Search

Background Vadadustat is an oral hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitor, a class of drugs that stabilize HIF and stimulate erythropoietin and red-cell production. Meth...

Background and Aims Vadadustat is a small-molecule inhibitor of hypoxia-inducible factor prolyl hydroxylases under development to treat anemia associated with chronic kidney disease (CKD). The vadadustat phase 3 program includes four efficacy and cardiovascular safety outcome trials of vadadustat versus the erythropoiesis-stimulating agent (ESA) darbepoetin alfa. Here we describe detailed results on hematologic efficacy in two phase 3, randomized trials (the PRO2TECT trials) in adult patients with non–dialysis-dependent (NDD) CKD and anemia, in which vadadustat met prespecified noninferiority criteria compared to darbepoetin alfa, with respect to hematologic efficacy (correction/maintenance of hemoglobin [Hb] target concentrations). Method The mean screening Hb level for the ESA-untreated NDD-CKD trial (NCT02648347) had to be Results A total of 3,476 patients (1751 ESA-untreated and 1725 ESA-treated) were randomized 1:1 to vadadustat or darbepoetin alfa. In both trials, vadadustat was noninferior to darbepoetin alfa with regard to the difference of mean change in Hb concentrations between baseline and PEP, as well as between baseline and SEP. The respective proportions of patients (vadadustat vs. darbepoetin alfa) with an average Hb value within the geography-specific target range in the PEP and SEP were 50.4% versus 50.2% and 43.1% versus 43.5% in the ESA-untreated trial and 60.1% versus 60.7% and 50.7% versus 49.0% in the ESA-treated trial. The proportion of patients (vadadustat vs darbepoetin alfa) who achieved an Hb increase >1.0 g/dL from baseline to week 52 was assessed only for the ESA-untreated trial and was 87.7% (95% CI: 85.4%, 89.8%) for vadadustat versus 88.0% (95% CI: 85.6%, 90.0%) for darbepoetin alfa. Hematologic parameters at time points within the PEP and SEP are presented in Table 1. In both the ESA-untreated and ESA-treated trials, the reticulocyte count trended up from baseline through week 52 for vadadustat and trended down from baseline for darbepoetin alfa. Trends in erythrocyte mean corpuscular volume and erythrocyte mean corpuscular Hb were largely unremarkable by week 52 in both treatment groups. Conclusion Vadadustat demonstrated similar profiles across erythrocyte parameters compared with darbepoetin alfa in the treatment of adults with anemia in CKD not on dialysis, whether ESA-untreated or ESA-treated at study entry.

Background and Aims Vadadustat is a small-molecule inhibitor of hypoxia-inducible factor prolyl hydroxylase being developed for treatment of anemia associated with chronic kidney disease (CKD). The vadadustat phase 3 program includes four efficacy and cardiovascular safety outcome trials of vadadustat versus the erythropoiesis-stimulating agent (ESA) darbepoetin alfa. Here we describe detailed results on hematologic efficacy in two of the four phase 3, randomized, open-label, sponsor-blind trials (the INNO2VATE trials) in adult patients with dialysis-dependent (DD) CKD and anemia, where vadadustat met prespecified noninferiority criteria compared with darbepoetin alfa with respect to cardiovascular safety and correction/maintenance of hemoglobin (Hb) target concentrations. Method The mean screening Hb range for the incident DD-CKD trial (NCT02865850) was 8.0-11.0 g/dL; for the prevalent DD-CKD trial (NCT02892149), it was 8.0-11.0 g/dL in the United States (US) and 9.0-12.0 g/dL for non-US. Patients in the incident and prevalent DD-CKD trials had initiated dialysis within 12 weeks with established ESA treatment prior to screening, respectively. Vadadustat starting dose was 300 mg/day for all patients, whereas initial darbepoetin alfa dose depended on each patient’s prior dose or product label. Both vadadustat and darbepoetin alfa doses were titrated according to prespecified dosing algorithms to achieve target Hb concentrations (US: 10-11 g/dL; non-US: 10-12 g/dL) during the primary evaluation period (PEP; weeks 24-36) and the secondary evaluation period (SEP; weeks 40-52). Herein, we present topline results from PEP and SEP endpoints, as well as other, more detailed hematologic erythrocyte parameters. Results A total of 3923 patients (369 with incident DD-CKD and 3554 with prevalent DD-CKD) were randomized 1:1 to vadadustat or darbepoetin alfa. Vadadustat was noninferior to darbepoetin alfa in achieving target-range Hb concentrations (primary efficacy endpoint) among patients who were new to, or established on, dialysis. The respective proportions of patients (vadadustat vs. darbepoetin alfa) with an average Hb value within the geography-specific target range in the PEP and SEP were 43.6% versus 56.9% and 39.8% versus 41.0% in the incident trial and 49.2% versus 53.2% and 44.3% versus 50.9% in the prevalent dialysis trial. The proportion of patients who achieved an Hb increase >1.0 g/dL from baseline to week 52 was assessed only for the incident trial and was 84.0% (95% CI: 77.8%, 89.0%) for vadadustat versus 89.9% (95% CI: 84.7%, 93.8%) for darbepoetin alfa. Hematologic erythrocyte parameters at time points within the PEP and SEP are presented in Table 1. In the incident trial, reticulocyte count was slightly increased from baseline at 28 and 52 weeks for vadadustat, whereas for darbepoetin alfa, reticulocyte count was slightly decreased or unchanged in both trials. Erythrocyte mean corpuscular volume and erythrocyte mean corpuscular Hb showed increases by week 52 for both groups. Conclusion Vadadustat demonstrated similar profiles across erythrocyte parameters compared with darbepoetin alfa in the treatment of anemia associated with CKD in adults in both incident dialysis and prevalent dialysis settings.

Vadadustat is an oral hypoxia-inducible factor prolyl hydroxylase inhibitor, a class of compounds that stimulate endogenous erythropoietin production.We conducted two randomized, open-label, noninferiority phase 3 trials to evaluate the safety and efficacy of vadadustat, as compared with darbepoetin alfa, in patients with anemia and incident or prevalent dialysis-dependent chronic kidney disease (DD-CKD). The primary safety end point, assessed in a time-to-event analysis, was the first occurrence of a major adverse cardiovascular event (MACE, a composite of death from any cause, a nonfatal myocardial infarction, or a nonfatal stroke), pooled across the trials (noninferiority margin, 1.25). A key secondary safety end point was the first occurrence of a MACE plus hospitalization for either heart failure or a thromboembolic event. The primary and key secondary efficacy end points were the mean change in hemoglobin from baseline to weeks 24 to 36 and from baseline to weeks 40 to 52, respectively, in each trial (noninferiority margin, -0.75 g per deciliter).A total of 3923 patients were randomly assigned in a 1:1 ratio to receive vadadustat or darbepoetin alfa: 369 in the incident DD-CKD trial and 3554 in the prevalent DD-CKD trial. In the pooled analysis, a first MACE occurred in 355 patients (18.2%) in the vadadustat group and in 377 patients (19.3%) in the darbepoetin alfa group (hazard ratio, 0.96; 95% confidence interval [CI], 0.83 to 1.11). The mean differences between the groups in the change in hemoglobin concentration were -0.31 g per deciliter (95% CI, -0.53 to -0.10) at weeks 24 to 36 and -0.07 g per deciliter (95% CI, -0.34 to 0.19) at weeks 40 to 52 in the incident DD-CKD trial and -0.17 g per deciliter (95% CI, -0.23 to -0.10) and -0.18 g per deciliter (95% CI, -0.25 to -0.12), respectively, in the prevalent DD-CKD trial. The incidence of serious adverse events in the vadadustat group was 49.7% in the incident DD-CKD trial and 55.0% in the prevalent DD-CKD trial, and the incidences in the darbepoetin alfa group were 56.5% and 58.3%, respectively.Among patients with anemia and CKD who were undergoing dialysis, vadadustat was noninferior to darbepoetin alfa with respect to cardiovascular safety and correction and maintenance of hemoglobin concentrations. (Funded by Akebia Therapeutics and Otsuka Pharmaceutical; INNO

