Kathryn L. Pepple

Wide-field optical coherence tomography based microangiography for retinal imaging

Optical coherence tomography angiography (OCTA) allows for the evaluation of functional retinal vascular networks without a need for contrast dyes. For sophisticated monitoring and diagnosis of retinal diseases, OCTA capable of providing wide-field and high definition images of retinal vasculature in a single image is desirable. We report OCTA with motion tracking through an auxiliary real-time line scan ophthalmoscope that is clinically feasible to image functional retinal vasculature in patients, with a coverage of more than 60 degrees of retina while still maintaining high definition and resolution. We demonstrate six illustrative cases with unprecedented details of vascular involvement in retinal diseases. In each case, OCTA yields images of the normal and diseased microvasculature at all levels of the retina, with higher resolution than observed with fluorescein angiography. Wide-field OCTA technology will be an important next step in augmenting the utility of OCT technology in clinical practice.

Caveats about QuantiFERON-TB gold in-tube testing for uveitis

In this Issue of the Journal, La Distia Nora and associates describe the features of uveitis in their cohort of patients with positive QuantiFERON–TB Gold In-Tube testing (QFT).1 This study highlights the continued challenge interpretation of QFT testing presents to the clinician treating uveitis in places where tuberculosis (TB) is not endemic. The United States Centers for Disease Control and Prevention recommendations for use of the QFT test include surveillance purposes or to identify persons likely to benefit from treatment. The reported specificity of the QFT for pulmonary and latent tuberculosis ranges from 91%–99%, but reported sensitivity is somewhat lower (89%–91%).2,3 As such, separating true-positives from false-positives and deciding when to initiate anti-TB treatment still requires a case-by-case analysis in patients with uveitis. As described by the authors, such an analysis was performed to determine the final group of 42 patients who were selected for anti-TB therapy out of the original 96 QFT-positive patients identified. False-positives are a result of the low positive predictive value of TB testing in areas that have very low pretest probability of disease (prevalence of TB exposure is ~4% in the US).4 Given a test sensitivity of 90% and a specificity of 99%, by Bayesian analysis, the positive predictive value of the test is ~78% (ie, 22% false-positive), while the negative predictive value is ~99%. Thus a negative test effectively rules out tuberculosis, but a substantial number of positive tests are false-positive. The reasons for false-positive results may include cross-reactivity with pulmonary5 and ocular6 Mycobacterium kansasii infection or exposure to a limited number of other nontuberculous mycobacteria. False-positives have also been reported in connection with a nonspecific defect in the assay vial.7 In the case where high suspicion for a false-positive result develops, one option open to the clinician is to repeat testing, as “reversion” to a negative test without treatment has been reported in uveitis patients with a positive QFT result and disease characteristics not suggestive of true mycobacterial infection.8 The current study suggests an interesting association of false-positive QFT results in patients with sarcoidosis. In the current study, 9 of 77 quantiferon-positive patients (12%) were confirmed to have a diagnosis of sarcoidosis based on radiologic and histologic features, as well as negative results of staining, cultures, and polymerase chain reaction (PCR) for M tuberculosis in lymph node biopsies. These results suggest that sarcoidosis may also be associated with elevated interferon gamma levels in some uveitis patients, resulting in a false-positive test. One of the authors of this Editorial has recently reported a similar case of positive QFT in a patient with histologic evidence of ocular sarcoidosis (but not TB) on chorioretinal biopsy.9 As the authors of La Distia Nora and associates’ study have pointed out, anassociation betweenTBand sarcoidosis has been described, and studies have suggested that mycobacterial antigens may represent the inciting agent in sarcoidosis.10 The QFT is based on the quantification of interferon gamma released from sensitized lymphocytes in whole blood incubated with peptides from M tuberculosis antigens and control antigens. Although the peptides used (ESAT-6, CFP-10, and TB7.7) are more specific for TB exposure than the antigens present in the purified protein derivative, they are not expressed exclusively by M tuberculosis. Although rare, a positive QFT can also be attributable to exposure to atypical mycobacteria including M kansasii, M szulgai, M gordonae, M flavescens, and M marinum.6 One of the QFT antigens is early secretory antigenic target 6 (ESAT-6). Interestingly, when tested in isolation (ie, not as part of the QFT), a cellular immune response to ESAT-6 has been reported in a significant number of patients with pulmonary sarcoidosis, including the induction of interferon gamma–producing T cell.11–13 However, these results have not been recapitulated using commercially available quantiferon testing, possibly because of a different number or combination of ESAT-6 peptides used in the assay. Cross-reactivity of blood leukocytes to ESAT-6 has also not been tested specifically in uveitic sarcoidosis (as opposed to pulmonary disease). In conclusion, the utility of routine screening using QFT testing in nonendemic areas remains problematic owing to the risk for false-positive results. The present study further adds to the complexity of quantiferon interpretation with the finding of false-positive QFT in a number of patients with uveitis from underlying sarcoidosis. These results suggest that sarcoidosis should be suspected in uveitis cases with positive QFT and no evidence of tuberculosis infection. The implications for therapy are significant, as treatment of true TB with immunosuppression can be catastrophic (although, conversely, several recent studies have suggested that cutaneous and pulmonary sarcoidosis may respond favorably to multidrug antitubercular treatment14,15). Ultimately, the diagnosis of TB or sarcoidosis in challenging cases must rely on pathologic examination of tissue specimens and not solely on the result of the QFT assay. Molecular studies, such as PCR testing of ocular fluids for M tuberculosis, may be useful in the diagnosis and treatment of these diagnostic dilemmas.

