CXCR1 Regulates Pulmonary Anti-Pseudomonas Host Defense.

Pseudomonas aeruginosa is a key opportunistic pathogen causing disease in cystic fibrosis (CF) and other lung diseases such as chronic obstructive pulmonary disease (COPD). However, the pulmonary host defense mechanisms regulating anti-P. aeruginosa immunity remain incompletely understood. Here we demonstrate, by studying an airway P. aeruginosa infection model, in vivo bioluminescence imaging, neutrophil effector responses and human airway samples, that the chemokine receptor CXCR1 regulates pulmonary host defense against P. aeruginosa. Mechanistically, CXCR1 regulates anti-Pseudomonas neutrophil responses through modulation of reactive oxygen species and interference with Toll-like receptor 5 expression. These studies define CXCR1 as a novel, noncanonical chemokine receptor that regulates pulmonary anti-Pseudomonas host defense with broad implications for CF, COPD and other infectious lung diseases.

Glycaemic control in insulin requiring diabetes patients receiving exclusive enteral tube feeding in an acute hospital setting.

AIMS: Optimising glycaemic control for insulin requiring individuals during enteral feeding is important but difficult. We compare 3 insulin regimens with the aim of improving glucose control and reducing hypoglycaemia. METHODS: Comparison of 3 insulin/feed regimens: (1) A 20 h feed using a 30:70 premixed insulin (2) Three bolus (4 h) feeds combined with short acting analogue insulin and a basal long acting insulin. (3) A 24 h feed combined with a long acting analogue insulin. The study combined a retrospective analysis of regimen (1) with consecutive prospective analyses of (2) and (3). RESULTS: Glucose concentrations were suboptimal with higher values during the feeds (12.6 mmol/L ± 4.4 vs 10.3 ± 4.1 p<0.001). Although there was no overall difference in glucose control between groups there was a reduction in hypoglycaemia during the feed in the bolus group (no hypoglycaemia during intermittent feeds p<0.001). CONCLUSIONS: Glucose concentrations were relatively high overall. Short bolus feeding appears to reduce the frequency of hypoglycaemia. This is of clinical significance for this patient group.

CCR5 deficiency and severe polio infection in the 1984 outbreak in Finland.

CCR5, a leukocyte chemoattractant receptor for chemokines CCL3, CCL4, and CCL5, promotes innate and adaptive immune responses by mediating leukocyte trafficking within lymph nodes and to peripheral tissues and is also known as a co-receptor for HIV cell entry. Homozygous inheritance of a complete loss-of-function mutation in CCR5 (CCR5Δ32/CCR5Δ32) is associated with symptomatic neuroinflammatory disease in humans with West Nile and Tickborne Encephalitis flavivirus infections. This study sought to establish whether CCR5 deficiency could also be a determinant of clinical outcome after infection by poliovirus which results in central nervous system damage in only a small proportion of cases. We analyzed serum samples from seven patients and 79 controls, collected during the 1984-1985 polio outbreak in Finland, where CCR5Δ32 is relatively common in the general population. The results excluded CCR5 deficiency as the sole determinant of severe neurologic disease after poliovirus infection in this population.

An investigation into the current peri-operative nutritional management of oesophageal carcinoma patients in major carcinoma centres in England.

INTRODUCTION: Patients with oesophageal carcinoma are at high risk of malnutrition. The aim of this study was to assess current practice for the nutritional management of patients following surgery for oesophageal carcinoma. PATIENTS AND METHODS: A postal questionnaire was sent to 82 dietetic departments of those hospitals in England identified as major centres for upper gastrointestinal surgery. RESULTS: Of the 66 (80%) responses received, 22 (33%) centres routinely perform pre-operative nutritional screening/assessment on oesophageal carcinoma patients. Centres with dietetic support dedicated to these patients are more likely to perform a pre-operative nutritional assessment (n = 17; 55%) than those without (n = 5; 14%; P < 0.001; chi(2) = 12.17). Pre-operative nutritional support is routinely provided in only 11 (17%) centres with the majority of centres (n = 50; 75%), providing it if patients are considered malnourished only. A total of 47 (70%) centres routinely provide postoperative nutritional support with jejunal feeding being the most commonly chosen route. Dedicated dietetic support is provided at 31 (47%) centres. Those centres with a dedicated dietitian are more likely to provide early postoperative nutritional support (n = 27; 87%) than those without (n = 20; 57%; P = 0.007; chi(2) = 7.195) and more likely to review patients routinely following discharge from hospital (n = 25 [81%] with a dietitian versus n = 17 [49%] without; P = 0.007; chi(2) = 7.2). CONCLUSIONS: The nutritional management of patients following surgery for upper gastrointestinal carcinoma is not uniform with practice varying considerably between centres. Those centres with a dedicated dietitian are more likely to assess patients' nutritional status and provide nutritional support.