Current clinical practice guidelines for anemia management in non–dialysis-dependent chronic kidney disease (NDD-CKD) recommend the use of erythropoiesis-stimulating agents (ESAs) as standard of care. Vadadustat, an investigational oral hypoxia-inducible factor prolyl-hydroxylase inhibitor, stimulates endogenous erythropoietin production. The PRO2TECT program comprises 2 global, Phase 3, randomized, open-label, active-controlled, sponsor-blind clinical trials to evaluate safety and efficacy of vadadustat vs darbepoetin alfa in adult patients with anemia associated with NDD-CKD. Patients recruited into the ESA-untreated NDD-CKD trial (N = 1751) had hemoglobin

Background Erythropoiesis-stimulating agents (ESAs) are currently the mainstay of treatment for anaemia of chronic kidney disease (CKD). Vadadustat is an investigational oral hypoxia-inducible factor prolyl-hydroxylase inhibitor that stimulates endogenous erythropoietin formation. The INNO2VATE programme comprises two studies designed to evaluate the safety and efficacy of vadadustat versus the ESA darbepoetin alfa in ameliorating anaemia in patients with dialysis-dependent CKD (DD-CKD). Here we describe the trial design along with patient demographics and baseline characteristics. Methods Two Phase 3, open-label, sponsor-blind, active-controlled trials enrolled adults with anaemia of CKD who recently initiated dialysis and had limited ESA exposure (incident DD-CKD trial) or were receiving maintenance dialysis with ESA treatment (prevalent DD-CKD trial). Study periods include correction/conversion (Weeks 0–23), maintenance (Weeks 24–52), long-term treatment (Weeks 53 to end of treatment) and safety follow-up. The primary safety endpoint is the time to the first major adverse cardiovascular event and the primary efficacy endpoint is the change in haemoglobin (baseline to Weeks 24–36). Results A total of 369 and 3554 patients were randomized in the incident DD-CKD and prevalent DD-CKD trials, respectively. Demographics and baseline characteristics were similar among patients in both trials and comparable to those typically observed in DD-CKD. Conclusions The two INNO2VATE trials will provide important information on the safety and efficacy of a novel approach for anaemia management in a diverse DD-CKD population. Demographics and baseline characteristics of enrolled patients suggest that study results will be representative for a large proportion of the DD-CKD population.

Applying transcriptional profiling analysis to myeloblasts from 59 adult patients with acute myeloid leukemia (AML) treated at our institution, we found that expression of the three-amino acid loop extension (TALE) homeobox gene TG-Interacting Factor 1 (TGIF1) correlated with overall and relapse-free survival, which was then confirmed in two other cohorts of patients.Moreover, TGIF1 expression correlated with survival for all cytogenetic risk groups and was an independent prognostic factor in multivariate analysis. To elucidate the mechanism, we used Tgif1 knockout mice in which acute or chronic myeloid leukemia was induced through retroviral transfer of the MLL-AF9 or BCR-ABL fusion genes into bone marrow cells. Loss of Tgif1 accelerated disease progression, shortened survival, attenuated the response to chemotherapy, and doubled the frequency of leukemia-initiating cells. RNA-based sequencing analysis showed that genes associated with transforming growth factor-β (TGF-β) and retinoic acid signaling pathways were differentially affected in Tgif1-/- compared to Tgif1+/+ leukemia cells.

Iron stores assuring optimal efficacy/safety for erythropoiesis are unknown in the dialysis population. Using multicenter trial data, we related safety profiles, erythropoiesis-stimulating agent (ESA), and intravenous iron dosing to achieved iron stores in 441 subjects randomized 2 : 1 to ferric citrate or active control as their phosphate binder over 52 weeks. Intravenous iron was given at each site's discretion if ferritin ≤ 1,000 ng/mL and transferrin saturation ≤ 30%. Multivariable time-dependent Cox regression jointly related the primary safety outcome (composite of cardiac, infection, gastrointestinal, and hepatobiliary serious adverse events) to moving averages of ferritin and transferrin saturation over the preceding 90 days with covariate adjustment. Multivariable generalized estimating equations related elevated ESA and intravenous iron doses to trailing 90-day averages of ferritin and transferrin saturation with covariate adjustment. The adjusted hazard ratio for the safety composite per 10% increase in transferrin saturation was 0.84 (95% confidence interval 0.68 - 1.02, p = 0.08) and 1.09 (0.86 - 1.35, p = 0.48) per 400 ng/mL increase in ferritin. The adjusted hazard ratio for the safety composite was 0.50 (0.29 - 0.88, p = 0.016) for the highest transferrin saturation tertile vs. the lowest. Adjusted odds ratios for higher intravenous iron dose were lower in the highest (0.23 [0.16 - 0.35], p 0.001) and middle transferrin saturation tertile (0.42 [0.31 - 0.57], p 0.001) vs. lowest. Incidence of elevated ESA dose was lower in the highest transferrin saturation tertile (p = 0.01). Ferritin did not predict clinical events or ESA dose. Transferrin saturation may be a better marker than serum ferritin to judge optimal iron stores in dialysis patients. Transferrin saturations 34% are safe and provide maximal efficacy. .

International audience; Drug resistance (DR) is a phenomenon characterized by the tolerance of a disease to pharmaceutical treatment. In cancer patients, DR is one of the main challenges that limit the therapeutic potential of the existing treatments. Therefore, overcoming DR by restoring the sensitivity of cancer cells would be greatly beneficial. In this context, mathematical modeling can be used to provide novel therapeutic strategies that maximize the efficiency of anti-cancer agents and potentially overcome DR. In this paper, we present a new multiscale model devoted to the interaction of potential treatments with multiple myeloma (MM) development. In this model, MM cells are represented as individual objects that move, divide, and die by apoptosis. The fate of each cell depends on intracellular and extracellular regulation, as well as the administered treatment. The model is used to explore the combined effects of a tyrosine-kinase inhibitor (TKI) with a pentose phosphate pathway (PPP) inhibitor. We use numerical simulations to tailor effective and safe treatment regimens that may eradicate the MM tumors. The model suggests that an interval for the daily dose of the PPP inhibitor can maximize the responsiveness of MM cells to the treatment with TKIs. Then, it demonstrates that the combination of high-dose pulsatile TKI treatment with high-dose daily PPP inhibitor therapy can potentially eradicate the tumor.The predictions of numerical simulations using such a model can be considered as testable hypotheses in future pre-clinical experiments and clinical studies.

