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OBJECTIVES: Currently, no systemic treatments are approved for patients with recurrent and/or metastatic (R/M) adenoid cystic carcinoma (ACC). PRT543, a protein arginine methyltransferase 5 inhibitor that downregulates NOTCH1 and MYB signalling in tumours, is a potential candidate for R/M ACC treatment. We report the safety, tolerability and preliminary efficacy of PRT543 in a dose-expansion cohort of patients with R/M ACC. MATERIALS AND METHODS: This phase I multicentre, open-label, sequential-cohort, dose-escalation and dose-expansion study (NCT03886831) enrolled patients with advanced solid tumours and select haematologic malignancies. Dose-escalation study design and results were reported previously. In the dose expansion, patients with R/M ACC received recommended phase II doses of 35 or 45 mg PRT543 orally on days 1-5 of each week. Primary objectives were to establish the safety and tolerability of PRT543. Secondary objectives included efficacy. RESULTS: Between February 2019 and May 2022, 56 patients with ACC were enrolled across 23 US sites and received either 35 mg (n = 28) or 45 mg (n = 28) of PRT543. Overall, 23% of patients experienced a grade 3 treatment-related adverse event, most commonly anaemia (16%) and thrombocytopaenia (9%). No grade 4/5 treatment-emergent adverse events were reported. Median progression-free survival was 5.9 months (95% CI: 3.8-8.3). The clinical benefit rate was 57% (95% CI: 43-70). Overall response rate (per Response Evaluation Criteria in Solid Tumours v1.1) was 2%, with 70% of patients having stable disease. CONCLUSION: In this analysis, PRT543 was tolerable, and the observed efficacy was limited in patients with R/M ACC.
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Carcinoma Adenoide Cístico , Humanos , Carcinoma Adenoide Cístico/tratamento farmacológico , Proteína-Arginina N-Metiltransferases , Recidiva Local de Neoplasia , Intervalo Livre de ProgressãoRESUMO
[This corrects the article DOI: 10.1038/s41378-023-00485-4.].
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The interactions that cells in Drosophila imaginal discs have with their neighbors are known to regulate their ability to survive. In a screen of genes encoding cell surface proteins for gene knockdowns that affect the size or shape of mutant clones, we found that clones of cells with reduced levels of echinoid (ed) are fewer, smaller, and can be eliminated during development. In contrast, discs composed mostly of ed mutant tissue are overgrown. We find that ed mutant tissue has lower levels of the anti-apoptotic protein Diap1 and has increased levels of apoptosis which is consistent with the observed underrepresentation of ed mutant clones and the slow growth of ed mutant tissue. The eventual overgrowth of ed mutant tissue results not from accelerated growth, but from prolonged growth resulting from a failure to arrest growth at the appropriate final size. Ed has previously been shown to physically interact with multiple Hippo-pathway components and it has been proposed to promote Hippo pathway signaling, to exclude Yorkie (Yki) from the nucleus, and restrain the expression of Yki-target genes. We did not observe changes in Yki localization in ed mutant tissue and found decreased levels of expression of several Yorkie-target genes, findings inconsistent with the proposed effect of Ed on Yki. We did, however, observe increased expression of several Yki-target genes in wild-type cells neighboring ed mutant cells, which may contribute to elimination of ed mutant clones. Thus, ed has two distinct functions: an anti-apoptotic function by maintaining Diap1 levels, and a function to arrest growth at the appropriate final size. Both of these are unlikely to be explained by a simple effect on the Hippo pathway.
