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J D Chambers

Publications and source records attributed to J D Chambers.

At least 19 recordsLinked to original sources
PubMed1995

C5a- and tumor necrosis factor-alpha-induced leukocytosis occurs independently of beta 2 integrins and L-selectin: differential effects on neutrophil adhesion molecule expression in vivo.

We previously demonstrated in rabbits that various neutrophil chemotactic factors share an ability to induce recruitment of polymorphonuclear leukocytes (PMN) from bone marrow when administered intravenously (Jagels and Hugli, J Immunol 148:1119, 1992). In the study reported here, we investigated the effects of chemotactic factors on the expression of beta 2 integrins and L-selectin in vivo and the roles of these adhesionmolecules in the recruitment process. Leukocytosis was induced by infusion of either C5a (5 micrograms/kg), N-formyl-methionyl-leucyl-phenylalanine (f-MLP; 2.5 micrograms/kg), or tumor necrosis factor alpha (TNF-alpha; 100 ng/kg). C5a increased the expression of CD18 (the common subunit of beta 2 integrins) on PMN by nearly twofold and decreased levels of L-selectin by 50% within 15 minutes after administration. Levels of beta 2 integrins returned to baseline 2 to 3 hours after induction of leukocytosis. L-selectin remained depressed for more than 5 hours, demonstrating that shedding was induced in the recruited bone marrow leukocytes as well as in circulating PMN. In contrast to the response to C5a, TNF-alpha did not cause upregulation of CD18 or shedding of L-selectin. Levels of L-selectin were consistently increased 60 minutes after administration of TNF-alpha, coinciding with a rapid rise in the number of band-form PMN in the circulation. Intact IgG and F(ab)2 forms of the anti-CD18 monoclonal antibody IB4 or the anti-L-selectin antibody DREG-200 were administered intravenously 15 minutes before induction of leukocytosis by the chemotactic factors. Neither IB4 nor its F(ab)2 fragments blocked leukocytosis induced by C5a, f-MLP, or TNF-alpha. DREG-200 also did not block leukocytosis induced by f-MLP, C5a, or TNF-alpha. These results suggest that leukocyte emigration from the bone marrow into the circulation proceeds through interactions distinct from those involved in neutrophil chemotaxis and diapedesis. Shedding of L-selectin from C5a-recruited bone marrow leukocytes demonstrates activation of these cells in the recruitment process and may reflect a potential mechanism for their release. The dissimilar effects of C5a and TNF-alpha on expression of adhesion molecules may result from distinct stimulatory pathways and suggests differential activation states for cellular recruitment by these inflammatory factors.

Animals↗
PubMed1995

Construction of a steric map of the binding pocket for cannabinoids at the cannabinoid receptor.

In order to gain information about the topology of the brain cannabinoid receptor (CB1), a Receptor Steric (RS) Map for cannabinoids at this receptor was calculated. The classical cannabinoids (-)-11-hydroxy-delta-9-tetrahydrocannabinol (K1 = 210 +/- 56 nM), (-)-9-nor-9-beta-hydroxy-hexahydrocannabinol (K1 = 124 +/- 17 nM), nabilone (K1 = 120 +/- 13 nM), and the non-classical cannabinoid, CP-55,244 (K1 = 1.4 +/- .3 nM) were used as template molecules. The RS map was obtained as the union of the van der Waals' volumes of only those accessible conformers identified by MMP2 calculations that were able to clear a region of steric interference at the CB1 receptor previously characterized by us [Reggio, P.H., Panu, A.M. and Miles, S. (1993), J. Med. Chem., 36, 1761-1771]. The utility of the RS Map was explored by screening the accessible conformers of the classical cannabinoid, cannabinol (CBN), (K1 = 3200 +/- 450 nM), for its ability to fit within the RS map. Only the global minimum energy conformer of CBN (53.2% abundance at 298K) was able to fit within the RS map. These results imply that one reason for the reduced affinity of CBN may be that only 53.2% of CBN molecules are shaped properly to fit in the binding pocket for cannabinoids at the CB1 receptor.

Brain Chemistry↗
PubMed1994

Influence of acridine tracer dyes on neutrophil function.

