Mutation in APOA1 predicts increased risk of ischaemic heart disease and total mortality without low HDL cholesterol levels.

OBJECTIVES: To determine whether mutations in APOA1 affect levels of high-density lipoprotein (HDL) cholesterol and to predict risk of ischaemic heart disease (IHD) and total mortality in the general population. BACKGROUND: Epidemiologically, risk of IHD is inversely related to HDL cholesterol levels. Mutations in apolipoprotein (apo) A-I, the major protein constituent of HDL, might be associated with low HDL cholesterol and predispose to IHD and early death. DESIGN: We resequenced APOA1 in 190 individuals and examined the effect of mutations on HDL cholesterol, risk of IHD, myocardial infarction (MI) and mortality in 10 440 individuals in the prospective Copenhagen City Heart Study followed for 31 years. Results were validated in an independent case-control study (n = 16 035). Additionally, we determined plasma ratios of mutant to wildtype (WT) apoA-I in human heterozygotes and functional effects of mutations in adenovirus-transfected mice. RESULTS: We identified a new mutation, A164S (1 : 500 in the general population), which predicted hazard ratios for IHD, MI and total mortality of 3.2 [95% confidence interval (CI): 1.6-6.5], 5.5 (95% CI: 2.6-11.7) and 2.5 (95% CI: 1.3-4.8), respectively, in heterozygotes compared with noncarriers. Mean reduction in survival time in heterozygotes was 10 years (P < 0.0001). Results for IHD and MI were confirmed in the case-control study. Furthermore, the ratio of mutant S164 to WT A164 apoA-I in plasma of heterozygotes was reduced. In addition, A164S heterozygotes had normal plasma lipid and lipoprotein levels, including HDL cholesterol and apoA-I, and this finding was confirmed in adenovirus-transfected mice. CONCLUSIONS: A164S is the first mutation in APOA1 to be described that predicts an increased risk of IHD, MI and total mortality without low HDL cholesterol levels.

Role of Esrrg in the fibrate-mediated regulation of lipid metabolism genes in human ApoA-I transgenic mice.

We have used a new ApoA-I transgenic mouse model to identify by global gene expression profiling, candidate genes that affect lipid and lipoprotein metabolism in response to fenofibrate treatment. Multilevel bioinformatical analysis and stringent selection criteria (2-fold change, 0% false discovery rate) identified 267 significantly changed genes involved in several molecular pathways. The fenofibrate-treated group did not have significantly altered levels of hepatic human APOA-I mRNA and plasma ApoA-I compared with the control group. However, the treatment increased cholesterol levels to 1.95-fold mainly due to the increase in high-density lipoprotein (HDL) cholesterol. The observed changes in HDL are associated with the upregulation of genes involved in phospholipid biosynthesis and lipid hydrolysis, as well as phospholipid transfer protein. Significant upregulation was observed in genes involved in fatty acid transport and beta-oxidation, but not in those of fatty acid and cholesterol biosynthesis, Krebs cycle and gluconeogenesis. Fenofibrate changed significantly the expression of seven transcription factors. The estrogen receptor-related gamma gene was upregulated 2.36-fold and had a significant positive correlation with genes of lipid and lipoprotein metabolism and mitochondrial functions, indicating an important role of this orphan receptor in mediating the fenofibrate-induced activation of a specific subset of its target genes.

Local control, toxicity, and cosmesis in women younger than 50 enrolled onto the American Society of Breast Surgeons MammoSite Radiation Therapy System registry trial.

BACKGROUND: The American Society of Breast Surgeons enrolled women onto a registry trial to prospectively study patients treated with the MammoSite Radiation Therapy System (RTS) breast brachytherapy device. This report examines local recurrence (LR), toxicity, and cosmesis as a function of age in women enrolled onto the trial. METHODS: A total of 1449 primary early-stage breast cancers were treated in 1440 women. Of these, 130 occurred in women younger than 50 years of age. Fisher's exact test was performed to correlate age (<50 vs. > or = 50 years) with toxicity and with cosmesis. The association of age with LR failure times was investigated by fitting a parametric model. RESULTS: Women younger than 50 were more likely to develop fat necrosis: 4.6% (6 of 130) vs. 1.8% (24 of 1319) (P = .0456). Other toxicities were comparable. At 2 years, cosmesis was excellent or good in 87% of assessable women aged <50 years (n = 74) and in 94% of assessable older women (n = 751) (P = .0197). At 3 years, this difference disappeared: excellent or good in 90% (56 of 62) of younger women vs. 93% (573 of 614) of older women (P = .2902). The crude LR rate for the group was 1.7% (25 of 1449). There was no statistically significant difference in LR as a function of age. In women <50, 3.1% (4 of 130) developed a LR; in the older patients, 1.6% (21 of 1319) developed LR (3-year actuarial LR rates, 2.9% vs. 1.7%, respectively; P = .2284). CONCLUSIONS: Accelerated partial breast irradiation with the MammoSite RTS results in low toxicity and produces similar cosmesis and local control at 3 years in women younger than 50 when compared with older women.

