Dual targeting of EGFR and ERBB2 pathways produces a synergistic effect on cancer cell proliferation and migration in vitro.

Members of the epidermal growth factor receptor (EGFR/ERBB) gene family are frequently dysregulated in a range of human cancers, and therapeutics targeting these proteins are in clinical use. We hypothesized that similar pathways are involved in feline and canine tumours and that the same drugs may be of clinical use in veterinary patients. We investigated EGFR and ERBB2 targeting using a panel of feline and canine cell lines. EGFR and ERBB2 were targeted with siRNAs or tyrosine kinase inhibitors (TKIs) and their effect on cellular proliferation, colony formation and migration was investigated in vitro. Here we report that EGFR and ERBB2 combined siRNA targeting produced synergistic effects in feline and canine cell lines similar to that reported in human cell lines. We conclude that dual EGFR and ERBB2 targeting using TKIs should be further evaluated as a potential new therapeutic strategy in feline head and neck and mammary tumours and canine mammary tumours.

A comparative life-table analysis of Sipha flava (Hemiptera: Aphididae) on two biofuel hosts, Miscanthus x giganteus and Saccharum spp.

Among the insects reported in biofuel crops, the yellow sugarcane aphid, Sipha flava (Forbes), is a potential pest of giant miscanthus, Miscanthus x giganteus Greef et Deu ex Hodkinson et Renvoize (M x g) and energy cane 'L79-1002', Saccharum spp. L. We studied the biology of S. flava on M x g and energy cane and estimated the development period, fecundity, longevity, intrinsic rate of increase, doubling time, reproductive value, and survivorship curves. To demonstrate the host suitability in a susceptible species, we studied the aphid life table on sorghum 'PL 18200,' Sorghum bicolor (L.) Moench. Life-table information was recorded under greenhouse conditions on the host plants. Our results suggested that both M x g and energy cane are suitable hosts for S. flava. We observed similar aphid development period on both hosts. Life-table estimates including longevity and fecundity suggested that M x g is a more suitable host for the aphid than energy cane. The intrinsic rate of increase for S. flava was lower on energy cane (0.231) than on M x g (0.258).

Relative feeding and development of armyworm on switchgrass and corn, and its potential effects on switchgrass grown for biomass.

To help assess the potential for damage by armyworms [Mythimna (Pseudaletia) unipuncta (Haworth) (Lepidoptera: Noctuidae)] to switchgrass (Panicum virgatum L.) and surrounding crops, survival and development were evaluated for larvae reared on leaves of switchgrass, corn (Zea mays L.), and miscanthus (Miscanthus x giganteus Greef and Deuter ex Hodkinson and Renvoize). Additional tests assessed the relationship between leaf position and the concentration of saponins (plant compounds which can provide protection from insect herbivores) and examined the effect of defoliation on switchgrass dry mass. Survival to adulthood was similar when larvae were reared on field-grown leaves of switchgrass and corn. However, lower larval mass (10 d) and delayed development of M. unipuncta (to pupation, adult emergence) suggest switchgrass is an inferior host relative to corn. When fed field-grown miscanthus, no larvae survived 10 d. Few differences were noted between switchgrass and corn grown under controlled (laboratory) conditions, but M. unipuncta survival seemed to decline rapidly when larvae were fed the fourth and fifth leaves of switchgrass. Switchgrass leaf samples collected from different leaf positions and stages of tiller maturity showed up to 10-fold differences in the concentration of the saponin protodioscin, with the greatest concentrations in the fourth and fifth leaves. However, other saponins showed an opposite pattern, indicating the role of protodioscin on insect development should be tested in isolation (e.g., by addition of the purified compound to an artificial diet). Defoliation trials indicated that extremely high M. unipuncta populations may be necessary to cause any significant reduction in switchgrass biomass. Collectively, results suggest M. unipuncta may not present a significant risk to biomass production in switchgrass, but that the spring emergence of switchgrass provides an alternate host for M. unipuncta before colonizing annual food and feed crops.

First Report of Basal Stem Rot and Foliar Blight Caused by Pythium sylvaticum on Miscanthus sinensis in Illinois.

