Dysregulated Retinoic Acid Signaling in the Pathogenesis of Pseudoexfoliation Syndrome.

Pseudoexfoliation (PEX) syndrome, a stress-induced fibrotic matrix process, is the most common recognizable cause of open-angle glaucoma worldwide. The recent identification of PEX-associated gene variants uncovered the vitamin A metabolic pathway as a factor influencing the risk of disease. In this study, we analyzed the role of the retinoic acid (RA) signaling pathway in the PEX-associated matrix metabolism and evaluated its targeting as a potential candidate for an anti-fibrotic intervention. We provided evidence that decreased expression levels of RA pathway components and diminished RA signaling activity occur in an antagonistic crosstalk with TGF-ß1/Smad signaling in ocular tissues and cells from PEX patients when compared with age-matched controls. Genetic and pharmacologic modes of RA pathway inhibition induced the expression and production of PEX-associated matrix components by disease-relevant cell culture models in vitro. Conversely, RA signaling pathway activation by natural and synthetic retinoids was able to suppress PEX-associated matrix production and formation of microfibrillar networks via antagonization of Smad-dependent TGF-ß1 signaling. The findings indicate that deficient RA signaling in conjunction with hyperactivated TGF-ß1/Smad signaling is a driver of PEX-associated fibrosis, and that restoration of RA signaling may be a promising strategy for anti-fibrotic intervention in patients with PEX syndrome and glaucoma.

Spatiotemporal dynamics of growth and death within spherical bacterial colonies.

Bacterial growth within colonies and biofilms is heterogeneous. Local reduction of growth rates has been associated with tolerance against various antibiotics. However, spatial gradients of growth rates are poorly characterized in three-dimensional bacterial colonies. Here, we report two spatially resolved methods for measuring growth rates in bacterial colonies. As bacteria grow and divide, they generate a velocity field that is directly related to the growth rates. We derive profiles of growth rates from the velocity field and show that they are consistent with the profiles obtained by single-cell-counting. Using these methods, we reveal that even small colonies initiated with a few thousand cells of the human pathogen Neisseria gonorrhoeae develop a steep gradient of growth rates within two generations. Furthermore, we show that stringent response decelerates growth inhibition at the colony center. Based on our results, we suggest that aggregation-related growth inhibition can protect gonococci from external stresses even at early biofilm stages.

[Isolated Unilateral Strabismus Sursoadductorius (Congenital Trochlear Palsy) with and without Hypotrophia of the Superior Oblique Muscle-Orthoptic Differences in Adulthood]. / Unilateraler dekompensierender Strabismus sursoadductorius mit und ohne Hypotrophie des M. obliquus superior ­ orthoptische Unterscheidungsmerkmale im Erwachsenenalter.

PURPOSE: The clinical feature of unilateral decompensating strabismus sursoadductorius (dSSA; often called congenital superior oblique palsy, CSOP) is not an etiologically uniform entity. Hypotrophy of the superior oblique muscle (HMOS) is a frequent and immediate cause of dSSA/CSOP. In this study, clinical characteristics of dSSA/CSOP with and without HMOS are compared. PATIENTS AND METHODS: Twenty-five consecutive patients (age 14 - 69 years; median 43 years) were included in this study, 14 with 3T MRI-proven HMOS (group 1) and 11 without HMOS (group 2). HMOS was defined as a reduction of the medio-lateral (ML) as well as based on ML and cranio-caudal (CC) diameter calculated area = (ML · CC/4) · π of the affected superior oblique muscle (SOM) < 80% in comparison to the contralateral SOM (measured on the single coronal image on which the muscle has its greatest extent). The two groups were compared in terms of head tilt, cyclo- and vertical deviation and the Bielschowsky head tilt test. Patients were classified according the Knapp's classification. RESULTS: Both the incidence of head tilt with 14/14 vs. 5/11 (χ2 = 0.003) and its degree: 11.1 ± 4.5° vs. 3.2 ± 4.1° (p < 0.001) was higher in group 1 than in group 2, as well as the Bielschowsky head tilt test: 9.3 ± 4.3° vs. 3.8 ± 4.9° (p = 0.008). The average amount of hypertropia was larger in group 1 than in group 2 during adduction: 16.7 ± 5.3° vs. 9.3 ± 3.4° (p < 0.001) as well as during adduction and downgaze of the affected eye: 14.6 ± 7.1 vs. 7.2 ± 3.7° (p = 0.03). In the sagittal plane, the increase of vertical deviation was larger in group 1 than in group 2: 2.8 ± 7.8 vs. - 2.4 ± 4.2 (p = 0.04); the excyclodeviation was larger in group 1 in all three planes (sagittal plane, adduction and abduction) in comparison to group 2: 1.3 ± 4.1 vs. - 2.9 ± 2.8 (p = 0.006), 2.4 ± 5.2 vs. - 2.2 ± 2.9 (p = 0.01), 0.5 ± 3.8 vs. - 2.7 ± 3.9 (p = 0.05). Knapp's class II was found in 6 of 15 patients in group 1 and only in this patient group (χ2 = 0.03). CONCLUSIONS: In both groups, the vertical deviation showed a great dispersion. In patients without HMOS (group 2), vertical deviation in adduction did not exceed 15°. Patients with HMOS (group 1) do not show the typical features of a later acquired trochlear palsy due to an early developed compensating innervation. A vertical deviation in adduction of more than 15°, increasing excyclodeviation towards downgaze in all three planes (sagittal plane, adduction and abduction) and Knapp's class II are relatively reliable predictors of a hypoplasia of the SOM. An internationally uniform term for this group of patients, such as superior oblique weakness or superior oblique hypotrophy, would be desirable.