Characterization of the promoter elements required for hepatic and intestinal transcription of the human apoB gene: definition of the DNA-binding site of a tissue-specific transcriptional factor.

The promoter elements important for intestinal and hepatic transcription of the human apoB gene have been localized downstream of nucleotide -150. Footprinting analysis using hepatic nuclear extracts identified four protected regions, -124 to -100, -97 to -93, -86 to -33, and +33 to +52. Gel electrophoretic mobility shift assays showed that multiple factors interact with the apoB sequence -86 to -33, while the region -88 to -61 binds a single nuclear factor. Methylation interference analysis and nucleotide substitution mutagenesis identified the binding site of the factor between residues -78 and -68. Binding competition experiments indicate that this factor recognizes the regulatory elements of other liver-specific genes.

The SP1 sites of the human apoCIII enhancer are essential for the expression of the apoCIII gene and contribute to the hepatic and intestinal expression of the apoA-I gene in transgenic mice.

We have generated transgenic mice carrying wild-type and mutant forms of the apolipoprotein (apo)A-I/apoCIII gene cluster. Mutations were introduced either in one or in three SP1 binding sites of the apoCIII enhancer. In mice carrying the wild-type transgene, major sites of apoA-I mRNA synthesis were liver and intestine and minor sites were kidney and, to a lesser extent, other tissues. The major site of chloramphenicol acetyl transferase (CAT) activity (used as a reporter for the apoCIII gene) was liver and minor sites intestine and kidney. A mutation in one SP1 binding site reduced the expression of the apoA-I gene to approximately 23 and 19% in the liver and intestine, respectively, as compared to the control wild-type. The hepatic expression of the CAT gene was not affected whereas the intestinal expression was nearly abolished. Mutations in three SP1 binding sites reduced the hepatic and intestinal expression of the apoA-I and CAT genes to 14 and 4%, respectively, as compared to the wild-type control, and abolished CAT expression in all tissues. The findings suggest that the SP1 sites of the apoCIII enhancer are required for the expression of the apoCIII gene and also contribute significantly to the hepatic and intestinal expression of the apoA-I gene in vivo.

Synthesis, modification, and flotation properties of rat hepatocyte apolipoproteins.

We have studied apolipoprotein synthesis, intracellular modification and secretion by primary adult rat hepatocyte cultures using continuous pulse or pulse chase labeling with [35S]methionine, immunoprecipitation and two-dimensional isoelectric focusing/polyacrylamide gel electrophoresis. The flotation properties of the newly secreted apolipoproteins were studied by discontinuous density gradient ultracentrifugation and one- and two-dimensional polyacrylamide gel electrophoresis. These studies showed that rat hepatocyte apoE is modified intracellularly to produce minor isoproteins that differ in size and charge. One of these minor isoproteins represents a monosialated apoE form (apoE3s1). Similarly, apoCIII is modified intracellularly to produce a disialated apoCIII form (apoCIIIs2), whereas newly synthesized apoA-I and apoA-IV are not glycosylated and overlap on two-dimensional gels with the proapoA-I and the plasma apoA-IV form, respectively. Both unmodified and modified apolipoproteins are secreted into the medium. Separation of secreted apolipoproteins by density gradient ultracentrifugation has shown that 50% of apoE, 80% of apoA-I, and more than 90% of apoA-IV and apoCIII are secreted in a lipid-poor form, whereas apoB-100 and apoB-48 are 100% associated with lipids. ApoB-100 floats in the VLDL and IDL regions, whereas apoB-48 is found in all lipoprotein fractions. ApoE and small amounts of apoA-I, apoA-IV and apoCIII float in the HDL region. Small amounts of apoE and apoCIII are also found in the VLDL and IDL regions, and apoE in the LDL region. Ultracentrifugation of nascent lipoproteins in the presence of rat serum promoted flotation of apoA-I and apoA-IV in the HDL fraction and resulted in increased flotation and distribution of apoE and apoCs in VLDL, IDL and LDL regions. These observations are consistent with the hypothesis that intracellular assembly of lipoproteins involves apoB-48 and apoB-100 forms, whereas a large portion of apoA-I, apoCIII and apoA-IV can be secreted in a lipid-poor form, which associates extracellularly with preexisting lipoproteins.

