Suggestions for the assessment of the allergenic potential of genetically modified organisms

The prevalence of allergic diseases has been increasing continuously and, accordingly, there is a great desire to evaluate the allergenic potential of components in our daily environment (e.g., food). Although there is almost no scientific evidence that genetically modified organisms (GMOs) exhibit increased allergenicity compared with the corresponding wild type significant concerns have been raised regarding this matter. In principle, it is possible that the allergenic potential of GMOs may be increased due to the introduction of potential foreign allergens, to potentially upregulated expression of allergenic components caused by the modification of the wild type organism or to different means of exposure. According to the current practice, the proteins to be introduced into a GMO are evaluated for their physiochemical properties, sequence homology with known allergens and occasionally regarding their allergenic activity. We discuss why these current rules and procedures cannot predict or exclude the allergenicity of a given GMO with certainty. As an alternative we suggest to improve the current evaluation by an experimental comparison of the wild-type organism with the whole GMO regarding their potential to elicit reactions in allergic individuals and to induce de novo sensitizations. We also recommend that the suggested assessment procedures be equally applied to GMOs as well as to natural cultivars in order to establish effective measures for allergy prevention.     

Frequent co-localization of Cox-2 and laminin-5 gamma2 chain at the invasive front of early-stage lung adenocarcinomas

Laminin-5 is an extracellular matrix protein that plays a key role in cell migration and tumor invasion. Cox-2 is an induced isoform of cyclooxygenases that plays an important role in carcinogenesis, suppression of apoptosis, angiogenesis, and metastasis of colon cancer. We report frequent co-expression of cox-2 and laminin-5 at the invasive front of early-stage lung adenocarcinomas. We investigated the expression of cox-2 and laminin-5 immunohistochemically in 102 cases of small-sized lung adenocarcinoma (maximum dimension, 2 cm or less). Cox-2 and laminin-5 were expressed in 97 (95.1%) and 82 (80.4%) cases, respectively. Both were preferentially localized in cancer cells at the cancer-stroma interface, although cox-2 tended to show a diffuse staining pattern in some cases. A comparison of their staining patterns revealed a striking similarity in their distribution in 24 cases, and a partial overlap between their localization in another 20 cases. Moreover, an overall correlation was found between the expression levels of cox-2 and laminin-5 (P = 0.018). To gain insight into the mechanisms that regulate the expression of these proteins, we additionally studied their expression in 58 cases of stage I lung adenocarcinoma, in which p53 status was determined by immunohistochemistry, polymerase chain reaction-single strand conformation polymorphism analysis, and direct sequencing. The results showed that tumors with mutant p53 tended to express more cox-2 than those with wild-type p53 (P = 0.080). Also, tumors that overexpressed p53 had higher levels of cox-2 and laminin-5 than those without p53 overexpression (P = 0.032 and 0.047, respectively). Further immunohistochemical analysis showed that tumors that overexpressed both epidermal growth factor receptor (EGFR) and erbB-2 had higher levels of cox-2 and laminin-5 than those without concomitant overexpression of these proteins (P = 0.014 and P = 0.018, respectively). To see whether EGFR signaling is involved in cox-2 and laminin-5 expression, we further conducted in vitro analyses using six lung adenocarcinoma cell lines (A549, HLC-1, ABC-1, LC-2/ad, VMRC-LCD, and L27). Western blot analyses showed that cox-2 mRNA levels, and to a lesser extent laminin-5 gamma2 mRNA levels, correlated with the expression levels of erbB-2 and the phosphorylated form of MAPK/ERK-1/2 protein. The addition of transforming growth factor-alpha increased both cox-2 and laminin-5 gamma2 mRNA levels in A549, ABC-1, and L27 with different kinetics; the induction of cox-2 occurred earlier than that of laminin-5 gamma2. Finally, the migration of ABC-1 cells was inhibited by MAP kinase kinase inhibitor PD98059 and a selective cox-2 inhibitor NS-398. In contrast, the migration of A549 cells was inhibited by PD98059, but much less effectively by NS-398. These results suggest that co-stimulatory mechanisms may exist that increase the expression of cox-2 and laminin-5 at the invasive front of lung adenocarcinomas and that EGFR signaling could be one of the mechanisms. Further investigations are warranted concerning the role of cox-2 and laminin-5 in cancer cell invasion and the significance of p53 and EGFR signaling in the regulation of cox-2 and laminin-5 expression.     

