Epitope analysis of the cerebrospinal fluid IgG in HTLV-I associated myelopathy patients using phage display method.

We, for the first time, analyzed the binding motifs of immunoglobulin G (IgG) in the cerebrospinal fluid (CSF) of human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients with a phage library displaying 12-mer random peptides. As a result, the sequences highly homologous to HTLV-I gp46 192-199, 237-243 and 255-261 were the common linear epitopes of high affinity- IgG exclusively detected in both CSF and sera of the patients. These IgG responses were confirmed with corresponding HTLV-I peptides and serum antibody titers significantly correlated with disease severity or duration. Gp46 237-243 has not been detected in previous enzyme-linked immunosorbent assay (ELISA) studies using bound longer peptides, suggesting the usefulness of the phage display method.

Synthesis of 8-C-glucosylflavones.

The syntheses of orientin, parkinsonin A, isoswertiajaponin, and parkinsonin B, which are 8-C-beta-D-glucopyranosyl-3',4',5,7-tetrahydroxyflavone, 5-methyl orientin, 7-methyl orientin, and 5,7-dimethyl orientin, respectively, are reported herein. The C-glucosyl phloroacetophenone derivatives were obtained via a regio- and stereoselective O-->C glycosyl rearrangement. Aldol condensation of the C-glucosyl phloroacetophenone derivatives with 3,4-bisbenzyloxybenzaldehyde afforded the corresponding C-glucosylchalcones. Construction of the flavone system by reaction with I(2)-Me(2)SO, followed by the elimination of the 5-benzyl protecting group in the flavone structure, yielded an orientin derivative and a isoswertiajaponin derivative. Methylation of the orientin derivatives with dimethyl sulfate afforded the parkinsonin A derivative, the isoswertiajaponin derivative, and the parkinsonin B derivative. Finally, hydrogenolysis of these C-glucosylflavone derivatives led to the four 8-C-glucosylflavones. The NMR spectra of these C-glucosylflavones showed a duplication of signals corresponding to a major rotamer, along with a minor one. Based on NOESY experiments in Me(2)SO at ambient temperature, they adopted conformations in which the H-2"and H-4" protons in the glucose moiety were oriented toward the B-ring in the flavone structure.

Cleavage of the C-C linkage between the sugar and the aglycon in C-glycosylphloroacetophenone, and the NMR spectral characteristics of the resulting di-C-glycosyl compound.

The treatment of unprotected mono-C-beta-D-glucopyranosylphloroacetophenone with a cation-exchange resin in anhydrous acetonitrile afforded both a phloroacetophenone and a di-C-beta-D-glucopyranosylphloroacetophenone. Treatment of an unprotected mono-C-(2-deoxy-beta-D-arabino-hexopyranosyl)phloroacetophenone (mono-C-2-deoxy-beta-D-glucopyranosylphloroacetophenone) also afforded both the aglycon and di-C-(2-deoxy-beta-D-arabino-hexopyranosyl)phloroacetophenone. The reaction mixtures were acetylated, and the structures of the isolated products were determined by NMR spectroscopy. This is the first demonstration of the formation of a di-C-glycosyl compound during the chemical cleavage of the C-C linkage between the sugar and the aglycon in an aryl C-glycosyl derivative.

Novel glycosylation of the nitroxyl radicals with peracetylated glycosyl fluorides using a combination of BF(3) x OEt(2) and an amine base as promoters.

Glycosylation of the nitroxyl radicals, 4-acetoxy-2,2,6,6-tetramethylpiperidin-1-oxyl (4-acetoxy-TEMPO) and 3-carbamoyl-2,2,5,5-tetramethylpyrollin-1-oxyl (3-carbamoyl-PROXYL) with peracetylglycosyl fluoride as the glycosyl donor, in the presence of boron trifluoride diethyl etherate (BF(3) x OEt(2)) and an amine base afforded the corresponding hydroxylamine-O-glycosides in 25-100% yields.

Synthesis of C-mannopyranosylphloroacetophenone derivatives and their anomerization.

