Age-dependent changes in the regulation of cyclooxygenases in the gastrointestinal tract after gram-negative endotoxemia.

BACKGROUND: Cyclooxygenases (COXs) modulate prostaglandin synthesis in the gastrointestinal tract. Prostaglandins have been shown to have a cytoprotective effect on bowel mucosa in adults, but no similar data are available in neonates. Thus, the purpose of the current study was to evaluate age-dependent changes in gastrointestinal tract COX regulation after Escherichia coli lipopolysaccharide exposure in rats. METHODS: Stomach, small bowel, and large bowel COX-1 and COX-2 mRNA levels (reverse transcription polymerase chain reaction technique) and protein content (Western blot) were obtained from neonates (younger than 3 days old) and adult rats 18 hours after exposure to E. coli O111:B4 lipopolysaccharide toxin. Untreated animals served as controls. RESULTS: Stomach, small bowel, and large bowel tissue COX-1 mRNA levels in the newborn were significantly lower (P < 0.01) than in the adult. No age-dependent differences were found for COX-2 mRNA levels. After lipopolysaccharide exposure, no significant changes in COX-1 levels were seen at either age, whereas COX-2 mRNA levels were increased only in the stomach for both ages. Western blot analysis of small bowel tissue for COX-1 and COX-2 showed no lipopolysaccharide-induced changes in protein content, but the COX-1 content was significantly lower in the newborn (P < 0.01). CONCLUSIONS: In the rat, COX expression in the gastrointestinal tract is regulated in an age-dependent fashion. Lower COX-1 expression and a lack of observable increase in COX-2 mRNA levels in the newborn small bowel after endotoxemia may render the bowel more susceptible to bowel injury early in life.

Glucose transport in amastigotes and promastigotes of Leishmania mexicana mexicana.

Promastigotes and amastigotes of Leishmania mexicana mexicana transported 2-deoxy-D-glucose (2-DOG) by a saturable process with a Km of 24 +/- 3 microM and Vmax of 2.21 nmol min-1 (mg protein)-1 for the promastigote and a Km of 29 +/- 8 microM and Vmax of 0.13 nmol min-1 (mg protein)-1 for the amastigote stage. Amastigotes incorporated 2-DOG maximally at pH 5.0, while for promastigotes the optimum was at pH 7.0. Mid-log phase promastigotes were found to accumulate 2-DOG via a stereospecific carrier-mediated process which was competitively inhibited by D-glucose and D-mannose but not L-glucose. Transport was dependent upon temperature, with a Q10 in promastigotes of 1.83 and an optimum rate at 35 degrees C (+/- 4 degrees C) with an activation energy of 50.12 kJ mol-1. Stationary phase promastigotes accumulated 2-DOG at approximately twice the rate of mid-log phase promastigotes. Cytochalasin B, forskolin and phloretin were all found to inhibit human erythrocyte 2-DOG uptake but only cytochalasin B was found significantly to inhibit promastigote 2-DOG uptake. Interestingly, leishmanial 2-DOG uptake was inhibited by a series of membrane potential antagonists including the ionophore monensin, the H+ATPase inhibitor N, N'-dicyclohexylcarbodiimide (DCCD) and uncoupling agent carbonylcyanide-4-(triflouromethoxy) phenylhydrazone (FCCP), as well as, the tricyclic drugs chlomipramine and imipramine, but was insensitive to the Na+/K+ATPase inhibitor ouabain and the antitrypanosomal drugs Pentostam and Suramin. We therefore conclude that there are significant structural and mechanistic differences between the D-glucose uptake systems of Leishmania and the mammalian host to merit the inclusion of glucose transporters as putative targets for rational drug design.

Childhood Guillain-Barré syndrome in Paraguay, 1990 to 1991.

During 1990 to 1991, through a national surveillance program for poliomyelitis, the Paraguayan Ministry of Health received reports of 50 children with incident acute flaccid paralysis (< 15 years old). On the basis of established criteria, 37 were diagnosed with Guillain-Barré syndrome. The average annual incidence rate for 1990 to 1991 was 1.1/100,000 children. The clinical course was more benign than reported in other pediatric series. There were low rates of hospitalization (57%), respiratory compromise (8%), and intubation (5%). The overall severity, however, was similar to that described in previous reports, with a 3% case-fatality rate and an 81% total recovery rate at 12 months. Seventy-six percent of patients had symptom onset during January to April, the warmest months of the year. Thirty percent of patients had definite or possible exposure to organophosphate pesticides, and the peak use coincides with the peak incidence of Guillain-Barré syndrome. There was no correlation between occurrence of Guillain-Barré syndrome and prior immunization.

A comparative study of the utilization of ether- and ester-linked phospholipid-containing liposomes by J774.E1 macrophage cell-line infected with Leishmania mexicana mexicana amastigotes.