Miscanthus sinensis Anderss., a perennial grass, is native to eastern Asia. It has been widely grown as an ornamental in temperate regions of the world, including the United States, and recently has become an important component of public and private sector bioenergy feedstock Miscanthus selection programs. In August 2008, stem rot and blight was observed on M. sinensis plants in two irregular patches, ~2 to 2.5 × 1 to 1.5 m each in a trial plot that was preceded by corn, at the University of Illinois Energy Farm near Urbana, IL. At the time of the observation, most plants were dead and the wilted tillers had black, soft rotted basal stems. A few plants were stunted and the crowns of the tillers had black-to-brown soft rot. Some tillers' leaves were dead and others had turned light brown. Sample tissue fragments were surface disinfested in 0.5% NaOCl and plated on 1% water agar (WA). After 3 days of incubation in the dark at 23°C, colonies were transferred to corn meal agar (CMA), potato dextrose agar (PDA), or 10% V8 juice agar and incubated at 23°C under continuous white light for up to 2 weeks. Morphological characteristics of the isolates correspond to those originally described for Pythium sylvaticum W.A. Campb. & J.W. Hendrix (1). The mycelia grew and covered the 10-cm-diameter plates within 5 days. On PDA, the culture was a creamy white mycelial mat of coenocytic hyphae. The isolates produced only globose, terminal or intercalary hyphal swellings ranging from 28 to 48 µm in diameter, but no oogonia were produced on any of the three growth media. No zoospores were produced when agar blocks bearing mycelium were flooded with distilled water or 1% soil water. Sequence analysis was performed with the internal transcribed spacer (ITS) region of the rDNA amplified with primer pair ITS1/ITS4 (3) and the mitochondrially encoded cytochrome c oxydase subunit II (cox II) gene using primers FM58/FM66 (2). The resulting 871-bp ITS nucleotide sequence (Accession No. HM991706) was identical among all three isolates analyzed and 99% identical (100% coverage) to ITS sequences of multiple isolates of P. sylvaticum in GenBank. Likewise, the 544-bp cox II sequence (Accession No. HQ454429) was 99% identical (97% coverage) to cox II sequences of multiple isolates of P. sylvaticum. Six pots of M. sinensis seedlings were inoculated by placing two CMA plugs of a 2-week-old culture of isolate F71 at the crown. The control pots were mock inoculated with sterile CMA plugs. The plants were incubated at ~90% relative humidity (RH) and 25°C day and 22°C night for 3 days, and thereafter left on the greenhouse bench at ~65% RH with alternating 9 h of darkness and 15 h of light. Three weeks after inoculation, two of the inoculated seedlings wilted, others were stunted with leaves wilting from the tip downwards and the stems rotting from the crown upward. A thick mat of mycelia was seen on the rotted basal stems. No symptoms were observed in the control. P. sylvaticum was reisolated from both the rotted basal stems and the wilted foliage. To our knowledge, this is the first report of P. sylvaticum on M. sinensis. Infestation of farm soils with P. sylvaticum could limit M. sinensis biomass production significantly by limiting seedling establishment. References: (1) W. A. Campbell and F. F. Hendrix. Mycologia 59:274, 1967. (2) F. M. Martin. Mycologia 92:711, 2000. (3) T. J. White et al. Page 38 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

Areawide suppression of European corn borer with Bt maize reaps savings to non-Bt maize growers.

Transgenic maize engineered to express insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) has become widely adopted in U.S. agriculture. In 2009, Bt maize was planted on more than 22.2 million hectares, constituting 63% of the U.S. crop. Using statistical analysis of per capita growth rate estimates, we found that areawide suppression of the primary pest Ostrinia nubilalis (European corn borer) is associated with Bt maize use. Cumulative benefits over 14 years are an estimated $3.2 billion for maize growers in Illinois, Minnesota, and Wisconsin, with more than $2.4 billion of this total accruing to non-Bt maize growers. Comparable estimates for Iowa and Nebraska are $3.6 billion in total, with $1.9 billion for non-Bt maize growers. These results affirm theoretical predictions of pest population suppression and highlight economic incentives for growers to maintain non-Bt maize refugia for sustainable insect resistance management.

First Report of Pithomyces chartarum Causing a Leaf Blight of Miscanthus × giganteus in Kentucky.

