Acute myeloid leukemia requires Hhex to enable PRC2-mediated epigenetic repression of Cdkn2a.

Unlike clustered HOX genes, the role of nonclustered homeobox gene family members in hematopoiesis and leukemogenesis has not been extensively studied. Here we found that the hematopoietically expressed homeobox gene Hhex is overexpressed in acute myeloid leukemia (AML) and is essential for the initiation and propagation of MLL-ENL-induced AML but dispensable for normal myelopoiesis, indicating a specific requirement for Hhex for leukemic growth. Loss of Hhex leads to expression of the Cdkn2a-encoded tumor suppressors p16(INK4a) and p19(ARF), which are required for growth arrest and myeloid differentiation following Hhex deletion. Mechanistically, we show that Hhex binds to the Cdkn2a locus and directly interacts with the Polycomb-repressive complex 2 (PRC2) to enable H3K27me3-mediated epigenetic repression. Thus, Hhex is a potential therapeutic target that is specifically required for AML stem cells to repress tumor suppressor pathways and enable continued self-renewal.

The diabetes gene Hhex maintains δ-cell differentiation and islet function.

The homeodomain transcription factor HHEX (hematopoietically expressed homeobox) has been repeatedly linked to type 2 diabetes mellitus (T2DM) using genome-wide association studies. We report here that within the adult endocrine pancreas, Hhex is selectively expressed in the somatostatin-secreting δ cell. Using two mouse models with Hhex deficiency in the endocrine pancreas, we show that Hhex is required for δ-cell differentiation. Decreased somatostatin levels in Hhex-deficient islets cause disrupted paracrine inhibition of insulin release from ß cells. These findings identify Hhex as the first transcriptional regulator specifically required for islet δ cells and suggest compromised paracrine control as a contributor to T2DM.

A crucial role for the homeodomain transcription factor Hhex in lymphopoiesis.

The hematopoietically expressed homeobox gene, Hhex, is a transcription factor that is important for development of definitive hematopoietic stem cells (HSCs) and B cells, and that causes T-cell leukemia when overexpressed. Here, we have used an Hhex inducible knockout mouse model to study the role of Hhex in adult hematopoiesis. We found that loss of Hhex was tolerated in HSCs and myeloid lineages, but resulted in a progressive loss of B lymphocytes in the circulation. This was accompanied by a complete loss of B-cell progenitors in the bone marrow and of transitional B-cell subsets in the spleen. In addition, transplantation and in vitro culture experiments demonstrated an almost complete failure of Hhex-null HSCs to contribute to lymphoid lineages beyond the common lymphoid precursor stage, including T cells, B cells, NK cells, and dendritic cells. Gene expression analysis of Hhex-deleted progenitors demonstrated deregulated expression of a number of cell cycle regulators. Overexpression of one of these, cyclin D1, could rescue the B-cell developmental potential of Hhex-null lymphoid precursors. Thus, Hhex is a key regulator of early lymphoid development, functioning, at least in part, via regulation of the cell cycle.

Endodermal and mesenchymal cross talk: a crossroad for the maturation of foregut organs.

The developmental stages of each foregut organ are intimately linked to the development of the other foregut organs such that the ultimate function of any one foregut organ, such as the metabolic function of the liver, depends on organizational changes associated with the maturation of multiple foregut organs. These changes include: (i) proliferation of the intrahepatic bile ducts and hepatoblasts within the liver coinciding with parenchymal expansion, (ii) elongation of extrahepatic bile ducts, which allows for proper gallbladder (GB) formation, and (iii) duodenal elongation and rotation, which coincides with all of the above to connect the intrahepatic, extrahepatic, and pancreatic ductal systems with the intestine. It is well established that cross talk between endodermal and mesenchymal components of the foregut occurs, particularly regarding the vascularization of developing organs. Furthermore, genetic mutations in mesenchymal and hepatic compartments of the developing foregut result in similar foregut pathologies: hypoplastic liver, absence of GB, biliary atresia (intrahepatic and/or extrahepatic), and failure of gut elongation and rotation. Finally, these shared pathologies can be linked to deficiencies in genes specific to the septum transversum mesenchyme (Hes1, Hlx, and Foxf1) or liver (Hhex and Hnf6), illustrating the complexity of such cross talk.