IgE.IgG4 antibody in eczema in infants between 5-7 months of age. I. Relationship to the degree of eczema involvement]

Assessments have been made of serum IgE and IgA value, specific IgE and IgG4 antibody titers to foods, house dust, mite and percent peripheral++ eosinophil count in 191 infants with and without eczema between 5-7 months of age. Eczema patients were divided into 4 groups according to the degree of eczema involvement and laboratory data were compared between these and not eczematous group. The results were as follows; 1) IgE antibody titers were higher as the degree of eczema involvement increased. 2) RAST positive rates to foods increased with the degree of eczema involvement. Positive rate was highest to egg white followed by milk, soybean, wheat and rice. 3) Only 9 cases were RAST-positive to rice or wheat and all of them had IgE antibodies to other allergens tested. 4) A value of 10 IU/ml, which is the lowest measurable value of IgE in infants was considered to be a little too high to speculate allergy to some foods. 5) Specific IgG4 antibodies were positive only to milk. 6) All the 18 cases with positive milk-specific IgG4 antibody were all negative in milk-specific IgE antibody, and conversely all the 14 cases with positive milk-specific IgE antibody were negative in milk-specific IgG4 antibody. From these results, it was concluded that food allergy is related to the degree of eczema involvement in infants between 5-7 months of age.     

Enhancement of lipopolysaccharide-stimulated cyclooxygenase-2 mRNA expression and prostaglandin E2 production in gingival fibroblasts from individuals with Down syndrome

It is well known that Down syndrome (DS) is a premature ageing syndrome. Periodontal disease in individuals with DS develops rapidly and extensively in a relatively younger age bracket compared with that in healthy controls. The mechanisms involved in the periodontal inflammatory processes in DS patients are not fully understood. In the present study, the non-inflamed gingival fibroblasts isolated from seven patients with DS (DGF) and seven healthy controls (NDGF) were stimulated with lipopolysaccharide (LPS) derived from Actinobacillus actinomycetemcomitans (A. a.). We measured the level of prostaglandin E2 (PGE2) production by DGF and NDGF by radioimmunoassay, and also measured the mRNA expression of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) by using the real-time PCR method. We found the higher levels of LPS-stimulated COX-2 mRNA expression and PGE2 production in DGF when compared with those in NDGF. This study may indicate that overexpression of LPS-stimulated COX-2 induced a greater ability of DGF to produce PGE2, and that these phenomena may be responsible for the severer periodontal disease in DS patients.     

Histochemical, immunohistochemical and ultrastructural investigations of giant cell tumors of bone

The origin and characteristics of so-called stromal cells (stromal cell) and the osteoclast-like giant cell series of 19 cases of giant cell tumor (G.C.T.) of bone were studied. Immunohistochemically, two interesting cases were found. The stromal cells of one case were alpha-1-antitrypsin positive and those of the other case were alpha-1-antichymotrypsin positive. The histiocytic stromal cells of the latter case seemed to be surely neoplastic since they showed mild to moderate cell atypism. There were foci consisting of fibroblastic cells or osteoid and osteoblasts within the tumor. Those cells in the foci were apparently continuous with the surrounding stromal cells, and they were, therefore, also considered to be neoplastic. These findings strongly indicate that the stromal cells originate from the undifferentiated mesenchymal cells in the bone marrow and may differentiate to osteoblastic, fibroblastic, and histiocytic cells. All cells of these three series were not stained for a high stable form of acid phosphatase (SAPhase). SAPhase activity was demonstrated only in osteoclast-like giant cells and some mononuclear cells, which are recently believed to be non-neoplastic. Therefore, the cell atypia of SAPhase negative stromal cells is considered to have a prognostic value.     

Analysis of motion of the visual field by direction, expansion/contraction, and rotation cells clustered in the dorsal part of the medial superior temporal area of the macaque monkey

1. The dorsal part of the medial superior temporal area (MST) is characterized by clusters of three types of visually responsive cells: Direction cells, which respond to a straight frontoparallel movement in a particular direction; Expansion/contraction cells, which selectively respond to either an expansion or contraction; and Rotation cells, which selectively respond to either a clockwise or counterclockwise rotation. To study their functional role, experiments were carried out on immobilized monkeys, anesthetized with N2O. 2. The areal extent of stimulation was crucial for activation: movements of a pattern extending over a wide visual field elicited a larger response than those elicited by a local pattern. 3. The shape, exact size, and sign of contrast of the texture components of the pattern were not important in determining the magnitude of response. 4. Different cells responded to different ranges of speed of movement. 5. Expansion/contraction cells were activated more strongly by a real (isotropic) expansion/contraction than by an "axial expansion/contraction" in which a pattern expanded or contracted along a particular axis. Rotation cells were activated more strongly by a circular rotation in the frontoparallel plane than by a shearing movement. 6. We discuss the possibility that the cells are involved in the detection and analysis of wide-field movements, which are generally caused by a movement of the animal itself. The mode (straight transfer, expansion/contraction, or rotation), direction, and speed of the relative movement of the animal and the external space may be represented by the activity of the cells.     

