Multiplex real-time PCR for exhaustive detection of diarrhoeagenic Escherichia coli.

AIMS: The source and routes of diarrhoeagenic Escherichia coli (DEC) have not been clarified because it is difficult to detect these organisms in samples with numerous coliform bacteria. We have developed multiplex real-time PCR assays for exhaustive detection of DEC. METHODS AND RESULTS: Primers and TaqMan probes were designed to amplify and quantify one gene (eae, stx1, stx2, elt, est, virB, aggR, astA, and afaB) from each of seven pathotypes of DEC, in duplex or triplex reactions under the same PCR cycling conditions. Specificity was confirmed using 860 strains including 88 DEC strains. The fluorescence threshold cycle and DNA concentrations correlated with decision coefficients of more than 0.99. Subsequently, meat samples and enrichment broths were spiked with DEC and the assays used to detect the genes. The detection limits varied from 7.1 x 10(2) to 1.1 x 10(4) CFU ml(-1), depending on the target genes. All meat samples spiked with a variety of DEC (more than 10 CFU 10 g(-1)) were found to be positive by the method. CONCLUSIONS: The present system allows for the efficient and simultaneous determination of various DEC pathotypes. SIGNIFICANCE AND IMPACT OF THE STUDY: This system makes epidemiological investigations for DEC sensitive and quick, and is a useful tool to clarify the source and routes of DEC.

Histamine inhibits chemotaxis, phagocytosis, superoxide anion production, and the production of TNFalpha and IL-12 by macrophages via H2-receptors.

Histamine is released from stimulated basophils and mast cells, and plays an important role in the pathogenesis of allergic inflammatory processes. In vitro treatment of macrophages with histamine resulted in inhibition of chemotaxis. Moreover, histamine at l0(-5) M markedly inhibited the production of superoxide anions by both opsonized zymosan-A and phorbol 12-myristate 13-acetate (PMA) stimulated macrophages and histamine at a concentration range of 10(-7) to 10(-5) M significantly inhibited phagocytosis of Escherichia coli by macrophages. In addition, H2-selective receptor agonist dimaprit resulted in inhibition of macrophage chemotaxis and markedly inhibited the production of superoxide anion by PMA-stimulated macrophages and phagocytosis of E. coli by macrophages. On the other hand, histamine and dimaprit both resulted in a concentration-dependent inhibition of lipopolysaccharide-induced production of TNFalpha and IL-12 by macrophages. These results suggest that histamine and dimaprit may inhibit chemotaxis, phagocytosis, superoxide anion production, and the production of TNFalpha and IL-12 by macrophages via H2-histamine receptors. reserved.

Helical CT scanning of laryngeal deviation.

OBJECTIVE: To evaluate helical computed tomography (CT) scanning in patients with laryngeal deviation. MATERIALS AND METHODS: Five patients with laryngeal deviation and three control subjects underwent helical CT. The laryngeal deviation was idiopathic in one patient and acquired in four. Three-dimensional (3D) images of the laryngeal cartilages and bones, 3D airway surface models, and sequential coronal and axial images were reconstructed for assessment. RESULTS: The thyroid cartilage was inclined and twisted to the right in three patients, inclined to the right and twisted to the left in one patient, and inclined to the left and twisted to the right in one patient. The infero-posterior part of the thyroid cartilage, and the cricoid and arytenoid cartilages were shifted to the left in four patients. The vocal cords were at the level of C4 approximately C4/C5 in two patients, C4/C5 in one, C4/C5 approximately C5 in one, and C5 approximately C5/C6 in one. In four patients, inclination of the laryngeal cavity to the right may have induced left false vocal cord protrusion. The levels of the two false vocal cords differed in all patients. When the inclination and twisting were corrected on the computer, 3D images of the laryngeal cartilages of the patients became almost the same as those of the control subjects, except for slight deformity of the thyroid cartilage. The width of the right and left thyroid alae was measured on the computer, and was almost equal in all patients. 3D airway surface models of the left hemilarynx resembled those of the right hemilarynx when the angle of view was changed on the computer. CONCLUSION: Three-dimensional images of the laryngeal cartilages and bones, 3D airway surface models, and sequential coronal and axial CT scans were obtained using helical CT. This method allows the 3D observation of laryngeal deviation, and viewing of images from various directions on the computer was useful to understand the characteristics of laryngeal deviation.

[Centipede stings notified by the "Centro de Informações Toxicológicas de Belém", over a 2-year period]. / Acidentes por centopéia notificados pelo "Centro de Informações Toxicológicas de Belém", num período de dois anos.

