Urinary extracellular vesicles as a monitoring tool for renal damage in patients not meeting criteria for chronic kidney disease.

Background: Current definition of chronic kidney disease (CKD) identifies only advanced stages, but effective management demands early detection. Urinary albumin-to-creatinine ratio (ACR) 30 mg/g is a cut-off point for CKD clinical diagnosis. Patients with lower values (normoalbuminuria) and eGFR > 60 mL/min/1.73 m2 are considered at no increased cardiorenal risk. However, higher incidence of renal function decline and cardiovascular events have been shown within the normoalbuminuria range. Novel subclinical indicators may help to identify higher-risk patients. Urinary extracellular vesicles (uEVs) are sentinels of renal function non-invasively. Here we aimed to approach the early assessment of cardiorenal risk by investigating the protein cargo of uEVs. Methods: Hypertensive patients were classified in control group (C) with ACR < 10 mg/g, and high-normal group (HN) with ACR 10-30 mg/g. Isolated uEVs were characterized by western blotting and electron microscopy and the protein cargo was analyzed by untargeted proteomics (LC-MS/MS) in a first discovery cohort. Protein confirmation was performed in a different cohort by ExoView. Immunohistochemistry of human kidney biopsies was also performed to evaluate the potential of uEVs to reflect renal damage. Results: HN albuminuria does not affect the uEVs concentration, size, or tetraspanin profile. Among >6200 uEVs proteins identified, 43 define a panel significantly altered in HN patients without variation in urine, mostly annotated in the tubule (39 out of 43). The tubular transporter long-chain fatty acid transport protein 2 (SLC27A2) and the apical membrane protein amnionless (AMN) confirmed their alteration in HN patients evidencing impaired tubular reabsorption. SLC27A2 showed tubular expression and significantly reduced levels in patients with diagnostic criteria for CKD. Conclusions: Alterations in the EV-mediated molecular profile are evident before pathological ACR levels are reached. Direct quantitation of SLC27A2 and AMN in uEVs helps identifying normoalbuminuric subjects with higher cardiorenal risk in early monitoring of CKD.

The Urinary Glycopeptide Profile Differentiates Early Cardiorenal Risk in Subjects Not Meeting Criteria for Chronic Kidney Disease.

Early diagnosis and treatment of chronic kidney disease (CKD) is a worldwide challenge. Subjects with albumin-to-creatinine ratio (ACR) ≥ 30 mg/g and preserved renal function are considered to be at no cardiorenal risk in clinical practice, but prospective clinical studies evidence increased risk, even at the high-normal (HN) ACR range (10-30 mg/g), supporting the need to identify other molecular indicators for early assessment of patients at higher risk. Following our previous studies, here we aim to stratify the normoalbuminuria range according to cardiorenal risk and identify the glycoproteins and N-glycosylation sites associated with kidney damage in subclinical CKD. Glycoproteins were analyzed in urine from hypertensive patients within the HN ACR range compared to control group (C; ACR < 10 mg/g) by mass spectrometry. A different cohort was analyzed for confirmation (ELISA) and sex perspective was evaluated. Patients' follow-up for 8 years since basal urine collection revealed higher renal function decline and ACR progression for HN patients. Differential N-glycopeptides and their N -glycosylation sites were also identified, together with their pathogenicity. N-glycosylation may condition pathological protein deregulation, and a panel of 62 glycoproteins evidenced alteration in normoalbuminuric subjects within the HN range. Haptoglobin-related protein, haptoglobin, afamin, transferrin, and immunoglobulin heavy constant gamma 1 (IGHG1) and 2 (IGHG2) showed increased levels in HN patients, pointing to disturbed iron metabolism and tubular reabsorption and supporting the tubule as a target of interest in the early progression of CKD. When analyzed separately, haptoglobin, afamin, transferrin, and IGHG2 remained significant in HN, in both women and men. At the peptide level, 172 N-glycopeptides showed differential abundance in HN patients, and 26 showed high pathogenicity, 10 of them belonging to glycoproteins that do not show variation between HN and C groups. This study highlights the value of glycosylation in subjects not meeting KDIGO criteria for CKD. The identified N-glycopeptides and glycosylation sites showed novel targets, for both the early assessment of individual cardiorenal risk and for intervention aimed at anticipating CKD progression.

