A new energy spectrum reconstruction method for time-of-flight diagnostics of high-energy laser-driven protons.

The Time-of-Flight (TOF) technique coupled with semiconductorlike detectors, as silicon carbide and diamond, is one of the most promising diagnostic methods for high-energy, high repetition rate, laser-accelerated ions allowing a full on-line beam spectral characterization. A new analysis method for reconstructing the energy spectrum of high-energy laser-driven ion beams from TOF signals is hereby presented and discussed. The proposed method takes into account the detector's working principle, through the accurate calculation of the energy loss in the detector active layer, using Monte Carlo simulations. The analysis method was validated against well-established diagnostics, such as the Thomson parabola spectrometer, during an experimental campaign carried out at the Rutherford Appleton Laboratory (UK) with the high-energy laser-driven protons accelerated by the VULCAN Petawatt laser.

Transversal dose distribution optimization for laser-accelerated proton beam medical applications by means of Geant4.

The main purpose of this paper is to quantitatively study the possibility of delivering dose distributions of clinical relevance with laser-driven proton beams. A Monte Carlo application has been developed with the Geant4 toolkit, simulating the ELIMED (MEDical and multidisciplinary application at ELI-Beamlines) transport and dosimetry beam line which is being currently installed at the ELI-Beamlines in Prague (CZ). The beam line will be used to perform irradiations for multidisciplinary studies, with the purpose of demonstrating the possible use of optically accelerated ion beams for therapeutic purposes. The ELIMED Geant4-based application, already validated against reference transport codes, accurately simulates each single element of the beam line, necessary to collect the accelerated beams and to select them in energy. Transversal dose distributions at the irradiation point have been studied and optimized to try to quantitatively answer the question if such kind of beam lines, and specifically the systems developed for ELIMED in Prague, will be actually able to transport ion beams not only for multidisciplinary applications, such as pitcher-catcher nuclear reactions (e.g. neutrons), PIXE analysis for cultural heritage and space radiation, but also for delivering dose patterns of clinical relevance in a future perspective of possible medical applications.

Myoepithelial carcinoma of parotid gland: a case report.

Myoepithelial carcinoma or malignant myoepithelioma are considered extremely rare malignant salivary gland neoplasms. Their clinical behaviour by literature seems variable: most studies reported good prognosis for low-grade malignancy and vice versa. Nevertheless long term survival has been described in patients with high-grade tumours, while patients with low-grade have been reported to develop metastases. We report a case of myoepithelial carcinoma of the parotid gland arising in pleomorphic adenoma in a 52-year-old woman with favourable course in long term follow-up.

The CC chemokine eotaxin (CCL11) is a partial agonist of CC chemokine receptor 2b.

Despite sharing considerable homology with the members of the monocyte chemoattractant protein (MCP) family, the CC chemokine eotaxin (CCL11) has previously been reported to signal exclusively via the receptor CC chemokine receptor 3 (CCR3). Using the monocyte cell line THP-1, we investigated the relative abilities of eotaxin and MCPs 1-4 to induce CCR2 signaling, employing assays of directed cell migration and intracellular calcium flux. Surprisingly, 1 microm concentrations of eotaxin were able to recruit THP-1 cells in chemotaxis assays, and this migration was sensitive to antagonism of CCR2 but not CCR3. Radiolabeled eotaxin binding assays performed on transfectants bearing CCR2b or CCR3 confirmed eotaxin binding to CCR2 with a K(d) of 7.50 +/- 3.30 nm, compared with a K(d) of 1.68 +/- 0.91 nm at CCR3. In addition, whereas 1 microm concentrations of eotaxin were able to recruit CCR2b transfectants, substimulatory concentrations of eotaxin inhibited MCP-1-induced chemotaxis of CCR2b transfectants and also inhibited MCP-1-induced intracellular calcium flux of THP-1 cells. Collectively, these findings suggest that eotaxin is a partial agonist of the CCR2b receptor. A greater understanding of the interaction of CCR2 with all of its ligands, both full and partial agonists, may aid the rational design of specific antagonists that hold great promise as future therapeutic treatments for a variety of inflammatory disorders.

