Encephalitogenic potential of the myelin basic protein peptide (amino acids 83-99) in multiple sclerosis: results of a phase II clinical trial with an altered peptide ligand.

Myelin-specific T lymphocytes are considered essential in the pathogenesis of multiple sclerosis. The myelin basic protein peptide (a.a. 83-99) represents one candidate antigen; therefore, it was chosen to design an altered peptide ligand, CGP77116, for specific immunotherapy of multiple sclerosis. A magnetic resonance imaging-controlled phase II clinical trial with this altered peptide ligand documented that it was poorly tolerated at the dose tested, and the trial had therefore to be halted. Improvement or worsening of clinical or magnetic resonance imaging parameters could not be demonstrated in this small group of individuals because of the short treatment duration. Three patients developed exacerbations of multiple sclerosis, and in two this could be linked to altered peptide ligand treatment by immunological studies demonstrating the encephalitogenic potential of the myelin basic protein peptide (a.a. 83-99) in a subgroup of patients. These data raise important considerations for the use of specific immunotherapies in general.

Association of human herpes virus 6 (HHV-6) with multiple sclerosis: increased IgM response to HHV-6 early antigen and detection of serum HHV-6 DNA.

Viruses have long been suggested to be involved in the etiology of multiple sclerosis (MS). This suggestion is based on (1) epidemiological evidence of childhood exposure to infectious agents and increase in disease exacerbations with viral infection; (2) geographic association of disease susceptibility with evidence of MS clustering; (3) evidence that migration to and from high-risk areas influences the likelihood of developing MS; (4) abnormal immune responses to a variety of viruses; and (5) analogy with animal models and other human diseases in which viruses can cause diseases with long incubation periods, a relapsing-remitting course, and demyelination. Many of these studies involve the demonstration of increased antibody titers to a particular virus, whereas some describe isolation of virus from MS material. However, no virus to date has been definitively associated with this disease. Recently, human herpesvirus 6 (HHV-6), a newly described beta-herpes virus that shares homology with cytomegalovirus (CMV), has been reported to be present in active MS plaques. In order to extend these observations, we have demonstrated increased IgM serum antibody responses to HHV-6 early antigen (p41/38) in patients with relapsing-remitting MS (RRMS), compared with patients with chronic progressive MS (CPMS), patients with other neurologic disease (OND), patients with other autoimmune disease (OID), and normal controls. Given the ubiquitous nature of this virus and the challenging precedent of correlating antiviral antibodies with disease association, these antibody studies have been supported by the detection of HHV-6 DNA from samples of MS serum as a marker of active viral infection.

Identification of candidate T-cell epitopes and molecular mimics in chronic Lyme disease.

Elucidating the cellular immune response to infectious agents is a prerequisite for understanding disease pathogenesis and designing effective vaccines. In the identification of microbial T-cell epitopes, the availability of purified or recombinant bacterial proteins has been a chief limiting factor. In chronic infectious diseases such as Lyme disease, immune-mediated damage may add to the effects of direct infection by means of molecular mimicry to tissue autoantigens. Here, we describe a new method to effectively identify both microbial epitopes and candidate autoantigens. The approach combines data acquisition by positional scanning peptide combinatorial libraries and biometric data analysis by generation of scoring matrices. In a patient with chronic neuroborreliosis, we show that this strategy leads to the identification of potentially relevant T-cell targets derived from both Borrelia burgdorferi and the host. We also found that the antigen specificity of a single T-cell clone can be degenerate and yet the clone can preferentially recognize different peptides derived from the same organism, thus demonstrating that flexibility in T-cell recognition does not preclude specificity. This approach has potential applications in the identification of ligands in infectious diseases, tumors and autoimmune diseases.

Impaired measles virus-specific cytotoxic T cell responses in multiple sclerosis.

To assess whether an virus-specific immune defect may be associated with multiple sclerosis (MS), we have examined the ability to generate measles virus-and influenza virus-specific cytotoxic T cells (CTL) in patients with MS, normal individuals, and other disease controls (ODC). The mean (+/- SEM) measles virus-specific CTL response for normal individuals and ODC was 26.9 +/- 2.9% (N = 17) and 26.7 +/- 2.8% (N = 13) specific lysis, respectively. In contrast, the capacity of MS patients to generate measles virus-specific CTL was markedly diminished. Peripheral blood lymphocytes from MS patients stimulated with measles virus lysed their measles virus-infected autologous B cell line at a group mean level of 6.0 +/- 1.4% (N = 16) specific lysis. MS patients had significantly lower measles virus-specific CTL responses than normal individuals (p less than 0.00001) or ODC (p less than 0.0001). Importantly, this lowered response did not reflect a generalized depressed cytolytic activity of MS patients, since influenza virus-specific CTL and NK activity from these patients were comparable to normals and ODC. Thus, in MS there is a significant depression of measles virus-specific CTL which suggests that this virus-specific immune dysfunction may play a role in the pathogenesis of this disorder.

