Development of a particle agglutination assay system for detecting Japanese encephalitis virus-specific human IgM,using hydroxyapatite-coated nylon beads

Japanese encephalitis virus-specific IgM is a reliable indicator for serodiagnosis of Japanese encephalitis. A particle agglutination (PA) assay system was developed to detect anti-Japanese encephalitis virus IgM in human serum samples. The newly developed PA assay consisted of hydroxyapatite-coated nylon beads and V-bottom 96-well microplates. Hydroxyapatite-coated nylon beads were coated with Japanese encephalitis virus antigens. Japanese encephalitis virus antigen-coated, hydroxyapatite-coated nylon beads agglutinated in the IgM-captured wells when anti-Japanese encephalitis virus IgM-positive serum samples were used. A button pattern was formed at the bottom of the wells when anti-Japanese encephalitis virus IgM-negative serum samples were used. Thirty anti-Japanese encephalitis virus IgM-positive serum samples from Japanese encephalitis-confirmed cases were tested by the PA assay. All these serum samples were determined to be Japanese encephalitis virus IgM-positive. IgM titers determined by the PA assay corresponded to those determined by enzyme-linked immunosorbent assay. The titers were consistent in two independent PA assays. These results indicate that the newly developed PA assay is a reliable method for detecting anti-Japanese encephalitis virus IgM in human serum samples and that this assay will be a suitable diagnostic system especially in rural areas of Asia.     

Saccular and utricular inputs to single vestibular neurons in cats

Saccular and utricular organs are essential for postural stability and gaze control. Although saccular and utricular inputs are known to terminate on vestibular neurons, few previous studies have precisely elucidated the origin of these inputs. We investigated the saccular and utricular inputs to single vestibular neurons in whole vestibular nuclei of decerebrated cats. Postsynaptic potentials were recorded from vestibular neurons after electrical stimulation of the saccular and utricular nerves. Ascending and descending axonal projections were examined by stimulating the oculomotor/trochlear nuclei and the cervical segment of the spinal cord, respectively. After each experiment, locations of recorded neurons were identified. The recorded neurons (140) were classified into vestibulo-spinal (79), vestibulo-oculo-spinal (9), and vestibulo-ocular (3) neurons based on antidromic responses; 49 other vestibular neurons were unidentified. The majority of recorded neurons were mainly located in the lateral vestibular nucleus. Most of the otolith-activated vestibular nuclei neurons seemed to participate in vestibulospinal reflexes. Of the total 140 neurons recorded, approximately one third (51) received saccular and utricular inputs (convergent neurons). The properties of these 51 convergent neurons were further investigated. Most (33/51) received excitatory postsynaptic potentials (EPSPs) after saccular and utricular nerve stimulation. These results implied that most of the convergent neurons in this study additively coded mixed information for vertical and horizontal linear acceleration. Based on the latencies of convergent neurons, we found that an early integration process for vertical and horizontal linear acceleration existed at the second-order level.     

Preferential expression of T(h)2-type chemokine and its receptor in atopic dermatitis

Lesional skin of patients with atopic dermatitis (AD) is histologically characterized by hypertrophy of the skin, and the infiltration of a large number of eosinophils and T cells into the dermis. Recent studies have indicated that Th2 cells play a crucial role in the pathogenesis of AD skin. Chemokines and their receptors are implicated in the development of symptoms of various skin diseases such as AD and psoriasis vulgaris (psoriasis). We have examined the in situ expression of a typical Th2-type chemokine, thymus- and activation-regulated chemokine (TARC), and its receptor (CCR4) using immunohistochemical techniques. TARC was found to be highly expressed in the basal epidermis of the lesional skin of AD patients and only slightly in the non-lesional skin. On the other hand, no positive cells were seen in the lesional skin of psoriasis. Consistently, CCR4+ cells were present predominantly in the lesional skin of AD patients, but not in the non-lesional skin. In contrast, in the lesional skin of psoriasis patients, cells positive for CCR5, which is expressed on Th1 cells, were abundantly present. Interestingly, psoralen plus ultraviolet A therapy reduced the number of CCR4+ cells in the AD skin lesions. These results suggest that Th2-type cytokines such as TARC are involved in the pathogenesis of skin lesions in AD patients through the preferential recruitment of Th2 cells.     