A study was performed to elucidate the effect of two commonly used fluorescent dyes in in vivo microscopic studies, acridine orange (AO) and acridine red (AR), on the ability of phorbol myristate acetate (PMA)- or formyl peptide (fMLP)-stimulated human neutrophils to adhere to a bovine serum albumin matrix and to generate superoxide anions (SOX). Unlabeled stimulated human neutrophils showed 36 +/- 9% (PMA, 10(-7) M) and 11 +/- 7% (fMLP, 10(-7) M) adherence to the matrix. This adhesion was CD18 dependent as evidence by 98% and 92% reduction, respectively, when the anti-CD18 antibody IB4 was included. A dose-dependent inhibition of stimulated human neutrophil adhesion was evident after 30 min of in vitro dye labeling and the EC50 was approximately 70 micrograms/ml (AR) and 145 micrograms/ml (AO). SOX generation by PMA-stimulated neutrophils was unaffected up to 100 micrograms/ml AR and AO but was reduced by 40-60% at higher doses. Rabbit neutrophils labeled in vivo or in vitro with 100 micrograms/ml AR exhibited 41% and 61% lower SOX generation, respectively. The study indicate that neutrophil function, in terms of ability to adhere to a BSA matrix using CD11/CD18 integrins and to generate SOX upon stimulation, is reduced depending on the dose and choice of fluorescent dye. Caution should be exercised when using these compounds at high concentrations in studies of PMN function.

Acridine Orange↗
PubMed1994

Technical aspects of recording evoked otoacoustic emissions using maximum length sequences.

Our previous work has demonstrated the feasibility of recording evoked otoacoustic emissions (EOAEs) using maximum length sequence (MLS) techniques. New equipment has been constructed, using standard DSP boards and computers, which permits stimulation at much higher rates. The technical aspects of analogue-to-digital converter (ADC) requirements, stimulus problems, artefacts, signal-to-noise ratio (SNR) computations and MLS application are considered here and illustrated with data from the new system.

Ear, Inner↗
PubMed1993

L-selectin mediates neutrophil rolling in inflamed venules through sialyl LewisX-dependent and -independent recognition pathways.

The glycoprotein (GP) L-selectin initiates adhesive interactions between leukocytes and endothelial cells (EC). It functions as a lymphocyte-lectin homing receptor recognizing carbohydrate determinants of the peripheral lymph node addressing on high endothelial venules. It also mediates neutrophil rolling, the earliest interaction of neutrophils with acutely inflamed venules. Neutrophil L-selectin presents sialyl-LewisX (sLe(X)) as a ligand to P- and E-selectin in vitro, and we have proposed that this is a major mechanism of L-selectin-mediated rolling in vivo. In contrast, the contribution of neutrophil L-selectin as a receptor protein recognizing one (or more) ligand(s) on inflamed EC is unclear. To address this question, an sLe(X)-negative murine pre-B cell line, L1-2, that can neither bind vascular selectins nor roll in inflamed rabbit venules, was transfected with human L-selectin cDNA. L-selectin expression in stable transfectants was sufficient to confer significant rolling in vivo. Rolling was unaffected by neuraminidase treatment but completely blocked by anti-L-selectin monoclonal antibody (MoAb) DREG-56. Thus, L-selectin can initiate leukocyte interactions with EC determinants potentially through recognition of endothelial carbohydrates. In contrast, when human neutrophils were tested, rolling was reduced, but not abolished, by MoAb DREG-56. Likewise, treatment with neuraminidase or anti-sLe(X) MoAbs decreased, but did not abrogate, neutrophil rolling, consistent with residual EC recognition via L-selectin. Combination of MoAb DREG-56 and neuraminidase resulted in almost complete loss of rolling, as did removal of glycosylated L-selectin by chymotrypsin. Together with the demonstrable rolling of L-selectin transfectants, our results support the concept of a bidirectional interaction between L-selectin bearing sLe(X) on neutrophils and activated EC in vivo. These findings also suggest that L-selectin may mediate rolling of lymphocytes that lack carbohydrate ligands for E- or P-selectin, although probably less efficiently than through bidirectional recognition.

Animals↗
PubMed1993

In vivo behavior of neutrophils from two patients with distinct inherited leukocyte adhesion deficiency syndromes.

