Palliative care for patients around the time of haematopoietic stem cell transplant: a qualitative study of patients' perceptions and experiences of unmet need and attitudes towards palliative care involvement.

BACKGROUND: Haemato-oncology patients are likely to be referred later to palliative care than patients with solid tumours, despite experiencing similar symptom burden. Patients prior to stem cell transplant may benefit from symptom control, advance care planning and shared decision-making, and previous studies have demonstrated feasibility and benefit of such a service. However, the views of patients are not yet established, and are vital to ensure acceptability of the service. AIMS: To identify areas where a palliative care team may help to support patients being considered for a stem cell transplant, and to explore the attitudes and perceptions of patients towards palliative care at this time. DESIGN: A qualitative study including interviews (N = 12) and a focus group (N = 4) for patients pre- and post-transplant, using a semi-structured format via telephone, online video-conferencing and face-to-face discussions. Recordings were transcribed and analysed using thematic analysis. SETTING: A tertiary cancer centre in the UK. FINDINGS: Themes identified were the following: Identified needs, Information and decision-making, Importance of relationships, Changing perceptions of what palliative care means, and The future. Patients associate palliative care with terminal care due to indirect experiences. Patients were open to palliative care once its purpose was explained and described emotional and physical needs relevant to early palliative care. CONCLUSIONS: The involvement of early palliative care alongside haematology treatment prior to stem cell transplant may improve quality of life for patients and facilitate shared decision-making at a crucial stage of treatment. Early palliative care should be offered alongside haematology care around the time of stem cell transplant, with information provided to patients regarding its role.

Foxp1 Negatively Regulates T Follicular Helper Cell Differentiation and Germinal Center Responses by Controlling Cell Migration and CTLA-4.

T follicular helper (Tfh) cells play an essential role in the formation of germinal centers (GC) and generation of high-affinity Abs. The homing of activated CD4+ T cells into B cell follicles and the involvement of key costimulatory and coinhibitory molecules are critical in controlling both the initiation and the magnitude of GC responses. Meanwhile, studies have shown that a high number of single clone B cells leads to intraclonal competition, which inhibits the generation of high-affinity Abs. Our previous work has shown that transcription factor Foxp1 is a critical negative regulator of Tfh cell differentiation. In this study, we report that the deletion of Foxp1 leads to a high proportion of activated CD4+ T cells homing into B cell follicles with faster kinetics, resulting in earlier GC formation. In addition, we show that Foxp1-deficient Tfh cells restore the generation of high-affinity Abs when cotransferred with high numbers of single clone B cells. We find that Foxp1 regulates the expression levels of cytotoxic T lymphocyte-associated Ag-4 (CTLA-4) in activated CD4+ T cells and that Ctla4 is a direct Foxp1 target. Finally, we demonstrate that CTLA-4 expression on conventional CD4+ T cells plays a cell-intrinsic role in Tfh cell differentiation in vivo, and CTLA-4 blockade helps abolish the intraclonal competition of B cells in generating high-affinity Abs.

Cutting Edge: Foxp1 Controls Naive CD8+ T Cell Quiescence by Simultaneously Repressing Key Pathways in Cellular Metabolism and Cell Cycle Progression.

Previously we have shown that transcription factor Foxp1 plays an essential role in maintaining naive T cell quiescence; in the absence of Foxp1, mature naive CD8(+) T cells proliferate in direct response to homeostatic cytokine IL-7. In this study, we report that the deletion of Foxp1 in naive CD8(+) T cells leads to enhanced activation of the PI3K/Akt/mammalian target of rapamycin signaling pathway and its downstream cell growth and metabolism targets in response to IL-7. We found that Foxp1 directly regulates PI3K interacting protein 1, a negative regulator of PI3K. Additionally, we found that deletion of Foxp1 in naive CD8(+) T cells results in increased expression levels of E2fs, the critical components for cell cycle progression and proliferation, in a manner that is not associated with increased phosphorylation of retinoblastoma protein. Taken together, our studies suggest that Foxp1 enforces naive CD8(+) T cell quiescence by simultaneously repressing key pathways in both cellular metabolism and cell cycle progression.

Influencing organisational culture: a leadership challenge.

In the wake of the Francis report, the need for NHS trusts and hospitals to adopt a culture of learning, safety and transparency has been highlighted. This article considers different aspects of culture in health care, and hones in on the link between culture and safety for patients in putting the patient first, embedding the 6Cs and considering the options to measure and influence organisational culture. The article reflects more deeply on how leadership across all levels can influence and inspire change in organisational culture, ensuring that the patient remains the focus of any changes in care delivery.

Mesenchymal stem cells enhance targeted bone growth from injectable hydrogels with BMP-2 peptides.

