scholarly journals Expedited gene delivery for osteochondral defect repair in a rabbit knee model: a one-year investigation

2021 ◽  
Author(s):  
Christopher V. Nagelli ◽  
Rodolfo E. De La Vega ◽  
Michael Coenen ◽  
Consuelo Padilla de Lopez ◽  
Joesph A. Panos ◽  
...  
Keyword(s):  
Transforming Growth Factor ◽  
Fluorescent Protein ◽  
Single Step ◽  
Transduction Efficiency ◽  
Osteochondral Lesions ◽  
In Vitro Experiments ◽  
Fibrin Gel ◽  
Fibrin Gels ◽  
Press Fit

Objective: To evaluate a single-step, gene-based procedure for repairing osteochondral lesions. Design: Osteochondral lesions were created in the patellar groove of skeletally mature rabbits. Autologous bone marrow aspirates were mixed with adenovirus vectors carrying cDNA encoding green fluorescent protein (Ad.GFP) or transforming growth factor-β (Ad.TGF-β) and allowed to clot. The clotted marrow was press-fit into the defects. Animals receiving Ad.GFP were euthanized at 2 weeks and intra-articular expression of GFP examined by fluorescence microscopy. Animals receiving Ad.TGF-β were euthanized at 3 months and 12 months; repair was compared to empty defects using histology and immunohistochemistry. Complementary in vitro experiments assessed transgene expression and chondrogenesis in marrow clots and fibrin gels. In a subsequent pilot study, repair at 3 months using a fibrin gel to encapsulate Ad.TGF-β was evaluated. Results: At 2 weeks, GFP expression was seen at variable levels within the cartilaginous lesion. At 3 months, there was a statistically significant improvement in healing of lesions receiving Ad.TGF-β, although variability was high. At 12 months, there was no difference between the empty defects and those receiving Ad.TGF-β in overall score and cartilage score, but the bone healing score remained higher. Variability was again high. In vitro experiments suggested that variability reflected variable transduction efficiency and chondrogenic activity of the marrow clots; using fibrin gels instead of marrow provided more uniformity in healing. Conclusions: This approach to improving the repair of osteochondral lesions holds promise but needs further refinement to reduce variability and provide a more robust outcome.

scholarly journals Helminth secretions induce de novo T cell Foxp3 expression and regulatory function through the TGF-β pathway

2010 ◽  
Vol 207 (11) ◽  
pp. 2331-2341 ◽  
Author(s):  
John R. Grainger ◽  
Katie A. Smith ◽  
James P. Hewitson ◽  
Henry J. McSorley ◽  
Yvonne Harcus ◽  
...  
Keyword(s):  
Transforming Growth Factor ◽  
Fluorescent Protein ◽  
De Novo ◽  
Foxp3 Expression ◽  
Dominant Negative ◽  
Regulatory Function ◽  
Intestinal Helminth ◽  
Helminth Parasites

Foxp3-expressing regulatory T (T reg) cells have been implicated in parasite-driven inhibition of host immunity during chronic infection. We addressed whether parasites can directly induce T reg cells. Foxp3 expression was stimulated in naive Foxp3− T cells in mice infected with the intestinal helminth Heligmosomoides polygyrus. In vitro, parasite-secreted proteins (termed H. polygyrus excretory-secretory antigen [HES]) induced de novo Foxp3 expression in fluorescence-sorted Foxp3− splenocytes from Foxp3–green fluorescent protein reporter mice. HES-induced T reg cells suppressed both in vitro effector cell proliferation and in vivo allergic airway inflammation. HES ligated the transforming growth factor (TGF) β receptor and promoted Smad2/3 phosphorylation. Foxp3 induction by HES was lost in dominant-negative TGF-βRII cells and was abolished by the TGF-β signaling inhibitor SB431542. This inhibitor also reduced worm burdens in H. polygyrus–infected mice. HES induced IL-17 in the presence of IL-6 but did not promote Th1 or Th2 development under any conditions. Importantly, antibody to mammalian TGF-β did not recognize HES, whereas antisera that inhibited HES did not affect TGF-β. Foxp3 was also induced by secreted products of Teladorsagia circumcincta, a related nematode which is widespread in ruminant animals. We have therefore identified a novel pathway through which helminth parasites may stimulate T reg cells, which is likely to be a key part of the parasite’s immunological relationship with the host.

