Web survey-based selection of controls for epidemiological analyses of a multi-prefectural outbreak of enterohaemorrhagic Escherichia coli O157 in Japan associated with consumption of self-grilled beef hanging tender.

An outbreak of enterohaemorrhagic Escherichia coli O157 occurred in multiple prefectures of Japan in November 2009. We conducted two case-control studies with trace-back and trace-forward investigations to determine the source. The case definition was met by 21 individuals; 14 (66.7%) were hospitalised, but no haemolytic uraemic syndrome, acute encephalopathy or deaths occurred. Median age was 23 (range 12-48) years and 14 cases were male (66.7%). No significant associations with food were found in a case-control study by local public health centres, but our matched case-control study using Internet surveys found that beef hanging tender (or hanger steak), derived from the diaphragm of the cattle, was significantly associated with illness (odds ratio = 15.77; 95% confidence interval, 2.00-124.11). Pulsed-field gel electrophoresis analysis of isolates from patients and the suspected food showed five different patterns: two in faecal and food samples, and another three in patient faecal samples only, although there were epidemiological links to the meat consumed at the restaurants. Trace-back investigation implicated a common food processing company from outside Japan. Examination of the logistics of the meat processing company suggested that contamination did not occur in Japan. We concluded that the source of the outbreak was imported hanging tender. This investigation revealed that Internet surveys could be useful for outbreak investigations.

Graft-Versus-Host Disease Following Liver Transplantation: Development of a High-Incidence Rat Model and a Selective Prevention Method.

Graft-versus-host disease (GvHD) following liver transplantation (LT) is a rare but serious complication with no presently available animal model and no preventive measures. To develop a rat model of GvHD after LT (LT-GvHD), we preconditioned hosts with sublethal irradiation plus reduction of natural killer (NK) cells with anti-CD8α mAb treatment, which invariably resulted in acute LT-GvHD. Compared with those in the peripheral counterpart, graft CD4+ CD25- passenger T cells showed lower alloreactivities in mixed leukocyte culture. Immunohistology revealed that donor CD4+ T cells migrated and formed clusters with host dendritic cells in secondary lymphoid organs, with early expansion and subsequent accumulation in target organs. For selectively preventing GvHD, donor livers were perfused ex vivo with organ preservation media containing anti-TCRαß mAb. T cell-depleted livers almost completely suppressed clinical GvHD such that host rats survived for >100 days. Our results showed that passenger T cells could develop typical LT-GvHD if resistant cells such as host radiosensitive cells and host radioresistant NK cells were suppressed. Selective ex vivo T cell depletion prevented LT-GvHD without affecting host immunity or graft function. This method might be applicable to clinical LT in prediagnosed high-risk donor-recipient combinations and for analyzing immunoregulatory mechanisms of the liver.

Endoscopic evaluation of food bolus formation and its relationship with the number of chewing cycles.

It is known that solid food is transported to the pharynx actively in parallel to it being crushed by chewing and mixed with saliva in the oral cavity. Therefore, food bolus formation should be considered to take place from the oral cavity to the pharynx. In previous studies, the chewed food was evaluated after the food had been removed from the oral cavity. However, it has been pointed out that spitting food out of the oral cavity interferes with natural food bolus formation. Therefore, we observed food boluses immediately before swallowing using an endoscope to establish a method to evaluate the food bolus-forming function, and simultaneously performed endoscopic evaluation of food bolus formation and its relationship with the number of chewing cycles. The subject was inserted the endoscope nasally and instructed to eat two coloured samples of boiled rice simultaneously in two ingestion conditions ('as usual' and 'chewing well'). The condition of the food bolus was graded into three categories for each item of grinding, mixing and aggregation and scored 2, 1 and 0. The score of aggregation was high under both ingestion conditions. The scores of grinding and mixing tended to be higher in subjects with a high number of chewing cycles, and the score of aggregation was high regardless of the number of chewing cycles. It was suggested that food has to be aggregated, even though the number of chewing cycles is low and the food is not ground or mixed for a food bolus to reach the swallowing threshold.

Human deltex is a conserved regulator of Notch signalling.

