PCR testing for herpesviruses in aqueous humor samples from patients with and without clinical corneal endothelial graft rejection.

To compare prevalence of positive PCR tests for herpesviruses between patients with and without a history of clinical corneal endothelial allograft rejection (AGR). Retrospective cross-sectional study with two-group comparison. A total of 307 aqueous humor (AH) samples from 235 Patients and 244 eyes who underwent penetrating keratoplasty or Descemet membrane endothelial keratoplasty or had a diagnostic AH aspiration due to clinical AGR between 2019 and 2023 were tested for DNA of herpes simplex virus (HSV), varicella-zoster virus (VZV), cytomegalovirus (CMV), and Epstein-Barr virus (EBV). PCR test results were compared between the two groups (with/without AGR). Another sub-analysis examined the results of patients without a history of herpetic keratitis. A total of 8% of eyes with clinical AGR (9/108) had a positive PCR result for one of the herpesviruses (HSV:3, CMV:3, EBV:2, VZV:1). All patients in the group without AGR had negative PCR results for all previous viruses (0/136). The difference was statistically significant (p < 0.001). The sub-analysis of eyes without a history of herpetic keratitis also revealed significantly more positive herpes PCR results (7/87) in eyes with AGR than in eyes without AGR (0/42, p = 0.005). Clinical AGR after keratoplasty shows a significant correlation to viral replication. Herpetic infection and AGR could occur simultaneously and act synergistically. Timely differentiation between active herpetic infection and/or AGR is pivotal for proper treatment and graft preservation.

Liquid-biopsy proteomics combined with AI identifies cellular drivers of eye aging and disease in vivo.

Single-cell analysis in living humans is essential for understanding disease mechanisms, but it is impractical in non-regenerative organs, such as the eye and brain, because tissue biopsies would cause serious damage. We resolve this problem by integrating proteomics of liquid biopsies with single-cell transcriptomics from all known ocular cell types to trace the cellular origin of 5,953 proteins detected in the aqueous humor. We identified hundreds of cell-specific protein markers, including for individual retinal cell types. Surprisingly, our results reveal that retinal degeneration occurs in Parkinson's disease, and the cells driving diabetic retinopathy switch with disease stage. Finally, we developed artificial intelligence (AI) models to assess individual cellular aging and found that many eye diseases not associated with chronological age undergo accelerated molecular aging of disease-specific cell types. Our approach, which can be applied to other organ systems, has the potential to transform molecular diagnostics and prognostics while uncovering new cellular disease and aging mechanisms.

Exquisite integration of quality-by-design and green analytical approaches for simultaneous determination of xylometazoline and antazoline in eye drops and rabbit aqueous humor, application to stability study.

This work implements a stability indicating HPLC method developed to simultaneously determine xylometazoline (XYLO) and antazoline (ANT) in their binary mixture, rabbit aqueous humor and cited drug's degradates by applying analytical quality-by-design (AQbD) combined with green analytical chemistry (GAC) experiment for the first time. This integration was designed to maximize efficiency and minimize environmental impacts, as well as energy and solvent consumption. Analytical quality-by-design was applied to achieve our aim starting with evaluation of quality risk and scouting analysis, tracked via five parameters chromatographic screening using Placket-Burman design namely: pH, temperature, organic solvent percentage, flow rate, and wavelength detection. Recognizing the critical method parameters was done followed by optimization employing central composite design and Derringer's desirability toward assess optimum conditions that attained best resolution with satisfactory peak symmetry with short run time. Optimal chromatographic separation was attained by means of an XBridge® C18 (4.6 × 250 mm, 5 µm) column through isocratic elution using a mobile phase consists of phosphate buffer (pH 3.0): ethanol (60:40, by volume) at a 1.6 mL/min flow rate and 230.0 nm UV detection. Linearity acquired over a concentration range of 1.0-100.0 µg/mL and 0.5-100.0 µg/mL for XYLO and ANT, respectively. Furthermore, imperiling cited drugs' stock solutions to stress various conditions and satisfactory peaks of degradation products were obtained indicating that cited drugs are vulnerable to oxidative degradation and basic hydrolysis. Degradates' structures were elucidated using mass spectrometry. Applying various assessment tools; namely: analytical greenness (AGREE), green analytical procedure index (GAPI), analytical eco-scale, and national environmental method index (NEMI), Greenness method's evaluation was applied and proved to be green. In fact, the developed method is established to be perceptive, accurate, and selective to assess cited drugs for routine analysis.

