Red Blood Cell Fraction Research Articles

Protective effects of sodium orthovanadate in diabetic reticulocytes and ageing red blood cells of Wistar rats.

The reticulocytes and the ageing red blood cells (RBCs) namely young (Y), middle-aged (M) and old RBCs (O) of female Wistar rats from different groups such as control animals (C), controls treated with vanadate (C + V), alloxan-induced diabetic (D), diabetic-treated with insulin (D + I) and vanadate (D + V), were fractionated on a percoll/BSA gradient. The following enzymes were measured - hexokinase (HK), glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R), glutathione-s-transferase (GST), alanine aminotransferase (AlaAT), aspartate aminotransferase (AsAT) and arginase in the hemolysates of all the RBCs fractions. Decreases in the activity of HK and AsAT by about 70%, arginase and GSH-Px by 30% in old RBCs were observed in comparison to reticulocytes of control animals. Increases in the activity of GSSG-R by 86%, AlaAT by more than 400% and GST by 70% were observed in old RBCs in comparison to reticulocytes of control animals. Alloxan diabetic animals showed a further decrease in the activities of HK in Y RBCs by 37%, M RBCs by 39% and O RBCs by 32%, GSH-Px activity in Y RBCs by 13%, M RBCs by 20% and O RBCs by 33% and GST activity in Y RBCs by 14%, M RBCs by 42% and O RBCs by 60% in comparison to their corresponding cells of control animals. An increase in the activity of all the enzymes studied was also observed in reticulocytes of diabetic animals in comparison to reticulocytes of controls. The GSSG-R activity was found to be increased in Y RBCs by 49%, M RBCs by 67% and O RBCs by 64% as compared to the corresponding age-matched cells of control animals. The activity of arginase also decreased in Y RBCs by about10%, M RBCs by 20% and O RBCs by 30% in comparison to the age-matched cells of control animals. A decrease in the activity of AsAT in Y and M RBCs by 30%, and O RBCs by 25% was observed in diabetic animals in comparison to the age-matched cells of control animals. The activity of AlaAT was found to be decreased by more than 10% in Y and M RBCs and 25% in O RBCs of diabetic animals in comparison to the age-matched cells of control animals. Insulin administration to diabetic animals reversed the altered enzyme activity to control values. Vanadate treatment also reversed the enzyme levels except for that of GST in old cells.

Study of phagocytosis of senescent erythrocytes in young and elderly individuals.

There is a decrease in the percentage contribution of a heavy density fraction of red blood cells to whole blood with increasing age. The aim of this study was to investigate in the young and elderly the interaction between monocytes and different erythrocyte suspensions: senescent red blood cells, erythrocytes stored with or without serum, and desialylated red blood cells. The results obtained with senescent red blood cells and erythrocytes stored with serum show the involvement of autologous IgG in the selective removal of erythrocytes. These values were higher in elderly individuals, indicating that this process increases with age. Our observation suggest that desialylation is not involved in the increased removal of erythrocytes observed in elderly individuals.

Lipoic Acid as an Antioxidant in Mature Thoroughbred Geldings: A Preliminary Study

alpha-Lipoic acid (LA) has demonstrated antioxidant effects in humans and laboratory animals. The objective of this study was to determine whether the effects of LA are similar in horses. Five Thoroughbred geldings were supplemented with 10 mg/kg/d DL-alpha-lipoic acid in a molasses and sweet feed carrier and five received only the carrier as a placebo (CON). Blood samples were obtained at baseline (0 d), after 7 and 14 d of supplementation, and 48 h postsupplementation (16 d). Blood fractions of red and white blood cells (RBC and WBC, respectively) and plasma were analyzed for glutathione (GSH), glutathione peroxidase (GPx) and total plasma lipid hydroperoxides (LPO). An experienced veterinarian observed no adverse clinical effects. Plasma LPO baselines differed between groups (P = 0.002). When covariates were used, there was a decrease over time in the LA group (P = 0.015) and concentrations were lower in the LA group than in the CON group at 7 and 14 d (P = 0.022 and P = 0.0002, respectively). At baseline, GSH concentration was 69 +/- 7 in WBC and 115 +/- 13 mmol/mg protein in the RBC, with no differences resulting from either time or treatment. The GPx activity was 47 +/- 4 and 26 +/- 5 U/g protein at baseline WBC and RBC, respectively, with a lower concentration in the LA group's WBC at 7 (P = 0.019) and 14 d (P = 0.013). The results show that 10 mg/kg LA had no evident adverse effects, and moderately reduced the oxidative stress of horses allowed light activity. These findings encourage studying of LA in horses subjected to strenuous exercise.

