3D holotomographic monitoring of Ca++ dynamics during ionophore-induced Neospora caninum tachyzoite egress from primary bovine host endothelial cells.

Neospora caninum represents an obligate intracellular parasite that belongs to the phylum Apicomplexa and is a major abortive agent in bovines. During merogony, N. caninum tachyzoites invade and proliferate in host cells in vivo, including endothelial cells of lymphatic and blood vessels. The egress at the end of the lytic cycle is tightly regulated in apicomplexans. Evidence in Toxoplasma gondii shows that Ca++ signalling governs tachyzoite egress. Much less is known on egress mechanisms of N. caninum. Here, we show, using 3D live cell holotomographic microscopy in fluo-4 AM-loaded N. caninum-infected BUVEC, that treatments with the calcium ionophore A23187 at 24- and 42-h post-infection (h p. i.) induced a fast and sustained increase in Ca++ signals in parallel to tachyzoite egress. A23187 treatments exclusively triggered tachyzoite release at 42-h p. i. but failed to do so at 24-h p. i. indicating a role for meront maturation in calcium-induced tachyzoite egress. Overall, we show that live cell 3D holotomographic analysis in combination with epifluorescence is a suitable tool to study calcium dynamics related to coccidian egress or other important cell functions.

Modulation of cholesterol-related sterols during Eimeria bovis macromeront formation and impact of selected oxysterols on parasite development.

Obligate intracellular apicomplexan parasites are considered as deficient in cholesterol biosynthesis and scavenge cholesterol from their host cell in a parasite-specific manner. Compared to fast proliferating apicomplexan species producing low numbers of merozoites per host cell, (e. g. Toxoplasma gondii), the macromeront-forming protozoa Eimeria bovis is in extraordinary need for cholesterol for offspring production (≥ 170,000 merozoites I/macromeront). Interestingly, optimized in vitro E. bovis merozoite I production occurs under low foetal calf serum (FCS, 1.2%) supplementation. To analyze the impact of extensive E. bovis proliferation on host cellular sterol metabolism we here compared the sterol profiles of E. bovis-infected primary endothelial host cells grown under optimized (1.2% FCS) and non-optimized (10% FCS) cell culture conditions. Therefore, several sterols indicating endogenous de novo cholesterol synthesis, cholesterol conversion and sterol uptake (phytosterols) were analyzed via GC-MS-based approaches. Overall, significantly enhanced levels of phytosterols were detected in both FCS conditions indicating infection-triggered sterol uptake from extracellular sources as a major pathway of sterol acquisition. Interestingly, a simultaneous induction of endogenous cholesterol synthesis based on increased levels of distinct cholesterol precursors was only observed in case of optimized parasite proliferation indicating a parasite proliferation-dependent effect. Considering side-chain oxysterols, 25 hydroxycholesterol levels were selectively found increased in E. bovis-infected host cells, while 24 hydroxycholesterol and 27 hydroxycholesterol contents were not significantly altered by infection. Exogenous treatments with 25 hydroxycholesterol, 27 hydroxycholesterol, and 7 ketocholesterol revealed significant adverse effects on E. bovis intracellular development. Thus, the number and size of developing macromeronts and merozoite I production was significantly reduced indicating that these oxysterols bear direct or indirect antiparasitic properties. Overall, the current data indicate parasite-driven changes in the host cellular sterol profile reflecting the huge demand of E. bovis for cholesterol during macromeront formation and its versatility in the acquisition of cholesterol sources.

Amiloride interferes with platelet- activating factor-induced respiratory burst and MMP-9 release in bovine neutrophils independent of Na+/H+ exchanger 1.

Cytoplasmic pH homeostasis is required for an appropriate response in polymorphonuclear neutrophils (PMNs). In these cells, chemotaxis and reactive oxygen species (ROS) production are reduced by the use of Na+/H+ exchanger (NHE-1) inhibitors, but these results are mainly obtained using amiloride, a non-selective NHE-1 inhibitor. In bovine PMNs, the role of NHE-1 in functional responses has not been confirmed yet. The aim of this study was to determine the role of NHE-1 using amiloride and zoniporide in pH regulation, ROS production, matrix metalloproteinase 9 (MMP-9) release and calcium flux in bovine PMNs induced by the platelet activation factor (PAF), additionally we evaluated the presence of NHE-1 and NHE-2 mRNA Our data show the presence only of NHE-1 but not NHE-2 in bovine PMNs. Amiloride or zoniporide inhibited the intracellular alkalization induced by PAF without affecting calcium flux. Amiloride diminished ROS production and MMP-9 release, while zoniporide enhanced ROS production without change the MMP-9 release induced by PAF. Our work led us to conclude that changes in intracellular pH induced by PAF are regulated by NHE-1 in bovine neutrophils, but the effects of amiloride on ROS production and MMP-9 release induced by PAF are not NHE-1 dependent.

Non-invasive detection of significant coronary artery disease with multi-section computed tomography angiography in patients with suspected coronary artery disease.

