Sarmad Jassim

Ocular Surface Extracellular DNA and Nuclease Activity Imbalance: A New Paradigm for Inflammation in Dry Eye Disease

PURPOSE We determined whether nucleases are deficient in the tear fluid of dry eye disease (DED) patients, and whether this causes extracellular DNA (eDNA) and neutrophil extracellular trap (NET) accumulation in the precorneal tear film, thus causing ocular surface inflammation. METHODS Exfoliated cells adhered to Schirmer test strips were collected on glass slides, and immunofluorescence confocal microscopy was used to evaluate neutrophils, eDNA, NETs, and their molecular components. Similar experiments were performed with mucoid films collected from the inferior conjunctival fornix or bulbar conjunctiva. We used quantitative PCR to evaluate eDNA signaling pathways and inflammatory cytokine expression. We also determined the amount of ocular surface eDNA and evaluated tear fluid nuclease activity. RESULTS eDNA, NETs, and neutrophils were present on the ocular surface in DED patients and abundant in mucoid films. NETs consisted of eDNA, histones, cathelicidin, and neutrophil elastase. Tear fluid nuclease activity was decreased significantly in DED patients, whereas the amount of eDNA on the ocular surface was increased significantly. Expression of genes downstream of eDNA signaling, such as TLR9, MyD88, and type I interferon, as well as the inflammatory cytokines interleukin-6 and tumor necrosis factor-α, was significantly increased in DED patients. CONCLUSIONS Extracellular DNA production and clearance mechanisms are dysregulated in DED. Nuclease deficiency in tear fluid allows eDNA and NETs to accumulate in precorneal tear film, and results in ocular surface inflammation. These findings point to novel therapeutic interventions in severe DED based on clearance of eDNA, NETs, and other molecular components from the ocular surface.

CD11b+GR1+ Myeloid Cells Secrete NGF and Promote Trigeminal Ganglion Neurite Growth: Implications for Corneal Nerve Regeneration

PURPOSE We characterized fluorescent bone marrow cells (YFP(+) BMCs) in the thy1-YFP mouse and determine if they promote trigeminal ganglion (TG) cell neurite growth. METHODS Excimer laser annular keratectomy was performed in thy1-YFP mice, and corneas were imaged. BMCs were harvested from femur and tibia, and the expression of surface markers on YFP(+) BMCs was analyzed by flow cytometry. The immunosuppressive action of BMCs (YFP(+) and YFP(-)) was evaluated in an allogenic mixed lymphocyte reaction (MLR). Neurotrophic action of BMCs (YFP(+) and YFP(-)) was determined in compartmental and transwell cultures of dissociated TG cells. RESULTS Following annular keratectomy, YFP(+) BMCs infiltrated the cornea. YFP(+) BMCs shared surface markers (CD11b+Gr1+Ly6C+Ly6G-F4/80(low)) with monocytic myeloid-derived suppressor cells (MDSCs), had similar morphology, and suppressed T-cell proliferation in allogenic MLR in a dose-dependent manner. YFP(+) BMCs, but not YFP(-) BMCs, significantly increased growth of TG neurites in vitro. When cultured in a transwell with TG neurites, YFP(+) BMCs expressed neurotrophins and secreted nerve growth factor (NGF) in conditioned medium. YFP(+) BMCs that infiltrated the cornea maintained their phenotype and actions (neuronal and immune). CONCLUSIONS YFP(+) BMCs in thy1-YFP mice have immunophenotypic features of MDSCs. They secrete NGF and promote neuroregeneration. Their immunosuppressive and neurotrophic actions are preserved after corneal infiltration. These findings increase our understanding of the beneficial roles played by leukocyte trafficking in the cornea and may lead to therapeutic strategies that use NGF-secreting myeloid cells to repair diseased or injured neurons.

