Development of a monoclonal anti-immunoglobulin E antibody (omalizumab) for the treatment of allergic respiratory disorders.

Immunoglobulin E (IgE) mediates many of the inflammatory processes that underlie the symptoms of asthma and other allergic respiratory disorders. Recently, a recombinant, humanized, monoclonal antibody (mAb) that binds to and neutralizes IgE has been developed for the treatment of these disorders. Preclinical and clinical studies have shown that this mAb, directed against IgE and known as omalizumab, inhibits the binding of IgE to its receptors on effector cells, reduces IgE synthesis by B cells in response to allergen exposure, decreases the expression of IgE receptors, and attenuates both immediate and delayed inflammatory airway responses following exposure to inhaled allergen. Omalizumab is nonanaphylactogenic, and clinical experience to date suggests that omalizumab is safe and well tolerated by patients. These results suggest that specific inhibition of IgE may be an important new therapeutic option for the treatment of asthma and related disorders.

Degranulation influences heparin-associated inhibition of RT-PCR in human lung mast cells.

BACKGROUND: The study of gene expression from human lung mast cells (HLMC) has been limited by the ability to reliably detect mRNA transcripts from scant quantities of mast cell RNA contaminated with heparin. OBJECTIVE: As heparin is granule-associated within the mast cell, we examined the role of degranulation in altering the intrinsic ability of this proteoglycan to inhibit reverse transcription polymerase chain reaction (RT-PCR) from HLMC RNA. We also explored alternative means of RNA isolation to eliminate primary heparin contamination. METHODS: Purified HLMC (> 90% pure) were challenged for 2 h with buffer, anti-IgE (3 microg/mL) and/or ionophore A23187 (100 ng/mL) or phorbol 12-myristate 13-acetate (50 ng/mL). Histamine release was measured using a spectroflourometric assay. Following challenge, RNA was isolated by either phenol-chloroform extraction or by nitrocellulose spin column. Parallel samples were either treated with heparinase or placed on ice for 2 h prior to reverse transcription. PCR was performed using primers specific for the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). RESULTS: In each of five studies, GAPDH bands were detected at greater intensity in total cellular RNA (tcRNA) derived from degranulated than from non-degranulated mast cells. However, when examining heparinase-treated tcRNA from the same mast cell samples, the intensity of GAPDH bands normalized between degranulated and non-degranulated cells. When comparing parallel samples of column-purified tcRNA, subsequent treatment of samples with heparinase had no effect on the detection of GAPDH, indicating that endogenous heparin was effectively removed by the technique. Moreover, no variability was noted in GAPDH signal detected from resting vs. degranulated mast cells (n = 3). CONCLUSIONS: Degranulation influences the degree of heparin-associated inhibition of RT-PCR in HLMC, and that spin column purification of tcRNA is a time-saving, effective alternative to heparinase pre-treatment.

Effects of purine and pyrimidine nucleotides on intracellular Ca2+ in human eosinophils: activation of purinergic P2Y receptors.

BACKGROUND: Extracellular adenosine 5'-triphosphate (ATP) increases human eosinophil intracellular Ca(2+) concentration; the mechanism of action is not fully known. ATP, a physiologic regulator, acts through 2 purinergic receptor types: cation channels (P2X) and G protein-coupled receptors (P2Y). OBJECTIVE: This study is aimed at identifying the functional purinergic receptors in human eosinophils. METHODS: The relative potency of ATP, uridine (UTP), cytidine (CTP), and inosine (ITP) 5'-triphosphates (P2Y agonists); 2-methylthio-ATP (P2Y(1) agonist); and 2 P2X agonists, alpha,beta-methylene-ATP and beta,gamma-methylene-ATP on intracellular Ca(2+) concentration was examined in Ca(2+)-sensitive Fura-2-labeled human eosinophils. For comparison, ATP effects were similarly studied in human neutrophils. P2X/P2Y mRNA expression in cells was examined by reverse transcription and PCR. RESULTS: The nucleotide potency order was UTP > or = ATP > ITP >>> 2-methylthio-ATP > alpha,beta-methylene-ATP = beta,gamma-methylene-ATP = CTP = 0 in eosinophils. Pertussis toxin (500 ng/mL) pretreatment abolished the effect of lower (10(-6) mol/L) but not higher (10(-5) mol/L) concentrations of ATP in eosinophils, whereas it attenuated the effects of 10(-4) mol/L ATP in neutrophils. The phospholipase C inhibitor U73122 (2 micromol/L) partially inhibited the effect of ATP in eosinophils but totally blocked it in neutrophils. Both cells constitutively express mRNA for P2X(1), P2X(4), P2X(5), P2Y(1), and P2Y(2), but not P2X(7), with much weaker expressions of P2X(4) and P2X(5) in neutrophils. Eosinophils cultured with the T(H)1 cytokine, IFN-gamma, expressed mRNA for P2X(7), a receptor linked to apoptosis. CONCLUSIONS: These results suggest that the P2 purinergic receptor signal transduction pathways in eosinophils and neutrophils are different and are mediated by more than 1 subtype of functional P2Y receptors.

