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1.
J Virol ; 96(8): e0012822, 2022 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-35343766

RESUMO

The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directs infection of the lungs and other tissues following its binding to the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2'. The "priming" of the surface S protein at S1/S2 (PRRAR685↓) [the underlined basic amino acids refer to critical residues needed for the furin recognition] by furin has been shown to be important for SARS-CoV-2 infectivity in cells and small-animal models. In this study, for the first time we unambiguously identified by proteomics the fusion activation site S2' as KPSKR815↓ (the underlined basic amino acids refer to critical residues needed for the furin recognition) and demonstrated that this cleavage was strongly enhanced by ACE2 engagement with the S protein. Novel pharmacological furin inhibitors (BOS inhibitors) effectively blocked endogenous S protein processing at both sites in HeLa cells, and SARS-CoV-2 infection of lung-derived Calu-3 cells was completely prevented by combined inhibitors of furin (BOS) and type II transmembrane serine protease 2 (TMPRSS2) (camostat). Quantitative analyses of cell-to-cell fusion and S protein processing revealed that ACE2 shedding by TMPRSS2 was required for TMPRSS2-mediated enhancement of fusion in the absence of S1/S2 priming. We further demonstrated that the collectrin dimerization domain of ACE2 was essential for the effect of TMPRSS2 on cell-to-cell fusion. Overall, our results indicate that furin and TMPRSS2 act synergistically in viral entry and infectivity, supporting the combination of furin and TMPRSS2 inhibitors as potent antivirals against SARS-CoV-2. IMPORTANCE SARS-CoV-2, the etiological agent of COVID-19, has so far resulted in >6.1 million deaths worldwide. The spike protein (S) of the virus directs infection of the lungs and other tissues by binding the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S protein is cleaved at two sites: S1/S2 and S2'. Cleavage at S1/S2 induces a conformational change favoring the S protein recognition by ACE2. The S2' cleavage is critical for triggering membrane fusion and virus entry into host cells. Our study highlights the complex dynamics of interaction between the S protein, ACE2, and the host proteases furin and TMPRSS2 during SARS-CoV-2 entry and suggests that the combination of a nontoxic furin inhibitor with a TMPRSS2 inhibitor significantly reduces viral entry in lung cells, as evidenced by an average synergistic ∼95% reduction of viral infection. This represents a powerful novel antiviral approach to reduce viral spread in individuals infected by SARS-CoV-2 or future related coronaviruses.


Assuntos
COVID-19 , Furina , SARS-CoV-2 , Serina Endopeptidases , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , COVID-19/patologia , COVID-19/virologia , Furina/metabolismo , Células HeLa , Humanos , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus
3.
Breast Cancer Res Treat ; 156(1): 21-32, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26907767

RESUMO

Little is known about mutational landscape of rare breast cancer (BC) subtypes. The aim of the study was to apply next generation sequencing to three different subtypes of rare BCs in order to identify new genes related to cancer progression. We performed whole exome and targeted sequencing of 29 micropapillary, 23 metaplastic, and 27 pleomorphic lobular BCs. Micropapillary BCs exhibit a profile comparable to common BCs: PIK3CA, TP53, GATA3, and MAP2K4 were the most frequently mutated genes. Metaplastic BCs presented a high frequency of TP53 (78 %) and PIK3CA (48 %) mutations and were recurrently mutated on KDM6A (13 %), a gene involved in histone demethylation. Pleomorphic lobular carcinoma exhibited high mutation rate of PIK3CA (30 %), TP53 (22 %), and CDH1 (41 %) and also presented mutations in PYGM, a gene involved in glycogen metabolism, in 8 out of 27 samples (30 %). Further analyses of publicly available datasets showed that PYGM is dramatically underexpressed in common cancers as compared to normal tissues and that low expression in tumors is correlated with poor relapse-free survival. Immunohistochemical staining on formalin-fixed paraffin-embedded tissues available in our cohort of patients confirmed higher PYGM expression in normal breast tissue compared to equivalent tumoral zone. Next generation sequencing methods applied on rare cancer subtypes can serve as a useful tool in order to uncover new potential therapeutic targets. Sequencing of pleomorphic lobular carcinoma identified a high rate of alterations in PYGM. These findings emphasize the role of glycogen metabolism in cancer progression.


