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.

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.

Isolation of Besnoitia besnoiti from infected cattle in Portugal

Besnoitia besnoiti, an obligate intracellular protozoan parasite belonging to the phylum apicomplexa, is the causative agent of bovine besnoitiosis. Besnoitiosis is responsible for significant losses in the cattle industry of Africa and Mediterranean countries due to the high morbidity rate, abortion and infertility in males. The acute stage of disease is associated with the proliferative forms (tachyzoites) and is characterized by fever, whimpery, general weakness and swelling of the superficial lymph nodes. During the following chronic stage, a huge number of cysts are formed mainly in the subcutaneous tissues. This process is non-reversible, and chronic besnoitiosis is characterized by hyper-sclerodermia, hyperkeratosis, alopecia and, in bulls, atrophy, sclerosis and focal necrosis that cause irreversible lesions in the testis. In this paper we report on the identification of large cysts in the skin of a cow and a bull in Portugal, which presented loss of hair and enlargement and pachydermis all over the body. The observation of a two-layered cyst wall within the host cell, the encapsulation of the host cell by a large outer cyst wall, and the subcutaneous localization of the cysts within the host, were characteristic for B. besnoiti. The parasites were isolated from the infected animals and successfully propagated in Vero cells without prior passages in laboratory animals. Morphological characterization of B. besnoiti tachyzoites and the amplification of the 149 bp segment from the internal transcribed spacer 1 (ITS1), aided with specific primers, confirmed the identification of B. besnoiti.

Magnetic field exposure and neurodegenerative diseases--recent epidemiological studies

OBJECTIVES: To analyse the results of recent studies not yet included in a 2003 report of the International Commission on Non-Ionizing Radiation Protection (ICNIRP) on occupational exposure to low-frequency electromagnetic fields as potential risk factor for neurodegenerative diseases. METHODS: A literature search was conducted in the online databases of PubMed, ISI Web of Knowledge, DIMDI and COCHRANE, as well as in specialised databases and journals. Eight studies published between January 2000 and July 2005 were included in the review. RESULTS: The findings of these studies contribute to the evidence of an association between occupational magnetic field exposure and the risk of dementia. Regarding amyotrophic lateral sclerosis, the recent results confirm earlier observations of an association with electric and electronic work and welding. Its relationship with magnetic field exposure remains unsolved. There are only few findings pointing towards an association between magnetic field exposure and Parkinson's disease. CONCLUSIONS: The epidemiological evidence for an association between occupational exposure to low-frequency electromagnetic fields and the risk of dementia has increased during the last five years. The impact of potential confounders should be evaluated in further studies.

Increased uptake of 111In-octreotide in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is characterized by an uncontrolled accumulation and activation of lung fibroblasts. A modulation of fibroblast activation has been observed in various systems with octreotide, a synthetic somatostatin analog with strong affinity for the somatostatin receptor subtype 2 (sst2). One aim of our study was to evaluate the expression of somatostatin receptors in the lungs of patients with IPF. A second aim was to evaluate the relationship between 111In-octreotide uptake and the effect of pulmonary fibrosis as assessed by lung function tests and parameters and by radiologic findings. METHODS: We investigated 11 patients with IPF, 6 patients with pulmonary fibrosis associated with systemic sclerosis (SSc), and 19 patients with disease not of the lung (control patients). The expression of somatostatin receptors was evaluated in vivo using 111In-octreotide scintigraphy. We evaluated the relationship between 111In-octreotide uptake and the activity of pulmonary fibrosis as assessed by lung function tests, bronchoalveolar lavage (BAL) cellularity, and high-resolution CT (HRCT) of the chest. Planar images and thoracic SPECT (24 h) were performed after injection of 222 MBq of 111In-octreotide. Lung uptake was quantified using the lung-to-background ratio (L/B). In addition, the expression of sst2 was evaluated in vitro, in frozen lung-tissue samples using autoradiography, and in human cultures of lung fibroblasts using a ligand-binding assay. RESULTS: Compared with lung uptake in control patients (median L/B, 1.25; range, 1.14-1.49), lung uptake was increased in all 11 IPF patients (median L/B, 2.63; range, 1.59-3.13; P < 0.001) and in 4 of 6 SSc patients (median L/B, 1.68; range, 1.42-2.16). The L/B was lower in SSc patients than in IPF patients (P = 0.011). Increased uptake correlated with the alteration of lung function (carbon monoxide diffusing capacity [rho = -0.655; P = 0.038], diffusing capacity for carbon monoxide and alveolar volume ratio [rho = -0.627; P = 0.047], vital capacity [rho = -0.609; P = 0.054], and total lung capacity [rho = -0.598; P = 0.058]) and with the intensity of alveolitis (total BAL cellularity [rho = 0.756; P = 0.045], neutrophil counts [rho = 0.738; P = 0.05]), and HRCT fibrosis score (rho = 0.673; P = 0.007). Autoradiography suggested that vascular structures were a prominent binding site. Lung fibroblasts expressed somatostatin receptors in vitro as measured by binding assay. CONCLUSION: Our preliminary results identified an increased expression of sst2 in (mainly idiopathic) pulmonary fibrosis. Lung uptake correlates with the alteration of lung function and with the intensity of alveolitis.

