scholarly journals Characterization, Stability, and In Vivo Efficacy Studies of Recombinant Human CNTF and Its Permeation into the Neural Retina in Ex Vivo Organotypic Retinal Explant Culture Models

Pharmaceutics ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 611 ◽  
Author(s):  
Jaakko Itkonen ◽  
Ada Annala ◽  
Shirin Tavakoli ◽  
Blanca Arango-Gonzalez ◽  
Marius Ueffing ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Disease Model ◽  
Neural Retina ◽  
Biologically Active ◽  
Target Cells ◽  
Therapeutic Effects ◽  
Duration Of Action ◽  
In Vivo Efficacy

Ciliary neurotrophic factor (CNTF) is one of the most studied neuroprotective agents with acknowledged potential in treating diseases of the posterior eye segment. Although its efficacy and mechanisms of action in the retina have been studied extensively, it is still not comprehensively understood which retinal cells mediate the therapeutic effects of CNTF. As with therapeutic proteins in general, it is poorly elucidated whether exogenous CNTF administered into the vitreous can enter and distribute into the retina and hence reach potentially responsive target cells. Here, we have characterized our purified recombinant human CNTF (rhCNTF), studied the protein’s in vitro bioactivity in a cell-based assay, and evaluated the thermodynamic and oligomeric status of the protein during storage. Biological activity of rhCNTF was further evaluated in vivo in an animal model of retinal degeneration. The retinal penetration and distribution of rhCNTF after 24 h was studied utilizing two ex vivo retina models. Based on our characterization findings, our rhCNTF is correctly folded and biologically active. Moreover, based on initial screening and subsequent follow-up, we identified two buffers in which rhCNTF retains its stability during storage. Whereas rhCNTF did not show photoreceptor preservative effect or improve the function of photoreceptors in vivo, this could possibly be due to the used disease model or the short duration of action with a single intravitreal injection of rhCNTF. On the other hand, the lack of in vivo efficacy was shown to not be due to distribution limitations; permeation into the retina was observed in both retinal explant models as in 24 h rhCNTF penetrated the inner limiting membrane, and being mostly observed in the ganglion cell layer, distributed to different layers of the neural retina. As rhCNTF can reach deeper retinal layers, in general, having direct effects on resident CNTF-responsive target cells is plausible.

scholarly journals An in-vitro assay using human spermatozoa to detect toxicity of biologically active substances

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Tino Vollmer ◽  
Börje Ljungberg ◽  
Vera Jankowski ◽  
Joachim Jankowski ◽  
Griet Glorieux ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Toxicity Testing ◽  
Biologically Active ◽  
Human Spermatozoa ◽  
Vitro Assay ◽  
Cellular Behavior ◽  
Novel Method ◽  
Set Up

Abstract Identifying the key toxic players within an in-vivo toxic syndrome is crucial to develop targeted therapies. Here, we established a novel method that characterizes the effect of single substances by means of an ex-vivo incubation set-up. We found that primary human spermatozoa elicit a distinct motile response on a (uremic) toxic milieu. Specifically, this approach describes the influence of a bulk toxic environment (uremia) as well as single substances (uremic toxins) by real-time analyzing motile cellular behavior. We established the human spermatozoa-based toxicity testing (HSTT) for detecting single substance-induced toxicity to be used as a screening tool to identify in-vivo toxins. Further, we propose an application of the HSTT as a method of clinical use to evaluate toxin-removing interventions (hemodialysis).

T-cell redirected killing and cure of pancreatic and gastric cancer xenografts by targeting Trop-2.

2015 ◽  
Vol 33 (3_suppl) ◽  
pp. 262-262
Author(s):  
David M. Goldenberg ◽  
Edmund A. Rossi ◽  
Diane L Rossi ◽  
Thomas M. Cardillo ◽  
Chien-Hsing Chang
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Ex Vivo ◽  
Control Group ◽  
Calcium Signal ◽  
Target Cells ◽  
Normal Tissues

