Kynurenic acid, an aryl hydrocarbon receptor ligand, is elevated in serum of Zucker fatty rats.

Obesity is an increasingly urgent global problem and the molecular mechanisms of obesity are not fully understood. Dysregulation of the tryptophan (Trp) - kynurenine (Kyn) metabolic pathway (TKP) have been suggested as a mechanism of obesity and described in obese humans and in animal models of obesity. However, to the best of our knowledge, TKP metabolism has not been studied in leptin-receptor-deficient Zucker fatty rats (ZFR) (fa/fa), the best-known and most widely used rat model of obesity. We were interested to determine if there are any deviations of TKP in ZFR. Concentrations of major TKP metabolites were evaluated (HPLC- MS method) in serum of ZFR (fa/fa) and age-matched lean rats (FA/-). Concentrations of kynurenic acid (KYNA) were 50% higher in ZFR than in lean rats (p<0.004, Mann-Whitney two-tailed test). Anthranilic acid (AA) concentrations, while elevated by 33%, did not reach statistical significance (p<0.04, one-tailed test). Elevated KYNA serum concentrations might contribute to development of obesity via KYNA-induced activation of aryl hydrocarbon receptor. Present results warrant further studies of KYNA and AA in ZFR and other animal models of obesity.

Recombinant LH supplementation to recombinant FSH during induced ovarian stimulation in the GnRH-antagonist protocol: a meta-analysis.

This study aims to compare the efficacy of recombinant LH (rLH) supplementation for ovarian stimulation in gonadotrophin-releasing hormone-antagonist protocol for IVF/intracytoplasmic sperm injection cycles. Search strategies included online surveys of databases. The fixed effects model was used for odds ratio (OR) and effect size (weighted mean difference, WMD). Five trials fulfilled the inclusion criteria. When the meta-analysis was carried out, advantages were observed for the LH supplementation protocol with respect to higher serum oestradiol concentrations on the day of human chorionic gonadotrophin administration P < 0.0001; WMD: 514, 95% CI 368, 660) and higher number of mature oocytes (P = 0.0098; WMD: 0.88, 95% CI 0.21, 1.54). However, these differences were not observed in the total amount of recombinant FSH (rFSH) administered, days of stimulation, number of oocyets retrieved, the clinical pregnancy rate per oocyte retrieval, the implantation rate and miscarriage rate. This result demonstrates that the association of rLH with rFSH may prevent any decrease in oestradiol after antagonist administration and that a significantly higher number of mature oocytes was available for laboratory work. Nevertheless, it failed to show any statistically significant difference in clinically significant end-points in IVF (implantation and pregnancy rates). Additional randomized controlled trials are needed to confirm these results further.

GnRH agonist versus GnRH antagonist in poor ovarian responders: a meta-analysis.

The aim of this meta-analysis was to compare the efficacy of gonadotrophin antagonist (GnRH-ant) versus GnRH agonist (GnRHa) as coadjuvant therapy for ovarian stimulation in poor ovarian responders in IVF/intracytoplasmic sperm injection cycles. Search strategies included on-line surveys of databases such as MEDLINE , EMBASE and others. A fixed effects model was used for odds ratio (OR) and effect size (weighted mean difference, WMD). Six trials fulfilled the inclusion criteria (randomized controlled trials). There was no difference between GnRH-ant and GnRHa (long and flare-up protocols) with respect to cycle cancellation rate, number of mature oocytes and clinical pregnancy rate per cycle initiated, per oocyte retrieval and per embryo transfer. When the meta-analysis was applied to the two trials that had used GnRH-ant versus long protocols of GnRHa, a significantly higher number of retrieved oocytes was observed in the GnRH-ant protocols [P=0.018; WMD: 1.12 (0.18, 2.05)]. However, when the meta-analysis was applied to the four trials that had used GnRH-ant versus flare-up protocols, a significantly higher number of retrieved oocytes (P=0.032; WMD: -0.51, 95% CI -0.99, -0.04) was observed in the GnRHa protocols. Nevertheless, additional randomized controlled trials with better planning are needed to confirm these results.

Macrophage-specific expression of class A scavenger receptors enhances granuloma formation in the absence of increased lipid deposition.

