Lipopolysaccharide Induces CD25-Positive, IL-10-Producing Lymphocytes Without Secretion of Proinflammatory Cytokines in the Human Colon: Low MD-2 mRNA Expression in Colonic Macrophages

Despite the huge number of colonized Gram-negative bacteria in the colon, the normal colon maintains its homeostasis without any excessive immune response. To investigate the potential mechanisms involved, human colonic lamina propria mononuclear cells (LPMCs) obtained from uninflamed mucosa were cultured with lipopolysaccharide (LPS) prepared from Bacteroides vulgatus (BV-LPS) or Bacteroides fragilis (BF-LPS), as representatives of indigenous flora, or pathogenic Salmonella minnesota (SM-LPS). Colonic LPMCs failed to produce inflammatory cytokines in response to any type of LPS. Colonic macrophages barely expressed mRNA for MD-2, an essential association molecule for LPS signaling via Toll-like receptor 4. Further, BV-LPS induced CD25 and Foxp3 expression in lymphocytes and CD4(+)CD25(+) cells expressed IL-10 mRNA. Thus, the low expression of functioning LPS receptor molecules and induction of IL-10-producing CD4(+)CD25(+) lymphocytes by indigenous LPS may play a central role in the maintenance of colonic immunological homeostasis.     

Treatment with aflatoxin B1 for immortalization of human fibroblasts from Li-Fraumeni patients

Immortalization of normal human fibroblasts is a very rare event. Multiple genes such as p53 and cellular senescence genes are possibly involved in immortalization of human fibroblasts, suggesting that multiple treatments with carcinogens are required for the immortalization. We describe here the procedure for immortalization of human fibroblasts (MDAH 087) from Li-Fraumeni cancer syndrome with a germ-line p53 mutation. The cells were subjected to multiple treatments with aflatoxin B₁ (AFB₁) in the presence of exogenous metabolic activation with rat liver post-mitochondrial supernatant (PMS), and 3 of 9 MDAH 087 cell cultures treated 1–3 times with 0.1–1 µg/ml AFB₁ became immortal, defined as continuous growth for over 300 population doublings after the first treatment. However, cultures of human fibroblasts from a normal embryo treated under the same conditions failed to escape senescence. The results indicate that the model of human fibroblasts with a mutated p53 allele exposed to AFB₁ is potentially useful for studying mechanisms of chemically induced immortalization.     

Infective endocarditis caused by Granulicatella elegans originating in the oral cavity

We studied the pheno- and genotypes of an oral Granulicatella elegans strain in comparison with those of a blood-derived isolate which caused infective endocarditis. The two isolates exhibited identical biochemical characteristics and had the same drug MICs. Their genotypes were indistinguishable, indicating that these were from the same clone. The transmission of G. elegans from the oral cavity thus should be noted as a possible cause of infective endocarditis.     

Immature dendritic cells (CD11c+ CD3- B220- cells) present in mouse peripheral blood

It is well known that dendritic cells (DCs) are developed from the peripheral blood of mice when peripheral blood mononuclear cells (PBMCs) are cultured with GM-CSF. We have previously found that immature DCs are present in the blood even in humans. In the present study, we show that CD11c+ CD3- B220- cells in the mouse peripheral blood are immature DCs. The percentage of CD11c+ CD3- B220- cells in the (PBMCs) of normal mice ranges from 0.5 to 2.5%. The CD11c+ CD3- B220- cells in the PBMCs show dendrites, similar in shape to the CD11c+ CD3- B220- cells in the spleen, which are thought to be DCs definitely. However, they have practically no capacity to stimulate the proliferation of allogeneic T cells, and show a lower expression of MHC class II, B7-1 and B7-2 than CD11c+ CD3- B220- cells in the spleen. When the CD11c+ CD3- B220- cells in the PBMCs are cultured with GM-CSF, they show not only the potent ability to stimulate the proliferation of allogeneic T cells but also a higher expression of MHC class II, B7-1 and B7-2. Moreover, they migrate into the spleen when they are injected intravenously. These results suggest that CD11c+ CD3- B220- cells in the PBMCs are immature DCs, and that they migrate into the spleen, where they mature.     

