Electro-antennographic response of Helopeltis theivora to synthetic pesticides used in tea plantations.

Helopeltis theivora is considered as one of the major pest in tea plantations causing considerable economic damage. Recent control strategies against this notorious polyphagous pest mainly depend on the application of insecticides. The study is focused on the antennal response of H. theivora on exposure to different insecticides using electroantenogram (EAG). The result showed that the insects perceive quinalphos as they are frequently exposed to it. The hierarchy of the EAG response of exposed and unexposed insects was quinalphos > bifenthrin > deltamethrin > thiamethoxam.

Four-dimensional computational ultrasound imaging of brain hemodynamics.

Four-dimensional ultrasound imaging of complex biological systems such as the brain is technically challenging because of the spatiotemporal sampling requirements. We present computational ultrasound imaging (cUSi), an imaging method that uses complex ultrasound fields that can be generated with simple hardware and a physical wave prediction model to alleviate the sampling constraints. cUSi allows for high-resolution four-dimensional imaging of brain hemodynamics in awake and anesthetized mice.

Swept-3-D Ultrasound Imaging of the Mouse Brain Using a Continuously Moving 1-D-Array-Part I: Doppler Imaging.

Volumetric 3-D Doppler ultrasound imaging can be used to investigate large scale blood dynamics outside of the limited view that conventional 2-D power Doppler images (PDIs) provide. To create 3-D PDIs, 2-D-matrix array transducers can be used to insonify a large volume for every transmission; however, these matrices suffer from low sensitivity, high complexity, and high cost. More typically, a 1-D-array transducer is used to scan a series of stationary 2-D PDIs, after which a 3-D volume is created by concatenating the 2-D PDIs in postprocessing, which results in long scan times due to repeated measurements. Our objective was to achieve volumetric 3-D Doppler ultrasound imaging with a high Doppler sensitivity, similar to that of a typical stationary recording using a 1-D-array transducer, while being more affordable than using 2-D-matrix arrays. We achieved this by mounting a 1-D-array transducer to a high-precision motorized linear stage and continuously translating over the mouse brain in a sweeping manner. For Part I of this article, we focused on creating the best vascular images by investigating how to best combine filtered beamformed ultrasound frames, which were not acquired at the same spatial locations, into PDIs. Part II focuses on the implications of sampling transient brain hemodynamics through functional ultrasound (fUS) while continuously translating over the mouse brain. In Part I, we show how the speed at which we sweep our 1-D-array transducer affects the Doppler spectrum in a flow phantom. In vivo recordings were performed on the mouse brain while varying the sweeping speed, showing how higher sweeping speeds negatively affect the PDI quality. A weighting vector is found to combine frames while continuously moving over the mouse brain, allowing us to create swept PDIs of similar sensitivity when compared with those obtained using a stationary 1-D-array while allowing a significantly higher 3-D Doppler volume rate and maintaining the benefits of having a low computational and monetary cost. We show that a vascular subvolume of 6 mm can be scanned in 2.5 s, with a PDI reconstructed every [Formula: see text], outperforming classical staged recording methods.

Swept-3-D Ultrasound Imaging of the Mouse Brain Using a Continuously Moving 1-D-Array-Part II: Functional Imaging.

Functional ultrasound (fUS) using a 1-D-array transducer normally is insufficient to capture volumetric functional activity due to being restricted to imaging a single brain slice at a time. Typically, for volumetric fUS, functional recordings are repeated many times as the transducer is moved to a new location after each recording, resulting in a nonunique average mapping of the brain response and long scan times. Our objective was to perform volumetric 3-D fUS in an efficient and cost-effective manner. This was achieved by mounting a 1-D-array transducer to a high-precision motorized linear stage and continuously translating over the mouse brain in a sweeping manner. We show how the speed at which the 1-D-array is translated over the brain affects the sampling of the hemodynamic response (HR) during visual stimulation as well as the quality of the resulting power Doppler image (PDI). Functional activation maps were compared between stationary recordings, where only one functional slice is obtained for every recording, and our swept-3-D method, where volumetric fUS was achieved in a single functional recording. The results show that the activation maps obtained with our method closely resemble those obtained during a stationary recording for that same location, while our method is not restricted to functional imaging of a single slice. Lastly, a mouse brain subvolume of ~6 mm is scanned at a volume rate of 1.5 s per volume, with a functional PDI reconstructed every [Formula: see text], highlighting swept-3-D's potential for volumetric fUS. Our method provides an affordable alternative to volumetric fUS using 2-D-matrix transducers, with a high SNR due to using a fully sampled 1-D-array transducer, and without the need to repeat functional measurements for every 2-D slice, as is most often the case when using a 1-D-array. This places our swept-3-D method as a potentially valuable addition to conventional 2-D fUS, especially when investigating whole-brain functional connectivity, or when shorter recording durations are desired.