Chromatographic Analysis of Aqueous Humor of Bupivacaine in Different Administration Approaches.

Different administration approaches were investigated for the selection of bupivacaine administration type and a sensitive high-performance liquid chromatographic (HPLC) method has been developed. Developed method was validated and applied for the determination of bupivacaine in rabbit aqueous humor. The separation was achieved using a XTerra, C8 (250 × 8 mm i.d., particle size 5 µm) analytical column with a mobile phase consisted of acetonitrile and sodium dihydrogen phosphate (pH = 3.0, 20 mM; 30:70, v/v). Bupivacaine detection was performed by Diode Array detector (DAD) at 220 nm. The retention times for bupivacaine is 15.886 min. HPLC-DAD method was linear in the range of 75-4000 ng/mL. The limit of detection was 25 ng/mL and the limit of quantification of bupivacaine was found to be 75 ng/mL (relative standard deviation, RSD ≤ 15%, n = 6). In intra-day and inter-day precision and accuracy analysis, the RSD was found to be in the range of 0.96 and 7.98%, the bias values were 0.64 and 3.33%. Method was carried out for three different type of bupivacaine application because of the investigation of effective drug administration. Twenty aqueous humor samples were in the range of 0.642 and 5.124 µg/mL.

Development and Validation of Facile RP-HPLC Method for Simultaneous Determination of Timolol Maleate, Moxifloxacin Hydrochloride, Diclofenac Sodium and Dexamethasone in Plasma, Aqueous Humor and Pharmaceutical Products.

The present study aimed to develop a validated RP-HPLC method for the simultaneous determination of timolol maleate (TM), moxifloxacin hydrochloride (MOXI), diclofenac sodium (DS) and dexamethasone (DEXA) in human plasma, bovine aqueous humor and pharmaceutical preparations. The chromatographic separation was studied using the C18 column. The chromatographic conditions, such as composition, pH, the flow rate of mobile phase, the temperature of column, wavelength of absorption and injection volume of the sample, were studied. The method was validated to confirm specificity, linearity and accuracy in accordance with an International Conference on Harmonization guidelines. The optimum conditions for separation included mobile phase 0.05 M monobasic phosphate buffer: acetonitrile (65:35 v/v), pH of buffer adjusted to 6.2 and the flow rate of 1 mL/minute. The optimum temperature of the column was found to be 35°C, absorption wavelength 265 nm and injection volume 50 µL. The baseline separation of all four drugs with good sensitivity, resolution, and a less than 15 min run time was achieved. The retention time of TM, MOXI, DS and DEXA were 4.3,5.7,9.9 and 13.5 minutes respectively. The limit of detection (LOD) values were 6.2, 4.8, 0.8 and 1.2 ng/mL for TM, MOXI, DS and DEXA, respectively, whereas their respective limit of quantification (LOQ) values was: were 42.6, 26.8, 5.6 and 6.2 ng/mL. The coefficient of variation for intra-day and inter-day were in the range of 0.32-1.57 and 1.29-3.07%, respectively. The method was found to be sensitive, precise and accurate in human plasma and bovine aqueous humor and can be applied for the quantification of these compounds in plasma, aqueous humor and pharmaceuticals.

Metabolomics Analysis of Aqueous Humor Based on Liquid Chromatography-Mass Spectrometry.

Aqueous humor (AH) is a transparent fluid that fills the anterior segment of the eye. The composition and level of metabolites in AH are important for understanding its physiology and changes caused by the occurrence of eye disease. A simple method for the preparation and analysis of AH samples was developed using the liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) technique. The analyses were performed using two types of chromatography: reversed-phase liquid chromatography-mass spectrometry (LC-RP-MS) and hydrophilic interaction liquid chromatography-mass spectrometry (LC-HILIC-MS), in the sample prepared by one protocol.

