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Involvement of β-chemokines in the development of inflammatory demyelination

The importance of β-chemokines (or CC chemokine ligands – CCL) in the development of inflammatory lesions in the central nervous system of patients with multiple sclerosis and rodents with experimental allergic encephalomyelitis is strongly supported by descriptive studies and experimental models. Our recent genetic scans in families identified haplotypes in the genes of CCL2, CCL3 and CCL11-CCL8-CCL13 which showed association with multiple sclerosis. Complementing the genetic associations, we also detected a distinct regional expression regulation for CCL2, CCL7 and CCL8 in correlation with chronic inflammation in multiple sclerosis brains. These observations are in consensus with previous studies, and add new data to support the involvement of CCL2, CCL7, CCL8 and CCL3 in the development of inflammatory demyelination. Along with our own data, here we review the literature implicating CCLs and their receptors (CCRs) in multiple sclerosis and experimental allergic encephalomyelitis. The survey reflects that the field is in a rapid expansion, and highlights some of the pathways which might be suitable to pharmaceutical interventions.

Introduction
Multiple sclerosis (MS) is a disabling disease of the central nervous system (CNS) with features of autoimmunity and neurodegeneration. Although the identity of primary antigenic determinant(s) is uncertain, an interaction between β-chemokine ligands and their receptors plays a central role in the recruitment and retention of inflammatory cells in the CNS. Thus, both the disease relevant chemokine ligands and their receptors represent potential therapeutic targets in MS.
Chemokines are a group of small, structurally related chemoattractant molecules that regulate cell trafficking through interactions with a set of receptors [1]. Evidence suggests that the migration of autoreactive immune cells via the blood-brain barrier (BBB) is an early and critical process during the development of inflammatory CNS lesions of experimental allergic encephalomyelitis (EAE) and MS, and that this transmigration is regulated by chemokines produced at the blood-brain barrier (BBB) and in the CNS. Subcellular signals induced by the binding of chemokines to their G-protein-coupled receptors leads to an increased avidity of integrins on leukocytes to their corresponding receptors on endothelial cells, followed by a facilitated migration of leukocytes towards the chemokine gradient in the CNS [2,3].
In addition to chemotaxis, chemokines are also involved in the regulation of T cell differentiation, apoptosis, cell cycle, angiogenesis and metastatic processes. Further, chemokines can control the generation of soluble inflammatory products such as free radicals, nitric oxide, cytokines and matrix metalloproteases [1,4]. Considering the predominantly T helper type 1 (TH1) mediated process of inflammatory demyelination and the TH2 driven suppression of inflammation, the differential effects of various chemokines on TH1 or TH2 polarization may have particular significance. The currently known, approximately 50 chemokine genes in humans are divided into four subfamilies on the basis of characteristic patterns of cysteine residues close to the N-terminal end of the products. The CC chemokine ligand family (CCL) (also known as β-chemokines or Small Cytokine Group A -SCYA in mice) is characterized by two adjacent cysteines, while the CXC (SCYB) and CX 3 C (SCYD or fractalkine) chemokine families have one or three intervening amino acids, respectively, between the two cysteines. In the XC family (SCYC or lymphotactin), only one cysteine is present [1]. All four classes of chemokines play important roles in the immune inflammatory network, but because of the complexity of interactions, here we only discuss the CC chemokine family. In humans, there are 27 CC chemokines, most of which including CCL2, CCL7, CCL11, CCL8, CCL13, CCL1, CCL5, CCL16, CCL14, CCL15, CCL23, CCL18, CCL3 and CCL4, respectively, are encoded as a cluster within chromosome 17q11. The genes for CCL27, CCL19 and CCL21 are located within chromosome 9p13, while CCL17 and CCL22 are encoded at 16q13. The remaining CCL genes can be found on chromosome 2 and 7 [1].
A functional classification was also proposed to distinguish between lymphoid and inflammatory chemokines [1,5]. Lymphoid or homeostatic chemokines (e.g. CCL21, CCL25, CXCL13) are constitutively expressed and control physiologic trafficking of cells of the adoptive immune system during hematopoiesis and immunosurveillance. Inflammatory or induced chemokines (e.g. CCL2, CCL3, CCL5, CCL7, CCL8, etc...) are transcriptionally regulated during inflammation and mediate the recruitment of inflammatory cells to target tissues.
The effects of chemokines are mediated by G-protein coupled receptors with seven-transmembrane-domains. Chemokine receptors tend to bind multiple chemokine ligands and vice versa. However, the biologically most efficient interaction often occurs between a receptor and its primary ligand (e.g. CCL2 -CCR2). The receptor binding involves high affinity interactions and signal transduction initiated by the dissociation of G-protein complex into Gα and Gβγ subunits. Gα induces the activation of the phosphoinositidine 3-kinase pathway, while the Gβγ subunits activate phospholipase C and induce Ca 2+ influx and protein kinase C activation. The involvement of MAP kinases as well as JAK/STAT signaling also has been shown [6]. As of today, 10 CC chemokine receptors (CCRs), 6 CXCRs, one CX 3 CR1 and one XCR1 are known [1,6].

Genetic evidence for the involvement of βchemokines in multiple sclerosis
A meta-analysis of raw genotype data from three genome scans in MS families revealed the highest nonparametric linkage (NPL) score = 2.58 at 17q11 [7]. Among several candidate genes (e.g. NOS2A, OMG, NF1), a cluster of evolutionarily closely related β-chemokine genes [CCL2, CCL7, CCL11, CCL8, CCL13, CCL1, CCL5, CCL16, CCL14, CCL15, CCL23, CCL18, CCL3 and CCL4, respectively] is encoded within a 1.85 Mb segment of 17q11.2-q12. Our recent linkage disequilibrium mapping confined the susceptibility regions to 3-30 kb haplotypes defined by single nucleotide polymorphisms (SNP) within the genes of CCL2, CCL11-CCL8, CCL8-CCL13, CCL13 and CCL3 [8]. A second study is under way to confirm and further refine the MS relevant haplotypes, and then, to identify the specific disease causing nucleotide variants in an independent set of families.
Within the orthologous mouse chromosome 11, two quantitative trait loci (QTL), eae6 and eae7 were identified. While these loci control the severity and duration of EAE, eae7 is also a susceptibility locus for the monophasic remitting / non-relapsing subtype of the disease [9]. Sequence polymorphisms within the genes of Scya1 (TCA-3 or CCL1), Scya2 (MCP-1 or CCL2) and Scya12 (MCP-5 or CCL12) in eae7 showed striking segregations among mouse strains resistant or susceptible to EAE.
Using an advanced intercross line in combination with congenic strains, Jagodic et al. [10] fine mapped eae18 and identified two adjacent QTLs, eae18a and eae18b, on the rat chromosome 10 in a myelin-oligodendrocyte glycoprotein (MOG)-induced, chronic relapsing EAE. The eae18b locus is also orthologous to human chromosome 17q11 and encodes a cluster of β-chemokine genes.
The recognition of β-chemokine genes as susceptibility and quantitative trait loci in mouse and rat EAE along with the human data revealing the β-chemokine gene cluster as a susceptibility locus in MS, strongly suggest the involvement of β-chemokine variants in the development of inflammatory demyelination.