262 Background: Trop-2 [also called tumor-associated calcium signal transducer 2 (TACSTD2), EGP-1 (epithelial glycoprotein-1), GA733-1, or M1S1]is a 35 kDa transmembrane glycoprotein that is overexpressed relative to normal tissues in a variety of human cancers, including pancreatic and gastric carcinomas, where increased expression correlates with poor prognosis. Trop-2 appears to be more tumor-specific than the related molecule, EpCAM (Trop-1). MT110, the EpCAM antibody x CD3 bispecific T-cell engager (BiTE), is currently undergoing a Phase I study in various solid tumors, including lung, gastric, colorectal, breast, prostate, and ovarian cancers. We produced a similar T-cell redirecting bispecific tandem scFv, E1-3, using the variable domains of hRS7 (humanized anti-Trop-2 mAb) and Okt-3 (anti-CD3 mAb). Methods: T-cell activation, cytokine induction and cytotoxicity were evaluated ex vivo using PBMCs or purified T cells with human pancreatic (Capan-1 and BxPC3) and gastric (NCI-N87) cancer cell lines as target cells. In vivo activity was assayed with NCI-N87 xenografts that were inoculated s.c. in a mixture with twice the number of human PBMCs and matrigel. Results: In the presence of target cells and PBMCs, E1-3 potently induced T-cell activation, proliferation, and dose-dependent cytokine production of IL-2 (>2 ng/mL), IL-6 (>1 ng/mL), IL-10 (>7 ng/mL), TNF-α (>1 ng/mL) and IFN-γ (>50 ng/mL). In vitro, E1-3 mediated a highly potent T-cell lysis of BxPC3 [IC50=0.09(±0.04) pM], Capan-1 [IC50=1.2(±1.1) pM] and NCI-N87 [IC50=1.2(±1.2) pM] target cells. In vivo, two 50-µg doses of E1-3 given three days apart cured all of the mice (N=8) bearing NCI-N87 xenografts (P=0.0005; Log-Rank). Tumors in the control group (PBMCs only) reached the endpoint (TV>1 cm3) with a median of 39.5 days. All mice remained tumor-free in the E1-3 group at 78 days. Conclusions: Trop-2 is an attractive target for T-cell-mediated killing of pancreatic, gastric and other epithelial cancers.

scholarly journals The Delivery of Biologically Active Agents into the Nuclei of Target Cells for the Purposes of Translational Medicine

Acta Naturae ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 47-56
Author(s):  
A. S. Sobolev
Keyword(s):  
Targeted Delivery ◽  
Translational Medicine ◽  
Intracellular Transport ◽  
Biologically Active ◽  
Target Cells ◽  
Subcellular Compartment ◽  
Core Technology ◽  
Dividing Cells

Development of vehicles for the subcellular targeted delivery of biologically active agents is very promising for the purposes of translational medicine. This review summarizes the results obtained by researchers from the Laboratory of Molecular Genetics of Intracellular Transport, Institute of Gene Biology RAS, which allowed them to design the core technology: modular nanotransporters. This approach ensures high efficacy and cell specificity for different anti-cancer agents, as they are delivered into the most vulnerable subcellular compartment within the cells of interest and makes it possible for antibody mimetics to penetrate into a compartment of interest within the target cells (diving antibodies). Furthermore, polyplexes, complexes of polycationic block copolymers of DNA, have been developed and characterized. These complexes are efficient both in vitro and in vivo and demonstrate predominant transfection of actively dividing cells.

scholarly journals A Semi Rigid Novel Hydroxamate AMPED-Based Ligand for 89Zr PET Imaging

Molecules ◽  
2021 ◽  
Vol 26 (19) ◽  
pp. 5819
Author(s):  
Lisa Russelli ◽  
Francesco De Rose ◽  
Loredana Leone ◽  
Sybille Reder ◽  
Markus Schwaiger ◽  
...  
Keyword(s):  
Pet Imaging ◽  
Ex Vivo ◽  
Small Animal ◽  
Biologically Active ◽  
Small Animal Pet ◽  
Organ Distribution ◽  
Complexing Agent ◽  
Distribution Studies

In this work, we designed, developed, characterized, and investigated a new chelator and its bifunctional derivative for 89Zr labeling and PET-imaging. In a preliminary study, we synthesized two hexadentate chelators named AAZTHAS and AAZTHAG, based on the seven-membered heterocycle AMPED (6-amino-6-methylperhydro-1,4-diazepine) with the aim to increase the rigidity of the 89Zr complex by using N-methyl-N-(hydroxy)succinamide or N-methyl-N-(hydroxy)glutaramide pendant arms attached to the cyclic structure. N-methylhydroxamate groups are the donor groups chosen to efficiently coordinate 89Zr. After in vitro stability tests, we selected the chelator with longer arms, AAZTHAG, as the best complexing agent for 89Zr presenting a stability of 86.4 ± 5.5% in human serum (HS) for at least 72 h. Small animal PET/CT static scans acquired at different time points (up to 24 h) and ex vivo organ distribution studies were then carried out in healthy nude mice (n = 3) to investigate the stability and biodistribution in vivo of this new 89Zr-based complex. High stability in vivo, with low accumulation of free 89Zr in bones and kidneys, was measured. Furthermore, an activated ester functionalized version of AAZTHAG was synthesized to allow the conjugation with biomolecules such as antibodies. The bifunctional chelator was then conjugated to the human anti-HER2 monoclonal antibody Trastuzumab (Tz) as a proof of principle test of conjugation to biologically active molecules. The final 89Zr labeled compound was characterized via radio-HPLC and SDS-PAGE followed by autoradiography, and its stability in different solutions was assessed for at least 4 days.

