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1.
Front Bioeng Biotechnol ; 12: 1379597, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38737536

RESUMO

Introduction: Engineered 3D models employing human induced pluripotent stem cell (hiPSC) derivatives have the potential to recapitulate the cell diversity and structure found in the human central nervous system (CNS). Therefore, these complex cellular systems offer promising human models to address the safety and potency of advanced therapy medicinal products (ATMPs), such as gene therapies. Specifically, recombinant adeno-associated viruses (rAAVs) are currently considered highly attractive for CNS gene therapy due to their broad tropism, low toxicity, and moderate immunogenicity. To accelerate the clinical translation of rAAVs, in-depth preclinical evaluation of efficacy and safety in a human setting is primordial. The integration of hiPSC-derived CNS models in rAAV development will require, amongst other factors, robust, small-scale, high-throughput culture platforms that can feed the preclinical trials. Methods: Herein, we pioneer the miniaturization and parallelization of a 200 mL stirred-tank bioreactor-based 3D brain cell culture derived from hiPSCs. We demonstrate the applicability of the automated miniaturized Ambr® 15 Cell Culture system for the maintenance of hiPSC-derived neurospheroids (iNSpheroids), composed of neuronal and glial cells. Critical process parameters were optimized, namely, cell density and agitation mode. Results: Under optimized conditions, stable iNSpheroid cultures were attained in the microbioreactors for at least 15 days, with high cell viability and astrocytic and neuronal phenotype maintenance. This culture setup allowed the parallelization of different rAAVs, in different multiplicity of infections (MOIs), to address rAAV-host interactions at a preclinical scale. The iNSpheroids were exposed to rAAV2- and rAAV9-eGFP in the microbioreactors. Transgene expression was detected 14 days post-transduction, revealing different astrocyte/neuron tropism of the two serotypes. Discussion: We advocate that the iNSpheroid cultures in miniaturized bioreactors are reliable and reproducible screening tools for addressing rAAV transduction and tropism, compatible with preclinical demands.

2.
J Biotechnol ; 384: 1-11, 2024 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-38340900

RESUMO

Host cell proteins (HCPs) are process-related impurities expressed by the host cells during biotherapeutics' manufacturing, such as monoclonal antibodies (mAbs). Some challenging HCPs evade clearance during the downstream processing and can be co-purified with the molecule of interest, which may impact product stability, efficacy, and safety. Therefore, HCP content is a critical quality attribute to monitor and quantify across the bioprocess. Here we explored a mass spectrometry (MS)-based proteomics tool, the sequential window acquisition of all theoretical fragment-ion spectra (SWATH) strategy, as an orthogonal method to traditional ELISA. The SWATH workflow was applied for high-throughput individual HCP identification and quantification, supporting characterization of a mAb purification platform. The design space of HCP clearance of two polishing resins was evaluated through a design of experiment study. Absolute quantification of high-risk HCPs was achieved (reaching 1.8 and 4.2 ppm limits of quantification, for HCP A and B respectively) using HCP-specific synthetic heavy labeled peptide calibration curves. Profiling of other HCPs was also possible using an average calibration curve (using labeled peptides from different HCPs). The SWATH approach is a powerful tool for HCP assessment during bioprocess development enabling simultaneous monitoring and quantification of different individual HCPs and improving process understanding of their clearance.


Assuntos
Anticorpos Monoclonais , Peptídeos , Cricetinae , Animais , Cricetulus , Anticorpos Monoclonais/química , Espectrometria de Massas/métodos , Ensaio de Imunoadsorção Enzimática , Células CHO
3.
Biomedicines ; 11(11)2023 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-38001891

