Emeritus Faculty, Acad Council, Pathology
Methods of evaluation of biopsies; differentiation and, classification of neoplasms; immunohistochemistry; scanning and, transmission electron microscopy; cardiovascular pathology;, histologic assessment of bone biopsies; heart preservation and, restoration; myocardial biopsies; venous autograft coronary artery, bypass; cardial and heart valve transplantation.
Chronic rejection, manifested as small airway fibrosis (obliterative bronchiolitis [OB]), is the main obstacle to long-term survival in lung transplantation. Recent studies demonstrate that the airways involved in a lung transplant are relatively hypoxic at baseline and that OB pathogenesis may be linked to ischemia induced by a transient loss of airway microvasculature. Here, we show that HIF-1α mediates airway microvascular repair in a model of orthotopic tracheal transplantation. Grafts with a conditional knockout of Hif1a demonstrated diminished recruitment of recipient-derived Tie2⁺ angiogenic cells to the allograft, impaired repair of damaged microvasculature, accelerated loss of microvascular perfusion, and hastened denudation of epithelial cells. In contrast, graft HIF-1α overexpression induced via an adenoviral vector prolonged airway microvascular perfusion, preserved epithelial integrity, extended the time window for the graft to be rescued from chronic rejection, and attenuated airway fibrotic remodeling. HIF-1α overexpression induced the expression of proangiogenic factors such as Sdf1, Plgf, and Vegf, and promoted the recruitment of vasoreparative Tie2⁺ cells. This study demonstrates that a therapy that enhances vascular integrity during acute rejection may promote graft health and prevent chronic rejection.
View details for DOI 10.1172/JCI46192
View details for Web of Science ID 000291234300032
View details for PubMedID 21606594
View details for PubMedCentralID PMC3104770
View details for DOI 10.1155/2011/562516
The specific mechanisms underlying Taenia solium oncosphere adherence and penetration in the host have not been studied previously. We developed an in vitro adhesion model assay to evaluate the mechanisms of T. solium oncosphere adherence to the host cells. The following substrates were used: porcine intestinal mucosal scrapings (PIMS), porcine small intestinal mucosal explants (PSIME), Chinese hamster ovary cells (CHO cells), epithelial cells from ileocecal colorectal adenocarcinoma (HCT-8 cells), and epithelial cells from colorectal adenocarcinoma (Caco-2 cells). CHO cells were used to compare oncosphere adherence to fixed and viable cells, to determine the optimum time of oncosphere incubation, to determine the role of sera and monolayer cell maturation, and to determine the effect of temperature on oncosphere adherence. Light microscopy, scanning microscopy, and transmission microscopy were used to observe morphological characteristics of adhered oncospheres. This study showed in vitro adherence of activated T. solium oncospheres to PIMS, PSIME, monolayer CHO cells, Caco-2 cells, and HCT-8 cells. The reproducibility of T. solium oncosphere adherence was most easily measured with CHO cells. Adherence was enhanced by serum-binding medium with >5% fetal bovine serum, which resulted in a significantly greater number of oncospheres adhering than the number adhering when serum at a concentration less than 2.5% was used (P < 0.05). Oncosphere adherence decreased with incubation of cells at 4 degrees C compared with the adherence at 37 degrees C. Our studies also demonstrated that T. solium oncospheres attach to cells with elongated microvillus processes and that the oncospheres expel external secretory vesicles that have the same oncosphere processes.
View details for DOI 10.1128/IAI.01175-06
View details for Web of Science ID 000250451900010
View details for PubMedID 17698575
Targeting of synaptic molecules to their proper location is essential for synaptic differentiation and plasticity. PSD-95/Dlg proteins have been established as key components of the postsynapse. However, the molecular mechanisms regulating the synaptic targeting, assembly, and disassembly of PSD-95/Dlg are not well understood. Here we show that PAR-1 kinase, a conserved cell polarity regulator, is critically involved in controlling the postsynaptic localization of Dlg. PAR-1 is prominently localized at the Drosophila neuromuscular junction (NMJ). Loss of PAR-1 function leads to increased synapse formation and synaptic transmission, whereas overexpression of PAR-1 has the opposite effects. PAR-1 directly phosphorylates Dlg at a conserved site and negatively regulates its mobility and targeting to the postsynapse. The ability of a nonphosphorylatable Dlg to largely rescue PAR-1-induced synaptic defects supports the idea that Dlg is a major synaptic substrate of PAR-1. Control of Dlg synaptic targeting by PAR-1-mediated phosphorylation thus constitutes a critical event in synaptogenesis.
View details for DOI 10.1016/j.neuron.2006.12.016
View details for Web of Science ID 000245126600007
View details for PubMedID 17224403
View details for PubMedCentralID PMC1855201
A 43-year-old woman underwent mitral valve replacement for severe mitral regurgitation nine years after orthotopic heart transplant. Histopathology showed chronic rejection of the mitral valve with lymphocytic infiltrates. The patient is well at one year follow-up. This report describes an identified case of chronic mitral valve rejection requiring valve replacement.
