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A Daugherty

Publications and source records attributed to A Daugherty.

At least 37 records · Page 2Linked to original sources
PubMed1998

Absence of T lymphocyte-derived cytokines fails to diminish macrophage 12/15-lipoxygenase expression in vivo.

IL-4 and IL-13 are the only known activators of 15-lipoxygenase (LO) expression in cultured macrophages. To determine whether these lymphocyte-derived cytokines regulate 15-LO expression in vivo, the abundance of the murine homologue (12/15-LO) was assessed in peritoneal macrophages from immune-deficient strains of mice. Macrophages were harvested from recombinase activator gene (RAG)-2-/- mice that do not develop mature lymphocytes and cannot secrete activation-dependent cytokines. Unexpectedly, 12/15-LO protein and activity were significantly increased in peritoneal macrophages from RAG-2-/- mice compared with strain-matched controls. This increase was related to phenotypic differences between cells from RAG-2+/+ and RAG-2-/- mice. After 3 h in culture, RAG-2+/+ macrophages were of two distinct sizes, with only the larger cells immunostaining for 12/15-LO. However, all RAG-2-/- cells were distributed in the large size range, and all were immunoreactive for the enzyme. The activation of 12/15-LO expression appears to be related to prolonged residence within the peritoneum, since there were fewer resident peritoneal macrophages in RAG-2-/- than in RAG-2+/+ mice, and newly recruited macrophages elicited by the administration of Sephacryl to RAG-2-/- mice did not immunostain for 12/15-LO. To determine whether 12/15-LO expression was due to IL-4 or IL-13 from nonlymphoid cells, the abundance of the enzyme was quantified in peritoneal macrophages from STAT6-/-mice that have attenuated responses to both cytokines. STAT6 deficiency did not influence the abundance of the protein in macrophages. Therefore, neither IL-4 nor IL-13 secretion is a requirement for macrophage 15-LO expression in vivo.

Animals↗
PubMed1998

A specific 15-lipoxygenase inhibitor limits the progression and monocyte-macrophage enrichment of hypercholesterolemia-induced atherosclerosis in the rabbit.

Oxidant signalling and lipoprotein oxidation may play important roles in atherosclerotic lesion development. Given coincident localization of 15-lipoxygenase (15-LO), stereospecific products of 15-LO and epitopes of modified LDL in atherosclerotic lesions, we hypothesized that inhibition of 15-LO by PD146176, an inhibitor of 15-LO with an IC50 in cells or isolated enzyme of 0.5-0.8 microM, may limit atherosclerotic lesion development through regulation of monocyte-macrophage enrichment. Rabbits exposed to chronic endothelial denudation of the iliac-femoral artery were meal-fed a 0.25% cholesterol (C), 3% peanut oil (PNO), 3% coconut oil (CNO) diet twice daily with and without 175 mg/kg PD146176 for 12 weeks. In a second study, atherosclerotic lesions were pre-established in rabbits through chronic endothelial denudation and meal-fed a 0.5% C, 3% PNO, 3% CNO diet for 9 weeks and a 0% C/fat diet for 6 weeks prior to an 8 week administration of PD146176 at 175 mg/kg, q.d. Plasma total and lipoprotein cholesterol exposure were similar in control and PD146176-treated animals in both studies but PD146176 increased plasma triglyceride exposure 2- to 4-fold. Plasma PD146176 concentrations ranged from 99 to 214 ng/ml at 2 h post-dose. In the progression study, the iliac-femoral monocyte-macrophage area was reduced 71%, cross-sectional lesion area was unchanged and cholesteryl ester (CE) content was reduced 63%. In the regression study, size and macrophage content of iliac-femoral, fibrous plaque-like lesions were decreased 34%, CE content was reduced 19% and gross extent of thoracic aortic lesions were reduced 41%. We conclude that PD146176 can limit monocyte macrophage enrichment of atherosclerotic lesions and can attenuate development of fibrofoamy and fibrous plaque lesions in the absence of changes in plasma total or lipoprotein cholesterol concentrations.

