Gracie Vargas

Use of an agent to reduce scattering in skin.

A method to increase light transport deeply into target areas of tissue would enhance both therapeutic and diagnostic laser applications. The effects of a hyperosmotic agent on the scattering properties of rat and hamster skin were investigated.

Influence of Acellular Natural Lung Matrix on Murine Embryonic Stem Cell Differentiation and Tissue Formation

We report here the first attempt to produce and use whole acellular (AC) lung as a matrix to support development of engineered lung tissue from murine embryonic stem cells (mESCs). We compared the influence of AC lung, Gelfoam, Matrigel, and a collagen I hydrogel matrix on the mESC attachment, differentiation, and subsequent formation of complex tissue. We found that AC lung allowed for better retention of cells with more differentiation of mESCs into epithelial and endothelial lineages. In constructs produced on whole AC lung, we saw indications of organization of differentiating ESC into three-dimensional structures reminiscent of complex tissues. We also saw expression of thyroid transcription factor-1, an immature lung epithelial cell marker; pro-surfactant protein C, a type II pneumocyte marker; PECAM-1/CD31, an endothelial cell marker; cytokeratin 18; alpha-actin, a smooth muscle marker; CD140a or platelet-derived growth factor receptor-alpha; and Clara cell protein 10. There was also evidence of site-specific differentiation in the trachea with the formation of sheets of cytokeratin-positive cells and Clara cell protein 10-expressing Clara cells. Our findings support the utility of AC lung as a matrix for engineering lung tissue and highlight the critical role played by matrix or scaffold-associated cues in guiding ESC differentiation toward lung-specific lineages.

Visualizing Hepatitis C Virus Infections in Human Liver by Two-Photon Microscopy

BACKGROUND & AIMS Although hepatitis C virus (HCV) is a common cause of cirrhosis and liver cancer, efforts to understand the pathogenesis of HCV infection have been limited by the low abundance of viral proteins expressed within the liver, which hinders the detection of infected cells in situ. This study evaluated the ability of advanced optical imaging techniques to determine the extent and distribution of HCV-infected cells within the liver. METHODS We combined 2-photon microscopy with virus-specific, fluorescent, semiconductor quantum dot probes to determine the proportion of hepatocytes that were infected with virus in frozen sections of liver tissue obtained from patients with chronic HCV infection. RESULTS Viral core and nonstructural protein 3 antigens were detected readily in liver tissues from patients with chronic infection without confounding tissue autofluorescence. Specificity was confirmed by blocking with specific antibodies and by tissue colocalization of distinct viral antigens. Between 7% and 20% of hepatocytes were infected in patients with plasma viral RNA loads of 10(5) IU/mL or greater. Infected cells were in clusters, which suggested spread of the virus from cell to cell. Double-stranded RNA, a product of viral replication, was abundant within cells at the center of such clusters, but often scarce in cells at the periphery, consistent with more recent infection of cells at the periphery. CONCLUSIONS Two-photon microscopy provides unprecedented sensitivity for the detection of HCV proteins and double-stranded RNA. Studies using this technology indicate that HCV infection is a dynamic process that involves a limited number of hepatocytes. HCV spread between cells is likely to be constrained by host responses.

Annexin A2 is involved in antiphospholipid antibody-mediated pathogenic effects in vitro and in vivo.

Antiphospholipid (aPL) antibodies recognize receptor-bound beta(2) glycoprotein I (beta(2)GPI) on target cells, and induce an intracellular signaling and a procoagulant/proinflammatory phenotype that leads to thrombosis. Evidence indicates that annexin A2 (A2), a receptor for tissue plasminogen activator and plasminogen, binds beta(2)GPI on target cells. However, whether A2 mediates pathogenic effects of aPL antibodies in vivo is unknown. In this work, we studied the effects of human aPL antibodies in A2-deficient (A2(-/-)) mice. A2(-/-) and A2(+/+) mice were injected with immunoglobulin G (IgG) isolated from either a patient with antiphospholipid syndrome (IgG-APS), a healthy control subject (IgG-normal human serum), a monoclonal anti-beta(2)GPI antibody (4C5), an anti-A2 monoclonal antibody, or monoclonal antibody of irrelevant specificity as control. We found that, after IgG-APS or 4C5 injections and vascular injury, mean thrombus size was significantly smaller and tissue factor activity was significantly less in A2(-/-) mice compared with A2(+/+) mice. The expression of vascular cell adhesion molecule-1 induced by IgG-APS or 4C5 in explanted A2(-/-) aorta was also significantly reduced compared with A2(+/+) mice. Interestingly, anti-A2 monoclonal antibody significantly decreased aPL-induced expression of intercellular cell adhesion molecule-1, E-selectin, and tissue factor activity on cultured endothelial cells. Together, these data indicate for the first time that A2 mediates the pathogenic effects of aPL antibodies in vivo and in vitro APS.

Morphological Changes in Blood Vessels Produced by Hyperosmotic Agents and Measured by Optical Coherence Tomography

Abstract Optical tissue clearing by hyperosmotic chemical agents significantly increases light depth penetration in skin and may improve light-based therapeutics such as laser treatment of cutaneous vascular lesions. A feasibility study was conducted to evaluate the potential role of optical clearing by glycerol in laser treatment of cutaneous vessels. Optical imaging was performed to investigate the morphological effects of glycerol on blood vessels of skin. Blood vessels were imaged using Doppler optical coherence tomography in in vivo hamster skin treated with glycerol. Images were obtained from the subdermal side to assess morphological changes in the blood vessels caused by glycerol and from the epidermal side to assess enhanced Doppler imaging of blood vessels. Application of glycerol to the subdermis resulted in venule stasis and for prolonged treatment times, arteriole stasis. In cases where flow remained in arterioles, an improved Doppler signal was detected from blood vessels when imaging transepidermally compared with the native condition. Intensity images indicated changes in blood optical properties and improved contrast of skin cross sections after glycerol application. The observed optical and morphological effects were reversed upon hydration of the skin with phosphate-buffered saline. The combination of increased depth of light penetration and the temporary slowing or cessation of flow in blood vessels could mean improved laser treatment of vessels.

