The anemia of end-stage renal disease: hematopoietic progenitor cell response

Patients with the anemia of end-stage renal disease (ESRD) fail to display an appropriate compensatory increase in red cell production. In order to investigate the extent to which the impaired erythropoietic response is determined at the progenitor cell level, we determined the frequencies of marrow colony-forming cells in 11 anemic and 3 non-anemic, dialysis-dependent ESRD patients and 10 healthy individuals. In addition, we measured serum levels of erythropoietin (Epo) by radioimmunoassay. There were no significant differences (P greater than 0.1) between normal and ESRD groups in the frequencies of primitive or late erythroid (BFU-E and CFU-E, respectively), granulocyte-macrophage, and megakaryocyte progenitors, CFU-E/BFU-E ratios, or serum Epo levels. In contrast, 5 non-uremic patients with chronic anemia comparable in severity to the anemic ESRD patients had serum Epo levels and CFU-E/BFU-E ratios that were significantly increased (P less than 0.05 and P less than 0.001, respectively) in comparison to the normal controls and ESRD patients. Pre-dialysis serum and plasma from both ESRD groups were as supportive of autologous erythroid and non-erythroid colony growth in vitro as normal serum and plasma; inhibition was not observed. We conclude that the relative numbers of erythroid and non-erythroid progenitors and the majority of serum Epo levels are unchanged from normal in patients with the anemia of ESRD. However, their normal CFU-E/BFU-E ratio reflects an inadequate compensatory erythropoietic response due to their inability to appropriately increase Epo production in response to anemia. Inhibitors of autologous erythroid colony formation were not detected in ESRD serum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Erythropoiesis after nonmyeloablative stem-cell transplantation is not impaired by inadequate erythropoietin production as observed after conventional allogeneic transplantation

BACKGROUND: It is now well established that after conventional allogeneic hematopoietic stem-cell transplantation (HSCT), erythropoietic recovery is impaired because erythropoietin (Epo) production remains inadequate for prolonged periods of time. However, erythropoietic reconstitution after nonmyeloablative SCT (NMSCT) has never been characterized. METHODS: Twelve patients received a nonmyeloablative conditioning regimen consisting of 2 Gy total body irradiation (TBI) alone (n=6), 2 Gy TBI and fludarabine (n=3), or cyclophosphamide and fludarabine (n=3), followed by transplantation of allogeneic peripheral blood stem cells. Graft-versus-host-disease (GvHD) prophylaxis was carried out with mycophenolate mofetil (from day -1 to day 28) plus cyclosporine (from day -1 to day 120 or longer in case of chronic GvHD). Erythropoiesis was quantitated by soluble transferrin receptor (sTfR) levels, and the adequacy of Epo production was evaluated by the observed-to-predicted Epo ratio (O/P Epo). RESULTS: Mean sTfR levels decreased following the conditioning regimen but remained well within the normal range throughout the posttransplant period. The O/P Epo ratio presented an initial surge quite similar to that observed after conventional conditioning. Thereafter, the O/P Epo ratio normalized rapidly, and Epo levels remained adequate during the whole observation period. CONCLUSION: Contrarily to what is observed after myeloablative transplant, Epo levels remained adequate after NMSCT, resulting in normal erythropoiesis. These results suggest that the administration of erythropoietin therapy (rHuEpo) could be less effective after NMSCT than after conventional allogeneic transplant.     

Expression of angiotensin II type I receptor on erythroid progenitors of patients with post transplant erythrocytosis

