A Randomized Trial of Photobiomodulation Therapy for Center-Involved Diabetic Macular Edema with Good Visual Acuity (Protocol AE).

PURPOSE: To determine if treatment with a photobiomodulation (PBM) device results in greater improvement in central subfield thickness (CST) than placebo in eyes with center-involved diabetic macular edema (CI-DME) and good vision. DESIGN: Phase 2 randomized clinical trial. PARTICIPANTS: Participants had CI-DME and visual acuity (VA) 20/25 or better in the study eye and were recruited from 23 clinical sites in the United States. METHODS: One eye of each participant was randomly assigned 1:1 to a 670-nm light-emitting PBM eye patch or an identical device emitting broad-spectrum white light at low power. Treatment was applied for 90 seconds twice daily for 4 months. MAIN OUTCOME MEASURES: Change in CST on spectral-domain OCT at 4 months. RESULTS: From April 2019 to February 2020, 135 adults were randomly assigned to either PBM (n = 69) or placebo (n = 66); median age was 62 years, 37% were women, and 82% were White. The median device compliance was 92% with PBM and 95% with placebo. OCT CST increased from baseline to 4 months by a mean (SD) of 13 (53) µm in PBM eyes and 15 (57) µm in placebo eyes, with the mean difference (95% confidence interval [CI]) being -2 (-20 to 16) µm (P = 0.84). CI-DME, based on DRCR Retina Network sex- and machine-based thresholds, was present in 61 (90%) PBM eyes and 57 (86%) placebo eyes at 4 months (adjusted odds ratio [95% CI] = 1.30 (0.44-3.83); P = 0.63). VA decreased by a mean (SD) of -0.2 (5.5) letters and -0.6 (4.6) letters in the PBM and placebo groups, respectively (difference [95% CI] = 0.4 (-1.3 to 2.0) letters; P = 0.64). There were 8 adverse events possibly related to the PBM device and 2 adverse events possibly related to the placebo device. None were serious. CONCLUSIONS: PBM as given in this study, although safe and well-tolerated, was not found to be effective for the treatment of CI-DME in eyes with good vision.

Evaluation of Stem Cell-Derived Red Blood Cells as a Transfusion Product Using a Novel Animal Model.

Reliance on volunteer blood donors can lead to transfusion product shortages, and current liquid storage of red blood cells (RBCs) is associated with biochemical changes over time, known as 'the storage lesion'. Thus, there is a need for alternative sources of transfusable RBCs to supplement conventional blood donations. Extracorporeal production of stem cell-derived RBCs (stemRBCs) is a potential and yet untapped source of fresh, transfusable RBCs. A number of groups have attempted RBC differentiation from CD34+ cells. However, it is still unclear whether these stemRBCs could eventually be effective substitutes for traditional RBCs due to potential differences in oxygen carrying capacity, viability, deformability, and other critical parameters. We have generated ex vivo stemRBCs from primary human cord blood CD34+ cells and compared them to donor-derived RBCs based on a number of in vitro parameters. In vivo, we assessed stemRBC circulation kinetics in an animal model of transfusion and oxygen delivery in a mouse model of exercise performance. Our novel, chronically anemic, SCID mouse model can evaluate the potential of stemRBCs to deliver oxygen to tissues (muscle) under resting and exercise-induced hypoxic conditions. Based on our data, stem cell-derived RBCs have a similar biochemical profile compared to donor-derived RBCs. While certain key differences remain between donor-derived RBCs and stemRBCs, the ability of stemRBCs to deliver oxygen in a living organism provides support for further development as a transfusion product.

HMGA1: a master regulator of tumor progression in triple-negative breast cancer cells.

