Jeremías G. Galletti

Improved keratoconus detection by ocular response analyzer testing after consideration of corneal thickness as a confounding factor.

PURPOSE To compare corneal hysteresis (CH) and corneal resistance factor (CRF) between normal eyes and eyes with keratoconus correcting for the effect of central corneal thickness (CCT) and to estimate keratoconus detection sensitivity and specificity of these parameters. METHODS Observational case series of 102 normal eyes (control group) and 77 eyes with keratoconus (keratoconus group). Examination included corneal topography, tomography, and biomechanical testing with the Ocular Response Analyzer (Reichert Technologies). The confounding effect of CCT was controlled by stratification (20-μm CCT intervals) and linear transformation. Receiver operating characteristic curves were used to identify optimal CH and CRF cutoff points for keratoconus detection. Main outcome measures were CCT, CH, CRF, and diagnostic performance of CH and CRF. RESULTS Corneal hysteresis and CRF were positively correlated to CCT in both groups. In the control versus keratoconus group, CH was 9.79 ± 1.51 vs 8.49 ± 1.48 (P<.0001) and CRF was 9.55 ± 1.64 vs 7.24 ± 1.43 (P<.0001), respectively. Only CRF remained significantly lower in eyes with keratoconus after CCT stratification in 20-μm intervals, and keratoconus grade influenced CH and CRF. Transformed CH and CRF data confirmed these results. Sensitivity and specificity of CRF cutoff points ranged between 83% and 94% and 69% and 83%, respectively. True positive rate for CRF in eyes with keratoconus with normal topography was 84%. CONCLUSIONS Corneal resistance factor was better than CH for detecting keratoconic corneas once the effect of CCT on ORA measurements was considered, even for topographically unaffected fellow eyes of patients with keratoconus. The CCT-corrected CRF cutoff values and transformed indices may be of clinical use.

Chronic lymphocytic leukemia cells bind and present the erythrocyte protein band 3: possible role as initiators of autoimmune hemolytic anemia.

The mechanisms underlying the frequent association between chronic lymphocytic leukemia (CLL) and autoimmune hemolytic anemia are currently unclear. The erythrocyte protein band 3 (B3) is one of the most frequently targeted Ags in autoimmune hemolytic anemia. In this study, we show that CLL cells specifically recognize B3 through a still unidentified receptor. B3 interaction with CLL cells involves the recognition of its N-terminal domain and leads to its internalization. Interestingly, when binding of erythrocyte-derived vesicles as found physiologically in blood was assessed, we observed that CLL cells could only interact with inside-out vesicles, being this interaction strongly dependent on the recognition of the N-terminal portion of B3. We then examined T cell responses to B3 using circulating CLL cells as APCs. Resting B3-pulsed CLL cells were unable to induce T cell proliferation. However, when deficient costimulation was overcome by CD40 engagement, B3-pulsed CLL cells were capable of activating CD4+ T cells in a HLA-DR-dependent fashion. Therefore, our work shows that CLL cells can specifically bind, capture, and present B3 to T cells when in an activated state, an ability that could allow the neoplastic clone to trigger the autoaggressive process against erythrocytes.

Benzalkonium chloride breaks down conjunctival immunological tolerance in a murine model

The impact of topical eye drops with benzalkonium chloride (BAK) as a preservative could involve more than the reported toxic effects on the ocular surface epithelium and ultimately affect the immune balance of the conjunctiva. We found that BAK not only impairs tolerance induction in a murine model, but leads to mild systemic immunization. Contrasting with antigen only–treated mice, there was no induction of interleukin 10–producing antigen-specific CD4+ cells in BAK-treated animals. Moreover, the tolerogenic capacity of migrating dendritic cells (DCs) was reduced, apparently involving differential conditioning by soluble epithelial factors. Accordingly, epithelial cells exposed in vitro to BAK were less suppressive and failed to induce tolerogenic DCs in culture. As this effect of BAK was dependent on epithelial nuclear factor κB pathway activation, our findings may provide new therapeutic targets. Thus, tolerance breakdown by BAK should be considered an important factor in the management of glaucoma and immune-mediated ocular surface disorders.

