Effector Unit Research Articles

Interacting mast cells and eosinophils acquire an enhanced activation state in vitro

Mast cells (MCs) and eosinophils (Eos), the key effector cells in allergy, are abundantly co-localized particularly in the late and chronic stages of allergic inflammation. Recent evidence has outlined a specialized 'allergic effector unit' in which MCs and Eos communicate via both soluble mediators and physical contact. However, the functional impact of this bi-directional crosstalk on the cells' effector activities has not yet been revealed. We aimed to investigate whether MC/eosinophil interactions can influence the immediate and late activation phenotypes of these cells. Human and murine MCs and Eos were co-cultured under various conditions for 1-2 h or 1-3 days, and in selected experiments cell-cell contact was blocked. Cell migration and mediator release were examined, and flow cytometry was applied to stain intracellular signaling molecules and surface receptors. Eosinophils enhanced basal MCs mediator release and co-stimulated IgE-activated MCs through physical contact involving CD48-2B4 interactions. Reciprocally, resting and IgE-stimulated MCs led to eosinophil migration and activation through a paracrine-dependent mechanism. Increased phosphorylation of activation-associated signaling molecules, and enhanced release of tumor necrosis factor α, was observed in long-term co-cultures. Eosinophils also showed enhanced expression of intercellular adhesion molecule 1, which depended on direct contact with MCs. Our findings reveal a new role for MC/eosinophil interplay in augmenting short- and long-term activation in both cells, in a combined physical/paracrine manner. This enhanced functional activity may thus critically contribute to the perpetuation of the inflammatory response in allergic conditions.

The design of selectively-activated anti-cancer prodrugs for use in antibody-directed and gene-directed enzyme-prodrug therapies.

Systemic anti-proliferative agents (cytotoxins) have been the most successful single design concept for anti-cancer drugs. However, they have inherent limitations (they target dividing cells rather than cancer cells) which limit their clinical efficacy, especially toward the more slowly-growing solid tumours. New concepts are required to improve the selectivity of their killing of tumour cells. One possibility is the use of prodrugs which can be activated selectively in tumour tissue. Several potential mechanisms for this are being explored, including tumour hypoxia, low extracellular pH, therapeutic radiation and tumour-specific endogenous or exogenous enzymes. In the last approach the exogenous enzyme can be delivered by attachment to monoclonal antibodies (ADEPT) or as DNA constructs containing the corresponding gene (GDEPT). A limitation of both approaches is that only a small proportion of the tumour cells become activation-competent, but this can be substantially overcome by the design of appropriate prodrugs capable of killing activation-incompetent cells via a bystander effect. We have proposed a modular approach to prodrug design in which a trigger unit determines tumour selectivity and an effector unit achieves the desired level of killing of cells when the trigger is activated. For ADEPT and GDEPT prodrugs the primary requirement of the trigger is efficient and selective activation by the appropriate enzyme; the released effector must be a potent, diffusible cytotoxin which fully exploits the small proportion of cells capable of activating the prodrug. A wide variety of chemistries has been used, but many of the existing effectors do not have all of these properties. We report work on two types of cytotoxin derived from very potent anti-tumour antibiotics (enediynes and amino-seco-cyclopropylindolines) as effectors in prodrugs for ADEPT and GDEPT applications.

Ultrastructural evidence for human mast cell-eosinophil interactions in vitro

We have hypothesized that mast cells (MC) and eosinophils (Eos), the main effectors of allergy, can form an effector unit. These cells co-exist in the inflamed tissues during the late and chronic stages of allergy and have been shown to be capable of influencing each other's survival and activity via soluble mediators. We have recently described couples of receptor-ligands that are expressed on either/both of these cells and that imply a physical interaction. In this study, we have investigated the existence of short-term (60 min) in vitro interactions between human peripheral blood Eos and cord-blood-derived MC by transmission electron microscopy. We have found that MC and Eos adhere to each other; the lipid body content and the granule morphology of co-cultured MC and Eos, respectively, are altered, and the level of Eos peroxidase (EPO) released by co-cultured Eos is elevated. Moreover, the transfer of EPO from Eos to MC and tryptase from MC to Eos has been observed. Our results thus indicate that, when co-cultured, MC and Eos show signs of physical contact and of reciprocal activation. This is the first in vitro evidence of functional physical interactions between human MC and Eos, interactions that might also occur in vivo during allergic diseases.

