Bone marrow- or vessel wall-derived osteoprotegerin is sufficient to reduce atherosclerotic lesion size and vascular calcification.

OBJECTIVE: Osteoprotegerin (OPG) is a decoy receptor for the osteoclast differentiation factor receptor activator of NF-κB ligand. OPG regulates bone homeostasis, and its inactivation in mice results in severe osteoporosis. OPG deficiency in apolipoprotein E (ApoE)(-/-) mice results in increased atherosclerotic lesion size and calcification. Furthermore, receptor activator of NF-κB ligand enhances macrophage-dependent smooth muscle cell calcification in vitro. Here, we hypothesized that reconstitution of ApoE(-/-)OPG(-/-) mice with ApoE(-/-)OPG(+/+) bone marrow (BM) would be sufficient to rescue lesion progression and vascular calcification. Conversely, reconstitution of ApoE(-/-)OPG(+/+) mice with ApoE(-/-)OPG(-/-) BM may accelerate lesion progression and vascular calcification. APPROACH AND RESULTS: ApoE(-/-)OPG(-/-) mice transplanted with ApoE(-/-)OPG(+/+) BM developed smaller atherosclerotic lesions and deposited less calcium in the innominate artery than that of ApoE(-/-)OPG(-/-) mice transplanted with ApoE(-/-)OPG(-/-) BM. There were no differences in lesion size and calcification in ApoE(-/-)OPG(+/+) mice transplanted with BM from ApoE(-/-)OPG(-/-) or ApoE(-/-)OPG(+/+) mice. The large lesions observed in the ApoE(-/-)OPG(-/-) mice transplanted with OPG(-/-) BM were rich in chondrocyte-like cells, collagen, and proteoglycans. Importantly, the ApoE(-/-)OPG(-/-) mice transplanted with OPG(+/+) BM remained osteoporotic, and the ApoE(-/-)OPG(+/+) mice did not show signs of bone loss regardless of the type of BM received. In coculture experiments, macrophages and mesenchymal stem cells derived from ApoE(-/-)OPG(-/-) BM induced more vascular smooth muscle cell calcification than cells derived from ApoE(-/-)OPG(+/+) mice. CONCLUSIONS: These results indicate that OPG derived either from the BM or from the vessel wall is sufficient to slow down lesion progression and vascular calcification independent of bone turnover.

Macrophage-derived foam cells freshly isolated from rabbit atherosclerotic lesions degrade modified lipoproteins, promote oxidation of low-density lipoproteins, and contain oxidation-specific lipid-protein adducts.

Pure macrophage-derived foam cells (MFC) were isolated from the aortas of rabbits made atherosclerotic by balloon deendothelialization followed by diet-induced hypercholesterolemia. The MFC were isolated under sterile conditions using an enzymatic digestion procedure and discontinuous density gradient centrifugation. The purity of the MFC preparations was verified immunocytochemically with the macrophage specific monoclonal antibody RAM-11. MFC plated in medium containing 0.5% FCS for 24 h contained approximately 600 micrograms cholesterol per mg cell protein, 80% of which was esterified cholesterol. The MFC specifically degraded low density lipoprotein (LDL), acetyl-LDL, copper oxidized LDL, and beta-very low density lipoprotein (beta-VLDL) at rates comparable to mouse peritoneal macrophages (MPM) in 5-h assays. MFC within sections of the atherosclerotic lesions from the ballooned rabbits as well as the MFC isolated from the same lesions in the presence of antioxidants, exhibited positive immunoreactivity with polyclonal guinea pig antisera and mouse monoclonal antibodies directed against malondialdehyde-LDL, and 4-hydroxynonal-LDL. The MFC also exhibited the capacity to induce the oxidation of LDL at rates comparable to those exhibited by MPM and rabbit aortic endothelial cells. These data provide direct evidence that arterial wall macrophages express modified LDL receptors in vivo, contain epitopes found in oxidized-LDL and are capable of oxidizing LDL even when maximally loaded with cholesterol.

Gene expression in macrophage-rich human atherosclerotic lesions. 15-lipoxygenase and acetyl low density lipoprotein receptor messenger RNA colocalize with oxidation specific lipid-protein adducts.

