Utility of human epididymis protein 4 in the differential diagnosis of ascites.

BACKGROUND AND AIMS: Human epididymal protein 4 (HE4) may be a useful tool in the differential diagnosis of malignant ascites. The aim of this study was to evaluate the diagnostic utility of HE4 for detecting malignant ascites, taking into account the possible false positives identified with adenosine deaminase (ADA), C-reactive protein (CRP), % polynuclear cells (%PMN) and glomerular filtration rate (eGFR). METHODS: Concentrations of HE4, ADA, %PMN and CRP were determined in 114 samples of peritoneal fluid and creatinine in serum in order to calculate eGFR. RESULTS: Concentrations of HE4 presented significant differences (P = 0.028) in benign [median (interquartile range)] [582(372)] pmol/L) and malignant ascites ([8241(367)] pmol/L. Sensitivity was 21.2% and specificity 100%. Significant differences were also observed for HE4 between tumors of gynecological origin ([3165(8769)] pmol/L) and others ([665(663)] pmol/L), with a sensitivity of 67% and a specificity of 100%. Classifying according to possible false positives (ADA > 45U/L, CRP > 50 mg/L, %PMN > 90 and eGFR < 30 mL/min/1.73 m2) at maximum specificity, a sensitivity of 33.3% was obtained for HE4, with a cut-off point of 2660 pmol/L. Without possible false positives (ADA < 45U/L, CRP < 50 mg/L, %PMN < 90 and eGFR ≥ 30 mL/min/1.73 m2), a sensitivity of 37.7% was obtained at 100% specificity for a cut-off point of 1041 pmol/L. Applying these criteria to the entire group, a sensitivity of 36.4% was obtained at maximum specificity. CONCLUSIONS: HE4 allows the identification of malignant ascites with moderate sensitivity at maximum specificity. HE4 levels can differentiate between tumors of gynecological origin and others. Classification according to possible false positives increases sensitivity without losing specificity.

Medication Assessment in an Older Population during Acute Care Hospitalization and Its Effect on the Anticholinergic Burden: A Prospective Cohort Study.

(1) Background: Anticholinergic and sedative drugs (ASDs) contribute to negative health outcomes, especially in the frail population. In this study, we aimed to assess whether frailty increases with anticholinergic burden and to evaluate the effects of medication reviews (MRs) on ASD regimens among patients attending an acute care for the elderly (ACE) unit. (2) Methods: A cohort study was conducted between June 2019 and October 2020 with 150 consecutive patients admitted to our ACE unit. Demographic, clinical, and pharmacological data were assessed. Frailty score was determined using the Frail-VIG index (FI-VIG), and ASD burden was quantified using the drug burden index (DBI). In addition, the MR was performed using the patient-centered prescription (PCP) model. We used a paired T-test to compare the DBI pre- and post-MR and univariate and multivariate regression to identify the factors associated with frailty. (3) Results: Overall, 85.6% (n = 128) of participants showed some degree of frailty (FI-VIG > 0.20) and 84% (n = 126) of patients received treatment with ASDs upon admission (pre-MR). As the degree of frailty increased, so did the DBI (p < 0.001). After the implementation of the MR through the application of the PCP model, a reduction in the DBI was noted (1.06 ± 0.8 versus 0.95 ± 0.7) (p < 0.001). After adjusting for covariates, the association between frailty and the DBI was apparent (OR: 11.42, 95% (CI: 2.77-47.15)). (4) Conclusions: A higher DBI was positively associated with frailty. The DBI decreased significantly in frail patients after a personalized MR. Thus, MRs focusing on ASDs are crucial for frail older patients.

Autologous Cultured Bone Marrow-Derived Mesenchymal Stem Cells in a Fibrin Spray to Treat Venous Ulcers: A Randomized Controlled Double-Blind Pilot Study.

