RESUMO
Type III interferon (IFNλ) and type I IFN (IFNα/ß) have overlapping antiviral activities in the lung. In this issue of Immunity, Galani et al. (2017) identify a critical early role for IFNλ, not shared by IFNα/ß, in protection of the lung following influenza virus infection.
Assuntos
Antivirais , Interferon-alfa , Humanos , Orthomyxoviridae , Infecções por Orthomyxoviridae/imunologiaRESUMO
BACKGROUND/AIMS: Maintenance of whole-body ascorbate levels and distribution is mediated via sodium-dependent vitamin C transporters (SVCTs). The kidney is one of a few organs that express both SVCT1 and SVCT2. Recent evidence suggests that accumulation of ascorbate may be different in tumour compared to normal tissue, but data on SVCT levels in tumours is sparse. METHODS: The role of the two SVCT isoforms in ascorbate uptake in renal cell carcinoma (RCC) was investigated in vitro and in clinical samples. In three human RCC cell lines, we investigated SVCT protein levels and cellular location in response to ascorbate supplementation and withdrawal. In clinical RCC samples (n=114), SVCT patterns of staining and protein levels were analysed and compared to ascorbate levels. RESULTS: In cell culture, transporter levels and cellular location were not modified by ascorbate availability at any time up to 8h, although basal SVCT2 levels governed maximal ascorbate accumulation. In clinical samples, SVCT1 protein levels in papillary RCC (pRCC) were similar to matched normal renal cortex, but were increased in clear-cell RCC (ccRCC). Native SVCT2 (72 kDa) was significantly decreased in both pRCC and ccRCC tissues compared to cortex (p<0.01), whereas a modified form of SVCT2 (100 kDa) was significantly increased (p<0.001). There was no association between the transporters (SVCT1, native or modified SVCT2) and ascorbate concentrations in either normal or tumour tissues. SVCT1 and SVCT2 displayed diffuse cytoplasmic staining in both pRCC and ccRCC tumour cells, with cortex showing distinct membrane staining for SVCT1. CONCLUSION: We observed a re-distribution of ascorbate transporters in tumour tissue compared to normal cortex and a shift from native to modified SVCT2 in cell culture and clinical samples. Data presented here show that SVCT protein levels do not appear to predict intracellular ascorbate accumulation in RCC.
Assuntos
Ácido Ascórbico/metabolismo , Carcinoma de Células Renais/metabolismo , Neoplasias Renais/metabolismo , Transportadores de Sódio Acoplados à Vitamina C/metabolismo , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Humanos , Neoplasias Renais/patologia , Transportadores de Sódio Acoplados à Vitamina C/análiseRESUMO
BACKGROUND: Diagnostic screening for pathogenic variants in breast cancer susceptibility genes, including BRCA1, BRCA2, PALB2, PTEN and TP53, may be offered to New Zealanders from suspected high-risk breast (and ovarian) cancer families. However, it is unknown how many high-risk pathogenic variant carriers in New Zealand are not offered genetic screening using existing triage tools and guidelines for breast (and ovarian) cancer patients. METHODS: Panel-gene sequencing of the coding and non-coding regions of the BRCA1 and BRCA2 genes, and the coding regions and splice sites of CDH1, PALB2, PTEN and TP53, was undertaken for an unselected cohort of 367 female breast cancer patients. A total of 1685 variants were evaluated using the ENIGMA and the ACMG/AMP variant classification guidelines. RESULTS: Our study identified that 13 (3.5%) breast cancer patients carried a pathogenic or likely pathogenic variant in BRCA1, BRCA2, PALB2, or PTEN. A significantly higher number of pathogenic variant carriers had grade 3 tumours (10/13) when compared to non-carriers; however, no other clinicopathological characteristics were found to be significantly different between (likely) pathogenic variant carriers and non-carriers, nor between variant of unknown significance carriers and non-carriers. Notably, 46% of the identified (likely) pathogenic variant carriers had not been referred for a genetic assessment and consideration of genetic testing. CONCLUSION: Our study shows a potential under-ascertainment of women carrying a (likely) pathogenic variant in a high-risk breast cancer susceptibility gene. These results suggest that further research into testing pathways for New Zealand breast cancer patients may be required to reduce the impact of hereditary cancer syndromes for these individuals and their families.
