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.

Modulation of inflammation response to murine cutaneous Leishmaniosis by homeopathic medicines: thymulin 5cH.

BACKGROUND: In previous studies, we observed that thymulin 5cH could modulate BCG (Bacillus Calmette-Guerin) induced chronic inflammation by increasing peritoneal B1 stem cells differentiation into phagocytes and improving phagocytosis efficiency. METHODS: We used the same protocol to study the effects of thymulin 5cH in the experimental murine Leishmaniasis, in order to elucidate some aspects of the parasite-host relation under this homeopathic treatment. Male Balb/c mice were orally treated with thymulin 5cH or vehicle during 60 days, after the subcutaneous inoculation of 2 × 10(6) units of Leishmania (L.) amazonensis into the footpad. Washied inflammatory cell suspension from peritoneal cavity, spleen, local lymph node and infected subcutaneous tissue were harvested after 2 and 60 days from infection to quantify the inflammation cells by flow cytometry and histometry methods. RESULTS: After a transitory increase of peritoneal T reg cells, treated mice presented, chronically, increase in the peritoneal and spleen B1 cells percentage (p = 0.0001) in relation to other cell types; more organized and exuberant inflammation response in the infection site, and decrease in the number of parasites per field inside the primary lesion (p = 0.05). No difference was seen in local lymph node histology. CONCLUSIONS: Thymulin 5cH is able to improve B1 cell activation and Leishmania (L) amazonensis phagocytosis efficiency in mice, similarly to that observed previously in BCG experimental infection.

Thymic peptides restrain the inflammatory response in mice with experimental autoimmune encephalomyelitis.

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 (15 µg 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.

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.

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, a thymic peptide, prevents the overproduction of pro-inflammatory cytokines and heat shock protein Hsp70 in inflammation-bearing mice.

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.

Effects of injecting thymulin into the anterior or medial hypothalamus or the pituitary on induced ovulation in prepubertal mice.

UNLABELLED: In prepubertal mice, subcutaneous thymulin injection before equine chorionic gonadotrophin (eCG) treatment simulates ovulation; seemingly, the thymulin could be acting at the hypothalamus-pituitary axis level. OBJECTIVE: This study was designed to analyze the effects of injecting thymulin into the hypothalamus or pituitary on induced ovulation of prepubertal mice. METHOD: Female mice, 19 days old, were anesthetized with ether and injected with saline solution or thymulin into the anterior or medial hypothalamus or the pituitary and treated with eCG when 20 days old. The ova shed were counted and serum concentrations of 17beta-estradiol were measured. In the ovaries, the morphometrical analysis was performed and the atresia evaluated. RESULTS: Ether anesthesia treatment blocked eCG-induced ovulation in almost all animals. Mice anesthetized and treated with eCG and gonadotrophin-releasing hormone (GnRH) or human chorionic gonadotrophin (hCG) ovulated a full quota of ova. Injecting saline solution into the anterior or medial hypothalamus or the pituitary did not reduce the blocking effects of ether anesthesia on induced ovulation, but the incidence of atretic follicles was higher. Injecting thymulin directly into the anterior hypothalamus did not restore ovulation, nor diminish the number of atretic follicles. In contrast, injecting thymulin into the medial hypothalamus restored the ovulation ratio and decreased the percentage of atretic follicles. Similar results were obtained by injecting thymulin into the pituitary, though thymulin treatment in the pituitary resulted in a higher number of ova shed and lower follicular atresia. CONCLUSION: The present results suggest that thymulin acts at the medial hypothalamus level, facilitating the release of GnRH and at the pituitary level regulating gonadotrophin release.

In vivo thymulin treatments enhance avian lung natural killer cell cytotoxicity in response to infectious bronchitis virus.

Previous work has shown that in vitro thymulin treatments have the ability to enhance natural killer (NK) cell cytotoxicity. The purpose of the experiments presented here was to examine the in vivo effects of thymulin on avian NK cell activity in response to a viral infection. Five and a half-week-old K-strain chickens infected with avian infectious bronchitis virus (IBV) served as the model for these experiments. Daily thymulin injections began at varying time points prior to or post-infection. The controls received daily injections of the ZnCl(2)-containing carboxymethyl-cellulose (CMC) diluent. A 51Cr-cytolytic release assay was used to determine the activity of the NK cells harvested via lung lavage from the respiratory tracts of infected chickens. The results of these experiments showed that in vivo thymulin treatments enhance NK cytotoxicity. The greatest enhancement of NK cytotoxicity was observed at 10 days post-infection in those chickens that began receiving thymulin after infection. These results suggest that thymulin may not only have a role in enhancing immunosurveillance but also in enhancing the response of the innate immune system following infection. Dose-response experiments found that the 50 ng/100 g body weight (Bwt) dose significantly depressed the cytolytic activity of the NK cells in comparison to either the 10 ng/100 g Bwt dose or the control.