BACKGROUND: The pathogenesis of posttransplant erythrocytosis (PTE) has been elusive. Angiotensin converting enzyme inhibitors (ACEI) are efficacious in lowering the hematocrit of patients with PTE and angiotensin II (AII) type I receptors (AT1R) were recently detected on red blood cell precursors, burst-forming unit-erythroid- (BFU-E) derived cells. The purpose of this study was to determine whether there is increased expression of the AT1R on BFU-E-derived cells of patients with PTE, which might contribute to the pathogenesis of PTE. METHODS: Twelve healthy volunteers and 25 transplant recipients (13 patients with and 12 without PTE) were studied. BFU-E from peripheral blood were cultured in methylcellulose and BFU-E-derived colonies were harvested on day 10. Western blotting was used to detect AT1R and erythropoietin receptor (EpoR) expression. Intracellular free calcium in response to AII and erythropoietin (Epo) was measured with digital video imaging. RESULTS: There were no differences between transplant patients, with and without PTE, with respect to weight, age, sex, blood pressure, serum creatinine, circulating renin, angiotensin II, and Epo levels. Hematocrit, red blood cell number, BFU-E-derived colony number,and size were significantly increased in PTE compared with other two groups. AT1R expression was increased by 44% on the erythroid progenitors of PTE versus non posttransplant erythrocytosis patients and by 32% in PTE patients versus normal volunteers. AT1R expression correlated significantly with the hematocrit in PTE (Spearman r=0.68, P=0.01). In contrast, EpoR expression was equivalent in all groups. The AT1R was functional since a significant increase in [Ca(i)] was observed in Fura-2 loaded day 10 cells when stimulated with AII (182%, P<0.0001). CONCLUSION: An increase in AT1R density was observed in erythroid precursors of transplant patients with PTE compared to those without PTE and normal volunteers, and the level of AT1R expression in PTE correlated significantly with the hematocrit. In contrast, EpoR expression was not different in PTE compared with non posttransplant erythrocytosis or normal controls. This study supports a role for the AT1 receptor signaling pathway in the pathogenesis of PTE.     

Determinants of circulating soluble transferrin receptor level in chronic haemodialysis patients.

BACKGROUND: The aim of this study was to identify the factors determining the circulating soluble transferrin receptor (sTfR) concentrations in haemodialysis (HD) patients on maintenance recombinant human erythropoietin (rHuEpo) treatment. METHODS: In a prospective cross-sectional study, 91 chronic HD patients and 18 anaemic controls with normal renal function were recruited. For each subject, blood samples were measured for complete blood count, reticulocyte count, percentage of hypochromic red cells (% HRC), serum ferritin, serum iron, transferrin saturation (TS), serum erythropoietin (sEpo), C-reactive protein (CRP), and sTfR. HD patients received constant rHuEpo doses and basal sEpo was measured > or = 86 h after the last injection. The age, gender, dialysis vintage, and the above-mentioned parameters were used as independent variables and logarithmic sTfR (log(10)sTfR) as a dependent variable in the forward stepwise multiple regression model. RESULTS: HD patients were similar to controls regarding haematocrit, serum ferritin, TS, and % HRC, but had significantly lower sTfR, sEpo, and reticulocyte index. Univariate analyses showed that the sTfR level strongly correlated with sEpo (r=0.60, P<0.001) and % HRC (r=0.60, P<0.001), and significantly with serum ferritin (r=-0.29, P<0.01), TS (r=-0.27, P<0.05), and dose of rHuEpo administered (r=0.27, P<0.05) in HD patients. sTfR also had a positive correlation with haematocrit (r=0.26, P<0.05), red blood cell (RBC) count (r=0.23, P<0.05), and reticulocyte count (r=0.24, P<0.05), but not with CRP (r=0.16, P>0.05). Multivariate regression analysis disclosed that sEpo, HRC, and serum ferritin were the independent predictors of sTfR level. Overall, the model explained 58.8% of the variability in sTfR (R(2)=0.588, P<0.001). CONCLUSIONS: Circulating sTfR is a good index of marrow erythropoietic activity in HD patients during rHuEpo treatment. Its level is also independently up-regulated by functional iron deficiency in the process of enhanced erythropoiesis. Our study showed that sTfR levels quantitatively reflect the integrated effects of iron availability, iron reserves, and erythropoietic stimulation.     

Red cell 2, 3-diphosphoglycerate levels among diabetic patents with and without vascular complications.

