In vivo analysis of uropod function during physiological T cell trafficking

Migrating lymphocytes acquire a polarized phenotype with a leading and a trailing edge, or uropod. Although in vitro experiments in cell lines or activated primary cell cultures have established that Rho-p160 coiled-coil kinase (ROCK)-myosin II-mediated uropod contractility is required for integrin de-adhesion on two-dimensional surfaces and nuclear propulsion through narrow pores in three-dimensional matrices, less is known about the role of these two events during the recirculation of primary, nonactivated lymphocytes. Using pharmacological antagonists of ROCK and myosin II, we report that inhibition of uropod contractility blocked integrin-independent mouse T cell migration through narrow, but not large, pores in vitro. T cell crawling on chemokine-coated endothelial cells under shear was severely impaired by ROCK inhibition, whereas transendothelial migration was only reduced through endothelial cells with high, but not low, barrier properties. Using three-dimensional thick-tissue imaging and dynamic two-photon microscopy of T cell motility in lymphoid tissue, we demonstrated a significant role for uropod contractility in intraluminal crawling and transendothelial migration through lymph node, but not bone marrow, endothelial cells. Finally, we demonstrated that ICAM-1, but not anatomical constraints or integrin-independent interactions, reduced parenchymal motility of inhibitor-treated T cells within the dense lymphoid microenvironment, thus assigning context-dependent roles for uropod contraction during lymphocyte recirculation.

CD69 modulates sphingosine-1-phosphate-induced migration of skin dendritic cells

In this study, we have investigated the role of CD69, an early inducible leukocyte activation receptor, in murine dendritic cell (DC) differentiation, maturation, and migration. Skin DCs and DC subsets present in mouse lymphoid organs express CD69 in response to maturation stimuli. Using a contact sensitization model, we show that skin DCs migrated more efficiently to draining lymph nodes (LNs) in the absence of CD69. This was confirmed by subcutaneous transfer of CD69-/- DCs, which presented an increased migration to peripheral LNs. Two-photon microscopy analysis showed that once DCs reached the LNs, CD69 deficiency did not alter DC interstitial motility in the LNs. Chemotaxis to sphingosine-1-phosphate (S1P) was enhanced in CD69-/- DCs compared with wild-type DCs. Accordingly, we detected a higher expression of S1P receptor type-1 (S1P(1)) by CD69-/- DCs, whereas S1P(3) expression levels were similar in wild-type and CD69-/- DCs. Moreover, in vivo treatment with S1P analogs SEW2871 and FTY720 during skin sensitization reduced skin DC migration to peripheral LNs. These results suggest that CD69 regulates S1P-induced skin DC migration by modulating S1P(1) function. Together, our findings increase our knowledge on DC trafficking patterns in the skin, enabling the development of new directed therapies using DCs for antigen (Ag) delivery.

Naive B-cell trafficking is shaped by local chemokine availability and LFA-1-independent stromal interactions.

It is not known how naive B cells compute divergent chemoattractant signals of the T-cell area and B-cell follicles during in vivo migration. Here, we used two-photon microscopy of peripheral lymph nodes (PLNs) to analyze the prototype G-protein-coupled receptors (GPCRs) CXCR4, CXCR5, and CCR7 during B-cell migration, as well as the integrin LFA-1 for stromal guidance. CXCR4 and CCR7 did not influence parenchymal B-cell motility and distribution, despite their role during B-cell arrest in venules. In contrast, CXCR5 played a nonredundant role in B-cell motility in follicles and in the T-cell area. B-cell migration in the T-cell area followed a random guided walk model, arguing against directed migration in vivo. LFA-1, but not α4 integrins, contributed to B-cell motility in PLNs. However, stromal network guidance was LFA-1 independent, uncoupling integrin-dependent migration from stromal attachment. Finally, we observed that despite a 20-fold reduction of chemokine expression in virus-challenged PLNs, CXCR5 remained essential for B-cell screening of antigen-presenting cells. Our data provide an overview of the contribution of prototype GPCRs and integrins during naive B-cell migration and shed light on the local chemokine availability that these cells compute.

