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Although the coronavirus disease 2019 pandemic has served as a flashlight, illuminating and unmasking deep socio-economic and health care divides in our country, the terrible events surrounding the horrific murder of Mr. George Floyd in Minneapolis has spawned even greater outrage. As we all know, Mr. Floyd’s death is not an isolated incident, as there have been a tragic string of such deaths in recent years that further reflect deep issues regarding racism and systemic underlying causes of injustice. Unfortunately, the country’s inability to fully address these systemic foundations of injustice persists.

Pediatricians play a key role in advancing child health research to best attain and improve the physical, mental, and social health and well-being of all infants, children, adolescents, and young adults. Child health presents unique issues that require investigators who specialize in pediatric research. In addition, the scope of the pediatric research enterprise is transdisciplinary and includes the full spectrum of basic science, translational, community-based, health services, and child health policy research. Although most pediatricians do not directly engage in research, knowledge of research methodologies and approaches promotes critical evaluation of scientific literature, the practice of evidence-based medicine, and advocacy for evidence-based child health policy. This statement includes specific recommendations to promote further research education and support at all levels of pediatric training, from premedical to continuing medical education, as well as recommendations to increase support and mentorship for research activities. Pediatric research is crucial to the American Academy of Pediatrics’ goal of improving the health of all children. The American Academy of Pediatrics continues to promote and encourage efforts to facilitate the creation of new knowledge and ways to reduce barriers experienced by trainees, practitioners, and academic faculty pursuing research.

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 β 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.

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.

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.

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 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.

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 G2 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.

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.

In November 2014, the American Academy of Pediatrics convened key stakeholders to discuss the feasibility of accelerating children's medical advances by creating an independent global Pediatric Clinical Trials Network. The Forum identified challenges posed by the U.S. and global clinical trial systems regarding testing and disseminating drugs and devices for pediatric patients. Stakeholders mapped a vision to improve the safety and efficacy of pediatric drugs, biological products, and medical devices by creating a global Pediatric Clinical Trials Network. Such a Network would act as a central infrastructure for pediatric subspecialties and enable dedicated staff to provide clinical research sites with scientific, medical, and operational support. A Network would facilitate development and availability of innovative, high-quality therapies to extend and enhance the lives of neonates, infants, children, adolescents, and young adults. Participants expressed strong interest in forming such a Network, since drugs and devices still come to market without adequate pediatric indications-particularly in neonatology and rare diseases. Participants developed a Consensus Statement expressing their shared vision for a Network: Attendees of the Pediatric Clinical Trials Stakeholder Forum resolved to establish a Global Pediatric Clinical Trials Network and are committed to engage in the work to create and sustain it.

Hhex encodes a homeodomain-containing protein that functions as both a transcriptional repressor and activator, and is necessary for normal embryonic development. We previously reported that a null mutation of Hhex leads to abnormalities in vasculogenesis and have focused on identifying the transcriptional targets of Hhex necessary for vascular development. Here we report that the expression of ESM-1 , a cysteine-rich protein expressed in the endothelium, is increased in Hhex −/− embryos. Overexpression of Hhex in endothelial cells down-regulates ESM-1. The results from transient cotransfection assay, electrophoretic-mobility shift assay, site-directed mutagenesis, and chromatin immunoprecipitation assay demonstrate that Hhex can directly bind to and repress ESM-1 via an evolutionarily conserved Hhex response element (HRE) 1. These findings indicate that ESM-1 is a direct target of Hhex and that Hhex functions as a transcriptional repressor of ESM-1 . We speculate that Hhex-mediated repression of ESM-1 is critical for the normal function of the vascular endothelium and for tumor vasculogenesis.