View details for DOI 10.1016/j.athoracsur.2004.06.036
View details for Web of Science ID 000232970500054
View details for PubMedID 16242482
Parkinson's disease (PD) is the most common movement disorder characterized by dopaminergic dysfunction and degeneration. The cause of most PD cases is unknown, although postmortem studies have implicated the involvement of oxidative stress. The identification of familial PD-associated genes offers the opportunity to study mechanisms of PD pathogenesis in model organisms. Here, we show that DJ-1A, a Drosophila homologue of the familial PD-associated gene DJ-1, plays an essential role in oxidative stress response and neuronal maintenance. Inhibition of DJ-1A function through RNA interference (RNAi) results in cellular accumulation of reactive oxygen species, organismal hypersensitivity to oxidative stress, and dysfunction and degeneration of dopaminergic and photoreceptor neurons. To identify other genes that may interact with DJ-1A in regulating cell survival, we performed genetic interaction studies and identified components of the phosphatidylinositol 3-kinase (PI3K)/Akt-signaling pathway as specific modulators of DJ-1A RNAi-induced neurodegeneration. PI3K signaling suppresses DJ-1A RNAi phenotypes at least in part by reducing cellular reactive oxygen species levels. Consistent with the genetic interaction results, we also found reduced phosphorylation of Akt in DJ-1A RNAi animals, indicating an impairment of PI3K/Akt signaling by DJ-1A down-regulation. Together with recent findings in mammalian systems, these results implicate impairments of PI3K/Akt signaling and oxidative stress response in DJ-1-associated disease pathogenesis. We also observed impairment of PI3K/Akt signaling in the fly parkin model of PD, hinting at a common molecular event in the pathogenesis of PD. Manipulation of PI3K/Akt signaling may therefore offer therapeutic benefits for the treatment of PD.
View details for DOI 10.1073/pnas.0504610102
View details for Web of Science ID 000232115100057
View details for PubMedID 16155123
View details for PubMedCentralID PMC1224636
The sympathetic nervous system enhances cardiac muscle function by activating beta adrenergic receptors (betaARs). Recent studies suggest that chronic betaAR stimulation is detrimental, however, and that it may play a role in the clinical deterioration of patients with congestive heart failure. To examine the impact of chronic beta1AR and beta2AR subtype stimulation individually, we studied the cardiovascular effects of catecholamine infusions in betaAR subtype knockout mice (beta1KO, beta2KO).Prospective, randomized, experimental study.Animal research laboratory.beta1KO and beta2KO mice and wild-type controls.The animals were subjected to 2 wks of continuous infusion of the betaAR agonist isoproterenol. Analyses of cardiac function and structure were performed during and 3 days after completion of the infusions. Functional studies included graded exercise treadmill testing, in vivo assessments of left ventricular function using Mikro-Tip catheter transducers, right ventricular pressure measurements, and analyses of organ weight to body weight ratios. Structural studies included heart weight measurements, assessments of myocyte ultrastructure using electron microscopy, and in situ terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling staining to quantitate myocyte apoptosis.We found that isoproterenol-treated beta2KO mice experienced greater mortality rates (p =.001, chi-square test using Fisher's exact method) and increased myocyte apoptosis at 3- and 7-day time points (p =.04 and p =.0007, respectively, two-way analysis of variance).The results of this study suggest that in vivo beta2AR activation is antiapoptotic and contributes to myocardial protection.
View details for DOI 10.1097/01.CCM.0000120049.43113.90
View details for Web of Science ID 000220872700021
View details for PubMedID 15071399
Whipple disease (WD) is a systemic disorder caused by the bacterium Tropheryma whipplei. Since the recognition of a bacterial etiology in 1961, many attempts have been made to cultivate this bacterium in vitro. It was eventually isolated, in 2000, from an infected heart valve, in coculture with human fibroblasts. Here we report the isolation of 2 new strains of T. whipplei from cerebrospinal fluid (CSF) of 2 patients with intestinal WD but no neurological signs or symptoms. One culture-positive specimen was obtained before treatment; the other was obtained 12 months after discontinuation of therapy, at a time of intestinal remission. In both cases, 15 passages of the cultures were completed over 17 months. Bacterial growth was measured by quantitative polymerase chain reaction, which suggested a generation time of 4 days. Staining with YO-PRO nucleic-acid dye showed characteristic rod-shaped bacteria arranged in chains. Fluorescent in situ hybridization with a T. whipplei-specific oligonucleotide probe, a broad-range bacterial probe, and a nonspecific nucleic-acid stain indicated that all visible bacteria were T. whipplei. Scanning electron microscopy and transmission electron microscopy showed both intracellular and extracellular bacteria. This first isolation of T. whipplei from CSF provides clear evidence of viable bacteria in the central nervous system in individuals with WD, even after prolonged antibiotic therapy.
View details for Web of Science ID 000185215600002
View details for PubMedID 12964110
To generate in vitro hyaline cartilage from cryogenically preserved human septal chondrocytes in a simulated microgravity environment on a 3-dimensional biodegradable scaffolding material.In this experiment, cryogenically frozen chondrocytes were thawed and cultured in a monolayer in serum-based chondrocyte media. They were seeded onto 3-dimensional biopolymer scaffolds in a spinner flask. The seeded constructs were then transferred to a bioreactor (an environment of solid-body rotation) for 6 weeks. Chondrocyte growth and extracellular matrix production in the constructs were confirmed by cell count, cell viability, and histologic analysis and by electron microscopy.Histologic sections stained with hematoxylin-eosin and Alcian blue (for acidic proteoglycans) confirmed the presence of hyaline cartilage in the cartilage constructs. Ultrastructural examination using transmission electron microscopy demonstrated matrix formation and chondrocyte viability.This study proves that chondrocytes that are cryogenically stored for extended periods can be used to grow cartilage in vitro. Cryogenically preserved chondrocytes retain their ability to grow in tissue culture, redifferentiate, and produce extracellular matrix.
