Zeng-Yu Wang

Mucosal Tolerance to Experimental Autoimmune Myasthenia Gravis Is Associated with Down‐regulation of AChR‐specific IFN‐γ‐expressing Th1‐like Cells and Up‐regulation of TGF‐β mRNA in Mononuclear Cells

Oral and nasal administration of nicotinic acetylcholine receptor (AChR) to Lewis rats prior to myasthenogenic immunization with AChR and complete Freunds adjuvant (CFA) resulted in prevention or marked decrease of the severity of experimental autoimmune myasthenia gravis (EAMG) and suppression of AChR-specific B-cell responses and of AChR-reactive T-cell function. To examine the involvement of immunoregulatory cytokines and the underlying mechanisms involved in tolerance induction, in situ hybridization with radiolabeled cDNA oligonucleotide proves was adopted to enumerate mononuclear cells (MNC) expressing mRNA for the proinflammatory cytokine interferon-gamma (IFN-gamma), the B cell-stimulating interleukin-4 (IL-4), and the immunosuppressive transforming growth factor-beta (TGF-beta). Popliteal and inguinal lymph nodes from EAMG rats contained elevated numbers of AChR-reactive IFN-gamma, IL-4, and TGF-beta mRNA-expressing cells, compared to control rats receiving PBS orally or nasally and injected with CFA only. Oral and nasal tolerance was accompanied by decreased numbers of AChR-reactive IFN-gamma and IL-4 mRNA-expressing cells and strong up-regulation of TGF-beta mRNA-positive cells in lymphoid organs when compared to nontolerized EAMG control rats. The results suggest that IFN-gamma and IL-4 are central effector molecules in the development of EAMG and that TGF-beta plays an important role in tolerance induction to EAMG.

B cell responses to acetylcholine receptor in rats orally tolerized against experimental autoimmune myasthenia gravis.

Oral administration of acetylcholine receptor (AChR) to Lewis rats prior to myasthenogenic immunization with AChR and complete Freunds adjuvant (CFA) results in the prevention of experimental autoimmune myasthenia gravis (EAMG), and decreased serum levels of anti-AChR antibodies. Using an ELISPOT assay, we have now determined numbers of cells in the popliteal, inguinal and mesenteric lymph nodes, spleen and thymus secreting anti-AChR IgG antibodies. Except for mesenteric lymph nodes, a marked diminution of such cells was detected in these lymphoid organs in rats orally tolerized with AChR compared to buffer-fed or vehicle-fed control rats with EAMG. Of note is that, after AChR feeding, the B cell response to AChR in thymus was diminished to the same low level as in CFA-injected, buffer-fed control rats. The relative affinity of serum anti-AChR IgG antibodies measured by KSCN-ELISA was lower in the orally tolerized rats compared to buffer-fed or vehicle-fed rats. The observations showed that oral administration of AChR, besides preventing clinical EAMG, also counteracts the development of AChR-specific B cells, especially those with high affinity antibody production, in most lymphoid organs.

Suppression of experimental autoimmune myasthenia gravis and experimental allergic encephalomyelitis by oral administration of acetylcholine receptor and myelin basic protein: double tolerance.

Oral administration of acetylcholine receptor (AChR) or myelin basic protein (MBP) to Lewis rat prior to immunization with AChr or MBP and complete Freunds adjuvant (CFA) has previously been shown to prevent or delay the onset of experimental autoimmune myasthenia gravis (EAMG) or experimental allergic encephalomyelitis (EAE), which represent animal models of myasthenia gravis and multiple sclerosis, respectively. Here we show that Lewis rats immunized with AChr+MBP+CFA developed both signs of muscular weakness seen in EAMG and paresis characteristic for EAE. This disease was associated with high levels of anti-AChR and anti-MBP antibody secreting cells and of AChR- and MBP-reactive INF-gamma secreting Th1-like cells in lymph nodes. The diseased rats also showed upregulation of AChR- and MBP-induced mRNA expression of IFN-gamma in lymph node cells. Oral tolerization with AChR and MBP in combination prior to immunization with AChR+MBP+CFA alleviated clinical disease as well as AChR- and MBP-specific B cell node cells. The results implicate that oral tolerization simultaneously to more than one autoimmune disease-related autoantigen is feasible, and that suppression of autoantigen-induced IFN-gamma and augmentation of TGF-beta are pivotal in tolerance induction.

