An outbreak of food poisoning due to Escherichia coli serotype O7:H4 carrying astA for enteroaggregative E. coli heat-stable enterotoxin1 (EAST1).

In June 2020, a large-scale food poisoning outbreak involving about 3000 elementary and junior high school students occurred in Yashio, Saitama, Japan. A school lunch was the only food stuff ingested by all of the patients. Escherichia coli serotype O7:H4 carrying the astA gene for enteroaggregative E. coli (EAggEC) heat-stable enterotoxin 1 (EAST1) was detected in faecal specimens from the patients, and sample inspection revealed its presence in a seaweed salad and red seaweed (Gigartina tenella) as one of the raw materials. Analysis of the antibiotic sensitivity of the isolates revealed resistance to ampicillin and cefotaxime. All isolates were confirmed to be of the same origin by pulsed-field gel electrophoresis after digestion with the restriction enzyme XbaI, and single nucleotide polymorphism analysis using whole genome sequencing. To our knowledge, this is the first report of a large-scale food poisoning caused by E. coli O7:H4, which lacks well-characterized virulence genes other than astA.

Comprehensive diagnostic ability of endocytoscopy compared with biopsy for colorectal neoplasms: a prospective randomized noninferiority trial.

BACKGROUND AND STUDY AIMS: Endocytoscopy enables observation at 450-fold magnification during gastrointestinal endoscopy, allowing on-site "optical biopsy." We compared the accuracies of endocytoscopy and standard biopsy for the diagnosis of colorectal neoplasms. PATIENTS AND METHODS: We performed a randomized, controlled, open-label trial of patients with colorectal lesions (≥ 5 mm) detected during colonoscopy in a tertiary referral center. We randomly assigned the 203 detected lesions of 170 eligible patients to either the endocytoscopy or standard biopsy group. An on-site endoscopist assessed the histopathology of the endocytoscopy group lesions according to the endocytoscopic findings, whereas a pathologist later assessed standard biopsy group lesions by microscopic examination of the biopsy specimens. We calculated the diagnostic accuracies in both groups with reference to the final histopathology of the resected specimens. The primary endpoint was to determine whether the diagnostic accuracy of endocytoscopy for neoplastic lesions was noninferior to that of standard biopsy (with a predefined noninferiority margin of 10%). Analyses were by intention-to-treat and per-protocol. The study is registered, number UMIN000003923. RESULTS: Overall, 102 lesions in the endocytoscopy group and 101 in the standard biopsy group were available for primary outcome analysis. There were no complications. The diagnostic accuracy of endocytoscopy for the discrimination of neoplastic lesions was 94.1% (95% confidence interval 87.6% to 97.8%), whereas that of standard biopsy was 96.0% (90.2% to 98.9%), which is within the noninferiority margin (absolute difference -1.9%, -8.6% to +5.0%). CONCLUSIONS: Endocytoscopy is noninferior to standard biopsy for the discrimination of neoplastic lesions. With its advantage of providing an on-site diagnosis, endocytoscopy could provide a novel alternative to standard biopsy in routine colonoscopy.

Evidence for extrathymic generation of intermediate T cell receptor cells in the liver revealed in thymectomized, irradiated mice subjected to bone marrow transplantation.