We treated a small cohort of venous ulcers that were very unresponsive to standard and advanced therapies with autologous cultured bone marrow-derived mesenchymal stem cells (MSCs). This pilot clinical trial was randomized, controlled, and double-blinded. Subjects were treated with either normal saline (Group A), fibrin spray alone (Group B), or MSCs in fibrin (1 million cells/cm2 of wound bed surface) (Group C). The control and test materials were applied to the wound using a double-barreled syringe with thrombin and fibrinogen (with or without MSCs) in each barrel, or saline alone in both barrels. The MSCs were separated, cultured in vitro, and expanded in a dedicated Good Manufacturing Practice (GMP) facility from 30-50 ml of bone marrow aspirate obtained from the iliac crest in Group C subjects. To ensure that the study remained controlled and blinded, subjects who were randomized to one of the two control arms (saline or fibrin) underwent sham bone marrow aspiration performed by a hematologist who anesthetized the iliac crest area down to and pushing against the periosteum, but without penetrating the bone marrow. Therefore, both the clinician who evaluated wound progress and the study subjects had no knowledge of whether bone aspiration was actually performed and what treatment had been applied to the wound. The study was performed after full FDA investigational new drug (IND) approval. The primary endpoint was the rate of healing (wound closure as linear healing from the wound margins in cm/week), as measured by the Gilman equation. One-way ANOVA was used to calculate the statistical significance of differences between the mean healing rates of each of the 3 treatment groups every 4 weeks and over the 24 weeks of treatment. Overall, treatment with MSCs accelerated the healing rate by about 10-fold compared to those in the saline and fibrin control groups. Although the total number of patients in this pilot study was small (n=11), the statistical significance was surprisingly promising: p<0.01 and f-ratio of 15.9358. No serious adverse events were noted. This small but carefully performed prospective, controlled, randomized, and double-blinded pilot study in a rare population of totally unresponsive patients adds to previous reports showing the promise of MSCs in the treatment of chronic wounds and provides proof of principle for how to approach this type of very demanding clinical and translational research.

Convolutional Neural Network for Skin Lesion Classification: Understanding the Fundamentals Through Hands-On Learning.

Deep learning architectures for the classification of images have shown outstanding results in a variety of disciplines, including dermatology. The expectations generated by deep learning for, e.g., image-based diagnosis have created the need for non-experts to become familiar with the working principles of these algorithms. In our opinion, getting hands-on experience with these tools through a simplified but accurate model can facilitate their understanding in an intuitive way. The visualization of the results of the operations performed by deep learning algorithms on dermatological images can help students to grasp concepts like convolution, even without an advanced mathematical background. In addition, the possibility to tune hyperparameters and even to tweak computer code further empower the reach of an intuitive comprehension of these processes, without requiring advanced computational and theoretical skills. This is nowadays possible thanks to recent advances that have helped to lower technical and technological barriers associated with the use of these tools, making them accessible to a broader community. Therefore, we propose a hands-on pedagogical activity that dissects the procedures to train a convolutional neural network on a dataset containing images of skin lesions associated with different skin cancer categories. The activity is available open-source and its execution does not require the installation of software. We further provide a step-by-step description of the algorithm and of its functions, following the development of the building blocks of the computer code, guiding the reader through the execution of a realistic example, including the visualization and the evaluation of the results.

Research Techniques Made Simple: Deep Learning for the Classification of Dermatological Images.

Deep learning is a branch of artificial intelligence that uses computational networks inspired by the human brain to extract patterns from raw data. Development and application of deep learning methods for image analysis, including classification, segmentation, and restoration, have accelerated in the last decade. These tools have been progressively incorporated into several research fields, opening new avenues in the analysis of biomedical imaging. Recently, the application of deep learning to dermatological images has shown great potential. Deep learning algorithms have shown performance comparable with humans in classifying skin lesion images into different skin cancer categories. The potential relevance of deep learning to the clinical realm created the need for researchers in disciplines other than computer science to understand its fundamentals. In this paper, we introduce the basics of a deep learning architecture for image classification, the convolutional neural network, in a manner accessible to nonexperts. We explain its fundamental operation, the convolution, and describe the metrics for the evaluation of its performance. These concepts are important to interpret and evaluate scientific publications involving these tools. We also present examples of recent applications for dermatology. We further discuss the capabilities and limitations of these artificial intelligence-based methods.

Research Techniques Made Simple: Analysis of Collective Cell Migration Using the Wound Healing Assay.

Collective cell migration is a hallmark of wound repair, cancer invasion and metastasis, immune responses, angiogenesis, and embryonic morphogenesis. Wound healing is a complex cellular and biochemical process necessary to restore structurally damaged tissue. It involves dynamic interactions and crosstalk between various cell types, interaction with extracellular matrix molecules, and regulated production of soluble mediators and cytokines. In cutaneous wound healing, skin cells migrate from the wound edges into the wound to restore skin integrity. Analysis of cell migration in vitro is a useful assay to quantify alterations in cell migratory capacity in response to experimental manipulations. Although several methods exist to study cell migration (such as Boyden chamber assay, barrier assays, and microfluidics-based assays), in this short report we will explain the wound healing assay, also known as the "in vitro scratch assay" as a simple, versatile, and cost-effective method to study collective cell migration and wound healing.

An in vitro priming step increases the expression of numerous epidermal growth and migration mediators in a tissue-engineering construct.