Optimizing the dose of intrathecal morphine in older patients undergoing hip arthroplasty.

UNLABELLED: Intrathecal (IT) morphine provides excellent postoperative analgesia but may result in many side effects, including postoperative nausea and vomiting, pruritus, and respiratory depression, particularly at larger doses. Older patients may be at particular risk. The optimal dose of spinal morphine in older patients undergoing hip arthroplasty is not known. We designed this prospective, randomized, controlled, double-blinded study to evaluate the analgesic efficacy and side effect profile of 50-200 microg of IT morphine in older patients undergoing elective hip arthroplasty. Sixty patients older than 65 years undergoing elective hip arthroplasty were enrolled. Patients were randomized to receive spinal anesthesia with 15 mg of bupivacaine and IT morphine in four groups: 1). 0 microg, 2). 50 microg, 3). 100 microg, and 4). 200 microg. IT morphine 100 and 200 microg produced effective pain relief and decreased the postoperative requirement for morphine compared with control. IT morphine 50 microg did not provide effective pain relief. Both 100 and 200 microg of IT morphine provided comparable levels of postoperative analgesia. There were no between-group differences in postoperative nausea and vomiting, sedation, respiratory depression, or urinary retention. Pruritus was significantly more frequent with 200 microg of IT morphine. In conclusion, 100 microg of IT morphine provided the best balance between analgesic efficacy and side effect profile in older patients undergoing hip arthroplasty. IMPLICATIONS: The dosage of intrathecal morphine that provides the best balance between analgesic efficacy and side effect profile in the older patient undergoing hip arthroplasty is not known. This prospective, randomized, controlled, double-blinded clinical trial demonstrates that a dose of 100 microg of intrathecal morphine provides the best balance between efficacy and side effects, compared with doses of 0, 50, and 200 microg of morphine, in this patient population.

The endogenous opioid spinorphin blocks fMet-Leu-Phe-induced neutrophil chemotaxis by acting as a specific antagonist at the N-formylpeptide receptor subtype FPR.

Spinorphin is an endogenous heptapeptide (leucylvalylvalyltyrosylprolyltryptophylthreonine), first isolated from bovine spinal cord, whose sequence matches a conserved region of beta-hemoglobin. Also referred to as LVV-hemorphin-4 and a member of the nonclassical opioid hemorphin family, spinorphin inhibits enkephalin-degrading enzymes and is analgesic. Recently, spinorphin was reported to block neutrophil activation induced by the chemotactic N-formylpeptide N-formylmethionylleucylphenylalanine (fMLF), suggesting a potential role as an endogenous negative regulator of inflammation. Here we use both gain- and loss-of-function genetic tests to identify the specific mechanism of spinorphin action on neutrophils. Spinorphin induced calcium flux in normal mouse neutrophils, but was inactive in neutrophils from mice genetically deficient in the fMLF receptor subtype FPR (N-formylpeptide receptor). Consistent with this, spinorphin induced calcium flux in human embryonic kidney 293 cells transfected with mouse FPR, but had no effect on cells expressing the closely related fMLF receptor subtype FPR2. Despite acting as a calcium-mobilizing agonist at FPR, spinorphin was a weak chemotactic agonist and effectively blocked neutrophil chemotaxis induced by fMLF at concentrations selective for FPR. Spinorphin did not affect mouse neutrophil chemotaxis induced by concentrations of fMLF that selectively activate FPR2. Thus, spinorphin blocks fMLF-induced neutrophil chemotaxis by acting as a specific antagonist at the fMLF receptor subtype FPR.