scholarly journals Self-complementarity in adeno-associated virus enhances transduction and gene expression in mouse cochlear tissues

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0242599
Author(s):  
Graham Casey ◽  
Charles Askew ◽  
Mark A. Brimble ◽  
R. Jude Samulski ◽  
Andrew M. Davidoff ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Transduction Efficiency ◽  
Sensory Cells ◽  
Adeno Associated Virus ◽  
Large Numbers ◽  
Green Fluorescent ◽  
Interest In Research ◽  
Tyrphostin 23

Sensorineural hearing loss is one of the most common disabilities worldwide. Such prevalence necessitates effective tools for studying the molecular workings of cochlear cells. One prominent and effective vector for expressing genes of interest in research models is adeno-associated virus (AAV). However, AAV efficacy in transducing cochlear cells can vary for a number of reasons including serotype, species, and methodology, and oftentimes requires high multiplicity of infection which can damage the sensory cells. Reports in other systems suggest multiple approaches can be used to enhance AAV transduction including self-complementary vector design and pharmacological inhibition of degradation. Here we produced AAV to drive green fluorescent protein (GFP) expression in explanted neonatal mouse cochleae. Treatment with eeyarestatin I, tyrphostin 23, or lipofectamine 2000 did not result in increased transduction, however, self-complementary vector design resulted in significantly more GFP positive cells when compared to single-stranded controls. Similarly, self-complementary AAV2 vectors demonstrated enhanced transduction efficiency compared to single stranded AAV2 when injected via the posterior semicircular canal, in vivo. Self-complementary vectors for AAV1, 8, and 9 serotypes also demonstrated robust GFP transduction in cochlear cells in vivo, though these were not directly compared to single stranded vectors. These findings suggest that second-strand synthesis may be a rate limiting step in AAV transduction of cochlear tissues and that self-complementary AAV can be used to effectively target large numbers of cochlear cells in vitro and in vivo.

scholarly journals Extracellular Vesicles-Loaded Fibrin Gel Supports Rapid Neovascularization for Dental Pulp Regeneration

2020 ◽  
Vol 21 (12) ◽  
pp. 4226
Author(s):  
Siyuan Zhang ◽  
Anja Lena Thiebes ◽  
Franziska Kreimendahl ◽  
Stephan Ruetten ◽  
Eva Miriam Buhl ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Delivery System ◽  
Dental Pulp ◽  
Fibrin Gel ◽  
Cell Growth And Migration ◽  
Fibrin Gels ◽  
Initial Stage ◽  
And Migration ◽  
Induced Apoptosis

Rapid vascularization is required for the regeneration of dental pulp due to the spatially restricted tooth environment. Extracellular vesicles (EVs) released from mesenchymal stromal cells show potent proangiogenic effects. Since EVs suffer from rapid clearance and low accumulation in target tissues, an injectable delivery system capable of maintaining a therapeutic dose of EVs over a longer period would be desirable. We fabricated an EV-fibrin gel composite as an in situ forming delivery system. EVs were isolated from dental pulp stem cells (DPSCs). Their effects on cell proliferation and migration were monitored in monolayers and hydrogels. Thereafter, endothelial cells and DPSCs were co-cultured in EV-fibrin gels and angiogenesis as well as collagen deposition were analyzed by two-photon laser microscopy. Our results showed that EVs enhanced cell growth and migration in 2D and 3D cultures. EV-fibrin gels facilitated vascular-like structure formation in less than seven days by increasing the release of VEGF. The EV-fibrin gel promoted the deposition of collagen I, III, and IV, and readily induced apoptosis during the initial stage of angiogenesis. In conclusion, we confirmed that EVs from DPSCs can promote angiogenesis in an injectable hydrogel in vitro, offering a novel and minimally invasive strategy for regenerative endodontic therapy.