Assuntos
Neoplasias da Mama/genética , Exoma , Sequenciamento de Nucleotídeos em Larga Escala/métodos , MAP Quinase Quinase 4/genética , Análise de Sequência de DNA/métodos , Neoplasias da Mama/patologia , Classe I de Fosfatidilinositol 3-Quinases , Feminino , Fator de Transcrição GATA3/genética , Humanos , Mutação , Fosfatidilinositol 3-Quinases/genética , Proteína Supressora de Tumor p53/genética
4.
Cell Rep Methods ; 4(1): 100690, 2024 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-38228152

RESUMO

Broadly applicable methods to identify and characterize antigen-specific CD4+ and CD8+ T cells are key to immunology research, including studies of vaccine responses and immunity to infectious diseases. We developed a multiplexed activation-induced marker (AIM) assay that presents several advantages compared to single pairs of AIMs. The simultaneous measurement of four AIMs (CD69, 4-1BB, OX40, and CD40L) creates six AIM pairs that define CD4+ T cell populations with partial and variable overlap. When combined in an AND/OR Boolean gating strategy for analysis, this approach enhances CD4+ T cell detection compared to any single AIM pair, while CD8+ T cells are dominated by CD69/4-1BB co-expression. Supervised and unsupervised clustering analyses show differential expression of the AIMs in defined T helper lineages and that multiplexing mitigates phenotypic biases. Paired and unpaired comparisons of responses to infections (HIV and cytomegalovirus [CMV]) and vaccination (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) validate the robustness and versatility of the method.


Assuntos
Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral , Antígenos/metabolismo , Citomegalovirus
5.
iScience ; 26(1): 105904, 2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36594081

RESUMO

Spacing the first two doses of SARS-CoV-2 mRNA vaccines beyond 3-4 weeks raised initial concerns about vaccine efficacy. While studies have since shown that long-interval regimens induce robust antibody responses, their impact on B and T cell immunity is poorly known. Here, we compare SARS-CoV-2 naive donors B and T cell responses to two mRNA vaccine doses administered 3-4 versus 16 weeks apart. After boost, the longer interval results in a higher magnitude and a more mature phenotype of RBD-specific B cells. While the two geographically distinct cohorts present quantitative and qualitative differences in T cell responses at baseline and after priming, the second dose led to convergent features with overall similar magnitude, phenotype, and function of CD4+ and CD8+ T cell responses at post-boost memory time points. Therefore, compared to standard regimens, a 16-week interval has a favorable impact on the B cell compartment but minimally affects T cell immunity.

6.
Cell Rep Med ; 4(3): 100955, 2023 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-36863335

RESUMO

Cellular immune defects associated with suboptimal responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccination in people receiving hemodialysis (HD) are poorly understood. We longitudinally analyze antibody, B cell, CD4+, and CD8+ T cell vaccine responses in 27 HD patients and 26 low-risk control individuals (CIs). The first two doses elicit weaker B cell and CD8+ T cell responses in HD than in CI, while CD4+ T cell responses are quantitatively similar. In HD, a third dose robustly boosts B cell responses, leads to convergent CD8+ T cell responses, and enhances comparatively more T helper (TH) immunity. Unsupervised clustering of single-cell features reveals phenotypic and functional shifts over time and between cohorts. The third dose attenuates some features of TH cells in HD (tumor necrosis factor alpha [TNFα]/interleukin [IL]-2 skewing), while others (CCR6, CXCR6, programmed cell death protein 1 [PD-1], and HLA-DR overexpression) persist. Therefore, a third vaccine dose is critical to achieving robust multifaceted immunity in hemodialysis patients, although some distinct TH characteristics endure.