Centipedes are arthropods of the class Chilopoda. The objective of this work was to study the incidence of accidents involving centipedes at "Centro de Informações Toxicológicas de Belém" (CIT-Belém) over a two-year period. Seventy-six patients were studied from March 30, 1998 until March 30, 2000. Centipede accidents occurred in 16.8% of all accidents by venomous animals at Centro de Informações Toxicológicas de Belém; compared to snake accidents, 44.4 % and scorpion accidents, 20.5%. The majority of cases occurred in the residence (86.8%). The most important age group was 20-49 years old (64.4%). The part of human body mostly affected was the superior members (47.4 %). Local pain and edema were found in 95.8% and 52.1% of the patients, respectively. Treatment was symptomatic. Healing occurred in 94.7%, although the outcome of 5.3% of cases was unknown. Centipede accidents are a benign accident, occurring within the residence and treatment consists of measures to decrease the pain.

Continuous local injection of carboplatin into the uterine cervix for cervical and endometrial cancer: preliminary study.

Platinum compounds are thought to be concentration- and time-dependent, but intravenous (i.v.) administration does not afford prolonged high platinum concentration in tumor tissue. In order to examine the influence of long-term local continuous (LC) injection of carboplatin, a pharmacokinetic study was performed. Twenty-six patients with uterine cancer were included. I.v. administration (11 patients): carboplatin (210 mg) was given i.v. and samples of target tissue were obtained at operation about 2 or 24 h after administration. LC administration (15 patients): the 21-gauge needle was implanted at the uterine cervix, and carboplatin was injected continuously (30 mg/day) for 3, 7 or 14 days using an external pump. The tissue platinum concentration was measured in the pelvic organs. The mean platinum levels at the cervix and vaginal wall in the LC (7 days) group were higher than those in the i.v. (2 h) group (p < 0.01). With LC injection, sustained platinum levels were maintained in the pelvic organs for a long time, with very few side effects. LC injection may be advantageous on the basis of pharmacokinetics.

Acute hepatic failure following transcatheter arterial embolization for the treatment of hepatocellular carcinoma.

We conducted a retrospective analysis to evaluate the risk factors associated with the occurrence of acute hepatic failure following transcatheter arterial embolization (TAE) for hepatocellular carcinoma. From 1984 to 1993 we performed a total of 623 embolization procedures in 369 patients with both hepatocellular carcinoma and chronic liver disease. Within 2 weeks after TAE, 13 patients (2.1%) experienced hepatic failure as characterized by a rapid increase in serum bilirubin levels and the development of hepatic encephalopathy of grade 2 or higher. These results indicated that the following are risk factors for acute hepatic failure after TAE: poor hepatic functional reserve; high-dose infusion of chemotherapeutic agents, and a history of multiple embolization procedures.

Value of combined technetium-99m hydroxy methylene diphosphonate and thallium-201 imaging in detecting bone metastases from thyroid carcinoma.

Detectability of bone metastases from differentiated thyroid carcinoma by technetium-99m hydroxymethylene diphosphonate ([99m]Tc-HMDP) bone scan is considered to be poor. Thallium-201 (201Tl) is also widely used for detecting metastatic lesions. Our present study was aimed at the evaluation of the combined use of (99m)Tc-HMDP and 201Tl imaging in successful detection of bone metastases from differentiated thyroid carcinoma. Twenty-seven thyroidectomized thyroid cancer patients (19 females, 8 males; 12 papillary type, 15 follicular type) with 77 bone lesions were included in this retrospective study. All of these patients received ablative doses of radioiodine. Thyroidal origin of the lesions was proved by positive iodine-131 (131I) uptake. In 131I-negative lesions, histological proof or absence of tumor markers other than thyroglobulin was considered when computed tomography (CT) and/or magnetic resonance imaging (MRI) suggested metastatic nature of the lesions. Of the 77 lesions, 58 (75.3%) were positive and 19 were negative in the (99m)Tc-HMDP bone scintigraphy, whereas 53 lesions (68.9%) could be detected by 201Tl scintigraphy. However, within the 19 (99m)Tc-HMDP-negative lesions, 14 showed abnormal accumulation of 201Tl, and within the 24 201Tl negative lesions, 19 were positive in (99m)Tc-HMDP scan. This resulted in a combined sensitivity of 93.5%. Our present study concludes that combined (99m)Tc-HMDP and 201Tl imaging is a sensitive and effective method for detecting bone metastases from thyroid carcinoma.

Angiographic A-V Shunt during Interventional Thrombolysis for Acute Cerebral Embolism. A New Predictive Sign for Hemorrhagic Complication.

