Microvascular and cellular responses in the retina of rats with acute experimental allergic encephalomyelitis (EAE)

The main mechanism causing catabolite repression in Escherichia coli is the dephosphorylation of enzyme IIAGlc, one of the enzymes of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The PTS is involved in the uptake of a large number of carbohydrates that are phosphorylated during transport, phosphoenolpyruvate (PEP) being the phosphoryl donor. Dephosphorylation of enzyme IIAGlc causes inhibition of uptake of a number of non-PTS carbon sources, a process called inducer exclusion. In this paper, we show that dephosphorylation of enzyme IIAGlc is not only caused by the transport of PTS carbohydrates, as has always been thought, and that an additional mechanism causing dephosphorylation exists. Direct monitoring of the phosphorylation state of enzyme IIAGlc also showed that many carbohydrates that are not transported by the PTS caused dephosphorylation during growth. In the case of glucose 6-phosphate, it was shown that transport and the first metabolic step are not involved in the dephosphorylation of enzyme IIAGlc, but that later steps in the glycolysis are essential. Evidence is provided that the [PEP]-[pyruvate] ratio, the driving force for the phosphorylation of the PTS proteins, determines the phosphorylation state of enzyme IIAGlc. The implications of these new findings for our view on catabolite repression and inducer exclusion are discussed.     

Microvascular and cellular responses in the optic nerve of rats with acute experimental allergic encephalomyelitis (EAE)

The optic nerve of rats with EAE was examined at various times to determine the integrity of the blood-brain barrier (BBB) and to assess monocyte-macrophage, T cell, and microglial responses. In naive control animals, leakage of horseradish peroxidase (HRP) and the presence of cells expressing major histocompatibility complex (MHC) class II antigen were evident in the meninges of the retrobulbar optic nerve. In rats with EAE, microglia in the region of the lamina cribrosa and in the regions adjacent to the meninges became activated from day 7 to 8 postinduction (pi). HRP leakage was also evident in the region of the lamina cribrosa from day 7 to 8 pi. On day 8 pi, infiltration of inflammatory cells and Monastral blue leakage were apparent in the myelinated region of the optic nerve. The intensity of these cellular and vascular changes peaked at day 12 pi, when signs of clinical disease became manifest. Monocytes-macrophages expressing MHC class II and the ED1 antigen, together with lymphocytes expressing the alphabetaT cell receptor, constituted the major proportion of cells associated with inflammatory lesions. Thus: (i) the inherent weakness of the BBB as well as the presence of both antigen (myelin) and MHC class II+ cells in the retrobulbar optic nerve are likely susceptibility factors for the frequent involvement of this region in EAE and multiple sclerosis; and (ii) activation of microglia occurs early in the pathogenesis of experimental optic neuritis.     

Persistent expression of experimental autoimmune encephalomyelitis (EAE)-specific Vbeta8.2 TCR spectratype in the central nervous system of rats with chronic relapsing EAE

Monitoring the TCR repertoire is indispensable for the assessment of T cell-associated autoimmune diseases and subsequent TCR-based immunotherapy. In the present study, we examined the TCR repertoire of spinal cord T cells of Lewis rats by CDR3 spectratyping during chronic relapsing experimental autoimmune encephalomyelitis (EAE) induced by immunization with spinal cord homogenate. It was found that Vbeta8.2 spectratype with the shortest CDR3 expanded oligoclonally throughout the course of the disease. In addition, Vbeta12 spectratype expansion was observed at the first and second attacks of EAE. Sequence analysis revealed that clones with the DSSYEQYF sequence, which is a representative sequence of myelin basic protein (MBP)-reactive T cell clones, constituted the predominant population in the Vbeta8.2 family. Surprisingly, Vbeta12 also used the identical amino acid sequence in the CDR3 region. These findings indicate that although infiltrating T cells in the central nervous system are activated polyclonally, the TCR repertoire remains unchanged throughout the course. Moreover, the finding that the predominant CDR3 amino acid sequence of Vbeta8.2 and Vbeta12 spectratypes is identical with that of MBP-induced EAE suggests that a single Ag in spinal cord homogenate, possibly MBP, is involved in disease development.     