Quantitative Assessment of Anterior Segment Inflammation in a Rat Model of Uveitis Using Spectral-Domain Optical Coherence Tomography.

Purpose To develop anterior segment spectral-domain optical coherence tomography (SD-OCT) and quantitative image analysis for use in experimental uveitis in rats. Methods Acute anterior uveitis was generated in Lewis rats. A spectral domain anterior segment OCT system was used to image the anterior chamber (AC) and ciliary body at baseline and during peak inflammation 2 days later. Customized MatLab image analysis algorithms were developed to segment the AC, count AC cells, calculate central corneal thickness (CCT), segment the ciliary body and zonules, and quantify the level of ciliary body inflammation with the ciliary body index (CBI). Images obtained at baseline and during peak inflammation were compared. Finally, longitudinal imaging and image analysis was performed over the 2-week course of inflammation. Results Spectral-domain optical coherence tomography identifies structural features of inflammation. Anterior chamber cell counts at peak inflammation obtained by automated image analysis and human grading were highly correlated (r = 0.961), and correlated well with the histologic score of inflammation (r = 0.895). Inflamed eyes showed a significant increase in average CCT (27 μm, P = 0.02) and an increase in average CBI (P < 0.0001). Longitudinal imaging and quantitative image analysis identified a significant change in AC cell and CBI on day 2 with spontaneous resolution of inflammation by day 14. Conclusions Spectral-domain optical coherence tomography provides high-resolution images of the structural changes associated with anterior uveitis in rats. Anterior chamber cell count and CBI determined by semi-automated image analysis strongly correlates with inflammation, and can be used to quantify inflammation longitudinally in single animals.

Optical coherence tomography based microangiography for quantitative monitoring of structural and vascular changes in a rat model of acute uveitis in vivo: a preliminary study

Abstract. Uveitis models in rodents are important in the investigation of pathogenesis in human uveitis and the development of appropriate therapeutic strategies for treatment. Quantitative monitoring of ocular inflammation in small animal models provides an objective metric to assess uveitis progression and/or therapeutic effects. We present a new application of optical coherence tomography (OCT) and OCT-based microangiography (OMAG) to a rat model of acute anterior uveitis induced by intravitreal injection of a killed mycobacterial extract. OCT/OMAG is used to provide noninvasive three-dimensional imaging of the anterior segment of the eyes prior to injection (baseline) and two days post-injection (peak inflammation) in rats with and without steroid treatments. OCT imaging identifies characteristic structural and vascular changes in the anterior segment of the inflamed animals when compared to baseline images. Characteristics of inflammation identified include anterior chamber cells, corneal edema, pupillary membranes, and iris vasodilation. In contrast, no significant difference from the control is observed for the steroid-treated eye. These findings are compared with the histology assessment of the same eyes. In addition, quantitative measurements of central corneal thickness and iris vessel diameter are determined. This pilot study demonstrates that OCT-based microangiography promises to be a useful tool for the assessment and management of uveitis in vivo.

