Human erythroid cells are affected by aluminium. Alteration of membrane band 3 protein

There is evidence that anaemia is associated with aluminium (Al). We have already reported on the sensitivity to Al, showed by erythroid cell populations of animals chronically exposed to the metal. In order to investigate whether Al could also affect human cells, experiments were carried out both on immature and mature human erythroid cells. Erythroid progenitors (CFU-E, colony-forming units-erythroid) concentrated from human peripheral blood were cultured in an Al-rich medium under erythropoietin stimulation and their development analysed. Human peripheral erythrocytes were aged in the presence of Al. Cells were examined using scanning electron microscopy, and membrane proteins analysed by polyacrylamide gel electrophoresis with sodium dodecyl sulphate and immunoblotting. The development of the Al-treated progenitors was 8750/6600-9200 CFU-E/10⁶ cells, a significantly lower median value (P<0.05) than that showed by non-treated cells (12?300/11?200-20?700 CFU-E/10⁶ cells). Erythrocyte morphological changes were induced by Al during the in vitro ageing. The cells lost their typical biconcave shape, turning into acanthocytes and stomatocytes. Simultaneously, an increased membrane protein breakdown compatible with band 3 degradation was detected. Besides, Al was found within the cells and attached to the membrane. The present in vitro results suggest that Al may disturb human erythropoiesis through combined effects on mature erythrocytes and cellular metabolism in late erythroid progenitors.     

Human erythroid cells are affected by aluminium. Alteration of membrane band 3 protein.

There is evidence that anaemia is associated with aluminium (Al). We have already reported on the sensitivity to Al, showed by erythroid cell populations of animals chronically exposed to the metal. In order to investigate whether Al could also affect human cells, experiments were carried out both on immature and mature human erythroid cells. Erythroid progenitors (CFU-E, colony-forming units-erythroid) concentrated from human peripheral blood were cultured in an Al-rich medium under erythropoietin stimulation and their development analysed. Human peripheral erythrocytes were aged in the presence of Al. Cells were examined using scanning electron microscopy, and membrane proteins analysed by polyacrylamide gel electrophoresis with sodium dodecyl sulphate and immunoblotting. The development of the Al-treated progenitors was 8750/6600-9200 CFU-E/10(6) cells, a significantly lower median value (P<0.05) than that showed by non-treated cells (12300/11200-20700 CFU-E/10(6) cells). Erythrocyte morphological changes were induced by Al during the in vitro ageing. The cells lost their typical biconcave shape, turning into acanthocytes and stomatocytes. Simultaneously, an increased membrane protein breakdown compatible with band 3 degradation was detected. Besides, Al was found within the cells and attached to the membrane. The present in vitro results suggest that Al may disturb human erythropoiesis through combined effects on mature erythrocytes and cellular metabolism in late erythroid progenitors.     

In vivo effect of aluminium upon the physical properties of the erythrocyte membrane

Aluminium (Al (III)) is a metal with no biological function. Its organic accumulation can lead to toxic effects. To elucidate the in vivo effect of Al (III) upon the rheological properties of the erythrocyte membrane, male adult Wistar rats have been submitted to periodical injections of Al(OH)3 during three months. Significant decreases in haematocrit (34+/-0.37% versus 36+/-0.20%, p<0.0001) and blood haemoglobin concentration (10.7+/-0.15 g/dl versus 12.3+/-0.49 g/dl, p<0.005) have been found. Haemolysis curves shifted towards the left, indicating that erythrocytes became more resistant to hypotonic haemolysis. Significant increments in rigidity index (29.6+/-1.59 versus 9.2+/-0.40, p<0.0001), relative viscosity at native haematocrit (3.6+/-0.03 versus 3.5+/-0.03, p<0.04), and relative viscosity at standard haematocrit (4.5+/-0.06 versus 3.9+/-0.05, p<0.0001) have been observed. The decrease in the erythrocyte aggregate size (1.6+/-0.01 versus 1.7+/-0.01, p<0.002) and the aggregation rate (0.5+/-0.02 versus 0.6+/-0.03, p<0.002) indicated a significantly dropped aggregability process. In conclusion, Al (III) disorganised the erythrocyte membrane by altering its mechanical properties, suggesting a reduction of the middle life of circulating erythrocytes, which could play a major role in the anaemia of these animals.     

