Nedocromil Research Articles

Effects of topical anti-inflammatory drugs on eosinophil survival primed by epithelial cells. Additive effect of glucocorticoids and nedocromil sodium

Eosinophil infiltration is a hallmark of the inflammatory response in rhinitis and in nasal polyposis. We studied the effect of steroids and nedocromil sodium on eosinophil survival primed by epithelial cells from healthy (nasal mucosa) and inflamed (nasal polyp) respiratory tissue. Blood eosinophils were incubated with increasing concentrations (10(-11)-10(-5) M) of topical steroids (fluticasone propionate, budesonide, triamcinolone acetonide and beclomethasone dipropionate) and/or nedocromil sodium prior to the addition of human epithelial cell conditioned media (HECM), eosinophil viability was measured and IC50 for each drug was calculated. All four steroids and nedocromil sodium caused a dose-related inhibition of HECM-induced eosinophil survival. The IC50 of steroids were lower in eosinophils primed by mucosa HECM than on those primed by polyp HECM (fluticasone, 4 nM vs 114 nM; budesonide, 21 nM vs 280 nM; triamcinolone, 7 nM vs 853 nM; and beclomethasone, 171 nM vs 181 nM). The combined inhibitory effect of 10(-7) M budesonide plus 10(-5) M nedocromil (43.8 +/- 10.8%, P<0.03) was significantly higher than budesonide (28.5 +/- 9.2%) or nedocromil (16.7 +/- 5.4%) alone and close to 10(-5) M budesonide (52.3 +/- 11%). No differences were found in cytokine (IL-8, IL-6, GM-CSF, TNF alpha, IL-1beta and RANTES) concentrations between HECM from mucosa and polyps. These results suggest that topical anti-inflammatory drugs may diminish airway eosinophilic infiltration by decreasing eosinophil viability, that nasal polyp epithelial cell secretions may induce steroid resistance in eosinophils, and that nedocromil sodium has additive effects with steroids.

Effects of topical anti-inflammatory drugs on eosinophil survival primed by epithelial cells. Additive effect of glucocorticoids and nedocromil sodium

Effects of topical anti-inflammatory drugs on eosinophil survival primed by epithelial cells. Additive effect of glucocorticoids and nedocromil sodium

The cellular and mediator basis of asthma in relation to natural history

The cellular and mediator basis of asthma in relation to natural history

Effect of topical anti-inflammatory drugs on epithelial cell-induced eosinophil survival and GM-CSF secretion.

Topical anti-inflammatory drugs decrease eosinophil infiltration. This action may be due to an effect on the release of epithelial cell products responsible for promoting eosinophil survival. We investigated the effect of fluticasone propionate, budesonide, beclomethasone dipropionate and nedocromil sodium on the release of granulocyte/macrophage colony-stimulating factor (GM-CSF) and on eosinophil survival induced by secretions from cultured nasal epithelial cells. Human epithelial cell-conditioned media (HECM) were generated by cultured epithelial cells obtained from healthy subjects undergoing corrective nasal surgery. Normodense eosinophils isolated from peripheral blood were incubated with HECM generated with and without the drugs. All of the drugs tested inhibited eosinophil survival, and response was dose-dependent. Fluticasone propionate had the highest inhibitory potency (25% inhibitory concentration (IC25) 1x10(-9) M), followed by budesonide (IC25 3.3x10(-8) M), beclomethasone dipropionate (IC25 1.5x10(-6) M), and nedocromil sodium IC25 5x10(-6) M). Likewise, fluticasone was the strongest steroid in inhibiting release of GM-CSF (IC25 8.4x10(-11) M), followed by budesonide (IC25 2x10(-9) M), beclomethasone dipropionate (IC25 13x10(-8) M), and nedocromil sodium (IC25 >10(-5) M). A significant correlation was found between both inhibitory effects (r=0.955; p<0.05). Topical anti-inflammatory drugs may decrease eosinophil survival by abrogating the promoting effect of epithelial cells. These drugs may exert part of their therapeutic effect by modulating GM-CSF release. The following rank of potency was observed: fluticasone propionate > budesonide > beclomethasone dipropionate > nedocromil sodium. The study of the interaction between epithelial cells and eosinophils may be a useful method for investigating and comparing the potency of topical drugs.

