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Journal of Extracellular Biology Mar 2024Extracellular vesicles (EVs) are intensively investigated for their therapeutic potential and application as drug delivery vehicle. A broad perception of favourable...
Extracellular vesicles (EVs) are intensively investigated for their therapeutic potential and application as drug delivery vehicle. A broad perception of favourable safety profiles and low immunogenicity make EVs an attractive alternative to synthetic nanoparticles. We recently showed that repeated intravenous administration of human cell-derived EVs into pig-tailed macaques unexpectedly elicited antibody responses after three or more injections. This coincided with decreasing EV circulation time, and may thus hamper successful EV-mediated cargo delivery into tissues. Here, we share the custom ELISA protocol that we used to measure such antibody responses. This protocol may help other researchers evaluate immune responses to EV-based therapies in preclinical studies.
PubMed: 38939411
DOI: 10.1002/jex2.129 -
Open Veterinary Journal May 2024Canine prostatic carcinoma (cPC) is a urogenital tumour with a poor prognosis, for which no effective treatment has been established. Recently, it has been shown that...
BACKGROUND
Canine prostatic carcinoma (cPC) is a urogenital tumour with a poor prognosis, for which no effective treatment has been established. Recently, it has been shown that human epidermal growth factor receptor type 2 (HER2) is overexpressed in cPC cells; however, the efficacy of HER2-targeted therapy remains unclear.
AIM
Investigate the anti-tumour effect of lapatinib on HER2-positive cPC cell lines.
METHODS
Two cell lines (muPC and bePC) were established from two dogs with cPC and the effects of lapatinib treatment on cell proliferation, apoptosis, and HER2 downstream signalling were investigated. Furthermore, muPC was used to generate tumour-bearing mice, and the anti-tumour effects of lapatinib were examined .
RESULTS
Lapatinib treatment inhibited the proliferation and phosphorylation of Erk1/2 and Akt, which are downstream signals of HER2. Furthermore, the TUNEL assay showed that lapatinib induced apoptosis in both cell lines. The muPC-engrafted nude mouse model showed that lapatinib significantly inhibited tumour growth and increased the area of necrotic tumour tissue compared to the vehicle-treated groups.
CONCLUSION
Lapatinib exerts anti-tumour effects on cPC cells by inhibiting HER-2 signalling.
Topics: Lapatinib; Animals; Dogs; Male; Cell Line, Tumor; Dog Diseases; Prostatic Neoplasms; Antineoplastic Agents; Receptor, ErbB-2; Mice; Mice, Nude; Apoptosis; Cell Proliferation; Quinazolines
PubMed: 38938437
DOI: 10.5455/OVJ.2024.v14.i5.21 -
Journal of Neuroinflammation Jun 2024Traumatic brain injury (TBI) is a significant risk factor for Alzheimer's disease (AD), and accumulating evidence supports a role for adaptive immune B and T cells in...
Traumatic brain injury alters the effects of class II invariant peptide (CLIP) antagonism on chronic meningeal CLIP + B cells, neuropathology, and neurobehavioral impairment in 5xFAD mice.
BACKGROUND
Traumatic brain injury (TBI) is a significant risk factor for Alzheimer's disease (AD), and accumulating evidence supports a role for adaptive immune B and T cells in both TBI and AD pathogenesis. We previously identified B cell and major histocompatibility complex class II (MHCII)-associated invariant chain peptide (CLIP)-positive B cell expansion after TBI. We also showed that antagonizing CLIP binding to the antigen presenting groove of MHCII after TBI acutely reduced CLIP + splenic B cells and was neuroprotective. The current study investigated the chronic effects of antagonizing CLIP in the 5xFAD Alzheimer's mouse model, with and without TBI.
