Elevation of Tear MMP-9 Concentration as a Biomarker of Inflammation in Ocular Pathology by Antibody Microarray Immunodetection Assays

Matrix metalloproteinases are a family of enzymes fundamental in inflammatory processes. Between them, MMP-9 is up-regulated during inflammation; thus, its quantification in non-invasive fluids is a promising approach for inflammation identification. To this goal, a biomarker quantification test was developed for ocular inflammation detection using anti-MMP-9 antibody microarrays (AbMAs). After validation with eight healthy control tear samples characterized by ELISA, 20 samples were tested from individuals diagnosed with ocular inflammation due to: cataracts, glaucoma, meibomian gland dysfunction, allergy, or dry eye. Concentration values of tear MMP-9 were obtained for each sample, and 12 patients surpassed the pathological threshold (30 ng/mL). A significant elevation of MMP-9 concentration in the tears of glaucoma patients compared with healthy controls was observed. In order to evaluate the diagnostic ability, an ROC curve analysis was performed using our data, determining the optimal threshold for the test at 33.6 ng/mL of tear MMP-9. In addition, a confusion matrix was applied, estimating sensitivity at 60%, specificity at 88%, and accuracy at 68%. In conclusion, we demonstrated that the AbMAs system allows the quantification of MMP-9 in pathologies that involve inflammation of the ocular surface.     

Can porous polymeric scaffolds be functionalized by stem cells leading to osteogenic differentiation? A systematic review of in vitro studies

The aim of this study was to analyze the influence of nanoporous structure of polymeric biomaterials on the in vitro osteogenic induction of human stem cells. An electronic search in three databases (MEDLINE, SCOPUS, and Web of Science) was performed for articles that were published before May 2018. In vitro studies were included if they met the following criteria: (1) the use of polymeric scaffolds (natural or synthetic); (2) the co-culture of human stem cells with the scaffold; and (3) cell viability, proliferation, and osteogenic differentiation assays. The main characteristics of the published studies were summarized, and a quality assessment tool was used to analyze methodological features. Eighty-eight potential articles were firstly retrieved. Thirteen were eligible for qualitative analysis. Only three studies characterized cell stemness. Nanostructure of the scaffolds showed a significant influence on viability, proliferation, and osteogenic differentiation of human stem cells. Combination of porosity between 72 and 93% and a large range diameter between 50 and 224 μm resulted in more remarkable cellular proliferation and differentiation. Porous polymeric scaffolds can be functionalized by stem cells leading to osteogenic induction. High standards of laboratory practice and accurate methodological reporting are essential for the credibility of the results.     

Molecular Dynamics of CYFIP2 Protein and Its R87C Variant Related to Early Infantile Epileptic Encephalopathy

The CYFIP2 protein (cytoplasmic FMR1-interacting protein 2) is part of the WAVE regulatory complex (WRC). CYFIP2 was recently correlated to neurological disorders by the association of the R87C variant with early infantile epileptic encephalopathy (EIEE) patients. In this set of syndromes, the epileptic spasms and seizures since early childhood lead to impaired neurological development in children. Inside the WRC, the variant residue is at the CYFIP2 and WAVE1 protein interface. Thus, the hypothesis is that the R87C modification weakens this interaction, allowing the WRC complex’s constant activation. This work aimed to investigate the impacts of the mutation on the structure of the WRC complex through molecular dynamics simulation. For that, we constructed WRC models containing WAVE1-NCKAP1 proteins complexed with WT or R87C CYFIP2. Our simulations showed a flexibilization of the loop comprising residues 80–110 due to the loss of contacts between internal residues in the R87C CYFIP2 as well as the key role of residues R/C87, E624, and E689 in structural modification. These data could explain the mechanism by which the mutation impairs the stability and proper regulation of the WRC.     

Genetic LGALS1 Variants Are Associated with Heterogeneity in Galectin-1 Serum Levels in Patients with Early Arthritis

Galectin 1 (Gal1) exerts immunomodulatory effects leading to therapeutic effects in autoimmune animal models. Patients with rheumatoid arthritis have been reported to show higher Gal1 serum levels than the healthy population. Our study aimed to find genetic variants on the Gal1 gene (LGALS1) modulating its expression and/or clinical features in patients with early arthritis (EA). LGALS1 was sequenced in 53 EA patients to characterize all genetic variants. Then, we genotyped rs9622682, rs929039, and rs4820293, which covered the main genetic variation in LGALS1, in 532 EA patients. Gal1 and IL-6 serum levels were measured by ELISA and Gal1 also by western blot (WB) in lymphocytes from patients with specific genotypes. Once disease activity improved with treatment, patients with at least one copy of the minor allele in rs9622682 and rs929039 or those with GG genotype in rs4820293 showed significantly higher Gal1 serum levels (p < 0.05). These genotypic combinations were also associated with higher Gal1 expression in lymphocytes by WB and lower IL-6 serum levels in EA patients. In summary, our study suggests that genetic variants studied in LGALS1 can explain heterogeneity in Gal1 serum levels showing that patients with higher Gal1 levels have lower serum IL-6 levels.     

