением синтеза белка теплового шока HSP70 в лимфоцитах селезенки, который, как известно, повышается при гиперактивации иммунных клеток при воспалении [61]. На моделях острого аутоиммунного энцефаломиелита, индуцированного основным миелиновым белком, у мышей NZW тимулин снижал степень выраженности заболевания путем влияния на каскад ядерного фактора NF kappaB (NF-9B), снижая уровень фосфорилирования сигнального белка IKK и продукции белка HSP72 [62]. ...
Article
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Interaction between thymus and thymus peptides with nervous and endocrine systems is discussed. Much attention is paid to the thymus hormone thymulin, its influence on the central nervous and endocrine systems, anti-inflammatory and analgesic properties. Prospects for therapeutic use of thymulin and its gene are described. The effects of thymosins α1 and β4 and prothymosin α on nervous and endocrine systems is reviewed. Neuroprotective and neurorestorative effects of thymosin β4 on damaged nerve cells are mentioned. Thymosin α1 is characterized by its influence on the nerve growth factor production, secretion of hypothalamic releasing hormones and reduction of chemotherapy associated neurotoxicity. The data on prothymosin α expression of in brain, its key role in inhibiting apoptosis and necrosis, induced by ischemia in brain and retina, is discussed. It was shown that the definite amino acid sequences of prothymosin α, consisting of 30 and 9 amino acids, exhibit neuroprotective effect.
... Anti-inflammatory effects of thymulin were shown using the model of endotoxin-induced inflammation: pretreatment with peptide analogue of thymulin (1, 5 or 25 μg/animal) prevented the hyperalgesia and maintained the body temperature within the normal range and resulted in downregulation of the levels of pro-inflammatory cytokines and prostaglandin E2 in the liver [14]. Also earlier we reported that preliminarily injected thymulin prevents the accumulation of LPS-induced proinflammatory cytokines (IL-1β, IL-2, IL-6, TNF-α and IFN-γ) in mouse plasma and also prevents LPS-induced upregulation of production of several cytokines by splenic lymphocytes and peritoneal macrophages [15]. Based on these results, the inhibitory dosage for the present study has been chosen (about 3 μg/animal). ...
... We have previously shown that the immunomodulatory potential mediated by thymulin is exhibited by downregulating an inflammatory signal through the differential reduction of the secretion of proinflammatory cytokines, including IL-1, IL-6, and TNF- [8][9][10][11]16,34]. This selective inhibition is accompanied by upregulating an anti-inflammatory response mediated by IL-10 [11], consistent with a counter-inflammatory signaling loop amplified by thymulin [40]. This leads to suggest that American Journal of Medical and Biological Research thymulin may exhibit an anti-inflammatory activity by acting as a novel dual regulator: It down-regulates a proinflammatory signal and, on the other hand, amplifies an anti-inflammatory response [1,2,11]. ...
... Our analysis of the available data [Lunin and Novoselova, 2010] showed that different thymic peptides have various activities on mature immune cells and central endocrine systems, including the so-called stress response system and its component, the hypothalamic-pituitary-adrenal axis. The thymic peptide thymulin, which is a metallopeptide consisting of a nonapeptide (Glu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asp) that couples with a zinc ion, exerts mostly inhibitory effects on immune responses Lunin et al., 2008]; on the other hand, thymulin stimulates endocrine systems, indicating its role in recovery from inflammatory conditions [Lunin and Novoselova, 2010]. Thymopoietin and its active fragment corresponding to residues 32-36, called thymopentin (Arg-Lys-Asp-Val-Tyr), stimulates immune cells in healthy humans and animals and inhibits immune responses under stress conditions; thus, it is very promising for clinical applications [Malaise et al., 1987;Novoselova et al., 2008]. ...
Article
Modulation of autoimmune inflammation by the thymic peptides thymulin and thymopentin was studied in mice with acute experimental autoimmune encephalomyelitis (EAE), which resembles multiple sclerosis in humans. EAE was induced in NZW mice by a single immunisation with myelin basic protein coupled with adjuvants. Visible signs of pathology appeared on days 12-14 after the immunisation, peaked on days 20-25, were retained up to day 45, and then reverted. A biphasic cytokine response was also detected. In the "early" phase, which started at day 35, increased levels of interferon-gamma and interleukin-6 in the blood were observed; during the "delayed" phase, which started at day 48, the levels of plasma interleukin-17 and tumour necrosis factor-alpha were also raised. In addition, the phosphorylation of NF-kappaB signalling proteins and the production of heat shock protein Hsp72 were significantly increased in splenic lymphocytes from EAE-bearing mice. When applied intraperitoneally every other day for 30 days, either thymulin or thymopentin (15μg per 100g of body weight) significantly reduced the disease severity compared to untreated EAE mice. The effect of thymulin but not thymopentin remained after its withdrawal. Thymulin reduced the cytokine response in both the early and the delayed phases, whereas thymopentin only reduced the "early phase cytokines" (IL-6 and interferon-gamma). Both peptides significantly reduced the level of phosphorylation of the NF-kappaB signalling protein IKK and the production of Hsp72 protein. The data presented here indicate the presence of time-dependent immune responses in EAE-bearing mice, which may be associated with the Th1 and Th17 subpopulations of T-cells. Thymulin and thymopentin demonstrated different patterns of activity, most likely via mechanisms involved in NF-kappa B signalling and Hsp72 expression.