METHODS
12-week-old male wild type (WT) and 5xFAD mice were administered either CLIP antagonist peptide (CAP) or vehicle, once at 30 min after either sham or a lateral fluid percussion injury (FPI). Analyses included flow cytometric analysis of immune cells in dural meninges and spleen, histopathological analysis of the brain, magnetic resonance diffusion tensor imaging, cerebrovascular analysis, and assessment of motor and neurobehavioral function over the ensuing 6 months.
RESULTS
9-month-old 5xFAD mice had significantly more CLIP + B cells in the meninges compared to age-matched WT mice. A one-time treatment with CAP significantly reduced this population in 5xFAD mice. Importantly, CAP also improved some of the immune, histopathological, and neurobehavioral impairments in 5xFAD mice over the ensuing six months. Although FPI did not further elevate meningeal CLIP + B cells, it did negate the ability of CAP to reduce meningeal CLIP + B cells in the 5xFAD mice. FPI at 3 months of age exacerbated some aspects of AD pathology in 5xFAD mice, including further reducing hippocampal neurogenesis, increasing plaque deposition in CA3, altering microgliosis, and disrupting the cerebrovascular structure. CAP treatment after injury ameliorated some but not all of these FPI effects.
Topics: Animals; Mice; Mice, Transgenic; Male; Brain Injuries, Traumatic; Histocompatibility Antigens Class II; Antigens, Differentiation, B-Lymphocyte; B-Lymphocytes; Meninges; Amyloid beta-Protein Precursor; Alzheimer Disease; Humans; Disease Models, Animal; Presenilin-1; Mice, Inbred C57BL
PubMed: 38937750
DOI: 10.1186/s12974-024-03146-z -
PloS One 2024To investigate the therapeutic effect and mechanism of sivelestat sodium on acute lung injury (AIL).
OBJECTIVE
To investigate the therapeutic effect and mechanism of sivelestat sodium on acute lung injury (AIL).
METHODS
A rat model for ALI/acute respiratory distress syndrome (ALI/ARDS) was established. Pathological examination of lung tissue was conducted to assess lung injury. Blood gas in the arteries was measured using a blood analyzer. Changes in PaO2, PaO2/FiO2, and lung wet/dry (W/D) weight ratio were carefully compared. ELISA assay was conducted to estimate cell adhesion and inflammation response. Finally, real-time reverse transcription polymerase chain reaction and western blotting assay was used to determine the activation of PI3K/AKT/mTOR pathway.
RESULTS
ARDS in vivo model was successfully constructed by LPS injection. Compared with the sham group, PaO2 and PaO2/FiO2 were significantly lower in the vehicle group, while the lung W/D ratio, the lung injury score, NE, VCAM-1, IL-8 andTNF-αwere significantly increased. After treatment with different doses of sivelestat sodium, we found PaO2, PaO2/FiO2 were prominently increased, while the lung W/D ratio, the lung injury score, NE, VCAM-1, IL-8, TNF-α levels were decreased in the dose-dependent manner. Meanwhile, compared with the vehicle group, the expression levels of Bax, PI3K, Akt and mTOR were significantly lower, and the expression of Bcl-2 was significantly higher after injection with sivelestat sodium.
CONCLUSION
Sivelestat sodium has an interventional effect on ALI in sepsis by inhibiting the PI3K/AKT/mTOR signalling pathway.
Topics: Animals; TOR Serine-Threonine Kinases; Acute Lung Injury; Signal Transduction; Proto-Oncogene Proteins c-akt; Phosphatidylinositol 3-Kinases; Rats; Male; Glycine; Sulfonamides; Rats, Sprague-Dawley; Lung; Disease Models, Animal
PubMed: 38935660
DOI: 10.1371/journal.pone.0302721 -
Investigative Ophthalmology & Visual... Jun 2024This study aimed to explore protective effects and potential mechanism of ectoine, a natural osmoprotectant, on ocular surface mucin production in dry eye disease.
PURPOSE
This study aimed to explore protective effects and potential mechanism of ectoine, a natural osmoprotectant, on ocular surface mucin production in dry eye disease.
