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Effective therapeutics are necessary for managing severe COVID-19 disease despite the availability of vaccines. Small interfering RNA (siRNA) can silence viral genes and restrict SARS-CoV-2 replication. Cell-penetrating peptides is a robust method for siRNA delivery, enhancing siRNA stability and targeting specific receptors. We developed a peptide HE25 that blocks SARS-CoV-2 replication by various mechanisms, including the binding of multiple receptors involved in the virus's internalization, such as ACE2, integrins and NRP1. HE25 not only acts as a vehicle to deliver the SARS-CoV-2 RNA-dependent RNA polymerase siRNA into cells but also facilitates their internalization through endocytosis. Once inside endosomes, the siRNA is released into the cytoplasm through the Histidine-proton sponge effect and the selective cleavage of HE25 by cathepsin B. These mechanisms effectively inhibited the replication of the ancestral SARS-CoV-2 and the Omicron variant BA.5 in vitro. When HE25 was administered in vivo, either by intravenous injection or inhalation, it accumulated in lungs, veins and arteries, endothelium, or bronchial structure depending on the route. Furthermore, the siRNA/HE25 complex caused gene silencing in lung cells in vitro. The SARS-CoV-2 siRNA/HE25 complex is a promising therapeutic for COVID-19, and a similar strategy can be employed to combat future emerging viral diseases.
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Background: Mutation-derived neoantigens are critical targets for tumor rejection in cancer immunotherapy, and better tools for neoepitope identification and prediction are needed to improve neoepitope targeting strategies. Computational tools have enabled the identification of patient-specific neoantigen candidates from sequencing data, but limited data availability has hindered their capacity to predict which of the many neoepitopes will most likely give rise to T cell recognition. Method: To address this, we make use of experimentally validated T cell recognition towards 17,500 neoepitope candidates, with 467 being T cell recognized, across 70 cancer patients undergoing immunotherapy. Results: We evaluated 27 neoepitope characteristics, and created a random forest model, IMPROVE, to predict neoepitope immunogenicity. The presence of hydrophobic and aromatic residues in the peptide binding core were the most important features for predicting neoepitope immunogenicity. Conclusion: Overall, IMPROVE was found to significantly advance the identification of neoepitopes compared to other current methods.
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Neoplasias , Linfócitos T , Humanos , Imunoterapia/métodosRESUMO
CD8 T cells provide immunity to virus infection through recognition of epitopes presented by peptide major histocompatibility complexes (pMHCs). To establish a concise panel of widely recognized T cell epitopes from common viruses, we combined analysis of TCR down-regulation upon stimulation with epitope-specific enumeration based on barcode-labeled pMHC multimers. We assess CD8 T cell binding and reactivity for 929 previously reported epitopes in the context of 1 of 25 HLA alleles representing 29 viruses. The prevalence and magnitude of CD8 T cell responses were evaluated in 48 donors and reported along with 137 frequently recognized virus epitopes, many of which were underrepresented in the public domain. Eighty-four percent of epitope-specific CD8 T cell populations demonstrated reactivity to peptide stimulation, which was associated with effector and long-term memory phenotypes. Conversely, nonreactive T cell populations were associated primarily with naive phenotypes. Our analysis provides a reference map of epitopes for characterizing CD8 T cell responses toward common human virus infections.
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Linfócitos T CD8-Positivos , Epitopos de Linfócito T , Humanos , Alelos , Regulação para Baixo , PeptídeosRESUMO
Merkel cell carcinoma (MCC) is a highly immunogenic skin cancer primarily induced by Merkel cell polyomavirus, which is driven by the expression of the oncogenic T antigens (T-Ags). Blockade of the programmed cell death protein-1 (PD-1) pathway has shown remarkable response rates, but evidence for therapy-associated T-Ag-specific immune response and therapeutic strategies for the nonresponding fraction are both limited. We tracked T-Ag-reactive CD8+ T cells in peripheral blood of 26 MCC patients under anti-PD1 therapy, using DNA-barcoded pMHC multimers, displaying all peptides from the predicted HLA ligandome of the oncoproteins, covering 33 class I haplotypes. We observed a broad T cell recognition of T-Ags, including identification of 20 T-Ag-derived epitopes we believe to be novel. Broadening of the T-Ag recognition profile and increased T cell frequencies during therapy were strongly associated with clinical response and prolonged progression-free survival. T-Ag-specific T cells could be further boosted and expanded directly from peripheral blood using artificial antigen-presenting scaffolds, even in patients with no detectable T-Ag-specific T cells. These T cells provided strong tumor-rejection capacity while retaining a favorable phenotype for adoptive cell transfer. These findings demonstrate that T-Ag-specific T cells are associated with the clinical outcome to PD-1 blockade and that Ag-presenting scaffolds can be used to boost such responses.
