The importance of the epithelial fibre cell interface to lens regeneration in an in vivo rat model and in a human bag-in-the-lens (BiL) sample.

Human lens regeneration and the Bag-in-the-Lens (BIL) surgical treatment for cataract both depend upon lens capsule closure for their success. Our studies suggest that the first three days after surgery are critical to their long-term outcomes. Using a rat model of lens regeneration, we evidenced lens epithelial cell (LEC) proliferation increased some 50 fold in the first day before rapidly declining to rates observed in the germinative zone of the contra-lateral, un-operated lens. Cell multi-layering at the lens equator occurred on days 1 and 2, but then reorganised into two discrete layers by day 3. E- and N-cadherin expression preceded cell polarity being re-established during the first week. Aquaporin 0 (AQP0) was first detected in the elongated cells at the lens equator at day 7. Cells at the capsulotomy site, however, behaved very differently expressing the epithelial mesenchymal transition (EMT) markers fibronectin and alpha-smooth muscle actin (SMA) from day 3 onwards. The physical interaction between the apical surfaces of the anterior and posterior LECs from day 3 after surgery preceded cell elongation. In the human BIL sample fibre cell formation was confirmed by both histological and proteome analyses, but the cellular response is less ordered and variable culminating in Soemmerring's ring (SR) formation and sometimes Elschnig's pearls. This we evidence for lenses from a single patient. No bow region or recognisable epithelial-fibre cell interface (EFI) was evident and consequently the fibre cells were disorganised. We conclude that lens cells require spatial and cellular cues to initiate, sustain and produce an optically functional tissue in addition to capsule integrity and the EFI.

Pterygium Pathology: A Prospective Case-Control Study on Tear Film Cytokine Levels.

Pterygium is a common eye disease, linked to an increased exposure to UV radiation and dry environments. The associated pathology culminates in visual impairment and, in some rare cases, blindness. However, there remains a lot of uncertainty concerning the pathogenesis of this fibrovascular lesion. As the composition of the tear film provides a reflection into the pathological changes at the ocular surface, tear analysis represents an ideal approach to gain insight in the progression of disease following pterygiectomy. This study enrolled 19 patients and age/gender-matched healthy controls. Tear film levels of interleukin- (IL-) 6, IL-8, and vascular endothelial growth factor (VEGF) were investigated over time, and preoperative concentrations were linked to corneal neovascularization and pterygium size. Diminished tear film levels were found in unilateral patients who show no clinical signs of pterygium recurrence over a period of one year. Hence, our results highlight the potential of using the course of IL-6, IL-8, and VEGF levels in tears as biomarkers for recovery. In addition, when focusing on the affected eyes (i.e., primary and recurrent pterygium), we detected fold changes in preoperative cytokine concentrations to correspond with disease severity. As our proposed biomarkers did not reveal a linear relationship with corneal neovascularization nor the invasive behaviour of pterygium, no exact role in the pterygium pathology could be established. Hence, our data point to these factors being contributors rather than decisive players in the pathological processes.

Disrupted function and axonal distribution of mutant tyrosyl-tRNA synthetase in dominant intermediate Charcot-Marie-Tooth neuropathy.

Charcot-Marie-Tooth (CMT) neuropathies are common disorders of the peripheral nervous system caused by demyelination or axonal degeneration, or a combination of both features. We previously assigned the locus for autosomal dominant intermediate CMT neuropathy type C (DI-CMTC) to chromosome 1p34-p35. Here we identify two heterozygous missense mutations (G41R and E196K) and one de novo deletion (153-156delVKQV) in tyrosyl-tRNA synthetase (YARS) in three unrelated families affected with DI-CMTC. Biochemical experiments and genetic complementation in yeast show partial loss of aminoacylation activity of the mutant proteins, and mutations in YARS, or in its yeast ortholog TYS1, reduce yeast growth. YARS localizes to axonal termini in differentiating primary motor neuron and neuroblastoma cultures. This specific distribution is significantly reduced in cells expressing mutant YARS proteins. YARS is the second aminoacyl-tRNA synthetase found to be involved in CMT, thereby linking protein-synthesizing complexes with neurodegeneration.

Hot-spot residue in small heat-shock protein 22 causes distal motor neuropathy.

