Linda R. Margraf

Endothelial nitric oxide synthase is expressed in cultured human bronchiolar epithelium

Nitric oxide (NO) is an important mediator of physiologic and inflammatory processes in the lung. To better understand the role of NO in the airway, we examined constitutive NO synthase (NOS) gene expression and function in NCI-H441 human bronchiolar epithelial cells, which are believed to be of Clara cell lineage. NOS activity was detected by [3H]arginine to [3H]citrulline conversion (1,070 +/- 260 fmol/mg protein per minute); enzyme activity was inhibited 91% by EGTA, consistent with the expression of a calcium-dependent NOS isoform. Immunoblot analyses with antisera directed against neuronal, inducible, or endothelial NOS revealed expression solely of endothelial NOS protein. Immunocytochemistry for endothelial NOS revealed staining predominantly in the cell periphery, consistent with the association of this isoform with the cellular membrane. To definitively identify the NOS isoform expressed in H441 cells, NOS cDNA was obtained by degenerate PCR. Sequencing of the H441 NOS cDNA revealed 100% identity with human endothelial NOS at the amino acid level. Furthermore, the H441 NOS cDNA hybridized to a single 4.7-kb mRNA species in poly(A)+ RNA isolated from H441 cells, from rat, sheep, and pig lung, and from ovine endothelial cells, coinciding with the predicted size of 4.7 kb for endothelial NOS mRNA. Guanylyl cyclase activity in H441 cells, assessed by measuring cGMP accumulation, rose 6.6- and 5.4-fold with calcium-mediated activation of NOS by thapsigargin and A23187, respectively. These findings indicate that endothelial NOS is expressed in select bronchiolar epithelial cells, where it may have autocrine effects through activation of guanylyl cyclase. Based on these observations and the previous identification of endothelial NOS in a kidney epithelial cell line, it is postulated that endothelial NOS may be expressed in unique subsets of epithelial cells in a variety of organs, serving to modulate ion flux and/or secretory function.

Nitric oxide synthase isoform expression in the developing lung epithelium

Nitric oxide (NO), generated by NO synthase (NOS), is an important mediator of physiological processes in the airway and lung parenchyma, and there is evidence that the pulmonary expression of the endothelial isoform of NOS (eNOS) is developmentally regulated. The purpose of the present study was to delineate the cellular distribution of expression of eNOS in the developing respiratory epithelium and to compare it with inducible (iNOS) and neuronal (nNOS) NOS. Immunohistochemistry was performed on fetal (125-135 days gestation, term 144 days), newborn (2-4 wk), and maternal sheep lungs. In fetal lung, eNOS expression was evident in bronchial and proximal bronchiolar epithelia but was absent in terminal and respiratory bronchioles and alveolar epithelium. Similar to eNOS, iNOS was detected in bronchial and proximal bronchiolar epithelia but not in alveolar epithelium. However, iNOS was also detected in terminal and respiratory bronchioles. nNOS was found in epithelium at all levels including the alveolar wall. iNOS and nNOS were also detected in airway and vascular smooth muscle. The cellular distribution of all three isoforms was similar in fetal, newborn, and adult lungs. Findings in the epithelium were confirmed by isoform-specific reverse transcription-polymerase chain reaction assays and NADPH diaphorase histochemistry. Thus the three NOS isoforms are commonly expressed in proximal lung epithelium and are differentially expressed in distal lung epithelium. All three isoforms may be important sources of epithelium-derived NO throughout lung development.

Adrenarche results from development of a 3β-hydroxysteroid dehydrogenase-deficient adrenal reticularis