In addition to the major intrathymic pathway of T cell differentiation, extrathymic pathways of such differentiation have been shown to exist in the liver and intestine. In particular, hepatic T cells of T cell receptors or CD3 of intermediate levels (i.e., intermediate T cell receptor cells) always contain self-reactive clones and sometimes appear at other sites, including the target tissues in autoimmune diseases and the tumor sites in malignancies. To prove their extrathymic origin and self reactivity, in this study we used thymectomized, irradiated (B6 x C3H/He) F1 mice subjected to transplantation of bone marrow cells of B6 mice. It was clearly demonstrated that all T cells generated under athymic conditions in the peripheral immune organs are intermediate CD3 cells. In the case of nonthymectomized irradiated mice, not only intermediate CD3 cells but also high CD3 cells were generated. Phenotypic characterization showed that newly generated intermediate CD3 cells were unique (e.g., interleukin 2 receptor alpha-/beta+ and CD44+ L-selectin-) and were, therefore, distinguishable from thymus-derived T cells. The precursor cells of intermediate CD3 cells in the bone marrow were Thy-1+ CD3-. The extrathymic generation of intermediate CD3 cells was confirmed in other combinations of bone marrow transplantation, C3H --> C3H and B10.Thy1.1 --> B6.Thy1.2. The generated intermediate CD3 cells in the liver contained high levels of self-reactive clones estimated by anti-V beta monoclonal antibodies in conjunction with the endogenous superantigen minor lymphocyte-stimulating system, especially the combination of B6 --> (B6 x C3H/He) (graft-versus-host-situation).(ABSTRACT TRUNCATED AT 250 WORDS)

Feasibility of stomach exploration with a guided capsule endoscope.

BACKGROUND AND STUDY AIMS: Video capsule endoscopy has been established in diagnosis of small-bowel disease and has been evaluated for esophageal pathology and recently for colorectal diagnostics. Gastric capsule endoscopy has not hitherto been feasible due to the stomach's large surface area and volume. We present the first application of a magnetically navigated capsule in the human stomach. PATIENTS AND METHODS: 29 volunteers and 24 patients (men 42, women 11; mean age 47.5 years) were included in a feasibility study. Low-level magnetic fields were used to maneuver the double-sensor video capsule within the human stomach with an air-water interface provided by ingestion of 1300 ml water within 1 hour before examination. Visualization of all parts of the stomach was attempted; time for visualization was recorded, and a subjective assessment of completeness of visualization was documented. RESULTS: There was technical failure in one individual; thus technical success rate was 98 %. In the 52 remaining cases, examiners assessed that the antrum, body, fundus, and cardia were fully visualized in 98 %, 96 %, 73 % and 75 %, respectively. Mean duration of examinations was 30 minutes (range 8 - 50), with a longer time (mean 37 minutes) for volunteers for study reasons. In total, 30 findings were identified: 14 were detected by both gastroscopy and capsule, 10 lesions were identified by guided capsule examination only, 6 by gastroscopy only. No significant capsule-related adverse events occurred. CONCLUSION: Magnetically navigated video capsule endoscopy appears to be feasible and sufficiently accurate for gastric examination. It may permit endoscopic examinations that are more patient-friendly and without sedation. Comparative studies are under way.

Salmonella prevalence in seafood imported into Japan.

A total of 353 samples of 29 types of seafood were tested for Salmonella prevalence and total microbial population. Salmonella enterica serotype Weltevreden was isolated from 2 of 47 black tiger prawn samples. The contamination levels of Salmonella were in a range of <30 to 40 most probable number per 100 g. In addition, one sample of black tiger prawns and two samples of white shrimp were positive for Salmonella invA gene on PCR assay. Although the mean aerobic bacterial count was greater than 4 log CFU/g in most of the sample types, those in the two Salmonella-isolated samples of black tiger prawn were 7.48 and 5.18 log CFU/g, respectively. These results indicate the possibility that shrimp and prawns contribute to foodborne infections. The improvement of seafood quality is an important issue, and the information on contamination by pathogens should be provided as feedback to the originating country, with the aim of increasing safety.

Role of heat shock cognate 70 protein in import of ornithine transcarbamylase precursor into mammalian mitochondria.