Interference of aluminium on iron metabolism in erythroleukaemia K562 cells

It has been suggested that aluminium (Al) has a deleterious effect on erythropoiesis. However, there is still uncertainty as to its action mechanism. The present work was designed to determine how Al could affect the iron (Fe) metabolism in the human erythroleukaemia cell line K562. These cells, that express surface transferrin receptors (TfRs), were induced to erythroid differentiation by either haemin or hydroxyurea in 72 h cultures in media containing apotransferrin (apoTf). In the presence of aluminium citrate, the number of benzidine-positive cells decreased 18% when the cultures were induced by haemin, and 30% when hydroxyurea was the inducer. Cell viability was always unaffected. From competition assays, surface binding of 125I-Tf-Fe2 was found to be inversely related (p < 0.05) to Tf-Al2 concentration (from 2.5 to 10 nM). The dissociation constants (Kd) of the binding reaction between TfRs and the ligands Tf-Fe2 and Tf-Al2 were calculated. Kd values of the same order of magnitude demonstrated that TfR has a similar affinity for Tf-Fe2 (Kd = 1.75 x 10(-9) M) and Tf-Al2 (Kd = 1.37 x 10(-9) M). The number of surface TfRs, measured by kinetic 125I-Tf-Fe2 binding assays, was higher in induced cells cultured in the presence of Al. Nevertheless, in spite of the inhibition of cell haemoglobinization observed, 59Fe incorporation values were not different from those measured in control cultures for 72 h. As a consequence, it can be suggested that cellular Fe utilisation, and not Fe uptake, might be the main metabolic pathway impaired by Al.     

Bioimpedance spectroscopy parameters of suspensions of young and old erythrocytes

The bioimpedance spectroscopy (BIS) parameters of the suspensions of young and old erythrocytes were studied. The separation of the erythrocytes by age was made by density gradient. The BIS parameters: extracellular (Re) and intracellular (Ri) fluid resistance, characteristic frequency (Fchar), cell membranes capacitance (Cm) and Alpha parameter of concentrate suspensions of young and old erythrocytes were measured on the BIA analyzer ABC-01 "Medass" in the frequency range 5-500 kHz. It was found that Re (300.4 +/- 30.0 Ohm and 261.2 +/- 21.8 Ohm for old and young respectively, p < 0.05), Ri (86.6 +/- 9.1 Ohm and 73.4 +/- 7.3 Ohm for old and young respectively, p < 0.001) and Alpha (0.305 +/- 0.003 and 0.302 +/- 0.001 for old and young respectively, p < 0.05) of the old erythrocytes suspensions were higher, than of the young one, and Fchar (308.3 +/- 42.0 kHz and 347.4 +/- 48.0 kHz for old and young respectively, p <0.05) and Cm (99.3 +/- 10.1 pF and 112.8 +/- 6.3 pF for old and young respectively, p < 0.01) of the old erythrocytes were lower, than of the young one. The found differences between electrical properties of the suspensions of young and old erythrocytes were obviously determined by the alterations of the red blood cells during aging (growth of intracellular hemoglobin concentration, erythrocytes rapprochement because of diminishing of surface negative charge, increase of red blood cell sphericity and cell membrane permeability for ions). Thus the BIS parameters are related to the erythrocyte aging.     

Ferrokinetics and erythropoiesis in mice after long-term inhalation of benzene

Ferrokinetics and erythropoiesis were examined in mice exposed for 6 or 7 weeks to an airborne concentration of 300 ppm of benzene, for 6 h per day, and 5 days per week. Ferrokinetic indicators showed only a slightly enhanced production of haeme and erythrocytes in the spleen (133% +/- 18% and 122% +/- 17%, respectively). Production did not change in the femoral marrow; a decline of CFU-C, BFU-E and especially CFU-E (34% +/- 8%) took place there and a shift of cellularity into less mature developmental classes in the erythroblast compartment, without this compartment as a whole being damaged. The erythrocytes produced have an enhanced MCV (109% +/- 0%) and MCH (109% +/- 1%) with an unchanged MCHC; their concentration in blood sank to 87% +/- 1%. The absolute reticulocyte count rose to 160% +/- 16%. 59Fe incorporation into the liver declined far below the level attributable to decreased accessibility of the tracer (84% +/- 4%). A shortening of the life span of late erythroblasts and circulating erythrocytes was deduced from these findings and methodological problems related to some of the seemingly controversial findings are discussed.     

