Familial neuroendocrine cell hyperplasia of infancy.

BACKGROUND: Neuroendocrine cell hyperplasia of infancy (NEHI) is a recently described children's interstitial lung disease (chILD) disorder of unknown etiology. It manifests clinically with tachypnea, retractions, hypoxemia, and crackles. The characteristic radiographic appearance consists of pulmonary hyperexpansion and ground-glass densities on high-resolution computed tomography (HRCT). Lung histology shows hyperplasia of bombesin-immunopositive neuroendocrine cells within distal bronchioles and alveolar ducts without other identifiable lung pathology or developmental anomaly. METHODS: We describe four families with multiple siblings diagnosed with NEHI. Cases were identified at three pediatric centers. Inclusion criteria included clinical findings consistent with NEHI, lung biopsy confirmation in the index case, and a diagnostic HRCT or biopsy in other siblings. RESULTS: Each family had a proband diagnosed with NEHI based upon pathologic review, and at least one additional sibling diagnosed either by pathologic review or HRCT. All patients presented between 2 and 15 months of age. Both male and female children were affected. The majority of the patients underwent both HRCT and lung biopsy. There were no deaths among affected children. No environmental exposures or other potential etiologies were identified as a cause of presenting symptoms. CONCLUSIONS: The familial occurrence of NEHI suggests the possibility of a genetic etiology for this disorder and highlights the importance of taking a complete family medical history for infants presenting with a suggestive clinical picture. Identification of familial NEHI patients allows for the opportunity to further our understanding of this disorder, its natural history, the phenotypic spectrum, and potential genetic causes.

Serum KL-6 differentiates neuroendocrine cell hyperplasia of infancy from the inborn errors of surfactant metabolism.

BACKGROUND: The study was conducted in order to determine if the glycoprotein KL-6 is a useful biomarker in differentiating neuroendocrine cell hyperplasia of infancy (NEHI), a benign form of children's interstitial lung disease, from the more severe inborn errors of surfactant metabolism (IESM), since their clinical presentation can be similar. METHODS: Serum KL-6 levels were measured in 10 healthy control children, 6 with NEHI and 13 with IESM (4 with surfactant protein C (SP-C) and 9 with ABCA3 mutations). The initial clinical presentation, findings on previous CT scans and interstitial lung disease (ILD) scores at the time of KL-6 testing were compared. Correlations of KL-6 levels with age and with interval from lung biopsy were evaluated. RESULTS: The median (range) KL-6 levels were 265 (1-409), 194 (47-352), 1149 (593-4407) and 3068 (726-9912) U/ml for the control, NEHI, SP-C and ABCA3 groups, respectively. When compared with the control and NEHI groups, median KL-6 levels were significantly higher in the SP-C (p<0.01; p = 0.01, respectively) and ABCA3 groups (p<0.001; p = 0.001, respectively); however, there was no difference between the control and NEHI groups (p = 0.91). An inverse relationship was seen between KL-6 levels and age in the IESM groups, but not in the NEHI or control groups. Children with NEHI had similar presenting clinical features and were equally symptomatic at the time of KL-6 measurement as those with IESM. CONCLUSIONS: Children with NEHI have normal KL-6 levels, in contrast to those with IESM, who have elevated serum KL-6 levels; serum KL-6 may be a useful biomarker in distinguishing between these entities when their clinical presentations overlap.

Bronchiolitis obliterans in the 1990s in Korea and the United States.