View details for Web of Science ID 000184620100015
View details for PubMedID 12925350
alpha(2A)-Adrenergic receptors (ARs) in the midbrain regulate sympathetic nervous system activity, and both alpha(2A)-ARs and alpha(2C)-ARs regulate catecholamine release from sympathetic nerve terminals in cardiac tissue. Disruption of both alpha(2A)- and alpha(2C)-ARs in mice leads to chronically elevated sympathetic tone and decreased cardiac function by 4 mo of age. These knockout mice have increased mortality, reduced exercise capacity, decreased peak oxygen uptake, and decreased cardiac contractility relative to wild-type controls. Moreover, we observed significant abnormalities in the ultrastructure of cardiac myocytes from alpha(2A)/alpha(2C)-AR knockout mice by electron microscopy. Our results demonstrate that chronic elevation of sympathetic tone can lead to abnormal cardiac function in the absence of prior myocardial injury or genetically induced alterations in myocardial structural or functional proteins. These mice provide a physiologically relevant animal model for investigating the role of the sympathetic nervous system in the development and progression of heart failure.
View details for DOI 10.1152/ajpheart.01063.2001
View details for Web of Science ID 000178625800012
View details for PubMedID 12384461
Small arteries exhibit tone, a partially contracted state that is an important determinant of blood pressure. In arterial smooth muscle cells, intracellular calcium paradoxically controls both contraction and relaxation. The mechanisms by which calcium can differentially regulate diverse physiological responses within a single cell remain unresolved. Calcium-dependent relaxation is mediated by local calcium release from the sarcoplasmic reticulum. These 'calcium sparks' activate calcium-dependent potassium (BK) channels comprised of alpha and beta1 subunits. Here we show that targeted deletion of the gene for the beta1 subunit leads to a decrease in the calcium sensitivity of BK channels, a reduction in functional coupling of calcium sparks to BK channel activation, and increases in arterial tone and blood pressure. The beta1 subunit of the BK channel, by tuning the channel's calcium sensitivity, is a key molecular component in translating calcium signals to the central physiological function of vasoregulation.
View details for Web of Science ID 000089901900041
View details for PubMedID 11057658
Rhinosporidium seeberi, a microorganism that can infect the mucosal surfaces of humans and animals, has been classified as a fungus on the basis of morphologic and histochemical characteristics. Using consensus polymerase chain reaction (PCR), we amplified a portion of the R. seeberi 18S rRNA gene directly from infected tissue. Analysis of the aligned sequence and inference of phylogenetic relationships showed that R. seeberi is a protist from a novel clade of parasites that infect fish and amphibians. Fluorescence in situ hybridization and R. seeberi- specific PCR showed that this unique 18S rRNA sequence is also present in other tissues infected with R. seeberi. Our data support the R. seeberi phylogeny recently suggested by another group. R. seeberi is not a classic fungus, but rather the first known human pathogen from the DRIPs clade, a novel clade of aquatic protistan parasites (Ichthyosporea).
View details for Web of Science ID 000087321300007
View details for PubMedID 10827117
The present study assessed whether impaired aerobic capacity previously observed in hypercholesterolemic mice is reversible by exercise training. Seventy-two 8-wk-old female C57BL/6J wild-type (+, n = 42) and apolipoprotein E-deficient (-, n = 30) mice were assigned to the following eight interventions: normal chow, sedentary (E+, n = 17; E-, n = 8) or exercised (E+ex, n = 13; E-ex, n = 7) and high-fat chow, sedentary (E+chol, n = 6; E-chol, n = 8) or exercised (E+chol-ex, n = 6; E-chol-ex, n = 7). Mice were trained on a treadmill 2 x 1 h/day, 6 days/wk, for 4 wk. Cholesterol levels correlated inversely with maximum oxygen uptake (r = -0.35; P < 0. 02), which was blunted in all hypercholesterolemic sedentary groups (all P < 0.05). Maximum oxygen uptake improved in all training groups but failed to match E+ex (all P < 0.05). Vascular reactivity and nitric oxide (NO) synthesis correlated with anaerobic threshold (r = 0.36; P < 0.025) and maximal distance run (r = 0.59; P < 0.007). We conclude that genetically induced hypercholesterolemia impairs aerobic capacity. This adverse impact of hypercholesterolemia on aerobic capacity may be related to its impairment of vascular NO synthesis and/or vascular smooth muscle sensitivity to nitrovasodilators. Aerobic capacity is improved to the same degree by exercise training in normal and genetically hypercholesterolemic mice, although there remains a persistent difference between these groups after training.
View details for Web of Science ID 000079554200030
View details for PubMedID 10199861
CCR5-utilizing (R5) and CXCR4-utilizing (X4) strains of human immunodeficiency virus type 1 (HIV-1) have been studied intensively in vitro, but the pathologic correlates of such differential tropism in vivo remain incompletely defined. In this study, X4 and R5 strains of HIV-1 were compared for tropism and pathogenesis in SCID-hu Thy/Liv mice, an in vivo model of human thymopoiesis. The X4 strain NL4-3 replicates quickly and extensively in thymocytes in the cortex and medulla, causing significant depletion. In contrast, the R5 strain Ba-L initially infects stromal cells including macrophages in the thymic medulla, without any obvious pathologic consequence. After a period of 3 to 4 weeks, Ba-L infection slowly spreads through the thymocyte populations, occasionally culminating in thymocyte depletion after week 6 of infection. During the entire time of infection, Ba-L did not mutate into variants capable of utilizing CXCR4. Therefore, X4 strains are highly cytopathic after infection of the human thymus. In contrast, infection with R5 strains of HIV-1 can result in a two-phase process in vivo, involving apparently nonpathogenic replication in medullary stromal cells followed by cytopathic replication in thymocytes.
