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

Pharmacokinetics of Natural and Engineered Secreted Factors Delivered by Mesenchymal Stromal Cells

Transient cell therapy is an emerging drug class that requires new approaches for pharmacological monitoring during use. Human mesenchymal stem cells (MSCs) are a clinically-tested transient cell therapeutic that naturally secrete anti-inflammatory factors to attenuate immune-mediated diseases. MSCs were used as a proof-of-concept with the hypothesis that measuring the release of secreted factors after cell transplantation, rather than the biodistribution of the cells alone, would be an alternative monitoring tool to understand the exposure of a subject to MSCs. By comparing cellular engraftment and the associated serum concentration of secreted factors released from the graft, we observed clear differences between the pharmacokinetics of MSCs and their secreted factors. Exploration of the effects of natural or engineered secreted proteins, active cellular secretion pathways, and clearance mechanisms revealed novel aspects that affect the systemic exposure of the host to secreted factors from a cellular therapeutic. We assert that a combined consideration of cell delivery strategies and molecular pharmacokinetics can provide a more predictive model for outcomes of MSC transplantation and potentially other transient cell therapeutics

Metabolic and lipidomic profiling of steatotic human livers during ex situ normothermic machine perfusion guides resuscitation strategies

There continues to be a significant shortage of donor livers for transplantation. One impediment is the discard rate of fatty, or steatotic, livers because of their poor post-transplant function. Steatotic livers are prone to significant ischemia-reperfusion injury (IRI) and data regarding how best to improve the quality of steatotic livers is lacking. Herein, we use normothermic (37°C) machine perfusion in combination with metabolic and lipidomic profiling to elucidate deficiencies in metabolic pathways in steatotic livers, and to inform strategies for improving their function. During perfusion, energy cofactors increased in steatotic livers to a similar extent as non-steatotic livers, but there were significant deficits in anti-oxidant capacity, efficient energy utilization, and lipid metabolism. Steatotic livers appeared to oxidize fatty acids at a higher rate but favored ketone body production rather than energy regeneration via the tricyclic acid cycle. As a result, lactate clearance was slower and transaminase levels were higher in steatotic livers. Lipidomic profiling revealed ω-3 polyunsaturated fatty acids increased in non-steatotic livers to a greater extent than in steatotic livers. The novel use of metabolic and lipidomic profiling during ex situ normothermic machine perfusion has the potential to guide the resuscitation and rehabilitation of steatotic livers for transplantation

Case Report Intraoperatively Diagnosed Tracheal Tear after Using an NIM EMG ETT with Previously Undiagnosed Tracheomalacia

Tracheal rupture is a rare complication of endotracheal intubation. We present a case of tracheal rupture that was diagnosed intraoperatively after the use of an NIM EMG endotracheal tube. A 66-year-old female with a recurrent multinodular goiter was scheduled for total thyroidectomy. Induction of anesthesia was uncomplicated. Intubation was atraumatic using a 6 mm NIM EMG endotracheal tube (ETT). Approximately 90 minutes into the surgery, a tracheal tear was suspected. After confirming the diagnosis, conservative treatment with antibiotic coverage was favored. The patient made a full recovery with no complications. Diagnosis of the tracheal tear was made intraoperatively, prompting early management

Propofol induces MAPK/ERK cascade dependant expression of cFos and Egr-1 in rat hippocampal slices

Background: Propofol is a commonly used intravenous anesthetic agent, which produce rapid induction of and recovery from general anesthesia. Numerous clinical studies reported that propofol can potentially cause amnesia and memory loss in human subjects. The underlying mechanism for this memory loss is unclear but may potentially be related to the induction of memory-associated genes such as c-Fos and Egr-1 by propofol. This study explored the effects of propofol on c-Fos and Egr-1 expression in rat hippocampal slices. Findings: Hippocampal brain slices were exposed to varying concentrations of propofol at multiple time intervals. The transcription of the immediate early genes, c-Fos and Egr-1, was quantified using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). MAPK/ERK inhibitors were used to investigate the mechanism of action. We demonstrate that propofol induced the expression of c-Fos and Egr-1 within 30 and 60 min of exposure time. At 16.8 μM concentration, propofol induced a 110% increase in c-Fos transcription and 90% decrease in the transcription of Egr-1. However, at concentrations above 100 μM, propofol failed to induce expression of c-Fos but did completely inhibit the transcription of Egr-1. Propofol-induced c-Fos and Egr-1 transcription was abolished by inhibitors of RAS, RAF, MEK, ERK and p38-MAPK in the MAPK/ERK cascade. Conclusions: Our study shows that clinically relevant concentrations of propofol induce c-Fos and down regulated Egr-1 expression via an MAPK/ERK mediated pathway. We demonstrated that propofol induces a time and dose dependant transcription of IEGs c-Fos and Egr-1 in rat hippocampal slices. We further demonstrate for the first time that propofol induced IEG expression was mediated via a MAPK/ERK dependant pathway. These novel findings provide a new avenue to investigate transcription-dependant mechanisms and suggest a parallel pathway of action with an unclear role in the activity of general anesthetics

Purification of antibodies to O antigen of Salmonella Typhimurium from human serum by affinity chromatography

