Magnetic resonance imaging and diseases of the liver and biliary tract. Part 2. Magnetic resonance cholangiography and angiography and conclusions

Magnetic resonance cholangiography (MRC) relies on the strong T-2 signal from stationary liquids, in this case bile, to generate images. No contrast agents are required, and the failure rate and risk of serious complications is lower than with endoscopic retrograde cholangiopancreatography (ERCP). Data from MRC can be summated to produce an image much like the cholangiogram obtained by using ERCP. In addition, MRC and conventional MRI can provide information about the biliary and other anatomy above and below a biliary obstruction. This provides information for therapeutic intervention that is probably most useful for hilar and intrahepatic biliary obstruction. Magnetic resonance cholangiography appears to be similar to ERCP with respect to sensitivity and specificity in detecting lesions causing biliary obstruction, and in the diagnosis of choledocholithiasis. It is also suited to the assessment of biliary anatomy (including the assessment of surgical bile-duct injuries) and intrahepatic biliary pathology. However, ERCP can be therapeutic as well as diagnostic, and MRC should be limited to situations where intervention is unlikely, where intrahepatic or hilar pathology is suspected, to delineate the biliary anatomy prior to other interventions, or after failed or inadequate ERCP. Magnetic resonance angiography (MRA) relies on the properties of flowing liquids to generate images. It is particularly suited to assessment of the hepatic vasculature and appears as good as conventional angiography. It has been shown to be useful in delineating vascular anatomy prior to liver transplantation or insertion of a transjugular intrahepatic portasystemic shunt. Magnetic resonance angiography may also be useful in predicting subsequent variceal haemorrhage in patients with oesophageal varices. (C) 2000 Blackwell Science Asia Pty Ltd.

Vesicle-associated proteins and transmitter release from sympathetic ganglionic boutons

A method is reported for introducing peptides derived from SNARE proteins that control exocytosis of vesicles at boutons formed by sympathetic ganglion cells in tissue culture. These peptides were coupled to the DNA binding domain of the Drosophila transcription factor antennapedia, called penetratin, This facilitated the passage of peptides across the bouton membrane. FMI-43 was used to monitor the exocytosis of transmitter from depolarized boutons after their exposure to the penetratin-peptide sequences IETRHNEIIKLETSIRELHD of syntaxin and KGFLSSLFGGSSK of alpha -SNAP. both of which blocked secretion, whereas the peptide sequences SELDDRA-DALQAGASQFETSAAKLKRK of synaptobrevin did not. This report introduces a readily applicable method for determining the effect of different peptide sequences of vesicle-associated proteins on secretion at vertebrate boutons and presents an account of the effects of a selection of such peptides on exocytosis. NeuroReport 12:607-610 (C) 2001 Lippincott Williams & Wilkins.

Expression of growth factors in Dictyostelium discoideum

Growth factors and their binding proteins are important proteins regulating mammalian cell proliferation and differentiation so there is considerable interest in producing them as recombinant proteins, especially in hosts that do not already produce a complex mixture of growth factors. Many growth factors require posttranslational modifications making them unsuitable for production in Escherichia coli or other prokaryotes. Since several expression vector systems have been recently developed for foreign protein production in the cellular slime mould, Dictyostelium discoideum, we attempted to use two of these systems to express human insulin-like growth factor binding protein 6 (hIGFBP6) and bovine beta-cellulin (bBTC) as secreted proteins. Although both proteins were successfully produced in stably transformed amoebae, no secretion was detected in spite of several attempts to facilitate this occurring.

Heavy chain ferritin activates regulatory T cells by induction of changes in dendritic cells

Heavy chain ferritin (H-ferritin) Is a component of the Iron-binding protein, ferritin. We have previously shown that H-ferritin Inhibits anti-CD3-stimulated lymphocyte proliferation and that this was due to Increased production of Interleukin-10 (IL-10). In the present study we have shown that Induction of IL-10 production was due to effects of H-ferritin on adherent antigen-presenting cells (APCs) In blood and monocyte-derived dendritic cells (MoDCs). IL-10 was produced by a subpopulation of CD4 T cells, which expressed the CD25 component of the IL-2 receptor and the CTLA-4 receptor characteristic of regulatory T cells. The changes Induced In MoDCs were compared with those Induced by CD40L and their significance tested by Inhibition with monoclonal antibodies. These studies Indicated that H-ferritin Induced relatively greater expression of CD86 and B7-H1 on MoDCs and that monoclonal antibodies against their receptors, CTLA-4 and programmed death receptor-1 (PD-1), Inhibited IL-10 production from the regulatory T cells. H-ferritin did not appear to Induce direct production of the cytokines IL-2, IL-4, IL-6, IL-10, IL-12, or Interferon-gamma from the DCs. These results are consistent with the thesis that H-ferritin Induces B7-H1 and CD86 (B7-2) on APCs, which In turn Induce IL-10 production from regulatory T cells. This is possibly one mechanism by which melanoma cells may Induce changes In APCs In the vicinity of the tumor and result in suppression of Immune responses by induction of regulatory T cells. (C) 2002 by The American Society of Hematology.

