Cleavage of L1 in exosomes and apoptotic membrane vesicles released from ovarian carcinoma cells.

PURPOSE: The L1 adhesion molecule (CD171) is overexpressed in human ovarian and endometrial carcinomas and is associated with bad prognosis. Although expressed as a transmembrane molecule, L1 is released from carcinoma cells in a soluble form. Soluble L1 is present in serum and ascites of ovarian carcinoma patients. We investigated the mode of L1 cleavage and the function of soluble L1. EXPERIMENTAL DESIGN: We used ovarian carcinoma cell lines and ascites from ovarian carcinoma patients to analyze soluble L1 and L1 cleavage by Western blot analysis and ELISA. RESULTS: We find that in ovarian carcinoma cells the constitutive cleavage of L1 proceeds in secretory vesicles. We show that apoptotic stimuli like C2-ceramide, staurosporine, UV irradiation, and hypoxic conditions enhance L1-vesicle release resulting in elevated levels of soluble L1. Constitutive cleavage of L1 is mediated by a disintegrin and metalloproteinase 10, but under apoptotic conditions multiple metalloproteinases are involved. L1 cleavage occurs in two types of vesicles with distinct density features: constitutively released vesicles with similarity to exosomes and apoptotic vesicles. Both types of L1-containing vesicles are present in the ascites fluids of ovarian carcinoma patients. Soluble L1 from ascites is a potent inducer of cell migration and can trigger extracellular signal-regulated kinase phosphorylation. CONCLUSIONS: We suggest that tumor-derived vesicles may be an important source for soluble L1 that could regulate tumor cell function in an autocrine/paracrine fashion.     

Blood viscosity and optimal hematocrit in preterm and full-term neonates in 50- to 500-micrometer tubes.

Blood viscosity is an important determinant of blood flow resistance. Because a substantial part of flow resistance arises in small arteries and arterioles with diameters of 100 microns and less, rheologic properties of blood from preterm infants (24 to 36 wk of gestation), full-term neonates, and adults were measured in glass tubes with diameters of 50, 100, and 500 microns for a wide range of adjusted feed hematocrits (0.15-0.70). At each of the feed hematocrits, blood viscosity decreased when going from a 500-microns tube to a 50-microns tube. The viscosity reduction increased with increasing hematocrit. Moreover, the viscosity reduction was more pronounced in the neonates than in the adults. At a hematocrit of 0.70, the viscosity reduction averaged 56% in preterm infants, 50% in full-term neonates, and 39% in adults (p less than 0.005). However, the viscosity reductions at a hematocrit of 0.30 were only 35, 29, and 19%, respectively (p less than 0.05). In all four groups, blood viscosity increased exponentially with increasing hematocrit. The steepness of the hematocrit-viscosity curves decreased with decreasing tube diameter and with decreasing maturity of the infants. Erythrocyte transport efficiency (hematocrit/blood viscosity) was calculated to estimate the optimal hematocrit (i.e. hematocrit with maximum erythrocyte transport). In 500-microns tubes, the optimal hematocrit was about 0.40 in all of the groups. In 100-microns tubes, the optimal hematocrit was 0.44 +/- 0.05 in the adults and 0.52 +/- 0.04 in the neonates (p less than 0.05). In 50-microns tubes, the optimal hematocrit was 0.51 +/- 0.04 in adults and 0.60 +/- 0.05 in the neonates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reactive hyperemia of skin microcirculation in septic neonates

Reactive hyperemia after 1 min of arterial occlusion was studied in back, thigh and heel skin of 40 preterm and full-term neonates using laser Doppler flowmetry. Twelve infants had clinical signs of septicemia, but normal laboratory tests at the time of fluxmetry. However, CRP, leukocyte count and the ratio of immature to total neutrophils increased during the following days and septicemia was confirmed by positive blood cultures (septic group). Seven neonates with clinical signs of septicemia had developed neither positive blood cultures nor laboratory signs (non-septic group). Fifteen were healthy neonates. In the septic neonates, time to reach maximal hyperemia, maximum post-occlusive hyperemia and recovery time of skin perfusion were increased significantly in back and thigh skin and the heal skin temperature was decreased when compared to healthy neonates. Healthy and non-septic neonates showed no significant difference in any of the parameters. We conclude that altered reactive hyperemia in the skin may be an earlier sign of neonatal septicemia than laboratory tests.     

