BR22, a 26 kDa thyroid transcription factor-1 associated protein (TAP26), is expressed in human lung cells.

The current authors have previously identified BR22, a thyroid transcription factor (TTF)-1 associated protein 26 (TAP26), which interacts with TTF-1 to enhance human surfactant protein (SP)-B promoter activity in transfected 293 cells. However, the expression of TAP26 in the lung cells and its biological relevance to the SP-B production under physiological conditions were not examined. In this study, endogenous co-immunoprecipitation and in situ immunohistochemical staining techniques were employed to explore the presence of TAP26 and TTF-1 complex in the lung epithelial cells. The correlation of TAP26, TTF-1 and SP-B expression was inspected in H441 cells in the presence of dexamethasone, a known positive effector of the SP-B promoter. Monoclonal antibody (mAb) against TAP26 can co-immunoprecipitate both TAP26 and TTF-1 from H441 cells. Using this antibody in in situ staining of human lung sections, the data show that TAP26 is present in the lung alveolar epithelial cells. Reverse transcriptase-polymerase chain reaction and Western blot analyses of type-II cells as well as dexamethasone-treated H441 cells suggest that TAP26 expression is modulated coordinately with SP-B and TTF-1 in these cells. In summary, the current study demonstrates that thyroid transcription factor-1 associated protein 26 is an associated protein of thyroid transcription factor-1 in the lung alveolar epithelial cells where surfactant protein gene expressions take place in vivo.

Prevalence and cellular reservoir of latent human herpesvirus 6 in tonsillar lymphoid tissue.

There are few studies that examine prevalence, quantity, and cellular proclivity of latent human herpesvirus 6 (HHV-6) in healthy populations. We examined 69 tonsils with paired blood specimens from children without evidence of acute infection. By polymerase chain reaction (PCR), HHV-6 was detected at low levels in 100% of tonsils and 39% of blood samples (n = 27), suggesting that prevalence of latent HHV-6 infection is high in children and may be underestimated by PCR analysis of blood. Although HHV-6A and HHV-6B were detected, HHV-6B predominated, being found in 97% of samples (n = 67). Tonsil sections from 7 cases were examined by in situ hybridization using 2 HHV-6 probes and immunohistochemical analysis. Using both in situ hybridization and immunohistochemical analysis, all tissues revealed marked HHV-6-specific staining in the squamous epithelium of the tonsillar crypts and rare positive lymphocytes. We conclude that HHV-6 is present universally in tonsils of children, and tonsillar epithelium may be an important viral reservoir in latent infection.

Proliferative activity in craniopharyngiomas: clinicopathological correlations in adults and children.

BACKGROUND: Craniopharyngiomas are slow-growing, locally invasive intracranial tumors that can generate considerable morbidity, and recurrences are often difficult to manage. Because reliable morphologic criteria for accurately predicting the clinical outcome of these tumors are lacking, we evaluated the growth potential of craniopharyngiomas by measuring their proliferative activity based on MIB-1 immunostaining for the Ki-67 antigen, which is expressed during all phases of the cell cycle except G(0.) METHODS: Paraffin sections from 37 cases of craniopharyngiomas were immunostained with the monoclonal antibody MIB-1, and a labeling index was derived in each case from an the with the highest labeling. RESULTS: MIB-1 immunoreactivity was mainly confined to the peripheral palisaded epithelium of craniopharyngiomas. In adult craniopharyngiomas, MIB-1 labeling indices (MIB-LI) varied from 0.1% to 34.6% (mean 8.9%; SD 9. 8), and in pediatric tumors the indices ranged from 1.8% to 15.0% (mean 6.3%; SD 3.7). MIB-LI was not found to be an independent predictor of recurrence, although in all the pediatric cases that recurred, MIB-LI in the second specimen was greater. CONCLUSIONS: The actively proliferating compartment in craniopharyngiomas seems to be the peripheral palisaded epithelium. Low MIB-LI observed in the majority of tumors is in concordance with the slow growth and low-grade invasiveness associated with craniopharyngiomas. However, unlike other intracranial neoplasms, where Ki-67 labeling indices have been useful in predicting tumor behavior, a clear relationship could not be demonstrated between MIB-1 immunoreactivity, morphological features and clinical outcomes in adults or children with craniopharyngiomas.

Synchronous wilms tumor and fibrolamellar hepatocellular carcinoma: report of a case.