SUMMARY: In order to predict the post-therapeutic hemorrhagic complication of interventional thrombolysis, we retrospectively examined angiographic findings and other factors in 44 patients with acute cerebral embolism. All patients were super selectively catheterized within 5 hours of onset and received a fixed regimen of urokinase or t-PA, unless recanalization was achieved or deterioration was apparent before total infusion. Immediate post-therapeutic CT scan revealed hematoma within the basal ganglia in 10 patients (group 1) and no hemorrhage in the remaining 34 (group 2). A series of angiograms during the therapy showed A-V shunt from lenticulostriate arteries to thalamostriate vein in 7 of 10 cases in group 1; no case in group 2 was accompanied by such abnormal shunt. Prognosis after the therapy was worse in group 1 than in group 2. No other factors, such as therapeutic timing after the disease onset or total doses of urokinase or t-PA infused, differed between the two groups. These findings indicate that angiographic A-V shunt during therapy is a predictive sign of a high incidence of hemorrhagic complication. We recommend performing several angiographic controls during therapy and when A-V shunt is detected, the therapy should be discontinued to prevent hemorrhage which leads to a poor prognosis.

Massive fetomaternal hemorrhage.

We evaluated the potential prognostic factors for fetomaternal hemorrhage (FMH). Sinusoidal heart rate pattern and decrease in fetal movement are considered important signs of FMH. However these findings were detected in only two of six cases of FMH and were thought to be late signs of FMH. Fetal hemoglobin levels in the maternal circulation for infants born alive following FMH were less than 5%. Our findings suggest that this amount of blood loss could cause fetal death.

[MR angiography in the evaluation of endoscopic injection sclerotherapy for esophago-gastric varices].

We evaluated the utility of magnetic resonance angiography (MRA) for the detection of esophago-gastric varices and assessment of their therapeutic response to endoscopic injection sclerotherapy (EIS). MRA was performed in a total of 12 patients with esophago (E)-gastric (G) varices (V) (9 EV and 3 GV patients) both before and two-weeks after EIS. 25-35 horizontal images were obtained during single breath holding and data were reconstructed by using the two dimension time of flight method. MRA detected varicose lesions in all GV patients and in 7 of 8 EV patients of the grades F2 or higher. Varicose lesion in grade F1 EV patients were initially undetectable before EIS but became evident on MRA after EIS. The portal collaterals were equally well displayed by MRA and the superior mesenteric arteriography at its portogram phase. MRA and endoscopy were concordant for the disappearance or persistence of varicose lesions after EIS. We conclude that MRA is useful for the detection of esophago-gastric varices and other portal collaterals. MRA provides a non-invasive and workable technique in evaluation of patients with esophago-gastric varices undergoing EIS.

Prevention of pregnancy-induced hypertension by calcium dietary supplement: a preliminary report.

OBJECTIVE: The purpose of this study is to clarify whether the taking of a calcium dietary supplement is able to prevent the development of pregnancy-induced hypertension (PIH) in Japanese people. STUDY DESIGN: Forty-five nulliparous women (Ca-group) with various high-risk factors for PIH started to take a calcium supplement (1 g/day in the form of calcium salts) from the 12th gestational week (GW). Obstetrical outcome of Ca-group was compared to that of the nullipara (n = 453) who did not take a calcium supplement during pregnancy (Non-Ca Group) and that of the nullipara (n = 413) who did not develop PIH among Non-Ca group (normotensive group). RESULTS: The incidence of PIH in the Ca-group was 2.2%, compared to 8.8% of Non-Ca group. The decrease in blood pressure after taking calcium during the first half of the pregnancy was much greater in Ca-group than in the normotensive group. The urinary excretion of sodium and calcium and serum free calcium at the 28th GW were much lower in a woman with PIH, despite of the taking of a calcium supplement, than in the normotensive calcium group. Also, the parathyroid hormone level at the 28th GW was higher in the calcium-taking woman with PIH than in the normotensive calcium group. CONCLUSION: Our preliminary study suggests that among the possible effects of taking a calcium supplement are a decrease in blood pressure and the prevention of PIH in Japanese people, and that calcium metabolism might be related to the development of PIH. However, more study is needed before it will be possible to reach a final conclusion.

A new constitutively activating point mutation in the luteinizing hormone/choriogonadotropin receptor gene in cases of male-limited precocious puberty.

