Evaluation of the PrioCHECK™ Trichinella AAD kit to detect Trichinella spiralis,T. britovi,and T. pseudospiralis larvae in pork using the automated digestion method Trichomatic-35

Trichinellosis is a potentially deadly parasitic zoonosis that is contracted by consuming undercooked infected meat. Reliable detection of infectious Trichinella spp. larvae in meat is therefore pivotal to ensure consumer's safety. The recently authorised PrioCHECK™ Trichinella Alternative Artificial Digestion (AAD) test kit appears promising when used with the standard magnetic stirrer method, but evaluation with other apparatus types is lacking.In this study, the performance of the AAD kit in an adapted Trichomatic-35 (TM35) instrument was evaluated, first, at the Swiss National Reference Laboratory for trichinellosis (NRL); second, in a ring trial involving four Swiss official laboratories. Proficiency pork samples spiked with larvae of Trichinella spiralis, T. britovi, or T. pseudospiralis were tested with the AAD kit and with the reference pepsin-HCl digestion method in TM35 instruments.At the NRL, both methods yielded identical qualitative and similar quantitative results independently of the Trichinella species. In the ring trial, satisfactory results were obtained for 47/50 (94.0%) (AAD) and 62/67 (92.5%) (reference method) of the analysed samples. Technical problems impairing analysis were more frequently observed with the AAD kit (n = 22) than with the reference method (n = 5) and were mainly (16/22) reported by one of the external labs. When no technical issues were recorded, the performance of both methods was comparable, in agreement with the observations at the NRL; however, these results suggest a need for further training with the kit and standardisation of the adapted TM35 instruments.     

Signal transduction via the T cell antigen receptor in naïve and effector/memory T cells

T cells play an indispensable role in immune defense against infectious agents, but can also be pathogenic. These T cells develop in the thymus, are exported into the periphery as naïve cells and participate in immune responses. Upon recognition of antigen, they are activated and differentiate into effector and memory T cells. While effector T cells carry out the function of the immune response, memory T cells can last up to the life time of the individual, and are activated by subsequent antigenic exposure. Throughout this life cycle, the T cell uses the same receptor for antigen, the T cell Receptor, a complex multi-subunit receptor. Recognition of antigen presented by peptide/MHC complexes on antigen presenting cells unleashes signaling pathways that control T cell activation at each stage. In this review, we discuss the signals regulated by the T cell receptor in naïve and effector/memory T cells.     

The association of rhodamine - labelled α-actinin with actin bundles in demembranated cells

Rhodamine-labelled α-actinin was specifically bound to actin containing filament bundles of demembranated fibroblasts, and was particularly associated with their termini. Optimal bi627-1ling of rhodamine-α-actinin occurred at pH 6.0–6.2 and could be a627-2ished by the addition of unlabeled α-actinin or myosin subfragment 1. The spatial relationships between the decorating α-actinin and several actin associated proteins was determined by double fluorescence microscopy.     

T cells redirected to interleukin-13Rα2 with interleukin-13 mutein–chimeric antigen receptors have anti-glioma activity but also recognize interleukin-13Rα1

Background aimsOutcomes for patients with glioblastoma remain poor despite aggressive multimodal therapy. Immunotherapy with genetically modified T cells expressing chimeric antigen receptors (CARs) targeting interleukin (IL)13Rα2, human epidermal growth factor receptor 2, epidermal growth factor variant III or erythropoietin-producing hepatocellular carcinoma A2 has shown promise for the treatment of glioma in preclinical models. On the basis of IL13Rα2 immunotoxins that contain IL13 molecules with one or two amino acid substitutions (IL13 muteins) to confer specificity to IL13Rα2, investigators have constructed CARS with IL13 muteins as antigen-binding domains. Whereas the specificity of IL13 muteins in the context of immunotoxins is well characterized, limited information is available for CAR T cells.MethodsWe constructed four second-generation CARs with IL13 muteins with one or two amino acid substitutions, and evaluated the effector function of IL13-mutein CAR T cells in vitro and in vivo.ResultsT cells expressing all four CARs recognized IL13Rα1 or IL13Rα2 recombinant protein in contrast to control protein (IL4R) as judged by interferon-γ production. IL13 protein produced significantly more IL2, indicating that IL13 mutein–CAR T cells have a higher affinity to IL13Rα2 than to IL13Rα1. In cytotoxicity assays, CAR T cells killed IL13Rα1- and/or IL13Rα2-positive cells in contrast to IL13Rα1- and IL13Rα2-negative controls. Although we observed no significant differences between IL13 mutein–CAR T cells in vitro, only T cells expressing IL13 mutein–CARs with an E13K amino acid substitution had anti-tumor activity in vivo that resulted in a survival advantage of treated animals.ConclusionsOur study highlights that the specificity/avidity of ligands is context-dependent and that evaluating CAR T cells in preclinical animal model is critical to assess their potential benefit.     

