James M. Killian

DNA duplication associated with Charcot-Marie-Tooth disease type 1A

Charcot-Marie-tooth disease type 1A (CMT1A) was localized by genetic mapping to a 3 cM interval on human chromosome 17p. DNA markers within this interval revealed a duplication that is completely linked and associated with CMT1A. The duplication was demonstrated in affected individuals by the presence of three alleles at a highly polymorphic locus, by dosage differences at RFLP alleles, and by two-color fluorescence in situ hybridization. Pulsed-field gel electrophoresis of genomic DNA from patients of different ethnic origins showed a novel SacII fragment of 500 kb associated with CMT1A. A severely affected CMT1A offspring from a mating between two affected individuals was demonstrated to have this duplication present on each chromosome 17. We have demonstrated that failure to recognize the molecular duplication can lead to misinterpretation of marker genotypes for affected individuals, identification of false recombinants, and incorrect localization of the disease locus.

Multi‐disciplinary clinical study of Smith‐Magenis syndrome (deletion 17p11.2)

Smith-Magenis syndrome (SMS) is a multiple congenital anomaly, mental retardation (MCA/MR) syndrome associated with deletion of chromosome 17 band p11.2. As part of a multi-disciplinary clinical, cytogenetic, and molecular approach to SMS, detailed clinical studies including radiographic, neurologic, developmental, ophthalmologic, otolaryngologic, and audiologic evaluations were performed on 27 SMS patients. Significant findings include otolaryngologic abnormalities in 94%, eye abnormalities in 85%, sleep abnormalities (especially reduced REM sleep) in 75%, hearing impairment in 68% (approximately 65% conductive and 35% sensorineural), scoliosis in 65%, brain abnormalities (predominantly ventriculomegaly) in 52%, cardiac abnormalities in at least 37%, renal anomalies (especially duplication of the collecting system) in 35%, low thyroxine levels in 29%, low immunoglobulin levels in 23%, and forearm abnormalities in 16%. The measured IQ ranged between 20-78, most patients falling in the moderate range of mental retardation at 40-54, although several patients scored in the mild or borderline range. The frequency of these many abnormalities in SMS suggests that patients should be evaluated thoroughly for associated complications both at the time of diagnosis and at least annually thereafter.

Increased CD8+ Cytotoxic T Cell Responses to Myelin Basic Protein in Multiple Sclerosis

Autoreactive T cells of CD4 and CD8 subsets recognizing myelin basic protein (MBP), a candidate myelin autoantigen, are thought to contribute to and play distinct roles in the pathogenesis of multiple sclerosis (MS). In this study we identified four MBP-derived peptides that had high binding affinity to HLA-A2 and HLA-A24 and characterized the CD8+ T cell responses and their functional properties in patients with MS. There were significantly increased CD8+ T cell responses to 9-mer MBP peptides, in particular MBP111–119 and MBP87–95 peptides that had high binding affinity to HLA-A2, in patients with MS compared with healthy individuals. The resulting CD8+ T cell lines were of the Th1 phenotype, producing TNF-α and IFN-γ and belonged to a CD45RA−/CD45RO+ memory T cell subset. Further characterization indicated that the CD8+ T cell lines obtained were stained with MHC class I tetramer (HLA-A2/MBP111–119) and exhibited specific cytotoxicity toward autologous target cells pulsed with MBP-derived peptides in the context of MHC class I molecules. These cytotoxic CD8+ T cell lines derived from MS patients recognized endogenously processed MBP and lysed COS cells transfected with genes encoding MBP and HLA-A2. These findings support the potential role of CD8+ CTLs recognizing MBP in the injury of oligodendrocytes expressing both MHC class I molecules and MBP.

