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Alexandru DP Papoiu,1 Hunza Chaudhry,2 Erin C Hayes,2 Yiong-Huak Chan,3 Kenneth D Herbst4 1Independent contractor, San Diego, CA, USA: 2Cosmederm Bioscience, Inc., San Diego, CA, USA; 3Biostatistics Unit, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; 4Department of Medicine, University of California, San Diego, San Diego, CA, USA Background: Itch is one of the most frequent skin complaints and its treatment is challenging. From a neurophysiological perspective, two distinct peripheral and spinothalamic pathways have been described for itch transmission: a histaminergic pathway and a nonhistaminergic pathway mediated by protease-activated receptors (PAR)2 and 4. The nonhistaminergic itch pathway can be activated exogenously by spicules of cowhage, a tropical plant that releases a cysteine protease named mucunain that binds to and activates PAR2 and PAR4. Purpose: This study was conducted to assess the antipruritic effect of a novel over-the-counter (OTC) steroid-free topical hydrogel formulation, TriCalm®, in reducing itch intensity and duration, when itch was induced with cowhage, and compared it with two other commonly used OTC anti-itch drugs. Study participants and methods: This double-blinded, vehicle-controlled, randomized, crossover study recorded itch intensity and duration in 48 healthy subjects before and after skin treatment with TriCalm hydrogel, 2% diphenhydramine, 1% hydrocortisone, and hydrogel vehicle, used as a vehicle control. Results: TriCalm hydrogel significantly reduced the peak intensity and duration of cowhage-induced itch when compared to the control itch curve, and was significantly superior to the two other OTC antipruritic agents and its own vehicle in antipruritic effect. TriCalm hydrogel was eight times more effective than 1% hydrocortisone and almost six times more effective than 2% diphenhydramine in antipruritic action, as evaluated by the reduction of area under the curve. Conclusion: TriCalm hydrogel has a robust antipruritic effect against nonhistaminergic pruritus induced via the PAR2 pathway, and therefore it could represent a promising treatment option for itch. Keywords: antipruritic treatment, nonhistaminergic pruritus, head-to-head comparison

The oxidation of various aryl and aliphatic thiols with the commercially available and environmentally benign reagent Bobbitt's salt ( 1 ) has been investigated. The reaction affords the corresponding disulfide products in good to excellent yields (71-99%) and can be accomplished in water, methanol, or acetonitrile solvent. Moreover, the process is highly chemoselective, tolerating traditionally oxidation-labile groups such as free amines and alcohols. Combined experimental and computational studies reveal that the oxidation takes place via a polar two-electron process with concomitant and unexpected deoxygenation of the oxoammonium cation through homolysis of the weak N-O bond, differing from prototypical radical-based thiol couplings. This unusual consumption of the oxidant has significant implications for the development of new nitroxide-based radical traps for probing S-centered radicals, the advancement of new electrochemical or catalytic processes involving nitroxide/oxoammonium salt redox couples, and applications to biological systems.

Herein we quantitatively investigate how metal ion Lewis acidity and steric properties influence the kinetics and thermodynamics of dioxygen binding versus release from structurally analogous Mn-O 2 complexes, as well as the barrier to Mn peroxo O-O bond cleavage, and the reactivity of Mn oxo intermediates. Previously we demonstrated that the steric and electronic properties of Mn III -OOR complexes containing N-heterocyclic (N Ar ) ligand scaffolds can have a dramatic influence on alkylperoxo O-O bond lengths and the barrier to alkylperoxo O-O bond cleavage. Herein, we examine the dioxygen reactivity of a new Mn II complex containing a more electron-rich, less sterically demanding N Ar ligand scaffold, and compare it with previously reported Mn II complexes. Dioxygen binding is shown to be reversible with complexes containing the more electron-rich metal ions. The kinetic barrier to O 2 binding and peroxo O-O bond cleavage is shown to correlate with redox potentials, as well as the steric properties of the supporting N Ar ligands. The reaction landscape for the dioxygen chemistry of the more electron-rich complexes is shown to be relatively flat. A total of four intermediates, including a superoxo and peroxo species, are observed with the most electron-rich complex. Two new intermediates are shown to form following the peroxo, which are capable of cleaving strong X-H bonds. In the absence of a sacrificial H atom donor, solvent, or ligand, serves as a source of H atoms. With TEMPOH as sacrificial H atom donor, a deuterium isotope effect is observed ( k H / k D = 3.5), implicating a hydrogen atom transfer (HAT) mechanism. With 1,4-cyclohexadiene, 0.5 equiv of benzene is produced prior to the formation of an EPR detected Mn III Mn IV bimetallic species, and 0.5 equiv after its formation.

