Leukotrienes (LTC4, LTD4) confer glass non-adherence on leukocytes of asthmatic individuals. Dependency on cycloxygenase products and calcium ion

It has been suggested that cysteinyl-containing leukotrienes (LT) are important mediators in bronchial asthma. Since leukotrienes have been shown to mediate the leukocyte adherence inhibition (LAI) phenomenon observed in cancer-bearing host we have devised a modified LAI assay which determines the acquisition of non-adherence properties of leukocytes following a challenge with pure synthetic LT. Our results demonstrate that peripheral blood leukocytes of asthmatic individuals acquire non-adherence properties when challenged with pure synthetic leukotriene C₄ and D₄, a property not shared by peripheral blood leukocytes of control healthy individuals. Furthermore, we demonstrate that LT activity as manifested by the LAI assay is dependent on cycloxygenase products, since 2×10⁻⁶ M Indomethacin abrogated the LT-induced LAI and is restored by the addition of 2×10⁻⁶ M prostaglandin E₂ which is also synergistic to LT activity. Our results further suggest the possibility that leukotriene activity is dependent on calcium ions since it was negated by known calcium antagonists. It is thus suggested that the LT-induced LAI may serve as a tool for the study of the interrelationship between the metabolic pathways of arachidonic acid and calcium ion homeostasis.     

Leukotriene B4, interleukin 1 and leucocyte accumulation in titanium and PTFE chambers after implantation in the rat abdominal wall.

The inflammatory reaction elicited after implantation of non-biologic materials was studied by analysis of the exudate in pure titanium or polytetrafluorethylene chambers 1–9 d after insertion in the abdominal wall of the rat. In chambers made of pure titanium, the number of leucocytes increased about twofold from 1.8 × 10⁶/ml to 4.1 × 10⁶/ml between 24 h and 6 d, whereas a larger increase from 2.1 × 10⁶/ml to 8.3 × 10⁶/ml was observed in the polytetrafluorethylene chambers. Irrespective of implant material, polymorphonuclear granulocytes constituted the vast majority of leucocytes (>81%). After 24 h the leukotriene B₄ content was slightly higher in polytetrafluorethylene chambers than in titanium chambers. In contrast to the titanium chambers, a marked increase in leukotriene B₄ levels was detected in polytetrafluorethylene chambers 6 d after insertion, whereas the greatest amount of leukotriene B₄ in titanium chambers was measured at 9 d. Interleukin 1 was only detected in both types of chambers after 6 d. The present study, together with previous findings, shows that the materials used in this study elicit different degrees of inflammatory reactions in the chamber exudate. Since the number of leucocytes in the exudate retrieved from the chambers correlated well with the leukotriene B₄ levels, it is suggested that leukotriene B₄, but not interleukin 1, may be an important mediator for polymorphonuclear granulocyte migration into the implant-tissue interface. The role of leukotriene B₄ and interleukin 1 for other parts of the inflammatory process around biomaterials in soft tissue, such as cell adhesion and activation and microvascular changes and their potential roles for the development of the peri-implant tissue remains to be established.     

Acetylcholine receptors in dissociated nucleus basalis of Meynert neurons of the rat

Electrical and pharmacological properties of acetylcholine (ACh)-induced currents in neurons dissociated from the nucleus basalis of Meynert (nBM) of immature (2-week-old) rats were investigated with the whole-cell mode of the patch-clamp technique. At a holding potential (V_H) of −50 mV, ACh (10⁻⁴M) evoked a transient inward current mimicked by nicotine (I_nACh), followed by a sustained outward current mimicked by carbamylcholine (I_mACh). The K_D values were 1.2 × 10⁻⁴ M for I_nACh) and 8.7 × 10⁻⁷ M for I_mACh. The reversal potenial of I_mACh was close to E_K. The I_mACh was determined to be elicited via the M₂ muscarinic receptor, based on the differences in sensitivity to muscarinic antagonists such as pirenzepine and AF-DX-116.     

Dual effect of verapamil on K-evoked release of endogenous dopamine from arcuate nucleus-median eminence complex

The effect of verapamil, a calcium-entrance blocker, on K⁺-evoked release of endogenous dopamine from tuberoinfundibular neurons incubated in vitro was studied. This compound, added to the incubation medium, at the dose of 10⁻⁶ M, significantly reinforced K⁺-induced dopamine release, whereas, at higher doses (10⁻⁵, 5 × 10⁻⁵ and 10⁻⁴ M), it completely prevented the stimulated dopamine release. The results obtained with the higher doses showed the calcium dependence of K⁺-evoked release of endogenous dopamine from central neurons. The opposite effect, seen with the lower dose of verapamil, could be due to different pharmacological properties of the drug.     