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Carcinoma de Célula de Merkel , Neoplasias Cutâneas , Humanos , Antígenos Virais de Tumores , Carcinoma de Célula de Merkel/tratamento farmacológico , Carcinoma de Célula de Merkel/genética , Receptor de Morte Celular Programada 1/genética , Linfócitos T CD8-Positivos , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genéticaRESUMO
Merkel cell carcinoma is a skin cancer often driven by Merkel cell polyomavirus (MCPyV) with high rates of response to anti-PD-1 therapy despite low mutational burden. MCPyV-specific CD8 T cells are implicated in anti-PD-1-associated immune responses and provide a means to directly study tumor-specific T cell responses to treatment. Using mass cytometry and combinatorial tetramer staining, we find that baseline frequencies of blood MCPyV-specific cells correlated with response and survival. Frequencies of these cells decrease markedly during response to therapy. Phenotypes of MCPyV-specific CD8 T cells have distinct expression patterns of CD39, cutaneous lymphocyte-associated antigen (CLA), and CD103. Correspondingly, overall bulk CD39+CLA+ CD8 T cell frequencies in blood correlate with MCPyV-specific cell frequencies and similarly predicted favorable clinical outcomes. Conversely, frequencies of CD39+CD103+ CD8 T cells are associated with tumor burden and worse outcomes. These cell subsets can be useful as biomarkers and to isolate blood-derived tumor-specific T cells.
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Carcinoma de Célula de Merkel , Poliomavírus das Células de Merkel , Oligossacarídeos , Antígeno Sialil Lewis X/análogos & derivados , Neoplasias Cutâneas , Humanos , Carcinoma de Célula de Merkel/tratamento farmacológico , Carcinoma de Célula de Merkel/metabolismo , Carcinoma de Célula de Merkel/patologia , Poliomavírus das Células de Merkel/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Linfócitos T CD8-Positivos , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/metabolismo , Biomarcadores/metabolismoRESUMO
INTRODUCTION AND HYPOTHESIS: The objective was to investigate the adherence to pessary treatment in women with pelvic organ prolapse (POP) who were found eligible for this treatment by the urogynecologist, at the first visit at the Department of Gynecology and Obstetrics, Odense University Hospital. METHODS: Data were extracted from the women's medical records. Frequency tabulations were performed to describe the women's reasons for pessary discontinuation by age group. Binominal logistic regression analysis was conducted to investigate how women's age, POP characteristics, urogynecological history, and their pessary experience and management were associated with continued pessary use. RESULTS: This study included 1,371 women treated with support pessary. Of these, 850 women continued pessary treatment and 521 women underwent surgical treatment. A history of hysterectomy (OR: 0.68, 95% CI: 0.51-0.90, p = 0.008), urinary incontinence (OR: 0.71, 95% CI: 0.56-0.89, p = 0.003), and previous pessary use (OR: 0.75, 95% CI: 0.56-0.99, p = 0.047) were significant factors associated with discontinuation. Further, women aged 81-99 years were significantly more likely to continue pessary treatment (OR: 1.77, 95% CI: 1.15-2.74, p = 0.009). "POP surgery," "prolapse stage," and "prolapse predominant compartment" were not associated with discontinuation. Approximately 38% of women aged 26-54 years discontinued owing to personal preference. CONCLUSIONS: Hysterectomy, incontinence, and previous pessary use are significant predictors of pessary discontinuation. Increasing age is significantly associated with pessary continuation.
