Ilka Martins Rosa

Bio: Ilka Martins Rosa is an academic researcher from University of Aveiro. The author has contributed to research in topics: Medicine & Dementia. The author has an hindex of 5, co-authored 7 publications receiving 160 citations.

Exosome isolation from distinct biofluids using precipitation and column-based approaches.

Abstract: The potential of exosomes as biomarker resources for diagnostics, prognostics and even for therapeutics is an area of intense research. Despite the various approaches available, there is no consensus with respect to the best methodology for isolating exosomes and to provide substantial yields with reliable quality. Differential centrifugation is the most commonly used method but it is time-consuming and requires large sample volumes, thus alternative methods are urgently needed. In this study two precipitation-based methods and one column-based approach were compared for exosome isolation from distinct biofluids (serum, plasma and cerebrospinal fluid). Exosome characterization included morphological analyses, determination of particle concentration, stability and exosome preparations’ purity, using different complementary approaches such as Nanoparticle Tracking Analysis, Electrophoretic Light Scattering, Transmission Electron Microscopy, EXOCET colorimetric assay, protein quantification methods and western blotting. The three commercial kits tested successfully isolated exosomes from the biofluids under study, although ExoS showed the best performance in terms of exosome yield and purity. Data shows that methods other than differential centrifugation can be applied to quickly and efficiently isolate exosomes from reduced biofluid volumes. The possibility to use small volumes is fundamental in the context of translational and clinical research, thus the results here presented contribute significantly in this respect.

Potential of FTIR Spectroscopy Applied to Exosomes for Alzheimer's Disease Discrimination: A Pilot Study.

Abstract: Alzheimer's disease (AD) diagnosis is based on psychological and imaging tests but can also include monitoring cerebrospinal fluid (CSF) biomarkers. However, CSF based-neurochemical approaches are expensive and invasive, limiting their use to well-equipped settings. In contrast, blood-based biomarkers are minimally invasive, cost-effective, and a widely accessible alternative. Blood-derived exosomes have recently emerged as a reliable AD biomarker source, carrying disease-specific cargo. Fourier-transformed infrared (FTIR) spectroscopy meets the criteria for an ideal diagnostic methodology since it is rapid, easy to implement, and has high reproducibility. This metabolome-based technique is useful for diagnosing a broad range of diseases, although to our knowledge, no reports for FTIR spectroscopy applied to exosomes in AD exist. In this ground-breaking pilot study, FTIR spectra of serum and serum-derived exosomes from two independent cohorts were acquired and analyzed using multivariate analysis. The regional UA-cohort includes 9 individuals, clinically diagnosed with AD, mean age of 78.7 years old; and the UMG-cohort comprises 12 individuals, clinically diagnosed with AD (based on molecular and/or imaging data), mean age of 73.2 years old. Unsupervised principal component analysis of FTIR spectra of serum-derived exosomes revealed higher discriminatory value for AD cases when compared to serum as a whole. Consistently, the partial least-squares analysis revealed that serum-derived exosomes present higher correlations than serum. In addition, the second derivative peak area calculation also revealed significant differences among Controls and AD cases. The results obtained suggest that this methodology can discriminate cases from Controls and thus be potential useful to assist in AD clinical diagnosis.

Screening Younger Individuals in a Primary Care Setting Flags Putative Dementia Cases and Correlates Gastrointestinal Diseases with Poor Cognitive Performance.

Abstract: Background/Aims: Diagnosing dementia is challenging in many primary care settings, given the limited human resources and the lack of current diagnostic tools. With this in mind, a primary care-based cohort was established in the Aveiro district of Portugal. Methods: A total of 568 participants were evaluated using cognitive tests and APOE genotyping. Results: The findings revealed a dementia prevalence of 12%. A strong correlation between increasing Clinical Dementia Rating (CDR) scores and education was clearly evident. Other highly relevant risk factors were activities of daily living (ADL), instrumental ADL, aging, depression, gender, the APOE e4 allele, and comorbidities (depression as well as gastrointestinal, osteoarticular, and neurodegenerative diseases). A hitherto unreported, significant correlation between gastrointestinal disease and high CDR score was clearly observable. Conclusions: This study shows the merit of carrying out a dementia screening on younger subjects. Significantly, 71 subjects in the age group of 50-65 years were flagged for follow-up studies; furthermore, these cases with a potentially early onset of dementia were identified in a primary care setting.

Exosomal Aβ-Binding Proteins Identified by "In Silico" Analysis Represent Putative Blood-Derived Biomarker Candidates for Alzheimer´s Disease.

