First Report of the Spittlebug Lepyronia quadrangularis and the Leafhopper Latalus sp. as Vectors of the Elm Yellows Associated Phytoplasma, Candidatus Phytoplasma ulmi in North America.

Elm yellows (EY) is a lethal disease of American (Ulmus americana L.) and other elm species (1). On the Pennsylvania State University campus, EY, together with Dutch elm disease, has killed 82 of about 400 mature elms since 2007, the year of first EY detection. Candidatus Phytoplasma ulmi, associated with EY, has been reported to be transmitted by the whitebanded elm leafhopper Scaphoideus luteolus Van Duzee, the meadow spittlebug Philaenus spumarius L., and the leafhopper Allygus atomarius Fabricius (1) in North America, but correlation of these insects with EY in the eastern United States has not been reported. Three Cicadellidae collections using sweep nets and aspirators were performed from July to September 2012 on branches of an EY infected red elm (U. rubra Muh; 40°48.408'N, 77°52.208'W) and on vegetation within a 0.5 km radius. The red elm is in close proximity to trees, shrubs, and a managed meadow and has repeatedly tested positive for EY since 2007. During each collection, about 200 cicadellids were captured in BioQuip No-See-Um catch bags with cups, and the bags were hung around the red elm branches, forcing the insects to feed on the infected tree for 24 h. Insects were transferred to BugDorm rearing tents containing wild grasses, elm seedlings, cowpeas, celery, carrots, and basil, all grown from seed, and were kept for 3 weeks in a controlled environment chamber at 28°C and 70% humidity with a 16-h photoperiod. Insects easily recognized in the same species or individual insects of uncertain identity were then isolated for about 1 week in cages each containing one 6-month-old healthy American elm seedling (grown from seed in growth chamber). Up to 10 morphospecies were found in each collection, with 1 to 20 individuals per morphospecies. The total number of unique morphospecies used in the three transmission trials and later identified as different species was 8. Dead insects collected daily were stored in 80% ethanol and later identified to genus or species level. About 70% insect mortality was recorded, but about 60 individuals from each collection survived the change of diet and environment. After 3 months, individual elm seedlings were tested by RT-PCR (3) for the presence of phytoplasmas using universal primers fU5/rU3 (2). PCR products were visualized on 1.5% agarose gel, and if DNA was amplified, it was cloned and sequenced. Three of 30 seedlings tested positive for phytoplasmas and sequencing of the cloned products (24 clones were sequenced per transformation, per each of the three positive seedlings) confirmed that only Ca. P. ulmi was present in the 3 infected seedlings but not in the remaining 27 or in 46 unexposed control seedlings. The 3 seedlings were each exposed to a single insect and the same insects that were used in the transmission trial were identified. One spittlebug (Cercopidae) Lepyronia quadrangularis Say, one P. spumarius, and one leafhopper in the genus Latalus (Cicadellidae: Deltocephalinae) were identified as vectors. The phytoplasma-positive seedlings showed stunting and yellowing, and died shortly after testing. Other insects captured and identified in the survey were A. atomarius, Neophilaenus lineatus L., Metcalfa pruinosa Say, Amblysellus curtisii Fitch and individuals in the genera Draeculacephala, Elymana, Empoasca, Mesamia, Stroggylocephalus, and Ceratagallia. S. luteolus was not captured during this sampling but was captured on yellow sticky traps and in light traps in previous years at other locations on the campus. This is the first report suggesting that L. quadrangularis and Latalus sp. can serve as natural vectors of EY. References: (1) P. Herath et al. Plant Dis. 94:1355, 2010. (2) H. Lorenz et al. Phytopathology 85:771, 1995. (3) P. Margaria et al. Plant Dis. 91:1496, 2007.

Serial propagation of human endothelial cells in vitro.

Human umbilical vein (HUV) endothelial cells were grown for 15 to 21 passages at a split ratio of 1:5 (at least 27 population doublings) on a human fibronectin (HFN) matrix in Medium 199 supplemented with fetal bovine serum (FBS) and endothelial-cell growth factor (ECGF). This system also permitted the growth of HUV endothelial cells at cell densities as low as 1.25 cells/cm2. In addition to delaying the premature senescence of HUV endothelial cells, ECGF also reduced the serum requirement for low-density HUV endothelial-cell growth; 2.5% serum and ECGF yields half-maximum growth as compared to high serum controls. Significant HUV endothelial-cell growth was also observed in medium supplemented with either ovine hypophysectomized (HYPOX) serum, plasma-derived serum (PDS), or HYPOX-PDS in the presence of ECGF, suggesting that neither the pituitary nor the platelet contributes to HUV endothelial-cell growth.

