In vitro evidence that immunuaffinity-purified MOG contains immunogenic quantities of contaminating mouse IgG; techniques for producing Ig-free MOG.

Myelin oligodendrocyte glycoprotein (MOG) has attracted considerable attention due to its possible role in multiple sclerosis (MS). Previous studies have shown that MOG-reactive T cells are more commonly detected in MS patients than controls. In this report, we studied human MOG-reactive T cell clones generated from healthy individuals as well as MS patients. Our results indicate that immunoaffinity-purified MOG, which was routinely used in prior studies, is contaminated by anti-MOG antibody (mouse IgG), despite the inability to detect IgG by Western blotting. Here, we used continuous SDS-PAGE, which enabled us to isolate highly purified MOG devoid of contaminating mouse IgG.

The 14-3-3 protein detectable in the cerebrospinal fluid of patients with prion-unrelated neurological diseases is expressed constitutively in neurons and glial cells in culture.

The 14-3-3 protein belongs to a family of 30-kD proteins originally identified by two-dimensional analysis of brain protein extracts. Recently, the detection of the 14-3-3 protein in the cerebrospinal fluid (CSF) is utilized as a highly reliable test for the premortem diagnosis of prion diseases such as Creutzfeldt-Jakob disease. For the initial step, to clarify the biological implication of the CSF 14-3-3 protein in these diseases, its expression was investigated in neural tissues and cultures and CSF samples from patients with a variety of neurological diseases by Western blot analysis and immunocytochemistry. The constitutive expression of the 14-3-3 protein was identified in all neural and nonneural tissues examined. It was expressed in all neurons, astrocytes, oligodendrocytes, and microglia in culture with its location in both cytoplasmic and nuclear regions. The 14-3-3 protein was detected in the CSF of 8 out of 71 patients, including 1 Gerstmann-Sträussler-Scheinker disease patient and 7 patients with prion-unrelated neurological diseases, such as meningoencephalitis of viral, bacterial, or tuberculous origin, multiple sclerosis, and mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes. These results suggest that the 14-3-3 protein expressed constitutively at substantial levels in both neurons and glial cells might be released into the CSF as a disease-nonspecific consequence of the extensive brain damage and indicate that the analysis of the 14-3-3 protein in the CSF is not useful as a screening test for prion diseases.

[A case of familial myoclonus showing extremely benign clinical course].

We report a patient with familial myoclonus showing an extremely benign clinical course. The patient was a 70-year-old woman, who first noticed shaking of hands at age of 25. The symptom did not worsen for more than 40 years. She visited our hospital at the age of 70 because of disturbance in chores because of worsening of her hand shaking in the past one year. A family history showed that 4 members had similar symptoms and that the two were afflicted with fits of loss of consciousness. On neurologic examination, rhythmic myoclonic jerks were noted in all the extremities, more in the upper limbs, both at rest and during action. Tandem gait was mildly disturbed. The remainings of neurologic examination were normal. SEP and jerk-locked back averaging provided evidence of cortical myoclonus. EEG showed multifocal polyspike discharges. Gene analysis for DRPLA, pyruvate and lactate levels in serum and the cerebrospinal fluid, serum amino acid levels, and CSF HVA and 5-HIAA levels were all normal. No brain atrophy was noted in cranial MRI. Myoclonus was markedly reduced after administration of clonazepam. The clinical features and electrophysiological data of our patient are consistent with the clinical diagnosis of familial essential myoclonus and epilepsy/benign adult familial myoclonic epilepsy.

Constitutive and heat-inducible expression of HSP105 in neurons and glial cells in culture.

The constitutive and heat-inducible expression of HSP105 was investigated in newborn mouse brain cell cultures by Northern blotting, Western blotting and immunocytochemistry. HSP105 was expressed most abundantly in the brain among the various tissues examined. HSP105 mRNA and protein were both present at substantial levels in brain cell cultures under unstressed conditions and up-regulated greatly during 3-48 h following exposure to heat stress (43 degrees C/20 min). HSP105 was expressed in nearly all neurons, oligodendrocytes, microglia and astrocytes with its location of both cytoplasmic and nuclear regions under unstressed and heat-stressed conditions. HSP105 expression was significantly down-regulated in astrocytes following treatment with IL-beta or TNF-alpha (50 ng/ml for 6 days), both of which are known growth-stimulatory cytokines for astrocytes. These results indicate that HSP105 is constitutive and heat-inducible HSP in neurons and glial cells in which its expression is under the control of both stressful stimuli and growth-regulatory factors.