Suicidal nonfatal impalement injury of the thorax.

We treated an impalement injury of the thorax resulting from a suicide attempt in the form of a road traffic accident. The patient survived and was discharged 5 weeks after his injury. The surgical management of thoracic impalement injuries and the rationale behind a multidisciplinary approach are discussed.

Contrasting evolution of the human leukocyte N-formylpeptide receptor subtypes FPR and FPRL1R.

N-formylpeptides are phagocyte chemoattractants that act by binding to two structurally related receptors, FPR (formylpeptide receptor) and FPRL1R (FPR-like-1 receptor), which are encoded by the human genes FPR1 and FPRL1. Single nucleotide polymorphisms (SNPs) in the FPR coding region have been reported and two have been associated with the disease juvenile periodontitis; however, their frequency and linkage relationships are unknown. Here we systematically analyzed polymorphism in the open reading frames of FPR1 and FPRL1 by direct sequencing of cloned alleles from random blood donors from North America. For FPR1 we detected five non-synonymous SNPs and two synonymous SNPs in a sample of 26 chromosomes one each from 17 Caucasian and nine black random blood donors. Although all five non-synonymous SNPs were common in Caucasians, Blacks, and Asians, notable differences in allele frequency were found for each SNP in the different racial groups, suggesting differential selective pressures. We found that the FPR1 polymorphisms are linked in 15 common haplotypes. No polymorphisms were detected in FPRL1 after sampling 44 chromosomes from 36 random blood donors from the same three racial groups. Thus FPR1 and FPRL1, though they originated from a common gene, appear to have undergone markedly different evolutionary events.

Association between polymorphism in the chemokine receptor CX3CR1 and coronary vascular endothelial dysfunction and atherosclerosis.

Fractalkine, a chemokine expressed by inflamed endothelium, induces leukocyte adhesion and migration via the receptor CX3CR1, and the CX3CR1 polymorphism V249I affects receptor expression and function. Here we show that this polymorphism is an independent risk factor for atherosclerotic coronary artery disease (CAD). Genotyping of the CX3CR1-V249I polymorphism was performed in a cohort of 339 white individuals who underwent cardiac catheterization (n=197 with and n=142 without CAD, respectively). In 203 patients, intracoronary acetylcholine 15 microg/min) and sodium nitroprusside (20 microg/min) were administered to test endothelium-dependent and -independent coronary vascular function, respectively. Change in coronary vascular resistance (DeltaCVR) was measured as an index of microvascular dilation. An association was observed between presence of the CX3CR1 I249 allele and reduced prevalence of CAD, independent of established CAD risk factors (odds ratio=0.54 [95% confidence interval, 0.30 to 0.96], P=0.03). Angiographic severity of CAD was also lower in these subjects (P=0.01). Furthermore, endothelium-dependent vasodilation was greater in these individuals compared with individuals homozygous for the CX3CR1-V249 allele (DeltaCVR during acetylcholine = -46+/-3% versus -36+/-3%, respectively, P=0.02), whereas DeltaCVR with sodium nitroprusside was similar in both groups (-55+/-2% versus -53+/-2%, P=0.45). The association between CX3CR1 genotype and endothelial function was independent of established risk factors and presence of CAD by multivariate analysis (P=0.02). Thus, the CX3CR1 I249 allele is associated with decreased risk of CAD and improved endothelium-dependent vasodilation. This suggests that CX3CR1 may be involved in the pathogenesis of CAD.

A membrane-proximal basic domain and cysteine cluster in the C-terminal tail of CCR5 constitute a bipartite motif critical for cell surface expression.