STUDY OBJECTIVES: Our current knowledge of pediatric bronchiolitis obliterans (BO) is based largely on a few small series of patients that were reported in the older literature. In these older cases, the mortality rate was high. This study was conducted to investigate the characteristics of pediatric BO cases in two different countries. DESIGN: We extracted specific information regarding predisposing factors, symptoms and signs, diagnostic studies, treatment, and outcome from the medical records of 31 children who received diagnoses of BO at four university medical centers in Korea and the United States in the 1990s. RESULTS: The large number of Asian children reflects a clustering of cases in Korea due to adenovirus and Mycoplasma pneumoniae epidemics. The characteristic diagnostic features of BO were present in 29 of 30 high-resolution CT (HRCT) studies. Seven of nine children who underwent biopsies had histologic confirmations of BO. In two patients whose biopsy results were nondiagnostic, the diagnosis was established by HRCT together with pulmonary function testing results that were consistent with nonreversible small airways obstruction. Fifteen children (48.4%) had evidence of hypoxemia. At present, all but one are alive. Patients with elevated severity-of-illness scores were observed to have increased likelihoods of lung transplantation or death. CONCLUSIONS: We conclude that BO has a good overall prognosis and that the mortality rate has declined over the past decade. This could be related primarily to the use of HRCT for accurate diagnosis and the availability of pediatric lung transplantation. BO cases in Korea were associated with infectious epidemics, whereas those in United States had variable predisposing factors.

KGF regulates pulmonary epithelial proliferation and surfactant protein gene expression in adult rat lung.

Keratinocyte growth factor (KGF, FGF-7) is a potent mitogen for epithelial cells. We instilled recombinant human KGF to determine the effects of KGF on alveolar epithelial cells. Left lungs of adult rats were instilled intrabronchially with KGF (5 mg/kg) or normal saline. KGF instillation resulted in epithelial cell hyperplasia, and the alveolar bromodeoxyuridine (BrdU) labeling index peaked at 35% on day 2 after instillation. The mRNA levels for the surfactant proteins (SPs) SP-A, SP-B, and SP-D were increased in whole lung tissue on days 1 and 2 after KGF treatment and then returned to control levels on days 3-7. SP-C mRNA levels were increased on days 2-5 after KGF instillation. However, all surfactant protein mRNAs were reduced in type II cells isolated from rats instilled with KGF 2 or 3 days before isolation. These observations were confirmed by in situ hybridization. Instillation of KGF also increased the amount of SP-A and SP-D in lavage fluid. Transcripts for CC10, the 10-kDa Clara cell protein, were decreased. KGF increases the mRNA for the surfactant proteins per lung because of type II cell hyperplasia, but the mRNA per cell is slightly diminished as measured in isolated cells or estimated by in situ hybridization.

Evaluation of a diagnostic approach to pediatric interstitial lung disease.

OBJECTIVE: To evaluate the value of a systematic approach to the diagnosis of pediatric interstitial lung disease (ILD). METHODS: In this descriptive, observational, prospective study, we evaluated 51 children presenting with ILD of unknown etiology during a 3-year period. Specific clinical information regarding history, physical examination, diagnostic evaluation, and final diagnosis was recorded on each patient. RESULTS: A specific diagnosis was established by history and physical examination alone in 1 patient, noninvasive tests alone in 8 others, and invasive tests, including lung biopsy, in another 26. Of the remaining patients, 8 had a suggestive diagnosis, and 8 had no specific diagnosis. CONCLUSIONS: A systematic approach to the diagnosis of pediatric ILD is useful, and not all patients need lung biopsy for diagnosis.

Prevention of bleomycin-induced lung injury in rats by keratinocyte growth factor.

Intratracheal instillation of bleomycin produces pulmonary fibrosis in rats. Alveolar type II cell proliferation is thought to minimize the fibrotic response after lung injury. Because keratinocyte growth factor (KGF) stimulates type II cell proliferation in the rat, we designed experiments to evaluate whether intratracheal KGF before or after intratracheal bleomycin would prevent pulmonary fibrosis. Intratracheal bleomycin without KGF resulted in moderate to severe lung injury and subsequent fibrosis. Conversely, intratracheal KGF pretreatment at 48 or 72 hr before bleomycin resulted in minimal to no visible lung injury. Rats pretreated with phosphate buffered saline before bleomycin had significantly more neutrophils and protein in bronchoalveolar lavage fluid at 4 and 6 days and higher hydroxyproline levels after bleomycin as compared to KGF-pretreated rats. Pretreatment with KGF at 48 hr protected against bleomycin-induced alterations in pulmonary physiology and increased surfactant protein C-positive (SP-C)-positive cells and SP-A, SP-B, SP-C, and SP-D mRNA levels after bleomycin instillation when compared to saline pretreated rats on day 1 or day 7. KGF posttreatment protocols did not prevent bleomycin lung injury and fibrosis. We conclude that KGF pretreatment attenuates bleomycin lung injury and increases type II cell proliferation and surfactant protein gene expression after bleomycin instillation in the rat.

