Simulating the influence of plasma protein on measured receptor affinity in biochemical assays reveals the utility of Schild analysis for estimating compound affinity for plasma proteins.

BACKGROUND AND PURPOSE: Plasma protein binding (PPB) influences the free fraction of drug available to bind to its target and is therefore an important consideration in drug discovery. While traditional methods for assessing PPB (e.g. rapid equilibrium dialysis) are suitable for comparing compounds with relatively weak PPB, they are not able to accurately discriminate between highly bound compounds (typically >99.5%). The aim of the present work was to use mathematical modelling to explore the potential utility of receptor binding and cellular functional assays to estimate the affinity of compounds for plasma proteins. Plasma proteins are routinely added to in vitro assays, so a secondary goal was to investigate the effect of plasma proteins on observed ligand-receptor interactions. EXPERIMENTAL APPROACH: Using the principle of conservation of mass and the law of mass action, a cubic equation was derived describing the ligand-receptor complex [LR] in the presence of plasma protein at equilibrium. KEY RESULTS: The model demonstrates the profound influence of PPB on in vitro assays and identifies the utility of Schild analysis, which is usually applied to determine receptor-antagonist affinities, for calculating affinity at plasma proteins (termed KP ). We have also extended this analysis to functional effects using operational modelling and demonstrate that these approaches can also be applied to cell-based assay systems. CONCLUSIONS AND IMPLICATIONS: These mathematical models can potentially be used in conjunction with experimental data to estimate drug-plasma protein affinities in the earliest phases of drug discovery programmes.

Potent and efficacious inhibition of CXCR2 signaling by biparatopic nanobodies combining two distinct modes of action.

Chemokines and chemokine receptors are key modulators in inflammatory diseases and malignancies. Here, we describe the identification and pharmacologic characterization of nanobodies selectively blocking CXCR2, the most promiscuous of all chemokine receptors. Two classes of selective monovalent nanobodies were identified, and detailed epitope mapping showed that these bind to distinct, nonoverlapping epitopes on the CXCR2 receptor. The N-terminal-binding or class 1 monovalent nanobodies possessed potencies in the single-digit nanomolar range but lacked complete efficacy at high agonist concentrations. In contrast, the extracellular loop-binding or class 2 monovalent nanobodies were of lower potency but were more efficacious and competitively inhibited the CXCR2-mediated functional response in both recombinant and neutrophil in vitro assays. In addition to blocking CXCR2 signaling mediated by CXCL1 (growth-related oncogene α) and CXCL8 (interleukin-8), both classes of nanobodies displayed inverse agonist behavior. Bivalent and biparatopic nanobodies were generated, respectively combining nanobodies from the same or different classes via glycine/serine linkers. Interestingly, receptor mutation and competition studies demonstrated that the biparatopic nanobodies were able to avidly bind epitopes within one or across two CXCR2 receptor molecules. Most importantly, the biparatopic nanobodies were superior over their monovalent and bivalent counterparts in terms of potency and efficacy.

Direct evidence of milk consumption from ancient human dental calculus.

Milk is a major food of global economic importance, and its consumption is regarded as a classic example of gene-culture evolution. Humans have exploited animal milk as a food resource for at least 8500 years, but the origins, spread, and scale of dairying remain poorly understood. Indirect lines of evidence, such as lipid isotopic ratios of pottery residues, faunal mortality profiles, and lactase persistence allele frequencies, provide a partial picture of this process; however, in order to understand how, where, and when humans consumed milk products, it is necessary to link evidence of consumption directly to individuals and their dairy livestock. Here we report the first direct evidence of milk consumption, the whey protein ß-lactoglobulin (BLG), preserved in human dental calculus from the Bronze Age (ca. 3000 BCE) to the present day. Using protein tandem mass spectrometry, we demonstrate that BLG is a species-specific biomarker of dairy consumption, and we identify individuals consuming cattle, sheep, and goat milk products in the archaeological record. We then apply this method to human dental calculus from Greenland's medieval Norse colonies, and report a decline of this biomarker leading up to the abandonment of the Norse Greenland colonies in the 15(th) century CE.

