BACKGROUND: The aim of study is to investigate the initial functional changes of muscle in rats induced to have myasthenia gravis, experimental autoimmune myasthenia gravis (EAMG). The authors investigated the functional changes of muscle evaluated by mechanomyography (MMG) and the expression of acetylcholine receptors (AChRs). METHODS: After the institutional approval, 39 male Lewis rats were randomly allocated into study. 26 animals were immunized to induce EAMG by Torpedo AChR (T-AChR) emulsified with complete Freund's adjuvant (CFA) and phosphate buffer saline (PBS)/bovine serum albumin (BSA) 0.01% at the base of tail, and received booster immunizations twice by T-AChR with incomplete Freund's adjuvant (IFA) and PBS/BSA 0.01% at all different site on the upper back. 13 animals were sham immunized as control group by the same method of EAMG except T-AChR. Clinical EAMG scores were examined. Anti T-AChR and anti rat-AChR (R-AChR) antibodies (Ab) were compared by using (125)I-alpha-bungarotoxin ((125)I-alpha-BuTx) radioimmunoassay. Under the anesthesia, neuromuscular functions were monitored by MMG using single twitch (ST) and TOF. AChRs were quantitated using (125)I-alpha-BuTx. RESULTS: Overall weight gain and final body mass, muscle force (ST), specific muscle force of ST, TOF fade ratio and AChRs were reduced in EAMG score 3 compared to control (P < 0.0001). Anti T-AChR Ab and anti R-AChR Ab were increased in score 3 EAMG (P < 0.0001). CONCLUSIONS: EAMG score 3 rats showed characteristic neuromuscular functions as depressed initial ST and its specific force, initial TOF fade and increased anti AChR Abs. Those above characteristics had significant correlations with the clinical EAMG scores. AChRs were significantly down-regulated according to their functional characteristics and clinical EAMG scores.
Acetylcholine* ; Anesthesia ; Animals ; Antibodies ; Freund's Adjuvant ; Humans ; Immunization, Secondary ; Male ; Myasthenia Gravis* ; Myasthenia Gravis, Autoimmune, Experimental ; Radioimmunoassay ; Rats* ; Receptors, Cholinergic* ; Serum Albumin ; Tail ; Torpedo ; Weight Gain

Umbilical cord mesenchymal stem cell (UCMSC) transplantation has been widely used in the treatment of a variety of diseases due to their advantages such as abundant resources, low immunogenicity and large ex vivo expansion capacity. This study was aimed to investigate the effects of UCMSC on experimental autoimmune myasthenia gravis (EAMG) rats. The distribution of human-derived cells was observed by immunofluorescence method, the effect of MSC on B-cell in situ-secreted antibodies was assayed by ELISPOT, the secreted IFN-γ level was detected by using Transwell test. The results showed that UCMSC were able to migrate to inflammation region and lymph nudes, moreover human-derived cells could be detected in medulla zone of lymph nudes. In vitro in situ detection of AchR specific antibody secretion revealed that the full contact of MSC with lymphnode-derived lymphocytes could effectively inhibit production of AchR antibody. Transwell test indicated that the direct contact of UCMSC with CD4 T cells could effectively decrease production of IFN-γ, which modulated the unbalance between Th1/Th2 to a certain extent. It is concluded that UCMSC can regulate the immune system by direct cell-cell contact or/and release of cytokines, which bring a new insight into knowledge about MSC-based therapy for EAMG.
Animals ; Cord Blood Stem Cell Transplantation ; Female ; Humans ; Mesenchymal Stem Cell Transplantation ; Myasthenia Gravis, Autoimmune, Experimental ; therapy ; Rats ; Rats, Inbred Lew

OBJECTIVETo study the prophylactic effects of nasal tolerance with a dual analogue, Lys262-Ala207, on the mouse model of experimental autoimmune myasthenia gravis (EAMG) and the underlying mechanism.

METHODSMouse model of EAMG was induced by intraperitoneal injection of mAb35. Lys262-Ala207 or PBS was given nasally before 10 days (study group A and control group A) or on the day (study group B and control group B) of immunization for 10 days. Clinical syndromes were evaluated after immunization. Serum level of acetylcholine receptor antibody (AChR-Ab) IgG was detected using ELISA. The number of monouclear cells expressing CD4+ and CD4+ CD25+T from spleen was measured using flow cytometry.

RESULTSCompared with the corresponding control groups, the clinical syndromes were improved (P<0.01) in mice from the study groups A and B. The positive rate of the repetitive nerve stimulation (RNS) test in the study groups A and B was significantly lower than that in the corresponding control groups (P<0.01). The study group A showed lower positive rate of RNS than the study group B (P<0.05). The serum levels of AChR-Ab IgG in the study groups A and B (15.01+/-1.09 and 19.23+/-1.31 microg/mL) decreased compared with that in the corresponding control groups (28.12+/-1.28 and 29.35+/-1.28 microg/mL) (P<0.01). The study group A mice had lower serum AChR-Ab IgG levels than the study group B (P<0.01). The number of CD4+ CD25+T cells in the study groups A (4.516+/-0.598%) and B (3.671+/-0.300%) increased significantly compared with that in the corresponding control groups (2.661+/-0.411% and 2.412+/-0.500%) (P<0.01) and more CD4+ CD25+T cells were found in the study group A than in the study group B (P<0.01).

CONCLUSIONSNasal administration with dual analogues may ameliorate clinical syndromes in EAMG rats, which may be associated with decreased serum AChR-Ab IgG levels and increased number of CD4+ CD25+T cells from spleen.

Administration, Intranasal ; Animals ; Female ; Immune Tolerance ; drug effects ; Immunoglobulin G ; blood ; Mice ; Mice, Inbred C57BL ; Myasthenia Gravis, Autoimmune, Experimental ; immunology ; prevention & control ; Receptors, Cholinergic ; immunology ; T-Lymphocytes, Regulatory ; physiology

Myasthenia gravis (MG) is a prototypical antibody-mediated neurological autoimmune disease with the involvement of humoral immune responses in its pathogenesis. T follicular helper (Tfh) cells have been implicated in many autoimmune diseases. However, whether and how Tfh cells are involved in MG remain unclear. Here, we established and studied a widely-used and approved animal model of human MG, the rat model with acetylcholine receptor alpha (AChRα) subunit (R-AChR)-induced experimental autoimmune myasthenia gravis (EAMG). This model presented mild body-weight loss 10 days after the first immunization (representing the early stage of disease) and more obvious clinical manifestations and body-weight loss 7 days after the second immunization (representing the late stage of disease). AChR-specific pre-Tfh cells and mature Tfh cells were detected in these two stages, respectively. In co-cultures of Tfh cells and B cells, the number of IgG2b-secreting B cells and the level of anti-AChR antibodies in the supernatant were higher in the cultures containing EAMG-derived Tfh cells. In immunohistochemistry and immunofluorescence assays, a substantial number of CD4/Bcl-6 T cells and a greater number of larger germinal centers were observed in lymph node tissues resected from EAMG rats. Based on these results, we hypothesize that an AChR-specific Tfh cell-mediated humoral immune response contributes to the development of EAMG.
Animals ; B-Lymphocytes ; immunology ; Disease Models, Animal ; Female ; Immunity, Humoral ; Lymph Nodes ; immunology ; Myasthenia Gravis, Autoimmune, Experimental ; immunology ; Protein Subunits ; immunology ; Proto-Oncogene Proteins c-bcl-6 ; immunology ; Rats, Inbred Lew ; Receptor Cross-Talk ; Receptors, Cholinergic ; immunology ; T-Lymphocytes, Helper-Inducer ; immunology