METHODS
A dry eye model was established in C57BL/6 mice exposed to desiccating stress (DS) with untreated (UT) mice as controls. DS mice were topically treated with 2.0% ectoine or PBS vehicle. Corneal epithelial defects were assessed by Oregon Green Dextran (OGD) fluorescent staining. Conjunctival goblet cells, ocular mucins, and T help (Th) cytokines were evaluated by immunofluorescent staining or ELISA, and RT-qPCR.
RESULTS
Compared with UT mice, corneal epithelial defects were detected as strong punctate OGD fluorescent staining in DS mice with vehicle, whereas ectoine treatment largely reduced OGD staining to near-normal levels. Conjunctival goblet cell density and cell size decreased markedly in DS mice, but was significantly recovered by ectoine treatment. The protein production and mRNA expression of two gel-forming secreted MUC5AC and MUC2, and 4 transmembrane mucins, MUC1, MUC4, MUC16, and MUC15, largely decreased in DS mice, but was restored by ectoine. Furthermore, Th2 cytokine IL-13 was inhibited, whereas Th1 cytokine IFN-γ was stimulated at protein and mRNA levels in conjunctiva and draining cervical lymph nodes (CLNs) of DS mice, leading to decreased IL-13/IFN-γ ratio. Interestingly, 2.0% ectoine reversed their alternations and restored IL-13/IFN-γ balance.
CONCLUSIONS
Our findings demonstrate that topical ectoine significantly reduces corneal damage, and enhances goblet cell density and mucin production through restoring imbalanced IL-13/IFN-γ signaling in murine dry eye model. This suggests therapeutic potential of natural osmoprotectant ectoine for dry eye disease.
Topics: Animals; Dry Eye Syndromes; Mice; Mice, Inbred C57BL; Disease Models, Animal; Goblet Cells; Interferon-gamma; Mucins; Interleukin-13; Conjunctiva; Enzyme-Linked Immunosorbent Assay; Female; Epithelium, Corneal; Real-Time Polymerase Chain Reaction; RNA, Messenger; Amino Acids, Diamino
PubMed: 38935032
DOI: 10.1167/iovs.65.6.39 -
Journal of the American Heart... Jul 2024Experimental preeclampsia (ePE) has been shown to have worsened outcome from stroke. We investigated the effect of low-dose aspirin, known to prevent preeclampsia, on...
BACKGROUND
Experimental preeclampsia (ePE) has been shown to have worsened outcome from stroke. We investigated the effect of low-dose aspirin, known to prevent preeclampsia, on stroke hemodynamics and outcome, and the association between the vasoconstrictor and vasodilator cyclooxygenase products thromboxane A and prostacyclin.
METHODS AND RESULTS
Middle cerebral artery occlusion was performed for 3 hours with 1 hour of reperfusion in normal pregnant rats on day 20 of gestation and compared with ePE treated with vehicle or low-dose aspirin (1.5 mg/kg per day). Multisite laser Doppler was used to measure changes in cerebral blood flow to the core middle cerebral artery and collateral vascular territories. After 30 minutes occlusion, phenylephrine was infused to increase blood pressure and assess cerebral blood flow autoregulation. Infarct and edema were measured using 2,3,5-triphenyltetrazolium chloride staining. Plasma levels of thromboxane A, prostacyclin, and inflammatory markers in plasma and cyclooxygenase levels in cerebral arteries were measured. ePE had increased infarction compared with normal pregnant rats (<0.05) that was reduced by aspirin (<0.001). ePE also had intact cerebral blood flow autoregulation and reduced collateral perfusion during induced hypertension that was also prevented by aspirin. Aspirin increased prostacyclin in ePE (<0.05) without reducing thromboxane B, metabolite of thromboxane A, or 8-isoprostane-prostaglandin-2α, a marker of lipid peroxidation. There were no differences in cyclooxygenase levels in cerebral arteries between groups.