Survival Analysis of Escherichia coli Encapsulated in a Hollow Fiber Membrane In Vitro and In Vivo: Preliminary Report

2005 ◽  
Vol 14 (5) ◽  
pp. 323-330 ◽  
Author(s):  
L. H. Granicka ◽  
M. Wdowiak ◽  
A. Kosek ◽  
S. Świezewski ◽  
D. Wasilewska ◽  
...  
Keyword(s):  
Hollow Fiber ◽  
Fluorescent Protein ◽  
Culture Media ◽  
Hollow Fiber Membrane ◽  
In Vitro Experiments ◽  
Bacteria Growth ◽  
E Coli ◽  
Green Fluorescent

The purpose of the observations was the viability and quality evaluation of E. coli bacteria encapsulated in hollow fiber membranes (HF) in short in vivo and in vitro experiments. A polypropylene, surface-modified hollow fiber was applied for immunoisolation of E. coli bacteria transfected with a green fluorescent protein (E. coli GFPI). The presence of GFP fluorescence of organisms was assessed with the use of flow cytometry. The E. coli GFPIs were then observed for the period of 5 days in in vitro experiments in the culture medium. A single IPTG (isopropyl β-D-1-thiogalactopyranoside) induction of GFP gene appeared to be adequate for an expression of GFP protein for 5 days. The GFP expression values observed for E. coli GFPs encapsulated in HF during culture in different culture media were comparable. The survival of E. coli GFPIs encapsulated in HF after 1, 2, 4, or 5 days of subcutaneous implantation into mice was evaluated. The explanted E. coli GFPIs exhibited mean expression 603 ± 17 (n = 32) units of fluorescence during the implantation period. The values obtained were comparable for selected days of observation. It was observed that the membranes applied ensured the bacteria growth within the HF's space only.

Ultrasound Increases Flow through Fibrin Gels

1995 ◽  
Vol 73 (03) ◽  
pp. 495-498 ◽  
Author(s):  
Farhan Siddiqi ◽  
Ales Blinc ◽  
Julie Braaten ◽  
Charles W Francis
Keyword(s):  
Tube Wall ◽  
Fibrin Gel ◽  
Fibrinolytic Enzymes ◽  
Fibrin Gels ◽  
Ultrasound Field ◽  
Flow Through ◽  
Induced Flow ◽  
Induced Changes ◽  
Measured Exposure

SummaryUltrasound accelerates fibrinolysis in vitro and in animal models of thrombosis. Since transport of fibrinolytic enzymes into clots by permeation may be an important determinant of the rate of fibrinolysis, we examined the effect of ultrasound on permeation through fibrin gels in vitro. Gels of purified fibrin were prepared in plastic tubes, and the rate of pressure-mediated fluid permeation was measured. Exposure to 1 MHz ultrasound at 2 W/cm2 and a duty cycle of 5 msec on, 5 msec off resulted in a significant (p = .005) increase in flow through the gel of 29.0 ± 4.2% (SEM). The ultrasound-induced flow increase was intensity-dependent, increasing from 17.0 ± 1.2% at 1 W/cm2 to 30.1 ± 1.9% at 2.3 W/cm2. Increased flow was not due to heating, detachment of fibrin from the tube wall or fragmentation of the gel resulting in channeling. However, degassing the fluid by autoclaving significantly reduced the ultrasound-induced increase in flow. We conclude that exposure of fibrin gels to ultrasound increases pressure-mediated permeation. This effect may be related to cavitation-induced changes in fibrin gel structure, and could contribute to the accelerated fibrinolysis observed in an ultrasound field.