Case report: High-resolution, intra-operative µDoppler-imaging of spinal cord hemangioblastoma.

Surgical resection of spinal cord hemangioblastomas remains a challenging endeavor: the neurosurgeon's aim to reach total tumor resections directly endangers their aim to minimize post-operative neurological deficits. The currently available tools to guide the neurosurgeon's intra-operative decision-making consist mostly of pre-operative imaging techniques such as MRI or MRA, which cannot cater to intra-operative changes in field of view. For a while now, spinal cord surgeons have adopted ultrasound and its submodalities such as Doppler and CEUS as intra-operative techniques, given their many benefits such as real-time feedback, mobility and ease of use. However, for highly vascularized lesions such as hemangioblastomas, which contain up to capillary-level microvasculature, having access to higher-resolution intra-operative vascular imaging could potentially be highly beneficial. µDoppler-imaging is a new imaging modality especially fit for high-resolution hemodynamic imaging. Over the last decade, µDoppler-imaging has emerged as a high-resolution, contrast-free sonography-based technique which relies on High-Frame-Rate (HFR)-ultrasound and subsequent Doppler processing. In contrast to conventional millimeter-scale (Doppler) ultrasound, the µDoppler technique has a higher sensitivity to detect slow flow in the entire field-of-view which allows for unprecedented visualization of blood flow down to sub-millimeter resolution. In contrast to CEUS, µDoppler is able to image high-resolution details continuously, without being contrast bolus-dependent. Previously, our team has demonstrated the use of this technique in the context of functional brain mapping during awake brain tumor resections and surgical resections of cerebral arteriovenous malformations (AVM). However, the application of µDoppler-imaging in the context of the spinal cord has remained restricted to a handful of mostly pre-clinical animal studies. Here we describe the first application of µDoppler-imaging in the case of a patient with two thoracic spinal hemangioblastomas. We demonstrate how µDoppler is able to identify intra-operatively and with high-resolution, hemodynamic features of the lesion. In contrast to pre-operative MRA, µDoppler could identify intralesional vascular details, in real-time during the surgical procedure. Additionally, we show highly detailed post-resection images of physiological human spinal cord anatomy. Finally, we discuss the necessary future steps to push µDoppler to reach actual clinical maturity.

High-resolution micro-Doppler imaging during neurosurgical resection of an arteriovenous malformation: illustrative case.

OBJECTIVE: Given the high-risk nature of arteriovenous malformation (AVM) resections, accurate pre- and intraoperative imaging of the vascular morphology is a crucial component that may contribute to successful surgical results. Surprisingly, current gold standard imaging techniques for surgical guidance of AVM resections are mostly preoperative, lacking the necessary flexibility to cater to intraoperative changes. Micro-Doppler imaging is a unique high-resolution technique relying on high frame rate ultrasound and subsequent Doppler processing of microvascular hemodynamics. In this paper the authors report the first application of intraoperative, coregistered magnetic resonance/computed tomograpy, micro-Doppler imaging during the neurosurgical resection of an AVM in the parietal lobe. OBSERVATIONS: The authors applied intraoperative two-dimensional and three-dimensional (3D) micro-Doppler imaging during resection and were able to identify key anatomical features including draining veins, supplying arteries and microvasculature in the nidus itself. Compared to the corresponding preoperative 3D-digital subtraction angiography (DSA) image, the micro-Doppler images could delineate vascular structures and visualize hemodynamics with higher, submillimeter scale detail, even at significant depths (>5 cm). Additionally, micro-Doppler imaging revealed unique microvascular morphology of surrounding healthy vasculature. LESSONS: The authors conclude that micro-Doppler imaging in its current form has clear potential as an intraoperative counterpart to preoperative contrast-dependent DSA, and the microvascular details it provides could build new ground to further study cerebrovascular pathophysiology.

Low prevalence of zoonotic Babesia in small mammals and Ixodes ricinus in Brittany, France.