There have been differences of opinion among authors concening in the levels of red cell 2,3-diphosphoglycerate (2,3-DPG) and nucleotides in nonacidotic diabetic patients. Our data suggest that abnormal levels of 2, 3-DPG in diabetic patients are related to the presence of vascular complications and not to the duration of the disease per sec. 2,3-DPG levels are normal in diabetic patients with no evidence of vascular complications (group A). In ambulatory patients with vascular complications (group B), significantly higher levels of 2,3-DPG are found than in normal subjects and patients in group A. In hospitalized diabetic patients with active peripheral vascular complications (group C), levels of 2,3-DPG are likewise significantly increased over those of normal subjects and patients of group A. 2,3-DPG was found to be significantly elevated in patients of group C as compared with group B. 2,3-DPG levels in venous blood from infected legs as compared with those of the peripheral venous blood were not significantly different, thereby ruling out local factors. There were no differences in the blood lactate levels in any of the group studied. The elevation of the 2,3-DPG levels may be a reflection of attempted red blood cell compensation for tissue hypoxia in the diabetic with vascular disease.     

In-vitro growth patterns of bone marrow erythroid progenitors from patients with post-renal transplant erythrocytosis

Background: Erythrocytosis is relatively common after renal transplantation and is associated with a higher risk of thromboembolism. Its aetiology is unclear and there is still debate about the most frequently suggested causes. The culture in vitro of erythroid progenitors is regarded as a useful tool for the differential diagnosis of patients with unclear erythrocytosis. We studied the growth in vitro of bone marrow erythroid progenitor from renal transplant patients with erythrocytosis and controls without erythrocytosis. Subjects and methods: Thirteen renal transplant patients with erythrocytosis and 12 normocythaemic renal transplant controls were studied. The clinical characteristics of these patients were evaluated and serum erythropoietin (Epo) and ferritin levels were determined. Bone marrow erythroid progenitors were cultured both with and without the addition of Epo to the medium. Results: Samples from six polycythaemic patients and seven controls did not grow spontaneously in the absence of exogenous Epo. Three cases of post-transplant erythrocytosis and five controls produced CFU-E, but not BFU-3. A few CFU-E and BFU-E grew spontaneously in samples from four polycythaemic patients but not in samples from the controls. Addition of 1 unit per millilitre Epo caused similar increases in the number of colonies in both polycythaemic patients and controls. Of the nine patients eligible for follow-up, all four with spontaneous growth of BFU-E had transient erythrocytosis and four of the five patients with no spontaneous growth or spontaneous growth of CFU-E only had persistent erythrocytosis requiring treatment with ACE inhibitors. Conclusions: Pathophysiology of post-transplant erythrocytes is heterogenous. In one-third of the patients, there was unexpected, spontaneous and transient growth of BFU-E which was not predictive of permanent erythrocytosis. The results of stem-cell studies suggest that in these cases erythrocytosis may be caused by defective regulation of erythroid progenitor proliferation, possibly due to particular cellular interactions or the effect of cyclosporin on erythropoiesis.     

Stimulation of erythropoietin in renal insufficiency by hypobaric hypoxia.

Patients with renal anaemia show inadequate levels of immunoreactive erythropoietin (Epo) related to the degree of anaemia. The purpose of our study is to compare the degree of stimulation of Epo by means of hypobaric hypoxia in normal controls and patients with renal anaemia. Baseline Epo concentrations were found to be 11.1 +/- 2.0 U/l in 10 healthy volunteers and 11.4 +/- 4.6 U/l in six patients with renal anaemia. After exposure to hypobaric hypoxia equivalent to 4560 m above sea level for a duration of 3.5 h, we observed a significant increase in serum Epo in healthy volunteers to 22.8 +/- 9.1 U/l (P < 0.005), while there was no increase in patients with renal anaemia: 12.3 +/- 5.2 U/l (P < 0.2). Our results show that in patients with renal anaemia serum Epo concentrations are comparable to those of normal controls, but inadequate in view of the concomitant degree of anaemia. Stimulation by acute hypobaric hypoxia was not possible in patients with renal insufficiency as opposed to normal controls. From these data it can be concluded that either Epo production is working at maximum capacity under baseline conditions, or an additional hybobaric stimulus is not able to influence a disturbed set point of the oxygen sensor regulating Epo synthesis.     

