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Biomedicines
Volume 10
Issue 2
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Biomedicines, Volume 10, Issue 2 (February 2022) – 318 articles

Cover Story (view full-size image): While patients with an early or locally advanced breast cancer can be cured, the metastatic disease remains a challenge and so far, palliative treatment is the only option. Metastasis formation is a critical step in breast cancer development. It involves activation of miscellaneous processes, including activation of angiogenesis, vasculogenesis, chemotaxis or coagulation. These are essential for subsequent events occurring during systemic cancer spread, including the escaping of tumor cells from the primary tumor, migration through the basement membrane, extracellular matrix and vascular walls into the circulation, and extravasation and homing to the target organs, finally resulting in growth of secondary tumors. Understanding the pathogenesis of breast cancer metastasis formation is crucial for development of new treatment strategies. View this paper
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12 pages, 5675 KiB  
Article
Ribose and Non-Ribose A2A Adenosine Receptor Agonists: Do They Share the Same Receptor Recognition Mechanism?
by Giovanni Bolcato, Matteo Pavan, Davide Bassani, Mattia Sturlese and Stefano Moro
Biomedicines 2022, 10(2), 515; https://doi.org/10.3390/biomedicines10020515 - 21 Feb 2022
Cited by 14 | Viewed by 3197
Abstract
Adenosine receptors have been a promising class of targets for the development of new therapies for several diseases. In recent years, a renewed interest in this field has risen, thanks to the implementation of a novel class of agonists that lack the ribose [...] Read more.
Adenosine receptors have been a promising class of targets for the development of new therapies for several diseases. In recent years, a renewed interest in this field has risen, thanks to the implementation of a novel class of agonists that lack the ribose moiety, once considered essential for the agonistic profile. Recently, an X-ray crystal structure of the A2A adenosine receptor has been solved, providing insights about the receptor activation from this novel class of agonists. Starting from this structural information, we have performed supervised molecular dynamics (SuMD) simulations to investigate the binding pathway of a non-nucleoside adenosine receptor agonist as well as one of three classic agonists. Furthermore, we analyzed the possible role of water molecules in receptor activation. Full article
(This article belongs to the Section Drug Discovery, Development and Delivery)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Binding modes of adenosine (<b>left</b>) and of LUF5833 (<b>right</b>) as observed in X-ray crystal structures (PDB code 2YDO and 7ARO, respectively). Please note that the AR used to obtain the crystal structure with LUF5833 present some thermostabilizing mutations, including Ser-277.Other AR agonists, such as CGS21680 and NECA, also interact with His-250 and Thr-88. The binding mode of these two ligands can be found in <a href="#biomedicines-10-00515-f002" class="html-fig">Figure 2</a>.</p> Full article ">Figure 2
<p>The crystallographic binding mode (green) and the final pose obtained using SuMD (orange) for the four agonists used in this study ((<b>A</b>): LUF5833; (<b>B</b>): NECA, (<b>C</b>): Adenosine; (<b>D</b>): CGS21680).</p> Full article ">Figure 3
<p>Per-residue energetic analysis of the SuMD trajectory of CGS21680.</p> Full article ">Figure 4
<p>Per-residue energetic analysis of the SuMD trajectory of adenosine.</p> Full article ">Figure 5
<p>Per-residue energetic analysis of the SuMD trajectory of NECA.</p> Full article ">Figure 6
<p>Per-residue energetic analysis of the SuMD trajectory of LUF5833.</p> Full article ">Figure 7
<p>In the upper part of the figure, the protein surface is colored according to the number of contacts with the ligand during the trajectory (scale white to red, from less contacted to more contacted residues). In the lower part, the SuMD trajectory is displayed superposing each frame. (<b>A</b>): LUF5833; (<b>B</b>): NECA; (<b>C</b>): Adenosine; (<b>D</b>): CGS21680.</p> Full article ">Figure 8
<p>Results of the AquaMMapS analysis for the “APO” trajectory (<b>left</b>) and for the prolonged SuMD trajectory of LUF5833 (<b>right</b>). The cells where the water molecules have an occupancy value higher than 25% are displayed as spheres coloured according to the occupancy value (from white to blue).</p> Full article ">
25 pages, 2808 KiB  
Review
Parthenolide and Its Soluble Analogues: Multitasking Compounds with Antitumor Properties
by Daniela Carlisi, Marianna Lauricella, Antonella D’Anneo, Anna De Blasio, Adriana Celesia, Giovanni Pratelli, Antonietta Notaro, Giuseppe Calvaruso, Michela Giuliano and Sonia Emanuele
Biomedicines 2022, 10(2), 514; https://doi.org/10.3390/biomedicines10020514 - 21 Feb 2022
Cited by 26 | Viewed by 4773
Abstract
Due to its chemical properties and multiple molecular effects on different tumor cell types, the sesquiterpene lactone parthenolide (PN) can be considered an effective drug with significant potential in cancer therapy. PN has been shown to induce either classic apoptosis or alternative caspase-independent [...] Read more.
Due to its chemical properties and multiple molecular effects on different tumor cell types, the sesquiterpene lactone parthenolide (PN) can be considered an effective drug with significant potential in cancer therapy. PN has been shown to induce either classic apoptosis or alternative caspase-independent forms of cell death in many tumor models. The therapeutical potential of PN has been increased by chemical design and synthesis of more soluble analogues including dimethylaminoparthenolide (DMAPT). This review focuses on the molecular mechanisms of both PN and analogues action in tumor models, highlighting their effects on gene expression, signal transduction and execution of different types of cell death. Recent findings indicate that these compounds not only inhibit prosurvival transcriptional factors such as NF-κB and STATs but can also determine the activation of specific death pathways, increasing intracellular reactive oxygen species (ROS) production and modifications of Bcl-2 family members. An intriguing property of these compounds is its specific targeting of cancer stem cells. The unusual actions of PN and its analogues make these agents good candidates for molecular targeted cancer therapy. Full article
(This article belongs to the Special Issue Biomarkers and New Therapeutical Strategies for Cancer Diagnosis and Treatment)
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Figure 1

Figure 1
<p>Chemical structures of (<b>A</b>) parthenolide (PN) and its analogue (<b>B</b>) dimethylaminoparthenolide (DMAPT).</p> Full article ">Figure 2
<p>Effects of PN on prosurvival/oncogenic transcriptional factors and relative signaling: (<b>A</b>) NF-κB; (<b>B</b>) JAK/STAT; (<b>C</b>) β-catenin.</p> Full article ">Figure 3
<p>The effects of PN on different signaling cascades.</p> Full article ">Figure 4
<p>Different types of cell death induced by PN treatment.</p> Full article ">Figure 5
<p>Mechanisms of oxidative stress induction by PN.</p> Full article ">Figure 6
<p>Chemical structures of (<b>A</b>) arglabin, (<b>B</b>) micheliolide (MCL) and its analogue (<b>C</b>) ACT001.</p> Full article ">
31 pages, 4417 KiB  
Article
Single Application of Low-Dose, Hydroxyapatite-Bound BMP-2 or GDF-5 Induces Long-Term Bone Formation and Biomechanical Stabilization of a Bone Defect in a Senile Sheep Lumbar Osteopenia Model
by Ines Hasenbein, André Sachse, Peter Hortschansky, Klaus D. Schmuck, Victoria Horbert, Christoph Anders, Thomas Lehmann, René Huber, Alexander Maslaris, Frank Layher, Christina Braun, Andreas Roth, Frank Plöger and Raimund W. Kinne
Biomedicines 2022, 10(2), 513; https://doi.org/10.3390/biomedicines10020513 - 21 Feb 2022
Cited by 8 | Viewed by 3533
Abstract
Effects of hydroxyapatite (HA) particles with bone morphogenetic BMP-2 or GDF-5 were compared in sheep lumbar osteopenia; in vitro release in phosphate-buffered saline (PBS) or sheep serum was assessed by ELISA. Lumbar (L) vertebral bone defects (Ø 3.5 mm) were generated in aged, [...] Read more.
Effects of hydroxyapatite (HA) particles with bone morphogenetic BMP-2 or GDF-5 were compared in sheep lumbar osteopenia; in vitro release in phosphate-buffered saline (PBS) or sheep serum was assessed by ELISA. Lumbar (L) vertebral bone defects (Ø 3.5 mm) were generated in aged, osteopenic female sheep (n = 72; 9.00 ± 0.11 years; mean ± SEM). Treatment was: (a) HA particles (2.5 mg; L5); or (b) particles coated with BMP-2 (1 µg; 10 µg) or GDF-5 (5 µg; 50 µg; L4; all groups n = 6). Untouched vertebrae (L3) served as controls. Three and nine months post-therapy, bone formation was assessed by osteodensitometry, histomorphometry, and biomechanical testing. Cumulative 14-day BMP release was high in serum (76–100%), but max. 1.4% in PBS. In vivo induction of bone formation by HA particles with either growth factor was shown by: (i) significantly increased bone volume, trabecular and cortical thickness (overall increase HA + BMP vs. control close to the injection channel 71%, 110%, and 37%, respectively); (ii) partial significant effects for bone mineral density, bone formation, and compressive strength (increase 17%; 9 months; GDF-5). Treatment effects were not dose-dependent. Combined HA and BMPs (single low-dose) highly augment long-term bone formation and biomechanical stabilization in sheep lumbar osteopenia. Thus, carrier-bound BMP doses 20,000-fold to 1000-fold lower than previously applied appear suitable for spinal fusion/bone regeneration and improved treatment safety. Full article
(This article belongs to the Special Issue Regenerative Medicine for Bone and Cartilage and Pathophysiology of Degenerative Diseases)
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Figure 1

Figure 1
<p>Representative image of OTC fluorescence double lines (arrows) at the interface between a bone trabecula (T) and the bone marrow (BM) close to the injection channel for the long-term, high-dose BMP-2 group.</p> Full article ">Figure 2
<p>In vitro evaluation of the dosages for BMP-2 (C2C12 cell line; upper panel) and GDF-5 (ATDC-5 cell line; lower panel) for application in animal experiments by direct coculture with the growth-factor-coated HA particles for 3 days and subsequent determination of their alkaline phosphatase (ALP) activity (<span class="html-italic">n</span> = 3–4 experiments each; one representative result is shown; y axes show the raw values of chemiluminescent reading). Derived therapeutic doses were 1 µg BMP-2/2.5 mg HA (low dose, lowest dose with a detectable effect; pharmaceutical formulation 2 µg BMP-2/5 mg HA; in vivo application of ½ dose) or 10 µg BMP-2/2.5 mg HA (high dose; half-maximal effect). Due to the presumably lower biological activity of GDF-5, doses for GDF-5 were 5 µg (low dose; half-maximal effect) and 50 µg (high dose; saturation effect).</p> Full article ">Figure 3
<p>In vitro release per day (respective broken lines; left axis) and cumulative release (respective solid lines; right axis) of the two different doses of BMP-2 ((<b>A</b>); squares for 2 µg, circles for 20 µg) or GDF-5 ((<b>B</b>); squares for 10 µg, circles for 100 µg) from the therapeutically applied hydroxyapatite (HA) particles in PBS; release and retention (%) of BMP-2 (<b>C</b>) or GDF-5 (<b>D</b>) in PBS; in vitro release per day and cumulative release of BMP-2 (<b>E</b>) or GDF-5 (<b>F</b>) from the therapeutically applied HA particles in sheep serum; release and retention (%) of BMP-2 (<b>G</b>) or GDF-5 (<b>H</b>) in sheep serum.</p> Full article ">Figure 4
<p>Light field (<b>A</b>,<b>B</b>,<b>D</b>) and phase contrast histology images (<b>C</b>,<b>E</b>) of therapeutically injected hydroxyapatite (HA) particles (see arrows in (<b>C</b>,<b>E</b>)) in plastic-embedded sections of lumbar vertebral bodies (L4 = HA/BMP-2; 10 μg; 3 months); trichrome stain according to Masson-Goldner, staining bone trabeculae including newly formed bone, as well as corticalis in blue, bone marrow including hematopoietic cells and fat marrow in brown/white, and HA particles in contrasted white (see arrows); (<b>F</b>) macro X-ray of the injected vertebral body demonstrating the placement of the injection channel (white arrow) and the spongiosa cylinder harvested for biomechanical analysis (red circle).</p> Full article ">Figure 5
<p>Paraffin sections of lumbar vertebral bodies from long-term animals (9 months) injected with either 10 µg BMP-2 (high dose; (<b>A</b>–<b>D</b>)) or with 5 µg GDF-5 (low dose; (<b>E</b>–<b>H</b>); for a low magnification overview of the injection channel in the vertebral body please compare with <a href="#biomedicines-10-00513-f004" class="html-fig">Figure 4</a>). Hematoxylin-eosin stain, staining bone trabeculae including newly formed bone with embedded osteocytes in red and bone marrow including hematopoietic cells and fat marrow in purple/white; (<b>A</b>) control (L3); (<b>B</b>) HA-injected vertebral body (L5; injection channel); (<b>C</b>) HA/BMP-2-injected vertebral body (L4; injection channel); (<b>D</b>) HA/BMP-2-injected vertebral body (L4; remote area); (<b>E</b>) control (L3); (<b>F</b>) HA-injected vertebral body (L5; injection channel); (<b>G</b>) HA/GDF-5-injected vertebral body (L4; injection channel); (<b>H</b>) HA/GDF-5-injected vertebral body (L4; remote area).</p> Full article ">Figure 6
<p>Increase of bone volume (%) in paraffin sections of lumbar vertebral bodies (L3 = control; L4 = HA/BMP; L5 = HA) of all investigated groups in the vicinity of the injection channel (<b>A</b>,<b>B</b>) and in the remote area (<b>C</b>,<b>D</b>). Results of the mixed linear model analysis (compare with <a href="#biomedicines-10-00513-t002" class="html-table">Table 2</a> and <a href="#app1-biomedicines-10-00513" class="html-app">Table S2</a>); *** <span class="html-italic">p</span> &lt; 0.001, * <span class="html-italic">p</span> ≤ 0.05 for HA compared to non-injected control; ### <span class="html-italic">p</span> &lt; 0.001, # <span class="html-italic">p</span> ≤ 0.05 for HA/BMP compared to non-injected control; and +++ <span class="html-italic">p</span> &lt; 0.001, + <span class="html-italic">p</span> ≤ 0.05 for HA/BMP compared to HA; &amp;&amp;&amp; <span class="html-italic">p</span> &lt; 0.001 for 9 months compared to 3 months. Please note: The linear mixed effects model was used to analyze global effects and overall interactions of the main effects treatment (group), time point (time), BMP dose (dose), and distance from the injection channel (area); subsequently, the non-parametric multigroup Kruskal–Wallis and two-sided Wilcoxon tests were applied for the comparison of paired samples within one group, and the multigroup Friedman and two-sided Mann-Whitney U-tests for statistical evaluation of differences between groups with the above-mentioned significance levels.</p> Full article ">Figure 7
<p>Increase of bone volume (%) in paraffin sections of lumbar vertebral bodies (L3 = control; L4 = HA/BMP; L5 = HA) of selected BMP-2 and GDF-5 groups in the vicinity of the injection channel. n.s. = not significant; <span class="html-italic">p</span> ≤ 0.05 Wilcoxon test, * for HA compared to non-injected control; # for HA/BMP compared to non-injected control; and + for HA/BMP compared to HA; brackets above the bars indicate the direct statistical comparison of the respective vertebral bodies in the BMP-2 and GDF-5 groups (§ <span class="html-italic">p</span> ≤ 0.01 U-test).</p> Full article ">Figure 8
<p>Increase of trabecular thickness (µm) in paraffin sections of lumbar vertebral bodies (L3 = control, L4 = HA/BMP; L5 = HA) of all investigated groups in the injection channel (<b>A</b>,<b>B</b>) and the remote area (<b>C</b>,<b>D</b>). Results of the mixed linear model analysis (compare with <a href="#biomedicines-10-00513-t002" class="html-table">Table 2</a> and <a href="#app1-biomedicines-10-00513" class="html-app">Table S2</a>); *** <span class="html-italic">p</span> &lt; 0.001, * <span class="html-italic">p</span> ≤ 0.05 for HA compared to non-injected control; ### <span class="html-italic">p</span> &lt; 0.001, ## <span class="html-italic">p</span> ≤ 0.01, # <span class="html-italic">p</span> ≤ 0.05 for HA/BMP compared to non-injected control; and +++ <span class="html-italic">p</span> &lt; 0.001, ++ <span class="html-italic">p</span> ≤ 0.01, + <span class="html-italic">p</span> ≤ 0.05 for HA/BMP compared to HA; &amp;&amp;&amp; <span class="html-italic">p</span> &lt; 0.001 for 9 months compared to 3 months. Please note: The linear mixed effects model was used to analyze global effects and overall interactions of the main effects treatment (group), time point (time), BMP dose (dose), and distance from the injection channel (area); subsequently, the non-parametric multigroup Kruskal-Wallis and two-sided Wilcoxon tests were applied for the comparison of paired samples within one group, and the multigroup Friedman and two-sided Mann-Whitney U-tests for statistical evaluation of differences between groups with the above-mentioned significance levels.</p> Full article ">Figure 9
<p>Increase of cortical thickness (µm) in paraffin sections of lumbar vertebral bodies (L3 = control, L4 = HA/BMP; L5 = HA) of all investigated groups in the ventral corticalis close to the tip of the injection channel (<b>A</b>,<b>B</b>) and the right corticalis (<b>C</b>,<b>D</b>). Results of the mixed linear model analysis (compare with <a href="#biomedicines-10-00513-t002" class="html-table">Table 2</a> and <a href="#app1-biomedicines-10-00513" class="html-app">Table S2</a>); *** <span class="html-italic">p</span> &lt; 0.001, * <span class="html-italic">p</span> ≤ 0.05 for HA compared to non-injected control; ### <span class="html-italic">p</span> &lt; 0.001, ## <span class="html-italic">p</span> ≤ 0.01, # <span class="html-italic">p</span> ≤ 0.05 for HA/BMP compared to non-injected control; and +++ <span class="html-italic">p</span> &lt; 0.001, ++ <span class="html-italic">p</span> ≤ 0.01, + <span class="html-italic">p</span> ≤ 0.05 for HA/BMP compared to HA. Please note: The linear mixed effects model was used to analyze global effects and overall interactions of the main effects treatment (group), time point (time), BMP dose (dose), and distance from the injection channel (area); subsequently, the non-parametric multigroup Kruskal-Wallis and two-sided Wilcoxon tests were applied for the comparison of paired samples within one group, and the multigroup Friedman and two-sided Mann-Whitney U-tests for statistical evaluation of differences between groups with the above-mentioned significance levels.</p> Full article ">Figure 10
<p>Relative increase of compressive strength (MPa; expressed as % of control) in lumbar vertebral bodies (L3 = control, L4 = HA/BMP; L5 = HA) of four selected groups (3 and 9 months; low-dose BMP-2 (<b>A</b>) or GDF-5 (<b>B</b>)); <span class="html-italic">p</span> ≤ 0.05 Wilcoxon test; # for HA/BMP compared to non-injected control; the HA/GDF-5 particles induced a numerical 14% or significant 17% increase at 3 and 9 months, respectively.</p> Full article ">
11 pages, 689 KiB  
Article
Comparative Study of the Aftereffect of CO2 Inhalation or Tiletamine–Zolazepam–Xylazine Anesthesia on Laboratory Outbred Rats and Mice
by Oksana N. Khokhlova, Natalya A. Borozdina, Elena S. Sadovnikova, Irina A. Pakhomova, Pavel A. Rudenko, Yuliya V. Korolkova, Sergey A. Kozlov and Igor A. Dyachenko
Biomedicines 2022, 10(2), 512; https://doi.org/10.3390/biomedicines10020512 - 21 Feb 2022
Cited by 11 | Viewed by 2865
Abstract
CO2 inhalation is currently the most common method of euthanasia for laboratory rats and mice, and it is often used for further terminal blood sampling for clinical biochemical assays. Lately, this method has been criticized due to animal welfare issues associated with [...] Read more.
