Mycosis fungoides-derived exosomes promote cell motility and are enriched with microRNA-155 and microRNA-1246, and their plasma-cell-free expression may serve as a potential biomarker for disease burden.

BACKGROUND: Literature regarding exosomes as mediators in intercellular communication to promote progression in mycosis fungoides (MF) is lacking. OBJECTIVES: To characterize MF-derived exosomes and their involvement in the disease. METHODS: Exosomes were isolated by ultracentrifugation from cutaneous T-cell lymphoma (CTCL) cell lines, and from plasma of patients with MF and controls (healthy individuals). Exosomes were confirmed by electron microscopy, NanoSight and CD81 staining. Cell-line exosomes were profiled for microRNA array. Exosomal microRNA (exomiRNA) expression and uptake, and plasma-cell-free microRNA (cfmiRNA) were analysed by reverse-transcriptase quantitative polymerase chain reaction. Exosome uptake was monitored by fluorescent labelling and CD81 immunostaining. Migration was analysed by transwell migration assay. RESULTS: MyLa- and MJ-derived exosomes had a distinctive microRNA signature with abundant microRNA (miR)-155 and miR-1246. Both microRNAs were delivered into target cells, but only exomiR-155 was tested, demonstrating a migratory effect on target cells. Plasma levels of cfmiR-1246 were significantly highest in combined plaque/tumour MF, followed by patch MF, and were lowest in controls (plaque/tumour > patch > healthy), while cfmiR-155 was upregulated only in plaque/tumour MF vs. controls. Specifically, exomiR-1246 (and not exomiR-155) was higher in plasma of plaque/tumour MF than in healthy controls. Plasma exosomes from MF but not from controls increased cell migration. CONCLUSIONS: Our findings show that MF-derived exosomes promote cell motility and are enriched with miR-155, a well-known microRNA in MF, and miR-1246, not previously reported in MF. Based on their plasma expression we suggest that they may serve as potential biomarkers for tumour burden.

Unilesional mycosis fungoides is associated with increased expression of microRNA-17~92 and T helper 1 skewing.

BACKGROUND: The molecular basis of unilesional mycosis fungoides (MF), characterized by a solitary lesion that is clinicopathologically indistinguishable from multifocal patch or plaque MF (early MF), is unknown. OBJECTIVES: To investigate the microRNA profile in unilesional MF distinguishing it from early MF. METHODS: Biopsy samples of unilesional MF and early MF were evaluated with the Affymetrix microRNA array, with further comparison with inflammatory dermatosis, using quantitative polymerase chain reaction. NanoString technology was applied to analyse the gene expression of T helper (Th)1 immune markers, and immunohistochemistry was used to evaluate CXCR3 and GATA-binding protein 3 (GATA3) markers for Th1 and Th2 cells, respectively. RESULTS: Unilesional MF had a significantly higher level of expression of all members of the microRNA miR-17~92 cluster than early MF. Specifically, unilesional MF had a higher miR-17 level than early MF and inflammatory dermatoses. There was downregulation of the expression of phosphatase and tensin homolog (PTEN) and CREB1, known targets of miR-17~92 members; higher gene expression of interleukin-2 and interferon-γ; and a statistically lower average percentage of GATA3+ dermal cells (6·7% vs. 42·3%), were detected in unilesional MF compared with early MF. High immunoreactivity of CXCR3 was noted in both unilesional and early MF. CONCLUSIONS: Unilesional MF exhibits a microRNA profile distinct from that of conventional early MF, with a higher level of miR-17~92 members along with Th1 skewing. These findings suggest a robust reactive T-cell immune response in unilesional MF and might account for the localized nature of this disease.

Oncogenic role of microRNA-155 in mycosis fungoides: an in vitro and xenograft mouse model study.