The reaction of 2,3,4-tri-O-benzyl-alpha-L-rhamnopyranosyl fluoride (6-deoxy-2,3,4-tri-O-benzyl-alpha-L-mannopyranosyl fluoride) with 2,4-dibenzylphloroacetophenone, in the presence of boron trifluoride.diethyl etherate, afforded both the 3-C-alpha-L- and the 3-C-beta-L-rhamnopyranosylphloroacetophenone derivatives. The 3-C-alpha-L-rhamnoside was produced as a major product, while the 3-C-beta-L-rhamnoside was produced as a minor product via anomerization of the 3-C-alpha-L-rhamnoside. Alternatively, the reaction of 2,3,4,6-tetra-O-benzyl-alpha-D-mannopyranosyl fluoride with 2,4-dibenzylphloroacetophenone afforded both the 3-C-alpha-D- and the 3-C-beta-D-mannnopyranosylphloroacetophenone derivatives under identical conditions. The 3-C-beta-D-mannoside was produced as a major product via anomerization of the 3-C-alpha-D-mannoside during the reaction. These differences in composition result apparently from the magnitude of the 1,3-diaxial interactions between the C-3 and C-5 positions in these sugar moieties.

Superoxide scavenging activities of sixty Chinese medicines determined by an ESR spin-trapping method using electrogenerated superoxide.

Superoxide-scavenging activities of 60 kinds of Chinese herbal medicines were determined accurately by an electron spin resonance (ESR) spin-trapping technique using 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) as a spin-trapping reagent. As a source of superoxide in this method, superoxide generated by one-electron reduction of the oxygen molecule in dimethyl sulfoxide solution was used. As a result of these studies, very powerful scavenging activity was found in Chinese medicines for inflammation, diseases of blood circulation and for tumors.

[A case of Tolosa-Hunt syndrome starting from abducens nerve palsy alone].

A 59-year-old man was admitted to our hospital because of right abducens nerve palsy without orbital pain. One month later right orbital pain appeared and two months later the pain changed to an aching severe pain and the right oculomotor, trochlear and trigeminal nerves were also involved. The administration of corticosteroid dramatically reduced these symptoms. The diagnosis was Tolosa-Hunt syndrome (THS) based on these findings. THS starting with abducens nerve palsy alone is rare, representing only 5.1% of 118 patients. In our patient, the newly developed dynamic MRI was able to reveal the right cavernous sinus lesion. The diagnostic usefulness of dynamic MRI for cavernous sinus lesions has been demonstrated for such cases.

Synthesis of C-pentopyranosylphloroacetophenone derivatives and their anomerization.

A 3-C-beta-D-xylopyranosylphloroacetophenone derivative was synthesized via reaction of 2,3,4-tri-O-benzyl-alpha-D-xylopyranosyl fluoride and 2,4-di-O-benzylphloroacetophenone in the presence of boron trifluoride diethyl etherate. Alternatively, the reaction of 2,3,4-tri-O-benzyl-beta-L-arabinopyranosyl fluoride with 2,4-di-O-benzylphloroacetophenone afforded both the 3-C-alpha-L- and the 3-C-beta-L-arabinopyranosylphloroacetophenone derivatives under identical reaction conditions. The C-beta-L-arabinoside, the thermodynanic product, was produced via anomerization of the C-alpha-L-arabinoside, the kinetic product during the reaction. The composition of this product mixture is apparently dictated by both 1,3-diaxial and 2,4-diaxial interactions.

An effective synthesis of isoorientin: the regioselective synthesis of a 6-C-glucosylflavone.

Isoorientin, a 6-C-beta-D-glucopyranosyl-3',4',5,7-tetrahydroxyflavone, was synthesized in a 15% overall yield, in ten steps, starting from the commercially available phloroacetophenone. The C-glucosyl phloroacetophenone derivative, a synthetic intermediate that contains a free hydroxyl group that is para to the glucosyl moiety, was obtained by hydrogenolysis by taking advantage of differences in the hydrogenolysis rates between a benzyl protecting group and a 2-methylbenzyl protecting group. Aldol condensation of the C-glucosyl phloroacetophenone derivative with 3,4-bis-benzyloxybenzaldehyde afforded the corresponding chalcone as a precursor of the 6-C-glucosyl flavone. Construction of the flavone system by application of I2-DMSO, followed by deprotection, yielded isoorientin.

Expression of OX40 and OX40 ligand (gp34) in the normal and myasthenic thymus.

