IκBNS induces Muc5ac expression in epithelial cells and causes airway hyper-responsiveness in murine asthma models.

BACKGROUND: In allergic asthma, environmental allergens including house dust mite (HDM) trigger pattern recognition receptors and activate downstream signaling pathways including NF-κB pathways not only in immune cells but also in airway epithelial cells. Recent studies have shown that NF-κB activation is regulated positively or negatively depending on the cellular context by IκBNS (encoded by the gene Nfkbid), one of atypical IκB proteins, in the nucleus. Therefore, we hypothesized that IκBNS expressed in immune cells or epithelial cells is involved in the regulation of asthmatic responses. AIM: To determine the roles of IκBNS in HDM-induced asthmatic responses. METHODS: Roles of IκBNS in HDM-induced airway inflammation and airway hyper-responsiveness (AHR) were examined by using IκBNS-deficient (Nfkbid-/- ) mice. Roles of IκBNS expressed in hematopoietic cells and nonhematopoietic cells were separately evaluated by bone marrow chimeric mice. Roles of IκBNS expressed in murine tracheal epithelial cells (mTECs) were examined by air-liquid interface culture. RESULTS: House dust mite-induced airway inflammation and AHR were exacerbated in mice lacking IκBNS in hematopoietic cells. In contrast, HDM-induced airway inflammation was exacerbated, but AHR was attenuated in mice lacking IκBNS in nonhematopoietic cells. The induction of Muc5ac, a representative mucin in asthmatic airways, was reduced in Nfkbid-/- mTEC, whereas the induction of Spdef, a master regulator of goblet cell metaplasia, was not impaired in Nfkbid-/- mTEC. Moreover, IκBNS bound to and activated the MUC5AC distal promoter in epithelial cells. CONCLUSION: IκBNS is involved in inducing Muc5ac expression in lung epithelial cells and causing AHR in HDM-induced asthma models.

MMP13 can be a useful differentiating marker between squamous cell carcinoma and benign hyperkeratotic lesions in recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe hereditary mechanobullous disease resulting from mutations in the COL7A1 gene, coding for type VII collagen. Patients with RDEB tend to develop squamous cell carcinomas (SCCs) at sites of chronic ulceration or scarring on the whole body. Distinguishing SCC from benign hyperkeratotic lesions is often difficult, not only clinically but also histologically in patients with RDEB. We investigated several matrix metallopeptidase (MMP) subtypes by comparing the DNA amplification microarray findings between evident SCCs and benign hyperkeratotic lesions in the same patient with RDEB. We report that MMP13 was found to be strongly positive in SCCs but negative in benign hyperkeratotic lesions. We found that there is an evident difference in the transitional area between SCCs and benign hyperkeratotic lesions. We propose that MMP13 may be a useful differentiating marker between SCC and benign hyperkeratotic lesions in RDEB.

Duration of norovirus excretion and the longitudinal course of viral load in norovirus-infected elderly patients.

To prevent dissemination of norovirus in semiclosed environments such as aged-care facilities, it is important to know the period of infectivity in norovirus-infected individuals. We recruited 13 elderly patients aged 60-98 years with norovirus gastroenteritis (11 residents in aged-care facilities and two healthy adults) for this study, and measured the viral loads for norovirus in a total of 63 follow-up faecal samples using a real-time quantitative polymerase chain reaction assay. The average period of norovirus excretion was 14.3 days (range: 9-32 days; median: 13 days). All of the follow-up samples collected between 7 and 10 days after the onset of symptoms tested positive. Viral loads in samples collected between 14 and 18 days after the onset of symptoms were divided into three groups: those testing negative, those with <10(4) copies/g stool, and those with >10(4) copies/g stool. Stools from the group with <10(4) copies/g stool were found to be negative for norovirus up to 21-24 days after the onset of symptoms; however, the group with >10(4) copies/g stool showed prolonged norovirus excretion (up to 32 days) in stools. Although the period of infectivity of excreted viruses has not yet been clarified, these results suggest that careful attention should be taken for at least 14 days after the onset of symptoms and that the measurement of viral load in stools around 16 days after onset might be a useful method for following the course of viral shedding for each patient infected with norovirus.

Cross-antigenicity among EV71 strains from different genogroups isolated in Yamagata, Japan, between 1990 and 2007.