The homeobox gene Hhex has recently been shown to be essential for normal liver, thyroid and forebrain development. Hhex–/– mice die by mid-gestation (E14.5) and the cause of their early demise remains unclear. Because Hhex is expressed in the developing blood islands at E7.0 in the endothelium of the developing vasculature and heart at E9.0-9.5, and in the ventral foregut endoderm at E8.5-9.0, it has been postulated to play a critical role in heart and vascular development. We show here, for the first time, that a null mutation of Hhex results in striking abnormalities of cardiac and vascular development which include: (1) defective vasculogenesis, (2)hypoplasia of the right ventricle, (3) overabundant endocardial cushions accompanied by ventricular septal defects, outflow tract abnormalities and atrio-ventricular (AV) valve dysplasia and (4) aberrant development of the compact myocardium. The dramatic enlargement of the endocardial cushions in the absence of Hhex is due to decreased apoptosis and dysregulated epithelial-mesenchymal transformation (EMT). Interestingly, vascular endothelial growth factor A (Vegfa) levels in the hearts of Hhex–/– mice were elevated as much as three-fold between E9.5 and E11.5, and treatment of cultured Hhex–/– AV explants with truncated soluble Vegfa receptor 1, sFlt-1, an inhibitor of Vegf signaling, completely abolished the excessive epithelial-mesenchymal transformation seen in the absence of Hhex. Therefore, Hhex expression in the ventral foregut endoderm and/or the endothelium is necessary for normal cardiovascular development in vivo, and one function of Hhex is to repress Vegfa levels during development.

Despite extensive research, bacterial sepsis and its associated systemic inflammation remain a major cause of morbidity and mortality in the pediatric intensive care unit. Advances in molecular biology, however, have improved our understanding of this disease process and have opened up new avenues of potential therapeutic approaches. One such exciting area has been the substantial and still growing evidence that the mammalian immune system uses a family of Toll-like receptors (TLRs) to generate a response to molecular patterns present on invading microorganisms. In particular, TLR4 is part of a recognition complex for bacterial lipopolysaccharide (LPS), thus raising the likelihood of its involvement in the inflammatory response to bacterial sepsis. This review highlights our understanding of the molecular biology of these receptors, focusing on the LPS response, and concluding with a summary of ongoing evaluation and potential therapeutic strategies for treating sepsis through blockade of TLR signaling.

Lmo2 is an oncogenic transcription factor that is frequently overexpressed in T-cell acute leukemias, in particular poor prognosis early T-cell precursor-like (ETP-) acute lymphoblastic leukemia (ALL). The primary effect of Lmo2 is to cause self-renewal of developing CD4(-)CD8(-) (double negative, DN) T cells in the thymus, leading to serially transplantable thymocytes that eventually give rise to leukemia. These self-renewing thymocytes are intrinsically radioresistant implying that they may be a source of leukemia relapse after therapy. The homeobox transcription factor, Hhex, is highly upregulated in Lmo2-transgenic thymocytes and can phenocopy Lmo2 in inducing thymocyte self-renewal, implying that Hhex may be a key component of the Lmo2-induced self-renewal program. To test this, we conditionally deleted Hhex in the thymi of Lmo2-transgenic mice. Surprisingly, this did not prevent accumulation of DN thymocytes, nor alter the rate of overt leukemia development. However, deletion of Hhex abolished the transplantation capacity of Lmo2-transgenic thymocytes and overcame their radioresistance. We found that Hhex regulates Kit expression in Lmo2-transgenic thymocytes and that abrogation of Kit signaling phenocopied loss of Hhex in abolishing the transplantation capacity and radioresistance of these cells. Thus, targeting the Kit signaling pathway may facilitate the eradication of leukemia-initiating cells in immature T-cell leukemias in which it is expressed.

A polyclonal antibody against a glutathione S:-transferase fusion protein containing the 76 COOH-terminal amino acids of Hex, a divergent homeobox gene, was raised in rabbits. Western blot and immunofluorescence reveal that Hex is a 35-37-kD soluble protein present both in the nucleus and cytoplasm of transfected and nontransfected cultured cells as well as in whole mouse embryo. Confocal microscopy of whole mount immunostained mouse embryos at E7. 5 and E8.5 demonstrates that Hex is differentially localized in the cytoplasm and nucleus of definitive endoderm, developing blood islands, and hepatic diverticulum. In particular, in the region of the foregut that gives rise to the liver, Hex expression is nuclear in the endodermal cells of the hepatic diverticulum, whereas expression is primarily cytoplasmic in cells lateral to the liver-forming region. This suggests that nuclear localization of Hex is involved in early hepatic specification and that compartmentalization of Hex protein plays an important role in its function during mouse development.