Assuntos
Vacinas contra COVID-19 , COVID-19 , Humanos , SARS-CoV-2/genética , COVID-19/prevenção & controle , Linfócitos T CD4-Positivos , Vacinas de mRNA
7.
Cell Rep ; 39(13): 111013, 2022 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-35732172

RESUMO

Spacing of BNT162b2 mRNA doses beyond 3 weeks raises concerns about vaccine efficacy. We longitudinally analyze B cell, T cell, and humoral responses to two BNT162b2 mRNA doses administered 16 weeks apart in 53 SARS-CoV-2 naive and previously infected donors. This regimen elicits robust RBD-specific B cell responses whose kinetics differs between cohorts, the second dose leading to increased magnitude in naive participants only. While boosting does not increase magnitude of CD4+ T cell responses further compared with the first dose, unsupervised clustering of single-cell features reveals phenotypic and functional shifts over time and between cohorts. Integrated analysis shows longitudinal immune component-specific associations, with early T helper responses post first dose correlating with B cell responses after the second dose, and memory T helper generated between doses correlating with CD8 T cell responses after boosting. Therefore, boosting elicits a robust cellular recall response after the 16-week interval, indicating functional immune memory.


Assuntos
COVID-19 , Vacinas Virais , Anticorpos Antivirais , Vacina BNT162 , Humanos , Imunidade Humoral , RNA Mensageiro , SARS-CoV-2
8.
bioRxiv ; 2021 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-34981046

RESUMO

Spacing of the BNT162b2 mRNA doses beyond 3 weeks raised concerns about vaccine efficacy. We longitudinally analyzed B cell, T cell and humoral responses to two BNT162b2 mRNA doses administered 16 weeks apart in 53 SARS-CoV-2 naïve and previously-infected donors. This regimen elicited robust RBD-specific B cell responses whose kinetics differed between cohorts, the second dose leading to increased magnitude in naïve participants only. While boosting did not increase magnitude of CD4 + T cell responses further compared to the first dose, unsupervised clustering analyses of single-cell features revealed phenotypic and functional shifts over time and between cohorts. Integrated analysis showed longitudinal immune component-specific associations, with early Thelper responses post-first dose correlating with B cell responses after the second dose, and memory Thelper generated between doses correlating with CD8 T cell responses after boosting. Therefore, boosting elicits a robust cellular recall response after the 16-week interval, indicating functional immune memory.

9.
mBio ; 11(5)2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32994328

RESUMO

Macrophages are a target of human immunodeficiency virus type 1 (HIV-1) and may serve as a viral reservoir during antiretroviral therapy (ART). Their susceptibility to HIV-1 infection is subject to variations from permissiveness to resistance depending on their origin, tissue localization, and polarization profile. This is in part due to the expression of regulatory microRNAs. Here, we identify two microRNA paralogs, microRNA 103 (miR-103) and miR-107, as regulators of CCR5 expression that are upregulated in noninfected bystander cells of HIV-1-infected-monocyte-derived macrophage (MDM) cultures. Transfection of microRNA 103 mimics in MDMs reduced CCR5 expression levels and inhibited CCR5-dependent HIV-1 entry, whereas the corresponding antagomirs enhanced virus spread in HIV-infected MDMs. Treatment of MDMs with interleukin-1ß (IL-1ß) enhanced microRNA 103 expression, a condition that we found contributed to the reduction of CCR5 mRNA in IL-1ß-exposed MDMs. Interestingly, we show that the induction of miR-103/107 expression is part of a tumor suppressor p53 response triggered by secreted IL-1ß that renders macrophages refractory to HIV-1 entry. In a more physiological context, the levels of microRNAs 103 and 107 were found enriched in tissue-resident colon macrophages of healthy donors and alveolar macrophages of individuals under antiretroviral therapy, conceivably contributing to their relative resistance to HIV-1 infection. Overall, these findings highlight the role of p53 in enforcing proinflammatory antiviral responses in macrophages, at least in part, through miR-103/107-mediated downmodulation of CCR5 expression and HIV-1 entry.IMPORTANCE Macrophages are heterogeneous immune cells that display varying susceptibilities to HIV-1 infection, in part due to the expression of small noncoding microRNAs involved in the posttranscriptional regulation of gene expression and silencing. Here, we identify microRNAs 103 and 107 as important p53-regulated effectors of the antiviral response triggered by the proinflammatory cytokine IL-1ß in macrophages. These microRNAs, which are enriched in colon macrophages of healthy donors and alveolar macrophages of HIV-infected individuals under antiretroviral therapy, act as inhibitors of HIV-1 entry through their capacity to downregulate the CCR5 coreceptor. These results highlight the important role played by miR-103/107 in modulating CCR5 expression and HIV-1 entry in macrophages. They further underscore a distinct function of the tumor suppressor p53 in enforcing proinflammatory antiviral responses in macrophages, thus providing insight into a cellular pathway that could be targeted to limit the establishment of viral reservoirs in these cells.