CO2 inhalation is currently the most common method of euthanasia for laboratory rats and mice, and it is often used for further terminal blood sampling for clinical biochemical assays. Lately, this method has been criticized due to animal welfare issues associated with some processes that develop after CO2 inhalation. The stress reaction and the value of the clinical laboratory parameters significantly depend on the used anesthetics, method, and the site of blood sampling. Especially in small rodents, an acute terminal state followed by a cascade of metabolic reactions that can affect the studied biochemical profile may develop and cause unnecessary suffering of animals. The aim of this study was to compare the stability of biochemical parameters of outbred Sprague Dawley rats and CD-1 mice serum collected after CO2 inhalation or the intramuscular injection of tiletamine–zolazepam–xylazine (TZX). The serum content of total protein and albumin, cholesterol, triglycerides, aspartate aminotransferase (AST), alanine aminotr ansferase (ALT), alkaline phosphatase (ALP), total bilirubin, and creatinine was decreased by the injection of TZX in comparison with CO2 inhalation. In addition, the levels of calcium, phosphates, chlorides and potassium were lowered by TZX vs. CO2 administration, while the level of sodium increased. Finally, the level of the majority of serum clinical biochemical parameters in rats and mice tend to be overestimated after CO2 inhalation, which may lead to masking the possible effect of anti-inflammatory drugs in animal tests. Injection anesthesia for small rodents with TZX is a more feasible method for terminal blood sampling, which also reduces the suffering of animals. Full article
(This article belongs to the Topic Animal Model in Biomedical Research)
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Figure 1
<p>Blood glucose levels in males and females of CD-1 mice (<b>A</b>), and Sprague Dawley rats (<b>B</b>), in the awake state and following 5 min after CO<sub>2</sub> inhalation or tiletamine–zolazepam–xylazine (TZX) anesthesia. # <span class="html-italic">p</span> &lt; 0.05 TZX versus awake, * <span class="html-italic">p</span> &lt; 0.05 TZX versus CO<sub>2</sub>.</p> Full article ">
14 pages, 3273 KiB  
Article
Preventive Effects of a Human Hematopoietic Mesenchymal Stem Cell (hHMSC) Therapy in Ovalbumin-Induced Food Allergy
by Dong-Geon Lee, Yu-Jin Lee, Song-Hee Park, Hye-Ree Park, Hoon Kang and Jung-Eun Kim
Biomedicines 2022, 10(2), 511; https://doi.org/10.3390/biomedicines10020511 - 21 Feb 2022
Cited by 2 | Viewed by 2853
Abstract
No effective therapeutic strategies have been developed against food allergies. Immunomodulation during early infant period could prevent the development of food allergies. We investigated the preventive effects of human hematopoietic mesenchymal stem cells (hHMSCs) in mice with ovalbumin (OVA)-induced food allergy. BALB/c mice [...] Read more.
No effective therapeutic strategies have been developed against food allergies. Immunomodulation during early infant period could prevent the development of food allergies. We investigated the preventive effects of human hematopoietic mesenchymal stem cells (hHMSCs) in mice with ovalbumin (OVA)-induced food allergy. BALB/c mice with OVA-induced food allergy were divided into 3 groups, and each group was treated with hHMSCs or hHMSC culture medium (hHMSC-CM) or saline. Ear thickness, allergy score, rectal temperature, and diarrhea occurrence were checked. Total IgE, OVA-specific IgE, and mucosal mast cell protease-1 (mMCP-1) were measured by ELISA. Other allergic parameters were analyzed using histology specimens, RT-PCR, and flow cytometry. Treatment with hHMSCs or hHMSC-CM significantly suppressed the frequency of anaphylactic response and rectal temperature decline, reduced diarrhea, total IgE, OVA-specific IgE, and mMCP-1. While the treatment decreased the level of Th2 cytokines, it enhanced IL-10 and TGF-β1 mRNA. Exposure to hHMSC or hHMSC-CM did not generate regulatory T cells, but reduced mast cells. The immunomodulatory effect on the Th2 cytokines was greater in hHMSC-CM than in hHMSCs. hHMSC treatment may be a promising preventive intervention against food allergy. Further studies are needed to elucidate the key substances released from hHMSC to induce immune tolerance. Full article
(This article belongs to the Special Issue New Insights in Gene and Cell Therapy)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Process of allergic sensitization to food in mice with OVA, MSC, or MSC CM is shown. MSC, mesenchymal stem cell; MSC CM, mesenchymal stem cell culture medium; OVA, Ovalbumin.</p> Full article ">Figure 2
<p>OVA challenge elevated immunoglobulin and mMCP-1 levels and MSC or MSC CM reversed the change. Concentrations of (<b>A</b>) total IgE, (<b>B</b>) OVA-specific IgE, (<b>C</b>) OVA-specific IgG, and (<b>D</b>) mMCP-1 in the serum measured by ELISA are shown. Each value indicates mean ± standard error of the mean. ## <span class="html-italic">p</span> &lt; 0.01 versus CTL, * <span class="html-italic">p</span> &lt; 0.05 versus OVA, ** <span class="html-italic">p</span> &lt; 0.01 versus OVA. CTL, control; Ig, immunoglobulin; mMCP, mucosal mast cell protease; MSC, mesenchymal stem cell; MSC CM, mesenchymal stem cell culture medium; OVA, Ovalbumin.</p> Full article ">Figure 3
<p>OVA challenge upregulated inflammatory cytokines and IL-10 transcription and MSC or MSC CM reversed the change in the ear skin. (<b>A</b>) IL-4, (<b>B</b>) IL-5, (<b>C</b>) IL-13, (<b>D</b>) IL-10, (<b>E</b>) TGF-β1, (<b>F</b>) IL-23α, and (<b>G</b>) IL-31 mRNA level changes in the ear skin measured by qPCR are shown. Each value indicates mean ± standard error of the mean. # <span class="html-italic">p</span> &lt; 0.05 versus CTL, ## <span class="html-italic">p</span> &lt; 0.01 versus CTL, * <span class="html-italic">p</span> &lt; 0.05 versus OVA, ** <span class="html-italic">p</span> &lt; 0.01 versus OVA. CTL, control; IL, interleukin; MSC, mesenchymal stem cell; MSC CM, mesenchymal stem cell culture medium; OVA, Ovalbumin; qPCR, quantitative polymerase chain reaction; TGF, transforming growth factor.</p> Full article ">Figure 4
<p>OVA challenge upregulated inflammatory cytokines and IL-10 transcription, downregulated TGF- β1 and MSC or MSC CM reversed the change in the SI. (<b>A</b>) IL-4, (<b>B</b>) IL-5, (<b>C</b>) IL-13, (<b>D</b>) IL-10, (<b>E</b>) TGF-β1, (<b>F</b>) IL-23α, (<b>G</b>) IL-31, (<b>H</b>) IL-12α, and (<b>I</b>) IFN-γ mRNA level changes in the SI measured by qPCR are shown. Each value indicates mean ± standard error of the mean. # <span class="html-italic">p</span> &lt; 0.05 versus CTL, * <span class="html-italic">p</span> &lt; 0.05 versus OVA, CTL, control; IL, interleukin; MSC, mesenchymal stem cell; MSC CM, mesenchymal stem cell culture medium; OVA, Ovalbumin; qPCR, quantitative polymerase chain reaction; SI, small intestine; TGF, transforming growth factor.</p> Full article ">Figure 5
<p>OVA challenge increased IgE+ c-kit+ mast cells and MSC or MSC CM reversed the change in the ear skin, mLN, and SI tissue. IgE+ c-kit+ mast cells in the (<b>A</b>) ear skin, (<b>B</b>) mLN, and (<b>C</b>) SI tissue measured by flow cytometry are shown. Mean percentage (mast cell counts/total cell counts) is shown in the right upper side. Ig, immunoglobulin; mLN, mesenteric lymph node; MSC, mesenchymal stem cell; MSC CM, mesenchymal stem cell culture medium; OVA, Ovalbumin; SI, small intestine.</p> Full article ">Figure 6
<p>OVA challenge increased CD4+ Foxp3+ T cells and MSC or MSC CM reversed the change in the ear skin, mLN, and SI tissue. CD4+ Foxp3+ T cells in the (<b>A</b>) ear skin, (<b>B</b>) mLN, and (<b>C</b>) SI tissue measured by flow cytometry are shown. Mean percentage (CD4+ Foxp3+ T cell counts/total cell counts) is shown in the right upper side. CD, cluster of differentiation; Fox, forkhead box; mLN, mesenteric lymph node; MSC, mesenchymal stem cell; MSC CM, mesenchymal stem cell culture medium; OVA, Ovalbumin; SI, small intestine.</p> Full article ">Figure 7
<p>OVA challenge induced inflammation and mast cell infiltration in the tissue and MSC or MSC CM attenuated the change in the ear skin, SI, and spleen. Histological changes of (<b>A</b>) ear skin, (<b>B</b>) SI, or (<b>C</b>) spleen are shown. Sections are stained with H&amp;E (hematoxylin and eosin, ear skin ×100, ×200, SI, ×100, ×200, spleen ×100) or Toluidine blue (×200). Mast cells are indicated by red arrows. H&amp;E, hematoxylin, and eosin; MSC, mesenchymal stem cell; MSC CM, mesenchymal stem cell culture medium; OVA, Ovalbumin; SI, small intestine.</p> Full article ">Figure 8
<p>No significant changes of villus/crypt length ratio were observed in the colon with OVA, MSC, or MSC CM. Villus/crypt length ratio of colon is shown. Each value indicates mean ± standard error of the mean. MSC, mesenchymal stem cell; MSC CM, mesenchymal stem cell culture medium; OVA, Ovalbumin.</p> Full article ">
16 pages, 802 KiB  
Review
Cigarette Smoking and Human Gut Microbiota in Healthy Adults: A Systematic Review
by Martina Antinozzi, Monica Giffi, Nicolò Sini, Francesca Gallè, Federica Valeriani, Corrado De Vito, Giorgio Liguori, Vincenzo Romano Spica and Maria Sofia Cattaruzza
Biomedicines 2022, 10(2), 510; https://doi.org/10.3390/biomedicines10020510 - 21 Feb 2022
Cited by 45 | Viewed by 6187
Abstract
The intestinal microbiota is a crucial regulator of human health and disease because of its interactions with the immune system. Tobacco smoke also influences the human ecosystem with implications for disease development. This systematic review aims to analyze the available evidence, until June [...] Read more.
The intestinal microbiota is a crucial regulator of human health and disease because of its interactions with the immune system. Tobacco smoke also influences the human ecosystem with implications for disease development. This systematic review aims to analyze the available evidence, until June 2021, on the relationship between traditional and/or electronic cigarette smoking and intestinal microbiota in healthy human adults. Of the 2645 articles published in PubMed, Scopus, and Web of Science, 13 were included in the review. Despite differences in design, quality, and participants’ characteristics, most of the studies reported a reduction in bacterial species diversity, and decreased variability indices in smokers’ fecal samples. At the phylum or genus level, the results are very mixed on bacterial abundance both in smokers and non-smokers with two exceptions. Prevotella spp. appears significantly increased in smokers and former smokers but not in electronic cigarette users, while Proteobacteria showed a progressive increase in Desulfovibrio with the number of pack-years of cigarette (p = 0.001) and an increase in Alphaproteobacteria (p = 0.04) in current versus never smokers. This attempt to systematically characterize the effects of tobacco smoking on the composition of gut microbiota gives new perspectives on future research in smoking cessation and on a new possible use of probiotics to contrast smoke-related dysbiosis. Full article
(This article belongs to the Section Immunology and Immunotherapy)
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<p>PRISMA 2020 flow diagram for study selection.</p> Full article ">
16 pages, 1627 KiB  
Article
Age and Intrinsic Fitness Affect the Female Rotator Cuff Tendon Tissue
by Manuela Thierbach, Estelle Heyne, Michael Schwarzer, Lauren G. Koch, Steven L. Britton and Britt Wildemann
Biomedicines 2022, 10(2), 509; https://doi.org/10.3390/biomedicines10020509 - 21 Feb 2022
Cited by 3 | Viewed by 2518
Abstract
The risk of the development of tendon disorders or ruptures increases with age, but it is unclear whether intrinsic fitness during lifetime might also affect tendon properties. To investigate this, a contrasting rat model of high-capacity runners (HCR with high intrinsic fitness) and [...] Read more.
The risk of the development of tendon disorders or ruptures increases with age, but it is unclear whether intrinsic fitness during lifetime might also affect tendon properties. To investigate this, a contrasting rat model of high-capacity runners (HCR with high intrinsic fitness) and low-capacity runners (LCR with low intrinsic fitness) was employed. Histological and molecular changes in rotator cuff (RC) tendons from 10 weeks old (young; HCR-10 and LCR-10) and 100 weeks old (old; HCR-100 and LCR-100) female rats were investigated. Age-dependent changes of RC tendons observed in HCR and LCR were increase of weight, decrease of tenocytes and RNA content, reduction of the wavy pattern of collagen and elastic fibers, repressed expression of Col1a1, Eln, Postn, Tnmd, Tgfb3 and Egr1 and reduction of the Col1:Col3 and Col1:Eln ratio. The LCR rats showed less physical activity, increased body weight, signs of metabolic disease and a reduced life expectancy. Their RC tendons revealed increased weight (more than age-dependent) and enlargement of the tenocyte nuclei (consistent with degenerative tendons). Low intrinsic fitness led to repressed expression of a further nine genes (Col3a1, Fbn1, Dcn, Tnc, Scx, Mkx, Bmp1, Tgfb1, Esr1) as well as the rise of the Col1:Col3 and Col1:Eln ratios (related to the lesser expression of Col3a1 and Eln). The intrinsic fitness influences the female RC tendons at least as much as age. Lower intrinsic fitness accelerates aging of RC tendons and leads to further impairment; this could result in decreased healing potential and elasticity and increased stiffness. Full article
(This article belongs to the Special Issue 10th Anniversary of Biomedicines—Advances in Tendon and Bone Regeneration)
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<p>Body weight [g] (<b>A</b>), weight of the RC tendon [mg] (<b>B</b>), ratio of the RC tendon weight to body weight [mg/g] (<b>C</b>), and RNA content per tendon [ng/mg] (<b>D</b>) from HCR-10, HCR-100, LCR-10 and LCR-100. Significant differences (analyzed using the Kruskal–Wallis test followed by the Mann–Whitney U-test) are marked and the <span class="html-italic">p</span>-value displayed above. n = 7 per group.</p> Full article ">Figure 2
<p>Histology of supraspinatus tendons from HCR-10 (<b>A</b>), HCR-100 (<b>B</b>), LCR-10 (<b>C</b>) and LCR-100 (<b>D</b>) using Hematoxylin Eosin (HE) staining. The tendons of the young rats showed a dense and aligned collagen structure with high cellularity. In the older rats, the structure was less dense with reduced number of cells. Scale bar: 100 µm. As marked in (<b>A</b>), the bone is always at the lower left corner.</p> Full article ">Figure 3
<p>Histological staining of supraspinatus tendons of HCR-10 (<b>A</b>), HCR-100 (<b>B</b>), LCR-10 (<b>C</b>) and LCR-100 (<b>D</b>) using Movat Pentachrome (MP) staining. The number of elastic fibers and the wavy structure of the tendons of the young rats were reduced in the old tendons. Cell nuclei were stained in red; collagen in yellow and elastic fibers in red. Scale bar: 50 µm. The bone would be on the left side and the muscle on the right side of the tendon samples shown.</p> Full article ">Figure 4
<p>mRNA expression of ECM markers <span class="html-italic">Col1a1</span> (<b>A</b>), <span class="html-italic">Col3a1</span> (<b>B</b>), <span class="html-italic">Fn1</span> (<b>C</b>), <span class="html-italic">Eln</span> (<b>D</b>), <span class="html-italic">Fbn1</span> (<b>E</b>) and <span class="html-italic">Postn</span> (<b>F</b>) and Ratio of the NE <span class="html-italic">Col1a1</span>:<span class="html-italic">Col3a1</span> (<b>G</b>) as well as <span class="html-italic">Col1a1:Eln</span> (<b>H</b>) in HCR-10, HCR-100, LCR-10 and LCR-100. Results are normalized to 18S rRNA and are shown as individual dot plots. Significant differences (analyzed using the Kruskal–Wallis test followed by the Mann–Whitney U-test and Bonferroni–Holm correction) are marked and the <span class="html-italic">p</span>-value displayed above. n = 7 per group.</p> Full article ">Figure 5
<p>mRNA expression of the tenocyte markers <span class="html-italic">Mkx</span> (<b>A</b>), <span class="html-italic">Scx</span> (<b>B</b>), <span class="html-italic">Tnmd</span> (<b>C</b>) and the transcription factor <span class="html-italic">Egr1</span> (<b>D</b>). Results are normalized to 18S rRNA and are shown as individual dot plots. Significant differences (analyzed using the Kruskal–Wallis test followed by the Mann–Whitney U-test and Bonferroni–Holm correction) are marked and the <span class="html-italic">p</span>-value displayed above. n = 7 per group.</p> Full article ">Figure 6
<p>mRNA expression of the metalloprotease <span class="html-italic">Bmp1</span> (<b>A</b>), the cytokines <span class="html-italic">Tgfb1</span> (<b>B</b>), <span class="html-italic">Tgfb3</span> (<b>C</b>) and the sex hormone receptor <span class="html-italic">Esr1</span> (<b>D</b>). Results are normalized to 18S rRNA and are shown as individual dot plots. Significant differences (analyzed using the Kruskal–Wallis test followed by the Mann–Whitney U-test and Bonferroni–Holm correction) are marked and the <span class="html-italic">p</span>-value displayed above. n = 7 per group.</p> Full article ">Figure 7
<p>Effect of intrinsic fitness on gene expression. Relative gene expression of 18 genes in HCR-100 (<b>A</b>) and in LCR-100 (<b>B</b>) given as fold change to HCR-10 (horizontal line, mean value = 1). Significant differences (Mann–Whitney U-test) between HCR-10 and HCR-100 and between HCR-10 and LCR-100 are marked with * (<span class="html-italic">p</span> ≤ 0.05) or ** (<span class="html-italic">p</span> ≤ 0.005). The vertical arrows in (<b>B</b>) mark reduction in gene expression of ≥60% in comparison to (<b>A</b>).</p> Full article ">
13 pages, 781 KiB  
Article
Familial Predisposition to Leiomyomata: Searching for Protective Genetic Factors
by Maria V. Kuznetsova, Nelly S. Sogoyan, Andrew J. Donnikov, Dmitry Y. Trofimov, Leila V. Adamyan, Natalia D. Mishina, Jekaterina Shubina, Dmitry V. Zelensky and Gennady T. Sukhikh
Biomedicines 2022, 10(2), 508; https://doi.org/10.3390/biomedicines10020508 - 21 Feb 2022
Cited by 7 | Viewed by 2391
Abstract
In order to determine genetic loci associated with decreasing risk of uterine leiomyomata (UL), a genome-wide association study (GWAS) was performed. We analyzed a group of patients with a family history of UL and a control group consisting of patients without uterine fibroids [...] Read more.