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Prolapso de Órgão Pélvico , Incontinência Urinária , Feminino , Humanos , Estudos Retrospectivos , Pessários , Prolapso de Órgão Pélvico/terapia , HisterectomiaRESUMO
BACKGROUND: Adoptive cell therapy (ACT) has shown promising results for the treatment of cancer and viral infections. Successful ACT relies on ex vivo expansion of large numbers of desired T-cells with strong cytotoxic capacity and in vivo persistence, which constitutes the greatest challenge to current ACT strategies. Here, in this study, we present a novel technology for ex vivo expansion of antigen-specific T-cells; artificial antigen-presenting scaffolds (Ag-scaffolds) consisting of a dextran-polysaccharide backbone, decorated with combinations of peptide-Major Histocompatibility Complex (pMHC), cytokines and co-stimulatory molecules, enabling coordinated stimulation of antigen-specific T-cells. METHODS: The capacity of Ag-scaffolds to expand antigen-specific T-cells was explored in ex vivo cultures with peripheral blood mononuclear cells from healthy donors and patients with metastatic melanoma. The resulting T-cell products were assessed for phenotypic and functional characteristics. RESULTS: We identified an optimal Ag-scaffold for expansion of T-cells for ACT, carrying pMHC and interleukin-2 (IL-2) and IL-21, with which we efficiently expanded both virus-specific and tumor-specific CD8+ T cells from peripheral blood of healthy donors and patients, respectively. The resulting T-cell products were characterized by a high frequency of antigen-specific cells with high self-renewal capacity, low exhaustion, a multifunctional cytokine profile upon antigen-challenge and superior tumor killing capacity. This demonstrates that the coordinated stimuli provided by an optimized stoichiometry of TCR engaging (pMHC) and stimulatory (cytokine) moieties is essential to obtain desired T-cell characteristics. To generate an 'off-the-shelf' multitargeting Ag-scaffold product of relevance to patients with metastatic melanoma, we identified the 30 most frequently recognized shared HLA-A0201-restricted melanoma epitopes in a cohort of 87 patients. By combining these in an Ag-scaffold product, we were able to expand tumor-specific T-cells from 60-70% of patients with melanoma, yielding a multitargeted T-cell product with up to 25% specific and phenotypically and functionally improved T cells. CONCLUSIONS: Taken together, the Ag-scaffold represents a promising new technology for selective expansion of antigen-specific CD8+ T cells directly from blood, yielding a highly specific and functionally enhanced T-cell product for ACT.
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Melanoma , Segunda Neoplasia Primária , Humanos , Imunoterapia Adotiva , Leucócitos Mononucleares , Melanoma/terapia , Citocinas , Receptores de Antígenos de Linfócitos TRESUMO
Aberrant hyperactivation of Wnt signaling, driven by nuclear ß-catenin in the colonic epithelium, represents the seminal event in the initiation and progression of colorectal cancer (CRC). Despite its established role in CRC tumorigenesis, clinical translation of Wnt inhibitors remains unsuccessful. Late SV40 factor (LSF; encoded by TFCP2) is a transcription factor and a potent oncogene. The current study identified a chemotype, named factor quinolinone inhibitors (FQIs), that specifically inhibits LSF DNA-binding, partner protein-binding, and transactivation activities. The role of LSF and FQIs in CRC tumor growth was examined. Herein, the study showed that LSF and ß-catenin interacted in several CRC cell lines irrespective of their mutational profile, which was disrupted by FQI2-34. FQI2-34 suppressed Wnt activity in CRC cells in a dose-dependent manner. Leveraging both allogeneic and syngeneic xenograft models showed that FQI2-34 suppressed CRC tumor growth, significantly reduced nuclear ß-catenin, and down-regulated Wnt targets such as axis inhibition protein 2 (AXIN-2) and SRY-box transcription factor 9, in the xenograft cells. FQI2-34 suppressed the proliferation of xenograft cells. Adenocarcinomas from a series of stage IV CRC patients revealed a positive correlation between LSF expression and Wnt targets (AXIN-2 and SRY-box transcription factor 9) within the CRC cells. Collectively, this study uncovers the Wnt inhibitory and CRC growth-suppressive effects of these LSF inhibitors in CRC cells, revealing a novel target in CRC therapeutics.