Miscanthus × giganteus is a warm-season perennial grass, native to eastern Asia. Brought into the United States as a landscape plant, it is currently being considered as a potential biomass fuel crop. In August 2009, a newly established and a 2-year-old M. × giganteus field research trial near Lexington, KY were found to have 100% incidence of severe leaf blight. Brown, mosaic-like, coalesced necrotic lesions covered leaf blades and sheaths on every stand, ultimately killing some leaves and tillers. The disease was more destructive in the newly established trial where 4- to 5-month-old M. × giganteus tillers were killed. No fruiting bodies were found immediately on diseased leaves. However, surface-disinfested diseased leaf tissue produced a sooty black mass of conidia after 1 week following incubation in a petri dish moisture chamber at 25°C in the dark. Single conidia isolations were made on half-strength potato dextrose agar (HSPDA) amended with 25 mg/liter of rifamycin and incubated at 25°C. Morphological characteristics of the fungus fit those originally described for Pithomyces chartarum (Berk. & Curt.) M.B. Ellis (2). Colonies were fast growing on HSPDA, at first hyaline, then shortly punctiform, grayish black, up to 1-mm diameter, and then became confluent, producing several dark brown multicellular conidia on small peg-like denticles on branched conidiophores. Every detached conidium had a small piece of the denticle attached to its base. The conidia were echinulate, broadly ellipsoidal, pyriform, 18 to 29 × 11 to 18 µm, with three transverse septa, and a longitudinal septum constricted at the transverse septa. The identity of the fungus was confirmed by sequence analysis of the internal transcribed spacers (ITS) region of the nuclear ribosomal DNA. The 615-bp cloned and sequenced amplicon (Accession No. GU195649) was 99% identical to sequences from multiple isolates of Leptosphaerulina chartarum (anamorph Pithomyces chartarum) in the GenBank. Five potted M. × giganteus plants (45 days old) were spray inoculated with an aqueous conidial suspension (2 × 106 conidia/ml) and incubated in one tier of a two-tiered-growth chamber at 86 to 90% relative humidity. Initial incubation was in the dark at 26°C for 48 h, and thereafter at alternating 15 h of light (320 µmol) at 25°C and 9 h of darkness at 23°C. Control plants were sprayed with sterile water and incubated in the second tier of the same growth chamber. A week after inoculation, leaf blight developed on all inoculated plants, but not the controls. P. chartarum was reisolated from infected leaves 2 weeks after inoculation. To our knowledge, this is the first report of P. chartarum causing a disease on Miscanthus (3). The fungus is cosmopolitan, usually saprophytic, but can cause diseases on a wide range of plants as well as produce mycotoxins (3). It has been reported to cause a leaf spot of smooth bromegrass (Bromus inermis) in Nebraska (1) and a leaf blight of wheat (Triticum aestivum) in Hungary (4). The observed disease severity suggests P. chartarum could potentially limit M. × giganteus production as an ethanol feedstock. References: (1) C. Eken et al. Plant Dis. 90:108, 2006. (2) M. B. Ellis. Dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Kew, Surrey, England, 1971. (3) D. F. Farr et al. Fungal Databases, Systematic Mycology and Microbiology Laboratory. Online publication. ARS, USDA, 2010. (4) B. Tóth et al. J. Plant Pathol. 89:405, 2007.

Development and feeding of fall armyworm on Miscanthus x giganteus and switchgrass.

Observations of fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), larvae infesting plots of Miscanthus x giganteus Greef and Deuter ex Hodkinson and Renvoize prompted laboratory-based tests of survival, development, and feeding preferences on leaf tissue from M. x giganteus and switchgrass, Panicum virgatum L. Survival from hatch to pupation was >70 and 50% for fall armyworms reared on switchgrass and M. x giganteus, respectively, although survival of the S. frugiperda rice strain was significantly greater than the corn strain on both crops. Developmental times from hatch to pupation or adult emergence showed effects of crop and S. frugiperda host strain, but analysis of an interaction revealed developmental times for the rice strain were similar on both crops, whereas corn strain larvae showed delayed development on M. x giganteus relative to switchgrass. Analysis of larval (10 d) and pupal masses showed a similar pattern, with effects of crop and an interaction (at 10 d), but only the mass of corn strain larvae feeding on M. x giganteus was reduced relative to the other crop and strain combinations. In choice tests, neonates of both corn and rice strains showed a strong preference for feeding on young tissues rather than mature leaves of M. x giganteus or switchgrass, but they also clearly favored corn, Zea mays L., leaves over either of the perennial grasses. Results indicate both plants are potential hosts for S. frugiperda, but additional information is needed to understand under which scenarios and to what degree fall armyworms may damage perennial grasses grown for biofuel production.

