Platelet distribution width (PDW) as a significant correlate of COVID-19 infection severity and mortality.

SARS-CoV-2 infection may cause a wide spectrum of symptoms, from asymptomatic, to mild respiratory symptoms and life-threatening sepsis. Among the clinical laboratory biomarkers analyzed during COVID-19 pandemic, platelet indices have raised great interest, due to the critical involvement of platelets in COVID-19-related thromboinflammation. Through an electronic literature search on MEDLINE, CINAHL, PubMed, EMBASE, Web of Science, and preprint servers we performed and updated a systematic review aimed at providing a detailed analysis of studies addressing the potential clinical utility of platelet distribution width, platelet distribution width (PDW), in laboratory medicine, exploring the possible association between increased PDW levels, disease severity, and mortality in COVID-19. Our systematic review revealed a wide heterogeneity of COVID-19 cohorts examined and a lack of homogenous expression of platelet indices. We found that 75 % of studies reported significantly elevated PDW values in COVID-19 infected cohorts compared to healthy/non-COVID-19 controls, and 40 % of studies reported that patients with severe COVID-19 showed increased PDW values than those with less-than-severe illness. Interestingly, 71.4 % of studies demonstrated significant increased PDW values in non survivors vs. survivors. Overall, these results suggest that platelets are critically involved as major players in the process of immunothrombosis in COVID-19, and platelet reactivity and morphofunctional alterations are mirrored by PDW, as indicator of platelet heterogeneity. Our results confirm that the use of PDW as prognostic biomarkers of COVID-19 sepsis still remains debated due to the limited number of studies to draw a conclusion, but new opportunities to investigate the crucial role of platelets in thrombo-inflammation are warranted.

Monocyte distribution width alterations and cytokine storm are modulated by circulating histones.

OBJECTIVES: Extracellular histone levels are associated with the severity of many human pathologies, including sepsis and COVID-19. This study aimed to investigate the role of extracellular histones on monocyte distribution width (MDW), and their effect on the release of cytokines by blood cells. METHODS: Peripheral venous blood was collected from healthy subjects and treated with different doses of a histone mixture (range 0-200 µg/mL) to analyze MDW modifications up-to 3 h and digital microscopy of blood smears. Plasma obtained after 3 h of histone treatment were assayed to evaluate a panel of 24 inflammatory cytokines. RESULTS: MDW values significantly increased in a time- and dose-dependent manner. These findings are associated with the histone-induced modifications of cell volume, cytoplasmic granularity, vacuolization, and nuclear structure of monocytes, promoting their heterogeneity without affecting their count. After 3 h of treatment almost all cytokines significantly increased in a dose-dependent manner. The most relevant response was shown by the significantly increased G-CSF levels, and by the increase of IL-1ß, IL-6, MIP-1ß, and IL-8 at the histone doses of 50, 100, and 200 µg/mL. VEGF, IP-10, GM-CSF, TNF-α, Eotaxin, and IL-2 were also up-regulated, and a lower but significant increase was observed for IL-15, IL-5, IL-17, bFGF, IL-10, IFN-γ, MCP-1, and IL-9. CONCLUSIONS: Circulating histones critically induce functional alterations of monocytes mirrored by MDW, monocyte anisocytosis, and hyperinflammation/cytokine storm in sepsis and COVID-19. MDW and circulating histones may be useful tools to predict higher risks of worst outcomes.

Deciphering the role of monocyte and monocyte distribution width (MDW) in COVID-19: an updated systematic review and meta-analysis.

