Biological activities of Bacteroides forsythus lipoproteins and their possible pathological roles in periodontal disease

Bacteroides forsythus is a gram-negative, anaerobic, fusiform bacterium and is considered to be an etiological agent in periodontal disease. A lipoprotein fraction prepared from B. forsythus cells by Triton X-114 phase separation (BfLP) activated human gingival fibroblasts and a human monocytic cell line, THP-1, to induce interleukin-6 production and tumor necrosis factor alpha production. BfLP was found to be capable of inducing nuclear factor-kappaB translocation in human gingival fibroblasts and THP-1 cells. By using Chinese hamster ovary K1 cells transfected with Toll-like receptor genes together with a nuclear factor-kappaB-dependent CD25 reporter plasmid, it was found that signaling by BfLP was mediated by Toll-like receptor 2 but not by CD14 or Toll-like receptor 4. BfLP induced apoptotic cell death in human gingival fibroblasts, KB cells (an oral epithelial cell line), HL-60 cells (a human myeloid leukemia cell line), and THP-1 cells but not in MOLT4 cells (a T-cell leukemia cell line). Caspase-8, an initiator caspase in apoptosis, was found to be activated in these cells in response to BfLP stimulation. Thus, this study suggested that BfLP plays some etiological roles in oral infections, especially periodontal disease, by induction of cell activation or apoptosis.     

Association of Actinobacillus actinomycetemcomitans leukotoxin with nucleic acids on the bacterial cell surface.

Actinobacillus actinomycetemcomitans, a periodontopathic gram-negative bacterium, produces a leukotoxin that is a member of the RTX cytotoxin family. Although genes may function in toxin secretion, the leukotoxin is not secreted extracellularly but remains associated with the bacterial cell surface. We report here that this toxin-cell surface association is mediated by nucleic acids and directly demonstrate that the extracellular secretion of toxin occurs in growing cultures with increased ionic strength of medium. All examinations were performed with freshly harvested A. actinomycetemcomitans 301-b from anaerobic fructose-limited chemostat cultures. The occurrence of cell surface-localized DNA was shown by directly digesting whole cells with the restriction endonuclease EcoRI or HindIII, which yielded many DNA fragments. The cell surface DNA constituted about 20% of the total cellular DNA. The leukotoxin was released from the whole cells by digestion with DNase I as well as restriction endonucleases. Because the leukotoxin binds ionically to DNA, it is dependent on the ionic strength of buffers or media. Accordingly, the toxin was released from cells suspended in saline at pH 7.5 in the presence of increasing amounts of MgCl2 (0 to 10 mM) or NaCl (0 to 50 mM). Moreover, a considerable quantity of leukotoxin was detected in the culture supernatant of fructose-limited chemostat cultures when sodium succinate solution was pumped into the steady state as an additional salt (30 and then 50 mM). This toxin-DNA association was also found in well-characterized strains including not only the leukotoxin-producing ATCC 29522 but also the toxin production-variable ATCC 29523 and the non-leukotoxin-producing ATCC 33384 when these strains were grown in the chemostat culture.     

The UDP-galactose translocator gene is mapped to band Xp11.23-p11.22 containing the Wiskott-Aldrich syndrome locus

We have cloned a segment of the human gene encoding UDP-galactose translocator by genetic complementation of its defective mutant in mouse FM3A cells. Chromosome mapping using fluorescentin situ hybridization revealed that the cloned gene hybridized to the Xp11.23-11.23 region of the X chromosome. This region is shared by the locus of Wiskott-Aldrich syndrome, an X-linked recessive immunodeficiency disorder, characterized by defective sugar chains on cell surface components. Genetic and phenotypic similarities suggest a possible link between UDP-galactose translocator and the Wiskott-Aldrich syndrome (WAS).     

Bovine serum albumin (BSA) nephritis in rats II. Histological findings and complement activation by immune complex in SHR rats

We introduced a mesangiopathic form of glomerulonephritis in spontaneously hypertensive (SHR) rats. Bovine serum albumin (BSA) was given i.v. to primed rats for 3 weeks and they were unilaterally nephrectomized (Nx). Then, they received rabbit anti-BSA- (Group A) or normal serum (Group B) for seven days, and half the rats were killed to obtain another kidney (Ex-1). The remainder were killed two weeks later and their kidneys were examined (Ex-2). In Nx kidneys, the glomerular lesions were characterized by leucocyte accumulation in the capillary lumina and by deposition of rat IgG, rat C3 and BSA both in the mesangial area and along the capillary walls. Glomeruli of Ex-1 kidneys manifested varying degrees of hypercellularity in the mesangium; a few leucocyte accumulations in the capillary lumina were noted and the immune deposits had decreased in the mesangium but not on the capillary walls. In Ex-2 kidneys, mesangial hypercellularity was conspicuous. There were no remarkable histological differences between Group A and B rats; in Ex-1 and Ex-2 kidneys of Group A, rabbit IgG was closely associated with rat IgG or C3. Serological evaluation revealed that the amount of circulating rat anti-BSA antibody was relatively small and that C3 was consumed by newly formed circulating immune complexes during BSA administration. Polymorphonuclear leucocyte (PMN) binding assay revealed that complement fixation to the immune deposits occurred in vitro and that this activity was highest in tissue from Nx kidneys.     

