Humans ; Candida albicans/genetics* ; CARD Signaling Adaptor Proteins/genetics* ; Mutation ; Encephalomyelitis

AIMTo observe the dynamic changes of heme oxygenase-1 (HO-1) mRNA and protein express in subfornical organ in rats with experimental allergic encephalomyelitis (EAE) to confirm that SFO is one of the sites for blood-bearing signaling molecules entering into brain.

METHODSEAE was induced by CFA-GPSCH on Wistar rats, we observed the levels of HO-1 mRNA and its protein expression with immunohistochemistry and in situ hybridization technology on 1 d, 7 d, 14 d, and 21 d after EAE induction in SFO of rats. The relationship between HO-1 and symptoms of EAE was also investigated.

RESULTSThe expression levels of HO-1 mRNA and its protein expression were very low in the brains of the control group, whereas they were enhanced gradually with pathological course in the brain and onsets of symptoms, signs of EAE. On 1 d after induction of EAE, positive cells of HO-1 mRNA and its protein expression were observed at SFO, but the labeled cells were rarely seen in the other brain regions. On 7 d, the positive cells increased markedly. On 14 d the levels of HO-1 mRNA and its protein expression in the brains reached the peak, the positive cells of HO-1 were mainly located at the choroid plexuses and SFO, as well as the regions around "sleeve-like" lesion foci, all of which were coincident with the locations of lesions of EAE. The changes of incidence, symptom, reduction of the body weight, and pathology lesions of EAE in rat brains were the most significant. On 21 d, the levels of HO-1 mRNA and its protein expression reduced gradually, which was in parallel with remitted symptoms of EAE. When a specific inhibitor of HO-1, Snpp9, was applied, the symptoms and pathological lesions of EAE in brains were mitigated markedly.

CONCLUSIONSFO may be one of the earliest sites for blood-bearing signaling molecules entering into brain. The dynamic changes of HO-1 mRNA and its protein expression are in parallel with the changes of symptoms and pathological lesions of EAE in the brains. Application of some inhibitors of HO-1 may be one of potential therapeutic methods for prevention and treatment of EAE.

Animals ; Encephalomyelitis, Autoimmune, Experimental ; metabolism ; Female ; Heme Oxygenase (Decyclizing) ; genetics ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar ; Subfornical Organ ; metabolism

Venezuelan equine encephalitis (VEE) is a zoonotic disease caused by the Venezuelan equine encephalitis virus (VEEV) complex. This disease has not yet been reported in China, and it is therefore essential to establish a rapid and accurate method for detection of the virus in order to prevent and control this disease. In this study, a one-step real-time quantitative RT-PCR method was developed for the detection of the VEEV complex. A pair of specific primers and a Taqman probe were designed corresponding to a conserved region of the VEEV gene nspl, allowing the detection of all known strains of different sub- types of the virus. Using RNA synthesized by in vitro transcription as template, the sensitivity of this method was measured at 3.27 x 10(2) copies/microL. No signal was generated in response to RNA from Chikungunya virus (CHIKV), nor to RNA encoding the nsp1 fragment of Eastern equine encephalitis virus (EE-EV) or Western equine encephalitis virus (WEEV), all of which belong to the same genus as VEEV. This indicates that the method has excellent specificity. These results show that this one-step real-time quantitative RT-PCR method may provide an effective tool for the detection of VEEV in China.
China ; DNA Primers ; genetics ; Encephalitis Virus, Venezuelan Equine ; classification ; genetics ; isolation & purification ; Encephalomyelitis, Venezuelan Equine ; virology ; Humans ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods

