Efficacy and safety of combined UV-light corneal crosslinking and fine-needle diathermy to regress pathological murine corneal (lymph)angiogenesis in vivo.

PURPOSE: To compare safety and efficacy of isolated and combined UV-light corneal crosslinking (CXL) and fine-needle diathermy (FND) to regress pathological corneal vessels in vivo. METHODS: Mice with inflamed and pathologically vascularized corneas received CXL or FND as monotherapy or a combination of both treatments. Corneal pathological blood and lymphatic vessels, immune cells and the morphology of anterior segment structures were evaluated. RESULTS: All three approaches were able to regress blood and lymphatic vessels in mice. A comparative analysis of the three methods revealed that the FND monotherapy and the CXL + FND combination were significantly more effective than the CXL monotherapy, one and 2 weeks after therapy and especially in regressing lymphatic vessels. Furthermore, the combination therapy induced significantly less immune cell recruitment compared to the monotherapies. All three methods were safe to use in regards of corneal integrity. CONCLUSIONS: A combination of FND and CXL led to regression of pathological corneal lymphatic and blood vessels and reduced the infiltration of immune cells into inflamed murine corneas. This approach offers a new effective, safe and clinically usable strategy to treat eyes with mature pathological blood vessels and even more so for lymphatic vessels, for example prior to high-risk corneal transplantation.

Blood-Aqueous Barrier Disruption in Penetrating and Posterior Lamellar Keratoplasty: Implications for Clinical Outcome. / Blut-Kammerwasser-Schranken-Störungen bei perforierender und posteriorer lamellärer Keratoplastik: Implikationen für das klinische Ergebnis.

BACKGROUND: The blood-aqueous barrier (BAB) separates immunoprivileged tissue of the eye from the blood circulation. Disruption of the BAB is therefore a risk factor for rejection after keratoplasty. PURPOSE: The present work provides a review of the work of our group and others on BAB disruption in penetrating and posterior lamellar keratoplasty and its implications for clinical outcome. METHODS: A PubMed literature search was performed to generate a review paper. RESULTS: Laser flare photometry provides an objective and reproducible method to assess the integrity of the BAB. Studies of the flare after penetrating and posterior lamellar keratoplasty demonstrate a mostly regressive disruption of the BAB in the postoperative course, which is influenced in extent and duration by multiple factors. Persistently elevated flare values or an increase in flare after initial postoperative regeneration may indicate an increased risk of rejection. DISCUSSION: In case of persistent or recurrent elevated flare values after keratoplasty, intensified (local) immunosuppression may potentially be useful. This could become important in the future, especially for the monitoring of patients after high-risk keratoplasty. Whether an increase of the laser flare is a reliable early indicator of an impending immune reaction after penetrating or posterior lamellar keratoplasty has to be shown in prospective studies.

The novel role of lymphatic vessels in the pathogenesis of ocular diseases.

Historically, the eye has been considered as an organ free of lymphatic vessels. In recent years, however, it became evident, that lymphatic vessels or lymphatic-like vessels contribute to several ocular pathologies at various peri- and intraocular locations. The aim of this review is to outline the pathogenetic role of ocular lymphatics, the respective molecular mechanisms and to discuss current and future therapeutic options based thereon. We will give an overview on the vascular anatomy of the healthy ocular surface and the molecular mechanisms contributing to corneal (lymph)angiogenic privilege. In addition, we present (i) current insights into the cellular and molecular mechanisms occurring during pathological neovascularization of the cornea triggered e.g. by inflammation or trauma, (ii) the role of lymphatic vessels in different ocular surface pathologies such as dry eye disease, corneal graft rejection, ocular graft versus host disease, allergy, and pterygium, (iii) the involvement of lymphatic vessels in ocular tumors and metastasis, and (iv) the novel role of the lymphatic-like structure of Schlemm's canal in glaucoma. Identification of the underlying molecular mechanisms and of novel modulators of lymphangiogenesis will contribute to the development of new therapeutic targets for the treatment of ocular diseases associated with pathological lymphangiogenesis in the future. The preclinical data presented here outline novel therapeutic concepts for promoting transplant survival, inhibiting metastasis of ocular tumors, reducing inflammation of the ocular surface, and treating glaucoma. Initial data from clinical trials suggest first success of novel treatment strategies to promote transplant survival based on pretransplant corneal lymphangioregression.