Characterization of CD28CD4 T cells in living kidney transplant patients with long-term allograft acceptance

CD28⁻CD4⁺ T-cell subpopulation is expanded in kidney allograft patients with long graft survival. To seek for the roles of CD28⁻CD4⁺ T cells in the long-term acceptance of kidney allografts, we characterized this population by analyzing cell surface molecules, TCR V_β repertoire, mixed lymphocyte reaction (MLR), and cytokine production. The number of CD28⁻CD4⁺ T cells increased correlatively with time after transplantation in this group of patients. The CD28⁻CD4⁺ T cells did not express detectable levels of CD25, CD69, V24, or CTLA-4 but expressed heterogeneous amounts of CD45 RA on the surface. Freshly sorted CD28⁻CD4⁺ T cells revealed a restricted V_β repertoire, whereas the V_β usage of CD28⁺CD4⁺ T cells from the same patients was much diversified. Expression levels of TGF-β and IFNγ gene were significantly higher in the CD28⁻ CD4⁺ T cells than in the CD28⁺CD4⁺ T cells from the kidney allograft patients. These findings suggest that an oligoclonal CD28⁻ CD4⁺ T-cell population is continuously activated in patients with long allograft survival, which may be linked with the long-term acceptance.     

The frequency of QAP2.1 is increased in psoriasis vulgaris patients

The heritage of psoriasis has a polygenic mode, the most essential antigens being Cw6,DR7,DQA1^*0201 in many ethnic groups. We have found a strong association between the high risk HLA haplotypes carrying DQA1^*0201 and psoriasis. Thus, the aim of this study was to further examine if the flanking promotor genes, URRs of DQ (QAP and QBP) are involved in the susceptibility to this disease. The series consisted of 62 patients and 50 control subjects and the PCR-SSO method was used. The frequency of the promoter gene QAP2.1 was significantly increased in the psoriatics (P_c=1.5×10⁻², RR=4.6) and the frequency of QAP4.1 was decreased in the patients group (P_c=3.9×10⁻²). The psoriasis risk allele DQA1^*0201 was associated always with QAP2.1, except once with QAP3.1.

: The combination of QAP2.1 and DQA1^*0201 is associated to psoriasis. Promotor region could exert its influence by gene expression or functionally through different inducibility by cytokines.     

A quantitative analysis of rat adrenal chromaffin tissue: Morphometric analysis at tissue and cellular level correlated with catecholamine content

A morphometric analysis of normal Wistar rat adrenal medulla following perfusion fixation and Araldite embedding, was correlated with catecholamine levels on fresh tissue, measured by high-performance liquid chromatography. The mean volume of whole adrenal is 13.2 mm³ and the mean medullary volume 1.3mm³. Volume density estimates showed that the medulla is composed of 63% chromaffin tissue with an adrenaline to noradrenaline storing cell ratio of 4.4:1. The vasculature occupies 20%, neuronal tissue 5% and interstitial tissues 12% of the medulla. A comparison was made of cell volumes, cell numbers and volume and surface density estimates of cytoplasmic organdies in adrenaline and noradrenaline storing cells. The mean cell volume of adrenaline storing cells at 1300 μm³ is larger than that of noradrenaline storing cells at 980 μm³. A single adrenal medulla contains4.4−5.7 × 10⁵ adrenaline cells and1.5−1.9 × 10⁵ noradrenaline cells. Chromaffin granules account for approximately 30% of the volume of the cytoplasm; the numerical density of granules at different sites in the cell was calculated for adrenaline cells. The volume density of mitochondria (4%) and the surface density of mitochondrial membranes (the ratio of outer to inner membrane being approximately 1:2.3) were similar in both cell types. Rough endoplasmic reticulum was the only organelle to show a significant difference in volume and surface density between the two cell types. Adrenaline storing cells have stacks of rough endoplasmic reticulum which have two to three times the surface and volume densities of that found diffusely scattered throughout noradrenaline cells. The adrenaline content of an adrenaline storing cell is0.14 × 10⁻⁶ μM and that of a granule 3.0 × 10⁻¹² or3.8 × 10⁻¹² μ moles depending on the method of calculation. The noradrenaline content of noradrenaline storing cells can only be calculated on the assumption that all noradrenaline is stored in this cell type though it is likely that some is contained within adrenaline cells. Based on this assumption the noradrenaline content is0.17 × 10⁻⁶μ moles per cell and5 × 10⁻¹² μ moles per granule. The present study provides baseline morphometric data on the rat adrenal medulla at tissue and cellular level correlated with amine levels in adrenaline and noradrenaline storing cells and granules.     