The SARS-CoV-2 infection is characterized by both systemic and organ hyper-thromboinflammation, with a clinical course ranging from mild up-to critical systemic dysfunction and death. In patients with coronavirus disease 2019 (COVID-19) the monocyte/macrophage population is deeply involved as both trigger and target, assuming the value of useful diagnostic/prognostic marker of innate cellular immunity. Several studies correlated morphological and immunophenotypic alterations of circulating monocytes with clinical outcomes in COVID-19 patients, concluding that monocyte distribution width (MDW) may retain clinical value in stratifying the risk of disease worsening. Through an electronic search in Medline and Scopus we performed an updated literature review and meta-analysis aimed to explore the association between increased MDW levels and illness severity in COVID-19 patients, deciphering role(s) and function(s) of monocytes in the harmful network underlining SARS-CoV-2 infection. We found that significantly elevated MDW values were frequently present in COVID-19 patients who developed unfavorable clinical outcomes, compounded by a significant association between monocyte anisocytosis and SARS-CoV-2 outcomes. These findings suggest that blood MDW index and its scatter plot could represent useful routine laboratory tools for early identification of patients at higher risk of unfavorable COVID-19 and for monitoring the progression of viral infection, clinical outcomes, and therapeutic efficacy throughout hospitalization. According to this evidence, therapeutic decisions in patients with SARS-CoV-2 infection could benefit from monitoring MDW value, with administration of drugs limiting thrombo-inflammation due to monocyte hyper-activation in patients with severe/critical COVID-19 disease.

What is the impact of circulating histones in COVID-19: a systematic review.

The infectious respiratory condition COVID-19 manifests a clinical course ranging from mild/moderate up-to critical systemic dysfunction and death linked to thromboinflammation. During COVID-19 infection, neutrophil extracellular traps participating in cytokine storm and coagulation dysfunction have emerged as diagnostic/prognostic markers. The characterization of NET identified that mainly histones, have the potential to initiate and propagate inflammatory storm and thrombosis, leading to increased disease severity and decreased patient survival. Baseline assessment and serial monitoring of blood histone concentration may be conceivably useful in COVID-19. We performed a literature review to explore the association among increased circulating levels of histones, disease severity/mortality in COVID-19 patients, and comparison of histone values between COVID-19 and non-COVID-19 patients. We carried out an electronic search in Medline and Scopus, using the keywords "COVID-19" OR "SARS-CoV-2" AND "histone" OR "citrullinated histones" OR "hyperhistonemia", between 2019 and present time (i.e., June 07th, 2022), which allowed to select 17 studies, totaling 1,846 subjects. We found that substantially elevated histone values were consistently present in all COVID-19 patients who developed unfavorable clinical outcomes. These findings suggest that blood histone monitoring upon admission and throughout hospitalization may be useful for early identification of higher risk of unfavorable COVID-19 progression. Therapeutic decisions in patients with SARS-CoV-2 based on the use of histone cut-off values may be driven by drugs engaging histones, finally leading to the limitation of cytotoxic, inflammatory, and thrombotic effects of circulating histones in viral sepsis.

Circulating histones contribute to monocyte and MDW alterations as common mediators in classical and COVID-19 sepsis.

OBJECTIVE: Histone proteins are physiologically involved in DNA packaging and gene regulation but are extracellularly released by neutrophil/monocyte extracellular traps and mediate thrombo-inflammatory pathways, associated to the severity of many human pathologies, including bacterial/fungal sepsis and COVID-19. Prominent and promising laboratory features in classic and viral sepsis emphasize monocyte distribution width (MDW), due to its ability to distinguish and stratify patients at higher risk of critical conditions or death. No data are available on the roles of histones as MDW modifiers. DESIGN: Comparison of MDW index was undertaken by routine hematology analyzer on whole blood samples from patients with COVID-19 and Sepsis. The impact of histones on the MDW characteristics was assessed by the in vitro time-dependent treatment of healthy control whole blood with histones and histones plus lipopolysaccharide to simulate viral and classical sepsis, respectively. MEASUREMENTS AND MAIN RESULTS: We demonstrated the breadth of early, persistent, and significant increase of MDW index in whole blood from healthy subject treated in vitro with histones, highlighting changes similar to those found in vivo in classic and viral sepsis patients. These findings are mechanistically associated with the histone-induced modifications of cell volume, cytoplasmic granularity and vacuolization, and nuclear structure alterations of the circulating monocyte population. CONCLUSIONS: Histones may contribute to the pronounced and persistent monocyte alterations observed in both acute classical and viral sepsis. Assessment of the biological impact of circulating histone released during COVID-19 and sepsis on these blood cells should be considered as key factor modulating both thrombosis and inflammatory processes, as well as the importance of neutralization of their cytotoxic and procoagulant activities by several commercially available drugs (e.g., heparins and heparinoids).