Distal hereditary motor neuropathies are pure motor disorders of the peripheral nervous system resulting in severe atrophy and wasting of distal limb muscles. In two pedigrees with distal hereditary motor neuropathy type II linked to chromosome 12q24.3, we identified the same mutation (K141N) in small heat-shock 22-kDa protein 8 (encoded by HSPB8; also called HSP22). We found a second mutation (K141E) in two smaller families. Both mutations target the same amino acid, which is essential to the structural and functional integrity of the small heat-shock protein alphaA-crystallin. This positively charged residue, when mutated in other small heat-shock proteins, results in various human disorders. Coimmunoprecipitation experiments showed greater binding of both HSPB8 mutants to the interacting partner HSPB1. Expression of mutant HSPB8 in cultured cells promoted formation of intracellular aggregates. Our findings provide further evidence that mutations in heat-shock proteins have an important role in neurodegenerative disorders.

Belgian Endothelial Surgical Transplant of the Cornea (BEST cornea) protocol: clinical and patient-reported outcomes of Ultra-Thin Descemet Stripping Automated Endothelial Keratoplasty (UT-DSAEK) versus Descemet Membrane Endothelial Keratoplasty (DMEK) - a multicentric, randomised, parallel group pragmatic trial in corneal endothelial decompensation.

OBJECTIVES: Corneal blindness is the third most frequent cause of blindness globally. Damage to the corneal endothelium is a leading indication for corneal transplantation, which is typically performed by lamellar endothelial keratoplasty. There are two conventional surgical techniques: Ultra-Thin Descemet Stripping Automated Endothelial Keratoplasty (UT-DSAEK) and Descemet Membrane Endothelial Keratoplasty (DMEK). The purpose of this study is to compare both techniques. METHODS AND ANALYSIS: The trial compares UT-DSAEK and DMEK in terms of clinical and patient reported outcomes using a pragmatic, parallel, multicentric, randomised controlled trial with 1:1 allocation with a sample size of 220 participants across 11 surgical centres. The primary outcome is the change in best-corrected visual acuity at 12 months. Secondary outcomes include corrected and uncorrected vision, refraction, proportion of high vision, quality of life (EQ-5D-5L and VFQ25), endothelial cell counts and corneal thickness at 3, 6 and 12 months follow-up appointments. Adverse events will also be compared 12 months postoperatively. ETHICS AND DISSEMINATION: The protocol was reviewed by ethical committees of 11 participating centres with the sponsor centre issuing the final definitive approval. The results will be disseminated at clinical conferences, by patient partner groups and open access in peer-reviewed journals. GOVERNANCE OF THE TRIAL: Both, trial management group and trial steering committee, are installed with representatives of all stakeholders involved including surgeons, corneal bankers, patients and external experts. TRIAL REGISTRATION NUMBER: NCT05436665.

Proteomic analysis of posterior capsular plaques in congenital unilateral cataract.

PURPOSE: To obtain insights on the protein composition of posterior capsular plaques (PCP) in congenital unilateral cataract with anterior vitreolenticular interface dysgenesis (AVLID). METHODS: Posterior capsular plaque's were collected during surgery in children presenting with congenital unilateral cataract. Surgeries were analysed focusing on the type of cataract, the integrity of the posterior capsule after peeling the PCP and the presence of vitreolenticular adherences when performing primary posterior capsulorhexis. Proteome analysis was performed on the collected PCPs. RESULTS: Posterior capsular plaques collection and proteome analysis were feasible from four children presenting with unilateral idiopathic congenital cataract and AVLID. A large portion of the proteins found in the PCPs was similar to the proteins known to be present in lens epithelial cells and fibres. Proteins like vimentin, fibronectin, collagen type I, collagen type VI and lumican were also found, which typically are present in mesenchymal tissue but not in lens tissue or capsule. CONCLUSION: Posterior capsular plaques in cases of unilateral idiopathic congenital cataract of the AVLID type present a protein composition of mainly proteins found in lens epithelial cells and fibres. Some proteins however are a specific for lens tissue and are typically seen in mesenchymal tissue.

Xeno-Free Cultivation of Mesenchymal Stem Cells From the Corneal Stroma.

