Western diet increases brain metabolism and adaptive immune responses in a mouse model of amyloidosis.

Diet-induced increase in body weight is a growing health concern worldwide. Often accompanied by a low-grade metabolic inflammation that changes systemic functions, diet-induced alterations may contribute to neurodegenerative disorder progression as well. This study aims to non-invasively investigate diet-induced metabolic and inflammatory effects in the brain of an APPPS1 mouse model of Alzheimer's disease. [18F]FDG, [18F]FTHA, and [18F]GE-180 were used for in vivo PET imaging in wild-type and APPPS1 mice. Ex vivo flow cytometry and histology in brains complemented the in vivo findings. 1H- magnetic resonance spectroscopy in the liver, plasma metabolomics and flow cytometry of the white adipose tissue were used to confirm metaflammatory condition in the periphery. We found disrupted glucose and fatty acid metabolism after Western diet consumption, with only small regional changes in glial-dependent neuroinflammation in the brains of APPPS1 mice. Further ex vivo investigations revealed cytotoxic T cell involvement in the brains of Western diet-fed mice and a disrupted plasma metabolome. 1H-magentic resonance spectroscopy and immunological results revealed diet-dependent inflammatory-like misbalance in livers and fatty tissue. Our multimodal imaging study highlights the role of the brain-liver-fat axis and the adaptive immune system in the disruption of brain homeostasis in amyloid models of Alzheimer's disease.

Acidosis-mediated increase in IFN-γ-induced PD-L1 expression on cancer cells as an immune escape mechanism in solid tumors.

Immune checkpoint inhibitors have revolutionized cancer therapy, yet the efficacy of these treatments is often limited by the heterogeneous and hypoxic tumor microenvironment (TME) of solid tumors. In the TME, programmed death-ligand 1 (PD-L1) expression on cancer cells is mainly regulated by Interferon-gamma (IFN-γ), which induces T cell exhaustion and enables tumor immune evasion. In this study, we demonstrate that acidosis, a common characteristic of solid tumors, significantly increases IFN-γ-induced PD-L1 expression on aggressive cancer cells, thus promoting immune escape. Using preclinical models, we found that acidosis enhances the genomic expression and phosphorylation of signal transducer and activator of transcription 1 (STAT1), and the translation of STAT1 mRNA by eukaryotic initiation factor 4F (elF4F), resulting in an increased PD-L1 expression. We observed this effect in murine and human anti-PD-L1-responsive tumor cell lines, but not in anti-PD-L1-nonresponsive tumor cell lines. In vivo studies fully validated our in vitro findings and revealed that neutralizing the acidic extracellular tumor pH by sodium bicarbonate treatment suppresses IFN-γ-induced PD-L1 expression and promotes immune cell infiltration in responsive tumors and thus reduces tumor growth. However, this effect was not observed in anti-PD-L1-nonresponsive tumors. In vivo experiments in tumor-bearing IFN-γ-/- mice validated the dependency on immune cell-derived IFN-γ for acidosis-mediated cancer cell PD-L1 induction and tumor immune escape. Thus, acidosis and IFN-γ-induced elevation of PD-L1 expression on cancer cells represent a previously unknown immune escape mechanism that may serve as a novel biomarker for anti-PD-L1/PD-1 treatment response. These findings have important implications for the development of new strategies to enhance the efficacy of immunotherapy in cancer patients.

Platelet-Derived MicroRNAs Regulate Cardiac Remodeling After Myocardial Ischemia.