Fibrolamellar hepatocellular carcinoma (FHCC) is a unique histologic variant of HCC that occurs in a younger subset of patients than classical HCC, and is associated with a better prognosis. Wilms tumor (WT) is a malignant embryonal neoplasm of the kidney and is one of the most common solid tumors of childhood, occurring at an estimated frequency of 1 in 8000 to 10,000 births. Although second malignant neoplasms (SMNs) following therapy for WTs have been reported in the liver, the coexistence of HCC and WT is extremely rare. We present the first report of a synchronous anaplastic WT and FHCC in a previously healthy 4-year-old girl. Despite the presence of focal immunohistochemical positivity for p53 in the WT, molecular analysis failed to reveal a germline or somatic p53 mutation, and was inconclusive in establishing a clonal relation between the two tumors.

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.

Parathyroid carcinoma in a child.

Parathyroid carcinoma is a rare cause of hypercalcemia in children but should be considered in a child presenting with an extremely elevated serum calcium level. The authors report the fifth case of parathyroid carcinoma in a child less than 16 years of age.

Tissue expression and subcellular localization of CLN3, the Batten disease protein.

Juvenile neuronal ceroid lipofuscinosis (Batten disease) is a progressive neurologic disorder which results from mutations in the CLN3 gene, which normally produces a 48-kDa polypeptide of unknown function. To help characterize the CLN3 protein, we have studied its tissue distribution and subcellular localization in human tissues using three epitope-specific polyclonal antibodies to human CLN3 by immunoblot, immunocytochemical, and immunoelectron microscopic analysis. The most abundant CLN3 protein expression was in the gray matter of the brain, where it was localized to astrocytes, capillary endothelium, and neurons. CLN3 was also evident in peripheral nerve, in pancreatic islet cells, and within the seminiferous tubules in the testis. Staining was generally diffuse within the cytoplasm with some nuclear reactivity. Subcellular localization identified the CLN3 protein within the nucleus and along cell membranes. These results were contrasted with the cellular distribution of palmitoyl-protein thioesterase (PPT), the enzyme whose deficiency is responsible for infantile neuronal ceroid lipofuscinosis (CLN1). PPT was most abundant in brain and visceral macrophages where it displayed a coarse granular staining pattern typical of lysosomal distribution. Immunoelectron microscopy confirmed that PPT immunoreactivity was limited to lysosomes.

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.

Surfactant protein A is decreased in a rat model of congenital diaphragmatic hernia.

We hypothesized that the expression of surfactant protein A (SP-A) would be altered in developing lungs from rat fetuses with congenital diaphragmatic hernia (CDH) induced by maternal ingestion of 2,4-dichlorophenyl-p-nitrophenyl ether (Nitrofen) on Day 9 of gestation. We compared our findings in fetuses exposed to Nitrofen with a CDH with those in Nitrofen-exposed fetuses without a CDH, and control fetuses whose mothers received olive oil only, the vehicle for Nitrofen. In late gestation, immunocytochemistry using a polyclonal rabbit antihuman SP-A antibody revealed decreased amounts of this protein in lungs from fetuses with CDH. Using immunoblotting, the relative amount of SP-A on Day 21 of gestation was also decreased in lung tissue from fetuses with CDH compared with the other groups. Abnormalities of mRNA for SP-A were observed in both groups of Nitrofen-exposed fetuses compared with control rats. These findings suggest that there is decreased expression of SP-A in rat fetuses with CDH secondary to Nitrofen exposure.

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.

Bcl-2 expression in Langerhans' cell histiocytosis.

Langerhans' cell histiocytosis (LCH) is an abnormal accumulation of dendritic histiocytes of unknown pathogenesis. It has recently been shown to be a clonal process. Bcl-2 is a proto-oncogene whose protein product is known to inhibit apoptosis. The overexpression of bcl-2 has been demonstrated in a number of neoplasms, presumably prolonging the survival of the neoplastic cells. We examined the expression of bcl-2 in normal Langerhans' cells in the skin and in LCH by immunohistochemistry for protein and in situ hybridization for mRNA to see if it could be implicated in the pathogenesis of this disorder. Additionally, we performed Southern analysis to determine if genomic rearrangement of the bcl-2 gene occurs in cases of LCH. Bcl-2 was not detected in normal skin Langerhans' cells. Eleven of thirteen cases of LCH demonstrated bcl-2 protein expression in the cytoplasm of the Langerhans' cells by immunohistochemistry, while 12 of 13 cases had evidence of bcl-2 mRNA by in situ hybridization. Southern analysis revealed a germline configuration of the bcl-2 gene in the five cases studied. These findings suggest that bcl-2 expression is present and up-regulated in pathologic Langerhans' cells, however, this overexpression does not appear to be due to genomic rearrangement.