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Neoplasias do Colo , Neoplasias Colorretais , Transplante de Células-Tronco Hematopoéticas , Proteína Axina/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias do Colo/tratamento farmacológico , Neoplasias Colorretais/patologia , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismoRESUMO
Factor quinolinone inhibitors (FQIs), a first-in-class set of small molecule inhibitors targeted to the transcription factor LSF (TFCP2), exhibit promising cancer chemotherapeutic properties. FQI1, the initial lead compound identified, unexpectedly induced a concentration-dependent delay in mitotic progression. Here, we show that FQI1 can rapidly and reversibly lead to mitotic arrest, even when added directly to mitotic cells, implying that FQI1-mediated mitotic defects are not transcriptionally based. Furthermore, treatment with FQIs resulted in a striking, concentration-dependent diminishment of spindle microtubules, accompanied by a concentration-dependent increase in multi-aster formation. Aberrant γ-tubulin localization was also observed. These phenotypes suggest that perturbation of spindle microtubules is the primary event leading to the mitotic delays upon FQI1 treatment. Previously, FQIs were shown to specifically inhibit not only LSF DNA-binding activity, which requires LSF oligomerization to tetramers, but also other specific LSF-protein interactions. Other transcription factors participate in mitosis through non-transcriptional means, and we recently reported that LSF directly binds α-tubulin and is present in purified cellular tubulin preparations. Consistent with a microtubule role for LSF, here we show that LSF enhanced the rate of tubulin polymerization in vitro, and FQI1 inhibited such polymerization. To probe whether the FQI1-mediated spindle abnormalities could result from inhibition of mitotic LSF-protein interactions, mass spectrometry was performed using as bait an inducible, tagged form of LSF that is biotinylated by endogenous enzymes. The global proteomics analysis yielded expected associations for a transcription factor, notably with RNA processing machinery, but also to nontranscriptional components. In particular, and consistent with spindle disruption due to FQI treatment, mitotic, FQI1-sensitive interactions were identified between the biotinylated LSF and microtubule-associated proteins that regulate spindle assembly, positioning, and dynamics, as well as centrosome-associated proteins. Probing the mitotic LSF interactome using small molecule inhibitors therefore supported a non-transcriptional role for LSF in mediating progression through mitosis.
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Proteínas Associadas aos Microtúbulos , Quinolonas , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose , Quinolonas/metabolismo , Quinolonas/farmacologia , Fuso Acromático/metabolismo , Fatores de Transcrição/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
PURPOSE: Sym013 contains six humanized monoclonal antibodies that bind to non-overlapping epitopes on three human epidermal growth factor receptors (HER1-3). Preclinical studies suggested Sym013 strongly suppresses growth of multiple epithelial tumors. This is a first-in-human study exploring safety and efficacy of Sym013 in patients with advanced epithelial malignancies. METHODS: Dose escalation used single-patient cohorts until the stopping rule was met, followed by 3 + 3 design. Dose levels planned were: 1, 2, 4, 6, 9, 12, 15, and 18 mg/kg. Treatment cycles were 28 days with imaging every eight weeks. Serum samples were collected at multiple time points for assessment of pharmacokinetics and development of anti-drug antibodies. RESULTS: Thirty-two patients were enrolled with multiple solid tumors, most common being colorectal cancer (CRC; 10/32, 31%). Due to mucositis, rash, and diarrhea at 4 mg/kg once-weekly, dosing was changed to biweekly (Q2W). Mandatory prophylaxis was added due to Grade 3 infusion-related reaction and oral mucositis at 9 mg/kg Q2W. The 15 mg/kg Q2W cohort was enrolling when the study was terminated for business reasons. Most common adverse events were skin (81%) and gastrointestinal (75%) disorders, including dermatitis/rash, stomatitis, and diarrhea. One patient with CRC achieved a partial response; 12 patients with varied malignancies had stable disease. CONCLUSION: During the conduct of the study, management of frequent infusion-related reactions, skin toxicities, and mucosal disorders, which are indicative of HER inhibition, necessitated multiple protocol amendments. The investigators, in concert with the Sponsor, agreed that achieving a tolerated regimen with acceptable target saturation was unlikely. TRIAL REGISTRY: www. CLINICALTRIALS: gov ; NCT02906670 (September 20, 2016).