Population dynamics of a Western corn rootworm (Coleoptera: Chrysomelidae) variant in east central Illinois commercial maize and soybean fields.

Three on-farm sites in Iroquois County, IL, each containing an adjacent 16.2-ha commercial production maize, Zea mays L., and soybean, Glycine max (L.) Merr., field, were monitored for western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), adults from June through September 1999-2001. Mean captures of D. v. virgifera adults as measured with Pherocon AM yellow sticky traps were significantly greater in maize than in soybean. Overall mean numbers of D. v. virgifera adults captured with vial traps were significantly greater in soybean than in maize. Emergence cage data revealed that after 50% emergence of D. v. virgifera adults occurred, peak captures of D. v. virgifera adults occurred in maize as measured with vial and Pherocon AM traps. After maize reached the R2 (blister stage, 10-14 d after silking) stage of development and 90% emergence of D. v. virgifera adults had occurred, peak captures of D. v. virgifera adults were observed in soybean by using vial and Pherocon AM traps. Also, after maize reached the R2 stage of development, numbers of females significantly increased in soybean and decreased in maize. Captures of female D. v. virgifera adults frequently exceeded published economic thresholds in soybean, regardless of trap type used. Estimated survival of variant D. v. virgifera (egg to adult) in these commercial rotated maize fields was 10.7 and 9.4% from 1999 to 2000 and from 2000 to 2001, respectively. This compares with nonvariant D. v. virgifera survival estimates in continuous maize production systems in Iowa of 6.7 and 11% from 1983 to 1984 and from 1984 to 1985, respectively.

Economic analysis of dynamic management strategies utilizing. Transgenic corn for control of western corn rootworm (Coleoptera: Chrysomelidae).

We studied management strategies for western corn rootworm, Diabrotica virgifera virgifera LeConte, using transgenic corn, Zea mays L., from both a biological and an economic perspective. In areas with and without populations adapted to a 2-yr rotation of corn and soybean (rotation-resistant), the standard management strategy was to plant 80% of a cornfield (rotated and continuous) to a transgenic cultivar each year. In each area, we also studied dynamic management strategies where the proportion of transgenic corn increased over time in a region. We also analyzed management strategies for a single field that is the first to adopt transgenic corn within a larger unmanaged region. In all areas, increasing the expression of the toxin in the plant increased economic returns. In areas without rotation-resistance, planting 80% transgenic corn in the continuous cornfield each year generated the greatest returns with a medium toxin dose or greater. In areas with alleles for rotation-resistance at low initial levels, a 2-yr rotation of nontransgenic corn and soybean, Glycine max (L.) Merr., may be the most economical strategy if resistance to crop rotation is recessive. If resistance to crop rotation is additive or dominant, planting transgenic corn in the rotated cornfield was the most effective strategy. In areas where rotation-resistance is already a severe problem, planting transgenic corn in the rotated cornfield each year was always the most economical strategy. In some cases the strategies that increased the proportion of transgenic corn in the region over time increased returns compared with the standard strategies. With these strategies the evolution of resistance to crop rotation occurred more rapidly but resistance to transgenic corn was delayed compared with the standard management strategy. In areas not managed by a regional norm, increasing the proportion of transgenic corn and increasing toxin dose in the managed field generally increased returns. In a sensitivity analysis, among the parameters investigated, only density-dependent survival affected the results.

Economics versus alleles: balancing integrated pest management and insect resistance management for rotation-resistant western corn rootworm (Coleoptera: Chrysomelidae).

