Application of the Beckman JE6-B Elutriator System in the isolation of human monocyte subpopulations

Beckman Instruments recently developed an improved version of their JE-6 Elutriator Rotor. At 3000 rpm (867 X g), lymphocytes were eluted from the chamber at flow rates of 10 to 18 ml/min. Small monocytes (362 mu 3) were collected at 18 ml/min over 400 ml while the larger monocytes (395 mu 3) were retained in the chamber until the flow rate was increased to 28 ml/min. These parameters differ from those previously reported by us when we used Beckman's original commercial version of the JE-6 Elutriator Rotor. The new JE-6B rotor required an increase of centrifugal force from 625 to 867 X g generated by an increase in rpm from 2500 to 3000, as well as an increase in flow rates to collect both small (17 to 18 ml/min) and large (26 to 28 ml/min) monocytes. The monocytes collected using the new rotor with the revised protocol have the same physical and functional properties as those collected with the earlier version using our previously published protocol.     

Preparation of thyrotroph cells from adult male rat pituitary glands by centrifugal elutriation

To study the metabolism of thyrotrophs and dynamics of TSH secretion in vitro, it is desirable to have a highly enriched population of thyrotrophs. For that purpose, centrifugal elutriation, a recently developed cell isolation method based on the size and density of cells, was used to prepare thyrotrophs from a cell suspension of adult male rat pituitary cells. Trypsin-dispersed cells (4-8 X 10(7] were loaded into the elutriation rotor (Beckman, JE-6) operating at 2800 rpm. Twelve cell fractions were collected at variable rotor speed (2000-2800 rpm) and increasing medium flow rate (10-103 ml/min). Cell recovery was 77-98%. The viability of the cells after elutriation was 90-95% based on trypan blue exclusion. Each fraction was analyzed for TSH, GH, and PRL content and for TRH-stimulated TSH release by RIA. Thyrotrophs were found predominantly in fractions 8-11 (flow rate 38-75 ml/min) based on TSH RIA. The mean TSH concentration in these fractions was 56 +/- 13.6 (+/- SD) microU/10(3) cells compared with 7.6 +/- 3.8 microU/10(3) cells in the initial cell suspension, representing a 7- to 8-fold enrichment of the thyrotrophs. Incubation with 20 nM TRH for 3 h increased the TSH release of cells eluted in fractions 8-11 by 3- to 5-fold; there was no significant increase in TSH release in fractions 3-6. Centrifugal elutriation may be used to prepare a uniform highly enriched thyrotroph fraction with excellent recovery from a suspension of rat pituitary cells. This technique should be valuable for study of the metabolism of thyrotrophs.     

Size and density characterization of human committed and multipotent hematopoietic progenitors

Physical characterization of human blood and bone marrow (BM) hematopoietic precursors is necessary for the design of adequate techniques to isolate these cells for experimental and possible clinical use. Elutriation and continuous Percoll gradient were used to define the size and density of committed (CFU-GM, BFU-E, and CFU-E) and multipotent (CFU-MIX) progenitors on paired BM and blood samples from normal donors. Cells were initially prepared over Ficoll-Hypaque (specific gravity 1.077 g/cm3). Elutriation was performed on a Beckman JE-6B rotor with a standard chamber at 2000 rpm using flow rates of 7-23 ml/min. Percoll gradients were centered at 1.070 g/cm3 using ultracentrifugation. Cell density was determined using marker beads. Density was slightly higher for BM than for blood progenitors of the same class. Progenitor sizes (proportionate to elutriation flow rate) were similar in BM and blood. CFU-MIX, BFU-E, and CFU-GM coseparated in blood and BM; CFU-E (BM) were larger and more dense than other BM progenitors. BM and blood E-rosette-positive cells were separated more effectively by size than by density. The similarity in size and small differences in density between blood and BM progenitors may allow adaption of blood separation techniques (apheresis) to processing of BM for cryopreservation and/or in vitro treatment. Similarly, the ability to separate T cells from progenitors in blood by elutriation (albeit only partially), as has been described for BM, may help in the modification of existing apheresis techniques to achieve such separations.     