A single point mutation that encodes an aspartic acid (Asp578) to glycine substitution in the LH/CG receptor (LH/CGR) gene, D578G, was recently found in American patients with familial male-limited precocious puberty and in a Japanese patient with a sporadic form of the disorder. Transfection of the mutant, compared to the wild-type, LH/CGR complementary DNA into COS-7 cells results in higher basal cAMP production, but a normal agonist-induced response; the mutation is, therefore, proposed to constitutively activate Leydig cells and elevate serum testosterone, despite low levels of gonadotropin. In the current study we examined two additional Japanese patients with male-limited precocious puberty without a family history of the disease. We describe a heterozygous cytosine (C) to thymine (T) transition at nucleotide 1715 in both; the mutation encodes an alanine to valine substitution in codon 572 of transmembrane helix 6, A572V. Transfected into COS-7 cells, the A572V mutant exhibited the same constitutively high basal cAMP levels and normal agonist-induced cAMP response as the D578G mutant. We conclude that the constitutively higher cAMP levels caused by the A572V mutation led to Leydig cell activation and male-limited precocious puberty, as in the previously described D578G mutation. As the mother of one of the two patients had the same heterozygous mutation, this patient represents the first recognized case of inherited male-limited precocious puberty in the Japanese population. The previously described D578G mutant did not increase basal or agonist-induced inositol phosphate production in transfected COS-7 cells, or the number of LH/CGRs or their affinity for LH/CG. In contrast, transfection of the A572V mutation in COS-7 cells exhibited significantly higher inositol phosphate levels basally and at 10(-11) mol/L hCG, but significantly lower inositol phosphate levels at 10(-7) mol/L hCG. These data suggest that the A572V mutation of the LH/CGR may have effects on the guanine nucleotide binding protein which activates phospholipase C (Gq) coupling and phospholipase-C activation in addition to its effects on Gs coupling and activation of adenylyl cyclase. A572V-transfected cells also exhibited a higher affinity, despite an apparent decrease in the number of binding sites, for [125I]hCG, compared to transfectants with the wild-type LH/CGR. We hypothesize that these differences between the A572V and D578G mutations reflect a greater impact of the A572V mutation on receptor conformation.

Augmentation of thyrotropin-induced iodide uptake in FRTL-5 cells by immunoglobulins containing blocking thyrotropin-receptor antibodies under NaCl-depleted conditions.

We have found recently that Graves' immunoglobulins (Igs) are more active under NaCl-free conditions to increase iodide uptake in FRTL-5 thyroid cells. The present study was designed to examine the effect of blocking-type thyrotropin (TSH)-receptor antibodies (TR-abs) in the same assay. FRTL-5 cells were incubated with crude Ig fractions from 13 patients with primary hypothyroidism having blocking TR-abs and 100 mU/l bovine TSH in NaCl-free Hanks' balanced salt solution (HBSS) supplemented with 274 mmol/l sucrose (HBSS(-)-sucrose) to keep isotonicity for 3 days, followed by the determination of 60 min of 125I uptake in the cells. Thyrotropin alone increased the uptake to 3.3- to 5.2-fold of the basal. When the highest concentration (250 ml serum equivalent (eq.)/l) of the Ig was used, the TSH-stimulated 125I uptake was decreased to 0-42% in 11 of the 13 cases. When the lower concentrations (0.4-50 ml serum eq./l) were used, however, 125I uptake was enhanced (1.4- to 11.4-fold) unexpectedly in all 13 cases. Such an enhancement was specific to blocking TR-abs, because the effect was duplicated by the purified IgG but not by Igs from 10 healthy control subjects or from three TR-ab-negative hypothyroid patients. When Graves' Igs, instead of TSH, were used as a stimulator, the similar stimulatory effect of the Ig with blocking activity was observed on the 125I uptake induced by three Graves' Igs but not on the response to one Graves' Ig. These Igs alone displayed no stimulating activity. When isotonic 5H medium was used instead of HBSS(-)-sucrose, effects of these 13 Igs were only inhibitory on the TSH-stimulated 125I uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

The thyrotropin receptor.