Survival of erythroid burst-forming units and erythroid colony-forming units in canine bone marrow cells exposed in vitro to 1 MeV neutron radiation or X rays

Conditioned media (CM) from allogeneic stimulated cultures of light density cells (less than 1.08 g/cm3) from the peripheral blood of normal dogs were used to stimulate the growth of erythroid burst-forming units (BFU-E) in bone marrow from normal dogs. Maximum numbers of BFU-E were obtained when 5% (vol/vol) 3 X CM and 2 U/ml erythropoietin were added to plasma clot cultures of bone marrow cells. In addition, the radiation sensitivity (D0 value) was determined for CFU-E and for BFU-E in bone marrow cells exposed in vitro to 1 MeV fission neutron radiation or 250 kVp X rays. BFU-E were more sensitive than CFU-E to the lethal effects of both types of radiation. For bone marrow cells exposed to 1 MeV neutron radiation, the D0 for CFU-E was 0.27 +/- 0.01 Gy, and the D0 for BFU-E was 0.16 +/- 0.03 Gy. D0 values for CFU-E and BFU-E were, respectively, 0.61 +/- 0.05 Gy and 0.26 +/- 0.09 Gy for cells exposed to X rays. The neutron RBE values for the culture conditions described were 2.3 +/- 0.01 for CFU-E and 1.6 +/- 0.40 for BFU-E.     

Determination of iron, copper, zinc, magnesium and selenium in plasma and erythrocytes in neurosurgical patients.

A direct method for determination of Fe, Cu, Zn, Mg and Se in erythrocytes was developed. The aim of the present study was to establish a method for examining perioperative levels of the above mentioned elements simultaneously in erythrocytes and plasma by atomic absorption spectrophotometry in 11 patients undergoing neurosurgery for acute spinal nerve compressions because of intervertebral disk prolapses. Reference values for erythrocytes were 11.49 +/- 3.48 mmol/mmol Hb; 0.82 +/- 0.087 mmol/mmol Hb; 9.01 +/- 2.20 mmol/mmol Hb; 0.104 +/- 0.032 mmol/mmol Hb; 0.07 +/- 0.050 mmol/mmol Hb for iron, copper, zinc, magnesium, and selenium, respectively. Postoperative erythrocyte concentrations did not differ significantly compared to those obtained preoperatively and remained within the reference ranges perioperatively. For plasma the following reference values were used: 19.0 +/- 8.0 mmol/l (Fe); 20.1 +/- 8.2 mmol/l (Cu); 15.4 +/- 4.6 mmol/l (Zn); 0.9 +/- 0.15 mmol/l (Mg); 1.02 +/- 0.3 mmol/l (Se). There was a significant decrease in the concentration of copper in plasma (13.41 +/- 3.46 mmol/l, p < 0.1) and zinc (10.73 +/- 2.73 mmol/l, p < 0.1) immediately postoperative, iron (10.56 +/- 3.91 mmol/l, p < 0.1) and zinc on day 1 (11.28 +/- 1.88 mmol/l, p < 0.10), and a significant postoperative increase of copper on day 5 (18.81 +/- 3.97 mmol/l, p < 0.1), postoperatively. The mean plasma concentrations of iron, copper, zinc magnesium and selenium remained within the reference ranges during the entire period.     