Effects of High Hydrostatic Pressurised Treatment on Porcine Blood Red Cells Fraction

The aim of this study was to determine the optimum conditions for the application of high hydrostatic pressure treatment (HHP) on the red blood cells (RBC) fraction from porcine blood. HHP was applied under varying conditions of pressure (400, 450 and 500 MPa), temperature (5, 20 and 40 °C) and time (5, 15 and 30 min), and the effects on the microbiological quality and certain functional characteristics were evaluated. High pressure affected the rheology of liquid RBC fraction. Treatments at 450 and 500 MPa produced effects ranging from the formation of a thick paste to the solidification of the RBC fraction, which would avoid any further processing. The effects of HHP treatments on microbiological counts and the growth capacity of the surviving microorganisms depended on the time and the temperature at which the treatment was performed. Pressurisation at 400 MPa for 15 min at 20 and 40 °C proved to be the most effective conditions for the improvement of the microbiological quality. L*, a and b colour parameters increased significantly after pressurisation at 400 MPa and the RBC fraction became lighter, redder and yellower than the nonpressurised samples. (A)

Implantation of human menstrual endometrium into nude mice

Objective: In the present study, human menstrual endometrium was transplanted into 66 nude mice in order to 1) examine the early stages of evolution of the endometriotic lesion, 2) to test whether the cellular, seric and red blood cell fractions from the menstrual effluent may have an influence on the development of endometriotic lesions in this animal model and 3) to investigate whether adhesion occurs on intact peritoneum or at the site of incision. Design: In a first series of experiments (n = 10), the timing of adhesion of menstrual endometrial tissue was analyzed by removal of the transplant on days 1, 3 and 5. In a second series (n = 25), 5 different fractions of menstrual effluent were transplanted by minilaparotomy performed on the ventral midline: 1) effluent as a whole, 2) seric fraction, 3) cellular + seric fractions 4) cellular + red blood cell fractions and 5) cellular fraction only. In a third series (n = 31), the same fractions, except the seric, were deposited by an incision on the ventral right side in order to test if the site of adhesion is influenced by the healing area. Materials/Methods: Menstrual effluent was obtained from women on day 2 or 3 of the cycle, by aspiration. The effluent was centrifuged and the seric fraction collected. Endometrial tissue was separated from red blood cells following two centifugations on Ficoll-Paque gradients and both fractions were resuspended in serum-free medium. Endometrial cell viability was checked by trypan blue exclusion. Minilaparotomy was performed in order to inject the various fractions into the peritoneal cavity of nude mice. Removal of the transplants was performed by laparotomy. Adhesion of endometrial cells and the early stages of development of endometrial lesions were studied morphologically and immunohistochemically. Staining of iron deposits and Ki67, a marker of cell proliferative activity, was carried out. The χ2 test was used for statistical analysis. Results: As early as day 1, stromal cells were found to adhere to the peritoneal mesothelium. A progressive reorganization of epithelial and stromal cells was observed. On day 5, typical endometriotic lesions, still with some necrotic areas but also large areas of normal-looking stroma and cystic glands with flattened or columnar epithelium, were present in 75% of cases. An increased rate was found when either total menstrual effluent or the cellular fraction associated with the red blood cell fraction was injected (82% and 89% respectively), when compared with rates observed after injection of the cellular fraction alone (50%) or associated with the soluble fraction (75%). A significant increase (p < 0.02) in glandular proliferative activity was observed on day 5 compared to values observed on days 1 and 3. The proliferative activity was similar whatever the injected fraction. Iron deposits were observed independently of the fraction injected. There was a tendency towards increased hemosiderin deposits when red blood cells were injected together with the cellular fraction. Implantation occurred on incision sites as well as intact mesothelium whether laparotomy was performed medially or on the right side. Conclusions: Implantation of endometrial cells can occur on intact mesothelium but may be favored by the presence of red blood cells as well as the occurrence of a peritoneal injury. Supported By: Grant 7.4530.98 from the “Fonds National de la PRecherche Scientifique de Belgique.”

