Mesenchymal Stem Cell-Derived Extracellular Vesicles Decrease Lung Injury in Mice.

Human mesenchymal stem cell (MSC) extracellular vesicles (EV) can reduce the severity of bacterial pneumonia, but little is known about the mechanisms underlying their antimicrobial activity. In the current study, we found that bacterial clearance induced by MSC EV in Escherichia coli pneumonia in C57BL/6 mice was associated with high levels of leukotriene (LT) B4 in the injured alveolus. More importantly, the antimicrobial effect of MSC EV was abrogated by cotreatment with a LTB4 BLT1 antagonist. To determine the role of MSC EV on LT metabolism, we measured the effect of MSC EV on a known ATP-binding cassette transporter, multidrug resistance-associated protein 1 (MRP1), and found that MSC EV suppressed MRP1 mRNA, protein, and pump function in LPS-stimulated Raw264.7 cells in vitro. The synthesis of LTB4 and LTC4 from LTA4 are competitive, and MRP1 is the efflux pump for LTC4 Inhibition of MRP1 will increase LTB4 production. In addition, administration of a nonspecific MRP1 inhibitor (MK-571) reduced LTC4 and subsequently increased LTB4 levels in C57BL/6 mice with acute lung injury, increasing overall antimicrobial activity. We previously found that the biological effects of MSC EV were through the transfer of its content, such as mRNA, microRNA, and proteins, to target cells. In the current study, miR-145 knockdown abolished the effect of MSC EV on the inhibition of MRP1 in vitro and the antimicrobial effect in vivo. In summary, MSC EV suppressed MRP1 activity through transfer of miR-145, thereby resulting in enhanced LTB4 production and antimicrobial activity through LTB4/BLT1 signaling.

Short-chain fatty acids activate GPR41 and GPR43 on intestinal epithelial cells to promote inflammatory responses in mice.

BACKGROUND & AIMS: Short-chain fatty acids (SCFAs), the most abundant microbial metabolites in the intestine, activate cells via G-protein-coupled receptors (GPRs), such as GPR41 and GPR43. We studied regulation of the immune response by SCFAs and their receptors in the intestines of mice. METHODS: Inflammatory responses were induced in GPR41(-/-), GPR43(-/-), and C57BL6 (control) mice by administration of ethanol; 2, 4, 6-trinitrobenzene sulfonic-acid (TNBS); or infection with Citrobacter rodentium. We examined the effects of C rodentium infection on control mice fed SCFAs and/or given injections of antibodies that delay the immune response. We also studied the kinetics of cytokine and chemokine production, leukocyte recruitment, intestinal permeability, and T-cell responses. Primary colon epithelial cells were isolated from GPR41(-/-), GPR43(-/-), and control mice; signaling pathways regulated by SCFAs were identified using immunohistochemical, enzyme-linked immunosorbent assay, and flow cytometry analyses. RESULTS: GPR41(-/-) and GPR43(-/-) mice had reduced inflammatory responses after administration of ethanol or TNBS compared with control mice, and had a slower immune response against C rodentium infection, clearing the bacteria more slowly. SCFAs activated intestinal epithelial cells to produce chemokines and cytokines in culture and mice after administration of ethanol, TNBS, or C rodentium. These processes required GPR41 and GPR43 and were required to recruit leukocytes and activate effector T cells in the intestine. GPR41 and GPR43 activated extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase signaling pathways in epithelial cells to induce production of chemokines and cytokines during immune responses. CONCLUSIONS: SCFAs activate GPR41 and GPR43 on intestinal epithelial cells, leading to mitogen-activated protein kinase signaling and rapid production of chemokines and cytokines. These pathways mediate protective immunity and tissue inflammation in mice.

Effective healing of diabetic skin wounds by using nonviral gene therapy based on minicircle vascular endothelial growth factor DNA and a cationic dendrimer.