Bone aluminum uptake in uremic rats receiving intraperitoneal iron

To assess the effect of concomitant iron and aluminum loads on bone aluminum accumulation and on the response to the deferoxamine test in rats with the same aluminum surcharge, Wistar rats with chronic renal failure were divided into three groups: iron-overloaded rats (N=6) (intraperitoneal iron); iron-depleted rats (N=6) (blood withdrawal two to three times per week); control rats (N=4) (no manipulation). All groups received intraperitoneal aluminum simultaneously. After 6 wk, a deferoxamine challenge test was performed. Thereafter, bone aluminum and iron were measured. The iron-overloaded rats showed higher bone iron content (iron overloaded: 147.7±55.4 μg/g; iron depleted: 7.9±1.0, and controls 13.3±9.9 μg/g, p<0.010) and lower bone aluminum content (iron overloaded: 14.2±4.0 μg/g; iron depleted: 70.9±35.1 μg/g; controls: 72.7±28.3 μg/g p<0.005). No differences were found between the iron-depleted and control rats. After the deferoxamine infusion, the iron-depleted rats tended to have higher serum aluminum increments (p=NS) and higher urinary aluminum excretion (p<0.012, p<0.020) than control rats despite similar amounts of aluminum in bone of the two groups. Aluminum bone accumulation was minor if iron and aluminum loads were given concomitantly. The iron depletion influenced the results of the deferoxamine challenge test in rats with similar bone aluminum burden.     

Systemic markers of the redox balance in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is highly prevalent and its pathogenesis is still not completely clarified. Clinically stable patients (n=21) and healthy subjects (n=24) were studied for blood markers of oxidative injury and antioxidant status. The plasma concentration of protein carbonyls was significantly increased in COPD patients, both ex-smokers (0.76 +/- 0.28 nmol mg(-1)) and smokers (0.99 +/- 020 nmol mg(-1)) versus controls (0.49 +/- 0.14 nmol mg(-1)) . The concentration of total thiols was slightly enhanced in plasma of the COPD patients (ex-smokers 492 +/- 23 micromol 1(-1) and smokers 505 +/- 36 micromol 1(-1) versus controls 450 +/- 67 micromol 1(-1); p < 0.05). The activity of the antioxidant enzyme superoxide dismutase was increased in erythrocytes (activity in U g(-1) haemoglobin; ex-smokers 4460 +/- 763 and smokers 4114+/- 1060 versus 3015 +/- 851 in controls; p > 0.01), while glutathione peroxidase activity was decreased in total blood (activity in U g(-1) haemoglobin: ex-smokers 27 +/- 9 and smokers 23 +/- 9 versus 47 +/- 25; p < 0.01). Lower levels of selenium in plasma were also found for COPD patients (concentration in mg 1(-1): ex-smokers 0.030 +/- 0.019 and smokers 0.032 +/- 0.024 versus 0.058 +/- 0.023 in controls; p < 0.01), being more evident in those with very low levels of arterial oxygen pressure. In addition, the levels of potassium and rubidium were increased in blood cells of the patient group. All these changes might reflect oxidant damage and an altered electrolytic homeostasis, and can be interpreted as markers of COPD rather than as indicators of smoking habits.     

Cyclin A2 regulates erythrocyte morphology and numbers

Cyclin A2 is an essential gene for development and in haematopoietic stem cells and therefore its functions in definitive erythropoiesis have not been investigated. We have ablated cyclin A2 in committed erythroid progenitors in vivo using erythropoietin receptor promoter-driven Cre, which revealed its critical role in regulating erythrocyte morphology and numbers. Erythroid-specific cyclin A2 knockout mice are viable but displayed increased mean erythrocyte volume and reduced erythrocyte counts, as well as increased frequency of erythrocytes containing Howell-Jolly bodies. Erythroblasts lacking cyclin A2 displayed defective enucleation, resulting in reduced production of enucleated erythrocytes and increased frequencies of erythrocytes containing nuclear remnants. Deletion of the Cdk inhibitor p27^Kip1 but not Cdk2, ameliorated the erythroid defects resulting from deficiency of cyclin A2, confirming the critical role of cyclin A2/Cdk activity in erythroid development. Loss of cyclin A2 in bone marrow cells in semisolid culture prevented the formation of BFU-E but not CFU-E colonies, uncovering its essential role in BFU-E function. Our data unveils the critical functions of cyclin A2 in regulating mammalian erythropoiesis.     

