Homophilic PECAM-1(CD31) interactions prevent endothelial cell apoptosis but do not support cell spreading or migration.

PECAM-1 (CD31) is a highly abundant cell surface glycoprotein expressed on haemopoietic and endothelial cells. As well as mediating homophilic (PECAM-1/PECAM-1) adhesion, PECAM-1 can also bind the integrin alphavbeta3. Both PECAM-1 and alphavbeta3 have been shown to have roles in regulating angiogenesis, endothelial tube formation and in the case of alphavbeta3, endothelial cell apoptosis. In this study we show that despite being expressed at equivalent levels, endothelial alphavbeta3 is not a ligand for PECAM-1. Rather, PECAM-1 supports homophilic binding on HUVEC with similar characteristics to those we have previously reported for leukocytes and becomes tyrosine phosphorylated after homophilic PECAM-1 and integrin/fibronectin engagement. Immunoprecipitation studies show that in addition to SHP-2, tyrosine phosphorylated PECAM-1 can interact with at least four other phosphoproteins in pervanadate stimulated HUVEC. While PECAM-1/PECAM-1 interactions support robust endothelial cell adhesion, they do not support cell spreading or migration. In addition PECAM-1 homophilic adhesion rescues HUVEC from serum deprivation-induced apoptosis. Taken together our results indicate that PECAM-1 homophilic interactions play an important role in interendothelial cell adhesion, survival and signalling.

Beagle 2: a proposed exobiology lander for ESA's 2003 Mars Express mission.

The aim of the proposed Beagle 2 small lander for ESA's 2003 Mars Express mission is to search for organic material on and below the surface of Mars and to study the inorganic chemistry and mineralogy of the landing site. The lander will have a total mass of 60kg including entry, descent, and landing system. Experiments will be deployed on the surface using a robotic arm. It will use a mechanical mole and grinder to obtain samples from below the surface, under rocks, and inside rocks. Sample analysis by a mass spectrometer will include isotopic analysis. An optical microscope, an X-ray spectrometer and a Mossbauer spectrometer will conduct in-situ rock studies.

Association between receptor density, cellular activation, and transformation of adhesive behavior of flowing lymphocytes binding to VCAM-1.

We investigated the ability of purified vascular cell adhesion molecule-1 (VCAM-1), adsorbed on plastic, to capture and immobilize flowing lymphocytes, and the dependence of adhesive behavior on activation of the counter-receptor, alpha 4 beta 1 integrin. This integrin/immunoglobulin interaction bound lymphocytes at a wall shear stress at which the beta 2-integrin family has previously been found ineffective (> 0.1 Pa), and whereas lymphocytes rolled on lower concentrations of VCAM-1 (10 micrograms/ml), they were stationary at high concentrations (100 micrograms/ml). Activation of alpha 4 beta 1 integrin by Mn2+ or by antibody 12G10 or treatment of lymphocytes with phorbol ester caused transformation to stationary adhesion, and increased binding significantly only at the lower concentrations of VCAM-1. We thus hypothesized that formation of a high density of ligand between VCAM-1 and alpha 4 beta 1 integrin actively transformed lymphocyte behavior. This concept was supported by the finding that the proportion of lymphocytes rolling on the higher concentrations of VCAM-1 increased if cells were pretreated with azide to block energy-dependent responses, or if intracellular Ca2+ was chelated. However, not all activation responses were equivalent: only phorbol ester induced marked spreading of immobilized cells, and if pretreatment was prolonged, this agent even reduced the efficiency of initial attachment of flowing lymphocytes. Azide treatment had no effect on transformation to stationary adhesion caused by Mn2+ or activating antibody. Thus, different forms of lymphocyte activation were identifiable: external modification of integrin converted rolling to stationary attachment, did not require ATP, and was reversible; high-density ligand binding induced an energy-dependent signal for conversion from rolling to stationary attachment, but not spreading; and protein kinase C activation promoted stationary attachment and spreading, but not necessarily capture. VCAM-1 is thus a versatile adhesion receptor capable of supporting all stages of leukocyte attachment, i.e. rolling, stationary, and spreading, and of ligand-induced transformation of adhesion, although an additional signal appears necessary to promote lymphocyte spreading and migration.

