Production of Mycobacterium bovis Antigens Included in Recombinant Occlusion Bodies of Baculovirus

2019 ◽  
Vol 29 (1-6) ◽  
pp. 83-90
Author(s):  
Luciana Villafañe ◽  
Marina Andrea Forrellad ◽  
María Gabriela López ◽  
Sergio Garbaccio ◽  
Carlos Garro ◽  
...  
Keyword(s):  
Mycobacterium Bovis ◽  
Intradermal Injection ◽  
In Vitro Assays ◽  
Chimeric Proteins ◽  
Purified Protein Derivative ◽  
E Coli ◽  
Occlusion Bodies ◽  
Ifn Γ ◽  
High Level

Bovine tuberculosis (bTB) is a disease produced by <i>Mycobacterium bovis</i> that affects livestock, wild animals, and humans. The classical diagnostic method to detect bTB is measuring the response induced with the intradermal injection of purified protein derivative of <i>M. bovis</i> (PPDb). Another ancillary bTB test detects IFN-γ produced in whole blood upon stimulation with PPDb, protein/peptide cocktails, or individual antigens. Among the most used <i>M. bovis</i> antigens in IFN-γ assays are the secreted proteins ESAT-6 and CFP-10, which together with antigen Rv3615c improve the sensitivity of the test in comparison to PPDb. Protein reagents for immune stimulation are generally obtained from <i>Escherichia coli</i>, because this bacterium produces a high level of recombinant proteins. However, <i>E. coli</i> recombinant antigens are in general contaminated with lipopolysaccharides and other components that produce non-specific IFN-γ secretion in in vitro assays. In this work, we produced the relevant ESAT-6, CFP-10, and Rv3615c <i>M. bovis</i> antigens as fusions to the polyhedrin protein from the baculovirus AcMNPV. We obtained chimeric proteins effectively incorporated to the occlusion bodies and easily purified the recombinant polyhedra with no reactive contaminants. In an IFN-γ assay, these fusion proteins showed equivalent sensibility but better specificity than the same <i>M. bovis</i> proteins produced in <i>E. coli</i>.

scholarly journals Optimization of a Whole-Blood Gamma Interferon Assay for Detection of Mycobacterium bovis-Infected Cattle

2009 ◽  
Vol 16 (8) ◽  
pp. 1196-1202 ◽  
Author(s):  
Irene Schiller ◽  
W. Ray Waters ◽  
H. Martin Vordermeier ◽  
Brian Nonnecke ◽  
Michael Welsh ◽  
...  
Keyword(s):  
Carbon Dioxide ◽  
Mycobacterium Bovis ◽  
Whole Blood ◽  
Intradermal Injection ◽  
Gamma Interferon ◽  
Blood Collection ◽  
Cell Mediated Immune Response ◽  
Delayed Initiation ◽  
Ifn Γ

ABSTRACT Antigens of Mycobacterium bovis elicit a cell-mediated immune response upon intradermal injection in cattle. In vitro, such antigens stimulate the production of gamma interferon (IFN-γ) by bovine T cells in whole-blood culture (IFN-γ assay). We have analyzed various parameters of the in vitro IFN-γ assay, ranging from blood sampling to execution of the IFN-γ test, in view of potential simplifications of the assay. Here, we show that IFN-γ responses may be reduced under certain animal handling/holding conditions and that a delayed time from blood collection to culture may lead to a reduced in vitro IFN-γ response. Delayed initiation of culture in a purified-protein-derivative-based assay (24 h compared to 8 h after blood collection), however, resulted in a significant improvement of specificity (97% compared to 85%), whereas there was only a modest reduction of sensitivity (from 96% to 90%), which was statistically not significant. Furthermore, we show that the stimulation temperature needs to be 33°C or higher; that carbon dioxide is not required for stimulation; and that various plate formats, ranging from 24 to 96 wells per plate, can be utilized. The produced IFN-γ is stable at 4°C for 28 days as well as after repeated freeze-thaw cycles. Thus, stimulation of samples may be initiated in the field without the need for a carbon dioxide source, and bovine IFN-γ is stable under various routine laboratory temperature scenarios. These findings demonstrate opportunities for improvements in the bovine IFN-γ test platform and flexibilities in test application.

scholarly journals Discovery and Optimisation of Bacterial Nitroreductases for Use in Anti-Cancer Gene Therapy

