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.

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.

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.

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.

Rhinovirus and respiratory syncytial virus in wheezing children requiring emergency care. IgE and eosinophil analyses.

This cross-sectional emergency department study of 70 wheezing children and 59 control subjects (2 mo to 16 yr of age) examined the prevalence of respiratory viruses and their relationship to age, atopic status, and eosinophil markers. Nasal washes were cultured for respiratory viruses, assayed for respiratory syncytial virus (RSV) antigen, and tested for coronavirus and rhinovirus RNA using reverse transcription-PCR (RT-PCR). Also evaluated were eosinophil numbers and eosinophil cationic protein (ECP) in both nasal washes and serum, along with total IgE and specific IgE antibody in serum. Respiratory viruses were detected in 82% (18 of 22) of wheezing infants younger than 2 yr of age and in 83% (40 of 48) of older wheezing children. The predominant pathogens were RSV in infants (detected in 68% of wheezing subjects) and rhinovirus in older wheezing children (71%), and both were strongly associated with wheezing (p < 0.005). RSV was largely limited to wheezing children younger than 24 mo of age, but rhinovirus was detected by RT-PCR in 41% of all infants and in 35% of nonwheezing control subjects older than 2 yr of age. After 2 yr of age the strongest odds for wheezing were observed among those who had a positive RT-PCR test for rhinovirus together with a positive serum radioallergosorbent testing (RAST), nasal eosinophilia, or elevated nasal ECP (odds ratios = 17, 21, and 25, respectively). Results from this study demonstrate that a large majority of emergent wheezing illnesses during childhood (2 to 16 yr of age) can be linked to infection with rhinovirus, and that these wheezing attacks are most likely in those who have rhinovirus together with evidence of atopy or eosinophilic airway inflammation.

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.

Chronic sinusitis: risk factors for extensive disease.

BACKGROUND: Chronic sinus disease is one of the most common diseases in the United States, and little is understood about its pathogenesis. OBJECTIVE: To characterize prospectively the immunologic parameters that accompany extensive chronic sinusitis. METHODS: Eighty adult patients with chronic sinus symptoms had a complete blood count with differential, coronal computed tomographic (CT) scan of the sinuses, and serum assays for total IgE, specific IgE, IgA, IgG, IgG subclasses, and pneumococcal titers. RESULTS: Thirty-seven (46%) patients had extensive sinusitis, defined as a CT score > or = 12. A highly significant correlation was noted between the extent of disease and the peripheral eosinophil count (r = 0.53, p < 0.0001). In keeping with this finding, an eosinophil count > or = 200/microl was strongly associated with extensive disease (odds ratio [OR] = 19.2, 95% confidence interval [CI] = 5.4 to 72.7). Asthma (OR = 6.8, 95% CI = 2.2 to 22.0), atopy (OR = 4.3, 95% CI, 1.5 to 12.8), and age > or = 50 years (OR = 6.5, 95% CI = 2.0 to 22.2) were also associated with extensive disease. However, the association of eosinophils with extent of disease was independent of asthma, atopy, or age. Levels of IgG1, IgG2, and IgG3 subclasses did not correlate with extent of disease seen on CT scan. Although total IgE and IgG4 levels did correlate with disease, on multiple stepwise regression they did not add to the predictive value of the eosinophil count in identifying patients with extensive disease. CONCLUSIONS: The association of asthma, atopy, eosinophilia, and elevated levels of IgE and IgG4 with extensive disease on CT scan is compatible with the hypothesis that chronic sinusitis may be a disease of immune activation of the T(H2) type.

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.

Eosinophil cationic protein in serum and nasal washes from wheezing infants and children.

OBJECTIVE: To compare eosinophil counts and concentrations of eosinophil cationic protein (ECP) in serum and nasal wash fluid from wheezing infants and children with those from age-matched children without respiratory tract symptoms. DESIGN: A case-control study of 71 children treated for wheezing and 59 control subjects in the University of Virginia Pediatric Emergency Department. The patients ranged from 2 months to 16 years of age. Eosinophil numbers and ECP concentrations were assessed in serum and nasal washes. Total serum IgE was measured and the radioallergosorbent test was used to measure IgE antibody to common inhalant allergens. RESULTS: Among children less than the age of 2 years, markedly elevated levels of ECP (> 200 ng/ml) were measured in nasal washes from 9 (41%) of 22 wheezing patients and 1 (6%) of 17 control subjects (p < 0.03). None of these children had a positive radioallergosorbent test result for IgE antibody to common aeroallergens or a nasal smear containing 10% eosinophils. Few of the wheezing children under 2 years of age had either increased concentrations of total IgE or ECP in their serum or an elevated total blood eosinophil count. After the age of 2 years, the percentage of patients with nasal ECP levels greater than 200 ng/ml was also significantly higher in wheezing children than in control subjects (p < 0.001), and a positive correlation was observed between ECP concentrations in their nasal washes and other eosinophil responses (total blood eosinophil counts, serum ECP levels, and nasal eosinophil counts). CONCLUSION: Increased concentrations of ECP were detected in nasal washes from wheezing infants and children, indicating that eosinophils may contribute to the pathogenesis of airway inflammation in some children who wheeze early in life.