Microcirculatory effects of fluid therapy and dopamine, associated or not to fluid therapy, in endotoxemic hamsters

Classically septic shock treatment takes into consideration only systemic parameters but failure in retaining arteriolar blood flow and functional capillary density (FCD) during sepsis worsens the outcome. Thus, we have investigated the effects of vigorous volume resuscitation (VR), two doses of dopamine and their combination upon the microcirculation during endotoxemia to evaluate if improvement on FCD and arteriolar blood flow would increase survival time. Sixty-seven adult male hamsters were studied using the window chamber model. Animals were randomized 1 h after the intravenous injection of 1 mg/kg of E. coli lipopolysaccharide (LPS) in LPS, no treatment; LPS/dopamine 3.0 μg/kg/min; LPS/dopamine 7.5 μg/kg/min; LPS/VR 20 ml/kg in 30 min followed by 20 ml/kg/h of saline; LPS/VR/Dopa 3.0, 20 ml/kg in 30 min followed by 20 ml/kg/h of saline associated to dopamine 3.0 μg/kg/min; LPS/VR/Dopa 7.5 (n = 6), 20 ml/kg in 30 min followed by 20 ml/kg/h of saline associated to dopamine 7.5 μg/kg/min and compared them to a Control group, no LPS. When present, treatment lasted 5 h. VR improved FCD and arteriolar blood flow score while dopamine did not. In conclusion, (1) improvement of FCD and arteriolar blood flow improved survival time; (2) VR recovered FCD and arteriolar blood flow and (3) in combination to VR, both dopamine doses reduced tissue perfusion (its low dose yielded the worst result).     

Inducible ventricular tachyarrhythmias in canine myocardial infarction

The hypothesis that local release of vasodilator prostaglandins mediates, in part, the decrease in coronary resistance after nitroglycerin (NG) administration was tested. NG (60 μg/min) and nitroprusside (NP) (30 μg/min) were infused for 10 minutes into the left circumflex coronary artery of 9 open-chest, chloralose-anesthetized dogs before and after blockade of prostaglandin synthesis with indomethacin (5 mg/kg). Also, to eliminate the effects of reflex tachycardia on the responses to NG and NP, these experiments were repeated in 6 dogs with heart rate held constant by pacing. NG infusion into dogs without pacing resulted in a maximal increase in coronary blood flow of 55% and after 8 minutes of infusion an increase of 20%, from a baseline of 57 ml/min. NP infusion resulted in a maximal increase in blood flow of 89% and after 8 minutes an increase of 71%, from a baseline of 64 ml/min. In dogs with heart rate held constant, NG infusion caused a maximal increase in coronary artery blood flow of 132% and after 8 minutes of infusion an increase of 18%, from a baseline of 48 ml/min; NP infusion resulted in increase from 51 ml/min of 132% and 62%, respectively. In neither group of dogs did indomethacin significantly change the increases in coronary blood flow or decreases in coronary resistance to NG or NP. Thus, both NG and NP, when infused into the coronary artery of dogs, cause increases in coronary blood flow and decreases in coronary resistance by a mechanism which is independent of prostaglandin release.     

Effect of intravenous glucose infusion on renal function in normal man and in insulin-dependent diabetics

Summary The effect of intravenous glucose infusion on glomerular filtration rate and renal plasma flow (constant infusion technique using ¹²⁵I-iothalamate and ¹³¹I-hippuran) and on urinary excretion of albumin and -2-microglobulin were studied in ten normal subjects and seven metabolically well-controlled insulin-dependent diabetics. Following glucose infusion in normal subjects (n = 10) blood glucose increased from 4.7±0.1 to 10.9±0.4 mmol/1 (SEM) (p 0.01). Glomerular filtration rate increased from 116±2 to 123±3ml/min x 1.73 m² (p 0,01), while no change in renal plasma flow was seen 552±11 versus 553±18 ml/min × 1.73 m². Volume expansion with intravenous saline infusion in six of the normal subjects induced no changes in blood glucose or kidney function. In seven strictly controlled insulin-dependent diabetics, blood glucose values were raised from 4.6±0.4 to 16.0±0.6 mmol/1 and clamped by means of an artificial beta cell. Glomerular filtration rate increased in all patients, from 133 ±5 to 140±6 ml/min × 1.73 m² (p 0.02), as did renal plasma flow from 576±26 to 623±38 ml/ min × 1.73 m² (p 0.02). Urinary albumin excretion remained unchanged in both normal subjects and diabetics. -2-microglobulin excretion rate increased significantly in the diabetics following glucose infusion, while no significant change was seen in the normal subjects. Our results show that hyperglycaemia per se contributes to the increased glomerular filtration rate and renal plasma flow in insulin-dependent diabetes.     

