Search

Preliminary investigations of human corneal tissues from coronavirus disease 2019 (COVID-19) cadaveric donors indicated that no severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA is present. Current eye banking guidelines do not recommend any type of routine testing for SARS-CoV‑2 RNA in post-mortem donor tissue. This is partly based on factors that can influence the test results of the reverse transcription polymerase chain reaction (RT-PCR).

Prostate cancer patients whose tumors develop resistance to conventional treatment often turn to natural, plant-derived products, one of which is sulforaphane (SFN). This study was designed to determine whether anti-tumor properties of SFN, identified in other tumor entities, are also evident in cultivated DU145 and PC3 prostate cancer cells. The cells were incubated with SFN (1-20 µM) and tumor cell growth and proliferative activity were evaluated. Having found a considerable anti-growth, anti-proliferative, and anti-clonogenic influence of SFN on both prostate cancer cell lines, further investigation into possible mechanisms of action were performed by evaluating the cell cycle phases and cell-cycle-regulating proteins. SFN induced a cell cycle arrest at the S- and G2/M-phase in both DU145 and PC3 cells. Elevation of histone H3 and H4 acetylation was also evident in both cell lines following SFN exposure. However, alterations occurring in the Cdk-cyclin axis, modification of the p19 and p27 proteins and changes in CD44v4, v5, and v7 expression because of SFN exposure differed in the two cell lines. SFN, therefore, does exert anti-tumor properties on these two prostate cancer cell lines by histone acetylation and altering the intracellular signaling cascade, but not through the same molecular mechanisms.

The estrogen receptor alpha (ERα) is expressed by the majority of breast cancers and plays an important role in breast cancer development and tumor outgrowth. Although ERα is well known to be a specific and efficient therapeutic target, the molecular mechanisms that are responsible for the control of ERα expression and function in the context of breast cancer initiation and progression are complex and not completely elucidated. In previous work, we have demonstrated that the tumor suppressor RASSF1A inhibits ERα expression and function in ERα-positive breast cancer cells through an AKT-dependent mechanism. Transcriptional activators such as forkhead box protein M1 (FOXM1) and forkhead transcription factor 3A (FOXO3A) and signaling pathways such as the Hippo pathway are also known to modulate ERα expression and activity. Here we report that RASSF1A acts as an inhibitor of ERα-driven breast cancer cell growth through a complex, hierarchically organized network that initially involves suppression of the Hippo effector Yes-associated protein 1 (YAP1), which is followed by inhibition of AKT1 activity, increased FOXO3A activity as well as a blockade of FOXM1 and ERα expression. Together our findings provide important new mechanistic insights into how the loss of RASSF1A contributes to ERα+ breast cancer initiation and progression.

Functional studies giving insight into the biology of circulating tumor cells (CTCs) remain scarce due to the low frequency of CTCs and lack of appropriate models. Here, we describe the characterization of a novel CTC-derived breast cancer cell line, designated CTC-ITB-01, established from a patient with metastatic estrogen receptor-positive (ER + ) breast cancer, resistant to endocrine therapy. CTC-ITB-01 remained ER + in culture, and copy number alteration (CNA) profiling showed high concordance between CTC-ITB-01 and CTCs originally present in the patient with cancer at the time point of blood draw. RNA-sequencing data indicate that CTC-ITB-01 has a predominantly epithelial expression signature. Primary tumor and metastasis formation in an intraductal PDX mouse model mirrored the clinical progression of ER + breast cancer. Downstream ER signaling was constitutively active in CTC-ITB-01 independent of ligand availability, and the CDK4/6 inhibitor Palbociclib strongly inhibited CTC-ITB-01 growth. Thus, we established a functional model that opens a new avenue to study CTC biology., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Metastasis is a multistep process, during which circulating tumor cells traffic through diverse anatomical locations. Stable inducible marking of tumor cells in a manner that is tightly spatially and temporally controlled would allow tracking the contribution of cells passing through specific locations to metastatic dissemination. For example, tumor cells enter the lymphatic system and can form metastases in regional lymph nodes, but the relative contribution of tumor cells that traffic through the lymphatic system to the formation of distant metastases remains controversial. Here, we developed a novel genetic switch based on mild transient warming (TW) that allows cells to be marked in a defined spatiotemporal manner in vivo. Prior to warming, cells express only EGFP. Upon TW, the EGFP gene is excised and expression of mCherry is permanently turned on. We employed this system in an experimental pancreatic cancer model and used localized TW to induce the genetic switch in tumor cells trafficking through tumor-draining lymph nodes. Thereby we found that tumor cells disseminating via the lymphatics make a major contribution to the seeding of lung metastases. The inducible genetic marking system we have developed is a powerful tool for the tracking of metastasizing cells in vivo., (© 2019 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Preliminary investigations of human corneal tissues from coronavirus disease 2019 (COVID-19) cadaveric donors indicated that no severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA is present. Current eye banking guidelines do not recommend any type of routine testing for SARS-CoV‑2 RNA in post-mortem donor tissue. This is partly based on factors that can influence the test results of the reverse transcription polymerase chain reaction (RT-PCR).

Purpose: To assess the developments in contrast sensitivity, color vision, and subjective perception after Descemet membrane endothelial keratoplasty (DMEK) in patients with Fuchs endothelial corneal dystrophy (FECD)., Methods: Included in this study were pseudophakic, unilateral DMEK patients with bilateral FECD having a follow-up period of 6 months (n = 23). The mean age at surgery was 70 years (range: 52-81 years). Pseudophakic eyes without history of other ocular pathology or surgery served as a control (n = 10). Pelli-Robson contrast sensitivity and Panel-D15 color vision tests were used. Best-corrected visual acuity, modified visual functioning questionnaire-25, central corneal thickness, and endothelial cell density were assessed. We visualized the subjective impression of patients with bilateral FECD after unilateral DMEK in a subgroup using Photoshop CS6., Results: Contrast sensitivity improved significantly from 1.35 ± 0.26 to 1.64 ± 0.17 (P = 0.002; control eyes: 1.92 ± 0.09). No difference in the color vision error score was observed for preoperative and postoperative eyes (P = 0.063). The best-corrected visual acuity improved significantly after surgery (P = 0.001). The average values in the Logarithm of the Minimum Angle of Resolution were 0.59 ± 0.42 preoperatively and 0.1 ± 0.10 postoperatively (control eyes: 0.01 ± 0.03). Examinations revealed a decrease of the central corneal thickness and endothelial cell density after surgery (P = 0.001; P = 0.001, respectively). Scores in the general and the driving questionnaire were significantly higher after surgery (P = 0.001; P = 0.005, respectively)., Conclusions: This study showed significant improvement in subjective patient satisfaction and contrast sensitivity. Spontaneous subjective color vision improvement might be explained by significantly improved contrast sensitivity. Contrast sensitivity might be considered as a parameter in preoperative decision-making and evaluation of surgical outcome.

The appearance of the novel severe acute respiratory syndrome-coronavirus-2 (SARS-CoV‑2) poses challenges in ophthalmology particularly for eye banks. A valid risk assessment for the removal and processing of donor corneas is difficult due to the lack of data. The risk to infect transplant recipients with SARS-CoV‑2 still appears very unlikely due to the experience with severe acute respiratory syndrome -coronavirus(‑1) (SARS-CoV(‑1)) and Middle East respiratory syndrome-coronavirus (MERS-CoV); however, due to the occurrence of angiotensin-converting enzyme 2 (ACE2) receptors in the cornea an infection of this tissue with SARS-CoV‑2 cannot be completely excluded. Therefore, routine testing of the organ culture medium used for donor corneas for SARS-CoV‑2 prior to transplantation during the coronavirus disease 2019 (COVID‑19) pandemic should be considered.