scholarly journals Ex vivo Ikkβ ablation rescues the immunopotency of mesenchymal stromal cells from diabetics with advanced atherosclerosis

2020 ◽  
Author(s):  
Ozge Kizilay Mancini ◽  
David N Huynh ◽  
Liliane Menard ◽  
Dominique Shum-Tim ◽  
Huy Ong ◽  
...  
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Inflammatory Responses ◽  
Ex Vivo ◽  
Therapeutic Effects ◽  
Atherosclerotic Cardiovascular Disease ◽  
Myocardial Repair ◽  
Iκb Kinase

Abstract Aims Diabetes is a conventional risk factor for atherosclerotic cardiovascular disease and myocardial infarction (MI) is the most common cause of death among these patients. Mesenchymal stromal cells (MSCs) in patients with type 2 diabetes mellitus (T2DM) and atherosclerosis have impaired ability to suppress activated T-cells (i.e. reduced immunopotency). This is mediated by an inflammatory shift in MSC-secreted soluble factors (i.e. pro-inflammatory secretome) and can contribute to the reduced therapeutic effects of autologous T2DM and atherosclerosis-MSC post-MI. The signalling pathways driving the altered secretome of atherosclerosis- and T2DM-MSC are unknown. Specifically, the effect of IκB kinase β (IKKβ) modulation, a key regulator of inflammatory responses, on the immunopotency of MSCs from T2DM patients with advanced atherosclerosis has not been studied. Methods and results MSCs were isolated from adipose tissue obtained from patients with (i) atherosclerosis and T2DM (atherosclerosis+T2DM MSCs, n = 17) and (ii) atherosclerosis without T2DM (atherosclerosis MSCs, n = 17). MSCs from atherosclerosis+T2DM individuals displayed an inflammatory senescent phenotype and constitutively expressed active forms of effectors of the canonical IKKβ nuclear factor-κB transcription factors inflammatory pathway. Importantly, this constitutive pro-inflammatory IKKβ signature resulted in an altered secretome and impaired in vitro immunopotency and in vivo healing capacity in an acute MI model. Notably, treatment with a selective IKKβ inhibitor or IKKβ knockdown (KD) (clustered regularly interspaced short palindromic repeats/Cas9-mediated IKKβ KD) in atherosclerosis+T2DM MSCs reduced the production of pro-inflammatory secretome, increased survival, and rescued their immunopotency both in vitro and in vivo. Conclusions Constitutively active IKKβ reduces the immunopotency of atherosclerosis+T2DM MSC by changing their secretome composition. Modulation of IKKβ in atherosclerosis+T2DM MSCs enhances their myocardial repair ability.

scholarly journals Cataract Preventive Role of Isolated Phytoconstituents: Findings from a Decade of Research

Nutrients ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 1580 ◽  
Author(s):  
Vuanghao Lim ◽  
Edward Schneider ◽  
Hongli Wu ◽  
Iok-Hou Pang
Keyword(s):  
Ex Vivo ◽  
Critical Evaluation ◽  
In Vivo Studies ◽  
Therapeutic Effects ◽  
Exclusion Criteria ◽  
Preventive Role ◽  
Selection Of

Cataract is an eye disease with clouding of the eye lens leading to disrupted vision, which often develops slowly and causes blurriness of the eyesight. Although the restoration of the vision in people with cataract is conducted through surgery, the costs and risks remain an issue. Botanical drugs have been evaluated for their potential efficacies in reducing cataract formation decades ago and major active phytoconstituents were isolated from the plant extracts. The aim of this review is to find effective phytoconstituents in cataract treatments in vitro, ex vivo, and in vivo. A literature search was synthesized from the databases of Pubmed, Science Direct, Google Scholar, Web of Science, and Scopus using different combinations of keywords. Selection of all manuscripts were based on inclusion and exclusion criteria together with analysis of publication year, plant species, isolated phytoconstituents, and evaluated cataract activities. Scientists have focused their attention not only for anti-cataract activity in vitro, but also in ex vivo and in vivo from the review of active phytoconstituents in medicinal plants. In our present review, we identified 58 active phytoconstituents with strong anti-cataract effects at in vitro and ex vivo with lack of in vivo studies. Considering the benefits of anti-cataract activities require critical evaluation, more in vivo and clinical trials need to be conducted to increase our understanding on the possible mechanisms of action and the therapeutic effects.

scholarly journals Effect of Mesenchymal Stem Cell-Derived Exosomes on Retinal Injury: A Review of Current Findings

