Johannes Pöschl

Endotoxin binding to erythrocyte membrane and erythrocyte deformability in human sepsis and in vitro

ObjectiveSeveral studies have shown that lipopolysaccharide and lipid A impair red blood cell deformability in vitro and in vivo. However, it is unclear whether impaired red blood cell deformability is associated with binding of lipopolysaccharide to the red blood cell membrane. DesignAnalysis of hydroxymyristic acid content in red blood cell membranes and red blood cell deformation in patients with Gram-negative septicemia and after in vitro incubation of red blood cells from healthy adults with 100 &mgr;g of Escherichia coli lipid A or 1 mg of E. coli lipopolysaccharide per milliliter of red blood cell in buffer solution and in whole blood. Hydroxymyristic acid is a fatty acid of the lipid A part of lipopolysaccharide in most Gram-negative bacteria. SettingUniversity research laboratories. SubjectsTen healthy adults and four patients with clinical and laboratory signs of septicemia. InterventionsBlood sampling. Measurements and Main ResultsRed blood cell deformation was measured with a laser-diffraction shearing device (Rheodyn) and a computerized micropore filtration system (CTA). Lipopolysaccharide and lipid A binding to red blood cell membranes was studied by measuring the amide-linked hydroxymyristic acid by gas chromatography.The detection rates of hydroxymyristic acid were 82% for lipopolysaccharide and 79% for lipid A in buffer solution. In membranes of washed red blood cell, the detection rates of lipopolysaccharide and lipid A were 0.26 ± 0.03% (2.6 ± 0.3 &mgr;g/mL) and 1.3 ± 0.5% (1.3 ± 0.5 &mgr;g/mL), and in red blood cell membranes of whole blood the detection rates were 2.6% (25.5 &mgr;g/mL) and 4.1% (4.1 &mgr;g/mL), respectively. The lipopolysaccharide content in red blood cell membranes of septic patients ranged from 47 to 103 &mgr;g/mL of red blood cell. Red blood cell deformation in the Rheodyn and in the CTA were not influenced by lipopolysaccharide incubated with washed red blood cells. In the Rheodyn, red blood cell deformation was significantly decreased by 18% after lipid A incubation in washed red blood cells, by 26% after lipopolysaccharide incubation in whole blood, and by 31% in septic patients. Similar effects were observed when we used the CTA. ConclusionsRed blood cell deformation is decreased in septic patients, after in vitro incubation of washed red blood cells with lipid A and of whole blood with lipopolysaccharide. Lipopolysaccharide did not influence red blood cell deformation after incubation with washed red blood cells. The decrease of red blood cell deformation was related to the amount of hydroxymyristic acid measured in red blood cell membranes, suggesting that endotoxin binding directly affects mechanical properties of red blood cells.

Intracerebral Human Regulatory T Cells: Analysis of CD4+CD25+FOXP3+ T Cells in Brain Lesions and Cerebrospinal Fluid of Multiple Sclerosis Patients

Impaired suppressive capacity of CD4+CD25+FOXP3+ regulatory T cells (Treg) from peripheral blood of patients with multiple sclerosis (MS) has been reported by multiple laboratories. It is, however, currently unresolved whether Treg dysfunction in MS patients is limited to reduced control of peripheral T cell activation since most studies analyzed peripheral blood samples only. Here, we assessed early active MS lesions in brain biopsies obtained from 16 patients with MS by FOXP3 immunohistochemistry. In addition, we used six-color flow cytometry to determine numbers of Treg by analysis of FOXP3/CD127 expression in peripheral blood and cerebrospinal fluid (CSF) of 17 treatment-naïve MS patients as well as quantities of apoptosis sensitive CD45ROhiCD95hi cells in circulating and CSF Treg subsets. Absolute numbers of FOXP3+ and CD4+ cells were rather low in MS brain lesions and Treg were not detectable in 30% of MS biopsies despite the presence of CD4+ cell infiltrates. In contrast, Treg were detectable in all CSF samples and Treg with a CD45ROhiCD95hi phenotype previously shown to be highly apoptosis sensitive were found to be enriched in the CSF compared to peripheral blood of MS patients. We suggest a hypothetical model of intracerebral elimination of Treg by CD95L-mediated apoptosis within the MS lesion.

Buprenorphine and norbuprenorphine concentrations in human breast milk samples determined by liquid chromatography-tandem mass spectrometry.

Buprenorphine (BUP) is considered to be safe during pregnancy. However, the extent of BUP transfer into breast milk has not been investigated thoroughly. Because the drug concentration in the milk is 1 of the determinants in the assessment of the exposure risk, a rapid and sensitive LC-MS/MS method has been developed and evaluated to measure BUP and norbuprenorphine (norBUP) concentrations in milk. A solid-phase and 2 liquid-liquid extraction procedures have been compared. The lower limits of detection and quantification were 0.05 ng/mL and 0.18 ng/mL for BUP and 0.05 ng/mL and 0.20 ng/mL for norBUP, respectively, using a sample volume of 0.5 mL milk. BUP and norBUP concentrations determined from 10 random breast milk samples collected over 4 successive days from a lactating woman during buprenorphine maintenance therapy ranged from 1.0 to 14.7 and 0.6 to 6.3 ng/mL, respectively. Drug exposure of the infant may be considered to be low. Further investigations may seek to extend these preliminary findings to evaluate an infants level of BUP exposure through breast milk.

