Prestin autoantibodies screening in idiopathic sudden sensorineural hearing loss.

OBJECTIVES: To define the clinical association of serum prestin autoantibodies and their impact on prognosis, as specific serum diagnostic markers in patients with idiopathic sudden sensorineural hearing loss (ISSNHL). DESIGN: Sera from 63 patients with ISSNHL were screened prospectively for the presence of prestin autoantibodies by an enzyme-linked immunosorbent assay (Elisa) test. Serum was assayed for anti-prestin IgG antibodies using recombinant human prestin (SLC26 A5). Demographic, clinical, and audiometric variables were analyzed. RESULTS: Two patients (3.17%) had demonstrable anti-prestin antibodies in serum (exact 95% CI: -1.16% to 7.5%). No statistically significant association was found between prestin autoantibodies and demographic or audiologic parameters. CONCLUSIONS: This preliminary and novel study does not support the presence of an active humoral immune reaction against prestin in ISSNHL.

Prevention and reversal of adoptively transferred, chronic relapsing experimental autoimmune encephalomyelitis with a single high dose cytoreductive treatment followed by syngeneic bone marrow transplantation.

A chronic relapsing form of experimental autoimmune encephalomyelitis (CR-EAE) was induced in SJL/J mice by adoptive transfer of lymph node cells (LNC) sensitized to guinea pig myelin basic protein (GMBP). We examined the efficacy of high dose immunosuppressive regimens (cyclophosphamide [CY] 300 mg/kg or total body irradiation [TBI] 900 cGy) followed by syngeneic bone marrow transplantation (SBMT) in prevention and treatment of already established CR-EAE. Treatment with TBI and SBMT on day 5 after the induction of CR-EAE, just before the onset of clinical signs, completely inhibited the appearance of the paralytic signs. The same treatment, applied 4 d after the clinical onset of the disease, led to a significant regression of the paralytic signs and to a total inhibition of spontaneous relapses during a follow-up period of 2 mo. Challenge of mice with GMBP+CFA 78 d after the passive induction of CR-EAE induced a relapse of the disease 7 d later in almost all of the untreated mice; in contrast, the same challenge given to TBI+SBMT-treated mice caused a delayed relapse (30 d later) in only a minority (3/7) of the challenged mice. In vitro lymphocytic proliferative responses to GMBP and purified protein derivative were significantly lower in TBI/SBMT-treated mice before and after the GMBP challenge, although these mice were fully immunocompetent, as evidenced by their normal lymphocytic proliferation to concanavalin A (ConA) and the FACS analysis of their lymphocytic subpopulations. A similar beneficial therapeutic effect was observed in mice treated with CY followed by SBMT, after the onset of CR-EAE. Our results could support possible clinical applications of similar therapeutic strategies, involving acute immunosuppression followed by stem cell transplantation and retolerization of the reconstituting immune cells in life-threatening neurological and multisystemic autoimmune diseases.

Copaxone interferes with the PrP Sc-GAG interaction.

The hallmark of prion disease-induced neurodegeneration is the accumulation of PrP(Sc), a misfolded form of PrP(C). In addition, several lines of evidence indicate a role for the immune system and, in particular, inflammation in prion disease pathogenesis. In this work, we tested whether Copaxone, an immunomodulatory agent currently used for the treatment of multiple sclerosis, can affect prion disease manifestation in scrapie-infected hamsters. We show here that Copaxone exerted no effect on prion disease incubation time when treatment commenced 2 weeks after i.p. prion infection. However, when Copaxone was mixed with the initial prion inoculum or administered to hamsters weekly starting on the day of infection, prion disease incubation time was prolonged by 30 days. This suggests that Copaxone may affect the initial infection process. In vitro experiments indicate that Copaxone significantly reduced PrP(Sc) binding to both Chinese hamster ovary (CHO) cells and heparin beads and also binds to heparin by itself. Interestingly, Copaxone also abolished PrP(Sc) accumulation in scrapie-infected cells. We propose that Copaxone delays prion infection by competing with the PrP(Sc)-glycosaminoglycans interaction. Whether the immunomodulating activity of Copaxone is related to its heparin binding and anti-prion properties remains to be established.

