Mutations in the peripheral myelin protein zero and connexin32 genes detected by non-isotopic RNase cleavage assay and their phenotypes in Japanese patients with Charcot-Marie-Tooth disease.

Mutations of myelin protein zero (MPZ) and connexin32 (Cx32) genes were examined in 70 unrelated Japanese patients with Charcot-Marie-Tooth disease (CMT) without PMP22 gene duplication. A new method, which could detect base pair mismatches with Rnase cleavage on agarose gel electrophoresis, identified 5 and 4 mutations of the MPZ and Cx32 genes, respectively, including one novel mutation (Ser128Ter) of Cx32. This non-isotopic RNase cleavage assay (NIRCA) employed in the present study is very suitable for exploring mutations of MPZ and Cx32 genes in a large number of CMT patients, as the phenotype of patients with CMT is greatly divergent from demyelinating to axonal pathology.

Elevated serum levels of manganese superoxide dismutase in polymyositis and dermatomyositis.

We studied serum concentrations of manganese superoxide dismutase (Mn SOD) and copper-zinc superoxide dismutase (Cu/Zn SOD) in 22 patients with polymyositis and dermatomyositis (PM/DM), 87 patients with four types of muscular dystrophy, 20 patients with amyotrophic lateral sclerosis, and 15 patients with collagen vascular diseases (CVD). Serum levels of Mn SOD were increased only in the patients with PM/DM and CVD, and the elevation was more prominent in those with PM/DM. Levels of Cu/Zn SOD were slightly elevated in some patients with PM/DM and Duchenne muscular dystrophy. In patients with PM/DM, the change in Mn SOD levels corresponded to disease activity as closely as or more closely than those of creatine kinase. The results indicate that serum Mn SOD may be a useful clinical marker for PM/DM.

Effects of microglia-derived cytokines on astrocyte proliferation.

Recent studies have suggested that cytokines, such as interleukin-l(IL-l), tumor necrosis factor(TNF)α, or interferon(IFN) γ, play a role in the development of astrocytic gliosis. In this study, we examined the effects of these cytokines on the proliferation of purified astrocytes in vitro, using the colorimetric assay, bromodeoxyuridine uptake by astrocytes, and changes in the amount of the S-100 ß protein as markers of astrocyte proliferation. The effects of a crude supernatant from microglia enriched cultures (Mi-Sup) also were examined. In contrast to previous reports, these recombinant cytokines did not induce proliferation of purified mouse astrocytes. However, stimulation of astrocytes with Mi-Sup increased all the markers for astrocyte proliferation, which could not be blocked by the addition of anti-IL-1, IL-6, IFNγ or TGFß antibodies. Thus, it appears that microglia produce factors, other than the above cytokines, which induce the proliferation of astrocytes in vitro. These factors may have a role in the development of gliosis in various pathologic conditions of the central nervous system.

Cultured rat Schwann cells express low affinity receptors for nerve growth factor.

Schwann cell cultures prepared from postnatal Sprague-Dawley rat sciatic nerves were used to demonstrate the presence of specific receptors for the beta-subunit of nerve growth factor (NGF) on rat Schwann cells. Indirect immunofluorescence microscopy with a monoclonal antineuronal NGF receptor (NGFR) antibody indicated that NGFR antigen was expressed on the surface of Schwann cells but not of endoneurial fibroblasts. Studies with 125I-NGF confirmed this distribution of NGFR in the cultures and showed that the Schwann cell NGFR had a single NGF binding affinity (Kd of 1.8 x 10(-9) M). 125I-NGF binding by the cultured Schwann cells increased with time in vitro, reaching a plateau level on the 4th day, but decreased with increasing age, reaching 40% of the neonatal value in Schwann cells isolated from 12-day-old rats. Treatment of the cultures with NGF did not alter Schwann cell phenotype, survival or proliferation.

Clinico-pathophysiological features of acute autonomic and sensory neuropathy: a long-term follow-up study.

