Specific binding of 1-[3H]-O-alkyl-2-acetyl-sn-glyceryl-3-phosphoryl choline (platelet-activating factor,PAF) by human polymorphonuclear neutrophils

1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (PAF) is a potent activator of polymorphonuclear neutrophil (PMN) aggregation, exocytosis and chemotaxis. Specific desensitization of PMN to PAF suggests a receptor-mediated interaction. The binding of 1-[³H]-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (³H-PAF) to human PMN and platelets was analysed and compared. Binding was saturable at 0.6 nM and 0.1 nM for 2×10⁶ PMN and 5×10⁷ platelets, respectively. The time course of binding at 22°C and 37°C for both cell types reached the plateau at 2 min. The averageK _d was 45.0±1.7 nM (mean ±1 SD of 4 experiments) for PMN (27.391±1381 sites for PMN) and 20.1±6.3 nM (4 experiments) for platelets (1577±461 sites for platelets). The Scatchard plot analysis revealed two distinct binding sites both on PMN and platelets: a high affinity binding site and a non-saturable binding site.This work was supported by C.N.R. Rome grant no. 81.00089.04.     

Inflammation, T-Cell Phenotype, and Inflammatory Cytokines in Chronic Kidney Disease Patients Under Hemodialysis and its Relationship to Resistance to Recombinant Human Erythropoietin Therapy

Background Resistance to recombinant human erythropoietin (rhEPO) occurs in some chronic kidney disease (CKD) patients, which may be due to enhanced systemic inflammatory response and to the erythropoiesis-suppressing effect of pro-inflammatory cytokines, some of which are produced by T cells. Aim of study The aim of this study was to investigate the relationship between resistance to rhEPO therapy in hemodialysis CKD patients and inflammatory markers [C-reactive protein (CRP), soluble interleukin (IL)-2 receptor (sIL2R), and serum albumin levels], blood cell counts, T-cell phenotype, cytokine production by T cells, and serum cytokine levels. Materials and Methods We studied 50 hemodialysis CKD patients, 25 responders and 25 nonresponders to rhEPO, and compared them to each other and with 25 healthy controls. When compared to controls, CKD patients showed increased serum levels of CRP, IL-6, and sIL2R and a T-cell lymphopenia, due to decreased numbers of both CD4⁺ and CD8⁺ T cells. T cells from CKD patients had an immunophenotype compatible with chronic T-cell stimulation as shown by the increased percentage of CD28⁻, CD57⁺, HLA-DR⁺, CD28⁻HLA-DR⁺, and CD57⁺ HLA-DR⁺ T cells and produce higher levels of IL-2, INF-γ, and TNF-α after short-term in vitro stimulation, although Th1 cytokines were not detectable in serum. Statistically significant differences were found between responders and nonresponders to rhEPO therapy for total lymphocyte and CD4⁺ T-lymphocyte counts, albumin (lower in nonresponders) and CRP (higher in nonresponders) levels. Conclusion CKD patients under hemodialysis present with raised inflammatory markers and decrease of total lymphocyte and CD4⁺ T-lymphocyte counts when compared with controls. Some of those markers are even further enhanced in nonresponders to rhEPO therapy patients, but resistance to this therapy cannot be justified by a Th1 polarized T-cell response.     

Epidermal growth factor and insulin short‐term increase hPepT1‐mediated glycylsarcosine uptake in Caco‐2 cells