... Furthermore, added in vitro, thymulin (5 ng/ml) reduced the peak of TNF-a production in macrophages cultivated with LPS, and this finding suggests that the effect on macrophages is not T-lymphocyte-mediated. Additionally, thymulin lowered the peak of heat-shock protein Hsp70 production by splenic lymphocytes induced by LPS treatment [44]. As we have shown further, the effect of thymulin may be related to the decrease of LPS-induced NF-kB cascade activation [26]. ...
Article
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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. 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. 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. In view of the role of thymic hormones in immune and neuroendocrine systems, they could be suitable as therapeutic agents for inflammation.
... In addition to their primary function as molecular chaperones, Heat shock proteins from the HSP60, HSP70 and HSP90 families, are potent activators of the innate immune system [168,169]. It has been shown that inflammatory responses mediated by LPS accumulate Hsp70 and plasma pro-inflammatory cytokines [170]. Hsp70 and cytokine effects are probably mediated via Toll-like receptor signal transduction pathways towards the activation of NF-κB and MAPKs [169]. ...
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An increasing amount of Expressed Sequence Tag (EST) and genomic data predominantly for the cnidarians Acropora, Hydra and Nematostella, reveals that despite being one of the morphologically simplest multicellular animals, cnidarians possess a high genomic complexity. In order to contribute towards a broader coverage of this phylum, an EST project was performed to analyze the transcriptome of Hydractinia echinata. Moreover, transcriptional profiling experiments were carried out to characterize the i-cell population and the immune system of the hydroid. In this work a cDNA-library containing about 20,000 clones was constructed, which covers the entire life cycle of the organism and also represents some stress-induced conditions. After randomly sequencing almost 9,000 clones, EST characterization revealed a broad diversity of genes, with higher sequence similarity to vertebrates than to ecdysozoan invertebrates. Furthermore, a significant number of sequences hitherto unknown in metazoans were detected. The identification of unique Hydractinia sequences is consistent with the suggested high diversity and complexity of genes within the phylum. To store all the acquired information a database aimed at making the data widely available was created, which is accessible at www.mchips.org/hydractinia_echinata.html. To further characterize Hydractinia genes, a cDNA-microarray was constructed including the already sequenced ESTs as well as PCR-products from almost 5,000 un-sequenced cDNAs. Genes associated with the i-cell lineage were identified by the analysis of the gene expression profile of colonies depleted from their i-cells using mitomycin-C and colonies after the recovery from the treatment. Microarray normalized data ended up with 162 significant differentially expressed genes. Several growth and transcription factors as well as genes associated with undifferentiated cells were identified including; BMPs, Bzip/Mafl and CnPL10. In addition, i-cell depleted organisms exhibited an activation of genes involved in detoxification and wound healing activities. These genes are good candidates to define the i-cell population of Hydractinia. Genes associated with the immune system of Hydractinia were identified by the analysis of the expression profile of organisms having a LPS mimicked Gram-negative bacterial infection as well as an allogeneic reaction. 245 candidate genes with a significantly different expression level were identified. Genes associated with an LPS response encode for e.g. HSP70, lipocalin-like proteins, serine protease inhibitors, proteins with TSR domains and lectins. In the case of allorecognition, a probable whole genome response with up-and down regulation of hundreds of genes was observed; demonstrating a complex process. Some of the identified genes encode for e.g. minicollagens, transcriptional and growth factors, proteins with a protective function against oxygen metabolites or with potent inflammatory and neurotoxicity effects. Gene expression pattern analysis provided insights towards the function of many genes which are still unknown. In the case of genes with a known functional annotation, the microarray experiments either corroborated their characterization or defined an alternative one for Hydractinia. This project is the first high-throughput effort aimed to identify and characterize the transcriptome of the colonial marine hydroid Hydractinia echinata. The combination of the EST dataset, database and the microarray, provides a solid platform to promote and facilitate molecular research not only in Hydractinia but also in other cnidarians. Die zunehmende Menge an EST und genomischen Daten über Cnidarier, allen voran über Acropora, Hydra und Nematostella