Specificity of the inflammatory response in viral encephalitis. I. Adoptive immunization of immunosuppressed mice infected with Sindbis virus.

The viral-induced perivascular inflammatory response in Sindbis virus encephalitis of mice was shown to be immunologically specific. Mice were inoculated intracerebrally with Sindbis virus, and 24 hr later a single dose of cyclophosphamide was given which ablated the inflammatory response. 3 days after virus inoculation, cells and/or sera from specifically and nonspecifically sensitized donor mice were given, and the inflammatory reactions, virus content, and antibody response of recipients were examined 5 days later. Reconstitution of the viral inflammatory response required virus-specific sensitized lymph node cells and was enhanced when these lymph node cells were combined with bone marrow cells. Reconstitution was not achieved with Sindbis virus immune serum even when combined with nonspecifically sensitized cells. Combination of immune serum with Sindbis virus-sensitized cells did not produce an accentuation of the reaction. In distinction, reconstitution of the inflammatory reaction surrounding the stab wound was reconstituted with bone marrow cells from mice inoculated with Sindbis virus or control antigens. Reconstitution of the perivascular reaction was associated with a reduction in brain virus content. Although the transfer of Sindbis virus-sensitized lymph node cells and bone marrow cells resulted in the limited production of neutralizing antibody in the immunosuppressed recipient, the reduction in virus was significantly greater with the transfers of Sindbis virus-sensitized lymph node cells than with the passive transfer of immune serum alone.

Identification of a specific HLA DR2 Ia molecule as a restriction element for measles virus-specific HLA class II-restricted cytotoxic T cell clones.

By using a panel of HLA-D-defined subtypes of HLA-DR2 HCL with known beta chain structural variabilities, we have demonstrated that HLA-DR2, OKT4+ cytotoxic T lymphocyte (CTL) clones specific for measles virus are apparently restricted to a distinct DR beta chain. The presence of this DR beta 2 molecule correlated precisely with the susceptibility of measles virus-infected HLA-DR2 HCL to lysis by these CTL clones. These studies demonstrate that delineation of HLA-DR2 into various subgroups can have a functional significance that parallels the structural differences within the HLA-D region. These results are discussed in the context of the possible association of HLA class II-restricted, measles virus-specific CTL and multiple sclerosis.

Identification of high potency microbial and self ligands for a human autoreactive class II-restricted T cell clone.

CD4+ class II-restricted T cells specific for self antigens are thought to be involved in the pathogenesis of most human autoimmune diseases and molecular mimicry between foreign and self ligands has been implicated as a possible mechanism for their activation. In this report we introduce combinatorial peptide libraries as a powerful tool to identify cross-reactive ligands for these T cells. The antigen recognition of a CD4+ T cell clone (TCC) specific for myelin basic protein peptide (MBP) (86-96) was dissected by the response to a set of 220 11-mer peptide sublibraries. Based on the results obtained with the libraries for each position of the antigen, artificial peptides were found that induced proliferative responses at much lower concentrations than MBP(86-96). In addition stimulatory ligands derived from protein sequences of self and microbial proteins were identified, some of them even more potent agonists than MBP(86-96). These results indicate that: (a) for at least some autoreactive CD4+ T cells antigen recognition is highly degenerate; (b) the autoantigen used to establish the TCC represents only a suboptimal ligand for the TCC; (c) a completely random and unbiased approach such as combinatorial peptide libraries can decrypt the spectrum of stimulatory ligands for a T cell receptor (TCR).

A myelin basic protein peptide is recognized by cytotoxic T cells in the context of four HLA-DR types associated with multiple sclerosis.

We have examined previously the peptide specificity of the T cell response to myelin basic protein (MBP) in patients with multiple sclerosis (MS) and healthy controls, and demonstrated that an epitope spanning amino acids 87-106 was frequently recognized. Because this region is encephalitogenic in some experimental animals, it has been postulated that the response to the epitope may have relevance to MS. In this study, the fine specificity of this response is studied using four well-characterized, monospecific T cell lines from three MS patients and an identical twin of a patient. Each of the lines recognized a peptide with the same core sequence, amino acids 89-99, although the responses were affected to various degrees by truncations at the COOH- or NH2 terminal ends of the 87-106 epitope. Importantly, the epitope was recognized in conjunction with four different HLA-DR molecules. Also, the T cell receptor beta chain usage was heterogeneous, and each line expressed a different VDJ sequence. The four HLA-DR molecules restricting the response to this epitope have been shown to be overrepresented in MS populations in various geographic areas, suggesting that the response to this region of the MBP molecule may be relevant to the pathogenesis of MS. These findings may have important implications in designing therapeutic strategies for the disease.

