[Fluorescence Angiography-assisted Management of Recurrences in Aggressive Posterior Retinopathy of Prematurity (APROP) after Intravitreal Monotherapy with 0.312 mg Bevacizumab]. / Fluoreszenzangiografieassistiertes Management von Rezidiven bei aggressiver posteriorer Frühgeborenenretinopathie (APROP) nach intravitrealer Monotherapie mit 0,312 mg Bevacizumab.

BACKGROUND: In cases of aggressive posterior retinopathy of prematurity (APROP), recurrences can occur after intravitreal injection of bevacizumab (IVB), in spite of successful treatment of the acute stage. Therefore, long-term examinations in extremely premature patients are needed. We defined recurrences as a relapse of plus disease and leakage (with or without proliferation) at the vascularisation border, but also anterior and posterior to it. METHODS: RetCam wide-field colour images and fluorescein angiography were performed before the first IVB (0.312 mg bevacizumab in 0.025 ml per eye), before each further therapy, i.e. additional intravitreal injection, laser- or cryocoagulation or pars-plana vitrectomy, and at the end of the therapy. We analysed the images of 18 eyes with APROP of 9 extreme premature patients treated between 08/2007 and 12/2017 (GA 21 - 27 weeks, BW 430 - 890 g). RESULTS: Long-term therapeutic success was achieved in only 4 eyes/2 children (22%) with one single injection. In 2 eyes/2 children (11%), a second and third injection was given within 2 weeks because of an insufficient therapeutic effect. Up to 3 injections together with laser coagulation were needed in 12 eyes/6 children (67%), in order to achieve complete resolution of ROP activity. In 6 eyes/2 children (33%), resolution of leakage at the original vascularisation border was achieved only with further laser coagulation. In one single eye, retinal detachment occurred after unsuccessful retinal surgery. Before IVB, fluorescein angiography disclosed leakage due to proliferation in most of the patients (12 eyes/6 children). In recurrences after IVB, a posterior shift of the leakage site was found (14 eyes/4 children), whereas after laser photocoagulation proliferative changes were also detected anterior to the vascularisation border (5 eyes/3 children). Treatment was indicated based on angiographic findings in 14 eyes/4 children where wide-field colour images did not show plus disease or proliferation. CONCLUSIONS: Intravitreal injection of 0.312 mg bevacizumab has been shown to be an effective therapy for the acute stage of APROP. Long-term success required consequent monitoring and treatment of APROP recurrences. Fluorescein angiography was particularly useful to detect recurrences that were not evident in wide-field colour images.

Oncostatin M induces RIG-I and MDA5 expression and enhances the double-stranded RNA response in fibroblasts.

Interleukin (IL)-6-type cytokines have no direct antiviral activity; nevertheless, they display immune-modulatory functions. Oncostatin M (OSM), a member of the IL-6 family, has recently been shown to induce a distinct number of classical interferon stimulated genes (ISG). Most of them are involved in antigen processing and presentation. However, induction of retinoic acid-inducible gene (RIG)-I-like receptors (RLR) has not been investigated. Here we report that OSM has the capability to induce the expression of the DExD/H-Box RNA helicases RIG-I and melanoma differentiation antigen 5 (MDA5) as well as of the transcription factors interferon regulatory factor (IRF)1, IRF7 and IRF9 in primary fibroblasts. Induction of the helicases depends on tyrosine as well as serine phosphorylation of STAT1. Moreover, we could show that the OSM-induced STAT1 phosphorylation is predominantly counter-regulated by a strong STAT3-dependent SOCS3 induction, as Stat3 as well as Socs3 knock-down results in an enhanced and prolonged helicase and IRF expression. Other factors involved in regulation of STAT1 or IRF1 activity, like protein tyrosine phosphatase, non-receptor type 2 (PTPN2), promyelocytic leukaemia protein (PML) or small ubiquitin-related modifier 1 (SUMO1), play a minor role in OSM-mediated induction of RLR. Remarkably, OSM and interferon-γ (IFN-γ) synergize to mediate transcription of RLR and pre-treatment of fibroblasts with OSM fosters the type I interferon production in response to a subsequent encounter with double-stranded RNA. Together, these findings suggest that the OSM-induced JAK/STAT1 signalling is implicated in virus protection of non-professional immune cells and may cooperate with interferons to enhance RLR expression in these cells.

RETINAL VASCULAR DEVELOPMENT WITH 0.312 MG INTRAVITREAL BEVACIZUMAB TO TREAT SEVERE POSTERIOR RETINOPATHY OF PREMATURITY: A Longitudinal Fluorescein Angiographic Study.