In order to evaluate the zoonotic risk due to Babesia spp., especially B. microti, we investigated their presence in 597 individuals of five small mammal species and in 2620 questing nymphs of Ixodes ricinus in rural landscapes of Western France (Brittany). Small mammals (rodents and shrews) are indeed suspected to be reservoir hosts for B. microti, and the tick I. ricinus is the vector of the three main zoonotic species in Europe, i.e. B. divergens, B. venatorum and B. microti. Only one bank vole carried B. microti (genotype "Munich") and only 13 and 2 nymphs of Ixodes ricinus ticks carried B. venatorum and B. capreoli respectively. According to these results, prevalences observed for zoonotic Babesia (0.17% for small mammals and 0.50% for ticks), indicate that exposure of humans to this infectious agent is probably low in western France.

Prevalence of Anaplasma phagocytophilum in small rodents in France.

Anaplasma phagocytophilum is an emerging zoonotic tick-borne pathogen affecting a wide range of mammals. Rodents are suspected to be natural reservoirs for this bacterium, but their role in the epidemiologic cycles affecting domestic animals and wild ungulates has not been demonstrated. This study aimed to improve our knowledge on A. phagocytophilum prevalence in Apodemus sylvaticus, A. flavicollis and Myodes glareolus using data collected in 2010 in one area in eastern France and in 2012-2013 in two others areas in western France. Rodents were captured in each site and infection was tested using qualitative real-time PCR assays on either blood or spleen samples. Prevalence showed high variability among sites. The highest prevalence was observed in the most eastern site (with an average infection rate of 22.8% across all species), whereas no rodent was found to be PCR positive in the south-west site and only 6.6% were positive in the north-west of France. Finally, a significant increase in prevalence was observed in autumn samples compared to spring samples in the north-west, but no change was found in the other two sites.

Stable expression of human kinin B1 receptor in 293 cells: pharmacological and functional characterization.

1. We compared the binding properties of [3H]-desArg10-[Leu9]-kallidin, a radiolabelled kinin B1 receptor antagonist, to membranes from IMR-90 human embryonic fibroblasts and from 293 cells transiently or stably transfected with the human B1 receptor. 2. The dissociation constant (KD) of [3H]-desArg10-[Leu9]-kallidin and the affinity of several kinin receptor agonists and antagonists were similar between the native and cloned receptor, either transiently or stably expressed in 293 cells. In IMR-90 cells, the rank order of potency was that expected for a kinin B1 receptor. 3. The receptors transiently or stably expressed in 293 cells were fully functional with respect to their signalling properties. Phosphoinositide hydrolysis was increased in a concentration-dependent manner by the B1 receptor agonist, desArg10-kallidin. Functional coupling to the calcium pathway was also demonstrated for the native and stably expressed human B1 receptor. 4. In conclusion, the established stable and functional 293 cell clone may provide an important tool for further analysis of the molecular mechanisms involved in binding, activation, and coupling of the kinin B1 receptor.

Transport of circulating IGF-I and LR3IGF-I from blood to extracellular wound fluid sites in rats.

Significant levels of IGF-I are found in wound fluid. The contribution that systemic IGF-I makes to the total IGF-I pool in wounds and the influence of IGF binding proteins (IGFBPs) on the delivery of systemic IGF-I to these wound sites has not been established. In the present series of experiments, we have shown that IGF-I flux across a model endothelial cell barrier is decreased in the presence of IGFBPs, whereas flux of Long R(3)IGF-I (LR(3)IGF-I, an IGF-I analogue with low affinity for IGFBPs) is unaffected. On the basis of these findings, the transport of IGF-I and LR(3)IGF-I from blood to extracellular wound fluid was assessed. Wound chambers were implanted subcutaneously in the backs of adult male rats and left in place for 14 days. A single i.v. bolus of either (125)I-IGF-I or (125)I-LR(3)IGF-I (10x10(6) c.p.m.) was administered via a jugular catheter and wound fluid and plasma samples taken at sequential time points between 5 and 240 min. (125)I-LR(3)IGF-I was removed from the circulation more rapidly than (125)I-IGF-I in both sham control and chamber implanted rats. Although implantation of the chambers did not alter the pharmacokinetic parameters of (125)I-IGF-I, significant increases in the steady state volume of distribution, clearance rate and half-life were recorded for (125)I-LR(3)IGF-I. In addition, significantly more intact (125)I-LR(3)IGF-I was recovered in wound fluid than (125)I-IGF-I at each time point, although only 0.08% of administered (125)I-LR(3)IGF-I was recovered per ml of wound fluid at 240 min. Compared with plasma, a greater proportion of wound fluid IGF-I radioactivity had distributed to the lower molecular weight IGFBPs or existed as free peptide. However, a small amount of wound fluid (125)I-IGF-I was detected in the 150 kDa region 30 min after injection. A greater proportion of (125)I-LR(3)IGF-I was associated with the lower molecular weight IGFBPs or existed as free peptide in both wound fluid and plasma. These data point to the importance of IGFBPs in determining the pharmacokinetic parameters of IGF-I in an extracellular fluid-expanded state. They also suggest only a minor role for endocrine IGF-I in surface wound repair.