Effect of muscular exercise on chronic relapsing experimental autoimmune encephalomyelitis

We examined whether physical exercise affected the development of an autoimmune response, experimental autoimmune encephalomyelitis (EAE), which is a demyelinating disease leading to paralysis. EAE was inducted on day 0, in rats of both sexes, by injecting them with spinal cord tissue in adjuvant. From days 1 to 10 after injection, exercised rats (n = 55) ran on a treadmill (60-120 min/day) before the onset of the paralytic disease. Clinical signs of the disease (ataxia, paralysis, and body mass loss) were examined in exercised and control rats (n = 54). Three types of EAE were induced: monophasic, acute, and chronic relapsing (CR)-EAE (3 bouts of disease, CR-EAE 1, 2, and 3, separated by remissions). Exercise significantly delayed the onset of CR-EAE 1 (P = 0.001) and the 1st day of maximum severity of CR-EAE 1 (P = 0.001) and CR-EAE 2 (P = 0.002). Moreover, the duration of CR-EAE 1 was significantly decreased in exercised rats compared with control rats (P = 0.004). The peak severity of the different types of EAE was not modified by exercise. The present study indicates that endurance exercise during the phase of induction of EAE diminished lightly only one type of EAE (CR-EAE) and therefore did not exacerbate the autoimmune disease.     

Ganglioside changes in brain in chronic relapsing experimental allergic encephalomyelitis induced in the Lewis rat

Chronic relapsing experimental allergic encephalomyelitis (CREAE) was induced in Lewis rats by inoculation with guinea-pig myelin and complete Freund's adjuvant followed by treatment with low-dose cyclosporin A. Rats were sacrificed at different phases of the disease (just before the onset of clinical signs, during the first clinical episode of CREAE and during the first recovery). Gangliosides were extracted from the brain, analysed after purification by HPTLC fractionation and quantified densitometrically. An increase of GM1, the main rat myelin ganglioside, and a decrease of GT1b, suggested to play a role in mediating the interactions between oligodendroglia and axons, were observed during the development of the CREAE. These findings indicating significant ganglioside changes in CREAE give further support to the concept concerning the involvement of gangliosides in autoimmune demyelination.     

2-Buten-4-olide (2-B4O) inhibits experimental allergic encephalomyelitis (EAE) in Lewis rats

Starvation is well known to induce immune suppression. Moreover, the concentration of 2-B4O, an endogenous sugar acid, is elevated in the circulation during starvation. To determine if these events are related, the influence of 2-B4O on experimental allergic encephalomyelitis (EAE) in Lewis rats, a model of human multiple sclerosis (MS), was studied. EAE, characterized by paralysis of hind legs, was induced by immunization with residues 68 to 84 (MB 68-84) of the guinea pig myelin basic protein (MBP) in complete adjuvant H37Ra. Interestingly, the daily administration of 2-B4O intraperitoneally from the day of MB 68-84 immunization (day 0) to day 20 dramatically suppressed the clinical severity of EAE. The daily administration of 2-B4O intraperitoneally from day 0 to day 7 also markedly reduced the clinical symptoms of EAE. In fact, passively induced EAE, using Con A activated spleen cells from rats immunized with MB 68-84 in H37Ra, was also inhibited by daily administration of 2-B4O. Histological examination confirmed clinical findings and revealed that mononuclear cell infiltration into the central nervous system was significantly inhibited by 2-B4O. To clarify the mechanism(s) responsible for suppression of EAE, the effects of 2-B4O on the immune responses to MB 68-84 were examined. When rats were treated daily with 2-B4O for 15 days after immunization with MB 68-84 in H37Ra, the delayed-type hypersensitivity (DTH) response to MB 68-84 was significantly reduced in 2-B4O treated rats as compared with saline treated rats. The proliferative response to MB 68-84 of spleen cells from 2-B4O treated rats was also significantly lower than that of saline treated rats. Our data demonstrate that 2-B4O has the potential to suppress autoimmune responses in both inductive and effector phases. 2-B4O may have significant potential to treat autoimmune diseases.     

