Biological components in a standardized derivative of bovine colostrum.

Products of different origin, time of collection, and activities fall under the general term of colostrum and, therefore, great variability in composition as well as in the concentration of its components has been reported in the literature. In the present study, we describe the standardization of a bovine colostrum derivative and the characterization of its bioactive components. Evaluation of the most representative agents (lactoferrin, transferrin, IL-2, IFN-γ, tumor necrosis factor, IgG, and IgA) showed that a marked decrease in active components occurs after the first few hours. Bovine colostrum was, therefore, collected up to the fifth hour after delivery from Holstein cows, in the presence of preservatives, and immediately frozen. A protocol of centrifugation, filtration, and lyophilization was then applied to pools of colostrum from at least 30 cows to obtain a stable, sterile, standardized product. Preservatives were removed by dialysis. Evaluation of the active biological components of colostrum showed that the final product of colostrums contained significant and reproducible amounts of bioactive factors, including cytokines, immunomodulating factors, growth factors, and immunoglobulins. The final product appeared, therefore, as a sterile, pyrogen-free, standardized derivative of bovine colostrum with a high concentration of bioactive components.

The effects of alcoholism pharmacotherapy on immune responses in alcohol-dependent patients.

Chronic alcohol use has profound modulatory effects on the immune system. Both the innate and the acquired immunity are compromised. The use of pharmacotherapy is increasingly applied to enhance the percentage of success in maintaining alcoholic patients in remission. Disulfiram, naltrexone and gamma hydroxybutiric acid are the drugs used for this purpose in Italian Addiction Services. In this study we analyze the effect of pharmacotherapy of alcohol dependence on immune responses in alcoholics. Six groups were studied. Group A included 10 patients who were still using alcohol. Group B consisted of 10 patients abstinent from alcohol in treatment only with group therapy. Groups C, D and E were composed of 10 patients each, treated for at least 6 months with oral doses of gamma hydroxybutiric acid, naltrexone or disulfiram respectively. Ten age- and sex-matched healthy volunteers who never misused alcohol were included as a control group. Lymphoproliferation and peripheral mononuclear cell production of the Th1 cytokines IL-2 and IFN-gamma, the Th2 cytokine IL-4, and of the pro-inflammatory cytokines IL-1 and TNF-alpha were evaluated in all the patients and controls. The level of activity of the hypothalamus pituitary adrenal axis was assessed. Both ACTH and cortisol levels in plasma were elevated in alcoholic patients with no treatment. In this group a significant alteration of cytokine production was observed. TNF and IFN-gamma were lower than controls, while the Th2 cytokine IL-4 was increased. These altered levels state for a Th1/Th2 unbalance characterized by decreased Th1 response in the presence of Th2 predominance. In patients undergoing pharmacological treatment, none of the immune parameters were different from those observed in healthy controls, independently of the type of drug administered. These data indicate that pharmacotherapy more than group therapy treatment is able to ameliorate the immune system functioning in alcoholic patients.

PPADS, a purinergic antagonist reduces Fos expression at spinal cord level in a mouse model of mononeuropathy.

Recent evidence suggest that ATP plays a role as an endogenous pain mediator generating and/or modulating pain signaling from the periphery to the spinal cord. In this study we evaluated the effects of intraperitoneal administration of P2 receptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), evaluating pain related behaviours and monitoring the expression of Fos, a marker of activated neurons, in an experimental mouse model of neuropathic pain (sciatic nerve tying). The PPADS administration decreased both tactile allodynia and thermal hyperalgesia in a time and dose dependent manner. The dose of 25 mg/kg PPADS completely reversed nociceptive hypersensitivity. Moreover, non-noxious stimulation induced an increase of Fos positive neurons in the spinal cord of animals with tying of sciatic nerve. PPADS administration partially reversed this increase. These results suggest that PPADS reduces neuronal activation at spinal cord level and that P2 receptors are involved in the retrograde signalling progress exciting sensory spinal neurons.