Differential expression of chemokine receptors on peripheral blood, synovial fluid, and synovial tissue monocytes/macrophages in rheumatoid arthritis.

OBJECTIVE: Since it is likely that monocytes utilize chemokines to migrate to the rheumatoid arthritis (RA) joint, we investigated the expression of C-C chemokine receptors (CCR) 1-6 and C-X-C receptor 3 (CXCR3) in the peripheral blood (PB), synovial fluid (SF), and synovial tissue of patients with RA as well as in the PB of normal subjects. METHODS: We compared chemokine receptor expression on CD14+ monocytes from normal PB, RA PB, and RA SF using 2-color flow cytometry. Correlations with patient clinical data were determined. Chemokine and receptor expression were investigated in RA synovial tissue by immunohistochemistry and 2-color immunofluorescence to identify CD68+ macrophages. RESULTS: Most normal PB monocytes expressed CCR1 (87%) and CCR2 (84%), but not CCRs 3, 4, 5, or 6 or CXCR3. RA PB monocytes expressed CCR1 (56%) and CCR2 (76%), with significantly more expressing CCR3 (18%), CCR4 (38%), and CCR5 (17%) compared with normal PB monocytes. Significantly fewer SF monocytes from RA patients expressed CCR1 (17%), CCR2 (24%), and CCR4 (6%) while significantly more expressed CCR3 (35%) and CCR5 (47%) compared with RA and normal PB monocytes; CCR6 and CXCR3 were rarely detected. Clinically, the erythrocyte sedimentation rate was inversely correlated with the expression of CCR1 and CCR4 by RA PB, and CCR5 expression by RA SF was correlated with the SF white blood cell count. CCR1-, CCR2-, and CCR5-immunoreactive cells were found in RA synovial tissue and colocalized with CD68+ macrophages. RA synovial tissue RANTES (regulated upon activation, normally T cell expressed and secreted chemokine)- and monocyte chemoattractant protein 1-immunoreactive cells colocalized with CCR1 and CCR2, respectively, on serial sections. Macrophage inflammatory protein 1alpha (MIP-1alpha) was principally restricted to vascular endothelium, and MIP-1beta+ macrophages were found throughout the sections. CONCLUSION: Monocytes mainly express CCR1 and CCR2 in normal and RA PB, CCR3 and CCR5 in RA PB and RA SF, and CCR4 in RA PB. The differential expression of chemokine receptors suggests that certain receptors aid in monocyte recruitment from the circulation while others are important in monocyte retention in the joint.

Malignant bronchial Abrikossoff's tumor and small cell lung cancer: a case report and review.

A 61-year-old male Caucasian smoker patient underwent chest radiography and CT scan for persistent non-inflammatory cough, which showed a left bronchial unresectable mass. Bronchoscopy showed an endobronchial mass; washing cytology was negative and histology findings suggested diagnosis of granular cell tumor (GCT), also called Abrikossoff's tumor. After 3 weeks a new washing cytology test revealed the presence of small cell lung cancer (SCLC). A CT-scan and chest radiography showed a 30% increase in the maximum diameter of the lesion, clinically defining the primary neoplasm as malignant. The patient was referred to our institution and started chemotherapy with cisplatin and etoposide. After 6 cycles of treatment, the CT scan showed complete, disappearance of the neoplasm and bronchoscopy examination showed no endobronchial lesion, defining the mucosal surface as normal. We have reviewed and summarized the international literature with regard to bronchial localization of malignant granular cell tumor and its association with SCLC, therefore concluding that our case is the first malignant endobronchial GCT linked to SCLC.

Selective lymphocyte chemokine receptor expression in the rheumatoid joint.

