Objective: B cells and the humoral immune system have been implicated in the pathogenesis of multiple sclerosis (MS). This study sought to evaluate the efficacy, safety, and tolerability of add-on therapy with rituximab, a monoclonal antibody that depletes circulating B cells, in subjects with relapsing MS with breakthrough disease defined by clinical and MRI activity (Class III evidence).Methods: Thirty subjects with a relapse within the past 18 months despite use of an injectable disease-modifying agent, and with at least 1 gadolinium-enhancing (GdE) lesion on any of 3 pretreatment MRIs, received rituximab administered at 375 mg/m(2) weekly x 4 doses. Three monthly posttreatment brain MRI scans were obtained beginning 12 weeks after the first infusion. Multiple Sclerosis Functional Composite (MSFC) and Expanded Disability Status Scale (EDSS) were obtained at baseline and throughout the posttreatment follow-up.Results: GdE lesions were reduced after treatment with rituximab, with 74% of posttreatment MRI scans being free of GdE activity compared with 26% free of GdE activity at baseline (p < 0.0001). Median GdE lesions were reduced from 1.0 to 0, and mean number was reduced from 2.81 per month to 0.33 after treatment (88% reduction). MSFC improved as well (p = 0.02). EDSS remained stable.Conclusion: Rituximab add-on therapy was effective based upon blinded radiologic endpoints in this phase II study. In combination with standard injectable therapies, rituximab was well-tolerated with no serious adverse events. B-cell-modulating therapy remains a potential option for treatment of patients with relapsing MS with an inadequate response to standard injectable therapies.Classification of evidence: This study provides Class III evidence that add-on rituximab reduces gadolinium-enhancing brain lesions in multiple sclerosis. Neurology (R) 2010; 74: 1860-1867

Suppressors of cytokine signaling (SOCS) are proteins that modulate cytokine responses in lymphoid cells. In these studies, cultured primary mouse astrocytes expressed SOCS-3 mRNA constitutively. Treatment with interferon-gamma (IFN-g) induced SOCS-1 and enhanced SOCS-3 expression, and was associated with decreased tumor necrosis factor-alpha (TNF) and increased leukemia inhibitory factor (LIF) in culture supernatants. Treatment with conditioned medium from myelin basic protein-stimulated encephalitogenic lymphoid cells (MBP-CM) increased SOCS-3 and induced SOCS-1 expression. The effects were largely due to IFN-g in MBP-CM, as anti-IFN-g antibody diminished induction. These findings suggest a role for IFN-g-induced SOCS expression in regulation of CNS inflammatory responses by astrocytes.