The nonpsychoactive component of marijuana cannabidiol modulates chemotaxis and IL-10 and IL-12 production of murine macrophages both in vivo and in vitro.

Cannabidiol is the main nonpsychoactive component of marijuana. We examined the ability of in vivo and in vitro cannabidiol to interfere with the production of interleukin (IL)-12 and IL-10 by murine macrophages and to modulate macrophage chemotaxis. Cannabidiol added in vitro to peritoneal macrophages significantly increased IL-12 and decreased IL-10 production. The CB1 and CB2 receptor antagonists prevented this modulation. Macrophages from animals treated with cannabidiol at the dose of 30 mg kg(-1) either orally or i.p. produced higher levels of IL-12 and lower levels of IL-10 in comparison to controls, and the CB receptor antagonists did not prevent these effects. Cannabidiol dose-dependently decreased fMLP-induced chemotaxis of macrophages, and the CB2 receptor antagonist prevented this decrease.

Effects of tramadol on synovial fluid concentrations of substance P and interleukin-6 in patients with knee osteoarthritis: comparison with paracetamol.

Both the analgesic drugs tramadol and paracetamol are widely used for the symptomatic therapy of osteoarthritis (OA). The aim of this double-blind, randomised study in patients with knee OA was to compare their effects on synovial fluid concentrations of interleukin (IL)-6 and substance P (SP). Moreover, we evaluated plasma and synovial fluid concentrations of tramadol and its active metabolite (O-desmethyl-tramadol, M1) after oral treatment with this drug. Twenty patients were enrolled. A group of 10 patients received tramadol (50 mg three times a day), and another group of 10 patients were treated with paracetamol (500 mg three times a day) for 7 days. Both drugs significantly reduced the intensity of joint pain. The synovial fluid concentrations of SP were significantly reduced only by the treatment with tramadol. In this group of patients, IL-6 synovial fluid concentrations were slightly, but not significantly, decreased. Paracetamol did not significantly change the synovial fluid concentrations of SP and IL-6. After oral administration, a considerable amount of tramadol was measurable in synovial fluid. Both in plasma and synovial fluid the concentrations of M1 were markedly lower than those of tramadol, with a T/M1 ratio of 14.7+/-4.6 and 9.3+/-3.9, respectively. These data demonstrate that the activity of tramadol may involve the modulation of inflammatory mediators. Moreover, they indicate that after oral treatment with tramadol, both the parent drug and its active metabolite can penetrate into synovial fluid.

Individual housing induces altered immuno-endocrine responses to psychological stress in male mice.

Social isolation and lack of social support have deleterious effects on health, thus being regarded as one of the most relevant causes of diseases in human and other mammalian species. However, only few are the studies aimed at evaluating the psychoneuroimmunological functions of individually housed subjects. The present study was designed to understand how the behavior and the physiology of male house mice might be affected by individual housing. We first analyzed whether individual housing of different duration (1-42 days) would result in immuno-endocrine dysfunction (experiment 1). Then we investigated whether housing conditions would affect the reaction to an acute mild psychological stress (experiments 2 and 3). There were three main findings: first, individually housing mice for increasing time periods did not induce any major immuno-endocrine effects compared to a stable sibling group housing. Therefore, prolonged isolation does not seem to dramatically impair mice immuno-endocrine functions. Second, when exposed to a mild acute stress, i.e. forced exposure to a novel environment, isolated mice showed higher basal corticosterone and lower type 1 (IL-2) and type 2 (IL-4) cytokines as well as splenocytes proliferation compared to group housed male mice. Finally, when faced with a free choice between a novel environment and their home cage, individually housed mice showed reduced neophobic responses resulting in increased exploration of the novel environment, thus suggesting a low anxiety profile. Altogether, our findings suggest that individual housing in itself does not change immunocompetence and corticosterone level, but does affect reactivity to a stressor. In fact, individually housed mice showed high behavioral arousal, as well as altered immuno-endocrine parameters, when challenged with mild psychological novelty-stress.

