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.

Primary retroperitoneal mucinous cystadenocarcinoma associated with pregnancy.

Primary retroperitoneal mucinous cystadenocarcinoma (PRMC) is an extremely rare tumor. Only 30 cases have been reported previously in the English literature, and little information is available concerning its treatment and prognosis. The patient was a 28-year-old woman, presenting with a right mid-abdominal tumor at 26 weeks of gestation. At 31 weeks of gestation, she underwent an exploratory laparotomy and was diagnosed with a PRMC. No disseminated tumor was observed, and an excision of only the tumor was performed. She had an uneventful vaginal delivery at 38 weeks of gestation and remains free of disease at 13 months after the operation. This report describes a case of PRMC associated with pregnancy. The optimal management of these retroperitoneal masses during pregnancy is discussed. Based on limited experience and the current literature, a PRMC with an intact capsule and no dissemination appears to have a good prognosis and can be treated by tumor excision alone in patients who wish to preserve fertility.

Primary malignant melanoma of the vagina.

Malignant melanoma originating in the vagina is considered extremely rare and has a very poor prognosis. We report a case of a 70-year-old woman with primary malignant melanoma of the vagina, and discuss the importance of prognostic factors and the efficacy of adjuvant chemotherapy.

Primary extramedullary plasmacytoma of the small intestine. A case report and review of the literature.

A case of primary extramedullary plasmacytoma of the small intestine in a 73-year-old Japanese woman was reported. The patient underwent local resection of the tumor, and showed no signs of local recurrence or dissemination of the disease after 28 months follow-up. The tumor cells had relatively large nuclei with distinct nucleoli and wide and slightly basophilic cytoplasm with a high N/C ratio which showed the morphology of atypical plasma cells. Immunohistochemical examination revealed that the tumor cells contained IgG gamma-type immunoglobulin in their cytoplasm but they did not contain IgA, IgM, IgD, and kappa-light chains. The tumor cells were also positive for CD79a and CD138 and negative for LCA, CD20 and CD45RO. These findings clearly indicated this case to be plasmacytoma.

Effect of granulocyte-colony stimulating factor on generation of oxygen-derived free radicals and myeloperoxidase activity in neutrophils from poorly controlled NIDDM patients.

To evaluate whether granulocyte-colony stimulating factor (G-CSF) improves an impaired production of oxygen-derived free radicals by neutrophils from poorly controlled NIDDM patients, we studied the effect of G-CSF on myeloperoxidase (MPO) activity and chemiluminescence amplified by a Cypridina luciferin analog (CLA-DCL), which is dependent on O2 generation, and luminol (L-DCL), which is dependent on OCl(-) generation, in response to formyl-methonyl-leucyl-phenylalanine. Both CLA-DCL and L-DCL by neutrophils from the diabetic group (n = 15, HbA(1c) >10%) were significantly decreased (26 and 37%, respectively: P < 0.01) compared with the age-matched normal control group (n = 15), and L-DCL was more sensitive to this inhibition than CLA-DCL (P < 0.05). In both control and diabetic neutrophils, G-CSF significantly enhanced both CLA-DCL (175% in control and 156% in diabetic) and L-DCL (283% in control and 346% in diabetic). In diabetic neutrophils, the enhancing effect of G-CSF on L-DCL was more sensitive than on CLA-DCL (P < 0.001). There was a positive correlation between HbA(1c) and the enhancing effect of G-CSF on L-DCL in diabetic patients (P < 0.05), but not on CLA-DCL. MPO activity was also decreased in the diabetic group (63%, P < 0.05), and G-CSF improved this impaired MPO activity (184%, P < 0.01). Furthermore, there was a positive correlation between HbA(1c) and the improving effect of G-CSF on MPO activity (P < 0.05). Because bacterial infection still accounts for an important cause of morbidity and mortality in diabetic patients, these data suggest that G-CSF may be useful as a drug to prevent the aggravation of bacterial infection by improving neutrophil function, especially through H2O2-MPO-OCl(-) mechanism, in poorly controlled diabetic patients.

Carcinosarcoma of the parotid gland: an unusual case with large-cell neuroendocrine carcinoma and rhabdomyosarcoma.

We report a case of carcinosarcoma of the parotid gland in a 72-year-old Japanese man. The patient noticed a rapidly enlarging hard mass in the right parotid gland. He underwent radical parotidectomy with cervical lymph node dissection. The resected tumor measured 3.5 x 4.5 cm and histopathologically showed carcinomatous and sarcomatous components. The carcinomatous component consisted of large-cell neuroendocrine carcinoma (LCNEC), squamous cell carcinoma and adenocarcinoma not otherwise specified, while the sarcomatous component included spindle cell sarcoma not otherwise specified, so-called myxosarcoma and rhabdomyosarcoma. The LCNEC component was predominant within the whole tumor. The diagnoses of LCNEC and rhabdomyosarcoma were also confirmed immunohistochemically. With regard to histopathogenesis, based on the lack of histopathological evidence and antecedent history of pleomorphic adenoma, we considered the present case to be de novo, not expleomorphic adenoma.