PAF-induced Secretory Hyperresponsiveness in the Ferret Trachea to Bradykinin and its Pharmacological Inhibition

Both PAF (10 μM) and bradykinin (0.1–10 μM) increased lysozyme (from submucosal gland serous cells (+138 and +45% for PAF, 10 μM, and bradykinin, 1 μM, respectively) and albumin (mainly active epithelial transport; +387 and +108%) outputs into the ferret tracheal lumen in vitro and reduced the negativity of the potential difference (PD: −33 and −17%) across the trachea. Since PAF can cause bronchial smooth muscle hyperresponsiveness, we tested whether these effects were interactive, and if PAF would increase the actions of bradykinin. The bradykinin-induced lysozyme and albumin outputs were more than trebled and the PD change was enhanced by PAF, after the immediate secretory effects of the latter had returned to baseline. The secretory and PD responses to PAF were all prevented by the PAF-antagonist WEB 2086 and by a combination of the free-radical scavengers catalase and SOD, indicating that PAF may act on specific receptors to release free-radicals. Nedocromil sodium inhibited the increase in lysozyme and albumin outputs produced by PAF, but had no effect on the PD response. None of the tracheal responses to bradykinin was modified by WEB 2086, catalase and SOD, or nedocromil sodium. The secretory and PD hyperresponsiveness to bradykinin caused by PAF was prevented by WEB 2086 and by catalase and SOD. Nedocromil sodium greatly inhibited the lysozyme and albumin hyperresponsiveness but had no effect on the PD response. Thus PAF may release more than one type of radical which have differential effects on serous cells and albumin transport compared with PD; nedocromil sodium may act only against the radical causing the secretory effects.

Preparation and subsequent degradation of poly( l-lactic acid) microspheres suitable for aerosolisation: a physico-chemical study

The encapsulation of nedocromil sodium and beclomethasone dipropionate with microspheres of poly( l-lactic acid) has been studied and preparation conditions optimised for entrapment efficiency and microsphere size suitable for inhalation. DSC was used to characterise the microspheres both in terms of drug/polymer interaction and influence of annealing conditions on the T g and degree of crystallinity. The absence of molecular interaction was confirmed by FTIR. Incubation of the microspheres in phosphate buffer at 37°C for 8 days demonstrated no chemical degradation of the polymer as evidenced by IR spectral analysis and estimates of percentage crystallinity. Surface morphology (SEM) and internal structure (TEM) were consistent with a homogeneous degradation pattern.

The effect of nedocromil sodium on human airway epithelial cell-induced eosinophil chemotaxis and adherence to human endothelial cell in vitro

Although some studies have shown that long-term treatment of asthmatics with nedocromil sodium can reduce airway hyperresponsiveness and improve symptoms and lung function, the mechanisms underlying its effects are not well understood. We have investigated the effect of nedocromil sodium on eosinophil chemotaxis, eosinophil adherence to human endothelial cells and release of soluble intercellular adhesion molecule-1 (sICAM-1) from endothelial cells, induced by conditioned medium collected from cultured human bronchial epithelial cells. Conditioned medium significantly increased eosinophil chemotaxis from a baseline median value of 2.1 (range 1.9-4.5) cells-high power field(-1) (HPF) to 10.5 (range 7.8-12.3) cells-HPF(-1) (p<0.05). Similarly, conditioned medium significantly increased eosinophil adherence to endothelial cells from a baseline value of 9 (range 8-12)% to 23 (range 21-30)% (p<0.05). Nedocromil sodium, at 10(-5) M concentration, significantly attenuated the eosinophil chemotaxis and adherence induced by conditioned medium. Conditioned medium also significantly increased the release of sICAM-1 from endothelial cells, from a baseline value of 11.5 (range 8.1-15.4) pg x microg(-1) protein to 67.6 (range 55.6-73.5) pg x microg(-1) protein (p<0.05). This was significantly attenuated by anti-tumour necrosis factor-alpha (TNF-alpha), anti-interleukin-1beta (IL-1beta) and 10(-5) M nedocromil sodium. These findings suggest that human bronchial epithelial cell-derived mediators may potentiate eosinophil activity, and that this can be modulated by nedocromil sodium, suggesting a possible mechanism underlying its anti-inflammatory effect.

Nedocromil sodium ameliorates skin manifestations in a murine model of chronic graft-versus-host disease.