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Antineoplásicos , Exantema , Neoplasias Epiteliais e Glandulares , Neoplasias , Anticorpos Monoclonais/efeitos adversos , Antineoplásicos/efeitos adversos , Diarreia/induzido quimicamente , Exantema/induzido quimicamente , Humanos , Dose Máxima Tolerável , Neoplasias/metabolismo , Neoplasias Epiteliais e Glandulares/induzido quimicamente , Neoplasias Epiteliais e Glandulares/tratamento farmacológicoRESUMO
When the Norwegian government closed down schools and kindergartens in response to the increased spread of COVID-19, the use of homeschooling raised concerns about students with school refusal behavior and the school system's ability to address their special needs in these circumstances. Six students referred to the school absenteeism team were interviewed about their circumstances, using an author-developed interview. The results indicate that the students rated homeschooling as very satisfactory. Students with school refusal behavior participated in homeschooling and their attendance continued during the initial reopening of schools.
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Linfócitos T CD8-Positivos/imunologia , Carcinoma de Célula de Merkel/imunologia , Poliomavírus das Células de Merkel/fisiologia , Infecções por Polyomavirus/imunologia , Neoplasias Cutâneas/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos Virais de Tumores/imunologia , Células Cultivadas , Mapeamento de Epitopos , Epitopos de Linfócito T/imunologia , Feminino , Antígenos HLA/metabolismo , Humanos , Vigilância Imunológica , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Peptídeos/imunologiaRESUMO
Factor quinolinone inhibitors are promising anti-cancer compounds, initially characterized as specific inhibitors of the oncogenic transcription factor LSF (TFCP2). These compounds exert anti-proliferative activity at least in part by disrupting mitotic spindles. Herein, we report additional interphase consequences of the initial lead compound, FQI1, in two telomerase immortalized cell lines. Within minutes of FQI1 addition, the microtubule network is disrupted, resulting in a substantial, although not complete, depletion of microtubules as evidenced both by microtubule sedimentation assays and microscopy. Surprisingly, this microtubule breakdown is quickly followed by an increase in tubulin acetylation in the remaining microtubules. The sudden breakdown and partial depolymerization of the microtubule network precedes FQI1-induced morphological changes. These involve rapid reduction of cell spreading of interphase fetal hepatocytes and increase in circularity of retinal pigment epithelial cells. Microtubule depolymerization gives rise to FH-B cell compaction, as pretreatment with taxol prevents this morphological change. Finally, FQI1 decreases the rate and range of locomotion of interphase cells, supporting an impact of FQI1-induced microtubule breakdown on cell motility. Taken together, our results show that FQI1 interferes with microtubule-associated functions in interphase, specifically cell morphology and motility.
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Benzodioxóis/farmacologia , Microtúbulos/efeitos dos fármacos , Quinolonas/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Forma Celular/efeitos dos fármacos , Forma Celular/fisiologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Hepatócitos/efeitos dos fármacos , Hepatócitos/fisiologia , Hepatócitos/ultraestrutura , Humanos , Interfase , Microtúbulos/fisiologia , Microtúbulos/ultraestrutura , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/fisiologia , Epitélio Pigmentado da Retina/ultraestrutura , Fatores de Transcrição/antagonistas & inibidores , Tubulina (Proteína)/metabolismoRESUMO
BACKGROUND: Stroke results in varying physical, cognitive, emotional and/or social disabilities in the short and long term alike. Motor impairments are important, persistent consequences of stroke and include, among others, decreased respiratory muscle function, decreased ability to expand the thorax and postural dysfunction. These deficits affect the patient's ability to perform daily activities, produce fatigue and reduce endurance and quality of life. Inspiratory muscle training (IMT) aims to improve the strength and endurance of the diaphragm and the external intercostal muscles. The objectives of this study are to investigate the effect of 3 weeks of IMT on (i) maximal inspiratory pressure (MIP) in adults having suffered a stroke, as well as (ii) functional activities and expiratory measurements. METHODS/DESIGN: This is a randomised controlled trial (RCT) comparing IMT with conventional neurorehabilitation (usual practice). The trial will include 80 patients with reduced MIP hospitalised at a specialised neurorehabilitation hospital in Denmark. The intervention group will receive IMT sessions, exercising at 30% of MIP. Patients in the intervention group will perform two daily sessions (one session of IMT with Threshold IMT consisting of two times 15 inspirations at normal breathing rhythm (5-10 min)), 7 days a week for 3 weeks. Training can be with or without physiotherapist supervision. STUDY OUTCOMES: MIP assessed by the Power Breath, Functional Independence Measurement, The 6-min walk test, the Fatigue Severity Scale and average voice volume. Expiratory function will be assessed using spirometry. All assessments will be conducted at baseline and 3 weeks (at termination of the intervention) and 3 months after the intervention has concluded. DISCUSSION: IMT is a promising and partly self-managed tool for rehabilitation to improve respiratory function. The introduction of IMT in combination with traditional physical therapy may enhance faster recovery after stroke and may at the same time demand little personnel resources to increase training intensity. This trial will provide further evidence of IMT to clinicians, patients and health managers. Hereby, this study accepts the call for further research. TRIAL REGISTRATION: ClinicalTrials.gov NCT04686019 . Registered on 28 December 2020.