PURPOSE: To report the outcome of intravitreal 0.312 mg bevacizumab (IVB) monotherapy in acute retinopathy of prematurity (ROP) and to describe the vascular development over time. METHODS: Seventeen prematurely born infants were treated with IVB (0.312 mg in 0.025 mL per eye) because of acute ROP in posterior Zone II or Zone I, including aggressive posterior ROP. Infants were examined by fluorescein angiography (FA) using RetCam II or III (Clarity Medical Systems Inc) before IVB (n = 21 eyes), within 6 weeks (n = 23 eyes), 8 to 13 weeks (n = 22 eyes), and up to 45 months (n = 10 eyes). RESULTS: Acute ROP regressed in 19 out of 27 analyzed eyes (70%), including 100% and 80% of posterior Zone II and Zone I eyes, respectively, but only 25% of aggressive posterior ROP eyes. Early recurrences (11%, all aggressive posterior ROP) and late reactivations (18%) were observed within 1 week and at 9 to 12 weeks, respectively. All eyes showed leakage at the junction of the vascularized zone and capillary malformation on FA before treatment. Vessel branching abnormalities and circumferential vessel formation were typical FA features after treatment. Vascular outgrowth after one IVB became complete in 87.5% of eyes for which FA was available up to at least 9 weeks after IVB. CONCLUSION: A single dose of 0.312 mg bevacizumab was efficient to induce regression of ROP in posterior Zone II and most of Zone I cases, but not in aggressive posterior ROP. FA describes vascular abnormalities, the importance of which warrants further investigation.

Endocytosis of pro-inflammatory cytokine receptors and its relevance for signal transduction.

The pro-inflammatory cytokines tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-6 (IL-6) are key players of the innate and adaptive immunity. Their activity needs to be tightly controlled to allow the initiation of an appropriate immune response as defense mechanism against pathogens or tissue injury. Excessive or sustained signaling of either of these cytokines leads to severe diseases, including rheumatoid arthritis, inflammatory bowel diseases (Crohn's disease, ulcerative colitis), steatohepatitis, periodic fevers and even cancer. Studies carried out in the last 30 years have emphasized that an elaborate control system for each of these cytokines exists. Here, we summarize what is currently known about the involvement of receptor endocytosis in the regulation of these pro-inflammatory cytokines' signaling cascades. Particularly in the last few years it was shown that this cellular process is far more than a mere feedback mechanism to clear cytokines from the circulation and to shut off their signal transduction.

The crystal structure of the protein-disulfide isomerase family member ERp27 provides insights into its substrate binding capabilities.

About one-third of all cellular proteins pass through the secretory pathway and hence undergo oxidative folding in the endoplasmic reticulum (ER). Protein-disulfide isomerase (PDI) and related members of the PDI family assist in the folding of substrates by catalyzing the oxidation of two cysteines and isomerization of disulfide bonds as well as by acting as chaperones. In this study, we present the crystal structure of ERp27, a redox-inactive member of the PDI family. The structure reveals its substrate-binding cleft, which is homologous to PDI, but is able to adapt in size and hydrophobicity. Isothermal titration calorimetry experiments demonstrate that ERp27 is able to distinguish between folded and unfolded substrates, only interacting with the latter. ERp27 is up-regulated during ER stress, thus presumably allowing it to bind accumulating misfolded substrates and present them to ERp57 for catalysis.

[10 years of screening for retinopathy of prematurity (2009-2019) : Results analysis of two German level-1 neonatal intensive care units (NICUs) with university on-site screening and a telemedical approach in the non-university NICU]. / 10 Jahre Screening auf Frühgeborenenretinopathie (2009­2019) : Ergebnisanalyse zweier deutscher Level-1-Perinatalzentren mit universitärem Vor-Ort-Screening und telemedizinischem Ansatz in dem nicht universitären Zentrum.

BACKGROUND: Retinopathy of prematurity (ROP) is one of the most common causes of childhood blindness in Germany and worldwide and adequate screening is essential. The telemedical approach with objective documentation of retinal findings opens up the possibility of reliably diagnosing all ROP stages independent of the examiner, if a team of ophthalmologists specialized in ROP evaluates the images. OBJECTIVE: A 10-year comparison of ROP screening at two level­1 neonatal intensive care units (NICU): university and on-site vs. non-university and telemedical. MATERIAL AND METHODS: Retrospective analysis of screened premature infants by gestational age (GA), birth weight (BW), sex, multiple births, time of ROP occurrence, treatment needs and time as well as examination frequency. RESULTS: From 2009 to 2019, we screened 1191 infants of whom 29 had been screened before by an external clinic. The internal 1162 infants were screened on-site with 3713 retinal examinations. We diagnosed 34% with ROP and treated 5.4% (3.7% in Giessen, 7.2% in Siegen). Mean GA was 28.9 weeks (±â€¯2.5 weeks); mean BW 1155 g (±â€¯417.5 g). The number of ROP diagnoses increased by 227.3% in Giessen and by 111.1% in Siegen due to the increasing number of premature births in neonatal care. CONCLUSION: Comparative analysis confirmed nationally and internationally increasing ROP screening and children with acute ROP. Telemedical screening was equivalent to on-site screening and safe. Both screening methods identified infants requiring treatment on time. No child with GA > 29 weeks required treatment, analogous to Swedish ROP registry results; however, in the German ROP registry some premature babies with GA ≥ 30 weeks required treatment.

