Doxycycline effects on the adherence of polymorphonuclear leukocytes to an albumin-coated glass surface

The effect of doxycycline on polymorphonuclear leukocyte (PMN) adherence to albumin-coated glass surfaces was studied in the absence and presence of the chemotactic peptide FMLP. Three concentrations of doxycycline were studied, one subtherapeutic (0.1 micrograms/ml), one therapeutic (1.0 micrograms/ml) and one supertherapeutic (10 micrograms/ml). PMN adherence was maximal after incubation for 5 min. FMLP did not affect PMN adherence in the present assay system and at the concentration studied (10(-7)M). PMN adherence remained stable and unaffected in the tested doxycycline concentrations. Thus, the present study could not confirm the reported and challenged doxycycline inhibition of PMN adherence.     

Activation of the bactericidal capacity of polymorphonuclear granulocytes after surgery, measured with a new in vitro assay.

A classic in vitro polymorphonuclear (PMN) granulocyte bactericidal system was used alongside a newly developed modification to see whether the new assay would increase the possibility to detect a stimulation of PMN bactericidal functions. In the new assay each granulocyte was provided with 30--40 bacteria, which is quite close to the maximal killing capacity (usually 60 bacteria per PMN). Granulocytes were obtained from 8 patients the day before, the day after and 2 d after they underwent thoracotomy with cardiopulmonary by-pass (CPS). The granulocytes from all patients showed an increased capacity to kill Staph. aureus in vitro 2 d after the operation, compared to before, when the submaximal bacterial concentration per granulocyte was used, whereas no change was observed with the standard bacterial concentration (3--4 bacteria per granulocyte). Thus, the new assay might make it possible to observe an enhanced PMN bactericidal ability.     

The ontogeny of a 57-Kd cationic antimicrobial protein of human polymorphonuclear leukocytes: localization to a novel granule population

The ontogeny of a 57-Kd cationic antimicrobial protein (CAP57) that has substantial similarities to bactericidal permeability increasing protein (BPI) has been determined immunocytochemically. CAP57 was detected in the granules of mature peripheral blood neutrophils. However, it was absent from other cells of the peripheral blood: eosinophils, red blood cells (RBCs), and mononuclear cells. In human bone marrow, CAP57 was confined to the neutrophilic series. The earliest stage of development of the myeloid cells at which CAP57 was demonstrated was the promyelocyte. Double immunofluorescent labeling showed that CAP57 was detected in cells positive for myeloperoxidase. The absence of lactoferrin in certain cells (promyelocytes) containing CAP57 indicated that CAP57 was synthesized and packaged in a population of granules prior to the development of granules that contain lactoferrin. CAP57 could not be demonstrated in HL60 cells either by enzyme-linked immunosorbent assay (ELISA) or by immunocytochemistry. However, the presence of another granule-associated cationic antimicrobial protein of molecular weight 37 Kd (CAP37) was readily detected in undifferentiated HL60 cells. Amino acid sequence analysis showed that CAP57 and BPI were identical. Further indication of the identity between CAP57 and BPI was that monoclonal anti-CAP57 antibodies cross reacted with BPI. Sucrose density-gradient centrifugations showed CAP57 was confined to a granule population that exhibited a buoyant density intermediate of the previously described light and heavy azurophil granules. Further resolution of the individual azurophil granule populations by Percoll density-gradient centrifugation revealed that CAP57 was most concentrated in the density range of 1.093 to 1.100 g/cc. These results strongly suggest the unique finding that CAP57 may be associated with a heretofore unreported granule type.     