Characterizing properties of the rock matrix that control retention and release of chlorinated solvents is essential in evaluating the extent of contamination and the application of remediation technologies in fractured rock. Core samples from seven closely spaced boreholes in a mudstone subject to trichloroethene (TCE) contamination were analyzed using Mercury Intrusion Porosimetry to investigate porosity and pore size distribution as a function of mudstone characteristics, and depth and lateral extent in the aquifer; organic carbon content was also evaluated to identify the potential for adsorption. Porosity and retardation factor varied over two orders of magnitude, with the largest porosities and largest retardation factors associated with carbon-rich mudstone layers. Larger porosities were also measured in the shallow rock that has been subject to enhanced groundwater flow. Porosity also varied over more than an order of magnitude in spatially continuous mudstone layers. The analyses of the rock cores indicated that the largest pore diameters may be accessible to entry of the nonaqueous form of TCE. Although the porosity associated with the largest pore diameters is small (~0.1%), that volume of TCE can significantly affect the total TCE that is retained in the rock matrix. The dimensions of the largest pore diameters may also be accessible to microbes responsible for reductive dechlorination; however, the small percentage of the pore space that can accommodate microbes may limit the extent of reductive dechlorination in the rock matrix., (Published by Elsevier B.V.)

The bulky 2,6-di-tert-butyl-4-nitrophenolate ligand forms complexes with [Tp tBu Cu II ] + and [Tp tBu Zn II ] + binding via the nitro group in an unusual nitronato-quinone resonance form (Tp tBu = hydro-tris(3-tert-butyl-pyrazol-1-yl)borate). The Cu complex in the solid state has a five-coordinate κ 2 -nitronate structure, while the Zn analogue has a four-coordinate κ 1 -nitronate ligand. 4-Nitrophenol, without the 2,6-di-tert-butyl substituents, instead binds to [Tp tBu Cu II ] + through the phenolate oxygen. This difference in binding is very likely due to the steric difficulty in binding a 2,6-di-tert-butyl-phenolate ligand to the [Tp tBu M II ] + unit. Tp tBu Cu II (κ 2 -O 2 N t Bu 2 C 6 H 2 O) reacts with the hydroxylamine TEMPO-H (2,2,6,6-tetramethylpiperidin-1-ol) by abstracting a hydrogen atom. This system thus shows an unusual sterically enforced transition metal-ligand binding motif and a copper-phenolate interaction that differs from what is typically observed in biological and chemical catalysis.

Spore photoproduct is a thymidine dimer formed when bacterial endospore DNA is exposed to ultraviolet (UV) radiation. The mechanism of formation of this thymidine dimer has been proposed to proceed through a radical-pair intermediate. The intermediate forms when a methyl-group hydrogen atom of one thymidine nucleobase is transferred to the C6 position of an adjacent thymidine nucleobase, forming two species, the TCH 2 and TH radicals, respectively. Using a series of thymidine isotopologues and electron paramagnetic resonance (EPR) spectroscopy, we show that microcrystals of thymidine exposed to UV radiation produce these two radical species. We observe three sources that donate the additional hydrogen at the C6 position of the TH radical. One of the three sources is the methyl group of another thymidine molecule in a significant fraction of the TH species. This lends support to the radical-pair intermediate proposed in the formation of spore photoproduct.

In the copper-catalyzed oxidation of alcohols to aldehydes, a Cu(II)-alkoxide (Cu(II)-OR) intermediate is believed to modulate the αC-H bond strength of the deprotonated substrate to facilitate the oxidation. As a structural model for these intermediates, we characterized the electronic structure of the stable compound Tp(tBu)Cu(II)(OCH2CF3) (Tp(tBu) = hydro-tris(3-tert-butyl-pyrazolyl)borate) and investigated the influence of the trifluoroethoxide ligand on the electronic structure of the complex. The compound exhibits an electron paramagnetic resonance (EPR) spectrum with an unusually large gzz value of 2.44 and a small copper hyperfine coupling Azz of 40 × 10(-4) cm(-1) (120 MHz). Single-crystal electron nuclear double resonance (ENDOR) spectra show that the unpaired spin population is highly localized on the copper ion (≈68%), with no more than 15% on the ethoxide oxygen. Electronic absorption and magnetic circular dichroism (MCD) spectra show weak ligand-field transitions between 5000 and 12,000 cm(-1) and an intense ethoxide-to-copper charge transfer (LMCT) transition at 24,000 cm(-1), resulting in the red color of this complex. Resonance Raman (rR) spectroscopy reveals a Cu-O stretch mode at 592 cm(-1). Quantum chemical calculations support the interpretation and assignment of the experimental data. Compared to known Cu(II)-thiolate and Cu(II)-alkylperoxo complexes from the literature, we found an increased σ interaction in the Cu(II)-OR bond that results in the spectroscopic features. These insights lay the basis for further elucidating the mechanism of copper-catalyzed alcohol oxidations.