Design and operation of ARGONTUBE: a 5 m long drift liquid argon TPC

The Liquid Argon Time Projection Chamber (LArTPC) is a prime type of detector for future large-mass neutrino observatories and proton decay searches. In this paper we present the design and operation, as well as experimental results from ARGONTUBE, a LArTPC being operated at the AEC-LHEP, University of Bern. The main goal of this detector is to prove the feasibility of charge drift over very long distances in liquid argon. Many other aspects of the LArTPC technology are also investigated, such as a voltage multiplier to generate high voltage in liquid argon (Greinacher circuit), a cryogenic purification system and the application of multi-photon ionization of liquid argon by a UV laser. For the first time, tracks induced by cosmic muons and UVlaser beam pulses have been observed and studied at drift distances of up to 5 m, the longest reached to date.

First measurements with ARGONTUBE, a 5m long drift Liquid Argon TPC

The Liquid Argon Time Projection Chamber (LAr TPC) technique is a promising technology for future neutrino detectors. At LHEP of the University of Bern (Switzerland), an R&D program towards large detectors are on-going. The main goal is to show the feasibility of long drift paths over many meters. Therefore, a liquid Argon TPC with 5m of drift distance was constructed. Many other aspects of the liquid Argon TPC technology are also investigated, such as a new device to generate high voltage in liquid Argon (Greinacher circuit), a recirculation filtering system and the multi-photon ionization of liquid Argon with a UV laser. Two detectors are built: a medium size prototype for specific detector technology studies, and ARGONTUBE, a 5m long device.

Development of light-responsive porous polycarbonate membranes for controlled caffeine delivery

For controlled caffeine release, light-responsive membranes were developed. It was possible to produce membranes that reduced their caffeine permeability resistance by about 97% when irradiated with UV-light compared to measurements at daylight. This was achieved by grafting polymers possessing photochromic units onto track-edged polycarbonate membranes. Covalently linked coatings on porous polycarbonate membranes were obtained by plasma activation of the membrane surface followed by plasma-induced graft polymerization. Copolymerization of spiro-compounds during the coating process as well as postmodification of preformed coatings with spiropyran resulted in photochromic membranes. For the copolymerization process, the synthesis of five photochromic methacrylic and acrylic spiropyrans and spirooxazines was successfully performed. Additionally, a spiropyran with carboxylic acid functionality was synthesized for the postmodification process. This enabled us to postmodify polymeric materials containing alcohol or amine groups to obtain photochromic materials. UV-irradiation of these light-responsive membranes resulted in a strong colouration of the membrane, in a reduction of surface tension, which resulted in a decreased caffeine permeability resistance. The membranes were characterized using XPS for the elemental composition of the coating, contact angle measurements for the surface tension, solid-state UV/VIS measurements for the determination of the kinetic and stability properties, and two-photon microscopy for the localisation of the photochromic substance in the porous membrane.

Local changes in neocortical circuit dynamics coincide with the spread of seizures to thalamus in a model of epilepsy

During the generalization of epileptic seizures, pathological activity in one brain area recruits distant brain structures into joint synchronous discharges. However, it remains unknown whether specific changes in local circuit activity are related to the aberrant recruitment of anatomically distant structures into epileptiform discharges. Further, it is not known whether aberrant areas recruit or entrain healthy ones into pathological activity. Here we study the dynamics of local circuit activity during the spread of epileptiform discharges in the zero-magnesium in vitro model of epilepsy. We employ high-speed multi-photon imaging in combination with dual whole-cell recordings in acute thalamocortical (TC) slices of the juvenile mouse to characterize the generalization of epileptic activity between neocortex and thalamus. We find that, although both structures are exposed to zero-magnesium, the initial onset of focal epileptiform discharge occurs in cortex. This suggests that local recurrent connectivity that is particularly prevalent in cortex is important for the initiation of seizure activity. Subsequent recruitment of thalamus into joint, generalized discharges is coincident with an increase in the coherence of local cortical circuit activity that itself does not depend on thalamus. Finally, the intensity of population discharges is positively correlated between both brain areas. This suggests that during and after seizure generalization not only the timing but also the amplitude of epileptiform discharges in thalamus is entrained by cortex. Together these results suggest a central role of neocortical activity for the onset and the structure of pathological recruitment of thalamus into joint synchronous epileptiform discharges.