Platelet-leukocyte interaction: selective binding of thrombin- stimulated platelets to human monocytes, polymorphonuclear leukocytes, and related cell lines

The association of platelets with leukocytes was investigated, using gel-filtered platelets stimulated with thrombin and then fixed with formaldehyde. Evidence is presented that stimulation of gel-filtered platelets with low concentrations of thrombin (0.01 to 0.1 U/mL) induces the expression of surface determinants interacting strongly with monocytes and polymorphonuclear leukocytes (PMNs) but only weakly with lymphocytes. Both monocyte-platelet binding and PMN-platelet binding occurred at 37 degrees C and more intensively at 0 degrees C; it was Ca2+-dependent and was unaffected by the addition of sodium azide. The binding also occurred with the monocytoid cell lines HL 60 and U 937 in exponential growth and was much less two days after induction of terminal differentiation by phorbol myristate acetate (PMA). No other tested cell lines (B cells, T cells, and myeloid cells) bound thrombin-stimulated platelets. Monocyte-derived macrophages kept in culture for one week also exhibited reduced binding of thrombin- stimulated platelets. IgG and fibronectin could be ruled out as ligands that mediate binding.     

SGP140: identification of a novel component on human polymorphonuclear leukocyte cell surface involved in chemotactic and phagocytic activities

SGP140, which has been isolated as the major sialoglycoprotein of 140 kd molecular weight from a human T-leukemic cell line, was identified on the cell surface of polymorphonuclear leukocytes (PMNs). Specific antibody against SGP140 was prepared in rabbits and was used to probe the function of SGP140 on PMNs in this study. PMNs from healthy donors that were treated with the anti-SGP140 IgG showed remarkably diminished chemotactic as well as phagocytic activity. Phagocytosis of both C3- and IgG-opsonized particles were affected similarly by the antibody treatment. The antibody neither affected cell adhesion and spreading over substrate, nor did it disturb the other functions of PMNs, i.e., enzyme release and superoxide generation. N-formyl-methionyl-leucyl-phenylalanine did not stimulate SGP140 expression on cell surfaces. We present evidence that SGP140 is distinct from the glycoprotein family (Mac-1, LFA-1, and p150/95 [CDw 18]) that performs adhesive reactions of leukocytes. We propose that SGP140 is a novel molecule on the human PMN cell surface that is involved both in chemotactic and phagocytic activities.     

Hepatic vasopressin receptor: differential effects of divalent cations, guanine nucleotides, and N-ethylmaleimide on agonist and antagonist interactions with the V1 subtype receptor

Previously, we reported that magnesium (Mg2+) enhanced the binding affinity of arginine vasopressin [( 3H]AVP) to a single class of sites in rat liver microsomes. In the present study we have examined the effects of divalent cations and guanine nucleotides on the binding characteristics of both the nonselective agonist and the V1 receptor-selective antagonist, d(CH2)5Tyr(Me)-[3H]AVP, to microsomal and plasma membrane fractions of rat liver. At a subsaturating concentration (100 pM) of [3H]AVP, divalent cations increased specific binding in a concentration-dependent manner with the following rank order of potency: Co2+ greater than Mn2+ greater than Ni2+ greater than Mg2+ greater than Ca2+ = control. The maximal effect for Mg2+ was evident at 1 mM, a physiologically relevant concentration. In contrast, binding of the V1 receptor antagonist (at a subsaturating concentration of 10 pM) was inhibited by divalent cations, the rank order of potency being Mn2+ greater than Co2+ greater than Ca2+ greater than Mg2+ greater than Ni2+. The inhibitory effects of divalent cations were of lesser magnitude (up to 60%) compared to the stimulation of agonist binding (up to 700%). Mg2+ enhanced the affinity of [3H]AVP (Kd was decreased from approximately 2 nM to 133 pM), while the affinity of the [3H]V1 antagonist was decreased (Kd was increased from 10 to 95 pM). Scatchard analysis of saturation data (Mg2+ present) revealed similar maximum binding values for the binding of radiolabeled agonist and antagonist, indicating that AVP receptors in rat liver are mostly of the V1 subtype. Competition experiments between V1/V2-specific AVP analogs with either the radiolabeled agonist or antagonist also indicated the presence of predominantly V1 receptor sites in rat liver microsomes. The properties of plasma membrane receptor sites were similar to those of the microsomal sites, except that the density of receptors was higher in the former. In both equilibrium and competitive inhibition experiments GTPase-resistant analogs of guanine nucleotides, GTP gamma S and GDP beta S, decreased the affinity of the agonist for the receptor, but not that of the antagonist. Treatment of membranes with 0.2 mM N-ethylmaleimide (NEM) reduced the maximum binding of [3H]AVP and abolished the GTP gamma S-evoked decrease in agonist-binding affinity. In contrast, antagonist binding was unaffected by NEM. NEM pretreatment failed to influence the divalent cation-dependent increase in agonist-binding affinity. The results provide direct evidence for the existence of a high and a low affinity state of the hepatic V1 receptor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comprehensive effects of lifestyle reform,adherence, and related factors on hypertension control: A review