Purpose/Objective: Heterotopic ossification (HO) is a term used to describe normal osseous tissue deposition at ectopic sites. Often this deposition is adjacent to or spans the elbow joint after traumatic injury, burns, neural axis trauma, and some genetic conditions. The treatment of HO depends on the volume of extra bone, its location, and the resultant limitation in range of motion around the joint. One method of preventing HO after injury or surgery is radiation therapy delivered to the area at risk. We describe the Cleveland Clinic's experience using radiation therapy to prevent heterotopic bone formation around the elbow joint.Materials/Methods: The records of 27 patients (29 elbows) treated with radiation therapy between 1993–1998 were reviewed. Twenty-one patients (23 elbows) had greater than 3 months follow-up available for review and are included in this analysis. All patients were classified using the classification system developed by Hastings and Graham prior to surgery. Pre-operative, intra-operative, and post-operative range of motion were recorded and compared. Radiation therapy dose ranged from 600-700cGy in one fraction delivered post-operatively.Results: The median follow-up was 10.2 months (range: 3.4-62.1 months). When comparing the pre-operative range of motion with the range of motion achieved intra-operatively, the median increase in range of motion achieved in the operating room was 75 degrees (range: 15-160 degrees). In comparing the post-operative range of motion with the pre-operative range of motion, the median increase in mobility was 57.5 degrees (range: 10-145 degrees). No patient experienced a post-operative wound complication.Conclusions: Radiation therapy consisting of 700 cGy in one fraction delivered post-operatively is successful in maintaining intra-operative range of motion gains in the majority of patients. This form of therapy should be considered in patients presenting with Hastings and Graham Class II or III heterotopic bone formation around the elbow joint or in patients with multiple prior surgeries who have exhibited a tendency towards subsequent heterotopic bone formation. Purpose/Objective: Heterotopic ossification (HO) is a term used to describe normal osseous tissue deposition at ectopic sites. Often this deposition is adjacent to or spans the elbow joint after traumatic injury, burns, neural axis trauma, and some genetic conditions. The treatment of HO depends on the volume of extra bone, its location, and the resultant limitation in range of motion around the joint. One method of preventing HO after injury or surgery is radiation therapy delivered to the area at risk. We describe the Cleveland Clinic's experience using radiation therapy to prevent heterotopic bone formation around the elbow joint. Materials/Methods: The records of 27 patients (29 elbows) treated with radiation therapy between 1993–1998 were reviewed. Twenty-one patients (23 elbows) had greater than 3 months follow-up available for review and are included in this analysis. All patients were classified using the classification system developed by Hastings and Graham prior to surgery. Pre-operative, intra-operative, and post-operative range of motion were recorded and compared. Radiation therapy dose ranged from 600-700cGy in one fraction delivered post-operatively. Results: The median follow-up was 10.2 months (range: 3.4-62.1 months). When comparing the pre-operative range of motion with the range of motion achieved intra-operatively, the median increase in range of motion achieved in the operating room was 75 degrees (range: 15-160 degrees). In comparing the post-operative range of motion with the pre-operative range of motion, the median increase in mobility was 57.5 degrees (range: 10-145 degrees). No patient experienced a post-operative wound complication. Conclusions: Radiation therapy consisting of 700 cGy in one fraction delivered post-operatively is successful in maintaining intra-operative range of motion gains in the majority of patients. This form of therapy should be considered in patients presenting with Hastings and Graham Class II or III heterotopic bone formation around the elbow joint or in patients with multiple prior surgeries who have exhibited a tendency towards subsequent heterotopic bone formation.

The role of B cells and antibody in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) remains controversial. We previously demonstrated that B cells are required for EAE to be induced by the 120-amino acid extracellular domain of myelin oligodendrocyte glycoprotein (MOG). In the present study, the role of B cells in MOG-induced EAE was further characterized. Passive transfer of activated B cells or serum from MOG-primed wild-type (WT) mice was found to reconstitute the ability for clinical and histological EAE to be induced in MOG-immunized B cell-deficient mice. MOG-induced EAE did not occur with transfer of B cells that had been nonspecifically activated by lipopolysaccharide or isolated from naive or myelin basic protein (MBP)-primed WT mice. Likewise, MOG-primed serum, but not naive serum or serum from MBP-, Hen egg Iysozyme-, or MOG(35-55)-primed mice, led to EAE in B cell(-/-) animals. While both MOG-primed B cells and serum reconstituted the ability for disease induction, MOG-primed serum was much more efficient, leading to clinical and histological EAE similar to that seen in the WT. Injection of MOG serum into healthy B cell(-/-) mice 30 days after MOG immunization led to rapid appearance of clinical signs and CNS inflammation, indicating that an antigen-specific factor is necessary for initiation of CNS inflammation, and not just demyelination. These data strongly suggest that MOG-specific antibody is critical to the initiation of MOG-induced murine EAE.

It was previously shown that BALB/c mice were susceptible to experimental autoimmune encephalomyelitis induced by immunization with proteolipid protein (PLP). To determine the encephalitogenic epitopes of PLP in BALB/c mice, mice were immunized with successively smaller pools of 20-mer peptides spanning the PLP molecule from amino acid 30 to amino acid 206. Immunization with PLP180–199 resulted in clinical EAE in 9/15 mice (mean max clinical score of 3.3), and immunization with PLP185–206 induced clinical EAE in 7/21 BALB/c mice (mean maximum score of 3.7). No relapses in disease were observed. No EAE was observed in BALB/c mice immunized with PLP185–199 (n=15), PLP178–191 (n=13) or other regions of PLP (n=15). Passive transfer of PLP180–199-primed lymph node cells into naı̈ve BALB/c mice resulted in EAE (2/2 mice, max score of 4.0). One-micron toluidine blue stained sections from the spinal cord of EAE-affected BALB/c mice revealed features typical of EAE in other strains, including mononuclear cell infiltration, myelin loss, and axonal loss.