Correlation of Clinical Fibrillar Layer Detection and Corneal Thickness in Advanced Fuchs Endothelial Corneal Dystrophy.

Central subendothelial geographic deposits are formed as a fibrillar layer (FL) in advanced Fuchs endothelial corneal dystrophy (FECD). Previous studies demonstrated a significant decrease in corneal endothelial cell (CEC) density and an increase in focal corneal backscatter in the FL area. The present study investigated the association of the FL with edema formation and its localization. Patients (n = 96) presenting for Descemet membrane endothelial keratoplasty (DMEK) for advanced FECD were included. Slit-lamp biomicroscopy with FECD grading was followed by Scheimpflug imaging with en face backscatter analysis and pachymetric analysis. FL dimensions were measured, and correlation with pachymetric values was performed. An FL was detected in 74% of all eyes (n = 71). Pachymetric values in FL-positive versus FL-negative eyes were for corneal thickness at the apex (ACT) 614 ± 52 µm and 575 ± 46 µm (p = 0.001), for peripheral corneal thickness at 1 mm (PCT1mm) 616 ± 50 µm and 580 ± 44 µm (p = 0.002), for PCT2mm 625 ± 48 µm and 599 ± 41 µm (p = 0.017), for PCT3mm 651 ± 46 µm and 635 ± 40 µm (p = 0.128) and for PCT4mm 695 ± 52 µm and 686 ± 43 µm (p = 0.435), respectively. Correlation analysis indicated a weak correlation for the FL maximum vertical caliper diameter with ACT and PCT1mm values but no further relevant correlations. In FL-positive eyes, increased focal corneal backscatter and increased corneal thickness showed primarily central and inferotemporal localization. In conclusion, Scheimpflug imaging shows an association of the FL with increased corneal thickness in advanced FECD and shows localization of the FL and increased corneal thickness in the central and inferotemporal region. This may provide important information for progression assessment and therapeutic decision making in FECD patients in the future.

Scheimpflug Backscatter Imaging of the Fibrillar Layer in Fuchs Endothelial Corneal Dystrophy.

A central collagen-rich subendothelial fibrillar layer (FL) correlates with areas of accentuated loss of corneal endothelial cells in advanced Fuchs endothelial corneal dystrophy (FECD). The present study sought to investigate whether the FL may be visualized by en face Scheimpflug backscatter imaging in vivo. DESIGN: Retrospective analysis of a prospective observational case series. METHODS: A total of 34 eyes (34 subjects) undergoing Descemet membrane endothelial keratoplasty (DMEK) surgery with preoperative high-quality Scheimpflug backscatter imaging data were included. The Descemet endothelium complex (DEC) was retrieved during DMEK surgery, and immunofluorescence staining was performed for collagens I, III, and IV. The FL morphology in en face Scheimpflug backscatter and immunofluorescence imaging was compared and agreement of FL parameters was analyzed using intraclass correlation coefficients (ICC) and Bland-Altman plots. RESULTS: Scheimpflug backscatter imaging delineated the FL in 26 eyes and was FL negative in 8 eyes with deviation compared to immunofluorescence in 1 case and good agreement of morphology characteristics. Horizontal caliper diameter ± SD was 4.84 ± 0.85 mm, vertical caliper diameter was 3.92 ± 0.78 mm, maximum caliper diameter was 5.12 ± 0.82 mm, and surface area was 12.43 ± 4.74 mm2. Compared to immunofluorescence imaging, mean difference (95% limits of agreement) and intraclass correlation coefficients were for horizontal caliper diameter 0.13 mm (-0.81 to 1.1 mm) and 0.88, vertical caliper diameter 0.23 mm (-0.76 to 1.2 mm) and 0.81, maximum caliper diameter 0.06 mm (-1.1 to 1.2 mm) and 0.86, and surface area 1.4 mm2 (-3.9 to 6.7 mm2) and 0.85. CONCLUSIONS: Scheimpflug backscatter imaging facilitates visualization of the FL in advanced FECD eyes, offering the potential to identify particularly diseased areas of the FECD endothelium in vivo.