Assuntos
Interleucina-1beta/genética , MicroRNAs/imunologia , Receptores CCR5/genética , Proteína Supressora de Tumor p53/genética , Internalização do Vírus , Regulação para Baixo , Regulação da Expressão Gênica , Infecções por HIV/virologia , HIV-1 , Humanos , Interleucina-1beta/imunologia , Macrófagos/imunologia , Macrófagos/virologia , MicroRNAs/genética , Receptores CCR5/imunologia , Proteína Supressora de Tumor p53/imunologia
10.
Clin Cancer Res ; 25(22): 6671-6682, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31439588

RESUMO

PURPOSE: Patients with anaplastic lymphoma kinase (ALK)-rearranged non-small-cell lung cancer (NSCLC) inevitably develop resistance to ALK inhibitors. New diagnostic strategies are needed to assess resistance mechanisms and provide patients with the most effective therapy. We asked whether single circulating tumor cell (CTC) sequencing can inform on resistance mutations to ALK inhibitors and underlying tumor heterogeneity in ALK-rearranged NSCLC. EXPERIMENTAL DESIGN: Resistance mutations were investigated in CTCs isolated at the single-cell level from patients at disease progression on crizotinib (n = 14) or lorlatinib (n = 3). Three strategies including filter laser-capture microdissection, fluorescence activated cell sorting, and the DEPArray were used. One hundred twenty-six CTC pools and 56 single CTCs were isolated and sequenced. Hotspot regions over 48 cancer-related genes and 14 ALK mutations were examined to identify ALK-independent and ALK-dependent resistance mechanisms. RESULTS: Multiple mutations in various genes in ALK-independent pathways were predominantly identified in CTCs of crizotinib-resistant patients. The RTK-KRAS (EGFR, KRAS, BRAF genes) and TP53 pathways were recurrently mutated. In one lorlatinib-resistant patient, two single CTCs out of 12 harbored ALK compound mutations. CTC-1 harbored the ALK G1202R/F1174C compound mutation virtually similar to ALK G1202R/F1174L present in the corresponding tumor biopsy. CTC-10 harbored a second ALK G1202R/T1151M compound mutation not detected in the tumor biopsy. By copy-number analysis, CTC-1 and the tumor biopsy had similar profiles, whereas CTC-10 harbored multiple copy-number alterations and whole-genome duplication. CONCLUSIONS: Our results highlight the genetic heterogeneity and clinical utility of CTCs to identify therapeutic resistance mutations in ALK-rearranged patients. Single CTC sequencing may be a unique tool to assess heterogeneous resistance mechanisms and help clinicians for treatment personalization and resistance options to ALK-targeted therapies.


Assuntos
Quinase do Linfoma Anaplásico/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Resistencia a Medicamentos Antineoplásicos/genética , Rearranjo Gênico , Neoplasias Pulmonares/genética , Células Neoplásicas Circulantes/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Adulto , Idoso , Antineoplásicos/farmacologia , Biomarcadores Tumorais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Biologia Computacional/métodos , Crizotinibe/farmacologia , Crizotinibe/uso terapêutico , Análise Mutacional de DNA , Feminino , Humanos , Imuno-Histoquímica , Imunofenotipagem , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Células Neoplásicas Circulantes/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Sequenciamento Completo do Genoma , Adulto Jovem
11.
Oncotarget ; 7(18): 25549-57, 2016 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-27016411