Identification of cellular changes associated with increased production of human growth hormone in a recombinant Chinese hamster ovary cell line

A proteomics approach was used to identify the proteins potentially implicated in the cellular response concomitant with elevated production levels of human growth hormone in a recombinant Chinese hamster ovary (CHO) cell line following exposure to 0.5 mM butyrate and 80 muM zinc sulphate in the production media. This involved incorporation of two-dimensional (2-D) gel electrophoresis and protein identification by a combination of N-terminal sequencing, matrix-assisted laser desorption/ionisation-time of flight mass spectrometry, amino acid analysis and cross species database matching. From these identifications a CHO 2-D reference,map and annotated database have been established. Metabolic labelling and subsequent autoradiography showed the induction of a number of cellular proteins in response to the media additives butyrate and zinc sulphate. These were identified as GRP75, enolase and thioredoxin. The chaperone proteins GRP78, HSP90, GRP94 and HSP70 were not up-regulated under these conditions.

Quantification of prolactin-releasing peptide (PrRP) mRNA expression in specific brain regions of the rat during the oestrous cycle and in lactation

Real-time Taqman(TM) RT-PCR was used to make quantitative comparisons of the levels of PrRP mRNA expression in micropunch brain samples from rats at different stages of the oestrous cycle and in lactation. The nucleus of the solitary tract and ventrolateral reticular nuclei of the medulla oblongata contained significantly (P < 0.05) greater levels of PrRP mRNA than any hypothalamic region. Within the hypothalamus, the highest level of PrRP expression was localised to the dorsomedial aspect of the ventromedial hypothalamus. All other hypothalamic regions exhibited significantly (P < 0.05) lower levels of expression, including the rostral and caudal dorsomedial hypothalamus. Very low levels of PrRP expression were observed in the arcuate nucleus, paraventricular nucleus, medial preoptic nucleus and ventrolateral aspect of the ventromedial hypothalamus. No significant changes in PrRP expression were noted in any sampled region between proestrus, oestrus or dioestrus. Similarly, PrRP expression in hypothalamic regions did not differ between lactating and non-lactating (dioestrous) animals. During validation of RT-PCR techniques we cloned and sequenced a novel splice variant of PrRP from the hypothalamus. This variant arises from alternative splicing of the donor site within exon 2, resulting in an insert of 64 base pairs and shift in the-codon:reading frame with the introduction of an early stop codon. In the hypothalamus and brainstem, mRNA expression of the variant was restricted to regions that expressed PrRP. These results suggest that PrRP expression in the hypothalamus may be more Widespread than previously reported. However, the relatively low level of PrRP in the hypothalamus and the lack of significant changes in expression during the oestrous cycle and lactation provides further evidence that PrRP is unlikely to be involved in the regulation of prolactin, secretion. (C) 2003 Elsevier Science B.V. All rights reserved.

Bovine mammary epithelial cells, initiators of innate immune responses to mastitis

Innate immunity plays a vital role in the protection of the bovine mammary gland against mastitis. Until recently, the migration of effector cells such as neutrophils and monocytes into the mammary gland was thought to provide the only defence against invading pathogens. However, mammary epithelial cells may also play an important role in the immune response, contributing to the innate defence of the mammary tissue through secretion of antimicrobial peptides and attraction of circulating immune effector cells. This paper reviews the innate immune pathways in mammary epithelial cells and examines their role in the initiation of an innate immune response to Gram-positive and Gram-negative bacteria.

Cholestanol: A serum marker to guide LDL cholesterol-lowering therapy

Statins have been the mainstay of lipid-lowering therapy since their introduction. However, as lower LDL cholesterol targets are sought, adjunct therapies are becoming increasingly important. Few patients reach new targets with statin monotherapy. We propose that the cholestanol: cholesterol ratio can be used to guide lipid-lowering therapy and result in greater numbers of patients reaching target LDL cholesterol. By determining whether a patient is mainly a synthesizer or absorber of cholesterol, customized regimens can be used and are expected to improve patient outcomes and minimize costs of treatment. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Compensation for obesity-induced insulin resistance in dogs: Causal web analysis of the associations of leptin and GLP-1.