Lipopolysaccharide induces proenkephalin gene expression in rat lymph nodes and adrenal glands.

The proenkephalin gene encodes a family of neuropeptides that was originally identified in brain tissue and adrenal glands. Recently, it was shown that proenkephalin is also expressed in cultured lymphoid cells. To elucidate the physiological significance of this expression, we examined the in vivo expression of proenkephalin in lymphoid tissues. We show here that exposing rats to the endotoxin lipopolysaccharide induces an intense and transient expression of proenkephalin in adrenal glands and lymph nodes. By using combined in situ hybridization and immunohistochemistry on tissue slices, we identified proenkephalin expression in macrophages located within the lymph nodes and in chromaffin cells within the adrenal glands. This in vivo expression of proenkephalin was enhanced by adrenaline. The present observations demonstrate that the immune system is a site of significant expression of proenkephalin and provide a basis for neuroimmune interactions.

Regulation of proenkephalin A messenger ribonucleic acid levels in normal B lymphocytes: specific inhibition by glucocorticoid hormones and superinduction by cycloheximide.

Proenkephalin A (PEA) encodes a group of small peptides known to function as neurotransmitters, neuromodulators, and neurohormones in the nervous and neuroendocrine systems. This gene has been shown to be expressed in lymphoid cells, supporting the concept of bidirectional communication between the immune system and the central nervous system. In the present study, we investigated the effect of steroids and the inhibition of protein and RNA syntheses on the regulation of PEA expression in normal rat B cells. The transient expression of PEA messenger (m) RNA levels occurring normally in B cells was markedly inhibited by the presence of either 50 nM prednisolone or dexamethasone, both of which are glucocorticoids; other steroids, such as testosterone or the steroid-inactive metabolite androsterone, were ineffective. In the presence of cycloheximide, a protein synthesis inhibitor, PEA mRNA was superinduced by a factor of 15-fold. Sorting by flow cytometry of cycloheximide-treated cells followed by in situ hybridization analysis revealed that the expression of PEA mRNA was exclusively confined to a small fraction of B cells. These results indicate that the mechanisms regulating PEA gene expression in B cells differ from those previously described in cells of the neuroendocrine and the nervous systems.

Prevention of experimental allergic encephalomyelitis by anterior hypothalamic lesion in rats.

Bilateral electrical lesions were performed in the anterior hypothalamus (AH) and hippocampus (HC) of female Lewis rats. AH but not HC lesions were found to inhibit the appearance of clinical signs typical of experimental allergic encephalomyelitis (EAE). The incidence of EAE was 17.2% and the duration was 1.33 +/- 0.07 days after AH lesions compared with an incidence of 85% and duration of 4.81 +/- 0.6 days in the controls. Destruction of the AH was followed by decreased levels of antibodies to myelin basic protein and increased reactivity of splenic lymphocytes to concanavalin A, but did not affect the extent of mononuclear cell infiltration within the brain and spinal cord.

Effects of antidepressant drugs on the behavioral and physiological responses to lipopolysaccharide (LPS) in rodents.