The T-Cell Repertoire in Myasthenia Gravis Involves Multiple Cholinergic Receptor Epitopes

Antibodies against the α‐subunit of the acetylcholine receptor (AChR) are found in most patients with myaslhenia gravis and are considered to contribute to the receptor damage which leads to the characteristic signs and symptoms of the disease. This B‐cell response is T‐cell driven. Elevated T‐cell reactivities to AChR and its α‐subunit have been described in myasthenia gravis, and AChR α‐subunit peptide reactive T‐cell lines and clones preferentially recognizing certain defined sequence segments have been reported, thereby disclosing the possibility of specific immunotherapy. We have defined the T‐cell repertoire to AChR, its α‐subunit and the synthetic peptide sequences 1OO‐117,113‐130,143‐163,161‐179,207‐225,221‐240, and 235‐255 of the α‐subunit in an immunospot assay which is based on secretion of interferon‐gamma (IFN‐γ) by individual memory T cells upon stimulation with specific antigen in short‐term cultures. Most patients with myasthenia gravis displayed T‐cell reactivities to 1 to 6 different peptides. The mean numbers of T cells recognizing individual peptides varied in the myasthenia gravis patients between 1 per 77,000 and 1 per 167,000 peripheral blood mononuclear cells. None of the seven peptides evaluated could be identified as an immunodominant T‐cell epitope, and any of them was found to dominate in individual patients. The numbers of T cells reacting with AChR and recombinant human AChR α‐subunit were slightly higher (mean numbers 1 per 26,000 and 1 per 50,000 mononuclear cells, respectively). Such cells, as well as AChR α‐subunit peptide reactive T cells, were also found in patients with other neurological diseases and in healthy subjects, but at lower frequencies and numbers. In myasthenia gravis, the elevated numbers of memory T cells recognizing multiple AChR α‐subunit peptides may be crucial for the development of the disease, and the IFN‐γ released by such T cells might be important for its perpetuation.

T-Cell Immunity to Acetylcholine Receptor and its Subunits in Lewis Rats over the Course of Experimental Autoimmune Myasthenia Gravis

Lymph nodes, spleen and thymus obtained from Lewis rats were examined over the course of experimental autoimmune myasthenia gravis (EAMG) for the distribution and the number of antigen‐reactive CD4+ T helper cells which, upon recognition of Torpedo acetylcholine receptor (AChR) or the α, β, γ or δ subunits of Torpedo AChR, responded by secretion of interferon‐gamma (IFN‐γ). T cells with these specificities were detected in these three immune organs. Numbers were highest in lymph nodes. In spleen and thymus, numbers of antigen‐reactive T cells did not differ. T cells reacting against the intact AChR were more frequent than T cells recognizing any of the subunits. The immunogenicity between the four subunits did not differ, with the exception that the α subunit induced a slightly higher T‐cell response. No restriction of the T‐cell repertoire to the four subunits was detected during early compared to late phases of EAMG. The AChR and subunit‐reactive T cells could—via secretion of effector molecules including IFN‐γ—play an important role in the initiation and perpetuation of EAMG. and consequently also of human myasthenia gravis. T cells with the same specificities were also detected in control animals injected with adjuvant only, but at much lower numbers which were within the range of T cells recognizing the control antigen myelin basic protein. They could represent naturally occurring autoimmune T cells.

Intrathecal Synthesis of Anti‐Borrelia burgdorferi Antibodies in Neuroborreliosis: a Study with Special Emphasis on Oligoclonal IgM Antibody Bands

Pre‐ and post‐treatment (up to 3–26 months after antibiotic therapy) humoral immune responses were investigated in five neuroborreliosis patients. Anti‐Borrelia (B.) burgdorferi IgG and IgM antibodies in CSF and serum were quantitated by capture ELISA. Agarose gel isoelectric focusing (AIF) and protein blotting were used to detect oligoclonal IgG and IgM bands as well as oligoclonal anti‐B. burgdorferi IgG and IgM antibodies. These latter components were visualized by transfer to antigen‐coated membranes (immunoblot) and immunoenzymatic staining. By ELISA, intrathecal anti‐B. burgdorferi IgG and IgM antibody synthesis was detected in all initial specimens and continued 3–26 months after antibiotic therapy in four and three cases, respectively. AIF with protein blotting showed oligoclonal bands of total IgG as well as total IgM in the initial CSF specimens of all patients and persistence of such components occurred in four and five cases, respectively. By AIF and immunoblot, oligoclonal anti‐B. burgdorferi IgG and IgM antibody bands could be detected in the CSF of every patient. IgG antibody bands were present in all initial CSF samples. The first specimen of one patient was negative for IgM antibody bands but such components appeared 3 weeks later. Oligoclonal CSF anti‐B. burgdorferi IgG antibody components persisted over the entire follow‐up periods in all but one case where they disappeared 6 weeks after treatment. The oligoclonal IgM antibodies in CSF vanished in two cases (after being present up to 4 and 11 months after antibiotic therapy) while they persisted over the entire (3–6 months after antibiotic therapy) follow‐up periods in three cases. The specificity of the IgM antibody immunoblot technique was corroborated by control experiments, including antibody absorption studies and use of 41 kDa flagellar antigen.