Factorial design-assisted reverse phase HPLC-UV approach for the concurrent estimation of cetirizine and azelastine in aqueous humor.

A new analytical quality by design-assisted HPLC-UV approach is presented, for the first time, for the concurrent determination of cetirizine (CTZ) and azelastine (AZE) in raw materials, commercial eye drops and aqueous humor. The two drugs are co-administered as eye drops in severe ocular allergies. A 23 full factorial design was adopted for the chromatographic optimization to ensure the best analytical performance and reliability, as well as to save time, effort and solvent consumption. The parameters, including pH, acetonitrile ratio, and flow rate, were selected as independent factors. The responses analyzed were resolution and tailing of peaks. The separation was achieved through isocratic elution on C8 column with mobile phase made up of acetonitrile: 0.3% triethylamine of pH 5 (60:40 v/v) at a flow rate of 1.2 mL min-1 and detection at 216 nm. The elution time was less than 6 min. The approach was fully validated in accordance with International Council for Harmonization (ICH) guidelines. Good linearity was achieved over the concentration ranges of 1.0-30 and 0.5-10 µg mL-1 with limits of detection of 0.310 and 0.158 µg mL-1 and limits of quantification of 0.940 and 0.479 µg mL-1 for CTZ and AZE, respectively, with correlation coefficients of 0.9998. The intra- and inter-day precisions were lower than 2%. The good sensitivity of the approach permits the analysis of CTZ and AZE in spiked aqueous humor with mean percentage recoveries of 100.93 ± 1.42 and 100.11 ± 1.55, respectively. The statistical comparison between results of the developed method and the comparison method revealed no differences, indicating the accuracy of the method.

Changes in the Lipid Profile of Aqueous Humor From Diabetic Cataract Patients.

Purpose: To identify the differential profiles of lipids in aqueous humor (AH) between control and diabetic cataract patients. Methods: The lipidomic profiles of 19 samples from patients with diabetic cataract (diabetic group) and 32 controls (control group) were analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS). Differentially expressed lipids between the two groups were determined through partial least-squares-discriminant analysis and principal component analysis. Receiver operating characteristic curve was calculated to evaluate the diagnostic value of differentially expressed lipids in distinguishing diabetic cataract patients from the control subjects. The correlations of the target lipids with the serum lipids were determined by Pearson correlation. Results: The results of LC-MS/MS analysis revealed a remarkable variation in lipid class composition. As compared with the control group, the percentage of triglycerides (TGs) was higher and that of Ceramide-1-phosphates was significantly lower in the AH of the diabetic group. A multivariate analysis of the lipid species showed that the expression levels of 16 of 639 lipids significantly changed in the diabetic group as compared to the control group. Among them, TG (42:6) (area under curve [AUC] = 0.985, P < 0.0001) and diacylglycerol (DG) (24:2) (AUC = 0.944, P < 0.0001) exhibited high prediction capability for diabetic cataract. The results of Pearson correlation revealed that the concentrations of TG (42:6) in AH were positively correlated with serum TG levels in diabetic patients. Conclusions: The lipid composition of AH of diabetic cataract patients showed a significant difference from that of the healthy subjects. Lipid changes, especially the high AH levels of TG (42:6) and DG (24:2), might contribute to cataractogenesis and possibly be involved in the development of diabetic cataract. Translational Relevance: Changes in the lipid profile of AH may partly account for the pathogenesis of diabetic cataracts and lead to a novel therapeutic strategy for diabetic cataract.

Aqueous Humor Analysis in Overlapping Clinical Diagnosis of Cytomegalovirus and Rubella Virus Anterior Uveitis.

