Characterization of a Helicobacter pylori neutrophil-activating protein.

Helicobacter pylori-associated gastritis is mainly an inflammatory cell response. In earlier work we showed that activation of human neutrophils by a cell-free water extract of H. pylori is characterized by increased expression of neutrophil CD11b/CD18 and increased adhesiveness to endothelial cells. The work reported here indicates that the neutrophil-activating factor is a 150,000-molecular-weight protein (150K protein). Neutrophil proadhesive activity copurified with this protein, which is a polymer of identical 15K subunits. Specific antibody, prepared against the purified 15K subunit, neutralized the proadhesive activity of the pure protein and of water extracts obtained from different strains of H. pylori. The gene (napA) for this protein (termed HP-NAP, for H. pylori neutrophil-activating protein) was detected, by PCR amplification, in all of the H. pylori isolates tested; however, there was considerable strain variation in the level of expression of HP-NAP activity in vitro. HP-NAP could play an important role in the gastric inflammatory response to H. pylori infection.     

Carcinosarcoma of the esophagus with osseous and cartilagenous production. A combined study of keratin immunohistochemistry and electron microscopy

A case of polypoid carcinosarcoma of the esophagus is presented. Histologically the bulk of the tumor consisted of a sarcomatous tissue having large foci of osseous and cartilagenous differentiation and infiltrating deeply the wall, whereas a superficially, invasive squamous cell carcinoma associated with in-situ carcinoma was located at the base and luminal surface of the polypoid tumor. Intermingling of the carcinomatous and sarcomatous elements was found only in areas where they appeared to be collided. Ultrastructurally the sarcomatous portion contained cells with fibroblastic features but with no typical epithelial characteristics. Immunoperoxidase staining of the paraffin-embedded histologic sections for keratin proteins revealed, however, some positive spindle cells indicative of epithelial nature in the sarcomatous area, but the great majority of the sarcoma cells were devoid of keratin. These combined findings strongly suggest that the sarcomatous component in our case of true carcinosarcoma is derived from mesenchymal transformation (metaplasia) of the squamous carcinoma cells. The findings were discussed in light of the previous pertinent literature.     

Biological properties and gene expression associated with metastatic potential of human osteosarcoma

Lung metastasis has a great influence on the prognosis of patients with osteosarcoma. We previously established two high-metastatic sublines, M112 and M132, from the HuO9 human osteosarcoma cell line by in vivo selection. In this study, we newly isolated a high-metastatic subline, H3, and three low-metastatic sublines, L6, L12 and L13, from HuO9 by the dilution plating method. Three high-metastatic sublines produced more than 200 metastatic nodules in the lung, while three low-metastatic sublines produced no or few nodules after injection of 2 × 10⁶ cells into the tail vein of nude mice. There were significant differences in the motility and invasiveness between high- and low-metastatic sublines, whereas the growth rates in vitro and the tumorigenicity in vivo showed no correlation with their metastatic abilities. Early adherence to culture plates was significantly lower in two of three low-metastatic sublines, which occupied smaller surface areas on the culture plates than other sublines did. Comparison of the expression of 637 cancer-related genes by cDNA microarray revealed that seven genes were differentially expressed between high- and low-metastatic sublines. Among them, five genes (AXL, TGFA, COLL7A1, WNT5A, and MKK6) were associated with adherence, motility, and/or invasiveness. These results suggest that the differences in motility/invasiveness and adhesive abilities are key determinants of lung metastasis in osteosarcoma. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification and characterization of PDZRN3 and PDZRN4 genes in silico

NUMB and NUMBL are implicated in cell fate determination through the inhibition of Notch signaling. LNX, binding to NUMB and CXADR (CAR), functions as E3 ubiquitin ligase at least for NUMB. LNX is the paralog of PDZRN1 (PDZ domain containing RING finger 1). Here, we identified two novel homologs of LNX and PDZRN1 by using bioinformatics, which were designated PDZRN3 (LNX3 or SEMCAP3) and PDZRN4 (LNX4 or SAMCAP3L), respectively. KIAA1095 cDNA (AB029018) was the representative PDZRN3 cDNA. Complete coding sequence of PDZRN4 cDNA was determined by assembling nucleotide sequences of ESTs (BF059062 and AW297403), FLJ33777 cDNA (AK091096) and IMAGE5767589 cDNA (BC040922). PDZRN4 gene, consisting of 11 exons, was found to encode two isoforms with N-terminal divergence (PDZRN4 and PDZRN4S) due to an alternative promoter. PDZRN3-CNTN3 locus at human chromosome 3p13-p12.3 and PDZRN4-CNTN1 locus at human chromosome 12q12 were paralogous regions within the human genome. PDZRN3 (1066 aa) and PDZRN4 (1036 aa) showed 59.9% total-amino-acid identity. Two bipartite nuclear localization signals (NLS) were located within the C-terminal region of PDZRN3 and PDZRN4. PR34H1 and PR34H2 domains were identified as the regions conserved among PDZRN3, PDZRN4 and Drosophila CG1783. PDZRN3 and PDZRN4 consist of RING, two PDZ, PR34H1, PR34H2 domains and two NLS, while PDZRN1 and LNX consist of RING and four PDZ domains. PDZRN family proteins were classified into the LNX-PDZRN1 subfamily and the PDZRN3-PDZRN4 subfamily. This is the first report on the PDZRN3 and PDZRN4 genes.     