MMP-2 and MMP-9 in Human Peripheral Blood: Optimizing Gelatinase Calibrator for Degradome Research and Discovering a Novel Gelatinolytic Enzyme.

Matrix metalloprotease-2 and -9 (gelatinase A and B, respectively) are enzymes crucially involved in a plethora of physiopathological conditions. Gelatin zymography is considered one of the major qualitative/semiquantitative assays for simultaneously determining zymogenic, active, and complexed forms of gelatinases. Critical steps are represented by variations in sample collection methods, molecular weight standard calibrators, and different zymography assay protocols. A normalization of these aspects is required for reducing discrepancies in technical procedures and interpreting results among different laboratories. In this study, we describe a novel protocol for gelatin zymography with increased pore size, which improves the separation of gelatinases with different molecular weights. A new method for obtaining gelatinase calibrator for gelatin zymography, by extracting MMP-2 and MMP-9 from peripheral blood, is also reported. Our method provides a gelatinase calibrator with enhanced stability both at room temperature and during multiple freeze-thaw cycles. This calibrator preparation is also suitable for in vitro post-translational modifications. For the first time, the improved zymography protocol allowed us to reveal in human peripheral blood samples new gelatinolytic bands resolved at very high molecular weight, likely complexes of MMP-9, undetectable with classical zymography protocols.

Validation of a Gravitational Model to Study Local Endogenous Biomarkers in Chronic Venous Insufficiency.

OBJECTIVE/BACKGROUND: Unlike most systemic chronic diseases, chronic venous insufficiency (CVI) is ideal to study using endogenous biomarkers. The stimulus causing damage can be turned on and off with gravitational positioning and venous blood samples can be taken locally. Annexin V (apoptosis) and microparticles (cell membrane debris) were used as markers of cell destruction, with matrix metalloproteinases (MMPs) as markers of tissue remodelling. The aim of this proof of concept study was to validate a gravitational model by investigating whether standing induced biochemical stress and whether recovery occurs on lying and after compression. METHODS: Fourteen patients (C4a-b) and 14 volunteers (C0-1) were tested under three supervised laboratory conditions for 1 h on separate days: (i) stationary standing on a small paper square; (ii) lying with both legs elevated 20°; (iii) compression standing using a 23-32 mmHg below knee stocking. Immediately after each condition, venous blood was withdrawn from the ankle. Commercial enzyme linked immunosorbent assay kits were used for batch analysis of the plasma samples. RESULTS: Median (interquartile range [IQR]) values of annexin V (AU/mL) and microparticles (nM) standing were as follows: volunteers 2.9 (2 - 3.4) and 10.2 (8.8 - 13.8), and patients 2.2 (1.3 - 6) and 11.3 (7.7 - 20), respectively. Significant reductions were observed lying: volunteers 2.1 (1.5 - 2.7; p = .019) and 8.5 (7.4 - 9.4; p = .041), patients 1.7 (1.2 - 2.7; p = .004) and 8.5 (7.0 - 11.4; p = .041), respectively. Globally, all median MMP values in the patients reduced with lying and with compression versus standing (p = .004). Individually, significant reductions occurred in MMPs 2 and 13 with compression and MMPs 3, 7, 8, 9, 10, and 12 on lying. Lying was more effective at reducing MMP levels than compression. CONCLUSION: Annexin V and microparticle concentrations are responsive to elevation and compression after 1 h. In the patients, all the tested MMPs decreased after lying and with compression versus standing. This model provides evidence supporting gravitational protection in the treatment of CVI.

Chronic Venous Disorders: The Dangerous, the Good, and the Diverse.

Chronic venous disorders are common vascular pathology of great medical and socioeconomic impact, characterized by a wide spectrum of clinical manifestations occurring with symptoms and/or signs that vary in type and severity. The predominant pathophysiological mechanisms of chronic venous disease start from the development of venous hypertension from shear stress and reflux, leading to endothelial dysfunction and venous wall dilatation. The altered hemodynamic transduces physical signals into harmful bio-molecular pathways, creating a vicious cycle among shear stress, proteolytic remodeling, and inflammatory processes. This intricate network is further exacerbated by the degradation of protective endothelial glycocalyx. In this special issue, at least three main aspects of these interactions are highlighted: the dangerous, the good, and the diverse, which may help to focus attention on the biomolecular mechanisms and the possible targeted therapy of chronic venous disorders (CVeD).