The work was done to study immunogenetic peculiarities of neuroinflammatory diseases among Korean children. A total of 13 children with neuroinflammatory diseases (8 males and 5 females; mean age 4.6+/-2.6 yr) were consecutively recruited. Genomic typing was performed on their HLA DRB/HLA DQB genes using PCR-SSOP/ SSP techniques with gel immunoelectrophoresis. The frequencies of HLA-DR1* 15 in children with acute disseminated encephalomyelitis (ADEM) (31%) and DQB1* 06 in other neuroinflammatory diseases (38%) were significantly increased compared with control subjects. The frequencies of HLA-DRB3*0202 (100%), HLA-DRB1*1302 (67%), HLA-DRB3*0301 (67%), and HLA-DQB1*0301 (67%) were significantly increased in children with multiple sclerosis and the frequencies of HLA-DRB1*1501 (40%) and HLA-DRB5*0101 (40%) were significantly increased in children with ADEM. HLA-DRB1*1401, HLA- DRB3*0202, and HLA-DQB1*0502 were found in children with acute necrotizing encephalopathy. In conclusion, HLA-DR1*15 and DQB1*06 may be involved in susceptibility to inflammation in Korean children. The frequencies of HLA-DRB1*1501, HLA-DRB5*0101, HLA-DRB3*0301, and HLADQB1* 0602 were not as high in Korean children with multiple sclerosis as in western children. However, HLA-DRB3*0202 was seen in all children with multiple sclerosis. Our data may provide further evidence that the immunogenetic background of neuroinflammatory diseases in Korean is distinctly different from the ones in western countries. Further studies are necessary to confirm this finding.
Alleles ; Child ; Child, Preschool ; Electrophoresis ; Encephalomyelitis/genetics ; Female ; Genes, MHC Class II/*genetics ; *Genetic Predisposition to Disease ; Genotype ; Human ; Inflammation/*genetics ; Male ; Multiple Sclerosis/genetics ; Neurons/*pathology ; Polymerase Chain Reaction ; Polymorphism, Single-Stranded Conformational ; Support, Non-U.S. Gov't

OBJECTIVE: To investigate the expression of EBI3 and p28 mRNA (the 2 subunits of IL-27) in the brain and spinal cord of the model of experimental autoimmune encephalomyelitis (EAE), and to explore their effect on EAE. METHODS: Seventy-two adult female SPF C57BL/6J mice (inbred strain) were randomly divided into a control group, an adjuvant group, and an EAE group. RT-PCR was performed to detect the expression of EBI3 mRNA and p28 mRNA in the brain and spinal cord. RESULTS: The expression of EBI3 mRNA and p28 mRNA was up-regulated at onset in the EAE group, which increased quickly during peak phase and maintained at a high level in the chronic phase. There was significant difference in the expression of EBI3 and p28 mRNA between the EAE group and the control/adjuvant group (P<0.01). Additionally, there was no remarkable difference in the expression of EBI3 and p28 mRNA in the brain and spinal cord between the control group and the adjuvant group (P>0.05). CONCLUSION IL-27 may play a role of promoting the morbility of EAE in the early stage, and sustain the inflammatory response in endgame.
Animals ; Brain ; metabolism ; Chronic Disease ; Encephalomyelitis, Autoimmune, Experimental ; metabolism ; Interleukins ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Minor Histocompatibility Antigens ; Protein Subunits ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Receptors, Cytokine ; genetics ; metabolism ; Spinal Cord ; metabolism

In order to investigate the role of heme oxygenase-1 (HO-1) in the molecular mechanism of experimental allergic encephalomyelitis (EAE), which was induced by guinea pig spinal cord homogenate + complete freund adjuvant on Wistar rats, we observed the gene of HO-1 and its protein expression with reverse transcriptase polymerase chain reaction(RT-PCR) and immunohistochemistry 1, 7, 14, and 21 d after EAE induction in rats. The relationship between HO-1 and the symptoms of EAE was also observed. The results showed that the levels of HO-1 mRNA and its protein expression were very low in the brains of the control group, whereas they were enhanced gradually with pathological course in the brain and onsets of symptoms, signs of EAE. On day 7, the level of HO-1 mRNA reached the peak, but the expression level of HO-1 protein in the brains reached the peak on day 14. The immunoreactive cells of HO-1 were mainly located at the choroid plexuses and subfornical organ (SFO), as well as in regions around the "sleeve-like" lesion foci, all of which were coincident with the locations of lesions of EAE. The levels of HO-1 mRNA and its protein expression were lowered gradually on day 21, which were in parallel with the severities of symptoms and signs of EAE. After a specific inhibitor of HO-1, Snpp-9, was applied, both of the symptoms and pathological lesions of EAE in the rat brains were mitigated markedly. Therefore, these results may suggest that the dynamic changes of HO-1 mRNA and its protein expression are in parallel with the changes of symptoms and pathological lesions of EAE in the brain. In conclusion, the levels of HO-1 mRNA and its protein expression in brains may play an important role in the pathogenesis of EAE, and application of inhibitors of HO-1 may be one of the potential therapeutic ways for the prevention and treatment of EAE.
Animals ; Brain ; enzymology ; metabolism ; Encephalomyelitis, Autoimmune, Experimental ; enzymology ; genetics ; physiopathology ; Female ; Heme Oxygenase (Decyclizing) ; biosynthesis ; genetics ; Heme Oxygenase-1 ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Subfornical Organ ; metabolism ; pathology

