Sequential Treatment with Temozolomide Plus Naturally Derived AT101 as an Alternative Therapeutic Strategy: Insights into Chemoresistance Mechanisms of Surviving Glioblastoma Cells.

Glioblastoma (GBM) is a poorly treatable disease due to the fast development of tumor recurrences and high resistance to chemo- and radiotherapy. To overcome the highly adaptive behavior of GBMs, especially multimodal therapeutic approaches also including natural adjuvants have been investigated. However, despite increased efficiency, some GBM cells are still able to survive these advanced treatment regimens. Given this, the present study evaluates representative chemoresistance mechanisms of surviving human GBM primary cells in a complex in vitro co-culture model upon sequential application of temozolomide (TMZ) combined with AT101, the R(-) enantiomer of the naturally occurring cottonseed-derived gossypol. Treatment with TMZ+AT101/AT101, although highly efficient, yielded a predominance of phosphatidylserine-positive GBM cells over time. Analysis of the intracellular effects revealed phosphorylation of AKT, mTOR, and GSK3ß, resulting in the induction of various pro-tumorigenic genes in surviving GBM cells. A Torin2-mediated mTOR inhibition combined with TMZ+AT101/AT101 partly counteracted the observed TMZ+AT101/AT101-associated effects. Interestingly, treatment with TMZ+AT101/AT101 concomitantly changed the amount and composition of extracellular vesicles released from surviving GBM cells. Taken together, our analyses revealed that even when chemotherapeutic agents with different effector mechanisms are combined, a variety of chemoresistance mechanisms of surviving GBM cells must be taken into account.

Degranulation of human cytotoxic lymphocytes is a major source of proteolytically active soluble CD26/DPP4.

Dipeptidyl peptidase 4 (DPP4, CD26) is a serine protease detected on several immune cells and on epithelial cells of various organs. Besides the membrane-bound enzyme, a catalytically active soluble form (sCD26/DPP4) is detected in several body fluids. Both variants cleave off dipeptides from the N-termini of various chemokines, neuropeptides, and hormones. CD26/DPP4 plays a fundamental role in the regulation of blood glucose levels by inactivating insulinotropic incretins and CD26/DPP4 inhibitors are thus routinely used in diabetes mellitus type 2 therapy to improve glucose tolerance. Such inhibitors might also prevent the CD26/DPP4-mediated inactivation of the T-cell chemoattractant CXCL10 released by certain tumors and thus improve anti-tumor immunity and immunotherapy. Despite its implication in the regulation of many (patho-)physiological processes and its consideration as a biomarker and therapeutic target, the cellular source of sCD26/DPP4 remains highly debated and mechanisms of its release are so far unknown. In line with recent reports that activated T lymphocytes could be a major source of sCD26/DPP4, we now demonstrate that CD26/DPP4 is stored in secretory granules of several major human cytotoxic lymphocyte populations and co-localizes with effector proteins such as granzymes, perforin, and granulysin. Upon stimulation, vesicular CD26/DPP4 is rapidly translocated to the cell surface in a Ca2+-dependent manner. Importantly, activation-induced degranulation leads to a massive release of proteolytically active sCD26/DPP4. Since activated effector lymphocytes serve as a major source of sCD26/DPP4, these results might explain the observed disease-associated alterations of sCD26/DPP4 serum levels and also indicate a so far unknown role of CD26/DPP4 in lymphocyte-mediated cytotoxicity.

Mechanistic peculiarities of activation-induced mobilization of cytotoxic effector proteins in human T cells.

It is widely accepted that cytotoxic T and NK cells store effector proteins including granzymes, perforin and Fas ligand (FasL) in intracellular granules, often referred to as secretory lysosomes. Upon target cell encounter, these organelles are transported to the cytotoxic immunological synapse, where they fuse with the plasma membrane to release the soluble effector molecules and to expose transmembrane proteins including FasL on the cell surface. We previously described two distinct species of secretory vesicles in T and NK cells that differ in size, morphology and protein loading, most strikingly regarding FasL and granzyme B. We now show that the signal requirements for the mobilization of one or the other granule also differ substantially. We report that prestored FasL can be mobilized independent of extracellular Ca2+, whereas the surface exposure of lysosome-associated membrane proteins (Lamps; CD107a and CD63) and the release of granzyme B are calcium-dependent. The use of selective inhibitors of actin dynamics unequivocally points to different transport mechanisms for individual vesicles. While inhibitors of actin polymerization/dynamics inhibit the surface appearance of prestored FasL, they increase the activation-induced mobilization of CD107a, CD63 and granzyme B. In contrast, inhibition of the actin-based motor protein myosin 2a facilitates FasL-, but impairs CD107a-, CD63- and granzyme B mobilization. From our data, we conclude that distinct cytotoxic effector granules are differentially regulated with respect to signaling requirements and transport mechanisms. We suggest that a T cell might 'sense' which effector proteins it needs to mobilize in a given context, thereby increasing efficacy while minimizing collateral damage.