Establishing In-House Cutoffs of CSF Alzheimer’s Disease Biomarkers for the AT(N) Stratification of the Alzheimer Center Barcelona Cohort

Background: Clinical diagnosis of Alzheimer’s disease (AD) increasingly incorporates CSF biomarkers. However, due to the intrinsic variability of the immunodetection techniques used to measure these biomarkers, establishing in-house cutoffs defining the positivity/negativity of CSF biomarkers is recommended. However, the cutoffs currently published are usually reported by using cross-sectional datasets, not providing evidence about its intrinsic prognostic value when applied to real-world memory clinic cases. Methods: We quantified CSF Aβ1-42, Aβ1-40, t-Tau, and p181Tau with standard INNOTEST^® ELISA and Lumipulse G^® chemiluminescence enzyme immunoassay (CLEIA) performed on the automated Lumipulse G600II. Determination of cutoffs included patients clinically diagnosed with probable Alzheimer’s disease (AD, n = 37) and subjective cognitive decline subjects (SCD, n = 45), cognitively stable for 3 years and with no evidence of brain amyloidosis in 18F-Florbetaben-labeled positron emission tomography (FBB-PET). To compare both methods, a subset of samples for Aβ1-42 (n = 519), t-Tau (n = 399), p181Tau (n = 77), and Aβ1-40 (n = 44) was analyzed. Kappa agreement of single biomarkers and Aβ1-42/Aβ1-40 was evaluated in an independent group of mild cognitive impairment (MCI) and dementia patients (n = 68). Next, established cutoffs were applied to a large real-world cohort of MCI subjects with follow-up data available (n = 647). Results: Cutoff values of Aβ1-42 and t-Tau were higher for CLEIA than for ELISA and similar for p181Tau. Spearman coefficients ranged between 0.81 for Aβ1-40 and 0.96 for p181TAU. Passing–Bablok analysis showed a systematic and proportional difference for all biomarkers but only systematic for Aβ1-40. Bland–Altman analysis showed an average difference between methods in favor of CLEIA. Kappa agreement for single biomarkers was good but lower for the Aβ1-42/Aβ1-40 ratio. Using the calculated cutoffs, we were able to stratify MCI subjects into four AT(N) categories. Kaplan–Meier analyses of AT(N) categories demonstrated gradual and differential dementia conversion rates (p = 9.815⁻²⁷). Multivariate Cox proportional hazard models corroborated these findings, demonstrating that the proposed AT(N) classifier has prognostic value. AT(N) categories are only modestly influenced by other known factors associated with disease progression. Conclusions: We established CLEIA and ELISA internal cutoffs to discriminate AD patients from amyloid-negative SCD individuals. The results obtained by both methods are not interchangeable but show good agreement. CLEIA is a good and faster alternative to manual ELISA for providing AT(N) classification of our patients. AT(N) categories have an impact on disease progression. AT(N) classifiers increase the certainty of the MCI prognosis, which can be instrumental in managing real-world MCI subjects.     

Ferroptosis Modulation: Potential Therapeutic Target for Glioblastoma Treatment

Glioblastoma multiforme is a lethal disease and represents the most common and severe type of glioma. Drug resistance and the evasion of cell death are the main characteristics of its malignancy, leading to a high percentage of disease recurrence and the patients’ low survival rate. Exploiting the modulation of cell death mechanisms could be an important strategy to prevent tumor development and reverse the high mortality and morbidity rates in glioblastoma patients. Ferroptosis is a recently described type of cell death, which is characterized by iron accumulation, high levels of polyunsaturated fatty acid (PUFA)-containing phospholipids, and deficiency in lipid peroxidation repair. Several studies have demonstrated that ferroptosis has a potential role in cancer treatment and could be a promising approach for glioblastoma patients. Thus, here, we present an overview of the mechanisms of the iron-dependent cell death and summarize the current findings of ferroptosis modulation on glioblastoma including its non-canonical pathway. Moreover, we focused on new ferroptosis-inducing compounds for glioma treatment, and we highlight the key ferroptosis-related genes to glioma prognosis, which could be further explored. Thereby, understanding how to trigger ferroptosis in glioblastoma may provide promising pharmacological targets and indicate new therapeutic approaches to increase the survival of glioblastoma patients.     

