In Vivo Comparison of 23-and 25-Gauge Sutureless Vitrectomy Incision Architecture Using Spectral Domain Optical Coherence Tomography

Purpose. To investigate the in vivo incision architecture using spectral domain optical coherence tomography (SD-OCT) in 23-gauge and 25-gauge transconjunctival sutureless pars plana vitrectomy (TSPPV). Methods. A prospective observational study of 22 eyes of 22 patients that underwent three-port 25-gauge (10 eyes) or 23-gauge (12 eyes) TSPPV was performed. the three sclerotomies sites in each eye were analyzed by Corneal Adapter Model (CAM) RTVue SD-OCT (Optovue Inc., Fremont, CA, USA) with wound cross-section images (longitudinal and transversal) on days 1, 7, and 30 postoperatively. Transversal and longitudinal length, location, angle between the conjunctival surface tangent and the incision plane, and architecture deformations were evaluated. Results. All patients (22 eyes) completed the study and surgeries lasted less than 60 minutes. All wounds were obliquely performed, 23-gauge mean angle was 23 +/- 5 degrees, and 25-gauge angule was 21 +/- 4 degrees. Twenty-three-gauge sclerotomy transversal mean length was 1122 +/- 242 mu m and 25-gauge transversal sclerotomy mean length was 977 +/- 174 mu m; 23-gauge longitudinal mean length was 363 +/- 42 mu m and 25-gauge longitudinal sclerotomy mean length was 234 +/- 19 mu m; 23-gauge open wound thickness mean was 61 +/- 28 mu m and 25-gauge open wound thickness mean was 22 +/- 6 mu m. All results were statistically significant (P < 0.05). No vitreous incarceration or silicone oil residue was observed in incision sites with both gauges. Conclusions. the 23-gauge and 25-gauge architectural wound constructions were well visualized using CAM SD-OCT. Statistical differences between the two gauges were observed throughout the study period.Teixeira Oftalmol, BR-70392901 Brasilia, DF, BrazilUniversidade Federal de São Paulo, Dept Ophthalmol, São Paulo, BrazilUniv Catolica Brasilia, Dept Ophthalmol, Brasilia, DF, BrazilUniv Montreal, Dept Ophthalmol, Hop Maisonneuve Rosemont, Montreal, PQ, CanadaPontificia Univ Catolica Rio de Janeiro, Dept Ophthalmol, Rio de Janeiro, RJ, BrazilUniv Illinois, Dept Ophthalmol, Chicago, IL 60680 USAUniversidade Federal de São Paulo, Dept Ophthalmol, São Paulo, BrazilWeb of Scienc

Isolation of Vascular Wall Mesenchymal Stem Cells from the Thoracic Aorta of Adult Göttingen Minipigs: A New Protocol for the Simultaneous Endothelial Cell Collection

Simple Summary It has been widely demonstrated that blood vessels are sources of multipotent progenitor cells, including mesenchymal stem cells. These stem cellular populations persist throughout adulthood and can be isolated from both microvascular and large vessels. Increasing evidence suggests that vascular stem cells, together with other cell populations residing in blood vessels, such as endothelial cells, are involved in physiological and pathological vascular remodeling. In the present paper, we described, for the first time, a new improved method to isolate a pure population of vascular wall cells showing a preserved mesenchymal tri-lineage differentiative potential from thoracic aorta of Gottingen Minipigs, preserving and also collecting endothelial cells. Considering the increasing interest in the use of Gottingen Minipigs as an animal model for cardiovascular diseases, the results obtained in the present research open the way to plan in vitro vascular remodeling experiments by using in co-culture system vascular mesenchymal stem cells and endothelial cells.Two main classes of perivascular multipotent populations have been described: the microvascular pericytes and the vascular wall mesenchymal stem cells (VW-MSCs). VW-MSCs are isolated from large vessels in many species and they participate in vascular remodeling together with other cellular components such as endothelial cells. Considering that the Gottingen Minipigs are widely used in Europe as a translational model in the field of cardiovascular diseases, the aim of the present research was to isolate VW-MSCs from the adult aorta of Gottingen Minipigs while preserving and also collecting endothelial cells. The results obtained in the present research demonstrated that this new protocol allows us to obtain a pure population of VW-MSCs and endothelial cells. VW-MSCs from Gottingen Minipigs responded fully to the MSC minima international criteria, being positive to CD105, CD90, and CD44 and negative to CD45 and CD34. Moreover, VW-MSCs presented a differentiative potential towards osteogenic, chondrogenic, and adipogenic lineages. Overall, the present protocol, preserving the viability and phenotypic features of the two isolated populations, opens future possibilities of using minipig VW-MSCs and endothelial cells in in vitro vascular remodeling studies