Manganese-Enhanced MRI in Patients with Multiple Sclerosis.

BACKGROUND AND PURPOSE: Cellular uptake of the manganese ion, when administered as a contrast agent for MR imaging, can noninvasively highlight cellular activity and disease processes in both animals and humans. The purpose of this study was to explore the enhancement profile of manganese in patients with multiple sclerosis. MATERIALS AND METHODS: Mangafodipir is a manganese chelate that was clinically approved for MR imaging of liver lesions. We present a case series of 6 adults with multiple sclerosis who were scanned at baseline with gadolinium, then injected with mangafodipir, and followed at variable time points thereafter. RESULTS: Fourteen new lesions formed during or shortly before the study, of which 10 demonstrated manganese enhancement of varying intensity, timing, and spatial pattern. One gadolinium-enhancing extra-axial mass, presumably a meningioma, also demonstrated enhancement with manganese. Most interesting, manganese enhancement was detected in lesions that formed in the days after mangafodipir injection, and this enhancement persisted for several weeks, consistent with contrast coming from intracellular uptake of manganese. Some lesions demonstrated a diffuse pattern of manganese enhancement in an area larger than that of both gadolinium enhancement and T2-FLAIR signal abnormality. CONCLUSIONS: This work demonstrates the first use of a manganese-based contrast agent to enhance MS lesions on MR imaging. Multiple sclerosis lesions were enhanced with a temporal and spatial profile distinct from that of gadolinium. Further experiments are necessary to uncover the mechanism of manganese contrast enhancement as well as cell-specific uptake.

Antiserums to neurons and to oligodendroglia from mammalian brain.

Two specific antiserums were produced, one to rat neurons and one to lamb oligodendroglia isolated in bulk from brain. On the basis of immunofluorescence and absorption studies with bulk isolated cells, the antiserums were produced to specific surface components. The antiserums are useful as markers for cell identification and for studying proteins in plasma membranes.

CD4+CD28- costimulation-independent T cells in multiple sclerosis.

Multiple lines of evidence suggest that CD4+ lymphocytes initiate autoimmune responses against myelin antigens in multiple sclerosis (MS). The increased frequency of activated myelin-specific cells in MS patients indicates that the activation of autoreactive cells represents a central event in the pathogenesis of the disease. We identified a CD4+ subpopulation that is characterized phenotypically by the persistent absence of surface CD28 expression and functionally by CD28-independent activation and Th1 cytokine secretion. Owing to their costimulation-independent activation and their expression of a full agonist signaling activation pattern, CD4+CD28- cells have the potential to initiate autoimmune responses in the central nervous system, a compartment devoid of professional antigen presenting cells. Long-term memory CD4+CD28- cells produce high amounts of IFN-gamma and maximally upregulate IFN-gamma and IL-12Rbeta2 chain expression in the absence of costimulation. They exhibit prominent growth characteristics and increased survival after activation, likely related to their persistent lack of CTLA-4 surface expression. The CD4+CD28- population is expanded in a subgroup of MS patients. Myelin basic protein-specific cells detected in this cell subset may play an important role in the inflammatory response within the central nervous system.

Homologies between T cell receptor junctional sequences unique to multiple sclerosis and T cells mediating experimental allergic encephalomyelitis.

The selection of T cell clones with mutations in the hypoxanthine guanine phosphoribosyltransferase (hprt) gene has been used to isolate T cells reactive to myelin basic protein (MBP) in patients with multiple sclerosis (MS). These T cell clones are activated in vivo, and are not found in healthy individuals. The third complementarity determining regions (CDR3) of the T cell receptor (TCR) alpha and beta chains are the putative contact sites for peptide fragments of MBP bound in the groove of the HLA molecule. The TCR V gene usage and CDR3s of these MBP-reactive hprt-T cell clones are homologous to TCRs from other T cells relevant to MS, including T cells causing experimental allergic encephalomyelitis (EAE) and T cells found in brain lesions and in the cerebrospinal fluid (CSF) of MS patients. In vivo activated MBP-reactive T cells in MS patients may be critical in the pathogenesis of MS.