OBJECTIVES: To examine the expression of OX40, an activated memory T-cell marker, and its ligand (OX40L), a set of molecules for T-cell-B-cell interaction, and other lymphocyte activation markers in the thymuses of myasthenia gravis (MG) and controls. MATERIAL AND METHODS: We studied the expression of OX40, OX40L, IL-2Ralpha and HLA-DR in the thymic tissues of MG and controls using immunocytochemistry and flowcytometry. RESULTS: In both hyperplastic thymus of MG and control thymus, OX40+ cells were scattered mainly in the medulla with much fewer OX40L+ cells being distributed in the corticomedullary junctions. IL-2Ralpha and HLA-DR were expressed in the medulla at higher frequencies as compared with OX40 in controls as well as MG. In contrast, the numbers of OX40+ cells around the germinal centers (GC) were significantly greater than those of control thymuses, and some mononuclear cells in GC were OX40L+. A considerable number of OX40+ cells were seen in the thymic tissues adjacent to thymomas. OX40+ cells were CD4+ CD8- or CD4+ CD8+ and were mostly HLA-DR-. (The coexpression of OX40 and IL-2Ralpha on activated CD4+ T cells was previously reported.) CONCLUSION: OX40, expressed in a fraction of activated CD4+ T cells, may be upregulated in thymic tissues adjacent to GC and thymoma in MG, and OX40 may interact with OX40L in GC to enhance anti-acetylcholine receptor antibody production in MG.

The conversion of 3-C-beta-D-galactopyranosyl phloroacetophenone to a spiroketal derivative.

Treatment of 3-C-beta-D-galactopyranosylphloroacetophenone in hot water with a catalytic amount of p-toluenesulfonic acid afforded a spiroketal compound as the main product. The chirality of the spiro carbon of the product was R, which is the opposite of the spiroketal obtained by the conversion of 3-C-beta-D-glucopyranosyl phloroacetophenone under identical conditions. The structure was determined by 1H-1H COSY, 1H-13C COSY, NOESY and HMBC spectroscopy.

Age-associated increase of spontaneous mutant frequency and molecular nature of mutation in newborn and old lacZ-transgenic mouse.

Accumulation of mutation has long been hypothesized to be a cause of aging and contribute to many of the degenerative diseases, which appear in the senescent phase of life. To test this hypothesis, age-associated changes in spontaneous mutation in different tissues of the body as well as the molecular nature of such changes should be examined. This kind of approach has become feasible only lately with a development of new transgenic mice suitable for mutation assay. Here, using one of these transgenic mice harboring lacZ gene, we have shown that the age-associated increase in spontaneous mutant frequency is common to all tissues examined; spleen, liver, heart, brain, skin and testis, while the rates of increase in mutant frequency differed among the tissues. DNA sequencing of the 496 lacZ mutants recovered from the tissues of newborn and old mice has revealed that spectra of mutations are similar at the two age points with G:C to A:T transition at CpG site being a predominant type of mutation. Furthermore, some mutations in old tissues are complex type and not found in tissues of newborn mice. These results suggest that similar mechanisms may be operating for mutation induction in fetal and postnatal aging process. In addition, the appearance of complex types of mutations in the old tissues suggests a unique cause for these mutations in aging tissues.

[Steroid-responsive diffuse cerebral white matter lesions in a case of intractable fungal meningoencephalitis].

We report a case of fungal meningoencephalitis with steroid-responsive diffuse cerebral white matter lesions. A 49-year-old male developed auditory hallucination, confusion and fever, on April, 1994. He was diagnosed as having cryptococcal meningoencephalitis based on the detection of cryptococcal antigens in the cerebrospinal fluid (CSF). Intravenous administration of fluconazole resulted in improvement of his neurologic symptoms and CSF findings. For the next seven months, he was treated with oral fluconazole and the neurological status was stable. However, soon after the dose of fluconazole was tapered, he became confused and febrile, which made him admitted to our hospital. Neurological examination on admission showed disturbance of consciousness, disorientation and meningeal irritation. The CSF examination revealed mild pleocytosis (mostly lymphocytes), elevated protein and normal glucose levels, although fungus was not detected. The T2-weighted image of brain MRI demonstrated diffuse hyperintense lesions in the bilateral cerebral white matters. GD-DTPA enhanced MRI showed spotty enhanced lesions in the periventricular white matters. The neurologic symptoms were once relieved after intravenous administration of fluconazole was started, but two months later, he became comatose and needed ventilatory support, despite amphotericine B therapy. Then, a needle brain biopsy targeting the white matter lesion was done. Histopathology of the specimen showed chronic inflammation with granuloma formation and T lymphycyte infiltrate around the small vessels, though fungus was not detected in the tissue. Combined therapy with corticosteroid and antifungal agents remarkably improved the neurological symptoms as well as the MRI findings. In the present case, fungal infection possibly induced an altered immune reactions which resulted in the steroid responsive diffuse cerebral white matter lesions.