Desiccating stress‐induced disruption of ocular surface immune tolerance drives dry eye disease

Dry eye is an allegedly autoimmune disorder for which the initiating mechanisms and the targeted antigens in the ocular surface are not known, yet there is extensive evidence that a localized T helper type 1 (Th1)/Th17 effector T cell response is responsible for its pathogenesis. In this work, we explore the reconciling hypothesis that desiccating stress, which is usually considered an exacerbating factor, could actually be sufficient to skew the ocular surfaces mucosal response to any antigen and therefore drive the disease. Using a mouse model of dry eye, we found that desiccating stress causes a nuclear factor kappa B (NF‐κB)‐ and time‐dependent disruption of the ocular surfaces immune tolerance to exogenous ovalbumin. This pathogenic event is mediated by increased Th1 and Th17 T cells and reduced regulatory T cells in the draining lymph nodes. Conversely, topical NF‐κB inhibitors reduced corneal epithelial damage and interleukin (IL)‐1β and IL‐6 levels in the ocular surface of mice under desiccating stress. The observed effect was mediated by an augmented regulatory T cell response, a finding that highlights the role of mucosal tolerance disruption in dry eye pathogenesis. Remarkably, the NF‐κB pathway is also involved in mucosal tolerance disruption in other ocular surface disorders. Together, these results suggest that targeting of mucosal NF‐κB activation could have therapeutic potential in dry eye.

Agreement between placido topography and Scheimpflug tomography for corneal astigmatism assessment.

PURPOSE To evaluate inter-device agreement between Placido topography (iTrace; Tracey Technologies, Houston, TX) and Scheimpflug tomography (Pentacam; Oculus Optikgeräte GmbH, Wetzlar, Germany) for measuring corneal power and cylinder and axis of astigmatism. METHODS Observational case series of 54 eyes from 54 subjects with no ocular disease. Main outcome measures were corneal power, cylinder power, and axis of astigmatism and their agreement was assessed by Bland–Altman analysis. RESULTS For corneal power and corneal cylinder, 95% limits of agreement (LoA) were considered good (−0.38 to 0.45 diopters [D] and −0.49 to 0.27 D, respectively). In contrast, the 95% LoA for corneal astigmatism axis exceeded the clinically relevant margins (−14.8 to 13.5): 28 eyes (52%) had a greater than 5° difference, 10 eyes (19%) had a greater than 10° difference, and 4 eyes (7%) had a greater than 20° difference between instruments. This absolute difference was significantly correlated with average corneal cylinder (Spearman’s r = −0.379, P = .005) but not with average corneal power. In eyes with corneal astigmatism 2 D or greater, the 95% LoA for axis were −8.7° to 6.7°, whereas in those with corneal astigmatism less than 1 D, the 95% LoA for axis were −19.1° to 16.6°. CONCLUSIONS Placido topography and Scheimpflug tomography show good agreement for corneal power and cylinder, but not for corneal astigmatism axis. These instruments could be used interchangeably only in eyes with corneal astigmatism of 2 D or greater.

Combining Ocular Response Analyzer Metrics for Corneal Biomechanical Diagnosis

PURPOSE To evaluate corneal biomechanical properties in non-keratoconic myopic eyes and to identify descriptors for improving the specificity of the Ocular Response Analyzer (ORA) (Reichert Ophthalmic Instruments, Depew, NY) testing for subclinical keratoconus detection. METHODS Observational case series of 52 non-keratoconic non-myopic eyes and 97 non-keratoconic myopic eyes (spherical equivalent < -5 diopters [D]) in dataset 1 and 87 non-keratoconic eyes and 73 eyes with subclinical keratoconus in dataset 2. Examination included corneal topography, tomography, and biomechanical testing with the ORA. Receiver operating characteristic curves and logistic regression were used to identify optimal combinations of biomechanical indices for keratoconus detection. Main outcome measures were corneal thickness-corrected hysteresis (DifCH) and resistance factor (DifCRF), the difference between these two (CH-CRF), and the diagnostic performance of their combinations. RESULTS Compared to non-keratoconic non-myopic eyes, non-keratoconic myopic eyes with flat corneas (average corneal power < 44.0 D) had reduced DifCH (mean ± standard deviation, 0.11 ± 1.27 vs -0.79 ± 1.50, P < .01) and DifCRF (0.24 ± 1.16 vs -0.70 ± 1.59, P < .01) values, whereas non-keratoconic myopic eyes with steep corneas showed no difference. Keratoconic eyes exhibited lower DifCH and DifCRF values than non-keratoconic myopic eyes. Combinations of DifCH, DifCRF, and CH-CRF had increased specificity (> 80%) for subclinical keratoconus compared to the DifCRF index alone (71%). CONCLUSIONS In biomechanical keratoconus screening, some non-keratoconic myopic eyes show altered ocular biomechanical properties and are identified as false-positive cases. The low specificity of DifCRF when dealing with these non-keratoconic eyes could be improved by considering additional biomechanical descriptors such as DifCH and CH-CRF, which seem to be indicative of the aforementioned biomechanical profile.