Electrostatic fields at the interface of the GTPase H-Ras (Ras) docked with the Ras binding domain of the protein Ral guanine nucleoside dissociation stimulator (Ral) were measured with vibrational Stark effect (VSE) spectroscopy. Nine residues on the surface of Ras that participate in the protein-protein interface were systematically mutated to cysteine and subsequently converted to cyanocysteine in order to introduce a nitrile VSE probe into the protein-protein interface. The absorption energy of the nitrile was measured both on the surface of Ras in its monomeric state, then after incubation with the Ras binding domain of Ral to form the docked complex. Boltzmann-weighted structural snapshots of the nitrile-labeled Ras protein were generated both in monomeric and docked configurations from molecular dynamics simulations using enhanced sampling of the cyanocysteine side chain's χ2 dihedral angle. These snapshots were used to determine that on average, most of the nitrile probes were aligned along the Ras surface, parallel to the Ras-Ral interface. The average solvent-accessible surface areas (SASA) of the cyanocysteine side chain were found to be <60 Å(2) for all measured residues, and was not significantly different whether the nitrile was on the surface of the Ras monomer or immersed in the docked complex. Changes in the absorption energy of the nitrile probe at nine positions along the Ras-Ral interface were compared to results of a previous study examining this interface with Ral-based probes, and found a pattern of low electrostatic field in the core of the interface surrounded by a ring of high electrostatic field around the perimeter of the interface. These data are used to rationalize several puzzling features of the Ras-Ral interface.

A novel class of indole ligands for estrogen receptor alpha have been discovered which exhibit potent affinity and high selectivity. Substitution of the bazedoxifene skeleton to the linker present in the HTS lead 1a provided 22b which was found to be 130-fold alpha-selective and acted as an antagonist of estradiol activity in uterine tissue and MCF-7 cancer cells.

Synthesis and derivatization of a series of substituted tetrahydrofluorenone analogs giving potent, ERbeta subtype selective ligands are described. Several analogs possessing ERbeta binding affinities comparable to 17beta-estradiol but with greater than 75-fold selectivity over ERalpha are reported.

Glycodelin modulates vascular endothelial growth factor (VEGF) production in cumulus cells in vitro. Patients with normal gonadotropin responses who were undergoing IVF demonstrated increased VEGF production to glycodelin, whereas poor responders had a decreased response to glycodelin.

Two novel side chains which had previously been found to enhance antagonist activity in the dihydrobenzoxathiin SERM series were applied to three existing platforms. The novel side chains did not improve the antagonist activity of the existing platforms.

An optimized side chain for dihydrobenzoxathiin SERAMs was discovered and attached to four dihydrobenzoxathiin platforms. The novel SERAMs show exceptional estrogen antagonist activity in uterine tissue and an MCF-7 breast cancer cell assay.

The discovery, synthesis, and SAR of chromanes as ER alpha subtype selective ligands are described. X-ray studies revealed that the origin of the ER alpha-selectivity resulted from a C-4 trans methyl substitution to the cis-2,3-diphenyl-chromane platform. Selected compounds from this class demonstrated very potent in vivo antagonism of estradiol in an immature rat uterine weight assay, effectively inhibited ovariectomy-induced bone resorption in a 42 days treatment paradigm, and lowered serum cholesterol levels in ovx'd adult rat models. The best antagonists 8F and 12F also exhibited potent inhibition of MCF-7 cell growth and were shown to be estrogen receptor down-regulators (SERDs).

The ring oxygen and sulfur analogs of lasofoxifene, 1a and 1b, were synthesized in an attempt to impart ERalpha selectivity, as found in the closely related dihydrobenzoxathiin compound I, recently discovered in these laboratories. The resulting isochroman and isothiochroman compounds were found to exhibit equipotent binding affinities to the ER isoforms and were less active in the inhibition of estradiol-triggered uterine growth when compared to I and lasofoxifene.

