Immediately sequential bilateral cataract surgery.

PURPOSE OF REVIEW: This review aims to clarify the advantages and disadvantages of immediately sequential bilateral cataract surgery (ISBCS) based on recent studies, illustrate the safety of this approach, the cost-effectiveness, and present the importance of inclusion protocols for the best results. RECENT FINDINGS: In recent studies, the authors found no evidence of an increased risk of bilateral devastating complications such as endophthalmitis with ISBCS based on descriptive evidence compared to delayed sequential bilateral cataract surgery (DSBCS). Furthermore, recent studies on cost analyses showed that ISBCS resulted in fewer costs and significant cost savings to third-party payers, patients, and society compared to DSBCS. SUMMARY: The ISBCS surgical approach decreases hospital visits, reduces costs, and provides rapid visual rehabilitation and neuro adaptation. The risk of bilateral simultaneous complications is now recognized to be very rare with intracameral antibiotics and compliance with correct protocols. With new generations of optical biometry and lens calculation formulas, refractive surprises are occasional for normal eyes. However, refractive surprise is controversial, especially in the implantation of presbyopia correction intra-ocular lenses, which must be evaluated carefully in the ISBCS approach.

Phakic intraocular Lens implantation in keratoconus patients.

BACKGROUND: Both the anterior chamber and posterior chamber phakic intraocular lenses (pIOLs) implantation are acceptable refractive surgical approaches in keratoconus patients with high anisometropia, contact lens intolerance, or who prefer spectacle and contact lens independent. They are beneficial for correcting anisometropia in stable keratoconus cases or following corneal procedures such as intrastromal corneal ring segments (ICRS), collagen cross-linking (CXL), and keratoplasty. They are suitable for eyes without advanced keratoconus with acceptable best-corrected distance visual acuity (BCDVA) or without highly irregular astigmatism, high comma, and higher-order aberrations (HOAs). Combined procedures for irregular astigmatism reduction and corneal regularization with either ICRS or topography/wavefront-guided transepithelial PRK (with or without CXL) can be associated in advance with pIOLs implantation to improve BCDVA in these cases. AIM: To study and report the evidence regarding the safety and efficacy of pIOLs for KC patients' visual and refractive rehabilitation, we have analyzed the scientific evidence published within the last 10 years (from 2012 onwards). RESULTS: No randomized controlled trials but only eleven retrospective case series and two prospective case series were identified. Satisfactory visual rehabilitation was achieved regarding uncorrected and corrected distance visual acuity (CDVA) and predictability of the refractive correction. Both types of pIOL (iris claw and posterior chamber pIOLs) offer very good results in terms of safety and efficacy with indexes close to or even exceeding 1. CONCLUSION: pIOLs implantation is a valid refractive therapeutic approach for correcting stable keratoconus with moderate-to-high refractive errors, especially anisometropia associated with regular or mildly irregular astigmatism, and good CDVA.

Visual Performance After Unilateral Implantation of an Extended Depth-of-Focus Intraocular Lens in Patients With Unilateral Cataract.

PURPOSE: To investigate the visual performance after unilateral implantation of an extended depth-of-focus intraocular lens (IOL) in patients with unilateral cataracts. METHODS: In this prospective study, uneventful phacoemulsification with LuxSmart IOL (Bausch & Lomb) implantation was performed in 25 eyes of 25 patients with unilateral cataracts. At postoperative 1, 4, 12, and 24 weeks, uncorrected and corrected visual acuity at far, intermediate, and near distances and the spherical equivalent in manifest refraction were measured. A Visual Function Index and modified Visual Function Index questionnaire were used to investigate glare, spectacle dependence, and satisfaction at 24 weeks in the eye that had surgery. RESULTS: At 6 months postoperatively, uncorrected distance visual acuity was 20/20 (0.0 logMAR) in 96% of cases, distance corrected intermediate visual acuity was 20/32 (0.2 logMAR) in all cases (60 cm), and distance corrected near visual acuity was 20/32 (0.2 logMAR) in 60% of cases (40 cm). The patient satisfaction score was 100% based on the Visual Function Index questionnaire for far and intermediate distance, respectively. No patients complained of the permanent photic phenomenon. No patients reported bilateral imbalance. All of the patients became spectacle independent for most of their intermediate activities at 60 cm. A total of 96% of the patients reported 100% contrast sensitivity in the Pelli-Robson test. CONCLUSIONS: The unilateral implantation of this EDOF IOL seems to be tolerated and effective in improving the visual function of patients with unilateral cataract with limited optical side effects such as halos or glare, providing spectacle-independent vision from far to intermediate object distances. [J Refract Surg. 2023;39(8):518-523.].