zeigt, dass Cnidarier trotz ihrer morphologischen Einfachheit eine große genomische Komplexität aufweisen. Um zu einem tieferen Verständnis des Phylums beizutragen, wurde ein EST Projekt realisiert, mit dessen Hilfe das Transkriptom des Hydroid Hydractinia echinata analysiert wurde. Zusätzlich wurden Experimente zur Analyse der Expressionsmuster durchgeführt, um die I-Zell Population und das angeborene Immunsystem des Hydroids zu charakterisieren. Im Rahmen dieser Arbeit wurde eine cDNA-Blibliothek mit insgesamt über 20.000 Genen erstellt, die den gesamten Lebenszyklus des Organismus abdeckt, und darüber hinaus einige stress-induzierte Konditionen repräsentiert. Die EST-Charakterisierung zeigt eine breite Vielfalt an Genen, wobei die Sequenzen eine größere Ähnlichkeit mit Vertebraten als mit Invertebraten der Ecdysozoen Gruppe aufweisen. Außerdem konnte eine signifikante Anzahl an Genen detektiert werden, die bisher in Metazoen unbekannt waren. Die Identifizierung der Hydractinia spezifischen Sequenzen unterstützt die Annahme einer großen Vielfalt und Komplexität der Gene innerhalb dieses Phylums. Zur Speicherung der gewonnen Informationen wurde eine allgemein zugängliche Datenbank angelegt, die unter www.mchips.org/hydractinia_echinata.html verfügbar ist. Um die Hydractinia Sequenzen näher zu untersuchen, wurde ein cDNA-Microarray erstellt, der die bereits sequenzierten ESTs sowie PCR-Produkte von fast 5.000 unsequenzierten cDNAs enthält. Die Identifizierung I-Zell assoziierter Gene erfolgte anhand der Genexpressionsprofile. Hierzu wurden die aufgrund der Mitomycin-C Behandlung I-Zell freien Kolonien mit Kolonien verglichen, die sich nach der Behandlung wieder regeneriert hatten. Aus den normalisierten Daten des Microarrays ergaben sich 162 signifikant unterschiedlich exprimierte Gene. Identifiziert wurden mehrere Wachstums- und Transkriptionsfaktoren, sowie Gene, die im Zusammenhang mit undifferenzierten Zellen stehen, einschließlich BMPs, Bzip/Mafl und CnPL10. Darüber hinaus zeigten I-Zell freie Organismen eine Aktivierung der in der Entgiftung und Wundheilung vorkommenden Gene. Diese Gene sind gute Kandidaten, um die I-Zell-Population von Hydractinia zu definieren. Zur Bestimmung der mit dem Immunsystem von Hydractinia assoziierten Gene, wurde eine Expressionsanalyse an Tieren durchgeführt, bei denen, mit LPS, eine bakterielle Infektion nachgeahmt wurde und die eine allogene Antwort zeigten. 245 Kandidatengene mit signifikant unterschiedlicher Expression konnten bestimmt werden. Gene, die mit einer LPS-Antwort in Verbindung stehen, kodieren für z. B. für HSP70, Lipocalin-ähnliche Proteine, Serin-Protease-Inhibitoren, Proteine mit TSR-Domänen und Lektinen. Im Falle der Allo-Erkennung wurde eine wahrscheinlich das ganze Genom umfassende Reaktion mit Hunderten von positiv und negativ regulierten Genen beobachtet, die einen komplexen Prozess vermuten lässt. Einige der identifizierten Gene kodieren bspw. für Minikollagene, Transkriptions-und Wachstumsfaktoren und für Proteine mit Schutzfunktion gegen Sauerstoff-Metaboliten oder mit stark entzündlichen und neurotoxischen Effekten. Die Analyse der Genexpressionsmuster lieferte Erkenntnisse über die Funktion vieler noch unbekannter Gene. Gene mit bereits bekannter Funktion, wurden durch die Microarray-Experimente entweder in ihre Annotation bestätigt, oder es konnte eine Alternativfunktion für Hydractinia definiert werden. Bei dieser Arbeit wurden erstmals Hochdurchsatztechnologien eingesetzt, um das Transkriptom der kolonialen marinen Hydroid, Hydractinia echinata zu identifizieren und charakterisieren. Die Kombination aus EST-Datensatz, Datenbank und Microarray, liefert eine zuverlässige Plattform um die molekulare Forschung an Hydractinia, aber auch an anderen Cnidariern zu fördern und zu vereinfachen.
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Rimulus cinnamon is the dried twig of Cinnamomum cassia Presl. It is widely used in China for the treatment of inflammatory processes, amenorrhea, and other diseases. We aimed to study the protective effects of ethyl acetate extracts of R. cinnamon (EAE) on systemic inflammation and lung injury in endotoxin-poisoned mice. EAE was administered 5 d prior to lipopolysaccharide (LPS) challenge with 15 mg/kg LPS. The administration of EAE increased the levels of interferon-γ (IFN-γ) and decreased the levels of interleukin-18 (IL-18) and tumor necrosis factor-α (TNF-α) in the serum. Additionally, EAE relieved the pathological changes in the tissues of the lungs and spleen, and significantly reduced the number of neutrophils in the lung tissues. In addition, treatment with EAE decreased the mRNA expression of the NLR family, pyrin domain-containing protein 3 (NLRP3), caspase-1, and interleukin-1β (IL-1β) in the lungs, as well as the expression of NLRP3, caspase-1 (p20), and pro-IL-1β proteins. These results demonstrated the promising anti-inflammatory effects of EAE in endotoxin-poisoned mice. Furthermore, EAE could alleviate the lung injury of endotoxin-poisoned mice by antagonizing the activation of the NLRP3 inflammasome.
