David F. O'Connor

Regulation of human metabolism by hypoxia- inducible factor

The hypoxia-inducible factor (HIF) family of transcription factors directs a coordinated cellular response to hypoxia that includes the transcriptional regulation of a number of metabolic enzymes. Chuvash polycythemia (CP) is an autosomal recessive human disorder in which the regulatory degradation of HIF is impaired, resulting in elevated levels of HIF at normal oxygen tensions. Apart from the polycythemia, CP patients have marked abnormalities of cardiopulmonary function. No studies of integrated metabolic function have been reported. Here we describe the response of these patients to a series of metabolic stresses: exercise of a large muscle mass on a cycle ergometer, exercise of a small muscle mass (calf muscle) which allowed noninvasive in vivo assessments of muscle metabolism using 31P magnetic resonance spectroscopy, and a standard meal tolerance test. During exercise, CP patients had early and marked phosphocreatine depletion and acidosis in skeletal muscle, greater accumulation of lactate in blood, and reduced maximum exercise capacities. Muscle biopsy specimens from CP patients showed elevated levels of transcript for pyruvate dehydrogenase kinase, phosphofructokinase, and muscle pyruvate kinase. In cell culture, a range of experimental manipulations have been used to study the effects of HIF on cellular metabolism. However, these approaches provide no potential to investigate integrated responses at the level of the whole organism. Although CP is relatively subtle disorder, our study now reveals a striking regulatory role for HIF on metabolism during exercise in humans. These findings have significant implications for the development of therapeutic approaches targeting the HIF pathway.

Perturbation of fetal liver hematopoietic stem and progenitor cell development by trisomy 21

The 40-fold increase in childhood megakaryocyte-erythroid and B-cell leukemia in Down syndrome implicates trisomy 21 (T21) in perturbing fetal hematopoiesis. Here, we show that compared with primary disomic controls, primary T21 fetal liver (FL) hematopoietic stem cells (HSC) and megakaryocyte-erythroid progenitors are markedly increased, whereas granulocyte-macrophage progenitors are reduced. Commensurately, HSC and megakaryocyte-erythroid progenitors show higher clonogenicity, with increased megakaryocyte, megakaryocyte-erythroid, and replatable blast colonies. Biased megakaryocyte-erythroid–primed gene expression was detected as early as the HSC compartment. In lymphopoiesis, T21 FL lymphoid-primed multipotential progenitors and early lymphoid progenitor numbers are maintained, but there was a 10-fold reduction in committed PreproB-lymphoid progenitors and the functional B-cell potential of HSC and early lymphoid progenitor is severely impaired, in tandem with reduced early lymphoid gene expression. The same pattern was seen in all T21 FL samples and no samples had GATA1 mutations. Therefore, T21 itself causes multiple distinct defects in FL myelo- and lymphopoiesis.

Changes in cerebral blood flow during and after 48 h of both isocapnic and poikilocapnic hypoxia in humans

During acclimatization to the hypoxia of altitude, the cerebral circulation is exposed to arterial hypoxia and hypocapnia, two stimuli with opposing influences on cerebral blood flow (CBF). In order to understand the resultant changes in CBF, this study examined the responses of CBF during a period of constant mild hypoxia both with and without concomitant regulation of arterial PCO2. Nine subjects were each exposed to two protocols in a purpose‐built chamber: (1) 48 h of isocapnic hypoxia (Protocol I), where end‐tidal PO2 (PET,O2) was held at 60 Torr and end‐tidal PCO2 (PET,CO2) at the subjects resting value prior to experimentation; and (2) 48 h of poikilocapnic hypoxia (Protocol P), where PET,O2 was held at 60 Torr and PET,CO2 was uncontrolled. Transcranial Doppler ultrasound was used to assess CBF. At 24 h intervals during and after the hypoxic exposure CBF was measured and the sensitivity of CBF to acute variations in PO2 and PCO2 was determined. During Protocol P, PET,CO2 decreased by 13% (P < 0.001) and CBF decreased by 6% (P < 0.05), whereas during Protocol I, PET,CO2 and CBF remained unchanged. The sensitivity of CBF to acute variations in PO2 and PCO2 increased by 103% (P < 0.001) and 28% (P < 0.01), respectively, over the 48 h period of hypoxia. These changes did not differ between protocols. In conclusion, CBF decreases during mild poikilocapnic hypoxia, indicating that there is a predominant effect on CBF of the associated arterial hypocapnia. This fall occurs despite increases in the sensitivity of CBF to acute variations in PO2/PCO2 arising directly from the hypoxic exposure.