Single sperm analysis of the CAG repeats in the gene for Machado-Joseph disease (MJD1): evidence for non-Mendelian transmission of the MJD1 gene and for the effect of the intragenic CGG/GGG polymorphism on the intergenerational instability

To investigate the mechanism of the meiotic instability of expanded CAG repeats in the gene for Machado-Joseph disease (MJD1), we analyzed the CAG repeat sizes of 1036 single sperm from six individuals with Machado- Joseph disease (MJD). The segregation ratio between single sperm with an expanded allele and those with a normal allele is significantly different (P <0.0001) from the expected 1:1 segregation ratio, which demonstrates segregation distortion of expanded alleles in male meiosis. In single sperm from individuals with the [expanded (CAG)n- CGG]/[normal (CAG)n-GGG] genotype, significantly greater instability of the CAG repeat was observed compared with single sperm from individuals with the [expanded (CAG)n-CGG]/[normal (CAG)n-CGG] genotype (F-test, P <0.001). These findings in single sperm confirm non-Mendelian transmission of the MJD1 gene and the effect of the intragenic CGG/GGG polymorphism on the intergenerational instability of the CAG repeats in the MJD1 gene, which have been observed in clinical and genetic studies. Our results indicate similarities and dissimilarities between MJD and Huntington's disease or myotonic dystrophy in terms of the inter-allelic interaction, segregation distortions and size distribution of trinucleotide repeats in mutant alleles. Further study is required to determine whether there is a common mechanism underlying the instability of the triplet repeats in 'triplet repeat diseases'.      

Thymic sarcomatoid carcinoma with skeletal muscle differentiation: report of two cases, one with cytogenetic analysis

AIMS: Malignant thymic tumour histologically resembling a soft tissue sarcoma is extremely rare and defined as sarcomatoid carcinoma in the recent World Health Organization (WHO) classification. We report two such cases in which the tumour cells showed a prominent rhabdomyoblastic differentiation and analyse whether these tumours retain an epithelial nature at least in part. METHODS AND RESULTS: One tumour occurred in a 51-year-old man (Case 1) and the other in a 40-year-old woman (Case 2). Microscopically, both tumours consisted essentially of two types of tumour cells: spindle and large round cells, with no apparent epithelial components. Osteosarcomatous small foci were also found in Case 2. Immunohistochemically, desmin and muscle-specific actin were positive in the majority of both types of tumour cells, whereas myogenin was predominant in the spindle cells and myoglobin in the large round cells. Some of both types of cells expressed cytokeratin with co-expression of myoglobin in the large round cells, but with no myogenin in the spindle cells. Some cytokeratin-positive spindle cells were also negative for desmin. Ultrastructural examination of a recurrent tumour in Case 2 revealed some epithelial features among the spindle cells. Cytogenetic study of the same tumour showed a complex abnormality including der(16)t(1;16)(q12;q12.1), an identical pattern previously reported in a case of thymic squamous cell carcinoma. CONCLUSIONS: The findings support the definition in the WHO classification of sarcomatoid carcinoma that includes purely sarcomatous tumour as in the present cases. Occurrence of this type of tumour may indicate a relationship between thymic epithelial cells and myoid cells and/or a potential for divergent differentiation in thymic epithelial tumours.     

Potential role of phosphodiesterase 7 in human T cell function: comparative effects of two phosphodiesterase inhibitors