Is transformation associated with an increased error frequency in mammalian cells?

Acute starvation of mammalian cells for amino acids results in translational errors that may be detected by two-dimensional polyacrylamide gel electrophoresis. Using this as an assay for error frequency in mammalian cells, we investigated the hypothesis that neoplastic transformation was associated with an increased error frequency which in turn leads to an increased mutation rate and a decreased efficiency of regulatory controls (phenomena of tumor progression). Although we found that transformation was not always associated with an increased level of mistranslation we showed that SV40 transformation increased the level of translational errors in all cell types tested.     

Large-scale expansion of Vγ9Vδ2 T cells with engineered K562 feeder cells in G-Rex vessels and their use as chimeric antigen receptor–modified effector cells

Vγ9Vδ2 T cells are a minor subset of lymphocytes in the peripheral blood that has been extensively investigated for their tolerability, safety and anticancer efficacy. A hindrance to the broad application of these cells for adoptive cellular immunotherapy has been attaining clinically appropriate numbers of Vγ9Vδ2 T cells. Furthermore, Vγ9Vδ2 T cells exist at low frequencies among cancer patients. We, therefore, sought to conceive an economical method that allows for a quick and robust large-scale expansion of Vγ9Vδ2 T cells. A two-step protocol was developed, in which peripheral blood mononuclear cells (PBMCs) from healthy donors or cancer patients were activated with Zometa and interleukin (IL)-2, followed by co-culturing with gamma-irradiated, CD64-, CD86- and CD137L-expressing K562 artificial antigen-presenting cells (aAPCs) in the presence of the anti-CD3 antibody OKT3. We optimized the co-culture ratio of K562 aAPCs to immune cells, and migrated this method to a G-Rex cell growth platform to derive clinically relevant cell numbers in a Good Manufacturing Practice (GMP)-compliant manner. We further include a depletion step to selectively remove αβ T lymphocytes. The method exhibited high expansion folds and a specific enrichment of Vγ9Vδ2 T cells. Expanded Vγ9Vδ2 T cells displayed an effector memory phenotype with a concomitant down-regulated expression of inhibitory immune checkpoint receptors. Finally, we ascertained the cytotoxic activity of these expanded cells by using nonmodified and chimeric antigen receptor (CAR)–engrafted Vγ9Vδ2 T cells against a panel of solid tumor cells. Overall, we report an efficient approach to generate highly functional Vγ9Vδ2 T cells in massive numbers suitable for clinical application in an allogeneic setting.     

Prominent expression of FRS2β protein in neural cells and its association with intracellular vesicles

The fibroblast growth factor receptor substrate (FRS)-2 protein family comprises FRS2α, a well-known central mediator for fibroblast growth factor signaling, and FRS2β, whose endogenous expression pattern and function are not yet defined. Immunohistochemical analysis revealed that expression of FRS2β was restricted to neural tissues and it colocalized with Tuj1, a neuronal marker. There are two distinct patterns of FRS2β expression in neural cells: punctate and cup/ring-shaped; moreover, some particles colocalized with lysosomes. Stimulation with brain-derived neurotrophic factor (BDNF) enhanced FRS2β phosphorylation and the cup/ring-shaped pattern. These results suggest a probable role of FRS2β in the intracellular degradation systems of neural cells, which involves lysosomes.     