Emerging evidence suggests that tumor cells metastasize by co-opting stem cell transcriptional networks, although the molecular underpinnings of this process are poorly understood. Here, we show for the first time that the high mobility group A1 (HMGA1) gene drives metastatic progression in triple negative breast cancer cells (MDA-MB-231, Hs578T) by reprogramming cancer cells to a stem-like state. Silencing HMGA1 expression in invasive, aggressive breast cancer cells dramatically halts cell growth and results in striking morphologic changes from mesenchymal-like, spindle-shaped cells to cuboidal, epithelial-like cells. Mesenchymal genes (Vimentin, Snail) are repressed, while E-cadherin is induced in the knock-down cells. Silencing HMGA1 also blocks oncogenic properties, including proliferation, migration, invasion, and orthotopic tumorigenesis. Metastatic progression following mammary implantation is almost completely abrogated in the HMGA1 knock-down cells. Moreover, silencing HMGA1 inhibits the stem cell property of three-dimensional mammosphere formation, including primary, secondary, and tertiary spheres. In addition, knock-down of HMGA1 depletes cancer initiator/cancer stem cells and prevents tumorigenesis at limiting dilutions. We also discovered an HMGA1 signature in triple negative breast cancer cells that is highly enriched in embryonic stem cells. Together, these findings indicate that HMGA1 is a master regulator of tumor progression in breast cancer by reprogramming cancer cells through stem cell transcriptional networks. Future studies are needed to determine how to target HMGA1 in therapy.

HMGA1 reprograms somatic cells into pluripotent stem cells by inducing stem cell transcriptional networks.

BACKGROUND: Although recent studies have identified genes expressed in human embryonic stem cells (hESCs) that induce pluripotency, the molecular underpinnings of normal stem cell function remain poorly understood. The high mobility group A1 (HMGA1) gene is highly expressed in hESCs and poorly differentiated, stem-like cancers; however, its role in these settings has been unclear. METHODS/PRINCIPAL FINDINGS: We show that HMGA1 is highly expressed in fully reprogrammed iPSCs and hESCs, with intermediate levels in ECCs and low levels in fibroblasts. When hESCs are induced to differentiate, HMGA1 decreases and parallels that of other pluripotency factors. Conversely, forced expression of HMGA1 blocks differentiation of hESCs. We also discovered that HMGA1 enhances cellular reprogramming of somatic cells to iPSCs together with the Yamanaka factors (OCT4, SOX2, KLF4, cMYC - OSKM). HMGA1 increases the number and size of iPSC colonies compared to OSKM controls. Surprisingly, there was normal differentiation in vitro and benign teratoma formation in vivo of the HMGA1-derived iPSCs. During the reprogramming process, HMGA1 induces the expression of pluripotency genes, including SOX2, LIN28, and cMYC, while knockdown of HMGA1 in hESCs results in the repression of these genes. Chromatin immunoprecipitation shows that HMGA1 binds to the promoters of these pluripotency genes in vivo. In addition, interfering with HMGA1 function using a short hairpin RNA or a dominant-negative construct blocks cellular reprogramming to a pluripotent state. CONCLUSIONS: Our findings demonstrate for the first time that HMGA1 enhances cellular reprogramming from a somatic cell to a fully pluripotent stem cell. These findings identify a novel role for HMGA1 as a key regulator of the stem cell state by inducing transcriptional networks that drive pluripotency. Although further studies are needed, these HMGA1 pathways could be exploited in regenerative medicine or as novel therapeutic targets for poorly differentiated, stem-like cancers.

The HMGA1-COX-2 axis: a key molecular pathway and potential target in pancreatic adenocarcinoma.

