Howard E. Savage

Evaluation of Topical Cyclosporine for the Treatment of Dry Eye Disease

OBJECTIVEnTo evaluate the use of topical cyclosporine, 0.05% (Restasis; Allergan Inc, Irvine, California), for the treatment of mild, moderate, and severe dry eye disease unresponsive to artificial tears therapy.nnnMETHODSnThis was a prospective clinical study. One hundred fifty-eight consecutive patients with dry eye disease unresponsive to artificial tears therapy were divided into 3 groups of disease severity: mild, moderate, and severe. Patients were evaluated using the Ocular Surface Disease Index for symptomatic improvement, tear breakup time, fluorescein staining, lissamine green staining, and Schirmer testing. Patients were observed for 3 to 16 months. The main outcome measure was improvement in disease.nnnRESULTSnForty-six of 62 patients with mild dry eye disease (74.1%), 50 of 69 with moderate disease (72.4%), and 18 of 27 with severe disease (66.7%) showed improvement, with 72.1% improving overall.nnnCONCLUSIONSnTopical cyclosporine shows beneficial effects in all categories of dry eye disease. Symptomatic improvement was greatest in the mild group and the best results in improvement of disease signs were in patients with severe dry eye disease.

Bacteria size determination by elastic light scattering

Light extinction and angular scattering measurements were performed on three species of bacteria with different sizes and shapes ( Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis). The Gaussian ray approximation of anomalous diffraction theory was used to determine the average bacteria size from transmission measurements. A rescaled spectra combining multiple angular data was analyzed in the framework of the Rayleigh-Gans theory of light scattering. Particle shape and size distribution is then obtained from the rescale spectra. Particle characteristics (size and/or shape) retrieved from both methods are in good agreement with size and shape measured under scanning electron microscopy. These results demonstrate that light scattering may be able to detect and identify microbial contamination in the environment.

Uveal Melanocytes, Ocular Pigment Epithelium, and Müller Cells in Culture: In Vitro Toxicology

Uveal melanocytes and the ocular pigment epithelium are located in the middle and inner layers of the eye. Müller cells (a type of glial cell) are located in the neural retina. Melanocytes, retinal pigment epithelium (RPE), and Müller cells do not participate directly in the detection or transfer of visual information, but they have various functions that support the neural retina and are essential for the maintenance of vision. Methods for the isolation and cultivation of melanocytes, RPE, and Müller cells have been established by us and other investigators. These cultured cells can be used as in vitro model systems for studying the toxicology of visible light, ultraviolet (UV) radiation, drugs, and other potentially toxic agents. Toxic effects on these cells may give rise to altered retinal function and result in impaired vision. Both melanocytes and pigment epithelium contain melanin, which has the ability to bind organic amines and metal ions. This results in the accumulation of these substances in the eye. Melanin may protect cells from chemical stress by binding toxic chemicals; but in chronic exposure, increased and lengthy binding may cause damage to these cells. Two different types of melanin are found in the eye: eumelanin and pheomelanin, which may have photoprotective and phototoxic effects, respectively. Pigment epithelium contains mainly eumelanin, whereas melanocytes contain both eumelanin and pheomelanin. Melanin is an antioxidant and with age, the antioxidant properties may diminish to the point that it may even become a prooxidant. There are also other functions of pigment epithelium and uveal melanocytes not related to melanin and there are also several functions of Müller cells that play a role in the toxicological aspects of the eye. Cultured uveal melanocytes, pigment epithelial cells, and Müller cells can be used to study the toxicology of these cells in vitro.

Innate cellular fluorescence reflects alterations in cellular proliferation

The objective of this study was to examine the question of whether unique spectral patterns were associated with cell proliferation and could be identified by comparing the fluorescence pattern of slow to rapid growing cells.

Native Fluorescence Spectroscopic Evaluation of Chemotherapeutic Effects on Malignant Cells using Nonnegative Matrix Factorization Analysis

The native fluorescence spectra of retinoic acid (RA)-treated and untreated human breast cancerous cells excited with the selective wavelengths of 300 nm and 340 nm were measured and analyzed using a blind source separation method namely Nonnegative Matrix Factorization (NMF). The results show that the fluorophores of human malignant breast cells change their compositions when they are treated with RA. The reduced contribution from tryptophan, NADH and flavin to the fluorescence of the treated breast cancerous cells was observed in comparison with that of the untreated cells. The results indicate that the decrease of adenosine triphosphate (ATP) in the RA-treated cells. The possible clinical applications of this native fluorescence study are discussed.