Characterization of the phosphatidylserine-exposing subpopulation of sickle cells

AbstractPhosphatidylserine (PS), exclusively present in the inner monolayer of the normal red blood cell (RBC) membrane, is exposed in subpopulations of sickle cells. PS-exposing RBCs were found predominantly among the densest and the very light sickle cells. Within the light RBC fraction, PS exposure was found on reticulocytes, transferrin receptor–expressing reticulocytes, and mature RBCs. The last subset contained low-density valinomycin-resistant RBCs, previously shown to have high Na+ and low K+content. This subpopulation contained the highest percentage of PS-exposing cells. The PS-exposing sickle cells did not show the sustained high cytosolic Ca++ levels that have been shown to activate scramblase activity. Data from this study indicate that PS exposure can occur at different stages in the life of the sickle RBC and that it correlates with the loss of aminophospholipid translocase activity, the only common denominator of the PS-exposing cells. The additional requirement of scramblase activation may occur during transient increases in cytosolic Ca++.

Derivated fetal haemoglobin as a marker for red cell age in the human fetus reflecting stimulated or impaired red blood cell production.

We have determined whether derivated fetal haemoglobin (dHbF, consisting of glycated and acetylated HbF) can be used as a cell age marker for fetal red blood cells (RBCs). Cord blood was obtained between 19 and 39 weeks of gestation from 28 alloimmunised anaemic fetuses (23 RhD+ and 5 Kell) and from 20 non-anaemic fetuses and newborns (controls). Density gradient centrifugation was applied to 36 samples (20 RhD+, 15 controls and 1 Kell) to obtain fractions of increasing cell age. Blood samples were used for measurements of mean cellular volume (MCV), mean cell haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), pyruvate kinase activity (PK) and derivated fetal haemoglobin (dHbF) by cation-exchange HPLC. Reticulocytes were counted only in the whole blood samples. In all density gradient separated RBC fractions, the values for MCV, MCH and PK activity decreased and those of MCHC and dHbF increased with increasing density (equivalent to increasing cell age). The mean density was lower for RBCs of the anaemic RHD group (1.072+/-0.007 g/ml) than for the non-anaemic controls (1.077+/-0.005 g/ml) (p<0.05) The RBC density of the Kell sensitised fetus did not differ from those of the controls. In the control group, the values of the cell age markers in whole blood changed significantly with the gestational age, showing an increase of mean age of the erythrocyte population. The best linear relationship was found for dHbF (y=6.28+0.17*weeks; r=0.84; p<0.001). In the anaemic RhD+ fetuses, the RBC age markers did not change with gestational age; the dHbF percentages were lower, and the MCV, MCH, PK values and the reticulocyte counts were higher than in the controls (0.05<p<0.001). The dHbF values of the Kell sensitised fetuses were above (p<0.01) and the reticulocyte counts were below normal (p<0.05) for gestational age. For the anaemic fetuses, a significant number of the dHbF values (86%) and of the reticulocyte counts (78%) differed from the values of the controls (p<0.01). The dHbF percentages in RhD+ fetuses showed the best correlation with the Hb deficit, which is a measure for anaemia (r=-0.81, p<0.0001). We conclude that the percentage derivated HbF may indicate whether the RBC production is normal for gestational age. It may in that sense reflect stimulated or impaired erythropoiesis in alloimmunised haemolytic anaemia.

Effect of arachidonic acid-rich oil on lipids and arachidonate metabolites in ethanol- treated rats

The effects of dietary arachidonic acid-rich oil (AAoil) on lipids and arachidonate metabolites in the liver and plasma were evaluated in ethanol-treated rats. Rats were fed a purified diet containing 10% weight of lard or AAoil for 14 days. Ethanol was administered by gavage at a single daily dose of 3 g/kg body weight. Comparing with the lard group, a decrease was observed in liver fatty vacuoles in the AAoil group. Plasma 6-keto-prostaglandin (PG) F1 α and thromboxane (TX) B2levels and the 6-keto-PGF1 α/TXB2ratio increased significantly in the AAoil group. Liver 6-keto-PGF1 α also increased but not leukotriene B4in the AAoil group. In the phospholipid fraction of liver tissue, plasma and red blood cells, arachidonic acid (20:4n-6) and docosatetraenoic acid (22:4n-6) increased and oleic acid (18:1n-9) and linoleic acid (18:2n-6) decreased significantly in the AAoil group compared with the lard group. These observations suggest that AAoil supplementation reduces liver injury of ethanol-treated rats, although longer observation will be necessary for confirmation.