Aims: Little is known about the physiological regulation of the human intestinal di/tri‐peptide transporter, hPepT1. In the present study we evaluated the effects of epidermal growth factor (EGF) and insulin on hPepT1‐mediated dipeptide uptake in the intestinal cell line Caco‐2. Methods: Caco‐2 cells were grown on filters for 23–27 days. Apical dipeptide uptake was measured using [¹⁴C]glycylsarcosine([¹⁴C]Gly‐Sar). HPepT1 mRNA levels were investigated using RT‐PCR, cytosolic pH was determined using the pH‐sensitive fluorescent probe BCECF. Results: Basolateral application of EGF increased [¹⁴C]Gly‐Sar uptake with an ED₅₀ value of 0.77 ± 0.25 ng mL^?1 (n = 3?6) and a maximal stimulation of 33 ± 2% (n = 3?6). Insulin stimulated [¹⁴C]Gly‐Sar uptake with an ED₅₀ value of 3.5 ± 2.0 ng mL^?1 (n = 3?6) and a maximal stimulation of approximately 18% (n = 3?6). Gly‐Sar uptake followed simple Michaelis‐Menten kinetics. K_m in control cells was 0.98 ± 0.11 mM (n = 8) and V_max was 1.86 ± 0.07 nmol cm^?2 min^?1 (n = 8). In monolayers treated with 200 ng mL^?1 of EGF, K_m was 1.11 ± 0.05 mM (n = 5) and V_max was 2.79 ± 0.05 nmol cm^?2 min^?1 (n = 5). In monolayers treated with 50 ng mL^?1 insulin, K_m was 1.03 ± 0.08 mM and V_max was 2.19 ± 0.06 nmol cm^?2 min^?1 (n = 5). Kinetic data thus indicates an increase in the number of active transporters, following stimulation. The incrased Gly‐Sar uptake was not accompanied by changes in hPepT1 mRNA, nor by measurable changes in cytosolic pH. Conclusions: Short‐term stimulation with EGF and insulin caused an increase in hPepT1‐mediated uptake of Gly‐Sar in Caco‐2 cell monolayers, which could not be accounted for by changes in hPepT1 mRNA or proton‐motive driving force.     

Relationship of megakaryocyte ploidy with platelet number and size in cats, dogs, rabbits and mice

Feline platelets are larger than platelets of many other species. The following parameters were examined in 13 normal cats in an attempt to determine a reason for the difference: platelet count, mean platelet volume (MPV), platelet mass and ploidy of mature megakaryocytes. Average ± SEM platelet count was 213 000 ± 15000/μl, MPV was 11.1 ± 0.2 fl, and platelet mass was 2.4 ± 0.2 × 10⁶ fl/μl. The predominant ploidy classes of feline megakaryocytes were 32N (41.6%) and 64N (47.1%). These findings were compared to existing data from normal dogs, rabbits, and mice. These species exhibited progressively higher platelet counts (313 000 ± 28 000, 568 000 ±35000, and 1328 000 ± 79 000/μl, respectively) and progressively smaller MPVs (7.2 ± 0.2, 4.4 ± 0.1 and 3.9 ± 0.1 fl, respectively) than cats. Platelet mass was the same in cats, dogs and rabbits, but it was much higher in mice (5.2 ± 0.3 × 10⁶ fl/μl) The modal megakaryocyte ploidy was 16N in mice and 32N in dogs and rabbits. The MPV was directly related to the ploidy of fully mature megakaryocytes except when comparing dogs and rabbits for which MPVs differed, but ploidy distributions did not. The findings suggested that ploidy of a megakaryocyte may be one of the determinants of the size and number of platelets it will produce during normal haemopoiesis.     

Relationship of megakaryocyte ploidy with platelet number and size in cats, dogs, rabbits and mice