In order to determine genetic loci associated with decreasing risk of uterine leiomyomata (UL), a genome-wide association study (GWAS) was performed. We analyzed a group of patients with a family history of UL and a control group consisting of patients without uterine fibroids and a family predisposition to this pathology. Six significant single nucleotide polymorphisms were selected for PCR-genotyping of a large data set of patients with UL. All investigated loci (rs3020434, rs11742635, rs124577644, rs12637801, rs2861221, and rs17677069) demonstrated the lower frequency of minor alleles within a group of women with UL, especially in a subgroup consisting of patients with UL and a familial history of leiomyomata. We also found that the minor allele frequencies of these SNPs in our control group were higher than those across the Caucasian population in all. Based on the obtained data, an evaluation of the common risk of UL was performed. Further work will pave the way to create a specific SNP-panel and allow us to estimate a genotype-based leiomyoma incidence risk. Subsequent studies of genetic variability in a group of patients with a familial predisposition to UL will allow us to make the prediction of the development and course of the disease more individualized, as well as to give our patients personalized recommendations about individual reproductive strategies. Full article
(This article belongs to the Special Issue Feature Papers in Molecular and Translation Medicine)
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<p>Selection of candidate single-nucleotide polymorphisms using <span class="html-italic">p</span>-value. The SNPs of interest are marked with a black dot. Their <span class="html-italic">p</span>-value reaches the maximum <span class="html-italic">p</span>-value. That is, the allelic differences between groups in these SNPs are the largest. (<b>a</b>) Selection of rs11742635 and rs17677069. Each point corresponds to a -log10 <span class="html-italic">p</span>-value of unique rs located on gene <span class="html-italic">FBN2</span> (<span class="html-italic">p</span>-value: 0.00014 for both SNPs). (<b>b</b>) Selection of rs124577644 and rs2861221. Each point corresponds to a -log10 <span class="html-italic">p</span>-value of unique rs located on gene <span class="html-italic">CELF4</span> (<span class="html-italic">p</span>-value: 0.00036 for both SNPs). (<b>c</b>) Selection of rs3020434 (bold point). Each point corresponds to a -log10 p-value of unique rs located on gene <span class="html-italic">ESR1</span> (<span class="html-italic">p</span>-value: 0.00036). (<b>d</b>) Selection of rs12637801 (bold point). Each point corresponds to a -log10 <span class="html-italic">p</span>-value of unique rs located on gene <span class="html-italic">KCNMB2</span> (<span class="html-italic">p</span>-value: 0.00038).</p> Full article ">Figure 1 Cont.
<p>Selection of candidate single-nucleotide polymorphisms using <span class="html-italic">p</span>-value. The SNPs of interest are marked with a black dot. Their <span class="html-italic">p</span>-value reaches the maximum <span class="html-italic">p</span>-value. That is, the allelic differences between groups in these SNPs are the largest. (<b>a</b>) Selection of rs11742635 and rs17677069. Each point corresponds to a -log10 <span class="html-italic">p</span>-value of unique rs located on gene <span class="html-italic">FBN2</span> (<span class="html-italic">p</span>-value: 0.00014 for both SNPs). (<b>b</b>) Selection of rs124577644 and rs2861221. Each point corresponds to a -log10 <span class="html-italic">p</span>-value of unique rs located on gene <span class="html-italic">CELF4</span> (<span class="html-italic">p</span>-value: 0.00036 for both SNPs). (<b>c</b>) Selection of rs3020434 (bold point). Each point corresponds to a -log10 p-value of unique rs located on gene <span class="html-italic">ESR1</span> (<span class="html-italic">p</span>-value: 0.00036). (<b>d</b>) Selection of rs12637801 (bold point). Each point corresponds to a -log10 <span class="html-italic">p</span>-value of unique rs located on gene <span class="html-italic">KCNMB2</span> (<span class="html-italic">p</span>-value: 0.00038).</p> Full article ">
26 pages, 1585 KiB  
Review
Oxidative Stress, Inflammation and Connexin Hemichannels in Muscular Dystrophies
by Arlek González-Jamett, Walter Vásquez, Gabriela Cifuentes-Riveros, Rafaela Martínez-Pando, Juan C. Sáez and Ana M. Cárdenas
Biomedicines 2022, 10(2), 507; https://doi.org/10.3390/biomedicines10020507 - 21 Feb 2022
Cited by 14 | Viewed by 4462
Abstract
Muscular dystrophies (MDs) are a heterogeneous group of congenital neuromuscular disorders whose clinical signs include myalgia, skeletal muscle weakness, hypotonia, and atrophy that leads to progressive muscle disability and loss of ambulation. MDs can also affect cardiac and respiratory muscles, impairing life-expectancy. MDs [...] Read more.
Muscular dystrophies (MDs) are a heterogeneous group of congenital neuromuscular disorders whose clinical signs include myalgia, skeletal muscle weakness, hypotonia, and atrophy that leads to progressive muscle disability and loss of ambulation. MDs can also affect cardiac and respiratory muscles, impairing life-expectancy. MDs in clude Duchenne muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy and limb-girdle muscular dystrophy. These and other MDs are caused by mutations in genes that encode proteins responsible for the structure and function of skeletal muscles, such as components of the dystrophin-glycoprotein-complex that connect the sarcomeric-actin with the extracellular matrix, allowing contractile force transmission and providing stability during muscle contraction. Consequently, in dystrophic conditions in which such proteins are affected, muscle integrity is disrupted, leading to local inflammatory responses, oxidative stress, Ca2+-dyshomeostasis and muscle degeneration. In this scenario, dysregulation of connexin hemichannels seem to be an early disruptor of the homeostasis that further plays a relevant role in these processes. The interaction between all these elements constitutes a positive feedback loop that contributes to the worsening of the diseases. Thus, we discuss here the interplay between inflammation, oxidative stress and connexin hemichannels in the progression of MDs and their potential as therapeutic targets. Full article
(This article belongs to the Special Issue Oxidative Stress and Inflammation: From Mechanisms to Therapeutic Approaches 3.0)
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<p>Inflammation in skeletal muscle healing and degeneration. (<b>A1</b>–<b>A3</b>) Upon muscle injury immune cells infiltrate, monocytes become macrophages with a proinflammatory M1-phenotype that release proinflammatory cytokines and growth factors promoting satellite cell proliferation (<b>A1</b>), myotube formation and muscle healing (<b>A2</b>) The transition from M1 to M2 pro-resolutive macrophages favors muscle regeneration (<b>A3</b>). (<b>B</b>) When these processes are deregulated the M1 to M2 transition is suppressed and inflammation becomes chronic, producing accumulation of fibrotic tissue and muscle dysfunction and atrophy. Proinflammatory cytokines bind to their receptors in inflammatory cells as well as in muscle cells, promoting the activation of the NFKB signaling, assembly of the NLRP3 inflammasome, activation of caspase-1 and cleavage of the immature forms of the IL1 family (pro-IL1), thus enhancing inflammation and potentially contributing to muscle damage. Damage signals also activate toll-like receptors (TLRs), promoting the same mechanism.</p> Full article ">Figure 2
<p>Mechanisms contributing to muscle dystrophy progression. Mutations in proteins that critically regulate skeletal muscle integrity and homeostasis can cause myofiber damage with consecutive accumulation of inflammatory cells that produce ROS. In muscular dystrophies (MDs), such as Duchenne muscular dystrophy (DMD), myofibers also display sarcolemma microtears that allow Ca<sup>2+</sup> entry. The de novo expression of non-selective channels, such as connexin hemichannels, can further contributes to excessive high cytosolic Ca<sup>2+</sup> concentrations, which in turn might lead to activation of Ca<sup>2+</sup>-activated proteases, mitochondria dysfunction and reactive oxygen species (ROS) generation from mitochondria, NADPH-oxidase (NOX) and oxidases (Oxs) such as xanthin oxidase. ROS overproduction leads to Ca<sup>2+</sup> leak from the sarcoplasmic reticulum via ryanodine receptors (RyR1), oxidation of lipids, proteins and DNA, and deregulation of the nuclear factor kappa B (NF-κB) and nuclear factor erythroid-derived 2-related factor 2 (Nrf2) signaling pathways, which regulate the expression of inflammatory mediators and antioxidant enzymes. All these elements contribute to muscle degeneration.</p> Full article ">
26 pages, 1181 KiB  
Review
Is Tissue Still the Issue? The Promise of Liquid Biopsy in Uveal Melanoma
by Daniël P. de Bruyn, Aaron B. Beasley, Robert M. Verdijk, Natasha M. van Poppelen, Dion Paridaens, Ronald O. B. de Keizer, Nicole C. Naus, Elin S. Gray, Annelies de Klein, Erwin Brosens and Emine Kiliç
Biomedicines 2022, 10(2), 506; https://doi.org/10.3390/biomedicines10020506 - 21 Feb 2022
Cited by 14 | Viewed by 4194
Abstract
Uveal melanoma (UM) is the second most frequent type of melanoma. Therapeutic options for UM favor minimally invasive techniques such as irradiation for vision preservation. As a consequence, no tumor material is obtained. Without available tissue, molecular analyses for gene expression, mutation or [...] Read more.
Uveal melanoma (UM) is the second most frequent type of melanoma. Therapeutic options for UM favor minimally invasive techniques such as irradiation for vision preservation. As a consequence, no tumor material is obtained. Without available tissue, molecular analyses for gene expression, mutation or copy number analysis cannot be performed. Thus, proper patient stratification is impossible and patients’ uncertainty about their prognosis rises. Minimally invasive techniques have been studied for prognostication in UM. Blood-based biomarker analysis has become more common in recent years; however, no clinically standardized protocol exists. This review summarizes insights in biomarker analysis, addressing new insights in circulating tumor cells, circulating tumor DNA, extracellular vesicles, proteomics, and metabolomics. Additionally, medical imaging can play a significant role in staging, surveillance, and prognostication of UM and is addressed in this review. We propose that combining multiple minimally invasive modalities using tumor biomarkers should be the way forward and warrant more attention in the coming years. Full article
(This article belongs to the Topic Cancer Biology and Therapy)
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<p>Overview of 774 UM-patients treated at the Erasmus MC and Rotterdam Eye Hospital, Rotterdam, The Netherlands from 1980 to 2021. At the Erasmus Medical Center, fractionated stereotactic radiotherapy (fSRT) was performed since December 1999 and proton beam therapy was available from January 2020. Plaque therapy was performed at the Leiden University Medical Center since January 1990. (<b>a</b>) the number and type of therapy conducted; and (<b>b</b>) the respective fraction of therapy type per time period. In 2020–2021, 24 patients were treated for UM. The gray and white striped bar marks the expected number of UMs in the Rotterdam Ocular Melanoma Study group (ROMS)-cohort from 2021 to 2025. Abbreviations: fSRT: fractionated stereotactic radiotherapy; PBT: proton beam therapy; TTT: transpupillary thermo therapy; PDT: photodynamic therapy.</p> Full article ">Figure 2
<p>Modified from Beasley et al. [<a href="#B26-biomedicines-10-00506" class="html-bibr">26</a>] showing CNV-profiles derived from the primary tumor (FFPE tissue) and CTCs isolated from 10 mL peripheral blood of the same patient after whole genome amplification and shallow Whole Genome Sequencing (genomic coverage: 0.37×, 0.30× and 0.26×, respectively).</p> Full article ">
18 pages, 11668 KiB  
Article
IGF-1 as a Potential Therapy for Spinocerebellar Ataxia Type 3
by Yong-Shiou Lin, Wen-Ling Cheng, Jui-Chih Chang, Ta-Tsung Lin, Yi-Chun Chao and Chin-San Liu
Biomedicines 2022, 10(2), 505; https://doi.org/10.3390/biomedicines10020505 - 21 Feb 2022
Cited by 10 | Viewed by 3719
Abstract
Although the effects of growth hormone (GH) therapy on spinocerebellar ataxia type 3 (SCA3) have been examined in transgenic SCA3 mice, it still poses a nonnegligible risk of cancer when used for a long term. This study investigated the efficacy of IGF-1, a [...] Read more.
Although the effects of growth hormone (GH) therapy on spinocerebellar ataxia type 3 (SCA3) have been examined in transgenic SCA3 mice, it still poses a nonnegligible risk of cancer when used for a long term. This study investigated the efficacy of IGF-1, a downstream mediator of GH, in vivo for SCA3 treatment. IGF-1 (50 mg/kg) or saline, once a week, was intraperitoneally injected to SCA3 84Q transgenic mice harboring a human ATXN3 gene with a pathogenic expanded 84 cytosine–adenine–guanine (CAG) repeat motif at 9 months of age. Compared with the control mice harboring a 15 CAG repeat motif, the SCA3 84Q mice treated with IGF-1 for 9 months exhibited the improvement only in locomotor function and minimized degeneration of the cerebellar cortex as indicated by the survival of more Purkinje cells with a more favorable mitochondrial function along with a decrease in oxidative stress caused by DNA damage. These findings could be attributable to the inhibition of mitochondrial fission, resulting in mitochondrial fusion, and decreased immunofluorescence staining in aggresome formation and ataxin-3 mutant protein levels, possibly through the enhancement of autophagy. The findings of this study show the therapeutic potential effect of IGF-1 injection for SCA3 to prevent the exacerbation of disease progress. Full article
(This article belongs to the Topic Neuroprotection by Drugs, Nutraceuticals and Physical Activity)
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<p>IGF-1 prevented impairment of the motor function in the SCA3 mice. (<b>a</b>) Latency to fall (time in seconds for which the mice persisted on the rotarod) for the SCA3 15Q mice and the saline- and IGF-1-treated SCA3 84Q mice during the 9 months of treatment. (<b>b</b>) Within the same group, the latency to fall at pretreatment was normalized to 100%. (<b>c</b>) EthoVision XT 7.0 software was used to analyze trajectories of the mice in the behavioral test. (<b>d</b>) The distance of movement, time of movement, frequency of zone change, and average velocity were included in transformed indices. (<b>e</b>) Captured images of the single stance for each paw. (<b>f</b>) Catwalk parameters included the step cycle, stride length, stand, and average speed. The data are presented as the means ± SEM. Note: # <span class="html-italic">p</span> &lt; 0.05 denotes statistical significance in the saline-treated SCA3 84Q mice compared with the SCA3 15Q mice; * <span class="html-italic">p</span> &lt; 0.05 indicates a significant difference.</p> Full article ">Figure 2
<p>IGF-1 prevented the death of the PCs in the cerebellum of the SCA3 84Q mice. (<b>a</b>) The arrows indicate the PCs located at the PCL (right panel). The average number of the PCs per 100 μm in the posterior lobules of the cerebellum is presented in the bar graph (mean ± SEM) (left panel). SCA3 15Q, n = 6; SCA3 84Q, n = 8; SCA3 84Q + IGF-1, n = 8. (<b>b</b>) Western blot analysis of calbindin (left panel). Relative expression levels of calbindin in the cerebellum (mean ± SEM) (right panel). SCA3 15Q, n = 4; SCA3 84Q, n = 4; SCA3 84Q + IGF-1, n = 4. (<b>c</b>) The lines indicate the distance from the tip of the granular layer (GL) to the white matter (right panel). Histogram showing the thickness of the GL (mean ± SEM) (left panel). SCA3 15Q, n = 5; SCA3 84Q, n = 6; SCA3 84Q + IGF-1, n = 8. (<b>d</b>) The frames are the sampling area of the Figure and the lines refer to the distance from the PCL to the edge of the molecular layer (ML) (right panel). Histogram showing the thickness of the ML (mean ± SEM) (left panel). SCA3 15Q, n = 5; SCA3 84Q, n = 6; SCA3 84Q + IGF-1, n = 8. Note: * <span class="html-italic">p</span> &lt; 0.05 indicates a significant difference.</p> Full article ">Figure 3
<p>IGF-1 reduced the ataxin-3 protein level in the cerebellum of the SCA3 84Q mice. (<b>a</b>) Immunochemical staining of ataxin-3 in the cerebellum. The black arrows indicate PCs (right panel). Histograms show the means ± SEM (left panel). SCA3 15Q, n = 4; SCA3 84Q, n = 4; SCA3 84Q + IGF-1, n = 5. (<b>b</b>) Western blot confirming ataxin-3 expression in the mouse cerebellum (left panel). Quantification of the ataxin-3 level relative to the total protein level (mean ± SEM) (right panel). SCA3 15Q, n = 4; SCA3 84Q, n = 4; SCA3 84Q + IGF-1, n = 5. (<b>c</b>) Slices of the cerebellum of two mice in each group were selected and double-labeled using an aggresome detection kit (red) and an Alexa 488-conjugated secondary IgG against the anti-ataxin-3 antibody (green), and fluorescence intensities of 30–40 PCs in each mouse were examined using the ImageJ software. The white arrows indicate PCs. SCA3 15Q, n = 2; SCA3 84Q, n = 2; SCA3 84Q + IGF-1, n = 2. Note: * <span class="html-italic">p</span> &lt; 0.05 indicates a significant difference.</p> Full article ">Figure 4
<p>Expression of the autophagic influx in the SCA3 mice. Representative Western blots of the autophagy-related markers (right panel). Quantitative results of the autophagy-related proteins were normalized to those of total protein (mean ± SEM) (left panel). SCA3 15Q, n = 4; SCA3 84Q, n = 4; SCA3 84Q + IGF-1, n = 4. Note: * <span class="html-italic">p</span> &lt; 0.05 indicates a significant difference.</p> Full article ">Figure 5
<p>Expression of the mitochondrial function in the SCA3 mice. (<b>a</b>) Typical trace of respirometry measurements recorded using an Oroboros O2k with 2 mg/mL of the cerebellum. The blue curve indicates the oxygen concentration in the sealed chamber, whereas the red curve shows the oxygen consumption of tissue cells (left panel). Oxygen consumption of cells at different mitochondrial stages was corrected for ROX, and the respiratory capacities in the routine, OXPHOS, Max-Ox, and Max-U states were plotted as the means ± SEM (right panel). SCA3 15Q, n = 3; SCA3 84Q, n = 3; SCA3 84Q + IGF-1, n = 2. (<b>b</b>) The 8-OHdG protein expression in the cerebellum sections by IHC staining analysis; the arrows indicate PCs (left panel). Histogram shows the mean ± SEM (right panel). SCA3 15Q, n = 4; SCA3 84Q, n = 5; SCA3 84Q + IGF-1, n = 5. (<b>c</b>) Western blot was performed to analyze the expression of mitochondrial dynamics-related proteins (right panel). Quantification of mitochondrial dynamics-related proteins (left panel). SCA3 15Q, n = 4; SCA3 84Q, n = 4; SCA3 84Q + IGF-1, n = 4. Note: * <span class="html-italic">p</span> &lt; 0.05 indicates a significant difference.</p> Full article ">Figure 6
<p>Plasma concentration of Nf-L. SCA3 15Q, n = 6; SCA3 84Q, n = 8; SCA3 84Q + IGF-1, n = 8. Note: * <span class="html-italic">p</span> &lt; 0.05 was considered a statistically significant difference.</p> Full article ">Figure 7
<p>Tissue sections of the liver, lung, and kidney. No significant histopathological findings of the kidneys, liver, and lungs were observed in the SCA3 15Q, saline-treated SCA3 84Q, and IGF-1-treated SCA3 84Q mice. n = 3 in all the groups.</p> Full article ">
17 pages, 742 KiB  
Review
Emerging Evidence and Treatment Perspectives from Randomized Clinical Trials in Systemic Sclerosis: Focus on Interstitial Lung Disease
by Caterina Oriana Aragona, Antonio Giovanni Versace, Carmelo Ioppolo, Daniela La Rosa, Rita Lauro, Maria Concetta Tringali, Simona Tomeo, Guido Ferlazzo, William Neal Roberts, Alessandra Bitto, Natasha Irrera and Gianluca Bagnato
Biomedicines 2022, 10(2), 504; https://doi.org/10.3390/biomedicines10020504 - 21 Feb 2022
Cited by 2 | Viewed by 4011
Abstract
Systemic sclerosis (SSc) is a complex rare autoimmune disease with heterogeneous clinical manifestations. Currently, interstitial lung disease (ILD) and cardiac involvement (including pulmonary arterial hypertension) are recognized as the leading causes of SSc-associated mortality. New molecular targets have been discovered and phase II [...] Read more.