The value of soluble transferrin receptor and hepcidin in the assessment of iron status in children with cystic fibrosis

BackgroundThe value of ferritin in the diagnosis of iron deficiency is limited in patients with CF since it increases in the presence of inflammation. We hypothesized that the soluble transferrin receptor (sTfR) and hepcidin may provide more information than ferritin in assessing iron status in children with CF.MethodsWe analyzed sTfR and hepcidin in relation to conventional iron status indicators in 49 children with CF.ResultsWe found no differences in sTfR concentration between children with and those without ID. sTfR concentrations were within the normal range in all children. Hepcidin concentrations were low, and concentrations below the limit of detection were observed in 25% of the clinically stable children.ConclusionThe sTfR is not useful to determine the iron status in this population, whereas hepcidin might serve as an early indicator of deficient iron stores in children with CF.     

Erythropoietin in patients with acute renal failure and continuous veno-venous haemofiltration

Erythropoietin (Epo) is a glycoprotein hormone produced in the kidney in response to hypoxia or anaemia. In acute renal failure (ARF) anaemia also occurs and current opinion is that Epo production is depressed with inappropriately low plasma levels throughout the uraemic phase. Our study was designed to determine the excretion of Epo in patients with ARF. Fifty-nine ventilated patients were studied, 39 with ARF and continuous veno-venous haemofiltration therapy (group 1) and 13 patients with normal renal function who served as a control group (group 2). All patients with ARF were anaemic and needed a mean transfusion of 0.6 units/day. Values for vitamin B12, folic acid, serum iron and ferritin were normal. While patients with normal renal function had Epo values within the normal range, patients with ARF had significantly higher values at the onset of haemofiltration therapy. Mean Epo (mean±SEM) values on days 0–2 were 92.6±11.7 mU/ml in group 1 and 16.5±6.4 mU/ml in group 2 (p<0.0002). Epo levels declined in group 1 to 49±10.5 mU/ml on days 9 and 10 compared to 23±9.1 mU/ml in group 2 (ns). These values were maintained until the end of the observation period. No differences were seen between oliguric and non-oliguric patients. Our data show that patients with ARF have increased Epo levels at the beginning of the disease with a strong tendency to decrease, suggesting that there might be inadequate Epo levels during the course of acute renal failure.     

Regulatory mechanisms of erythropoietin levels in dialysis patients

We described a radioimmunoassay system for measuring blood erythropoietin (Epo) levels using recombinant human Epo and evaluated the regulatory mechanisms of Epo in dialysis patients. A satisfactory dose-response relationship for Epo levels was observed within the wide range of 3-250 mU/ml with a sensitivity of approximately 5 mU/ml. The Epo levels were found to be 16.1 +/- 8.6 mU/ml (m +/- SD) in 422 uremic patients on maintenance dialysis and 17.1 +/- 7.2 mU/ml in 86 normal subjects. The Epo levels were not statistically different between the two groups, although the hematocrit was significantly lower in the dialysis patients. No correlation was observed between the Epo levels and hematocrit in the dialysis patients. Patients with polycystic kidneys had higher hematocrits and Epo levels than patients with chronic nephritis. No changes in Epo levels were observed with age and with the period of dialysis. Diurnal variations in the Epo levels revealed a significant increase after hemodialysis, and an acute reduction in hematocrit following massive hemorrhage raised the Epo levels up to 3,180 mU/ml. These findings indicate that the Epo levels in dialysis patients are inappropriately low for the severity of anemia and suggest that a negative feedback mechanism of the hematocrit on the Epo secretion may exist in uremic patients as well as in normal subjects and that the threshold for Epo secretion might be 'reset' at a low hematocrit level.     

