Vitreous Samples Research Articles

15 years of PVRL‐experience of Prague uveitis Centre

AbstractIntravitreal lymphoma is a malignant masquerade syndrome which is crucial to identify among uveitis patients because of its life‐threatening nature.A total of 35 immunocompetent patients, 15 men and 20 women, were observed with vitreoretinal lymphoma in the Centre for Diagnosis and Treatment of Uveitis in Prague between the years 2003 to 2019. Primary vitreoretinal lymphoma (PVRL) was diagnosed in 15 patients, 6 patients from this group did not show any CNS involvement nor had an intraocular relapse during the observation period. The clinical manifestation of lymphoma in our patients was usually the vitreous haze, sometimes accompanied by retinal lesions and anterior chamber cells with or without keratic precipitates. In our observation, the diagnosis was determined by cytology or/and flow cytometry from vitreous analysis in all patients with exception of 4 patients with clinically highly suspicious finding of vitreous. Beside the vitritis, retinal infiltration was present in 3 of these 4 patients. From this group of 4 patients, the diagnosis was confirmed from brain biopsy in 3 patients and lung biopsy in 1 patient. The vitreous analysis was negative for lymphoma in 2 patients and in 2 patients' vitreous analysis was not performed. Over the years, the sensitivity of VRL detection from vitreous sample became very high, in our observation 94%.All patients diagnosed with VRL by vitreous analysis were sent to haemato‐oncologist work‐up. Treatment was decided by the consensus of a haemato‐oncologist and an ophthalmologist according to the clinical manifestation. Combination of intravitreal and systemic chemotherapy is recommended in many reports. There has been a decline in ocular and brain radiotherapy in recent years because of the unpredictable and irreversible nature of its side effects. Autologous stem cell transplantation (ASCT) following systemic chemotherapy was indicated in 3 patients. The relapse of lymphoma was observed in 13 patients, first relapse was after 11–120 months (median 32 months).Supported by research project AZV MZ CR NU20‐03‐00253.

Low expression of RBP4 in the vitreous humour of patients with proliferative diabetic retinopathy who underwent Conbercept intravitreal injection.

Intravitreal injection of anti-VEGF antibodies has been widely used in the treatment of proliferative diabetic retinopathy (PDR). However, anti-VEGF drugs can exacerbate fibrosis and eventually lead to retinal detachment. To explore proteins closely related to fibrosis, we conducted proteomic analysis of human vitreous humour collected from PDR patients who have or have not intravitreal Conbercept (IVC) injection. Sixteen vitreous humour samples from PDR patients with preoperative IVC and 20 samples from those without preoperative IVC were examined. An immunodepletion kit was used to remove high-abundance vitreous proteins. Conbercept-induced changes were determined using a tandem mass tag-based quantitative proteomic strategy. Enzyme-linked immunosorbent assays were performed to confirm the concentrations of selected proteins and validate the proteomic results. Based on a false discovery rate between 0.05% and -0.05% and a fold-change > 1.5, 97 proteins were altered (49 higher levels and 48 lower levels) in response to IVC. Differentially expressed proteins were found in the extracellular and intracellular regions and were found to be involved in VEGF binding and VEGF-activated receptor activity. Protein-protein interactions indicated associations with fibrosis, neovascularisation and inflammatory signalling pathways. We found the low levels of RBP4 in the vitreous humour of PDR patients with IVC injection, as revealed by ELISA and proteomic profiling. Moreover, RBP4 significantly restored the mitochondrial function of HRMECs induced by AGEs and down regulated the level of glycolysis. Our study is the first to report that RBP4 decreases in the vitreous humour of PDR patients who underwent Conbercept treatment, thereby verifying the role of RBP4 in glucose metabolism. Results provide evidence for the potential mechanism underlying Conbercept-related fibrosis.

