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).

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.

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 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.

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.

Intestinal lipoprotein synthesis in control and hypercholesterolemic rats.

Previous studies in our laboratory have shown that very-low-density lipoproteins (VLDL) synthesized by the intestine of the diet-induced hypercholesterolemic rat are enriched in cholesteryl esters and unesterified cholesterol compared with intestinal VLDL from control rats. In these studies, we isolated and characterized nascent intestinal Golgi intermediate-density lipoproteins (IDL, d 1.006-1.040 g/ml) and studied isotope incorporation into apoliproteins of Golgi VLDL from control and hypercholesterolemic rats. IDL were triacylglycerol-rich lipoproteins but contained more cholesteryl ester and protein than the corresponding Golgi VLDL fractions. IDL from hypercholesterolemic rats were enriched in cholesteryl esters to a greater extent than IDL from control rats. The apolipoprotein patterns of IDL fractions were the same as those of intestinal Golgi VLDL, consisting of apolipoproteins (apo) B-48, A-I and A-IV. Time-course isotope incorporation curves for apo A-I and A-IV in Golgi VLDL were similar, but they differed from curves for apo B-48. None of these curves was markedly altered in the hypercholesterolemic rat. We conclude that the major effect of increased dietary cholesterol on intestinal lipoprotein biosynthesis is to increase the percentage of cholesteryl esters in Golgi lipoproteins. Dietary cholesterol does not alter the apolipoprotein composition of Golgi lipoproteins, nor does it have a significant effect on the pattern of isotope incorporation into apolipoproteins of Golgi VLDL. The effect of cholesteryl ester enrichment on the subsequent metabolism of these particles in the circulation and the effect of these particles on hepatic lipoprotein production remain to be determined.

The lipid composition of isolated rat spermatids and spermatocytes.

The lipids composition of enriched fractions of spermatids and spermatocytes, isolated from rat testicular tissue, has been investigated. More than 20% of the total fatty acids of spermatids but only 10% of those of spermatocytes, isolated from testes of mature rats, was 4,7,10,13,16-docosapentaenoic acid. Spermatocyte-enriched fractions isolated from testes of immature rats had fatty acid compositions similar to those isolated from testes of mature rats. On the other hand, spermatids isolated from immature rats had a level of docosapentaenoic acid which was intermediate between the level found in spermatocytes and that of spermatids from mature rats. Major phospholipid classes and the triacylglycerols of spermatids contained much more of the docosapentaenoic acid than the corresponding lipid types from spermatocytes. Differences in content of total phospholipids, individual classes of phospholipids and triacylglycerols among spermatocytes, spermatids and late spermatids were also observed.

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.

Calcitonin gene-related peptide in human fetal lung and in neonatal lung disease.

The ontogeny of calcitonin gene-related peptide immunoreactivity (CGRP-IR) was evaluated immunohistochemically in 67 human fetal or newborn lungs previously analyzed for calcitonin immunoreactivity (CT-IR). CGRP-IR was present by 10 weeks of gestation in rare, solitary neuroendocrine (NE) cells of developing conducting airways in two of eight first-trimester lungs. During the second trimester, cells with CGRP-IR were found consistently (21/23 fetuses). However, the numbers of positively staining cells did not appear to increase in these fetuses or in third-trimester infants dying of non-pulmonary causes. The highest concentrations of CGRP-IR cells were seen in lungs of premature infants with advancing chronic lung disease associated with bronchopulmonary dysplasia (BPD). CGRP-IR was seen earlier in gestation and in greater numbers of NE cells than was calcitonin immunoreactivity (CT-IR) reported previously in these same fetal lungs (Lab Invest 52:52, 1985). Its presence paralleled that of CT-IR in postnatal chronic lung disease.

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.

The ontogeny and distribution of surfactant protein B in human fetuses and newborns.

The distribution of immunoreactive surfactant-associated protein B (IR-SP-B) was studied immunohistochemically in 120 subjects from 10 weeks of gestation to 7 postnatal months with a polyclonal antibody against human SP-B. Electron microscopy (EM) was done in 72 subjects to document the presence of Type II cells containing lamellar bodies. Fetuses of less than 18 weeks' gestation showed no immunostaining. Beginning at 18 weeks, non-mucous cells of tracheal glands immunostained in a few instances. Fetuses of 19 through 23 weeks showed progressive immunostaining of cells lining terminal airways. Infants 26-40 weeks who died with or without pulmonary pathology showed immunostaining of Type II cells and bronchioloalveolar (BA) portal cells of the respiratory bronchioles. In infants with hyaline membrane disease (HMD) who died less than 12 days after birth, occasional tracheal gland cells, BA portal cells, and mature and relining Type II cells immunostained. In bronchopulmonary dysplasia (BPD), BA portal cells, relining Type II cells, macrophages, and luminal material immunostained. Occasional tracheal and bronchial gland cells and Clara cells immunostained. The appearance of IR-SP-B at mid-gestation correlated with differentiation of Type II cells. There was good correlation of immunostaining with the presence of lamellar bodies on EM. Accelerated maturation of the lung was often associated with premature rupture of membranes (PROM).

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.

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 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)

Immunocytochemical localization of human epidermal growth factor/urogastrone in several human tissues.