Characterization of metabolic profiles and lipopolysaccharide effects on porcine vascular wall mesenchymal stem cells

The link between metabolic remodeling and stem cell fate is still unclear. To explore this topic, the metabolic profile of porcine vascular wall mesenchymal stem cells (pVW-MSCs) was investigated. At the first and second cell passages, pVW-MSCs exploit both glycolysis and cellular respiration to synthesize adenosine triphosphate (ATP), but in the subsequent (third to eighth) passages they do not show any mitochondrial ATP turnover. Interestingly, when the first passage pVW-MSCs are exposed to 0.1 or 10 μg/ml lipopolysaccharides (LPSs) for 4 hr, even if ATP synthesis is prevented, the spare respiratory capacity is retained and the glycolytic capacity is unaffected. In contrast, the exposure of pVW-MSCs at the fifth passage to 10 μg/ml LPS stimulates mitochondrial ATP synthesis. Flow cytometry rules out any reactive oxygen species (ROS) involvement in the LPS effects, thus suggesting that the pVW-MSC metabolic pattern is modulated by culture conditions via ROS-independent mechanisms

Ex vivo effect of vascular wall stromal cells secretome on enteric ganglia

BACKGROUND Mesenchymal stromal cell (MSC)-based therapy is currently under study to treat inflammatory bowel diseases. MSC bioactive products could represent a valid alternative to overcome issues associated with systemic whole-cell therapies. However, MSC anti-inflammatory mechanisms differ between rodents and humans, impairing the reliability of preclinical models. AIM To evaluate the effect of conditioned medium (CM) derived from porcine vascular wall MSCs (pVW-MSCs) on survival and differentiation of porcine and Guinea pig enteric ganglia exposed to lipopolysaccharide (LPS). METHODS Primary cultures of enteric ganglia were obtained by mechanic and enzymatic digestion of ileum resections from Guinea pigs (Cavia porcellus) (GPEG) and pigs (Suus scrofa) (PEG). pVW-MSCs were derived by enzymatic digestion from vascular wall resections of porcine aorta and tested by immunoflowcytometry for MSC immune profile. Enteric ganglia were treated with increasing concentrations of LPS, CM derived by pVW-MSCs or a combination of CM and LPS 1 \u3bcg/mL. Cell count and morphometric analysis of HuD positive neurons and glial fibrillary acidic protein positive glial cells were performed by immunofluorecent staining of cultured ganglia. RESULTS PEG showed a higher number of neurons compared to GPEG. Overall, CM exerted a protective role on LPS-treated enteric ganglia. CM in combination with LPS increased the number of glial cells per ganglion in both cultures evoking glial cells differentiation in porcine cultures. CONCLUSION These findings suggest an immunomodulating activity of pVW-MSCs mediators on the enteric nervous system in inflammatory conditions

Constitutive and LPS-stimulated secretome of porcine Vascular Wall-Mesenchymal Stem Cells exerts effects on in vitro endothelial angiogenesis

Background: MSCs secretome is under investigation as an alternative to whole-cell-based therapies, since it is enriched of bioactive molecules: growth factors, cytokines and chemokines. Taking into account the translational value of the pig model, the leading aim of the present paper was to characterize the secretome of porcine Vascular Wall-Mesenchymal Stem Cells (pVW-MSCs) and its change in presence of LPS stimulation. Moreover, considering the importance of angiogenesis in regenerative mechanisms, we analysed the effect of pVW-MSCs secretome on in vitro angiogenesis. Results: Our results demonstrated that conditioned medium from unstimulated pVW-MSCs contained high levels of IL-8, GM-CSF, IFN-\u3b3 and other immunomodulatory proteins: IL-6 IL-18 IL-4 IL-2 IL-10. LPS modulates pVW-MSCs gene expression and secretome composition, in particular a significant increase of IL-6 and IL-8 was observed; conversely, the amount of GM-CSF, IFN-\u3b3, IL-2, IL-4, IL-10 and IL-18 showed a significant transient decrease with the LPS stimulation. Conditioned medium from unstimulated pVW-MSCs induced in vitro endothelial angiogenesis, which is more evident when the conditioned medium was from LPS stimulated pVW-MSCs. Conclusions: The lines of evidence here presented shed a light on possible future application of secretome derived by pVW-MSCs on research studies in translational regenerative medicine

