Miryam A. Fragoso

Poly(α-hydroxyacids) for application in the spinal cord: Resorbability and biocompatibility with adult rat schwann cells and spinal cord

Future surgical strategies to restore neurological function in the damaged human spinal cord may involve replacement of nerve tissue with cultured Schwann cells using biodegradable guiding implants. We have studied the in vitro and in vivo degradability of various aliphatic polyesters as well as their effects on rat Schwann cells in vitro and on spinal cord tissue in vivo. In vitro, cylinders made of poly(D,L-lactic-co-glycolic acid) 50:50 (PLA25GA50) started to degrade at 7 days, compared with 28 days for cylinders made of poly(D,L-lactic acid) (PLA50). This faster degradation of PLA25GA50 was reflected by a much higher absorption of water. In vivo, after implantation of PLA25GA50 or PLA50 cylinders between the stumps of a completely transected adult rat spinal cord, the decrease in molecular weight of both polymers was similar to that found in vitro. In vitro degradation of poly(L-lactic acid) (PLA100) mixed with increasing amounts of PLA100 oligomers also was determined. The degradation rate of PLA100 mixed with 30% oligomers was found to be similar to that of PLA50. In vitro, PLA25GA50 and the breakdown products had no adverse effect on the morphology, survival, and proliferation of cultured rat Schwann cells. In vivo, PLA25GA50 cylinders were integrated into the spinal tissue 2 weeks after implantation, unlike PLA50 cylinders. At all time points after surgery, the glial and inflammatory response near the lesion site was largely similar in both experimental and control animals. At time points later than 1 week, neurofilament-positive fibers were found within PLA25GA50 cylinders or the remains thereof. Growth-associated protein 43, which is indicative of regenerating axons, was observed in fibers in the vicinity of the injury site and in the remains of PLA25GA50 cylinders. The results suggest that poly(alpha-hydroxyacids) are likely candidates for application in spinal cord regeneration paradigms involving Schwann cells.

Transcellular thiocyanate transport by human airway epithelia

Human airway mucosa synthesizes and secretes lactoperoxidase (LPO). As H2O2 and thiocyanate (SCN−) are also present, a functional LPO antibacterial defence system exists in the airways. SCN− concentrations in several epithelial secretions are higher than in serum, although the mechanisms of transepithelial transport and accumulation in these secretions are unknown. To examine SCN− accumulation in secretions, human airway epithelial cells, re‐differentiated at the air–liquid interface, were used in open‐circuit conditions. [14C]SCN−, in the basolateral medium, was transported across the epithelium and concentrated tenfold at the apical surface. Measurement of the transepithelial potential showed that the basolateral compartment was positive relative to the apical surface (13.7 ± 1.8 mV) and therefore unfavourable for passive movement of SCN−. Transport was dependent on basolateral [SCN−] and saturable (Km,app= 69 ± 25 μm); was inhibited by increased apical [SCN−]; and was dependent on the presence of basolateral Na+. Perchlorate (Ki,app= 0.6 ± 0.05 μm) and iodide (Ki,app= 9 ± 8 μm) in the basolateral medium reversibly inhibited transport, but furosemide did not. Iodide was also transported (Km,app= 111 ± 69 μm). RT‐PCR and immunohistochemistry confirmed expression of Na+−I− symporter (NIS) in the airways. SCN− transport was insensitive to apical disulphonic acid Cl− channel blockers, but sensitive to apical glibenclamide and arylaminobenzoates. Forskolin and dibutyryl cAMP increased transport. These data suggest SCN− transport may occur through basolateral NIS‐mediated SCN− concentration inside cells, followed by release through an apical channel, perhaps cystic fibrosis transmembrane conductance regulator.

Oxidative epithelial host defense is regulated by infectious and inflammatory stimuli