Despite the effectiveness of currently available antihypertensive medications, there is still a need for new treatment strategies that are more effective in certain groups of hypertensive and for additional resources to combat hypertension. However, medication non-adherence was previously recognized as a major problem in the treatment of hypertension. The mechanisms behind the positive impacts of lifestyle changes might occur in different ways. In comparison with other studies, the efficacy and effectiveness of lifestyle modifications and antihypertensive pharmaceutical treatment for the prevention and control of hypertension and concomitant cardiovascular disease have been demonstrated in randomized controlled trials. However, in this review, the attitudinal lifestyle modifications and barriers to blood pressure control were elaborated on. An effective method for reducing blood pressure (BP) and preventing cardiovascular events with antihypertensive medications has been outlined. Maintaining healthy lifestyle factors (body mass index, diet, smoking, alcohol consumption, sodium excretion, and sedentary behavior) could lower systolic blood pressure BP by 3.5 mm Hg and reduce the risk of cardiovascular disease (CVD) by about 30%, regardless of genetic susceptibility to hypertension. Conducting a lifestyle intervention using health education could improve lifestyle factors, such as reducing salt, sodium, and fat intake, changing eating habits to include more fruits and vegetables, not smoking, consuming less alcohol, exercising regularly, maintaining healthy body weight, and minimizing stressful conditions. Each behavior could affect BP by modulating visceral fat accumulation, insulin resistance, the renin-angiotensin-aldosterone system, vascular endothelial function, oxidative stress, inflammation, and autonomic function. Evidence of the joint effect of antihypertensive medications and lifestyle reforms suggests a pathway to reduce hypertension.     

Amidolytic assay of human factor XI in plasma: comparison with a coagulant assay and a new rapid radioimmunoassay

The traditional coagulant assay for plasma factor XI suffers from a relatively high coefficient of variation, the need for rare congenitally deficient plasma, and a poor correlation between precision and sensitivity. We have developed a simple functional amidolytic assay for factor XI in plasma using the chromogenic substrate PyrGlu-Pro-Arg- p-nitroanilide (S-2366). After inactivation of alpha 1-antitrypsin, CI inhibitor, and other plasma protease inhibitors with CHCI3, plasma was incubated with kaolin, in the absence of added calcium, which limited the enzymes formed to those dependent on contact activation. Soybean trypsin inhibitor was used to minimize the action of kallikrein on the substrate. Once the reaction was complete, corn trypsin inhibitor was used to inactive factor XIIa, the enzyme generated by exposure of plasma to negatively charged surfaces, which had activated the factor XI. The assay is highly specific for factor XI, since plasma totally deficient in that zymogen yielded only 1%-3% of the enzymatic activity in normal plasma under identical conditions. The requirements for complete conversion of factor XI to XIa in plasma within 60 min were, respectively, factor XII, 0.6 U/ml, and high molecular weight kininogen, 0.2 U/ml. Prekallikrein was not an absolute requirement for complete activation but did accelerate the reaction. The intraassay coefficient of variation was 3.4%, and the mean of 35 normal plasmas was 1.00 U +/- 0.24 SD. In addition, a new rapid radioimmunoassay was devised using staphylococcal protein A as the precipitating agent for a complex of factor XI antigen with monospecific rabbit antibody. The mean was 1.01 U +/- 0.30 SD. The correlation coefficients for amidolytic versus coagulant and amidolytic versus radioimmunoassay were r = 0.95 for the former and 0.96 for the latter. Thus, a simple, accurate amidolytic assay and a radioimmunoassay have been devised for measuring factor XI in plasma that correlate well with the coagulant activity of factor XI, as determined in our laboratory.     