Trophic action of pharmacological substances with a guanidine group on mouse neuroblastoma cells and chick ganglionic neurons in culture

A series of substances (designated CTQ compounds) with a guanidine group have been synthesized and tested for their ability to promote neuronal survival and neurite outgrowth. Mouse neuroblastoma clonal cell lines grown in serum-containing medium for 10 days as well as primary cultures of embryonic chicken ganglion neurons grown in serum-free defined medium for 1 or 2 days have been used for the experiments. Among the various CTQ compounds (CTQ1–CTQ20) tested, only CTQ8 exerted positive neurotrophic effects on these peripheral neuronal cells. At a concentration of 10⁻⁴ M, CTQ8 enhanced neuritogenesis of neuroblastoma cells. However, the most striking influence of CTQ8 was its promoting effect (6- to 10-fold) on the survival of chicken ciliary and dorsal root ganglionic neurons at concentrations ranging from 10⁻³ M to 5×10⁻⁴ M.     

Human extracellular proteins display a different pattern of local sequence similarity with the four classes of human T-cell receptor V-regions than foreign proteins and human intracellular proteins: a preliminary report

A pool of 110 randomly selected/generated amino acids sequences was used to perform specific local sequence similarity alignment analysis with the pool of 279 reported sequences of human T-cell receptor (TCR) V-regions. The 110 analyzed sequences were divided, according to their origin and nature, into six protein groups, as: human intracellular (hi), extracellular/transmembrane (he) and extracellular adhesive matrix (ha) proteins, ‘average' human proteins (hum), proteins of non-human origin (nhum) and randomly generated quasi-protein sequences (r). These sequences were decomposed into all their overlapping 11-mer segments, generating a total of 56 836 derived peptides (at least 8000 per group). Each derived peptide was aligned with the 279 human TCR V-regions and assigned to the category (-like, β-like, γ-like or δ-like) corresponding to the class (V, Vβ, Vγ or Vδ) of the V-region encompassing the most similar segment, as determined by the performed similarity-search. The six protein groups were found to differ significantly in their distribution of derived peptides among the four categories. According to the binomial tests results, human proteins from the extracellular compartment (he, ha) comprise a higher proportion of δ-like segments (P=2.3×10⁻² and P<10⁻⁸, respectively) than the ‘average' human proteins (hum). In addition, and in accordance with this finding, proteins that are normally not found in that topological compartment comprise a lower proportion of δ-like peptides (P=1.4×10⁻⁵ and P<10⁻⁸ for groups nhum and hi, respectively) than the ‘average' human proteins (hum). In contrast, these proteins comprise a higher proportion of γ-like segments (P=8.3×10⁻³, P=1.4×10⁻³ and P=1.7×10⁻⁴, for groups r, nhum and hi, respectively) than the ‘average' human proteins (hum). These findings indicate significant differences between proteins encountered in the extracellular compartment—that are normally immunologically tolerated—and those the presence of which is usually non-tolerated. The results suggest that the discrimination and the reaction of the human immune network to proteins found in the extracellular compartment correlate with the proteins' pattern of preferential local sequence similarity with the Vγ and Vδ classes of human TCR V-regions, implying a specific and an important role of γδ-cells in the maintenance of the immune homeostasis. Whether this implication represents a rule associated with self-tolerance, will be investigated by future analyses.     

Effect of epitalon on the lifespan increase in Drosophila melanogaster

The geroprotector activity of epitalon, a synthetic tetrapeptide Ala–Glu–Asp–Gly, was studied on the Drosophila melanogaster wild strain Canton-S. The substance was added to the culture medium only at the developmental stage (from egg to larva). Epitalon significantly increased the lifespan (LS) of imagoes by 11–16% when applied at unprecedented low concentrations — from 0.001×10^–6 to 5×10^–6 wt.% of culture medium for males and from 0.01×10^–6 to 0.1×10^–6 wt.% of culture medium for females. The increase in LS did not depend on the substance dose. Effective concentrations of epitalon were 16 000–80 000 000 times lower than those of melatonin. The possible mechanisms of the antioxidant and regulatory effects of epitalon are discussed.     

