Multiple batch extraction test to estimate contaminant release parameters using a Bayesian approach.

Industrial activities produce vast amounts of weakly contaminated materials which are commonly reused as filling materials on natural ground. There is a strong demand to define guidelines for the application of these materials, to estimate the leaching potential of contaminants from the materials, and to assess the potential hazard for groundwater pollution. We present a multiple batch experiment, where measurements of liquid-phase concentrations at varying liquid/solid ratios are used to estimate the total mass of contaminant that can be extracted from a contaminated material with a mild extractant like water. Furthermore, the experiment yields estimates of the isotherm describing the partitioning of the contaminant between the solid and liquid phases, and a concentration that might be expected under soil hydraulic conditions representative for the field situation. Model parameters are estimated from liquid-phase concentrations within a Bayesian framework by applying the Shuffled Complex Evolution Metropolis Algorithm (SCEM-UA), an efficient Markov Chain Monte Carlo sampler. A sensitivity analysis and inversions of synthetically generated data corrupted with noise show the general suitability of the proposed method. An uncertainty analysis for model parameters and model predictions shows the expected accuracy of the estimates. An application to concentration measurements obtained from a multiple batch extraction test illustrates the applicability of the approach for a real situation.

Granzyme B is expressed in mouse mast cells in vivo and in vitro and causes delayed cell death independent of perforin.

Mast cells respond to pathogens and allergens by secreting a vast array of preformed and newly synthesized mediators, including enzymes, vasoactive amines, lipid mediators, cytokines and chemokines, thereby affecting innate and adaptive immune responses and pathogenesis. Here, we present evidence that skin-, but not lung-associated primary mast cells as well as in vitro-differentiated bone marrow-derived mast cells (BMMC) express granzyme (gzm) B, but not gzmA or perforin (perf). GzmB is associated with cytoplasmic granules of BMMC and secreted after Fcepsilon-receptor-mediated activation. BMMC from wild type but not gzmB-deficient mice cause cell death in susceptible adherent target cells, indicating that the perf-independent cytotoxicity of BMMC is executed by gzmB. Furthermore, gzmB induces a disorganization of endothelial cell-cell contacts. The data suggest that activated mast cells contribute, via secreted gzmB, to cell death, increased vascular permeability, leukocyte extravasation and subsequent inflammatory processes in affected tissues.

Improving access to and utilization of genetic services in Arizona's Hispanic population.

This abstract presents a model project aimed to train community lay health workers about genetics, increase cultural competency of genetic services providers, and provide local access to genetic services in primarily Hispanic communities in the state of Arizona. Health Start, a community-based prenatal outreach program, served as the basis for providing genetic education and services. A genetics training curriculum was developed and training of community lay health workers was provided. Cultural and Spanish language training was provided for all genetic services providers. Pediatric genetics outreach clinics were established in eight communities. Community-based lay health workers eagerly incorporate genetic information into their public health knowledge base, but this may not lead to acceptance of these personnel by local health care providers as sources of referrals for specialized health services such as genetics. Cultural competence training of genetic service providers is enthusiastically accepted and utilized in the provision of locally accessible genetics clinics.

Activation of the human neutrophil secretory process with 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid.

Exposure of human neutrophils to 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (leukotriene B4, LTB4) resulted in a time- and concentration- (10(-9)-10(-6) M) dependent extracellular release of granule-associated lysozyme and myeloperoxidase (MPO). Enzyme extrusion was negligible if cells were not pretreated with cytochalasin B prior to exposure to LTB4. A time-dependent deactivation of granule exocytosis was observed in neutrophils which were stimulated with LTB4 prior to contact with cytochalasin B. LTB4-induced enzyme release was markedly enhanced in the presence of extracellular calcium. Nevertheless, significant enzyme discharge occurred in the absence of extracellular calcium, and the percent of total activity released was not altered in the presence of EGTA. The calmodulin antagonist, trifluoperazine (TFP), and the intracellular calcium antagonist, 8-(N,N-diethylamino)-octyl-(3,4,5-trimethoxy)benzoate hydrochloride (TMB-8), caused a dose-related inhibition of enzyme release from LTB4-stimulated neutrophils. Degranulation was suppressed by the glycolytic inhibitor, 2-deoxy-D-glucose (2-DG), and the sulfhydryl reagents iodoacetic acid (IA) and N-ethylmaleimide (NEM). Sodium cyanide was inactive. Two inhibitors of transmethylation, 3-deazaadenosine (3-DZA) and L-homocysteine thiolactone (HCTL), alone or in combination, had no effect on LTB4-elicited degranulation. The protein synthesis inhibitor, cycloheximide, was inactive. Neutrophils pretreated with LTB4 or 5(S),12(R),20-trihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (20-OH-LTB4, an omega-oxidation metabolite of LTB4) were desensitized to the subsequent exposure to LTB4. Cross-desensitization was also demonstrated between LTB4 and 20-OH-LTB4. The stimulus specific nature of LTB4-induced desensitization of neutrophil degranulation was demonstrated by the fact that cells exposed to 1-O-hexadecyl/octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC) or N-formyl-methionyl-leucyl-phenylalanine (FMLP) were capable of inducing granule exocytosis from LTB4-pretreated neutrophils. Enzyme release from LTB4-treated cells was suppressed with the phospholipase inhibitor, 4-bromophenacyl bromide (4-BPB), the cyclooxygenase/lipoxygenase inhibitor, ETYA, and the 5-lipoxygenase inhibitor, U-60, 257. However, the cyclooxygenase inhibitor, flurbiprofen, exerted a weak suppressive effect on LTB4-induced degranulation.

