Biological evaluation of intervertebral disc cells in different formulations of gellan gum‐based hydrogels

Gellan gum (GG)‐based hydrogels are advantageous in tissue engineering not only due to their ability to retain large quantities of water and provide a similar environment to that of natural extracellular matrix (ECM), but also because they can gelify in situ in seconds. Their mechanical properties can be fine‐tuned to mimic natural tissues such as the nucleus pulposus (NP). This study produced different formulations of GG hydrogels by mixing varying amounts of methacrylated (GG‐MA) and high‐acyl gellan gums (HA‐GG) for applications as acellular and cellular NP substitutes. The hydrogels were physicochemically characterized by dynamic mechanical analysis. Degradation and swelling abilities were assessed by soaking in a phosphate buffered saline solution for up to 170 h. Results showed that as HA‐GG content increased, the modulus of the hydrogels decreased. Moreover, increases in HA‐GG content induced greater weight loss in the GG‐MA/HA‐GG formulation compared to GG‐MA hydrogel. Potential cytotoxicity of the hydrogel was assessed by culturing rabbit NP cells up to 7 days. An MTS assay was performed by seeding rabbit NP cells onto the surface of 3D hydrogel disc formulations. Viability of rabbit NP cells encapsulated within the different hydrogel formulations was also evaluated by Calcein‐AM and ATP assays. Results showed that tunable GG‐MA/HA‐GG hydrogels were non‐cytotoxic and supported viability of rabbit NP cells. Copyright © 2012 John Wiley & Sons, Ltd.     

Solid Organ Transplantation From Hepatitis B Virus–Positive Donors: Consensus Guidelines for Recipient Management

Use of organs from donors testing positive for hepatitis B virus (HBV) may safely expand the donor pool. The American Society of Transplantation convened a multidisciplinary expert panel that reviewed the existing literature and developed consensus recommendations for recipient management following the use of organs from HBV positive donors. Transmission risk is highest with liver donors and significantly lower with non‐liver (kidney and thoracic) donors. Antiviral prophylaxis significantly reduces the rate of transmission to liver recipients from isolated HBV core antibody positive (anti‐HBc+) donors. Organs from anti‐HBc+ donors should be considered for all adult transplant candidates after an individualized assessment of the risks and benefits and appropriate patient consent. Indefinite antiviral prophylaxis is recommended in liver recipients with no immunity or vaccine immunity but not in liver recipients with natural immunity. Antiviral prophylaxis may be considered for up to 1 year in susceptible non‐liver recipients but is not recommended in immune non‐liver recipients. Although no longer the treatment of choice in patients with chronic HBV, lamivudine remains the most cost‐effective choice for prophylaxis in this setting. Hepatitis B immunoglobulin is not recommended.     

