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The World Health Organization estimates that nearly 500 million malaria tests are performed annually. While microscopy and rapid diagnostic tests (RDTs) are the main diagnostic approaches, no single method is inexpensive, rapid, and highly accurate. Two recent studies from our group have demonstrated a prototype computer vision platform that meets those needs. Here we present the results from two clinical studies on the commercially available version of this technology, the Sight Diagnostics Parasight platform, which provides malaria diagnosis, species identification, and parasite quantification. We conducted a multisite trial in Chennai, India (Apollo Hospital [n = 205]), and Nairobi, Kenya (Aga Khan University Hospital [n = 263]), in which we compared the device to microscopy, RDTs, and PCR. For identification of malaria, the device performed similarly well in both contexts (sensitivity of 99% and specificity of 100% at the Indian site and sensitivity of 99.3% and specificity of 98.9% at the Kenyan site, compared to PCR). For species identification, the device correctly identified 100% of samples with Plasmodium vivax and 100% of samples with Plasmodium falciparum in India and 100% of samples with P. vivax and 96.1% of samples with P. falciparum in Kenya, compared to PCR. Lastly, comparisons of the device parasite counts with those of trained microscopists produced average Pearson correlation coefficients of 0.84 at the Indian site and 0.85 at the Kenyan site.
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Testes Diagnósticos de Rotina/métodos , Malária Falciparum/diagnóstico , Malária Vivax/diagnóstico , Plasmodium falciparum/isolamento & purificação , Plasmodium vivax/isolamento & purificação , Humanos , Índia , Quênia , Carga Parasitária/métodos , Plasmodium falciparum/classificação , Plasmodium vivax/classificação , Estudos Prospectivos , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Microscopy has long been considered to be the gold standard for diagnosis of malaria despite the introduction of newer assays. However, it has many challenges like requirement of trained microscopists and logistic issues. A vision based device that can diagnose malaria, provide speciation and estimate parasitaemia was evaluated. METHODS: The device was evaluated using samples from 431 consented patients, 361 of which were initially screened by RDT and microscopy and later analysed by PCR. It was a prospective, non-randomized, blinded trial. Quantification of parasitaemia was performed by two experienced technicians. Samples were subjected to diagnosis by Sight Dx digital imaging scanning. RESULTS: The sensitivity and specificity of the SightDx P1 device for analysed samples were found to be 97.05 and 96.33%, respectively, when compared to PCR. When compared to microscopy, sensitivity and specificity were found to be 94.4 and 95.6%, respectively. The device was able to speciate 73.3% of the PCR Plasmodium falciparum positive samples and 91.4% of PCR Plasmodium vivax positive samples. CONCLUSION: The ability of the device to detect parasitaemia as compared with microscopy, was within 50% in 71.3% of cases and demonstrated a correlation coefficient of 0.89.
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Testes Diagnósticos de Rotina/métodos , Processamento de Imagem Assistida por Computador/métodos , Malária Falciparum/diagnóstico , Malária Vivax/diagnóstico , Imagem Óptica/métodos , Parasitemia/diagnóstico , Custos e Análise de Custo , Testes Diagnósticos de Rotina/economia , Testes Diagnósticos de Rotina/instrumentação , Humanos , Processamento de Imagem Assistida por Computador/economia , Processamento de Imagem Assistida por Computador/instrumentação , Imagem Óptica/economia , Imagem Óptica/instrumentação , Estudos Prospectivos , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Accurate detection of graft-versus-host disease (GVHD) is a major challenge in the management of patients undergoing hematopoietic stem cell transplantation (HCT). Here, we demonstrated the use of circulating cell-free DNA (cfDNA) for detection of tissue turnover and chronic GVHD (cGVHD) in specific organs. METHODS: We established a cocktail of tissue-specific DNA methylation markers and used it to determine the concentration of cfDNA molecules derived from the liver, skin, lungs, colon, and specific immune cells in 101 patients undergoing HCT. RESULTS: Patients with active cGVHD showed elevated concentrations of cfDNA, as well as tissue-specific methylation markers that agreed with clinical scores. Strikingly, transplanted patients with no clinical symptoms had abnormally high levels of tissue-specific markers, suggesting hidden tissue turnover even in the absence of evident clinical pathology. An integrative model taking into account total cfDNA concentration, monocyte/macrophage cfDNA levels and alanine transaminase was able to correctly identify GVHD with a specificity of 86% and precision of 89% (AUC of 0.8). CONCLUSION: cfDNA markers can be used for the detection of cGVHD, opening a window into underlying tissue dynamics in patients that receive allogeneic stem cell transplants. FUNDING: This work was supported by grants from the Ernest and Bonnie Beutler Research Program of Excellence in Genomic Medicine, The Israel Science Foundation, the Waldholtz/Pakula family, the Robert M. and Marilyn Sternberg Family Charitable Foundation and the Helmsley Charitable Trust (to YD).