Assuntos
Neoplasias da Mama , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/epidemiologia , Neoplasias da Mama/genética , Feminino , Genes BRCA2 , Predisposição Genética para Doença , Testes Genéticos , Mutação em Linhagem Germinativa , Heterozigoto , Humanos , Nova Zelândia/epidemiologiaRESUMO
Human norovirus (HNoV) is the leading cause of acute gastroenteritis and is spread by fecal shedding that can often persist for weeks to months after the resolution of symptoms. Elimination of persistent viral reservoirs has the potential to prevent outbreaks. Similar to HNoV, murine norovirus (MNV) is spread by persistent shedding in the feces and provides a tractable model to study molecular mechanisms of enteric persistence. Previous studies have identified non-structural protein 1 (NS1) from the persistent MNV strain CR6 as critical for persistent infection in intestinal epithelial cells (IECs), but its mechanism of action remains unclear. We now find that the function of CR6 NS1 is regulated by apoptotic caspase cleavage. Following induction of apoptosis in infected cells, caspases cleave the precursor NS1/2 protein, and this cleavage is prevented by mutation of caspase target motifs. These mutations profoundly compromise CR6 infection of IECs and persistence in the intestine. Conversely, NS1/2 cleavage is not strictly required for acute replication in extra-intestinal tissues or in cultured myeloid cells, suggesting an IEC-centric role. Intriguingly, we find that caspase cleavage of CR6 NS1/2 reciprocally promotes caspase activity, potentiates cell death, and amplifies spread among cultured IEC monolayers. Together, these data indicate that the function of CR6 NS1 is regulated by apoptotic caspases, and suggest that apoptotic cell death enables epithelial spread and persistent shedding.
Assuntos
Mucosa Intestinal/virologia , Norovirus/patogenicidade , Proteínas não Estruturais Virais/metabolismo , Animais , Apoptose , Infecções por Caliciviridae/etiologia , Infecções por Caliciviridae/patologia , Infecções por Caliciviridae/virologia , Caspases/metabolismo , Células Cultivadas , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Células Epiteliais/virologia , Feminino , Gastroenterite/etiologia , Gastroenterite/patologia , Gastroenterite/virologia , Interações entre Hospedeiro e Microrganismos , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Camundongos , Camundongos Knockout , Modelos Biológicos , Células Mieloides/metabolismo , Células Mieloides/patologia , Células Mieloides/virologia , Norovirus/genética , Norovirus/fisiologia , Proteínas não Estruturais Virais/genética , Replicação Viral , Eliminação de Partículas ViraisRESUMO
During immature capsid assembly, HIV-1 genome packaging is initiated when Gag first associates with unspliced HIV-1 RNA by a poorly understood process. Previously, we defined a pathway of sequential intracellular HIV-1 capsid assembly intermediates; here we sought to identify the intermediate in which HIV-1 Gag first associates with unspliced HIV-1 RNA. In provirus-expressing cells, unspliced HIV-1 RNA was not found in the soluble fraction of the cytosol, but instead was largely in complexes ≥30S. We did not detect unspliced HIV-1 RNA associated with Gag in the first assembly intermediate, which consists of soluble Gag. Instead, the earliest assembly intermediate in which we detected Gag associated with unspliced HIV-1 RNA was the second assembly intermediate (~80S intermediate), which is derived from a host RNA granule containing two cellular facilitators of assembly, ABCE1 and the RNA granule protein DDX6. At steady-state, this RNA-granule-derived ~80S complex was the smallest assembly intermediate that contained Gag associated with unspliced viral RNA, regardless of whether lysates contained intact or disrupted ribosomes, or expressed WT or assembly-defective Gag. A similar complex was identified in HIV-1-infected T cells. RNA-granule-derived assembly intermediates were detected in situ as sites of Gag colocalization with ABCE1 and DDX6; moreover these granules were far more numerous and smaller than well-studied RNA granules termed P bodies. Finally, we identified two steps that lead to association of assembling Gag with unspliced HIV-1 RNA. Independent of viral-RNA-binding, Gag associates with a broad class of RNA granules that largely lacks unspliced viral RNA (step 1). If a viral-RNA-binding domain is present, Gag further localizes to a subset of these granules that contains unspliced viral RNA (step 2). Thus, our data raise the possibility that HIV-1 packaging is initiated not by soluble Gag, but by Gag targeted to a subset of host RNA granules containing unspliced HIV-1 RNA.