Mutations in ARL2BP, encoding ADP-ribosylation-factor-like 2 binding protein, cause autosomal-recessive retinitis pigmentosa.

Retinitis pigmentosa (RP) is a genetically heterogeneous retinal degeneration characterized by photoreceptor death, which results in visual failure. Here, we used a combination of homozygosity mapping and exome sequencing to identify mutations in ARL2BP, which encodes an effector protein of the small GTPases ARL2 and ARL3, as causative for autosomal-recessive RP (RP66). In a family affected by RP and situs inversus, a homozygous, splice-acceptor mutation, c.101-1G>C, which alters pre-mRNA splicing of ARLBP2 in blood RNA, was identified. In another family, a homozygous c.134T>G (p.Met45Arg) mutation was identified. In the mouse retina, ARL2BP localized to the basal body and cilium-associated centriole of photoreceptors and the periciliary extension of the inner segment. Depletion of ARL2BP caused cilia shortening. Moreover, depletion of ARL2, but not ARL3, caused displacement of ARL2BP from the basal body, suggesting that ARL2 is vital for recruiting or anchoring ARL2BP at the base of the cilium. This hypothesis is supported by the finding that the p.Met45Arg amino acid substitution reduced binding to ARL2 and caused the loss of ARL2BP localization at the basal body in ciliated nasal epithelial cells. These data demonstrate a role for ARL2BP and ARL2 in primary cilia function and that this role is essential for normal photoreceptor maintenance and function.

Impact of Clonal Architecture on Clinical Course and Prognosis in Patients With Myeloproliferative Neoplasms.

Myeloproliferative neoplasms (MPNs) are caused by a somatic gain-of-function mutation in 1 of the 3 disease driver genes JAK2, MPL, or CALR. About half of the MPNs patients also carry additional somatic mutations that modify the clinical course. The order of acquisition of these gene mutations has been proposed to influence the phenotype and evolution of the disease. We studied 50 JAK2-V617F-positive MPN patients who carried at least 1 additional somatic mutation and determined the clonal architecture of their hematopoiesis by sequencing DNA from single-cell-derived colonies. In 22 of these patients, the same blood samples were also studied for comparison by Tapestri single-cell DNA sequencing (scDNAseq). The clonal architectures derived by the 2 methods showed good overall concordance. scDNAseq showed higher sensitivity for mutations with low variant allele fraction, but had more difficulties distinguishing between heterozygous and homozygous mutations. By unsupervised analysis of clonal architecture data from all 50 MPN patients, we defined 4 distinct clusters. Cluster 4, characterized by more complex subclonal structure correlated with reduced overall survival, independent of the MPN subtype, presence of high molecular risk mutations, or the age at diagnosis. Cluster 1 was characterized by additional mutations residing in clones separated from the JAK2-V617F clone. The correlation with overall survival improved when mutation in such separated clones were not counted. Our results show that scDNAseq can reliably decipher the clonal architecture and can be used to refine the molecular prognostic stratification that until now was primarily based on the clinical and laboratory parameters.