We isolated and identified six subgenogroups (B2, B4, B5, C1, C2, and C4) of enterovirus 71 (EV71) between 1990 and 2007 in Yamagata, Japan. We measured neutralizing antibody (NT Ab) titers against those subgenogroup strains and the BrCr reference strain for antigenic analysis. Serological analysis of 83 residents in Yamagata in 2004 showed that differences in the NT Ab titer of each individual against the different subgenogroups were mostly within 4-fold. Furthermore, sera from guinea pigs, immunized with the B2 and C1 strains indicated cross-antigenicity among the seven different subgenogroups. In conclusion, our results showed that cross-antigenicity exists among EV71 strains from different subgenogroups circulating in the community through genomic evolution. Our results also suggest that eliciting neutralizing antibodies against one genotype is likely to confer cross-neutralization against other genotypes.

Neurovirulence of H7N7 influenza A virus: brain stem encephalitis accompanied with aspiration pneumonia in mice.

A mouse-adapted influenza A virus, A/equine/London/1416/73-MA (H7N7) caused viral pneumonia, ganglionitis and encephalitis after intranasal inoculation in mice. Virological and pathological data suggested that this virus spreads to the brain by both hematogenous and transneuronal routes, and produces encephalitic lesions similar to those seen in mice infected with H5 highly pathogenic avian influenza A viruses by intranasal infection. Some mice infected with this strain were affected by aspiration pneumonia, which may be caused by neurogenic dysfunction of the pharyngeal/laryngeal reflex due to brain stem encephalitis.

IgE-dependent enhancement of Th2 cell-mediated allergic inflammation in the airways.

T helper 2 (Th2) cell-derived cytokines, including interleukin (IL)-4, IL-5 and IL-13, play important roles in causing allergic airway inflammation. In contrast to Th2 cells, however, the role of IgE and mast cells in inducing allergic airway inflammation is not understood fully. In the present study, we addressed this point using transgenic mice expressing trinitrophenyl (TNP)-specific IgE (TNP-IgE mice), which enable us to investigate the role of IgE without the influence of antigen-specific T cell activation and other immunoglobulins. When the corresponding antigen, TNP-BSA, was administered intranasally to TNP-IgE mice, a large number of CD4+ T cells were recruited into the airways. In contrast, TNP-BSA administration did not induce eosinophil recruitment into the airways or airway hyperreactivity. Furthermore, when ovalbumin (OVA)-specific Th2 cells were transferred to TNP-IgE mice and the mice were challenged with inhaled OVA, TNP-BSA administration increased OVA-specific T cell recruitment and then enhanced Th2 cell-mediated eosinophil recruitment into the airways. These results indicate that IgE-induced mast cell activation principally induces CD4+ T cell recruitment into the airways and thus plays an important role in enhancing Th2 cell-mediated eosinophilic airway inflammation by recruiting Th2 cells into the site of allergic inflammation.

Mechanical model of normal and anomalous diffusion.

The overdamped dynamics of a charged particle driven by an uniform electric field through a random sequence of scatterers in one dimension is investigated. Analytic expressions of the mean velocity and of the velocity power spectrum are presented. These show that above a threshold value of the field normal diffusion is superimposed to ballistic motion. The diffusion constant can be given explicitly. At the threshold field, the transition between conduction and localization is accompanied by an anomalous diffusion. Our results exemplify that, even in the absence of time-dependent stochastic forces, a purely mechanical model equipped with a quenched disorder can exhibit normal as well as anomalous diffusion, the latter emerging as a critical property. Via another interpretation, as the motion of a particle on an inclined rough surface, our results are relevant for the problem of segregation by flow.

Bcl-2 expression in breast cancer is down-regulated by trans-arterial administration of chemotherapeutic agents.

Bcl-2 is one of the cytoplasmic oncoproteins, and has been shown to suppress apoptotic cell death. In this study, we investigated the relationship between Bcl-2 expression and effects of chemotherapeutic agents on human breast cancer cells. We examined 26 surgically resected breast tumors with preoperative trans-arterial administration of chemotherapeutic agents and 30 control cases using immunohistochemical methods. In all 26 cases in the chemotherapy group, the breast cancer cells were focally degenerated to various degrees, associated with inflammation and stromal desmoplastic changes. Bcl-2 expression was found in 46% (12/26) of the chemotherapy group and in 67% (20/30) of controls. Of the 12 Bcl-2-positive cases in the chemotherapy group, 5 were diffusely positive [Bcl-2(2+)] and 7 were focally positive [Bcl-2(+)]. Of the 20 Bcl-2-positive cases in the control group, 18 were diffusely positive and 2 were focally positive. We speculate that Bcl-2 expression was down-regulated by trans-arterial administration of chemotherapeutic agents and was associated with apoptosis and degeneration of breast cancer cells.

Role of CD4(+) CD25(+) regulatory T cells in T helper 2 cell-mediated allergic inflammation in the airways.