Lentiviral-Mediated RNAi Knockdown of SBDS in Murine Hematopoietic Progenitors Impairs Their Engraftment Potential and Granulocytic Differentiation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 635-635
Author(s):  
Amy S. Rawls ◽  
Jill R. Woloszynek ◽  
Daniel C. Link
Keyword(s):  
Fluorescent Protein ◽  
Lentiviral Vector ◽  
Yellow Fluorescent Protein ◽  
Cathepsin G ◽  
Transduction Efficiency ◽  
Hematopoietic Progenitors ◽  
Granulocytic Differentiation ◽  
Post Transplantation ◽  
Exocrine Insufficiency

Abstract Shwachman-Bodian Diamond Syndrome (SDS) is an autosomal recessive disorder characterized by congenital neutropenia, pancreatic exocrine insufficiency and bony abnormalities. About 90% of SDS patients have mutations in the novel SBDS gene. The SBDS protein is conserved from archaea to mammals; however, the cellular functions of SBDS, as well as its contribution to the molecular pathogenesis of this disease, are not yet known. In this study, we developed an RNAi knockdown model to investigate the consequences of a reduction in SBDS protein on hematopoiesis. Three lentiviral RNAi constructs directed against SBDS were generated and tested in BAF3 cells, a murine pro-B cell line. SBDS protein reduction of 78 ± 11%, 63 ± 14%, and 27 ± 6% was observed. The most efficient lentiviral SBDS RNAi construct was used to transduce primary murine hematopoietic cells, with knockdown similar to that observed in BAF3 cells. In addition, empty lentiviral and Cathepsin G (CG) RNAi lentiviral constructs were studied to control for non-specific effects of lentivirus infection and/or induction of the RNAi machinery. In each case, the lentiviral vector contained a yellow fluorescent protein (YFP) expression cassette to track transduced cells. Murine hematopoietic progenitors (Kit+, Lin-) were transduced with these lentiviral vectors and transplanted into irradiated syngeneic recipients. Initially, a high multiplicity of infection (MOI) was used, resulting in a transduction efficiency of ~75%. Interestingly, 5/5 animals transplanted with SBDS RNAi transduced cells died by 19 days post-transplantation due to engraftment failure, compared to 0/5 animals transplanted with empty lentivirus transduced cells. To study the long-term consequence of SBDS knockdown, cells were transduced at a lower MOI, resulting in a transduction efficiency of ~15%. Engraftment of SBDS RNAi cells (N=8) was modestly (3-fold), yet consistently, reduced compared to both empty lentivirus (N=8) and CG RNAi (N=5) controls. However, once engraftment of SBDS RNAi cells was established, the percentage of transduced (YFP+) cells remained stable for at least 3 months. Moreover, there was no evidence of lineage skewing, as a similar percentage of YFP+ cells was observed in all hematopoietic lineages. Importantly, the SBDS knockdown effect was maintained in YFP+ cells recovered from mice 3 months post-transplantation (~70% reduction in SBDS expression). Neutropenia is the most common hematopoietic abnormality in patients with SDS. Thus, we also characterized the effect of reduced SBDS function on granulocytic differentiation. Specifically, the ability of SBDS RNAi transduced Kit+, Lin- progenitors to generate hematopoietic colonies and differentiate into mature granulocytes was assessed. No defect in the ability of transduced progenitors to form CFU-GM or CFU-G was observed. However, a modest, yet significant, delay in neutrophil maturation was observed. Mature neutrophils constitute only 34.6 ± 7.3% of SBDS RNAi granulocyte cultures by day 7, compared to 59.7 ± 6.9% and 60.1 ± 12.9% in empty lentivirus and CG RNAi cultures, respectively. In summary, we established an RNAi model for SDS and have shown that a 70% reduction in SBDS protein levels in hematopoietic progenitors is sufficient for (i) impaired engraftment upon bone marrow transplantation and (ii) modest delay in neutrophil maturation in vitro. These findings provide the first experimental evidence that reduced SBDS function may drive the hematopoietic defects seen in SDS patients.