CCL and CCR molecules in inflammatory demyelination
Experimental allergic encephalomyelitis EAE is a valuable model for studying the effector arm of immune response in inflammatory demyelination. It can be induced in susceptible strains of inbred and outbred species by active immunization with myelin related proteins and their peptides (myelin basic protein -MBP, proteolipid lipoprotein -PLP, myelin oligodendrocyte glycoprotein -MOG) emulsified in Freund's complete adjuvant along with intravenous Pertussis toxin, or with a passive transfer of myelin antigen specific T cell lines into naïve recipients. Using various immunization protocols, acute and chronic relapsing (CR-EAE) models have been developed. In both the active immunization and the passive transfer models of EAE, the efferent arm of immune response involves the migration of monocytes / macrophages, dendritic cells and activated myelin-antigen-specific T lymphocytes from the blood circulation into the CNS, where a reactivation of specific lymphocytes by myelin-antigen-presenting dendritic cells, macrophages and residential microglia takes place, and the sequential development of perivascular and parenchymal inflammation is followed by demyelination and neuronal degeneration.
Encephalitogenic T lymphocytes have CD4+ TH1 phenotype characterized by the production of interleukin (IL)-2 and interferon-γ. TH2 lymphocytes producing IL4, IL5, IL6 and IL10 cytokines are involved in the counter-regulation of TH1 effects, and promote clinical recovery. The TH1 / TH2 polarization is regulated by cytokines and chemokines. The transmigration of immune competent cells via the blood-brain barrier is aided by a temporal and spatial regulation of adhesion molecules on T lymphocytes and their counterparts on endothelial cells, and of chemokine ligands and their receptors in the residential CNS and hematogenous mononuclear cells.
One of the most comprehensively studied CC chemokines in inflammatory demyelination is MCP-1 (CCL2). MCP-1 (CCL2) influences both innate immunity through its chemoattractant effect on monocytes / macrophages, and adaptive immunity through its effect on T cell polarization towards the TH2 subtype [11]. CCL2 primarily acts via the CCR2 receptor [1].
Our understanding concerning the role of these CC chemokines and their receptors in inflammatory demyelination was greatly advanced by studies in the EAE model. In mice, the increased expression of MCP-1 (CCL2) by CNS immune cells is closely associated with the clinical activity of EAE [12][13][14]. Some studies, however, suggest that the presence of leukocytes is necessary for the production of CCL2 by astrocytes, as the expression of CCL2 prior to the accumulation of inflammatory mononuclear cells has not been observed in the CNS. Substantial MCP-1 (CCL2) expression may only occur in the late phase of acute disease and in the relapsing phases of CR-EAE. It was therefore postulated, that CCL2 is involved in the amplification rather than in the initiation of EAE [4]. In contrast, the MIP-1α (CCL3) expression correlates with the severity of acute disease and also is elevated during relapses in CR-EAE. RANTES (CCL5) is expressed in the CNS throughout the course, but does not correlate with the severity of acute or CR-EAE [13].
Jee et al [15] compared the histological features and MCP-1 (CCL2) and CCR2 expression levels in the lesions of Lewis rats during the acute attack of monophasic EAE and during the first two clinical events of CR-EAE. In concert with the mouse data [4,13], not only higher numbers of macrophages infiltrated the spinal cord during the first and second attacks of CR-EAE as compared to those at the peak of acute EAE in these rats, but the expression of MCP-1 (CCL2) was also significantly higher in the lesion of CR-EAE as compared to that of acute EAE. Similarly, CCR2, the main receptor for CCL2, was expressed by astrocytes, macrophages and T cells in higher amounts during CR-EAE than at the peak of acute EAE. This observation confirmed the role of CCL2 -CCR2 interaction in the development of relapses.
Youseff et al [16] observed an increased mRNA transcription not only for MCP-1 (CCL2), but also for MIP-1α (CCL3) and MIP-1β (CCL4) at the onset of EAE in rat brains. MIP-1α (CCL3) and MCP-1 (CCL2) declined in two days even though the clinical disease further progressed. MIP-1β (CCL4) mRNA declined in correlation with the clinical recovery. RANTES (CCL5) mRNA, in contrast, increased in the brains only after recovery. The full length, reverse transcribed and PCR amplified DNA product for each of these four CCL molecules was transferred into a plasmid vector and injected as naked DNA vaccine into rats. Both the transcription of a relevant chemokine and the induced antibody response against it was monitored. The in vivo immune response to these CCL molecules differentially influenced the evolution of EAE. MIP-1α (CCL3) and MCP-1 (CCL2) DNA vaccines prevented EAE, while MIP-1β (CCL4) aggravated the disease and RANTES (CCL5) did not have an effect on the course of EAE. This study emphasizes the importance of CCL2 and CCL3 in the development of active EAE in rats.
CCL1 also attracted attention in EAE. Teutscher et al [9] identified eae7 encoding CCL1 and other chemokines as a susceptibility locus and QTL in murine EAE. SNPs in CCL1 differentially segregated in mouse strains susceptible or resistant to EAE. mRNA molecules for both CCL1 and its receptor CCR8 were detected in spinal cord lesions of EAE, in correlation with the expression of tumor necrosis factor (TNF)-α by inflammatory leukocytes [17][18][19]. As both CCL1 and CCR8 were detected in microglia, an autocrine signaling mechanism was postulated. CCR8 (-/-) mice showed marked delay in the onset and reduced severity of EAE as compared to controls. Leukocyte infiltration in the spinal cord was not diminished in the CCR8 (-/-) mice, suggesting that that a defective microglial activation might have altered the clinical phenotype.
Recent studies addressed the role of chemokines at the blood-brain barrier. Using intravital fluorescence videomicroscopy, Vajkoczy et al [20] demonstrated that the interaction between encephalitogenic T cells and endothelial cells of the BBB involves α4-integrin (VLA-4) which mediates a G-protein-independent capture (arrest) followed by G-protein-dependent adhesion strengthening of circulating T cells to VCAM-1 on endothelial cells. Postulating the involvement of chemokines in the integrin-mediated arrest of autoreactive T cells at the BBB, the investigators [3] subsequently aimed to identify the specific chemokines by performing in situ hybridization and immunohistochemistry on brain and spinal cord sections of mice with EAE. Constitutive expression of the lymphoid chemokine called EBV-induced molecule 1 ligand chemokine (ELC) / CCL19 in a subpopulation of CNS venules and induced expression of the secondary lymphoid chemokine (SLC) / CCL21 in inflamed CNS venules was detected. CCR7, the common receptor for these two chemokines was expressed on a subpopulation of cells in the perivascular cuffs. Encephalitogenic T cells in vitro showed expression of CCR7 and CXCR3, the alternative receptor for CCL21, and chemotaxed towards both CCL19 and CCL21 in a concentration-dependent and a Pertussis toxin-sensitive manner similar to naïve T cells. Functional deletion of CCR7 and CXCR3 or immune blockade of CCL19 and CCL21 reduced the binding of encephalitogenic T cells to inflamed venules in frozen brain sections. Altogether, these data suggest that CCL19 and CCL21 are expressed in cerebral endothelial cells and are involved in α4-integrin mediated adhesion strengthening of autoreactive T cells and subsequently of other inflammatory cells to the endothelial layer of the BBB. These molecular interactions may lead to permanent inflammatory cell immigration into the CNS in chronic autoimmune disease. CCL20 or MIP-3α (exodus-3 / LARC) is a chemokine active on dendritic cells and lymphocytes that express CCR6 [1]. Serafini et al [21]demonstrated the occurrence of dendritic cells in the spinal cord of mice immunized with the PLP139-151 peptide. Although dendritic cells were present during early acute, chronic and relapsing EAE, most prominent infiltration of spinal cord by mature dendritic cells was noted in relapsing disease. In all stages of EAE, CCL20 and CCR6 were upregulated in the CNS. This study emphasizes the importance of dendritic cells in antigen presentation and T cell restimulation, and links the immigration of dendritic cells to the expression of CCL20 in the CNS during EAE.