BACKGROUND: Platelets can infiltrate ischemic myocardium and are increasingly recognized as critical regulators of inflammatory processes during myocardial ischemia and reperfusion (I/R). Platelets contain a broad repertoire of microRNAs (miRNAs), which, under certain conditions such as myocardial ischemia, may be transferred to surrounding cells or released into the microenvironment. Recent studies could demonstrate that platelets contribute substantially to the circulating miRNA pool holding the potential for so far undiscovered regulatory functions. The present study aimed to determine the role of platelet-derived miRNAs in myocardial injury and repair following myocardial I/R. METHODS: In vivo model of myocardial I/R, multimodal in vivo and ex vivo imaging approaches (light-sheet fluorescence microscopy, positron emission tomography and magnetic resonance imaging, speckle-tracking echocardiography) of myocardial inflammation and remodeling, and next-generation deep sequencing analysis of platelet miRNA expression. RESULTS: In mice with a megakaryocyte/platelet-specific knockout of pre-miRNA processing ribonuclease Dicer, the present study discloses a key role of platelet-derived miRNAs in the tightly regulated cellular processes orchestrating left ventricular remodeling after myocardial I/R following transient left coronary artery ligation. Disruption of the miRNA processing machinery in platelets by deletion of Dicer resulted in increased myocardial inflammation, impaired angiogenesis, and accelerated development of cardiac fibrosis, culminating in an increased infarct size by d7 that persisted through d28 of myocardial I/R. Worsened cardiac remodeling after myocardial infarction in mice with a platelet-specific Dicer deletion resulted in an increased fibrotic scar formation and distinguishably increased perfusion defect of the apical and anterolateral wall at day 28 post-myocardial infarction. Altogether, these observations culminated in an impaired left ventricular function and hampered long-term cardiac recovery after experimental myocardial infarction and reperfusion therapy. Treatment with the P2Y12 (P2Y purinoceptor 12) antagonist ticagrelor completely reversed increased myocardial damage and adverse cardiac remodeling observed in DicerPf4∆/Pf4∆ mice. CONCLUSIONS: The present study discloses a critical role of platelet-derived miRNA in myocardial inflammation and structural remodeling processes following myocardial I/R.

Fat Grafts Show Higher Hypoxia, Angiogenesis, Adipocyte Proliferation, and Macrophage Infiltration than Flaps in a Pilot Mouse Study.

BACKGROUND: Over 137,000 breast reconstructions are performed annually by American Society of Plastic Surgeons (ASPS) members. Vascularized flaps and avascular lipofilling each account for over 33,000 autologous reconstructions. Although clinical and experimental observations suggest biologic differences with diverging effects on locoregional tumor control, comparative animal models are lacking. The authors standardized existing techniques in immunocompetent mice, laying the foundation for in vivo models of autologous breast reconstruction combinable with orthotopic tumor implantations. METHODS: Twenty-five groin flaps and 39 fat grafts were transferred in female BALB/c-mice. Adipocytes were tracked via Hoechst-Calcein-DiI staining ( n = 2 per group), and postoperative volume retentions were compared via magnetic resonance imaging ( n = 3 per group) on days 1, 11, 21, and 31. Proliferation indices, microvessel densities, tissue hypoxia, and macrophage infiltrates were compared via Ki67, CD31, pimonidazole, and hematoxylin-eosin staining on days 5, 10, 15, 20, and 30 ( n = 4 per group). RESULTS: Viable adipocytes were present in both groups. Graft volumes plateaued at 42.7 ± 1.2% versus 81.8 ± 4.0% of flaps ( P < 0.001). Initially, grafts contained more hypoxic cells (day 5: 15.192 ± 1.249 versus 1.157 ± 192; P < 0.001), followed by higher proliferation (day 15: 25.2 ± 1.0% versus 0.0 ± 0.0%; P < 0.001), higher microvessel numbers (day 30: 307.0 ± 13.2 versus 178.0 ± 10.6; P < 0.001), and more pronounced macrophage infiltrates (graded 3 versus 2; P < 0.01). CONCLUSION: This comparative murine pilot study of vascularized flaps versus avascular lipofilling suggests differences in volume retention, proliferation, angiogenesis, hypoxia, and inflammation. CLINICAL RELEVANCE STATEMENT: The biological differences of fat grafting versus flap transfer are not fully understood because no single comparative experimental model has been established to date. The authors present the first comparative small animal model of both techniques, which will allow the gaining of deeper insights into their biological effects.

Preclinical Identification Of Tumor-Draining Lymph Nodes Using a Multimodal Non-invasive In vivo Imaging Approach.