Effect of activators and the phosphodiesterase inhibitors pentoxifylline and enoximone on the deformability of neutrophils in neonates and adults

BACKGROUND: Stimulated polymorphonuclear leukocytes (PMN) are extremely rigid compared to resting PMN. They may obstruct narrow vessels and contribute to ischaemic organ injury. Deformability is a prerequisite for both active and passive movement in the microcirculation. AIM: The investigation was designed to study whether stimulators and inhibitors of stimulation show different effects on deformability of neonatal and adult PMN. METHODS: Deformability of PMN was assessed by complete aspiration of a PMN into a micropipette with an internal diameter of 5 microm. Blood samples from 20 neonates and 20 adults were studied before and after stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP), interleukin-8 (IL-8) or tumour necrosis factor-alpha (TNF-alpha). Moreover, effects of the phosphodiesterase inhibitors Pentoxifylline (PTX) and Enoximone on the deformability of stimulated PMN were investigated. RESULTS: fMLP, IL-8 and TNF-alpha significantly delayed aspiration times of PMN in relation to the concentrations of the stimulators. The addition of PTX or Enoximone to stimulated PMN increased the deformability up to 60% depending on the concentration of the inhibitors. No significant differences in the aspiration times were found between neonatal and adult PMN at any of the experimental conditions after activation with the three stimulators and treatment with the two inhibitors. CONCLUSION: Neonatal and adult PMN show similar reduction of passive deformability when stimulated with either fMLP, IL-8 or TNF-alpha compared to resting PMN and a similar improvement of deformability in response to PTX or Enoximone.     

Renal phosphate handling of premature infants of 23–25 weeks gestational age

Premature infants with low serum phosphate concentrations (<2 mmol/l) are at risk for osteopenia. Therefore, serum phosphate levels in premature infants should be kept above 2 mmol/l. Premature infants of 26-31 weeks gestational age (GA) have renal phosphate threshold concentrations (Tp/GFR) in the range of normal serum phosphate values (2 mmol/l). Therefore, these infants show significant urinary phosphate excretion only when serum phosphate levels are normal, and urinary phosphate excretion can be used to monitor phosphate supplementation. However, few data are available on extremely premature infants of 23-25 weeks GA. The objective of this study was to compare Tp/GFR levels in infants of 23-25 weeks GA to those in infants of 26-31 weeks GA. We retrospectively evaluated case notes of 12 infants of 23-25 weeks GA and compared them to 19 infants of 26-31 weeks GA. Tp/GFR was calculated from simultaneous measurements of urinary phosphate, urinary creatinine, serum phosphate, and serum creatinine. Tp/GFR values 3-5 weeks postnatally were lower in infants of 23-25 weeks GA (1.06+/-0.36 mmol/l, p<0.001) than in infants of 26-31 weeks GA (1.76+/-0.26 mmol/l). Near term (35-37 weeks postmenstrual age), there was no significant difference between Tp/GFR values in infants of 23-25 weeks GA (1.83+/-0.32 mmol/l) and in infants of 26-31 weeks GA (2.05+/-0.22 mmol/l). We conclude that at 3-5 weeks postnatally, infants of 23-25 weeks GA are at risk for low Tp/GFR values, leading to urinary phosphate excretion even in the presence of low serum phosphate levels. In these infants, serum phosphate levels should be monitored, and phosphate supplementation should be adjusted to keep serum phosphate levels above 2 mmol/l.     

Geometric, osmotic, and membrane mechanical properties of density- separated human red cells

Although there is evidence that the deformability of the entire red blood cell (RBC) decreases during aging, reports on changes in relevant specific properties associated with the aging process are limited and not in total agreement. The purpose of this study was to evaluate some of the factors that might contribute to this decreased deformability. Geometric, osmotic, and membrane mechanical properties of unfractionated, top ("young") and bottom ("old") RBC from 5 healthy adult donors were measured using micropipette techniques. Surface area, volume, and diameter of RBC were measured at osmolalities of 297, 254, 202, and 153 mosm/kg. Two membrane mechanical properties, surface shear modulus of elasticity (mu) and time constant (tc) of viscoelastic recovery, were studied only in isotonic media. At each of the osmolalities, volume and surface area of the bottom cells were about 25% lower than those of the top cells. Bottom cells showed smaller increases in volume with decreasing osmolality than top cells; the surface area remained constant with changing osmolality for all three groups. The surface area-to-volume ratio and the minimum cylindrical diameter of the bottom cells were essentially identical to the top cells. However, both the surface area index (actual are of RBC divided by area of a sphere of same volume) and the swelling index (maximal volume divided by actual volume) of the bottom cells were significantly lower than top RBC. The shear modules of elasticity (mu) was about 0.006 dyne/cm in all 3 RBC populations, indicating that the forces necessary to deform a portion of the membrane did not change with RBC aging. The viscoelastic time constant (tc) was 0.148 +/- 0.020 (SD) sec for the bottom RBC and 0.099 +/- 0.017 sec for the top cells. This difference indicates that shape recovery following membrane deformation is delayed in old RBC. The membrane surface viscosity (eta), calculated as the product of tc times mu was 0.95 +/- 0.22 x 10(-3) dyne-sec/cm for the bottom cells and 0.54 +/- 0.15 x 10(-3) for the top RBC. These data indicate that the relative deficit in membrane surface area and the increased membrane viscosity of old RBC may be important determinants for their decreased deformability and their eventual removal from the circulation.     

Pain reduction in children during port-à-cath catheter puncture using local anaesthesia with EMLA™

Introduction  

Alleviating pain is of high importance for children undergoing chemotherapy. Eutectic mixture of lidocain-prilocain cream (EMLA) is assumed to require 60 min application time.