[Effect of thymostimulin on endocrine thymus function in thyroidectomized rats during suppressive hormone therapy]. / Vplyv tymostymulinu na endokrynnu funktsiiu tymusa shchuriv pisla tyreoïdektomiï za umov supresyvnoï hormonoterapiï tyroksynom.

Due to disorders of hormonal balance in the organism, a decrease in thymic endocrine function occurred in rats after thyroidectomy. After removing the thyroid gland, we observed 1,3-2,2-fold decrease in the level of thymic hormone thymulin in the blood serum. When thyroxin was applied at a suppressive dose, endocrine function of the thymus did not restore. Injections of thymostimulin (Tp1) or its combination with thyroxin to thyroidectomized animals restored the level of thymulin up to the level in the intact rats due to effects of either injected preparation or induction of substances possessing thymosine-like activity.

FTS reduces bleomycin-induced cytokine and chemokine production and inhibits pulmonary fibrosis in mice.

Bleomycin (BLM), an antitumour drug, is known to cause interstitial pneumonia followed by pulmonary fibrosis, and has often been used to produce an animal model of pulmonary fibrosis. In the present study, we examined the effect of a nonapeptide thymic hormone, facteur thymique serique (FTS), on the murine lung fibrosis induced by intratracheal instillation of BLM. Treatment with FTS ameliorated BLM-induced fibrotic changes in a dose-dependent manner, as indicated by the reduced accumulation of hydroxyproline (HP). In addition, FTS suppressed BLM-induced cellular inflammatory response in the lungs, as evidenced by inhibition of increased lung weight, reduced accumulation of inflammatory leucocytes, including lymphocytes and neutrophils, but not macrophages, and less pronounced histopathological changes. Finally, BLM challenge increased the local synthesis of proinflammatory cytokines, TNF-alpha and IL-1beta and chemokines, MCP-1, MIP-1alpha RANTES, MIP-2 and KC, while administration of FTS suppressed the production of these cytokines, except for MCP-1. These effects of FTS were observed only when mice received intratracheal instillation with BLM. Considered collectively, our results indicated that FTS treatment ameliorated the cellular inflammatory responses and fibrotic changes in the lungs caused by BLM and such inhibition was well correlated with reduced synthesis of several fibrosis-related cytokines, and suggested that FTS may be potentially useful for the treatment of pulmonary fibrosis.

Cytokine-mediated or direct effects of thymulin on the nervous system as assessed by pain-related behavior.

Thymulin is a thymic hormone with known immunomodulatory activities. Recent evidence has indicated a signaling role for this peptide in the interaction between the immune, endocrine and the nervous system. In this report, we review recent experimental findings on the analgesic actions of thymulin (high doses) in rats with endotoxin-induced localized inflammation and the hyperalgesic actions (low doses) of this peptide in intact animals. These actions involve both proinflammatory cytokines and PGE2. The possibility of a dual role played by thymulin as a hormone that might also involve a direct effect on the nervous system is discussed.

Growth hormone-releasing activity of thymulin on pituitary somatotropes is age dependent.

Thymulin is a Zn-bound nonapeptide produced by the thymic epithelial cells (TEC) whose secretion is modulated by growth hormone (GH), among others. We assessed the ability of thymulin to influence the release of GH from dispersed anterior pituitary (AP) cells from young, middle-aged and senescent Sprague-Dawley female rats. Perifused and incubated AP cells were used in different sets of experiments and GH release was measured by RIA. Perifusion of young and senescent AP cells with thymulin doses, ranging from 10(-8) to 10(-5) M, gave a logarithmic dose-response pattern of GH. Supernatants from TEC lines also showed GH secretagogue activity. The GH release was always lower in the senescent cells. AP cells incubated with 10(-8)-10(-3) M thymulin showed a time- and dose-dependent response, the latter being bell-shaped with a maximum at 10(-7) M thymulin. Preincubation of thymulin with an antithymulin serum completely quenched the secretagogue activity of the hormone. Coincubation of thymulin with GHRH revealed a semiadditive release of GH in young and middle-aged AP cells and an additive effect in senescent cells. In middle-aged AP cells, the synthetic GH secretagogue GHRP-6 showed a synergistic effect with thymulin on GH release. The calcium chelator EGTA, but not the calcium ionophore A23187, blocked the GH-releasing activity of thymulin in AP cells. The cAMP enhancers, caffeine, NaF and forskolin significantly increased the thymulin-stimulated release of GH while trifluoperazine, a protein kinase C inhibitor, had no effect. The inositol phosphate enhancer LiCl potentiated the action of thymulin on GH release. The data suggest that the GH-releasing activity of thymulin is receptor-mediated and involves calcium, cAMP and inositol phosphates. In addition, senescence appears to impair somatotrope responsiveness to thymulin.

