Association of human herpesvirus 6 with the demyelinative lesions of progressive multifocal leukoencephalopathy.

Progressive Multifocal Leukoencephalopathy (PML) is a primary demyelinating disease of the central nervous system occurring almost exclusively in individuals with impaired cell-mediated immunity. The JC polyoma virus has been accepted as the etiologic agent ofPML. Using a two-step in-situ polymerase chain reaction procedure to amplify and detect genomic DNA of human herpesvirus-6 (HHV6) in formalin-fixed paraffin-embedded archival brain tissues, a high frequency of infected cells was consistently detected in PML white matter both within and surrounding demyelinative lesions and HHV6 genome was found mainly within oligodendrocytes. Lesser amounts of HHV6 genome were detected in most normal, AIDS, and other neurological disease control tissues. Immunocytochemistry for HHV6 antigens showed actively infected nuclei of swollen oligodendrocytic morphology only within the demyelinative lesions of PML but not in adjacent uninvolved tissue. In addition, no HHV6 antigens were detectable in control tissues including brains of individuals with HIV-1 encephalopathy but without PML. Double immunohistochemical staining for JC virus large T antigen and HHV6 antigens demonstrated co-labeling of many swollen intralesional oligodendrocytes in the PML cases. The evidence suggests that HHV6 activation in conjunction with JC virus infection is associated with the demyelinative lesions of PML.

Interferon-beta-1-b (IFN-B) decreases induced nitric oxide (NO) production by a human astrocytoma cell line.

Inducible nitric oxide synthase (iNOS) is expressed by astrocytes in demyelinating regions of multiple sclerosis (MS) brain plaques, suggesting that NO contributes to MS pathology. Since the immunosuppressive cytokine IFN-B ameliorates MS disease activity, it is of interest to assess the modulatory role of IFN-B on NO production. We studied the effects of IFN-B, as well as dexamethasone, IL-10, and transforming growth factor-beta (TGF-B), on cytokine-induced NO production by the human astrocytoma cell line, A172. L-NMMA and aminoguanidine, competitive inhibitors of iNOS suppressed NO production as measured by the NO byproduct, nitrite, as did IFN-B. Dexamethasone enhanced NO production, and IFN-B decreased the amount of the enhancement. Neither IL-10 nor TGF-B inhibited nitrite production. The therapeutic effect of IFN-B in MS may be partly due to suppression of pathogenic NO production.

The measurement of ambulatory impairment in multiple sclerosis.

The objective of this study was to examine the relationships between continuous measures of ambulatory impairment in MS patients and their ordinal counterparts. Much of the disability caused by MS is due to ambulatory impairment. The Expanded Disability Severity Scale (EDSS) and the Ambulation Index (AI) are ordinal measures of MS severity based largely on the maximal distance subjects can walk (Dmax) and the time to walk 8 m (T8), respectively. At EDSS levels 6.0 to 7.0 and AI levels 3 to 6, scores are defined more by the use of ambulatory aids, rather than by Dmax or T8. We determined Dmax (up to 500 m), T8, the EDSS score, and the AI in 237 ambulatory MS patients. The maximal distance subjects could walk and T8 were strongly related to their ordinal counterparts (Spearman r = 0.65 and 0.91, respectively), but the continuous measures showed considerable variability within EDSS and AI levels that the ordinal scales did not reflect. Most of the variability occurred at EDSS levels 6.0 to 7.0 and AI levels 3 to 6. Because the use of an aid did not clearly predict Dmax or T8, many patients in these ranges had better ambulatory function based on the continuous measures than those with less disability according to the ordinal scales. We found that Dmax and T8 provide more precise information about ambulatory impairment in MS than do the EDSS and AI, allowing better discrimination of differences between patients and potentially greater sensitivity to detect therapeutic effects in clinical trials.

Autoimmune hyperthyroidism in patients with multiple sclerosis treated with interferon beta-1b.