Beta endorphin concentrations in PBMC of patients with different clinical phenotypes of multiple sclerosis.

The possible link between the opioid peptide beta endorphin and the heterogeneity of the clinical course of multiple sclerosis (MS) was investigated. Peripheral blood mononuclear cells (PBMC) concentrations of beta endorphin were measured in 50 patients in different phases of MS. Thirty nine patients also underwent post-contrast magnetic resonance imaging of the brain. Among MS forms, the highest beta endorphin concentrations were found in PBMC from patients with relapsing remitting MS and the lowest in patients with the progressive forms. Average beta endorphin concentrations were lower, although not significantly, in patients with than in those without magnetic resonance imaging enhanced lesions. These data suggest that beta endorphin may have a role in the downregulation of the inflammatory process.

MFP14, a multifunctional emerging protein with immunomodulatory properties, prevents spontaneous and recurrent autoimmune diabetes in NOD mice.

AIMS/HYPOTHESIS: To test the effects of multifunctional protein 14 (MFP14), which shares structural homology with heat shock proteins (HSPs), on the development of Type I (insulin-dependent) diabetes mellitus in NOD mice. METHODS: MFP14 was given to euglycaemic female NOD mice from either the 4th to the 25th or from the 12th until the 35th week, or commencing one day before islet transplantation and until the reappearance of hyperglycaemia. Pancreata from NOD mice treated with multifunctional protein 14 for 14 consecutive weeks until 18 weeks of age were examined histologically for insulitis. Anti-CD3 and/or lipopolysaccharide (LPS)-induced blood levels of interferon (IFN)-gamma, interleukin (IL)-4, IL-10, IL-12 and tumour necrosis factor (TNF)-alpha were measured by ELISA in 10 week-old female NOD mice treated for 6 consecutive weeks with either MFP14 or PBS. Unless otherwise stated, multifunctional protein 14 was administered daily 5 times a week at a dose of 25 microg. Control mice received PBS or, in selected experiments, heat-inactivated MFP14. RESULTS: MFP 14 treated mice had a significantly lower incidence of spontaneous diabetes compared to control mice. The MFP14 was equally effective both upon early and late prophylaxis and the protection persisted until week 50 in mice treated from weeks 4 to 25. Insulitis was significantly reduced by the MFP14. The MFP14 also delayed recurrence of hyperglycaemia in syngeneic islet-transplanted NOD mice. Although MFP14 reduced anti-CD3 and/or LPS-induced blood levels of IFN-gamma, TNF-alpha and IL-12 it increased IL-4 and IL-10. CONCLUSION/INTERPRETATION: The MFP14 could be a possible candidate for the prevention or early treatment of human Type I (insulin-dependent) diabetes mellitus.

Social status in mice: behavioral, endocrine and immune changes are context dependent.

The aim of the present study was to investigate the effect of social status on the endocrine, immune and behavior response of male mice. We found that in mice reared in a group of siblings since weaning, no difference exists between dominants and subordinates in basal corticosterone level, in behavior in the open-field test (OFT) and in a series of immune parameters. These results suggest that living with siblings is not a stressful condition for either dominant or subordinate mice. Therefore, group-housed siblings can be regarded as a valid control group in social stress studies. When mice were subjected to chronic psychosocial stress for 21 days, four types of social outcome occurred: residents becoming dominants, intruders becoming subordinates, residents becoming subordinates and intruders becoming dominants. Interestingly, the behavioral profile in the OFT revealed a status-dependent effect, with resident dominants (RD) and intruder dominants (InD) showing the highest locomotor and exploratory activity, whereas the corticosterone level was higher than control for all four categories. In addition, a context-dependent effect emerged at the immune level: resident subordinates (RS) had a reduced splenocyte proliferation and IL-4 and IL-10 production. Mice in all the other three social ranks showed no immune alterations. Therefore, the loss of an individual's social rank position seems a promising field of study to investigate the psychological impact of stressful events.