The paper is devoted to multi-scale modelling of erythropoiesis and hemoglobin production. Red blood cells, which carry oxygen from the lungs to the other body tissues, are produced in the bone marrow of adult humans in cell units called erythroblastic islands. Erythroblastic islands are composed by a central macrophage surrounded by erythroid cells in different stages of maturation. Immature cells, the colony-forming units-erythroid, make a choice between self-renewal, differentiation and apoptosis determined by the intracellular proteins and extracellular substances. Moreover, this choice is regulated by erythropoietin and other hormones. Erythropoietin is produced in the kidney in response to hypoxia from decreased numbers of red blood cells, and it is delivered in the plasma to the bone marrow. Erythropoietin stimulates differentiation of erythroid cells and increases their proliferation by downregulating apoptosis. The rate of erythropoietin production depends on the level of hemoglobin in blood which is function of the number of circulating red blood cells. Hemoglobin is produced in the erythroid cells within the bone marrow in the process of their terminal differentiation. Thus, there is a feedback between production of red blood cells by the bone marrow, the level of hemoglobin contained in these cells and the level of erythropoietin. The multi-scale model developed in this work includes erythroid cells in the bone marrow, their intracellular and extracellular regulations, hemoglobin production, and the feedback by erythropoietin. This model describes normal functioning of erythropoiesis and its response to anemia resulting from the loss of red blood cells.

Multiple myeloma (MM) infiltrates bone marrow and causes anemia by disrupting erythropoiesis, but the effects of marrow infiltration on anemia are difficult to quantify. Marrow biopsies of newly diagnosed MM patients were analyzed before and after four 28-day cycles of non-erythrotoxic remission induction chemotherapy. Complete blood cell counts and serum paraprotein concentrations were measured at diagnosis and before each chemotherapy cycle. At diagnosis, marrow area infiltrated by myeloma correlated negatively with hemoglobin, erythrocytes, and marrow erythroid cells. After successful chemotherapy, patients with less than 30% myeloma infiltration at diagnosis had no change in these parameters, whereas patients with more than 30% myeloma infiltration at diagnosis increased all three parameters. Clinical data were used to develop mathematical models of the effects of myeloma infiltration on the marrow niches of terminal erythropoiesis, the erythroblastic islands (EBIs). A hybrid discrete-continuous model of erythropoiesis based on EBI structure/function was extended to sections of marrow containing multiple EBIs. In the model, myeloma cells can kill erythroid cells by physically destroying EBIs and by producing proapoptotic cytokines. Following chemotherapy, changes in serum paraproteins as measures of myeloma cells and changes in erythrocyte numbers as measures of marrow erythroid cells allowed modeling of myeloma cell death and erythroid cell recovery, respectively. Simulations of marrow infiltration by myeloma and treatment with non-erythrotoxic chemotherapy demonstrate that myeloma-mediated destruction and subsequent reestablishment of EBIs and expansion of erythroid cell populations in EBIs following chemotherapy provide explanations for anemia development and its therapy-mediated recovery in MM patients.

Ferric citrate (FC) is a phosphate binder with shown efficacy and additional effects on iron stores and use of intravenous (iv) iron and erythropoiesis-stimulating agents (ESAs). We provide detailed analyses of changes in iron/hematologic parameters and iv iron/ESA use at time points throughout the active control period of a phase 3 international randomized clinical trial. In all, 441 subjects were randomized (292 to FC and 149 to sevelamer carbonate and/or calcium acetate [active control (AC)]) and followed for 52 weeks. Subjects on FC had increased ferritin and transferrin saturation (TSAT) levels compared with subjects on AC by week 12 (change in ferritin, 114.1±29.35 ng/ml; P

International audience; Multiple myeloma (MM) is a genetically complex hematological cancer that is characterized by proliferation of malignant plasma cells in the bone marrow. MM evolves from the clonal premalignant disorder monoclonal gammopathy of unknown significance (MGUS) by sequential genetic changes involving many different genes, resulting in dysregulated growth of multiple clones of plasma cells. The migration, survival, and proliferation of these clones require the direct and indirect interactions with the non-hematopoietic cells of the bone marrow. We develop a hybrid discrete-continuous model of MM development from the MGUS stage. The discrete aspect of the model is observed at the cellular level: cells are represented as individual objects which move, interact, divide, and die by apoptosis. Each of these actions is regulated by intracellular and extracellular processes as described by continuous models. The hybrid model consists of the following submodels that have been simplified from the much more complex state of evolving MM: cell motion due to chemotaxis, intracellular regulation of plasma cells, extracellular regulation in the bone marrow, and acquisition of mutations upon cell division. By extending a previous, simpler model in which the extracellular matrix was considered to be uniformly distributed, the new hybrid model provides a more accurate description in which cytokines are produced by the marrow microenvironment and consumed by the myeloma cells. The complex multiple genetic changes in MM cells and the numerous cell-cell and cytokine-mediated interactions between myeloma cells and their marrow microenviroment are simplified in the model such that four related but evolving MM clones can be studied as they compete for dominance in the setting of intraclonal heterogeneity.

Most dialysis patients require phosphate binders to control hyperphosphatemia. Ferric citrate has been tested in phase 2 trials as a phosphate binder. This trial was designed as a dose-response and efficacy trial.Prospective, phase 3, multicenter, open-label, randomized clinical trial.151 participants with hyperphosphatemia on maintenance hemodialysis therapy.Fixed dose of ferric citrate taken orally as a phosphate binder for up to 28 days (1, 6, or 8 g/d in 51, 52, and 48 participants, respectively).Primary outcome is dose-response of ferric citrate on serum phosphorus level; secondary outcomes are safety and tolerability.Serum chemistry tests including phosphorus, safety data.151 participants received at least one dose of ferric citrate. Mean baseline phosphorus levels were 7.3 ± 1.7 (SD) mg/dL in the 1-g/d group, 7.6 ± 1.7 mg/dL in the 6-g/d group, and 7.5 ± 1.6 mg/dL in the 8-g/d group. Phosphorus levels decreased in a dose-dependent manner (mean change at end of treatment, -0.1 ± 1.3 mg/dL in the 1-g/d group, -1.9 ± 1.7 mg/dL in the 6-g/d group, and -2.1 ± 2.0 mg/dL in the 8-g/d group). The mean difference in reduction in phosphorus levels between the 6- and 1-g/d groups was 1.3 mg/dL (95% CI, 0.69 to 1.9; P0.001), between the 8- and 1-g/d groups was 1.5 mg/dL (95% CI, 0.86 to 2.1; P0.001), and between the 8- and 6-g/d groups was 0.21 mg/dL (95% CI, -0.39 to 0.81; P = 0.5). The most common adverse event was stool discoloration.Sample size and duration confirm efficacy, but limit our ability to confirm safety.Ferric citrate is efficacious as a phosphate binder in a dose-dependent manner. A phase 3 trial is ongoing to confirm safety and efficacy.

