Biomedical subjects
Publications and source records attributed to J D Smith.
Malformations of the tongue are rare. There are < 50 cases of tongue hypoplasia reported in the literature since the first case described by de Jussieu in 1718. These are usually associated with the hypoglossia-hypodactaly syndrome. We describe a newborn with a hypoplastic anterior tongue unfused to the posterior tongue and airway obstruction. Its embryology, theories of pathogenesis, treatment and clinical course are discussed.
Using the rat balloon catheter denudation model, we examined the role of transforming growth factor-beta (TGF-beta) isoforms in vascular repair processes. By en face in situ hybridization, proliferating and quiescent smooth muscle cells in denuded vessels expressed high levels of mRNA for TGF-beta1, TGF-beta2, TGF-beta3, and lower levels of TGF-beta receptor II (TGF-betaRII) mRNA. Compared with normal endothelium, TGF-beta1 and TGF-beta2, as well as TGF-betaRII, mRNA were upregulated in endothelium at the wound edge. Injected recombinant soluble TGF-betaRII (TGF-betaR:Fc) localized preferentially to the adventitia and developing neointima in the injured carotid artery, causing a reduction in intimal lesion formation (up to 65%) and an increase in lumen area (up to 88%). The gain in lumen area was largely due to inhibition of negative remodeling, which coincided with reduced adventitial fibrosis and collagen deposition. Four days after injury, TGF-betaR:Fc treatment almost completely inhibited the induction of smooth muscle alpha-actin expression in adventitial cells. In the vessel wall, TGF-betaR:Fc caused a marked reduction in mRNA levels for collagens type I and III. TGF-betaR:Fc had no effect on endothelial proliferation as determined by reendothelialization of the denuded rat aorta. Together, these findings identify the TGF-beta isoforms as major factors mediating adventitial fibrosis and negative remodeling after vascular injury, a major cause of restenosis after angioplasty.
The epsilon 4 allele of apolipoprotein E (APOE) has been found to be a risk factor for late-onset Alzheimer's disease (AD). While the pathogenic mechanism of APOE in AD is not yet clear, APOE isoforms appear to differentially influence the aggregation of A beta, the principal component of Alzheimer-associated beta-amyloid deposits. To date, no data are available for the propensity of A beta to aggregate in the presence of APOE under conditions where these components are at physiological concentrations (in cerebrospinal fluid, APOE and A beta are approximately 100 nM and approximately 5 nM, respectively). We employed a novel in vitro filtration assay for detecting zinc(II)- and copper(II)-induced aggregation of A beta in solutions containing concentrations of the peptide that are similar to those reported for human cerebrospinal fluid. The potential for resolubilization with EDTA and the relative densities of zinc- and copper-induced A beta aggregates were also compared. Zinc-induced A beta aggregates were found to be denser and less easily resolubilized than copper-induced precipitates. Metal-induced aggregation of A beta was studied in the presence of purified apolipoprotein E2, apolipoprotein E3, and apolipoprotein E4 under conditions that approximate the physiological concentrations and ratios of these proteins. In the presence of all three APOE isoforms, zinc-induced aggregation of A beta was attenuated, while precipitation with copper was enhanced. Consistent with the increased risk for AD associated with the epsilon 4 allele of APOE, metal-induced aggregation of A beta was highest for both zinc and copper in the presence of apolipoprotein E4. Our data are consistent with a role for APOE as an in vivo molecular chaperone for A beta.
