Natural and cryptic peptides dominate the immunopeptidome of atypical teratoid rhabdoid tumors

BACKGROUND: Atypical teratoid/rhabdoid tumors (AT/RT) are highly aggressive CNS tumors of infancy and early childhood. Hallmark is the surprisingly simple genome with inactivating mutations or deletions in the SMARCB1 gene as the oncogenic driver. Nevertheless, AT/RTs are infiltrated by immune cells and even clonally expanded T cells. However, it is unclear which epitopes T cells might recognize on AT/RT cells. METHODS: Here, we report a comprehensive mass spectrometry (MS)-based analysis of naturally presented human leukocyte antigen (HLA) class I and class II ligands on 23 AT/RTs. MS data were validated by matching with a human proteome dataset and exclusion of peptides that are part of the human benignome. Cryptic peptide ligands were identified using Peptide-PRISM. RESULTS: Comparative HLA ligandome analysis of the HLA ligandome revealed 55 class I and 139 class II tumor-exclusive peptides. No peptide originated from the SMARCB1 region. In addition, 61 HLA class I tumor-exclusive peptide sequences derived from non-canonically translated proteins. Combination of peptides from natural and cryptic class I and class II origin gave optimal representation of tumor cell compartments. Substantial overlap existed with the cryptic immunopeptidome of glioblastomas, but no concordance was found with extracranial tumors. More than 80% of AT/RT exclusive peptides were able to successfully prime CD8(+) T cells, whereas naturally occurring memory responses in AT/RT patients could only be detected for class II epitopes. Interestingly, >50% of AT/RT exclusive class II ligands were also recognized by T cells from glioblastoma patients but not from healthy donors. CONCLUSIONS: These findings highlight that AT/RTs, potentially paradigmatic for other pediatric tumors with a low mutational load, present a variety of highly immunogenic HLA class I and class II peptides from canonical as well as non-canonical protein sources. Inclusion of such cryptic peptides into therapeutic vaccines would enable an optimized mapping of the tumor cell surface, thereby reducing the likelihood of immune evasion

Therapy with lopinavir/ritonavir and hydroxychloroquine is associated with acute kidney injury in COVID-19 patients.

BackgroundAcute kidney injury (AKI) is an independent risk factor for mortality, which affects about 5% of hospitalized coronavirus disease-2019 (COVID-19) patients and up to 25-29% of severely ill COVID-19 patients. Lopinavir/ritonavir and hydroxychloroquine show in vitro activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and have been used for the treatment of COVID-19. Both, lopinavir and hydroxychloroquine are metabolized by cytochrome P450 (CYP) 3A4. The impact of a triple therapy with lopinavir/ritonavir and hydroxychloroquine (triple therapy) on kidney function in COVID-19 is currently not known.MethodsWe retrospectively analyzed both non-ICU and ICU patients with COVID-19 receiving triple therapy for the incidence of AKI. Patients receiving standard therapy served as a control group. All patients were hospitalized at the University Hospital of Freiburg, Germany, between March and April 2020. A matched-pair analysis for the National Early Warning Score (NEWS) 2 was performed to control for the severity of illness among non-intensive care unit (ICU) patients.ResultsIn non-ICU patients, the incidence of AKI was markedly increased following triple therapy (78.6% vs. 21.4% in controls, p = 0.002), while a high incidence of AKI was observed in both groups of ICU patients (triple therapy: 80.0%, control group: 90.5%). ICU patients treated with triple therapy showed a trend towards more oliguric or anuric kidney injury. We also observed a linear correlation between the duration of the triple therapy and the maximum serum creatinine level (p = 0.004, R2 = 0.276, R = 0.597).ConclusionTriple therapy is associated with an increase in the incidence of AKI in non-ICU COVID-19 patients. The underlying mechanisms may comprise a CYP3A4 enzyme interaction, and may be relevant for any future therapy combining hydroxychloroquine with antiviral agents

CNTF Attenuates Vasoproliferative Changes Through Upregulation of SOCS3 in a Mouse-Model of Oxygen-Induced Retinopathy

