Comparison of surfactant protein B polymorphisms of healthy term newborns with preterm newborns having respiratory distress syndrome.

Polymorphisms and mutations in the surfactant protein B (SP-B) gene have been associated with the pathogenesis of respiratory distress syndrome (RDS). The objective of the present study was to compare the frequencies of SP-B gene polymorphisms between preterm babies with RDS and healthy term newborns. We studied 50 preterm babies with RDS (inclusion criteria - newborns with RDS and gestational age between 28 and 33 weeks and 6 days), and 100 healthy term newborns. Four SP-B gene polymorphisms were analyzed: A/C at nucleotide -18, C/T at nucleotide 1580, A/G at nucleotide 9306, and G/C at nucleotide 8714, by PCR amplification of genomic DNA and genotyping by cRFLP. The healthy newborns comprised 42 female and 58 male neonates; 39 were white and 61 non-white. The RDS group comprised 21 female and 29 male preterm neonates; 28 were white and 22 non-white. Weight ranged from 640 to 2080 g (mean: 1273 g); mean gestational age was 31 weeks and 2 days (range: 28-33 weeks and 6 days). When white children were analyzed separately, a statistically significant difference in the G/C polymorphism at 8714 was observed between groups (P = 0.028). All other genotype frequencies were similar for both groups when sex and race were analyzed together. Analysis of the SP-B polymorphism G/C at nucleotide 8714 showed that among white neonates the GG genotype was found only in the RDS group at a frequency of 17% and the GC genotype was more frequently found in healthy term newborns. These data demonstrate an association of GG genotype with RDS.

Unsuspected transection of lung by pigtail catheter in a premature infant.

Thoracostomy tubes are commonly required to treat pnuemothoraces in premature infants. Evidence of impalement of the lungs by tube thoracostomy has been seen in autopsy studies. In neonates, there has been described a surprisingly high incidence of lung perforation. The premature lung is thought to be at greater risk for this complication owing to the pliant, thin chest wall, the proximity of vital tissues and the fragility of the lung tissue itself. The modified Fuhrman catheter, or polyurethane pigtail catheter, has been developed for the drainage of pneumothorax in premature infants. In a study of complications of the placement of pigtail catheters, no instance of penetration of the lungs was reported. We report the case of a premature infant with pigtail catheter placement that, at autopsy, was found to have impaled the lung and discuss the incidence of lung injury associated with invasive management of pnuemothoraces.

Morphologic changes in alveolar macrophages in response to UVEC-activated pulmonary Type II epithelial cells.

We hypothesize that Type II epithelial cells, which line the distal airspaces of the lung, are early responders to invading pathogens and release a signal, which activates and alters the phenotype and phagocytosis properties of alveolar macrophages even at a distance. The T(7) cell line is a conditionally immortalized murine Type II epithelial cell line developed in our laboratory. Using an in vitro transwell model we have previously shown that UV-irradiated Escherichia coli (UVEC)-stimulated T(7) cells cultured in the lower transwell chamber, release a diffusible signal which activates MH-S cells (immortalized murine alveolar macrophages) cultured in the upper transwell chamber, to produce nitric oxide. Using scanning electron microscopy, we show that MH-S cells activated in this manner exhibit increased cell surface ruffling, numerous long filopodia, increased lamellipodia and cell flattening. DynaBead uptake studies show that these morphologic changes are accompanied by increased phagocytosis. These findings indicate that a diffusible signal released at a distance by UVEC-stimulated Type II epithelial cells initiates changes in morphology and phagocytosis reflective of macrophage activation concomitant with the functional activation we previously reported.

Diffusible signal to murine alveolar macrophages from lipopolysaccharide- and Escherichia coli-stimulated lung Type II epithelial cells.

OBJECTIVE AND DESIGN: To demonstrate a diffusible intercellular macrophage activation factor secreted by Type II alveolar epithelial cells (AECs) in transwell co-cultures. MATERIALS: T(7), our Type II conditionally immortalized AEC line; MH-S, an alveolar macrophage cell line; Lipopolysaccharide (LPS) or uv-killed Escherichia coli (UVEC) for antigen presentation. METHODS: LPS or UVEC stimulation of T(7) cells in the lower chamber was investigated for ability to activate MH-S cells in the upper chamber, as assayed by nitric oxide production and western blots for inducible nitric oxide synthase-2. RESULTS: Both transwell and UVEC-conditioned medium experiments showed secretion of an MH-S activation factor by T(7) cells. Many common inflammatory cytokines were ruled out as this immunoactivator. CONCLUSION: Demonstration of a diffusible activation factor produced by Type II AECs supports their potential role as first responders of innate immunity in the lung.

Atelectasis--an unusual and late complication of lung transplant.