Assuntos
Infecções por HIV/virologia , HIV-1/genética , Splicing de RNA , RNA Viral/metabolismo , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Citoplasma/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Infecções por HIV/genética , Humanos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Viral/genética , Vírion , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genéticaRESUMO
BACKGROUND: The transcription factor hypoxia inducible factor (HIF) -1 drives tumor growth and metastasis and is associated with poor prognosis in breast cancer. Ascorbate can moderate HIF-1 activity in vitro and is associated with HIF pathway activation in a number of cancer types, but whether tissue ascorbate levels influence the HIF pathway in breast cancer is unknown. In this study we investigated the association between tumor ascorbate levels and HIF-1 activation and patient survival in human breast cancer. METHODS: In a retrospective analysis of human breast cancer tissue, we analysed primary tumor and adjacent uninvolved tissue from 52 women with invasive ductal carcinoma. We measured HIF-1α, HIF-1 gene targets CAIX, BNIP-3 and VEGF, and ascorbate content. Patient clinical outcomes were evaluated against these parameters. RESULTS: HIF-1 pathway proteins were upregulated in tumor tissue and increased HIF-1 activation was associated with higher tumor grade and stage, with increased vascular invasion and necrosis, and with decreased disease-free and disease-specific survival. Grade 1 tumors had higher ascorbate levels than did grade 2 or 3 tumors. Higher ascorbate levels were associated with less tumor necrosis, with lower HIF-1 pathway activity and with increased disease-free and disease-specific survival. CONCLUSIONS: Our findings indicate that there is a direct correlation between intracellular ascorbate levels, activation of the HIF-1 pathway and patient survival in breast cancer. This is consistent with the known capacity of ascorbate to stimulate the activity of the regulatory HIF hydroxylases and suggests that optimisation of tumor ascorbate could have clinical benefit via modulation of the hypoxic response.
Assuntos
Antígenos de Neoplasias/metabolismo , Ácido Ascórbico/metabolismo , Neoplasias da Mama/patologia , Anidrase Carbônica IX/metabolismo , Carcinoma Ductal de Mama/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Antígenos de Neoplasias/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Anidrase Carbônica IX/genética , Hipóxia Celular , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Proteínas Proto-Oncogênicas/genética , Estudos Retrospectivos , Análise de Sobrevida , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
BRCA1 and BRCA2 spliceogenic variants are often associated with an elevated risk of breast and ovarian cancers. Analyses of BRCA1 and BRCA2 splicing patterns have traditionally used technologies that sample a population of cells but do not account for the variation that may be present between individual cells. This novel proof of concept study utilises RNA in situ hybridisation to measure the absolute expression of BRCA1 and BRCA2 mRNA splicing events in single lymphoblastoid cells containing known spliceogenic variants (BRCA1c.671-2 A>G or BRCA2c.7988 A>T). We observed a large proportion of cells (>42%) in each sample that did not express mRNA for the targeted gene. Increased levels (average mRNA molecules per cell) of BRCA2 ∆17_18 were observed in the cells containing the known spliceogenic variant BRCA2c.7988 A>T, but cells containing BRCA1c.671-2 A>G were not found to express significantly increased levels of BRCA1 ∆11, as had been shown previously. Instead, we show for each variant carrier sample that a higher proportion of cells expressed the targeted splicing event compared to control cells. These results indicate that BRCA1/2 mRNA is expressed stochastically, suggesting that previously reported results using RT-PCR may have been influenced by the number of cells with BRCA1/2 mRNA expression and may not represent an elevation of constitutive mRNA expression. Detection of mRNA expression in single cells allows for a more comprehensive understanding of how spliceogenic variants influence the expression of mRNA isoforms. However, further research is required to assess the utility of this technology to measure the expression of predicted spliceogenic BRCA1 and BRCA2 variants in a diagnostic setting.
Assuntos
Processamento Alternativo , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/genética , RNA Mensageiro , Alelos , Linhagem Celular Tumoral , Feminino , Genótipo , Humanos , Análise de Célula ÚnicaRESUMO
UNLABELLED: The major homology region (MHR) is a highly conserved motif that is found within the Gag protein of all orthoretroviruses and some retrotransposons. While it is widely accepted that the MHR is critical for assembly of HIV-1 and other retroviruses, how the MHR functions and why it is so highly conserved are not understood. Moreover, consensus is lacking on when HIV-1 MHR residues function during assembly. Here, we first addressed previous conflicting reports by confirming that MHR deletion, like conserved MHR residue substitution, leads to a dramatic reduction in particle production in human and nonhuman primate cells expressing HIV-1 proviruses. Next, we used biochemical analyses and immunoelectron microscopy to demonstrate that conserved residues in the MHR are required after assembling Gag has associated with genomic RNA, recruited critical host factors involved in assembly, and targeted to the plasma membrane. The exact point of inhibition at the plasma membrane differed depending on the specific mutation, with one MHR mutant arrested as a membrane-associated intermediate that is stable upon high-salt treatment and other MHR mutants arrested as labile, membrane-associated intermediates. Finally, we observed the same assembly-defective phenotypes when the MHR deletion or conserved MHR residue substitutions were engineered into Gag from a subtype B, lab-adapted provirus or Gag from a subtype C primary isolate that was codon optimized. Together, our data support a model in which MHR residues act just after membrane targeting, with some MHR residues promoting stability and another promoting multimerization of the membrane-targeted assembling Gag oligomer. IMPORTANCE: The retroviral Gag protein exhibits extensive amino acid sequence variation overall; however, one region of Gag, termed the major homology region, is conserved among all retroviruses and even some yeast retrotransposons, although the reason for this conservation remains poorly understood. Highly conserved residues in the major homology region are required for assembly of retroviruses; however, when these residues are required during assembly is not clear. Here, we used biochemical and electron microscopic analyses to demonstrate that these conserved residues function after assembling HIV-1 Gag has associated with genomic RNA, recruited critical host factors involved in assembly, and targeted to the plasma membrane but before Gag has completed the assembly process. By revealing precisely when conserved residues in the major homology region are required during assembly, these studies resolve existing controversies and set the stage for future experiments aimed at a more complete understanding of how the major homology region functions.