Satellite laser ranging to GPS and GLONASS

Satellite laser ranging (SLR) to the satellites of the global navigation satellite systems (GNSS) provides substantial and valuable information about the accuracy and quality of GNSS orbits and allows for the SLR-GNSS co-location in space. In the framework of the NAVSTAR-SLR experiment two GPS satellites of Block-IIA were equipped with laser retroreflector arrays (LRAs), whereas all satellites of the GLONASS system are equipped with LRAs in an operational mode. We summarize the outcome of the NAVSTAR-SLR experiment by processing 20 years of SLR observations to GPS and 12 years of SLR observations to GLONASS satellites using the reprocessed microwave orbits provided by the center for orbit determination in Europe (CODE). The dependency of the SLR residuals on the size, shape, and number of corner cubes in LRAs is studied. We show that the mean SLR residuals and the RMS of residuals depend on the coating of the LRAs and the block or type of GNSS satellites. The SLR mean residuals are also a function of the equipment used at SLR stations including the single-photon and multi-photon detection modes. We also show that the SLR observations to GNSS satellites are important to validate GNSS orbits and to assess deficiencies in the solar radiation pressure models. We found that the satellite signature effect, which is defined as a spread of optical pulse signals due to reflection from multiple reflectors, causes the variations of mean SLR residuals of up to 15 mm between the observations at nadir angles of 0∘ and 14∘. in case of multi-photon SLR stations. For single-photon SLR stations this effect does not exceed 1 mm. When using the new empirical CODE orbit model (ECOM), the SLR mean residual falls into the range 0.1–1.8 mm for high-performing single-photon SLR stations observing GLONASS-M satellites with uncoated corner cubes. For best-performing multi-photon stations the mean SLR residuals are between −12.2 and −25.6 mm due to the satellite signature effect.

Rapid Sequestration of Leishmania mexicana by Neutrophils Contributes to the Development of Chronic Lesion.

The protozoan Leishmania mexicana parasite causes chronic non-healing cutaneous lesions in humans and mice with poor parasite control. The mechanisms preventing the development of a protective immune response against this parasite are unclear. Here we provide data demonstrating that parasite sequestration by neutrophils is responsible for disease progression in mice. Within hours of infection L. mexicana induced the local recruitment of neutrophils, which ingested parasites and formed extracellular traps without markedly impairing parasite survival. We further showed that the L. mexicana-induced recruitment of neutrophils impaired the early recruitment of dendritic cells at the site of infection as observed by intravital 2-photon microscopy and flow cytometry analysis. Indeed, infection of neutropenic Genista mice and of mice depleted of neutrophils at the onset of infection demonstrated a prominent role for neutrophils in this process. Furthermore, an increase in monocyte-derived dendritic cells was also observed in draining lymph nodes of neutropenic mice, correlating with subsequent increased frequency of IFNγ-secreting T helper cells, and better parasite control leading ultimately to complete healing of the lesion. Altogether, these findings show that L. mexicana exploits neutrophils to block the induction of a protective immune response and impairs the control of lesion development. Our data thus demonstrate an unanticipated negative role for these innate immune cells in host defense, suggesting that in certain forms of cutaneous leishmaniasis, regulating neutrophil recruitment could be a strategy to promote lesion healing.

Intravital and whole-organ imaging reveals capture of melanoma-derived antigen by lymph node subcapsular macrophages leading to widespread deposition on follicular dendritic cells.

Aberrant antigens expressed by tumor cells, such as in melanoma, are often associated with humoral immune responses, which may in turn influence tumor progression. Despite recent data showing the central role of adaptive immune responses on cancer spread or control, it remains poorly understood where and how tumor-derived antigen (TDA) induces a humoral immune response in tumor-bearing hosts. Based on our observation of TDA accumulation in B cell areas of lymph nodes (LNs) from melanoma patients, we developed a pre-metastatic B16.F10 melanoma model expressing a fluorescent fusion protein, tandem dimer tomato, as a surrogate TDA. Using intravital two-photon microscopy (2PM) and whole-mount 3D LN imaging of tumor-draining LNs in immunocompetent mice, we report an unexpectedly widespread accumulation of TDA on follicular dendritic cells (FDCs), which were dynamically scanned by circulating B cells. Furthermore, 2PM imaging identified macrophages located in the subcapsular sinus of tumor-draining LNs to capture subcellular TDA-containing particles arriving in afferent lymph. As a consequence, depletion of macrophages or genetic ablation of B cells and FDCs resulted in dramatically reduced TDA capture in tumor-draining LNs. In sum, we identified a major pathway for the induction of humoral responses in a melanoma model, which may be exploitable to manipulate anti-TDA antibody production during cancer immunotherapy.