We report a previously unrecognized late complication of allograft lung transplantation - persistent recurrent atelectasis of the transplanted lung. The patient developed sudden, severe respiratory distress about 2 yr after a right lung transplant, because of acute atelectasis of her transplanted lung. Multiple transbronchial biopsies at the time revealed minimal inflammation and no evidence of rejection. She was treated with surfactant replacement therapy, and her collapsed lung fully expanded following surfactant installation. To eliminate the possibility of acquired deficiency of surfactant lipids or proteins, ultrastructural examination and immunostains for surfactant proteins were performed in a transbronchial lung biopsy. No deficiency of surfactant lipids or proteins was found. On ultrastructural examination of the lung biopsy, the number of Type II cells per alveolus and the number of lamellar bodies per square micron of Type II cell cross-sectional area was increased compared with an age-matched control. We conclude that synthesis of surfactant lipids and proteins was unimpaired and because of the patient's response to surfactant replacement therapy, that the increase in number of lamellar bodies could reflect a compensatory mechanism for a surfactant functional defect. The patient later developed breast carcinoma to which she succumbed. We raise the possibility that the functional surfactant defect is a hitherto unrecognized non-metastatic manifestation of malignancy.

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 result from mutations in the gene for SP-B. The common mutation, 121ins2, is present in about two-third 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.

Immunolocalization of CC10 in Clara cells in mouse and human lung.

Two antisera, denoted R41 and R42, were raised against a synthetic peptide from the murine Clara cell-specific protein CC10, and one antiserum, denoted R40, was raised against human recombinant uteroglobin, the human homolog of murine CC10. Purified antigen-specific antisera, denoted R40AP, R41AP, and R42AP were prepared using peptide columns. The purified antisera were characterized by dot blots, immunohistochemistry, and immunoblots. Immunohistochemistry of mouse lung showed specific labeling of Clara cells in distal bronchioles by all three antisera. In human lung, the antiuteroglobin antiserum specifically labeled Clara cells, while the anti-mouse peptide antisera had weak crossreactivity and higher background staining. Electron microscopy revealed immunogold labeling of CC10 granules in Clara cells of mouse lung with all antisera. All antisera also labeled a 5-kDa protein on immunoblots of mouse lung homogenates. The surface epithelium of the alveolar air spaces around the distal bronchioles were CC10 positive suggesting a functional activity for CC10 in the lung parenchyma distal to Clara cells. R40AP immunohistochemical staining of sections of normal human lungs and lungs from patients with surfactant protein B deficiency, bronchopneumonia, and idiopathic alveolar proteinosis illustrate the utility of the anti-human CC10 antibody for diagnostic pathology.

Focal congenital alveolar proteinosis associated with abnormal surfactant protein B messenger RNA.

Two siblings presented with typical clinical features of congenital pulmonary alveolar proteinosis (PAP). Necropsy of one sibling revealed scattered foci of the diagnostic histologic changes in the lung tissue. In contrast to infantile and adult PAP, focal distribution is uncommon in congenital PAP. Defective expression of the granulocyte-macrophage colony-stimulating factor receptor was ruled out. The surfactant protein B (SP-B) content in the lung tissue of the autopsied patient was low, and a deletion in the SP-B messenger RNA was detected. We speculate that the PAP in our patients was related to the reduced quantity and/or to the altered quality of SP-B.

Differential expression of VEGF isoforms in mouse during development and in the adult.

Vascular endothelial growth factor (VEGF), a factor that is critical for development of the vascular system in mouse embryos, exists as at least three isoforms, VEGF120, VEGF164, and VEGF188. The isoforms have different affinities for heparan sulfate as well as for the three known VEGF receptors, VEGFR-1 (Flt-1), VEGFR-2 (Flk-1), and neuropilin-1, suggesting that different VEGF isoforms may play distinct roles in vascular development. To determine whether there are differences in the organ-specific expression patterns that would support this concept, we used a quantitative RNase protection assay (RPA) to determine the distribution of different VEGF isoform mRNA in developing and adult mouse organs. Results revealed that the ratios of the three VEGF isoforms changed during organ development and that adult organs expressed different levels of the three VEGF isoforms. Because the lung expressed the highest levels of VEGF188 isoform, we used VEGF isoform-specific in situ hybridization in the developing lung and determined that type II alveolar epithelial cells were expressing high levels of VEGF188 mRNA. Finally, targeted exon deletion of the VEGF gene revealed that mice that developed in the absence of the heparan sulfate binding isoforms VEGF164 and VEGF188, displayed a variety of vascular defects, including abnormal pulmonary vascular development. Our results support the concept that different VEGF isoforms have distinct functions in vascular development.

Generation of an immortal differentiated lung type-II epithelial cell line from the adult H-2K(b)tsA58 transgenic mouse.

