Evaluation of colonisation resistance in stool of human donors using ex vivo, in vitro and in vivo assays

2017 ◽  
Vol 8 (2) ◽  
pp. 217-230 ◽  
Author(s):  
M.F. Galvão ◽  
R.W. Bastos ◽  
L.B. Acurcio ◽  
B.B. Nascimento ◽  
S.H.C. Sandes ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Faecal Microbiota ◽  
Healthy Human ◽  
In Vivo Assays ◽  
Enteropathogenic Bacteria ◽  
Histological Data ◽  
Lactobacillus Ruminis ◽  
Colonisation Resistance

The indigenous microbiota is the population of microorganisms normally present on the surface and mucosa of an individual, where it performs essential health functions, including the colonisation resistance (CR) against pathogens. To identify the bacteria responsible and the mechanisms involved in the CR, the germ-free (GF) animal model has been used, because in vitro studies cannot always be extrapolated to what occurs in vivo. In this study, ex vivo antagonism assays against seven enteropathogenic bacteria using stools from 15 healthy human donors confirmed that the CR showed individual variation. Using in vitro antagonism assays, 14 strains isolated from dominant faecal microbiota of donors with elevated CR were selected for mono-association in GF mice to test the in vivo antagonism against Salmonella enterica ser. Typhimurium. Mice mono-associated with Enterococcus hirae strain 8.2, Bacteroides thetaiotaomicron strain 16.2 and Lactobacillus ruminis strain 18.1 had significant reductions in faecal counts of the pathogen during the challenge. After five days of infection, the group associated with E. hirae 8.2 showed a reduction in the translocation of S. Typhimurium to the spleen, while the group associated with L. ruminis 18.1 presented an increased translocation to the liver. The histological data confirmed these results and revealed that the mice associated with E. hirae 8.2 showed fewer lesions on ileum and liver, compared to the damage caused by S. Typhimurium alone, while in mice associated with L. ruminis 18.1 there was significantly worse lesions. Concluding, from the dominant faecal microbiota from healthy human with high CR, through ex vivo, in vitro and in vivo assays, a bacterium was characterised for its high CR potential, being a candidate for probiotic use.

scholarly journals Evaluation of persistence and fate of ex vivo edited-HSC modified with donor template and Its role in correcting Sickle Cell Disease

2021 ◽  
Author(s):  
Sowmya Pattabhi ◽  
Samantha N Lotti ◽  
Mason P Berger ◽  
David J Rawlings
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Ex Vivo ◽  
Cell Disease ◽  
Gene Correction ◽  
Healthy Human ◽  
Peripheral Blood Stem Cells ◽  
Donor Template

Sickle cell disease (SCD) is caused by a single nucleotide transversion in exon 1 of the HBB gene that changes the hydrophobicity of adult globin (βA), leading to substantial morbidity and reduced lifespan. Ex vivo autologous gene editing utilizing co-delivery of a designer nuclease along with a DNA donor template allows for precise homology-directed repair (HDR). These gene corrected cells when engrafted into the bone marrow (BM) can prove to be therapeutic and serves as an alternative to HLA-matched BM transplantation. In the current study, we extensively explored the role of single stranded oligonucleotide (ssODN) and recombinant adeno-associated 6 (rAAV6) donor template delivery to introduce a codon-optimized change (E6optE) or a sickle mutation (E6V) change following Crispr/Cas9-mediated cleavage of HBB in healthy human mobilized peripheral blood stem cells (mPBSCs). We achieved efficient HDR in vitro in edited cells and observed robust human CD45+ engraftment in the BM of NBSGW mice at 16-17 weeks. Notably, recipients of ssODN-modified HSC exhibited a significantly higher proportion of HDR-modified cells within individual BM, CD34+ and CD235+ compartments of both E6optE and E6V cohorts. We further assessed key functional outcomes including RNA transcripts analysis and globin sub-type expression. Our combined findings demonstrate the capacity to achieve clinically relevant HDR in vitro and in vivo using both donor template delivery method. The use of ssODN donor template-delivery is consistently associated with higher levels of gene correction in vivo as demonstrated by sustained engraftment of HDR-modified HSC and erythroid progeny. Finally, the HDR-based globin protein expression was significantly higher in the E6V ssODN-modified animals compared to the rAAV6-modified animals confirming that the ssODN donor template delivery outperforms rAAV6-donor template delivery.

