RESUMEN
Multiplex Immunochemistry/Immunofluorescence (mIHC/IF) aims to visualise multiple biomarkers in a single tissue section and is especially powerful when used on slide scanners coupled with digital analysis tools. mIHC/IF is commonly employed in immuno-oncology to characterise features of the tumour microenvironment (TME) and correlate them with clinical parameters to guide prognostication and therapy. However, mIHC/IF can be applied to a wide range of organisms in any physiological or disease context. Recent innovation has extended the number of markers that can be detected using slide scanners well beyond the 3-4 markers typically reported in traditional fluorescence microscopy. However, these methods often require sequential antibody staining and stripping, and are not compatible with frozen tissue sections. Using fluorophore-conjugated antibodies, we have established a simple mIHC/IF imaging workflow that enables simultaneous staining and detection of seven markers in a single section of frozen tissue. Coupled with automated whole slide imaging and digital quantification, our data efficiently revealed the tumour-immune complexity in metastatic melanoma. Computational image analysis quantified the immune and stromal cell populations present in the TME as well as their spatial interactions. This imaging workflow can also be performed with an indirect labelling panel consisting of primary and secondary antibodies. Our new methods, combined with digital quantification, will provide a valuable tool for high-quality mIHC/IF assays in immuno-oncology research and other translational studies, especially in circumstances where frozen sections are required for detection of particular markers, or for applications where frozen sections may be preferred, such as spatial transcriptomics.
Asunto(s)
Secciones por Congelación , Melanoma , Humanos , Inmunoquímica , Color , Biomarcadores de Tumor/análisis , Técnica del Anticuerpo Fluorescente , Anticuerpos , Microambiente TumoralRESUMEN
The current standard of care for hepatitis C virus (HCV) infection, pegylated alpha interferon in combination with ribavirin, has a limited response rate and adverse side effects. Drugs targeting viral proteins are in clinical development, but they suffer from the development of high viral resistance. The inhibition of cellular proteins that are essential for viral amplification is thought to have a higher barrier to the emergence of resistance. Three cyclophilin inhibitors, the cyclosporine analogs DEBIO-025, SCY635, and NIM811, have shown promising results for the treatment of HCV infection in early clinical trials. In this study, we investigated the frequency and mechanism of resistance to cyclosporine (CsA), NIM811, and a structurally unrelated cyclophilin inhibitor, SFA-1, in replicon-containing Huh7 cells. Cross-resistance between all clones was observed. NIM811-resistant clones were selected only after obtaining initial resistance to either CsA or SFA-1. The time required to select resistance against cyclophilin inhibitors was significantly longer than that required for resistance selection against viral protein inhibitors, and the achievable resistance level was substantially lower. Resistance to cyclophilin inhibitors was mediated by amino acid substitutions in NS3, NS5A, and NS5B, with NS5A mutations conferring the majority of resistance. Mutation D320E in NS5A mediated most of the resistance conferred by NS5A. Taken together, the results indicate that there is a very low frequency and level of resistance to cyclophilin-binding drugs mediated by amino acid substitutions in three viral proteins. The interaction of cyclophilin with NS5A seems to be the most critical, since the NS5A mutations have the largest impact on resistance.
Asunto(s)
Ciclofilinas/antagonistas & inhibidores , Ciclosporina/farmacología , Farmacorresistencia Viral/fisiología , Hepacivirus/genética , Replicón/genética , Antivirales/farmacología , Línea Celular , Ciclosporinas/farmacología , Inhibidores Enzimáticos/farmacología , Hepacivirus/crecimiento & desarrollo , Hepacivirus/metabolismo , Humanos , Lactonas/farmacología , Mutagénesis Sitio-Dirigida , ARN Viral/metabolismo , Compuestos de Espiro/farmacología , Transfección , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
Host factor pathways are known to be essential for hepatitis C virus (HCV) infection and replication in human liver cells. To search for novel host factor proteins required for HCV replication, we screened a subgenomic genotype 1b replicon cell line (Luc-1b) with a kinome and druggable collection of 20,779 siRNAs. We identified and validated several enzymes required for HCV replication, including class III phosphatidylinositol 4-kinases (PI4KA and PI4KB), carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), and mevalonate (diphospho) decarboxylase. Knockdown of PI4KA could inhibit the replication and/or HCV RNA levels of the two subgenomic genotype 1b clones (SG-1b and Luc-1b), two subgenomic genotype 1a clones (SG-1a and Luc-1a), JFH-1 genotype 2a infectious virus (JFH1-2a), and the genomic genotype 1a (FL-1a) replicon. In contrast, PI4KB knockdown inhibited replication and/or HCV RNA levels of Luc-1b, SG-1b, and Luc-1a replicons. The small molecule inhibitor, PIK93, was found to block subgenomic genotype 1b (Luc-1b), subgenomic genotype 1a (Luc-1a), and genomic genotype 2a (JFH1-2a) infectious virus replication in the nanomolar range. PIK93 was characterized by using quantitative chemical proteomics and in vitro biochemical assays to demonstrate PIK93 is a bone fide PI4KA and PI4KB inhibitor. Our data demonstrate that genetic or pharmacological modulation of PI4KA and PI4KB inhibits multiple genotypes of HCV and represents a novel druggable class of therapeutic targets for HCV infection.
