ABSTRACT
Intratumour heterogeneity (ITH) fuels lung cancer evolution, which leads to immune evasion and resistance to therapy1. Here, using paired whole-exome and RNA sequencing data, we investigate intratumour transcriptomic diversity in 354 non-small cell lung cancer tumours from 347 out of the first 421 patients prospectively recruited into the TRACERx study2,3. Analyses of 947 tumour regions, representing both primary and metastatic disease, alongside 96 tumour-adjacent normal tissue samples implicate the transcriptome as a major source of phenotypic variation. Gene expression levels and ITH relate to patterns of positive and negative selection during tumour evolution. We observe frequent copy number-independent allele-specific expression that is linked to epigenomic dysfunction. Allele-specific expression can also result in genomic-transcriptomic parallel evolution, which converges on cancer gene disruption. We extract signatures of RNA single-base substitutions and link their aetiology to the activity of the RNA-editing enzymes ADAR and APOBEC3A, thereby revealing otherwise undetected ongoing APOBEC activity in tumours. Characterizing the transcriptomes of primary-metastatic tumour pairs, we combine multiple machine-learning approaches that leverage genomic and transcriptomic variables to link metastasis-seeding potential to the evolutionary context of mutations and increased proliferation within primary tumour regions. These results highlight the interplay between the genome and transcriptome in influencing ITH, lung cancer evolution and metastasis.
Subject(s)
Evolution, Molecular , Genome, Human , Lung Neoplasms , Neoplasm Metastasis , Transcriptome , Humans , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Genomics , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Mutation , Neoplasm Metastasis/genetics , Transcriptome/genetics , Alleles , Machine Learning , Genome, Human/geneticsABSTRACT
PURPOSE: A major challenge to developing new therapies for patients with malignant brain tumors is that relatively few small molecule anticancer drugs penetrate the blood-brain barrier (BBB) well enough to provide therapeutically effective concentrations in brain tissue before drug exposure in non-CNS tissues results in unacceptable toxicity. METHODS: KX2-361, a member of a novel family of compounds with Src-kinase and tubulin polymerization inhibitory activity, demonstrates good oral bioavailability and readily crosses the BBB in mice. The objective of this study was to investigate the activity of KX2-361 against human and murine glioma cells and assess its therapeutic effect in a syngeneic orthotopic model of glioblastoma. RESULTS: In addition to reducing the level of Src autophosphorylation in the GL261 murine glioblastoma cell line, KX2-361 binds directly to tubulin and disrupts microtubule architecture in glioma cells maintained in culture. CONCLUSIONS: The drug is active in vivo against orthotopic GL261 gliomas in syngeneic C57BL/6 mice. Long term survival is not observed in mice lacking an adaptive immune system, indicating that KX2-361 works in concert with the host immune system to control tumor growth and promote long-term survival in the GL261 glioma model.
Subject(s)
Acetamides/administration & dosage , Antineoplastic Agents/administration & dosage , Brain Neoplasms/metabolism , Glioblastoma/metabolism , Morpholines/administration & dosage , Pyridines/administration & dosage , Tubulin Modulators/administration & dosage , src-Family Kinases/antagonists & inhibitors , Animals , Apoptosis , Blood-Brain Barrier/metabolism , Brain Neoplasms/drug therapy , Cell Cycle Checkpoints , Cell Line, Tumor , Disease Models, Animal , Glioblastoma/drug therapy , Humans , Mice, Inbred C57BL , Phosphorylation , Protein Kinase Inhibitors/administration & dosageABSTRACT
Despite a demonstrated role for TNF-α in promoting muscle wasting and cachexia, the associated molecular mechanisms and signaling pathways of myoblast differentiation dysregulated by TNF-α remain poorly understood. This study presents well-controlled proteomic profiling as a means to investigate the mechanisms of TNF-α-regulated myogenic differentiation. Primary human muscle precursor cells (MPCs) cultured in growth medium (GM), differentiation medium (DM) to induce myogenic differentiation, and DM with 20 ng/mL of TNF-α (n = 5/group) were comparatively analyzed by an ion current-based quantitative platform consisting of reproducible sample preparation/on-pellet digestion, a long-column nano-LC separation, and ion current-based differential analysis. The inhibition of myogenic differentiation by TNF-α was confirmed by reduced formation of multinucleated myotubes and the recovered expression of altered myogenic proteins such as MYOD and myogenin during myogenic differentiation. Functional analysis and validation by immunoassay analysis suggested that the cooperation of NF-κB and STAT proteins is responsible for dysregulated differentiation in MPCs by TNF-α treatment. Increased MHC class I components such as HLA-A, HLA-B, HLA-C, and beta-2-microglobulin were also observed in cultures in DM treated with TNF-α. Interestingly, inhibition of the cholesterol biosynthesis pathway during myogenic differentiation induced by serum starvation was not recovered by TNF-α treatment, which combined with previous reports, implies that this process may be an early event of myogenesis. This finding could lay the foundation for the potential use of statins in modulating myogenesis through cholesterol, for example, in stem cell-based myocardial infarction treatment, where differentiation of myoblasts and stem cells into force-generating mature muscle cells is a key step to the therapeutic capacity. In conclusion, the landscapes of altered transcription regulators, metabolic processes, and signaling pathways in MPCs are revealed in the regulation of myogenic differentiation by TNF-α, which is valuable for myogenic cellular therapeutics.