The macrophage cell-line J774.E1 and Leishmania m. mexicana infection was used to investigate the uptake of liposomes, which differed in their bulk phospholipid: ester- or ether-analogue of phosphatydilcholine (PC). The receptor-mediated uptake of both species of liposomes, containing native or acetylated LDL as ligands was also evaluated. Uninfected and infected J774.E1 cell-line accumulated more ester- and ether-liposomes alone than mixed type (50:50, ester/ether). The utilization was significantly enhanced when both types of liposomes contained native LDL. The highest uptake was recorded for liposomes bearing acetylated LDL by infected J774.E1 cells. Accumulation of ester- and ether-liposomes with the same ligand was not markedly affected by different chemical nature of PC. Finally, ether-liposomes alone possessed certain activity against Leishmania m. mexicana amastigotes. The results presented here demonstrated the usefulness of ether-liposomes with specific ligands in site-specific delivery of antileishmanial compounds in vitro.

A survey of observation units in the United States.

Observation units have been proposed as a tool in lowering over-all health care costs and increasing the quality of care in outpatient facilities. Emergency department (ED) use of these units has been evaluated at single facilities but never at a national level. A survey of 250 facilities across the United States was performed to gather information about the observation unit phenomenon. Of the 250 hospitals in the survey group, 27% had operational observation or holding units and another 16% planned units within 1 year. A statistically significant increase in the use of these units was noted in nonteaching facilities when compared with their teaching counterparts. A trend toward higher use of observation units in suburban/urban settings was noted when compared with rural locations, although the difference was not statistically significant. Of the units in existence, 93% were located within the ED, staffed by emergency physicians, and administrated by the ED director. Most are staffed by ED nurses and ancillary help. No hospital had both an ED unit and a non-ED unit, and many units functioned as both holding and observation areas. The units are perceived to be beneficial in patient care and in lowering health care casts, although objective documentation to validate these beliefs is lacking. Further prospective research is needed to evaluate these units scientifically before broad recommendations can be made.

Perturbation of sterol biosynthesis by itraconazole and ketoconazole in Leishmania mexicana mexicana infected macrophages.

The azole antifungals ketoconazole and itraconazole possess in vitro antileishmanial activity against Leishmania mexicana mexicana amastigotes in macrophages (cell line J774G8). As in yeast and fungi, the activity is likely to be due to inhibition of the cytochrome P-450-dependent 14 alpha-demethylation of lanosterol and/or 24,25-dihydrolanosterol. Indeed, 50% inhibition of ergosterol synthesis was observed at 0.21 microM ketoconazole and 0.15 microM itraconazole. At 5 microM ketoconazole, traces of ergosterol could be found, whereas no ergosterol could be detected in cells treated with 5 microM itraconazole. The inhibition of ergosterol biosynthesis was concomitant with an accumulation of the 14 alpha-methylsterols lanosterol and 24,25-dihydrolanosterol. Fifty percent inhibition of cholesterol synthesis in uninfected macrophages was achieved at 0.95 microM and 1.5 microM itraconazole and ketoconazole, respectively. In infected macrophages all [14C]acetate was incorporated in ergosterol, suggesting an inhibition in cholesterol synthesis in the host cells. An inhibition of ergosterol synthesis coincided with increasing cholesterol synthesis. The latter synthesis was inhibited at concentrations greater than 1 microM. However, even at 5 microM cholesterol synthesis was higher than under control conditions.

Leishmania mexicana: drug sensitivities of promastigotes and transforming amastigotes.

A comparison has been made of the effects of a range of antiprotozoal drugs and other metabolic inhibitors upon growing promastigotes and transforming amastigotes of Leishmania mexicana mexicana. Amastigotes transforming to promastigotes in vitro were highly susceptible to 2-mercaptoacetate, 4-pentenoate, alpha-difluoromethylornithine, ethidium bromide, acridine orange, pentamidine isethionate, allopurinol, amphotericin B, menoctone and pentostam. Promastigotes were in most cases less sensitive to these inhibitors. The results reiterate that the biochemical differences between the two developmental forms are reflected in their sensitivities to inhibitors. The transformation in vitro of Leishmania amastigotes to promastigotes may be a useful model for use as a primary screen for antileishmanial agents.

A comparative study of Leishmania mexicana amastigotes and promastigotes. Enzyme activities and subcellular locations.

Leishmania mexicana mexicana amastigotes have been shown to contain greater activities than promastigotes of the enzymes that catalyse the beta-oxidation of fatty acids, but lower activities of several glycolytic enzymes, with the activity of pyruvate kinase being especially low. The results suggest the beta-oxidation of fatty acids is relatively more important to Leishmania amastigotes than promastigotes, whereas the reverse is true for glycolysis. Succinic dehydrogenase and peptidase activities were much higher in promastigotes than amastigotes. The activities of glucose-6-phosphatase, fructose-1,6-bisphosphatase, acid phosphatase and glucose-6-phosphate dehydrogenase varied less, although in each case the activity was significantly lower in the mammalian stage. A method for lysing and fractionating L. m. mexicana promastigotes has been developed. Using this procedure it has been established that many of the glycolytic and functionally related enzymes are located in cell organelles, that hexokinase is intimately connected with the particulate part of the parasite, and that the microsomal fraction of L. m. mexicana is very different in composition from the microsomes of mammalian liver cells.