Keratinocyte growth factor reduces lung damage due to acid instillation in rats.

Acid aspiration is a serious complication of anesthesia and other forms of unconsciousness that can result in the adult respiratory distress syndrome (ARDS), which continues to have a very high mortality despite our current therapeutic interventions. This type of injury damages the alveolar epithelium, principally alveolar type I cells, and requires proliferation of alveolar type II cells to restore gas exchange units. Since keratinocyte growth factor (KGF) has been shown to be a potent mitogen for alveolar type II cells, we evaluated whether intrabronchial administration of KGF would minimize lung injury due to the unilateral instillation of 0.1 N hydrochloric acid (HCl). Rats were pretreated or post-treated by intrabronchial instillation of KGF (5 mg/kg) into the left lung before HCl instillation. All rats receiving KGF at 48 or 72 h before HCl instillation survived for the 7-day observation period, whereas the mortality rate for those receiving HCl alone or saline followed by HCl was 31% and 33%, respectively. Pretreatment with KGF at 72 h but not at 24 or 48 h considerably ameliorated morphologic damage produced by HCl. Inflammatory cells in bronchoalveolar lavage were markedly decreased 3 and 7 days after HCl instillation by the 72-h KGF pretreatment. Pretreatment with KGF at 72 h also attenuated the reduction of total lung capacity, decreased the alpha 1(I) procollagen mRNA levels, and diminished hydroxyproline accumulation due to HCl instillation. Saline pretreatment at 72 h had no significant effect on the HCl injury and subsequent physiologic abnormalities. Our attempts to improve outcome with post-treatment instillation of KGF were unsuccessful. We conclude that KGF pretreatment reduces lung injury due to acid instillation and can prevent subsequent pulmonary fibrosis.

Acidic fibroblast growth factor and keratinocyte growth factor stimulate fetal rat pulmonary epithelial growth.

We have shown that pulmonary epithelial growth and differentiation can occur if pulmonary mesenchyme is replaced with a mixture of growth factors [total growth medium (TGM)] that consists of adult rat bronchoalveolar lavage fluid, insulin, epidermal growth factor (EGF), cholera toxin (CT), acidic fibroblast growth factor (aFGF), and fetal bovine serum. In the present study, we have defined the importance of specific components of TGM. Day 14 fetal rat distal lung epithelium, devoid of mesenchyme, was enrobed in growth factor-depleted Matrigel and cultured for 5 days in various soluble factors. We found that deleting aFGF or CT from TGM significantly reduced DNA synthesis. Epithelial proliferation was not significantly different when keratinocyte growth factor (KGF) replaced aFGF in TGM. KGF, however, required the presence of a basal medium containing CT, insulin, and serum for optimal proliferation. We then added specific growth factors to the basal medium and showed that aFGF and KGF were more potent mitogens than EGF, transforming growth factor-alpha, and hepatocyte growth factor. Additionally, basal medium + KGF also allowed progression to a distal alveolar phenotype. We conclude that aFGF and KGF may be important mediators in epithelial-mesenchymal interactions.

Proliferation and differentiation of fetal rat pulmonary epithelium in the absence of mesenchyme.

