In vitro transendothelial migration of blood T lymphocytes from HIV-infected individuals.

OBJECTIVE: We hypothesized that differential extravasation of circulating CD4+ or CD8+ T lymphocytes contributes to HIV-associated CD8+ lymphocytic alveolitis. Differences in T-cell transendothelial migration may be intrinsic or emerge at sites where vascular endothelium is activated by overexpression of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma. DESIGN: We used an in vitro model of lymphocyte extravasation to assess transendothelial migration of peripheral blood mononuclear cells (PBMC) from HIV-positive individuals. We assayed bronchoalveolar lavage (BAL) fluid from HIV-positive and normal individuals to determine if increased levels of TNF-alpha and IFN-gamma were present in the lungs of HIV-infected individuals. METHODS: Transendothelial migration was assessed by determining the number and flow cytometric phenotype of PBMC adherent to or migrating across unstimulated or TNF-alpha and IFN-gamma-activated endothelial cell monolayers. We measured BAL fluid cytokine concentrations using standard antigen-capture enzyme-linked immunosorbent assays for TNF-alpha and IFN-gamma. RESULTS: T cells migrating across unactivated endothelial cells were significantly enriched for CD4+ T cells. Cytokine activation of endothelial cells allowed significantly greater transendothelial migration of CD8+ T cells compared to unactivated endothelial cells. TNF-alpha was increased in BAL fluid from HIV-positive individuals relative to controls. CONCLUSIONS: These data suggest that, in HIV-positive individuals, CD4+ T cells are migration competent and blood CD8+ T cells do not have enhanced migration competence relative to CD4+ T cells. CD8+ T cell extravasation is aided by TNF-alpha and IFN-gamma-induced endothelial cells activation.

Distinct effect of hypoxia on endothelial cell proliferation and cycling.

Endothelial cells (EC) occupy a strategic location in the vasculature as a barrier between the intravascular compartment and underlying tissues; as such, they are often exposed to stresses, such as decreases in ambient oxygen, diminished metabolic substrate, or changes in temperature, that could affect their ability to divide and proliferate. The present study characterizes cell counts, cell cycle distribution, and bromodeoxyuridine incorporation in pulmonary artery and aortic EC exposed to acute and/or chronic hypoxia and other cellular stresses. During hypoxia, EC division slows but does not arrest; progression through the G1-to-S transition point and/or progression from S to G2/M is altered with an increased percent of EC in S phase. These changes in EC cell cycle distribution with hypoxia are dependent on the origin of the EC as well as the ambient oxygen concentration; moreover, they are distinct from changes observed with elevated temperature or glucose deprivation. and differ from the quiescent pattern induced by serum deprivation or high-density confluence. These findings demonstrate that hypoxia exerts a distinct effect on the cell cycle distribution and proliferation of EC.

Ultrastructural features of danazol-induced cholestasis: a case study.

A case study of a severe cholestatic syndrome induced by danazol, with ultrastructural description of liver morphology, is reported. Cholestasis appeared after 3 months treatment with danazol (300 mg/daily) and completely resolved 2 months after withdrawal. In spite of the severe increase in serum bilirubin and total serum bile salts, transaminases were only slightly elevated and GGT and alkaline phosphatases were almost normal. Light microscopy shows a pattern of predominantly centrolobular cholestasis without necrosis, with minimal inflammatory infiltrate and with no sign of bile ductule involvement. At the ultrastructural level very dilated bile canaliculus predominate with stunted or loss of microvilli and dense bile material in the lumen. Nonspecific alterations were seen in hepatocyte intracellular organelles. It is suggested that danazol may cause a rare but severe hepatocanalicular cholestasis, differing from the "bland" cholestasis frequently described during therapy with other anabolic steroids.

Effect of glucagon on intracellular pH regulation in isolated rat hepatocyte couplets.

To elucidate mechanisms of glucagon-induced bicarbonate-rich choleresis, we investigated the effect of glucagon on ion transport processes involved in the regulation of intracellular pH (pHi) in isolated rat hepatocyte couplets. It was found that glucagon (200 nM), without influencing resting pHi, significantly stimulates the Cl-/HCO3- exchange activity. The effect of glucagon was associated with a sevenfold increase in cAMP levels in rat hepatocytes. The activity of the Cl-/HCO3- exchanger was also stimulated by DBcAMP + forskolin. The effect of glucagon on the Cl-/HCO3- exchange was individually blocked by two specific and selective inhibitors of protein kinase A, Rp-cAMPs (10 microM) and H-89 (30 microM), the latter having no influence on the glucagon-induced cAMP accumulation in isolated rat hepatocytes. The Cl- channel blocker, NPPB (10 microM), showed no effect on either the basal or the glucagon-stimulated Cl-/HCO3 exchange. In contrast, the protein kinase C agonist, PMA (10 microM), completely blocked the glucagon stimulation of the Cl-/HCO3- exchange; however, this effect was achieved through a significant inhibition of the glucagon-stimulated cAMP accumulation in rat hepatocytes. Colchicine pretreatment inhibited the basal as well as the glucagon-stimulated Cl-/HCO3- exchange activity. The Na+/H+ exchanger was unaffected by glucagon either at basal pHi or at acid pHi values. In contrast, glucagon, at basal pHi, stimulated the Na(+)-HCO3- symport. The main findings of this study indicate that glucagon, through the cAMP-dependent protein kinase A pathway, stimulates the activity of the Cl-/HCO3- exchanger in isolated rat hepatocyte couplets, a mechanism which could account for the in vivo induced bicarbonate-rich choleresis.