Chronic kidney disease associated mineral and bone disorders arise as a result of aberrant bone mineral metabolism in patients with advancing levels of renal dysfunction and end-stage renal disease. One of the cornerstones of treatment is the use of phosphate-binding agents. We describe the rationale and study design for a clinical trial to assess the safety and efficacy of ferric citrate as a phosphate binder. This trial is a three-period, international, multicenter, randomized, controlled clinical trial to assess the safety and efficacy of ferric citrate as a phosphate binder, consisting of a 2-week washout period, a 52-week safety assessment period in which subjects are randomized to ferric citrate or active control, and a 4-week efficacy assessment period in which subjects randomized to ferric citrate in the safety assessment period are randomized to ferric citrate or placebo. Eligible subjects include end-stage renal disease patients who have been treated with thrice-weekly hemodialysis or peritoneal dialysis for at least 3 months in dialysis clinics in the United States and Israel. Primary outcome measure will be the effect of ferric citrate vs. placebo on the change in serum phosphorus. Safety assessments will be performed by monitoring adverse events, concomitant medication use, and sequential blood chemistries (including iron parameters, phosphorus, and calcium). This three-period trial will assess the efficacy of ferric citrate as a phosphate binder. If proven safe and efficacious, ferric citrate will likely provide an additional phosphate binder to treat chronic kidney disease associated mineral and bone disorders.

It is widely accepted that elevated plasma total homocysteine is an independent risk factor for vascular disease. The relation is believed to be causal, but there is no generally accepted mechanism for the pathophysiology involved. The metabolic precursor of homocysteine in all tissues is S-adenosylhomocysteine (AdoHcy). AdoHcy is present in normal human plasma at concentrations approximately 1-500th of those of homocysteine, a fact that presents difficulties in measurement. The requirement for specialized equipment, complicated time-consuming methodology, or both is a reason that measurement of plasma AdoHcy has not generally been carried out in large studies. A recently published rapid immunoassay for AdoHcy in human plasma should make measurement of this important metabolite available for general use. Advantages of the measurement of plasma AdoHcy include 1) a smaller overlap of values between control subjects and patients, and thus the possibility of observing significant differences in fewer samples, 2) an accepted mechanism of metabolic activity as an inhibitor of all S-adenosylmethionine-mediated methyltransferases, and 3) evidence (from recent studies) that a higher plasma concentration of AdoHcy is a more sensitive indicator of vascular disease than is a higher plasma concentration of homocysteine.

Red blood cell production rates increase rapidly following blood loss or hemolysis, but the expansion of erythropoiesis in these anemic states is tightly regulated such that rebound polycythemia does not occur. The erythroid cells that respond to erythropoietic stimulation or suppression are the progenitor stages of burst-forming units–erythroid (BFU-Es) and colony-forming units–erythroid (CFU-Es). Results from an early study of the changes in the size, location, and cell cycling status of BFU-E and CFU-E populations in mice under normal conditions, erythropoietic stimulation, and erythropoietic suppression are used as reference points to review subsequent developments related to erythroid progenitor populations and regulation of their size. The review concerns development of erythroid progenitor populations mainly in mice and humans, with a focus on the mechanisms related to the rapid but highly regulated expansion of erythropoiesis in spleens of erythropoietically stimulated mice. Current knowledge is used as a model of erythroid progenitor populations in mice under normal, erythropoietically suppressed, and erythropoietically stimulated conditions. Clinical applications of information learned from studies of erythropoietic expansion, in terms of current therapies for anemia, are reviewed.

Improved understanding of the oxygen-dependent regulation of erythropoiesis has provided new insights into the pathogenesis of anaemia associated with renal failure and has led to the development of novel therapeutic agents for its treatment. Hypoxia-inducible factor (HIF)-2 is a key regulator of erythropoiesis and iron metabolism. HIF-2 is activated by hypoxic conditions and controls the production of erythropoietin by renal peritubular interstitial fibroblast-like cells and hepatocytes. In anaemia associated with renal disease, erythropoiesis is suppressed due to inadequate erythropoietin production in the kidney, inflammation and iron deficiency; however, pharmacologic agents that activate the HIF axis could provide a physiologic approach to the treatment of renal anaemia by mimicking hypoxia responses that coordinate erythropoiesis with iron metabolism. This Review discusses the functional inter-relationships between erythropoietin, iron and inflammatory mediators under physiologic conditions and in relation to the pathogenesis of renal anaemia, as well as recent insights into the molecular and cellular basis of erythropoietin production in the kidney. It furthermore provides a detailed overview of current clinical experience with pharmacologic activators of HIF signalling as a novel comprehensive and physiologic approach to the treatment of anaemia.

Background Phosphate binders are the cornerstone of hyperphosphatemia management in dialysis patients. Ferric citrate is an iron-based oral phosphate binder that effectively lowers serum phosphorus levels. Study Design 52-week, open-label, phase 3, randomized, controlled trial for safety-profile assessment. Setting & Participants Maintenance dialysis patients with serum phosphorus levels ≥6.0mg/dL after washout of prior phosphate binders. Intervention 2:1 randomization to ferric citrate or active control (sevelamer carbonate and/or calcium acetate). Outcomes Changes in mineral bone disease, protein-energy wasting/inflammation, and occurrence of adverse events after 1 year. Measurements Serum calcium, intact parathyroid hormone, phosphorus, aluminum, white blood cell count, percentage of lymphocytes, serum urea nitrogen, and bicarbonate. Results There were 292 participants randomly assigned to ferric citrate, and 149, to active control. Groups were well matched. For mean changes from baseline, phosphorus levels decreased similarly in the ferric citrate and active control groups (−2.04±1.99 [SD] vs −2.18±2.25mg/dL, respectively; P =0.9); serum calcium levels increased similarly in the ferric citrate and active control groups (0.22±0.90 vs 0.31±0.95mg/dL; P =0.2). Hypercalcemia occurred in 4 participants receiving calcium acetate. Parathyroid hormone levels decreased similarly in the ferric citrate and active control groups (−167.1±399.8 vs −152.7±392.1pg/mL; P =0.8). Serum albumin, bicarbonate, serum urea nitrogen, white blood cell count and percentage of lymphocytes, and aluminum values were similar between ferric citrate and active control. Total and low-density lipoprotein cholesterol levels were lower in participants receiving sevelamer than those receiving ferric citrate and calcium acetate. Fewer participants randomly assigned to ferric citrate had serious adverse events compared with active control. Limitations Open-label study, few peritoneal dialysis patients. Conclusions Ferric citrate was associated with similar phosphorus control compared to active control, with similar effects on markers of bone and mineral metabolism in dialysis patients. There was no evidence of protein-energy wasting/inflammation or aluminum toxicity, and fewer participants randomly assigned to ferric citrate had serious adverse events. Ferric citrate is an effective phosphate binder with a safety profile comparable to sevelamer and calcium acetate.