Western corn rootworm, Diabrotica virgifera virgifera LeConte, has overcome crop rotation in several areas of the central United States. We expanded a simple model of adult behavior and population genetics to explain how rotation resistance may have developed and to study ways to manage the western corn rootworm in a landscape of corn, soybean, and winter wheat where evolution of resistance may occur. We modeled six alternative management strategies over a 15-yr time horizon, as well as a strategy involving a 2-yr rotation of corn and soybean in 85% of the landscape, to investigate their effectiveness from both a biological and economic perspective. Generally, resistance to crop rotation evolves in fewer than 15 yr, and the rate of evolution increases as the level of rotated landscape (selection pressure) increases. When resistance is recessive, all six alternative strategies were effective at preventing evolution of rotation resistance. The two most successful strategies were the use of transgenic rotated corn in a 2-yr rotation and a 3-yr rotation of corn, soybean, and wheat with unattractive wheat (for oviposition) preceding corn. Results were most sensitive to increases in the initial allele frequency and modifications of the density-dependent survival function. Economically, three alternative strategies were robust solutions to the problem, if technology fees were not too high. Repellant soybean, attractive rotated corn, and transgenic rotated corn, all in 2-yr rotations, were economically valuable approaches. However, even the currently common 2-yr rotation was economical when resistance was recessive and the actual costs of resistance would not be paid until far in the future.

Modeling the dynamics of adaptation to transgenic corn by western corn rootworm (Coleoptera: Chrysomelidae).

A simulation model of the population dynamics and genetics of the western corn rootworm, Diabrotica virgifera virgifera LeConte, was created for a landscape of corn, soybean, and other crops. Although the model was created to study a 2-locus problem for beetles having genes for resistance to both crop rotation and transgenic corn, during this first phase of the project, the model was simulated to evaluate only resistance management plans for transgenic corn. Allele expression in the rootworm and toxin dose in the corn plant were the two most important factors affecting resistance development. A dominant resistance allele allowed quick evolution of resistance to transgenic corn, whereas a recessive allele delayed resistance >99 yr. With high dosages of toxin and additive expression, the time required to reach 3% resistance allele frequency ranged from 13 to >99 yr. With additive expression, lower dosages permitted the resistant allele frequency to reach 3% in 2-9 yr with refuges occupying 5-30% of the land. The results were sensitive to delays in emergence by susceptible adults and configuration of the refuge (row strips versus blocks).

Predicting western corn rootworm (Coleoptera: Chrysomelidae) larval injury to rotated corn with Pherocon AM traps in soybeans.

Crop rotation for portions of east central Illinois and northern Indiana no longer adequately protects corn (Zea mays L.) roots from western corn rootworm, Diabrotica virgifera virgifera LeConte. Seventeen growers in east central Illinois monitored western corn rootworm adults in soybean (Glycine max L.) fields with unbaited Pherocon AM traps during 1996 and 1997. In the following years (1997 and 1998), growers left untreated strips (no insecticide applied) when these fields were planted with corn. Damage to rotated corn by rootworms was more severe in untreated than in treated strips of rotated corn, ranging from minor root scarring to a full node of roots pruned. Densities of western corn rootworms in soybean fields from 1996 were significantly correlated with root injury to rotated corn the following season. Adult densities from 1997 were not significantly correlated with root injury in 1998, due to heavy precipitation throughout the spring of 1998 and extensive larval mortality. Twenty-eight additional growers volunteered in 1998 to monitor rootworm adults in soybean fields with Pherocon AM traps based on recommendations that resulted from our research efforts in 1996 and 1997. In 1999, these 28 fields were rotated to corn, and rootworm larval injury was measured in untreated strips. Based on 1996-1997 and 1998-1999 data, a regression analysis revealed that 27% of the variation in root injury to rotated corn could be explained by adult density in soybeans the previous season. We propose a sampling plan for soybean fields and a threshold for predicting western corn rootworm larval injury to rotated corn.

Temporal-spatial distribution of hepatocyte nuclear factor-3beta in developing human lung and other foregut derivatives.