BACKGROUND: MicroRNA (miR)-155 contributes to the proliferation of mycosis fungoides (MF) in vitro and is upregulated in tumours of MF compared with early MF lesions. OBJECTIVES: To investigate the contribution of miR-155 to the cancerous phenotype and drug resistance of MF/Sézary cell lines. METHODS: miR-155 was inhibited in MF cell lines (MyLa and MJ) by transduction of miRZip anti-miR-155, and overexpressed in Hut78 cells by transduction of miRVec-miR-155; empty plasmids served as controls. Cells were analysed for response to inducers of apoptosis and cell-cycle arrest, using fluorescence-activated cell sorting. Transduced MyLa cells were subcutaneously injected into severe combined immunodeficient mice, and tumours were analysed immunohistochemically and for final size. RESULT: MyLa and MJ cells expressed a high level of miR-155; Hut78 cells expressed a low level. MF cell lines stably expressing miR-155 inhibitor showed increased G2/M arrest in response to N-p-tolyl-2-(3,4,5-trimethoxyphenyl quinazolin-4-amine) (SL111), an inducer of cell-cycle arrest, followed by increased apoptosis. Additionally, they showed increased apoptosis in response to suberoylanilide hydroxamic acid (SAHA). Tumours formed in mice from injected anti-miR-155-expressing MyLa cells had a significantly lower volume and higher occurrence of apoptosis than controls. Stable overexpression of miR-155 in Hut78 cells had no effect. CONCLUSIONS: Oncogenic miR-155 appears to contribute to the cancerous phenotype of MyLa and MJ cells, but not of Hut78 cells, by interrupting activation of the G2/M checkpoint in response to SL111, and decreasing apoptosis in response to SL111 and SAHA, thereby facilitating tumour growth. These findings have implications for the potential development of novel therapeutic modalities for MF incorporating miR-155 inhibitors.

Enhanced phosphorylation of p53 by ATM in response to DNA damage.

The ATM protein, encoded by the gene responsible for the human genetic disorder ataxia telangiectasia (A-T), regulates several cellular responses to DNA breaks. ATM shares a phosphoinositide 3-kinase-related domain with several proteins, some of them protein kinases. A wortmannin-sensitive protein kinase activity was associated with endogenous or recombinant ATM and was abolished by structural ATM mutations. In vitro substrates included the translation repressor PHAS-I and the p53 protein. ATM phosphorylated p53 in vitro on a single residue, serine-15, which is phosphorylated in vivo in response to DNA damage. This activity was markedly enhanced within minutes after treatment of cells with a radiomimetic drug; the total amount of ATM remained unchanged. Various damage-induced responses may be activated by enhancement of the protein kinase activity of ATM.

[Boston Keratoprosthesis with temporal aponeurosis graft: A solution when there seems to be no more]. / Kératoprothèse de Boston avec greffe d'aponévrose temporale : une solution quand il semble ne plus y en avoir.

PURPOSE: To report cases of patients with severe bilateral corneal blindness and recurrent refractory perforation to keratoplasty and conventional treatment, for whom Boston keratoprosthesis (KP) was a satisfactory alternative when combined with a temporalis aponeurosis graft. DESCRIPTION OF CASES: The first patient had progressive Lyell syndrome with spontaneous corneal perforation. The second had a severe graft vs. host reaction with a persistent Seidel-positive descemetocele. Despite repeated penetrating keratoplasties, amniotic membrane (AM) transplantations, and buccal mucosal (BM) grafts, they both experienced recurrent corneal perforation. The only solution thus appeared to be Boston Type I KP surgery. One month postoperatively, the first patient had to receive a temporalis aponeurosis (TA) graft, due to thinning of the recipient graft. Six months postoperatively, his visual acuity (VA) was 1/10 without correction, and the corneal status had been stabilized. The second patient underwent KP and TA graft concurrently. Six months after surgery, VA was 2/10 uncorrected, and the local inflammation had been stabilized. OBSERVATION: Boston type I keratoprostheses constitute an alternative in cases of severe bilateral corneal blindness with perforation refractory to conventional treatment and surgery, with satisfactory visual results. DISCUSSION: Patients with preoperative severe ocular surface disease are at greater risk of postoperative keratolysis. For our patients with a higher risk, TA graft prevented corneal melt. TA seems to be more effective than AM or BM in preventing corneal thinning or melt. CONCLUSION: We would recommend performing a TA graft in combination with Boston KP surgery concurrently as first line treatment in eyes with severe ocular surface inflammation.

[Ocular graft-versus-host disease: An often misdiagnosed etiology of dry eye syndrome]. / Réaction oculaire du greffon contre l'hôte : une étiologie de syndrome sec à ne pas méconnaître.