Antidepressants produce various immunomodulatory effects, as well as an attenuation of the behavioral responses to immune challenges, such as lipopolysaccharide (LPS). To explore further the effects of antidepressants on neuroimmune interactions, rats were treated daily with either fluoxetine (Prozac) or saline for 5 weeks, and various behavioral, neuroendocrine, and immune functions were measured following administration of either LPS or saline. Chronic fluoxetine treatment significantly attenuated the anorexia and body weight loss, as well as the depletion of CRH-41 from the median eminence and the elevation in serum corticosterone levels induced by LPS. Chronic treatment with imipramine also attenuated LPS-induced adrenocortical activation. In rats and in mice, which normally display a biphasic body temperature response to LPS (initial hypothermia followed by hyperthermia), chronic treatment with fluoxetine completely abolished the hypothermic response and facilitated and strengthened the hyperthermic response. The effects of antidepressants on the responsiveness to LPS are probably not mediated by their effects on peripheral proinflammatory cytokine production, because LPS-induced expression of TNFalpha and IL-1beta mRNA in the spleen (assessed by semiquantitative in situ hybridization) was not altered following chronic treatment with either fluoxetine or imipramine. The effects of antidepressants on the acute phase response may have important clinical implications for the psychiatric and neuroendocrine disturbances that are commonly associated with various medical conditions.

Albumin magnetic microspheres: a novel carrier for myelin basic protein.

Myelin basic protein (MBP) of guinea pig origin was incorporated into magnetically responsive albumin microspheres. Protein-protein bonding and stabilization of the GPMBP microspheres by heating at 120 degrees C did not adversely influence their capacity to bind anti-MBP antibodies or demonstrably alter the encephalitogenic activity of the incorporated GPMBP. The magnetic properties of the particles and the fact that immunodeterminants of some of the incorporated MBP fortuitously were distributed on the exterior surfaces of the microspheres allowed a number of experiments to be carried out in Lewis rats for the first time: (a) selective capture and deletion of that particular subpopulation of lymphoid cells responsible for transfer of experimental allergic encephalomyelitis (EAE) represented within the lymph node cells (LNC) of donor animals sensitized to neutral antigen, (b) enhancement of in vivo uptake of MBP by macrophages (M phi s) contained in oil-induced peritoneal cell exudates and exposed briefly to MBP microspheres, and (c) preparation of cell suspensions specifically enriched with respect to MBP-containing M phi s.

Normal immunosuppressive protein purification and quantitative estimation experiments.

An improved method for the preparation and purification of normal immunosuppressive protein (NIP) is described. The purified material has a molecular weight between 10,000 and 25,000. Its biological and serological activity is approximately 10--20 times higher than that of the crude fraction. An antibody to normal immunosuppressive protein prepared in rabbits made the quantitative estimation of NIP by a haemaggluination inhibition test possible. Similarly, a very sensitive assay for the quantitative determination of NIP by its inhibitory effect on the proliferation of EL-4 tumor cells is also described. Eluates prepared from polyacrylamide gels were active in inhibiting EL-4 tumor cell proliferation and neutralized the anti-NIP activity in the haemagglutination inhibition test.

Cellular and secretory mechanisms related to delayed radiation-induced microvessel dysfunction in the spinal cord of rats.

PURPOSE: This study aimed to investigate long-term, radiation-induced changes in microvessel permeability, the profile of the vasoactive mediators endothelin and nitric oxide, and the response of specific cell systems in the irradiated spinal cord of rats. METHODS AND MATERIALS: The thoracolumbar spinal cords of Fischer rats were irradiated to a dose of 15 Gy, and the rats were sacrificed at various times afterward. Endothelin levels and nitric oxide-synthase (NOS) activity were assayed in extracts of spinal cords. Microvascular permeability and the effect of treatment with recombinant human manganese superoxide dismutase (r-hMnSOD) were assessed quantitatively. Immunohistochemistry evaluated astrocytes, microglia, vascular basal membrane, and neurofilaments. RESULTS: None of the rats developed neurologic dysfunction. Endothelin levels were significantly reduced at 18 h after irradiation and markedly attenuated after 10 days (p < 0.007). Thereafter, endothelin levels returned to normal values at 56 days after radiation and escalated to markedly high levels after 120 and 180 days (p < 0.002). NOS activity remained very low throughout the period of follow-up and failed to counterbalance the shifts in endothelin levels. Treatment with r-hMnSOD had no effect on normal vascular permeability but it abolished the abnormally increased permeability measured at 18 h after radiation and again after 120 and 180 days. Standard microscopic evaluation failed to reveal abnormalities in the irradiated spinal cord, but immunohistochemical staining showed a progressive increase in the number of microglial cells per field after 120 and 180 days (p < 0003). A similar increase in the number of astrocytic cells per field was noted after more than 180 days, but an earlier short lasting peak was also noted at 14 days after radiation. No abnormalities were found in blood vessel configuration, density, diameter, and basal membrane staining, or in the neurofilaments. CONCLUSION: Marked imbalance in the regulatory function of endothelium-derived mediators of the vascular tone is present after radiation therapy probably inducing chronic vasoconstriction. This imbalance favors localized procoagulation that may enhance the consequent loss of function measured as increased permeability. Microglial proliferation may account for continuous release of superoxide that may enhance disruption of normal permeability. The latter is corrected by SOD treatment. Astrocytic proliferation may present a response to the mitogenic effect of endothelin and to microglial-derived paracrine effect of cytokines.