We assessed the temporal-spatial distribution of hepatocyte nuclear factor-3beta (HNF-3beta) in developing human lung and other foregut derivatives. Tissue from 31 fetuses (10-40 weeks) and 24 infants with hyaline membrane disease (HMD) or bronchopulmonary dysplasia (BPD) (2 days to 7 months) was studied. HNF-3beta was detected in nuclei of epithelial cells of trachea and of conducting and terminal airways at 10 weeks. Thereafter, epithelial nuclei were immunolabeled more widely in peripheral than proximal airways. HNF-3beta was confined to bronchiolo-alveolar portals and Type II cells in nonfetal lung. In infants with BPD, HNF-3beta was expressed abundantly in regenerating epithelial cells at the periphery of lung lobules. HNF-3beta was also detected in fetal esophagus, pancreas, duodenum, stomach, and gallbladder, suggesting that it is a marker for progenitor cells in foregut derivatives. The pattern of expression of HNF-3beta in the lung was similar to that of thyroid transcription factor-1 (TTF-1) at all ages. The temporal-spatial patterns of HNF-3beta and TTF-1 in the developing and regenerating lung are consistent with their proposed role in epithelial cell differentiation, regeneration, and surfactant protein gene expression. (J Histochem Cytochem 46:955-962, 1998)

Immunogold EM localization of neurochemicals in human pulmonary neuroendocrine cells.

Antibodies against the pulmonary neuroendocrine cell peptides gastrin-releasing peptide (bombesin), calcitonin and calcitonin gene-related peptide (CGRP) have been labeled with colloidal gold spheres for immunocytochemical localization in human fetal and newborn lung tissue. In general, the presence and amount of immunolabeling increased with increasing gestational age, with only calcitonin appearing late in fetal life. The largest percentage of neuroepithelial body (NEB) cells labeled and the largest number of labeled dense core vesicles (DCV) were in infants with chronic lung disease (bronchopulmonary dysplasia). Serial ribbons allowed identification of more than one peptide in a single NEB cell. The use of two antibodies labeled with colloidal gold spheres of different sizes allowed the identification of two peptides in the same DCV. Quantification of relative amounts of labeled peptides was not possible, as the peptide labeling with the larger size gold sphere was consistently underestimated. Colocalization to the same DCV has been shown in humans for bombesin and calcitonin, calcitonin and CGRP, bombesin and CGRP and, by others for cholecystokinin (CCK) and serotonin. Colocalization of two or more peptides or an amine to a single DCV within the same cell implies simultaneous discharge by exocytosis. The action of the two (or more) substances might be in concert, perhaps with one acting in a paracrine fashion, and the second in an autocrine fashion. In this case, the second peptide or amine might have a regulatory function in the parent cell, influencing DCV storage or rate of release.

Ontogeny of Clara cell-specific protein and its mRNA: their association with neuroepithelial bodies in human fetal lung and in bronchopulmonary dysplasia.

Clara cell-specific 10-KD protein (CCSP) is an abundant product of nonciliated bronchiolar epithelial (Clara) cells in the lung. We have determined the temporal-spatial distribution of CCSP and its mRNA in developing human lung and in neonatal lung disease, using immunohistochemistry and in situ hybridization. CCSP immunoreactivity was found in nonciliated bronchiolar epithelial cells from 12 weeks of gestation onward. Tracheal and bronchial epithelia showed positive immunoreactivity at each gestational week after 15 weeks and 14 weeks, respectively. CCSP mRNA was seen in the bronchial and bronchiolar epithelia from 16 weeks onward and was detected in the trachea from 19 through 23 weeks of gestation. CCSP immunoreactivity and mRNA were present in nonciliated single cells of bronchial and bronchiolar epithelia in fetuses and in infants with and without lung disease. CCSP- and CCSP mRNA-containing epithelial cells also formed dusters around neuroepithelial bodies (NEBs), especially at airway branch points, suggesting that NEBs and Clara cells might interact during development and during pulmonary regeneration. Because of evidence of overlapping of some but not all cells expressing CCSP, SP-A, and pro-SP-B during lung development, a common cell lineage is proposed, with subsequent divergence of phenotypes.

Respiratory syncytial virus infection enhances the response to laryngeal chemostimulation and inhibits arousal from sleep in young lambs.

To evaluate the effect of respiratory syncytial virus (RSV) infection on the response to laryngeal chemostimulation (LCS) with water, five lambs were inoculated with human RSV and three lambs were given control media at an age of 3-5 days. During RSV infection, LCS resulted in increased inhibition of minute ventilation and delayed recovery of regular breathing. Sleep further increased the response, and arousal was less likely to occur in active sleep. Two of the five infected lambs needed resuscitation after LCS when arousal was absent. Histological studies showed bronchiolitis and pneumonitis. Laryngeal tastebud morphology was unchanged at 8 days after inoculation. However, infected lambs had disrupted tastebuds 4-6 weeks after infection. Failure to arouse and to terminate reflex apnea may play a role in the pathogenesis of the sudden infant death syndrome associated with respiratory tract infection.