Feline platelets are larger than platelets of many other species. The following parameters were examined in 13 normal cats in an attempt to determine a reason for the difference: platelet count, mean platelet volume (MPV), platelet mass and ploidy of mature megakaryocytes. Average ± SEM platelet count was 213 000 ± 15000/μl, MPV was 11.1 ± 0.2 fl, and platelet mass was 2.4 ± 0.2 × 10⁶ fl/μl. The predominant ploidy classes of feline megakaryocytes were 32N (41.6%) and 64N (47.1%). These findings were compared to existing data from normal dogs, rabbits, and mice. These species exhibited progressively higher platelet counts (313 000 ± 28 000, 568 000 ±35000, and 1328 000 ± 79 000/μl, respectively) and progressively smaller MPVs (7.2 ± 0.2, 4.4 ± 0.1 and 3.9 ± 0.1 fl, respectively) than cats. Platelet mass was the same in cats, dogs and rabbits, but it was much higher in mice (5.2 ± 0.3 × 10⁶ fl/μl) The modal megakaryocyte ploidy was 16N in mice and 32N in dogs and rabbits. The MPV was directly related to the ploidy of fully mature megakaryocytes except when comparing dogs and rabbits for which MPVs differed, but ploidy distributions did not. The findings suggested that ploidy of a megakaryocyte may be one of the determinants of the size and number of platelets it will produce during normal haemopoiesis.     

Glutathione-dependent modulation of exhausting exercise-induced changes in neutrophil function of rats

Reduced glutathione (GSH) plays a central role in maintaining an effective synergism between various physiological and exogenous antioxidants. We tested the effects of GSH andN-acetylcysteine (NAC, a pro-GSH clinical drug), intraperitoneal (i.p.) supplementation and GSH deficiency on exercise-induced leucocyte margination and neutrophil oxidative burst activity. GSH, NAC (1g · kg^–1) or placebo saline was i.p. injected (one or eight times) to male rats (n seven per group). The GSH-deficient rats were prepared by i.p. injections ofl-buthionine-[SR]-sulphoximine (BSO, 6 mmol · 1^–1 · kg^–1) twice daily for 4 days. Exercised animals were subjected to treadmill run to exhaustion. Exhausting treadmill exercise significantly decreased peripheral blood leucocyte count in the controls (P < 0.001). Such exercise-associated leucocyte margination was prevented by GSH supplementation. Peripheral blood neutrophil counts were significantly higher (P < 0.02) in the GSH-supplemented groups compared to the placebo control groups. Exercise-induced increase in peripheral blood neutrophil oxidative burst activity as measured by luminol-enhanced chemiluminescence per volume of blood tended to be higher in the GSH-supplemented group (P < 0.10), and lower in the GSH-deficient rats (P < 0.02). In these experiments, for the first time we have shown that GSH supplementation can induce neutrophil mobilization and decrease exercise-induced leucocyte margination, and that exogenous and endogenous GSH can regulate exercise-induced stimulation of the neutrophil oxidative burst.     

Thrombocytosis in preterm infants: a possible involvement of thrombopoietin receptor gene expression

Transient thrombocytosis is commonly observed in preterm infants after birth, but its physiological mechanism is still unknown. To understand the mechanism of the transient thrombocytosis in preterm infants we firstly evaluated a correlation between platelet counts and thrombopoietin (TPO) levels in preterm infants and next c-mpl mRNA levels on platelets in healthy preterm infants longitudinally during a half-year of life. The mean platelet counts in 45 very low birth weight infants (mean gestational age 27.4±1.8 weeks, mean birth weight 1047±249 g) was 230±71×10⁹/l just after birth and thereafter gradually increased to 579±178×10⁹/l by 5 weeks of age. The platelet counts continued this level for about next 8 weeks. Serum TPO levels soon after birth and at 1 month of age were significantly higher than those at the age of 2–6 months. There was a significant negative correlation between platelet counts and serum TPO values. The c-mpl expression levels on platelets at birth and at 1 month of age tended to be lower than those on platelets from adults, and the c-mpl levels gradually increased through 6 months of age, although they were still lower than those of adults. Our results suggest that low expression of TPO receptor on platelets until 1 month after birth cause a decreased TPO clearance and keep a high level of free TPO in blood, thereby promoting platelet production from megakaryocytes or their progenitors in bone marrow, resulting in the subsequent thrombocytosis in preterm infants.     