Activation of the human neutrophil respiratory burst with zymosan-activated serum.

Zymosan-activated serum ( ZAS ) stimulated a time- and concentration-dependent generation of superoxide anion (O-2) by human neutrophils. O-2 production was rapid with maximum generation occurring 2 minutes after cell exposure to ZAS . O-2 generation is markedly reduced if cells are not preincubated with cytochalasin B prior to contact with ZAS . The amount of O-2 produced by ZAS stimulated neutrophils was enhanced in the presence of extra-cellular calcium. However, the intracellular calcium antagonist, 8-(N,N-diethylamino)-octyl-(3,4,5-trimethoxy) benzoate hydrochloride (TMB-8), caused a dose-related inhibition of ZAS -elicited O-2 production. Neutrophils pretreated with ZAS were desensitized to the subsequent exposure to this stimulus. The fact that pretreatment of neutrophils with ZAS did not diminish the capacity of these cells to generate O-2 in response to 1-O-hexadecyl/octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC),N-formyl-methionyl-leucyl-phenylalanine (FMLP) or 5(5),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB4), demonstrates the stimulus specific nature of ZAS -induced desensitization. Thus, ZAS , which contains the complement-derived neutrophil activator, C5a, a naturally occurring phlogistic mediator, represents a relevant probe for investigating neutrophil function.

Effects of an anion channel blocker, 4, 4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS), on human neutrophil function.

We investigated the capacity of DIDS to influence degranulation and superoxide anion (O-2) generation by human neutrophils exposed to soluble, surface-active stimuli. DIDS caused a concentration-related (25-200 microM) inhibition of myeloperoxidase (MPO) release from N-formyl-methionyl-leucyl-phenylalanine (FMLP), 5(S), 12(R)-dihydroxy-6, 14-cis-8, 10-trans-eicosatetraenoic acid (LTB4), 1-0-hexadecyl/octadecyl-2-0-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC) and phorbol myristate acetate (PMA)-stimulated neutrophils. In contrast, DIDS had no effect on O-2 production by FMLP and PMA activated cells, whereas it enhanced LTB4 and AGEPC-elicited O-2 generation. The respective effects of DIDS on these neutrophil activities were reversed by washing the cells prior to exposure to stimulus. Thus, DIDS represents a useful pharmacologic probe for investigating the role(s) of anions in the mechanisms of neutrophil activation with structurally and chemically dissimilar stimuli.

Stimulation of the human neutrophil superoxide anion-generating system with 1-O-hexadecyl/octadecyl-2-acetyl-sn-glyceryl-3- phosphorylcholine.

1-O-Hexadecyl/octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC) stimulated a time- and concentration-dependent generation of superoxide anion (O-2) by human neutrophils. Maximum production of O-2 occurred 60 sec subsequent to cell contact with AGEPC. Superoxide generation was reduced significantly if cells were not preincubated with cytochalasin B prior to exposure to AGEPC (0.01 to 10 microM). A time-dependent desensitization for O-2 production was demonstrated in neutrophilis which were stimulated with AGEPC prior to contact with cytochalasin B. The rate and amount of O-2 generated by AGEPC-activated neutrophils were enhanced significantly in the presence of extra-cellular calcium. However, incubation of neutrophils with ethyleneglycol-bis(beta-amino-ethyl ether) N,N'-tetra-acetate (EGTA) in calcium-free medium had no effect on the O-2 generating system. AGEPC-induced O-2 production was suppressed by the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) and the sulfhydryl reagents N-ethylmaleimide (NEM) and iodoacetic acid (1A). Sodium cyanide was inactive. Pretreatment of neutrophils with AGEPC reduced cell responsiveness to subsequent exposure to this stimulus. Desensitization of the O-2-generating system activated with AGEPC appears to be stimulus specific for leukotriene B4 (LTB4) and N-formyl-methionyl-leucyl-phenylalanine (FMLP) in that these stimuli were capable of inducing O-2 production by cells pretreated with AGEPC. However, neutrophils pretreated with AGEPC were desensitized to zymosan-activated serum (ZAS).