Adrenarche is the increased adrenal production of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) that occurs during the prepubertal period. To date, the exact mechanism initiating adrenarche is unknown, although many factors have been postulated. In the present study, we examined the hypothesis that alterations in intra-adrenal expression of 3beta-hydroxysteroid dehydrogenase (3betaHSD) or 21-hydroxylase (CYP21) within the inner reticularis zone leads to the increased production of 19-carbon (C19) steroids. After conversion of cholesterol to pregnenolone, 17alpha-hydroxylase/17,20-lyase (CYP17) can metabolize pregnenolone through to DHEA. The enzyme 3betaHSD competes for substrate with CYP17 and effectively removes steroid precursor from the pathway leading to DHEA. On the other hand, deficiency in CYP21 expression is known to cause excessive production of adrenal C19 steroids, suggesting that CYP21 could play a role in adrenarche. Thus, a decrease in 3betaHSD or CYP21 expression would allow substrate to flow toward the synthesis of DHEA. To determine whether adrenarche results from a decreased expression of 3betaHSD or CYP21 in the reticularis, immunohistochemical localization of 3betaHSD and CYP21 was performed, and staining intensities compared using adrenal glands from children ages 4 months to 4 yr (n = 12), ages 5-7 yr (n = 9), ages 8-13 yr (n = 9), and adults ages 25-56 yr (n = 8). There were no differences in the zonal expression of CYP21. No difference in 3betaHSD staining was observed between the glomerulosa and fasciculata from any age group. However, children age 8 yr and older show a significant decrease in 3betaHSD expression in reticularis as compared with the fasciculata. No significant difference was noted for 3betaHSD levels between the fasciculata and reticularis for children age 7 yr or younger. The level of 3betaHSD expression in the reticularis continued to decrease in the adult adrenals examined. These findings suggest that as children mature there is a decreased level of 3betaHSD in the adrenal reticularis that may contribute to the increased production of DHEA and DHEAS seen during adrenarche.

Genomic analysis of diffuse pediatric low-grade gliomas identifies recurrent oncogenic truncating rearrangements in the transcription factor MYBL1

Pediatric low-grade gliomas (PLGGs) are among the most common solid tumors in children but, apart from BRAF kinase mutations or duplications in specific subclasses, few genetic driver events are known. Diffuse PLGGs comprise a set of uncommon subtypes that exhibit invasive growth and are therefore especially challenging clinically. We performed high-resolution copy-number analysis on 44 formalin-fixed, paraffin-embedded diffuse PLGGs to identify recurrent alterations. Diffuse PLGGs exhibited fewer such alterations than adult low-grade gliomas, but we identified several significantly recurrent events. The most significant event, 8q13.1 gain, was observed in 28% of diffuse astrocytoma grade IIs and resulted in partial duplication of the transcription factor MYBL1 with truncation of its C-terminal negative-regulatory domain. A similar recurrent deletion-truncation breakpoint was identified in two angiocentric gliomas in the related gene v-myb avian myeloblastosis viral oncogene homolog (MYB) on 6q23.3. Whole-genome sequencing of a MYBL1-rearranged diffuse astrocytoma grade II demonstrated MYBL1 tandem duplication and few other events. Truncated MYBL1 transcripts identified in this tumor induced anchorage-independent growth in 3T3 cells and tumor formation in nude mice. Truncated transcripts were also expressed in two additional tumors with MYBL1 partial duplication. Our results define clinically relevant molecular subclasses of diffuse PLGGs and highlight a potential role for the MYB family in the biology of low-grade gliomas.

Effects of the MYC oncogene antagonist, MAD, on proliferation, cell cycling and the malignant phenotype of human brain tumour cells

To investigate how overexpression of MAD, an antagonist of MYC oncogenes influences the malignant phenotype of human cancer cells, an adenovirus vector system was used to transfer the human MAD gene (AdMAD) into human astrocytoma cells. Decreased growth potential of AdMAD-infected cells was evidenced by a decrease in [3H]thymidine incorporation, an increase in cell doubling time and alteration of cell-cycle distribution. Diminished malignant potential of AdMAD-infected cells was manifested by their loss of anchorage-independent growth in soft agar and by their inability, in general, to induce tumorigenesis in a xenograft animal model. These studies indicate that adenovirus constructs encoding MAD dramatically inhibit the proliferation and tumorigenicity of human astrocytoma cells and support the use of MAD for gene therapy of human tumours.