S-Adenosylhomocysteine (SAH) is the metabolic precursor of all the homocysteine (Hcy) produced in the body. It is formed by the enzyme SAH hydrolase in a reversible reaction. In a previous study we have shown that plasma SAH is a more sensitive indicator of the risk for cardiovascular disease, and in a second study involving patients with renal disease, we also showed that it is a more sensitive indicator of renal insufficiency than plasma Hcy. However, in the latter study, the patients with renal disease were older and had a variety of other diseases such as diabetes and primary hypertension, which are associated with vascular disease and which could reduce renal function by involvement of the kidneys. Our objective was to rule out these complicating factors as the cause of the elevated SAH in renal disease and determine whether renal insufficiency alone was the cause of the elevated SAH. We therefore measured SAH, Hcy, folate, and vitamin B12 in 23 patients between the ages of 1 and 18 years with a wide range of renal function, but who had none of these complicating factors. Glomerular filtration rate (GFR) was calculated using serum creatinine according to the Schwartz formula. None of the children were deficient in folate or vitamin B12. After adjusting for age, folate, and vitamin B12, there was a modest and insignificant decrease of 0.033 micromol/L of Hcy associated with an increase of 1 mL/min of GFR (95% confidence interval, -0.066 to 0.0002). However, there was a strong and statistically significant association between log(SAH) and log(GFR): P < .0005, R2 = 0.76. This result suggests that plasma SAH rather than Hcy is the metabolite primarily affected in renal disease. We suggest that plasma Hcy elevations that have been linked to vascular disease may be due to elevated SAH resulting from renal insufficiency.

Patients on dialysis require phosphorus binders to prevent hyperphosphatemia and are iron deficient. We studied ferric citrate as a phosphorus binder and iron source. In this sequential, randomized trial, 441 subjects on dialysis were randomized to ferric citrate or active control in a 52-week active control period followed by a 4-week placebo control period, in which subjects on ferric citrate who completed the active control period were rerandomized to ferric citrate or placebo. The primary analysis compared the mean change in phosphorus between ferric citrate and placebo during the placebo control period. A sequential gatekeeping strategy controlled study-wise type 1 error for serum ferritin, transferrin saturation, and intravenous iron and erythropoietin-stimulating agent usage as prespecified secondary outcomes in the active control period. Ferric citrate controlled phosphorus compared with placebo, with a mean treatment difference of -2.2±0.2 mg/dl (mean±SEM) (P

BACKGROUND Although plasma total homocysteine has been identified as an independent risk factor for vascular disease in a multitude of studies, there is a considerable overlap in values between patients at risk and control subjects. The difference in values can be used to distinguish statistically between the 2 groups, provided each group is large enough; however, discriminating between individual patients at risk and control subjects is difficult. OBJECTIVE We investigated whether the precursor of homocysteine, S-adenosylhomocysteine, is a more sensitive indicator of risk. DESIGN We measured plasma total homocysteine, S-adenosylhomocysteine, S-adenosylmethionine, creatinine, folate, and vitamin B-12 in 30 patients with proven cardiovascular disease and 29 age- and sex-matched control subjects. RESULTS The homocysteine values (+/-SD) were 12.8 +/- 4.9 (95% CI: 11.0, 14.7) micromol/L for patients and 11.0 +/- 3.2 (9.8, 12.2) micromol/L for control subjects. The S-adenosylhomocysteine values were 40.0 +/- 20.6 (32.3, 47.7) nmol/L for patients and 27.0 +/- 6.7 (24.5, 30.0) nmol/L for control subjects (P = 0.0021). The S-adenosylmethionine values were 121.8 +/- 42.9 (105.8, 137.8) nmol/L for patients and 103.9 +/- 21.8 (95.6, 112.2) nmol/L for control subjects (P = 0.0493). The creatinine values were 110 +/- 27 (97, 120) micromol/L for patients and 97 +/- 9 (80, 100) micromol/L for control subjects (P = 0.0025). Values for folate and vitamin B-12 did not differ significantly between groups. CONCLUSIONS Plasma S-adenosylhomocysteine appears to be a much more sensitive indicator of the difference between patients with cardiovascular disease and control subjects than is homocysteine. Both plasma total homocysteine and S-adenosylhomocysteine are significantly correlated with plasma creatinine in patients.

Deficiency of folate or vitamin B12 (cobalamin) causes megaloblastic anemia, a disease characterized by pancytopenia due to the excessive apoptosis of hematopoietic progenitor cells. Clinical and experimental studies of megaloblastic anemia have demonstrated an impairment of DNA synthesis and repair in hematopoietic cells that is manifested by an increased percentage of cells in the DNA synthesis phase (S phase) of the cell cycle, compared with normal hematopoietic cells. Both folate and cobalamin are required for normal de novo synthesis of thymidylate and purines. However, previous studies of impaired DNA synthesis and repair in megaloblastic anemia have concerned mainly the decreased intracellular levels of thymidylate and its effects on nucleotide pools and misincorporation of uracil into DNA. An in vitro model of folate-deficient erythropoiesis was used to study the relationship between the S-phase accumulation and apoptosis in megaloblastic anemia. The results indicate that folate-deficient erythroblasts accumulate in and undergo apoptosis in the S phase when compared with control erythroblasts. Both the S-phase accumulation and the apoptosis were induced by folate deficiency in erythroblasts fromp53 null mice. The complete reversal of the S-phase accumulation and apoptosis in folate-deficient erythroblasts required the exogenous provision of specific purines or purine nucleosides as well as thymidine. These results indicate that decreased de novo synthesis of purines plays as important a role as decreased de novo synthesis of thymidylate in the pathogenesis of megaloblastic anemia.

Chemotherapeutic agents exert their antitumor effects by inducing apoptosis. The microculture kinetic (MiCK) assay provides an automated, continuous means of monitoring apoptosis in a cell population. We used the MiCK assay to determine the chemosensitivities of the human promyelocytic HL-60 and lymphoblastic CEM cell lines and leukemia cells freshly isolated from patients with acute nonlymphocytic (ANLL) or acute lymphocytic (ALL) leukemias. Continuous monitoring of apoptosis in the MiCK assay permits determination of the time to the maximum apoptosis (Tm) and its two components which are initiation time (Ti) and development time (Td). Duration of the three timing components of apoptosis varies from hours to days depending on the drug, drug concentration, and type of target cells. In the MiCK assay, the extent of apoptosis is reported in kinetic units of apoptosis. Kinetic units are determined by the slope of the curve created when optical density caused by cell blebbing is plotted as a function of time. Using the leukemia cell lines, we define the relationship between kinetic units determined by the MiCK assay and the percentage of morphologically apoptotic cells in the culture. Flow cytometry analysis of apoptosis in Annexin-V-fluorescein isothiocyanate–labeled preparations of HL-60 and CEM cells was also used to compare with data obtained by the MiCK assay. The feasibility of the MiCK assay of apoptosis as a chemosensitivity test was confirmed by its comparison with a 3H-thymidine incorporation assay. We show that samples from 10 ANLL and ALL patients patients tested for sensitivity to various doses of idarubicin (IDR), daunorubicin (DNR), or mitoxantrone (MTA) gave the same percentages of apoptotic cells when calculated by the MiCK assay as when determined by morphological analysis. The MiCK assay was used for dose-response analyses of the sensitivities to IDR, DNR, and MTA of leukemia cells from 4 other patients (2 ANLL and 2 ALL). The results from both cell lines and patient samples indicate that ANLL cells are more sensitive than ALL cells to all three of these chemotherapeutic agents. However, for individual patients the chemosensitivities varied significantly among the three chemotherapeutic agents. These varying responses to IDR, DNR, and MTA indicate that the MiCK assay results can be of potential use in designing a treatment regimen for a specific patient with acute leukemia. Among several drugs of presumed similar efficacy, the MiCK assay can permit the selection of the specific chemotherapeutic agent that causes the most apoptosis in the patient's leukemic cells.© 1998 by The American Society of Hematology.

