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Background: Studies in adults showed a relationship between low hemoglobin (Hb) and acute kidney injury (AKI). We performed this study to evaluate this association in newborns., Methods: We evaluated 1891 newborns from the Assessment of Worldwide AKI Epidemiology in Neonates (AWAKEN) database. We evaluated the associations for the entire cohort and 3 gestational age (GA) groups: <29, 29-<36, and ≥36 weeks' GA., Results: Minimum Hb in the first postnatal week was significantly lower in neonates with AKI after the first postnatal week (late AKI). After controlling for multiple potential confounders, compared to neonates with a minimum Hb ≥17.0 g/dL, both those with minimum Hb ≤12.6 and 12.7-14.8 g/dL had an adjusted increased odds of late AKI (aOR 3.16, 95% CI 1.44-6.96, p = 0.04) and (aOR 2.03, 95% CI 1.05-3.93; p = 0.04), respectively. This association was no longer evident after controlling for fluid balance. The ability of minimum Hb to predict late AKI was moderate (c-statistic 0.68, 95% CI 0.64-0.72) with a sensitivity of 65.9%, a specificity of 69.7%, and a PPV of 20.8%., Conclusions: Lower Hb in the first postnatal week was associated with late AKI, though the association no longer remained after fluid balance was included., Impact: The current study suggests a possible novel association between low serum hemoglobin (Hb) and neonatal acute kidney injury (AKI). The study shows that low serum Hb levels in the first postnatal week are associated with increased risk of AKI after the first postnatal week. This study is the first to show this relationship in neonates. Because this study is retrospective, our observations cannot be considered proof of a causative role but do raise important questions and deserve further investigation. Whether the correction of low Hb levels might confer short- and/or long-term renal benefits in neonates was beyond the scope of this study.

The current vaccine against tuberculosis, live attenuated Mycobacterium bovis BCG, has variable efficacy, but development of an effective alternative is severely hampered by the lack of an immune correlate of protection. There has been a recent resurgence of interest in functional in vitro mycobacterial growth inhibition assays (MGIAs), which provide a measure of a range of different immune mechanisms and their interactions. We identified a positive correlation between mean corpuscular haemoglobin and in vitro growth of BCG in whole blood from healthy UK human volunteers. Mycobacterial growth in peripheral blood mononuclear cells (PBMC) from both humans and macaques was increased following the experimental addition of haemoglobin (Hb) or ferric iron, and reduced following addition of the iron chelator deferoxamine (DFO). Expression of Hb genes correlated positively with mycobacterial growth in whole blood from UK/Asian adults and, to a lesser extent, in PBMC from South African infants. Taken together our data indicate an association between Hb/iron levels and BCG growth in vitro, which may in part explain differences in findings between whole blood and PBMC MGIAs and should be considered when using such assays.

Lysophosphatidic acid (LPA), a growth factor-like phospholipid, regulates numerous physiological functions, including cell proliferation and differentiation. In a previous study, we have demonstrated that LPA activates erythropoiesis by activating the LPA 3 receptor subtype (LPA3) under erythropoietin (EPO) induction. In the present study, we applied a pharmacological approach to further elucidate the functions of LPA receptors during red blood cell (RBC) differentiation. In K562 human erythroleukemia cells, knockdown of LPA2 enhanced erythropoiesis, whereas knockdown of LPA3 inhibited RBC differentiation. In CD34(+) human hematopoietic stem cells (hHSC) and K526 cells, the LPA3 agonist 1-oleoyl-2-methyl-sn-glycero-3-phosphothionate (2S-OMPT) promoted erythropoiesis, whereas the LPA2 agonist dodecyl monophosphate (DMP) and the nonlipid specific agonist GRI977143 (GRI) suppressed this process. In zebrafish embryos, hemoglobin expression was significantly increased by 2S-OMPT treatment but was inhibited by GRI. Furthermore, GRI treatment decreased, whereas 2S-OMPT treatment increased RBC counts and amount of hemoglobin level in adult BALB/c mice. These results indicate that LPA2 and LPA3 play opposing roles during RBC differentiation. The pharmacological activation of LPA receptor subtypes represent a novel strategies for augmenting or inhibiting erythropoiesis.