Mast cells and eosinophils: the two key effector cells in allergic inflammation

The allergic inflammatory response is composed of two main phases--the early and the late. The early phase initiates when an allergen activates the tissue resident mast cell, triggering the release of a variety of granule-stored and newly formed mediators. As the inflammatory response progresses, blood borne inflammatory cells--in particular, eosinophils--are recruited into the inflamed tissue. Eosinophil activation and consequent release and production of several pro-inflammatory mediators results in the late phase reaction. A chronic allergic inflammation always features prominent tissue eosinophilia. In this review, we will discuss the possible channels of communication, both soluble and physical, between mast cells and eosinophils that can occur in the late and chronic stages of allergy. Such interactions, that we have termed "the allergic effector unit", may modulate the severity and/or duration of the allergic inflammatory reaction.

Design of Self-Immolative Linkers for Tumour-Activated Prodrug Therapy

The main drawback of most cancer chemotherapy is its relatively low ability to target tumour cells versus normal cells. As a consequence, chemotherapy is usually connected with severe side effects due to the toxicity of traditional cytostatic agents towards normal tissues. A few years ago, the site-specific activation of non-toxic prodrugs in tumours has been proposed in order to enhance the selectivity for the killing of cancer cells. Within this framework, most of the prodrugs that have been designed were three part compounds comprising trigger, linker and effector units. The main function of the linker is to release the effector unit after selective trigger activation via a spontaneous chemical breakdown. However, its structure also affects significantly many prodrug properties such as stability, pharmacokinetic, organ distribution, bioavailability or trigger activation. This review, focussed on the linker unit, is an update of our previous article published in 2002. It deals with recent advances in the design of prodrug linkers including new delivery systems such as elongated linkers or self-immolative dendrimers.

Identification of a Novel Ras-Regulated Proapoptotic Pathway

The Ras-GTPase controls cell fate decisions through the binding of an array of effector molecules, such as Raf and PI 3-kinase, in a GTP-dependent manner. NORE1, a noncatalytic polypeptide, binds specifically to Ras-GTP and to several other Ras-like GTPases. NORE is homologous to the putative tumor suppressor RASSF1 and to the Caenorhabditis elegans polypeptide T24F1.3. We find that all three NORE-related polypeptides bind selectively to the proapoptotic protein kinase MST1, a member of the Group II GC kinases. Endogenous NORE and MST1 occur in a constitutive complex in vivo that associates with endogenous Ras after serum stimulation. Targeting recombinant MST1 to the membrane, either through NORE or myristoylation, augments the apoptotic efficacy of MST1. Overexpression of constitutively active Ki-RasG12V promotes apoptosis in a variety of cell lines; Ha-RasG12V is a much less potent proapoptotic agent; however, a Ha-RasG12V effector loop mutant (E37G) that binds NORE, but not Raf or PI 3-kinase, exhibits proapoptotic efficacy approaching that of Ki-RasG12V. The apoptotic action of both Ki-RasG12V and Ha-RasG12V, E37G is suppressed by overexpression of the MST1 carboxy-terminal noncatalytic segment or by the NORE segment that binds MST1. MST1 is a phylogenetically conserved partner of the NORE/RASSF polypeptide family, and the NORE-MST1 complex is a novel Ras effector unit that mediates the apoptotic effect of Ki-RasG12V.

Angiotensin II releases 20-HETE from rat renal microvessels.

We studied hydroxyeicosatetraenoic acid (HETE) release in response to ANG II from preglomerular microvessels (PGMVs), the vascular segment governing changes in renal vascular resistance. PGMVs were isolated from Sprague-Dawley rats and incubated with NADPH and hormones at 37 degrees C. Eicosanoids were extracted, and cytochrome P-450 (CYP)-derived HETEs were purified and quantitated by negative chemical ionization gas chromatography-mass spectroscopy. PGMVs produced primarily 20- and 19-HETEs, namely, 7.9 +/- 1.7 and 2.2 +/- 0.5 ng/mg protein, respectively. ANG II (5 nM) increased CYP-HETE release by two- to threefold; bradykinin, phenylephrine, and Ca(2+) ionophore were without effect. [Sar(1)]ANG II (0.1-100 microM) dose dependently stimulated 19- and 20-HETEs, an effect blocked by the AT(2)-receptor antagonist PD-123319 as well as by U-73122, a phospholipase C inhibitor. Microvascular 20-HETE release was increased more than twofold by the third day in response to ANG II (120 ng. kg(-1). min(-1)) infused subcutaneously for 2 wk; it was not further enhanced after 14 days, although blood pressure continued to rise. Thus an AT(2)-phospholipse C effector unit is associated with synthesis of a vasoconstrictor product, 20-HETE, in a key renovascular segment.

