Functional Deactivation Research Articles

In vivo Assessment of Mast Cell Functional Alteration Induced by <i>L</i>-Leucine Methyl Ester, Using the Passive Cutaneous Anaphylaxis Technique

We have recently reported that in vitro, mast cells were sensitive to the action of L-leucine methyl ester (Leu-OMe), a lysosomotropic compound. We now report the in vivo effect of Leu-OMe on mast cells, qualitatively assessed by using the passive cutaneous anaphylaxis (PCA) reaction. The L- but not the D-stereoisomer of Leu-OMe (25 mM) injected together with Dactylis glomerata, pollen-specific, IgE-containing serum inhibited the PCA reaction in rat skin triggered by a subsequent challenge with the corresponding allergen. When specific IgE antibodies were injected in the rat skin 3 days after the L-Leu-OMe, subsequent challenge with the antigen displayed a recovery of the PCA reaction. Thus, an in vivo L-Leu-OMe treatment, at a concentration which did not lead to any macroscopic tissue injury, elicited either an alteration of mast cell mediator release or an inhibition of allergen-induced vasoactive mediator release through a functional deactivation and/or a depletion of mast cells.

Comparison of [99mTc]HMPAO SPECT with [18F]Fluoromethane PET in Cerebrovascular Disease

Positron emission tomography (PET) of [18F]fluoromethane (FM) and single-photon emission tomography (SPECT) of [99mTc]hexamethylpropyleneamine oxime (HMPAO) were performed under identical conditions within 2 h in 22 patients suffering from cerebrovascular disease (8 ischemic infarction, 2 intracerebral hemorrhages, 7 transient ischemic attacks, and 5 multi-infarct syndrome). While gross pathological changes could be seen in the images of either procedure, focal abnormalities corresponding to transient ischemic deficits or to lesions in multi-infarct syndrome and areas of functional deactivation were sometimes missed on SPECT images. Overall, HMPAO SPECT images showed less contrast between high and low activity regions than the FM PET images, and differences between lesions and contralateral regions were less pronounced (6.4 vs 13.3% difference). Regional cerebral blood flow (rCBF) was calculated from FM PET studies in 14 large territorial regions and the pathological lesion, and the regional values relative to mean flow were compared to the relative HMPAO uptake in an identical set of regions defined on the SPECT images. Among individual patients, the Spearman rank-correlation coefficient between relative rCBF and HMPAO uptake varied between 0.48 and 0.89, with a mean of 0.70. While an underestimation of high flow with SPECT--which was demonstrated in a curvilinear relationship between all relative regional PET and SPECT values--could be corrected by linearization taking into account HMPAO efflux from the brain before metabolic trapping, correspondence of SPECT data with PET rCBF values was not improved since this procedure also increased the variance in high flow areas. In the cerebellum, however, a high HMPAO uptake in SPECT always overestimated CBF in relation to forebrain values; this finding might be due to high capillary density in the cerebellum. The differences observed between SPECT and PET data may be explained by technical and physical properties of the methods and by the incomplete first-pass extraction of HMPAO. Additionally, HMPAO or its metabolites may leak through a damaged blood-brain barrier (as observed in one infarct and in the surrounding of hemorrhages), impairing the contrast between lesion and normal tissue. The presented data indicate that the quantification of rCBF by HMPAO SPECT is limited.

The tumor necrosis factor receptor and human neutrophil function. Deactivation and cross-deactivation of tumor necrosis factor-induced neutrophil responses by receptor down-regulation.