Obesity affects aspects of glucose homeostasis such as insulin secretion and insulin sensitivity. Hormones secreted by adipocytes like leptin mediate the metabolic consequences of obesity. Incretin hormones like glucagon-like peptide-1 (GLP-1) increase insulin secretion in response to changes in blood glucose concentration and have been proposed to regulate insulin secretion in fasting, overweight dogs. The aim of this study was to examine hormonal mechanisms by which adiposity alters glucose homeostasis, plasma insulin concentration, and insulin sensitivity in spontaneously overweight dogs.

Subpopulations of corticotrophs in the sheep pituitary during late gestation: Effects of development and placental restriction

The prepartum surge in fetal plasma cortisol is essential for the normal timing of parturition in sheep and may result from an increase in the ratio of ACTH to proopiomelanocortin (POMC) in the fetal circulation. In fetuses subjected to experimental induction of placental restriction, the prepartum surge in fetal cortisol is exaggerated, whereas pituitary POMC mRNA levels are decreased, and in vitro, unstimulated ACTH secretion is elevated in corticotrophs nonresponsive to CRH. We therefore investigated the changes in the relative proportions of cells expressing POMC, ACTH, and the CRH type 1 receptor (CRHR1) shortly before birth and during chronic placental insufficiency. Placental restriction (PR) was induced by removal of the majority of placental attachment sites in five ewes before mating. Pituitaries were collected from control and PR fetal sheep at 140 d (control, n = 4; PR, n = 4) and 144 d (control, n = 6; PR, n = 4). Pituitary sections were labeled with specific antisera raised against POMC, ACTH, and CRHR1. Three major subpopulations of corticotrophs were identified that expressed POMC + ACTH + CRHR1, ACTH + CRHR1, or POMC only. The proportion of pituitary corticotrophs expressing POMC + ACTH + CRHR1 decreased (P < 0.05) between 140 (control, 60 +/- 1%; PR, 66 +/- 4%) and 144 (control, 45 +/- 2%; PR, 56 +/- 6%) d. A significantly higher (P < 0.05) proportion of corticotrophs expressed POMC + ACTH + CRHR1 in the pituitary of the PR group compared with controls. This study is the first to demonstrate subpopulations of corticotrophs in the fetal sheep pituitary that differentially express POMC, ACTH, and CRHR1 and the separate effects of gestational age and placental restriction on these subpopulations of corticotrophs.

Use of methotrexate in older patients - A risk-benefit assessment

Methotrexate is eliminated almost entirely by the kidneys. The risk of methotrexate toxicity is therefore increased in patients with poor renal function, most likely as a result of drug accumulation. Declining renal function with age may thus be an important predictor of toxicity to methotrexate. Up to 60% of all patients who receive methotrexate for rheumatoid arthritis (RA) discontinue taking it because of adverse effects, most of which occur during the first year of therapy. Gastrointestinal complications are the most common adverse effects of methotrexate, but hepatotoxicity, haematological toxicity, pulmonary toxicity, lymphoproliferative disorders and exacerbation of rheumatic nodules have all been reported, Decreased renal function as a result of disease and/or aging appears to be an important determinant of hepatic, lymphoproli ferative and haematological toxicity, Concomitant use of low doses of folic acid has been recommended as an approach to limiting toxicity. Interactions between methotrexate and several nonsteroidal anti-inflammatory drugs have been reported, but they may not be clinically significant. However, caution is advised in the use of such combinations in patients with reduced renal function. More serious toxicities (e.g. pancytopenia) may result when other inhibitors of folate utilisation [e.g. cotrimoxazole (trimethoprim-sulfamethoxazole)] or inhibitors of renal tubular secretion (e.g. probenecid) are combined with methotrexate. Before starting low dose methotrexate therapy in patients with RA, a full blood count, liver function tests, renal function tests and chest radiography should be performed. Blood counts and liver function tests should be repeated at regular intervals. Therapeutic drug monitoring of methotrexate has also been suggested as a means of limiting toxicity. Patients with RA usually respond very favourably to low dose methotrexate therapy, and the probability of patients continuing their treatment beyond 5 years is greater than for other slow-acting antirheumatic drugs. Thus, given its sustained clinical utility and relatively predictable toxicity profile, low dose methotrexate is a useful addition to the therapy of RA.