Neutrophil and monocyte adherence to and migration across monolayers of cytokine-activated endothelial cells: the contribution of CD18, ELAM-1, and VLA-4.

Pretreatment of endothelial cells with cytokines enhances the adherence of leukocytes, a process that is mediated by surface proteins expressed on both cell types. A three-dimensional model system for the simultaneous determination of leukocyte adherence and migration was used to study the contribution of CD11/CD18, endothelial leukocyte-adhesion molecule-1 (ELAM-1) and VLA-4 in neutrophil and monocyte adherence to and migration through cytokine-activated endothelial cells. Pretreatment of endothelial cells for 4 hours with recombinant interleukin-1 beta (rIL-1 beta) was found to enhance neutrophil adherence and migration to a much greater extent than monocyte adherence and migration. Neutrophil adherence was almost completely prevented by the combined use of monoclonal antibodies (MoAbs) against ELAM-1 and CD18. Although ELAM-1 has been designated an endothelial cell-specific cytokine-inducible receptor for neutrophils, we observed that ENA2, an anti-ELAM-1 MoAb, significantly reduced monocyte adherence about 30%. MoAbs against VLA-4, the ligand of the cytokine-inducible receptor VCAM-1, did not affect monocyte adherence. However, the combined use of the MoAbs against CD18, ELAM-1, and VLA-4 had a very strong and additive inhibitory effect on rIL-1 beta-induced monocyte adherence. The anti-CD18 MoAb reduced both rIL-1 beta-induced neutrophil and monocyte migration far below the level of the unstimulated controls, whereas neither the anti-ELAM-1 nor the anti-VLA-4 MoAb significantly affected the process of migration. Our results indicate that neutrophils and monocytes initially adhere to cytokine-activated endothelial cells by CD18-independent and (to a lesser extent) by CD18-dependent mechanisms and subsequently change gears to a completely CD18-dependent migratory mechanism.     

Human polymorphonuclear leukocytes have dual effects on endothelin-1: The induction of endothelin-1 mRNA expression in vascular endothelial cells and modification of the endothelin-1 molecule

Summary The effect of human polymorphonuclear leukocytes (PMNs) on the expression of the endothelin-1 (ET-1) gene and the production of ET-1 peptide was investigated. Human PMNs were separated from venous blood with Mono-Poly Resolving Medium and activated by incubation with formyl-methionyl-lencyl-phenylalanine (FMLP) (1 μM). Then PMN suspension was added to cultured porcine endothelial cell monolayers and coincubated for various periods. Following the coincubation, ET-1 mRNA in endothelial cells was examined by Northern blotting and immunoreactive ET-1 (irET-1) peptide levels in the conditioned media were measured by an enzyme-linked immunosorbent assay (ELISA). Similar experiments were also carried out with cell-free PMN supernatant. Untreated and activated PMNs led to a 1.4-fold and 6.3-fold increase in ET-1 mRNA levels in endothelial cells, respectively, at 6 h, while irET-1 peptide levels did not significantly increase as compared with control. In contrast, when PMNs were coincubated in the presence of an Intercell chamber without direct contact, to endothelial cells, PMNs did not induce ET-1 mRNA expression in endothelial cells, and significantly decreased irET-1 peptide levels in the conditioned media. Cell-free PMN supernatant did not have all these effects on ET-1. These findings suggest that direct PMN-endothelial cell contact was essential for PMN-induced expression of the ET-1 gene and that PMNs may decrease irET-1 through some modification of the ET-1 molecule.     