Butyrophilin 3A/CD277-Dependent Activation of Human γδ T Cells: Accessory Cell Capacity of Distinct Leukocyte Populations.

Human Vγ9Vδ2 T cells recognize in a butyrophilin 3A/CD277-dependent way microbial (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) or endogenous pyrophosphates (isopentenyl pyrophosphate [IPP]). Nitrogen-bisphosphonates such as zoledronic acid (ZOL) trigger selective γδ T cell activation because they stimulate IPP production in monocytes by inhibiting the mevalonate pathway downstream of IPP synthesis. We performed a comparative analysis of the capacity of purified monocytes, neutrophils, and CD4 T cells to serve as accessory cells for Vγ9Vδ2 T cell activation in response to three selective but mechanistically distinct stimuli (ZOL, HMBPP, agonistic anti-CD277 mAb). Only monocytes supported γδ T cell expansion in response to all three stimuli, whereas both neutrophils and CD4 T cells presented HMBPP but failed to induce γδ T cell expansion in the presence of ZOL or anti-CD277 mAb. Preincubation of accessory cells with the respective stimuli revealed potent γδ T cell-stimulating activity of ZOL- or anti-CD277 mAb-pretreated monocytes, but not neutrophils. In comparison with monocytes, ZOL-pretreated neutrophils produced little, if any, IPP and expressed much lower levels of farnesyl pyrophosphate synthase. Exogenous IL-18 enhanced the γδ T cell expansion with all three stimuli, remarkably also in response to CD4 T cells and neutrophils preincubated with anti-CD277 mAb or HMBPP. Our study uncovers unexpected differences between monocytes and neutrophils in their accessory function for human γδ T cells and underscores the important role of IL-18 in driving γδ T cell expansion. These results may have implications for the design of γδ T cell-based immunotherapeutic strategies.

SDF1α-induced interaction of the adapter proteins Nck and HS1 facilitates actin polymerization and migration in T cells.

Noncatalytic region of tyrosine kinase (Nck) is an adapter protein that comprises one SH2 (Src homology) domain and three SH3 domains. Nck links receptors and receptor-associated tyrosine kinases or adapter proteins to proteins that regulate the actin cytoskeleton. Whereas the SH2 domain binds to phosphorylated receptors or associated phosphoproteins, individual interactions of the SH3 domains with proline-based recognition motifs result in the formation of larger protein complexes. In T cells, changes in cell polarity and morphology during T-cell activation and effector function require the T-cell receptor-mediated recruitment and activation of actin-regulatory proteins to initiate cytoskeletal reorganization at the immunological synapse. We previously identified the adapter protein HS1 as a putative Nck-interacting protein. We now demonstrate that the SH2 domain of Nck specifically interacts with HS1 upon phosphorylation of its tyrosine residue 378. We report that in human T cells, ligation of the chemokine receptor CXCR4 by stromal cell-derived factor 1α (SDF1α) induces a rapid and transient phosphorylation of tyrosine 378 of HS1 resulting in an increased association with Nck. Consequently, siRNA-mediated downregulation of HS1 and/or Nck impairs SDF1α-induced actin polymerization and T-cell migration.

Tetraspanin 8 is an interactor of the metalloprotease meprin ß within tetraspanin-enriched microdomains.