Background and Objectives: A cross-sectional single-center study was conducted to investigate the etiology in hypertensive anterior uveitis whose clinical features are not fully distinctive from cytomegalovirus or from rubella virus and to demonstrate the possible coexistence of both these viruses in causing anterior uveitis. Materials and Methods: The clinical charts of a cohort of patients with hypertensive viral anterior uveitis of uncertain origin consecutively seen in a single center from 2019 to 2022 were retrospectively reviewed; data on the clinical features, aqueous polymerase chain reaction, and antibody response to cytomegalovirus and rubella virus were collected. Results: Forty-three eyes of as many subjects with viral anterior uveitis of uncertain origin were included. Thirty-two patients had an aqueous polymerase chain reaction or antibody index positive to cytomegalovirus only, while 11 cases had an aqueous antibody response to both cytomegalovirus and rubella virus. This latter overlapping group had a statistically significant higher rate of hypochromia and anterior vitritis (p-value: 0.02 and < 0.001, respectively). Conclusions: The simultaneous presence of intraocular antibodies against cytomegalovirus and rubella virus could redefine the differential diagnosis of hypertensive viral anterior uveitis, demonstrating a possible "converged" immune pathway consisting in a variety of stimuli.

APRIL and BAFF play a key role in differentiating vitreoretinal lymphoma from uveitis.

BACKGROUND: Early diagnosis of vitreoretinal lymphoma (VRL) is critical for the successful treatment of this rare intraocular malignancy. However, fast and reliable diagnosis of VRL in patients presenting with intermediate or posterior non-infectious uveitis remains a challenge. A proliferation-inducing ligand (APRIL) and B-cell activating factor (BAFF) are vital factors in the pathophysiology, diagnosis, and prognosis of primary central nervous system lymphoma (PCNSL) and systemic autoimmune diseases. However, their utility as biomarkers for the diagnosis of VRL and uveitis remains unclear. METHODS: In this retrospective study, we analyzed APRIL and BAFF levels in the aqueous humor (AH) of 43 eyes of 40 patients, including 23 eyes of 20 patients with VRL, eight eyes of eight patients with uveitis, and 12 eyes of 12 patients with other ocular diseases (OODs). Additionally, we measured their levels after induction of chemotherapy in five eyes of five patients with VRL. RESULTS: AH levels of APRIL reliably distinguished VRL from uveitis, with a specificity of 78.3% and sensitivity of 75%. BAFF also showed similar potential. Serial AH analysis of patients with VRL during chemotherapy demonstrated a corresponding decline in AH levels of APRIL and BAFF. CONCLUSION: This study extends the spectrum of valuable diagnostic biomarkers for VRL and uveitis. In patients with uveitis, the assessment of AH APRIL may help accelerate the diagnosis of VRL. Moreover, our results underline the important role of APRIL and BAFF in therapeutic monitoring of VRL.

Study on Factors Associated with High Myopia CNV in Aqueous Humor and Serum.

Objective: The objective is to investigate the relationship and correlation between PEDF and TGF-ß in aqueous humor and serum and high myopia CNV lesions. Methods: For each group of patients (namely, group A: patients with high myopia CNV (mCNV); group B: patients with high myopia without CNV; group C: patients with CNV caused by other eye diseases; and group D (control group): patients with simple cataract (without CNV and high myopia)), 20 patients were collected. A total of 40 patients have been collected since the beginning of the study in December 2020, including 7 patients in group A, 13 patients in group B, 10 patients in group C, and 10 patients in group D. Serum and aqueous humor samples were collected, and PEDF and TGF-ß levels in serum and aqueous humor were detected by enzyme-linked immunosorbent assay (ELISA). SPSS 26.0 statistical software was used to process the data. Independent sample t-test was used to compare the data of the same factor in the same group between serum and aqueous humor. Comparisons of the same factors between different groups were performed using a one-way analysis of variance (ANOVA). Correlation analysis was conducted by the Pearson correlation coefficient test. P < 0.05 indicated that the difference was statistically significant. Results: There were no significant differences in age, gender, and course of disease among all groups (P > 0.05). The concentration of PEDF in aqueous humor in group A and group C was higher than that in group B and group D. There was no significant correlation between serum PEDF content and the above-mentioned diseases. The concentration of TGF-ß in aqueous humor in groups A, B, and C was significantly higher than that in group D. However, there was no significant correlation between TGF-ß content in serum and the above-mentioned diseases. There was no significant correlation between aqueous humor and serum PEDF. There was no significant correlation between the content of TGF-ß in aqueous humor and serum. Conclusion: TGF-ß in aqueous humor may be involved in the development of high myopia and intraocular CNV disease. However, PEDF in aqueous humor may be involved in the development of intraocular CNV disease and has no significant correlation with high myopia. At the same time, TGF-ß and PEDF in serum had no significant correlation with high myopia and intraocular CNV disease. There was no significant correlation between the concentrations of TGF-ß and PEDF in aqueous humor and serum.

