Daphne E. deMello

Embryonic and early fetal development of human lung vasculature and its functional implications.

Recently, we have identified in the mouse three processes involved in the early development of pulmonary vasculature: angiogenesis for branching of central vessels, vasculogenesis (lakes in the mesenchyme) for peripheral vessels, and a lytic process to establish luminal connection between the two. We have established that these three processes also operate in the human by studying serial sections of human embryos and early fetuses. Vascular lakes of hematopoietic cells appear at stage 13, i.e., 4+ weeks gestational age (GA), the first intrapulmonary vascular structure to appear. At stage 20 (50.5 days GA), a venous network with luminal connections to central pulmonary veins (PV) is present. Airways have not yet reached these regions of lung.At its first intrapulmonary appearance, the pulmonary artery (PA) is small and thick walled: it runs with the airway but its branching is slower, so many peripheral airways are not accompanied by a PA branch. By contrast, the PV has a peripheral patent network well before the PA.In the pseudoglandular phase, airway branching continues, and the PA catches up so that small PA branches are found with all airways. Later in this phase small nonmuscular vessels lie in the mesenchyme close to airway epithelium.By the early canalicular phase and the age of viability, continuity between pulmonary artery and the peripheral capillary network must be established. In a 10-week fetus several structures suggesting a breakthrough site were seen. Air-blood barrier structure is first seen at 19 weeks. Thus in the lung, the PA and PV are dissociated in their timing and pattern of branching. Early veins are present diffusely through the mesenchyme and establish central luminal connection to the main pulmonary vein before airway or artery are present at this level.

Pulmonary alveolar proteinosis: a review.

Pulmonary alveolar proteinosis (PAP) is a disorder that rapidly leads to respiratory failure, because the alveolar spaces fill with a lipid-rich, proteinaceous material that impedes gas exchange. The pathogenesis of this life-threatening process remained an enigma for decades. Recent analysis of the lung pathology and molecular genetics of affected families has provided a molecular basis for some cases of PAP—deficiency of surfactant protein SP-B. This lack results from mutations in the gene for SP-B. The common mutation, 121ins2, is present in about two-thirds of the patients with SP-B deficiency. Additional insights into the mechanism for this lipoproteinaceous accumulation within alveoli were contributed by serendipity in a granulocyte-macrophage – colony stimulating factor (GM–CSF) knock-out mouse model developed to study basal hematopoiesis. In this model, hematopoiesis was unaffected, but the animals developed pulmonary alveolar proteinosis. Subsequently, mutations in the genes for GM–CSF or its receptor were identified as the cause for pulmonary alveolar proteinosis in some patients. In our review, we discuss the known clinical, pathologic, and molecular genetic aspects of pediatric PAP and consider avenues for future research.

Molecular Mechanisms of Pulmonary Vascular Development

In this era of rapidly advancing vascular biology research, a vast array of growth factors and signaling molecules have been recognized as key players in the mechanisms that control lung vascular development. In the lung, vascular development is a complex, multistep process that includes specialization of primitive cells to vascular progenitors; formation of primitive vascular networks; remodeling with local regression and branching; specialization toward arteries, veins, and lymphatics; stabilization of vessels by matrix production and recruitment of supporting cells; and maintenance of the vascular structure. This complex, highly organized process requires exquisite orchestration of the regulatory activity of multiple molecules in a specific temporospatial order. Most of these molecules are members of 3 major growth factor families that have been recently identified. They are the vascular endothelial growth factor, angiopoietin, and ephrin families. Understanding the functional reach of several members of these growth factor families is integral to an appreciation of the etiology and pathogenesis of developmental lung vascular disorders affecting newborns. This review summarizes recent advances in the molecular bases of lung vascular development and some of the pulmonary diseases resulting from aberrant vascular growth, including bronchopulmonary dysplasia, alveolar capillary dysplasia, congenital cystic pulmonary disorders, congenital pulmonary hemangiomatosis, and lung hypoplasia.

Propylthiouracil-induced fulminant hepatitis: case report and review of the literature.

Propylthiouracil (PTU), a thyroid hormone inhibitor, is widely used for the treatment of hyperthyroidism. Rarely, the drug has been associated with severe hepatotoxicity. We present the case of a 13-year-old girl who developed jaundice and profound liver dysfunction with rapid progression to metabolic encephalopathy while receiving PTU therapy. She died despite extensive therapeutic measures including orthotopic liver transplantation. Her rapid clinical course and fatal outcome show that in spite of regular monitoring, severe, rare, rapidly occurring complications of PTU therapy may still occur.