Transmitter-induced changes of the membrane voltage of HT29 cells

The colonic carcinoma cell line HT₂₉ was used to examine the influence of agonists increasing cytosolic cAMP and Ca²⁺ activity on the conductances and the cell membrane voltage (V _m). HT₂₉ cells were grown on glass cover-slips. Cells were impaled by microelectrodes 4–10 days after seeding, when they had formed large plaques. In 181 impalements V _m was –51±1 mV. An increase in bath K⁺ concentration from 3.6 mmol/l to 18.6 mmol/l or 0.5 mmol/l Ba²⁺ depolarized the cells by 10±1 mV (n=49) or by 9±2 mV (n=3), respectively. A decrease of bath Cl^– concentration from 145 to 30 mmol/l depolarized the cells by 11±1 mV (n=24). Agents increasing intracellular cAMP such as isobutylmethylxanthine (0.1 mmol/l), forskolin (10 mol/l) or isoprenaline (10 mol/l) depolarized the cells by 6±1 (n=13), 15±3 (n=5) and 6±2 (n=3) mV, respectively. In hypoosmolar solutions (225 mosmol/l) cells depolarized by 9±1 mV (n=6). Purine and pyrimidine nucleotides depolarized the cells dose-dependently with the following potency sequence: UTP > ATP > ITP > GTP > TIP > CTP = 0. The depolarization by ATP was stronger than that by ADP and adenosine. The muscarinic agonist carbachol led to a sustained depolarization by 27±6 mV (n=5) at 0.1 mmol/l, and to a transient depolarization by 12±4 mV (n=5) at 10 mol/l. Neurotensin depolarized with a half-maximal effect at around 5 nmol/l. The depolarization induced by nucleotides and neurotensin was transient and followed by a hyperpolarization. We confirm that HT₂₉ cells possess Cl^–- and K⁺-conductive pathways. The Cl^– conductance is regulated by intracellular cAMP level, cytosolic Ca²⁺ activity, and cell swelling. The K⁺ conductance in HT₂₉ cells is regulated by intracellular Ca²⁺ activity.Supported by DFG Gre 480/10 and GIF Proj. no. I-86-100.10/ 88     

Lack of transport of erythropoietin across the human placenta as studied by an in vitro perfusion system

The transfer of human recombinant erythropoietin (rhEPO) from the maternal to the fetal side was investigated using the technique of in vitro perfusion of an isolated cotyledon of human placenta, with recirculation of the perfusate (130 ml) in separate closed maternal and fetal circuits. rhEPO (221–512 U), together with [¹⁴C]BSA (bovine serum albumin, 44.8 kBq or 2,688,000 dpm), was added to the maternal circuit only. Despite a considerably lower molecular weight of EPO mol. wt.=30,400 Da) compared to BSA (mol. wt.= 69,000 Da), no difference was found in their transfer across the placenta from the maternal to the fetal side, which was very low for both macromolecules. The total transfer of rhEPO derived from the concentration measured in the samples taken from the fetal circuit at the end of 4–5 h of perfusion, was in the range of 0.04% of the amount initially added to the maternal compartment. A similar amount of transfer was determined for [¹⁴C]BSA (0.04–0.07%,n=12). In conclusion, by direct determination in a dually in vitro perfused human placental cotyledon, no significant transfer of rhEPO from the maternal to the fetal side could be shown.     

Haematology, red cell enzymes, and red cell metabolic intermediates of 20 wild southern elephant seals (Mirounga leonina) from Macquarie Island

Blood was collected for haematological, red cell enzyme and red cell metabolic intermediate studies from 20 Southern elephant seals Mirounga leonina. Mean haematological values were: haemoglobin (Hb) 22.4 ± 1.4 g/dl, packed cell volume (PCV) 54.2 ± 3.8%, mean cell volume (MCV) 213.0 ± 5.0 fl and red cell count (RCC) 2.5 × 10¹²/l. Red cell morphology was unremarkable. Most of the red cell enzymes showed low activity in comparison with human red cells. Haemoglobin electrophoresis showed a typical pinniped pattern, i.e. two major components. Total leucocyte counts, platelet counts, and coagulation studies were within expected mammalian limits. Eosinophil counts varied from 0.5 × 10⁹/l to 7.7 × 10⁹/l(5%–49%), and there was a very wide variation in erythrocyte sedimentation rates, from 3 to 60 mm/h.     

