Tun Kuan Yeo

Cytokine analysis of aqueous humor in HIV patients with cytomegalovirus retinitis

PURPOSE Cytomegalovirus retinitis (CMVR) is the most common opportunistic ocular infection in patients with AIDS. Comprehensive analysis of aqueous humor for immunologic factors has yet to be performed in patients with CMVR. This study aims to perform comprehensive immune factor analysis of aqueous humor in CMVR patients to determine the presence of any characteristic immunological profile in the aqueous humor. METHODS Comparative prospective analysis of aqueous humor was performed across three groups: (1) AIDS patients with CMVR (CMVR group) (n=20), (2) HIV-positive patients without CMVR (HIV group) (n=6) and (3) patients undergoing cataract surgery with no underlying ocular infection or inflammation (control group) (n=11). At least 100μl of aqueous humor was drawn from all subjects and fractionated prior to analysis for 41 cytokines, chemokines and growth factors with the FlexMAP 3D (Luminex®) platform using the Milliplex Human Cytokine® kit. RESULTS Three distinct immunologic signatures were observed in the aqueous humor of the three groups. Statistically significant differences (p<0.05) were observed across the three groups with the HIV group having lower levels and CMVR group having raised levels for the following factors: IP-10, fractalkine, PDGF-AA, G-CSF, Flt-3L and MCP-1. CONCLUSION Aqueous humor though clinically quiescent in CMVR revealed a unique immunologic signature consistent with a combined Th-1 and monocyte-macrophage mediated response. Subsequent longitudinal analysis of aqueous cytokine levels of CMVR through the course of treatment would allow better understanding of the immunopathogenetic mechanisms of CMVR. This may also be used to better prognosticate the disease, predict complications and allow better assessment of treatment response and individualization of treatment in the future.

Retinal vascular parameter variations in patients with human immunodeficiency virus.

PURPOSE To compare the retinal vascular parameters in patients with human immunodeficiency virus (HIV) infection with normal controls, and to determine the relationship between retinal vascular parameters and HIV-related blood biomarkers (CD4(+) T-lymphocytes count, presence of HIV RNA). METHODS Case-control study of eighty-five patients with HIV on follow-up at the Communicable Disease Center, Singapore, and 251 age-, sex-, and race-matched normal healthy controls (case: control matching ≈ 1:3) selected from the Singapore Epidemiology of Eye Disease program were included in this study. Standardized retinal photographs were taken from patients and controls. Trained technicians measured quantitative retinal vascular parameters (retinal vascular caliber, branching angle, tortuosity, and fractal dimension) with a semiautomated computer-based program following a standardized protocol. RESULTS HIV-patients had more tortuous arterioles (0.77 × [10(4)] vs. 0.59 × [10(4)], P < 0.001) and venules (0.90 × [10(4)] vs. 0.74 × [10(4)], P < 0.001), compared with healthy normal subjects. Amongst the HIV-patients, increasing HIV viral loads were associated with decreased retinal arteriolar caliber (P trend = 0.009) and decreased arteriolar-venular ratio (P trend = 0.025). CONCLUSIONS Our study showed that patients with HIV have significant variations in retinal vasculature. Retinal vascular imaging may offer further insight into the pathophysiology behind HIV-related vascular disease in future.

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.

A prospective case-control study to investigate retinal microvascular changes in acute dengue infection.

Dengue infection can affect the microcirculation by direct viral infection or activation of inflammation. We aimed to determine whether measured retinal vascular parameters were associated with acute dengue infection. Patients with acute dengue were recruited from Communicable Diseases Center, Singapore and age-gender-ethnicity matched healthy controls were selected from a population-based study. Retinal photographs were taken on recruitment and convalescence. A spectrum of quantitative retinal microvascular parameters (retinal vascular caliber, fractal dimension, tortuosity and branching angle) was measured using a semi-automated computer-based program. (Singapore I Vessel Assessment, version 3.0). We included 62 dengue patients and 127 controls. Dengue cases were more likely to have wider retinal arteriolar and venular calibers (158.3 μm vs 144.3 μm, p < 0.001; 227.7 μm vs 212.8 μm, p < 0.001; respectively), higher arteriolar and venular fractal dimensions (1.271 vs 1.249, p = 0.002; 1.268 vs. 1.230, p < 0.001, respectively), higher arteriolar and venular tortuosity (0.730 vs 0.546 [x104], p < 0.001; 0.849 vs 0.658 [x104], p < 0.001; respectively), compared to controls. Resolution of acute dengue coincided with decrease in retinal vascular calibers and venular fractal dimension. Dengue patients have altered microvascular network in the retina; these changes may reflect pathophysiological processes in the immune system.

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.

Retinal vascular changes and immune restoration in a cohort of HIV/AIDS patients on highly active antiretroviral therapy

Abstract Background Retinal microvascular changes have been shown to reflect systemic inflammation in vivo. We aimed to investigate whether retinal microvascular changes are correlated with, and predictive of, CD4 and CD8 cell count changes in patients with HIV/AIDS being treated with highly active antiretroviral therapy (HAART). Methods We did a longitudinal hospital-based study. 50 HIV/AIDS patients being treated with HAART were sequentially recruited from March, 2011, to September, 2011, from the Communicable Disease Center, Singapore, and then followed up at a 9 month visit. Demographic and socioeconomic information and history of HIV infection were collected at baseline. Blood pressure and anthropometric measurements, blood tests for immune status assessment (CD4 and CD8 cell counts), and retinal photography were done at baseline and at 9 months. Retinal vascular parameters (calibre, tortuosity, branching angle, and fractal dimension) were assessed by a semiautomated computer-based programme (Singapore I vessel assessment [SIVA], version 3.0, Singapore Eye Research Institute, Singapore). Changes of retinal vascular parameters and CD4 and CD8 cell counts were defined as the difference between baseline and 9 months. Findings The mean age of the 50 participants was 46·36 years (SD 8·67). Participants were ethnically Chinese, Malay, and Indian; most (82%) were Chinese and most male (96%). 35 of 50 patients had CD4 counts of fewer than 200 cells per μL at baseline. Years of HAART ranged from 0 to 11 years. There were significant increments in CD4 cell counts (153·42 vs 227·74 cells per μL; p vs 1014·1 cells per μL; p=0·012) between baseline and the 9 month visit, respectively. There were no temporal changes in retinal vascular parameters during this period. After adjustment for age, sex, ethnicity, and years of HAART, each 10 μm reduction in retinal venular calibre at baseline was associated with a 191·08 cells per μL increase in CD8 count (SE 63·54; p=0·004), but not in CD4 cell count, during the 9 month period. Changes of retinal arteriolar calibre and other retinal vascular geometric parameters were not associated with CD4 or CD8 cell count changes. Interpretation Retinal venular narrowing was associated with an increase in CD8 cell counts over time. Our findings suggest that improved retinal venular health was predictive of immune restoration in HIV/AIDS patients who had been on HAART for at least 9 months. Longer follow-up is warranted to monitor the retinal vascular calibre changes in response to HAART treatment. Funding Singapore Medical Research Counsel SIG/11016.