Oxidatively modified low density lipoprotein (LDL) exhibits several potentially atherogenic properties, and inhibition of LDL oxidation in rabbits decreases the rate of the development of atherosclerotic lesions. In vitro studies have suggested that cellular lipoxygenases may be involved in LDL oxidation, and we have shown previously that 15-lipoxygenase and oxidized LDL are present in rabbit atherosclerotic lesions. We now report that epitopes of oxidized LDL are also found in macrophage-rich areas of human fatty streaks as well as in more advanced human atherosclerotic lesions. Using in situ hybridization and immunostaining techniques, we also report that 15-lipoxygenase mRNA and protein colocalize to the same macrophage-rich areas. Moreover, these same lesions express abundant mRNA for the acetyl LDL receptor but no detectable mRNA for the LDL receptor. We suggest that atherogenesis in human arteries may be linked to macrophage-induced oxidative modification of LDL mediated by 15-lipoxygenase, leading to subsequent enhanced macrophage uptake, partly by way of the acetyl LDL receptor.

Expression of suppressors of cytokine signaling during liver regeneration.

The cytokines TNF and IL-6 play a critical role early in liver regeneration following partial hepatectomy (PH). Since IL-6 activates signal transducers and activators of transcription (STATs), we examined whether the suppressors of cytokine signaling (SOCS) may be involved in terminating IL-6 signaling. We show here that SOCS-3 mRNA is induced 40-fold 2 hours after surgery. SOCS-2 and CIS mRNA are only weakly induced, and SOCS-1 is not detectable. SOCS-3 induction after PH is transient and correlates with a decrease in STAT-3 DNA binding and a loss of tyrosine 705 phosphorylation. This response is markedly reduced in IL-6 knockout (KO) mice. TNF injection induces SOCS-3 mRNA in wild-type mice (albeit weakly compared with the increase observed after PH) but not in TNF receptor 1 or IL-6 KO mice. In contrast, IL-6 injection induces SOCS-3 in these animals, demonstrating a requirement for IL-6 in SOCS-3 induction. IL-6 injection into wild-type mice also induces SOCS-1, -2, and CIS mRNA, in addition to SOCS-3. Together, these results suggest that SOCS-3 may be a key component in downregulating STAT-3 signaling after PH and that SOCS-3 mRNA levels in the regenerating liver are regulated by IL-6.

Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man.

Three lines of evidence are presented that low density lipoproteins gently extracted from human and rabbit atherosclerotic lesions (lesion LDL) greatly resembles LDL that has been oxidatively modified in vitro. First, lesion LDL showed many of the physical and chemical properties of oxidized LDL, properties that differ from those of plasma LDL: higher electrophoretic mobility, a higher density, higher free cholesterol content, and a higher proportion of sphingomyelin and lysophosphatidylcholine in the phospholipid fraction. A number of lower molecular weight fragments of apo B were found in lesion LDL, similar to in vitro oxidized LDL. Second, both the intact apo B and some of the apo B fragments of lesion LDL reacted in Western blots with antisera that recognize malondialdehyde-conjugated lysine and 4-hydroxynonenal lysine adducts, both of which are found in oxidized LDL; plasma LDL and LDL from normal human intima showed no such reactivity. Third, lesion LDL shared biological properties with oxidized LDL: compared with plasma LDL, lesion LDL produced much greater stimulation of cholesterol esterification and was degraded more rapidly by macrophages. Degradation of radiolabeled lesion LDL was competitively inhibited by unlabeled lesion LDL, by LDL oxidized with copper, by polyinosinic acid and by malondialdehyde-LDL, but not by native LDL, indicating uptake by the scavenger receptor(s). Finally, lesion LDL (but not normal intimal LDL or plasma LDL) was chemotactic for monocytes, as is oxidized LDL. These studies provide strong evidence that atherosclerotic lesions, both in man and in rabbit, contain oxidatively modified LDL.

Targeted gene delivery by tropism-modified adenoviral vectors.

The utility of adenoviral vectors for gene therapy is currently limited due, in part, to the widespread distribution of the cellular receptor for the adenovirus fiber that precludes the targeting of specific cell types. In order to develop a targeted adenovirus, it is therefore necessary both to ablate endogenous viral tropism and to introduce novel tropism. We hypothesized that these two goals could be achieved by employing a neutralizing anti-fiber antibody, or antibody fragment, chemically conjugated to a cell-specific ligand. To test this concept, we chose to target the folate receptor, which is overexpressed on the surface of a variety of malignant cells. Therefore, we conjugated folate to the neutralizing Fab fragment of an anti-fiber monoclonal antibody. This Fab-folate conjugate was complexed with an adenoviral vector carrying the luciferase reporter gene and was shown to redirect adenoviral infection of target cells via the folate receptor at a high efficiency. Furthermore, when complexed with an adenoviral vector carrying the gene for herpes simplex virus thymidine kinase, the Fab-folate conjugate mediated the specific killing of cells that overexpress the folate receptor. This work thus represents the first demonstration of the retargeting of a recombinant adenoviral vector via a non-adenoviral cellular receptor.