Effect of basal epicardial tone on endothelium-independent coronary flow reserve measurement

Increased basal epicardial tone may attenuate the coronary flow reserve (CFR) by causing vasodilatation of resistance vessels. We examined the effect of basal epicardial tone on the endothelium-independent CFR measurements in subjects with nonobstructive coronary disease. Patients underwent evaluation of endothelium-independent CFR using adenosine (18–36 μg) and endothelium-dependent CFR using acetylcholine (10⁻⁶M-10⁻⁴M), both administered intracoronary. CFR to adenosine, presented as the ratio of Doppler flow velocities post- and pre-adenosine, was measured at baseline and after intracoronary nitroglycerin (200 μg). Nitroglycerin increased the coronary artery diameter by 19.7 ± 2.5%, and decreased the coronary vascular resistance from 3.0 ± 0.2 mm Hg/ml/min to 1.8 ± 0.1 mm Hg/ml/min (p < 0.0001). The response to adenosine at baseline and after nitroglycerin was similar (CFR ratio of 2.52 ± 0.09 and 2.57 ± 0.10, respectively, p = NS). The effect of nitroglycerin on the response to adenosine did not correlate with coronary endothelial function (r² = 0.06, p = 0.13). The basal epicardial tone does not affect CFR measurements in patients with angina and nonobstructive coronary disease. Cathet. Cardiovasc. Diagn. 44:392–396, 1998. © 1998 Wiley-Liss, Inc.     

Haemodynamic changes during dobutamine stress echocardiography in patients with and without ischaemia

We studied haemodynamic changes during dobutamine stress echocardiography in 69 patients (mean age 58 years, 6 female, 63 male) referred for investigation of chest pain. We used a standard protocol of 3 min stages using infusion rates of 5, 10, 20, 30 and 40 μg/kg/min. Heart rate rose from 74 (13) to 123 (21) beats per min with the major increment occurring during the high dose phase of the study (>20 μg/kg/min). Stroke volume was calculated as the product of left ventricular outflow tract cross-sectional area and the velocity integral of the continuous wave aortic signal. Mean stroke volume increased from 67.5 (22) ml pre-test to 82 (22) ml at 20 μg/kg/min dose (P<0.0001) and 85 (21) ml at 40 μg/kg/min (P<0.00001). Only 15 patients (26%) reached their maximal stroke volume by 10 μg/kg/min, 38 patients (65%) reached maximal stroke volume by 20 μg/kg/min. Patients with ischaemic responses tended to have a blunted rise in stroke volume from 67 (22) ml to 85 (22) ml at maximum compared with a rise from 69 (23) to 92 (19) ml in those without ischaemia (P=0.09). In conclusion, the early rise in cardiac output during dobutamine stress was mainly due to a rise in stroke volume and the later rise due to an increase in heart rate. Individual increases in stroke volume did not adequately differentiate between ischaemic and non-ischaemic results.     

Paired Plateletpheresis with the CS 3000 Plus Blood Cell Separator Using the New 30 ml Low-Volume Collection Chamber and the A-35 Collection Chamber

Eight blood donors underwent paired plateletpheresis using the CS 3000 Plus blood cell separator. For each donor, two samples were run: one with the TNX-6^TM separation and the new 30-ml low-volume collection chamber (LVCC) and one with TNX-6 separation and the A-35 collection chamber. With the LVCC, 4.8±0.5 times 10¹¹ thrombocytes could be harvested, whereas 4.4.±1.4 times 10¹¹ platelets (PLT) were collected with the A-35 chamber. The separation efficiencies were 51.7±6.6% for the LVCC and 43.8±10.7% for the A-35 chamber. The leukocyte contamination of the platelet concentrates was not statistically different.     