Animals↗
PubMed1998

Development of a single-shot subunit vaccine for HIV-1. 5. programmable in vivo autoboost and long lasting neutralizing response.

The subunit vaccine for HIV-1, recombinant glycoprotein 120 (rgp120), was used as a model antigen to evaluate the potential for a pulsatile single immunization vaccine formulation consisting of poly(lactic-co-glycolic) acid (PLGA) microspheres. We designed rgp120 PLGA microsphere formulations that provide a pulse of rgp120 at 1 to 6 months (depending on the polymer) after administration, mimicking another immunization. In these studies, the in vitro pulse of rgp120 correlated well with the observed in vivo autoboost as measured by an increase in anti-gp120 antibodies in guinea pigs. The immune response to the rgp120 PLGA microsphere formulations was increased by adding the soluble form of the saponin-derived adjuvant, QS-21. The use of small microspheres, however, did not increase the humoral response to rgp120. A single immunization with rgp120 PLGA microspheres resuspended in soluble rgp120 and QS-21 elicited neutralizing antibody titers that were comparable to titers obtained from two immunizations of rgp120 and QS-21 at the same total dose. Administration of rgp120 PLGA microspheres in baboons resulted in high, long-lasting neutralizing antibody titers that were greater than repeated immunizations with soluble rgp120 and QS-21. These studies also indicated that a continuous release of QS-21 at the injection site may provide a greater immune response than a bolus injection. Overall, this work demonstrated that PLGA microsphere formulations may be designed to provide in vivo pulses of an antigen eliminating the need for repeated immunizations.

AIDS Vaccines↗
PubMed1998

Effects of heterozygous lipoprotein lipase deficiency on diet-induced atherosclerosis in mice.

Heterozygous lipoprotein lipase deficiency (LPL+/-) is common and has been implicated in premature atherosclerosis in epidemiologic studies. However, in vitro data suggest that LPL deficiency in the vascular wall may be antiatherogenic. To address the role of LPL in atherosclerosis, LPL+/- mice in the C57BL/6J background were fed an atherogenic diet for 8 months. LPL+/- mice were more dyslipidemic than +/+ animals due to increased concentrations of non-HDL lipoproteins. There was no difference in aortic origin atherosclerosis between LPL+/- (n=56) and +/+ (n=55) mice. LPL+/- mice in the low density lipoprotein receptor knockout (LDLR-/-) background were fed the same atherogenic diet for 3 months. LPL+/-LDLR-/- mice were more dyslipidemic than LPL+/+LDLR-/- animals. There was no difference in atherosclerosis assayed for the entire aorta and no difference in aortic sterol content between LPL+/-LDLR-/- (n=28) and LPL+/+LDLR-/- (n=15) mice. LPL protein was detected in murine lesions in a consistent layered pattern. More luminal, lipid-laden macrophages generally did not stain for LPL, but deeper, lipid-poor macrophages as well as necrotic core regions contained immunoreactive LPL. LPL protein was more abundant in lesions from LPL+/+ LDLR-/- than LPL+/-LDLR-/- mice. After eating an atherogenic diet, LPL+/- as compared to LPL+/+ mice have more dyslipidemia, but no more atherosclerosis, and less LPL protein in atherosclerotic lesions. These data suggest that lipoprotein lipase deficiency in the vascular wall could prevent the retention of atherogenic lipoproteins.

Animals↗
PubMed1998

Apolipoprotein E-deficient mice have impaired innate immune responses to Listeria monocytogenes in vivo.