scholarly journals The safety profile of Bald’s eyesalve for the treatment of bacterial infections

2020 ◽  
Author(s):  
Blessing O Anonye ◽  
Valentine Nweke ◽  
Jessica Furner-Pardoe ◽  
Rebecca Gabrilska ◽  
Afshan Rafiq ◽  
...  
Keyword(s):  
Topical Application ◽  
Safety Profile ◽  
Bacterial Infections ◽  
Ex Vivo ◽  
Corneal Opacity ◽  
Healthy Human ◽  
Patch Tests ◽  
Human Volunteers

AbstractThe rise in antimicrobial resistance has prompted the development of alternatives, such as plant-derived compounds, to combat bacterial infections. Bald’s eyesalve, a remedy used in the Early Medieval period, has previously been shown to have efficacy against Staphylococcus aureus grown in an in vitro model of soft tissue infection. This remedy also had bactericidal activity against methicillin-resistant S. aureus (MRSA) in a chronic mouse wound. However, the safety profile of Bald’s eyesalve has not yet been demonstrated, and this is vital before testing in humans. Here, we determined the safety potential of Bald’s eyesalve using in vitro, ex vivo, and in vivo models representative of skin or eye infections. We also confirmed that Bald’s eyesalve is active against an important eye pathogen, Neisseria gonorrhoeae. Low levels of cytotoxicity were observed in eyesalve-treated cell lines representative of skin and immune cells. Results from a bovine corneal opacity and permeability test demonstrated slight irritation to the cornea that resolved within 10 minutes. The slug mucosal irritation assay revealed that a low level of mucus was secreted by slugs exposed to eyesalve, indicating mild mucosal irritation. We obtained promising results from mouse wound closure experiments; no visible signs of irritation or inflammation were observed. Our results suggest that Bald’s eyesalve could be tested further on human volunteers to assess safety for topical application against bacterial infections.ImportanceAlternative treatment for bacterial infections are needed to combat the ever increasing repertoire of bacteria resistant to antibiotics. A medieval plant-based remedy, Bald’s eyesalve, shows promise as a substitute for the treatment of these infections. For any substance to be effective in the treatment of bacterial infections in humans, it is important to consider the safety profile. This is a key consideration in order to have the necessary regulatory approval. We demonstrate the safety profile of Bald’s eyesalve using a variety of models, including whole-organ and whole-animal models. Our results show that Bald’s eyesalve is mildly toxic to cultured human cells, but potentially suitable for patch tests on healthy human volunteers to assess safety for later clinical trials. Our work has the potential to transform the management of diseases caused by bacterial infections, such as diabetic foot ulcers, through topical application of a natural product cocktail based on Bald’s eyesalve.

Potency comparison in in vitro, ex vivo, and in vivo assays of commercially available botulinum neurotoxin serotypes A1, B1, and F1

Toxicon ◽  
2018 ◽  
Vol 156 ◽  
pp. S25
Author(s):  
Sarah Donald ◽  
Mark Elliott ◽  
Bryony Gray ◽  
Fraser Hornby ◽  
Agnieszka Lewandowska ◽  
...  
Keyword(s):  
Botulinum Neurotoxin ◽  
Ex Vivo ◽  
In Vivo Assays

scholarly journals In vivo NAD assay reveals the intracellular NAD contents and redox state in healthy human brain and their age dependences