Meprin ß is a dimeric type I transmembrane protein and acts as an ectodomain sheddase at the cell surface. It was shown that meprin ß cleaves the amyloid precursor protein (APP), thereby releasing neurotoxic amyloid ß peptides and implicating a role of meprin ß in Alzheimer's disease. In order to identify non-proteolytic regulators of meprin ß, we performed a split ubiquitin yeast two-hybrid screen using a small intestinal cDNA library. In this screen we identified tetraspanin 8 (TSPAN8) as interaction partner for meprin ß. Since several members of the tetraspanin family were described to interact with metalloproteases thereby affecting their localization and/or activity, we hypothesized similar functions of TSPAN8 in the regulation of meprin ß. We employed cell biological methods to confirm direct binding of TSPAN8 to meprin ß. Surprisingly, we did not observe an effect of TSPAN8 on the catalytic activity of meprin ß nor on the specific cleavage of its substrate APP. However, both proteins were identified being present in tetraspanin-enriched microdomains. Therefore we hypothesize that TSPAN8 might be important for the orchestration of meprin ß at the cell surface with impact on certain proteolytic processes that have to be further identified.

Tetraspanin 8 is an interactor of the metalloprotease meprin ß within tetraspanin-enriched microdomains.

Meprin ß is a dimeric type I transmembrane protein and acts as an ectodomain sheddase at the cell surface. It has been shown that meprin ß cleaves the amyloid precursor protein (APP), thereby releasing neurotoxic amyloid ß peptides and implicating a role of meprin ß in Alzheimer's disease. In order to identify non-proteolytic regulators of meprin ß, we performed a split ubiquitin yeast two-hybrid screen using a small intestinal cDNA library. In this screen we identified tetraspanin 8 (TSPAN8) as interaction partner for meprin ß. As several members of the tetraspanin family were described to interact with metalloproteases thereby affecting their localization and/or activity, we hypothesized similar functions of TSPAN8 in the regulation of meprin ß. We employed cell biological methods to confirm direct binding of TSPAN8 to meprin ß. Surprisingly, we did not observe an effect of TSPAN8 on the catalytic activity of meprin ß nor on the specific cleavage of its substrate APP. However, both proteins were identified as present in tetraspanin-enriched microdomains. Therefore we hypothesize that TSPAN8 might be important for the orchestration of meprin ß at the cell surface with impact on certain proteolytic processes that have to be further identified.

Nuclear death receptor TRAIL-R2 inhibits maturation of let-7 and promotes proliferation of pancreatic and other tumor cells.

BACKGROUND & AIMS: Tumor necrosis factor-related apoptosis inducing ligand (TRAIL-R1) (TNFRSF10A) and TRAIL-R2 (TNFRSF10B) on the plasma membrane bind ligands that activate apoptotic and other signaling pathways. Cancer cells also might have TRAIL-R2 in the cytoplasm or nucleus, although little is known about its activities in these locations. We investigated the functions of nuclear TRAIL-R2 in cancer cell lines. METHODS: Proteins that interact with TRAIL-R2 initially were identified in pancreatic cancer cells by immunoprecipitation, mass spectrometry, and immunofluorescence analyses. Findings were validated in colon, renal, lung, and breast cancer cells. Functions of TRAIL-R2 were determined from small interfering RNA knockdown, real-time polymerase chain reaction, Drosha-activity, microRNA array, proliferation, differentiation, and immunoblot experiments. We assessed the effects of TRAIL-R2 overexpression or knockdown in human pancreatic ductal adenocarcinoma (PDAC) cells and their ability to form tumors in mice. We also analyzed levels of TRAIL-R2 in sections of PDACs and non-neoplastic peritumoral ducts from patients. RESULTS: TRAIL-R2 was found to interact with the core microprocessor components Drosha and DGCR8 and the associated regulatory proteins p68, hnRNPA1, NF45, and NF90 in nuclei of PDAC and other tumor cells. Knockdown of TRAIL-R2 increased Drosha-mediated processing of the let-7 microRNA precursor primary let-7 (resulting in increased levels of mature let-7), reduced levels of the let-7 targets (LIN28B and HMGA2), and inhibited cell proliferation. PDAC tissues from patients had higher levels of nuclear TRAIL-R2 than non-neoplastic pancreatic tissue, which correlated with increased nuclear levels of HMGA2 and poor outcomes. Knockdown of TRAIL-R2 in PDAC cells slowed their growth as orthotopic tumors in mice. Reduced nuclear levels of TRAIL-R2 in cultured pancreatic epithelial cells promoted their differentiation. CONCLUSIONS: Nuclear TRAIL-R2 inhibits maturation of the microRNA let-7 in pancreatic cancer cell lines and increases their proliferation. Pancreatic tumor samples have increased levels of nuclear TRAIL-R2, which correlate with poor outcome of patients. These findings indicate that in the nucleus, death receptors can function as tumor promoters and might be therapeutic targets.