The X-Ray CCDs Developed for the Joint European X-Ray Telescope.

The charge coupled devices (CCDs) developed for the Joint European X-ray Telescope (JET-X) are described in detail. A history of the development program and device performance is given. We present results from a comprehensive study to characterize the x-ray response of the flight model focal plane detectors. The goal of the program is to calibrate the efficiency, energy resolution, gain, etc. down to a precision of ~1%. Final calibration data sets will be based on combinations of measurements and calculations. For example, the CCD quantum efficiency will be composed of discrete line measurements made at the University of Leicester test facility and calculation and synchrotron measurements from the Daresbury Synchrotron Radiation Source (SRS). The absolute normalizations will be provided by x-ray long beam pipe measurements at the Max Planck Institut für Extraterrestrische Physik (MPE) Panter test facility in Munich. Using the available data, it is shown that it is possible to calibrate the quantum efficiency, the FWHM energy resolution, and the system gain of the flight devices to better than 1%.

Mapping the homotypic binding sites in CD31 and the role of CD31 adhesion in the formation of interendothelial cell contacts.

CD31 is a member of the immunoglobulin superfamily consisting of six Ig-related domains. It is constitutively expressed by platelets, monocytes, and some lymphocytes, but at tenfold higher levels on vascular endothelial cells. CD31 has both homotypic and heterotypic adhesive properties. We have mapped the homotypic binding sites using a deletion series of CD31-Fc chimeras and a panel of anti-CD31 monoclonal antibodies. An extensive surface of CD31 is involved in homotypic binding with domains 2 and 3 and domains 5 and 6 playing key roles. A model consistent with the experimental data is that CD31 on one cell binds to CD31 on an apposing cell in an antiparallel interdigitating mode requiring full alignment of the six domains of each molecule. In addition to establishing intercellular homotypic contacts. CD31 binding leads to augmented adhesion via beta 1 integrins. The positive cooperation between CD31 and beta 1 integrins can occur in heterologous primate cells (COS cells). The interaction is specific to both CD31 and beta 1 integrins. Neither intercellular adhesion molecule-1 (ICAM-1)/leukocyte function-associated antigen-1 (LCAM-1) nor neural cell adhesion molecule (NCAM)/NCAM adhesion leads to recruitment of beta 1 integrin adhesion pathways. Establishment of CD31 contacts have effects on the growth and morphology of endothelial cells. CD31(D1-D6)Fc inhibits the growth of endothelial cells in culture. In addition, papain fragments of anti-CD31 antibodies (Fab fragments) disrupt interendothelial contact formation and monolayer integrity when intercellular contacts are being formed. The same reagents are without effect once these contacts have been established, suggesting that CD31-CD31 interactions are critically important only in the initial phases of intercellular adhesion.

Recognition of cryptic sites in human and mouse laminins by rat osteoclasts is mediated by beta 3 and beta 1 integrins.

Laminins may be encountered by osteoclasts and their precursors in basement membranes when they migrate from periosteal vasculature during skeletal development and in pathological situations. We have examined the recognition by osteoclasts of intact laminins and their proteolytic derivatives, and analysed the mechanism of adhesion. Rat osteoclasts fail to bind intact mouse Engelbreth-Holm-Swarm (EHS) laminin (3% adhesion relative to adhesion to foetal calf serum proteins) and bind only weakly to native human placental laminin (13%) or human merosin (9%). Pepsin treatment of native mouse EHS and human laminins increased osteoclast adhesion. Rat osteoclasts adhered to mouse EHS laminin-derived P1 fragment (70%), but failed to bind the E8 fragment, which contains adhesion sites recognised by some integrins. Binding to human and mouse P1 laminins was abolished by treatment with RGD-containing peptides and required divalent cations, but not by YIGSR peptide. Combinations of monoclonal antibodies to rat beta 3 and alpha v integrins reduced binding to P1 fragment by 91% and to human laminin by 72%, demonstrating that the major integrin involved in rat osteoclast adhesion to proteolysed laminin is alpha v beta 3. Antiserum to beta 1 integrin inhibited adhesion to human laminin by 40%, but to P1 fragment by only 8%; this suggests that beta 1 integrins(s) contribute to osteoclast adhesion to human laminin but probably not to P1 fragment. The involvement of alpha v beta 3 integrin was confirmed using a recombinant human alpha v beta 3 solid phase binding assay, alpha v beta 3 bound to mouse P1 fragment and proteolytically digested human laminin, but not intact laminins.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies of lymphocyte transendothelial migration: analysis of migrated cell phenotypes with regard to CD31 (PECAM-1), CD45RA and CD45RO.