Reference Material Production and Milk Protein Concentration as Elements to Improve Bluetongue Serological Diagnosis in Bulk Tank Milk.

The serological surveillance of bluetongue in bulk tank milk is an efficient and cost-effective method for the early detection of bluetongue virus incursions in unvaccinated free areas of the disease. In addition, the availability of standardized and reliable reagents and refined diagnostic procedures with high sensitivity and specificity are essential for surveillance purposes. However, no available reference materials for bluetongue virus serological surveillance in bulk tank milk exist. This study shows the production and characterization of reference material for the implementation of a commercially available bluetongue milk ELISA test in official laboratories, as well as the evaluation of a procedure to increase the sensitivity in samples with low levels of antibodies. This procedure, based on milk protein concentration, allowed us to notably increase the ELISA test's analytical sensitivity, which is useful for milk samples from farms with low within-herd prevalence or pools of bulk tank milk samples. The standardized milk reference material produced here, together with the evaluated procedure to improve analytical sensitivity, could be applied as tools to ensure an accurate diagnosis by official laboratories in bluetongue unvaccinated free areas.

Complex Karyotype Detection in Chronic Lymphocytic Leukemia: A Comparison of Parallel Cytogenetic Cultures Using TPA and IL2+DSP30 from a Single Center.

Current CLL guidelines recommend a two parallel cultures assessment using TPA and IL2+DSP30 mitogens for complex karyotype (CK) detection. Studies comparing both mitogens for CK identification in the same cohort are lacking. We analyzed the global performance, CK detection, and concordance in the complexity assessment of two cytogenetic cultures from 255 CLL patients. IL2+DSP30 identified more altered karyotypes than TPA (50 vs. 39%, p = 0.031). Moreover, in 71% of those abnormal by both, IL2+DSP30 identified more abnormalities and/or abnormal metaphases. CK detection was similar for TPA and IL2+DSP30 (10% vs. 11%). However, 11/33 CKs (33%) were discordant, mainly due to the detection of a normal karyotype or no metaphases in the other culture. Patients requiring treatment within 12 months after sampling (active CLL) displayed significantly more CKs than those showing a stable disease (55% vs. 12%, p < 0.001). Disease status did not impact cultures' concordance (κ index: 0.735 and 0.754 for stable and active). Although CK was associated with shorter time to first treatment (TTFT) using both methods, IL2+DSP30 displayed better accuracy than TPA for predicting TTFT (C-index: 0.605 vs. 0.580, respectively). In summary, the analysis of two parallel cultures is the best option to detect CKs in CLL. Nonetheless, IL2+DSP30 could be prioritized above TPA to optimize cytogenetic assessment in clinical practice.

Analysis of Vascular Smooth Muscle Cells from Thoracic Aortic Aneurysms Reveals DNA Damage and Cell Cycle Arrest as Hallmarks in Bicuspid Aortic Valve Patients.

Thoracic aortic aneurysm (TAA) is mainly sporadic and with higher incidence in the presence of a bicuspid aortic valve (BAV) for unknown reasons. The lack of drug therapy to delay TAA progression lies in the limited knowledge of pathophysiology. We aimed to identify the molecular hallmarks that differentiate the aortic dilatation associated with BAV and tricuspid aortic valve (TAV). Aortic vascular smooth muscle cells (VSMCs) isolated from sporadic TAA patients with BAV or TAV were analyzed by mass spectrometry. DNA oxidative damage assay and cell cycle profiling were performed in three independent cohorts supporting proteomics data. The alteration of secreted proteins was confirmed in plasma. Stress phenotype, oxidative stress, and enhanced DNA damage response (increased S-phase arrest and apoptosis) were found in BAV-TAA patients. The increased levels of plasma C1QTNF5, LAMA2, THSB3, and FAP confirm the enhanced stress in BAV-TAA. Plasma FAP and BGN point to an increased inflammatory condition in TAV. The arterial wall of BAV patients shows a limited capacity to counteract drivers of sporadic TAA. The molecular pathways identified support the need of differential molecular diagnosis and therapeutic approaches for BAV and TAV patients, showing specific markers in plasma which may serve to monitor therapy efficacy.

