Veonice Bijin Au

Physiological Doses of Red Light Induce IL‐4 Release in Cocultures between Human Keratinocytes and Immune Cells

Phototherapy is routinely used for the treatment of various skin conditions and targeted therapy of superficial cancers. However, the molecular mechanisms behind their biological effects and the need for efficacy enhancing photosensitizers are not well addressed. Particularly, not much is known about the inherent effect of light from the visible spectrum on cytokine release and its downstream effects in keratinocytes and immune cells located in skin and therefore exposed to light. To address this, we delivered calibrated doses of well‐defined light qualities (380 to 660 nm) to cocultures of human keratinocytes and macrophage/dendritic cells in the absence or presence of the commonly used photosensitizer 8‐methoxypsoralen (8‐MOP). The experiments identified IL‐4 as a key effector cytokine released by this coculture model with need for 8‐MOP in the UVA1/blue (380 nm) and no requirement for photosensitizer in the red light spectrum (627 nm). 3D organotypic skin cultures treated with IL‐4 showed thickening of the epidermal layer and delayed differentiation. However unlike IL‐4 and UVA1/blue light treatment, red light did not reduce the expression of keratinocyte differentiation markers or increase signs of photo‐oxidative damage. This supports the application of isolated red light as a possible alternative for photo‐immunotherapy without need for additional photosensitizers.

A distinct cytokines profile in tear film of dry eye disease (DED) patients with HIV infection.

PURPOSE To investigate the tear cytokine profile in HIV patients with dry eye disease (DED) and study the association between the severity of ocular inflammatory complications and tear cytokines levels. We postulate that HIV-mediated inflammation may be the underlying pathogenic mechanism for HIV-associated DED. METHODS The current prospective case-control study compared tear film cytokine profiles in DED patients with HIV infection (n=34) and age/gender-matched DED patients without HIV infection [controls (n=32)]. Participants were recruited from tertiary referral eye care centre and communicable disease clinics, Singapore. Ocular surface health was documented using tear film, Schirmers test, corneal staining, and conjunctival injection measurements. Tear samples were collected using Schirmers strips and analysed for the levels of 41 cytokines using Luminex bead assay. Logistic regression models were performed to determine correlation and significance. RESULTS Among the 41 cytokines analysed, statistically significant differences were observed in the mean values of epithelial growth factor (EGF), growth related oncogene (GRO) and interferon gamma-induced protein 10 (IP-10). EGF and IP-10 levels were higher and GRO levels were lower in the tears of DED patients with HIV infection compared to DED patients without HIV infection. No significant association was found between varying levels of ocular surface parameters and cytokine concentrations in HIV patients with DED (p>0.05). CONCLUSIONS EGF and IP-10 were significantly elevated and GRO levels were lower in the tear profile of HIV patients with DED compared to immunocompetent patients with DED. This study suggests a novel cytokine driven paradigm for ocular inflammatory complications of HIV infection. Additional studies in large organised cohorts can validate the results.

Aqueous humor immune factors and cytomegalovirus (CMV) levels in CMV retinitis through treatment - The CRIGSS study.

PURPOSE This study aims to perform comprehensive longitudinal immune factor analysis of aqueous humor in relation to the aqueous CMV viral load and systemic CD4 counts during treatment of patients with co-infection of HIV and CMVR. METHODS Aqueous humor samples were collected from 17 HIV-positive patients with CMVR scheduled to undergo weekly intravitreal ganciclovir therapy as part of the prospective CMV Retinitis Intravitreal Ganciclovir Singapore Study (CRIGSS) over the course of 1year. Full data across all the 4 time points was obtained and analyzed for CMV DNA viral load, 41 cytokine and chemokine factors using real-time PCR with the FlexMAP 3D (Luminex®) platform and assessed using the Milliplex Human Cytokine® kit. RESULTS The following immune factors (Spearman correlation coefficient r value in parenthesis, p<0.05) showed strong correlation with CMV DNA load in the aqueous - MCP-1 (0.80, IFN-g (0.83), IP-10 (0.82), IL-8 (0.81), fractalkine (0.73), RANTES (0.68) - while the following showed moderate correlation - PDGF-AA (0.58), Flt-3L (0.59) and G-CSF (0.53). Only PDGF-AA revealed a statistically significant negative correlation with serum CD4 levels (r=-0.74). CONCLUSION Immune factors that correlate with intraocular CMV DNA load are identified. They are indicative of a Th1 and monocyte-macrophage mediated response, and exhibit a decreasing trend longitudinally through the course of treatment. These factors may be an important new consideration in individualizing the treatment of patients with CMVR.

Dataset of tear film cytokine levels in dry eye disease (DED) patients with and without HIV infection

The tear film cytokine profiling data in this article was obtained from a prospective case-control study with a sample size of 34 dry eye disease (DED) patients with HIV infection and 32 DED patients without HIV infection, see “A distinct cytokines profile in tear film of dry eye disease (DED) patients with HIV infection” (R. Agrawal, P.K. Balne, A. Veerappan, V.B. Au, B. Lee, E. Loo, A. Ghosh, L. Tong, S.C. Teoh, J. Connolly, P. Tan, 2016) [1]. Tear samples were collected from all the subjects using Schirmer׳s strips and cytokine profiling was done using the Luminex bead based multiplex assay with a panel of 41 analytes. The cytokine level differences in each group of subjects were analyzed using logistic regression models.