Main partner factors associated with worse adherence to HAART among women in Baltimore, Maryland: a preliminary study

Compared to US men, US women have worse HAART and HIV health outcomes. The study examined main partner factors associated with women's HAART adherence. The community sample comprised 85% African-Americans; 63% had a main partner and 32% relied on their partner for emotional support. Adherence was highest (92%) among those without a main partner and lowest (57%) among those with an HIV seropositive main partner. In adjusted analysis, adherence was 75% less likely among women with an HIV seropositive main partner and 78% less likely among those relying on their partner for emotional support. Furthermore, HIV seropositive versus other serostatus main partners were most likely to provide medication taking assistance and to be preferred in helping participants deal with HIV, yet were no more likely to be nominated as the most helpful to them. Findings reveal women's perceived unmet support needs from HIV seropositive main partners in this population and the need for interventions to promote their HAART adherence. Seroconcordant couples-focused intervention that enhances mutual support of HAART adherence may be an effective approach to improving women's HAART adherence and reducing US gender disparities in HIV health outcomes.     

Characterization of a new potent, in vivo neutralizing monoclonal antibody to human vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is an important mediator of tumor-induced angiogenesis and represents a potential target for anticancer therapy. Therefore, we prepared a panel of monoclonal antibodies (mAb) against both the VEGF₁₂₁ and VEGF₁₆₅ isoforms. Three of them completely neutralized the mitogenic stimulation by VEGF of human umbilical vein endothelial cells at mAb concentrations below 0.1 μg/ml. The most potent one, with a dissociation constant (K _d) of 8 pM, inhibited, in a dose-dependent manner, VEGF-induced angiogenesis in a growth factor implant model in mice. A complete inhibition of the angiogenic response was obtained by daily intraperitoneal injections of 10 μg mAb/mouse. Angiogenesis induced by basic fibroblast growth factor was not inhibited by the mAb. Epitope mapping of the mAb, performed by competitive enzyme-linked immunosorbent assay and Western blot analysis, showed that it did not bind to the reduced and denatured monomer of VEGF. Substitutions of three residues (Q87R, G88K, Q89K), located on the major surface loop β5 to β6 of VEGF, resulted in the complete loss of binding (more than 400-fold reduction). The results suggest that the mAb binds primarily to a conformation-dependent epitope on the VEGF dimeric form, encompassing one of the loop regions involved in KDR receptor binding. The mAb with its strong neutralizing properties represents a useful agent for effective blocking of VEGF-mediated tumor neovascularization. Received: 11 November 1998 / Accepted: 16 December 1998     

The effects of adiponectin and leptin on human endothelial cell proliferation: a live-cell study

The effect of adiponectin and leptin on the proliferation of the human microvascular endothelial cell line (HMEC-1) was studied in the absence or presence of fetal bovine serum (FBS). The participation of extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase/Akt (PI-3K/Akt) pathways in this effect were evaluated. We studied the effect of both adipokines on the motility, mitosis, proliferation and cell death processes of HMEC-1 cells using live-cell imaging techniques. Adiponectin but not leptin further increased the proliferative effect induced by FBS on HMEC-1. This effect seems to be the consequence of an increase in the mitotic index in adiponectin-treated cells when compared to untreated ones. The presence of either the mitogen-activated protein kinase (MAPK) inhibitor (PD98059), or PI-3K inhibitor (LY294002), reduced the effect of adiponectin in a dose-dependent manner. Neither adipokine was able to affect HMEC-1 proliferation in FBS-free conditions. Duration of mitosis, cell motility and the cell death process were similar in all conditions. These data suggest that adiponectin and leptin exert different effects on endothelial cell function. Adiponectin was able to potentiate proliferation of HMEC-1. This effect involves the activation of both PI3-K/Akt and ERK/MAPK pathways. However, it seems to exert minimal effects on HMEC-1 function in the case of leptin.     

