Experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis (MS), reflects pathophysiologic steps in MS such as the influence of T cells and antibodies reactive to the myelin sheath, and the cytotoxic effect of cytokines. Galectin-9 (Gal-9) is a member of animal lectins that plays an essential role in various biological functions. The expression of Gal-9 is significantly enhanced in MS lesions; however, its role in autoimmune disease has not been fully elucidated. To identify the role of Gal-9 in EAE, we measured changes in mRNA and protein expression of Gal-9 as EAE progressed. Expression increased with disease progression, with a sharp rise occurring at its peak. Gal-9 immunoreactivity was mainly expressed in astrocytes and microglia of the central nervous system (CNS) and macrophages of spleen. Flow cytometric analysis revealed that Gal-9+CD11b+ cells were dramatically increased in the spleen at the peak of disease. Increased expression of tumor necrosis factor (TNF)-R1 and p-Jun N-terminal kinase (JNK) was observed in the CNS of EAE mice, suggesting that TNF-R1 and p-JNK might be key regulators contributing to the expression of Gal-9 during EAE. These results suggest that identification of the relationship between Gal-9 and EAE progression is critical for better understanding Gal-9 biology in autoimmune disease.
Animals ; Antibodies ; Astrocytes ; Autoimmune Diseases ; Biology ; Central Nervous System ; Cytokines ; Disease Progression ; Encephalomyelitis, Autoimmune, Experimental* ; Humans ; Lectins ; Macrophages ; Mice ; Microglia ; Models, Animal ; Multiple Sclerosis ; Myelin Sheath ; Phosphotransferases ; RNA, Messenger ; Spleen ; T-Lymphocytes ; Tumor Necrosis Factor-alpha

OBJECTIVETo set up an animal model of autoimmune auditory neuropathy and to observe the auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) in guinea pigs.

METHODSThe spiral ganglion and the cochlear nerve were obtained and purified by electrophoresis from 250 normal guinea pigs. The purified cochlear nerve antigen was mixed with an equal volume of complete Freunds adjuvant for immunization. Seventy guinea pigs were divided into three groups: experiment group (50 guinea pigs), control group (10 guinea pigs), normal group (10 guinea pigs). ABR, DPOAE, serum IgG levels, and morphological changes of spiral ganglion cells and the cochlear nucleus were observed. The protein expressions of the antigen were examined by immunohistochemistry and the super-structure of the auditory nerve were observed.

RESULTSThe threshold of ABR response increased ranged from 10 to 25 dB in 32% (32/100 ears) of the guinea pigs. The peak latencies of waves I , III and the interpeak latency I approximately III were prolonged in the hearing loss group of guinea pigs. Prolonged peak latency of wave III was noted in hearing loss group at 2 and 3 weeks post immunization and slowly decreased to normal peak latency. The amplitude of DPOAE was no difference in the guinea pigs. The levels of serum IgG increased significantly compared with those of the control group. Inflammatory cell infiltration was observed in the cochlear nerve and the number of spiral ganglion cells detected. On the contrary, inflammatory cell infiltration was not observed in the cochlear nucleus. The cell densities and the across-sectional areas of neurons in anteroventral cochlear nucleus and posteroventral cochlear nucleus were no difference in the guinea pigs. The antigen protein distributed strictly in cochlear nerve and the spiral ganglion. Some demyelinated areas in cochlear nerve was observed in this group. The threshold of ABR response in 68% guinea pigs (68/100 ears) did not increase. The data of DPOAE and the serum IgG levels show no difference compared with the control group. There were not pathological observation in spiral ganglion cells, cochlear nucleus and cochlear nerve.

CONCLUSIONAn animal model of autoimmune auditory neuropathy has been set up successfully and the character of the ABR and DPOAE was observed.

