mRNA amplification system by viral replicase in transgenic plants.

We have constructed transgenic tobacco plants (M1x2-FCP2IFN plants) expressing viral RNA replication genes of brome mosaic virus (BMV) and BMV RNA3 derivative (FCP2IFN) carrying the human gamma interferon (IFN-gamma) gene. In M1x2-FCP2IFN plants the RNA3 derivative expressed from the integrated cDNA was replicated and subgenomic RNA (i.e. mRNA of IFN-gamma) was produced by BMV replicase. The accumulation level of the mRNA of IFN-gamma was approximately 5-fold higher than that by the cauliflower mosaic virus (CaMV) 35S RNA promoter. In addition IFN-gamma accumulated in M1x2-FCP2IFN plants.

The frequency of patients with dystrophin abnormalities in a limb-girdle patient population.

Of the 3,048 diagnostic muscle biopsies processed by the National Institute of Neuroscience, Tokyo, over 12 years, 41 cases carried the clinical diagnosis of limb-girdle muscular dystrophy. We have analyzed all 41 cases for dystrophin content in muscle by both immunofluorescence and immunoblot. We identified five male patients with an abnormal dystrophin pattern diagnostic of Becker muscular dystrophy, and two female patients with dystrophin patterns consistent with a manifesting carrier of Duchenne muscular dystrophy diagnosis. Thus, 17% of our limb-girdle patients showed a dystrophinopathy, indicating that they in fact had a disorder related to Duchenne/Becker muscular dystrophy. Misclassification of isolated male limb-girdle patients was 31% (4/13), while misclassification of isolated female limb-girdle patients was 13% (2/15). Using multiplex polymerase chain reaction analyses of small amounts of muscle biopsy DNA confirmed a dystrophin gene deletion in all five male Becker dystrophy patients identified. This study emphasizes the clinical overlap between limb-girdle muscular dystrophy and dystrophinopathies, and reinforces the necessity of dystrophin protein and gene studies for the accurate clinical diagnosis of isolated cases of muscular dystrophy.

Undetectable dystrophin can still result in a relatively benign phenotype of dystrophinopathy.

We present here a 28-year-old male patient with Becker muscular dystrophy whose skeletal muscle showed an absence of dystrophin. He has had progressive and predominantly proximal muscular wasting since 5 years of age, but was able to walk until 26 years of age. He showed hypertrophic calves, cardiomyopathy, and an elevated serum creatine kinase level (934 U/1). A skeletal muscle biopsy revealed advanced chronic myopathic changes. Immunohistochemical examination using anti-dystrophin antibodies against C-terminus showed deficiency of the protein. Rod domain and N-terminus were also absent in almost all muscle fibers, but only in a small part of the sample, they were faintly stained. beta-Dystroglycan and utrophin were present only in a small number of muscle fibers. DNA and RT-PCR analysis showed a frame-shift deletion of exons 3-7 in the dystrophin gene. In such an exceptional case as this one, it is important to investigate the factors which determine the severity of dystrophinopathy.

Evidence of long-term survival of donor-derived cells after limbal allograft transplantation.

PURPOSE: Severe destruction of the corneal limbus causes conjunctival invasion and subsequent visual loss. Limbal allograft transplantation (LAT) was recently proposed for the treatment of these disorders. However, whether the method functions as a stem cell transplantation of the corneal epithelium remains unclear. This study provided evidence that donor-derived corneal epithelial cells survive long after LAT. METHODS: Epithelial cells on the paracentral cornea in patients who have undergone LAT were subjected to fluorescence in situ hybridization (FISH) and polymerase chain reaction restriction fragment length polymorphism (RFLP) analysis. X and Y chromosomes were detected using sex chromosome-specific probes in the FISH analysis, and HLA-DPBI antigens were examined in the RFLP analysis. Eyes receiving conventional penetrating keratoplasty (PKP) served as controls. RESULTS: Donor-derived epithelial cells were detected in three of five eyes (60.0%) in the FISH analysis and in seven of nine eyes (77.8%) in the RFLP analysis. Among these eyes, one and three eyes in the FISH and RFLP analysis, respectively, had both donor- and recipient-derived cells. In control PKP eyes, none of the eyes in the FISH analysis and one of eight eyes (12.5%) in the RFLP analysis had donor-derived cells. CONCLUSIONS: These results suggest that donor-derived cells survive much longer after LAT than those after PKP, and that LAT may function as stem cell transplantation of the corneal epithelium.