Development of a PCR method for rapid identification of new Streptococcus mutans serotype k strains

In a previous study, we isolated and characterized a new serotype k of Streptococcus mutans from human blood and oral cavities. Analysis of the genes involved in biosynthesis of the serotype-specific polysaccharide of serotype k strains revealed that the serotype k-specific nucleotide alignment was commonly present in the 5' region of the rgpF gene (350 bp from the initial sequence) compared to the reference strains, and then a method for rapid identification of serotype k strains was developed by use of PCR with primers designed on the basis of the sequence of the variable region. PCR assays with primers specific for amplification of serotype k strains showed a negative reaction with serotype c, e, and f strains and a positive reaction with serotype k strains, with the sensitivity for identification of the serotype k strains shown to range from 5 to 50 cells. Next, the frequency of positive reactions for serotype k-specific primers was surveyed with DNA taken from saliva samples from 200 subjects (2 to 18 years of age), and 10 of those showed a positive reaction, which was higher than the frequency in our previous survey with a serological method. In addition, all saliva samples from subjects with serotype k strains in our previous study were shown to be positive with the serotype k-specific primers. These results indicate that this new PCR method is effective for identification of subjects with S. mutans serotype k.     

Primary pulmonary low-grade marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue type with prominent hyalinosis. A case report

We report a case of primary pulmonary low-grade marginal zone B-cell lymphoma of the mucosa-associated lymphoid tissue (MALT)-type with prominent sclerosis, which morphologically resembled pulmonary hyalinizing granuloma (PHG) or inflammatory pseudotumor (IPT) of the lung. The patient, a 66-year-old Japanese female with a history of Sj?gren's syndrome and primary biliary cirrhosis, presented with a lower left lobe mass 6.8 cm in diameter. Histologically, the lesion is characterized by dense bundles of collagen with scattered plasma cells, mature small lymphocytes, and histiocytes among the collagen bundles. Only the peripheral area of the nodule contained dense lymphoplasmacytoid and histiocytoid infiltrates. A few centrocyte-like cells were obscured by the numerous plasma cells and plasmacytoid cells. In addition, lymphoepithelial lesions and colonalized lymphoid follicles were identified by immunohistochemistry alone. Although PHG and IPT are unlikely to be confused with pulmonary MALT-type lymphomas, the present case suggests that MALT-type lymphoma should be added to the list of differential diagnoses for PHG and IPT.     

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.     

Use of conventional or replicating nucleic acid-based vaccines and recombinant Semliki forest virus-derived particles for the induction of immune responses against hepatitis C virus core and E2 antigens

Replicating and nonreplicating nucleic acid-based vaccines as well as Semliki Forest-recombinant Viruses (rSFVs) were evaluated for the development of a vaccine against hepatitis C virus (HCV). Replicating SFV-DNA vaccines (pSFV) and rSFVs expressing HCV core or E2 antigens were compared with classical CMV-driven plasmids (pCMV) in single or bimodal vaccine protocols. In vitro experiments indicated that all vaccine vectors produced the HCV antigens but to different levels depending on the antigen expressed. Both replicating and nonreplicating core-expressing plasmids induced, upon injection in mice, specific comparable CTL responses ranging from 10 to 50% lysis (E:T ratio 100:1). Comparison of different injection modes (intramuscular versus intraepidermal) and the use of descalating doses of DNA (1-100 microgram) did not show an increased efficacy of the core-SFV plasmid compared with the CMV-driven one. Surprisingly, rSFVs yielded either no detectable anticore CTL or very low anti-E2 antibody titers following either single or bimodal administration together with CMV-expressing counterparts. Prime-boost experiments revealed, in all cases, the superiority of DNA-based only vaccines. The anti-E2 antibody response was evaluated using three different assays which indicated that all generated anti-E2 antibodies were targeted at similar determinants. This study emphasizes the potential of DNA-based vaccines for induction of anti-HCV immune responses and reveals an unexpected and limited benefit of SFV-based vaccinal approaches in the case of HCV core and E2.     

Activities of a soluble extract from lymphoid cells of MRL mice. Effect on B cell differentiation in vitro

Soluble extract (sEx) was prepared from lymphoid cells of MRL/Mp-lpr/lpr(MRL/l) mice with early lupus nephritis and also of MRL/Mp-+/+ (MRL/n) mice. sEx from lymph node and spleen T cells of MRL/l mice had an activity for B cells to differentiate into immunoglobulin-producing cells but that of MRL/n mice did not show such an activity. sEx of MRL/l mice also enhanced the in vitro response of B cells to a suboptimal dose of lipopolysaccharide. Implication of these phenomena in the development of lupus nephritis is discussed.