Chronic venous disease - Part II: Proteolytic biomarkers in wound healing.

Venous leg ulcers (VLU) are characterized by sustained proteolytic microenvironment impairing the healing process. Wound fluid (WF) reflect the biomolecular activities occurring within the wound area; however, it is unclear if WF from different healing phases have different proteolytic profiles and how VLU microenvironment affects the wound healing mechanisms. We investigated the proteolytic network of WF from distinct VLU phases, and in WF- and LPS-stimulated THP-1 monocytes treated with glycosaminoglycan sulodexide, a well known therapeutic approach for VLU healing. WF were collected from patients with VLU during inflammatory (Infl) and granulating (Gran) phases. WF and THP-1 supernatants were analyzed for nine matrix metalloproteinases (MMP) and four tissue inhibitors of metalloproteinases (TIMP) by multiplex immunoassays. Our results demonstrated that: 1) WF from Infl VLU contained significantly increased concentrations of MMP-2, MMP-9, MMP-12, TIMP-1, and TIMP-2 compared to Gran WF; 2) WF from Gran VLU showed significantly increased levels of MMP-1, MMP-7, MMP-13, and TIMP-4 compared to Infl WF; 3) LPS- and WF-stimulation of THP-1 cells significantly increased the expression of several MMP compared to untreated cells; 4) Sulodexide treatment of both LPS- and WF-stimulated THP-1 significantly down-regulated the release of several MMPs. Our study provides evidence-based medicine during treatment of patients with VLU. WF from Infl and Gran VLU have different MMP and TIMP signatures, consistent with their clinical state. The modulation of proteolytic pathways in wound microenvironment by glycosaminoglycan sulodexide, provide insights for translating research into clinical practice during VLU therapy.

Chronic venous disease - Part I: Inflammatory biomarkers in wound healing.

Venous leg ulcers (VLUs) produce wound fluid (WF), as a result of inflammatory processes within the wound. It is unclear if WF from different healing phases of VLU has a peculiar biochemical profile and how VLU microenvironment affects the wound healing mechanisms. This study was conducted to evaluate the cytokine/chemokine profiles in WF from distinct VLU phases, in WF- and LPS-stimulated monocytes and treated with glycosaminoglycan Sulodexide, a therapeutic option for VLU healing. WF and plasma were collected from patients with VLU during active inflammatory (Infl) and granulating (Gran) phases. Demographics, clinical characteristics and pain measurements were evaluated. WF, plasma, and THP-1 supernatants were analyzed for 27 inflammatory mediators by multiplex immunoassay. Our results demonstrated that: 1) pain was significantly increased in patients with Infl compared to Gran VLU; 2) cytokine profile of Infl WF was found to be statistically different from that Gran WF, as well significantly increased respect to plasma; 3) LPS- and WF-stimulation of THP-1 cells significantly increased the expression of several cytokines compared to untreated cells; 4) Sulodexide treatment of both LPS- and WF-stimulated THP-1 monocytes was able to significantly down-regulate the release of peculiar inflammatory mediators. Our study highlighted the importance to understand biomolecular processes underlying CVI when providing treatment for chronic VLU. Identification of inflammatory biomarkers in leg ulcer microenvironment, may provide useful tools for predicting healing outcome and developing targeted therapies.

Glutathione Depletion Is Linked with Th2 Polarization in Mice with a Retrovirus-Induced Immunodeficiency Syndrome, Murine AIDS: Role of Proglutathione Molecules as Immunotherapeutics.

