Novel enzyme immunoassay of anti-insulin igG in human serum

A novel enzyme immunoassay of anti-insulin IgG in human serum is described. A serum sample containing anti-insulin IgG was treated with dextran-charcoal at pH 6.0 to remove endogenous insulin and subsequently incubated with dinitrophenyl biotinyl nonspecific rabbit IgG-insulin conjugate. The reaction mixture was further incubated with a rabbit (antidinitrophenyl bovine serum albumin) IgG-coated polystyrene ball to trap the complex formed between anti-insulin IgG and the conjugate. After washing to eliminate nonspecific IgG in the test serum, the polystyrene ball was incubated with dinitrophenyl-L-lysine to elute the complex. The eluate was incubated with an avidin-coated polystyrene ball. Finally, the amount of human anti-insulin IgG in the complex trapped onto the avidin-coated polystyrene ball was measured by incubation with rabbit (antihuman IgG (γ-chain)) Fab'-peroxidase conjugate. This enzyme immunoassay was 1,000-fold more sensitive than the conventional enzyme immunoassay, in which an insulin-bovine serum albumin-coated polystyrene ball was incubated with a serum sample containing anti-insulin IgG and subsequently with rabbit (antihuman IgG (γ-chain)) Fab'-peroxidase conjugate. The principle of the novel enzyme immunoassay can be used to more sensitively measure antibodies for most kinds of haptens and antigens than the conventional enzyme immunoassay.     

Role of macrophage and smooth muscle cell apoptosis in association with oxidized low-density lipoprotein in the atherosclerotic development.

To examine the role of the apoptosis of macrophages and smooth muscle cells in the development of atherosclerosis, human aortic tissues with intimal lesions were immunostained with antibodies against terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL), single-stranded DNA (clone F7-26), and active caspase-3. Apoptotic cells were detected in the intima using both TUNEL and single-stranded DNA, however, the latter method was the more sensitive one for detecting apoptotic cells in the early stages of atherosclerosis. The number of apoptotic cells increased as the disease progressed. It implies that the apoptosis of intimal cells is involved in the formation of atherosclerotic lesions. In addition, quantitative analyses of the cell types undergoing apoptosis using double-immunostaining revealed that the susceptibility of macrophages and smooth muscle cells to apoptosis was greater specifically in atheroma than in the other atherosclerotic lesions, and macrophages were more susceptible to apoptosis than smooth muscle cells. The frequency and spatial distribution of oxidized low-density lipoprotein (oxLDL) (FOH1a/DLH3)-positive cells were examined by immunohistochemistry, and the results resembled those of apoptotic cells. The number of oxLDL-positive cells in the intima significantly correlated with the susceptibility of smooth muscle cells, but not with that of macrophages, to apoptosis. These results suggest that oxLDL affects the apoptosis of smooth muscle cells during the atherosclerotic development.     

Hemangiopericytoma of the Greater Omentum

A 41-year-old Chinese woman was admitted to our hospital with epigastric pain. Computed tomography detected a heterogeneous enhancement tumor fed by the left gastroepiploic artery in the left lower quadrant and cholelithiasis. Excision of the tumor in the greater omentum and cholecystectomy were performed laparoscopically. Histological findings confirmed a diagnosis of hemangiopericytoma with low-grade malignancy. To our knowledge, hemangiopericytoma of the greater omentum is very rare, and only 12 cases were reported in English literature. We report a case of hemangiopericytoma arising in the greater omentum and review the literature.     

Complete nucleotide sequence of the Japanese isolate S of beet necrotic yellow vein virus RNA and comparison with European isolates

Summary The complete nucleotide sequences of beet necrotic yellow vein virus RNA-1 to RNA-4 of the Japanese isolate S (BNYVV-S) were determined and compared with those of French isolate (BNYVV-F2). The nucleotide sequences of the two isolates were very similar, differing by only 1.7% (RNA-1), 4.1% (RNA-2), 2.9% (RNA-3) and 3.6% (RNA-4), respectively. The differences of the amino acid sequences of the two isolates depended upon the open reading frames (ORF) as follows: P237, 1.4%; P22 (coat protein), 2.1%; 54k ORF, 3.4%; P42, 0.5%; P13, 1.7%; P15, 3.0%; P14, 7.0% P25, 6.4%; P31, 3.5%. Comparison of the coat protein and triple gene block (P42, P13 and P15) regions of RNA-2 with other isolates revealed that BNYVV-S was much more similar to the Yugoslavian isolate (BNYVV-Yu2) than to BNYVV-F2. The nucleotide differences between BNYVV-S and BNYVV-Yu2 were less than 1%. Based upon the grouping of BNYVV variants reported by Kruse et al. [10], BNYVV-S is thus considered to belong to the A type along with BNYVV-Yu2, whereas BNYVV-F2 is classified in the B type. Our data suggest that the Japanese isolate S may have been derived from European countries other than France or Germany.The BNYVV-S nucleotide sequences have been assigned the accession numbers D84410, D84411, D84412 and D84413 in the DDBJ.     