Primary cell culture of human type II pneumonocytes: maintenance of a differentiated phenotype and transfection with recombinant adenoviruses.

Studies of the regulation of surfactant lipoprotein metabolism and secretion and surfactant protein gene expression have been hampered by the lack of a cell culture system in which the phenotypic properties of type II cells are maintained. We have developed a primary culture system that facilitates the maintenance of a number of morphologic and biochemical properties of type II pneumonocytes for up to 2 wk. Cells were isolated by collagenase digestion of midgestation human fetal lung tissue that had been maintained in organ culture in the presence of dibutyryl cyclic AMP (Bt2cAMP) for 5 days. The isolated cells were enriched for epithelial components by treatment with DEAE-dextran, plated on an extracellular matrix (ECM) derived from Madin-Darby canine kidney (MDCK) cells, and incubated at an air/liquid interface in a minimal amount of culture medium containing Bt2cAMP. The cell cultures were comprised of islands of round epithelial-like cells containing numerous dense osmiophilic granules, surrounded by sparse spindle-shaped cells with the appearance of fibroblasts. Ultrastructural examination revealed that the osmiophilic granules had the appearance of lamellar bodies, the distinguishing feature of type II pneumonocytes. Additionally, the cultures maintained elevated levels of SP-A gene expression for up to 2 wk. The expression of mRNAs encoding SP-A, SP-B, and SP-C were regulated in the cultured cells by glucocorticoids and cyclic AMP in a manner similar to that observed in fetal lung tissue in organ culture. The differentiated phenotype was most apparent when the cells were cultured at an air/liquid interface. In order to utilize the cultured type II cells for study of the effects of overexpression of various proteins and for promoter analysis, it is of essence to transfect DNA constructs into these cells with high efficiency. Unfortunately, we found the cells to be refractory to efficient transfer of DNA using conventional methods (i.e., lipofection, electroporation, or calcium phosphate-mediated transfection). However, replication-defective recombinant human adenoviruses were found to provide a highly efficient means of introducing DNA into the type II pneumonocytes. Furthermore, we observed in type II cell-enriched cultures infected with recombinant adenoviruses containing the lacZ gene under control of a cytomegalovirus promoter, that beta-galactosidase was expressed uniformly in the islands of type II cells and surrounding fibroblasts. By contrast, in cultures infected with recombinant adenoviruses containing the human growth hormone (hGH) gene under control of the SP-A gene promoter and 5'-flanking region, hGH was expressed only in the type II cells. Thus, this culture system provides an excellent means for identifying genomic elements that mediate type II cell-specific gene expression.

Transcription factor C/EBPdelta in fetal lung: developmental regulation and effects of cyclic adenosine 3',5'-monophosphate and glucocorticoids.

Pulmonary surfactant is a developmentally and hormonally regulated lipoprotein synthesized exclusively in alveolar type II cells. Surfactant protein-A (SP-A) gene transcription in human fetal lung in culture is stimulated by glucocorticoids and cAMP; cAMP also enhances the rate of type II cell differentiation. The CCAAT/enhancer-binding protein (C/EBP) family of transcription factors serves an important role in the regulation of genes involved in energy metabolism, lipid biosynthesis, and cellular differentiation. The gene encoding C/EBPdelta, which is induced by glucocorticoids during the early phases of adipocyte differentiation, is expressed at relatively high levels in lung compared with other tissues. In the present study we have analyzed developmental changes in C/EBPdelta messenger RNA levels in fetal rabbit lung as well as changes in the levels of immunoreactive C/EBPdelta in human fetal lung during differentiation in organ culture and after treatment with cAMP and glucocorticoids. We observed that C/EBPdelta messenger RNA is detectable in fetal rabbit lung on day 19 of gestation and is increased approximately 3.7-fold to maximum levels on day 28 of gestation, the time when SP-A gene transcription increases to maximum levels. Immunohistochemical analysis of C/EBPdelta in midgestation human fetal lung before culture revealed trace nuclear staining in epithelial and occasional stromal cells. After 12 h of organ culture in serum-free medium, nuclear staining of C/EBPdelta was markedly increased in epithelial cells lining the prealveolar ducts of the human fetal lung tissue. By immunoblot analysis, it was found that C/EBPdelta levels were induced rapidly during organ culture in control medium and were increased further by treatment with dexamethasone and (Bt)2cAMP. C/EBPdelta levels were maximally induced during the first 24 h of culture and declined thereafter; after 72 h of incubation in control or cAMP-containing medium, C/EBPdelta was reduced markedly. By contrast, in fetal lung tissues incubated in medium containing dexamethasone or dexamethasone plus (Bt)2cAMP, the decline in C/EBPdelta was more modest, so that levels remained elevated throughout the 96-h culture period. Our findings that C/EBPdelta is localized primarily to alveolar epithelial cells, rapidly induced during differentiation of human fetal lung in culture, and increased by cAMP and glucocorticoids suggest a possible role in the regulation of type II cell differentiation and in the synthesis of surfactant phospholipids and proteins.