RESUMO

Monoclonal antibody-based therapy has shown efficacy against cancer, autoimmune, infectious, and inflammatory diseases. Multispecific antibodies (MsAbs), including trispecifics (tsAbs), offer enhanced therapeutic potential by targeting different epitopes. However, when co-expressed from three or more different polypeptide chains, MsAb production can lead to incorrect chain assembly and co-production of mispaired species with impaired biological activity. Moreover, mispairing carries significant challenges for downstream purification, decreasing yields and increasing the cost of bioprocess development. In this study, quantitative transcriptomics and proteomics analyses were employed to investigate which signaling pathways correlated with low and high mispairing clone signatures. Gene and protein expression profiles of Chinese hamster ovary (CHO) clones producing an tsAb were analyzed in the exponential growth and stationary (tsAb production) phase of fed-batch culture. Functional analysis revealed activated endoplasmic reticulum stress in high mispairing clones in both culture phases, while low mispairing clones exhibited expression profiles indicative of activated protein translation, as well as higher endocytosis and target protein degradation, suggesting the clearance of unfolded proteins through ubiquitin-mediated mechanisms. In addition, through transcriptomic profiling, we identified a group of genes that have the potential to be used as a biomarker panel tool for identifying high mispairing levels in the early stages of bioprocess development.

4.
Biotechnol Bioeng ; 120(9): 2578-2587, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37027346

RESUMO

The majority of recombinant adeno-associated viruses (rAAV) approved for clinical use or in clinical trials areproduced by transient transfection using the HEK293 cell line. However, this platform has several manufacturing bottlenecks at commercial scales namely, low product quality (full to empty capsid ratio <20% in most rAAV serotypes), lower productivities obtained after scale-up and the high cost of raw materials, in particular of Good Manufacturing Practice grade plasmid DNA required for transfection. The HeLa-based stable cell line rAAV production system provides a robust and scalable alternative to transient transfection systems. Nevertheless, the time required to generate the producer cell lines combined with the complexity of rAAV production and purification processes still pose several barriers to the use of this platform as a suitable alternative to the HEK293 transient transfection. In this work we streamlined the cell line development and bioprocessing for the HeLaS3-based production of rAAV. By exploring this optimized approach, producer cell lines were generated in 3-4 months, and presented rAAV2 volumetric production (bulk) > 3 × 1011 vg/mL and full to empty capsids ratio (>70%) at 2 L bioreactor scale. Moreover, the established downstream process, based on ion exchange and affinity-based chromatography, efficiently eliminated process related impurities, including the Adenovirus 5 helper virus required for production with a log reduction value of 9. Overall, we developed a time-efficient and robust rAAV bioprocess using a stable producer cell line achieving purified rAAV2 yields > 1 × 1011 vg/mL. This optimized platform may address manufacturing challenges for rAAV based medicines.


Assuntos
Dependovirus , Vetores Genéticos , Humanos , Dependovirus/genética , Células HEK293 , Células HeLa , Transfecção
5.
Viruses ; 14(11)2022 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-36423148

RESUMO

Virus-based biopharmaceutical products are used in clinical applications such as vaccines, gene therapy, and immunotherapy. However, their manufacturing remains a challenge, hampered by the lack of appropriate analytical tools for purification monitoring or characterization of the final product. This paper describes the implementation of a highly sensitive method, capillary electrophoresis (CE)-sodium dodecyl sulfate (SDS) combined with a laser-induced fluorescence (LIF) detector to monitor the impact of various bioprocess steps on the quality of different viral vectors. The fluorescence labelling procedure uses the (3-(2-furoyl) quinoline-2-carboxaldehyde dye, and the CE-SDS LIF method enables the evaluation of in-process besides final product samples. This method outperforms other analytical methods, such as SDS-polyacrylamide gel electrophoresis with Sypro Ruby staining, in terms of sensitivity, resolution, and high-throughput capability. Notably, this CE-SDS LIF method was also successfully implemented to characterize enveloped viruses such as Maraba virus and lentivirus, whose development as biopharmaceuticals is now restricted by the lack of suitable analytical tools. This method was also qualified for quantification of rAAV2 according to the International Council for Harmonisation guidelines. Overall, our work shows that CE-SDS LIF is a precise and sensitive analytical platform for in-process sample analysis and quantification of different virus-based targets, with a great potential for application in biomanufacturing.