Plasma clearance and tissue distribution of labelled insulin-like growth factor-I (IGF-I) and an analogue LR3IGF-I in pregnant rats.

To determine whether the changes in insulin-like growth factor-binding proteins (IGFBPs) and IGF-I levels during pregnancy in rats affect the clearance of IGFs from the circulation, we have measured pharmacokinetic parameters and the tissue distribution of radiolabelled IGF-I and LR3IGF-I, a potent analogue which has a markedly reduced affinity of binding to the IGFBPs. Intravenous boluses of radiolabelled growth factors were administered to catheterized virgin and age-matched pregnant rats on day 18 of gestation when plasma IGFBPs, in particular IGFBP-3, are dramatically reduced. IGF-I was cleared more rapidly from plasma in pregnant rats compared with the virgins; metabolic clearance rate (MCR) = 2.88 +/- 0.12 (S.E.M.) and 0.90 +/- 0.05 ml/min per kg respectively. Although LR3IGF-I was cleared more rapidly than IGF-I from plasma, similar clearance rates for the analogue were obtained in both pregnant and virgin animals, MCR = 9.19 +/- 0.15 and 9.84 +/- 0.28 ml/min per kg respectively. In virgin rat plasma, labelled IGF-I was mainly associated with the 150 kDa complex, whereas in pregnant rat plasma IGF-I was predominantly associated with lower M(r) IGFBPs (approximately 30-50 kDa). The majority of LR3IGF-I was detected as free peptide. A larger proportion of the tracer was detected as small M(r) breakdown products in plasma from rats under conditions of reduced binding to IGFBPs, e.g. during pregnancy or when LR3IGF-I was the labelled tracer, suggesting greater rates of IGF degradation. More LR3IGF-I tracer was detected in kidneys, ovaries and adrenals of virgin rats and in the ovaries and adrenals of pregnant rats, compared with IGF-I tracer. IGF-I radioactivity was greater than LR3IGF-I in caecum, brain, liver and heart in virgin rats and in kidneys, caecum, brain, liver, heart, placenta, fetus and fetal plasma of the pregnant rats. These results show that the reduction in IGFBP during pregnancy dramatically increased the clearance of IGF-I from the circulation towards that obtained with LR3IGF-I. The observation that less LR3IGF-I was detected in placenta, fetus and fetal plasma compared with IGF-I raises the possibility that the ability to bind IGFBPs during pregnancy may enhance IGF uptake by the conceptus.

Differences in the association of insulin-like growth factor-I (IGF-I) and IGF-I variants with rat, sheep, pig, human and chicken plasma-binding proteins.

Associations between labelled insulin-like growth factors (IGFs) and IGF-binding proteins in plasma have been compared in the rat, sheep, human, pig and chicken. The IGFs tested were recombinant human IGF-I, the truncated variant, des(1-3)IGF-I, and LR3IGF-I, an extended form that had been engineered so as to minimize interactions with IGF-binding proteins. Marked species differences were demonstrated, notably that the IGF-I variants which exhibited extremely weak binding in rat plasma bound significantly in plasma from the other species. This result was shown both by size-exclusion chromatography of labelled IGFs added to plasma, in which the extent of variant IGF-I binding decreased in the order sheep > human > pig = chicken > rat, and by competition for labelled IGF-I binding in vitro, in which the order was pig = chicken > sheep > human > rat. Notwithstanding these differences, the two IGF-I variants showed only slight between-species binding differences when tested with purified rat, sheep and human IGF-binding protein-3. Ligand blotting experiments with plasma from the five species similarly showed a consistent pattern in that IGF-I binding was much greater than des(1-3)IGF-I binding, which in turn was greater than LR3IGF-I binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of betacellulin levels in bovine serum, colostrum and milk.