Assuntos
HIV-1/fisiologia , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/virologia , HIV-1/genética , Interações Hospedeiro-Patógeno , Humanos , Modelos Biológicos , Mutação de Sentido Incorreto , Ligação Proteica , Multimerização Proteica , Deleção de SequênciaRESUMO
Remarkable progress has been made over the past decade in cancer medicine. Personalized medicine, driven by biomarker predictive factors, novel biotherapy, novel imaging, and molecular targeted therapeutics, has improved outcomes. Cancer is becoming a chronic disease rather than a fatal disease for many patients. However, despite this progress, there is much work to do if patients are to receive continuous high-quality care in the appropriate place, at the appropriate time, and with the right specialized expert oversight. Unfortunately, the rapid expansion of therapeutic options has also generated an ever-increasing burden of emergency care and encroaches into end-of-life palliative care. Emergency presentation is a common consequence of cancer and of cancer treatment complications. It represents an important proportion of new presentations of previously undiagnosed malignancy. In the U.K. alone, 20%-25% of new cancer diagnoses are made following an initial presentation to the hospital emergency department, with a greater proportion in patients older than 70 years. This late presentation accounts for poor survival outcomes and is often associated with poor patient experience and poorly coordinated care. The recent development of acute oncology services in the U.K. aims to improve patient safety, quality of care, and the coordination of care for all patients with cancer who require emergency access to care, irrespective of the place of care and admission route. Furthermore, prompt management coordinated by expert teams and access to protocol-driven pathways have the potential to improve patient experience and drive efficiency when services are fully established. The challenge to leaders of acute oncology services is to develop bespoke models of care, appropriate to local services, but with an opportunity for acute oncology teams to engage cancer care strategies and influence cancer care and delivery in the future. This will aid the integration of highly specialized cancer treatment with high-quality care close to home and help avoid hospital admission.
Assuntos
Serviços Médicos de Emergência , Neoplasias/mortalidade , Neoplasias/terapia , Serviço Hospitalar de Oncologia , Medicina de Precisão , Humanos , Prognóstico , Reino UnidoRESUMO
UNLABELLED: The interferon (IFN) response is the earliest host immune response dedicated to combating viral infection. As such, viruses have evolved strategies to subvert this potent antiviral response. Two closely related gammaherpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV) and rhesus macaque rhadinovirus (RRV), are unique in that they express viral homologues to cellular interferon regulatory factors (IRFs), termed viral IRFs (vIRFs). Cellular IRFs are a family of transcription factors that are particularly important for the transcription of type I IFNs. Here, we demonstrate a strategy employed by RRV to ensure rapid inhibition of virus-induced type I IFN induction. We found that RRV vIRF R6, when expressed ectopically, interacts with a transcriptional coactivator, CREB-binding protein (CBP), in the nucleus. As a result, phosphorylated IRF3, an important transcriptional regulator in beta interferon (IFN-ß) transcription, fails to effectively bind to the IFN-ß promoter, thus inhibiting the activation of IFN-ß genes. In addition, we found R6 within RRV virion particles via immunoelectron microscopy and, furthermore, that virion-associated R6 is capable of inhibiting the type I IFN response by preventing efficient binding of IRF3/CBP complexes to the IFN-ß promoter in the context of infection. The work shown here is the first example of a vIRF being associated with either the KSHV or RRV virion. The presence of this immunomodulatory protein in the RRV virion provides the virus with an immediate mechanism to evade the host IFN response, thus enabling the virus to effectively establish an infection within the host. IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) and the closely related rhesus macaque rhadinovirus (RRV) are the only viruses known to encode viral homologues to cellular interferon regulatory factors (IRFs), known as vIRFs. In KSHV, these proteins have been shown to play major roles in a variety of cellular processes and are particularly important in the evasion of the host type I interferon (IFN) response. In this study, we delineate the immunomodulatory mechanism of an RRV vIRF and its ability to assist the virus in rapid immune evasion by being prepackaged within the virion, thus providing evidence, for the first time, of a virion-associated vIRF. This work further contributes to our understanding of the mechanisms behind immunomodulation by the RRV vIRFs during infection.