In vitro and in vivo effects of thymulin on rat testicular steroid synthesis.

Although the stimulatory effects of immunological hormone thymulin on ovarian function are documented, responses of the testis to thymulin are unknown. Effects of thymulin and thymulin augmentation of human chorionic gonadotropin (hCG) stimulation of testicular steroidogenesis were analyzed via incubation of testis from 3-, 6-, and 9-week-old rats with 0.1, 1, 10, and 100 ng/ml of thymulin or thymulin plus hCG (1 IU/ml). At three weeks of age, thymulin decreased testosterone and estrone synthesis. By 6 and 9 weeks of age, lower doses of thymulin (0.1 and 1 ng/ml) stimulated testosterone and estrone synthesis. Increased doses of thymulin (100 ng/ml) resulted in decreased testicular steroid synthesis. No thymulin augmentation of hCG stimulation of testicular steroidogenesis was noted in vitro. Responses in vitro may have been confounded with the endocrine status at the time of incubation. Thymulin levels are increased at 3 weeks of age and decline at 6 and 9 weeks of age whereas gonadotropin levels are just the opposite pattern; these in vivo endocrine changes may have influenced the in vitro responses. Analysis of in vivo response to thymulin was pursued in pituitary intact, hypophysectomized and hCG primed (12 h before thymulin injection) hypophysectomized rats (5 weeks of age) with 1 and 10 microg of thymulin injected intravenously. Thymulin had no effect on testicular steroidogenesis in hypophysectomized rats 30, 60, and 120 min post injection). In pituitary intact rats, thymulin reduced testosterone and estrone concentrations. In hCG primed hypophysectomized rats, thymulin injection was followed by significant increases in testosterone levels and declines in estrone concentrations. These studies indicated that any increases in testicular steroidogenesis from thymulin required gonadotropin stimulation and increased concentrations of thymulin had inhibitory effects on testicular steroidogenesis.

Hyperalgesia induced by low doses of thymulin injections: possible involvement of prostaglandin E2.

Thymulin injection into rats (20-150 ng) i.p. caused a significant reduction in both mechanical (paw pressure test) and thermal (hot plate and tail flick tests) nociceptive thresholds. Thymulin injection also doubled IL-1beta level in the liver of these animals. Induced hyperalgesia was reversed completely by alpha-MSH related tripeptide, Lys-D-Pro-Val in low doses, which is known to antagonize IL-1beta and PGE2 induced hyperalgesia, but was only partly reversed by IL-1beta related tripeptide, Lys-D-Pro-Thr at high doses, which is known to antagonize IL-1beta induced hyperalgesia only. We conclude from these results that thymulin causes hyperalgesia and that this effect is at least in part mediated via PGE2 and its effectiveness at low concentration implies a physiological role for this thymic hormone.

Involvement of interleukin-1 beta, nerve growth factor, and prostaglandin-E2 in the hyperalgesia induced by intraplantar injections of low doses of thymulin.

The effect of various doses of intraplantar thymulin injection, on nociceptive thresholds, in the hind paw of rats was assessed using different pain tests. As little as 0.5 ng thymulin resulted in localized mechanical hyperalgesia as assessed by the paw pressure test and thermal hyperalgesia as assessed by the paw immersion, hot plate, and tail flick tests. The highest dose of thymulin (10 ng) reduced both paw pressure and paw immersion latencies in the noninjected paw also. Thymulin (5 ng) also resulted in significant elevation in the levels of interleukin-1 beta (IL-1 beta) and nerve growth factor (NGF) levels in the injected paw. Both dexamethasone and indomethacin reversed thymulin-induced hyperalgesia. Also interleukin-1 receptor antagonist (IL-1ra) and a polyclonal anti-NGF antiserum significantly reduced thymulin-induced hyperalgesia. On the other hand, the tripeptide lys-D-pro-val (known to antagonize IL-1 beta and PGE2 induced hyperalgesia) reversed the hyperalgesia due to thymulin. In conclusion, thymulin induces localized hyperalgesia which is mediated by PGE2-dependent mechanisms and this pathway could be either partially dependent on or totally independent of IL-1 beta mechanisms.