Function of the syndecan-4 cytoplasmic domain in oligomerization and association with α-actinin in turkey muscle satellite cells

Syndecan-4 (S4) is a cell membrane heparan sulfate proteoglycan that plays a role in satellite cell mediated myogenesis. S4 modulates the proliferation of myogenic satellite cells, but the mechanism of how S4 functions during myogenesis is not well understood. In other cell systems, S4 has been shown to form oligomers in the cell membrane and interact through its cytoplasmic domain with the cytoskeletal protein α-actinin. This study addressed if S4 forms oligomers and interacts with α-actinin in muscle. The S4 cytoplasmic domain was found to interact with α-actinin in a phosphatidylinositol-4,5-bisphosphate dependent manner, but did not associate with vinculin. Through confocal microscopy, both S4 and syndecan-4 without the cytoplasmic domain were localized to the cell membrane. Although the cytoplasmic domain was necessary for the interaction with α-actinin, S4 oligomer formation occurred in the absence of the cytoplasmic domain. These data indicated that S4 function in skeletal muscle is mediated through the formation of oligomers and interaction with the cytoskeletal protein α-actinin.     

Clinorotation prevents differentiation of rat myoblastic L6 cells in association with reduced NF-κB signaling

In this study, we examined effects of the three-dimensional (3D)-clinorotation, a simulated-model of microgravity, on proliferation/differentiation of rat myoblastic L6 cells. Differentiation of L6 cells into myotubes was significantly disturbed in the 3D-clinorotation culture system, although the 3D-clinorotation had no effect on the proliferation. The 3D-clinorotation also suppressed the expression of myogenesis marker proteins, such as myogenin and myosin heavy chain (MHC), at the mRNA level. In association with this reduced differentiation, we found that the 3D-clinorotation prevented accumulation of ubiquitinated proteins, compared with non-rotation control cells. Based on these findings, we focused on the ubiquitin-dependent degradation of IκB, a myogenesis inhibitory protein, to clarify the mechanism of this impaired differentiation. A decline in the amount of IκB protein in L6 cells was significantly prevented by the rotation, while the amount of the protein in the non-rotated cells decreased along with the differentiation. Furthermore, the 3D-clinorotation reduced the NF-κB-binding activity in L6 cells and prevented the ubiquitination of IκB proteins in the IκB- and ubiquitin-expressing Cos7 cells. Other myogenic regulatory factors, such as deubiquitinases, cyclin E and oxygen, were not associated with the differentiation impaired by the clinorotation. Our present results suggest that simulated microgravity such as the 3D-clinorotation may disturb skeletal muscle cell differentiation, at least in part, by inhibiting the NF-κB pathway.     

CD19 chimeric antigen receptor–redirected T cells combined with epidermal growth factor receptor pathway substrate 8 peptide–derived dendritic cell vaccine in leukemia

BackgroundChimeric antigen receptor (CAR)–T cell therapy opens a new era for cancer treatment. However, in prolonged follow-up, relapse has emerged as one of the major obstacles. Dendritic cell (DC) vaccination is a promising treatment to eradicate tumor cells and prevent relapse. The epidermal growth factor receptor (EGFR) pathway substrate 8 (Eps8) gene is involved in regulating cancer progression and is considered an attractive target for specific cancer immunotherapy. The purpose of this study was to explore a combinatorial therapy using CAR-T cells and a DC vaccine such as Eps8-DCs to increase leukemia treatment efficacy.MethodsWe pulsed DCs with Eps8-derived peptides to generate Eps8-DCs, engineered T cells to express a second-generation CAR specific for CD19, and analyzed the effects of the Eps8-DCs on the in vitro expansion, phenotype and effector functions of the CD19 CAR-T cells.ResultsThe Eps8-DCs significantly reduced the activation-induced cell death and enhanced the proliferative potential of CAR-T cells during in vitro expansion. In addition, the expanded T cells co-cultured with the Eps8-DCs exhibited an increased percentage of central memory T cells (Tcms) and a decreased percentage of effector memory T cells (Tems). The Eps8-DCs enhanced CD19 CAR-T cell immune functions, including cytokine production, CD107a degranulation activity and cytotoxicity.DiscussionThis study demonstrates that Eps8-DCs exert synergistic effect on CD19 targeting CAR-T cells and paves the way for clinical trials using the combination of DC vaccination and engineered T cells in relapsed leukemia.     

