Capture, crawl, cross: the T cell code to breach the blood-brain barriers

The central nervous system (CNS) is an immunologically privileged site to which access of circulating immune cells is tightly controlled by the endothelial blood-brain barrier (BBB; see Glossary) localized in CNS microvessels, and the epithelial blood-cerebrospinal fluid barrier (BCSFB) within the choroid plexus. As a result of the specialized structure of the CNS barriers, immune cell entry into the CNS parenchyma involves two differently regulated steps: migration of immune cells across the BBB or BCSFB into the cerebrospinal fluid (CSF)-drained spaces of the CNS, followed by progression across the glia limitans into the CNS parenchyma. With a focus on multiple sclerosis (MS) and its animal models, this review summarizes the distinct molecular mechanisms required for immune cell migration across the different CNS barriers.

The anatomical and cellular basis of immune surveillance in the central nervous system

The central nervous system (CNS) comprises the brain, spinal cord, optic nerves and retina, and contains post-mitotic, delicate cells. As the rigid coverings of the CNS render swelling dangerous and destructive, inflammatory reactions must be carefully controlled in CNS tissues. Nevertheless, effector immune responses that protect the host during CNS infection still occur in the CNS. Here, we describe the anatomical and cellular basis of immune surveillance in the CNS, and explain how this shapes the unique immunology of these tissues. The Review focuses principally on insights gained from the study of autoimmune responses in the CNS and to a lesser extent on models of infectious disease. Furthermore, we propose a new model to explain how antigen-specific T cell responses occur in the CNS.

Live cell imaging techniques to study T cell trafficking across the blood-brain barrier in vitro and in vivo

BACKGROUND The central nervous system (CNS) is an immunologically privileged site to which access for circulating immune cells is tightly controlled by the endothelial blood-brain barrier (BBB) located in CNS microvessels. Under physiological conditions immune cell migration across the BBB is low. However, in neuroinflammatory diseases such as multiple sclerosis, many immune cells can cross the BBB and cause neurological symptoms. Extravasation of circulating immune cells is a multi-step process that is regulated by the sequential interaction of different adhesion and signaling molecules on the immune cells and on the endothelium. The specialized barrier characteristics of the BBB, therefore, imply the existence of unique mechanisms for immune cell migration across the BBB.

Targeting the insulin-like growth factor-1 receptor in human cancer

The insulin-like growth factor (IGF) signaling system plays a crucial role in human cancer and the IGF-1 receptor (IGF-1R) is an attractive drug target against which a variety of novel anti-tumor agents are being developed. Deregulation of the IGF signaling pathway frequently occurs in human cancer and involves the establishment of autocrine loops comprising IGF-1 or IGF-2 and/or IGF-1R over-expression. Epidemiologic studies have documented a link between elevated IGF levels and the development of solid tumors, such as breast, colon, and prostate cancer. Anti-cancer strategies targeting the IGF signaling system involve two main approaches, namely neutralizing antibodies and small molecule inhibitors of the IGF-1R kinase activity. There are numerous reports describing anti-tumor activity of these agents in pre-clinical models of major human cancers. In addition, multiple clinical trials have started to evaluate the safety and efficacy of selected IGF-1R inhibitors, in combination with standard chemotherapeutic regimens or other targeted agents in cancer patients. In this mini review, I will discuss the role of the IGF signaling system in human cancer and the main strategies which have been so far evaluated to target the IGF-1R.

Io volcano observer (IVO) integrated approach to optimizing system design for radiation challenges