OBJECTIVE: To report symptomatic autoimmune hyperthyroidism developing in patients with multiple sclerosis (MS) treated with interferon beta-1b (IFN-beta-1b). REPORT OF CASES: A 44-year-old woman experienced gradually worsening fatigue, depression, and motor function several months after beginning therapy with IFN-beta-1b for MS. Graves disease associated with episodic palpitations, shortness of breath, hair loss, increased appetite, weight loss, and insomnia was confirmed with endocrinologic studies. Increased fatigue and weakness developed in a 52-year-old woman several months after starting IFN-beta-1b therapy. She also noted sweats, heat intolerance, palpitations, increased appetite, and irritability, and endocrinologic evaluation supported a diagnosis of Graves disease. CONCLUSIONS: To our knowledge, this is the first report of autoimmune thyroid disease associated with IFN-beta-1b treatment in patients with MS. Psoriasis has also been reported during interferon therapy for MS, and similar phenomena occur during interferon therapy for hepatitis C. Since some symptoms of thyroid dysfunction may be difficult to distinguish from typical MS-related symptoms, thyroid hormone levels should be checked when unexplained constitutional symptoms occur during IFN-beta-1b therapy.

Frequency and significance of antinuclear antibodies in multiple sclerosis.

In a prospective sample of patients with multiple sclerosis (MS) we found a high frequency of antinuclear antibodies (ANA), 22.5%, confirming results from previous studies. ANA occurrence did not correlate with gender, age, duration of MS, MS course, or disability in either the prospective and retrospective samples of MS patients. In 16 patients with MS tested at two time points, ANA occurrence did correlate with MS disease activity. This suggests that the high frequency of ANA in MS reflects ongoing immune dysregulation.

Analysis of a sequenced cDNA library from multiple sclerosis lesions.

To identify genes that are expressed in MS pathogenesis, we have analyzed a normalized cDNA library made from mRNA obtained from CNS lesions of a patient with primary progressive MS. Complementary DNA clones obtained from this library were subjected to automated DNA sequencing to generate expressed sequence tags. Analysis of this MS cDNA library revealed the presence of 54 cDNAs that were associated with immune activation and indicated the presence of an ongoing inflammatory response with evidence of both cell-mediated and humoral immune responses. The surprising finding was that 16 of the cDNAs encoded autoantigens associated with seven other autoimmune disorders, while only three of these 16 autoantigen cDNAs were present in a similarly constructed adult brain library. Such aberrant autoantigen expression could provide a source of secondary autoimmune stimulation that could contribute to the ongoing inflammatory response in MS. In addition, two cDNAs were found that mapped to a known MS susceptibility locus (5p14-p12): one encoded an excitatory amino acid transporter and the other a human homologue of the Drosophila disabled gene. This approach to the molecular biology of MS pathogenesis may help to illuminate previously unappreciated aspects of this disease.

The cost-effectiveness of magnetic resonance imaging for patients with equivocal neurological symptoms.

OBJECTIVE: To determine the incremental cost-effectiveness of magnetic resonance imaging (MRI) and computed tomography (CT) in young adults presenting with equivocal neurological signs and symptoms. DESIGNS AND METHODS: A decision analysis of long-term survival using accuracy data from a diagnostic technology assessment of MRI and CT in patients with suspected multiple sclerosis, information from the medical literature, and clinical assumptions. MAIN RESULTS: In the baseline analysis, at 30% likelihood of an underlying neurologic disease, MRI use has an incremental cost of $101,670 for each additional quality-adjusted life-year saved compared with $20,290 for CT use. As the probability of disease increases, further MRI use becomes a cost-effective alternative costing $30,000 for each quality-adjusted life-year saved. If a negative MRI result provides reassurance, the incremental costs of immediate MRI use decreases and falls below $25,000 for each quality-adjusted life-year saved no matter the likelihood of disease. CONCLUSIONS: For most individuals with neurological symptoms or signs, CT imaging is cost-effective while MR imaging is not. The cost-effectiveness of MRI use, however, improves as the likelihood of an underlying neurological disease increases. For selected patients who highly value diagnostic information, MRI is a reasonable and cost-effective use of medical resources when even the likelihood of disease is quite low (5%).

Sporadic corticosteroid pulses and osteoporosis in multiple sclerosis.

BACKGROUND: Bone mineral density is reduced in patients with multiple sclerosis (MS), but the reduction has not been shown to correlate with steroid use retrospectively. OBJECTIVE: To prospectively measure bone density following a single corticosteroid pulse using dual energy x-ray absorptiometry. PATIENTS AND METHODS: Thirty acutely relapsing patients with MS were given 1000 mg of methylprednisolone intravenously daily for 3 days followed by an oral prednisone taper for 2 weeks. The bone density was determined at the lumbar spine and femoral neck prior to treatment. Seventeen patients were reevaluated 2,4, and 6 months following treatment. RESULTS: Prior to treatment, bone density in patients with MS was already reduced at the femoral neck compared with an age-matched reference population, but the degree of this reduction did not correlate with prior steroid exposure. Lumbar density, in contrast, was normal. Following the steroid pulse, lumbar bone density increased, becoming 1.7% greater than baseline 6 months later (P = .02). Femoral bone density did not change on average, but the patients who required a cane or walker for ambulation had a 1.6% decrease in femoral bone density, while those with better ambulation had a 2.9% increase (P = .04). CONCLUSIONS: Bone density is decreased in MS. A single corticosteroid pulse did not reduce bone density in fully ambulatory patients with MS and multiple pulses did not have a cumulative effect on bone density in retrospective analysis. The change in femoral density in poorly ambulatory patients may have been related to inactivity rather than the steroid pulse.