Apolipoprotein E (apoE) influences both innate and acquired immunity in cultured cells. To determine whether apoE affects the immune system in vivo, Listeria monocytogenes (LM) was administered intraperitoneally (10(4) c.f.u.) to congenic C57BL/6 apoE-/- and +/+ mice (n = 12 in each group). Survival was assessed daily for 5 days. Deficiency of apoE significantly increased death by day 5 (P = 0.03). The majority of deaths occurred at day 4. Extent of infection after LM administration was assessed at day 3 by determining colony counts in hepatic and splenic extracts. ApoE+/+ mice had very low colony counts in both spleen and liver [mean +/- SE: 2.0 +/- 0.5 and 0.7 +/- 0.2 (x 10(4)), respectively, n = 8 in each group]; while apoE-/- mice had significantly increased counts in both spleen and liver [64 +/- 51 and 98 +/- 93 (x 10(4)), P = 0.05 and 0.03]. Serum concentrations of TNF-alpha were significantly increased in apoE-/- mice at day 3 compared to apoE+/+ mice (127 +/- 43 pg/ml versus 20 +/- 17, P = 0.003). LM induced more hepatic damage in apoE-/- mice compared to apoE+/+ mice as judged by increased serum concentrations of alanine aminotransferase at day 1 (apoE-/- 301 +/- 45 U/ml, apoE+/+ 101 +/- 9 U/ml, P = 0.01). The increased proliferation and mortality from LM in apoE-/- mice occurred prior to the initiation of acquired immune responses. Therefore, apoE-deficient mice have an impaired innate response to infection by LM.

Animals↗
PubMed1997

The effects of total lymphocyte deficiency on the extent of atherosclerosis in apolipoprotein E-/- mice.

Activated T lymphocytes are present in human atherosclerotic lesions and autoantibodies to antigens within lesions have been detected in serum, but the roles of the cellular and humoral immune systems in atherogenesis have not been determined. The effect of total lymphocyte deficiency on atherogenesis was investigated by crossing apo E-deficient mice (which develop atherosclerosis resembling human disease) with mice deficient in RAG2 (which is required for normal B and T lymphocyte development). Mice were placed on a fat- and cholesterol-enriched diet for 12 wk. RAG2-deficient mice had no serum autoantibodies, in contrast to the high titers in RAG2+/- littermates. There were no T lymphocytes and a markedly reduced number of MHC class II-positive macrophages in atherosclerotic lesions of RAG2-deficient mice. Despite these differences, RAG2-deficient mice developed atherosclerosis similar in extent to that in immunocompetent littermates, based on quantification by two independent methods. In conclusion, the absence of autoantibodies and T lymphocytes did not influence the extent of aortic atherosclerotic lesions in apo E-deficient mice.

Animals↗
PubMed1997

Attenuation of diet-induced atherosclerosis in rabbits with a highly selective 15-lipoxygenase inhibitor lacking significant antioxidant properties.

1. 15-Lipoxygenase (15-LO) has been implicated in the pathogenesis of atherosclerosis because of its localization in lesions and the many biological activities exhibited by its products. To provide further evidence for a role of 15-LO, the effects of PD 146176 on the development of atherosclerosis in cholesterol-fed rabbits were assessed. This novel drug is a specific inhibitor of the enzyme in vitro and lacks significant non specific antioxidant properties. 2. PD 146176 inhibited rabbit reticulocyte 15-LO through a mixed noncompetitive mode with a Ki of 197 nM. The drug had minimal effects on either copper or 2,2'-azobis(2-amidinopropane)hydrochloride (ABAP) induced oxidation of LDL except at concentrations 2 orders higher than the Ki. 3. Control New Zealand rabbits were fed a high-fat diet containing 0.25% wt./wt. cholesterol; treated animals received inhibitor in this diet (175 mg kg-1, b.i.d.). Plasma concentrations of inhibitor were similar to the estimated Ki (197 nM). During the 12 week study, there were no significant differences in weight gain haematocrit, plasma total cholesterol concentrations, or distribution of lipoprotein cholesterol. 4. The drug plasma concentrations achieved in vivo did not inhibit low-density lipoprotein (LDL) oxidation in vitro. Furthermore, LDL isolated from PD 146176-treated animals was as susceptible as that from controls to oxidation ex vivo by either copper or ABAP. 5. PD 146176 was very effective in suppressing atherogenesis, especially in the aortic arch where lesion coverage diminished from 15 +/- 4 to 0% (P < 0.02); esterified cholesterol content was reduced from 2.1 +/- 0.7 to 0 micrograms mg-1 (P < 0.02) in this region. Immunostainable lipid-laden macrophages present in aortic intima of control animals were totally absent in the drug-treated group. 6. Results of these studies are consistent with a role for 15-LO in atherogenesis.