PURPOSE. Retinal vascular disease represents a major cause for vision loss in the Western world. Recent research has shown that neuronal and vascular damage are closely related in retinal disease. Ciliary neurotrophic factor (CNTF) is a well-studied neurotrophic factor that is currently being tested in clinical trials for the treatment of retinal degenerative diseases and macular telangiectasia. However, little is known about its effect on retinal vasculature. In this study, we investigate the effects of CNTF in retinal neovascular disease using the mouse model of oxygen-induced retinopathy (OIR). METHODS. Newborn pups were exposed to 75% oxygen from postnatal day (P) 7 to P12 and subsequently returned to room air. Ciliary neurotrophic factor was injected intravitreally at OIR P12 and the vaso-obliterated and neovascular areas were quantified at OIR P17. Immunohistochemistry, RNA, and protein analysis were used to identify CNTF-responsive cells. In vitro experiments were performed to analyze the effect of CNTF on endothelial and astroglial cells. RESULTS. In the OIR model, CNTF facilitated capillary regrowth and attenuated preretinal neovascularization in a dose-dependent manner. The protective effect of CNTF was mediated via activation of the JAK/STAT3/SOCS3 signaling pathway. Immunohistochemical studies identified endothelial cells among others as CNTF-responsive cells in the retina. In vitro studies confirmed the anti-angiogenic effect of CNTF on endothelial cell sprouting. CONCLUSIONS. This study provides evidence for a therapeutic potential of CNTF beyond degenerative retinal disease. Vasoproliferative retinopathies may benefit from a CNTF-dependent and SOCS3-mediated angiomodulatory effect

Pre-Analytical Parameters Affecting Vascular Endothelial Growth Factor Measurement in Plasma: Identifying Confounders

Background Vascular endothelial growth factor-A (VEGF-A) is intensively investigated in various medical fields. However, comparing VEGF-A measurements is difficult because sample acquisition and pre-analytic procedures differ between studies. We therefore investigated which variables act as confounders of VEGF-A measurements. Methods Following a standardized protocol, blood was taken at three clinical sites from six healthy participants (one male and one female participant at each center) twice one week apart. The following pre-analytical parameters were varied in order to analyze their impact on VEGF-A measurements: analyzing center, anticoagulant (EDTA vs. PECT/CTAD), cannula (butterfly vs. neonatal), type of centrifuge (swing-out vs. fixed-angle), time before and after centrifugation, filling level (completely filled vs. half-filled tubes) and analyzing method (ELISA vs. multiplex bead array). Additionally, intrapersonal variations over time and sex differences were explored. Statistical analysis was performed using a linear regression model. Results The following parameters were identified as statistically significant independent confounders of VEGF-A measurements: analyzing center, anticoagulant, centrifuge, analyzing method and sex of the proband. The following parameters were no significant confounders in our data set: intrapersonal variation over one week, cannula, time before and after centrifugation and filling level of collection tubes. Conclusion VEGF-A measurement results can be affected significantly by the identified pre-analytical parameters. We recommend the use of CTAD anticoagulant, a standardized type of centrifuge and one central laboratory using the same analyzing method for all samples

Impact of angiogenic activation and inhibition on miRNA profiles of human retinal endothelial cells

Background: Human retinal microvascular endothelial cells (HRMVECs) are involved in the pathogenesis of retinopathy of prematurity. In this study, the microRNA (miRNA) expression profiles of HRMVECs were investigated under resting conditions, angiogenic stimulation (VEGF treatment) and anti-VEGF treatment. Materials and methods: The miRNA profiles of HRMVECs under resting and angiogenic conditions (VEGF treatment), as well as after addition of aflibercept, bevacizumab or ranibizumab were evaluated by analyzing the transcriptome of small non-coding RNAs. Differentially expressed miRNAs were validated using qPCR and classified using Gene Ontology enrichment analysis. Results: Ten miRNAs were found to be significantly changed more than 2-fold. Seven of these miRNAs were changed between resting conditions and angiogenic stimulation. Four of these miRNAs (miR-139-5p/-3p and miR-335-5p/-3p) were validated by qPCR in independent experiments and were found to be associated with angiogenesis and cell migration in Gene Ontology analysis. In addition, analysis of the most abundant miRNAs in the HRMVEC miRNome (representing at least 1% of the miRNome) was conducted and identified miR-21-5p, miR-29a.3p, miR.100-5p and miR-126-5p/-3p to be differently expressed by at least 15% between resting conditions and angiogenic conditions. These miRNAs were found to be associated with apoptotic signaling, regulation of kinase activity, intracellular signal transduction, cell surface receptor signaling and positive regulation of cell differentiation in Gene Ontology analysis. No differentially regulated miRNAs between angiogenic stimulation and angiogenic stimulation plus anti-VEGF treatment were identified. Conclusion: In this study we characterized the miRNA profile of HRMVECs under resting, angiogenic and antiangiogenic conditions and identified several miRNAs of potential pathophysiologic importance for angioproli-ferative retinal diseases. Our results have implications for possible miRNA-targeted angiomodulatory approaches in diseases like diabetic retinopathy or retinopathy of prematurity

Semaphorin 3F Modulates Corneal Lymphangiogenesis and Promotes Corneal Graft Survival