View details for Web of Science ID 000076892100078
View details for PubMedID 9811751
Apoptosis (programmed cell death) regulates cell population size. To determine the mechanisms whereby hematopoietic growth factors (HGFs) modulate apoptosis in human myeloid leukemic cells, we evaluated the roles of protein and mRNA synthesis for altering apoptosis in growth factor-stimulated vs. quiescent leukemic TF1 cells. Lysates of cells from the granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent myeloid leukemic cell line TF1 were separated into high molecular weight (HMW) pellets of intact DNA and supernatants of fragmented low MW (LMW) DNA, and the DNA purified from these fractions was quantified. In the absence of both GM-CSF and fetal bovine serum (FBS), 70% of the DNA was fragmented after 3 days in culture, with a characteristic apoptotic ladder-like pattern on agarose gel electrophoresis, whereas this proportion had initially been < 5%. In contrast, less than 5% of the DNA was fragmented in cells incubated with GM-CSF plus FBS or GM-CSF alone. Delayed addition of GM-CSF, but not FBS, permitted partial rescue of the cells, inhibiting increasing rates of accumulation of fragmented DNA. When the macro-molecular synthesis inhibitor cycloheximide (CHX) or actinomycin D (Act D) was present for 26 hours in the absence of GM-CSF and FBS, apoptosis was inhibited. In contrast, in the presence of GM-CSF or FBS, apoptosis was enhanced upon addition of CHX or Act D. The latter effect persisted even with the late addition of CHX. These findings indicate that disparate mechanisms of enhancing or inhibiting apoptosis exist in myeloid leukemic cells related to environmental conditions, including HGF-regulated cellular synthesis of distinct proteins and mRNA.
View details for Web of Science ID A1995QK72300014
View details for PubMedID 7875243
Poor patency rates have limited the success of biological vascular grafts in the coronary artery position. Recently, two bovine internal mammary arterial grafts have been developed for possible use as coronary artery bypass graft (CABG) conduits: (1) Denaflex grafts (Baxter Health-care Co, 3-mm ID) treated with polyepoxy compounds and with heparin ionically bound to the luminal surface and (2) Bioflow grafts (Bio-Vascular, Inc, 3-mm ID) treated with dialdehyde starch.Thirty dogs underwent CABG with either a Denaflex (n = 20) or Bioflow (n = 10) graft to the left circumflex coronary artery (LCx). The left main coronary artery (n = 12) or proximal LCx (n = 18) was then ligated. Six-month patency (Kaplan-Meier) for Denaflex grafts was 44 +/- 13% (+/- SEM), compared with 12 +/- 11% for Bioflow grafts, but this difference did not reach statistical significance (P = .56). Among grafts open at 14 days, however, there were no occlusions among six Denaflex grafts versus five occlusions among seven Bioflow grafts. At 6 months, all six surviving Denaflex grafts appeared normal, while the only remaining patent Bioflow graft was angiographically dilated and had diffuse luminal irregularities. At 1 year, three Denaflex grafts angiographically had no dilation, stenosis, or luminal irregularities. Macroscopically, all explanted long-term (6 to 12 months) Denaflex grafts had a smooth, clean luminal surface, whereas the only patent Bioflow graft had multifocal thrombi. Microscopically, all Denaflex grafts had minimal degenerative changes, but the Bioflow graft had transmural linear cracks and medial deterioration.These data suggest that long-term (> 6-month) patency is possible with small-caliber, low-flow biological grafts in the canine coronary position, although both types of grafts are prone to early occlusion. If these early failures are excluded, the Denaflex graft appears to be associated with better long-term patency and an absence of degenerative changes.
View details for PubMedID 7955246
We describe a 40-year-old white man with a red-brown, indurated plaque on the proximal aspect of his right thigh. The lesion had been present since birth, and the patient had a 20-year clinical history of recurrent cellulitis in the same area. The histopathologic features of the lesion included permeation of the dermis by flattened, endothelium-lined channels without cellular atypia, hemorrhage, or inflammation. The endothelial cells were stained intensely with monoclonal antibody anti-CD34 (clone MY10). In addition, antibodies to factor VIII antigen, HLA-DR, smooth muscle actin, ICAM-1, and the lectin Ulex europaeus labeled the luminal cells. The basement membrane of the channels stained with anti-type IV collagen and laminin. Desmin-positive cells were abundant adjacent to the channels. Factor XIIIa stained both mononuclear cells and occasional dendritic cells in the perivascular area. Ki-67 immunolabeling could not be demonstrated on fresh or frozen tissue. Electron microscopy revealed the presence of both tight junctions and a well-formed, continuous basement membrane but the absence of Weibel-Palade bodies.