The selectins and the beta 2-integrins (CD11/CD18) mediate distinct adhesive interactions between neutrophils and endothelial cells. Selectins are believed to initiate binding by mediating neutrophil rolling, whereas beta 2-integrins are required for subsequent activation-induced firm sticking and emigration. In vitro evidence suggests that two endothelial cell selectins, P- and E-selectin, can mediate rolling by binding to the carbohydrate ligand sialyl-Lewisx (sLex) on neutrophil surface glycoconjugates. To test the relative contribution of selectins and beta 2-integrins in vivo we used intravital microscopy to study the behavior of neutrophils from two patients with distinct inherited leukocyte adhesion deficiency syndromes. Neutrophils from a patient suffering from CD18 deficiency showed normal rolling behavior but were incapable of sticking or emigrating upon chemotactic stimulation. Neutrophils from a second patient with a newly described adhesion deficiency had normal CD18 but did not express sLex. These neutrophils rolled poorly and also failed to stick in venules under shear force. Under static conditions, however, chemoattractant-induced sticking and emigration could be observed. This demonstrates that both selectin-carbohydrate-mediated initiation of adhesion and subsequent activation-induced beta 2-integrin engagement are essential for the normal function of human neutrophils in vivo.

Animals↗
PubMed1992

Neutrophil aggregation is beta 2-integrin- and L-selectin-dependent in blood and isolated cells.

Neutrophil aggregation in response to formyl peptide was analyzed in blood and isolated cells by fluorescence flow cytometry. The isolated leukocyte aggregates and the leukocytes in blood were identified with the vital nucleic acid stain LDS-751. This method enabled us to discriminate nucleated cells from other blood cells and to detect granulocyte aggregates without isolation or E lysis. Cells isolated in the absence of endotoxin retained the characteristics of cells in blood and exhibited similar aggregation kinetics and dose-response to formyl peptide. We show that it is possible to analyze epitope expression in blood with homogeneous flow cytometric assays and that carefully isolated neutrophils retain the expression characteristics of those in blood. The expression of CD18 was at its lowest levels in unstimulated cells, while the rate of formyl peptide stimulated aggregation was most rapid in these cells. Aggregation in isolated cells as well as blood preceded an increase in receptor expression. After stimulation, L-selectin expression decreased in both blood and isolated cells over a time frame similar to disaggregation. The aggregation response in blood was blocked by pretreatment with antibody to CD18 over a concentration range consistent with the amount of antibody bound. Aggregation was also blocked in isolated cells and blood by antibodies DREG-200 and DREG-56 to L-selectin, but not by isotype controls or anti-LFA-1. The results are discussed in terms of the roles of adhesive receptor expression and recognition in neutrophil aggregation. The methods validated here permit linkage between isolated cells and in vivo studies.

Antigens, CD↗
PubMed1992

L-selectin function is required for beta 2-integrin-mediated neutrophil adhesion at physiological shear rates in vivo.

In vivo interactions between neutrophils and endothelial cells (EC) follow a multistep process involving two distinct neutrophil adhesion receptors. L-selectin, constitutively functional on resting neutrophils, mediates an activation-independent primary interaction resulting in rolling along the venular wall. Subsequent activation of rolling neutrophils induces upregulation and functional activation of beta 2-integrins (CD11/CD18) leading to firm attachment. Based on previous findings we hypothesized that, under shear force, rolling may be essential for successful neutrophil-EC recognition. Here we report results of our studies of human neutrophil behavior in interleukin (IL)-1-activated rabbit mesentery venules, an interaction that requires both L-selectin and beta 2-integrins. Rolling of human neutrophils is L-selection mediated; it was strongly reduced by monoclonal antibody inhibition or enzymatic removal of L-selectin. Furthermore, activation induced L-selectin shedding and, in a dose- and time-dependent fashion, rendered neutrophils unable to recognize inflamed EC despite expression of active beta 2-integrins, which promoted adhesion in vitro. Neutrophils activated for 5 min or longer lost most of their ability to roll. However, 1-3 min after activation, rolling was reduced (not abolished), and cells that were still able to roll displayed a significant tendency for a CD18-dependent transition from rolling to sticking. The whole sequence of events, rolling, sticking, and transendothelial migration, could be observed if an extravascular chemotactic stimulus was applied by superfusing mesenteries with leukotriene B4. Under such conditions, sticking and emigration was blocked when rolling was inhibited by enzymatic removal of L-selectin. Our results indicate that primary neutrophil interaction with inflamed EC through the L-selectin is a prerequisite for neutrophil function at physiological shear rates in vivo.