Subarachnoid hemorrhage (SAH) usually results from ruptured aneurysm, but how leaked hemoglobin regulates the microcirculation in the pathophysiology of early brain injury after SAH is still unclear. In the present study, we sought to investigate the role and possible mechanism of hemoglobin induced pericyte phenotype transformation in the regulation of microcirculation after SAH. Endovascular perforation SAH rat model, brain slices and cultured pericytes were used, and intervened with endothelial nitric oxide synthase (eNOS) antagonist L-NNA and its agonist scutellarin, hemoglobin, DETA/NO (nitric oxide(NO) donor), PITO (NO scavenger), 8-Br-cGMP (cGMP analog). We found modulating eNOS regulated pericyte α-SMA phenotype transformation, microcirculation, and neurological function in SAH rats. Modulating eNOS also affected eNOS expression, eNOS activity and NO availability after SAH. In addition, we showed hemoglobins penetrated into brain parenchyma after SAH. And hemoglobins significantly reduced the microvessel diameters at pericyte sites, due to the effects of hemoglobin inducing α-SMA expressions in cultured pericytes and brain slices via inhibiting NO/cGMP pathway. In conclusion, pericyte α-SMA phenotype mediates acute microvessel constriction after SAH possibly by hemoglobin suppressing NO/cGMP signaling pathway. Therefore, by targeting the eNOS and pericyte α-SMA phenotype, our present data may shed new light on the management of SAH patients.

The generation of induced pluripotent stem cells (iPSCs) from differentiated somatic cells by over-expression of several transcription factors has the potential to cure many genetic and degenerative diseases currently recalcitrant to traditional clinical approaches. One such genetic disease is β-thalassemia major (Cooley's anemia). This disease is caused by either a point mutation or the deletion of several nucleotides in the β-globin gene, and it threatens the lives of millions of people in China. In the present study, we successfully generated iPSCs from fibroblasts collected from a 2-year-old patient who was diagnosed with a homozygous 41/42 deletion in his β-globin gene. More importantly, we successfully corrected this genetic mutation in the β-thalassemia iPSCs by homologous recombination. Furthermore, transplantation of the genetically corrected iPSCs-derived hematopoietic progenitors into sub-lethally irradiated immune deficient SCID mice showed improved hemoglobin production compared with the uncorrected iPSCs. Moreover, the generation of human β-globin could be detected in the mice transplanted with corrected iPSCs-derived hematopietic progenitors. Our study provides strong evidence that iPSCs generated from a patient with a genetic disease can be corrected by homologous recombination and that the corrected iPSCs have potential clinical uses.

Anemia is one of the most common and morbid complications of chronic kidney disease, causing unpleasant symptoms and reducing the quality of life. The availability of recombinant human erythropoietin (rHuEPO) in 1989 has been one of the most important developments in the care of this population in the past several decades. Treatment with erythropoiesis-stimulating agents (ESAs) has improved patients' lives, but recent studies have found that higher hemoglobin (Hgb) targets cause harm, resulting in more cautious treatment. Despite widespread recognition by clinicians and patients of the value of this biological agent, the high cost and new concerns over safety have led to a reexamination of its use. Although rHuEPO is prescribed by individual physicians and target Hgb is guided by current evidence in the context of individual patients, critics within and outside the medical community have charged that rHuEPO is being overused, that financial motives are driving its use, and that patients are suffering from adverse consequences. Regulatory agencies, including the Centers for Medicare and Medicaid Services and the US Food and Drug Administration, have weighed in as well. In this review article, issues related to the current and future status of ESA treatment will be considered with a view to assessing factors that result in a lack of clarity and need for further study. It is essential that the renal community vigorously support additional rigorous research to expand the evidence base for optimal anemia management so that the debate over appropriate ESA use remains where it belongs, in the scientific domain.