Subject(s)
Cell Differentiation/drug effects , Muscle Development/drug effects , Proteomics/methods , Tumor Necrosis Factor-alpha/pharmacology , Cells, Cultured , Humans , Metabolism/drug effects , Myoblasts , Proteins/analysis , Signal Transduction/drug effects , Transcription, Genetic/drug effectsABSTRACT
The initial composition of acrylic bone cement along with the mixing and delivery technique used can influence its final properties and therefore its clinical success in vivo. The polymerisation of acrylic bone cement is complex with a number of processes happening simultaneously. Acrylic bone cement mixing and delivery systems have undergone several design changes in their advancement, although the cement constituents themselves have remained unchanged since they were first used. This study was conducted to determine the factors that had the greatest effect on the final properties of acrylic bone cement using a pre-filled bone cement mixing and delivery system. A design of experiments (DoE) approach was used to determine the impact of the factors associated with this mixing and delivery method on the final properties of the cement produced. The DoE illustrated that all factors present within this study had a significant impact on the final properties of the cement. An optimum cement composition was hypothesised and tested. This optimum recipe produced cement with final mechanical and thermal properties within the clinical guidelines and stated by ISO 5833 (International Standard Organisation (ISO), International standard 5833: implants for surgery-acrylic resin cements, 2002), however the low setting times observed would not be clinically viable and could result in complications during the surgical technique. As a result further development would be required to improve the setting time of the cement in order for it to be deemed suitable for use in total joint replacement surgery.
Subject(s)
Bone Cements/chemistry , Cementation , Equipment Failure Analysis/methods , Polymethyl Methacrylate/chemistry , Calibration , Cementation/methods , Cementation/standards , Compressive Strength , Glass Ionomer Cements/chemistry , Materials Testing , Porosity , Prostheses and Implants , Research Design , Stress, MechanicalABSTRACT
ABSTRACT: Paging and text messaging to request new orders remain common means of communication between clinicians and nurses in the hospital setting. However, sending and triaging multiple pages can lead to interruptions in other clinical duties. A medication order delegation protocol allowing for nurse-driven ordering of low-risk medications was developed with an objective of decreasing potentially avoidable pages. The aim of this study was to evaluate the impact of implementing this protocol on nurse and clinician perceptions of clerical burden and satisfaction. A survey assessing satisfaction with the process of obtaining medications in this protocol and the perception of clerical burden associated with ordering them before and after delegation protocol implementation was completed by over 160 clinicians and nurses. Survey respondents reported increased satisfaction and decreased clerical burden associated with the implementation of the delegation protocol. These results suggest the potential for delegation protocols to limit clerical burden associated with paging.