View details for Web of Science ID A1994NZ81600014
View details for PubMedID 7518477
This study was designed to test the hypothesis that long-term oral supplementation of dietary L-arginine (to provide a sustained elevation of nitric oxide activity) would inhibit atherogenesis in hypercholesterolemic rabbits, as assessed by histomorphometric measurements.Endothelium-derived nitric oxide inhibits a number of processes that are critical in atherogenesis. Hypercholesterolemia reduces endothelial nitric oxide activity, and we postulate that this may promote atherogenesis. This reduction in nitric oxide activity can be reversed acutely by intravenous infusion of L-arginine, the precursor of nitric oxide. We show that dietary supplementation of L-arginine abrogates the development of coronary atheroma in hypercholesterolemic rabbits.Male New Zealand White rabbits were fed normal rabbit chow, 1% cholesterol chow or 1% cholesterol chow with dietary arginine or methionine supplementation to increase their intake of these amino acids sixfold. After 1 or 10 weeks of dietary intervention, the left main and left anterior descending coronary arteries were harvested for histologic study. Plasma cholesterol measurements were elevated to the same degree in all groups of rabbits receiving the 1% cholesterol diet, whereas plasma arginine levels were doubled in the arginine-treated group. High density lipoprotein (HDL) cholesterol values were not affected by arginine treatment.In rabbits receiving the 1% cholesterol diet, with or without methionine supplementation, light and electron microscopy revealed a marked increase from 1 to 10 weeks in the intimal accumulation of macrophages, associated with an increase in the intimal area of the left main coronary artery. By contrast, in arginine-treated hypercholesterolemic rabbits, there was a near absence of adherent monocytes and tissue macrophages and no progression of intimal thickness from 1 to 10 weeks.Dietary supplements of L-arginine prevent intimal thickening in the coronary arteries of hypercholesterolemic rabbits. This antiatherogenic effect is not due to an alteration in plasma total cholesterol, HDL cholesterol or caloric or nitrogen balance. The data are consistent with the hypothesis that nitric oxide has antiatherogenic properties.
View details for Web of Science ID A1994NR75200026
View details for PubMedID 8294700
We compared the effectiveness of pulsed magnetization transfer contrast (MTC) magnetic resonance imaging (MRI) and spin-echo MRI in detecting tumor necrosis.Adenocarcinoma cells were transplanted in the livers of 12 syngenic BDIX rats. To induce various degrees of tumor necrosis, the rats were randomly assigned to the following groups: 1) control; 2) localized hyperthermia; 3) intralesional cisplatin; and 4) hyperthermia plus intralesional cisplatin. At day 7 after treatment, the rats were imaged using a 1.5-T imager with 1) multiplanar gradient-recalled echo sequence (MPGR) 500/8/20 degrees with and without magnetization transfer contrast (MTC); 2) spin-echo 2500/20,80, and 3) spin-echo 300/20 pulse sequences. The rats were then sacrificed and pathologic specimens were prepared using MR images as guidance. T2 and ratios of signal intensity after saturation to signal intensity before saturation (Ms/Mo ratios) of the necrotic and granulation tissues and viable tumors were determined in 10 rats.Compared with standard MPGR images, MPGR images with MTC provided better contrast between the pathologic tissues and normal liver. However, T2 values were more useful than Ms/Mo ratios in distinguishing necrotic areas from viable tumor. The T2 values of coagulative necrosis and granulation tissue were significantly different from that of viable tumor. No significant difference between the Ms/Mo ratios of the different pathologic tissues and normal liver was found.Pulsed magnetization transfer contrast MRI was inferior to spin-echo MRI in distinguishing necrotic from viable tumors in rat livers using the pulse sequences described, and none of the sequences studied was thought to be reliable enough for this purpose.
View details for Web of Science ID A1993MD17700004
View details for PubMedID 8262743
The efficacy of purified cross-linked human hemoglobin solution in maintaining the metabolic integrity of perfused livers from fed rats was determined and compared with that of livers recirculated with red blood cells, perfluorocarbon, and Krebs' Ringer bicarbonate buffer media at normal and accelerated flow rates. The data indicate that oxygen utilization was comparable in livers perfused with red blood cell medium (53.5 +/- 4.0 microL.g liver-1.min-1), hemoglobin (45.7 +/- 1.9), and perfluorocarbon (57.2 +/- 6.1) and less in livers perfused with Krebs' Ringer bicarbonate buffer solution at normal (17.4 +/- 1.4) and high (27.7 +/- 1.4) flow rates. Bile flow, the outflow of glucose and lactic acid, and residual glycogen levels were similar when livers were perfused with red blood cells (hematocrit, 19) and hemoglobin solutions containing 7 g hemoglobin/dL at flow rates of 1.1-1.2 mL.g liver-1.min-1. However, livers perfused with perfluorocarbon at 1.1 mL.g-1.min-1 showed a significantly greater (P less than 0.01) decline in bile flow and outflow of glucose and lactic acid. Livers perfused with Krebs' Ringer bicarbonate buffer at normal (1.3 mL.g-1.min-1) and accelerated flow rates (3.0 mL.g-1.min-1) also showed a progressive decrease in bile flow and marked glycogenolysis as well as depletion of adenosine triphosphate content. In addition, morphological studies (light and electron microscopy) showed more vacuoles, membrane alterations, and increased mitochondrial swelling in livers perfused with Krebs' Ringer bicarbonate buffer and perfluorocarbon. These findings suggest that hepatocyte function in livers from fed rats is maintained equally well with hemoglobin solutions and with red blood cell medium, suggesting that cross-linked hemoglobin solution may serve as an effective blood substitute for maintaining adequate oxygenation and metabolic integrity of the isolated perfused rat liver.
View details for Web of Science ID A1991GM00800026
View details for PubMedID 1936806
An in vitro system was established in which single-cell suspensions of lymphocytes and synovial cells from the joints of patients with rheumatoid arthritis were cultured and produced an outgrowth of an organized inflammatory tissue with an extracellular matrix and capsule. The tissue outgrowth, which had histologic features of pannus, required the addition of mycobacterial antigen and interleukin-2 to the tissue culture medium and was dependent upon the presence of T lymphocytes and their interaction with synovial fibroblasts.