Immunoglobulin G from anti-glucose-6-phosphate isomerase antibodies positive patient with rheumatoid arthritis induces synovitis in cynomolgus monkeys

Anti-glucose-6-phosphate isomerase (GPI) antibodies (Abs) solely induce arthritis in mice. High titers of anti-GPI Abs are found in some patients with rheumatoid arthritis (RA), but their pathogenic role remains elusive. The aim of this study was to evaluate the pathogenic role of anti-GPI Abs in cynomolgus monkeys. IgG fractions were separated from sera of anti-GPI Abs-positive RA patients and healthy subjects and directly injected into the metacarpophalangeal joints of 4 cynomolgus monkeys. At day 16, the joints were harvested and examined histologically and immunohistochemically. The expression of C5a receptor (C5aR) molecule in the synovium was quantified by real-time PCR using cDNA from monkey joints. In monkey joints, IgG including anti-GPI Abs resulted in recruitment of granulocytes and mononuclear cells, strong deposition of human IgG on the articular surface, and overexpression of C5aR, but no joint swelling. No infiltrated cells or IgG deposition were observed in monkeys injected with IgGs from healthy subjects. Our results suggest that IgG fraction from RA patients including anti-GPI Abs may play a crucial role in the generation of synovitis in monkeys, although the pathogenesis of anti-GPI Abs in RA patients is still uncertain.     

Effect of Bcl-2 antisense oligonucleotide on drug-sensitivity in association with apoptosis in undifferentiated thyroid carcinoma

Although attempts have been made to treat undifferentiated thyroid carcinoma using multidisciplinary therapeutic procedures including surgery, radiotherapy, and chemotherapy, the prognosis of undifferentiated thyroid carcinoma remains quite poor. New approaches to increase the sensitivity of patients to anticancer drugs and radiation will be needed to improve the survival rate for undifferentiated thyroid carcinoma. We examined the effect of Bcl-2 antisense oligonucleotide on drug-sensitivity in association with apoptosis in the 8305C undifferentiated thyroid carcinoma cell line. The drug sensitivity was evaluated by MTT assay for 48 h, while apoptosis was assessed according to the formation of internucleosomal DNA ladders. The Bcl-2 antisense was introduced into 8305C cells by using a 18-mer phosphorothioate oligonucleotide by lipopolyamine-mediated transfection twice for 12 h. The expression of apoptosis genes was assessed by Western blotting. The 8305C cells were sensitive to adriamycin (ADM), mitomycin (MMC), docetaxel (TXT), and paclitaxel (TXL), showing mean IC50 values of 0.72, 1.1, 1.3, and 4.1 microM, respectively. In contrast, the 8305C cells were resistant to cisplatin (CDDP) and 5-fluorouracil (5-FU), with mean IC50 values of 42.0 and 48.0 microM, respectively. Treatment with Bcl-2 antisense suppressed the protein level of Bcl-2 in 8305C cells in a dose-dependent manner up to 1.0 microM. Drug-sensitivity was increased by pretreatment with Bcl-2 antisense as assessed by the IC50 (x-fold): 0.48 (1.5-fold) in ADM; 0.42 (2.6-fold) in MMC, 0.56 (2.3-fold) in TXT, 1.5 (2.7-fold) in TXL, 8.6 (4.9-fold) in CDDP, and 25.0 (1.9-fold) in 5-FU, respectively. The increased drug-sensitivity was associated with the induction of apoptosis-related proteins, Fas, caspase 8, cytochrome c, caspase 3, and to subsequent apoptosis, as determined by the formation of internucleosomal DNA ladders and PARP in the treated cells. Susceptibility in apoptotic cell death following treatment with anticancer drugs was associated with induction of apoptosis-related genes in undifferentiated thyroid carcinoma cells, and induction of apoptosis was enhanced by pretreatment with Bcl-2 antisense oligonucleotide. These results imply a potential new strategy targeting an antiapoptotic protein, Bcl-2, by its antisense oligonucleotide for enhancement of chemotherapeutic efficacy in undifferentiated thyroid carcinomas.     