Characteristics of 1-O-hexadecyl- and 1-O-octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine-stimulated granule enzyme release from human neutrophils.

1-O-Hexadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (C16-AGEPC) and 1-O-octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (C18-AGEPC) stimulated a time- and concentration-dependent release of granule-associated lysozyme and beta-glucuronidase from human neutrophils. Maximum discharge of granule enzymes occurred between 30 and 60 sec after neutrophil exposure to C16- or C18-AGEPC (0.01-10 microM). Less than 10% of total enzyme activity is released when cells are not preincubated with cytochalasin B prior to interaction with the AGEPC analogs. A time-dependent desensitization for granule exocytosis was observed in neutrophils which were stimulated with C18-AGEPC prior to contact with cytochalasin B. The rate and amount of enzyme released by C16- and C18-AGEPC activated neutrophils was significantly enhanced in the presence of extracellular calcium. Trifluoperazine, an inhibitor of calmodulin, caused a dose-related suppression of C18-AGEPC-induced degranulation. Granule enzyme extrusion from C18-AGEPC-treated neutrophils was inhibited by the sulfhydryl reagents, N-ethylmaleimide and iodoacetic acid, and by the glycolytic inhibitor, 2-deoxy-D-glucose. Sodium cyanide was inactive. Pretreatment of neutrophils with C16- or C18-AGEPC rendered the cells unresponsive to subsequent exposure to either AGEPC analog. C18-AGEPC-induced desensitization of neutrophil degranulation appears to be stimulus specific in that serum-treated zymosan and N-formyl-methionyl-leucyl-phenylalanine were capable of eliciting granule enzyme release from C18-AGEPC-pretreated cells.

Biochemical, metabolic and morphological characteristics of human neutrophil activation with pepstatin A.

Pepstatin A, a chemotactic pentapeptide, elicited a concentration-dependent extracellular release of granule-associated beta-glucuronidase and lysozyme from, and generation of superoxide anion (O2-) by, cytochalasin B (CB)-treated human neutrophils. Prior exposure of neutrophils to pepstatin A before the addition of CB, suppressed, in a time-dependent fashion, the subsequent production of O2- and exocytotic response. The rate and amount of enzymes released and O2- generated by pepstatin A-activated neutrophils were significantly enhanced in the presence of extracellular calcium. Pepstatin A-elicited degranulation and O2- production were suppressed by the intracellular calcium antagonist, 8-(N,N-diethylamino)-octyl-(3, 4, 5-trimethoxy) benzoate hydrochloride (TMB-8). Granule exocytosis and O2- generation by pepstatin A-treated neutrophils were suppressed by the sulphydryl reagents, N-ethylmaleimide (NEM) and iodoacetic acid (IA), and by the glycolytic inhibitor, 2-deoxy-D-glucose (2-DG). Sodium cyanide was inactive. Preincubation of neutrophils with pepstatin A "desensitized' the cells to a subsequent exposure to pepstatin A or the chemotactic tripeptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP). Pepstatin A-induced desensitization of granule enzyme release and O2- generation appears to be stimulus-specific in that phorbol myristate acetate (PMA) was capable of eliciting normal responses from pepstatin A-pretreated cells. The morphological changes observed in pepstatin A-treated neutrophils are reminiscent of those seen in cells exposed to FMLP.

A model of Arthus pleurisy: modulation by various pharmacologic and therapeutic agents.

A reversed passive Arthus reaction was elicited in the rat pleural cavity. The kinetics of this inflammatory response indicate that exudate volume (cells and fluid) reaches a maximum level approximately 2 to 4 hr postantibody challenge. The neutrophil is the major cellular constituent of the pleural exudate during the first 12 hr of this reaction, reaching peak values at 4 hr; whereas, the monocyte predominates between 15 and 24 hr. Lymphocytes, eosinophils, and mast cells were also identified in the pleural exudates. The serotonin antagonists, cyproheptadine and methylsergide, and the antihistamine, chlorpheniramine, demonstrated marginal activity in the Arthus pleurisy model. The histamine antagonist, metiamide, was inactive. The nonsteroidal anti-inflammatory agents, flurbiprofen, ibuprofen, indomethacin, and benoxaprofen caused a modest suppression of exudate volume (18-32%) and cell accumulation (28-34%). The fluid and cellular components of the Arthus reaction were significantly inhibited by dexamethasone, triamcinolone, paramethasone, and prednisolone. The oral gold preparation, auranofin, had a pronounced effect on exudate volume; whereas, other antirheumatic agents such as D-penicillamine, azathioprine, and chloroquine had no effect on the Arthus pleurisy reaction. The immunomodulator, levamisole, suppressed exudate volume, but had no effect on cell accumulation in the pleural cavity.

Effects of trifluoperazine on human neutrophil function.

The interaction of cytochalasin B-treated human neutrophils with the synthetic tripeptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP) results in a time- and concentration-dependent generation of superoxide anion (O2-) by an extracellular release of granule-associated beta-glucuronidase and lysozyme from these cells. Granule exocytosis was not accompanied by significant cytoplasmic lactate dehydrogenase extrusion. FMLP-stimulated O2- production occurs but is significantly curtailed in the absence of extracellular calcium. Nevertheless, incubation of neutrophils with EGTA in calcium-free medium had no effect on the O2- -generating system. Trifluoperazine (TFP), an inhibitor of calmodulin (a calcium-binding protein), caused a dose-related inhibition of FMLP-elicited degranulation and O2- production in the presence of absence of extracellular calcium. This effect TFP could be reversed by washing the cells before contact with FMLP. These data suggest that TFP represents a useful tool for defining the relevance of calmodulin and calcium to neutrophil function.

Properties of calcium ionophore-induced generation of superoxide anion by human neutrophils.

Exposure of human neutrophils to the calcium ionophore, A23187, eventuates in a time- and concentration-dependent generation of superoxide anion (O2-) in the presence but not absence of extracellular calcium. The selective requirement for calcium is demonstrated by the observation that magnesium caused a dose-related inhibition of A23187-stimulated O2- generation. Preincubation of neutrophils with cytochalasin B prior to their interaction with A23187 results in a significant enhancement of O2- production. The activity of the O2--generating system was maximum at 37 degrees C and was significantly curtailed at lower and higher temperatures. A23187-induced O2- generation was inhibited by the sulfhydryl reagents. N-ethyl maleimide (NEM) and iodoacetic acid (IA), and by the metabolic inhibitor 2-deoxy-D-glucose (2-DG) in the absence of glucose and cytochalasin B. Cyanide was inactive. Therefore, A23187 represents a useful pharmacologic probe for investigating the divalent cation and metabolic requirements of the neutrophil O2--generating system.

Properties of concanavalin A-elicited granule exocytosis from human polymorphonuclear neutrophils.

Exposure of human neutrophils to concanavalin A (Con A) resulted in a time- and concentration-dependent extracellular release of granule-associated lysozyme but not beta-glucuronidase or cytosolic lactate dehydrogenase. Maximum extrusion of lysozyme occurred 30 min after cell contact with Con A. The percent of total granule enzyme activity discharged is insignificant when cells are not preincubated with cytochalasin B prior to being exposed to Con A (5-50 micrograms/ml). Granule enzyme release from Con A-treated cells is markedly inhibited by alpha-methyl-D-mannoside. Con A-elicited extrusion of lysozyme is reduced significantly, but not abolished, in the absence of extracellular calcium. However, contact between neutrophils and EGTA in calcium-free medium had no effect on Con A-stimulated release of granule enzymes. 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), an antagonist of intracellular calcium, caused a dose-dependent inhibition of lysozyme discharge from Con A-treated neutrophils. The activity of TMB-8 could be abrogated with the addition of calcium, but not magnesium, to the extracellular medium. Therefore, Con A and TMB-8 should serve as useful tools for elucidating the mechanism of granule enzyme release from neutrophils.

Characteristics of N-formyl-methionyl-leucyl-phenylalanine as an inducer of lysosomal enzyme release from human neutrophils.

N-formyl-methionyl-leucyl-phenylalanine (FMLP) stimulated a time- and concentration-dependent release of granule associated beta-glucuronidase and lysozyme but not cytoplasmic lactate dehydrogenase from human neutrophils. Maximum discharge of lysome activity released is insignificant when cells are not preincubated with cytochalasin B prior to being exposed to FMLP (10(-10)-10(-7) M); although 11.2 +/- 1.3 and 12.4 +/- 1.1% of total activity for beta-glucuronidase and lysozyme, respectively, is secreteer, had no effect on FMLP-elicited lysosomal enzyme extrusion. 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), a purported antagonist of intracidating the role of calcium in the mechanism of lysosomal enzyme release.