Regenerative potential of human muscle stem cells in chronic inflammation

Introduction

Chronic inflammation is a profound systemic modification of the cellular microenvironment which could affect survival, repair and maintenance of muscle stem cells. The aim of this study was to define the role of chronic inflammation on the regenerative potential of satellite cells in human muscle.

Methods

As a model for chronic inflammation, 11 patients suffering from rheumatoid arthritis (RA) were included together with 16 patients with osteoarthritis (OA) as controls. The mean age of both groups was 64 years, with more females in the RA group compared to the OA group. During elective knee replacement surgery, a muscle biopsy was taken from the distal musculus vastus medialis. Cell populations from four RA and eight OA patients were used for extensive phenotyping because these cell populations showed no spontaneous differentiation and myogenic purity greater than 75% after explantation.

Results

After mononuclear cell explantation, myogenic purity, viability, proliferation index, number of colonies, myogenic colonies, growth speed, maximum number of population doublings and fusion index were not different between RA and OA patients. Furthermore, the expression of proteins involved in replicative and stress-induced premature senescence and apoptosis, including p16, p21, p53, hTERT and cleaved caspase-3, was not different between RA and OA patients. Mean telomere length was shorter in the RA group compared to the OA group.

Conclusions

In the present study we found evidence that chronic inflammation in RA does not affect the in vitro regenerative potential of human satellite cells. Identification of mechanisms influencing muscle regeneration by modulation of its microenvironment may, therefore, be more appropriate.     

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.Download video file.^{(95M, mov)}     

Biologically active human GM-CSF produced in the seeds of transgenic rice plants

Rice flour is a well-known and characterized source of pharmaceutical ingredients, which are gluten-free and incorporated in many drug delivery applications such as excipient starch. To further exploit this uniqueness, the synthetic capacity of rice endosperm tissue, the basis of rice flour, was extended by genetic transformation. Recombinant human GM-CSF, a cytokine used in treating neutropenia and with other potential clinical applications, has been expressed in transgenic rice seeds using a rice glutelin promoter. Rice seeds accumulated human GM-CSF to a level of 1.3% of total soluble protein. The rice seed-produced human GM-CSF was found to be biologically active when tested using a human cell line TF-1. Use of rice as a host plant offers not only attractive features of safe production in seeds but also self-containment of foreign genes, as rice is primarily a self-pollinated crop plant. Ravinder Sardana and Anil K. Dudani contributed equally to this work.     

Novel Giant Siphovirus from Bacillus anthracis Features Unusual Genome Characteristics

Here we present vB_BanS-Tsamsa, a novel temperate phage isolated from Bacillus anthracis, the agent responsible for anthrax infections in wildlife, livestock and humans. Tsamsa phage is a giant siphovirus (order Caudovirales), featuring a long, flexible and non-contractile tail of 440 nm (not including baseplate structure) and an isometric head of 82 nm in diameter. We induced Tsamsa phage in samples from two different carcass sites in Etosha National Park, Namibia. The Tsamsa phage genome is the largest sequenced Bacillus siphovirus, containing 168,876 bp and 272 ORFs. The genome features an integrase/recombinase enzyme, indicative of a temperate lifestyle. Among bacterial strains tested, the phage infected only certain members of the Bacillus cereus sensu lato group (B. anthracis, B. cereus and B. thuringiensis) and exhibited moderate specificity for B. anthracis. Tsamsa lysed seven out of 25 B. cereus strains, two out of five B. thuringiensis strains and six out of seven B. anthracis strains tested. It did not lyse B. anthracis PAK-1, an atypical strain that is also resistant to both gamma phage and cherry phage. The Tsamsa endolysin features a broader lytic spectrum than the phage host range, indicating possible use of the enzyme in Bacillus biocontrol.     