CONTEXT: Although pancreatic cancer is a common, highly lethal malignancy, the molecular events that enable precursor lesions to become invasive carcinoma remain unclear. We previously reported that the high-mobility group A1 (HMGA1) protein is overexpressed in >90% of primary pancreatic cancers, with absent or low levels in early precursor lesions. METHODS: Here, we investigate the role of HMGA1 in reprogramming pancreatic epithelium into invasive cancer cells. We assessed oncogenic properties induced by HMGA1 in non-transformed pancreatic epithelial cells expressing activated K-RAS. We also explored the HMGA1-cyclooxygenase (COX-2) pathway in human pancreatic cancer cells and the therapeutic effects of COX-2 inhibitors in xenograft tumorigenesis. RESULTS: HMGA1 cooperates with activated K-RAS to induce migration, invasion, and anchorage-independent cell growth in a cell line derived from normal human pancreatic epithelium. Moreover, HMGA1 and COX-2 expression are positively correlated in pancreatic cancer cell lines (r(2) = 0.93; p < 0.001). HMGA1 binds directly to the COX-2 promoter at an AT-rich region in vivo in three pancreatic cancer cell lines. In addition, HMGA1 induces COX-2 expression in pancreatic epithelial cells, while knock-down of HMGA1 results in repression of COX-2 in pancreatic cancer cells. Strikingly, we also discovered that Sulindac (a COX-1/COX-2 inhibitor) or Celecoxib (a more specific COX-2 inhibitor) block xenograft tumorigenesis from pancreatic cancer cells expressing high levels of HMGA1. CONCLUSIONS: Our studies identify for the first time an important role for the HMGA1-COX-2 pathway in pancreatic cancer and suggest that targeting this pathway could be effective to treat, or even prevent, pancreatic cancer.

High mobility group A1 and cancer: potential biomarker and therapeutic target.

The High Mobility Group A1 (HMGA1, formerly HMG-I/Y) gene is highly expressed during embryogenesis and in virtually all aggressive human cancers studied to date, although its role in these settings is only beginning to emerge. Moreover, high levels of expression portend a poor prognosis in some tumors. Increasing evidence suggests that the HMGA1 protein functions as a master regulator with a critical role in normal development and tumor progression in diverse malignancies. These proteins contain AT-hook DNA binding domains that mediate binding to AT-rich regions of chromatin. After binding to DNA, HMGA1 alters DNA structure, and orchestrates the assembly of a transcriptional complex or "enhanceosome" to regulate gene expression. Previous studies indicate that HMGA1 participates in regulating fundamental cellular processes, including transcription, cell cycle progression, embryonic development, neoplastic transformation, differentiation, senescence, viral integration, and DNA repair by virtue of its ability to interact with other proteins, bind to DNA, and modulate gene expression. Recent studies also link HMGA1 expression to poor differentiation status and a refractory, stem cell-like state in aggressive cancers. Together, these findings suggest that HMGA1 could serve as a useful biomarker and therapeutic target in advanced malignancies. Here, we focus on prior studies implicating HMGA1 in the pathogenesis of refractory human tumors arising from diverse tissues and its potential role as a biomarker. We also review previous attempts to target HMGA1 pathways in cancer. Further study of HMGA1 promises to have a major impact on our ability to understand and treat cancer.

HMGA1 drives stem cell, inflammatory pathway, and cell cycle progression genes during lymphoid tumorigenesis.

BACKGROUND: Although the high mobility group A1 (HMGA1) gene is widely overexpressed in diverse cancers and portends a poor prognosis in some tumors, the molecular mechanisms that mediate its role in transformation have remained elusive. HMGA1 functions as a potent oncogene in cultured cells and induces aggressive lymphoid tumors in transgenic mice. Because HMGA1 chromatin remodeling proteins regulate transcription, HMGA1 is thought to drive malignant transformation by modulating expression of specific genes. Genome-wide studies to define HMGA1 transcriptional networks during tumorigenesis, however, are lacking. To define the HMGA1 transcriptome, we analyzed gene expression profiles in lymphoid cells from HMGA1a transgenic mice at different stages in tumorigenesis. RESULTS: RNA from lymphoid samples at 2 months (before tumors develop) and 12 months (after tumors are well-established) was screened for differential expression of > 20,000 unique genes by microarray analysis (Affymetrix) using a parametric and nonparametric approach. Differential expression was confirmed by quantitative RT-PCR in a subset of genes. Differentially expressed genes were analyzed for cellular pathways and functions using Ingenuity Pathway Analysis. Early in tumorigenesis, HMGA1 induced inflammatory pathways with NFkappaB identified as a major node. In established tumors, HMGA1 induced pathways involved in cell cycle progression, cell-mediated immune response, and cancer. At both stages in tumorigenesis, HMGA1 induced pathways involved in cellular development, hematopoiesis, and hematologic development. Gene set enrichment analysis showed that stem cell and immature T cell genes are enriched in the established tumors. To determine if these results are relevant to human tumors, we knocked-down HMGA1 in human T-cell leukemia cells and identified a subset of genes dysregulated in both the transgenic and human lymphoid tumors. CONCLUSIONS: We found that HMGA1 induces inflammatory pathways early in lymphoid tumorigenesis and pathways involved in stem cells, cell cycle progression, and cancer in established tumors. HMGA1 also dyregulates genes and pathways involved in stem cells, cellular development and hematopoiesis at both early and late stages of tumorigenesis. These results provide insight into HMGA1 function during tumor development and point to cellular pathways that could serve as therapeutic targets in lymphoid and other human cancers with aberrant HMGA1 expression.