Structure of TcpG, the DsbA protein folding catalyst from Vibrio cholerae

The efficient and correct folding of bacterial disulfide bonded proteins in vivo is dependent upon a class of periplasmic oxidoreductase proteins called DsbA, after the Escherichia coli enzyme. In the pathogenic bacterium Vibrio cholerae, the DsbA homolog (TcpG) is responsible for the folding, maturation and secretion of virulence factors. Mutants in which the tcpg gene has been inactivated are avirulent; they no longer produce functional colonisation pill and they no longer secrete cholera toxin. TcpG is thus a suitable target for inhibitors that could counteract the virulence of this organism, thereby preventing the symptoms of cholera. The crystal structure of oxidized TcpG (refined at a resolution of 2.1 Angstrom) serves as a starting point for the rational design of such inhibitors. As expected, TcpG has the same fold as E. coli DsbA, with which it shares similar to 40% sequence identity. Ln addition, the characteristic surface features of DsbA are present in TcpG, supporting the notion that these features play a functional role. While the overall architecture of TcpG and DsbA is similar and the surface features are retained in TcpG, there are significant differences. For example, the kinked active site helix results from a three-residue loop in DsbA, but is caused by a proline in TcpG (making TcpG more similar to thioredoxin in this respect). Furthermore, the proposed peptide binding groove of TcpG is substantially shortened compared with that of DsbA due to a six-residue deletion. Also, the hydrophobic pocket of TcpG is more shallow and the acidic patch is much less extensive than that of E. coli DsbA. The identification of the structural and surface features that are retained or are divergent in TcpG provides a useful assessment of their functional importance in these protein folding catalysts and is an important prerequisite for the design of TcpG inhibitors. (C) 1997 Academic Press Limited.

A method for drug infusion into the lateral median eminence and arcuate nucleus of sheep

The role of catecholamines in the control of the GnRH pulse generator is unclear as studies have relied on the use of peripheral or intracerebroventricular injections, which lack specificity in relation to the anatomical site of action. Direct brain site infusions have been used, however, these are limited by the ability to accurately target small brain regions. One such area of interest in the control of GnRH is the median eminence and arcuate nucleus within the medial basal hypothalamus. Here we describe a method of stereotaxically targeting this area in a large animal (sheep) and an infusion system to deliver drugs into unrestrained conscious animals. To test our technique we infused the dopamine agonist, quinpirole or vehicle into the medial basal hypothalamus of ovariectomised ewes. Quinpirole significantly suppressed LH pulsatility only in animals with injectors located close to the lateral median eminence. This in vivo result supports the hypothesis that dopamine inhibits GnRH secretion by presynaptic inhibition in the lateral median eminence. Also infusion of quinpirole into the medial basal hypothalamus suppressed prolactin secretion providing in vivo evidence that is consistent with the hypothesis that there are stimulatory autoreceptors on tubero-infundibular dopamine neurons. (C) 1997 Elsevier Science B.V.

Expression of recombinant human follicle stimulating hormone mRNA in Chinese hamster ovary cells under different promoters

Levels of recombinant human follicle stimulating hormone (r-hFSH) mRNA expressed under butyrate and zinc treatment were compared in two CHO-K1 derived cell lines. In King cells under the metallothionein promoter, butyrate induced the increase in both r-hFSH productivity (q(FSH)) and mRNA levels proportionally. In the presence of 1 mM butyrate and 40 mu M zinc, a 4-fold increase in q(FSH) and mRNA levels was achieved as compared to zinc (40) alone; this wasa approximately 6 times higher than in serum free medium. In Darren cells under the beta-actin promotor butyrate induced an increase in q(SFH) but not in mRNA levels.

In vivo effects of chicken interferon-gamma during infection with Eimeria

Newly hatched chickens are highly susceptible to infection by opportunistic pathogens during the first 1 or 2 weeks of life, The use of cytokines as therapeutic agents has been studied in animal models as well as in immunosuppressed patients, This approach has become more feasible in livestock animals, in particular poultry, with the recent cloning of cytokine genes and the development of new technologies, such as live delivery vectors, We have recently cloned the gene for chicken interferon-gamma (Ch-IFN-gamma), Poly-HIS-tagged recombinant Ch-IFN-gamma was expressed in Escherichia coil, was purified by Ni chromatography, and was found to be stable at 4 degrees C and an ambient temperature for at least several months and Several weeks, respectively, Ch-IFN-gamma was capable of protecting chick fibroblasts from undergoing virus-mediated lysis, induced nitrite secretion from chicken macrophages in vitro, and enhanced MHC class II expression on macrophages, Administration of recombinant Ch-IFN-gamma to chickens resulted in enhanced weight gain over a 12-day period, Furthermore, the therapeutic potential of Ch-IFN-gamma was assessed using a coccidial challenge model, Birds were treated with Ch-IFN-gamma or a diluent control and then infected with Eimeria acervulina. Infected birds treated with Ch-IFN-gamma showed improved weight gain relative to noninfected birds, The ability of Ch-IFN-gamma to enhance weight gain in the face of coccidial infection makes it an excellent candidate as a therapeutic agent.