Roles of individual kringle domains in the functioning of positive and negative effectors of human plasminogen activation.

In order to identify the individual contributions of the kringle (K) domains of human plasminogen (Pg) to the epsilon-aminocaproic acid (EACA) induced stimulation of Pg activation by low-molecular-weight urokinase-type plasminogen activator (LMW-uPA) and inhibition of this same activation by Cl-, we constructed the most conservative recombinant- (r-) Pg mutants possible that would greatly reduce the strength of the EACA binding site in the omega-amino acid binding kringles, [K1Pg] ([D139-->N]r-Pg), [K4Pg] ([D413-->N]r-Pg), and [K5Pg] ([D515--N]r-Pg). In each case, this involved mutation of a critical Asp (to Asn) within these three kringle domains in intact Pg. The three r-mutants were expressed in r-baculovirus-infected lepidopteran insect (Trichoplusia ni) cells. In the presence of Cl-, the positive activation effector, EACA, first stimulated and then inhibited the LMW-uPA-catalyzed initial activation of wild-type (wt) r-[Glu1]Pg and, to a lesser extent, the [K5Pg] mutant, [D518-->N/Glu1]r-Pg. The concentration of EACA that produced 50% stimulation of activation (C50) occurred at 3.3 mM for wtr-[Glu1]Pg and at 0.7 mM for [D518-->N/Glu1]r-Pg. Subsequent inhibition by EACA occurred with a C50 of approximately 15 mM and is likely due to inhibition of the amidolytic activity of plasmin generated during the activation. Similar initial activation rates of both [D139-->N]r-Pg and [D413N]r-Pg did not display this initial EACA-mediated stimulatory phase but did undergo ultimate inhibition with a C50 for this process that was similar to wtr-[Glu1]Pg and [D518-->N/Glu1]r-Pg.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of glycosphingolipids by fluorophore-assisted carbohydrate electrophoresis using ceramide glycanase from Mercenaria mercenaria.

Polyacrylamide gel electrophoresis of fluorophore-labeled saccharides is a simple and sensitive separation method for glycan analysis. This method requires an aldehydic reducing carbon on the saccharide in order to react with the amine group of the fluorophore (8-aminonaphthalene-1,3,6-trisulfonic acid or 2-aminoacridone). We have used exoglycosidase-free ceramide glycanase from hard-shelled clam to expose the reducing terminal of the glycan in glycosphingolipid by cleaving the linkage between the glycan and the ceramide. The released glycan was used for fluorophore-assisted carbohydrate electrophoresis, in order to qualitatively determine its monosaccharide composition, chain length, and glycosidic linkages using linkage-specific glycosidases, including a beta 1-3,4 galactosidase from clam.

Amino acids of the recombinant kringle 1 domain of human plasminogen that stabilize its interaction with omega-amino acids.

A series of strategically designed recombinant (r) mutants of the kringle 1 region of human plasminogen ([K1HPg]) have been constructed and the resulting gene products employed to reveal the identities of the residues that contribute to stabilization of the binding of omega-amino acid ligands to this domain. On the basis of determinations of the binding constants of the ligands, 6-aminohexanoic acid and trans-4-(aminomethyl)cyclohexane-1-carboxylic acid, to a variety of these mutants, we find that the anionic site of the polypeptide responsible for stabilization of the amino group of the ligands consists of both D54 and D56 and the cationic site of the polypeptide that interacts with the carboxylate group of the ligand is composed solely of R70. The main hydrophobic interactions that stabilize binding of these ligands, likely by interactions with the ligand hydrophobic regions, are principally due to W61, Y63, and Y71. The results obtained are consistent with conclusions that could be made from analysis of the X-ray crystal structure of r-[K1HPg] and from previous studies from this laboratory regarding the binding of ligands of this type to the kringle 2 region of tissue-type plasminogen activator ([K2tPA]). It thus appears as though a common ligand binding site has evolved in different kringles with ligand specificity differences between r-[K2tPA] and r-[K1HPg] perhaps explainable by the different nature of the cationic sites on these polypeptides that are involved in coordination to the ligand carboxylate groups.

Initial experience with a second generation locking femoral nail: the Russell-Taylor reconstruction nail.

A series of 36 patients with 20 subtrochanteric fractures, 12 ipsilateral neck/shaft fractures, and five intertrochanteric fractures with shaft extension underwent closed intramedullary nailing with the Russell-Taylor reconstruction (RECON) nail. The average Injury Severity Score was 16, and seven of the fractures were open. All fractures were acute injuries, and all but one were treated within 24 hours of admission. Follow-up was obtained at three, six, nine, 12, and 24 months or until the fracture healed. The range of follow-up was one to three years. Complete follow-up was obtained in 33 of 36 patients. Union was achieved in all acute fractures. Shortening occurred in two cases and chondrolysis and avascular necrosis occurred in another patient. Excellent hip and knee range of motion were obtained except in a few cases of ipsilateral limb injuries. While many complex femoral shaft fractures can be treated successfully with first generation locking nails, this study demonstrates that second generation locking nails, such as the RECON nail, offer the added strength and design features necessary for more effective treatment of complex proximal and ipsilateral femoral neck/shaft fractures.

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.

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.

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.

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.

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.