Inactivation of T cell receptor peptide-specific CD4 regulatory T cells induces chronic experimental autoimmune encephalomyelitis (EAE)

T cell receptor (TCR)-recognizing regulatory cells, induced after vaccination with self-reactive T cells or TCR peptides, have been shown to prevent autoimmunity. We have asked whether this regulation is involved in the maintenance of peripheral tolerance to myelin basic protein (MBP) in an autoimmune disease model, experimental autoimmune encephalomyelitis (EAE). Antigen-induced EAE in (SJL x B10.PL)F1 mice is transient in that most animals recover permanently from the disease. Most of the initial encephalitogenic T cells recognize MBP Ac1-9 and predominantly use the TCR V beta 8.2 gene segment. In mice recovering from MBP-induced EAE, regulatory CD4+ T cells (Treg) specific for a single immunodominant TCR peptide B5 (76-101) from framework region 3 of the V beta 8.2 chain, become primed. We have earlier shown that cloned B5-reactive Treg can specifically downregulate responses to Ac1-9 and also protect mice from EAE. These CD4 Treg clones predominantly use the TCR V beta 14 or V beta 3 gene segments. Here we have directly tested whether deletion/blocking of the Treg from the peripheral repertoire affects the spontaneous recovery from EAE. Treatment of F1 mice with appropriate V beta-specific monoclonal antibodies resulted in an increase in the severity and duration of the disease; even relapses were seen in one-third to one-half of the Treg-deleted mice. Interestingly, chronic disease in treated mice appears to be due to the presence of Ac1-9-specific T cells. Thus, once self-tolerance to MBP is broken by immunization with the antigen in strong adjuvant, TCR peptide-specific CD4 Treg cells participate in reestablishing peripheral tolerance. Thus, a failure to generate Treg may be implicated in chronic autoimmune conditions.     

Nitric oxide and the immunomodulation of experimental allergic encephalomyelitis

Previous studies examining the effect of nitric oxide synthase (NOS) inhibition on the course of experimental allergic encephalomyelitis (EAE) have yielded conflicting results. This may relate to the use of nonspecific inhibitors and to differences between active and adoptive EAE. We examined the effect of treatment with L-N-(1-iminoethyl)lysine (L-NIL), a selective inhibitor of the cytokine-inducible isoform of NOS, on the clinical course of active and adoptive EAE in Lewis rats. We find that while L-NIL treatment of recipients is protective in adoptive EAE, treatment of active EAE with L-NIL leads to a marked accentuation of disease expression. In L-NIL-treated animals treated with myelin basic protein/complete Freund's adjuvant (MBP/CFA), disease onset is accelerated and clinical symptoms are more severe. Accentuation of integrated disease scores is seen even if L-NIL treatment is started 5 days following immunization. The histological findings in involved spinal cords from L-NIL-treated animals with active EAE are similar to those from untreated animals with similar clinical scores. L-NIL treatment of MBP/CFA-immunized animals does not prevent recovery from clinical symptoms, nor does it allow for reinduction of disease in animals previously immunized with MBP/CFA. Treatment of F344 rats, a strain which is relatively nonsusceptible for EAE, with L-NIL results in consistent evidence of EAE following immunization with MBP/CFA. These findings, together with our previous work on interstitial nephritis, support a role for endogenously generated NO in immunoregulation of T cell responses following immunization with antigen in CFA, and suggest that inducibility of NOS expression may be an important susceptibility factor for autoimmunity.     