Dietary thiamine influences l-asparaginase sensitivity in a subset of leukemia cells.

Tumor environment influences anticancer therapy response but which extracellular nutrients affect drug sensitivity is largely unknown. Using functional genomics, we determine modifiers of l-asparaginase (ASNase) response and identify thiamine pyrophosphate kinase 1 as a metabolic dependency under ASNase treatment. While thiamine is generally not limiting for cell proliferation, a DNA-barcode competition assay identifies leukemia cell lines that grow suboptimally under low thiamine and are characterized by low expression of solute carrier family 19 member 2 (SLC19A2), a thiamine transporter. SLC19A2 is necessary for optimal growth and ASNase resistance, when standard medium thiamine is lowered ~100-fold to human plasma concentrations. In addition, humanizing blood thiamine content of mice through diet sensitizes SLC19A2-low leukemia cells to ASNase in vivo. Together, our work reveals that thiamine utilization is a determinant of ASNase response for some cancer cells and that oversupplying vitamins may affect therapeutic response in leukemia.

Breast Carcinoma Metastasis to the Medial Rectus Muscle: Case Report

A 63-year-old woman with metastatic breast carcinoma presented to the ophthalmology clinic with diplopia and right abduction deficit. Magnetic resonance imaging showed isolated enlargement of the right medial rectus muscle. Biopsy of the enlarged muscle revealed metastasis of breast carcinoma. Ocular motility deficit in a patient with breast carcinoma should raise suspicion of metastasis to the orbit involving the extraocular muscles. Orbital imaging and biopsy are necessary for diagnosis and appropriate treatment.

INTRAOPERATIVE OCULAR MANOMETRY IN SILICONE OIL-FILLED EYES WITH A BOSTON TYPE 1 KERATOPROSTHESIS.

PURPOSE: To demonstrate a novel technique to measure the intraocular pressure in silicone oil (SO)-filled eyes with Boston Type 1 keratoprosthesis (KPro) during intraocular surgery. METHODS: In this retrospective case series, an ocular manometer that is predicated on a continuous fluid column between a pressure sensor and interior of the eye was designed and used to directly measure intraocular pressure during intraocular surgery in SO-filled eyes with KPro. RESULTS: Six eyes of six patients were included in the study. The indications for SO injection with ocular manometry were hypotony in five patients, and endophthalmitis and complex retinal detachment with proliferative vitreoretinopathy in one patient. All patients had a successful reinflation of their globes without any evidence of SO underfill, without evidence of SO overfill, and without progression of glaucomatous optic neuropathy. Visual acuity increased in five eyes and was maintained in one eye. CONCLUSION: Intraoperative ocular manometry is a safe and effective technique in determining intraocular pressure in SO-filled eyes with KPro.

Falcine Myxoid Chondrosarcoma: A Rare Aggressive Case.

Chondrosarcoma is the second most common primary malignancy of bone after osteosarcoma. Cranial primary chondrosarcomas mostly originate from the skull base cartilage formation zones. Parasagittal falcine origin is very rare for primary extra-skeletal intracranial chondrosarcomas. We report a rare case of primary myxoid chondrosarcoma at falx cerebri. The patient was a 35-year-old lady with right arm and leg weakness. Her brain magnetic resonance imaging depicted a left parasagittal mass lesion attached to the falx cerebri. En bloc resection via left frontal craniotomy was performed. Three more local recurrences occurred in 9 months' time since the index surgery, which were all managed with re-surgeries and/or adjuvant stereotactic radiosurgeries. This is the second case of myxoid type parasagittal chondrosarcoma but with the most protracted disease course. Even though surgery remains the mainstay of treatment for parasagittal chondrosarcomas, adjuvant therapy might be necessary in aggressive ones.

Recurrence of acute lymphoblastic leukemia manifesting as serous retinal detachments and optic disc swelling.