Animals ; Disease Models, Animal ; Evoked Potentials, Auditory, Brain Stem ; Guinea Pigs ; Nervous System Autoimmune Disease, Experimental ; physiopathology ; Otoacoustic Emissions, Spontaneous ; Vestibulocochlear Nerve Diseases ; etiology ; physiopathology

OBJECTIVETo construct the recombinant plasmid of human cardiac C protein (CCP) peptide with immunogenicity and to express, purification and renature fusion protein. The fusion protein was injected to Lewis rats to establish experimental autoimmune myocarditis (EAM) model.

METHODSTotal RNA was extracted from human heart and used as the template for reverse transcriptase-directed cDNA synthesis. The cDNA was then amplified by polymerase chain reaction (PCR) using oligonucleotide primers specific for CCP peptide with immunogenicity. Subsequently, the purified CCP peptide gene was cloned into PEASY-T1 vector and the ligated product was identified by PCR and DNA sequence analysis. Then the CCP target gene of positive clone was inserted into the pQE30, a prokaryotic expression vector, and the inserting plasmid was transformed into Escherichia coli. host M15. The positive clone extracted from the bacterium liquid was sieved by insertional inactivation sieve method and identified by PCR of bacterium liquid, CCP immunological peptide was purified and renatured in semipermeable membrane. EAM model in Lewis rats was induced by injection of mixture of 100 µg CCP fusion protein immunological peptide and 2.5 g/L completed Freund adjuvant from two double foot pad and subsequent abdominal injection of 0.5 µg pertussis toxin. Two, four, six, and eight weeks after immunization, hemodynamic evaluation was made and hearts underwent histological examination.

RESULTSThe DNA sequence analysis for cloning vector extraction revealed that the CCP target gene was cloned into pQE30 exactly. The DNA of 1000 bp length was obtained by PCR examination of bacterium liquid with transformation of express recombinants which were consistent with the expected size. Purified fusion protein in vertical slab gel electrophoresis showed 35 000 as expected. The recombinant CCP fusion protein existed in inclusion bodies of E. coli and amounted to 80% - 90% of the total protein. Hemodynamic and histological evaluations showed typical acute inflammatory responses at 2 weeks, subacute inflammatory and fibrosis changes at 4 weeks after injection, and signs of chronic dilated cardiomyopathy at 6 weeks post injection.

CONCLUSIONCombination of gene clone technique and histidine tag protein purification technique can be used to synthesize human cardiac C protein to induce EAM model in Lewis rat.

Animals ; Carrier Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Humans ; Myocarditis ; Nervous System Autoimmune Disease, Experimental ; Plasmids ; Rats ; Recombinant Fusion Proteins ; biosynthesis ; genetics

Erythropoietin (EPO) is known to have numerous biological functions. While its primary function is during haematopoiesis, recent studies have shown that EPO plays important role in cytoprotection, immunomodulation, and antiapoptosis. These secondary functions of EPO are integral to tissue protection following hypoxic injury, ischemia-reperfusion injury, and spinal cord injury in the central nervous system. This review focuses on experimental evidence documenting the neuroprotective effects of EPO in organ-specific autoimmune nervous system disorders such as experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune neuritis (EAN). In addition, the immunomodulatory role of EPO in the pathogenesis of EAE and EAN animal models of human multiple sclerosis and Guillain-Barre syndrome, respectively, will be discussed.
Autoimmune Diseases ; Central Nervous System ; Cytoprotection ; Encephalomyelitis ; Encephalomyelitis, Autoimmune, Experimental ; Erythropoietin ; Guillain-Barre Syndrome ; Hematopoiesis ; Humans ; Immunomodulation ; Models, Animal ; Multiple Sclerosis ; Nervous System Diseases ; Neuritis, Autoimmune, Experimental ; Neuroprotective Agents ; Reperfusion Injury ; Spinal Cord Injuries

OBJECTIVETo investigate the expression of the integrin alpha6beta4 in experimental allergic neuritis (EAN) and the relationship of the integrin alpha6beta4 with functional states of Schwann cells (Sc) as well as the injury and repair of the myelin during EAN.