We evaluated the clinico-pathophysiological features of three patients with acute autonomic and sensory neuropathy (AASN) who were followed for over 3 years. Signs of an autonomic disturbance including vomiting, anhidrosis, urinary disturbances, orthostatic hypotension and reduced coefficient of variation of the R-R interval on electrocardiography gradually improved about 1 year after onset. However, all three exhibited severe generalized sensory impairment for all modalities with the development of persistent sensory ataxia. No sensory nerve action potentials could be elicited and no somatosensory evoked potentials could be obtained. Sural nerve biopsy revealed severe axonopathy. In two patients, a high-intensity area was observed in the posterior column of the spinal cord on T2*-weighted axial magnetic resonance images. The level of neuron-specific enolase in cerebrospinal fluid was markedly elevated in two patients, indicating spinal nerve root or sensory neuron damage. Motor nerve function was well preserved in all patients. Our findings suggests that the major lesion in patients with AASN, particularly those with a sensory deficit, is present in the dorsal root ganglion neurons, that is there is a ganglioneuronopathy.

Serum muscle-specific enolase in progressive muscular dystrophy and other neuromuscular diseases.

Serum muscle-specific enolase ( MSE , beta beta and alpha beta enolases) levels were determined in 162 patients with progressive muscular dystrophy (PMD) and other neuromuscular diseases by means of an enzyme immunoassay method. The relationships were examined between serum MSE , creatine kinase (CK) and other markers of muscle disease. Serum MSE was strikingly increased in Duchenne muscular dystrophy, and this elevation was more prominent in younger patients. Serum MSE was also increased in other types of PMD and certain other diseases. Serum MSE showed the highest correlation with CK. In the PMD group, the frequency of cases with elevated MSE was the same as in CK. These results indicated that serum MSE may well be a specific marker of muscle disease on a par with CK.

Serum carbonic anhydrase III in progressive muscular dystrophy.

Serum carbonic anhydrase III (CA-III) levels were determined by means of an enzyme immunoassay method and compared with serum creatine kinase (CK) and muscle-specific enolase (MSE) levels in 143 patients with four types of progressive muscular dystrophy (PMD), namely, Duchenne muscular dystrophy (DMD), limb-girdle dystrophy, facioscapulohumeral dystrophy and congenital dystrophy. Serum CA-III levels were raised in the majority of patients, especially in those with DMD. In DMD patients, the gradual decline in the CA-III level was observed with age. High correlations were found between CA-III, CK and MSE levels. The frequency of cases with elevated CA-III levels was the same as or greater than that of elevated CK or MSE levels in four types of PMD. These results suggest that serum CA-III may be a useful marker of muscle disease.

Elevated cerebrospinal fluid levels of manganese superoxide dismutase in bacterial meningitis.

We examined the mechanism of increase of manganese superoxide dismutase (Mn SOD) in the cerebrospinal fluid (CSF) in bacterial meningitis (BM). The elevated levels of Mn SOD in the CSF in BM, measured with an enzyme immunoassay method, were more prominent than those in aseptic meningitis (AM) and encephalitis (EN). In AM and EN Mn SOD levels well correlated with levels of neuron-specific enolase and S-100b protein, which are markers of damages to nervous tissues, but did not with any of them in BM. CSF concentrations of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 alpha (IL-1 alpha) were higher in BM than in AM and EN. From the serial measurements in BM, the peak values of these cytokines chronologically preceded or corresponded to those of Mn SOD. Immunohistochemically, a large number of the glial cells were stained for Mn SOD in the cerebral cortex from a patient with BM. By contrast, in the normal cerebral cortex, the glial cells were negative for Mn SOD staining. These results suggest that the marked increase of Mn SOD in the CSF in BM may be related to the increase of such cytokines as TNF-alpha and IL-1 alpha and that these cytokines may play a role in the induction of Mn SOD in nervous tissues.

Cerebrospinal fluid 28-kDa calbindin-D as a possible marker for Purkinje cell damage.

To examine the clinical value of 28-kDa calbindin-D (CaBP) in cerebrospinal fluid (CSF) as a marker for the damage to Purkinje cells, we measured CSF CaBP levels using an enzyme immunoassay method in 107 patients with cerebellar and other neurological diseases, and 26 controls. The mean CaBP level was markedly elevated in patients with cerebellar diseases, and the elevation of CaBP level was more frequent in the diseases involving Purkinje cells, such as multiple system atrophy (MSA) and subacute cerebellar degeneration in association with lung cancer. Further, in MSA patients, the CaBP levels decreased with duration of illness. The mean levels of CaBP were also elevated in some of the other diseases. We conclude that the elevations of CaBP levels are not specific for cerebellar diseases, but CSF CaBP may be a useful marker for examining the Purkinje cell involvement in cerebellar diseases.