Tim-3 enhances FcεRI-proximal signaling to modulate mast cell activation

T cell (or transmembrane) immunoglobulin and mucin domain protein 3 (Tim-3) has attracted significant attention as a novel immune checkpoint receptor (ICR) on chronically stimulated, often dysfunctional, T cells. Antibodies to Tim-3 can enhance antiviral and antitumor immune responses. Tim-3 is also constitutively expressed by mast cells, NK cells and specific subsets of macrophages and dendritic cells. There is ample evidence for a positive role for Tim-3 in these latter cell types, which is at odds with the model of Tim-3 as an inhibitory molecule on T cells. At this point, little is known about the molecular mechanisms by which Tim-3 regulates the function of T cells or other cell types. We have focused on defining the effects of Tim-3 ligation on mast cell activation, as these cells constitutively express Tim-3 and are activated through an ITAM-containing receptor for IgE (FcεRI), using signaling pathways analogous to those in T cells. Using a variety of gain- and loss-of-function approaches, we find that Tim-3 acts at a receptor-proximal point to enhance Lyn kinase-dependent signaling pathways that modulate both immediate-phase degranulation and late-phase cytokine production downstream of FcεRI ligation.T cell, or transmembrane, immunoglobulin domain and mucin domain (Tim-3) is a type I membrane protein expressed on a variety of innate and adaptive immune cell types. Tim-3 is often referred to as a checkpoint receptor due to its apparent inhibitory function on T cells and its association with activation-induced T cell exhaustion in tumors and chronic viral infection (Sánchez-Fueyo et al., 2003; Jones et al., 2008; Fourcade et al., 2010; Jin et al., 2010; Sakuishi et al., 2010). Recent studies, however, suggest a more nuanced picture of Tim-3 function in T cells, depending on the setting, e.g., acute versus chronic stimulation (Ferris et al., 2014; Gorman and Colgan, 2014). In addition to CD4 and CD8 T cells, Tim-3 is also expressed on other immune cell types, such as NK cells, macrophages, DCs, and mast cells, but its function on these cell types is less clear. Tim-3 blockade was shown to enhance macrophage function in response to sepsis (Yang et al., 2013), and also to regulate antigen (Ag) presentation by DCs, partly through Btk and c-Src (Maurya et al., 2014). On the other hand, Tim-3 expression on monocytes infiltrating the CNS during EAE was shown to promote inflammation (Anderson et al., 2007).Mast cells are first-line defenders against allergens and invading pathogens as a result of their proximity to the external environment. Cross-linking of IgE bound to the high-affinity IgE receptor FcεRI by Ag leads to the release of preformed mediators and de novo synthesis of proinflammatory and antiinflammatory mediators and cytokines, which together serve to regulate hypersensitivity, autoimmunity, cardiovascular disease, and tumor progression (Kalesnikoff and Galli, 2008). In addition to their well-known pathological roles in allergic responses, mast cells also contribute to defense against bacteria, helminthes, and tumors (Abraham and St John, 2010). It was reported that mast cells constitutively express cell surface Tim-3, and that cross-linking of Tim-3 could enhance cytokine production of IgE-sensitized and Ag-stimulated BM-derived mast cells (BMMCs) and peritoneal mast cells (pMCs) without affecting degranulation (Nakae et al., 2007). TGF-β has been shown to up-regulate expression of Tim-3 in tumor-infiltrating mast cells and a human mast cell line, through a mitogen-activated protein kinase Erk-kinase (MEK)–dependent pathway (Wiener et al., 2006; Yoon et al., 2011). Although previous data suggest that Tim-3 is a positive regulator of mast cell activation, the molecular mechanisms behind the contribution of Tim-3 to mast cell function are still unknown. Importantly, there was until now no genetic evidence addressing the function of Tim-3 in these cells. Given the important role of mast cells as sentinels in both allergic and nonallergic diseases, it is of interest to explore Tim-3 activity on this cell type and how antibody (Ab) modulation can affect its function.Here, we demonstrate through multiple approaches that Tim-3 functions to enhance proximal FcεRI signaling in mast cells. Cross-linking of Tim-3 with multiple independent antibodies enhanced mast cell degranulation and cytokine release in a dose-dependent manner. Acute knock-down or genetic deficiency of Tim-3 rendered mast cells less responsive to Ag cross-linking of FcεRI, resulting in decreased degranulation and cytokine production. The cytoplasmic tail of Tim-3 was required for co-stimulatory signal transduction in mast cells, together with FcεRI signaling pathways. This was shown in part with the use of recently reported Nur77-GFP transgenic models, which have not previously been used for the study of FcεRI signaling. Collectively, our data demonstrate that Tim-3 acts at a receptor-proximal level to intensify activation of FcεRI-dependent signaling pathways upon Ag cross-linking, while maintaining the threshold for negative signaling of Lyn.     

Impact of the Combination of Daptomycin and Trimethoprim-Sulfamethoxazole on Clinical Outcomes in Methicillin-Resistant Staphylococcus aureus Infections