PURPOSE: Resection of the tumor-draining lymph -node (TDLN) represents a standard method to identify metastasis for several malignancies. Interestingly, recent preclinical studies indicate that TDLN resection diminishes the efficacy of immune checkpoint inhibitor-based cancer immunotherapies. Thus, accurate preclinical identification of TDLNs is pivotal to uncovering the underlying immunological mechanisms. Therefore, we validated preclinically, and clinically available non-invasive in vivo imaging approaches for precise TDLN identification. PROCEDURES: For visualization of the lymphatic drainage into the TDLNs by non-invasive in vivo optical imaging, we injected the optical imaging contrast agents Patent Blue V (582.7 g mol-1) and IRDye® 800CW polyethylene glycol (PEG; 25,000-60,000 g mol-1), subcutaneously (s.c.) in close proximity to MC38 adenocarcinomas at the right flank of experimental mice. For determination of the lymphatic drainage and the glucose metabolism in TDLNs by non-invasive in vivo PET/magnetic resonance imaging (PET/MRI), we injected the positron emission tomography (PET) tracer (2-deoxy-2[18F]fluoro-D-glucose (18F-FDG) [181.1 g mol-1]) in a similar manner. For ex vivo cross-correlation, we isolated TDLNs and contralateral nontumor-draining lymph nodes (NTDLNs) and performed optical imaging, biodistribution, and autoradiography analysis. RESULTS: The clinically well-established Patent Blue V was superior for intraoperative macroscopic identification of the TDLNs compared with IRDye® 800CW PEG but was not sensitive enough for non-invasive in vivo detection by optical imaging. Ex vivo Patent Blue V biodistribution analysis clearly identified the right accessory axillary and the proper axillary lymph node (LN) as TDLNs, whereas ex vivo IRDye® 800CW PEG completely failed. In contrast, functional non-invasive in vivo 18F-FDG PET/MRI identified a significantly elevated uptake exclusively within the ipsilateral accessory axillary TDLN of experimental mice and was able to differentiate between the accessory axillary and the proper LN. Ex vivo biodistribution and autoradiography confirmed our in vivo 18F-FDG PET/MRI results. CONCLUSIONS: When taken together, our results demonstrate the feasibility of 18F-FDG-PET/MRI as a valid method for non-invasive in vivo, intraoperative, and ex vivo identification of the lymphatic drainage and glucose metabolism within the TDLNs. In addition, using Patent Blue V provides additive value for the macroscopic localization of the lymphatic drainage both visually and by ex vivo optical imaging analysis. Thus, both methods are valuable, easy to implement, and cost-effective for preclinical identification of the TDLN.

A Single-Pot Template Reaction Towards a Manganese-Based T1 Contrast Agent.

Manganese-based contrast agents (MnCAs) have emerged as suitable alternatives to gadolinium-based contrast agents (GdCAs). However, due to their kinetic lability and laborious synthetic procedures, only a few MnCAs have found clinical MRI application. In this work, we have employed a highly innovative single-pot template synthetic strategy to develop a MnCA, MnLMe , and studied the most important physicochemical properties in vitro. MnLMe displays optimized r1 relaxivities at both medium (20 and 64 MHz) and high magnetic fields (300 and 400 MHz) and an enhanced r1b =21.1 mM-1 s-1 (20 MHz, 298 K, pH 7.4) upon binding to BSA (Ka =4.2×103  M-1 ). In vivo studies show that MnLMe is cleared intact into the bladder through renal excretion and has a prolonged blood half-life compared to the commercial GdCA Magnevist. MnLMe shows great promise as a novel MRI contrast agent.

Non invasive in vivo monitoring of dimethyl fumarate treatment in EAE by assessing the glucose metabolism in secondary lymphoid organs.

[18 F]FDG-PET/CT is a high sensitive functional diagnostic imaging modality to monitor tumor but also immune cell activation by determination of the glucose metabolism. Our results show that the anti-inflammatory effects of immunotherapeutics like DMF can be assessed non invasively in vivo during Th1/Th17 cell-mediated encephalomyelitis (EAE) by [18 F]FDG-PET/CT imaging of the draining lymph nodes.

A Single-Pot Template Reaction Towards a Manganese-Based T1 Contrast Agent.

Manganese-based contrast agents (MnCAs) have emerged as suitable alternatives to gadolinium-based contrast agents (GdCAs). However, due to their kinetic lability and laborious synthetic procedures, only a few MnCAs have found clinical MRI application. In this work, we have employed a highly innovative single-pot template synthetic strategy to develop a MnCA, MnLMe, and studied the most important physicochemical properties in vitro. MnLMe displays optimized r 1 relaxivities at both medium (20 and 64 MHz) and high magnetic fields (300 and 400 MHz) and an enhanced r 1 b=21.1 mM-1 s-1 (20 MHz, 298 K, pH 7.4) upon binding to BSA (K a=4.2×103 M-1). In vivo studies show that MnLMe is cleared intact into the bladder through renal excretion and has a prolonged blood half-life compared to the commercial GdCA Magnevist. MnLMe shows great promise as a novel MRI contrast agent.