CD31 is a 130,000 MW cell-surface glycoprotein expressed on endothelial cells, polymorphonuclear leucocytes, monocytes and about 50% of peripheral blood lymphocytes, and it has been proposed that it plays a role in transendothelial migration. If it is involved in endothelial transmigration of lymphocytes then the proportion of CD31+ cells should be increased in the lymphocyte population which has crossed an endothelial monolayer. This was tested using two endothelial types, namely human umbilical vein endothelial cells (HUVEC) and rat high endothelial venule (RHEV) cells. As a control, lymphocyte CD45RA and CD45RO expression was also determined since there is a correlation between lymphocytes bearing these isoforms and different migratory patterns. Double labelling techniques showed a close correlation between CD31 and CD45RA expression. With HUVEC monolayers, the transmigrated lymphocyte population was depleted of CD31+ cells. This depletion was even more marked if the HUVEC monolayers had been stimulated with interleukin-1 beta (IL-1 beta). The migrated lymphocytes were enriched for CD31-CD45RO+ cells but depleted of CD31+CD45RA+ cells. In addition, lymphocyte populations depleted of CD31+ cells by immunopanning were also able to migrate across HUVEC monolayers. Taken together these data suggest that lymphocyte CD31 expression is not necessary for transmigration across HUVEC monolayers and, if anything, is negatively correlated with transmigration. With the second endothelial cell type, RHEV cells, there was no consistent change in the proportion of CD31+ lymphocyte in the transmigrated population, suggesting neither a positive nor a negative correlation between CD31+ expression and lymphocyte transmigration across RHEV cells. However, with both endothelial cell types, the migrated lymphocyte populations were enriched for the marker CD45RO. In conclusion, lymphocyte surface expression of CD31 is not necessary for transmigration across the endothelial cell types used in this study, but with both cell types an enrichment of CD45RO+ lymphocytes is seen in the migrated population.

Immunohistochemical determination of complement activation in joint tissues of patients with rheumatoid arthritis and osteoarthritis using neoantigen-specific monoclonal antibodies.

Murine monoclonal antibodies specific for neoepitopes expressed by C9 incorporated into membrane attack complexes and by membrane-bound C3b and iC3b have been prepared and characterised. These reagents were used to determine the extent and locus of complement activation in synovial-tissues obtained from patients with rheumatoid arthritis and osteoarthritis. In the four rheumatoid arthritis patients there was extensive deposition of C3 activation products and C5b-9 complexes onto the synovial membrane and the pattern of deposition of both neoantigens in serial tissue sections was very similar. There was less extensive staining for C3 and, particularly, C9 neoepitopes on the apical surface of vessel endothelia. In two of four osteoarthritic patients a similar pattern of C3 and C9 neoepitope deposition was found; in the remaining patients no C5b-9 could be located. Synovial vessel walls, but not synovial cells, from both groups of patients stained extensively for the complement regulatory protein CD59. In synovial membranes from patients with osteoarthritis, C9 appeared to be present predominantly in SC5b-9 complexes whereas in rheumatoid arthritis patients no evidence of S-protein incorporation into membrane attack complexes could be demonstrated, suggesting that in rheumatoid arthritis there is damage to the synovial membrane as a result of complement activation and C5b-9 deposition.

Monoclonal antibodies specific for the C-terminus of the laminin B2 subunit. Evidence for glycosylation differences between murine and human laminin.