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Oral vaccines have a distinctive advantage of stimulating immune responses in the mucosa, where numerous pathogens gain entry and cause disease. Although various efforts have been attempted to create recombinant mucosal vaccines that provoke strong immunogenicity, the outcomes in clinical trials have been weak or inconsistent. Therefore, next-generation mucosal vaccines are needed that are more immunogenic. Here, we discuss oral vaccines with an emphasis on a next-generation mucosal vaccine that utilizes a nonreplicating human recombinant adenovirus type-5 (rAd5) vector. Numerous positive clinical results investigating oral rAd5 vaccines are reviewed, with a summary of the immunogenicity and efficacy results for specific vaccine indications of influenza, norovirus, and SARS-CoV-2. The determination of correlates of protection for oral vaccination and the potential impact this novel vaccine formulation may have on disease transmission are also discussed. In summary, successful oral vaccination can be accomplished and would have major public health benefits if approved.
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COVID-19 , Humanos , COVID-19/prevenção & controle , SARS-CoV-2 , Adenoviridae/genética , Vacinas Sintéticas , Vacinação , Anticorpos AntiviraisRESUMO
Caenorhabditis elegans embryos have been widely used to study cellular processes and developmental regulation at early stages. However, most existing microfluidic devices focus on the studies of larval or adult worms rather than embryos. To accurately study the real-time dynamics of embryonic development under different conditions, many technical barriers must be overcome; these can include single-embryo sorting and immobilization, precise control of the experimental environment, and long-term live imaging of embryos. This paper reports a spiral microfluidic device for effective sorting, trapping, and long-term live imaging of single C. elegans embryos under precisely controlled experimental conditions. The device successfully sorts embryos from a mixed population of C. elegans at different developmental stages via Dean vortices generated inside a spiral microchannel and traps the sorted embryos at single-cell resolution through hydrodynamic traps on the sidewall of the spiral channel for long-term imaging. Through the well-controlled microenvironment inside the microfluidic device, the response of the trapped C. elegans embryos to mechanical and chemical stimulation can be quantitatively measured. The experimental results show that a gentle hydrodynamic force would induce faster growth of embryos, and embryos developmentally arrested in the high-salinity solution could be rescued by the M9 buffer. The microfluidic device provides new avenues for easy, rapid, high-content screening of C. elegans embryos.
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Serologic tests to detect specific IgGs to antigens related to viral infections are urgently needed for diagnostics and therapeutics. We present a diagnostic method for serotype-specific IgG identification of dengue infection by a competitive enzyme-linked immunosorbent assay (ELISA), using high-affinity unnatural-base-containing DNA (UB-DNA) aptamers that recognize the four categorized serotypes. Using UB-DNA aptamers specific to each serotype of dengue NS1 proteins (DEN-NS1), we developed our aptamer-antibody sandwich ELISA for dengue diagnostics. Furthermore, IgGs highly specific to DEN-NS1 inhibited the serotype-specific NS1 detection, inspiring us to develop the competitive ELISA format for dengue serotype-specific IgG detection. Blood samples from Singaporean patients with primary or secondary dengue infections confirmed the highly specific IgG detection of this format, and the IgG production initially reflected the serotype of the past infection, rather than the recent infection. Using this dengue competitive ELISA format, cross-reactivity tests of 21 plasma samples from Singaporean Zika virus-infected patients revealed two distinct patterns: 8 lacked cross-reactivity, and 13 were positive with unique dengue serotype specificities, indicating previous dengue infection. This antigen-detection ELISA and antibody-detection competitive ELISA combination using the UB-DNA aptamers identifies both past and current viral infections and will facilitate specific medical care and vaccine development for infectious diseases.