Hydrogen peroxide release from human eosinophils on fibronectin: scopoletin enhances eosinophil activation.

We have examined the release of H(2)O(2) from PAF or TNFalpha-stimulated human eosinophils on fibronectin (FN)-coated polystyrene plates. H(2)O(2) release was measured by the standard scopoletin-horseradish peroxidase (SCOP-HRP) method and compared with that measured by a new microplate fluorescent assay for H(2)O(2) using a novel HRP substrate A6550. We observed that the SCOP-HRP method gave a 25-fold higher estimate of H(2)O(2) release from eosinophils than did the A6550-HRP method. Microscopic examination of PAF or TNFalpha-stimulated eosinophils in buffer alone or A6550-HRP reaction mixture showed that the cells remained generally round, while eosinophils in SCOP-HRP reaction mixture were spread on the fibronectin-coated surface. Measurement of the cellular ATP content after PAF-stimulation showed that only eosinophils activated in SCOP-HRP had a 50% fall in ATP content. This supported our conclusion that measurement of H(2)O(2) release from eosinophils in SCOP-HRP reaction mixture is problematic since the SCOP-HRP system activates eosinophils. However, we also found that A6550-HRP, when present throughout the incubation, resulted in a lower estimate of H(2)O(2) release than expected. The method used to detect eosinophil H(2)O(2) release greatly influences the absolute amount of H(2)O(2) detected.

ATP modulates anti-IgE-induced release of histamine from human lung mast cells.

Adenosine 5'-triphosphate (ATP) is released from the cytoplasm under physiologic and pathophysiologic conditions and enters the extracellular space, where it acts on a group of recently cloned cell-surface receptors termed P2-purinoceptors (subtypes P2X and P2Y). We examined the effects of extracellular ATP, uridine triphosphate (UTP), the stable ATP analogues alpha,betamethylene-ATP (alpha,betamATP), beta,gammamethylene-ATP (beta,gammamATP), and 2-methylthio-ATP (2mSATP), and adenosine (10(-6)-10(-3) M) on histamine release from human lung mast cells (HLMC) induced by anti-IgE and the calcium ionophore A23187. None of the nucleotides or adenosine directly induced histamine release. Adenosine exhibited a bimodal effect, enhancing histamine release at 10(-6) to 10(-4) M (P > 0.05, NS) and inhibiting it at 10(-3) M (P < 0.05). ATP (10(-4) M) enhanced anti-IgE-induced histamine release (10.9 +/- 2.7% to 19. 2 +/- 2.9%, n = 20, P < 0.01), but not ionophore A23187-induced histamine release (n = 10). The adenine nucleotides consistently enhanced anti-IgE-induced histamine release; the rank order for this action was: ATP > 2mSATP > alpha,betamATP > beta,gammamATP, suggesting mediation by a P2Y-purinoceptor subtype. The selective P2X purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2', 4'-disulfonic acid failed to influence the effect of ATP, further supporting P2Y-purinoceptor mediation of anti-IgE-induced histamine release. UTP, an agonist at P2Y-purinoceptors, also significantly enhanced anti-IgE-induced histamine release. Application of the reverse transcription-polymerase chain reaction indicated that HLMC constitutively express the messenger RNAs encoding the P2Y1- and P2Y2-purinoceptor subtypes, and not that encoding the P2X7-purinoceptor (i.e., P2Z), a subtype implicated in ATP-induced histamine release in rodent peritoneal mast cells. The data produced in the study suggest that ATP plays an important modulatory role in histamine release from HLMC, and that it may therefore be mechanistically involved in human allergic/asthmatic reactions.

A highly sensitive fluorescent micro-assay of H2O2 release from activated human leukocytes using a dihydroxyphenoxazine derivative.