A fundamental cell-fate control mechanism regulating multicellular development is defined by the Notch-signalling pathway. Developmental and genetic studies of wild type and activated Notch-receptor expression in diverse organisms suggest that Notch plays a general role in development by governing the ability of undifferentiated precursor cells to respond to specific signals. Notch signalling has been conserved throughout evolution and controls the differentiation of a broad spectrum of cell types during development. Genetic studies in Drosophila have led to the identification of several components of the Notch pathway. Two of the positive regulators of the pathway are encoded by the suppressor of hairless [Su(H)] and deltex (dx) genes. Drosophila dx encodes a ubiquitous, novel cytoplasmic protein of unknown biochemical function. We have cloned a human deltex homologue and characterized it in parallel with its Drosophila counterpart in biochemical assays to assess deltex function. Both human and Drosophila deltex bind to Notch across species and carry putative SH3-binding domains. Using the yeast interaction trap system, we find that Drosophila and human deltex bind to the human SH3-domain containing protein Grb2 (ref. 10). Results from two different reporter assays allow us for the first time to associate deltex with Notch-dependent transcriptional events. We present evidence linking deltex to the modulation of basic helix-loop-helix (bHLH) transcription factor activity.

A novel migration pathway for rat dendritic cells from the blood: hepatic sinusoids-lymph translocation.

The migration pathways for dendritic cells (DC) from the blood are not yet completely resolved. In our previous study, a selective recruitment of DC progenitors from the blood to the liver was suggested. To clarify the role of the hepatic sinusoids in the migration of blood DC, relatively immature DC and mature DC were isolated from hepatic and intestinal lymph, and intravenously transferred to allogeneic hosts. It was then possible to detect small numbers of DC within secondary lymphoid tissues either by immunostaining for donor type major histocompatibility complex class I antigen or, at much higher sensitivity, for bromodeoxyuridine incorporated by proliferating cells (mainly T lymphocytes), which responded to the alloantigen presented by the administered DC. The intravenously injected DC accumulated in the paracortex of regional lymph nodes of the liver via a lymph-borne pathway. Intravenously injected fluorochrome-labeled syngeneic DC behaved similarly. In contrast, very few DC were found in spleen sections and were hardly detectable in other lymph nodes or in other tissues. An in situ cell binding assay revealed a significant and selective binding of DC to Kupffer cells in liver cryosections. It is concluded that rat DC can undergo a blood-lymph translocation via the hepatic sinusoids, but not via the high endothelial venules of lymph nodes. Hence the hepatic sinusoids may act as a biological concentrator of blood DC into the regional hepatic nodes. Kupffer cells may play an important role in this mechanism.

A life stage of particle-laden rat dendritic cells in vivo: their terminal division, active phagocytosis, and translocation from the liver to the draining lymph.

Initiation of an adoptive immune response against pathogenic organisms, such as bacteria and fungi, may involve phagocytic activity of dendritic cells (DC) or their immature precursors as a prelude to antigen processing and presentation. After intravenous injection of rats with particulate matter, particle-laden cells were detected in the peripheral hepatic lymph. Since it has been known there is a constant efflux of DC from nonlymphoid organs into the draining peripheral lymph, we examined whether these particle-laden cells belonged to the DC or macrophage lineage. The majority of particle-laden cells in lymph showed immature monocyte-like cytology, and the amount of ingested particles was small relative to typical macrophages. We identified these particle-laden cells as DC based on a number of established criteria: (a) they had a phenotype characteristic of rat DC, that is, major histocompatibility complex class Ihigh+ and IIhigh+, intercellular adhesion molecule 1+ and 80% positive with the rat DC-specific mAb OX62; (b) they showed strong stimulating capacity in primary allogeneic mixed leukocyte reaction; (c) in vitro, they had little phagocytic activity; and (d) the kinetics of translocation was similar to that of lymph DC in that they migrated to the thymus-dependent area of the regional nodes. Furthermore, bromodeoxyuridine feeding studies revealed that most of the particle-laden DC were recently produced by the terminal division of precursor cells, at least 45% of them being <5.5 d old. The particle-laden DC, defined as OX62+ latex-laden cells, were first found in the sinusoidal area of the liver, in the liver perfusate, and in spleen cell suspensions, suggesting that the site of particle capture was mainly in the blood marginating pool. It is concluded that the particle-laden cells in the hepatic lymph are recently produced immature DC that manifest a temporary phagocytic activity for intravascular particles during or after the terminal division and that the phagocytic activity is downregulated at a migratory stage when they translocate from the sinusoidal area to the hepatic lymph.