Low-dose AtropIne for Myopia Control in Children (AIM): protocol for a randomised, controlled, double-blind, multicentre, clinical trial with two parallel arms.

INTRODUCTION: Myopia is a major cause of degenerative eye disease and increases the risk of secondary visual impairment. Mitigating its progression therefore has great potential of clinically relevant benefit as shown by using highly diluted atropine eye drops in children of Asian origin. However, limited evidence is available regarding the efficacy and safety of low-dose atropine therapy in non-Asian populations. Hence, the Low-dose AtropIne for Myopia Control in Children (AIM) study will test the efficacy and safety of 0.02% atropine vs placebo in a German population. METHODS AND ANALYSIS: AIM is a national, multicentre, prospective, randomised, placebo-controlled, double-blind trial with two parallel arms. The primary objective is to assess the efficacy of atropine 0.02% eyedrops for myopia control in children of Caucasian origin. The primary outcome is the change in cycloplegic refraction after 1 year of treatment (D/year). Secondary and tertiary outcome measures comprise the change in axial length (mm/year) in children treated with 0.02% atropine compared with placebo, the myopic progression of participants treated with 0.01% compared with 0.02% atropine (D/year and mm/year), and the safety profile of both 0.02% and 0.01% atropine. Furthermore, the myopic progression 1 year after cessation of therapy with 0.02% atropine will be evaluated. Inclusion criteria are an age of 8-12 years and myopia of -1 D to -6 D with an estimated annual myopia progression of ≥0.5 D. After randomisation, patients will receive either atropine 0.02% (arm A) or placebo eye drops (arm B) in the first year of treatment. In the second year, they will continue to receive atropine 0.02% (arm A) or switch to atropine 0.01% (arm B). In the third year, they will switch to placebo (arm A) or continue with atropine 0.01% (arm B). To achieve a statistical power of 80%, the calculated sample size is 300. The trial has started in October 2021 with a planned recruitment period of 18 months. ETHICS AND DISSEMINATION: AIM has been approved by the Central Ethics Committee of the University Medical Center Freiburg (21-1106), local ethics committees of each participating centre and the German Federal Institute for Drugs and Medical Devices (61-3910-4044659). It complies with the Declaration of Helsinki, local laws and ICH-GCP. Results and underlying data from this trial will be disseminated through peer-reviewed publications and conference presentations. TRIAL REGISTRATION NUMBER: NCT03865160.

Inhibition of interleukin-1ß reduces myelofibrosis and osteosclerosis in mice with JAK2-V617F driven myeloproliferative neoplasm.

Interleukin-1ß (IL-1ß) is a master regulator of inflammation. Increased activity of IL-1ß has been implicated in various pathological conditions including myeloproliferative neoplasms (MPNs). Here we show that IL-1ß serum levels and expression of IL-1 receptors on hematopoietic progenitors and stem cells correlate with JAK2-V617F mutant allele fraction in peripheral blood of patients with MPN. We show that the source of IL-1ß overproduction in a mouse model of MPN are JAK2-V617F expressing hematopoietic cells. Knockout of IL-1ß in hematopoietic cells of JAK2-V617F mice reduces inflammatory cytokines, prevents damage to nestin-positive niche cells and reduces megakaryopoiesis, resulting in decrease of myelofibrosis and osteosclerosis. Inhibition of IL-1ß in JAK2-V617F mutant mice by anti-IL-1ß antibody also reduces myelofibrosis and osteosclerosis and shows additive effects with ruxolitinib. These results suggest that inhibition of IL-1ß with anti-IL-1ß antibody alone or in combination with ruxolitinib could have beneficial effects on the clinical course in patients with myelofibrosis.

Independent NF1 and PTPN11 mutations in a family with neurofibromatosis-Noonan syndrome.