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Fisioterapeutas , Acidente Vascular Cerebral , Adulto , Exercício Físico , Humanos , Modalidades de Fisioterapia , Ensaios Clínicos Controlados Aleatórios como Assunto , Músculos Respiratórios , Acidente Vascular Cerebral/diagnósticoRESUMO
Tubulin polyglutamylation is a polymeric modification that extends from the carboxyl-terminus of tubulins. Molecular description of amino acids and their branching polyglutamyls is a hallmark of tubulin in microtubules. There are different chemical approaches for detecting these polymeric structures, mostly reported prior to development of nESI peptide analysis. Here we demonstrate a novel and simple approach to detect shared regions of amino acid ions from tubulin polyglutamylated peptides in nanoLC-MS/MS. This involves two parallel in gel digestions with trypsin and subtilisin followed by mapping of di- and triglutamyl modifications of α- and ß-tubulins using a routine proteomics assay. We present three levels of information: i) identification of proteomics MS/MS data, ii) description of internal fragment ion series common across digests, and iii) extracted ion chromatograms mapped relative to retention time standards for confirmation of relative hydrophobicity values. Our nanoLC assay positive ion ESI detects up to 3 conjugated glutamates in tubulins. We implemented an analytical column only bottom up approach that characterizes molecular features of polyglutamylated tubulins.
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Cromatografia Líquida de Alta Pressão/métodos , Nanotecnologia/métodos , Ácido Poliglutâmico/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Tubulina (Proteína)/química , Sequência de Aminoácidos , Animais , Interações Hidrofóbicas e Hidrofílicas , Íons/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ácido Poliglutâmico/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise , Suínos , Tubulina (Proteína)/metabolismoRESUMO
BACKGROUND: The oncogene LSF (encoded by TFCP2) has been proposed as a novel therapeutic target for multiple cancers. LSF overexpression in patient tumors correlates with poor prognosis in particular for both hepatocellular carcinoma and colorectal cancer. The limited treatment outcomes for these diseases and disappointing clinical results, in particular, for hepatocellular carcinoma in molecularly targeted therapies targeting cellular receptors and kinases, underscore the need for molecularly targeting novel mechanisms. LSF small molecule inhibitors, Factor Quinolinone Inhibitors (FQIs), have exhibited robust anti-tumor activity in multiple pre-clinical models, with no observable toxicity. METHODS: To understand how the LSF inhibitors impact cancer cell proliferation, we characterized the cellular phenotypes that result from loss of LSF activity. Cell proliferation and cell cycle progression were analyzed, using HeLa cells as a model cancer cell line responsive to FQI1. Cell cycle progression was studied either by time lapse microscopy or by bulk synchronization of cell populations to ensure accuracy in interpretation of the outcomes. In order to test for biological specificity of targeting LSF by FQI1, results were compared after treatment with either FQI1 or siRNA targeting LSF. RESULTS: Highly similar cellular phenotypes are observed upon treatments with FQI1 and siRNA targeting LSF. Along with similar effects on two cellular biomarkers, inhibition of LSF activity by either mechanism induced a strong delay or arrest prior to metaphase as cells progressed through mitosis, with condensed, but unaligned, chromosomes. This mitotic disruption in both cases resulted in improper cellular division leading to multiple outcomes: multi-nucleation, apoptosis, and cellular senescence. CONCLUSIONS: These data strongly support that cellular phenotypes observed upon FQI1 treatment are due specifically to the loss of LSF activity. Specific inhibition of LSF by either small molecules or siRNA results in severe mitotic defects, leading to cell death or senescence - consequences that are desirable in combating cancer. Taken together, these findings confirm that LSF is a promising target for cancer treatment. Furthermore, this study provides further support for developing FQIs or other LSF inhibitory strategies as treatment for LSF-related cancers with high unmet medical needs.