Animals↗
PubMed1997

Lipopolysaccharide decreases scavenger receptor mRNA in vivo.

Lipopolysaccharide (LPS) downregulates scavenger receptor (ScR) activity in cultured macrophages through release of tumor necrosis factor-alpha (TNF-alpha). However, LPS administration in vivo stimulates cytokine release from both macrophages and lymphocytes, the combined effects of which could alter ScR expression differently from TNF-alpha in isolation. To investigate whether LPS regulates ScR in vivo, 10 microg/g was injected i.p. into Swiss Webster mice. Administration of LPS produced a profound decrease in hepatic ScR mRNA, with reductions of 74% +/- 8% at 2 h that returned to baseline levels by 6 h. Changes in ScR mRNA abundance coincided with changes in serum concentrations of TNF-alpha, which peaked at 2 h (1320 +/- 309 pg/ml) and returned to preinjection concentrations at 4 h. Serum concentrations of interferon-gamma (IFN-gamma) did not increase until 4 h after injection of LPS. There was no effect on ScR mRNA abundance following LPS administration to LPS-resistant strains of mice, C3H/HeJ and IFN-gamma receptor-/-. The LPS-induced reduction in ScR mRNA in Swiss Webster mice was not sufficiently sustained to affect receptor function, as determined by the kinetics of [(125)I]-acetylated LDL clearance from plasma. Therefore, as observed in cultured cells, LPS administration to mice decreases ScR mRNA despite the release of several cytokines in vivo.

Animals↗
PubMed1997

Scavenger receptors are present on rabbit aortic endothelial cells in vivo.

Endothelial cells metabolize modified LDL, but attempts to detect scavenger receptors in this cell type in vitro have been unsuccessful. To determine whether scavenger receptors are present on endothelial cells in vivo, species-specific reagents were developed to detect rabbit scavenger receptor protein. Antiserum against the rabbit scavenger receptor was generated with the use of synthetic peptides of two distinct regions: residues 3 to 21 in the cytoplasmic tail and residues 282 to 304 in the collagen-like region. Reactivity of antiserum against the synthetic peptides was confirmed with an enzyme-linked immunosorbent assay. Positive reactivity was also observed against fragments of scavenger receptor protein expressed in bacteria. Antiserum to both regions reacted with liver membrane proteins of sizes consistent with the scavenger receptor, as confirmed by Western blotting under reduced and nonreduced conditions. Immunocytochemical examination of rabbit aortic tissue by use of antiserum to both regions of scavenger receptor protein produced striking and identical patterns of staining of aortic endothelium. Immunostaining was abolished for both antisera by preadsorption with the specific peptide region used as immunogen. In contrast, incubation of scavenger receptor antiserum with a peptide of a region of the rabbit LDL receptor failed to influence immunoreactivity against endothelium. These data demonstrate the presence of scavenger receptors in rabbit endothelium in vivo, which may have fundamental implications for lipoprotein metabolism by the arterial wall.

Amino Acid Sequence↗
PubMed1996

Apolipoprotein E-containing high density lipoprotein promotes neurite outgrowth and is a ligand for the low density lipoprotein receptor-related protein.