Neurofibromatosis-Noonan syndrome (NFNS), an entity which combines both features of Noonan syndrome (NS) and neurofibromatosis type 1 (NF1), was etiologically unresolved until recent reports demonstrated NF1 mutations in the majority of patients with NFNS. The phenotypic overlap was explained by the involvement of the Ras pathway in both disorders, and, accordingly, clustering of the NF1 mutations in the GTPase-activating protein (GAP) domain of neurofibromin was observed in individuals with NFNS. We report on an 18-month-old girl with typical findings suggestive of NS in combination with multiple café-au-lait spots and bilateral optic gliomas suggestive of NF1. The patient was found to carry a de novo PTPN11 mutation p.T2I as well as the maternally inherited NF1 mutation c.4661+1G>C. Her otherwise healthy mother and brother, who also had the NF1 mutation, showed few café-au-lait spots as the only sign of neurofibromatosis. Since our patient's unique NF1 mutation results in skipping of exon 27a and thus involves the same region, Gap-related domain, that had been shown to be associated with NFNS, her phenotype could have been misleadingly attributed to the NF1 mutation only. Contrarily, absence of both cutaneous neurofibromas and NS features in her relatives with the same NF1 mutation, suggests that the index patient's typical NFNS phenotype is caused by an additive effect of mutations in both NF1 and PTPN11. In contrast to previous findings, we speculate that absence of cutaneous neurofibromas is not solely associated with the recurrent 3-bp in-frame deletion in exon 17.

In-center intermittent peritoneal dialysis: retrospective ten-year single-center experience with thirty consecutive patients.

BACKGROUND: In-center intermittent peritoneal dialysis (IPD) is sometimes performed in elderly and multimorbid patients that have failed hemodialysis and that are unable to perform peritoneal dialysis (PD) at home. Complications, frequency of hospital admission, and survival are often claimed to be dismal although current data are lacking. METHODS: We performed a retrospective cohort study of patients that underwent IPD at Hannover Medical School, Germany, between 1997 and 2007. Underlying renal disorders, comorbidity, and circumstances that precluded hemodialysis and home PD were recorded. Survival, cause of death, episodes of hospitalization, and episodes of peritonitis were calculated. Laboratory values at baseline and after 3 months of IPD were also retrieved. RESULTS: We identified 30 patients with severe comorbidity (median Charlson Comorbidity Index of 6; n = 30) who underwent IPD for 439 months in total. The majority of patients had vascular/hypertensive nephropathy (n = 12; 40%); congestive heart failure was the leading cause for choosing PD (n = 13; 43.3%); 73.3% of our patients had either no partner or at least one disease that precluded home therapy. Hospitalization rate was 1.39 admissions per patient-year and there was 1 episode of peritonitis per 48.8 IPD-months. Mean survival was 26.6 months (median 17 months; n = 30); sepsis was the leading cause of death (n = 13; 59.1%). CONCLUSIONS: IPD is associated with fewer episodes of peritonitis, fewer admissions, and longer survival than is often believed. Suitable patients in whom palliative care alone seems inappropriate should not be denied a trial of IPD if they so choose.

Initial graft size and not the innate immune response limit survival of engrafted neural stem cells.

Transplantation of neural stem cells (NSCs) appears to be a promising regenerative therapy for a variety of neurological disorders. Nevertheless, NSC engraftment is limited by the number of surviving cells. To maximize stem cell-mediated effects, timing of implantation and cell number have to be precisely evaluated. Here, a transgenic murine NSC line was optimized for high expression levels of the imaging reporters Luc2 and copGFP. NSCs of 150 000, 75 000, 15 000 or 1500 cells or Hanks buffered salt solution were implanted into the striatum of nude mice. The survival of NSCs was monitored with in vivo bioluminescence imaging (BLI) over 2 weeks and brain sections were histologically analysed for glial cells of the innate immune system. The longitudinal in vivo BLI data revealed a significantly reduced viability with the highest rate for 150 000 engrafted NSCs. The cell loss was not correlated with the number of Iba-1+ immune cells nor GFAP+ astrocytes. Histological quantification of copGFP+ cells at 14 days postimplantation confirmed the in vivo data with the highest density of copGFP+ cells in the 150 000-cell graft and the highest survival rate for 1500 cells/graft. In conclusion, regenerative therapies should strictly evaluate the maximal number of stem cells to be transplanted in one location, as the results suggest that there is a critical limit of cells able to survive in the adult brain. Survival is limited by availability of oxygen and nutrients but not the inflammatory response induced by the implantation.

Imaging Reporter Strategy to Monitor Gene Activation of Microglia Polarisation States under Stimulation.