This study describes a simple, reliable, highly sensitive and quantitative fluorescence microplate-assay of H2O2 from activated leukocytes using a novel horse radish peroxidase (HRP) substrate N-acetyl-3,7-dihydroxyphenoxazine (A6550). Unlike the widely used fluorescent HRP substrate scopoletin, A6550 is non-fluorescent and becomes highly fluorescent upon HRP-catalyzed H2O2 oxidation. Using 50 microM A6550, the change in fluorescence due to H2O2 generated from phorbol 12-myristate 13-acetate-activated human eosinophils and neutrophils is found to have a linear cell dose response up to 1.5 x 10(4) and 5 x 10(4) cells, respectively. The increase in fluorescence from A6550 is specifically due to H2O2 generation since it is inhibitable by catalase. Oxidized A6550 is found to be highly stable and the H2O2 dose response is linear as long as the ratio of A6550:H2O2 in the reaction mixture is higher than five. Unlike scopoletin, A6550 has a very low background, which changes little with time. In addition, the high fluorescent yield of oxidized A6550 results in an increased sensitivity for the detection of H2O2. When the concentrations of A6550 and HRP were 10 microM and 0.2 U/ml, respectively, as low as 2 pmol of H2O2 could be reliably measured. The sensitivity of A6550/H2O2 assay is found to be at least 10-fold higher than with scopoletin as the HRP substrate. The protocol described in this study using A6550 to measure H2O2 release from activated granulocytes can be easily adapted to other cell types which generate H2O2.

Human lung mast cell activation leads to IL-13 mRNA expression and protein release.

Using reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), we studied the generation of the recently described Th2 cytokine interleukin-13 (IL-13) by anti-IgE-activated lung fragments (LF), lung mast cells (LMC), and the mast cell line HMC-1. We found that IL-13 messenger ribonucleic acid (mRNA) was constitutively expressed in LF and rapidly increased after anti-IgE challenge, persisting throughout a 16-h period. Quantitative-competitive PCR (QCPCR) demonstrated an increase from 1.2 fg to 120 fg of IL-13 mRNA/micrograms LF total cellular RNA. Time-course experiments showed that IL-13 protein was not increased in supernatants at 2 h after activation, but was upregulated by 8 h. Anti-IgE-activated LF supernatants contained 592.1 +/- 314.8 pg IL-13/g wet weight of tissue at 24 h (mean +/- SE; n = 11). LMC demonstrated upregulation of IL-13 mRNA expression following treatment with A23187 (n = 4), with maximal upregulation by 3 h; anti-IgE or phorbol myristate acetate (PMA) also led to increased IL-13 mRNA expression. QCPCR analysis of LMC IL-13 mRNA expression at 4 h after activation showed a 7-, 13.8-, and 13.2-fold increase after A23187, anti-IgE, and PMA, respectively. Quantities of IL-13 released from optimally activated LMC and peripheral blood T cells were comparable. HMC-1 also showed enhanced IL-13 mRNA beginning 30 min after A23187 activation, with peak expression from 1 to 10 h, followed by waning over the subsequent 24 h. A23187 stimulation of HMC-1 led to 100-fold upregulation of IL-13 mRNA within 4 h and detectable IL-13 in 24-h supernatants. These results demonstrate that activation of LF and LMC through multiple signal-transduction pathways results in increased IL-13 mRNA and protein expression temporally consistent with a potential role in chronic allergic inflammation.

Human lung mast cell IL-5 gene and protein expression: temporal analysis of upregulation following IgE-mediated activation.