One of the major challenges for a mission to the Jovian system is the radiation tolerance of the spacecraft (S/C) and the payload. Moreover, being able to achieve science observations with high signal to noise ratios (SNR), while passing through the high flux radiation zones, requires additional ingenuity on the part of the instrument provider. Consequently, the radiation mitigation is closely intertwined with the payload, spacecraft and trajectory design, and requires a systems-level approach. This paper presents a design for the Io Volcano Observer (IVO), a Discovery mission concept that makes multiple close encounters with Io while orbiting Jupiter. The mission aims to answer key outstanding questions about Io, especially the nature of its intense active volcanism and the internal processes that drive it. The payload includes narrow-angle and wide-angle cameras (NAC and WAC), dual fluxgate magnetometers (FGM), a thermal mapper (ThM), dual ion and neutral mass spectrometers (INMS), and dual plasma ion analyzers (PIA). The radiation mitigation is implemented by drawing upon experiences from designs and studies for missions such as the Radiation Belt Storm Probes (RBSP) and Jupiter Europa Orbiter (JEO). At the core of the radiation mitigation is IVO's inclined and highly elliptical orbit, which leads to rapid passes through the most intense radiation near Io, minimizing the total ionizing dose (177 krads behind 100 mils of Aluminum with radiation design margin (RDM) of 2 after 7 encounters). The payload and the spacecraft are designed specifically to accommodate the fast flyby velocities (e.g. the spacecraft is radioisotope powered, remaining small and agile without any flexible appendages). The science instruments, which collect the majority of the high-priority data when close to Io and thus near the peak flux, also have to mitigate transient noise in their detectors. The cameras use a combination of shielding and CMOS detectors with extremely fast readout to mi- imize noise. INMS microchannel plate detectors and PIA channel electron multipliers require additional shielding. The FGM is not sensitive to noise induced by energetic particles and the ThM microbolometer detector is nearly insensitive. Detailed SNR calculations are presented. To facilitate targeting agility, all of the spacecraft components are shielded separately since this approach is more mass efficient than using a radiation vault. IVO uses proven radiation-hardened parts (rated at 100 krad behind equivalent shielding of 280 mils of Aluminum with RDM of 2) and is expected to have ample mass margin to increase shielding if needed.

Regulation of immune cell entry into the central nervous system

The central nervous system (CNS) has long been regarded as an immune privileged organ implying that the immune system avoids the CNS to not disturb its homeostasis, which is critical for proper function of neurons. Meanwhile, it is accepted that immune cells do in fact gain access to the CNS and that immune responses can be mounted within this tissue. However, the unique CNS microenvironment strictly controls these immune reactions starting with tightly controlling immune cell entry into the tissue. The endothelial blood-brain barrier (BBB) and the epithelial blood-cerebrospinal fluid (CSF) barrier, which protect the CNS from the constantly changing milieu within the bloodstream, also strictly control immune cell entry into the CNS. Under physiological conditions, immune cell migration into the CNS is kept at a very low level. In contrast, during a variety of pathological conditions of the CNS such as viral or bacterial infections, or during inflammatory diseases such as multiple sclerosis, immunocompetent cells readily traverse the BBB and likely also the choroid plexus and subsequently enter the CNS parenchyma or CSF spaces. This chapter summarizes our current knowledge of immune cell entry across the blood CNS barriers. A large body of the currently available information on immune cell entry into the CNS has been derived from studying experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Therefore, most of this chapter discussing immune cell entry during CNS pathogenesis refers to observations in the EAE model, allowing for the possibility that other mechanisms of immune cell entry into the CNS might apply under different pathological conditions such as bacterial meningitis or stroke.

Dysferlin is a new marker for leaky brain blood vessels in multiple sclerosis

Dysferlin is a muscle protein involved in cell membrane repair and its deficiency is associated with muscular dystrophy. We describe that dysferlin is also expressed in leaky endothelial cells. In the normal central nervous system (CNS), dysferlin is only present in endothelial cells of circumventricular organs. In the inflamed CNS of patients with multiple sclerosis (MS) or in animals with experimental autoimmune encephalomyelitis, dysferlin reactivity is induced in endothelial cells and the expression is associated with vascular leakage of serum proteins. In MS, dysferlin expression in endothelial cells is not restricted to vessels with inflammatory cuffs but is also present in noninflamed vessels. In addition, many blood vessels with perivascular inflammatory infiltrates lack dysferlin expression in inactive lesions or in the normal-appearing white matter. In vitro, dysferlin can be induced in endothelial cells by stimulation with tumor necrosis factor-alpha. Hence, dysferlin is not only a marker for leaky brain vessels, but also reveals dissociation of perivascular inflammatory infiltrates and blood-brain barrier disturbance in multiple sclerosis.