Binding specificity of a class II-restricted hepatitis B epitope by DR molecules from responder and nonresponder vaccine recipients

A small but significant proportion of people who receive the hepatitis B vaccine do not produce anti-hepatitis B antibodies, a phenomenon associated with certain human leukocyte antigen (HLA) class II haplotypes. We were interested in determining whether natural allelic differences between two HLA-DR4 molecules associated with responder versus nonresponder subtypes differed with respect to binding of an immunodominant hepatitis B surface antigen (HBsAg) peptide as measured using a resonant mirror biosensor. In contrast to our original hypothesis, we found a ten-fold difference in the affinity in favor of the nonresponder DRB1*0401 allele, with a K_D of 6.89 × 10⁻⁸ M versus a K_D of 6.71 × 10⁻⁷ M for the responder DRB1*0404 allele. Half-times of dissociation were 1.3 min and 7.7 min, respectively, although association rate constants for both HLA class II molecules were similar (approximately 10⁴ M⁻¹s⁻¹). Of particular interest was the observation of different on-rates during the association phase, suggesting that stoichiometry of binding was not 1:1 or that different structural forms of the HLA-peptide complex exist. Our observations indicate that whereas HBsAg peptide binding to HLA class II molecules is influenced by HLA polymorphism, the nonresponse to hepatitis B vaccine associated with this HLA-DR4 subtype is not a result of failure of processed HBsAg to bind HLA class II molecules.     

Heterogeneity in terms of interleukin 4-dependent regulation of Fce receptor/CD23 expression on chronic B-lymphocytic leukemia cells

To discriminate the stages of maturation arrest of leukemic B cells, we have investigated the cell surface expression of FcεR_1l (H107 antigen) on leukemic B cells from 6 patients with chronic type B-lymphocytic leukemia(B-CLL) by a double staining method combined with cytoflorometry, and their production of soluble FceR_ll ⁺ by an ELISA technique. FceR_ll was expressed onμ⁺/δ⁻ cells of case 5 as well as on μ⁺/δ⁺cells of cases 1,2 and 4, but not on μ⁺/δ⁺cells in cases 3 and 6. The cultivation of leukemic cells with IL-4 not only increased the percentage of FceR_ll⁺cells but also enhanced the production of soluble Fcer_llin most cases. However, IL-4 had no effects on μ⁺/δ⁻/Fcεr_ll⁺ cells of cases 5, which appeared to correspond to a rather late     

Mechanism of histamine-induced calcium efflux from cultured bovine adrenal chromaffin cells: possible involvement of an Na/Ca exchange mechanism

The effect of stimulation of the histamine receptor on Ca²⁺ mobilization in cultured bovine adrenal chromaffin cells was examined. Histamine (10⁻⁵ M) increased the intracellular free Ca²⁺ ([Ca²⁺]_i) to a peak in the presence or absence of extracellular Ca²⁺, followed by decrease with time. Histamine (10⁻⁸–10⁻⁵ M) also stimulated ⁴⁵Ca²⁺ efflux from cultured bovine adrenal chromaffin cells in a concentration dependent manner. Its stimulatory effect on ⁴⁵Ca²⁺ efflux was inhibited by the specific histamine H₁ receptor antagonist mepyramine. The increase in histamine-stimulated ⁴⁵Ca²⁺ efflux was inhibited by deprivation of extracellular Na⁺ and by the Na⁺/Ca²⁺ exchange inhibitor amiloride. In addition, histamine stimulated ²²Na⁺ influx into the cells, and this action was inhibited by amiloride. These results suggest that stimulation of the histamine H₁ receptor regulates Na⁺/Ca²⁺ exchange in cultured bovine adrenal chromaffin cells.     