Serum FDP assay with latex photometric immunoassay and its analysis by immunoblotting

Sixty patients' sera with FDP-E value ranged from 200 to 2,500 ng/ml by LPIA system were assayed for FDP-Total (FDP-T) by the same system and FDP-DD by ELISA. Correlations of values between FDP-E, FDP-T and FDP-DD were excellent and ratio of FDP-T/FDP-E (T/E) and FDP-DD/FDP-E (DD/E) from same 60 sera were 32.4 + 7.4 and 1.55 + 0.50, respectively. Ratio of T/E from sera of FDP-E value demonstrating more than 500 ng/ml were classified into three groups, namely, 25.0-35.0, less than 18.0, and more than 50.0. Ten sera in each group were analyzed for FDP fragments of D, DD, Y, DY or X (DY/X) and high molecular weight (HMW) with SDS-PAGE and immunoblot method. It was found that relative concentration and proportion of D and DD.E fragments showed its characteristic pattern in each group.     

Primary renal angiosarcoma: A case report and review of the literature

Primary renal angiosarcoma is very rare. To our knowledge, only 15 cases have been reported to date. A 77-year-old Japanese man with a unilateral kidney presented with massive hematuria followed by renal failure. A renal tumor was suspected and a left nephrectomy was performed. The histopathological diagnosis was angiosarcoma of the kidney. A hemorrhagic tumor measuring 10 × 5 cm and clotted blood was found in the modularly area. The atypical tumor cells had a sinusoidal and solid appearance, and showed Immunohistochemically positive reactions for some of the endothelial markers. The patient died about 21 months after the nephrectomy and the autopsy revealed massive metastases to the liver and retroperitoneum. One of the differential diagnoses of the case was anglomyolipoma, because the tumor cells were relatively bland in their histological appearance with entrapped fat cells in the pelvic area. Fifteen case reports with titles that included the term 'hemangiosarcoma/anglosarcoma', 'hemangioendothelloma/endothelloma' or 'vascular sarcoma' of the kidney were reviewed and compared to the present case.     

Antigen-induced elevation of immunoreactive endothelin-1 (ET-1) levels in ovalbumin-sensitized guinea pig airway tissue

Changes in the immunoreactive ET-1 levels during the anaphylactic reaction of airway tissue from ovalbumin-sensitized guinea pigs were investigated. ET-1-immunoreactivity (ET-IR) was detected in the epithelial and smooth muscle layers of tracheal sections from normal guinea pigs and it was enhanced slightly by phosphoramidon (1 microM) treatment. The ET-IR level of the epithelial layer of ovalbumin-treated tissue from actively sensitized animals was slightly higher than that from normal animals, but it was enhanced markedly by phosphoramidon (1 microM) treatment. Furthermore, the mean ET-IR level of homogenates of antigen-treated tracheal tissues from sensitized guinea pigs (22.8 +/- 1.55 fmol mg-1 protein, n = 5) was significantly higher than the corresponding normal level (12.3 +/- 1.21 fmol mg-1 protein, n = 5). These results suggest that increased epithelial airway ET-1 levels contribute to the anaphylactic reaction of guinea pig airways.     

Cloning and characterization of the cDNA encoding the HA protein of a hemagglutination-defective measles virus strain

cDNA clones corresponding to the mRNA for the hemagglutinin of the hemagglutination-defective strain AK-1 of measles virus were isolated and characterized. Compared with the prototype Edmonstron strain, 60 nucleotide substitutions that resulted in 18 amino acid changes were detected. An additional potential N-linked glycosylation site was added by point mutation, which was supported by the observation that the hemagglutinin of the AK-1 strain was stained more heavily after NaDodSO₄PAGE and periodic acid-Schiff (PAS) staining than the Edmonston strain. Computer-assisted analysis revealed that three reverse turns in the secondary structure had disappeared in the hemagglutinin of the AK-1 strain. Moreover, one of these structural changes occurred in the closely glycosylated region at amino acid residues 168–240, which appeared to be a biologically important functional domain. The isoelectric point calculated from the predicted amino acid sequence became about 1 pH unit more basic in the AK-1 strain than the Edmonston strain. This present study is the first sequence analysis of the hemagglutinin gene in a hemagglutination-defective strain of the measles virus.