BACKGROUND: The development of an efficient method to improve the wound healing process is urgently required for diabetic patients suffering a threat of limb amputations. Various growth factors have been proposed for treatment; however, more research still has to be carried out to maintain their curative effect. In the present study, we describe a simple nonviral gene therapy method for improving wound healing. METHODS: Minicircle plasmid DNA encoding vascular endothelial growth factor (VEGF) was combined with an arginine-grafted cationic dendrimer, PAM-RG4. The formed complexes were injected subcutaneously into the skin wounds of diabetic mice. RESULTS: Actively proliferating cells in wound tissue were efficiently transfected, resulting in a high level of VEGF expression. Within 6 days after injection, skin wounds in the diabetic mice were generally healed and displayed a well-ordered dermal structure, which was confirmed by histological staining. CONCLUSIONS: This simple and effective gene therapy method may represent a powerful tool for the treatment of diabetic foot ulcers and other diseases that are refractory to treatment.

IAEA Activities on 67Cu, 186Re, 47Sc Theranostic Radionuclides and Radiopharmaceuticals.

Despite interesting properties, the use of 67Cu, 186Re and 47Sc theranostic radionuclides in preclinical studies and clinical trials is curtailed by their limited availability due to a lack of widely established production methods. An IAEA Coordinated Research Project (CRP) was initiated to identify important technical issues related to the production and quality control of these emerging radionuclides and related radiopharmaceuticals, based on the request from IAEA Member States. The international team worked on targetry, separation, quality control and radiopharmaceutical aspects of the radionuclides obtained from research reactors and cyclotrons leading to preparation of a standard recommendations for all Member States. The CRP was initiated in 2016 with fourteen participants from thirteen Member States from four continents. Extraordinary results on the production, quality control and preclinical evaluation of selected radionuclides were reported in this project that was finalized in 2020. The outcomes, outputs and results of this project achieved by participating Member States are described in this minireview.

Unexpected extensive hemorrhage from a subcapsular hematoma of the liver during emergent laparotomy in a premature neonate.

A subcapsular hematoma of the liver is often found during autopsy in stillborn infants rather than clinically. It is usually asymptomatic unless ruptured; thus, the diagnosis is often delayed or missed. Rupture of a subcapsular hematoma in a premature neonate causes massive intraabdominal hemorrhage, which is associated with high mortality. Thus, early recognition and treatment to avoid rupture are imperative. We describe a case of life-threatening hemorrhage from a subcapsular hematoma of the liver during emergent laparotomy for mechanical obstruction in an 860 g premature neonate and discuss the appropriate preoperative preparation and anesthetic management for this case.

Nateglinide and acarbose for postprandial glucose control after optimizing fasting glucose with insulin glargine in patients with type 2 diabetes.

AIMS: Basal insulin treatment is frequently used in type 2 diabetes, but the successful control of postprandial glucose is challenging. We compared the effect of preferential postprandial glucose targeting drugs for postprandial glucose control after optimizing fasting glucose with basal insulin. METHODS: This study was performed in 58, insulin naïve type 2 diabetes. After fasting glucose was optimized by insulin glargine, nateglinide or acarbose was initiated and then crossed over after second wash out period. 75 g oral glucose tolerance test and 7 point self monitoring blood glucose for 3 days at the end of each period was performed. RESULTS: Both drugs effectively reduced postprandial glucose levels compared with the insulin glargine monotherapy. No significant differences were found between nateglinide and acarbose in terms of mean glucose level, standard deviation of glucose levels, mean average glucose excursion and average daily risk range. Homeostasis model analysis (HOMA)% ß, corrected insulin response and insulin-to-glucose ratio were significantly higher in the responder group compared with the non-responder. There was no episode of severe hypoglycemia. CONCLUSIONS: Nateglinide and acarbose are equally effective in type 2 diabetes for postprandial glucose excursions during basal insulin treatment. The markers of beta cell function might be used for predicting response. (Clinical trial reg. no. NCT 00437918, clinicaltrial.gov.).

Ultrasound-mediated gene delivery.