Advanced atherosclerotic lesions in the innominate artery of the ApoE knockout mouse.

Most previous studies of atherosclerosis in hyperlipidemic mouse models have focused their investigations on lesions within the aorta or aortic sinus in young animals. None of these studies has demonstrated clinically significant advanced lesions. We previously mapped the distribution of lesions throughout the arterial tree of apolipoprotein E knockout (apoE(-/-)) mice between the ages of 24 and 60 weeks. We found that the innominate artery, a small vessel connecting the aortic arch to the right subclavian and right carotid artery, exhibits a highly consistent rate of lesion progression and develops a narrowed vessel characterized by atrophic media and perivascular inflammation. The present study reports the characteristics of advanced lesions in the innominate artery of apoE(-/-) mice aged 42 to 60 weeks. In animals aged 42 to 54 weeks, there is a very high frequency of intraplaque hemorrhage and a fibrotic conversion of necrotic zones accompanied by loss of the fibrous cap. By 60 weeks of age, the lesions are characterized by the presence of collagen-rich fibrofatty nodules often flanked by lateral xanthomas. The processes underlying these changes in the innominate artery of older apoE(-/-) mice could well be a model for the critical processes leading to the breakdown and healing of the human atherosclerotic plaque.

Adenoviral-mediated delivery of the herpes simplex virus thymidine kinase gene selectively sensitizes human ovarian carcinoma cells to ganciclovir.

One strategy used for gene therapy of cancer is molecular chemotherapy. This approach is based on selective expression of an encoded toxin in cancer cells to achieve their eradication. One potential advantage of this strategy derives from a phenomenon, termed the bystander effect, whereby only a fraction of cells needs to be transduced to eradicate a tumor population. Despite the theoretical advantages of this phenomenon, it has only been described in a few cellular targets. Therefore, we undertook strategies to develop a molecular chemotherapy approach for ovarian carcinoma utilizing the herpes simplex virus thymidine kinase (HSV-TK) gene. Initially, we established that human ovarian carcinoma cell lines could be transduced at high efficiency with adenoviral vectors encoding reporter genes. We next determined that the human ovarian cell line SKOV3 could exhibit bystander killing by stably transducing it to express HSV-TK and performing cell mixing experiments with varying percentages of HSV-TK-expressing and HSV-TK-nonexpressing cells. Based on these findings, we constructed a recombinant adenovirus encoding HSV-TK and utilized it to induce human ovarian carcinoma cell lines to the sensitizing effects of ganciclovir. In addition, primary cultures of ovarian carcinoma cells were found to be highly transducible with recombinant adenoviral vectors and could be induced to the sensitizing effects of ganciclovir after induction of HSV-TK expression by the adenoviral vector. These studies indicate that molecular chemotherapy using a recombinant adenoviral vector expressing HSV-TK may provide a rational strategy for human ovarian carcinoma.

Adenoviral-mediated delivery of gastrin-releasing peptide receptor results in specific tumor localization of a bombesin analogue in vivo.

Radioimmunotherapy is hindered by a variety of factors linked to the utilization of monoclonal antibodies. These limitations include restricted tumor penetration as well as low levels of intratumoral antigen expression. To address the latter problem, we used a gene therapy approach to induce tumor cells to express enhanced levels of receptor with high binding affinity for a radiolabeled peptide. In this regard, a radiolabeled bombesin analogue was used in conjunction with a recombinant adenoviral vector encoding the murine gastrin-releasing peptide receptor (mGRPr). A panel of human carcinoma cell lines was infected in vitro with the recombinant adenoviral vector encoding the mGRPr vector to examine the induced binding of a 125I-labeled bombesin peptide. All cell lines examined displayed high levels of induced peptide binding, with approximately 60-80% of the radioactivity bound to the cells, in a live-cell binding assay. The human ovarian carcinoma cell line SKOV3.ip1 was chosen for in vivo analysis of radiolabeled bombesin analogue tumor localization in biodistribution and pharmacokinetic studies in athymic nude mice. Genetic induction of mGRPr in vivo resulted in selective tumor uptake of the radiolabeled peptide and high tumor:blood ratios. The biodistribution results compared favorably to those obtained with 131I-labeled e21 anti-erbB-2 monoclonal antibody in animals bearing i.p. SKOV3.ip1 tumors that endogenously express erbB-2. Thus, a novel method to combine gene transfer and radioimmunotherapy may result in augmented tumor cell targeting of radiopharmaceuticals.

