Characterization of the 1H-cyclopentapyrimidine-2,4(1H,3H)-dione derivative (S)-CPW399 as a novel, potent, and subtype-selective AMPA receptor full agonist with partial desensitization properties.

(S)-CPW399 (2b) is a novel, potent, and subtype-selective AMPA receptor full agonist that, unlike (S)-willardiine and related compounds, in mouse cerebellar granule cells, stimulated an increase in [Ca(2+)](i), and induced neuronal cell death in a time- and concentration-dependent manner. Compound 2b appears to be a weakly desensitizing, full agonist at AMPA receptors and therefore represents a new pharmacological tool to investigate the role of AMPA receptors in excitotoxicity and their molecular mechanisms of desensitization.

Photogenotoxicity of skin phototumorigenic fluoroquinolone antibiotics detected using the comet assay.

The fluoroquinolone (FQ) antibiotics photosensitize human skin to solar UV radiation and are reported to photosensitize tumor formation in mouse skin. As tumor initiation will not occur without genotoxic insult, we examined the potential of ciprofloxacin, lomefloxacin, fleroxacin, BAYy3118 (a recently developed monofluorinated quinolone) and a nalidixic acid to photosensitize DNA damage in V79 hamster fibroblasts in vitro. Cells were exposed to 37.5 kJ/m2 UVA (320-400 nm; glass filtered Sylvania psoralen + UVA (PUVA) tubes; calibrated Waldmann radiometer) at 4 degrees C in the presence of FQ and immediately afterwards embedded in agarose, lysed and placed in an electrophoretic field at pH 12. Under these denaturing conditions, the presence of DNA single-strand breaks (SSB), alkali-labile sites (ALS) and double-strand breaks (DSB) can be visualized as DNA migrating away from the nucleus (characteristic "comet" appearance) after staining with a specific fluorochrome. At FQ concentrations that induced minimal loss of cell viability (neutral red uptake assay) the compounds tested induced comets with a rank order of BAYy3118 > norfloxacin > ciprofloxacin > lomefloxacin > fleroxacin > nalidixic acid. If cells were incubated after treatment for 1 h at 37 degrees C, the comet score decreased, suggesting efficient removal of SSB/ALS/DSB. Addition of the DNA polymerase(alpha) inhibitor, aphidicolin, to cells treated with either ciprofloxacin alone or ciprofloxacin + UVA resulted in an accumulation of SSB due to the endo/exonuclease steps of excision repair. We have demonstrated that the FQ are photogenotoxic in mammalian cells but the FQ-photosensitized SSB are efficiently repaired. Preliminary evidence that ciprofloxacin photosensitizes the formation of DNA lesions warranting excision repair may indicate production of more mutagenic lesions.

Metabolizing systems in cell culture cytotoxicity tests.

1. The addition of 9000 g supernatant of rat liver homogenate (S9) or rat liver microsomal fractions to a cytotoxicity test system using BCL-D1 cells has been investigated. 2. The choice of culture medium influenced the intrinsic cytotoxicity of the metabolising system to the BCL-D1 cells. Use of Ham's F10 nutrient mixture resulted in greater cytotoxicity compared with several other media. 3. Microsomal fractions provided greater cytochrome P-450 dependent activation of cyclophosphamide and were less cytotoxic than S9. 4. Direct-acting toxic compounds, such as p-aminophenol, were less toxic in the presence of a metabolising system. This was due to protein-binding rather than enzymic detoxification.

Erythrocyte radiolabelling: in vitro comparison of chromium, technetium, and indium in undamaged and heat damaged cells.

The stability of the commonly used red cell radio labels chromium-51, indium-(111 or 113m), and technetium-99m, within intact red cells and stroma and their distribution within the cell were compared in undamaged and heat damaged red cells in relation to the clinical use of heat damaged cells in the assessment of splenic function. Chromium-51 labelled haemoglobin both in undamaged and heat damaged cells; indium predominantly labelled haemoglobin in undamaged cells but labelled stroma in heat damaged cells, even when the cells were labelled before heating; technetium-99m predominantly labelled haemoglobin in undamaged cells but only labelled stroma in heat damaged cells if these were heated before labelling. Indium was more firmly bound by stroma prepared from heat damaged cells, and technetium-99m showed a high rate of elution both from cells and stroma, although this rate was lower for heat damaged cells.

The kinetics of 111indium distribution following injection of 111indium labelled autologous granulocytes in man.

The kinetics of human autologous granulocytes, separated and labelled with 111In without isolation from plasma, have been studied in subjects with and without sepsis with the aim of identifying the fate and sites of destruction of granulocytes in man. In subjects without inflammatory disease, 111In granulocyte recovery in faeces, urine and saliva over 4 d was less than 1% of the dose, so that the activity visualized by the gamma camera represented almost 100% of the dose. On images taken at 24 and 48 h, this activity was distributed between spleen, bone marrow and liver, with foci of additional abnormal activity in subjects with inflammatory disease. Splenic activity fell between 40 min and 24 h, consistent with the presence of a splenic granulocyte pool, but remained constant after 24 h. Since granulocyte clearance from the blood was predominantly completed by 24 h, the residual splenic activity at that time reflected splenic granulocyte destruction. In patients with sepsis, the fall in splenic activity was greater than in those without, implying diversion of granulocytes from splenic destruction to tissue utilization when inflammation is present. Bone marrow activity increased between 40 min and 24 h and then remained stable. Granulocytes that were extensively manipulated in saline prior to labelling failed to localize in marrow, suggesting that visualization of the latter reflected destruction of intact, normal granulocytes. Although the changes in splenic and marrow activities terminated at 24 h, at which time granulocyte clearance from blood was at least 80% completed, plasma 111In remained essentially unchanged between 40 min and 48 h at less than 5% of the dose, discounting it as the source of splenic and marrow activities.

A simple quantitative estimate of the function of radiolabelled platelets.

The function ex vivo of autologous platelets, labelled with 111In, was evaluated at various times following injection by comparing the retention on foreign surfaces of radioactivity with the retention of total platelets (represented almost entirely by unlabelled and therefore unmanipulated platelets) after the application of fresh un-anticoagulated whole blood to (a) filter paper and (b) glass bead columns. Relative retentions were similar for the two materials, with labelled platelet retention being about 80% that of native platelet retention. Prior to injection, when labelled platelets were returned to an excess of freshly drawn whole blood, retention of radioactivity was greater than that of native platelets with a ratio significantly greater than that seen with labelled platelets tested ex vivo. Filter paper retention provides a simple technique which may be useful for the evaluation of labelled platelet function as a function of platelet age and also, with further standardisation, as an inexpensive rapid test of unlabelled platelet function.

The comparative blood clearance kinetics of modified radiolabelled erythrocytes.

The rate of clearance from blood of 111In-labelled heat damaged autologous erythrocytes (HD-RBC) has been compared with that of simultaneously injected autologous 99mTc-labelled erythrocytes (IgG-RBC) coated with a Rhesus anti-D antibody. In 17 studies, the number of antibody molecules coating the erythrocytes was 9000 (high coating) and in nine studies the number was 5000 (low coating). On gamma camera imaging, IgG-RBC uptake, at both levels of coating, could be visualized only in the spleen. HD-RBC were predominantly taken up by the spleen, although slight 111In activity was visible in the liver. The blood clearance of IgG-RBC was monoexponential, whereas that of HD-RBC was biexponential. The reciprocal of the t1/2 (the time taken for the 3 min value to fall by 50%) of the HD-RBC clearance correlated rather poorly with the rate constant of the simultaneous IgG-RBC clearance (r = 0.47, P greater than 0.05 at high coating; r = 0.75, P less than 0.05 at low coating). The rate constant of the second exponential of the HD-RBC clearance showed a correlation with the rate constant of IgG-RBC clearance that was closer than the reciprocal of the t1/2 of HD-RBC clearance (r = 0.89, P less than 0.001 at high coating; r = 0.76, P less than 0.05 at low coating) but significantly closer only at high coating. Splenic blood flow, measured using indium labelled platelets in ten subjects, correlated closely with the initial slope of HD-RBC clearance (r = 0.93, P less than 0.001).