CCL22 or macrophage-derived chemokine (MDC) is chemoattractant for monocytes, dendritic and NK cells, and T lymphocytes of the TH2 subtype. MDC / CCL22 acts via CCR4 which is preferentially detected on TH2 type, memory and regulatory T cells [22]. While MDC / CCL22 is considered to be predominantly involved in TH2 mediated immunity, Columba-Cabezas et al [22] demonstrated mRNA expression for MDC / CCL22 in the CNS of mice with relapsing-remitting and chronic-relapsing EAE induced by PLP139-151 or whole spinal cord homogenate. Immunohistochemistry demonstrated that MDC / CCL22 is produced by infiltrating leukocytes and residential microglia, while CCR4 is expressed by infiltrating leukocytes. In vitro activation of microglia resulted in secretion of bioactive MDC / CCL22 that induced chemotaxis of TH2 lymphocytes. This study concludes that MDC / CCL22 produced by microglia may play a role in a TH1 mediated CNS inflammation by inducing the homing of TH2 regulatory cells into the lesion site.
To further clarify the role of chemokine receptors involved in EAE, Fife et al [23] examined CCR expression in normal (unprimed), PLP139-151 primed non-activated, PLP139-151 primed and reactivated lymph node derived T cells, and CNS-isolated CD4+ T cells from SJL mice receiving PLP139-151 specific, in vitro reactivated T cells. Normal resting CD4+ T cells and primed non-activated T cells expressed mRNA for CCR1, CCR2, CCR3, CCR5, CCR6, CCR7 and CCR8. In vitro activated T cells expressed in higher amounts most of the CCRs found in normal T cells as well as CCR4. After passive transfer of encephalitogenic activated T cells in naïve recipients, the donor derived encephalitogenic cells and the host-derived CD4+ T cells isolated only from the CNS lesions but not from spleen expressed mRNA for CCR1. This latter observation was confirmed at protein level, and appeared to be specific for acute EAE. Neutralization of the CCR1 ligand CCL3 (MIP-1α) diminished the inflammatory infiltrate in the CNS.
The effects of anti-chemokine treatments in the mouse EAE is summarized by Elhofy et al [24] and Karpus et al [25] and is in consensus with data in the rat model. Although various strains and protocols were used, overall anti-RANTES (CCL5) had no effect in these models, anti-MIP-1α (CCL3) decreased the severity of acute EAE and anti-MCP-1 (CCL2) reduced the severity of both acute EAE and the relapses in CR-EAE. However, it is important noting that in some respect, these observations are model specific. While the impact of anti-CCL5 immune treatment was unremarkable in the autoantigen-induced forms of EAE, antibody treatment targeting CCL5 in a mouse hepatitis virus-induced inflammatory demyelination model resulted in diminished leukocyte infiltration and reduced neurological disability [26].
Genetic manipulations of the murine model provide further insights in the characterization of CCL / CCR molecules in EAE. In mice with the CCL2 transgene under the control of the lck (which directs the expression of transgene to cortical thymocytes) or MBP promoters (which directs the expression of transgene to the CNS), a spontaneous infiltration of monocytes / macrophages in the thymus and CNS was observed, respectively [27]. LPS injection induced higher CCL2 expression in the brain and markedly enhanced the mononuclear cell (MNC) infiltrate. The relationship between LPS treatment, CCL2 expression and MNC recruitment into the CNS remains partially understood, and seems to involve a complex immune regulatory mechanism rather than just a selective effect mediated by the upregulation of the CCL2 transgene. Nevertheless, these transgenic mice were clinically normal both before and after LPS injection. More recently, Elhofy et al [28] examined TH1 lymphocytes in a PLPinduced EAE model using a transgenic mouse strain that constitutively expressed low CCL2 levels in the CNS under the control of the astrocyte-specific glial fibrillary acidic protein promoter. CCL2 transgenic mice developed milder EAE than the littermate controls, despite similar numbers of CD4 and CD8 T cells in the CNS infiltrates and an increased number of monocytes in the CNS of the CCL2 transgenic animals. Functional studies revealed that encephalitogenic T cells from the CCL2 transgenic mice produced significantly less interferon-γ and proliferated less in the presence of PLP peptides than those of the nontransgenic controls. Increased CCL2 expression in the CNS also resulted in a decreased IL-12 receptor expression by PLP-specific T cells. Thus in this model, the overexpression of CCL2 in the CNS resulted in a suppression of the TH1 response and a milder clinical phenotype of EAE, despite the enhanced effect on monocytes.
The clinical phenotype of CCR2 (-/-) genotype was similar to that of the CCL2 (-/-) genotype, characterized by a reduced macrophage infiltration in the spinal cord and a decreased susceptibility to actively (MOG35-55) induced acute EAE in the studies by Fife et al [31] and Izikson et al [32]. T cells from CCR2 (-/-) immunized mice produced similar levels of interferon-γ and IL2 as those from controls, and were capable of transferring EAE in a naïve recipient. In contrast, T cells from wild type mice did not cause EAE in a CCR2 (-/-) recipient [31]. However, these observations again appeared to be model specific. Gaupp et al [33] reported that, even though the disease was milder or delayed, three CCR2 (-/-) mouse strains retained susceptibility to EAE in their experiments. Histological analyses revealed an abundance of neutrophils in lesions of the CCR2 (-/-) mice in contrast to the monocyte abundance in EAE lesions of wild-type mice. The development of compensatory immune mechanisms for the lack of CCR2 was evidenced by the increased mRNA expression for other CCL and CCR molecules (most notably IL8 and its receptor involved in neutrophil recruitment). This study emphasizes that promiscuity of chemokines and their receptors may overcome the deletion of a single CCR receptor with a resultant mild modification of the clinical and more profound modification of the histological phenotype.
Further studies demonstrated an approximately 50% reduction of clinical EAE activity in the CCR1 (-/-) mice, likely involving the altered migration of monocytes and lymphocytes [34]. In contrast to the observed EAE suppression in the CCR1 (-/-) and CCR2 (-/-) models, the CCR5 knockout mice had the same disease severity as the wild-type controls [35]. These studies underscore the importance of CCR1 and CCR2 in the development of inflammatory demyelination and give support to novel alternative strategies targeting these CCR molecules. Such strategies include the development of small functional CCR antagonists, amongst which the most significant progress has been made with CCR1 antagonists [36,37]. CCR1 antagonist compounds were shown to inhibit CCL3 and CCL5 induced migration of MNCs in a dose dependent manner, and to reduce clinical EAE in rat [36][37][38].
In sum, CCL and CCR data from rodent EAE models using inbred, transgenic and knockout strains along with data from chemokine-specific antibody treatments or CCL DNA immunization in EAE suggest that concentration gradients of CCL2 and CCL3 decreasing from the CNS to the peripheral circulation are involved in the spatially and temporally regulated recruitment of mononuclear cells into the CNS which correlates with the course of clinical disease. CCL2 may play a more significant role during relapses than during the induction phase of the disease. CCL5 is expressed by mononuclear cells in the perivascular space during the recovery phase of an acute event, and may therefore be involved in the regulation of recovery rather than in the initiation of the disease. In addition, CCL19 and CCL21 are expressed by endothelial cells of the BBB, and are involved in the strengthening of leukocyte adhesion to inflamed venules followed by homing of encephalitogenic T lymphocytes to the CNS. CCL20 can control the recruitment of dendritic cells into lesions, whereas CCL22 may be involved in a TH2 mediated regulatory process during EAE. CCL1 is likely playing an important role in the autocrine regulation of activation of macrophages and microglia in EAE lesions. Thus, the functional involvement of CCL chemokines during EAE is not only restricted to a well orchestrated recruitment of dendritic cells, monocytes, macrophages, T effector and regulatory cells into the CNS, but also includes a temporal and spatial regulation of TH1 (CCL3, CCL5) or TH2 (CCL2, CCL22) polarization, and monocyte, macrophage and microglial activation (CCL1, CCL2, CCL7, CCL8). Their receptors, the CCRs play equally important roles in these processes. Experimental evidence now suggests that CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8 and CXCR3 on hematogenous mononuclear cells recognize these chemoattractant and regulatory molecules to induce cell differentiation, adhesion or migration of distinct inflammatory cells in peripheral lymphoid organs, at the BBB and in the CNS during the course of EAE. Even taking into consideration the complex and promiscuous nature of the CCL -CCR network, certain pathways may be associated with distinct biological function amenable to intervention. Targeting CCR molecules either by monoclonal antibodies or by small functional antagonists has become a novel and realistic strategy in the treatment and prevention of autoimmune diseases.