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Síndrome de Bronquiolite Obliterante , Ácidos Nucleicos Livres , Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Humanos , Metilação de DNA , Ácidos Nucleicos Livres/genética , Doença Enxerto-Hospedeiro/diagnóstico , Biomarcadores , Marcadores Genéticos , Doença CrônicaRESUMO
Pancreatic beta cell proliferation has emerged as the principal mechanism for homeostatic maintenance of beta cell mass during adult life. This underscores the importance of understanding the mechanisms of beta cell replication and suggests novel approaches for regenerative therapy to treat diabetes. Here we use an in vivo pulse-chase labeling assay to investigate the replication dynamics of adult mouse beta cells. We find that replicated beta cells are able to re-enter the cell division cycle shortly after mitosis and regain their normal proliferative potential after a short quiescence period of several days. This quiescence period is lengthened with advanced age, but shortened during injury-driven beta cell regeneration and following treatment with a pharmacological activator of glucokinase, providing strong evidence that metabolic demand is a key determinant of cell cycle re-entry. Lastly, we show that cyclin D2, a crucial factor in beta cell replication, is downregulated during cell division, and is slowly upregulated post-mitosis by a glucose-sensitive mechanism. These results demonstrate that beta cells quickly regain their capacity to re-enter the cell cycle post-mitosis and implicate glucose control of cyclin D2 expression in the regulation of this process.
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Senescência Celular , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Envelhecimento , Animais , Ciclo Celular , Proliferação de Células , Ciclina D2/metabolismo , Células Secretoras de Insulina/citologia , CamundongosRESUMO
Introduction: Ex vivo organ cultures (EVOC) were recently optimized to sustain cancer tissue for 5 days with its complete microenvironment. We examined the ability of an EVOC platform to predict patient response to cancer therapy. Methods: A multicenter, prospective, single-arm observational trial. Samples were obtained from patients with newly diagnosed bladder cancer who underwent transurethral resection of bladder tumor and from core needle biopsies of patients with metastatic cancer. The tumors were cut into 250 µM slices and cultured within 24 h, then incubated for 96 h with vehicle or intended to treat drug. The cultures were then fixed and stained to analyze their morphology and cell viability. Each EVOC was given a score based on cell viability, level of damage, and Ki67 proliferation, and the scores were correlated with the patients' clinical response assessed by pathology or Response Evaluation Criteria in Solid Tumors (RECIST). Results: The cancer tissue and microenvironment, including endothelial and immune cells, were preserved at high viability with continued cell division for 5 days, demonstrating active cell signaling dynamics. A total of 34 cancer samples were tested by the platform and were correlated with clinical results. A higher EVOC score was correlated with better clinical response. The EVOC system showed a predictive specificity of 77.7% (7/9, 95% CI 0.4-0.97) and a sensitivity of 96% (24/25, 95% CI 0.80-0.99). Conclusion: EVOC cultured for 5 days showed high sensitivity and specificity for predicting clinical response to therapy among patients with muscle-invasive bladder cancer and other solid tumors.