CONCLUSIONS
Low-dose aspirin in ePE reduced infarction that was associated with increased vasodilator prostacyclin and improved collateral perfusion during induced hypertension. The beneficial effect of aspirin on the brain and cerebral circulation is likely multifactorial and worth further study.
Topics: Animals; Female; Pregnancy; Aspirin; Cerebrovascular Circulation; Pre-Eclampsia; Homeostasis; Rats, Sprague-Dawley; Disease Models, Animal; Collateral Circulation; Thromboxane A2; Infarction, Middle Cerebral Artery; Blood Flow Velocity; Rats; Epoprostenol; Laser-Doppler Flowmetry
PubMed: 38934871
DOI: 10.1161/JAHA.124.035990 -
Journal of the American Heart... Jul 2024Current protocols generate highly pure human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro that recapitulate characteristics of mature...
BACKGROUND
Current protocols generate highly pure human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro that recapitulate characteristics of mature in vivo cardiomyocytes. Yet, a risk of arrhythmias exists when hiPSC-CMs are injected into large animal models. Thus, understanding hiPSC-CM maturational mechanisms is crucial for clinical translation. Forkhead box (FOX) transcription factors regulate postnatal cardiomyocyte maturation through a balance between FOXO and FOXM1. We also previously demonstrated that p53 activation enhances hiPSC-CM maturation. Here, we investigate whether p53 activation modulates the FOXO/FOXM1 balance to promote hiPSC-CM maturation in 3-dimensional suspension culture.
METHODS AND RESULTS
Three-dimensional cultures of hiPSC-CMs were treated with Nutlin-3a (p53 activator, 10 μM), LOM612 (FOXO relocator, 5 μM), AS1842856 (FOXO inhibitor, 1 μM), or RCM-1 (FOXM1 inhibitor, 1 μM), starting 2 days after onset of beating, with dimethyl sulfoxide (0.2% vehicle) as control. P53 activation promoted hiPSC-CM metabolic and electrophysiological maturation alongside FOXO upregulation and FOXM1 downregulation, in n=3 to 6 per group for all assays. FOXO inhibition significantly decreased expression of cardiac-specific markers such as TNNT2. In contrast, FOXO activation or FOXM1 inhibition promoted maturational characteristics such as increased contractility, oxygen consumption, and voltage peak maximum upstroke velocity, in n=3 to 6 per group for all assays. Further, by single-cell RNA sequencing of n=2 LOM612-treated cells compared with dimethyl sulfoxide, LOM612-mediated FOXO activation promoted expression of cardiac maturational pathways.
CONCLUSIONS
We show that p53 activation promotes FOXO and suppresses FOXM1 during 3-dimensional hiPSC-CM maturation. These results expand our understanding of hiPSC-CM maturational mechanisms in a clinically-relevant 3-dimensional culture system.
Topics: Myocytes, Cardiac; Forkhead Box Protein M1; Tumor Suppressor Protein p53; Humans; Induced Pluripotent Stem Cells; Cell Differentiation; Cell Culture Techniques, Three Dimensional; Cells, Cultured; Signal Transduction; Forkhead Box Protein O1
PubMed: 38934864
DOI: 10.1161/JAHA.123.033155 -
Journal of Molecular and Cellular... Jun 2024Heart failure remains one of the largest clinical burdens globally, with little to no improvement in the development of disease-eradicating therapeutics. Integrin...