Multiple sclerosis
The complexity of disease pathogenesis, difficulties accessing the site of pathology and the descriptive nature of studies explain why the available CCL / CCR data are less comprehensive in MS as compared to those in EAE. Nevertheless, new observations support the generally accepted views that MS is a predominantly TH1 lymphocyte mediated disease, and CCL -CCR molecules play a significant part in the regulation of intercellular interactions in the peripheral lymphoid organs, at the BBB and in the CNS. In addition to defining chemotaxis, CCL-CCR interactions are involved in TH1 / TH2 polarization and regulation in MS. Recent studies also raise the possibility that distinct molecular mechanisms with characteristic CCL-CCR kinetics correlate with the development of histological subtypes of the disease.

CCRs in the multiple sclerosis brain
A recent review of chemokines and their receptors [39] suggests that every CC chemokine receptor (CCR1-CCR5) interact with multiple CCLs and vice versa. While control brain specimens had only scarce appearance of CCR positive (microglial) cells throughout the CNS, foamy macrophages, microglia, perivascular lymphocytes and occasionally, astrocytes were positive for CCR2, CCR3 and CCR5 in chronic active plaques [39,40]. In other studies, CCR1, CCR2, CCR3 and CCR5 were detected on mononuclear cells and macrophages in demyelinating plaques [41,42].
Trebst et al [43] investigated the kinetics of CCR expression. In early demyelinating lesions, CCR1+/CCR5+ hematogenous monocytes and CCR1-/CCR5-microglial cells were detected. In later stages, macrophages became CCR1-/CCR5+, while microglia upregulated CCR5. This observation suggest that CCR1+/CCR5+ hematogenous monocytes enter into the CNS and stay there in the presence of appropriate ligands. During evolution of lesions, these cells down-regulate CCR1 while retain the CCR5 expression. A more recent study [44] reveals that this distinct temporal pattern, namely the decrease in CCR1+ and increase in CCR5+ cells, may be restricted to the histological type II demyelinating lesions characterized by mononuclear cell infiltration and immunoglobulin plus complement deposition, and is not seen in type III lesions characterized by oligodendrocytopathy and apoptosis [45].
CCR2 may also play a key role in the lesion development based on more indirect information. CCR2 is the main, but not exclusive, functional receptor for CCL2 [4], and as discussed above, the CCL2 -CCR2 interaction appears to play a key role in the development of EAE lesions. Microglia, macrophages and perivascular mononuclear cells show some degrees of immune reactivity for CCR2 in chronic active plaques in several studies, but the expression of CCR2 is generally low in MS lesions. Nevertheless, the data from EAE and observations in MS suggest that the CCR2 -CCL2 interaction is important in the development of plaques. This view was recently proposed and will be discussed below.
CCR8, the receptor for CCL1, has been detected in vitro on TH2 and regulatory lymphocytes, macrophages and microglia. Using immunohistochemistry, Trebst et al [19] detected CCR8 on phagocytic macrophages and activated microglia in type II and type III demyelinating MS lesions. CCR8 expression correlated with the demyelinating activity, but was not restricted to the MS pathology. Phagocytic macrophages and activated microglia in stroke and progressive multifocal leukoencephalopathy also expressed CCR8. Thus, CCR8 seems to identify a subset of activated microglia in different CNS pathologies.

CCLs in the multiple sclerosis brain
Using methods of immunohistochemistry and in situ hybridization, McManus et al [46] investigated the expression of three monocyte chemoattractant proteins, MCP-1 (CCL2), MCP-2 (CCL8) and MCP-3 (CCL7) in correlation with the temporal evolution of plaques. All three proteins were detected in high amounts in the center, but sharply decreased at the edges of acute and chronic active lesions. Hypertrophic astrocytes showed the strongest expresion, while infiltrating mononuclear cells showed variable reactivity in plaques. MCP-3 (CCL7) was also detected in the extracellular matrix. Reactivity for these CC chemokine ligands outside of plaques was otherwise restricted to hypertrophic astrocytes. In situ hybridization confirmed the observation for CCL2 at mRNA level. There seemed to be an inverse correlation between the age of plaques and expression of these three CCL molecules, with only a scanty appearance of immunoreactive astrocytes in chronic silent lesions. These methods did not detect MCP chemokines in the brains of normal controls.
While most of the above studies used the method of immunohistochemistry, we recently assessed the mRNA expression levels for CCL2, CCL3, CCL5, CCL7, CCL8, CCL13 and CCL15 relative to β-actin in corresponding normal appearing white matter (NAWM), normal appearing gray matter (NAGM) and chronic active plaque containing specimens from ten post mortem MS brains. These specimens were characterized by hematoxyllin & eosin, Luxol Fast Blue and immune staining specific for CD68 and β 2 -microglobulin [51]. In addition, the expression distribution for pro-and anti-apoptotic molecules in these specimens was also assessed by real-time PCR [51]. The selection of the above listed CC chemokines was based on two considerations. First, we detected MS associated SNP haplotypes in the genes of CCL2, CCL11-CCL8-CCL13, CCL15 and CCL3 [8]. Second, previous studies suggested the involvement of CCL2, CCL7, CCL8, CCL5 and CCL3 molecules in the development of plaques [39,46]. While neither our genetic nor our mRNA studies revealed positive findings for CCL5, the three MCP chemokines CCL2 (MCP-1), CCL7 (MCP-3) and CCL8 (MCP-2) showed altered regional expressions in MS brains. We detected an increased expression of CCL2 in plaques as compared to NAWMs, and an increased expression of CCL7 in both plaques and NAWMs as compared to NAGMs. In contrast, the expression level of CCL8 was decreased in plaques as compared to NAWM or NAGM specimens (Banisor and Kalman, unpublished observation). This analysis of CCL mRNA molecules in various regions of MS brain complements the data from previous immunohistochemical studies, and further confirms the involvement of CCL2 and CCL7 (and possibly of CCL8) in the development of pathology. In consensus with others, however, we also note an increased CCL7, CCL8 and CCL13 expression in the white matter as compared to the gray matter in 5 other neurological disease controls (1 viral and 1 post-infectious encephalitis, 2 Alzheimer disease and 1 Parkinson disease). No differences were observed for any of these molecules in the white and gray matters of normal controls. We postulate that the expression of CCL molecules may be detected in various inflammatory conditions of the CNS, however, the temporal and cell specific upregulation of certain CCL and CCR molecules is pathology specific. Therefore, further exploration of the expression kinetics of these molecules may facilitate a better understanding of MS pathogenesis.