Dermal fibrosis, loss of cutaneous appendages, and loss of subcutaneous fat are the main manifestations of chronic graft-versus-host disease (cGVHD) in mice. Mast cells (MC) may have a role in cGVHD. To investigate this point we have evaluated the effect of a MC stabilizer (nedocromil sodium) and a MC activator (compound 48/80) on skin manifestations in a murine model of cGVHD (B10.D2-->BALB/c). Mice were treated with nedocromil sodium (5 mg/day) or compound 48/80 (10 micrograms/day) from day -3 until day 15. Nedocromil ameliorated the skin features of cGVHD while compound 48/80 caused skin changes reminiscent of mild cGVHD in control mice. In addition nedocromil normalized peritoneal MC numbers and skin MC numbers and their activation state in the cGVHD mice. On the other hand compound 48/80 caused a complete disappearance of toluidine blue stainable peritoneal MC from cGVHD and control mice and decreased skin MC in controls. In summary, MC activation may play a negative role in the skin changes seen in cGVHD while MC stabilization ameliorates the skin manifestations of cGVHD and therefore may have a therapeutic benefit.

Physicochemical Characterization of Nedocromil Bivalent Metal Salt Hydrates. 2. Nedocromil Zinc

Salts are usually considered as alternatives for drug delivery when the physicochemical characteristics of the acidic or basic parent drug are unsuitable or inadequate for a satisfactory formulation. The physical, chemical, and biological characteristics of nedocromil sodium, which is used in the treatment of reversible obstructive airways diseases such as asthma, can be altered by its conversion to other salt forms. Nedocromil zinc (NZ), a bivalent metal salt, was found to exist in several hydration states, an octahydrate, a heptahydrate, and a pentahydrate, which itself exists in two modifications, designated as A and B. The relationships between these NZ hydrates and the nature of the water interactions in the solid phases were studied through characterization by differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), Karl Fischer titrimetry (KFT), hot-stage microscopy (HSM), ambient- or variable-temperature powder X-ray diffraction (PXRD), Fourier-transform infrared (FTIR) spectroscopy, solid-state nuclear magnetic resonance (SSNMR) spectroscopy, environmental scanning electron microscopy (ESEM), water uptake at various relative humidities (RH), intrinsic dissolution rate (IDR), and solubility measurements. The integral water stoichiometries of the NZ hydrates were deduced from KFT and TGA and were confirmed by elemental analysis. For the heptahydrate, the loss of 1mol of water at a higher temperature than for the others is attributed to an identifiable water molecule that is linked directly to the zinc and to two carboxylate oxygen atoms but not to the other water molecules, as deduced from the crystal structure previously determined. Similarly, for both pentahydrate modifications, 1mol of water was also lost at a higher temperature than the others. Results from studies using DSC, TGA, HSM, PXRD, SSNMR, and FTIR suggested that the octahydrate contains loosely bound water in its structure and is partially amorphous. The course of the dehydration processes depended on the water vapor pressure and temperature. The octahydrate and heptahydrate underwent an apparently irreversible phase transformation to the pentahydrate at an elevated temperature and water vapor pressure. Pentahydrate modifications A and B differ in their long-range order (deduced from differences in their PXRD pattern and their thermal analytical behavior), but their short-range order (i.e., molecular environments) are identical (deduced by identical SSNMR spectra). The rank order of both IDR and solubility in water at 25°C was octahydrate>heptahydrate>pentahydrate modification A ≈ pentahydrate modification B, corresponding to the rank order of free energy with respect to the aqueous solution and the order of preparation according to Ostwald's rule of stages.

Development and validation of an ion-pair liquid chromatographic method for the quantitation of sodium cromoglycate in urine following inhalation.