SNAP II index: an alternative to the COMFORT scale in assessing the level of sedation in mechanically ventilated pediatric patients.

Sedation monitoring is essential in pediatric patients on ventilatory support to achieve comfort and safety. The COMFORT scale was designed and validated to assess the level of sedation in intubated pediatric patients. However, it remains unreliable in pharmacologically paralyzed patients. The SNAP II index is calculated using an algorithm that incorporates high-frequency (80-420 Hz) electroencephalogram (EEG) components, known to be useful in discriminating between awake and unconscious states, unlike other measurements that only include low-frequency EEG segments such as the bispectral index score. Previous studies suggested that the SNAP II index is a reliable and sensitive indicator of the level of consciousness in adult patients. Despite its potential, no data are currently available in the pediatric critically ill population on ventilatory support. This is the first pilot study assessing the potential application of the SNAP II index in critically ill pediatric patients by comparing it to the commonly used COMFORT scale.

The homeobox gene Hhex regulates the earliest stages of definitive hematopoiesis.

The development and emergence of the hematopoietic stem cell involves a series of tightly regulated molecular events that are not well characterized. The hematopoietically expressed homeobox (Hhex) gene, a member of the homeobox gene family, is an essential regulator of embryogenesis and hematopoietic progenitor development. To investigate the role of Hhex in hematopoiesis we adapted a murine embryonic stem (ES) cell coculture system, in which ES cells can differentiate into CD41(+) and CD45(+) hematopoietic progenitors in vitro. Our results show that in addition to delayed hemangioblast development, Hhex(-/-) ES-derived progeny accumulate as CD41(+) and CD41(+)c-kit(+) cells, or the earliest definitive hematopoietic progenitors. In addition, Hhex(-/-) ES-derived progeny display a significantly reduced ability to develop into mature CD45(+) hematopoietic cells. The observed reduction in hematopoietic maturation was accompanied by reduced proliferation, because Hhex(-/-) CD41(+)CD45(-)c-kit(+) hematopoietic progenitors accumulated in the G(2) phase of the cell cycle. Thus, Hhex is a critical regulator of hematopoietic development and is necessary for the maturation and proliferation of the earliest definitive hematopoietic progenitors.

Relationship between hypoglycemia and mortality in critically ill children.

OBJECTIVES: To determine the prevalence of hypoglycemia in critically ill nondiabetic children and the association of hypoglycemia with mortality and worsening organ function in critically ill children. DESIGN: Retrospective cohort study with matched-cohort analysis. SETTING: Academic pediatric intensive care unit. PATIENTS: A total of 899 nondiabetic patients <18 yrs old admitted to the pediatric intensive care unit for >1 day with at least one blood glucose measurement. Forty-two patients with a blood glucose level of <50 mg/dL (<2.8 mmol/L) were matched with 126 nonhypoglycemic patients. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Hypoglycemia, based on point-of-care blood glucose measurements, occurred in 2.2% (<40 mg/dL [<2.2 mmol/L]) to 7.5% (<60 mg/dL [<3.3 mmol/L]) of the patients. Hypoglycemia was more common in patients on mechanical ventilation and/or vasopressor support. Severity of hypoglycemia correlated with an increased mortality rate. The highest odds ratio of mortality was 4.49 (95% confidence interval [CI], 1.69-11.96; p < .01) at a blood glucose level of <40 mg/dL (<2.2 mmol/L). In the matched analysis, hypoglycemia was an independent risk factor for mortality. The unadjusted, covariate-adjusted, and propensity score-adjusted odds ratios of mortality were 3.69 (95% CI, 1.78-7.68; p < .01), 4.16 (95% CI, 1.53-11.32; p < .01), and 8.45 (95% CI, 1.75-40.86; p < .01), respectively. Hypoglycemia was associated with worsening organ function in the covariate-adjusted model (odds ratio, 2.37; 95% CI, 1.12-5.01; p = .02) but not in the unadjusted and propensity-score adjusted models. CONCLUSIONS: Hypoglycemia is common in critically ill children. It is associated with increased mortality rates in critically ill nondiabetic children. Our data suggest that hypoglycemia is also associated with worsening organ function. Hypoglycemia may merely be a marker of severity of illness. Further investigations are needed to establish the mortality risk with hypoglycemia due to insulin compared to spontaneous hypoglycemia.