Prolonged bleeding time during nitric oxide inhalation in the rabbit

During a study on the modulatory effect of inhaled nitric oxide (NO) on the airway, we observed an increased bleeding tendency. Therefore, we studied bleeding time and blood rheology in rabbits during inhalation of 3, 30 and 300 parts per million (ppm) NO. The rabbits were intubated during neurolept anaesthesia and were ventilated mechanically. The bleeding time was significantly increased after 15 min of inhalation of 30 ppm NO, from 51 + 5 to 72 + 7 s (mean + SEM, P < 0.001, n= 7). However, there were no changes in haematocrit, whole blood or plasma viscosity, erythrocyte aggregation tendency, or erythrocyte deformability. Inhalation of 3 ppm NO increased bleeding time from 46+11 to 59 + 8 s (n.s., n= 4) and 300 ppm NO from 48 + 12 to 78 + 17s(P<0.05, n= 4). In another group of rabbits mean arterial pressure (MAP) was monitored using NO inhalation. A non-significant decrease was seen with 3 ppm and 30 ppm NO, from 63 + 2 to 59 + 3 mmHg (n= 6) and from 65 + 2 to 61 + 1 mmHg (n= 6) respectively. Inhalation with 300 ppm NO decreased MAP from 62 + 3 to 55 + 2 mmHg (P < 0.05, n= 6). We conclude from these data that inhalation of NO, 30 ppm or more exerts systemic effects.     

The Influence of Recombinant Human Erythropoietin on Apoptosis and Cytokine Production of CD4+ lymphocytes from Hemodialyzed Patients

Recombinant human erythropoietin (rhEPO) treatment of hemodialyzed (HD) patients normalizes the altered phenotype of CD4⁺ lymphocytes and restores the balance of Th1/Th2 cytokines. We decided to test how the presence of rhEPO in cell culture modulates cytokine production of CD4⁺ lymphocytes in HD patients with stable hemoglobin level and expression of activation antigens of stimulated CD4⁺ lymphocytes similar to those observed in healthy individuals. We also tested whether the presence of rhEPO in cell culture protects stimulated CD4⁺ lymphocytes of HD patients from apoptosis. Peripheral blood mononuclear cells (PBMC) of HD patients were stimulated with an immobilized anti-CD3 antibody with or without addition of rhEPO. The percentage of apoptotic CD4⁺ lymphocytes and the level of Th1/Th2 cytokines in culture supernatants were measured with flow cytometry. HD patients showed a decrease in the percentage of apoptotic CD4⁺ cells after stimulation with the anti-CD3 antibody combined with rhEPO. The level of IFN-γ and IL-10 was increased while the level of TNF-α was decreased in the presence of rhEPO in cell culture from HD patients. These results confirm the role of rhEPO signaling in T lymphocytes of HD patients.     

Effect of Angiostrongylus costaricensis extract on eosinophilic pulmonary response in BALB/c mice

Epidemiological and experimental studies have demonstrated an association between parasitic infections and the allergic diseases. A protective effect in asthma was shown in animals infected with helminths. The aim of this study was to determine the effect of Angiostrongylus costaricensis extract on inflammatory lung response to ovalbumin (OVA) in mice. Four BALB/c mice received A. costaricensis extract by intraperitoneal (i.p.) injection on the first day. Mice were immunised against OVA by i.p. injection on day (D) 5 and D12 and received a daily intranasal OVA challenge (40 μl) between the D19 and D21. On D23, we performed a bronchoalveolar lavage (BAL) on the mice. Four BALB/c mice (control group) were immunised against OVA using the same protocol, but did not receive parasite extract. Total cell counts (TCC) and differential cell counts were performed in BAL fluid samples. Eosinophil cell counts in BAL fluid were lower in the group that received A. costaricensis extract when compared with the control group (0.04×10⁶ cells/ml and 0.01×10⁶ cells/ml, respectively; p=0.04). TCC were not different between the groups studied. A. costaricensis extract in mice decreases eosinophilic response to OVA in BAL fluid.     