Comparative Proteomic Analysis of Two Uveitis Models in Lewis Rats

PURPOSE Inflammation generates changes in the protein constituents of the aqueous humor. Proteins that change in multiple models of uveitis may be good biomarkers of disease or targets for therapeutic intervention. The present study was conducted to identify differentially-expressed proteins in the inflamed aqueous humor. METHODS Two models of uveitis were induced in Lewis rats: experimental autoimmune uveitis (EAU) and primed mycobacterial uveitis (PMU). Differential gel electrophoresis was used to compare naïve and inflamed aqueous humor. Differentially-expressed proteins were separated by using 2-D gel electrophoresis and excised for identification with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Expression of select proteins was verified by Western blot analysis in both the aqueous and vitreous. RESULTS The inflamed aqueous from both models demonstrated an increase in total protein concentration when compared to naïve aqueous. Calprotectin, a heterodimer of S100A8 and S100A9, was increased in the aqueous in both PMU and EAU. In the vitreous, S100A8 and S100A9 were preferentially elevated in PMU. Apolipoprotein E was elevated in the aqueous of both uveitis models but was preferentially elevated in EAU. Beta-B2-crystallin levels decreased in the aqueous and vitreous of EAU but not PMU. CONCLUSIONS The proinflammatory molecules S100A8 and S100A9 were elevated in both models of uveitis but may play a more significant role in PMU than EAU. The neuroprotective protein β-B2-crystallin was found to decline in EAU. Therapies to modulate these proteins in vivo may be good targets in the treatment of ocular inflammation.

Primed Mycobacterial Uveitis (PMU): Histologic and Cytokine Characterization of a Model of Uveitis in Rats

PURPOSE The purpose of this study was to compare the histologic features and cytokine profiles of experimental autoimmune uveitis (EAU) and a primed mycobacterial uveitis (PMU) model in rats. METHODS In Lewis rats, EAU was induced by immunization with interphotoreceptor binding protein peptide, and PMU was induced by immunization with a killed mycobacterial extract followed by intravitreal injection of the same extract. Clinical course, histology, and the cytokine profiles of the aqueous and vitreous were compared using multiplex bead fluorescence immunoassays. RESULTS Primed mycobacterial uveitis generates inflammation 2 days after intravitreal injection and resolves spontaneously 14 days later. CD68+ lymphocytes are the predominant infiltrating cells and are found in the anterior chamber, surrounding the ciliary body and in the vitreous. In contrast to EAU, no choroidal infiltration or retinal destruction is noted. At the day of peak inflammation, C-X-C motif ligand 10 (CXCL10), IL-1β, IL-18, and leptin were induced in the aqueous of both models. Interleukin-6 was induced 2-fold in the aqueous of PMU but not EAU. Cytokines elevated in the aqueous of EAU exclusively include regulated on activation, normal T cell expressed and secreted (RANTES), lipopolysaccharide-induced CXC chemokine (LIX), growth-related oncogene/keratinocyte chemokine (GRO/KC), VEGF, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), and IL-17A. In the vitreous, CXCL10, GRO/KC, RANTES, and MIP-1α were elevated in both models. Interleukin-17A and IL-18 were elevated exclusively in EAU. CONCLUSIONS Primed mycobacterial uveitis generates an acute anterior and intermediate uveitis without retinal involvement. Primed mycobacterial uveitis has a distinct proinflammatory cytokine profile compared with EAU, suggesting PMU is a good complementary model for study of immune-mediated uveitis. CXCL10, a proinflammatory cytokine, was increased in the aqueous and vitreous of both models and may be a viable therapeutic target.

In Vivo Bioluminescence Imaging for Longitudinal Monitoring of Inflammation in Animal Models of Uveitis