Cross-regulation of cytokine signalling: pro-inflammatory cytokines restrict IL-6 signalling through receptor internalisation and degradation.

The inflammatory response involves a complex interplay of different cytokines which act in an auto- or paracrine manner to induce the so-called acute phase response. Cytokines are known to crosstalk on multiple levels, for instance by regulating the mRNA stability of targeted cytokines through activation of the p38-MAPK pathway. In our study we discovered a new mechanism that answers the long-standing question how pro-inflammatory cytokines and environmental stress restrict immediate signalling of interleukin (IL)-6-type cytokines. We show that p38, activated by IL-1beta, TNFalpha or environmental stress, impairs IL-6-induced JAK/STAT signalling through phosphorylation of the common cytokine receptor subunit gp130 and its subsequent internalisation and degradation. We identify MK2 as the kinase that phosphorylates serine 782 in the cytoplasmic part of gp130. Consequently, inhibition of p38 or MK2, deletion of MK2 or mutation of crucial amino acids within the MK2 target site or the di-leucine internalisation motif blocks receptor depletion and restores IL-6-dependent STAT activation as well as gene induction. Hence, a novel negative crosstalk mechanism for cytokine signalling is described, where cytokine receptor turnover is regulated in trans by pro-inflammatory cytokines and stress stimuli to coordinate the inflammatory response.

A role for upstream binding factor in organizing ribosomal gene chromatin.

Human ribosomal genes are located in NORs (nucleolar organizer regions) on the short arms of acrocentric chromosomes. During metaphase, previously active NORs appear as prominent chromosomal features termed secondary constrictions, which are achromatic in chromosome banding and positive in silver staining. The architectural RNA polymerase I transcription factor UBF (upstream binding factor) binds extensively across the ribosomal gene repeat throughout the cell cycle. Evidence that UBF underpins NOR structure is provided by an examination of cell lines in which large arrays of a heterologous UBF binding sequences are integrated at ectopic sites on human chromosomes. These arrays efficiently recruit UBF even to sites outside the nucleolus, and during metaphase form novel silver-stainable secondary constrictions, termed pseudo-NORs, that are morphologically similar to NORs.

UBF-binding site arrays form pseudo-NORs and sequester the RNA polymerase I transcription machinery.

Human ribosomal genes (rDNA) are located in nucleolar organizer regions (NORs) on the short arms of acrocentric chromosomes. Metaphase NORs that were transcriptionally active in the previous cell cycle appear as prominent chromosomal features termed secondary constrictions that are achromatic in chromosome banding and positive in silver staining. The architectural RNA polymerase I (pol I) transcription factor UBF binds extensively across rDNA throughout the cell cycle. To determine if UBF binding underpins NOR structure, we integrated large arrays of heterologous UBF-binding sequences at ectopic sites on human chromosomes. These arrays efficiently recruit UBF even to sites outside the nucleolus and, during metaphase, form novel silver stainable secondary constrictions, termed pseudo-NORs, morphologically similar to NORs. We demonstrate for the first time that in addition to UBF the other components of the pol I machinery are found associated with sequences across the entire human rDNA repeat. Remarkably, a significant fraction of these same pol I factors are sequestered by pseudo-NORs independent of both transcription and nucleoli. Because of the heterologous nature of the sequence employed, we infer that sequestration is mediated primarily by protein-protein interactions with UBF. These results suggest that extensive binding of UBF is responsible for formation and maintenance of the secondary constriction at active NORs. Furthermore, we propose that UBF mediates recruitment of the pol I machinery to nucleoli independently of promoter elements.

On the formation of amplified nucleoli during early Xenopus oogenesis.

In Xenopus the genes for ribosomal RNA are selectively amplified during the early stages of oogenesis and give rise to over 1000 extrachromosomal nucleoli. These oocyte nucleoli are unique in that they contain very high copy numbers of rRNA genes, are not attached to chromosomes, and lack nonribosomal DNA. How the amplified rRNA genes induce the formation of multiple nucleoli is as yet poorly understood. To gain some more insight into this assembly process we have studied nucleolar development in early previtellogenic Xenopus oocytes. By using light and electron microscopic immunocytochemistry, in situ hybridization, and the TdT-method for detection of DNA, we have identified three separate structural entities which are related to the three components of the functionally active nucleoli present in midsized oocytes. Besides fibrillar and granular bodies we describe a novel rod-like structure which contains the pol I-specific transcription factor UBF. Notably, rDNA emerges from these rods forming a filamentous layer. These results reinforce UBF's role as an architectural element involved in the spatial organization of the rRNA genes. We consider the rod-like rDNA/UBF complexes as extrachromosomal nucleolar organizers which are first juxtaposed to preformed fibrillar bodies before both elements gradually fuse into a coherent nucleolar structure.