Microglial cells as innate immune key players have a critical and unique role in neurodegenerative disorders. They strongly interact with their microenvironment in a complex manner and react to changes by switching their phenotype and functional activation states. In order to understand the development of brain diseases, it is imperative to elucidate up- or down-regulation of genes involved in microglia polarisation in time-profile by a simple-to-use strategy. Here, we present a new imaging strategy to follow promoter activity of genes involved in microglia polarisation. We lentivirally transduced BV-2 microglia cells in culture with constructs consisting of the induced nitric oxide synthase (iNOS), Fc gamma receptor III (Fcgr3) (both resembling the pro-inflammatory M1-like phenotype) or Chitinase-like 3 (Chil3/Ym1) (resembling the anti-inflammatory M2-like phenotype) promoters and stimulated transgenic cells with potent activators for pro- or anti-inflammatory response, such as lipopolysaccharide (LPS) + interferon gamma (IFN-γ) or interleukin (IL)-4, respectively. Promoter activities upon polarisation phases were quantitatively assessed by the two imaging reporters Luc2 for bioluminescence and eGFP for fluorescence.

Posttranscriptional Regulation of LOXL1 Expression Via Alternative Splicing and Nonsense-Mediated mRNA Decay as an Adaptive Stress Response.

Purpose: Alternative mRNA splicing coupled to nonsense-mediated decay (NMD) is a common mRNA surveillance pathway also known to dynamically modulate gene expression in response to cellular stress. Here, we investigated the involvement of this pathway in the regulation of lysyl oxidase-like 1 (LOXL1) expression in response to pseudoexfoliation (PEX)-associated pathophysiologic factors. Methods: Transcript levels of LOXL1 isoforms were determined in ocular tissues obtained from donor eyes without and with PEX syndrome. Pseudoexfoliation-relevant cell types, including human Tenon's capsule fibroblasts (hTCF) and trabecular meshwork cells (hTMC), were exposed to puromycin, caffeine, TGF-ß1, homocysteine, IL-6, retinoic acid, UV-B radiation, oxidative stress, and mechanical stress for up to 48 hours. Western blot analysis was carried out using antibodies against LOXL1, (phosphorylated-) eukaryotic initiation factor 2-α (eIF2-α), and regulator of nonsense transcripts 2 (UPF2). RNA interference was used to knockdown UPF1-3 and Serine/threonine-protein kinase (SMG1). Results: Constitutive expression of wild-type LOXL1 and alternatively spliced LOXL1-a transcripts was detected in all ocular tissues showing highest levels in trabecular meshwork and differential expression between PEX and control specimens. LOXL1-a transcripts were upregulated in hTCF and hTMC by NMD inhibitors puromycin and caffeine (≥6-fold; P < 0.01) or after knockdown of NMD core factors (≥2-fold; P < 0.05), whereas mRNA and protein levels of LOXL1 were reduced (≤0.8 fold; P < 0.05). Exposure of cells to various PEX-associated (stress) factors, including TGF-ß1, UV-B light, oxidative stress, mechanical stress, and retinoic acid enhanced LOXL1-a transcript levels (≥1.5-fold; P < 0.05), while partially downregulating LOXL1 levels (≤0.7-fold; P < 0.05). Stress-induced inhibition of NMD was dependent on phosphorylation of eIF2α. Conclusions: These findings provide evidence for a functional role of alternative splicing coupled to NMD in the posttranscriptional regulation of LOXL1 gene expression and suggest this mechanism to represent a dynamic mode of adapting LOXL1 expression to PEX-associated environmental and nutritional cues.

In Vivo Non-Invasive Tracking of Macrophage Recruitment to Experimental Stroke.