Betacellulin, a member of the epidermal growth factor (EGF) family, was originally isolated and identified from the conditioned medium from a murine pancreatic beta-cell carcinoma cell line. Recently, we isolated bovine betacellulin from a growth factor enriched cheese whey extract, but there is no information on the presence of betacellulin in other biological fluids. We have cloned the cDNA for bovine betacellulin, produced recombinant betacellulin and shown that it has a similar potency to the purified native molecule in stimulating the proliferation of Balb/c3T3 fibroblasts. We have produced a polyclonal antiserum to bovine betacellulin which did not cross-react with EGF or transforming growth factor-alpha (TGF-alpha). The antibody was used in a homologous RIA that was able to detect betacellulin in pooled bovine colostrum sampled during the first 3 days after calving (2.30+/-0.11 ng/ml mean+/-s.e.m.; n=6), in bovine milk soluble fraction (1.93+/-0.64 ng/ml mean+/-s.e.m.; n=5) and in bovine cheese whey (2.59+/-0.16 ng/ml mean+/-s.e.m.; n=3). The betacellulin concentration in foetal bovine serum (FBS) (3.68+/-0.59 ng/ml mean+/-s.e.m.; n=6) greatly exceeded that of betacellulin in serum from male calves 1 and 5 weeks of age (0.53+/-0.15 ng/ml and 0.70+/- 0.09 ng/ml respectively; mean+/-s.e.m.; n=9). Betacellulin measured in the serum of these same animals when aged between 27 and 43 weeks was below the detection limits of the RIA. Sera from 10 out of 36 unmated heifers contained betacellulin levels within the detection limits of the assay (0.433+/-0.06 ng/ml mean+/-s.e.m.; n=10). The presence of betacellulin in bovine colostrum and milk suggests that it plays a role in the growth and development of the neonate and/or mammary gland function. The results also show that betacellulin is undetectable in the castrated adult male circulation. Additionally, although present in very low amounts, serum betacellulin could be under hormonal regulation in the female, since betacellulin was detected in sera from 27% of the unmated heifers examined in this study. The high levels of betacellulin detected in FBS relative to newborn and adult serum suggests a possible endocrine role for this growth factor in the bovine foetus.

Transport of IGF-I across epithelial cell monolayers.

Epithelial cells line the lumens of organs including the gastrointestinal tract, kidney tubules and respiratory airways, where they regulate the transport of electrolytes and the movement of macromolecules. The current study aimed to investigate the transport of IGF-I across epithelial cell barriers. Epithelial cell lines derived from gut (IEC-6), kidney (MDBK) and lung (Mv1Lu) were shown to possess high-affinity, functional receptors for IGF-I and formed tight junctions in monolayer culture. To investigate the transport of IGF-I, the three cell lines were grown on microporous filters in a bi-chamber system. In comparison with filters without cells, IEC-6 and Mv1Lu epithelial cell monolayers restricted the passage of (125)I-IGF-I and [(3)H]inulin, whereas the MDBK cells virtually occluded any passage of these molecules. Transport of (125)I-IGF-I across the epithelial cell monolayers was significantly less than that of [(3)H]inulin, suggesting that the binding of (125)I-IGF-I to high-affinity IGF receptors or IGF-binding proteins retarded its transport. Moreover, (125)I-IGF-I transport was not inhibited by the presence of excess unlabelled IGF-I. Our findings provide evidence for the restricted diffusion of intact, free IGF-I across gut, kidney and lung epithelial cell monolayers via a paracellular or low-affinity transcellular pathway.

Comparison of 14 PCR systems for the detection and subtyping of stx genes in Shiga-toxin-producing Escherichia coli.