Surface CD107a/LAMP-1 protects natural killer cells from degranulation-associated damage.

Cytotoxic lymphocytes are important for immune responses against viral infections and cancer. They are able to kill target cells through the release of cytotoxic granules (CGs) without being harmed in the process. Because the lysosomal-associated membrane proteins (LAMPs) appear on the cell surface after CG exocytosis, we hypothesized that some of these proteins might be involved in transiently protecting cytotoxic lymphocytes from self-destruction. Intracellular expression of CD107a/LAMP-1, and to a lesser extent that of CD107b/LAMP-2, correlated with lymphocyte CG content. Engineered surface expression of CD107a/LAMP-1, but not of CD107b/LAMP-2, reduced the granule-mediated killing of transfected target cells. This was dependent on glycosylation of the CD107a/LAMP-1 hinge. Moreover, surface expression of CD107a/LAMP-1 reduced binding of perforin to cells. Importantly, knockdown of CD107a/LAMP-1 in primary human natural killer (NK) cells and deficiency of CD107a/LAMP-1 in mice resulted in increased NK cell apoptosis upon target cell-induced degranulation. Thus, our data support a novel role of CD107a/LAMP-1 in the protection of NK cells from degranulation-associated suicide, which may represent a general mechanism to transiently limit self-destruction by cytotoxic lymphocytes upon target cell killing.

Differential protein-protein interactions of full length human FasL and FasL fragments generated by proteolysis.

Fas ligand (FasL) is a death factor of the tumor necrosis factor superfamily. Like other members of this family of type II transmembrane proteins, FasL is subject to ectodomain shedding by a disintegrin and metalloproteinases (ADAMs) liberating soluble FasL and leaving membrane-integral N-terminal fragments (NTFs). These NTFs are further processed by intramembrane proteolysis through signal peptide peptidase-like 2a (SPPL2a), releasing intracellular domains (ICDs) which might translocate to the nucleus to regulate transcription. Previous work established that the proline-rich domain within the cytosolic N-terminus of FasL is required for protein-protein interactions with different Src homology 3 (SH3) or WW domain proteins. Distinct binding partners regulate FasL storage and surface appearance or are involved in other aspects of FasL biology. Given the large number of FasL interactors, we asked whether proteolytically processed FasL fragments associate with the same or distinct sets of SH3 domain proteins. To address this, we performed co-precipitation experiments using a monoclonal antibody directed against the FasL N-terminus for subsequent protein detection of full length FasL and NTFs/ICDs in Western blots. We demonstrate that members of the sorting nexin (SNX) family bind full length FasL and its N-terminal fragments whereas members of the Pombe Cdc15 homology (PCH) protein family bind full length FasL, but fail to associate with processed FasL. Thus, we provide first evidence that full length FasL and FasL fragments display selectivity regarding their association with intracellular binding partners. The differential binding most likely governs the fate and function of the intracellular FasL fragments.

Breakpoint characterization of the der(19)t(11;19)(q13;p13) in the ovarian cancer cell line SKOV-3.

About 20% of ovarian carcinomas show alterations of 19p13 and/or 19q13 in the form of added extra material whose origin often is from chromosome 11. Based on earlier spectral karyotype analysis of the ovarian cancer cell line SKOV-3, which shows an unbalanced translocation der(19)t(11;19), the aim of this study was to determine the precise breakpoints of that derivative chromosome. After rough delimitation of the breakpoints of microdissected derivative chromosomes by array analysis, we designed a matrix of primers spanning 11q13.2 and 19p13.2 detecting multiple amplicons on genomic and cDNA. Sequencing the amplicons, accurate localization of both breakpoints on both chromosomes was possible and we found that exon 14 of HOOK2 from chromosome 19 and exon 2 of ACTN3 from chromosome 11 were fused in the derivative chromosome. The breakpoint in the HOOK2 gene was in an intrinsic triplet of nucleic acids leading to a shift in the ACTN3 reading frame in the derivative chromosome. This frameshift alteration should give rise to an early stop codon causing a loss of function of ACTN3. Signals in two-dimensional Western blotting exactly match to calculated molecular mass and the isoelectric point of the fusion protein.