PURPOSE: The aim of this case report is to describe a patient with acute lymphoblastic leukemia (ALL) who developed bilateral serous retinal detachments and unilateral optic disc swelling. METHODS: A 23-year-old woman with ALL presented to the ophthalmology clinic with bilateral subacute visual loss. RESULTS: Ophthalmologic examination revealed bilateral serous retinal detachments and unilateral optic disc swelling. Magnetic resonance imaging for differential diagnosis was inconclusive; however, cerebrospinal fluid sampling demonstrated leukemic involvement of the central nervous system. The patient's vision improved and fundus findings resolved with the institution of systemic and intrathecal chemotherapy. CONCLUSIONS: Serous retinal detachment and optic disc swelling are unusual ocular manifestations of ALL. They may occur due to leukemic infiltration of ocular structures and may indicate extramedullary recurrence of the disease. Early recognition and treatment is crucial to improve prognosis.

Low-Grade Mucoepidermoid Carcinoma of the Lacrimal Gland in a Teenaged Patient.

Mucoepidermoid carcinoma is the most common malignant, locally invasive tumour of the salivary glands and accounts for approximately 35% of all malignancies of the major and minor salivary glands. Mucoepidermoid carcinoma that originated from the lacrimal gland is exceedingly rare in teenage patients, with only a few cases reported. Herein, we report clinical and radiological findings of a mucoepidermoid carcinoma arising from the lacrimal gland in a 15-year-old boy. We suggest that since preoperative imaging findings are not diagnostic for mucoepidermoid carcinoma, histopathologic examination should be performed for definitive diagnosis. Complete surgical resection is the treatment of choice for low-grade lacrimal gland mucoepidermoid carcinoma.

Virtual screening of small molecules databases for discovery of novel PARP-1 inhibitors: combination of in silico and in vitro studies.

Poly(ADP-ribose) polymerase-1 (PARP-1) enzyme has critical roles in DNA replication repair and recombination. Thus, PARP-1 inhibitors play an important role in the cancer therapy. In the current study, we have performed combination of in silico and in vitro studies in order to discover novel inhibitors against PARP-1 target. Structure-based virtual screening was carried out for an available small molecules database. A total of 257,951 ligands from Otava database were screened at the binding pocket of PARP-1 using high-throughput virtual screening techniques. Filtered structures based on predicted binding energy results were then used in more sophisticated molecular docking simulations (i.e. Glide/standard precision, Glide/XP, induced fit docking - IFD, and quantum mechanics polarized ligand docking - QPLD). Potential high binding affinity compounds that are predicted by molecular simulations were then tested by in vitro methods. Computationally proposed compounds as PARP-1 inhibitors (Otava Compound Codes: 7111620047 and 7119980926) were confirmed by in vitro studies. In vitro results showed that compounds 7111620047 and 7119980926 have IC50 values of 0.56 and 63 µM against PARP-1 target, respectively. The molecular mechanism analysis, free energy perturbation calculations using long multiple molecular dynamics simulations for the discovered compounds which showed high binding affinity against PARP-1 enzyme, as well as structure-based pharmacophore development (E-pharmacophore) studies were also studied.

The signaling pathway of dopamine D2 receptor (D2R) activation using normal mode analysis (NMA) and the construction of pharmacophore models for D2R ligands.

G-protein-coupled receptors (GPCRs) are targets of more than 30% of marketed drugs. Investigation on the GPCRs may shed light on upcoming drug design studies. In the present study, we performed a combination of receptor- and ligand-based analysis targeting the dopamine D2 receptor (D2R). The signaling pathway of D2R activation and the construction of universal pharmacophore models for D2R ligands were also studied. The key amino acids, which contributed to the regular activation of the D2R, were in detail investigated by means of normal mode analysis (NMA). A derived cross-correlation matrix provided us an understanding of the degree of pair residue correlations. Although negative correlations were not observed in the case of the inactive D2R state, a high degree of correlation appeared between the residues in the active state. NMA results showed that the cytoplasmic side of the TM5 plays a significant role in promoting of residue-residue correlations in the active state of D2R. Tracing motions of the amino acids Arg219, Arg220, Val223, Asn224, Lys226, and Ser228 in the position of the TM5 are found to be critical in signal transduction. Complementing the receptor-based modeling, ligand-based modeling was also performed using known D2R ligands. The top-scored pharmacophore models were found as 5-sited (AADPR.671, AADRR.1398, AAPRR.3900, and ADHRR.2864) hypotheses from PHASE modeling from a pool consisting of more than 100 initial candidates. The constructed models using 38 D2R ligands (in the training set) were validated with 15 additional test set compounds. The resulting model correctly predicted the pIC50 values of an additional test set compounds as true unknowns.