A series of dihydrobenzoxathiin SERAMs with alkylated pyrrolidine side chains or alkylated linkers was prepared. Minor modifications in the side chain or linker resulted in significant effects on biological activity, especially in uterine tissue.

A series of benzoxathiin SERAMs with bicyclic amine side chains was prepared. Minor modifications in the side chain resulted in significant effects on biological activity, especially in uterine tissue.

A series of benzoxathiin SERAMs with heteroatom-substituted amine side chains was prepared. Minor modifications in the side chain resulted in significant effects on biological activity, especially in uterine tissue.

Dihydrobenzoxathiin analogs (1-11) with modifications on the basic side chain region were prepared and evaluated for estrogen/anti-estrogen activity in both in vitro and in vivo models. The compounds generally maintained a high degree of selectivity for ERalpha over ERbeta, similar to the original lead compound I. Many of the compounds also maintained high potency in the inhibition of human carcinoma MCF-7 cell growth. However, all were less potent in the inhibition of estradiol-triggered uterine growth. This work demonstrates the sensitive nature of modification to the antagonist basic side chain region.

Dihydrobenzodithiin compounds (1-6) were prepared to explore the expansion of the dihydrobenzoxathiin lead compounds I-III as SERAMs (Selective Estrogen Receptor Alpha Modulators). The dihydrobenzodithiin compounds generally maintained a high degree of selectivity for ERalpha over ERbeta, however, they lacked the in vivo antagonism/agonism activity exhibited by the lead class in an immature rat uterine growth model.

Objective: To describe the presentation and clinical course of two patients after development of vaginal agglutination associated with chronic graft-versus-host disease., Design: Case report., Setting: Tertiary referral center for pelvic reconstructive surgery., Patient(s): Two patients with the diagnosis of chronic graft-versus-host disease who later developed vaginal agglutination requiring treatment., Intervention(s): Surgical lysis of vaginal adhesions and postoperative use of vaginal dilators., Main Outcome Measure(s): Successful treatment of vaginal adhesions., Result(s): Both patients underwent successful surgical lysis of vaginal adhesions and maintained vaginal patency with postoperative use of vaginal dilators., Conclusion(s): Prompt diagnosis of vaginal agglutination in patients with chronic graft-versus-host disease and appropriate surgical correction of this complication rather than prophylaxis is the correct treatment course.

Unlabelled: This review summarizes current knowledge about the roles of cyclooxygenases and prostaglandins in reproductive medicine. With the development of COX-2 specific inhibitors, new therapeutic options are available to obstetricians and gynecologists, offering better-tolerated alternatives to conventional NSAIDs. The analgesic effectiveness of COX-2 specific inhibitors is well established, and they are already in use in a range of painful conditions. Both celecoxib and valdecoxib are indicated for the treatment of primary dysmenorrhea, and may be effective in postoperative pain, including hysterectomy, and pain associated with endometriosis. There is also speculation that COX-2 specific inhibitors may be effective tocolytic agents without the risks to the fetus seen with conventional NSAIDs. The role of COX-2 in oncogenesis is also under investigation, and COX-2 specific inhibitors may eventually be used in the prevention and treatment of gynecologic malignancies., Target Audience: Obstetricians & Gynecologists, Family Physicians Learning Objectives: After completion of this article, the reader will be able to describe the two types of cylooxygenase enzymes (COX), to list the effects and side effects of NSAIDs and COX-2 medications, and to outline the various changes in COX expression during pregnancy.

Objective: The purpose of this study was to compare complication rates of patients who undergo dilation and evacuation or medical abortion between 14 and 24 weeks of gestation., Study Design: We present a retrospective cohort study of 297 women who underwent either dilation and evacuation or medical abortion. Statistical methods included the Student t test, the chi(2) test, the Fisher exact test (where appropriate), and logistic regression., Results: The overall complication rate was significantly lower in patients who underwent dilation and evacuation than in patients who underwent medical abortion (4% vs 29%; P <.001). Medical abortions with misoprostol resulted in a lower complication rate than abortions with other medications (odds ratio, 0.2; 95% CI, 0.1-0.4). More Laminaria was associated with a decreased risk of complications with surgical abortions (odds ratio, 0.9; 95% CI, 0.7-1.0)., Conclusion: Dilation evacuation is the safest method of second-trimester abortion. Misoprostol is safer than other methods for medical abortion. Maximal use of Laminaria will decrease complication rates in surgical abortion.