Despite numerous reports, the role of tumor necrosis factor (TNF) in polymorphonuclear leukocyte (PMN) function remains controversial. We found TNF to be a potent, pertussis toxin-independent stimulator of PMN adhesion (ED50 2.6 pM). TNF-stimulated PMN under adherent conditions released up to 65% of their transcobalamine content (ED50 3.9 pM) and increased their burst activity 10-fold (ED50 3.2 pM) as measured by the hexose monophosphate shunt, whereas PMN held in suspension hardly degranulated at all and only little burst activity was demonstrable. However, preincubation of PMN with TNF in suspension led to a decrease in cellular adhesiveness, degranulation, and burst activity in response to a secondary stimulus of TNF under adherent conditions, although cells remained fully responsive toward phorbol myristate acetate. A concomitant dose-dependent decline of TNF receptor numbers that correlated well with the inhibition of PMN function (r = 0.91) suggests receptor down-regulation as the mechanism of functional PMN deactivation. Remarkably, preincubation with other PMN stimuli such as N-formyl-methionyl-leucyl-phenylalanine, platelet-activating factor, leukotriene B4, complement component fragment 5a (C5a)/C5a (desarginated), and endotoxin also led to a reduction of TNF-specific PMN responses (cross-deactivation) from 35% (LTB4) to 90% (endotoxin), corresponding with the down-regulation of TNF receptors. Deactivation and receptor down-regulation are independent of pertussis toxin-sensitive G proteins and protein kinase C but seemed to depend on changes in calcium metabolism. Granulocyte hyporesponsiveness towards TNF in sepsis (with elevated blood levels of endotoxin and TNF) might be a mechanism of self-protection or, to the contrary, might impair a possibly central mode of host defense.

Acetazolamide effect on cerebellar blood flow in crossed cerebral-cerebellar diaschisis.

We studied the effect of acetazolamide on cerebellar blood flow in 11 stroke patients with large, unilateral cerebral hemispheric infarcts and no evidence of cerebellar infarction, but with cerebrocerebellar diaschisis of cerebral blood flow. Blood flow was determined with xenon-133 inhalation and dynamic single-photon emission computed tomography at rest and 20 minutes after the intravenous injection of 1.0 g acetazolamide. After acetazolamide, the mean +/- SD increases in blood flow in the affected and contralateral cerebellar hemispheres were 11.1 +/- 3.7 and 12.0 +/- 5.3 ml/100 g/min, respectively; the difference between hemispheres was not significant. The absolute increase in cerebellar flow in these 11 patients was of the same magnitude as that in 12 healthy controls. We conclude that cerebellar vasoreactivity is intact in stroke patients with crossed cerebrocerebellar diaschisis of cerebral blood flow. Our results lend further support to the concept that reduced cerebellar blood flow is secondary to functional deactivation. Our patients were studied 2 weeks to 5 years after their stroke, indicating that this phenomenon may be persistent.

Monocyte chemotactic peptide receptor. Functional characteristics and ligand-induced regulation.

Monocytes, macrophages, and neutrophils will demonstrate several important cellular functions in response to synthetic formylated oligopeptides. N-formyl-norleucyl-leucyl-phenylalanyl-norleucyl-tyrosyl-lysine (fNLPNTL) was a potent chemoattractant for human blood monocytes; a 1.0-nM concentration induced a maximal chemotactic response. Binding of 125I-labeled fNLPNTL to the monocyte formyl peptide receptor was rapid, specific, and saturable at 4, 24, or 37 degrees C. At 4 degrees C, monocytes from several different donors demonstrated between 10,000 and 18,000 receptors/cell with a dissociation constant (Kd) of 1.7-2.7 nM. The association of the 125I peptide with the cells was irreversible at the elevated temperatures and exceeded the amount of surface receptor by approximately four-fold, suggesting receptor-mediated peptide endocytosis. Processing of rhodamine-labeled fNLPNTL by monocytes was observed directly by video intensification microscopy. At 37 degrees C, diffuse membrane fluorescence was seen initially, followed by rapid aggregation and internalization of the peptide. Monocytes incubated with fNLPNTL displayed a temperature dependent loss of surface binding capacity (receptor down-regulation). This decrease was due to a decrease in surface receptor number rather than to a decrease in receptor affinity. A dose-response curve for peptide-induced receptor down-regulation correlated with a dose-response curve for 125I-labeled fNLPNTL uptake, suggesting that each uptake event led to the loss of one surface receptor. Surface receptor replenishment following down-regulation was rapid and not dependent on new protein synthesis, but was inversely related to both the time and peptide concentration used to induce down-regulation. An exact correlation between receptor down-regulation and functional deactivation of the chemotactic response could not be demonstrated.