METHODSEAN was induced in Lewis rats and sciatic nerves were resected in 18 EAN and 3 normal rats. The expression of tissue integrin alpha6beta4 was analyzed during the course of EAN induction and in controls by in situ hybridization and semi-quantitative RT-PCR.

RESULTSThe detection of integrin alpha6 and beta4 subunit by hybridization in situ demonstrated that expression of alpha6 subunit present no significant changes during the course of EAN, while expression of beta4 declined in the early phase, showing less positive signals than those of the control, and restored its expression in the later or recovery phase. The changes of expression of integrin alpha6 and beta4 in EAN were confirmed by semi-quantitative PT-PCR, using GAPDH as the internal standard.

CONCLUSIONSThe degeneration and injury of Sc caused by inflammation affect the expression of integrin, which shows similar changes in Sc during embryogenesis, indicating alpha6beta4 may be a marker of Sc differentiation and at least an important molecule to mark the course of EAN. The expression of alpha6beta4 correlate with the injury and repair of myelin during EAN.

Animals ; Disease Models, Animal ; In Situ Hybridization ; Integrin alpha6beta4 ; genetics ; physiology ; Neuritis, Autoimmune, Experimental ; metabolism ; pathology ; RNA, Messenger ; analysis ; Rats ; Rats, Inbred Lew ; Reverse Transcriptase Polymerase Chain Reaction

To investigate the pattern of expression of osteopontin (OPN) in tissues of the central nervous system (CNS) responding to peripheral immunological stimulation, the expression of OPN was studied in the spinal cord of rats with experimental autoimmune neuritis (EAN). In this model system, the sciatic nerves and spinal nerve roots are the target organs of EAN and the spinal cord is a remote organ that may be indirectly affected. OPN was constitutively expressed in some astrocytes adjacent to the pia mater and neurons in normal rats. In rats with EAN, OPN was increased in the same cells and in some inflammatory cells, including macrophages in the subarachnoid space. Expression of CD44, a receptor of OPN, was weak in normal spinal cord tissue and increased in the entire spinal cord parenchyma in rats with EAN, as well as in inflammatory cells. These findings suggest that inflammatory cells as well as reactive astrocytes are major sources of OPN and CD44 in the spinal cord of rats with EAN. Further study is needed to elucidate the functional role of OPN in the spinal cord affected by EAN.
Animals ; Antigens, CD44/metabolism ; Astrocytes/metabolism ; Ectodysplasins ; Female ; Immunohistochemistry ; Macrophages/metabolism ; Membrane Proteins ; Neuritis, Autoimmune, Experimental/*metabolism ; Neuroglia/metabolism ; Neurons/metabolism ; Osteopontin ; Rats ; Rats, Inbred Lew ; Sciatic Nerve/metabolism ; Sialoglycoproteins/*metabolism ; Spinal Cord/*metabolism ; Spinal Nerve Roots/metabolism

OBJECTIVE: To examine the expression of mRNA of Oxford 40(OX40) and Oxford 40 ligand(OX40L) in the sciatic nerve, spleen, peripheral blood mononuclear cells and lymph nodes of experimental allegic neuritis (EAN). METHODS: Thirty-six Lewis rats were randomly assigned into an EAN group and a CFA group. The rats were sacrificed on 9th, 17th, and 26th day after immunization. OX40 and OX40L mRNA was detected by reverse transcription polymerase chain reaction in the spleen, sciatic nerves, peripheral blood mononuclear cells and lymphonodes. RESULTS: The peak of clinical course came on 17th day after the immunization in EAN. The mRNA expression of OX40/OX40L was higher on 8th day and 17th day than that on 26th day after the immunization (P<0.05). There was significant difference between the EAN group and the CFA group at the 3 time points (P<0.05); rats in the CFA group didn't have any clinical manifestations. The mRNA expression of OX40 and OX40L in the EAN group raised in the sciatic nerves and lymph nodes at the above 3 time points (P<0.05). Weak expression was seen in the peripheral blood mononuclear cells. CONCLUSION OX40 and OX40L may play a role in the pathogenesis of experimental allegic neuritis.
Animals ; Female ; Membrane Glycoproteins ; genetics ; metabolism ; Neuritis, Autoimmune, Experimental ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Inbred Lew ; Receptors, OX40 ; genetics ; metabolism ; Sciatic Nerve ; metabolism ; Tumor Necrosis Factors ; genetics ; metabolism