PURPOSE. Corneal vascularization significantly increases the risk for graft rejection after keratoplasty. Semaphorin 3F (Sema3F) is a known modulator of physiologic avascularity in the outer retina. The aim of this study was to investigate whether Sema3F is involved in maintaining corneal avascularity and can reduce the risk for corneal graft rejection. METHODS. Corneal Sema3F expression was investigated using immunohistochemistry and qPCR in human and murine tissue. Pathologic invasion of blood and lymph vessels into corneal tissue was analyzed in the murine corneal suture and high-risk keratoplasty model. The anti-lymphangiogenic effects of Sema3F were further investigated using an in vitro spheroidal sprouting model with supernatant from isolated primary human corneal epithelial cells (hCECs). RESULTS. Sema3F is constitutively expressed in human and murine corneal epithelium. In the corneal suture model, lymphangiogenesis was significantly suppressed by topical Sema3F treatment (P = 0.0003). In the murine high-risk keratoplasty model, pretreatment by topical Sema3F in the inflammation phase significantly promoted subsequent graft survival (P = 0.0006). In this model, both lymph-and blood angiogenesis were reduced (P < 0.05). In vitro, hCEC supernatant had a direct anti-lymphangiogenic effect on human lymphatic endothelial cells (P < 0.01). This effect was completely abolished by addition of anti-Sema3F antibodies. CONCLUSIONS. Sema3F is a novel mediator of corneal avascularity with potent antilymphangiogenic properties. Topical treatment with Sema3F eye drops may help to limit corneal vascularization and improve outcomes in high-risk keratoplasty patients

Comparing Alternative Ranibizumab Dosages for Safety and Efficacy in Retinopathy of Prematurity

IMPORTANCE: Anti-vascular endothelial growth factor (VEGF) therapies are a novel treatment option in retinopathy of prematurity (ROP). Data on dosing, efficacy, and safety are insufficient. OBJECTIVE: To investigate lower doses of anti-VEGF therapy with ranibizumab, a substance with a significantly shorter systemic half-life than the standard treatment, bevacizumab. DESIGN, SETTING, AND PARTICIPANTS: This randomized, multicenter, double-blind, investigator-initiated trial at 9 academic medical centers in Germany compared ranibizumab doses of 0.12 mg vs 0.20 mg in infants with bilateral aggressive posterior ROP; ROP stage 1 with plus disease, 2 with plus disease, or 3 with or without plus disease in zone I; or ROP stage 3 with plus disease in posterior zone II. Patients were recruited between September 2014 and August 2016. Twenty infants were screened and 19 were randomized. INTERVENTIONS: All infants received 1 baseline ranibizumab injection per eye. Reinjections were allowed in case of ROP recurrence after at least 28 days. MAIN OUTCOMES AND MEASURES: The primary end point was the number of infants who did not require rescue therapy at 24 weeks. Key secondary end points included time-to-event analyses, progression of physiologic vascularization, and plasma VEGF levels. Stages of ROP were photodocumented and reviewed by an expert committee. RESULTS: Nineteen infants with ROP were enrolled (9 [47.4%] female; median [range] postmenstrual age at first treatment, 36.4 [34.7-39.7] weeks), 3 of whom died during the study (1 in the 0.12-mg group and 2 in the 0.20-mg group). Of the surviving infants, 8 (88.9%) (17 eyes [94.4%]) in the 0.12-mg group and 6 (85.7%) (13 eyes [92.9%]) in the 0.20-mg group did not require rescue therapy. Both ranibizumab doses were equally successful in controlling acute ROP (Cochran-Mantel-Haenszel analysis; odds ratio, 1.88; 95% CI, 0.26-13.49; P = .53). Physiologic intraretinal vascularization was superior in the 0.12-mg group. The VEGF plasma levels were not systematically altered in either group. CONCLUSIONS AND RELEVANCE: This pilot study demonstrates that ranibizumab is effective in controlling acute ROP and that 24% of the standard adult dose (0.12 mg) appears equally effective as 40% (0.20 mg). Superior vascularization of the peripheral retina with 0.12 mg of ranibizumab indicates that the lower dose may be favorable. Unchanged plasma VEGF levels point toward a limited systemic drug exposure after ranibizumab

Sema3f Protects Against Subretinal Neovascularization In Vivo

Pathological neovascularization of the outer retina is the hallmark of neovascular age-related macular degeneration (nAMD). Building on our previous observations that semaphorin 3F (Sema3f) is expressed in the outer retina and demonstrates anti-angiogenic potential, we have investigated whether Sema3f can be used to protect against subretinal neovascularization in two mouse models. Both in the very low-density lipid-receptor knockout (Vldlr−/−) model of spontaneous subretinal neovascularization as well as in the mouse model of laser-induced choroidal neovascularization (CNV), we found protective effects of Sema3f against the formation of pathologic neovascularization. In the Vldlr−/− model, AAV-induced overexpression of Sema3f reduced the size of pathologic neovascularization by 56%. In the laser-induced CNV model, intravitreally injected Sema3f reduced pathologic neovascularization by 30%. Combined, these results provide the first evidence from two distinct in vivo models for a use of Sema3f in protecting the outer retina against subretinal neovascularization