Thomas B. Casale

Induction of human cutaneous mast cell degranulation by opiates and endogenous opioid peptides: Evidence for opiate and nonopiate receptor participation

In order to examine the capacity of pharmacologically useful opiates to stimulate human mast cell secretion, subjects were skin tested with morphine, codeine, or meperidine hydrochloride. All three agents acted equipotently in eliciting positive immediate skin reactions from all subjects tested. Each agent demonstrated 10 mm of net whealing at 5 to 10 micrograms base (16.7 to 40.4 nmol) injected intradermally. The ability to elicit immediate skin test reactions with endogenous opioid peptides was examined with the use of dynorphin, [D-Ala, 2-D-Leu5] enkephalin, beta-endorphin, and morphiceptin . All four compounds induced wheal-and-flare reactions with the order of potency: dynorphin, greater than beta-endorphin, and greater than [D-Ala, 2-D-Leu5] enkephalin approximately equal to morphiceptin at dose ranges of 0.3 to 8.45 nmol. The inhibition of reactivity by hydroxyzine and the demonstration of mast cell degranulation by electron microscopy suggest that the immediate skin responses to opioid stimulation occur as a consequence of mast cell degranulation. Experiments with the opioid receptor antagonist, naloxone, suggest that both opioid and nonopioid receptors may be involved. These results imply that endogenous opioid peptides possibly may play a role in mast cell function and/or degradulation .

Chronic Granulomatous Disease of Childhood and Chromobacterium violaceum Infections in the Southeastern United States

Patients with chronic granulomatous disease are predisposed to infections by catalase-positive organisms in the environment. Chromobacterium violaceum is a catalase-positive bacterium whose saprophytic source in this country is the subtropical soil and water of the southeastern United States. Two patients with chronic granulomatous disease, followed at the National Institutes of Health in Maryland, acquired C. violaceum infections in Florida. All 10 cases previously reported were acquired in Florida and Louisiana, and reports for which dates were available showed that all infections were acquired from June to September. Seven of 10 patients died; one patient was studied and found to have chronic granulomatous disease. Thus, at least three of the 12 known patients have had underlying chronic granulomatous disease. We suggest that C. violaceum infections occur with unusual frequency in patients with a common underlying predisposing disorder; C. violaceum poses a potential threat to patients with chronic granulomatous disease living in or visiting the endemic states.

Characterization of histamine H-1 receptors on human peripheral lung

Histamine H-1 receptors in human peripheral lung were characterized by radioligand and biochemical assays employing binding of the H-1 receptor antagonist [3H]pyrilamine to plasma membrane preparations. Simultaneous computerized analyses of the data from fourteen separate equilibrium-binding assays indicated the presence of three distinct classes of binding sites with Kd values of 81 +/- 35 pM, 7 +/- 3 microM, and 320 +/- 167 microM and binding capacities of 23 +/- 3 pmoles, 10 +/- 5 nmoles, and 297 +/- 119 nmoles/mg protein respectively. Dissociation kinetics of [3H]pyrilamine binding also supported the presence of three binding sites or states. Further, competition binding curves for histamine receptor agonists and antagonists also indicated the presence of multiple binding sites for the H-1 receptor. The effect of exogenous stimulation of histamine H-1 receptors on human cyclic nucleotides was also examined. Both histamine and the H-1 agonist 2-methyl histamine caused dose-related increases in the cyclic guanosine monophosphate (GMP) content of human lung. The effects of 2-methyl histamine were selective for cyclic GMP. The histamine-induced increase in cyclic GMP peaked within 1.0 min and was effectively prevented by the H-1 antagonist pyrilamine. Thus, human lung possesses a large number of H-1 receptors which exhibit three binding states and produce cyclic GMP, but not cyclic adenosine monophosphate (AMP), when stimulated.

Demonstration that circulating human blood cells have no detectable alpha1-adrenergic receptors by radioligand binding analysis

The mechanisms underlying the autonomic nervous system abnormalities reported in allergic asthma have not been defined. In order to determine if these abnormalities reflect abnormal alpha-adrenergic receptor numbers or drug affinities, we attempted to identify alpha-receptors on circulating human blood cells. Platelets, red blood cells, polymorphonuclear leukocytes, and mononuclear cells were examined by use of radioligand binding techniques with the [3H]antagonists, dihydro-alpha-ergocryptine, prazosin hydrochloride, and yohimbine as ligands. The presence of alpha 2-receptors was confirmed on platelets, but no detectable alpha-receptors were identified on red blood cells or polymorphonuclear leukocytes. Preliminary observations suggested the presence of specific alpha-receptor binding to mononuclear cells; however, this binding was determined to reflect directly the presence of contaminating platelets. By use of a newly developed isolation technique to obtain platelet-depleted mononuclear cells, no alpha-adrenergic receptors could be identified on platelet-depleted mononuclear cells. Therefore, since no alpha 1-receptors could be identified on circulating human blood cells, these cells are not a suitable model for the study of the mechanisms underlying abnormal alpha-adrenergic responsiveness, and it may be necessary to reanalyze previous reports of alpha-adrenergic responsiveness on human blood cells with the use of platelet-depleted cell preparations.

A rapid method for isolation of human mononuclear cells free of significant platelet contamination

In order to obtain mononuclear cells from peripheral human blood for the study of cell surface receptors, it was necessary to effectively eliminate contaminating platelets. The usual Hypaque-Ficoll isolation procedures were found to produce mononuclear cells contaminated with 10-1000 platelets per mononuclear cell (by phase microscopy). Multiple slow speed centrifugations reduced the contamination to 5-10 platelets per mononuclear cell. However, centrifugation of EDTA-anticoagulated blood through Hypaque (D20(20) 1.060) at 400 X g for 5 min at 22 degrees C followed by the usual Hypaque-Ficoll gradient reduced platelet contamination to less than 1 platelet per 2 mononuclear cells. Thus, a rapid and simple gradient procedure is capable of significantly reducing platelet contamination of mononuclear cell preparations and should facilitate the analysis of mononuclear cell receptors and functions.