Immediately sequential bilateral cataract surgery importance during the COVID-19 pandemic.

Cataract surgery is one of the most frequently performed types of surgery in the world. Most patients suffer from bilateral cataract and while cataract surgery of only one eye is effective in restoring functional vision, second eye surgery leads to further improvements in health-related quality of life, and is cost effective. At present, most patients undergo cataract surgery in both eyes on separate days, referred to as delayed sequential bilateral cataract surgery (DSBCS). An alternative procedure involves operating both eyes on the same day, but as separate procedures, known as immediately sequential bilateral cataract surgery (ISBCS). The aim of this study is to evaluate the effectiveness and costs of ISBCS compared to DSBCS. ISBCS is an important topic in ophthalmology, especially during the recent COVID-19 pandemic as it is necessary to decrease the hospital visits in order to prevent the contagious risk of this disease. There are well-documented advantages in terms of reduced costs for patients and health-care systems as well as more rapid visual rehabilitation and neuroadaptation. Based on recent studies, the risk of bilateral simultaneous complications is now recognized to be rare with the advent of intracameral antibiotics and strict protocols in this surgical approach. With the use of more sophisticated optical biometry and the newest generation lens calculation, refractive surprises are rare for normal eyes. A widely recognized protocol from the International Society of Bilateral Cataract Surgeons needs to adhere in order to prevent any further complications and obtaining better outcomes.

Plasticity within Aldehyde Dehydrogenase-Positive Cells Determines Prostate Cancer Radiosensitivity.

Tumor heterogeneity and cellular plasticity are key determinants of tumor progression, metastatic spread, and therapy response driven by the cancer stem cell (CSC) population. Within the current study, we analyzed irradiation-induced plasticity within the aldehyde dehydrogenase (ALDH)-positive (ALDH+) population in prostate cancer. The radiosensitivity of xenograft tumors derived from ALDH+ and ALDH-negative (ALDH-) cells was determined with local tumor control analyses and demonstrated different dose-response profiles, time to relapse, and focal adhesion signaling. The transcriptional heterogeneity was analyzed in pools of 10 DU145 and PC3 cells with multiplex gene expression analyses and illustrated a higher degree of heterogeneity within the ALDH+ population that even increases upon irradiation in comparison with ALDH- cells. Phenotypic conversion and clonal competition were analyzed with fluorescence protein-labeled cells to distinguish cellular origins in competitive three-dimensional cultures and xenograft tumors. We found that the ALDH+ population outcompetes ALDH- cells and drives tumor growth, in particular upon irradiation. The observed dynamics of the cellular state compositions between ALDH+ and ALDH- cells in vivo before and after tumor irradiation was reproduced by a probabilistic Markov compartment model that incorporates cellular plasticity, clonal competition, and phenotype-specific radiosensitivities. Transcriptional analyses indicate that the cellular conversion from ALDH- into ALDH+ cells within xenograft tumors under therapeutic pressure was partially mediated through induction of the transcriptional repressor SNAI2. In summary, irradiation-induced cellular conversion events are present in xenograft tumors derived from prostate cancer cells and may be responsible for radiotherapy failure. IMPLICATIONS: The increase of ALDH+ cells with stem-like features in prostate xenograft tumors after local irradiation represents a putative cellular escape mechanism inducing tumor radioresistance.