2021 ◽  
Author(s):  
◽  
Gareth Adrian Prosser
Keyword(s):  
Dna Damage ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Alkylating Agents ◽  
Dna Lesions ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
E Coli ◽  
Anti Cancer

<p>Nitroaromatic prodrugs are biologically inert compounds that are attractive candidates for anti-cancer therapy by virtue of their ability to be converted to potent DNA alkylating agents by nitroreductase (NTR) enzymes. In gene-directed enzyme-prodrug therapy (GDEPT), NTR-encoding therapeutic transgenes are delivered specifically to tumour cells, whereupon their expression confers host cell sensitivity to subsequent systemic administration of a nitroaromatic prodrug. The most well studied NTR-GDEPT system involves reduction of the aziridinyl dinitrobenzamide prodrug CB1954 by the Escherichia coli NTR NfsB. However, low affinity of this enzyme for CB1954 has so far limited the clinical efficacy of this GDEPT combination. The research described in this thesis has primarily sought to address this limitation through identification and optimisation of novel NTR enzymes with improved nitroaromatic prodrug reductase activity. Efficient assessment of NTR activity from large libraries of candidate enzymes requires a rapid and reliable screening system. An E. coli-based assay was developed to permit indirect assessment of relative rates of prodrug reduction by over-expressed NTRs via measurement of SOS response induction resulting from reduced prodrug-induced DNA damage. Using this assay in concert with other in vitro and in vivo tests, more than 50 native bacterial NTRs of diverse sequence and origin were assessed for their ability to reduce a panel of clinically attractive nitroaromatic prodrugs. Significantly, a number of NTRs were identified, particularly in the family of enzymes homologous to the native E. coli NTR NfsA, which displayed substantially improved activity over NfsB with CB1954 and other nitroaromatic prodrugs as substrates. This work also examined the roles of E. coli DNA damage repair pathways in processing of adducts induced by various nitroaromatic prodrugs. Of particular interest, nucleotide excision repair was found to be important in the processing of DNA lesions caused by 4-, but not 2-nitro group reduction products of CB1954, which suggests that there are some parallels in the mechanisms of CB1954 adduct repair in E. coli and mammalian cells. Finally, a lead NTR candidate, YcnD from Bacillus subtilis, was selected for further activity improvement through site-directed mutagenesis of active site residues. Using SOS screening, a double-site mutant was identified with 2.5-fold improved activity over the wildtype enzyme in metabolism of the novel dinitrobenzamide mustard prodrug PR-104A. In conclusion, novel NTRs with substantially improved nitroaromatic prodrug reducing activity over previously documented enzymes were identified and characterised. These results hold significance not only for the field of NTR-GDEPT, but also for other biotechnological applications in which NTRs are becoming increasingly significant, including developmental studies, antibiotic discovery and bioremediation. Furthermore, the in vitro assays developed in this study have potential utility in the discovery and evolution of other GDEPT-relevant enzymes whose prodrug metabolism is associated with genotoxicity.</p>

scholarly journals Discovery and Optimisation of Bacterial Nitroreductases for Use in Anti-Cancer Gene Therapy

2021 ◽  
Author(s):  
◽  
Gareth Adrian Prosser
Keyword(s):  
Dna Damage ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Alkylating Agents ◽  
Dna Lesions ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
E Coli ◽  
Anti Cancer