Molecular mechanisms involved in T cell migration across the blood-brain barrier

In the healthy individuum lymphocyte traffic into the central nervous system (CNS) is very low and tightly controlled by the highly specialized blood-brain barrier (BBB). In contrast, under inflammatory conditions of the CNS such as in multiple sclerosis or in its animal model experimental autoimmune encephalomyelitis (EAE) circulating lymphocytes and monocytes/macrophages readily cross the BBB and gain access to the CNS leading to edema, inflammation and demyelination. Interaction of circulating leukocytes with the endothelium of the blood-spinal cord and blood-brain barrier therefore is a critical step in the pathogenesis of inflammatory diseases of the CNS. Leukocyte/endothelial interactions are mediated by adhesion molecules and chemokines and their respective chemokine receptors. We have developed a novel spinal cord window preparation, which enables us to directly visualize CNS white matter microcirculation by intravital fluorescence videomicroscopy. Applying this technique of intravital fluorescence videomicroscopy we could provide direct in vivo evidence that encephalitogenic T cell blasts interact with the spinal cord white matter microvasculature without rolling and that alpha4-integrin mediates the G-protein independent capture and subsequently the G-protein dependent adhesion strengthening of T cell blasts to microvascular VCAM-1. LFA-1 was found to neither mediate the G-protein independent capture nor the G- protein dependent initial adhesion strengthening of encephalitogenic T cell blasts within spinal cord microvessel, but was rather involved in T cell extravasation across the vascular wall into the spinal cord parenchyme. Our observation that G-protein mediated signalling is required to promote adhesion strengthening of encephalitogenic T cells on BBB endothelium in vivo suggested the involvement of chemokines in this process. We found functional expression of the lymphoid chemokines CCL19/ELC and CCL21/SLC in CNS venules surrounded by inflammatory cells in brain and spinal cord sections of mice afflicted with EAE suggesting that the lymphoid chemokines CCL19 and CCL21 besides regulating lymphocyte homing to secondary lymphoid tissue might be involved in T lymphocyte migration into the immuneprivileged CNS during immunosurveillance and chronic inflammation. Here, I summarize our current knowledge on the sequence of traffic signals involved in T lymphocyte recruitment across the healthy and inflamed blood-brain and blood-spinal cord barrier based on our in vitro and in vivo investigations.

A new microbiopsy system enables rapid preparation of tissue for high-pressure freezing

A microbiopsy system was developed to overcome long sampling times for tissues before they are cryo-fixed by high-pressure freezing. A commercially available biopsy gun was adapted to the needs of small-organ excisions, and biopsy needles were modified to allow small samples (0.6 mm x 1.2 mm x 0.3 mm) to be taken. Specimen platelets with a central slot of the same dimensions as the biopsy are used. A self-made transfer device (in the meantime optimized by Leica-Microsystems [Vienna, Austria]) coordinates the transfer of the excised sample from the biopsy needle into the platelet slot and the subsequent loading in a specimen holder, which is then introduced into a high-pressure freezer (Leica EM PACT; Leica Microsystems, Vienna, Austria). Thirty seconds preparation time is needed from excision until high-pressure freezing. Brain, liver, kidney and muscle excisions of anesthetised rats are shown to be well frozen.

Examining the gateway to the limbic system with diffusion tensor imaging: the perforant pathway in dementia

Current treatments for Alzheimer's disease (AD) are only able to slow the progression of mental deterioration, making early and reliable diagnosis an essential part of any promising therapeutic strategy. In the initial stages of AD, the first neuropathological alterations occur in the perforant pathway (PP), a large neuronal fiber tract located at the entrance to the limbic system. However, to date, there is no sensitive diagnostic tool for performing in vivo assessments of this structure. In the present bimodal magnetic resonance imaging (MRI) study, we examined 10 elderly controls, 10 subjects suffering from mild cognitive impairment (MCI), and 10 AD patients in order to evaluate the sensitivity of diffusion tensor imaging (DTI), a new MRI technique, for detecting changes in the PP. Furthermore, the diagnostic explanatory power of DTI data of the PP should be compared to high-resolution MRI volumetry and intervoxel coherences (COH) of the hippocampus and the entorhinal cortex, two limbic regions also involved in the pathophysiology of early AD. DTI revealed a marked decrease in COH values in the PP region of MCI (right side: 26%, left side: 29%, as compared to controls) and AD patients (right side: 37%, left side: 43%, as compared to controls). Reductions in COH values of the PP region were significantly correlated with cognitive impairment. DTI data of the PP zone were the only parameter differing significantly between control subjects and MCI patients, while the volumetric measures and the COH values of the hippocampus and the entorhinal cortex did not. DTI of medial temporal brain regions is a promising non-invasive tool for the in vivo diagnosis of the early/preclinical stages of AD.

