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.     

Antigenic Characterization of Hantaan and Seoul Virus Nucleocapsid Proteins Expressed by Recombinant Baculovirus: Application of a Truncated Protein, Lacking an Antigenic Region Common to the Two Viruses, as a Serotyping Antigen

Hantaan virus (HTN) and Seoul virus (SEO) are members of the genus Hantavirus in the family Bunyaviridae and are causative agents of hemorrhagic fever with renal syndrome. The complete and truncated nucleocapsid proteins (NP) of HTN and SEO were expressed by a recombinant baculovirus system. Antigenic characterization of the NP using monoclonal antibodies (MAbs) indicated that the binding sites for the serotype-specific MAbs were located between amino acids (aa) 155 and 429. A Western blot assay indicated that the serotype-specific epitopes were conformation dependent. An indirect immunofluorescence antibody (IFA) assay with the truncated NP (aa 155 to 429) was able to distinguish convalescent-phase sera from HTN and SEO patients. However, the antibody titers with the truncated NP were lower than those with the whole NP. The truncated NP of SEO (aa 155 to 429) could be used as an enzyme-linked immunosorbent assay (ELISA) antigen, but the truncated NP from HTN lost its reactivity when used for ELISA. The IFA assay using baculovirus-expressed truncated NP as an antigen is a rapid, simple, and safe test for distinguishing between HTN and SEO infections by serotype.     

The effect of exhaustive exercise on the antioxidant enzyme system in skeletal muscle from calcium-deficient rats

 The aim of the current study was to elucidate the synergism of dietary calcium restriction and exhaustive exercise in the antioxidant enzyme system of rat soleus muscle, and to investigate the involvement of neutrophils in exercise-induced muscle damage. Forty-eight male Wistar rats were assigned to the following groups: control (C) or calcium-restricted [1 month (1 M) or 3 months (3 M)]. Each group was subdivided into acutely exercised or non-exercised groups. Soleus muscle from each rat was analysed to determine the levels of antioxidant enzymes [Mn-superoxide dismutase (SOD), Cu,Zn-SOD, glutathione peroxidase (GPX), and catalase (CAT)]. Dietary calcium restriction resulted in calcium deficiency and upregulated the antioxidant enzymes examined except GPX. Conversely, exhaustive exercise significantly decreased GPX and CAT, but not SODs activities in the calcium-restricted (1 M and/or 3 M) rats. Contents of immunoreactive Mn-SOD and Cu,Zn-SOD were only increased in the 3 M rats. During calcium restriction, the mRNA expression of both forms of SOD showed initial upregulation, followed by downregulation. Exhaustive exercise significantly increased the mRNA expressions only in the 3 M rats. Moreover, exhaustive exercise markedly increased myeloperoxidase activity in soleus muscles from the 1 M and 3 M rats compared with the C rats, and significantly enhanced the ability of neutrophils to generate superoxide in the 3 M rats. The results demonstrate that dietary calcium restriction upregulates certain antioxidant enzyme activities in rat soleus muscle, indicating an enhanced resistance to potential increases in intracellular reactive oxygen species. The results also suggest that exhaustive exercise may cause oxidative damage in soleus muscle of calcium-deficient rats through the activation of neutrophils. Received: 4 August 1997 / Received after revision: 29 September 1997 / Accepted: 26 November 1997     

Characterization of a neurotoxigenic Clostridium butyricum strain isolated from the food implicated in an outbreak of food-borne type E botulism.

Neurotoxigenic Clostridium butyricum was isolated from the food implicated in an outbreak of clinically diagnosed type E botulism in China. PCR assay showed that the isolate (LCL 155) contained the type E botulinum toxin gene. This appears to be the first report of neurotoxigenic C. butyricum causing food-borne botulism.     

Reduction of cell proliferative activities of gastric stump adenomatous hyperplasias after bile reflux diversion in rats

Previously we reported the majority of lesions induced by bilereflux, in the absence of chemical carcinogens, in the rat remnantstomach to consist primarily of gastric type and secondarilyof intestinal type cells, and that they are reversible afterdiversion of bile reflux. The present study was designed toevaluate changes in proliferative activities in cells of eachtype under these conditions. The frequency of adenomatous hyperplasia(AH) induced in the gastric stump mucosa by duodenal contentreflux after Billroth II partial gastrectomy (BII) increaseduntil the 54th week of the experiment. Roux-en-Y (RY) surgicalprocedure which prevents duodenal reflux performed at the 24thor 36th week after BII led to a decrease in AH. Cell contentof the lesions was analyzed using routine H&E staining,immunohistochemical staining for pepsinogen isoenzyme 1 andhistochemical procedures for mucins (paradoxical concanavalinA, galactose oxidase Schiff and sialidase galactose oxidaseSchiff reactions) and proliferation in each compartment evaluatedby an immunohistochemical method using bromodeoxyuridine (BrdU)and a monoclonal antibody against BrdU. At the 54th week thenumber of BrdU-labeled cells per normal pyloric column was significantly(P < 0.05) increased to 10.63/pit after the BII operation,while it diminished to 5.23/pit after RY diversion, this beingthe same level as with the RY procedure alone. AH maintaineda high rate of BrdU incorporation at 12.7% after BII operation,which was also significantly reduced (P < 0.01) to 7.0% bythe RY surgery. The intestinal type cell showed highest (22.2%),the surface mucous type cell showed the next (16.5%) and thepyloric gland type cell showed lowest (5.2%) BrdU labeling indicesafter BII operation. All the cell types in AH showed similarproportional decreases in BrdU incorporation after RY diversion.Thus surgical intervention reverses the cell proliferation causedby bile reflux in the gastric stump.     