Experimental autoimmune encephalomyelitis was induced in macaques. T cell clones infiltrated into the brain lesion area were compared with those in blood. Intradermal immunization of macaques with brain white matter derived from healthy macaque in combination with pertussis toxin, induced neurological symptoms in two macaques. One died on day 25 after immunization, whereas the other survived. Gross examination of the brain from the dead macaque, showed clear hemorrhagic lesions in the white matter. Hematological analysis showed that drastic T cell response was induced in macaques immunized with white matter, but not in control macaques. Flow cytometric analysis of blood cells from the affected macaques demonstrated an increase of CD4 and CD8 T cell populations expressing the CD69 early activation marker. Single strand conformation polymorphism (SSCP) analysis of T cell receptor beta chain showed T cell clones infiltrated into the brain lesion, which were different from those found in the peripheral blood of the same monkey. The present paper shows that SSCP analysis of TCR is useful in studying clonality of T cells infiltrating into the brain tissue of macaque with EAE.
Animals ; Antigens, CD3/analysis ; Disease Models, Animal ; Encephalomyelitis, Autoimmune, Experimental/immunology/*pathology ; Flow Cytometry/veterinary ; Leukocyte Count/veterinary ; *Macaca fascicularis ; Male ; Polymorphism, Single-Stranded Conformational ; Receptors, Antigen, T-Cell, alpha-beta/genetics ; T-Lymphocytes/cytology/immunology

BACKGROUNDTh9 cells are a newly discovered CD4+ T helper cell subtype, characterized by high interleukin (IL)-9 secretion. Growing evidences suggest that Th9 cells are involved in the pathogenic mechanism of multiple sclerosis (MS). Mast cells are multifunctional innate immune cells, which are perhaps best known for their role as dominant effector cells in allergies and asthma. Several lines of evidence point to an important role for mast cells in MS and its animal models. Simultaneously, there is dynamic "cross-talk" between Th9 and mast cells. The aim of the present study was to examine the IL-9-mast cell axis in experimental autoimmune encephalomyelitis (EAE) and determine its interaction after neutralizing anti-IL-9 antibody treatment.

METHODSFemale C57BL/6 mice were randomly divided into three groups (n = 5 in each group): mice with myelin oligodendrocyte glycoprotein (MOG)-induced EAE (EAE group), EAE mice treated with anti-IL-9 antibody (anti-IL-9 Abs group), and EAE mice treated with IgG isotype control (IgG group). EAE clinical score was evaluated. Mast cells from central nervous system (CNS) were detected by flow cytometry. The production of chemokine recruiting mast cells in the CNS was explored by reverse transcription-polymerase chain reaction (RT-PCR). In mice with MOG-induced EAE, the expression of IL-9 receptor (IL-9R) complexes in CNS and spleen mast cells was also explored by RT-PCR, and then was repeating validated by immunocytochemistry. In vitro, spleen cells from EAE mice were cultured with anti-IL-9 antibody, and quantity of mast cells was counted by flow cytometry after co-culture.

RESULTSCompared with IgG group, IL-9 blockade delayed clinical disease onset and ameliorated EAE severity (t = -2.217, P = 0.031), accompany with mast cells infiltration decreases (day 5: t = -8.005, P < 0.001; day 15: t = -11.857, P < 0.001; day 20: t = -5.243, P = 0.001) in anti-IL-9 Abs group. The messenger RNA expressions of C-C motif chemokine ligand 5 (t = -5.932, P = 0.003) and vascular cell adhesion molecule-1 (t = -4.029, P = 0.004) were significantly decreased after IL-9 neutralization in anti-IL-9 Abs group, compared with IgG group. In MOG-induced EAE, the IL-9R complexes were expressed in CNS and spleen mast cells. In vitro, splenocytes cultured with anti-IL-9 antibody showed significantly lower levels of mast cells in a dose-dependent manner, compared with splenocytes cultured with anti-mouse IgG (5 μg/ml: t = -0.894, P = 0.397; 10 μg/ml: t = -3.348, P = 0.019; 20 μg/ml: t = -7.639, P < 0.001).

