Active Induction of Experimental Autoimmune Encephalomyelitis (EAE) with MOG35-55 in the Mouse.

Experimental autoimmune encephalomyelitis (EAE) is one of the most popular animal models of multiple sclerosis (MS). There are a number of EAE models, being actively induced EAE in strains such as C57Bl/6 mice very robust and reproducible. We herewith present details of the materials and methods for active EAE. Mice are immunized with an emulsion of myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) + complete Freund's adjuvant (supplemented with Mycobacterium tuberculosis), and treated with Bordetella pertussis toxin, to induce EAE. Sham-EAE mice are immunized with bovine serum albumin instead of MOG35-55.

Myelin Protein Zero180-199 Peptide Induced Experimental Autoimmune Neuritis in C57BL/6 Mice.

Mouse models of peripheral demyelinating neuropathy play an important role in enabling the study of disease pathogenesis. Further, induction in transgenic mice allows for the precise interrogation of disease mechanisms, as well as the analysis of the efficacy and mechanisms of potential new therapies. Here we describe a method to successfully induce experimental autoimmune neuritis (EAN) using myelin protein zero (P0)180-199 peptide in combination with Freund's complete adjuvant and pertussis toxin in the C57BL/6 mouse strain. We also outline a sensitive paradigm of accurately assessing the extent of functional deficits occurring in murine EAN.

Towards replacement of the acellular pertussis vaccine safety test: Comparison of in vitro cytotoxic activity and in vivo activity in mice.

Because of the exquisite sensitivity of the murine histamine sensitization test (HIST) in detecting minute amounts of active pertussis toxin (PTx), this animal-based test has been used to assure the safety of acellular pertussis vaccines in the United States and other countries around the world. Prompted by humane considerations, efforts are underway to find a suitable in vitro replacement assay that has critical attributes comparable to that of the HIST. In this study, we compared the sensitivity of the in vivo HIST with an in vitro Chinese Hamster Ovary (CHO) cell-based assay. Using vaccine samples that had been spiked with PTx, we found that both assays were capable of detecting as little as 4-10 ng of active pertussis toxin per dose of vaccine; thus, the sensitivities of the two assays are comparable. Because the strength of adsorption of PTx to the vaccine adjuvant could change over time, we also used both assays to examine the bioavailability of PTx in spiked vaccine samples that had been stored at 25 °C for 9 weeks, mimicking long term vaccine storage conditions. We found that both assays detected similar amounts of active PTx in these samples, indicating that bioavailability of the toxin in stored samples was similar. Taken together, our results indicate that critical attributes of the HIST are met by the CHO cell assay used in this study and provide proof of concept that the CHO cell assay may be further considered as a replacement for the in vivo HIST.

Safety testing of acellular pertussis vaccines: Use of animals and 3Rs alternatives.

The current test of acellular Bordetella pertussis (aP) vaccines for residual pertussis toxin (PTx) is the Histamine Sensitization test (HIST), based on the empirical finding that PTx sensitizes mice to histamine. Although HIST has ensured the safety of aP vaccines for years, it is criticized for the limited understanding of how it works, its technical difficulty, and for animal welfare reasons. To estimate the number of mice used worldwide for HIST, we surveyed major aP manufacturers and organizations performing, requiring, or recommending the test. The survey revealed marked regional differences in regulatory guidelines, including the number of animals used for a single test. Based on information provided by the parties surveyed, we estimated the worldwide number of mice used for testing to be 65,000 per year: ∼48,000 by manufacturers and ∼17,000 by national control laboratories, although the latter number is more affected by uncertainty, due to confidentiality policies. These animals covered the release of approximately 850 final lots and 250 in-process lots of aP vaccines yearly. Although there are several approaches for HIST refinement and reduction, we discuss why the efforts needed for validation and implementation of these interim alternatives may not be worthwhile, when there are several in vitro alternatives in various stages of development, some already fairly advanced. Upon implementation, one or more of these replacement alternatives can substantially reduce the number of animals currently used for the HIST, although careful evaluation of each alternative's mechanism and its suitable validation will be necessary in the path to implementation.

Toward a mechanism-based in vitro safety test for pertussis toxin.

Pertussis vaccines are routinely administered to infants to protect them from whooping cough. Still, an adequate safety test for pertussis toxin (PT), one of the main antigens in these vaccines, is not available. The histamine sensitization test is currently the only assay accepted by regulatory authorities to test for the absence of active PT in vaccines. This is however, a lethal animal test with poor reproducibility. In addition, it is not clear whether the assumed underlying mechanism, i.e., ADP-ribosylation of G proteins, is the only effect that should be considered in safety evaluation of PT. The in vitro safety test for PT that we developed is based on the clinical effects of PT in humans. For this, human cell lines were chosen based on the cell types involved in the clinical effects of PT. These cell lines were exposed to PT and analyzed by microarray. In this review, we discuss the clinical effects of PT and the mechanisms that underlie them. The approach taken may provide as an example for other situations in which an in vitro assay based on clinical effects in humans is required.