Insulin-like growth factor I plays a role in regulating erythropoiesis in patients with end-stage renal disease and erythrocytosis

Erythroid progenitor growth, the serum hormones that regulate erythropoiesis, and the effect of patient's serum on the growth of normal erythroid progenitors were assessed in eight patients with end-stage renal disease (ESRD) and erythrocytosis. All patients were male and had been on maintenance dialysis, they had a hematocrit >50% and/or a red blood cell count >6 x 10(12)/L and an arterial oxygen saturation >95%. Four had acquired cystic disease of the kidney (ACDK), and four other non-ACDK patients did not have known causes of secondary erythrocytosis after appropriate investigations and long-term follow-up. The methylcellulose culture technique was used to assay the erythroid progenitor (BFU-E/CFU-E) growth. Serum erythropoietin (EPO) and insulin-like growth factor I (IGF-I) levels were measured by RIA. Paired experiments were performed to determine the effects of 10% sera from ESRD patients and control subjects on normal marrow CFU-E growth. The numbers of EPO-dependent BFU-E in marrow and/or blood of patients with ESRD and erythrocytosis were higher than those of normal controls. No EPO-independent erythroid colonies were found. Serum EPO levels were constantly normal in one patient and elevated in three patients with ACDK; for non-ACDK patients, EPO levels were normal or low in two patients and persistently increased in one, but fluctuated in the remaining one on serial assays. There was no correlation between serum EPO levels and hematocrit values. The serum IGF-I levels in patients with ESRD and erythrocytosis were significantly increased compared with normal subjects or ESRD patients with anemia. We found an inverse correlation between serum EPO and IGF-I levels. Sera from patients with ESRD and erythrocytosis exhibited a stimulating effect on normal marrow CFU-E growth. The stimulating effect of sera from patients who had a normal serum EPO level and an elevated IGF-I level could be partially blocked by anti-IGF-I. The present study suggests that IGF-I plays an important role in the regulation of erythropoiesis in patients with ESRD and erythrocytosis who did not have an increased EPO production.     

Does circulating erythropoietin reflect progression of IgA nephropathy? Comparison with urinary N-acetyl-beta-D-glucosaminidase.

BACKGROUND: Recent reports describe that erythropoietin (Epo) is produced by peritubular interstitial fibroblast-like cells in response to a hypoxic stimulus. We studied serum Epo levels as a possible marker of tubulointerstitial damage in the progression of IgA nephropathy (IgAN), in comparison with urinary (u-) levels of N-acetyl-beta-D-glucosaminidase (NAG), which is mainly derived from proximal tubular cells and is used as a marker of tubular damage. METHODS: Thirty-eight patients with IgA nephropathy (IgAN) with relatively preserved renal function (serum creatinine: sCr, 0.5-2.2 mg/dl) were examined. The severity of glomerulosclerosis and interstitial fibrosis of the renal biopsy tissue was expressed by semiquantitative grading scores. Clinical parameters including serum creatinine (sCr), blood pressures, and 24-h proteinuria levels were obtained at the renal biopsy. Epo was measured by a radioimmunoassay (RIA) of sera obtained in the morning and u-NAG was measured by colorimetric method of 24-h urine samples. RESULTS: The mean Epo level of the patients (17.7+/-6.3 mU/ml) was not different from the control level (19.3+/-3.7 mU/ml). There were no significant correlations between Epo levels and red blood cell (RBC) counts, haematocrit (Hct), or haemoglobin (Hb) levels. The mean u-NAG level of the patients (6.7+/-6.2 U/gCr) was significantly higher than the control level (1.9+/-0.5 U/gCr). There was an inverse quantitative correlation between Epo and u-NAG levels in the patients (P<0.02). The u-NAG levels showed quantitative positive correlations with sCr (P<0.001), u-proteins (P<0.001), systolic (SBP) (P<0.001), and diastolic blood pressures (DBP) (P<0.05). Conversely, Epo levels were inversely correlated with sCr, SBP and DBP (each P<0.05). The patients with higher u-proteins (>2.0 g/day) showed significantly decreased Epo levels (P<0.05) than those with lower u-proteins (<2.0 g/day). The both scores of glomerulosclerosis and interstitial fibrosis were positively correlated with the u-NAG levels (each P<0.001), but were not correlated with the Epo levels. CONCLUSIONS: The significant correlation between u-NAG and serum Epo levels suggests that tubular damage and interstitial cell dysfunction are associated each other in the progression of IgAN. Serum Epo levels bearing inverse correlations with sCr, blood pressure levels and heavy proteinuria seem to reflect clinical severity of IgAN, whereas u-NAG can be more useful progression marker of IgAN bearing correlations with both clinical and histological findings.     