Autofluorescence imaging for diagnosis and follow‐up of serpiginous choroiditis

AbstractPurpose: To report by multimodality imaging the follow‐up of a patient with posterior uveitis presenting serpiginous choroiditis (SC). To highlight the importance of autofluorescence in the follow‐up of patients with atrophic scarring due to CS.Methods: We present an observational case of SC documented photographically. Clinical data were gathered retrospectively from several visits. The data included corrected visual acuity (BCVA), fundus examination, optical coherence tomography (OCT) and autofluorescence (AF).Results: Periodic revision of a 49‐year‐old female being followed up for SC secondary to tuberculosis. Mantoux test was positive in 2010 and she was treated correctly. Twelve years later, BCVA was hand movements in the right eye and in the left eye 20/40. Fundus examination revealed no presence of vitritis and atrophic scars post choroiditis serpiginosa. OCT displayed disruption of outer retinal layers. AF revealed atrophy plaque with a new diffuse hypoautofluorescence halo surrounding hyperautofluorescent rim. Aqueous humour and vitreous sample results were negatives. Finally, the lesion was diagnosed as a serpiginous choroiditis. She was treated with corticosteroid and biological therapy to improve her visual acuity. It should suggest a reactivation of SC the appearance of a new atrophy plaque with ill‐defined creamy rim, early block and diffuse oozing at late times, choroidal hypoperfusion, diffuse hypoautofluorescence halo surrounding hyperautofluorescent rim and disruption of outer retinal layers with ellipsoid loss.Conclusions: A differential diagnosis should be made between SC and posterior uveitis secondary to tuberculosis. Multimodal imaging by autofluorescence is important for the diagnosis and follow‐up of patients with atrophic scarring due to SC. Differential diagnosis of cause posterior uveitis is essential for the monitoring and treatment. In inflammatory diseases always rule out infection prior to corticosteroid treatment or biological therapy.

Acute retinal necrosis in a patient on immunosuppressive treatment for COVID-19 pneumonia: a case report

BackgroundPatients with coronavirus disease 2019 (COVID-19) occasionally develop ocular complications. We report a case of acute retinal necrosis (ARN) caused by Epstein–Barr Virus (EBV) that developed in a patient who had severe acute respiratory syndrome due to SARS-CoV-2 infection.Case presentationA 68-year-old woman complained of floaters and blurred vision in her right eye as she was receiving systemic prednisolone for COVID-19 pneumonia under isolation in our hospital. The patient visited an ophthalmologist following her discharge from the hospital and after the 2 weeks of isolation had ended. At the initial examination, her best-corrected visual acuity (BCVA) was 20/100 in the right eye, and the eye showed moderate anterior segment inflammation and vitreous opacities. Treatment was initiated with topical 0.1% betamethasone and 1.5% levofloxacin. After 1 month, the inflammation in the right eye decreased and her BCVA improved to 20/40. However, on day 48 from her initial visit, the inflammation in her right eye worsened and her BCVA decreased to 20/2000 by day 80. Pars plana vitrectomy with silicone oil tamponade was performed to remove the vitreous opacities, and expanded white exudates peripherally and retinal vessels with white sheathing suggestive of acute retinal necrosis (ARN) were seen intraoperatively. Analysis of the vitreous sample revealed EBV positivity on polymerase chain reaction. The patient was diagnosed with EBV-associated ARN and treated with systemic steroids and valaciclovir. The ocular inflammation gradually decreased, and she was discharged from the hospital. However, a week later, the inflammation in the right eye markedly worsened. Despite another course of steroids, the inflammation worsened, resulting in total retinal detachment and absolute glaucoma. Because of the severe pain, the right eye was enucleated.ConclusionsClinicians should be aware that COVID-19 and immunosuppressive treatment can reactivate EBV in the eye.

Upregulation of PCED1B-AS1 in proliferative diabetic retinopathy and its involvement in retinal vascular endothelial cell proliferation

BackgroundThis study was to assess the diagnostic value of PCED1B-AS1 for proliferative diabetic retinopathy (PDR) and investigate the involvement of PCED1B-AS1 in PDR.MethodsThe vitreous and blood specimens from 37 subjects with PDR and 21 non-diabetics were examined by reverse transcription quantitative PCR to determine the PCED1B-AS1 level. The two groups were age- and gender-matched. Receiver operating characteristic (ROC) curves were plotted to visually illustrate the diagnostic ability of PCED1B-AS1. Human retinal Müller glial cells were studied by ELISA. Proliferation and migration of human retinal microvascular endothelial cells (HRMECs) were assessed in vitro.ResultsSignificant increases of PCED1B-AS1 levels were observed in the vitreous samples and CD34 + VEGFR-2 + cells from blood samples of diabetic subjects with PDR, compared with those of non-diabetics. The ROC curve based on the vitreous PCED1B-AS1 levels revealed an AUC of 0.812, while the ROC curve based on the PCED1B-AS1 levels in CD34 + VEGFR-2 + cells from blood samples revealed an AUC of 0.870. In Müller cell cultures, PCED1B-AS1 siRNA significantly attenuated VEGF and MCP-1 upregulation which were induced by CoCl2 and TNF-α. Additionally, PCED1B-AS1 siRNA attenuated VEGF-induced proliferation and migration in HRMECs.ConclusionThis study revealed the potential of PCED1B-AS1 as a diagnostic biomarker for PDR. In vitro data point to the anti-angiogenic and anti-proliferation effects of PCED1B-AS1.

