The aim of this study is to identify a set of microRNAs (miRNAs) as prognostic molecular biomarkers for the progression of Barrett esophagus (BE) to esophageal adenocarcinoma (EAC) to rationalize the ...surveillance programs in patients with BE.
Histological dysplasia is currently used as the main biomarker to identify the BE patients at high risk for developing EAC. Although miRNA expression profiles in BE and EAC have been reported, it has not been established which set of miRNAs could constitute a robust diagnostic test to predict the progression of BE to EAC.
miRNAs associated with progression of BE to EAC were identified using miRNA sequencing analysis. Further validation by quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed in 2 groups of BE patients who either developed or did not develop adenocarcinoma after at least 5 years of follow-up.
Twenty-three miRNAs were identified by miRNA sequencing analysis in the carcinogenesis process associated with BE. qRT-PCR analysis using independent tissue samples confirmed differential expression for 19 of them (miR-let-7c, 7, 146a, 149, 153, 192, 192*, 194, 194*, 196a, 196b, 200a, 203, 205, 215, 424, 625, 625*, and 944). However, only miR-192, 194, 196a, and 196b showed a significantly higher expression in BE samples from patients with progression to EAC compared with those who did not progress to EAC.
These findings suggest that the expression pattern of a modest number of miRNAs in metaplasia biopsies could identify the BE patients at high risk for developing EAC. Therefore, it has potential use for the control and treatment of this malignancy.
Transfusion of washed platelet concentrates (W-PC) is recommended for some patients, such as those who have had previous severe allergic transfusion reactions. However, we still lack a standardised ...method for preparing these products. Here, we assessed the effect of a manual washing procedure on in vitro platelet quality and on the transfusion efficacy of W-PCs.
Buffy coat-derived W-PC in Composol solution were prepared by one-step centrifugation. Platelet activation and function were evaluated before and after washing by means of: (i) CD62 expression by flow cytometry; (ii) platelet aggregation (LTA); and (iii) the VerifyNow
P2Y12 test. A pilot prospective transfusion study was carried out in 11 onco-hematology patients receiving, in a short time, two consecutive transfusions: one with standard PC (S-PC) and one with W-PC. The post-transfusion platelet increment, the 1 h and 24 h corrected count increment (CCI) and occurrence of bleeding events were used as indices of transfusion efficacy.
Platelet recovery in W-PC was 84.8±5.4%. Washing slightly increased platelet activation in W-PC vs pre-washed samples (% CD62+ platelets 23.6±7 vs 14.8±1; p=0.03). As compared to prewash samples, platelet reactivity of W-PC as measured by VerifyNow
P2Y12 was significantly lower with ADP (PRU 32.2±37.7 vs 4.2±2.4, p=0.027), but similar using TRAP. Platelet aggregation responses to TRAP, collagen, ristocetin and arachidonic acid were maintained in W-PC. The pilot transfusion trial showed similar 1 h (13.5±5.6 vs 11.5±7.3, p=0.49) and 24 h (11±7.2 vs 9±6.5, p=0.48) CCI for S-PC and W-PC. Transfusion of W-PC was not associated with an increased number of bleeding events.
We have set up a simple method to obtain buffy-coat-derived W-PC, which has minor effects on in vitro platelet quality and transfusion effectiveness. This procedure can be easily implemented in transfusion centres for on-demand preparation of washed platelets.
Background & Aims Patients with cirrhosis are at high risk for developing hepatocellular carcinoma (HCC), and their liver tissues have abnormal levels of S -adenosylmethionine (SAMe). Glycine N ...-methyltransferase (GNMT) catabolizes SAMe, but its expression is down-regulated in HCC cells. Mice that lack GNMT develop fibrosis and hepatomas and have alterations in signaling pathways involved in carcinogenesis. We investigated the role of GNMT in human HCC cell lines and in liver carcinogenesis in mice. Methods We studied hepatoma cells from GNMT knockout mice and analyzed the roles of liver kinase B1 (LKB1, STK11) signaling via 5′-adenosine monophosphate–activated protein kinase (AMPK) and Ras in regulating proliferation and transformation. Results Hepatoma cells from GNMT mice had defects in LKB1 signaling to AMPK, making them resistant to induction of apoptosis by adenosine 3′,5′-cyclic monophosphate activation of protein kinase A and calcium/calmodulin-dependent protein kinase kinase 2. Ras-mediated hyperactivation of LKB1 promoted proliferation of GNMT-deficient hepatoma cells and required mitogen-activated protein kinase 2 (ERK) and ribosomal protein S6 kinase polypeptide 2 (p90RSK). Ras activation of LKB1 required expression of RAS guanyl releasing protein 3 (RASGRP3). Reduced levels of GNMT and phosphorylation of AMPKα at Thr172 and increased levels of Ras, LKB1, and RASGRP3 in HCC samples from patients were associated with shorter survival times. Conclusions Reduced expression of GNMT in mouse hepatoma cells and human HCC cells appears to increase activity of LKB1 and RAS; activation of RAS signaling to LKB1 and RASGRP3, via ERK and p90RSK, might be involved in liver carcinogenesis and be used as a prognostic marker. Reagents that disrupt this pathway might be developed to treat patients with HCC.