Conditioning of cyclophosphamide-induced leukopenia in humans.

This study evaluated whether decrements in peripheral leukocyte counts induced by cyclophosphamide can be conditioned in humans. Ten subjects being treated for multiple sclerosis received four intravenous treatments with cyclophosphamide (unconditioned stimulus) paired with a conditioned stimulus. Subjects received conditioned stimulus plus 10 mg of cyclophosphamide during one of the next two treatments in a double-blind manner. Eight of 10 subjects (P = 0.044) displayed decreased peripheral leukocyte counts following conditioned stimulus. This change may have resulted from classical conditioning processes.

Frequency of anti-nuclear antibodies in multiple sclerosis.

We found anti-nuclear antibodies (ANA) in 26.7% of 150 relapsing-remitting and in 30.4% of 23 chronic progressive definite multiple sclerosis (MS) patients by retrospective chart review. These patients did not have systemic lupus erythematosus. Since ANA are not pathogenically relevant in MS, they are false-positive, and likely reflect systemic immune dysregulation in MS.

Oligodendrocyte-specific expression and autoantigenicity of transaldolase in multiple sclerosis.

Although the etiology of multiple sclerosis (MS) is unknown, there is compelling evidence that its pathogenesis is mediated through the immune system. Molecular mimicry, i.e., crossreactivity between self-antigens and viral proteins, has been implicated in the initiation of autoimmunity and MS. Based on homology to human T cell lymphotropic virus type I (HTLV-I) a novel human retrotransposon was cloned and found to constitute an integral part of the coding sequence of the human transaldolase gene (TAL-H). TAL-H is a key enzyme of the nonoxidative pentose phosphate pathway (PPP) providing ribose-5-phosphate for nucleic acid synthesis and NADPH for lipid biosynthesis. Another fundamental function of the PPP is to maintain glutathione at a reduced state and, consequently, to protect sulfhydryl groups and cellular integrity from oxygen radicals. Immunohistochemical analyses of human brain sections and primary murine brain cell cultures demonstrated that TAL is expressed selectively in oligodendrocytes at high levels, possibly linked to production of large amounts of lipids as a major component of myelin, and to the protection of the vast network of myelin sheaths from oxygen radicals. High-affinity autoantibodies to recombinant TAL-H were detected in serum (25/87) and cerebrospinal fluid (15/20) of patients with MS. By contrast, TAL-H antibodies were absent in 145 normal individuals and patients with other autoimmune and neurological diseases. In addition, recombinant TAL-H stimulated proliferation and caused aggregate formation of peripheral blood lymphocytes from patients with MS. Remarkable amino acid sequence homologies were noted between TAL-H and core proteins of human retroviruses. Presence of crossreactive antigenic epitopes between recombinant TAL-H and HTLV-I/human immunodeficiency virus type 1 (HIV-1) gas proteins was demonstrated by Western blot analysis. The results suggest that molecular mimicry between viral core proteins and TAL-H may play a role in breaking immunological tolerance and leading to a selective destruction of oligodendrocytes in MS.

Clinical diagnosis of multiple sclerosis. The impact of magnetic resonance imaging and ancillary testing. Rochester-Toronto Magnetic Resonance Study Group.