Effects of local continuous release of brain derived neurotrophic factor (BDNF) on peripheral nerve regeneration in a rat model

The purpose of this study was to evaluate the effect of continuously released BDNF on peripheral nerve regeneration in a rat model. Initial in vitro evaluation of calcium alginate prolonged-release-capsules (PRC) proved a consistent release of BDNF for a minimum of 8 weeks. In vivo, a worst case scenario was created by surgical removal of a 20-mm section of the sciatic nerve of the rat. Twenty-four autologous fascia tubes were filled with calcium alginate spheres and sutured to the epineurium of both nerve ends. The animals were divided into 3 groups. In group 1, the fascial tube contained plain calcium alginate spheres. In groups 2 and 3, the fascial tube contained calcium alginate spheres with BDNF alone or BDNF stabilized with bovine serum albumin, respectively. The autocannibalization of the operated extremity was clinically assessed and documented in 12 additional rats. The regeneration was evaluated histologically at 4 weeks and 10 weeks in a blinded manner. The length of nerve fibers and the numbers of axons formed in the tube was measured. Over a 10-week period, axons have grown significantly faster in groups 2 and 3 with continuously released BDNF compared to the control. The rats treated with BDNF (groups 2 and 3) demonstrated significantly less autocannibalization than the control group (group 1). These results suggest that BDNF may not only stimulate faster peripheral nerve regeneration provided there is an ideal, biodegradable continuous delivery system but that it significantly reduces the neuropathic pain in the rat model.

PEGylation and multimerization of the anti-p185HER-2 single chain Fv fragment 4D5: effects on tumor targeting

A major goal in antibody design for cancer therapy is to tailor the pharmacokinetic properties of the molecule according to specific treatment requirements. Key parameters determining the pharmacokinetics of therapeutic antibodies are target specificity, affinity, stability, and size. Using the p185HER-2 (HER-2)-specific scFv 4D5 as model system, we analyzed how changes in molecular weight and valency independently affect antigen binding and tumor localization. By employing multimerization and PEGylation, four different antibody formats were generated and compared with the scFv 4D5. First, dimeric and tetrameric miniantibodies were constructed by fusion of self-associating, disulfide-linked peptides to the scFv 4D5. Second, we attached a 20-kDa PEG moiety to the monovalent scFv and to the divalent miniantibody at the respective C terminus. In all formats, serum stability and full binding reactivity of the scFv 4D5 were retained. Functional affinity, however, did change. An avidity increase was achieved by multimerization, whereas PEGylation resulted in a 5-fold decreased affinity. Nevertheless, the PEGylated monomer showed an 8.5-fold, and the PEGylated dimer even a 14.5-fold higher tumor accumulation than the corresponding scFv, 48 h post-injection, because of a significantly longer serum half-life. In comparison, the non-PEGylated bivalent and tetravalent miniantibodies showed only a moderate increase in tumor localization compared with the scFv, which correlated with the degree of multimerization. However, these non-PEGylated formats resulted in higher tumor-to-blood ratios. Both multimerization and PEGylation represent thus useful strategies to tailor the pharmacokinetic properties of therapeutic antibodies and their combined use can additively improve tumor targeting.