Genetic aberrations in the development and subsequent progression of myelodysplastic syndrome.

We performed longitudinal analyses of chromosomes and studied the configuration of NRAS, TP53, NF1, and cFMS genes in 70 patients with myelodysplastic syndrome(MDS). The NRAS mutations were detected in 6 patients(9%) at codons 12 or 13. The TP53 mutations were found in 10 patients(14%) in exons 4 through 8. Longitudinal studies revealed that the NRAS mutation was a late-appearing event, while the TP53 mutations were detectable at the presentation of MDS. No patients had both NRAS and TP53 mutations, simultaneously. NF1 and cFMS genes showed any mutational event among these 70 patients. Patients with a TP53 mutation had a significantly shorter survival time than those with an NRAS mutation or those without NRAS or TP53 mutation. However, patients who showed an NRAS mutation had a shorter survival time once the mutation emerged, similar to that of patients with a TP53 mutation.

Rapid detection of lymphoma-specific translocations in interphase nuclei of non-Hodgkin's lymphoma by fluorescence in situ hybridization.

We have recently developed a method to detect tumor-specific rearrangement of the IgH gene in interphase nuclei by fluorescence in situ hybridization. Tumor-specific IgH gene rearrangement is equivalent to 14q32.33 translocation. Using this approach, we detected 14q32.33 translocation in 29 of 70 patients with B-cell non-Hodgkin's lymphoma (NHL). Chromosome t(3;14) was found in 10 of these 29 patients, and were demonstrated as a fusion signal of BCL6 and VH gene probes in interphase nuclei. Furthermore, in another series of 11 patients and a NHL cell line, we demonstrated t(14;18) and t(11;14) in interphase and metaphase cells with a combination of BCL2 (or PRAD1) with IgH gene probes. Interphase FISH with lymphoma-associated gene probes is a rapid procedure for cytogenetic diagnosis of B-cell NHL.

N-ras mutation and karyotypic evolution are closely associated with leukemic transformation in myelodysplastic syndrome.

We performed a longitudinal analysis of the karyotypes and N-ras gene configuration of bone marrow cells in 35 patients with myelodysplastic syndrome (MDS). Karyotypic evolution was found in eight patients, and was associated with disease progression, including leukemic transformation, in all the patients. We identified N-ras mutations in six patients, using a polymerase chain reaction (PCR) technique, in which oligonucleotide primers were constructed with induced mismatches, followed by endonuclease digestion. Direct sequencing confirmed single base substitutions at codon 12 in two patients and at codon 13 in four. The incidence of N-ras gene mutations was significantly higher in the karyotypically evolved group (five of eight patients) than in the stable group (one of 27 patients). All of five patients harboring both karyotypic evolution and an N-ras mutation showed concomitant disease progression to overt leukemia or refractory anemia with excess of blasts in transformation (RAEB-T). Two of four patients with either karyotypic evolution or N-ras mutation and six of 26 patients without any of these alterations also progressed to overt leukemia. Our results indicate that the accumulation of these genetic alterations is closely associated with leukemic transformation of MDS, although other genetic alterations may also play a key role in the remaining patients.

Detection and quantification of TEL/AML1 fusion transcripts by polymerase chain reaction in childhood acute lymphoblastic leukemia.

We investigated TEL/AML1 fusion mRNA in 108 children with acute lymphoblastic leukemia (ALL) (86 B-lineage ALL, 15 T-ALL, two mixed lineage ALL, and five other phenotypes) using reverse transcriptase-polymerase chain reaction (RT-PCR). TEL/AML1 transcripts were found in 14 patients (13%) including three relapsed patients, and were unexceptionally limited to B-lineage ALL patients. The incidence of TEL/AML1 transcripts among B-lineage ALL was 16% (14/86). The reciprocal AML1/TEL transcripts were detected in 12 (86%) of the 14 cases expressing a TEL/AML1 transcript. In three cases, the TEL gene was fused to exon 3 of the AML1 gene, and to exon 2 in the remaining cases. To evaluate the amount of TEL/AML1 molecules for the quantification of a minimal residual disease (MRD), a plasmid vector which contained either a long TEL/AML1 PCR product (464 bp) or a short one (425 bp) was used as a competitor. We amplified RNAs obtained from bone marrow (BM) at complete remission or from peripheral blood stem cell (PBSC) harvests in two representative cases. For one PBSC harvest showing a positive result, a competitive PCR was carried out to quantify the amount of MRD. A 1:4 dilution series of competitor vectors was constructed, and each vector was added to a PCR reaction which contain a constant amount of cDNA obtained from the PBSC harvest. An equivalent point was compared to that of corresponding samples at diagnosis. Using this method, MRD in the PBSC harvest was 3.9:10(3). Our results elucidated the incidence, lineage-specificity, and variant forms of TEL/AML1 fusion transcripts in childhood ALL. Since the percentage of other chromosomal translocations in childhood ALL is not more than 5%, TEL/AML1 transcript would be the most feasible clone-specific marker for these patients. In addition, our method could be a powerful tool for quantification of the TEL/AML1 transcript and for the detection of MRD.