Hex is a divergent homeobox gene expressed as early as E4.5 in the mouse and in a pattern that suggests a role in anterior-posterior patterning. Later in embryogenesis, Hex is expressed in the developing thyroid, lung, and liver. We now show Hex expression during thymus, gallbladder, and pancreas development and in the adult thyroid, lung, and liver. At E10.0, Hex is expressed in the 3rd pharyngeal pouch, from which the thymus originates, the endodermal cells of liver that are invading the septum transversum, the thyroid, the dorsal pancreatic bud, and gallbladder primoridum. At E13.5, expression is maintained at high levels in the thyroid, liver, epithelial cells lining the pancreatic and extrahepatic biliary ducts and is present in both the epithelial and mesenchymal cells of the lung. Expression in the thymus at this age is less than in the other organs. In the E16.5 embryo, expression persists in the thyroid, pancreatic, and bile duct epithelium, lung, and liver, with thymic expression dropping to barely detectable levels. By E18.5, expression in the thyroid and bile ducts remains high, whereas lung expression is markedly decreased. At this age, expression in the pancreas and thymus is no longer present. Finally, we show the cell types in the adult thyroid, lung, and liver that express Hex in the mature animal. Our results provide more detail on the potential role of Hex in the development of several organs derived from foregut endoderm and in the maintenance of function of several of these organs in the mature animal.

Diabetes is known to be associated with delayed lung development in humans and in experimental animals. This includes delayed expression of surfactant apoproteins. An important component of the metabolic abnormalities in diabetes is elevated levels of analogs of butyric acid, and the effects of diabetes on surfactant apoproteins can be reproduced by exposure of fetal rat lung explants to butyrate. Dexamethasone has the opposite effects on lung development. In humans, antenatal exposure to dexamethasone results in a lower incidence of RDS, whereas in experimental animals, dexamethasone increases the expression of surfactant apoproteins. A subset of Hox genes are expressed in developing lung, and their level of expression decreases with advancing gestation. We hypothesized that: 1) lungs of fetuses of rats with streptozotocin-induced diabetes would have altered levels of expression of Hox genes, 2) the effect would be mediated in part through elevated levels of butyrate, and 3) dexamethasone would reverse the effect. We tested our hypotheses in vivo using fetuses from streptozotocin-treated rats and in vitro by treating lung explants from normal rats with sodium butyrate. Streptozotocin treatment increased expression of Hoxb-5 at 18 d of gestation, but did not affect Hoxa-5 expression. This was associated with a 20-fold increase in alpha-aminobutyrate levels. Dexamethasone tended to reverse this effect. In contrast, butyrate treatment of explants decreased the expression of Hoxa-5 and Hoxb-5. We conclude that diabetes alters expression of Hox genes, but that the effect of butyrate on lung development, and in particular on surfactant apoprotein expression, is independent of its effects on Hox genes.

Over the past several years, many new strategies have evolved for improving the care of patients with acute lung injury and respiratory failure. Although many of these new modalities remain unproven, some show much promise for decreasing the morbidity and mortality seen in critically ill patients who need assisted ventilation. In this review, we discuss recent data concerning four of these modalities: high frequency ventilation, prone positioning, tracheal gas insufflation, and partial liquid ventilation.

In this chapter, we discuss some of the more serious syndromes associated with infectious diseases that are encountered by clinicians in the PICU. The syndromes discussed include necrotizing fasciitis, hemorrhagic shock and encephalopathy, Rocky Mountain spotted fever and Lemierre’s syndrome. All of these conditions are serious, potentially life-threatening syndromes for which early recognition and treatment is critical to obtaining a positive outcome for the patient. We discuss the history and epidemiology, pathogenesis, clinical manifestations and management of each condition with the goal of providing clinicians with the most current information about each syndrome.

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.