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Thymic peptides are immune regulators produced mainly in the thymus. However, thymic peptides such as thymosin-α and thymopoietin have precursors widely expressed outside the thymus, localized in cell nuclei, and involved in vital nuclear functions. In stress-related conditions, they can relocalize. We hypothesized that another thymic peptide, thymulin, could be similarly produced by non-thymic cells during stress and have a precursor therein. Non-thymic cells, including macrophages and fibroblasts, were exposed to oxidative stress, heat, apoptosis, or necrosis. Extracellular thymulin was identified in media of both cell types 2 h after exposure to stress or lethal signals. Therefore, thymulin is released by non-thymic cells. To examine possible thymulin precursors in non-thymic cells, macrophage lysates were analyzed by western blotting. Bands stained with anti-thymulin antibody were detected in two locations, approximately 60 kDa and 10 kDa, which may be a possible precursor and intermediate. All of the exposures except for heat were effective for induction of the 10 kDa protein. BLAST search using thymulin sequence identified SPATS2L, an intranucleolar stress-response protein with molecular weight of 62 kDa, containing thymulin-like sequence. Comparisons of blots stained with anti-thymulin and anti-SPATS2L antibodies indicate that SPATS2L may be a possible candidate for the precursor of thymulin.
Article
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.
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Lysophosphatidylcholine (lysoPC) with polyunsaturated fatty acyl chains has been known to be anti-inflammatory in vivo. In the present study, we examined the effect of docosahexaenoyl-lysophosphatidylcholine (DHE-lysoPC) and 17-hydroxydocosahexaenoyl-lysophosphatidylcholine (17-HDHE-lysoPC) on spleen weight and cytokine level in spleen of mice treated with lipopolysaccharide (LPS). For this purpose, mice were administrated i.p. with DHE-lysoPC or 17-HDHE-lysoPC 1 h before i.p. injection of LPS. First, DHE-lysoPC (50–400 µg/kg) was found to suppress the LPS-induced increase of spleen weight dose-dependently, and such a suppressive effect was greater for 17-HDHE-lysoPC, compared to DHE-lysoPC. Next, in an attempt to see the effect of DHE-lysoPC on cytokine levels in spleen of mice treated with LPS, DHE-lysoPC was found to suppress LPS-induced increase in the levels of cytokines such as TNF-α, IL-1β, or IL-6 in a dose dependent manner (50–400 µg/kg), in contrast to DHA showing a significant action at a high dose (400 µg/kg) only. The greater suppressive effect of 17-HDHE-lysoPC (15–150 µg/kg) than DHE-lysoPC suggested that action of DHE-lysoPC may be enhanced through lipoxygenation process. Presumably in support of this, when the interval time between 17-HDHE-lysoPC administration and LPS challenge was varied, the cytokine-suppressing effect was found to be augmented in a time-dependent manner. Taken all together, it is suggested that DHE-lysoPC and 17-HDHE-lysoPC may be beneficial in suppressing the inflammation in spleen tissue.
Article
The aim of this study was to reveal T-lymphocyte-independent mechanisms of thymic peptide-mediated immunomodulation. 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. 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. 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.
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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. The physiological features of thymulin and data that support its potential as an anti-inflammatory treatment for lung diseases are reviewed. 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. 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.
Article
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.
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To explore regulation of potentially lethal responses to bacterial lipopolysaccharide (LPS), we used differential display under LPS-free conditions to compare macrophage cell lines from two strains of mice congenic for a locus affecting LPS sensitivity. LPS-hyporesponsive cells, primary macrophages, and polymorphonuclear leukocytes transcribed secretory leukocyte protease inhibitor (SLPI), a known epithelial cell-derived inhibitor of leukocyte serine proteases. Transfection of macrophages with SLPI suppressed LPS-induced activation of NF-kappa B and production of nitric oxide and TNF alpha. The ability of interferon-gamma (IFN gamma) to restore LPS responsiveness is a hallmark of the LPS-hyporesponsive phenotype. IFN gamma suppressed expression of SLPI and restored LPS responsiveness to SLPI-producing cells. Thus, SLPI is an LPS-induced IFN gamma-suppressible phagocyte product that serves to inhibit LPS responses.
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Previously, the changes in phagocyte functions such as adherence, chemotaxis or TNFalpha production were found to be associated with oxidative stress in endotoxin-induced septic shock. However, in this type of oxidative stress the lymphocyte involvement has rarely been studied. In the present report, we analyzed the above functions in peritoneal lymphocytes from male and female BALB/c mice with a lethal endotoxic shock caused by intraperitoneal injection of E. coli lipopolysaccharide (LPS) (100 mg/kg), male and female Swiss mice with lethal endotoxic shock caused by intraperitoneal injection of LPS (150 and 250 mg/kg, respectively) or non-lethal endotoxic shock (100 mg/kg). In peritoneal lymphocytes obtained at 0, 2, 4, 12 or 24 h after LPS injection, the first two functions of these cells in the immune response, i.e. adherence to tissues and directed migration (chemotaxis), were studied. At 0, 0.5, 1, 1.5, 2, 4, 12 and 24 h after LPS injection, TNFalpha released by lymphocytes was also analyzed. The results show that endotoxic shock increases the adherence and TNFalpha release, and decreases the chemotaxis of peritoneal lymphocytes. These changes were more significant in mice with lethal than with non-lethal endotoxic shock, a fact that confirms the important role of lymphocytes during endotoxic shock.