CD98 regulates the phosphorylation of HER2 and a bispecific anti-HER2/CD98 antibody inhibits the growth signal of human breast cancer cells

Human epidermal growth factor receptor (HER) family proteins are currently major targets of therapeutic monoclonal antibodies against various epithelial cancers. However, the resistance of cancer cells to HER family-targeted therapies, which may be caused by cancer heterogeneity and persistent HER phosphorylation, often reduces overall therapeutic effects. We herein showed that a newly discovered molecular complex between CD98 and HER2 affected HER function and cancer cell growth. The immunoprecipitation of the HER2 or HER3 protein from lysates of SKBR3 breast cancer (BrCa) cells revealed the HER2-CD98 or HER3-CD98 complex. The knockdown of CD98 by small interfering RNAs inhibited the phosphorylation of HER2 in SKBR3 cells. A bispecific antibody (BsAb) that recognized the HER2 and CD98 proteins was constructed from a humanized anti-HER2 (SER4) IgG and an anti-CD98 (HBJ127) single chain variable fragment, and this BsAb significantly inhibited the cell growth of SKBR3 cells. Prior to the inhibition of AKT phosphorylation, BsAb inhibited the phosphorylation of HER2, however, significant inhibition of HER2 phosphorylation was not observed in anti-HER2 pertuzumab, trastuzumab, SER4 or anti-CD98 HBJ127 in SKBR3 cells. The dual targeting of HER2 and CD98 has potential as a new therapeutic strategy for BrCa.     

The effect on the bones of condensed phosphate when used as food additives: Its Importance in Relation to Preventive Medicine

Based on the fact that chemical products such as binding agents are produced by mixing three kinds of phosphates with different ratios, we mixed metaphosphate, polyphosphate and pyrophosphate. Each was made to Na-phosphate, K-phosphate, and Ca-phosphate and each was mixed with commercial feeds so that the content of P would be approximately 0.1, 0.15, 0.3, 0.4, 0.6 and 1.0%. The prepared pellets were given to ICR, CF # 1 and AKR strains of mice at 29 days of age for 680 days and observations were made through this experimental period at different stages. The observations were also carried out on the mice administered with the experimental feeds for 1.5 months from 9 to 10.5 months of age. The observations were compared with those of the control group at all times. As a result, plasma 1 α, 25 (OH)₂ D₃ and P levels were always significantly higher in the phosphate administered groups relative to the control. Urine P and Fe increased while urine Ca decreased in the phosphate-treated groups. The effect of phosphates on the bones was studied taking soft X-ray pictures of hind legs and applying microdensitometry to them. Through these observations we recognized thinning of the cortex of bones, reduction of marrow trabecules and development of osteophyte. Histological observations disclosed that changes in knee joint tissues were apparent; that is, a decrease in or an irregular loss of the number of cells in superficial, intermediate, and radial strata of the joint cartilage, proliferation of subchondral bone, and the development of osteophytes were noted. As for muscles, diameters of musclar fibers became smaller; in particular, type II fibers showed greater shrinkage. Regarding kidneys, swelling and atrophy of glomerular capillaries, proliferation of mesangial cells, nephroselerosis, swelling, thinning, and loss of tubular epithelium, interstitial tissue inflammation, development of cylindruria, and deposition of calcium were observed. All these changes seem to be a particularly advanced aspect of the changes which are more pronounced with increasing dose and age. These changes were found even in the group administered with the feed containing 0.1% phosphorus, and, these changes were dependent on the concentration level of P. It was observed that administration to older subjects for a short term (1.5 months) produced effects stronger than those to younger subjects administered for a long term (10.5 months). The effects of condensed Ca-phosphate on bones were similar to those of condensed Na- and K-phosphates, and, hence, it was supposed that these effects were caused by phosphate radicals. An erratum to this article is available at .     

Immunocytochemical localization of aromaticl-amino acid decarboxylase and catechol-O-methyltransferase in blood vessel wall of the human dental pulp

Relatively large amounts of DOPA as compared with the concentration of norepinephrine are found in human dental pulp. AADC and COMT are localized in blood vessel walls of human dental pulp. This localization suggests a functional relationship between COMT and AADC with regard to the metabolism of DOPA.     

Catecholamine Release caused by Carbon Dioxide Insufflation during Laparoscopic Surgery

Purpose

We evaluated plasma catecholamine levels during pneumoperitoneum in laparoscopic surgery.

Materials and Methods

Plasma epinephrine and norepinephrine were evaluated in 29 patients who underwent laparoscopic retroperitoneal surgery in a half lateral decubitus position (group 1) or laparoscopic varicocelectomy in a Trendelenburg position (group 2).

Results

The levels of epinephrine and norepinephrine increased significantly 5 minutes after carbon dioxide insufflation compared to levels after Veress needle insertion and just before insufflation. The elevation of catecholamine levels during laparoscopic procedures was greater in group 1.

Conclusions

Our results indicate that carbon dioxide insufflation may cause catecholamine release during laparoscopic surgery. Careful monitoring of hemodynamics is mandatory at the beginning of the procedure.