Regulation by chemokines of circulating dendritic cell precursors, and the formation of portal tract-associated lymphoid tissue, in a granulomatous liver disease.

We have studied the recruitment and roles of distinct dendritic cell (DC) precursors from the circulation into Propionibacterium acnes-induced granulomas in mouse liver. During infection, F4/80(-)B220(-)CD11c(+) DC precursors appeared in the circulation, migrated into the perisinusoidal space, and matured within newly formed granulomas. Recruited DCs later migrated to the portal area to interact with T cells in what we term "portal tract-associated lymphoid tissue" (PALT). Macrophage inflammatory protein 1alpha attracted blood DC precursors to the sinusoidal granuloma, whereas secondary lymphoid organ chemokine (SLC) attracted mature DCs to the newly identified PALT. Anti-SLC antibody diminished PALT expansion while exacerbating granuloma formation. Therefore, circulating DC precursors can migrate into a solid organ like liver, and participate in the granulomatous reaction in response to specific chemokines.

Notch signaling.

The Notch/Lin-12/Glp-1 receptor family mediates the specification of numerous cell fates during development in Drosophila and Caenorhabditis elegans. Studies on the expression, mutant phenotypes, and developmental consequences of unregulated receptor activation have implicated these proteins in a general mechanism of local cell signaling, which includes interactions between equivalent cells and between different cell types. Genetic approaches in flies and worms have identified putative components of the signaling cascade, including a conserved family of extracellular ligands and two cellular factors that may associate with the Notch Intracellular domain. One factor, the Drosophila Suppressor of Hairless protein, is a DNA-binding protein, which suggests that Notch signaling may involve relatively direct signal transmission from the cell surface to the nucleus. Several vertebrate Notch receptors have also been discovered recently and play important roles in normal development and tumorigenesis.

Elongation of oligopeptides in a simulated submarine hydrothermal system.

Oligomerization of a peptide was attempted in a flow reactor that simulated a submarine hydrothermal system. When fluid containing glycine repeatedly circulated through the hot and cold regions in the reactor, oligopeptides were made from glycine. When divalent ions (such as copper ions) were added under acidic conditions, oligoglycine was elongated up to hexaglycine. This observation suggests that prebiotic monomers could have oligomerized in the vicinity of submarine hydrothermal vents on primitive Earth.

Interaction between Wingless and Notch signaling pathways mediated by dishevelled.

In Drosophila, the Wingless and Notch signaling pathways function in m any of the same developmental patterning events. Genetic analysis demonstrates that the dishevelled gene, which encodes a molecule previously implicated in implementation of the Winglass signal, interacts antagonistically with Notch and one of its known ligands, Delta. A direct physical interaction between Dishevelled and the Notch carboxyl terminus, distal to the cdc10/ankyrin repeats, suggests a mechanism for this interaction. It is proposed that Dishevelled, in addition to transducing the Wingless signal, blocks Notch signaling directly, thus providing a molecular mechanism for the inhibitory cross talk observed between these pathways.

Isolation and characterization of avian influenza viruses from raw poultry products illegally imported to Japan by international flight passengers.