<p>Nitroaromatic prodrugs are biologically inert compounds that are attractive candidates for anti-cancer therapy by virtue of their ability to be converted to potent DNA alkylating agents by nitroreductase (NTR) enzymes. In gene-directed enzyme-prodrug therapy (GDEPT), NTR-encoding therapeutic transgenes are delivered specifically to tumour cells, whereupon their expression confers host cell sensitivity to subsequent systemic administration of a nitroaromatic prodrug. The most well studied NTR-GDEPT system involves reduction of the aziridinyl dinitrobenzamide prodrug CB1954 by the Escherichia coli NTR NfsB. However, low affinity of this enzyme for CB1954 has so far limited the clinical efficacy of this GDEPT combination. The research described in this thesis has primarily sought to address this limitation through identification and optimisation of novel NTR enzymes with improved nitroaromatic prodrug reductase activity. Efficient assessment of NTR activity from large libraries of candidate enzymes requires a rapid and reliable screening system. An E. coli-based assay was developed to permit indirect assessment of relative rates of prodrug reduction by over-expressed NTRs via measurement of SOS response induction resulting from reduced prodrug-induced DNA damage. Using this assay in concert with other in vitro and in vivo tests, more than 50 native bacterial NTRs of diverse sequence and origin were assessed for their ability to reduce a panel of clinically attractive nitroaromatic prodrugs. Significantly, a number of NTRs were identified, particularly in the family of enzymes homologous to the native E. coli NTR NfsA, which displayed substantially improved activity over NfsB with CB1954 and other nitroaromatic prodrugs as substrates. This work also examined the roles of E. coli DNA damage repair pathways in processing of adducts induced by various nitroaromatic prodrugs. Of particular interest, nucleotide excision repair was found to be important in the processing of DNA lesions caused by 4-, but not 2-nitro group reduction products of CB1954, which suggests that there are some parallels in the mechanisms of CB1954 adduct repair in E. coli and mammalian cells. Finally, a lead NTR candidate, YcnD from Bacillus subtilis, was selected for further activity improvement through site-directed mutagenesis of active site residues. Using SOS screening, a double-site mutant was identified with 2.5-fold improved activity over the wildtype enzyme in metabolism of the novel dinitrobenzamide mustard prodrug PR-104A. In conclusion, novel NTRs with substantially improved nitroaromatic prodrug reducing activity over previously documented enzymes were identified and characterised. These results hold significance not only for the field of NTR-GDEPT, but also for other biotechnological applications in which NTRs are becoming increasingly significant, including developmental studies, antibiotic discovery and bioremediation. Furthermore, the in vitro assays developed in this study have potential utility in the discovery and evolution of other GDEPT-relevant enzymes whose prodrug metabolism is associated with genotoxicity.</p>

scholarly journals Kinetics of Targeted Phage Rescue in a Mouse Model of SystemicEscherichia coliK1

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
György Schneider ◽  
Nikolett Szentes ◽  
Marianna Horváth ◽  
Ágnes Dorn ◽  
Alysia Cox ◽  
...  
Keyword(s):  
Capsular Polysaccharide ◽  
Protective Efficacy ◽  
Lytic Bacteriophage ◽  
E Coli ◽  
Causative Agents ◽  
High Level ◽  
Systemic Infections ◽  
Kinetics Of

Escherichia (E.) coliK1 strains remain common causative agents of neonatal sepsis and meningitis. We have isolated a lytic bacteriophage (ΦIK1) againstE. colistrain IHE3034 and tested its specificityin vitro, as well as distribution and protective efficacyin vivo. The phage was shown to be specific to the K1 capsular polysaccharide. In the lethal murine model, a high level of protection was afforded by the phage with strict kinetics. A single dose of 1 x 108phage particles administered 10 and 60 minutes following the bacterial challenge elicited 100 % and 95 % survival, respectively. No mice could be rescued if phage administration occurred 3 hours postinfection. Tissue distribution surveys in the surviving mice revealed that the spleen was the primary organ in which accumulation of active ΦIK1 phages could be detected two weeks after phage administration. These results suggest that bacteriophages have potential as therapeutic agents in the control of systemic infections.

Προβιοτικές και τεχνολογικές ιδιότητες οξυγαλακτικών βακτηρίων. In vitro και in vivo επίδραση στο ελικοβακτήριο του πυλωρού

2004 ◽  
Author(s):  
Πέτρος-Αχιλλέας Μαραγκουδάκης
Keyword(s):  
E Coli ◽  
Tnf Α ◽  
Ifn Γ