High level of expression of functional human platelet alpha 2-adrenergic receptors in a stable mouse C127 cell line.

We have generated, by transfection and proper selection, a stable mouse C127 cell line which expresses the human alpha 2-adrenergic receptor gene. The size of the mRNA produced by the cloned gene is 1.8 kb. Electrophoretic analysis and autoradiography of cell membrane proteins photoaffinity labeled with p-[3H]azidoclonidine gave a broad protein band of molecular mass of approx. 64 kDa. Saturation binding with [3H]rauwolscine as ligand gave an equilibrium dissociation constant of 1.29 +/- 0.46 nM (mean +/- S.D.) and binding capacity range of 18-35 pmol/mg membrane protein, with (3-6) x 10(6) receptors per cell. Antagonist competition experiments displayed the order of potency: yohimbine greater than rauwolscine greater than phentolamine much greater than prazosin. Agonist competitions demonstrated the order of potency: p-aminoclonidine greater than (-)epinephrine much greater than (+)epinephrine much greater than (-)isoproterenol. This pharmacological profile is characteristic of the human platelet alpha 2-adrenergic receptor. The expressed receptor is able to couple to the Gi protein. Thus, when epinephrine competition for specific binding of [3H]rauwolscine was performed in the presence of 1 mM MgCl2, 1 mM Gpp[NH]p increased the Ki for epinephrine from 164 to 315 nM. Following preincubation of cultures with 1 mM isobutylmethylxanthine, 1 microM epinephrine decreased forskolin-stimulated cellular cyclic AMP accumulation by 72%. The response was biphasic, and the attenuation effect disappeared at 100 microM epinephrine. A transfected clone which did not demonstrate detectable alpha 2-adrenergic receptor mRNA displayed low levels of alpha 2-adrenergic receptor, (less than 50 fmol/mg membrane protein), similar to those found in the parent C127 cell line. In this clone, epinephrine did not attenuate but, rather, enhanced forskolin-stimulated cyclic AMP accumulation. This new C127 cell line expressing high levels of alpha 2-adrenergic receptor provides an abundant source of a single human adrenergic receptor subtype in membrane-bound conformation which is able to couple to the Gi protein and inhibit forskolin-stimulated adenylate cyclase activity. This cell line will facilitate studies of the structure: function relationship of the alpha 2-adrenergic receptor and should aid in separating the components of various signal transduction mechanisms putatively attributed to this receptor.

Detailed molecular model of apolipoprotein A-I on the surface of high-density lipoproteins and its functional implications.

The major apolipoprotein (apo) A-I containing lipoprotein, high- density lipoprotein, is a negative risk factor for cardiovascular disease. An atomic resolution molecular model for lipid-associated apo A-I was recently proposed in which two apo A-I molecules are wrapped beltwise around a small discoidal patch of phospholipid bilayer. Because of its detailed predictions of lipid-associated apo A-I structure, this molecular belt model, if confirmed, provides a blueprint for understanding the molecular mechanisms of reverse cholesterol transport, and thus for the rational design of new classes of drugs for reversal of atherosclerosis and cardiovascular disease. The details and implications of the model are currently being explored by site-directed mutagenesis.

Regulation of renin gene expression in hypertensive rats.

A carboxy terminal renin complementary DNA (cDNA) clone from rat kidney was isolated, characterized, and used as a probe for renin messenger RNA (mRNA) quantification in normotensive and hypertensive rats. RNA blotting analysis detected renin mRNA in control kidney and brain. Deoxycorticosterone acetate (DOCA) and high salt (1%) treatment of experimental animals resulted in a greater than 95% decrease in the content of renin mRNA in the kidney, as compared with values in control rats receiving 0.4% NaCl in their diet. In contrast, high salt (1%) treatment alone caused only a twofold decrease in kidney renin mRNA content, as compared with values in controls. DOCA and low salt (0.04%) or low salt (0.04%) treatment alone caused a 1.5-fold increase in the kidney renin mRNA content, as compared with values in control rats. These results indicate that DOCA and salt have a synergistic effect in depressing renin mRNA levels in kidney. Clipping of the left renal artery caused a threefold increase in the steady state level of renin mRNA in the ischemic kidney and a 0.5-fold decrease in the hypertrophied kidney. The data are consistent with the hypothesis that blood pressure and other stimuli regulate the expression of the renin gene in vivo.