In vivo administration of serum thymic factor (FTS) prevents EMC-D virus-induced diabetes and myocarditis in BALB/cAJcl mice.

The effect of serum thymic factor (FTS) on the D-variant of encephalomyocarditis (EMC-D) virus-induced diabetes and myocarditis in BALB/cAJcl mice was investigated. Mice pretreated with 50 or 10 micrograms of FTS were infected with 10 or 10(3) PFU of EMC-D virus. In the mice inoculated with 10 PFU of virus, 40% developed diabetes on post-infection day (PID) 14, whereas those treated with FTS (50 micrograms/administration) on day 2 and 1 before infection did not develop diabetes. FTS (10 micrograms)-pretreated mice developed diabetes. In histological observation, FTS non-treated mice which developed diabetes showed severe necrosis and inflammation of mononuclear cells in the islets of Langerhans and myocardia on 19 PID. Mice pretreated with 50 micrograms of FTS, however, manifested mild islet degeneration without any myocardial inflammation. Furthermore, in FTS non-treated mice, immunohistological staining showed a loss of insulin granules. This loss was markedly reversed and insulin granules remained largely intact in FTS-pretreated mice. Viral titers in pancreas of FTS-pretreated mice approximated well to those of non-treated mice on PID 4, 7 and 19. In mice inoculated with higher titer of EMC-D virus (10(3) PFU), however, 50 micrograms of FTS pretreatment did not change the course of these acute pathological developments (diabetes and myocarditis observed from PID 4).

Protective effects of serum thymic factor to Leptospira interrogans serovar Copenhageni infection in Mongolian gerbils.

The susceptibility to Leptospira interrogans serovar copenhageni in Mongolian gerbils treated with 10 micrograms of serum thymic factor (FTS) 1 day before infection was examined. Susceptibility of gerbils treated 5 times with 10 micrograms of FTS was also investigated. Mortality of FTS-treated gerbils was significantly lower than that of controls when small challenge doses were used. To analyse the FTS-induced resistance to leptospiral infection, natural killer (NK) cell activity and macrophage activity were studied. Macrophage activity was unaltered but NK cell activity was enhanced in FTS-treated gerbils, with or without leptospiral infection. Since no side-effects of FTS were observed, this compound should be considered for the treatment of leptospirosis.

The humoral activity of the avian thymic microenvironment.

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

Radioprotective effects of serum thymic factor in mice.

Serum thymic factor (FTS) reduced mortality of mice after total-body irradiation with 7.56 Gy X rays. The radioprotective effect was achieved by daily repeated subcutaneous injections of 3-100 micrograms FTS, while doses higher than 300 micrograms/day/mouse were neither radioprotective nor toxic. Similarly, degeneration of the spleen was moderated by 3-100 micrograms FTS but not by 500 micrograms FTS in sublethally (3.78 Gy) irradiated mice. Histological examination showed that hematopoiesis was enhanced in the spleen by daily injections of 10 micrograms FTS. Spleen cells from the FTS-treated mice incorporated more [3H]thymidine in culture with or without concanavalin A. The treatment with FTS increased the production of colony-stimulating factor in the spleen as well as in peritoneal macrophage-like cells, and caused a significant increase in the number of granulocyte-macrophage colony-forming cells both in the spleen and in the femoral bone marrow. Furthermore, FTS prevented a decrease in circulating neutrophils in the sublethally irradiated mice. Prominent overshoot recovery of myelopoiesis, which occurred occasionally in sublethally irradiated mice, did not occur in the FTS-treated mice. The decrease in blood erythrocytes was also significantly reduced. These observations imply that this thymic hormone has potential as a radioprotector.

Serum thymic factor as a radioprotective agent promoting survival after X-irradiation.

Serum thymic factor (FTS, zinc-free thymulin) protected mice from death after whole-body X-irradiation. It was significantly radioprotective even when administered after irradiation, but it was more effective when administered both before and after irradiation. The protective effect appears to be due to the enhancement of hematologic recovery in the animals.