Class A scavenger receptors (SR-A) have several proposed functions that could impact atherosclerosis and inflammatory processes. To define the function of SR-A in vivo, we created C57BL/6 transgenic mice that expressed bovine SR-A under the control of the restricted macrophage promoter, lysozyme (lyso-bSR-A). bSR-A mRNA was present in cultured peritoneal macrophages of transgenic mice and tissues that contain significant macrophages including spleen, lung, and ileum. Functional overexpression of SR-A was demonstrated in peritoneal macrophages both by augmented cholesterol ester deposition in response to AcLDL and enhanced adhesion in transgenic mice compared with nontransgenic littermates. To determine whether macrophage-specific expression of bSR-A regulated inflammatory responses, granulomas were generated by subcutaneous injection of carrageenan. Granuloma size was significantly increased in lyso-bSR-A transgenic mice compared with wild-type littermates [421 +/- 51 mg (n = 11) vs. 127 +/- 22 mg (n = 10), P < 0.001]. However, the larger granulomas in lyso-bSR-A transgenic mice were only associated with an increase in unesterified cholesterol, and not cholesterol esters. Furthermore, granulomas from transgenic mice had an increase in the number of macrophages within the tissue.Therefore, macrophage expression of bSR-A increased presence of this cell type in granulomas without enhancing the deposition of cholesterol esters, consistent with a role of the adhesive property of the protein.

Evaluation of the activity and localization of 15-lipoxygenase-1 after introduction into human colorectal carcinoma Caco-2 cells.

In human colorectal carcinoma Caco-2 cells, sodium butyrate (NaBT) induces the expression of the reticulocyte, 15-lipoxygenase-1 (15-LO-1) and causes these cells to undergo differentiation and apoptosis. 15-LO-1 is also expressed in human colorectal epithelium with a significant higher expression observed in colorectal tumors. In this study, we have prepared stable Caco-2 cells that expressed 15-LO-1 under control of an inducible promoter. These cells provide a model system to study regulation of 15-LO-1 activity in colorectal cells without the interfering presence of NaBT and are useful to study the biological function of 15-LO-1. The expressed 15-LO-1 was highly active as measured in cell lysates, but we were unable to detect metabolism in intact cells. The addition of calcium to the media for the Caco-2 cells was required for 15-LO-1 to translocate from the cytosol to the membrane which is frequently a requirement for lipoxygenase activity. Despite the addition of calcium and translocation, little lipoxygenase activity was detected with intact cells. However, after removal of phenol red, a common constituent of cell culture media, we were able to detect 15-LO-1 activity in the transfected Caco-2 cultured cells. Thus the presence of calcium and the absence of antioxidants present in commonly used culture media are required for expressed 15-LO-1 to be catalytically active and to permit an examination of its biological effects.

Interleukin-4 augments acetylated LDL-induced cholesterol esterification in macrophages.

Activated subpopulations of lymphocytes and mast cells have been detected in atherosclerotic lesions. Interleukin-4 (IL-4) is a prominent cytokine released during activation of both cell types and its transcripts have been detected in both human and mouse atherosclerotic lesions. To define whether this local release of IL-4 influences macrophage lipid metabolism, we examined the effects of this cytokine on intracellular cholesterol esterification during incubation with modified low density lipoprotein (LDL). IL-4 greatly augmented cholesterol esterification induced by acetylated LDL (AcLDL) in both mouse peritoneal macrophages and the murine macrophage cell line, J774. This augmentation was maximal at a concentration of 1 ng/ml after incubation for 48 h. This was not a generalized effect on lipoprotein metabolism as IL-4 had no effect on cholesterol esterification in the presence of either LDL or beta-VLDL. Determination of binding isotherms demonstrated that IL-4 increased the number of cell surface binding sites for AcLDL. The IL-4-augmented AcLDL-induced cholesterol esterification was attenuated by the scavenger receptor class A (SR-A) antagonist, fucoidan, and the anti-mouse SR-A monoclonal antibody, 2F8. These data, combined with the known receptor specificity of AcLDL interactions, imply a role of SR-A in the IL-4 induced responses. Two cytokines that have been demonstrated previously to down-regulate SR-A, TNF-alpha and TGF-beta, antagonized the IL-4-induced augmentation of cholesterol esterification. Therefore, local release of IL-4 within atherosclerotic lesions could have a profound effect on macrophage lipid metabolism and the subsequent atherogenic process.

15-Lipoxygenase and its inhibition: a novel therapeutic target for vascular disease.