Somatostatin (SST) regulates growth hormone (GH) secretion from pituitary somatotrophs by interacting with members of the SST family of G-protein-coupled receptors (sst1-5). We have used potent, nonpeptidyl SST agonists with sst2 and sst5 selectivity to determine whether these receptor subtypes are involved in regulating growth hormone releasing hormone (GHRH) stimulated secretion. GHRH stimulated GH release from pituitary cells in a dose-dependent manner, and this secretion was inhibited by Tyr(11)-SST-14, a nonselective SST analog. A sst2 selective agonist, L-779,976, potently inhibited GHRH-stimulated GH release. In addition, L-817, 818, a potent sst5 receptor selective agonist, also inhibited GH secretion, but was approximately 10-fold less potent (P < 0.01, ANOVA) in inhibiting GH release than either Tyr(11)-SST-14 or L-779, 976. These results show that both sst2 and sst5 receptor subtypes regulate GHRH-stimulated GH release from rat pituitary cells., (Copyright 1999 Academic Press.)

Nonpeptide agonists of each of the five somatostatin receptors were identified in combinatorial libraries constructed on the basis of molecular modeling of known peptide agonists. In vitro experiments using these selective compounds demonstrated the role of the somatostatin subtype-2 receptor in inhibition of glucagon release from mouse pancreatic alpha cells and the somatostatin subtype-5 receptor as a mediator of insulin secretion from pancreatic beta cells. Both receptors regulated growth hormone release from the rat anterior pituitary gland. The availability of high-affinity, subtype-selective agonists for each of the somatostatin receptors provides a direct approach to defining their physiological functions.

A series of nonpeptide somatostatin agonists which bind selectively and with high affinity to somatostatin receptor subtype 2 (sst2) have been synthesized. One of these compounds, L-054,522, binds to human sst2 with an apparent dissociation constant of 0.01 nM and at least 3,000-fold selectivity when evaluated against the other somatostatin receptors. L-054,522 is a full agonist based on its inhibition of forskolin-stimulated adenylate cyclase activity in Chinese hamster ovary-K1 cells stably expressing sst2. L-054,522 has a potent inhibitory effect on growth hormone release from rat primary pituitary cells and glucagon release from isolated mouse pancreatic islets. Intravenous infusion of L-054,522 to rats at 50 microgram/kg per hr causes a rapid and sustained reduction in growth hormone to basal levels. The high potency and selectivity of L-054, 522 for sst2 will make it a useful tool to further characterize the physiological functions of this receptor subtype.

High affinity avermectin binding sites have been identified and partially characterized in membranes from two insect species, Drosophila melanogaster and the locus Schistocerca americana. There is a 10-fold increase in the density of ivermectin binding sites associated with membranes isolated from Drosophila heads (a neuronally enriched tissue source) compared to the bodies (Bmax values were 3.5 and 0.22 pmol/mg, respectively) with only a small difference in the apparent dissociation constant (Kd values of 0.20 and 0.34 nM for heads and bodies, respectively). Membranes prepared from metathoracic ganglia of the locust, Schistocerca americana, were highly enriched in high affinity avermectin binding sites (Kd = 0.2 nM and Bmax = 42 pmol/mg). Using an [125I]arylazido-avermectin analog as a photoaffinity probe, a 45 kDa protein was identified in both the Drosophila head and body tissue preparations. A 45 kDa protein was also specifically labeled with [125I]azido-avermectin in the locust neuronal membranes.

Avermectin-binding proteins from the free-living nematode worm Caenorhabditis elegans and from the fruitfly Drosophila melanogaster were purified to homogeneity via a three-step procedure. The binding proteins were covalently labelled using a radioactive photoaffinity probe and then partially purified on a Sephacryl S-300 gel-filtration column. The radiolabelled binding proteins were then purified by immunoaffinity chromatography using a monoclonal antibody to avermectin covalently attached to Protein A-Sepharose beads. Three affinity-labelled Drosophila proteins with molecular masses between 45 and 50 kDa were isolated in this way and then separated from each other by electroelution. This three-step protocol provides a rapid technique for receptor purification which may be of use in the purification of other binding proteins.

The design and synthesis of a series of avermectin affinity probes used in the identification and purification of the avermectin binding proteins is described. These modified avermectins fall into two design classes: ligands to covalently modify specific avermectin binding proteins [an 125I-labeled aryl azide photoprobe (15) and a tritiated aziridine analog (6)] and ligands for affinity chromatography applications [three biotinylated compounds (10, 12, and 13) and one resin-bound derivative (9)]. The binding affinities of these compounds for the Caenorhabditis elegans avermectin binding protein is presented as well as their biological activities against C. elegans and Artemia salina.