Hyperbaric oxygen treatment in acute experimental allergic encephalomyelitis. Contribution of magnetic resonance imaging study

Magnetic resonance imaging (MRI) has been performed to determine the efficacy of hyperbaric oxygen treatment (HBO) on experimental allergic encephalomyelitis (EAE) in Lewis rats. The animals were exposed for 2 x 3 h in a 24-h period at 2 ATA pure O2. Three HBO treatment protocols were used: a 10-day preventive treatment (beginning on the 1st day postimmunization), a 3-day preventive treatment (beginning on the 1st day postimmunization), and a 10-day symptomatic treatment (beginning on the 11th day postimmunization). Based on clinical and MRI observations, this study demonstrates: (i) that HBO treatment does not reduce the blood-brain barrier (BBB) disturbance and cerebral edema in EAE, (ii) that in an opposite way, it provokes reversible BBB breakdown, and (iii) that preventive HBO treatments results in modification of the course of EAE, possibly by immunosuppression effect during the initial sensitization step.     

Study of relapsing remitting experimental allergic encephalomyelitis SJL mouse model using MION-46L enhanced in vivo MRI: early histopathological correlation

Constitutive expression of VEGF after implantation of genetically engineered myoblasts into non-ischemic muscle led to an increase in vascular structures. Previously, effects of VEGF delivery to adult muscle have only been reported in ischemic tissues. The resulting vascular structures were reminiscent of those formed during embryonic vasculogenesis, rather than angiogenesis, sprouting from preexisting vessels. Initially, VEGF caused an accumulation of endothelial cells and macrophages, followed by networks of vascular channels and hemangiomas with locally high serum VEGF levels. No effects were evident in adjacent tissue or contralateral legs, where low serum VEGF was detected. These data suggest that the induction by VEGF of angiogenesis or vasculogenesis may be dose-dependent. Furthermore, VEGF expression must be carefully modulated, as overexpression is deleterious.     

Uric acid, a natural scavenger of peroxynitrite, in experimental allergic encephalomyelitis and multiple sclerosis

Uric acid, the naturally occurring product of purine metabolism, is a strong peroxynitrite scavenger, as demonstrated by the capacity to bind peroxynitrite but not nitric oxide (NO) produced by lipopolysaccharide-stimulated cells of a mouse monocyte line. In this study, we used uric acid to treat experimental allergic encephalomyelitis (EAE) in the PLSJL strain of mice, which develop a chronic form of the disease with remissions and exacerbations. Uric acid administration was found to have strong therapeutic effects in a dose-dependent fashion. A regimen of four daily doses of 500 mg/kg uric acid was required to promote long-term survival regardless of whether treatment was initiated before or after the clinical symptoms of EAE had appeared. The requirement for multiple doses is likely to be caused by the rapid clearance of uric acid in mice which, unlike humans, metabolize uric acid a step further to allantoin. Uric acid treatment also was found to diminish clinical signs of a disease resembling EAE in interferon-gamma receptor knockout mice. A possible association between multiple sclerosis (MS), the disease on which EAE is modeled, and uric acid is supported by the finding that patients with MS have significantly lower levels of serum uric acid than controls. In addition, statistical evaluation of more than 20 million patient records for the incidence of MS and gout (hyperuricemic) revealed that the two diseases are almost mutually exclusive, raising the possibility that hyperuricemia may protect against MS.     

Genetic analysis of inflammation, cytokine mRNA expression and disease course of relapsing experimental autoimmune encephalomyelitis in DA rats