PURPOSE: To report a case of severe ocular graft-versus-host disease (GVHD) after cataract surgery. METHODS: Observational case report. RESULTS: We describe the case of a 59-year-old man with postoperative corneal ulcer on his only functional eye. His past history reported allogenic bone marrow transplant. His visual acuity (VA) was limited to hand motions. Slit lamp examination revealed diffuse conjunctival hyperemia, severe blepharitis, Meibomian dysfunction, total corneal opacification with epithelial and stromal keratitis and neovascular invasion. Because of the severe dry eye symptoms and history of allogenic hematological stem cell transplantation, ocular GVHD was diagnosed. Functional and anatomical improvement occurred rapidly with topical cyclosporine 2%, with improved VA after treatment. CONCLUSION: With any severe dry eye syndrome in the context of allogenic bone marrow transplant, ocular GVHD must be considered. For planned ocular surgery, we recommend adding cyclosporine 0.1% treatment before and after surgery to prevent severe ocular GVHD.

[Interest of optical coherence tomography performed immediately before intravitreal injection of anti-VEGF in exudative AMD]. / Intérêt de la tomographie à cohérence optique réalisée immédiatement avant injection intra-vitréenne d'anti-VEGF sur récidive exsudative dans la DMLA.

INTRODUCTION: Two or three systematic intravitreal injections (IVT) may be prescribed in a PRN approach to treat an exudative recurrence of neovascular age-related macular degeneration (AMD), according to the phenotype. Optical coherence tomography (OCT) may be performed immediately before the 2nd or the 3rd scheduled IVT, making it possible to cancel the procedure in the absence of exudation. The aim of the study was to evaluate the usefulness of this OCT examination and to assess the percentage of IVT cancelled, in order to evaluate a potential medico-economic benefit. METHODS: Monocentric retrospective study, in which were included 292 consecutive eyes with exudative recurrence of AMD, for which 2 or 3 IVT were scheduled between January 1st and April 30th, 2014. All patients received a first systematic IVT in the seven days following the diagnosis. Then, on the days of the 2nd and 3rd scheduled IVT, each patient had a visual acuity measurement and a Spectral domain-OCT (Spectralis, HRA Heidelberg Engineering). This measurement allowed for the IVT to be either performed as scheduled or cancelled. Both ranibizumab and aflibercept were used. A Chi(2) test was used to compare the qualitative variables and an adjusted Wilcoxon test for the quantitative values. RESULTS: Two hundred and ninety-two consecutive eyes were included; 172 in the "2 scheduled IVT" group (group A) and 120 in the "3 scheduled IVT" group (group B). At the first follow-up, 37.6% of scheduled IVT were cancelled after the OCT (44.1% in group A and 28.3% in group B). At the second follow-up, 33.3% of IVT were cancelled in group B. Overall, 150/412 (36.4%) IVT were avoided in this series. Presence of serous retinal detachment, retinal edema and increased central macular thickness were statistically correlated with confirmation of the scheduled IVT at the two follow-ups (P<0.001, P<0.001 and P=0.002, respectively). A savings of 429.80 € per patient was calculated during this short period of follow-up. CONCLUSION: An average non-injection rate of 36.4% of scheduled IVT was found in this protocol of management of recurrences with OCT performed the day of IVT. This protocol allowed to avoid unnecessary IVT one-third of the time and appeared highly cost-effective.

[Comparison of two different surgical treatments of presbyopia for hyperopic patients over 55 years old: Presbylasik (Supracor) and Prelex (presbyopic lens exchange)]. / Chirurgie de la presbytie chez l'hypermétrope de plus de 55ans : comparaison entre Presbylasik (Supracor) et Prelex (presbyopic lens exchange).