It has recently been shown that CD4(+) CD25(+) T cells are immunoregulatory T cells that prevent CD4(+) T cell-mediated organ-specific autoimmune diseases. To determine whether CD4(+) CD25(+) T cells downregulate Th2 cell-mediated allergic inflammation in the airways, we studied antigen-induced eosinophil recruitment in the airways in BALB/c Rag-2(-)(/-) mice transferred with CD4(+) CD25(+) T cell-depleted or unfractionated T cells from ovalbumin-specific TCR transgenic mice. Antigen-induced eosinophil recruitment into the airways was significantly decreased in the mice transferred with CD4(+) CD25(+) T cell-depleted splenocytes as compared with those transferred with unfractionated splenocytes. On the other hand, the depletion of CD4(+) CD25(+) T cells increased antigen-induced neutrophil and T cell recruitment in the airways of the mice. The depletion of CD4(+) CD25(+) T cells also decreased antigen-induced IL-4 and IL-5 production in the airways of the mice. Finally, the depletion of CD4(+) CD25(+) T cells prevented antigen-induced Th2 cell differentiation in vitro but increased the differentiation of Th1 cells. These results indicate that CD4(+) CD25(+) T cells modulate the Th1 and Th2 cell balance toward Th2 cells and thus upregulate Th2 cell-mediated allergic inflammation in the airways.

First confirmed canine case of Ehrlichia canis infection in Japan.

An 11-year-old castrated Pekinese dog that had been moved from Indonesia to Japan eight years previously was diagnosed with an Ehrlichia canis infection by haematological characteristics (normocytic anaemia, mild thrombocytopenia and hypergammaglobulinaemia) and serological findings (antibody titre to E canis 1:3,200 or more). The dog did not respond to treatment with tetracycline and died from renal failure. The diagnosis was confirmed postmortem by pathological evaluation and polymerase chain reaction (PCR) followed by sequencing of the 16S rRNA gene. Typical morulae of Ehrlichia were detected in the cytoplasm of macrophages in spleen tissue by immunohistological staining. Ehrlichia-like organisms were also detected in the spleen by electron microscopy. E canis-specific PCR analysis of DNA extracted from the spleen gave a positive signal, and sequence analysis of the fragment revealed that it was identical to part of the 16s rRNA gene of E canis. The dog was the first confirmed clinical case of E canis infection in Japan.

Stat5a regulates T helper cell differentiation by several distinct mechanisms.

We have previously shown that CD4(+) T cell-mediated allergic inflammation is diminished in signal transducer and activator of transcription (Stat)5a-deficient (Stat5a(-/-)) mice. To determine whether Stat5a regulates T helper cell differentiation, we studied T helper (Th)1 and Th2 cell differentiation of Stat5a(-/-)CD4(+) T cells at single-cell levels. First, Th2 cell differentiation from antigen-stimulated splenocytes was significantly decreased in Stat5a(-/-) mice as compared with that in wild-type mice. Further, Th2 cell differentiation was also impaired in Stat5a(-/-) mice even when purified CD4(+) T cells were stimulated with anti-CD3 plus anti-CD28 antibodies in the presence of interleukin-4. Moreover, the retrovirus-mediated gene expression of Stat5a in Stat5a(-/-)CD4(+) T cells restored the Th2 cell differentiation at the similar levels to that in wild-type CD4(+) T cells. In addition, interleukin-4 normally phosphorylated Stat6 in CD4(+) T cells from Stat5a(-/-) mice. Second, the development of CD4(+)CD25(+) immunoregulatory T cells was impaired in Stat5a(-/-) mice, as indicated by a significant decrease in the number of CD4(+)CD25(+) T cells in Stat5a(-/-) mice. Furthermore, the depletion of CD4(+)CD25(+) T cells from wild-type splenocytes significantly decreased Th2 cell differentiation but increased Th1 cell differentiation, whereas the depletion of CD4(+)CD25(+) T cells from Stat5a(-/-) splenocytes had no significant effect on the Th1 and Th2 cell differentiation. Together, these results indicate that the intrinsic expression of Stat5a in CD4(+) T cells is required for Th2 cell differentiation and that Stat5a is involved in the development of CD4(+)CD25(+) immunoregulatory T cells that modulate T helper cell differentiation toward Th2 cells.

Resistant mechanisms of anthracyclines--pirarubicin might partly break through the P-glycoprotein-mediated drug-resistance of human breast cancer tissues.