Subject(s)
Hospitals , Text Messaging , Humans , Communication , Surveys and QuestionnairesABSTRACT
Resistance to currently available therapies is a major impediment to the successful treatment of hematological malignancies. Here, we used a model of therapy-resistant B-cell non Hodgkin lymphoma (B-NHL) developed in our laboratory along with primary B-NHL cells to study basic mechanisms of bortezomib activity. In resistant cells and a subset of primary B-NHLs, bortezomib treatment led to stabilization of Bak and subsequent Bak-dependent activation of apoptosis. In contrast to sensitive cells that die strictly by apoptosis, bortezomib was capable of killing resistant cells through activation of apoptosis or caspase-independent mechanism(s) when caspases were pharmacologically inhibited. Our data demonstrate that bortezomib is capable of killing B-NHL cells via multiple mechanisms, regardless of their basal apoptotic potential, and contributes to growing evidence that proteasome inhibitors can act via modulation of B-cell lymphoma 2 (Bcl-2) family proteins. The capacity of bortezomib to act independently of the intrinsic apoptotic threshold of a given B-NHL cell suggests that bortezomib-based therapies could potentially overcome resistance and result in relevant clinical activity in a relapsed/refractory setting.
Subject(s)
Antineoplastic Agents/therapeutic use , Boronic Acids/therapeutic use , Drug Resistance, Neoplasm , Lymphoma, B-Cell/drug therapy , Lymphoma, B-Cell/pathology , Proto-Oncogene Proteins c-bcl-2/metabolism , Pyrazines/therapeutic use , Blotting, Western , Bortezomib , Caspases/metabolism , Enzyme Activation/drug effects , Humans , Immunoprecipitation , Lymphoma, B-Cell/metabolism , Prognosis , Tumor Cells, Cultured , UbiquitinationABSTRACT
The SH2 domain containing leukocyte phosphoprotein of 76 kD (SLP-76) is critical for pre-TCR-mediated maturation to the CD4+CD8+ double positive (DP) stage in the thymus. The absolute block in SLP-76null mice at the CD4-CD8-CD44-CD25+ (double-negative 3, DN3) stage has hindered our understanding of the role of this adaptor in alphabeta TCR-mediated signal transduction in primary thymocytes and peripheral T lymphocytes. To evaluate the requirements for SLP-76 in these events, we used a cre-loxP approach to generate mice that conditionally delete SLP-76 after the DN3 checkpoint. These mice develop DP thymocytes that express the alphabeta TCR on the surface, but lack SLP-76 at the genomic DNA and protein levels. The DP compartment has reduced cellularity in young mice and fails to undergo positive selection to CD4+ or CD8+ single positive (SP) cells in vivo or activation-induced cell death in vitro. A small number of CD4+SP thymocytes are generated, but these cells fail to flux calcium in response to an alphabeta TCR-generated signal. Peripheral T cells are reduced in number, lack SLP-76 protein, and have an abnormal surface phenotype. These studies show for the first time that SLP-76 is required for signal transduction through the mature alphabeta TCR in primary cells of the T lineage.
Subject(s)
Cell Differentiation/immunology , Cell Lineage/immunology , Gene Deletion , Phosphoproteins/genetics , Signal Transduction/immunology , T-Lymphocyte Subsets/immunology , Thymus Gland/cytology , Adaptor Proteins, Signal Transducing , Animals , Blotting, Southern , Blotting, Western , Calcium/metabolism , Female , Flow Cytometry , Mice , Mice, Inbred C57BL , Stem Cells/immunology , T-Lymphocyte Subsets/cytologyABSTRACT
Dendritic cells (DCs) are the most potent APCs for activating naive T cells, a process facilitated by the ability of immature DCs to mature and home to lymph nodes after encountering an inflammatory stimulus. Proteins involved in cytoskeletal rearrangement play an important role in regulating the adherence and motility of DCs. Vav1, a guanine nucleotide exchange factor for Rho family GTPases, mediates cytoskeletal rearrangement in hematopoietic cells following integrin ligation. We show that Vav1 is not required for the normal maturation of DCs in vitro; however, it is critical for DC binding to fibronectin and regulates the distribution but not the formation of podosomes. We also found that DC Vav1 was an important component of a signaling pathway involving focal adhesion kinase, phospholipase C-gamma2, and ERK1/2 following integrin ligation. Surprisingly, Vav1(-/-) DCs had increased rates of migration in vivo compared with wild-type control DCs. In vitro findings show that the presence of adhesive substrates such as fibronectin resulted in inhibition of migration. However, there was less inhibition in the absence of Vav1. These findings suggest that DC migration is negatively regulated by adhesion and integrin-mediated signaling and that Vav1 has a central role in this process.