The roles of the 70-kDa cytosolic heat shock protein (hsp70) in import of precursor proteins into the mitochondria were postulated to be related to (i) unfolding of precursor proteins in the cytosol, (ii) maintenance of the import-competent state, and (iii) unfolding and transport of precursor proteins through contact sites, in cooperation with matrix hsp70. We examined roles of cytosolic hsp70 family members in import of ornithine transcarbamylase precursor (pOTC) into rat liver mitochondria, using an in vitro import system and antibodies against hsp70. Immunoblot analysis using an hsc70 (70-kDa heat shock cognate protein)-specific monoclonal antibody and a polyclonal antibody that reacts with both hsc70 and hsp70 showed that hsc70 is the only or major form of hsp70 family members in the rabbit reticulocyte lysate. The hsc70 antibody did not inhibit pOTC import when added prior to import assay. However, when pOTC was synthesized in the presence of the antibody and then subjected to import assay, pOTC import was markedly decreased. pOTC import was also decreased when the precursor was synthesized in the lysate depleted for hsc70 by treatment with hsc70 antibody-conjugated Sepharose. This reduction was almost completely restored by readdition of purified mouse hsc70 during pOTC synthesis. The readdition of hsc70 after pOTC synthesis and only during the import assay was not effective. Thus, once import competence of pOTC was lost, hsc70 was ineffective for restoration. Newly synthesized pOTC lost import competence in the absence of hsc70 somewhat more rapidly than in its presence. These results indicate that hsc70 is required during pOTC synthesis and not during import into the mitochondria. hsc70 presumably binds to pOTC polypeptide and maintains it in an import-competent form.

The requirement of heat shock cognate 70 protein for mitochondrial import varies among precursor proteins and depends on precursor length.

The cytosolic heat shock cognate 70-kDa protein (hsc70) is required for efficient import of ornithine transcarbamylase precursor (pOTC) into rat liver mitochondria (K. Terada, K. Ohtsuka, N. Imamoto, Y. Yoneda, and M. Mori, Mol. Cell. Biol. 15:3708-3713, 1995). The requirement of hsc70 for mitochondrial import of various precursor proteins and truncated pOTCs was studied by using an in vitro translation import system in which hsc70 was completely depleted. hsc70-dependent import of pOTC was about 60% of the total import, while import of the aspartate aminotransferase precursor, the serine:pyruvate aminotransferase precursor, and 3-oxoacyl coenzyme A thiolase was about 50, 30, and 0%, respectively. The subunit sizes of these four precursor proteins were 40 to 47 kDa. When pOTC was serially truncated from the COOH terminal, the hsc70 requirement decreased gradually and was not evident for the shortest truncated pOTCs of 90 and 72 residues. These truncated pOTCs were imported and proteolytically processed rapidly in 0.5 to 2 min at 25 degrees C, and the processed mature portions and the presequence portion were rapidly degraded. Sucrose gradient centrifugation analysis followed by import assay showed that pOTC synthesized in rabbit reticulocyte lysate forms an import-competent complex of about 11S in an hsc70-dependent manner. S values of import-competent forms of aspartate aminotransferase precursor, serine:pyruvate aminotransferase precursor, and 3-oxoacyl coenzyme A thiolase were 9S, 9S, and 4S, respectively. Thus, the S value decreased as the hsc70 dependency decreased. Precursor proteins were coimmunoprecipitated from the reticulocyte lysate containing the newly synthesized precursor proteins with an hsc70 antibody. The amount of coimmunoprecipitated proteins was much larger in the absence of ATP than in its presence. Among the four precursor proteins, the amount of coimmunoprecipitated protein decreased as the hsc70 dependency decreased.

Spatiotemporal treadmill gait measurements using a laser range scanner: feasibility study of the healthy young adults.