UNLABELLED: Injection of the LP-BM5 murine leukemia virus into mice causes murine AIDS, a disease characterized by many dysfunctions of immunocompetent cells. To establish whether the disease is characterized by glutathione imbalance, reduced glutathione (GSH) and cysteine were quantified in different organs. A marked redox imbalance, consisting of GSH and/or cysteine depletion, was found in the lymphoid organs, such as the spleen and lymph nodes. Moreover, a significant decrease in cysteine and GSH levels in the pancreas and brain, respectively, was measured at 5 weeks postinfection. The Th2 immune response was predominant at all times investigated, as revealed by the expression of Th1/Th2 cytokines. Furthermore, investigation of the activation status of peritoneal macrophages showed that the expression of genetic markers of alternative activation, namely, Fizz1, Ym1, and Arginase1, was induced. Conversely, expression of inducible nitric oxide synthase, a marker of classical activation of macrophages, was detected only when Th1 cytokines were expressed at high levels. In vitro studies revealed that during the very early phases of infection, GSH depletion and the downregulation of interleukin-12 (IL-12) p40 mRNA were correlated with the dose of LP-BM5 used to infect the macrophages. Treatment of LP-BM5-infected mice with N-(N-acetyl-l-cysteinyl)-S-acetylcysteamine (I-152), an N-acetyl-cysteine supplier, restored GSH/cysteine levels in the organs, reduced the expression of alternatively activated macrophage markers, and increased the level of gamma interferon production, while it decreased the levels of Th2 cytokines, such as IL-4 and IL-5. Our findings thus establish a link between GSH deficiency and Th1/Th2 disequilibrium in LP-BM5 infection and indicate that I-152 can be used to restore the GSH level and a balanced Th1/Th2 response in infected mice. IMPORTANCE: The first report of an association between Th2 polarization and alteration of the redox state in LP-BM5 infection is presented. Moreover, it provides evidence that LP-BM5 infection causes a decrease in the thiol content of peritoneal macrophages, which can influence IL-12 production. The restoration of GSH levels by GSH-replenishing molecules can represent a new therapeutic avenue to fight this retroviral infection, as it reestablishes the Th1/Th2 balance. Immunotherapy based on the use of pro-GSH molecules would permit LP-BM5 infection and probably all those viral infections characterized by GSH deficiency and a Th1/Th2 imbalance to be more effectively combated.

Chronic Venous Insufficiency: Transforming Growth Factor-ß Isoforms and Soluble Endoglin Concentration in Different States of Wound Healing.

Venous leg ulcer (VLU) is a huge healthcare problem with poorly understood pathophysiology. Transforming growth factor-ß (TGF-ß) and endoglin (Eng), are inflammatory and wound healing mediators. Eng, co-receptor for TGF-ß type-II receptors, may be cleaved forming soluble Eng (sEng), antagonizing TGF-ß signaling, a crucial process in vascular pathologies. We evaluated the accumulation in wound fluid (WF) of TGF-ß isoforms and sEng in healing stages, showing the effects of sulodexide treatments, a glycosaminoglycan with clinical efficacy in VLU healing. Patients with inflammatory (Infl) and granulating (Gran) VLU were recruited. WFs and THP-1 monocytes exposed to Infl and Gran WF (treated/untreated with sulodexide) were analyzed for TGF-ß isoforms and sEng by multiplex immunoassay. In both Infl and Gran WF, TGF-ß1 and ß2 were similar; TGF-ß3 was significantly increased in Infl compared to Gran WFs (p = 0.033). sEng was significantly elevated in Gran compared to Infl WFs (p = 0.002). In THP-1 monocytes there was a significant increase in sEng after co-treatment of WF and sulodexide. The increase in TGF-ß3 found in Infl WF highlights its negative effect on wound healing, while the increased levels of sEng in Gran WF affects the leukocyte adhesion/transmigration through the endothelium, reducing the inflammatory response and favoring the wound healing. Glycosaminoglycan sulodexide potentiates the effects of sEng release from monocyte, representing an important therapeutic option for wound healing.

Breast cyst fluid heparan sulphate is distinctively N-sulphated depending on apocrine or flattened type.