Treatment of experimental encephalomyelitis with a novel chimeric fusion protein of myelin basic protein and proteolipid protein.

It has been shown that peripheral T cell tolerance can be induced by systemic antigen administration. We have been interested in using this phenomenon to develop antigen-specific immunotherapies for T cell-mediated autoimmune diseases. In patients with the demyelinating disease multiple sclerosis (MS), multiple potentially autoantigenic epitopes have been identified on the two major proteins of the myelin sheath, myelin basic protein (MBP) and proteolipid protein (PLP). To generate a tolerogenic protein for the therapy of patients with MS, we have produced a protein fusion between the 21.5-kD isoform of MBP (MBP21.5) and a genetically engineered form of PLP (deltaPLP4). In this report, we describe the effects of treatment with this agent (MP4) on clinical disease in a murine model of demyelinating disease, experimental autoimmune encephalomyelitis (EAE). Treatment of SJL/J mice with MP4 after induction of EAE either by active immunization or by adoptive transfer of activated T cells completely prevented subsequent clinical paralysis. Importantly, the administration of MP4 completely suppressed the development of EAE initiated by the cotransfer of both MBP- and PLP-activated T cells. Prevention of clinical disease after the intravenous injection of MP4 was paralleled by the formation of long-lived functional peptide-MHC complexes in vivo, as well as by a significant reduction in both MBP- and PLP-specific T cell proliferative responses. Mice treated with MP4 were resistant to disease when rechallenged with an encephalitogenic PLP peptide emulsified in CFA, indicating that MP4 administration had a prolonged effect in vivo. Administration of MP4 was also found to markedly ameliorate the course of established clinical disease. Finally, MP4 therapy was equally efficacious in mice defective in Fas expression. These results support the conclusion that MP4 protein is highly effective in suppressing disease caused by multiple neuroantigen epitopes in experimentally induced demyelinating disease.

Immunodominance of a low-affinity major histocompatibility complex-binding myelin basic protein epitope (residues 111-129) in HLA-DR4 (B1*0401) subjects is associated with a restricted T cell receptor repertoire.

The pathogenesis of multiple sclerosis (MS) is currently ascribed in part to a T cell-mediated process targeting myelin components. The T cell response to one candidate autoantigen, myelin basic protein (MBP), in the context of HLA-DR15Dw2, has been previously studied in detail. However, the characteristics of cellular immunity in the context of other MS-associated HLA-DR haplotypes are scarcely known. MBP-specific T cell lines (TCL) were generated from HLA-DR4 (B1*0401)-positive MS subjects. Out of 275 MBP-specific TCL, 178 (64. 7%) specifically recognized region MBP(111-129), predominantly in the context of DRB1*0401. The major T cell epitope for MBP recognition corresponded to residues MBP(116-123). These TCL expressed disparate profiles of cytokine secretion and cytotoxicity. T cell receptor analysis, on the other hand, revealed a strikingly limited heterogeneity of rearrangements. In contrast to MBP(81-99), which binds with high affinity to HLA-DR15 and is recognized by a diverse T cell repertoire, MBP(111-129) binds weakly to DRB1*0401, suggesting that only high affinity T cell receptors might be able to efficiently engage such unstable MHC/peptide complexes, thus accounting for the T cell receptor restriction we observed. This study provides new insight about MBP recognition and proposes an alternative mechanism for immunodominance of self-antigen T cell epitopes in humans.

In vivo detection of cortical plaques by MR imaging in patients with multiple sclerosis.

BACKGROUND AND PURPOSE: In vivo detection of cortical lesions in patients with multiple sclerosis (MS) by MR imaging is hampered by several factors. Among them is the low contrast between small cortical lesions and surrounding cortical gray matter offered by present techniques. METHODS: T1-weighted 3D spoiled gradient-recalled-echo (SPGR) volumes and 2D fluid-attenuated inversion recovery (FLAIR) sequences of 22 patients with MS who had 12 monthly brain MR imaging examinations at 1.5T, using a quadrature head coil, were retrospectively analyzed. These serial studies were coregistered and averaged to generate a single high signal-to-noise ratio (SNR) mean image, which was used to identify cortical lesions. The means of 12 FLAIRs and SPGRs from 14 age- and sex-matched healthy volunteers were analyzed as well. RESULTS: No cortical lesions were found on images of healthy subjects. Eighty-six cortical lesions were identified in 13 (59.1%) patients, predominantly in the frontal lobe (73.3%); 23.3% of cortical lesions lay entirely in the cortex, whereas the remaining lesions invaded the white matter underneath. CONCLUSION: Averaging multiple SPGRs created a single high SNR volume, allowing identification of cortical lesions. Because data were obtained monthly for 1 year, the average image does not account for transient lesion activity. However, for cortical lesions that remained stable during this time, the findings are valid in demonstrating the importance of high SNR images for detecting cortical brain abnormalities in MS.