New modulated metallic macrocycles: Electrochemistry and their interaction with calf thymus DNA

New modulated pentacoordinate complexes [C₁₇H₃₄N₇O₂Cu]ClO₄ (5), [C₁₇H₃₄N₇O₂Co]ClO₄ (6), [C₁₇H₃₄N₇O₂Ni] ClO₄ (7) have been synthesized by the interaction of 1,8-dihydro-1,3,6,8,10,13 Cu(II) (2), Co(II) (3), Ni(II) (4) hexaazacyclotetradecane complexes and Hsalea N-(2-hydroxy benzyl)-2-amino-1-ethanol ligand (1). All the complexes have been characterized by infrared, electron paramagnetic resonance, ¹H and ¹³C nuclear magnetic resonance (NMR), 2D correlation spectroscopy NMR and ultraviolet–visible (UV–vis) spectroscopy. In all the complexes, the metal center is encapsulated by the Hsalea ligand in a pentacoordinate environment. Conductance measurements show that the complexes are ionic in nature. UV–vis absorption titration and viscometric studies have been carried out to ascertain the interaction of complexes 5 and 6 with calf thymus DNA (CT DNA). The experimental results suggest that complex 5 binds to CT DNA through partial intercalation of the aromatic ring into the base pair of DNA while complex 6 binds to CT DNA by electrostatic mode. The intrinsic binding constants K_b of complex 5 and 6 were found to be 6.8 × 10⁻⁵ M⁻¹ and 1.8 × 10⁻⁴ M⁻¹, respectively. The binding of complexes 5 and 6 with CT DNA has also been investigated by cyclic voltammetry.     

Biomechanics of the saphenous artery and vein in spontaneous hypertension in rats

Incremental compliance and distensibility of certain muscular arteries were recently reported to be normal or slightly increased in hypertension at the same pressure levels. In this work biomechanical properties of isolated perfused and superfused veins and large muscular arteries from saphenous bed from male spontaneously hypertensive (SHR) and Wistar–Kyoto (WKY) rats were compared in vitro. Outer diameter of cylindrical vessel segments was measured and intraluminal pressure (IP) was changed cyclically. We found larger contractile response to methoxamine (1.06×10⁻⁵ mol/l) in SHR arteries compared to WKY (active strain e.g. at 100 mmHg IP: 7.12±4.1 vs 0.35±0.46%). Resting incremental distensibility was higher (e.g. 100 mmHg IP: 3.4±0.4×10⁻⁶ vs 1.2±0.3×10⁻⁷ m²/N), elastic modulus lower (e.g. 100 mmHg IP: 3.7±0.6×10⁵ vs 27±7.6×10⁵ N/m²) in the arteries from SHR in pressure range of 60–110 mmHg. After papaverine administration (2.8×10⁻⁴ mol/l) the artery became more rigid, thus the increased incremental elasticity of SHR artery might be due to the enhanced smooth muscle tone. However, compared at in vivo pressure levels the differences were negligible suggesting a shift in the elastic parameters toward the higher operation pressures. Saphenous vein of SHR had larger diameter, than that of WKY, while in the wall thicknesses no difference were found (therefore external radius-wall thickness ratio was larger, e.g. at 6 mmHg IP: 15.9±3.0 vs 8.1±0.7). Consequently, lumen capacity of the vein was also higher in SHR, however, elastic parameters did not exhibit significant differences. We conclude that pressure-distensibility curve of muscular type arteries like SHR saphenous artery is shifted to higher pressure levels compared with that of normotensive controls. This shift is due to the enhanced smooth muscle contractility. The unchanged elasticity of veins suggests that the arterial deformations in SHR are not primary but secondary alterations.     

Permeability of amalgam restorative materials

The beneficial effect of fluoride-containing amalgam in preventing recurrent dental caries depends on the ability of the material to deliver fluoride (F). A two-chamber diffusion cell has been employed to monitor the diffusion of F in freshly prepared amalgam sections (membranes) as well as in amalgam sections stored in F solution for 15 d. The diffusion of ¹²⁵I was also monitored, as a reference. Five and ten successive measurements at 72 h intervals were made on the fresh and stored amalgam specimens, respectively. The average diffusion coefficient, D, of F and ¹²⁵I in fresh amalgam was 2.39 × 10⁻¹⁰ and 1.85 × 10⁻¹⁰ cm²/s, respectively. For stored amalgam, the average D of F during a 30 d experiment was 1.35 × 10⁻¹⁰ cm²/s. The average D of F in stored amalgam, during the first 15 d of the experiment, was 31 % less than in fresh amalgam (p < 0.01). A decline in the diffusion process was observed during the course of the experiments. During 15 d diffusion in fresh amalgam and 30 d diffusion in stored amalgam the cumulative diffused F were 0.79 and 0.88% of the F in the source. SEM findings revealed the deposition of corrosion products on amalgam stored for 3 months in 0.19% F solution.     