Detection of occludable angle with anterior segment optical coherence tomography and Pentacam as non-contact screening methods.

PURPOSE: To evaluate diagnostic capacity for occludable anterior chamber angle detection with anterior segment optical coherence tomography (AS-OCT) and Pentacam. METHODS: Observational cross-sectional study with AS-OCT and Pentacam. AS-OCT measures: angle opening distance from Schwalbe line (SL) perpendicular (AOD-SL-Perp) and vertical to iris (AOD-SL-Vert), and iridotrabecular angle (ITA). Pentacam measures: anterior chamber depth (ACD), anterior chamber volume (ACV), and anterior chamber angle (ACA). We analysed Spearman's correlation with gonioscopic classification. Area under receiver operating characteristic curves (AUCs) for occludable angle detection were compared. Agreement between iridocorneal values of methods was evaluated. RESULTS: Seventy-four left eyes of 74 patients. Correlation between temporal AS-OCT and gonioscopy: 0.83 (p < 0.0001) AOD-SL-Perp temporal, 0.82 (p < 0.0001) AOD-SL-Vert temporal, and 0.69 (p < 0.0001) ITA temporal. Correlation between AS-OCT nasal and gonioscopy: 0.74 (p < 0.0001) AOD-SL-Perp nasal, 0.74 (p < 0.0001) AOD-SL-Vert nasal, and 0.70 (p < 0.0001) ITA nasal. Correlation of Pentacam with temporal gonioscopy: 0.57 (p < 0.0001) ACD, 0.56 (p < 0.0001) ACV, and 0.63 (p < 0.0001) ACA. Correlation of Pentacam with nasal gonioscopy: 0.47 (IC 0.27-0.73, p < 0.0001) ACD, 0.49 (p < 0.0001) ACV, and 0.56 (CI 0.38-0.7, p < 0.0001) ACA. AS-OCT AUCs: AOD-SL-Perp temporal 0.89 (CI 0.80-0.95), AOD-SL-Vert 0.87 (CI 0.77-0.94), ITA temporal 0.88 (CI 0.78-0.94), AOD-SL-Perp nasal 0.83 (CI 0.72-0.91), AOD-SL-Vert nasal 0.87 (CI 0.77-0.94), and ITA nasal 0.91 (IC 0.81-0.96). Pentacam AUCs: ACD 0.76 (CI 0.64-0.85), ACV 0.75 (CI 0.63-0.84), and ACA 0.84 (CI 0.74-0.92). No statistical differences between different AUCs. Intraclass correlation coefficient (ICC) of ACA (Pentacam) with ITA temporal (AS-OCT) 0.59 and with nasal ITA nasal (AS-OCT) 0.65. CONCLUSION: Both systems show high capacity for non-contact occludable angle detection. But agreement between methods is moderate or low.

High-Complexity cellular barcoding and clonal tracing reveals stochastic and deterministic parameters of radiation resistance.

It is elusive whether clonal selection of tumor cells in response to ionizing radiation (IR) is a deterministic or stochastic process. With high resolution clonal barcoding and tracking of over 400 000 HNSCC patient-derived tumor cells the clonal dynamics of tumor cells in response to IR was analyzed. Fractionated IR induced a strong selective pressure for clonal reduction which significantly exceeded uniform clonal survival probabilities indicative for a strong clone-to-clone difference within tumor cell lines. IR induced clonal reduction affected the majority of tumor cells ranging between 96% and 75% and correlated to the degree of radiation sensitivity. Survival to IR is driven by a deterministic clonal selection of a smaller population which commonly survives radiation, while increased clonogenic capacity is a result of clonal competition of cells which have been selected stochastically. A 2-fold increase in radiation resistance results in a 4-fold (P < .05) higher deterministic clonal selection showing that the ratio of these parameters is amenable to radiation sensitivity which correlates to prognostic biomarkers of HNSCC. Evidence for the existence of a rare subpopulation with an intrinsically radiation resistant phenotype commonly surviving IR was found at a frequency of 0.6% to 3.3% (P < .001, FDR 3%). With cellular barcoding we introduce a novel functional heterogeneity associated qualitative readout for tracking dynamics of clonogenic survival in response to radiation. This enables the quantification of intrinsically radiation resistant tumor cells from patient samples and reveals the contribution of stochastic and deterministic clonal selection processes in response to IR.