Isokinematic muscle mechanics in four groups of women of increasing age

Summary The purpose of this study was to evaluate the effects of age on dynamic muscle attributes of the knee extensors and flexors in postmenopausal women. Young healthy women (3rd decade,n = 15; 4th decade,n = 5) and older healthy women (6th decade,n = 9; 7th decade,n = 6) were tested at six angular velocities from 60° · s^–1 to 400° · s^–1. The 3rd and 4th decade groups produced greater extensor and flexor values for strength related variables at all angular velocities (peak torque, angle specific torque, work, power) than both the 6th and 7th decade groups (P<0.05). However, relative changes in these variables, with increments in angular velocity, were equivalent among the groups. Analysis of the flexor: extensor ratios for these variables demonstrated a differential loss in flexor function with increased age, perhaps indicative of type II motor unit loss or muscle fibre atrophy. It is suggested that such changes may be present even within 4th decade subjects.     

Evaluation of the CELL-DYN® 3500 haematology instrument for the analysis of the mouse and rat blood

The objective of this study was to evaluate the performance of the CELL-DYN^® 3500 for rat and mouse blood analysis in a routine environment. The WBC (white blood cells), RBC (red blood cells), PLT (platelets) counts and the WBC differential were determined. In addition, the following aspects were studied: within-run precision, day-to-day precision, biasfree paired difference precision; extended ranges of linearity for RBC, HCT (haematocrit), WBC, PLT; carry-over, the fffect of blood ageing, cell stability with different anticoagulants; and the normal ranges, the out of range flagging and some typical pathology cases. The CELL-DYN^® 3500 is a multiparameter flow cytometer which counts and differentiates WBC, based on the principle of multi-angle polarised light scatter separation. RBC and PLT are determined by the impedance method. The WBC count is evaluated by both, optical and impedance methods. Reference methods used were according to the ICSH recommendations on blood cell analysis, including manual counts of WBC and platelets, a centrifugal microhaematocrit method and a haemoglobin measurement by spectrophotometry using the WHO haemoglobin standard. All cell counts were compared with the results obtained by our routine blood cell analyser (Contraves AL820), and the WBC differential was compared with the manual microscopic differentiation of the 400 WBC (200 cells differentiated by two technicians). The following coefficients of variation were obtained: within-run precision was 1.2% and 2.7% for WBC; 1.0% and 1.0% for RBC; 1.3% and 0.9% for haematocrit; 2.1% and 2.7% for platelets (rats and mice respectively). Day-to-day precision was performed using human trilevel control blood, and the CVs were found to be <1.7% for WBC, <1.4% for RBC, <1.2% for haemoglobin and <6.3% for platelets. The following ranges of measurement were found to be linear in the rat: WBC: 0.10–20.20×10³/μl; RBC: 0.016–14.3×10⁶/μl; haemoglobin: 0.08–26.8 g/dl; haematocrit: 5.0%–77%; platelets: 14.0–1670.0×10³/μl. Equal ranges were observed for mouse blood. Carry-over in rat blood was found to be 0.12% for WBC, 0.05% for RBC, 0.15% for haemoglobin and 0.46% for platelets. In mice, similar carry-over results were obtained. The correlation coefficients (Pearson, correlation coefficient) between the CELL-DYN^® 3500 and Contraves AL 820 using linear regression analysis were as follows: 0.988 and 0.997 for WBC; 0.986 and 0.920 for RBC; 0.995 and 0.984 for haemoglobin; 0.958 and 0.85 for haematocrit; 0.958 and 0.963 for platelets, for rats and mice, respectively. Correlation coefficients between the CELL-DYN^® 3500 and the manual differential of NEU (neutrophils) and LYM (lymphocytes) were higher than 0.8 in rats and higher than 0.9 in mice. Due to the relatively low absolute counts of MONO (monocytes), EOS (eosinophils) and BASO (basophils), only moderate correlation of methods was found. The CELL-DYN^® 3500 was judged to be reliable, accurate and easy-to-use for counting and identifying normal and most of the pathological blood specimens obtained from mice and rats. By using the CELL-DYN^® 3500, the time for blood sample analysis can be shortened significantly and provides extensive opportunities to characterise pathological samples.     