Comprehensive assessment of growth factors, inflammatory mediators, and cytokines in vitreous from patients with proliferative diabetic retinopathy

To assess alterations in growth factors, inflammatory mediators, and cytokines associated with vitreous-retinal diseases in vitreous humor from patients with proliferative diabetic retinopathy (PDR), and to identify potential new treatment targets and strategies. Control vitreous samples were collected from patients with macular hole, epiretinal membranes, or rhegmatogenous retinal detachments, and PDR samples from patients with complications of PDR, who required pars plana vitrectomy. Specimens were stored at -80°C and then investigated by Luminex multi-factor assay. Parametric and nonparametric analyses of demographic characteristics and cytokine expression levels were conducted using SPSS. There were no significant differences in demographic characteristics between patients with and without PDR. Expression levels of growth factors [platelet-derived growth factor (PDGF)-AA, glial cell line-derived neurotrophic factor (GDNF), and vascular endothelial growth factor A (VEGFA)], inflammatory mediators [interleukin (IL)-8, IL-11, and tumor necrosis factor-α (TNF-α)] and cytokines [chemokine C-X-C ligand (CXCL)10, interferon-γ (IFN-γ), and granulocyte macrophage-colony stimulating factor (GM-CSF)] were significantly elevated in vitreous humor from patients with PDR compared with those in the control group (all P<0.05). Further, VEGFA levels were lower in patients with PDR treated with anti-VEGF injection than those who were not (P<0.05), and there was no difference between the PDR group treated with anti-VEGF and controls (P>0.05). This proof-of-concept study demonstrates the potential for combinational therapeutic strategies to ameliorate diabetic retinopathy progression by targeting growth factors, inflammatory factors, and cytokines, in addition to VEGFA.

Decreased expression of Glucagon-like peptide-1 receptor and Sodium-glucose co-transporter 2 in patients with proliferative diabetic retinopathy.

To investigate the expression of Glucagon-like peptide-1 receptor (GLP-1R), sodium-glucose co-transporter (SGLT) 1, SGLT2, Glucose transporter type 1 (GLUT1) and GLUT2 in patients with diabetic retinopathy (DR). We obtained peripheral blood mononuclear cells (PBMCs) and vitreous samples from 26 proliferative DR (PDR) patients, 25 non-proliferative DR (NPDR) patients, 25 non-DR (NDR) patients, and 26 nondiabetic patients with idiopathic epiretinal membranes (ERMs, control). The protein level and mRNA expression level of GLP-1R were quantified by immunoblot and qRT-PCR and the levels of SGLT1, SGLT2, GLUT1, and GLUT2 expression were determined by PCR. Their association with clinical parameters and PBMCs/vitreous cytokine was analyzed. Furthermore, immunofluorescence staining of GLP-1R and SGLT2 was carried out on samples of fibrovascular membranes (FVMs) retrieved from 26 patients with PDR and 26 patients with ERMs. The transcriptional levels of GLP-1R and SGLT2 in PBMCs were significantly more decreased in PDR patients than in patients without DR and controls, which was simultaneously associated with an increased level of expression of tumor necrosis factor (TNF)-α and interferon (IFN)-γ. The expression levels of GLUT1 and GLUT2 were tightly correlated with their SGLT partners, respectively. Further, Immunofluorescence staining showed no positive staining of GLP-1R and SGLT2 was detected in the FVMs from PDR. GLP-1R and SGLT2 were significantly decreased in PDR patients which was associated with an increased level of expression of TNF-α and IFN-γ. These findings implicate that defective GLP-1R and SGLT2 signaling may potentially correlate with immune response cytokines in patients with PDR.