Status of higher TGF-ß1 and TGF-ß2 levels in the aqueous humour of patients with diabetes and cataracts.

BACKGROUND: Transforming growth factor (TGF) is a cytokine that acts on the proliferation, migration, differentiation, and apoptosis of cells and the accumulation of extracellular matrix components. Very few studies have precisely evaluated the concentration of TGF-ß in the aqueous humour (AH) of diabetic and cataract (DMC) eyes due to the low expression of proteins in the AH or other reasons. The concentrations of TGF-ß1, -ß2, and -ß3 in the AH of the DMC group were compared with those of the age-related cataract (ARC) group. METHODS: We collected AH and lens epithelium samples from 33 DMC patients and 36 ARC patients. Luminex liquid suspension chip detection was applied to detect the concentration of TGF-ß1, -ß2, and -ß3 in the AH samples. The expression of TGFB1/2/3 in lens epithelium samples was determined by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: The concentrations of TGF-ß1 and TGF-ß2 in AH samples of DMC eyes were higher than those of ARC eyes. The differences in TGF-ß1 and TGF-ß2 between the two groups were statistically significant (P value = 0.001 for TGF-ß1, P value = 0.023 for TGF-ß2). The difference of the correlation between TGF-ß1 and glycosylated haemoglobin was significant (P value = 0.011, and Pearson correlation coefficient = 0.306). The difference of the correlation between TGF-ß2 and glycosylated haemoglobin was significant (P value = 0.026, and Pearson correlation coefficient = 0.269). The mRNA expression levels of TGFB1 and TGFB2 were upregulated in DMC epithelium samples compared with ARC epithelium samples. The differences in TGFB1 and TGFB2 between the two groups were statistically significant (P value for TGFB1 = 0.041, P value for TGFB2 = 0.021). CONCLUSIONS: The concentrations of TGF-ß1 and TGF-ß2 in AH samples were significantly higher in DMC eyes than in ARC eyes. The higher the glycosylated haemoglobin was, the higher the concentrations of TGF-ß1 and -ß2 were. The mRNA expression of TGFB1 and TGFB2 was significantly upregulated in DMC epithelial samples compared with ARC epithelial samples, suggesting the proinflammatory status of the anterior chamber of DMC eyes.

Cytokine Changes in the Aqueous Humor in Rubella-Related Fuchs Heterochromic Iridocyclitis.

This retrospective study is aimed at determining the correlation between cytokine levels and virus status in the aqueous humor of 38 patients with Fuchs heterochromic iridocyclitis (FHI) with/without a viral presence between May 2017 and January 2020. The levels of cytokines were analyzed in the groups with and without virus-related FHI. Among the patients, 50% had rubella virus, 5.26% had cytomegalovirus, and 2.63% had herpes simplex virus infections. The expression of interleukin-6 (IL-6) and IL-8 was significantly higher, and that of basic fibroblast growth factor (bFGF) was significantly lower in the virus-positive group than in the virus-negative group (P = 0.015, P = 0.001, and P = 0.001, respectively). Although there was no significant difference in the mean expression of vascular cell adhesion protein 1 (VCAM-1), IL-10, and vascular endothelial growth factor (VEGF), that of VCAM-1 and IL-10 was higher (M = 1338 and M = 1390, respectively; M = 6.225 and 10.600, respectively) and that of VEGF was lower (M = 134.5 and M = 38.70, respectively) in the virus-positive group than in the virus-negative group. Similar findings were observed for the expressions of IL-6, IL-8, and bFGF in the rubella-positive and rubella-negative groups. Viral presence was highly related to FHI, especially that of the rubella virus. High levels of inflammatory cytokines and low levels of neovascularization-related factors are involved in rubella-related FHI. These study findings could be helpful in the diagnosis and treatment of FHI.