Measurement of splenic blood flow in the anaesthetised dog using electromagnetic flowmetry and indium labelled platelets.

Splenic blood flow has been measured in the dog using indium labelled autologous platelets (SBFp) and compared with splenic blood flow measured with an electromagnetic flowmeter placed on the splenic vein (SVBF). The overall correlation between SBFp and SVBF was only moderately close (r = 0.54, P less than 0.01) and SBFp was about half SVBF. When expressed as a change between sequential measurements in an individual animal, the correlation between SBFp and SVBF was closer (r = 0.85, P less than 0.001). It was concluded that SBFp is a specific measure of splenic pulp blood flow and that the discrepancy between SBFp and SVBF reflects the degree of splenic blood flow bypassing the pulp.

Measurement of granulocyte migration and accumulation in inflammation in man.

A method has been developed for studying granulocyte kinetics in inflammation. Autologous granulocytes isolated from whole blood and labelled with 111indium, without separation from plasma enriched media, localized rapidly in intra-abdominal abscesses and inflammatory bowel disease following intravenous reinjection. Utilizing a gamma camera interfaced to a computer for dynamic imaging, uptake of activity in the site of inflammation could be demonstrated from 10 min following injection. A quantitative estimate of the rate of granulocyte uptake in inflammatory foci, (early granulocyte accumulation index EGAI) was made by subtracting the gradient of the time activity profile, recorded over the inflammatory focus from that of the profile recorded over a similar sized control region, and expressing the difference in relation to the zero time intercept of the profile over the inflammatory focus. The EGAI showed a positive correlation with the ESR (sigma = 0.574; P less than 0.05) and, in the single patient studied twice, decreased following steroid therapy. This technique should prove useful in studying granulocyte kinetics in inflammation in man.

Lung transit of 111Indium-labelled granulocytes. Relationship to labelling techniques.

The early in vivo distribution of 111Indium-labelled granulocytes, recorded by dynamic imaging using a gamma camara and computer, varied according to the separation and labelling technique. Following i.v. bolus injection, 4 kinetic patterns could be identified: (A) rapid transit through the pulmonary vasculature, (B) delayed transit through the lung with clearance by about 30 min, (C) complete retention by the lung, for up to 10 min, followed by slow release over a period of 1 to 2 h, (D) delayed transit through the lung with a similar time course to (B) but with subsequent heavy liver uptake. Granulocytes labelled with 111In-tropolonate and maintained in plasma throughout the labelling procedure, whether injected as a 'pure' (separated by plasma-enriched density gradient centrifugation) or 'crude' (separated by differential centrifugation) preparation, displayed type A kinetics, thought to most closely represent the normal behaviour of granulocytes. 'Crude' cells labelled in saline with 111In-acetylacetonate displayed type B kinetics. 'Pure' cells isolated on Percoll-saline and labelled in saline with 111In-acetylacetonate displayed type C kinetics, thought to represent granulocyte 'stimulation' and/or damage, or type D kinetics, thought to represent severe damage. The importance is stressed of labelling granulocytes for kinetic studies with a technique that results in minimal alteration of cell behaviour.

Imaging of inflammation with indium-111 tropolonate labeled leukocytes.

Indium-111 tropolonate has recently been introduced as a new cell-labeling agent. It has the interesting property of labeling cells in plasma with high efficiency, and may therefore promote an improvement in viability of labeled cells. This paper describes our initial experience with In-111 tropolonate as a leukocyte label for abscess imaging. Pure populations of separated granulocytes, as well as crude leukocyte preparations, have been labeled. Of 101 studies performed, 51 were positive (no false positives) and 50 negatives, of which only two were false negatives. Localization in sites of inflammation was prominent and rapid. Of 36 positive studies, 27 were already positive at 40 min following injection and an additional nine at 3 hr. Of the other 15 positive studies, 11 were scanned for the first time at 3 hr, when they were positive. Granulocytes labeled with this agent in plasma showed minimal sequestration in lungs and liver, interpreted as indicating improved viability in comparison with cells displaying prolonged lung sequestration.