OBJECTIVE: Magnetic resonance imaging, computed tomography, cerebrospinal fluid analysis, and evoked potential testing are used to assist in the diagnosis of patients suspected to have multiple sclerosis (MS). The impact of these tests on a clinician's diagnosis of patients suspected to have MS has not been studied systematically. DESIGN: Clinicians made a diagnosis of each patient following clinical evaluation, again after reviewing the results of magnetic resonance imaging, and finally after reviewing information from other laboratory testing. These diagnoses were compared with the criterion standard of a masked "gold standard" panel reviewing all information after a mean follow-up of 0.9 year. SETTING: The General Neurology Clinic and Multiple Sclerosis Clinic of the University of Rochester (NY). PATIENTS: A consecutive sample of 62 patients diagnosed as having either possible or probable MS following clinical evaluation. MAIN OUTCOME MEASURE: Changes in diagnostic certainty of clinicians following incremental presentation of new laboratory data and the accuracy of such diagnoses. RESULTS: Clinicians used magnetic resonance imaging findings to diagnose definite MS or to eliminate MS from diagnostic consideration in 44% of cases. In these cases, further laboratory testing did not alter clinicians' decisions. In the remaining 56% of cases, in which magnetic resonance imaging did not lead to a diagnosis of definite MS or eliminate MS from diagnostic consideration, further laboratory testing led to such diagnoses in an additional 13% of cases. Gold standard diagnoses were in agreement with the clinician's assessments. CONCLUSIONS: Magnetic resonance imaging aids in the evaluation of patients suspected to have MS; other subsequent studies (computed tomography, cerebrospinal fluid analysis, and evoked potential testing) have less impact. After all studies are performed, about half of such patients still have a tentative diagnosis.

Perils and pitfalls of magnetic resonance imaging in the diagnosis of multiple sclerosis. The Rochester-Toronto MRI Study Group.

Purpose. Magnetic resonance imaging (MRI) has come to assume a position of major importance in the diagnostic process for multiple sclerosis (MS). The authors believe that a tendency toward overreliance on MRI results in isolation from clinical findings continues to result in both false-positive and false-negative diagnostic errors. Methods. To evaluate this, MRI results in newly referred patients with clinical findings suggestive, but not diagnostic, for MS, were studied prospectively. Results. Of 99 consecutive referrals for suspected MS, there were 3 false-positive diagnoses of MS and 7 false-negatives, when the MRIs were read in isolation from specific clinical data. None of the scans in the false-negative groups were normal. Representative images of both groups are provided. Conclusion. In newly referred patients who fall short of criteria for definite MS, it remains dangerous for both clinicians and radiologists to rely too heavily only on MRI results.

Human T-cell lymphotropic virus (HTLV)-related endogenous sequence, HRES-1, encodes a 28-kDa protein: a possible autoantigen for HTLV-I gag-reactive autoantibodies.

The presence of a human T-cell lymphotropic virus (HTLV)-related endogenous sequence, HRES-1, in the human genome has been documented. The HRES-1 genomic locus is transcriptionally active and contains open reading frames. Antibodies 232 and 233, specific for synthetic peptides pep14-24 and pep117-127, corresponding to two nonoverlapping HTLV-related regions in the longer open reading frame of HRES-1, recognize an identical 28-kDa protein in H9 human T cells. Thus, HRES-1 is a human endogenous retroviral sequence capable of protein expression. HRES-1/p28 is localized to the cytoplasm and nuclear bodies. While HTLV-I-specific antibodies react with HRES-1 peptides, antibody 233 cross-reacts with HTLV-I gag p24 protein. Three consecutive highly charged amino acid residues, Arg-Arg-Glu, present in both HRES-1 pep117-127 and HTLV-I gag p24 are likely to be the core of cross-reactive epitopes. The prevalence of antibodies to HRES-1 peptides pep14-24 and pep117-127 was determined in 65 normal blood donors and 146 patients with immunological disorders. Sera of patients with multiple sclerosis (19 out of 65, 29%), progressive systemic sclerosis (4 out of 17, 23%), systemic lupus erythematosus (4 out of 19, 21%), and Sjogren syndrome (2 out of 19, 10%) contained significantly higher HRES-1 peptide binding activity than sera of normal donors. Sera of patients with AIDS showed no specific binding to HRES-1 peptides. Nine of 30 HRES-1-seropositive patients showed immunoreactivity to HTLV-I gag p24. The data indicate that HRES-1/p28 may serve as an autoantigen eliciting autoantibodies cross-reactive with HTLV-I gag antigens.

The kinetics of peptide binding to HLA-A2 and the conformation of the peptide-A2 complex can be determined by amino acid side chains on the floor of the peptide binding groove.