Assuntos
Infecções por Herpesviridae/imunologia , Fatores Reguladores de Interferon/imunologia , Interferon beta/imunologia , Rhadinovirus/imunologia , Proteínas Virais/imunologia , Vírion/imunologia , Animais , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/imunologia , Linhagem Celular , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/virologia , Interações Hospedeiro-Patógeno , Humanos , Evasão da Resposta Imune , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Fatores Reguladores de Interferon/genética , Interferon beta/genética , Macaca mulatta , Regiões Promotoras Genéticas , Ligação Proteica , Rhadinovirus/genética , Proteínas Virais/genética , Vírion/genéticaRESUMO
UNLABELLED: During HIV-1 assembly, Gag polypeptides target to the plasma membrane, where they multimerize to form immature capsids that undergo budding and maturation. Previous mutational analyses identified residues within the Gag matrix (MA) and capsid (CA) domains that are required for immature capsid assembly, and structural studies showed that these residues are clustered on four exposed surfaces in Gag. Exactly when and where the three critical surfaces in CA function during assembly are not known. Here, we analyzed how mutations in these four critical surfaces affect the formation and stability of assembly intermediates in cells expressing the HIV-1 provirus. The resulting temporospatial map reveals that critical MA residues act during membrane targeting, residues in the C-terminal CA subdomain (CA-CTD) dimer interface are needed for the stability of the first membrane-bound assembly intermediate, CA-CTD base residues are necessary for progression past the first membrane-bound intermediate, and residues in the N-terminal CA subdomain (CA-NTD) stabilize the last membrane-bound intermediate. Importantly, we found that all four critical surfaces act while Gag is associated with the cellular facilitators of assembly ABCE1 and DDX6. When correlated with existing structural data, our findings suggest the following model: Gag dimerizes via the CA-CTD dimer interface just before or during membrane targeting, individual CA-CTD hexamers form soon after membrane targeting, and the CA-NTD hexameric lattice forms just prior to capsid release. This model adds an important new dimension to current structural models by proposing the potential order in which key contacts within the immature capsid lattice are made during assembly in cells. IMPORTANCE: While much is known about the structure of the completed HIV-1 immature capsid and domains of its component Gag proteins, less is known about the sequence of events leading to formation of the HIV-1 immature capsid. Here we used biochemical and ultrastructural analyses to generate a temporospatial map showing the precise order in which four critical surfaces in Gag act during immature capsid formation in provirus-expressing cells. Because three of these surfaces make important contacts in the hexameric lattices that are found in the completed immature capsid, these data allow us to propose a model for the sequence of events leading to formation of the hexameric lattices. By providing a dynamic view of when and where critical Gag-Gag contacts form during the assembly process and how those contacts function in the nascent capsid, our study provides novel insights into how an immature capsid is built in infected cells.
Assuntos
Antígenos HIV/metabolismo , Proteína do Núcleo p24 do HIV/metabolismo , HIV-1/fisiologia , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Capsídeo/metabolismo , RNA Helicases DEAD-box/metabolismo , Análise Mutacional de DNA , Antígenos HIV/genética , Proteína do Núcleo p24 do HIV/genética , HIV-1/genética , Modelos Biológicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Tempo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genéticaRESUMO
AIMS: The proliferation marker Ki67 has been extensively investigated as a prognostic factor in breast cancer, but has not gained widespread clinical acceptance. Phosphohistone H3 is a new immunohistochemical marker for quantifying mitoses; however, there is limited information on its prognostic value in breast cancer. In this study, we performed a head-to-head comparison of Ki67 and phosphohistone H3 to establish the marker with the greatest prognostic value. METHODS AND RESULTS: Tissue microarrays from 108 breast cancer patients were immunohistochemically stained for Ki67 and phosphohistone H3. Our results showed that phosphohistone H3 had a greater prognostic value than Ki67 in a multivariable model that adjusted for traditional prognostic variables in breast cancer. Phosphohistone H3 staining was a stronger predictor of survival at 5 years after diagnosis [hazard ratio (HR) 4.35, P < 10(-5) ] than Ki67 (HR 2.44, P = 0.004), and better separated the risk of death in patients aged >45 years. Importantly, phosphohistone H3 consistently showed strong unequivocal staining, in contrast to the variable staining intensities associated with Ki67. CONCLUSIONS: Our study suggests that phosphohistone H3 staining is a stronger and more robust prognostic indicator than Ki67 staining in breast cancer patients, and has the potential for use in routine diagnostic laboratories.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/patologia , Histonas/análise , Antígeno Ki-67/análise , Adulto , Idoso , Neoplasias da Mama/mortalidade , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Fosforilação , Prognóstico , Modelos de Riscos Proporcionais , Análise Serial de TecidosRESUMO
BACKGROUND: Dose intense chemotherapy may improve efficacy with acceptable toxicity. A phase II study was conducted to determine the feasibility of a dose-intense two weekly schedule of capecitabine, oxaliplatin, and bevacizumab in metastatic colorectal cancer (mCRC). METHODS: 49 patients with previously untreated mCRC were recruited. Nineteen received capecitabine (1750 mg/m(2) oral BD days 1-7)oxaliplatin (85 mg/m(2)i.v. day 1) and bevacizumab (5 mg/kg i.v. day 1) using a 14-day cycle (C1750). Following toxicity concerns capecitabine was reduced to 1500 mg/m2oral BD (C1500) and 30 further patients recruited. RESULTS: Over 80% of patients received at least 75% of planned chemotherapy doses over the first two cycles. At C1750 Grade 3 or higher toxicity occurred in 74% (95% CI 49% to 91%) and on C1500 in 70% (95% CI 51% to 85%). The median progression-free survival was 6.9 months (95% CI 4.7 to 8.7) for C1750 dose and 8.9 months (95% CI 4.1 to 12.4) for C1500. 3 treatment-related deaths occurred. CONCLUSIONS: Dose intense capecitabine and oxaliplatin with bevacizumab does not show additional efficacy and has potentially significant toxicity. Its use outside of clinical trials is not recommended. TRIAL REGISTRATION: ISRCTN41540878.
Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Neoplasias Colorretais/tratamento farmacológico , Idoso , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Bevacizumab , Capecitabina , Desoxicitidina/administração & dosagem , Desoxicitidina/efeitos adversos , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Fluoruracila/administração & dosagem , Fluoruracila/efeitos adversos , Fluoruracila/análogos & derivados , Fluoruracila/uso terapêutico , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Compostos Organoplatínicos/administração & dosagem , Compostos Organoplatínicos/efeitos adversos , Compostos Organoplatínicos/uso terapêutico , Oxaliplatina , Análise de Sobrevida , Resultado do TratamentoRESUMO
The treatment of metastatic melanoma has been revolutionised by immunotherapy, yet a significant number of patients do not respond, and many experience autoimmune adverse events. Associations have been reported between patient outcome and monocyte subsets, whereas vitamin C (ascorbate) has been shown to mediate changes in cancer-stimulated monocytes in vitro. We therefore investigated the relationship of ascorbate with monocyte subsets and epigenetic modifications in patients with metastatic melanoma receiving immunotherapy. Patients receiving immunotherapy were compared to other cancer cohorts and age-matched healthy controls. Ascorbate levels in plasma and peripheral blood-derived mononuclear cells (PBMCs), monocyte subtype and epigenetic markers were measured, and adverse events, tumour response and survival were recorded. A quarter of the immunotherapy cohort had hypovitaminosis C, with plasma and PBMC ascorbate levels significantly lower than those from other cancer patients or healthy controls. PBMCs from the immunotherapy cohort contained similar frequencies of non-classical and classical monocytes. DNA methylation markers and intracellular ascorbate concentration were correlated with monocyte subset frequency in healthy controls, but correlation was lost in immunotherapy patients. No associations between ascorbate status and immune-related adverse events or tumour response or overall survival were apparent.
RESUMO
Kaposi's sarcoma-associated herpesvirus and rhesus macaque rhadinovirus (RRV), two closely related gammaherpesviruses, are unique in their expression of viral homologs of cellular interferon regulatory factors (IRFs), termed viral IRFs (vIRFs). To assess the role of vIRFs during de novo infection, we have utilized the bacterial artificial chromosome clone of wild-type RRV(17577) (WT(BAC) RRV) to generate a recombinant virus with all 8 of the vIRFs deleted (vIRF-ko RRV). The infection of primary rhesus fibroblasts and peripheral blood mononuclear cells (PBMCs) with vIRF-ko RRV resulted in earlier and increased induction of type I interferon (IFN) (IFN-α/ß) and type II IFN (IFN-γ). Additionally, plasmacytoid dendritic cells maintained higher levels of IFN-α production in PBMC cultures infected with vIRF-ko RRV than in cultures infected with WT(BAC) RRV. Moreover, the nuclear accumulation of phosphorylated IRF-3, which is necessary for the induction of type I IFN, was also inhibited following WT(BAC) RRV infection. These findings demonstrate that during de novo RRV infection, vIRFs are inhibiting the induction of IFN at the transcriptional level, and one potential mechanism for this is the disruption of the activation and localization of IRF-3.