Shedding of endogenous MHC class I-related chain molecules A and B from different human tumor entities: heterogeneous involvement of the "a disintegrin and metalloproteases" 10 and 17.

The interaction of the MHC class I-related chain molecules A and B (MICA and MICB) with the corresponding natural killer group 2, member D (NKG2D) receptor triggers cytotoxic effector activity of natural killer cells and certain T-cell subsets and provides a costimulatory signal for cytokine production. Thus, the presence of MICA/B on transformed cells contributes to tumor immunosurveillance. Consequently, the proteolytic cleavage of MICA/B is regarded as an important immune escape mechanism of various cancer cells. To investigate the molecular machinery responsible for the shedding of endogenous MICA/B, we analyzed different human tumor entities including mammary, pancreatic and prostate carcinomas. Flow cytometry and enzyme-linked immunosorbent assay (ELISA) revealed that all tested tumor cells constitutively expressed MICA and MICB on the cell surface and also released NKG2D ligands into the supernatant. We demonstrate that the "a disintegrin and metalloproteases" (ADAMs) 10 and 17 are largely responsible for the generation of soluble MICA/B. Pharmacological inhibition of metalloproteases reduced the level of released MICA/B and increased cell surface expression. Studies using RNA interference not only revealed a prominent role of ADAM10 and ADAM17 in NKG2D ligand shedding but also a tumor cell-specific role of ADAM10 and/or ADAM17 in shedding of MICA or MICB. Moreover, we report that in the prostate carcinoma cell line PC-3, MICA was not shed at all but rather was secreted in exosomes. These data indicate that the release of NKG2D ligands from individual tumor entities is by far more complex than suggested in previously reported MICA/B transfection systems.

Down-regulation of the cancer/testis antigen 45 (CT45) is associated with altered tumor cell morphology, adhesion and migration.

BACKGROUND: Due to their restricted expression in male germ cells and certain tumors, cancer/testis (CT) antigens are regarded as promising targets for tumor therapy. CT45 is a recently identified nuclear CT antigen that was associated with a severe disease score in Hodgkin's lymphoma and poor prognosis in multiple myeloma. As for many CT antigens, the biological function of CT45 in developing germ cells and in tumor cells is largely unknown. METHODS: CT45 expression was down-regulated in CT45-positive Hodgkin's lymphoma (L428), fibrosarcoma (HT1080) and myeloma (U266B1) cells using RNA interference. An efficient CT45 knock-down was confirmed by immunofluorescence staining and/or Western blotting. These cellular systems allowed us to analyze the impact of CT45 down-regulation on proliferation, cell cycle progression, morphology, adhesion, migration and invasive capacity of tumor cells. RESULTS: Reduced levels of CT45 did not coincide with changes in cell cycle progression or proliferation. However, we observed alterations in cell adherence, morphology and migration/invasion after CT45 down-regulation. Significant changes in the distribution of cytoskeleton-associated proteins were detected by confocal imaging. Changes in cell adherence were recorded in real-time using the xCelligence system with control and siRNA-treated cells. Altered migratory and invasive capacity of CT45 siRNA-treated cells were visualized in 3D migration and invasion assays. Moreover, we found that CT45 down-regulation altered the level of the heterogeneous nuclear ribonucleoprotein syncrip (hnRNP-Q1) which is known to be involved in the control of focal adhesion formation and cell motility. CONCLUSIONS: Providing first evidence of a cell biological function of CT45, we suggest that this cancer/testis antigen is involved in the modulation of cell morphology, cell adherence and cell motility. Enhanced motility and/or invasiveness of CT45-positive cells could contribute to the more severe disease progression that is correlated to CT45-positivity in several malignancies.

The proteases HtrA2/Omi and UCH-L1 regulate TNF-induced necroptosis.

