production and characterization of excretory/secretory products of

Onchocerca volvulus excretory/secretory products (ESP) free of host contaminants are often needed for immunologic and biochemical studies. Since prolonged in vitro survival of O. volvulus in culture requires the presence of human serum, as a supplement, it was necessary to investigate other culture medium supplements in which pure ESP could be generated. Thus heat-inactivated normal rabbit serum, fetal calf serum and the synthetic serum substitute Ultroser-G were tested for their abilities to sustain O. volvulus adult females and nodular microfilariae in vitro and their abilities to promote the synthesis and release of ESP. Using [35S]-methionine as the radioactive precursor, O. volvulus nodular microfilariae and adult females were shown to actively synthesize and release excretory/secretory proteins with increasing efficiency in human serum, rabbit serum, Ultroser-G and fetal calf serum. Analysis of the ESP by SDS-PAGE revealed that at least 30 polypeptides in the 7-174 kDa range were continuously released. About 14 of these components were immunogenic to the human host as shown by immunoprecipitation. Prominent antigenic polypeptides were those with relative molecular weights of 13, 16, 33, 68 and 170. Rabbit serum, fetal calf serum and Ultroser-G could therefore, conveniently replace human serum in cultures of O. volvulus nodular microfilariae and adult females

A cDNA coding for a novel antigen from Onchocerca volvulus

info:eu-repo/semantics/publishe

Supplementary Material for: PML-Nuclear Bodies Regulate the Stability of the Fusion Protein Dendra2-Nrf2 in the Nucleus

Background/Aims: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a basic leucine-zipper transcription factor essential for cellular responses to oxidative stress. Degradation of Nrf2 in the cytoplasm, mediated by Keap1-Cullin3/RING box1 (Cul3-Rbx1) E3 ubiquitin ligase and the proteasome, is considered the primary pathway controlling the cellular abundance of Nrf2. Although the nucleus has been implicated in the degradation of Nrf2, little information is available on how this compartment participates in degrading Nrf2. Methods: Here, we fused the photoconvertible fluorescent protein Dendra2 to Nrf2 and capitalized on the irreversible change in color (green to red) that occurs when Dendra2 undergoes photoconversion to study degradation of Dendra2-Nrf2 in single live cells. Results: Using this approach, we show that the half-life (t1/2) of Dendra2-Nrf2 in the whole cell, under homeostatic conditions, is 35 min. Inhibition of the proteasome with MG-132 or induction of oxidative stress with tert-butylhydroquinone (tBHQ) extended the half-life of Dendra2-Nrf2 by 6- and 28-fold, respectively. By inhibiting nuclear export using Leptomycin B, we provide direct evidence that degradation of Nrf2 also occurs in the nucleus and involves PML-NBs (Promyelocytic Leukemia-nuclear bodies). We further demonstrate that co-expression of Dendra2-Nrf2 and Crimson-PML-I lacking two PML-I sumoylation sites (K65R and K490R) changed the decay rate of Dendra2-Nrf2 in the nucleus and stabilized the nuclear derived Nrf2 levels in whole cells. Conclusion: Altogether, our findings provide direct evidence for degradation of Nrf2 in the nucleus and suggest that modification of Nrf2 in PML nuclear bodies contributes to its degradation in intact cells

Human alpha defensin 5 is a candidate biomarker to delineate inflammatory bowel disease

Inability to distinguish Crohn’s colitis from ulcerative colitis leads to the diagnosis of indeterminate colitis. This greatly effects medical and surgical care of the patient because treatments for the two diseases vary. Approximately 30 percent of inflammatory bowel disease patients cannot be accurately diagnosed, increasing their risk of inappropriate treatment. We sought to determine whether transcriptomic patterns could be used to develop diagnostic biomarker(s) to delineate inflammatory bowel disease more accurately. Four patients groups were assessed via whole-transcriptome microarray, qPCR, Western blot, and immunohistochemistry for differential expression of Human α-Defensin-5. In addition, immunohistochemistry for Paneth cells and Lysozyme, a Paneth cell marker, was also performed. Aberrant expression of Human α-Defensin-5 levels using transcript, Western blot, and immunohistochemistry staining levels was significantly upregulated in Crohn’s colitis, p< 0.0001. Among patients with indeterminate colitis, Human α-Defensin-5 is a reliable dif-ferentiator with a positive predictive value of 96 percent. We also observed abundant ectopic crypt Paneth cells in all colectomy tissue samples of Crohn’s colitis patients. In a retrospective study, we show that Human α-Defensin-5 could be used in indeterminate colitis patients to determine if they have either ulcerative colitis (low levels of Human α-Defensin-5) or Crohn’s colitis (high levels of Human α-Defensin-5). Twenty of 67 patients (30 percent) who underwent restorative proctocolectomy for definitive ulcerative colitis were clinically changed to de novo Crohn’s disease. These patients were profiled by Human α-Defensin-5 immunohistochemistry. All patients tested strongly positive. In addition, we observed by both hematoxylin and eosin and Lysozyme staining, a large number of ectopic Paneth cells in the colonic crypt of Crohn’s colitis patient samples. Our experiments are the first to show that Human α-Defensin-5 is a potential candidate biomarker to molecularly differentiate Crohn’s colitis from ulcerative colitis, to our knowledge. These data give us both a potential diagnostic marker in Human α-Defensin-5 and insight to develop future mechanistic studies to better understand crypt biology in Crohn’s colitis. © 2017 Williams et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited