Breakthrough instruments and products steady-state and time-resolved photoluminescence using the FluoTime 300 spectrometer with a FluoMic add-on.

This report highlights the combination of the FluoTime 300 photoluminescence spectrometer with a FluoMic add-on as a powerful tool for photophysical research and applications, yielding spectral, temporal, and spatial information on a wide range of samples. The steady-state and time-resolved measurement capabilities of this combination are demonstrated reflecting a broad range of applications.

Radioligand binding of antagonists of platelet-activating factor to intact human platelets.

Two new antagonists of platelet-activating factor (PAF), the pyrrolothiazole derivative 52770 RP and the triazolodiazepine WEB 2086, have been studied as radioligands in intact human platelets. [3H]52770 RP and [3H]WEB 2086 bound specifically to high-affinity sites with dissociation constants (Kd) of 14.8 and 6.1 nM, respectively. The maximal number of sites for [3H]52770 RP binding was approx. 15-fold higher than for [3H]PAF and [3H]WEB 2086. In addition, C16-PAF, lyso-PAF, WEB 2086 and 52770 RP had Ki values which were nearly identical for both [3H]PAF and [3H]WEB 2086, whereas only 52770 RP competed for [3H]52770 RP-binding sites. These results demonstrate that in human platelets the sites of [3H]WEB 2086 binding are identical to [3H]PAF-binding sites, whereas those of [3H]52770 RP are not. [3H]WEB 2086 appears, therefore, to be a suitable antagonist radioligand for labelling PAF receptors.

Inhibition of a store-operated Ca2+ entry pathway in human endothelial cells by the isoquinoline derivative LOE 908.

1. The novel cation channel blocker, LOE 908, was tested for its effects on Ca2+ entry and membrane currents activated by depletion of intracellular Ca2+ stores in human endothelial cells. 2. LOE 908 inhibited store-operated Ca2+ entry induced by direct depletion of Ca2+ stores with 100 nM thapsigargin or 100 nM ionomycin with an EC50 of 2 microM and 4 microM, respectively. 3. LOE 908 did not affect thapsigargin- or ionomycin-induced Ca2+ release from intracellular stores up to concentrations of 3 microM. 4. LOE 908 reversibly suppressed thapsigargin- as well as ionomycin-induced whole-cell membrane currents. 5. The LOE 908-sensitive membrane conductance corresponded to a cation permeability of 5.5 and 6.9 fold selectivity for Ca2+ over K+ in the presence of thapsigargin and ionomycin, respectively. 6. Our results suggest that the isoquinoline, LOE 908 is a novel, potent inhibitor of the store-operated (capacitive) Ca2+ entry pathway in endothelial cells.

Inhibition by the PAF antagonist WEB 2086 of PAF induced inositol-1,4,5-trisphosphate production in human platelets.

Platelet-activating factor (PAF) activates human platelets by binding to a putative PAF receptor which evokes the rapid formation of inositol-1,4,5-trisphosphate (IP3) by phospholipase C mediated phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis. Stimulation of [3H]inositol-labeled human platelets by PAF (1 nM-1 microM) resulted in a concentration-dependent increase of intracellular IP3, IP2 and inositolmonophosphate (IP1). IP1 levels increased up to three-fold upon maximum stimulation by 100 nM PAF. The EC50 concentration for PAF was 1.2 +/- 0.3 nM. Addition of the hetrazepinoic PAF antagonist, WEB 2086, inhibited PAF stimulated hydrolysis of PIP2 in a dose-dependent manner. WEB 2086 (100 microM) blocked inositol-1,4,5-trisphosphate formation down to baseline levels (IC50 = 33 +/- 12 microM WEB 2086). In thrombin and ADP stimulated platelets, inositol phosphate (IP) generation was not influenced by WEB 2086. It is concluded that WEB 2086 selectively antagonizes PAF-induced increases in IP and does not interfere directly with intracellular signal transduction. Instead, WEB 2086, which has been shown to bind specifically and with high affinity (Ki 15 nM) to human platelets, acts as a competitive antagonist at the PAF receptor level.