White clover (Trifolium repens) was germinated and grown in microgravity aboard the Space Shuttle (STS-60, 1994; STS-63, 1995), on Earth in stationary racks and in a slow-rotating two-axis clinostat. The objective of this study was to determine if normal root cap development and early plant gravity responses were dependent on gravitational cues. Seedlings were germinated in space and chemically fixed in orbit after 21, 40, and 72 h. Seedlings 96 h old were returned viable to earth. Germination and total seedling length were not dependent on gravity treatment. In space-flown seedlings, the number of cell stories in the root cap and the geometry of central columella cells did not differ from those of the Earth-grown seedlings. The root cap structure of clinorotated plants appeared similar to that of seedlings from microgravity, with the exception of three-day rotated plants, which displayed significant cellular damage in the columella region. Nuclear polarity did not depend on gravity; however, the positions of amyloplasts in the central columella cells were dependent on both the gravity treatment and the age of the seedlings. Seedlings from space, returned viable to earth, responded to horizontal stimulation as did 1 g controls, but seedlings rotated on the clinostat for the same duration had a reduced curvature response. This study demonstrates that initial root cap development is insensitive to either chronic clinorotation or microgravity. Soon after differentiation, however, clinorotation leads to loss of normal root cap structure and plant graviresponse while microgravity does not.
The epsilon4 allele of apolipoprotein E gene is a major risk factor for Alzheimer's disease. However, the mechanism by which the E4 isoform of apolipoprotein E increases the risk of Alzheimer's disease is poorly understood. To determine whether the isoform-specific effects of apolipoprotein E may be mediated via clearance of bound beta-amyloid, we examined the uptake of beta-amyloid 1-40 into Chinese hamster ovary cells in the presence or absence of the apolipoprotein E isoforms E2, E3 and E4. Apolipoprotein E2 and E3 treatments were associated with higher association of beta-amyloid with cells as compared to treatment with E4. Heparin blocked the association of beta-amyloid with cells, as did an antibody to one of the apolipoprotein E receptors (the low-density lipoprotein receptor-related protein). Thus, the apolipoproteins E2 and E3, but not E4, may play important roles in the clearance of beta-amyloid from the extracellular space via the low-density lipoprotein receptor-related protein.
BACKGROUND: Aptamers are single-stranded oligonucleotides derived from an in vitro evolution protocol called systematic evolution of ligands by exponential enrichment (SELEX). They bind tightly and specifically to target molecules; most aptamers to proteins bind with Kds (equilibrium dissociation constant) in the range of 1 pM to 1 nM. METHODS AND RESULTS: The SELEX protocol has been automated; therefore, hundreds to thousands of aptamers can be made in an economically feasible fashion. Blood and urine can be analyzed on chips that capture and quantitate proteins. SELEX has been adapted to the use of 5-bromo (5-Br) and 5-iodo (5-I) deoxyuridine residues. These halogenated bases can be specifically cross-linked to proteins. Selection pressure during in vitro evolution can be applied for both binding specificity and specific photo-cross-linkability. These are sufficiently independent parameters to allow one reagent, a photo-cross-linkable aptamer, to substitute for two reagents, the capture antibody and the detection antibody, in a typical sandwich array. After a cycle of binding, washing, cross-linking, and detergent washing, proteins will be specifically and covalently linked to their cognate aptamers. CONCLUSIONS: Because no other proteins are present on the chips, protein-specific stain will now show a meaningful array of pixels on the chip. Learning algorithms and retrospective studies should lead to a robust, simple, diagnostic chip.
Degloving injuries range from the occult, easily missed injury to obvious massive tissue damage. The serious nature of these wounds is exacerbated by mismanagement. It is generally accepted that the degloved tissue should be excised, defatted, fenestrated, and reapplied as a full-thickness skin graft. Dressings are required that provide gentle, evenly distributed pressure and avoid shear stress to the newly grafted skin. Numerous types of dressings have been devised but all are cumbersome and time-consuming. We have found the Vacuum-Assisted Closure device to be a rapid, effective, and easy-to-use alternative to traditional methods. The authors examine their experience using a vacuum-assisted closure device to treat nine degloving injuries in 5 patients and discuss the important aspects in using this technique.
Giant congenital pigmented nevi pose a substantial reconstructive challenge for the treating physician. Due to the increased risk of malignant transformation in such lesions, complete excision with tissue expansion or skin grafting is the generally accepted treatment. These modalities can, however, leave the patient with secondary deformities that also require complex reconstructive procedures. The following case details a patient requiring secondary reconstruction with large-volume tissue expansion 12 years after excision of a giant nevus, and split-thickness skin grafting. This patient illustrates a severe secondary deformity and the usefulness of large-volume serial expansion in such patients.