Interferon beta induces T-helper 2 immune deviation in MS

Objective: To define the in vitro effects of interferon beta 1a (IFN-β1a) on myelin basic protein (MBP)-reactive T cells and to determine its regulatory mechanism on cytokine networks in patients with MS. Methods: The proliferation and cytokine production of MBP-reactive T-cell clones were measured in thymidine uptake assays and ELISA respectively. The precursor frequency of MBP-reactive T cells was estimated in a microwell culture system. Results: IFN-β inhibited the proliferation of established MBP-reactive T-cell clones, which correlated with enhanced production of anti-inflammatory interleukin (IL)-4 and IL-10, and a decrease in tumor necrosis factor alpha (TNF-α) and IFN-γ. When examined with peripheral blood mononuclear cells (PBMCs), IFN-β was found to reduce the in vitro T-cell responses to MBP, as indicated by the significantly decreased frequency of MBP-reactive T cells. The decreased frequency of MBP-reactive T cells corresponded to an augmented production of IL-4 and IL-10. Although the level of TNF-α and IFN-γ was generally unaltered or decreased, IFN-β appeared to enhance the production of IFN-γ in PBMCs derived from some individuals with MS. Conclusion: Interferon beta 1a (IFN-β) suppresses myelin basic protein (MBP)-reactive T cells and induces immune deviation toward the production of T-helper 2 cytokines, which may contribute to its therapeutic benefit in MS. The study also suggests some heterogeneity in MBP-reactive T-cell responses to IFN-β in different individuals with MS.

IMPAIRED APOPTOTIC DELETION OF MYELIN BASIC PROTEIN-REACTIVE T CELLS IN PATIENTS WITH MULTIPLE SCLEROSIS

T cell responses to myelin basic protein (MBP) may play an important role in the pathogenesis of multiple sclerosis (MS). If MBP‐reactive T cells are involved in the disease processes and undergo clonal activation and expansion, their precursor frequency would be increased in patients with MS. The frequency of MBP‐reactive T cells is also influenced by regulatory mechanisms in vivo, including apoptotic deletion. In this study, we examined changes in the frequency of MBP‐reactive T cells in patients with MS as a function of the apoptotic deletional mechanism in vivo, using a cell culture‐based assay. A significantly increased frequency of MBP‐reactive T cells was found in patients with MS relative to healthy individuals only when Fas‐ligand antibody was used to block apoptosis. This result indicates that a significant proportion of MBP‐reactive T cells are sensitive to apoptosis and are not deleted in vivo in patients with MS, as opposed to healthy individuals, thus suggesting a functional deficit in apoptotic deletional mechanism. Surviving Fas‐sensitive MBP‐reactive T cell lines represent distinct subpopulations preferentially recognizing the 111–139 region of MBP and exhibiting a Th2 cytokine profile. The findings are relevant to our understanding of regulation of MBP‐reactive T cells in vivo in MS.

Familial tic disorder, parkinsonism, motor neuron disease, and acanthocytosis A new syndrome

We report two brothers who were of consanguineous parents and who displayed a unique association of motor and vocal tics, parkinsonism, distal muscular atrophy, and acanthocytosis. In the older brother, leg weakness and muscle wasting started at age 13, and he became wheelchair bound at 40. Electrophysiologic studies and muscle biopsy confirmed diffuse denervation. Involuntary vocalizations and facial tics began at age 36, but within 5 years the tics were replaced by progressive parkinsonism with supranuclear ophthalmoparesis. CSF studies implied impaired central dopamine and serotonin turnover. In the younger brother, orofacial tics started at age 36, vocalizations and fasciculations in the legs began 1 year later, and parkinsonian findings were present at age 40. This is the first report of an association of Tourettism, parkinsonism, motor neuron disease, and acanthocytosis occurring as an autosomal recessive syndrome.

Regulatory effects of IFN-β on production of osteopontin and IL-17 by CD4+ T Cells in MS

IFN‐β currently serves as one of the major treatments for MS. Its anti‐inflammatory mechanism has been reported as involving a shift in cytokine balance from Th1 to Th2 in the T‐cell response against elements of the myelin sheath. In addition to the Th1 and Th2 groups, two other important pro‐inflammatory cytokines, IL‐17 and osteopontin (OPN), are believed to play important roles in CNS inflammation in the pathogenesis of MS. In this study, we examined the potential effects of IFN‐β on the regulation of OPN and IL‐17 in MS patients. We found that IFN‐β used in vitro at 0.5–3 ng/mL significantly inhibited the production of OPN in primary T cells derived from PBMC. The inhibition of OPN was determined to occur at the CD4+ T‐cell level. In addition, IFN‐β inhibited the production of IL‐17 and IL‐21 in CD4+ T cells. It has been described that IFN‐β suppresses IL‐17 production through the inhibition of a monocytic cytokine, the intracellular translational isoform of OPN. Our further investigation demonstrated that IFN‐β also acted directly on the CD4+ T cells to regulate OPN and IL‐17 expression through the type I IFN receptor‐mediated activation of STAT1 and suppression of STAT3 activity. Administration of IFN‐β to EAE mice ameliorated the disease severity. Furthermore, spinal cord infiltration of OPN+ and IL‐17+ cells decreased in IFN‐β‐treated EAE mice along with decreases in serum levels of OPN and IL‐21. Importantly, decreased OPN production by IFN‐β treatment contributes to the reduced migratory activity of T cells. Taken together, the results from both in vitro and in vivo experiments indicate that IFN‐β treatment can down‐regulate the OPN and IL‐17 production in MS. This study provides new insights into the mechanism of action of IFN‐β in the treatment of MS.