The expression of ion channel mRNAs in skeletal muscles from patients with myotonic muscular dystrophy.

We investigated gene expression patterns of ion channels including the apamin-sensitive small-conductance Ca(2+)-activated K(+) (SK3) channel, the adult isoform of the skeletal muscle Na(+) channel (SkM1), the fetal isoform of skeletal muscle Na(+) channel (H1), and the Cl(-) channel (ClC-1) by using the semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) for muscle samples from patients with adult onset myotonic dystrophy (DM), amyotrophic lateral sclerosis, and polymyositis. Patients with DM showed a significant increase in SK3 mRNA but not in mRNAs for other ion channels. The increased expression of SK3 gene in DM did not correlate with H1, the marker of muscle denervation, or the percentage of type 2C fiber, the marker of muscle regeneration.

A new surgical technique for deep lamellar keratoplasty with single running suture adjustment.

PURPOSE: To improve the technique of deep lamellar keratoplasty. METHODS: For the recipient eye, a divide-and-conquer technique was applied to deep lamellar keratoplasty. After trephinization, the recipient cornea within the trephine was divided into four quadrants to facilitate lamellar dissection at approximately 70% depth. This procedure of division was continued until the Descemet membrane was exposed in the central area. The corneal graft was placed with an adjusted single running suture. Seventeen eyes were treated with this technique. RESULTS: In 17 eyes of 15 patients, the mean visual acuity 6 months or more after deep lamellar keratoplasty was 20/52 with eyeglass correction and 20/80 without eyeglass correction. At 6 months or more after deep lamellar keratoplasty, the mean +/- SD keratometric astigmatism in 17 eyes was 3.2 +/- 2.3 diopters. CONCLUSION: This technique facilitates deep lamellar keratoplasty and prevents high or excessive astigmatism after surgery.

HTLV-I-associated myelopathy manifested after renal transplantation.

We report a patient with HTLV-I-associated myelopathy (HAM), who developed symptoms of myelopathy 4 years after cadaveric renal transplantation. Since he was seronegative before the transplantation, it is suggested that HTLV-I infection was transmitted via renal graft transplantation. He has been treated with immunosuppressive agents such as cyclosporin A (CsA), mycophenolate mofetil (MMF), and prednisolone (PSL) to prevent graft rejection. This case suggested that these immunosuppressive agents are poorly effective in suppressing either the onset or progression of HAM/TSP.

Clinical and magnetic resonance image correlation in idiopathic cerebellar ataxia.

Sixty-one patients who fulfilled the clinical criteria for idiopathic cerebellar ataxia and who had symptoms at least for 3 years were examined clinically and by magnetic resonance imaging (MRI). Based on the clinical signs, they were divided into patients with pure cerebellar signs (Group 1), patients with additional mild rigidity and/or hyperreflexia (Group 2) and patients with additional severe rigidity and hypokinesia (Group 3). Patients in Group 1 had milder disability and better prognosis than patients in Group 2 or Group 3 (ataxic score: 14.9 vs. 28.6 and 36.0; annual progression ratio: 0.26 vs. 0.65 and 0.70, respectively). We measured the area of the cerebellar vermis, ventral pons and dorsal brainstem on midsagittal T1-weighted MR images for all patients and age- and sex-matched controls. The cerebellar vermis as well as the ventral pons of patients were significantly smaller than corresponding structures in controls (p < 0.001). The ventral pons of patients in Group 2 and Group 3 was significantly smaller than that of patients in Group 1 (p < 0.0001, respectively), and the dorsal brainstem of patients in Group 2 and Group 3 was also significantly smaller than that of patients in Group 1 (p < 0.001, respectively). The ventral pons of patients in Group 3 was significantly smaller than that of patients in Group 2 (p < 0.05) as well. There was a significant correlation between the area of the ventral pons and the annual progression ratio (p < 0.001). With MRI, slight but definite hyperintensities were demonstrated in the pontine base and the medulla of 22 patients on proton density images. In the longitudinal study, patients in Group 2 and Group 3 had atrophy of the ventral pons already at an early stage. The ventral pons of patients in Group 3 was smaller at the initial MR examination than that of patients in Group 2. These observations suggest that patients with smaller ventral pons may have rapid progression and poor prognosis. Thus, even a relatively simple quantitation of the area of the ventral pons may be useful to predict the prognosis of patients, in addition to neurologic assessment at intervals.