The Design of Selectively-Activated Prodrugs for Cancer Chemotherapy

Abstract: Tumor-selective activation of anticancer prodrugs is of increasing interest, given several new concepts for such activation which have recently emerged. These include chronic hypoxia and low external pH as consistent properties of solid tumor tissue, and new antibody- and genetic-based methods for specifically delivering activating enzymes to tumors. Such prodrugs are conceptually considered as comprising three separate domains (a trigger which determines the tumor-selective activation, an effector which provides the cytotoxic event following trigger activation, and a linker mechanism which deactivates the effector in the prodrug and efficiently transmits the activating signal). This concept allows independent consideration of the types of structures most suitable for each domain, and the criteria which they must meet. The existing classes of compounds designed as selectively-activated anticancer prodrugs are reviewed, showing that the primary design criterion for the effector unit is high cytotoxicity, whereas the requirements of trigger units depend critically on the mode of selective activation.

The relationship between receptor-effector unit heterogeneity and the shape of the concentration-effect profile: pharmacodynamic implications.

The apparent concentration-effect relationship is the ensemble of many effector units (such as individual cells or channels) that do not always exhibit a uniform stimulus-effect relationship. This concept is substantiated by many observations of heterogeneity in receptor-effector populations including hormone secreting cells, response to hormonal stimuli, activity pattern of second messengers, stimulus-evoked synaptic currents, and single ion channels. The relationship between drug concentration and magnitude of pharmacologic response is commonly described by the sigmoidal Emax model which was derived from the Hill equation. The sigmoidicity factor (N) in this model is assumed to be a pure mathematical parameter without physiological connotations. This work demonstrates that the numerical value of N (measured empirically) is the product of two factors: (i) the degree of heterogeneity of the effector subunits, i.e., the elemental component that upon drug stimulus contributes its pharmacological effect independently and does not interact with other subunits (it could range from a single receptor up to a whole tissue), and (ii) value of N*--the shape factor of the subunits' concentration-effect relationship. A special case of this approach occurs when N* > 5, which is an on-off case. Here N is determined by the distribution (density equation) of the subunit values. In case of heterogeneity of the microparameters of the effector subunits the apparent N will always have a lower value than N*. According to this theory it can be concluded that without knowledge of the distribution of the microparameters no mechanistic interpretation can be deduced from the apparent N value. If in the future N* can be determined by theoretical or experimental methods, the distribution function relating N* to N can be calculated. The relevance of this theory is increased in view of the progress being made in advanced research techniques which may enable us to determine the concentration-effect relationship at the level of the individual effector unit.

Effects of adrenaline on the blood flow through the spleen of rainbow trout ( Salmo gairdneri)

1. 1. Effects of adrenaline on the blood flow through the spleen were examined with rainbow trout, Salmo gairdneri. 2. 2. Administration of adrenaline induced an initial increase and an ensuing decrease in the flow rate. 3. 3. The dose producing 50% response of the maximum one was 1.2 ± 0.7 μmol/l. 4. 4. Observation of the intrasplenic vascular system by a corrosion casting method showed blind ended arteries and dramatic constriction of the arteries. 5. 5. The splenic arteries are an important site for control of blood flow and cause decreased flow rate after adrenaline administration. 6. 6. The possible effector unit causing rapid expulsion of stored blood cells is discussed.

Change of insulin action with aging in conscious rats determined by euglycemic clamp.

To clarify the mechanism responsible for age-related changes in insulin action, the euglycemic clamp technique was performed with graded doses of insulin in conscious rats aged 2, 4, 10, and 20 mo. Insulin binding (IB) to muscle membranes was also studied. Maximal response of insulin-induced glucose disappearance rate (Rd) was decreased significantly between 2 and 4 mo of age. Dose-response curves shifted to the right progressively up to 20 mo of age. However, IB to the muscle membrane diminished between 1 and 4 mo of age without a decrease thereafter. When Rd was plotted against insulin bound to the membranes, the resulting curves shifted to the right with aging, suggesting a coupling defect between the binding and effector unit. In conclusion, insulin action alters in rats between 2 and 20 mo of age. The most pronounced impairment in IB and maximal response of insulin-induced Rd occurs during early life stage (through maturation) and then a coupling defect seems to be superimposed with further aging. However, we cannot exclude the possibility that these changes may be secondary to obesity or reduced physical activity, rather than aging per se.