In vitro maturation of immature oocytes from ovarian tissue prior to shipment to a cryobank.

PURPOSE: Female fertility preservation prior to gonadotoxic therapies can be achieved by the cryopreservation of ovarian cortical tissue. Immature oocytes may be recovered during the preparation, matured in vitro and lead to live births, thereby providing an additional option for fertility preservation. The purpose of this study was to test the feasibility of this approach in a setting with unilateral biopsy of a small piece of ovarian tissue and minimal tissue preparation prior to shipment to an external cryobank. METHODS: A prospective observational clinical study in an academic center was performed from January 2018 through December 2019. Ovarian tissue was obtained laparoscopically. Immature oocytes were recovered by minimal preparation of the tissue before shipment to an external cryobank for cryopreservation. In vitro maturation was performed on recovered immature oocytes. RESULTS: Twelve patients were enrolled. Immature oocytes could be recovered for all. The maturation rate was 38.9% (n = 14/36). Metaphase II (MII) were either directly used for intracytoplasmic sperm injection (ICSI) with a fertilization rate of 66.6% (n = 4/6) or vitrified (n = 8). PNs were cryopreserved (n = 4). Vitrified MII were warmed with a post-warming vitality rate of 75.0% (n = 3/4) and used for ICSI with a fertilization rate of 33.3% (n = 1/3). CONCLUSIONS: Immature oocytes can be successfully retrieved from ovarian tissue through minimal tissue preparation prior to shipment to a cryobank, matured in vitro, fertilized and cryopreserved for potential future fertility treatments. The total number of oocytes available for fertility preservation can be increased even without controlled ovarian stimulation in a situation where only ovarian biopsy for cryopreservation is performed. TRIAL REGISTRATION: German Clinical Trials Register (DRKS), DRKS00013170. Registered 11 December 2017, https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00013170 .

Visualization and quantification of in vivo homing kinetics of myeloid-derived suppressor cells in primary and metastatic cancer.

Myeloid-derived suppressor cells (MDSCs) are immunosuppressive cells of the myeloid compartment and major players in the tumor microenvironment (TME). With increasing numbers of studies describing MDSC involvement in cancer immune escape, cancer metastasis and the dampening of immunotherapy responses, MDSCs are of high interest in current cancer therapy research. Although heavily investigated in the last decades, the in vivo migration dynamics of MDSC subpopulations in tumor- or metastases-bearing mice have not yet been studied extensively. Therefore, we have modified our previously reported intracellular cell labeling method and applied it to in vitro generated MDSCs for the quantitative in vivo monitoring of MDSC migration in primary and metastatic cancer. MDSC migration to primary cancers was further correlated to the frequency of endogenous MDSCs. Methods: Utilizing a 64Cu-labeled 1,4,7-triazacyclononane-triacetic acid (NOTA)-modified CD11b-specific monoclonal antibody (mAb) (clone M1/70), we were able to label in vitro generated polymorphonuclear (PMN-) and monocytic (M-) MDSCs for positron emission tomography (PET) imaging. Radiolabeled PMN- and M-MDSCs ([64Cu]PMN-MDSCs and [64Cu]M-MDSCs, respectively) were then adoptively transferred into primary and metastatic MMTV-PyMT-derived (PyMT-) breast cancer- and B16F10 melanoma-bearing experimental animals, and static PET and anatomical magnetic resonance (MR) images were acquired 3, 24 and 48 h post cell injection. Results: The internalization of the [64Cu]NOTA-mAb-CD11b-complex was completed within 3 h, providing moderately stable radiolabeling with little detrimental effect on cell viability and function as determined by Annexin-V staining and T cell suppression in flow cytometric assays. Further, we could non-invasively and quantitatively monitor the migration and tumor homing of both [64Cu]NOTA-αCD11b-mAb-labeled PMN- and M-MDSCs in mouse models of primary and metastatic breast cancer and melanoma by PET. We were able to visualize and quantify an increased migration of adoptively transferred [64Cu]M-MDSCs than [64Cu]PMN-MDSCs to primary breast cancer lesions. The frequency of endogenous MDSCs in the PyMT breast cancer and B16F10 melanoma model correlated to the uptake values of adoptively transferred MDSCs with higher frequencies of PMN- and M-MDSCs in the more aggressive B16F10 melanoma tumors. Moreover, aggressively growing melanomas and melanoma-metastatic lesions recruited higher percentages of both [64Cu]PMN- and [64Cu]M-MDSCs than primary and metastatic breast cancer lesions as early as 24 h post adoptive MDSC transfer, indicating an overall stronger recruitment of cancer-promoting immunosuppressive MDSCs. Conclusion: Targeting of the cell surface integrin CD11b with a radioactive mAb is feasible for labeling of murine MDSCs for PET imaging. Fast internalization of the [64Cu]NOTA-αCD11b-mAb provides presumably enhanced stability while cell viability and functionality was not significantly affected. Moreover, utilization of the CD11b-specific mAb allows for straightforward adaptation of the labeling approach for in vivo molecular imaging of other myeloid cells of interest in cancer therapy, including monocytes, macrophages or neutrophils.