Generation of an immortal differentiated lung type-II epithelial cell line from the adult H-2KbtsA58 transgenic mouse

SummaryThis paper describes a new fully differentiated Type-II alveolar epithelial cell line designated T7, derived from transgenic H-2Kb-tsA58 mice, capable of being passaged as an immortalized cloned cell line in culture. H-2Kb-tsA58 mice harbor a temperature-sensitive (ts) mutant of the simian virus 40 (SV40) large tumor antigen (T antigen) under the control of the γ-interferon (INF)-inducible mouse major histocompatibility complex H-2Kb promoter. When cultured under permissive conditions (33°C and in the presence of γ-INF) cells isolated from H-2Kb-tsA58 mice express the large T antigen, which drives the cells to proliferate. However, upon withdrawal of the γ-INF and transfer of the cells to a higher temperature (39°C), T antigen expression is turned off, the cells stop proliferating and differentiate. The T7 cell line is a clonal cell line originally derived from a Type-II cell-rich fraction isolated from lungs of H-2Kb-tsA58 mice. The T7 cells form confluent monolayers, and have a polarized epithelial cell morphology with tight junctions and apical microvilli. In addition, the T7 cells have distinct cytoplasmic lamellar bodies, which become more numerous and pronounced when the cells are grown under nonpermissive conditions. The T7 cells synthesize and secrete phosphatidylcholine and the three surfactant proteins, SP-A, SP-B, and SP-C. The T7 cell line is unique in that it is the first non-tumor-derived Type-II cell line capable of synthesizing and secreting the major components of surfactant. Based on the criteria studied, the T7 cell line is phenotypically very similar to normal Type-II cells. The T7 cell line, therefore, should prove a valuable experimental system to advance the study of the cell biology/physiology of surfactant metabolism and secretion as well as serve as a model for other studies of Type-II cell physiology.

An alternatively spliced surfactant protein B mRNA in normal human lung: disease implication

We identified an alternatively-spliced surfactant protein B (SP-B) mRNA from normal human lung with a 12 nt deletion at the beginning of exon 8. This deletion causes a loss of four amino acids in the SP-B precursor protein. Sequence comparison of the 3 splice sites reveals only one difference in the frequency of U/C in the 11 predominantly-pyrimidine nucleotide tract, 73% for the normal and 45% for the alternatively-spliced SP-B mRNA (77-99% for the consensus sequence). Analysis of SP-B mRNA in lung indicates that the abundance of the alternatively-spliced form is very low and varies among individuals. Although the relative abundance of the deletion form of SP-B mRNA remains constant among normal lungs, it is found with relatively higher abundance in the lungs of some individuals with diseases such as congenital alveolar proteinosis, respiratory distress syndrome, bronchopulmonary dysplasia, alveolar capillary dysplasia and hypophosphatasia. This observation points to the possibility that the alternative splicing is a potential regulatory mechanism of SP-B and may play a role in the pathogenesis of disease under certain circumstances.

The development of vaccines against SARS corona virus in mice and SCID-PBL/hu mice

n Abstractn n We have investigated to develop novel vaccines against SARS CoV using cDNA constructs encoding the structural antigen; spike protein (S), membrane protein (M), envelope protein (E), or nucleocapsid (N) protein, derived from SARS CoV. Mice vaccinated with SARS-N or -M DNA using pcDNA 3.1(+) plasmid vector showed T cell immune responses (CTL induction and proliferation) against N or M protein, respectively. CTL responses were also detected to SARS DNA-transfected type II alveolar epithelial cells (T7 cell clone), which are thought to be initial target cells for SARS virus infection in human. To determine whether these DNA vaccines could induce T cell immune responses in humans as well as in mice, SCID-PBL/hu mice was immunized with these DNA vaccines. As expected, virus-specific CTL responses and T cell proliferation were induced from human T cells. SARS-N and SARS-M DNA vaccines and SCID-PBL/hu mouse model will be important in the development of protective vaccines.n n

Recurring digital fibroma of childhood

A recurrent digital fibroma of childhood is reported. This case illustrates difficulties in the management of these recurrent tumors. Despite tumor-free margins on the excised tumors, recurrence occurred at other sites. Recurrences or new primary lesions are reported in 75% of the cases. Because of the rare tendency of these lesions to regress and the high recurrence rate, an individualized approach based on lesion location and behavior is recommended.