The absence of DNA polymerase kappa does not affect somatic hypermutation of the mouse immunoglobulin heavy chain gene

During the immune response to T cell-dependent antigen, somatic hypermutation (SHM) is introduced into immunoglobulin (Ig) genes. The variable region is the target for SHM and it is here that DNA lesions are introduced and mutations are generated. It has been suggested that error-prone DNA polymerase(s) may play an important role in this mutagenesis phase. Recently, DNA polymerase kappa (Polkappa), which belongs to the Y-family of DNA polymerases, was identified. Since a hot spot of SHMs (RGYW motif) is also a hot spot of mutations by human Polkappa, this enzyme was suggested to be an SHM instigator. In order to address the question whether Polkappa is involved in SHM, we immunized Polkappa-deficient mice and analyzed the SHM of the Ig heavy chain gene. We found that the SHM frequency and spectrum were indistinguishable between the Polkappa knockout mice and control mice. These results suggested that Polkappa is not essential for this process.     

Abdominal angiography is associated with reduced in-hospital mortality among pediatric patients with blunt splenic and hepatic injury: A propensity-score-matching study from the national trauma registry in Japan

PurposeThe aim of this study was to assess the association between the implementation of abdominal angiography and outcome among pediatric patients with blunt splenic or hepatic injury.MethodsThis was a retrospective observational study, with a study period of 14 years, from January 2004 to December 2017. Blunt-trauma patients with splenic or hepatic injury who were less than 19 years old were included in this study. We used propensity-score-(PS) matching analysis to assess the relationship between abdominal angiography and in-hospital mortality.ResultsIn total, 639 patients were eligible for analysis, with 257 patients included in the abdominal-angiography group and 382 patients in the no-abdominal-angiography group. After PS matching, 224 patients from each group were selected. In the PS matched patients, in-hospital mortality was lower in the abdominal-angiography group than in the no-abdominal-angiography group (4.9% vs. 11.2%, odds ratio 0.416, 95% confidence interval 0.177–0.903).ConclusionIn this population, the implementation of abdominal angiography was significantly associated with lower in-hospital mortality among pediatric patients with blunt splenic or hepatic injury compared with nonimplementation of abdominal angiography.Type of studyPrognosis study.Level of evidenceIII     

Progression of renal failure with anaemia and multiple effects of angiotensin-converting enzyme inhibitor in rats with renal mass reduction.

Several factors such as proteinuria and renal fibrosis may be important in the progression of many forms of chronic renal diseases. The purposes of the current study were to investigate the progressive renal failure of the rats with surgical renal mass reduction (RMR) and the effect of the angiotensin-converting enzyme (ACE) inhibitor, lisinopril, and to document correlation of several factors associated with progressive renal failure. Rats were subtotal (5/6) nephrectomized by resection of the renal poles and sham-operated. The functional, histological and haematological changes of the rats were studied for up to 10 weeks. After 2 weeks of RMR, oral administration of lisinopril (10 mg kg(-1) per day) was performed for 8 weeks. RMR resulted in progressive renal failure with proteinuria, monocyte/macrophage (ED1+) infiltration, anaemia as assessed by haemoglobin and haematocrit (Htc), renal hypertrophy as assessed by left kidney to body weight ratio (BKW/BW), and renal fibrosis as assessed by glomerular lesions and tubulointerstitial changes. Lisinopril exhibited renoprotection with antiproteinuric effect and inhibition of monocyte/macrophage (ED1+) infiltration. However, beneficial effect of lisinopril on anaemia was not observed. At 10 weeks after surgery, severity of proteinuria positively correlated with plasma creatinine (Pcr), BKW/BW, histological damage, and systolic blood pressure, and negatively correlated with haemoglobin. Severity of tubulointerstitial changes positively correlated with Pcr and blood urea nitrogen, and negatively correlated with haemoglobin and Htc. Moreover, monocyte/macrophage (ED1+) infiltration positively correlated with severity of proteinuria and tubulointerstitial changes. These findings strongly support that proteinuria, monocyte/macrophage infiltration and renal fibrosis appear to play principal roles in the progressive renal failure with anaemia and renoprotection of ACE inhibition may be mediated by multiple actions of ACE inhibitor. The present study confirms that rats with RMR is useful to explore target molecules for renoprotective drugs and evaluate renoprotective effect of new molecular entities.     