Symptom burden of patients with dry eye disease: A four domain analysis

Purpose To determine which sensory (symptom persistence and intensity) and reactive (activity and affective interference) domains of symptom analysis are essential for assessing symptom burden in dry eye disease (DED) patients. Methods A symptom domain tool was developed to investigate all four symptom domains in DED. In a cross-sectional pilot study, we administered the symptom burden tool and the Ocular Surface Disease Index (OSDI) questionnaire to 48 DED patients. Total and domain scores from the symptom burden tool and the OSDI were normalized to achieve comparability. Spearman correlation coefficients were calculated to measure the relationship between domains and subscales. Agreement between the symptom burden tool and OSDI was assessed by Bland-Altman plot. Assigned treatments were compared by symptom burden to determine whether treatment aggressiveness is linked to symptom intensity. Results There was high agreement between the symptom burden tool and the OSDI. Symptom persistence had a stronger correlation with affective interference (r  =  0.62 for the symptom burden tool and r = 0.73 for the OSDI) than activity interference (r = 0.58 for the symptom burden tool and r = 0.60 for the OSDI). Symptom intensity correlated weakly with affective interference (r = 0.38) and activity interference (r = 0.37) in the symptom burden tool (OSDI does not have a subscale for intensity). In patients with equal persistence of symptoms, those having high symptom intensity were receiving more aggressive treatment (66.7%) than those with lower symptom intensity (33.3%). Conclusions Persistence of symptoms correlates better with affective interference than activity interference. Intensity of symptoms may be important for treatment decisions.

Histatin-1 Expression in Human Lacrimal Epithelium

Background Study of human lacrimal cell biology is limited by poor access to tissue samples, heterogeneous cell composition of tissue and a lack of established lacrimal epithelial markers. In order to further our understanding of lacrimal cell biology, we sought to find a better marker for human lacrimal epithelial cells, compared to what has been reported in the literature. Methods We utilized human Muller’s muscle conjunctival resection (MMCR) specimens containing accessory lacrimal gland (ALG) and cadaveric main lacrimal gland (MLG) as sources of lacrimal tissue. Candidate markers were sought using human ALG tissue from MMCR specimens, isolated by laser capture microdissection (LCM). Affymetrix® analysis was performed on total RNA isolated from FFPE samples to profile transcription in ALG. MMCR tissue sections were assessed by immunofluorescence using antibodies for histatin-1, lactoferrin, E-cadherin (E-cad) and alpha-smooth muscle actin (ASMA). Reverse transcriptase polymerase chain reaction (RT-PCR) analysis was performed to analyze the expression of histatin-1, E-cad and lactoferrin from cadaveric MLG. Results Histatin-1 is expressed in ALG and MLG, localizes to lacrimal epithelium, and to a greater degree than do other putative lacrimal epithelial markers. Conclusions Histatin-1 is a good marker for human lacrimal epithelium in ALG and MLG and can be used to identify lacrimal cells in future studies.

Bacteria Colonizing the Ocular Surface in Eyes With Boston Type 1 Keratoprosthesis: Analysis of Biofilm-Forming Capability and Vancomycin Tolerance.

PURPOSE To analyze the bacterial microbiota colonizing the ocular surface of patients with Boston type 1 keratoprostheses (K-Pros) for antibacterial resistance patterns and capacity to form biofilms. METHODS Twenty-seven eyes with a Boston type 1 K-Pro and 16 fellow control eyes from 26 patients were enrolled. The surface of the K-Pro optic and/or the inferior conjunctival fornix was swabbed and plated separately on culture media. Positive cultures were processed to assess for biofilm-forming capability. Microtiter plate adherence assay and polymerase chain reaction for ica and atlE genes were used. An in vitro assay of vancomycin tolerance was performed on isolated strains and compared to standard controls with and without biofilm-forming capability. RESULTS Eighty-five percent of K-Pro eyes and 69% of control eyes had positive cultures (P = 0.20). All Gram-positive strains exhibited susceptibility to vancomycin by standard testing. Biofilm-forming bacterial isolates were detected in 57.7% of K-Pro eyes and 53.3% of control eyes. A vancomycin tolerance assay showed that the antibiotic susceptibility of coagulase-negative staphylococcus (CNS) within biofilms was significant in only three of five biofilm-forming strains (P < 0.05). In all strains, bacterial cells in planktonic form were more susceptible to vancomycin than in biofilm form (P < 0.001). CONCLUSIONS Coagulase-negative staphylococcus can be isolated from K-Pro surfaces despite the use of vancomycin prophylaxis. In this study, the majority of isolated strains had biofilm-forming capability. In vitro vancomycin tolerance assays suggest that biofilm formation decreases susceptibility to vancomycin. This may contribute to higher rates of infectious complications observed in these patients.