An in vitro investigation of the acetabular labral seal in hip joint mechanics

Labrum pathology may contribute to early joint degeneration through the alteration of load transfer between, and the stresses within, the cartilage layers of the hip. We hypothesize that the labrum seals the hip joint, creating a hydrostatic fluid pressure in the intra-articular space, and limiting the rate of cartilage layer consolidation. The overall cartilage creep consolidation of six human hip joints was measured during the application of a constant load of 0.75 times bodyweight, or a cyclic sinusoidal load of 0.75+/-0.25 times bodyweight, before and after total labrum resection. The fluid pressure within the acetabular was measured. Following labrum resection, the initial consolidation rate was 22% greater (p=0.02) and the final consolidation displacement was 21% greater (p=0.02). There was no significant difference in the final consolidation rate. Loading type (constant vs. cyclic) had no significant effect on the measured consolidation behaviour. Fluid pressurisation was observed in three of the six hips. The average pressures measured were: for constant loading, 541+/-61kPa in the intact joint and 216+/-165kPa following labrum resection, for cyclic loading, 550+/-56kPa in the intact joint and 195+/-145kPa following labrum resection. The trends observed in this experiment support the predictions of previous finite element analyses. Hydrostatic fluid pressurisation within the intra-articular space is greater with the labrum than without, which may enhance joint lubrication. Cartilage consolidation is quicker without the labrum than with, as the labrum adds an extra resistance to the flow path for interstitial fluid expression. However, both sealing mechanisms are dependent on the fit of the labrum against the femoral head.     

Iron loading induces cholesterol synthesis and sensitizes endothelial cells to TNFα-mediated apoptosis

In plasma, iron is normally bound to transferrin, the principal protein in blood responsible for binding and transporting iron throughout the body. However, in conditions of iron overload when the iron-binding capacity of transferrin is exceeded, non–transferrin-bound iron (NTBI) appears in plasma. NTBI is taken up by hepatocytes and other parenchymal cells via NTBI transporters and can cause cellular damage by promoting the generation of reactive oxygen species. However, how NTBI affects endothelial cells, the most proximal cell type exposed to circulating NTBI, has not been explored. We modeled in vitro the effects of systemic iron overload on endothelial cells by treating primary human umbilical vein endothelial cells (HUVECs) with NTBI (ferric ammonium citrate [FAC]). We showed by RNA-Seq that iron loading alters lipid homeostasis in HUVECs by inducing sterol regulatory element-binding protein 2–mediated cholesterol biosynthesis. We also determined that FAC increased the susceptibility of HUVECs to apoptosis induced by tumor necrosis factor-α (TNFα). Moreover, we showed that cholesterol biosynthesis contributes to iron-potentiated apoptosis. Treating HUVECs with a cholesterol chelator hydroxypropyl-β-cyclodextrin demonstrated that depletion of cholesterol was sufficient to rescue HUVECs from TNFα-induced apoptosis, even in the presence of FAC. Finally, we showed that FAC or cholesterol treatment modulated the TNFα pathway by inducing novel proteolytic processing of TNFR1 to a short isoform that localizes to lipid rafts. Our study raises the possibility that iron-mediated toxicity in human iron overload disorders is at least in part dependent on alterations in cholesterol metabolism in endothelial cells, increasing their susceptibility to apoptosis.     

Sustained Expression of Human Cytomegalovirus Glycoprotein B (UL55) in the Seeds of Homozygous Rice Plants

Production of recombinant subunit vaccines in transgenic plants may be a means of reducing vaccine costs while increasing availability and safety. A plant-derived product found safe and effective for oral administration would provide additional advantages when used as a vaccine. Outstanding issues with the technology include transgene stability through successive generations and consistent bioproduction. We previously reported expression of glycoprotein B (gB) of human cytomegalovirus in seeds of transgenic tobacco. Here the goal was to determine if gB could be similarly expressed in rice, and if so, to examine expression over several plant generations. Results show that immunoreactive gB was successfully expressed in transgenic rice seeds, with sustained expression over three generations. The gB contained several neutralizing epitopes and was stable over 27 months.