Presence of the epsilon4 allele of apolipoprotein E (apoE) is a risk factor for Alzheimer's disease (AD), although the mechanism(s) by which it confers this risk is unknown. ApoE may play a direct role in AD neuropathology by modulating neuronal structure. We previously showed that apoE3-containing beta-very low density lipoprotein (beta-VLDL) can stimulate neurite outgrowth to a significantly greater extent than apoE4-enriched beta-VLDL in a central nervous system-derived neuronal cell line and that this effect is mediated by interaction with the low density lipoprotein receptor-related protein (LRP). To determine whether similar differences exist when apoE is associated with other lipoprotein particles, the effects of high density lipoprotein (HDL) derived from plasma and cerebrospinal fluid were defined. ApoE3-enriched HDL significantly enhanced neurite outgrowth as compared with apoE4-enriched HDL, and the majority of this stimulation was blocked in the presence of the receptor-associated protein or a neutralizing antibody to LRP. We also found that cholesterol esterification in the presence of apoE-containing plasma HDL was attenuated in fibroblasts lacking LRP. Therefore, apoE-containing HDL can serve as an LRP ligand, and apoE isoform-specific effects on neurite outgrowth are observed when HDL is the carrier particle.

Apolipoproteins E↗
PubMed1996

Development of a single-shot subunit vaccine for HIV-1. 2. Defining optimal autoboost characteristics to maximize the humoral immune response.

The design of a single-shot subunit vaccine for HIV-1 with polylactic-coglycolic acid (PLGA) sustained-release technology to effect an autoboost of antigen (MN gp120) at a given time after the primary immunization requires in-depth knowledge about the timing, the duration, and the need for coadjuvant in the autoboost. These questions cannot be answered unambiguously with PLGA microspheres, so we have conducted studies using Alzet minipumps to release antigen at prescribed times to mimic a PLGA autoboost. The results show that a discrete autoboost is preferred over continuous release of antigen, that the time profile of the autoboost (whether pulsatile or a 2-week continuous release) does not affect the booster immune response, and that only antigen is required in the booster immunization (a coadjuvant in the boost does not give higher titers).

AIDS Vaccines↗
PubMed1996

Development of a single-shot subunit vaccine for HIV-1. 3. Effect of adjuvant and immunization schedule on the duration of the humoral immune response to recombinant MN gp120.

HIV-1 prophylaxis may require "sterilizing immunity" (i.e., the prevention of infection), and this is likely to demand a vaccine that gives high, long-lasting antibody titers. Although it is known that vaccine adjuvants and immunization schedule affect the magnitude of the immune response, there are few reports on antibody decay rates and persistence. Guinea pigs were immunized with recombinant gp120 using different adjuvants and immunization schedules, and the anti-gp120 and HIV-1 neutralization titers were determined over time following the last booster immunization. As observed previously in the literature, a longer time between boosting gave higher titers, with a slight increase in the decay half-life as the booster was spaced farther out from the primary immunization. The decay rate of the antibody titers showed surprisingly little effect of adjuvant, except for sustained-release polymer-based formulations. Adjuvants that gave high titers initially after boosting showed the greatest persistence of antibody titers (persistence defined as the residual titers at long times). These data show that high, long-lasting titers may be achieved by using sustained-release formulations, and these are likely the prime vaccine candidates for prophylaxis requiring prolonged sterilizing immunity.

AIDS Vaccines↗
PubMed1996

Mouse peritoneal macrophages contain abundant omega-6 lipoxygenase activity that is independent of interleukin-4.

The action of an omega-6 lipoxygenase (LO) has been implicated in the development of atherosclerosis through a mechanism involving oxidation of LDL, and its regulation in macrophages may have important implications for the disease process. Human monocyte-derived macrophages (HMDMs) showed no demonstrable LO protein or activity unless they were incubated with interleukin-4 (IL-4). In contrast, mouse peritoneal macrophages (MPMs) possessed significant basal levels of LO activity and protein that were augmented by IL-4 treatment. Interferon gamma prevented the induction of LO in both HMDMs and MPMs. Whereas interferon gamma could completely block the IL-4 induction of LO in human cells, it did not suppress basal LO activity in MPMs. Both HMDMs and MPMs exhibited similar concentration-response relationships for stimulation of LO activity and protein, with maximal induction at 1 ng/mL IL-4. The time course of IL-4 induction of LO activity was markedly different in human and murine cells. IL-4 induced LO activity and protein in human cells by 48 hours that were maximal by 72 hours; there was a decline to a new baseline by 96 hours. MPMs have a significant amount of LO activity at baseline, which declined with time by nearly 10-fold in the absence of IL-4, IL-4 blunted the decline of LO activity with time and restored activity to that found at baseline by 48 hours. IL-4 was not responsible for the LO activity present in freshly isolated MPMs since both activity and protein content were similar in cells harvested from IL-4+/+ and IL-/- mice. Therefore, whereas IL-4 may be an important modulator of LO production in vitro, it is not essential for the in vivo expression of this protein. Further, these studies demonstrate that significant differences exist between monocyte-derived macrophages matured in vitro and tissue macrophages that have matured in vivo.