Clinical significance of serum and vitreous soluble interleukin-2 receptor in patients with intraocular lymphoma

BackgroundIntraocular lymphoma (IOL) is a masquerade syndrome that mimics uveitis, making diagnosis difficult. The serum soluble interleukin-2 receptor (sIL-2R), which is cleaved by matrix metalloproteinase (MMP) -2 and MMP-9, has been recognized as a tumor-related biomarker of malignant lymphomas. The aim of this study was to review the reliability of serum and vitreous sIL-2R for distinguishing IOL from uveitis.MethodsPatients who underwent diagnostic vitrectomy for marked vitreous haze at Hokkaido University Hospital between April 2014 and June 2019 were enrolled. The patients were divided into an IOL group and a uveitis group, according to the pathology of their vitreous samples. The IOL group was further divided at the time of vitrectomy into patients who already had extraocular involvement (IOL with extraocular involvement group) and patients with no evidence of having extraocular involvement (IOL without extraocular involvement group). Serum sIL-2R, and intravitreal sIL-2R, MMP-2, and MMP-9 levels were assessed.ResultsTwenty-five eyes of 25 patients, and 15 eyes of 15 patients were included in the IOL group and uveitis group, respectively. The serum sIL-2R levels were significantly lower in the IOL group than in the uveitis group (P < 0.05), and 20.0% and 66.7% in the IOL and the uveitis group showed high sIL-2R value above the normal range. Vitreous sIL-2R tended to be higher in the IOL group than in the uveitis group (P = 0.80). Serum sIL-2R was significantly lower in the IOL without extraocular involvement group than in the IOL with extraocular involvement group (P < 0.05); 5.9% in the IOL without extraocular involvement group and 50.0% in the IOL with extraocular involvement group showed high sIL-2R value above the normal range. Vitreous sIL-2R, MMP-2, and MMP-9 tended to be higher in the IOL with extraocular involvement group than in the IOL without extraocular involvement group (P = 0.30, < 0.05, 0.16).ConclusionsSerum sIL-2R is often within the normal range in IOL patients. Even if it is within the normal range, the possibility of IOL should be considered. Serum sIL-2R is not a reliable biomarker for IOL, whereas vitreous sIL-2R may be useful for the diagnosis of IOL.

CULTURE POSITIVITY RATES OF DEEP VITREOUS BIOPSY UNDER AIR VIS-À-VIS CONVENTIONAL ANTERIOR VITREOUS BIOPSY IN ENDOGENOUS ENDOPHTHALMITIS.

To describe the comparison of the culture positivity rates of deep vitreous biopsy under air and conventional anterior vitreous biopsy in endogenous endophthalmitis. A retrospective, consecutive, comparative series including cases of endogenous endophthalmitis from January 2014 to January 2021. They were divided into those where conventional anterior vitreous biopsy was taken and those where a deep biopsy was taken under air (DBA group). The culture positivity rate among the two groups was compared, and factors affecting the same were analyzed by a regression analysis. There were 140 eyes in the conventional anterior vitreous biopsy group and 44 eyes in the DBA group. Sex, age, duration of symptoms, underlying systemic illness, presenting vision in logMAR, and total number of vitreous interventions were comparable between the two groups. Visual outcome was better in patients undergoing DBA as compared with those who underwent conventional anterior vitreous biopsy , (1.28 ± 1.01 logMAR, 20/380 vs. 1.88 ± 1.33 logMAR, 20/1,500, P = 0.03). No case in the DBA group developed retinal detachment or any complication attributable to the technique. In the conventional anterior vitreous biopsy group, the culture positivity was seen in 43/140 samples (30.71%), and in the DBA group, it was noted in 29/44 samples (65.9%). Taking a DBA was the only factor that was significant both in bivariate (odds ratio 4.36, 95% confidence interval 2.12-8.95, P < 0.0001) and multivariate analysis (odds ratio 4.02, 95% confidence interval 3.1-7.43, P < 0.0001). Vitreous sampling can be performed from the deep vitreous cavity in endogenous endophthalmitis safely and effectively under air infusion, improving the culture positivity rates.

Intraocular Lymphoma: Correlation of Cytology and Flow Cytometry Results in Vitreous Fluid Samples

Intraocular Lymphoma: Correlation of Cytology and Flow Cytometry Results in Vitreous Fluid Samples

Optimal timing of preoperative intravitreal anti-VEGF injection for proliferative diabetic retinopathy patients.