Hox genes are sequence-specific DNA transcription factors, which are important in embryonic development and are expressed in a number of fetal tissues, including the lung. Additionally, retinoic acid (RA) has been shown to modulate Hox gene expression in a number of cell types. The specific aims of this study were to 1) identify those Hox genes expressed in newborn mouse lung using reverse transcription-polymerase chain reaction (RT-PCR), 2) study the ontogeny of Hox gene expression in fetal mouse and rat lung by Northern analysis using cDNAs for mouse Hox genes, and 3) study the effects of RA on whole lung Hox mRNA levels in cultured fetal rat lung explants. Our data show that 16 different homeobox genes are expressed in newborn mouse lung. This includes seven Hox genes not previously identified in lung, as well as the divergent homeobox gene Hex. Steady-state mRNA levels of Hox A5 (Hox 1.3), B5 (Hox 2.1), B6 (Hox 2.2), and B8 (Hox 2.4) decrease with advancing gestational age in mouse lungs (E14 to adult). Similarly, Hox A5, B5, and B6 follow the same decreasing pattern of expression with advancing gestational age in rat lungs (E15 to adult). RA treatment of E17 rat lung explants in culture resulted in a significant dose- and time-dependent increase in Hox A5, B5, and B6 mRNA levels. The highest mRNA levels were seen in explants treated with 1 x 10(-5) M RA for 4-16 h. We conclude that there are many homeobox genes expressed in developing rodent lung and that their mRNA levels are affected by both gestational age and RA.

esults of recent studies exam-ining glycemic control in crit-ically ill patients have high-lighted the significance ofhypoglycemia. In a landmark study byVan den Berghe et al (1), tightly control-ling blood glucose (BG) levels at 80–110mg/dL (4.4–6.1 mmol/L) with intrave-nous insulin achieved an absolute reduc-tion of 3.4% in mortality rate in mechan-ically ventilated adult surgical patients.The rate of severe hypoglycemia, which isthe most important adverse outcome withinsulin use, with BG at 40 mg/dL ( 2.2mmol/L), was six-fold higher in the inter-vention group compared to the controlgroupinwhichBGwasmaintainedat180–200 mg/dL (10.0–11.1 mmol/L). Subse-quent trials (2–4) designed to confirm thevalidity of the Van den Berghe et al studyresults failed to demonstrate any improve-ment in mortality rate. Higher hypoglyce-mia rates in the insulin-treated inter-vention groups have been postulated tonegate the survival benefit of glycemiccontrol (4, 5). Despite these findings,professional organizations (6–8) con-tinue to recommend glycemic controlin critically ill adults. In children, onthe basis of the adult experience and inconjunction with nominal pediatricdata, intravenous insulin is commonlyused to achieve glycemic control in selectsubsets of critically ill children (9–12). Al-thoughhypoglycemiaisasignificantconcernamong pediatric intensivists (9, 13), the risksandoutcomesofhypoglycemiaincriticallyillchildren are uncertain.The prevalence of hypoglycemia in crit-ically ill children has been previously re-ported (14, 15). However, the publishedrates were derived from all patients admit-ted to the pediatric intensive care unit(ICU). Hypoglycemia rates in critically illchildren receiving vasopressor supportand/or on mechanical ventilation, whichare the target populations for glycemiccontrol in children (11, 12, 16, 17), areunknown. Vasopressor support and me-chanical ventilation are significant risk fac-tors for hypoglycemia in critically ill adults(18, 19) and are also expected to affect therates of hypoglycemia in children.The relationship between hypoglyce-mia and mortality in children is unclear.Although implicated as a cause of “death-in-bed” syndrome in diabetic children,the risk of mortality has not been ade-quately quantified (20, 21). Wintergerstet al (14) reported an increased risk ofmortality in critically ill nondiabetic chil-dren with hypoglycemia. However, therisk of mortality was only adjusted forhyperglycemia and glucose variability.Other factors, particularly severity of ill

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

Hhex is required for early development of the liver. A null mutation of Hhex results in a failure to form the liver bud and embryonic lethality. Therefore, Hhex null mice are not informative as to whether this gene is required during later stages of hepatobiliary morphogenesis. To address this question, we derived Hhex conditional null mice using the Cre-loxP system and two different Cre transgenics (Foxa3-Cre and Alfp-Cre). Deletion of Hhex in the hepatic diverticulum (Foxa3-Cre;Hhex(d2,3/-)) led to embryonic lethality and resulted in a small and cystic liver with loss of Hnf4alpha and Hnf6 expression in early hepatoblasts. In addition, the gall bladder was absent and the extrahepatic bile duct could not be identified. Loss of Hhex in the embryonic liver (Alfp-Cre;Hhex(d2,3/-)) caused irregular development of intrahepatic bile ducts and an absence of Hnf1beta in many (cystic) biliary epithelial cells, which resulted in a slow, progressive form of polycystic liver disease in adult mice. Thus, we have shown that Hhex is required during multiple stages of hepatobiliary development. The altered expression of Hnf4alpha, Hnf6 and Hnf1beta in Hhex conditional null mice suggests that Hhex is an essential component of the genetic networks regulating hepatoblast differentiation and intrahepatic bile duct morphogenesis.