Cerebrospinal fluid levels of superoxide dismutases in neurological diseases detected by sensitive enzyme immunoassays.

We measured cerebrospinal fluid (CSF) levels of Cu/Zn superoxide dismutase (Cu/Zn SOD) and Mn superoxide dismutase (Mn SOD) using enzyme immunoassays in 196 neurological patients and 44 controls. The mean Cu/Zn SOD level was 55.8 +/- 27.6 (SD) ng/ml and the Mn SOD, 8.0 +/- 2.5 ng/ml in the controls. Cu/Zn SOD or Mn SOD levels showed neither age-nor sex-related differences in the controls. Both SODs were markedly elevated in cerebrovascular diseases, bacterial meningitis and encephalitis. Mn SOD alone was significantly elevated in neurodegenerative diseases. We compared SODs with CSF levels of neuron-specific enolase (NSE) and S-100b protein (S-100b) in cerebral infarction and bacterial meningitis. Both SODs were correlated with NSE and S-100b in patients with cerebral infarction, but not in those with bacterial meningitis. This means that elevations of SODs in CSF may not only be due to leakage from damaged nervous tissues, but also to the induction of SOD in lesions. We conclude that the mean SOD levels were elevated in various neurological diseases, and their varied magnitudes may be associated with the underlying diseases.

Neuron-specific enolase and S-100 protein levels in cerebrospinal fluid of patients with various neurological diseases.

Neuron-specific enolase (NSE) and S-100 protein (S-100) levels in cerebrospinal fluid (CSF) were determined in 129 patients with various neurological diseases. The chronological changes of these nervous system-specific proteins in CSF were also examined in 3 patients with acute disorders. NSE and S-100 levels were elevated in many cases with acute conditions. These specific proteins did not increase simultaneously but independently. These results suggested that NSE and S-100 in CSF would be useful markers for damage of the nervous system and that measurement of both NSE and S-100 might positively indicate whether the damage was neuronal, glial or mixed in origin. Moreover, from the serial determination of these substances, they would be better markers than cell counts and total protein in CSF for the active injury for the nervous tissues.

Distribution of immunoreactive carbonic anhydrase III in various human tissues determined by a sensitive enzyme immunoassay method.

A sensitive sandwich enzyme immunoassay method for measurement of carbonic anhydrase III (CA-III) was established by use of purified antibodies to CA-III. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from E. coli. The assay was highly sensitive and pg levels of CA-III were measureable. Coefficients of variation in within-run and between-run precision studies for serum CA-III were less than 10%. Serum CA-III levels in healthy subjects of various ages ranged from 0.8 to 24 ng/ml. Concentrations of immunoreactive CA-III in the extracts of various human tissues were also determined. Tissues composed of striated muscle contained more than 10 micrograms/mg protein of CA-III, whereas other tissues, including heart muscle, contained less than 0.5 microgram/mg protein. These results were consistent with other data showing that serum CA-III levels were raised in patients with progressive muscular dystrophy but not in those with acute myocardial infarction.

Highly sensitive enzyme immunoassay for human creatine kinase BB isozyme.

A sensitive sandwich-type enzyme immunoassay method for measurement of brain-type isozyme of human creatine kinase (CK-BB) was developed using purified antibodies specific to the B subunit. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labelled with beta-D-galactosidase from Escherichia coli. The assay was highly sensitive and 1 pg of CK-BB was measurable. The assay was specific to the B subunit of creatine kinase (CK-B), and it cross-reacted about 25% with CK-MB, the heart-type isozyme. However, the assay showed no cross-reactivity with CK-MM, the muscle type-isozyme or with neuron-specific gamma gamma enolase. Coefficients of variation in within-run and between-run precision studies for serum CK-B were less than 8%. Serum CK-B levels in healthy adults of various ages (16-59 yr old) ranged from 0.25-1.44 ng/ml, whereas the CK-B concentrations in children (less than 10 yr old) were relatively high, ranging from 1.3-7.4 ng/ml. The CK-B levels in the cerebrospinal fluids (CSF) could be determined by the present method, and they ranged from 0.10-0.76 ng/ml in the samples from patients with non-neuronal disorders. Determination of immunoreactive CK-B in the extracts of various human tissues confirmed previous reports that CK-B was distributed at high concentrations in the central nervous tissue, prostate, uterus, bladder, gastrointestinal tract and heart muscle.