Improved oxygen delivery to the myocardium during hypothermia by perfusion with 2,3 DPG-enriched red blood cells

The oxygen affinity of red cells increases stepwise with temperature reductions below 37 degrees C. In vitro studies demonstrated that biochemically modified red cells with increased 2,3 diphosphoglycerate (2,3 DPG) (150% and 250% of normal) exhibited significantly less oxygen affinity at 24 degrees C than did unmodified cells. At 15 degrees C, significant attenuation of affinity was observed with 250%, but not 150%, of normal 2,3 DPG cells. Measurements made of isolated fibrillating dog hearts during perfusion at 24 degrees C alternately with unmodified (80% of normal 2,3 DPG) and modified (300% of normal 2,3 DPG) red cells demonstrated significantly greater oxygen consumption, higher coronary sinus partial pressures of oxygen and carbon dioxide, higher in vitro P50 values, and lower arterial and coronary sinus lactate levels during perfusion with modified as compared with unmodified cells. This evidence, indicating improved oxygen delivery to hypothermic dog hearts by red cells with 300% of normal 2,3 DPG activity, suggests that high 2,3 DPG cells might protect myocardial tissue in patients undergoing hypothermic cardiac operation.     

Erythropoietin deficiency and relative resistance cause anaemia in post-renal transplant recipients with normal renal function

BACKGROUND: Following successful renal transplantation, blood erythropoietin(Epo) levels peak in two phases during the first 2–3 months,and blood haemoglobin/haematocrit (Hb/Hct) levels are restoredto normal in a period of 2–6 months. However, some transplantrecipients continue to remain anaemic in spite of normal graftfunction and in the absence of recognizable causes. The roleof endogenous Epo production in the causation of anaemia insuch patients is poorly understood and has been investigatedin this study. METHODS: Twenty-three post-renal transplant recipients with stable normalrenal function were studied. Eleven of these patients had normalHb/Hct levels (group 1) and served as control for the rest 12patients with anaemia (group 2). Patients included in group2 had no readily recognizable cause for their anaemia. Otherlaboratory and clinical findings were similar in both groups.Patients with erythrocytosis were excluded. Serum Epo levelswere measured in all patients. Five patients in group 2 weretreated with recombinant human erythropoietin (rHuEpo) and theirerythropoietic response was assessed. rHuEpo was discontinuedwhen the target Hb/Hct levels (lowest normal range) were achievedand the patients were followed up for a further period of 9–12months. RESULTS: Five patients in group 1 had normal expected serum Epo levelswhereas the other six patients had inappropriately high serumEpo levels with respect to their Hb/Hct status suggestive ofrelative ‘Epo resistance’. Serum Epo levels in allpatients except two in group 2 were low indicative of ‘Epodeficiency’. The two exceptional patients in group 2 hadhigher serum Epo levels in the presence of anaemia suggestiveof relative ‘Epo resistance’. All five patients treated with rHuEpo responded adequately byachieving normal Hb/Hct levels. Three of them were originally‘Epo deficient’ and they reached target Hb/Hct levelsin a mean period of 4 weeks, requiring a mean cumulative rHuEpodose of 428.3 units/kg. The other two patients with higher initialserum Epo levels, and considered to be ‘Epo resistant’,required an average of 11 weeks of treatment and a mean cumulativerHuEpo dose of 1582.5 units/ kg, indicating an increased Epodemand. On cessation of therapy the Hb/Hct levels fell in allfive patients to pretreatment values in 6 months. CONCLUSIONS: There are important variations in the endogenous Epo productionin renal transplant patients with normal renal function, thecause of which is not clear. Epo deficiency and relative Eporesistance play a causative role for anaemia in some post-renaltransplant recipients with stable normal renal function. Theyrespond adequately to rHuEpo administration.     