Assuntos
Eletroforese Capilar , Vírion , Eletroforese Capilar/métodos , Dodecilsulfato de Sódio , Eletroforese em Gel de Poliacrilamida
6.
Biomedicines ; 10(11)2022 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-36428511

RESUMO

Heart failure with preserved ejection fraction (HFpEF) is a highly prevalent but still poorly understood clinical entity. Its current pathophysiological understanding supports a critical role of comorbidities and their chronic effect on cardiac function and structure. Importantly, despite the replication of some HFpEF phenotypic features, to this day, experimental models have failed to bring new effective therapies to the clinical setting. Thus, the direct investigation of HFpEF human myocardial samples may unveil key, and possibly human-specific, pathophysiological mechanisms. This study employed quantitative proteomic analysis by advanced mass spectrometry (SWATH-MS) to investigate signaling pathways and pathophysiological mechanisms in HFpEF. Protein-expression profiles were analyzed in human left ventricular myocardial samples of HFpEF patients and compared with a mixed control group. Functional analysis revealed several proteins that correlate with HFpEF, including those associated with mitochondrial dysfunction, oxidative stress, and inflammation. Despite the known disease heterogeneity, proteomic profiles could indicate a reduced mitochondrial oxidative phosphorylation and fatty-acid oxidation capacity in HFpEF patients with diabetes. The proteomic characterization described in this work provides new insights. Furthermore, it fosters further questions related to HFpEF cellular pathophysiology, paving the way for additional studies focused on developing novel therapies and diagnosis strategies for HFpEF patients.

7.
Vaccines (Basel) ; 10(10)2022 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-36298449

RESUMO

Rotavirus A infection is a global leading cause of severe acute gastroenteritis associated with life-threatening diarrheal episodes in infants and young children. The disease burden is being reduced, namely due to a wider access to rotavirus vaccines. However, there is a demand to expand rotavirus vaccination programs, and to achieve this, it is critical to improve high-throughput in-process product quality control and vaccine manufacturing monitoring. Here, we present the development of an analytical method for the quantification of rotavirus particles contained in a licensed vaccine. The binding of rotavirus proteins to distinct glycoconjugate receptors and monoclonal antibodies was evaluated using biolayer interferometry analysis, applied on an Octet platform. The antibody strategy presented the best results with a linear response range within 2.5 × 107-1.0 × 108 particles·mL-1 and limits of detection and quantification of 2.5 × 106 and 7.5 × 106 particles·mL-1, respectively. Method suitability for the quantification of in-process samples was shown using samples from different manufacturing stages and their titers were comparable with the approved CCID(50) method. This cell-free method enables a fast and high-throughput analysis, compatible with time constraints during bioprocess development and it is suitable to be adapted to other viral particle-based drug products.

8.
Cancers (Basel) ; 14(18)2022 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-36139619

RESUMO

Predicting patient response to treatment and the onset of chemoresistance are still major challenges in oncology. Chemoresistance is deeply influenced by the complex cellular interactions occurring within the tumor microenvironment (TME), including metabolic crosstalk. We have previously shown that ex vivo tumor tissue cultures derived from ovarian carcinoma (OvC) resections retain the TME components for at least four weeks of culture and implemented assays for assessment of drug response. Here, we explored ex vivo patient-derived tumor tissue cultures to uncover metabolic signatures of chemosensitivity and/or resistance. Tissue cultures derived from nine OvC cases were challenged with carboplatin and paclitaxel, the standard-of-care chemotherapeutics, and the metabolic footprints were characterized by LC-MS. Partial least-squares discriminant analysis (PLS-DA) revealed metabolic signatures that discriminated high-responder from low-responder tissue cultures to ex vivo drug exposure. As a proof-of-concept, a set of potential metabolic biomarkers of drug response was identified based on the receiver operating characteristics (ROC) curve, comprising amino acids, fatty acids, pyrimidine, glutathione, and TCA cycle pathways. Overall, this work establishes an analytical and computational platform to explore metabolic features of the TME associated with response to treatment, which can leverage the discovery of biomarkers of drug response and resistance in OvC.