CCL and CCR detected in blood and CSF
Relatively limited numbers of studies are available regarding chemokines and their receptors in the blood circulation and in the cerebrospinal fluid (CSF) in MS patients. The expression of CCR5 was found to be higher on circulating T lymphocytes from MS patients than on those from normal controls. These T cells showed an increased migration towards CCL3 and CCL5, suggesting a functional significance of the altered receptor expression [42,52]. The migratory population represented predominantly TH1 / TH0 cells, while the non-migratory population was enriched for TH2 cells. The aberrant migration of T cells towards CCL3 and CCL5 was related to the increased expression of the CCR5 receptor, and could be blocked by anti-CCR5 antibodies. A fluctuation of CCR5 expression by T cells was also suggested in correlation with relapses and remissions in a small group of patients [53].
Sorensen and Sellebjerg [54] assessed the CCR expression profile on peripheral T cells of patients with relapse, remission or secondary progressive disease, and detected a higher percentage of CCR2-expressing T cells in secondary progressive MS (SPMS) than in other patient groups. CCR2-positive T cells displayed TH2 profile producing IL5 and tumor necrosis factor-α. The CCR5 expression associated with TH1 profile was significantly lower in SPMS than in patients with relapsing-remitting MS (RRMS) during relapse. Thus, the authors conclude that patients with SPMS have a high expression of CCR2, a chemokine receptor associated with TH2 profile, whereas patients with RRMS preferentially display T cells with CCR5 expression and TH1 profile. More CCR5 positive T cells produced tumor necrosis factor-α in patients with RRMS than those in patients with SPMS. CCR2 is known to be predominantly expressed on monocytes. However, when expressed on T cells, CCR2 is associated with the TH2 subtype as CCL2 induces differentiation of T cells into TH2 phenotype [11]. While Sorensen and Sellebjerg [54] detected significant differences in the CCR5 and CCR2 expression profile between RRMS and SPMS, they noted no differences in CCR expression between RRMS and controls. The observation regarding the association of CCR5 with RRMS is consistent with a previous study revealing that patients with the defective CCR5 receptor (CCR5 ∆32 deletion) have prolonged relapse free periods, but the long term prognosis of MS did not seem to correlate with the CCR5 ∆32 genotype [55]. Besides establishing the CCR characteristics in RRMS and SPMS, this study also suggests that targeting the CCL2-CCR2 axis with specific CCR2 antagonist or a combination of CCR2 and CCR5 antagonists might be an option in SPMS, whereas CCR5 antagonists alone may be considered in RRMS.
However, CCR5 on peripheral MNCs was not uniformly found to be differentially expressed in MS subtypes [56]. MNCs from blood constitutively expressed CCL4 and CCL5, the ligands for CCR5, in all patient groups and controls. This study also failed to detect CCL2 and CCL3 by ribonuclease protection assay in peripheral blood MNCs. Further, the complexity of information regarding CCR5 is reflected by a recent study suggesting the association of the CCR5 ∆32 genotype with early death in MS [57].
There is relatively limited information available regarding CCR and CCL expression levels in the CSF. Some investi-gators showed that CCR5+ mononuclear cells of MS patients were enriched in the CSF, representing a significant proportion of monocytes and only a minority of T cells. However, neither cell population differed quantitatively from those of controls, suggesting that CSF leukocytes may not be fully reflective of CNS inflammation [39,55].
Giunti et al [58] detected CCR5, CCR7 and CXCR3 positive T cells in the CSF of patients with MS and other inflammatory neurological disease (IND) (meningitis, encephalitis, CIDP, neuroborreliosis). Coexpression of these receptors was noted on a subset of memory cells. The increased ratio of CXCR3 / CCR4 was suggested as a molecular correlate of disease activity by Nakajima et al. [59] TH1 clones established from the CSF of patients with IND and of controls similarly migrated in vitro towards CXCL10, CXCL12 and CCL5. CXCL10, CXCL12 and CCL19 were increased in the CSF of these patients [58].
Amongst CC chemokines, CCL3 and CCL5 were most consistently found to be elevated in the CSF of MS patients during relapses as compared to normal controls [59][60][61]. In contrast, decreased CCL2 was found in the CSF in all clinical forms of MS by Scarpini et al. [62] More consistently, however, low CCL2 levels were detected only during relapses by others [41,59,61,63,64]. The drop of CCL2 in the CSF was not found during relapses of neuromyelitis optica [65]. Mahad et al [64] also found that CCL2 in the CSF was decreased not only in patients with MS but also in patients with IND when compared to those of non-inflammatory CNS disease controls. In contrast, Bartosik-Psujek and Stelmasiak [61] observed an increase in both CCL2 and CCL5 in the CSF of patients with IND, and suggested that the drop of CCL2 during relapses is characteristic only of MS. Further, CCL2 concentration increased as time from the last relapse increased and following corticosteroid therapy [63,64]. With the exception of well defined changes in the CCL2, CCL3 and CCL5 levels in the CSF during relapses, most investigators observed no differences in various clinical forms of the disease [56,61,64,66].
Pashenkov et al [67] studied two secondary lymphoid organ chemokines, CCL19 (exodus-3, MIP-3β) and CCL21 (exodus-2, SCL) in CSF and sera of patients with MS, clinically isolated syndrome (CIS) presenting as optic neuritis (ON), isolated ON, IND and non-inflammatory neurological disease controls (NINC). CSF of the NINC group contained CCL19 but not CCL21, while both chemokines were elevated in the CSF of patients with MS, CIS-ON and IND. The authors postulate that CCL19 and CCL21 may control the retention of dendritic cells and the recruitment of naïve T cells and activated B cells, or a de novo formation of lymphoid structures in plaques. These cells are known to express CCR7, the receptor for CCL19 and CCL21. EAE studies also support the notion that CCL19 and CCL21 play important roles both at the BBB and in the CNS [3].
To correlate previous data on CCL concentrations in the CSF of MS patients, Kivisakk et al [68] measured mRNA for CCL2 / MCP-1 and CCL5 / RANTES in MNCs in the CSF and blood of patients with MS, acute meningitis and normal controls. While high numbers of MNCs expressing CCL2 and CCL5 were found in some patients, overall no differences were observed between MS and acute meningitis. This study would argue that there is no systemic dysregulation of CC chemokines contributing to MS pathogenesis.
In sum, the above data suggest that CCL1, CCL2, CCL3, CCL4, CCL5, CCL7 and CCL8 are expressed by residential glia and perivascular leukocytes in plaques. Expression of the corresponding CCR1, CCR2, CCR3, CCR5 and CCR8 receptors has been demonstrated on infiltrating leukocytes, but also on microglia, dendritic cells and astrocytes.
While the expression kinetics of CCR1 and CCR5 may discriminate between histological type II and type III lesions of MS, CCR8 is similarly expressed in both lesions types ( Table 1).
The increase of CCL3 and CCL5 in the CSF during a relapse correlates with the increase in the expression of their receptor, CCR5 on TH1 lymphocytes, which results in an enhanced migratory activity of these cells towards CCL3 and CCL5. The consistently observed decrease in CCL2 levels in the CSF during or even prior to a relapse generated alternative considerations. The first consideration suggests, that the decreased CCL2 level likely relate to a decreased TH2 lymphocyte activity, as CCL2 induces TH2 polarization. Vice versa, CCL2 expression is controlled by TH2 cytokines such as IL4. The concept of CCL2 -TH2 coregulation is supported by the observation that clinical improvement and normalization of the inflammatory CSF profile after corticosteroid treatment correlate with the normalization of CCL2 in the CSF. Thus, measurements of CCL2 in the CSF may also reflect the fluctuation of TH2 activity during the course of MS. The second consideration was proposed by Dr. Ransohoff (oral pres- entation at the ECTRIMS meeting 2004) [4,69]. This interpretation reconciles the complex observations from the EAE model suggesting a key role for CCR2 -CCL2 in the development of inflammatory lesions, and from MS suggesting a low expression of CCR2 and increased expression of CCL2 in active plaques, but a decreased CCL2 level in the CSF. Based on this model: 1) CCL2 -CCR2 play an important role in the development of inflammatory demyelinating lesions both in EAE and MS; 2) CCL2 expressed in the CNS attracts CCR2+ monocytes and T cells into the developing plaque; 3) while CCR2 binds and internalizes CCL2 molecules in large amounts, CCL2 will be consumed resulting in a reduced CCL2 level in the intercellular fluids and the CSF; 4) when CCR2 encoun-Interaction between CCL and CCR molecules at the blood-brain barrier Figure 1 Interaction between CCL and CCR molecules at the blood-brain barrier. This figure depicts CCL-CCR interactions at the BBB (endothelial cells and astrocytic processes) interfacing a venule and the CNS. CCL molecules (most prominently CCL2, CCL3, CCL7 and CCL8, but also CCL1, CCL4, CCL19 and CCL21) are produced by residential microglia, astrocytes and endothelial cells throughout the course of lesion development, and by infiltrating MNCs (CCL5) during late phases of plaque formation, and attract functionally different subsets of monocytes / macrophages, dendritic cells and T lymphocytes from the circulation via the BBB into the CNS. The temporal and spatial regulation of molecular events, the association of distinct CCR molecules with different histological subtypes of demyelination and the involvement of different CCL-CCR interactions in T cell polarization are detailed in the text. Here we illustrate in a simplified and cross-sectional manner the main groups of interacting receptors on various hematogenous cells and ligands released by residential immune cells of the CNS or by components of the BBB. Group A of receptors and ligands expressed by and acting on monocytes / macrophages, respectively: CCR1 / CCR2 / CCR3-CCL7, CCR2-CCL2, CCR3-CCL8, CCR4-CCL22; Group B of receptors and ligands expressed by and acting on dendritic cells, respectively: CCR4-CCL22, CCR6-CCL20, CCR7-CCL19 / CCL21; Group C of receptors and ligands expressed by and acting on T lymphocyes, respectively: CCR1-CCL3 / CCL5, CCR2-CCL2, CCR4-CCL22, CCR5-CCL3 / CCL4 / CCL5, CCR7-CCL19 / CCL21, CCR8-CCL1.
ters its ligand, the CCR2 / CCL2 complex will be internalized and CCR2 will be downregulated on the surface of inflammatory cells in the lesion.

Conclusion
Studies on EAE and MS suggest that CC chemokine ligands (most prominently CCL2, CCL3, CCL5, CCL7, CCL8, but also CCL1, CCL4, CCL19 and CCL21) expressed by residential immune cells in the CNS or by endothelial cells at the BBB are major chemoattractants for hematogenic immune cells (primarily monocytes / macrophages [CCL2, CCL7, CCL8, CCL22] dendritic cells [CCL19, CCL20, CCL21, CCL22] and T lymphocytes [CCL1, CCL2, CCL3, CCL4, CCL5, CCL19, CCL21, CCL22]) via interactions with their G-protein-coupled receptors (CCR1-CCR10). These CCL -CCR interactions play a key role in the recruitment, activation and retention of immune competent cells in the CNS, with the CCL1 -CCR8, CCL2 -CCR2, CCL3 -CCR1 / CCR5, CCL5 -CCR1 / CCR5, CCL7 -CCR1 / CCR2 / CCR3, CCL8 -CCR3, CCL20 -CCR6, CCL19 / CCL21 -CCR7, CCL22 -CCR4 interactions being the best characterized among them ( Figure 1). The EAE model suggests that CCL19 and CCL21 produced by endothelial cells induce G-proteinmediated signaling via their receptor CCR7. This signaling leads to an enhanced adhesion of the leukocyte α4integrin (VLA-4) to the endothelial VCAM-1 and results in a facilitated transmigration of leukocytes via the BBB. CCL-CCR interactions also define the differentiation and chemotaxis of T cell subpopulations, and thus may control the dynamic changes in the local balance of TH1 (CCL3 -CCR1 / CCR5, CCL5 -CCR1 / CCR5) and TH2 (CCL1 -CCR8, CCL2 -CCR2, CCL22 -CCR4) cell populations in lesion. Different CCL -CCR expression kinetics may characterize the different (initial, height, selflimiting) phases and histological subtypes (type II or type III) of inflammatory demyelination. This differential involvement of chemokines and their receptors in various stages and forms of MS, and the arising information concerning the involvement of genetic variants of CCLs suggest that small CCR antagonists may represent a realistic strategy in controlling the inflammatory activity that may have to be adjusted to individual disease characteristics.

