Clinical and biochemical improvements in a patient with MNGIE following enzyme replacement.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive metabolic disorder caused by a deficiency of thymidine phosphorylase (TP, EC2.4.2.4) due to mutations in the nuclear gene TYMP. TP deficiency leads to plasma and tissue accumulations of thymidine and deoxyuridine which generate imbalances within the mitochondrial nucleotide pools, ultimately leading to mitochondrial dysfunction.1 MNGIE is characterized clinically by leukoencephalopathy, external ophthalmoplegia, peripheral polyneuropathy, cachexia, and enteric neuromyopathy manifesting as gastrointestinal dysmotility. The condition is relentlessly progressive, with patients usually dying from a combination of nutritional and neuromuscular failure at an average age of 37 years.2 Allogeneic hematopoietic stem cell transplantation (AHSCT) offers a permanent cure. Clinical and biochemical improvements following AHSCT have been reported but it carries a high mortality risk and is limited by matched donor availability.3 A consensus proposal for standardizing AHSCT recommends treatment of patients without irreversible end-stage disease and with an optimally matched donor; a majority of patients are ineligible and thus there is a critical requirement for an alternative treatment

Prospective study of biliary strictures to determine the predictors of malignancy

ORIGINAL ARTICLE BACKGROUND: There have been few prospective studies regarding the investigation of biliary strictures, principally because of rapid technological change. The present study was designed to determine the sensitivity of various imaging studies for the detection of biliary strictures. Serum biochemistry and imaging studies were evaluated for their role in distinguishing benign from malignant strictures. METHODS: Thirty-one patients with suspected noncalculus biliary obstruction were enrolled consecutively in the study. A complete biochemical profile, ultrasound, Disida scan and cholangiogram (endoscopic retrograde cholangiopancreatography [ERCP] or percutaneous cholangiogram) were obtained at study entry. Stricture etiology was determined based on cytology, biopsy and/or clinical follow-up at one year. RESULTS: Twenty-nine of 31 patients had biliary strictures, of which 15 were malignant. The mean age of the malignant cohort was 73.9 years versus 53.9 years in the benign cohort (P<0.001). Statistically significant differences between the malignant and benign groups, respectively, were as follows: alanine transaminase 235.2 versus 66.9 U/L (P=0.004), aspartate transaminase 189.8 versus 84.5 U/L (P=0.011), alkaline phosphatase 840.2 versus 361.1 U/L (P=0.002), bilirubin 317.8 versus 22.1 µmol/L (P<0.001) and bile acids 242.5 versus 73.2 µmol/L (P=0.001). Threshold analysis using receiver operative characteristic (ROC) curves demonstrated that a bilirubin level of 75 µmol/L was most predictive of malignant strictures. Intrahepatic duct dilation was present in 93% of malignant strictures versus 36% of benign strictures (P=0.002). Common hepatic duct dilation was less discriminatory (malignant 13.5 versus benign 9.6 mm; P=0.11). Ultrasound was highly sensitive (93%) in the detection of the primary tumour in the bile duct or pancreas, or in the visualization of nodal or liver metastases. In benign disease, ultrasound failed to detect evidence of intrahepatic or extrahepatic biliary dilation in most cases. Disida scans were not able to distinguish between malignant or benign strictures and could not accurately localize the level of obstruction. The sensitivity of Disida scan for the diagnosis of obstruction was 50%. Cholangiographic characterization of strictures revealed an equal distribution of smooth (eight of 13) and irregular (five of 13) strictures in the malignant group. Ten of 13 benign strictures were characterized as smooth. Malignant strictures were significantly longer than benign ones -30.3 versus 9.2 mm (P=0.001). Threshold analysis using ROC curves showed that strictures greater than or equal to 14 mm were predictive of malignancy (sensitivity 78%, specificity 75%, log odds ratio 11.23). CONCLUSIONS: A serum bilirubin level of 75 µmol/L or higher, or a stricture length of greater than 14 mm was highly predictive of malignancy in patients with a biliary stricture. Ultrasound was useful in predicting malignant strictures by detecting either intrahepatic duct dilation or by visualizing the tumour (primary or metastases). Strictures with a 'benign' cholangiographic appearance are frequently malignant. Disida scan did not add additional information. ERCP is necessary to diagnose benign strictures, which tend to be less extensive at presentation. B iliary strictures are a challenging problem for the clinician. By the time that patients with biliary strictures are referred to a specialist, the diagnosis is usually already known or strongly suspected because clinical evaluation and noninvasive investigations alone have a high specificity and sensitivity (1-4). The job of the medical or surgical specialist is not only to confirm the diagnosis of biliary stricture but also, importantly, to define the etiology and the exact anatomic location, which is vital to therapeutic planning. The differentiation between benign and malignant strictures can be difficult but is of obvious importance in regard to prognosis and optimal therapy. Numerous imaging modalities are available for the investigation of biliary strictures, including abdominal ultrasound, computed tomographic (CT) scanning, nuclear imaging, percutaneous transhepatic cholangiography (PTC), endoscopic retrograde cholangiopancreatography (ERCP) and most recently magnetic resonance cholangiopancreatography (MRCP). Comparative and descriptive studies in this area are lacking, primarily because rapid technological improvements and developments outdate them. We, therefore, embarked on a prospective descriptive trial with the following aims: · Determine the predictive value of liver enzymes, serum bilirubin, serum bile acids, ultrasound and diethyl-iminodiacetic acid (Disida) nuclear imaging for the presence of malignant biliary strictures. · Measure the ability of ultrasound and nuclear imaging to localize the level of obstruction using direct cholangiography as the gold standard. · Determine