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Dysregulation of the immuno‐inflammatory response, as seen in sepsis, may culminate in host cell and organ damage. Lipopolysaccharide from Gram‐negative bacterial cell walls induces gene activation and subsequent inflammatory mediator expression. Gene activation is regulated by a number of transcription factors at the nuclear level, of which nuclear factor κB appears to have a central role. The redox (reduction–oxidation) cellular balance is important for normal cellular function, including transcription factor regulation. In sepsis, a state of severe oxidative stress is encountered, with host endogenous antioxidant defences overcome. This has implications for cellular function and the regulation of gene expression. This review gives an overview of the mechanisms by which transcription factor activation and inflammatory mediator overexpression occur in sepsis, together with the events surrounding the state of oxidative stress encountered and the effects on the host’s antioxidant defences. The effect of oxidative stress on transcription factor regulation is considered, together with the role of antioxidant repletion in transcription factor activation and in sepsis in general. Other interventions that may modulate transcription factor activation are also highlighted. Br J Anaesth 2003; 90: 221–32
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Thymulin is a thymic peptide possessing hypophysiotropic activity and antiinflammatory effects in the brain. We constructed a synthetic DNA sequence encoding met-FTS, a biologically active analog of thymulin, and subsequently cloned it into different expression vectors. A sequence optimized for expression of met-FTS in rodents, 5'-ATGCAGGCCAAGTCGCAGGGGGGGTCGAACTAGTAG-3', was cloned in the mammalian expression vectors pCDNA3.1(+) and phMGFP (which expresses the Monster Green Fluorescent Protein), thus obtaining pcDNA3.1-metFTS and p-metFTS-hMGFP, which express met-FTS and the fluorescent fusion protein metFTS-hMGFP, respectively. The synthetic sequence was also used to construct the adenoviral vector RAd-metFTS, which expresses met-FTS. Transfection of HEK293 and BHK cells with pcDNA3.1-metFTS (experimental groups) or pcDNA3.1 (control), led to high levels of thymulin bioactivity (>600 versus <0.1 pg/ml in experimental and control supernatants, respectively). Transfection of HEK293 and BHK cells with pmetFTS-hMGFP revealed a cytoplasmic and nuclear distribution of the fluorescent fusion protein. A single intramuscular (i.m.) injection (10(7) plaque forming units (PFU)/mouse or 10(8) PFU/rat) of RAd-metFTS in thymectomized animals (nondetectable serum thymulin) restored serum thymulin levels for at least 110 and 130 days post-injection in mice and rats, respectively. We conclude that RAd-metFTS constitutes a suitable biotechnological tool for the implementation of thymulin gene therapy in animal models of chronic brain inflammation.
Article
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Article
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Article
Medium obtained by the perifusion of fragments of adult male CSE rat thymuses stimulates the release of luteinizing hormone (LH) from the anterior pituitary gland in vitro. The release of the stimulatory factor/s from the thymus appears to be stimulated by the depolarizing concentration of K+ and to be Ca++-dependent. Perifusates from heart, liver and spleen did not elicit the response suggesting that the effect is specific to the thymus gland. Neither LH nor its releasing hormone, LHRH, were detectable in the thymus perifusates by RIA. Levels of facteur thymique serique (FTS), as measured by bioassay are highest in K+ stimulated fractions that exhibit the greatest LH releasing activity. In separate experiments in vitro FTS, but not two other thymic peptides, thymopoietin and thymosin alpha 1, caused a dose-related release of LH from pituitary tissue. That ability of thymic perifusates to cause the release of LH was age-related because tissue from neonatal and ageing males failed to release compounds with any significant effect on the pituitary release of LH. However, when the thymus was enlarged in ageing rats as a result of orchidectomy 1 month before sacrifice LH release was similar to that observed in young male adults.
Article
We investigated the in vitro effects (kinetics and dose-response) of adrenal and sexual steroid hormones on the secretion of thymulin, a thymic hormone, by human thymic epithelial cells in primary cultures as well as in a rat epithelial cell line. We demonstrated that all steroids tested, in a range of physiological doses, stimulated thymulin production to various extents. Progesterone and estradiol, however, were revealed to be the most efficient. Specific steroid antagonists abrogated the steroid-induced stimulation of thymulin production. These findings confirm our previous in vivo results and demonstrate that steroid hormones can act directly on thymic epithelial cells to modulate their endocrine production.