HHEX is a transcriptional regulator of the VEGFC/FLT4/PROX1 signaling axis during vascular development.

Formation of the lymphatic system requires the coordinated expression of several key regulators: vascular endothelial growth factor C (VEGFC), its receptor FLT4, and a key transcriptional effector, PROX1. Yet, how expression of these signaling components is regulated remains poorly understood. Here, using a combination of genetic and molecular approaches, we identify the transcription factor hematopoietically expressed homeobox (HHEX) as an upstream regulator of VEGFC, FLT4, and PROX1 during angiogenic sprouting and lymphatic formation in vertebrates. By analyzing zebrafish mutants, we found that hhex is necessary for sprouting angiogenesis from the posterior cardinal vein, a process required for lymphangiogenesis. Furthermore, studies of mammalian HHEX using tissue-specific genetic deletions in mouse and knockdowns in cultured human endothelial cells reveal its highly conserved function during vascular and lymphatic development. Our findings that HHEX is essential for the regulation of the VEGFC/FLT4/PROX1 axis provide insights into the molecular regulation of lymphangiogenesis.

Decompensated hyperglycemic hyperosmolarity without significant ketoacidosis in the adolescent and young adult population.

AIM: To identify patients aged 10-30 years with probable hyperglycemic hyperosmolar syndrome (HHS), to describe demographic and clinical profiles, and to attempt to assess risk factors for poor outcomes. STUDY DESIGN: Retrospective cohort study (medical records review). SETTING: A 944-bed tertiary care teaching and research hospital and a 425-bed affiliated facility. PATIENTS: 10-30 year-old patients with a primary or secondary discharge diagnosis of HHS or diabetic ketoacidosis (DKA). Patients with a serum glucose >600 mg/dl in the absence of significant ketoacidosis (possible HHS) were profiled. Further stratification based on measured or calculated serum osmolarity >320 mOsm/kg (probable HHS) was undertaken. INTERVENTIONS: Patients received treatment for hyperglycemic crises, consisting primarily of fluids, electrolyte replacement and insulin. MEASUREMENTS AND MAIN RESULTS: Of the 629 admissions, 10 with a diagnosis of HHS and 33 with a diagnosis of DKA met the initial study criteria for HHS. 60% were African Americans and 89% were new-onset diabetics. From this group, 20 admissions had serum osmolarity > or =320 mOsm/kg. Fisher's exact test and Pearson coefficients were used to examine associations between risk factor and poor outcomes and correlations between admission data and length of hospital stay, respectively. Serious complications occurred in four patients (including two deaths, 10% mortality) and were limited to those with unreversed shock over the first 24 hours of admission and who received <40 ml/kg of intravenous fluids over the first 6 hours of treatment. CONCLUSIONS: HHS was underdiagnosed in this population and occurred disproportionately in African Americans. Serious complications occurred exclusively in those with unreversed shock and inadequate fluid resuscitation.

The Next 7 Great Achievements in Pediatric Research.