An FDA-approved, prototypic, living, bilayered skin construct (BSC) has been used for non-healing wounds. Using this particular construct as proof of principle, we hypothesized that an in vitro 'priming' step may enhance its repertoire of expression of key mediators and genes. The priming step used here was incubation in Dulbecco's modified Eagle's medium (DMEM) for 24 h at 37°C and 5% CO2 , with or without construct meshing. Microarray and ingenuity pathway analysis (IPA) showed that >1000 genes were overexpressed by the priming step, including interleukin 6 (IL-6), which plays important roles in wound healing. Genes highly overexpressed by priming were those involved in epidermal proliferation and migration. Quantitative real-time PCR (qRT-PCR), immunostaining and western blots verified the results. An epiboly assay (epidermal migration over dermis) showed that BSC epiboly was inhibited by IL-6 neutralizing antibody. Back wounds of nude mice were treated with primed or control BSCs for 3 days prior to harvesting; primed BSCs showed a significantly (p = 0.006) greater level of epidermal migration vs unprimed. Our study demonstrates that an in vitro priming step induces wound healing-related genes in the BSC, leading to a construct that could prove more effective in stimulating wound healing. Copyright © 2014 John Wiley & Sons, Ltd.

Sterol regulatory element binding proteins downregulate LDL receptor-related protein (LRP1) expression and LRP1-mediated aggregated LDL uptake by human macrophages.

OBJECTIVE: In the extracellular intima, extracellular matrix proteoglycans favor LDL retention and aggregation (agLDL). In contrast to native LDL (nLDL), agLDL induces high intracellular cholesteryl ester (CE) accumulation in macrophages. It has been suggested that LDL receptor-related protein (LRP1) is involved in agLDL binding and internalization by macrophages. The aim of this work was to analyze whether sterol regulatory element binding proteins (SREBPs) modulate LRP1 expression and LRP1-mediated agLDL uptake by human monocyte-derived macrophages (HMDM). METHODS AND RESULTS: The treatment of HMDM with small anti-LRP1 interfering RNA (siRNA-LRP1) led to the specific inhibition of LRP1 mRNA expression and also to the inhibition of LRP1 protein expression in these cells. In siRNA-LRP1-treated HMDM, CE accumulation from agLDL uptake (84.66+/-5 microg CE/mg protein) was reduced by 95.76+/-5.22%. This suggests that LRP1 plays a pivotal role in agLDL uptake by HMDM. N-acetyl-leucyl-leucyl-norleucinal (ALLN), an inhibitor of SREBP catabolism, maintained high levels of active SREBP-2 and SREBP-1 even in the presence of nLDL and agLDL. Therefore, ALLN induced LDL receptor (LDLR) upregulation. Concomitantly, a strong downregulation of LRP1 mRNA and LRP1 protein was observed in ALLN-treated macrophages. By decreasing LRP1 expression levels, ALLN reduced CE accumulation from agLDL at all tested concentrations. CONCLUSIONS: These results suggest that high levels of active SREBPs downregulate LRP1 expression and intracellular CE accumulation in HMDM.

Aggregated low-density lipoprotein uptake induces membrane tissue factor procoagulant activity and microparticle release in human vascular smooth muscle cells.

BACKGROUND: Tissue factor (TF) is the main initiator of the arterial blood coagulation system, and aggregated LDL (agLDL) are found in the arterial intima. Our hypothesis is that agLDL internalization by vascular smooth muscle cells (VSMCs) may trigger TF-procoagulant activity. METHODS AND RESULTS: Cultured human VSMCs were obtained from human coronary arteries of explanted hearts during transplant operations. VSMCs were incubated with native LDL (nLDL) or agLDL. TF mRNA was analyzed by real-time polymerase chain reaction, and cellular and released TF protein antigen were analyzed by Western blot. TF microparticle (MP) content was analyzed by flow cytometry and TF activity by a factor Xa generation test. Both nLDL and agLDL strongly and equally increased TF mRNA and cell membrane protein expression, by approximately 5- and 9-fold, respectively. A sustained TF procoagulant activity was induced by agLDL but not by nLDL (agLDL 2.46+/-0.22 versus nLDL 0.72+/-0.12 mU/mg protein at 12 hours). AgLDL increased TF antigen release (agLDL 5.64+/-0.4 versus nLDL 3.28+/-0.22 AU) and TF MP release (agLDL 89.85+/-8.51 versus nLDL 19.69+/-4.59 TF MP/10(3) cells). TF activation and release induced by agLDL is not related to apoptosis. Blockade of LDL receptor-related protein, a receptor for agLDL, prevented the agLDL-induced release of TF protein and TF MP. CONCLUSIONS: VSMC-TF expression is upregulated by both nLDL and agLDL. However, only agLDL engagement to LDL receptor-related protein induced cellular TF procoagulant activity and TF release by human VSMCs.