Giant macular hole in Alport syndrome.

PURPOSE: To describe the evolution of a giant macular hole in a patient with Alport syndrome and review the literature. METHODS: An observational case report is presented with serial clinical examination, visual acuity, fundus photographs, and ocular coherence tomography performed. RESULTS: A man with Alport syndrome and a giant macular hole in one eye developed multiple, small lamellar macular holes which coalesced into a giant full thickness macular hole in the contralateral eye. CONCLUSIONS: Giant macular holes may occur in Alport syndrome. The mechanism and clinical progression appear to differ from that of idiopathic macular holes and, is likely related to an abnormality in Type IV collagen in basement membrane of retinal Muller cells. Anomalous vitreoretinal adhesion may also play a role. Previous cases of giant macular holes in the literature may not have been properly associated with Alport syndrome.

Defective mismatch repair, microsatellite mutation bias, and variability in clinical cancer phenotypes.

Microsatellite instability is associated with 10% to 15% of colorectal, endometrial, ovarian, and gastric cancers, and has long been used as a diagnostic tool for hereditary nonpolyposis colorectal carcinoma-related cancers. Tumor-specific length alterations within microsatellites are generally accepted to be a consequence of strand slippage events during DNA replication, which are uncorrected due to a defective postreplication mismatch repair (MMR) system. Mutations arising within microsatellites associated with critical target genes are believed to play a causative role in the evolution of MMR-defective tumors. In this review, we summarize current evidence of mutational biases within microsatellites arising as a consequence of intrinsic DNA sequence effects as well as variation in MMR efficiency. Microsatellite mutational biases are generally not considered during clinical testing; however, we suggest that such biases may be clinically significant as a factor contributing to phenotypic variation among microsatellite instability-positive tumors.

DNA structure and the Werner protein modulate human DNA polymerase delta-dependent replication dynamics within the common fragile site FRA16D.

Common fragile sites (CFS) are chromosomal regions that exhibit instability during DNA replication stress. Although the mechanism of CFS expression has not been fully elucidated, one known feature is a severely delayed S-phase. We used an in vitro primer extension assay to examine the progression of DNA synthesis through various sequences within FRA16D by the replicative human DNA polymerases delta and alpha, and with human cell-free extracts. We found that specific cis-acting sequence elements perturb DNA elongation, causing inconsistent DNA synthesis rates between regions on the same strand and complementary strands. Pol delta was significantly inhibited in regions containing hairpins and microsatellites, [AT/TA](24) and [A/T](19-28), compared with a control region with minimal secondary structure. Pol delta processivity was enhanced by full length Werner Syndrome protein (WRN) and by WRN fragments containing either the helicase domain or DNA-binding C-terminal domain. In cell-free extracts, stalling was eliminated at smaller hairpins, but persisted in larger hairpins and microsatellites. Our data support a model whereby CFS expression during cellular stress is due to a combination of factors--density of specific DNA secondary-structures within a genomic region and asymmetric rates of strand synthesis.

Phenotypic heterogeneity and lesion size measurements in Stargardt macular dystrophy.