The transportation of poultry and related products for international trade contributes to transboundary pathogen spread and disease outbreaks worldwide. To prevent pathogen incursion through poultry products, many countries have regulations about animal health and poultry product quarantine. However, in Japan, animal products have been illegally introduced into the country in baggage and confiscated at the airport. Lately, the number of illegally imported poultry and the incursion risk of transboundary pathogens through poultry products have been increasing. In this study, we isolated avian influenza viruses (AIVs) from raw poultry products illegally imported to Japan by international passengers. Highly (H5N1 and H5N6) and low (H9N2 and H1N2) pathogenic AIVs were isolated from raw chicken and duck products carried by flight passengers. H5 and H9 isolates were phylogenetically closely related to viruses isolated from poultry in China, and haemagglutinin genes of H5N1 and H5N6 isolates belonged to clades 2.3.2.1c and 2.3.4.4, respectively. Experimental infections of H5 and H9 isolates in chickens and ducks demonstrated pathogenicity and tissue tropism to skeletal muscles. To prevent virus incursion by poultry products, it is important to encourage the phased cleaning based on the disease control and eradication and promote the reduction in contamination risk in animal products.

Notch inhibition of E47 supports the existence of a novel signaling pathway.

E47 is a widely expressed transcription factor that activates B-cell-specific immunoglobulin gene transcription and is required for early B-cell development. In an effort to identify processes that regulate E47, and potentially B-cell development, we found that activated Notch1 and Notch2 effectively inhibit E47 activity. Only the intact E47 protein was inhibited by Notch-fusion proteins containing isolated DNA binding and activation domains were unaffected-suggesting that Notch targets an atypical E47 cofactor. Although overexpression of the coactivator p300 partially reversed E47 inhibition, results of several assays indicated that p300/CBP is not a general target of Notch. Notch inhibition of E47 did not correlate with its ability to activate CBF1/RBP-Jkappa, the mammalian homolog of Suppressor of Hairless, a protein that associates physically with Notch and defines the only known Notch signaling pathway in drosophila. Importantly, E47 was inhibited independently of CBF1/RPB-Jkappa by Deltex, a second Notch-interacting protein. We provide evidence that Notch and Deltex may act on E47 by inhibiting signaling through Ras because (i) full E47 activity was found to be dependent on Ras and (ii) both Notch and Deltex inhibited GAL4-Jun, a hybrid transcription factor whose activity is dependent on signaling from Ras to SAPK/JNK.

A human protein with sequence similarity to Drosophila mastermind coordinates the nuclear form of notch and a CSL protein to build a transcriptional activator complex on target promoters.

Mastermind (Mam) has been implicated as an important positive regulator of the Notch signaling pathway by genetic studies using Drosophila melanogaster. Here we describe a biochemical mechanism of action of Mam within the Notch signaling pathway. Expression of a human sequence related to Drosophila Mam (hMam-1) in mammalian cells augments induction of Hairy Enhancer of split (HES) promoters by Notch signaling. hMam-1 stabilizes and participates in the DNA binding complex of the intracellular domain of human Notch1 and a CSL protein. Truncated versions of hMam-1 that can maintain an association with the complex behave in a dominant negative fashion and depress transactivation. Furthermore, Drosophila Mam forms a similar complex with the intracellular domain of Drosophila Notch and Drosophila CSL protein during activation of Enhancer of split, the Drosophila counterpart of HES. These results indicate that Mam is an essential component of the transcriptional apparatus of Notch signaling.

A cross-sectional study to quantify the prevalence of avian influenza viruses in poultry at intervention and non-intervention live bird markets in central Vietnam, 2014.

In Vietnam, live bird markets are found in most populated centres, providing the means by which fresh poultry can be purchased by consumers for immediate consumption. Live bird markets are aggregation points for large numbers of poultry, and therefore, it is common for a range of avian influenza viruses to be mixed within live bird markets as a result of different poultry types and species being brought together from different geographical locations. We conducted a cross-sectional study in seven live bird markets in four districts of Thua Thien Hue Province in August and December, 2014. The aims of this study were to (i) document the prevalence of avian influenza in live bird markets (as measured by virus isolation); and (ii) quantify individual bird-, seller- and market-level characteristics that rendered poultry more likely to be positive for avian influenza virus at the time of sale. A questionnaire soliciting details of knowledge, attitude and avian influenza practices was administered to poultry sellers in study markets. At the same time, swabs and faecal samples were collected from individual poultry and submitted for isolation of avian influenza virus. The final data set comprised samples from 1,629 birds from 83 sellers in the seven live bird markets. A total of 113 birds were positive for virus isolation; a prevalence of 6.9 (95% CI 5.8-8.3) avian influenza virus-positive birds per 100 birds submitted for sale. After adjusting for clustering at the market and individual seller levels, none of the explanatory variables solicited in the questionnaire were significantly associated with avian influenza virus isolation positivity. The proportions of variance at the individual market, seller and individual bird levels were 6%, 48% and 46%, respectively. We conclude that the emphasis of avian influenza control efforts in Vietnam should be at the individual seller level as opposed to the market level.