Juliano and Ling initially reported the expression of a 170 kDa glycoprotein in the membrane of Chinese hamster ovarian cells in 1976, and named this glycoprotein P-glycoprotein (P-gp) based on its predicted role of causing "permeability" of the cell membrane. After much research on anthracycline-resistance, this P-gp was finally characterized as a multidrug-resistant protein coded by the mdr1 gene. Multidrug resistance associated protein (MRP) was initially cloned from H69AR, a human small cell-lung carcinoma cell line which is resistant to doxorubicin (DXR) but does not express P-gp. MRP also excretes substrates through the cell membrane using energy from ATP catabolism. The substrate of MRP is conjugated with glutathione before active efflux from cell membrane. Recently, membrane transporter proteins were re-categorized as members of "ATP-Binding Cassette transporter"(ABC-transporter) superfamily, as shown at http://www.med.rug.nl/mdl/humanabc.htm and http://www.gene.ucl.ac.uk/nomenclature/genefamily/abc.html. A total of ABC transporters have been defined, and MDR1 and multidrug resistance associated protein 1 (MRP1) were reclassified as ABCB1 and ABCC1, respectively. Their associated superfamilies include 11 and 13 other protein, in addition to ABCB and ABCC, respectively. Lung resistance-related protein (LRP) is not a member of the superfamily of ABC transporter proteins, because it shows nuclear membrane expression and transports substrate between nucleus and cytoplasm. LRP was initially cloned from a non-small cell lung carcinoma cell line, SW1573/2R120 which is resistant to DXR, vincristine, etoposide and gramicidin D and does not express P-gp. The mechanisms of resistance remains unclear, and why some resistant cell lines express P-gp and others express MRP and/or LRP is likewise unclear.

Role of common cytokine receptor gamma chain (gamma(c))- and Jak3-dependent signaling in the proliferation and survival of murine mast cells.

The regulatory roles of the common cytokine receptor gamma chain (gamma(c))- and Jak3-dependent signaling in the proliferation and survival of mast cells were determined using gamma(c)-deficient (gamma(c)(-)) and Jak3-deficient (Jak3(-)) mice. Although the mast cells in gamma(c)(-) and Jak3(-) mice were morphologically indistinguishable from those in wild-type mice, the number of peritoneal mast cells was decreased in gamma(c)(-) and Jak3(-) mice as compared with that in wild-type mice. Among gamma(c)-related cytokines, interleukin (IL)-4 and IL-9, but not IL-2, IL-7, or IL-15, enhanced the proliferation and survival of bone marrow-derived mast cells (BMMCs) from wild-type mice. However, the effects of IL-4 and IL-9 were absent in BMMCs from gamma(c)(-) and Jak3(-) mice. In addition, IL-4Ralpha, gamma(c), and Jak3, but not IL-2Rbeta or IL-7Ralpha, were expressed in BMMCs. In contrast, IL-13 did not significantly induce the proliferation and survival of BMMCs even from wild-type mice, and IL-13Ralpha1 was not expressed in BMMCs. Furthermore, IL-4 phosphorylated the 65-kd isoform of Stat6 in BMMCs from wild-type mice but not from gamma(c)(-) and Jak3(-) mice. These results indicate that gamma(c)- and Jak3-dependent signaling is essential for IL-4- and IL-9-induced proliferation and survival of murine mast cells, that the effects of IL-4 are mediated by type I IL-4R and that type II IL-4R is absent on mast cells, and that IL-4 phosphorylates the 65-kd isoform of Stat6 in mast cells in a gamma(c)- and Jak3-dependent manner.

Both stat5a and stat5b are required for antigen-induced eosinophil and T-cell recruitment into the tissue.

Antigen-induced eosinophil recruitment into the airways of sensitized mice is mediated by CD4(+) T cells and their cytokines, especially IL-5. In this study, we found that the antigen-induced airway eosinophilia was diminished in Stat5a-deficient (Stat5a(-/-)) mice and Stat5b-deficient (Stat5b(-/-)) mice. We also found that antigen-induced CD4(+) T-cell infiltration and IL-5 production in the airways were diminished in Stat5a(-/- )mice and Stat5b(-/-) mice. Moreover, antigen-induced proliferation of splenocytes was diminished in Stat5a(-/- )mice and Stat5b(-/-) mice, suggesting that the generation of antigen-primed T cells may be compromised in Stat5a(-/-) mice and Stat5b(-/-) mice and this defect may account for the diminished antigen-induced T-cell infiltration into the airways. Interestingly, IL-4 and IL-5 production from anti-CD3-stimulated splenocytes was diminished in Stat5a(-/-) mice and Stat5b(-/-) mice. However, antigen-specific IgE and IgG1 production was diminished in Stat5a(-/-) mice but not in Stat5b(-/-) mice, whereas antigen-specific IgG2a production was increased in Stat5a(-/-) mice, suggesting the enhanced Th1 responses in Stat5a(-/-) mice. Finally, we found that eosinophilopoiesis induced by the administration of recombinant IL-5 was also diminished in Stat5a(-/-) mice and Stat5b(-/-) mice. Together, these results indicate that both Stat5a and Stat5b are essential for induction of antigen-induced eosinophil recruitment into the airways and that the defects in antigen-induced eosinophil recruitment in Stat5a(-/-) mice and Stat5b(-/-) mice result from both impaired IL-5 production in the airways and diminished IL-5 responsiveness of eosinophils. (Blood. 2000;95:1370-1377)