The ability of amino acid side chains in the floor of the peptide binding groove of HLA-A2 to affect the presentation of a viral peptide to peptide-specific cytotoxic T lymphocytes (CTL) has been examined. HLA-A2 molecules with naturally occurring single amino acid substitutions of Phe to Tyr at position 9 (HLA-A2.4a, Tyr9) and Tyr to Cys at position 99 (HLA-A2.4b, Cys99) and a site directed mutant with a Val to Leu substitution at position 95 (Leu95) were examined for their ability to present the influenza virus matrix M1 55-73 peptide and several sequence variants of the M1 peptide to a panel of 36 M1 55-73-specific HLA-A2.1-restricted CTL lines. The Leu95 molecule demonstrated enhanced kinetics of M1 peptide presentation and the ability to be sensitized by lower concentrations of the M1 peptide than the A2.1 molecule. The Tyr9 and Cys99 molecules exposed to M1 peptide were not recognized by 33 out of 36 CTL lines. The Tyr9 and Cys99 HLA-A2 molecules could bind the M1 55-73 peptide because at least one CTL line was found that could recognize each of these molecules that were exposed to the M1 peptide. CTL recognition patterns of variant M1 peptides presented by the Tyr9 molecule demonstrated that the amino acid at position 9 can be a critical determinant of the conformation of the peptide-A2 complex, and indicated that a particular peptide can bind in the HLA-A2 peptide binding groove in more than one conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of amino acid substitutions in the beta-sheet floor and alpha-2 helix of HLA-A2 on recognition by alloreactive viral peptide-specific cytotoxic T lymphocytes.

Crystallographic studies of the HLA-A2 molecule have led to the assignment of a putative peptide binding site that consists of a groove with a beta-pleated sheet floor bordered by two alpha-helices. A CTL-defined variant of HLA-A2, termed HLA-A2.2F, differs from the common A2.1 molecule by three amino acids: a Leu to Trp substitution at position 156 in the alpha-2 helix, a Val to Leu substitution at position 95 in the beta-sheet floor of the groove, and a Gln to Arg substitution at position 43 in a loop outside of the groove. Another HLA-A2 variant, termed CLA, has a single Phe to Tyr substitution at position 9 that is sterically located adjacent to position 95 in the beta-sheet floor of the groove. We have determined which of the amino acid substitutions at positions 9, 43, 95, or 156 could individually affect recognition by panels of A2.1 allospecific and A2.1-restricted influenza viral matrix peptide-specific CTL lines, using a panel of site-directed mutants and CLA. Recognition by allospecific CTL lines was generally unaffected by any one of the amino acid substitutions, but was eliminated by the double substitution at positions 95 and 156. Allorecognition by some CTL lines was eliminated by a single substitution at position 9 or 95. In contrast, recognition by A2.1-restricted matrix peptide specific CTL was totally eliminated by a single substitution at position 9 or 156. The substitution at position 43 in a loop away from the peptide binding groove had no effect on allorecognition or matrix peptide recognition. These results indicate that amino acid residues in the floor or alpha-2 helical wall of the peptide binding groove of the HLA-A2 molecule can differentially affect allorecognition and viral peptide recognition.

Beta-chain gene rearrangements and V beta gene usage in DPw2-specific T cells.

The diversity of human T cell receptor beta-chain gene rearrangements and variable region gene usage in the T cell response to a single allogeneic class II HLA gene product has been investigated. Nine clones of cytotoxic T lymphocytes (8.2 to 8.10) that are specific for the class II specificity DPw2 were analyzed for their T cell receptor beta-chain gene rearrangements using a constant-region probe. A minimum of seven different clonotypes were present in this panel of clones. The beta-gene expressed by one clone, 8.9, was isolated and the variable (V), diversity (D), and joining (J) segments were sequenced. The sequence of the V beta 8.9 segment is identical to the V beta 14 sequence, and is joined to D beta 1.1 and J beta 1.1 segments. Northern analysis revealed that three of the eight clones analyzed, 8.5, 8.7, and 8.9, expressed a 1.3 kb transcript that hybridized with the V beta 8.9 probe, indicating that these clones were using the same V beta gene (these clones shared common DNA rearrangements). The remaining five clones did not express V beta 8.9. Southern analysis of DNA obtained from a DPw2-specific tertiary mixed lymphocyte reaction bulk culture from which the clones were derived showed a prominent rearrangement of the V beta 8.9 gene that was indistinguishable from those observed for clones 8.5, 8.7, and 8.9. This prominent rearrangement of V beta 8.9 was not observed in DNA obtained from normal peripheral blood lymphocytes. These results suggest that although the number of V beta genes which can contribute to a DPw2 specificity may be relatively large, only a limited number of clonotypes ultimately predominate in the response to certain class II HLA antigens.