To analyze concentrations of vascular endothelial growth factor (VEGF) and fibrosis-related factors in vitreous fluid of proliferative diabetic retinopathy (PDR) patients pre-treated with intravitreal anti-VEGF injections (IVI) at different time periods prior to pars plana vitrectomy (PPV), and their correlation with the degree of vitreoretinal fibrosis and explore the optimal timing of preoperative IVI. The prospective case-control study from January 2019 to July 2020 included 31 eyes with PDR-related complications (PDR group) and 21 eyes with non-diabetic ocular disease (control group) requiring PPV. PDR eyes were divided into four groups based on timing of PPV: 3d after IVI (3-day group); 5d after IVI (5-day group); 7 or more days after IVI (≥7-day group); and no IVI. Vitreous fluid samples (0.5-1.0 mL) were collected prior to switching on the infusion before routine 23-G PPV. Concentrations of VEGF, basic fibroblast growth factor (bFGF), periostin (PN), interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α were measured by immunoassay, and concentration differences for each cytokine were compared among the groups. The degree of vitreoretinal fibrosis was graded intraoperatively, and the correlation between the changes in cytokine levels and the severity of vitreoretinal fibrosis was analyzed by univariate ordinal logistic regression analysis. PDR eyes without IVI had significantly higher VEGF, bFGF, PN, and IL-6 concentrations than non-diabetic eyes (all P<0.05), and had a significantly higher concentration of VEGF (P<0.05) and a significantly lower concentration of IL-8 (P<0.05) than PDR eyes with IVI. Statistically significant differences were also observed for concentrations of VEGF, bFGF, PN, IL-6, and IL-8 among 3-day, 5-day, and ≥7-day groups (all P<0.05); meanwhile there was no significant difference in TNF-α among groups (P=0.226). The 5-day group had the lowest concentration of VEGF and the ≥7-day group had the highest concentration of bFGF and PN. The degree of vitreoretinal fibrosis was significantly higher in the ≥7-day group compared to the 3-day (P=0.015) and 5-day group (P=0.039), and vitreoretinal fibrosis correlated significantly with concentrations of bFGF, PN, IL-6, and IL-8 (all P<0.05). Univariate ordinal logistic regression analysis showed that bFGF was an independent risk factor for the severity of vitreoretinal fibrosis in PDR patients pre-treated with IVI. The vitreous concentrations of VEGF, bFGF, PN, IL-6, and IL-8 change after pretreatment with IVI before PPV in PDR patients. The degree of vitreoretinal fibrosis is higher in patients with a longer time between IVI treatment and PPV, which may be related to the angio-fibrosis switch. The results suggest that PPV should be performed 5d after IVI administration in PDR patients.

Effect of pan-retinal laser photocoagulation on intravitreal vascular endothelial growth factor concentration in proliferative diabetic retinopathy.

Objective: This study aimed to determine the intravitreal concentration of VEGF in eyes with PDR and to evaluate the effects of previous PRP on its level. Methods: It was a cross-sectional study performed at the Clinical Centre University. It included 90 eyes surgically treated with PPV, divided into three groups, group A - patients with PDR with previous PRP, group B - patients with PDR without previous PRP, and group C - PPV performed due to the indication unrelated to diabetes. A vitreous sample was obtained during PPV, and the VEGF concentration was determined using an Enzyme-linked immunosorbent assay test (ELISA). Shapiro-Wilk, nonparametric tests Kruskal-Wallis, Mann-Whithney U test, ANOVA and Spearman's correlation test were used. Results: The highest vitreous VEGF concentration was in group B - 972.96 (743.33-1149.13) and was higher than in group A - 69.22 (37.33-225.15) and in group C - 19.93 (1.15-32.17) (p<0.001). There was a positive correlation between VEGF vitreous concentration and glucose level in group A patients (Rho=0.410; p=0.027). Conclusion: As a treatment before PPV surgery, PRP showed to be effective in the reduction of VEGF levels, which also highlighted a decrease in complications during and postoperatively. Abbreviations: DRS = Diabetic Retinopathy Study, PDR = proliferative diabetic retinopathy, VEGF = vascular endothelial growth factor, PRP = pan-retinal photocoagulation, PPV = pars plana vitrectomy, HbA1c = glycosylated hemoglobin, ETDRS = Early treatment diabetic retinopathy study, ESR = erythrocyte sedimentation rate, BCVA = best corrected visual acuity, OCT = optical coherent tomography, ILM = internal limiting membrane, PHACO = phacoemulsification, IOL = intraocular lens, ELISA = Enzyme-linked immunosorbent assay test, AUC = area under the curve, DME = diabetic macular oedema, TDR = tractional retinal detachment, VMT = vitreomacular traction.

Plasma and vitreous selenium concentrations in patients with type 2 diabetes and diabetic retinopathy.