Adenoviral-mediated delivery of herpes simplex virus thymidine kinase results in tumor reduction and prolonged survival in a SCID mouse model of human ovarian carcinoma.

The herpes simplex virus thymidine kinase gene is the most widely utilized toxin for selective killing of carcinoma cells. Expression of the viral thymidine kinase gene renders cells sensitive to the toxic effects of nucleoside analogs such as ganciclovir. An advantage of this system is the "bystander effect" whereby thymidine kinase transduced tumor cells elicit a toxic effect on surrounding nontransduced tumor cells. Ovarian carcinoma appears to be an ideal candidate for gene therapy as the majority of women present with advanced stage disease, have poor prognosis for long-term survival and have the disease confined within the peritoneal cavity. Therefore the utility of an adenoviral vector to elicit an in vitro bystander effect in ovarian carcinoma cells and the therapeutic efficacy of such a system in vivo was undertaken. Immunocompetent animals were utilized to determine the maximum dose of adenovirus that could be administered without any undesirable side effects and that preimmunization had no effects on subsequent challenge. SCID mice were orthotopically transplanted with human ovarian carcinoma cells and, after establishment of tumor, given a recombinant adenovirus expressing either the herpes simplex virus thymidine kinase or the Escherichia coli beta-galactosidase gene. Half the animals from each viral group were treated with either a ganciclovir regiment (50 mg/kg daily for 14 days) or an equal volume of serum-free media. A subset of mice were killed following drug treatment and analyzed for tumor reduction. The remaining animals were followed daily for survival. The animals treated with the recombinant adenovirus expressing the herpes simplex virus thymidine kinase gene and ganciclovir had significant reduction in overall tumor burden and demonstrated statistically significant prolongation in overall survival.

Accuracy of IgM immunoblotting to confirm the clinical diagnosis of early Lyme disease.

BACKGROUND: A 2-test approach for the serologic diagnosis of Lyme disease has recently been proposed. A positive or equivocal result on a first-stage test (eg, an enzyme immunoassay) is followed by a Western immunoblot test. For a sample to be considered seropositive for Lyme disease, the immunoblot result must be positive. OBJECTIVES: To assess the accuracy of IgM immunoblotting for detection of early Lyme disease and to establish interpretative criteria for a commercially available immunoblot assay. METHODS: Serum samples from 44 patients with erythema migrans were tested by an IgM immunoblot assay. All patients were culture-positive for Borrelia burgdorferi. Serum samples from 2 different control groups were also tested. Interpretative criteria were developed using receiver operating characteristic curves. RESULTS: The presence of any 2 IgM bands was found to be the optimal criterion for a positive test result, and in patients with illness of less than 7 days' duration, this was significantly more sensitive than the criterion of any 2 of the 3 specific bands defined by the Centers for Disease Control and Prevention/Association of State and Territorial Public Health Laboratory Directors Lyme Disease Workgroup (P < .05). Specificity of the criterion of any 2 bands was 100% for 1 group of controls but only 96% for the more clinically relevant control group; this small difference had a large impact on the positive predictive value in populations at low risk for Lyme disease. CONCLUSIONS: Using a commercially available immunoblot test kit, the presence of any 2 IgM bands is proposed as a positive result. The predictive value of a positive IgM immunoblot result, however, is poor in patients with minimal clinical evidence for Lyme disease.

Laboratory diagnostic techniques for patients with early Lyme disease associated with erythema migrans: a comparison of different techniques.