Immune cell migration across the blood–brain barrier: molecular mechanisms and therapeutic targeting

The endothelial blood–brain barrier (BBB) and the epithelial blood–cerebrospinal fluid barrier protect the CNS from the constantly changing milieu within the bloodstream. The BBB strictly controls immune cell entry into the CNS, which is rare under physiological conditions. 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 subsequently enter the CNS parenchyma. Most of the available information on immune cell entry into the CNS is derived from studying experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Consequently, our current knowledge on traffic signals mediating immune cell entry across the BBB during immunosurveillance and disease results mainly from experimental data in the EAE model. Therefore, a large part of this review summarizes these findings. Similarly, the potential benefits and risks associated with therapeutic targeting of immune cell trafficking across the BBB will be discussed in the context of multiple sclerosis, since elucidation of the molecular mechanisms relevant to this disease have largely relied on the use of its in vivo model, EAE.

MODELING FUNCTIONAL DATA WITH SPATIALLY HETEROGENEOUS SHAPE CHARACTERISTICS

We propose a novel class of models for functional data exhibiting skewness or other shape characteristics that vary with spatial or temporal location. We use copulas so that the marginal distributions and the dependence structure can be modeled independently. Dependence is modeled with a Gaussian or t-copula, so that there is an underlying latent Gaussian process. We model the marginal distributions using the skew t family. The mean, variance, and shape parameters are modeled nonparametrically as functions of location. A computationally tractable inferential framework for estimating heterogeneous asymmetric or heavy-tailed marginal distributions is introduced. This framework provides a new set of tools for increasingly complex data collected in medical and public health studies. Our methods were motivated by and are illustrated with a state-of-the-art study of neuronal tracts in multiple sclerosis patients and healthy controls. Using the tools we have developed, we were able to find those locations along the tract most affected by the disease. However, our methods are general and highly relevant to many functional data sets. In addition to the application to one-dimensional tract profiles illustrated here, higher-dimensional extensions of the methodology could have direct applications to other biological data including functional and structural MRI.

PENALIZED FUNCTIONAL REGRESSION

We develop fast fitting methods for generalized functional linear models. An undersmooth of the functional predictor is obtained by projecting on a large number of smooth eigenvectors and the coefficient function is estimated using penalized spline regression. Our method can be applied to many functional data designs including functions measured with and without error, sparsely or densely sampled. The methods also extend to the case of multiple functional predictors or functional predictors with a natural multilevel structure. Our approach can be implemented using standard mixed effects software and is computationally fast. Our methodology is motivated by a diffusion tensor imaging (DTI) study. The aim of this study is to analyze differences between various cerebral white matter tract property measurements of multiple sclerosis (MS) patients and controls. While the statistical developments proposed here were motivated by the DTI study, the methodology is designed and presented in generality and is applicable to many other areas of scientific research. An online appendix provides R implementations of all simulations.