9.
Front Physiol ; 13: 926528, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35784878

RESUMO

Extracellular Vesicles (EV) play a critical role in the regulation of regenerative processes in wounded tissues by mediating cell-to-cell communication. Multiple RNA species have been identified in EV, although their function still lacks understanding. We previously characterized the miRNA content of EV secreted over hiPSC-cardiomyocyte differentiation and found a distinct miRNA expression in hiPSC-EV driving its in vitro bioactivity. In this work, we investigated the piRNA profiles of EV derived from key stages of the hiPSC-CM differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors (CPC-EV), immature (CMi-EV), and mature (CMm-EV) cardiomyocytes, demonstrating that EV-piRNA expression differs greatly from the miRNA profiles we previously identified. Only four piRNA were significantly deregulated in EV, one in hiPSC-EV, and three in CPC-EV, as determined by differential expression analysis on small RNA-seq data. Our results provide a valuable source of information for further studies aiming at defining the role of piRNA in the bioactivity and therapeutic potential of EV.

10.
Pharmaceutics ; 14(4)2022 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-35456687

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) homotrimeric spike (S) protein is responsible for mediating host cell entry by binding to the angiotensin-converting enzyme 2 (ACE2) receptor, thus being a key viral antigen to target in a coronavirus disease 19 (COVID-19) vaccine. Despite the availability of COVID-19 vaccines, low vaccine coverage as well as unvaccinated and immune compromised subjects are contributing to the emergence of SARS-CoV-2 variants of concern. Therefore, continued development of novel and/or updated vaccines is essential for protecting against such new variants. In this study, we developed a scalable bioprocess using the insect cells-baculovirus expression vector system (IC-BEVS) to produce high-quality S protein, stabilized in its pre-fusion conformation, for inclusion in a virosome-based COVID-19 vaccine candidate. By exploring different bioprocess engineering strategies (i.e., signal peptides, baculovirus transfer vectors, cell lines, infection strategies and formulation buffers), we were able to obtain ~4 mg/L of purified S protein, which, to the best of our knowledge, is the highest value achieved to date using insect cells. In addition, the insect cell-derived S protein exhibited glycan processing similar to mammalian cells and mid-term stability upon storage (up to 90 days at -80 and 4 °C or after 5 freeze-thaw cycles). Noteworthy, antigenicity of S protein, either as single antigen or displayed on the surface of virosomes, was confirmed by ELISA, with binding of ACE2 receptor, pan-SARS antibody CR3022 and neutralizing antibodies to the various epitope clusters on the S protein. Binding capacity was also maintained on virosomes-S stored at 4 °C for 1 month. This work demonstrates the potential of using IC-BEVS to produce the highly glycosylated and complex S protein, without compromising its integrity and antigenicity, to be included in a virosome-based COVID-19 vaccine candidate.