Evidence for the involvement of the central adrenergic system in interleukin 1-induced adrenocortical response.

The role of central catecholamines in the mediation of adrenocortical activation, induced by interleukin 1 (IL-1), was investigated by measuring ACTH and corticosterone in serum. Adult male rats were injected with either vehicle or the neurotoxin 6-hydroxydopamine (6-OHDA) into the lateral ventricle or the ventral noradrenergic ascending bundle. In vehicle-injected rats, 2 U of IL-1, injected intraventricularly, produced a 5- and 15-fold increase in ACTH and CS, respectively, in serum, 120 min after the injection of IL-1. In contrast, 6-OHDA, injected either intraventricularly or into the ventral noradrenergic ascending bundle, abolished the response to an intracerebral injection of IL-1. In addition, in rats pretreated with the alpha 1-adrenergic antagonist, prazosin, IL-1 failed to activate the adrenocortical axis. In other rats pretreated with the beta-adrenergic antagonist, propranolol, the adrenocortical response did not significantly differ from that of vehicle-pretreated rats. These results suggest that central adrenergic transmission, originating at the ventral noradrenergic ascending bundle and acting through alpha 1-adrenergic receptors, is involved in the adrenocortical response to IL-1.

Characterization of the hypothermic effect of the synthetic cannabinoid HU-210 in the rat. Relation to the adrenergic system and endogenous pyrogens.

In the present study we have characterized the hypothermic effect of the psychoactive cannabinoid HU-210, by investigating its interaction with the endogenous pyrogens, IL-1 and PGE2. We also studied the involvement of the adrenergic system in mediation of this hypothermic effect. Injection of HU-210 directly into the preoptic area caused a dose dependent reduction of rectal temperature from 37 to 32.1 degrees C. Injection of the non-psychoactive analog, HU-211 which does not bind to brain cannabinoid receptor, did not affect body temperature. Injection of the adrenergic agonists, CGP-12177 and clonidine (beta, and alpha adrenergic agonists, respectively) abrogated the hypothermia induced by HU-210. Injection of the adrenergic antagonists, prazosin (alpha 1) and propranolol (beta) enhanced the hypothermic effect of HU-210. Intracerebral administration of IL-1 or PGE2 to rats pretreated with HU-210 caused a transient inhibition of the hypothermia. The ex vivo rate of basal or bacterial endotoxin-induced synthesis of PGE2 by different brain regions, including the preoptic area was not affected by HU-210 administration. These results suggest that the synthetic cannabinoid HU-210 acts in the preoptic area, probably via the brain cannabinoid receptor to induce hypothermia. The hypothermic effect can be antagonized by adrenergic agonists and enhanced by adrenergic antagonists. HU-210 does not interfere with the pyrogenic effect of IL-1 or PGE2.

Evidence for the involvement of the central adrenergic system in the febrile response induced by interleukin-1 in rats.