Sustained expression of the Spi-1/PU.1 and Fli-1 oncoproteins blocks globin gene activation in mouse erythroleukemia cells; however, only Spi-1/PU.1 expression inhibits the inclusion of exon 16 in the mature 4.1R mRNA. This splicing event is crucial for a functional 4.1R protein and, therefore, for red blood cell membrane integrity. This report demonstrates that Spi-1/PU.1 downregulation induces the activation of TRIM10/hematopoietic RING finger 1 (HERF1), a member of the tripartite motif (TRIM)/RBCC protein family needed for globin gene transcription. Additionally, we demonstrate that TRIM10/HERF1 is required for the regulated splicing of exon 16 during late erythroid differentiation. Using inducible overexpression and silencing approaches, we found that: (1) TRIM10/HERF1 knockdown inhibits hemoglobin production and exon splicing and triggers cell apoptosis in dimethylsulfoxide (DMSO)-induced cells; (2) TRIM10/HERF1 upregulation is required but is insufficient on its own to activate exon retention; (3) Fli-1 has no effect on TRIM10/HERF1 expression, whereas either DMSO-induced downregulation or shRNA-knockdown of Spi-1/PU.1 expression is sufficient to activate TRIM10/HERF1 expression; and (4) Spi-1/PU.1 knockdown triggers both the transcription and the splicing events independently of the chemical induction. Altogether, these data indicate that primary Spi-1/PU.1 downregulation acts on late erythroid differentiation through at least two pathways, one of which requires TRIM10/HERF1 upregulation and parallels the Spi-1/PU.1-induced Fli-1 shutoff regulatory cascade.

Estimating the risk of in-hospital mortality in the newborn intensive care unit can provide important information for health-care providers, and illness severity scores have been devised to provide mortality risk estimates. Calculation of illness severity scores is time-consuming, and the information used to predict mortality is collected only for the first 12 to 24 h of life. A noninvasive continuous measure that uses information collected throughout the hospitalization and that requires no data entry could be less costly and more informative. We have previously shown that the abnormal heart rate characteristics (HRC) of reduced variability and transient decelerations accompany neonatal illness such as late-onset sepsis. We hypothesized that more frequent and severe abnormal HRC are associated with an increased risk of death. We tested this hypothesis in two ways. Using data on infants older than 7 d of age, we first determined the association of the HRC index with death in the next week. Second, we devised a cumulative HRC score and determined its association with in-hospital death. There were 37 deaths in the 685 patients. The major findings were 1) the HRC index showed highly significant association with death in the succeeding 7 d (receiver-operating characteristic area > 0.7, p < 0.001), and 2) the cumulative HRC was highly significantly associated with neonatal in-hospital mortality (receiver-operating characteristic area > 0.80, p < 0.001). In both analyses, HRC added information to birth weight, gestational age, and postnatal age (p < 0.01). The HRC index provides independent information about the risk of neonatal death in the upcoming 7 d, and the cumulative HRC is an estimate of the risk of in-hospital neonatal mortality.

Friend murine erythroleukemia (MEL) cells are transformed erythroid precursors that are held in an immature and proliferating state but can be induced to differentiate in vivo by treatment with a variety of chemical agents such as N, N-hexamethylene bisacetamide (HMBA). To investigate the role of Myb proteins in maintaining MEL cells in an immature and proliferating state we have produced stable transfectants in the C19 MEL cell line that contain a dominant interfering Myb allele (MEnT) under the control of an inducible mouse metallothionein I promoter. When expression of MEnT protein was induced with ZnCl2, the stable transfectants differentiated with kinetics that were similar to wild type C19 MEL cells treated with HMBA, including induction of alpha-globin mRNA expression, assembly of hemoglobin and growth arrest. Expression of endogenous c-myb and c-myc was also decreased in response to MEnT. Expression of mad-1 mRNA was rapidly increased in response to expression of MEnT resulting in a shift from predominantly c-Myc/Max complexes to predominantly Mad/Max containing complexes. These results strongly suggest that C19 MEL cells are held in an immature and proliferating state by a pathway that is dependent on Myb activity.