Breast cyst fluid (BCF) contained in gross cists is involved with its many biomolecules in different stages of breast cystic development. Type I apocrine and type II flattened cysts are classified based on biochemical, morphological and hormonal differences, and their different patterns of growth factors and active biocompounds may require different regulation. In a previous paper, hyaluronic acid in a very low content and chondroitin sulphate/dermatan sulphate were identified and characterized in BCF. In this new study, various apocrine and flattened BCFs were analyzed for HS concentration and disaccharide pattern. Apocrine HS was found specifically constituted of N-acetyl groups contrary to flattened HS richer in N-sulphate disaccharides with an overall N-acetylated/N-sulphated ratio significantly increased in apocrine compared with flattened (13.5 vs 3.7). Related to this different structural features, the charge density significantly decreased (~-30%) in apocrine versus flattened BCFs. Finally, no significant differences were observed for HS amount (~0.9-1.3 µg ml(-1) ) between the two BCF types even if a greater content was determined for flattened samples. The specifically N-sulphated sequences in flattened BCF HS can exert biologic capacity by regulating growth factors activity. On the other hand, we cannot exclude a peculiar regulation of the activity of biomolecules in apocrine BCF by HS richer in N-acetylated disaccharides. In fact, the different patterns of growth factors and active biocompounds in the two types of cysts may require different regulation by specific sequences in the HS backbone possessing specific structural characteristics and distinctive chemical groups.

Characterization of oversulfated chondroitin sulfate rich in 4,6-O-disulfated disaccharides in breast cyst fluids collected from human breast gross cysts.

Glycosaminoglycans (GAGs) from breast cyst fluid (BCF) of gross cysts, subdivided into apocrine and flattened, directly collected from 27 gross-cystic-breast-disease (GCBD)-affected women were analysed. Heparan sulfate, not further investigated, and chondroitin sulfate were identified. This last polysaccharide, in a content of 25-27 µg ml(-1) BCF and having a high molecular mass (~20 000-22 000), was found rich in glucuronic acid (~96%-98%) and mainly sulfated in position 4 of the N-acetyl-galactosamine (~60%-64%). Moreover, the presence of ~19%-24% of uncommon 4,6-O-disulfated disaccharides CS-E inside the polysaccharide chains with a high charge density of ~1.15-1.20 was determined. No substantial differences between apocrine and flattened cysts were observed. The current study describes the first effort to examine the yield and distribution of complex macromolecules like GAGs in BCF, and the understanding of their structure may help explain some functions associated with physiological and pathological conditions.

Transcriptional signatures in human macrophage-like cells infected by Leishmania infantum, Leishmania major and Leishmania tropica.

BACKGROUND: In the Mediterranean basin, three Leishmania species have been identified: L. infantum, L. major and L. tropica, causing zoonotic visceral leishmaniasis (VL), zoonotic cutaneous leishmaniasis (CL) and anthroponotic CL, respectively. Despite animal models and genomic/transcriptomic studies provided important insights, the pathogenic determinants modulating the development of VL and CL are still poorly understood. This work aimed to identify host transcriptional signatures shared by cells infected with L. infantum, L. major, and L. tropica, as well as specific transcriptional signatures elicited by parasites causing VL (i.e., L. infantum) and parasites involved in CL (i.e., L. major, L. tropica). METHODOLOGY/PRINCIPAL FINDINGS: U937 cells differentiated into macrophage-like cells were infected with L. infantum, L. major and L. tropica for 24h and 48h, and total RNA was extracted. RNA sequencing, performed on an Illumina NovaSeq 6000 platform, was used to evaluate the transcriptional signatures of infected cells with respect to non-infected cells at both time points. The EdgeR package was used to identify differentially expressed genes (fold change > 2 and FDR-adjusted p-values < 0.05). Then, functional enrichment analysis was employed to identify the enriched ontology terms in which these genes are involved. At 24h post-infection, a common signature of 463 dysregulated genes shared among all infection conditions was recognized, while at 48h post-infection the common signature was reduced to 120 genes. Aside from a common transcriptional response, we evidenced different upregulated functional pathways characterizing L. infantum-infected cells, such as VEGFA-VEGFR2 and NFE2L2-related pathways, indicating vascular remodeling and reduction of oxidative stress as potentially important factors for visceralization. CONCLUSIONS: The identification of pathways elicited by parasites causing VL or CL could lead to new therapeutic strategies for leishmaniasis, combining the canonical anti-leishmania compounds with host-directed therapy.

Resolving breast cancer heterogeneity by searching reliable protein cancer biomarkers in the breast fluid secretome.