We have raised a panel of monoclonal antibodies against a beta-galactosidase fusion protein (XLB2.1) containing the C-terminal 153 amino acids of the murine laminin B2 subunit. Five of the nine antibodies characterized recognize human placental laminin as well as murine Engelbreth-Holm-Swarm (EHS)-tumour laminin. Only two of the antibodies recognize both rat parietal-yolk-sac laminin and murine EHS-tumour laminin. Two antibodies recognize an epitope on the human laminin B2 subunit which is masked by N-linked oligosaccharide in murine EHS-tumour laminin. These antibodies also fail to bind to laminin from adult-mouse tissues. These results demonstrate a species-specific difference in the glycosylation of the laminin B2 subunit.

The effects of cyclosporin A on eicosanoid excretion in patients with rheumatoid arthritis.

Alterations in renal eicosanoid levels have been postulated as a factor in cyclosporin A (CSA) nephrotoxicity. The effects of CSA on renal eicosanoid excretion in rheumatoid arthritis were studied over a 24-week period, during which treatment with nonsteroidal antiinflammatory drugs was discontinued. The initial dosage of CSA was 4 mg/kg/day; at week 24, the mean dosage of CSA was 3.9 mg/kg/day. At week 24, the mean (+/- SD) serum creatinine level (1.04 +/- 0.24 mg/dl) was 32% above the baseline value; renal blood flow had decreased by 21% (P less than 0.03) and the glomerular filtration rate had decreased by 16%. There was a significant increase (P less than 0.03) in the 2,3-dinor thromboxane B2 level at week 2, but there was no significant change in the levels of the other eicosanoids. This study demonstrates that after CSA treatment, there is a selective increase in a thromboxane metabolite that parallels an increase in renal vascular resistance, even in the absence of nonsteroidal antiinflammatory drugs, and with unimpaired formation of other vasodilator eicosanoids.

Identification of the B1 and B2 subunits of human placental laminin and rat parietal-yolk-sac laminin using antisera specific for murine laminin-beta-galactosidase fusion proteins.

Antisera raised against fusion proteins consisting of murine laminin B1 and B2 subunit sequences fused to the C-terminus of Escherichia coli beta-galactosidase were tested for their subunit specificity on Western blots of deglycosylated murine Engelbreth-Holm-Swarm (EHS) laminin. The antisera raised against B2 subunit sequences (anti-XLB2.1 and anti-XLB2.2) bound only to the EHS laminin B2 subunit. One of the antisera raised against B1 subunit sequences (anti-XLB1.2) was specific for the B1 subunit, whereas two others (anti-XLB1.1 and anti-XLB1.3) cross-reacted with the EHS laminin B2 subunit. Gold-labelled heparin-albumin was shown to bind specifically to the A subunit of deglycosylated EHS laminin on Western blots. These reagents were used to identify the homologous subunits in rat parietal-yolk-sac laminin and human placental laminin. The anti-(fusion protein) antisera identified the B1 and B2 subunits of the rat laminin, and these were similar in size to the murine EHS B subunits. Human placental laminin gave bands of 400, 340, 230, 190 and 180 kDa on reducing SDS/PAGE. The anti-(fusion protein) antisera identified the 230 and 190 kDa bands as the B1 and B2 subunits respectively. Gold-labelled heparin-albumin bound to the 400, 340 and 190 kDa bands of human placental laminin and so did not unambiguously identify a single A subunit. The human placental laminin may contain a mixture of isoforms, with alternative subunits substituting for the A subunit.

Does lithium carbonate affect the ion transport abnormality in cystic fibrosis?

Lithium is known to affect several aspects of cellular regulation which may be related to ion channel function in epithelial cells. To determine whether the ion transport abnormality in cystic fibrosis (CF) is affected by lithium with resultant changes in clinical status, 36 CF patients, 12-37 years old, were enrolled in a 14 week, double-blind, placebo-controlled trial. Eighteen patients were randomly assigned to receive lithium carbonate for 10 weeks. At the end of therapy their average serum lithium concentration was 0.56 +/- 0.06 mmol (SEM) per liter. Their sweat chloride concentration fell from 92.1 +/- 4.8 mmol per liter to 87.4 +/- 4.0 mmol per liter after 10 weeks of therapy (P = 0.07) and rose to 94.4 +/- 3.5 mmol per liter 4 weeks after end of therapy (P less than 0.001 compared to results at end of therapy). Their forced vital capacity (FVC) fell from 72 +/- 5.3% of predicted to 66 +/- 5.1% of predicted after 4 weeks of therapy (P less than 0.01), and their forced expiratory volume in one second (FEV1) fell from 56 +/- 5.5% of predicted to 51 +/- 5.5% of predicted after 4 weeks of therapy (P less than 0.01). In a non-blind assessment, performed 19 weeks after the end of therapy, their FVC and FEV1 had risen and were not significantly different from baseline. Sweat chloride, FVC, and FEV1 remained unchanged in the placebo group throughout the period of study.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of cyclosporine on urinary kallikrein excretion in patients with rheumatoid arthritis.