Osteoporosis is the most common chronic metabolic bone disease, and the prevalence of osteoporotic fractures is rapidly increasing with the aging population. While bisphosphonates can reduce bone loss and risk of fracture, these drugs are systemic, rely on long-term use, and patient compliance is low. Recombinant human bone morphogenetic protein-2 (BMP-2) is an FDA-approved protein that can offer a more targeted therapeutic than systemic treatments. DWIVA is a peptide sequence corresponding to the wrist epitope of BMP-2, and DWIVA-functionalized hydrogels feature osteoinductive propertiesin vitro and in vivo. This study reports that self-forming DWIVA-functionalized hydrogels injected into the intramedullary canal of rat femurs induce a local increase in trabecular bone in as little as 2 weeks. Increases in bone volume, trabecular thickness, and trabeculae count from DWIVA-laden hydrogels persist for at least 4 weeks, and the inclusion of mesenchymal stem cells (MSCs) significantly enhances the development of mineralized bone. Histological analysis of decalcified femurs also shows that hydrogel injections containing DWIVA peptide and MSCs stimulate unmineralized bone tissue formation and induce an increased count of osteoblasts and osteoclasts at the injection site after 4 weeks. Overall, the MSC-laden DWIVA peptide-functionalized hydrogels presented rapidly induce targeted bone formation and have the potential to form nascent bone within bones in jeopardy of an osteoporotic fracture such as the femur.

Development of Potent and Selective Monoacylglycerol Lipase Inhibitors. SARs, Structural Analysis, and Biological Characterization.

New potent, selective monoacylglycerol lipase (MAGL) inhibitors based on the azetidin-2-one scaffold ((±)-5a-v, (±)-6a-j, and (±)-7a-d) were developed as irreversible ligands, as demonstrated by enzymatic and crystallographic studies for (±)-5d, (±)-5l, and (±)-5r. X-ray analyses combined with extensive computational studies allowed us to clarify the binding mode of the compounds. 5v was identified as selective for MAGL when compared with other serine hydrolases. Solubility, in vitro metabolic stability, cytotoxicity, and absence of mutagenicity were determined for selected analogues. The most promising compounds ((±)-5c, (±)-5d, and (±)-5v) were used for in vivo studies in mice, showing a decrease in MAGL activity and increased 2-arachidonoyl-sn-glycerol levels in forebrain tissue. In particular, 5v is characterized by a high eudysmic ratio and (3R,4S)-5v is one of the most potent irreversible inhibitors of h/mMAGL identified thus far. These results suggest that the new MAGL inhibitors have therapeutic potential for different central and peripheral pathologies.

A mature ovarian teratoma from New Kingdom Amarna, Egypt.

OBJECTIVE: This paper describes the fifth case of a mature ovarian teratoma reported in the bioarchaeological literature, contributing to the temporal and geographical distribution of known examples of this unusual pathology. MATERIALS: An 18-21-year-old female found in situ within a multi-chambered subterranean tomb in the North Desert Cemetery at Amarna, Egypt (founded c. 1345 BCE) was recovered associated with a multi-lobed roughly ovoid calcified mass and two associated teeth identified within the pelvic cavity. METHODS: Macroscopic evaluation alongside careful differential diagnosis. CONCLUSIONS: The presence of multiple teeth and their degree of development provided sufficient evidence to identify this mass as an ovarian teratoma found in association with a young woman buried in one of the most richly adorned burials in the non-elite cemeteries at the New Kingdom site of Amarna. The teratoma is interpreted within our understanding of the broader social context of ancient Egyptian medical knowledge. SIGNIFICANCE: This case is the only reported example of a mature ovarian teratoma from Pharaonic Egypt, and Africa more broadly, predating other reported cases by several centuries. It adds considerable temporal and geographical depth to our understanding of this condition in the past. LIMITATIONS: This study was limited to macroscopic examination of remains and would likely benefit from either x-ray or CT-scanning of the object to examine the internal structure. SUGGESTIONS FOR FURTHER RESEARCH: Further consideration of the likely physical implications of this pathology, along with the broader social aspects of burial with objects of potential magico-medical significance is necessary.

A monoacylglycerol lipase inhibitor showing therapeutic efficacy in mice without central side effects or dependence.