Changes in the levels of some acute-phase proteins in human immunodeficiency virus-1 infected patients, following interleukin-2 treatment

Intermittent interleukin (IL)‐2 administration to human immunodeficiency virus (HIV)‐1 infected patients is well documented and generally used, but there is limited information about the changes of acute‐phase protein (APP) levels in response to this treatment. Fifteen patients undergoing highly active anti‐retroviral therapy (HAART) treatment, with undetectable viral load, but low CD4⁺ cell count (<300/µl), have been treated with 3·6 M IU Proleukine® administered twice daily by subcutaneous injection over 5 days. C‐reactive protein (CRP), d ‐dimer, C3, C9, C1‐inh and alpha‐2HS glycoprotein levels were measured immediately before IL‐2 administration, as well as on day 5 and 2–3 weeks thereafter. After IL‐2 administration, both mean d ‐dimer and CRP levels increased significantly (P < 0·001), but returned (P < 0·001) to baseline within the subsequent 2–3 weeks. Alpha‐2HS glycoprotein decreased immediately after IL‐2 administration. No significant differences were detected in the levels of C3, C9 and C1‐inh. A significant, positive correlation (r = 0·5178, P = 0·0008) was ascertained between the changes of CRP level, measured immediately before as well as 5 days after IL‐2 administration, and changes in CD4 T cell counts measured 2–3 weeks before and after treatment, respectively. IL‐2 administration induces rapid elevation of two major APPs (CRP, d ‐dimer). The positive correlation observed between the changes of CRP levels and CD4⁺ cell counts after IL‐2 administration may indicate that the abrupt, but transitory overproduction of CRP might contribute to the CD4⁺ cell count‐increasing effect of the drug and/ or may be associated with serious side effects.     

Influence of catheter intervention on platelet function and activation in a pig model for endovascular embolization of arteriovenous malformations

Catheter interventions are associated with the risk of thromboembolism; however, the extent of platelet activation is not known. Samples from an arteriovenous malformation model (n?=?21 pigs) were examined. The pigs received a continuous infusion of 66?IU?kg^?1?h^?1 (n?=?11) or 100?IU?kg^?1?h^?1 (n?=?10) heparin applied 20?min after an initial bolus of 100?IU/kg. Platelet aggregation according to Born and ADVIA 120? platelet activation indices were used to study platelet function and activation. Samples were taken previous to vascular puncture, following vascular puncture, 20?min after application of heparin bolus, following placement of microcatheters and after endovascular embolization. Reactivity of platelets was increased after puncture of the vessels (ADP: P?<?0. 0001, collagen: P?=?0.0053). Further on activity of platelets was constrained by heparinization (ADP: P?<?0.0001, collagen P?<?0.0001). It can be concluded that the puncture of vessels yields the highest risk of thromboembolic complications.     