This paper describes a new fully differentiated Type-II alveolar epithelial cell line designated T7, derived from transgenic H-2K(b)-tsA58 mice, capable of being passaged as an immortalized cloned cell line in culture. H-2K(b)-tsA58 mice harbor a temperature-sensitive (ts) mutant of the simian virus 40 (SV40) large tumor antigen (T antigen) under the control of the gamma-interferon (INF)-inducible mouse major histocompatibility complex H-2Kb promoter. When cultured under permissive conditions (33 degrees C and in the presence of gamma-INF) cells isolated from H-2Kb-tsA58 mice express the large T antigen, which drives the cells to proliferate. However, upon withdrawal of the gamma-INF and transfer of the cells to a higher temperature (39 degrees 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.

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.

Aberrant SP-B mRNA in lung tissue of patients with congenital alveolar proteinosis (CAP).

Mutations in the surfactant protein (SP)-B gene are responsible for SP-B deficiency in congenital alveolar proteinosis (CAP) (Nogee et al. J Clin Invest 1994: 93: 1860-1883; Lin et al. Mol Genet Metab 1998: 64: 25-35; Klein et al. Pediatrics 1998: 132: 244-248; Ballard et al. Pediatrics 1995: 96: 1046-1052). The multigenerational consanguineous pedigree under study does not carry any of the known mutations, although this pedigree had 14 infant deaths following respiratory distress at birth. Immunostaining of the lungs from three such infants revealed decreased or absent SP-B. By sequencing of SP-B exons, exon-intron junctions, and the 5' and 3' flanking regions, nine polymorphisms were found in this pedigree, but none of them could explain the observed SP-B deficiency. Further analysis of SP-B mRNA by reverse transcription-polymerase chain reaction from paraffin-embedded lung tissue of CAP patients showed that SP-B mRNA is not intact. Although the sequence of mRNA from exon 1-exon 7 and from exon 8-exon 10 could be amplified, the region between exons 7 and 8 could not. From fluorescence in situ hybridization of the short arm of chromosome 2p, only 2 signals were identified, eliminating the possibility of translocation as the cause of the SP-B mRNA aberrance. Although the nature of the genetic basis of SP-B deficiency in this family is currently unknown, the existence of aberrant SP-B mRNA may, at least in part, be responsible for the SP-B deficiency in this pedigree.

Population-based estimates of surfactant protein B deficiency.

OBJECTIVE: Surfactant protein B deficiency is a lethal cause of respiratory distress in infancy that results most commonly from a homozygous frameshift mutation (121ins2). Using independent clinical ascertainment and molecular methods in different populations, we sought to determine allele frequency. STUDY DESIGN: Using clinical characteristics of the phenotype of affected infants, we screened the Missouri linked birth-death database (n = 1 052 544) to ascertain potentially affected infants. We used molecular amplification and restriction enzyme digestion of DNA samples from a metropolitan New York birth cohort (n = 6599) to estimate allele frequency. RESULTS: The point estimate and 95% confidence interval of the 121ins2 allele frequency in the Missouri cohort are 1/1000 individuals (.03-5.6/1000) and in the New York cohort are.15/1000 (. 08-.25/1000). These estimates are not statistically different. CONCLUSIONS: The close approximation of these independent estimates suggests accurate gene frequency (approximately one 121ins2 mutation per 1000-3000 individuals) despite its rare occurrence and that this mutation does not account for the majority of full-term infants with lethal respiratory distress.

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.

Computerized morphometry of the pulmonary vasculature over a range of intravascular pressures.

Recent availability of computerized image analysis has fostered hope that barium injection and landmarking of pulmonary arteries would be unnecessary for morphometric assessment when using this technique. We reasoned that if barium injection altered morphometric variables, it would do so in a linear fashion correlating with incremental increases in injection pressure of the barium. The two goals of the present study were to determine whether barium injection into arteries affected morphometric measurements and to determine whether incremental increases in injection pressure correlated with alterations in morphometric measurements in a linear fashion. Computerized image analysis was used to measure the internal elastic lamina (IEL) and external elastic lamina (EEL). Medial area (MA), luminal area (LA), percentage of medial thickness, IEL square root of MA, and idealized LA were calculated. Barium injection did not alter morphometric variables in a linear fashion correlating with incremental increases in injection pressure of the barium except the percentage of arteries that filled with barium. Maximum recruitment for pre-acinar arteries occurred at 40 mmHg pressure and 60 mmHg distending pressure for intra-acinar arteries. Incremental increases in injection pressure did not affect IEL, EEL, or calculated morphometric variables. However, IEL, medial thickness, and MA were all smaller in injected vessels than in uninjected vessels. IEL square root of MA and the ratio of measured vs. idealized LA were both increased in injected lungs. We suspect that vascular injection selects for evaluation, a population of smaller, thin-walled vessels, which in the uninjected lungs are collapsed and hence excluded from analysis.