Induction of cathelicidin in normal and CF bronchial epithelial cells by 1,25-dihydroxyvitamin D3

BACKGROUND: Antimicrobial peptides (AMPs) such as cathelicidins contribute to initial defense of the airway against inhaled pathogens. Recent studies have shown that the hormonally active form of vitamin D(3), 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) up-regulates AMP gene expression in several established cell lines. Furthermore, serum levels of vitamin D are often deficient in cystic fibrosis (CF) patients. METHODS: We investigated the effect of 1,25(OH)(2)D(3) on AMP mRNA levels in primary cultures of normal human bronchial epithelial (NHBE) cells by real-time PCR, and protein levels by Western blot. Antimicrobial activity of airway surface fluid from these cells was measured by in vitro assay against laboratory strains of bacteria. RESULTS: Treatment of NHBE cells with 1,25(OH)(2)D(3) (10(-8)M), resulted in a 10-fold up-regulation of cathelicidin mRNA levels after 12 h, which was augmented 2-fold with co-incubation of 1 mM Calcium. Moreover, 1,25(OH)(2)D(3) induced antimicrobial activity against the airway pathogens Bordetella bronchiseptica and Pseudomonas aeruginosa. 1,25(OH)(2)D(3) induced cathelicidin mRNA expression equally in both normal and CF bronchial epithelial cells. CONCLUSIONS: Elucidation of the effect of 1,25(OH)(2)D(3) on cathelicidin expression in NHBE cells and CF bronchial epithelial cells will aid in the development of novel therapeutic agents for treatment of airway infections in CF.     

Association between bone mineral density and erythropoiesis in Thai children and adolescents with thalassemia syndromes

Increased marrow erythropoiesis in patients with thalassemia syndromes results in the expansion of bone marrow cavities and consequently decreases bone tissues, leading to osteoporosis. Whether the soluble transferrin receptor (sTfR), a marker of erythropoietic activity, correlates with the bone mineral density (BMD) in thalassemic patients has not previously been addressed. Forty-six children and adolescents with thalassemia syndromes, who were either not transfused or suboptimally transfused, were studied. BMD was determined by dual-energy X-ray absorptiometry. Blood samples were obtained in order to determine sTfR and hemoglobin. The patients were categorized into four groups: 1, β-thalassemia/hemoglobin E (β-thal/E) with transfusion-dependency (TD) (n = 18); 2, β-thal/E with transfusion-independency (TI) (n = 15); 3, β-thalassemia major (β-major) (n = 6); 4, hemoglobin H (HbH) (n = 7). All patients had normal serum free thyroxine (FT₄) and thyroid-stimulating hormone (TSH), and intact parathyroid hormone (PTH), serum calcium (Ca), phosphate (P), and 25-OH-vitamin D levels. The BMD of patients in the β-major and β-thal/E with TD groups were not significantly different. In comparison with the β-major and β-thal/E with TD groups, the β-thal/E with TI and HbH groups had significantly higher BMD of the total body (TB), femoral neck (FN), and lumbar spine (LS), as well as higher levels of hemoglobin. In contrast, the sTfR levels of the β-major, β-thal/E with TI, and HbH groups were significantly lower than those of the β-thal/E with TD group. The BMD of TB, FN, and LS was negatively correlated with the sTfR level, but positively correlated with the hemoglobin level. In conclusion, increased marrow erythropoiesis is one of the major determinants of reduced bone mass in thalassemic patients with either no transfusion or suboptimal transfusion.     

Poor response to recombinant erythropoietin is associated with loss of T-lymphocyte CD28 expression and altered interleukin-10 production.