Cultured skin fibroblasts isolated from mice devoid of the prion protein gene express major heat shock proteins in response to heat stress.

Recent evidence has suggested that molecular chaperones participate in the conformational change between the normal cellular prion protein (PrPC) and its scrapie isoform (PrPSc). To study a role of PrPC in the regulation of expression of heat shock proteins (HSPs), a group of molecular chaperones, heat-induced expression of major HSPs (HSP105, HSP90alpha, HSP72, HSC70, HSP60, and HSP25) was investigated in cultured skin fibroblasts isolated from the mice homogeneous for a disrupted PrP gene (PrP-/- mice) by Western blot analysis and immunocytochemistry. Two lines of fibroblasts were established and designated SFK derived from the PrP-/- mice and SFH derived from the PrP+/+ mice, respectively. In both SFK and SFH cells, HSP105, HSP72, and HSP25 were expressed at low levels under unstressed conditions but they were induced markedly following exposure to heat stress (43 degreesC/20 min) at 3-72 h postrecovery. In both cell types, HSC70 and HSP60 were expressed at high levels under unstressed conditions and their levels remained unchanged after heat shock treatment. HSP90alpha was undetectable in both cell types under any conditions examined. The pattern of expression, induction, and subcellular location of HSP105, HSP72, HSC70, HSP60, and HSP25 was not significantly different between SFK and SFH cells under unstressed and heat-stressed conditions. Furthermore, the levels of constitutive expression of HSP105, HSC70, HSP60, and HSP25 were similar between the brain tissues isolated from the PrP-/- and PrP+/+ mice. These results indicate that HSP induction is not affected by either the existence or the absence of PrPC in the cells.

Constitutive and cytokine-inducible expression of prion protein gene in human neural cell lines.

Prion diseases are a group of neurodegenerative disorders characterized by intracerebral accumulation of a protease-resistant prion protein (PrP(Sc)) that causes extensive neuronal degeneration and astrogliosis. The regulation of prion protein (PrP) gene expression by a panel of glial and neuronal cytokines (TNF-alpha, IFN-gamma, IL-1beta, IL-10, and TGF-beta1) was investigated in human neural cell lines by reverse transcription-polymerase chain reaction and Northern blot analysis. The constitutive expression of PrP mRNA was identified in all human neural cell lines and tissues examined including Y79 retinoblastoma, IMR-32 neuroblastoma, SK-N-SH neuroblastoma, U-373MG astrocytoma, KG-1-C glioma, NTera2 teratocarcinoma, NTera2-derived differentiated neurons (NTera2-N), peripheral nerve, and cerebral and cerebellar tissues. In SK-N-SH cells, a 48 hour (h) treatment with 100 ng/ml IL-1beta, 100 ng/ml TNF-alpha, or 100 nM phorbol 12-myristate 13-acetate induced a 2.7- to 4.2-fold increase in the level of PrP mRNA, while the exposure to 100 ng/ml IFN-gamma resulted in a 50% decrease. By contrast, none of these cytokines significantly altered the levels of PrP mRNA in IMR-32, NTera2-N, or U-373MG cells. These results indicate that the PrP gene expression is constitutive in a wide range of human neural cell lines and tissues where it is controlled by cell type-specific regulatory mechanisms.

Spinocerebellar ataxia type 6: MRI of three Japanese patients.

We describe the MRI findings in three Japanese patients with spinocerebellar ataxia type 6 (SCA6) in which a polymorphic CAG repeat was identified in the gene encoding the alpha 1A voltage-dependent P/Q-type Ca2+ channel subunit (CACNL1A4). All showed slowly progressive cerebellar ataxia and mild pyramidal signs. Neuroradiologically, they had moderate cerebellar atrophy, most prominently in the superior vermis, whereas the brain stem appeared to be spared. No abnormal signal intensity was identified.

Interleukin-15, a T-cell growth factor, is expressed in human neural cell lines and tissues.

