Nancy A. McNamara

American College of Rheumatology classification criteria for Sjögren's syndrome: a data-driven, expert consensus approach in the Sjögren's International Collaborative Clinical Alliance cohort.

We propose new classification criteria for Sjögrens syndrome (SS), which are needed considering the emergence of biologic agents as potential treatments and their associated comorbidity. These criteria target individuals with signs/symptoms suggestive of SS.

A link between tear instability and hyperosmolarity in dry eye.

PURPOSE Tear film instability and tear hyperosmolarity are considered core mechanisms in the development of dry eye. The authors hypothesize that evaporation and instability produce transient shifts in tear hyperosmolarity that lead to chronic epithelial stress, inflammation, and symptoms of ocular irritation. The purpose of this study was to provide indirect evidence of short-term hyperosmolar conditions during tear instability and to test whether the corneal epithelium responds to transient hyperosmolar stress. METHODS Five subjects kept one eye open as long as possible, and overall discomfort and sensations associated with tear break-up were scaled. Later, the same subjects used the same scales to report discomfort sensations after instillation of NaCl and sucrose hyperosmolar drops (300-1000 mOsM/kg). A two-alternative, forced-choice experiment was used to obtain osmolarity thresholds. In the second experiment, primary cultured bovine corneal epithelial cells were transiently stressed with the same range of hyperosmolar culture medium, and proinflammatory mitogen-activated protein kinase (MAPKs) were measured by Western blot analysis. RESULTS Tear instability led to an average discomfort rating of 6.13 and sensations of burning and stinging. These sensations also occurred with hyperosmolar solutions (thresholds, 450-460 mOsM/kg) that required 800 to 900 mOsM/kg to generate the same discomfort levels reported during tear break-up. MAPK was activated at 600 mOsM/kg of transient hyperosmolar stress. CONCLUSIONS These experiments provide a link between hyperosmolarity and tear instability, suggesting that hyperosmolar levels in the tear film may transiently spike during tear instability, resulting in corneal inflammation and triggering sensory neurons.

A simplified quantitative method for assessing keratoconjunctivitis sicca from the Sjögren's Syndrome International Registry.

PURPOSE To describe, apply, and test a new ocular grading system for assessing keratoconjunctivitis sicca (KCS) using lissamine green and fluorescein. DESIGN Prospective, observational, multicenter cohort study. METHODS The National Institutes of Health-funded Sjögrens Syndrome International Registry (called Sjögrens International Collaborative Clinical Alliance [SICCA]) is developing standardized classification criteria for Sjögren syndrome (SS) and is creating a biospecimen bank for future research. Eight SICCA ophthalmologists developed a new quantitative ocular grading system (SICCA ocular staining score [OSS]), and we analyzed OSS distribution among the SICCA cohort and its association with other phenotypic characteristics of SS. The SICCA cohort includes participants ranging from possibly early SS to advanced disease. Procedures include sequenced unanesthetized Schirmer test, tear break-up time, ocular surface staining, and external eye examination at the slit lamp. Using statistical analyses and proportional Venn diagrams, we examined interrelationships between abnormal OSS (>or=3) and other characteristics of SS (labial salivary gland [LSG] biopsy with focal lymphocytic sialadenitis and focus score >1 positive anti-SS A antibodies, anti-SS B antibodies, or both). RESULTS Among 1208 participants, we found strong associations between abnormal OSS, positive serologic results, and positive LSG focus scores (P < .0001). Analysis of the overlapping relationships of these 3 measures defined a large group of participants who had KCS without other components of SS, representing a clinical entity distinct from the KCS associated with SS. CONCLUSIONS This new method for assessing KCS will become the means for diagnosing the ocular component of SS in future classification criteria. We find 2 forms of KCS whose causes may differ.

Tear mixing under a soft contact lens: effects of lens diameter.

PURPOSE Tear exchange under a soft contact lens is modest, and higher exchange rates may be necessary to reduce extended-wear complications; what is not known is the optimal soft lens design to increase tear mixing. We explored the effect of lens diameter on tear mixing. METHODS Twenty-three subjects wore four different soft contact lenses with diameters of 12.0, 12.5, 13.0, and 13.5 mm. Tear mixing was quantified by placing fluorescein isothiocyanate-dextran on the posterior lens surface, inserting the lens, and monitoring the changes in fluorescence intensity in the postlens tear film. Tear mixing, expressed as the percentage decrease in fluorescence intensity per blink, was estimated using an exponential model. Lens movement was videotaped and lens comfort was graded on a 50-point scale (50 = excellent comfort). Subjects reporting a comfort level of less than 35 were excluded. RESULTS The mean +/- SE tear mixing rates were 1.82% +/- 0.17%, 1.61% +/- 0.16%, 1.34% +/- 0.17%, and 1.24% +/- 0.17% per blink for the 12.0-, 12.5-, 13.0-, and 13.5-mm diameter lenses, respectively. By regression analysis we found that, on average, mixing under the 12.0-mm lens was 0.59% per blink greater than with the 13.5-mm lens (P = .0024). Lens diameter was a significant predictor of lens comfort, and adjusting for the effects of comfort weakened the relationship between diameter and tear replenishment rate, although the mean rate under the 12.0-mm lens was still 0.43% per blink greater than with the 13.5-mm lens (P = .0468). CONCLUSIONS These data suggest that smaller-diameter soft lenses provide substantially better tear mixing than larger lenses; however, even small lenses provide modest tear mixing compared with rigid contact lenses.