BACKGROUND: In apoptosis, proteolysis by caspases is the primary mechanism for both initiation and execution of programmed cell death (PCD). In contrast, the impact of proteolysis on the regulation and execution of caspase-independent forms of PCD (programmed necrosis, necroptosis) is only marginally understood. Likewise, the identity of the involved proteases has remained largely obscure. Here, we have investigated the impact of proteases in TNF-induced necroptosis. RESULTS: The serine protease inhibitor TPKC protected from TNF-induced necroptosis in multiple murine and human cells systems whereas inhibitors of metalloproteinases or calpain/cysteine and cathepsin proteases had no effect. A screen for proteins labeled by a fluorescent TPCK derivative in necroptotic cells identified HtrA2/Omi (a serine protease previously implicated in PCD) as a promising candidate. Demonstrating its functional impact, pharmacological inhibition or genetic deletion of HtrA2/Omi protected from TNF-induced necroptosis. Unlike in apoptosis, HtrA2/Omi did not cleave another protease, ubiquitin C-terminal hydrolase (UCH-L1) during TNF-induced necroptosis, but rather induced monoubiquitination indicative for UCH-L1 activation. Correspondingly, pharmacologic or RNA interference-mediated inhibition of UCH-L1 protected from TNF-induced necroptosis. We found that UCH-L1 is a mediator of caspase-independent, non-apoptotic cell death also in diseased kidney podocytes by measuring cleavage of the protein PARP-1, caspase activity, cell death and cell morphology. Indicating a role of TNF in this process, podocytes with stably downregulated UCH-L1 proved resistant to TNF-induced necroptosis. CONCLUSIONS: The proteases HtrA2/Omi and UCH-L1 represent two key components of TNF-induced necroptosis, validating the relevance of proteolysis not only for apoptosis, but also for caspase-independent PCD. Since UCH-L1 clearly contributes to the non-apoptotic death of podocytes, interference with the necroptotic properties of HtrA2/Omi and UCH-L1 may prove beneficial for the treatment of patients, e.g. in kidney failure.

Chemotherapy-induced release of ADAM17 bearing EV as a potential resistance mechanism in ovarian cancer.

Ovarian cancer (OvCa) is the gynaecological disorder with the poorest prognosis due to the fast development of chemoresistance. We sought to connect chemoresistance and cancer cell-derived extracellular vesicles (EV). The mechanisms of how chemoresistance is sustained by EV remained elusive. One potentially contributing factor is A Disintegrin and Metalloprotease 17 (ADAM17)-itself being able to promote chemoresistance and inducing tumour cell proliferation and survival via the Epidermal Growth Factor Receptor (EGFR) pathway by shedding several of its ligands including Amphiregulin (AREG). We now demonstrate that upon chemotherapeutic treatment, proteolytically active ADAM17 is released in association with EV from OvCa cells. In terms of function, we show that patient-derived EV induce AREG shedding and restore chemoresistance in ADAM17-deficient cells. Confirming that ADAM17-containing EV transmit chemoresistance in OvCa, we propose that ADAM17 levels (also on EV) might serve as an indicator for tumour progression and the chemosensitivity status of a given patient.

Isolation of erythrocytes infected with viable early stages of Plasmodium falciparum by flow cytometry.

The erythrocytic life cycle of Plasmodium falciparum is highly associated with severe clinical symptoms of malaria that causes hundreds of thousands of death each year. The parasite develops within human erythrocytes leading to the disruption of the infected red blood cell (iRBC) prior to the start of a new cycle of erythrocyte infection. Emerging mechanisms of resistance against antimalarial drugs require improved knowledge about parasite's blood stages to facilitate new alternative antimalarial strategies. For the analysis of young blood stages of Plasmodium at the molecular level, the isolation of ring stages is essential. However, early stages can hardly be separated from both, late stages and non-infected red blood cells using conventional methods. Here, iRBCs were stained with the DNA-binding dyes Vybrant® DyeCycle™ Violet and SYBR® Green I. Subsequently, cells were subjected to flow-cytometric analysis. This enabled the discrimination of early stage iRBCs as well as late-stage iRBCs from non-infected erythrocytes and the properties of the used dyes were evaluated. Moreover, early stage iRBCs were isolated with high purity (>98%) by FACS. Subsequently, development of sorted early stages of the parasite was monitored over time and compared with control cultures. The described flow cytometry method, based on staining with Vybrant DyeCycle Violet, allows the isolation of viable ring stages of the malarial agent P. falciparum, and thereby provides the basis for new, broad-range molecular investigations of the parasite.