Clinical studies have indicated that folate deficiency may enhance the development of various malignancies. In animal studies that examined the effect of folate deficiency on malignancies, conflicting results have been reported. In some studies, folate deficiency increased the development and growth of malignant tumors; in others, it decreased the development and growth of malignancies. We examined the effect of transient folate deficiency on the development of leukemia in mice infected with the anemia-inducing strain of Friend leukemia virus. Friend virus disease can be considered as a model for human acute leukemias that are preceded by a preleukemic period. These include leukemias that develop in patients who received previous chemotherapy and/or radiation therapy, as well as patients with chronic granulocytic leukemia or myelodysplasia. Folate deficiency around the time of Friend virus-infection delayed the onset but increased the incidence of leukemia. The rates of rearrangement of the Spi-1 (PU.1 ) oncogene by provirus integration and alteration of the p53 tumor-suppressor gene were the same in leukemia cell lines derived from folate-deficient mice as they were in cell lines from control mice. These results indicate that folate deficiency did not exert its enhancement of leukemogenesis through changes in either Spi-1 or p53, even though these two genes have been found to be the most frequently altered ones in Friend virus-induced leukemias. Our results suggest that folate deficiency may enhance the development of acute leukemia in patients who are at high risk for this disease.

An in vitro model of folate-deficient erythropoiesis has been developed using proerythroblasts isolated from the spleens of Friend virus-infected mice fed an amino acid-based, folate-free diet. Control proerythroblasts were obtained from Friend virus-infected mice fed the same diet plus 2 mg folic acid/kg diet. Our previous studies showed that, after 20 to 32 hours of culture in folate-deficient medium with 4 U/mL of erythropoietin, the folate-deficient proerythroblasts underwent apoptosis, whereas control erythroblasts survived and differentiated into reticulocytes over a period of 48 hours. The addition of folic acid or thymidine to the folate-deficient medium prevented the apoptosis of the folate-deficient erythroblasts, thereby implicating decreased thymidylate synthesis as the main cause of apoptosis in the folate-deficient erythroblasts. In the study reported here, we examined intracellular folate levels, uracil misincorporation into DNA, p53 and p21 proteins, and reticulocyte formation in erythroblasts cultured in folate-deficient or control medium. In all experiments, the folate-deficient erythroblasts cultured in folate-deficient medium gave results that varied significantly from folate-deficient erythroblasts cultured in control medium or control erythroblasts cultured in either folate-deficient or control media. Folate-deficient erythroblasts cultured in folate-deficient medium had marked decreases in all coenzyme forms of folate that persisted throughout culture, increased uracil misincorporation into DNA, persistent accumulations of p53 and p21, and decreased reticulocyte production but increased size of individual reticulocytes. A model of folate-deficient erythropoiesis based on apoptosis of late stage erythroblasts is presented. This model provides explanations for the clinical findings in megaloblastic anemia.

Deficiency of the vitamin folic acid causes pancytopenia by decreasing the production of new blood cells. Although impaired DNA synthesis and destruction of hematopoietic cells have been implicated, the mechanism by which folate deficiency decreases blood cell production is uncertain. An in vitro model of folate-deficient erythropoiesis was developed by using proerythroblasts isolated from folate-deficient mice that were infected with Friend leukemia virus. Proerythroblasts from folate-deficient mice had one-tenth the total folate as did proerythroblasts from control mice. The folate-deficient proerythroblasts underwent apoptosis, a form of programmed cell death, after 20-32 h in culture in folate-deficient medium. At the time of apoptosis the cells had differentiated into the later erythroblast stages and some had begun hemoglobin synthesis. Addition of either folic acid or thymidine, but not deoxycytidine or inosine, to the folate-deficient medium prevented the apoptosis and permitted proliferation and differentiation of the proerythroblasts into reticulocytes. The prevention of apoptosis by thymidine indicates (i) that decreased thymidylate synthesis plays a role in erythroblast apoptosis and the anemia of folate deficiency and (ii) that DNA cleavage is likely to be a primary event in the apoptosis of folate-deficient erythroblasts. Apoptosis of erythroblasts in the late stages of differentiation leads to decreased erythrocyte production and to anemia. The increased erythropoietin produced in response to the anemia increases the number of erythroid progenitor cells in the differentiation stages preceding those in which the cells undergo apoptosis. This population shift to earlier stage erythroblasts and proerythroblasts is characteristic of bone marrows of individuals with folate deficiency anemia.

Erythropoietin (EPO) is the glycoprotein hormone which controls the production of erythrocytes in mammals. Erythrocytes contain hemoglobin which delivers oxygen from the lungs to other tissues throughout the body. The amount of oxygen delivered to the tissues is controlled by the number of erythrocytes in the bloodstream. Erythrocytes have a finite life span, and, therefore, maintenance of normal oxygen delivery requires continuous replacement of those aged erythrocytes normally lost from the circulation. Adjustments in this basal rate of erythrocyte production are made when tissue oxygenation becomes either decreased or increased compared to the normal level. Development of anemia due to blood loss or inspiration of decreased atmospheric oxygen at high altitude results in decreased oxygen delivery to the tissues.

There is no known clinical association between chronic myelogenous leukemia (CML) and membranoproliferative glomerulonephritis (MPGN). We present a patient who was followed in the renal clinic for proteinuria of unknown etiology (3.2 g/24 h) and normal renal function who was diagnosed with CML as well as MPGN and acute renal failure at the same time. The patient's renal function and proteinuria improved when his CML was treated with imatinib mesylate, suggesting that CML either caused or exacerbated existing MGPN. To the best of our knowledge, this is the first reported case of MPGN associated with CML that improved with imatinib mesylate therapy.