Splanchnic nerve Stimulation increases the lymphocyte output in mesenteric efferent lymph

Mesenteric efferent lymph was collected from anaesthetized sheep. Lymph flow rate and leucocyte content (> 95% lymphocytes) were measured under control conditions and during stimulation of the left greater splanchnic nerve. During the first 5 min of nerve stimulation at 4 Hz lymph flow was increased by 128±57% and lymph white cell count by 44±15% (P<0.05, n= 8 in both cases). This produced an overall increase in the white cell output of 228±151% (P<0.05, n=8). The response was repeatable but short lived, with no significant differences from control being observed after the first 5 min of stimulation. There was a rise in the red cell count in arterial blood during nerve stimulation (from 3.21±0.24 · 10¹² l^–1 to 4.48±0.22 · 10¹² l^–1, P<0.05, n=9) but no statistically significant changes in the white cell count or percentage of lymphocytes. The increase in lymph white cell output could be mimicked by intravenous injection of noradrenaline while phentolamine blocked the nerve-induced increases in both lymph flow and white cell concentration. The possible mechanisms and immunological consequences of this -adrenoceptormediated increase in lymphocyte traffic are discussed.Part of this work has been communicated to the Physiological Society of Great Britain and Ireland [10]     

IL-1β and IL-6 stimulate the production of platelet-activating factor (PAF) cultured rabbit synovial cells

The aim of this study was to determine whether synovial cells are capable of producing PAF in the presence of cytokines such as IL-1βand IL-6 and other stimuli. Synovial cells were obtained from joints of healthy rabbits. PAF production was assayed by measurement of serotonin release in rabbit platelets and the incorporation of ³H-acetate into PAF. Synovial cells produced PAF after 5 min of incubation with ionophore A23187, reaching the maximal amount at 15 min (4·3 ± 0·7 ± 10^?3 pmo1 of PAF/mg protein, P < 0·005, n= 4), and declining afterwards. The treatment of synoviocytes with IL-1β and IL-6 induced synthesis of PAF after 5 min of stimulation, reaching the greatest production at 15 min with IL-6 and 30 min with IL-1β (3·6 ± 1·1 ± 10^?3 and 3·3 ± 1·2 pmol of PAF/mg protein, respectively, P < 0·05, n= 4). The incubation of the cells with PMSF, an inhibitor of acetylhydrolase, before the addition of the stimuli, increased the incorporation rate of ³H-acetate, suggesting a rapid degradation of PAF by synoviocytes. These results demonstrate that synovial cells can produce PAF after stimulation with agonists, such as ionophore, and cytokines. Thus, PAF, acting alone or with other mediators, could amplify the inflammatory joint reaction.     

Lymphocyte subsets in hemodialysis patients treated with recombinant human erythropoietin

We investigated whether recombinant human erythropoietin (rhEPO) therapy affected the lymphocyte subsets in patients on long-term maintenance hemodialysis (HD) with severe anemia. Before treatment, the numbers of peripheral blood lymphocyte, CD3⁺, CD4⁺, CD8⁺, and CD20⁺ cells were decreased in HD patients compared to those in healthy subjects, while the number of CD3⁺ HLA-DR⁺ cells was increased in HD patients compared to that in healthy subjects. Furthermore, the number of CD4⁺CD45RA⁺ (naive T) cells was markedly decreased in HD patients (112±77 vs 241±146/µl;P<0.01). The number of CD8⁺S6F1⁺ (cytotoxic T) cells in HD patients was also less than that in healthy subjects (247±104 vs 122±83/µl; NS). During a 6-month period of rhEPO therapy, we found that the low level of CD4⁺CD45RA⁺ cells gradually increased (from 112±18 to 163±24/µl;P<0.01) with the elevation of hematocrit values (from 21.5±1.7 to 28.2±3.5%;P<0.05). The number of CD3⁺HLA-DR⁺ cells decreased after 1 month of rhEPO therapy (from 93±14 to 46±13/µl) and gradually declined throughout the 6-month study period. In ourin vitro study, we demonstrated that no effects were observed on [³H]thymidine uptake in the T cell subsets at various concentrations of rhEPO. These results suggest that rhEPO-induced immunoregulation is mediated by an indirect stimulatory effect on the immune system, this stimulation being accompanied by an improvement in physical condition.