scholarly journals Gran1: A Granulysin-Derived Peptide with Potent Activity against Intracellular Mycobacterium tuberculosis

2021 ◽  
Vol 22 (16) ◽  
pp. 8392
Author(s):  
Reiner Noschka ◽  
Fanny Wondany ◽  
Gönül Kizilsavas ◽  
Tanja Weil ◽  
Gilbert Weidinger ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Mycobacterium Tuberculosis ◽  
Developmental Toxicity ◽  
Super Resolution ◽  
In Vivo Models ◽  
Large Size ◽  
Acid Fragment ◽  
Powerful Approach ◽  
Target Effects

Granulysin is an antimicrobial peptide (AMP) expressed by human T-lymphocytes and natural killer cells. Despite a remarkably broad antimicrobial spectrum, its implementation into clinical practice has been hampered by its large size and off-target effects. To circumvent these limitations, we synthesized a 29 amino acid fragment within the putative cytolytic site of Granulysin (termed “Gran1”). We evaluated the antimicrobial activity of Gran1 against the major human pathogen Mycobacterium tuberculosis (Mtb) and a panel of clinically relevant non-tuberculous mycobacteria which are notoriously difficult to treat. Gran1 efficiently inhibited the mycobacterial proliferation in the low micro molar range. Super-resolution fluorescence microscopy and scanning electron microscopy indicated that Gran1 interacts with the surface of Mtb, causing lethal distortions of the cell wall. Importantly, Gran1 showed no off-target effects (cytokine release, chemotaxis, cell death) in primary human cells or zebrafish embryos (cytotoxicity, developmental toxicity, neurotoxicity, cardiotoxicity). Gran1 was selectively internalized by macrophages, the major host cell of Mtb, and restricted the proliferation of the pathogen. Our results demonstrate that the hypothesis-driven design of AMPs is a powerful approach for the identification of small bioactive compounds with specific antimicrobial activity. Gran1 is a promising component for the design of AMP-containing nanoparticles with selective activity and favorable pharmacokinetics to be pushed forward into experimental in vivo models of infectious diseases, most notably tuberculosis.

scholarly journals Three-dimensional in situ morphometrics of Mycobacterium tuberculosis infection within lesions by optical mesoscopy and novel acid-fast staining

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Robert J. Francis ◽  
Gillian Robb ◽  
Lee McCann ◽  
Bhagwati Khatri ◽  
James Keeble ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Three Dimensional ◽  
Large Field ◽  
Staining Procedure ◽  
3D Analysis ◽  
Mycobacterium Tuberculosis Infection ◽  
In Vivo Models ◽  
Novel Approach

AbstractTuberculosis (TB) preclinical testing relies on in vivo models including the mouse aerosol challenge model. The only method of determining colony morphometrics of TB infection in a tissue in situ is two-dimensional (2D) histopathology. 2D measurements consider heterogeneity within a single observable section but not above and below, which could contain critical information. Here we describe a novel approach, using optical clearing and a novel staining procedure with confocal microscopy and mesoscopy, for three-dimensional (3D) measurement of TB infection within lesions at sub-cellular resolution over a large field of view. We show TB morphometrics can be determined within lesion pathology, and differences in infection with different strains of Mycobacterium tuberculosis. Mesoscopy combined with the novel CUBIC Acid-Fast (CAF) staining procedure enables a quantitative approach to measure TB infection and allows 3D analysis of infection, providing a framework which could be used in the analysis of TB infection in situ.

scholarly journals Targeting redox heterogeneity to counteract drug tolerance in replicating Mycobacterium tuberculosis

2019 ◽  
Vol 11 (518) ◽  
pp. eaaw6635 ◽  
Author(s):  
Richa Mishra ◽  
Sakshi Kohli ◽  
Nitish Malhotra ◽  
Parijat Bandyopadhyay ◽  
Mansi Mehta ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Tolerance ◽  
Antibiotic Tolerance ◽  
Lung Pathology ◽  
In Vivo Models ◽  
Drug Efflux Pumps ◽  
Major Drug ◽  
Hiv 1 ◽  
Multiple Antibiotics