Detailed analysis of biased histamine H4 receptor signalling by JNJ 7777120 analogues.

BACKGROUND AND PURPOSE: The histamine H4 receptor, originally thought to signal merely through Gαi proteins, has recently been shown to also recruit and signal via ß-arrestin2. Following the discovery that the reference antagonist indolecarboxamide JNJ 7777120 appears to be a partial agonist in ß-arrestin2 recruitment, we have identified additional biased hH4R ligands that preferentially couple to Gαi or ß-arrestin2 proteins. In this study, we explored ligand and receptor regions that are important for biased hH4R signalling. EXPERIMENTAL APPROACH: We evaluated a series of 48 indolecarboxamides with subtle structural differences for their ability to induce hH4R-mediated Gαi protein signalling or ß-arrestin2 recruitment. Subsequently, a Fingerprints for Ligands and Proteins three-dimensional quantitative structure-activity relationship analysis correlated intrinsic activity values with structural ligand requirements. Moreover, a hH4R homology model was used to identify receptor regions important for biased hH4R signalling. KEY RESULTS: One indolecarboxamide (75) with a nitro substituent on position R7 of the aromatic ring displayed an equal preference for the Gαi and ß-arrestin2 pathway and was classified as unbiased hH4R ligand. The other 47 indolecarboxamides were ß-arrestin2-biased agonists. Intrinsic activities of the unbiased as well as ß-arrestin2-biased indolecarboxamides to induce ß-arrestin2 recruitment could be correlated with different ligand features and hH4R regions. CONCLUSION AND IMPLICATIONS: Small structural modifications resulted in diverse intrinsic activities for unbiased (75) and ß-arrestin2-biased indolecarboxamides. Analysis of ligand and receptor features revealed efficacy hotspots responsible for biased-ß-arrestin2 recruitment. This knowledge is useful for the design of hH4R ligands with biased intrinsic activities and aids our understanding of the mechanism of H4R activation.

Pilot evaluation of anterior dynamic ultrasound screening for developmental dysplasia of the hip in an Australian regional hospital.

INTRODUCTION: Developmental Dysplasia of the Hip (DDH) is the most common notifiable musculoskeletal birth defect in South Australia (SA). Despite routine screening by physical examination of the hips in the neonatal period and at 6 weeks of age, the risk of late diagnosis is increased in rural areas. It is assumed this is due to the examining doctors' reduced clinical expertise. Introducing Anterior Dynamic Ultrasound (ADUS) has reduced the late detection rates in Sweden to almost zero, and may benefit Australian infants in rural areas if routine screening was introduced. This study reports on a small implementation pilot in a SA regional hospital where volunteer postnatal mothers consented to their babies having ADUS examinations. METHODS: The pilot was evaluated by collecting results of physical examination, ADUS, and surveying parental impressions of the screening test. RESULTS: Hips of 86 infants underwent ADUS during the implementation pilot. Parents' perceptions were mainly very positive and indicated ADUS was an accessible and acceptable screening test. Of the hips scanned, three were found to have maximum movement of the femoral head of >3 mm and were deemed to demonstrate increased laxity. Four hips described as loose or mobile on clinical examination were found to be within normal limits of maximum mobility on ADUS. CONCLUSIONS: This study has demonstrated that a larger scale implementation project would be feasible in regional Australia, and would enable researchers to better understand how to reduce the late diagnosis rate of DDH in rural areas.

Reassessment of the pharmacology of Sphingosine-1-phosphate S1P3 receptor ligands using the DiscoveRx PathHunter™ and Ca2+ release functional assays.