the sensitivity of abdominal ultrasound and Disida nuclear scanning for the detection of biliary strictures. · Investigate the utility of various cholangiographic features to distinguish malignant from benign strictures. PATIENTS AND METHODS Patients: All patients with biliary strictures referred to the Division of Gastroenterology at the University of Alberta Hospitals for investigation between January 1, 1995 and December 31, 1995 were prospectively entered into the trial. The inclusion criteria were age 16 years or older and noncalculus biliary obstruction. Patients were excluded if subsequent evaluation did not show a stricture. Ethics committee approval was obtained. sente dans 93 % des sténoses malignes par rapport à 36 % des sténoses béni-gnes (P = 0,002). Une dilatation du canal hépatique commun était moins discriminatoire (13,5 mm en cas de malignité par rapport à 9,6 mm en cas de bénignité, P = 0,11). L'échographie était hautement sensible (93 %) pour déceler la tumeur primaire dans le canal biliaire ou le pancréas, ou pour visualiser les nodules ou les métastases hépatiques. Dans le cas d'une maladie bénigne, l'échogra-phie n'a pas réussi à déceler la présence d'une dilatation biliaire intra-hépa-tique ou extra-hépatique dans la plupart des cas. Les scintigraphies au disida n'ont pas pu différencier les sténoses malignes des sténoses bénignes ou localiser précisément le niveau de l'obstruction. La sensibilité des scintigraphies au disida pour établir un diagnostic d'obstruction était de 50 %. La caractérisation cholangiographique des sténoses a révélé une distribution égale de sténoses lisses (huit sur 13) et irrégulières (cinq sur 13) dans le groupe des sténoses malignes. Dix des 13 sténoses bénignes ont été qualifiées de lisses. Les sténoses malignes étaient nettement plus longues que les sténoses bénignes, soit 30,3 mm par rapport à 9,2 mm (P=0,001). L'analyse des seuils au moyen des courbes ROC a révélé que des sténoses supérieures ou égales à 14 mm étaient un prédicteur de malignité (sensibilité de 78 %, spécificité de 75 %, risque relatif logarithmique de 11,23). CONCLUSIONS : Un niveau de bilirubine sérique de 75 µmol/L ou une longueur de sténose de 14 mm étaient fortement prédictifs de malignité chez les patients atteints d'une sténose biliaire. L'échographie était utile pour prédire les sténoses malignes en décelant soit une dilatation du canal intra-hépatique ou en visualisant la tumeur (primaire ou métastases). Les sténoses d'apparence « bénigne » à la cholangiographie s'avèrent souvent malignes. La scintigraphie au disida n'apportait pas d'informations supplé-mentaires. Une CPRE est nécessaire pour diagnostiquer des sténoses bénignes, qui ont tendance à être moins étendues à l'examen. · Abdominal ultrasound examination with particular attention to intrahepatic biliary dilation, extrahepatic duct calibre, presence or absence of gallbladder and other relevant pathology such as tumour mass or ductal stones. · Disida scan. Patients were examined after a 4 h fast. Opiates were withheld for the proceeding 24 h. In addition to the standard scan, data were collected for deconvolutional analysis to determine hepatic extraction fraction and time activity curve so that the half-life of biliary excretion and time to peak activity could be analyzed. · Cholangiography. ERCP was attempted first in all patients with failures proceeding to PTC. Cefazoline 1 g was administered intravenously 30 to 60 mins before cholangiography. The biliary system was filled as completely as possible using 50% Conray 60 (Mallenchrodt, St Louis, Missouri) contrast injected under low pressure. The information obtained from each cholangiogram included site of stricture, multiplicity, character (smoothly tapered versus irregular or shouldered), stricture(s) length, minimal stricture width, maximal proximal biliary dilation and other information (ampullary mass, primary sclerosing cholangitis, cancer of the pancreas). All data were to be collected within five working days so that the different imaging modalities tested would be comparable. All imaging studies were interpreted by radiologists blinded to the results of the patients' other diagnostic studies. The ERCP data were obtained last so that a biliary stent could be inserted if indicated. The cholangiographic measurements were confirmed by two independent observers. Stricture etiology was defined by cytology or biopsy histology or by clinical outcome after one year. Statistical analysis: Statistical analysis was performed using SPSS. Between-groups differences in mean values of continuous variables were tested by independent samples t tests or by nonparametric Mann-Whitney Rank Sum tests when the distributions were not normal. The differences in frequencies of categorical variables were tested by c 2 test with Yates' correction for continuity or by Fisher's exact test when the expected number of observations per cell was less than five. Associations between continuous variables were assessed by Pearson correlation coefficient. Logistic regression analysis was used to analyze the association of dichotomous outcome variable (malignant versus benign) with continuous and categorical predictor variables. The statistical inferences were based on the level of significance P<0.05. Receiver operating characteristic (ROC) curves were constructed for the biochemical variables. To determine optimal threshold levels for each diagnostic parameter, ROC plots were constructed using the observed true and false positive rates at each potential threshold level. A best fit ROC curve was constructed according to methods published elsewhere (5,6). The threshold value providing the best compromise between true and false positive rates was estimated from the ROC plot. Likelihood ratios were calculated from the fitted ROC curve. RESULTS Thirty-one patients were enrolled in the study. Two patients were excluded -one because he did not have a stricture and one whose suspected stricture was unevaluable because of previous biliary bypass and contraindication for PTC as a result of coagulopathy. Of the remaining 29 patients, 15 were diagnosed with malignant strictures and 14 with benign strictures. Two patients had primary sclerosing cholangitis, both of whom had multiple strictures. Patient demographics and underlying diagnosis are shown i