Diseases that infiltrate the bone marrow disrupt erythropoiesis leading to anemia. In multiple myeloma (MM), anemia severity can be correlated with degree of marrow infiltration by myeloma cells. Infiltrating MM may impair the function and structure of erythroblastic islands (EBIs), the marrow erythropoietic niches. An EBI consists of a central macrophage surrounded by colony-forming units-erythroid/proerythroblasts (CFU-E/pro-EBs) and their progeny, the differentiating erythroblasts. Cytokines produced by MM cells, such as Fas ligand (FL), tumor necrosis factor (TNF), and TNF-related apoptosis-inducing ligand (TRAIL), can induce erythroid cell apoptosis. Physical displacement of the erythroid cells away from central macrophages by MM can destroy the EBIs. Non-erythrotoxic therapies that kill MM cells while sparing erythropoietic cells allow quantification of erythropoiesis and marrow MM infiltration before and after treatment of newly diagnosed MM patients. Marrow biopsies from 15 newly diagnosed MM patients were obtained before and after 4 courses of non-erythrotoxic induction therapy with bortezomib, dexamethasone, and lenalidomide (Richardson et al, Blood 2010). CBCs and serum MM paraprotein quantifications were obtained with the marrow biopsies and before each course of therapy. No patient had renal insufficiency, iron or cobalamin deficiency, erythropoietin (EPO) therapy, or RBC transfusion. At diagnosis, percentages of marrow space occupied by MM and erythroid cells were negatively correlated. Percentages of marrow space infiltrated by MM (range = 2.3 - 72.3%) were also negatively correlated with hemoglobin (Hb), hematocrit (Hct) and RBCs. One patient had a partial response: marrow myeloma decreasing from 27.5% to 5.3%. All other patients had reductions in marrow myeloma to < 2.2%. The 8 patients with < 30% MM infiltration at diagnosis had no change (-1.4% to 1.8%) in marrow space occupied by erythroid cells following therapy, whereas 7 patients with > 35% MM infiltration at diagnosis increased marrow space occupied by erythroid cells following therapy (3.4 to 19.2%). Hb, Hct, and RBCs did not change during therapy in patients with < 30% MM infiltration, but those with > 35% myeloma infiltration at diagnosis had progressive increases in Hb, Hct, and RBCs during therapy. These clinical data were used to study the relationship between marrow infiltration by MM and erythropoiesis. Mathematical models of MM infiltration effects on marrow EBI structure/function were developed and tested in simulations. A previously developed hybrid discrete-continuous model of erythropoiesis based on EBI (Eymard et al, J Math Biol 2015) was extended to a larger area of marrow containing multiple EBIs. In the model, CFU-E/proEBs have 3 fates-- self-renewal, differentiation, and apoptosis--that depend upon factors produced systemically, such as glucocorticoids and EPO, and locally, such as stem cell factor and bone morphogenetic protein 4 by central macrophages and FL by mature erythroblasts. Intracellular regulatory networks were modeled with ordinary differential equations and extracellular concentrations by partial differential equations. Under normal conditions, EBIs achieve a steady-state that produces new RBCs at rates which maintain normal Hb, Hct and RBCs. At early times after the section of bone marrow is infiltrated by small foci of proliferating MM cells, EBI function is not affected. With further proliferation, infiltrating MM cells occupy more marrow space, inducing erythroid cell apoptosis by producing FL, TNF or TRAIL and by displacing erythroid cells from central macrophages, thereby destroying EBIs. However, central macrophages of destroyed islands persist or are replaced by differentiation of monocyte-macrophage precursors. After MM cells are killed by therapy, the residual macrophages can interact with burst-forming units-erythroid (BFU-E), thereby reestablishing EBIs. If the MM infiltrate is not sufficiently reduced after a course of therapy, it can physically interfere with the macrophage-BFU-E interaction, preventing the reestablishment of an EBI and full recovery of RBC production until a subsequent therapy reduces the infiltrate sufficiently that the EBI is reestablished. The model is consistent with the clinical data and may apply to other marrow infiltrative diseases including myelofibrosis, systemic infections, or other malignancies. Disclosures No relevant conflicts of interest to declare.

We develop a hybrid multi-scale model of normal and pathological erythropoiesis, the process of red blood cell production in the bone marrow. Cells are described by a soft sphere model. They can move, divide and die by apoptosis. Their fate is determined by intracellular proteins, by extracellular substances and surrounding cells. We apply this model to describe normal function of erythropoiesis and the development of multiple myeloma in the bone marrow. It is a malignant disease characterized by proliferation of mutated plasma cells which form multiple tumors in the bone marrow. They disrupt erythroblastic islands by direct mechanical contact and due to apoptotic cytokines produced by myeloma cells. The development of the disease leads to decrease of red blood cell production and to anemia.

MicroRNAs are small non-coding RNAs that regulate gene expression through mRNA degradation or translational inhibition. MicroRNAs are emerging as key regulators of normal hematopoiesis and hematologic malignancies. Several miRNAs are differentially expressed during hematopoiesis and their specific expression regulates key functional proteins involved in hematopoietic lineage differentiation. This study focused on the functional role of microRNA-223 (miR-223) on erythroid differentiation.Purified cord blood CD34+ hematopoietic progenitor cells were grown in strictly controlled conditions in the presence of saturating dosage of erythropoietin to selectively induce erythroid differentiation. The effects of enforced expression of miR-223 in unilin-eage erythroid cultures were evaluated in liquid phase culture experiments and clonogenic studies.In unilineage erythroid culture of cord blood CD34+ hematopoietic progenitor cells miR-223 is down-regulated, whereas LMO2, an essential protein for erythroid differentiation, is up-regulated. Functional studies showed that enforced expression of miR-223 reduces the mRNA and protein levels of LMO2, by binding to LMO2 3' UTR, and impairs differentiation of erythroid cells. Accordingly, knockdown of LMO2 by short interfering RNA mimics the action of miR-223. Furthermore, hematopoietic progenitor cells transduced with miR-223 showed a significant reduction of their erythroid clonogenic capacity, suggesting that downmodulation of this miRNA is required for erythroid progenitor recruitment and commitment.These results show that the decline of miR-223 is an important event for erythroid differentiation that leads to the expansion of erythroblast cells at least partially mediated by unblocking LMO2 protein expression.

The anemia of chronic disease (ACD) is commonly encountered in medical practice and can be the first sign of disease. Inflammatory cytokines such as tumor necrosis factor α (TNFα), interleukin-6, and interferon γ are produced in the bone marrow or other organs of patients with chronic diseases.

Erythropoietin (EPO) is the glycoprotein hormone that controls the production of erythrocytes in mammals. If one administers EPO to an animal, shortly thereafter, new erythrocytes (RBCs) appear in the circulation. The manner by which new RBCs are formed has been surmised by in vitro culture of bone marrow or spleen cells in semisolid medium in the presence of EPO. In such cultures, colonies of erythroblasts arise from committed erythroid progenitor cells that divide and mature simultaneously. The committed erythroid progenitor cells constitute continuous stages of differentiation that have been defined by the time in vitro required for them to form colonies of mature erythroblasts and by the number of cells in the colonies (1). Burst-forming units-erythroid (BFU-E) are the most immature committed erythroid progenitor cells; they give rise to colonies of thousands of mature erythroblasts in 7 days for murine BFU-E and 14 days for human BFU-E. How the BFU-E arise by “commitment” to erythroid differentiation of pluripotent hematopoietic cells is not understood. Colony-forming units-erythroid (CFU-E) are intermediate in their level of differentiation, giving rise to colonies of 8–32 erythroblasts after 2 days of culture for murine CFU-E and 7 days for human CFU-E. The immediate descendants of CFU-E are the proerythroblasts that are the earliest morphologically recognizable, committed erythroid cells and are capable of very limited proliferation.