List of abbreviations
BBB -blood brain barrier


extfieldsofstudy: Medicine Biology

text:
SARS Proteomics Reveals Viral Secrets

Worldwide cooperative efforts to understand the biology of the SARS coronavirus have already born significant fruit. In a further advance, the X-ray structure of a domain of nonstructural protein 3 is reported by Saikatendu et al. (2005) in this issue of Structure.

, an A family DNA polymerase, the molecular interactions and events leading to the bypass of a mutagenic DNA lesion are emerging.
In the DNA binary complexes of A family polymerases, the template base is typically positioned outside of the DNA helix with an aromatic side chain stacking with the template base immediately upstream of the coding base (Beard and Wilson, 2000). In the Bacillus fragment structure, 8oG is in a syn conformation in this extrahelical position (Hsu et al., 2004). A binary complex with T7 DNA polymerase shows 8oG to be extrahelical as well, but disordered suggesting conformational flexibility (Brieba et al., 2004).
In the newly solved ternary complex, the K536A mutant of T7 DNA polymerase facilitates closing of the fingers subdomain with 8oG in the syn conformation, which is further stabilized by dATP binding. Mechanistically, insertion of dATP and subdomain opening promotes translocation to position the (syn)8oG-(anti)A base pair at the primer terminus. The syn conformation of 8oG positions O8 in the minor groove at an equivalent position expected for O2 of thymidine thereby preserving the minor groove hydrogen bonding interactions expected for a Watson-Crick base pair. This permits escape of the mutagenic base pair from the intrinsic proofreading exonuclease activities of these polymerases (Brieba et al., 2004;Hsu et al., 2004).
The structural/kinetic/biochemical approach utilized by Brieba et al. (2005) has provided new insights into the molecular interactions that modulate the anti-syn and open-closed equilibriums of 8oG and the fingers subdomain, respectively. The results underscore the many dynamic aspects of DNA synthesis, and the importance of precise template base positioning for effi-cient and faithful replication. It remains an exciting challenge to decipher the general as well as the specific strategies utilized by DNA polymerases to make the right decisions about substrate selection and insertion. The structure of the mutagenic base pair in the confines of a closed polymerase complex exposes some of those strategies.

SARS Proteomics Reveals Viral Secrets
Worldwide cooperative efforts to understand the biology of the SARS coronavirus have already born significant fruit. In a further advance, the X-ray structure of a domain of nonstructural protein 3 is reported by Saikatendu et al. (2005)  In a worldwide cooperative research effort involving a multidisciplinary approach, structural and functional characterization of the SARS virus and its host interactions has been swiftly pursued. Four months after the first case of SARS, the virus was identified, and determination of its complete sequence was finished only 2 weeks later. The virus genome was found to code for 28 proteins, and a month after its publication, the structure of the first SARS protein, the 3CL protease, was placed in the Protein Data Bank (Anand et al., 2003). This protease represented a prime target for design of anti-SARS drugs (Yang et al., 2003) and was followed by the structures of other SARS proteins, including parts of the SARS CoV replicase polyprotein: nsP7 (Peti et al., 2005) by NMR and nsP9 (Egloff et al., 2004) (Li et al., 2005), and the sar-s7a (ORF7a) transmembrane protein of so far unknown function .
In this issue of Structure, Kuhn and coworkers (Saikatendu et al., 2005) describe the X-ray crystal structure of a domain of the nsP3 (nonstructural protein 3: the virus has 16 nsPs), the first example of this particular domain to be solved from a ssRNA virus. Intact nsP3 is a 1922 amino acid multidomain protein with an N-terminal Glu-rich domain, an ADP-ribose-1 00 -phosphate phosphatase (ADRP) domain, a protease active domain, the papain-like protease (PLP) domain, and finally two or three domains of unknown function. The structure of the ADRP domain, consisting of 182 amino acids, was solved using the selenium-SAD (single-wavelength anomalous dispersion) method of phase determination to 1.4 Å , a resolution that allowed detailed analysis of the molecular interactions.
The structure was determined as part of an integrated structural and functional proteomics program aimed at understanding emerging infectious diseases. Previously, little was known about the function of the SARS ADRP domain, or of its role in virulence, although it is conserved across the four known groups of coronaviridae. The tremendous impact that knowledge of such structures can have on the functional characterization of unknown but conserved ''hypothetical'' proteins was exemplified by this study.
The overall fold of the ADRP domain was found to be that of the three-layered a/b/a core of a macro-H2A-like domain, a unique class of phosphatases that possesses the ability to dephosphorylate ADRP. By sequence alignment and structural comparison with all known H2A domains, as well as examination of functional data, the authors conjecture that proteins from this superfamily form an emerging group of nucleotide phosphatases, all with similar functionality. The putative active site of ARDP has been identified by sequence alignment with its three structural homologs; yeast Ymx7, E. coli hypothetical protein Er58, and AF1521 from Archeoglobus fulgidus. The phosphatase activity of ADRP was subsequently confirmed by in vitro experiments on enzymatically synthesized substrate (provided by E. Phizicky, University of Rochester).
This functional discovery was surprising, because the presence of such a phosphatase in SARS had not been anticipated, and also, the original identification of a phosphatase with this substrate specificity was in an analogous tRNA maturation pathway in higher eukaryotes. It raises the interesting possibility that the SARS virus has a fairly sophisticated mechanism, which perhaps involves multiple components of the replicase machinery, to process newly synthesized genomic and subgenomic RNA. If this theory is validated, it will provide another avenue for intervention by targeted small-molecule ligands that could have therapeutic potential.
The integration of structural information with insights into the virology of SARS CoV is critical for an understanding of viral infection, its lifecycle, and the potential for intervention. A complete structural picture of the proteome of this virus would impact the understanding of many other viruses of this type.
However, there are still substantial challenges ahead, not least because structural characterization of viral proteins presents a number of severe obstacles. First, there is a paucity of genetic information available to assist with the identification of the putative protein function, since rather few viruses have been fully characterized. Second, the protocols for identifying domains, a prerequisite for expressing and purifying crystallizationgrade protein, are often unsuccessful. Third, for a virus such as SARS with only 28 proteins, the success rate for structural genomics must be high for understanding of the viral biology to advance, unlike for a bacterial system where there are a many more proteins to investigate and a higher attrition rate in the pipeline can be tolerated. Last but not least, viruses are finely tuned biological systems with a large number of interdependent molecular interactions and little redundancy. This has two pivotal consequences for understanding the biology of the virus: A systematic approach is essential, and, even more importantly, a deeper structural and functional knowledge of the many complexes that the SARS CoV proteins form with one another and with proteins of the host organisms will be required-research that is still in its infancy.
Following the development of suitable antiviral drugs and vaccines, a global strategy for their administration must be agreed upon and implemented. A similar public health challenge is currently being faced for H5N1 influenza pandemic planning by governments worldwide.

Elspeth Garman
Department of Biochemistry University of Oxford South Parks Road Oxford OX1 3QU United Kingdom


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Chemistry

text:
Proximity ligation assays with peptide conjugate ‘burrs’ for the sensitive detection of spores

The proximity ligation assay (PLA) has previously been used for the sensitive and specific detection of single proteins. In order to adapt PLA methods for the detection of cell surfaces, we have generated multivalent peptide–oligonucleotide–phycoerythrin conjugates (‘burrs’) that can bind adjacent to one another on a cell surface and be ligated together to form unique amplicons. Real-time PCR detection of burr ligation events specifically identified as few as 100 Bacillus anthracis, 10 Bacillus subtilis and 1 Bacillus cereus spore. Burrs should prove to be generally useful for detecting and mapping interactions and distances between cell surface proteins.