Tracking the fate of adoptively transferred myeloid-derived suppressor cells in the primary breast tumor microenvironment.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid progenitor cells that are expanded in cancer and act as potent suppressors of the anti-tumor immune response. MDSCs consist of two major subsets, namely monocytic (M-) MDSCs and granulocytic (G-) MDSCs that differ with respect to their phenotype, morphology and mechanisms of suppression. Here, we cultured bone marrow cells with IL-6 and GM-CSF in vitro to generate a population of bone marrow MDSCs (BM-MDSCs) similar to G-MDSCs from tumor-bearing mice in regards to phenotype, morphology and suppressive-function. Through fluorescent labeling of these BM-MDSCs and optical imaging, we could visualize the recruitment and localization of BM-MDSCs in breast tumor-bearing mice in vivo. Furthermore, we were able to demonstrate that BM-MDSCs home to primary and metastatic breast tumors, but have no significant effect on tumor growth or progression. Ex vivo flow cytometry characterization of BM-MDSCs after adoptive transfer demonstrated both organ-and tumor-specific effects on their phenotype and differentiation, demonstrating the importance of the local microenvironment on MDSC fate and function. In this study, we have developed a method to generate, visualize and detect BM-MDSCs in vivo and ex vivo through optical imaging and flow cytometry, in order to understand the organ-specific changes rendered to MDSCs in breast cancer.

Comparison of the Accuracy of FMT/CT and PET/MRI for the Assessment of Antibody Biodistribution in Squamous Cell Carcinoma Xenografts.

Noninvasive imaging technologies are increasingly used in preclinical drug research for the pharmacokinetic analysis of therapeutic compounds in living animals over time. The different preclinical imaging modalities available differ intrinsically in their detection principle and thus might exhibit limitations for a specific application. Here, we systematically investigated the performance of advanced fluorescence-mediated tomography (FMT)/CT in comparison to PET/MRI for quantitative analysis of the biodistribution of different antibody formats and dependence on the required imaging label in squamous cell carcinoma xenografts. Methods: Different formats of an antibody (monoclonal antibody and the antigen binding fragments F(ab')2 and Fab) targeting epidermal growth factor receptor were labeled with Alexa750 or 64Cu-NODAGA and injected intravenously into separate cohorts of nude mice bearing subcutaneous A-431 tumors. Two and 24 h after injection, the mice were measured by FMT/CT and PET/MRI. Probe accumulation was quantitatively assessed in organs and tumors. In vivo data were compared between modalities and correlated with ex vivo fluorescence, γ-counting, and electrochemiluminescence immunoassay. Results: Both imaging methods faithfully monitored the biodistribution and elimination routes of the compounds, and organ accumulation measured by FMT/CT and PET/MRI correlated significantly with ex vivo measurements. In addition, the accumulation in kidney, muscle, and tumor tissue correlated between FMT/CT and PET/MRI. However, the pharmacokinetics of the Alexa750-labeled antibody formats showed shorter blood half-times and higher liver uptake than the radiolabeled counterparts. Conclusion: FMT/CT imaging allows quantifying the biodistribution of antibodies in nude mice and provides an alternative to PET analysis in preclinical drug research. However, even for large molecules, such as monoclonal antibodies, Alexa750 labeling can change pharmacokinetics and trigger liver uptake.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT.