BACKGROUND AND PURPOSE: DiscoverRx's PathHunter™ assay measures GPCR agonist potency, via the recruitment of ß-arrestin, independent of the subtype of G(α) protein activated. This assay is frequently used in drug discovery although little is known about the agonist pharmacology generated. Here we have compared agonist potency, efficacy and affinity values obtained in PathHunter™ assays with those from more established radioligand binding and functional techniques. EXPERIMENTAL APPROACH: Using cells expressing the human sphingosine-1-phosphate S1P(3) receptor at four different densities, we compared pharmacological affinity and efficacy values of four structurally distinct ligands - FTY720-P, VPC24191, CYM5442 and the endogenous agonist S1P - obtained from competition binding, functional Ca(2+) release and PathHunter™ assays. KEY RESULTS: The pK(i) values for S1P were significantly different (9.34 ± 0.10 and 8.92 ± 0.15) in clones expressing different receptor levels using the binding assay. In the PathHunter™ and Ca(2+) assays, S1P and CYM5442 were full agonists, FTY720-P was a partial agonist, while the efficacy of VPC24191 could not be detected in PathHunter™ assays. VPC23019, previously described as a S1P(1/3) receptor antagonist, behaved as an S1P(3) receptor partial agonist in the Ca(2+) release assay. CONCLUSIONS AND IMPLICATIONS: Comparison of data from the PathHunter™ assay with binding and functional Ca(2+) assays suggest that PathHunter™ assays measured a different agonist-bound receptor conformation. While this assay has great utility in drug discovery, care must be taken as high-efficacy, low-affinity agonist compounds would not be detected. Therefore highly amplified, more traditional assays are necessary to identify agonists with low efficacy.

Use of microwells to investigate the effect of quorum sensing on growth and antigen production in Bacillus anthracis Sterne 34F2.

AIM: The aim of this study was to investigate the role of quorum sensing in Bacillus anthracis growth and toxin production. METHODS AND RESULTS: A microwell plate culture method was developed to simulate the normal UK-licensed anthrax vaccine production run. Once established, sterile supernatant additions from a previous B. anthracis culture were made, and reductions in lag phase and early stimulation of the anthrax toxin component protective antigen (PA) were monitored using ELISA. The addition of the quorum-sensing inhibitor, fur-1, prolonged the lag phase and impeded PA production. Spin filters of various sizes were used to identify the molecular weight fraction of the sterile supernatant responsible for the autoinducer effect. A weight fraction between 5 and 10 kDa was responsible for the autoinducer effect; however, further identification using mass spectroscopy proved inconclusive. CONCLUSIONS: Quorum sensing mediated by the autoinducer two molecule plays a significant role in both B. anthracis growth and toxin production. SIGNIFICANCE AND IMPACT OF THE STUDY: While genomic analysis has eluded to the importance of LuxS and quorum sensing in anthrax, this is the first analysis using a production strain of B. anthracis and a quorum-sensing inhibitor to monitor the effect on growth and toxin production. This gives insights into anthrax pathogenicity and vaccine manufacture.

A common intracellular allosteric binding site for antagonists of the CXCR2 receptor.

BACKGROUND AND PURPOSE: We have previously shown that SB265610 (1-(2-bromo-phenyl)-3-(7-cyano-3H-benzotriazol-4-yl)-urea) behaves as an allosteric, inverse agonist at the C-X-C chemokine (CXCR)2 receptor. The aim of this study was to determine whether SB265610, in addition to two other known antagonists, bind to either of the two putative, topographically distinct, allosteric binding sites previously reported in the Literature. EXPERIMENTAL APPROACH: Ten single point mutations were introduced into the CXCR2 receptor using site-directed mutagenesis. Three CXCR2 antagonists were investigated, SB265610, Pteridone-1 (2-(2,3 difluoro-benzylsulphanyl)-4-((R)-2-hydroxy-1-methyl-ethylamino)-8H-pteridin-7-one) and Sch527123 (2-hydroxy-N,N-dimethyl-3-{2-[[(R)-1-(5-methyl-furan-2-yl)-propyl]amino]-3,4-dioxo-cyclobut-1enylamino}-benzamide), and the effect of these mutations on their binding affinity and ability to inhibit interleukin-8-stimulated binding of [(35)S]GTPgammaS was examined. KEY RESULTS: Seven of the nine mutations introduced into the C-terminal domain and intracellular loops of the receptor produced a significant reduction in affinity at least one of the antagonists tested. Of those seven mutations, three produced a significant reduction in the affinity of all three antagonists, namely K320A, Y314A and D84N. In all but one mutation, the changes observed on antagonist affinity were matched with effects on inhibition of interleukin-8-stimulated [(35)S]GTPgammaS binding. CONCLUSIONS AND IMPLICATIONS: These antagonists bind to a common intracellular, allosteric, binding site of the CXCR2 receptor, which has been further delineated. As many of these mutations are close to the site of G protein coupling or to a region of the receptor that is responsible for the transduction of the activation signal, our results suggest a molecular mechanism for the inhibition of receptor activation.