The "7 Great Achievements in Pediatric Research" campaign noted discoveries in the past 40 years that have improved child and adult health in the United States and around the globe. This article predicts the next 7 great pediatric research advancements, including new immunizations, cancer immunotherapy, genomic discoveries, identification of early antecedents of adult health, impact of specific social-environmental influences on biology and health, quality improvement science, and implementation and dissemination research to reduce global poverty. It is an extraordinary time of new research tools that include electronic health records, technological ability to manage big data and measure "omics," and new functional and structural imaging modalities. These tools will discern mechanisms leading to health and disease with new prevention targets and cures. This article further discusses the challenges and opportunities to accelerate these exciting pediatric research discoveries to improve the lives of children and the adults they will become.

Thyroid-specific transcription factors control Hex promoter activity.

The homeobox-containing gene Hex is expressed in several cell types, including thyroid follicular cells, in which it regulates the transcription of tissue- specific genes. In this study the regulation of Hex promoter activity was investigated. Using co- transfection experiments, we demonstrated that the transcriptional activity of the Hex gene promoter in rat thyroid FRTL-5 cells is approximately 10-fold greater than that observed in HeLa and NIH 3T3 cell lines (which do not normally express the Hex gene). To identify the molecular mechanisms underlying these differences, we evaluated the effect of the thyroid- specific transcription factor TTF-1 on the Hex promoter activity. TTF-1 produced 3-4-fold increases in the Hex promoter activity. Gel- retardation assays and mutagenesis experiments revealed the presence of functionally relevant TTF-1 binding sites in the Hex promoter region. These in vitro data may also have functional relevance in vivo, since a positive correlation between TTF-1 and Hex mRNAs was demonstrated in human thyroid tissues by means of RT-PCR analysis. The TTF-1 effect, however, is not sufficient to explain the difference in Hex promoter activity between FRTL-5 and cells that do not express the Hex gene. For this reason, we tested whether Hex protein is able to activate the Hex promoter. Indeed, co-transfection experiments indicate that Hex protein is able to increase the activity of its own promoter in HeLa cells approximately 4-fold. TTF-1 and Hex effects are additive: when transfected together in HeLa cells, the Hex promoter activity is increased 6-7-fold. Thus, the contemporary presence of both TTF-1 and Hex could be sufficient to explain the higher transcriptional activity of the Hex promoter in thyroid cells with respect to cell lines that do not express the Hex gene. These findings demonstrate the existence of direct cross-regulation between thyroid-specific transcription factors.

Hhex Is Necessary for the Hepatic Differentiation of Mouse ES Cells and Acts via Vegf Signaling.

Elucidating the molecular mechanisms involved in the differentiation of stem cells to hepatic cells is critical for both understanding normal developmental processes as well as for optimizing the generation of functional hepatic cells for therapy. We performed in vitro differentiation of mouse embryonic stem cells (mESCs) with a null mutation in the homeobox gene Hhex and show that Hhex(-/-) mESCs fail to differentiate from definitive endoderm (Sox17(+/)Foxa2(+)) to hepatic endoderm (Alb(+)/Dlk(+)). In addition, hepatic culture elicited a >7-fold increase in Vegfa mRNA expression in Hhex(-/-) cells compared to Hhex(+/+) cells. Furthermore, we identified VEGFR2(+)/ALB(+/)CD34(-) in early Hhex(+/+) hepatic cultures. These cells were absent in Hhex(-/-) cultures. Finally, through manipulation of Hhex and Vegfa expression, gain and loss of expression experiments revealed that Hhex shares an inverse relationship with the activity of the Vegf signaling pathway in supporting hepatic differentiation. In summary, our results suggest that Hhex represses Vegf signaling during hepatic differentiation of mouse ESCs allowing for cell-type autonomous regulation of Vegfr2 activity independent of endothelial cells.

Spontaneous Pancreatitis Caused by Tissue-Specific Gene Ablation of Hhex in Mice.