Development and Validation of Three Triplex Real-Time RT-PCR Assays for Typing African Horse Sickness Virus: Utility for Disease Control and Other Laboratory Applications.

The African horse sickness virus (AHSV) belongs to the Genus Orbivirus, family Sedoreoviridae, and nine serotypes of the virus have been described to date. The AHSV genome is composed of ten linear segments of double-stranded (ds) RNA, numbered in decreasing size order (Seg-1 to Seg-10). Genome segment 2 (Seg-2) encodes outer-capsid protein VP2, the most variable AHSV protein and the primary target for neutralizing antibodies. Consequently, Seg-2 determines the identity of the virus serotype. An African horse sickness (AHS) outbreak in an AHS-free status country requires identifying the serotype as soon as possible to implement a serotype-specific vaccination program. Considering that nowadays 'polyvalent live attenuated' is the only commercially available vaccination strategy to control the disease, field and vaccine strains of different serotypes could co-circulate. Additionally, in AHS-endemic countries, more than one serotype is often circulating at the same time. Therefore, a strategy to rapidly determine the virus serotype in an AHS-positive sample is strongly recommended in both epidemiological situations. The main objective of this study is to describe the development and validation of three triplex real-time RT-PCR (rRT-PCR) methods for rapid AHSV serotype detection. Samples from recent AHS outbreaks in Kenia (2015-2017), Thailand (2020), and Nigeria (2023), and from the AHS outbreak in Spain (1987-1990), were included in the study for the validation of these methods.

Spatial Metabolomics Identifies LPC(18:0) and LPA(18:1) in Advanced Atheroma With Translation to Plasma for Cardiovascular Risk Estimation.

BACKGROUND: The metabolic alterations occurring within the arterial architecture during atherosclerosis development remain poorly understood, let alone those particular to each arterial tunica. We aimed first to identify, in a spatially resolved manner, the specific metabolic changes in plaque, media, adventitia, and cardiac tissue between control and atherosclerotic murine aortas. Second, we assessed their translatability to human tissue and plasma for cardiovascular risk estimation. METHODS: In this observational study, mass spectrometry imaging (MSI) was applied to identify region-specific metabolic differences between atherosclerotic (n=11) and control (n=11) aortas from low-density lipoprotein receptor-deficient mice, via histology-guided virtual microdissection. Early and advanced plaques were compared within the same atherosclerotic animals. Progression metabolites were further analyzed by MSI in 9 human atherosclerotic carotids and by targeted mass spectrometry in human plasma from subjects with elective coronary artery bypass grafting (cardiovascular risk group, n=27) and a control group (n=27). RESULTS: MSI identified 362 local metabolic alterations in atherosclerotic mice (log2 fold-change ≥1.5; P≤0.05). The lipid composition of cardiac tissue is altered during atherosclerosis development and presents a generalized accumulation of glycerophospholipids, except for lysolipids. Lysolipids (among other glycerophospholipids) were found at elevated levels in all 3 arterial layers of atherosclerotic aortas. LPC(18:0) (lysophosphatidylcholine; P=0.024) and LPA(18:1) (lysophosphatidic acid; P=0.025) were found to be significantly elevated in advanced plaques as compared with mouse-matched early plaques. Higher levels of both lipid species were also observed in fibrosis-rich areas of advanced- versus early-stage human samples. They were found to be significantly reduced in human plasma from subjects with elective coronary artery bypass grafting (P<0.001 and P=0.031, respectively), with LPC(18:0) showing significant association with cardiovascular risk (odds ratio, 0.479 [95% CI, 0.225-0.883]; P=0.032) and diagnostic potential (area under the curve, 0.778 [95% CI, 0.638-0.917]). CONCLUSIONS: An altered phospholipid metabolism occurs in atherosclerosis, affecting both the aorta and the adjacent heart tissue. Plaque-progression lipids LPC(18:0) and LPA(18:1), as identified by MSI on tissue, reflect cardiovascular risk in human plasma.