Thiopurine methyltransferase activity in the Jewish population of Israel

6-Mercaptopurine is used therapeutically for its immunosuppressant and cytotoxic properties. It is deactivated by thiopurine methyltransferase (TPMT), which shows genetic polymorphism in many populations. In North American populations, TMPT activity exhibits a trimodal activity pattern. In Oriental populations, TPMT shows almost a unimodal pattern of activity. The purpose of the present study was to assess the activity of TPMT in a Jewish male population sample in Israel. The study was approved by the Israeli Ministry of Health. Blood samples of 2.5 ml were collected in heparinized tubes from 134 males. The red blood cell (RBC) fraction of each individual was washed and hemolyzed. TPMT activity in the RBC hemolysate was determined using a radioactive assay with tritiated S-adenosyl methionine as a methyl donor. The activity of TPMT ranged from 3.2 nmol/h/ml to 42.9 nmol/h/ml packed RBCs with mean and median activities of 18.6 nmol/h/ml and 17.9 nmol/h/ml packed RBCs, respectively. The distribution frequency of TPMT was very close to the unimodal by analysis of normal distribution. The pattern of distribution of TPMT in the Jewish population of Israel is closer to that of East Asian populations than European and North American populations. This observation may have relevance for the usage of 6-mercaptopurine and azathioprine as therapeutic agents in the Jewish population.

Quantitation of suspensions (MESED). Application of MESED-gC/MS in the quantitation of ifosfamide mustard in erythrocytes, plasma, and plasma water

Compounds associated with particles in suspension are difficult to quantitate in the analysis of drugs in red blood cells. Breakthrough experiments were performed for the determination of a mustard gas related substance, ifosfamide mustard, originating from the anticancer agent ifosfamide. It is shown that the use of the measurement of sediment (MESED) instrument enables a comparison of ifosfamide mustard levels in red blood cells, total plasma, and plasma water. Red blood cell sediments were analysed by GC/MS together with plasma and plasma water extracts. The MESED maintains a direct relationship between the volume and weight of red blood cell sediments without disturbance of the equilibrium of a substance between the cells and the plasma, the latter being the suspending fluid. The MESED GC/MS combination has been applied successfully in clinical settings, estimating the importance of the red blood cell, plasma, and plasma water fractions in the presentation of ifosfamide mustard to other tissues by the blood. The presence of ifosfamide and ifosfamide mustard in red blood cells, total plasma, and plasma water was confirmed by negative chemical ionisation (NCI) mass spectrometry of the trifluoroacetylated derivatives following capillary GC. The NCI-Mass spectra of ifosfamide, ifosfamide mustard, and the internal standard cyclophosphamide provide specific fragment ion masses, which can be used in single or double ion monitoring of the substances for quantitation of cancer patient samples.

In vitro characterization of the occurrence of hemolysis during extracorporeal blood circulation using a mini hemodialyzer.

A minimodule dialyzer was made of polyacrylonitrile (PAN) hollow fibers and tested in an in vitro circuit under varying flow rates and blood volumes. Hemolysis occurred in the open circuit in the inlet and outlet chambers. The module was measured to investigate the hemolysis effect during dialysis. The results show that hemolysis ratio (HR) increased with blood flow rate. When the blood speed was increased from 27 to 70 cm/sec, the damaged fraction of red blood cells increased from 0.8% to 1.5%. Shearing in the hollow fiber contributed approximately 13% of the overall HR. Approximately 55% of the overall HR occurred at the inlet and outlet chambers. With increasing blood volume, the contacting surface area per unit blood volume (S/V) and the frequency of pump squeezing of red cells were decreased; hence the damage to the red blood cells and thus reduction of HR. When S/V was 2 cm(-1), the damaged fraction of red cells was approximately 0.7%. In addition, the tubing and connectors of the circuit caused further damage to the red blood cells. By referring to the linear velocity and blood volume, we can predict the HR in an actual protocol from the results of this experiment. This should improve the quality of hemodialysis and benefit the patient.