Degradation of CCNK/CDK12 is a druggable vulnerability of colorectal cancer.

Novel treatment options for metastatic colorectal cancer (CRC) are urgently needed to improve patient outcome. Here, we screen a library of non-characterized small molecules against a heterogeneous collection of patient-derived CRC spheroids. By prioritizing compounds with inhibitory activity in a subset of-but not all-spheroid cultures, NCT02 is identified as a candidate with minimal risk of non-specific toxicity. Mechanistically, we show that NCT02 acts as molecular glue that induces ubiquitination of cyclin K (CCNK) and proteasomal degradation of CCNK and its complex partner CDK12. Knockout of CCNK or CDK12 decreases proliferation of CRC cells in vitro and tumor growth in vivo. Interestingly, sensitivity to pharmacological CCNK/CDK12 degradation is associated with TP53 deficiency and consensus molecular subtype 4 in vitro and in patient-derived xenografts. We thus demonstrate the efficacy of targeted CCNK/CDK12 degradation for a CRC subset, highlighting the potential of drug-induced proteolysis for difficult-to-treat types of cancer.

Central retinal vein occlusion in a patient using the antipsychotic drug olanzapine: a case report.

BACKGROUND: We report our findings in a patient who developed central retinal vein occlusion (CRVO) and was a chronic user of olanzapine, an antipsychotic medication. CASE PRESENTATION: A 50-year-old Caucasian man, non-smoker, was referred to our clinic with the chief complaint of floater appearance in his left eye for the past 3 days. His past medical history indicated that he had been taking antipsychotic drugs (olanzapine) for about 3 years, with no other systemic disease or risk factors for CRVO. In the examination, his best-corrected visual acuity (BCVA) was 0.7 in the left eye. The fundus showed signs of nonischemic CRVO with subhyaloid hemorrhage and intraretinal hemorrhage in the posterior pole and superior and inferior retina, without macular edema, confirmed by optical coherence tomography (OCT). We ruled out other probable differential diagnoses and risk factors which lead to CRVO through a complete physical exam and blood analysis (complete blood count, glucose, urea, creatinine, lipid profile, erythrocyte sedimentation rate, C-reactive protein, prothrombin time, partial thromboplastin time, Bleeding time (BT), fibrinogen level, proteins, antiphospholipid antibodies, homocysteine blood level, antithrombin III, protein C and S, factor V Leiden, prothrombin mutation, angiotensin-converting enzyme level, other autoantibodies, and human leukocyte antigen [HLA]-B51). Finally, we confirmed the probable side effect of olanzapine in CRVO, which has not been previously reported. A possible pro-thrombogenic mechanism of olanzapine at the molecular level is an affinity for 5-HT2Aserotonin receptors. Blocking these receptors results in increased platelet aggregation and increased blood coagulability. CONCLUSIONS: These results indicate that CRVO can be a complication of chronic use of antipsychotic medications such as olanzapine, as shown for the first time in our case report. Clinicians should question patients who develop a sudden CRVO whether they are using antipsychotic medications such as olanzapine.

Differential transcriptome response to proton versus X-ray radiation reveals novel candidate targets for combinatorial PT therapy in lymphoma.