Obesity in Pregnancy as a Risk Factor in Maternal and Child Health-A Retrospective Cohort Study.

The prevalence of overweight and obesity has risen dramatically in the last few years. This has led to an increase in both conditions in pregnant women. Obesity and overweight are associated with complications for both the mother and the newborn. The aim of this study is to determine the prevalence of obesity and its association with the risk of complications during pregnancy. Materials and Methods: We conducted a retrospective cohort study of pregnant women who delivered from 1 January 2012 to 31 December 2018. Results: A higher prevalence of obesity is observed in the group of women aged 35 or older. Women with a BMI > 25 present a higher risk of cesarean section (aOR 1.49, 95% CI: 1.37-1.61), preeclampsia (aOR 1.64, 95% CI: 1.19-2.26), high-risk pregnancy (aOR 2.34, 95% CI: 1.68-2.6), Apgar < 7 at one minute (aOR 1.53, 95% CI: 1.25-1.89) and macrosomia (aOR 2.08, 95% CI: 1.83-2.37). Maternal overweight and obesity are important determinants of the risk of complications for both the mother and the newborn.

Immunohistochemical markers as predictors of prognosis in multifocal prostate cancer.

The impact of tumor focality on prostate cancer (PCa) prognosis has been addressed in several studies with conflicting results. Tumor foci from multifocal (MF) PCa can show highly heterogeneous molecular features. Our aim was to analyze the protein expression of PTEN, SPOP, SLC45A3, ETV1, ERG and the "triple hit" (ERG overexpression, PTEN plus SLC45A3 loss) in unifocal (UF) and MF PCa, to evaluate their value as prognostic markers according to focality, and the role of tumor heterogeneity in MF disease. PTEN, SPOP, SLC45A3, ETV1 and ERG immunohistochemical expression was evaluated in 185 PCa from 9 TMAs, 51 UF and 134 MF. In a subset of 69 MF cases, the dominant and secondary foci (DF and SF) were compared. Heterogeneity was considered when both tumor foci presented different expression patterns. Relationship with clinicopathological features was also analyzed. MF PCa was diagnosed in significantly younger patients when compared to UF ones (p = 0.007). ETV1 overexpression was associated with UF disease (p = 0.028). A shorter time to PSA recurrence was related to SLC45A3 wt expression in UF PCa (p = 0.052), and to SPOP expression loss (p = 0.043) or "triple hit" phenotype in MF PCa (p = 0.041). In MF cases, PTEN loss, SLC45A3 loss and "triple hit" phenotype were associated with the DF and had significant heterogeneity. In conclusion, our results indicate that UF and MF PCa have relevant and consistent molecular differences. The analysis of an immunohistochemical panel, composed by PTEN, SPOP, SLC45A3, ETV1 and ERG, could be useful to predict outcome in MF cases.

Metabolomics Analysis of Urinary Extracellular Vesicles by Nuclear Magnetic Resonance and Liquid Chromatography-Mass Spectrometry.

Extracellular vesicle (EV) release and their content are influenced by diverse clinical conditions. EVs participate in inter-cellular communication and have been postulated as reflectors of the pathophysiology of the cells, tissues, organs or the whole system with which they are in contact. Urinary EVs have been proved to reflect pathophysiology not only of renal system related diseases constituting an additional source of potential biomarkers easily accessible in a non-invasive way. The interest in EVs cargo has been mostly focused on proteins and nucleic acids and more recently it has been extended to metabolites. Metabolites represent the downstream changes in the genome, transcriptome, and proteome as a reflection of processes occurring in living organisms. For their study, nuclear magnetic resonance (NMR) and mass spectrometry in tandem (LC-MS/MS) are widely used. NMR is a reproducible and non-destructive technique and we show here methodological protocols for the metabolomics analysis of urinary EVs by NMR. Additionally, we also describe the workflow for a targeted LC-MS/MS analysis that is extensible to untargeted studies.

Donor liquid biopsy and outcomes in kidney transplantation.