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1-Fosfatidilinositol 4-Quinasa/metabolismo , Hepacivirus/genética , Hepacivirus/metabolismo , Hígado/virología , Replicación Viral , 1-Fosfatidilinositol 4-Quinasa/química , Antivirales/farmacología , Unión Competitiva , Línea Celular , Silenciador del Gen , Genotipo , Humanos , Concentración 50 Inhibidora , Espectrometría de Masas/métodos , Proteómica/métodos , ARN Interferente Pequeño/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tiazoles/farmacologíaRESUMEN
Metastasis of human tumors to lymph nodes (LN) is a universally negative prognostic factor. LN stromal cells (SC) play a crucial role in enabling T-cell responses, and because tumor metastases modulate their structure and function, this interaction may suppress immune responses to tumor antigens. The SC subpopulations that respond to infiltration of malignant cells into human LNs have not been defined. Here, we identify distinctive subpopulations of CD90+ SCs present in melanoma-infiltrated LNs and compare them with their counterparts in normal LNs. The first population (CD90+ podoplanin+ CD105+ CD146+ CD271+ VCAM-1+ ICAM-1+ α-SMA+) corresponds to fibroblastic reticular cells that express various T-cell modulating cytokines, chemokines, and adhesion molecules. The second (CD90+ CD34+ CD105+ CD271+) represents a novel population of CD34+ SCs embedded in collagenous structures, such as the capsule and trabeculae, that predominantly produce extracellular matrix. We also demonstrated that these two SC subpopulations are distinct from two subsets of human LN pericytes, CD90+ CD146+ CD36+ NG2- pericytes in the walls of high endothelial venules and other small vessels, and CD90+ CD146+ NG2+ CD36- pericytes in the walls of larger vessels. Distinguishing between these CD90+ SC subpopulations in human LNs allows for further study of their respective impact on T-cell responses to tumor antigens and clinical outcomes.
Asunto(s)
Biomarcadores de Tumor/inmunología , Ganglios Linfáticos/inmunología , Melanoma/inmunología , Pericitos/inmunología , Células del Estroma/inmunología , Biomarcadores de Tumor/metabolismo , Diferenciación Celular/inmunología , Humanos , Inmunofenotipificación/métodos , Ganglios Linfáticos/patología , Melanoma/clasificación , Melanoma/patología , Metástasis de la Neoplasia , Pericitos/patología , Células del Estroma/patología , Escape del TumorRESUMEN
Chronic hepatitis C virus (HCV) infection remains a major global health burden while current interferon-based therapy is suboptimal. Efforts to develop more effective antiviral agents mainly focus on two viral targets: NS3-4A protease and NS5B polymerase. However, resistant mutants against these viral specific inhibitors emerge quickly both in vitro and in patients, particularly in the case of monotherapy. An alternative and complementary strategy is to target host factors such as cyclophilins that are also essential for viral replication. Future HCV therapies will most likely be combinations of multiple drugs of different mechanisms to maximize antiviral activity and to suppress the emergence of resistance. Here, the effects of combining a host cyclophilin inhibitor NIM811 with other viral specific inhibitors were investigated in vitro using HCV replicon. All of the combinations led to more pronounced antiviral effects than any single agent, with no significant increase of cytotoxicity. Moreover, the combination of NIM811 with a nucleoside (NM107) or a non-nucleoside (thiophene-2-carboxylic acid) polymerase inhibitor was synergistic, while the combination with a protease inhibitor (BILN2061) was additive. Resistant clones were selected in vitro with these inhibitors. Interestingly, it was much more difficult to develop resistance against NIM811 than viral specific inhibitors. No cross-resistance was observed among these inhibitors. Most notably, NIM811 was highly effective in blocking the emergence of resistance when used in combination with viral protease or polymerase inhibitors. Taken together, these results illustrate the significant advantages of combining inhibitors targeting both viral and host factors as key components of future HCV therapies.
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Antivirales/farmacología , Ciclosporina/farmacología , Farmacorresistencia Viral/efectos de los fármacos , Hepacivirus/efectos de los fármacos , Inhibidores de Proteasas/farmacología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Antivirales/química , Línea Celular , Ciclofilinas/antagonistas & inhibidores , Ciclosporina/química , Sinergismo Farmacológico , Quimioterapia Combinada , Hepacivirus/enzimología , Hepacivirus/fisiología , Humanos , Inhibidores de Proteasas/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Replicación Viral/efectos de los fármacosRESUMEN
INTRODUCTION For general practitioners, practice nurses and community pharmacists in New Zealand, a core duty is to educate patients about sun protection. We aimed to evaluate compliance of locally available sunscreens with regional clinical practice guidelines and sunscreen labelling standards, to assist clinicians in advising consumers on sunscreen selection. METHODS We audited all sunscreens available at two Auckland stores for three New Zealand sunscreen retailers. We then assessed compliance with accepted regional clinical practice guidelines for sun protection from the New Zealand Guidelines Group. We further assessed compliance with regional Australia/New Zealand consumer standards for sunscreen labelling. RESULTS All sunscreens satisfied clinical guidelines for broad-spectrum protection, and 99% of sunscreens met or exceeded clinical guidelines for minimal Sun Protection Factor. Compliance with regional standardized labelling guidelines is voluntary in New Zealand and 27% of audited sunscreens were not fully compliant with SPF labelling standards. DISCUSSION Sunscreens were generally compliant with clinical guidelines for minimal sun protection. However there was substantial noncompliance with regional recommendations for standardized sunscreen labelling. Primary health care clinicians should be aware that this labelling noncompliance may mislead patients into thinking some sunscreens offer more sun protection than they do. Mandatory compliance with the latest regional labelling standards would simplify sunscreen selection by New Zealand consumers. KEYWORDS Sunscreen; Sun Protection Factor; SPF; Skin Neoplasms; Melanoma; Skin Cancer Prevention.