Functional recovery following traumatic brain injury in rats is enhanced by oral supplementation with bovine thymus extract.

Traumatic brain injury (TBI) is one of the leading causes of death worldwide. There are currently no effective treatments for TBI, and trauma survivors suffer from a variety of long-lasting health consequences. With nutritional support recently emerging as a vital step in improving TBI patients' outcomes, we sought to evaluate the potential therapeutic benefits of nutritional supplements derived from bovine thymus gland, which can deliver a variety of nutrients and bioactive molecules. In a rat model of controlled cortical impact (CCI), we determined that animals supplemented with a nuclear fraction of bovine thymus (TNF) display greatly improved performance on beam balance and spatial memory tests following CCI. Using RNA-Seq, we identified an array of signaling pathways that are modulated by TNF supplementation in rat hippocampus, including those involved in the process of autophagy. We further show that bovine thymus-derived extracts contain antigens found in neural tissues and that supplementation of rats with thymus extracts induces production of serum IgG antibodies against neuronal and glial antigens, which may explain the enhanced animal recovery following CCI through possible oral tolerance mechanism. Collectively, our data demonstrate, for the first time, the potency of a nutritional supplement containing nuclear fraction of bovine thymus in enhancing the functional recovery from TBI.

ThymicPeptides Reverse Immune Exhaustion in Patients with Reactivated Human Alphaherpesvirus1 Infections.

Recurrent infection with human alphaherpesvirus 1 (HHV-1) may be associated with immune exhaustion that impairs virus elimination. Thymic peptides enhance immune function and thus could overcome immune exhaustion. In this study, we investigated whether reactivation of herpes infections was associated with immune exhaustion. Moreover, we examined the impact of treatment with thymostimulin on the expression of programmed cell death protein 1 (PD-1) and its ligand (PD-L1) on T and B lymphocytes in patients suffering from recurrent HHV-1 reactivation. We also assessed the effector function of peripheral blood mononuclear cells (PBMCs) after stimulation with thymic peptides. We enrolled 50 women with reactivated HHV-1 infections and healthy volunteers. We measured the expression of various activation and exhaustion markers on the surface of PBMCs using flow cytometry. In ex vivo experiments, we measured the secretion of inflammatory cytokines by PBMCs cultured with thymostimulin. Compared with controls, patients with reactivated HHV-1 infections had increased percentages of CD3+ co-expressing CD25, an activation marker (p < 0.001). Moreover, these patients had increased percentages of CD4+ and CD8+ cells co-expressing the inhibitory markers PD-1 and PD-L1. In cultures of PBMCs from the patients, thymostimulin increased the secretion of interferon gamma (p < 0.001) and interleukin (IL)-2 (p = 0.023), but not IL-4 or IL-10.Two-month thymostimulin therapy resulted in no reactivation of HHV-1 infection during this period and the reduction of PD-1 and PD-L1 expression on the surface of T and B lymphocytes (p < 0.001). In conclusion, reactivation of herpes infection is associated with immune exhaustion, which could be reversed by treatment with thymic peptides.

Thymus Polypeptide Preparation Tactivin Restores Learning and Memory in Thymectomied Rats.

We studied the effects of tactivin and splenic polypeptides on learning and memory of thymectomized animals. In 3-week rats, thymectomy blocked active avoidance conditioning. Injections of tactivin (0.5 mg/kg) during 1 month after surgery restored learning capacity; splenic polypeptides were ineffective.

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

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

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

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

Quantitative evaluation of learning and memory trace in studies of mnemotropic effects of immunotropic drugs.

Apart from restoration of disordered immunological parameters, tactivin and derinat exhibit a pronounced effect on the higher integrative functions of the brain. Experiments on Wistar rats have shown that these drugs accelerated conditioning of food and defense responses. New methods for quantitative evaluation of memory trace consolidation are proposed.

[Pharmacological correction of nonspecific resistance and production of proinflammatory cytokines during chronic intoxication with organophosphorus compound VX].