Kidney transplantation is the treatment of choice for patients with kidney failure. Priority on the waiting list and optimal donor-recipient matching are guided by mathematical scores, clinical variables and macroscopic observation of the donated organ. Despite the increasing rates of successful kidney transplantation, maximizing the number of available organs while ensuring the optimum long-term performance of the transplanted kidney remains both key and challenging, and no unequivocal markers are available for clinical decision making. Moreover, the majority of studies performed thus far has focused on the risk of primary non-function and delayed graft function and subsequent survival and have mainly analysed recipients' samples. Given the increasing use of donors with expanded criteria and/or cardiac death, predicting whether grafts will provide sufficient kidney function is increasingly more challenging. Here we compile the available tools for pre-transplant kidney evaluation and summarize the latest molecular data from donors that may predict short-term (immediate or delayed graft function), medium-term (6 months) and long-term (≥12 months) kidney function. The use of liquid biopsy (urine, serum, plasma) to overcome the limitations of the pre-transplant histological evaluation is proposed. Novel molecules and approaches such as the use of urinary extracellular vesicles are also reviewed and discussed, along with directions for future research.

An optimized MALDI MSI protocol for spatial detection of tryptic peptides in fresh frozen prostate tissue.

MALDI MS imaging (MSI) is a powerful analytical tool for spatial peptide detection in heterogeneous tissues. Proper sample preparation is crucial to achieve high quality, reproducible measurements. Here we developed an optimized protocol for spatially resolved proteolytic peptide detection with MALDI time-of-flight MSI of fresh frozen prostate tissue sections. The parameters tested included four different tissue washes, four methods of protein denaturation, four methods of trypsin digestion (different trypsin densities, sprayers, and incubation times), and five matrix deposition methods (different sprayers, settings, and matrix concentrations). Evaluation criteria were the number of detected and excluded peaks, percentage of high mass peaks, signal-to-noise ratio, spatial localization, and average intensities of identified peptides, all of which were integrated into a weighted quality evaluation scoring system. Based on these scores, the optimized protocol included an ice-cold EtOH+H2 O wash, a 5 min heating step at 95°C, tryptic digestion incubated for 17h at 37°C and CHCA matrix deposited at a final amount of 1.8 µg/mm2 . Including a heat-induced protein denaturation step after tissue wash is a new methodological approach that could be useful also for other tissue types. This optimized protocol for spatial peptide detection using MALDI MSI facilitates future biomarker discovery in prostate cancer and may be useful in studies of other tissue types.

Machine learning for grading and prognosis of esophageal dysplasia using mass spectrometry and histological imaging.

BACKGROUND: Barrett's esophagus (BE) is a precursor lesion of esophageal adenocarcinoma and may progress from non-dysplastic through low-grade dysplasia (LGD) to high-grade dysplasia (HGD) and cancer. Grading BE is of crucial prognostic value and is currently based on the subjective evaluation of biopsies. This study aims to investigate the potential of machine learning (ML) using spatially resolved molecular data from mass spectrometry imaging (MSI) and histological data from microscopic hematoxylin and eosin (H&E)-stained imaging for computer-aided diagnosis and prognosis of BE. METHODS: Biopsies from 57 patients were considered, divided into non-dysplastic (n = 15), LGD non-progressive (n = 14), LGD progressive (n = 14), and HGD (n = 14). MSI experiments were conducted at 50 × 50 µm spatial resolution per pixel corresponding to a tile size of 96x96 pixels in the co-registered H&E images, making a total of 144,823 tiles for the whole dataset. RESULTS: ML models were trained to distinguish epithelial tissue from stroma with area-under-the-curve (AUC) values of 0.89 (MSI) and 0.95 (H&E)) and dysplastic grade (AUC of 0.97 (MSI) and 0.85 (H&E)) on a tile level, and low-grade progressors from non-progressors on a patient level (accuracies of 0.72 (MSI) and 0.48 (H&E)). CONCLUSIONS: In summary, while the H&E-based classifier was best at distinguishing tissue types, the MSI-based model was more accurate at distinguishing dysplastic grades and patients at progression risk, which demonstrates the complementarity of both approaches. Data are available via ProteomeXchange with identifier PXD028949.