PET study of striatal fluorodopa uptake and dopamine D2 receptor binding in a patient with juvenile parkinsonism.

We studied pre-synaptic and post-synaptic function in the striatum of a patient with juvenile parkinsonism (JP) using positron emission tomography (PET). [18F]6-fluorodopa (18FDOPA), 11C-YM-09151-2 and [18F]fluoro-2-deoxy-d-glucose (18FDG) were used to measure fluorodopa uptake, dopamine D2 receptor binding and glucose metabolism, respectively. In this patient, 18FDOPA accumulation was decreased markedly in the caudate nucleus and the putamen bilaterally. In the images of 11C-YM-09151-2 and 18FDG, in contrast, no conspicuous changes were observed in the striatum. Thus our PET studies using 18FDOPA, 11C-YM-09151-2 and 18FDG provide a useful approach for assisting the diagnosis of JP, because the present findings are different from the results in patients with dopa-responsive dystonia and hereditary progressive dystonia with marked diurnal fluctuation. Furthermore, our findings are of particular interest in relation to the pathogenesis of JP.Copyright Lippincott-Raven Publishers

Primary central nervous system leukemia with a novel chromosomal translocation.

A case of central nervous system (CNS) leukemia with normal bone marrow, associated with a novel chromosomal abnormality, is described. A 58 year-old woman complained of hearing disturbance, severe headache and vomiting, and showed signs of meningeal irritation, as well as papilledema and bilateral dysacusis. Immature atypical cells were found in the cerebrospinal fluid (CSF) with elevated pressure, pleocytosis, increased protein and decreased glucose levels. She was diagnosed as having neoplastic meningitis. In spite of intensive investigations, including bone marrow puncture, malignancies were not found in organs other than intra-cranial site. The symptoms and CSF findings were temporarily improved with chemotherapy and irradiation, but she relapsed into neoplastic meningitis. The anaplastic cells in CSF were positive with CD45 by immunocytochemistry, and were positive by peroxidase staining. Thus, the anaplastic cells were considered to be myelocytic leukemic cells. Chromosomal analysis showed that these leukemic cells had a novel chromosomal abnormality: 46XX, 4q+, 10q-, 16q-. There has been no report of leukemic meningitis without bone marrow abnormalities. It is possible that this peculiar abnormal chromosome is related to the primary infiltration of the central nervous system. With this novel chromosomal abnormality, this case is important for considering the mechanism of primary leukemic meningitis.

[Pregnancy after augmentation cystoplasty. A case report].

A 31-year-old woman with a history of spina bifida occulta became pregnant after ileocystoplasty for her neurogenic bladder. During the pregnancy she had frequent episodes of febrile urinary tract infections, and progressive hydronephrosis appeared in the second trimester. At 25 weeks gestation she was complicated by severe pyelonephritis requiring the intervention with the placement of double pigtail ureteral stent. However, long term efficacy of ureteral stent was questionable and this indwelling catheter caused bacteriuria which was not eradicated by intravenous antibiotics. Classical cesarean section was performed at 32 weeks of gestation due to the fear of fetal distress. Neobladder and mesenteric blood supply were adherent to the anterior surface of the uterus. Urinary tract infection is extremely common during pregnancy after enterocystoplasty. The most important point is prophylactic antibiotics throughout the pregnancy. At the time of cesarean section, a reconstructive urological surgeon should be part of the operative team and take great care to avoid injury to the blood supply of cystoplasty.

[A juvenile case of Lambert-Eaton myasthenic syndrome with severe emaciation].

