A Simple Pre-Operative Nuclear Classification Score (SPONCS) for Grading Cataract Hardness in Clinical Studies

Background: The aim of this study was to evaluate whether a simplified pre-operative nuclear classification score (SPONCS) was valid, both for clinical trials and real-world settings. Methods: Cataract classification was based on posterior nuclear color: 0 (clear), 1 (subcapsular/posterior cataract with clear nucleus), 2 (mild “green nucleus” with plus sign for yellow reflection of the posterior cortex), 3 (medium “yellow nucleus” with plus sign for brown/red posterior cortex reflection), 4 (advanced with 4 being “red/brown nucleus” and 4+ white nucleus), and 5 (hypermature/Morgagnian nucleus). Inter- and intra-observer validity was assessed by 30 Ophthalmologists for 15 cataract cases. The reliability of the cataract grading score in a surgical setting was evaluated. Correlation of nuclear scores was compared with phacoemulsification cumulative dissipated energy (CDE) in 596 patients. Results: Analysis of mean intra-observer Cohen kappa agreement was 0.55 with an inter-observer score of 0.54 for the first assessment and 0.49 for the repeat assessment one week later. When evaluating results by nuclear color alone, there was a substantial agreement for both the intra-observer (0.70) and inter-observer parameters: 0.70 for the first test, and 0.66 on repetition with randomization of the cases after a week. CDE levels were found to be significantly different between all SPONCS score groups (p < 0.001), with a lower CDE related to a lower SPONCS score. A strong correlation was found between the SPONCS score and CDE (Spearman′s rho = 0.8, p < 0.001). Conclusion: This method of grading cataract hardness is both simple and repeatable. This system can be easily incorporated in randomized controlled trials to lower bias and confounding effects regarding nuclear density along with application in the clinical setting

Inflammatory mediators in breast cancer: Coordinated expression of TNFα & IL-1β with CCL2 & CCL5 and effects on epithelial-to-mesenchymal transition

<p>Abstract</p> <p>Background</p> <p>The inflammatory chemokines CCL2 (MCP-1) & CCL5 (RANTES) and the inflammatory cytokines TNFα & IL-1β were shown to contribute to breast cancer development and metastasis. In this study, we wished to determine whether there are associations between these factors along stages of breast cancer progression, and to identify the possible implications of these factors to disease course.</p> <p>Methods</p> <p>The expression of CCL2, CCL5, TNFα and IL-1β was determined by immunohistochemistry in patients diagnosed with: (1) Benign breast disorders (=healthy individuals); (2) Ductal Carcinoma <it>In Situ </it>(DCIS); (3) Invasive Ducal Carcinoma without relapse (IDC-no-relapse); (4) IDC-with-relapse. Based on the results obtained, breast tumor cells were stimulated by the inflammatory cytokines, and epithelial-to-mesenchymal transition (EMT) was determined by flow cytometry, confocal analyses and adhesion, migration and invasion experiments.</p> <p>Results</p> <p>CCL2, CCL5, TNFα and IL-1β were expressed at very low incidence in normal breast epithelial cells, but their incidence was significantly elevated in tumor cells of the three groups of cancer patients. Significant associations were found between CCL2 & CCL5 and TNFα & IL-1β in the tumor cells in DCIS and IDC-no-relapse patients. In the IDC-with-relapse group, the expression of CCL2 & CCL5 was accompanied by further elevated incidence of TNFα & IL-1β expression. These results suggest progression-related roles for TNFα and IL-1β in breast cancer, as indeed indicated by the following: (1) Tumors of the IDC-with-relapse group had significantly higher persistence of TNFα and IL-1β compared to tumors of DCIS or IDC-no-relapse; (2) Continuous stimulation of the tumor cells by TNFα (and to some extent IL-1β) has led to EMT in the tumor cells; (3) Combined analyses with relevant clinical parameters suggested that IL-1β acts jointly with other pro-malignancy factors to promote disease relapse.</p> <p>Conclusions</p> <p>Our findings suggest that the coordinated expression of CCL2 & CCL5 and TNFα & IL-1β may be important for disease course, and that TNFα & IL-1β may promote disease relapse. Further <it>in vitro </it>and <it>in vivo </it>studies are needed for determination of the joint powers of the four factors in breast cancer, as well as analyses of their combined targeting in breast cancer.</p