Subject(s)
Cell Movement/immunology , Dendritic Cells/cytology , Dendritic Cells/immunology , Proto-Oncogene Proteins c-vav/physiology , Animals , Cell Adhesion/genetics , Cell Adhesion/immunology , Cell Differentiation/genetics , Cell Differentiation/immunology , Cell Movement/genetics , Cells, Cultured , Dendritic Cells/metabolism , Fibronectins/metabolism , Integrins/metabolism , Ligands , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Binding/genetics , Protein Binding/immunology , Proto-Oncogene Proteins c-vav/biosynthesis , Proto-Oncogene Proteins c-vav/deficiency , Proto-Oncogene Proteins c-vav/genetics , Pseudopodia/genetics , Pseudopodia/immunology , Pseudopodia/metabolism , Signal Transduction/genetics , Signal Transduction/immunologyABSTRACT
Major histocompatibility complex class II (MHCII) antigen expression is directly correlated with immunogenicity, and inversely correlated with tumorigenicity, in clones of the L1210 murine B lymphoma. Moreover, loss of MHCII expression on human diffuse large B-cell lymphoma is associated with dramatic decreases in patient survival. Thus, the role that MHCII antigens play in the progression of B-cell lymphomas is clinically important. In this study, we investigated the basis for the immunogenicity of MHCII(+) L1210 clones. Immunogenic, but not tumorigenic L1210 clones stimulated the proliferation of naïve T cells and their interleukin (IL)-2 production, which indicates that the immunogenic clones can function as antigen-presenting cells (APCs). However, subclonal variants of the immunogenic L1210 clones, which form tumours slowly in mice, could not activate T cells. The costimulatory molecules B7-1, B7-2 and CD40 were expressed on the immunogenic L1210 clones, but not the tumorigenic clones. Importantly, the tumour-forming subclonal variants expressed MHCII and B7-1, but lacked B7-2 and CD40. These results suggest that MHCII and B7-1 expression on L1210 cells is insufficient to activate naïve T cells, and, furthermore, loss of B7-2 and/or CD40 expression contributes to the decreased immunogenicity of L1210 subclones. Blocking B7-1 or B7-2 function on immunogenic L1210 cells reduced their capacity to activate naïve T cells. Furthermore, incubation of immunogenic L1210 cells with CD40 antibodies significantly enhanced APC function. Therefore, the immunogenicity of L1210 cells directly correlates (i) with their ability to stimulate naïve T cells, and (ii) with the concomitant expression of MHCII, B7-1, B7-2, and CD40.
Subject(s)
Antigen-Presenting Cells/immunology , Lymphocyte Activation , Lymphoma, Large B-Cell, Diffuse/immunology , T-Lymphocytes/immunology , Animals , Antibodies, Monoclonal/immunology , Antigen-Presenting Cells/drug effects , B7-1 Antigen/drug effects , B7-1 Antigen/immunology , B7-2 Antigen/drug effects , B7-2 Antigen/immunology , CD40 Antigens/antagonists & inhibitors , CD40 Antigens/immunology , CD40 Antigens/metabolism , Cell Line, Tumor , Cells, Cultured , Clone Cells/drug effects , Clone Cells/immunology , Histocompatibility Antigens Class II/immunology , Humans , Interleukin-2/biosynthesis , Interleukin-2/immunology , Mice , Mice, Inbred DBAABSTRACT