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Germline BRCA-associated pancreatic ductal adenocarcinoma (glBRCA PDAC) tumors are susceptible to platinum and PARP inhibition. The clinical outcomes of 125 patients with glBRCA PDAC were stratified based on the spectrum of response to platinum/PARP inhibition: (i) refractory [overall survival (OS) <6 months], (ii) durable response followed by acquired resistance (OS <36 months), and (iii) long-term responders (OS >36 months). Patient-derived xenografts (PDX) were generated from 25 patients with glBRCA PDAC at different clinical time points. Response to platinum/PARP inhibition in vivo and ex vivo culture (EVOC) correlated with clinical response. We deciphered the mechanisms of resistance in glBRCA PDAC and identified homologous recombination (HR) proficiency and secondary mutations restoring partial functionality as the most dominant resistant mechanism. Yet, a subset of HR-deficient (HRD) patients demonstrated clinical resistance. Their tumors displayed basal-like molecular subtype and were more aneuploid. Tumor mutational burden was high in HRD PDAC and significantly higher in tumors with secondary mutations. Anti-PD-1 attenuated tumor growth in a novel humanized glBRCA PDAC PDX model. This work demonstrates the utility of preclinical models, including EVOC, to predict the response of glBRCA PDAC to treatment, which has the potential to inform time-sensitive medical decisions. SIGNIFICANCE: glBRCA PDAC has a favorable response to platinum/PARP inhibition. However, most patients develop resistance. Additional treatment options for this unique subpopulation are needed. We generated model systems in PDXs and an ex vivo system (EVOC) that faithfully recapitulate these specific clinical scenarios as a platform to investigate the mechanisms of resistance for further drug development. This article is highlighted in the In This Issue feature, p. 1749.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Platina/farmacologia , Platina/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Mutação , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Neoplasias PancreáticasRESUMO
Neutrophils play critical roles in a broad spectrum of clinical conditions. Accordingly, manipulation of neutrophil function may provide a powerful immunotherapeutic approach. However, due to neutrophils characteristic short half-life and their large population number, this possibility was considered impractical. Here we describe the identification of peptides which specifically bind either murine or human neutrophils. Although the murine and human neutrophil-specific peptides are not cross-reactive, we identified CD177 as the neutrophil-expressed binding partner in both species. Decorating nanoparticles with a neutrophil-specific peptide confers neutrophil specificity and these neutrophil-specific nanoparticles accumulate in sites of inflammation. Significantly, we demonstrate that encapsulating neutrophil modifying small molecules within these nanoparticles yields specific modulation of neutrophil function (ROS production, degranulation, polarization), intracellular signaling and longevity both in vitro and in vivo. Collectively, our findings demonstrate that neutrophil specific targeting may serve as a novel mode of immunotherapy in disease.
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Nanopartículas , Neutrófilos , Camundongos , Humanos , Animais , Neutrófilos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Inflamação/metabolismoRESUMO
Lung and bladder cancers are mostly incurable because of the early development of drug resistance and metastatic dissemination. Hence, improved therapies that tackle these two processes are urgently needed to improve clinical outcome. We have identified RSK4 as a promoter of drug resistance and metastasis in lung and bladder cancer cells. Silencing this kinase, through either RNA interference or CRISPR, sensitized tumor cells to chemotherapy and hindered metastasis in vitro and in vivo in a tail vein injection model. Drug screening revealed several floxacin antibiotics as potent RSK4 activation inhibitors, and trovafloxacin reproduced all effects of RSK4 silencing in vitro and in/ex vivo using lung cancer xenograft and genetically engineered mouse models and bladder tumor explants. Through x-ray structure determination and Markov transient and Deuterium exchange analyses, we identified the allosteric binding site and revealed how this compound blocks RSK4 kinase activation through binding to an allosteric site and mimicking a kinase autoinhibitory mechanism involving the RSK4's hydrophobic motif. Last, we show that patients undergoing chemotherapy and adhering to prophylactic levofloxacin in the large placebo-controlled randomized phase 3 SIGNIFICANT trial had significantly increased (P = 0.048) long-term overall survival times. Hence, we suggest that RSK4 inhibition may represent an effective therapeutic strategy for treating lung and bladder cancer.
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Neoplasias Pulmonares , Neoplasias da Bexiga Urinária , Animais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Humanos , Pulmão/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Camundongos , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/genéticaRESUMO
Accurate malaria diagnosis is critical to prevent malaria fatalities, curb overuse of antimalarial drugs, and promote appropriate management of other causes of fever. While several diagnostic tests exist, the need for a rapid and highly accurate malaria assay remains. Microscopy and rapid diagnostic tests are the main diagnostic modalities available, yet they can demonstrate poor performance and accuracy. Automated microscopy platforms have the potential to significantly improve and standardize malaria diagnosis. Based on image recognition and machine learning algorithms, these systems maintain the benefits of light microscopy and provide improvements such as quicker scanning time, greater scanning area, and increased consistency brought by automation. While these applications have been in development for over a decade, recently several commercial platforms have emerged. In this review, we discuss the most advanced computer vision malaria diagnostic technologies and investigate several of their features which are central to field use. Additionally, we discuss the technological and policy barriers to implementing these technologies in low-resource settings world-wide.