Variations in erythropoiesis throughout a lifetime. Studies in a high-leukaemic mouse strain, the AKR/O strain, and a non-leukaemic strain, the WLO strain

We have studied the development of some haematological variables: erythropoiesis stimulating factor(s) (ESF), investigated with an in vitro cell culture assay; and the content of bone marrow and spleen erythroid colony forming unit(s) (CFU-E) and erythroid burst forming unit(s) (BFU-E) throughout the lifetime of 2 different mouse strains: the high-leukaemic, retrovirus infected AKR/O strain, and the non-leukaemic WLO strain. During the recovery phase of the postnatal anaemia, a peak in plasma ESF occurs in both strains. In young adult mice of both strains another peak in plasma ESF occurs at 70-110 days of age, associated with an increased number of bone marrow CFU-E, in a period when packed cell volume (PCV) remains stable. As the animals grow older PCV decreases, whereas plasma ESF and bone marrow CFU-E concentration increase. These results, together with in vitro dose-response studies, suggest reduced sensitivity to erythropoietin (Epo) of the ageing erythron. Throughout, the AKR/O strain has higher levels of plasma ESF and bone marrow CFU-E concentrations than the WLO strain, indicating both a reduced Epo responsiveness and some degree of ineffective erythropoiesis in the AKR/O strain. At all ages the AKR/O strain has a high concentration of Epo independent bone marrow CFU-E, possibly caused by the virus infection of precursor cells.     

Interleukine-17-induced inhibitory effect on late stage murine erythroid bone marrow progenitors

Recent studies have shown that the T cell-derived cytokine, interleukin-17 (IL-17), stimulates hematopoiesis, specifically granulopoiesis inducing expansion of committed and immature progenitors in bone marrow. Our previous results pointed to its role in erythropoiesis too, demonstrating significant stimulation of BFU-E and suppression of CFU-E growth in the bone marrow from normal mice. As different sensitivities of erythroid and myeloid progenitor cells to nitric oxide (NO) were found, we considered the possibility that the observed effects of IL-17 were mediated by NO. The effects of recombinant mouse IL-17, NO donor (sodium nitroprusside - SNP) and two NO synthases inhibitors (L-NAME and aminoguanidine) on erythroid progenitor cells growth, as well as the ability of IL-17 to induce nitric oxide production in murine bone marrow cells, were examined. In addition, we tested whether the inhibition of CFU-E colony formation by IL-17 could be corrected by erythropoietin (Epo), the principal regulator of erythropoiesis. We demonstrated that IL-17 can stimulate low level production of NO in murine bone marrow cells. Exogenously added NO inhibited CFU-E colony formation, whereas both L-NAME and aminoguanidine reversed the CFU-E suppression by IL-17 in a dose-dependent manner. The inhibition of CFU-E by IL-17 was also corrected by exposure to higher levels of Epo. The data obtained demonstrated that at least some of the IL-17 effects in bone marrow related to the inhibition of CFU-E, were mediated by NO generation. The fact that Epo also overcomes the inhibitory effect of IL-17 on CFU-E suggests the need for further research on their mutual relationship and co-signalling.     

Changes in hemopoietic and regulator levels in mice during fatal or nonfatal malarial infections. I. Erythropoietic populations

Erythroid precursors BFU-E and CFU-E and erythroblasts (ERB) were monitored in the marrow and spleen of mice during fatal or nonfatal malaria. Transient depletions of marrow CFU-E and ERB without modification of BFU-E or erythropoietin (Epo) levels were found as early events in fatal infections. Before anemia development, erythropoiesis was reduced in the bone marrow but increased in the spleen. During the anemic phase, for comparable levels of anemia, plasma Epo levels were elevated to a similar degree in fatal and nonfatal malaria. In the bone marrow, CFU-E increased twofold and BFU-E were usually reduced as expected in severe anemia. ERB populations increased but remained below or within normal values, suggesting an impairment of marrow erythropoiesis related to early events following infection. In contrast, in the spleen, ERB production was strongly simulated but amplification of ERB, CFU-E, and BFU-E populations was 2.5-fold lower in fatal than in nonfatal malaria. The results suggest that a defect in amplification of splenic erythropoiesis is a crucial determinant of the fatal outcome of malarial infection. This may have been mediated by a defective stem cell migration or multiplication. Some evidence obtained during recovery stages suggested that a factor(s) other than Epo may control splenic erythropoiesis during the anemia associated with malaria.     