...immunothrombosis and neutrophil extracellular trap (NET) release contribute to inflammation-associated lung damage and thrombosis, and second, endothelial cell dysfunction participates in the ...disease-associated coagulopathy.1,2 In addition, cytokine release syndrome (CRS) dictates adverse clinical outcomes, with interleukin (IL)-6 playing a central role.3 However, the sequence of events leading to a vicious cycle of synergistic pathways causing unchecked immune activation and thrombus formation have to date not been clearly defined. In sepsis, ADAMTS13 has been found highly citrullinated, a posttranscriptional modification implicated in NET formation, due to the action of peptidylarginine deiminase type IV (PAD4).6 Interestingly, we found that severe decreased ADAMTS13 activity was inversely correlated with citH3-DNA levels in plasma (Figure S4). ...we hypothesize that low ADAMTS13 activity observed in COVID-19 patients might be due in part to high levels of PAD4, or by the action of specific autoantibodies.7 Overall, these results are in agreement with previous reports associating NETs and VWF with severity in COVID-19.1,2,8 Additionally, our study showed that VWF/ADAMTS13 imbalance contributed to adverse outcomes, influencing patient's survival. Different studies have provided evidences of the connection between neutrophils/NETs, CRS, and VWF/ADAMTS13: (i) α-defensins, released from neutrophils, bind to A2 domain of VWF inhibiting its proteolytic cleavage by ADAMTS13; (ii) VWF directly binds to leukocytes and NETs; (iii) IL-6 inhibits VWF cleavage by ADAMTS13 in vitro; (iv) CRS is exacerbated in inflammatory diseases when abnormal NET levels are generated; and finally (v) histones from NETs induce VWF release from endothelial cells in vitro and in vivo.10 In conclusion, our results support that the increase of IL-6 due to the CRS synchronizes a feedback mechanism between NETosis and VWF/ADAMTS13 axis, reflected by the correlation dynamics between plasma levels of NETs and VWF that, in turn, may perpetuate endothelium damage, amplifying immunothrombosis and worsening the course of the disease (Figure 3).
The diagnostic evaluation of inherited platelet disorders (IPDs) is complicated and time-consuming, resulting in a relevant number of undiagnosed and incorrectly classified patients. In order to ...evaluate the spectrum of IPDs in individuals with clinical suspicion of these disorders, and to provide a diagnostic tool to centers not having access to specific platelets studies, we established the project "Functional and Molecular Characterization of Patients with Inherited Platelet Disorders" under the scientific sponsorship of the Spanish Society of Thrombosis and Haemostasis.
Subjects were patients from a prospective cohort of individuals referred for clinical suspicion of IPDs as well as healthy controls. Functional studies included light transmission aggregation, flow cytometry, and when indicated, Western-blot analysis of platelet glycoproteins, and clot retraction analysis. Genetic analysis was mainly performed by sequencing of coding regions and proximal regulatory regions of the genes of interest.
Of the 70 cases referred for study, we functionally and molecularly characterized 12 patients with Glanzmann Thrombasthenia, 8 patients with Bernard Soulier syndrome, and 8 with other forms of IPDs. Twelve novel mutations were identified among these patients. The systematic study of patients revealed that almost one-third of patients had been previously misdiagnosed.
Our study provides a global picture of the current limitations and access to the diagnosis of IPDs, identifies and confirms new genetic variants that cause these disorders, and emphasizes the need of creating reference centers that can help health care providers in the recognition of these defects.