Focal cytochrome c oxidase deficiency in the brain and dorsal root ganglia in a case with mitochondrial encephalomyopathy (tRNA(Ile) 4269 mutation): histochemical, immunohistochemical, and ultrastructural study.

This is the first report with histochemical and immunohistochemical techniques of an autopsy case with mitochondrial encephalomyopathy caused by the mitochondrial tRNA(Ile) (nt4269) A to G mutation showing focal cytochrome c oxidase (COX) deficiency of neuronal cells. The 18-year-old male patient had cardiomyopathy, hearing disability, mental retardation, and seizures. Muscle biopsy exhibited many ragged-red fibers and focal COX deficiency. A postmortem histochemical study on frozen sections of the cerebral cortex, cerebellum, brain stem, and dorsal root ganglia revealed a loss of COX activity in some neuronal cells. On immunohistochemical staining, COX was also defective in a mosaic pattern. Focal COX deficiency may cause variable neurological manifestations in mitochondrial encephalomyopathies.

Diseases associated with ocular surface abnormalities: the importance of reflex tearing.

AIM: To investigate the correlation between tear function tests and ocular surface integrity in patients with dry eye. METHODS: 297 dry eye patients (55 Sjögren's syndrome, two male and 53 female, average age 52.4 (SD 15.0) years, and 242 non-Sjögren's syndrome, 41 male and 201 female, average age 53.5 (14.1) years) were examined. The following tear function tests were performed: (1) cotton thread test, (2) Schirmer test with topical anaesthesia, (3) Schirmer test without anaesthesia, (4) Schirmer test with nasal stimulation, (5) tear clearance test, and (6) tear break up time (BUT). The ocular surface was evaluated by rose bengal and fluorescein staining. Correlation analysis was performed between each tear function index and vital staining scores. RESULTS: Among the six tear function tests, the Schirmer test with nasal stimulation correlated most with both of the vital stains (rho = 0.530 for rose bengal and 0.393 for fluorescein). The Schirmer test with or without anaesthesia correlated slightly with rose bengal staining, whereas tear clearance test and tear break up time slightly correlated with fluorescein staining. CONCLUSION: Vital staining of the ocular surface correlates most with reflex tearing measured by the Schirmer test with nasal stimulation.

Dry eye and Meige's syndrome.

AIMS: To determine the relation between dry eye and Meige's syndrome. METHODS: 325 patients with dry eye were divided into those responsive to topical and other forms of treatment (n = 276) and those who were not (n = 49). A neuropsychiatric examination was performed to check for Meige's syndrome in the latter group. RESULTS: Twenty eight (57%) of the treatment unresponsive patients were diagnosed with Meige's syndrome. CONCLUSIONS: There is a subgroup of patients with dry eye who do not respond to simple therapy. More than half of these patients have Meige's syndrome and need psychiatric, as well as ophthalmic, care.

Clinical utility of somatosensory evoked potentials in diabetes mellitus.

The posterior tibial nerve and median nerve somatosensory evoked potentials (PTN-SEPs and MN-SEPs) were investigated in 34 patients with diabetes mellitus (DM). We measured the latency of the first positive cortical potential (the cortical P37) of PTN-SEPs and that of the first negative cortical potential (the cortical N18) and Erb's potential of MN-SEPs. In 18 patients (52.9%), the cortical P37 latency was more than 3 SD longer than normal in the tibial nerve. There were positive correlations between the latency of cortical P37 and the duration of DM and the motor nerve conduction velocity of the posterior tibial nerve. Sensory action potentials of the posterior tibial nerve were not detectable in 21 patients, though cortical P37 potential was unambiguously recorded by stimulating the posterior tibial nerve even in those subjects. Diabetic retinopathy and nephropathy also tended to rise with increasing latency of cortical P37. The latency of cortical P37 is an important parameter in assessing diabetic neuropathy.