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Anticorpos Antivirais/imunologia , Afinidade de Anticorpos , Aptâmeros de Nucleotídeos/imunologia , Vírus da Dengue/imunologia , Dengue/imunologia , Imunoglobulina G/imunologia , Sorogrupo , Testes Sorológicos/métodos , Proteínas não Estruturais Virais/imunologia , Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Reações Cruzadas , Dengue/sangue , Dengue/diagnóstico , Dengue/virologia , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Imunoglobulina G/sangue , Sensibilidade e Especificidade , Zika virus/imunologia , Infecção por Zika virus/sangue , Infecção por Zika virus/diagnóstico , Infecção por Zika virus/imunologia , Infecção por Zika virus/virologiaRESUMO
Genetic alphabet expansion of DNA by introducing unnatural bases (UBs), as a fifth letter, dramatically augments the affinities of DNA aptamers that bind to target proteins. To determine whether UB-containing DNA (UB-DNA) aptamers obtained by affinity selection could spontaneously achieve high specificity, we have generated a series of UB-DNA aptamers (KD: 27-182 pM) targeting each of four dengue non-structural protein 1 (DEN-NS1) serotypes. The specificity of each aptamer is remarkably high, and the aptamers can recognize the subtle variants of DEN-NS1 with at least 96.9% amino acid sequence identity, beyond the capability of serotype identification (69-80% sequence identities). Our UB-DNA aptamers specifically identified two major variants of dengue serotype 1 with 10-amino acid differences in the DEN-NS1 protein (352 aa) in Singaporeans' clinical samples. These results suggest that the high-affinity UB-DNA aptamers generated by affinity selection also acquire high target specificity. Intriguingly, one of the aptamers contained two different UBs as fifth and sixth letters, which are essential for the tight binding to the target. These two types of unnatural bases with distinct physicochemical properties profoundly expand the potential of DNA aptamers. Detection methods incorporating the UB-DNA aptamers will facilitate precise diagnoses of viral infections and other diseases.
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Aptâmeros de Nucleotídeos/química , Dengue/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Proteínas não Estruturais Virais/metabolismo , Aptâmeros de Nucleotídeos/genética , Dengue/virologia , Humanos , Mutação , Ligação Proteica , Técnica de Seleção de Aptâmeros , Sorogrupo , Proteínas não Estruturais Virais/genéticaRESUMO
Dengue virus (DENV) and Zika virus (ZIKV) belong to the Flaviviridae family of viruses spread by Aedes aegypti mosquitoes in tropical and subtropical areas. Accurate diagnostic tests to differentiate the 2 infections are necessary for patient management and disease control. Using characterized ZIKV and DENV patient plasma in a blind manner, we validated an ELISA and a rapid immunochromatographic test for ZIKV detection. We engineered the ZIKV nonstructural protein 1 (NS1) for sensitive serologic detection with low cross reactivity against dengue and developed monoclonal antibodies specific for the ZIKV NS1 antigen. As expected, the serologic assays performed better with convalescent than acute plasma samples; the sensitivity ranged from 71% to 88%, depending on the performance of individual tests (IgM/IgG/NS1). Although serologic tests were generally less sensitive with acute samples, our ZIKV NS1 antibodies were able to complement the serologic tests to achieve greater sensitivity for detecting early infections.
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Vírus da Dengue , Dengue , Infecção por Zika virus , Zika virus , Anticorpos Antivirais , Ensaio de Imunoadsorção Enzimática , Humanos , Sensibilidade e Especificidade , Testes Sorológicos , Proteínas não Estruturais ViraisRESUMO
A short tetramer peptide, Ac-IVKC, spontaneously formed a hydrogel in water. Disulfide bonds were introduced via hydrogen peroxide (H2O2)-assisted oxidation, resulting in (Ac-IVKC)2 dimers. The extent of disulfide bond formation and gel stiffness increased with the amount of H2O2 used and 100% dimerization was achieved with 0.2% H2O2. The resultant gel achieved an elastic modulus of â¼0.9â¯MPa, which to our knowledge, has not been reported for peptide-based hydrogels. The enhanced mechanical property enabled the fabrication of thin and transparent membranes. The hydrogel could also be handled with forceps at mm thickness, greatly increasing its ease of physical manipulation. Excess H2O2 was removed and the membrane was then infused with cell culture media. Various cells, including primary human corneal stromal and epithelial cells, were seeded onto the hydrogel membrane and demonstrated to remain viable. Depending on the intended application, specific cell combination or membrane stacking order could be used to engineer layered biostructures. STATEMENT OF SIGNIFICANCE: A short tetramer peptide - Ac-IVKC - spontaneously formed a hydrogel in water and disulfide bonds were introduced via hydrogen peroxide (H2O2)-assisted oxidation. The extent of disulfide-bond formation and gel stiffness were modulated by the amount of H2O2. At maximum disulfide-bond formation, the hydrogel achieved an elastic modulus of â¼0.9â¯MPa, which to our knowledge, has not been reported for peptide-based hydrogels. The enhanced mechanical property enabled the fabrication of thin transparent membranes that can be physically manipulated at mm thickness. The gels also supported 3D cell growth, including primary human corneal stromal and epithelial cells. Depending on the intended application, specific combination of cells or individual membrane stacking order could be used to engineer layered biostructures.