11.
NPJ Parkinsons Dis ; 8(1): 51, 2022 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-35468899

RESUMO

Alpha-synuclein (aSyn) is a central player in the pathogenesis of synucleinopathies due to its accumulation in typical protein aggregates in the brain. However, it is still unclear how it contributes to neurodegeneration. Type-2 diabetes mellitus is a risk factor for Parkinson's disease (PD). Interestingly, a common molecular alteration among these disorders is the age-associated increase in protein glycation. We hypothesized that glycation-induced neuronal dysfunction is a contributing factor in synucleinopathies. Here, we dissected the impact of methylglyoxal (MGO, a glycating agent) in mice overexpressing aSyn in the brain. We found that MGO-glycation potentiates motor, cognitive, olfactory, and colonic dysfunction in aSyn transgenic (Thy1-aSyn) mice that received a single dose of MGO via intracerebroventricular injection. aSyn accumulates in the midbrain, striatum, and prefrontal cortex, and protein glycation is increased in the cerebellum and midbrain. SWATH mass spectrometry analysis, used to quantify changes in the brain proteome, revealed that MGO mainly increase glutamatergic-associated proteins in the midbrain (NMDA, AMPA, glutaminase, VGLUT and EAAT1), but not in the prefrontal cortex, where it mainly affects the electron transport chain. The glycated proteins in the midbrain of MGO-injected Thy1-aSyn mice strongly correlate with PD and dopaminergic pathways. Overall, we demonstrated that MGO-induced glycation accelerates PD-like sensorimotor and cognitive alterations and suggest that the increase of glutamatergic signaling may underly these events. Our study sheds new light into the enhanced vulnerability of the midbrain in PD-related synaptic dysfunction and suggests that glycation suppressors and anti-glutamatergic drugs may hold promise as disease-modifying therapies for synucleinopathies.

12.
Adv Sci (Weinh) ; 9(15): e2104296, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35322574

RESUMO

Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications.


Assuntos
Vesículas Extracelulares , Células-Tronco Pluripotentes Induzidas , MicroRNAs , Vesículas Extracelulares/metabolismo , Humanos , MicroRNAs/genética , Miócitos Cardíacos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo
13.
Curr Opin Biotechnol ; 74: 271-277, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35007989

RESUMO

Recombinant adeno-associated (rAAV) vector-based gene therapy has been the focus of intense research driven by the safety profile and several recent clinical breakthroughs. As of April 2021, there are two rAAV-based gene therapies approved and more than two-hundred active clinical trials (approximately thirty in Phase III). However, the expected increase in demand for rAAV vectors still poses several challenges. Discussed herein are key aspects related to R&D needs and Chemistry, Manufacturing and Control (CMC) efforts required to attend this growing demand. Authors provide their perspective on strategic topics for rAAV-based therapies success: scalability and productivity; improved safety; increased process understanding combined with development of orthogonal bioanalytics that are able to identify, monitor and control Critical Quality Attributes (CQAs) during bioprocessing.


Assuntos
Dependovirus , Vetores Genéticos , Dependovirus/genética , Terapia Genética , Vetores Genéticos/genética
14.
PLoS One ; 17(1): e0262711, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35085302

RESUMO

Quality control of biopharmaceuticals such as monoclonal antibodies (mAbs) has been evolving and becoming more challenging as the requirements of the regulatory agencies increase due to the demanding complexity of products under evaluation. Mass Spectrometry (MS)-based methods such as the multi-attribute method (MAM) are being explored to achieve a deeper understanding of the attributes critical for the safety, efficacy, and quality of these products. MAM uses high mass accuracy/high-resolution MS data that enables the direct and simultaneous monitoring of relevant product quality attributes (PQAs, in particular, chemical modifications) in a single workflow, replacing several orthogonal methods, reducing time and costs associated with these assays. Here we describe a MAM implementation process using a QTOF high resolution platform. Method implementation was accomplished using NIST (National Institute for Standards and Technology) mAb reference material and an in-process mAb sample. PQAs as glycosylation profiles, methionine oxidation, tryptophan dioxidation, asparagine deamidation, pyro-Glu at N-terminal and glycation were monitored. Focusing on applications that require batch analysis and high-throughput, sample preparation and LC-MS parameters troubleshooting are discussed. This MAM workflow was successfully explored as reference analytical tool for comprehensive characterization of a downstream processing (DSP) polishing platform and for a comparability study following technology transfer between different laboratories.