Generation and characterization of a conditionally immortalized lung clara cell line from the H-2Kb-tsA58 transgenic mouse.

SummaryThe Clara cell is believed to be the progenitor of the peripheral airway epithelium, and it produces the surfactant proteins SP-A and SP-B, in addition to the 10-kDa Clara cell secretory protein (CCSP or CC10). To date, attempts to develop Clara cell lines have been unsuccessful. Most such attempts have involved the in vitro insertion of a transforming viral oncogene. We have reported previously the characterization of a differentiated conditionally immortalized murine lung Type II epithelial cell line, T7, from the H-2Kb-tsA58 transgenic mouse. We have also used this mouse model to derive Clara cell lines. In this model, the need for in vitro gene insertion is circumvented by the creation of a transgene, in which the large tumor antigen of a temperature-sensitive strain (tsA58) of the simian virus 40 (SV40) is fused with the major histocompatibility complex promoter H-2Kb. The promoter is active in a wide range of tissues and is induced by interferons (IFN). From the lungs of animals harboring the hybrid construct, we isolated and characterized Clara cells. The cells contain dense secretory granules and mitochondria typical of Clara cells, and express SP-A, SP-B, SP-D, and the Clara cell secretory protein, CC10. Withdrawal of the IFN and elevation of the incubation temperature permit normal cell differentiation similar to that of Clara cells in vivo. This cell line should be very useful for the investigation of normal Clara cell function and gene expression.

A matter of life and breath.

Analysis of the hypoplastic lung poses a challenge to the pediatric pathologist. Growth of the major components of the lung—airways, alveoli, and vessels—needs to be carefully studied because development of any or all of these structures could be impaired. In 1 example of lung hypoplasia, congenital diaphragmatic hernia (CDH), abnormal development of the pulmonary vasculature causes the serious clinical condition known as persistent pulmonary hypertension of the newborn. In persistent pulmonary hypertension of the newborn, whether idiopathic or secondary (as in CDH), the lung vascular abnormalities range from decreased preacinar branching and sparse intraacinar arteries to structural alterations such as decreased luminal diameter, medial hypertrophy, and thickened adventitia. These growth aberrations raise questions about the mechanisms that control lung vascular development. Two processes, angiogenesis, i.e., the sprouting of blood vessels from pre-existing ones, and vasculogenesis, i.e., the formation of blood vessels from blood islands within mesenchyme, contribute to the vasculature of the lung. These events involve specification and commitment of primitive mesenchymal cells to form endothelial cells, with subsequent migration, sprouting, proliferation, alignment, tube formation, cell differentiation, and anastomosis. Understandably, angiogenesis and vasculogenesis require the coordinated action of a plethora of genes, growth factors, and transcription factors (Fig. 1). Deciphering the functions and interactions between these genes and their products is the aim of vascular biologists. However, relating this vast sea of information to impaired lung vascular growth is the task of the pediatric pathologist. In this issue of Pediatric and Developmental Pathology, de Rooij et al. [1] examine angiogenesis factors in the lungs of patients with CDH and report that expression of the von Hippel-Lindau (VHL) gene product (pVHL) is increased, whereas hypoxia inducible factor-a (HIF-1a) is decreased. The VHL gene is so named because a germline mutation in this tumor suppressor gene results in vascular (and other) tumors. HIF is a transcription factor, which is activated in response to hypoxia to turn on the transcription of ‘‘angiogenic’’ genes such as vascular endothelial growth factor (VEGF). In normoxia, pVHL binds to the asubunits of HIF-1 and promotes HIF degradation by ubiquitination, which in turn suppresses angiogenesis. In short, ‘‘VHL takes HIF’s breath away’’ [2]. In this context, the findings of de Rooij et al. suggest a molecular basis for the ‘‘pruned’’ vascular tree in CDH. For processes as complex as angiogenesis and vasculogenesis, additional mechanisms are likely to be involved. Nakatsu et al. [3], in an in vitro angiogenesis model, found that low concen*Corresponding author, e-mail: demellde@slu.edu Pediatric and Developmental Pathology 7, 422–424, 2004 DOI: 10.1007/s10024-004-6063-9 a 2004 Society for Pediatric Pathology