Mass transfer and metabolic reactions in hepatocyte spheroids cultured in rotating wall gas-permeable membrane system

Isolated hepatocytes in spheroid configuration exhibit a high degree of cell–cell contacts, which are important in the maintenance of viability and liver specific functions. In the absence of a vascular network, the cells in a large spheroid size experience mass transfer limitations of metabolites and oxygen in the core of aggregates. In this paper transport phenomena related to the diffusion and reaction of oxygen, glucose and lactate are mathematically described and experimentally verified for hepatocyte spheroids cultured in a rotating-wall polystyrene system (RWPS) not permeable for gases and in a rotating-wall membrane system (RWMS) with oxygen-permeable membrane. The concentration profiles of glucose, oxygen and lactate in the hepatocyte spheroids were estimated for different diameters of aggregates by solving the mass transfer equations for simultaneous diffusion and reaction, by finite element method. Simulation results evidenced that, for aggregates with size lower than 300 μm cultured in both RWPS and RWMS systems, the concentration profiles of glucose and lactate towards the core of spheroids (effective diffusion coefficients in the order of 10⁻¹¹ m²/s) are not significantly affected by the metabolic rate (c.a 10⁻⁶ μg/mm³/s for glucose and about one order of magnitude less for lactate). On the contrary, the transport of oxygen (diffusion coefficient: 3.4×10⁻¹⁰ m²/s, reaction rate: 1.5×10⁻⁵ μg/mm³/s) is critically affected by the size of the multicellular spheroids and significant gradients in oxygen concentration may develop in spheroids. Aggregates with a size greater than 200 μm suffer severe oxygen limitation in the most part of its size attaining the lowest partial pressure in the centre. The improved viability predicted by the model culturing hepatocyte spheroids in the RWMS, characterized by a higher O₂ permeability with respect to RWPS, was experimentally confirmed. The results demonstrated that the mathematical model used in this study represents a useful support to experimental procedures in order to obtain hepatocyte spheroids with optimal size.     

Interferon modulates the leukotriene C4-induced non-adherence properties of leukocytes: acquisition of an asthmatic phenotype

We have previously shown that peripheral blood leukocytes (PBL) of asthmatic patients acquire non-adherence properties after challenge with leukotriene C4 (LTC4), whereas PBL of normal individuals do not. Hence the use of the LTC4-induced leukocyte-adherence-inhibition (LAI) assay enables one to recognise an asthmatic phenotype on the basis of the ability of PBL to respond in vitro to LTC4. To examine the possibility that alpha interferon (IFN alpha) may have relevance to the pathogenesis of bronchial asthma, various concentrations of IFN were incubated with normal PBL and the acquisition of non-adhering properties was measured. We found that following 24 h incubation with 500 U/ml IFN, normal PBL were induced to respond to a standard dose of LTC4, and this reaction was abrogated by FPL 55712 and cyclohexamide.     

An assay to quantitate the binding of leishmania amastigotes to macrophages

A leishmania amastigote radiobinding assay has been developed using organisms labeled with tritiated uracil. These labeled amastigotes resemble freshly isolated unlabeled amastigotes in metabolic activity, bouyant density, morphology, viability and their ability to transform into promastigotes. Organisms routinely incorporate between 5 × 10⁻³ and 3 × 10⁻² cpm per amastigote, which allows the detection of as little as 1 × 10⁴ amastigotes per assay well. This radiolabeling technique has been used to quantitate the attachment of amastigotes to macrophages adherent to either 13 mm coverslips or to 96 well plates. It can also be used to screen monoclonal antibodies to macrophage surface proteins involved in amastigote binding. Once incorporated, the label remains amastigote associated, even after intact organisms have been internalized by macrophages. It remains parasite associated until the organisms have been degraded by macrophages, at which time label is released into the supernatant. Thus, a small adaptation of the binding assay can be used to compare the intracellular survival of amastigotes in macrophages following various experimental manipulations. This amastigote radiolabeling assay, therefore, represents an important step toward determining the receptors on macrophages involved in amastigote recognition and can also be used to study the degradation of intracellular pathogens by macrophages.