Rat blood neutrophils express very late antigen 4 and it mediates migration to arthritic joint and dermal inflammation

Blood neutrophils contribute to joint injury in human and experimental models of arthritis. Neutrophil migration out of the blood in joint inflammation involves both the CD18 (beta2) integrins and a CD18 integrin-independent pathway. To investigate this migration, radiolabeled rat blood neutrophils were used to measure neutrophil accumulation in the inflamed joints of rats with adjuvant arthritis and the role of leukocyte integrins in migration to these joints and to dermal inflammation was determined. Neutrophils migrated rapidly (<2 h) to the inflamed joints 14-18 d after immunization with adjuvant. Blocking monoclonal antibodies (mAbs) to both LFA-1 and Mac-1 together, as well as a mAb to CD18, inhibited neutrophil accumulation in the inflamed joints by 50-75%. However, migration to dermal inflammation induced by C5a(des Arg)' tumor necrosis factor alpha, lipopolysaccharide, and poly-inosine:cytosine was inhibited by approximately 90%. Flow cytometry revealed the expression of low levels of very late antigen 4 (VLA-4) on nearly all rat blood neutrophils. Treatment with anti-VLA-4 plus anti-LFA-1 but neither mAb alone, strongly (60-75%) inhibited neutrophil accumulation in arthritic joints. This mAb combination also inhibited neutrophil migration to dermal inflammatory reactions by 30-70%. Blocking VLA-4 together with the CD18 integrins inhibited neutrophil accumulation by 95-99%, virtually abolishing neutrophil accumulation in cutaneous inflammation. A similar blockade of VLA-4 and CD18 decreased neutrophil accumulation in the inflamed joints by 70-83%, but a significant portion of the neutrophil accumulation to these joints still remained. In conclusion, rat blood neutrophils express functional VLA-4 that can mediate neutrophil migration to both inflamed joints and dermal inflammatory sites. VLA-4 appears to be able to substitute for LFA-1 in this migration and is particularly important for accumulation in inflamed joints. However, there exists an additional CD18- and VLA-4-independent pathway of neutrophil migration to arthritic joints that is not involved in acute dermal inflammation.     

Optical amplification of ligand-receptor binding using liquid crystals

Liquid crystals (LCs) were used to amplify and transduce receptor-mediated binding of proteins at surfaces into optical outputs. Spontaneously organized surfaces were designed so that protein molecules, upon binding to ligands hosted on these surfaces, triggered changes in the orientations of 1- to 20-micrometer-thick films of supported LCs, thus corresponding to a reorientation of approximately 10(5) to 10(6) mesogens per protein. Binding-induced changes in the intensity of light transmitted through the LC were easily seen with the naked eye and could be further amplified by using surfaces designed so that protein-ligand recognition causes twisted nematic LCs to untwist. This approach to the detection of ligand-receptor binding does not require labeling of the analyte, does not require the use of electroanalytical apparatus, provides a spatial resolution of micrometers, and is sufficiently simple that it may find use in biochemical assays and imaging of spatially resolved chemical libraries.     

Laminin directs growth cone navigation via two temporally and functionally distinct calcium signals

During development, growth cones navigate to their targets via numerous interactions with molecular guidance cues, yet the mechanisms of how growth cones translate guidance information into navigational decisions are poorly understood. We have examined the role of intracellular Ca2+ in laminin (LN)-mediated growth cone navigation in vitro, using chick dorsal root ganglion neurons. Subsequent to contacting LN-coated beads with filopodia, growth cones displayed a series of stereotypic changes in behavior, including turning toward LN-coated beads and a phase of increased rates of outgrowth after a pause at LN-coated beads. A pharmacological approach indicated that LN-mediated growth cone turning required an influx of extracellular Ca2+, likely in filopodia with LN contact, and activation of calmodulin (CaM). Surprisingly, fluorescent Ca2+ imaging revealed no LN-induced rise in intracellular Ca2+ in filopodia attached to their parent growth cone. However, isolation of filopodia by laser-assisted transection unmasked a rapid, LN-specific rise in intracellular Ca2+ (+73 +/- 11 nM). Additionally, a second, sustained rise in intracellular Ca2+ (+62 +/- 8 nM) occurred in growth cones, with a distinct delay 28 +/- 3 min after growth cone filopodia contacted LN-coated beads. This delayed, sustained Ca2+ signal paralleled the phase of increased rates of outgrowth, and both events were sensitive to the inhibition of Ca2+/CaM-dependent protein kinase II (CaM-kinase II) with 2 microM KN-62. We propose that LN-mediated growth cone guidance can be attributed, in part, to two temporally and functionally distinct Ca2+ signals linked by a signaling cascade composed of CaM and CaM-kinase II.     

Paradoxical hypertension after reversal of heart failure in patients treated with intensive vasodilator therapy

Hypertension is a major cause of heart failure, evolving from left ventricular hypertrophy to systolic and diastolic dysfunction. Although effective heart failure therapy has been associated with a lowering or no change in systemic arterial blood pressure in long-term follow-up, this study describes the symptomatic, clinical, and left ventricular functional response of a subgroup of heart failure patients with a prior history of hypertension who demonstrated a paradoxical hypertensive response despite high-dose vasodilator therapy. We prospectively identified 45 patients with a past history of hypertension who had become normotensive with symptomatic heart failure. Of these 45 heart failure patients, 12 became hypertensive while receiving therapy in follow-up, with systolic blood pressure > or = 140 mm Hg (Group A). The remaining 33 patients did not have a hypertensive response to therapy (Group B). In the 12 Group A patients, 60+/-10 years old, with symptomatic heart failure for 6.3+/-4.3 years, vasodilator therapy was intensified in the 2.0+/-0.5 years of follow-up, achieving final doses of enalapril 78+/-19 mg and isosorbide dinitrate 293 +/-106 mg per day. New York Heart Association classification improved from 2.9+/-0.8 to 1.3+/-0.5 (P < or = .0001), with a reduction in heart-failure-related hospitalizations. Left ventricular ejection fraction increased from 17+/-6% to 40+/-10% (P < .0001). Follow-up blood pressure at 1 to 3 months was unchanged. However, both systolic and diastolic blood pressure increased at final follow-up, rising from 116+/-14 to 154+/-13 mm Hg (P = .0001) and from 71+/-9 to 85+/-14 mm Hg (P = .004), respectively. Renal function remained unchanged. Although both groups had similar clinical responses, there were more blacks and women in the hypertensive Group A. Effectively, 12 of 45 (27%) heart failure patients with an antecedent history of hypertension demonstrated a paradoxical hypertensive response to vasodilator therapy. The recurrence of hypertension in a significant portion of patients successfully treated for heart failure has important clinical implications.     