Diurnal rhythms are altered in a mouse model of multiple sclerosis.

Our earlier studies described a disruption of heart rate and blood pressure diurnal rhythms in mice with experimental autoimmune encephalomyelitis (EAE). The present study investigates whether these observations could be extended to additional clock-regulated rhythms in mice with EAE. Analysis of clock gene expression in the liver of EAE mice demonstrated significant variability associated with Per2 rhythmic expression. Corticosterone and leptin hormone rhythms were also altered in EAE mice. The results presented here indicate that disturbances in clock-regulated rhythms are associated with EAE and present a suitable model for investigating the relationship between circadian disruption and autoimmune inflammatory disease.

Inflammation modulates anxiety in an animal model of multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) characterized by inflammation, but also degenerative changes. Besides neurological deficits, the rate of affective disorders such as depression and anxiety is at least six fold increased. Many aspects of MS can be mimicked in the animal model of myelin oligodendrocyte glycoprotein experimental autoimmune encephalomyelitis (MOG-EAE). Here we investigate behavioral changes in C57BL/6 mice suffering from mild MOG-EAE. In the later phase of the disease, mice were subjected to behavioral tests including the light-dark-box (LD Box), the acoustic startle response (SR) with a pre-pulse inhibition protocol as well as the learned helplessness (LH) paradigm. Behavioral data were correlated with the motor performance in an open field and rotarod test (RR). In the RR and open field, there was no significant difference in the motor performance between controls and mice suffering from mild MOG-EAE. Yet EAE mice displayed an increased anxiety-like behavior with a 23% reduction of the time spent in the bright compartment of the LD Box as well as an increased SR. In the LH paradigm, mice suffering from MOG-EAE were twice as much prone to depressive-like behavior. These changes correlate with an increase of hippocampal tissue tumor necrosis factor alpha levels and neuronal loss in the hippocampus. Modulation of monoaminergic transmission by chronic application of the antidepressant amitriptyline resulted in a decreased startle reaction and increased hippocampal norepinephrine levels. These data imply that chronic inflammation in the CNS may impact on emotional responses in rodent models of anxiety.

Suppression of neuro inflammation in experimental autoimmune encephalomyelitis by glia maturation factor antibody.

Glia maturation factor (GMF), a protein primarily localized in the central nervous system (CNS) was isolated, sequenced and cloned in our laboratory. We previously demonstrated that GMF mediates the experimental autoimmune encephalomyelitis (EAE)-induced production of pro-inflammatory cytokines and chemokines in the central nervous system of mice. In the present study we show that immunization with myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) caused an early onset (days 7-9 post immunization) and severe EAE with a mean peak score of 3.5 ± 0.5 in mice. Neutralization of GMF with four injections of anti-GMF antibody 5 to 11 days post immunization delayed the time of onset (days 12-14 post immunization) and significantly reduced the severity of EAE (mean peak score of 1.5 ± 0.4). Consistent with these clinical scores, histological examination of the CNS of these mice revealed profound differences between GMF-antibody treated mice and isotype matched control-antibody treated mice. Histological analysis of the spinal cord and brain showed severe inflammation and demyelination in the control antibody-treated mice whereas significantly reduced inflammation and demyelination was detected in GMF-antibody-treated mice at days 8, 16, and 24 post immunization. The decreased incidence and reduced severity of EAE in GMF-antibody-treated mice was consistent with the significantly reduced expressions of pro-inflammatory cytokines and chemokines. Our overall results demonstrate that neutralization of endogenous GMF with an affinity purified GMF antibody significantly decreased the inflammation, severity and progression of immunization induced active, passive and relapsing-remitting EAE. Treatment of mice with isotype-matched control antibody did not have any effect on EAE. Taken together, these results demonstrate the critical role of GMF in EAE, and GMF antibody as a potent anti-inflammatory therapeutic agent for effectively suppressing EAE in mouse models of major types of multiple sclerosis (MS).

Blood-brain barrier disruption and enhanced vascular permeability in the multiple sclerosis model EAE.