Previous studies have shown that pulmonary mesenchyme is required to maintain epithelial viability and to support branching morphogenesis and cytodifferentiation. We have examined whether pulmonary mesenchyme can be replaced by a medium containing a combination of soluble factors. Day 13-14 fetal rat distal lung epithelium was enzymatically separated from its mesenchyme, enrobed in EHS tumor matrix, and cultured for 5 d in medium containing concentrated bronchoalveolar lavage, EGF, acidic fibroblast growth factor, cholera toxin, insulin, and FBS (TGM), or in control medium containing only FBS. After 5 d in culture, marked growth and morphological changes occurred in epithelial rudiments cultured in TGM, whereas no changes were seen in controls. [3H]Thymidine incorporation and nuclear labeling indices during the last 24 h of culture confirmed that epithelial rudiments cultured in TGM had significant proliferative capacities. Evaluation of surfactant protein gene expression by Northern analysis, in situ hybridization, and immunocytochemistry demonstrated that distal lung epithelial differentiation progressed in TGM. Ultrastructural analysis demonstrated that fetal distal lung epithelium cultured in TGM contained lamellar bodies and deposited a basal lamina. These results are the first demonstration that sustained proliferation and differentiation of glandular stage distal pulmonary epithelium can proceed in the absence of mesenchyme.

Hepatocyte growth factor is a growth factor for rat alveolar type II cells.

Proliferation of alveolar type II cells is thought to be critical for restoration of gas exchange units after diffuse alveolar damage. However, the factors that regulate type II cell proliferation are not well understood. Hepatocyte growth factor (HGF) is a potentially important mitogen because it causes epithelial cells but not fibroblasts to proliferate and is found in the lung. We used rat alveolar type II cells in primary culture to demonstrate that HGF stimulates DNA synthesis in a concentration-dependent manner. The half maximal effect on stimulation of thymidine incorporation was less than 1 ng/ml. By autoradiography, HGF increased nuclear labeling from 1.3% of type II cells with medium alone to 9.4% with 5 ng/ml HGF. During this time, HGF modestly increased cell number in comparison to control media. However, in an assay of colony formation in low-density cultures, HGF did not consistently increase colony formation by alveolar type II cells and was less effective than acidic fibroblast growth factor or bronchoalveolar lavage fluid in this assay. The receptor for HGF (c-met proto-oncogene) was expressed in rat type II cells and whole lung but not in macrophages. In contrast, the mRNA for HGF was detected in rat macrophages and lung but not in type II cells. However, HGF message was not detected in human alveolar macrophages under conditions in which the HGF message was detected in rat alveolar macrophages and in human fibroblasts. Hence, HGF is a potential paracrine growth factor for alveolar type II cells, but there may be important species differences in the relative level of expression.

Regulation of surfactant protein D expression by glucocorticoids in vitro and in vivo.

SP-D is a recently described lung-associated protein that is produced by alveolar type II cells and may function in pulmonary host defenses. Since little is known regarding the hormonal regulation of SP-D, and since the other surfactant proteins (SP-A, SP-B, and SP-C) are known to be regulated by glucocorticoids, we sought to determine the effects of glucocorticoids on SP-D mRNA and protein expression, both in vitro and in vivo, in the fetal rat lung. In vitro experiments were performed on lung explants from fetuses on gestational day 15 or 18. Explants were cultured in serum-free conditions with or without hydrocortisone for 3 days. SP-D mRNA expression was evaluated by Northern blot analysis. SP-D protein expression was analyzed using a polyclonal antibody against SP-D and standard immunohistochemical techniques. The expression of SP-D mRNA increased in fetal day 15 explants but remained unchanged in fetal day 18 explants cultured without the addition of hydrocortisone, compared with in vivo controls. The addition of hydrocortisone resulted in increases in SP-D mRNA expression at both gestational ages. This pattern of SP-D mRNA expression was compared with the expression of the other surfactant proteins and found to be most similar to that of SP-B. In vivo experiments were performed using maternal administration of dexamethasone (1 mg/kg) or an equal volume of saline on fetal days 15, 16, and 17 or on fetal day 17 with sacrifice on fetal day 18.(ABSTRACT TRUNCATED AT 250 WORDS)