Epithelia express oxidative antimicrobial protection that uses lactoperoxidase (LPO), hydrogen peroxide (H(2)O(2)), and thiocyanate to generate the reactive hypothiocyanite. Duox1 and Duox2, found in epithelia, are hypothesized to provide H(2)O(2) for use by LPO. To investigate the regulation of oxidative LPO-mediated host defense by bacterial and inflammatory stimuli, LPO and Duox mRNA were followed in differentiated primary human airway epithelial cells challenged with Pseudomonas aeruginosa flagellin or IFN-gamma. Flagellin upregulated Duox2 mRNA 20-fold, but upregulated LPO mRNA only 2.5-fold. IFN-gamma increased Duox2 mRNA 127-fold and upregulated LPO mRNA 10-fold. DuoxA2, needed for Duox2 activity, was also upregulated by flagellin and IFN-gamma. Both stimuli increased H(2)O(2) synthesis and LPO-dependent killing of P. aeruginosa. Reduction of Duox1 by siRNA showed little effect on basal H(2)O(2) production, whereas Duox2 siRNA markedly reduced basal H(2)O(2) production and resulted in an 8-fold increase in Nox4 mRNA. In conclusion, large increases in Duox2-mediated H(2)O(2) production seem to be coordinated with increases in LPO mRNA and, without increased LPO, H(2)O(2) levels in airway secretion are expected to increase substantially. The data suggest that Duox2 is the major contributor to basal H(2)O(2) synthesis despite the presence of greater amounts of Duox1.

Efficacy of various drugs in the prevention of posterior capsule opacification: Experimental study of rabbit eyes

Purpose: To assess the efficacy of various drugs in the prevention of posterior capsule opacification (PCO) in a closed capsular bag technique. Setting: Ophthalmic Biophysics Center, Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, Florida, USA. Methods: Lens material was removed using phacoaspiration or phacoemulsification through a microcapsulorhexis according to the hardness of the crystalline lens correlated with the weight and age of the rabbits. A mixture of an ophthalmic viscosurgical device (sodium hyaluronate 1.4% [SHA]) and a drug was injected into the empty capsular bag, allowed to remain inside for 3 minutes, and removed. The capsular bag was rinsed with balanced salt solution (BSS®) and refilled with SHA. In a group of rabbits, the capsulorhexis was sealed with a minicapsulorhexis valve (MCV). Rabbits were treated with 1 of the following: SHA (control), BSS, mitomycin‐C (MMC, 0.2 mg/mL), ethylenediaminetetraacetic acid (EDTA) (10 mM and 15 mM), 5‐fluorouracil (5‐FU, 33 mg/mL), acetic acid (3%, 0.3%, and 0.003%), and distilled water. Results: Upon completion of the study, the control and treated eyes had PCO and new lens material (not residual). Anterior capsule proliferation was observed in eyes treated with 5‐FU. The order of PCO appearance (earliest to latest) was as follows: 15 mM EDTA, SHA, MMC, acetic acid 0.3%, acetic acid 3%, BSS, distilled water (small animals; no MCV), acetic acid 0.003%, 5‐FU, 10 mM EDTA, and distilled water (large animals; MCV). The earliest appearance was day 1 postoperatively and the latest, day 47. Conclusions: Distilled water and 10 mM EDTA treatments were the most efficient in retarding the appearance of PCO.

The Wnt/β-Catenin Pathway Cross-Talks with STAT3 Signaling to Regulate Survival of Retinal Pigment Epithelium Cells

Wnt/β-catenin signaling is an essential pathway that regulates numerous cellular processes, including cell survival. The molecular mechanisms contributing to pro-survival Wnt signaling are mostly unknown. Signal transducer and activator of transcription proteins (STATs) are a well-described family of transcription factors. STAT3 induces expression of anti-apoptotic genes in many tissues and is a downstream mediator of protective growth factors and cytokines. In this study, we investigated whether pro-survival Wnt signaling is mediated by STAT3. The Wnt3a ligand activated Wnt signaling in the retinal pigment epithelium ARPE-19 cell line and significantly increased the viability of cells exposed to oxidative stress. Furthermore, Wnt3a increased STAT3 activation and nuclear translocation, as measured by an antibody against phosphorylated STAT3. Reducing STAT3 levels with siRNA eliminated Wnt3a-dependent protection from oxidative stress. Together, these data demonstrate a previously unknown link between Wnt3a-mediated activation of STAT3 and cell survival, and indicate cross-talk between two important pro-survival signaling pathways.