2015 ◽  
Vol 112 (9) ◽  
pp. 2876-2881 ◽  
Author(s):  
Xiao-Hong Zhu ◽  
Ming Lu ◽  
Byeong-Yeul Lee ◽  
Kamil Ugurbil ◽  
Wei Chen
Keyword(s):  
Human Brain ◽  
Redox State ◽  
Ex Vivo ◽  
Healthy Human ◽  
Nad Metabolism ◽  
Mitochondrial Functions ◽  
Age Dependent ◽  
Nadh Redox State

NAD is an essential metabolite that exists in NAD+or NADH form in all living cells. Despite its critical roles in regulating mitochondrial energy production through the NAD+/NADH redox state and modulating cellular signaling processes through the activity of the NAD+-dependent enzymes, the method for quantifying intracellular NAD contents and redox state is limited to a few in vitro or ex vivo assays, which are not suitable for studying a living brain or organ. Here, we present a magnetic resonance (MR) -based in vivo NAD assay that uses the high-field MR scanner and is capable of noninvasively assessing NAD+and NADH contents and the NAD+/NADH redox state in intact human brain. The results of this study provide the first insight, to our knowledge, into the cellular NAD concentrations and redox state in the brains of healthy volunteers. Furthermore, an age-dependent increase of intracellular NADH and age-dependent reductions in NAD+, total NAD contents, and NAD+/NADH redox potential of the healthy human brain were revealed in this study. The overall findings not only provide direct evidence of declined mitochondrial functions and altered NAD homeostasis that accompany the normal aging process but also, elucidate the merits and potentials of this new NAD assay for noninvasively studying the intracellular NAD metabolism and redox state in normal and diseased human brain or other organs in situ.

The Role of Polyphenols in the Modulation of Plasma Non-Enzymatic Antioxidant Capacity (NEAC)

2012 ◽  
Vol 82 (3) ◽  
pp. 228-232 ◽  
Author(s):  
Mauro Serafini ◽  
Giuseppa Morabito
Keyword(s):  
Antioxidant Capacity ◽  
Dietary Intervention ◽  
Ex Vivo ◽  
The Body ◽  
Dietary Polyphenols ◽  
Enzymatic Antioxidant ◽  
Endogenous Antioxidant

Dietary polyphenols have been shown to scavenge free radicals, modulating cellular redox transcription factors in different in vitro and ex vivo models. Dietary intervention studies have shown that consumption of plant foods modulates plasma Non-Enzymatic Antioxidant Capacity (NEAC), a biomarker of the endogenous antioxidant network, in human subjects. However, the identification of the molecules responsible for this effect are yet to be obtained and evidences of an antioxidant in vivo action of polyphenols are conflicting. There is a clear discrepancy between polyphenols (PP) concentration in body fluids and the extent of increase of plasma NEAC. The low degree of absorption and the extensive metabolism of PP within the body have raised questions about their contribution to the endogenous antioxidant network. This work will discuss the role of polyphenols from galenic preparation, food extracts, and selected dietary sources as modulators of plasma NEAC in humans.

Significance of Platelet β-Adrenoceptors for Platelet Responses In Vivo and In Vitro

1992 ◽  
Vol 68 (06) ◽  
pp. 687-693 ◽  
Author(s):  
P T Larsson ◽  
N H Wallén ◽  
A Martinsson ◽  
N Egberg ◽  
P Hjemdahl
Keyword(s):  
Ex Vivo ◽  
High Dose ◽  
Platelet Aggregability ◽  
Adrenoceptor Agonist ◽  
Dose Infusion ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Factor Antigen

SummaryThe significance of platelet β-adrenoceptors for platelet responses to adrenergic stimuli in vivo and in vitro was studied in healthy volunteers. Low dose infusion of the β-adrenoceptor agonist isoprenaline decreased platelet aggregability in vivo as measured by ex vivo filtragometry. Infusion of adrenaline, a mixed α- and β-adrenoceptor agonist, increased platelet aggregability in vivo markedly, as measured by ex vivo filtragometry and plasma β-thromboglobulin levels. Adrenaline levels were 3–4 nM in venous plasma during infusion. Both adrenaline and high dose isoprenaline elevated plasma von Willebrand factor antigen levels β-Blockade by propranolol did not alter our measures of platelet aggregability at rest or during adrenaline infusions, but inhibited adrenaline-induced increases in vWf:ag. In a model using filtragometry to assess platelet aggregability in whole blood in vitro, propranolol enhanced the proaggregatory actions of 5 nM, but not of 10 nM adrenaline. The present data suggest that β-adrenoceptor stimulation can inhibit platelet function in vivo but that effects of adrenaline at high physiological concentrations are dominated by an α-adrenoceptor mediated proaggregatory action.