Pharmacogenetic characterization of indacaterol, a novel beta 2-adrenoceptor agonist.

BACKGROUND AND PURPOSE: Indacaterol is a novel beta(2)-adrenoceptor agonist in development for the treatment of chronic obstructive pulmonary disease. The aim of this study was to investigate the comparative pharmacology of indacaterol in recombinant cells expressing the common polymorphic variants of the human beta(2)-adrenoceptor and in human primary airway smooth muscle (ASM) cells. EXPERIMENTAL APPROACH: Chinese hamster ovarian-K1 cell lines expressing high and low levels of the common human beta(2)-adrenoceptor variants were generated [Gly16-Glu27-Val34-Thr164(GEVT), RQVT, GQVT] and also the rare GQVI variant. Human primary ASM cells were isolated from explants of trachealis muscle. Adenosine-3',5'-cyclic-monophosphate production was used as an outcome measure. KEY RESULTS: In both the low- and high-expression recombinant GEVT 'wild type' cell lines indacaterol is a high-efficacy agonist. Salmeterol and formoterol were identified as low- and high-efficacy agonists, respectively, and showed similar potencies to indacaterol irrespective of the beta(2)-adrenoceptor genotype. The I164 variant cell line was associated with a reduced capacity to generate adenosine-3',5'-cyclic-monophosphate in response to beta(2)-adrenoceptor agonist. In the human primary ASM cells indacaterol gave a maximal response intermediate between that of salmeterol and formoterol. CONCLUSIONS AND IMPLICATIONS: These data demonstrate that indacaterol is a high-efficacy agonist in recombinant cell systems but acts with lower efficacy in human primary ASM cells. No marked genotype-dependent effects were observed for common variants; however, changes in I164 receptor activity were identified, which were dependent on the level of expression of beta(2)-adrenoceptors.

SB265610 is an allosteric, inverse agonist at the human CXCR2 receptor.

BACKGROUND AND PURPOSE: In several previous studies, the C-X-C chemokine receptor (CXCR)2 antagonist 1-(2-bromo-phenyl)-3-(7-cyano-3H-benzotriazol-4-yl)-urea (SB265610) has been described as binding competitively with the endogenous agonist. This is in contrast to many other chemokine receptor antagonists, where the mechanism of antagonism has been described as allosteric. EXPERIMENTAL APPROACH: To determine whether it displays a unique mechanism among the chemokine receptor antagonists, the mode of action of SB265610 was investigated at the CXCR2 receptor using radioligand and [(35)S]-GTPgammaS binding approaches in addition to chemotaxis of human neutrophils. KEY RESULTS: In equilibrium saturation binding studies, SB265610 depressed the maximal binding of [(125)I]-interleukin-8 ([(125)I]-IL-8) without affecting the K(d). In contrast, IL-8 was unable to prevent binding of [(3)H]-SB265610. Kinetic binding experiments demonstrated that this was not an artefact of irreversible or slowly reversible binding. In functional experiments, SB265610 caused a rightward shift of the concentration-response curves to IL-8 and growth-related oncogene alpha, but also a reduction in maximal response elicited by each agonist. Fitting these data to an operational allosteric ternary complex model suggested that, once bound, SB265610 completely blocks receptor activation. SB265610 also inhibited basal [(35)S]-GTPgammaS binding in this preparation. CONCLUSIONS AND IMPLICATIONS: Taken together, these data suggest that SB265610 behaves as an allosteric inverse agonist at the CXCR2 receptor, binding at a region distinct from the agonist binding site to prevent receptor activation, possibly by interfering with G protein coupling.