Internal tandem duplication of the flt3 gene found in acute myeloid leukemia.

We analyzed mRNA expression of the flt3 gene in 30 patients with acute myeloid leukemia (AML) and 50 with acute lymphoblastic leukemia (ALL). Using reverse transcriptase-polymerase chain reaction (RT-PCR), expression of flt3 was observed in 61 patients; 22 (73%) with AML and 39 (78%) with ALL. Among these, five patients with AML (one M2, two M4, and two M5) showed unexpected longer transcripts with a primer combination which could amplify the transmembrane (TM) domain through the juxtamembrane (JM) domain. For those patients who expressed flt3 mRNA, the extracellular domain of the flt3 gene was also examined by RT-PCR, but no length abnormality was seen in this region. We further analyzed the TM domain through the second tyrosine kinase domain by genomic amplifications. The five patients who showed aberrant flt3 transcripts exhibited abnormal longer PCR products in addition to the germline products at a region corresponding to the JM through the first TK (TK1) domains. Sequence analyses of the abnormal RT-PCR products demonstrated that partial sequences were tandemly duplicated. Because all these altered transcripts were in-frame, deduced protein products could be expected. Sequence analyses of the genomic DNA revealed that three of the five patients showed a simple internal duplication within exon 11; one had an internal duplication (26 bp) with a 4-bp insertion; and in the fifth patient, a 136-bp sequence from the 3' part of exon 11 to intron 11 and the first 16-bp sequence of exon 12 were each duplicated with 1-bp insertion. In order to confirm the tumor specificity of these alterations, DNA samples obtained at complete remission were also analyzed in the three patients harboring an flt3 duplication, but no abnormal PCR product other than germline was detected in any of the samples. Our results suggest that an internal tandem duplication at the JM/TK1 domains of the flt3 gene is a somatic change detected preferentially in AML, possibly containing a monocytic component.

Tandem duplications of the FLT3 receptor gene are associated with leukemic transformation of myelodysplasia.

We recently reported an internal tandem duplication of the human flt3 receptor gene (FLT3) as a somatic mutation in 17% of acute myelogenous leukemia (AML). The present study revealed the duplication at the juxtamembrane and the first tyrosine kinase domains of FLT3 in seven of 92 (8%) patients with myelodysplastic syndrome (MDS) and AML with trilineage myelodysplasia (AML/TMDS), the diseases which may represent neoplastic changes of pluripotent stem cells. A tandem duplication of exon 11 of FLT3 was harbored by two of 58 (3%) patients with MDS and five of 34 (15%) with overt leukemia, including MDS-derived leukemia, AML/TMDS and therapy-related leukemia. Although the duplicated regions varied within exon 11 in each case, they occurred in-frame, and altered mRNA expressions were demonstrated by reverse-transcription polymerase chain reaction. Two cases of MDS with a FLT3 duplication transformed to overt leukemia within a few months. Longitudinal analyses in two other patients with leukemia revealed that the duplication was a late genetic event during the disease course; one of whom showed two independent duplications of FLT3 at the terminal therapy-resistant phase. Of seven patients with the FLT3 duplication, six had abnormal karyotypes, and four harbored a point mutation of the N-RAS and/or TP53 genes. Patients with FLT3 mutations have poor prognoses. This study uncovered the fact that the accumulation of genetic events, including FLT3 duplication, correlates with leukemic transformation from antecedent myelodysplasia and with subsequent disease progression.

Distinct genetic involvement of the TP53 gene in therapy-related leukemia and myelodysplasia with chromosomal losses of Nos 5 and/or 7 and its possible relationship to replication error phenotype.