Elevated frequencies of 6-thioguanine-resistant lymphocytes in multiple sclerosis patients treated with cyclophosphamide: a prospective study.

An autoradiographic assay for 6-thioguanine-resistant (TGr) lymphocytes was used to determine the frequency of in vivo derived variant T lymphocytes in peripheral blood from multiple sclerosis (MS) patients treated with monthly intravenous infusions of 750 mg/m2 of cyclophosphamide (CP). To analyze the time-course of response to CP, the MS patients were studied prospectively. Samples were obtained from the patients before the beginning of CP therapy, 4-5 times during the course of treatment, and, finally, 2 or 3 months after the completion of therapy. 2 weeks after the first CP infusion, the variant frequencies (Vfs) of the MS patients were significantly increased (p less than 0.05) above their pre-treatment values, but by 4 weeks following the first CP infusion the Vfs had fallen to normal or near-normal levels. After subsequent treatments, the frequencies of variant TGr cells were again higher than pre-treatment Vfs. However, within 7-13 weeks after the cessation of CP therapy, the Vfs of all subjects had returned to normal levels. The transient nature of the response indicates rapid in vivo selection against CP-induced TGr mutant cells. The mean pre-treatment Vf of the 4 MS patients who were cigarette smokers was 6.56 X 10(-6) which was significantly greater (p less than 0.05) than the mean Vf (1.52 X 10(-6) of the 4 MS patients who were non-smokers. The mean Vf from 8 assays of healthy non-smokers was 1.92 X 10(-6).

Preferential recognition of TCR hypervariable regions by human anti-idiotypic T cells induced by T cell vaccination.

T cell responses to myelin basic protein (MBP) are potentially involved in the pathogenesis of multiple sclerosis (MS). Immunization with irradiated MBP-reactive T cells (T cell vaccination) induces anti-idiotypic T cell responses that suppress circulating MBP-reactive T cells. This T cell-T cell interaction is thought to involve the recognition of TCR expressed on target T cells. The study was undertaken to define the idiotypic determinants responsible for triggering CD8+ cytotoxic anti-idiotypic T cell responses by T cell vaccination in patients with MS. A panel of 9-mer synthetic TCR peptides corresponding to complementarity-determining region 2 (CDR2) and CDR3 of the immunizing MBP-reactive T cell clones were used to isolate anti-idiotypic T cell lines from immunized MS patients. The resulting TCR-specific T cell lines expressed exclusively the CD8 phenotype and recognized preferentially the CDR3 peptides. CDR3-specific T cell lines were found to lyze specifically autologous immunizing MBP-reactive T cell clones. The findings suggest that CDR3-specific T cells represented anti-idiotypic T cell population induced by T cell vaccination. In contrast, the CDR2 peptides were less immunogenic and contained cryptic determinants as the CDR2-specific T cell lines did not recognize autologous immunizing T cell clones from which the peptide sequence was derived. The study has important implications in our understanding of in vivo idiotypic regulation of autoimmune T cells and the regulatory mechanism underlying T cell vaccination.

Clinical utility of dorsal sural nerve conduction studies.

A technique of testing sensory nerve conduction of the dorsal sural nerve in the foot was used in 38 normal subjects and 70 patients with peripheral neuropathies. The normal dorsal sural sensory nerve action potential (SNAP) had a mean amplitude of 8.9 μV (range 5–15 μV), mean latency to negative peak of 4.0 ms (range 3.2–4.7 ms), and mean conduction velocity of 34.8 m/s (range 30–44 m/s). Optimal placement of the recording electrodes to obtain a maximal nerve action potential was proximal to digits 4 and 5. Cooling to below 25°C prolonged the latency but did not decrease the SNAP amplitude. Among the patients with peripheral neuropathy, dorsal sural SNAP was absent in 68 (97%), whereas only 54 (77%) showed abnormalities of sural sensory conduction. The diagnostic sensitivity of sensory nerve conduction studies in peripheral neuropathies may be significantly improved by the use of this technique for evaluating the action potential of the dorsal sural nerve.