Bradykinin Stimulates Type II Alveolar Cells to Release Neutrophil and Monocyte Chemotactic Activity and Inflammatory Cytokines

In the present study, we evaluated the potential of bradykinin (BK) to induce the release of neutrophil and monocyte chemotactic activity (NCA and MCA) and cytokines from an alveolar type II epithelial cell line, A549 cells. BK stimulated A549 cells to release NCA and MCA in a dose- and time-dependent manner (P < 0.001). Checkerboard analysis revealed that both NCA and MCA involved chemotactic and chemokinetic activity. Molecular sieve column chromatography showed three molecular weight masses (near 19 kd, 8 kd, and 400 d) for NCA and several molecular weight peaks (near 66 kd, 25 kd, 19 kd, 16 kd, and 400 d) for MCA. The release of NCA and MCA was inhibited by cycloheximide and lipoxygenase inhibitors (P < 0.01). The NCA and MCA were inhibited by leukotriene B4 (LTB4) receptor antagonist (P < 0.01), and the concentration of LTB4 was high enough for NCA and MCA. Antibodies to interleukin (IL)-8 and granulocyte colony-stimulating factor (G-CSF) attenuated NCA (P < 0.01), and antibodies to monocyte chemotactic protein-1 (MCP-1), G-CSF, and transforming growth factor (TGF)-β attenuated MCA (P < 0.01). The levels of IL-8, G-CSF, MCP-1, and TGF-β increased time dependently (P < 0.01). BK also stimulated the release of ILeukin-6 from A549 cells (P < 0.001). The receptors responsible for the release of NCA, MCA, and individual chemokines involved both BKB1 and BKB2 receptors. These data suggest that BK may stimulate alveolar type II pneumocytes to release inflammatory cytokines, which then may modulate the lung inflammation.     

SF-1/Ad4BP transforms primary long-term cultured bone marrow cells into ACTH-responsive steroidogenic cells

Bone marrow stem cells develop into haematopoietic and mesenchymal lineages, but have not been known to participate in steroidogenic cell production. Steroidogenic factor 1 (SF-1), also designated adrenal 4 binding protein (Ad4BP), is an essential orphan nuclear receptor for steroidogenesis as well as for adrenal and gonadal gland development. In the present study, we revealed that the adenovirus-mediated forced expression of SF-1 can transform cultured primary long-term cultured bone marrow cells into steroidogenic cells, showing the de novo synthesis of multiple steroid hormones in response to adrenocorticotropic hormone (ACTH). This finding may provide an initial step in innovative autograft cell transfer therapy for steroid hormone deficiencies.     

Induction of Thymus-Derived γδ T Cells by Escherichia coli Enterotoxin B Subunit in Peritoneal Cavities of Mice

We examined the activation of intraperitoneal T cells in BALB/c mice by the Escherichia coli enterotoxin B subunit, which induced a specific Th2 type of T-cell response to intraperitoneally coadministered bovine immunoglobulin G. The numbers of both γδ and αβ T cells increased significantly after intraperitoneal administration of the B subunit in a time-dependent manner; these numbers were not affected by the B-subunit G33D mutant, which is defective in GM₁ ganglioside-binding ability. Early after administration a small number of γδ T cells produced either interleukin-4 (IL-4) or gamma interferon, while late after administration primarily IL-10-producing γδ T cells were detected. γδ T cells induced by the B subunit did not express a characteristic V gene over the time course of the study. The induction of γδ T cells did not occur in athymic nu/nu mice but could be induced upon transplantation of fetal AKR thymus-like αβ T cells. γδ T cells in athymic nu/nu mice with a fetal thymic graft predominantly expressed the donor Thy-1.1 antigen but not the host Thy-1.2 antigen. The induction of these T cells, however, could not be restored by coadministration of the B subunit with peritoneal cells from normal mice. These results suggest that the B subunit activates intraperitoneal γδ and αβ T cells in a manner dependent upon its ability to bind to GM₁ ganglioside. γδ T cells induced by the B subunit are Th2-type cells derived from the thymus. These γδ T cells may be functionally involved in specific Th2 responses to the B subunit, which possibly acts as an adjuvant through the influence of αβ T cells.     

Over-expression of Aurora-A targets cytoplasmic polyadenylation element binding protein and promotes mRNA polyadenylation of Cdk1 and cyclin B1

Aurora-A is a centrosomal serine-threonine kinase that regulates mitosis. Over-expression of Aurora-A has been found in a wide range of tumors and has been implicated in oncogenic transformation. However, how Aurora-A over-expression contributes to promotion of carcinogenesis remains elusive. Immunohistochemical analysis of breast tumors revealed that over-expressed Aurora-A is not restricted to the centrosomes but is also found in the cytoplasm. This over-expressed Aurora-A appeared to be phosphorylated on Thr288, which is known to be required for its enzymatic activation. In analogy to Aurora-A's role in oocyte maturation and the early embryonic cell cycle, here we investigated whether ectopically over-expressed Aurora-A can similarly stimulate polyadenylation of mRNA in human somatic cultured cells by interacting with a human ortholog of cytoplasmic polyadenylation element binding protein, h-CPEB. In vitro experiments revealed that Aurora-A binds directly to, and phosphorylates, h-CPEB. We found that polyadenylation of mRNA tails of cyclin B1 and Cdk1 was synergistically stimulated when Aurora-A and h-CPEB were over-expressed, and they were further promoted in the presence of an Aurora-A activator Ajuba. Our results suggest a function of ectopically over-expressed Aurora-A that might be relevant for carcinogenesis.     