Expression of thyroid transcription factor-1(TTF-1) in fetal and neonatal human lung.

We assessed the immunohistochemical localization of thyroid transcription factor-1 (TTF-1) in the lungs of 24 human fetuses (11-23 weeks), three infants without pulmonary pathology (36-42 weeks), and 24 infants (2 days-6.5 months) with hyaline membrane disease (HMD) or bronchopulmonary dysplasia (BPD). TTF-1 was detected in fetal lung epithelial cell nuclei by 11 weeks' gestation. Budding tips of terminal airways had prominently labeled nuclei. By 17 weeks, labeling was present in scattered nonciliated columnar and cuboidal cells. Throughout gestation, TTF-1 nuclear staining was prominent in airways abutting pleural, peribronchial, or perivascular connective tissue, being less prominent in centers of lobules. By 23 weeks, many cells in cuboidal but not columnar cell-lined airways had labeled nuclei. At term, TTF-1 was detected primarily in Type II epithelial cells. In HMD with alveolar hemorrhage, edema, or airway collapse, little or no TTF-1 was present except in open terminal airways. In BDP lungs, TTF-1 was absent in areas of alveolar collapse or infection, being present in regenerating open airways. The temporal-spatial distribution of TTF-1, in general, follows patterns of distribution of surfactant protein-B in developing and pathological lungs, consistent with its role in the regulation of epithelial cell gene expression in the lung.

Temporal-spatial distribution of SP-B and SP-C proteins and mRNAs in developing respiratory epithelium of human lung.

We determined the temporal and spatial distribution of surfactant protein B (pro-SP-B) and C (pro-SP-C) mRNAs and proteins by immunohistochemistry and in situ hybridization in fetal, neonatal, and adult human lung. Pro-SP-B and SP-B mRNA were detected in bronchi and bronchioles by 15 weeks' gestation. After 25 weeks, pro-SP-B, active SP-B peptide, and SP-B mRNA were co-localized in bronchiolo-alveolar portal cells and in Type II epithelial cells. In adult lung, pro-SP-B and SP-B mRNA were detected primarily in non-ciliated bronchiolar epithelial cells and in Type II cells in the alveolus. Pro-SP-C and SP-C mRNA were detected in cells lining terminal airways from 15 weeks' gestation and thereafter. After 25 weeks, SP-C mRNA and precursor protein were detected in epithelial cells of the bronchiolo-alveolar portals and in Type II cells, where expression increased with advancing gestational age. Distinct cellular patterns of staining for pro-SP-B compared with SP-B active peptide support the concept that its proteolytic processing or cellular routing may be influenced by cell type and/or cell differentiation. SP-B and SP-C are expressed primarily in distal conducting and terminal airway epithelium of human fetal lung well in advance of surfactant lipid synthesis or physiologic requirements to produce pulmonary surfactant at the time of birth.

Developmental expression of SP-A and SP-A mRNA in the proximal and distal respiratory epithelium in the human fetus and newborn.

We used immunolocalization and in situ hybridization to determine the distribution of SP-A and SP-A mRNA in lungs of human fetuses and normal newborn infants. Early in the second fetal trimester a few immunostained cells were observed in tracheal epithelium, often in mucosal folds near the origin of submucosal gland ducts. Non-mucous tracheal gland cells were immunostained for SP-A as they became differentiated. Expression of SP-A mRNA was similar to that of immunolocalization in the second trimester. Immunostained cells and SP-A mRNA also appeared about the same time in gestation in isolated cells of bronchial epithelium and glands. SP-A mRNA was seen in bronchiolar cells and pre-Type II cells lining terminal airways of fetuses at 19-20 weeks of gestation. Only in liveborn infants did cells of bronchioloalveolar portals and mature Type II cells contain SP-A mRNA or immunostain for SP-A. In postnatal infants, luminal material was also stained for SP-A. Although some alveolar macrophages contained immunoreactive material, SP-A mRNA was never detected. The abundance of SP-A in tracheal and bronchial glands and epithelium of conducting airways supports the importance of non-surfactant-associated functions for SP-A and may be related to a role in host defense.