BACKGROUND AND PURPOSE: Knowledge of biological responses to proton therapy (PT) in comparison to X-ray remains in its infancy. Identification of PT specific molecular signals is an important opportunity for the discovery of biomarkers and synergistic drugs to advance clinical application. Since PT is used for the treatment of lymphoma, we report here transcriptomic responses of lymphoma cell lines to PT vs X-ray and identify potential therapeutic targets. MATERIALS AND METHODS: Two lymphoma cell lines of human (BL41) and murine (J3D) origin were irradiated by X-ray and PT. Differential transcriptome regulation was quantified by RNA sequencing for each radiation type at 12 hours post irradiation. Gene-set enrichment analysis revealed deregulated molecular pathways and putative targets for lymphoma cell sensitization to PT. RESULTS: Transcriptomic gene set enrichment analyses uncovered pathways that contribute to the unfolded protein response (UPR) and mitochondrial transport. Functional validation at multiple time points demonstrated increased UPR activation and decreased protein translation, perhaps due to increased oxidative stress and oxidative protein damage after PT. PPARgamma was identified as a potential regulator of the PT transcriptomic response. Inhibition of PPARgamma by two compounds, T0070907 and SR2595, sensitized lymphoma cells to PT. CONCLUSIONS: Proton vs X-ray radiation leads to the transcriptional regulation of a specific subset of genes in line with diminished protein translation and UPR activation that may be due to oxidative stress. This study demonstrates that different radiation qualities trigger distinct cellular responses in lymphoma cells, and identifies PPARgamma inhibition as a potential strategy for the sensitization of lymphoma to PT.

Peripheral vascular disease - a new vascular disease associated with normal tension glaucoma: a case report.

INTRODUCTION: Normal-tension glaucoma is known as a multifactorial optic neuropathy. A number of lines of evidence suggested that vascular factors played a significant role in the development of normal-tension glaucoma. The mechanisms underlying the abnormal ocular blood flow in normal-tension glaucoma are still not clear. Peripheral vascular disease seems to be associated with glaucoma populations independent of other cardiovascular risk factors. We found this presentation, for the first time, to our knowledge, as another probable vascular abnormality related to our patient with normal-tension glaucoma, although it is necessary to confirm its pathological effect in future studies. CASE PRESENTATION: Our patient was a 48-year-old Spanish man without any personal and family history of interest except for circulatory problems of the lower limbs with repetitive ulcers at the frontal and lateral aspects of his legs. His chief complaint was vision loss when he came to consult us. In exploration, his best corrected visual acuity was 20/20 in both eyes; initial intraocular pressure in the right eye was 14-16 mmHg and in the left eye was 16-18 mmHg, with a mild sclerosis of the lens in slit-lamp examination. No inflammation or pigmented lesion was detected in the anterior chamber. Open angle confirmed by Goldman four quadrants gonioscopy. Funduscopic examination revealed a vertical cup disc ratio of 0.6 in the right eye and 0.8 in the left eye. The patient's neuroretinal rim was normal in the right eye, and superior thinning in the left eye was determined. Examination of the patient's visual field showed inferior mild probable nasal scotoma in the right eye and an inferior deep arcuate scotoma defect in the left eye. His optical coherence tomography examination revealed thinning of the peripapillary nerve fiber layer thickness in the left eye and superior loss of macular retinal ganglion cells in the left eye. Normal intraocular pressure values were measured on the intraocular pressure curve without treatment (maximum value, 18-20 mmHg), discarding higher intraocular pressures measured out of office. Ultrasonic pachymetry measured 515/520 µm, and normal intraocular pressure measured with a PASCAL tonometer ruled out probable corneal biomechanical underestimations. The patient's polysomnography study was normal and excluded sleep apnea syndrome. The patient's serial mean blood pressure was normal, especially in the lower limbs (mean value, 125/70 mmHg), ruling out the possibility of systemic hypotension. Thyroidal and coagulation abnormalities, autoimmune disease, and inflammatory disease were excluded. Normal immunologic study and normal vascular biopsy were observed, as well as normal brain magnetic resonance imaging and a normal carotid vascular study. The primary diagnosis was moderate medium peripheral arterial disease in the lower limbs, which was confirmed by echography after ruling out other probable vascular abnormalities related to normal-tension glaucoma. CONCLUSION: After ruling out other systemic diseases and vascular abnormalities related to normal-tension glaucoma, we found peripheral arterial disease as a probable vascular abnormality related to normal-tension glaucoma in our patient. To our knowledge, this is the first time such a case has been reported. Thus, further research is needed to determine the relevance of these results to the general population.