PURPOSE: Targeting malignant B cells using rituximab (anti-CD20) has improved the efficacy of chemotherapy regimens used to treat patients with non-Hodgkin's lymphoma. Despite the promising clinical results obtained using rituximab, many patients relapse with therapy-resistant disease following rituximab-based treatments. We have created a cell line model of rituximab resistance using three B-cell non-Hodgkin's lymphoma-derived cell lines (Raji, RL, and SUDHL-4). In an attempt to define strategies to overcome rituximab resistance, we sought to determine the chemotherapy sensitivity of our rituximab-resistant cell lines (RRCL). EXPERIMENTAL DESIGN: Parental, rituximab-sensitive cell lines (RSCL) Raji, RL, and SUDHL-4, along with RRCLs derived from them, were exposed to several chemotherapeutic agents with different mechanisms of action and the ability of these agents to induce apoptotic cell death was measured. Expression of multidomain Bcl-2 family proteins was studied as potential mediators of chemotherapy/rituximab resistance. RESULTS: We found that RRCLs are resistant to multiple chemotherapeutic agents and have significantly decreased expression of the Bcl-2 family proteins Bax, Bak, and Bcl-2. RRCLs do not undergo rituximab- or chemotherapy-induced apoptosis but die in a caspase-dependent manner when either wild-type Bax or Bak is exogenously expressed. Furthermore, forced expression of Bak sensitized RRCL to chemotherapy-induced apoptosis. CONCLUSIONS: Whereas a single or limited exposure of lymphoma cells to rituximab may lead to a favorable ratio of proapoptotic to antiapoptotic Bcl-2 family proteins, repeated exposure to rituximab is associated with a therapy-resistant phenotype via modulation of Bax and Bak expression.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm , Lymphoma, B-Cell/drug therapy , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-2-Associated X Protein/metabolism , Antibodies, Monoclonal, Murine-Derived , Antibody-Dependent Cell Cytotoxicity , Apoptosis/drug effects , Blotting, Western , Caspases/metabolism , Humans , Lymphoma, B-Cell/immunology , Lymphoma, B-Cell/metabolism , Membrane Potential, Mitochondrial/drug effects , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Rituximab , Tumor Cells, Cultured , bcl-2 Homologous Antagonist-Killer Protein/genetics , bcl-2-Associated X Protein/geneticsABSTRACT
Intracellular signaling initiated by ligation of the TCR influences cell fate at multiple points during the lifespan of a T cell. This is especially evident during thymic selection, where the nature of TCR-dependent signaling helps to establish a MHC-restricted, self-tolerant T cell repertoire. The Src homology 2 domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76) adaptor protein is a required intermediate in multiple signaling pathways triggered by TCR engagement, several of which have been implicated in dictating the outcome of thymic selection (e.g., intracellular calcium flux and activation of ERK family MAPKs). To determine if thymocyte maturation and selection at later stages of development are sensitive to perturbations in SLP-76 levels, we analyzed these crucial events using several transgenic (Tg) lines of mice expressing altered levels of SLP-76 in the thymus. In Tg mice expressing low levels of SLP-76 in preselection thymocytes, the CD4:CD8 ratio in the thymus and spleen was skewed in a manner consistent with impaired selection and/or maturation of CD4+ thymocytes. Low SLP-76 expression also correlated with reduced CD5 expression on immature thymocytes, consistent with reduced TCR signaling potential. In contrast, reconstitution of SLP-76 at higher levels resulted in normal thymic CD5 expression and CD4:CD8 ratios in the thymus and periphery. It is curious that thymic deletion of TCR-Tg (HY) thymocytes was markedly impaired in both lines of Tg-reconstituted SLP-76-/- mice. Studies using chimeric mice indicate that the defect in deletion of HY+ thymocytes is intrinsic to the developing thymocyte, suggesting that maintenance of sufficient SLP-76 expression from the endogenous locus is a key element in the selection process.