The late-phase of allergic asthma is characterized by infiltration of the airway with eosinophils within 6 h of mast cell activation. Pro-eosinophilic/pro-allergic (TH2) cytokines, originally described as T-lymphocyte products, have recently been ascribed to mast cells as well. To date, however, it is unknown if TH2 cytokine gene expression by the human mast cells is subject to receptor-mediated regulation analogous to that of T-cells, and if messenger RNA (mRNA) expression results in protein secretion occurring in a temporal context consistent with the late-phase response. We examined interleukin-4 (IL-4), IL-5, and IL-6 mRNA expression induced by anti-IgE activation of human lung explants as assessed using reverse transcription/polymerase chain reaction (RT-PCR). Anti-IgE stimulation resulted in rapid and sustained upregulation of IL-5 message, but did not have analogous effects on IL-4 or IL-6. Using quantitative-competitive PCR, we demonstrated that 100 ng of total cellular RNA from human lung contained 1 fg of IL-5 mRNA; this increased to 100 fg 4 h after anti-IgE activation. The source of the anti-IgE-enhanced IL-5 mRNA is likely the mast cell itself, as anti-CD3 activation of lung led to a dissimilar array of cytokine expression. In addition, human lung mast cells purified to near homogeneity expressed IL-5 mRNA after activation, as shown by both RT-PCR and in situ hybridization. In both lung fragments and purified human lung mast cells, the modulation of IL-5 mRNA expression preceded the secretion of IL-5 protein, detected as early as 4 h after activation. Neither isolated purified mast cells nor purified peripheral blood T cells could be induced to secrete detectable amounts of IL-5 protein when activated only with antibodies against IgE or CD3-T cell receptor complex, respectively. However, mast cells (n = 4) and T cells (n = 6) cultured at comparable concentrations (4 x 10(6)/ml) activated through their respective antigen receptors in the presence of phorbol ester yielded comparable IL-5 production (253 +/- 126 pg/ml versus 183 +/- 75 pg/ml, mean +/- SE). We conclude that mast cells are analogous to T cells in the requirement of co-stimuli for the production of IL-5 protein. Moreover, the rapid kinetics of IgE-mediated IL-5 transcription and protein elaboration are consistent with a primary role for mast cell activation directly leading to late-phase airway eosinophilia.

IgE-dependent expression of interleukin-5 mRNA and protein in human lung: modulation by dexamethasone.

mRNA and protein expression of the Th2 cytokines IL-4 and IL-5 from human lung were examined during the first 4 hr following IgE-mediated triggering, a time representative of the evolving late-phase reaction (LPR). Lung explants were incubated for 16 hr at 37 degrees C in culture media alone or with added dexamethasone (10(-6) M), washed, and then challenged with buffer or anti-IgE (3 micrograms/ml). Using RNase protection assays, in 16/16 individual lungs IL-5 mRNA expression was observed at 4 hr following anti-IgE and at no points following buffer challenge. Fragments released 1129 +/- 499 ng of IL-5/g wet wt over a 24-hr period (mean +/- SEM, n = 5). Neither IL-4 transcripts nor protein were detected in any anti-IgE challenges. Both the IgE-mediated IL-5 mRNA and protein responses were below the limits of detection following dexamethasone preincubation, suggesting a mechanism for the potent inhibitory effects of these agents observed in the LPR.

Effects of colchicine on IgE-mediated early and late airway reactions.

BACKGROUND: The pathogenesis of bronchial asthma is thought to involve elements of both acute and chronic inflammation. Hence, there is growing interest in the potential of immunomodulatory drugs in asthma therapy. This study examines the effects of the anti-inflammatory compound colchicine on early and late allergen-induced, IgE-mediated airway reactions. METHODS: Nine mildly allergic asthmatic subjects were evaluated in a single-blind, two-way crossover study designed to examine the effects of colchicine and placebo on early and late airway reactions to ragweed allergen and related changes in nonspecific responsiveness to methacholine. RESULTS: Compared with placebo, colchicine provided 19% (p = 0.036) and 40% (p = 0.004) inhibition of early and late airway reactions to allergen, respectively. Allergen-induced increases in methacholine responsiveness were observed with both types of treatment, although there was a trend toward a smaller increase after administration of colchicine (p = 0.13). We also found that methacholine responsiveness per se was not directly altered by colchicine (n = 7). In 6 subjects, we found suppression of neutrophil leukotriene B4 generation after colchicine treatment, suggesting that the colchicine dose (0.6 mg twice daily) was sufficient to produce an anti-inflammatory effect. Further in vitro studies using purified human lung tissue mast cells failed to demonstrate inhibition of mediator release at concentrations corresponding to achievable tissue or blood levels during the in vivo trial. CONCLUSION: Colchicine partially inhibits IgE-mediated early and late airway reactions at conventional clinical doses. This inhibitory effect may be mediated via suppression of some cell species other than the lung tissue mast cell. Controlled studies to examine the benefits of colchicine in clinically evidenced asthma are warranted.

Pharmacologic characterization of a novel histamine receptor on human eosinophils.