Personalized Assessment of Normal Tissue Radiosensitivity via Transcriptome Response to Photon, Proton and Carbon Irradiation in Patient-Derived Human Intestinal Organoids.

Radiation-induced normal tissue toxicity often limits the curative treatment of cancer. Moreover, normal tissue relative biological effectiveness data for high-linear energy transfer particles are urgently needed. We propose a strategy based on transcriptome analysis of patient-derived human intestinal organoids (HIO) to determine molecular surrogates for radioresponse of gastrointestinal (GI) organs at risk in a personalized manner. HIO were generated from induced pluripotent stem cells (iPSC), which were derived from skin biopsies of three patients, including two patients with FANCA deficiency as a paradigm for enhanced radiosensitivity. For the two Fanconi anemia (FA) patients (HIO-104 and 106, previously published as FA-A#1 IND-iPS1 and FA-A#2 IND-iPS3), FANCA expression was reconstituted as a prerequisite for generation of HIO via lentiviral expression of a doxycycline inducible construct. For radiosensitivity analysis, FANCA deficient and FANCA rescued as well as wtHIO were sham treated or irradiated with 4Gy photon, proton or carbon ions at HIT, respectively. Immunofluorescence staining of HIO for 53BP1-foci was performed 1 h post IR and gene expression analyses was performed 12 and 48 h post IR. 53BP1-foci numbers and size correlated with the higher RBE of carbon ions. A FANCA dependent differential gene expression in response to radiation was found (p < 0.01, ANOVA; n = 1071 12 h; n = 1100 48 h). Pathways associated with FA and DNA-damage repair i.e., transcriptional coupled nucleotide excision repair, homology-directed repair and translational synthesis were found to be differentially regulated in FANCA deficient HIO. Next, differential regulated genes were investigated as a function of radiation quality (RQ, p < 0.05, ANOVA; n = 742 12 h; n = 553 48 h). Interestingly, a gradual increase or decrease of gene expression was found to correlate with the three main qualities, from photon to proton and carbon irradiation. Clustering separated high-linear energy transfer irradiation with carbons from proton and photon irradiation. Genes associated with dual incision steps of TC-NER were differentially regulated in photon vs. proton and carbon irradiation. Consequently, SUMO3, ALC1, POLE4, PCBP4, MUTYH expression correlated with the higher RBE of carbon ions. An interaction between the two studied parameters FA and RQ was identified (p < 0.01, 2-way ANOVA n = 476). A comparison of genes regulated as a function of FA, RQ and RBE suggest a role for p53 interacting genes BRD7, EWSR1, FBXO11, FBXW8, HMGB1, MAGED2, PCBP4, and RPS27 as modulators of FA in response to radiation. This proof of concept study demonstrates that patient tailored evaluation of GI response to radiation is feasible via generation of HIO and comparative transcriptome profiling. This methodology can now be further explored for a personalized assessment of GI radiosensitivity and RBE estimation.

Large scale in vivo micro-RNA loss of function screen identified miR-29a, miR-100 and miR-155 as modulators of radioresistance and tumor-stroma communication.