Acute disseminated encephalomyelitis(ADEM) and acute relapsing disseminated encephalomyelitis(ARDEM) are representative demyelination diseases that occur among young children with a fulminant onset similar to encephalitis or meningitis. The diseases often occur after some viral infection of immunization and the etiology of these diseases is considered to be an autoimmune response because of the similarity in pathologic findings to experimental allergic encephalomyelitis. Cerebral computed tomography(CT) findings of demyelination in ADEM or ARDEM show normal to low density areas in the white matter. In cerebral MRI findings, a scattered distinct high intensity lesion considered to be demyelination is observed in 72-weighted imaging even in the early stages. ADEM is usually monophasic, but recurrent episodes may occure. When ADEM is reccurent, the distinction from multiple sclerosis becomes difficult. We report here a case of acute relapsing disseminated encephalomyelitis(ARDEM) in a 9 years old male child who experence ADEM, 3 times.
Autoimmunity ; Child ; Demyelinating Diseases ; Encephalitis ; Encephalomyelitis, Acute Disseminated* ; Encephalomyelitis, Autoimmune, Experimental ; Humans ; Immunization ; Magnetic Resonance Imaging ; Male ; Meningitis ; Multiple Sclerosis

Experimental autoimmune encephalomyelitis (EAE) in Lewis rats is characterized by transient paralysis followed by recovery. To evaluate whether transient paralysis in EAE affects bone density, tibiae of EAE rats were morphologically investigated using micro-computed tomography and histology. The parameters of bone health were significantly reduced at the peak stage of EAE rats relative to those of controls (p < 0.05). The reduction of bone density was found to remain unchanged, even in the recovery stage. Collectively, the present data suggest that osteoporosis occurs in paralytic rats with monophasic EAE, possibly through the disuse of hindlimbs and/or autoimmune inflammation.
Animals ; Autoimmunity ; Bone Density ; Encephalomyelitis, Autoimmune, Experimental* ; Hindlimb ; Inflammation ; Osteoporosis* ; Paralysis ; Rats* ; Tibia

OBJECTIVETo observe the pathological changes of axonal injury in a rat model of experimental allergic encephalomyelitis (EAE).

METHODSWith HE, luxol fast blue and Bielschowsky staining, the expression of APP, MBP, SMI-32 and MBP in the brain and spinal cord of EAE rats using double-labeling indirect immunofluorescence.

RESULTSExtensive cuffing lesions of inflammatory cell infiltrations were found in the brain and spinal cord of the rats, accompanied by multiple lesions of demyelination, axonal disarrangement with vesicular loss. SMI-32 staining identified numerous nonphosphorylated neurofilament, indicating the presence of axonal injury. Axonal oval bodies formed by APP accumulation were found in the white matters of the spinal cord 14 days after EAE, suggesting that neuraxial damage occurred in the early stage of EAE which was not synchronous with myelin loss.

CONCLUSIONDifferent levels of inflammation occur in different stages of EAE, and inflammatory cell infiltration is the most obvious at the peak of EAE. Axonal injury occurs in the early stage of EAE and progresses over the entire disease course.

Animals ; Axons ; pathology ; Brain ; pathology ; Encephalomyelitis, Autoimmune, Experimental ; pathology ; Female ; Rats ; Rats, Wistar ; Spinal Cord ; pathology