Is the new coronavirus SARS-CoV‑2 able to infect ocular tissue and thus poses a risk of infection through the tissue in addition to the risk of contact? This is the question that has occupied ophthalmologists since the beginning of the outbreak. In order to infect a certain type of tissue specific receptors for each virus and sometimes also coreceptors or other proteins must be present. The aim of this review was to summarize and reflect the current state of research with the help of the currently available literature as of 28 May 2020. At the time of the research, angiotensin-converting enzyme 2 (ACE2) was clearly identified as the receptor and transmembrane serine protease 2 (TMPRSS2) as the necessary protease to enable the infection of human cells with SARS-CoV‑2. In the eye both ACE2 and TMPRSS2 are expressed, although sometimes very weakly and with varying degrees in different tissues. It is noteworthy that very different results were obtained with different methods. Several reasons can account for this effect: Firstly, the method of detection or preservation of the tissue, secondly, the possibly different expression of the tested tissue samples and thirdly, a possibly rapid loss of receptor expression post-mortem. Therefore, an infection of the eye seems possible, which has already been reported in various publications. The amount of virus or receptor expression necessary to cause an infection is not known. According to current state of knowledge the eye is not considered to be a high-risk tissue due to the low ACE2 and TMPRSS2 expression. Nevertheless, appropriate protective measures are necessary for both medical personnel and patients. In cases of corneal transplantation an infection of the donor tissue with SARS-CoV‑2 must be excluded.

Purpose: To evaluate the status of ocular donor tissues of a COVID-19 postmortem donor., Methods: SARS-CoV-2 was detected via a pharyngeal swab and broncho-alveolar lavage in the COVID-19 suspect. Postmortem tissue procurement and preparation were performed with personal protective equipment (PPE) and the necessary protective measures. qRT-PCR-testing was performed for the following ocular tissues and fluids: conjunctival fluid swabs, bulbar conjunctiva, corneal epithelium, corneal stroma, corneal endothelium, anterior chamber fluid, lens, iris, vitreous, retina, uvea, sclera, and optic nerve. Informed consent and Institutional Review Board approval was obtained prior to this study (196/2020BO2; Date of approval: 03/26/2020; Ethics Committee of the University of Tuebingen)., Results: In all ocular tissue and fluid samples no SARS-CoV-2 RNA was detected via qRT-PCR of the confirmed COVID-19 postmortem donor., Conclusions and Importance: Late-stage COVID-19 patients might not harbor an ocular reservoir of SARS-CoV-2. The risk of transmitting SARS-CoV-2 via ocular tissues and fluids might be low. This may bear future implications for patient management in ophthalmological practice, surgery and transplantation., Competing Interests: Tarek Bayyoud, MD: Conflicts of interest: none. Angelika Iftner: Conflicts of interest: none. Thomas Iftner, PhD: Conflicts of interest: none. Karl Ulrich Bartz-Schmidt, MD: Conflicts of interest: none. Marius Ueffing, PhD: Conflicts of interest: none. Michael Schindler, PhD: Conflicts of interest: none. Sebastian Thaler, MD: Conflicts of interest: none., (© 2020 The Authors.)

Kynurenines, products of tryptophan (TRP) metabolism, display neurotoxic (e.g., 3-hydroxykynurenine; 3-HK), or neuroprotective (e.g., kynurenic acid; KYNA) properties. Imbalance between the enzymes constituting the kynurenine pathway (KP) plays a role in several disease, including neurodegeneration. In this study, we track changes in concentrations of tryptophan and its selected metabolites after damage to retinal ganglion cells and link this data with expression of KP enzymes. Brown-Norway rats were subjected to intravitreal N -methyl-D-aspartate (NMDA) injection or partial optic nerve crush (PONC). Retinas were collected 2 and 7 days after the completion of PONC or NMDA injection. Concentrations of TRP, kynurenine (KYN), and KYNA were determined by high performance liquid chromatography (HPLC). Data on gene expression in the rat retina were extracted from GEO, public microarray experiments database. Two days after NMDA injection concentration of TRP decreased, while KYN and KYNA increased. At day 7 compared to day 2 decrease of KYN, KYNA and further reduction of TRP concentration were observed, but on day 7 KYN concentration was still elevated when compared to controls. At day 2 and 7 after NMDA injection no statistically significant alterations of 3-HK were observed. TRP and 3-HK concentration was higher in PONC group than in controls. However, both KYN and KYNA were lower. At day seven concentration of TRP, 3-HK, and KYN was higher, whereas concentration of KYNA declined. In vivo experiments showed that retinal damage or optic nerve lesion affect TRP metabolism via KP. However, the pattern of changes in metabolite concentrations was different depending on the model. In particular, in PONC KYNA and KYN levels were decreased and 3-HK elevated. These observations correspond with data on expression of genes encoding KP enzymes assessed after optic nerve crush or transection. After intraorbital optic nerve crush downregulation of KyatI and KyatIII between 24 h and 3 days after procedure was observed. Kmo expression was transiently upregulated (12 h after the procedures). After intraorbital optic nerve transsection (IONT) Kmo expression was upregulated after 48 h and 7 days, KyatI and KyatIII were downregulated after 12, 48 h, 7 days and upregulated after 15 days. Collected data point to the conclusion that development of therapeutic strategies targeting the KP could be beneficial in diseases involving retinal neurodegeneration., (Copyright © 2019 Fiedorowicz, Choragiewicz, Thaler, Schuettauf, Nowakowska, Wojtunik, Reibaldi, Avitabile, Kocki, Turski, Kaminska, Grieb, Zrenner, Rejdak and Toro.)

Endothelial lipase (LIPG) is a cell surface associated lipase that displays phospholipase A1 activity towards phosphatidylcholine present in high-density lipoproteins (HDL). LIPG was recently reported to be expressed in breast cancer and to support proliferation, tumourigenicity and metastasis. Here we show that severe oxidative stress leading to AMPK activation triggers LIPG upregulation, resulting in intracellular lipid droplet accumulation in breast cancer cells, which supports survival. Neutralizing oxidative stress abrogated LIPG upregulation and the concomitant lipid storage. In human breast cancer, high LIPG expression was observed in a limited subset of tumours and was significantly associated with shorter metastasis-free survival in node-negative, untreated patients. Moreover, expression of PLIN2 and TXNRD1 in these tumours indicated a link to lipid storage and oxidative stress. Altogether, our findings reveal a previously unrecognized role for LIPG in enabling oxidative stress-induced lipid droplet accumulation in tumour cells that protects against oxidative stress, and thus supports tumour progression., (© 2019 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Background: We report the first histopathologically proven occurrence of a retrocorneal membrane after Descemet's membrane endothelial keratoplasty., Case Presentation: A white Caucasian 76-year-old woman received penetrating keratoplasty on her right eye 2 years after Descemet's membrane endothelial keratoplasty surgery with combined cataract extraction and intraocular lens implantation for Fuchs' endothelial corneal dystrophy due to an allograft rejection with ensuing graft failure. Her preoperative vision was counting fingers (20/2000) caused by immunological debris, corneal edema, and secondary membrane formation. Her postoperative vision at 3 months was 20/125. The histopathological evaluation showed a membranous structure overlying the denuded Descemet membrane., Conclusions: We report a case of a histopathologically proven retrocorneal membrane after Descemet's membrane endothelial keratoplasty surgery.