The disease process known as atherosclerosis is the leading cause of morbidity and mortality in the Western world. Current therapies have focused on treating the major risk factors identified to date including plasma lipid derangements, hypertension, clotting disorders, and diabetes. However, a significant number of individuals will be diagnosed with this malady in the apparent absence of known risk factors. Recent attention has turned toward treating the disease at the level of the vessel wall. In this review, we assess the relevancy of the oxygenating enzyme 15-lipoxygenase (15-LO) as a therapeutic target. In vitro studies suggest that this enzyme may be involved in processes that modify native LDL in such a way as to be avidly taken up by tissue macrophages. In support of this contention are reports demonstrating the colocalization of 15-LO with macrophage-rich arterial lesions and epitopes of modified LDL. Investigations using transgenic animals also suggest that the site of 15-LO expression may be an important factor in the development of the disease. The alteration of important cellular fatty acids may also generate intracellular signals that promote a pro-atherogenic phenotype in the absence of measurable changes in bulk lipid peroxidation. A limited number of studies have examined 15-LO inhibitors and those structural determinants necessary for inhibition of the enzyme. These include natural products and synthetic analogs. Structure activity relationships have been defined for a number of compounds including caffeic acid derivatives, propargyl ethers, and catechols. A novel, potent, specific inhibitor of 15-LO that lacks significant antioxidant activity was tested for its ability to inhibit atherosclerotic lesion formation in vivo. This benzothiopyranoindole virtually eliminated lesion formation in two animal models in the absence of significant changes in plasma lipids. Further, it prevented the progression of pre-established lesions in another study. Collectively, these data provide a strong scientific rationale for exploring the inhibition of 15-LO as a therapeutic strategy.

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.

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.

15-lipoxygenase inhibitors as anti-atherosclerosis agents.

15-lipoxygenase (15-LO) has been implicated in the process of atherogenesis. Numerous studies have suggested a role for this enzyme in lipoprotein oxidation and modification, resulting in the generation of an LDL particle that is avidly metabolized by monocytes/macrophages. Whilst biochemical studies have demonstrated the induction of 15-LO by cytokines, in vivo work has shown that the induction of the enzyme alone is not sufficient to promote lesion formation. The discovery of novel and specific inhibitors of 15-LO, in addition to animals created using genetic technologies, is allowing the evaluation of this hypothesis in vivo.

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.

Is there a role for 15-lipoxygenase in atherogenesis?

15-Lipoxygenase has been suggested to play a role in atherogenesis. The proposed action of this enzyme is to oxidize low density lipoprotein (LDL) to the extent that LDL becomes a ligand for the macrophage scavenger receptor. 15-Lipoxygenase and oxidized LDL are co-localized in atherosclerotic lesions; antioxidant drugs that block the lipoxygenase also block oxidation of LDL and the progression of experimental atherosclerosis. Biochemical experiments have demonstrated that the lipoxygenase can be induced by cytokines and/or another factor(s) associated with hypercholesterolemia. However, molecular biological work has shown that induction of the enzyme alone is not sufficient to induce lesion formation. Furthermore, the mechanism of action of 15-lipoxygenase in atherogenesis remains unclear. Predictions of the stereochemistry of enzyme-oxidized linoleate products appear to conflict with the available data. In fact, most studies have discovered substantial levels of racemic 13-hydroxyoctadecadienoic acid (13-HODE) in arterial lesions rather than the stereochemically pure 13(S)-HODE expected from purified enzyme. The possibility that the generation of products of 15-lipoxygenase metabolism must occur in a specific cellular location and during a brief time window in the development of the disease has been discussed. It is also possible that the true function of the linoleate metabolites is to modulate gene expression and regulate mitogenesis, and that oxidation of LDL may play a secondary role. The advent of transgenic species that both develop atherosclerosis and either fail to express or overexpress the lipoxygenase presents an opportunity to clarify some of these issues in the near future.

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.

Metabolic effects of troglitazone in the Goto-Kakizaki rat, a non-obese and normolipidemic rodent model of non-insulin-dependent diabetes mellitus.