An ion-pair liquid high-performance chromatography method with solid-phase extraction for measuring urinary concentrations of sodium cromoglycate following inhalation has been developed and validated. Sodium cromoglycate was extracted from urine on a 100-mg phenyl cartridge (Isolute, Jones Chromatography) and then quantified on a 25-cm C8 Spherisorb 5 microns stationary phase with a mobile phase of methanol-0.045 M phosphate buffer-0.05 M dodecyl triethyl ammonium phosphate (550:447.6:2.4, v/v) pH 2.3, at 0.85 ml min-1 using nedocromil sodium as an internal standard and UV detection at 238 nm. The inter- and intra-day reproducibilities were 8.33 and 13.63%, respectively, at 0.25 mg l-1. The limit of determination for sodium cromoglycate was 0.25 mg l-1 (with a signal-to-noise ratio of greater than 10:1). Following oral and inhaled administration of 20 mg of sodium cromoglycate to eight healthy volunteers, the mean and S.D. of sodium cromoglycate excreted in the urine at 0.5, 1 and 24 h post-dose were 0.02, 0.05 and 0.33%, and 0.16, 0.30 and 1.55% of the dose, respectively. The urinary recovery of sodium cromoglycate at 0.5 and 1 h following inhalation can therefore be used to compare the amount of drug reaching the respiratory tract using different sodium cromoglycate inhaled products or inhalation methods.

Comparison of effects of topical levocabastine and nedocromil sodium on the early response in a conjunctival provocation test with allergen

BACKGROUND:Multiple ocular challenges or seasonal trials have demonstrated the efficacy of levocabastine and nedocromil sodium in the treatment of allergic conjunctivitis. OBJECTIVE: This study was designed to compare the protective effect of levocabastine eye drops with that of nedocromil in a conjunctival provocation test with allergen. METHODS: Twenty-four patients with allergic conjunctivitis to grass pollen were recruited. After a preliminary provocation to determine conjunctival reaction threshold (erythema of at least 50% of the conjunctiva with ocular itching), patients were randomized to receive either topical levocabastine (0.05%) or nedocromil (2%) 15 minutes before provocation. Erythema and pruritus intensity were recorded at each concentration of allergen up to the reaction threshold. RESULTS: The allergen concentration level necessary to reach reaction threshold was increased ( p < 0.001) after treatment with both drugs. Comparison between screening and each treatment indicated that the shift in allergen concentration was significantly greater after levocabastine treatment than after nedocromil treatment ( p = 0.019). Conjunctival itching (symptom score) and erythema (percent conjunctival surface) were also better controlled by levocabastine than by nedocromil during provocation ( p < 0.05). CONCLUSION: In a provocation test with allergen, levocabastine and nedocromil were both effective in increasing the conjunctival tolerance to allergen, with better protection provided by levocabastine. (J Allergy Clin Immunol 1996;98:1045-50.)

Ozone-induced mediator release from human bronchial epithelial cells in vitro and the influence of nedocromil sodium.

Although animal and human studies have demonstrated that ozone inhalation leads to airway epithelial inflammation and damage, the underlying mechanisms are not fully understood. We cultured human bronchial epithelial cells as explant cultures and investigated the effect of 6 h of exposure to 0-500 parts per billion (ppb) O3 with or without 10(-5) M nedocromil sodium on: 1) epithelial cell membrane integrity; and 2) release of inflammatory cytokines and soluble intercellular adhesion molecule-1 (sICAM-1), as assessed by enzyme-linked immunosorbent assay (ELISA). O3 exposure led to significant epithelial cell damage at concentrations of 10-500 ppb O3, as indicated by increased release of [51Cr]-labelled sodium chromate. At concentrations of 10-100 ppb, O3 induced maximal release of interleukin-8 (IL-8), granulocyte/macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor-alpha (TNF-alpha) and sICAM-1. The IL-8 and GM-CSF release increased significantly from 5.64+/-0.58 and 0.04+/-0.03 pg x microg(-1) cellular protein, respectively, from control cells exposed to air, to 20.16+/-2.56 and 0.20+/-0.04 pg x microg(-1) cellular protein, respectively, from cells exposed to 50 ppb O3. 10(-5) M nedocromil sodium significantly attenuated the O3-induced release of both IL-8 and GM-CSF (p<0.01). The TNF-alpha and sICAM-1 increases after exposure to 10-50 ppb O3, were also abrogated by treatment of the cells with 10(-5) M nedocromil sodium (p<0.05). Similarly, the antioxidant, glutathione, at concentrations of 400-600 microM, significantly reduced the O3-induced release of IL-8 (p<0.05). In conclusion, these studies indicate that ambient concentrations of ozone may induce airway inflammation, through release of proinflammatory mediators from airway epithelial cells. This effect may be inhibited both by the anti-inflammatory drug, nedocromil sodium, and the naturally occurring antioxidant glutathione.