Most of the patients with Lambert-Eaton myasthenic syndrome (LEMS) are over 40 years of age at onset and have a malignant neoplasm, especially pulmonary small cell carcinoma. Juvenile cases of LEMS are rare and have not been reported in Japan. We report a 21-year-old women whose onset of LEMS was at 17 years of age. In the early stage of the disease, she had limb muscle weakness and was diagnosed has having seronegative myasthenia gravis (MG) based on decrementing response in low frequency repetitive nerve stimulation, positive edrophonium test, and negative serum anti-acetylcholin receptor antibodies. Serum anti-voltage-dependent-calcium-channel (VGCC) antibody was negative with an available assay system. She was thymectomized and received anti-cholinesterases, corticosteroids, and cyclophosphamide, but their therapeutic effects were not evident. Two years later, she was confined to bed due to sever emaciation (Her body weight reduced from 47 Kg to 27 Kg.), bulbar palsy, and limb muscle weakness. She needed respiratory support and tube feeding. Type II fiber atrophy, which is non-specific, but is a common finding in LEMS, was seen in the biopsied muscle of the patient. On the other hand, postsynaptic folds and clefts of all seven motor end-plates in the specimen were normal, which is against the diagnosis of MG. Recently, she was found to have anti-VGCC antibody with an improved assay system. Despite vigorous examination, no malignancy or other autoimmune disorders have been found. 3,4-Diaminopyridine was effective to improve limb muscle powers, but she is still bedridden.

[A case of Ki-1 lymphoma-associated eosinophilic polymyositis].

We report on a patient of Ki-1 lymphoma-associated hypereosinophilic syndrome and eosinophilic polymyositis. A 22-year-old male was admitted to a hospital because of anterior chest pain, when he had a white cell count of 12,000 with 33% of eosinophils. He first noticed muscle weakness in the right shoulder at age 23 years. At the age of 25, he experienced marked muscle weakness in his arms and neck, which brought him to our hospital. Neurological examination on admission revealed moderate muscle atrophy and weakness in the neck flexors and both upper extremities, and marked muscle hypertrophy in the sternocleidomastoideus and trapezius muscles. Laboratory studies showed a white blood cell count of 17,700 with 56% of eosinophils. His serum creatine kinase (CK) level was elevated to 347 units. A muscle biopsy specimen showing active myositis infiltrated by eosinophils confirmed the diagnosis of eosinophilic myositis. Although eosinophilia and serum CK activity decreased markedly with corticosteroid treatment, his muscle weakness was unchanged. At the age of 27 years, lymphadenopathy developed. Immunocytochemical examination on a biopsied neck lymph node indicated Ki-1 lymphoma with positive Ki-1, CD45 and epithelial membrane antibodies. In spite of chemotherapy and local irradiation treatment, he died four months after the emergence of Ki-1 lymphoma. We discussed the relationship between hypereosinophilic syndrome, eosinophilic polymyositis and Ki-1 lymphoma on pathogenetic mechanism.

Melanin-laden macrophages in cerebrospinal fluid in Vogt-Koyanagi-Harada syndrome.

OBJECTIVE: To identify basophilic granules in the cytoplasm of macrophages in cerebrospinal fluid (CSF) smears from patients with Vogt-Koyanagi-Harada (VKH) syndrome as melanin pigment. DESIGN: Morphological and immunocytochemical studies were performed on CSF smears obtained from 7 patients with VKH syndrome. Specimens were stained with May-Grünwald and evaluated by a silver impregnation method (Fontana-Masson staining). For immunocyto-chemical study, the smear was reacted with mouse anti-human melanoma cell (HMB-45) monoclonal antibody. RESULTS: Basophilic granules were detected in the cytoplasm of macrophages in CSF cell smears obtained from 6 of 7 patients with VKH syndrome in the early stages (within 25 days). Using the silver impregnation method, these basophilic granules were strongly stained black. Moreover, they were identified as melanin by positive immunocytochemical staining. The total number of cells containing cytoplasmic granules ranged from 3 to 8 per 10,000 total cells in CSF. In contrast, no cells containing cytoplasmic granules were found in control CSF cell smears from patients with viral, bacterial, or noninflammatory diseases. CONCLUSION: The identification of melanin-laden macrophages in the CSF of patients with VKH syndrome suggests that these cells are responsible for pleocytosis in the immunopathologic process of VKH syndrome.