OBJECTIVE: In patients with rheumatoid arthritis (RA), chemokines and their receptors are important for lymphocyte trafficking into the inflamed joint. This study was undertaken to characterize the expression of chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CXCR3, and CX3CR1 in normal (NL) peripheral blood (PB), RA PB, and RA synovial fluid (SF). METHODS: Using flow cytometry, immunohistochemistry, and 2-color immunofluorescence, we defined the expression of chemokine receptors on CD3+ T lymphocytes in RA synovial tissue (ST), RA SF, RA PB, and NL PB. RESULTS: The percentage of CD3+ lymphocytes expressing CCR2, CCR4, CCR5, and CX3CR1 was significantly elevated in RA PB compared with that in NL PB, while the percentage of CD3+ lymphocytes expressing CCR5 was significantly enhanced in RA SF compared with that in NL and RA PB. In contrast, similar percentages of CD3+ lymphocytes in NL PB, RA PB, and RA SF expressed CCR6 and CXCR3. Immunohistochemistry of RA ST showed lymphocyte expression of CCR4, and 2-color immunofluorescence staining revealed RA ST CD3+ lymphocytes intensely immunoreactive for CXCR3, suggesting that these 2 receptors may be particularly important for CD3+ lymphocyte trafficking to the inflamed joint. In comparisons of chemokine receptor expression on naive (CD45RA+) and memory (CD45RO+) CD3+ lymphocytes, there were greater percentages of memory CD3+/CD4+ lymphocytes expressing CCR4, CCR5, and CXCR3 than naive CD3+/CD4+ lymphocytes in RA PB and RA SF, and greater percentages of memory CD3+/CD8+ lymphocytes expressing CCR4, CCR5, and CXCR3 than naive CD3+/CD8+ lymphocytes in RA SF, suggesting receptor up-regulation upon lymphocyte activation. In contrast, percentages of CD3+/CD8+ memory lymphocytes expressing CX3CR1 were significantly less than percentages of naive CD3+/CD8+ lymphocytes in RA PB, suggesting that this receptor may be down-regulated upon lymphocyte activation. A major difference between the RA PB and NL PB groups was significantly more CCR4+ memory leukocytes and memory CCR5+/ CD3+/CD8+ lymphocytes in RA PB than NL PB, further suggesting that these receptors may be particularly important for lymphocyte homing to the RA joint. CONCLUSION: These results identify CCR4, CCR5, CXCR3, and CX3CR1 as critical chemokine receptors in RA.

Basophil responses to chemokines are regulated by both sequential and cooperative receptor signaling.

To investigate human basophil responses to chemokines, we have developed a sensitive assay that uses flow cytometry to measure leukocyte shape change as a marker of cell responsiveness. PBMC were isolated from the blood of volunteers. Basophils were identified as a single population of cells that stained positive for IL-3Ralpha (CDw123) and negative for HLA-DR, and their increase in forward scatter (as a result of cell shape change) in response to chemokines was measured. Shape change responses of basophils to chemokines were highly reproducible, with a rank order of potency: monocyte chemoattractant protein (MCP) 4 (peak at <1 nM) >/= eotaxin-2 = eotaxin-3 >/= eotaxin > MCP-1 = MCP-3 > macrophage-inflammatory protein-1alpha > RANTES = MCP-2 = IL-8. The CCR4-selective ligand macrophage-derived chemokine did not elicit a response at concentrations up to 10 nM. Blocking mAbs to CCR2 and CCR3 demonstrated that responses to higher concentrations (>10 nM) of MCP-1 were mediated by CCR3 rather than CCR2, whereas MCP-4 exhibited a biphasic response consistent with sequential activation of CCR3 at lower concentrations and CCR2 at 10 nM MCP-4 and above. In contrast, responses to MCP-3 were blocked only in the presence of both mAbs, but not after pretreatment with either anti-CCR2 or anti-CCR3 mAb alone. These patterns of receptor usage were different from those seen for eosinophils and monocytes. We suggest that cooperation between CCRs might be a mechanism for preferential recruitment of basophils, as occurs in tissue hypersensitivity responses in vivo.

Functional differences between monocyte chemotactic protein-1 receptor A and monocyte chemotactic protein-1 receptor B expressed in a Jurkat T cell.