Cationic detergents enable the separation of membrane proteins of Plasmodium falciparum-infected erythrocytes by 2D gel electrophoresis.

The intraerythrocytic stage of Plasmodium falciparum alters the characteristics of its host cell by exporting selected plasmodial proteins. Although it is clear that the physicochemical and immunobiological properties of the host cell are modulated during parasite development, the involved plasmodial proteins and their mode of action are not completely known. Using cetyltrimethylammonium bromide (CTAB) or benzyldimethyl-n-hexadecylammonium chloride (16-BAC) for the first dimension and SDS for the second dimension, we separated proteins from membranes of human erythrocytes and of erythrocytes infected with the malaria parasite P. falciparum. Protein spots were analyzed by MALDI-TOF/TOF MS and annotated in respective 2D master gels. By using the alternative 2D approach, characteristic host cell membrane proteins and, more importantly, membrane-associated and exported plasmodial proteins were identified that might play a role in parasite-induced host cell modulation.

The challenge to quantify proteins with charge trains due to isoforms or conformers.

Single proteins separated by 2-DE often show multiple spots spreading along the first dimension. In many cases, such charge trains are explained by isoform differences or by putative post-translational modifications including phosphorylation, glycosylation and others. We now report that individual spots of such charge trains on 2-D gels in fact often represent the same protein, but, apparently due to conformational changes, segregate to different isoelectric points. If MS analysis reveals protein identity, we therefore suggest integrating all individual spots within a charge train for quantification. Especially in quality control of pharmaceutical proteins, the integration of the spot groups of all active contents is preferable in order to obtain reproducible and reasonable quantitative results. However, most commercial software packages for gel analysis integrate the signals spot-wise. We provide an improved quantification tool for proteins with charge train groups. This calculation can be implemented using the MATLAB software and the self-developed "Correct Integration Software System" or the commercial software package Delta2D.

Meeting report: Signal transduction meets systems biology.

In the 21st century, systems-wide analyses of biological processes are getting more and more realistic. Especially for the in depth analysis of signal transduction pathways and networks, various approaches of systems biology are now successfully used. The EU FP7 large integrated project SYBILLA (Systems Biology of T-cell Activation in Health and Disease) coordinates such an endeavor. By using a combination of experimental data sets and computational modelling, the consortium strives for gaining a detailed and mechanistic understanding of signal transduction processes that govern T-cell activation. In order to foster the interaction between systems biologists and experimentally working groups, SYBILLA co-organized the 15th meeting "Signal Transduction: Receptors, Mediators and Genes" together with the Signal Transduction Society (STS). Thus, the annual STS conference, held from November 7 to 9, 2011 in Weimar, Germany, provided an interdisciplinary forum for research on signal transduction with a major focus on systems biology addressing signalling events in T-cells. Here we report on a selection of ongoing projects of SYBILLA and how they were discussed at this interdisciplinary conference.

Analysis of the Seasonal Fluctuation of γδ T Cells and Its Potential Relation with Vitamin D3.

In addition to its role in bone metabolism, vitamin D3 exerts immunomodulatory effects and has been proposed to contribute to seasonal variation of immune cells. This might be linked to higher vitamin D3 levels in summer than in winter due to differential sun exposure. γδ T cells comprise a numerically small subset of T cells in the blood, which contribute to anti-infective and antitumor immunity. We studied the seasonal fluctuation of γδ T cells, the possible influence of vitamin D3, and the effect of the active metabolite 1α,25(OH)2D3 on the in vitro activation of human γδ T cells. In a retrospective analysis with 2625 samples of random blood donors, we observed higher proportions of γδ T cells in winter when compared with summer. In a prospective study over one year with a small cohort of healthy adults who did or did not take oral vitamin D3 supplementation, higher proportions of γδ T cells were present in donors without oral vitamin D3 uptake, particularly in spring. However, γδ T cell frequency in blood did not directly correlate with serum levels of 25(OH)D3. The active metabolite 1α,25(OH)2D3 inhibited the in vitro activation of γδ T cells at the level of proliferation, cytotoxicity, and interferon-γ production. Our study reveals novel insights into the seasonal fluctuation of γδ T cells and the immunomodulatory effects of vitamin D3.