Assuntos
Produtos Biológicos/química , Espectrometria de Massas/métodos , Anticorpos Monoclonais/química , Cromatografia Líquida/métodos , Controle de Qualidade , Projetos de Pesquisa , Tripsina/química , Fluxo de Trabalho
15.
Clin Exp Rheumatol ; 40(2): 267-273, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34874829

RESUMO

OBJECTIVES: We aimed to investigate muscle physical properties, strength, mass, physical performance, and the prevalence of sarcopenia in patients with axial spondylarthritis (axSpA) compared to the healthy controls (HC). METHODS: We performed a cross-sectional study on 54 participants: 27 patients with axSpA and 27 HC, matched by age, gender, and level of physical activity. Muscle physical properties (stiffness, tone and elasticity), muscle strength (five-times sit-to-stand [5STS] test), muscle mass, physical performance (measured through gait speed) and sarcopenia were compared between the groups. Linear regression models were conducted allowing adjustment for relevant variables. RESULTS: Patients with axSpA (mean age 36.5 (SD 7.5) years, 67% males, mean disease duration 6.5 (3.2) years) had no significant difference in segmental muscle stiffness, tone or elasticity, compared with the HC, despite showing a slight numerically higher lower lumbar (L3-L4) stiffness [median 246.5 (IQR 230.5-286.5) vs. 232.5 (211.0-293.5), p=0.38]. No participants presented sarcopenia. Patients with axSpA, compared to the HC, had lower total strength [B=1.88 (95% CI 0.43;3.33)], as well as lower strength in the upper (B=-17.02 (-27.33;-6.70)] and lower limbs [B=-11.14 (-18.25;-4.04)], independently of muscle physical properties. Patients had also significantly lower gait speed than the HC [B=-0.11 (-0.21;-0.01)], adjusted for muscle mass, strength and muscle physical properties. CONCLUSIONS: Young axSpA patients with a relatively short disease duration presented similar segmental muscle physical properties as the HC and had no sarcopenia. Patients with axSpA had reduced physical performance and lower strength compared to the HC, despite normal muscle mass, suggesting a possible muscle dysfunction. Gait characteristics may be a potential biomarker of interest in axSpA.


Assuntos
Espondiloartrite Axial , Sarcopenia , Espondilartrite , Adulto , Biomarcadores , Estudos Transversais , Feminino , Humanos , Masculino , Força Muscular/fisiologia , Músculos , Sarcopenia/diagnóstico , Sarcopenia/epidemiologia , Sarcopenia/etiologia , Espondilartrite/diagnóstico , Espondilartrite/epidemiologia
16.
Acta Reumatol Port ; 46(4): 342-349, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34962249

RESUMO

BACKGROUND: Axial Spondyloarthritis (axSpA) is a chronic, inflammatory rheumatic disease that affects the axial skeleton, causing pain, stiffness, and fatigue. Genetics and environmental factors such as microbiota and microtrauma are known causes of disease susceptibility and progression. Murine models of axSpA found a decisive role for biomechanical stress as an inducer of enthesitis and new bone formation. Here, we hypothesize that muscle properties in axSpA patients are compromised and influenced by genetic background. OBJECTIVES: To improve our current knowledge of axSpA physiopathology, we aim to characterize axial and peripheral muscle properties and identify genetic and protein biomarker that might explain such properties. METHODS: A cross-sectional study will be conducted on 48 participants aged 18-50 years old, involving patients with axSpA (according to ASAS classification criteria, symptoms duration < 10 years) and healthy controls matched by gender, age, and levels of physical activity. We will collect epidemiological and clinical data and perform a detailed, whole body and segmental, myofascial characterization (focusing on multifidus, brachioradialis and the gastrocnemius lateralis) concerning: a) Physical Properties (stiffness, tone and elasticity), assessed by MyotonPRO®; b) Strength, by a dynamometer; c) Mass, by bioimpedance; d) Performance through gait speed and 60-second sit-to-stand test; e) Histological and cellular/ molecular characterization through ultrasound-guided biopsies of multifidus muscle; f) Magnetic Resonance Imaging (MRI) characterization of paravertebral muscles. Furthermore, we will perform an integrated transcriptomics and proteomics analysis of peripheral blood samples. DISCUSSION: The innovative and multidisciplinary approaches of this project rely on the elucidation of myofascial physical properties in axSpA and also on the establishment of a biological signature that relates to specific muscle properties. This hitherto unstudied link between gene/protein signatures and muscle properties may enhance our understanding of axSpA physiopathology and reveal new and useful diagnostic and therapeutic targets.