scholarly journals ABCG2pos lung mesenchymal stem cells are a novel pericyte subpopulation that contributes to fibrotic remodeling

2014 ◽  
Vol 307 (8) ◽  
pp. C684-C698 ◽  
Author(s):  
Shennea Marriott ◽  
Rubin S. Baskir ◽  
Christa Gaskill ◽  
Swapna Menon ◽  
Erica J. Carrier ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Transforming Growth Factor ◽  
Fluorescent Protein ◽  
Intratracheal Administration ◽  
Adult Lung ◽  
Inflammatory Phase ◽  
Tgf Β1

Genesis of myofibroblasts is obligatory for the development of pathology in many adult lung diseases. Adult lung tissue contains a population of perivascular ABCG2pos mesenchymal stem cells (MSC) that are precursors of myofibroblasts and distinct from NG2 pericytes. We hypothesized that these MSC participate in deleterious remodeling associated with pulmonary fibrosis (PF) and associated hypertension (PH). To test this hypothesis, resident lung MSC were quantified in lung samples from control subjects and PF patients. ABCG2pos cell numbers were decreased in human PF and interstitial lung disease compared with control samples. Genetic labeling of lung MSC in mice enabled determination of terminal lineage and localization of ABCG2 cells following intratracheal administration of bleomycin to elicit fibrotic lung injury. Fourteen days following bleomycin injury enhanced green fluorescent protein (eGFP)-labeled lung MSC-derived cells were increased in number and localized to interstitial areas of fibrotic and microvessel remodeling. Finally, gene expression analysis was evaluated to define the response of MSC to bleomycin injury in vivo using ABCG2pos MSC isolated during the inflammatory phase postinjury and in vitro bleomycin or transforming growth factor-β1 (TGF-β1)-treated cells. MSC responded to bleomycin treatment in vivo with a profibrotic gene program that was not recapitulated in vitro with bleomycin treatment. However, TGF-β1 treatment induced the appearance of a profibrotic myofibroblast phenotype in vitro. Additionally, when exposed to the profibrotic stimulus, TGF-β1, ABCG2, and NG2 pericytes demonstrated distinct responses. Our data highlight ABCG2pos lung MSC as a novel cell population that contributes to detrimental myofibroblast-mediated remodeling during PF.

Neurofilaments and Paired Helical Filaments

1985 ◽  
Vol 43 ◽  
pp. 740-743
Author(s):  
J. Metuzals
Keyword(s):  
Animal Model ◽  
Posttranslational Modifications ◽  
Posttranslational Modification ◽  
Giant Axon ◽  
Paired Helical Filaments ◽  
Squid Giant Axon ◽  
In Vitro Experiments ◽  
Thin Sections ◽  
Structural Relationships

It has been demonstrated that the neurofibrillary tangles in biopsies of Alzheimer patients, composed of typical paired helical filaments (PHF), consist also of typical neurofilaments (NF) and 15nm wide filaments. Close structural relationships, and even continuity between NF and PHF, have been observed. In this paper, such relationships are investigated from the standpoint that the PHF are formed through posttranslational modifications of NF. To investigate the validity of the posttranslational modification hypothesis of PHF formation, we have identified in thin sections from frontal lobe biopsies of Alzheimer patients all existing conformations of NF and PHF and ordered these conformations in a hypothetical sequence. However, only experiments with animal model preparations will prove or disprove the validity of the interpretations of static structural observations made on patients. For this purpose, the results of in vitro experiments with the squid giant axon preparations are compared with those obtained from human patients. This approach is essential in discovering etiological factors of Alzheimer's disease and its early diagnosis.

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