Effect of S-adenosyl-L-methionine on ethanol cholestasis and hepatotoxicity in isolated perfused rat liver.

We investigated whether S-adenosyl-L-methionine (SAMe) influences the inhibitory effect of ethanol on bile secretion and ethanol hepatotoxicity in the isolated perfused rat liver. SAMe (25 mg/kg intramuscularly three times a day) was administered for three days consecutively. Liver was then isolated and perfused with taurocholate to stabilize bile secretion and exposed to 1% ethanol for 70 min. The effect of ethanol on bile flow, bile salt biliary secretion, oxygen liver consumption, AST and LDH release in the perfusate, and hepatic concentration of glutathione, malondialdehyde, and diene conjugates was compared between SAMe-treated livers (N = 11) and paired controls (N = 11). Control experiments without ethanol were also performed (N = 6). Exposure to 1% ethanol induced a significantly (P < 0.03) higher inhibition of bile flow (-35% vs 17%) and bile salt secretion (-28% vs 16%) in untreated compared with SAMe-treated livers. During 1% ethanol exposure, the release of LDH and AST in the perfusate was significantly lower (P < 0.02) in SAMe-treated livers. Oxygen liver consumption was markedly inhibited by 1% ethanol administration (P < 0.02 vs controls without ethanol), an effect almost totally prevented by SAMe treatment (P < 0.02 vs ethanol controls). The hepatic concentration of total glutathione was significantly (P < 0.02) decreased by 1% ethanol exposure, but this effect was less pronounced in SAMe-treated than in untreated controls (P < 0.02). The hepatic levels of malondialdehyde and diene conjugates were not significantly changed by ethanol exposure in either SAMe-treated or control livers in comparison to ethanol-free controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute ethanol hepatotoxicity is modulated by bile salt hydrophilic-hydrophobic properties.

The influence of the hydrophobic-hydrophilic properties of bile salts (BS) on acute ethanol hepatotoxicity was investigated. Bile flow, biliary BS secretion and enzyme (LDH,AST) release in the perfusate were measured before and after exposure to low (0.1%) or high (1%) doses of ethanol in in vitro isolated livers perfused with 1 microM/min taurocholate (TCA), tauroursodeoxycholate (TUDCA) or taurodeoxycholate (TDCA). Ethanol promotes a rapid decrease of basal bile flow and BS secretion in TCA-perfused livers [-28% of basal values with 0.1% (N = 6), and -35% with 1% ethanol (N = 6)]. Bile flow and BS secretion were minimally decreased by ethanol in livers perfused with a hydrophilic BS (TUDCA) [-8% decrease of basal values with 0.1% ethanol (N = 6), and -10% with 1% ethanol (N = 9); p < 0.02 vs TCA-perfused livers]. In contrast, when livers were perfused with a hydrophobic BS (TDCA), ethanol showed a higher cholestatic effect than either TCA- or TUDCA-perfused livers. Enzyme release in the perfusate was not modified by 0.1% ethanol, while 1% ethanol promoted a 4-5 fold increase in LDH and AST release in the perfusate of TCA-perfused livers with respect to a mere 2-fold increase in TUDCA-perfused livers and a 6-7 fold increase in TDCA perfused livers (p < 0.03). In conclusion, we showed that TUDCA almost completely counteracts the cholestatic and cytolitic effects promoted by ethanol in the isolated perfused rat liver.

Effect of Brefeldin A on transcytotic vesicular pathway and bile secretion: a study on the isolated perfused rat liver and isolated rat hepatocyte couplets.