Recently, a number of refinements in diagnostic modalities for detection of Borrelia burgdorferi infection have been developed. These include large-volume blood cultures, quantitative polymerase chain reaction (PCR) techniques, and 2-stage serologic testing. In the present study, we compared 6 diagnostic modalities in 47 adult patients who had a clinical diagnosis of erythema migrans. Quantitative PCR on skin biopsy-derived material was the most sensitive diagnostic method (80.9%), followed by 2-stage serologic testing of convalescent-phase samples (66.0%), conventional nested PCR (63.8%), skin culture (51.1%), blood culture (44.7%), and serologic testing of acute-phase samples (40.4%). Results of all assays were negative for 3 patients (6.4%). We conclude that the clinical diagnosis of erythema migrans is highly accurate in an area where B. burgdorferi is endemic if it is made by experienced health care personnel, but some patients with this diagnosis may not have B. burgdorferi infection. No single diagnostic modality is suitable for detection of B. burgdorferi in every patient with erythema migrans.

Chlamydia pneumoniae infection accelerates hyperlipidemia induced atherosclerotic lesion development in C57BL/6J mice.

Considerable evidence of an association between Chlamydia pneumoniae infections and cardiovascular disease has emerged. Animal models using genetically altered mice and hypercholesterolemic rabbits have shown a pathogenic role of C. pneumoniae in accelerating atherosclerotic plaque development. In the present study, we evaluated the effect of chronic C. pneumoniae infection on atherosclerosis in C57BL/6J mice, fed either a regular chow diet or a high fat, high cholesterol diet. Infected animals on an atherogenic diet developed significantly larger lesion areas compared with control mice at 18 weeks (2.5-fold increase; 4177+/-777 vs. 1650+/-808 microm(2); P<0.05) and 24 weeks of age (3.3-fold increase; 14139+/-4147 vs. 4298+/-869 microm(2); P<0.02). This study shows that chronic C. pneumoniae infection accelerates atherosclerotic lesion development in diet induced hypercholesterolemic mice, indicating that C. pneumoniae is a co-risk factor of hyperlipidemia in atherogenesis.

Low level expression of hormone-sensitive lipase in arterial macrophage-derived foam cells: potential explanation for low rates of cholesteryl ester hydrolysis.

Conversion of arterial macrophages into foam cells is a key process involved in both the initiation and progression of atherosclerotic lesions. Foam cell formation involves the progressive accumulation and storage of lipoprotein-derived cholesteryl esters. The resulting imbalance in cholesterol metabolism in arterial foam cells may be due in part to an inadequately low level of cytoplasmic neutral cholesteryl ester hydrolase (NCEH) activity. In this study, we have demonstrated that hormone-sensitive lipase (HSL) mRNA is expressed at very low levels in macrophage-derived foam cells, using the unique approach of extracting mRNA from macrophage-derived foam cells purified from human and rabbit atherosclerotic plaques coupled with reverse transcriptase polymerase chain reaction (RT-PCR). We also demonstrate that macrophage-derived foam cells isolated from rabbit atherosclerotic lesions exhibit a resistance to high density lipoprotein (HDL)-mediated cholesterol efflux along with reduced levels of NCEH activity compared to lipid-loaded mouse peritoneal macrophages. Thus, low level expression of HSL may partially account for the reduced NCEH activity observed in arterial foam cells isolated from atherosclerosis-susceptible species.

Localization of iodine-125-mIP-Des-Met14-bombesin (7-13)NH2 in ovarian carcinoma induced to express the gastrin releasing peptide receptor by adenoviral vector-mediated gene transfer.