Improving Compliance with Medical Treatment Using Eye Drop Aids

Achieving optimal treatment outcomes in glaucoma requires patients to adhere to their medication regimens. Possible barriers to patients’ cooperation include the misunderstanding of a treatment’s importance or errors in applying instructions, forgetfulness, financial constraints and others. Due to the fact that glaucoma usually causes no apparent symptoms or pain, on the one hand, and the significant inconvenience that the eye drops used for glaucoma treatment can cause due to local irritation, on the other, patient compliance is a challenge. To address this challenge, we require strategies for improving adherence to glaucoma treatment. The importance of proper eye drop administration techniques cannot be overstated, particularly for vulnerable populations such as the elderly, the sick and the visually handicapped. Studies have shown that failure to comply with glaucoma treatment is a significant factor affecting disease progression, emphasizing the need for interventions that improve patient compliance. Educational interventions, medication reminders and the use of assistive devices such as eye drop aids have been shown to improve adherence to glaucoma treatment. By promoting strategies that can be used to enhance treatment adherence, healthcare providers can ensure that glaucoma patients receive the full benefits of their treatment plans, reducing the risk of disease progression. Many patients struggle with the complexity of their treatment regimens and the challenges of administering eye drops. This entry provides a comprehensive overview of the different barriers to patient adherence to glaucoma eye drop treatment, emphasizing the difficulties associated with eye drop instillation. This entry examines a range of eye drop aids available to patients, evaluating their modes of action, benefits, drawbacks and effectiveness in improving patient compliance. By providing detailed information on the barriers to adherence and the range of eye drop aids available, this entry aims to support healthcare providers in helping glaucoma patients to achieve better treatment adherence and outcomes

TGF-β1 induced transdifferentiation of rpe cells is mediated by TAK1.

Proliferative vitreoretinopathy (PVR) is an active process that develops as a complication upon retinal detachment (RD), accompanied by formation of fibrotic tissue. The main cells involved in the development of fibrotic tissue during PVR are the retinal pigment epithelial (RPE) cells. The RPE cells undergo epithelial-mesenchymal transition (EMT) which leads to complex retinal detachment and loss of vision. Transforming growth factor-β1 (TGF-β1) is considered as the main player in the EMT of RPE cells, even though the mechanism is not fully understood. This study was performed to determine the possible involvement of transforming growth factor β activated kinase 1 (TAK1) in the EMT process of the RPE cells.ARPE-19 Cells were treated with 5Z-7 oxozeaenol (TAK1 inhibitor) or SB431542 (TGF-β1 receptor kinase inhibitor) followed by TGF-β1 stimulation. Immunofluorescence, scratch assay Real time PCR and collagen contraction assay assessed the EMT features. The phosphorylation of Smad2/3 and p38 was examined using western blots analysis.This study demonstrates that stimulation of RPE cells with TGF-β1 increases α-SMA expression, cell migration and cell contractility, all of which are EMT features. Remarkably, addition of TAK1 inhibitor abolishes all these processes. Furthermore, we show hereby that TAK1 regulates not only the activation of the non-canonical cascade of TGF-β1 (p38), but also the canonical cascade, the Smad2/3 activation. Thus, the outcome of the TGF-β response in RPE cells is TAK1 dependent.This work demonstrated TAK1, a component of the non-canonical pathway of TGF-β1, is a key player in the EMT process, thus provides deep insight into the pathogenesis of PVR. The ability to halt the process of EMT in RPE cells may reduce the severity of the fibrotic response that occurs upon PVR, leading to a better prognosis and increase the probability of success in RD treatment

Inhibition of TAK1 abolishes the activation of TGF-β cascades.

<p><b>A</b>: Serum-starved RPE cells were pretreated with or without with 5Z-7-oxozeaenol (1μM) for 1 hour and then with TGF-β (2.5ng/ml) for the indicated times. Total protein extracts were analyzed by western blot using the indicated antibodies. The blot shows a representative result of four independent experiments. <b>B:</b> Levels of phospho-Smad2/3 were quantified and normalized to total Smad2/3 <b>C:</b> Statistical analysis of p-p38 and p-Smad3 activation normalized to p38 and Smad3 respectively, with or without TGF-β stimulation. The histograms present results of 5 independent experiments. Statistics were computed using student t-test (Two tailed distribution equal variance). Data is expressed as the Mean±SD.</p

TAK1 is activated upon TGF-β1 stimulation in RPE cells.