Bovine fibroblast growth factor: comparison of brain and pituitary preparations.

Bovine brain and pituitary fibroblast growth factors (FGF) have been compared with regard to their chemical and biological properties. Pituitary and one preparation of brain FGF (Prep A) contain a basic mitogenic activity, which migrates to the same position on electrophoresis in acid pH gels as detected by incorporation of [methyl-3H]-thymidine into BALB/c 3T3 cells. In contrast, another preparation of brain FGF (Prep B) contains two mitogens, one (20-30%) indistinguishable from the basic components in pituitary and brain (Prep A) FGF preparations and an acidic activity (70-80%), pl 5-6, that migrates more slowly on acid gels, corresponding to the acidic component of brain FGF described previously (Thomas, K. A., M. C. Riley, S. K. Lemmon, N. C. Baglan, and R. A. Bradshaw. 1980. J. Biol. Chem. 255:5517-5520.) In agreement with that report, none of the mitogens comigrates with fragments of myelin basic protein. Pituitary FGF was virtually inactive, brain (Prep A) FGF had a small amount of activity, and brain (Prep B) FGF was highly potent (50% maximal stimulation at 15-30 ng/ml) in stimulating the growth of human umbilical vein endothelial (HUVE) cells. The acidic component of brain FGF, which is much more unstable at pH 8.5 than the basic one, can be protected by reducing agents, whereas the basic constituent of brain FGF as well as pituitary FGF is unaffected by reducing conditions. Thus, brain FGF preparations may contain two distinct mitogenic activities, one that is acidic and contains HUVE cell activity, and a basic mitogen that is similar to and may be identical with pituitary FGF.

Blood reflux in Schlemm's canal. I. Normal findings.

Blood may be refluxed into Schlemm's canal in the normal eye by occlusion of recipient veins in a sector of the conjunctiva and episclera. The blood makes the anterior margin of the canal distinct near the midportion of the trabecular mesh at the same place in which it is seen after hypotony. Circumferential flow in segments of the canal is possible when a pressure head exists to promote such flow. Evidence was found that the canal is not partially collapsed in the normal eye.

A system for counting endothelial cells from transparencies and negatives.

We have developed a system for endothelial cell density calculation and morphologic examination, which has proven to be convenient and reproducible, by using a currently available viewing unit, a few inexpensive materials, and simple arithmetic.

What the pulmonologist needs to know about allergy.

A majority of children and young adults with asthma are allergic to common allergens in their environment. Over the last 10 years, it has become increasingly clear that chronic exposure to these foreign proteins is an important cause of the chronic eosinophilic inflammation of the lungs that characterizes patients with asthma. The main allergens relevant to asthma are those found indoors, that is, mite, cat, dog, and cockroach. Reducing exposure to these common indoor allergens is an important anti-inflammatory treatment for asthma; furthermore, the techniques of avoidance are increasingly well defined and allergen specific.

Angiogenic activity of the atherosclerotic carotid artery plaque.

Neovascularization is observed in complicated atherosclerotic plaques associated with cellular proliferation, plaque hemorrhage, and thrombosis. The angiogenic activity of 278 plaque fragments was tested; the fragments were taken from 12 patients with cerebral ischemia who underwent carotid endarterectomy. Angiogenesis, determined by the sustained ingrowth of new vessels in the rabbit cornea, was induced in 125 (45%) of these fragments. By contrast, angiogenesis was found in only two (2.4%) of 80 control tissues (p less than 0.001): in none of 22 samples of boiled atherosclerotic plaque; in two of 26 samples of normal rabbit carotid artery; and in none of 32 samples of nonatherosclerotic human uterine artery. Histological evaluation revealed that the cellular zones (composed mainly of smooth-muscle cells) were highly angiogenic, with 97 (76%) of 127 samples showing angiogenesis compared with 23 (17%) of 132 acellular fragments that consisted of amorphic, necrotic, calcific, lipid-laden material (p less than 0.001). These results indicate that angiogenesis in vivo is a function of the cellular component of the advanced atherosclerotic plaque, and is not expressed in the normal, stable arterial wall. The fragile new vessels could promote the growth of the plaque or be a source of hemorrhages, microinfarcts, and plaque fissures that convert a stable, silent lesion to an expanding, ulcerated, thrombotic, symptomatic plaque.

Salivary cotinine levels in children presenting with wheezing to an emergency department.