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.

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.

The divergent homeobox gene Hex is expressed in both developing and mature liver. A putative Hex binding site was identified in the promoter region of the liver-specific Na+-bile acid cotransporter gene ( ntcp), and we hypothesized that Hex regulates the ntcp promoter through this site. Successive 5′-deletions of the ntcp promoter in a luciferase reporter construct transfected into Hep G2 cells confirmed a Hex response element (HRE) within the ntcppromoter (nt −733/−714). Moreover, p-CMHex transactivated a heterologous promoter construct containing HRE multimers (p4xHRELUC), whereas a 5-bp mutation of the core HRE eliminated transactivation. A dominant negative form of Hex (p- Hex-DN) suppressed basal luciferase activity of p-4xHRELUC and inhibited activation of this construct by p-CMHex. Interestingly, p-CMHex transactivated the HRE in Hep G2 cells but not in fibroblast-derived COS cells, suggesting the possibility that Hex protein requires an additional liver cell-specific factor(s) for full activity. Electrophoretic mobility shift assays confirmed that liver and Hep G2 cells contain a specific nuclear protein that binds the native HRE. We have demonstrated that the liver-specific ntcp gene promoter is the first known target of Hex and is a useful tool for evaluating function of the Hex protein.

The orphan homeobox gene, Hex, has a limited domain of expression which includes the developing and adult mouse liver. Hex is expressed in the developing liver coincident with the forkhead/winged helix transcription factor, Hepatocyte Nuclear Factor 3beta (HNF3beta). Although preliminary characterization of the mouse Hex promoter has recently been reported, the identity of the molecular regulators that drive liver expression is not known. We hypothesized that putative HNF3beta and GATA-4 elements within the Hex promoter would confer liver-enriched expression. A series of Hex promoter-driven luciferase reporter constructs were transfected in liver-derived HepG2 and fibroblast-like Cos cells+/-HNF3beta or GATA expression plasmids. The Hex promoter region from nt -235/+22 conferred basal activity in both HepG2 and Cos cells, with the region from -103/+22 conferring liver-enriched activity. HNF3beta and GATA-4 transactivated the promoter via response elements located within nt -103/+22, whereas Sp1 activated the -235/+22 construct. Mutation of the HNF3 element significantly reduced promoter activity in HepG2 cells, whereas this element in isolation conferred HNF3beta responsiveness to a heterologous promoter. Electrophoretic mobility shift assays were performed to confirm transcription factor:DNA binding. We conclude that HNF3beta and GATA-4 contribute to liver-enriched expression of Hex.

Over the last 10 years, there has been a fundamental change in physicians' attitudes toward analgesia and sedation in pediatrics. In this time, basic and clinical research have provided a wealth of information. In this paper we review important advances registered in the past year, including new molecular and physiological mechanisms of antinociception and sedation, behavioral and psychoemotional implications of pain, and advances in the clinical practice of pediatric analgesia and sedation. Fortunately, the attitude of physicians toward these matters has changed significantly and much more attention is now paid to the alleviation of pain and provision of adequate sedation. However, there remains, according to most estimates, incongruity between these advances and what is practiced clinically.