Heart failure remains one of the largest clinical burdens globally, with little to no improvement in the development of disease-eradicating therapeutics. Integrin targeting has been used in the treatment of ocular disease and cancer, but little is known about its utility in the treatment of heart failure. Here we sought to determine whether the second generation orally available, αvβ3-specific RGD-mimetic, , was cardioprotective. Male mice were subjected to transverse aortic constriction (TAC) and treated with 50 μg/kg or volume-matched saline as Vehicle control. At 3 weeks post-TAC, echocardiography showed that treatment significantly restored cardiac function and structure indicating the protective effect of treatment in this model of heart failure. Importantly, treatment improved cardiac function giving improved fractional shortening, ejection fraction, heart weight and lung weight to tibia length fractions, together with partial restoration of Ace and Mme levels, as markers of the TAC insult. At a tissue level, reduced cardiomyocyte hypertrophy and interstitial fibrosis, both of which are major clinical features of heart failure. RNA sequencing identified that, mechanistically, this occurred with concomitant alterations to genes involved molecular pathways associated with these processes such as metabolism, hypertrophy and basement membrane formation. Overall, targeting αvβ3 with provides a novel strategy to attenuate pressure-overload induced cardiac hypertrophy and fibrosis, providing a possible new approach to heart failure treatment.
PubMed: 38933087
DOI: 10.1016/j.jmccpl.2024.100069 -
Pharmaceutics Jun 2024In the past several decades, polymeric microparticles (MPs) have emerged as viable solutions to address the limitations of standard pharmaceuticals and their...
In the past several decades, polymeric microparticles (MPs) have emerged as viable solutions to address the limitations of standard pharmaceuticals and their corresponding delivery methods. While there are many preclinical studies that utilize polymeric MPs as a delivery vehicle, there are limited FDA-approved products. One potential barrier to the clinical translation of these technologies is a lack of understanding with regard to the manufacturing process, hindering batch scale-up. To address this knowledge gap, we sought to first identify critical processing parameters in the manufacturing process of blank (no therapeutic drug) and protein-loaded double-emulsion poly(lactic-co-glycolic) acid MPs through a quality by design approach. We then utilized the design of experiments as a tool to systematically investigate the impact of these parameters on critical quality attributes (e.g., size, surface morphology, release kinetics, inner occlusion size, etc.) of blank and protein-loaded MPs. Our results elucidate that some of the most significant CPPs impacting many CQAs of double-emulsion MPs are those within the primary or single-emulsion process (e.g., inner aqueous phase volume, solvent volume, etc.) and their interactions. Furthermore, our results indicate that microparticle internal structure (e.g., inner occlusion size, interconnectivity, etc.) can heavily influence protein release kinetics from double-emulsion MPs, suggesting it is a crucial CQA to understand. Altogether, this study identifies several important considerations in the manufacturing and characterization of double-emulsion MPs, potentially enhancing their translation.
PubMed: 38931917
DOI: 10.3390/pharmaceutics16060796 -
Pharmaceutics May 2024This review explores the intersection of microfluidic technology and complex emulsion development as a promising solution to the challenges of formulations in multi-drug... (Review)
Review
This review explores the intersection of microfluidic technology and complex emulsion development as a promising solution to the challenges of formulations in multi-drug therapy (MDT) and polypharmacy. The convergence of microfluidic technology and complex emulsion fabrication could herald a transformative era in multi-drug delivery systems, directly confronting the prevalent challenges of polypharmacy. Microfluidics, with its unparalleled precision in droplet formation, empowers the encapsulation of multiple drugs within singular emulsion particles. The ability to engineer emulsions with tailored properties-such as size, composition, and release kinetics-enables the creation of highly efficient drug delivery vehicles. Thus, this innovative approach not only simplifies medication regimens by significantly reducing the number of necessary doses but also minimizes the pill burden and associated treatment termination-issues associated with polypharmacy. It is important to bring forth the opportunities and challenges of this synergy between microfluidic-driven complex emulsions and multi-drug therapy poses. Together, they not only offer a sophisticated method for addressing the intricacies of delivering multiple drugs but also align with broader healthcare objectives of enhancing treatment outcomes, patient safety, and quality of life, underscoring the importance of dosage form innovations in tackling the multifaceted challenges of modern pharmacotherapy.
PubMed: 38931830
DOI: 10.3390/pharmaceutics16060707