Dose-dependent effects on tissue distribution and metabolism of dimethylarsinic acid in the mouse after intravenous administration

Most mammals methylate inorganic arsenic to dimethylarsinic acid (DMA). This organic arsenical causes organ-specific toxicity and is a multi-organ tumor promoter. The objective of this study was to examine whether dose could affect the distribution and metabolism of DMA. Female B6C3F1 mice (3–4/time point) were administered 1.11 or 111 mg/kg of DMA (1 μCi of [ 14C] or unlabeled) intravenously and killed serially (5–480 min). Blood was separated into plasma and red blood cell fractions and liver, kidney and lung were removed, weighed and homogenized. Tissue samples were oxidized and analyzed for DMA-derived radioactivity. Blood and several organs of the non-radioactive DMA-treated animals were digested in acid and analyzed by hydride generation atomic absorption spectrophotometry for DMA and metabolites. Concentration–time profiles showed a biexponential decrease of DMA-derived radioactivity in all tissues examined. Kidney had the highest concentration (1–20% dose/gm) of radioactivity of all tissues up to 60 min post-administration. Concentration of radioactivity was greater in plasma than red blood cells at 5 and 15 min and then was similar for the remaining time points. A dose-dependent effect on the concentration of radioactivity was observed in the lung. The retention of radioactivity in the lung was altered compared with liver and kidney, with a much longer t 1 2 β and a disproportionate increase in area under the curve with increased dose. No methylated or demethylated products of DMA were detected in blood or any organ up to 8 h post-exposure. The dose-dependent distribution of DMA in the lung may have a role in the toxic effects DMA elicits in this organ.

Disposition of propofol between red blood cells, plasma, brain and cerebrospinal fluid in rabbits.

The disposition of propofol in the blood and brain of New Zealand rabbits was studied in three groups of six rabbits. One group received a single anaesthetic dose; a second group received a 1-h infusion; and a third group was studied after the rabbits were judged to have recovered from a 1-h infusion. There was a high concentration of propofol in the red blood cell fraction and in the brain, however, the red blood cell concentration largely exceeded the one found in the brain in all groups of animals. This is consistent with the high fat solubility of diisopropylphenol. The possible effects of propofol sequestered in red blood cells is discussed.

Disposition of propofol between red blood cells, plasma, brain and cerebrospinal fluid in rabbits

The disposition of propofol in the blood and brain of New Zealand rabbits was studied in three groups of six rabbits. One group received a single anaesthetic dose; a second group received a 1-h infusion; and a third group was studied after the rabbits were judged to have recovered from a 1-h infusion. There was a high concentration of propofol in the red blood cell fraction and in the brain, however, the red blood cell concentration largely exceeded the one found in the brain in all groups of animals. This is consistent with the high fat solubility of diisopropylphenol. The possible effects of propofol sequestered in red blood cells is discussed.

The Low Potential for Drug Interactions with Zanamivir

The objective of this study was to assess the potential of zanamivir, a specific inhibitor of influenza A and B virus neuraminidase, to interact with other coadministered therapies in the clinical setting. Potential interactions with zanamivir were examined in a series of in vitro and in vivo model systems. The expression of microsomal cytochrome P450 (CYP) isoenzymes was examined after daily treatment of rats with intravenous zanamivir. The ability of zanamivir to inhibit the metabolism of CYP probe substrates was studied in human liver microsomes. The binding of zanamivir to human and animal red blood cell fractions and plasma proteins was measured. Finally, the effect of commonly coadministered drugs on the ability of zanamivir to inhibit viral replication in vitro was tested. Zanamivir had no effect on the expression of microsomal CYP isoenzymes after daily intravenous treatment of rats with zanamivir 1, 9 or 90 mg/kg for 5 weeks. Zanamivir at concentrations up to 500 mumol/L (150 mg/L) had no effect on the metabolism of the CYP probe substrates bufuralol, chlorzoxazone, coumarin, ethoxyresorufin, mephenytoin, midazolam, phenacetin and tolbutamide by human liver microsomes. The binding of zanamivir 0.05 to 10 mg/L to human, dog and rat red blood cells and plasma proteins was low. The in vitro potency of zanamivir against influenza virus in Madin Darby canine kidney cells was not adversely affected by aspirin (acetylsalicylic acid) 1.2 mmol/L, paracetamol (acetaminophen) 6.6 mmol/L, ibuprofen 243 mumol/L, phenylephrine 6 mmol/L, oxymetazoline 380 mumol/L, promethazine 35 mumol/L and co-amoxiclav (amoxicillin-clavulanic acid) 1.66 mmol/L. These data suggest the following: (i) there is no theoretical basis for expecting metabolic interactions between zanamivir and other coadministered compounds; (ii) zanamivir is unlikely to interact with coadministered compounds that are protein bound; and (iii) commonly coadministered drugs will not interfere with the antiviral activity of zanamivir in vivo. Although none of these in vitro or in vivo studies were exhaustive, and although none were performed in humans, all the data are consistent with zanamivir having a very low potential for interactions with coadministered drugs in the clinical setting.