Modulation of experimental allergic encephalomyelitis in Lewis rats by administration of a peptide of Fas ligand.

The effects of modulation of apoptosis in experimental allergic encephalomyelitis (EAE) in Lewis rats have been investigated using a peptide of the Fas-Ligand protein (FasL-p). The peptide was administered both subcutaneously and intra-cerebro-ventricularly (i.c.v.) after EAE induction. Rats treated subcutaneously with FasL-p showed a worse clinical score as compared to saline treated animals, while i.c.v. treatment with FasL-p did not modify significantly the severity of EAE. Apoptotic lymphomonocytes (identified by TUNEL) infiltrating the brain and the spinal cord were decreased in rats treated i.c.v. with FasL-p. The data suggest that the Fas/Fas-ligand pathway may be modulated by treatments with peptides of Fas-Ligand and that it may be at work within the central nervous system in EAE.

In vivo and in vitro treatment with the synthetic cannabinoid CP55, 940 decreases the in vitro migration of macrophages in the rat: involvement of both CB1 and CB2 receptors.

Cannabinoids have been shown to affect immune responses, acting on different populations of immune cells. In the present paper we analyze the ability of in vivo and in vitro treatment with the potent synthetic cannabinoid CP55,940 to interfere with an important function of rat peritoneal macrophages, i.e. spontaneous migration and formyl-metionyl-leucine-phenylalanine (fMLP)-induced chemotaxis, that were assessed by the use of a Boyden-modified microchemotaxis chamber. When added in vitro, CP55,940 induced a significant and dose-dependent inhibition of both spontaneous migration and fMLP-induced chemotaxis. Both the Cannabinoid Receptor 1 (CB1) and the Cannabinoid Receptor 2 (CB2) antagonists were able to block the CP55,940-induced inhibition of spontaneous migration, although the CB2 antagonist was more potent and only the CB2 antagonist was able to reverse the effect of CP55,940 on fMLP-induced chemotaxis. Similarly, in the in vivo experiments, 1 h after the acute subcutaneous administration of 0.4 mg/kg of CP55,940, both spontaneous motility and chemotaxis were reduced. The pretreatment with the CB2 antagonist, but not with the CB1 antagonist, was able to prevent this effect. Our data confirm that cannabinoids can affect some macrophage functions, mainly throughout CB2 receptors, and suggest that the development of specific CB2 ligands may lead to an interesting new class of anti-inflammatory drugs.

Beta-endorphin concentrations in peripheral blood mononuclear cells of patients with multiple sclerosis: effects of treatment with interferon beta.

CONTEXT: It has been reported that the opioid peptide beta-endorphin (BE) has immunosuppressive effects. Interferon beta (IFN-beta) is a well-established therapy for multiple sclerosis (MS), but immunological mechanisms underlying its beneficial effects in MS are partially undefined. OBJECTIVES: To determine BE levels in peripheral blood mononuclear cells (PBMCs) of patients with relapsing-remitting MS during different phases of disease activity and the possible modulating effects of IFN-beta treatment on PBMC BE synthesis in patients with MS. DESIGN: We measured BE levels in blood samples collected from 6 patients with MS who had not experienced clinical changes during the previous 3 months (patients with stable MS) and from 7 patients with MS during a clinical relapse. We also surveyed BE levels in PBMC samples from 8 patients with MS before treatment and for 6 months after the beginning of IFN-beta administration. The control group was 13 healthy subjects. RESULTS: Low PBMC BE levels were detected in patients with stable MS and in those entering IFN-beta treatment compared with control subjects. Increased BE concentrations were observed in MS patients experiencing a clinical relapse compared with patients with stable MS. During IFN-beta treatment, the levels of BE in PBMC samples from patients with MS increased significantly (after 1 month, P =.02; after 3 months, P =.007; and after 6 months, P =.16). CONCLUSIONS: A reduction of BE levels was present in patients with clinically inactive MS. Treatment with IFN-beta seems to induce an increase of this opioid in PBMCs of MS patients. The increase of BE concentration during a clinical relapse may represent a possible control mechanism aimed at counterbalancing the inflammatory phase of the disease. Arch Neurol. 2000;57:1178-1181

The effects of tramadol and morphine on immune responses and pain after surgery in cancer patients.