Multiple sclerosis (MS) is a demyelinating disease characterized by the breakdown of the blood-brain barrier (BBB), and accumulation of inflammatory infiltrates in the central nervous system. Tight junctions are specialized cell-cell adhesion structures and critical components of the BBB that have previously been shown to be abnormally distributed in MS tissue. To evaluate whether experimental autoimmune encephalomyelitis (EAE) provides a suitable model for this aspect of MS disease, we examined the expression and distribution of ZO-1 over the course of disease in EAE. We observed a dramatic relocalization of ZO-1 which precedes overt clinical disease and correlates with the sites of inflammatory cell accumulation. Treatment of in vitro cultures of murine brain endothelial cells with components of EAE induction provided similar findings, with relocalization of ZO-1 and increased permeability of endothelial monolayers. BBB disruption in the EAE model appears to parallel disease progression in MS, with direct effects on the cerebrovascular endothelium, making it an ideal tool for future evaluation of tight junction breakdown and repair in MS-like pathology.

A Lecinoxoid, an oxidized phospholipid small molecule, constrains CNS autoimmune disease.

Oxidized phospholipids (Ox-PLs) are generated in abundance at sites of inflammation. Recent studies have indicated that Ox-PLs may also exhibit anti-inflammatory activities. In this study, we investigated the beneficial effect of VB-201, a pure synthetic Ox-PL analog that we synthesized, on the development of a central nervous system (CNS) autoimmune inflammatory disease, in vivo. Oral administration of VB-201 ameliorated the severity of experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) peptide MOG35-55, and restrained the encephalogenicity of MOG35-55-specific T-cells. Our data presents a novel prospect for the role of Ox-PL analogs in CNS inflammatory diseases.

Therapeutic approach by Aloe vera in experimental model of multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) that leads to an inflammatory demyelination, axonal damage, and progressive neurologic disability that affects approximately 2.5 million people worldwide. The aim of the present research was to test the therapeutic effect of Aloe vera in experimental model of MS. All experiments were conducted on C57BL/6 male mice aged 6-8 weeks. To induce the experimental autoimmune encephalomyelitis (EAE), 250 microg of the myelin oligodendrocyte glycoprotein 35-55 peptide emulsified in complete freund's adjuvant was injected subcutaneously on day 0 over two flank areas. In addition, 200 ng of pertussis toxin in 100 microL phosphate buffered saline was injected intraperitoneally on days 0 and 2. The therapeutic protocol was carried out intragastrically using 120 mg/kg/day Aloe vera from 7 days before to 21 days after EAE induction. The mice were killed 21 days after EAE induction. The brains of mice were removed for histological analysis and their isolated splenocytes were cultured. The results indicated that treatment with Aloe vera caused a significant reduction in severity of the disease in experimental model of MS. Histological analysis showed 3 +/- 2 plaques in Aloe vera-treated mice compared with 5 +/- 1 plaques in control group. The density of mononuclear infiltration in the CNS of Aloe vera-treated mice (500 +/- 200) was significantly less in comparison to 700 +/- 185 cells in control group. Moreover, the serum level of nitric oxide in treatment group was significantly less than control animals. The level of interferon-gamma in cell culture supernatant of treated mice splenocytes was lower than control group, whereas decrease in serum level of interleukin-10 in treatment group was not significant in comparison with control mice. These data indicate that Aloe vera therapy can attenuate the disease progression in experimental model of MS.

A two-phased population epidemiological study of the safety of thimerosal-containing vaccines: a follow-up analysis.

BACKGROUND: Thimerosal is an ethylmercury-containing preservative in vaccines. Toxicokinetic studies have shown children received doses of mercury from thimerosal-containing vaccines (TCVs) that were in excess of safety guidelines. Previously, an ecological study showing a significant association between TCVs and neurodevelopmental disorders (NDs) in the US was published in this journal. MATERIAL/METHODS: A two phased population-based epidemiological study was undertaken. Phase one evaluated reported NDs to the Vaccine Adverse Event Reporting System (VAERS) following thimerosal-containing Diphtheria-Tetanus-acellular-Pertussis (DTaP) vaccines in comparison to thimerosal-free DTaP vaccines administered from 1997 through 2001. Phase two evaluated the automated Vaccine Safety Datalink (VSD) for cumulative exposures to mercury from TCVs at 1-, 2-, 3-, and 6-months-of-age for infants born from 1992 through 1997 and the eventual risk of developing NDs. RESULTS: Phase one showed significantly increased risks for autism, speech disorders, mental retardation, personality disorders, and thinking abnormalities reported to VAERS following thimerosal-containing DTaP vaccines in comparison to thimerosal-free DTaP vaccines. Phase two showed significant associations between cumulative exposures to thimerosal and the following types of NDs: unspecified developmental delay, tics, attention deficit disorder (ADD), language delay, speech delay, and neurodevelopmental delays in general. CONCLUSIONS: This study showed that exposure to mercury from TCVs administered in the US was a consistent significant risk factor for the development of NDs. It is clear from these data and other recent publications linking TCVs with NDs that additional ND research should be undertaken in the context of evaluating mercury-associated exposures and thimerosal-free vaccines should be made available.