Subject(s)
Adaptor Proteins, Signal Transducing/physiology , Clonal Deletion/physiology , Phosphoproteins/physiology , T-Lymphocyte Subsets/cytology , Thymus Gland/cytology , Adaptor Proteins, Signal Transducing/deficiency , Adaptor Proteins, Signal Transducing/genetics , Animals , CD4-Positive T-Lymphocytes/cytology , CD5 Antigens/analysis , CD8-Positive T-Lymphocytes/cytology , Cell Differentiation , Immunologic Memory , Mice , Mice, Congenic , Mice, Knockout , Mice, Transgenic , Phosphoproteins/deficiency , Phosphoproteins/genetics , Radiation Chimera , Receptors, Antigen, T-Cell, alpha-beta/analysis , Receptors, Antigen, T-Cell, gamma-delta/analysis , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/physiology , Signal Transduction/physiology , Spleen/cytologyABSTRACT
The discovery of potent, peptide site directed, tyrosine kinase inhibitors has remained an elusive goal. Herein we describe the discovery of two such clinical candidates that inhibit the tyrosine kinase Src. Compound 1 is a phase 3 clinical trial candidate that is likely to provide a first in class topical treatment for actinic keratosis (AK) with good efficacy and dramatically less toxicity compared to existing standard therapy. Compound 2 is a phase 1 clinical trial candidate that is likely to provide a first in class treatment of malignant glioblastoma and induces 30% long-term complete tumor remission in animal models. The discovery strategy for these compounds iteratively utilized molecular modeling, along with the synthesis and testing of increasingly elaborated proof of concept compounds, until the final clinical candidates were arrived at. This was followed with mechanism of action (MOA) studies that revealed tubulin polymerization inhibition as the second MOA.
Subject(s)
Acetamides/pharmacology , Drug Discovery , Morpholines/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyridines/pharmacology , Tubulin Modulators/pharmacology , src-Family Kinases/antagonists & inhibitors , Acetamides/metabolism , Amino Acid Sequence , Catalytic Domain , Cell Line, Tumor , Humans , Molecular Docking Simulation , Morpholines/metabolism , Protein Kinase Inhibitors/metabolism , Pyridines/metabolism , Signal Transduction/drug effects , Tubulin Modulators/metabolism , src-Family Kinases/chemistry , src-Family Kinases/metabolismABSTRACT
Antigen receptors and integrins are structurally and functionally distinct, but both play key roles in regulating immune cell activation and function. Understanding the molecular basis of the signaling pathways utilized by antigen receptors and integrins is fundamental to identifying the mechanisms underlying immune system function and dysfunction (e.g. autoimmune disease) and identifying potential targets for modifying the immune response with therapy. Recently, several key regulators of antigen receptor signaling have also been revealed to be important molecular intermediates in integrin-triggered signaling pathways. These include the protein tyrosine kinase Syk, the guanine nucleotide exchange factor Vav, and the adaptor protein SLP-76. While antigen-receptor signaling is generally associated with leukocyte activation and differentiation, integrins are most commonly thought of as adhesive receptors. This raises the interesting question of how common molecular intermediates may regulate diverse cellular processes such as activation versus adhesion and migration, and provides a framework for defining potentially unique mechanisms utilized by cells of the immune system to regulate integrin-dependent cell function.
Subject(s)
Integrins/metabolism , Receptors, Antigen, T-Cell/metabolism , Signal Transduction , T-Lymphocytes/immunology , Adaptor Proteins, Signal Transducing/metabolism , Animals , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Lymphocyte Activation , Protein-Tyrosine Kinases/metabolism , Syk Kinase , T-Lymphocytes/enzymologyABSTRACT
Proteasome activator 200 kDa (PA200) forms nuclear foci after exposure of cells to ionizing radiation and enhances proteasome activity in vitro. Within cells, it is unclear whether PA200 responds to radiation alone or in association with proteasomes. In the present study, we identified three forms of cellular PA200 (termed PA200i, ii and iii) at the mRNA and protein levels. Neither PA200ii nor PA200iii appears to associate with proteasomes. All detectable PA200i is associated with proteasomes, which indicates that PA200i and proteasomes function together within the cell. Consistent with this idea, we find that exposure of cells to radiation leads to an equivalent accumulation of both PA200i and core proteasomes on chromatin. This increase in PA200 and proteasomes on chromatin is not specific to the stage of cell cycle arrest since it occurs in cells that arrest in G(2)/M and cells that arrest in G(1)/S after exposure to radiation. These data provide evidence that PA200 and proteasomes function together within cells and respond to a specific radiation-induced damage independent of the stage of cell cycle arrest.