Subcellular localization of hydrogen peroxide production in human polymorphonuclear leukocytes stimulated with lectins, phorbol myristate acetate, and digitonin: an electron microscopic study using CeCl3

The ultrastructural H2O2-producing site in human polymorphonuclear leukocytes (PMN) stimulated with soluble stimuli was studied using a CeCl3-technique. CeLlular aggregation and formation of small vacuoles were observed when PMN were stimulated with 100 microgram/ml concanavalin-A, 1 mg/ml phytohemagglutinin, or 100 microgram/ml wheat germ agglutinin for 10 min at 37 degrees C. Electron-dense deposits formed from the reaction of H2O2 and CeCl3 were observed on the contact surface of the plasma membrane of aggregated PMN stimulated with lectins. Treatment with 5 microgram/ml cytochalasin-B before lectin- stimulation induced an enhanced formation of vacuoles, degranuLation, rounding of the contour, cellular aggregation, and enhancement of the deposits. Phorbol myristate acetate (PMA; 100 ng/ml) induced strong leukocyte aggregation, the formation of multiple huge vacuoles, degranulation, and H2O2 production at almost all of the contact surface between adjoining PMN and between PMN and erythrocytes, mononuclear cells, or thrombocytes. In PMN stimulated with digitonin (B microgram/ml), vacuolar formation, degranulation, multiple projections on the surface, and H2O2 production on the whole surface membrane were demonstrated. It is shown that cellular aggregation and cell-to-cell contact have an important role in the induction of O2- production induced by lectins or PMA and that O2- production induced by the detergent is not dependent on leukocyte aggregation.     

Aging process,adherence to Mediterranean diet and nutritional status in a large cohort of nonagenarians: Effects on endothelial progenitor cells

Background and aim

Adherence to the Mediterranean Diet (MD) has been associated with a longer and better life. The aim of this study was to examine the effects of adherence to the MD, and of nutritional habits on endothelial progenitor (EPCs) and circulating progenitor (CPCs) cells in a cohort of nonagenarians enrolled within the Mugello Study, an epidemiological study aimed at investigating both clinically relevant geriatric items and various health issues, including those related to nutritional status.

An autoantibody directed against human thrombin anion-binding exosite in a patient with arterial thrombosis: effects on platelets, endothelial cells, and protein C activation

An autoantibody, developed by a patient with severe and recurrent arterial thrombosis, was characterized to be directed against the anion- binding exosite of thrombin, and inhibited all thrombin interactions requiring this secondary binding site without interfering with the catalytic site. The effect of the antibody was studied on thrombin interactions with platelets and endothelial cells from human umbilical veins (HUVEC). The autoantibody specifically and concentration- dependently inhibited alpha-thrombin-induced platelet activation and prostacyclin (PGI2) synthesis from HUVEC. It had no effect when gamma- thrombin or the thrombin receptor activation peptide SFLLR were the inducers. The effect of the antibody on protein C activation has been studied. The antibody blocked the thrombin-thrombomodulin activation of protein C. The inhibition of the activation was maximal with a low concentration of thrombomodulin. The fact that the autoantibody inhibited concentration-dependent alpha-thrombin-induced platelet and endothelial cell functions emphasizes the crucial role of the anion- binding exosite of thrombin to activate its receptor. In regard to the pathology, the antibody inhibited two vascular processes implicated in thrombin-antithrombotic functions, PGI2 secretion, and protein C activation, which could be implicated in this arterial thrombotic disease.     

Determinants of endothelial function in human immunodeficiency virus infection: a complex interplay among therapy, disease, and host factors

In the era of highly active antiretroviral therapy (HAART), human immunodeficiency virus (HIV) infection has become a chronic disease in which patients may develop significant metabolic complications and risk factors for cardiovascular disease (CVD), including insulin resistance, visceral fat deposition, and increases in atherogenic cholesterol and triglyceride levels. Epidemiologic studies have found that persons infected with HIV are likely to be at higher risk for premature CVD compared with the general population, and clinical studies examining endothelial function in HIV-infected cohorts have supported such conclusions. The mechanisms underlying the regulation of endothelial function in HIV-infected persons appear to be multifactorial, including direct effects of HIV on the endothelium, indirect effects of HIV on lipids and inflammatory cytokines, HAART-related effects, and traditional/host factors. Better understanding of these processes can lead to improved strategies for the long-term management of HIV infection.