Proinflammatory Cytokine Profile Differences between Primary Open-Angle and Pseudoexfoliative Glaucoma.

INTRODUCTION: Few studies have investigated glaucoma biomarkers in aqueous humor and tear and have found elevations of proinflammatory cytokines in patients with primary open-angle glaucoma (POAG) and pseudoexfoliative glaucoma (PXG). In this study, we investigate differences in inflammatory cytokines between POAG and PXG patients to find specific disease biomarkers. METHODS: For this purpose, tear and aqueous humor samples of 14 eyes with POAG and 15 eyes with PXG undergoing cataract surgery were immunoassayed for 27 proinflammatory cytokines. The concentrations of cytokines in tear and aqueous humor and their association with clinical variables were analyzed, correlated, and compared between the groups. RESULTS: We found that the levels of three cytokines differed significantly in the aqueous humor of POAG and PXG patients: IL-12 and IL-13 were higher in the POAG group, while monocyte chemoattractant protein-1 (monocyte chemotactic and activating factor) was higher in the PXG group. The number of topical hypotensive medications was correlated with diminished levels of two cytokines (IL-7 and basic fibroblast growth factor) in aqueous humor in the POAG group and with diminished levels of IL-12 in tear in the PXG group. CONCLUSION: We conclude that both POAG and PXG show elevated concentrations of proinflammatory cytokines in tear and aqueous humor that could be used as biomarkers for these types of glaucoma and that the concentrations in aqueous humor of three cytokines, IL-12, IL-13, and monocyte chemoattractant protein-1 (monocyte chemotactic and activating factor), could be used to differentiate POAG and PXG.

Dysregulated expression of microRNAs in aqueous humor from intraocular tuberculosis patients.

BACKGROUND: Systemic Mycobacterium tuberculosis (Mtb) infection alters microRNA's expression that controls cellular processes and modulates host defense mechanisms. However, the role of miRNAs in intraocular tuberculosis (IOTB) remains unknown. Therefore, this study aims to identify dysregulated miRNAs in the aqueous humor (AH) of patients with IOTB. METHODS: AH from intraocular tuberculosis patients (n = 2) and cataract controls (n = 2) were used for small RNA deep sequencing using HiSeq Illumina sequencing platform. Differentially expressed miRNAs and their targets were identified by the bioinformatics approach, and their regulatory functions were predicted by pathway enrichment analysis. The expression of selected miRNAs and their binding targets were further validated by real-time quantitative PCR (RT-qPCR). RESULTS: In total, we identified 56 differentially expressed miRNAs in the AH of intraocular tuberculosis (IOTB) patients compared to controls. We selected four significantly dysregulated miRNAs (miR-423-5p, miR-328-3p, miR-21-5p, and miR-16-5p) based on the RT-qPCR validation and predicted their gene targets. We developed a miRNA-targets regulatory network by combining pathways of interest and genes associated with TB. We identified that these four miRNAs might play an important role in IOTB pathogenesis via tuberculosis-associated pathways; PI3K-Akt signaling, autophagy and MAPK pathway. CONCLUSIONS: For the first time, this study identifies the dysregulation of four miRNAs in the AH of IOTB patients using the ultra-low input small-RNA sequencing approach. Further target prediction and validation identify the role of these miRNAs in tuberculosis pathogenesis via tuberculosis-related pathways. This study identifies miRNAs as potentially ideal biomarkers in the aqueous humor of IOTB patients.

A novel, sensitive, and widely accessible besifloxacin quantification method by HPLC-fluorescence: Application to an ocular pharmacokinetic study.