There is increased recognition that lung mast cell mediators not only produce the symptoms of acute asthma, but also result in the recruitment and activation of additional proinflammatory cells, such as eosinophils. Histamine, one of the major mast cell mediators, is known to have numerous effects on eosinophil function. These effects of histamine are mediated by distinct receptors on the surface of eosinophils, only some of which have been characterized. Prior studies have suggested that eosinophils have non-H1, non-H2 histamine receptors which mediate the chemotactic effects of histamine. We observed previously that the histamine-induced increase in cytosolic calcium in human eosinophils could not be blocked by classic H1 or H2 antagonists, but could be inhibited by the H3 antagonist thioperamide. The purpose of this study was to further characterize the pharmacologic properties of this calcium-linked histamine receptor. Using Fura-2 loaded eosinophils to measure the concentration of cytosolic calcium, we examined the effect of additional histamine receptor antagonists and agonists. We found that the pKb for the H3 antagonists thioperamide, impromidine, and burimamide (8.1, 7.6, and 7.2, respectively), were similar to those reported for H3 receptors in the central nervous system, suggesting that the eosinophil histamine receptor was similar to H3 receptors. However, when the known H3 agonists were tested for activity ([R]-alpha-methylhistamine, N alpha-methylhistamine), the potencies of these compounds were much less than the potency of histamine itself, indicating a significant difference between H3 receptors and this eosinophil histamine receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of 5-lipoxygenase products in preclinical models of asthma.

BACKGROUND: The action of 5-lipoxygenase on arachidonic acid generates potent inflammatory mediators that may contribute to the pathophysiology of asthma. METHODS: Using the potent and selective 5-lipoxygenase inhibitor BI-L-239, we have examined the role of 5-lipoxygenase products in three animal models of asthma. RESULTS: In vitro BI-L-239 inhibited 5-lipoxygenase product generation from human lung mast cells, alveolar macrophages, and peripheral blood leukocytes with a concentration that would provide 50% inhibition values of 28 to 340 nmol/L. A 36-fold selectivity for immunoreactive leukotriene C4 versus immunoreactive prostaglandin D2 inhibition was demonstrated in mast cells. In anesthetized cynomolgus monkeys, inhaled BI-L-239 provided dose-dependent inhibition of the inhaled Ascaris-induced immunoreactive leukotriene C4 release (maximum, 73%; bronchoalveolar lavage [BAL], 20 minutes), late-phase bronchoconstriction (maximum, 41%; +6 to 8 hours), and neutrophil infiltration (maximum, 63%; BAL, +8 hours). In conscious sheep, inhaled BI-L-239 provided dose-dependent inhibition of the inhaled Ascaris-induced late-phase bronchoconstriction (maximum, 66%; +6 to 8 hours) and increase in airway responsiveness (maximum, 82%; carbachol, +24 hours). The acute bronchoconstriction was shortened, and neutrophil infiltration diminished (maximum, 61%; BAL, +8 hours) in this model. Finally in conscious actively sensitized guinea pigs pretreated with pyrilamine and indomethacin, inhaled BI-L-239 attenuated acute bronchoconstriction (maximum, 80%; +5 to 15 minutes), leukocyte infiltration (58%; BAL, +3 days) and increase in airway responsiveness (100%; methacholine, +3 days) induced by three alternate-day ovalbumin inhalations. CONCLUSIONS: In conclusion, results in these three animal models indicate that 5-lipoxygenase products may be major contributors to the bronchoconstriction (especially late phase), leukocyte infiltration, and airway hyperresponsiveness that characterize asthma.

The role of mast cells in inflammatory responses in the lung.

Human lung mast cells (HLMC), by virtue of their anatomic localization and release of potent chemical mediators, are well suited to produce characteristic pathophysiological inflammatory reactions in the lung. Much has been learned about the nature of these cells over the past 10 years, following the development of techniques to disperse them from lung tissue and ultimately purify them to near homogeneity. Recent work focusing on the origin, ultrastructure, proliferation, differentiation, heterogeneity, biochemistry of release, mediators, and pharmacological control of HLMC secretion is reviewed. Current knowledge of HLMC involvement in asthma and interstitial lung diseases, characterized by fibrosis, is summarized with an emphasis on potential areas of therapeutic intervention.

Surfactant: clinical applications.

