Open food challenge with milk, egg white and wheat

Background: A method for open food challenge test to determine food allergy has not been established in an evidence-based manner. Methods: We conducted an analysis of 438 open food challenges of raw milk (n=133, mean age 2.7+/-1.9 years), boiled egg white (n=216, 2.8+/-2.1 years) or udon noodles (n=89, 2.7+/-1.7 years) for the patients aged 1 year or more. Doses were increased (trace amounts, 1 g, 2 g, 5 g, 10 g, 20-30 g) every 20 minutes. Results: In total, 151 (38.5%) of food challenges were positive. The positive rates of milk, egg and wheat challenges were 35.8%, 42.4% and 33.3%, respectively. Of these, 76.2%, 32.5%, 27.8% and 0.7% had, respectively, skin, respiratory, gastrointestinal and cardiovascular symptoms. Although the prevalence of positive challenge increased with level of specific IgE, it did not correlate with the threshold amount of positive food challenge or the severity of symptoms. Among the challnege positive patients, 10.6% required injection of antihistamines, corticosteroids or adrenalines for the treatment of the symptoms. Conclusions: This challenge protocol seemed to be appropriate and safe.     

Modification of an <Emphasis Type="Italic">in vivo</Emphasis> Lung Metastasis Model of Hepatocellular Carcinoma by Low Dose N-nitrosomorpholine and Diethylnitrosamine

Previously, we established the in vivo lung metastasis model of rat HCC induced by two hepatocarcinogens, diethylnitrosamine (DEN) and N-nitrosomorpholine (NMOR) at a dose of 120 ppm. This model allows us to investigate modifying factors leading to the inhibition of metastasis formation. However, low survival rates made the evaluation of metastasis formation difficult. The current experiments were conducted to modify the experimental protocol to improve survival and to establish a better animal metastasis model. Lower doses of NMOR (80 or 40 ppm in drinking water) were given to F344 rats for 14 weeks after DEN treatment. Survival rates in the 80 ppm group and in the 40 ppm group were 57% and 81%, respectively and these values were significantly higher than that in 120 ppm. Incidences of lung metastasis in the 40 ppm group steadily increased up to 67% by week 36 while that in the 80 ppm increased sharply up to 86% by week 24. Severity of lung metastases in the 40 ppm group at week 36 was mild compared with the 80 ppm group at week 24. In the second experiment, in order to characterize HCC development and lung metastasis in the 40 ppm group, rats given DEN and then followed with 40 ppm NMOR were killed sequentially. Development of HCC was observed at week 14 and reached 100% incidence at week 20. First lung metastatic lesions were evident at week 22, and incidence of lung metastasis reached 100%. Tumor cells were identified in the blood at week 20 by RT-PCR. The current study revealed that 40 ppm NMOR for 14 weeks after DEN treatment developed HCC without lung metastases at week 22, then HCC with a frequent lung metastasis at week 40. Thus, it can be said that this system is a more appropriate model for elucidation of mechanisms of metastasis and also for analysis of factors to inhibit natural metastasis.     

Mutation in saposin D domain of sphingolipid activator protein gene causes urinary system defects and cerebellar Purkinje cell degeneration with accumulation of hydroxy fatty acid-containing ceramide in mouse

The sphingolipid activator proteins (saposins A, B, C and D) are small homologous glycoproteins that are encoded by a single gene in tandem within a large precursor protein (prosaposin) and are required for in vivo degradation of some sphingolipids with relatively short carbohydrate chains. Human patients with prosaposin or specific saposin B or C deficiency are known, and prosaposin- and saposin A-deficient mouse lines have been generated. Experimental evidence suggests that saposin D may be a lysosomal acid ceramidase activator. However, no specific saposin D deficiency state is known in any mammalian species. We have generated a specific saposin D(-/-) mouse by introducing a mutation (C509S) into the saposin D domain of the mouse prosaposin gene. Saposin D(-/-) mice developed progressive polyuria at around 2 months and ataxia at around 4 months. Pathologically, the kidney of saposin D(-/-) mice showed renal tubular degeneration and eventual hydronephrosis. In the nervous system, progressive and selective loss of the cerebellar Purkinje cells in a striped pattern was conspicuous, and almost all Purkinje cells disappeared by 12 months. Biochemically, ceramides, particularly those containing hydroxy fatty acids accumulated in the kidney and the brain, most prominently in the cerebellum. These results not only indicate the role of saposin D in in vivo ceramide metabolism, but also suggest possible cytotoxicity of ceramide underlying the cerebellar Purkinje cell and renal tubular cell degeneration.     

Myopathy phenotype in transgenic mice expressing mutated PABPN1 as a model of oculopharyngeal muscular dystrophy

Autosomal dominant oculopharyngeal muscular dystrophy (OPMD)is a late-onset disorder characterized clinically by progressiveptosis, dysphagia and limb weakness, and by unique intranuclearinclusions in the skeletal muscle fibers. The disease is causedby the expansion of a 10-alanine stretch to 12–17 alanineresidues in the poly(A)-binding protein, nuclear 1 (PABPN1;PABP2). While PABPN1 is a major component of the inclusionsin OPMD, the exact cause of the disease is unknown. To elucidatethe molecular mechanism and to construct a useful model fortherapeutic trials, we have generated transgenic mice expressingthe hPABPN1. Transgenic mice lines expressing a normal hPABPN1with 10-alanine stretch did not reveal myopathic changes, whereaslines expressing high levels of expanded hPABPN1 with a 13-alaninestretch showed an apparent myopathy phenotype, especially inold age. Pathological studies in the latter mice disclosed intranuclearinclusions consisting of aggregated mutant hPABPN1 product.Furthermore, some TUNEL positive nuclei were shown around degeneratingfibers and a cluster of it in the lesion in necrotic musclefibers. Interestingly, the degree of myopathic changes was moreprominent in the eyelid and pharyngeal muscles. Further, muscleweakness in the limbs was apparent as shown by the fatigabilitytest. Nuclear inclusions seemed to develop gradually with aging,at least after 1 week of age, in model mouse muscles. We establishedthe first transgenic mouse model of OPMD by expressing mutatedPABPN1, and our model mice appear to have more dramatic alternationsin myofiber viability. ^* To whom correspondence should be addressed. Tel: +81 963736083; Fax: +81 963736599; Email: yamamura{at}gpo.kumamoto-u.ac.jp     

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.     

Non-invasive fibrosis assessments of non-alcoholic fatty liver disease associated with low estimated glomerular filtration rate among CKD patients: the Fukuoka Kidney disease Registry Study

Clinical and Experimental Nephrology - A growing body of evidence has shown that non-alcoholic fatty liver disease (NAFLD) is associated with chronic kidney disease (CKD). Non-invasive fibrosis...     

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.