Brain-infiltrating monocyte-derived macrophages are one of the key players in the local immune response after stroke. It is now widely accepted that the inflammatory response is not an exclusively destructive process. However, the underlying molecular mechanisms needed for proper regulation still remain to be elucidated. Here, we propose an in vitro labelling strategy for multimodal in vivo observation of macrophage dynamics distinguished from brain-residing microglia response. Prior to intracerebral transplantation into the striatum of recipient mice or systemic administration, monocytes and macrophages, isolated from luciferase-expressing mice, were labelled with superparamagnetic iron oxide particles. Temporo-spatial localization was monitored by magnetic resonance imaging, whereas survival of grafted cells was investigated using bioluminescence imaging. The labelling procedure of the isolated cells did not significantly influence cell characteristics and resulted in detection of as few as 500 labelled cells in vivo. Two weeks after stereotactic transplantation, the luciferase signal was sustained traceable, with approximately 18% of the original luciferase signal detectable for monocytes and about 30% for macrophages. Hypointensity in MRI of the graft appeared unaltered in spatial location. In a therapeutically relevant approach, systemic cell administration after stroke resulted in accumulation mostly in thoracic regions, as could be visualized with BLI. For detection of homing to ischemic brain tissue more cells need to be administered. Nevertheless, during parallel MRI sessions recruitment of i.v. injected cells to the lesion site could be detected by day 2 post stroke as scattered hypointense signal voids. With further increase in sensitivity, our multi-facetted labelling strategy will provide the basis for in vivo tracking and fate specification of tissue-infiltrating macrophages and their distinct role in stroke-related neuro-inflammation.

Human neural stem cell intracerebral grafts show spontaneous early neuronal differentiation after several weeks.

Human neural stem cells (hNSCs) hold great promise for the treatment of neurological diseases. Considerable progress has been made to induce neural differentiation in the cell culture in vitro and upon transplantation in vivo [2] in order to explore restoration of damaged neuronal circuits. However, in vivo conventional strategies are limited to post mortem analysis. Here, we apply our developed first fate mapping platform to monitor neuronal differentiation in vivo by magnetic resonance imaging, bioluminescence imaging, and fluorescence imaging. Ferritin, Luciferase and GFP under neuronal-specific promoters for immature and mature neurons, respectively, were used to generate transgenic hNSCs. Differentiation-linked imaging reporter expression was validated in vitro. The time profile of spontaneous neuronal maturation after transplantation into mouse brain cortex demonstrated early neuronal differentiation within 6 weeks. Fully mature neurons expressing synaptogenesis were observed only after three months or longer. Our trimodal fate mapping strategy represents a unique non-invasive tool to monitor the time course of neuronal differentiation of transplanted stem cells in vivo.

Novel bimodal iron oxide particles for efficient tracking of human neural stem cells in vivo.

AIMS: We validated novel bimodal iron oxide particles as substitute of ferumoxides for efficient labeling of human neural stem cells (NSCs). The dextrane-coated FeraTrack Direct (FTD)-Vio particles have additional far-red fluorophores for microscopic cell analysis. METHODS: MR relaxometry, spectrophotometric iron determination and microscopy are used for characterization in vitro and in vivo. RESULTS: Efficient uptake is not transfection agent-dependent. FTD-Vio594 labeling had no influence on viability, proliferation, migration and differentiation capacity. It allows MRI-based tracking of engrafted NSCs in mouse brain up to 11 days, complemented by bioluminescence imaging of firefly luciferase expressed by the engrafted cells. CONCLUSION: Our results highlight the FTD-Vio594 particles as safe and sensitive substitute of ferumoxides for longitudinal tracking of NSCs in preclinical studies.

The Leber congenital amaurosis protein AIPL1 and EB proteins co-localize at the photoreceptor cilium.

PURPOSE: The aim of this study was to investigate the interaction and co-localization of novel interacting proteins with the Leber congenital amaurosis (LCA) associated protein aryl hydrocarbon receptor interacting protein-like 1 (AIPL1). METHODS: The CytoTrapXR yeast two-hybrid system was used to screen a bovine retinal cDNA library. A novel interaction between AIPL1 and members of the family of EB proteins was confirmed by directed yeast two-hybrid analysis and co-immunoprecipitation assays. The localization of AIPL1 and the EB proteins in cultured cells and in retinal cryosections was examined by immunofluorescence microscopy and cryo-immunogold electron microscopy. RESULTS: Yeast two-hybrid (Y2H) analysis identified the interaction between AIPL1 and the EB proteins, EB1 and EB3. EB1 and EB3 were specifically co-immunoprecipitated with AIPL1 from SK-N-SH neuroblastoma cells. In directed 1:1 Y2H analysis, the interaction of EB1 with AIPL1 harbouring the LCA-causing mutations A197P, C239R and W278X was severely compromised. Immunofluorescent confocal microscopy revealed that AIPL1 did not co-localize with endogenous EB1 at the tips of microtubules, endogenous EB1 at the microtubule organising centre following disruption of the microtubule network, or with endogenous ß-tubulin. Moreover, AIPL1 did not localize to primary cilia in ARPE-19 cells, whereas EB1 co-localized with the centrosomal marker pericentrin at the base of primary cilia. However, both AIPL1 and the EB proteins, EB1 and EB3, co-localized with centrin-3 in the connecting cilium of photoreceptor cells. Cryo-immunogold electron microscopy confirmed the co-localization of AIPL1 and EB1 in the connecting cilia in human retinal photoreceptors. CONCLUSIONS: AIPL1 and the EB proteins, EB1 and EB3, localize at the connecting cilia of retinal photoreceptor cells, but do not co-localize in the cellular microtubule network or in primary cilia in non-retinal cells. These findings suggest that AIPL1 function in these cells is not related to the role of EB proteins in microtubule dynamics or primary ciliogenesis, but that their association may be related to a specific role in the specialized cilia apparatus of retinal photoreceptors.