Study of the MIB-1 Labeling Index as a Predictor of Tumor Progression in Pilocytic Astrocytomas in Children and Adolescents

PURPOSE The pilocytic astrocytoma (PA) is the most common childhood brain tumor. This report examines the MIB-1 labeling index (LI) as a predictor of progression-free survival (PFS) among childhood PAs. PATIENTS AND METHODS Consecutive PAs were examined to determine whether the MIB-1 LI was associated with tumor progression. Other variables evaluated included tumor location, use of adjuvant therapy, extent of resection, and age at diagnosis. RESULTS One hundred forty-one children were identified (mean +/- SD age, 7.6 +/- 4.7 years; range, 0.43 to 18.56 years); 118 children had adequate tissue for MIB-1 immunohistochemistry. The 5-year PFS was 61.25%. By log-rank analysis, an MIB-1 LI of more than 2.0 was associated with shortened PFS (P =.035). Patients with PAs who underwent complete surgical resection, had tumors located in the cerebellum, and were treated with surgery only also had more prolonged PFS (P =.001 for all). Tumors in the optic pathways were associated with a shorter PFS (P =.001). Restricting the evaluation of MIB-1 LI to only incompletely resected tumors revealed an insignificant trend of MIB-1 LI of more than 2.0 having a shortened PFS. Multivariate analysis demonstrated completely resected tumors and tumors located in the cerebellum as less likely to progress (P =.001 and.019, respectively). CONCLUSION Children with PAs with an MIB-1 LI of more than 2.0 have a shortened PFS. PAs that are completely resected and are located in the cerebellum have a prolonged PFS. This initial study suggests that the MIB-1 LI identifies a more aggressive subset of PAs. Further work should focus on elucidating features of pilocytic astocytomas that will identify prospectively children at risk for progression.

Cytomegalovirus and Human Herpesvirus 6, but Not Human Papillomavirus, Are Present in Neonatal Giant Cell Hepatitis and Extrahepatic Biliary Atresia

The purpose of our study was to confirm reports of an association of human papillomavirus (HPV) with neonatal giant cell hepatitis (GCH) and biliary atresia (BA), and to expand these studies to include cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV6), and parvovirus B19 (PVB19). Frozen hepatic tissue was available for polymerase chain reaction (PCR) analysis in 19 cases of GCH or BA and 8 controls. Nested PCR to detect HPV types 6, 16, 18, and 33 was followed by 32P hybridization with generic probes. PCR followed by hybridization with a digoxigenin-labeled probe was used for all other viruses. HPV, EBV, and PVB19 were not detected in cases or controls. Two cases of GCH and 1 case of BA were PCR positive for CMV; controls were negative. HHV6 was detected in 6 cases: 2 GCH, 2 BA, and 2 controls. We conclude that HPV is not associated with GCH or BA. Detection of CMV in BA and GCH confirms other reports of this association. HHV6 requires further study to determine the significance of a positive PCR test in the livers of infants.

Ontogeny of cyclooxygenase-1 and cyclooxygenase-2 gene expression in ovine lung

Prostacyclin is a key mediator of pulmonary vascular and parenchymal function during late fetal and early postnatal life, and its synthesis in whole lung increases during that period. The rate-limiting enzyme in prostacyclin synthesis in the developing lung is cyclooxygenase (COX). We investigated the ontogeny and cellular localization of COX-1 (constitutive) and COX-2 (inducible) gene expression in lungs from late-gestation fetal lambs, 1-wk-old newborn lambs (NB1), and 1- to 4-mo-old newborn lambs (NB2). COX-1 mRNA abundance rose progressively from fetal to NB1 to NB2, increasing 12-fold overall. In parallel, immunoblot analysis revealed a progressive increase in COX-1 protein, rising fourfold from fetal lambs to NB2. COX-2 mRNA levels increased fivefold from fetal to NB1 but were similar in NB1 and NB2. However, COX-2 protein was not detectable by immunoblot analysis in any age group. Immunohistochemistry for COX-1 showed intense immunostaining in endothelial cells at all ages. COX-1 was also expressed in airway epithelium at all ages, with a greater number of epithelial cells staining positively in NB2 compared with fetal and NB1 groups. In addition, COX-1 was expressed in airway smooth muscle from NB1. COX-2 immunostaining was absent in all age groups. These findings indicate that there is differential expression of COX-1 and COX-2 in the developing lung and that the enzymes are expressed in a cell-specific manner. The developmental upregulation in COX-1 may optimize the capacity for prostaglandin-mediated vasodilation, bronchodilation, and surfactant synthesis in the newborn lung.Prostacyclin is a key mediator of pulmonary vascular and parenchymal function during late fetal and early postnatal life, and its synthesis in whole lung increases during that period. The rate-limiting enzyme in prostacyclin synthesis in the developing lung is cyclooxygenase (COX). We investigated the ontogeny and cellular localization of COX-1 (constitutive) and COX-2 (inducible) gene expression in lungs from late-gestation fetal lambs, 1-wk-old newborn lambs (NB1), and 1- to 4-mo-old newborn lambs (NB2). COX-1 mRNA abundance rose progressively from fetal to NB1 to NB2, increasing 12-fold overall. In parallel, immunoblot analysis revealed a progressive increase in COX-1 protein, rising fourfold from fetal lambs to NB2. COX-2 mRNA levels increased fivefold from fetal to NB1 but were similar in NB1 and NB2. However, COX-2 protein was not detectable by immunoblot analysis in any age group. Immunohistochemistry for COX-1 showed intense immunostaining in endothelial cells at all ages. COX-1 was also expressed in airway epithelium at all ages, with a greater number of epithelial cells staining positively in NB2 compared with fetal and NB1 groups. In addition, COX-1 was expressed in airway smooth muscle from NB1. COX-2 immunostaining was absent in all age groups. These findings indicate that there is differential expression of COX-1 and COX-2 in the developing lung and that the enzymes are expressed in a cell-specific manner. The developmental upregulation in COX-1 may optimize the capacity for prostaglandin-mediated vasodilation, bronchodilation, and surfactant synthesis in the newborn lung.