Micro RNAs (miR) are master regulators of cellular transcriptome. We aimed to investigate the role of miR regulation on tumor radiosensitivity and development of local tumor recurrence by a novel large-scale in vivo loss of function screen. For stable miR silencing, human A431 tumor cells were transduced with lentiviral constructs against 170 validated human miR (miRzip library). Fractionated radiotherapy (5x6Gy) was applied to A431 miRzip library growing s.c. in NCr nude mice. Enrichment of miRZip and miR expression was assessed using multiplexed qRT-PCR. The modulatory effect of miR on tumor and tumor microenvironment response to ionizing radiation was further evaluated by clonogenic survival, apoptosis (Caspase 3/7), DNA double-strand breaks (DSB, nuclear γH2AX foci), tumor microvessel density (MVD), transcriptome and protein analysis. Fractionated irradiation of the A431 miRzip library led to regression of tumors. However, after a latency period, tumors ultimately progressed and formed local recurrences indicating the survival of a subpopulation of miRzip expressing tumor clones. Among the selected miR for subsequent validation studies, loss of miR-29a, miR-100 and miR-155 was found to enhance clonogenic survival, reduce apoptosis and residual γH2AX foci of irradiated tumor cells. Moreover, knockdown of miR increased tumor angiogenesis correlating with elevated VEGF and TGFα expression levels. This phenomenon was most evident after tumor irradiation in vivo suggesting a critical role for tumor-stroma communication in development of the radioresistant phenotype. Engineering radioresistant tumors in vivo by modulating miR expression may lead to identification of critical targets for conquering local therapy failure.

Cellular Barcoding Identifies Clonal Substitution as a Hallmark of Local Recurrence in a Surgical Model of Head and Neck Squamous Cell Carcinoma.

Local recurrence after surgery for head and neck squamous cell carcinoma (HNSCC) remains a common event associated with a dismal prognosis. Improving this outcome requires a better understanding of cancer cell populations that expand from postsurgical minimal residual disease (MRD). Therefore, we assessed clonal dynamics in a surgical model of barcoded HNSCC growing in the submental region of immunodeficient mice. Clonal substitution and massive reduction of clonal heterogeneity emerged as hallmarks of local recurrence, as the clones dominating in less heterogeneous recurrences were scarce in their matched primary tumors. These lineages were selected by their ability to persist after surgery and competitively expand from MRD. Clones enriched in recurrences exhibited both private and shared genetic features and likely originated from ancestors shared with clones dominating in primary tumors. They demonstrated high invasiveness and epithelial-to-mesenchymal transition, eventually providing an attractive target for obtaining better local control for these tumors.

AAVvector-mediated in vivo reprogramming into pluripotency.

In vivo reprogramming of somatic cells into induced pluripotent stem cells (iPSC) holds vast potential for basic research and regenerative medicine. However, it remains hampered by a need for vectors to express reprogramming factors (Oct-3/4, Klf4, Sox2, c-Myc; OKSM) in selected organs. Here, we report OKSM delivery vectors based on pseudotyped Adeno-associated virus (AAV). Using the AAV-DJ capsid, we could robustly reprogram mouse embryonic fibroblasts with low vector doses. Swapping to AAV8 permitted to efficiently reprogram somatic cells in adult mice by intravenous vector delivery, evidenced by hepatic or extra-hepatic teratomas and iPSC in the blood. Notably, we accomplished full in vivo reprogramming without c-Myc. Most iPSC generated in vitro or in vivo showed transcriptionally silent, intronic or intergenic vector integration, likely reflecting the increased host genome accessibility during reprogramming. Our approach crucially advances in vivo reprogramming technology, and concurrently facilitates investigations into the mechanisms and consequences of AAV persistence.

Identification of T cell target antigens in glioblastoma stem-like cells using an integrated proteomics-based approach in patient specimens.

Glioblastoma (GBM) is a highly aggressive brain tumor and still remains incurable. Among others, an immature subpopulation of self-renewing and therapy-resistant tumor cells-often referred to as glioblastoma stem-like cells (GSCs)-has been shown to contribute to disease recurrence. To target these cells personalized immunotherapy has gained a lot of interest, e.g. by reactivating pre-existing anti-tumor immune responses against GSC antigens. To identify T cell targets commonly presented by GSCs and their differentiated counterpart, we used a proteomics-based separation of GSC proteins in combination with a T cell activation assay. Altogether, 713 proteins were identified by LC-ESI-MS/MS mass spectrometry. After a thorough filtering process, 32 proteins were chosen for further analyses. Immunogenicity of corresponding peptides was tested ex vivo. A considerable number of these antigens induced T cell responses in GBM patients but not in healthy donors. Moreover, most of them were overexpressed in primary GBM and also highly expressed in recurrent GBM tissues. Interestingly, expression of the most frequent T cell target antigens could also be confirmed in quiescent, slow-cycling GSCs isolated in high purity by the DEPArray technology. Finally, for a subset of these T cell target antigens, an association between expression levels and higher T cell infiltration as well as an increased expression of positive immune modulators was observed. In summary, we identified novel immunogenic proteins, which frequently induce tumor-specific T cell responses in GBM patients and were also detected in vitro in therapy-resistant quiescent, slow-cycling GSCs. Stable expression of these T cell targets in primary and recurrent GBM support their suitability for future clinical use.