Preclinical Assessment of the Combination of PSMA-Targeting Radionuclide Therapy with PARP Inhibitors for Prostate Cancer Treatment

Prostate specific membrane antigen targeted radionuclide therapy (PSMA-TRT) is a promising novel treatment for prostate cancer (PCa) patients. However, PSMA-TRT cannot be used for curative intent yet, thus additional research on how to improve the therapeutic efficacy is warranted. A potential way of achieving this, is combining TRT with poly ADP-ribosylation inhibitors (PARPi), which has shown promising results for TRT of neuroendocrine tumor cells. Currently, several clinical trials have been initiated for this combination for PCa, however so far, no evidence of synergism is available for PCa. Therefore, we evaluated the combination of PSMA-TRT with three classes of PARPi in preclinical PCa models. In vitro viability and survival assays were performed using PSMA-expressing PCa cell lines PC3-PIP and LNCaP to assess the effect of increasing concentrations of PARPi veliparib, olaparib or talazoparib in combination with PSMA-TRT compared to single PARPi treatment. Next, DNA damage analyses were performed by quantifying the number of DNA breaks by immunofluorescent stainings. Lastly, the potential of the combination treatments was studied in vivo in mice bearing PC3-PIP xenografts. Our results show that combining PSMA-TRT with PARPi did not synergistically affect the in vitro clonogenic survival or cell viability. DNA-damage analysis revealed only a significant increase in DNA breaks when combining PSMA-TRT with veliparib and not in the other combination treatments. Moreover, PSMA-TRT with PARPi treatment did not improve tumor control compared to PSMA-TRT monotherapy. Overall, the data presented do not support the assumption that combining PSMA-TRT with PARPi leads to a synergistic antitumor effect in PCa. These results underline that extensive preclinical research using various PCa models is imperative to validate the applicability of the combination strategy for PCa, as it is for other cancer types.     

Technical Validation and Clinical Implications of Ultrasensitive PCR Approaches for EGFR-Thr790Met Mutation Detection in Pretreatment FFPE Samples and in Liquid Biopsies from Non-Small Cell Lung Cancer Patients

In pretreatment tumor samples of EGFR-mutated non-small cell lung cancer (NSCLC) patients, EGFR-Thr790Met mutation has been detected in a variable prevalence by different ultrasensitive assays with controversial prognostic value. Furthermore, its detection in liquid biopsy (LB) samples remains challenging, being hampered by the shortage of circulating tumor DNA (ctDNA). Here, we describe the technical validation and clinical implications of a real-time PCR with peptide nucleic acid (PNA-Clamp) and digital droplet PCR (ddPCR) for EGFR-Thr790Met detection in diagnosis FFPE samples and in LB. Limit of blank (LOB) and limit of detection (LOD) were established by analyzing negative and low variant allele frequency (VAF) FFPE and LB specimens. In a cohort of 78 FFPE samples, both techniques showed an overall agreement (OA) of 94.20%. EGFR-Thr790Met was detected in 26.47% of cases and was associated with better progression-free survival (PFS) (16.83 ± 7.76 vs. 11.47 ± 1.83 months; p = 0.047). In LB, ddPCR was implemented in routine diagnostics under UNE-EN ISO 15189:2013 accreditation, increasing the detection rate of 32.43% by conventional methods up to 45.95%. During follow-up, ddPCR detected EGFR-Thr790Met up to 7 months before radiological progression. Extensively validated ultrasensitive assays might decipher the utility of pretreatment EGFR-Thr790Met and improve its detection rate in LB studies, even anticipating radiological progression.     

Effects of the Treatment with Flavonoids on Metabolic Syndrome Components in Humans: A Systematic Review Focusing on Mechanisms of Action

Diets high in bioactive compounds, such as polyphenols, have been used to mitigate metabolic syndrome (MetS). Polyphenols are a large group of naturally occurring bioactive compounds, classified into two main classes: non-flavonoids and flavonoids. Flavonoids are distributed in foods, such as fruits, vegetables, tea, red wine, and cocoa. Studies have already demonstrated the benefits of flavonoids on the cardiovascular and nervous systems, as well as cancer cells. The present review summarizes the results of clinical studies that evaluated the effects of flavonoids on the components of the MetS and associated complications when offered as supplements over the long term. The results show that flavonoids can significantly modulate several metabolic parameters, such as lipid profile, blood pressure, and blood glucose. Only theaflavin and catechin were unable to affect metabolic parameters. Moreover, only body weight and body mass index were unaltered. Thus, the evidence presented in this systematic review offers bases in support of a flavonoid supplementation, held for at least 3 weeks, as a strategy to improve several metabolic parameters and, consequently, reduce the risk of diseases associated with MetS. This fact becomes stronger due to the rare side effects reported with flavonoids.