Two strains of ApoE-deficient mice were found to have markedly different plasma lipoprotein profiles and susceptibility to atherosclerosis when fed either a low-fat chow or a high-fat Western-type diet. FVB/NJ ApoE-deficient (FVB E0) mice had higher total cholesterol, HDL cholesterol, ApoA1, and ApoA2 levels when compared with C57BL/6J ApoE-deficient (C57 E0) mice. At 16 weeks of age, mean aortic root atherosclerotic lesion area was 7- to 9-fold higher in chow diet-fed C57 E0 mice and 3.5-fold higher in Western diet-fed C57 E0 mice compared with FVB E0 mice fed similar diets. Lesion area in chow diet-fed first-generation mice from a strain intercross was intermediate in size compared with parental values. The distribution of the lesion area in 150 chow diet-fed second-generation progeny spanned the range of the lesion area in both parental strains. There were no correlations between total cholesterol, non-HDL cholesterol, HDL cholesterol, ApoA1, ApoA2, ApoJ, or anti-cardiolipin antibodies and lesion area in the second-generation progeny. Thus, a genomic approach may succeed in identifying the genes responsible for the variation in atherosclerosis susceptibility in these 2 strains of ApoE-deficient mice, which could not be explained by measured plasma parameters.
We have previously shown that expression of the human apo A-I transgene on the apo E-deficient background increases HDL cholesterol and greatly diminishes fatty streak lesion formation. To examine the mechanism, prelesional events in atherosclerotic plaque development were examined in 6- to 8-week-old apo E-deficient and apo E-deficient/human apo A-I transgenic mice. A quantitative assessment of subendothelial lipid deposition by freeze-fracture and deep-etch electron microscopy indicated that elevated apo A-I did not affect the distribution or amount of aortic arch subendothelial lipid deposits. Immunohistochemical staining for VCAM-1 demonstrated similar expression on endothelial cells at prelesional aortic branch sites from both apo E-deficient and apo E-deficient/human apo A-I transgenic mice. Transmission electron microscopy revealed monocytes bound to the aortic arch in mice of both genotypes, and immunohistochemical staining demonstrated that the area occupied by bound mononuclear cells was unchanged. Serum paraoxonase and aryl esterase activity did not differ between apo E-deficient and apo E-deficient/human apo A-I transgenic mice. These data suggest that increases in apo A-I and HDL cholesterol inhibit foam cell formation in apo E-deficient/human apo A-I transgenic mice at a stage following lipid deposition, endothelial activation, and monocyte adherence, without increases in HDL-associated paraoxonase.
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Systemic mast cell disease (SMCD) has a highly variable clinical expression and course. That SMCD is associated with hematologic disorders has been widely described. We report an unusual case of systemic mast cell disease and concurrent chronic myelomonocytic leukemia in a 60 year old male.
OBJECTIVE: To identify clinical variables that indicate postoperative pain in cats after ovariohysterectomy in a veterinary hospital setting. ANIMALS: 40 cats. PROCEDURE: Cats were anesthetized and ovariohysterectomized by senior veterinary students. Butorphanol (0.1 mg/kg [n = 20] or 0.3 mg/kg [20] of body weight) was administered IM after surgery. Blood samples were obtained before, during, and after the anesthetic period for measurements of PCV and blood glucose and cortisol concentrations. Clinical variables measured included heart rate, systolic blood pressure, respiratory rate, and rectal temperature. Data for these variables were compared with changes in cortisol concentrations and with similar data-which was used as historical control data-obtained from 20 cats in another study (10 that had been ovariohysterectomized but had not received butorphanol and 10 that had only been anesthetized). RESULTS: Surgical durations were longer in this study, and cats had higher cortisol concentrations, compared with historical control cats. Objective clinical variables did not consistently correlate with changes in cortisol concentration. CONCLUSIONS: Cortisol concentration increased in response to surgical stress and pain. This response was greater in cats in which duration of surgery was longer. CLINICAL RELEVANCE: The objective clinical variables evaluated in this study were not consistent indicators of pain in an uncontrolled, clinical situation.