A study of the physiology of Bacillus anthracis Sterne during manufacture of the UK acellular anthrax vaccine.

AIM: To analyse the growth of Bacillus anthracis during simulations of the UK anthrax vaccine manufacturing process. METHODS AND RESULTS: Simulated vaccine production runs were performed using the toxigenic, acapsulate Sterne 34F(2) strain of B. anthracis in semi-defined medium. After rising during the logarithmic growth phase, the pH of the culture starts to fall at about 18 h from pH 8.7 to reach <7.6 at 26 h, coincident with consumption of glucose and optimal production of protective antigen (PA; 7.89 g ml(-1), SD 1.0) and lethal factor (LF; 1.85 g ml(-1), SD 0.29). No increased breakdown of toxin antigens was seen over the 26-32 h period. When glucose was exhausted, amino acids (principally serine) were utilized as an alternative carbon source. Sporulation was not observed during the 32 h. CONCLUSIONS: PA and LF, the principal constituents in the UK anthrax vaccine, undergo little degradation during vaccine fermentation. The vaccine manufacturing process is robust and reproducible. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first detailed analysis of the manufacturing process used for the UK acellular anthrax vaccine; insight gained into the process will support continued and safe vaccine manufacture.

Analysis of second messenger pathways stimulated by different chemokines acting at the chemokine receptor CCR5.

The chemokine receptor, CCR5, responds to several chemokines leading to changes in activity in several signalling pathways. Here, we investigated the ability of different chemokines to provide differential activation of pathways. The effects of five CC chemokines acting at CCR5 were investigated for their ability to inhibit forskolin-stimulated 3'-5'-cyclic adenosine monophosphate (cAMP) accumulation and to stimulate Ca(2+) mobilisation in Chinese hamster ovary (CHO) cells expressing CCR5. Macrophage inflammatory protein 1alpha (D26A) (MIP-1alpha (D26A), CCL3 (D26A)), regulated on activation, normal T-cell expressed and secreted (RANTES, CCL5), MIP-1beta (CCL4) and monocyte chemoattractant protein 2 (MCP-2, CCL8) were able to inhibit forskolin-stimulated cAMP accumulation, whilst MCP-4 (CCL13) could not elicit a response. CCL3 (D26A), CCL4, CCL5, CCL8 and CCL13 were able to stimulate Ca(2+) mobilisation through CCR5, although CCL3 (D26A) and CCL5 exhibited biphasic concentration-response curves. The Ca(2+) responses induced by CCL4, CCL5, CCL8 and CCL13 were abolished by pertussis toxin, whereas the response to CCL3 (D26A) was only partially inhibited by pertussis toxin, indicating G(i/o)-independent signalling induced by this chemokine. Although the rank order of potency of chemokines was similar between the two assays, certain chemokines displayed different pharmacological profiles in cAMP inhibition and Ca(2+) mobilisation assays. For instance, whilst CCL13 could not inhibit forskolin-stimulated cAMP accumulation, this chemokine was able to induce Ca(2+) mobilisation via CCR5. It is concluded that different chemokines acting at CCR5 can induce different pharmacological responses, which may account for the broad spectrum of chemokines that can act at CCR5.

Nasal administration of an angiotensin antagonist in the rat model: effect of bioadhesive formulations on the distribution of drugs to the systemic and central nervous systems.