There is much evidence to implicate B cells, plasma cells, and their products in the pathogenesis of MS. Despite unequivocal evidence that the animal model for MS, EAE, is initiated by myelin-specific T cells, there is accumulating evidence of a role for B cells, plasma cells, and their products in EAE pathogenesis. The role(s) played by B cells, plasma cells, and antibodies in CNS inflammatory demyelinating diseases are likely to be multifactorial and complex, involving distinct and perhaps opposing roles for B cells versus antibody.

T cell co-stimulation through the CD28 receptor on T cells is critical to the induction of experimental autoimmune encephalomyelitis (EAE). In this study, expression of the co-stimulatory ligands B7-1 (CD80) and B7-2 (CD86), as well as the receptors CD28 and CTLA-4, were quantitated in central nervous system (CNS) tissues from mice at various stages of EAE. Immunohistochemistry and flow cytometry of CNS-infiltrating cells revealed a high percentage of infiltrating T cells expressing B7-1 and B7-2 during acute, chronic and relapsing EAE. Of the infiltrating cells 10-20% were CTLA-4(+), most of which were CD4(+) T cells. B7-1 and B7-2 expression within the CNS during active EAE might increase the potential for local activation of autoimmune T cells; however, the high level of expression of B7 molecules may also provide a mechanism for the autoregulation of activated CTLA-4(+) T cells.

While the pathology of multiple sclerosis implicates a role for B cells and antibodies in the disease process, results from animal models have yielded conflicting results. To further characterize the role of B cells in experimental allergic encephalomyelitis (EAE), wild-type and B cell-deficient C57BL/6 mice were immunized with either a recombinant form of myelin oligodendrocyte glycoprotein (MOG) or with the encephalitogenic MOG(35-55) peptide. B cell-deficient mice did not develop EAE when immunized with MOG, although they were susceptible to MOG(35-55)-induced disease. In contrast, wild-type mice were fully susceptible to both MOG and MOG(35-55)-induced EAE. B cell-deficient mice immunized with MOG were primed to the encephalitogenic MOG(35-55) epitope, as their spleen cells responded with Th1 cytokine production in a fashion similar to WT cells when challenged in vitro with MOG protein or MOG(35-55) peptide. These results demonstrate that the form of inducing antigen (protein vs. peptide) plays a role in the pathogenesis of EAE and may be relevant when applying results from the EAE model to multiple sclerosis.

The present study was designed to assess the pattern of cytokine expression over the course of disease in the central nervous system (CNS) of recipients of an encephalitogenic T-cell clone specific for proteolipid protein (PLP) peptide 139-151. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses of CNS mRNA from samples taken during the onset of acute disease demonstrated upregulation of message for cytokines involved in the recruitment and activation of macrophages (GM-CSF, interleukin (IL)-3, IL-9) and the inflammatory cytokines tumor necrosis factor (TNF)-alpha and iNOS as well as message for IL-10 and transforming growth factor (TGF)beta. During the recovery stage message for most cytokines was absent, but during relapse inflammatory cytokine messages were again detectable. Message for the accessory molecules B7-2 and CTLA-4 was observed only on the day of onset of acute experimental allergic encephalomyelitis (EAE) and at relapse. The messages for these molecules were downregulated at the onset of recovery. These results illustrate the dynamic nature of the immune response during the course of EAE, and support a model of disease in which T-cells are involved in the regulation of disease while a nonspecific inflammatory reaction is responsible for the CNS damage observed during EAE.

Kinetics of entry into the CNS of donor- and host-derived T-cells during the onset of acute murine EAE induced by the passive transfer of an encephalitogenic PLP(139-151)-specific T-cell clone was investigated. RT-PCR and spectratypic analysis of total RNA recovered from recipient mice demonstrated the presence in the CNS of donor- and host-derived T-cells 24 h post adoptive transfer. Donor-derived T-cells detected in the CNS decreased days 2-6 post transfer while host-derived T-cells persisted during this time. Beginning 3 days before clinical onset, an increase in the CNS of both T-cell populations was observed which persisted through disease onset. Similar analysis performed on recipients of an nonencephalitogenic PLP(139-151)-specific T-clone demonstrated a transient infiltration of donor- and host-derived T-cells beginning 4 days post transfer (dpt) and returning to background levels by day 7 post transfer. Results presented here suggest the importance of host-derived T-cells in the onset of acute passive murine EAE. (C) 1998 Elsevier Science B.V. All rights reserved.