Interleukin-15 (IL-15) is a novel cytokine which shares activities and receptor components with IL-2. To investigate the biological roles of IL-15 in the human nervous system, we examined the expression of mRNAs for IL-15 and the IL-15 receptor three subunits (IL-15alpha, IL-2Rbeta and IL-2Rgamma) in human neural cell lines and tissues using reverse transcription-polymerase chain reaction and Southern blot analysis. The constitutive expression of high levels of IL-15 mRNA was observed in all the cell lines examined, including Y79 retinoblastoma, IMR-32 neuroblastoma, SK-N-SH neuroblastoma, U-373MG glioma, KG-1-C glioma, NTera2 teratocarcinoma and neurons derived from NTera2 cells following treatment with retinoic acid (RA). Among these cell lines, IL-15 protein was detectable at high levels in culture supernatants of SK-N-SH cells and NTera2-derived neurons. The expression of an alternatively-spliced transcript of the IL-15 gene was up-regulated in NTera2 cells during RA-induced neuronal differentiation, suggesting the existence of differentiation-dependent transcriptional regulation. The expression of IL-15 mRNA was also identified in the human cerebral and cerebellar tissues, peripheral nerve and skeletal muscle, while the mRNAs for the complete set of IL-15R components were detectable only in U-373MG cells, cerebral and cerebellar tissues at significant levels. These results indicate that the expression of IL-15 but not of IL-15R mRNA is universal in human neural cell lines and tissues and raise the possibility that IL-15 acts as a neuroimmune regulatory factor in the human central nervous system.

Adult-onset Krabbe disease with homozygous T1853C mutation in the galactocerebrosidase gene. Unusual MRI findings of corticospinal tract demyelination.

A 51-year-old woman developed a slowly progressive spastic paraparesis and diminished vibration sense beginning at age 38. Intellectual capacity was normal. Krabbe disease was confirmed by markedly reduced leukocyte galactocerebrosidase (GALC) activity, typical inclusions in Schwann cell cytoplasm, and an identification of the homozygous point mutation T1835C (Leu618Ser) in the GALC gene. T2-weighted MRI of the brain showed symmetric high-signal-intensity lesions in the bilateral frontoparietal white matter, the centrum semiovale, and the posterior limb of the internal capsule with sparing of the periventricular white matter. This case is unusual because of the late onset, protracted clinical course, and MRI findings of demyelination confined to the corticospinal tracts.

Homozygosity for an allele carrying intermediate CAG repeats in the dentatorubral-pallidoluysian atrophy (DRPLA) gene results in spastic paraplegia.

We report a family with autosomal recessive spastic paraplegia. Patient 1 was a 37-year-old woman and patient 2 was her 35-year-old sister. They showed spastic paraplegia with mild truncal ataxia and dysarthria but no dementia, epilepsy, myoclonus, or other involuntary movements. They were the products of a consanguineous marriage but the parents were neurologically normal. We analyzed the CAG repeats of the dentatorubral-pallidoluysian atrophy (DRPLA) gene in the family members. The patients were homozygous for an allele carrying an intermediate size of CAG repeats (41 or 40 repeats) in the DRPLA gene; the parents were heterozygous for an intermediate allele and a normal allele in this gene. Homozygosity for an intermediate allele in the DRPLA gene appears to have resulted in spastic paraplegia different from any DRPLA phenotype.

Spinal cord herniation: report of two cases and review of the literature.

Idiopathic herniation of the spinal cord is an extremely rare disorder which may cause progressive myelopathy. Two cases of this entity reported herein were both examined using MRI and CT myelography. The typical appearance of this disease with or without a dorsal intradural arachnoid cyst is focal ventral displacement of the mid-thoracic spinal cord, mimicking an isolated intradural spinal arachnoid cyst on MRI. CT myelography using thin slice sections is useful in the diagnosis of this disease.

Occurrence of Borrelia garinii oculo-neuroborreliosis in Japan.

We report a 64-year-old Japanese man with oculo-neuroborreliosis. His clinical features consisted of polyarthralgia, keratoconjunctivitis, chorioretinitis, optic neuritis, confusion, and polyradiculitis. Assay of antibodies to Borrelia species detected IgG-antibody to B. garinii in both serum and CSF. Progressive declining of serum IgG antibody titer against Borrelia garinii, in parallel with clinical improvement, was observed after administration of ceftriaxone.

[Improvement of ophthalmoplegia by 5-hydroxytryptophan in two cases of progressive supranuclear palsy].