The effect of bioadhesive formulations on the direct transport of an angiotensin antagonist drug ((14)C-GR138950) from the nasal cavity to the central nervous system was evaluated in a rat model. Three different bioadhesive polymer formulations (3% pectin LM-5, 1.0% pectin LM-12 and 0.5% chitosan G210) containing the drug were administered nasally to rats by inserting a dosing cannula 7mm into the nasal cavity after which the plasma and brain tissue levels were measured. It was found that the polymer formulations provided significantly higher plasma levels and significantly lower brain tissue levels of drug than a control, in the form of a simple drug solution. Changing the depth of insertion of the cannula from 7 to 15mm, in order to reach the olfactory region in the nasal cavity significantly decreased plasma levels and significantly increased brain tissue levels of drug for the two formulations studied (1.0% pectin LM-12 and a simple drug solution). There was no significant difference between the drug availability for the bioadhesive formulation and the control in the brain when the longer cannula was used for administration. It is suggested that the conventional rat model is not suitable for evaluation of the effects of bioadhesive formulations in nose-to-brain delivery.

Distribution and clearance of bioadhesive formulations from the olfactory region in man: effect of polymer type and nasal delivery device.

There is an increasing need to identify novel approaches by which to improve the efficiency of drug transport from the nasal cavity (olfactory region) to the CNS, especially for treatment of central nervous system disorders. It is suggested, that one approach is the combination of active targeting of a bioadhesive formulation, that will retain the drug at the absorption site, potentially in combination with, an absorption enhancer. Two low methylated pectins, LM-5 and LM-12 were selected for evaluation as drug delivery systems, due to their ability to gel in the nasal cavity and their bioadhesive characteristics, together with chitosan G210, which acts both as a bioadhesive material and as an efficient absorption enhancer. It was found that all of the bioadhesive formulations were able to reach the olfactory region in the nasal cavity of human volunteers when delivered using a simple nasal drop device. Furthermore, the formulations displayed a significantly increased residence time on the epithelial surface. This was in contrast to a non-bioadhesive control delivered with the same device. In contrast, a pectin formulation administered with a nasal spray system did not show an increase in residence time in the olfactory region. It was further shown that the reproducibility of olfactory delivery of a polymer formulation was significantly better intra-subject than inter-subject.

Evaluation of bioadhesive polymers as delivery systems for nose to brain delivery: in vitro characterisation studies.

There is an increasing need for nasal drug delivery systems that could improve the efficiency of the direct nose to brain pathway especially for drugs for treatment of central nervous system disorders. Novel approaches that are able to combine active targeting of a formulation to the olfactory region with controlled release bioadhesive characteristics, for maintaining the drug on the absorption site are suggested. If necessary an absorption enhancer could be incorporated. Low methylated pectins have been shown to gel and be retained in the nasal cavity after deposition. Chitosan is known to be bioadhesive and also to work as an absorption enhancer. Consequently, two types of pectins, LM-5 and LM-12, together with chitosan G210, were selected for characterisation in terms of molecular weight, gelling ability and viscosity. Furthermore, studies on the in vitro release of model drugs from candidate formulations and the transport of drugs across MDCK1 cell monolayers in the presence of pectin and chitosan were also performed. Bioadhesive formulations providing controlled release with increased or decreased epithelial transport were developed. Due to their promising characteristics 3% LM-5, 1% LM-12 pectin and 1% chitosan G210 formulations were selected for further biological evaluation in animal models.

Application of low dose rate pulsed fluoroscopy in cardiac pacing and electrophysiology: patient dose and image quality implications.

The performance of a low dose rate pulsed fluoroscopy option and its successful application to cardiac pacing and electrophysiology is reported. Low dose rate 6.25 frames per second pulsed fluoroscopy was made available in two catheter laboratories at a specialist cardiac centre in February 2003, and was adopted as the standard imaging technique for cardiac pacing procedures. The image quality was found to be considerably poorer than conventional modern units, being very similar to that which would have been accepted as adequate performance 20 years ago, but at less than one-tenth of the dose rate. No problems with the clinical acceptance of this imaging mode for cardiac pacing and electrophysiology have been reported. The already low median patient dose-area product for pacing at this cardiac centre was further reduced by 50% with the introduction of this fluoroscopy option.