2020 ◽  
Vol 2020 ◽  
pp. 1-9 ◽  
Author(s):  
Raffaele Nuzzi ◽  
Paolo Caselgrandi ◽  
Alessandro Vercelli
Keyword(s):  
Current Knowledge ◽  
Target Cells ◽  
Therapeutic Effects ◽  
Retinal Damage ◽  
Phospholipid Membrane ◽  
Retinal Injury ◽  
Immunological Rejection ◽  
Reduction Effect

In recent years, various studies have followed in the literature on the therapeutic effects of mesenchymal stem cells (MSC) on damage in retinal cells. The evidence that MSCs exert their regenerative and damage reduction effect in a paracrine way, through the release of soluble factors and exosomes, is now consolidated. Exosomes are microvesicles formed by a double layer of phospholipid membrane and carry proteins and RNA, through which they play a therapeutic role on target cells. Scientific research has recently focused on the use of exosomes derived from MSC in various models of retinal damage in vitro and in vivo as they, compared to MSCs, have similar functions and at the same time have different advantages such as greater stability and handling, a lower chance of immunological rejection and no risk of malignant transformation. The purpose of this review is to summarize current knowledge on the therapeutic use of exosomes derived from MSCs in retinal damage and to stimulate new clinical perspectives regarding their use.

scholarly journals Seaweed Components as Potential Modulators of the Gut Microbiota

Marine Drugs ◽  
2021 ◽  
Vol 19 (7) ◽  
pp. 358
Author(s):  
Emer Shannon ◽  
Michael Conlon ◽  
Maria Hayes
Keyword(s):  
Gut Microbiota ◽  
Large Scale ◽  
Current Knowledge ◽  
Ex Vivo ◽  
Short Chain Fatty Acids ◽  
In Vivo Studies ◽  
Therapeutic Effects ◽  
Oral Bioaccessibility

Macroalgae, or seaweeds, are a rich source of components which may exert beneficial effects on the mammalian gut microbiota through the enhancement of bacterial diversity and abundance. An imbalance of gut bacteria has been linked to the development of disorders such as inflammatory bowel disease, immunodeficiency, hypertension, type-2-diabetes, obesity, and cancer. This review outlines current knowledge from in vitro and in vivo studies concerning the potential therapeutic application of seaweed-derived polysaccharides, polyphenols and peptides to modulate the gut microbiota through diet. Polysaccharides such as fucoidan, laminarin, alginate, ulvan and porphyran are unique to seaweeds. Several studies have shown their potential to act as prebiotics and to positively modulate the gut microbiota. Prebiotics enhance bacterial populations and often their production of short chain fatty acids, which are the energy source for gastrointestinal epithelial cells, provide protection against pathogens, influence immunomodulation, and induce apoptosis of colon cancer cells. The oral bioaccessibility and bioavailability of seaweed components is also discussed, including the advantages and limitations of static and dynamic in vitro gastrointestinal models versus ex vivo and in vivo methods. Seaweed bioactives show potential for use in prevention and, in some instances, treatment of human disease. However, it is also necessary to confirm these potential, therapeutic effects in large-scale clinical trials. Where possible, we have cited information concerning these trials.

scholarly journals iAmideV-Deep: Valine Amidation Site Prediction in Proteins Using Deep Learning and Pseudo Amino Acid Compositions

Symmetry ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 560
Author(s):  
Sheraz Naseer ◽  
Rao Faizan Ali ◽  
Amgad Muneer ◽  
Suliman Mohamed Fati
Keyword(s):  
Amino Acid ◽  
Computational Models ◽  
Ex Vivo ◽  
Biologically Active ◽  
Peptide Sequence ◽  
Post Translational Modification ◽  
Amino Acid Compositions ◽  
Efficient Alternative

Amidation is an important post translational modification where a peptide ends with an amide group (–NH2) rather than carboxyl group (–COOH). These amidated peptides are less sensitive to proteolytic degradation with extended half-life in the bloodstream. Amides are used in different industries like pharmaceuticals, natural products, and biologically active compounds. The in-vivo, ex-vivo, and in-vitro identification of amidation sites is a costly and time-consuming but important task to study the physiochemical properties of amidated peptides. A less costly and efficient alternative is to supplement wet lab experiments with accurate computational models. Hence, an urgent need exists for efficient and accurate computational models to easily identify amidated sites in peptides. In this study, we present a new predictor, based on deep neural networks (DNN) and Pseudo Amino Acid Compositions (PseAAC), to learn efficient, task-specific, and effective representations for valine amidation site identification. Well-known DNN architectures are used in this contribution to learn peptide sequence representations and classify peptide chains. Of all the different DNN based predictors developed in this study, Convolutional neural network-based model showed the best performance surpassing all other DNN based models and reported literature contributions. The proposed model will supplement in-vivo methods and help scientists to determine valine amidation very efficiently and accurately, which in turn will enhance understanding of the valine amidation in different biological processes.

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