Background:This work aimed to determine and compare plasma and vitreous selenium (Se) concentrations in patients with type 2 diabetes and diabetic retinopathy (DR).Methods:A total of 60 type-2-diabetes patients including 20 without DR, 20 with non-proliferative DR (NPDR), and 20 with proliferative diabetic retinopathy (PDR), were involved in this study. Blood plasma samples were collected from above 60 patients and 20 normal controls (without diabetes). Twenty control vitreous samples were obtained from the eyes presenting a macular hole and epimacular membrane. Vitreous samples were also collected from PDR patients receiving one-week intravitreal anti-VEGF therapy or not. Plasma and vitreous Se concentrations were determined by inductively coupled plasma mass spectrometry.Results:Plasma Se concentrations in PDR patients (163.74 ± 32.68 μg/L) were significantly higher than those in normal control patients (121.59 ± 28.33 μg/L), NPDR patients (130.34 ± 29.11 μg/L), and the patients without DR (81.23 ± 20.59 μg/L) (all P < .001). Similarly, Se concentrations in vitreous samples of PDR patients (56.30 ± 12.03 μg/L) were consistently higher than those in control vitreous samples (26.26 ± 6.53 μg/L). In addition, vitreous Se concentrations in PDR patients decreased to 47.76 ± 9.72 μg/L after intravitreal injection of the anti-VEGF drug ranibizumab for one week, which was significantly lower than those before injection (P = .02). Plasma VEGF levels of diabetic patients were lower than those of the normal controls (P < .001). On the contrary, the vitreous VEGF level in the PDR group (913.61 ± 193.32 pg/mL) was significantly higher than that of the normal control group (101.23 ± 21.33 pg/mL) (P < .001).Conclusion:The elevation of Se concentrations may be an important risk factor in plasma and vitreous with diabetic retinopathy among type-2-diabetes patients. The elevated VEGF may be also closely related to the intraocular Se concentration in PDR patients.

Bioanalytical method for simultaneous determination of benzodiazepines in vitreous humor using liquid chromatography-tandem mass spectrometry

The use of vitreous humor (VH) in forensic casework has been growing in the last years due to numerous advantages. Several compounds can be evaluated in this matrix, including benzodiazepines whose determination is essential due to their great availability and potential of dependance and misuse. Postmortem toxicological analyses are required to determine the influence of benzodiazepines in deaths. However, most of the analytical methods which determine these drugs in VH are laborious and time consuming. This article describes a simple method based on protein precipitation for the determination of eight benzodiazepines in VH samples. Samples were prepared through a protein precipitation method and analyzed by liquid chromatography tandem mass spectrometry. Solvent choice and sample and solvent volumes for precipitation were optimized using chemometric approaches. The method was validated for selectivity, lower limit of quantification (LLOQ), linearity, carryover, precision, bias, matrix effect and dilution integrity. In order to verify the applicability, 62 vitreous humor samples were analyzed. LLOQs were 1 ng/mL and calibration curves were linear from 1 to 25 ng/mL (r2 > 0,99) for all analytes. Bias, precision and dilution integrity results were satisfactory according to proper guidelines. Ionization suppression was significant with values ranging from 8 to 37%. Two samples from real cases were positive for diazepam with the following concentrations: 6.80 ng/mL and 47.68 ng/mL, approximately 10 times lower than those found in peripheral blood. The procedure described here can be used as a straightforward and low cost method for the quantitation of multiple benzodiazepines in VH.

Primary Vitreoretinal Lymphoma: Current Diagnostic Laboratory Tests and New Emerging Molecular Tools.

Primary vitreoretinal lymphoma (PVRL), a rare aggressive malignancy primarily involving the retina and/or the vitreous, is a major diagnostic challenge for clinicians (who commonly misdiagnose it as chronic uveitis) as well as for pathologists (for biological and technical reasons). Delays in diagnosis and treatment are responsible for visual impairments and life-threatening consequences, usually related to central nervous system involvement. The identification of lymphoma cells in vitreous fluid, obtained by vitrectomy, is required for diagnosis. Of note, the scarcity of neoplastic cells in small volumes of vitreous sample, and the fragility of lymphoma cells with degenerative changes caused by previous steroid use for presumed uveitis makes diagnosis based on cytology plus immunophenotyping difficult. Interleukin levels, immunoglobulin heavy chain or T-cell receptor gene rearrangements, and MYD88 mutation are applied in combination with cytology to support diagnosis. We aim to describe the current laboratory technologies for PVRL diagnosis, focusing on the main issues that these methods have. In addition, new emerging diagnostic strategies, such as next-generation sequencing analysis, are discussed. The genetic profile of PVRL remains largely unexplored. Better knowledge of genetic alterations is critical for precision medicine interventions with target-based treatments of this lymphoma for which no standardised treatment protocol currently exists.