This study investigated the effect of Brefeldin A (BFA) on the transcytotic vesicular pathway labeled with horseradish peroxidase (HRP) in both isolated rat hepatocyte couplets (IRHC) and the isolated perfused rat liver (IPRL). To evaluate the role of the transcytotic vesicular pathway on bile secretion, the effect of BFA on bile secretion in the IPRL was then investigated. In the basolateral area of IRHC, BFA showed no effect on the density and percentage of area of HRP-labeled vesicles. However, HRP-labeled vesicles tended to accumulate in the juxtanuclear area of BFA-treated hepatocytes (P < .001 vs. controls). In the pericanalicular area, on the other hand, HRP-labeled vesicles were depleted compared with controls (P < .001). In keeping with these findings, although the early peak remained unchanged, BFA inhibited as much as 50% of the late peak of HRP excretion in bile, after a pulse load of HRP in the IPRL. Bile flow and the biliary secretion of bile salts (BS) and phospholipids were not modified by BFA in isolated livers perfused without BS in the perfusate or with 1 mumol/min taurocholate (TCA). In BFA-treated livers, peak bile flow and BS output decreased by 20% (P < .05 vs. controls) only when a 5 mumol TCA bolus was administered. In conclusion, this study demonstrates that BFA inhibits the transcytotic vesicular pathway in the liver. However, BFA has no significant effect on bile secretion either in basal conditions or during perfusion with physiological amounts of BS. BFA slightly decreases bile flow and BS output only after an overload of BS, providing evidence against the physiological relevance of the transcytotic vesicular pathway in the process of bile formation.

Efficacy of cromoglycate in persistently wheezing infants.

A prospective study was undertaken to evaluate the efficacy of (sodium) cromoglycate in the treatment of persistent wheezing in 31 children between 4 and 12 months of age. The subjects were randomised to receive either 40 mg of cromoglycate (n = 16) or physiological saline as placebo (n = 15) three times a day by wet nebulisation in a double blind fashion for a period of six weeks. The patients were evaluated with daily symptom scores and respiratory function testing measuring maximal expiratory flow at functional residual capacity (VmaxFRC) before initiating treatment and upon completion. At baseline, mean (SD) symptom scores between the two groups were comparable (cromoglycate 99.5 (29.8), placebo 104.5 (29.7)) as were VmaxFRC expressed as per cent of predicted normals (cromoglycate 48 (28), placebo 46 (20)). Upon completion of the treatment protocol, no significant difference could be found between the two groups for either symptom score (cromoglycate 67.6 (40.2), placebo 58.6 (41.4)), or VmaxFRC (cromoglycate 52 (24), placebo 60 (32)). It is concluded, therefore, that 40 mg of cromoglycate three times a day administered via facemask and wet nebulisation was no more effective than placebo in the treatment of our sample of persistently wheezing infants under 1 year of age.

Arterial catheter-related infections in children. A 1-year cohort analysis.

To determine the incidence of infection secondary to arterial catheterization in children as well as the risk markers, we prospectively evaluated, during a 1-year period, all arterial catheters installed in children admitted to the pediatric intensive care unit. A total of 340 cannulas were placed in 310 children aged 80 +/- 4 months (mean +/- SEM) for a period of 64 +/- 4 hours. Most catheters were inserted percutaneously (99%) in the radial artery (86.5%). Ninety-two percent (313/340) of the catheters were sterile (group 1), 5% (17/340) were contaminated (less than 10 colony-forming units on semiquantitative culture) (group 2), and 3% (10/340) were considered either locally infected (ie, greater than or equal to 10 colony-forming units) (eight of 10) or associated with a possible catheter-related sepsis (two of 10) (group 3, or infected group). The incidence of local inflammation at the insertion site was higher in group 2 than in group 1 (18% vs 2.9%) but not statistically different between groups 3 and 1 (10% vs 2.9%). The duration of arterial catheterization was longer in group 3 than in group 1 (125 +/- 31 vs 61 +/- 4 hours). The risk of infection was nonexistent in the first 48 hours of catheterization. Thereafter it was calculated as being 6.2% (10/161), but it correlated poorly with the duration of arterial catheterization. These results confirm the very low incidence of infection related to arterial catheterization in children. Thus, routine catheter reinsertion is, in our opinion, unjustified.

Hemoglobin oxygen dissociation (P50) in bronchopulmonary dysplasia.

A study was devised to determine the P50 in infants with bronchopulmonary dysplasia (BPD). Other factors such as red blood cell 2,3-diphosphoglycerate (2,3-DPG) level proportions of adult (HbA) to fetal (HbF) hemoglobins which could affect P50 were also measured. Fourteen infants with clinical and radiological signs of BPD with a mean post-conceptional age of 42.2 +/- 4.7 weeks born at a mean gestational age of 29.3 +/- 2.0 weeks were evaluated. The percentage of HbF determined in 8 infants was 40.1 +/- 20.3% and the mean 2,3-DPG concentrations was 13.1 +/- 2.2 mumol/g Hb. The P50 was 25.1 +/- 2.7 mm Hg (range 18-29.5 mm Hg). When a HbO2 curve was established based on a large volume of blood consisting of adult blood and newborn cord blood mixed to attain a P50 of 25.1 mm Hg, the PaO2 at 90% O2 saturation was 52 mm Hg. Since there can be a wide range in HbO2 in infants with chronic BPD, pulse oximetry remains the most prudent method of monitoring oxygen therapy in BPD infants.