Mortality from neurodegenerative disease and exposure to extremely low-frequency magnetic fields: 31 years of observations on Swiss railway employees

AIMS: The objective of the present study was to investigate the relationship between extremely low-frequency magnetic field (ELF-MF) exposure and mortality from several neurodegenerative conditions in Swiss railway employees. METHODS: We studied a cohort of 20,141 Swiss railway employees with 464,129 person-years of follow-up between 1972 and 2002. For each individual, cumulative exposure was calculated from on-site measurements and modelling of past exposure. We compared cause-specific mortality in highly exposed train drivers (mean exposure: 21 microT) with less exposed occupational groups (for example station masters: 1 microT). RESULTS: The hazard ratio for train drivers compared to station masters was 1.96 [95% confidence interval (CI) = 0.98-3.92] for senile dementia and 3.15 (95% CI = 0.90-11.04) for Alzheimer's disease. For every 10 microT years of cumulative exposure senile dementia mortality increased by 5.7% (95% CI = 1.3-10.4), Alzheimer's disease by 9.4% (95% CI = 2.7-16.4) and amyotrophic lateral sclerosis by 2.1% (95% CI = -6.8 to 11.7). There was no evidence for an increase in mortality from Parkinson's disease and multiple sclerosis. CONCLUSIONS: This study suggests a link between exposure to ELF-MF and Alzheimer's disease and indicates that ELF-MF might act in later stages of the disease process.

Homozygous Cys542-->Arg substitution in GPIIIa in a Swiss patient with type I Glanzmann's thrombasthenia

Glanzmann's thrombasthenia (GT) arises from a qualitative or quantitative defect in the GPIIb-IIIa complex (integrin alphaIIbbeta3), the mediator of platelet aggregation. We describe a patient in whom clinical and laboratory findings typical of type I GT were found together with a second pathology involving neurological and other complications symptomatic of tuberous sclerosis. Analysis of platelet proteins by Western blotting revealed trace amounts of normally migrating GPIIb and equally small amounts of GPIIIa of slightly slower than normal migration. Flow cytometry confirmed a much decreased binding to platelets of monoclonal antibodies to GPIIb, GPIIIa or GPIIb-IIIa, and an antibody to the alphav subunit also showed decreased binding. Nonradioactive PCR single-strand conformation polymorphism analysis followed by direct sequencing of PCR-amplified DNA fragments showed a homozygous point mutation (T to C) at nucleotide 1722 of GPIIIa cDNA and which led to a Cys542-->Arg substitution in the GPIIIa protein. The mutation gave rise to a HinP1 I restriction site in exon 11 of the GPIIIa gene and allele-specific restriction enzyme analysis of family members confirmed that a single mutated allele was inherited from each parent. This amino acid substitution presumably changes the capacity for disulphide bond formation within the cysteine-rich core region of GPIIIa and its study will provide new information on GPIIb-IIIa and alphavbeta3 structure and biosynthesis.

E- and P-selectin are not required for the development of experimental autoimmune encephalomyelitis in C57BL/6 and SJL mice

In multiple sclerosis and in its animal model experimental autoimmune encephalomyelitis (EAE), inflammatory cells migrate across the endothelial blood-brain barrier (BBB) and gain access to the CNS. It is well-established that alpha4 integrins are actively involved in leukocyte recruitment across the BBB during EAE. In contrast, the role of endothelial E- and P-selectin in this process has been a controversial issue. In this study, we demonstrate that P-selectin protein can be detected in meningeal blood vessel endothelial cells in healthy SJL and C57BL/6 mice and on rare parenchymal CNS blood vessels in C57BL/6, but not SJL, mice. During EAE, expression of P-selectin but not E-selectin was found up-regulated on inflamed CNS microvessels surrounded by inflammatory infiltrates irrespective of their meningeal or parenchymal localization with a more prominent immunostaining detected in C57BL/6 as compared with SJL mice. P-selectin immunostaining could be localized to CNS endothelial cells and to CD41-positive platelets adhering to the vessel wall. Despite the presence of P-selectin in wild-type mice, E/P-selectin-deficient SJL and C57BL/6 mice developed clinical EAE indistinguishable from wild-type mice. Absence of E- and P-selectin did neither influence the activation of myelin-specific T cells nor the composition of the cellular infiltrates in the CNS during EAE. Finally, endothelial-specific tetracycline-inducible expression of E-selectin at the BBB in transgenic C57BL/6 mice did not alter the development of EAE. Thus, E- and P-selectin are not required for leukocyte recruitment across the BBB and the development of EAE in C57BL/6 and in SJL mice.