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Benzodioxóis/farmacologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Quinolonas/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Apoptose/genética , Benzodioxóis/uso terapêutico , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Senescência Celular/efeitos dos fármacos , Senescência Celular/genética , Cromossomos Humanos/efeitos dos fármacos , Cromossomos Humanos/genética , Cromossomos Humanos/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Humanos , Microscopia Intravital , Terapia de Alvo Molecular/métodos , Neoplasias/genética , Neoplasias/patologia , Quinolonas/uso terapêutico , RNA Interferente Pequeno/metabolismo , Imagem com Lapso de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Aims: The aim of this study was to investigate the relationship between diabetes stigma as experienced by adults with type 1 diabetes and diabetes outcomes using the novel, validated measure of the Type 1 Diabetes Stigma Assessment Scale. Methods: A total of 1594 adults with type 1 diabetes completed a questionnaire on socio-economic factors, psychosocial health, and diabetes stigma and these self-reported data were linked with data from electronic clinical records on glycaemic control, diabetes duration, age, and diabetes-related complications. Bivariate analyses and multivariate linear regressions were performed to assess the relationship between diabetes stigma as measured by three subscales, Identity concern, Blame and judgement, and Treated differently on the one hand, and patient characteristics and diabetes outcomes on the other. Results: Endorsement of the stigma statements ranged from 3.6-78.3% of respondents. Higher stigma scores in relation to Identity concern and Blame and judgement were significantly associated with being female, of lower age, lower diabetes duration, and having at least one complication. Those who reported higher levels of perceived stigma reported significantly higher levels of diabetes distress (ß = 0.37 (95% CI: 0.33-0.40), 0.35 (95% CI: 0.30-0.39), 0.41 (95% CI: 0.35-0.46)), and HbA1c levels (ß = 0.11 (95% CI: 0.02-0.21), 0.28 (95% CI: 0.16-0.40), 0.26 (95% CI: 0.14-0.42) for Identity concern, Blame and judgement, and Treated differently, respectively). Conclusions: The findings demonstrated that diabetes stigma is negatively associated with both diabetes distress and glycaemic control and should be considered part of the psychosocial burden of adults with type 1 diabetes.