We set out to evaluate salivary cotinine concentrations to judge tobacco smoke exposure among infants and children, and to examine the results in relation to age and wheezing. This was a case-control study of wheezing children (n = 165) and children without respiratory tract symptoms (n = 106) who were enrolled in the Pediatric Emergency Department at the University of Virginia. The age range of both wheezing and control patients was 2 months to 16 years. Questionnaires were combined with cotinine assays in saliva to evaluate exposure to environmental tobacco smoke (ETS) for each child. The prevalence of exposure to one or more smokers at home was high (68%); and 43% of the children enrolled were exposed to ETS from their mothers. According to the questionnaires, and after adjusting for age and race, a wheezing child in this study was more likely than a control to be exposed to at least one smoker at home (odds ratio = 1.9; 95% CI = 1.1-3.4). However, the odds of exposure to ETS from smoking mothers did not differ significantly between wheezing and control patients, and no significant association was found between the presence of wheezing and salivary cotinine levels. Among children exposed to ETS at home, cotinine levels were significantly higher in saliva from those under the age of two years, and from toddlers aged 2 and 3 years, compared to values from children over age 4 years. Moreover, the number of smokers in the home strongly influenced cotinine levels from children under age 4 years. In addition, higher cotinine levels were observed in saliva from children under age 2 years who were exposed to ETS from their mothers. Cotinine levels were similar and significantly correlated in paired samples of saliva and serum from children under 4 years of age (n = 54), (r = 0.92, P < 0.001). Based on information gathered from questionnaires, the results indicate that wheezing children were more likely than controls to be exposed to ETS at home. However, significant differences in ETS exposure between wheezing and control groups with respect to maternal smoke exposure or comparisons of salivary cotinine levels were not apparent. It was clear that determinations of salivary cotinine for monitoring the prevalence and intensity of household smoke exposure in this study were most valuable during the first 4 years of life.

Avian air sac and plasma proteins that bind surface polysaccharides of Escherichia coli O2.

Some serovars of Escherichia coli, mainly O2 and O78, are responsible for air sac and systemic infections in farm-raised turkeys (Meleagris gallopavo) and chickens (Gallus gallus). We looked in air sac surface fluid from young turkeys to identify proteins that bind surface polysaccharides of pathogenic respiratory E. coli O2. Turkey air sac surface fluid was subjected to affinity chromatography on Toyopearl AF-Epoxy-650M, coupled with either lipopolysaccharide (LPS) or lipid-free polysaccharide (LFP) purified from an avian pathogenic E. coli O2 isolate. A multimeric protein termed lipid-free polysaccharide binding protein-40 (LFPBP-40) composed of six covalently associated subunits of approximately 40 kDa was isolated by elution from LFP by EDTA or L-rhamnose. An analogous protein in air sac fluid proteins bound to intact E. coli O2 and eluted with L-rhamnose or N-acetylglucosamine (GlcNAc). The N-terminal amino acid sequence of LFPBP-40 DINGGGATLPQHLYLTPDV was related to the N-terminus of fragment 3 of a partially characterized human protein possessing T cell stimulation activity in synovial membrane of rheumatoid arthritis patients. However, endogenous amino acid sequences were unrelated to other known proteins. LFPBP-40 was immunoreactively distinct from pulmonary collectins and ficolins. These studies demonstrate a novel avian respiratory soluble lectin that can bind surface polysaccharides of pathogenic E. coli responsible for respiratory disease.

Plasma proteins of rainbow trout (Oncorhynchus mykiss) isolated by binding to lipopolysaccharide from Aeromonas salmonicida.