An efficient framework for photon Monte Carlo treatment planning

Currently photon Monte Carlo treatment planning (MCTP) for a patient stored in the patient database of a treatment planning system (TPS) can usually only be performed using a cumbersome multi-step procedure where many user interactions are needed. This means automation is needed for usage in clinical routine. In addition, because of the long computing time in MCTP, optimization of the MC calculations is essential. For these purposes a new graphical user interface (GUI)-based photon MC environment has been developed resulting in a very flexible framework. By this means appropriate MC transport methods are assigned to different geometric regions by still benefiting from the features included in the TPS. In order to provide a flexible MC environment, the MC particle transport has been divided into different parts: the source, beam modifiers and the patient. The source part includes the phase-space source, source models and full MC transport through the treatment head. The beam modifier part consists of one module for each beam modifier. To simulate the radiation transport through each individual beam modifier, one out of three full MC transport codes can be selected independently. Additionally, for each beam modifier a simple or an exact geometry can be chosen. Thereby, different complexity levels of radiation transport are applied during the simulation. For the patient dose calculation, two different MC codes are available. A special plug-in in Eclipse providing all necessary information by means of Dicom streams was used to start the developed MC GUI. The implementation of this framework separates the MC transport from the geometry and the modules pass the particles in memory; hence, no files are used as the interface. The implementation is realized for 6 and 15 MV beams of a Varian Clinac 2300 C/D. Several applications demonstrate the usefulness of the framework. Apart from applications dealing with the beam modifiers, two patient cases are shown. Thereby, comparisons are performed between MC calculated dose distributions and those calculated by a pencil beam or the AAA algorithm. Interfacing this flexible and efficient MC environment with Eclipse allows a widespread use for all kinds of investigations from timing and benchmarking studies to clinical patient studies. Additionally, it is possible to add modules keeping the system highly flexible and efficient.

Evaluation of a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-conjugated bombesin-based radioantagonist for the labeling with single-photon emission computed tomography, positron emission tomography, and therapeutic radionuclides

PURPOSE: G protein-coupled receptor agonists are being used as radiolabeled vectors for in vivo localization and therapy of tumors. Recently, somatostatin-based antagonists were shown to be superior to agonists. Here, we compare the new [111In/68Ga]-labeled bombesin-based antagonist RM1 with the agonist [111In]-AMBA for targeting the gastrin-releasing peptide receptor (GRPR). EXPERIMENTAL DESIGN: IC50, Kd values, and antagonist potency were determined using PC-3 and HEK-GRPR cells. Biodistribution and imaging studies were done in nude mice transplanted with the PC-3 tumor. The antagonist potency was assessed by evaluating the effects on calcium release and on receptor internalization monitored by immunofluorescence microscopy. RESULTS: The IC50 value of [(nat)In]-RM1 was 14 +/- 3.4 nmol/L. [(nat/111)In]-RM1 was found to bind to the GRPR with a Kd of 8.5 +/- 2.7 nmol/L compared with a Kd of 0.6 +/- 0.3 nmol/L of [111In]-AMBA. A higher maximum number of binding site value was observed for [111In]-RM1 (2.4 +/- 0.2 nmol/L) compared with [111In]-AMBA (0.7 +/- 0.1 nmol/L). [(nat)Lu]-AMBA is a potent agonist in the immunofluorescence-based internalization assay, whereas [(nat)In]-RM1 is inactive alone but efficiently antagonizes the bombesin effect. These data are confirmed by the calcium release assay. The pharmacokinetics showed a superiority of the radioantagonist with regard to the high tumor uptake (13.4 +/- 0.8% IA/g versus 3.69 +/- 0.75% IA/g at 4 hours after injection. as well as to all tumor-to-normal tissue ratios. CONCLUSION: Despite their relatively low GRPR affinity, the antagonists [111In/68Ga]-RM1 showed superior targeting properties compared with [111In]-AMBA. As found for somatostatin receptor-targeting radiopeptides, GRP-based radioantagonists seem to be superior to radioagonists for in vivo imaging and potentially also for targeted radiotherapy of GRPR-positive tumors.