Effects of melatonin on carbonic anhydrase from human erythrocytes in vitro and from rat erythrocytes in vivo

The in vitro effects of melatonin (N-acetyl-5-methoxy-tryptamine) on human carbonic anhydrase isozymes (HCA-I and HCA-II) from human erythrocytes and in vivo effects on rat erythrocytes carbonic anhydrase (CA) were determined. Human erythrocyte carbonic anhydrase isozymes were purified by haemolysate preparation and Sepharose-4B-L tyrosine-sulfanilamide affinity gel chromatography. The HCA-I enzyme, having a specific activity of 7337.5 EU/mg protein, was purified 843-fold with a yield of 60% and the HCA-II enzyme, having a specific activity of 17067EU/mg protein, was purified 1962-fold with a yield of 22.7%. For in vitro experiments, the enzyme activity was minimal at 2 x 10(-4) M melatonin concentration and increased above this concentration. Ten mgkg(-1) melatonin was administered intraperitoneally and showed a stimulatory effect on the enzyme. Time-dependent in vivo studies were conducted for melatonin in Sprague-Dawley type rats. It was found that CA activity in the rat erythrocytes was decreased by the melatonin after 1 and 3 hours to 2500 +/- 500.0 and 1875 +/- 239.4 respectively which were statistically significant (p < 0.05) differences to the control (2660 +/- 235.8). However, CA activity was restored to its normal level after 6h (2666 +/- 235.7) (p > 0.05) probably due to metabolism of the melatonin. The findings indicate that melatonin may be pharmacologically useful in some diseases.     

Comparative effects of inhibitors of arachidonic acid metabolism on erythropoiesis

The effects of a variety of inhibitors of the arachidonic acid metabolic pathway have been tested on the growth of early erythroid progenitor cell-derived colonies (CFU-E and BFU-E) in an attempt to discern whether products of the cyclo-oxygenase pathway or lipoxygenase pathway are essential for erythropoiesis. Murine erythroid progenitor cells obtained from fetal livers were cultured in the presence of erythropoietin for CFU-E and of interleukin 3 for BFU-E colony formation in response to the cyclo-oxygenase inhibitors, aspirin or sodium meclofenamate, and the lipoxygenase inhibitors, BW755C, nordihydroguiaretic acid (NDGA), phenidone, and butylated hydroxyanisole (BHA). The most potent inhibitor of colony formation (both CFU-E and BFU-E) was the selective lipoxygenase inhibitor, BW755C, followed by NDGA, phenidone and BHA. Neither aspirin nor sodium meclofenamate (10(-4) - 10(-6)M) significantly (p less than 0.05) inhibited CFU-E or BFU-E formation. These results support the hypothesis that lipoxygenase products of arachidonic acid metabolism may be essential for erythroid cell proliferation/differentiation.     

Urea-selective concentrating defect in transgenic mice lacking urea transporter UT-B.