The monocyte chemotactic protein-1 (MCP-1) receptor (MCP-1R) is expressed on monocytes, a subpopulation of memory T lymphocytes, and basophils. Two alternatively spliced forms of MCP-1R, CCR2A and CCR2B, exist and differ only in their carboxyl-terminal tails. To determine whether CCR2A and CCR2B receptors function similarly, Jurkat T cells were stably transfected with plasmids encoding the human CCR2A or CCR2B gene. Nanomolar concentrations of MCP-1 induced chemotaxis in the CCR2B transfectants that express high, intermediate, and low levels of MCP-1R. Peak chemotactic activity was shifted to the right as receptor number decreased. Five-fold more MCP-1 was required to initiate chemotaxis of the CCR2A low transfectant, but the peak of chemotaxis was similar for the CCR2A and CCR2B transfectants expressing similar numbers of receptors. MCP-1-induced chemotaxis was sensitive to pertussis toxin, implying that both CCR2A and CCR2B are G(i)alpha protein coupled. MCP-1 induced a transient Ca(2+) flux in the CCR2B transfectant that was partially sensitive to pertussis toxin. In contrast, MCP-1 did not induce Ca(2+) flux in the CCR2A transfectant. Since MCP-1 can stimulate chemotaxis of the CCR2A transfectant without inducing Ca(2+) mobilization, Ca(2+) flux may not be required for MCP-1-induced chemotaxis in the Jurkat transfectants. These results indicate that functional differences exist between the CCR2A and CCR2B transfectants that can be attributed solely to differences in the carboxyl-terminal tail.

Defective expression of the monocyte chemotactic protein-1 receptor CCR2 in macrophages associated with human ovarian carcinoma.

Monocyte chemotactic protein-1 (MCP-1, CCL2) is an important determinant of macrophage infiltration in tumors, ovarian carcinoma in particular. MCP-1 binds the chemokine receptor CCR2. Recent results indicate that proinflammatory and anti-inflammatory signals regulate chemokine receptor expression in monocytes. The present study was designed to investigate the expression of CCR2 in tumor-associated macrophages (TAM) from ovarian cancer patients. TAM isolated from ascitic or solid ovarian carcinoma displayed defective CCR2 mRNA (Northern blot and PCR) and surface expression and did not migrate in response to MCP-1. The defect was selective for CCR2 in that CCR1 and CCR5 were expressed normally in TAM. CCR2 gene expression and chemotactic response to MCP-1 were decreased to a lesser extent in blood monocytes from cancer patients. CCR2 mRNA levels and the chemotactic response to MCP-1 were drastically reduced in fresh monocytes cultured in the presence of tumor ascites from cancer patients. Ab against TNF-alpha restored the CCR2 mRNA level in monocytes cultured in the presence of ascitic fluid. The finding of defective CCR2 expression in TAM, largely dependent on local TNF production, is consistent with previous in vitro data on down-regulation of chemokine receptors by proinflammatory molecules. Receptor inhibition may serve as a mechanism to arrest and retain recruited macrophages and to prevent chemokine scavenging by mononuclear phagocytes at sites of inflammation and tumor growth. In the presence of advanced tumors or chronic inflammation, systemic down-regulation of receptor expression by proinflammatory molecules leaking in the systemic circulation may account for defective chemotaxis and a defective capacity to mount inflammatory responses associated with advanced neoplasia.

Human G protein-coupled receptor GPR-9-6/CC chemokine receptor 9 is selectively expressed on intestinal homing T lymphocytes, mucosal lymphocytes, and thymocytes and is required for thymus-expressed chemokine-mediated chemotaxis.

TECK (thymus-expressed chemokine), a recently described CC chemokine expressed in thymus and small intestine, was found to mediate chemotaxis of human G protein-coupled receptor GPR-9-6/L1.2 transfectants. This activity was blocked by anti-GPR-9-6 monoclonal antibody (mAb) 3C3. GPR-9-6 is expressed on a subset of memory alpha4beta7(high) intestinal trafficking CD4 and CD8 lymphocytes. In addition, all intestinal lamina propria and intraepithelial lymphocytes express GPR-9-6. In contrast, GPR-9-6 is not displayed on cutaneous lymphocyte antigen-positive (CLA(+)) memory CD4 and CD8 lymphocytes, which traffic to skin inflammatory sites, or on other systemic alpha4beta7(-)CLA(-) memory CD4/CD8 lymphocytes. The majority of thymocytes also express GPR-9-6, but natural killer cells, monocytes, eosinophils, basophils, and neutrophils are GPR-9-6 negative. Transcripts of GPR-9-6 and TECK are present in both small intestine and thymus. Importantly, the expression profile of GPR-9-6 correlates with migration to TECK of blood T lymphocytes and thymocytes. As migration of these cells is blocked by anti-GPR-9-6 mAb 3C3, we conclude that GPR-9-6 is the principal chemokine receptor for TECK. In agreement with the nomenclature rules for chemokine receptors, we propose the designation CCR-9 for GPR-9-6. The selective expression of TECK and GPR-9-6 in thymus and small intestine implies a dual role for GPR-9-6/CCR-9, both in T cell development and the mucosal immune response.