UNLABELLED: The gastrin releasing peptide receptor (GRPr) has a high affinity for the 14 amino acid bombesin peptide. For this analysis, [125I]-Tyr4-bombesin was compared with [125I]-mIP-bombesin (a seven amino acid bombesin analog) for in vitro binding and internalization into tumor cells and for tumor localization in vivo. Also, a recombinant adenoviral vector (AdCMVGRPr) was used for gene transfer to induce the expression of GRPr in human ovarian cancer cells for binding and tumor localization with these radiolabeled peptides. METHODS: [125I]-mIP-bombesin was synthesized and compared with [125I]-Tyr4-bombesin in internalization assays using BNR-11 cells (mouse fibroblast cells stably transfected with GRPr) over a 24-hr period. In vitro binding assays used BNR-11, and A427, HeLa and SKOV3.ip1 human cancer cells, which were either uninfected or infected with AdCMVGRPr. Biodistribution studies were performed in normal BALB/c mice and in athymic nude mice bearing orthotopic SKOV3.ip1 ovarian cancer tumors. The SKOV3.ip1 tumors were induced to express GRPr with the AdCMVGRPr adenoviral vector. RESULTS: Internalization assays showed that [125I]-Tyr4-bombesin was rapidly internalized and catabolized at 37 degrees C with approximately 10% of the radioactivity remaining intracellularly at 4 hr, compared with approximately 30% with [125I]-mIP-bombesin. HeLa, A427 and SKOV3.ip1 cells were all induced to express levels of GRPr that were higher than those seen with the positive control BNR-11 cells. Normal mice showed a lower level of radioactivity in both the blood and thyroid for [125I]-mIP-bombesin [0.26% +/- 0.10% injected dose per gram (ID/g) and 0.24% +/- 0.05% ID] than for [125I]-Tyr4-bombesin (3.5% +/- 1.6% ID/g and 5.2% +/- 4.4% ID) at 4 hr postinjection. Mice bearing intraperitoneal (i.p.) SKOV3.ip1 tumors and given AdCMVGRPr i.p. 5 days after tumor cell inoculation followed by [125I]-mIP-bombesin i.p. at day 7 showed 16.5% +/- 4.8% ID/g in tumor compared with 5.9% +/- 3.0% ID/g with [125I]-Tyr4-bombesin at 4 hr postinjection. Tumor bearing mice given saline or a control adenovirus expressing the beta-galactosidase (LacZ) gene showed significantly lower tumor uptake values of both bombesin peptides. CONCLUSION: Internalization assays showed that [125I]-mIP-bombesin has favorable characteristics compared with [125I]-Tyr4-bombesin with regards to cellular internalization and retention. The results demonstrate successful in vitro and in vivo transduction of human tumor cells with a recombinant adenoviral vector-expressing GRPr. Additionally, tumors transduced in vivo to express GRPr demonstrated significantly greater localization of [125I]-mIP-bombesin when compared with [125I]-Tyr4-bombesin.

In vivo and in vitro studies on Anaplasma phagocytophilum infection of the myeloid cells of a patient with chronic myelogenous leukaemia and human granulocytic ehrlichiosis.

AIMS: The occurrence of human granulocytic ehrlichiosis (HGE) in a patient with chronic myelogenous leukaemia (CML) provided an opportunity to study whether Anaplasma phagocytophilum, the aetiological agent of HGE, infects mature or immature cells, both in vivo and in vitro. METHODS: Diagnosis of HGE was confirmed by culture, polymerase chain reaction (PCR), detection of intragranulocytic inclusions, and serology. The infection rates of different myelogenous stages of granulocytic differentiation were determined by microscopy. Anaplasma phagocytophilum infection of the bone marrow was analysed by PCR, culture, and microscopy. In addition, the in vitro growth of A phagocytophilum in the patient's granulocytes and in HL-60 cells (a promyelocytic leukaemia cell line) was compared. RESULTS: Pretreatment blood smears showed that mature granulocytic cells had a higher infection rate with A phagocytophilum than did immature cells. In the original inoculation of the patient's cells into HL-60 cells to isolate A phagocytophilum, the bacterium grew faster in the patient's leukaemic cells than in HL-60 cells. Anaplasma phagocytophilum inclusions were rarely seen in bone marrow granulocytes and PCR was negative. In vitro, two A phagocytophilum isolates grew faster in the patient's granulocytes than in HL-60 cells. CONCLUSIONS: The superior growth in CML cells compared with HL-60 cells suggests that A phagocytophilum preferentially infects mature granulocytes. The higher infection rate of the patient's mature versus immature granulocytes before treatment and the minimal level of infection of the patient's bone marrow support this. It is possible that the primary site of infection in HGE is the peripheral mature granulocytic population.

Cellular mechanisms in the development of atherosclerosis.