Restoring Conjunctival Tolerance by Topical Nuclear Factor–κB Inhibitors Reduces Preservative-Facilitated Allergic Conjunctivitis in Mice

PURPOSE To evaluate the role of nuclear factor-κB (NF-κB) activation in eye drop preservative toxicity and the effect of topical NF-κB inhibitors on preservative-facilitated allergic conjunctivitis. METHODS Balb/c mice were instilled ovalbumin (OVA) combined with benzalkonium chloride (BAK) and/or NF-κB inhibitors in both eyes. After immunization, T-cell responses and antigen-induced ocular inflammation were evaluated. Nuclear factor-κB activation and associated inflammatory changes also were assessed in murine eyes and in an epithelial cell line after BAK exposure. RESULTS Benzalkonium chloride promoted allergic inflammation and leukocyte infiltration of the conjunctiva. Topical NF-κB inhibitors blocked the disruptive effect of BAK on conjunctival immunological tolerance and ameliorated subsequent ocular allergic reactions. In line with these findings, BAK induced NF-κB activation and the secretion of IL-6 and granulocyte-monocyte colony-stimulating factor in an epithelial cell line and in the conjunctiva of instilled mice. In addition, BAK favored major histocompatibility complex (MHC) II expression in cultured epithelial cells in an NF-κB-dependent fashion after interaction with T cells. CONCLUSIONS Benzalkonium chloride triggers conjunctival epithelial NF-κB activation, which seems to mediate some of its immune side effects, such as proinflammatory cytokine release and increased MHC II expression. Breakdown of conjunctival tolerance by BAK favors allergic inflammation, and this effect can be prevented in mice by topical NF-κB inhibitors. These results suggest a new pharmacological target for preservative toxicity and highlight the importance of conjunctival tolerance in ocular surface homeostasis.

Mucosal immune tolerance at the ocular surface in health and disease

The ocular surface is constantly exposed to environmental irritants, allergens and pathogens, against which it can mount a prompt immune response to preserve its integrity. But to avoid unnecessary inflammation, the ocular surfaces mucosal immune system must also discriminate between harmless and potentially dangerous antigens, a seemingly complicated task. Despite its unique features, the ocular surface is a mucosal lining, and as such, it shares some homeostatic and pathophysiological mechanisms with other mucosal surfaces. The purpose of this review is to explore the mucosal homeostatic immune function of the ocular surface in both the healthy and diseased states, with a special focus on mucosal immunology concepts. The information discussed in this review has been retrieved by PubMed searches for literature published from January 1981 to October 2016.

Multivariate Analysis of the Ocular Response Analyzer’s Corneal Deformation Response Curve for Early Keratoconus Detection

Purpose. To thoroughly analyze corneal deformation responses curves obtained by Ocular Response Analyzer (ORA) testing in order to improve subclinical keratoconus detection. Methods. Observational case series of 87 control and 73 subclinical keratoconus eyes. Examination included corneal topography, tomography, and biomechanical testing with ORA. Factor analysis, logistic regression, and receiver operating characteristic curves were used to extract combinations of 45 corneal waveform descriptors. Main outcome measures were corneal-thickness-corrected corneal resistance factor (ccCRF), combinations of corneal descriptors, and their diagnostic performance. Results. Thirty-seven descriptors differed significantly in means between groups, and among them ccCRF afforded the highest individual diagnostic performance. Factor analysis identified first- and second-peak related descriptors as the most variable one. However, conventional biomechanical descriptors corneal resistance factor and hysteresis differed the most between control and keratoconic eyes. A combination of three factors including several corneal descriptors did not show better diagnostic performance than a combination of conventional indices. Conclusion. Multivariate analysis of ORA signals did not surpass simpler models in subclinical keratoconus detection, and there is considerable overlap between normal and ectatic eyes irrespective of the analysis model. Conventional biomechanical indices seem to already provide the best performance when appropriately considered.

Surface localization of high-mobility group nucleosome-binding protein 2 on leukemic B cells from patients with chronic lymphocytic leukemia is related to secondary autoimmune hemolytic anemia

Abstract Chronic lymphocytic leukemia (CLL) is the main cause of autoimmune hemolytic anemia (AHA). However, the cellular basis underlying this strong association remains unclear. We previously demonstrated that leukemic B cells from patients with CLL recognize the erythrocyte protein Band 3, a prevalent autoantigen in AHA. Here we show that the major binding site of Band 3 on leukemic cells is an extrinsic protein identified as high-mobility group nucleosome binding protein 2 (HMGN2), a nucleosome-interacting factor which has not been previously reported at the cell surface. T lymphocytes do not express HMGN2 or bind Band 3. Removal of HMGN2 from the cell membrane abrogated the capacity of Band 3-pulsed CLL cells to induce CD4 + T cell proliferation. We conclude that surface HMGN2 in leukemic B cells is involved in Band 3 binding, uptake and presentation to CD4 + T lymphocytes, and as such may favor the initiation of AHA secondary to CLL.