We examined the structural requirements for cell surface expression, signaling, and human immunodeficiency virus co-receptor activity for the chemokine receptor, CCR5. Serial C-terminal truncation of CCR5 resulted in progressive loss of cell surface expression; mutants truncated at the 317th position and shorter were not detected at the cell surface. Alanine substitution of basic residues in the membrane-proximal domain (residues 314-322) in the context of a full-length C-tail resulted in severe reduction in surface expression. C-terminal truncation that excised the three cysteines in this domain reduced surface expression, but further truncation of upstream basic residue(s) abolished surface expression. Substituting the carboxyl-terminal domain of CXCR4 for that of CCR5 failed to rectify the trafficking defect of the tailless CCR5. In contrast, tailless CXCR4 or a CXCR4 chimera that exchanged the native cytoplasmic domain for that of wild type CCR5 was expressed at the cell surface. Deletion mutants that expressed at the cell surface responded to chemokine stimulation and mediated human immunodeficiency virus entry. Substitution of all serine and threonine residues in the C-terminal tail of CCR5 abolished chemokine-mediated receptor phosphorylation but preserved downstream signaling (Ca(2+) flux), while substitutions of tyrosine residues in the C-tail affected neither phenotype. CCR5 mutants that failed to traffic to the plasma membrane did not exhibit obvious changes in metabolic turnover and were retained in the Golgi or pre-Golgi compartments(s). Thus, the basic domain (-KHIAKRF-) and the cysteine cluster (-CKCC-) in the C-terminal tail of CCR5 function cooperatively for optimal surface expression.

CX3C chemokine mimicry by respiratory syncytial virus G glycoprotein.

Chemokines are chemoattractant proteins that are divided into subfamilies based upon cysteine signature motifs termed C, CC, CXC and CX3C. Chemokines have roles in immunity and inflammation that affect cell trafficking and activation of T cells as well as cells of the innate immune system. We report here CX3C chemokine mimicry for the G glycoprotein of respiratory syncytial virus (RSV) and show binding to CX3CR1--the specific receptor for the CX3C chemokine fractalkine--and induction of leukocyte chemotaxis. We also show that CX3CR1 facilitates RSV infection of cells. Thus, G glycoprotein interaction with CX3CR1 probably plays a key role in the biology of RSV infection.

Synthetic peptide MMK-1 is a highly specific chemotactic agonist for leukocyte FPRL1.

Human phagocytic leukocytes express the seven-transmembrane G-protein-coupled receptors formyl peptide receptor (FPR) and FPR-like 1 (FPRL1). MMK-1, a synthetic peptide derived from a random peptide library, is reported to induce calcium mobilization specifically in human FPRL1 gene-transfected cells. However, its actions on human phagocytic leukocytes remain poorly defined. We found that MMK-1 is a potent chemotactic and calcium-mobilizing agonist for human monocytes, neutrophils, and FPRL1-transfected human embryonic kidney (HEK) 293 cells but is inactive in cells transfected with FPR. MMK-1 also activated HEK 293 cells transfected with FPR2, a mouse counterpart of human FPRL1. Furthermore, MMK-1 increased pertussis toxin-sensitive production of inflammatory cytokines in human monocytes. MMK-1 signaling in human phagocytes was completely desensitized by a well-defined FPRL1 agonist, suggesting that FPRL1 is likely a receptor that mediates the action of MMK-1 in primary cells. Since MMK-1 is one of the most potent FPRL1-specific agonists identified so far, it can serve as a modulator of the host defense and a useful agent for further studying the signaling and function of FPRL1.

Involvement of the receptor for formylated peptides in the in vivo anti-migratory actions of annexin 1 and its mimetics.

An innovative avenue for anti-inflammatory therapy is inhibition of neutrophil extravasation by potentiating the action of endogenous anti-inflammatory mediators. The glucocorticoid-inducible protein annexin 1 and derived peptides are effective in inhibiting neutrophil extravasation. Here we tested the hypothesis that an interaction with the receptor for formylated peptide (FPR), so far reported only in vitro, could be the mechanism for this in vivo action. In a model of mouse peritonitis, FPR antagonists abrogated the anti-migratory effects of peptides Ac2-26 and Ac2-12, with a partial reduction in annexin 1 effects. A similar result was obtained in FPR (knock-out) KO mice. Binding of annexin 1 to circulating leukocytes was reduced (>50%) in FPR KO mice. In vitro, annexin binding to peritoneal macrophages was also markedly reduced in FPR KO mice. Finally, evidence of direct annexin 1 binding to murine FPR was obtained with HEK-293 cells transfected with the receptor. Overall, these results indicate a functional role for FPR in the anti-migratory effect of annexin 1 and derived peptides.

Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor constitutively activates NF-kappa B and induces proinflammatory cytokine and chemokine production via a C-terminal signaling determinant.

Kaposi's sarcoma-associated herpesvirus (KSHV) is believed to be the causative agent of Kaposi's sarcoma (KS), a multicentric growth factor-dependent tumor common in AIDS patients characterized histopathologically by spindle cell proliferation, angiogenesis, and leukocyte infiltration. Recently, open reading frame 74 of KSHV has been implicated as a major viral determinant of KS. Open reading frame 74 encodes KSHV G protein-coupled receptor (GPCR), a constitutively active chemokine receptor that directly transforms NIH 3T3 cells in vitro and induces multifocal KS-like lesions in KSHV-GPCR-transgenic mice. Interestingly, receptor-positive cells are very rare in lesions from these mice, implicating an indirect mechanism of tumorigenesis. In this regard, here we report that expression of KSHV-GPCR in transfected epithelial, monocytic, and T cell lines induced constitutive activation of the immunoregulatory transcription factors AP-1 and NF-kappaB. This was associated with constitutive induction of the proinflammatory NF-kappaB-dependent cytokines IL-1beta, IL-6, and TNF-alpha, and chemokines monocyte chemoattractant protein-1 and IL-8, as well as the AP-1-dependent basic fibroblast growth factor. In addition, IL-2 and IL-4 production was induced in transfected Jurkat T cells. Truncation of the final five amino acids in the cytoplasmic tail of KSHV-GPCR caused complete loss of its transforming and NF-kappaB-inducing activities, without affecting receptor expression or ligand binding. These data suggest that KS results in part from KSHV-GPCR induction of proinflammatory cytokine and growth factor gene expression, mediated by a signaling determinant within the last five amino acids of the C terminus, a domain that is also critical for direct cell transformation.

Amyloid-beta induces chemotaxis and oxidant stress by acting at formylpeptide receptor 2, a G protein-coupled receptor expressed in phagocytes and brain.

Amyloid-beta, the pathologic protein in Alzheimer's disease, induces chemotaxis and production of reactive oxygen species in phagocytic cells, but mechanisms have not been fully defined. Here we provide three lines of evidence that the phagocyte G protein-coupled receptor (N-formylpeptide receptor 2 (FPR2)) mediates these amyloid-beta-dependent functions in phagocytic cells. First, transfection of FPR2, but not related receptors, including the other known N-formylpeptide receptor FPR, reconstituted amyloid-beta-dependent chemotaxis and calcium flux in HEK 293 cells. Second, amyloid-beta induced both calcium flux and chemotaxis in mouse neutrophils (which express endogenous FPR2) with similar potency as in FPR2-transfected HEK 293 cells. This activity could be specifically desensitized in both cell types by preincubation with a specific FPR2 agonist, which desensitizes the receptor, or with pertussis toxin, which uncouples it from G(i)-dependent signaling. Third, specific and reciprocal desensitization of superoxide production was observed when N-formylpeptides and amyloid-beta were used to sequentially stimulate neutrophils from FPR -/- mice, which express FPR2 normally. Potential biological relevance of these results to the neuroinflammation associated with Alzheimer's disease was suggested by two additional findings: first, FPR2 mRNA could be detected by PCR in mouse brain; second, induction of FPR2 expression correlated with induction of calcium flux and chemotaxis by amyloid-beta in the mouse microglial cell line N9. Further, in sequential stimulation experiments with N9 cells, N-formylpeptides and amyloid-beta were able to reciprocally cross-desensitize each other. Amyloid-beta was also a specific agonist at the human counterpart of FPR2, the FPR-like 1 receptor. These results suggest a unified signaling mechanism for linking amyloid-beta to phagocyte chemotaxis and oxidant stress in the brain.

Cloning, mRNA distribution, and functional expression of an avian counterpart of the chemokine receptor/HIV coreceptor CXCR4.