[A case of spinal muscular atrophy with marked calf hypertrophy and adolescent onset].

We report on a 41-year-old male patient with spinal muscular atrophy (SMA). He had slowly progressive muscular weakness and hypertrophic calves since 14 years of age. The upper arms were slightly, and the thighs moderately atrophic, but the calves were remarkably hypertrophic. There was muscle weakness of both the upper and lower limbs, being more proximal in distribution. He had a positive Gowers' sign and his gait was slightly waddling. Serum creatine kinase level was elevated (518IU/l). Electromyogram revealed a neurogenic pattern. Muscle biopsy of the left biceps brachii showed chronic neurogenic changes. Immunohistochemical examination and Western blot analysis using anti-dystrophin antibodies showed no abnormality. DNA analysis with multiplex PCR proved no deletion in the dystrophin gene, while deletions of exons 7 and 8 of the telomeric copy of survival motor neuron gene were detected. In 1978, Pearn et al. described a new variant syndrome of SMA, characterized by adolescent onset, gross hypertrophy of calves, and a slowly progressive clinical course. The present case is compatible with this syndrome. Therefore, it is suggested that this syndrome, mimicking Becker muscular dystrophy, is not an independent clinical entity, although the phenotype of this syndrome is different from that of typical SMA.

[A patient presented with atypical paroxysmal kinesigenic choreoathetosis and Becker muscular dystrophy].

A 22-year-old man had choreatic movements in upper limbs, neck and trunk for over twelve years which were associated with dystonia in lower limbs upon initiating voluntary movements. The choreatic movement lasted for a few seconds and the dystonia lasted for a few minutes. He also had high serum CK levels and hypertrophic calf muscles. His muscle strength and deep tendon reflexes were normal. His choreatic movements fulfill the criteria for paroxysmal kinesigenic choreoathetosis (PKC). However, it was unclear what the symptom of dystonia was due to. From a muscle biopsy and DNA analysis, he was diagnosed as having Becker muscular dystrophy. Administration of anticonvulsant improved the dystonia as well as the choreatic movement, which showed that the dystonia was a symptom of PKC. Coincidence of choreatic movements and dystonias which had different lasting time in a patient of PKC was atypical and had not previously reported.

[Evaluation of testicular damage by flow cytometry: testicular atrophy caused by ethylene oxide].

The new method using flow cytometry was applied to analyse the testicular toxicity of ethylene oxide, and the usefulness of this method is discussed. When Wistar male rats were exposed to ethylene oxide for six hours a day, three times a week for six weeks, the testicular weights of the exposed group significantly decreased. When the cells of these testes were stained by propidium iodide and analysed by flow cytometry, four peaks which corresponded to maturation phase spermatids (less than C), the other haploid cells (C), diploid cells (2C) and tetraploid cells (4C) were obtained. Calculating the ratio of the percentage of less than C, C and 4C to that of 2C, the ratio of less than C of the exposed group decreased by 72.9%, 2C by 53.5% and 4C by 5.1% when compared with the control group. As these changes were almost consistent with that of histopathological examinations, we are able to conclude that more mature germ cells were affected by ethylene oxide. This method by flow cytometry is thought to be objective, quantitative and convenient to evaluate testicular damage by chemicals.

Testicular damage induced by megadoses of pyridoxine.

Pyridoxine hydrochloride, 125 mg/kg, 250 mg/kg, 500 mg/kg or 1,000 mg/kg, daily, was intraperitoneally injected into Wistar male rats and its effects on weights and mature spermatid or sperm counts in the testis and the epididymis were investigated. After six weeks administration, weights of the testis and the epididymis in the 500 mg/kg and 1,000 mg/kg groups dramatically decreased and weights of the epididymis in the 125 mg/kg and 250 mg/kg groups also decreased significantly. Mature spermatid counts in the testis and sperm counts in the epididymis decreased in the 500 mg/kg and 1,000 mg/kg groups, and sperm counts in the tail plus body of the epididymis also decreased in the 250 mg/kg group. From these results, it was elucidated that megadoses of pyridoxine induced testicular damage in rats.