Up-regulation of CCR2 chemokine receptor expression and increased susceptibility to the multitropic HIV strain 89.6 in monocytes exposed to glucocorticoid hormones.

Glucocorticoid hormones (GC) are potent antiinflammatory agents widely used in the treatment of diverse human diseases. The present study was aimed at assessing the effect of GC on chemokine receptor expression in human monocytes. Dexamethasone (Dex) up-regulated mRNA expression of the monocyte chemotactic protein (MCP-1, CCL2) chemokine receptor CCR2. The effect was selective in that other chemokine receptors were not substantially affected. Stimulation by Dex was observed after 4 h of exposure at concentrations of 10(-7) to 10(-5) M. Steroids devoid of GC activity were inactive, and the GC receptor antagonist, RU486, inhibited stimulation. Dex did not affect the rate of nuclear transcription, but augmented the CCR2 mRNA half-life. Augmentation of CCR2 expression by Dex was associated with increased chemotaxis. Finally, Dex treatment induced productive replication of the HIV strain 89.6, which utilizes CCR2 as entry coreceptor, in freshly isolated monocytes. Together with previous findings, these results indicate that at least certain pro- and antiinflammatory molecules have reciprocal and divergent effects on expression of a major monocyte chemoattractant, MCP-1, and of its receptor (CCR2). Augmentation of monocyte CCR2 expression may underlie unexplained in vivo effects of GC as well as some of their actions on HIV infection.

[Elastic intramedullary nailing of the tibia with the Marchetti-Vicenzi nail. 43 treated cases]. / L'enclouage élastique du tibia à foyer fermé par clou de Marchetti-Vicenzi. A propos de 43 cas traités.

PURPOSE OF THE STUDY: The purpose of this study was to analyse the results of tibial intramedullary nailing using an unreamed "Universal Elastic Bundle Nail". MATERIAL AND METHODS: Forty-three intramedullary nailing of tibial shaft were done in 43 patients with recents fractures, from May 1993 and May 1996. There were 36 males and 7 females. The average age was 31.5 years (range 17-68 years). Thirty-three were injured in a traffic accident (20 motorcycles, 5 pedestrians and 8 car passengers), seven were injured in a home accident (fall) and three had a sport injury. There were 13 open fractures according to Gustilo: 5 grade I, 7 grade II and one grade III B. Eight fractures involved the proximal metaphyseal part of the tibia, 16 the distal metaphyseal part and 14 the tibial shaft; in five cases there were segmental fractures. According to AO classification there were: 10 fractures type A, 24 fractures type B and 9 fractures type C (5 segmental fractures). In 5 cases there were associated femoral fractures: three ipsilaterals and two controlaterals. All were treated in the same time: four by UEBN device and one by AO's nail. All the patients with type B and C fractures were positioned on a Maquet table with a boot traction or transcalcaneal pin traction (in the distal fractures). The nail was introduced after closed reduction through a vertical transpatellar tendon incision, without reaming procedure. RESULTS: Forty one fractures healed after an average time of 96 days (60-120). In 11 open fractures bone union occurred after 98 days (85-120). The distal fractures healed after a mean time of 86 days (60-120), proximal fractures in 123 days and mid shaft fractures in 98 days. In type A fractures bone union occurred after an average time of 68 days, while bone union occurred after a mean time of 100 days in type B and C fractures. Two patients with an open proximal type B fracture, had a delayed union: both healed after proximal screws removal. Two fractures healed with a valgus angulaton 5 degrees and 10 degrees. No infection, no loss of reduction and no bundle migration has been noted. DISCUSSION: The Marchetti-Vicenzi's nail (UEBN) permitted a stable fixation in tibial fractures. The use of this unreamed nailing coupled with an automatic distal locking in the metaphyseal cancellous bone, reduced operative time and shortened X Ray's radiation exposure. At the follow-up fracture healing occurred in 41 cases 95.3 p. 100 at four months. Two delayed union occurred after four months, the two cases were open fractures grade II. All the two cases healed after secondary procedure without any loss of function. Malunion occurred in two patients (in only one case there was a major valgus angulation 10 degrees), the two cases were related to technical error. We had no cases of infection or leg shortening or bundle migration in the ankle joint. CONCLUSION: We believe that Universal Elastic Bundle Nail allows a stable and safety fixation in open or closed tibial fractures without pseudarthrosis and without infection (in our series).