Interplay of nutrients and hormones in the regulation of insulin release.

Single pancreatic B cells are purified by autofluorescence-activated cell sorting, and their secretory activity is measured after overnight culture. Compared to intact islets, the isolated cells release 2-fold more insulin under basal conditions and 5-fold less during nutrient stimulation. Their secretory activity can be induced by glucose, leucine, or arginine, but only 0.3-1.7% of their hormone content is liberated at 20 mM nutrient concentrations. This poor nutrient-induced insulin release from purified B cells is attributed to their low cAMP levels and is markedly increased after addition of (Bu)2cAMP, of glucagon, or of pancreatic A cells. These results strongly support the concept that the potent in vivo insulin-releasing action of glucose and leucine is not only dependent on their fuel capacity in pancreatic B cells but also on the concurrent cAMP levels in these cells. In isolated islets, endogenously released glucagon apparently determines the cAMP production in B cells and thus participates in the nutrient-induced secretory process. Somatostatin and epinephrine were shown to exert their suppressive effects via the glucagon-dependent messenger system. It is concluded that nutrients and hormones interact with two different messenger systems which amplify each others' stimulatory effect upon insulin release. cAMP might represent the hormone-induced messenger which sets the B cell's sensitivity and secretory capacity for nutrient stimuli such as glucose. The higher insulin secretory response observed after reaggregation of single B cells could not be attributed to an altered activity in the nutrient or hormonal regulatory units, raising the possibility that the aggregated state of the cells is rather responsible for a better organization or cooperation of the secretory effector unit.

Functional integrity of desensitized beta-adrenergic receptors.

The adenylate cyclase-coupled beta 2-adrenergic receptor of the frog erythrocyte has served as a useful model system for elucidating the mechanisms of catecholamine-induced densensitization. In this system, it has been previously demonstrated that agonist-induced refractoriness is associated with sequestration of the beta-adrenergic receptors in vesicles away from the cell surface and from their effector unit, the adenylate cyclase system (Stadel, J.M., Strulovici, B., Nambi, P., Lavin, T.N., Briggs, M.M., Caron, M.G., and Lefkowitz, R.J. (1983) J. Biol. Chem. 258, 3032-3038). These internalized beta-adrenergic receptors appear to be structurally intact as assessed by photoaffinity labeling, but their functional status has previously been unknown. In the present studies, we sought to assess the functionality of the sequestered vesicular receptors by fusing them to Xenopus laevis erythrocytes. This cell is suitable for such studies, since it has almost no detectable beta-adrenergic receptor or catecholamine-sensitive adenylate cyclase, but contains prostaglandin E1-stimulable adenylate cyclase. Fusion of beta-adrenergic receptor-containing vesicles from desensitized frog erythrocytes with X. laevis erythrocytes results in a 30-fold stimulation of the hybrid adenylate cyclase by the beta-adrenergic agonist isoproterenol. This effect was entirely blocked by the beta-antagonist propranolol. The catecholamine-sensitive adenylate cyclase activity established in the vesicle-Xenopus hybrids showed the characteristic agonist potency series of the donor frog erythrocyte beta 2-adrenergic receptor. Fusion of vesicles from desensitized frog erythrocytes in which the beta-adrenergic receptors had been inactivated with the group specific reagent dicyclohexylcarbodiimide, or of vesicles derived from control frog erythrocytes, which contain low amounts of beta-adrenergic receptor, did not establish catecholamine-sensitive adenylate cyclase activity in the hybrids. These data demonstrate that beta-adrenergic receptors internalized during desensitization retain their functionality when recoupled to an adenylate cyclase system from a different source. The functional uncoupling of these receptors during desensitization is thus more likely due to their sequestration away from the other components of the adenylate cyclase than to any alterations in the receptors themselves.