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described. In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-γ-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-γ enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented. The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Flux balance analysis of cyanobacterial metabolism: the metabolic network of Synechocystis sp. PCC 6803.

Cyanobacteria are versatile unicellular phototrophic microorganisms that are highly abundant in many environments. Owing to their capability to utilize solar energy and atmospheric carbon dioxide for growth, cyanobacteria are increasingly recognized as a prolific resource for the synthesis of valuable chemicals and various biofuels. To fully harness the metabolic capabilities of cyanobacteria necessitates an in-depth understanding of the metabolic interconversions taking place during phototrophic growth, as provided by genome-scale reconstructions of microbial organisms. Here we present an extended reconstruction and analysis of the metabolic network of the unicellular cyanobacterium Synechocystis sp. PCC 6803. Building upon several recent reconstructions of cyanobacterial metabolism, unclear reaction steps are experimentally validated and the functional consequences of unknown or dissenting pathway topologies are discussed. The updated model integrates novel results with respect to the cyanobacterial TCA cycle, an alleged glyoxylate shunt, and the role of photorespiration in cellular growth. Going beyond conventional flux-balance analysis, we extend the computational analysis to diurnal light/dark cycles of cyanobacterial metabolism.

The influence of the chloride currents on action potential firing and volume regulation of excitable cells studied by a kinetic model.

In excitable cells, the generation of an action potential (AP) is associated with transient changes of the intra- and extracellular concentrations of small ions such as Na(+), K(+) and Cl(-). If these changes cannot be fully reversed between successive APs cumulative changes of trans-membrane ion gradients will occur, impinging on the cell volume and the duration, amplitude and frequency of APs. Previous computational studies focused on effects associated with excitation-induced changes of potassium and sodium. Here we present a model based study on the influence of chloride on the fidelity of AP firing and cellular volume regulation during excitation. Our simulations show that depending on the magnitude of the basal chloride permeability two complementary types of responsiveness and volume variability exist: (i) At high chloride permeability (typical for muscle cells), large excitatory stimuli are required to elicit APs; repetitive stimuli of equal strength result in almost identical spike train patterns (Markovian behavior), however, long excitation may lead to after discharges due to an outward directed current of intracellular chloride ions which accumulate during excitation; cell volume changes are large. (ii) At low chloride permeability (e.g., neurons), small excitatory stimuli are sufficient to elicit APs, repetitive stimuli of equal strength produce spike trains with progressively changing amplitude, frequency and duration (short-term memory effects or non-Markovian behavior); cell volume changes are small. We hypothesize that variation of the basal chloride permeability could be an important mechanism of neuronal cells to adapt their responsiveness to external stimuli during learning and memory processes.

2B4 engagement mediates rapid LFA-1 and actin-dependent NK cell adhesion to tumor cells as measured by single cell force spectroscopy.

Adhesion to tumor target cells is essential for initiation and execution of cellular cytotoxicity. In this study, we use single cell force spectroscopy to determine the exact biophysical values of the interaction forces between NK cells and tumor cells. We show that engagement of the activating NK cell receptor 2B4 can rapidly mediate an increase in the force necessary to separate NK cells from tumor cells, starting from 1 nN and increasing to 3 nN after only 120 s tumor cell contact. This early adhesion was mediated by the integrin LFA-1 and dependent on the actin cytoskeleton. The ability of NK cells to rapidly adhere to tumor target cells is consistent with their function in innate immune responses. Our data further suggest that a killing decision is already made within 120- 300 s of tumor cell contact, supporting the essential function of cell adhesion during the early phase of cellular cytotoxicity.

FASIMU: flexible software for flux-balance computation series in large metabolic networks.