CONCLUSIONSThis study revealed that IL-9 neutralization reduced mast cell infiltration in CNS and ameliorated EAE, which might be relate to the interaction between IL-9 and mast cells.

Animals ; Antibodies ; therapeutic use ; Central Nervous System ; metabolism ; Encephalomyelitis, Autoimmune, Experimental ; drug therapy ; metabolism ; Female ; Immunohistochemistry ; Interleukin-9 ; antagonists & inhibitors ; immunology ; metabolism ; Mast Cells ; metabolism ; Mice ; Mice, Inbred C57BL ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo analyze the molecular characters of the W135 Neisseria meningitidis strain firstly isolated from patients in Guangdong province.

METHODSBiochemical profile by using the API NH system (bio-Merieux, France) was used for confirmation,and sero-grouping of the meningococcal isolates including one serogroup W135, one serogroup C and three serogroups of a Neisseria meningitidis isolated from patients in Guangdong province in recent two years were performed. The subtype was determined after amplifying porA and porB respectively from the genome DNA of Neisseria meningitidis. Multilocus sequence typing (MLST) was performed for determining the allele profiles and the sequence types (STs). The polygenetic tree was obtained by analyzing the allele profiles with program Splits tree online. The molecular characters of the serogroup W135 Neisseria meningitidis was analyzed by its evolution relationship and the variable regions in porA and porB which encoding the outer membranes proteins (OMPs).

RESULTSThe subtype determined by porA variable regions of the serogroup W135 Neisseria meningitidis was P1.5,2, which was one of the most invasive types. The types of variable regions (VRs) I, IV, V, VII with porB were 1, 1, 1, 17, and there was no VI and VIII in porB. The allele profile of the W135 strain in this study was 2, 123, 4, 3, 8, 4, 6, and its sequence type was ST-2960, which belonged to ST-11/ET-37 clone complex. The subtypes of the serogroup C and serogroup A strains were P1.20, while their types of VR IV were all 7, and they all hadn't VR VII in porB. The strain serogroup C belonged to ST-4821 clone complex, and the 3 serogroup A strains belonged to ST-5 clone complex.

CONCLUSIONThe molecular character of the serogroup W135 Neisseria meningitidis should be the same with the strains isolated in foreign country, and be different from the epidemic types in the area. This serogroup W135 Neisseria meningitis isolated from patients in Guangdong for the first time was thought to be a new type appearing in the local area.

Bacterial Typing Techniques ; China ; epidemiology ; DNA, Bacterial ; Disease Outbreaks ; Encephalomyelitis ; epidemiology ; microbiology ; Genotype ; Humans ; Molecular Sequence Data ; Neisseria meningitidis, Serogroup W-135 ; classification ; genetics ; isolation & purification ; Sequence Analysis, DNA

Objective To investigate the therapeutic effect of SPK1 gene transfected adipose derived mesenchymal stem cells(ADMSC)on experimental autoimmune encephalomyelitis mice and the effect on T helper cell 17(Th17)/regulatory T(Treg) cells balance. Methods EAE was induced by myelin oligodendrocyte glycoprotein 35-55 in mice.Totally 44 mice were randomly divided into four groups:normal control group(NC group),model group(EAE group),ADMSC group,and ADMSC-SPK1 group.Forty days after injection,the pathological changes of brain and spinal cord,Th17/Treg-related inflammatory markers in brain tissue,expressions of interleukin-17A(IL-17A)and forkhead box protein p3(Foxp3)in brain and spinal cord tissue,and flow cytometric results of spleen immune cells were detected. Results Forty days after the injection,serious inflammatory cell infiltration and demyelination occurred in the brain and spinal cord of EAE group,whereas demyelination and axonal injury were improved in ADMSC group and ADMSC-SPK1 group.Compared with EAE group,the ADMSC group and ADMSC-SPK1 group had significantly improved levels of IL-17A(
Adipose Tissue/cytology* ; Animals ; Cytokines ; Encephalomyelitis, Autoimmune, Experimental/therapy* ; Interleukin-17 ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stem Cells/cytology* ; Mice ; Mice, Inbred C57BL ; Phosphotransferases (Alcohol Group Acceptor)/genetics* ; T-Lymphocytes, Regulatory/cytology* ; Th17 Cells/cytology* ; Transfection