The capacity of Mycobacterium tuberculosis (Mtb) to tolerate multiple antibiotics represents a major problem in tuberculosis (TB) management. Heterogeneity in Mtb populations is one of the factors that drives antibiotic tolerance during infection. However, the mechanisms underpinning this variation in bacterial population remain poorly understood. Here, we show that phagosomal acidification alters the redox physiology of Mtb to generate a population of replicating bacteria that display drug tolerance during infection. RNA sequencing of this redox-altered population revealed the involvement of iron-sulfur (Fe-S) cluster biogenesis, hydrogen sulfide (H2S) gas, and drug efflux pumps in antibiotic tolerance. The fraction of the pH- and redox-dependent tolerant population increased when Mtb infected macrophages with actively replicating HIV-1, suggesting that redox heterogeneity could contribute to high rates of TB therapy failure during HIV-TB coinfection. Pharmacological inhibition of phagosomal acidification by the antimalarial drug chloroquine (CQ) eradicated drug-tolerant Mtb, ameliorated lung pathology, and reduced postchemotherapeutic relapse in in vivo models. The pharmacological profile of CQ (Cmax and AUClast) exhibited no major drug-drug interaction when coadministered with first line anti-TB drugs in mice. Our data establish a link between phagosomal pH, redox metabolism, and drug tolerance in replicating Mtb and suggest repositioning of CQ to shorten TB therapy and achieve a relapse-free cure.

IN VITRO MODELS OF MYCOBACTERIUM TUBERCULOSIS DORMANCY, AND IN VIVO MODELS OF LATENT TUBERCULOSIS INFECTION

2019 ◽  
Vol 64 (5) ◽  
pp. 299-307
Author(s):  
M. V. Fursov ◽  
I. A. Dyatlov ◽  
V. D. Potapov
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Latent Tuberculosis Infection ◽  
Latent Tuberculosis ◽  
In Vitro Models ◽  
Tuberculosis Infection ◽  
Published Data ◽  
In Vivo Models ◽  
Dormant State

Modeling of tuberculosis infection is carried out in order to clarify various aspects of the tuberculosis pathogenesis, as well as the testing of new anti-tuberculosis drugs. The characteristic of in vitro models (n = 16) for Mycobacterium tuberculosis dormant state and in vivo models (n = 14) for the latent tuberculosis infection involving several animal species published to date are presented in this review. A brief description of the models and the results obtained by the authors are presented. The analysis of the published data reflects the list of methodological procedures that allow researchers to study the mechanism of the transition of M. tuberculosis cells to a dormant state and reverse to metabolically active state, as well as the process of conversion of active tuberculosis infection to a latent tuberculosis and reactivation.

scholarly journals Preclinical Testing of the Nitroimidazopyran PA-824 for Activity against Mycobacterium tuberculosis in a Series of In Vitro and In Vivo Models

2005 ◽  
Vol 49 (6) ◽  
pp. 2294-2301 ◽  
Author(s):  
Anne J. Lenaerts ◽  
Veronica Gruppo ◽  
Karen S. Marietta ◽  
Christine M. Johnson ◽  
Diane K. Driscoll ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Bacterial Load ◽  
Multidrug Resistant ◽  
Infection Model ◽  
Significant Activity ◽  
Dependent Manner ◽  
In Vivo Models ◽  
Long Term Treatment

ABSTRACT This study extends earlier reports regarding the in vitro and in vivo efficacies of the nitroimidazopyran PA-824 against Mycobacterium tuberculosis. PA-824 was tested in vitro against a broad panel of multidrug-resistant clinical isolates and was found to be highly active against all isolates (MIC < 1 μg/ml). The activity of PA-824 against M. tuberculosis was also assessed grown under conditions of oxygen depletion. PA-824 showed significant activity at 2, 10, and 50 μg/ml, similar to that of metronidazole, in a dose-dependent manner. In a short-course mouse infection model, the efficacy of PA-824 at 50, 100, and 300 mg/kg of body weight formulated in methylcellulose or cyclodextrin/lecithin after nine oral treatments was compared with those of isoniazid, rifampin, and moxifloxacin. PA-824 at 100 mg/kg in cyclodextrin/lecithin was as active as moxifloxacin at 100 mg/kg and isoniazid at 25 mg/kg and was slightly more active than rifampin at 20 mg/kg. Long-term treatment with PA-824 at 100 mg/kg in cyclodextrin/lecithin reduced the bacterial load below 500 CFU in the lungs and spleen. No significant differences in activity between PA-824 and the other single drug treatments tested (isoniazid at 25 mg/kg, rifampin at 10 mg/kg, gatifloxacin at 100 mg/kg, and moxifloxacin at 100 mg/kg) could be observed. In summary, its good activity in in vivo models, as well as its activity against multidrug-resistant M. tuberculosis and against M. tuberculosis isolates in a potentially latent state, makes PA-824 an attractive drug candidate for the therapy of tuberculosis. These data indicate that there is significant potential for effective oral delivery of PA-824 for the treatment of tuberculosis.