Doxorubicin treatment modulates chemoresistance and affects the cell cycle in two canine mammary tumour cell lines

Background: Doxorubicin (DOX) is widely used in both human and veterinary oncology although the onset of multidrug resistance (MDR) in neoplastic cells often leads to chemotherapy failure. Better understanding of the cellular mechanisms that circumvent chemotherapy efficacy is paramount. The aim of this study was to investigate the response of two canine mammary tumour cell lines, CIPp from a primary tumour and CIPm, from its lymph node metastasis, to exposure to EC50(20h) DOX at 12, 24 and 48 h of treatment. We assessed the uptake and subcellular distribution of DOX, the expression and function of P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP), two important MDR mediators. To better understand this phenomenon the effects of DOX on the cell cycle and Ki67 cell proliferation index and the expression of p53 and telomerase reverse transcriptase (TERT) were also evaluated by immunocytochemistry (ICC). Results: Both cell lines were able to uptake DOX within the nucleus at 3 h treatment while at 48 h DOX was absent from the intracellular compartment (assessed by fluorescence microscope) in all the surviving cells. CIPm, originated from the metastatic tumour, were more efficient in extruding P-gp substrates. By ICC and qRT-PCR an overall increase in both P-gp and BCRP were observed at 48 h of EC50(20h) DOX treatment in both cell lines and were associated with a striking increase in the percentage of p53 and TERT expressing cells by ICC. The cell proliferation fraction was decreased at 48 h in both cell lines and cell cycle analysis showed a DOX-induced arrest in the S phase for CIPp, while CIPm had an increase in cellular death without arrest. Both cells lines were therefore composed by a fraction of cells sensible to DOX that underwent apoptosis/necrosis. Conclusions: DOX administration results in interlinked modifications in the cellular population including a substantial effect on the cell cycle, in particular arrest in the S phase for CIPp and the selection of a subpopulation of neoplastic cells bearing MDR phenotype characterized by P-gp and BCRP expression, TERT activation, p53 accumulation and decrease in the proliferating fraction. Important information is given for understanding the dynamic and mechanisms of the onset of drug resistance in a neoplastic cell population

Discontinuation of alpha-interferon treatment in patients with chronic myeloid leukemia in long-lasting complete molecular response

To evaluate follow-up after α-interferon (IFN) discontinuation, 23 patients with chronic myeloid leukemia (CML) in stable complete molecular response (CMolR) with IFN were revisited. After a median IFN treatment of 105.8 months (IR 56.1 - 127.3), all patients discontinued IFN for prolonged CMolR (12), intolerance (8) or planned ABMT (3). After 12.5 months, one patient developed an extramedullar blast crisis. Four patients needed to start imatinib, all achieving again molecular response. Eighteen patients are still off-therapy (median time from IFN discontinuation 125.5 months, IR 86.9-205.3); among these, five are BCR-ABL negative, six present with a sporadic positivity (BCR-ABL ratio &lt; 0.1) and seven show a stable and long-lasting mild positivity (BCR-ABL ratio &lt; 0.5). Patients in prolonged CMolR with IFN have low risk of recurrence after discontinuation; the reappearance of a BCR-ABL positivity &lt; 0.5 did not always precede a relapse, suggesting mechanisms of immunological control induced by IFN

Development of a pig mammary epithelial cell culture model as a non‐clinical tool for studying epithelial barrier— a contribution from the imi‐conception project

The ConcePTION project aims at generating further knowledge about the risks related to the use of medication during breastfeeding, as this information is lacking for most commonly used drugs. Taking into consideration multiple aspects, the pig model has been considered by the consortium as the most appropriate choice. The present research was planned to develop an efficient method for the isolation and culture of porcine Mammary Epithelial Cells (pMECs) to study the mammary epithelial barrier in vitro. Mammary gland tissues were collected at a local slaughterhouse, dissociated and the selected cellular population was cultured, expanded and characterized by morphology, cell cycle analysis and immunophenotyping. Their ability to create a barrier was tested by TEER measurement and sodium fluorescein transport activity. Expression of 84 genes related to drug transporters was evaluated by a PCR array. Our results show that primary cells express epithelial cell markers: CKs, CK18, E‐Cad and tight junctions molecules ZO‐1 and OCL. All the three pMEC cellular lines were able to create a tight barrier, although with different strengths and kinetics, and express the main ABC and SLC drug transporters. In conclusion, in the present paper we have reported an efficient method to obtain primary pMEC lines to study epithelial barrier function in the pig model