An SP-B gene mutation responsible for SP-B deficiency in fatal congenital alveolar proteinosis: evidence for a mutation hotspot in exon 4.

Mutations and polymorphisms within the human SP-B locus have been linked to fatal congenital alveolar proteinosis (CAP) and associated with respiratory distress syndrome (RDS), respectively. In the present study we used PCR and direct sequence analysis of the SP-B gene of three individuals from a family with CAP to search for additional SP-B mutations resulting in CAP and/or polymorphisms that could be used as markers in association studies of RDS and/or CAP. We found three novel mutations/polymorphisms in this family. One is a C/A substitution at nt 1013 at the splice junction of intron 2-exon 3. A second one is a single base T deletion at nt 1553 in exon 4. The single base (T) deletion at nucleotide 1553 (1553delT) shifts the reading frame at amino acid 122(122delT) and creates a premature termination codon at amino acid 214 in exon 6. The mutated gene produces no mature SP-B protein. Genotype analysis from the nuclear family carrying this mutation showed that both parents and three of the four living children are heterozygous for the mutation. One of the four living children is homozygous for the normal allele and a child that died in the perinatal period from CAP is homozygous for the mutation. A third change is a C/T substitution at nt 1580 in exon 4 that changes amino acid 131 from threonine to isoleucine (Thr131Ile). The location of a previously reported mutation, 121ins2 (1), is only 4 nt upstream of 122delT, and the missense mutation Thr131Ile (exon 4) is only 27 nt downstream of 122delT. These changes are within or in close proximity to a CCTG sequence and a poly(C) tract, both of which are shown in other systems to be mutation hotspots. The 122delT occurs within the CCTG and the poly(C) tract sequences, the Thr131Ile occurs 26 nt downstream from the CCTG sequence, and the 121ins2 occurs 2 nt upstream from CCTG sequence and within the poly(C) tract. The present observations suggest that the short SP-B sequence containing the CCTG motif and the poly(C) tract is a mutation hotspot.

Spontaneous resolution of myelodysplastic cytogenetic abnormality developed during the treatment of leukemia.

PURPOSE: We describe the spontaneous resolution of a myelodysplastic cytogenetic abnormality developing during the treatment of acute lymphocytic leukemia. PATIENTS AND METHODS: A 6-year-old girl with acute lymphocytic leukemia had a clinical picture of myelodysplasia 18 months after diagnosis. The clonal cytogenetic abnormality, 46,XX,del(5)(q12q12), resolved spontaneously 4 months after the discontinuation of chemotherapy. Maintenance chemotherapy was resumed 1 month later and continued for an additional 9 months. Currently, she has been off therapy for 10 months. CONCLUSION: A myelodysplastic clonal cytogenetic abnormality developing during treatment may cause some confusion for management. This study demonstrates that spontaneous resolution is possible and that bone marrow transplantation or other intensive treatment may not be necessary.

The effect of intrauterine growth restriction upon fetal and postnatal hepatic glucose transporter and glucokinase proteins.

Employing immunohistochemical and Western blot analyses, we investigated the cellular localization (22-d fetal and 14-d postnatal animals) and concentrations (22-d fetal to 21-d postnatal animals) of rat hepatic glucose transporters (Glut 1 and Glut 2) and glucokinase in response to development and uteroplacental insufficiency with IUGR. Glut 1, the predominant fetal hematopoietic cellular isoform, persisted in postnatal hematopoietic islands and was noted minimally in fetal hepatic cellular membranes. A approximately 40% extrauterine decline in Glut 1 levels paralleled the decline in hematopoietic cells. IUGR increased the fetal hepatic Glut 1 levels in parallel with an expanded hematopoietic cell mass (p < 0.05). In contrast, IUGR failed to alter the 2-fold increase in extrauterine Glut 2 concentrations (1-7-d postnatal animals), the isoform found in fetal and postnatal hepatocytic cell membranes. Glucokinase, the nuclear enzyme, increased 25% postnatally. IUGR caused a 16% increase in fetal glucokinase levels and a approximately 25% decline at postnatal d 1 (p < 0.05) without a comparable change in the hepatocytic cell number (92 +/- 6 versus 86 +/- 4). We conclude that hepatic Glut 1 concentrations reflect the extramedullary hematopoietic cellular mass, whereas extrauterine Glut 2 changes herald the need for enhanced flexibility in hepatocytic glucose transport with the initiation of food ingestion. The age-related alteration along with the IUGR-induced compensatory changes in the nuclear-mitochondrial glucokinase levels attributes a critical role for this enzyme in perinatal hepatocytic glucose homeostasis.