Morphologic and immunocytochemical studies of hypercholesterolemic animal models have now clearly established the chronological patterns of cellular interactions that occur during the initial and transitional phases of the atherogenic process. These include: adherence of leukocytes to the endothelial surface, chemotactic attraction of the leukocytes into the arterial intima, conversion of monocytes to foam cells, stimulation of smooth muscle cell migration, connective tissue synthesis and proliferation, inflammatory and immune activation of macrophages and T lymphocytes, and the necrosis or apoptosis of cells within the developing lesions. Recent studies have begun to provide-mechanistic explanations for these observed cellular events. For example, the adherence of leukocytes to the endothelium appears to be dependent on the increased expression of adherence molecules by endothelial cells. The formation of foam cells is likely dependent on an increase in the expression of modified lipoprotein receptors. An increase in the migration and proliferation of macrophages, T lymphocytes, and smooth muscle cells appears to be in response to the inflammatory activation of cells with a resulting increase in the secretion of cytokines, chemoattractants, and growth regulatory molecules. However, it is still unclear how cells within atherosclerotic lesions initially become activated and whether there are common stimulatory factors. In this regard, immunocytochemical staining of human and rabbit lesions with antibodies recognizing oxidation-specific epitopes suggests that many of the cells involved in these key events in the atherogenic process contain these lipid-protein adducts and that it is these products of oxidation that activate the cells. Furthermore, we have also recently demonstrated that components of oxidized LDL maximally induce the production of IL-1 by macrophage-derived foam cells. These observations suggest that there may be a common intracellular signal transduction pathway that is responsive to oxidative mechanisms and which underlies some of the key cellular events in the atherogenic process.

Detection of dissection and remodeling of atherosclerotic lesions in rabbits after balloon angioplasty by magnetic-resonance imaging.

OBJECTIVE: To assess the usefulness of magnetic-resonance imaging (MRI) for monitoring acute changes after angioplasty of preexisting lesions in rabbits with basal lesions similar to those observed in humans. METHODS: A combination of Fogarty balloon injury (1 week after initiation of diet) and a mildly hypercholesterolemic diet (0.2% cholesterol and 5% peanut oil) was used to promote the rapid formation of atherosclerotic lesions in 16 New Zealand white rabbits. After 5 months of the diet, angioplasty was performed on these lesions with a Grüntzig catheter in both iliac arteries and the abdominal aorta. MRI was used to monitor the initial formation of lesions after 3 and 5 months of the diet, and 2 days, 2 weeks, and 1 and 2 months after angioplasty. RESULTS: The combination of early Fogarty injury and mildly hypercholesterolemic diet induced fibroproliferative lesions similar to type Vb atherosclerotic lesions seen in humans. Angioplasty induced deep dissections at the shoulders of lesions in the majority of animals. These dissections often extended into the media. The cellular, proliferative response after angioplasty was localized and limited to sites of dissection. A significant increase in area of arterial wall was observed after angioplasty at sites of dissection without any loss of lumen. In contrast, proximal and distal to the sites of injury, there was no change in wall area but a transient reduction in lumen area. CONCLUSIONS: Comparison of MRI results with histology confirmed that changes in the wall and lumen, including small linear dissections in the lesions and arterial remodeling, are detectable by MRI.

Lyme carditis: complete AV dissociation with episodic asystole presenting as syncope in the emergency department.

We report a case of Lyme carditis in an otherwise-healthy young male who presented to the Emergency Department (ED) with syncope and a possible seizure. This patient, without documented history of Lyme disease, acutely developed third-degree atrioventricular (AV) block with episodic asystole, which required placement of a transvenous pacemaker in the ED and resolved only after the patient had been placed on ceftriaxone. We discuss the significance of Lyme carditis and its increasing prevalence, and review the current literature. We also recommend appropriate screening modalities for patients with known Lyme disease, or an atypical profile for cardiac abnormalities.

Clinical and laboratory evolution of a culture-confirmed case of human granulocytic ehrlichiosis.

A 74-year-old man from suburban New York City, who was hospitalized because of chest pain and fever, was diagnosed as having human granulocytic ehrlichiosis on the eighth hospital day. Although leukocyte and platelet counts were normal on admission, they fell to abnormally low values then normalized prior to specific therapy against the human granulocytic ehrlichiosis agent. Intracytoplasmic inclusions suggestive of Ehrlichia were observed in up to six percent of granulocytes, and the human granulocytic ehrlichiosis bacterium was cultured in an HL 60 human promyelocytic cell line. The patient improved dramatically within 24 hours of doxycycline treatment, after failing to improve on various beta lactam antimicrobial agents. He was discharged from the hospital 14 days after admission. Because human granulocytic ehrlichiosis was not diagnosed until his eight hospital day, clinical and laboratory parameters prior to specific treatment were available. This case illustrates the clinical and laboratory evolution of the infection with human granulocytic ehrlichiosis agent in humans.