Purpose: To assess the influence of donor, environment and storage factors on the contamination rate of organ-cultured corneas, to consider the microbiological species causing corneal contamination and to investigate the corresponding sensitivities., Methods: Data from 1340 consecutive donor corneas were analysed retrospectively. Logistic regression analysis was used to assess the influence of different factors on the contamination rate of organ-cultured corneas for transplantation., Results: The mean annual contamination rate was 1.8 ± 0.4% (range: 1.3-2.1%); 50% contaminations were of fungal origin with exclusively Candida species, and 50% contaminations were of bacterial origin with Staphylococcus species being predominant. The cause of donor death including infection and multiple organ dysfunction syndrome increased the risk of bacterial or fungal contamination during organ culture (p = 0.007 and p = 0.014, respectively). Differentiating between septic and aseptic donors showed an increased risk of contamination for septic donors (p = 0.0020). Mean monthly temperature including warmer months increased the risk of contamination significantly (p = 0.0031). Sex, donor age, death to enucleation, death to corneoscleral disc excision and storage time did not increase the risk of contamination significantly., Conclusion: The genesis of microbial contamination in organ-cultured donor corneas seems to be multifactorial. The main source of fungal or bacterial contamination could be resident species from the skin flora. The rate of microbial contamination in organ-cultured donor corneas seems to be dependent on the cause of donor death and mean monthly temperature., (© 2017 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

To assess the influence of donor, environment, and logistical factors on the results of virological testing of blood samples from cornea donors.Data from 670 consecutive cornea donors were analyzed retrospectively. Logistic regression analysis was used to assess the influence of different factors on the results of virological testing of blood samples from cornea donors.The mean annual rate of donors with serology-reactive or not evaluable result was 14.8% (99 of 670) (range 11.9%-16.9%). The cause of donor death by cancer increased the risk of serology-reactive or not evaluable result (P = .0300). Prolonged time between death and post mortem blood removal was associated with a higher rate of serology-reactive or not evaluable result (P < .0001). Mean monthly temperature including warmer months, differentiating between septic and aseptic donors, sex, and donor age had no significant impact on the results of virological testing of blood samples from cornea donors.The cause of donor death by cancer and a prolonged time between death and post mortem blood removal seem to be mainly responsible for serology-reactive or not evaluable result of blood samples from cornea donors. The percentage of discarded corneas caused by serology-reactive or not evaluable result may be reduced by shortening the period of time between death and post mortem blood removal., (Copyright © 2017 The Authors. Published by Wolters Kluwer Health, Inc. All rights reserved.)

Inherited retinal diseases (IRDs) are often associated with variable clinical expressivity (VE) and incomplete penetrance (IP). Underlying mechanisms may include environmental, epigenetic, and genetic factors. Cis -acting expression quantitative trait loci ( cis -eQTLs) can be implicated in the regulation of genes by favoring or hampering the expression of one allele over the other. Thus, the presence of such loci elicits allelic expression imbalance (AEI) that can be traced by massive parallel sequencing techniques. In this study, we performed an AEI analysis on RNA-sequencing (RNA-seq) data, from 52 healthy retina donors, that identified 194 imbalanced single nucleotide polymorphisms(SNPs) in 67 IRD genes. Focusing on SNPs displaying AEI at a frequency higher than 10%, we found evidence of AEI in several IRD genes regularly associated with IP and VE ( BEST1 , RP1 , PROM1 , and PRPH2 ). Based on these SNPs commonly undergoing AEI, we performed pyrosequencing in an independent sample set of 17 healthy retina donors in order to confirm our findings. Indeed, we were able to validate CDHR1 , BEST1 , and PROM1 to be subjected to cis -acting regulation. With this work, we aim to shed light on differentially expressed alleles in the human retina transcriptome that, in the context of autosomal dominant IRD cases, could help to explain IP or VE., Competing Interests: The authors declare no conflict of interest.

Amplification and/or overexpression of the human epidermal growth factor 2 (HER2) oncogene occurs in about 13-15% of invasive breast cancer and triggers breast cancer cell proliferation, survival and metastatic progression. Around half of all breast cancers with HER2 overexpression co-express hormone receptors (HR) such as those for estrogen and progesterone. Aberrant signaling through HER2 and other members of the HER-family mediates endocrine-resistance in estrogen receptor alpha (ERα) positive breast cancer. On the other hand, ERα co-expression has been shown to attenuate the efficiency of anti-HER2 therapies. These findings indicate that HER2 and ERα synergize to escape from both anti-ERα and anti-HER2-targeted therapies. Rationally designed clinical trials that combine endocrine therapy with anti-HER2 agents to interfere with HER2/ERα cross-talk have been conducted. However, the outcome of these trials suggests that novel therapeutic approaches are needed to further improve inhibition of HER2 and other HER-family members in conjunction with a more efficient ERα blockade. Here, we demonstrate that carfilzomib and bortezomib stabilize the HER2-specific protein tyrosine phosphatase BDP1 leading to decreased HER2 autophosphorylation, reduced HER2 activity and subsequently attenuated activation of the PI3K/Akt-pathway, together with blockade of ERα expression. We further observed that proteasome inhibitors (PIs) reverse autophosphorylation and thereby inhibit the activity of constitutively active mutant HER2. We also demonstrate that PIs cause cell death in lapatinib and endocrine-resistant HER2+/ER+ breast cancer cells. These findings suggest that PIs might have the potential to improve the management of HER2+/ER+ breast cancer patients by efficiently disrupting the bi-directional HER2/ERα cross-talk., Competing Interests: CONFLICTS OF INTEREST The authors have no conflicts of interest to disclose.

BACKGROUND The purpose of this study was to assess the effect of an organ transplantation scandal on the rate of corneal donations and organ donors at the University Hospital Tübingen. MATERIAL AND METHODS Data from all hospital deaths from January 2012 to December 2013 were analyzed retrospectively. Each deceased patient was considered as a potential corneal donor. The corneal donor procurement was handled by an ophthalmic resident on a full-time basis with standard used unchanged set of procedures. Each patient who died due to cerebral complications was considered as a potential organ donor. During the two-year period, a German transplantation scandal occurred at the end of 2012 and received worldwide attention. The rates of corneal donation and organ donation in 2012 and 2013 were examined and evaluated. RESULTS Among the 1685 deceased patients, approval for corneal donation was received in 220 cases (13.1%): 124 cases (15.0%) in 2012 and 96 cases (11.2%) in 2013. This corresponds to a decline of 23%. The leading causes of nonfulfillment of corneal donations were refusal to donation: 401 cases (48.4%) in 2012 and 445 cases (52.0%) in 2013; and medical contraindications: 201 cases (24.2%) in 2012 and 212 cases (24.8%) in 2013. During the two-year period, consent for organ donation was obtained in 25 cases (1.5%): 15 cases (1.8%) in 2012 and 10 cases (1.2%) in 2013. The number of realized organ donors was 20 cases (1.2%): 12 cases (1.4%) in 2012 and 8 cases (0.9%) in 2013. This corresponds to a decline of 33%. CONCLUSIONS After a transplantation scandal, the number of realized corneal donors and realized organ donors decreased significantly. It seems that increasing professional performance is very important to gaining trust inside and outside the hospital and improving corneal and organ donation rate.

Reestablishing tissue organization of breast cancer cells into acini was previously shown to override their malignant phenotype. In our study, we demonstrate that alpha(v)beta(3) integrin (Int-αvβ3), previously shown to play a role in cancer progression, promoted differentiation and growth arrest of organoids derived from luminal A breast cancer cells grown in their relevant three-dimensional microenvironment. These organoids differentiated into normal-like acini resembling a benign stage of breast tissue. Likewise, we demonstrate that Int-αvβ3 is selectively expressed in the epithelium of the benign stage of breast tissues, and is lost during the early stages of luminal A breast cancer progression. Notably, the organoids' reversion into normal-like acini was mediated by cancer luminal progenitor-like cells expressing both EpCAM high CD49f low CD24 + and Int-αvβ3. Furthermore, downregulation of Notch4 expression and downstream signaling was shown to mediate Int-αvβ3-induced reversion. Intriguingly, when luminal A breast cancer cells expressing Int-αvβ3 were injected into a humanized mouse model, differentiated tumors developed when compared with that generated by control cells. Hence, our data suggest that promoting differentiation of luminal A breast cancer cells by signaling emanating from Int-αvβ3 can potentially promote 'normalization' of their malignant phenotype and may prevent the malignant cells from progressing.

Oncogene-induced senescence is thought to act as a barrier to tumorigenesis by arresting cells at risk of malignant transformation. Nevertheless, numerous findings suggest that senescent cells may conversely promote tumor progression through the development of the senescence-associated secretome they produce. It is likely that the composition and the physiological consequences mediated by the senescence secretome are dependent on the oncogenes that trigger the senescence program. Breast cancer represents a heterogenous disease that can be divided into breast cancer subtypes due to different subsets of genetic and epigenetic abnormalities. As tumor initiation and progression of these breast cancer subtypes is triggered by diverse oncogenic stimuli, differences in the senescence secretomes within breast tumors might be responsible for tumor initiation, progression, metastasis and therapeutic response. Many studies have addressed the role of senescence as a barrier to tumor progression using murine xenograft models. However, few investigations have been performed to elucidate the degree to which senescent tumor cells are present within untreated human tumors, and if present, whether these senescent tumor cells may play a role in disease progression. In the present study we analysed the appearance of senescent cells within invasive breast cancers. Detection of cellular senescence by the use of SAβ-galactosidase (SAβ-gal) staining within invasive breast carcinoms from 129 untreated patients revealed differences in the amount of SAβ-gal+ tumor cells between breast cancer subtypes. The highest percentages of SAβ-gal+ tumor cells were found in HER2-positive and luminal A breast carcinomas whereas triple negative tumors showed either little or no positivity.