CCR2-64I polymorphism is not associated with altered CCR5 expression or coreceptor function.

A polymorphism in the gene encoding CCR2 is associated with a delay in progression to AIDS in human immunodeficiency virus (HIV)-infected individuals. The polymorphism, CCR2-64I, changes valine 64 of CCR2 to isoleucine. However, it is not clear whether the effect on AIDS progression results from the amino acid change or whether the polymorphism marks a genetically linked, yet unidentified mutation that mediates the effect. Because the gene encoding CCR5, the major coreceptor for HIV type 1 primary isolates, lies 15 kb 3' to CCR2, linked mutations in the CCR5 promoter or other regulatory sequences could explain the association of CCR2-64I with slowed AIDS pathogenesis. Here, we show that CCR2-64I is efficiently expressed on the cell surface but does not have dominant negative activity on CCR5 coreceptor function. A panel of peripheral blood mononuclear cells (PBMC) from uninfected donors representing the various CCR5/CCR2 genotypes was assembled. Activated primary CD4(+) T cells of CCR2 64I/64I donors expressed cell surface CCR5 at levels comparable to those of CCR2 +/+ donors. A slight reduction in CCR5 expression was noted, although this was not statistically significant. CCR5 and CCR2 mRNA levels were nearly identical for each of the donor PBMC, regardless of genotype. Cell surface CCR5 and CCR2 levels were more variable than mRNA transcript levels, suggesting that an alternative mechanism may influence CCR5 cell surface levels. CCR2-64I is linked to the CCR5 promoter polymorphisms 208G, 303A, 627C, and 676A; however, in transfected promoter reporter constructs, these did not affect transcriptional activity. Taken together, these findings suggest that CCR2-64I does not act by influencing CCR5 transcription or mRNA levels.

Characterization of genes which exhibit reduced expression during the retinoic acid-induced differentiation of F9 teratocarcinoma cells: involvement of cyclin D3 in RA-mediated growth arrest.

In the presence of retinoic acid (RA), F9 murine teratocarcinoma cells differentiate into cells resembling the extra-embryonic endoderm of the early mouse embryo. Using differential hybridization, we have cloned and characterized six cDNAs corresponding to mRNAs that exhibit reduced expression in F9 cells following RA treatment. Two of these cDNAs encode novel genes (REX-2 and REX-3). The other isolated cDNAs encode genes that have been previously described in other contexts: 1-4 (cyclin D3); 2-10 (pyruvate kinase); 2-12 (glutathione S-transferase); and 2-17 (GLUT 3). The mRNA levels of these genes are reduced by RA or RA plus theophylline and cAMP (RACT) only after 48 h of treatment, and continue to decrease at 96 h. The half-lives of these mRNAs are not changed by RA treatment, indicating that these mRNAs may be regulated through a transcriptional mechanism. In isoleucine-deprived cells, which are growth arrested but do not differentiate, the steady state mRNA levels of genes Rex 2, Rex 3, pyruvate kinase and GLUT 3 are not reduced, in contrast to cyclin D3 and glutathione S-transferase. The expression of the REX-2, REX-3, pyruvate kinase, glutathione S-transferase and GLUT 3 genes is reduced by RACT to the same extent in F9 RARgamma-/- and RARalpha-/- lines as in F9-Wt. In contrast, cyclin D3 exhibits lower mRNA expression in F9 RARgamma-/- and RARalpha-/- stem cells, and this mRNA is not decreased by RACT treatment. Overexpression of cyclin D3 blocks the RA-induced growth arrest of F9 cells, indicating that the downregulation of this gene following RA treatment may constitute a necessary step in the cascade of events leading to growth inhibition by RA.