OBJECTIVE: Spatio-temporal parameters are typically used for gait analysis. Although these parameters are measured by sophisticated systems such as 3D motion capture system or optoelectronic bars, these systems cannot be deployed easily because of their high costs, large space requirements and elaborate set-up. The purpose of this study is to develope a system for measuring spatiotemporal gait parameters using a laser range scanner during treadmill gait. APPROACH: To calculate accurate spatiotemporal parameters, the differences between the laser range scanner measured values and the reference values obtained from a 3D motion capture system were investigated in thirty subjects. From measurements in time and position at foot contact/off, adjustments to compensate for the differences in time and position were derived. Then, to determine the validity of the proposed system, values from the proposed system and the reference system were compared in four additional subjects. MAIN RESULTS: The results indicate that the data from the laser range scanner demonstrate certain differences in time and position compared with reference values. However, when compensation values were introduced, each spatiotemporal parameter correlated well with the reference values. SIGNIFICANCE: This newer system is smaller, is easier to deploy and requires less training than the 3D motion capture system.

Murine cDNA encoding a novel type I HSP40/DNAJ homolog, mmDjA4(1).

We have cloned a cDNA encoding a novel type I HSP40/DNAJ protein from the mouse EST database, and designated it mmDjA4 (Mus musculus type I DnaJ homolog 4). This cDNA encodes 397 amino acid residues whose sequence shows 67 and 51% identity with the previously identified murine Hsj2 and mDj3, respectively. The sequence of mmDjA4 contains the four repeats of CxxCxGxG motif which are characteristic of type I HSP40/DNAJ proteins, and a CaaX prenylation motif at the carboxy terminus. Northern blot analysis showed that mmDjA4 is specifically expressed in mouse testis and heart. This is the fourth member of the mammalian type I HSP40/DNAJ family to be identified.

Characterization of HSE sequences in human Hsp40 gene: structural and promoter analysis.

We have recently cloned a gene of Hsp40, a human homologue of bacterial DnaJ. Here we describe the structural and promoter analysis of human Hsp40 gene. Analysis of Hsp40 transcripts by 5' and 3' RACE suggested that they have different 3' ends, and primer extension studies revealed that the major transcription initiation site was localized 47 bp upstream of the ATG translation initiation codon. Promoter analysis using deletion derivatives defined a minimal region which was active in response to heat shock. The region contained the consensus heat shock element (HSE) sequences. The factor bound to these sequences was suggested to be a heat shock factor 1 (HSF1) by gel mobility supershift assay. In vivo footprinting and promoter analysis revealed that the HSEs in 5' upstream region of human Hsp40 gene were composed of eight contiguous (A/G)GAAN motifs and were essential for heat shock response. These results indicate that Hsp40 is a real heat shock protein. It is also shown that the HSE found in the first intron might not be the essential element for heat shock response.

Co-expression of human chaperone Hsp70 and Hsdj or Hsp40 co-factor increases solubility of overexpressed target proteins in insect cells.

The insect-baculovirus expression system has proved particularly useful for producing recombinant proteins that are biologically active. Overexpression of foreign proteins using the recombinant baculovirus system is often accompanied by aggregation of the overexpressed protein, which is thought to be due to a limitation of the translated protein folding in the infected cells. Co-infection of a recombinant baculovirus capable of expressing the human chaperone Hsp70 slightly increased the solubility of the overexpressed Epstein-Barr virus replication protein, BZLF1. Co-expression of Hsp70 and its co-factor, Hsdj or Hsp40, was here found to improve the solubility of the target protein several fold. Thus, a baculovirus expression system producing these molecular chaperones may find application for improved production of target foreign gene products in insect cells.

Role of FMRFamide-activated brain sodium channel in salt-sensitive hypertension.