We examined chromosomes and molecular aberrations in 21 patients with therapy-related leukemias (t-AML) or myelodysplastic syndromes (t-MDS). All patients showed abnormal karyotypes, and chromosomal losses of No. 5 and/or No. 7 (-5/5q- and/or -7/7q-) were identified in 12 patients. Among these 12, six patients (50%) harbored a TP53 mutation, and two of five examined showed microsatellite instability, suggesting replication error (RER+) phenotype. Meanwhile, among the other nine patients without -5/5q- and/or -7/7q-, none harbored a TP53 mutation, and none of five examined showed RER+ phenotype. Thus, TP53 mutations and RER+ phenotype were preferentially associated with specific chromosomal losses in t-AML/MDS. We then screened for mutational events in representative DNA mismatch repair genes; exons 5-7 and 12-15 of the hMSH2 gene and exon 9 of hMLH1. Notably, two unrelated patients showing RER+ phenotype had an identical missense alteration at codon 419 of hMSH2 in their marrow cells and fibroblasts, which were not found in 120 DNA samples from healthy volunteers or patients with other hematological disorders. Consequently, this study revealed a possible relationship of RER+ phenotype accompanying an hMSH2 alteration to the development of therapy-related AML/MDS in association with TP53 mutations and specific chromosomal losses, and suggests that some patients may be predisposed to myelodysplasia after chemotherapy for their primary tumor.

Lack of involvement of the G-CSF gene by chromosomal translocation t(15;17) in acute promyelocytic leukemia.

Using a human G-CSF cDNA as a probe, we analyzed the t(15;17) breakpoint by Southern blot analysis with conventional and/or pulsed-field gel electrophoresis in 12 patients with acute promyelocytic leukemia. The results did not show the rearrangement, deletion, or restriction fragment length polymorphism within the gene and in the surrounding sequences.

Expression of beta-catenin, E-cadherin and cyclin D1 in adenoid cystic carcinoma of the salivary gland.

We evaluated the expression of beta-catenin, E-cadherin, and cyclin-D1 in 23 cases of adenoid cystic carcinoma (ACC) of the salivary gland. We detected beta-catenin on the cell membranes in all ACCs, but its distribution was irregular, as compared to that on normal structures. In three out of the 23 cases, beta-catenin was detected in the nuclei, as well as on cell membranes. Polymerase chain reaction (PCR) and direct sequencing revealed a missense mutation in one case in which beta-catenin had been detected in the nuclei of tumor cells. We also detected E-cadherin on cell membranes with a similar irregular distribution to that of beta-catenin. In 11 cases (almost 48%) of ACC, cyclin D1 was localized in cell nuclei but there was no correlation with the nuclear staining of the beta-catenin. Our results suggest that disturbances in the distribution of beta-catenin and E-cadherin might affect the morphology ofACC and that a small fraction of cases of ACC are characterized by a mutation in the beta-catenin gene, which is associated with the nuclear accumulation of the product of this gene but does not affect the transcription of the gene for cyclin-D1.

Epalrestat, an aldose reductase inhibitor, improves an impaired generation of oxygen-derived free radicals by neutrophils from poorly controlled NIDDM patients.

OBJECTIVE: To study the in vivo effect of epalrestat (Epa), an aldose reductase inhibitor, on the generation of oxygen-derived free radicals by neutrophils from poorly controlled NIDDM patients (HbA1c > 10%). RESEARCH DESIGN AND METHODS: A total of 31 diabetic patients were randomly divided into two groups: an Epa(+) group of 16 patients treated with 150 mg/day epalrestat and an Epa(-) group of 15 patients treated without epalrestat. A control group of 20 age- and sex-matched normal healthy subjects also participated. HbA1c, postprandial plasma glucose (PPG), and neutrophil bactericidal function were measured before and at the end of the drug treatment period (4 weeks). Neutrophil bactericidal function was measured as chemilu-minescence amplified by a Cypridina luciferin analog (CLA), which is dependent on O2- generation, and by luminol (L), which is highly dependent on OCl- generation, in response to formyl-methonyl-leucyl-phenylalanine (fMLP). RESULTS: At the start of the experiment, both CLA-dependent chemiluminescence (CLA-DCL) and L-dependent chemiluminescence (L-DCL) were clearly decreased in diabetic subjects (64 and 54%, respectively; P < 0.05) compared with control subjects (2,182 +/- 144 and 3,221 +/- 173 kc.min-1.10(-6) cells, respectively). At the end of the experiment, CLA-DCL and L-DCL in the Epa(+) group were significantly improved by 44 and 46%, respectively; however, these values were still lower than the corresponding results in the control group. HbA1c and PPG in both the Epa(+) and Epa (-) groups were significantly higher than in the control group, and treatment had no effect on either HbA1c or PPG. CONCLUSIONS: These data suggest that epalrestat may be a useful drug to prevent infection by improving the impaired O2- and OCl- generation by neutrophils from poorly controlled NIDDM patients.