RESUMO

PURPOSE: This phase I trial aimed to assess the safety and determine the recommended Phase II dose (RP2D) of Cidofovir combined with chemoradiotherapy in patients with stage IB2-IVA cervical cancer. EXPERIMENTAL DESIGN: Incremental doses (1, 2.5, 5 and 6.5 mg/kg) of IV Cidofovir were administered weekly for two weeks, and then every 2 weeks from the start of chemoradiotherapy to the initiation of utero-vaginal brachytherapy. Biological expression of HPV was analyzed during treatment and tumor response was assessed according to RECIST v1.0 criteria. RESULTS: A total of 15 patients were treated with Cidofovir. Dose-limiting toxicities occurred in 2/6 patients at the 6.5 mg/kg dose level (G3 proteinuria, and G3 acute pyelonephritis with G3 febrile neutropenia). No toxicity occurred at the 5 mg/kg dose level, but only 3 patients received this dose due to trial interruption because of low accrual. The most frequent G3-4 adverse effects observed during the trial were: abdominal pain (n=3), infection (n=2), leuckoneutropenia (n=2), and others (n=6). No toxic death or major renal side effect occurred. The best response was that 8/9 evaluable patients achieved a complete response (89%). In the intention to treat population, the 2-year overall and progression-free survival rates were 93% and 76%, respectively. Biological monitoring of HPV-related markers (decreased p16 expression, and increased p53 and pRb levels) was possible on sequential tumor biopsy samples. The genomic alterations identified were PIK3CA (n=5; one also had a KRAS mutation), and HRAS (n=1) mutations. CONCLUSIONS: Cidofovir at a dose of 5mg/kg combined with chemoradiotherapy appeared tolerable and yielded tumor regressions. Due to early trial interruption, the RP2D was not confirmed.


Assuntos
Antivirais/administração & dosagem , Quimiorradioterapia/métodos , Citosina/análogos & derivados , Organofosfonatos/administração & dosagem , Neoplasias do Colo do Útero/tratamento farmacológico , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Antivirais/efeitos adversos , Braquiterapia/métodos , Carboplatina/administração & dosagem , Cidofovir , Citosina/administração & dosagem , Citosina/efeitos adversos , Feminino , Humanos , Pessoa de Meia-Idade , Organofosfonatos/efeitos adversos , Radiossensibilizantes/administração & dosagem , Radiossensibilizantes/efeitos adversos , Neoplasias do Colo do Útero/radioterapia , Neoplasias do Colo do Útero/virologia
12.
Cancer Res ; 76(6): 1476-84, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26825172

RESUMO

BRAF inhibitors (BRAFi) elicit therapeutic responses in metastatic melanoma, but alarmingly, also induce the formation of secondary benign and malignant skin tumors. Here, we report the emergence and molecular characterization of 73 skin and extracutaneous tumors in 31 patients who underwent BRAFi therapy. The majority of patients presented with classic epidermal tumors such as verrucous papillomas, keratoacanthomas, and squamous cell carcinomas (SCC). However, 15 patients exhibited new or rapidly progressing tumors distinct from these classic subtypes, such as lymph node metastasis, new melanomas, and genital and oral mucosal SCCs. Genotyping of the tumors revealed that oncogenic RAS mutations were found in 58% of the evaluable tumor samples (38/66) and 49% of the control tumors from patients not treated with BRAFi (30/62). Notably, proximity ligation assays demonstrated that BRAF-CRAF heterodimerization was increased in fixed tumor samples from BRAFi-treated patients compared with untreated patients. Our findings reveal that BRAF-CRAF complex formation is significantly associated with BRAFi treatment, and may therefore serve as a useful biomarker of BRAFi-induced cutaneous and extracutaneous tumor formation.


Assuntos
Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/metabolismo , Linhagem Celular Tumoral , Dimerização , Genótipo , Humanos , Mutação/efeitos dos fármacos , Proteínas Proto-Oncogênicas B-raf/metabolismo
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