The effect of cyclosporine (6 to 8 mg/kg/24 hr) on urinary kallikrein excretion is summarized for 9 patients with rheumatoid arthritis using a specific kallikrein radioimmunoassay. Baseline serum creatinine and BUN values were within the normal range for all patients, and baseline kallikrein excretion rates were either normal (n = 5) or more than two S.D. below the mean of the normal group (n = 4). The patients with normal baseline values excreted significantly less urinary kallikrein three and six months after cyclosporine therapy was started, but all of them completed the six-month protocol. Patients in the subgroup with low baseline values also decreased their kallikrein excretion in response to cyclosporine therapy, and two of the four in this group experienced elevations of BUN such that therapy was terminated. In a low-dose (3 mg/kg/24 hr) open extension that followed the initial trial, kallikrein excretion decreased by almost 50% at least one month before any change in serum creatinine was observed. The data suggest that changes in urinary kallikrein excretion rates may be an indicator or predictor of cyclosporine nephrotoxicity. Decreased kallikrein excretion rates could also be a factor in the diminished renal blood flow reported in patients treated with cyclosporine.

In vivo inhibition of tissue kallikreins by kininogen sequence analogue peptides.

Kininogen sequence analogs containing amino acid residues around the Arg-Ser cleavage site of bovine kininogens were prepared with bulky aliphatic residues in P3 position. KKI-7 (containing a cyclohexylacetyl group) and KKI-8 (containing an adamantaneacetyl group) both inhibited human urinary kallikrein (HUK) with Ki of 4 microM. These inhibitors were 40 times more potent than the corresponding peptide containing the naturally occurring Pro at P3 and were one-seventh as susceptible to hydrolysis by HUK. Rat submaxillary kallikrein (RSK) and porcine pancreatic kallikrein (PPK) were also inhibited by these analogs. Both analogs were poor inhibitors of human plasma kallikrein, while their capacity to inhibit bovine trypsin was 1/3 and 1/17, respectively, that to inhibit HUK. In a rat blood flow study, KKI-7 infusion depressed the response to injected RSK. The response gradually returned toward normal 30 to 60 min after the infusion was terminated. Blood flow increase of dog jejunal artery in response to infused PPK was blunted by the simultaneous local infusion of Trasylol, KKI-7, or KKI-8, whereas these infusions did not alter the response to infused bradykinin. The vehicle infusion did not attenuate the response either to PPK or bradykinin. These analogs appear to have greater specificity and stability than those previously developed and to be appropriate for the in vivo inhibition of glandular kallikreins.

Inhibition of human and rat tissue kallikreins by peptide analog antagonists of bradykinin.

Three representative bradykinin receptor antagonists have been examined for their capacity to inhibit purified human and rat urinary kallikreins. In an amidolytic assay, the Ki values for the inhibition of human urinary kallikrein were 0.5, 0.3, and 2.5 microM for B4307 (DArg-Arg-Pro-Hyp-Gly-Thi-Ser-DPhe-Thi-Arg), B4308 (Lys-Lys-Arg-Hyp-Hyp-Gly-Thi-Ser-DPhe-Thi-Arg) and B3852 (Arg-Pro-Hyp-Gly-Phe-Ser-DPhe-Phe-Arg), respectively. B4308 and B3852 inhibited rat urinary kallikrein with Ki's of 1.0 and 5.5 microM, respectively. B4308 (0.4 to 1.6 microM) also inhibited the kininogenase activity of human urinary kallikrein on human low molecular weight kininogen in a dose-dependent fashion. These bradykinin receptor antagonists may inhibit the kallikrein-kinin system both by blocking the binding of kinins to B2 receptors and by inhibiting the cleavage of kinins from kininogens.