BACKGROUND: Recombinant erythropoietin (Epo) therapy is well established as an effective treatment for the anaemia of end-stage renal disease. However, 5-10% of such patients do not respond adequately and an important contributory factor to this is chronic inflammation. METHODS: The present study compares the circulating T-cell phenotypes of haemodialysis patients who respond poorly to Epo with those who respond well, along with normal controls. Isolated peripheral blood mononuclear cells were labelled with immunofluorescent monoclonal antibodies to surface antigens and analysed by flow cytometry. In vitro mononuclear cell cytokine secretion was also studied in the three subject groups. The cells were cultured for 48 h either without stimulus, with lipopolysaccharide or with monoclonal antibodies to CD3 and CD28. RESULTS: C-reactive protein levels were increased in poor responders to Epo (18.6 +/- 20.7 mg/l) compared with good responders (8.7 +/- 8.0 mg/l) and normal controls (3.8 +/- 1.1 mg/l). Patients responding poorly to Epo had increased circulating levels of CD4+/CD28- and CD8+/CD28- T-cells compared with patients responding well to Epo and normal controls. Unstimulated mononuclear cells from poor responders showed increased in vitro generation of interleukin-10 (IL-10) compared with both patients responding well to Epo and normal controls. Additionally, IL-10 generation stimulated by monoclonal antibodies to CD3 and CD28 was increased in poor responders compared with normal controls. CONCLUSIONS: These findings suggest that patients responding poorly to Epo may show enhanced immune activation as manifest by changes in both T-cell function and phenotype.     

Erythropoietin stimulates growth and STAT5 phosphorylation in human prostate epithelial and prostate cancer cells

BACKGROUND: Erythropoietin (Epo), the principal regulator of erythroid progenitor survival, growth, and differentiation, initiates its action by binding to its cognate cell surface receptor (EpoR). EpoR have been identified on a variety of non-hematopoietic cells, both normal and malignant, however, little is known about the function of EpoR on malignant cells. METHODS: RT-PCR, Western blotting, and immunohistochemistry were used to demonstrate that prostate cancer cells express EpoR at both the gene and protein level. Cell proliferation assays and STAT5 phosphorylation were used to demonstrate Epo's mitogenic action and intracellular signaling, respectively. RESULTS: We have demonstrated that transformed prostate epithelial and prostate cancer cell lines, as well as primary prostate tissue, express the EpoR. Importantly, the EpoR on prostate cells are functional, as demonstrated by the observation that each of the cell lines exhibited a dose-dependent proliferative response to Epo, and that Epo triggered STAT5b phosphorylation in the cells. CONCLUSION: Human prostatic epithelial cells and prostate cancer cells express functional EpoR, and Epo serves as a growth factor for these cells. These results have implications for our understanding of normal prostatic growth and development and of the pathobiology of human prostate cancer.     

Prognostic significance of erythropoietin expression in human renal cell carcinoma

OBJECTIVES: To investigate, in a retrospective study, the expression of erythropoietin (Epo) in human renal cell carcinoma (RCC) and its correlation with overall survival, as Epo (an haematopoietic cytokine that regulates the production of red blood cells), with its receptor, was recently localized in non-haematopoietic tissues, e.g. liver, uterus, central nervous system, vascular endothelial cells and solid tumours. PATIENTS AND METHODS: We used data from 113 patients who had radical nephrectomy for RCC between 1990 and 2000, taking sections from formalin-fixed and paraffin wax-embedded tissue blocks. The association between Epo staining and the patients' characteristics was assessed by either chi-squared tests (for categorical variables) or two-sample independent t-tests (for continuous variables). RESULTS: Tissue from 37 patients (33%) was positive for cytoplasmic Epo expression; 76 (67%) samples were negative. Univariate hazard ratio analysis confirmed that those with positive Epo staining were more than twice as likely to die as those with negative staining (hazard ratio 2.34, 95% confidence interval 1.27-4.3). CONCLUSION: This study shows that the expression of Epo in RCC is adversely associated with overall survival. This is the first report of such an association, and might be explained by the loss of Von Hippel-Lindau protein function in clear cell RCC. The expression of Epo might have potential use in clinical trials when stratifying high-risk patients for adjuvant therapy after nephrectomy.