Assuntos
Regulação para Baixo , Infecções por Herpesviridae/veterinária , Fatores Reguladores de Interferon/metabolismo , Interferon Tipo I/genética , Interferon gama/genética , Doenças dos Primatas/genética , Rhadinovirus/fisiologia , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/metabolismo , Infecções por Herpesviridae/virologia , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Fatores Reguladores de Interferon/genética , Interferon Tipo I/metabolismo , Interferon gama/metabolismo , Macaca mulatta , Doenças dos Primatas/metabolismo , Doenças dos Primatas/virologia , Rhadinovirus/genética , Proteínas Virais/genéticaRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) and the closely related gamma-2 herpesvirus rhesus macaque (RM) rhadinovirus (RRV) are the only known viruses to encode viral homologues of the cellular interferon (IFN) regulatory factors (IRFs). Recent characterization of a viral IRF (vIRF) deletion clone of RRV (vIRF-knockout RRV [vIRF-ko RRV]) demonstrated that vIRFs inhibit induction of type I and type II IFNs during RRV infection of peripheral blood mononuclear cells. Because the IFN response is a key component to a host's antiviral defenses, this study has investigated the role of vIRFs in viral replication and the development of the immune response during in vivo infection in RMs, the natural host of RRV. Experimental infection of RMs with vIRF-ko RRV resulted in decreased viral loads and diminished B cell hyperplasia, a characteristic pathology during acute RRV infection that often develops into more severe lymphoproliferative disorders in immune-compromised animals, similar to pathologies in KSHV-infected individuals. Moreover, in vivo infection with vIRF-ko RRV resulted in earlier and sustained production of proinflammatory cytokines and earlier induction of an anti-RRV T cell response compared to wild-type RRV infection. These findings reveal the broad impact that vIRFs have on pathogenesis and the immune response in vivo and are the first to validate the importance of vIRFs during de novo infection in the host.
Assuntos
Modelos Animais de Doenças , Infecções por Herpesviridae/imunologia , Fatores Reguladores de Interferon/imunologia , Macaca mulatta , Rhadinovirus/imunologia , Proteínas Virais/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/virologia , Infecções por Herpesviridae/virologia , Humanos , Fatores Reguladores de Interferon/genética , Interferons/imunologia , Rhadinovirus/genética , Proteínas Virais/genéticaRESUMO
BACKGROUND AND OBJECTIVE: Increasing evidence implicates lymphocytes in pulmonary arterial hypertension (PAH) pathogenesis. Rats deficient in T-lymphocytes show increased propensity to develop PAH but when injected with endothelial progenitor cells are protected from PAH (a mechanism dependent on natural killer (NK) cells). A decreased quantity of circulating cytotoxic CD8+ T-lymphocytes and NK cells are now reported in PAH patients; however, the effect of lymphocyte depletion on disease outcome is unknown. METHODS: This prospective study analysed the lymphocyte profile and plasma brain natriuretic peptide (BNP) levels of patients with idiopathic PAH (IPAH), connective tissue disease-associated PAH (CTD-APAH) and matched healthy controls. Lymphocyte surface markers studied include: CD4+ (helper T-cell marker), CD8+ (cytotoxic T-cell marker), CD56/CD16 (NK cell marker) and CD19+ (mature B-cell marker). Lymphocyte deficiencies and plasma BNP levels were then correlated with clinical outcome. RESULTS: Fourteen patients with PAH (9 IPAH, 5CTD) were recruited. Three patients were deceased at 1-year follow-up; all had elevated CD4 : CD8 ratios and deficiencies of NK cells and cytotoxic CD8+ T-lymphocytes at recruitment. Patients with normal lymphocyte profiles at recruitment were all alive a year later, and none were on the active transplant list. As univariate markers, cytotoxic CD8+ T-cell and NK cell counts were linked to short-term survival. CONCLUSIONS: Deficiencies in NK cells and cytotoxic CD8+ T-cells may be associated with an increased risk of death in PAH patients. Further research is required in larger numbers of patients and to elucidate the mechanism of these findings.
Assuntos
Linfócitos T CD8-Positivos/patologia , Hipertensão Pulmonar/mortalidade , Hipertensão Pulmonar/patologia , Células Matadoras Naturais/patologia , Adulto , Idoso , Biomarcadores/sangue , Estudos de Casos e Controles , Contagem de Células , Hipertensão Pulmonar Primária Familiar , Feminino , Humanos , Hipertensão Pulmonar/sangue , Masculino , Pessoa de Meia-Idade , Peptídeo Natriurético Encefálico/sangue , Prognóstico , Estudos Prospectivos , Taxa de SobrevidaRESUMO
BACKGROUND: Discrepancies have been reported between what is being researched, and what patients/families deem important to be investigated. Our aim was to understand research priorities for those who live with cancer in Aotearoa/New Zealand, with emphasis on Maori. METHODS: Adult outpatients with cancer and their whanau/family completed a survey (demographics, selecting keywords, free-text comments) at Christchurch hospital. Quantitative and qualitative data were evaluated using standard statistical and thematic analyses, respectively. RESULTS: We recruited 205 participants, including both turoro/patients (n = 129) and their whanau/family/carer (n = 76). Partnership with Maori health workers enabled greater recruitment of Maori participants (19%), compared to the proportion of Maori in Canterbury (9%). Cancer research was seen as a priority by 96% of participants. Priorities were similar between Maori and non-Maori participants, with the keywords 'Cancer screening', 'Quality of Life' and 'Development of new drugs' chosen most often. Free-text analysis identified three themes; 'Genetics and Prevention', 'Early Detection and Treatment', and 'Service Delivery', with some differences by ethnicity. CONCLUSIONS: Cancer research is a high priority for those living with cancer. In addition, participants want researchers to listen to their immediate and practical needs. These findings may inform future cancer research in Aotearoa. MaORI TERMS AND TRANSLATION: Aotearoa (New Zealand) he aha o whakaaro (what are your thoughts) hui (gathering) mate pukupuku (cancer) mokopuna (descendent) Otautahi (Christchurch) rongoa (traditional healing) tane (male) te reo (Maori language) Te Whatu Ora (weaving of wellness, Health New Zealand) tikanga (methods, customary practices) turoro (patients) (alternative terms used: whanau affected by cancer or tangata whaiora (person seeking health)) wahine (female) Waitaha (Canterbury) whakapapa (genealogy) whanau ((extended) family, based on whakapapa, here also carer).