It is established in experiments on noninbred rats that the use of imunofan (20 mg/kg daily) and polyoxidonium (150 mg/kg daily) for 7 days on the background of chronic intoxication with organophosphorus agent VX (0.01 LD50, single daily treatment for 30 days) resulted in almost complete recovery of phagocytic-metabolic activity of neutrophils, the content of lysozyme, cationic protein of platelet, and levels of proinflammatory cytokines TNFa, IL-1b and IL-6 in the blood. The administration of T-activin (20 mg/kg daily for 7 days) restores these parameters insignificantly. The maximum overall stimulatory effect was produced by polyoxidonium, while the minimum effect was observed for T-activin.

The effect of immunomodulating drugs on the percentage of apoptotic and necrotic lymphocytes in inflammatory infiltrations in the muscle tissue of mice infected with Trichinella spiralis.

In order to determine the effect of apoptosis and necrosis on the intensity of the muscular phase of infection by Trichinella spiralis, male CFW mice were orally infected with T. spiralis larvae and treated with some immunomodulating drugs: calf thymus extract (TFX), lipopolysaccharide from Escherichia coli (LPS), and dexametasone (DEX). Treatment with TFX increased the proportion of apoptotic lymphocytes and decreased the proportion of necrotic lymphocytes from 14 to 60 days after infection in mice infected with T. spiralis. Treatment with LPS increased proportions of both apoptotic and necrotic lymphocytes from 21 to 60 days after infection, especially at 28 days after infection. Treatment with DEX increased the proportion of apoptotic lymphocytes only at 28 days after infection, and significantly increased the proportion of necrotic lymphocytes at 21 days after infection. Parasite load in the affected muscle tissue was significantly lower than the control in mice treated with TFX, not significantly different from the control in mice treated with LPS, and significantly higher than the control in mice treated with DEX. The results of the study suggest that the parasite made an effort to reduce the effectivity of the host immune response in order to ensure its own survival.

[Effect of tactivin on the behaviour and learning in rats].

The action of the immunocorrective agent tactivin on CNS functional activity was studied in an experiment using outbred white rats. The drug was shown to affect the animals' behaviour in an elevated cross-shaped maze including learning capacity and ability to overcome functional disorders of conditioned active avoidance response. The data obtained suggest anti-stress activity of tactivin.

Trichinella spiralis: Effect of thymus factor X on apoptosis and necrosis in mice.

The aim of the study was to determine the effect of thymus factor X (TFX-Jelfa) on the percentage of apoptotic and necrotic lymphocytes in the spleen, mesenteric lymph nodes, and muscle tissue of mice infected with 200 larvae of Trichinella spiralis. TFX was administered subcutaneously at a dose of 15mg/kg. On days 7, 14, 21, 28, 35, 42, and 60 after infection, apoptotic and necrotic cells were detected by flow cytometry after staining with the Annexin V-Fluos Staining Kit. TFX increased the percentage of apoptotic lymphocytes in the spleen, mesenteric lymph nodes, and muscle tissue of mice infected with T. spiralis. The effect of TFX on the percentage of necrotic lymphocytes was weaker and less clear. Parasite load was lower in infected mice treated with TFX than in the untreated control mice. The effect of TFX on the host immune response and the survival of parasite larvae was therefore probably affected by the extent of inflammatory infiltrates, and not by the percentage of lymphocytes undergoing apoptosis.

Phenotypic correction of Age-associated functional decline in murine immune cells by Thymax, a thymic extract.