Immunoassay of human muscle enolase subunit in serum: a novel marker antigen for muscle diseases.

A sandwich enzyme immunoassay method for measurement of beta subunit of muscle enolase in human serum was developed by use of purified antibodies to enolase beta subunit and beta-D-galactosidase from Escherichia coli as label. The assay was specific to the beta subunit with no cross-reaction with the alpha and gamma subunits of human enolase. The measurable range was from 10 pg to 10 ng per assay tube or 1 to 1000 ng/ml serum. Coefficients of variation within-run and between-run for the assay of serum beta subunit were less than 14%. Normal adult sera contained about 6 ng/ml of the beta subunit, and the levels were significantly elevated in sera of patients with muscular dystrophy and those with myocardial infarction. Serum levels of the beta subunit correlated well with those of creatine phosphokinase, but poorly with those of myoglobin in the same samples. The specific distribution of beta subunit in skeletal muscle and heart was confirmed by measuring the levels in various tissue extracts.

[Features of MRI diffusion weighted image in early stage of lateral medullary infarction presenting Wallenberg syndrome].

We described early features of MRI diffusion weighted images (DWIs) in three patients with lateral medullary infarction. Patient 1. A 65-year-old woman who complained of vertigo was admitted. Then, DWIs showed a high signal intensity in the right lateral medulla, though T2 weighted images (T2WIs) did not show any abnormalities. On the next day, when the typical symptoms and signs of lateral medullary infarction appeared, the lesion was recognized on both DWIs and T2WIs. Patient 2. A 48-year-old man was admitted 9 hours after he had dizziness, nausea, and repeated vomitings. When a diagnosis of Wallenberg syndrome was made on the neurological examination, MRI DWIs demonstrated a high signal intensity in the right lateral medulla. The lesion became apparent on T2WIs 82 hours after the onset. Patient 3. A 71-year-old man was admitted for nausea, dizziness, and repeated vomitings. A diagnosis of Wallenberg syndrome was made fifty eight hours after the onset, a lesion with high signal intensity in the right lateral medulla was evident on DWIs, but it was faint on T2WIs. We concluded that DWIs is useful in early diagnosis of lateral medullary infarction presenting Wallenberg syndrome.

[The clinical usefulness of MRI diffusion weighted images in herpes simplex encephalitis-like cases].

We examined the serial MRI diffusion weighted images (DWIs) in two patients with acute viral encephalitis similar to herpes simplex encephalitis (HSE). Patient 1. A 27-year-old woman was admitted to the psychiatry ward for her confusional state and convulsions. Because of abnormal CSF findings she was transferred to the neurology ward, and the infusion of acyclovir was started. On disease day 5. MRI demonstrated high signal intensity in the left lateral lobe both on T2 weighted images (T2WIs) and DWIs. On day 18, MRI showed progression of the lesions, so acyclovir was changed to ara-A. On day 26, no improvement was seen clinically or radiologically. Then, combination therapy with acyclovir, ara-A and gamma-globulin was began. On day 36, she recovered completely, and abnormal intensity in MRI disappeared both on T2WIs and DWIs. Therefore, antiviral agent therapy was discontinued. Patient 2. A 31-year-old man was admitted for headache, fever and aphasia. On the next day, acyclovir was started and both DWIs and T2WIs of MRI demonstrated a high signal intensity in the left temporal lobe. Ten days later, he became perfectly well, and the increased signal intensity disappeared on DWIs, but not on T2WIs. Treatment was therefore discontinued. No relapse was in either patient. We concluded that serial MRI DWIs may be useful to determine when to discontinue the treatment in encephalitis.

[Neuron-specific enolase (NSE) and S-100b protein in cerebrospinal fluid of patients with cervical spondylosis--the relations with MRI findings and the changes of NSE and S-100b protein levels through Glisson's traction].

We have previously reported that NSE and S-100b protein (S-100) could be used as reliable markers to evaluate the damage of the spinal cord in cervical spondylosis (CS) and ossification of posterior longitudinal ligament (OPLL). In the present study we made MRI in 21 patients with CS. There was a positive correlation between the NSE level in CSF and the degree of the spinal cord compression shown by MRI. In 10 cases of CS we examined these specific protein levels in CSF before and after the Glisson's traction therapy for one month. In seven of them the NSE levels decreased with the clinical improvements. On the other hand, 2 cases showed the rise of NSE levels after the traction. One of them became clinically worse during the therapy, while in another case the NSE levels changed within the normal range. The level of NSE in the other case was unchanged. In 57-year-old patient with CS myelopathy we examined the NSE levels chronologically. The NSE level changed in parallel with his clinical features. We suggest that NSE level in CSF may be a useful marker to estimate the change of the degree of the spinal cord damage in CS.