Implantation of a new accommodative posterior chamber intraocular lens.

PURPOSE: A new, potentially accommodative posterior chamber lens (PCIOL) was designed based on principles elaborated by Hanna using finite element computer simulation methods. We report 3-month postoperative results in patients. METHODS: In a prospective study, 12 eyes of 12 patients (age 45 to 87 yr) underwent phacoemulsification for cataracts and PCIOL implantation. The PCIOL, 1 CU, has haptics designed for anterior optic movement following ciliary muscle contraction. Patients were examined postoperatively after 1 and 2 days, 1, 2 and 6 weeks, and 3 months, and results were compared with a control group of 12 eyes that received standard PMMA or acrylic PCIOLs. RESULTS: Surgery was uncomplicated and all PCIOLs were well-tolerated and stable with good centration in the capsular bag. The results were (mean +/- SD [range] and median; 1 CU versus control PCIOL): near visual acuity (Birkhäuser reading chart at 35 cm) with best distance correction 0.34 +/- 0.17 (0.2 to 0.6), 0.3 (J10-J1, median J7) versus 0.15 +/- 0.07 (0.1 to 0.3), 0.15 (J16-J7, median J13), P=.001; subjective near point 59 +/- 10 cm (40 to 100 cm), 53.5 cm versus 93 +/- 20 cm (64 to 128 cm), 86 cm, P=.004; retinoscopic accommodative range 1.2 +/- 0.4 D (0.63 to 1.5 D), 1.2 D versus 0.2 +/- 0.19 D (-0.25 to 0.5 D), 0.25 D, P < .001; decrease of anterior chamber depth after 2% pilocarpine 0.63 +/- 0.16 mm (0.40 to 0.91 mm), 0.63 mm versus 0.15 +/- 0.05 mm (0.08 to 0.20 mm), 0.17 mm, P < .001. CONCLUSIONS: The new PCIOL appears to be safe at short to medium term. Our results indicate pseudophakic accommodation secondary to focus shift with this PCIOL. Additional larger and long-term studies are necessary for exact evaluation of safety and accommodative power of this new PCIOL.

Measurement of accommodation after implantation of an accommodating posterior chamber intraocular lens.