Assuntos
Neoplasias , Aranhas , Adulto , Animais , Humanos , Feminino , Masculino , Cuidadores , Nova Zelândia , Pesquisa , Pacientes Ambulatoriais , Pessoal de Saúde , Neoplasias/terapiaRESUMO
BACKGROUND: Enzyme prodrug gene therapy is designed as a targeted cancer treatment, destroying gene-modified and bystander cells via exogenous enzyme-generated cytotoxins. Targeting of tumour blood vessels using gene therapy is attractive, although optimal enzyme prodrug combinations have yet to be identified. METHODS: Seven enzyme prodrug combinations were ranked in two endothelial (HUVEC, HMEC-1) and one tumour cell line (T24) for their ability to reduce proliferation and viability. The ability to destroy bystander cells in two dimensions (2D) and three dimensions (3D), mode of cell kill, and the ability to disrupt vascular networks were measured. RESULTS: Endothelial cell proliferation (bromodeoxyuridine uptake) was reduced most effectively by Herpes simplex virus thymidine kinase (TK) with ganciclovir (GCV), followed by Escherichia coli nitroreductase NfsB (NTR) with CB1954; viability [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay] was reduced most efficiently by NTR/CB1954 followed by TK/GCV. Of the seven combinations, only NTR/CB1954 displayed measurable bystander effects in 2D monolayers, and none demonstrated bystander killing in coated spheroids, a 3D spatially distinct model with tissue-like cell density. NTR-expressing endothelial cells displayed increased apoptosis, necrosis and caspase-3 activity after CB1954 treatment. Despite good antiproliferative activity, TK/GCV was ineffective at disrupting vascular network-like structures of endothelial cells, whereas NTR/CB1954 was efficient. NTR/metronidazole and the vascular disrupting agent, combretastatin A-4 phosphate, were the only other effective agents. CONCLUSIONS: Collectively, these data demonstrate that cytotoxic rather than cytostatic activity is necessary for efficient vascular disruption in vitro, and bystander killing is not essential. We identify NTR/CB1954 and NTR/metronidazole as candidates for in vivo investigation of vascular-targeted gene therapy.
Assuntos
Vasos Sanguíneos/citologia , Vasos Sanguíneos/efeitos dos fármacos , Enzimas/farmacologia , Terapia Genética/métodos , Pró-Fármacos/farmacologia , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacos , Efeito Espectador/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Clonais , Terapia Combinada , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , TransfecçãoRESUMO
Monkeypox virus (MPXV) is an orthopoxvirus closely related to variola virus, the causative agent of smallpox. Human MPXV infection results in a disease that is similar to smallpox and can also be fatal. Two clades of MPXV have been identified, with viruses of the central African clade displaying more pathogenic properties than those within the west African clade. The monkeypox inhibitor of complement enzymes (MOPICE), which is not expressed by viruses of the west African clade, has been hypothesized to be a main virulence factor responsible for increased pathogenic properties of central African strains of MPXV. To gain a better understanding of the role of MOPICE during MPXV-mediated disease, we compared the host adaptive immune response and disease severity following intrabronchial infection with MPXV-Zaire (n = 4), or a recombinant MPXV-Zaire (n = 4) lacking expression of MOPICE in rhesus macaques (RM). Data presented here demonstrate that infection of RM with MPXV leads to significant viral replication in the peripheral blood and lungs and results in the induction of a robust and sustained adaptive immune response against the virus. More importantly, we show that the loss of MOPICE expression results in enhanced viral replication in vivo, as well as a dampened adaptive immune response against MPXV. Taken together, these findings suggest that MOPICE modulates the anti-MPXV immune response and that this protein is not the sole virulence factor of the central African clade of MPXV.