Presentation of tumor antigens by phagocytic dendritic cell clusters generated from human CD34+ hematopoietic progenitor cells: induction of autologous cytotoxic T lymphocytes against leukemic cells in acute myelogenous leukemia patients.

The use of antigen-presenting dendritic cells (DCs) is currently proposed for tumor immunotherapy through generation of CTLs to tumor antigens in cancer patients. In this study, DCs were differentiated using granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-alpha from CD34+ hematopoietic progenitor cells that had been mobilized into the peripheral blood. To use the phagocytic activity of DCs for processing and presentation of tumor antigens, we established DC clusters containing immature DCs by preserving proliferating cell clusters without mechanical disruption. After an 11-day culture, the developed clusters contained not only typical mature DCs but also immature DCs that showed active phagocytosis of latex particles, suggesting that the clusters consisted of DCs of different maturational stages. These heterogeneous clusters could present an exogenous protein antigen, keyhold limpet hemocyanin, to both CD4+ and CD8+ T lymphocytes. Furthermore, in three acute myelogeneous leukemia patients, clusters pulsed with autologous irradiated leukemic cells could also induce antileukemic CTLs. The mechanical disruption of clusters abrogated the induction of CTLs to leukemic cells as well as to hemocyanin. This observation gives an important information for the use of heterogeneous DC clusters derived from autologous peripheral blood CD34+ cells in the case of immunotherapy for leukemia.

Association of matrilysin expression with recurrence and poor prognosis in human esophageal squamous cell carcinoma.

Matrix metalloproteinase-7 (matrilysin) has been implicated in tumor invasion and metastasis as well as tumor initiation and growth. In this study, we analyzed an association between immunohistochemically detected matrilysin expression at the invasive front in esophageal squamous cell carcinomas and clinicopathological characteristics and determined whether matrilysin predicts recurrence and/or survival Matrilysin expression at the invasive front was detected in 49% of 100 carcinoma tissues and was associated with the depth of invasion (P < 0.0001), advanced tumor stage (P = 0.0159), recurrences (P = 0.0002), and recurrences within the first postoperative year (P = 0.002). Patients with matrilysin-positive carcinoma had a significantly shorter disease-free and overall survival time than did those with a matrilysin-negative one (P < 0.0001). Matrilysin remained a significant predictive value for disease-free and overall survival in multivariate analysis, including conventional clinicopathological factors (P = 0.0007 and 0.0004, respectively). Our results suggest that matrilysin may play a key role in the progression of esophageal carcinoma and that its detection may be useful for the prediction of recurrence and poor prognosis and, possibly, for selecting patients for anti-matrix metalloproteinase therapy.

Decorating actin filaments with troponin T-I complexes and acceleration of their sliding movement on myosin molecules.

Actin filaments, when partially decorated with troponin T-I complexes, can slide faster on myosin heads than those with no decoration. Purified troponin T-I complexes bind to actins, and inhibit the actin activated myosin adenosine 5'-triphosphatase activity completely when the molar ratio of troponin T-I complex to actin is increased to 1 to 1. Those actin filaments decorated with troponin T-I complexes up to 20 to 50% of their molar ratio exhibit enhancement of the velocity of sliding on myosins up to 20% compared to those without such decoration. As the molar ratio of decoration further increases, the sliding velocity decreases. These results are consistent with the observation that even if some of actin monomers do not participate in the ATPase activity directly, they can interact with myosin heads and take part in the sliding movement.

Dendritic cell dynamics in the liver and hepatic lymph.