B cell autoimmunity to acetylcholine receptor and its subunits in Lewis rats over the course of experimental autoimmune myasthenia gravis.

Experimental autoimmune myasthenia gravis (EAMG) is induced by a single injection of acetylcholine receptor (AChR) with complete Freunds adjuvant and represents a useful animal model for studying the mechanisms by which autoimmune responses to AChR and its subunits are coupled to the development of human myasthenia gravis. Using an immunospot assay, we enumerated cells secreting IgG antibodies against Torpedo AChR and the alpha-, beta-, gamma- and delta-subunits of Torpedo AChR in lymph nodes, spleen and thymus from Lewis rats over the course of EAMG. Cells secreting IgG antibodies to AChR and to all four subunits were detected at higher numbers in the three immune organs in EAMG compared to controls. Numbers were highest in lymph nodes followed by spleen and thymus. Cells secreting IgG antibodies against native AChR were always higher than those against individual subunits. The immunogenicity between the four subunits did not differ, with the exception that the alpha-subunit induced a slightly higher B cell response in thymus and lymph nodes. The patterns of B cell responses were similar when analyzed over the course of EAMG from week 2 to week 5, and there was no restriction of the B cell repertoire early in EAMG. Anti-AChR and anti-subunit antibody-secreting cells were also detected in control animals immunized with adjuvant only, but at numbers which were much lower, and which were within the same level as numbers of cells secreting IgG antibodies to the control antigen myelin basic protein, probably reflecting naturally occurring autoimmune B cells.

β-Bungarotoxin binding protein is immunogenic but lacks myasthenogenicity in rats

Myasthenia gravis is an autoimmune disease resulting from autoaggressive immunity to the nicotinic acetylcholine receptor (AChR) of the neuromuscular junction. Immunization with AChR leads to experimental autoimmune myasthenia gravis (EAMG). AChR binds to alpha-bungarotoxin, and this property is utilized to purify AChR. The utilization of beta-bungarotoxin (beta-BGT) allows the purification of a different protein of striated muscle. To examine the myasthenogenic activity of the beta-BGT binding protein, we immunized Lewis rats with the beta-BGT binding protein or AChR from Torpedo. While AChR-immunized rats developed typical signs of EAMG, beta-BGT binding protein immunized rats showed no definite clinical signs of muscular weakness during observation periods up to 14 weeks after immunization. High levels of anti-AChR IgG antibodies and low levels of antibodies against beta-BGT binding protein were present in the AChR-immunized rats, while high levels of IgG antibodies to the beta-BGT binding protein and low anti-AChR IgG antibody levels were found in the rats immunized with the beta-BGT binding protein. No relationship was observed between antibody levels and severity of EAMG. The anti-AChR and anti-beta-BGT binding protein IgG antibodies showed only low degrees of cross-reactivity. We conclude that the beta-BGT binding protein is immunogenic also in EAMG, while it is still an open question whether it also has myasthenogenic properties.

Antigen-induced cytokine patterns in double mucosal tolerance to EAE and EAMG

The prmmv atm of phase 3 trials IS to demonstrate eficacy by assessmg the cl&al outcome. In consideration of the time, manpower and tinanc~al resources associated wth a multi-center phase 3 tnal It IS prudent to invest in a teaching program to maxlmize the reltabihty of the clmical outcome-assessment. In the European multi-centre Interferon P-lb (Betaferona) trial in secondary progressive MS a sustamed increase in the Kurt&e Expanded Disability Status Scale (EDSS) is the primary outcome parameter Baaed on experience in previous trials a standardized documentation form for the neurological examination was developed with detailed instructtons on how to assign the functtonal systemand EDSSscores. 126 neurologists and residents m neurology from I2 different countries participated in one out of 6 training sewons that were conducted by the same person and mcluded a special training video. At the end of the sessions participants evaluated a neurological examinaoon demonstrated in a second video usmg the study documentation forms 561126 d&red by 1 pomt in the assessment of the optic fimcttonal system (FS), 60 by 1 or 2 pomts in the assessment of bramstem FS. I2 by 1 or 2 points in pyramIdal FS, 26 by I in ccrebellar FS, 35 by 1 point in sensory FS, I4 by I pomt in cerebral FS and 14 by I point in vegetative FS Only 181126 forms dtffered m the tinal EDSS (by 0.5-1.5 pomts) Most dcviatlons were found m the ambulation index (90/126 differed by a! least I pomt) The variance m the scoring of ambulation Index IS most likely due to mcomplete information m the v&n The EDSS inter-ratmg reliability found in this study 1s equal or even bettor than that reported from smglc centre studies. This training approach using wdeos seems to contribute to m a high degree of concordance behueen parhcipants with different experience and natIonal background, although differences in the assessment of some of the functional systems are evident