Article
The influence of different endocrinological manipulations on the blood concentration of serum thymic factor (FTS) was studied in young-adult and old mice. Among the experimentally induced endocrinopathies in youth, hypothyroidism and diabetes caused strong reductions of FTS levels, which were restored to normal by the appropriate hormonal substitutive therapy. Removal of adrenals or gonads has no significant effect on FTS level. Old mice, which show undetectable levels of FTS and low levels of thyroxine, can regain the capacity to produce FTS, provided they are treated with thyroxine. The variations of FTS blood levels in the course of endocrinological manipulations were due to a direct or indirect effect exerted on the recipient thymus. Hormonal treatment of thymectomized mice did not induce any FTS-like activity in their sera, nor did hormones interfere in vitro with the bioassay used to test for FTS. These data suggest that the neuroendocrine balance modulates the synthesis and/or the release of FTS from the thymus during the whole life of the organism and that the decline of FTS production with advancing age is largely dependent on age-associated endocrinological imbalances.
Article
Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
Article
The serum thymic factor (FTS) utilized in its synthetic or natural form loses its biological activity in a rosette assay after treatment with a metal ion-chelating agent, Chelex 100. This activity is restored by the addition of Zn salts and, to a lesser extent, certain other metal salts. FTS activation is secondary to the binding of the metal to the peptide. The metal-to-peptide molar ratio of 1:1 provides the best activation. These data indicate the existence of two forms of FTS. The first one lacks Zn and is biologically inactive; the second one contains Zn and is biologically active, for which we propose the name of "thymulin" (FTS-Zn). The presence of Zn in synthetic FTS was confirmed by atomic absorption spectrometry. The interaction between Zn and FTS was further suggested by microanalysis demonstrating the presence of this metal in thymic reticuloepithelial cells.
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This prospective, multicenter, epidemiological study was carried out in 99 Italian ICUs, distributed throughout the country, from April 1993 to March 1994. In the study, we applied the new ACCP/SCCM classification system for sepsis (SIRS, sepsis, severe sepsis and septic shock) and determined the prevalence, incidence, evolution and outcome of these categories in critically ill patients. The preliminary analysis of 1101 patients showed that on admission SIRS accounted for about half of the diagnoses (52%) with sepsis, severe sepsis and septic shock accounting for 4.5%, 2.1% and 3% of patients, respectively. Patients with severe sepsis or septic shock more frequently had high SAPS scores than patients without sepsis. Mortality rates were similar in patients with SIRS (26.5%) and without SIRS or infection (24%), but rose to 36% in patients with sepsis, to 52% in those with severe sepsis and to 81.8% in those with septic shock. Sepsis, severe sepsis and septic shock were more common in patients with medical diagnoses, and neither severe sepsis nor septic shock was observed in trauma patients. With respect to evolution, the incidence of septic shock was progressively higher in patients admitted with more severe "sepsis-related" diagnoses, while only a trivial difference in rates of incidence was observed between SIRS patients and those admitted without SIRS or any septic disorder (nil). The breakdown of the various ACCP/SCCM "sepsis-related" diagnoses at any time during the study was: SIRS in 58% of the population, sepsis in 16.3%, severe sepsis in 5.5% and septic shock in 6.1%. It seems reasonable to expect from the final evaluation of our study answers to the questions raised by the ACCP/SCCM Consensus Conference about the correlations between "sepsis-related" diagnosis, severity score, organ dysfunction score and outcome.
Article
A significant fraction of murine small intestinal intraepithelial lymphocytes (i-IELs) mature in local sites outside the thymus. However, there is evidence suggesting that extrathymic differentiation of i-IELs is still influenced by the thymus or thymus-derived factors. Facteur thymique serique (FTS), a nonapeptide thymic hormone, is involved in several aspects of intra- and extrathymic T cell differentiation in vivo. In this study, we investigated the effects of FTS on the kinetics of i-IELs in mice following a single administration of 5-fluorouracil (5-FU). FTS treatment significantly accelerated the recovery in cell number of i-IELs after administration of 5-FU. Flow cytometric analysis revealed that this accelerated recovery was mainly due to a rapid increase in CD8 alpha alpha+ i-IELs. Similar findings were also evident in adult thymectomized (ATX) mice, indicating that FTS treatment caused a rapid recovery of CD8 alpha alpha+ i-IELs following 5-FU administration in the absence of a functional thymus. Furthermore, expression levels of the mRNAs for interleukin-2, interferon-gamma, and transforming growth factor beta 1 in the i-IELs were augmented by FTS treatment. Notably, FTS treatment protected mice from 5-FU-induced lethal toxicity, accompanied with an inhibition of the translocation of Enterobacteriaceae. These results suggest that FTS has an important function in the extrathymic maturation and activation of i-IELs in the small intestine following 5-FU administration, which may contribute at least partly to the protection against 5-FU-induced lethal toxicity.