The chemokine signaling system, which coordinates the basal and emergency trafficking of leukocytes, presumably coevolved with the hematopoietic system. To study its phylogenetic origins, we used the open reading frame (ORF) of the human chemokine receptor CXCR4 as a genomic probe, since in mammals it is the most highly conserved chemokine receptor known. CXCR4 cross-hybridized to genomic DNA from mouse and chicken, but not zebrafish, Drosophila, or Caenorhabditis elegans. Accordingly, we cloned the corresponding chicken cDNA. The ORF is 359 codons long versus 352 for human CXCR4, and encodes a protein 82% identical to human CXCR4. In a calcium flux assay of receptor function, CHO-K1 cells stably transfected with the chicken cDNA responded specifically to human SDF-1, the specific ligand for CXCR4, but not to a panel of other chemokines tested at 100 nM. SDF-1 activated the cells in a dose-dependent manner (EC50 approximately 5 nM), whereas parental CHO-K1 cells did not respond. The CHO-K1 cell transfectants also bound 125I-SDF-1 specifically. Leukocytes from chicken peripheral blood expressed chCXCR4 mRNA and responded to human SDF-1 in a calcium flux assay with an EC50 similar to that for chCXCR4-transfected CHO cells, suggesting that this response is mediated by native chCXCR4. Analysis of chicken genomic DNA with the chicken cDNA as probe revealed a pattern consistent with a single copy gene, and the absence of any closely related genes. mRNA was detected in brain, bursa, liver, small and large intestine, embryonal fibroblasts, and blood leukocytes, but not in stomach or pancreas. These results, which identify the first functional non-viral, non-mammalian chemokine receptor, suggest that the origins of a functional chemokine system extend at least to birds and suggest that, as in mammals, CXCR4 functions in many avian tissues.

Polymorphism in the fractalkine receptor CX3CR1 as a genetic risk factor for coronary artery disease.

Coronary atherosclerosis is a major cause of death in industrialized countries. Monocytes, which play a key role in atherosclerosis, migrate into the vessel wall, presumably guided by specific chemoattractant and adhesion molecules. A compelling candidate for this role is the chemokine receptor CX3CR1, which is expressed on monocytes and acts as either a chemotactic receptor or an adhesion molecule, depending on whether its ligand, fractalkine, is presented free or membrane bound. A common variant of CX3CR1 was recently identified, encoded by the alleles I249 and M280, which form a common I(249)M(280) haplotype. When CX3CR1 genotypes were analyzed in 151 patients with acute coronary syndromes and in 249 healthy controls, CX3CR1 I249 heterozygosity was associated with a markedly reduced risk of acute coronary events, independent of established acquired coronary risk factors (eg, smoking, diabetes). The adjusted odds ratio for this allele was 0.43 (95% confidence interval, 0.26-0.72; P =.001). Consistent with this, functional analysis of peripheral blood mononuclear cells showed that CX3CR1 I249 heterozygosity was associated with a significant decrease in the number of fractalkine binding sites per cell. The results show that CX3CR1 I249 is an independent genetic risk factor for coronary artery disease and that CX3CR1 may be involved in the pathogenesis of atherosclerotic disease. (Blood. 2001;97:1925-1928)

Coreceptor choice and T cell depletion by R5, X4, and R5X4 HIV-1 variants in CCR5-deficient (CCR5delta32) and normal human lymphoid tissue.

Coreceptor utilization by HIV-1 is an important determinant of pathogenesis. However, coreceptor selectivity is defined in vitro, while in vivo critical pathogenic events occur in lymphoid tissues. Using pharmacological inhibitors, we recently provided evidence that coreceptor selectivity by the R5X4 dual-tropic isolate 89.6 was more restricted in ex vivo infected lymphoid tissue than in vitro [S. Glushakova, Y. Yi, J. C. Grivel, A. Singh, D. Schols, E. De Clercq, R. G. Collman, and L. Margolis (1999). J. Clin. Invest. 104, R7-R11]. Here we extend those observations using CCR5-deficient (CCR5Delta32) lymphoid tissue as well as additional primary isolates. We definitively show that neither CCR5 nor secondary coreceptors used in vitro mediate 89.6 infection in lymphoid tissue. We also demonstrate that restricted coreceptor use in lymphoid tissue ex vivo compared with in vitro utilization occurs with other dual-tropic primary isolates and is not unique to 89.6. For all strains tested that are dual tropic in vitro, severe CD4 T cell depletion in lymphoid tissue correlated with preferential CXCR4 use in this ex vivo system.