Animals↗
PubMed1996

Lymphocyte populations in atherosclerotic lesions of apoE -/- and LDL receptor -/- mice. Decreasing density with disease progression.

Lymphocytes are prominent components of human atherosclerotic lesions, but their presence in murine models of disease has not been confirmed. Lymphocyte subpopulations have been identified in apoE -/- and LDL receptor -/- mice fed a cholesterol-enriched diet for up to 3 months. ApoE -/- mice had higher serum cholesterol concentrations than did LDL receptor -/- mice during most of the feeding period, primarily due to large increases in VLDL concentrations. Total area of atherosclerotic lesions was greater at all times in apoE -/- than LDL receptor -/- mice (lesion area after 3 months on cholesterol-enriched diet: apoE -/-, 993 +/- 193 and LDL receptor -/-, 560 +/- 131 microns2 x 10(3), mean +/- SEM, n = 6 in each group). Lesions in apoE -/- mice contained larger macrophage-rich necrotic cores and more calcification than did those in LDL receptor -/- mice. Immunocytochemical analyses of tissue sections of ascending aortas performed with monoclonal antibodies to T and B lymphocytes and macrophages revealed that T lymphocytes immunoreactive for Thy 1.2, CD5, CD4, and CD8 were observed in lesions from both strains, but no B lymphocytes were detected. The density of Thy 1.2+ T lymphocytes in lesions was greatest at 1 month (apoE -/-, 98 +/- 23 and LDL receptor -/-, 201 +/- 40 lymphocytes/mm2, n = 6 in each group), decreasing in apoE -/- mice to 12 +/- 3 and in LDL receptor -/- mice to 51 +/- 20 lymphocytes/mm2 at 3 months. The presence of T lymphocytes in murine atherosclerotic lesions makes these animals potentially useful for studying the involvement of the immune system in atherogenesis.

Animals↗
PubMed1995

Augmented urokinase receptor expression in atheroma.

Smooth muscle cell proliferation and migration into neointima are hallmarks of atherogenesis. However, mechanisms responsible have not yet been fully elucidated. One potential mediator of both smooth muscle cell proliferation and migration is activation of plasminogen by activators bound to receptors on cells within the vessel wall. To determine whether vascular smooth muscle cells within atheroma express the receptor for urokinase-type plasminogen activator (uPA-R), we characterized atheroma in cholesterol-fed New Zealand White rabbits and human subjects by immunostaining. Intense immunostaining of uPA-R was observed throughout the neointima in both rabbit and human atherosclerotic lesions with the use of a monoclonal antibody to uPA-R. uPA-R was not detectable in normal arterial tissues. uPA-R was localized to macrophages and neointimal smooth muscle cells identified by immunostaining in serial sections. Furthermore, uPA-R protein in extracts from atheroma was present in at least a ninefold greater quantity compared with extracts from normal vessels, as shown by Western blotting. Expression of uPA-R mRNA in migrating vascular smooth muscle cells did not increase significantly. Thus, altered posttranscriptional regulation may be contributing to the increased uPA-R. In vitro, antibodies to uPA-R delayed the migration of cultured vascular smooth muscle cells. Our results suggest that increased cell-surface uPA-R contributes to pericellular proteolysis and consequently increased neointimalization secondary to increased vascular smooth muscle cell migration in atheroma.

Animals↗