Past history of major depression is more common in smokers than in non-smokers. We have shown in a previous study that lifetime prevalence of major depression is higher in dependent smokers and they have lower monoamine oxidase-A and -B activities than non-smokers. Because several studies have found an association between MAO-B activity and depression we analysed data of these smokers to assess whether past history of major depression is associated with reduced monoamine oxidase activities (A and B) or not. Further, we tried to characterize smokers with past history of major depression and its effect on withdrawal symptoms. The data of 88 dependent smokers (Fagerstr?m Tolerance Questionnaire score > or = 6 and smoking > or = 20 cigarettes/day) who participated in a smoking cessation study were analysed. Smokers with past history of major depression but without current illness did not differ in demographic and smoking characteristics from smokers without past history of major depression. Smokers with past history of major depression were mainly women and had lower body mass index. Adjusted for gender and body mass index dependent smokers with or without past history of depression had similar MAO-A and MAO-B activities but smokers with past history of major depression had significantly lower resting plasma norepinephrine levels. Smokers with past history of depression had not significantly higher ratings for depression (Montgomery-Asberg Depression Rating Scales) and anxiety (Hamilton Anxiety Scales) and smoking cessation did not exacerbate these ratings (assessed up to 3 months) and none had depressive episode during the postcessation period up to one year. Past history of depression was associated with higher scores on 'expressed sadness' and 'depressive mood'. Abstinent smokers with past history of depression had significantly higher ratings in one of the seven ratings of a 6 months period for craving (day 28), anxiety (day 7) and total withdrawal symptom score (day 7) when compared to those who had no past history of major depression. It is concluded that (i) past history of major depression is more frequent in female smokers; (ii) smokers with past history of depression may have more intense withdrawal symptoms (craving and anxiety) at some time after cessation: and (iii) past history of depression does not affect monoamine oxidase activities, therefore, reduced monoamine oxidase activities found in previous studies are possibly characteristic features of smoking.     

IFN-tau suppresses both the autoreactive humoral and cellular immune responses and induces stable remission in mice with chronic experimental allergic encephalomyelitis

PURPOSE: The purpose of this study was to compare attitudes toward violence and weapon-carrying among seventh-grade students in three dissimilar U.S. communities. A second focus was to determine students' understanding of their parents' violence-related guidance and behavior. METHODS: Five hundred sixty-seven seventh-grade students (48% male, 46% white, 35% African-American, 13% Latino) completed a self-administered questionnaire in May or October 1991. RESULTS: Thirty-four percent of the students had fought at least once, and 7% more than four times during the previous month. Also, within that period, 5% had skipped school owing to fear of violence. Students whose parents used nonviolent disciplinary techniques fought less frequently than those whose parents relied on hitting and more violent disciplinary methods (p < 0.001). Fighting was significantly more common among students who believe their parents want them to fight if insulted (p = 0.001). Students who reported that they try to stay out of fights usually succeeded (p = 0.001). Those students who more frequently participated in and observed fighting were more likely to carry a weapon (p = 0.001). CONCLUSIONS: Fighting is a frequent occurrence in the lives of seventh-grade students. Students' understanding of their parents' attitudes and behavior correlate strongly with violent behavior. While many students feel that weapons confer safety, those students who actually carry weapons are much more likely to fight.     

The effect of gammadelta T cell depletion on cytokine gene expression in experimental allergic encephalomyelitis

In experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, we showed previously that depletion of gammadelta T cells using the mAb GL3 immediately before disease onset, or during the chronic phase, significantly ameliorated clinical severity. We now report on the effect of gammadelta T cell depletion on expression of five cytokine genes, IL-1, IL-6, TNF, lymphotoxin, and IFN-gamma in spinal cords of mice during the pre-onset, onset, height, and recovery phases of EAE, and on expression of type II nitric oxide synthase. In control animals, the mRNAs for IL-1 and IL-6 rose dramatically at disease onset and peaked before disease height, whereas the mRNAs for TNF, lymphotoxin, and IFN-gamma rose more slowly and peaked with peak of disease. In GL3-treated animals, a dramatic reduction in all five cytokines was noted at disease onset, but only IFN-gamma remained significantly reduced at a time point equivalent to height of disease in control animals. ELISA data confirmed the reduced levels of IL-1 and IL-6 at disease onset in GL3-treated animals, and pathologic analysis demonstrated a marked reduction in meningeal infiltrates at the same time point. Studies of type II NOS also demonstrated a significant reduction in both mRNA and protein expression at the height of disease in GL3-treated animals. These results suggest that gammadelta T cells contribute to the pathogenesis of EAE by regulating the influx of inflammatory cells into the spinal cord and by augmenting the proinflammatory cytokine profile of the inflammatory infiltrates.     