Article
The neuroendocrine system plays a key role in the regulation of the secretion of the thymic peptide, thymulin, but it remains to be determined whether thymulin exerts reciprocal regulatory actions on the functional activity of the hypothalamo-pituitary axis. In the present study, we have used a well established in vitro preparation to examine the influence of thymulin on cyclic nucleotide formation and hormone secretion by the rat anterior pituitary gland. Thymulin-Zn2+(0.5–50 p M) stimulated the release of immmunoreactive corticotrophin (ir-ACTH), producing effects which were maximal at 10 pM (p M), italso produced small but significant increases in immunoreactive luteinising hormone (ir-LH) release (p 0.05) while that of immunoreactive prolactin (ir-PRL) was reduced (p M) raised the cyclic 3′,5′-adenosine monophosphate (cyclic AMP) content of the pituitary tissue (p M), it also increased cyclic 3′,5′-guanosine monophosphate (cyclic GMP; p 2+(0.5–5.0 pM) were potentiated markedly by rolipram (1µM; p M), a selective inhibitor of cyclic-GMP-specific phosphodiesterase, attenuated the corticotrophic responses to higher concentrations of the peptide (10 and 50 pM;p M) nor zaprinast (10µM) influenced the release of ir-LH, ir-PRL or ir-GH in the presence or absence of thymulin-Zn2+(0.5–50 pM; p > 0.05). The results suggest that thymulin modulates the secretion of ACTH and possibly LH by the anterior pituitary gland and that its actions are associated with increased cyclic nucleotide formation; in addition, it appears to exert an inhibitory influence on ir-PRL release.
Article
The thymus gland is a central lymphoid organ in which bone marrow-derived T cell precursors undergo differentiation, eventually leading to migration of positively selected thymocytes to the peripheral lymphoid organs. This differentiation occurs along with cell migration in the context of the thymic microenvironment, formed of epithelial cells, macrophages, dendritic cells, fibroblasts, and extracellular matrix components. Various interactions occurring between microenvironmental cells and differentiating thymocytes are under neuroendocrine control. In this review, we summarize data showing that thymus physiology is pleiotropically influenced by hormones and neuropeptides. These molecules modulate the expression of major histocompatibility complex gene products by microenvironmental cells and the extracellular matrix-mediated interactions, leading to enhanced thymocyte adhesion to thymic epithelial cells. Cytokine production and thymic endocrine function (herein exemplified by thymulin production) are also hormonally controlled, and, interestingly in this latter case, a bidirectional circuitry seems to exist since thymic-derived peptides also modulate hormonal production. In addition to their role in thymic cell proliferation and apoptosis, hormones and neuropeptides also modulate intrathymic T cell differentiation, influencing the generation of the T cell repertoire. Finally, neuroendocrine control of the thymus appears extremely complex, with possible influence of biological circuitry involving the intrathymic production of a variety of hormones and neuropeptides and the expression of their respective receptors by thymic cells.
Article
We utilized a line of transgenic mice expressing Photinus luciferase complementary DNA (cDNA) under the control of a nuclear factor kappa B (NF-kappaB)-dependent promoter (from the 5' human immunodeficiency virus-1 [HIV-1] long terminal repeat) to examine the role of NF-kappaB activation in the pathogenesis of systemic inflammation induced by bacterial endotoxin (lipopolysaccharide [LPS]). After intraperitoneal injection of E. coli LPS, these mice displayed a time- and dose-dependent, organ-specific pattern of luciferase expression, showing that NF-kappaB-dependent gene transcription is transiently activated in multiple organs by systemic LPS administration. Luciferase expression in liver could be specifically blocked by intravenous administration of replication-deficient adenoviral vectors expressing a dominant inhibitor of NF-kappaB (IkappaB-alphaDN), confirming that luciferase gene expression is a surrogate marker for NF-kappaB activation in this line of mice. After treatment with intraperitoneal LPS, the mice were found to have increased lung tissue messenger RNA (mRNA) expression of a variety of cytokines that are thought to be NF-kappaB-dependent, as well as elevated serum concentrations of presumed NF-kappaB-dependent cytokines. In lung tissue homogenates, a close correlation was identified between luciferase activity and KC levels. These studies show that systemic treatment with LPS orchestrates a multiorgan NF-kappaB-dependent response that likely regulates the pathobiology of systemic inflammation.
Article
The effect of thymulin on IL-2 receptor (IL-2R) expression by avian splenocytes was examined in the functionally hypothyroid sex-linked dwarf (SLD) and in normal euthyroid K strain chickens. Daily thymulin injections of 0, 0.05 and 5.0 ng/100 g body weight were given from hatching until 4 weeks of age. ConA-treated and non-stimulated splenocytes from these animals were analyzed by flow cytometry for their expression of IL-2Ralpha, CD4 and CD8 cell surface molecules. ConA activation increased the number of IL-2R+ cells within K strain more than in the SLD. Thymulin treatment increased the number of IL-2R+ cells in the SLD but had the opposite effect in K strain chickens. Mitogen activation or thymulin treatment had little effect on the IL-2R density within small cell populations. In contrast, mitogen activation increased the density of IL-2R on larger cell populations in both K and SLD. IL-2R densities on non-stimulated larger cells decreased in the SLD after thymulin exposure. Thymulin treatment produced no effect on the mean IL-2R densities for large activated cells. ConA stimulation increased the number of CD4+ cells in both strains. The density of CD4 expression was modulated by both mitogen activation and thymulin treatment. ConA stimulation produced an increase in the number of CD8+ cells. The SLD had fewer CD8+ cells than did the K strain and thymulin treatment had little effect on this population in either strain. Mitogen stimulation increased the density of CD8 on CD8+ cells but again thymulin treatment had little effect. These results suggest that thymulin can modulate IL-2R expression on splenocytes and that this effect may be dependent upon the thyroidal status of the animal. Further, these data suggest that thymulin has a differential effect on the CD4 and CD8 T-cell subpopulations.