Adenosine up-regulation of the mucin gene, MUC2, in asthma

Mucus hypersecretion is a hallmark of asthma that contributes to airway obstruction. While the etiology is not well understood, hypersecretion has been linked to the presence of cytokines such as IL‐4, IL‐5, IL‐9, and IL‐13 in the inflamed airway. The presence of adenosine has also been noted in asthmatic airways, and adenosine‐mediated signaling in mast cells has been implicated in the severe bronchoconstriction and inflammation prevalent in these patients (1, 2). Here we examine the possibility that adenosine also contributes to mucus hypersecretion by airway epithelial cells. Results in cultured airway epithelial cells showed that MUC2 mucin expression increased in response to adenosine. This appeared to be mediated by a pathway initiated at the adenosine A1 receptor that transduced signals through a Ca2+‐activated Cl− channel and EGFR. That this signaling cascade is relevant to asthmatic hypersecretion was indicated by results showing that mucin induction by asthmatic tracheal aspirates was reduced by A1, CLCA1, and EGFR inhibitors. These results suggest that adenosine cooperates with inflammatory cytokines to stimulate mucin production in the asthmatic airway and supports the use of A1, CLCA1, and EGFR inhibitors in the treatment of asthma.

Small Proline-Rich Protein 1B (SPRR1B) Is a Biomarker for Squamous Metaplasia in Dry Eye Disease

PURPOSE Squamous metaplasia occurs in ocular surface diseases like Sjögrens syndrome (SS). It is a phenotypic change whereby epithelial cells initiate synthesis of squamous cell-specific proteins such as small proline-rich protein 1B (SPRR1B) that result in pathologic keratin formation on the ocular surface. The authors hypothesized that inflammation is a key inducer of pathologic keratinization and that SPRR1B represents an analytical biomarker for the study of the molecular mechanisms. METHODS Real-time quantitative RT-PCR and immunohistochemistry were used to examine SPRR1B mRNA and protein in two different mouse models of dry eye and patients with SS. Adoptive transfer of mature lymphocytes from mice lacking the autoimmune regulator (aire) gene was performed to examine the role of inflammation as an inducer of squamous metaplasia. SPRR1B expression in response to several cytokines was examined in vitro, whereas the expression of cytokines IL1beta and IFNgamma was quantified in ocular tissues of aire-deficient mice and patients with SS. RESULTS SPRR1B was increased across the ocular surface of mice with both desiccating stress and autoimmune-mediated, aqueous-deficient dry eye and in patients with SS. Adoptive transfer of CD4(+) T cells from aire-deficient mice to immunodeficient recipients caused advanced ocular surface keratinization. IL1alpha, IL1beta, IL6, IFNgamma, and TNFalpha induced SPRR1B expression in vitro and the local expression of IL1beta and IFNgamma was elevated in ocular tissues of patients with SS and aire-deficient mice. CONCLUSIONS SPRR1B is a valid biomarker for the study of the molecular mechanisms of squamous metaplasia. There is a definitive link between inflammation and squamous metaplasia in autoimmune-mediated dry eye disease, with IL1beta and IFNgamma likely acting as key participants.

Interleukin-1 as a phenotypic immunomodulator in keratinizing squamous metaplasia of the ocular surface in Sjögren's syndrome.

Chronic inflammation of the ocular surface in Sjögrens syndrome (SS) is associated with a vision-threatening, phenotypic change of the ocular surface, which converts from a nonkeratinized, stratified squamous epithelium to a nonsecretory, keratinized epithelium. This pathological process is known as squamous metaplasia. Based on a significant correlation between ocular surface interleukin (IL)-1beta expression and squamous metaplasia in patients with SS, we investigated the role of IL-1 in the pathogenesis of squamous metaplasia in an animal model that mimics the clinical characteristics of SS. Using autoimmune-regulator (aire)-deficient mice, we assessed lacrimal gland and ocular surface immunopathology by quantifying the infiltration of major histocompatibility complex class II(+) (I-A(d+)) dendritic cells and CD4(+) T cells. We examined squamous metaplasia using a biomarker of keratinization, small proline-rich protein 1B. We used lissamine green staining as a readout for ocular surface epitheliopathy and Alcian blue/periodic acid-Schiff histochemical analysis to characterize goblet cell muco-glycoconjugates. Within 8 weeks, the eyes of aire-deficient mice were pathologically keratinized with significant epithelial damage and altered mucin glycosylation. Although knockdown of IL-1 receptor 1 did not attenuate lymphocytic infiltration of the lacrimal gland or eye, it significantly reduced ocular surface keratinization, epitheliopathy, and muco-glycoconjugate acidification. These data demonstrate a phenotypic modulation role for IL-1 in the pathogenesis of squamous metaplasia and suggest that IL-1 receptor 1-targeted therapies may be beneficial for treating ocular surface disease associated with SS.