Pulmonary surfactant is an important chemical component of the lung. It decreases surface tension in the alveolar cells to help stabilize the alveoli, and it may help prevent pulmonary edema. Currently, naturally and synthetically derived surfactants are being used to treat neonatal respiratory distress syndrome, a leading cause of death in premature infants. Surfactant is recommended for prophylactic therapy in infants weighing less than 1,350 g (3 lb) and in infants weighing more than 1,350 g who show signs of pulmonary immaturity and for rescue therapy in infants with respiratory distress syndrome. Surfactant is administered by endotracheal tube, and the recommended dose is 5 mg per kg. Three doses, given 12 hours apart, is the recommended regimen for prophylactic therapy. Rescue therapy consists of one dose of surfactant given at the onset of respiratory distress and another dose given 12 hours later.

Mast cell mediators prostaglandin-D2 and histamine activate human eosinophils.

Airway damage secondary to eosinophil activation is thought to contribute to the development of asthma. Using the fluorescent dye FURA-2 to measure the concentration of cytosolic calcium, we found that supernatants from anti-IgE-stimulated human lung mast cells increased cytosolic calcium in human eosinophils. We then examined the major mast cell mediators (histamine, PGD2, platelet-activating factor (PAF), eosinophil chemotactic factor of anaphylaxis (ECF-A), leukotriene (LT)C4 and LTB4) for their ability to increase cytosolic calcium in eosinophils. We found that both PAF (5 x 10(-9) to 5 x 10(-6) M) and PGD2 (two of five donors responsive at 1 x 10(-9) M) were potent stimuli for calcium mobilization. LTB4 (10(-8), 10(-7) M) and histamine were also active, although higher concentrations of histamine were required to see a response (3 x 10(-7) to 10(-5) M). LTC4, val-ECF-A, and ala-ECF-A were inactive. The effects of PGD2 and histamine were specific for eosinophils, although LTB4 and PAF increased calcium in both neutrophils and eosinophils. The histamine-induced increase in intracellular calcium was not blocked by the H1 or H2 antagonists pyrilamine or cimetidine (10(-4) M), respectively; however, the response to 10(-6) M histamine was completely blocked by the specific H3 antagonist thioperamide (10(-6) M). To evaluate the relative contribution of these stimulatory mast cell mediators on the calcium mobilizing activity in supernatants from anti-IgE-stimulated human lung mast cell (HLMC), we examined the effect of supernatants from HLMC pretreated with indomethacin and/or the 5-lipoxygenase pathway inhibitor MK886. These supernatants were added to FURA-2-loaded eosinophils that had been preincubated with thioperamide and/or the PAF antagonist WEB-2086. We found that the increase in eosinophil calcium in response to supernatants from anti-IgE-stimulated-HLMC was totally inhibited only when the mast cells were challenged in the presence of indomethacin and MK886, and the eosinophils were preincubated with thioperamide. WEB-2086 had little effect. When we examined the effect of these mediators on eosinophil secretory function, we found that PGD2 (not histamine) primed eosinophils for enhanced release of LTC4 in response to the calcium ionophore A23187. We conclude that the activation of eosinophils by PGD2 and other mast cell products may contribute to airways inflammation that is characteristic of asthma.

Elevated plasma histamine in chronic uremia. Effects of ketotifen on pruritus.

A role for histamine in the pathogenesis of uremic pruritus was investigated in maintenance hemodialysis patients. Venous plasma histamine levels, as determined by radioenzymatic assay, were significantly higher (p less than 0.05) in hemodialysis patients with pruritus (368 +/- 103 pg/ml [mean +/- SEM], n = 6) than in those without pruritus (146 +/- 22 pg/ml, n = 5) and in normal controls (142 +/- 16, n = 5). Arteriovenous fistula histamine levels (202 +/- 52 pg/ml, n = 6) were significantly lower (p less than 0.05) than simultaneously drawn venous samples. Markedly elevated histamine-degrading enzyme (histaminase) activities were found in both hemodialysis patients with (2.95 +/- 0.18 pg histamine degraded/minute) and without (2.44 +/- 0.28) pruritus, but was undetectable in normal controls. Histaminase activities did not significantly differ in simultaneously drawn venous and fistula samples. With hemodialysis, histaminase activities fell significantly (p less than 0.01), whereas plasma histamine did not change. We further examined the effects of ketotifen, a putative mast cell stabilizer, on severe uremic pruritus. Five of five patients had significant (p less than 0.01) reductions in pruritus, as judged on a six-point pruritus index, after 8 weeks of drug (x = 2.3), as compared to conventional therapy (x = 5.9). Despite these improvements, no significant differences were noted in pre- versus post-drug plasma histamine levels, histaminase activities, or the histamine content per gram of skin biopsy specimen. These data support prior hypotheses that mast cell activation contributes to the pruritus of uremia.