Purpose: The human cornea has recently been described as a source of corneal stroma-derived mesenchymal stem cells (hMSCs). In vitro expansion of these cells involves basal medium supplemented with fetal bovine serum (FBS). As animal-derived serum can confer a risk of disease transmission and can be subject to considerable lot-to-lot variability, it does not comply with newer Good Manufacturing Practice (GMP)-required animal component-free culture protocols for clinical translation. Methods: This study investigated animal-free alternatives to FBS for cultivation of human corneal stromal MSCs. Proliferative capacity was studied for cultures supplemented with different concentrations (2.5%, 5%, and 10%) of FBS, human AB serum, human platelet lysate (HPL), and XerumFree. Unsupplemented basal medium was used as a control. The expression of specific hMSC markers (CD73+, CD90+, CD105+, CD19-, CD34-, CD79α-, CD11b-, CD14-, CD45-, and HLA-DR-) and trilineage differentiation (adipogenesis, osteogenesis, and chondrogenesis) were compared for the two outperforming supplements: 10% FBS and HPL. Results: HPL is the only consistent non-xeno supplement where hMSC cultures show significantly higher proliferation than the 10% FBS-supplemented cultures. Both FBS- and HPL-supplemented hMSC cultures showed plastic adherence and trilineage differentiation, and no significant differences were found in the expression of the hMSC marker panel. No significant differences in stemness were detected between FBS and HPL cultures. Conclusions: We conclude that HPL is the best supplement for expansion of human corneal stromal MSCs. HPL significantly outperforms human AB serum, the chemically defined XerumFree, and even the gold standard, FBS. The xeno-free nature of HPL additionally confers preferred standing for use in GMP-regulated clinical trials using human corneal stromal MSCs.

A method for quantifying limbal stem cell niches using OCT imaging.

AIMS: To evaluate the efficacy of Fourier domain-optical coherence tomography (FD-OCT) in imaging and quantifying the limbal palisades of Vogt and to correlate these images with histological findings. METHODS: The superior and inferior limbal region of both eyes of 50 healthy volunteers were imaged by FD-OCT. Images were processed and analysed using Matlab software. In vitro immunofluorescent staining of a cadaveric donor limbus was analysed to correlate the presence of stem cells in the visualised structures. RESULTS: FD-OCT could successfully visualise limbal crypts and the palisades of Vogt in the limbus region. Fluorescent labelling confirmed the presence of stem cells in these structures. The mean palisade ridge width (ΔPR) and the mean interpalisade epithelial rete peg width (ΔERP) were both of the order of 72 µm, leading to a palisade density (PD) of about 7.4 palisades/mm. A significant difference in ΔPR, ΔERP and PD was seen between the inferior and superior sides of the right eye and the superior sides of the left and right eye(p<0.05.). A significant influence of iris colour on parameters ΔPR, ΔERP and PD was found, and of age on PD and ΔERP (p<0.05). CONCLUSIONS: In vivo OCT imaging is a safe and effective modality to image the limbus and can be used to visualise the palisades of Vogt. Image processing using Matlab software enabled quantification and density calculation of imaged limbal palisades of Vogt. This technique may enhance targeted limbal biopsies for transplantation.

Novel frameshift and splice site mutations in the neurotrophic tyrosine kinase receptor type 1 gene (NTRK1) associated with hereditary sensory neuropathy type IV.

Congenital insensitivity to pain with anhidrosis or hereditary sensory and autonomic neuropathy type IV (HSAN IV) is the first human genetic disorder implicated in the neurotrophin signal transduction pathway. HSAN IV is characterized by absence of reaction to noxious stimuli, recurrent episodes of fever, anhidrosis, self-mutilating behavior and often mental retardation. Mutations in the neurotrophic tyrosine kinase, receptor, type 1 (NTRK1) are associated with this disorder. Here we report four homozygous mutations, two frameshift (p.Gln626fsX6 and p.Gly181fsX58), one missense (p.Arg761Trp) and one splice site (c.359+5G>T) mutation in four HSAN IV patients. The splice site mutation caused skipping of exons 2 and 3 in patient's mRNA resulting in an in-frame deletion of the second leucine-rich motif. NTRK1 mutations are only rarely reported in the European population. This report extends the spectrum of NTRK1 mutations observed in patients diagnosed with HSAN IV.

Immunohistochemical characteristics of the vitreolenticular interface in congenital unilateral posterior cataract.

PURPOSE: To gain insight into the histology of the vitreolenticular interface in congenital unilateral posterior cataract. SETTING: Antwerp University Hospital, Department of Ophthalmology, Edegem, and the University of Antwerp, Faculty of Medicine and Health Sciences, Antwerp, Belgium. DESIGN: Prospective case study. METHODS: Samples of the posterior lens capsule of patients with congenital posterior cataract (including opaque plaque on the anterior and adhesion to the vitreous on the posterior surface) were collected during the posterior capsulorhexis procedure. Staining for collagen types II and IV was performed using indirect immunohistochemistry. Results were compared with those of control posterior lens capsules of 3 children and 3 adults. RESULTS: Samples were collected from 3 patients. All posterior lens capsules contained collagen type IV. Samples from congenital posterior cataract patients all showed a narrow band of collagen type II on the outer surface, indicating strong adherence of the anterior hyaloid membrane to the center of the posterior lens capsule. Surprisingly, collagen type II was also found in the posterior capsule plaques. Collagen type II was not found in any control posterior lens capsule. CONCLUSION: The adherence of collagen type II to the center of the posterior lens capsule histologically supports the hypothesis that this subgroup of congenital cataract hints at an abnormality at the vitreolenticular interface. FINANCIAL DISCLOSURE: None of the authors has a financial or proprietary interest in any material or method mentioned.