Effectiveness and side effects of an escalating, stepwise approach to indomethacin treatment for symptomatic patent ductus arteriosus in premature infants below 33 weeks of gestation.

Objective. Symptomatic patent ductus arteriosus (sPDA) is a common problem in premature infants and can be treated effectively with intravenous indomethacin, leading to permanent ductal closure in 70% to 80% of infants. Infants who do not respond to pharmacologic closure of the duct ultimately have to undergo surgical or interventional closure of the PDA. Optimizing the pharmacologic treatment could offer an interesting approach to reduce the number of infants who need surgical closure of the duct. Methods. We conducted a retrospective analysis in infants who were <33 weeks’ gestation, had sPDA, and were treated with high-dose intravenous indomethacin. From 1993 to 2002, 129 infants with sPDA received indomethacin after diagnosis of sPDA was confirmed by echocardiography. Treatment was started in all infants with intravenous indomethacin (0.2 mg/kg given 5 times at 0 hours, 12 hours, 24 hours, 48 hours, and 72 hours). When the ductus was still open at 36 hours, indomethacin every 12 hours was continued and single doses increased up to 1 mg/kg until ductal closure was achieved. Results. In 68 (53%) of 129 infants who were treated with indomethacin, ductal closure occurred during intermediate-dose indomethacin therapy (up to 1.5 mg/kg total dose). In the 61 initial nonresponders, the continuation of indomethacin led to ductal closure in 59 infants. When infants who were treated with an intermediate dose were compared with the initial nonresponders, no differences in the incidences of renal or electrolyte abnormalities, gastrointestinal bleeding, intraventricular hemorrhage, or periventricular leukomalacia were found. Conclusions. High-dose indomethacin after intermediate-dose therapy resulted in an overall closure rate of 98.5% (127 of 129). Although single indomethacin doses of up to 1 mg/kg were given, high-dose indomethacin was safe.

Acylcarnitine profiles of preterm infants over the first four weeks of life

Measurement of free carnitine and acylcarnitines allows the detection of several inborn errors of metabolism in neonatal screening. Because available data for premature infants is limited, we studied longitudinal changes in acylcarnitine profiles of full-term and preterm neonates over the first 4 weeks of life. One hundred twenty infants were divided into four groups of 30: A, gestational age 22 to 27 wk; B, 28 to 31 wk; C, 32 to 36 wk; and D, 37 to 41 wk. Blood samples spotted on a Guthrie card were taken on days 5 and 28. Additional specimens (groups A and B only) were collected on days 1, 3, 7, and 14. Carnitine and its acyl esters were detected by looking for the precursor ions of m/z = 85 using a PE Sciex API 365 electrospray ionization tandem mass spectrometer. Concentrations of free carnitine and most acylcarnitines were significantly higher in group A compared with group D postnatally. Groups B and C displayed intermediate values. Carnitine levels in infants from group A and B decreased steadily from day 1 to day 7, and recovered up to day 14 in group B only. On day 28 carnitine concentrations had further decreased in group A, while reaching postnatal levels again in group B. Postnatal carnitine levels are higher in very immature preterm infants compared with full-term infants, but become lower on day 28. However, the commonly used metabolite ratios should still allow the detection of inborn errors of metabolism.

Dietary docosahexaenoic acid improves red blood cell deformability in rats

Abstract Epidemiologic studies have shown that consumption of fish rich in the two n-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), decreases the mortality from cardiovascular disease (1,2). The n-3 fatty acids alter the formation of eicosanoids, modulators involved in regulation of blood vessel diameter and inflammation. By ingestion of n-3 fatty acids, the eicosanoid system is shifted toward vasodilation, antiaggregation of platelets, and less proinflammation by leukotrienes B (3). The mechanisms involved are many-fold varying from actions on platelets, plasma lipid pattern and endothelial function. Red blood cell (RBC) deformability may also be improved by fish oil as suggested by filtration studies (4,5,6). However, fish oil contains both n-3 fatty acids, i.e. EPA and DHA. Studies on the effect of individual omega-3 fatty acids on RBC deformability are lacking. This study was designed to investigate the effect of diets supplemented with purified n-3 fatty acids (EPA and DHA) alone or in combination on RBC membrane n-3 fatty acids and RBC deformation in rats.

A haemophagocytic lymphohistiocytosis (HLH)-like picture following breastmilk transmitted cytomegalovirus infection in a preterm infant.