In VitroReduction Kinetics of Hexavalent Chromium in Human Blood

This study examines time- and concentration-dependent changes in distribution of hexavalent chromium [Cr(VI)] and total chromium [Cr-(TOT)] in reconstituted human blood following addition of potassium dichromate. Fresh human blood stabilized with EDTA was obtained from human volunteers soon after meal ingestion and at 2.5 h after a light meal (herein defined as “2.5-h fasted” conditions). Cr(VI) spiked into plasma under 2.5-h fasting conditions at 3.0–12.5 μg/L was stable for several hours, indicating a lack of appreciable reductive capacity in isolated plasma. Spiked plasma following a recent meal exhibited immediate but variable reduction of Cr(VI) up to 300 μg/L. When the spiked plasma was recombined with the red blood cell (RBC) fraction, rapid reduction occurred in both the plasma and the RBC fractions based on measurement of Cr(VI) and Cr(TOT). The data indicate that plasma reduction capacity is enhanced by a recent meal, but may be overwhelmed at Cr(VI) concentrations between 2000 and 10,000 μg/L. These data also suggest that the RBC fraction apparently has the capacity to reduce Cr(VI) at concentrations in blood up to 15,000 μg/L, and that the rate of Cr(VI) uptake into RBCs may not exceed the rate of intracellular reduction at these concentrations.

Erythrocyte membrane band 3 profile imposed by cellular aging, by activated neutrophils and by neutrophilic elastase

The aim of this study was to evaluate in vitro the modifications in the profile of erythrocyte membrane protein band 3 (monomer, aggregates and fragments) imposed by cellular aging, by activated neutrophils (AN) and by neutrophilic elastase. The modifications imposed by cellular aging were evaluated in fractions of less dense and dense red blood cells (RBCs). To evaluate the changes imposed by AN, suspensions of RBCs and AN were used, in a ratio similar to or higher than that described elsewhere for individuals post-myocardial infarction (Santos-Silva A et al. Atherosclerosis 1995; 116:199–209). Neutrophilic elastase was used at increased concentrations. We found that the modifications imposed upon band 3 profile by AN and neutrophilic elastase were significantly correlated with AN and elastase concentration, and were similar to those presented by cellular aging. We propose band 3 profile as a useful cumulative marker of oxidative and/or proteolytic stress conditions, namely those arising from normal RBC aging or from an accelerated aging process imposed by stressful situations, such as inflammatory diseases.

Quantitative measurement of protein S gene expression by reverse transcription‐competitive PCR method; determination of mRNA level in fractionated human peripheral blood cells and various cultured cell lines

AbstractWe developed the measurement system for human protein S mRNA (cDNA) by a reverse transcription‐competitive polymerase chain reaction (RT‐competitive PCR) method with a compact digital camera. The sensitivity of the method was 100,000 fold more sensitive than the traditional northern blot analysis. We confirmed the accuracy and usefulness of the system by measuring the absolute values of protein S mRNA in fractionated peripheral blood cells, namely platelets, mononuclear cells, granulocytes and red blood cell from healthy human and six different cultured disease cell lines tested, namely, lung carcinoma, stomach carcinoma, hepatoma, highly differentiated hepatoma, promyelocytic leukemia and monocytic leukemia. The amount of protein S mRNA in the platelet rich fraction (6.89×10‐19 mol/μg total RNA) was the largest of all fractions tested and that in the mononuclear cells was the second largest. In the fractions of granulocytes and red blood cells, no protein S mRNA was detected. The detected values in five cell lines ranged from approximately 5.6×10‐19 to 97.2×10‐19 mol/μg total RNA. The content was reduced in the order, highly differentiated hepatoma&gt;hepatoma&gt;lung carcinoma&gt;stomach carcinoma&gt;monocytic leukemia. Promyelocytic leukemia cell line had no expressed protein S mRNA. Thus, the assay system described here was sensitive, reproducible, simple and useful for the quantitative measurement of an infinitesimal level of protein S mRNA to characterize normal and/or patho‐physiological states of human blood and somatic cells.