Effects of autonomic denervation on canine exocrine pancreatic secretion and blood flow

Summary The effect of autonomic denervation on the exocrine pancreatic secretion and blood flow was studied in a group of dogs. Pancreatic secretion was collected and analyzed for volume and bicarbonate by direct cannulation of the main papilla through a duodenotomy prior to and following truncal vagotomy and celiac plexus denervation. Pancreatic blood flow was determined by the radioisotope distribution method (¹⁴¹Ce). Truncal vagotomy causes a reduction in pancreatic secretion of volume and bicarbonate by 25–30%, while celiac denervation caused a reduction of 70% in the secretion. The mean baseline pancreatic blood flow was 0.5 ml/g pancreas/min. Truncal vagotomy did not cause any significant flow changes, while celiac denervation caused a significant increase in blood flow of 350% (to 1.75 ml/g/min). These results suggest that both the parasympathetic and the sympathetic system affect pancreatic secretion independently of their effect upon pancreatic blood flow.     

Prostacyclin in experimental myocardial ischemia: Effects on hemodynamics,regional myocardial blood flow,infarct size and mortality

To determine the actions of prostacyclin in acute myocardial ischemia, the left anterior descending coronary artery was ligated in 26 anesthetized dogs. At 15 and 45 minutes after coronary ligation, regional myocardial blood flow (ml/min per 100 g) and cardiac output (ml/min) were measured by the radiolabeled microsphere technique (strontium-85 or cerium-141 18 to 10μ]). The dogs were randomly allocated 17 minutes after coronary ligation to a control group (n = 17) or to treatment with prostacyclin infusion at 0.32 μg/kg per min for 6 hours (n = 9). Heart rate and cardiac output were unchanged (p > 0.05) by prostacyclin; mean systemic arterial pressure decreased from 121 ± 8 to 90 ± 3 mm Hg (mean ± standard error of the mean) (p < 0.01) and systemic vascular resistance from 2,690 ± 339 to 2,372 ± 398 dynes · s · cm^?5 (p < 0.05). Prostacyclin reduced blood flow in nonischemic myocardium from 116 ± 12 to 80 ± 3 ml/min (p < 0.01); flow in the ischemic zone was unchanged (p > 0.05) from 22 ± 5 to 20 ± 4 ml/min. Thus, the ratio of ischemic to nonischemic flow was increased by prostacyclin from 0.20 ± 0.03 to 0.25 ± 0.05 (p < 0.05); neither the ischemic nor the nonischemic endocardial/epicardial flow ratio was altered (p > 0.05). In the control dogs, all variables remained constant (p > 0.05) from 15 to 45 minutes. Mortality to 6 hours after coronary ligation was 0 of 9 in prostacyclin-treated versus 6 of 17 (35 percent) in control dogs (p < 0.06). Surviving dogs were killed 24 hours after ligation and infarct size, determined by planimetry and weight techniques, was 18 ± 2 percent of left ventricle in prostacyclin-treated dogs versus 22 ± 2 percent in control dogs (p > 0.05). Thus, prostacyclin in acute myocardial ischemia decreases myocardial oxygen demands, maintains ischemic myocardial blood flow while decreasing nonischemic flow, and greatly reduces early mortality.     

Correlation of plasma propylthiouracil levels with inhibition of thyroid hormone synthesis in the rat

The inhibition of thyroid hormone synthesis was studied in relation to plasma levels of 6-n-propyl-2-thiouracil (PTU). Na ¹²⁵I (5 μCi) was injected i.p. into adult male Sprague-Dawley rats. After 30 min, graded doses of PTU (0.2 mg/kg, 0.1 mg/kg, and 0.05 mg/kg) were similarly injected. Thyroid hormone synthesis was followed by the accumulation of radioactivity into thyroid glands, which were removed at specified time intervals. PTU levels were measured spectrophotometrically at the time of sacrifice. Additionally, PTU (³⁵S) was used to confirm blood levels of PTU and also to follow intrathyroidal PTU levels. Plasma PTU levels in excess of 0.18 μg/ml completely inhibited thyroid hormone synthesis. Levels between 0.14 and 0.09 μg/ml had a partial effect, and PTU levels less than 0.09 μg/ml had no effect on thyroid hormone synthesis.     