Troglitazone (TRG) is an orally active antidiabetic agent that increases insulin sensitivity in models of non-insulin-dependent diabetes mellitus (NIDDM), subsequently reducing hyperinsulinemia and hyperglycemia. We examined the effects of TRG on the development and severity of diabetes in the Goto-Kakizaki (GK) rat, a spontaneous, non-obese model of NIDDM. TRG was administered at a dose of 30 mg/kg/d beginning at 4 weeks of age. TRG-treated GK rats were evaluated against Wistar and untreated GK rats at 8, 12, and 16 weeks of age. Untreated GK rats were nonketotic, normolipidemic, hyperglycemic, and had normal fasting insulin levels compared with Wistar rats. TRG treatment decreased glycosylated hemoglobin levels in the GK rat independently of its effects on plasma insulin. In untreated GK rats, intravenous glucose tolerance tests (IVGTTs) showed a hyperglycemic response to glucose loading with severely impaired glucose disposal relative to Wistar controls. TRG treatment was successful in decreasing the glucose area under the curve (AUC) (P < .03) but did not improve glucose disposal, suggesting a direct hepatic effect. Ex vivo evaluation of hepatic glucose output (HGO) further supported a direct hepatic action, with 50% reduction in HGO in TRG-treated GK rats (P < .004). A euglycemic-hyperinsulinemic clamp performed at 16 weeks of age showed severe insulin resistance in the untreated GK rat, with a glucose infusion rate (GIR) 33% lower than in Wistar rats (P < .004). TRG treatment had no effect on this insulin resistance. These results indicate that TRG selectively decreases hepatic glucose production in this unique model of NIDDM independently of its action on peripheral insulin sensitivity or hyperlipidemia.

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.

Transient overexpression of human 15-lipoxygenase in aortic endothelial cells enhances tumor necrosis factor-induced vascular cell adhesion molecule-1 gene expression.

15-lipoxygenase (15-LO) expression in artery wall cells has been demonstrated during the development of atherosclerosis in various animal models. We examined whether the expression of 15-LO in aortic endothelial cells affects the gene expression of the adhesion molecule, vascular cell adhesion molecule-1 (VCAM-1). Transient transfection of human 15-LO cDNA into bovine aortic endothelial cells led to the expression of 15-LO protein and enzymatic activity. We studied the induction of VCAM-1 mRNA in these cells. 15-LO expressing cells showed no detectable levels of VCAM-1 message. However, when TNF was added to these cells there was a synergistic increase in VCAM-1 expression relative to cells that were transfected with control plasmid pcDNA I. Our data suggest that 15-LO expression in aortic endothelium may amplify the expression of VCAM-1 induced by inflammatory stimulus during atherogenesis.

Hepatic overexpression of bovine scavenger receptor type I in transgenic mice prevents diet-induced hyperbetalipoproteinemia.

Hepatic scavenger receptors (SR) may play a protective role by clearing modified lipoproteins before they target the artery wall. To gain insight into this hypothesized function, transgenic mice expressing hepatic bovine SR (TgSR) were created and studied when fed chow, and during diet-induced hyperlipidemia. SR overexpression resulted in extensive hepatic parenchymal cell uptake of fluorescently labeled acetylated human low density lipoprotein (DiI ac-hLDL) and a twofold increase in 125I-acetylated-LDL clearance. Food intake and cholesterol absorption was indistinguishable between control and TgSR mice. In chow-fed mice, lipoprotein cholesterol was similar in control and TgSR mice. However, on a 3-wk high fat/cholesterol (HFHC) diet, the rise in apoB containing lipoproteins was suppressed in TgSR+/- and TgSR+/+ mice. The rise in HDL was similar in control and TgSR+/- mice, but significantly elevated in the TgSR+/+ mice. Overall, on chow, the ratio of apo-B containing lipoprotein cholesterol to HDL cholesterol was similar for all groups (control = 0.33; TgSR+/- = 0.32; TgSR+/+ = 0.38). However, after 3 wk on the HFHC diet, this ratio was markedly higher in control (2.34 +/- 0.21) than in either TgSR+/- (1.00 +/- 0.24) or TgSR+/+ (1.00 +/- 0.19) mice. In TgSR+/- mice, hepatic cholesteryl esters were reduced by 59%, 7 alpha-hydroxylase mRNA levels were elevated twofold, and a significant increase in fecal bile acid flux was observed after the 3-wk HFHC diet. These results suggest SR may play a protective role in liver by preventing diet-induced increases in apoB containing lipoproteins.

Fine needle aspiration for flow cytometry immunophenotyping of non Hodgkin lymphoma.