Monoacylglycerol lipase (MAGL) regulates endocannabinoid 2-arachidonoylglycerol (2-AG) and eicosanoid signalling. MAGL inhibition provides therapeutic opportunities but clinical potential is limited by central nervous system (CNS)-mediated side effects. Here, we report the discovery of LEI-515, a peripherally restricted, reversible MAGL inhibitor, using high throughput screening and a medicinal chemistry programme. LEI-515 increased 2-AG levels in peripheral organs, but not mouse brain. LEI-515 attenuated liver necrosis, oxidative stress and inflammation in a CCl4-induced acute liver injury model. LEI-515 suppressed chemotherapy-induced neuropathic nociception in mice without inducing cardinal signs of CB1 activation. Antinociceptive efficacy of LEI-515 was blocked by CB2, but not CB1, antagonists. The CB1 antagonist rimonabant precipitated signs of physical dependence in mice treated chronically with a global MAGL inhibitor (JZL184), and an orthosteric cannabinoid agonist (WIN55,212-2), but not with LEI-515. Our data support targeting peripheral MAGL as a promising therapeutic strategy for developing safe and effective anti-inflammatory and analgesic agents.

Chemical Proteomics Reveals Off-Targets of the Anandamide Reuptake Inhibitor WOBE437.

Anandamide or N-arachidonoylethanolamine (AEA) is a signaling lipid that modulates neurotransmitter release via activation of the type 1 cannabinoid receptor (CB1R) in the brain. Termination of anandamide signaling is thought to be mediated via a facilitated cellular reuptake process that utilizes a purported transporter protein. Recently, WOBE437 has been reported as a novel, natural product-based inhibitor of AEA reuptake that is active in cellular and in vivo models. To profile its target interaction landscape, we synthesized pac-WOBE, a photoactivatable probe derivative of WOBE437, and performed chemical proteomics in mouse neuroblastoma Neuro-2a cells. Surprisingly WOBE437, unlike the widely used selective inhibitor of AEA uptake OMDM-1, was found to increase AEA uptake in Neuro-2a cells. In line with this, WOBE437 reduced the cellular levels of AEA and related N-acylethanolamines (NAEs). Using pac-WOBE, we identified saccharopine dehydrogenase-like oxidoreductase (SCCPDH), vesicle amine transport 1 (VAT1), and ferrochelatase (FECH) as WOBE437-interacting proteins in Neuro-2a cells. Further genetic studies indicated that SCCPDH and VAT1 were not responsible for the WOBE437-induced reduction in NAE levels. Regardless of the precise mechanism of action of WOB437 in AEA transport, we have identified SSCPHD, VAT1, and FECH as unprecedented off-targets of this molecule which should be taken into account when interpreting its cellular and in vivo effects.

Multi-parameter optimization: Development of a morpholin-3-one derivative with an improved kinetic profile for imaging monoacylglycerol lipase in the brain.

Monoacylglycerol lipase (MAGL) is a gatekeeper in regulating endocannabinoid signaling and has gained substantial attention as a therapeutic target for neurological disorders. We recently discovered a morpholin-3-one derivative as a novel scaffold for imaging MAGL via positron emission tomography (PET). However, its slow kinetics in vivo hampered the application. In this study, structural optimization was conducted and eleven novel MAGL inhibitors were designed and synthesized. Based on the results from MAGL inhibitory potency, in vitro metabolic stability and surface plasmon resonance assays, we identified compound 7 as a potential MAGL PET tracer candidate. [11C]7 was synthesized via direct 11CO2 fixation method and successfully mapped MAGL distribution patterns on rodent brains in in vitro autoradiography. PET studies in mice using [11C]7 demonstrated its improved kinetic profile compared to the lead structure. Its high specificity in vivo was proved by using MAGL KO mice. Although further studies confirmed that [11C]7 is a P-glycoprotein (P-gp) substrate in mice, its low P-gp efflux ratio on cells transfected with human protein suggests that it should not be an issue for the clinical translation of [11C]7 as a novel reversible MAGL PET tracer in human subjects. Overall, [11C]7 ([11C]RO7284390) showed promising results warranting further clinical evaluation.

Mechanical injury and cytokines cause loss of cartilage integrity and upregulate proteins associated with catabolism, immunity, inflammation, and repair.

The objectives of this study were to perform a quantitative comparison of proteins released from cartilage explants in response to treatment with IL-1beta, TNF-alpha, or mechanical compression injury in vitro and to interpret this release in the context of anabolic-catabolic shifts known to occur in cartilage in response to these insults in vitro and their implications in vivo. Bovine calf cartilage explants from 6-12 animals were subjected to injurious compression, TNF-alpha (100 ng/ml), IL-1beta (10 ng/ml), or no treatment and cultured for 5 days in equal volumes of medium. The pooled medium from each of these four conditions was labeled with one of four iTRAQ labels and subjected to nano-2D-LC/MS/MS on a quadrupole time-of-flight instrument. Data were analysed by ProQuant for peptide identification and quantitation. k-means clustering and biological pathways analysis were used to identify proteins that may correlate with known cartilage phenotypic responses to such treatments. IL-1beta and TNF-alpha treatment caused a decrease in the synthesis of collagen subunits (p < 0.05) as well as increased release of aggrecan G2 and G3 domains to the medium (p < 0.05). MMP-1, MMP-3, MMP-9, and MMP-13 were significantly increased by all treatments compared with untreated samples (p < 0.10). Increased release of proteins involved in innate immunity and immune cell recruitment were noted following IL-1beta and TNF-alpha treatment, whereas increased release of intracellular proteins was seen most dramatically with mechanical compression injury. Proteins involved in insulin-like growth factor and TGF-beta superfamily pathway modulation showed changes in pro-anabolic pathways that may represent early repair signals. At the systems level, two principal components were sufficient to describe 97% of the covariance in the data. A strong correlation was noted between the proteins released in response to IL-1beta and TNF-alpha; in contrast, mechanical injury resulted in both similarities and unique differences in the groups of proteins released compared with cytokine treatment.