Evaluation of Accessory Lacrimal Gland in Muller’s Muscle Conjunctival Resection Specimens for Precursor Cell Markers and Biological Markers of Dry Eye Disease

ABSTRACT Purpose: The accessory lacrimal glands (ALGs) are an understudied component of the tear functional unit, even though they are important in the development of dry eye syndrome (DES). To advance our understanding of aging changes, regenerative potential, and histologic correlates to human characteristics, we investigated human ALG tissue from surgical samples to determine the presence or absence of progenitor cell markers and lacrimal epithelial markers and to correlate marker expression to relevant patient characteristics. Materials and Methods: ALG tissues obtained from Muller’s muscle conjunctival resection (MMCR) specimens were created using tissue microarrays (TMAs). Immunofluorescence staining of MMCR sections was performed using primary antibodies specific to cell protein markers. Cell marker localization in TMAs was then assessed by two blinded observers using a standardized scoring system. Patient characteristics including age, race, and status of ocular surface health were then compared against expression of stem cell markers. Results: Human ALG expressed a number of epithelial markers, and in particular, histatin-1 was well correlated with the expression of epithelial markers and was present in most acini. In addition, we noted the presence of precursor cell markers nestin, ABCG2, and CD90 in ALG tissue. There was a decrease in precursor cell marker expression with increasing age. Finally, we noted that a negative association was present between histatin-1 expression and DES. Conclusions: Thus, we report for the first time that human ALG tissues contain precursor marker-positive cells and that this marker expression may decrease with increasing age. Moreover, histatin-1 expression may be decreased in DES. Future studies will be performed to use these cell markers to isolate and culture lacrimal epithelial cells from heterogeneous tissues, determine the relevance of histatin-1 expression to DES, and isolate candidate precursor cells from ALG tissue.

Biofilm Formation on Bandage Contact Lenses Worn by Patients with the Boston Type 1 Keratoprosthesis: A Pilot Comparison Study of Prophylactic Topical Vancomycin 15 mg/mL and Linezolid 0.2%

Objectives: To determine the rate of biofilm formation on bandage contact lenses worn by patients with the Boston type 1 keratoprosthesis (K-Pro) while on prophylactic topical vancomycin versus linezolid. Methods: Patients wearing a bandage contact lens (BCL) with a K-Pro were eligible for enrollment. After irrigation of the ocular surface with 5% povidone-iodine solution, each patient was placed on either topical vancomycin 15 mg/mL or linezolid 0.2% BID for one month. At the one-month visit, the BCL was collected and stored in fixative solution. Standard photographs were taken of each lens at high magnification using scanning electron microscopy (SEM), which were subsequently analyzed for evidence of biofilm. Results: Nineteen contact lenses were obtained from 12 K-Pro patients at the Illinois Eye and Ear Infirmary. Zero of eight (0%; 95% CI=0 to 37%) contact lenses from patients treated with topical vancomycin, and 1 of 11 (9%; 95% CI=0 to 41%; P-value=1.00) contact lenses from patients treated with topical linezolid were found to have biofilm formation at one month as detected by SEM. None of the patients developed a clinically significant infection while on either prophylactic vancomycin or linezolid during the study period. Conclusions: Overall, the rate of biofilm formation as detected by SEM on the surface of bandage contact lenses was low. These results suggest that vancomycin and linezolid are both relatively effective in reducing biofilm-forming bacterial growth at one month. Accordingly, linezolid may be an effective alternative to vancomycin in patients with allergy or intolerance. However, further investigation is required to develop evidence-based antibiotic prophylaxis regimens.