The aim was to evaluate the usefulness of lymph node biopsies obtained by fine needle aspiration (FNA) for immunophenotyping of non Hodgkin lymphoma (NHL). Seventeen superficial and deep lymph node samples were fractioned for conventional cytological examination and immunophenotyping studies. Out of ten NHL, nine were readily detected by flow cytometry (FC), while failure on the remaining case was due to selective loss of large cell population, which is liable to occur with this procedure. A single case, which proved negative for all markers employed, was finally diagnosed by immunohistochemistry as germ cell tumor. The other six cases, presenting lymphoid population without phenotypic abnormalities, were diagnosed by cytology and/or histology as Hodgkin disease or hyperplasic disorders. To conclude, FC immunophenotyping seems to improve the efficacy of FNA in NHL diagnosis, whereas for Hodgkin disease and hyperplasic disorders, classic morphological criteria are more useful for differential diagnosis. Although FNA for FC immunophenotyping cannot replace histopathological examination for NHL diagnosis, it proves to be a useful tool for staging and follow up, making surgical procedures for sample collection unnecessary.

Fine needle aspiration for flow cytometry immunophenotyping of non Hodgkin lymphoma.

The aim was to evaluate the usefulness of lymph node biopsies obtained by fine needle aspiration (FNA) for immunophenotyping of non Hodgkin lymphoma (NHL). Seventeen superficial and deep lymph node samples were fractioned for conventional cytological examination and immunophenotyping studies. Out of ten NHL, nine were readily detected by flow cytometry (FC), while failure on the remaining case was due to selective loss of large cell population, which is liable to occur with this procedure. A single case, which proved negative for all markers employed, was finally diagnosed by immunohistochemistry as germ cell tumor. The other six cases, presenting lymphoid population without phenotypic abnormalities, were diagnosed by cytology and/or histology as Hodgkin disease or hyperplasic disorders. To conclude, FC immunophenotyping seems to improve the efficacy of FNA in NHL diagnosis, whereas for Hodgkin disease and hyperplasic disorders, classic morphological criteria are more useful for differential diagnosis. Although FNA for FC immunophenotyping cannot replace histopathological examination for NHL diagnosis, it proves to be a useful tool for staging and follow up, making surgical procedures for sample collection unnecessary.

Divergent pharmacologic activities of PD 132301-2 and CL 277,082, urea inhibitors of acyl-CoA:cholesterol acyltransferase.

The in vitro potencies and hypocholesterolemic properties of CL 277,082 and PD 132301-2, two urea inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT) were compared. PD 132301-2 was several-fold more potent at inhibiting ACAT in microsomes from rat and rabbit tissues and in cultured cells (murine macrophages and the human HepG2 cell line). This disubstituted urea was also relatively specific for ACAT as other cholesterol esterifying enzymes (e.g., lecithin:cholesterol acyltransferase, pancreatic cholesterol ester hydrolase), as well as intestinal diglyceride synthesis (acyl-CoA:monoglyceride acyltransferase), were unaffected in vitro at relevant concentrations. In normal chow-fed rats, both compounds reduced plasma triglycerides at doses > 50 mg/kg, but only PD 132301-2 reduced plasma cholesterol. In rat and rabbit models of hypercholesterolemia the greater in vitro potency of PD 132301-2 translated into greater in vivo efficacy (i.e., ED50 values 2- to 3-fold higher for CL 277,082 in both acute and chronic rate models). Of particular note was the greater elevation of high-density lipoprotein-cholesterol and parenteral activity of PD 132301-2 compared to CL 277,082 in the chronic rat model. Inhibition of cholesterol absorption in rats was also greater with PD 132301-2. In guinea pigs, in which 77% of plasma cholesterol was transported in low-density lipoprotein, PD 132301-2 potently reduced plasma total cholesterol (lowest significant dose = 1 mg/kg) as well as plasma triglycerides. CL 277,082 only reduced cholesterol at doses > 100 mg/kg in this low-density lipoprotein model. In a canine model of hypercholesterolemia CL 277,082 was inactive at doses up to 50 mg/kg, but PD 132301-2 was active at 3 mg/kg. Moreover, efficacy in dogs with PD 132301-2 was positively correlated with plasma drug concentration, an observation not previously demonstrated for other hypolipidemic drugs. The combined data illustrate that pharmacologic activities can vary widely among ACAT inhibitors of the same general class. In addition, the unique observation of proportionality between efficacy and blood drug levels in nonrodent animal models may not only help to simplify early stages in drug development but also may help to predict or monitor a direct action of the drug on vascular disease.