Chromobactehum violaceum Infectious and Chronic Granulomatous Disease

Excerpt To the editor: In July 1982 we (1) reported the association ofChromobacterium violaceuminfections and underlying chronic granulomatous disease of childhood in patients who live or visit in ...

Preparation of a human lung purified plasma membrane fraction: Confirmation by enzyme markers, electron microscopy, and histamine H1 receptor binding

SummaryA simple and rapid method of isolating plasma membranes from human peripheral lung tissue is described. The method involves homogenization of tissue in 0.25m sucrose-buffered medium followed by differential and sucrose density gradient centrifugation. Enzymatic and morphological characterization of the plasma membrane fraction revealed minimal contamination by nonplasma membrane fragments. The isolated plasma membranes showed an 18-fold purification of 5′-nucleotidase activity compared to the original homogenate. Electronmicroscopic studies of the plasma membrane fraction revealed the presence of small membrane vesicles having a trilaminar membrane structure. To further examine the purity of the plasma membrane preparation, the binding of the H1 receptor antagonist,3H pyrilamine, to the plasma membrane-enriched fraction was compared to the binding to crude membrane preparations. Both the plasma membrane-enriched fraction and the crude membrane preparation had similar Kds for the histamine antagonist, but the plasma membrane-enriched fraction had a threefold greater binding capacity, reflecting the relative enrichment of plasma membranes of the preparation. Thus, a method has been developed for the isolation of plasma membranes from human peripheral lung which should provide material for a variety of biochemical and pharmacological studies.

Dopamine inhibition of histamine-mediated cutaneous responses

Several patients receiving dopamine for hypotension were skin tested for possible penicillin sensitivity. Not only were the penicillin skin tests negative but also the histamine control. On the possibility that dopamine might affect cutaneous histamine responses, we examined the effect of dopamine on histamine, antigen, morphine, and compound 48/80 skin responses. Both intradermal and intravenous dopamine selectively inhibited histamine but not antigen, morphine, or compound 48/80 skin responses, and the inhibition was in a dose-related fashion. This observation indicates that histamine should not be used to demonstrate dermal reactivity in patients receiving dopamine. The results of this study also suggest that histamine may not be the sole mast cell-derived mediator involved in the wheal-and-flare reaction characteristic of immediate-type skin tests since dopamine did not affect skin reactions caused by endogenous mast cell degranulation. Finally, the possible use of dopaminergic drugs in diseases with histamine-associated symptoms is discussed.

Detection of beta-adrenergic receptors on rabbit mononuclear cells isolated free of significant contamination by other cell types☆

In order to study rabbit mononuclear cell surface receptors, it was necessary to develop a procedure to isolate mononuclear cell preparations that are free of significant contamination by other cell types, especially platelets. Centrifugation of dextran-sedimented, anti-coagulated whole blood through Hypaque (density 1.060) at 600 X g for 5 min at 22 degrees C eliminated greater than 93% of starting platelets. A second 5-min Hypaque centrifugation of Hypaque-Ficoll-isolated mononuclear cells (MNC) (approximately 80% lymphocytes) at 450 X g for 5 min at 22 degrees C reduced platelet contamination to less than one platelet per three MNC, and resulted in the overall removal of greater than 99.5% of starting platelets. These relatively pure MNC which were isolated in less than 2 hr were identified as having beta-adrenergic receptors by radioligand binding techniques using [125I]iodohydroxybenzylpindolol [( 125I]IHYP). Binding of [125I]IHYP to intact rabbit MNC was a saturable, stereospecific, and rapid process with a dissociation constant (KD) of 0.53 +/- 0.18 nM and a binding capacity of 3,461 +/- 235 sites/cell.

Complete hematologic and hepatic recovery in a patient with chloramphenicol hepatitis-pancytopenia syndrome.

1. Jacobs AH: Eruptions in the diaper area, Pediatr Clin North Am 25:209, 1978. 2. Gellis SE: Eruptions in the diaper area. In Gellis SE, Kagan BM, editors: Current pediatric therapy, ed. 9, Philadelphia, 1980, W. B. Saunders Co., p 466. 3. Dixon PN, Warin RP, English MP: Role of Candida albicans infection in napkin rashes, Br Med J 2:23, 1969. 4. Kozinn P J, Taschdijana CL: Enteric candidiasis: Diagnosis and clinical considerations, Pediatrics 30:71, 1962. 5. Leyden J J, Kligman AM: The role of microorganisms in diaper dermatitis, Arch Dermatol 114:56, 1978. 6. Rebora A, Marples RR, Kligman AM: Experimental infection with Candida albicans, Arch Dermatol 108:69, 1973. 7. Silva-Hutner M, Cooper BH: Medically important yeasts. In Lennette EH, Spaulding EH, Truant JP, editors: Manual of clinical microbiology, Washington D.C., 1974, American Society of Microbiology, p 491. 8. Montes LF, Pittillo RF, Hunt D, Narkates A J, Dillon HC: Microbial flora of infants skin: Comparison of types of microorganisms between normal skin and diaper dermatitis, Arch Dermatol 103:640, 1971. 9. Leyden J J, Katz S, Stewart R, Kligman AM: Urinary ammonia and ammonia-producing microorganisms in infants with and without diaper dermatitis, Arch Dermatol 113:1678, 1977. 10. Kucers A, Bennett NM: The use of antibiotics, ed. 3, Philadelphia, 1979, J. B. Lippincott Co., p 924.