[An autopsy case of acute interstitial pneumonia].

An autopsy case of a 77-year-old male with acute interstitial pneumonia (AIP) is reported. The patient died of respiratory failure in the extremely rapid course of 8 days. The histogenesis of the thickening of the alveolar wall and the intraluminal lesions were noticed. Incorporation of the hyaline membrane as well as the intraseptal edema and septal cell proliferation played an important role in the fibrous thickening of the alveolar wall. The intraluminal granulation tissue was observed in the alveolar space and the alveolar duct, often extending into the respiratory bronchioles. Intraluminal granulation tissues in the alveoli frequently had the appearance of intraluminal buds of mesenchymal cells, so-called Masson's bodies, and there was little remodeling of pulmonary structures in this case. These findings suggest an acute, diffuse and severe damage of the peripheral respiratory tract and are consistent with bronchiolitis obliterans with classical interstitial pneumonia (BIP) by Liebow and AIP by Katzenstein. This patient had suffered from suspected "Bronchiolitis obliterans organizing pneumonia" (BOOP) one and half years ago. The relationship between AIP, BIP and BOOP is discussed.

[Effects of inhalation of ethylene oxide on female rats].

The effects of systemic toxicity including reproductive toxicity of ethylene oxide on female rats were studied. When Wistar female rats were exposed to 250 ppm of ethylene oxide for six hours a day, five days a week for ten weeks, they showed inhibition of body weight gain and paralysis of the hindlegs. Hematological examination revealed macrocytic and normochromic anemia with high reticulocyte counts. The estrus cycle of the exposed group was prolonged and the percentage of the diestrus stage increased. There was no atrophy in the ovary or the uterus. However, the activity of glutathione reductase in the ovary decreased by 18% and that of glutathione-S-transferase increased by 30%. These results indicate that ethylene oxide has a similar effect on both female and male rats and that the female reproductive system is also affected.

[A case of inflammatory myopathy worsened by miscarriage with autoimmune disorder-associated disease].

The patient was 42-year-old woman who had exhibited elevated levels of serum creatine kinase(CK) and intermittent weakness of proximal muscles since her thirties. She had a history of palmoplantar pustulosis, Mondor's disease and recurrent miscarriages. Basedow's disease, which had been treated with antithyroid drugs since 37 years of age, recurred during the fourth pregnancy. After the pregnancy was terminated in the sixth week, weakness and grasp pain in the proximal muscles developed. The biopsy of biceps brachii muscle showed necrosis and reconstruction of muscle fibers with equivocal inflammatory cells, which was compatible with the findings for inflammatory myopathy such as polymyositis(PM). She was treated with prednisolone and the weakness and grasp pain in the proximal muscles were resolved. PM beginning during a woman's reproductive period is rare, and few studies have examined the association between PM and pregnancy. In this case, pregnancy and her past diseases were considered to be linked to an autoimmune abnormality that might have contributed to the inflammatory myopathy.

Reversibility of ethylene oxide-induced testicular damage evaluated by the hemiorchiectomy method.

In the present study, we tried to use the hemiorchiectomy method to examine a reversibility of the testicular damage induced by ethylene oxide (EtO). Wistar male rats were exposed to EtO at a concentration of 500 ppm, 6 hours a day, 3 days a week for 8 weeks. Thereafter we evaluated the reversibility after 13-week recovery period. From the result about a propriety of the hemiorchiecyomy method for recovery study, it was suggested that hemiorchiectomy did not affect remaining testis and epididymis after the recovery period and there was no laterality about the testicular damage just after the exposure period. Therefore we concluded that the hemiorchiectomy method could be used for recovery study for EtO-induced testicular damage. The reversibility was evaluated by comparing the damages of hemiorchiectomized testis just after the exposure period and those of remaining testis removed after the recovery period in the same rat. The result indicated that the reversibility of EtO-induced testicular damage may depend on a degree of damage just after the exposure.