Pulsed Laser-Induced Thermal Damage in Whole Blood

An investigation of the effects of laser irradiation with a wavelength of 532 nm and pulse duration of 10 ms on whole blood was performed in vitro. Threshold radiant exposures for coagulation were quantified and transient radiometric temperatures were measured. The progression of effects with increasing radiant exposure--from evaporation to coagulation-induced light scattering to aggregated coagulum formation to ablation--is described. Results indicate that coagulation and ablation occur at temperatures significantly in excess of those assumed in previous theoretical studies. An Arrhenius rate process analysis based on hemoglobin data indicates good agreement with experimental results.

High resolution imaging of epithelial injury in the sheep cervicovaginal tract: a promising model for testing safety of candidate microbicides.

Background: Access to readily available large animal models and sensitive noninvasive techniques that can be used for the evaluation of microbicide-induced changes in tissue could significantly facilitate preclinical evaluations of microbicide safety. The sheep cervicovaginal tract, with stratified squamous epithelium similar to humans, holds promise as a large animal model used before nonhuman primates. In addition, optical coherence tomography (OCT) could enable high resolution visualization of tissue morphology and noninvasive assessment of microbicide-induced epithelial injury. Methods: We evaluated the dose response of sheep cervicovaginal tract to benzalkonium chloride (BZK). Twenty sheep received treatment with phosphate-buffered saline or BZK solution (2%, 0.2%, or 0.02%). Pre- and posttreatment colposcopy and OCT images were collected and graded based on World Health Organization criteria and a previously reported scoring system, respectively. Biopsies were collected and the degree of epithelial injury and its thickness was assessed based on histology and OCT. Results: The sheep cervicovagina exhibited anatomic and microscopic features similar to the human. Extensive loss of the epithelium was noted on colposcopy and OCT after application of 2% BZK. Colposcopy detected findings in half of sheep and OCT in all sheep treated with 0.2% BZK. OCT detected differences in the 0.02% BZK-treated group compared with controls, whereas colposcopy failed to detect any changes. Conclusions: The sheep cervicovagina is similar to humans, and exhibits dose dependent epithelial changes after BZK treatment. These findings suggest that the sheep model and OCT may become valuable tools for the safety evaluation of candidate microbicides, and warrant continued development.

In vivo multimodal nonlinear optical imaging of mucosal tissue

We present a multimodal nonlinear imaging approach to elucidate microstructures and spectroscopic features of oral mucosa and submucosa in vivo. The hamster buccal pouch was imaged using 3-D high resolution multiphoton and second harmonic generation microscopy. The multimodal imaging approach enables colocalization and differentiation of prominent known spectroscopic and structural features such as keratin, epithelial cells, and submucosal collagen at various depths in tissue. Visualization of cellular morphology and epithelial thickness are in excellent agreement with histological observations. These results suggest that multimodal nonlinear optical microscopy can be an effective tool for studying the physiology and pathology of mucosal tissue.

Use of hyperosmotic chemical agent to improve the laser treatment of cutaneous vascular lesions

A method is presented for decreasing radiant exposures required for photocoagulation of cutaneous blood vessels using a hyperosmotic agent applied to skin prior to laser irradiation. The 50% probability for a given radiant exposure (RE50) to result in photocoagulation of vessels classified by type (arteriole, venule) and diameter was determined following direct (subcutaneous) laser irradiation of 84 vessels in a dorsal skin preparation pretreated with glycerol. Values were compared against results without glycerol pretreatment. A second set of experiments involved irradiation of blood vessels through the skin from the epidermal surface after application of glycerol. Subcutaneous RE50s for vessels treated with glycerol were typically several factors lower than untreated vessels. For example, arterioles in the 80- to 110-microm-diam range in untreated skin had RE50 values approximately 12 J/cm(2), compared to approximately 2 J/cm(2) in glycerol-treated cases. Results from epidermal irradiations also indicate that pretreatment with glycerol decreases radiant exposures required for photocoagulation. Vessels were successfully coagulated from the epidermal side in glycerol-treated samples using radiant exposures ranging from 1.6 J/cm(2) to 5 Jcm(2), compared to the 12 to >16 J/cm(2) range for control cases. We believe that this method could be a powerful technique for reducing the radiant exposures required for vessel photocoagulation.

Estradiol Improves Genital Herpes Vaccine Efficacy in Mice

Herpes Simplex Virus type 2 causes genital herpes but is frequently transmitted asymptomatically; therefore, a prophylactic vaccine is desirable. A candidate vaccine in clinical trials has only shown efficacy in preventing disease in women. Using this subunit vaccine candidate, we were able to demonstrate infection prophylaxis, improved disease prevention and modulated antibody production by complimenting vaccination with estradiol in our murine model. Findings of estradiol-enhanced vaccine efficacy are the first of their kind using a vaccine of this type and have potential clinical relevance to the development of other vaccines and our understanding of gender differences in vaccine efficacy.