The metabolic and genetic determinants of HDL cholesterol (HDL-C) levels and HDL turnover were studied in 36 normolipidemic female subjects on a whole-food low-fat metabolic diet. Lipid, lipoprotein, and apolipoprotein levels, lipoprotein size, and apolipoprotein turnover parameters were determined, as were genetic variation at one site in the hepatic lipase promoter and six sites in the apolipoprotein AI/CIII/AIV gene cluster. Menopause had no significant effect on HDL-C or turnover. Stepwise multiple regression analysis revealed that HDL-C was most strongly correlated with HDL size, apolipoprotein A-II (apoA-II), and apolipoprotein A-I (apoA-I) levels, which together could account for 90% of the variation in HDL-C. HDL size was inversely correlated with triglycerides, body mass index, and hepatic lipase activity, which together accounted for 82% of the variation in HDL size. The hepatic lipase promoter genotype had a strong effect on hepatic lipase activity and could account for 38% of the variation in hepatic lipase activity. The apoA-I transport rate (AI-TR) was the major determinant of apoA-I levels, but AI-TR was not associated with six common genetic polymorphism in the apoAI/CIII/AIV gene cluster.A simplified model of HDL metabolism is proposed, in which A-I and apoA-II levels combined with triglycerides, and hepatic lipase activity could account for 80% of the variation in HDL-C.
Endoglin (CD105) is a cell surface component of the transforming growth factor-beta (TGF-beta) receptor complex highly expressed by endothelial cells. Mutations in the endoglin gene are responsible for the hereditary hemorrhagic telangiectasia type 1 (HHT1), also known as Osler-Weber-Rendu syndrome (OMIM 187300). This is an autosomal dominant vascular disorder probably caused by a haploinsufficiency mechanism displaying low levels of the normal protein. To understand the mechanisms underlying the regulated expression of endoglin, a genomic DNA clone containing 3.3 kb of the 5'-flanking sequence of the human endoglin gene has been isolated. The 5'-flanking region of the endoglin gene lacks consensus TATA and CAAT boxes, but contains two GC-rich regions and consensus motifs for Sp1, ets, GATA, AP-2, NFkappaB, and Mad, as well as TGF-beta-, glucocorticoid-, vitamin D-, and estrogen-responsive elements. As determined by primer extension and 5' RACE experiments, a cluster of transcriptional start sites was found to be located 350 bp upstream from the translation initiation codon. To analyze the endoglin promoter activity, the upstream -400/+341 fragment was fused to the luciferase gene and transient transfections were conducted in several cell types. This construct displayed a tissue-specific activity in human and bovine endothelial cells. Analysis of various deletion constructs showed the existence of a basal promoter region within the -81/+350 fragment as well as major transcriptional regulatory elements within the -400/-141 fragment. Electrophoretic mobility shift assays demonstrated the specific interaction of a member of the ets family with a consensus motif located at position -68. A promoter construct mutated at this ets sequence showed a much reduced activity as compared with the wild-type construct, supporting the involvement of this ets motif in the basal activity of the promoter. The endoglin promoter exhibited inducibility in the presence of TGF-beta1, suggesting possible therapeutic treatments in HHT1 patients, in which the expression level of the normal endoglin allele might not reach the threshold required for its function. Isolation and characterization of the human endoglin promoter represents an initial step in elucidating the controlled expression of the endoglin gene.