Histopathology of tumour associated sarcoid-like stromal reaction in breast cancer. An analysis of 5 cases with immunohistochemical investigations.

In 5 cases of invasive ductal and lobular carcinoma of the breast multiple epithelioid and giant cell containing granulomas were detected, localized mainly in circumferential regions, but also in the center of the carcinomas. These granulomas were interpreted as sarcoid-like stromal reactions, occurring as sarcoid-like lesions in uni- and bilateral primaries, in a recurrent tumour, and also in axillary lymph nodes. Histopathologically, these granulomas were not quite uniform, some of them corresponding to typical sarcoidosis, others showing marked proliferations of epithelioid or giant cells or containing fibrinoid exudate or necroses. The granulomas were surrounded by dense infiltrates of mononuclear cells. Tuberculosis and mycosis was excluded. There were no hints of generalized sarcoidosis. Pathogenetically, these are reactions in the tumour stroma of varying intensity, and are not caused by necroses of the tumour tissue nor by microbial infections. Such tumour-associated sarcoid-like stroma reactions are interpreted as a T-cell mediated immune response to an antigen expression of the carcinoma acting as the local trigger; in 2 cases they were connected with sarcoid-like lesions of the axillary lymph nodes. Their occurrence in bilateral carcinoma of the breast points to an immunological disposition for this special kind of host-versus-tumour response. The intensity of these changes in a recurrent tumour reflects an immunological hypersensitivity reaction. The pathogenetic and differential diagnostic aspects of epithelioid granulomas of the female breast in chronic granulomatous mastitis, panniculitis, foreign body reaction, rare infections, and in therapeutically induced sarcoidosis are described and discussed.

Hetrazepine PAF antagonists.

Hetrazepines such as WEB-2086 or WEB-2170 are potent, specific, competitive PAF antagonists binding at the same site as the PAF molecule. Despite their structural similarity to known hypnotic drugs they lack hypnotic actions. They almost certainly bind not only cell-surface but also intracellular PAF receptors, which may explain their activity in disease models in which adherence of neutrophils or other cell types to the vascular endothelium plays a role.

Serum PAF-acetylhydrolase in severe renal or hepatic disease in man: relationship to circulating levels of PAF and effects of nephrectomy or transplantation.

(1) PAF-acetylhydrolases form a major pathway for the degradation of platelet-activating factor (PAF). Here we investigate the role of the kidney and the liver in the control of PAF-acetylhydrolase levels by comparing normal subjects to patients with abnormal liver or kidney function. These patients had either severe chronic liver disease, chronic renal failure or were anephric. In a few cases PAF was also measured. (2) In those patients where PAF was measured there was no evidence that circulating PAF levels determined PAF-acetylhydrolase release. (3) In anephric patients serum PAF-acetylhydrolase levels were normal or even raised. Therefore the kidney is unlikely to be the usual major source of serum PAF-acetylhydrolase in man. (4) Liver patients with chronic cholestasis had elevated serum PAF-acetylhydrolase especially in stage III or IV primary biliary cirrhosis, as well as in a patient with secondary biliary cirrhosis and one with cholangiocarcinoma. Since normalisation of liver function following liver transplantation was accompanied by a reduction to normal or near normal PAF-acetylhydrolase levels, it is likely that the liver can play an important role in regulating levels of this enzyme in serum.

In vitro and in vivo pharmacological characterization of BIIL 284, a novel and potent leukotriene B(4) receptor antagonist.

BIIL 284 is a new LTB(4) receptor antagonist. It is a prodrug and has negligible binding to the LTB(4) receptor. However, ubiquitous esterases metabolize BIIL 284 to the active metabolites BIIL 260 and BIIL 315, the glucuronidated form of BIIL 260. Both metabolites have high affinity to the LTB(4) receptor on isolated human neutrophil cell membranes with K(i) values of 1.7 and 1.9 nM, respectively. On vital human neutrophilic granulocytes K(i) was around 1 nM. BIIL 260 and BIIL 315 interact with the LTB(4) receptor in a saturable, reversible, and competitive manner. BIIL 260 and its glucuronide BIIL 315 also potently inhibited LTB(4)-induced intracellular Ca(2+) release in human neutrophils (IC(50) values of 0.82 and 0.75 nM, respectively) as measured with Fura-2. High efficacy of BIIL 284 has been demonstrated in various in vivo models. BIIL 284 inhibited LTB(4)-induced mouse ear inflammation with ED(50) = 0.008 mg/kg p.o., LTB(4)-induced transdermal chemotaxis in guinea pigs with ED(50) = 0.03 mg/kg p.o., LTB(4)-induced neutropenia in various species (monkey: ED(50) = 0.004 mg/kg p.o.), and LTB(4)-induced Mac1-expression in monkeys (ED(50) = 0.05 mg/kg p.o. in Tylose). Full blockade of LTB(4) receptors over 24 h was achieved by 0.3 mg/kg BIIL 284 after single oral dose as measured by LTB(4)-induced neutropenia or Mac1-expression in the monkey model. BIIL 284 is an unusually potent and long-acting orally active LTB(4) antagonist, and is therefore under clinical development as a novel anti-inflammatory principle.

Pharmacodynamics, pharmacokinetics and safety profile of the new platelet-activating factor antagonist apafant in man.

Platelet-activating factor (PAF) is a unique phospholipid mediator with multifunctional properties. Evidence generated in experimental studies suggests that PAF plays a pathogenetic role in anaphylactic, inflammatory and immunogenic reactions. Apafant (WEB 2086, CAS 105219-56-5), a novel synthetic PAF receptor antagonist, was administered to a total of 101 healthy volunteers within 5 studies to investigate its pharmacologic activity, pharmacokinetic behaviour and safety profile. Pharmacologic activity was monitored by inhibition of 5 x 10(-8) mol/l PAF-induced platelet aggregation ex vivo. The following treatment schedules were studied: oral single dose 1.25 to 400 mg; oral multiple dose 100 mg t.i.d. over 7 days; i.v. infusion 0.5 to 50 mg (over 30 min); inhalative administration up to 1.0 mg. PAF induced platelet aggregation was virtually completely inhibited by single oral doses of 20 mg upwards, throughout during the multiple oral dose study, at all dose levels tested in the i.v. study and (significantly but not completely) at 0.5 and 1.0 mg in the inhalative study. Following oral administrations (capsules) apafant is absorbed rapidly (tmax 1 to 2 h), there is linear pharmacokinetics for the mean plasma concentrations of apafant measured by RIA as well as for the areas under the curve (AUCs). Approximately 60% of apafant is bound to plasma protein, the mean volume of distribution is 28 l, about 44% of an oral dose is excreted in the urine, the mean renal clearance is 192 ml/min. No accumulation of the drug occurred in volunteers with normal kidney function. No clinically relevant drug related adverse events or changes in laboratory or vital parameters such as blood pressure, heart rate, respiratory rate and ECG were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of human HL-60 cell responses to chemotactic factors by antisense messenger RNA depletion of G proteins.

Chemotactic factors bound to receptors of the seven-transmembrane domain family signal leukocytes through associated guanine nucleotide-binding (G) proteins. Human leukocytes of the HL-60 line, which express G protein-coupled receptors for leukotriene B4 (LTB4) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) after differentiation with vitamin D3 and transforming growth factor-beta, were transfected with expression plasmids containing antisense-oriented cDNAs encoding the alpha-chains of Go, Gi1, Gi2, and Gi3. Antisense mRNA for Go and Gi2 alpha-chains suppressed by over 80% the level of the respective G protein. Go-deficient HL-60 cells had depressed functional and intracellular calcium responses to LTB4 and fMLP, but no alterations in the responses of cyclic adenosine 3',5'-monophosphate (cAMP). In contrast, HL-60 cells deficient in Gi2 lost only responses of the intracellular concentration of cAMP. Antisense mRNA suppression of distinct G proteins thus may delineate some transductional requirements for cellular responses.