Besifloxacin has been embraced for the treatment of ocular bacterial infections. While LC-MS/MS has been used in investigating BSF pharmacokinetics, those costly instruments are not universally available and have complicated requirements for operation and maintenance. Additionally, pharmacokinetics of besifloxacin in dose-intense regimens are still unknown. Herein, a new quantification method was developed employing the widely accessible HPLC with fluorescence detection and applied to an ocular pharmacokinetic study with an intense regimen. Biosamples were pre-treated using protein precipitation. Chromatographic separation was achieved on a C18 column using mobile phase of 0.1% trifluoroacetic acid and acetonitrile. To address the weak fluorescence issue of besifloxacin, effects of detection parameters, elution pattern, pH of mobile phase, and reconstitution solvents were investigated. The method was fully validated per US-FDA guidelines and demonstrated precision (<13%), accuracy (91-112%), lower limit of quantification (5 ng/mL), linearity over clinically relevant concentrations (R2 > 0.999), matrix-effects (93-105%), recoveries (95-106%), and excellent selectivity. The method showed agreement with agar disk diffusion assays for in vitro screening and comparable in vivo performance to LC-MS/MS (Deming Regression, y = 1.010x + 0.123, r = 0.997; Bland-Altman analysis, mean difference was -6.3%; n = 21). Pharmacokinetic parameters suggested superior surface-retentive properties of besifloxacin. Maximum concentrations were 1412 ± 1910 and 0.15 ± 0.12 µg/mL; area under the curve was 1,637 and 1.08 µg·h/g; and half-life was 4.9 and 4.1 h; and pharmacokinetic-to-pharmacodynamic ratios were ≥ 409 and ≤ 17.8 against ocular pathogens in tears and aqueous humor, respectively. This readily available method is sensitive for biosamples and practical for routine use, facilitating besifloxacin therapy development.

Intra-population differences of apolipoproteins in the aqueous humor.

BACKGROUND: Evidence suggests that proteins related to lipid metabolism, such as apolipoproteins, play an important role in the maintenance of normal vision. While several members of the apolipoprotein family are abundant in human aqueous humor (AH), their study remains difficult due to the AH's small volume, low protein concentration, and the invasive nature of sample collection. In this study, we report the use of Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) to discover associations between AH apolipoproteins and race, gender, and ocular structure in patients with and without primary open angle glaucoma (POAG). METHODS: AH samples were collected from 231 patients undergoing phacoemulsification or glaucoma incisional surgery at the Medical College of Georgia, Augusta University and subsequently analyzed via LC-MS/MS. The number of peptide spectrum matches (PSMs) for each protein was used as a semi-quantitative measure of relative protein levels. Parameters related to ocular structure were determined using Optical Coherence Tomography (OCT) and Heidelberg Retinal Tomography (HRT). These data sets were probed for relationships between apolipoprotein levels and POAG, demographics (gender and race), and ocular structure. RESULTS: A total of ten apolipoproteins were detected in the 231 collected AH samples, with six detected in 100% of the samples, one detected in almost 57% of the samples and three detected in less than 10% of the samples. The levels of APOA1, APOC3, and APOD were higher among POAG subjects. Stratification by gender and race revealed demographic-specific variations. The levels of five apolipoproteins (APOA1, APOA2, APOA4, APOC3, and APOD) were higher in female POAG patients, whereas no apolipoprotein levels were altered in male POAG patients. The levels of APOA1, APOA2, APOA4, and APOD were increased in glaucomatous African American patients, whereas APOE and APOH levels were decreased in glaucomatous Caucasian patients. We also found distinct associations between apolipoprotein levels and OCT and HRT parameters in patients with and without POAG. CONCLUSIONS: The intra-population variation in apolipoprotein levels highlights the heterogeneity of glaucoma as a disease, suggesting the importance of personalized treatments. Gender and race-specific alterations may be associated with higher risks of POAG in females and members of the African American population.

Reduced 25-hydroxyvitamin D concentration in the aqueous humor of cataract patients with open-angle glaucoma.