Urea transporter UT-B has been proposed to be the major urea transporter in erythrocytes and kidney-descending vasa recta. The mouse UT-B cDNA was isolated and encodes a 384-amino acid urea-transporting glycoprotein expressed in kidney, spleen, brain, ureter, and urinary bladder. The mouse UT-B gene was analyzed, and UT-B knockout mice were generated by targeted gene deletion of exons 3-6. The survival and growth of UT-B knockout mice were not different from wild-type littermates. Urea permeability was 45-fold lower in erythrocytes from knockout mice than from those in wild-type mice. Daily urine output was 1.5-fold greater in UT-B- deficient mice (p < 0.01), and urine osmolality (U(osm)) was lower (1532 +/- 71 versus 2056 +/- 83 mosM/kg H(2)O, mean +/- S.E., p < 0.001). After 24 h of water deprivation, U(osm) (in mosM/kg H(2)O) was 2403 +/- 38 in UT-B null mice and 3438 +/- 98 in wild-type mice (p < 0.001). Plasma urea concentration (P(urea)) was 30% higher, and urine urea concentration (U(urea)) was 35% lower in knockout mice than in wild-type mice, resulting in a much lower U(urea)/P(urea) ratio (61 +/- 5 versus 124 +/- 9, p < 0.001). Thus, the capacity to concentrate urea in the urine is more severely impaired than the capacity to concentrate other solutes. Together with data showing a disproportionate reduction in the concentration of urea compared with salt in homogenized renal inner medullas of UT-B null mice, these data define a novel "urea-selective" urinary concentrating defect in UT-B null mice. The UT-B null mice generated for these studies should also be useful in establishing the role of facilitated urea transport in extrarenal organs expressing UT-B.     

Haematological disturbances and osmotic shifts in rainbow trout,Oncorhynchus mykiss (Walbaum) under acid and aluminium exposure

Summary Elevated values of haematocrit were observed in rainbow trout (Oncorhynchus mykiss) during combined acid and aluminium exposure. Possible causes of this, i.e., decreased plasma volume, swelling of erythrocytes, and/or mobilization of erythrocytes into the blood were investigated. Slight haematocrit increases (10–20%) during mild acid stress (pH 5.0, 25 mol Ca²⁺·1^-1) were mostly caused by osmotic shifts. Both swelling of erythrocytes rocytes and a significant decrease of the plasma volume were demonstrated in fish at pH 5.0. These osmotic disturbined were greater during acid exposure (pH 5.0) combined with Al (60 g·1^-1; 200 g·1^-1). In addition, numbers of erythrocytes increased by 40% compared to acid exposure, which contributed to the severe haematocrit rise (35%) during Al exposure. A contraction of the spleen releasing erythrocytes into the blood is suggested to occur as an adrenergic response to hypoxia, which is observed in fish acutely exposed to Al.Abbreviations BV blood volume - ECFV extracellular fluid volume - ICFV intracellular fluid volume - ISFV interstitial fluid volume - MCHC mean cell haemoglobin concentration - MCV mean cell volume - PV plasma volume     

Long term evaluation of disease progression through the quantitative magnetic resonance imaging of symptomatic knee osteoarthritis patients: correlation with clinical symptoms and radiographic changes

The objective of this study was to further explore the cartilage volume changes in knee osteoarthritis (OA) over time using quantitative magnetic resonance imaging (qMRI). These were correlated with demographic, clinical, and radiological data to better identify the disease risk features. We selected 107 patients from a large trial (n = 1,232) evaluating the effect of a bisphosphonate on OA knees. The MRI acquisitions of the knee were done at baseline, 12, and 24 months. Cartilage volume from the global, medial, and lateral compartments was quantified. The changes were contrasted with clinical data and other MRI anatomical features. Knee OA cartilage volume losses were statistically significant compared to baseline values: -3.7 +/- 3.0% for global cartilage and -5.5 +/- 4.3% for the medial compartment at 12 months, and -5.7 +/- 4.4% and -8.3 +/- 6.5%, respectively, at 24 months. Three different populations were identified according to cartilage volume loss: fast (n = 11; -13.2%), intermediate (n = 48; -7.2%), and slow (n = 48; -2.3%) progressors. The predictors of fast progressors were the presence of severe meniscal extrusion (p = 0.001), severe medial tear (p = 0.005), medial and/or lateral bone edema (p = 0.03), high body mass index (p < 0.05, fast versus slow), weight (p < 0.05, fast versus slow) and age (p < 0.05 fast versus slow). The loss of cartilage volume was also slightly associated with less knee pain. No association was found with other Western Ontario McMaster Osteoarthritis Index (WOMAC) scores, joint space width, or urine biomarker levels. Meniscal damage and bone edema are closely associated with more cartilage volume loss. These data confirm the significant advantage of qMRI for reliably measuring knee structural changes at as early as 12 months, and for identifying risk factors associated with OA progression.