Histological grade in invasive ductal carcinoma of breast correlates with the proliferative activity evaluated by BrdU: An immunohistochemical study including correlations with p53, c-erbB-2 and estrogen receptor status

Thirty cases of invasive ductal carcinoma of the breast were classified to histological subtype according to the General Rules for Clinical and Pathological Recording of Breast Cancer of the Japanese Breast Cancer Society and histologically graded using the Nottingham method and the correlation of histology with proliferative activity was investigated using bromodeoxyuridine (BrdU). In addition, the overexpression of p53 protein, c-erbB-2 oncoprotein and estrogen receptor (ER) were immunohistochemically examined in order to discuss the relationship with histological subtype and histological grade. Histological grade correlated positively to the BrdU labeling index (LI) and overexpression of p53. High grade carcinoma demonstrated c-erbB-2 more frequently and exhibited a low incidence of ER. However, no significant relationship was found between BrdU LI, overexpression of p53 and c-erbB-2 and histological subtype. These results suggest that the histological grade does represent the proliferative activity of tumor cells and that adding the histological grade to the pathological diagnosis in invasive ductal breast carcinoma may be useful from the clinicopathological aspect concerning tumor behavior.     

Production of thymocyte-stimulating activity by cultured human thyroid epithelial cells.

Since thyroid follicular epithelial cells (thyrocytes) have been shown to express a number of functions similar to monocytes, they were further examined for their potency in secreting thymocyte-stimulating activity (TSA). Although spontaneous production of TSA could not be detected when thyrocytes were cultured in the culture medium, TSA was demonstrated in the culture supernatants after stimulation with the immune adjuvant lentinan. The release of TSA was found in the culture supernatants collected 24 h after stimulation and was maintained for the 4 days of culture. Maximum levels of TSA release were achieved by 2 days. In addition, when culture supernatants of thyrocytes stimulated with lentinan or monocyte-derived interleukin 1 (IL-1) were incubated with a rabbit antibody to human IL-1, a parallel reduction in TSA was observed, suggesting that the active product in the thyrocyte culture supernatant shared a common antigenic site with IL-1. The demonstration of the production of IL-1 like activity by thyrocytes provides additional evidence that these cells, in addition to their functions as endocrine cells, may also participate in the local immune responses under appropriate conditions.     

Isolation of amantadine-resistant influenza a viruses (H3N2) from patients following administration of amantadine in Japan

In Japan, the use of amantadine for treatment of influenza A virus infection was not accepted until November 1998, although it was widely used for treatment of Parkinsonism. Since then, we have monitored the emergence of amantadine-resistant viruses and isolated two viruses from patients on long-term treatment with amantadine.     

Defects in regulation of apoptosis in caspase-2-deficient mice

During embryonic development, a large number of cells die naturally to shape the new organism. Members of the caspase family of proteases are essential intracellular death effectors. Herein, we generated caspase-2-deficient mice to evaluate the requirement for this enzyme in various paradigms of apoptosis. Excess numbers of germ cells were endowed in ovaries of mutant mice and the oocytes were found to be resistant to cell death following exposure to chemotherapeutic drugs. Apoptosis mediated by granzyme B and perforin was defective in caspase-2-deficient B lymphoblasts. In contrast, cell death of motor neurons during development was accelerated in caspase-2-deficient mice. In addition, caspase-2-deficient sympathetic neurons underwent apoptosis more effectively than wild-type neurons when deprived of NGF. Thus, caspase-2 acts both as a positive and negative cell death effector, depending upon cell lineage and stage of development.     

Fate redirection of hippocampal astrocytes toward neuronal lineage by aggregate culture

Mammalian cells that have been committed to a certain cell lineage cannot be directed to other lineages. However, some astrocytes in the mammalian brains have been reported to represent plasticity to redirect to other cell lineages. We found that mouse hippocampal astrocytes cultured in aggregate forms of "astrosphere", redirected to MAP2-positive immature neurons. In astrospheres, basic HLH factors positively regulating neuronal differentiation were up-regulated and Id3 inhibiting basic HLH factors was down-regulated. Ectopic Id3 induction repressed redirection of astrocytes to a neuronal lineage, suggesting that astrosphere formation induced plasticity of astrocytes by changing the gene expression patterns.