The stable introduction of a functional beta-globin gene in haematopoietic stem cells could be a powerful approach to treat beta-thalassaemia and sickle-cell disease. Genetic approaches aiming to increase normal beta-globin expression in the progeny of autologous haematopoietic stem cells might circumvent the limitations and risks of allogeneic cell transplants. However, low-level expression, position effects and transcriptional silencing hampered the effectiveness of viral transduction of the human beta-globin gene when it was linked to minimal regulatory sequences. Here we show that the use of recombinant lentiviruses enables efficient transfer and faithful integration of the human beta-globin gene together with large segments of its locus control region. In long-term recipients of unselected transduced bone marrow cells, tetramers of two murine alpha-globin and two human betaA-globin molecules account for up to 13% of total haemoglobin in mature red cells of normal mice. In beta-thalassaemic heterozygous mice higher percentages are obtained (17% to 24%), which are sufficient to ameliorate anaemia and red cell morphology. Such levels should be of therapeutic benefit in patients with severe defects in haemoglobin production.

Friend murine erythroleukaemia (F-MEL) cells are a useful model for studying the processes that regulate erythroid differentiation since exposure of these cells to chemical inducers (DMSO or HMBA) results in commitment to terminal cell division and synthesis of haemoglobin. This study examined the relationship between differentiation and apoptosis in DMSO sensitive and resistant F-MEL cells. Clear apoptosis was not observed in DMSO-treated sensitive F-MEL (strain 745A) cells during the induction of differentiation. In contrast, DMSO-induced 745A cells exhibited delayed apoptosis compared to uninduced cells. Since the Bcl-2 family members play a major role in the control of apoptosis and/or differentiation, we determined their expression before and after DMSO or HMBA treatment. Neither untreated nor chemically-induced 745A cells expressed the Bcl-2 protein. The levels of Bax and Bad proteins remained relatively constant during DMSO-induced differentiation. DMSO or HMBA treatment of 745A cells induced a marked increase of Bcl-XL expression during the late phase of differentiation which persisted even when the cells began to die. This upregulation of Bcl-XL was independent of cell density but was correlated with cell arrest in G0/G1. DMSO treatment induced a similar delay of apoptosis and enhancement of Bcl-XL expression in F-MEL (strain TFP10) cells which fail to synthesize haemoglobin in the presence of DMSO. Dexamethasone, which blocks DMSO-induced differentiation of F-MEL cells, prevented the induction of Bcl-XL. Inhibitors such as imidazole or succinylacetone, which inhibit haemoglobin synthesis but not commitment to terminal cell division, did not suppress Bcl-XL induction in DMSO-induced cells. Taken together, these results indicate that DMSO treatment of F-MEL cells induces a marked increase in Bcl-XL expression suggesting a role for this anti-apoptotic protein in the process of erythroid differentiation in F-MEL cells. Moreover, induction of Bcl-XL during this process seems to be associated with loss of proliferative capacity rather than with haemoglobin synthesis.

In this study, the effect of -158 (C-->T) (XmnI) polymorphism on the synthesis of fetal Hb and its G gamma component during the switchover from fetal to adult Hb was examined using cord blood samples from normal Caucasian term infants. The presence of -158(C-->T) mutation was determined by amplification of G gamma and A gamma-globin gene promoter fragments from the DNA isolated from cord blood samples, followed by XmnI restriction enzyme digestion. The syntheses of fetal and adult Hb in cord blood were measured by [3H]leucine incorporation in globin synthesis, separation of the globin polypeptides by HPLC, and scintillation counting of the fractions. The presence of -158(C-->T) substitution in the G gamma-globin promoter region was positively correlated with elevated synthesis of fetal Hb and its G gamma-globin component in term newborn infants and is associated with delayed switchover from fetal to adult Hb. In addition analysis of cord blood samples from 100 normal Caucasian French Canadian term infants revealed that the frequency of -158(C-->T) substitution in G gamma-promoter was 0.32.

The need to develop a blood substitute is now urgent because of the increasing concern over blood-transmitted viral and bacterial pathogens. Cell-free haemoglobin solutions and human haemoglobin synthesized in Escherichia coli and Saccharomyces cerevisiae have been investigated as potential oxygen-carrying substitutes for red blood cells. But these haemoglobins cannot be used as a blood substitute because (1) the oxygen affinity in the absence of 2,3-bisphosphoglycerate is too high to allow unloading of enough oxygen in the tissues, and (2) they dissociate into alpha beta dimers that are cleared rapidly by renal filtration, which can result in long-term kidney damage. We have produced a human haemoglobin using an expression vector containing one gene encoding a mutant beta-globin with decreased oxygen affinity and one duplicated, tandemly fused alpha-globin gene. Fusion of the two alpha-globin subunits increases the half-life of this haemoglobin molecule in vivo by preventing its dissociation into alpha beta dimers and therefore also eliminates renal toxicity.