UNLABELLED: There has been growing interest in determining the possible immune consequences of opioid administration for the management of postoperative pain. We studied the effects of morphine and tramadol on pain and immune function during the postoperative period in 30 patients undergoing abdominal surgery for uterine carcinoma. Phytohemoagglutinin-induced T lymphocyte proliferation and natural killer cell activity were evaluated immediately before and after surgery, and 2 h after the acute administration of either 10 mg of morphine IM or 100 mg tramadol IM for pain. In all patients, phytohemagglutinin-induced lymphoproliferation was significantly depressed by surgical stress. However, in the morphine-treated group, proliferative values remained lower than basal levels for 2 h after treatment, whereas in tramadol-administered patients proliferative values returned to basal levels. Natural killer cell activity was not significantly affected by surgery nor by morphine administration, whereas tramadol significantly enhanced the activity of natural killer cells. Both drugs produced a comparable reduction in postoperative pain. We conclude that, as previously observed in the experimental animal, tramadol and morphine, when administered in analgesic doses, induce different immune effects. IMPLICATIONS: Recent studies suggest that opioids can have an adverse impact on the immune system. Because surgical stress also induces immune dysfunction, the search for analgesic drugs devoid of immunosuppressive effects is of import. This study compared the effects on immune responses of morphine and of the atypical opioid analgesic, tramadol, given for postoperative pain to gynecological cancer patients. Tramadol and morphine showed comparable analgesic activity; however, tramadol, in contrast to morphine, induced an improvement of postoperative immunosuppression and, therefore, may be preferred to morphine for the treatment of postoperative pain.

The opioid antagonist naloxone induces a shift from type 2 to type 1 cytokine pattern in BALB/cJ mice.

Opioid peptides affect different immune functions. We present evidence that these effects could be mediated by the modulation of T(H)1/T(H)2 cytokine production. BALB/cJ mice were immunized with 50 or 100 microg of the protein antigen keyhole-limpet hemocyanin (KLH), and treated acutely or chronically with the opioid antagonist naloxone. One and 2 weeks after immunization, the production of cytokines by splenocytes was evaluated by in vitro restimulation with KLH. The acute and chronic treatment with the opioid receptor antagonist naloxone decreased the production of interleukin (IL)-4 by splenocytes of BALB/cJ mice. In contrast, IL-2 and interferon-gamma levels increased after naloxone treatment. Finally, the opioid antagonist diminished the serum immunoglobulin G anti-KLH antibody titers. These results suggest that naloxone increases T(H)1 and decreases T(H)2 cytokine production. The effect of naloxone could be ascribed to the removal of the regulatory effects exerted by endogenous opioid peptides, which could therefore activate T(H)2 and suppress T(H)1 cytokines. (Blood. 2000;95:2031-2036)

The opioid antagonist naloxone induces a shift from type 2 to type 1 cytokine pattern in normal and skin-grafted mice.

Opioid peptides affect different immune functions. We present evidence that these effects could be mediated by the modulation of Th1/Th2 cytokine production. The acute and chronic treatment with the opioid receptor antagonist naloxone decreased the production of IL-4 by splenocytes of C57BL/6 and Balb/cJ mice, that present a Th1/Th2 dominance, respectively, immunized with the protein antigen KLH. In contrast, IL-2 and IFN-gamma levels were increased after naloxone treatment. These results indicate that naloxone increases Th1 and decreases Th2 cytokine production. Moreover in C57BL/6 mice, naloxone treatment was able to accelerate skin-graft rejection, a Th1-mediated phenomenon, by increasing Th1 cytokine production. The effect of naloxone could be ascribed to the removal of the regulatory effects exerted by endogenous opioid peptides, which could activate Th2 and suppress Th1 cytokines.