Molecular Targets and Early Response Biomarkers for the Prediction of Developmental Toxicity In Vitro

2007 ◽  
Vol 35 (3) ◽  
pp. 335-342 ◽  
Author(s):  
Michael Stigson ◽  
Kim Kultima ◽  
Måns Jergil ◽  
Birger Scholz ◽  
Henrik Alm ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Developmental Toxicity ◽  
Molecular Targets ◽  
Toxicity Testing ◽  
Early Response ◽  
In Vitro Tests ◽  
In Vivo Models ◽  
Response Biomarkers

There is an urgent need for new in vitro methods to predict the potential developmental toxicity of candidate drugs in the early lead identification and optimisation process. This would lead to a reduction in the total number of animals required in full-scale developmental toxicology studies, and would improve the efficiency of drug development. However, suitable in vitro systems permitting robust high-throughput screening for this purpose, for the most part, remain to be designed. An understanding of the mechanisms involved in developmental toxicity may be essential for the validation of in vitro tests. Early response biomarkers — even a single one — could contribute to reducing assay time and facilitating automation. The use of toxicogenomics approaches to study in vitro and in vivo models in parallel may be a powerful tool in defining such mechanisms of action and the molecular targets of toxicity, and also for use in finding possible biomarkers of early response. Using valproic acid as a model substance, the use of DNA microarrays to identify teratogen-responsive genes in cell models is discussed. It is concluded that gene expression in P19 mouse embryocarcinoma cells represents a potentially suitable assay system, which could be readily used in a tiered testing system for developmental toxicity testing.

scholarly journals Antimycobacterial activity of peptide conjugate of pyridopyrimidine derivative against Mycobacterium tuberculosis in a series of in vitro and in vivo models

Tuberculosis ◽  
2015 ◽  
Vol 95 ◽  
pp. S207-S211 ◽  
Author(s):  
Kata Horváti ◽  
Bernadett Bacsa ◽  
Nóra Szabó ◽  
Kinga Fodor ◽  
Gyula Balka ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Antimycobacterial Activity ◽  
In Vivo Models

Tuberkulose - Screening

2011 ◽  
Vol 68 (7) ◽  
pp. 381-387
Author(s):  
Otto Schoch
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Interferon Gamma ◽  
Wird Eine ◽  
Interferon Gamma Release Assays

Das primäre Ziel der Aktivitäten zur bevölkerungsbezogenen Tuberkulosekontrolle ist die Identifizierung von Patienten mit sputummikroskopisch positiver Lungentuberkulose. Wenn diese Patienten umgehend therapiert werden, haben sie nicht nur eine optimale Heilungschance, sondern übertragen auch den Krankheitserreger nicht weiter auf andere Personen. Das Screening, die systematische Suche nach Tuberkulose, erfolgt in der Regel radiologisch bei der Suche nach Erkrankten, während immunologische Teste bei der Suche nach einer Infektion mit Mycobacterium tuberculosis zur Anwendung kommen. Diese Infektion, die ein erhöhtes Risiko für die Entwicklung einer Tuberkulose-Erkrankung mit sich bringt, wird im Rahmen der Umgebungsuntersuchungen oder bei Hochrisikogruppen gesucht. Neben dem traditionellen in vivo Mantoux Hauttest stehen heute die neueren in vitro Blutteste, die sogenannten Interferon Gamma Release Assays (IGRA) zur Verfügung, die unter anderem den Vorteil einer höheren Spezifität mit sich bringen, weil die verwendeten Antigene der Mykobakterien-Wand beim Impfstamm Bacille Calmitte Guerin (BCG) und bei den meisten atypischen Mykobakterien nicht vorhanden sind. Zudem kann bei Immunsupprimierten dank einer mitgeführten Positivkontrolle eine Aussage über die Wahrscheinlichkeit eines falsch negativen Testresultates gemacht werden. Bei neu diagnostizierter Infektion mit Mycobacterium tuberculosis wird eine präventive Chemotherapie mit Isoniazid während 9 Monaten durchgeführt.