View details for Web of Science ID A1991FR47300006
View details for PubMedID 1905130
We assessed the reversibility of functional and morphological changes of arterialized vein segments by returning them to the venous circulation. Thirteen dogs underwent right carotid and femoral veno-arterial grafting. After 12 weeks, veno-arterial grafts were removed for contractility (norepinephrine [NE] and 5-hydroxytryptamine [5-HT]), luminal prostacyclin (PGI2), and morphometric analyses; the remaining segments were used as left jugular and femoral veno-venous grafts. After another 12 weeks, the veno-venous grafts were harvested. To NE, veno-arterial grafts (ED50, 5.4 +/- 0.1 [-log M]) were less sensitive than control veins (ED50, 6.0 +/- 0.2) or veno-venous grafts (ED50, 6.4 +/- 0.2) but were more sensitive than control arteries (ED50, 4.0 +/- 0.1); the maximum tension of veno-arterial grafts (6.2 +/- 0.6 g) was greater than that of veins, less than that of arteries (9.8 +/- 1.0 g), and comparable with that of veno-venous grafts (5.1 +/- 1.1 g). To 5-HT, veno-arterial (ED50, 7.5 +/- 0.1) and veno-venous (ED50, 7.3 +/- 0.2) grafts were more sensitive than arteries (ED50, 6.0 +/- 0.3), while the vein was unresponsive; the maximum tension of veno-arterial grafts (5.0 +/- 0.7 g) was less than that of arteries (6.9 +/- 0.9 g) and greater than that of veno-venous grafts (1.4 +/- 0.3 g). PGI2 production in veins (3.6 +/- 0.8 ng/ml), veno-arterial grafts (3.9 +/- 0.8 ng/ml), and veno-venous grafts (3.3 +/- 0.9 ng/ml) was comparable and less than that of arteries (6.4 +/- 0.9 ng/ml). Veno-arterial graft intimal thickness (127 +/- 8 microns) and intimal area (15.6 +/- 1.8 x 10(3) microns 2) tended to be greater than that in the veno-venous graft (113 +/- 9 microns and 12.4 +/- 1.8 x 10(3) microns 2); also, the veno-arterial graft medial area (103.0 +/- 7.3 x 10(3) microns 2) was greater than that of the veno-venous graft (80.3 +/- 6.9 x 10(3) microns 2), thereby resulting in a similar relative intimal area (13 +/- 1%). Therefore, some changes associated with arterialization, for example, adrenergic sensitivity, maximum tension to 5-HT, medial thickening, and perhaps intimal hyperplasia, reverted toward venous values when replaced in the venous environment, possibly due to variations in pressure, flow, shear stress, and/or graft preparation techniques. Luminal PGI2 was unchanged in the grafts, implying that graft contractility was not modulated by luminal PGI2.
View details for PubMedID 2225440
Favorable changes in lipoproteins, inhibition of platelet aggregation, reduction of serum thromboxane (TX), altered plasma-membrane fluidity, and reduced production of growth factors (mitogens) have all been implicated as possibly being involved in the inhibition of arteriosclerosis by fish oil (FO), which is rich in omega 3 fatty acids; however, causal relations are mostly lacking. Several putative mechanisms responsible for the salutary effects of FO were investigated in a canine model of accelerated vein-graft arteriosclerosis. Venoarterial autografts (N = 192) were implanted in 48 hypercholesterolemic dogs divided into six groups: group A, control; B, FO (as MaxEPA, 200 mg/kg/day eicosapentaenoic acid); C, aspirin (ASA, 50 mg/kg/day); D, TX synthetase inhibitor (TXSI [CGS-12970], 10 mg/kg/day); E, FO + ASA; and F, FO + TXSI. At sacrifice 3 months later, there was no significant difference in plasma lipoproteins, hepatic low density lipoprotein-receptor concentration, red blood cell fragility, bleeding time, or platelet count compared with controls; the decrease in platelet aggregation (30 +/- 5% [mean +/- SEM]) was similar in all treatment groups. Arterialized vein-graft intimal thickening was significantly inhibited by FO (with or without ASA), while ASA alone was ineffective. Conversely, serum TX was significantly lower only in the ASA and FO + ASA groups. Serum mitogenic activity was higher at 3 months in the control group versus all treatment groups. Compared with baseline values, serum mitogenic activity rose significantly over time in the control and the TXSI groups, and an increase or rising trend was present in all other treatment groups except for the FO-treated animals. Thus, the salutary biologic effect of FO in this hypercholesterolemic model of arterialized vein grafts may have been more related to in vivo inhibition of platelet-mitogen growth factor release than to changes in lipoproteins, low density lipoprotein receptors, platelet function, or eicosanoid metabolism. These observations underscore the need for further studies to clarify the interactions between FO (omega 3 fatty acids) and paracrine cellular mitogenic factors in the context of atherosclerosis prevention.