Assuntos
Espondiloartrite Axial , Espondilartrite , Espondilite Anquilosante , Adolescente , Adulto , Animais , Estudos Transversais , Humanos , Camundongos , Pessoa de Meia-Idade , Músculos , Adulto Jovem
17.
Curr Opin Biotechnol ; 71: 175-181, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34425321

RESUMO

Stem cells hold outstanding potential to model and treat disease and are valuable tools in pharmacology and toxicology. Characterization of stem cells and derivatives still poses many challenges to ensure safe, efficacious, and reliable therapies. Regulatory agencies have defined key mandatory attributes related to identity, purity, sterility, and genomic integrity, however robust analytics to determine cell's potency are still a major challenge, in most cases assessed case-by-case. Importantly, the application of high-throughput 'omic tools is opening new perspectives on stem cell's research and development. Here, analytical methodologies currently employed to characterize stem cells' quality attributes are discussed, with special focus on 'omics as relevant tools for definition of cell's mechanism of action, and for potency assay development and assessment.


Assuntos
Genômica , Células-Tronco , Genoma
18.
Acta Reumatol Port ; 46(2): 110-119, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34226434

RESUMO

OBJECTIVE: The human resting myofascial tone maintains the body tone in a neutral posture, the assessment of this and other muscle physical properties (MPP) is relevant, since, it is altered in many pathological states. PATIENTS AND METHODS: Seventeen healthy subjects (8 males), between 18-50 years old, were assessed. The MPP of lower lumbar muscles was evaluated on right and left sides during prone resting position using two devices; myotonometry (stiffness, elasticity and tone) and ultrasound-based shear-wave elastography (SWE) (shear modulus). MTM measurements were performed at two anatomic points (ANp), selected by an experienced reader and at an adjacent ultra-sound determined point (USp). Myotonometry measurements of the erector spinae and SWE measurements of multifidus muscles at the L3-4 level were compared between genders and sides. The intra-reader reliability (IRR) for each device and correlations between techniques were analysed. MTM measurements performed at ANp and USp were compared. The intraclass correlation coefficient (ICC) was assessed for both devices. Correlations between stiffness (myotonometry) and shear modulus (SWE) at the respective muscle depths were assessed with Spearman correlation. RESULTS: Males had greater stiffness and tone than females, particularly on the dominant side. MPP assessed by myotonometry were not different between ANp and USp. Good/Excellent IRR was documented for measurements by MTM (ICC≥0.90) and SWE (ICC≥0.85). No correlation in myotonometry stiffness and SWE shear modulus was found. For myotonometry assessments, the addition of ultrasonography was not different from anatomic localizations. No correlation of measurements was found between devices assessing respective L3-4 level muscles. CONCLUSIONS: Gender and side differences must be considered when assessing MPP in axial muscles. For MTM assessments, the addition of ultrasonography was not different to anatomic references. No correlation was found between devices.


Assuntos
Técnicas de Imagem por Elasticidade , Adolescente , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Músculos Paraespinais/diagnóstico por imagem , Reprodutibilidade dos Testes , Ultrassonografia , Adulto Jovem
19.
Biotechnol Adv ; 49: 107755, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33895330