Σκοπός: Ο σκοπός της παρούσας διατριβής ήταν η εξέταση του προβιοτικού δυναμικού στελεχών οξυγαλακτικών βακτηρίων μέσω μίας σειράς in vitro δοκιμών, και της μετέπειτα εφαρμογής επιλεγμένων στελεχών σε in vivo μοντέλα ποντικών έναντι της λοίμωξης του Η. pylori και της ελκώδους κολίτιδας, αλλά και σε τεχνολογικό επίπεδο στην παρασκευή γιαουρτιού.Υλικά και μέθοδοι: Στη διατριβή αυτή μελετήθηκαν 51 στελέχη Lactobacillus του Εργαστηρίου Γαλακτοκομίας του Γεωπονικού Πανεπιστημίου Αθηνών, απομονωμένα κυρίως από γαλακτοκομικά προϊόντα και κόπρανα προβάτων, καθώς και 22 στελέχη Enterococcus και Streptococcus, απομονωμένα από κόπρανα προβάτων. Τα στελέχη αυτά εξετάστηκαν σε μία σειρά in vitro δοκιμών που αφορούν την επιβίωσή τους σε συνθήκες εξομοίωσης του ανθρώπινου γαστρεντερικού συστήματος, οι οποίες περιελάμβαναν επιβίωση σε χαμηλό pH, επιβίωση παρουσία των πρωτεολυτικών ενζύμων πεψίνη και παγκρεατίνη, και επιβίωση παρουσία χολικών αλάτων.Στην συνέχεια τα στελέχη εξετάστηκαν σε δοκιμές βασικές για την ασφάλεια χρήσης τους στον ανθρώπινο οργανισμό. "Ολα τα στελέχη δοκιμάστηκαν ως προς την αιμολυτική τους δραστικότητα, και τα στελέχη γαλακτοβακίλλων συγκεκριμένα για την ανθεκτικότητά τους έναντι επιλεγμένων αντιβιοτικών.Παράλληλα, εξετάστηκαν in vitro βασικές ιδιότητες που θεωρούνται επιθυμητές για τα προβιοτικά στελέχη, όπως υδρόλυση των χολικών αλάτων, μελέτη της υδροφοβίας και της ικανότητας πρόσδεσης των γαλακτοβακίλλων σε κύτταρα Caco-2, ικανότητά διέγερσης κυττάρων του ανθρώπινου οργανισμού (PBMCs), καθώς και μελέτη της αντιμικροβιακής δράσης έναντι παθογόνων βακτηρίων και της δυνατότητάς αναστολής της πρόσδεσης παθογόνων σε κύτταρα Caco-2.Στην συνέχεια πραγματοποιήθηκαν in vivo μελέτες σε ποντίκια, για την εξακρίβωση του προβιοτικού χαρακτήρα επιλεγμένων στελεχών. Τα στελέχη L. casei Shirota ACA-DC 6002 και L. paracasei subsp. tolerans ACA-DC 4037 χορηγήθηκαν σε ποντίκια επιμολυσμένα με Η. pylori και αξιολογήθηκε η επίδραση των γαλακτοβακίλλων αυτών στον αποικισμό του παθογόνου και στην αξιολόγηση του βαθμού και της δραστικότητας στο στομάχι. Επίσης, τα στελέχη αυτά χρησιμοποιήθηκαν και για την πρόληψη TNBS- κολίτιδας σε ποντίκια.Τέλος, επιλεγμένα στελέχη γαλακτοβακίλλων αξιολογήθηκαν για την τεχνολογική τους εφαρμογή, μελετώντας την ικανότητά οξίνϊσης του γάλακτος και τη χρήση τους ως καλλιέργειες για την παρασκευή γιαούρτηςΑποτελέσματα: Ορισμένα στελέχη βρέθηκαν να έχουν ικανοποιητική επιβίωση σε συνθήκες εξομοίωσης του ανθρώπινου πεπτικού συστήματος. Κανένα στέλεχος δεν βρέθηκε να έχει ισχυρή (β-) αιμολυτική δράση, ενώ η αντοχή των γαλακτοβακίλλων έναντι επιλεγμένων αντιβιοτικών δεν παρουσιάστηκε διαφορετική από την έμφυτη και αναμενόμενη, ανάλογα το είδος των εξεταζόμενων γαλακτοβακίλλων, ενώ αρκετά στελέχη παρουσίασαν ικανότητα υδρόλυσης χολικών αλάτων. Τα στελέχη παρουσίασαν ποικιλόμορφη πρόσδεση στους οργανικούς διαλύτες, αλλά γενικά τα περισσότερα