Recombination Based Part Assembly

Here we propose a new recombination-based assembly standard, optimized to allow for efficient cloning of mammalian vectors

Enriched protein screening of human bone marrow mesenchymal stromal cell secretions reveals MFAP5 and PENK as novel IL-10 modulators

The secreted proteins from a cell constitute a natural biologic library that can offer significant insight into human health and disease. Discovering new secreted proteins from cells is bounded by the limitations of traditional separation and detection tools to physically fractionate and analyze samples. Here, we present a new method to systematically identify bioactive cell-secreted proteins that circumvent traditional proteomic methods by first enriching for protein candidates by differential gene expression profiling. The bone marrow stromal cell secretome was analyzed using enriched gene expression datasets in combination with potency assay testing. Four proteins expressed by stromal cells with previously unknown anti-inflammatory properties were identified, two of which provided a significant survival benefit to mice challenged with lethal endotoxic shock. Greater than 85% of secreted factors were recaptured that were otherwise undetected by proteomic methods, and remarkable hit rates of 18% in vitro and 9% in vivo were achieved

Resistance to checkpoint blockade therapy through inactivation of antigen presentation

Treatment with immune checkpoint blockade (CPB) therapies often leads to prolonged responses in patients with metastatic melanoma, but the common mechanisms of primary and acquired resistance to these agents remain incompletely characterized and have yet to be validated in large cohorts. By analyzing longitudinal tumor biopsies from 17 metastatic melanoma patients treated with CPB therapies, we observed point mutations, deletions or loss of heterozygosity (LOH) in beta-2-microglobulin (B2M), an essential component of MHC class I antigen presentation, in 29.4% of patients with progressing disease. In two independent cohorts of melanoma patients treated with anti-CTLA4 and anti-PD1, respectively, we find that B2M LOH is enriched threefold in non-responders (~30%) compared to responders (~10%) and associated with poorer overall survival. Loss of both copies of B2M is found only in non-responders. B2M loss is likely a common mechanism of resistance to therapies targeting CTLA4 or PD1

High Efficiency and Low Migration Hyperbranched Silicone Contain Macrophotoinitiators for UV-Cured Transparent Coatings

A kind of hyperbranched silicone containing macrophotoinitiators (HBSMIs) were synthesized from 2-hydroxy-2-methyl-1-phenyl propanone (HMPP) and the UV-curing behaviors of HBSMIs were investigated in UV-cured transparent polyurethane-acrylate (PUA) coatings. HBSMIs show higher UV-initiating efficiency than HMPP. The migration of HBSMIs from the UV-cured coatings can be as low as 1.7–6.0 wt%, which is obviously lower than the migration of HMPP. There is a remarkable improvement of the tensile strength of the UV-cured materials initiated by HBSMI in comparison to that of the materials prepared with the same PUA initiated by HMPP. Especially for the UV-cured materials prepared from PUA with 20 wt% 1,1,1-tris(hydroxymethyl)propane (TMP), the tensile strength and the strain at break increased from 6.81 MPa to 12.14 MPa and from 43.0% to 71.9%, respectively. The fraction of improvement for the tensile strength and the strain at break is as high as 78.9% and 67.2%, respectively. The coatings prepared with HBSMI also have better UV resistance ability than those coatings prepared with HMPP because they turn slightly yellow when they are aged by UV for about 15 min while the coating prepared with 4 wt% of HMPP will turn yellow only aged by UV for 2 min. These results suggest that preparation hyperbranched silicone containing macrophotoinitiators will be one of the good strategies to improve the curing efficiency of the UV-curing system, reduce the migration of UV initiator from cured material, improve the mechanical and UV resistance performance of UV-cured materials

Secreted Factors from Bone Marrow Stromal Cells Upregulate IL-10 and Reverse Acute Kidney Injury