PURPOSE: To analyze techniques of measuring accommodation after implantation of an accommodating posterior chamber intraocular lens (PC IOL). SETTING: Department of Ophthalmology and University Eye Hospital, University Erlangen-Nürnberg, Erlangen, Germany. METHODS: This prospective study analyzed 23 eyes of 23 patients (aged 41 to 87 years) after cataract surgery and PC IOL implantation (1 CU, HumanOptics) 4 weeks and 3 and 6 months after surgery. The results were compared to those in an age-matched control group (n = 20) 6 months after surgery. The following methods were used to measure accommodation: dynamic with objective techniques (PlusOptix PowerRefractor videorefractometry, streak retinoscopy) and subjective techniques (subjective near point [push-up test, accommodometer], defocusing); static with pharmacologic stimulation after pilocarpine 2% eyedrops directly (conventional refractometry); indirectly (change in the anterior chamber depth [ACD] with Zeiss IOLMaster). RESULTS: Results at 6 months, given as mean +/- SD (range), in the study and control groups, respectively, were as follows: near visual acuity (Birkhäuser reading charts at 35 cm) with distance correction, 0.32 +/- 0.11 (0.20 to 0.60) and 0.14 +/- 0.10 (0.05 to 0.30); accommodation amplitude (diopters) by PowerRefractor, 1.00 +/- 0.44 (0.75 to 2.13) and 0.35 +/- 0.26 (0.10 to 0.65), by retinoscopy, 0.99 +/- 0.48 (0.13 to 2.00) and 0.24 +/- 0.21 (-0.13 to +0.75), by subjective near point, 1.60 +/- 0.55 (0.50 to 2.56) and 0.42 +/- 0.25 (0.00 to 0.75), and by defocusing, 1.46 +/- 0.53 (1.00 to -2.50) and 0.55 +/- 0.33 (0.25 to 0.87). The mean ACD decrease (mm) was 0.78 +/- 0.12 (0.49 to 1.91) and 0.16 +/- 0.09 (0.00 to 0.34) after pilocarpine 2% eyedrops, indicating a mean accommodation of 1.40 D and 0.29 D, respectively, based on Gullstrand's model eye (P =.001). The lowest fluctuation between follow-ups was with the subjective near point and the defocusing techniques followed by ACD decrease with the IOLMaster. CONCLUSIONS: Accommodation after implantation of an accommodating PC IOL should be assessed with several techniques, including subjective and objective, to differentiate true pseudophakic accommodation from pseudoaccommodation. Researchers should be aware of the different variability and consistency of measurements with each technique over time.

Annular crystalline keratopathy in association with immunoglobulin therapy for pyoderma gangrenosum.

PURPOSE: To report on a patient with a clinical presentation of annular crystalline keratopathy after immunoglobulin therapy for pyoderma gangrenosum. METHODS: Case report of a 6-year-old boy with biopsy-proven history of pyoderma gangrenosum who had undergone several cycles of systemic immunoglobulin therapy. The literature on ocular manifestations associated with pyoderma gangrenosum was reviewed. RESULTS: One year after the last cycle, the patient complained of the sudden onset of photophobia. Slit-lamp biomicroscopy revealed bilateral symmetric crystalline deposits in an annular region of the cornea. Because visual acuity was not reduced, specific therapy was not initiated. Symptoms could be reduced by the prescription of medical edged filter lenses. CONCLUSIONS: Annular crystalline corneal deposits may be associated with immunoglobulin therapy or represent a hitherto unknown ocular complication of pyoderma gangrenosum.

In vivo bioluminescence imaging of vascular remodeling after stroke.

Thrombolysis remains the only beneficial therapy for ischemic stroke, but is restricted to a short therapeutic window following the infarct. Currently research is focusing on spontaneous regenerative processes during the sub-acute and chronic phase. Angiogenesis, the formation of new blood vessels from pre-existing ones, was observed in stroke patients, correlates with longer survival and positively affects the formation of new neurons. Angiogenesis takes place in the border zones of the infarct, but further insight into the temporal profile is needed to fully apprehend its therapeutic potential and its relevance for neurogenesis and functional recovery. Angiogenesis is a multistep process, involving extracellular matrix degradation, endothelial cell proliferation, and, finally, new vessel formation. Interaction between vascular endothelial growth factor and its receptor 2 (VEGFR2) plays a central role in these angiogenic signaling cascades. In the present study we investigated non-invasively the dynamics of VEGFR2 expression following cerebral ischemia in a mouse model of middle cerebral artery occlusion (MCAO). We used a transgenic mouse expressing firefly luciferase under the control of the VEGFR2 promotor to non-invasively elucidate the temporal profile of VEGFR2 expression after stroke as a biomarker for VEGF/VEGFR2 signaling. We measured each animal repetitively up to 2 weeks after stroke and found increased VEGFR2 expression starting 3 days after the insult with peak values at 7 days. These were paralleled by increased VEGFR2 protein levels and increased vascular volume in peri-infarct areas at 14 days after the infarct, indicating that signaling via VEGFR2 leads to successful vascular remodeling. This study describes VEGFR2-related signaling is active at least up to 2 weeks after the infarct and results in increased vascular volume. Further, this study presents a novel strategy for the non-invasive evaluation of angiogenesis-based therapies.