Immunocytochemical characterization of murine Hex, a homeobox-containing protein.

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 expression suggests a role in the development and function of organs derived from foregut endoderm.

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.

Divergent homeobox gene hex regulates promoter of the Na(+)-dependent bile acid cotransporter.

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 ntcp promoter (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.

HNF3beta and GATA-4 transactivate the liver-enriched homeobox gene, Hex.

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.

Pain management and weaning from narcotics and sedatives.

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.

Use of high-frequency and nonconventional ventilation for respiratory failure.

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.

Fetal lung mRNA levels of Hox genes are differentially altered by maternal diabetes and butyrate in rats.

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.

Retinoic acid increases surfactant protein mRNA in fetal rat lung in culture.

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.

Expression of Hoxb genes in the developing mouse foregut and lung.

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.

Identification of Hox genes in newborn lung and effects of gestational age and retinoic acid on their expression.

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.

Antibiotic therapy for cat-scratch disease?

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.