Structure-Activity Relationship Studies of α-Ketoamides as Inhibitors of the Phospholipase A and Acyltransferase Enzyme Family.

The phospholipase A and acyltransferase (PLAAT) family of cysteine hydrolases consists of five members, which are involved in the Ca2+-independent production of N-acylphosphatidylethanolamines (NAPEs). NAPEs are lipid precursors for bioactive N-acylethanolamines (NAEs) that are involved in various physiological processes such as food intake, pain, inflammation, stress, and anxiety. Recently, we identified α-ketoamides as the first pan-active PLAAT inhibitor scaffold that reduced arachidonic acid levels in PLAAT3-overexpressing U2OS cells and in HepG2 cells. Here, we report the structure-activity relationships of the α-ketoamide series using activity-based protein profiling. This led to the identification of LEI-301, a nanomolar potent inhibitor for the PLAAT family members. LEI-301 reduced the NAE levels, including anandamide, in cells overexpressing PLAAT2 or PLAAT5. Collectively, LEI-301 may help to dissect the physiological role of the PLAATs.

Identification of α,ß-Hydrolase Domain Containing Protein 6 as a Diacylglycerol Lipase in Neuro-2a Cells.

The endocannabinoid 2-arachidonoylglycerol (2-AG) is involved in neuronal differentiation. This study aimed to identify the biosynthetic enzymes responsible for 2-AG production during retinoic acid (RA)-induced neurite outgrowth of Neuro-2a cells. First, we confirmed that RA stimulation of Neuro-2a cells increases 2-AG production and neurite outgrowth. The diacylglycerol lipase (DAGL) inhibitor DH376 blocked 2-AG production and reduced neuronal differentiation. Surprisingly, CRISPR/Cas9-mediated knockdown of DAGLα and DAGLß in Neuro-2a cells did not reduce 2-AG levels, suggesting another enzyme capable of producing 2-AG in this cell line. Chemical proteomics revealed DAGLß and α,ß-hydrolase domain containing protein (ABHD6) as the only targets of DH376 in Neuro-2a cells. Biochemical, genetic and lipidomic studies demonstrated that ABHD6 possesses DAGL activity in conjunction with its previously reported monoacylglycerol lipase activity. RA treatment of Neuro-2a cells increased by three-fold the amount of active ABHD6. Our study shows that ABHD6 exhibits significant DAG lipase activity in Neuro-2a cells in addition to its known MAG lipase activity and suggest it is involved in neuronal differentiation.

Solution structure and backbone dynamics of the catalytic domain of matrix metalloproteinase-2 complexed with a hydroxamic acid inhibitor.

MMP-2 is a member of the matrix metalloproteinase family that has been implicated in tumor cell metastasis and angiogenesis. Here, we describe the solution structure of a catalytic domain of MMP-2 complexed with a hydroxamic acid inhibitor (SC-74020), determined by three-dimensional heteronuclear NMR spectroscopy. The catalytic domain, designated MMP-2C, has a short peptide linker replacing the internal fibronectin-domain insertion and is enzymatically active. Distance geometry-simulated annealing calculations yielded 14 converged structures with atomic root-mean-square deviations (r.m.s.d.) of 1.02 and 1.62 A from the mean coordinate positions for the backbone and for all heavy atoms, respectively, when 11 residues at the N-terminus are excluded. The structure has the same global fold as observed for other MMP catalytic domains and is similar to previously solved crystal structures of MMP-2. Differences observed between the solution and the crystal structures, near the bottom of the S1' specificity loop, appear to be induced by the large inhibitor present in the solution structure. The MMP-2C solution structure is compared with MMP-8 crystal structure bound to the same inhibitor to highlight the differences especially in the S1' specificity loop. The finding provides a structural explanation for the selectivity between MMP-2 and MMP-8 that is achieved by large inhibitors.