[A case of Guillain-Barré syndrome with bulbar palsy showing the elevations of the anti-GD1a and GT1b antibodies].

We reported a 44-year-old woman with Guillain-Barré syndrome (GBS) showing elevations of serum anti-GD1a and anti-GT1b antibody levels. A few days after an upper respiratory infection, she felt numbness in her hands and feet, dysphagia and dysarthria, and weakness in her limbs. On admission, examination showed the paralysis of pharynx and neck, moderate weakness of face and upper limbs, and mild weakness of lower limbs. Sensory deficits were minimal on the distal side of extremities. Deep reflexes were decreased or absent. Laboratory examinations revealed the albumino-cytological dissociation in cerebrospinal fluid and the increase of anti-GD1a and anti-GT1b antibodies in serum. Nerve conduction studies demonstrated axonal damage to the motor nerves. With immunoadsorption therapy, she gradually recovered and the anti-GD1a and anti-GT1b antibodies were normalized. It was reported that the anti-GT1a antibody may be associated with a pharyngeal-cervical-brachial (PCB) variant of GBS (Ropper) and the similar cases including the present case. However, in the present case, the serum anti-GT1a antibody level was not increased, whereas those of anti-GD1a and anti-GT1b antibodies did. Therefore, these antibodies may also play a role in the development of PCB signs in GBS.

[Central nervous system disorders in myotonic dystrophy--with special reference to neuron-specific enolase, S-100b protein and creatine kinase BB isoenzyme levels in CSF].

In order to evaluate central nervous system disorders in myotonic dystrophy (MyD), neuron-specific enolase (NSE), S-100b protein and creatine kinase BB (CK-BB) isoenzyme were measured using enzyme immunoassay in MyD. Intelligence quotient (IQ) test (WAIS, 17 cases), electro-encephalography (17 cases) and brain computed tomography (18 cases) were examined. In patients with MyD, NSE level was significantly elevated in comparison with 25 age-sex matched control subjects. In some cases of MyD levels of S-100b protein and CK-BB in CSF were elevated. IQ test disclosed intellectual impairment in 70.6% of the patients examined and EEG study demonstrated slowing of basic rhythm in the majority of the cases. On brain CT both enlarged ventricles and dilated sulci were commonly found. The results of the present study suggest that in MyD the CNS is involved not only functionally but structurally as well. Since NSE, S-100b and CK-BB are localized in neuronal and glia cells, their elevated levels in CSF indicate existence of organic lesions in the central nervous tissue in patients with MyD.

[Cerebrospinal fluid levels of 28 kDa calcium-binding protein in patients with neurological diseases].

Vitamin D-dependent calcium-binding protein (Calbindin-D: CaBP) (MW 28 kDa) is present in high concentrations in the central nervous system (CNS), especially in the cerebellum. In the cerebellum, CaBP is localized in Purkinje cells. By using an enzyme immunoassay method, we measured the cerebrospinal fluid (CSF) levels of CaBP in 96 neurological patients and 24 control subjects, and evaluated the clinical usefulness of determining CaBP, especially in the diagnosis of cerebellar damage. CSF CaBP levels were strikingly elevated in patients with cerebellar lesions, including primary spinocerebellar degeneration (SCD), subacute cerebellar degeneration with lung cancer, Wernicke-Korsakoff syndrome, as well as in patients with cerebrovascular disease (CVD) without involvement of the cerebellum. In these 2 groups, neuron-specific enolase (NSE) levels were determined. The ratio of CaBP to NSE (CaBP/NSE) in patients with cerebellar lesions was higher than that in CVD, reflecting differences in lesions between these 2 groups. In the SCD group, multiple system atrophy (MSA) showed higher CaBP levels than the other types of SCD. Probably it reflects remarkable damage of the Purkinje cells in MSA. In MSA, the CaBP levels decreased with the duration of illness. In myelopathy, neuropathy, meningitis, multiple sclerosis, and other degenerative diseases, the increase of CaBP was not remarkable. Our results suggested that CSF CaBP is considered to be a marker of cerebellar damage.