We have studied the effect of noradrenaline- and serotonin-depleting agents on the febrile response induced by an intracerebroventricular (i.c.v.) injection of interleukin-1 (IL-1) in rats. Pretreatment with an injection into the lateral ventricle of the catecholamine-depleting agent, 6-hydroxydopamine (6-OHDA), abolished the febrile response induced by IL-1. Injection of 6-OHDA into the ventral noradrenergic ascending bundle (VNAB) did not affect the pyrogenic effect of IL-1. Pretreatment with the serotonin-depleting agent, 5,7-dihydroxytryptamine (5,7-DHT), did not inhibit the febrile response to IL-1. In addition, pretreatment with a beta-adrenergic blocker (propranolol) but not an alpha-adrenergic blocker (yohimbine) attenuated the fever induced by an i.c.v. injection of IL-1. These results suggest that the integrity of the central catecholaminergic system is important in mediating the IL-1-induced fever in rats. The central serotonergic system, as well as noradrenergic neurotransmission at the hypothalamus, do not appear to participate in this endogenous pyrogen-induced febrile response.

Characterization of opiate binding sites on membranes of rat lymphocytes.

In view of the importance of membrane receptors for the interconnection between the central nervous system and the immune system, we carried out a study to characterize opiate binding sites on membranes of rat lymphocytes. We found that mitogen-activated spleen cells, but not thymocytes, possess specific and displaceable binding sites for [3H]naloxone. The binding was equally effective and intense while using B-cell-depleted spleen cells. The binding showed two sites of saturation, one at 10 nM and the other at concentrations greater than 20 nM of [3H]naloxone. Computer analysis of the binding data obtained with the lowest concentrations of naloxone revealed a unique site with high affinity binding to opiates. Displacement was achieved with morphine sulphate and naloxone but not with opioid peptides. The binding of the antagonist, [3H]naloxone, was profoundly inhibited by the co-presence of 120 mM NaCl and up to 100 microM guanosine 5'-O-(3-thiotriphosphate (GTP gamma S). Other metal ions and cyclic nucleotides were not able to interfere with the specific binding. This specificity for GTP analogues is consistent with the hypothesis that a GTP-binding regulatory protein that couples receptors to adenylate cyclase is involved in the process of binding of opiates to lymphocytes. The existence of binding sites on lymphoid cells, analogous to receptors for agents known to affect brain functions, may be another link between the immune system and the central nervous system.

Molecular characterization of immune derived proenkephalin mRNA and the involvement of the adrenergic system in its expression in rat lymphoid cells.

Proenkephalin (PENK), a classically defined opioid gene, was originally thought to be expressed almost exclusively in the mature nervous and neuroendocrine systems. In the last few years, it was demonstrated, however, that significant levels of PENK mRNA and PENK-derived peptides are transiently expressed in cells of the immune system. Very little is known about the molecular mechanisms regulating this transient expression. In order to investigate those mechanisms, we examined the in vivo expression of PENK mRNA in mesenteric lymph nodes after exposing rats to lipopolysaccharide. In the present study we demonstrate that: (i) promoter usage and splicing of PENK mRNA function similarly in mesenteric lymph nodes as in neural cells; (2) PENK expression in mesenteric lymph nodes is modulated by adrenaline via adrenergic receptors; and (3) the adrenergic system participates in the modulation of the LPS induced PENK mRNA expression. These results provide more evidence for the involvement of opioids in neuro-immune interactions.

The EAE-associated behavioral syndrome: II. Modulation by anti-inflammatory treatments.