The ATP synthase of Trypanosoma brucei is developmentally regulated by an inhibitor peptide

The Trypanosoma brucei ATP synthase, like those of other organisms, is composed of two moieties, the membrane bound F0 and the catalytic F1 with each of these parts comprised of multiple subunits. In addition, an endogenous inhibitor peptide of the ATP synthase has been identified from a variety of sources. Previous reports have suggested that the Trypanosoma brucei ATPase may not possess such an inhibitor. Recently, we have isolated an inhibitor peptide fraction from the procyclic form of Trypanosoma brucei by modification of a previously published procedure. This fraction is composed of two dominant polypeptides with estimated molecular weights of 14,000 and 12,000 and an additional polypeptide of 15,000 that may or may not be functionally required. Antibodies raised to the smallest polypeptide showed strong cross reactivity with the other two polypeptides, suggesting that they are related. Antibodies to rat liver inhibitor peptide show cross reactivity with the same polypeptides in crude fractions. The inhibitor peptide fraction strongly suppresses the ATPase activity of membrane bound ATPase in a Mg(2+)-dependent manner and is cold and heat stable. Using antibodies to the smallest polypeptide and rat liver inhibitor peptide we have shown in crude extracts from the three experimental life cycle stages of T. brucei that the inhibitor peptide(s) is developmentally regulated to a modest extent. The pattern of regulation is opposite of the pattern seen for the ATP synthase complex. This suggests that the ATP synthase is stringently controlled in T. brucei in a unique way.     

Continuous monitoring of global left ventricular ejection fraction during percutaneous transluminal coronary angioplasty

Continuous monitoring of left ventricular (LV) function during percutaneous transluminal coronary angioplasty (PTCA) was performed in 40 patients (53 +/- 2 years) with a miniature, nuclear detector system after labeling the patients' red blood cells with technetium-99m. Balloon dilation (113 seconds, range 60 to 240) induced on average a 0.12 ejection fraction (EF) unit (19%) decrease in the LVEF, which was explained by a 34% increase in end-systolic counts. Balloon dilation of the left anterior descending artery (n = 23) produced a decrease in the LVEF of 0.17 +/- 0.13 EF units compared with the decrease of 0.06 +/- 0.07 EF units in patients undergoing dilation of the left circumflex artery (n = 9) and 0.05 +/- 0.04 EF units in patients treated for a stenosis of the right coronary artery (n = 8), (p = 0.02). Balloon deflation was associated with an immediate return to pre-PTCA levels. In 10 patients with 2 identical balloon occlusions, the second occlusion led to a significantly less decrease in the LVEF (0.41 +/- 0.14 vs 0.44 +/- 0.15) and electrocardiographic ST-segment deviation (88 +/- 54 microV vs 65 +/- 42 microV) than the first. We conclude that PTCA is associated with an abrupt transient decrease in the LVEF. The effect of balloon occlusion of the left anterior descending artery is more pronounced than balloon occlusion of the left circumflex and the right coronary arteries. Neither single nor multiple balloon occlusions were associated with post-PTCA global LV dysfunction, whereas the lesser degree of LV dysfunction and electrocardiographic signs of myocardial ischemia during the second of 2 identical balloon occlusions suggests that preconditioning can be induced during PTCA.     

Platelet adhesion, release and aggregation in flowing blood: effects of surface properties and platelet function

Platelet adhesion to natural and artificial surfaces and adhesion-induced aggregation were investigated in vitro using an annular perfusion chamber. The surfaces were exposed to anticoagulated blood under identical flow conditions (approximately arterial shear rates). The initial attachment of platelets (contact) appeared less surface specific than spreading and release. Fibrillar collagen was the most powerful inducer of platelet degranulation whereas elastin, microfibrils and epon were virtually inactive. Fibrillar collagen caused release also in the absence of spreading. Surface coverage with platelets did not exceed 25% unless spreading occurred. Perfusion with platelet-free plasma or platelet-poor blood did not remove adhering platelets. However, platelets were translocated from mural thrombi to the surface by such perfusion. In addition, platelets which detached from mural thrombi adhered more readily to elastin or microfibrils than platelets from the circulating blood. The initial attachment of platelets to subendothelium was inhibited in von Willebrand's disease, the Bernard-Soulier syndrome and at high concentrations of dipyridamole; spreading was inhibited in storage pool disease of rats, at low temperature (20 degrees C), with EDTA (3 MM) and Prostaglandin E1 (1 muM); and adhesion-induced aggregation was inhibited in thrombasthenia, storage pool disease and after ingestion of sulfinpyrazone or Aspirin. It is concluded that the initial attachment (contact) of platelets, spreading and surface-induced release of platelet constituents are at least partially indendent phenomena, the latter two being highly surface specific. At flow conditions which cause the disappearance of platelet adhesion appears as an irreversible process.