στελέχη δεν παρουσίασαν μεγάλη υδροφοβία, ενώ αρκετά στελέχη γαλακτοβακίλλων παρουσίασαν ικανοποιητική πρόσδεση σε κύτταρα Caco-2 (>5%), με το στέλεχος L. plantarum ACA-DC 146 (25% πρόσδεση) να ξεχωρίζει. Το ίδιο στέλεχος, μαζί με το L. paracasei subsp. tolerans ACA-DC 4037 βρέθηκαν να προκαλούν την έκκριση υψηλών επιπέδων φλεγμονωδών κυτταροκινών (IL-12, TNF-α και IFN-γ) από PBMCs, ενώ αντίθετα το στέλεχος L. casei Shirota ACA-DC 6002 προκάλεσε την έκκριση της αντιφλεγμονώδους κυτταροκίνης IL-10. Παρόλο που κανένα από τα υπερκείμενα των στελεχών δεν παρεμπόδισε τα στελέχη E. coli, S. typhimurium και Η. pylori, ορισμένα στελέχη, όπως τα L. casei Shirota, L. plantarum ACA-DC 146 και L. paracasei subsp. tolerans ACA-DC 4037 προκάλεσαν την αναστολή της πρόσδεσης εντερικών παθογόνων (E. coli και S. typhimurium) σε κύτταρα Caco-2 σε επίπεδα μέχρι και 50%.In vivo, η χορήγηση του L. casei Shirota ACA-DC 6002 σε ποντίκια οδήγησε στην μείωση του βαθμού και της δραστικότητας της γαστρίτιδας αλλά και στον αποικισμό του Η. pylori. Παρόμοια αποτελέσματα, αλλά σε μικρότερη έκταση, παρατηρήθηκαν με την χορήγηση του στελέχους L. paracasei subsp. tolerans ACA-DC 4037. Παράλληλα, το στέλεχος L. casei Shirota ACA-DC 6002 δείχνει να παρέχει προστασία σε ποντίκια κατά την πρόκληση ελκώδους κολίτιδας με την χορήγηση TNBS.Σε τεχνολογικό επίπεδο, κανένα από τα εξεταζόμενα στελέχη δεν παρουσίασε υψηλή ικανότητα οξύνισης στο γάλα και κατά συνέπεια δεν ήταν δυνατόν να χρησιμοποιηθούν ως εναρκτήριες καλλιέργειες για την παρασκευή γιαούρτης. Από την άλλη, καθώς κανένα στέλεχος δεν παρεμποδίζει αλλά και δεν παρεμποδίζεται από τις παραδοσιακές εναρκτήριες καλλιέργειες γιαούρτης, ήταν δυνατή η χρήση τους ως συμπληρωματικές καλλιέργειας για την παρασκευή γιαούρτης. Στην συνέχεια επιλεγμένα στελέχη χρησιμοποιήθηκαν για το σκοπό αυτό και το στέλεχος L. paracasei subsp. tolerans ACA- DC 4037 ξεχώρισε όταν χρησιμοποιήθηκε ως συμπληρωματική καλλιέργεια για την παρασκευή γιαούρτης με εμβόλιο πηγμένου γάλακτος (1% ν/ν) στους 42°C, λόγω της υψηλής μικροβιακής, φυσικοχημικής και οργανοληπτικής ποιότητας του προϊόντος. Συμπεράσματα: Τα στελέχη L. casei Shirota ACA-DC 6002, L. plantarum ACA-DC 146 και L. paracasei subsp. tolerans ACA-DC 4037, επέδειξαν υποσχόμενο προβιοτικό δυναμικό κατά την διάρκεια εκτεταμένων in vitro και in vivo δοκιμών. Η μελέτη αυτή επιβεβαιώνει τις προβιοτικές ιδιότητες του L. casei Shirota και επιδεικνύει την πιθανή εφαρμογής του στη λοίμωξη του Η. pylori και στην ελκώδη κολίτιδα στον άνθρωπο. Επιπλέον, το στέλεχος L paracasei subsp. tolerans ACA-DC 4037 διαθέτει επίσης δυναμικό χρήσης έναντι του Η. pylori αλλά και στην παρασκευή ενός επιτυχημένου προβιοτικού γιαουρτιού. Τέλος, το στέλεχος L. plantarum ACA-DC 146 μπορεί να εξεταστεί in vivo σε μοντέλα αλλεργικών αντιδράσεων, λόγω της υψηλής αντιφλεγμονώδους επίδρασής τους σε ανθρώπινα περιφερειακά μονοπύρηνα (PBMCs.)