Fundus autofluorescence has been used in Stargardt macular dystrophy and fundus flavimaculatus to better understand disease status and progression. Fundus autofluorescence images delineate atrophic regions with great clarity and high contrast. The authors investigated whether fundus autofluorescence images revealed more atrophy than color fundus photographs by studying a family of three siblings with long-standing disease that demonstrated a heterogeneous fundus appearance. Findings on fundus autofluorescence were compared with color fundus photographs and fluorescein images. Optical coherence tomography images were also analyzed. Areas of atrophic lesions were measured and compared on fundus autofluorescence and color fundus photographs. Autofluorescence images delineated atrophic regions more clearly than color fundus photographs. However, the measured areas of atrophy were only modestly larger in fundus autofluorescence images than in color fundus photographs. The optical coherence tomography images showed severe outer retinal atrophy and loss of foveal contour.

Transconjunctival 20-gauge pars plana vitrectomy using a single entry cannulated sutureless system.

PURPOSE: Pars plana vitrectomy with a 20-gauge transconjunctival cannulated sutureless (TCS) system has the potential of combining the advantages of smaller-gauge vitrectomy systems with the economical advantage of not needing to purchase any additional handheld instruments. However, the sclerotomy size is much larger, and self-sealing sclerotomies may be more difficult to construct. Therefore, we evaluated the need for sclerotomy suturing after performing 20-gauge TCS vitrectomy. METHODS: A retrospective chart review was performed on the first consecutive 55 eyes of 54 patients who underwent 20-gauge TCS vitrectomy. The main outcome measure was the number of sclerotomies requiring suturing and complications. RESULTS: Of the 164 sclerotomies made, 101 sclerotomies (62%) were not sutured, whereas the remaining 63 sclerotomies (38%) were closed with a single transconjunctival- scleral suture. The reasons for suturing included leakage and gaping at the sclerotomy, conjunctiva not covering the sclerotomy site, and prevention of gas leak. Complications noted include premature dislodging of cannulas, retinal tear, hypotony, hemorrhagic choroidals, subconjunctival gas, and less than full gas fill. CONCLUSION: Twenty-gauge transconjunctival sutureless vitrectomy is associated with risks similar to other cannulated systems while retaining most of the functionality and handheld instrumentation of the 20-gauge approach. A possibly higher sclerotomy suturing rate relative to smaller-gauge approaches is a disadvantage of this technique.

Human postmeiotic segregation 2 exhibits biased repair at tetranucleotide microsatellite sequences.

The mismatch repair (MMR) system plays a major role in removing DNA polymerization errors, and loss of this pathway results in hereditary cancers characterized by microsatellite instability. We investigated microsatellite stability during DNA replication within human postmeiotic segregation 2 (hPMS2)-deficient and proficient human lymphoblastoid cell lines. Using a shuttle vector assay, we measured mutation rates at reporter cassettes containing defined mononucleotide, dinucleotide, and tetranucleotide microsatellite sequences. A mutator phenotype was observed in the hPMS2-deficient cell line. The mutation rate of vectors containing [G/C](10) or [GT/CA](10) alleles was elevated 20-fold to 40-fold in hPMS2-deficient cells, relative to an hPMS2-expressing cell line. We observed a 6-fold and 12-fold relative increase in mutation rate of [TTTC/AAAG](9) and [TTCC/AAGG](9) sequences, respectively, in hPMS2-deficient cells. Mutational specificity analyses suggested that repair by hPMS2 is biased. In the absence of hPMS2, a greater number of microsatellite expansion versus deletion mutations was observed, and expansion rates of the tetranucleotide alleles were similar. In the presence of hPMS2, we observed a 29-fold decrease in the [TTCC/AAGG](9) expansion rate but only a 6-fold decrease for the [TTTC/AAAG](9) allele. Our data indicate that hPMS2 is more protective of tetranucleotide expansions than deletions and that hPMS2 displays a sequence bias, wherein [TTCC/AAGG] sequences are stabilized to a greater extent than [TTTC/AAAG]. Our results allow for greater accuracy during identification of MMR defects by providing a mutational signature characteristic of hPMS2 defect. This study also provides clues to possible mechanisms of repair by hPMS2 in the context of the MMR system.