Human colorectal adenocarcinoma cells: differential nitric oxide synthesis determines their ability to aggregate platelets.

The existence and role of an L-arginine:nitric oxide (NO) pathway in two human colorectal adenocarcinoma cell lines, SW-480 and SW-620, were investigated. Both cell lines, which derive from the same patient, SW-480 from the primary tumor and SW-620 from its metastatic lesion, were shown to have a cytosolic, Ca(2+)-independent, NADPH-dependent NO synthase, the activity of which was lower in the cytosol of SW-620. These cells were more potent inducers of platelet aggregation. In contrast, SW-480, which had more NO synthase activity, were less potent inducers of platelet aggregation. Pretreatment of both cell lines with NG-monomethyl-L-arginine, an inhibitor of NO synthase, potentiated their proaggregating effect and made them equally active. Exogenous L-arginine, NO, and related nitrovasodilators all inhibited platelet aggregation induced by SW-620. The antiaggregating activity of NO was further potentiated by prostacyclin and by M&B22948, a selective inhibitor of cyclic GMP phosphodiesterase. We propose that the generation of NO by tumor cells inversely correlates with their metastatic potential. Furthermore, we show that the lower activity of NO synthase in metastatic cells is due to the presence in these cells of a low molecular weight inhibitor of the NO synthase. In addition, agents which modulate platelet function by a cyclic GMP-dependent mechanism may be useful in the prevention of tumor metastasis.

Epilepsy, myasthenia gravis, and effect of plasmapheresis on antiepileptic drug concentrations.

A 28-year-old woman developed complex partial seizures at the age of 17 years and was treated with phenytoin sodium. Five years later she developed myasthenia gravis, and phenytoin was replaced by valproic acid and phenobarbital. She required plasmapheresis (PP). During one course of PP, total and unbound concentrations of valproic acid and phenobarbital were measured in serum sampled before, during, and after PP and in plasma removed by PP. It was determined that the magnitude of loss of valproic acid or phenobarbital by PP was small, and the changes of unbound/total ratio did not reach clinical importance.

Colorimetric quantitation of filarial viability.

A simple three-step colorimetric assay based on the tetrazolium salt MTT (3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) has been developed for quantifying filarial viability. Living (but not dead) filariae take up MTT and rapidly reduce it to formazan, so staining themselves dark blue. This colour change which is easily seen provides a rapid qualitative test for filarial viability. Quantitative data can be obtained by solubilizing formazan out of the worm with DMSO and measuring the absorbance of the resulting solution at 510 nm. To date the technique has been demonstrated in several species of filariae including Onchocerca volvulus. MTT reduction is thought to be selective for NADH-dependent dehydrogenase activity in viable worms. The reaction occurs readily in all developmental stages of Dipetalonema viteae including fragments of filarial tissue. Enzyme activity in viable intact D. viteae appears to be primarily associated with the hypodermis/muscle cells, with minimal formazan formation in the gut and reproductive tracts. The application of this MTT assay as a parameter for quantifying in vitro drugs effects is described. Assay procedures have been developed and optimized with D. viteae and Brugia pahangi for the assessment of effects of macrofilariae and microfilarial release, and the activity of a range of antifilarial standards reported. Several potential applications of the technique to studies on filarial biology are discussed.

The use of blood and blood components in 1,769 patients undergoing open-heart surgery.

There has been a decrease in the use of whole blood and red cell transfusions during and after open-heart operations in the greater Kansas City area from an average of slightly more than 9 units per patient from 1969 through 1971, to just over 3 units per patient from 1975 through 1977. In 1977, 1,256 patients, or 71% of 1,769 patients, underwent coronary artery bypass exclusively and had an average transfusion utilization of 2.6 units. All other open-heart operations averaged 4.7 units per patient. Hemodilution and the acceptance of hematocrits between 25 and 30% in open-heart operations are probably the main factors responsible for lower transfusion use per patient, while the increased proportion of patients undergoing coronary artery bypass accounts for a further decrease in the average amount of blood used per patient. It is of note that blood transfused to patients having an open-heart operation was not significantly fresher than blood for routine use, yet hemostasis was not a problem as evidenced by the small use of fresh-frozen plasma in 67 patients (3.8%) and platelet concentrates in 42 patients (2.4%).

Trichostrongylus colubriformis: in vitro culture of parasitic stages and their use for the evaluation of anthelmintics.