Antibodies, Monoclonal↗
PubMed1992

Inhibition of polymorphonuclear leukocyte adherence suppresses no-reflow after focal cerebral ischemia in baboons.

BACKGROUND AND PURPOSE: While polymorphonuclear leukocytes may contribute to the "no-reflow" phenomenon after focal cardiac and skeletal muscle ischemia/reperfusion, their contribution to acute focal cerebral ischemia is unresolved. We have examined the role of polymorphonuclear leukocytes in microvascular perfusion defects after focal cerebral ischemia/reperfusion in a baboon model of reversible middle cerebral artery occlusion with the anti-CD18 monoclonal antibody IB4, which inhibits neutrophil adherence to endothelium. METHODS: Microvascular patency in the basal ganglia after 3-hour middle cerebral artery occlusion and 1-hour reperfusion (by india ink tracer perfusion) was quantified by computerized video imaging. Animals were randomized to receive intravenous IB4 infusion 15 minutes before reperfusion (n = 7) or to receive no treatment (n = 6). Binding of IB4 to baboon leukocytes was maximal within 5 minutes of infusion. RESULTS: In the untreated group, a significant reduction in patency was observed in microvessels less than 30 microns diameter: mean percent reflow was 51% in the capillary diameter class (4.0-7.5 microns) and 39% in the precapillary arteriole and postcapillary venule diameter class (7.5-30 microns). Infusion of IB4 before middle cerebral artery reperfusion increased reflow in microvessels of all size classes, most significantly in those 7.5-30 microns (p = 0.049) and 30-50 microns (p = 0.034) in diameter. CONCLUSIONS: These results suggest that CD18-mediated polymorphonuclear leukocyte-endothelium adherence contributes to no-reflow predominantly in noncapillary microvessels and at least partially to that in capillaries.

Animals↗
PubMed1991

Two-step model of leukocyte-endothelial cell interaction in inflammation: distinct roles for LECAM-1 and the leukocyte beta 2 integrins in vivo.

The lectin homing receptor LECAM-1 (LAM-1, Leu8) and the beta 2 integrins, particularly Mac-1 (CD11b/CD18), participate in leukocyte-endothelial cell interactions in inflammation. LECAM-1 is rapidly shed while Mac-1 expression is dramatically increased upon neutrophil activation, suggesting functionally distinct roles for these molecules. Using intravital video microscopy, we have compared the effect of antibodies against LECAM-1 and CD18 on leukocyte interactions with rabbit mesenteric venules. Anti-LECAM-1 monoclonal antibody and its Fab fragments inhibited initial reversible leukocyte rolling along the vascular wall. Anti-CD18 monoclonal antibody had no effect on rolling but prevented subsequent firm attachment of leukocytes to venular endothelium. These results support a two-step model of leukocyte-endothelial cell interactions: reversible rolling mediated in part by LECAM-1 facilitates leukocyte recruitment by the local microenvironment and precedes activation-dependent firm attachment involving beta 2 integrins.

Animals↗
PubMed1990

Flow cytometric analysis and modeling of cell-cell adhesive interactions: the neutrophil as a model.

The immune function of granulocytes, monocytes, lymphocytes, and other specialized cells depends upon intercellular adhesion. In many cases the molecules mediating leukocyte cell adhesion belong to the Leu-CAM superfamily of adhesive molecules. To elucidate the events of homotypic aggregation in a quantitative fashion, we have examined the aggregation of neutrophils stimulated with formyl peptides, where aggregate formation is a transient reversible cell function. We have mathematically modeled the kinetics of aggregation using a linear model based on particle geometry and rates of aggregate formation and breakup. The time course was modeled as a three-phase process, each phase with distinct rate constants. Aggregate formation was measured on the flow cytometer; singlets and larger particles were distinguished using the intravital stain LDS-751. Aggregation proceeded rapidly after stimulation with formyl peptide (CHO-nle-leu-phe-nle-tyr-lys). The first phase lasted 30-60 s; this was modeled with the largest aggregation rate and smallest rate of disaggregation. Aggregate formation plateaued during the second phase which lasted up to 2.5 min. This phase was modeled with an aggregation rate nearly an order of magnitude less than that of the initial fast phase, whereas the disaggregation rate for this phase did not change significantly. A third phase where disaggregation predominated, lasted the remaining 2-3 min and was modeled with a four to fivefold increase of the disaggregation rate. The mechanism of cell-cell adhesion in the plateau phase was probed with the monoclonal antibody IB4 to the CD18 subunit of the adhesive receptor CR3. Based on these studies it appears that new aggregates do not form to a large degree after the first phase of aggregate formation is complete. However, new adhesive contact sites may form within the contact region of these adherent cells to keep the aggregates together.