AbstractNontyphoidal Salmonellae (NTS) are a common cause of bacteraemia in children and HIV-infected adults in Sub-Saharan Africa. We have previously shown that antibodies play a key role in both bactericidal and cellular mechanisms of immunity to NTS, but found that high concentrations of antibody to Salmonella Typhimurium O antigen (OAg) in the serum of some HIV-infected African adults is associated with impaired killing of NTS. To further investigate the function of antibodies to the OAg of NTS, we developed a method to purify these antibodies from human serum by affinity chromatography. Purified Salmonella Typhimurium OAg was activated with adipic acid dihydrazide (ADH) via two different chemistries before linking to N-hydroxysuccinamide-Sepharose resin: one ADH molecule was introduced per OAg chain on its terminal 3-deoxy-D-manno-octulosonic acid sugar (OAg–ADH), or multiple ADH molecules were attached along the OAg chain after oxidation with sodium periodate (OAgoxADH). Both resulting columns worked well when tested with commercial polyclonal anti-O:4,5 antibodies from rabbit serum. Over 90% of the applied antibodies bound to the resin and 89% of these antibodies were then eluted as detected by ELISA. OAg–ADH was preferred as the method for OAg derivatisation as it does not modify the saccharide chain and can be applied to OAg from different bacteria. Both columns were able to bind OAg-specific antibodies in human serum, but antibody recovery was initially low. Different elution buffers were tested and different amounts of OAg–ADH were linked to the resin to improve the yield. Optimal recovery (51%) was obtained by loading 1mg of activated OAg per ml of resin and eluting with 0.1M glycine, 0.1M NaCl pH2.4. The column matrix could be regenerated following elution with no detectable loss in performance for over ten uses. This method offers the potential to purify antibodies to Salmonella OAg from polyclonal serum following vaccination or natural exposure to Salmonella and so investigate the functionality and diversity of the antibody response to OAg

Three-dimensional Numerical Modeling and Computational Fluid Dynamics Simulations to Analyze and Improve Oxygen Availability in the AMC Bioartificial Liver

A numerical model to investigate fluid flow and oxygen (O(2)) transport and consumption in the AMC-Bioartificial Liver (AMC-BAL) was developed and applied to two representative micro models of the AMC-BAL with two different gas capillary patterns, each combined with two proposed hepatocyte distributions. Parameter studies were performed on each configuration to gain insight in fluid flow, shear stress distribution and oxygen availability in the AMC-BAL. We assessed the function of the internal oxygenator, the effect of changes in hepatocyte oxygen consumption parameters in time and the effect of the change from an experimental to a clinical setting. In addition, different methodologies were studied to improve cellular oxygen availability, i.e. external oxygenation of culture medium, culture medium flow rate, culture gas oxygen content (pO(2)) and the number of oxygenation capillaries. Standard operating conditions did not adequately provide all hepatocytes in the AMC-BAL with sufficient oxygen to maintain O(2) consumption at minimally 90% of maximal uptake rate. Cellular oxygen availability was optimized by increasing the number of gas capillaries and pO(2) of the oxygenation gas by a factor two. Pressure drop over the AMC-BAL and maximal shear stresses were low and not considered to be harmful. This information can be used to increase cellular efficiency and may ultimately lead to a more productive AMC-BAL

Towards a three-dimensional microfluidic liver platform for predicting drug efficacy and toxicity in humans

Although the process of drug development requires efficacy and toxicity testing in animals prior to human testing, animal models have limited ability to accurately predict human responses to xenobiotics and other insults. Societal pressures are also focusing on reduction of and, ultimately, replacement of animal testing. However, a variety of in vitro models, explored over the last decade, have not been powerful enough to replace animal models. New initiatives sponsored by several US federal agencies seek to address this problem by funding the development of physiologically relevant human organ models on microscopic chips. The eventual goal is to simulate a human-on-a-chip, by interconnecting the organ models, thereby replacing animal testing in drug discovery and development. As part of this initiative, we aim to build a three-dimensional human liver chip that mimics the acinus, the smallest functional unit of the liver, including its oxygen gradient. Our liver-on-a-chip platform will deliver a microfluidic three-dimensional co-culture environment with stable synthetic and enzymatic function for at least 4 weeks. Sentinel cells that contain fluorescent biosensors will be integrated into the chip to provide multiplexed, real-time readouts of key liver functions and pathology. We are also developing a database to manage experimental data and harness external information to interpret the multimodal data and create a predictive platform. © 2013 BioMed Central Ltd

Nest Making and Oxytocin Comparably Promote Wound Healing in Isolation Reared Rats

Background: Environmental enrichment (EE) fosters attachment behavior through its effect on brain oxytocin levels in the hippocampus and other brain regions, which in turn modulate the hypothalamic-pituitary axis (HPA). Social isolation and other stressors negatively impact physical healing through their effect on the HPA. Therefore, we reasoned that: 1) provision of a rat EE (nest building with Nestlets®) would improve wound healing in rats undergoing stress due to isolation rearing and 2) that oxytocin would have a similar beneficial effect on wound healing. Methodology/Principal Findings: In the first two experiments, we provided isolation reared rats with either EE or oxytocin and compared their wound healing to group reared rats and isolation reared rats that did not receive Nestlets or oxytocin. In the third experiment, we examined the effect of Nestlets on open field locomotion and immediate early gene (IEG) expression. We found that isolation reared rats treated with Nestlets a) healed significantly better than without Nestlets, 2) healed at a similar rate to rats treated with oxytocin, 3) had decreased hyperactivity in the open field test, and 4) had normalized IEG expression in brain hippocampus. Conclusions/Significance: This study shows that when an EE strategy or oxytocin is given to isolation reared rats, the peripheral stress response, as measured by burn injury healing, is decreased. The findings indicate an association between the effect of nest making on wound healing and administration of the pro-bonding hormone oxytocin. Further elucidation of this animal model should lead to improved understanding of how EE strategies can ameliorate poor wound healing and other symptoms that result from isolation stress