BACKGROUND: We have recently demonstrated that Thymax, a gross thymic extract, induces the functional activity of human dendritic cells (DCs) in vitro. In this study, the role of Thymax in phenotypic correction of age-associated functional decline in immune cells in mice was evaluated. MATERIALS AND METHODS: C57BL/6 mice (13 months old) were treated with Thymax orally (20% v/v) for 4 weeks. Different splenic cell types, dendritic cells (DCs), B-cells, T-cells and natural killer (NK) cells, were analyzed using flow cytometry. RESULTS: Treatment with Thymax resulted in: i) a significant increase in the percentages of DCs (1.6-fold), B-cells (7-fold) and T-cells (5-fold) over the control (p<0.05); ii) an increase in the percentages of activation markers (CD25 and CD69) of CD4(+) and CD8(+) T-cells; and iii) an enhancement in NK activity. Thymax showed no adverse side-effects. CONCLUSION: Thymax might have a role in reversing immune dysfunction in the elderly.

Aqueous extracts from dietary supplements influence the production of inflammatory cytokines in immortalized and primary T lymphocytes.

BACKGROUND: Congaplex and Immuplex are dietary supplements that have been traditionally used to support immune system function. The purpose of these experiments was to determine whether Congaplex and Immuplex affect immune function using primary and immortalized T lymphocytes. METHODS: Immortalized CEM and Jurkat T lymphocytes and primary peripheral mononuclear blood cells (PBMCs) were treated with the aqueous extracts from Congaplex and Immuplex to determine the effects of these products on cytokine production in activated T lymphocytes. RESULTS: Congaplex enhanced phytohemagglutinin/phorbol 12-myristate 13-acetate (PHA/PMA) stimulation of both CEM and Jurkat cells as measured by the production of cytokines, while Immuplex suppressed PHA/PMA-induced production of cytokines, with the exception of interleukin (IL)-8 which was enhanced by Immuplex. In vitro treatment of PBMCs from 10 healthy donors with Congaplex or Immuplex decreased PHA-stimulated production of interferon (IFN)-gamma but increased the production of IL-13. CONCLUSIONS: While the effects of Congaplex and Immuplex differed in these two models, these data demonstrate that the aqueous extracts from these two dietary supplements can affect the inflammatory response of T lymphocytes.

Gross thymic extract, Thymax, induces apoptosis in human breast cancer cells in vitro through the mitochondrial pathway.

Previous studies have shown that thymic extracts possess antitumor and antimetastatic properties, but the mechanisms are not completely understood. Therefore, in this study the ability of the gross thymic extract Thymax to induce apoptosis in human breast cancer cell line (MCF-7) cells in vitro was evaluated. Tumor cells were cultured with different concentrations of Thymax for 24 h and the apoptotic response was assessed by propidium iodide and TUNEL assays. Activation of caspases and changes in mitochondrial membrane potential (MMP) were monitored by flow cytometry and the expression of Bcl-2 and Bax was determined by Western blot analysis. Thymax induced apoptosis in monolayer MCF-7 cells in a dose-dependent manner; at concentrations of 2.5, 5 and 10% (v/v) it caused 9%, 10% and 25% apoptosis, respectively, as compared to 6% for control cancer cells without treatment. The induction of apoptosis by Thymax was associated with activation of caspases 8 and 9, and the addition of a pan caspase inhibitor partially inhibited Thymax-induced apoptosis by 20%. In addition, the MMP was decreased significantly at Thymax concentrations of 5%-20%, which was associated with a decrease in the protein expression of Bcl-2 and an increase in Bax. These results suggest that Thymax exerts its effects via the mitochondrial pathway of apoptosis and may represent a new class of adjuvants for the treatment of breast cancer.

Modulation of immune response and tumor development in tumor-bearing mice treated by the thymic factor thymostimulin.