Respiration of Leishmania mexicana amastigotes and promastigotes.

Promastigotes of Leishmania mexicana mexicana recently derived from amastigotes by transformation in vitro respired at a rate (17 nmol O2/min per 10(8) parasites) 4-5 times higher than that of amastigotes, but when the difference in cell protein content between the two preparations was taken into account the rates were not significantly different (32 nmol O2/min per mg protein). The respiration of both amastigotes and promastigotes was sensitive to cyanide, azide, antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide and high concentrations of amytal, but insensitive to rotenone and salicyl-hydroxamic acid, indicating that the two developmental forms possess a similar cytochrome-containing respiratory chain. D-Glucose and non-esterified fatty acids stimulated promastigote respiration and amastigote transformation to promastigotes in vitro; possibly these substances are important exogenous energy substrates for both forms of the parasites. Amino acids (incuding L-proline) and proteins did not appear to be used as energy substrates. The respiration rate of promastigotes was found to rise significantly upon continued sub-culture in vitro; at the same time cell size and protein content increased.

The effects of carbon dioxide and oxygen upon the growth and in vitro transformation of Leishmania mexicana mexicana.

Growth of Leishmania mexicana promastigotes is highly dependent upon O2 tension. There was a strong positive correlation between the level of O2, growth rate and maximum parasite density. Promastigotes under low oxygen tension decreased in size, protein content and motility, and deaths occurred. Changes in the carbon dioxide concentration (0.1-5.0%) had little effect on promastigote growth. Transformation in vivo of L. mexicana amastigotes to promastigotes also required oxygen, but a low level (0.4%) was sufficient for a high percentage of the amastigotes to transform. At high O2 concentrations, transformation was a little speedier but the number of parasites transforming was little affected. A greater effect was found with CO2. At 5%, transformation was much more rapid than at 0.1% and also an even greater percentage of amastigotes transformed within 48 h. The results give some indication that amastigotes are adpated for growth at low oxygen tensions encountered in vivo and that high carbon dioxide levels may act as a trigger for transformation of the amastigote to promastigote after it is taken up by the sandfly.

A quick, simple method for purifying Leishmania mexicana amastigotes in large numbers.

A rapid method for the bulk isolation of purified Leishmania mexicana mexicana amastigotes from parasite-induced lesions in experimentally infected mice is described. The procedure includes purification steps based on differences in net cell charge, lysis susceptibility and buoyant density between parasite and host cells. Yields of up to 2 x 10(10) untransformed amastigotes with minimal contamination with host cells and cell debris can be obtained. At least 90% of the purified amastigotes are viable as judged by light and electron microscopy, the staining of their lysosomes with acridine orange, their ability to transform to promastigotes and their infectivity to macrophages in vivo and in vitro.

Solitary pyogenic lung abscesses in the Bahamas over a ten-year period - abstract

Sixty-five cases of solitary pyogenic lung abscess treated in the Princess Margaret Hospital, Nassau, Bahamas, between January, 1961, and December, 1970, are reviewed. There were 52 males with an average age of 40 years and 13 females averaging 32 1/2 years old. Ages ranged from 11 to 68 years. Thirty-three men and 9 women were chronic alcoholics and 3 other men had idiopathic grand mal epilepsy. Illness had been noted for an average of 31/2 weeks before admission to hospital. All patients complained of cough and a majority had putrid sputum and chest pain. Seventy per cent of abscesses were located in the right lung with near equal distribution between upper and lower lobes. The middle lobe and lingular were least involved and the most frequently affected bronchopulmonary segments were the superior segments of both lower lobes, and the posterior segment of the right upper lobe. Organisms were isolated from 42 cases of which 13 had mixed infections. The most common organisms were staphylococcus, pneumococcus, klebsiella and haemolytic streptococcus. Sixty-one patients were treated for an average of 39 days with benzyl penicillin and stromycin regardless of the reported antibiotic sensitivities and when satisfactory clinical and radiological response occurred oral tetracycline was frequently substituted for the remainder of the stay in hospital. The average length of time spent in hospital was 62 days. Thirty-one per cent of patients left hospital with risidual cavities and many did not return for review. Four of these became re-infected and 3 patients were submitted for surgical resection of persistant cavities. One patient refused surgery and eventually died of a metastatic brain abscess. The results of this surgery indicate that lung abscesses in the Bahamas occur most frequently in chronic alcoholics. Bronchial aspiration of oral contents in the supine position is suggested by the distribution of the lesions in many of the cases. A wide range of organisms were isolated and treatment with intramuscular penicillin and streptomycin appear to give excellent results in most cases with relatively few sequelae. Surgery is seldom necessary if antibiotic treatment and postural drainage are given for prolonged periods of time (AU)