In an attempt to find plasma proteins that might be involved in the constitutive resistance of rainbow trout to furunculosis, a disease caused by Aeromonas salmonicida (AS), we purified serum and plasma proteins based on their calcium- and carbohydrate-dependent affinity for A. salmonicida lipopolysaccharide (LPS) coupled to an epoxy-activated synthetic matrix (Toyopearl AF Epoxy 650M). A multimeric family of high molecular weight (96 to 200-kDa) LPS-binding proteins exhibiting both calcium and mannose dependent binding was isolated. Upon reduction the multimers collapsed to subunits of approximately 16-kDa as estimated by 1D-PAGE and exhibited pI values of 5.30 and 5.75 as estimated from 2D-PAGE. Their N-terminal sequences were related to rainbow trout ladderlectin (RT-LL), a Sepharose-binding protein. Polyclonal antibodies to the LPS-purified 16-kDa subunits recognized both the reduced 16-kDa subunits and the non-reduced multimeric forms. A calcium- and N-acetylglucosamine (GlcNAc)-dependent LPS-binding multimeric protein (approximately 207-kDa) composed of 34.5-kDa subunits was purified and found to be identical to trout serum amyloid P (SAP) by N-terminal sequence (DLQDLSGKVFV). A protein of 24-kDa, in reduced and non-reduced conditions, was isolated and had N-terminal sequence identity with a known C-reactive protein (CRP) homologue, C-polysaccharide-binding protein 2 (TCBP2) of rainbow trout. A novel calcium-dependent LPS-binding protein was purified and termed rainbow trout lectin 37 (RT-L37). This protein, composed of dimers, tetramers and pentamers of 37 kDa subunits (pI 5.50-6.10) with N-terminal sequence (IQE(D/N)GHAEAPGATTVLNEILR) showed no close homology to proteins known or predicted from cDNA sequences. These findings demonstrate that rainbow trout have several blood proteins with lectin properties for the LPS of A. salmonicida; the biological functions of these proteins in resistance to furunculosis are still unknown.

Treatment of ipsilateral femoral neck and shaft fractures with the Russell-Taylor reconstructive nail.

Twenty-seven ipsilateral femoral neck and shaft fractures were treated with the Russell-Taylor reconstructive nail. Follow-up ranged from 6-48 months (average: 23.6 months). Femoral neck fractures healed within an average of 3.7 months and femoral shaft fractures healed within an average of 4.8 months. Complications included one case of avascular necrosis of the femoral head, a varus healing of one femoral neck fracture, and a rotational malalignment of the femoral shaft in another case. There were no cases of hardware failure. The Russell-Taylor reconstructive nail allows concomitant hip and shaft fractures to be fixed with a single implant.

Aortic intimal response to endothelial removal in cebus and squirrel monkeys.

Whereas squirrel monkeys have an inherent susceptibility to atherosclerosis, cebus monkeys are relatively resistant. To assess whether this difference might lie in their response to endothelial injury, the acute morphologic changes in the aortic intima after endothelial removal were examined in the two species. The endothelium of the lower thoracic aorta was removed with an embolectomy catheter, and the intimal response was compared with the uninjured upper thoracic aorta in each monkey. By 21 days after aortic denudation, regrowth of the endothelium (assessed by in vivo Evans' blue dye staining) was significantly greater in squirrel monkeys (90% +/- 5% of aortic surface) than in cebus (56% +/- 11%). Squirrel monkeys had comparable sudanophilic surface in the nonballooned, control aorta (25% +/- 7%) and the ballooned, lower thoracic segment (17% +/- 6%). Cebus monkey aortas had no sudanophilia in either segment. The intima/media ratios (IMR) in all regions of the aorta were significantly greater in squirrel monkeys than in cebus, but in both species the IMR of the ventral ballooned segment was two to three times the IMR of the nonballooned control segment. In the dorsal aorta, where endothelial regrowth was more rapid, the IMR was similar to the control aortic segment. By electron microscopy the thickened aortic intima in both species contained a marked increase in modified smooth muscle cells, but lipid accumulation did not result from endothelial removal or regrowth in either species. Thus, although the squirrel monkey aorta had atherosclerotic lesions before endothelial removal, the acute intimal response to endothelial injury was similar in degree and kind in both cebus and squirrel monkeys. This suggests that factors other than those controlling the initial intimal thickening following endothelial injury are responsible for the observed difference in arterial lipid accumulation between cebus and squirrel monkeys.

Influence of collagen gel substratum on response to low-density lipoprotein by cultured human skin fibroblasts.