Comparative Intravenous Toxicity of Cisplatin Solution and Cisplatin Encapsulated in Long-Circulating, Pegylated Liposomes in Cynomolgus Monkeys

The toxicity of cisplatin encapsulated in pegylated, long-circulating liposomes (SPI-077) was compared with nonliposomal cisplatin in male and female cynomolgus monkeys (n = 2-4 per sex per group) treated with intravenous infusions of 2.5 or 25 mg/kg SPI-077, 2.5 mg/kg cisplatin, placebo liposomes, or saline once every 3 weeks for total of five treatments. All animals survived until scheduled necropsy at 3 days after the final treatment or after a treatment-free 4-week recovery period. Emesis occurred after each treatment in all cisplatin-treated monkeys, but only once in one monkey treated with high-dose SPI-077. Dose-related mild decreases in red blood cell (RBC) count, hemoglobin, and hematocrit to or slightly below low normal range occurred in the high-dose SPI-077 and placebo liposome treatment groups after each treatment, with partial to complete recovery between treatments and no signs of correlating bone marrow toxicity. Decreases were similar in cisplatin-treated monkeys, but resolved only slightly between treatments and after the end of treatment (continuing to decrease in females) and were accompanied by bone marrow hypocellularity. Indirect, but not direct, bilirubin levels were cyclically elevated in the high-dose SPI-077 and placebo-treated animals, but not in the other treatment groups. Levels had either fully resolved or were near baseline and/or saline group values prior to the next treatment. Serum cholesterol levels were cyclically increased in SPI-077- and placebo liposome-treated animals, and minimally increased numbers of foam cells were seen in the liver, spleen, kidney, and other organs; both were considered related to the lipid dose administered. Cisplatin-treated monkeys exhibited sensory polyneuropathy and moderate irreversible toxic tubular nephrosis, but no neuropathy or nephrotoxicity was seen in either SPI-077 treatment group. Microscopically, treatment-related cell death was seen in dorsal root ganglia (DRG), affecting 15% of the cells in cisplatin-treated animals, compared to 8 and 12% in the low- and high-dose SPI-077 treatment groups. Neither drug was ototoxic. In summary, repeated administration of SPI-077 produced minimal, reversible effects related to the lipid dose administered, mostly limited to the 25 mg/kg dose group. The most notable effects in this group were cyclical decreases in hematology parameters thought to be related to increased recycling of a small fraction of RBCs and limited cell death in the DRG in the absence of any neurophysiological changes. Animals treated with a 10-fold lower dose of cisplatin (2.5 mg/kg), in contrast, exhibited myelo-, nephro-, and neurotoxicity, including sensory neuropathy, and were emetic after every dose. The SPI-077 liposomal formulation of cisplatin may provide a less toxic alternative to standard cisplatin solution.

Thalassaemia trait, red blood cell age and oxidant stress: effects on Plasmodium falciparum growth and sensitivity to artemisinin

Knowledge of innate mechanisms of protection against malaria could be used to bolster the existing limited treatments. Oxidant stress may play a role in the protective mechanism and the effect of red blood cell (RBC) age has recently been recognized. This study investigated the role of oxidant stress in the protection against malaria in thalassaemic trait RBC (α and β) using an experimental approach which controlled for cell age. ‘Young’, ‘intermediate’ and ‘old’ RBC obtained by Percoll® fractionation and whole blood were used to set up malaria cultures. Antioxidants (vitamin E and dithiothreitol) and prooxidants (riboflavin, menadione and artemisinin) were added to modulate oxidant stress effect. Antioxidants improved parasite growth. The degree of improvement was significantly greater with increasing RBC age ( P < 0·0001), and relatively greater in thalassaemic RBC ( P < 0·0001). Pro-oxidants had a parasiticidal effect. With the exception of the ‘old’ RBC fraction, the median inhibitory concentration (IC 50) for riboflavin and menadione was significantly higher in normal RBC. In contrast, the IC 50 for artemisinin was significantly higher in ‘old’ thalassaemic RBC but was similar in the ‘young’ and ‘intermediate’ fractions and whole blood. These findings suggest that oxidant stress plays a role in mediating the protection against malaria in thalassaemic RBC. Vitamin E and other antioxidant supplementation could feasibly exacerbate clinical malaria. Conversely, pro-oxidant agents could act as useful adjuvants to therapy. It is important to confirm the reduced sensitivity to artemisinin in ‘old’ thalassaemic trait RBC, as such an effect may promote selective pressure for the emergence of resistant parasite strains with widespread use of artemisinin.