The specificity of 14 polymerase chain reaction (PCR) systems designed for the detection and subtyping of stx genes was tested on a set of Escherichia coli strains with known sequences of stx genes. Systems designed for the detection of genes of the stx1 type did not detect any variant genes of the stx2 type and conversely, no stx2 type-specific systems detected stx1 variant genes. Among five stx2 type-specific systems, none detected the stx2ev gene, and two detected the stx2e gene. Among systems designed for screening genes of the both stx1 and stx2 types with a single primer pair, only one system (the Lin system) was able to detect stx genes in all studied strains. Shiga-toxin-producing E. coli frequently carry more than one stx variant gene. Coamplification of stx genes present in the same strain was demonstrated by restriction of PCR products with endonucleases generating fragments of variant-specific size. The amplification product obtained by the Lin system restricted by Hincll yielded fragments of different size for stx1, stx2, stx2c, stx2e and stx2ev. Thus it was possible to identify different genes carried in a single strain with a simple two-step PCR/endonuclease restriction protocol.

Evaluation of a sensitive colorimetric FXIII incorporation assay. Effects of FXIII Val34Leu, plasma fibrinogen concentration and congenital FXIII deficiency.

There is an increasing interest in the role of coagulation factor XIII (FXIII) in cardio- and cerebrovascular diseases. It has recently been reported that a common G-->T point mutation in the A-subunit gene of FXIII, which codes for a valine (val) to leucine (leu) change (FXIIIVal34Leu), is protective against thrombotic diseases but seems to increase the risk of intracerebral bleeding. We developed a colorimetric incorporation assay for detection of FXIII activity based on incorporation of 5-(biotinamido) pentylamine (BAPA) into fibrin or fibrinogen. With this new assay, we studied the effects of FXIIIVal34Leu mutation, plasma fibrinogen concentration and congenital FXIII deficiency on FXIII activity. There are no data available about the ability of different FXIII assays to detect altered activity in FXIIIVal34Leu genotypes. We therefore compared our results determined by the incorporation method with a commonly used photometric method based on ammonia release after cross-linking of glycine-ethylester to a specific glutamine containing peptide substrate. We also determined FXIII A-subunit antigen (Ag) levels using enzyme-linked immunosorbent assay (ELISA) technique. The FXIIIVal34Leu genotype could not be detected either by the photometric method nor by the FXIII A-subunit ELISA. The incorporation assay showed an increased specific FXIII activity in subjects possessing the leu allele. The photometric assay and ELISA gave similar results independent from genotype. In patients with congenital FXIII deficiency before and after substitution, however, ELISA and the incorporation assay gave similar results, whereas the photometric assay showed consistently higher values. Our results show that the incorporation assay, not the photometric assay based on ammonia release, can be used for detection of elevated activity in subjects with FXIIIVal34Leu. Because of specificity and over a wide range sensitivity, the assay can also be used for determination of FXIII deficiency and monitoring of FXIII substitution therapy.

Neuromuscular adaptations in isokinetic, isotonic, and agility training programs.

Weight training is an integral part of most athletic conditioning programs; yet, the effect of these programs on neuromuscular function remains unclear. To examine the neuromuscular effects of training and conditioning at the knee joint, 32 volunteers (16 men and 16 women; average age, 25.4 years) were placed into one of four groups: isokinetic, isotonic, agility, or control. Each group trained 3 days per week for 6 weeks. The knee function of all participants was evaluated just before and after the 6-week training period. The agility-trained group significantly improved the spinal reflex times of the lateral and medial quadriceps muscles in response to anterior tibial translation. The cortical response time of the agility group also significantly improved in the gastrocnemius, medial hamstring, and the lateral quadriceps muscles. Interestingly, the cortical response time of the medial hamstring and the medial quadriceps muscles in the isokinetic group slowed significantly, by 39.1 and 32.4 msec, respectively, after 6 weeks of training. Isotonic and isokinetic strength training of the lower extremities do not appear to improve muscle reaction time to anterior tibial translation, whereas agility exercises potentially improve this parameter.

Review of epidemiological surveys on the prevalence of contamination of healthy cattle with Escherichia coli serogroup O157:H7.