Safety and Efficacy of Erythrocyte Encapsulated Thymidine Phosphorylase in Mitochondrial Neurogastrointestinal Encephalomyopathy.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an ultra-rare autosomal recessive disorder of nucleoside metabolism that is caused by mutations in the nuclear thymidine phosphorylase gene (TYMP) gene, encoding for the enzyme thymidine phosphorylase. There are currently no approved treatments for MNGIE. The aim of this study was to investigate the safety, tolerability, and efficacy of an enzyme replacement therapy for the treatment of MNGIE. In this single centre study, three adult patients with MNGIE received intravenous escalating doses of erythrocyte encapsulated thymidine phosphorylase (EE-TP; dose range: 4 to 108 U/kg/4 weeks). EE-TP was well tolerated and reductions in the disease-associated plasma metabolites, thymidine, and deoxyuridine were observed in all three patients. Clinical improvements, including weight gain and improved disease scores, were observed in two patients, suggesting that EE-TP is able to reverse some aspects of the disease pathology. Transient, non-serious adverse events were observed in two of the three patients; these did not lead to therapy discontinuation and they were managed with pre-medication prior to infusion of EE-TP. To conclude, enzyme replacement therapy with EE-TP demonstrated biochemical and clinical therapeutic efficacy with an acceptable clinical safety profile