Investigation of inhibition of human glucose 6-phosphate dehydrogenase by some 99mTc chelators by in silico and in vitro methods.

The inhibitory effects of methoxyisobutylisonitrile (MIBI), diethylene triamine pentaacetic acid (DTPA), dimercaptosuccinic acid (DMSA) and metilendifosfonat (MDP) on human erythrocyte glucose 6-phosphate dehydrogenase (hG6PD) activity were investigated. For this purpose, hG6PD was initially purified 557-fold at a yield of 51.43% using 2',5'-adenosine diphosphate (ADP) sepharose 4B affinity gel chromatography. The in vitro effects of these chelators on hG6PD enzyme were studied. IC50 values of MIBI, DTPA, DMSA and MDP were 0.056, 0.172, 0.274 and 0.175 mM, of hG6PD, respectively. It was detected in in vitro studies that the hG6PD enzyme is inhibited due to these radiopharmaceutical chelators. In addition to in vitro studies, in order to better understand the molecular mechanism of studied compounds, combined in silico approaches, including molecular docking and molecular dynamics (MD), simulations were successfully performed. MD simulations shed light on inhibition mechanisms of the individual inhibitors into the ligand-binding pocket of hG6PD. Essential amino acids for binding are also investigated using per-residue interaction analysis studies.

Mutated form (G52E) of inactive diphtheria toxin CRM197: molecular simulations clearly display effect of the mutation to NAD binding.

Mutated form (G52E) of diphtheria toxin (DT) CRM197 is an inactive and nontoxic enzyme. Here, we provided a molecular insight using comparative molecular dynamics (MD) simulations to clarify the influence of a single point mutation on overall protein and active-site loop. Post-processing MD analysis (i.e. stability, principal component analysis, hydrogen-bond occupancy, etc.) is carried out on both wild and mutated targets to investigate and to better understand the mechanistic differences of structural and dynamical properties on an atomic scale especially at nicotinamide adenine dinucleotide (NAD) binding site when a single mutation (G52E) happens at the DT. In addition, a docking simulation is performed for wild and mutated forms. The docking scoring analysis and docking poses results revealed that mutant form is not able to properly accommodate the NAD molecule.

Kinetic and in silico studies of hydroxy-based inhibitors of carbonic anhydrase isoforms I and II.

A series of hydroxy and phenolic compounds have been assayed for the inhibition of two physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isozymes, the cytosolic human isozymes I and II. The investigated molecules showed inhibition constants in the range of 1.07-4003 and 0.09-31.5 µM at the hCA I and hCA II enzymes, respectively. In order to investigate the binding mechanisms of these inhibitors, in silico studies were also applied. Molecular docking scores of the studied compounds are compared using three different scoring algorithms, namely Glide/SP, Glide/XP and Glide/IFD. In addition, different ADME (absorption, distribution, metabolism and excretion) analysis was performed. All the examined compounds were found within the acceptable range of pharmacokinetic profiles.

Molecular Simulations of Solved Co-crystallized X-Ray Structures Identify Action Mechanisms of PDEδ Inhibitors.

PDEδ is a small protein that binds and controls the trafficking of RAS subfamily proteins. Its inhibition protects initiation of RAS signaling, and it is one of the common targets considered for oncological drug development. In this study, we used solved x-ray structures of inhibitor-bound PDEδ targets to investigate mechanisms of action of six independent all-atom MD simulations. An analysis of atomic simulations combined with the molecular mechanic-Poisson-Boltzmann solvent accessible surface area/generalized Born solvent accessible surface area calculations led to the identification of action mechanisms for a panel of novel PDEδ inhibitors. To the best of our knowledge, this study is one of the first in silico investigations on co-crystallized PDEδ protein. A detailed atomic-scale understanding of the molecular mechanism of PDEδ inhibition may assist in the design of novel PDEδ inhibitors. One of the most common side effects for diverse small molecules/kinase inhibitors is their off-target interactions with cardiac ion channels and human-ether-a-go-go channel specifically. Thus, all of the studied PDEδ inhibitors are also screened in silico at the central cavities of hERG1 potassium channels.