Metabolic Alterations Identified in Urine, Plasma and Aortic Smooth Muscle Cells Reflect Cardiovascular Risk in Patients with Programmed Coronary Artery Bypass Grafting.

Atherosclerosis is the predominant pathology associated to premature deaths due to cardiovascular disease. However, early intervention based on a personalized diagnosis of cardiovascular risk is very limited. We have previously identified metabolic alterations during atherosclerosis development in a rabbit model and in subjects suffering from an acute coronary syndrome. Here we aim to identify specific metabolic signatures which may set the basis for novel tools aiding cardiovascular risk diagnosis in clinical practice. In a cohort of subjects with programmed coronary artery bypass grafting (CABG), we have performed liquid chromatography and targeted mass spectrometry analysis in urine and plasma. The role of vascular smooth muscle cells from human aorta (HA-VSMCs) was also investigated by analyzing the intra and extracellular metabolites in response to a pro-atherosclerotic stimulus. Statistically significant variation was considered if p value < 0.05 (Mann-Whitney test). Urinary trimethylamine N-oxide (TMAO), arabitol and spermidine showed higher levels in the CVrisk group compared with a control group; while glutamine and pantothenate showed lower levels. The same trend was found for plasma TMAO and glutamine. Plasma choline, acetylcholine and valine were also decreased in CVrisk group, while pyruvate was found increased. In the secretome of HA-VSMCs, TMAO, pantothenate, glycerophosphocholine, glutathion, spermidine and acetylcholine increased after pro-atherosclerotic stimulus, while secreted glutamine decreased. At intracellular level, TMAO, pantothenate and glycerophosphocholine increased with stimulation. Observed metabolic deregulations pointed to an inflammatory response together with a deregulation of oxidative stress counteraction.

TCA Cycle and Fatty Acids Oxidation Reflect Early Cardiorenal Damage in Normoalbuminuric Subjects with Controlled Hypertension.

Moderately increased albuminuria, defined by an albumin to creatinine ratio (ACR) > 30 mg/g, is an indicator of subclinical organ damage associated with a higher risk of cardiovascular and renal disease. Normoalbuminuric subjects are considered at no cardiorenal risk in clinical practice, and molecular changes underlying early development are unclear. To decipher subjacent mechanisms, we stratified the normoalbuminuria condition. A total of 37 hypertensive patients under chronic renin-angiotensin system (RAS) suppression with ACR values in the normoalbuminuria range were included and classified as control (C) (ACR < 10 mg/g) and high-normal (HN) (ACR = 10-30 mg/g). Target metabolomic analysis was carried out by liquid chromatography and mass spectrometry to investigate the role of the cardiorenal risk urinary metabolites previously identified. Besides this, urinary free fatty acids (FFAs), fatty acid binding protein 1 (FABP1) and nephrin were analyzed by colorimetric and ELISA assays. A Mann-Whitney test was applied, ROC curves were calculated and Spearman correlation analysis was carried out. Nine metabolites showed significantly altered abundance in HN versus C, and urinary FFAs and FABP1 increased in HN group, pointing to dysregulation in the tricarboxylic acid cycle (TCA) cycle and fatty acids ß-oxidation. We showed here how cardiorenal metabolites associate with albuminuria, already in the normoalbuminuric range, evidencing early renal damage at a tubular level and suggesting increased ß-oxidation to potentially counteract fatty acids overload in the HN range.

Proteomic and Biological Analysis of an In Vitro Human Endothelial System in Response to Drug Anaphylaxis.