Carcinosarcoma of the ampulla of Vater: a case report with immunohistochemical and ultrastructural studies.

Carcinosarcoma of the duodenum has not been reported previously, although this type of tumor has been detected in other organs. We present here a case of carcinosarcoma of the duodenum, including immunohistochemical and electron microscopical findings. An ulcerating tumor, located in the duodenal ampullary region, contained two divergent components: ordinary differentiated tubular adenocarcinoma, and sarcomatoid tissue composed of spindle tumor cells. Immunohistochemically, the adenocarcinoma cells were stained with antibodies against epithelial markers including keratin and CA19-9. In contrast, the sarcomatoid tissue was strongly positive for vimentin and was focally positive for myoglobin, keratin, and CA19-9. We speculate that the sarcomatoid element of the carcinosarcoma arose from part of the ordinary adenocarcinoma tissue.

In Vitro and In Vivo Modulation of Growth Regulation in the Human Breast Cancer Cell Line MCF-7 by Estradiol Metabolites.

BACKGROUND: The natural estrogen 17 beta-estradiol (E2) functions as a potent tumor promoter during tumorigenic transformation of the mammary gland. From amongst the various pathways of E2 metabolism upregulation of C16 alpha-hydroxylation of E2 has been associated with carcinogenesis. In the present study in vitro and in vivo experiments were performed on estrogen receptor positive human breast cancer MCF-7 cells to examine whether the natural estrogen E2 and its metabolites 16 alpha-hydroxyestrone (16 alpha-OHE1) and 2-hydroxyestrone (2-OHE1)function as modulators of tumor cell growth. METHODS: An anchorage-independent growth assay was used for in vitro study bycounting the number of tri-dimensional colonies formed by MCF-7 cells suspendedin 0.33% agar. In vivo experiments examined the effect of implanting metabolitematerial pellets into female nude mice. RESULTS: In the anchorage-independent growth assay (AIG), continuous 14-day exposure to E2 and to 16 alpha-OHE1 at 200 ng/ml induced a 59.4% and a 105.9% increase (P= 0.001)respectively in the number of colonies of MCF-7 cells. Identical treatment with 2-OHE1, however, failed to increase AIG relative to that seen in the solvent treated control cultures. In the in vivo tumorigenicity assay, treatment of nude mice with 1.5 mg E2 or 16 alpha-OHE1 resulted in a 335.4% and a 384.1% increase (P< 0.0002) in tumor growth, while identical treatment with 2-OHE1 failed to exhibit any increase relative to the control group. CONCLUSION: These results suggest that the 16 alpha- and 2-hydroxylated metabolites of E2 may directly affect in vitro growth of MCF-7 cells via an autocrine mechanism and in vivo growth via paracrine mechanisms. Thus, E2-mediated growth regulation in MCF-7 cells may in part be due to distinct effects of specific E2 metabolites on the breast cancer cells.

Heterogeneous expression of nm23 gene product as a predictor of lymph nodal status in human breast cancer.

The nm23 gene was originally identified by differential hybridization of metastatic murine melanoma cell lines. Some experimental studies demonstrated a significantly low metastatic potential of melanoma cell lines transfected with the nm23 gene. In this study, we clarified the relationship between intracellular nm23-immunoreactivity and lymph nodal status of human breast cancer. We analyzed 82 surgically removed breast tumors including 67 invasive carcinomas (ductal, lobular and mucinous carcinomas). The nm23 expression was diffusely positive in the benign tumors and non-invasive carcinomas. Of the invasive ductal carcinomas, lymph node metastasis was found in 67.7% (21/31) of the focally positive/negative cases and in 18.2% (4/22) of the diffusely positive cases (p<0.001). Immunohistochemically, advanced margins of invasive carcinomas with lymph node metastasis were shown to be negative for nm23 expression, while intraductal carcinoma components were positive. This observation suggested that focally positive/negative nm23 expression can be a predictor of lymph node metastasis of invasive ductal breast carcinoma.