Epidermal growth factor (EGF) stimulates the growth of many tissues and inhibits stimulated gastric acid secretion. Its primary tissue of origin in man is still unknown. We used polyclonal anti-human EGF sera in the peroxidase-antiperoxidase immunocytochemical staining technique to identify immunoreactive human EGF (ihEGF) in tissue sections from 29 subjects ranging from fetuses to 63 years in age. In addition to acinar cells in the submandibular salivary glands and cells of Brunner's duodenal glands, previously reported to contain ihEGF, we found ihEGF in most anterior pituitary glycopeptide hormone-secreting cells, in gastric and pyloric gland cells of the stomach, and in bone marrow cells that resembled mononuclear phagocytes in subjects of all ages. The eccrine sweat glands in the skin of adults also contained ihEGF. Cells containing ihEGF were found singly or in clusters in the trachea of the fetus only. No fetal pancreatic islet cells stained, but occasional cells in neonates and a majority of islet cells in older subjects contained ihEGF; there was no constant association with insulin, glucagon, or somatostatin. Only the lactating breast contained ihEGF. In adults, outer adrenomedullary cells contained ihEGF. Intense immunostaining was observed in the renal medulla, apparently limited to the extracellular area between the renal tubules, and increased with age; the cortex was devoid of ihEGF. No ihEGF was detected in posterior pituitary gland, thyroid gland, heart, lung, or liver at any age. An adult prostate contained ihEGF only in an area of local injury, and some primordial follicles from the ovary of a newborn appeared to contain ihEGF. Thus, many tissues appear to synthesize hEGF, which may exert exocrine, endocrine, or paracrine functions in different tissues and at different ages.

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.

Lipocortin-1 immunoreactivity in the human pituitary gland.

We evaluated the distribution of lipocortin-1 immunoreactivity in 118 immature or mature human hypophyses using the peroxidase-antiperoxidase (PAP) technique with a polyclonal rabbit antiserum against lipocortin-1. Serial sections were evaluated for five pituitary hormones and S-100 protein immunoreactivity to compare their distributions with that of lipocortin-1. Scattered or moderate numbers of cells exhibited lipocortin-1 immunoreactivity in the pars distalis of 89 subjects ranging in age from 27 weeks' gestation to 83 years. Seven immature and seven aged specimens exhibited no immunostaining, while 15 specimens from older individuals exhibited only rare immunostaining. Immunostaining did not appear to co-localize selectively with any specific pituitary hormone, although the distribution of immunoreactivity did overlap that of some corticotrophs and was seen in elongated processes of S-100-containing folliculostellate cells. Lipocortin-1 was also found in epithelial cells lining colloid cysts of the residual pars intermedia in 115 of 118 pituitaries ranging in age from 23 weeks' gestation to 83 years. In many intermediate lobe cysts, lipocortin-1 exhibited a pattern of immunoreactivity that partially overlapped the distribution of S-100 protein immunostaining, although the pattern was not identical. Pre-absorption of anti-lipocortin-1 antiserum with lipocortin-1-coupled Sepharose-4B immunoreactivity resulted in loss of immunoreactivity in both lobes. No lipocortin-1 immunoreactivity was seen in the neurohypophysis.

Ontogeny of epidermal growth factor receptor and lipocortin-1 in fetal and neonatal human lungs.

The ontogeny and distribution of the epidermal growth factor (EGF) receptor and lipocortin-1, a major cellular substrate of the EGF receptor, were evaluated in a developmental series of fetal and neonatal human lungs (8 to 41 weeks' gestation and stillborn to 16 days' postnatal age). The peroxidase anti-peroxidase technique with two polyclonal antibodies recognizing the EGF receptor and one polyclonal antibody recognizing lipocortin-1 were used for immunohistochemical localization. Extensive or scattered bronchiolar EGF receptor immunoreactivity appeared in the entire series of frozen lung specimens from 15 to 32 weeks' gestation. Bronchial glands exhibited EGF receptor immunostaining from 19 weeks onward, and immunoreactivity in bronchial epithelium was detected from 23 weeks onward. Most tracheas showed extensive lipocortin-1 immunoreactivity in the epithelium beginning at 10 weeks' gestation. Immunostaining was also seen in cells lining the ducts of submucosal glands after 15 weeks' gestation and in nonmucous acinar cells of tracheal glands after their appearance at 18 weeks' gestation. Bronchial epithelium exhibited lipocortin-1 immunoreactivity from 12 weeks' gestation onward. Bronchial gland necks became immunostained from 16 weeks' gestation onward, followed by acinar immunostaining as they subsequently developed. Bronchiolar epithelium was immunostained as early as 12 weeks, beginning with the largest airways, and by 24 weeks extending distally to the bronchioloalveolar portals. Lipocortin-1 immunostaining of larger conducting airway epithelium was primarily confined to ciliated cells. Neither EGF receptor nor lipocortin-1 immunoreactivity was detected in alveolar type I or type II cells, fibrocytes, chondrocytes, or smooth muscle cells at any gestational age. These developmental patterns suggest that the EGF receptor and lipocortin-1 may participate in normal growth factor-induced proliferation of the conducting airways and their glands in the human fetal lung and trachea.