EAE is associated with sickness behavior symptoms that are temporally correlated with inflammatory processes. To further elucidate the role of inflammatory mediators in the behavioral syndrome, EAE mice were injected daily with anti-inflammatory drugs, beginning at disease onset. Dexamethasone or interleukin-1 (IL-1) receptor antagonist or the prostaglandins synthesis inhibitor indomethacin attenuated the behavioral symptoms. Administration of the tumor necrosis-factor alpha (TNF-alpha) synthesis inhibitor pentoxifylline or targeted deletion of the type I TNF receptor had no behavioral effects whereas administration of pentoxifylline in IL-1ra-treated mice further reversed the behavioral depression. These findings demonstrate the critical involvement of pro-inflammatory cytokines and prostaglandins in the EAE-associated behavioral syndrome, and may have implications for understanding and treating the neuropsychiatric disturbances in multiple sclerosis (MS) patients.

The EAE-associated behavioral syndrome: I. Temporal correlation with inflammatory mediators.

To elucidate the mechanisms underlying the EAE-associated behavioral syndrome (EBS), we examined the temporal correlation between the behavioral alterations and inflammatory processes. Onset of the behavioral syndrome was associated with the onset of brain infiltration, as well as mRNA expression of interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) and elevated production of interleukin 1 beta protein and prostaglandin E(2) (PGE(2)). Sickness behavior symptoms coincided with peak cytokine expression. Behavioral recovery was associated with a reduction of cytokine expression, but not infiltration, PGE(2) production or motor disturbances. These results suggest that inflammatory processes in general, and the production of pro-inflammatory cytokines in particular, are involved in mediating EAE-associated sickness behavior.

Experimental allergic encephalomyelitis: passive transfer of resistance during lactation.

Pregnant rats challenged with encephalitogenic antigen in complete Freund's adjuvant (CFA) during pregnancy, transferred a resistance to induction of experimental allergic encephalomyelitis (EAE) in encephalitogenic challenged offspring. The resistance to induction of EAE was transferred during the whole lactation period, until weaning, and not during pregnancy. Through the milk, anti-myelin basic protein antibodies were transferred to the newborn animals. The degree of protection against EAE decayed with age and was not influenced by EAE occurrence in the mothers. In addition, the course of EAE in the rats was not affected by pregnancy. We believe that such transfer of resistance and antibodies may serve as a model for the study of milk-transmitted maternal immunocompetent factors, as well as a model for the mechanisms involved in the resistance of EAE.

Behavioral aspects of experimental autoimmune encephalomyelitis.

Acute inflammation is known to induce a depressive-like sickness behavior syndrome in humans and in experimental animals. In the present study, we sought to determine whether a chronic neuroautoimmune inflammation is also associated with a similar behavioral syndrome. Experimental autoimmune encephalomyelitis (EAE) was induced in SJL/J female mice by adoptive transfer of lymph node cells, and sickness behavior symptoms, including anorexia, loss of body weight, reduced social exploration, and decreased preference for sucrose solution were measured. We report that these components of sickness behavior were induced during the acute phase of the disease, and recovered in later phases. Moreover, the onset and recovery of the behavioral symptoms preceded the onset and recovery of the neurological signs, respectively. Since EAE is considered a model for multiple sclerosis (MS), it is suggested that EAF-induced behavioral changes may serve as a model for the depressive symptomatology that characterizes most MS patients.

Linomide activates the adrenocortical axis in the rat: inhibition of experimental autoimmune encephalomyelitis by linomide is not related to the increase of corticosterone.

Linomide is a synthetic compound that affects various immunological functions and inhibits experimental autoimmune encephalomyelitis (EAE). In the present study we evaluated the effect of linomide on the HPA axis functions under basal and stress-induced conditions and examined whether the effect of linomide on the HPA axis is involved in linomide-induced amelioration of EAE in rats. Linomide caused a significant increase of serum ACTH and corticosterone (CS). The adrenocortical response to various stress modalities as well as the negative feedback exerted by glucocorticoids was not affected. The marked reduction of thymus weight following linomide treatment was abrogated in adrenalectomized rats. The induction of EAE in adrenalectomized rats was completely inhibited by linomide treatment. These results suggest that the increased CS levels induced by linomide are responsible for the decrease in thymus weight but do not play a role in the therapeutic effect of this drug in EAE.