Purpose We develop a quantitative bioluminescence assay for in vivo longitudinal monitoring of inflammation in animal models of uveitis. Methods Three models of experimental uveitis were induced in C57BL/6 albino mice: primed mycobacterial uveitis (PMU), endotoxin-induced uveitis (EIU), and experimental autoimmune uveitis (EAU). Intraperitoneal injection of luminol sodium salt, which emits light when oxidized, provided the bioluminescence substrate. Bioluminescence images were captured by a PerkinElmer In Vivo Imaging System (IVIS) Spectrum and total bioluminescence was analyzed using Living Image software. Bioluminescence on day zero was compared to bioluminescence on the day of peak inflammation for each model. Longitudinal bioluminescence imaging was performed in EIU and EAU. Results In the presence of luminol, intraocular inflammation generates detectable bioluminescence in three mouse models of uveitis. Peak bioluminescence in inflamed PMU eyes (1.46 × 105 photons/second [p/s]) was significantly increased over baseline (1.47 × 104 p/s, P = 0.01). Peak bioluminescence in inflamed EIU eyes (3.18 × 104 p/s) also was significantly increased over baseline (1.09 × 104 p/s, P = 0.04), and returned to near baseline levels by 48 hours. In EAU, there was a nonsignificant increase in bioluminescence at peak inflammation. Conclusions In vivo bioluminescence may be used as a noninvasive, quantitative measure of intraocular inflammation in animal models of uveitis. Primed mycobacterial uveitis and EIU are both acute models with robust anterior inflammation and demonstrated significant changes in bioluminescence corresponding with peak inflammation. Experimental autoimmune uveitis is a more indolent posterior uveitis and generated a more modest bioluminescent signal. In vivo imaging system bioluminescence is a nonlethal, quantifiable assay that can be used for monitoring inflammation in animal models of uveitis.

Tubulointerstitial nephritis and uveitis

Purpose of review Tubulointerstitial nephritis and uveitis (TINU) is an important yet underrecognized ocular inflammatory syndrome. This review summarizes key historical publications that identified and defined the syndrome, and more recent literature that reveal the importance of urinary &bgr;2-microglobulin testing and kidney biopsy in the diagnostic evaluation of patients with TINU. Additionally, research studies providing new insights into disease pathogenesis are highlighted. Recent findings In contrast with initial reports of TINU manifesting exclusively as an anterior uveitis in pediatric patients, more recent reports have identified TINU in patients of all ages with a wide range of ocular manifestations. Urinary &bgr;2-microglobulin has emerged as a sensitive and specific laboratory screening test, and the role of kidney biopsy in differentiating TINU from sarcoidosis continues to evolve. Genetic studies have identified HLA-DQA1*01, HLA-DQB1*05, and HLA-DRB1*01 as high-risk alleles and the identification of antimonomeric C-reactive protein antibodies suggests a role for humoral immunity in disease pathogenesis. Management strategies have evolved to include systemic anti-inflammatory treatment as a result of important outcome studies in patients with significant renal and ocular disease. Summary With greater recognition, understanding, and treatment of this syndrome, both ocular inflammation and renal disease can be better addressed.

Urinary β2-Microglobulin Testing in Pediatric Uveitis: A Case Report of a 9-Year-Old Boy with Renal and Ocular Sarcoidosis.

We present here a case of a 9-year-old boy with bilateral anterior uveitis and an extremely elevated urinary β2-microglobulin level (25,400 μg/l). The normal range for urinary excretion of β2-microglobulin is 0-300 μg/l. In patients with tubulointerstitial nephritis and uveitis syndrome (TINU), elevations typically range from 1,260 to 5,160 μg/l. Renal biopsy was pursued, and significant granulomatous interstitial nephritis consistent with sarcoidosis was identified. Systemic immune modulation was required for control of ocular inflammation. This case highlights the importance of urinary β2-microglobulin testing in the pediatric patient uveitis population, and additionally the need to pursue kidney biopsy in the presence of extreme elevations in urinary β2-microglobulin to differentiate between TINU and sarcoidosis.

Automated three-dimensional cell counting method for grading uveitis of rodent eye in vivo with optical coherence tomography

In preclinical vision research, cell grading in small animal models is essential for the quantitative evaluation of intraocular inflammation. Here, we present a new and practical optical coherence tomography (OCT) image analysis method for the automated detection and counting of aqueous cells in the anterior chamber (AC) of a rodent model of uveitis. Anterior segment OCT images are acquired with a 100 kHz swept-source OCT system. The proposed method consists of 2 steps. In the first step, we first despeckle and binarize each OCT image. After removing AS structures in the binary image, we then apply area thresholding to isolate cell-like objects. Potential cell candidates are selected based on their best fit to roundness. The second step performs the cell counting within the whole AC, in which additional cell tracking analysis is conducted on the successive OCT images to eliminate redundancy in cell counting. Finally, 3D cell grading using the proposed method is demonstrated in longitudinal OCT imaging of a mouse model of anterior uveitis in vivo. Rendering of anterior segment (orange) of mouse eye and automatically counted anterior chamber cells (green). Inset is a top view of the rendering, showing the cell distribution across the anterior chamber.