BACKGROUND & AIMS: Perturbations in pancreatic ductal bicarbonate secretion cause chronic pancreatitis. The physiologic mechanism of ductal secretion is known, but its transcriptional control is not. We determine the role of the transcription factor hematopoietically expressed homeobox protein (Hhex) in ductal secretion and pancreatitis. METHODS: We derived mice with pancreas-specific, Cremediated Hhex gene ablation to determine the requirement of Hhex in the pancreatic duct in early life and in adult stages. Histologic and immunostaining analyses were used to detect the presence of pathology. Pancreatic primary ductal cells were isolated to discover differentially expressed transcripts upon acute Hhex ablation on a cell autonomous level. RESULTS: Hhex protein was detected throughout the embryonic and adult ductal trees. Ablation of Hhex in pancreatic progenitors resulted in postnatal ductal ectasia associated with acinar-to-ductal metaplasia, a progressive phenotype that ultimately resulted in chronic pancreatitis. Hhex ablation in adult mice, however, did not cause any detectable pathology. Ductal ectasia in young mice did not result from perturbation of expression of Hnf6, Hnf1ß, or the primary cilia genes. RNA-seq analysis of Hhex-ablated pancreatic primary ductal cells showed mRNA levels of the G-protein coupled receptor natriuretic peptide receptor 3 (Npr3), implicated in paracrine signaling, up-regulated by 4.70-fold. CONCLUSIONS: Although Hhex is dispensable for ductal cell function in the adult, ablation of Hhex in pancreatic progenitors results in pancreatitis. Our data highlight the critical role of Hhex in maintaining ductal homeostasis in early life and support ductal hypersecretion as a novel etiology of pediatric chronic pancreatitis.

Genetic Models in Applied Physiology. Functional genomics in the mouse: powerful techniques for unraveling the basis of human development and disease.

Now that near-complete DNA sequences of both the mouse and human genomes are available, the next major challenge will be to determine how each of these genes functions, both alone and in combination with other genes in the genome. The mouse has a long and rich history in biological research, and many consider it a model organism for the study of human development and disease. Over the past few years, exciting progress has been made in developing techniques for chromosome engineering, mutagenesis, mapping and maintenance of mutations, and identification of mutant genes in the mouse. In this mini-review, many of these powerful techniques will be presented along with their application to the study of development, physiology, and disease.

Hypoglycemia in critically ill children.

BACKGROUND: The practice of glycemic control with intravenous insulin in critically ill patients has brought clinical focus on understanding the effects of hypoglycemia, especially in children. Very little is published on the impact of hypoglycemia in this population. We aimed to review the existing literature on hypoglycemia in critically ill neonates and children. METHODS: We performed a systematic review of the literature up to August 2011 using PubMed, Ovid MEDLINE and ISI Web of Science using the search terms "hypoglycemia or hypoglyc*" and "critical care or intensive care or critical illness". Articles were limited to "all child (0-18 years old)" and "English". RESULTS: A total of 513 articles were identified and 132 were included for review. Hypoglycemia is a significant concern among pediatric and neonatal intensivists. Its definition is complicated by the use of a biochemical measure (i.e., blood glucose) for a pathophysiologic problem (i.e., neuroglycopenia). Based on associated outcomes, we suggest defining hypoglycemia as <40-45 mg/dl in neonates and <60-65 mg/dl in children. Below the suggested threshold values, hypoglycemia is associated with worse neurological outcomes, increased intensive care unit stay, and increased mortality. Disruptions in carbohydrate metabolism increase the risk of hypoglycemia incritically ill children. Prevention of hypoglycemia, especially in the setting of intravenous insulin use, will be best accomplished by the combination of accurate measuring techniques, frequent or continuous glucose monitoring, and computerized insulin titration protocols. CONCLUSION: Studies on hypoglycemia in critically ill children have focused on spontaneous hypoglycemia. With the current practice of maintaining blood glucose within a narrow range with intravenous insulin, the risk factors and outcomes associated with insulin-induced hypoglycemia should be rigorously studied to prevent hypoglycemia and potentially improve outcomes of critically ill children.