FMRFamide, a cardioexcitatory neuropeptide, directly activates a newly cloned amiloride-sensitive sodium channel that is expressed specifically in the brain and blocked by benzamil hydrochloride. In the present study, we investigated the effects of short- and long-term intracerebroventricular infusion of FMRFamide on arterial pressure, sympathetic activity, vasopressin release, and brain renin-angiotensin system genes in rats and studied the role of FMRFamide-activated brain sodium channels in salt-sensitive hypertension. The intracerebroventricular preinjection of FMRFamide and subsequent intracerebroventricular infusion of 0.15 mol/L NaCl increased mean arterial pressure (FMRFamide: 30 nmol/kg +13+/-2.6 mm Hg, P<0.01; 100 nmol/kg +21+/-1.8 mm Hg, P<0.01), heart rate, abdominal sympathetic activity, and plasma vasopressin concentration compared with vehicle. The intracerebroventricular copreinjection with either benzamil or CV-11974 abolished these increases. In rats administered a high-salt diet (8% NaCl), the continuous intracerebroventricular infusion of FMRFamide (50 and 200 nmol. kg(-1). d(-1)) for 5 days increased mean arterial pressure, heart rate, urinary excretion of vasopressin and norepinephrine, and mRNAs of renin, angiotensin I-converting enzyme, and angiotensin II type 1 receptor in hypothalamus and brain stem compared with vehicle. These increases were abolished by intracerebroventricular coinfusion of benzamil. In rats administered a low-salt diet (0.3% NaCl), however, increases in these variables were smaller than those in rats receiving a high-salt diet. Together, these findings suggest that brain FMRFamide-activated sodium channels may be involved in the mechanism of salt-sensitive hypertension through regulation of the brain renin-angiotensin system.

Peripheral plasma concentrations of somatostatin-like immunoreactivity in newborns and infants.

Postprandial peripheral plasma concentrations of somatostatin-like immunoreactivity (SLI) were measured by RIA after extraction with acetone-petroleum ether in 98 normal newborns and infants. In 62 subjects, plasma concentrations of gastrin were determined simultaneously. The plasma levels of SLI increased progressively with age during the neonatal period. The mean plasma levels of SLI increased significantly from 19.5 +/- 7.5 (+/- SD) pg/ml in 29 cord blood samples to 29.0 +/- 17.2 pg/ml in 36 newborns aged 1-5 days. The mean postprandial level of plasma SLI was significantly higher in 18 infants aged 1-3 months (45.5 +/- 25.8 pg/ml) than in newborns and tended to decrease in infants aged 7-10 months after weaning (31.6 +/- 7.9 pg/ml). The mean postprandial levels of plasma SLI were significantly higher in newborns and infants less than 10 months old than in older children aged 8-12 yr (10.2 +/- 3.7 pg/ml). The gel filtration patterns of SLI of plasma extracts were similar in infants and older children. The changing pattern of mean plasma gastrin with age was similar to that of SLI, and individual plasma levels of SLI were significantly correlated with those of gastrin during the neonatal period. High concentrations of SLI are present in the peripheral circulation during the first 10 months of life. Thus, this peptide may play a significant role as a hormone in nutrient homeostasis in infants.

Increased serum concentrations of human hepatocyte growth factor in proliferative diabetic retinopathy.

Human hepatocyte growth factor (hHGF) is a powerful inducer of angiogenesis. We investigated the relationship between serum hHGF concentrations and proliferative diabetic retinopathy, the major characteristic of which is retinal neovascularization. Serum hHGF concentrations were measured in diabetic (n = 135) and nondiabetic subjects (n = 80). The mean serum hHGF concentration in diabetic subjects without retinopathy was lower than that in nondiabetic subjects [0.041 +/- 0.003 ng/mL (n = 62) vs. 0.080 +/- 0.010 ng/mL (n = 80); P < 0.05], but was not different from that in diabetic subjects with background retinopathy (0.058 +/- 0.007 ng/mL; n = 26) or preproliferative retinopathy (0.048 +/- 0.010 ng/mL; n = 10). The mean serum hHGF concentration was increased in subjects with proliferative retinopathy who had not undergone photocoagulation (0.213 +/- 0.025 ng/mL; n = 24), but not in those who had undergone photocoagulation (0.040 +/- 0.008 ng/mL; n = 13). Circulating hHGF may be involved in the mechanism of neovascularization in the proliferative diabetic retinopathy, and measurement of serum hHGF may be helpful in predicting the presence of proliferative retinopathy in diabetic subjects.

Serum hepatocyte growth factor as a possible indicator of vascular lesions.