The role of T cells in multiple sclerosis: implications for therapies targeting the T cell receptor.

The cause of multiple sclerosis (MS) is unknown, but an immunopathological process with both endogenous and exogenous factors contributing to disease seems likely. Considerable recent attention, triggered predominantly by findings in the animal model, experimental allergic encephalomyelitis (EAE), which resembles MS, has focused on the role of T cells in MS. Findings in the animal model have raised the possibility that demyelination could be produced by CD4+ T cells specific for myelin proteins and expressing a limited set of T cell receptor (TCR) molecules. Thus, specific therapies targeting T cells or more specifically the TCR could represent an effective treatment of MS as has been demonstrated in EAE. However, current studies of patients with MS indicate that the immunological mechanisms in MS are considerably more complicated than in EAE. The evidence for a pivotal role for T cells in MS and the characteristics of these T cells particularly with respect to TCR usage and potential for therapies directed at the TCR will be examined in this review.

Chronic relapsing experimental autoimmune encephalomyelitis: effects of insulin-like growth factor-I treatment on clinical deficits, lesion severity, glial responses, and blood brain barrier defects.

Chronic relapsing experimental autoimmune encephalomyelitis (crEAE), a model for multiple sclerosis, was used to test 2 regimens of insulin-like growth factor-I (IGF-I) treatment. We induced crEAE by injecting 3x10(7) myelin basic protein-(MBP) sensitized lymph node cells into adult female SJL/J mice. Fifty-one mice, divided randomly into 4 groups, were used in the first trial. Two groups received IGF-I (a gift of Cephalon, Inc.) 0.6 mg/kg/d subcutaneously from day 7 to day 16 and the other two groups received placebo injections. IGF-I treatment reduced clinical deficits during the first attack and during 2 subsequent relapses. Image analysis of immunostained and histological sections showed that IGF-I treatment reduced BBB defects and both the numbers and sizes of inflammatory, demyelinating, and demyelinated lesions. Twelve mice that had recovered from their first attack were used in our second trial to evaluate possible adverse effects of prolonged treatment with a higher dose of IGF-I. Six received 1.2 mg/kg/d for 6 weeks (days 19-63). No adverse effects of IGF-I treatment were identified. The eyes, hearts, livers, and kidneys of IGF-I-treated mice were normal histologically and their spleens also appeared normal except for mild to moderate microscopic increases in lymphopoesis. Our results suggest that prolonged IGF-I treatment is well tolerated and that the anti-inflammatory effects of IGF-I have a major role in reducing clinical deficits and lesion severity in crEAE. These effects, if present in multiple sclerosis, may benefit patients with this disease.

Cerebrospinal fluid somatostatin, mood, and cognition in multiple sclerosis.

BACKGROUND: Cerebrospinal fluid (CSF) somatostatin (SS) levels have been shown to be decreased in multiple sclerosis (MS) during relapse as well as in disorders characterized by depression or cognitive impairment. Since MS is often associated with depression and cognitive impairment, we examined both the effect of course of illness on CSF SS as well as the variance in SS attributable to associated features (e.g., depression or cognitive impairment). METHODS: Fifteen patients with chronic progressive MS participating in a 2-year cyclosporine trial underwent lumbar punctures for CSF SS at baseline and at 12 and 24 months. Additionally, patients were evaluated by neuropsychological testing, and physical disability and mood ratings. Baseline CSF SS levels were also obtained in a group of control subjects (n = 10). RESULTS: At baseline, CSF SS levels were lower in MS patients than control subjects (p < .001). Decreased CSF SS at 24 months was correlated with decreased cognitive performance on several measures and was best and significantly predicted by cognitive deterioration at 24 months. CONCLUSIONS: Our data support those from previous studies that found lower levels of CSF SS in MS during relapse and suggest that changes in CSF SS are related to the process responsible for diminished cognitive function in MS.

Amusia due to right temporoparietal infarct.

An accomplished organist, who could neither read nor write music, suddenly lost his ability to play familiar melodies as the result of an infarction of the right superior temporal and supramarginal gyri. Although the patient had minor difficulty recognizing familiar melodies and coordinating his hands, he had mainly an expressive instrumental amusia. This case provides further evidence of right hemispheric dominance for musical execution, relatively independent of musical knowledge and training.