Immune modulation by zoledronic acid in human myeloma: an advantageous cross-talk between Vγ9Vδ2 T cells, αβ CD8+ T cells, regulatory T cells, and dendritic cells

Vγ9Vδ2 T cells play a major role as effector cells of innate immune responses against microbes, stressed cells, and tumor cells. They constitute <5% of PBLs but can be expanded by zoledronic acid (ZA)-treated monocytes or dendritic cells (DC). Much less is known about their ability to act as cellular adjuvants bridging innate and adaptive immunity, especially in patients with cancer. We have addressed this issue in multiple myeloma (MM), a prototypic disease with several immune dysfunctions that also affect γδ T cells and DC. ZA-treated MM DC were highly effective in activating autologous γδ T cells, even in patients refractory to stimulation with ZA-treated monocytes. ZA inhibited the mevalonate pathway of MM DC and induced the intracellular accumulation and release into the supernatant of isopentenyl pyrophosphate, a selective γδ T cell activator, in sufficient amounts to induce the proliferation of γδ T cells. Immune responses against the tumor-associated Ag survivin (SRV) by MHC-restricted, SRV-specific CD8(+) αβ T cells were amplified by the concurrent activation of γδ T cells driven by autologous DC copulsed with ZA and SRV-derived peptides. Ancillary to the isopentenyl pyrophosphate-induced γδ T cell proliferation was the mevalonate-independent ZA ability to directly antagonize regulatory T cells and downregulate PD-L2 expression on the DC cell surface. In conclusion, ZA has multiple immune modulatory activities that allow MM DC to effectively handle the concurrent activation of γδ T cells and MHC-restricted CD8(+) αβ antitumor effector T cells.     

Satellite 2 methylation patterns in normal and ICF syndrome cells and association of hypomethylation with advanced replication

Mutation in the DNMT3B DNA methyltransferase gene is a common cause of ICF (immunodeficiency, centromeric heterochromatin, facial anomalies) immunodeficiency syndrome and leads to hypomethylation of satellites 2 and 3 in pericentric heterochromatin. This hypomethylation is associated with centromeric decondensation and chromosomal rearrangements, suggesting that these satellite repeats have an important structural role. In addition, the satellite regions may have functional roles in modifying gene expression. The extent of satellite hypomethylation in ICF cells is unknown because methylation status has only been determined with restriction enzymes that cut infrequently at these loci. We have therefore developed a bisulfite conversion-based method to determine the detailed cytosine methylation patterns at satellite 2 sequences in a quantitative manner for normal and ICF samples. From our sequence analysis of unmodified DNA, the internal repeat region analyzed for methylation contains an average of 17 CpG sites. The average level of methylation in normal lymphoblasts and fibroblasts is 69% compared with 20% in such cells from ICF patients with DNMT3B mutations and 29% in normal sperm. Although the mean satellite 2 methylation values for these groups do not overlap, there is considerable overlap at the level of individual DNA strands. Our analysis has also revealed a pattern of methylation specificity, suggesting that some CpGs in the repeat are more prone to methylation than other sites. Variation in satellite 2 methylation among lymphoblasts from different ICF patients has prompted us to determine the frequency of cytogenetic abnormalities in these cells. Although our data suggest that some degree of hypomethylation is necessary for pericentromeric decondensation, factors other than DNA methylation appear to play a major role in this phenomenon. Another such factor may be altered replication timing because we have discovered that the hypomethylation of satellite 2 in ICF cultures is associated with advanced replication.     