Native fluorescence and excitation spectroscopic changes in Bacillus subtilis and Staphylococcus aureus bacteria subjected to conditions of starvation

Fluorescence emission and excitation spectra were measured over a 7-day period for Bacillus subtilis (Bs), a spore-forming, and Staphylococcus aureus (Sa), a nonspore-forming bacteria subjected to conditions of starvation. Initially, the Bs fluorescence was predominantly due to the amino acid tryptophan. Later, a fluorescence band with an emission peak at 410 nm and excitation peak at 345 m, from dipicolinic acid, appeared. Dipicolinic acid is produced during spore formation and serves as a spectral signature for detection of spores. The intensity of the 410-nm band continued to increase over the next 3 days. The Sa fluorescence was predominantly from tryptophan and did not change over time. In 6 of the 17 Bs specimens studied, an additional band appeared with a weak emission peak at 460 cm and excitation peaks at 250, 270, and 400 nm. The addition of beta-hydroxybutyric acid to the Bs or the Sa cultures resulted in a two-order of magnitude increase in the 460-nm emission. The addition of Fe2+ quenched the 460 emission, indicating that a source of the 460-nm emission was a siderophore produced by the bacteria. We demonstrate that optical spectroscopy-based instrumentation can detect bacterial spores in real time.

Time resolved degree of polarization for human breast tissue

Polarized light propagating through the tissue retains the polarization information. The temporal profiles of the degree of polarization of the output light depends on the wavelength of the light and the type of the tissue that it propagates.

Tissue autofluorescence as an intermediate endpoint in NMBA-induced esophageal carcinogenesis

The capacity to identify subclinical neoplastic disease of the upper aerodigestive tract (UADT) using tissue auto-fluorescent spectroscopy would significantly contribute to cancer screening. Rats received N-nitrosomethyl benzylamine (NMBA), a carcinogen shown to cause esophageal malignancies. Following sacrifice at early weekly intervals, gross assessment of esophageal mucosa of NMBA-exposed rats was indistinguishable from saline-treated controls. Histopathologic evaluation, however, revealed NMBA-induced preneoplastic changes in the epithelium. Concurrent with these changes, the NMBA-exposed rats demonstrated specific alterations in autofluorescence. These results demonstrate that NMBA-induced esophageal premalignancy can be distinguished by autofluorescent properties. The capacity to detect alterations in autofluorescence may allow more sensitive screening of UADT mucosa at risk for cancer development.

Native cellular fluorescence can identify changes in epithelial thickness in-vivo in the upper aerodigestive tract

BACKGROUNDnChange in epithelial thickness is part of the neoplastic transformation process of the upper aerodigestive tract. The quantitation of native cellular fluorescence (NCF) may represent a noninvasive means of distinguishing such a change.nnnPATIENTS AND METHODSnNineteen patients with squamous neoplasms and 12 surgical specimens from cancer patients were analyzed for NCF using a hand-held fiber optic probe attached to a fluorescent spectrometer. Tumors and normal sites were analyzed for fluorescence, and tissue samples were obtained. Ratios of intensities of various emitted wavelengths were computed to quantitate and compare various spectral patterns. These ratios were then correlated with mucosal thickness.nnnRESULTSnThe 330 nm peak in the excitation scan (lambda Ex 200 to 360 nm, lambda Em 380 nm) was lost in the tumors compared with the normal sites. The 390 nm peak in the emission scan (lambda Ex 340 nm, lambda Em 360 to 660 nm) was also lost. The 290 nm/330 nm ratio in the in-vivo excitation scan (lambda Ex 200 to 360 nm, lambda Em 380 nm) correlated with changes in epithelial thickness. The 390/450 ratio in the emission scan (lambda Ex 340 nm, lambda Em 360 to 660 nm) correlated negatively with the mean epithelial thickness.nnnCONCLUSIONSnNative cellular fluorescence analysis can identify changes in neoplastic tissues, including changes in epithelial thickness.

Detecting retinoic acid-induced biochemical alterations in squamous cell carcinoma using intrinsic fluorescence spectroscopy.

Intrinsic fluorescence spectroscopy offers a new method for diagnosing head and neck cancers. By establishing a unique spectral fingerprint for benign tissue, one can readily identify subtle changes in tissue based on altered spectral patterns. The authors applied this technology to a multicellular tumor spheroid (MTS) model and obtained baseline spectral data. A cohort of MTS was treated with the chemopreventive agent retinoic acid (RA) to determine its effect on tumor cells. Excitation and emission spectroscopy were performed on the samples. Spectroscopic scans demonstrated consistently that RA‐treated MTS exhibit a decrease in the peak associated with reduced nicotinamide‐adenine dinucleotide (NADH) and an increase in the peaks associated with flavins, tryptophan, and cytokeratins when compared to controls. These findings are suggestive of alterations in cellular electron transport, an increase in proteins incorporating tryptophan, and a decrease in adenosine triphosphate (ATP) in the RA‐treated cells. A discussion of the potential clinical applications of intrinsic fluorescence spectroscopy is included.