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Materiais Biocompatíveis/farmacologia , Hidrogéis/farmacologia , Membranas Artificiais , Peptídeos/farmacologia , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Fenômenos Biomecânicos , Substância Própria/citologia , Dissulfetos/metabolismo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Endotélio Corneano/citologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/metabolismo , CamundongosRESUMO
BACKGROUND: 1-α-D-(5-Deoxy-5-[18F]fluoroarabinofuranosyl)-2-nitroimidazole ([18F]FAZA) is manufactured by nucleophilic radiofluorination of 1-α-D-(2',3'-di-O-acetyl-5'-O-toluenesulfonylarabinofuranosyl)- 2-nitroimidazole (DiAcTosAZA) and alkaline deprotection to afford [18F]FAZA. High yields (>60%) under optimized conditions frequently revert to low yields (<20%) in large scale, automated syntheses. Competing side reactions and concomitant complex reaction mixtures contribute to substantial loss of product during HPLC clean-up. OBJECTIVE: To develop alternative precursors for facile routine clinical manufacture of [18F]FAZA that are compatible with current equipment and automated procedures. METHODS: Two new precursors, 1-α-D-(2',3'-di-O-acetyl-5'-O-(4-nitrobenzene)sulfonyl-arabinofuranosyl)-2- nitroimidazole (DiAcNosAZA) and 1-α-D-(2',3'-di-O-acetyl-5'-iodo-arabinofuranosyl)-2-nitroimidazole (DiAcIAZA), were synthesized from commercially-available 1-α-D-arabinofuranosyl-2-nitroimidazole (AZA). A commercial automated synthesis unit (ASU) was used to condition F-18 for anhydrous radiofluorination, and to radiofluorinate DiAcNosAZA and DiAcIAZA using the local standardized protocol to manufacture [18F]FAZA from AcTosAZA. RESULTS: DiAcNosAZA was synthesized via two pathways, in recovered yields of 29% and 40%, respectively. The nosylation of 1-α-D-(2',3'-di-O-acetyl-arabinofuranosyl)-2-nitroimidazole (DiAcAZA) featured a strong competing reaction that afforded 1-α-D-(2',3'-di-O-acetyl-5'-chloro-arabinofuranosyl)-2- nitroimidazole (DiAcClAZA) in 55% yield. Radiofluorination yields were better from DiAcNosAZA and DiAcIAZA than from DiAcTosAZA, and the presence of fewer side products afforded higher purity [18F]FAZA preparations. Several radioactive and non-radioactive by products of radiofluorination were assigned tentative chemical structures based on co-chromatography with authentic reference compounds. CONCLUSION: DiAcClAZA, a major side-product in the preparation of DiAcNosAZA, and its deprotected analogue (ClAZA), are unproven hypoxic tissue radiosensitizers. DiAcNosAZA and DiAcIAZA provided good radiofluorination yields in comparison to AcTosAZA and could become preferred [18F]FAZA precursors if the cleaner reactions can be exploited to bypass HPLC purification.