An azido-avermectin analog [4'' alpha-(4-azidosalicylamido-epsilon-caproylamido-beta-alan ylamido)-4''-deoxyavermectin B1a; azido-AVM] was synthesized and used to photoaffinity label avermectin binding sites present in the membranes of Caenorhabditis elegans and Drosophila melanogaster. Azido-AVM was biologically active and behaved like a competitive inhibitor of [3H]ivermectin binding to C. elegans membranes (Ki = 0.2 nM). Radiolabeled azido-AVM bound specifically and with high affinity to C. elegans membranes (Kd = 0.14 nM) and, upon photoactivation, became covalently linked to three C. elegans polypeptides of 53, 47, and 8 kDa. Photoaffinity labeling of a membrane preparation from D. melanogaster heads resulted in labeling of a single major polypeptide of approximately 47 kDa. The proteins that were covalently tagged in these experiments are believed to be associated with avermectin-sensitive chloride channels present in the neuromuscular systems of C. elegans and D. melanogaster. Azido-AVM did not bind to rat brain membranes and therefore was selective for the nematode and insect receptors.

A procedure using high performance liquid chromatography (HPLC) is described for the separation of major primary cyclooxygenase metabolites (prostacyclin metabolite-6ketoPGF1 alpha, thromboxane B2, and prostaglandins F2 alpha, E2, and D2), leukotrienes (C4, B4, and D4), monohydroxyeicosatetraenoic acids (15-, 11-, 12-, and 5HETEs), and free arachidonic acid. It is therefore possible to quantitate major arachidonic acid metabolites by a single chromatographic procedure. Using this technique we have determined that a major arachidonic acid metabolite of human lung macrophages co-elutes with leukotriene B4.

Rabbits were immunized with leukotriene C4 (LTC4) coupled to thiolated keyhole limpet hemocyanin (KLH) by using 6-N-maleimidohexanoic acid as a spacer molecule. Immune serum was obtained with 7.9 nmol of LTC4-specific immunoglobulin per milliliter and a mean association constant of 2.1 X 10(9) M-1. A radioimmunoassay was developed that detected 0.1 pmol of LTC4 per 1-ml sample. LTD4 and LTE4, three isomers of LTC4, the sulfones of LTC4, LTD4, and LTE4, and one isomer of LTD4 reacted to varying degrees in the assay. A number of other structurally related compounds, such as LTB4 and 5-HETE, did not react. Conditions were established to determine LTC4 levels in human plasma without loss of LTC4 during sample preparation and without the need for extraction procedures before the measurement of LTC4.

Arachidonic acid metabolism has been explored in preparations of purified human lung mast cells prelabeled with arachidonic acid (AA). Cells were of 83 to greater than 96% purity, and each experiment was performed with four to six different preparations of mast cells. After overnight culture of the purified cells in the presence of 3H-AA, followed by extensive washing in buffer, mast cell uptake of labeled AA was 61.4 +/- 14.8 pmol/10(6) cells with 21 +/- 2.4% of the label in phospholipids, 73 +/- 2.1% in neutral lipids, and 3.6 +/- 0.8% as free AA. Analysis of the distribution of radioactivity in phospholipid classes revealed 51.4 +/- 5.5% of the label in phosphatidylcholine, 14.5 +/- 1.6% in phosphatidylinositol, 12.0 +/- 3.0% in phosphatidylethanolamine, and 9.1 +/- 2.4% in sphingomyelin, with the rest in other phospholipid classes. Challenge of these cells with an optimal concentration of anti-IgE led to the release of 20 +/- 4.0% of cellular histamine and to a reduction in labeled phosphatidylcholine and phosphatidylinositol to 75.5 +/- 8.8% and 84.2 +/- 4.5% of the control levels, respectively, (p less than 0.05); anti-IgE challenge produced no statistically significant change in the quantities of other labeled phospholipids. Activation of human lung mast cells with anti-IgE led to the release of 3.4 +/- 1.3% of the cellular 3H as AA and AA metabolites (1.5 +/- 0.6% as unmetabolized AA) in conjunction with 16 +/- 4.3% of the cellular histamine. Although activation of human lung mast cells with ionophore A23187 caused 70 +/- 1.1% histamine release, a similar quantity of AA and AA metabolites was released (a total of 4.0 +/- 0.8% with 2.3 +/- 1.5% as unmetabolized AA). Analysis of the released metabolites by liquid scintillation spectrometry after high performance liquid chromatography separation showed that approximately equal amounts of metabolites were produced after mast cell activation with anti-IgE and ionophore A23187. In this series of experiments approximately equal amounts of cyclooxygenase and lipoxygenase products were generated.(ABSTRACT TRUNCATED AT 400 WORDS)