Limbal Stem Cell Deficiency: Current Treatment Options and Emerging Therapies.

Severe ocular surface disease can result in limbal stem cell deficiency (LSCD), a condition leading to decreased visual acuity, photophobia, and ocular pain. To restore the ocular surface in advanced stem cell deficient corneas, an autologous or allogenic limbal stem cell transplantation is performed. In recent years, the risk of secondary LSCD due to removal of large limbal grafts has been significantly reduced by the optimization of cultivated limbal epithelial transplantation (CLET). Despite the great successes of CLET, there still is room for improvement as overall success rate is 70% and visual acuity often remains suboptimal after successful transplantation. Simple limbal epithelial transplantation reports higher success rates but has not been performed in as many patients yet. This review focuses on limbal epithelial stem cells and the pathophysiology of LSCD. State-of-the-art therapeutic management of LSCD is described, and new and evolving techniques in ocular surface regeneration are being discussed, in particular, advantages and disadvantages of alternative cell scaffolds and cell sources for cell based ocular surface reconstruction.

The phenotype of motor neuropathies associated with BSCL2 mutations is broader than Silver syndrome and distal HMN type V.

Silver syndrome is a rare autosomal dominant neurodegenerative disorder characterized by marked amyotrophy and weakness of small hand muscles and spasticity in the lower limbs. The locus for Silver syndrome (SPG17) was assigned to a 13 cM region on chromosome 11q12-q14 in a single large pedigree. We recently found heterozygous mutations in the Berardinelli-Seip congenital lipodystrophy (BSCL2, seipin) gene causing SPG17 and distal hereditary motor neuropathy type V (distal HMN V). Here we report the clinical features of two families with heterozygous BSCL2 mutations. Interestingly, both families show a clinical phenotype different from classical Silver syndrome, and in some patients the phenotype is also different from distal HMN V. Patients in the first family had marked spasticity in the lower limbs and very striking distal amyotrophy that always started in the legs. Patients in the second family had distal amyotrophy sometimes starting and predominating in the legs, but no pyramidal tract signs. These observations broaden the clinical phenotype of disorders associated with BSCL2 mutations, having consequences for molecular genetic testing.

Mutations in the small GTP-ase late endosomal protein RAB7 cause Charcot-Marie-Tooth type 2B neuropathy.

Charcot-Marie-Tooth type 2B (CMT2B) is clinically characterized by marked distal muscle weakness and wasting and a high frequency of foot ulcers, infections, and amputations of the toes because of recurrent infections. CMT2B maps to chromosome 3q13-q22. We refined the CMT2B locus to a 2.5-cM region and report two missense mutations (Leu129Phe and Val162Met) in the small GTP-ase late endosomal protein RAB7 which causes the CMT2B phenotype in three extended families and in three patients with a positive family history. The alignment of RAB7 orthologs shows that both missense mutations target highly conserved amino acid residues. RAB7 is ubiquitously expressed, and we found expression in sensory and motor neurons.

Slowed conduction and thin myelination of peripheral nerves associated with mutant rho Guanine-nucleotide exchange factor 10.

Slowed nerve-conduction velocities (NCVs) are a biological endophenotype in the majority of the hereditary motor and sensory neuropathies (HMSN). Here, we identified a family with autosomal dominant segregation of slowed NCVs without the clinical phenotype of HMSN. Peripheral-nerve biopsy showed predominantly thinly myelinated axons. We identified a locus at 8p23 and a Thr109Ile mutation in ARHGEF10, encoding a guanine-nucleotide exchange factor (GEF) for the Rho family of GTPase proteins (RhoGTPases). Rho GEFs are implicated in neural morphogenesis and connectivity and regulate the activity of small RhoGTPases by catalyzing the exchange of bound GDP by GTP. Expression analysis of ARHGEF10, by use of its mouse orthologue Gef10, showed that it is highly expressed in the peripheral nervous system. Our data support a role for ARHGEF10 in developmental myelination of peripheral nerves.