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Radioisótopos de Flúor/química , Nitroimidazóis/química , Tomografia por Emissão de Pósitrons , Radioquímica/métodos , Compostos Radiofarmacêuticos/síntese química , Hipóxia TumoralRESUMO
We aimed to bioengineer a scaffold that can facilitate the transplantation of corneal endothelial cells (CEC), given the global shortage of cadaveric donor tissues. Although agarose (A) has outstanding biocompatibility and mechanical properties, it natively does not permit cell adhesion. In this study, agarose was modified with different attachment signals: GRGD (giving AR as product), lysine (AK), poly lysine (AP), and fish-derived gelatin (AG). Samples with varying conjugation ratios were prepared. All products formed bulk hydrogels, which were then collapsed into ultrathin membranes in a controlled environment. Membranes were evaluated for their ability to support attachment of various cell types. Cells, however, preferred the AG series of membrane. Notably, primary rabbit CEC remained attached and viable for ⩾4 weeks. The cells also stained positive for CD166, ZO-1 and Na+/K+ ATPase, indicative of function. The hydrated AG membranes allowed >96% transmittance of visible light. The membranes were typically â¼15 µm thick and did not swell significantly after immersion in PBS. Tensile strength was 49-60 MPa, while young's modulus was 525-596 MPa. This membrane thus offers great promise as a scaffold for CEC during endothelial keratoplasty.
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Antineoplásicos/uso terapêutico , Policitemia Vera/tratamento farmacológico , Pirazóis/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Resistencia a Medicamentos Antineoplásicos , Humanos , Hidroxiureia/uso terapêutico , Pessoa de Meia-Idade , Nitrilas , Pirimidinas , Resultado do TratamentoRESUMO
BACKGROUND: Myelofibrosis (MF) is a life-shortening complication of myeloproliferative neoplasms associated with ineffective hematopoiesis, splenomegaly, and progressive bone marrow (BM) fibrosis. The oral Janus kinase (JAK) 1/JAK2 inhibitor ruxolitinib has been shown to improve splenomegaly, symptom burden, and overall survival in patients with intermediate-2 or high-risk MF compared with placebo or best available therapy (BAT). METHODS: The effects of ruxolitinib therapy for up to 66 months on BM morphology in 68 patients with advanced MF with variable BM fibrosis grade were compared with those in 192 matching patients treated with BAT. Available trephine biopsies underwent independent, blinded review by three hematopathologists for consensus-based adjudication of grades for reticulin fibrosis, collagen deposition, and osteosclerosis. RESULTS: Ruxolitinib treatment versus BAT was associated with greater odds of BM fibrosis improvement or stabilization and decreased odds of BM fibrosis worsening based on changes from baseline in reticulin fibrosis grade. Generally, these changes were accompanied by a sustained higher level of individual spleen size reduction and regression of leukoerythroblastosis. Patients with more advanced baseline fibrosis showed lower spleen size response. CONCLUSIONS: The finding that long-term ruxolitinib therapy may reverse or markedly delay BM fibrosis progression in advanced MF suggests that sustained JAK inhibition may be disease-modifying. TRIAL REGISTRATION: INCB18424-251, ClinicalTrials.gov identifier NCT00509899 .