Article
The ability of thymulin to directly enhance NK cell-mediated cytotoxicity was examined. Specific cell population depletions were done in K and SLD chicken splenocyte preparations using anti-CD3, CD4, and CD8 monoclonal antibodies and secondary complement-fixing polyclonal antibodies. The remaining cells were incubated overnight with in vitro treatments of thymulin and IFN-gamma, either separately or together, followed by an assay for cytotoxicity. Although the control K-strain had higher overall NK cell-mediated cytotoxicity than the thymulin-deficient SLD-strain, the following trends were seen in both strains. Thymulin continued to enhance NK activity following CD4 or CD3 cell depletion, but not after CD8 or CD8 and CD4 cell depletion. Since avian NK cells express CD8 alpha, but not CD3 or CD4 on their surface, these results suggest that the ability of in vitro thymulin treatments to enhance NK activity is not mediated by T-cells but may be due to direct effects on NK cells.
Article
The present study examines the effect of PAT (peptide analogue of thymulin) in two rat models of inflammatory hyperalgesia induced by either i.pl. (1.25 μg in 50 μl saline) or i.p. (50 μg in 100 μl) injections of endotoxin ET. Pretreatment with PAT (1, 5 or 25 μg in 100 μl saline, i.p.) decreased, in a dose dependent manner, both mechanical hyperalgesia, determined by the paw pressure (PP) test and thermal hyperalgesia determined by the hot plate (HP), the paw immersion (PI) and the tail flick (TF) tests. Compared to the tripeptides K(D)PT and K(D)PV, known to antagonize interleukin (IL)-1β or IL-1β and PGE2 mechanisms, PAT, at lower dosages, exerted stronger anti-hyperalgesic effects. When compared with the effect of a steroidal (dexamethasone) and a non-steroidal (indomethacin) anti-inflammatory drugs (NSAID), PAT demonstrated equal analgesic actions. Pretreatment with PAT, reduced significantly the increased concentration of IL-1β, IL-6, TNF-α and NGF due to i.pl. injection of ET. Injection of i.p. ET produced sickness behaviour characterized by hyperalgesia and fever. Pretreatment with PAT prevented the hyperalgesia and maintained the body temperature within the normal range and was accompanied by a down-regulation of the levels of pro-inflammatory cytokines and PGE2 in the liver. PAT, in all doses used, did not result in any evident changes in the physiological parameters or in the normal behaviour of the rats. The anti-hyperalgesic and anti-inflammatory effects of PAT can be attributed, at least partially, to the down-regulation of pro-inflammatory mediators. British Journal of Pharmacology (2002) 136, 947–955. doi:10.1038/sj.bjp.0704793
Article
The contemporary views on the clinical application and efficiency of the most well-known peptide preparations isolated from thymus are presented in this review. Among these preparations are thymosins, thymulin, thymopoetin, thymostimulin, thymopentin, thymalin, tactivin, and others. The results of clinical trials, dosages and indications of thymus peptide preparations, their advantages and disadvantages and perspectives of their therapeutical use are discussed.
Article
Expression of heat shock proteins Hsp27, Hsp90, and Hsp70 and production of tumor necrosis factors (TNF-alpha, TNF-beta), interferon-gamma (IFN-gamma), interleukin-2, -3, -6, and nitric oxide (NO) were studied under conditions of acute and chronic intoxication of animals with lipopolysaccharides. Injection of endotoxin increased expression of heat shock proteins Hsp70 and Hsp90-alpha in mouse cells. Acute toxic stress also provoked a sharp increase in the production of TNF-alpha, TNF-beta, and NO in mouse cells. The production of other cytokines (interleukins and IFN-gamma) was changed insignificantly. In the model of chronic toxic stress, changes in the production of Hsp70, Hsp90, TNF, and NO were followed during 11 days after the beginning of the toxin injections. The expression of Hsp70 and Hsp90 in acute stress was significantly higher than at the final stage of the chronic exposure. The changes in the TNF and NO productions, on one hand, and the production of heat shock proteins, on the other hand, were synchronous. The findings indicate that repeated injections of increasing endotoxin doses result in a decreased ability of the body cells to respond to stress by overproduction of heat shock proteins, TNF, and NO.
UK) was used to develop the immunostaining of blots following the manufacturer's REFERENCES Arion Contemporary views on the nature and clinical application of thymus preparations
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ECL-plus chemiluminescent cocktail (Amersham/GE Healthcare, UK) was used to develop the immunostaining of blots following the manufacturer's REFERENCES Arion, V.Y., Zimina, I.V., Lopuchin, Y.M. (1997). Contemporary views on the nature and clinical application of thymus preparations. Russ. J. Immunol. 2: 157–166.