Assuntos
Diabetes Mellitus Tipo 1/psicologia , Diabetes Mellitus Tipo 1/terapia , Controle Glicêmico/estatística & dados numéricos , Estigma Social , Adulto , Estudos Transversais , Dinamarca/epidemiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Angústia Psicológica , Inquéritos e Questionários , Resultado do TratamentoRESUMO
Mutation-derived neoantigens are important targets for T cell-mediated reactivity toward tumors and, due to their unique tumor expression, an attractive target for immunotherapy. Neoepitope-specific T cells have been detected across a number of solid cancers with high mutational burden tumors, but neoepitopes have been mostly selected from single nucleotide variations (SNVs), and little focus has been given to neoepitopes derived from in-frame and frameshift indels, which might be equally important and potentially highly immunogenic. Clear cell renal cell carcinomas (ccRCCs) are medium-range mutational burden tumors with a high pan-cancer proportion of frameshift mutations. In this study, the mutational landscape of tumors from six RCC patients was analyzed by whole-exome sequencing (WES) of DNA from tumor fragments (TFs), autologous tumor cell lines (TCLs), and tumor-infiltrating lymphocytes (TILs, germline reference). Neopeptides were predicted using MuPeXI, and patient-specific peptide-MHC (pMHC) libraries were created for all neopeptides with a rank score < 2 for binding to the patient's HLAs. T cell recognition toward neoepitopes in TILs was evaluated using the high-throughput technology of DNA barcode-labeled pMHC multimers. The patient-specific libraries consisted of, on average, 258 putative neopeptides (range, 103-397, n = 6). In four patients, WES was performed on two different sources (TF and TCL), whereas in two patients, WES was performed only on TF. Most of the peptides were predicted from both sources. However, a fraction was predicted from one source only. Among the total predicted neopeptides, 16% were derived from frameshift indels. T cell recognition of 52 neoepitopes was detected across all patients (range, 4-18, n = 6) and spanning two to five HLA restrictions per patient. On average, 21% of the recognized neoepitopes were derived from frameshift indels (range, 0-43%, n = 6). Thus, frameshift indels are equally represented in the pool of immunogenic neoepitopes as SNV-derived neoepitopes. This suggests the importance of a broad neopeptide prediction strategy covering multiple sources of tumor material, and including different genetic alterations. This study, for the first time, describes the T cell recognition of frameshift-derived neoepitopes in RCC and determines their immunogenic profile.
Assuntos
Antígenos de Neoplasias/imunologia , Carcinoma de Células Renais/imunologia , Neoplasias Renais/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos T/imunologia , Antígenos de Neoplasias/genética , Carcinoma de Células Renais/genética , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Mutação da Fase de Leitura , Humanos , Neoplasias Renais/genética , Mutação PuntualRESUMO
Microtubules are cytoskeletal structures critical for mitosis, cell motility, and protein and organelle transport and are a validated target for anticancer drugs. However, how tubulins are regulated and recruited to support these distinct cellular processes is incompletely understood. Posttranslational modifications of tubulins are proposed to regulate microtubule function and dynamics. Although many of these modifications have been investigated, only one prior study reports tubulin methylation and an enzyme responsible for this methylation. Here we used in vitro radiolabeling, MS, and immunoblotting approaches to monitor protein methylation and immunoprecipitation, immunofluorescence, and pulldown approaches to measure protein-protein interactions. We demonstrate that N-lysine methyltransferase 5A (KMT5A or SET8/PR-Set7), which methylates lysine 20 in histone H4, bound α-tubulin and methylated it at a specific lysine residue, Lys311 Furthermore, late SV40 factor (LSF)/CP2, a known transcription factor, bound both α-tubulin and SET8 and enhanced SET8-mediated α-tubulin methylation in vitro In addition, we found that the ability of LSF to facilitate this methylation is countered by factor quinolinone inhibitor 1 (FQI1), a specific small-molecule inhibitor of LSF. These findings suggest the general model that microtubule-associated proteins, including transcription factors, recruit or stimulate protein-modifying enzymes to target tubulins. Moreover, our results point to dual functions for SET8 and LSF not only in chromatin regulation but also in cytoskeletal modification.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Fatores de Transcrição/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Células COS , Chlorocebus aethiops , Proteínas de Ligação a DNA/genética , Células HEK293 , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Humanos , Metilação , Ligação Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Fatores de Transcrição/genéticaRESUMO
Expansile nanoparticles (eNPs) are a promising pH-responsive polymeric drug delivery vehicle, as demonstrated in multiple intraperitoneal cancer models. However, previous delivery routes were limited to intraperitoneal injection and to a single agent, paclitaxel. In this study, we preliminarily evaluate the biodistribution and in vivo toxicity of eNPs in mice after intravenous injection. The eNPs localize predominantly to the liver, without detectable acute toxicity in the liver or other key organs. On the basis of these results, we encapsulated FQI1, a promising lead compound for treatment of hepatocellular carcinoma, in eNPs. eNPs are taken up by cancerous and noncancerous human liver cells in vitro, although at different rates. FQI1-loaded eNPs release FQI1 in a pH-dependent manner and limit proliferation equivalently to unencapsulated FQI1 in immortalized hepatocytes in vitro. eNPs are a versatile platform delivery system for therapeutic compounds and have potential utility in the treatment of liver disease.