<p><b>A:</b> RPE cells were treated with TGF-β1 (2.5ng/ml) for the indicated times or left untreated. The cells were then immunostained with phospho-Thr 187 TAK1 antibodies (green) and DAPI (blue) as described in Materials and Methods. Representative photographs of three independent experiments. Scale bar is 10μm for all images. <b>B</b>: The histogram demonstrating pixel intensity, measured using Image-J software, is based on three independent experiments. (Number of cells: Control 0 = 24, Control 4 hours = 28, control 24 hours = 21, control 48 hours = 25; TGF-β1 4 hours = 26, TGF-β1 24 hours = 21, TGF-β1 48 hours = 22). Statistics were computed using student t-test (Two tailed distribution equal variance). Data is expressed as the Mean±SD.</p

TAK1 is a general regulator of the EMT process in RPE cells.

<p><b>A:</b> RPE cells were pre-treated with or without 5Z-7-oxozeaenol (1μM) and seeded in collagen lattices in full medium. The experiments were performed in triplicates. Lattices were photo-documented after 24 hours and measured using Image-J software. <b>B:</b> The histogram demonstrates the percentage of lattice area (marked by white line) relative to initial gel area, based on three independent experiments. Statistics were computed using student t-test (Two tailed distribution equal variance). Data is expressed as the Mean±SD.</p

TGF-β regulates morphological and transcription changes in RPE cells through TAK1.

<p><b>A:</b> RPE cells were plated on fibronectin-coated glass coverslips, serum-starved for 16 hours, pretreated with 5Z-7-oxozeaenol (1μM) or SB431542 (10μM), or DMSO for 1 hour, then treated with or without TGF-β1 for 2 days. Following treatment the cells were stained with rhodamine-phalloidin (actin fibers-red) and DAPI (blue) and visualized by confocal microscopy. Scale bars: 20μM. The histogram represents quantification of cells size <b>B:</b> Serum-starved RPE cells pretreated with 5Z-7-oxozeaenol (1μM) DMSO for 1 hour, were exposed to TGF-β as in A. Total RNA was extracted at each time point and qPCR was performed. Transcription levels of CTGF were determined after 6h, 16h and 24h. Bars represent the specific mRNA amount relative to GAPDH mRNA in the same samples. All experiments were performed in triplicates. Representative histogram from two independent experiments. Statistics were computed using student t-test (Two tailed distribution equal variance). Data is expressed as the Mean±SD.</p

TAK1 regulates RPE cells EMT phenotype upon TGF-β stimulation.

<p><b>A:</b> RPE cells were serum-starved for 16 hours, pretreated with 5Z-7-oxozeaenol (1μM) or left untreated for 1 hour. The cells were then treated with TGF-β1 (2.5ng/ml) as described in Materials and Methods for 24h and immunostained with α-SMA antibodies (red) and DAPI (blue). Representative photographs of three independent experiments. The histogram represent quantification of pixel intensity <b>B:</b> RPE cells were serum-starved for 16 hours, then treated with mitomycin C (10ng/ml) for 3h. Thereafter, pretreated with 5Z-7-oxozeaenol (1μM) or Dimethyl sulfoxide (DMSO) for 1 hour. Following this process a scratch was performed in the cell monolayer and the serum free medium was supplemented with TGF-β1 (2.5ng/ml) or left unsupplemented. Scratches were photo-documented at the indicated times (top panel) and their width was measured using Image-J software. The histogram (bottom panel) demonstrates the percentage of remaining gap at each time point, relative to initial gap width, based on three independent experiments. Bars are Mean±SD. <b>C:</b> RPE cells were serum-starved for 16 hours then pretreated with 5Z-7-oxozeaenol (1μM) or SB431542 (10μM), or DMSO for 1 hour. Finally, the medium was replaced with serum free medium with or without TGF-β1 (2.5ng/ml) and supernatants from the different treatments were collected after 24 hours. Total MMP-9 activities were processed by gelatin zymography (top panel) and the band intensity values were calculated by Quantity One 1-D analysis software. Histogram demonstrating secretion levels of MMP-9 in the different treatments (bottom panel). Statistics were computed using student t-test (Two tailed distribution equal variance). Data is expressed as the Mean±SD.</p