RESUMO

Research in stem cells paved the way to an enormous amount of knowledge, increasing expectations on cardio regenerative therapeutic approaches in clinic. While the first generation of clinical trials using cell-based therapies in the heart were performed with bone marrow and adipose tissue derived mesenchymal stem cells, second generation cell therapies moved towards the use of cardiac-committed cell populations, including cardiac progenitor cells and pluripotent stem cell derived cardiomyocytes. Despite all these progresses, translating the aptitudes of R&D and pre-clinical data into effective clinical treatments is still highly challenging, partially due to the demanding regulatory and safety concerns but also because of the lack of knowledge on the regenerative mechanisms of action of these therapeutic products. Thus, the need of analytical methodologies that enable a complete characterization of such complex products and a deep understanding of their therapeutic effects, at the cell and molecular level, is imperative to overcome the hurdles of these advanced therapies. Omics technologies, such as proteomics and glyco(proteo)mics workflows based on state of the art mass-spectrometry, have prompted some major breakthroughs, providing novel data on cell biology and a detailed assessment of cell based-products applied in cardiac regeneration strategies. These advanced 'omics approaches, focused on the profiling of protein and glycan signatures are excelling the identification and characterization of cell populations under study, namely unveiling pluripotency and differentiation markers, as well as paracrine mechanisms and signaling cascades involved in cardiac repair. The leading knowledge generated is supporting a more rational therapy design and the rethinking of challenges in Advanced Therapy Medicinal Products development. Herein, we review the most recent methodologies used in the fields of proteomics, glycoproteomics and glycomics and discuss their impact on the study of cardiac progenitor cells and pluripotent stem cell derived cardiomyocytes biology. How these discoveries will impact the speed up of novel therapies for cardiovascular diseases is also addressed.


Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Mesenquimais , Células-Tronco Pluripotentes , Diferenciação Celular , Terapia Baseada em Transplante de Células e Tecidos , Miócitos Cardíacos , Proteômica
20.
Biotechnol Bioeng ; 118(6): 2202-2219, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33624859

RESUMO

Serological assays are valuable tools to study SARS-CoV-2 spread and, importantly, to identify individuals that were already infected and would be potentially immune to a virus reinfection. SARS-CoV-2 Spike protein and its receptor binding domain (RBD) are the antigens with higher potential to develop SARS-CoV-2 serological assays. Moreover, structural studies of these antigens are key to understand the molecular basis for Spike interaction with angiotensin converting enzyme 2 receptor, hopefully enabling the development of COVID-19 therapeutics. Thus, it is urgent that significant amounts of this protein became available at the highest quality. In this study, we produced Spike and RBD in two human derived cell hosts: HEK293-E6 and Expi293F™. We evaluated the impact of different and scalable bioprocessing approaches on Spike and RBD production yields and, more importantly, on these antigens' quality attributes. Using negative and positive sera collected from human donors, we show an excellent performance of the produced antigens, assessed in serologic enzyme-linked immunosorbent assay (ELISA) tests, as denoted by the high specificity and sensitivity of the test. We show robust Spike productions with final yields of approx. 2 mg/L of culture that were maintained independently of the production scale or cell culture strategy. To the best of our knowledge, the final yield of 90 mg/L of culture obtained for RBD production, was the highest reported to date. An in-depth characterization of SARS-CoV-2 Spike and RBD proteins was performed, namely the antigen's oligomeric state, glycosylation profiles, and thermal stability during storage. The correlation of these quality attributes with ELISA performance show equivalent reactivity to SARS-CoV-2 positive serum, for all Spike and RBD produced, and for all storage conditions tested. Overall, we provide straightforward protocols to produce high-quality SARS-CoV-2 Spike and RBD antigens, that can be easily adapted to both academic and industrial settings; and integrate, for the first time, studies on the impact of bioprocess with an in-depth characterization of these proteins, correlating antigen's glycosylation and biophysical attributes to performance of COVID-19 serologic tests.


Assuntos
Antígenos Virais/biossíntese , Glicosilação , Glicoproteína da Espícula de Coronavírus/biossíntese , Temperatura Baixa , Ensaio de Imunoadsorção Enzimática/normas , Congelamento , Células HEK293 , Humanos , Conformação Proteica , Estabilidade Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/normas , SARS-CoV-2 , Testes Sorológicos/normas , Glicoproteína da Espícula de Coronavírus/normas
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