Infection-related mortality in children with acute lymphoblastic leukemia: an analysis of infectious deaths on UKALL2003

Although infection is the major cause of treatment-related mortality (TRM) in childhood acute lymphoblastic leukemia, factors associated with infection-related mortality (IRM) are poorly understood. To address this, we report an analysis of all 75 cases of IRM in the United Kingdom Childhood Acute Lymphoblastic Leukaemia Randomised Trial 2003 (UKALL 2003). The 5-year cumulative incidence of IRM was 2.4% (95% confidence interval [CI], 1.9%-3.0%), accounting for 75 (30%) of 249 trial deaths and 75 (64%) of 117 TRM deaths. Risk for IRM as a proportion of TRM was greater in induction than other phases (77% vs 56%; P = .02). Sixty-eight percent of cases were associated with bacterial infection (64% Gram-negative) and 20% with fungal infection. Down syndrome was the most significant risk factor for IRM (odds ratio [OR], 12.08; 95% CI, 6.54-22.32; P < .0001). In addition, there was a trend toward increased IRM in girls (OR, 1.63; 95% CI, 1.02-2.61; P = .04), as well as increasing treatment intensity (regimen B vs A: OR, 2.11 [95% CI, 1.24-3.60]; regimen C vs A: OR, 1.41 [95% CI, 0.76-2.62]; P = .02). Importantly, patients with Down syndrome were at significantly higher risk for IRM during maintenance (P = .048). Our results confirm Down syndrome as a major risk factor for IRM. Enhanced supportive care and prophylactic antibiotics should be considered in high-risk patient groups and during periods of increased risk. This study was registered at http://www.controlled-trials.com/ as #ISRCTN07355119.

An assessment of central-peripheral ventilatory chemoreflex interaction using acid and bicarbonate infusions in humans.

1. The object of this study was to investigate the effect of central chemoreceptor stimulation on the ventilatory responses to peripheral chemoreceptor stimulation. 2. The level of central chemoreceptor stimulation was varied by performing experiments at two different levels of end‐tidal CO2 pressure (PCO2). Variations in peripheral chemoreceptor stimulus were achieved by varying arterial pH (at constant end‐tidal PCO2) and by varying end‐tidal O2 pressure (PO2). 3. Two protocols were each performed on six human subjects. In one protocol ventilatory measurements were made during eucapnia, when the arterial pH was lowered from 7.4 to 7.3. The variation in pH was achieved by the progressive infusion of acid (0.1 M HCl). In the other protocol ventilatory measurements were made during hypercapnia, when the arterial pH was increased from 7.3 to 7.4. The variation in pH was achieved by the progressive infusion of 1.26% NaHCO3. In each protocol ventilatory responses were measured during euoxia (end‐tidal PO2, 100 Torr), hypoxia (end‐tidal PO2, 50 Torr) and hyperoxia (end‐tidal PO2, 300 Torr), with end‐tidal PCO2 held constant. 4. The increase in ventilatory sensitivity to arterial pH induced by hypoxia (50 Torr) was not significantly different between protocols (acid protocol, ‐104 +/‐ 31 l min‐1 (pH unit)‐1 vs. bicarbonate protocol, ‐60 +/‐ 44 l min‐1 (pH unit)‐1; mean +/‐ S.E.M.; not significant (n.s.)). The ventilatory sensitivity to hypoxia at an arterial pH of 7.35 was not significantly different between protocols (acid protocol, 14.7 +/‐ 3.3 l min‐1 vs. bicarbonate protocol, 15.6 +/‐ 2.4 l min‐1; mean +/‐ S.E.M.; n.s.). The results provide no evidence to suggest that peripheral chemoreflex ventilatory responses are modulated by central chemoreceptor stimulation.

An assessment of central-peripheral ventilatory chemoreflex interaction in humans.

The independence of the central and peripheral chemoreflexes has been tested in humans. Acute metabolic acidosis generated by a prior bout of brief, hard exercise was used to stimulate primarily the peripheral chemoreceptors, and respiratory acidosis generated by inhaled CO2 was used to stimulate both central and peripheral chemoreceptors. Seven healthy young men were studied. Ventilation and arterial pH, PCO2 and PO2 were recorded. Peripheral chemoreflex sensitivity to hypoxia during acute metabolic acidosis was repeatedly determined by measuring ventilation in euoxia (PETO2 = 100 Torr) and hypoxia (PETO2 = 50 Torr) as the subject recovered from exercise-induced acidosis. Peripheral chemoreflex sensitivity to hypoxia during CO2 inhalation was repeatedly determined by measuring ventilation in euoxia and hypoxia at two levels of hypercapnia (PETCO2 = 45 Torr and PETCO2 = 50 Torr). The ventilatory sensitivity to hypoxia at matched arterial pH values was not significantly different between conditions of high (CO2 inhalation) and low (metabolic acidosis) central chemoreceptor activity. We therefore conclude that interaction between central and peripheral chemoreflexes was non-significant in all subjects.