Severe thrombocytopenic purpura occurred in seven patients who had histories of recent or current intravenous cocaine use. All of the patients denied heroin use and all were seronegative for antibodies to the human immunodeficiency virus (HIV). The seven patients had normal or increased numbers of megakaryocytes in their bone marrows. While receiving corticosteroid therapy their platelet counts rose to 10(11)/L over periods ranging from 5 to 11 days. In view of the widespread use of cocaine, severe thrombocytopenia in the HIV-seronegative patient should suggest the possibility of current or recent intravenous use of cocaine.

Bcl-x is a protein in the outer mitochondrial membrane. A member of the Bcl-2 family, Bcl-x protects developing erythroid cells from apoptosis. The exact stage of erythroid development at which Bcl-x exerts its anti-apoptotic effect is not known, but induction of Bcl-x has been proposed to be the mediator of erythropoietin’s (EPO) anti-apoptotic effect in erythroid differentiation. EPO is the principal trophic hormone that controls red blood cell production by regulating apoptosis of erythroid progenitor cells at the CFU-E and early erythroblast stages. Bcl-x has also been reported to be necessary for heme synthesis. In mice, Bcl-x deficiency is embryonically lethal; when Bcl-x deficiency is acquired postnatally by conditional knockout technology, it is associated with splenomegaly, thrombocytopenia, and a profound anemia that is thought to be hemolytic in origin. Objectives: 1)To characterize the defect of erythroid differentiation in conditional Bcl-x −/− mice. 2)To determine whether Bcl-x is the mediator of EPO’s anti-apoptotic action. 3)To determine whether Bcl-x is necessary for heme synthesis. Methods: Phlebotomized or unbled littermate controls and anemic adult Bcl-x −/− mice obtained by cre-lox conditional knockout were bled, sacrificed, and splenectomized. Purified populations of splenic erythroblasts were isolated by sedimentation at unit gravity, cultured with or without EPO, and harvested at 0, 8, 20, 32, and 44 hours for cell counts, cytospin preparations for morphology, flow cytometry analyses for apoptosis (TUNEL) and cell cycle phases, and 59FeCl3 incorporation into heme. Results: Compared to littermate controls, Bcl-x −/− mice were severely anemic (Hgb 2.8 g/dL vs 15.4 g/dL in unbled controls and 7.2 g/dL in bled controls), thrombocytopenic (platelets 23x103/microL vs 905x103/microL in unbled controls and 984x103/microL in bled controls), and reticulocytopenic (82.8x103/microL vs 281x103/microL in unbled controls and 1410x103/microL in bled controls), while WBCs were unaffected. Expanded erythropoiesis led to massive splenomegaly (spleens =4.3gm vs 0.1gm in unbled controls and 0.3gm in bled controls). After 44 hours of culture with EPO, purified erythroblasts from bled controls proliferated 4-fold and differentiated such that the majority enucleated, producing 200–250 reticulocytes per 100 erythroblasts plated, whereas Bcl-x −/− erythroblast numbers doubled during the first 20 hours in culture, but the large majority died by apoptosis between 20 and 44 hours, producing only 9–12 reticulocytes per 100 erythroblasts plated. Bcl-x −/− erythroblast apoptosis occurred after the initiation of heme synthesis and proportionally in all phases of cell cycle. Compared to culture with EPO, Bcl-x −/− erythroblasts cultured without EPO underwent increased apoptosis at earlier times of culture-- at 8 hours (45% vs 29%), 20 hours (71% vs 42%) and 32 hours (83% vs 57%). Conclusions: 1)Bcl-x is required for the survival and differentiation of the late-stage erythroblasts in all phases of cell cycle. Thus, Bcl-x deficiency results in ineffective erythropoiesis rather than hemolytic anemia. 2)Bcl-x is not required for heme synthesis, but has its anti-apoptotic effect during the stage of hemoglobin synthesis. 3)Bcl-x does not mediate EPO’s anti-apoptotic effect in early-stage erythroblasts.

In situ hybridization was used to quantitate the cells that produce erythropoietin (EP) in the renal cortices of mice with varying severities of acute anemia and of mice recovering from severe, acute anemia. The number of EP-producing cells in the renal cortex increased in an exponential manner as hematocrit was decreased. Individual EP- producing cells had very similar densities of silver grains in autoradiograms regardless of whether they were from normal mice or from slightly, moderately or severely anemic animals. With increasingly severe anemia, total renal EP mRNA levels and serum EP concentrations showed increases that correlated with the number of renal EP-producing cells. These results indicate that as mice become more anemic, additional cells are recruited to produce EP rather than the cells already producing EP being stimulated to increase their individual production. In mildly and moderately anemic animals, small clusters of EP-producing cells were found in the inner cortex with large areas of cortex containing no EP-producing cells. In severely anemic mice, EP- producing cells were found throughout the inner cortex with only a very few found scattered in the outer cortex and outer medulla. The data indicate that only a subset of total renal interstitial cells produce EP. During recovery from severe, acute anemia, the numbers of EP- producing cells decreased exponentially as hematocrits rose and correlated with decreases in total renal EP mRNA and serum EP concentrations. These results suggest that following an acute blood loss and during the recovery from a blood loss, the capacity to deliver oxygen, as represented by hematocrit, is the major regulator of EP production.

Murine retroviruses which cause sarcomas also induce abnormal proliferation of haematopoietic cells leading to death in newborn1–5 and, in some cases, adult4 mice. A murine sarcoma virus is a complex of a replication-defective sarcoma virus and a replication-competent murine leukaemia virus (MuLV). Cloned MuLV isolates also cause abnormal haematopoietic cell proliferations6–9. It is unknown whether such proliferative responses are direct effects of retroviruses on haematopoietic progenitor cells or indirect effects on other cells, which in turn provide the stimuli for the proliferation of haematopoietic cells. We demonstrate here that specific fibroblastic cell lines produce low levels of granulocyte–macrophage colony stimulating activity (GM–CSA) and that infection of certain fibroblast lines with murine retroviruses results in large increases in GM–CSA. Other cell lines which do not produce detectable CSA when uninfected do not produce it after retroviral infection.

SummaryWhen the marrow and spleen cells of plethoric mice are cultured with 1.0 unit/ml of limulus-lysate-absorbed Ep, the predominant erythroid progenitor cell assayed is the Day 3 BFU-E. Erythropoiesis by these cells can be studied by enumeration of bursts at 3 days or by 24-hr 59Fe incorporation into heme. Endotoxin markedly increased the number of Day 3 BFU-E in the spleens of these mice and decreased the number in the marrow, but it had little effect on erythropoietic maturation. In contrast, Ep produced differentiation of the erythroid progenitor cells resulting in increased splenic CFU-E and a decline in the more primitive erythroid precursor cells.We wish to thank Ms. A. Brockman for excellent technical assistance. Mark J. Koury is the recipient of a research fellowship supported by an NIH Postdoctoral Training Program in Hematology, Grant T32 AM07186. This work was supported in part by Grant AM15555 from the National Institute of Arthritis, Metabolism and Digestive Diseases, by the Medical Resear...