The murine T-lymphocyte response to tyrosine-azobenzenearsonate. Characteristics of a low responder haplotype T-cell clone.

An I-Ab-restricted, L3T4+ Ly2- T-cell clone, 5R-4F3, specific for ABAtyr was established in culture from a B10.A(5R) mouse. Since b haplotype mice respond weakly to ABAtyr compared to other haplotypes, this is a candidate clone of low responder phenotype. In support of this contention, 5R-4F3 grew very poorly under conditions that supported the vigorous growth of E beta bE alpha k-restricted T-cell clones from the same mouse. The I-A (low responder) and I-E (high responder) restricted T-cell clones also differed in their responses to apc pre-pulsed with antigen, compared to apc with antigen present continuously during culture. The low and high responder clones responded comparably to IL-2. Attempts to elevate the response of C57BL/6 mice to ABAtyr in vivo by injecting them with human recombinant IL-2 and antigen together were only partially successful: C57BL/6 mice treated in this way showed a 3-5-fold increase in their proliferative responses to ABAtyr, which was at best only one quarter of the level of response shown by high responder A/J mice to the same antigen dose.

Angioedema induced by a peptide derived from complement component C2.

Synthetic peptides that correspond to the COOH-terminal portion of C2b enhance vascular permeability in human and guinea pig skin. In human studies, 1 nmol of the most active peptide of 25-amino acid residues produced substantial local edema. A pentapeptide and a heptapeptide corresponding to the COOH-terminal sequence of C2b each induced contraction of estrous rat uterus in the micromole range; a peptide of 25 amino acids from this region induced a like contraction of rat uterus at a concentration 20-fold lower than the smaller peptides. The vascular permeability of guinea pig skin was enhanced by doses of these synthetic peptides in a similar fashion as that observed for the concentration of rat uterus. The induction of localized edema by intradermal injection in both the guinea pig and the human proceeds in the presence of antihistaminic drugs, suggesting that there is a histamine-independent component to the observed increase in vascular permeability. Cleavage of C2 with the enzymic subcomponent of C1, C1s, yields only C2a and C2b, and no small peptides, whereas cleavage of C2 with C1s and plasmin yields a set of small peptides. These plasmin-cleaved peptides are derived from the COOH terminus of C2b, and they induce the contraction of estrous rat uterus.

Effect of cyclosporine on urinary kallikrein excretion in patients with rheumatoid arthritis.

The effect of cyclosporine (6 to 8 mg/kg/day) on urinary kallikrein excretion was examined in 10 patients with rheumatoid arthritis by using a radioimmunoassay for kallikrein, a product of renal tubular biosynthesis. All patients had baseline values of serum creatinine and blood urea nitrogen (BUN) within the normal range. The group had a mean baseline kallikrein excretion of 98.30 +/- 29.98 micrograms/24 hours (mean +/- SEM), and 3 and 6 months after therapy was initiated, kallikrein excretion was 44% and 46% of baseline, respectively (p less than 0.01). The five patients who had a normal mean baseline kallikrein excretion rate (106.60 +/- 15.21 micrograms/24 hr) excreted significantly less (p less than 0.05) kallikrein 3 and 6 months after therapy was initiated (56.60 +/- 3.98 micrograms/24 hr and 34.50 +/- 11.02 micrograms/24 hr, respectively), as did one patient with an elevated baseline kallikrein. All six of these individuals completed the protocol. In a subgroup of four patients with low baseline levels (28.25 +/- 5.06 micrograms/24 hr), two individuals experienced elevations of BUN such that cyclosporine was discontinued; in the two who completed the protocol, there was some further decrement in kallikrein excretion. Kallikrein excretion increased in all patients after a 3-month washout period. During a low-dose (3 mg/kg/day) open extension study that followed the initial trial, kallikrein excretion was determined monthly. Seven episodes in which kallikrein excretion decreased in six patients by 44% +/- 18% over a 1-month interval preceded any increase in serum creatinine by 1 to 4 months.(ABSTRACT TRUNCATED AT 250 WORDS)