INTRODUCTION: The management of presbyopic patients is a medical, surgical and economic issue. We wondered which procedure, whether a Presbylasik technique called Regular Supracor or the intraocular surgery of clear lens replacement by a trifocal diffractive lens (presbyopic lens exchange [Prelex]), provided the best results (in visual acuity and satisfaction) for our hyperopic and presbyopic patients between 55 and 70. The aim of this study was to compare the safety and efficacy of the two techniques (at one week and three months) and to assess patients' quality of life. METHODS: This study is a retrospective monocentric clinical trial conducted between June 2011 and March 2014, on 21 hyperopic presbyopic patients (mean age 60.9 years), in the "hôpital d'instruction des armées-Percy" (Clamart); 13 patients underwent the corneal treatment of Presbylasik (with the Intralase FS60 femtosecond laser [AMO, USA] and the Technolas 217P excimer laser [Technolas Perfect Vision, GmbH] following the Zyoptix Tissue-Saving algorithm adjusted with a nomogram and the Regular Supracor mode), and 8 patients underwent clear lens extraction (Prelex), through bimanual phacoemulsification and implantation of diffractive trifocal intraocular lenses (Finevision Micro F, PhysIOL(*)). RESULTS: Mean uncorrected binocular distance and near vision visual acuity in the Supracor group were respectively 0.03 (-0.2-0.1) LogMar and 0.23 (0.06-0.36) one week postoperatively and 0.031 (-0.2-0.1) and 0,166 (0.06-0.36) three months postoperatively. In the Prelex group, the mean uncorrected one week binocular VA was 0.025 (0-0.1) for distance vision and 0.165 (0.06-0.18) for near distance and the three months visual acuity was 0 (-0.1-0.1) and 0.105 (0.06-0.18) for distance and near vision. All Prelex patients were spectacle-free at all distances, whereas 4 Supracor patients required spectacles for near vision postoperatively. Seven of 11 patients in the Supracor group and 100% of the Prelex patients were completely satisfied. Both groups experienced halos, but patients reported more halos in the Prelex group (75%). One eye required intraocular lens exchange and four eyes (16.7%) required a new corneal procedure in the Supracor group. CONCLUSION: Both surgeries are safe and effective modalities in the management of hyperopic and presbyopic patients. However, the Prelex procedure seems to be more appropriate for patients over 55 years of age.

[Results of fractionated targeted proton beam therapy in the treatment of primary optic nerve sheath meningioma]. / Résultats de la protonthérapie ciblée fractionnée dans le traitement des méningiomes primitifs de la gaine du nerf optique.

INTRODUCTION: Optic nerve sheath meningioma (ONSM) is a common benign neoplasm arising from the arachnoid tissue encapsulating the optic nerve and affects mainly middle aged women. It is a slow-growing tumor for which there is still no consensus on treatment. PURPOSE: To evaluate the safety and efficacy of fractionated targeted proton beam therapy (PBT) in the treatment of ONSM and to define its role in the treatment of these tumors. METHODS AND MATERIALS: We performed a retrospective analysis of 15 patients (13 women and 2 men), mean age 41.8 years, presenting with primary ONSM, followed at the Fondation Ophtalmologique Adolphe de Rothschild (Paris) between September 2006 and August 2013. After a multidisciplinary consultation, all were treated with PBT at a total dose of 52.2 Gy Eco, in fractions of 1.8 Gy Eco, at the Institut Curie (Paris). Patients underwent standardized follow-up including ophthalmologic examinations, visual field testing and imaging every 6 months. Study parameters were post-treatment visual acuity, tumor size on MRI, and treatment side effects RESULTS: We separated the patients into 3 distinct groups: patients treated by PBT after an observation period (4/15), patients treated by PBT after primary surgery (5/15) and patients treated by PBT as primary treatment (6/15). Visual acuity improved in 3 cases, deteriorated in 1 and remained stable in 11 cases. Tumor size on MRI remained stable in 100 % of cases following PBT. No serious adverse effects were recorded after a mean follow-up of 22.4 months (8-79 months). CONCLUSION: Our experience confirms the efficacy and the safety of proton beam therapy in patients with ONSM. PBT presents a promising alternative to surgery and conventional radiotherapy in the treatment of the MGNO. It seems to be effective in controlling tumor size and stabilizing visual function, at the cost of very low toxicity. Additional studies are needed to accurately determine the decision-making criteria and the ideal timing of this treatment.

Nuclear retention of ATM at sites of DNA double strand breaks.

The ATM protein kinase mediates a rapid induction of cellular responses to DNA double strand breaks (DSBs). ATM kinase activity is enhanced immediately after exposure of cells to DSB-inducing agents, but no changes in its amount or subcellular location following that activation have been reported. We speculated that some of the ATM molecules associate with sites of DSBs, while the rest of the nuclear ATM pool remains in the nucleoplasm, masking detection of the damage-associated ATM fraction. Using detergent extraction to remove nucleoplasmic proteins, we show here that immediately following induction of DSBs, a fraction of the ATM pool becomes resistant to extraction and is detected in nuclear aggregates. Colocalization of the retained ATM with the phosphorylated form of histone H2AX (gamma-H2AX) and with foci of the Nbs1 protein suggests that ATM associates with sites of DSBs. The striking correlation between the appearance of retained ATM and of gamma-H2AX, and the rapid association of a fraction of ATM with gamma-H2AX foci, are consistent with a major role for ATM in the early detection of DSBs and subsequent induction of cellular responses.