Retinoic acid has both early or immediate (within hours) and late (after days) effects on gene expression. We studied the early effects of retinoic acid on the surfactant protein (SP) genes. Exposure of fetal rat lung explants to all trans-retinoic acid for 4 h resulted in a significant dose-dependent increase in SP-A, -B, and -C mRNA with markedly different dose-response characteristics. The maximal (2.5x) increase in SP-A mRNA was observed with 10(-10) M retinoic acid, whereas treatment with 10(-5) M resulted in a tendency to decreased levels. In contrast, maximal stimulation of SP-C (6x) was noted at 10(-5) M retinoic acid and that of SP-B (2x) at 10(-7) to 10(-5) M retinoic acid. Similar differences in the dose-response characteristics of SP-A and SP-C were observed with 9-cis-retinoic acid. A retinoic acid response element consensus sequence was identified in the rat SP-A gene; we hypothesize that retinoic acid-receptor complexes act directly on the SP-A gene via this response element.

Lung development in the mouse begins at embryonic day 9.5 (E9.5) when lung buds form in the foregut. Subsequently, there is extensive branching and cellular differentiation that depends upon specific epithelial-mesenchymal interactions. Homeobox genes are expressed in specific temporo-spatial patterns in the developing embryo and are known to be involved in axial patterning and specification of regional identity. Using whole mount in situ hybridization and immunohistochemistry, we studied the expression of Hoxb-1, b-2, b-3, b-4 and b-5 in the E9.5-E14.5 foreguts and lungs. Our results show that in E9.5 branchial arches and foregut, Hoxb genes are expressed in overlapping spatial domains and the anterior boundaries of these domains correspond to the position of a particular gene in the cluster-genes on the 3' end of the cluster are expressed more anteriorly in the branchial arches and foregut and those on the 5' end are expressed more posteriorly. Three of the genes, Hoxb-3, b-4, and b-5, are highly expressed in the foregut where the lung buds form. In contrast, in E10.5-E14.5 lung, there are two patterns of Hoxb gene expression. Hoxb-3 and b-4 are expressed in the mesenchyme of the trachea, mainstem bronchi, and distal lung, whereas Hoxb-2 and b-5 mRNA are present only in the mesenchyme of the distal lung buds. These results suggest that specific combinations of Hoxb gene expression are important in lung development and that Hoxb genes may be involved in specifying the differences between proximal (trachea and main bronchi) and distal (lung bud) mesenchyme.

The proline-rich homeodomain protein, PRH/HEX, participates in the early development of the brain, thyroid, and liver and in the later regenerative processes of damaged liver, vascular endothelial, and hematopoietic cells. A virulent strain of lymphocytic choriomeningitis virus (LCMV-WE) that destroys hematopoietic, vascular, and liver functions also alters the transcription and subcellular localization of PRH. A related virus (LCMV-ARM) that does not cause disease in primates can infect cells without affecting PRH. Biochemical experiments demonstrated the occurrence of binding between the viral RING protein (Z) and PRH, and genetic experiments mapped the PRH-suppressing phenotype to the large (L) segment of the viral genome, which encodes the Z and polymerase genes. The Z protein is clearly involved with PRH, but other viral determinants are needed to relocate PRH and to promote disease. By down-regulating PRH, the arenavirus is able to eliminate the antiproliferative effects of PRH and to promote liver cell division. The interaction of an arenavirus with a homeodomain protein suggests a mechanism for viral teratogenic effects and for the tissue-specific manifestations of arenavirus disease.

Multiple Factors Regulate the Promoter Region of the Liver-Enriched Orphan Homeobox Protein, Hex

Hex Expression During Development Suggests an Important Role in Both Gastrulation and Organogenesis • 251

Homeobox transcription factors influence the expression of multiple genes, thereby regulating patterns of differentiation and cell fate. The Hoxa5, b5 and Hex homeobox genes are actively expressed in fetal rat lung, but little is known of their regulation.

Cat-scratch disease is usually a benign, self-limited disease that causes regional lymphadenopathy. Occasionally, it may present with systemic symptoms and have a prolonged course. To date, antibiotic therapy has not been proved to be of value. We describe three patients with cat-scratch disease who were treated successfully with gentamicin sulfate. Two patients had extensive hepatic involvement, and one patient had regional lymphadenopathy. All three patients responded within 48 hours to intravenous gentamicin. Extensive follow-up has shown no recurrence of symptoms. These cases suggest that gentamicin may be efficacious in shortening the course of cat-scratch disease. Prospective, randomized trials should be performed to confirm these results. (JAMA. 1989;262:813-816)