Two-dimensional NMR lineshape analysis

NMR titration experiments are a rich source of structural, mechanistic, thermodynamic and kinetic information on biomolecular interactions, which can be extracted through the quantitative analysis of resonance lineshapes. However, applications of such analyses are frequently limited by peak overlap inherent to complex biomolecular systems. Moreover, systematic errors may arise due to the analysis of two-dimensional data using theoretical frameworks developed for one-dimensional experiments. Here we introduce a more accurate and convenient method for the analysis of such data, based on the direct quantum mechanical simulation and fitting of entire two-dimensional experiments, which we implement in a new software tool, TITAN (TITration ANalysis). We expect the approach, which we demonstrate for a variety of protein-protein and protein-ligand interactions, to be particularly useful in providing information on multi-step or multi-component interactions

Inhibitor of DNA Binding 3 Limits Development of Murine Slam-Associated Adaptor Protein-Dependent “Innate” γδ T cells

Id3 is a dominant antagonist of E protein transcription factor activity that is induced by signals emanating from the alphabeta and gammadelta T cell receptor (TCR). Mice lacking Id3 were previously shown to have subtle defects in positive and negative selection of TCRalphabeta+ T lymphocytes. More recently, Id3(-/-) mice on a C57BL/6 background were shown to have a dramatic expansion of gammadelta T cells.Here we report that mice lacking Id3 have reduced thymocyte numbers but increased production of gammadelta T cells that express a Vgamma1.1+Vdelta6.3+ receptor with restricted junctional diversity. These Vgamma1.1+Vdelta6.3+ T cells have multiple characteristics associated with "innate" lymphocytes such as natural killer T (NKT) cells including an activated phenotype, expression of the transcription factor PLZF, and rapid production of IFNg and interleukin-4. Moreover, like other "innate" lymphocyte populations, development of Id3(-/-) Vgamma1.1+Vdelta6.3+ T cells requires the signaling adapter protein SAP.Our data provide novel insight into the requirements for development of Vgamma1.1+Vdelta6.3+ T cells and indicate a role for Id3 in repressing the response of "innate" gammadelta T cells to SAP-mediated expansion or survival

Aggregated a-synuclein and complex I deficiency: exploration of their relationship in differentiated neurons

α-Synuclein becomes misfolded and aggregated upon damage by various factors, for example, by reactive oxygen species. These aggregated forms have been proposed to have differential toxicities and their interaction with mitochondria may cause dysfunction within this organelle that contributes to the pathogenesis of Parkinson’s disease (PD). In particular, the association of α-synuclein with mitochondria occurs through interaction with mitochondrial complex I and importantly defects of this protein have been linked to the pathogenesis of PD. Therefore, we investigated the relationship between aggregated α-synuclein and mitochondrial dysfunction, and the consequences of this interaction on cell survival. To do this, we studied the effects of α-synuclein on cybrid cell lines harbouring mutations in either mitochondrial complex I or IV. We found that aggregated α-synuclein inhibited mitochondrial complex I in control and complex IV-deficient cells. However, when aggregated α-synuclein was applied to complex I-deficient cells, there was no additional inhibition of mitochondrial function or increase in cell death. This would suggest that as complex I-deficient cells have already adapted to their mitochondrial defect, the subsequent toxic effects of α-synuclein are reduced