Melatonin modifies tumor hypoxia and metabolism by inhibiting HIF-1α and energy metabolic pathway in the in vitro and in vivo models of breast cancer

2019 ◽  
Vol 2 (4) ◽  
pp. 83-98 ◽  
Author(s):  
André De Lima Mota ◽  
Bruna Vitorasso Jardim-Perassi ◽  
Tialfi Bergamin De Castro ◽  
Jucimara Colombo ◽  
Nathália Martins Sonehara ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Glucose Metabolism ◽  
Tumor Growth ◽  
Tumor Cells ◽  
Carbonic Anhydrases ◽  
In Vivo Models ◽  
Ca Ix ◽  
Gene And Protein Expression

Breast cancer is the most common cancer among women and has a high mortality rate. Adverse conditions in the tumor microenvironment, such as hypoxia and acidosis, may exert selective pressure on the tumor, selecting subpopulations of tumor cells with advantages for survival in this environment. In this context, therapeutic agents that can modify these conditions, and consequently the intratumoral heterogeneity need to be explored. Melatonin, in addition to its physiological effects, exhibits important anti-tumor actions which may associate with modification of hypoxia and Warburg effect. In this study, we have evaluated the action of melatonin on tumor growth and tumor metabolism by different markers of hypoxia and glucose metabolism (HIF-1α, glucose transporters GLUT1 and GLUT3 and carbonic anhydrases CA-IX and CA-XII) in triple negative breast cancer model. In an in vitro study, gene and protein expressions of these markers were evaluated by quantitative real-time PCR and immunocytochemistry, respectively. The effects of melatonin were also tested in a MDA-MB-231 xenograft animal model. Results showed that melatonin treatment reduced the viability of MDA-MB-231 cells and tumor growth in Balb/c nude mice (p <0.05). The treatment significantly decreased HIF-1α gene and protein expression concomitantly with the expression of GLUT1, GLUT3, CA-IX and CA-XII (p <0.05). These results strongly suggest that melatonin down-regulates HIF-1α expression and regulates glucose metabolism in breast tumor cells, therefore, controlling hypoxia and tumor progression. 

ANTIMICROBIAL ACTIVITY OF Aloe vera EXTRACT ON CASES OF KERATOCONJUNCTIVITISIN SHEEP (IN VIVO AND INVITRO STUDY)AND COMPARED WITH PENICILLIN –STREPTOMYCIN.

2016 ◽  
Vol 15 (2) ◽  
pp. 227-245
Author(s):  
A H.H.Al-Ahbabi ◽  
M. H. Al-Ahbab ◽  
Marwa amer ◽  
S. O. Hasson ◽  
A W.F.abed ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Aloe Vera

Programmable in vitro Co-Encapsidation of Guest Proteins Inside Protein Nanocages

2018 ◽  
Author(s):  
Noor H. Dashti ◽  
Rufika S. Abidin ◽  
Frank Sainsbury
Keyword(s):  
Self Assembly ◽  
Mammalian Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Super Resolution ◽  
Cell Engineering ◽  
Particle Analysis ◽  
Protein Cages

Bioinspired self-sorting and self-assembling systems using engineered versions of natural protein cages have been developed for biocatalysis and therapeutic delivery. The packaging and intracellular delivery of guest proteins is of particular interest for both <i>in vitro</i> and <i>in vivo</i> cell engineering. However, there is a lack of platforms in bionanotechnology that combine programmable guest protein encapsidation with efficient intracellular uptake. We report a minimal peptide anchor for <i>in vivo</i> self-sorting of cargo-linked capsomeres of the Murine polyomavirus (MPyV) major coat protein that enables controlled encapsidation of guest proteins by <i>in vitro</i> self-assembly. Using Förster resonance energy transfer (FRET) we demonstrate the flexibility in this system to support co-encapsidation of multiple proteins. Complementing these ensemble measurements with single particle analysis by super-resolution microscopy shows that the stochastic nature of co-encapsidation is an overriding principle. This has implications for the design and deployment of both native and engineered self-sorting encapsulation systems and for the assembly of infectious virions. Taking advantage of the encoded affinity for sialic acids ubiquitously displayed on the surface of mammalian cells, we demonstrate the ability of self-assembled MPyV virus-like particles to mediate efficient delivery of guest proteins to the cytosol of primary human cells. This platform for programmable co-encapsidation and efficient cytosolic delivery of complementary biomolecules therefore has enormous potential in cell engineering.

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