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Janus Quinases/uso terapêutico , Mielofibrose Primária/tratamento farmacológico , Pirazóis/uso terapêutico , Idoso , Feminino , Humanos , Janus Quinases/farmacologia , Masculino , Nitrilas , Mielofibrose Primária/patologia , Pirazóis/farmacologia , Pirimidinas , Resultado do TratamentoRESUMO
BACKGROUND: Myelofibrosis (MF) is associated with a variety of burdensome symptoms and reduced survival compared with age-/sex-matched controls. This analysis evaluated the long-term survival benefit with ruxolitinib, a Janus kinase (JAK)1/JAK2 inhibitor, in patients with intermediate-2 (int-2) or high-risk MF. METHODS: This was an exploratory analysis of 5-year data pooled from the phase 3 COMFORT-I and -II trials. In both trials, patients could cross over to ruxolitinib from the control group (COMFORT-I, placebo; COMFORT-II, best available therapy). All continuing patients in the control groups crossed over to ruxolitinib by the 3-year follow-up. Overall survival (OS; a secondary endpoint in both trials) was evaluated using pooled intent-to-treat data from patients randomized to ruxolitinib or the control groups. OS was also evaluated in subgroups stratified by baseline anemia and transfusion status at week 24. RESULTS: A total of 528 patients were included in this analysis; 301 were originally randomized to ruxolitinib (COMFORT-I, n = 155; COMFORT-II, n = 146) and 227 to control (n = 154 and n = 73, respectively). The risk of death was reduced by 30% among patients randomized to ruxolitinib compared with patients in the control group (median OS, 5.3 vs 3.8 years, respectively; hazard ratio [HR], 0.70 [95% CI, 0.54-0.91]; P = 0.0065). After correcting for crossover using a rank-preserving structural failure time (RPSFT) method, the OS advantage was more pronounced for patients who were originally randomized to ruxolitinib compared with patients who crossed over from control to ruxolitinib (median OS, 5.3 vs 2.3 years; HR [ruxolitinib vs RPSFT], 0.35 [95% CI, 0.23-0.59]). An analysis of OS censoring patients at the time of crossover also demonstrated that ruxolitinib prolonged OS compared with control (median OS, 5.3 vs 2.4 years; HR [ruxolitinib vs censored at crossover], 0.53 [95% CI, 0.36-0.78]; P = 0.0013). The survival benefit with ruxolitinib was observed irrespective of baseline anemia status or transfusion requirements at week 24. CONCLUSIONS: These findings support ruxolitinib treatment for patients with int-2 or high-risk MF, regardless of anemia or transfusion status. Further analyses will be important for exploring ruxolitinib earlier in the disease course to assess the effect on the natural history of MF. TRIAL REGISTRATION: ClinicalTrials.gov identifiers, NCT00952289 and NCT00934544 .
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Anemia/induzido quimicamente , Mielofibrose Primária/tratamento farmacológico , Pirazóis/uso terapêutico , Progressão da Doença , Método Duplo-Cego , Feminino , Humanos , Masculino , Nitrilas , Mielofibrose Primária/mortalidade , Mielofibrose Primária/patologia , Pirazóis/efeitos adversos , Pirazóis/farmacologia , Pirimidinas , Análise de Sobrevida , Resultado do TratamentoRESUMO
BACKGROUND: The phase III COMFORT (Controlled Myelofibrosis Study With Oral JAK inhibitor Treatment)-I and COMFORT-II trials in patients with intermediate-2 or high-risk myelofibrosis (MF) showed that ruxolitinib was superior to placebo and best available therapy, respectively, for improvements in spleen volume, MF-related symptoms, and overall survival (OS). However, patients managed in community settings might not have access to the methods used in the COMFORT trials. In this exploratory analysis we summarize efficacy findings of COMFORT-I using practical, community-oriented measures of patient outcomes. PATIENTS AND METHODS: In this post hoc analysis of data from COMFORT-I we evaluated changes from baseline to week 12 in spleen size (palpable length and volume), patient-reported outcomes (Patient Global Impression of Change; Myelofibrosis Symptom Assessment Form; Patient-Reported Outcomes Measurement System Fatigue Scale), body weight, and serum albumin levels in 5 subgroups of ruxolitinib-treated patients on the basis of week 12 spleen length changes from baseline: (1-4) ≥ 50%, 25% to < 50%, 10% to < 25%, or < 10% reduction; and (5) worsening. OS was evaluated in ruxolitinib-treated patients with week 12 spleen length reductions from baseline ≥ 50%, 25% to < 50%, or < 25% (including worsening). RESULTS: In all spleen length subgroups, including patients with worsening spleen length at week 12, ruxolitinib (n = 150) was associated with improvements in spleen volume, patient-reported symptom burden, body weight, and serum albumin levels. Greater reductions in spleen length were associated with prolonged OS. CONCLUSION: A variety of assessment methods beyond palpable spleen length that are easily accessible in the community setting might be useful in evaluating the clinical benefit of ruxolitinib over time in patients with MF.