Hypertonic glucose impairs glucose-induced increases in cytosol Ca2+ concentration and insulin secretion by HIT-T 15 cells.

To evaluate the mechanism of an impaired insulin secretion under diabetic state, we studied the influence of graded degrees of isotonic and hypertonic glucose on the rise in cytosol Ca2+ concentration ([Ca2+]i) and insulin secretion by HIT-T 15 cells. [Ca2+]i was monitored with Fura-2 and insulin secretion was measured with static culture. Both isotonic and hypertonic glucose induced a dose-dependent [Ca2+]i rise and insulin secretion. Although low degrees of isotonic and hypertonic glucose (5 and 15 mM) were equally effective to cause both increases in [Ca2+]i and insulin secretion, high degrees of isotonic glucose (30 and 60 mM) were clearly more effective than the same degrees of hypertonic glucose. The addition of 30 mM sucrose in isotonic 30 mM glucose also decreased the induced increases in [Ca2+]i and insulin secretion, and isotonic sucrose was ineffective in inducing either an increase in [Ca2+]i or insulin secretion. Removal of medium Ca2+ inhibited the 30 mM isotonic glucose-induced [Ca2+]i rise and insulin secretion. These data suggest that an impaired rise in [Ca2+]i, which was caused by blocking of Ca2+ influx due to hyperosmolarity, may be one mechanism by which glucose can not stimulate appropriate insulin secretion under diabetic state.

Effect of epalrestat, an aldose reductase inhibitor, on the generation of oxygen-derived free radicals in neutrophils from streptozotocin-induced diabetic rats.

Neutrophil function is impaired by a known mechanism in diabetic patients, thus increasing susceptibility to infections. We studied the effect of epalrestat, an aldose reductase inhibitor, on the generation of oxygen-derived free radicals and cytosolic sorbitol concentration in neutrophils from streptozotocin-induced diabetic rats. There were four groups: treated and untreated control and diabetic rats. Treated groups were given 0.075% epalrestat in their diet for 4 weeks from the induction of diabetes and were untreated for the subsequent 4 weeks. Oxygen radicals were measured as chemiluminescence amplified by a luciferin analog [Cypridina luciferin analog-dependent chemiluminescence (CLA-DCL), which is dependent on O2- generation] and luminol (L)-DCL, which is highly dependent on OCl- generation) in response to formyl-methonyl-leucyl-phenylalanine. Diabetes resulted in a significant decrease in CLA/L-DCL and a significant increase in sorbitol (P < 0.01); there was a negative correlation between sorbitol and CLA-DCL (P < 0.05) in diabetic groups. The 4-week treatment with epalrestat in the diabetic group completely prevented the increase in sorbitol and partially improved the CLA-DCL, although L-DCL was not significantly affected. After 4 weeks off treatment, CLA-DCL decreased and sorbitol increased. Treatment had no effect on serum insulin or glucose concentration. We conclude that an increase in sorbitol in neutrophils causes, in part, an impaired generation of O2-. Epalrestat improves the impaired O2- generation by preventing the sorbitol increase in streptozotocin-induced diabetic rats.

Tyrosine kinase-dependent modulation by interferon-alpha of the ATP-sensitive K+ current in rabbit ventricular myocytes.

We examined the effects of interferon-alpha on the ATP-sensitive K+ current (IK,ATP) in rabbit ventricular cells using the patch-clamp technique. IK,ATP was induced by NaCN. Whole-cell experiments indicated that interferon-alpha (5 x 10(2) - 2.4 x 10(4) U/ml) inhibited IK,ATP in a concentration-dependent manner (60.7+/-7.5% with 2.4 x 10(4) U/ml). In cell-attached configuration, interferon-alpha (2.4 x 10(4) U/ml) applied to the external solution also inhibited the activity of the single ATP-sensitive K+ (KATP) channel by 56.0+/-5.8% without affecting the single channel conductance. The inhibitory effect of IK,ATP by interferon-alpha was blocked by genistein and herbimycin A, tyrosine kinase inhibitors, but was not affected by N-(2-metylpiperazyl)-5-isoquinolinesulfoamide (H-7), an inhibitor of protein kinase C and cAMP-dependent protein kinase. These findings suggest that interferon-alpha inhibits the cardiac KATP channel through the activation of tyrosine kinase. The tyrosine kinase-mediated inhibition of IK,ATP by cytokines may aggravate cell damage during myocardial ischemia.