Atrial Natriuretic Peptide Reduces the Severe Consequences of Coronary Artery Occlusion in Anaesthetized Dogs

The aim of the present study was to examine the effects of atrial natriuretic peptide (ANP) on the responses to coronary artery occlusion. In chloralose-urethane anaesthetised mongrel dogs either saline (controls) or human synthetic ANP was infused intravenously (10 g kg^–1 + 0.1 g kg^–1 min^–1), starting 30 min before and continuing 10 min during a 25 min occlusion of the left anterior descending coronary artery (LAD). ANP infusion resulted in a fall in mean arterial blood pressure (by 17 ± 2 mmHg, p < 0.05), a transient (max. at 5 min) increase in coronary blood flow (by 24 ± 5 ml min^–1, P < 0.05), and a reduction in coronary vascular resistance (by 0.27 ± 0.05 mmHg ml^–1, p < 0.05). When the LAD coronary artery was occluded, there was a less marked elevation in left ventricular end-diastolic pressure (LVEDP) in the ANP-treated dogs than in the controls (9.0 ± 0.9 versus 12.2 ± 0.8 mmHg, p < 0.05). Compared to the controls, ANP reduced the number of ventricular premature beats (VPBs, 26 ± 12 versus 416 ± 87, p < 0.05), the number of episodes of ventricular tachycardia per dogs (VT, 0.7 ± 0.3 versus 12.4 ± 4.2, p < 0.05), and the incidences of VT (45% versus 100%, p < 0.05) and ventricular fibrillation (VF 18% versus 57%, p < 0.05) during occlusion. Reperfusion of the ischaemic myocardium at the end of the occlusion period led to VF in all the control dogs (survival from the combined ischaemia-reperfusion insult was therefore 0%), but VF following reperfusion was much less in the dogs given ANP (survival 64%; p < 0.05). The severity of myocardial ischaemia, as assessed from changes in the epicardial ST-segment and the degree of inhomogeneity, was significantly less marked in dogs given ANP. We conclude that ANP protects the myocardium from the consequences of myocardial ischaemia resulting from acute coronary artery occlusion and reperfusion in anaesthetized dogs.     

G-CSF-mobilized peripheral blood progenitor cells for allogeneic transplantation: safety,kinetics of mobilization,and composition of the graft

Allogeneic transplantation of peripheral blood progenitor cells (PBPC) markes the general anaesthesia of the donor unnecessary and may result in more rapid engraftment and faster recovery of the immune system. We have studied G-CSF-mediated PBPC mobilization in healthy donors and analysed the cellular composition of the resulting PBPC grafts. PBPC grafts were obtained from nine healthy donors (18-67 years old) for allogeneic or syngeneic transplantation. Six donors received 10 μg/kg G-CSF per day, the others 5-6 μg/kg. Mobilization and harvesting were well tolerated except for moderate bone pain which occurred in all donors primed with 10 μg/kg. With 10 μg/kg, a 31-fold (9-62) enrichment of circulating CD34⁺ cells was observed with peak values constantly occurring on day 5 after the start of G-CSF administration. Starting harvest on day 5, one to three collections on consecutive days yielded 5.5 x 10⁶/kg (0.9-10.7) CD34⁺ cells, 219 x 10⁶/kg (106–314) T cells, and 34 x 10⁶/kg (23–67) NK cells per 10 litres leukapheresis volume. Altogether, PBPC grafts contained 3 times more CD34⁺ cells, 7 times more T cells, and 20 times more NK cells than five allogeneic marrow grafts that were analysed for comparison. The yield of CD34⁺ cells per 10 litres apheresis volume as well as the height of the CD34⁺ peak in peripheral blood were inversely correlated to the age of the donor. In the donors primed with 5–6μg/kg G-CSF the increase of circulating CD34⁺ cells (4–7-fold enrichment) and the CD34⁺ cell yield per 10 litres leukapheresis volume (1 x 10⁶/kg [0.8-2-2]) was much smaller compared with the 10μg/kg group. In conclusion, sufficient amounts of PBPC capable of restoring haemopoiesis in allogeneic recipients can be mobilized safely by administration of G-CSF (10 μg/kg s.c. for 5 d) in healthy donors, and harvested with one or two leukapheresis procedures. Whether the large numbers of T-cells and NK cells that are contained in the collection products may influence graft-versus-host and graft-versus-leukaemia reactivities of PBPC grafts remains to be determined.     