A single-electron transistor made from a cadmium selenide nanocrystal

The techniques of colloidal chemistry permit the routine creation of semiconductor nanocrystals, whose dimensions are much smaller than those that can be realized using lithographic techniques. The sizes of such nanocrystals can be varied systematically to study quantum size effects or to make novel electronic or optical materials with tailored properties. Preliminary studies of both the electrical and optical properties of individual nanocrystals have been performed recently. These studies show clearly that a single excess charge on a nanocrystal can markedly influence its properties. Here we present measurements of electrical transport in a single-electron transistor made from a colloidal nanocrystal of cadmium selenide. This device structure enables the number of charge carriers on the nanocrystal to be tuned directly, and so permits the measurement of the energy required for adding successive charge carriers. Such measurements are invaluable in understanding the energy-level spectra of small electronic systems, as has been shown by similar studies of lithographically patterned quantum dots and small metallic grains.Comment: 3 pages, PDF forma

Diversity in sound pressure levels and estimated active space of resident killer whale vocalizations

Author Posting. © The Author, 2005. This is the author's version of the work. It is posted here by permission of Springer for personal use, not for redistribution. The definitive version was published in Journal of Comparative Physiology A: Sensory, Neural, and Behavioral Physiology 192 (2006): 449-459, doi:10.1007/s00359-005-0085-2.Signal source intensity and detection range, which integrates source intensity with propagation loss, background noise and receiver hearing abilities, are important characteristics of communication signals. Apparent source levels were calculated for 819 pulsed calls and 24 whistles produced by free-ranging resident killer whales by triangulating the angles-of-arrival of sounds on two beamforming arrays towed in series. Levels in the 1-20 kHz band ranged from 131-168 dB re 1μPa @1m, with differences in the means of different sound classes (whistles: 140.2 ± 4.1 dB; variable calls: 146.6 ± 6.6 dB; stereotyped calls: 152.6 ± 5.9 dB), and among stereotyped call types. Repertoire diversity carried through to estimates of active space, with “long-range” stereotyped calls all containing overlapping, independently-modulated high-frequency components (mean estimated active space of 10-16km in sea state zero) and “short-range” sounds (5-9 km) included all stereotyped calls without a high-frequency component, whistles, and variable calls. Short-range sounds are reported to be more common during social and resting behaviors, while long-range stereotyped calls predominate in dispersed travel and foraging behaviors. These results suggest that variability in sound pressure levels may reflect diverse social and ecological functions of the acoustic repertoire of killer whales.Funding was provided by WHOI’s Ocean Ventures Fund and Rinehart Coastal Research Center and a Royal Society fellowship

Preparation and Characterization of Covalently Binding of Rat Anti-human IgG Monolayer on Thiol-Modified Gold Surface

The 16-mercaptohexadecanoic acid (MHA) film and rat anti-human IgG protein monolayer were fabricated on gold substrates using self-assembled monolayer (SAM) method. The surface properties of the bare gold substrate, the MHA film and the protein monolayer were characterized by contact angle measurements, atomic force microscopy (AFM), grazing incidence X-ray diffraction (GIXRD) method and X-ray photoelectron spectroscopy, respectively. The contact angles of the MHA film and the protein monolayer were 18° and 12°, respectively, all being hydrophilic. AFM images show dissimilar topographic nanostructures between different surfaces, and the thickness of the MHA film and the protein monolayer was estimated to be 1.51 and 5.53 nm, respectively. The GIXRD 2θ degrees of the MHA film and the protein monolayer ranged from 0° to 15°, significantly smaller than that of the bare gold surface, but the MHA film and the protein monolayer displayed very different profiles and distributions of their diffraction peaks. Moreover, the spectra of binding energy measured from these different surfaces could be well fitted with either Au4f, S2p or N1s, respectively. Taken together, these results indicate that MHA film and protein monolayer were successfully formed with homogeneous surfaces, and thus demonstrate that the SAM method is a reliable technique for fabricating protein monolayer