Even though the existence of phosphodiesterase (PDE) 7 in T cells has been proved, the lack of a selective PDE7 inhibitor has confounded an accurate assessment of PDE7 function in such cells. In order to elucidate the role of PDE7 in human T cell function, the effects of two PDE inhibitors on PDE7A activity, cytokine synthesis, proliferation and CD25 expression of human peripheral blood mononuclear cells (PBMC) were determined. Recombinant human PDE7A was obtained and subjected to cyclic AMP-hydrolysis assay. PBMC of Dermatophagoides farinae mite extract (Df)-sensitive donors were stimulated with the relevant antigen or an anti-CD3 monoclonal antibody (MoAb). PBMC produced IL-5 and proliferated in response to stimulation with Df, while stimulation with anti-CD3 MoAb induced CD25 expression and messenger RNA (mRNA) synthesis of IL-2, IL-4 and IL-5 in peripheral T cells. A PDE inhibitor, T-2585, which suppressed PDE4 isoenzyme with high potency (IC50 = 0.00013 microM) and PDE7A with low potency (IC50 = 1.7 microM) inhibited cytokine synthesis, proliferation and CD25 expression in the dose range at which the drug suppressed PDE7A activity. A potent selective inhibitor of PDE4 (IC50 = 0.00031 microM), RP 73401, which did not effectively suppress PDE7A (IC50 > 10 microM), inhibited the Df- and anti-CD3 MoAb-stimulated responses only weakly, even at 10 microM. PDE7 may play a critical role in the regulation of human T cell function, and thereby selective PDE7 inhibitors have the potential to be used to treat immunological and inflammatory disorders.     

Contribution to the morphology of mitochondria with prismatic cristae in astrocytes of the inferior olivary nucleus of the cat

The internal structure of mitochondria with prismatic cristae in astrocytes of the inferior olivary nucleus of the adult cat was examined. The interior of the mitochondria with prismatic cristae cut in cross-section can be divided into 2 areas: (1) a peripheral, rather structureless area, and (2) a central, highly organized area. The former is composed of the inner mitochondrial membrane and a small number of peripheral cristae protruding from the membrane and scattered dots. The latter is composed of numerous prismatic cristae arranged in almost hexagonal spacing and many dots which probably represent the transverse configuration of filaments oriented parallel to the cristae embedded in the matrix. For future comparative analysis, various quantitative observations on the fine structure of the central, highly organized area are described and discussed.     

Cavernous structures composed of hepatocytes in experimentally induced hepatocellular carcinoma

Cavernous structures composed of hepatocytes were observed for the first time in trabecular-patterned hepatocellular carcinomas by light, scanning and transmission electron microscopy. The carcinomas were induced by a single dose of diethylnitrosamine combined with subsequent administrations of 2-acetylaminofluorene and partial hepatectomy. The cavernous structures had numerous cavities walled by flat hepatocytes. In the cavities, dendritic hepatocytes, connected with each other by their long and slender processes, formed a network and were attached to the fenestrated sinusoidal endothelium via intercellular spaces between the flat hepatocytes. Dendritic hepatocytes had smooth surfaces, prominent nucleoli, decreased glycogen, and well organized cisternae. Dilated bile canaliculi were sometimes seen among flat and dendritic cells. Dendritic cells showed more morphological dearrangement than flat cells. Blood cells were observed in the cavities. Kupffer cells were not seen in the sinusoids among the cavernous structures. The cavernous structures in the hepatocellular carcinomas resembled the hepatocyte arrangement in fetal liver in which intercellular spaces of hepatocytes are extremely widened.     

Disseminated Atypical Mycobacteriosis

Two cases of disseminated infection caused by Mycobacterium intracellulare were reported and discussed. In the first case, the patient was a fifty-seven-year-old male who complained of general fatigue, weight loss, and fever. Biopsy of the right inguinal lymph nodes and the liver revealed infiltration by histiocytes engulfing many acid-fast bacilli. At autopsy an egg-sized abscess was found is the region of the right Iliac lymph nodes. Histological examination showed histiocytic infiltration in the abscess wall, neighboring lymph nodes, liver, and spleen. In the second case, the patient was a four-year-old boy, who had persistent fever and splenomegaly. Splenectomy was performed and histological examination of the spleen revealed multiple nodular infiltration by swollen histiocytes with many acid-fast bacilli in their cytoplasm. The bone marrow aspirates and liver tissue obtained in the necropsy also showed many histiocytes containing many acid-fast bacilli. The authors emphasized the importance of paying special attention to atypical mycobacteriosis in feverish patients having lesions with a proliferation of histiocytes.