Subject(s)
Cell Cycle/radiation effects , Chromatin/physiology , Chromatin/radiation effects , Nuclear Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Proteasome Endopeptidase Complex/radiation effects , Dose-Response Relationship, Radiation , HeLa Cells , Humans , Radiation Dosage , Radiation, Ionizing , Signal Transduction/radiation effectsABSTRACT
IL-17 (IL-17A or CTLA-8) is the founding member of a novel family of inflammatory cytokines, and emerging evidence indicates that it plays a central role in inflammation and autoimmunity. IL-17 is made primarily, if not exclusively by T cells, but relatively little is known about how its expression is regulated. In the present study, we examined the requirements and mechanisms for IL-17 expression in primary mouse lymphocytes. Like many cytokines, IL-17 is induced rapidly in primary T cells after stimulation of the T cell receptor (TCR) through CD3 crossinking. Surprisingly, however, the pattern of regulation of IL-17 is different in mice than in humans, because "costimulation" of T cells through CD28 only mildly enhanced IL-17 expression, whereas levels of IL-2 were dramatically enhanced. Similarly, several other costimulatory molecules such as ICOS, 4-1BB and CD40L exerted only very weak enhancing effects on IL-17 production. In agreement with other reports, IL-23 enhanced CD3-induced IL-17 expression. However, IL-17 production can occur autonomously in T cells, as neither dendritic cells nor IL-23 were necessary for promoting short-term production of IL-17. Finally, to begin to characterize the TCR-mediated signaling pathway(s) required for IL-17 production, we showed that IL-17 expression is sensitive to cyclosporin-A and MAPK inhibitors, suggesting the involvement of the calcineurin/NFAT and MAPK signaling pathways.
Subject(s)
Dendritic Cells/physiology , Interleukin-17/biosynthesis , Interleukins/immunology , Receptors, Antigen, T-Cell/metabolism , Signal Transduction/physiology , T-Lymphocytes/immunology , Animals , CD28 Antigens/metabolism , CD3 Complex/pharmacology , Calcineurin/metabolism , Cyclosporine/pharmacology , Enzyme Inhibitors/pharmacology , Gene Expression Regulation/immunology , Humans , Interleukin-2/metabolism , Interleukin-2/pharmacology , Interleukin-23 , Interleukin-23 Subunit p19 , Mice , Mice, Inbred C57BL , Mitogen-Activated Protein Kinases/metabolism , NFATC Transcription Factors/metabolismABSTRACT
Management of pancreatic ascites poses significant therapeutic challenges. Treatment usually consists of either conservative management or interventional therapy with little consensus between the two options. Conservative therapy is the most common initial treatment option but has high failure rates hence arguing for interventional therapy as a preferred primary treatment option. Endoscopic treatment is particularly appealing due to lower failure rates and mortality than conservative therapy or surgery. We describe a patient with recurrent pancreatic ascites who was successfully managed with endoscopic transpapillary stenting. This report contributes to the limited but growing literature on the management of pancreatic ascites.
Subject(s)
Ascites/surgery , Cholangiopancreatography, Endoscopic Retrograde , Pancreatic Diseases/surgery , Stents , Adult , Anti-Bacterial Agents/therapeutic use , Bacterial Infections/drug therapy , Cholangiopancreatography, Endoscopic Retrograde/adverse effects , Humans , Male , Meropenem , Recurrence , Thienamycins/therapeutic use , Treatment OutcomeABSTRACT
Here we describe an ultra-low-cost origami-based approach for large-scale manufacturing of microscopes, specifically demonstrating brightfield, darkfield, and fluorescence microscopes. Merging principles of optical design with origami enables high-volume fabrication of microscopes from 2D media. Flexure mechanisms created via folding enable a flat compact design. Structural loops in folded paper provide kinematic constraints as a means for passive self-alignment. This light, rugged instrument can survive harsh field conditions while providing a diversity of imaging capabilities, thus serving wide-ranging applications for cost-effective, portable microscopes in science and education.
Subject(s)
Microscopy/instrumentation , Equipment Design , PaperABSTRACT
Inpatient hyperglycemia is increasingly recognized as a contributor to in-hospital complications and prolonged hospital stays. Protocols to assist in management of hyperglycemia are becoming more widely used and have been shown to improve outcomes for hyperglycemic patients. In this article, several evidence-based protocols are reviewed for use by hospital-based clinicians, both for subcutaneous and intravenous insulin. Clinicians should consider implementing protocols for hyperglycemia management in the inpatient setting.