Cell Adhesion↗
PubMed1986

Multiwell cap assay: a simple objective method for the assessment of leukocyte locomotion in vitro.

A simple method for evaluating leukocyte locomotion in vitro has been developed and validated for several chemoattractants. The multiwell cap assay (MWCA) comprises chambers constructed from readily available disposable plastics and is quickly assembled, permitting large experimental protocols. Leukocytes which have migrated through a micropore filter are recovered and counted electronically yielding a precise, objective result. Coefficients of variation are approximately 6%.

Chemotaxis, Leukocyte↗
PubMed1986

Human immune responses to herpes simplex virus, varicella-zoster and cytomegalovirus in vitro.

The cell principally responsible for lymphocyte proliferation to herpes simplex virus (HSV), varicella-zoster (VZ) and cytomegalovirus (CMV) has been shown to be a T cell of helper phenotype. Lymphocytes from a proportion of proliferation-positive normal individuals produced anti-viral antibody in vitro. Although in some cases, and at some time-points, the antibody was specific for the priming virus, in others, antibodies to more than one virus were detected. Similarly, some T-cell clones proliferated specifically to the priming virus, whereas others were not specific for the virus used in the priming culture. Two clones helped the production of HSV-specific antibody, one by autologous, the other by both autologous and allogeneic non-T cells.

Antibodies, Viral↗
PubMed1985

Use of donors sharing one genetic haplotype for bone marrow transplantation.

Matched sibling transplants enjoy over 95% survival of the grafting procedure, but are only available for 1:5 patients. A sibling sharing one genetic haplotype is today our next choice of donor (67% survival) faring better than other relatives (50% survival), providing total body irradiation (of the thymus) has been avoided. The latter, without increasing the attack rate (64%) of GvHD more than doubles the deaths (57% as against 27%) attributable to it. Rejection is avoided by (a) suicide of host responders to donor buffy coat; (b) Cyclosporin-A; (c) displacement induction; (d) a higher dose of marrow. Prevention of GvHD is essential, using either Cyclosporin-A or removing donor T-cells from marrow prior to infusion or, probably better, both. Autoblast immunisation should be further explored. Tolerization seems an active process, easier in the very young, and nonirradiation of the thymus is believed important. An assay to assess tolerization (to guide cessation of immunosuppressive measures) is badly needed. Selection of a donor whose lymphocytes can deal with intracellular infections of the host's fibroblasts is now possible. The required increased immunosuppressive measures appear to increase the risk of leukemic relapse, and perhaps should be first improved in the more cost-effective fields of inborn error transplants.

Bone Marrow Transplantation↗
PubMed1979

Timing of cyclosporin-A therapy for abrogation of HVG and GVH responses in rats.

Treatment with cyclosporin A was most effective in abrogating popliteal-lymph-node enlargement induced by host-versus-graft and graft-versus-host reactivity in rats when started before injection of donor-strain lymphocytes. Popliteal lymph-node enlargement was never completely abolished, and splenic lymphocytes from recipients treated with cyclosporin A showed no significant reduction in their response to donor-strain lymphocytes in mixed lymphocyte cultures, suggesting that clonal deletion had not taken place. Mixed lymphocyte cultures also indicated that cyclosporin treatment had not reduced the antigenicity of recipient lymphocytes towards donor strain.

Animals↗
PubMed1979

Double-blind trial of the use of transfer factor in the treatment of Crohn's disease.

We have undertaken a double-blind controlled trial of the use of transfer factor in Crohn's disease. Thirty-three patients with known Crohn's disease completed the trial in which half the patients had three injections of transfer factor and the other half were given saline. After six months there was no significant difference in the clinical condition of either of the two groups compared with before receiving treatment. There was also no difference in their in vitro lymphocyte function, although a number of patients exhibited altered responsiveness to skin testing with tuberculin or streptokinase/streptodornase. A signficant fall on Crohn's disease activity index score occurred over the initial 'acclimatising period' before the trial was started, probably related to overcoming initial introspection and the placebo effect of being part of a trial.

Adult↗