Thymostimulin (TS), a partially purified thymic factor, has a significant impact on tumor development in C57B1/6 mice inoculated with Lewis lung carcinoma (3LL) cells, as judged by its effect on time of tumor appearance after tumor cell transplantation. In a previous study, we determined the conditions under which survival rate of the tumor-bearing mice can be significantly increased by TS treatment. In the study communicated here we analyzed host defense mechanisms that are modified by TS treatment in the tumor-bearing mice. In general, immune parameters that were increased or stimulated by the presence of the tumor were further increased in the TS-treated animals (number of lymphoid spleen cells, their response in mixed lymphocyte tumor cultures, their natural killer cell activity, and their ability to produce colony-stimulating factor), or reached earlier maximum levels (spontaneous [3H]thymidine incorporation, a reflection of in vivo spleen cell activation). Responses which reflect tumor-induced immunosuppression (proliferative response induced by phytohemagglutinin or concanavalin A stimulation) were restored to normal level by TS. Specific tumor-related reactions (specific cell-mediated cytotoxicity) were preserved in the TS-treated animals. The wide spectrum of TS effects had, nevertheless, certain elements of selectivity; e.g. colony-stimulating factor, but no interferon production is enhanced by TS in the tumor-bearing mice in diametric contrast to TS effect in Mengo virus-infected mice. The spectrum of TS effects was also dependent on the type of tumor cell used. The results indicate that the significant effect of TS on 3LL tumor development in mice is associated with a strong, multifaceted effect of TS on the immune system.

Thymic hormone modulation of leukemogenic virus replication.

The effect of purified calf thymus extract, thymosin, on infection with murine leukemia virus (MuLV) (Rauscher) was studied in adult thymectomized BALB/c mice. The course of infection was determined by virus titer in the blood plasma, by enumeration of spleen cells replicating MuLV (i.e., infectious centers), and by the examination of cells for expression of virus-induced cell membrane antigen. Thymectomy (performed 1 week prior to the virus injection) decreased MuLV infection by all three parameters. However, administration of thymosin (500 mug, given in equal doses 2 days before and at the time of injection of virus) caused a marked increase of plasma titer of virus, as well as an increased number of spleen cells generating MuLV as compared to controls treated either with purified spleen fraction or with diluent (buffered salt solution). More than two doses of thymic extract (up to nine, given every 2 days after infection) had no further effect on plasma virus titer or number of splenic infectious centers. Furthermore, treatment of mice with either thymic or splenic extracts increased the incidence of virus related membrane antigen appearing on the surface of spleen cells, as compared to diluent-treated control animals. These results indicate that thymosin may enhance infection by or replication of murine oncornaviruses.

The thymus extract Thymex-L potentiates the retinoic acid-induced differentiation of the human myeloid leukemia cell line HL-60.

The human promyelocytic cell line HL-60 can be differentiated with retinoic acid (RA) along the granulocytic pathway. Numerous studies have identified many synergistic combinations of RA with cytostatics, cytokines and other inducers. A combination of RA with the crude thymus extract Thymex-L increased differentiation of HL-60 cells as confirmed by two functional assays and morphology, whereas the extract itself did not show any effect. The functional markers phagocytosis-associated chemiluminescence and nitroblue tetrazolium reduction were more enhanced (up to 4-fold with 1000 micrograms/ml Thymex-L) than morphology. The effect was found over a wide RA concentration range (10(-11) - 10(-6) M) and was dependent on extract concentration. The half-maximal induction of both functional markers was reached at 400 micrograms/ml. To achieve the same effect with the combination in comparison with RA alone, an RA dose reduction of about 100-fold was estimated. The effect was also seen when the cells were pretreated with the thymus extract for two days. The enhancement of RA action by Thymex-L was not correlated with an increase of extracellular or intracellular RA concentration. The active compound in Thymex-L is heat stable and bigger than 5 kDa as confirmed by gelfiltration. The defined thymus peptides thymosin alpha 1, prothymosin alpha 1 and thymopentin were unable to synergistically enhance HL-60 differentiation. These data suggest that the treatment with a thymus extract can increase the sensitivity of HL-60 cells for RA. This may have clinical implications.

The in vivo effect of thymic factor (thymostimulin) administration on circulating immune complexes and serum lysozyme levels in untreated Hodgkin's disease patients.