Systemic sclerosis (SSc) is a complex rare autoimmune disease with heterogeneous clinical manifestations. Currently, interstitial lung disease (ILD) and cardiac involvement (including pulmonary arterial hypertension) are recognized as the leading causes of SSc-associated mortality. New molecular targets have been discovered and phase II and phase III clinical trials published in the last 5 years on SSc-ILD will be discussed in this review. Details on the study design; the drug tested and its dose; the inclusion and exclusion criteria of the study; the concomitant immunosuppression; the outcomes and the duration of the study were reviewed. The two most common drugs used for the treatment of SSc-ILD are cyclophosphamide and mycophenolate mofetil, both supported by randomized controlled trials. Additional drugs, such as nintedanib and tocilizumab, have been approved to slow pulmonary function decline in SSc-ILD. In this review, we discuss the therapeutic alternatives for SSc management, offering the option to customize the design of future studies to stratify SSc patients and provide a patient-specific treatment according to the new emerging pathogenic features of SSc-ILD. Full article
(This article belongs to the Special Issue Frontiers of Targeted Therapy and Predictors of Treatment Response in Systemic Sclerosis)
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<p>Schematic representation of the mechanism of action of nintedanib, pomalidomide, tocilizumab, rituximab, romilikimab, pirfenidone, lenabasum, and riociguat.</p> Full article ">
15 pages, 3173 KiB  
Article
Cytoprotective Effect of Idebenone through Modulation of the Intrinsic Mitochondrial Pathway of Apoptosis in Human Retinal Pigment Epithelial Cells Exposed to Oxidative Stress Induced by Hydrogen Peroxide
by Maria Elisabetta Clementi, Michela Pizzoferrato, Giada Bianchetti, Anna Brancato, Beatrice Sampaolese, Giuseppe Maulucci and Giuseppe Tringali
Biomedicines 2022, 10(2), 503; https://doi.org/10.3390/biomedicines10020503 - 21 Feb 2022
Cited by 29 | Viewed by 5305
Abstract
Idebenone is a ubiquinone short-chain synthetic analog with antioxidant properties, which is believed to restore mitochondrial ATP synthesis. As such, idebenone is investigated in numerous clinical trials for diseases of mitochondrial aetiology and it is authorized as a drug for the treatment of [...] Read more.
Idebenone is a ubiquinone short-chain synthetic analog with antioxidant properties, which is believed to restore mitochondrial ATP synthesis. As such, idebenone is investigated in numerous clinical trials for diseases of mitochondrial aetiology and it is authorized as a drug for the treatment of Leber’s hereditary optic neuropathy. Mitochondria of retinal pigment epithelium (RPE) are particularly vulnerable to oxidative damage associated with cellular senescence. Therefore, the aim of this study was to explore idebenone’s cytoprotective effect and its underlying mechanism. We used a human-RPE cell line (ARPE-19) exposed to idebenone pre-treatment for 24 h followed by conditions inducing H2O2 oxidative damage for a further 24 h. We found that idebenone: (a) ameliorated H2O2-lowered cell viability in the RPE culture; (b) activated Nrf2 signaling pathway by promoting Nrf2 nuclear translocation; (c) increased Bcl-2 protein levels, leaving unmodified those of Bax, thereby reducing the Bax/Bcl-2 ratio; (d) maintained the mitochondrial membrane potential (ΔΨm) at physiological levels, preserving the functionality of mitochondrial respiratory complexes and counteracting the excessive production of ROS; and (e) reduced mitochondrial cytochrome C-mediated caspase-3 activity. Taken together, our findings show that idebenone protects RPE from oxidative damage by modulating the intrinsic mitochondrial pathway of apoptosis, suggesting its possible role in retinal epitheliopathies associated with mitochondrial dysfunction. Full article
(This article belongs to the Special Issue Mitochondrial Dysfunction and Oxidative Stress in Aging and Disease)
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<p>Idebenone pre-treatment diminishes H<sub>2</sub>O<sub>2</sub>–induced cell death. ARPE-19 cell viability: after treatment with different concentrations of H<sub>2</sub>O<sub>2</sub> (range 200–800 µM) for 24 h (<b>A</b>); after idebenone (range 0.01–10 µM) pre-treatment for 24 h, both under baseline conditions (<b>B</b>); and following stimulation with 300 µM H<sub>2</sub>O<sub>2</sub> (<b>C</b>). Phase-contrast micrography shows morphological changes of ARPE-19 cells treated for 24 h with 300 µM H<sub>2</sub>O<sub>2</sub> in the absence or presence of idebenone (1 µM) pre-treatment (24 h), compared to the control. Image of idebenone alone is also shown, (<b>D</b>). The results are from two independent experiments, each including five replicates per experimental group. Data are expressed as a percentage relative to the untreated cells (control = 100%), the means ± SEM of 10 replicates per group. * <span class="html-italic">p</span> &lt; 0.05 and *** <span class="html-italic">p</span> &lt; 0.001 vs. control; °° <span class="html-italic">p</span> &lt; 0.01 and °°° <span class="html-italic">p</span> &lt; 0.001 vs. H<sub>2</sub>O<sub>2</sub> alone.</p> Full article ">Figure 2
<p>Idebenone stimulates Nrf2 nuclear translocation in ARPE-19 cells. Idebenone significantly increased NRF2 nuclear activation in both basal and 300 μM H<sub>2</sub>O<sub>2</sub> stimulated conditions. All of the optical density values (see Materials and Methods) are expressed as a percentage relative to the non-treated cells (control 100%) ± SEM of three independent experiments performed in triplicate (nine in all, for each experimental group). *** <span class="html-italic">p</span> &lt; 0.001 vs. control; °°° <span class="html-italic">p</span> &lt; 0.001 vs. H<sub>2</sub>O<sub>2</sub> alone.</p> Full article ">Figure 3
<p>Idebenone effect on the protein levels of Bcl-2 and Bax. Idebenone pre-treatment promotes Bcl-2 expression (<b>A</b>) but inhibits Bax expression (<b>B</b>) after H<sub>2</sub>O<sub>2</sub> induced oxidative stress for 24 h. Both histograms represent the means ± SEM of two independent experiments performed in quadruplicate (eight replicates per experimental group). *** <span class="html-italic">p</span> &lt; 0.001 vs. control; °°° <span class="html-italic">p</span> &lt; 0.001 vs. H<sub>2</sub>O<sub>2</sub> alone.</p> Full article ">Figure 4
<p>The role of idebenone against H<sub>2</sub>O<sub>2</sub>-induced mitochondrial depolarization. The panel shows, in the first row, representative images of ARPE-19 cells stained with JC-1 under different conditions (untreated, CTRL; 300 µM H<sub>2</sub>O<sub>2</sub>, H-300; Idebenone, IDB; and pre-treated with idebenone 24 h before H<sub>2</sub>O<sub>2</sub>, IDB + H-300), together with the ratio maps of mitochondrial membrane potential, in the second row. In the composite confocal images, the fluorescence signal from monomers is represented in green (emission: 525/50 nm), while the red channel (emission: 595/50 nm) collects the signal from aggregates. In the mitochondrial membrane potential maps, each pixel is colored according to the red/green fluorescence intensity ratio, with black pixels representing low R/G values (i.e., mitochondrial depolarization) and white pixels representing high values of the R/G ratio (i.e., mitochondrial hyperpolarization). The scale bar is 20 µm. The distribution of the R/G values, corresponding to the mitochondrial membrane potential, are reported in the box plot (y-axis) for each sample (x-axis). One-way ANOVA analysis was carried out with post hoc Tukey’s test for multiple group comparisons (* <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; ns = not statistically significant).</p> Full article ">Figure 5
<p>Idebenone reduces mitochondrial ROS production in ARPE-19 cells treated with H<sub>2</sub>O<sub>2</sub>. Idebenone pre-treatment (24 h) blocks H<sub>2</sub>O<sub>2</sub>-induced mitochondrial ROS accumulation. Mitochondrial ROS levels are quantified by measurement of DCF fluorescence (see Materials and Methods) and the results are expressed as a percentage of the untreated control (control = 100%). Results are from two independent experiments, the means ± SEM of 10 replicates per group. *** <span class="html-italic">p</span> &lt; 0.001 vs. control; °°° <span class="html-italic">p</span> &lt; 0.001 vs. H<sub>2</sub>O<sub>2</sub> alone.</p> Full article ">Figure 6
<p>Idebenone supports the functions of mitochondrial respiratory chain complexes (I and III). The graphs show the ability of the idebenone pre-treatment (24 h) to restore complex I (<b>A</b>) and complex III (<b>B</b>) mitochondrial respiratory functions, which were depressed after oxidative stress induced by 24 h of treatment with H<sub>2</sub>O<sub>2</sub>. Both graphs are the result of two independent experiments performed in quintuplicate (<span class="html-italic">n</span> = 10/group). The results are expressed as a percentage of the untreated control (control = 100%). Results are from two independent experiments, the means ± SEM of 10 replicates per group. ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001 vs. control; °° <span class="html-italic">p</span> &lt; 0.01 and °°° <span class="html-italic">p</span> &lt; 0.001 vs. H<sub>2</sub>O<sub>2</sub> alone.</p> Full article ">Figure 7
<p>Idebenone inhibits cytochrome c release and Caspase-3 enzymatic activity. Effect of the idebenone pre-treatment (1 µM for 24 h) both on the release of Cytochrome-c (<b>A</b>) and the activity of Caspase-3 (<b>B</b>) in the presence or absence of 300 µM H<sub>2</sub>O<sub>2</sub>-induced oxidative stress for a further 24 h. Results are quantified by measurement of OD (see Materials and Methods) and are reported as a percentage of the ARPE-19 untreated cells (arbitrarily set to 100%). The graphs are the result of three independent experiments performed in triplicate, the means ± SEM of 9 replicates per group. ** <span class="html-italic">p</span> &lt; 0.01 and °° <span class="html-italic">p</span> &lt; 0.01 vs. control and H<sub>2</sub>O<sub>2</sub> alone, respectively.</p> Full article ">
16 pages, 1419 KiB  
Review
Drugs Modulating Renin-Angiotensin System in COVID-19 Treatment
by Jose L. Labandeira-Garcia, Carmen M. Labandeira, Rita Valenzuela, Maria A. Pedrosa, Aloia Quijano and Ana I. Rodriguez-Perez
Biomedicines 2022, 10(2), 502; https://doi.org/10.3390/biomedicines10020502 - 21 Feb 2022
Cited by 16 | Viewed by 6110
Abstract
A massive worldwide vaccination campaign constitutes the main tool against the COVID-19 pandemic. However, drug treatments are also necessary. Antivirals are the most frequently considered treatments. However, strategies targeting mechanisms involved in disease aggravation may also be effective. A major role of the [...] Read more.
A massive worldwide vaccination campaign constitutes the main tool against the COVID-19 pandemic. However, drug treatments are also necessary. Antivirals are the most frequently considered treatments. However, strategies targeting mechanisms involved in disease aggravation may also be effective. A major role of the tissue renin-angiotensin system (RAS) in the pathophysiology and severity of COVID-19 has been suggested. The main link between RAS and COVID-19 is angiotensin-converting enzyme 2 (ACE2), a central RAS component and the primary binding site for SARS-CoV-2 that facilitates the virus entry into host cells. An initial suggestion that the susceptibility to infection and disease severity may be enhanced by angiotensin type-1 receptor blockers (ARBs) and ACE inhibitors (ACEIs) because they increase ACE2 levels, led to the consideration of discontinuing treatments in thousands of patients. More recent experimental and clinical data indicate that ACEIs and, particularly, ARBs can be beneficial for COVID-19 outcome, both by reducing inflammatory responses and by triggering mechanisms (such as ADAM17 inhibition) counteracting viral entry. Strategies directly activating RAS anti-inflammatory components such as soluble ACE2, Angiotensin 1-7 analogues, and Mas or AT2 receptor agonists may also be beneficial. However, while ACEIs and ARBs are cheap and widely used, the second type of strategies are currently under study. Full article
(This article belongs to the Special Issue Non-antiviral Agents for Treatment of COVID-19)
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<p>The renin-angiotensin system (RAS) consists of two axes that counteract each other: a pro-inflammatory axis (red arrows) mainly constituted by Angiotensin II acting on AT1 receptors (AT1R), and an anti-inflammatory axis (green arrows) constituted by Angiotensin II acting on AT2 receptors, and particularly Angiotensin 1-7 acting on Mas receptors. Angiotensin II is produced by the action the enzyme prorenin/renin on the precursor protein angiotensinogen, producing Angiotensin I, which is transformed by the angiotensin-converting enzyme (ACE) into Angiotensin II. Renin and its precursor prorenin (PR) can also activate specific PR receptors. Angiotensin-converting enzyme 2 (ACE2) plays a central role in the RAS balance, as ACE2 (with the aid of peptidases such as Neprilysin, NE) converts compounds of the pro-inflammatory arm (Angiotensin I and, particularly, Angiotensin II) into compounds of the anti-inflammatory arm (i.e., Angiotensin 1-9 and, particularly Angiotensin 1-7).</p> Full article ">Figure 2
<p>Both SARS-CoV-2 binding to cell membrane ACE2 and activation of the RAS pro-inflammatory arm decrease levels of membrane ACE2 and increase ADAM17 and TMPRSS2 activities. A decrease in transmembrane ACE2 activity leads to a decrease in anti-inflammatory RAS activity. Treatment with ARBs or ACEIs reduces the activity of the pro-inflammatory RAS axis, upregulating ACE2 transmembrane levels and the anti-inflammatory axis activity. Inhibition of the RAS pro-inflammatory axis also inhibits ADAM17 and TMPRSS2 activities, which are necessary for membrane ACE2 shedding and viral entry. Membrane ACE2 shedding further reduces membrane ACE2 and releases circulating soluble ACE2 that may bind/neutralize circulating viruses.</p> Full article ">Figure 3
<p>Current therapeutical strategies with drugs that inhibit components of the RAS pro-inflammatory axis or enhance components of the RAS anti-inflammatory axis.</p> Full article ">
15 pages, 3595 KiB  
Article
Identification of Altered Evoked and Non-Evoked Responses in a Heterologous Mouse Model of Endometriosis-Associated Pain
by Miguel A. Tejada, Ana I. Santos-Llamas, Lesley Escriva, Juan J. Tarin, Antonio Cano, Maria J. Fernández-Ramírez, Paulina Nunez-Badinez, Bianca De Leo, Philippa T. K. Saunders, Victor Vidal, Florent Barthas, Katy Vincent, Patrick J. Sweeney, Rowland R. Sillito, James Douglas Armstrong, Jens Nagel and Raúl Gomez
Biomedicines 2022, 10(2), 501; https://doi.org/10.3390/biomedicines10020501 - 21 Feb 2022
Cited by 8 | Viewed by 3797
Abstract
The aim of this study was to develop and refine a heterologous mouse model of endometriosis-associated pain in which non-evoked responses, more relevant to the patient experience, were evaluated. Immunodeficient female mice (N = 24) were each implanted with four endometriotic human lesions [...] Read more.
The aim of this study was to develop and refine a heterologous mouse model of endometriosis-associated pain in which non-evoked responses, more relevant to the patient experience, were evaluated. Immunodeficient female mice (N = 24) were each implanted with four endometriotic human lesions (N = 12) or control tissue fat (N = 12) on the abdominal wall using tissue glue. Evoked pain responses were measured biweekly using von Frey filaments. Non-evoked responses were recorded weekly for 8 weeks using a home cage analysis (HCA). Endpoints were distance traveled, social proximity, time spent in the center vs. outer areas of the cage, drinking, and climbing. Significant differences between groups for von Frey response, climbing, and drinking were detected on days 14, 21, and 35 post implanting surgery, respectively, and sustained for the duration of the experiment. In conclusion, a heterologous mouse model of endometriosis-associated evoked a non-evoked pain was developed to improve the relevance of preclinical models to patient experience as a platform for drug testing. Full article
(This article belongs to the Special Issue Advanced Research in Endometriosis 2.0)
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<p>Experimental design: image shows schematic representation of timeline. Recipients that were 6–8-week-old immunocompromised animals were implanted with a 17β-E2 pellet in the neck and, if required, a microtracker chip in the groin. 4 days later, mice underwent surgery to place human tissue (endometriotic lesions or fat) in the peritoneal cavity. Behavioral non-evoked and evoked pain responses were monitored every week starting on day 7 after surgery.</p> Full article ">Figure 2
<p>Schematic image of nesting test. (<b>A</b>) Example of a cage divided into 6 equivalent areas in which a piece of cotton is placed in each area. After 3 h, the total number of cleared spaces was counted (<b>B</b>). Illustrative examples of different nest quality scores: 1 = cotton not broken, 2 = cotton partially broken, 3 = cotton totally broken (<b>C</b>).</p> Full article ">Figure 3
<p>Evaluation of abdominal and hind paw mechanical pain thresholds in a heterologous model of endometriosis. Graphs show abdominal (<b>A</b>) and hind paw (<b>B</b>) mechanical threshold in animals implanted with human endometriotic lesion (red line) or fat tissue (blue line) at several time points after implanting surgery. Data in each time point are expressed as mean ± SEM values of each group (N = 6 per group). A one-way repeated ANOVA followed by Student Newman–Keuls post-hoc test was performed to analyze comparisons between groups. ** <span class="html-italic">p</span> &lt; 0.01 = statistically significant differences between lesion and control groups at each time point.</p> Full article ">Figure 4
<p>Nest building time course in animals with endometriosis or its control. (<b>A</b>) Nest building was evaluated by counting the number of cleared areas or (<b>B</b>) with a quality score (1 = intact bedding, 2 = bedding partially broken, 3 = bedding totally broken). Lesion and control groups are represented by red and blue lines. Data in each time point express mean ± SEM values of each group (N = 6 per group). A one-way repeated ANOVA followed by Student Newman–Keuls post-hoc test was performed to analyze comparisons between groups. Statistically significant differences between groups were not detected.</p> Full article ">Figure 5
<p>Comparison of temporal pattern of home cage activity between animals implanted with endometriosis lesions or fat. Parameters analyzed were distance traveled (<b>A</b>), time isolated (<b>B</b>), time spent in centers zone (<b>C</b>), thigmotactic (<b>D</b>), and the time spent climbing (<b>E</b>) and drinking (<b>F</b>). The lesion and control groups are represented by the red and blue lines, respectively. Each point and vertical line represent the mean ± SEM of the values obtained of 6 animals. A one-way repeated ANOVA followed by Student Newman–Keuls post-hoc test was performed to analyze comparisons between groups during the time course. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 = Statistically significant differences between lesion and control groups at each time point.</p> Full article ">Figure 6
<p>Image in (<b>A</b>) shows representative histological section of an endometriotic lesion recovered from human patients before being implanted in mice. Image in (<b>B</b>) shows representative observation of the macroscopic appearance of lesions (white arrows) recovered from mice at sacrifice at the end of the study period. Image in (<b>C</b>) shows representative histological section of an endometriotic lesion recovered from mice at the end of the study period. Note the parallels in the overall tissue architecture between the fresh and implanted tissues in regards to dense stromal tissue and few/absent glands. Note also detail of presence of immune cell infiltrate (i.e., giant cells, black arrows) in implanted tissue. Scale bar is represented in histological images.</p> Full article ">Figure 7
<p>Staining of nerve fibers in human endometrial lesion before and after implantation in mice. Nuclei are stained with DAPI (blue) and fibers with anti-Beta III tubulin (red). Scale bar = 25 µm.</p> Full article ">
15 pages, 711 KiB  
Review
Gastrointestinal Microbiome and Neurologic Injury
by Eric J. Panther, William Dodd, Alec Clark and Brandon Lucke-Wold
Biomedicines 2022, 10(2), 500; https://doi.org/10.3390/biomedicines10020500 - 21 Feb 2022
Cited by 41 | Viewed by 6705
Abstract
Communication between the enteric nervous system (ENS) of the gastrointestinal (GI) tract and the central nervous system (CNS) is vital for maintaining systemic homeostasis. Intrinsic and extrinsic neurological inputs of the gut regulate blood flow, peristalsis, hormone release, and immunological function. The health [...] Read more.