IMPORTANCE OF THE FIELD: The use of ultrasound with microbubbles raises the possibility of an efficient and safe gene delivery. AREAS COVERED IN THIS REVIEW: This review summarizes the current state of the art of gene delivery by sonoporation under the following topics. First, the basic ultrasound parameters and the characteristics of microbubble in biological systems are discussed. Second, the extensions of sonoporation to other fields of gene delivery such as viral and non-viral vector are briefly reviewed. Finally, recent applications in an animal model for various diseases are introduced. WHAT THE READER WILL GAIN: Information and comments on gene delivery by sonoporation or enhanced cell membrane permeability by means of ultrasound. TAKE HOME MESSAGE: Ultrasound-mediated gene delivery combined with microbubble agents provides significant safety advantages over other methods of local gene delivery.

The effect of chromium on rat insulinoma cells in high glucose conditions.

AIMS: it has been suggested that Chromium (Cr), one of the essential minerals, can be beneficial to type 2 diabetic patients because it lowers blood glucose levels by improving various steps in insulin action. A few studies reported that Cr might also have some beneficial effects in people with type 1 diabetes mellitus and in streptozotocin-treated rats, but direct beneficial effects of Cr on pancreatic beta cells have not been proven. We performed this study to determine whether Cr could have direct protective effects on INS-1 cells in high glucose conditions that mimic the actual diabetic state. MAIN METHODS: INS-1 cells were cultured for 48h in RPMI medium with 33mM glucose as the stress condition and 11mM glucose as a control. CrCl(3) was used to verify whether Cr could protect INS-1 cells from glucotoxic stress. Cell viability and apoptosis were evaluated by MTT assay and FACS. The level of insulin mRNA, by semi-quantitative RT-PCR, was significantly reduced at 33mM glucose concentration after 48h of incubation. KEY FINDINGS: cell viability was reduced by 50%, and 35% of the cells underwent apoptosis at the same culture condition. Addition of various concentrations of CrCl(3) to INS-1 cells in 33mM glucose for different durations of time did not reveal any beneficial effects on cell viability, degree of apoptosis, insulin mRNA levels, and glucose stimulated insulin secretion. SIGNIFICANCE: we could not find any evidence that Cr had direct beneficial effects on INS-1 cells in high glucose induced stress conditions.

EGCG and quercetin protected INS-1 cells in oxidative stress via different mechanisms.

EGCG and quercetin are known as beneficial dietary flavonoid for various diseases including diabetes mellitus. But it is not certain whether they could protect pancreatic beta cell directly. We performed this study to test both EGCG and quercetin could directly protect beta cell line under oxidative stress, and verify the action mechanisms. The protective effect of quercetin on INS-1 cells against oxidative stress was concentration dependent, but EGCG showed specific concentration zone for the protection. The protective effect of EGCG was more pronounced in pre-treatment before oxidative stress, while quercetin showed dramatic improvement of viability in simultaneous incubation with H2O2. In EGCG pre-treatment, antioxidant enzymes and activity were decreased, but the phosphorylated PI3K and Akt were significantly increased. PI3K inhibitor significantly reduced cell viability in EGCG pre-treatment. In conclusion, EGCG and quercetin have protective effect on INS-1 cells against oxidative stress through both antioxidant effect and anti-apoptosis signaling. In EGCG, pre-treatment make its effect better by the enhancement of anti-apoptosis signaling. Quercetin protected INS-1 cells more in simultaneous incubation via strong antioxidant defense.

Comparison of the efficiency and toxicity of sonoporation with branched polyethylenimine-mediated gene transfection in various cultured cell lines.

OBJECTIVE: The objective of this study is to evaluate transfection efficiency and safety for gene delivery by sonoporation in comparison with cationic polymer gene carrier branched polyethylenimine (BPEI). METHODS: The cDNA expressing VEGF(165) was cloned under chicken beta-actin promoter. The plasmid DNA was transfected into the CHO, HEK293, and NIH3T3 cells using microbubble-based sonoporation and BPEI (25 kDa) under various conditions. Enzyme-linked immunosorbent assay (ELISA) was used to determine the expressed protein level. Cytotoxicities of transfection methods were compared by Cell Counting Kit-8. RESULTS: At 1 MHz intensity, transfection efficiency of sonoporation was enhanced by microbubble concentration with no detrimental effects. By contrast, BPEI exacerbated cell viability, despite its high transgene expression efficiency. CONCLUSION: Sonoporation gene therapy might be the safest technique to be used in actual clinical practice.