The chemokine receptor CXCR3 mediates rapid and shear-resistant adhesion-induction of effector T lymphocytes by the chemokines IP10 and Mig.

Integrin-mediated adhesion to the vascular endothelium is an essential step in leukocyte diapedesis. We show that the chemokines 10-kDa inflammatory protein (IP10) and monokine induced by IFN (Mig) induce rapid and transient adhesion of human IL-2-stimulated T lymphocytes (IL-2 T cells) to immobilized integrin ligands through their receptor CXCR3, which is selectively expressed on activated T cells. Induction of adhesion by IP10 and Mig was already observed at subnanomolar concentrations and was maximal at 5-10 nM, resulting in three- to sixfold increase in adhesion of IL-2 T cells over background. No effect was seen with resting naive/memory T cells which lack CXCR3 and migration responses to IP10 and Mig. Both chemokines are produced in human umbilical vein endothelial cells (HUVEC) upon stimulation with IFN-gamma and TNF-alpha. These chemokines induce IL-2 T cell adhesion also when captured on the surface of endothelial cells. Under conditions of flow, IL-2 T cells roll and rapidly adhere to IP10/Mig-expressing HUVEC, and anti-CXCR3 mAb treatment reduces arrest and firm adhesion. This is the first study that shows chemokine-induced adhesion in activated memory/effector T cells which represent the fraction of T cells that are selectively mobilized in inflammation. The critical role of IFN-gamma as inducer of IP10/Mig production in HUVEC indicates that these chemokines are essential mediators of effector T cell recruitment to IFN-gamma-dependent pathologies.

Interaction of chemokine receptor CCR5 with its ligands: multiple domains for HIV-1 gp120 binding and a single domain for chemokine binding.

CCR5 is a chemokine receptor expressed by T cells and macrophages, which also functions as the principal coreceptor for macrophage (M)-tropic strains of HIV-1. To understand the molecular basis of the binding of chemokines and HIV-1 to CCR5, we developed a number of mAbs that inhibit the various interactions of CCR5, and mapped the binding sites of these mAbs using a panel of CCR5/CCR2b chimeras. One mAb termed 2D7 completely blocked the binding and chemotaxis of the three natural chemokine ligands of CCR5, RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta, to CCR5 transfectants. This mAb was a genuine antagonist of CCR5, since it failed to stimulate an increase in intracellular calcium concentration in the CCR5 transfectants, but blocked calcium responses elicited by RANTES, MIP-1alpha, or MIP-1beta. This mAb inhibited most of the RANTES and MIP-1alpha chemotactic responses of activated T cells, but not of monocytes, suggesting differential usage of chemokine receptors by these two cell types. The 2D7 binding site mapped to the second extracellular loop of CCR5, whereas a group of mAbs that failed to block chemokine binding all mapped to the NH2-terminal region of CCR5. Efficient inhibition of an M-tropic HIV-1-derived envelope glycoprotein gp120 binding to CCR5 could be achieved with mAbs recognizing either the second extracellular loop or the NH2-terminal region, although the former showed superior inhibition. Additionally, 2D7 efficiently blocked the infectivity of several M-tropic and dual-tropic HIV-1 strains in vitro. These results suggest a complicated pattern of HIV-1 gp120 binding to different regions of CCR5, but a relatively simple pattern for chemokine binding. We conclude that the second extracellular loop of CCR5 is an ideal target site for the development of inhibitors of either chemokine or HIV-1 binding to CCR5.

The second extracellular loop of CCR5 is the major determinant of ligand specificity.