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Both type 1 and type 2 diabetes patients would greatly benefit from transplantation of insulin-producing pancreatic beta cells; however, a severe shortage of transplantable beta cells is a major current limitation in the use of such therapy. Understanding the mechanisms by which beta cells are naturally formed is therefore a central challenge for modern pancreas biology, in the hope that insights will be applicable for regenerative cell therapy strategies for diabetes. In particular, the cellular origins of pancreatic beta cells pose an important problem, with significant basic and therapeutic implications. This chapter discusses the current controversy regarding the identity of the cells that give rise to new beta cells in the adult mammal. Whereas numerous models suggest that beta cells can originate from adult stem cells, proposed to reside in the pancreas or in other locations, more recent work indicates that the major source for new beta cells during adult life is the proliferation of preexisting, differentiated beta cells. We present these different views, with emphasis on the methodologies employed. In particular, we focus on genetic lineage tracing using the Cre-lox system in transgenic mice, a technique considered the "gold standard" for addressing in vivo problems of cellular origins.
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Células-Tronco Adultas , Células Secretoras de Insulina , Pâncreas/citologia , Animais , Técnicas de Cultura de Células/métodos , HumanosRESUMO
Activity-based probes are small molecules that can be used to monitor enzyme activity by covalently binding to specific residues in the active site. In this issue of Chemistry & Biology, Lu and colleagues developed a specific fluorescent activity-based probe that targets the papain-like cysteine bacterial type III effector protease AvrPphB and used it to demonstrate the regulation of the protease secretion and pathogenesis.
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The frequency of pancreatic ß-cell replication declines dramatically with age, potentially contributing to the increased risk of type 2 diabetes in old age. Previous studies have shown the involvement of cell-autonomous factors in this phenomenon, particularly the decline of polycomb genes and accumulation of p16/INK4A. Here, we demonstrate that a systemic factor found in the circulation of young mice is able to increase the proliferation rate of old pancreatic ß-cells. Old mice parabiosed to young mice have increased ß-cell replication compared with unjoined old mice or old mice parabiosed to old mice. In addition, we demonstrate that old ß-cells transplanted into young recipients have increased replication rate compared with cells transplanted into old recipients; conversely, young ß-cells transplanted into old mice decrease their replication rate compared with young cells transplanted into young recipients. The expression of p16/INK4A mRNA did not change in heterochronic parabiosis, suggesting the involvement of other pathways. We conclude that systemic factors contribute to the replicative decline of old pancreatic ß-cells.
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Proliferação de Células , Células Secretoras de Insulina/fisiologia , Fatores Etários , Envelhecimento/fisiologia , Animais , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Células Secretoras de Insulina/citologia , Camundongos , Parabiose , Transdução de Sinais/fisiologiaRESUMO
Understanding the molecular triggers of pancreatic ß-cell proliferation may facilitate the development of regenerative therapies for diabetes. Genetic studies have demonstrated an important role for cyclin D2 in ß-cell proliferation and mass homeostasis, but its specific function in ß-cell division and mechanism of regulation remain unclear. Here, we report that cyclin D2 is present at high levels in the nucleus of quiescent ß-cells in vivo. The major regulator of cyclin D2 expression is glucose, acting via glycolysis and calcium channels in the ß-cell to control cyclin D2 mRNA levels. Furthermore, cyclin D2 mRNA is down-regulated during S-G(2)-M phases of each ß-cell division, via a mechanism that is also affected by glucose metabolism. Thus, glucose metabolism maintains high levels of nuclear cyclin D2 in quiescent ß-cells and modulates the down-regulation of cyclin D2 in replicating ß-cells. These data challenge the standard model for regulation of cyclin D2 during the cell division cycle and suggest cyclin D2 as a molecular link between glucose levels and ß-cell replication.