α-Dystrobrevin distribution and association with other proteins in human promyelocytic NB4 cells treated for granulocytic differentiation

Dystrobrevins (DBs) bind directly to dystrophin and are prominent components of the dystrophin-associated protein complex (DAPC) that links the cytoskeleton to the extracellular matrix. They are involved in brain development, synapse formation and plasticity, as well as water and ion homeostasis. However, the role of DB in non-muscular cells is not clear. In this study, we show that different α-dystrobrevin isoforms are present in promyelocytic leukemia (NB4) cells. Only the biggest α-dystrobrevin isoform (DB-α), which can be important for its function, was expressed in the membrane fraction of NB4 cells; the other α-DB isoforms were found in the hydrophilic cell fractions. Employing the immunoprecipitation and mass spectrometry, we identified novel α-DB-interacting proteins involved in cytoskeleton reorganization (actin, tropomyosin, gelsolin, tubulin) and signal transduction process (stathmin, prohibitin, RIBA) during proliferation and differentiation of NB4 cells. Our results suggest that α-DB isoforms play a central role in cytoskeleton reorganization via their multiple interactions with actin and actin-associating proteins and may participate in signal transduction process during NB4 cell granulocytic differentiation via directly and non directly associated proteins.     

Proteomics analysis of the ezrin interactome in B cells reveals a novel association with Myo18aα

The molecular regulation of recruitment and assembly of signalosomes near the B cell receptor (BCR) is poorly understood. We have previously demonstrated a role for the ERM family protein ezrin in regulating antigen-dependent lipid raft coalescence in B cells. In this study, we addressed the possibility that ezrin may collaborate with other adaptor proteins to regulate signalosome dynamics at the membrane. Using mass spectrometry-based proteomics analysis, we identified Myo18aα as a novel binding partner of ezrin. Myo18aα is an attractive candidate as it has several protein-protein interaction domains and an intrinsic motor activity. The expression of Myo18aα varied during B cell development in the bone marrow and in mature B cell subsets suggesting functional differences. Interestingly, BCR stimulation increased the association between ezrin and Myo18aα, and induced co-segregation of Myo18aα with the BCR and phosphotyrosine-containing proteins. Our data raise an intriguing possibility that the Myo18aα/ezrin complex may facilitate BCR-mediated signaling by recruiting signaling proteins that are in close proximity of the antigen receptor. Our study is not only significant with respect to understanding the molecular regulation of BCR signaling but also provides a broader basis for understanding the mechanism of action of ezrin in other cellular systems.     

An expanded family of proteins with BPI/LBP/PLUNC-like domains in trypanosome parasites: an association with pathogenicity?

Trypanosomatids are protozoan parasites that cause human and animal disease. Trypanosoma brucei telomeric ESs (expression sites) contain genes that are critical for parasite survival in the bloodstream, including the VSG (variant surface glycoprotein) genes, used for antigenic variation, and the SRA (serum-resistance-associated) gene, which confers resistance to lysis by human serum. In addition, ESs contain ESAGs (expression-site-associated genes), whose functions, with few exceptions, have remained elusive. A bioinformatic analysis of the ESAG5 gene of T. brucei showed that it encodes a protein with two BPI (bactericidal/permeability-increasing protein)/LBP (lipopolysaccharide-binding protein)/PLUNC (palate, lung and nasal epithelium clone)-like domains and that it belongs to a multigene family termed (GR)ESAG5 (gene related to ESAG5). Members of this family are found with various copy number in different members of the Trypanosomatidae family. T. brucei has an expanded repertoire, with multiple ESAG5 copies and at least five GRESAG5 genes. In contrast, the parasites of the genus Leishmania, which are intracellular parasites, have only a single GRESAG5 gene. Although the amino acid sequence identity between the (GR)ESAG5 gene products between species is as low as 15-25%, the BPI/LBP/PLUNC-like domain organization and the length of the proteins are highly conserved, and the proteins are predicted to be membrane-anchored or secreted. Current work focuses on the elucidation of possible roles for this gene family in infection. This is likely to provide novel insights into the evolution of the BPI/LBP/PLUNC-like domains.     