INTRODUCTION
The sensitive and accurate detection of spores is of key importance for both clinical and biodefense applications. Because of their extraordinary sensitivities (1), PCR-based methods are widely utilized for the detection and identification of nucleic acid sequences associated with spores (2)(3)(4). However, the accurate identification of specific bacterial species often requires that multiple gene targets be detected in parallel (3); otherwise, the amplification of genes from closely related, non-target organisms can occur (5). In addition, the detection of protein as well as nucleic acid targets can help to guard against the detection of false positives. Alternative methods for spore detection have relied upon either ELISA (6) or the binding of fluorescently labeled antibodies or peptides to spore surfaces, followed by microscopy or FACS (7)(8)(9).
Since most protein detection methods are not as sensitive as PCR, it would be useful to couple methods for the identification of spore surfaces with PCR amplification. Although an immuno-PCR approach should be possible (10), such methods require that unbound antibody-DNA conjugates be separated from bound conjugates, and are inherently prone to generating false positive results owing to non-specific binding. Therefore, we have adapted the proximity ligation assay (PLA) (11,12) to couple spore coat recognition and realtime PCR amplification. Proximity ligation is an innovative technique in which small DNA tags are co-localized on a protein surface and subsequently ligated together, creating a unique amplicon that can be sensitively detected using realtime PCR. PLA has previously been used to detect zeptomole amounts of proteins (11).
We reasoned that co-localization of DNA tags on a cell surface, rather than on a single protein molecule, might lead to the specific and sensitive detection of cells. In the present study, we have adapted peptides that bind specifically to either Bacillus anthracis, Bacillus subtilis or Bacillus cereus spores (9,13) to PLA. Peptides and DNA tags were conjugated to the fluorescent protein phycoerythrin (PE), creating multivalent 'burrs' that could detect spore surfaces. Following ligation, the amplicons associated with burrs could be used to specifically detect as few as 100 B.anthracis and 10 B.subtilis spores, and down to 1 B.cereus spore.

Bacterial strains and spores
The Bacillus strains used in this study and their sources were as follows: B.subtilis (ATCC 6051) and B.cereus (ATCC 14579) were obtained from the American Type Culture Collection. Spores were produced by growing the respective bacteria (50 ml) in 500 ml of Luria-Bertani (LB) broth for 3 days to an optical density at 600 nm (OD 600 ) of 1.6-2.0. The culture was then diluted to an OD 600 of 0.4-0.5 in synthetic replacement sporulation media (SRSM) (14) and incubated at 37 C on a shaker at 250 r.p.m. for 2 days. The culture was centrifuged at 10 000 g for 10 min using the JLA 16.250 rotor, and the pellet resuspended and lysed in 2 ml of the detergent B-Per (Pierce Biotechnology, Rockford, IL) and lysozyme (5 mg/ml). The lysate was placed on a lab rotator for 30 min at room temperature and then sonicated twice using a sonic dismembrator (Fisher Scientific, Hampton, NH) with a Branson model 102D horn fitted with a microtip at an *To whom correspondence should be addressed. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org amplitude of 15% for two 5 min intervals. The lysate was placed on ice between the two sonications. The sonicated lysate was centrifuged at 18 000 g for 15 min and washed twice with 3 ml of phosphate-buffered saline (PBS). During the last wash, the pellet was divided into five aliquots. Bacterial spores were separated from cell debris by density gradient centrifugation with sodium diatrizoate (15). Optimal conditions for separation were determined by resuspending the pellets in 2 ml of 25, 30, 35, 40 or 45% sodium diatrizoate in ddH 2 O. The 2 ml solutions were layered over 20 ml of 50% sodium diatrizoate and centrifuged at 11 000 r.p.m. for 45 min at 4 C. The broken vegetative cell debris floating in the supernatant was removed and the spores were washed three times with 2 ml of ddH 2 O. Although all the five concentrations of sodium diatrizoate could be used to separate spores from the broken vegetative cells, optimal separation was observed for pellets that were resuspended in 35% sodium diatrizoate.
Bacillus anthracis Sterne (BA) was purchased in the form of a vaccine from Colorado Serum (Denver, CO). The detergentbased spore suspension was centrifuged at 10 000 g for 45 min to pellet the BA spores. The spores were washed three times with 1 ml of 1· PBS and resuspended in 1 ml of ddH 2 O.

Peptides and primers
The spore-binding peptides used for PLA were synthesized by Biosynthesis Incorporated (Lewisville, TX). The sequences of the peptides were NH (B.subtilis specific), NHFLPKVGGGC-OH; A-TY (B.anthracis specific), ATYPLPIRGGGC-OH; and S (B.cereus specific), SLLPGLPGGGC-OH. Fluorescently labeled peptides were synthesized by conjugation of the C-terminal cysteine with fluorescein maleimide, followed by reverse-phase HPLC purification.
PLA probe synthesis R-PE was obtained from Prozyme (San Leandro, CA) and was purified from its ammonium sulfate buffer using a Microcon YM-100 filter (Millipore, MA) and resuspended in 250 ml of 1· PBS. The protein was activated using the heterobifunctional crosslinker sulfosuccinimidyl-4-(-N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC, Pierce, IL) as described previously (16). In short, 0.1 mg of PE was incubated with 0.2 mg of Sulfo-SMCC for 1 h at room temperature. The activated PE was then desalted using a NAP-5 column and resuspended in 1 ml of 1· PBS. The final concentration of the activated PE was calculated using a Nanodrop ND-1000 (Nanodrop, Wilmington, DE).
Probe conjugation was mediated through the terminal cysteine residue on the peptide and either 3 0 or 5 0 terminal thiol modifications on the oligonucleotides. Before conjugation, the DNA probes were treated with 10 mM of DTT for 30 min at room temperature, desalted using a NAP-5 column, and resuspended in 1· PBS to the desired concentration. Conjugation was achieved by incubating 56 pmol of activated PE with a mixture of a spore specific peptide (400 pmol) and either the 5 0 or 3 0 DNA probe (400 pmol) in 20 ml of PBS overnight in the dark at 4 C. The PE conjugates were desalted using a Microcon YM-100 filter and resuspended in 100 ml of 1· PBS. Probe concentrations were measured using the Nanodrop ND-1000. The approximate stoichiometry of oligonucleotide-peptide-PE was determined for the ATY-conjugate probes by comparing the absorbance of the conjugates at 260, 280 and 566 nm. The stoichiometry was estimated to be 5:3:1.

Fluorescence microscopy
Spore binding assays were prepared by combining 10 8 spores with 40 nM fluorescein-labeled monovalent peptidefluorescein or polyvalent peptide-PE conjugates in a 20 ml reaction. Samples were incubated for an hour at room temperature in 1· PBS and then washed three times with 100 ml of 1· PBS and 0.5% Tween-20. After each wash, the spores were centrifuged at 820 g at 4 C for 5 min. After the final centrifugation step, the spores were resuspended in 50 ml of PBS and fluorescence was detected using a Nikon Eclipse E800 microscope. Single band length excitation filters for FITC (501/16; 535/30) and Texas Red (568/24; 610/40) (Chroma, VT) were used to observe the monovalent peptide-fluorescein-and polyvalent peptide-PE-labeled spores, respectively. Spores incubated either without peptides or with unlabeled peptides served as controls for all microscopy experiments. PLA PLA reactions minus enzymes were assembled at room temperature in 48.3 ml of optimized PCR buffer. After the addition of Platinum Taq polymerase (0.3 ml at 5 U/ml), ligation reactions were initiated by the addition of T4 DNA ligase (0.4 ml at 1 U/ml). The reaction mixtures were incubated for an additional 5 min and then placed in the thermocycler. Experiments in which burrs and spores were preincubated for 1 h in PBS before the addition of enzymes showed no apparent effect on signal or detection.

Real-time PCR amplification and optimization
All reactions were repeated a minimum of three times and were conducted with at least two independent preparations of PE-conjugated probes. The cycle differences reported in all figures represents the cycle difference (C[T]) between the background amplification reaction (no spores) and amplification in the presence of varying amounts of target spores.
PLA optimizations were carried out with reactions containing 100 (Figures 3-5) and 10 spores (data not shown). The optimal probe concentration was determined for reactions containing 10 pM splint and probe concentrations of 100, 50, 10, 1 and 0.1 pM. The optimal splint concentration was determined for reactions containing 1 pM probe and splint concentrations of 100, 50, 10, 1 and 0.5 pM. Spore detection assays were conducted using optimized conditions, 10 pM PLA probe and 10 pM splint. Reactions contained 10 000, 1000, 100, 10, 1 or 0 spores.