High-resolution analysis of the human T-cell receptor repertoire.

Unbiased dissection of T-cell receptor (TCR) repertoire diversity at the nucleotide level could provide important insights into human immunity. Here we show that TCR ligation-anchored-magnetically captured PCR (TCR-LA-MC PCR) identifies TCR α- and ß-chain diversity without sequence-associated or quantitative restrictions in healthy and diseased conditions. TCR-LA-MC PCR identifies convergent recombination events, classifies different stages of cutaneous T-cell lymphoma in vivo and demonstrates TCR reactivation after in vitro cytomegalovirus stimulation. TCR-LA-MC PCR allows ultra-deep data access to both physiological TCR diversity and mechanisms influencing clonality in all clinical settings with restricted or distorted TCR repertoires.

Lentiviral-Mediated Gene Therapy in Fanconi Anemia-A Mice Reveals Long-Term Engraftment and Continuous Turnover of Corrected HSCs.

Fanconi anemia is a DNA repair-deficiency syndrome mainly characterized by cancer predisposition and bone marrow failure. Trying to restore the hematopoietic function in these patients, lentiviral vector-mediated gene therapy trials have recently been proposed. However, because no insertional oncogenesis studies have been conducted so far in DNA repair-deficiency syndromes such as Fanconi anemia, we have carried out a genome-wide screening of lentiviral insertion sites after the gene correction of Fanca(-/-) hematopoietic stem cells (HSCs), using LAM-PCR and 454-pyrosequencing. Our studies first demonstrated that transduction of Fanca(-/-) HSCs with a lentiviral vector designed for clinical application efficiently corrects the phenotype of Fanconi anemia repopulating cells without any sign of toxicity. The identification of more than 6,500 insertion sites in primary and secondary recipients showed a polyclonal pattern of reconstitution, as well as a continuous turnover of corrected Fanca(-/-) HSC clones, without evidences of selection towards specific common integration sites. Taken together our data show, for the first time in a DNA repair-deficiency syndrome, that lentiviral vector-mediated gene therapy efficiently corrects the phenotype of affected HSCs and promotes a healthy pattern of clonal turnover in vivo. These studies will have a particular impact in the development of new gene therapy trials in patients affected by DNA repair syndromes, particularly in Fanconi anemia.

Comparative next-generation sequencing of adeno-associated virus inverted terminal repeats.

The inverted terminal repeats (ITRs) of adeno-associated virus (AAV) are notoriously difficult to sequence owing to their high GC-content (70%) and palindromic sequences that result in the formation of a very stable, 125 bp long, T-shaped hairpin structure. Here we evaluate the performance of two widely used next-generation sequencing platforms, 454 GS FLX (Roche) and MiSeq Benchtop Sequencer (Illumina), in analyzing ITRs in comparatively sequencing linear amplification-meditated PCR (LAM-PCR) amplicons derived from AAV-concatemeric structures. While our data indicate that both platforms can sequence complete ITRs, efficiencies (MiSeq: 0.11% of sequence reads; 454: 0.02% of reads), frequencies (MiSeq: 171 full ITRs, 454: 3 full ITRs), and rates of deviation from the derived ITR consensus sequence (MiSeq: 0.8%-1.3%; 454: 0.5%) did differ. These results suggest that next-generation sequencing platforms can be used to specifically detect ITR mutations and sequence complete ITRs.