DPP9 is an endogenous and direct inhibitor of the NLRP1 inflammasome that guards against human auto-inflammatory diseases

The inflammasome is a critical immune complex that activates IL-1 driven inflammation in response to pathogen- and danger-associated signals. Nod-like receptor protein-1 (NLRP1) is a widely expressed inflammasome sensor. Inherited gain-of-function mutations in NLRP1 cause a spectrum of human Mendelian diseases, including systemic autoimmunity and skin cancer susceptibility. However, its endogenous regulation and its cognate ligands are still unknown. Here we apply a proteomics screen to identify dipeptidyl dipeptidase, DPP9 as a novel interacting partner and a specific endogenous inhibitor of NLRP1 inflammasome in diverse primary cell types from human and mice. DPP9 inhibition via small molecule drugs, targeted mutations in its catalytic site and CRISPR/Cas9-mediated genetic deletion potently and specifically activate the NLRP1 inflammasome leading to pyroptosis and IL-1 processing via ASC and caspase-1. Mechanistically, DPP9 maintains NLRP1 in its monomeric, inactive state by binding to the auto-cleaving FIIND domain. NLRP1-FIIND is a self-sufficient DPP9 binding module and its disruption by a single missense mutation abrogates DPP9 binding and explains the aberrant inflammasome activation in NAIAD patients with arthritis and dyskeratosis. These findings uncover a unique peptidase enzyme-based mechanism of inflammasome regulation, and suggest that the DPP9-NLRP1 complex could be broadly involved in human inflammatory disorders.

Dataset of plasma and aqueous humor cytokine profiles in patients with exudative age related macular degeneration and polypoidal choroidal vasculopathy

In this report the data was obtained from a prospective case-control study with a sample size of sixteen patients with exudative age related macular degeneration (AMD) due to choroidal neovascularization (CNV) and eighteen patients with polypoidal choroidal vasculopathy (PCV) and fifty controls (cataract patients without any other ocular diseases). Luminex bead based multiplex assay with a panel of 41 analytes was used to study the cytokine levels in plasma and aqueous humor.

Bead Based Multiplex Assay for Analysis of Tear Cytokine Profiles

Tear film is a complex mixture of lipids, proteins and minerals which covers the external surface of the eye, thereby providing lubrication, nutrition and protection to the underlying cells. Analysis of tears is an emerging area for the identification of biomarkers for the prediction, diagnosis, and prognosis of various ocular diseases. Tears are easily accessible and their collection is non-invasive. Therefore, advancing technologies are gaining prominence for identification of multiple analytes in tears to study changes in protein or metabolite composition and its association with pathological conditions. Tear cytokines are ideal biomarkers for studying the health of the ocular surface and also help in understanding the mechanisms of different ocular surface disorders like dry eye disease and vernal conjunctivitis. Bead based multiplex assays have the capability of detecting multiple analytes in a small amount of sample with a higher sensitivity. Here we describe a standardized protocol of tear sample collection, extraction and analysis of cytokine profiling using a bead based multiplex assay.

Dataset of longitudinal analysis of tear cytokine levels, CD4, CD8 counts and HIV viral load in dry eye patients with HIV infection

The data presented in this article shows the longitudinal analysis of tear fluid cytokine profiles, blood CD4 and CD8 counts and HIV viral load in 34 dry eye patients with HIV infection during the HAART therapy. Clinical samples were collected from HIV patients with dry eye disease at the time of presentation to the clinic (visit 1), three months (visit 2) and 6 months (visit 3) after the presentation. At each time point tear samples were evaluated for 41 cytokines using Luminex bead based multiplex assay and blood samples were tested for HIV viral load and CD4 and CD8 counts.

Dataset of aqueous humor cytokine profile in HIV patients with Cytomegalovirus (CMV) retinitis.

The data shows the aqueous humor cytokine profiling results acquired in a small cohort of 17 HIV patients clinically diagnosed with Cytomegalovirus retinitis using the FlexMAP 3D (Luminex®) platform using the Milliplex Human Cytokine® kit. Aqueous humor samples were collected from these patients at different time points (pre-treatment and at 4-weekly intervals through the 12-week course of intravitreal ganciclovir treatment) and 41 cytokine levels were analyzed at each time point. CMV DNA viral load was assessed in 8 patients at different time points throughout the course of ganciclovir treatment. The data described herein is related to the research article entitled “Aqueous humor immune factors and cytomegalovirus (CMV) levels in CMV retinitis through treatment - The CRIGSS study” (Iyer et al., 2016) [1]. Cytokine levels against the different time points which indicate the response to the given treatment and against the CMV viral load were analyzed.