Acute kidney injury is a devastating syndrome that afflicts over 2,000,000 people in the US per year, with an associated mortality of greater than 70% in severe cases. Unfortunately, standard-of-care treatments are not sufficient for modifying the course of disease. Many groups have explored the use of bone marrow stromal cells (BMSCs) for the treatment of AKI because BMSCs have been shown to possess unique anti-inflammatory, cytoprotective, and regenerative properties in vitro and in vivo. It is yet unresolved whether the primary mechanisms controlling BMSC therapy in AKI depend on direct cell infusion, or whether BMSC-secreted factors alone are sufficient for mitigating the injury. Here we show that BMSC-secreted factors are capable of providing a survival benefit to rats subjected to cisplatin-induced AKI. We observed that when BMSC-conditioned medium (BMSC-CM) is administered intravenously, it prevents tubular apoptosis and necrosis and ameliorates AKI. In addition, we observed that BMSC-CM causes IL-10 upregulation in treated animals, which is important to animal survival and protection of the kidney. In all, these results demonstrate that BMSC-secreted factors are capable of providing support without cell transplantation, and the IL-10 increase seen in BMSC-CM-treated animals correlates with attenuation of severe AKI.National Human Genome Research Institute (U.S.) (Grant number T32 HG002295)National Institutes of Health (U.S.) (Grant K01DK087770)National Institutes of Health (U.S.) (Grant R21DK085267)National Institutes of Health (U.S.) (Grant DK39773)National Institutes of Health (U.S.) (Grant DK72381)Shriners Hospitals for Childre

Enriched Protein Screening of Human Bone Marrow Mesenchymal Stromal Cell Secretions Reveals MFAP5 and PENK as Novel IL-10 Modulators

The secreted proteins from a cell constitute a natural biologic library that can offer significant insight into human health and disease. Discovering new secreted proteins from cells is bounded by the limitations of traditional separation and detection tools to physically fractionate and analyze samples. Here, we present a new method to systematically identify bioactive cell-secreted proteins that circumvent traditional proteomic methods by first enriching for protein candidates by differential gene expression profiling. The bone marrow stromal cell secretome was analyzed using enriched gene expression datasets in combination with potency assay testing. Four proteins expressed by stromal cells with previously unknown anti-inflammatory properties were identified, two of which provided a significant survival benefit to mice challenged with lethal endotoxic shock. Greater than 85% of secreted factors were recaptured that were otherwise undetected by proteomic methods, and remarkable hit rates of 18% in vitro and 9% in vivo were achieved. © 2014 The American Society of Gene and Cell Therapy.National Human Genome Research Institute (U.S.) (Grant T32 HG002295

Risk factors for secondary hemophagocytic lymphohistiocytosis in severe coronavirus disease 2019 adult patients

International audienceBackground: Secondary hemophagocytic lymphohistiocytosis (sHLH) is a life-threatening hyperinflammatory event and a fatal complication of viral infections. Whether sHLH may also be observed in patients with a cytokine storm induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is still uncertain. We aimed to determine the incidence of sHLH in severe COVID-19 patients and evaluate the underlying risk factors.Method: Four hundred fifteen severe COVID-19 adult patients were retrospectively assessed for hemophagocytosis score (HScore). A subset of 7 patients were unable to be conclusively scored due to insufficient patient data.Results: In 408 patients, 41 (10.04%) had an HScore ≥169 and were characterized as "suspected sHLH positive". Compared with patients below a HScore threshold of 98, the suspected sHLH positive group had higher D-dimer, total bilirubin, alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, serum creatinine, triglycerides, ferritin, interleukin-6, C-reactive protein, procalcitonin, lactate dehydrogenase, creatine kinase isoenzyme, troponin, Sequential Organ Failure Assessment (SOFA) score, while leukocyte, hemoglobin, platelets, lymphocyte, fibrinogen, pre-albumin, albumin levels were significantly lower (all P 1922.58 ng/mL), low platelets (2.28 mmol/L) were independent risk factors for suspected sHLH in COVID-19 patients. Importantly, COVID-19 patients that were suspected sHLH positive had significantly more multi-organ failure. Additionally, a high HScore (>98) was an independent predictor for mortality in COVID-19.Conclusions: HScore should be measured as a prognostic biomarker in COVID-19 patients. In particular, it is important that HScore is assessed in patients with high ferritin, triglycerides and low platelets to improve the detection of suspected sHLH