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Mielofibrose Primária/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis/uso terapêutico , Idoso , Biomarcadores , Transfusão de Sangue , Terapia Combinada , Gerenciamento Clínico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Nitrilas , Mielofibrose Primária/diagnóstico , Mielofibrose Primária/mortalidade , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos , Pirazóis/administração & dosagem , Pirazóis/efeitos adversos , Pirimidinas , Qualidade de Vida , Baço/patologia , Análise de Sobrevida , Resultado do TratamentoRESUMO
With the discovery of the CRISPR/Cas9 technology, genome editing could be performed in a rapid, precise and effective manner. Its potential applications in functional interrogation of cancer-causing genes and cancer therapy have been extensively explored. In this study, we demonstrated the use of the CRISPR/Cas9 system to directly target the oncogene HER2. Directing Cas9 to exons of the HER2 gene inhibited cell growth in breast cancer cell lines that harbor amplification of the HER2 locus. The inhibitory effect was potentiated with the addition of PARP inhibitors. Unexpectedly, CRISPR-induced mutations did not significantly affect the level of HER2 protein expression. Instead, CRISPR targeting appeared to exert its effect through a dominant negative mutation. This HER2 mutant interfered with the MAPK/ERK axis of HER2 downstream signaling. Our work provides a novel mechanism underlying the anti-cancer effects of HER2-targeting by CRISPR/Cas9, which is distinct from the clinical drug Herceptin. In addition, it opens up the possibility that incomplete CRISPR targeting of certain oncogenes could still have therapeutic value by generation of dominant negative mutants.
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Neoplasias da Mama/terapia , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Proliferação de Células , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Marcação de Genes , Terapia Genética/métodos , Mutação , Receptor ErbB-2/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas Associadas a CRISPR/metabolismo , Proliferação de Células/efeitos dos fármacos , Quimioterapia Adjuvante , Éxons , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Genótipo , Humanos , Células MCF-7 , Fenótipo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Receptor ErbB-2/metabolismo , Transdução de Sinais , Transfecção , Trastuzumab/farmacologiaAssuntos
Anemia/etiologia , Anemia/mortalidade , Mielofibrose Primária/complicações , Pirazóis/uso terapêutico , Idoso , Anemia/induzido quimicamente , Anemia/tratamento farmacológico , Feminino , Humanos , Masculino , Nitrilas , Mielofibrose Primária/tratamento farmacológico , Mielofibrose Primária/mortalidade , Pirazóis/efeitos adversos , PirimidinasRESUMO
Cell surface antigens are important targets for monoclonal antibodies, but they are often difficult to work with due to their association with the cell membrane. Phage display is a versatile technique that can be applied to generate binders against difficult targets. Here we used antibody phage display to isolate a binder for a rare and specialized cell, the human corneal endothelial cell. The human corneal endothelium is a medically important cell layer; defects in this layer account for about half of all corneal transplants. Despite its importance, no specific antigens have been found to mark this cell type. By panning a phage library directly on human corneal endothelial cells, we isolated an antibody that bound to these cells and not the other types of corneal cells. Subsequently, we identified the antibody's putative target to be CD166 by immunoprecipitation and mass spectrometry. This approach can be used to isolate antibodies against other poorly-characterized cell types, such as stem cells or cancer cells, without any prior knowledge of their discriminating markers.