Epstein-Barr virus lymphoproliferative disorder in children with leukemia: case report and review of the literature.

PURPOSE: The purpose of this study is to report a case of Epstein-Barr virus-related lymphoproliferative disorder (EBV-related LPD) in a child with leukemia and present a review of literature on the subject. PATIENTS AND METHODS: A 6-year-old boy with acute lymphoblastic leukemia (ALL) undergoing maintenance chemotherapy presented with fever, abdominal pain, and vomiting. Imaging studies revealed multiple mass lesions in the liver, kidneys, and lungs. Liver biopsy was obtained. Immunocytochemistry for T and B lymphocytes and in situ hybridization for evidence of latent EBV infection was performed. RESULTS: Hepatic portal tracts were infiltrated with lymphocytes, monocytes, eosinophils, and large atypical mononuclear cells. Both T and B lymphocytes were present, but the large atypical cells were of B origin and were positive for latent EBV infection. Chemotherapy was discontinued, and the patient was treated with intravenous gammaglobulin, interferon, and acyclovir. He had an excellent response and has been free of disease for 19 months. CONCLUSION: EBV-related LPD can complicate the course of patients with ALL in remission. The clinical findings and diagnosis in patients with ALL are similar to other immunocompromised patients. Withdrawal of chemotherapy and treatment with immune modulators should be strongly considered.

Calcifying fibrous pseudotumor of the chest wall.

This report concerns the finding of a clinically benign 4-cm mass in the anterior thorax of a 12-year-old girl. The tumor was inseparable from the chest wall. A wide excision was performed. Microscopic examination proved it to be a rare but distinctive lesion recently entitled "calcifying fibrous pseudotumor" primarily involving the chest wall. Wide excision is thought necessary to preclude local recurrence.

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.

Pulmonary gastrin-releasing peptide expression in anencephaly.

Gastrin-releasing peptide (GRP) is a developmentally regulated bioactive peptide believed to function as a pulmonary growth factor. It is produced by pulmonary neuroendocrine cells, found within the conducting and respiratory epithelium, as isolated cells and in clusters known as neuroepithelial bodies (NEBs). Deficient GRP expression has been reported in pulmonary hypoplasia (PH) associated with oligohydramnios and diaphragmatic hernia. To assess further the role of GRP in maldeveloped lung we reviewed the postmortem records and histologic lung sections, stained with H&E and anti-GRP antiserum, from 11 infants with anencephaly and 11 age-matched controls. Cells immunoreactive for GRP were quantified (isolated versus NEBs) in airways and airspaces per mm2 for a standard area. PH was present in five anencephalic infants. There was no difference in the total number of GRP-positive cells, number of NEBs, size of NEBs, or number of GRP-positive cells in airways or alveoli in either group regardless of lung development. A greater proportion of the GRP-positive cells was present in the airways in anencephalic infants with PH (58%) compared with anencephalic infants without PH (40%) (P = .018). There were no differences when comparing these groups with control infants and no differences in the density of airways in each of these groups. We conclude that deficient GRP expression is not a feature of lung hypoplasia in anencephalic infants. The altered distribution of GRP-positive cells in anencephalic infants with PH may be a reflection of the structural abnormalities or accompanying altered cellular maturity.

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.