Abstract: The potential of exosomes as biomarker resources for diagnostics and even for therapeutics has intensified research in the field, including in the context of Alzheimer´s disease (AD). The search for disease biomarkers in peripheral biofluids is advancing mainly due to the easy access it offers. In the study presented here, emphasis was given to the bioinformatic identification of putative exosomal candidates for AD. The exosomal proteomes of cerebrospinal fluid (CSF), serum and plasma, were obtained from three databases (ExoCarta, EVpedia and Vesiclepedia), and complemented with additional exosomal proteins already associated with AD but not found in the databases. The final biofluids’ proteomes were submitted to gene ontology (GO) enrichment analysis and the exosomal Aβ-binding proteins that can constitute putative candidates were identified. Among these candidates, gelsolin, a protein known to be involved in inhibiting Abeta fibril formation, was identified, and it was tested in human samples. The levels of this Aβ-binding protein, with anti-amyloidogenic properties, were assessed in serum-derived exosomes isolated from controls and individuals with dementia, including AD cases, and revealed altered expression patterns. Identification of potential peripheral biomarker candidates for AD may be useful, not only for early disease diagnosis but also in drug trials and to monitor disease progression, allowing for a timely therapeutic intervention, which will positively impact the patient’s quality of life.

Progress, opportunity, and perspective on exosome isolation - efforts for efficient exosome-based theranostics.

Abstract: Exosomes are small extracellular vesicles with diameters of 30-150 nm. In both physiological and pathological conditions, nearly all types of cells can release exosomes, which play important roles in cell communication and epigenetic regulation by transporting crucial protein and genetic materials such as miRNA, mRNA, and DNA. Consequently, exosome-based disease diagnosis and therapeutic methods have been intensively investigated. However, as in any natural science field, the in-depth investigation of exosomes relies heavily on technological advances. Historically, the two main technical hindrances that have restricted the basic and applied researches of exosomes include, first, how to simplify the extraction and improve the yield of exosomes and, second, how to effectively distinguish exosomes from other extracellular vesicles, especially functional microvesicles. Over the past few decades, although a standardized exosome isolation method has still not become available, a number of techniques have been established through exploration of the biochemical and physicochemical features of exosomes. In this work, by comprehensively analyzing the progresses in exosome separation strategies, we provide a panoramic view of current exosome isolation techniques, providing perspectives toward the development of novel approaches for high-efficient exosome isolation from various types of biological matrices. In addition, from the perspective of exosome-based diagnosis and therapeutics, we emphasize the issue of quantitative exosome and microvesicle separation.

Application of exosomes as liquid biopsy in clinical diagnosis.

Abstract: Liquid biopsy refers to the sampling and molecular analysis of the biofluids of circulating tumor cells, extracellular vesicles, nucleic acids, and so forth. Exosomes are small extracellular vesicles with sizes between 30–150 nm. They are secreted by multivesicular bodies through exocytosis in live cells and can participate in intercellular communication due to their contents, including nucleic acids, proteins, and lipids. Herein, we investigate publication frequencies on exosomes over the past 10 years, and review recent clinical studies on liquid biopsy of exosomes in the fields of oncology, pregnancy disorders, cardiovascular diseases, and organ transplantation. We also describe the advantages of exosomes as an effective liquid biopsy tool and the progression of exosome extraction methods. Finally, we depict the commercial development of exosome research and discuss the future role of exosomes in liquid biopsy.

Quality and efficiency assessment of six extracellular vesicle isolation methods by nano-flow cytometry

Abstract: Extracellular vesicles (EVs) have sparked tremendous interest owing to their prominent potential in diagnostics and therapeutics. Isolation of EVs from complex biological fluids with high purity is...

Exosome isolation from distinct biofluids using precipitation and column-based approaches.

Abstract: The potential of exosomes as biomarker resources for diagnostics, prognostics and even for therapeutics is an area of intense research. Despite the various approaches available, there is no consensus with respect to the best methodology for isolating exosomes and to provide substantial yields with reliable quality. Differential centrifugation is the most commonly used method but it is time-consuming and requires large sample volumes, thus alternative methods are urgently needed. In this study two precipitation-based methods and one column-based approach were compared for exosome isolation from distinct biofluids (serum, plasma and cerebrospinal fluid). Exosome characterization included morphological analyses, determination of particle concentration, stability and exosome preparations’ purity, using different complementary approaches such as Nanoparticle Tracking Analysis, Electrophoretic Light Scattering, Transmission Electron Microscopy, EXOCET colorimetric assay, protein quantification methods and western blotting. The three commercial kits tested successfully isolated exosomes from the biofluids under study, although ExoS showed the best performance in terms of exosome yield and purity. Data shows that methods other than differential centrifugation can be applied to quickly and efficiently isolate exosomes from reduced biofluid volumes. The possibility to use small volumes is fundamental in the context of translational and clinical research, thus the results here presented contribute significantly in this respect.