Studies on the improvement of leucodepletion performance of the Haemonetics MCS+ for production of leucodepleted platelet concentrate

With the implementation of universal leucodepletion, an in-line, negatively charged LRF6H leucodepleting filter became an essential part of the Haemonetics MCS+ plateletpheresis system. A larger-scale (968) study using the standard protocol revealed a 2.79% leucodepletion failure rate (standard < 5 x 10(6) leucocytes per adult therapeutic dose). Factors influencing the efficacy of the filter were investigated. The pH of the filtrate was 7.0, the temperature 28 degrees C and filtration rate 80 ml/min. Reduction of the filtration rate to 30 ml/min (784 doses) reduced leucodepletion failure to 0.38%. Measurement of the leucocyte count, pre- and post-filtration of the platelet products, revealed that donations from 1% of donors contained substantially larger numbers of leucocytes in pre-filter samples (300-1500/microl) than in control samples (35-70/microl). This number tends to increase progressively with subsequent donations in these individuals, leading to leucodepletion failure, whilst peripheral leucocyte counts remain normal. The new continuous filtration protocol (version C) using a less impact filter LRF-XL and a lower (7 ml/min) head pressure was also effective but failure still occurred twice on one of the donors who persistently showed high pre-filter count. We conclude that leucodepletion failures in the Haemonetics system are related to both donor leucocyte (i.e., being light and non-adherent) and operational/filter performance.     

Intracoronary delivery of mesenchymal stem cells at high flow rates after myocardial infarction improves distal coronary blood flow and decreases mortality in pigs

Objectives: Evaluate the effects of pressure and duration of intracoronary (IC) infusion of mesenchymal stem cells (MSCs) on delivery efficiency and safety after myocardial infarction (MI). Background: Standard IC delivery of MSCs can lead to intravascular plugging and reduced coronary blood flow. The optimal delivery pressure and duration is unknown. Methods: Immediately after MI pigs were randomized to 1 of 3 delivery protocols of 5 × 10⁷ iron‐fluorescent microspheres labeled MSCs, control received 2 ml infusions at 1 ml/min (five times), very high flow rate (VHFR) a single 10 ml infusion at 60 ml/min and the high flow rate (HFR) group a single 10 ml infusion at 20 ml/min. TIMI grade flow was assessed throughout the procedure and at sacrifice (day 14). MSCs distribution was analyzed in isolated hearts by 4.7T MRI. Delivery efficiency was quantified via fluorescent microsphere recovery using a magnetic separation technique and by light microscopy. Results: TIMI grade flow did not change following MI (all groups TIMI 3). However, following MSCs delivery only 18% (2/11) of control animals had TIMI 3 blood flow vs. 56% (5/9) in VHFR and 67% (4/6) in HFR (P = 0.03). As a consequence, 63% of control animals died within 24 hr, 33% in VHFR and none in HFR (P = 0.02). MSCs delivery in the infarct tissue did not differ between the groups (P = 0.06). Conclusions: A single MSCs infusion at 20 ml/min resulted in improved coronary blood flow and decreased mortality, without sacrificing delivery efficiency. © 2008 Wiley‐Liss, Inc.     

Studies on the improvement of leucodepletion performance of the Haemonetics MCS+ for production of leucodepleted platelet concentrate

With the implementation of universal leucodepletion, an in-line, negatively charged LRF6H leucodepleting filter became an essential part of the Haemonetics MCS+ plateletpheresis system. A larger-scale (968) study using the standard protocol revealed a 2.79% leucodepletion failure rate (standard < 5 2 10 6 leucocytes per adult therapeutic dose). Factors influencing the efficacy of the filter were investigated. The pH of the filtrate was 7.0, the temperature 28°C and filtration rate 80 ml/min. Reduction of the filtration rate to 30 ml/min (784 doses) reduced leucodepletion failure to 0.38%. Measurement of the leucocyte count, pre- and post-filtration of the platelet products, revealed that donations from 1% of donors contained substantially larger numbers of leucocytes in pre-filter samples (300-1500/ w l) than in control samples (35-70/ w l). This number tends to increase progressively with subsequent donations in these individuals, leading to leucodepletion failure, whilst peripheral leucocyte counts remain normal. The new continuous filtration protocol (version C) using a less impact filter LRF-XL and a lower (7 ml/min) head pressure was also effective but failure still occurred twice on one of the donors who persistently showed high pre-filter count. We conclude that leucodepletion failures in the Haemonetics system are related to both donor leucocyte (i.e., being light and non-adherent) and operational/filter performance.     