Slow-reacting substance of anaphylaxis (composed of leukotrienes C, D, and E) is released in vitro by the interaction of antigen and IgE antibody on human mast cells and basophils. When we challenged ragweed-sensitive patients intranasally with pollen grains, their clinical response was significantly correlated with the release of the peptide leukotrienes (P less than 0.001). Nonallergic subjects had neither symptoms nor leukotriene release. The leukotrienes were released in a dose-dependent fashion, with a peak mean level of 827 +/- 234 pg per 0.1 ml of a 10-ml nasal wash. High-performance liquid chromatography revealed the presence of leukotrienes C, D, and E, suggesting that nasal cells or fluids had the ability to degrade leukotriene C enzymatically. The in vivo release of these potent inflammatory mediators after exposure to pollen suggests that leukotrienes may have an important role in human allergic reactions.

Platelet-activating factor (PAF) and leukotrienes, newly described classes of vasoactive lipids, may play a role in anaphylaxis. It has recently been suggested that the vasoconstrictor effects of PAF in isolated rat lung are related to release of leukotrienes C4 and D4. Thyrotropin-releasing hormone (TRH), a tripeptide, has potent antihypotensive activity in experimental shock, including that resulting from either leukotriene D4 administration or antigen-induced anaphylaxis. We utilized an unanesthetized guinea pig model to study the relationships among PAF, leukotrienes, and TRH and their potential interactions on the cardiovascular system. PAF (1 nmol/600 g body weight i.v.) produced profound hypotension which was completely blocked by TRH (2 mg/kg i.v.). Nafazatrom or FPL 55712, a presumed receptor antagonist of leukotrienes, was ineffective, whereas U-60257, a leukotriene synthesis inhibitor, displayed incomplete blockade. Moreover, leukotriene-like immunoreactivity in plasma did not increase following PAF administration. Thus, hypotension produced by PAF does not appear to result secondarily from release of cysteinyl leukotrienes. Moreover, the ability of TRH to block the hypotensive effects of PAF may partially account for its beneficial effects in experimental anaphylaxis and provides further rationale for the therapeutic evaluation of this peptide in anaphylactic shock.

Although cyclooxygenase and lipoxygenase inhibitors have been widely used in studies of allergic and inflammatory disorders, the effect of these agents has not been determined on mediators released from purified human lung mast cells. The release of histamine, prostaglandin D2, leukotriene C4, leukotriene B isomers, 5-hydroxyeicosatetraenoic acid (5-HETE), and free arachidonic acid (AA) from preparations of purified human lung mast cells (90 +/- 2% pure) prelabeled with 3H-AA as modified by indomethacin, eicosatriynoic acid (ETI), and diethylcarbamazine (DEC) was determined. Indomethacin (3 microM) markedly (greater than 90%) inhibited the anti-IgE- and ionophore A23187-induced release of PGD2 from these cells but had no effect on the release of histamine or other AA metabolites. Specifically, no increase in LTC production was noted, and no large amount of shunting of products into those produced by lipoxygenases was noted. The ETI, a lipoxygenase inhibitor in many systems, inhibited the release of histamine, PGD2, LTC, LTB isomers, 5-HETE, and free AA, all with an IC50 between 3 and 10 microM. The lack of specificity for lipoxygenase products together with a similar potency for inhibition of histamine release suggests that ETI may act at an early event in the activation process of these cells, apparently prior to the action of phospholipase(s), which releases AA from cellular stores. The DEC inhibited PGD2 release in purified mast cells and histamine release in pure and crude preparations of mast cells, both with IC50 values ranging from 1 to 3 mM. No specificity for enzymes responsible for the production of LTA4 was observed in these cells, unlike the results reported in other systems.(ABSTRACT TRUNCATED AT 250 WORDS)

The increased capacity of lymphocytes from patients with multiple sclerosis to adhere to human epithelial cells persistently infected with measles virus has provided an accurate blood test for multiple sclerosis. When lymphocytes from affected patients were mixed with measles-infected human epithelial cells, the lymphocytes formed rosettes around a mean (+/-S.E.) of 69.2 +/-1.7% of the measles-infected cells. In contrast, lymphocytes from controls, either healthy or with other neurologic and non-neurologic diseases, formed rosettes around a mean of only 28.2+/-2.1% of the measles-infected cells. Of greater importance was the complete absence of overlap between multiple sclerosis and control values, thus indicating the diagnostic potential of the rosetting phenomenon. The severity, duration and activity of the disease had no effect on the degree of rosette formation.