MiR-145, miR-92a and miR-375 Show Differential Expression in Serum from Patients with Diabetic Retinopathies.

Diabetic retinopathies are important disabling conditions. Micro-RNAs (miRNAs) are regulators of gene expression and diseases can change their expression. Our aim was to analyze the expression of miRNAs in serum and vitreous samples from patients with diabetic retinopathies. The following groups and number of individuals were included: proliferative diabetic retinopathy (PDR) (n = 16), diabetic macular edema (DME) (n = 17), and idiopathic epiretinal membrane (IEM) as non-diabetic controls (n = 23). The initial miRNA expression was explored using TaqMan low-density arrays (TLDAs) with subsequent validation through a quantitative polymerase chain reaction (qPCR). Target genes were identified through bioinformatic tools for enrichment analysis. The TLDAs revealed the following miRNAs with differential expression in terms of PDR vs. IEM: miR-320a-3p, miR-92a-3p, and miR-375-3p in the serum, with miR-541-5p and miR-223-5p in the vitreous samples. DME vs IEM: miR-486-5p, miR-145-5p, miR-197-3p, and miR-125b-5p in the serum, and miR-212-3p in vitreous samples. PDR vs. DME: miR-486-5p, miR-100-5p, miR-328-3p, miR-660-5p, and miR-145 in the serum and none in the vitreous samples. Validation was confirmed only for miR-145, miR-92a, and miR-375 in the serum. The relevant enriched pathways for these three validated miRNAs, miR-145, miR-92a, and miR-375 were the vascular endothelial growth factor and its receptor, hepatocyte growth factor receptor, epidermal growth factor, focal adhesion, and phosphoinositide 3-kinase. Our results support the involvement of miRNAs in the pathophysiology of diabetic retinopathies and reinforce their potential as biomarkers or therapeutic resources.

TRNA-derived fragment tRF-1020 ameliorates diabetes-induced retinal microvascular complications.

Transfer RNA (tRNA)‐derived fragments are the non‐coding single‐stranded RNAs involved in several physiological and pathological processes. Herein, we investigated the role of tRF‐1020, a tRNA fragment, in diabetes‐induced retinal microvascular complications. The results showed that the levels of tRF‐1020 expression were down‐regulated in diabetic retinal vessels and retinal endothelial cells following high glucose or H2O2 stress. Overexpressing tRF‐1020 led to decreased endothelial cell viability, proliferation, migration, and tube formation and alleviated retinal vascular dysfunction as shown by decreased retinal acellular capillaries, vascular leakage, and inflammation. By contrast, tRF‐1020 silencing displayed the opposite effects. tRF‐1020 regulated endothelial angiogenic functions and retinal vascular dysfunction by targeting Wnt signalling. Moreover, the levels of tRF‐1020 expression were reduced in aqueous humour and vitreous samples of the patients with diabetic retinopathy. Collectively, tRF‐1020 is a potential target for the diagnosis and treatment of diabetic retinopathy.

Trends in antibiotic resistance in ocular samples in a tertiary eye centre in Saudi Arabia in 2003–2019

ObjectifLa présente étude visait à analyser l'antibiorésistance dans des échantillons oculaires recueillis pendant 16 ans. MéthodesIl s'agissait d'une étude de cohorte rétrospective réalisée à l'hôpital King Khaled, spécialisé en ophtalmologie, situé à Riyad, en Arabie Saoudite, entre 2003 et 2019. On y a examiné des écouvillonnages cornéens et conjonctivaux de même que des échantillons aqueux et vitréens. RésultatsOn a observé une tendance significative vers une augmentation de la résistance des staphylocoques à coagulase négative au fil du temps envers l’érythromycine (p < 0,001), l'oxacilline (p < 0,001), l'acide fusidique (p < 0,001) et la moxifloxacine (p = 0,003). Staphylococcus aureus a également présenté une hausse significative de ses profils de résistance envers l'oxacilline (p = 0,001), l'ofloxacine (p = 0,003) et la moxifloxacine (p = 0,001). Streptococcus pneumoniæ, pour sa part, a manifesté une augmentation significative de ses profils de résistance envers l’érythromycine (p = 0,01) et l'ofloxacine, soit de 0,80 % en 2003 à 80 % en 2019 (p = 0,015). On n'a enregistré aucune hausse statistiquement significative de l'antibiorésistance de Pseudomonas. ConclusionsOn a observé une augmentation des profils d'antibiorésistance, notamment dans le cas des microorganismes gram-positifs. Compte tenu de ces résultats, il serait judicieux de réviser les stratégies habituelles de prescription des antibiotiques à usage oculaire et d'envisager le recours à des antibiotiques différents.