Following the passage of the Health Information Technology for Economic and Clinical Health (HITECH) Act of 2009, electronic health records were widely adopted by eligible physicians and hospitals in the United States. Stage 2 meaningful use menu objectives include a digital family history but no stipulation as to how that information should be used. A variety of data mining techniques now exist for these data, which include artificial neural networks (ANNs) for supervised or unsupervised machine learning. In this pilot study, we applied an ANN-based simulation to a previously reported digital family history to mine the database for trends. A graphical user interface was created to display the input of multiple conditions in the parents and output as the likelihood of diabetes, hypertension, and coronary artery disease in male and female offspring. The results of this pilot study show promise in using ANNs to data mine digital family histories for clinical and research purposes.

Around 70% of breast cancers express the estrogen receptor α (ERα) and depend on estrogen for growth, survival and disease progression. The presence of hormone sensitivity is usually associated with a favorable prognosis. Use of adjuvant anti-endocrine therapy has significantly decreased breast cancer mortality in patients with early-stage disease, and anti-endocrine therapy also plays a central role in the treatment of advanced stages. However a subset of hormone receptor-positive breast cancers do not benefit from anti-endocrine therapy, and nearly all hormone receptor-positive metastatic breast cancers ultimately develop resistance to anti-hormonal therapies. Despite new insights into mechanisms of anti-endocrine therapy resistance, e.g., crosstalk between ERα and Her2/neu, the management of advanced hormone-receptor-positive breast cancers that are resistant to anti-endocrine agents remains a significant challenge. In the present study, we demonstrate that the proteasome inhibitor Bortezomib strongly inhibits ERα and HER2/neu expression, increases expression of cyclin-dependent kinase inhibitors, inhibits expression of multiple genes associated with poor prognosis in ERα+ breast cancer patients and induces cell death in ER+ breast cancer cells in both the presence and absence of functional p53. Although Bortezomib increased the levels of p53 and increased the expression of pro-apoptotic target genes in ERα+ breast cancer cells harboring wild-type p53, Bortezomib also exerts anti-tumoral effects on ERα+ breast cancer cells through suppression of ERα expression and inhibition of PI3K/Akt/mammalian target of rapamycin (mTOR) and ERK signaling independently of functional p53. These findings suggest that Bortezomib might have the potential to improve the management of anti-endocrine therapy resistant ERα+ breast cancers independently of their p53 status., (© 2014 UICC.)

Background: Open globe injuries complicated with the presence of an intraocular foreign body constitute a vision threatening condition., Purpose: To present the results of pars plana vitrectomy in patients with intraocular foreign body., Material and Methods: Medical records of 22 patients were analyzed. Retrospective analysis of data included visual acuity, age, gender and type of injury., Results: All patients were men and the mean age was 37 years. All injuries occurred while working with a hammer. All patients were treated with pars plana vitrectomy combined with intraocular foreign body removal and internal limiting membrane peeling. The visual acuities improved in 9 cases (41%), in 13 cases (59%) the deterioration of visual acuity was observed, no eye was enucleated. In 14 eyes pars plana vitrectomy was combined with lens removal, in 14 eyes silicone oil was used as a tamponade., Conclusions: Surgical intervention with pars plana vitrectomy combined with intraocular foreign body removal and cataract extraction may preserve severely traumatized eyes and maintain or even improve vision. ocular trauma, vitrectomy, intraocular foreign body.

Objective: This study was conducted to investigate neuroprotective effects of a high fat/low carbohydrate and protein diet (ketogenic diet, KD) in a model of N-methyl D-aspartate (NMDA)-induced retinal ganglion cell (RGC) damage in juvenile and young adult rats., Methods: Juvenile (30-35 days old) and young adult (56-70 days old) female Brown Norway rats were fed the KD for 21 days; rats exposed to a standard rodent diet (SRD) served as controls. The main constituents of the KD used in the present study were approximately 80% fats, 8% proteins, and less than 1% carbohydrates. On day 14 of exposure to the KD (or the SRD in the control group), each rat received a single intravitreal injection of NMDA; RGCs were then retrogradely labelled by hydroxystilbamidine on day 19 and collected on day 21 to assess the degree of damage induced by NMDA. Blood biomarkers to confirm the expected metabolic response to the KD (i.e. ketosis and hypoglycaemia) were also assessed., Results: Although both the juvenile and young adult rats developed comparable ketosis and hypoglycaemia when fed the KD, NMDA-induced loss in RGCs was significantly attenuated only in juvenile rats exposed to the KD in comparison with those fed the SRD; exposure to the KD had no protective effect in young adult rats. In summary, exposure to the KD had a neuroprotective effect in NMDA-induced RGC damage in juvenile rats, but not in young adult rats., Conclusion: These results support further exploration of metabolic interventions to treat optic neuropathies associated with neurodegeneration., (© 2015 S. Karger AG, Basel.)

Background: Cataract surgery is the most commonly performed surgical procedure in developed countries. The annual number of cataract surgeries in Germany is about 600,000. Acute postoperative endophthalmitis is a very severe and the most dreaded complication of cataract surgery. Various operative and non-operative measures have been suggested to prevent this serious complication. The European Society of Cataract & Refractive Surgeons (ESCRS) study of intracameral cefuroxime was the first prospective, randomised and partially placebo-controlled clinical trial showing the efficacy of antibiotic prophylaxis to prevent endophthalmitis in 2007. The aim of this retrospective study is to investigate a possible reduction of intracameral cefuroxime to prevent postoperative endophthalmitis at the University Eye Hospital Tübingen., Patients and Methods: During the period from January 2002 to August 2013, 2 time periods were determined based on the adoption of intracameral cefuroxime injections after cataract surgery. From January 2002 to May 2009 patients received at the end of cataract surgery a subconjunctival administration of 50 mg of mezlocillin and postoperative antibiotic eye drops (gentamicin) without intracameral injection. From June 2009 to August 2013, patients received an intracameral injection of cefuroxime while antibiotic drops (moxifloxacin) were used too. The rates of postoperative infectious endophthalmitis during these 2 periods were calculated., Results: 31 cases of endophthalmitis occurred in 31,386 cataract surgeries. The overall cumulative incidence was 0.99 per 1000 patients. The incidence in the first period without intracameral cefuroxime injection was 1.38 (95 % confidence interval [CI]: 1.03-1.72) per 1000 patients and in the second period 0.44 (95 % CI: 0.34-0.54) per 1000 patients (p < 0.001)., Conclusion: Intracameral injection of cefuroxime reduces the rate of postoperative infectious endophthalmitis in cataract surgery significantly., (Georg Thieme Verlag KG Stuttgart · New York.)

Purpose: Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxyl) is a membrane-permeable superoxide dismutase and potentially neuroprotective substance. This study evaluates the retinal tolerance of 0.5 mM, 1 mM, 2 mM, and 5 mM tempol measured by the electroretinogram (ERG) of an isolated and perfused retina whole mount., Methods: For functionality testing, bovine retinas were prepared and perfused with an oxygen-saturated standard solution, and the ERG was recorded until stable b-wave amplitudes were reached. Tempol concentrations of 0.5 mM, 1 mM, 2 mM, and 5 mM were tested for 45 minutes. To investigate the effects on photoreceptor function, 1 mM aspartate was added to suppress the b-wave and obtain isolated a-waves. ERG amplitudes were monitored for 100 minutes., Results: While no toxic effects for concentrations of 0.5 mM and 1 mM tempol could be detected, concentrations of 2 mM tempol and higher caused statistically significant negative effects on the b-wave amplitude (-38 %, p = 0.02 for 2 mM; -54 %, p = 0.02 for 5 mM). The a-wave amplitude remained stable even at higher concentrations., Conclusions: Although the photoreceptors seem to have a tolerance to high concentrations of tempol, higher intravitreal concentrations than 1 mM should be considered critical.