Antibody protein array analysis of the tear film cytokines.

Purpose. Many bioactive proteins including cytokines are reported to increase in dry eye disease although the specific profile and concentration of inflammatory mediators varies considerably from study to study. In part, this variability results from inherent difficulties in quantifying low abundance proteins in a limited sample volume using relatively low sensitivity dot ELISA methods. Additional complexity comes with the use of pooled samples collected using a variety of techniques and intrinsic variation in the diurnal pattern of individual tear proteins. The current study describes a recent advance in the area of proteomics that has allowed the identification of dozens of low abundance proteins in human tear samples. Methods. Commercially available stationary phase antibody protein arrays were adapted to improve suitability for use in small volume biological fluid analysis with particular emphasis on tear film proteomics. Arrays were adapted to allow simultaneous screening for a panel of inflammatory cytokines in low volume tear samples collected from individual eyes. Results. A preliminary study comparing tear array results in a small population of Sjögren’s syndrome patients was conducted. The multiplex microplate array assays of cytokines in tear fluid present an unanticipated challenge due to the unique nature of tear fluid. The presence of factors that exhibit an affinity for plastic, capture antibodies and IgG and create a complex series of matrix effects profoundly impacting the reliability of dot ELISA, including with elevated levels of background reactivity and reduction in capacity to bind targeted protein. Conclusions. Preliminary results using tears collected from patients with Sjögren’s syndrome reveal methodological advantages of protein array technology and support the concept that autoimmune-mediated dry eye disease has an inflammatory component. They also emphasize the inherent difficulties one can face when interpreting the results of micro-well arrays that result from blooming effects, matrix effects, image saturation and cross-talk between capture and probe antibodies that can greatly reduce signal-to-noise and limit the ability to obtain meaningful results.

Signaling networks controlling mucin production in response to Gram-positive and Gram-negative bacteria

Human lung cells exposed to pathogenic bacteria upregulate the production of mucin, the major macromolecular component of mucus. Generally this upregulation is beneficial for the host, however, in the lungs of cystic fibrosis patients, overproduction of mucin can lead to the plugging of pulmonary airways. Mucus plugging impedes airflow and creates an environment that is highly compartmentalized: those bacteria within the mucus layer are shielded from high doses of antibiotics whereas those outside the mucus are exposed. These conditions augment mutation rate and the development of drug resistance in bacteria that colonize the lungs of cystic fibrosis patients. While therapeutic inhibition of mucin induction would improve airflow and reduce antibiotic resistance in these patients, the challenge is to develop drugs that block excessive mucin production while leaving beneficial aspects of the response intact. To do this, we must understand the molecular mechanisms underlying mucin production. Here we review the signal transduction pathways that control mucin production in response to Gram-positive and Gram-negative bacteria.

Soft lens extended wear affects epithelial barrier function.

OBJECTIVE To explore the impact of eye closure and soft contact lens extended wear (SCLEW) on epithelial permeability to fluorescein (Pdc). DESIGN A prospective cohort study. PARTICIPANTS Thirty-one noncontact lens (CL) wearers participated. INTERVENTION The effects of eye closure on Pdc were evaluated by comparing morning (AM) and afternoon (PM) measurements on non-CL subjects. The effects of SCLEW on Pdc were determined by measuring Pdc before beginning SCLEW and then after 2, 4, and 12 weeks of SCLEW. MAIN OUTCOME MEASURES Pdc measured in the morning versus the afternoon and before versus after SCLEW was examined. RESULTS Analyses of Pdc were done using the natural logarithm (ln). The mean +/- standard error (SE) ln(Pdc) measured in the AM versus PM on 16 non-CL wearers did not differ significantly (-2.56+/-0.16 vs. -2.69+/-0.15, respectively; P = 0.46). In contrast, the mean +/- SE ln(Pdc) in 15 subjects after 2 (-1.73+/-0.183, P < 0.001), 4 (- 1.59+/-0.188, P < 0.001), and 12 weeks (-1.99+/-0.206, P = 0.02) of SCLEW was substantially greater than that measured before lens wear (-2.42+/-0.159 ln(nm/sec)). Interestingly, the mean+/-SE ln(Pdc) measured in the afternoon on seven subjects after 13 weeks of SCLEW was similar to their average baseline ln(Pdc) (-2.62+/-0.27 vs. -2.52+/-0.243, respectively; P = 0.54). Further analysis showed that Pdc was highest in the morning and decreased approximately 12.5%/hour (P < 0.001) during the day. CONCLUSIONS Sleeping without CLs does not appear to alter Pdc; however, 2 weeks of SCLEW caused a 99% increase in permeability without observable changes by slit-lamp examination. Increases in Pdc appear greatest in the morning after SCLEW and then decrease exponentially during the day. Whether changes in Pdc will predict CL-associated keratopathy needs further study.