Communication between the enteric nervous system (ENS) of the gastrointestinal (GI) tract and the central nervous system (CNS) is vital for maintaining systemic homeostasis. Intrinsic and extrinsic neurological inputs of the gut regulate blood flow, peristalsis, hormone release, and immunological function. The health of the gut microbiome plays a vital role in regulating the overall function and well-being of the individual. Microbes release short-chain fatty acids (SCFAs) that regulate G-protein-coupled receptors to mediate hormone release, neurotransmitter release (i.e., serotonin, dopamine, noradrenaline, γ-aminobutyric acid (GABA), acetylcholine, and histamine), and regulate inflammation and mood. Further gaseous factors (i.e., nitric oxide) are important in regulating inflammation and have a response in injury. Neurologic injuries such as ischemic stroke, spinal cord injury, traumatic brain injury, and hemorrhagic cerebrovascular lesions can all lead to gut dysbiosis. Additionally, unfavorable alterations in the composition of the microbiota may be associated with increased risk for these neurologic injuries due to increased proinflammatory molecules and clotting factors. Interventions such as probiotics, fecal microbiota transplantation, and oral SCFAs have been shown to stabilize and improve the composition of the microbiome. However, the effect this has on neurologic injury prevention and recovery has not been studied extensively. The purpose of this review is to elaborate on the complex relationship between the nervous system and the microbiome and to report how neurologic injury modulates the status of the microbiome. Finally, we will propose various interventions that may be beneficial in the recovery from neurologic injury. Full article
(This article belongs to the Special Issue 10th Anniversary of Biomedicines: Neuroinflammation and Neuroprotection)
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<p>Innervation of the gut through the enteric nervous system (ENS). Mechanical and chemical sensory information is detected by the intrinsic primary afferent neurons (IPANs). The gut microbiota secretes short-chain fatty acids (SCFAs), neurotransmitters, and gaseous factors (e.g., NO). The signal is then transported by ascending (projected orally) and descending (projected anally) interneurons. The signal is transferred to excitatory motor neurons (releasing acetylcholine (Ach) and substance P (SP) to contract the enteric musculature) and inhibitory motor neurons (releasing vasoactive intestinal peptide (VIP) and nitric oxide (NO)) [<a href="#B41-biomedicines-10-00500" class="html-bibr">41</a>]. Created with <a href="http://BioRender.com" target="_blank">BioRender.com</a> (accessed on 23 January 2022).</p> Full article ">
9 pages, 538 KiB  
Editorial
Chronic Obstructive Pulmonary Disease: The Present and Future
by Aditya Krishnan and Alice M. Turner
Biomedicines 2022, 10(2), 499; https://doi.org/10.3390/biomedicines10020499 - 20 Feb 2022
Cited by 6 | Viewed by 3167
Abstract
Chronic obstructive pulmonary disease (COPD) is a highly prevalent condition associated with smoking and is predicted to become a leading cause of death in the current decade [...] Full article
(This article belongs to the Special Issue Chronic Obstructive Pulmonary Disease (COPD): From Pathophysiology to Novel Therapeutic Approaches)
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<p>The heterogeneous spectrum of COPD. The x-axis (red) demonstrates the radiological dichotomy of characterising syndromes in COPD; the y-axis (blue) represents the nosological complexity of COPD’s physiologic overlap with asthma; and the z-axis (green) shows the cellular mediation driving the disease. Overarching environmental factors (gold) perpetuate the disease.</p> Full article ">
22 pages, 1961 KiB  
Review
Orchestrated Cytokines Mediated by Biologics in Psoriasis and Its Mechanisms of Action
by Aina Akmal Mohd Noor, Maryam Azlan and Norhanani Mohd Redzwan
Biomedicines 2022, 10(2), 498; https://doi.org/10.3390/biomedicines10020498 - 20 Feb 2022
Cited by 30 | Viewed by 12565
Abstract
Psoriasis is an autoimmune disease mediated by disturbed T cells and other immune cells, and is defined by deep-red, well-demarcated skin lesions. Due to its varied etiologies and indefinite standard pathogenesis, it is challenging to consider the right treatment exclusively for each psoriasis [...] Read more.
Psoriasis is an autoimmune disease mediated by disturbed T cells and other immune cells, and is defined by deep-red, well-demarcated skin lesions. Due to its varied etiologies and indefinite standard pathogenesis, it is challenging to consider the right treatment exclusively for each psoriasis patient; thus, researchers yearn to seek even more precise treatments other than topical treatment and systemic therapy. Using biologics to target specific immune components, such as upregulated cytokines secreted by activated immune cells, is the most advanced therapy for psoriasis to date. By inhibiting the appropriate pro-inflammatory cytokines, cellular signaling can be altered and, thus, can inhibit further downstream inflammatory pathways. Herein, the roles of cytokines with their mechanisms of action in progressing psoriasis and how the usage of biologics alleviates cellular inflammation are discussed. In addition, other potential pro-inflammatory cytokines, with their mechanism of action, are presented herein. The authors hope that this gathered information may benefit future research in expanding the discovery of targeted psoriasis therapy. Full article
(This article belongs to the Section Immunology and Immunotherapy)
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<p>The summarized pathogenesis of psoriasis. Upon triggers, pDCs receive the signal via TLR8 and present the antigens to mDCs. mDCs activate Th1 and Th22 cells to release cytokines to generate hyperproliferative keratinocytes in the epidermis region. mDCs also present cytokines to Th17 cells to initiate keratinocyte hyperproliferation and the assembling of neutrophils to create Munro’s microabscesses. It is thought that once neutrophils undergo degranulation, this produces granular compounds, such as LL-37 and proteinase 3, which create an information loop to be detected again by pDCs. The cycle repeats. (AMP: antimicrobial proteins; <span class="html-italic">PSORS</span>: psoriasis susceptibility loci).</p> Full article ">Figure 2
<p>TNF-α inhibitors. Infliximab and adalimumab depict almost similar molecular structures with different Fv regions. Etanercept has specialized extracellular portions of human TNFR2. Certolizumab pegol is the most unique, as its Fc region is replaced with PEG molecules to lengthen its half-life.</p> Full article ">Figure 3
<p>Cytokine inhibitors and their respective blocking interests based on the postulated psoriasis pathogenesis. The overexpressed cytokines released by respective cells can be blocked, and this can downregulate the further inflammatory pathway.</p> Full article ">
14 pages, 447 KiB  
Review
Concept of the Number Needed to Treat for the Analysis of Pain Relief Outcomes in Patients Treated with Spinal Cord Stimulation
by Ashley Bailey-Classen, Amar Parikh, Nima Adimi, Deborah Edgar, Alice Yan, Anand Rotte and David Caraway
Biomedicines 2022, 10(2), 497; https://doi.org/10.3390/biomedicines10020497 - 20 Feb 2022
Cited by 6 | Viewed by 2695
Abstract
In the rapidly evolving field of spinal cord stimulation (SCS), measures of treatment effects are needed to help understand the benefits of new therapies. The present article elaborates the number needed to treat (NNT) concept and applies it to the SCS field. We [...] Read more.
In the rapidly evolving field of spinal cord stimulation (SCS), measures of treatment effects are needed to help understand the benefits of new therapies. The present article elaborates the number needed to treat (NNT) concept and applies it to the SCS field. We reviewed the basic theory of the NNT, its calculation method, and its application to historical controlled trials of SCS. We searched the literature for controlled studies with ?20 implanted SCS patients with chronic axial back and/or leg pain followed for ?3 months and a reported responder rate defined as ?50% pain relief. Relevant data necessary to estimate the NNT were extracted from the included articles. In total, 12 of 1616 records were eligible for inclusion. The records reported 10 clinical studies, including 7 randomized controlled trials, 2 randomized crossover trials, and 1 controlled cohort study. The studies investigated traditional SCS and more recently developed SCS modalities, including 10 kHz SCS. In conclusion, the NNT estimate may help SCS stakeholders better understand the effect size difference between compared treatments; however, interpretation of any NNT should take into account its full context. In addition, comparisons across trials of different therapies should be avoided since they are prone to interpretation biases. Full article
(This article belongs to the Special Issue Neuropathic Pain: Therapy and Mechanisms 2.0)
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<p>Literature search flow chart.</p> Full article ">
14 pages, 1665 KiB  
Article
The lncRNAs/miR-30e/CHI3L1 Axis Is Dysregulated in Systemic Sclerosis
by Valentin Dichev, Nikolay Mehterov, Maria Kazakova, Rositsa Karalilova, Anastas Batalov and Victoria Sarafian
Biomedicines 2022, 10(2), 496; https://doi.org/10.3390/biomedicines10020496 - 19 Feb 2022
Cited by 11 | Viewed by 2855
Abstract
Systemic sclerosis (SSc) is an autoimmune disease with completely undefined etiology and treatment difficulties. The expression of both protein coding and non-coding RNAs is dysregulated during disease development. We aimed to examine a possible regulatory axis implemented in the control of chitinase-3 like [...] Read more.
Systemic sclerosis (SSc) is an autoimmune disease with completely undefined etiology and treatment difficulties. The expression of both protein coding and non-coding RNAs is dysregulated during disease development. We aimed to examine a possible regulatory axis implemented in the control of chitinase-3 like protein 1 (CHI3L1) or YKL-40, an inflammation-associated glycoprotein, shown to be elevated in SSc. A panel of seven miRNAs and three lncRNAs potentially involved in the control of CHI3L1 were selected on the basis of in silico analysis. TagMan assay was used to evaluate the expression levels of miRNAs and RT-qPCR for lncRNAs in white blood cells (WBCs) and plasma from SSc patients and healthy controls. Among the eight screened miRNAs, miR-30e-5p (p = 0.04) and miR-30a-5p (p = 0.01) were significantly downregulated in WBCs and plasma of SSc patients, respectively. On the contrary, the expression of the metastasis associated lung adenocarcinoma transcript 1 (MALAT1) (p = 0.044) and the Nuclear enriched abundant transcript 1 (NEAT1) (p = 0.008) in WBCs was upregulated compared to the controls. Increased levels of MALAT1 and NEAT1 could be associated with the downregulation of miR-30e-5p and miR-30a-5p expression in WBCs and plasma. We present novel data on the involvement of a possible regulatory axis lncRNAs/miR-30e/CHI3L1 in SSc and hypothesize that MALAT1 and NEAT1 could act as miR-30e-5p and miR-30a-5p decoys. This may be a reason for the increased serum levels of CHI3L1 in SSc patients. Full article
(This article belongs to the Special Issue MicroRNAs, tRNA Fragments, and Circular RNAs: Pivotal Regulators of Gene Expression and Their Roles in Human Diseases)
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<p>miR-30a-5p and miR-30e-5p are downregulated in SSc. Box plot of (<b>A</b>) miR-24-3p, (<b>C</b>) miR-30a-5p, (<b>D</b>) miR-30b-5p, (<b>E</b>) miR-30c-5p, (<b>G</b>) miR-30e-5p, (<b>F</b>) miR-30d-5p and (<b>B</b>) miR-125a-3p measured in both WBC and plasma of SSc (WBC, <span class="html-italic">n</span> = 31; plasma, <span class="html-italic">n</span> = 30) and healthy controls (WBC, <span class="html-italic">n</span> = 12; plasma, <span class="html-italic">n</span> = 9). Expression levels were determined by TagMan assay after total RNA extraction and presented as fold difference. snoRNA U48 and U6 were used as miRNA normalizers. The data are summarized from two technical replicates for each examined individual. <span class="html-italic">p</span> &gt; 0.05 was marked with *.</p> Full article ">Figure 2
<p>miR-30e-5p and miR-30a-5p are differentially expressed in SSc subgroups. Box plot of miR-30e-5p (<b>A</b>) in WBCs of lcSSc patients (<span class="html-italic">n</span> = 12) and miR-30a-5p (<b>B</b>) in plasma of dcSSc patients (<span class="html-italic">n</span> = 14), compared to healthy controls (WBC, <span class="html-italic">n</span> = 12; plasma, <span class="html-italic">n</span> = 9). Expression levels were determined by TagMan assay after total RNA extraction and presented as fold difference. snoRNA U48 and U6 were used as miRNA normalizers. The data are summarized from two technical replicates for each examined individual. <span class="html-italic">p</span> &gt; 0.05 was marked with *; <span class="html-italic">p</span> &gt; 0.01 was marked with **.</p> Full article ">Figure 3
<p>Binding sites of miR-30e-5p (<b>A</b>) and miR-30a-5p (<b>B</b>) in CHI3L1mRNA and MALAT1. DIANA-LncBase v3 and Starbas bioinformatical tools were applied to predict the binding sequence of the two down regulated miRNAs: miR-30e-5p and miR-30a-5p in the 3’UTR of CHI3L1mRNA and lncRNA MALAT1. The in silico analysis revealed that both have a conserved seed region by which they could bind to the complementary sites in CHI3L1mRNA and lncRNA MALAT1 molecules.</p> Full article ">Figure 4
<p>MALAT1 and NEAT1 are upregulated in SSc WBCs. MALAT1 and NEAT1 are induced in WBCs of SSc patients, compared with the control subjects. Expression levels of MALAT1, NEAT1 and UCA1 were evaluated in WBCs of patients (<span class="html-italic">n</span> = 31) and controls (<span class="html-italic">n</span> = 12) by RT-qPCR after total RNA extraction and presented as fold difference. GAPDH, ACTINβ and hUBC were used as RNA normalizers. The data are summarized from two technical replicates for each examined individual. <span class="html-italic">p</span> &gt; 0.05 was marked with *; <span class="html-italic">p</span> &gt; 0.01 was marked with **.</p> Full article ">Figure 5
<p>Discriminatory potential of plasma miR-30a-5p levels. ROC analysis was performed to access the discriminatory power of miR-30a-5p in plasma. The AUC values show that miR-30a-5p has the potential as a biomarker for distinguishing SSc from healthy subjects.</p> Full article ">Figure 6
<p>Possible lncRNAs/miR30e/CHI3L1regulatory axis in SSc. In SSc, the expression of MALAT1 and NEAT1 in WBCs is upregulated. MALAT1 and NEAT1 can act as competitive endogenous RNAs for miR-30e. Interaction between MALAT1/NEAT1 and miR-30e-5p would abolish the suppressive effect of miR-30e-5p over CHI3L1production, leading to increased serum levels of the glycoprotein in patients with SSc. ↑ indicates induction, whereas ↓ shows downregulation.</p> Full article ">
18 pages, 1660 KiB  
Article
FCGR2A-HH Gene Variants Encoding the Fc Gamma Receptor for the C-Reactive Protein Are Associated with Enhanced Monocyte CD32 Expression and Cardiovascular Events’ Recurrence after Primary Acute Coronary Syndrome
by Pascale Paul, Christophe Picard, Luc Lyonnet, Noémie Resseguier, Lucas Hubert, Laurent Arnaud, Julie Di Cristofaro, Marc Laine, Franck Paganelli, Françoise Dignat-George, Corinne Frère, Florence Sabatier, Regis Guieu and Laurent Bonello
Biomedicines 2022, 10(2), 495; https://doi.org/10.3390/biomedicines10020495 - 19 Feb 2022
Cited by 3 | Viewed by 2830
Abstract
Fcγ receptors (FcγRs) interact with the C-reactive protein (CRP) and mediate activation of inflammation-related pathogenic mechanisms affecting cardiovascular health. Our study evaluated whether FcγRIIA and FcγRIIIA profiles are associated with the recurrence of adverse cardiovascular events during the first year after a primary [...] Read more.