Approximately 50 per cent of fourth stage larvae of Trichostrongylus colubriformis taken from the gerbil, Meriones unguiculatus, on day 8 after infection, moulted to the young adult stage when cultured in a complex medium over a seven day period in vitro. Larvae at the late fourth stage of development were highly susceptible to certain benzimidazole, prebenzimidazole, imidazothiazole, pyrimidine, quaternary ammonium, organophosphorus and macrocyclic lactone anthelmintics when any of these were included at very low concentrations in the culture medium. However, few anthelmintics lacking activity against T colubriformis in vivo affected these larvae. An assay employing these larvae in vitro should offer a means for assessing the intrinsic activity of new compounds against T colubriformis in the absence of any complicating host pharmacokinetic factors, and could also be adapted for use as a high capacity preliminary screen. Thus it should now be possible to employ a target parasite at the earliest stages of a lead discovery programme obviating the need to use less relevant free-living nematodes or ones that are natural parasites of rodents.

The influence of Nematospiroides dubius on subsequent Nippostrongylus brasiliensis infections in mice.

The fecundity and longevity of Nippostrongylus brasiliensis was prolonged in mice previously infected with Nematospiroides dubius only when the former developed from the larval stage in those mice. Such worms appeared to be less immunogenic than worms which developed in mice never exposed to N. dubius. It is proposed that prolonged fecundity and longevity resulted from an adaptation undertaken by the worms in the face of host antibodies which had been developed against the pre-existing N. dubius infection.

Nematospiroides dubius: the course of primary, secondary and tertiary infections in high and low responder Biozzi mice.

Biozzi mice of Selections 1 and 3, each exhibiting either high- or low-antibody responses to a wide range of antigens, were exposed to primary, secondary and tertiary infections of the nematode, Nematospiroides dubius. The course of these infections was followed by monitoring egg output and by examination of the worm burdens on the 14th day after each exposure to the parasite. When exposed to a primary infection, high and low responders were equally susceptible to the parasite, and the egg production of the infections was similar in all groups. However, when the mice were exposed to a 2nd infection of N. dubius, marked differences in the susceptibility of the hosts were seen. The high-responder mice of both Selections 1 and 3 were significantly more resistant to infection, far less adult worms being found in these. Resistance to a 3rd and final infection of the parasite was amplified appreciably in all groups but, again, the high-responder mice were significantly more resistant than the low responders. These results imply that host antibodies play an essential role in immunity to this parasite and that resistance cannot be attributed solely to non-specific macrophage activity or cell-mediated immune reactions, since the latter are equivalent in high and low lines and the macrophages of the Ab/L line of Selection 1 are hyperactive.

An in vitro screening test for compounds active against the parenteral stages of Trichinella spiralis.

A new in vitro screening test for compounds showing activity against the tissue stages of Trichinella spiralis is described. In this test freshly decapsulated larvae of the parasite are exposed to low concentrations of experimental compound in a medium capable of supporting th partial development of the worms. The screen which does not require elaborate continuous flow equipment nor special gas phase detects the activity of those compounds known to be effective against the parenteral stages of the parasite in mice. It has been shown to be a highly selective test giving rise to very few irrelevant or misleading results.

Nippostrongylus brasiliensis and Ascaridia galli: mitochondrial respiration in free-living and parasitic stages.

Aerobic respiratory pathways have been delineated and respiratory efficiency has been assessed in mitochondria isolated from embryonated eggs, infective larvae, and adult Nippostrongylus brasiliensis and Ascaridia galli. Mitochondrial respiration in free-living stages of N. brasiliensis is mediated mainly by a mammalian-like antimycin A- and cyanide-sensitive pathway; specific respiratory activity is high and oxidative phosphorylation efficient. In mitochondria of adult N. brasiliensis, antimycin A- and cyanide-sensitive respiration is decreased relative to respiration though an alternative pathway, and specific respiratory activity and mitochondrial efficiency are lower. Respiration in mitochondria from embryonated eggs and tissues of adult A. galli is comparable, and apparently mediated by an antimycin A- and cyanide-insensitive alternative respiratory pathway; no evidence for the presence of a mammalian-like respiratory pathway in embryonated eggs of A. galli was found. The results of this study are compared to mitochondrial respiration in eggs, larvae, and adult body wall muscle of Ascaris suum.

Nematoda: aerobic respiratory pathways of adult parasitic species.

Aerobic respiratory pathways have been compared in adult parasitic nematodes, including Trichostrongylus colubriformis, Nippostrongylus brasiliensis, Ostertagia ostertagi, Cooperia oncophora, Haemonchus contortus, Oesophagostomum venulosum, Chabertia ovina, Dictyocaulus filaria, Dictyocaulus viviparus, and Ascaridia galli. Respiration was measured in both whole worm or tissue homogenates and isolated mitochondrial fractions, and delineated into the mammalian type or alternative respiratory pathways on the basis of their inhibition by antimycin A. The alternative, antimycin A-insensitive respiratory pathway was of comparable activity in all parasitic nematodes studied, irrespective of the body diameter or habitat of the worm. The mammalian-type, antimycin A-sensitive respiratory pathway showed variations; the extent of this pathway correlated with both the body diameter and habitat of the worm, being greater in thinner worms and those worms whose habitat is supposedly more aerobic.