Dendritic cells (DC) are bone-marrow-derived cells that function as professional antigen-presenting cells (APC). Liver is an essential organ for a host defense. It not only is armed with a powerful macrophage system but also is constantly surveyed by a heavy traffic of DC and lymphocytes. In case of emergency, such as infection and inflammation, DC traffic in the liver is accelerated. DC in the liver (interstitial DC) capture and process antigens, enter the draining lymph (DC in hepatic lymph) and accumulate in the T-cell area of hepatic lymph nodes (LN). DC in the LN present antigens to T and B cells to initiate immune responses. In accelerated states, DC precursors are recruited to the liver and soon translocate to hepatic lymph. Even mature lymph DC can undergo a blood-lymph translocation from the liver to hepatic LN after i.v. injection into normal rats. Rat Kupffer cells in the hepatic sinusoids are capable of selectively trapping DC from the blood in vivo and in vitro, suggesting involvement of certain adhesion molecules. Kupffer cells presumably elaborate chemokines to attract and trap the recruited DC via selective adhesion, leading to DC extravasation. The accelerated traffic and the presence of blood-lymph translocation would induce rapid and efficient immune responses and thus contribute to the local defense to antigens within liver tissues as well as systemic defense to blood-borne antigens. DC progenitors are also present in the liver, and these may play an important role in tolerance induction in liver transplantation.

Fibroin gene promoter contains a cluster of homeodomain binding sites that interact with three silk gland factors.

By nuclease protection and mobility shift assays, we have shown that the 5' flanking sequence of the Bombyx mori fibroin gene, which is known to be required for a maximal level of transcription in vitro, contains five regions (A to E) that bind protein factors from the posterior silk gland extract. A silk gland-specific factor (SGF-1) and a ubiquitous factor (FBF-A1) were found to interact with the proximal A region, while the related B region can only bind SGF-1. The three distal regions (C, D and E) bind one posterior silk gland-specific factor (SGF-2) and two ubiquitous factors (SGF-3 and -4). SGF-1 might play an important role in the expression of silk protein genes because it also binds to a similar site in the sericin-1 gene and potential SGF-1 binding sites can be found in two other silk protein genes of Bombyx mori and the fibroin gene of Antheraea yamamai. The three distal regions for SGF-2, -3 and -4 contain ten base-pairs of A + T-rich repeats that resemble the consensus binding site (TCAATTAAAT) of a number of homeodomain proteins. The TAAT motif in the core of these regions is shown to be important for the binding of these three proteins and, as described elsewhere, two Drosophila homeodomain proteins, ZEN (zerknüllt) and EVE (even-skipped). Interestingly, SGF-3 appears to be a Bombyx octamer binding protein and may also be involved in the regulation of the sericin-1 gene. The possibility that the fibroin gene and other silk protein genes may be a group of target genes for some members of the homeobox gene family is discussed.

In vivo roles of donor and host dendritic cells in allogeneic immune response: cluster formation with host proliferating T cells.

Possible roles of dendritic cells (DCs) in allogeneic immune responses in host lymphoid tissues were characterized in situ by using rat DC transfer and cardiac transplantation models. When allogeneic DCs were intravenously injected, these cells selectively migrated to the T-cell area of hepatic lymph nodes, with peak accumulation at 18 h after injection. Donor DCs and proliferating host T cells formed clusters (rosettes) in which the T-cell proliferative response started. The donor DCs were CD80(+) CD86(+) and, ultrastructurally, were in intimate contact with lymphoblasts within the rosettes. As a novel finding, some of the migrated donor DCs were quickly phagocytosed by putative host interdigitating DCS: By 48 h, the remaining donor DCs had disintegrated within the rosettes. Host interdigitating DCs also formed rosettes throughout the T-cell area, and their kinetics correlated well with that of the T-cell proliferation. In the cardiac allograft model, a few donor DCs selectively migrated to the host spleen and hepatic nodes. Rosette formation by donor and host DCs, phagocytosis of donor DCs, and the T-cell proliferative response occurred in much the same fashion as they did in the first experiment. We conclude that the donor rosettes at the early stage represent the sites of direct allosensitization and those at the late stage represent donor-DC killing. Host rosettes are the sites of T-cell proliferation. In this structure, phagocytosed donor-DC-derived antigens are presumably indirectly presented.