ECHOvirus 33 isolated from the cerebrospinal fluid of a patient with agammaglobulinemia was shown to replicate in vitro in peripheral leukocyte suspensions from a normal antibody negative donor. Replication was demonstrated by use of sequential dilution experiments. Washed glass adherent mononuclear cell cultures from a normal antibody negative donor were also capable of supporting ECHOvirus 33 replication in vitro. Leukocyte suspensions from the infected agammaglobulinemic patient extinguished detectable infectious virus in vitro.

Nucleocapsids in quantities approaching 1 mg were purified from 109 measles virus-infected cells. They contained one polypeptide species with a molecular weight of 59,000. Antiserum was raised in rabbits against purified nucleocapsids and used in a competitive radioimmunoassay. Because of their instability, purified nucleocapsids were not suitable for use in such an assay. Instead partially purified nucleocapsids from HEp-2 cells persistently infected with measles virus and labeled in vitro with [35S]methionine were used as the source of radioactive antigen. The radioimmunoassay thus developed measured less than 5 ng of nucleocapsids in infected cells.

Radioimmunoassays for leukotriene C4 (LTC4) and for leukotriene B4 (LTB4) have been developed. LTC4 was conjugated with thiolated hemocyanin (Keyhole Limpet) (KLH) using 6-(N-maleimido)hexanoic acid chloride as coupling agent. LTB4 was converted to its hydrazide derivative, via the delta-lactone and the hydrazide was similarly coupled with thiolated KLH using 6-(N-maleimido)hexanoic acid chloride as coupling agent. These conjugates were used to consistently raise high titres of anti-leukotriene antibodies in rabbits. 14,15-[3H]-LTC4 was prepared by total synthesis via two routes. 14,15-[3H]-LTB4 was prepared by total synthesis. The assay for LTC4 recognizes LTC4, LTD4 and LTF4, and to a lesser extent, LTE4 with a detection limit of ca. 0.1 pmoles LTC4 per mL of sample. The assay for LTB4 is highly specific and has a similar detection limit.

Receptor binding proteins of Treponema pallidum were identified by incubation of [35S]methionine-labeled, soluble T. pallidum preparations with formaldehyde-fixed HEp-2 cells. Three major treponemal proteins (bands 1--3) that avidly bound to the eucaryotic cell surface were detected by sodium dodecylsulfate-polyacrylamide gel electrophoresis and fluorography. Brief trypsin treatment of HEp-2 cells before formaldehyde fixation reduced the extent of the interaction of these treponemal macromolecules, which implicated receptor-mediated attachment mechanisms. The presence of unlabeled T. pallidum preparations directly competed with radiolabeled T. pallidum samples for the available HEp-2 cells, which suggested a limiting number of membrane binding sites. Samples of unlabeled avirulent Reiter treponeme did not compete. T. Pallidum immunogens were examined by radioimmunoprecipitation with human and rabbit syphilitic sera. Of interest were the similarities and extent of the humoral response represented by the detection of antigen-antibody complexes against numberous treponemal proteins, including bands 1--3. T. pallidum portein band 1 appeared to be the major antigenic stimulus. Formation of antigen-antibody complexes between 35S-labeled T. pallidum proteins and human syphilitic sera was prevented by unlabeled T. pallidum but not by T. phagedenis preparations, which demonstrated specificity of the reaction. Gel profiles of radioimmunoprecipitation assays using radiolabeled T. pallidum antigens and human syphilitic and yaws sera delineated both the similarities and differences in the humoral response to these two spirochetes. The latter suggested both overlapping and distinguishing antigenic properties between T. pallidum and T. pertenue. Detection in yaws sera of specific antibody against T. pallidum protein bands 1--3 further incriminates the role of these three treponemal proteins as virulence determinants.

Viral polypeptides in extracts prepared from [35S]methionine-labeled human epithelial carcinoma cells persistently infected with measles virus were reacted with virus-specific antisera. Microcolumns of staphylococcal protein A linked to Sepharose were used to isolate antigen-antibody complexes that contained few contaminating host polypeptides. Measles virus polypeptides in these complexes could be identified readily on sodium dodecyl sulfate-polyacrylamide gels even when antiserum with low reactivity toward the viral antigens was used.