The effect of low-density lipoprotein (LDL) on accumulation of glycosaminoglycans (GAG) was compared in cultures of human skin fibroblasts on a conventional plastic substratum and in a native type I collagen gel. The 24-h incorporation of [3H]glucosamine and Na2(35)SO4 into GAG secreted into the medium or associated with the substratum and cell surface (SCA) was measured in cells at subconfluent densities. When cells were grown on plastic, 13-25% of the labeled GAG was in the SCA pool. Cells cultured within a collagen gel matrix incorporated three times more [3H]glucosamine and up to five times more [35S]sulfate into this pool. The addition of LDL (300 micrograms protein/mL) to the medium increased the level of total GAG incorporation of [3H]glucosamine by 40-50% and of [35S]sulfate by 15-20% on both substrata. For cells on plastic the relative increase in the medium and SCA pool was similar, whereas for cells in collagen gel the response to LDL was twice as great in the SCA pool as in the medium. The distribution of GAG types was unaffected by LDL; hyaluronic acid remained the principal GAG in the media pools of both substrata, heparan sulfate remained the main SCA GAG in cultures on plastic, and dermatan sulfate remained the dominant GAG in the SCA pool of collagen gel cultures. LDL degradation was measured at intervals up to 48 h after the addition of 125I-labeled LDL. The rate of accumulation of degraded LDL products was lower in collagen gel cultures, but the final levels achieved were the same in the two substrata. Concentrations of total cell cholesterol were similar, although the increases in free cholesterol induced by LDL were 26% greater in cells within collagen gel than in those on plastic. We conclude that fibroblasts grown within a collagen gel, as compared with those on a plastic substratum, (i) accumulate more GAG that remain attached to the substratum and cell surface; (ii) respond to LDL with a similar degree of increase in GAG accumulation, but more of the increase is found in the substratum and cell surface compartment; and (iii) accumulate more intracellular free cholesterol in response to LDL.

Interaction of native and cell-modified low density lipoprotein with collagen gel.

We have examined the binding of native and cell-modified low density lipoprotein (LDL) to gels of Type I collagen. Diffusion of native 125I-LDL into the collagen gel was slow, reaching equilibrium after 24 to 48 hours, while L-3H-glucose, a low molecular weight marker, equilibrated in 6 hours. Binding of 125I-LDL was measured at 48 hours as the amount associated with the collagen after extensive washing. Binding was saturable with an increasing concentration of LDL. Prior incubation with cell-free culture medium resulted in modest, but progressive, increases in electrophoretic mobility and binding to collagen. Incubation with cells produced a marked increase in electrophoretic mobility and a 5- to 10-fold increase in collagen binding; the presence of butylated hydroxytoluene during incubation prevented both effects. These changes in LDL were induced by porcine aortic endothelial cells, smooth muscle cells, human skin fibroblasts, and a variety of cell lines, as well as by acetylation. There was a curvilinear relationship between the amount of LDL protein bound and the net negative charge of the LDL; increasing net charge was associated with progressively greater increases in binding. These results suggest a potential role for collagen in trapping lipid in the extracellular matrix of arterial intima by slowing the diffusion of and by binding LDL. The data also demonstrate that binding of LDL to collagen is enhanced by modifications that increase its net negative charge.

Effects of porcine aortic smooth muscle cell conditioned medium on endothelial cell replication.

Previous studies suggested that arterial smooth muscle cells (SMC) may be involved in regulating the growth of capillaries into atherosclerotic plaques. In the present study, we determined the effect of SMC products on porcine aortic endothelial cell (EC) replication in vitro. Quiescent or slowly growing EC in medium without endothelial cell growth factor (ECGF) were stimulated to proliferate in the presence of porcine aortic SMC conditioned medium, while the same conditioned medium inhibited the growth of rapidly dividing EC in high serum concentrations or with ECGF. The magnitude of both activities depended on SMC conditioned medium concentration. The dose-dependent increase in EC number stimulated by ECGF was completely inhibited by SMC conditioned medium. This effect was not due to a direct interaction of conditioned medium with ECGF because SMC conditioned medium inhibited the growth of EC that were rapidly proliferating in 10% serum without ECGF. The inhibitory activity was retained by an ultrafiltration membrane with an exclusion limit of 1000 daltons; the stimulatory activity was recovered in the ultrafiltrate and remained stable after boiling, treatment with acid or base and trypsin, and repeated freezing and thawing, but was removed by activated charcoal. The growth-promoting activity could not be accounted for by release of cell contents from lysed cells or of thymidine into the medium. Conditioned medium from SMC incubated in the presence of serum contained less EC growth-stimulatory activity but more growth-inhibitory activity than that from SMC in serum-free medium.

Infrared absorption by overlapping bands of atmospheric gases.

The spectral transmission of carbon monoxide, nitrous oxide, and mixtures of the two has been studied in the 2200-cm(-1) region, where overlapping absorption bands occur. With spectral slit widths sufficiently large to include several absorption lines, it was found that the observed spectral transmittance of a mixture is equal to the product of the transmittances of the components measured separately, provided that sufficient nitrogen is added to give the same total pressure for all samples. This result was also obtained for overlapping bands of nitrous oxide and methane in the 1300-cm(-1) region. The present work confirms Burch's earlier studies of overlapping bands of CO(2) and water vapor. An investigation of the possible breakdown of the multiplicative property of transmission for narrow spectral slit widths was inconclusive.