The chemokine receptor CCR5 binds macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and regulated on activation, normal T-cell expressed and secreted (RANTES), and constitutes the major co-receptor allowing infection of CD4(+) T lymphocytes, macrophages, and microglial cells by macrophage-tropic strains of human and simian immunodeficiency virus. CCR5 is most closely related to CCR2b, another chemokine receptor that responds to monocyte chemoattractant protein (MCP)-1, MCP-2, MCP-3, and MCP-4. We have investigated by mutagenesis the regions of CCR5 and CCR2b involved in the specificity of binding and functional response to their respective ligands. We demonstrate that the key region of CCR5 involved in its specific interaction with MIP-1alpha, MIP-1beta, and RANTES, and its subsequent activation, lies within the second extracellular loop (and possibly the adjacent transmembrane segments). Conversely, the NH2-terminal domain of CCR2b is responsible for the high affinity binding of MCP-1, but is not sufficient to confer activation of the intracellular cascades. Extracellular loops of the receptor, among which the second loop plays a prominent role, are necessary to achieve efficient signaling of the receptor. These data complement our previous mapping of CCR5 domains functionally involved in the fusion process with the human immunodeficiency virus envelope, and will help in the development of agents able to interfere with the early steps of viral infection.

High expression of the chemokine receptor CCR3 in human blood basophils. Role in activation by eotaxin, MCP-4, and other chemokines.

Eosinophil leukocytes express high numbers of the chemokine receptor CCR3 which binds eotaxin, monocyte chemotactic protein (MCP)-4, and some other CC chemokines. In this paper we show that CCR3 is also highly expressed on human blood basophils, as indicated by Northern blotting and flow cytometry, and mediates mainly chemotaxis. Eotaxin and MCP-4 elicited basophil migration in vitro with similar efficacy as regulated upon activation normal T cells expressed and secreted (RANTES) and MCP-3. They also induced the release of histamine and leukotrienes in IL-3-primed basophils, but their efficacy was lower than that of MCP-1 and MCP-3, which were the most potent stimuli of exocytosis. Pretreatment of the basophils with a CCR3-blocking antibody abrogated the migration induced by eotaxin, RANTES, and by low to optimal concentrations of MCP-4, but decreased only minimally the response to MCP-3. The CCR3-blocking antibody also affected exocytosis: it abrogated histamine and leukotriene release induced by eotaxin, and partially inhibited the response to RANTES and MCP-4. In contrast, the antibody did not affect the responses induced by MCP-1, MCP-3, and macrophage inflammatory protein-1alpha, which may depend on CCR1 and CCR2, two additional receptors detected by Northern blotting with basophil RNA. This study demonstrates that CCR3 is the major receptor for eotaxin, RANTES, and MCP-4 in human basophils, and suggests that basophils and eosinophils, which are the characteristic effector cells of allergic inflammation, depend largely on CCR3 for migration towards different chemokines into inflamed tissues.

Induction of monocyte chemoattractant protein-1 in the small veins of the ischemic and reperfused canine myocardium.

BACKGROUND: Healing after myocardial infarction is characterized by the presence of macrophages in the infarcted area. Since augmented monocyte influx has been implicated as a potential mechanism for improved healing after reperfusion, we wished to study the induction of monocyte chemoattractant protein-1 (MCP-1) during reperfusion. METHODS AND RESULTS: The cDNA for MCP-1 was cloned from a canine jugular vein endothelial cell (CJVEC) library and exhibited 78% identity with the deduced amino acid sequence of human MCP-1. Samples of myocardium were taken from control and ischemic segments after 1 hour of ischemia and various times of reperfusion; total RNA was isolated from myocardial samples and probed with a cDNA probe for canine MCP-1. Induction of MCP-1 mRNA occurred only in previously ischemic segments within the first hour of reperfusion, peaked at 3 hours, and persisted throughout the first 2 days of reperfusion. In the absence of reperfusion, no significant MCP-1 induction was seen. Both ischemic (but not preischemic) cardiac lymph and human recombinant TNF-alpha induced MCP-1 in CJVECs. MCP-1 was identified by immunostaining on infiltrating cells and venular (but not arterial) endothelium by 3 hours. In contrast, in situ hybridization showed MCP-1 mRNA to be confined to the endothelium of small veins (venules) 10 to 70 microns in diameter. CONCLUSIONS: MCP-1 mRNA is induced in the endothelium of a specific class of small veins immediately after reperfusion. MCP-1 induction is confined to the previously ischemic area that has been reperfused. We suggest a significant role for MCP-1 in monocyte trafficking in the reperfused myocardium.