View details for PubMedID 2766520
The effects of fish oil on the development of arteriosclerosis were assessed using a special susceptible strain (SEA) of Japanese quail (Coturnix coturnix japonica). Sixty four quail were randomly divided into two groups and placed on isocaloric and approximately isocholesterolic (2% by weight) diets. Group A (control) was supplemented with 10% beef tallow oil, while group B received 10% Menhaden fish oil. The birds were sacrificed at 10 weeks (early) and 15-16 weeks (late). Based on semiquantitative histological grading of the arteriosclerotic lesions in the proximal aorta and brachiocephalic arteries, a score from 1 (no lesion) to 5 (severe, diffuse lesions) was assigned. A total of 57 quail were evaluated (seven died prior to scheduled sacrifice). At the early period, the mean arteriosclerosis scores for group A (n = 8) and group B (n = 8) were 3.3 (SD 1.0) and 1.9(1.0) respectively (p less than 0.017); 63% of the quail in group A and 13% of those in group B had a score greater than or equal to 3 (p less than 0.25, NS). At the late period, the scores for group A (n = 20) and group B (n = 21) were 3.8(0.6) and 2.6(0.9), respectively (p less than 0.001); 95% of the birds in group A and 43% of those in group B had a score greater than or equal to 3 (p less than 0.005). Histopathological examination of the arteriosclerotic lesions revealed disruption of the innermost elastic lamina, increased proteoglycan deposition in the medial interlamellar spaces, and the distinct involvement of macrophage like cells. Compared to human disease, arteriosclerosis in the quail is marked by distinct similarities, as well as differences. The SEA strain of Japanese quail appears to be a practical model for the study of arteriosclerosis; fish oil reduces the severity of disease in these birds when fed a high cholesterol diet.
View details for PubMedID 2598217
In an attempt to study the effects of allogeneic lymphocytes on endothelial cells (EC) and analyze the mechanism whereby such lymphocytes traverse an EC barrier, we have established human microvascular EC monolayers, in vitro, and analyzed the effects of lymphocyte subpopulations on such monolayers. Previous studies have shown that CD16+ (natural killer) and CD8+ (cytotoxic) lymphocytes but not CD4+ (helper) cells bind and induce the appearance of class II major histocompatibility complex antigens on allogeneic EC. The current findings indicate that these same lymphocyte subsets induce marked swirling and elongation of allogeneic EC, and traverse intact EC monolayers. In contrast, none of the functional consequences of the initial lymphocyte-EC adhesion were observed using autologous combinations, despite the presence of significant intercellular binding. Scanning and electron micrographs demonstrate extensive areas of lymphocyte-EC surface contact and EC-coated pit formation, whereas a panel of recombinant cytokines known to alter the surface phenotype of EC fail to induce the same morphologic changes whether used singly or in combination. We postulate that the cellular interactions observed here, in vitro, may represent the initial steps in the rejection of vascularized allografts in vivo.
View details for Web of Science ID A1989AB98900026
View details for PubMedID 2660343
The effects of the administration of aspirin (ASA), dipyridamole (DPM), and cod liver oil (CLO) on graft patency rate and degree of intimal hyperplasia were investigated in a canine, hypercholesterolemic veno-arterial allograft model in an attempt to modify this immunologically mediated vascular injury. The drug regimens were ASA 1 mg/kg/day, DPM 10 mg/kg/day, combined ASA and DPM (ASA + DPM), and CLO (1.8 g/day eicosapentanoic acid [EPA] and 1.2 g/day docosahexanoic acid [DHA]), and control. The early angiographic patency rate (1-3 weeks) was 81% +/- 10% (+/- 70% confidence limits); the 90-day overall patency rate was 60% +/- 4% (87/144), with no statistically significant differences among the groups (range 46 +/- 10-71 +/- 9%). Qualitatively, there was no difference in luminal thrombus, intimal hemorrhage, or lesion eccentricity. Considering the relatively short time of graft implantation, an extensive amount of microscopic disease was observed; quantitatively, the mean intimal thickness was 515 +/- 17 microgram overall but was not statistically different between the groups. The fraction of potential lumenal area occupied by intimal thickening was 0.37 +/- 0.01 but again did not differ significantly between the groups. These doses of ASA, DPM, ASA + DPM, and CLO did not alter graft occlusion or retard the marked degree of subintimal myointimal cell hyperplasia that was generated in this hypercholesterolemic canine veno-arterial allograft preparation. Possible explanations for these negative findings include inadequate dosage or form of omega-3 fatty acids and the antiplatelet drugs administered, excessive variability in graft response due to uncharacterized immunologic histocompatibility, and the possible influence of non-platelet-mediated mechanisms. Nevertheless, this preparation is attractive as a reproducible model of accelerated (immunologically mediated) experimental arteriosclerosis.
View details for PubMedID 3196097
A carcinogen bioassay of isoflurane was performed in groups of Swiss/Webster mice exposed to either air (n = 181), 0.1% isoflurane (n = 167), or 0.4% isoflurane (n = 165), for 4 h per day, 5 days per week. After 78 weeks of exposure, mice were left untreated for 3 weeks and were then killed. Mice killed at this time when they were 86 weeks of age, and those killed or dying at other times during the study were subjected to complete gross and microscopic examination. Throughout most of the study, mean body weights of mice exposed to 0.1% isoflurane and 0.4% isoflurane were less by 1-5% and 5-8%, respectively, than that of mice exposed to air alone. Otherwise, no gross toxic treatment effects were noted. The first neoplastic lesion was detected 23 weeks after starting treatment and, by the end of the study, 190 tumors had been detected in 179 mice. However, there were no statistical differences among the groups in the number of mice with a particular tumor at a specific site, the ratio of benign to malignant tumors, or the time to tumor appearance. It was concluded that isoflurane is unlikely to have carcinogenic potential and is a remarkably non-toxic anesthetic in mice.