This chapter has outlined the complex process required for thyroid growth and function. Both events are regulated by TSHR via a multiplicity of signals, with the aid of and requirement for a multiplicity of hormones that regulate the TSHR via receptor cross-talk: insulin, IGF-I, adrenergic receptors, and purinergic receptors. Cross-talk appears to regulate G-protein interactions or activities induced by TSH as well as TSHR gene expression. The TSHR structure and its mechanism of signal transduction is being rapidly unraveled in several laboratories, since the recent cloning of the receptor. In addition, the epitopes for autoantibodies against the receptor that can subvert the normal regulated synthesis and secretion of thyroid hormones, causing hyper- or hypofunction, have been defined. Studies of regulation of the TSHR minimal promotor have uncovered a better understanding of the mechanisms by which TSH regulates both growth and function of the thyroid cell. A key novel component of this phenomenon involves TSH AMP positive and negative regulation of the TSHR. Negative transcriptional regulation is a common feature of MHC class I genes in the thyroid. Subversion of negative regulation or too little negative regulation is suggested to result in autoimmune disease. Methimazole and iodide at autoregulatory levels may be important in reversing this process and returning thyroid function to normal. Their action appears to involve factors that react with the IREs on both the TSHR and the TG promoter. Too much negative regulation, as in the case of ras transformation, results in abnormal growth without function. TTF-1 is implicated as a critical autoregulatory component in both positive and negative regulation of the TSHR and appears to be the link between TSH, the TSHR, TSHR-mediated signals, TG and TPO biosynthesis, and thyroid hormone formation. Differentially regulated expression of the TSHR and TG by cAMP and insulin depend on differences in the specificity of the TTF-1 site, that is, the lack of Pax-8 interactions with the TSHR, and the IRE sites. Single-strand binding proteins will become important in determining how TSHR transcription is controlled mechanistically.

Comparison between transabdominal and transvaginal ultrasonography for identifying endometrial malignancies.

Transvaginal ultrasound (TVS) is generally considered superior to transabdominal ultrasound (TAS) in the estimation of endometrial abnormalities. A disadvantage of TVS is the limited field of view. The purpose of this study is a comparison between TAS and TVS for endometrial cancer screening. Ninety-one postmenopausal women were included. They were evaluated by TAS, TVS, and endometrial tissue sampling. The mean endometrial thickness estimated by TVS was larger than that obtained by TAS (p < 0.0001). Sensitivity and specificity of TAS were 83.3 and 58.8% and of TVS 100 and 54.1%, respectively. We conclude that TVS might be superior to TAS, especially in patients with a retroflexed uterus.

A sporadic case of male-limited precocious puberty has the same constitutively activating point mutation in luteinizing hormone/choriogonadotropin receptor gene as familial cases.

Familial male-limited precocious puberty (FMPP) is an autosomal dominant disorder characterized by marked elevation of serum testosterone despite low levels of gonadotropin. Recently, a single point mutation in the LH/hCG receptor (LH/CGR) gene was found in FMPP families that constitutively activates the LH/CGR, causing Leydig cell activation and precocious puberty. Among the Japanese population, only four sporadic cases of male-limited precocious puberty have been reported. In the current study, we examined one of the four reported Japanese patients with sporadic male-limited precocious puberty and found the same mutation as that in the FMPP families. Genomic DNA was isolated, and the polymerase chain reaction (PCR) was performed to amplify a fragment of LH/CGR DNA encoding amino acid residues that include transmembrane helixes 5 and 6. Sequencing of the PCR products revealed a heterozygous adenosine-guanine transition at nucleotide 1733 in codon 578. The mutation encodes an aspartic acid578-glycine substitution in transmembrane helix 6. The mutant LH/CGR, created by site-directed mutagenesis in vitro, exhibited constitutively higher cAMP levels in transfected COS-7 cells than the wild-type LH/CGR, as described previously; however, basal inositol phosphate levels were not increased by transfection with complementary DNA for the mutant receptor. The concentration and affinity of [125I]hCG-binding sites were similar in cells transfected with the mutant and wild-type LH/CGR complementary DNAs, indicating that the mutant did not alter the production of receptor or its ability to bind human LH/CG. The sporadic occurrence of this case was confirmed by further studies. The mutation creates a recognition site for the restriction endonuclease MspI. Restriction digestion was positive for the mutant not digested by MspI, indicating that the patient's mutant allele was not inherited from his parents. DNA analysis of the patient and the parents, using microsatellite repeat markers, was compatible with biological paternity and maternity. We conclude that the aspartic acid578-->glycine mutation in the LH/CGR has arisen in the Japanese population and is the cause of a sporadic case of male-limited precocious puberty.

Prediction of pregnancy-induced hypertension by isometric exercise.

The purpose of this study is to evaluate an isometric exercise (Hand-Grip test) as a method to predict pregnancy-induced hypertension (PIH). One hundred and twenty-five pregnant women were given the Hand-Grip (HG) test before the 15th gestational week. The Hand-Grip test was rated positive when the systolic blood pressure increased 15 mmHg or more during isometric exercise or decreased 14 mmHg or more immediately after isometric exercise. As a result, the Hand-Grip test had the highest sensitivity (81.8%) and specificity (68.4%) for predicting PIH, compared to other risk factors. The positive predictive value was 20% (second highest among risk factors, the actual incidence of hypertension was 8.8%), and the negative predictive value was 97.5% (highest). In conclusion, by use of a very simple Hand-Grip test early in gestation, we are able to predict PIH with the highest sensitivity.