AIM: The objective of this prospective study was to compare the accuracy of multi-section computed tomography (MSCT) coronary angiography with invasive selective coronary angiography in the detection of significant coronary stenosis (> or =50% lumen diameter narrowing). METHODS: Thirty consecutive patients (mean age 59+/-10 years) with suspected coronary artery disease underwent both invasive coronary angiography and MSCT using a 40-section multidetector row machine with temporal resolution of 53ms. Reconstruction images were performed in eight phases of the cardiac cycle. Images of MSCT and invasive coronary angiography were analysed using the 16-segment model of the American Heart Association. RESULTS: A total of 480 segments from 30 patients were evaluated. Coronary segments distal to a vessel occlusion and segments with coronary stent were not considered for analysis (20 segments in total). Ninety-four (20.4%) segments showed significant (> or =50%) stenosis by invasive coronary angiogram. The accuracy of coronary MSCT was computed on a per segment basis. Average sensitivity, specificity, positive predictive value, and negative predictive value of MSCT were 99, 98, 94, and 99%, respectively. CONCLUSION: This study demonstrated that MSCT is as reliable as coronary angiography at detecting significant obstructive coronary artery disease. In selected groups of patients, it may replace the more invasive and potentially more dangerous conventional coronary angiography.

[Cardiac arrhythmia in patients with chronic obstructive pulmonary disease]. / Arritmias cardíacas en pacientes con enfermedad pulmonar obstructiva crónica.

A retrospective study of all patients diagnosed as having obstructive pulmonary disease (POD), who were admitted to our department during 1989, was carried out. The presence of cardiac arrhythmia (CA) in standard ECG having been correlated to clinical and laboratory parameters on admission. Out ot 101 admissions, 67.3% had a type of CA, the most frequent being sinus tachycardia (54.4%). 71.9% of the patients who had cardio-respiratory decompensation, 72.8% of those admitted in basal situation III/IV and 86% who were regularly taking theophylline (p less than 0.01), had a type of CA. Our data suggests that patients with worst functional situation and those with cardiorespiratory decompensation had a higher incidence of CA and that regular theophylline intake can play a role in the etiopathogenesis of CA.

Pulse oximetry in very low birth weight infants with acute and chronic lung disease.

With improved survival of very low birth weight infants, the incidence of bronchopulmonary dysplasia has significantly increased. Pulse oximetry appears to be an adequate alternative to transcutaneous PO2, for continuous arterial oxygen saturation (SaO2) monitoring in neonates; however, its usefulness has not been very well documented in very low birth weight infants. We studied 68 patients with birth weight less than 1,250 g; 44 neonates had respiratory distress syndrome and 24 had bronchopulmonary dysplasia. Using a Nellcor N-100 pulse oximeter, we compared transcutaneous oxygen saturation with simultaneous arterial samples analyzed for SaO2 (range 78% to 100%) using an IL 282 co-oximeter. Fetal hemoglobin was measured in 66 patients. We also evaluated the accuracy of transcutaneous PO2 in reflecting arterial PO2 in patients with bronchopulmonary dysplasia. Over a wide range of PO2, PCO2, pH, heart rate, BP, hematocrit, and fetal hemoglobin, linear regression analysis revealed a close correlation between pulse oximeter values and co-oximeter measured SaO2 in patients with acute (r = .88, Y = 19.41 + 0.79X) and chronic (r = .90, Y = 9.72 + 0.92X) disease. Regression analysis of transcutaneous v arterial PO2 in infants with bronchopulmonary dysplasia showed an r value of .78. In addition, in these patients with chronic disease, the mean difference between pulse oximeter SaO2 and co-oximeter measured SaO2 was 2.7 +/- 1.9% (SD); whereas the mean difference between transcutaneous and arterial PO2 was -14 +/- 10.7 mm Hg. Our findings indicate that pulse oximetry can be used reliably in very low birth weight infants with acute and chronic lung disease, for SaO2 values greater than 78%.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of metaproterenol on pulmonary mechanics, oxygenation, and ventilation in infants with chronic lung disease.

Changes in pulmonary resistance, dynamic compliance, tidal volume, and transcutaneous PO2 and PCO2 after nebulized administration of metaproterenol were evaluated in eight newborn infants (birth weight 650 to 1060 g, gestational age 25 to 28 weeks) with chronic lung disease receiving mechanical ventilation. The infants were monitored continuously before and for 15 minutes after nebulization of metaproterenol during 3 consecutive days at mean age 34 days. There were significant increases in compliance, tidal volume, and tcPO2, and significant decreases in pulmonary resistance and tcPCO2. These data show that bronchospasm contributes significantly to the high pulmonary resistance in preterm infants with chronic lung disease and that metaproterenol is beneficial in the therapy of infants with chronic lung disease requiring mechanical ventilation.

Hemodynamic variables in infants weighing less than 1000 grams.

VVLBW infants pose a challenge in the management of their hemodynamic changes. Data are just accumulating regarding the hemodynamic variables in this very special group. We have described several newly developed methods of monitoring hemodynamic variables in infants less than 1000 gm. In the coming decade, some of these methods may become more refined and routine.