To investigate the role of human hepatocyte growth factor (hHGF) in vascular lesions associated with endothelial injury, we measured serum hHGF concentrations in subjects with retinal arteriosclerosis, coronary atherosclerosis, or arteriolitis due to Henoch-Schönlein purpura. Individuals with more advanced grades of retinal arteriosclerosis showed higher serum hHGF concentrations [grade 0, 0.053+/-0.005 ng/mL (n = 68); grade 1, 0.144+/-0.022 ng/mL (n = 21; P<0.01 vs. grade 0); grade 2, 0.338+/-0.036 ng/mL (n = 20; P<0.01 vs. grade 0 or 1); grade 3, 0.526+/-0.051 ng/mL (n = 9; P<0.01 vs. grade 0, 1, or 2)]. Patients with active arteriolitis due to Henoch-Schönlein purpura showed higher (P<0.01) serum hHGF concentrations (0.347+/-0.038 ng/mL; n = 14) than those in the remission phase (0.097+/-0.017 ng/mL; n = 19). Mean serum hHGF concentrations were higher in subjects with coronary atherosclerosis than in those without, but a significant overlap in serum hHGF concentrations was found between subjects with and those without coronary atherosclerosis. Serum hHGF may be an indicator of the presence or development of arteriolar lesions.

Effect of DFP on loading of fura 2/AM and quin 2/AM into single smooth muscle cells prepared from guinea pig taenia coli.

The effect of diisopropyl fluorophosphate (DFP), a potent cholinesterase (ChE) inhibitor, on loading quin 2 acetoxymethyl ester (quin 2/AM) and fura 2/AM into smooth muscle cells isolated from guinea pig taenia coli was investigated spectrofluorometrically. The presence of DFP during the loading permitted the incorporation of quin 2 into the cells, so that it became possible to measure intracellular Ca2+ concentrations using the ester of this dye. Also, DFP significantly enhanced the incorporation of fura 2 into the cells. These results indicate that loading of quin 2/AM and fura 2/AM into the smooth muscle cells may depend on the suppression of ChE or various serine protease activities outside cells.

Interaction between hsp70 and hsp40, eukaryotic homologues of DnaK and DnaJ, in human cells expressing mutant-type p53.

We have recently identified a novel 40-kDa heatshock protein hsp40 as a mammalian homologue of bacterial DnaJ protein. Here we demonstrate the physical interaction between hsp70 (DnaK homologue) and hsp40 in human cells as determined by immunoprecipitation methods. Co-immunoprecipitation of hsp70 with hsp40 was dependent on the presence of ATP or unfolded protein (reduced carboxymethylated alpha-lactalbumin). A mutant type of tumor suppressor gene product, mtp53, was co-immunoprecipitated not only with hsp70 but also with hsp40. These results suggest the existence of a hsp70(DnaK)/hsp40(DnaJ) chaperone system in mammalian cells.

Role of NK cells and TGF-beta in the regulation of T-cell-dependent antibody production in health and autoimmune disease.

Natural killer (NK) cells are a third lymphocyte population especially important in innate immunity. NK cells may also have an important role in the regulation of acquired immunity. These lymphocytes spontaneously produce large amounts of both active and latent transforming growth factor-beta (TGF-beta). NK-cell-derived TGF-beta1 enabled activated CD8(+) T cells to inhibit antibody production by blocking the induction of this response. Production of lymphocyte-derived TGF-beta is decreased in systemic lupus erythematosus. Insufficient levels of this cytokine in SLE and other autoimmune diseases may contribute to defective T regulatory cell function characteristic of this and other autoimmune diseases. NK cells are found in mucosal tissues and the TGF-beta spontaneously released by these cells could contribute to the usual tolerogenic response of T cells to antigens presented at these sites. Thus, in addition to its well known immunosuppressive effects, TGF-beta could have an equally important role in the generation of regulatory T cells.