BACKGROUND: Flux-balance analysis based on linear optimization is widely used to compute metabolic fluxes in large metabolic networks and gains increasingly importance in network curation and structural analysis. Thus, a computational tool flexible enough to realize a wide variety of FBA algorithms and able to handle batch series of flux-balance optimizations is of great benefit. RESULTS: We present FASIMU, a command line oriented software for the computation of flux distributions using a variety of the most common FBA algorithms, including the first available implementation of (i) weighted flux minimization, (ii) fitness maximization for partially inhibited enzymes, and (iii) of the concentration-based thermodynamic feasibility constraint. It allows batch computation with varying objectives and constraints suited for network pruning, leak analysis, flux-variability analysis, and systematic probing of metabolic objectives for network curation. Input and output supports SBML. FASIMU can work with free (lp_solve and GLPK) or commercial solvers (CPLEX, LINDO). A new plugin (faBiNA) for BiNA allows to conveniently visualize calculated flux distributions. The platform-independent program is an open-source project, freely available under GNU public license at http://www.bioinformatics.org/fasimu including manual, tutorial, and plugins. CONCLUSIONS: We present a flux-balance optimization program whose main merits are the implementation of thermodynamics as a constraint, batch series of computations, free availability of sources, choice on various external solvers, and the flexibility on metabolic objectives and constraints.

Single cell force spectroscopy of T cells recognizing a myelin-derived peptide on antigen presenting cells.

T-cell recognition of peptide-MHC complexes on APCs requires cell-cell interactions. The molecular events leading to T-cell activation have been extensively investigated, but the underlying physical binding forces between T-cells and APCs are largely unknown. We used single cell force spectroscopy for quantitation of interaction forces between T-cells and APCs presenting a tolerogenic peptide derived from myelin basic protein. When T-cells were brought into contact with peptide-loaded APCs, interaction forces increased with time from about 0.5nN after 10s interaction to about 15nN after 30min. In the absence of antigen, or when ICAM-1-negative APC was used, no increase in binding forces was observed. The temporal development of interaction forces correlated with the kinetics of immune synapse formation, as determined by LFA-1 and TCR enrichment at the interface of T-cell/APC conjugates using high throughput multispectral imaging flow cytometry. Together, these results suggest that ICAM-1/LFA-1 redistribution to the contact area is mainly responsible for development of strong interaction forces. High forces will keep T-cells and APCs in tight contact, thereby providing a platform for optimal interaction between TCRs and peptide-MHC complexes.

Endothelial cell ephrinB2-dependent activation of monocytes in arteriosclerosis.

OBJECTIVE: The expression of ephrinB2 in endothelial cells delineates their arterial phenotype and is a prerequisite for the development of the embryonic vasculature. Whereas the role of ephrinB2 in the microcirculation has been studied extensively, its expression and function in adult arteries is hardly understood. METHODS AND RESULTS: Our analyses showed that in mouse arteries, ephrinB2 is located on the luminal surface of endothelial cells and may physically interact with monocyte EphB receptors. Moreover, transdifferentiation of human monocytes into macrophages was associated with an increase in EphB2 expression, and exposing monocytes to immobilized ephrinB2 resulted in phosphorylation of the receptor followed by an increased expression of proinflammatory chemokines such as interleukin-8 and monocyte chemotactic protein-1/CCL2. Relating to the (patho)physiological relevance of these findings, immunofluorescence analyses revealed that ephrinB2 is most abundantly expressed in endothelial cells at arteriosclerosis predilection sites of the mouse aorta. Subsequent analyses indicated that monocyte adhesion to aortic segments abundantly expressing ephrinB2 is strongly enhanced and that endothelial cell ephrinB2 forward signaling is sufficient to upregulate cytokine expression in monocytes. CONCLUSIONS: These observations suggest a hitherto unknown link between vascular ephrinB2 expression and the proinflammatory activation of monocytes that may contribute to the pathogenesis of arteriosclerosis.

Functional analysis of bispecific antibody (EpCAMxCD3)-mediated T-lymphocyte and cancer cell interaction by single-cell force spectroscopy.

The atomic force microscopy (AFM) is a powerful tool to analyze forces generated on cellular interactions on the single-cell level. This highly sensitive device can record changes in force in the pico-Newton range, which equals single molecule bonds. Here, we have used single-cell force spectroscopy by AFM to investigate the interaction between T cells and tumor cells that is induced by the bispecific antibody HEA125xOKT3 (specificity anti-EpCAMxCD3). We show that HEA125xOKT3 induces a specific increase in adhesion force between T cells and cancer cells. The adhesive force that is generated on cell-cell contact is dependent on the applied force on initial contact and the duration of this initial contact. In summary, HEA125xOKT3 has been found to mediate contact formation by distinct processes. It induces direct cell-cell interaction, which results in the activation of T-cell signaling, facilitates the formation of supramolecular activation clusters and ultimately of an immune synapse.