Due to septic illness, neutro- and thrombocytopenia, blocked myelopoiesis, CMV-positive breast milk and CMV-pp65 antigen in bone marrow mononuclear cells, CMV-related HLH was presumed in a breast fed neonate (gestational age 24 weeks). Treatment was successful with foscarnet and methylprednisolone. HLH may be a complication of post-natal CMV-infection acquired from breast milk.

Effects of dietary supplementation of saturated fatty acids and of n−6 or n−3 polyunsaturated fatty acids on plasma and red blood cell membrane phospholipids and deformability in weanling guinea pigs

The fatty acid composition of plasma cholesteryl esters, plasma phospholipids, red blood cell (RBC) membrane phosphatidylcholine (corresponding to the outer membrane leaflet), and phosphatidylethanolamine (corresponding to the inner membrane leaflet) was investigated in weanling guinea pigs fed with diets of cacao (saturated fatty acids), sunflower oil [n−6 polyunsaturated fatty acids (PUFA)] or fish oil (n−3 PUFA) for 20 wk. RBC deformation was measured by means of a cell-transit analyzer (filtration) and a cone-plate rheoscope. The contents of saturated fatty acids in plasma phospholipids and RBC membrane leaflets were similar in all three groups. Diets with sunflower oil resulted in a high content of linoleic acid in plasma cholesteryl esters and in the outer leaflet of RBC membranes. Fatty acids of fish oil were mainly incorporated in plasma phospholipids and in the inner leaflet of RBC membranes. The arachidonic acid content was high in all groups in the plasma phospholipids and in the inner leaflet. The n−6 and n−3 PUFA were mainly incorporated in the inner leaflet. In all groups the polyunsaturated/saturated fatty acid ratio and the total PUFA content were similar in the inner RBC membrane. The RBC filtration times and the RBC deformation indices were not affected by the dietary treatment.

Immunohistochemical localization of α-tocopherol transfer protein and lipoperoxidation products in human first-trimester and term placenta

OBJECTIVE Pregnancy is described as a state of oxidative stress arising from the high metabolic turnover taking place during feto-placental development and little is known about the balance of oxidation and antioxidation in early human pregnancy. The aim of this study was to analyze placental expression of alpha-tocopherol transfer protein (alpha-TTP) as the major transport protein for the antioxidant alpha-tocopherol as well as the placental expression of two lipoperoxidation products, malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE) in early first-trimester and term human placenta. STUDY DESIGN Placental tissue was obtained from 10 pregnancy interruptions at 6-8 weeks gestational age and 10 samples were obtained from term pregnancies after routine cesarean section. The placental expression of alpha-TTP, MDA and HNE has been investigated with immunohistochemistry by the use of specific human alpha-TTP, MDA and HNE antibodies. RESULTS While MDA and HNE showed similar expression in first-trimester and term placenta, alpha-TTP expression was less in first-trimester syncytiotrophoblast as compared to term. In first-trimester specimen, alpha-TTP showed major expression in extravillous trophoblast. In amniotic epithelial cells, a rising tendency in all three parameters investigated from immature to mature cells could be documented. No direct correlation between alpha-TTP, MDA and HNE expression was detected. CONCLUSIONS Our study shows the presence of alpha-TTP not only in term, but in first-trimester extravillous trophoblast, syncytiotrophoblast and amniotic epithelium. Furthermore, lipoperoxidation products MDA and HNE are also present in first-trimester and term placenta, documenting the presence of oxidative processes in the placenta from early on. It therefore seems possible that scavenging of reactive oxygen species (ROS) by alpha-tocopherol is already required in first-trimester human pregnancy, but the missing correlation to MDA and HNE expression leads to the speculation that alpha-TTP and its ligand alpha-tocopherol have functions beyond the antioxidative capacity of alpha-tocopherol in early pregnancy.

Oxidative stress stimulates α-tocopherol transfer protein in human trophoblast tumor cells BeWo.

Abstract α-Tocopherol transfer protein (α-TTP) has been identified as the major intracellular transport protein for the antioxidant vitamin E (α-tocopherol). Expression of α-TTP on the reproductive system has been described both in mouse uterus and lately in the human placenta. The aim of this study was to clarify if placental expression of α-TTP can be modified by substances causing oxidative reactions. The human choriocarcinoma cell line BeWo was, therefore, treated with two known pro-oxidants. α-TTP expression was determined with immunocytochemistry and evaluated by applying a semiquantitative score. The presence of pro-oxidants in BeWo cells induced α-TTP expression. We thus hypothesize that stimulation of α-TTP expression by oxidative stress, as this was induced by pro-oxidants, could be part of an antioxidant process occurring in the placenta in the aim of enhancing the supply of α-tocopherol. This process could occur both in normal pregnancies, as well as in pregnancy disorders presented with intensified oxidative stress. In that view, this model is proposed for further oxidative stress studies on trophoblast and placenta, on the grounds of clarifying the role of α-tocopherol in pregnancy physiology and pathophysiology.