RESULTS AND DISCUSSION
The concept of peptide conjugate 'burrs' for spore recognition and PLA PLAs have previously been shown to be a sensitive and specific method for protein detection and analysis. The method relies on two independent affinity reagents that bear oligonucleotide tails binding in proximity to one another; the oligonucleotides can then be ligated together, yielding an amplicon that can be detected by PCR or other amplification methods. PLA was initially developed using DNA aptamers that either bound to individual subunits of a dimeric protein or to different epitopes on the same protein (11). The method has since been expanded to include antibody-DNA conjugates (11,12). We now further expand PLA to the use of peptide-based affinity reagents that can bind specifically not to proteins, but to the surfaces of spores.
Phage-displayed peptides have been selected that bind with high specificity to several different Bacillus spores [Table 1; (9,13)]. It was known that the peptides bound poorly as isolated, synthetic monomers (8,17), and our own preliminary studies with fluorescent peptide derivatives indicated that there was a significant degree of cross-reactivity between different spores ( Figure 1A). However, polyvalent presentation of the peptides either in the context of a fluorescently labeled phage or as PE conjugates was known to support specific recognition of spores, and we therefore decided to use PE as the basis for PLA affinity reagents. As Turnbough and co-workers previously observed, polyvalent peptide-PE conjugates proved to be highly specific for spores from Bacillus species ( Figure 1B).
PLA affinity reagents were further developed by conjugating both peptides and oligonucleotides to PE, creating 'burrs' that had multiple opportunities to bind both to the spore surface and to position oligonucleotides for ligation reactions (Figure 2A). Peptides and oligonucleotides bearing thiol linkers were mixed with one another and then with PE activated with sulfo-SMCC. This joint immobilization procedure allows us to control the ratio of peptide-oligonucleotide. Starting with an equimolar ratio of peptide and oligonucleotide resulted in the conjugation of 5 oligonucleotides and  3 peptides per PE. When two burrs bind adjacent to one another on a spore surface, the pendant oligonucleotides can be aligned by an external template (splint) and ligated by T4 DNA ligase. The ligation event can be detected and quantified by real-time PCR ( Figure 2B).

Spore detection via burrs and PLA
Spore-specific burrs were mixed with B.subtilis (BS), B.cereus (BC) or B.anthracis (BA) and incubated for 5 min in optimized PLA buffer before the addition of T4 DNA ligase and Taq polymerase. Since we wished to preferentially capture proximity events, ligation was carried out for a very short period of time (5 min), and then ligated sequences were amplified via real-time PCR. In addition, since the splint can potentially promote the ligation of the burrs even in the absence of spores we carried out negative controls without spores. After PCR, the spore-dependent signal is represented as the shift in the number of PCR cycles required for amplification to a given cycle threshold [C[T] value (18)  Initially, we did not know how much burr would be necessary to achieve a significant shift in the cycle threshold. We, therefore, varied burr concentration from 0.5 to 100 pM while keeping the splint concentration (10 pM) and other variables constant. PLA reactions with only 100 BS and BC spores were conducted with burrs bearing either the NH-(BS-specific) or S-peptide (BC-specific), while reactions with 100 BA (Sterne) spores were conducted using probes bearing either the NH-, S-or the ATY-peptide (BA-specific). A single dataset generated with B.cereus spores is shown in Figure 3. A substantive real-time PCR signal was observed when the PLA reaction was conducted using a 10 pM concentration of burrs. Similar experiments were conducted with spores from all three bacterial species a minimum of three times. The averaged data from these experiments are shown in Figure 4. Again, spore-specific signals, indicated by a positive C[T] value, were reproducibly observed at some burr concentrations. PLA reactions in which the burrs and spores were pre-incubated for 1 h in PBS before the addition of enzymes gave similar results (data not shown).
The fact that only some burr concentrations should give large changes in C[T] values is not surprising; too many burrs in solution will yield a background of ligated templates that is not spore-dependent, whereas too few burrs will not generally bind adjacent to one another on a spore surface, will not ligate and again will not yield a spore-dependent signal. For 100 spores, 10 pM burr generally seemed to give a reliable signal. Gratifyingly, the BS-specific peptide never yielded a significant, positive C[T] value with BC and BA, and the BC-specific peptide did not give a positive C[T] value with BS or BA. Additional optimizations ( Figure 6) revealed that the BA-specific peptide did not produce a signal in the presence of BS or BC spores. In some cases, a negative cycle difference (1-4 cycles) was observed when reactions were conducted in the presence of spores. These negative C[T] differences may reflect the general inhibition of PCR reactions by spores or attendant organics in solution, and further emphasize the validity of the reproducible, positive C[T] values seen with cognate burr-spore pairs.
In addition to the affinity reagent concentration, the concentration of the splint oligonucleotide has been shown to be an important factor in the optimization of PLA detection (11,12). Therefore, we performed a series of assays in which we varied the splint concentration. Assays were conducted using a constant amount (10 pM) of burr and 100 BS, BC or BA (Sterne) spores. As shown in Figure 5, optimal spore detection was observed for reactions conducted with either 10 or 50 pM splint. The decrease in the observed cycle difference at the higher splint concentrations can be attributed to a decrease in the number of amplification cycles necessary to generate a signal in the absence of spores, indicating an increase in the number of spore-independent ligation events (data not shown). Most importantly, though, all reactions conducted with non-cognate spores again showed no positive signal.
Finally, we carried out PLA reactions to examine the limits of detection with burrs. As shown in Figure 6, specific amplification is once again only observed for each burr with its cognate spore. The observed detection limits for optimized reaction conditions are as few as 10 BC or BS spores, and 100 BA (Sterne) spores. It should again be emphasized that these are detection limits for the detection of the spore coat, not the spore genome, and thus that PLA with burrs is likely the single most sensitive method for the detection of spores themselves currently available.
The loss of a positive signal at higher concentrations of spores is likely due to dilution of the burrs on the spore surface. At higher spore concentrations (10 3 -10 4 spores/50 ml the number of burrs binding to adjacent sites on the spore coat is decreased, leading to fewer or no ligation events. In keeping with this hypothesis, we reasoned that at lower spore concentrations there would be fewer spore-dependent ligation events but the same level of background ligation. If so, positive signals would be harder to acquire. Based on this, we attempted to rationally optimize the PLA detection method. Splint concentrations were lowered from 10 to 1 pM, in order to reduce the level of background ligation. As shown in Figure 7, this modification resulted in a further decrease in the detection limit to a single BC spore.
Although there appears to be a relatively narrow window in which specific spore-dependent amplification can be achieved, this window can be rationally manipulated and a variety of spore concentrations could potentially be detected by using several different burr-splint pairs in parallel. Each burr-splint pair would form a unique amplicon and would be present at a concentration that had previously been optimized for a given spore concentration. Thus, in a multiplex PCR, each burr-splint pair could detect a particular concentration range of a spore. Additionally, it may prove possible to Figure 6. Specificity of spore detection assays. Reactions were carried out with 10 pM probe and 10 pM splint, and contained burrs bearing one of the three spore-specific peptides. Cycle difference is as in Figure 4.  . Optimization of the limits of detection for BC spores. Reactions were carried out with 10 pM probe and 1 pM splint, and contained burrs bearing one of the three spore-specific peptides. Cycle difference is as in Figure 4.
improve detection by generating burrs that bear two different peptides for the same spore, or by synthesizing burrs with optimal oligonucleotide-peptide ratios.
The use of burrs is not merely an incredibly sensitive assay for cell surface epitopes, but should be an extremely powerful technique to probe the surfaces of cells. Although previous implementations of the PLA have indicated that multiple epitopes on the same protein or protein oligomer can be simultaneously detected, we have now extended this technique to multiple epitopes on the surfaces of cells. To the extent that type, number, or distribution of protein or other epitopes that can be identified by affinity reagents is diagnostic for a given cell or cell type, burr-based PLA may provide novel and interesting information about cell biology. For example, proteins that are ensconced within lipid rafts could be readily detected by burrs, even if the total concentration of proteins on the cell surface did not change. Similarly, burrs made from Annexin V could be used to identify when phosphotidylserine began to make an appearance on the cell surface, and thus could be used to monitor the earliest stages of apoptosis. As more applications for burr-based PLA are explored, it is even possible that oligonucleotides of differing lengths could be used as molecular rulers for probing the distances between target antigens on a cell surface.