In Vivo Effect of Suloctidil as an Antiplatelet Agent

1979 ◽  
Vol 41 (03) ◽  
pp. 465-474 ◽  
Author(s):  
Marcia R Stelzer ◽  
Thomas S Burns ◽  
Robert N Saunders
Keyword(s):  
Ex Vivo ◽  
Antiplatelet Agent ◽  
Ratio Method ◽  
Platelet Aggregate ◽  
Permanent Effect ◽  
Platelet Aggregates ◽  
5 Ht Uptake ◽  
High Doses

SummaryThe relationship between the effects of suloctidil in vivo as an antiplatelet agent and in vitro as a modifier of platelet serotonin (5-HT) parameters was investigated. Suloctidil was found to be effective in reducing platelet aggregates formation in the retired breeder rat as determined using the platelet aggregate ratio method (PAR) with an ED50 of 16.1 mg/kg 24 hours post administration. In contrast to the hypothesis that 5-HT depletion is involved in the anti-aggregatory mechanism of suloctidil, no correlation was found between platelet 5- HT content and this antiplatelet activity. Reduction of platelet 5-HT content required multiple injections of high doses (100 mg/kg/day) of suloctidil. Suloctidil administration for 8 days at 100 mg/kg/day, which lowered platelet 5-HT content by 50%, resulted in no permanent effect on ex vivo platelet 5-HT uptake or thrombin-induced release, nor alteration in the plasma 5-HT level. However, these platelets exhibited a short-lived, significant increase in percent leakage of 5-HT after 30 minutes of incubation. Therefore, suloctidil treatment at high doses may with time result in platelet 5-HT depletion, however this effect is probably not related to the primary anti-aggregatory activity of the drug.

A Comparative Ex Vivo Study of Plasminogen Activators and Proteases for Fibrinolytic Activity and Side Effects in Rabbits

1977 ◽  
Vol 37 (01) ◽  
pp. 154-161 ◽  
Author(s):  
B. A Janik ◽  
S. E Papaioannou
Keyword(s):  
Side Effects ◽  
Effective Dose ◽  
Fibrinolytic Activity ◽  
Ex Vivo ◽  
Plasminogen Activators ◽  
Assay System ◽  
Coagulation Parameters ◽  
Ex Vivo Assay

SummaryUrokinase, streptokinase, Brinase, trypsin, and SN 687, a bacterial exoprotease, have been evaluated in an ex vivo assay system. These enzymes were injected into rabbits and the fibrinolytic activity as well as other coagulation parameters were measured by in vitro techniques. Dose-response correlations have been made using the euglobulin lysis time as a measure of fibrinolytic activity and the 50% effective dose has been determined for each enzyme. Loading doses, equal to four times the 50% effective dose, were administered to monitor potential toxicity revealing that Brinase, trypsin, and SN 687 were very toxic at this concentration.Having established the 50% effective dose for each enzyme, further testing was conducted where relevant fibrinolytic and coagulation parameters were measured for up to two days following a 50% effective dose bolus injection of each enzyme. Our results have demonstrated that urokinase and streptokinase are plasminogen activators specifically activating the rabbit fibrinolytic system while Brinase, trypsin and SN 687 increase the general proteolytic activity in vivo.The advantages of this ex vivo assay system for evaluating relative fibrinolytic potencies and side effects for plasminogen activators and fibrinolytic proteases have been discussed.

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