A system for the isolation and functional evaluation of fetal liver erythroblasts is described. Isolated erythroblasts were prepared from 14-day embryonic avian livers and incubated at various oxygen tension (0, 5, 12, and 95%). The concentration of ATP in erythroblasts remained constant for at least 4 hr at 37degree, but was rapidly reduced by incubation in nitrogen. Protein synthesis as measured by L-[14C]leucine incorporation into cell protein occurred at a linear rate in 5%, 12%, and 95% oxygen, whereas little protein synthesis occurred at 0% oxygen. The effect of hypoxia on the type of hemoglobin synthesized was studied in this system by isolating the hemoglobin A, hemoglobin D, and hemoglobin H fractions and determining the incorporation of L-[14C]leucine. The major fraction, hemoglobin A, contained most of the radioactivity; smaller amounts were present in hemoglobin D and hemoglobin H, respectively. The relative proportion of each hemoglobin synthesized was not altered by oxygen from 5% to 95%. These results argue against a direct effect of oxygen on the type of hemoglobin synthesized at this stage of development.

A temperature sensitive mutant has been isolated for the first time from a replication defective acute leukaemia virus, AEV. In vivo, at 41 degrees C, the mutant shows a reduced leukaemogenic potential. In vitro, in erythroblasts transformed at 35 degrees C, haemoglobin synthesis can be induced by a shift to 41 degrees C. This indicates that the continuous expression of a viral gene product is necessary to maintain the undifferentiated state of the virus-transformed leukaemia cells.

The biogenesis of erythropoietin is incompletely understood. One hypothesis maintains that erythropoietin is synthesized primarily in the kidney while according to another theory an erythropoietin precursor present in plasma is activated by a renal factor, erythrogenin. An attractive candidate for the erythropoietin precursor is renin substrate (angiotensinogen) which has chemical similarities with erythropoietin. We show here that purified renin substrate from human plasma is immunologically related to human erythropoietin. Moreover, purified renin substrate, like erythropoietin, causes the dose-dependent increase of haemoglobin F in cultured human erythroid leukaemia K562 cells. We conclude that renin substrate is a likely precursor of erythropoietin.

The activity of heme synthetase (ferrochelatase), the enzyme(s) which catalyzes the formation of heme from iron and protoporphyrin IX, was studied in the various fractions of a cell-free reticulocyte system which synthesizes hemoglobin. The ribosomal fraction contained heme synthetase activity and its characteristics were similar to that described in avian erythrocytes, human and rat liver, and rabbit reticulocytes. It has a pH optimum of 7.5, required sulfhydryl reagents, was denatured by heat and was unstable on freezing. Heme synthetase is a mitochondrial enzyme. Fragments of mitochondrial membrane were identified in the ribosome fraction by both electron microscopy and the presence of cytochrome oxidase activity.

The pattern of haemoglobin production changes at the embryonic, fetal and postnatal stages of human development, reflecting the expression of different globin genes in both the alpha-like and beta-like gene clusters. Recent studies have identified alterations in the state of DNA methylation and sensitivity to nuclease digestion associated with developmental expression of the globin genes in red blood cell precursors, but the mechanism initiating these changes remains unknown. Despite the screening of large numbers of blood samples from newborn infants, no mutants have been found which affect the timing of these changes (with one possible exception involving a chromosomal translocation), thus necessitating alternative approaches to analysing the cellular basis for the timing of haemoglobin switching. Although many mechanisms are possible, the initiation of the switch from fetal to adult haemoglobin could be regulated essentially either by a developmental clock inherent to haematopoietic stem cells or by an inductive environment, and in an attempt to distinguish between these possibilities, we have transplanted sheep fetal haematopoietic tissue into adult animals. Although previous experiments of this type produced conflicting results, the accumulated results presented here demonstrate that the pattern of haemoglobin production after transplantation is determined largely by the gestational age of the fetal donor cells.