Inhibitory effect of antiserum to surface antigen P50 of Babesia gibsoni on growth of parasites in severe combined immunodeficiency mice given canine red blood cells

The inhibitory effect of an antiserum to surface protein P50 of Babesia gibsoni on the growth of the parasite was determined with severe combined immunodeficiency mice given canine red blood cells. The antiserum to the recombinant P50 protein significantly inhibited the parasite growth, indicating that P50 might be a useful vaccine candidate.     

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.     

Severe Neutropenia in Japanese Patients with X-Linked Agammaglobulinemia

X-linked agammaglobulinemia (XLA) is clinically characterized by recurrent bacterial infections during early infancy. Although it is not a phagocytic disorder, XLA is sometimes associated with neutropenia. We conducted a nation-wide survey to determine the frequency of neutropenia among Japanese XLA patients. Responses were received from 87 (86%) of 101 patients in which BTK mutations were previously identified, and of these, 16 (18%) had neutropenia. All episodes of neutropenia occurred before initiation of intravenous immunoglobulin (IVIG) replacement therapy. Two XLA patients died of multiple organ failure caused by severe neutropenia and Pseudomonas sepsis before initiation of IVIG replacement therapy. These results suggest that, in some cases, severe bacterial infections in XLA patients might be caused not only by antibody deficiencies but also by neutropenia.     

Micromechanical properties of demineralized dentin collagen with and without adhesive infiltration

In a previous study, we reported the upper limit of Young's modulus of the unprotected protein at the dentin/adhesive interface to be 2 GPa. In this study, to obtain a more exact value of the moduli of the components at the d/a interface, we used demineralized dentin collagen with and without adhesive infiltration. The prepared samples were analyzed using micro-Raman spectroscopy (micro RS) and scanning acoustic microscopy (SAM). Using an Olympus UH3 SAM (Olympus Co., Tokyo), measurements were recorded with a 400 MHz burst mode lens (120 degrees aperture angle; nominal lateral resolution, 2.5 microm). A series of calibration curves were prepared using the relationship between the ultrasonically measured elastic moduli of a set of known materials and their SAM response. Finally, both the bulk and bar wave elastic moduli were computed for a set of 13 materials, including polymers, ceramics, and metals. These provided the rationale for using extensional wave measurements of the elastic moduli as the basis for extrapolation of the 400 MHz SAM data to obtain Young's moduli for the samples: E = 1.76 +/- 0.00 GPa for the collagen alone; E = 1.84 +/- 0.65 GPa for the collagen infiltrated with adhesive; E = 3.4 +/- 1.00 GPa for the adhesive infiltrate.     

Role of complement in acute tubulointerstitial injury of rats with aminonucleoside nephrosis.

The present work was designed to elucidate the in vivo role of complement in the proteinuria-associated tubulointerstitial injury. Rats were intravenously injected with puromycin aminonucleoside, and massive proteinuria was observed within 5 days. Prominent tubulointerstitial injury characterized by proximal tubular degeneration, tubular dilatation, and leukocyte infiltration were observed 7 days after injection. C3 and C5b-9 were observed in the luminal side of proximal tubular cells. Renal function, assessed by inulin and para-aminohippurate clearance, was significantly decreased. To-assess the role of complement in this model, rats were injected with either cobra venom factor or soluble recombinant human complement receptor type 1 starting at day 3. These manipulations significantly improved tubulointerstitial pathology and para-aminohippurate clearance without affecting the degree of proteinuria. Deposition of C3 and C5b-9 was not detected in the kidney of rats depleted of complement by cobra venom factor. In rats treated with soluble complement receptor, C3 was still detected in the tubules, but deposition of C5b-9 was not observed. Soluble complement receptor was detected at the site of C3 deposition and in the urine. These data strongly suggest that complement plays a pivotal role in proteinuria-associated tubulointerstitial injury and that systemic complement depletion or inhibition of complement in the tubular lumen may diminish the tubulointerstitial damage.     

Morphogenesis of nonpolypoid colorectal adenomas and early carcinomas assessed by cell proliferation and apoptosis

Nonpolypoid neoplasms, as well as ordinary polypoid tumours, are occasionally found in the colorectum. To clarify whether cell kinetic status affects the macroscopic morphology of colorectal neoplasms, we investigated proliferative indices (PI), apoptotic indices (AI), and the expression of apoptosis-related gene products. We examined 110 colorectal neoplasms comprised of 36 polypoid, 38 flat elevated and 36 depressed tumours. According to WHO’s criteria these tumours consisted of 61 adenomas with low grade dysplasia (LGD), 30 adenomas with high grade dysplasia (HGD) and 19 carcinomas with submucosal invasion. Apoptotic cells were detected by TUNEL staining. Proliferating cells and apoptosis-related gene products were assessed by immunohistochemistry for Ki-67, p53, Bcl-2, and Bax antigens. AI were closely associated with macroscopic morphology in adenomas but not in carcinomas. PI were relatively constant among the three macroscopic types in adenomas and carcinomas. Median AI values of polypoid, flat elevated and depressed tumours were 1.8%, 2.1% and 4.6% for adenomas with LGD, 0.8%, 2.4% and 6.2% for adenomas with HGD and 2.9%, 4.0% and 3.6% for carcinomas, respectively. Overall PI were significantly higher in carcinomas than in adenomas with LGD, whereas AI were not different. Although the incidence of expression was significantly higher in carcinomas for p53 and in adenomas for Bcl-2 than the others, the expression of apoptosis-related gene products (p53, Bcl-2 and Bax) was similar among polypoid, flat elevated and depressed tumours. Macroscopic morphology of colorectal adenomas is determined by the apoptosis not by proliferation, and high apoptosis found in depressed adenomas implies their low net growth. Received: 1 July 1999 / Accepted: 17 January 2000