An association between the CTLA4 exon 1 polymorphism and early rheumatoid arthritis with autoimmune endocrinopathies.

OBJECTIVE: To examine the allelic association of the single nucleotide polymorphism (CTLA4A/G) in exon 1 of the cytotoxic T lymphocyte antigen-4 (CTLA4) gene with early rheumatoid arthritis (RA). METHODS: One hundred and twenty-three unrelated white probands with early RA from the north-east of England and 349 local ethnically matched controls were studied. The CTLA4A/G polymorphism was genotyped with a polymerase chain reaction (PCR) method and digestion with the restriction enzyme Bst71I. Probands were also screened by allele-specific PCR for alleles HLA DRB1*01 and DRB1*04. RESULTS: The frequency of the G allele at CTLA4A/G was significantly increased in probands with early RA compared with controls [43 vs 36%; P=0.028, odds ratio (OR) 1.35, 95% confidence interval (CI) 1.01-1.82]. Most of this increased frequency was attributable to RA individuals with coexisting autoimmune thyroid disease or type 1 diabetes (58 vs 36% in controls; P=0.005, OR 2.50, CI 1.29-4.84). The frequency of the G allele in RA patients without autoimmune endocrinopathy was 40%, which was not significantly different from that in controls (P=0.140). CONCLUSION: The association between the CTLA4 G allele and early RA is largely explained by individuals with RA who have coexisting autoimmune endocrinopathies.

Expression and functional analysis of chemokine receptors in human peripheral blood leukocyte populations.

Emerging evidence indicates that chemokine receptor expression patterns are critical in determining the spectrum of action of the chemokines. We have analysed the expression patterns of 17 chemokine receptors and two orphan chemokine receptor-like genes in various freshly prepared human peripheral blood leucocyte populations, including neutrophils, lymphocytes, and naïve and differentiated monocytes using real-time quantitative polymerase chain reaction (TaqMan). This is the first comprehensive study of chemokine receptor expression in such a wide variety of cell types. Human peripheral blood leukocyte populations were found to express a wide range of chemokine receptors that varies depending on cell type and differentiation state. Novel expression patterns of certain chemokine receptors were seen during our analysis. For example, the orphan chemokine receptor HCR was expressed at very high levels by both primary neutrophils and primary monocytes, and was further upregulated on neutrophil activation and during monocyte to macrophage differentiation. When neutrophil calcium transients were measured in response to a panel of 30 different chemokines the results clearly correlated with the chemokine receptor expression profile. For example strong calcium responses were seen in neutrophils following stimulation with the CXCR1 and CXCR2 ligands, interleukin (IL-)8, GCP-2 and Gro-beta. These data have implications for the study of the functional responses of leukocytes to external stimuli and will aid in our understanding of general leukocyte biology.

Characterization of the exosporium of Bacillus cereus.

Exosporium components from endospores of Bacillus cereus ATCC 10876 were purified and separated by gel electrophoresis. Several of the proteins for which N-terminal sequences were recovered were found to have homologues in protein databases which have been demonstrated to have enzymic activity in other organisms. Amongst these is a zinc metalloprotease, immune inhibitor A, already described in B. thuringiensis. This has been shown to be present in an active 73 kDa form on the exosporium of B. cereus. Other proteins associated with the exosporium include the molecular chaperone GroEL and a homologue of RocA (1-pyrroline-5-carboxylate dehydrogenase (EC 1.5.1.12)) of B. subtilis. Although these are unlikely to represent integral structural proteins of the exosporium, the observation that they are selectively present in the spore surface layer suggests that this layer may have functional significance.