Comparison of Vibrio and Firefly Luciferases as Reporter Gene Systems for Use in Bacteria and Plants

1996 ◽  
Vol 23 (1) ◽  
pp. 75 ◽  
Author(s):  
SR Mudge ◽  
WR Lewis-Henderson ◽  
RG Birch
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Ccd Camera ◽  
Reporter Gene Expression ◽  
In Vitro Assays ◽  
E Coli ◽  
Cell Extracts ◽  
Cooled Ccd

Luciferase genes from Vibrio harveyi (luxAB) and firefly (luc) were introduced into E. coli, Agrobacteriurn, Arabidopsis and tobacco. Transformed bacteria and plants were quantitatively assayed for luciferase activity using a range of in vitro and in vivo assay conditions. Both lux and luc proved efficient reporter genes in bacteria, although it is important to be aware that the sensitive assays may detect expression due to readthrough from distant promoters. LUX activity was undetectable by liquid nitrogen-cooled CCD camera assays on intact tissues of plants which showed strong luxAB expression by in vitro assays. The decanal substrate for the lux assay was toxic to many plant tissues, and caused chemiluminescence in untransformed Arabidopsis leaves. These are serious limitations to application of the lux system for sensitive, non-toxic assays of reporter gene expression in plants. In contrast, LUC activity was readily detectable in intact tissues of all plants with luc expression detectable by luminometer assays on cell extracts. Image intensities of luc-expressing leaves were commonly two to four orders of magnitude above controls under the CCD camera. Provided adequate penetration of the substrate luciferin is obtained, luc is suitable for applications requiring sensitive, non-toxic assays of reporter gene expression in plants.

scholarly journals Coordinated In Vitro Release of Granulysin, Perforin and IFN-γ in TB and HIV/TB Co-Infection Associated with Clinical Outcomes before and after Anti-TB Treatment

Pathogens ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 655
Author(s):  
Nada Pitabut ◽  
Panadda Dhepakson ◽  
Shinsaku Sakurada ◽  
Naoto Keicho ◽  
Srisin Khusmith
Keyword(s):  
Clinical Outcomes ◽  
Mononuclear Cells ◽  
Granzyme B ◽  
In Vitro Release ◽  
Peripheral Blood Mononuclear ◽  
Purified Protein Derivative ◽  
Tb Treatment ◽  
Before And After ◽  
Ifn Γ

Granule-associated killing molecules released from cytotoxic T lymphocytes participate as a crucial step in immunity against tuberculosis (TB), but the role of coordinated production remains controversial. Coordinated release of effector molecules in vitro after stimulating peripheral blood mononuclear cells (PBMCs) of active TB or HIV/TB coinfection patients with PPD, purified protein derivative of tuberculin and avirulent Mtb, H37Ra, an attenuated strain were investigated in association with clinical outcomes. Perforin, granzyme-B, granulysin and IFN-γ were measured using ELISA. Before anti-TB treatment, PBMCs of TB stimulated with PPD or H37Ra released higher perforin, granzyme-B, and granulysin levels than in HIV/TB and released significantly higher IFN-γ (p = 0.045, p = 0.022). Granulysin positively correlated with perforin in TB (p = 0.042, r = 0.385), HIV/TB coinfection (p = 0.003, r = 0.941) after PPD stimulation, and after H37Ra stimulation in TB (p = 0.005, r = 0.549), but negatively correlated with granzyme B in TB (p = 0.042, r = −0.386), HIV/TB coinfection (p = 0.042, r = 0.754) were noted. After anti-TB treatment, increased levels of perforin, granulysin and IFN-γ in TB or HIV/TB upon PPD or H37Ra stimulation, and decreased granzyme-B levels after PPD (p = 0.003) or H37Ra (p = 0.028) stimulation in TB were observed. These results suggest that granulysin may act synergistic with perforin and IFN-γ in TB, indicating its crucial function in host immunity to tuberculosis. Future studies with larger numbers of patients ought to be conducted in the future.

scholarly journals Increased Numbers of ESAT-6- and Purified Protein Derivative-Specific Gamma Interferon-Producing Cells in Subclinical and Active Tuberculosis Infection

2000 ◽  
Vol 68 (10) ◽  
pp. 6073-6076 ◽  
Author(s):  
Timo Ulrichs ◽  
Peter Anding ◽  
Steven Porcelli ◽  
Stefan H. E. Kaufmann ◽  
Martin E. Munk
Keyword(s):  
Mycobacterium Bovis ◽  
Synthetic Peptides ◽  
Gamma Interferon ◽  
Active Tuberculosis ◽  
Tuberculosis Infection ◽  
Potential Candidate ◽  
Tuberculosis Patients ◽  
Purified Protein Derivative ◽  
Healthy Donors ◽  
Ifn Γ