The A4VAR is a variant antigen expressed by a clonal line that binds CD36 and intercellular adhesion molecule-1, ICAM-1. We have cloned and sequenced the extracellular domain coded by the A4var gene. To probe the relationship between A4var expression and parasite adhesion to ICAM-1, var mRNA and protein expression were analyzed in an enriched population of A4 parasites that displayed higher ICAM-1 binding. By Northern analyses, A4var was the predominant var message and antisera raised against a recombinant A4VAR protein reacted with the majority of infected erythrocytes, reinforcing previous conclusions that A4VAR binds ICAM-1. A4VAR contains five Duffy-binding like (DBL) domains, and two cysteine-rich interdomain regions (CIDR) domains. DBL and CIDR domains from A4VAR were expressed in mammalian cells to determine which regions mediate binding to CD36 and ICAM-1. Using several different binding assays, the A4VAR CIDR1 was the only domain found to bind CD36. In contrast, the same assays were unable to identify the ICAM-1 binding domain in A4VAR. This is the first time that each of the DBL and CIDR domains from a Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) have been systematically expressed and tested for binding. These results confirm that CIDR1 is sufficient to bind CD36 without any apparent contribution from other domains.
BACKGROUND: It has been suggested that antibodies which are associated with chronic pathological conditions such as chronic rejection and autoimmune diseases have the capacity to activate endothelial cells by induction and up-regulation of adhesion molecules. It has also been suggested that HLA antibodies formed by patients awaiting transplantation can activate endothelial cells. These antibodies include HLA and those that bind to endothelial cells. METHODS: We have further investigated this phenomenon using monoclonal antibodies against HLA class I determinants and sera from aortic valve graft recipients, containing strong HLA antibodies. The effect of 24-hr incubation of antibodies/serum with human umbilical vein endothelial cells (HUVECs) on adhesion molecule expression was measured by flow cytometry. RESULTS: HLA monoclonal antibodies had no effect on ICAM-1 expression on HUVECs. Five of 31 (16%) patients' sera caused strong up-regulation of adhesion molecules (ICAM-1, vascular cell adhesion molecule-1, and E-selectin) but this did not correlate with HLA specificity, IgG, or IgM binding to HUVECs. The activity, found in whole serum and IgG-depleted fractions was inhibited by neutralizing antibodies against interleukin (IL)-1beta and tumor necrosis factor-alpha. Examination of patient sera for presence of IL-1beta demonstrated high levels of IL-1beta in all five sera (range, 30 -500 U/ml) as well as in samples from an additional three patients. CONCLUSION: The ability to activate endothelial cells detected in our patient sera was caused by cytokines and not antibody. Our observation that addition of cytokines to sera before separation into large and low molecular weight fractions demonstrated retention of cytokines in both fractions may be a confounding issue when investigating endothelial cell activation by patients' sera.
The regulation of dietary cholesterol absorption was examined in C57BL/6 and transgenic mice with liver overexpression of the scavenger receptor BI (SR-BI Tg). In C57BL/6 animals, feeding 0.02 to 1% (wt/wt) dietary cholesterol resulted in a dose-dependent decrease in the percentage of dietary cholesterol absorbed. A plot of total daily mass of dietary cholesterol absorbed versus the percentage by weight of cholesterol in the diet yielded a curve suggesting a saturable process with a Km of 0.4% (wt/wt) and a Vmax of 0.65 mg cholesterol/g body weight per day. Dietary cholesterol suppressed hepatic 3-hydroxy-3-methylglutaryl CoA reductase activity, stimulated cholesterol 7alpha-hydroxylase activity, and enhanced fecal excretion of bile acids, but none of these changes correlated with the percentage of dietary cholesterol absorption. Dietary cholesterol also caused an increase in biliary cholesterol concentration, and in this case the concentration of biliary cholesterol was strongly and inversely correlated with the percentage dietary cholesterol absorption (r = -0.63, P < 0.0001). Biliary cholesterol concentration was also directly correlated with daily cholesterol intake, dietary cholesterol mass absorption, and liver cholesterol ester content. Transgene-induced overexpression of SR-BI resulted in a stimulation of excretion of cholesterol into the bile and suppressed percentage dietary cholesterol absorption. Furthermore, biliary cholesterol levels in SR-BI Tg mice were strongly and inversely correlated with the percentage of dietary cholesterol absorbed (r = -0.99, P < 0.0008). In summary, these results suggest that the excretion of cholesterol into the bile plays an important role in regulating the percentage absorption of dietary cholesterol.