The relationship between somatostatin binding and cyclic AMP-stimulated protein kinase inhibition

Somatostatin binding and the ability to inhibit cyclic AMP stimulated protein kinase were investigated utilizing isolated pancreatic islets, anterior pituitary plasma membranes, adipocytes, erythrocyte ghosts, hepatic plasma membranes, and anterior pituitary secretion vesicles. Three types of response were observed. With type I response, somatostatin bound specifically to pancreatic islets and anterior pituitary secretion vesicles and inhibited cyclic AMP stimulated protein kinase. In type II response, adipocytes and anterior pituitary plasma membranes exhibited somatostatin binding but not effect of the ligand on the kinase. In erythrocyte membrane ghosts and hepatic plasma membranes, there was neither specific somatostatin binding nor protein kinase inhibition (type III response). The absence of somatostatin binding in erythrocytes or hepatic plasma membranes cannot be explained by degradation of the ligand per se. Secretory vesicles isolated from the anterior pituitary gland bind somatostatin with an average affinity which exceeds that observed in plasma membranes (for pituitary secretory vesicles Kd 1 = 8.5 × 10 −8 M, Kd 2 = 5.2 × 10 −7 M; for pituitary membranes Kd 1 = 1.9 × 10 −8 M, Kd 2 = 8.1 × 10 −7 M). The molar concentration of high affinity binding sites ( Ro) for plasma membranes was 6.9 × 10 −10 M: for secretory vesicles 3.6 × 10 −9 M. Calculated in terms of somatostatin binding per U 5′nucleotidase activity, the binding for plasma membranes becomes 8.4 × 10 −14 mole/U 5′nucleotidase; secretory vesicles 4.4 × 10 −13 mole/U 5′nucleotidase. Thus, secretory vesicles are fivefold richer in high affinity receptor sites than plasma membranes. It is suggested that in order for somatostatin to act, both a receptor and an effector unit must be present. In the case of tissues secreting polypeptide hormones by granule extrusion, the secretory vesicle may possess both the receptor and the effector units. It is postulated that during the process of fusion of the plasma and secretory vesicle membranes, a high affinity binding site for somatostatin is incorporated into the plasma membrane, thereby allowing somatostatin to act at a specific locus in the cell in inhibiting hormone release.

The two-step model of ligand-receptor interaction

The theoretical properties of a ‘two-step’ receptor, consisting of two independent molecular entities, a regulatory (R) and an effector (E) unit, which are supposed to diffuse freely in the plane of a fluid and mosaic membrane, are presented. The ligand (L)-hormone, drug, neurotransmitter-binds to the regulatory unit (first step), so that it then becomes able to interact with the effector unit (second step) which is then activated. R + L = RL RL + E = RLE → effect This type of receptor can exhibit remarkable properties. (1) The relationship between binding, and to a lesser extent effect, and ligand concentration is negatively cooperative; (2) The degree of negative cooperativity varies biphasically as function of the total concentrations of regulatory and effector units; (3) The relationship between binding and effect is non-linear: the ‘two-step’ model constitutes a plausible explanation for the existence of a ‘receptor reserve’; (4) Kinetics of activation and inactivation can be more or less rapid than kinetics of association and dissociation of the ligand. This model can account for and integrate in a coherent framework many data on membrane receptors. It could also be used to describe the two-step interaction of steroids with their nuclear receptors.

Characteristics of a cutter-loader with a percussive effector unit

1. From the results of pit investigations the authors have determined approximate values for the energy parameters of the effector units of a machine with high-energy percussive rock breaking. The effector unit of an entry-driving machine for hard rocks cutting workings 15 m2 in cross section should have an impact energy of 20,000 kgf·m and a frequency of 60 min−1. 2. The authors give analytical relations for the size of the effector unit and the frame of an entry-driving machine with high-energy breaking.

Results of bench tests on the USD-2 dynamic plow

The productivity of coal-cutting machines can be increased and the quality indices of the cut coal improved by means of new, less energy-consuming methods of coal cutting. In certain conditions the percussive method of coal cutting satisfies these requirements. In this connection, increasing attention has recently been paid to finding or devising efficient designs for percussive effector units for dynamic plows. In the plans for attacking this problem, on the basis of combined investigations of cutting Karaganda coals by impulsive mechanical loads, the Giprouglegormash and Karaganda Polytechnic Institutes have together developed a design for an experimental dynamic plow, the USD-2, which was built by the Kargormash group. The USD-2 includes a cutting machine--the SD-1 dynamic plow with bilateral percussive effector unit--together with feed mechanism and face conveyer taken from the commercially produced USB-67 static plow. The USD-2 is intended for cutting coal from flat-lying seams 1.2 to 1.4 m thick with cutting resistances of up to 300 kgf/cm with individual or mechanized supports.

Finding the working loads in the effector unit of a mining machine by statistical modeling on a computer

Finding the working loads in the effector unit of a mining machine by statistical modeling on a computer