Selection of Human Ovarian Carcinoma Cells with High Dissemination Potential by Repeated Passage of the Cells in vivo into Nude Mice, and Involvement of Lex-determinant in the Dissemination Potential

Cells of the human tumor cell line RMG-1, derived from a clear-cell adenocarcinoma of the ovary, were injected intraperitoneally into nude mice, and the cells obtained from the tumor nodules in the mesenterium were found to form a larger number of, and larger-sized, tumor nodules than the original RMG-1 cells. The RMG-1-h cells, transferred into culture from the tumor nodules after a 4th in vivo passage, showed a dissemination potential as high as that of cells disseminating directly from the tissues, and exceedingly higher than that of RMG-1 cells. To assess the molecular bases of the different biological properties of RMG-1 and RMG-1-h cells, we compared the content and expression of various carbohydrate antigens in both cells. The chromosomal profile of RMG-1-h cells revealed their human origin and was identical to that of the original RMG-1 cells. In contrast to the broad histogram for the Le^x-bearing cells among RMG-1 cells in flow cytometry, the weakly and moderately positive cells toward anti-Le^x antibody were found to be eliminated from the histogram for the RMG-1-h cells, resulting in the enrichment of cells strongly expressing Le^x, which may account for the high dissemination potential. In addition, the adhesion of RMG-1 cells to mesothelial cells was found to be significantly inhibited by pretreatment of the cells with anti-Le^x antibody, indicating Le^x-mediated cell-to-cell interaction between ovarian cancer cells and mesothelial cells. By TLC-immunostaining, two Le^x-glycolipids, III³Fucα-nLc₄Cer and V³Fucα-nLc₆Cer were detected in both RMG-1 and RMG-1-h cells, and their total concentrations were not significantly different from each other. However, the hydrophobic moieties of Le^x-glycolipids in RMG-1-h cells were different from those in RMG-1 cells, suggesting that a difference in the structure of the hydrophobic moieties of Le^x is partly involved in the enhanced reactivity of RMG-1-h cells toward anti-Le^x antibody. Thus, the high dissemination potential of ovarian cancer cells was shown to be mediated by the Le^x-determinant and the Le^x-bearing cells are enriched by repeated in vivo passage of the cells into nude mice.     

Chemopreventive and therapeutic efficacy of orally active tyrosine kinase inhibitors in a transgenic mouse model of gallbladder carcinoma.

Biliary tract cancer (BTC) is the second most common primary hepatobiliary cancer after hepatocellular cancer. At the time of diagnosis, most BTC are at an advanced stage and are unresectable. There is presently no effective curative treatment of the advanced disease nor is there any effective clinical therapy that will prevent the development of BTC. All of these factors render gallbladder cancer nearly incurable with a poor survival rate. The aim of our study was to provide a better understanding of the mechanisms involved in the development of gallbladder carcinoma as the advancement of more effective treatment options would significantly improve prognosis. In the present study, we examined the effect of gefitinib, a selective epidermal growth factor receptor/tyrosine kinase inhibitor (EGFR/TKI), on the development of gallbladder carcinoma in BK5.erbB2 mice. In addition, we examined the effect of another quinazoline derivative, GW2974, which is able to block the activation of both the EGFR and erbB2, in this model. Animals were treated with either 400 ppm gefitinib or 200 ppm GW2974 as a supplement in the diet using either a chemopreventive or therapeutic protocol. The results show that both compounds were potent chemopreventive and therapeutic agents in this mouse model of human BTC. The results also suggest that activation of the EGFR plays an important role in development of BTC in this model and that targeting both the EGFR and erbB2 may be an effective strategy for treatment of this disease.