The Identification of CD163 Expressing Phagocytic Chondrocytes in Joint Cartilage and Its Novel Scavenger Role in Cartilage Degradation

<div><h3>Background</h3><p>Cartilage degradation is a typical characteristic of arthritis. This study examined whether there was a subset of phagocytic chondrocytes that expressed the specific macrophage marker, CD163, and investigated their role in cartilage degradation.</p> <h3>Methods</h3><p>Cartilage from the knee and temporomandibular joints of Sprague-Dawley rats was harvested. Cartilage degradation was experimentally-induced in rat temporomandibular joints, using published biomechanical dental methods. The expression levels of CD163 and inflammatory factors within cartilage, and the ability of CD163⁺ chondrocytes to conduct phagocytosis were investigated. Cartilage from the knees of patients with osteoarthritis and normal cartilage from knee amputations was also investigated.</p> <h3>Results</h3><p>In the experimentally-induced degrading cartilage from temporomandibular joints, phagocytes were capable of engulfing neighboring apoptotic and necrotic cells, and the levels of CD163, TNF-α and MMPs were all increased (<em>P</em><0.05). However, the levels of ACP-1, NO and ROS, which relate to cellular digestion capability were unchanged (<em>P</em>>0.05). CD163⁺ chondrocytes were found in the cartilage mid-zone of temporomandibular joints and knee from healthy, three-week old rats. Furthermore, an increased number of CD163⁺ chondrocytes with enhanced phagocytic activity were present in Col-II⁺ chondrocytes isolated from the degraded cartilage of temporomandibular joints in the eight-week experimental group compared with their age-matched controls. Increased number with enhanced phagocytic activity of CD163⁺ chondrocytes were also found in isolated Col-II⁺ chondrocytes stimulated with TNF-α (<em>P</em><0.05). Mid-zone distribution of CD163⁺ cells accompanied with increased expression of CD163 and TNF-α were further confirmed in the isolated Col-II⁺ chondrocytes from the knee cartilage of human patients with osteoarthritis, in contrast to the controls (both <em>P</em><0.05).</p> <h3>Conclusions</h3><p>An increased number of CD163⁺ chondrocytes with enhanced phagocytic activity were discovered within degraded joint cartilage, indicating a role in eliminating degraded tissues. Targeting these cells provides a new strategy for the treatment of arthritis.</p> </div

CD163⁺ chondrocytes in normal joint cartilage of 3-week old rats.

<p>A: Immunohistochemical staining of CD163 in cartilage from the TMJ and knee. The CD163⁺ cells located below the superior zone of the TMJ and knee cartilage, show intense membrane and cytoplasmic staining (arrows). Rat liver and muscle were selected as positive and negative controls, respectively, for the detection of CD163. Membrane staining of CD163⁺ cells was observed in liver (indicated by arrows), but no CD163⁺ cells were detected in muscle. As additional controls, TMJ and knee cartilage was also stained with an isotype control antibody. B: Flow cytometric analysis and graphical representation of the percentage of total CD163⁺ cells and CD163⁺ cells with phagocytic activity within the Col-II⁺ cells isolated from TMJ cartilage (n = 3; *<i>P</i><0.05).</p

TNF-α increased the phagocytic and migratory activities of CD163⁺ cells isolated from rat TMJ cartilage.

<p>A–B: Confocal microscope images (A) and graphical representation (B) of the numbers of CD163⁺ cells and their phagocytic activity within primary cells isolated from TMJ cartilage and treated with vehicle, TNF-α alone, or TNF-α and a CD163 neutralizing antibody. The co-localization of the CD163⁺ cell with DiO-labeled cell debris (arrows), indicates that the cell debris is undergoing phagocytosis by the CD163⁺ cells, as shown in the insets. Bar: 50 µm. C: Transwell assay combined with immunohistochemical staining of CD163 indicates the migratory potential of CD163⁺ cells in response to 10 ng/ml TNF-α, which is impaired in the presence of the TNF-α neutralizing antibody (AT, 1 µg/ml). Arrows indicate the migrating CD163⁺ cells. Five fields were selected at random (at 200× magnification), and the number of CD163⁺ cells and total cells in each image were counted. **<i>P</i><0.01.</p