T cell-intrinsic factors contribute to the differential ability of CD8+ T cells to rapidly secrete IFN-γ in the absence of antigen

A subset of CD44(hi)CD8(+) T cells isolated from C57BL/6/J (B6) mice, but not BALB/c/By/J (BALB/c) mice, rapidly secrete IFN-γ within 16 h of infection with Listeria monocytogenes. This Ag-independent response requires the presence of both IL-12 and IL-18. Previous studies showed that dendritic cells from B6 mice produced more Th1-type cytokines such as IL-12 than did those from BALB/c mice in response to L. monocytogenes infection. In this report, we demonstrate that the microenvironment in L. monocytogenes-infected BALB/c mice is sufficient to induce responsive B6 CD8(+) T cells to rapidly secrete IFN-γ. Furthermore, BALB/c CD8(+) T cells did not rapidly secrete IFN-γ even when they were exposed to high concentrations of IL-12 plus IL-18 in vitro. In the presence of IL-12 and IL-18, B6 CD44(hi)CD8(+) T cells upregulated expression of the receptor subunits for these cytokines more rapidly than did BALB/c T cells. In comparing particular subsets of memory phenotype CD8(+) T cells, we found that virtual memory cells, rather than true Ag-experienced cells, had the greatest level of impairment in BALB/c mice. These data suggest that the degree of cytokine-driven bystander activation of CD8(+) T cells that occurs during infection depends on both APCs and T cell-intrinsic properties that can vary among mouse strains.     

Interleukin-17A- or tumor necrosis factor α-mediated increase in proliferation of T cells cocultured with synovium-derived mesenchymal stem cells in rheumatoid arthritis

Introduction

Mesenchymal stem cells (MSCs) represent promising applications in rheumatoid arthritis (RA). However, the inflammatory niche in the RA synovium could adversely affect MSC function. This study was designed to investigate biologic and immunologic properties of synovium-derived MSCs (SMSCs) in RA, with particular focus on whether cytokines can mediate increase of proliferation of T cells cocultured with SMSCs in RA.

Methods

Compared with SMSCs from eight healthy donors (HDs), SMSCs from 22 patients with RA (RAp) were evaluated. The methyl thiazolyl tetrazolium (MTT) assay was used to assess cell-population doubling and viability. Multipotentiality of SMSCs was examined by using appropriate culture conditions. Flow cytometry was used to investigate the marker phenotype of SMSCs. Immunomodulation potential of SMSCs was examined by mixed peripheral blood mononuclear cells (PBMCs) reactions, and then by PBMCs or synovial T cells with or without the addition of inflammatory cytokines (interleukin-17A (IL-17A), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ)) after stimulation with phytohemagglutinin (PHA), respectively.

Results

SMSCs from RA patients (RA-SMSCs) showed normal population doubling, cell viability, multiple differentiation characteristics, and surface markers. In either mixed PBMC reactions or PBMC proliferation stimulated with PHA, RA-SMSCs showed normal immunomodulation function compared with SMSCs from healthy donors (HD-SMSCs). However, the increase in proliferation of T cells was observed when IL-17A and TNF-α were added alone or in combination.

Conclusions

Our data suggest that the inflammatory niche, especially these cytokines, may increase the proliferation of T cells cocultured with SMSCs in RA.     

Dendritic cells transduced with lentiviral vector targeting RelB gene using RNA interference induce hyporesponsiveness in memory CD4(+) T cells and naïve CD4(+) T cells

The ability of DCs to induce immune tolerance depends on its maturation status. RelB plays a pivotal role in DCs differentiation. A therapeutic protocol of DCs-based not only induces hyporesponsiveness in T(N)s, but also in alloreactive T(M)s is required. Thus, it is urgent to assess modulatory effects of RelB-silenced DCs on T(M)s and T(N)s. In this study, we constructed lentiviral vector which could efficiently silenced the RelB in DCs (DCs-miR RelB) to keep them immature. These DCs induced antigen-specific hyporesponsiveness in CD4(+) T(N)s. In contrast, upon re-stimulation with mature DCs, CD4(+) T(M)s primed by DCs-miR RelB maintained hyporesponsiveness in terms of proliferation and cytokine production. And these may be associated with micro155 and micro181a expression levels in T(M)s and T(N)s. These results may help developing the DCs-based therapeutical protocols by inducing hyporesponsiveness in CD4(+) T(N)s and T(M)s.