Background: The efficacy of tempol and its acyl derivative tempol-C8 as retinoprotective agents was compared in a rat model of NMDA-induced retinal ganglion cell (RGC) damage., Material and Methods: Tempol or tempol-C8 in different doses was administered intraperitoneally to 6 weeks old (pre-adolescent) and 9-10 weeks old (young adult) rats before and after an intravitreous NMDA injection. Retinal ganglion cell were retrogradely labeled with the fluorescent tracer hydroxystilbamidine and RGC counting was performed on retinal flatmounts., Results: Intravitreal NMDA reduced RGC counts by about 90%, independently of age < 0.001). In pre-adolescent animals tempol-C8, but not tempol unmodified, showed a significant, dose-dependent RGC rescue effect, with peak activity at 5.8 µmol/kg (p < 0.001). In young adult animals, however, no neuroprotective effect was found for either tempol or tempol-C8., Conclusions: In contrast to tempol itself, tempol-C8 acyl ester was neuroprotective in pre-adolescent rats in the NMDA- induced RGC damage model. Therefore, neuroprotection by tempol acyl esters seems to be superior to that of tempol under certain conditions.

We have recently demonstrated that the anthocyanidin delphinidin (DEL), one of the most abundant dietary flavonoids, inhibits activation of ErbB and vascular endothelial growth factor receptor family members. These receptors play crucial roles in the context of tumor progression and the outgrowth of blood and lymphatic vessels. Here, we have developed an improved chemical synthesis for DEL in order to study the effects of the aglycon and its degradation product gallic acid (GA) on endothelial and tumor cells in vitro and in vivo. We found that DEL blocked the proliferation in vitro of primary human blood and lymphatic endothelial cells as well as human HT29 colon and rat MT-450 mammary carcinoma cells in a dose-dependent manner. In contrast, its degradation product GA had little effect. At higher concentrations, DEL induced apoptosis of endothelial and tumor cells. Furthermore, DEL potently blocked the outgrowth of lymphatic capillaries in ex vivo lymphangiogenesis assays. In the MT-450 rat syngeneic breast tumor model, it also significantly reduced angiogenesis and tumor-induced lymphangiogenesis when administered in vivo. These data reveal DEL to be a novel antilymphangiogenesis reagent. Surprisingly, however, the application of DEL unexpectedly promoted tumor growth and metastasis in the MT-450 tumor model, suggesting that the antiproliferative effect of DEL on cultured cells does not necessarily reflect the response of tumors to this anthocyanidin in vivo. Furthermore, while DEL may have utility as a cancer chemopreventative agent, its ability to promote tumor growth once a neoplasm develops also needs to be taken into consideration.

Purpose: To investigate the presence and distribution of l-kynurenine aminotransferases immunoreactivity in human and animal lenses during cataract formation., Methods: Immunohistochemistry was conducted using polyclonal antibodies against KAT I, KAT II and KAT III on sections of 26 anterior capsules from patients undergoing surgical treatment of anterior subcapsular cataract (ASC) and 22 cataractous lenses from human eyes enucleated because of choroidal malignant melanoma. Additionally, the eyes of 11-month-old DBA/2J mice (6 eyes) were investigated (with KAT I and II). Ten clear human lenses and four BL6 mice lenses were used as controls. Spatial immunoreactivity patterns of enzymes were compared with Periodic Acid - Schiff (PAS)-stained sections., Results: Immunohistochemical analysis revealed presence of KAT I, KAT II and KAT III in extracellular structures of all studied types of cataract in human eyes showing specific pattern of the stain. In cortical cataract, immunoreactivity was observed on cortical lens fibres. In nuclear cataract, KAT II revealed stronger and diffused staining than KAT I. Additionally, both KAT showed more pronounced staining at the edge of small clefts. In normal human lenses, KAT I, II and III, immunoreactivity was not observed. Presence of KAT I and KAT II in the intercellular substance of DBA/2J mice cataract was observed. In BL6 mice lenses without cataract, only weak KAT I and KAT II staining was observed., Conclusions: Presence of l-kynurenine aminotransferases in extracellular matrix (ECM) during human cataract formation suggests that products of l-kynurenine pathway might be involved in mechanisms of cataractogenesis., (© 2013 Acta Ophthalmologica Scandinavica Foundation. Published by Blackwell Publishing Ltd.)

Purpose: Indocyanine green (ICG) has been widely used as a vital dye for macular surgery. However, ICG can be toxic to retinal cells. Here we evaluate whether tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl), a free radical scavenger, can protect against ICG-induced retinal damage in rats., Methods: Brown Norway rats received intravitreal injections of ICG 0.5 % or BSS as controls. Tempol (20 mg/kg BW) or PBS as a control was administered intraperitoneally 24 h and 30 min before ICG and once daily for 7 consecutive days. Tempol was detected in the retina using electron paramagnetic resonance (EPR) spectroscopy. One week after ICG injections, the effects of tempol on retinal toxicity were assessed by retinal ganglion cell (RGC) back-labeling and by light microscopy. Electroretinography (ERG) was performed after 1 and 2 weeks., Results: ICG administration reduced RGC numbers by 17 % (1,943 ± 45 vs. 2,342 ± 31 RGCs/mm(2)). Tempol treatment rescued RGCs in a significant manner (2,258 ± 36, p < 0.01) and diminished morphological changes detected by light microscopy. ICG-injected eyes showed a significant reduction of ERG potentials only in PBS-treated animals (V(max) 530 ± 145 µV vs. 779 ± 179 µV, p = 0.0052), but not in the tempol-treated group., Conclusions: Tempol significantly attenuates ICG-induced toxicity in rat retinas and may therefore be considered for further evaluation as accompanying treatment in ICG-assisted chromovitrectomy.

Targeting mechanisms that result in increased concentrations of kynurenic acid (KYNA) in the brain has been considered as a therapeutic approach for the treatment of epilepsy and certain neurodegenerative disorders. Recently, KYNA has been implicated in the effects produced by the high-fat and low-protein/carbohydrate ketogenic diet (KD) in a report demonstrating an increased production of KYNA in vitro by one of the ketone bodies, β-hydroxybutyrate, elevated by the KD. To further explore this association, brain concentrations of KYNA were compared in young (3 weeks old) and adult (8-10 weeks old) rats that were chronically exposed to the KD and regular diet. Exposure to the KD resulted in the anticipated elevations of β-hydroxybutyrate with accompanying decreases in glucose concentrations. In comparison to rats fed the regular diet, KYNA concentrations were significantly (p < 0.05) increased in the hippocampus (256 and 363% increase in young and adult rats, respectively) and in the striatum (381 and 191% increase in young and adult rats, respectively) in KD-fed rats. KD-induced increases in KYNA concentrations in young versus adult rats in the hippocampus and striatum were comparable (p > 0.05). Exposure to the KD had no effect on KYNA concentrations in the cortex of young and adult rats (p > 0.05). In summary, chronic exposure to the KD resulted in several-fold increases in KYNA concentrations in discrete brain structures in the rats. Thus, the relevant clinical question for further exploration is whether KD-induced increases in KYNA concentrations can translate into clinically significant improvements in neuropsychiatric diseases associated with KYNA hypofunction.