Novel FOXF1 Mutations in Sporadic and Familial Cases of Alveolar Capillary Dysplasia with Misaligned Pulmonary Veins Imply a Role for its DNA Binding Domain

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a rare and lethal developmental disorder of the lung defined by a constellation of characteristic histopathological features. Nonpulmonary anomalies involving organs of gastrointestinal, cardiovascular, and genitourinary systems have been identified in approximately 80% of patients with ACD/MPV. We have collected DNA and pathological samples from more than 90 infants with ACD/MPV and their family members. Since the publication of our initial report of four point mutations and 10 deletions, we have identified an additional 38 novel nonsynonymous mutations of FOXF1 (nine nonsense, seven frameshift, one inframe deletion, 20 missense, and one no stop). This report represents an up to date list of all known FOXF1 mutations to the best of our knowledge. Majority of the cases are sporadic. We report four familial cases of which three show maternal inheritance, consistent with paternal imprinting of the gene. Twenty five mutations (60%) are located within the putative DNA‐binding domain, indicating its plausible role in FOXF1 function. Five mutations map to the second exon. We identified two additional genic and eight genomic deletions upstream to FOXF1. These results corroborate and extend our previous observations and further establish involvement of FOXF1 in ACD/MPV and lung organogenesis.

Placental histopathology of congenital syphilis.

OBJECTIVE To evaluate the contribution of placental histopathology to the diagnosis of congenital syphilis. METHODS From January 1, 1986, through December 31, 1998, all pregnant women presenting to a large, urban Dallas County labor and delivery unit with untreated syphilis at delivery and who had placental evaluation performed were identified. Women were clinically staged, and the infants were evaluated for congenital syphilis using a standard protocol. Each placenta was evaluated by two independent pathologists. Histologic characteristics of the placenta related to congenital syphilis in live‐born and stillborn infants were then analyzed. RESULTS Sixty‐seven women met the study criteria: 33 (49%) stillborn and 18 (27%) live‐born infants with congenital syphilis, 15 (22%) uninfected live‐born infants, and one uninfected stillborn fetus diagnosed by current criteria. There were no differences between the groups with regard to demographic characteristics, prenatal care, or stage of syphilis. Stillborn infants were more likely to deliver pre‐term (P < .001). Controlling for gestational age, histopathology revealed necrotizing funisitis, villous enlargement, and acute villitis associated with congenital syphilis. Erythroblastosis was more common in stillborn infants with congenital syphilis than all live‐born infants (odds ratio 16, 95% confidence interval 1, 370). The addition of histologic evaluation to conventional diagnostic evaluations improved the detection rate for congenital syphilis from 67% to 89% in live‐born infants, and 91% to 97% in stillborn infants. CONCLUSION Our results show that histopathologic examination of the placenta is a valuable adjunct to the contemporary diagnostic criteria used to diagnose congenital syphilis.