BACKGROUND: One of the major goals in cancer research is to find and evaluate the early presence of biomarkers in human fluids and tissues. To resolve the complex cell heterogeneity of a tumor mass, it will be useful to characterize the intricate biomolecular composition of tumor microenvironment (the so called cancer secretome), validating secreted proteins as early biomarkers of cancer initiation and progression. This approach is not broadly applicable because of the paucity of well validated and FDA-approved biomarkers and because most of the candidate biomarkers are mainly organ-specific rather than tumor-specific. For these reasons, there is an urgent need to identify and validate a panel of biomarker combinations for early detection of human tumors. This is especially important for breast cancer, the cancer spread most worldwide among women. It is well known that patients with early diagnosed breast cancer live longer, require less extensive treatment and fare better than patients with more aggressive and/or advanced disease. RESULTS: In the frame of searching breast cancer biomarkers (especially using nipple aspirate fluid mirroring breast microenvironment), studies have highlighted an optimal combination of well-known biomarkers: uPA + PAI-1 + TF. When individually investigated they did not show perfect accuracy in predicting the presence of breast cancer, whereas the triple combination has been demonstrated to be highly predictive of pre-cancer and/or cancerous conditions, approaching 97-100% accuracy. CONCLUSION: Despite the heterogeneous composition of breast cancer and the difficulties to find specific breast cancer biomolecules, the noninvasive analysis of the nipple aspirate fluid secretome may significantly improve the discovery of promising biomarkers, helping also the differentiation among benign and invasive breast diseases, opening new frontiers in early oncoproteomics.

The influence of redox modulation on hypoxic endothelial cell metabolic and proteomic profiles through a small thiol-based compound tuning glutathione and thioredoxin systems.

Reduction in oxygen levels is a key feature in the physiology of the bone marrow (BM) niche where hematopoiesis occurs. The BM niche is a highly vascularized tissue and endothelial cells (ECs) support and regulate blood cell formation from hematopoietic stem cells (HSCs). While in vivo studies are limited, ECs when cultured in vitro at low O2 (<5%), fail to support functional HSC maintenance due to oxidative environment. Therefore, changes in EC redox status induced by antioxidant molecules may lead to alterations in the cellular response to hypoxia likely favoring HSC self-renewal. To evaluate the impact of redox regulation, HUVEC, exposed for 1, 6, and 24 h to 3% O2 were treated with N-(N-acetyl-l-cysteinyl)-S-acetylcysteamine (I-152). Metabolomic analyses revealed that I-152 increased glutathione levels and influenced the metabolic profiles interconnected with the glutathione system and the redox couples NAD(P)+/NAD(P)H. mRNA analysis showed a lowered gene expression of HIF-1α and VEGF following I-152 treatment whereas TRX1 and 2 were stimulated. Accordingly, the proteomic study revealed the redox-dependent upregulation of thioredoxin and peroxiredoxins that, together with the glutathione system, are the main regulators of intracellular ROS. Indeed, a time-dependent ROS production under hypoxia and a quenching effect of the molecule were evidenced. At the secretome level, the molecule downregulated IL-6, MCP-1, and PDGF-bb. These results suggest that redox modulation by I-152 reduces oxidative stress and ROS level in hypoxic ECs and may be a strategy to fine-tune the environment of an in vitro BM niche able to support functional HSC maintenance.

Deciphering the single-cell omic: innovative application for translational medicine.

Traditional technologies to investigate system biology are limited by the detection of parameters resulting from the averages of large populations of cells, missing cells produced in small numbers, and attempting to uniform the heterogeneity. The advent of proteomics and genomics at a single-cell level has set the basis for an outstanding improvement in analytical technology and data acquisition. It has been well demonstrated that cellular heterogeneity is closely related to numerous stochastic transcriptional events leading to variations in patterns of expression among single genetically identical cells. The new-generation technology of single-cell analysis is able to better characterize a cell's population, identifying and differentiating outlier cells, in order to provide both a single-cell experiment and a corresponding bulk measurement, through the identification, quantification and characterization of all system biology aspects (genomics, transcriptomics, proteomics, metabolomics, degradomics and fluxomics). The movement of omics into single-cell analysis represents a significant and outstanding shift.