Background: The aim of this work is to investigate the biocompatibility and staining properties of DSS: 3,3'-Di-(4-sulfobutyl)-1,1,1',1'-tetramethyl-di-1H-benz[e]indocarbocyanine (DSS)., Methods: Dye concentrations of 0.5, 0.25, and 0.1% were evaluated (290 and 295 mOsm). Toxicity was assessed using a colorimetric test measuring the inhibition of ARPE 19 cell, human primary RPE cell, and human Müller cell proliferation. Exposure time was 30, 60, 120, and 300 s. Indocyanine green (ICG) (0.5, 0.25, and 0.1%) served as a control. Cells were also illuminated with plain white light (750 mW/cm(2)) for 10 min to assess phototoxic effects. Besides staining of porcine and human lens capsule, internal limiting membrane (ILM)-staining was assessed by applying 0.25 and 0.5% DSS over the macula in two human post-mortem eyes., Results: DSS of 0.25 and 0.1% showed no toxic effect on primary RPE cells and MIO-M1cells, and 0.5, 0.25, and 0.1% for ARPE-19 cells. In MIO-M1cells, 0.5% dye showed a significant reduction of mitochondrial dehydrogenase activity only following an exposure time of 300 s. Following illumination, ICG showed a significantly more pronounced effect on cell viability in primary RPE cells and MIO-M1cells compared to DSS. The absorption maximum is found at 591 nm; the even more bathochromic fluorescence proceeds with a common Stokes' shift where maxima at 620 and 660 nm with a quantum yield of 32% were found. The fluorescence is sufficiently hypsochromic and the fluorescence quantum yield high enough for an easy visual detection. The contrast and staining properties at the ILM were excellent and allowed for a controlled removal of the ILM during surgery. No penetration into deeper retinal layers was noted., Conclusions: Our results indicate that this new cyanine dye DSS may represent an alternative for ILM staining due to its matched absorption concerning visibility and fluorescence qualities as well as its good biocompatibility.

Purpose: To evaluate the potential of decellularized bovine corneas (DBCs) as a carrier matrix for cultivating and transplanting human corneal endothelial cells (HCECs)., Methods: Posterior lamellae of ten bovine corneas were decellularized using ethylene diamin tetra-acetic acid (EDTA, 0.1%), aprotinin (10 KIU/mL) and 0.3% sodium dodecyl sulphate (SDS). Hematoxylin-eosin (HE) and 4,6-diamidino-2-phenylindole (DAPI) staining was done to confirm the absence of bovine cells. Quantitative analysis was performed to determine levels of desoxyribonucleic acid (DNA) using a DNA Purification Kit. HCECs were harvested from human donor eyes and seeded on the Descemet's membrane of the DBCs. Cell morphology was assessed after 6 h of incubation, and at days 1, 4, 7, 10 and 14. Expression of zonula occludens-1 (ZO-1), connexin-43 (CX-43), Na(+)/K(+)-adenosine triphosphatase (Na(+)/K(+)-ATPase), natrium hydrogen carboanhydrase (Na(+)/HCO(3)(-)), collagen type VIII, collagen type IV and cytokeratin-3 (AE5) were analyzed by immunohistochemistry., Results: HE staining and DAPI staining showed that bovine cells were substantially removed from the stroma and Descemet's membrane. A significant DNA reduction (mean before decelluraziation 365.3 ± 88.6 ng/mg, mean after decelluarization 23.2 ± 7.9 ng/mg, p < 0.001) was observed. HCECs formed a continuous, viable, predominantly polygonal monolayer with a mean cell density of 2380 ± 179 cells/mm(2) on DBCs. Immunohistochemistry analysis demonstrated positive staining for AE5, collagen type VIII, ZO-1, CX-43, Na(+)/HCO(3)(-), and Na(+)/K(+)-ATPase., Conclusions: Phenotypical properties of HCECs on DBCs imply that the HCEC sheets are capable of maintaining an intact barrier and ionic pump function in vitro. DBCs might, therefore, be a promising scaffold for ex vivo expansion of HCECs. This xenogeneic substrate might be used for therapy of isolated corneal endothelial diseases.

Purpose: The aim of this study is to search for more effective derivatives of the superoxide dismutase mimetic tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl). Although tempol is neuroprotective in a rat partial optic nerve crush (PONC) model, relatively high doses are required to exert this effect., Methods: Tempol acyl esters with different-length fatty acids (tempol-C4, tempol-C8, tempol-C12 and tempol-C16) were synthesized and the following properties were evaluated: water-octanol partition coefficient, liposome-liposome energy transfer, and electron paramagnetic resonance (EPR). Brown Norway rats underwent PONC and received tempol or acyl esters intraperitoneally once daily for 7 consecutive days. We then compared the effects of tempol and its four esters on retinal ganglion cell (RGC) damage using a retrograde labelling method., Results: The water-octanol partition coefficient increased with increasing length of attached acyl chain. However, the energy of the liposome-liposome transfer seemed to be optimal for tempol-C8 and tempol-C12. The EPR signal was very similar for all tested compounds, suggesting similar efficiency of superoxide scavenging. Partial optic nerve crush in vehicle-treated animals reduced RGC numbers by approx. 59% when compared with sham-operated eyes. Tempol did not affect RGC loss at a dose of 1 mg/kg. In contrast, at molar doses equivalent to 1 mg/kg of tempol, tempol-C8 showed a significant neuroprotective effect, whereas tempol-C4, tempol-C12 and tempol-C16 did not act neuroprotectively., Conclusion: Manipulating the hydrophobicity of tempol seems to be a promising tool for developing more potent neuroprotectants in the PONC degeneration model. However, the resulting compounds need further pharmacological evaluation., (© 2011 The Authors. Acta Ophthalmologica © 2011 Acta Ophthalmologica Scandinavica Foundation.)

Background: Behçet's disease is a systemic vasculitis disorder of unknown aetiology. Ocular involvement, especially with vasculitis, is detected in up to 80 % of the cases. Anterior segment involvement such as cataract is also seen in the follow-up of patients who are then treated surgically. In this study, we aimed to analyze the outcomes of cataract surgery in patients with Behçet's disease retrospectively., Patients and Methods: The records of 9 patients (12 eyes) with Behçet's disease who underwent phacoemulsification with IOL implantation in 11 eyes and extracapsular cataract extraction (ECCE) with IOL implantation in one eye between June 2001 and September 2009 were evaluated retrospectively. The visual outcome and complications were analysed., Results: The mean follow-up was 33.8 months (range 3 to 88 months). The mean preoperative LogMAR BCVA was 1.15 ± 0.53 (95 % CI: 0.81 - 1.49) and increased to 0.36 ± 0.32 (95 % CI: 0.15 - 0.56) at last medical visit (p < 0.001). The most frequent postoperative complication was posterior capsular opacification, which developed in 2 eyes (17 %). Other complications were mild fibrinous reaction in 1 eye (8 %)., Conclusions: The outcomes of cataract surgery in patients with Behçet's disease were satisfactory. The great majority of the patients regained and retained a good visual outcome and had fewer postoperative complications., (© Georg Thieme Verlag KG Stuttgart · New York.)

Background: Apoptosis is a major mechanism of cell death in glutamate-induced excitotoxicity and caspases as the executors of apoptosis play an important role in the development of various central nervous system and eye diseases. We studied the involvement of certain caspases in excitotoxic retinal ganglion cell death, which was experimentally induced in Brown Norway Rats by application of the glutamate receptor agonist N-methyl-D-aspartate (NMDA)., Methods: Animals were injected intravitreally with one of six caspase inhibitors (against caspases 1, 3, 4, 6, 8 and 9). Seven hours later, NMDA or phosphate-buffered saline as a control was injected intravitreally into the respective eyes. The neuroprotective potential against NMDA toxicity was assessed by retinal ganglion cell quantification. Additionally, wholemount TUNEL was performed., Results: Statistical analysis revealed significant neuroprotective effects for the inhibitors of caspases 3, 6, 8 and 9, but not for those of caspases 1 and 4. The inhibitors of caspases 6 and 9 showed greater neuroprotective potential than those of caspases 3 and 8, although cell death was not entirely averted in any case. Results of ganglion cell counts were confirmed for the most pronounced treatment groups using wholemount TUNEL., Conclusion: Excitotoxic retinal ganglion cell death after NMDA injection is mediated mainly through apoptosis, whereby extrinsic as well as intrinsic pathways of caspase activation play a role., (© 2011 The Authors. Clinical and Experimental Ophthalmology © 2011 Royal Australian and New Zealand College of Ophthalmologists.)