Anaphylaxis is a life-threatening systemic hypersensitivity reaction. During anaphylaxis, mediator release by effector cells causes endothelial barrier breakdown, increasing vascular permeability and leakage of fluids, which may lead to tissue edema. Although endothelial cells (ECs) are key players in this context, scant attention has been paid to the molecular analysis of the vascular system, and further analyses of this cell type are necessary, especially in humans. The protein expression pattern of human microvascular ECs was analyzed in response to sera from anaphylactic patients (EC-anaphylaxis) and sera from non-allergic subjects (EC-control) after 2 hours of contact. Firstly, a differential quantitative proteomic analysis of the protein extracts was performed by mass spectrometry using an isobaric labeling method. Second, the coordinated behavior of the identified proteins was analyzed using systems biology analysis (SBA). The proteome of the EC-anaphylaxis system showed 7,707 proteins, of which 1,069 were found to be significantly altered between the EC-control and EC-anaphylaxis groups (p-value < 0.05). Among them, a subproteome of 47 proteins presented a high rate of change (|ΔZq| ≥ 3). This panel offers an endothelial snapshot of the anaphylactic reaction. Those proteins with the highest individual changes in abundance were hemoglobin subunits and structural support proteins. The interacting network analysis of this altered subproteome revealed that the coagulation and complement systems are the main biological processes altered in the EC-anaphylactic system. The comprehensive SBA resulted in 5,512 functional subcategories (biological processes), 57 of which were significantly altered between EC-control and EC-anaphylaxis. The complement system, once again, was observed as the main process altered in the EC system created with serum from anaphylactic patients. Findings of the current study further our understanding of the underlying pathophysiological mechanisms operating in anaphylactic reactions. New target proteins and relevant signaling pathways operating in the in vitro endothelial-serum system have been identified. Interestingly, our results offer a protein overview of the micro-EC-anaphylaxis environment. The relevance of the coagulation, fibrinolytic, contact and complement systems in human anaphylaxis is described. Additionally, the untargeted high-throughput analysis used here is a novel approach that reveals new pathways in the study of the endothelial niche in anaphylaxis.

Early renal and vascular damage within the normoalbuminuria condition.

OBJECTIVE: A continuous association between albuminuria and cardiorenal risk exists further below moderately increased albuminuria ranges. If only based in albumin to creatinine ratio (ACR) higher than 30 mg/g, a significant percentage of individuals may be out of the scope for therapeutic management. Despite epidemiological outcomes, the identification of biochemical changes linked to early albuminuria is underexplored, and normoalbuminuric individuals are usually considered at no risk in clinical practice. Here, we aimed to identify early molecular alterations behind albuminuria development. METHODS: Hypertensive patients under renin-angiotensin system (RAS) suppression were classified as control, (ACR < 10 mg/g) or high-normal (ACR = 10-30 mg/g). Urinary protein alterations were quantified and confirmed by untargeted and targeted mass spectrometry. Coordinated protein responses with biological significance in albuminuria development were investigated. Immunohistochemistry assays were performed in human kidney and arterial tissue to in situ evaluate the associated damage. RESULTS: A total of 2663 identified proteins reflect inflammation, immune response, ion transport and lipids metabolism (P value ≤ 0.01). A1AT, VTDB and KNG1 varied in high-normal individuals (P value < 0.05), correlated with ACR and associated with the high-normal condition (odds ratio of 20.76, 6.00 and 7.04 were found, respectively (P value < 0.001)). After 12 months, protein variations persist and aggravate in progressors to moderately increased albuminuria. At tissue level, differential protein expression was found in kidney from individuals with moderately increased albuminuria and atherosclerotic aortas for the three proteins, confirming their capacity to reflect subclinical organ damage. CONCLUSION: Early renal and vascular damage is molecularly evidenced within the normoalbuminuria condition.

Urinary Spermidine Predicts and Associates with In-Hospital Acute Kidney Injury after Cardiac Surgery.

Acute Kidney Injury (AKI) affects up to 30% of the patients who undergo cardiac surgery (CVS) and is related to higher mortality. We aim to investigate molecular features associated with in-hospital AKI development and determine the predictive value of these features when analyzed preoperatively. This is a case-control study. From an initial cohort of 110 recruited subjects, a total of 60 patients undergoing cardiac surgery were included: 20 (33%) developed in-hospital AKI (CVS-AKI) and 40 did not (controls, CVS-C). Pre- and post-surgery samples were collected and a prospective study was carried out. A total of 312 serum samples and 258 urine samples were analyzed by nuclear magnetic resonance, mass spectrometry and ELISA. Six features predicted AKI development in pre-surgery samples: urinary kidney functional loss marker kidney injury molecule-1 (uKIM-1), 2-hydroxybutyric acid, 2-hydroxyphenylacetic acid, hippuric acid, phosphoethanolamine and spermidine. Two of them stood out as powerful predictors. Pre-surgery uKIM-1 levels were increased in CVS-AKI vs. CVS-C (AUC = 0.721, p-value = 0.0392) and associated strongly with the outcome (OR = 5.333, p-value = 0.0264). Spermidine showed higher concentration in CVS-AKI (p-value < 0.0001, AUC = 0.970) and had a strong association with the outcome (OR = 69.75, p-value < 0.0001). uKIM-1 and particularly spermidine predict in-hospital AKI associated with CVS in preoperative samples. These findings may aid in preventing postoperative AKI and improve prognosis of CVS.