New genetic loci that control susceptibility and symptoms of experimental allergic encephalomyelitis in inbred mice

Experimental allergic encephalomyelitis (EAE), the principal animal model of multiple sclerosis, is a genetically determined phenotype. In this study, analyses of the cumulative disease frequencies in parental, F1 hybrid, and F2 mice, derived from the EAE-susceptible SJL/J strain and the EAE-resistant B10.S/DvTe strain, confirmed that susceptibility to EAE is not inherited as a simple Mendelian trait. Whole genome scanning, using 150 informative microsatellite markers and a panel of 291 affected and 390 unaffected F2 progeny, revealed significant linkage of EAE susceptibility to marker loci on chromosomes 7 (eae4) and 17, distal to H2 (eae5). Quantitative trait loci for EAE severity, duration, and onset were identified on chromosomes 11 (eae6, and eae7), 2 (eae8), 9 (eae9), and 3 (eae10). While each locus reported in this study is important in susceptibility or disease course, interactions between marker loci were not statistically significant in models of genetic control. One locus, eae7, colocalizes to the same region of chromosome II as Orch3 and Idd4, susceptibility loci in autoimmune orchitis and insulin-dependent diabetes mellitus, respectively. Importantly, eae5 and eae7 are syntenic with human chromosomes 6p21 and 17q22, respectively, two regions of potential significance recently identified in human multiple sclerosis genome scans.     

Chronic relapsing experimental autoimmune encephalomyelitis: effects of insulin-like growth factor-I treatment on clinical deficits, lesion severity, glial responses, and blood brain barrier defects

Chronic relapsing experimental autoimmune encephalomyelitis (crEAE), a model for multiple sclerosis, was used to test 2 regimens of insulin-like growth factor-I (IGF-I) treatment. We induced crEAE by injecting 3x10(7) myelin basic protein-(MBP) sensitized lymph node cells into adult female SJL/J mice. Fifty-one mice, divided randomly into 4 groups, were used in the first trial. Two groups received IGF-I (a gift of Cephalon, Inc.) 0.6 mg/kg/d subcutaneously from day 7 to day 16 and the other two groups received placebo injections. IGF-I treatment reduced clinical deficits during the first attack and during 2 subsequent relapses. Image analysis of immunostained and histological sections showed that IGF-I treatment reduced BBB defects and both the numbers and sizes of inflammatory, demyelinating, and demyelinated lesions. Twelve mice that had recovered from their first attack were used in our second trial to evaluate possible adverse effects of prolonged treatment with a higher dose of IGF-I. Six received 1.2 mg/kg/d for 6 weeks (days 19-63). No adverse effects of IGF-I treatment were identified. The eyes, hearts, livers, and kidneys of IGF-I-treated mice were normal histologically and their spleens also appeared normal except for mild to moderate microscopic increases in lymphopoesis. Our results suggest that prolonged IGF-I treatment is well tolerated and that the anti-inflammatory effects of IGF-I have a major role in reducing clinical deficits and lesion severity in crEAE. These effects, if present in multiple sclerosis, may benefit patients with this disease.     

Prostacyclin production in acute, chronic, and long-term experimental portal hypertension

Curcumin, bis(4-hydroxy-3-methoxyphenyl)-1,6-diene-3,5-dione, is a yellow-orange dye derived from the rhizome of the plant Curcuma longa. Curcumin has demonstrated phototoxicity to several species of bacteria under aerobic conditions (Dahl, T. A., et al., 1989, Arch. Microbiol. 151 183), denoting photodynamic inactivation. We have now found that curcumin is also phototoxic to mammalian cells, using a rat basophilic leukemia cell model, and that this phototoxicity again requires the presence of oxygen. The spectral and photochemical properties of curcumin vary with environment, resulting in the potential for multiple or alternate pathways for the exertion of photodynamic effects. For example, curcumin photogenerates singlet oxygen and reduced forms of molecular oxygen under several conditions relevant to cellular environments. In addition, we detected carbon-centered radicals, which may lead to oxidation products (see accompanying paper). Such products may be important reactants in curcumin's phototoxicity since singlet oxygen and reduced oxygen species alone could not explain the biological results, such as the relatively long lifetime (t1/2 = 27 s) of the toxicant responsible for decreased cell viability.