Purpose: To evaluate capsule-staining properties and biocompatibility of the triarylmethane dyes methyl blue and aniline blue compared with patent blue and trypan blue on cultured human corneal endothelial cells., Setting: Centre for Ophthalmology, University of Tuebingen, Tuebingen, Germany., Design: Experimental study., Methods: Human corneal endothelial cell cultures were harvested from human donor cells and exposed to various concentrations (0.025 to 5.0 mg/mL) of methyl blue, aniline blue, patent blue, and trypan blue. Cytotoxicity was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test after 24 hours of incubation. Calcein live cell staining was performed at the same time point. The dyes were also used to stain pig lens capsules in vitro by incubating the lenses for 1 minute with 3 concentrations (0.5, 1.5, and 2.5 mg/mL) of dye, after which the staining properties were evaluated., Results: No significant cytotoxicity was detected for patent blue and methyl blue at any tested concentration. However, aniline blue exerted significant cytotoxicity at concentrations of 1.5 mg/mL or higher and trypan blue at 2.5 mg/mL or higher. Capsule staining of the tested triarylmethane dyes was suitable for performing capsulorhexis, but only at higher concentrations than with trypan blue., Conclusions: High concentrations and long incubation times of trypan blue and aniline blue showed significant cytotoxicity to human cultured endothelial cells in contrast to patent blue and methyl blue. All tested dyes were able to stain lens capsules sufficiently for capsulorhexis creation., Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned., (Copyright © 2011 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.)

The kynurenine aminotransferases (KATs) KAT I and KAT II are pivotal to the synthesis of kynurenic acid (KYNA), the only known endogenous glutamate receptor antagonist and neuroprotectant. KAT I and II have been found in avian, rodent, and human retina. Expression of KAT I in Müller cell endfeet and KAT II in retinal ganglion cells has been documented. Developmental changes in KAT expression and KYNA concentration in the avian and rodent retina have also been found. Studies of retinal neurodegeneration have shown alterations in KYNA synthesis in the retina in response to retinal ganglion cell loss. In DBA/2J mice, a model of ocular hypertension, an age-dependent decrease of retinal KYNA and KATs was found. In the corpora amylacea in the human retina intensive KAT I and II immunoreactivity was demonstrated. In summary, these findings point to the potential involvement of KYNA in the mechanisms of retinal aging and neurodegeneration.

Background: Corpora amylacea (CAm) are a hallmark of aging and neurodegeneration. The presence of kynurenine aminotransferases I and II (KAT I and II) in CAm in the human retina and optic nerve has been already shown. The present study aimed to examine kynurenine aminotransferase III (KAT III) immunoreactivity in CAm in the human retina and optic nerve., Material and Methods: Polyclonal antibody against KAT III was used on sections of human eyes enucleated due to malignant uveal melanoma. PAS-stained sections of CAm were compared with KAT III stained ones., Results: KAT III immunoreactivity was observed in CAm in the retina, prelaminar, laminar and retrolaminar region of the optic nerve with similar location to PAS-stained sections. The most intense staining was observed in the retrolaminar part of the optic nerve. KAT III immunoreactivity was also present in the cytoplasm of retinal ganglion cells., Conclusions: Expression of KAT III in CAm in the human retina and optic nerve indicates that this enzyme may be relevant in mechanisms of neurodegeneration leading to CAm formation.

Purpose: This study investigated the effects of systemically administered lithium acetoacetate (ACA) and sodium β-hydroxybutyrate (BHB) in a rat model of N-methyl-D-aspartate (NMDA)-induced damage of retinal ganglion cells (RGC). Additionally, the influence of ACA and BHB on kynurenic acid (KYNA) production was assessed in vitro in bovine retinal slices., Methods: Female adult Brown-Norway rats in groups of 5-8 animals were used. ACA and BHB were administered intraperitoneally once a day for 21 consecutive days, and phosphate buffered saline (PBS) was administered to control animals. After 2 weeks, the animals received intraocular NMDA (2 μl of a 10 mM solution in PBS) or intraocular PBS as a control. On day 19, retinal ganglion cells were labeled retrogradely with hydroxystilbamidine. Two days later, RGC density (cells per mm(2)) was assessed on retinal flatmounts. Additionaly, bovine retinal slices were incubated with NMDA and ACA or BHB at concentrations of 1.0 mM and 3.0 mM, and de novo KYNA production was measured using HPLC., Results: Intraperitoneal ACA (250 mg/kg) or BHB (291.2 mg/kg) significantly protected RGC against NMDA-induced neurodegeneration. De novo KYNA production in bovine retinal slices was lowered by NMDA. Both ACA and BHB at a concentration of 3.0 mM significantly reduced the effects of NMDA., Conclusions: ACA and BHB had a significant dose-dependent neuroprotective effect on RGC in a rat model of NMDA-induced RGC damage. Both ketone bodies also significantly attenuated NMDA-induced reduction of retinal KYNA production in vitro, suggesting that this mechanism may be essential for the neuroprotective effects of ACA and BHB in vivo. Our results imply that ketone bodies may represent an additional treatment option in chronic neurodegenerative disorders of the eye.

Purpose: To evaluate the effects of intravitreally introduced vascular endothelial growth factor (VEGF) inhibitors in rat eyes with healthy retinal ganglion cells (RGC) and into others with N-methyl-D-aspartate (NMDA)-induced RGC damage., Methods: Bevacizumab, ranibizumab and pegaptanib were intravitreally injected each at two different concentrations. Respective vehicles of the three substances served as controls. In a different group, additionally a rat anti-VEGF antibody was injected after NMDA treatment. Retrogradely labelled RGC were counted on retinal wholemounts 1 week or 2 months after intravitreal introduction of the VEGF inhibitors. Electron microscopy (EM) was performed on normal rat eyes 2 months after introduction of the VEGF inhibitors., Results: RGC counts in healthy rat eyes were essentially unchanged from those of the control animals after the administration of both low and high concentrations of bevacizumab, ranibizumab or pegaptanib. Compared to the other two substances, however, high doses of pegaptanib and its respective vehicle significantly decreased RGC after 1 week and led to a marked increase of mitochondrial swelling in EM. In eyes with NMDA-induced RGC damage, no changes of RGC numbers were detected after rat anti-VEGF antibody or bevacizumab, ranibizumab and pegaptanib at both tested concentrations., Conclusions: Even at higher doses, bevacizumab and ranibizumab showed no toxic effects on RGC in vivo in either untreated rats or in the NMDA-induced RGC damage model. Also a rat anti-VEGF antibody showed no adverse effects after NMDA. Anti-VEGF therapy therefore appears safe even for eyes with additional excitotoxic RGC damage. Potential harm from the pegaptanib carrier solution at very high local concentrations cannot be excluded.