Nedocromil sodium and airway inflammation in vivo and in vitro

We conducted a series of studies investigating the antiinflammatory effects of nedocromil sodium, with particular reference to its effects on human bronchial epithelial cells and eosinophils in vitro and on eosinophils in vivo. Nedocromil sodium produced a dose-related inhibition of ozone-induced IL-8 release from human bronchial epithelial cells and also attenuated the release of granulocyte macrophage colony-stimulating factor, tumor necrosis factor–α, and soluble intercellular adhesion molecule 1. The culture medium from human bronchial epithelial cell cultures, containing the proinflammatory cytokines IL-8, granulocyte macrophage colony-stimulating factor, “regulated on activation, normal T expressed and secreted,” IL-1β, and tumor necrosis factor–α, increased eosinophil chemotaxis and eosinophil adhesion to cultured human endothelial cells. The chemotaxis and increased adhesion were blocked in the presence of nedocromil sodium. The drug also abrogated the epithelial cell dysfunction (assessed as ciliary beat frequency) induced by the presence of activated eosinophils and blocked the release of eosinophil cationic protein from the eosinophils. We also conducted a double-blind placebo-controlled study of the effects of regular albuterol 200 μg or nedocromil sodium 4 mg, both given four times daily for 16 weeks, on inflammatory cell numbers in bronchial biopsy and bronchoalveolar lavage samples. Assessed in terms of total and activated eosinophils in biopsy samples, inflammation decreased with nedocromil sodium and was significantly different from a deterioration with albuterol, although neither of these changes was significantly different from that with placebo treatment. Levels of eosinophil cationic protein in bronchoalveolar lavage samples showed a similar trend. (J A LLERGY C LIN I MMUNOL 1996;98:S51-7.)

Clinical effects of nedocromil sodium on allergen-related mechanisms

Many studies have demonstrated the ability of nedocromil sodium to block the acute response to allergen in persons who are sensitive to allergen. One study with subjects sensitive to ragweed showed a significant protective effect after a single 4 mg dose of nedocromil sodium, with no further protection achieved at higher doses. Numerous studies have also shown the ability of the drug to reduce or abolish the late asthmatic response in sensitive subjects when it was given before allergen challenge. Nedocromil sodium has also been shown to prevent the subsequent development of bronchial hyperresponsiveness. In addition, nedocromil sodium prevented the late asthmatic response when given after allergen challenge and an intact early response. These findings suggest that nedocromil sodium may be preventing the inflammatory events initiated by mast cell mediator and cytokine release. (J A LLERGY C LIN I MMUNOL 1996;98:S118-23.)

Effects of nedocromil sodium on airway neurogenic mechanisms

Evidence shows that nedocromil sodium has a major inhibitory effect on sensory nerve activation. Animal models in which inhibitory effects have been demonstrated include bradykinin- or ovalbumin-induced plasma extravasation; cigarette smoke– or sulfur dioxide–induced bronchial hyperresponsiveness and increase in inflammatory cells in the airway; and bradykinin-induced airway vasodilatation and nasal mucosal edema. Nedocromil sodium has prevented the edema in human skin induced by substance P and neurokinin A, and, in the isolated rabbit trachea, has prevented substance P–induced potentiation of cholinergic neural responses at preganglionic (but not postganglionic) sites. In vitro, the drug also has inhibited nonadrenergic noncholinergic bronchoconstriction in guinea pig bronchi. Although a protective effect against citric acid–induced cough in the dog has been reported, no data are available from models of enhanced cough reflex, such as that in asthma. Inhibition of sensory nerve activation and prevention of tachykinin release by nedocromil sodium probably contribute to its beneficial effects in the treatment of asthma. (J A LLERGY C LIN I MMUNOL 1996;98:112-7.)

Effects of nedocromil sodium on inflammation and symptoms in therapeutic studies

This review cites clinical evidence demonstrating that nedocromil sodium can reverse the clinical features of airway inflammation in asthma, as shown by decreased bronchial responsiveness, decreased diurnal variation in peak expiratory flow, improved baseline lung function, and a decrease in the frequency and severity of chronic symptoms. Clinical trials have also shown that nedocromil sodium has a statistically significant and clinically relevant effect on daytime and nighttime asthma symptoms within 3 days of starting treatment. On an analysis of relevant trials, the effect on asthmatic cough was significant within 24 hours. This rapid effect on symptoms, together with a reversal of the underlying airways inflammation, is a beneficial combination in the management of asthma. (J A LLERGY C LIN I MMUNOL 1996;98:S143-50.)