View details for Web of Science ID A1988Q772100018
View details for PubMedID 3189921
The envelope protein of human immunodeficiency virus (HIV) is synthesized as a polyprotein (gp160) and cleaved intracellularly to a gp120-gp41 heterodimer. In this study, the tryptic-like endoproteolytic cleavage site was removed by site-directed mutagenesis and replaced with a chymotryptic-like site. The resultant mutant, RIP7/mut10, was found to be indistinguishable from wild-type HIV when analyzed at the level of proviral replication, RNA processing, protein expression, and viral assembly. However, the gp160 polyprotein was not cleaved and the mutated virions were biologically inactive, until and unless they were exposed to limiting concentrations of chymotrypsin. As is the case for other enveloped mammalian viruses, endoproteolytic cleavage of the HIV envelope protein and release of a unique hydrophobic domain appear to be necessary for the full expression of viral infectivity.
View details for Web of Science ID A1988M922100008
View details for PubMedID 2450679
Marine lipids containing omega-3 fatty acids (chiefly, eicosapentanoic acid [EPA] and docosahexanoic acid [DHA]) may inhibit the development of atherosclerotic vascular disease, but the mechanisms responsible for this putative beneficial effect are unknown. We investigated the effects of EPA and DHA in a canine model of accelerated vein graft arteriosclerosis during a 3-month period. Twenty-five dogs were divided into three dietary groups: group I (control), group II (2.5% cholesterol), and group III (2.5% cholesterol plus 2 gm EPA/day [as MaxEPA]). The effects of EPA on vein graft intimal thickening, platelet and vascular prostaglandin metabolism, lipid and lipoprotein receptor metabolism, and hematologic parameters were assessed. Cholesterol feeding caused a significant 54% increase in graft intimal thickness compared with control animals (124.9 +/- 50.4 vs 81.2 +/- 32.4 micron; p = 0.013), which was prevented by supplementation with EPA in group III (56.9 +/- 30.0 micron; p = 0.001 vs group II). Intimal thickness in group III was not significantly different from that of control. EPA supplementation was also associated with a 38% decline in serum thromboxane levels from 457.0 +/- 129.3 pg/0.1 ml in group II to 283.5 +/- 96.9 pg/0.1 ml in group III (p = 0.007). The alterations in lipoprotein metabolism associated with cholesterol feeding were not affected by EPA: in both groups II and III, serum cholesterol and high-density lipoproteins and liver cholesterol content were elevated and hepatic low-density lipoproteins (LDL) receptor content was reduced. There were no differences between the three groups in terms of vein graft or native vessel prostacyclin production, hematocrit, platelet count, or coagulation parameters. In this canine model, dietary supplementation with marine omega-3 fatty acids reduced the extent and magnitude of accelerated vein graft intimal thickening induced by hypercholesterolemia; moreover, this beneficial effect was associated with lower serum thromboxane production and appeared to be independent of alterations in lipoprotein metabolism or LDL receptor density.
View details for PubMedID 3336117
The standard coronary ligation model for experimental myocardial infarction results in variable areas and patterns of necrosis; therefore, the healing of such infarctions is also variable. The authors developed an experimental myocardial injury model using simple cryoinjury, which allows standardization of the size, depth, and location of the wound. Thirty-eight left ventricular cryolesions were created in 19 dogs, which were then killed from 3 to 35 days after injury. A consistent decrease in the depth of scar (p less than 0.005) and accumulation of collagen (p less than 0.0001) over time characterized this healing myocardial wound. Histologic examination revealed that the cellular pattern of healing myocardial cryolesions is similar to that of a healing myocardial infarction but with less variability. The authors advocate the use of cardiac cryolesions as a model of experimental myocardial wound healing.
View details for PubMedID 3689016
Biochemical (or functional) adaptation of venoarterial grafts has been demonstrated recently. We reexamined one aspect of this biochemical "arterialization" process: prostacyclin (PGI2) production by canine venoarterial autologous grafts and the responsiveness of this biosynthetic pathway to maximal stimulation with substrate enhancement. Four reversed autologous grafts (femoral vein) were interposed into both carotid and femoral arteries in eight dogs. After 12 weeks, the grafts were removed, and radioimmunoassay was used to determine luminal surface production of 6-keto-PGF1 alpha (the stable metabolite of PGI2) in both the basal and stimulated (27 mumol/L arachidonic acid [AA]) states. PGI2 production by the venous autologous grafts was compared with that of control native artery and vein. We confirmed that PGI2 production (measured in nanograms per milliliter) by control artery was greater than vein under both basal conditions (5.8 +/- 0.4 [+/- SEM] vs. 2.7 +/- 0.5, p less than 0.001) and stimulated conditions (8.8 +/- 0.8 vs. 5.5 +/- 0.4, p = 0.002); moreover, AA stimulation significantly increased PGI2 production in both native artery and vein compared with basal PGI2 production. Under basal conditions, graft PGI2 production (6.3 +/- 1.6 ng/ml) was not significantly different than basal arterial levels (p = 0.8) but was higher than basal venous levels (p = 0.05). However, in marked contrast to both native artery and vein, the vein graft flow surface showed no significant response to substrate enhancement with AA: basal (6.3 +/- 1.6 ng/ml) vs. stimulated (5.9 +/- 0.9 ng/ml) (p = 0.8). These observations confirm that canine venoarterial autologous grafts undergo biochemical "arterialization"; however, this process appears to be an incomplete one.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for PubMedID 3312650
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