Iron overload can contribute to oxidative stress in many tissues. We studied the effects of pretreatment with iron dextran on RGC loss in a calibrated partial optic nerve crush (PONC) model in rats, along with the protection offered by tempol (4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxyl, a membrane-permeable superoxide dismutase mimetic and free-radical scavenger), in the same experimental paradigm. A total of 40 rats in 6 groups of 5-8 animals each underwent PONC in one eye and sham crush in the other. Animals were pretreated with a single iron dextran load 24 h prior to PONC, and treated with tempol 6 h before and then once daily after PONC. Control animals were treated with PBS. RGC were retrogradely labeled with a fluorescent marker; all data are expressed in percent of the RGC count in the respective sham-treated eye. Immunohistochemistry was performed to visualize 3-nitrotyrosine, a marker of nitroxidative stress. PONC without iron pretreatment resulted in the survival of only 31.4% of labeled RGC after 7 days. Even fewer RGC (12.7%) survived after PONC with iron pretreatment. However, tempol in doses of 20 mg/kg of body weight (BW) significantly attenuated this effect when given as described above; in the group without iron pretreatment the number of surviving RGC doubled from 31.4% to 62.1%. In the group with iron pretreatment the survival rate of RGC increased even more pronouncedly, from 12.7% without tempol to 46.2% with tempol. Tempol in doses of 1 mg/kg BW and 5 mg/kg BW showed no significant rescue of RGC. Immunostaining showed nitrotyrosine-positive RGCs in PONC but not in sham-treated eyes and an increase in positive cells after iron load. Tempol treatment reduced nitrotyrosine staining in both the iron and non-iron groups. Our results demonstrate that PONC results in significantly greater RGC damage when iron pretreatment is performed, and that the compound tempol may provide additional protection for RGC in cases of neuronal damage both with and without prior iron treatment., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Background: This laboratory study was undertaken to investigate the influence of bevacizumab on apoptosis, Na(+)-K(+)-adenosine triphosphatase (Na(+)-K(+)-ATPase) and zonula occludens 1 (ZO-1) expression on cultured human corneal endothelial cells (HCECs)., Methods: Annexin V binding combined with propidium iodide (PI) costaining was used to distinguish viable, early and late apoptotic cells. Immunolocalization of ZO-1 and Na(+)-K(+)-ATPase was performed to analyze intercellular cell integrity after exposure to 5.0 mg/ml bevacizumab for 24 h., Results: No significant induction of apoptosis or necrosis was seen in HCECs after exposure to 5.0 mg/ml bevacizumab (p = 0.689, p = 0.516, respectively). The mean number of annexin-V-FITC- and PI-positive cells did not change significantly. Additionally, no significant changes in expression were detectable, neither for ZO-1 nor for Na(+)-K(+)-ATPase in comparison with the control. For ZO-1, 70.0% of the cells stained intensely, 24.7% stained moderately, and 5.3% stained weakly in the control group. After exposure to 5.0 mg bevacizumab, only minor changes were observable: 68.8% stained intensely, 25.4% moderately and 5.8% weakly (p = 0.524). For Na(+)-K(+)-ATPase, 19.3% of the cells stained intensely, 59.4% moderately, and 21.3% weakly in the control group. After exposure to 5.0 mg bevacizumab, again only minor changes were observable in the expression pattern: 18.2% stained intensely, 60.3% moderately and 21.5% weakly. The changes were not significant compared with the control (p = 0.492)., Conclusions: Bevacizumab, at concentrations used clinically, did not induce apoptosis or necrosis in HCECs in vitro. Additionally, no alteration of ZO-1 or Na(+)/K(+)-ATPase expression was detected after exposure to 5.0 mg/ml bevacizumab for 24 h., ((c) 2010 S. Karger AG, Basel.)

The anthropogenic nitrogen turnover of Western societies is highly unbalanced. As a consequence, a permanent supply of nitrogen via mineral fertiliser (produced with high energy inputs) and/or imported fodder are required and result in significant impacts of nitrogen emissions on the environment (air, water, soil). Key factors to an improved nitrogen balance on a national scale are the nutrition habits of the population. According to primary calculations a shift from the actual animal-based nutrition to a "healthier nutrition" (mainly characterised by 2/3 vegetable protein supply) would lead to a reduction of needed nitrogen supply as well as of nitrogen emissions to the environment by about one quarter to one third on a national scale. More detailed investigations are required in this respect in order to arrive at more specific conclusions and have commenced by an interdisciplinary team led by the authors.

Retinal degeneration leads to release of cell-type specific proteins into the adjacent compartment. Here we investigated whether the neurofilament heavy chain protein (NfH) could be measured from the vitreous body and anterior chamber fluid. This prospective study included 85 patients who underwent vitrectomy (44 retinal detachment, 12 macular hole, 15 epiretinal gliosis, 8 organ donors) or trabelectomy (six glaucoma). The cut-off level was calculated from the organ donors. An established enzyme-linked immunosorbent assay (ELISA, SMI35) was used for quantification of NfH (190-210 kDa). Measurable levels of NfH were detected from the vitreous body homogenate, but not from the anterior chamber fluid. The cut-off level was 0.29 ng/mL. A significant proportion of patients suffering from retinal detachment (43.2%, mean 0.74 ng/mL) had vitreous body NfH levels above cut-off when compared to organ donors (0%, 0.12 ng/mL, p = 0.02), epiretinal sclerosis (1.6%, 0.05 ng/mL, p = 0.01), macular hole (0%, 0.04 ng/mL, p = 0.004). Following retinal detachment, vitreous NfH-SMI35 levels correlated with time from onset (R = -0.3, p < 0.05), persisting for up to 2 years. This study shows that NfH can be quantified from the human vitreous body and may be a useful novel biomarker for retinal degeneration. The method can be applied for investigating the dynamics of retinal degeneration and the response to neuroprotective strategies in a broad range of retinal diseases in either clinical or experimental research.

Purpose: The purpose of this study was to investigate the biocompatibility of aniline and methyl blue in a well-established cell culture model and assess the staining properties of these dyes at the level of the internal limiting membrane (ILM) in human donor eyes., Methods: Dye-related toxicity was evaluated by a colorimetric test (MTT) measuring the inhibition of retinal pigment epithelium (ARPE-19 and primary human retinal pigment epithelium) cell proliferation. Cell viability was also quantified based on a two-color fluorescence assay (Life-Dead Assay). Aniline blue and methyl blue at a concentration of 0.2% was applied over the macula during vitrectomy in human donor eyes to evaluate the staining properties at the level of the ILM., Results: Both dyes and dye concentrations of 0.1% and 0.2% showed no toxic effect on ARPE-19 and primary human retinal pigment epithelium cell proliferation for exposure times of 1 and 10 minutes, respectively. Cell viability was also not affected at all. Both dyes provided a good contrast at the level of the ILM and allowed for a controlled removal of the ILM during surgery. No penetration into deeper retinal layers was noted., Conclusion: Our results indicate that aniline blue and methyl blue might be applicable for intraocular surgery, providing a very good biocompatibility and required selective staining characteristics at the level of the ILM.

Purpose: To investigate the biocompatibility of methyl blue and aniline blue as vital dyes for vitreoretinal surgery in an in vivo rat model and to evaluate the effect of these dyes on retinal structure and function., Methods: Adult Brown-Norway rats received intravitreal injections of 0.1%, 0.2%, and 2% methyl blue or aniline blue dissolved in balanced salt solution with balanced salt solution serving as a control. Retinal toxicity was assessed 7 days thereafter by means of retinal ganglion cell counts, light microscopy, and electroretinography., Results: No significant decrease in retinal ganglion cell counts at concentrations up to 0.2% was observed. At 2%, however, a significant retinal ganglion cell loss was detected with both dyes (more pronounced for aniline blue). Light microscopy showed no structural changes in the central retina for concentrations up to 0.2%. Electroretinographies detected no adverse effects of methyl blue or aniline blue on rod- or cone-driven responses at concentrations up to 0.2%., Conclusion: Methyl blue and aniline blue are very biocompatible and may, therefore, be usable for intraocular surgery. Further testing with other animal models will be necessary to confirm this. The safety margin of methyl blue is possibly higher than that of aniline blue.

Promoter hypermethylation preventing expression of the RAS association domain family 1 isoform A (RASSF1A) gene product is among the most abundant epigenetic deregulations in human cancer. Restoration of RASSF1A inhibits tumor cell growth in vitro and in murine xenograft models. Rassf1a-deficient mice feature increased spontaneous and carcinogen-induced tumor formation. Mechanistically, RASSF1A affects several cellular functions, such as microtubule dynamics, migration, proliferation, and apoptosis; however, its tumor-suppressive mechanism is incompletely understood. To study the functional consequences of RASSF1A expression in human cancer cells, we made use of a doxycycline-inducible expression system and a RASSF1A-deficient lung cancer cell line. We observed that RASSF1A induces cell cycle arrest in G(1) phase and senescence in vitro and in tumors established in immunodeficient mice. RASSF1A-mediated growth inhibition was accompanied by the up-regulation of the cyclin-dependent kinase inhibitor p21(Cip1/Waf1) and proceeded independently of p53, p14(Arf), and p16(Ink4a). Loss of p21(Cip1/Waf1) or coexpression of the human papilloma virus 16 oncoprotein E7 was found to override RASSF1A-induced cell cycle arrest and senescence. Conditional RASSF1A affected mitogen-activated protein kinase and protein kinase B/Akt signaling to up-regulate p21(Cip1/Waf1) and to facilitate its nuclear localization. In summary, RASSF1A can mediate cell cycle arrest and senescence in human cancer cells by p53-independent regulation of p21(Cip1/Waf1).