ABSTRACT Numbers of gamma interferon (IFN-γ)-producing cells reactive to ESAT-6 antigen were increased in recent converters to purified protein derivative positivity and in tuberculosis patients but not in unvaccinated or Mycobacterium bovis BCG-vaccinated healthy donors. ESAT-6-reactive IFN-γ-producing cells in recent converters and tuberculosis patients recognized similar synthetic peptides. Thus, ESAT-6 is a potential candidate for use in detection of early, as well as active, tuberculosis and for control of the disease.

scholarly journals In Vitro Activities of the Ketolides Telithromycin (HMR 3647) and HMR 3004 Compared to Those of Clarithromycin against Slowly Growing Mycobacteria at pHs 6.8 and 7.4

2000 ◽  
Vol 44 (10) ◽  
pp. 2848-2852 ◽  
Author(s):  
Nalin Rastogi ◽  
Khye Seng Goh ◽  
Mylene Berchel ◽  
André Bryskier
Keyword(s):  
Animal Models ◽  
Mycobacterium Bovis ◽  
Experimental Animal ◽  
Intracellular Accumulation ◽  
Experimental Animal Models ◽  
High Level

ABSTRACT The in vitro activities of HMR 3647 (telithromycin) and HMR 3004, two novel semisynthetic ketolides, were investigated and compared with that of the reference macrolide drug, clarithromycin, against 34 strains of slowly growing mycobacteria at pHs 6.8 and 7.4, as determined radiometrically. The MICs at pH 7.4 were about 1 to 2 dilutions lower than those observed at pH 6.8. In terms of the highest to the lowest activity, the three antibiotics could be classified as follows: clarithromycin > HMR 3004 > HMR 3647. Among the species tested, Mycobacterium bovis BCG, M. ulcerans, M. avium, and M. paratuberculosis were moderately susceptible to HMR 3004 and HMR 3647 (MICs at pH 7.4, ≤5.0 and ≤20.0 μg/ml, respectively, versus ≤1.25 μg/ml for clarithromycin), whereas M. tuberculosis, M. africanum, M. bovis, andM. simiae were resistant (MICs, ≥10.0 and ≥40.0 μg/ml, respectively, at pH 7.4). Although not more active than clarithromycin in vitro, the high level of intracellular accumulation of the two ketolides inside phagocytes warrants further screening in experimental animal models.

Virulent bacteriophage infection of Rhizobium leguminosarum

1972 ◽  
Vol 18 (4) ◽  
pp. 375-384 ◽  
Author(s):  
A. N. Ley ◽  
H. R. Warner ◽  
Phyllis L. Kahn
Keyword(s):  
Molecular Weight ◽  
Rna Synthesis ◽  
Rhizobium Leguminosarum ◽  
Deoxyribonucleic Acid ◽  
In Vitro Assays ◽  
Tail Fiber ◽  
Bacteriophage Infection ◽  
Cell Extracts ◽  
High Level

Bacteriophage 317, which virulently infects Rhizobium leguminosarum, has an eclipse period of 60–70 min and a latent period of 100 min at 30°. Electron micrographs of the phage indicated head, tail, and tail-fiber structural components.Base analysis of phage 317 deoxyribonucleic acid (DNA) indicated the presence of equimolar amounts of adenine and thymine and of guanine and cytosine, which suggests that the DNA is double stranded. The DNA has a molecular weight of 41 × 106 daltons as determined from electron micrographs.The results of 14C-uracil incorporation studies showed that net ribonucleic acid (RNA) synthesis was markedly inhibited after infection. There was a slight stimulation in DNA synthesis after infection as indicated by 14C-thymidine incorporation.The results of in vitro assays of enzymes involved in the biosynthesis of DNA showed that deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase) and deoxythymidine 5′-monophosphate kinase (dTMP kinase) increased 50- and 30-fold respectively, after infection. The reason for the increased dUTPase activity is not readily apparent. Phage 317 DNA contains only the standard bases, unlike the DNA of other phages that induce an increase in this enzyme after infection. A high level of deoxythymidine 5′-monophosphatase (dTMPase) was observed in both uninfected and infected crude cell extracts. Further work is necessary to see if similar changes occur in Rhizobium during the establishment of symbiosis with legumes.

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