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Anticorpos Monoclonais/isolamento & purificação , Antígenos CD/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Células Endoteliais/metabolismo , Endotélio Corneano/metabolismo , Proteínas Fetais/metabolismo , Biblioteca de Peptídeos , Anticorpos de Cadeia Única/isolamento & purificação , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Antígenos CD/genética , Biomarcadores/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Células Endoteliais/citologia , Endotélio Corneano/citologia , Proteínas Fetais/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Imunoprecipitação , Especificidade de Órgãos , Cultura Primária de Células , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismoRESUMO
BACKGROUND: In the COMFORT (COntrolled MyeloFibrosis Study with ORal JAK Inhibitor Therapy)-I study, the Janus kinase (JAK)1/JAK2 inhibitor ruxolitinib provided significant reductions in splenomegaly, improvements in myelofibrosis (MF)-related symptoms, and a survival advantage relative to placebo in patients with intermediate-2 or high-risk MF. In this post hoc analysis, we assessed the effects of ruxolitinib treatment on measures of metabolic and nutritional status. PATIENTS AND METHODS: Patients were randomized to receive ruxolitinib (n = 155; 15 or 20 mg twice a day for patients with baseline platelet counts of 100-200 × 10(9)/L or > 200 × 10(9)/L, respectively) or placebo (n = 154). The primary end point was the proportion of patients with a ≥ 35% spleen volume reduction from baseline to week 24. A secondary end point was the proportion of patients with ≥ 50% improvement in Total Symptom Score (TSS) from baseline to week 24, measured using the modified Myelofibrosis Symptom Assessment Form version 2.0. Weight, cholesterol, and albumin were measured at specified time points throughout the study. RESULTS: Compared with placebo, ruxolitinib treatment was associated with increased weight (mean change: 3.9 kg vs. -1.9 kg), total cholesterol (mean percentage change: 26.4% vs. -3.3%), and albumin levels (mean percentage change: 5.8% vs. -1.7%) at week 24; sustained improvements were observed with longer-term ruxolitinib therapy. Relative to placebo, increases in mean weight, total cholesterol, and albumin levels were observed with ruxolitinib treatment regardless of the degree of spleen volume and TSS reductions at 24 weeks. CONCLUSION: Treatment with ruxolitinib improved measures of metabolic and nutritional status of patients with intermediate-2 or high-risk MF.
Assuntos
Mielofibrose Primária/tratamento farmacológico , Mielofibrose Primária/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis/uso terapêutico , Biomarcadores , Peso Corporal , Colesterol/sangue , Colesterol/metabolismo , Humanos , Janus Quinases/antagonistas & inibidores , Nitrilas , Tamanho do Órgão , Mielofibrose Primária/diagnóstico , Pirimidinas , Albumina Sérica , Baço/anatomia & histologia , Baço/patologia , Resultado do TratamentoRESUMO
In the phase III COMFORT-I study, the Janus kinase 1 (JAK1)/JAK2 inhibitor ruxolitinib provided significant improvements in splenomegaly, key symptoms, and quality-of-life measures and was associated with an overall survival benefit relative to placebo in patients with intermediate-2 or high-risk myelofibrosis. This planned analysis assessed the long-term efficacy and safety of ruxolitinib at a median follow-up of 149 weeks. At data cutoff, approximately 50% of patients originally randomized to ruxolitinib remained on treatment whereas all patients originally assigned to placebo had discontinued or crossed over to ruxolitinib. At week 144, mean spleen volume reduction was 34% with ruxolitinib. Previously observed improvements in quality-of-life measures were sustained with longer-term ruxolitinib therapy. Overall survival continued to favor ruxolitinib despite the majority of placebo patients crossing over to ruxolitinib [hazard ratio 0.69 (95% confidence interval: 0.46-1.03); P = 0.067]. Exploratory analyses suggest that crossover may have contributed to an underestimation of the true survival difference between the treatment groups. Ruxolitinib continued to be generally well tolerated; there was no pattern of worsening grade ≥ 3 anemia or thrombocytopenia with longer-term ruxolitinib exposure. These longer-term data continue to support the efficacy and safety of ruxolitinib in patients with myelofibrosis. The study is registered at clinicaltrials.gov: NCT00952289.