The in vivo effect of a calf thymus extract, thymostimulin, on the levels of circulating immune complexes (CIC) and serum lysozyme was evaluated in 32 patients with untreated Hodgkin's disease. Using the platelet aggregation test for detecting CICs, 12 patients (37%) had positive titers before thymostimulin treatment; 3 patients (10%) remained positive following therapy. Serum levels of Clq-binding immune complexes were evaluated (greater than 24.5 micrograms/ml) in 8 patients prior to thymostimulin therapy (mean value: 42.3 micrograms/ml); 3 patients continued to have elevated levels after treatment. Serum lysozyme levels for Hodgkin's patients was similar to control values (10.6 vs. 8.3 micrograms/ml); however, the Hodgkin's patients with initially elevated CICs had a lower serum lysozyme level than patients with initially normal CICs (12.9 vs. 7.3, p less than 0.02). Thymostimulin increased serum lysozyme levels in the Hodgkin's patients in whom the CICs were initially elevated (7.3 vs. 10.4 micrograms/ml, p less than 0.05). These data suggest that thymostimulin exerts an effect on the nonspecific immune system of Hodgkin's disease patients.

Effect of calf thymus extract and zinc supplementation on the cellular response of mice exposed to restraint stress.

The studies were carried out on Balb/c mice exposed to restraint stress twice for 12 h at 24 h intervals. Prior to stress exposure, the mice were treated with calf thymus extract (TFX - Jelfa) i.p. at a dose of 10 mg/kg, ten times at 24 h intervals. TFX was used per se or with zinc ions interaction, by adding zinc ions (as sulfate salt) to drinking water at a dose of 72 microg/mouse per day. The results obtained show that restraint stress dramatically decreased the total number of thymocytes and splenocytes which is also accompanied by decreasing weight ratio of the thymus and spleen. The decreasing number of thymic and spleen cells corresponded to a diminishing percentage of immature, double-positive CD4+CD8+ thymocytes, mature single-positive CD4+ thymic cells and CD4+, CD8+ and CD19+ splenocytes. Changes in the number of thymic cells affect their activity, which is expressed as a decreased proliferative response of thymocytes stimulated in vitro with concanavalin A (Con A) and phytohaemagglutinin (PHA). Besides, exposure to the restraint stress decreased interleukin-1 (IL-1) production by murine intraperitoneal macrophages stimulated in vitro with lipopolisacharide (LPS) from E. coli. Previous treatment with TFX counteracted restraint stress-induced immunosuppression, which is expressed as partial normalisation of the total number of thymic and spleen cells, accelerated regeneration of these two lymphatic organs, shortned suppressive action of restraint stress on the percentage of immature CD4+CD8+ thymocytes and CD4+ splenocytes and in total normalisation of the CD4+ thymocytes and CD8+ splenocytes. TFX administered prior to restraint stress not only counteracted the suppresive effects of stress on the proliferative activity of thymic cells stimulated in vitro with Con A and PHA, but also augmented the proliferative response of these cells to two mitogens. The immunorestorative effect of TFX was augmented by zinc supplementation.

The role of a thymus humoral factor in the proliferation of bone marrow CFU-S from thymectomized mice.

The colony forming capacity of bone marrow cells from thymectomized mice was shown to be reduced as compared to that of bone marrow cells from normal donors. A further indication of changes in the proliferative capacity of colony forming cells (CFU-S), following thymectomy, was given by examination of the sensitivity of these cells to chlorambucil and by 3H-thymidine 'suicide' experiments; both showed that CFU-S from thymectomized mice were not cycling at the same rate as normals. It was also found that in late pregnancy of thymectomized females, there is an elevation in the number of bone marrow CFU-S and an increase in cell cycling. Such an increase could also be achieved by implantation, into thymectomized mice, of thymus lobes in closed diffusion chambers. Finally, in vitro administration of a thymus hormone (THF) reversed the suppressive effect of thymectomy on DNA synthesis in bone marrow CFU-S. Since the action of THF was restricted to bone marrow cells of thymectomized mice it is plausible that normal bone marrow contains at least two subpopulations of CFU-S, one of which is dependent upon a humoral product of the thymus.