A review identifies and classifies reasons for ordering diagnostic tests

OBJECTIVE: To consider the reasons and context for test ordering by doctors when faced with an undiagnosed complaint in primary or secondary care. STUDY DESIGN AND SETTING: We reviewed any study of any design that discussed factors that may affect a doctor's decision to order a test. Articles were located through searches of electronic databases, authors' files on diagnostic methodology, and reference lists of relevant studies. We extracted data on: study design, type of analysis, setting, topic area, and any factors reported to influence test ordering. RESULTS: We included 37 studies. We carried out a thematic analysis to synthesize data. Five key groupings arose from this process: diagnostic factors, therapeutic and prognostic factors, patient-related factors, doctor-related factors, and policy and organization-related factors. To illustrate how the various factors identified may influence test ordering we considered the symptom low back pain and the diagnosis multiple sclerosis as examples. CONCLUSIONS: A wide variety of factors influence a doctor's decision to order a test. These are integral to understanding diagnosis in clinical practice. Traditional diagnostic accuracy studies should be supplemented with research into the broader context in which doctors perform their work.

Nutrient-responsive mTOR signalling grows on Sterile ground

The control of cell growth, that is cell size, is largely controlled by mTOR (the mammalian target of rapamycin), a large serine/threonine protein kinase that regulates ribosome biogenesis and protein translation. mTOR activity is regulated both by the availability of growth factors, such as insulin/IGF-1 (insulin-like growth factor 1), and by nutrients, notably the supply of certain key amino acids. The last few years have seen a remarkable increase in our understanding of the canonical, growth factor-regulated pathway for mTOR activation, which is mediated by the class I PI3Ks (phosphoinositide 3-kinases), PKB (protein kinase B), TSC1/2 (the tuberous sclerosis complex) and the small GTPase, Rheb. However, the nutrient-responsive input into mTOR is important in its own right and is also required for maximal activation of mTOR signalling by growth factors. Despite this, the details of the nutrient-responsive signalling pathway(s) controlling mTOR have remained elusive, although recent studies have suggested a role for the class III PI3K hVps34. In this issue of the Biochemical Journal, Findlay et al. demonstrate that the protein kinase MAP4K3 [mitogen-activated protein kinase kinase kinase kinase-3, a Ste20 family protein kinase also known as GLK (germinal centre-like kinase)] is a new component of the nutrient-responsive pathway. MAP4K3 activity is stimulated by administration of amino acids, but not growth factors, and this is insensitive to rapamycin, most likely placing MAP4K3 upstream of mTOR. Indeed, MAP4K3 is required for phosphorylation of known mTOR targets such as S6K1 (S6 kinase 1), and overexpression of MAP4K3 promotes the rapamycin-sensitive phosphorylation of these same targets. Finally, knockdown of MAP4K3 levels causes a decrease in cell size. The results suggest that MAP4K3 is a new component in the nutrient-responsive pathway for mTOR activation and reveal a completely new function for MAP4K3 in promoting cell growth. Given that mTOR activity is frequently deregulated in cancer, there is much interest in new strategies for inhibition of this pathway. In this context, MAP4K3 looks like an attractive drug target since inhibitors of this enzyme should switch off mTOR, thereby inhibiting cell growth and proliferation, and promoting apoptosis.

Brain endothelial PPARgamma controls inflammation-induced CD4+ T cell adhesion and transmigration in vitro

An important step in the pathogenesis of multiple sclerosis is adhesion and transmigration of encephalitogenic T cells across brain endothelial cells (EC) which strongly relies on interaction with EC-expressed adhesion molecules. We provide molecular evidence that the transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) is a negative regulator of brain EC inflammation. The PPARgamma agonist pioglitazone reduces transendothelial migration of encephalitogenic T cells across TNFalpha-stimulated brain EC. This effect is clearly PPARgamma mediated, as lentiviral PPARgamma overexpression in brain EC results in selective abrogation of inflammation-induced ICAM-1 and VCAM-1 upregulation and subsequent adhesion and transmigration of T cells. We therefore propose that PPARgamma in brain EC may be exploited to target detrimental EC-T cell interactions under inflammatory conditions.