Complicated Staphylococcus aureus infections, including bacteremia, are often associated with treatment failures, prolonged hospital stays, and the emergence of resistance to primary and even secondary therapies. Daptomycin is commonly used as salvage therapy after vancomycin failure for the treatment of methicillin-resistant S. aureus (MRSA) infections. Unfortunately, the emergence of daptomycin resistance, especially in deep-seated infections, has been reported, prompting the need for alternative or combination therapy. Numerous antibiotic combinations with daptomycin have been investigated clinically and in vitro. Of interest, the combination of daptomycin and trimethoprim-sulfamethoxazole (TMP-SMX) has proved to be rapidly bactericidal in vitro to strains that are both susceptible and nonsusceptible to daptomycin. However, to date, there is limited clinical evidence supporting the use of this combination. This was a multicenter, retrospective case series of patients treated with the combination of daptomycin and TMP-SMX for at least 72 h. The objective of this study was to describe the safety and effectiveness of this regimen in clinical practice. The most commonly stated reason that TMP-SMX was added to daptomycin was persistent bacteremia and/or progressive signs and symptoms of infection. After the initiation of combination therapy, the median time to clearance of bacteremia was 2.5 days. Microbiological eradication was demonstrated in 24 out of 28 patients, and in vitro synergy was demonstrated in 17 of the 17 recovered isolates. Further research with this combination is necessary to describe the optimal role and its impact on patient outcomes.     

Identification of an updated set of prescribing-safety indicators for GPs

Background

Medication error is an important contributor to patient morbidity and mortality and is associated with inadequate patient safety measures. However, prescribing-safety tools specifically designed for use in general practice are lacking.

Aim

To identify and update a set of prescribing-safety indicators for assessing the safety of prescribing in general practice, and to estimate the risk of harm to patients associated with each indicator.

Design and setting

RAND/UCLA consensus development of indicators in UK general practice.

Method

Prescribing indicators were identified from a systematic review and previous consensus exercise. The RAND Appropriateness Method was used to further identify and develop the indicators with an electronic-Delphi method used to rate the risk associated with them. Twelve GPs from all the countries of the UK participated in the RAND exercise, with 11 GPs rating risk using the electronic-Delphi approach.

Results

Fifty-six prescribing-safety indicators were considered appropriate for inclusion (overall panel median rating of 7–9, with agreement). These indicators cover hazardous prescribing across a range of therapeutic indications, hazardous drug–drug combinations and inadequate laboratory test monitoring. Twenty-three (41%) of these indicators were considered high risk or extreme risk by 80% or more of the participants.

Conclusion

This study identified a set of 56 indicators that were considered, by a panel of GPs, to be appropriate for assessing the safety of GP prescribing. Twenty-three of these indicators were considered to be associated with high or extreme risk to patients and should be the focus of efforts to improve patient safety.     

Simultaneous quantification of ten key Kratom alkaloids in Mitragyna speciosa leaf extracts and commercial products by ultra‐performance liquid chromatography−tandem mass spectrometry

Kratom (Mitragyna speciosa) is a psychoactive plant popular in the United States for the self‐treatment of pain and opioid addiction. For standardization and quality control of raw and commercial kratom products, an ultra‐performance liquid chromatography?tandem mass spectrometry (UPLC?MS/MS) method was developed and validated for the quantification of ten key alkaloids, namely: corynantheidine, corynoxine, corynoxine B, 7‐hydroxymitragynine, isocorynantheidine, mitragynine, mitraphylline, paynantheine, speciociliatine, and speciogynine. Chromatographic separation of diastereomers, or alkaloids sharing same ion transitions, was achieved on an Acquity BEH C18 column with a gradient elution using a mobile phase containing acetonitrile and aqueous ammonium acetate buffer (10mM, pH 3.5). The developed method was linear over a concentration range of 1–200 ng/mL for each alkaloid. The total analysis time per sample was 22.5 minutes. The analytical method was validated for accuracy, precision, robustness, and stability. After successful validation, the method was applied for the quantification of kratom alkaloids in alkaloid‐rich fractions, ethanolic extracts, lyophilized teas, and commercial products. Mitragynine (0.7%–38.7% w/w), paynantheine (0.3%–12.8% w/w), speciociliatine (0.4%–12.3% w/w), and speciogynine (0.1%–5.3% w/w) were the major alkaloids in the analyzed kratom products/extracts. Minor kratom alkaloids (corynantheidine, corynoxine, corynoxine B, 7‐hydroxymitragynine, isocorynantheidine) were also quantified (0.01%–2.8% w/w) in the analyzed products; however mitraphylline was below the lower limit of quantification in all analyses.