The impact of trisomy 21 on foetal haematopoiesis

The high frequency of a unique neonatal preleukaemic syndrome, transient abnormal myelopoiesis (TAM), and subsequent acute myeloid leukaemia in early childhood in patients with trisomy 21 (Down syndrome) points to a specific role for trisomy 21 in transforming foetal haematopoietic cells. N-terminal truncating mutations in the key haematopoietic transcription factor GATA1 are acquired during foetal life in virtually every case. These mutations are not leukaemogenic in the absence of trisomy 21. In mouse models, deregulated expression of chromosome 21-encoded genes is implicated in leukaemic transformation, but does not recapitulate the effects of trisomy 21 in a human context. Recent work using primary human foetal liver and bone marrow cells, human embryonic stem cells and iPS cells shows that prior to acquisition of GATA1 mutations, trisomy 21 itself alters human foetal haematopoietic stem cell and progenitor cell biology causing multiple abnormalities in myelopoiesis and B-lymphopoiesis. The molecular basis by which trisomy 21 exerts these effects is likely to be extremely complex, to be tissue-specific and lineage-specific and to be dependent on ontogeny-related characteristics of the foetal microenvironment.

Effect of raised potassium on ventilation in euoxia, hypoxia and hyperoxia at rest and during light exercise in man.

The purpose of this study was to determine whether changes in arterial plasma potassium concentration [K+]a affect expired ventilation (VE) in euoxia, hypoxia and hyperoxia during rest and light exercise in humans. Three periods of ventilatory measurements were undertaken in eight healthy subjects at rest and in seven other subjects during cycle ergometry (70 W). The first period of measurement was before the ingestion of 64 mmol of potassium chloride (KCl), the second 20 min after ingestion of KCl when [K+]a levels were elevated, and the third 3 h after the ingestion of KCl when [K+]a had returned substantially to normal. During each period, end‐tidal PO2 was cycled between euoxia, hypoxia and hyperoxia, whilst the end‐tidal PCO2 was maintained constant. The acute ventilatory response to hypoxia (AHVR) was calculated as the difference in VE during hypoxia and hyperoxia within each period of measurement. Oral KCl produced a 1.3 +/‐ 0.2 mM (mean +/‐ S.E.M.) increase in [K+]a at rest and a 0.8 +/‐ 0.2 mM increase during exercise. There was no significant difference in ventilation during euoxia between the three periods of measurement at rest or during exercise. There was a significant increase in AHVR with the rise in [K+]a of 21 min‐1 mM‐1 at rest (arterial PO2 during hypoxia ca 57 Torr) and 10 l min‐1 mM‐1 during exercise (arterial PO2 during hypoxia ca 52 Torr). There was a significant difference in the absolute increase in AHVR with [K+]a between rest and exercise, but this difference was not significant if the increase in AHVR with [K+]a was expressed as a percentage of the initial AHVR. We conclude that changes in [K+]a of the order of 1 mM have little effect on euoxic ventilation at rest or during light exercise in humans. We also conclude that [K+]a changes of this order increase AHVR at rest and during light exercise and that increases in [K+]a contribute to the increase in AHVR with exercise in humans.

Transcriptional and epigenetic basis for restoration of G6PD enzymatic activity in human G6PD-deficient cells.

HDAC inhibitors (HDACi) increase transcription of some genes through histone hyperacetylation. To test the hypothesis that HDACi-mediated enhanced transcription might be of therapeutic value for inherited enzyme deficiency disorders, we focused on the glycolytic and pentose phosphate pathways (GPPPs). We show that among the 16 genes of the GPPPs, HDACi selectively enhance transcription of glucose 6-phosphate dehydrogenase (G6PD). This requires enhanced recruitment of the generic transcription factor Sp1, with commensurate recruitment of histone acetyltransferases and deacetylases, increased histone acetylation, and polymerase II recruitment to G6PD. These G6PD-selective transcriptional and epigenetic events result in increased G6PD transcription and ultimately restored enzymatic activity in B cells and erythroid precursor cells from patients with G6PD deficiency, a disorder associated with acute or chronic hemolytic anemia. Therefore, restoration of enzymatic activity in G6PD-deficient nucleated cells is feasible through modulation of G6PD transcription. Our findings also suggest that clinical consequences of pathogenic missense mutations in proteins with enzymatic function can be overcome in some cases by enhancement of the transcriptional output of the affected gene.