Previous epidemiological studies have demonstrated that the lower serum concentration of vitamin D was associated with elevated risk of open-angle glaucoma (OAG). However, few studies have examined the association between aqueous humor vitamin D concentrations and OAG. Hence, we investigated the relationship between 25-hydroxyvitamin D (25(OH)D) concentrations in aqueous humor and OAG. We measured 25(OH)D concentrations in aqueous humor and serum of 126 patients who underwent cataract surgery. 36 were patients with OAG and 90 were control patients. The 25(OH)D concentrations were measured using Elecsys Vitamin D Total Kits with the Cobas e602 module (Roche Diagnostics, Mannheim, Germany), an electrochemiluminescence assay. Multiple linear regression analysis was performed to investigate factors associated with serum and aqueous humor 25(OH)D concentrations. Patients with OAG had significantly lower 25(OH)D concentrations in aqueous humor than control patients. Serum 25(OH)D concentrations were higher in patients with OAG than in the control, but this was not statistically significant. 25(OH)D concentrations in aqueous humor of patients with OAG were significantly associated with axial length but not with glaucoma severity, which was determined by the retinal nerve fiber layer thickness or mean deviation. Vitamin D concentrations in aqueous humor of patients with OAG were significantly lower than those in patients without OAG.

Utilization of Localized Surface Plasmon Resonance of Silver Nanoparticles for the Spectrofluorimetric Estimation of Oxymetazoline in Dosage Forms: Application to Aqueous Humor.

A simple, novel, cost-effective and highly sensitive spectrofluorimetric method was developed for estimation of the nasal decongestant oxymetazoline (OMZ) whether per se or in its pharmaceutical preparations using colloidal silver nanoparticles (AgNPs). The method is based on the high catalytic potential activity of AgNPs on the fluorescence intensity of OMZ leading to 12-fold increase in its fluorescence intensity. The response was linear over the range of 20.0 to 700.0 ng/mL with lower detection limit of 5.0 ng/mL and limit of quantification of 14.0 ng/mL. The proposed method was applied to the assay of commercial nasal drops, nasal spray and synthetic aqueous humor. Interference likely to be encountered from co-administered drugs was studied. The developed method was optimized and validated as per International Council of Harmonization (ICH). An explanation for the drug-AgNPs interaction was proposed.

High irisin and low BDNF levels in aqueous humor of high myopia.

BACKGROUND: The pathogenesis of myopia remains unclear. Both genetic and environmental factors play a role in the disease progression. Reasons including reduced physical activity (PA) and low-grade intraocular inflammation may be involved in the development of myopia. OBJECTIVES: To analyze the levels of irisin, brain-derived neurotrophic factor (BDNF) and other intraocular cytokines in aqueous humor of high myopia patients, and to evaluate the roles of PA and inflammation in developing myopia. MATERIAL AND METHODS: We collected aqueous humor samples from patients with axial length (AL) over 26 mm (n = 35) or shorter than 25 mm (n = 38) during cataract extraction surgery. Samples were assayed using the enzyme-linked immunosorbent assay (ELISA) kit for irisin and a multiplex immunoassay kit for BDNF, interleukin (IL)-6, IL-8 and IL-10, and tumor necrosis factor alpha (TNF-α). RESULTS: Irisin levels in the aqueous samples of the highly myopic eyes were significantly higher than in the control group (p = 0.027). The BDNF levels of the highly myopic group were significantly lower than in the control group (p = 0.043). Median level of leukemia inhibitory factor (LIF) for highly myopic group (2.035 pg/mL) was statistically significantly higher than in the control group (0.750 pg/mL) (U = 210.5, Z = -4.495, p < 0.001). Interleukin 1 receptor antagonist (IL-1ra) level in the aqueous samples of the highly myopic group was significantly lower than in the shorter AL group (p = 0.049). Interleukin 6, IL-8 and IL-10 levels were not significantly different between the 2 groups (p = 0.501, p = 0.059 and p = 0.192, respectively). Tumor necrosis factor α levels could only be detected in 30 samples and median levels in the 2 groups were not statistically significantly different (U = 99, Z = -0.482, p = 0.650). No correlation was found between IL-6, IL-8, IL-10 and TNF-α, and the AL (p > 0.05). Irisin was positively correlated with AL (p = 0.028, r = 0.287). The BDNF was negatively correlated with AL (p = 0.040, r = -0.246). Interleukin 1ra was negatively correlated with AL (p = 0.038, r = -0.276). There was also a correlation between LIF and AL (p < 0.001, r = 0.486). CONCLUSIONS: Higher irisin level in high myopia group opens a new direction to discover the relationship between PA and myopia. The decreased BDNF in high myopia group probably demonstrates the connection between myopia and neurodegenerative disease.