Fcγ receptors (FcγRs) interact with the C-reactive protein (CRP) and mediate activation of inflammation-related pathogenic mechanisms affecting cardiovascular health. Our study evaluated whether FcγRIIA and FcγRIIIA profiles are associated with the recurrence of adverse cardiovascular events during the first year after a primary acute coronary syndrome (ACS). The primary endpoint was the recurrence of cardiovascular events (RCE), identified as a composite outcome comprising acute heart failure (AHF) and major adverse cardiovascular events (MACE). We obtained blood samples of 145 ACS patients to measure hsCRP circulating levels, to identify FcγRIIA-131RH rs1801274 and FcγRIIIA-158FV rs396991 polymorphisms, to analyze circulating monocytes and NK cell subsets expressing CD16 and CD32, and to detect serum-mediated FCGR2A-HH activation by luciferase reporter assays. The hsCRP, CD32-expression, and Fc-R mediated activation levels were similar in all patients regardless of their MACE risk. In contrast, the hsCRP levels and the proportion of CD14+ circulating monocytes expressing the CD32 receptor for CRP were significantly higher in the patients who developed AHF. The FCGR2A rs1801274 HH genotype was significantly more common in patients who developed RCE and MACE than in RCE-free patients and associated with an enhanced percentage of circulating CD32+CD14+ monocytes. The FCGR2A-HH genotype was identified as an independent predictor of subsequent RCE (OR, 2.7; p = 0.048; CI, 1.01–7.44) by multivariate analysis. These findings bring preliminary evidence that host FCGR2A genetic variants can influence monocyte CD32 receptor expression and may contribute to the fine-tuning of CD32-driven chronic activating signals that affect the risk of developing RCEs following primary ACS events. Full article
(This article belongs to the Section Cell Biology and Pathology)
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<p>High circulating CRP levels are associated with AHF recurrence. (<b>A</b>) Comparative analysis of circulating hsCRP levels was analyzed in RCE (RCE+, <span class="html-italic">n</span> = 27) and non-RCE patients (RCE−, <span class="html-italic">n</span> = 118). HsCRP levels were then analyzed in the subgroups of patients experiencing MACE (MACE+ group, <span class="html-italic">n</span> = 18, among which 14 patients were MACE+AHF− and 4 patients experienced both MACE and AHF episodes during the first year post PCI) or AHF (AHF+ group, <span class="html-italic">n</span> = 13, 9 AHF+MACE− and 4 AHF+MACE+ patients) analyzed in reference to patients without MACE (MACE−, <span class="html-italic">n</span> = 127) or free of AHF (AHF−, <span class="html-italic">n</span> = 132) at one year post ACS. hsCRP variables are described in box and whiskers plots as medians and (10–90 percentile) using GraphPad Prism. Comparison between groups were performed using Mann–Whitney non-parametric unpaired <span class="html-italic">t</span> tests. <span class="html-italic">p</span> values &lt; 0.05 were reported as significant. (<b>B</b>) ROC curve analysis of CRP levels associated with recurrent AHF episodes during the first year post initial ACS. Receiver operating characteristic (ROC) curves were used to define the best threshold value of hsCRP that allows us to discriminate patients with and without AHF, according to the Youden’s method (which allows for the maximization of both sensitivity and specificity).</p> Full article ">Figure 2
<p>Flow cytometry analysis of circulating CD32+ and CD16+ monocyte. (<b>A</b>) Comparative analysis of circulating hsCRP levels was analyzed in RCE (RCE+, <span class="html-italic">n</span> = 27) and non-RCE patients (RCE−, <span class="html-italic">n</span> = 118). HsCRP levels were then analyzed in the subgroups of patients experiencing MACE (MACE+ group, <span class="html-italic">n</span> = 18, among which 14 patients were MACE+AHF− and 4 patients experienced both MACE and AHF episodes during the first year post PCI) or AHF (AHF+ group, <span class="html-italic">n</span> = 13, 9 AHF+MACE− and 4 AHF+MACE+ patients) analyzed in reference to patients without MACE (MACE−, <span class="html-italic">n</span> = 127) or free of AHF (AHF−, <span class="html-italic">n</span> = 132) at one year post ACS. hsCRP variables are described in box and whiskers plots as medians and (10–90 percentile) using GraphPad Prism. Comparison between groups were performed using Mann–Whitney non-parametric unpaired <span class="html-italic">t</span> tests. <span class="html-italic">p</span> values &lt; 0.05 were reported as significant. (<b>B</b>) Flow cytometry analysis of the percentage of CD14+ monocytes expressing the CD16 receptor. Peripheral blood monocytes expression of CD16 could be analyzed in 24 RCE patients analyzed in reference to a group of 24 RCE-free patients (RCE−) without a MACE or AHF event, matched for age, gender, and CKD status. Monocyte CD16 expression in RCE patients was further analyzed in a subgroup of 12 MACE+AHF− patients and 12 AHF+ patients (among which 3 were AHF+MACE+ and also experienced a MACE event, and 9 were AHF+MACE− and only experienced AHR during the first-year post ACS). Groups of RCE+, MACE+, and AHR+ patients were analyzed using Mann–Whitney non-parametric unpaired <span class="html-italic">t</span> tests and compared to proportion of CD16+CD14+ monocytes observed in gender-, age-, and CKD status-matched groups of AHR−MACE− patients. <span class="html-italic">p</span> values &lt; 0.05 were reported as significant. <span class="html-italic">p</span> values ≥ 0.05 and &lt;0.2 were indicated as a trend for significance.</p> Full article ">Figure 3
<p><span class="html-italic">FCGR2A</span> polymorphisms are associated with baseline hsCRP levels and CD32 expression in CD14+ monocytes. (<b>A</b>) Baseline hsCRP levels were analyzed in group of patients with ACS according to the presence of the H (<span class="html-italic">n</span> = 113) or R allelic variants (<span class="html-italic">n</span> = 102). HH (<span class="html-italic">n</span> = 43), HR (<span class="html-italic">n</span> = 70), and RR (<span class="html-italic">n</span> = 32) refer to the homozygous of heterozygous combinations of FCGR2A allelic variants at the extracellular amino acid position 131: arginine (R) or histidine (H). Groups of RCE+, MACE+, and AHR+ patients were analyzed using Mann–Whitney non-parametric unpaired <span class="html-italic">t</span> tests and compared to the control group of AHR−MACE− patients. <span class="html-italic">p</span> values &lt; 0.05 were reported as significant. hsCRP variables are described as box and whiskers plots representing medians and (10–90%) using GraphPad Prism. Comparison between groups were performed using Mann–Whitney non-parametric unpaired <span class="html-italic">t</span> tests. <span class="html-italic">p</span> values &lt; 0.05 were reported as significant. (<b>B</b>) The expression of CD32 on circulating CD14+ monocytes (%CD32+CD14+ cells) were analyzed in a subgroup of 58 patients with ACS according to the presence of the H or R allelic variants. HH, HR, and RR refer to the homozygous of heterozygous combinations of FCGR2A allelic variants at the extracellular amino acid position 131: arginine (R) or histidine (H). Proportion of CD32+ monocytes are described using GraphPad Prism as box and whiskers plots representing medians and (10–90%). Comparison between groups were performed using Mann–Whitney non-parametric unpaired <span class="html-italic">t</span> tests. <span class="html-italic">p</span> values &lt; 0.05 were reported as significant, and <span class="html-italic">p</span> values ≥ 0.05 and &lt;0.2 were indicated as a trend for significance.</p> Full article ">Figure 4
<p>Evaluation of serum-induced activation of CD32-H-dependent activation in patients who bear the <span class="html-italic">FCGR2A</span> H allelic variants (HH and H/R genotypes). We assessed the serum-induced ADCP activation potential of serum from patients with the H allele at position 131 of the CD32 protein using a reporter luciferase assay in Jurkat cells transfected with the CD32-H variant. Results are expressed as fold RLU induction analyzed in reference to human AB serum of a non-immunized healthy male control donor. Recombinant CRP was diluted in the human male SAB control serum using different concentration (400 mg/mL, 100 mg/L, 25 mg/L, 6.25 mg/L). Variables that quantify serum-induced ADCP were compared in the group of RCE+ (<span class="html-italic">n</span> = 15) and REC− patients (<span class="html-italic">n</span> = 19) that bear the FCGR2A H allelic variant. The RCE-positive group was further stratified as MACE+AHF− patients (<span class="html-italic">n</span> = 7) and AHF+MACE− patients (<span class="html-italic">n</span> = 8). Serum-induced ADCP activation is described as box and whiskers plots representing medians and (10–90%) using GraphPad Prism. Comparison between groups were performed using Mann–Whitney non-parametric unpaired <span class="html-italic">t</span> tests. <span class="html-italic">p</span> values &lt; 0.05 were reported as significant. <span class="html-italic">p</span> values ≥ 0.05 and &lt;0.2 were indicated as a trend for significance.</p> Full article ">Figure 5
<p>Distribution of FCGR2A rs1801274 and FCGR3A Rs 396,991 genetic allelic variants associated with development of RCE during the first year after initial ACS diagnosis. (<b>A</b>) Distribution of the FCGR2A genotype in the whole cohort of patients with ACS (all patients) subdivided into patients who developed RCE and those who remained RCE free (no RCE). HH, HR, and RR refer to the homozygous or heterozygous combinations of FCGR2A allelic variants at the extracellular amino acid position 131: arginine (R) or histidine (H). Frequencies in patient groups were analyzed in reference to those in healthy control groups. (<b>B</b>) FF, VF, and VV refer to the combinations of FCGR3A allelic variants at position 158: valine (V) or phenylalanine (F). FF indicates the homozygous phenylalanine genotype; VV indicates the homozygous alanine genotype; VF indicates the heterozygous valine/phenylalanine genotype. RCE, recurrence of cardiovascular events; CTL, healthy control individuals.</p> Full article ">
12 pages, 1535 KiB  
Article
Obesity Reshapes the Microbial Population Structure along the Gut-Liver-Lung Axis in Mice
by Apostolos Galaris, Dionysios Fanidis, Elli-Anna Stylianaki, Vaggelis Harokopos, Alexandra-Styliani Kalantzi, Panagiotis Moulos, Antigone S. Dimas, Pantelis Hatzis and Vassilis Aidinis
Biomedicines 2022, 10(2), 494; https://doi.org/10.3390/biomedicines10020494 - 19 Feb 2022
Cited by 7 | Viewed by 2961
Abstract
The microbiome is emerging as a major player in tissue homeostasis in health and disease. Gut microbiome dysbiosis correlates with several autoimmune and metabolic diseases, while high-fat diets and ensuing obesity are known to affect the complexity and diversity of the microbiome, thus [...] Read more.
The microbiome is emerging as a major player in tissue homeostasis in health and disease. Gut microbiome dysbiosis correlates with several autoimmune and metabolic diseases, while high-fat diets and ensuing obesity are known to affect the complexity and diversity of the microbiome, thus modulating pathophysiology. Moreover, the existence of a gut-liver microbial axis has been proposed, which may extend to the lung. In this context, we systematically compared the microbiomes of the gut, liver, and lung of mice fed a high-fat diet to those of littermates fed a matched control diet. We carried out deep sequencing of seven hypervariable regions of the 16S rRNA microbial gene to examine microbial diversity in the tissues of interest. Comparison of the local microbiomes indicated that lung tissue has the least diverse microbiome under healthy conditions, while microbial diversity in the healthy liver clustered closer to the gut. Obesity increased microbial complexity in all three tissues, with lung microbial diversity being the most modified. Obesity promoted the expansion of Firmicutes along the gut-liver-lung axis, highlighting staphylococcus as a possible pathologic link between obesity and systemic pathophysiology, especially in the lungs. Full article
(This article belongs to the Special Issue Microbial Ecology in Health and Disease 2.0)
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<p>Mice fed with a high-fat diet (HFD) developed obesity and non-alcoholic liver fatty liver disease (NAFLD). Mice fed with HFD for 16 weeks presented with (<b>A</b>) statistically significant higher body weight from the tenth week of HFD onwards, (<b>B</b>) elevated ALT levels, and (<b>C</b>) decreased AST/ALT ratio in plasma after 16 weeks of HFD. (<b>D</b>) Representative images from the histopathology (hematoxylin &amp; eosin staining) of gut, liver, and lung tissues, illustrating lipid deposition in the liver and gut after 16 weeks of HFD. Hematoxylin stains cell nuclei (purple) and eosin stains the extracellulal matrix (pink). The “bubbles” appearing in HDF liver and gut samples are lipid droplets. Statistical significance was assessed through the Friedman test followed by pairwise Mann-Whitney tests (in <b>A</b>) and Mann-Whitney tests (in <b>B</b> and <b>C</b>); * <span class="html-italic">p</span> &lt; 0.05 was considered significant.</p> Full article ">Figure 2
<p>HFD-driven obesity triggers changes in the microbiome composition of all tissues examined. (<b>A</b>) HFD-driven obesity results in a greater number of detected Amplicon Sequence Variants (ASVs) in all tissues. (<b>B</b>) α-diversity per sample and diet. Shannon’s index was used to evaluate sample biodiversity per tissue and diet. (<b>C</b>) Similarity of samples per tissue and diet as described by β-diversity. Aitchison distance was used to account for the compositional nature of 16S rRNA sequencing data. (<b>D</b>) Venn diagrams of common phyla or (<b>E</b>) families upon CD or HFD in gut, liver, and lung.</p> Full article ">Figure 3
<p>Relative abundance of phyla detected in gut, liver, and lung of control and HFD-fed mice. Heatmap and respective dendrogram of non-zero abundance phyla under CD (<b>A</b>) and HFD (<b>B</b>). (<b>C</b>) Relative abundance of all detected phyla. Relative abundance calculations for all panels were based on GCN values. For panels A and B, only phyla with an abundance greater than zero after value rounding to two decimal places were considered. Heatmaps are scaled per phylum. Manhattan distance was used to perform hierarchical clustering of the tissues with complete linkage in panels A and B.</p> Full article ">Figure 4
<p>HFD consistently affects specific microbial families and genera. (<b>A</b>) Heatmap and tissue dendrogram of HFD-to-CD differences in family relative abundance (<b>B</b>) Heatmap of HFD-to-CD difference in genera relative abundance. For both panels, only those taxa with HFD-to-CD relative abundance differences other than zero in at least one tissue after rounding to two decimal places were considered. Heatmaps are scaled per taxon. Manhattan distance was used to perform hierarchical clustering of tissues with complete linkage. Relative abundance was calculated based on GCN abundance values. Families and respective genera are coded with the same color in both panels.</p> Full article ">
18 pages, 7442 KiB  
Article
Synthesis and Characterization of Fatty Acid Grafted Chitosan Polymeric Micelles for Improved Gene Delivery of VGF to the Brain through Intranasal Route
by Richard Nii Lante Lamptey, Avinash Gothwal, Riddhi Trivedi, Sanjay Arora and Jagdish Singh
Biomedicines 2022, 10(2), 493; https://doi.org/10.3390/biomedicines10020493 - 19 Feb 2022
Cited by 23 | Viewed by 3685
Abstract
Multifunctional fatty acid grafted polymeric micelles are an effective and promising approach for drug and gene delivery to the brain. An alternative approach to bypass the blood–brain barrier is administration through intranasal route. Multifunctional fatty acid grafted polymeric micelles were prepared and characterized [...] Read more.
Multifunctional fatty acid grafted polymeric micelles are an effective and promising approach for drug and gene delivery to the brain. An alternative approach to bypass the blood–brain barrier is administration through intranasal route. Multifunctional fatty acid grafted polymeric micelles were prepared and characterized for pVGF delivery to the brain. In vitro pVGF expression was analyzed in bEnd.3 cells, primary astrocytes, and neurons. Comparative in-vivo pVGF expression was analyzed to evaluate the effective route of administration between intranasal and intravenous. Biocompatible, multifunctional polymeric micelles were prepared, having an average size of 200 nm, and cationic zeta potential. Modified polymers were found to be hemo- and cyto-compatible. When transfected with the different modified chitosan formulations, significantly (p < 0.05) higher VGF expression was observed in primary astrocytes and neurons using the mannose, Tat peptide, and oleic acid grafted chitosan polymer. Compared to intravenous administration, intranasal administration of pVGF in polyplex formulation led to significantly (p < 0.05) higher pVGF expression. Developed multifunctional polymeric micelles were an effective pVGF delivery platform to the brain. Mannose and Tat ligand tagging improved the pVGF delivery to the brain. Full article
(This article belongs to the Special Issue Lipid-Based Nanocarriers)
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<p>The <sup>1</sup>H-NMR spectra of CS and synthesized OA-g-CS, OA-g-CS-Man, OA-g-CS-Tat, and OA-g-CS-Man-Tat conjugates.</p> Full article ">Figure 2
<p>FT-IR spectra of CS and synthesized OA-g-CS, OA-g-CS-Man, OA-g-CS-Tat, and OA-g-CS-Man-Tat conjugates.</p> Full article ">Figure 3
<p>(<b>A</b>) Analysis of OA-g-CS-Man-Tat–pVGF complex formation at different weight ratios by agarose gel electrophoresis. (<b>B</b>) DNase I protection assay showing protection of DNA from degradation by polymers at the different N/P ratios.</p> Full article ">Figure 4
<p>DNase protecting effect of different polymeric micelles containing pVGF. Column (<b>A</b>) Untreated pVGF, column (<b>B</b>) pVGF treated with DNase, column (<b>C</b>) CS–pVGF column (<b>D</b>) OA-g-CS–pVGF, column (<b>E</b>) OA-g-CS-Man–pVGF, column (<b>F</b>) OA-g-CS-Tat–pVGF, Column (<b>G</b>) OA-g-CS-Man-Tat–pVGF.</p> Full article ">Figure 5
<p>Hemolytic activity of chitosan-modified polymers at different concentrations. A total of 1% (<span class="html-italic">v</span>/<span class="html-italic">v</span>) Triton X-100 was used as a positive control (hemolytic activity was set as 100%) at 37 °C.</p> Full article ">Figure 6
<p>Images of hemolysis caused following 1 h incubation with different polymers at different concentrations.</p> Full article ">Figure 7
<p>In vitro cytocompatibility of CS, OA-g-CS, OA-g-CS-Man, OA-g-CS-Tat, and OA-g-CS-Man-Tat polymeric micelles at different concentrations (0.125–1000 µg/mL) in bEnd.3 cells. MTT was used to evaluate cell viability, data presented as mean ± SD (n = 4).</p> Full article ">Figure 8
<p>Qualitative analysis of in vitro transfection efficiency of naked pGFP, CS–pGFP, OA-g-CS–pGFP, OA-g-CS-Man–pGFP, OA-g-CS-Tat–pGFP, and OA-g-CS-Man-Tat–pGFP against primary astrocytes, primary neurons, and bEnd.3 cells. Images were taken at 20× magnification.</p> Full article ">Figure 9
<p>VGF expression levels in primary astrocytes and primary neurons after treatment with CS–pVGF, OA-g-CS–pVGF, and OA-g-CS-Man-Tat–pVGF polyplexes. Results presented as mean ± SD (<span class="html-italic">n</span> = 4). (“*” indicates significantly (<span class="html-italic">p</span> &lt; 0.05) different from naked pDNA “#” indicates significantly (<span class="html-italic">p</span> &lt; 0.01) different from respective naked pDNA and “<span>$</span>” indicates significantly (<span class="html-italic">p</span> &lt; 0.001) different from respective naked pDNA).</p> Full article ">Figure 10
<p>In-vivo VGF expression after treating C75BI/6 J mice with CS–pVGF, OA-g-CS–pVGF, and OA-g-CS-Man-Tat–pVGF polyplexes using the intranasal and intravenous route. Results are presented as mean ± SD (<span class="html-italic">n</span> = 6). (“*” indicates significantly (<span class="html-italic">p</span> &lt; 0.05) different from intravenous CS “&amp;” indicates significantly (<span class="html-italic">p</span> &lt; 0.05) different from intravenous OA-g-CS-Man-Tat–pVGF). NS represents non significance.</p> Full article ">Figure 11
<p>H and E staining of heart, spleen, kidney, lung, liver, and brain, showing no toxicity from different groups after drug administration. Images were taken at 20× magnification and the scale bar is 75 µm.</p> Full article ">
10 pages, 1387 KiB  
Article
LL-37 and Double-Stranded RNA Synergistically Upregulate Bronchial Epithelial TLR3 Involving Enhanced Import of Double-Stranded RNA and Downstream TLR3 Signaling
by Sara Bodahl, Samuel Cerps, Lena Uller and Bengt-Olof Nilsson
Biomedicines 2022, 10(2), 492; https://doi.org/10.3390/biomedicines10020492 - 19 Feb 2022
Cited by 5 | Viewed by 2998
Abstract
The human host defense peptide LL-37 influences double-stranded RNA signaling, but this process is not well understood. Here, we investigate synergistic actions of LL-37 and synthetic double-stranded RNA (poly I:C) on toll-like receptor 3 (TLR3) expression and signaling, and examine underlying mechanisms. In [...] Read more.