Effect of regular nedocromil sodium or albuterol on bronchial inflammation in chronic asthma

Nedocromil sodium is recommended for daily treatment of mild persistent asthma but its effect on cellular changes in asthmatic airways is poorly understood. We compared the antiinflammatory effects of nedocromil sodium and albuterol in 32 patients with asthma who received either nedocromil sodium 4 mg or albuterol 0.2 mg four times daily for 12 weeks according to a double-blind protocol. Patients underwent fiberoptic bronchoscopy, and lung function and bronchial responsiveness to histamine were measured. Numbers of inflammatory cells were studied by immunohistochemistry and image analysis. We were unable to identify any statistical differences between treatment groups for any of the variables measured. We conclude that our immunohistochemical study does not support the concept of nedocromil sodium as a potent/antiinflammatory drug. (J A LLERGY C LIN I MMUNOL 1996;98:S58-66.)

Effect of nedocromil sodium on neurogenic mechanisms in vitro

We have developed a guinea pig model that allows monitoring of single afferent nerve fiber activity after stimulation of tracheal nerve endings with various stimuli. Action potentials from nodose or jugular neurons are recorded extracellularly. Previous experiments have shown that the excitability of the nerve endings to, for example, mechanical stimuli can be increased by antigen challenge of presensitized guinea pigs. Nedocromil sodium 10 -4 mol/L significantly reduced by more than 50% the generation of action potentials in C fibers in response to stimulation by capsaicin. The precise mechanism is not known, although the membrane depolarization induced by capsaicin was reduced in the presence of nedocromil sodium. This agent did not have a generalized inhibitory effect on afferent excitability because it had no effect on sensitivity to electrical or mechanical stimuli. Nedocromil sodium did not affect the generation of action potentials in Aδ and C fibers in the presence of hypertonic saline solution. (J A LLERGY C LIN I MMUNOL 1996;98:S107-11.)

Clinical effects of nedocromil sodium on challenges invoking neuronal mechanisms and on virally induced symptoms

Although sensory nerve activity may be important to the human airway in numerous possible ways, the relevance of “neurogenic inflammation” to the onset and development of asthma is unknown. However, several of the symptoms of asthma (bronchoconstriction, cough, and dyspnea) have a neuronal component that can be modeled in the laboratory by various stimuli that are thought to invoke sensory nerve activation. Nedocromil sodium is highly effective against bronchoconstriction induced by bradykinin, the tachykinins substance P and neurokinin A, and sulfur dioxide and metabisulfite. The results for induced cough in healthy subjects are equivocal, although the drug is effective on spontaneously occurring cough in patients with asthma. Nedocromil sodium had a modest but significant effect on symptoms associated with episodes of viral infection. (J A LLERGY C LIN I MMUNOL 1996;98:S135-42.)

Effect of nedocromil sodium pretreatment on the immediate and late responses of the airway to segmental antigen challenge

In many patients with asthma, exposure to allergen results in worsening symptoms, pulmonary dysfunction, and increased airway inflammation. In this study, segmental allergen challenge and bronchoalveolar lavage were used to evaluate the airway responses at 5 minutes and 48 hours and to study the extent of inhibition of the response by pretreatment with nedocromil sodium in eight subjects with allergic asthma in a double-blind, placebo-controlled trial. At 5 minutes after challenge, nedocromil sodium pretreatment significantly reduced histamine concentrations, with a trend toward a concomitant reduction in tryptase levels. Nedocromil sodium did not affect the increase in total protein, total cell counts, or cell concentrations that occurred 48 hours after challenge, but it did significantly reduce eosinophil recruitment. Eosinophil activation, assessed as release of granule proteins, was unaffected. A significant positive correlation was shown between the degree of histamine reduction at 5 minutes and the degree of reduction of eosinophil influx at 48 hours, raising the possibility that eosinophil influx into the airway may depend on mediators or cytokines released during the immediate response to allergen. (J A LLERGY C LIN I MMUNOL 1996;98:S46-50.)