P16-immunostaining pattern as a predictive marker of lymph node metastasis and recurrence in early uterine cervical cancer.

OBJECTIVES: We investigated the relationship between P16-immunostaining patterns and clinicopathological factors in early uterine cervix cancers and assessed whether P16-immunostaining patterns predict the prognosis of the patients with early uterine cervix cancers. METHODS: Twenty-nine early squamous cell carcinoma (SCC) specimens of the uterus were examined using immunohistochemistry for P16 expression. The P16-immunostaining pattern was classified into two groups: the homogeneous type and the heterogeneous type. P16-immunostaining patterns were evaluated in different parts of the carcinoma in situ (CIS): the center of the tumor and the front interface of the infiltrating tumor. RESULTS: All specimens were of the homogeneous type in CIS. The P16-immunostaining pattern was significantly of the heterogeneous type in the front interface of the infiltrating tumor with lymphatic invasion, vascular invasion, lymph node metastasis, and recurrence. Regarding the P16-immunostaining patterns in the front interface of the infiltrating tumor, the patients with the heterogeneous type showed a significantly worse prognosis than the patients with the homogeneous type. CONCLUSIONS: The prognosis of patients with early uterine cervical SCC may be predicted by evaluating the P16-immunostaining pattern in the front interface of the infiltrating tumor.     

Terminal deoxynucleotidyltransferase forms a ternary complex with a novel chromatin remodeling protein with 82 kDa and core histone

BACKGROUND: Terminal deoxynucleotidyltransferase (TdT) is a DNA polymerase that enhances the Ig and TcR gene diversity in the N region at the junctions of variable (V), diversity (D) and joining (J) segments in B- and T-cells. TdT synthesizes the N region in concert with many proteins including DNA-PKcs, Ku70 and Ku86. To elucidate the molecular mechanism of the N region synthesis, we first attempted to isolate the genes with products that directly interact with TdT. RESULTS: Using a yeast two-hybrid system, we isolated a cDNA clone encoding a novel nuclear protein that interacts with TdT. This protein was designated as TdT interacting factor 2 (TdIF2). The confined region of the C-terminal in TdIF2 is involved in specific interaction with the entire C-terminal in TdT. TdIF2 contains an acidic region comprised of 42 residues. TdIF2 was shown to bind specifically to a core histone by pull down assay using specific antibodies against TdIF2. When a TdT/TdIF2 complex was applied on to a DNA-cellulose column, only TdT bound to the column while TdIF2 passed through. TdIF2 reduces the TdT activity to 46% of its maximum value in vitro assay system using activated DNA as primer. CONCLUSIONS: TdIF2 binds directly to TdT and core histone. Furthermore, TdT, TdIF2 and core histone form a ternary complex. TdIF2 liberates H2A/H2B from a core histone in correlation with PCNA. The enzymatic consequence of the TdIF2/TdT complex is the reduction of TdT activity in vitro. TdIF2 would function as a chromatin remodeling protein at the N region synthesis.     

Renal glomerulogenesis in medaka fish, Oryzias latipes

We provide an overview of glomerulogenesis in medaka from the embryo to the adult by means of in situ hybridization with the wt1 gene as a marker as well as histology and three-dimensional images. The pronephric glomus starts to develop in the intermediate mesoderm during early somitogenesis, is completed before hatching, and persists throughout the lifetime of the fish. Within 5 days after hatching, mesonephric glomerulus formation begins in the caudomedial end of the pronephric sinus and duct area. The number of glomeruli reaches approximately 200-300 in each kidney within 2 months after hatching. wt1 expression during nephron maturation served as a marker for the formation of the mesenchymal condensate and the nephrogenic body. Existence of mesenchymal condensates and persistence of wt1 expression in the adult kidney suggest that the mesonephros retains precursor cells that may be capable of contributing to neoglomerulogenesis during adulthood. Developmental Dynamics 237:2342-2352, 2008. (c) 2008 Wiley-Liss, Inc.