Histochemical heterogeneity of dispersed human lung mast cells.

Cytocentrifuge preparations of enzymatically dispersed human lung parenchymal mast cells were examined by light microscopy after fixation in either Mota's basic lead acetate or 10% neutral buffered formalin followed by toluidine blue staining at pH 0.5. Fixation in Mota's basic lead acetate allowed detection of all mast cells. However, after formalin fixation only 10.8 +/- 1.3%, range 4.7 to 17%, n = 8 remained detectable (i.e., formalin "resistant"). Therefore, the vast majority of human lung mast cells lose their metachromatic staining after formalin fixation (i.e., are formalin "sensitive"). Mast cells were then separated on the basis of diameter by countercurrent elutriation and on the basis of density by discontinuous Percoll gradients. Histochemically distinct populations of mast cell types emerged in all lungs studied. The proportion of formalin-resistant mast cells increased as a function of diameter: less than 5% at diameters of less than or equal to 11 mu and densities less than or equal to 1.063 g/ml, to 30 to 40% in cells of diameters greater than or equal to 16 mu and densities greater than or equal to 1.100 g/ml. Maximum anti-IgE challenge of nearly homogeneous formalin-sensitive mast cells (94.3 +/- 2.1% purity, n = 6) caused the generation of both leukotriene C4 (64.6 +/- 26.4 pg/mast cell) and PGD2 (114.8 +/- 37.5 pg/mast cell). Six- to eight-fold enrichment of formalin-resistant mast cells did not significantly alter the histamine release response or profiles of arachidonate metabolites. Similar results were obtained for the nonimmunologic stimulus ionophore A23187. We conclude that two histochemically distinct subpopulations, of mast cells are present in human lung suspensions. Although formalin-sensitive cells account for almost 90% of lung mast cells, formalin-resistant cells are separable by their large diameters and higher densities. Both subtypes show similar histamine release responses and arachidonate oxidation profiles.

Raised histamine concentrations in chronic cholestatic liver disease.

Pruritus is a frequent symptom in chronic cholestatic liver disease. To date, no single causative mechanism has been identified. We examined venous plasma concentrations of the known pruritogen, histamine, using a highly sensitive radioenzymatic assay in 42 patients with chronic cholestatic liver disease, and in normal controls. The mean plasma histamine level was significantly greater in chronic cholestatic liver disease patients (275 (117) pg/ml; X (SD) than in controls (140 (72) pg/ml, n = 20) (p less than 0.0001). No significant differences were found between histamine concentrations in the two chronic cholestatic liver disease subgroups: primary biliary cirrhosis and sclerosing cholangitis. Histamine concentrations were significantly greater (p less than 0.01) in the pruritic (319 (132) pg/ml) as compared with the non-pruritic (227 (75) pg/ml) chronic cholestatic liver disease patients. The histaminase activity was equivalent in patients and controls. The finding of raised histamine concentrations in chronic cholestatic liver disease suggests in vivo mast cell activation and a potential role for its mediators in the pruritus characteristic of these disorders.

Alpha 1-PI for emphysema due to alpha 1-antitrypsin deficiency.

Alpha 1-antitrypsin deficiency is a genetic disorder that may result in premature pulmonary emphysema. Affected persons are deficient in the protective protein, alpha 1-proteinase inhibitor (alpha 1-PI). The diagnosis should be considered in patients with signs of emphysema by age 40 (especially nonsmokers), those with predominantly lower lobe disease and those with a family history of premature emphysema. Recently, human alpha 1-PI has been purified and is available to prevent the development of disabling emphysema in affected individuals. Weekly infusion of the concentrate is effective in raising serum levels and is very safe, with only rare minor adverse effects.

Treatment of tracheobronchial granular cell myoblastomas with endoscopic bipolar cautery.

Granular cell myoblastomas of the trachea and right upper lobe bronchus were discovered incidentally during therapeutic bronchoscopy. Because of their propensity to cause airway compromise and distal atelectasis, ablation of both lesions was undertaken. This is the first reported case of bipolar cautery of GCM through a flexible fiberoptic bronchoscope. Small tumor size and lack of atelectasis permitted utilization of this technique. Long-term follow-up is necessary to compare this therapy with other nonresectional therapies.