Effects of tramadol and its enantiomers on Concanavalin-A induced-proliferation and NK activity of mouse splenocytes: involvement of serotonin.

The centrally acting analgesic drug tramadol is a 1:1 racemic mixture of two enantiomers, with different pharmacological properties. The (-)-tramadol preferentially inhibits noradrenaline uptake, whereas the (+)-tramadol inhibits serotonin uptake and binds to opioid receptors. Since tramadol has been shown to stimulate some immune responses in mice, in the present work we analyzed the effects of its enantiomers on the same parameters, with the aim to better characterize the mechanisms involved in such action of tramadol. The acute administration of 20 and 40 mg/kg of racemic tramadol and of 10 and 20 mg/kg of (+)-tramadol induced a significant and comparable stimulation of Concanavalin-A (Con-A) induced proliferation and of Natural Killer (NK) activity of splenocytes. On the contrary, the (-)-tramadol was devoid of any effect. The pretreatment with the serotoninergic antagonist metergoline (3.0 mg/kg) completely blocked the effects of both tramadol and (+)-tramadol. We suggest that the enhancement of the serotoninergic tone could be at the basis of the stimulatory effects exerted by tramadol on Con-A induced lymphoproliferation and NK activity.

Hypothalamic beta-endorphin concentrations are decreased in animals models of autoimmune disease.

Complex interactions between the neuroendocrine and the immune systems are present in autoimmune diseases. The central opioid peptide beta-endorphin (BE) has been shown to modulate peripheral immune responses in normal animals. In the present study we analyze the hypothalamic concentrations of this peptide in two models of spontaneous autoimmune disease, the MRL [corrected] lpr/lpr mouse, that develops a lupus-like autoimmune disease, and the obese strain (OS) chickens afflicted with spontaneous autoimmune thyroiditis. In both instances, hypothalamic concentrations of BE are significantly lower than normal controls. In MRL [corrected] lpr/lpr mice, BE is already lower at 1 month of age, when no clinical sign of the disease is yet present. Similarly, low levels of BE are observed in OS chickens before the onset of thyroiditis, i.e., already at the embryonic stage. Moreover, a further decrease of BE is observed in OS chickens in correspondence with the first signs of thyroid mononuclear infiltration. Considering the immunosuppressive effects exerted by central BE, these results are suggestive of the fact that in autoimmune disease prone animals the low hypothalamic concentrations may be one of several factors predisposing for the development of autoimmune disease.

Chronic administration of UK-114, a multifunctional emerging protein, modulates the Th1/Th2 cytokine pattern and experimental autoimmune diseases.

UK-114 is a 14-kDa ubiquitous protein recently sequenced by several groups throughout the world. Its activity ranges from being a tumor antigen, a protein synthesis inhibitor or a specific mu-calpain activator. UK-114 shows structural homologies also with proteins of the MHC-1 binding proteins, and heat shock proteins (HSPs). We investigated the possible effects of UK-114 on T helper cells cytokine profile and the development and progression of experimental autoimmune diseases. Homogeneous recombinant UK-114 was used in all experiments. Treatment of Balb/c male mice for two weeks resulted in the increase of IL-4, and the decrease of TNF-alpha, IFN-gamma, and IL-2 release from stimulated splenocytes, suggesting that UK-114 modulates the Th1/Th2 cytokine profile toward Th2. Similar to that observed with HSP60/65, a single pretreatment of Lewis rats with UK-114 significantly blunted the development of adjuvant-induced arthritis, whereas chronic treatment of 4-week-old female NOD mice dose dependently inhibited the development of diabetes.