Plasticity in the adult language system: a longitudinal electrophysiological study on second language learning

Event-related potentials (ERPs) were used to trace changes in brain activity related to progress in second language learning. Twelve English-speaking exchange students learning German in Switzerland were recruited. ERPs to visually presented single words from the subjects' native language (English), second language (German) and an unknown language (Romansh) were measured before (day 1) and after (day 2) 5 months of intense German language learning. When comparing ERPs to German words from day 1 and day 2, we found topographic differences between 396 and 540 ms. These differences could be interpreted as a latency shift indicating faster processing of German words on day 2. Source analysis indicated that the topographic differences were accounted for by shorter activation of left inferior frontal gyrus (IFG) on day 2. In ERPs to English words, we found Global Field Power differences between 472 and 644 ms. This may due to memory traces related to English words being less easily activated on day 2. Alternatively, it might reflect the fact that--with German words becoming familiar on day 2--English words loose their oddball character and thus produce a weaker P300-like effect on day 2. In ERPs to Romansh words, no differences were observed. Our results reflect plasticity in the neuronal networks underlying second language acquisition. They indicate that with a higher level of second language proficiency, second language word processing is faster and requires shorter frontal activation. Thus, our results suggest that the reduced IFG activation found in previous fMRI studies might not reflect a generally lower activation but rather a shorter duration of activity.

Spastin in the human and mouse central nervous system with special reference to its expression in the hippocampus of mouse pilocarpine model of status epilepticus and temporal lobe epilepsy

In the present in situ hybridization and immunocytochemical studies in the mouse central nervous system (CNS), a strong expression of spastin mRNA and protein was found in Purkinje cells and dentate nucleus in the cerebellum, in hippocampal principal cells and hilar neurons, in amygdala, substantia nigra, striatum, in the motor nuclei of the cranial nerves and in different layers of the cerebral cortex except piriform and entorhinal cortices where only neurons in layer II were strongly stained. Spastin protein and mRNA were weakly expressed in most of the thalamic nuclei. In selected human brain regions such as the cerebral cortex, cerebellum, hippocampus, amygdala, substania nigra and striatum, similar results were obtained. Electron microscopy showed spastin immunopositive staining in the cytoplasma, dendrites, axon terminals and nucleus. In the mouse pilocarpine model of status epilepticus and subsequent temporal lobe epilepsy, spastin expression disappeared in hilar neurons as early as at 2h during pilocarpine induced status epilepticus, and never recovered. At 7 days and 2 months after pilocarpine induced status epilepticus, spastin expression was down-regulated in granule cells in the dentate gyrus, but induced expression was found in reactive astrocytes. The demonstration of widespread distribution of spastin in functionally different brain regions in the present study may provide neuroanatomical basis to explain why different neurological, psychological disorders and cognitive impairment occur in patients with spastin mutation. Down-regulation or loss of spastin expression in hilar neurons may be related to their degeneration and may therefore initiate epileptogenetic events, leading to temporal lobe epilepsy.

Multimodal augmented reality system for surgical microscopy

Image-guided, computer-assisted neurosurgery has emerged to improve localization and targeting, to provide a better anatomic definition of the surgical field, and to decrease invasiveness. Usually, in image-guided surgery, a computer displays the surgical field in a CT/MR environment, using axial, coronal or sagittal views, or even a 3D representation of the patient. Such a system forces the surgeon to look away from the surgical scene to the computer screen. Moreover, this kind of information, being pre-operative imaging, can not be modified during the operation, so it remains valid for guidance in the first stage of the surgical procedure, and mainly for rigid structures like bones. In order to solve the two constraints mentioned before, we are developing an ultrasoundguided surgical microscope. Such a system takes the advantage that surgical microscopy and ultrasound systems are already used in neurosurgery, so it does not add more complexity to the surgical procedure. We have integrated an optical tracking device in the microscope and an augmented reality overlay system with which we avoid the need to look away from the scene, providing correctly aligned surgical images with sub-millimeter accuracy. In addition to the standard CT and 3D views, we are able to track an ultrasound probe, and using a previous calibration and registration of the imaging, the image obtained is correctly projected to the overlay system, so the surgeon can always localize the target and verify the effects of the intervention. Several tests of the system have been already performed to evaluate the accuracy, and clinical experiments are currently in progress in order to validate the clinical usefulness of the system.