The human host defense peptide LL-37 influences double-stranded RNA signaling, but this process is not well understood. Here, we investigate synergistic actions of LL-37 and synthetic double-stranded RNA (poly I:C) on toll-like receptor 3 (TLR3) expression and signaling, and examine underlying mechanisms. In bronchial epithelial BEAS-2B cells, LL-37 potentiated poly I:C-induced TLR3 mRNA and protein expression demonstrated by qPCR and Western blot, respectively. Interestingly, these effects were associated with increased uptake of rhodamine-tagged poly I:C visualized by immunocytochemistry. The LL-37/poly I:C-induced upregulation of TLR3 mRNA expression was prevented by the endosomal acidification inhibitor chloroquine, indicating involvement of downstream TLR3 signaling. The glucocorticoid dexamethasone reduced LL-37/poly I:C-induced TLR3 expression on both mRNA and protein levels, and this effect was associated with increased IκBα protein expression, suggesting that dexamethasone acts via attenuation of NF-κB activity. We conclude that LL-37 potentiates poly I:C-induced upregulation of TLR3 through a mechanism that may involve enhanced import of poly I:C and that LL-37/poly I:C-induced TLR3 expression is associated with downstream TLR3 signaling and sensitive to inhibition of NF-κB activity. Full article
(This article belongs to the Special Issue Antimicrobial Peptides Aka Host Defense Peptides – from Basic Research to Therapy)
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<p>LL-37/poly I:C-induced stimulation of TLR3 expression is reduced by dexamethasone in BEAS-2B cells. (<b>A</b>,<b>B</b>) TLR3 mRNA expression was determined using quantitative real-time RT-PCR in cells treated with or without poly I:C (10 µg/mL) alone, or LL-37 (1 µM) and poly I:C (10 µg/mL) in combination in the presence or absence of dexamethasone (1 µM) for 6 h (<b>A</b>) and 24 h (<b>B</b>). (<b>C</b>) TLR3 protein expression was assessed using Western blot in cells treated with or without LL-37 (1 µM) and poly I:C (10 µg/mL) in combination (LL-37/poly I:C) in the presence or absence of dexamethasone (1 µM) for 24 h. The TLR3 immunoreactive band was observed at the expected molecular weight of 115–130 kDa and normalized to GAPDH (37 kDa), serving as internal control. (<b>A</b>–<b>C</b>) Data are presented as mean ± SEM, <span class="html-italic">n</span> = 7–8 (<b>A</b>,<b>B</b>) and <span class="html-italic">n</span> = 6 (<b>C</b>) in each group. Statistical significance was calculated using a one-way ANOVA, followed by Tukey’s post hoc test. ** and *** represent <span class="html-italic">p</span> &lt; 0.01 and <span class="html-italic">p</span> &lt; 0.001, respectively, vs. control. For comparisons indicated by the bars, * and *** represent <span class="html-italic">p</span> &lt; 0.05 and <span class="html-italic">p</span> &lt; 0.001, respectively.</p> Full article ">Figure 2
<p>Dexamethasone reduces NF-κB activity in BEAS-2B cells by increasing expression of IκBα. (<b>A</b>–<b>C</b>) Western blot analysis of IκBα (<b>A</b>), NF-κB phosphorylated p65 (<b>B</b>) and NF-κB phosphorylated p105 (<b>C</b>) was performed in cells stimulated with or without poly I:C (10 µg/mL) and LL-37 (1 µM) in combination in the presence or absence of dexamethasone (1 µM) for 24 h. The immunoreactive bands were observed at the expected molecular weight of 39 kDa (IκBα), 65 kDa (phosphorylated p65) and 105 kDa (phosphorylated p105), and the intensity of each band was normalized to GAPDH (37 kDa), serving as internal control. Data are presented as mean ± SEM, <span class="html-italic">n</span> = 7–8 in each group. Statistical significance was calculated using a one-way ANOVA followed by Tukey’s post hoc test. * represents <span class="html-italic">p</span> &lt; 0.05 vs. control. For comparisons indicated by the bars, ** represents <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 3
<p>LL-37/poly I:C-induced stimulation of TLR3 mRNA expression is abolished by chloroquine in BEAS-2B cells. (<b>A</b>,<b>B</b>) TLR3 mRNA expression was analyzed using quantitative real-time RT-PCR in cells treated with or without poly I:C (0.2 or 2 µg/mL) alone, LL-37 (1 µM) alone, or the two in combination for 6 (<b>A</b>) and 24 h (<b>B</b>). (<b>C</b>) TLR3 mRNA expression was determined in cells treated with poly I:C (0.2 µg/mL), LL-37 (1 µM), or the two in combination in the presence or absence of chloroquine (2 µg/mL) for 6 h. (<b>A</b>–<b>C</b>) Data are presented as mean ± SEM, <span class="html-italic">n</span> = 8–12 (<b>A</b>), <span class="html-italic">n</span> = 6–8 (<b>B</b>) and <span class="html-italic">n</span> = 4 (<b>C</b>) in each group. Statistical significance was calculated using a one-way ANOVA, followed by Tukey’s post hoc test. *** represents <span class="html-italic">p</span> &lt; 0.001 vs. control or <span class="html-italic">p</span> &lt; 0.001 for comparisons indicated by the bars.</p> Full article ">Figure 4
<p>High but not low concentration of LL-37 stimulates poly I:C-induced upregulation of TLR3 in BEAS-2B cells. (<b>A</b>) TLR3 mRNA expression was evaluated using quantitative real-time RT-qPCR in cells treated with poly I:C (0.2 µg/mL), LL-37 (4 µM) or LL-37 (1 and 4 µM) and poly I:C (0.2 µg/mL) in combination for 24 h. (<b>B</b>) TLR3 protein expression was evaluated using Western blot in cells treated with poly I:C (0.2 µg/mL) alone or poly I:C in combination with LL-37 (4 µM) for 24 h. The TLR3 immunoreactive band was observed at the expected molecular weight of 115-130 kDa and normalized to GAPDH (37 kDa) serving as internal control. (<b>C</b>) Cell viability was assessed using the MTT assay in cells treated with LL-37 (0.1, 1, 4 and 10 µM) for 4 h. (<b>A</b>-<b>C</b>) Data are presented as mean ± SEM, <span class="html-italic">n</span> = 8 (<b>A</b>,<b>B</b>) and <span class="html-italic">n</span> = 6–8 (<b>C</b>) in each group. Statistical significance was calculated using a one-way ANOVA, followed by Tukey’s post hoc test. *** represents <span class="html-italic">p</span> &lt; 0.001 vs. control. For comparisons indicated by the bars, ** and *** represent <span class="html-italic">p</span> &lt; 0.01 and <span class="html-italic">p</span> &lt; 0.001, respectively.</p> Full article ">Figure 5
<p>LL-37 triggers import of poly I:C in BEAS-2B cells. (<b>A</b>–<b>D</b>) Cells were treated with fluorescent rhodamine-tagged poly I:C (4 µg/mL) alone or in combination with LL-37 (4 µM) for 6 (<b>A</b>,<b>B</b>) and 24 h (<b>C</b>,<b>D</b>). The intracellular fluorescence signal (red) and the nuclei staining with DAPI (blue) were analyzed and photographed using a fluorescence microscope equipped with a digital camera. The bars in panel <b>A</b> and <b>C</b> represent 40 µm. The fluorescence intensity of five cells was measured in three different areas on each coverslip, and an average cellular fluorescence intensity was calculated for each experiment. Data are presented as mean ± SEM, <span class="html-italic">n</span> = 4 in each group representing the number of independent experiments. Statistical significance was calculated using a one-way ANOVA, followed by Tukey’s post hoc test. ** represents <span class="html-italic">p</span> &lt; 0.01 vs. control. For comparisons indicated by the bars, ** represents <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">
21 pages, 8506 KiB  
Article
PTML Modeling for Pancreatic Cancer Research: In Silico Design of Simultaneous Multi-Protein and Multi-Cell Inhibitors
by Valeria V. Kleandrova and Alejandro Speck-Planche
Biomedicines 2022, 10(2), 491; https://doi.org/10.3390/biomedicines10020491 - 18 Feb 2022
Cited by 25 | Viewed by 3371
Abstract
Pancreatic cancer (PANC) is a dangerous type of cancer that is a major cause of mortality worldwide and exhibits a remarkably poor prognosis. To date, discovering anti-PANC agents remains a very complex and expensive process. Computational approaches can accelerate the search for anti-PANC [...] Read more.
Pancreatic cancer (PANC) is a dangerous type of cancer that is a major cause of mortality worldwide and exhibits a remarkably poor prognosis. To date, discovering anti-PANC agents remains a very complex and expensive process. Computational approaches can accelerate the search for anti-PANC agents. We report for the first time two models that combined perturbation theory with machine learning via a multilayer perceptron network (PTML-MLP) to perform the virtual design and prediction of molecules that can simultaneously inhibit multiple PANC cell lines and PANC-related proteins, such as caspase-1, tumor necrosis factor-alpha (TNF-alpha), and the insulin-like growth factor 1 receptor (IGF1R). Both PTML-MLP models exhibited accuracies higher than 78%. Using the interpretation from one of the PTML-MLP models as a guideline, we extracted different molecular fragments desirable for the inhibition of the PANC cell lines and the aforementioned PANC-related proteins and then assembled some of those fragments to form three new molecules. The two PTML-MLP models predicted the designed molecules as potentially versatile anti-PANC agents through inhibition of the three PANC-related proteins and multiple PANC cell lines. Conclusions: This work opens new horizons for the application of the PTML modeling methodology to anticancer research. Full article
(This article belongs to the Special Issue Pancreatic Cancer: From Mechanisms to Therapeutic Approaches)
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<p>The different <span class="html-italic">D</span>(<span class="html-italic">GTI</span>)<span class="html-italic">cj</span> descriptors and their relative significances in <span class="html-italic">Model 1</span>.</p> Full article ">Figure 2
<p>Relative importance of the different <span class="html-italic">D</span>(<span class="html-italic">GTI</span>)<span class="html-italic">cj</span> descriptors in <span class="html-italic">Model 2</span>.</p> Full article ">Figure 3
<p>Generic molecular fragments directly extracted from the physicochemical and structural interpretation of the descriptors in <span class="html-italic">Model 2</span>. The descriptors are associated with different fragments. The symbols mean A = amino, hydroxyl, alkylamino, or alkoxy; G = halogen; Q = amino, hydroxyl, alkylamino, alkoxy, or a non-substituted secondary carbon; X = O, -NH-, or a secondary carbon; Z = N or aromatic carbon.</p> Full article ">Figure 4
<p>New molecules designed from suitable molecular fragments by using the physicochemical and structural interpretations as guidelines.</p> Full article ">
22 pages, 1127 KiB  
Review
The Mitochondrial Genome in Aging and Disease and the Future of Mitochondrial Therapeutics
by Sanjana Saravanan, Caitlin J. Lewis, Bhavna Dixit, Matthew S. O’Connor, Alexandra Stolzing and Amutha Boominathan
Biomedicines 2022, 10(2), 490; https://doi.org/10.3390/biomedicines10020490 - 18 Feb 2022
Cited by 5 | Viewed by 5962
Abstract
Mitochondria are intracellular organelles that utilize nutrients to generate energy in the form of ATP by oxidative phosphorylation. Mitochondrial DNA (mtDNA) in humans is a 16,569 base pair double-stranded circular DNA that encodes for 13 vital proteins of the electron transport chain. Our [...] Read more.
Mitochondria are intracellular organelles that utilize nutrients to generate energy in the form of ATP by oxidative phosphorylation. Mitochondrial DNA (mtDNA) in humans is a 16,569 base pair double-stranded circular DNA that encodes for 13 vital proteins of the electron transport chain. Our understanding of the mitochondrial genome’s transcription, translation, and maintenance is still emerging, and human pathologies caused by mtDNA dysfunction are widely observed. Additionally, a correlation between declining mitochondrial DNA quality and copy number with organelle dysfunction in aging is well-documented in the literature. Despite tremendous advancements in nuclear gene-editing technologies and their value in translational avenues, our ability to edit mitochondrial DNA is still limited. In this review, we discuss the current therapeutic landscape in addressing the various pathologies that result from mtDNA mutations. We further evaluate existing gene therapy efforts, particularly allotopic expression and its potential to become an indispensable tool for restoring mitochondrial health in disease and aging. Full article
(This article belongs to the Special Issue Mitochondrial Genetics and Pathologies)
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<p>Schematic for Allotopic Expression. The various steps involved in the successful implementation of the allotopic expression strategy are depicted, beginning with design of the optimal DNA expression construct and ending with incorporation of the exogenous protein into the correct RC complex. HR: homologous region, UTR: untranslated region, MTS: mitochondrial targeting sequence, IMS: intermembrane space, IM: inner membrane, RC: respiratory chain.</p> Full article ">Figure 2
<p>Frequency of codon use in human nuclear and mitochondrial genomes. Left column: nuclear codon usage; right column: mitochondrial codon usage for respective amino acids.</p> Full article ">
21 pages, 5501 KiB  
Article
The Inflammatory Pattern of Chronic Limb-Threatening Ischemia in Muscles: The TNF-α Hypothesis
by Diego Caicedo, Clara V. Alvarez, Sihara Perez-Romero and Jesús Devesa
Biomedicines 2022, 10(2), 489; https://doi.org/10.3390/biomedicines10020489 - 18 Feb 2022
Cited by 4 | Viewed by 2699
Abstract
Background: Vascular inflammation plays a crucial role in peripheral arterial disease (PAD), although the role of the mediators involved has not yet been properly defined. The aim of this work is to investigate gene expression and plasma biomarkers in chronic limb-threating ischemia [...] Read more.
Background: Vascular inflammation plays a crucial role in peripheral arterial disease (PAD), although the role of the mediators involved has not yet been properly defined. The aim of this work is to investigate gene expression and plasma biomarkers in chronic limb-threating ischemia (CLTI). Methods: Using patients from the GHAS trial, both blood and ischemic muscle samples were obtained to analyze plasma markers and mRNA expression, respectively. Statistical analysis was performed by using univariate (Spearman, t-Student, and X2) and multivariate (multiple logistic regression) tests. Results: A total of 35 patients were available at baseline (29 for mRNA expression). Baseline characteristics (mean): Age: 71.4 ± 12.4 years (79.4% male); TNF-α: 10.7 ± 4.9 pg/mL; hsCRP:1.6 ± 2.2 mg/dL; and neutrophil-to-lymphocyte ratio (NLR): 3.5 ± 2.8. Plasma TNF-α was found elevated (≥8.1) in 68.6% of patients, while high hsCRP (≥0.5) was found in 60.5%. Diabetic patients with a high level of inflammation showed significantly higher levels of NOX4 expression at baseline (p = 0.0346). Plasma TNF-α had a negative correlation with NOS3 (eNOS) expression (−0.5, p = 0.015) and plasma hsCRP with VEGFA (−0.63, p = 0.005). The expression of NOX4 was parallel to that of plasma TNF-α (0.305, p = 0.037), especially in DM. Cumulative mortality at 12 months was related to NLR ≥ 3 (p = 0.019) and TNF-α ≥ 8.1 (p = 0.048). The best cutoff point for NLR to predict mortality was 3.4. Conclusions: NOX4 and TNF-α are crucial for the development and complications of lower limb ischemia, especially in DM. hsCRP could have a negative influence on angiogenesis too. NLR and TNF-α represent suitable markers of mortality in CLTI. These results are novel because they connect muscle gene expression and plasma information in patients with advanced PAD, deepening the search for new and accurate targets for this condition. Full article
(This article belongs to the Special Issue Vascular Inflammation: From Target Molecules to Therapeutic Approaches)
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<p><b>mRNA expression of different genes at baseline in both groups, GH, and placebo) in DM and non-DM patients</b>. (<b>A</b>). <span class="html-italic">NOX4</span>: NADPH (Nicotinamide adenine dinucleotide phosphate oxidase) 4; (<b>B</b>). <span class="html-italic">VEGFA</span>: Vascular endothelial growth factor A; (<b>C</b>). <span class="html-italic">MSTN</span>: Myostatin; (<b>D</b>). <span class="html-italic">KDR</span>: VEGFA receptor 1. (<b>E</b>). <span class="html-italic">NOS3</span> or <span class="html-italic">eNOS</span>: Nitric oxide synthase 3 or endothelial NOS; (<b>F</b>). <span class="html-italic">MYOG</span>: Myogenin; (<b>G</b>). <span class="html-italic">IL6</span>: Interleukin 6; (<b>H</b>). <span class="html-italic">IGF1</span>: Insulin-like growth factor I; GH: Growth hormone; Placebo: control group; Basal: Baseline. * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 2
<p><b>Basal to final mRNA expression of different genes.</b> <span class="html-italic">NOX4</span> (<b>A</b>), <span class="html-italic">TNF</span> (<span class="html-italic">TNF</span>-α) (<b>B</b>), and <span class="html-italic">KDR</span> (<b>C</b>). Findings in both groups, GH, and placebo, with DM and without DM. * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 3
<p><b>Baseline plasma levels of TNF-α related to different muscle gene expressions in DM patients.</b> Both groups: GH and placebo. Correlation between plasma TNF-α and muscle <span class="html-italic">TNF</span> (<b>A</b>), <span class="html-italic">NOS3</span> (<b>B</b>), <span class="html-italic">VEGFA</span> (<b>C</b>), and <span class="html-italic">IGF-1</span> (<b>D</b>) mRNA expressions. TBP: housekeeping gene used as an expression control gene. * <span class="html-italic">p</span> &lt; 0.05; and ** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 4
<p><b>Relationship between high levels of plasma TNF-α (≥8.1 pg/mL) and <span class="html-italic">NOX4</span> expression in all patients of the study</b>. (<b>A</b>). Left: expression of muscle <span class="html-italic">NOX4</span> mRNA in relation to plasma TNF-α. Striped bar: TNF-α &lt; 8.1 pg/mL. White bar: TNF-α ≥ 8.1 pg/mL. Right: Spearman correlation between both markers. (<b>B</b>). Trend of this correlation differentiating by TNF-α values: ≥8.1 (green color) and &lt;8.1 (blue color). * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 5
<p><b>Correlation between plasma hsCRP and muscle gene expression in diabetic patients.</b> (<b>A</b>). Baseline expression levels of hsCRP and <span class="html-italic">NOS3</span> in the GH group. (<b>B</b>). Baseline hsCRP and <span class="html-italic">VEGFA</span> levels in both groups. (<b>C</b>). Final hsCRP and redox stress levels measured by final <span class="html-italic">NOX4</span> in the GH-treated patients. (<b>D</b>). Final hsCRP levels were related to final <span class="html-italic">MSTN</span> mRNA expression in GH group. Striped bars: hsCRP &lt; 0.5; White bars: hsCRP ≥ 0.5. * <span class="html-italic">p</span> &lt; 0.05; and ** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 6
<p><b>Correlation of plasma HbA1C and muscle mRNA expression in diabetic patients.</b> (<b>A</b>). <span class="html-italic">TNF</span>. (<b>B</b>)<b>.</b> <span class="html-italic">IGF-1</span>. <b>C.</b> <span class="html-italic">VEGFA</span>. (<b>A</b>,<b>B</b>) represent correlations at baseline, while (<b>C</b>) represents final time point. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 7
<p><b>Cumulative 12-month mortality and plasma markers</b>. (<b>A</b>). Basal TNF-α; (<b>B</b>). Basal NLR (Neutrophil-to-lymphocyte ratio). * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 8
<p><b>Relationship between NLR and plasma TNF-</b>α <b>with mortality.</b> (<b>A</b>). Kaplan-Meier analysis of NLR and mortality. (<b>B</b>). AUC-ROC (Area Under the Curve-Receiver Operating Characteristics) curve of NLR related to mortality. (<b>C</b>). AUC-ROC curve of plasma TNF-α related to mortality. Color blue and yellow represent confidence intervals. Values in brackets represents sensitivity and specificity, respectively (<b>D</b>). Comparation between plasma TNF-α and NLR as predictors of mortality showed no significant differences (<span class="html-italic">p</span> = 0.9264).</p> Full article ">Figure 9
<p><b>Relationship between basal levels of NLR and plasma hsCRP.</b> (<b>A</b>). Spearman correlation. (<b>B</b>). Histogram comparing hsCRP ≥ 0.5 and NLR ≥ 2.5. Green bars: NLR ≥ 2.5; White bars: NLR &lt; 2.5. *** <span class="html-italic">p</span> &lt; 0.0001.</p> Full article ">Figure 10
<p>(<b>A</b>): Relation between different muscle genes and clinical variables in both groups. (<b>B</b>): Graph showing the link between <span class="html-italic">NOX4</span> mRNA expression and Rutherford class, grouped in 1: less severity of ischemia, and 2: more severity of ischemia. White box: basal level of <span class="html-italic">NOX4</span>; Green bars: final level of <span class="html-italic">NOX4</span> in the GH group; and Blue bars: final level of <span class="html-italic">NOX4</span> in the placebo group. Striped bars: group with severe ischemia. ** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">
7 pages, 678 KiB  
Communication
A Comparison of Pharyngeal Swabs and Tracheal Secretions for the Diagnosing of COVID-19
by Maibritt Meldgaard Arildsen, Sif Bay Glenting, Anette Marianne Fedder, Bettina Jørgensen, Svend Ellermann-Eriksen, Marianne Kragh Thomsen, Christina Catherine Dahm and Michael Pedersen
Biomedicines 2022, 10(2), 488; https://doi.org/10.3390/biomedicines10020488 - 18 Feb 2022
Cited by 1 | Viewed by 2547
Abstract
The aim of this study was to compare the test results from patients who, within a short timescale, have been tested for COVID-19 using both a pharyngeal swab and tracheal secretion. Data were collected from the database of AUH, from patients hospitalized between [...] Read more.