Background Immune thrombocytopenia (ITP) is characterised by low platelet counts and often leads to bleeding, fatigue, and reduced health-related quality of life. Methods This observational, ...retrospective, population-based study using BIG-PAC® database included Spanish paediatric and adult patients with primary ITP diagnosed in primary care and hospitals between 2014 and 2020 (median follow-up: 4 years). Epidemiology, baseline/clinical characteristics, treatment trends, healthcare resources and costs were analysed. Results The BIG-PAC® database contains records of 1,818,588 patients; 170 adults and 27 children with ITP were included in our analysis. ITP prevalence and annual incidence per 100,000 were estimated in 10.8 (2.8 in chronic ITP cITP patients) and 1.5 (0.3 in cITP patients), respectively. Epistaxis was the most common bleeding event, followed by genitourinary and gastrointestinal bleeding; >50%/> 75% of ITP/cITP patients reported fatigue. Chronic patients had lower platelet counts at baseline and required more transfusions. Corticosteroids, immunosuppressants, and thrombopoietin receptor agonists were the most used agents in first-, second- and third-line treatment, respectively. Thirty-five patients, all of them in chronic phase, underwent splenectomy. Patients had on average 13.9, 6.6, and 1.2 visits/year to primary care, haematology/internal medicine, and emergency departments, respectively. More than one-fourth of adult patients took on average 16.3 days of sick leave annually. Mean annual total health care costs were €10,741 (ITP patients) and €19,809 (cITP patients). Conclusion This is the first study to provide an overall perspective on the situation of the Spanish ITP population in terms of epidemiology, treatment trends, health care resources and costs, highlighting unmet patient needs, and direct and indirect costs/resource use between 2014 and 2020.
BACKGROUND/AIMS:The aim of this exploratory trial was to establish if the probiotic Bifidobacterium natren life start (NLS) strain strain may affect the clinical course and pathophysiological ...features of patients with untreated celiac disease (CD). Positive findings would be helpful in directing future studies.
METHODS:Twenty-two adult patients having 2 positives CD-specific tests were enrolled. Patients were randomized to receive 2 capsules before meals for 3 weeks of either Bifidobacterium infantis natren life start strain super strain (Lifestart 2) (2×10 colony-forming units per capsule) (n=12) or placebo (n=10), whereas they also consumed at least 12 g of gluten/day. A biopsy at the end of the trial confirmed CD in all cases. The primary outcome was intestinal permeability changes. Secondary endpoints were changes in symptoms and the Gastrointestinal Symptom Rating Scale, and in immunologic indicators of inflammation.
RESULTS:The abnormal baseline intestinal permeability was not significantly affected by either treatment. In contrast to patients on placebo, those randomized to B. infantis experienced a significant improvement in Gastrointestinal Symptom Rating Scale (P=0.0035 for indigestion; P=0.0483 for constipation; P=0.0586 for reflux). Final/baseline IgA tTG and IgA DGP antibody concentration ratios were lower in the B. infantis arm (P=0.055 for IgA tTG and P=0.181 for IgA DGP). Final serum macrophage inflammatory protein-1β increased significantly (P<0.04) only in patients receiving B. infantis. The administration of B. infantis was safe.
CONCLUSIONS:The study suggests that B. infantis may alleviate symptoms in untreated CD. The probiotic produced some immunologic changes but did not modify abnormal intestinal permeability. Further studies are necessary to confirm and/or expand these observations.
A new strategy for the determination of riboflavin (RF, or vitamin B2) in human urine samples has been developed, combining excitation-emission fluorescence matrix (EEFM) data in standard addition ...mode and second-order chemometric analysis. The method is simple, fast and eco-friendly because it complies with the green analytical chemistry principles, avoiding the need of previous clean up and separation steps that consume high amounts of organic solvents. Successful results were obtained by different chemometric algorithms, namely parallel factor analysis (PARAFAC), unfolded partial least-squares/residual bilinearization (U-PLS/RBL) and multidimensional partial least-squares/residual bilinearization (N-PLS/RBL), all in the modified standard addition. These algorithms allowed us to achieve selectivity in a system, which requires standard addition and shows a significant background spectral overlapping with the studied vitamin. The quality of the proposed strategy was evidenced on the basis of the analytical recoveries from urine samples spiked with RF. The detection limits achieved in urine samples are encouraging compared to those obtained using chromatographic approaches. The relative prediction errors were lower than 5.6%.
Immune thrombocytopenia is an acquired autoimmune disorder, which can affect both adults and children, characterized by lower than normal platelet counts (below 100 × 109/l). Thrombocytopenia may ...result in an increased risk of bleeding and puts patients at risk for serious complications. In the last decade, the multifactorial pathogenesis of ITP has become apparent, leading to greater understanding that different immune-mediated mechanisms could be involved in each patient, explaining the variable clinical presentation and response to therapies. The management of ITP patients has changed considerably in these past 10 years, but diagnosis of the disease has changed little, and remains clinical and possible only with the exclusion of other causes of thrombocytopenia. Although the search for such a test continues, to date, there is no reliable biomarker or gold-standard diagnostic test, which contributes to the high rate of misdiagnosis of the disease. This review presents the current limitations in the identification of the molecular disease underlying this disorder.
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