Pharmacokinetics and safety of roledumab, a novel human recombinant monoclonal anti-RhD antibody with an optimized Fc for improved engagement of FCγRIII, in healthy volunteers

Background and Objectives A human recombinant monoclonal anti‐RhD IgG may be useful to prevent RhD allo‐immunization. Roledumab is such an antibody with a glycosylation pattern optimized for biological activity. The objective of the study was to assess the safety and pharmacokinetics of roledumab in healthy RhD‐negative volunteers. Materials and Methods A total of 46 subjects received doses of 30–3000 μg i.v. of roledumab or placebo using a double‐blind escalating single‐dose design; 12 of these subjects also received 300 μg i.m. of roledumab. Subjects were followed for 6 months after administration. Serum roledumab concentrations were determined using flow cytometry. Results Fourteen treatment‐emergent adverse events related to treatment were reported in nine subjects, with no apparent difference in their frequency or nature after placebo or roledumab administration. No anti‐roledumab antibodies were detected. AUC_last increased from 4·4 ng/ml.day at 30 μg i.v. to 2257 ng/ml.day at 3000 g i.v. The t_½ ranged from 18 to 22 days, and the absolute bioavailability after i.m. administration was between 73% and 80%. Conclusion Roledumab is safe and well tolerated in healthy RhD‐negative volunteers and shows a pharmacokinetic profile similar to that of polyclonal anti‐RhD immunoglobulin.     

Purification of hepatocyte couplets by centrifugal elutriation

An initial preparation of rat hepatocytes containing approximately 30% couplets was enriched by centrifugal elutriation. Of the couplets loaded onto the elutriator, 87% were eluted at medium flow rates of 60 to 80 ml/min at a rotor speed of 1,100 rpm; cells eluted in this range maintained a viability of more than 95%. Peak fractions were enriched in couplets to 84.5% +/- 2.5%. After elutriation, couplets retained the ability to secrete fluorescent cholephiles into sealed canalicular vacuoles. The preparation can now be used in hepatobiliary and hepatotoxicity studies not possible with preparations in which they are minor components.     

Fibrin degradation product D-dimer induces the synthesis and release of biologically active IL-1β, IL-6 and plasminogen activator inhibitors from monocytes in vitro

Summary Disseminated intravascular coagulation, characterized by circulating fibrin(ogen) degradation products (FDP), is associated with both acute and chronic inflammatory conditions. Since the association of FDP with monocytes could influence the release of cytokines and other regulatory proteins with significant clinical ramifications, we have studied cytokine synthesis and release following the interaction of D-dimer (DD), a terminal degradation product of fibrin, with human monocytes in vitro.
Adherent peripheral blood monocytes were incubated with purified DD for 24 and 48 h and secreted or cell-associated IL-1β and IL-6 antigen levels and activity determined. DD (50 μg/ml) boosted the secretion of IL-1β antigen from median control levels of 659 pg/ml to 2704 pg/ml and that of IL-6 antigen from 806 pg/ml to > 3000 pg/ml at 48 h ( P <0.05). Similar increases in extracellular biologically active IL-1 and IL-6 were observed. Although DD increased cell associated IL-1 β antigen levels from median values of 188 to 1600 pg/106 cells and IL-6 antigen from 660 to 2215 pg/106 cells ( P < 0.05). cell-associated IL-1 functional activity decreased from control levels of 98 inhibitor units ml to 65 units/ml for cells exposed to DD. Secreted plasminogen activator inhibitor (PAI) bioactivity and PAI type 2 antigen levels were significantly increased following exposure of monocytes to DD. This may explain the decreased cell associated IL-1 activity observed in our study as PAI are known to inhibit biologically active membrane bound IL-1.
Our finding that DD enhances monocyte release of biologically active cytokines suggests the presence of positive feedback pathways for fibrinogen synthesis by hepatocytes. Furthermore, the association of monocytes with DD may potentiate localized coagulation processes by subsequent alterations in pericellular proteolysis.