This paper gathers and critically analyses the results of 26 published epidemiological surveys on the prevalence of contamination of cattle with verocytotoxin-producing E. coli (VTEC) serogroup O157:H7. These surveys have been conducted since 1986 on farms in North America (10 studies), on farms in Europe (6 studies) and at slaughterhouses prior to or just after slaughter (7 studies) or after skinning and evisceration (3 studies). The purpose of this review is to understand the first stages of the epidemiology of the infection in animals and humans (the infection process being obscure in many points) and to prepare herd-based control measures to reduce the risk of O157:H7 human infection. The different statistical methods employed in these surveys, as well as the various laboratory screening methods used for detecting positive animals are presented. The observed frequencies of infected animals (animal prevalence) and herds (herd prevalence) are given as a function of localisation, year, type of industry (beef or dairy) and age. From these measured prevalence values, the risk of contamination of ground beef by E. coli O157:H7 in the first stages of the farm-to-fork continuum is assessed. First, we follow the evolution of contamination frequencies from the living animal on-farm to carcasses before transformation. Then, within each set of measurements (i.e., on farm or at slaughterhouse), we identify the effects of the following factors: target population, sampling strategies and laboratory procedures. We argue that the prevalence values inferred from these measurements are very likely underestimated, due to insufficient sampling and not enough sensitive laboratory procedures (one exception being the immunomagnetic bead separation technique). No firm conclusion can be drawn as to the effects of geographical localisation and season. In those surveys, the effect of hygiene level at slaughterhouse on prevalence values is not quantitatively assessed. In addition, there is growing evidence of other sources of E. coli O157:H7 than live cattle in the farm environment, such as feed, water and water-troughs.

Paracellular transport of insulin-like growth factor-I (IGF-I) across human umbilical vein endothelial cell monolayers.

Insulin-like growth factors (IGFs) are well defined mitogens and growth promoters, which are found in blood associated with high affinity IGF binding proteins (IGFBPs). In vivo, the endothelium is potentially the primary site of uptake of IGFs or IGF-IGFBP complexes from blood for transport to the extravascular space. However, the pathway and mechanisms by which IGFs cross the endothelial cell barrier are not known. The presence of high affinity receptors for IGF-I and IGF-II on human umbilical vein endothelial (HUVE) cells was demonstrated by (i) radio-receptor assays using both IGF-I and IGF-II and (ii) affinity label cross-linking studies. In addition, Western ligand blotting and immunoblotting revealed that IGFBP-2, -3, and -4 are secreted into serum-free media conditioned by confluent HUVE cell monolayers. To study transendothelial migration of IGF-I, HUVE cells were grown on microporous membranes in a bichamber system. When compared with membranes without cells, HUVE monolayers restricted the passage of 125I-IGF-I and [3H]inulin, whereas the control Madin Darby canine kidney (MDCK) cell line virtually excluded all passage of these molecules. Transport of 125I-IGF-I across HUVE cell monolayers was not significantly different to that of [3H]inulin, a paracellular probe. Moreover, 125I-IGF-I transport was not inhibited by either excess unlabelled IGF-I or a monoclonal antibody to the type I IGF receptor at a concentration shown to inhibit 125I-IGF-I binding to HUVE cell monolayers. Our findings show that the movement of free IGF-I across HUVE cell monolayers occurs via a paracellular route and not by a receptor-mediated, transcellular pathway.

Identification of a key region of kinin B(1) receptor for high affinity binding of peptide antagonists.

To investigate the molecular basis for the specificity of ligand recognition in human kinin B(1) (B(1)R) and B(2) (B(2)R) receptors, we constructed a series of chimeric receptors by progressively replacing, from the N to the C terminus, the human B(2)R domains by their B(1) counterparts. The chimeric construct possessing the C-terminal tail and the transmembrane domain VII (TM VII) of the B(2)R (construct 6) displayed 7- and 20- fold decreased affinities for the B(1) agonist [(3)H]desArg(10)-kallidin (desArg(10)-KD) and the B(1) antagonist [(3)H]desArg(10)-[Leu(9)]-KD respectively, as compared with the wild-type B(1)R. Moreover, the substitution of the B(1) TM VII by its B(2) homologue TM increased the affinity for the pseudopeptide antagonists, Hoe140 and NPC 567. High affinity for desArg(10)-KD binding was fully regained when the B(2) residue Thr(287) was replaced in construct 6 by the corresponding B(1) Leu(294) residue. When the B(2) residue Tyr(295) was exchanged with the corresponding B(1) Phe(302), high affinity binding for both agonist and antagonist was recovered. Moreover, the L294T and F302Y mutant B(1)R exhibited 69- and 6.5-fold increases, respectively, in their affinities for the B(2) receptor antagonist, Hoe140. Therefore we proposed that Leu(294) and Phe(302) residues, which may not be directly involved in the binding of B(1)R ligands and, hence, their Thr(287) and Tyr(295) B(2) counterparts, are localized in a receptor region, which plays a pivotal role in the binding selectivity of the peptide or pseudopeptide kinin ligands.