Gene transfer of integration defective anti-HSV-1 meganuclease to human corneas ex vivo.

Corneal graft rejection is a major problem in chronic herpetic keratitis (HK) patients with latent infection. A new class of antiviral agents targeting latent and active forms of herpes simplex virus type 1 (HSV-1) is importantly required. Meganucleases are sequence-specific homing endonucleases capable of inducing DNA double-strand breaks. A proof-of-concept experiment has shown that tailor-made meganucleases are efficient against HSV-1 in vitro. To take this work a step forward, we hypothesized that the pre-treatment of human corneas in eye banks using meganuclease-encoding vectors will allow HK patients to receive a medicated cornea to resist the recurrence of the infection and the common graft rejection problem. However, this strategy requires efficient gene delivery to human corneal endothelium. Using recombinant adeno-associated virus, serotype 2/1 (rAAV2/1), efficient gene delivery of a reporter gene was demonstrated in human corneas ex vivo. The optimum viral dose was 3.7 × 10(11) VG with an exposure time of 1 day, followed by 6 days incubation in de-swelling medium. In addition, 12 days incubation can result in transgene expression in excess of 70%. Using similar transduction conditions, meganuclease transgene expression was detected in 39.4% of the endothelial cells after 2 weeks in culture. Reduction of the total viral load in the media and the endothelial cells of corneas infected with HSV-1 was shown. Collectively, this work provides information about the optimum conditions to deliver genetic material to the cornea, and demonstrates for the first time the expression of meganuclease in human corneas ex vivo and its antiviral activity. In conclusion, we demonstrate that the treatment of human corneas in eye banks before transplantation is a new approach to address the unmet clinical needs in corneal diseases.

Development of a hemicornea from human primary cell cultures for pharmacotoxicology testing.

We report the reconstruction and characterization of a hemicornea (epithelialized stroma), using primary human cells, for use in research and as an alternative to the use of animals in pharmacotoxicology testing. To create a stromal equivalent, keratocytes from human corneas were cultured in collagen-glycosaminoglycan-chitosan foams. Limbal stem cell-derived epithelial cells were seeded on top of these, giving rise to hemi-corneas. The epithelium appeared morphologically similar to its physiological counterpart, as shown by the basal cell expression of p63 isoforms including, in some cases, the stem cell marker p63DeltaNalpha, and the expression of keratin 3 and 14-3-3sigma in the upper cell layers. In addition, the cuboidal basal epithelial cells were anchored to a basement membrane containing collagen IV, laminin 5, and hemidesmosomes. In the stromal part, the keratocytes colonized the porous scaffold, formed a network of interconnecting cells, and synthesized an ultrastructurally organized extracellular matrix (ECM) containing collagen types I, V, and VI. Electron microscopy showed the newly synthesized collagen fibrils to have characteristic periodic striations, with diameters and interfibril spacings similar to those found in natural corneas. Compared to existing models for corneal pharmacotoxicology testing, this new model more closely approaches physiological conditions by including the inducing effects of mesenchyme and cell-matrix interactions on epithelial cell morphogenesis.

Nonlinear coupling among heart rate, blood pressure, and respiration in patients susceptible to neuromediated syncope.

The aim of this study is to evaluate the degree of coupling between the cardiovascular variability series and the respiration in subjects susceptible to neurally mediated syncope. Twenty-one informed patients susceptible to syncope and ten sex- and age-matched control subjects were enrolled in the study. ECG, respiration activity, and arterial blood pressure were simultaneously recorded at rest (controlled and free breathing) and during the 70 degrees head-up TILT test (free breathing). The degree of nonlinear coupling among heart rate variability (HRV), blood pressure variability (BPV), and respiration was quantified by means of two indices according to a multivariate embedding-based approach. Eleven patients developed syncope during the TILT test. We found that during the late TILT phase, the TILT-positive group experienced a significant increase in nonlinear coupling respect to the mid TILT phase (p < 0.01, Wilcoxon nonparametric test for pair data) while the TILT-negative group did not (p < 0.01, Mann-Whitney U-test). If the proposed nonlinear coupling indexes can be considered expression of the coupling mechanisms involved in the vagal regulation of the cardiovascular system, an increase in vagal tone accompanied by a decrease in sympathetic activity seem to occur before a vasovagal event.

A mathematical model of the fetal cardiovascular system based on genetic algorithms as identification technique.

The development of fetal cardiac surgery, considered the ultimate goal in the treatment of congenital cardiac malformations, needs to be supported by detailed knowledge of the blood circulation in the fetal cardiovascular system. The hemodynamic behavior in distal territories is usually inferred from vessel resistance indices, which give limited physiological information. This study presents a mathematical model of the human fetal global cardiovascular system, developed to clarify the relationships and differences existing between upper and lower body circulation. We modelled the heart with two time-varying capacitances, each representing the respective ventricle's pressure-volume relationship. The fetal vascular system was represented using two six-element Windkessel models, for the upper and lower body respectively. We obtained the identification of the set of circuital and elastance function parameters of the model using Genetic Algorithms (GAs), which follow the laws of evolutionary theory. We compared the results of our numerical study on the model identified with data collected from measurements and literature, to validate the proposed global cardiovascular system model of the human fetus. This model is intended as an instrument to investigate the differences in blood distribution between the different vascular districts in the upper and lower fetal body and the role of the aortic isthmus, the small tract of vessel connecting upper and lower fetal vascular beds; it may also represent a useful tool in the assessment of dynamic balance during mechanical assistance of circulation.

Principal stress analysis in LDA measurement of the flow field downstream of 19-mm Sorin Bicarbon heart valve.

Heart valve replacement has become, since many years, a common surgical practice. Along with the improvement that the patients' health has derived from it, however, a certain amount of risk could not be avoided, bound to the inevitable hemodynamic disturbances that an artificial device generates. A major shortcoming, often reported, is the formation of thrombus on the edge of the prosthetic valve, with a possible obstruction of the orifices through which blood should normally flow undisturbed. Hemolysis is another possible consequence of the implantation of a mechanical heart valve, generally correlated to turbulence downstream of prosthetic heart valves (PHV). As it is agreed upon by many researchers, the risk of thrombogenicity or hemolysis is higher in those valves that are more subject to promote turbulence and flow separation in the flow through them. In the following paper, we present a study of the turbulence-related shear stress downstream of a bileaflet valve of minimum size (19 mm external diameter) Sorin Bicarbon. This size was chosen, accordingly to the Food & Drug Administration (FDA) draft guidance suggestion to investigate the worst case in turbulence promoted by PHVs, in order to have the highest velocity gradients and shear stresses for the FDA-stated cardiac output (6 1/min), related to maximum Reynolds number conditions. Velocity data were collected with the two-dimensional laser Doppler anemometry (LDA) technique; whereas this approach does not investigate directly all three components of the flow field, in the present case (bileaflet valves) it is not a limitation to the assessment of the maximum turbulence shear stress (TSS), thanks to the two-dimensional flow nature downstream of bileaflet models. Data taken in coincident mode were elaborated in order to determine the maximum shear stress in the measured points in the flow field, using the 2D Principal Stress Analysis (PSA). The consequences of a variable principal normal stress direction all along the measured profile will be illustrated in terms of differences between measured and maximum shear stresses. Results show the need to estimate the maximum values for the TSS and the direction along which it is obtained to correctly define the turbulent flow field downstream of PHVs.