We report two patients with clinically diagnosed progressive supranuclear palsy (PSP): a 69-year-old man and a 73-year-old woman. Both patients showed supranuclear ophthalmoplegia, postural instability, pseudobulbar palsy, and Parkinsonism. In the first patient, we administered L-dopa/carbidopa (300 mg/30 mg/ day), which moderately improved gait disturbance, but exerted no beneficial effects on gaze palsy. Then, we administered amitriptyline, bromocriptine, pergolide, l-threo-DOPS or 5-hydroxytryptophan (5-HTP) in addition to L-dopa/carbidopa. The second patient was treated by the monotherapy of L-dopa/carbidopa, amitriptyline, l-threo-DOPS or 5-HTP. We interposed two to three weeks between administration of each drug. In both patients, amitriptyline (75 mg/day) markedly improved both gait disturbance and horizontal gaze palsy. 5-HTP (600 mg/day) also improved horizontal gaze palsy, but failed to alleviate gait disturbance. The results suggest the involvement of impaired serotonergic system in ophthalmoplegia of PSP.

A follow-up study on spastic paraparesis in Japanese HAM/TSP.

We followed 24 patients with HTLV-I-associated myelopathy (HAM) for 4-12 years (mean 7 years) and assessed longitudinal changes of their spastic paraparesis by Kurtzke's Disability Status Scale (DSS). The DSS score of spastic paraparesis was unchanged in 18 patients (75%), advanced (worsened) by two in 1 patient and by one in 3 patients, and declined (improved) by one in 2 patients during the follow-up period. The results demonstrated that HAM presents with a variety of clinical courses including spontaneous improvement.

Assessment of MRI criteria for MS in Japanese MS and HAM/TSP.

We evaluated the usefulness of the MRI criteria for multiple sclerosis (MS) proposed by Paty et al and Fazekas et al in 36 Japanese MS patients, using HTLV-I-associated myelopathy (HAM)/tropical spastic paraparesis (TSP) as the control. Although 30 of 36 HAM/TSP patients had multiple white matter lesions on T2-weighted cranial MRI, only two fulfilled the MRI criteria for MS. At the same time, 31 of the 36 MS patients fulfilled the primary MRI criterion, yielding 93% specificity and 86% sensitivity for the criterion. MS has disease-specific MRI abnormalities.

In situ demonstration of the HTLV-I genome in the spinal cord of a patient with HTLV-I-associated myelopathy.

Using polymerase chain reaction (PCR) and in situ hybridization, we investigated the HTLV-I genome in the CNS of an HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patient with a 20-year disease duration. Neuropathologically, there was severe white matter degeneration throughout the spinal cord, but lymphocytic infiltrates were not evident in any lesion. PCR amplification of the pX region of HTLV-I DNA detected its sequence in the spinal cord and all extra-CNS tissue samples. In situ hybridization using probes complementary to the pX and gag regions detected the HTLV-I genome in the cytoplasm and nucleus of cells in the thoracic cord. The findings indicate a direct involvement of HTLV-I in the neurodegeneration of HAM/TSP.

[Two cases of Paragonimus westermani in one family].

A 68-year-old woman and her 28-year-old son ate some rare fresh-water crabs (Eriocheir japonicus). The son became ill one month later with pleural effusion. After 10 months the mother's symptoms included cough, hemosputum and a nodular shadow on her chest X-ray. Eosinophilia was present and the Ouchterlony test showed strong bands toward Paragonimus westermani antigen in both patients. They were diagnosed as having Paragonimiasis westermani. The symptoms stopped after Bithionol administration and the bands of the Ouchterlony test also disappeared. The difference in the latent stage and clinical symptoms of these patients, who were infected at the same time, is interesting as it highlights the importance of observing other people who may have been infected with Paragonimus westermani.

Development of HTLV-I-associated myelopathy after blood transfusion in a patient with aplastic anemia and a recipient of a renal transplant.

We report the development of rapid progressive HTLV-I-associated myelopathy (HAM) after blood transfusion in two immunosuppressed patients, one of whom had aplastic anemia and the other was the recipient of a renal transplant receiving immunosuppressive chemotherapy. Spastic paraparesis developed 11 or 16 months after transfusion and rapidly progressed to a wheelchair-bound state. The present 2 cases suggest that the coexistent immunosuppression may play an important role in the rapid development of HAM in transfusion-acquired cases.