Differential Expression and Localization of ADAMTS Proteinases in Proliferative Diabetic Retinopathy.

We analyzed the expression of ADAMTS proteinases ADAMTS-1, -2, -4, -5 and -13; their activating enzyme MMP-15; and the degradation products of proteoglycan substrates versican and biglycan in an ocular microenvironment of proliferative diabetic retinopathy (PDR) patients. Vitreous samples from PDR and nondiabetic patients, epiretinal fibrovascular membranes from PDR patients, rat retinas, retinal Müller glial cells and human retinal microvascular endothelial cells (HRMECs) were studied. The levels of ADAMTS proteinases and MMP-15 were increased in the vitreous from PDR patients. Both full-length and cleaved activation/degradation fragments of ADAMTS proteinases were identified. The amounts of versican and biglycan cleavage products were increased in vitreous from PDR patients. ADAMTS proteinases and MMP-15 were localized in endothelial cells, monocytes/macrophages and myofibroblasts in PDR membranes, and ADAMTS-4 was expressed in the highest number of stromal cells. The angiogenic activity of PDR membranes correlated significantly with levels of ADAMTS-1 and -4 cellular expression. ADAMTS proteinases and MMP-15 were expressed in rat retinas. ADAMTS-1 and -5 and MMP-15 levels were increased in diabetic rat retinas. HRMECs and Müller cells constitutively expressed ADAMTS proteinases but not MMP-15. The inhibition of NF-κB significantly attenuated the TNF-α-and-VEGF-induced upregulation of ADAMTS-1 and -4 in a culture medium of HRMECs and Müller cells. In conclusion, ADAMTS proteinases, MMP-15 and versican and biglycan cleavage products were increased in the ocular microenvironment of patients with PDR.

Comparison of Two Leptospira Type Strains of Serovar Grippotyphosa in Microscopic Agglutination Test (MAT) Diagnostics for the Detection of Infections with Leptospires in Horses, Dogs and Pigs.

Simple SummaryLeptospires are bacteria of major health concern as they can cause severe systemic diseases in humans and animals alike. In routine diagnostics, the detection of a leptospirosis heavily relies on the analysis of specific antibodies that were generated by the immune system of an infected species. The most widely used test for the detection of such antibodies is the microscopic agglutination test which itself depends on defined types of laboratory grown Leptospires, so-called serovars. A good judgment of the behaviour of these serovars is vital for interpreting test results and clearly defining what type of Leptospires the patient is infected with as this may have implications for therapy and prognosis. In the following, a study on the reaction pattern of a certain type of serovars named ‘Grippotyphosa’ that itself can be divided into two differently reacting subtypes was conducted. It turned out that, depending on the animal species sampled, a differing reaction patterns to the two Grippotyphosa subtypes in the diagnostic test reflected different distribution of these subtypes in the respective populations. In the future, these insights will further improve test performance and assessment or results.The MAT test is of great importance in the diagnosis of leptospiral infections. Based on various differences, the serovar Grippotyphosa has been divided into two types, Moskva V and Duyster. Differences or similarities of the two type strains in the context of leptospiral diagnostics have not yet been elucidated in more detail; therefore both strains were analysed in MAT diagnostics for the detection of leptospiral infections in pigs, dogs and horses. Serum samples from 2996 pigs, 55 dogs and 35 horses, as well as vitreous and/or aqueous fluid samples from these and 13 additional horses were analysed by MAT; available supplementary samples were tested for leptospires by PCR. In pigs, 92.6% of the samples with both strains received an identical titre result in the MAT test, whereas in dogs and horses only 53.0% and 43.6% had concordant results. Since infections with the serovar Grippotyphosa occur more frequently in dogs and horses overall, more differences were observed here. In the case of discrepant serological results, supplementary samples and PCR examinations were not able to add information on the true status. Further analyses of follow-up studies or at least serum pairs from dogs and horses infected with the serovar Grippotyphosa are necessary.