The Addition of Other Fecal Biomarkers Does Not Improve the Diagnostic Accuracy of Immunochemical Fecal Occult Blood Test Alone in a Colorrectal Cancer Screening Cohort.

Background: Screening with fecal occult blood test reduces colorectal cancer (CRC) incidence and mortality, and is currently implemented in most countries. However, around 40% of screening colonoscopies are normal. Thus, strategies to avoid these colonoscopies are highly necessary. Adding other fecal biomarkers, such as fecal calprotectin (FC), lactoferrin, and transferrin may be useful, but evidence is scarce. Aims: To evaluate the diagnostic accuracy of fecal occult blood immunochemical test (FIT), FC, and a one-step combo card test for the simultaneous semi-qualitative detection of human hemoglobin (hHb), transferrin (hTf), calprotectin (hCp) and lactoferrin (hLf) in a CRC screening program population. Methods: Single-center, prospective observational study, enrolling patients included in a CRC screening program, referred for a colonoscopy due to a positive FIT test. Participants collected a stool sample prior to bowel preparation, and FIT, FC and the combo semi-qualitative tests were performed on the sample. Sensitivity, specificity, positive and negative predictive values and area under receiver operator curve (AUC) for diagnosis of advanced neoplasia, advanced adenoma and CRC were estimated for each biomarker and their combinations. The primary endpoint of the study was to assess whether these biomarkers could improve the diagnostic accuracy of FIT alone. Results: 336 consecutive patients (64% males) were recruited. Advanced neoplasia was found in 129/336 (38.4%) patients, and of these, 22/336 (6.5%) were diagnosed of CRC. 153/336 (45.5%) colonoscopies were completely normal. The AUC for the diagnosis of advanced neoplasia were 0.725 (95%CI 0.665-0.784) for FIT, 0.477 (95%CI 0.413-0.541) for FC and 0.732 (95%CI 0.674-0.791) for the combination of both (FIT + FC) quantitative tests. The AUCs for the combo test were 0.70 (95%CI 0.641-0.760) for hHb, 0.625 (95%CI 0.562-0.698) for hTf, 0.532 (95%CI 0.469-0.595) for hCp, 0.531 (95%CI 0.466-0.595 ) for hLf and 0.681 (95%CI 0.620-0.741) for the combination of the four biomarkers. Conclusion: In average-risk population, FIT appears to be the best fecal marker for the diagnosis of CRC and advanced adenoma. None of the other biomarkers explored or their combinations provided a better diagnostic accuracy. Only hTF showed an acceptable diagnostic accuracy. FC and hLF were not useful in this setting.

Non-syndromic thoracic aortic aneurysm: cellular and molecular insights.

Thoracic aortic aneurysm (TAA) develops silently and asymptomatically and is a major cause of mortality. TAA prevalence is greatly underestimated, it is usually diagnosed incidentally, and its treatment consists mainly of prophylactic surgery based on the aortic diameter. The lack of effective drugs and biological markers to identify and stratify TAAs by risk before visible symptoms results from scant knowledge of its pathophysiological mechanisms. Here we integrate the structural impairment affecting non-syndromic non-familial TAA with the main cellular and molecular changes described so far and consider how these changes are interconnected through specific pathways. The ultimate goal is to define much-needed novel markers of TAA, and so the potential of previously identified molecules to aid in early diagnosis/prognosis is also discussed. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.