Pertussis toxin-induced histamine sensitisation: an aspecific phenomenon independent from the nitric oxide system?

Mechanisms were studied initially to develop an in vitro safety test for detecting pertussis toxin toxicity in acellular pertussis vaccines based on the histamine sensitisation test. Maximal contractions and sensitivities to different agonists and adrenoceptor-induced contractions in Ca2+-free medium of isolated rat small mesenteric resistance arteries were significantly reduced by in vivo [30 microg/kg, intravenously (i.v.), day 5] or in vitro (10 microg/ml, 2 h) pertussis toxin pretreatment. Pertussis toxin-induced decrease in sensitivity of small mesenteric resistance arteries to noradrenaline was endothelium-dependent. Nomega-nitro-L-arginine methyl ester (L-NAME) (100 microM, 20 min) did not reestablish the sensitivity to noradrenaline. In vivo L-NAME treatment (0, 1, 10 or 30 mg/kg) of pertussis toxin-pretreated (15 microg/kg) rats did not reduce pertussis toxin-induced enhancement of the histamine-induced decrease in blood pressure and histamine (10, 30, 100 or 300 mg/kg) induced mortality. Finally, in vivo pertussis toxin pretreatment sensitises rats for sodium nitroprusside (50 microg/kg/min). We conclude that pertussis toxin-induced histamine sensitisation is caused by an interference of pertussis toxin with the contractile mechanisms of vascular smooth muscle of resistance arteries which indicates only an indirect role for histamine in the histamine sensitisation test.

Role of alpha2-adrenergic receptors and cyclic adenosine monophosphate-dependent protein kinase in the regulation of norepinephrine release in the central nervous system of spontaneously hypertensive rats.

There has been much evidence showing that the central sympathetic nervous system may be involved in the control of blood pressure. In the present study, we investigated the role of the presynaptic alpha2-adrenergic receptors and the cyclic adenosine monophosphate-dependent protein kinase (protein kinase A) in the regulation of norepinephrine release in the central nervous system in hypertension. The alpha2-adrenergic receptor agonists UK 14, 304 and clonidine inhibited the stimulation-evoked [3H]norepinephrine release in a dose-dependent manner in the medulla oblongata of Sprague-Dawley rats. Pretreatment of pertussis toxin (a potent inhibitor of the Gi-protein) attenuated the suppression of NE release by UK 14, 304. The protein kinase A inhibitor H-8 also reduced the stimulation-evoked [3H]norepinephrine release in rat medulla oblongata. In spontaneously hypertensive rats, the inhibitory effect of UK 14, 304 on the stimulation-evoked norepinephrine release was significantly less than in age-matched normotensive Wistar-Kyoto rats. By contrast, the protein kinase A inhibitor H-8 reduced the stimulation-evoked norepinephrine release to a greater extent in hypertension than in normotensive controls. The results of the present study showed that the alteration in the presynaptic alpha2-receptor-protein kinase A system might actively participate in the regulation of norepinephrine release in the central nervous system in hypertension.

Influence of pertussis toxin on superficial bladder carcinoma in rats.

The proliferation and migration of cells is a fundamental process for the metastasis of malignant tumour cells. In several in vitro studies, pertussis toxin (PTX) inhibited cell proliferation and cell motility in the human transitional cell carcinoma cell line J82. The present study investigated the effect of the intravesical application of PTX on the development of superficial bladder cancer in rats. We used the model of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN, 0.05% via drinking water x10 weeks) to induce superficial bladder carcinomas in 40 female rats. After 16 weeks the rats were treated in two groups with 0.4 ml PTX (1 microg/ml) or 0.4 ml phosphate buffered saline (PBS) by intravesical application (once a week for 10 weeks). In the 25th week urine cytology was determined and all rats were killed at week 26 followed by histological evaluation. In the control group, the urine cytology was positive for G2/G3 cells in ten of 17 rats. In the PTX group G2/G3 cells were determined in five of 20 rats ( P two tailed <0.05). Histopathologically 12 rats (71%) of the control group and 11 rats (55%) of the PTX group developed T1-T2 transitional-cell carcinomas. No local or systemic side effects were disclosed. PTX treatment reduces the development of G3 transitional cell carcinomas in rats and may represent a new approach for local therapy in superficial bladder cancer.