Active Trafficking of Alpha 1 Antitrypsin across the Lung Endothelium

The homeostatic lung protective effects of alpha-1 antitrypsin (A1AT) may require the transport of circulating proteinase inhibitor across an intact lung endothelial barrier. We hypothesized that uninjured pulmonary endothelial cells transport A1AT to lung epithelial cells. Purified human A1AT was rapidly taken up by confluent primary rat pulmonary endothelial cell monolayers, was secreted extracellularly, both apically and basolaterally, and was taken up by adjacent rat lung epithelial cells co-cultured on polarized transwells. Similarly, polarized primary human lung epithelial cells took up basolaterally-, but not apically-supplied A1AT, followed by apical secretion. Evidence of A1AT transcytosis across lung microcirculation was confirmed in vivo by two-photon intravital microscopy in mice. Time-lapse confocal microscopy indicated that A1AT co-localized with Golgi in the endothelium whilst inhibition of the classical secretory pathway with tunicamycin significantly increased intracellular retention of A1AT. However, inhibition of Golgi secretion promoted non-classical A1AT secretion, associated with microparticle release. Polymerized A1AT or A1AT supplied to endothelial cells exposed to soluble cigarette smoke extract had decreased transcytosis. These results suggest previously unappreciated pathways of A1AT bidirectional uptake and secretion from lung endothelial cells towards the alveolar epithelium and airspaces. A1AT trafficking may determine its functional bioavailablity in the lung, which could be impaired in individuals exposed to smoking or in those with A1AT deficiency.

Endocapsular hyperthermia probe to prevent posterior capsular opacification

Purpose: To evaluate the feasibility to induce lens epithelial cell death with intraoperative hyperthermia for prevention of secondary cataract. Methods: A prototype miniature resistive hyperthermia probe was designed. The probe contained a thermocouple for temperature feed-back. A timer allowed monitoring of the electrical driving of the hyperthermia probe and the temperature induced as a function of time. To model the heating response, a simple model of the lens capsule was constructed using a thin acrylic plastic shell embedded in a sponge immersed in a water bath at 37°C. The shell was filled with sodium hyaluronate. The probe was positioned at the center of the shell with the thermocouple next to the wall. An experimental protocol was developed to assess the feasibility of increasing the temperature of the human lens to hyperthermia levels in fresh cadaver eyes: An annular metal ring was bonded just below the limbus, the cornea and iris were sectioned, the lens material was removed through a central 5mm diameter capsulorhexis, the capsule was filled with SHA and the globe was set on a temperature-controlled cylindrical vial. Preliminary Results: At 3.3W (2.2V, 1.5A) the shells content increases from 37°C to 51°C in 30s. At that temperature, LEC death is expected to occur within 1sec. Conclusion: This preliminary study demonstrates the feasibility of increasing the temperature of the capsular bag to kill LECs by hyperthermia.

Regulation of Cell Proliferation: Role of A Chromosome-Matrix Protein

INTRODUCTION. LFM-1 is a recently described chromosomal-matrix component cell-cycle dependent 1 . Preliminary experiments have suggested a regulatory role of LFM-1 in normal cell proliferation in human epithelia 2 . Lens Posterior Capsule Opacification (PCO) is a common and disabling health condition due to proliferation of Lens Epithelial Cells (LECs) to the posterior capsule of the lens 3 after cataract surgery. PCO is a widely studied condition with no effective medical treatment up to date. The purpose of this work is to develop an assay to control the proliferation of Lens Epithelial Cells by blocking LFM-1 protein synthesis and its potential function(s) in this mechanism. METHODS. LECs were harvested from rabbit’s lens capsules, dissociated and cultured under standard tissue culture conditions to be used as biological model for experiments. Phosphorothioate ODNs 4 (S-oligos, 23-mer) were designed, synthesized in the specific antisense (AS-ODNs) and random directions of the LFM-1 mRNA, and employed for treatment of cells during 4 days with replacement of fresh media/oligos every other day. Indirect immunofluorescence and western blot were performed to study the LFM-1 molecular specie(s), its rate of synthesis and sub-cellular distribution. Cell proliferation was simultaneously asserted by quantization of number of cells, and DNA cellular content measured by spectrofluorometry and video analysis as a function of the LFM-1 knocked translation. RESULTS AND DISCUSSION. Cell proliferation was analyzed as a function of the expression of LFM-1 protein at the translational level. Indirect immunofluorescence images of cells treated with specific LFM-1 antisense oligonucleotides showed inhibition of LFM-1 endogenous synthesis and DNA replication with a 2n DNA content, and concomitant arrest of cell proliferation after 48hs of treatment. Approximately 70% of the cultures consisted of sparse cells, and no groups exceeding 4 cells per islet were observed with a viability rating between 90-96%. In contrast, LFM-1 protein displayed sub-cellular nuclear distribution (sparse fluorescent dots) in untreated and random controls during G1 and S phases of the cell cycle suggesting a co-localization in areas of DNA replication and an involvement in the DNA synthesis machinery. No LFM-1 electrophoresis bands (87kDa precursor, 65-intermedia and 58kDa chromosome polypeptides) were detected in parallel samples treated with LFM-1 AS ODNs confirming the immunofluorescence results and supporting our previous reports in other human epithelia.