The aim of this study was to compare the test results from patients who, within a short timescale, have been tested for COVID-19 using both a pharyngeal swab and tracheal secretion. Data were collected from the database of AUH, from patients hospitalized between 1 March 2020 and 1 March 2021 who, due to symptoms of COVID-19, were tested by a pharyngeal swab and by tracheal secretion. We found great agreement between oropharyngeal swab and tracheal secretion RT-PCR testing for the diagnosis of COVID-19, with 98.5% of double tests being concordant and only 1.5% being discordant. This finding may advocate a single-test strategy being either an oropharyngeal swab RT-PCR testing or tracheal secretion, although this study revealed 15.9% false negative oropharyngeal swabs. Full article
(This article belongs to the Section Molecular and Translational Medicine)
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<p>(<b>A</b>) Danish regional guidelines for the COVID-19 test-based diagnostics of hospitalized symptomatic patients. (<b>B</b>) COVID-19 weekly incidence. Adapted from <a href="https://www.ecdc.europa.eu" target="_blank">https://www.ecdc.europa.eu</a> (accessed on 27 December 2021); European Centre for Disease Prevention and Control (ECDC), 2022.</p> Full article ">
22 pages, 42052 KiB  
Article
Testicular Macrophages Produce Progesterone De Novo Promoted by cAMP and Inhibited by M1 Polarization Inducers
by Sawako Yamauchi, Kousuke Yamamoto and Kazushige Ogawa
Biomedicines 2022, 10(2), 487; https://doi.org/10.3390/biomedicines10020487 - 18 Feb 2022
Cited by 9 | Viewed by 2929
Abstract
Tissue-resident macrophages (Mø) originating from fetal precursors are maintained via self-renewal under tissue-/organ-specific microenvironments. Herein, we developed a propagation method of testicular tissue-resident Mø in mixed primary culture with interstitial cells composed of Leydig cells from the mouse testis. We examined Mø/monocyte marker [...] Read more.
Tissue-resident macrophages (Mø) originating from fetal precursors are maintained via self-renewal under tissue-/organ-specific microenvironments. Herein, we developed a propagation method of testicular tissue-resident Mø in mixed primary culture with interstitial cells composed of Leydig cells from the mouse testis. We examined Mø/monocyte marker expression in propagated testicular Mø using flow cytometry; gene expression involved in testosterone production as well as spermatogenesis in testicular Mø and interstitial cells propagated by mixed culture via RT-PCR; and progesterone (P4) de novo production in propagated testicular Mø treated with cyclic adenosine monophosphate, isoproterenol, and M1 polarization inducers using ELISA. Mø marker expression patterns in the propagated Mø were identical to those in testicular interstitial Mø with a CD206-positive/major histocompatibility complex (MHC) II-negative M2 phenotype. We identified the genes involved in P4 production, transcription factors essential for steroidogenesis, and androgen receptors, and showed that P4 production de novo was upregulated by cyclic adenosine monophosphate and β2-adrenergic stimulation and was downregulated by M1 polarization stimulation in Mø. We also demonstrated the formation of gap junctions between Leydig cells and interstitial Mø. This is the first study to demonstrate de novo P4 production in tissue-resident Mø. Based on previous studies revealing inhibition of testosterone production by P4, we propose that local feedback machinery between Leydig cells and adjacent interstitial Mø regulates testosterone production. The results presented in this study can facilitate future studies on immune-endocrine interactions in gonads that are related to infertility and hormonal disorders. Full article
(This article belongs to the Special Issue Macrophages in Health and Non-infectious Disease 3.0)
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<p>Propagation and separation of testicular macrophages (Mø) in co-culture with testicular interstitial cells and their phagocytic property. Testicular interstitial cells from two testes of one mouse were collected by dispersing the cells with collagenase, seeding in a 10 cm tissue culture dish, and culturing with standard culture medium. Testicular interstitial cells propagated and reached over-confluence within 10 days. Cells harvested from an over-confluent dish were seeded in a bacteriological Petri dish to separate highly adhesive Mø from other interstitial cells. (<b>A</b>) Phase-contrast images of primary testicular interstitial cells cultured in a tissue culture dish for the indicated days (upper panels). Testicular tissue-resident Mø selectively adhere to the Petri dish surface (lower panels), and nonadherent cells formed cell aggregates (lower left panel), which were easily removed by tapping and washing with conditioned medium (lower right panel). (<b>B</b>) Phagocytotic properties assessed by incubation with fluorescent beads in testicular Mø segregated by adhesion to bacteriological Petri dishes. A green fluorescence image (Fl) merged with the phase-contrast image (PhC) (left panel) and differential interference contrast (DIC) images of the same fields (right panel). (<b>C</b>) Bar graph presenting the percentage of testicular tissue-resident Mø in the cells adherent to the bacteriological Petri dishes, presented as mean ± SD. More than 700 cells per sample were counted, and the percentage of phagocytic cells from three mice was determined from three independent experiments.</p> Full article ">Figure 2
<p>Macrophage (Mø) marker expressions in propagated Mø identical to properties of testicular interstitial Mø. Representative histograms from flow cytometric analyses revealing the expression of nine Mø/monocyte markers in propagated testicular Mø by mixed culture (blue histogram, specific antibody; grey histogram, isotype control). Cell suspensions were pretreated with an anti-mouse CD16/32 antibody and further treated with a fluorochrome-labeled test antibody, or the same amount of fluorochrome-labeled isotype control antibody. Propagated testicular Mø present characteristic expression patterns of highly CD206-positive and MHC II-negative cells, which are identical to those of testicular interstitial Mø.</p> Full article ">Figure 3
<p>Expression profiles of transcription factors and molecules characterizing testicular tissue-resident macrophages (Mø) propagated by mixed culture. mRNA expression in primary Mø propagated by mixed culture with interstitial cells was analyzed using a reverse transcription-polymerase chain reaction (RT-PCR). (<b>A</b>) Expression profiles of 14 transcription factors that are tissue-/organ-specific for resident Mø and the lineage-determining transcription factor of Mø, <span class="html-italic">Pu.1</span>, in propagated testicular Mø. (<b>B</b>) Expression profiles of molecules characterizing testicular tissue-resident Mø in propagated Mø by mixed culture.</p> Full article ">Figure 4
<p>Properties of testicular interstitial cells propagated in mixed culture and depletion of phagocytic cells. Testicular interstitial cells were propagated by mixed culture, treated with clodronate liposomes for 3 days to deplete testicular macrophages (Mø), and used as samples to examine the expression of molecules related to Mø propagation and sex steroid production by reverse transcription-polymerase chain reaction (RT-PCR). (<b>A</b>) Phase-contrast images (upper panel) and expression profiles of growth factors for Mø (lower panel) in testicular interstitial cells propagated by mixed culture and treated with or without clodronate liposomes. Small round/oval/fusiform cells, identical to the morphological features of Mø, disappeared in the interstitial cells treated with clodronate liposomes. These cells do not express <span class="html-italic">Pu.1</span> as a universal marker of Mø, albeit they clearly express <span class="html-italic">Csf1</span> and <span class="html-italic">Il34</span>. (<b>B</b>) Schematic drawing demonstrating pathways and molecules involved in androgen synthesis from cholesterol. (<b>C</b>) Expression profiles of molecules involved in androgen production by RT-PCR in the interstitial cells propagated by mixed culture and treated with or without clodronate liposomes.</p> Full article ">Figure 5
<p>Gap junction localization between interstitial macrophages (Mø) and Leydig cells and HSD3B expression in interstitial Mø. (<b>A</b>) Immunofluorescence micrographs demonstrating the distribution of gap junctions in the adult mouse testis. Fluorescence images are merged with differential interference contrast images. Cryostat sections were stained with the indicated antibodies and 4′,6-diamidino-2-phenylindole (DAPI) (blue). Punctate GJA1 immunoreactivity (green) is localized in CD206-positive (red) interstitial Mø and interstitial cells (arrows) with a large round nucleus (*), an identical profile of Leydig cells. ST, seminiferous tubule. (<b>B</b>) Immunofluorescence micrographs demonstrating HSD3B immunoreactivity (green) in interstitial cells. HSD3B immunoreactivity is localized not only in Leydig cells but also weakly in F4/80-positive interstitial Mø (arrows).</p> Full article ">Figure 6
<p>Progesterone (P4) is produced de novo specifically in testicular macrophages (Mø), albeit not in the liver, spleen, and lung Mø that are propagated by mixed culture. (<b>A</b>) Reverse transcription-polymerase chain reaction (RT-PCR) amplification of molecules related to androgen production. <span class="html-italic">Star</span>, <span class="html-italic">Cyp11a1</span>, and <span class="html-italic">Hsd3b1</span> are clearly expressed in testicular Mø, albeit not in the liver, spleen, and lung tissue-resident Mø that are propagated by mixed culture with the respective organ-specific cells. Testicular Mø also express <span class="html-italic">Sf-1</span> and <span class="html-italic">Gata4</span>. (<b>B</b>) P4 concentrations in the cultured testis, liver, spleen, or lung Mø. Mø propagated by mixed culture were seeded at a density of 4 × 10<sup>5</sup> cells/2 mL in six-well plates, and the medium cultured for 24 h and 72 h was collected and assayed for P4 concentrations by ELISA. P4 concentrations were determined from three independent experiments in propagated testicular Mø derived from three mice. P4 was detected only in the testicular Mø samples. Data are presented as the mean ± SD.</p> Full article ">Figure 7
<p>Cyclic adenosine monophosphate (cAMP) induces upregulation of progesterone (P4) production and molecules related to P4 production in testicular macrophages (Mø). Testicular macrophages (Mø) were seeded at a density of 4 × 10<sup>5</sup> cells/2 mL in six-well plates and cultured in DMEM-FBS with (cAMP) or without (Con) 50 µM dibutyryl cAMP. P4 concentration in the medium and molecules involved in P4 production were measured using enzyme-linked immunosorbent assay (ELISA) and quantitative polymerase chain reaction (qPCR), respectively, in the testicular Mø at 24 h and 72 h after addition. Data were determined from three independent experiments in propagated testicular Mø derived from three mice and are presented as mean ± SD (** <span class="html-italic">p &lt;</span> 0.01). (<b>A</b>) Relative P4 concentration in the culture medium of testicular Mø treated with or without dibutyryl cAMP. (<b>B</b>,<b>C</b>) The expression levels of <span class="html-italic">Star</span>, <span class="html-italic">Cyp11a1</span>, and <span class="html-italic">Hsd3b1</span> as well as <span class="html-italic">Sf-1</span> and <span class="html-italic">Gata4</span> in the testicular Mø treated with (cAMP) or without (Con) dibutyryl cAMP were determined by qPCR with the 2<sup>−∆∆CT</sup> method and are normalized to those of <span class="html-italic">Actb</span>. The mRNA expression levels relative to the untreated controls were upregulated and significantly different between the two groups in most cases.</p> Full article ">Figure 8
<p>Progesterone (P4) production is upregulated by isoproterenol and inhibited by M1 polarization inducers in testicular macrophages (Mø). Testicular Mø were seeded at a density of 4 × 10<sup>5</sup> cells/2 mL in six-well plates and cultured in DMEM-FBS containing 1 µM isoproterenol (Iso), 1 µM Iso plus 100 nM β2-adrenergic antagonist ICI-118551 (Iso + ICI), and 20 ng/mL lipopolysaccharide plus 50 ng/mL interferon-γ (M1). At 24 h and/or 72 h after the addition, the medium was collected, and P4 concentration was measured using an ELISA kit. P4 concentrations relative to the medium without reagents were calculated. Data were determined from more than three independent experiments in propagated testicular Mø derived from more than three mice and presented as mean ± SD (* <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01). (<b>A</b>) Expression of β-adrenergic receptors in testicular Mø were examined by reverse transcription-polymerase chain reaction (RT-PCR). (<b>B</b>) P4 production levels were enhanced by the addition of Iso. The relative P4 concentration levels in the Iso + ICI group tended to attenuate the enhancing effect of the Iso group (<span class="html-italic">p</span> = 0.076). (<b>C</b>) P4 concentration levels in the M1 group relative to the untreated controls (Con) were significantly different between the two groups at 72 h.</p> Full article ">Figure 9
<p>A schematic drawing showing the properties of progesterone (P4) production regulated by cyclic adenosine monophosphate (cAMP), β-adrenergic stimulation, and M1-polarization inducers in testicular interstitial macrophages (Mø). The dotted lines indicate signal cascades deduced from previous studies. The solid lines indicate well-accepted signal cascades and are derived from data in the present study.</p> Full article ">
31 pages, 49644 KiB  
Review
Functionalized Liposome and Albumin-Based Systems as Carriers for Poorly Water-Soluble Anticancer Drugs: An Updated Review
by Sofia Teixeira, Maria Alice Carvalho and Elisabete M. S. Castanheira
Biomedicines 2022, 10(2), 486; https://doi.org/10.3390/biomedicines10020486 - 18 Feb 2022
Cited by 22 | Viewed by 4546
Abstract
Cancer is one of the leading causes of death worldwide. In the available treatments, chemotherapy is one of the most used, but has several associated problems, namely the high toxicity to normal cells and the resistance acquired by cancer cells to the therapeutic [...] Read more.
Cancer is one of the leading causes of death worldwide. In the available treatments, chemotherapy is one of the most used, but has several associated problems, namely the high toxicity to normal cells and the resistance acquired by cancer cells to the therapeutic agents. The scientific community has been battling against this disease, developing new strategies and new potential chemotherapeutic agents. However, new drugs often exhibit poor solubility in water, which led researchers to develop functionalized nanosystems to carry and, specifically deliver, the drugs to cancer cells, targeting overexpressed receptors, proteins, and organelles. Thus, this review is focused on the recent developments of functionalized nanosystems used to carry poorly water-soluble drugs, with special emphasis on liposomes and albumin-based nanosystems, two major classes of organic nanocarriers with formulations already approved by the U.S. Food and Drug Administration (FDA) for cancer therapeutics. Full article
(This article belongs to the Special Issue Advances in Nanomaterials for Drug Delivery)
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Figure 1

Figure 1
<p>Representation of the active targeting of cancer cells by functionalized nanocarriers. Specific ligands on the nanocarrier surface bind to cell surface receptors. Upon recognition of the ligands, the uptake of the functionalized nanocarriers occurs by receptor-mediated endocytosis. The nanocarriers can also be functionalized with ligands to target specific cell organelles.</p> Full article ">Figure 2
<p>Schematic representation of SALT method for liposome preparation. DMSO is used as solvent for curcumin, to increase bilayer permeability and allow active curcumin loading into the inner aqueous core of liposomes. Formation of hydrogen bonds between the buffers used (Meglumine or Myo-inositol) and curcumin allows trapping the drug in the aqueous core. Adapted from [<a href="#B36-biomedicines-10-00486" class="html-bibr">36</a>] with permission from Elsevier, 2022.</p> Full article ">Figure 3
<p>Cytotoxicity of modified Tripalm-NPs-PTX with MAC, OEG and β-CD. Cell viability (%) of breast cancer MCF7 cells (<b>A</b>), corresponding normal cells MCF-10A (<b>B</b>), lung cancer A549 cells (<b>C</b>) and non-tumor cells L132 (<b>D</b>), after treatment with modified Tripalm-NPs-PTX and free PTX. Data represent the mean value ± SD of quadruplicate cultures. (*) Significant differences (<span class="html-italic">p</span> &lt; 0.001) between free PTX and modified Tripalm-NPs-PTX. Reprinted from [<a href="#B85-biomedicines-10-00486" class="html-bibr">85</a>] with permission.</p> Full article ">Figure 4
<p>Representation of the synthesis of PTX-loaded a-OMPC micelles (<b>A</b>); Cellular internalization with recognition of the PTX-loaded a-OMPC micelles by sigma-1 receptor overexpressed in prostate tumor cell, and the release of the PTX by pH-responsive release (<b>B</b>). Reprinted from [<a href="#B37-biomedicines-10-00486" class="html-bibr">37</a>] with permission from Elsevier, 2021.</p> Full article ">Figure 5
<p>Structural representation of some natural, semi-synthetic and synthetic polymers.</p> Full article ">Figure 6
<p>Cell viability of (<b>A</b>) A875 and (<b>B</b>) B16/F10 cells after incubation with different concentrations of DTIC@HMLBFs, DTIC@HMSNs, and free DTIC for 48 h. The experiments were conducted in triplicate (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, results were given as Mean ± SD). Reprinted from [<a href="#B56-biomedicines-10-00486" class="html-bibr">56</a>] with permission from American Chemical Society, 2021.</p> Full article ">Figure 7
<p>Schematic representation of in vivo targeting with nanocarrier DTIC@HMLBFs. First, by EPR effect, the nanocarrier can achieve the tumor tissue, with higher local accumulation, and then target folate-receptors of melanoma cells and tumor-associated macrophage (TAM). Reprinted from [<a href="#B56-biomedicines-10-00486" class="html-bibr">56</a>] with permission from American Chemical Society, 2021.</p> Full article ">Figure 8
<p>Different formulations of liposomes: conventional liposomes; PEGylated liposomes and ligand targeted liposomes for active targeting.</p> Full article ">Figure 9
<p>In vitro (above) and in vivo (below) application of PEGylated DOX-loaded liposomes coupled to a high-affinity EGFR-antagonist affibody (Z<sub>EGFR</sub>), named AS-DOX, to target A431 tumor cells overexpressing EGFR. LS-DOX: Non-targeted liposomes; f-DOX: free DOXorubicin; * <span class="html-italic">p</span> &lt; 0.05. Reprinted from [<a href="#B134-biomedicines-10-00486" class="html-bibr">134</a>] with permission from Elsevier, 2021.</p> Full article ">Figure 10
<p>Comparison of efficacy, in vivo, of docetaxel (DTX)-loaded nanosystems compared with free-DTX in the treatment of hepatocellular carcinoma. Tumor growth until 20 days: phosphate buffer (PBS), effect of free docetaxel (free-DTX), effect of DTX-loaded nanosystems (<sup>D</sup>T7-LIP, <sup>L</sup>T7-LIP, Tf-LIP and PEG-LIP); * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 versus the <sup>D</sup>T7-LIP group. Reprinted from [<a href="#B143-biomedicines-10-00486" class="html-bibr">143</a>] with permission from Elsevier, 2021.</p> Full article ">Figure 11
<p>Enhanced anti-tumor effects of folate-targeted liposomal nitrooxy-DOXorubicin against resistant tumors. (<b>A</b>) Tumor growth until 21 days: control (ctrl); effect of free DOX (D); effect of Caelyx; effect of nitrooxy-DOXorubicin (ND); liposomal nitrooxy-DOXorubicin (LND); folate-targeted liposomal nitrooxy-DOXorubicin (LNDF). Data are presented as means ± SD. ND/LND/LNDF vs. ctrl group: * <span class="html-italic">p</span> &lt; 0.01; ND/LND/LNDF vs. D: ° <span class="html-italic">p</span> &lt; 0.01; LNDF vs. ND: # <span class="html-italic">p</span> &lt; 0.001; LNDF vs. LND: § <span class="html-italic">p</span> &lt; 0.05; (<b>B</b>) Pictures of representative tumors from each treatment group after mice sacrifice. Reprinted from [<a href="#B39-biomedicines-10-00486" class="html-bibr">39</a>] with permission from Elsevier, 2021.</p> Full article ">Figure 12
<p>Schematic representation of mitochondria-targeting liposomes carrying resveratrol into mitochondria cancer cell. Reprinted from reference [<a href="#B168-biomedicines-10-00486" class="html-bibr">168</a>].</p> Full article ">Figure 13
<p>Release and accumulation of DOX in the cancer cell nucleus mediated by a modified liposome loaded with an aptamer-DOX complex. Reprinted from reference [<a href="#B172-biomedicines-10-00486" class="html-bibr">172</a>].</p> Full article ">Figure 14
<p>General representation of albumin with targeting ligands.</p> Full article ">Figure 15
<p>Fluorescence microscopy images of folate-conjugated ARM-HSA NPs in treated MDA-MB-231 cells (high FRα-expressing cell line) and SK-BR-3 cells (low FRα-expressing cell line). Reprinted from [<a href="#B130-biomedicines-10-00486" class="html-bibr">130</a>] with permission from Elsevier, 2021.</p> Full article ">Figure 16
<p>Illustration of the formation of cRGD-BSA/KALA/DOX nanoparticles and of the efficient delivery of DOX, including cellular uptake mediated by cRGD, cell internalization and pH-triggered disassembly to accelerate DOX release. Reprinted from [<a href="#B197-biomedicines-10-00486" class="html-bibr">197</a>] with permission from American Chemical Society, 2021.</p> Full article ">
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