Expression of multiple alpha 2-adrenergic receptor messenger RNA species in rat tissues.

We used a human platelet alpha 2-adrenergic receptor probe to study the tissue distribution and messenger RNA (mRNA) forms of the rat alpha 2-adrenergic receptor. Under stringent conditions of hybridization and washing, we detected an mRNA species of 3.8 kb. The abundance of this form follows the order spleen, kidney, brain stem and cortex, and skeletal muscle and lung and is consistent with the reported abundance and tissue distribution of the alpha 2 receptor activity. A 3.0 kb mRNA form was also detected in cerebral cortex and brain stem and a 4.1 kb mRNA form was observed in kidney under less stringent hybridization conditions. The tissue distribution of the 3.0 kb form is different from that of alpha 1- and beta-adrenergic receptors and the D2 dopaminergic receptor. The mRNA analysis combined with Southern blot analysis of rat and human genomic DNA indicate that: 1) in addition to a 3.8 kb rat alpha 2-adrenergic receptor transcript, there are other mRNA forms in the rat that do not correspond to previously described adrenergic receptor mRNA species and 2) more than one alpha 2-adrenergic receptor gene in the rat is expressed in a tissue-specific manner.

Isolation and characterization of a third isoform of human hepatocyte nuclear factor 4.

Hepatocyte nuclear factor 4 (HNF-4) is an essential positive regulator of a large number of liver-specific genes. We report here the isolation of three HNF-4 isoforms from a human liver cDNA library. hHNF-4A and hHNF-4B, differing by the insertion of 10 amino acids in the C-terminal region, have been previously identified in mouse, rat and human liver. The novel isoform, hHNF-4C, is identical to hHNF-4A and B in the regions encompassing the DNA-binding and dimerization domains, but contains a different C-terminal domain. Similar to the other isoforms, hHNF-4C is produced in a limited number of tissues and represents 2.6-13% of the total hHNF-4 mRNA population, depending on the cell type. The chromosomal origin of all three isoforms has been localized to human chromosome 20. hHNF-4C can form heterodimers with hHNF-4A and B in vitro, and exhibits similar transactivation potential as hHNF-4A or B in transient transfection assays, suggesting that the divergent C-terminal region is not part of the transactivation domain.

Transcriptional regulation of the human apolipoprotein genes.

This review provides experiments and putative mechanisms which underlie the transcription of the human apolipoprotein genes in vitro and in vivo. Summarized below are the key findings for individual genes and gene clusters. ApoA-II. 1- The -911/+29 promoter is sufficient to direct expression of a reporter gene exclusively in the liver and thus represents a liver-specific promoter. 2- Important factors for the activity of this promoter are hormone nuclear receptors and the ubiquitous factor USF. 3. SREBP-1 and SREBP-2 bind to five and four sites respectively and transactivate the apoA-II promoter. Their role in the in vivo transcription of the apoA-II gene has not been established. ApoB. 1. Regulatory sequence extending 5 Kb upstream and 1.5 Kb downstream of the apoB promoter are sufficient to direct hepatic expression of the apoB gene. The intestinal expression of the apoB gene requires in addition a 315 bp intestinal enhancer located 56 Kb upstream of the apoB gene. 2. Important factors for apoB gene transcription appear to be C/EBP, HNF-3, HNF-4 and other nuclear receptors which bind both on the proximal promoter and the intestinal enhancer. ApoE/ApoCI/ApoCIV/ApoCII Cluster. 1. The expression of the genes of the apoE/apoCI/apoCII/apoE cluster are controlled by two homologous hepatic control regions designated HCR-1 and HCR-2 of approximately 600 bp located 15 and 27 Kb 3? of the apoE gene. Either region is sufficient to direct gene expression in vivo, although HCR-1 appears to have a dominant effect on apoE and apoCI and HCR-2 has a dominant effect on apoCIV and apoCII gene expression. 2. Two other homologous regulatory regions designated ME-1 and ME-2 located 3.3 and 15.9 Kb downstream of the apoE gene can direct independently the expression of the apoE gene in macrophages and adipocytes. 3. Important factors for apoE gene regulation appear to be SP1 on the proximal promoter, and possibly HNF-3, C/EBP and hormone nuclear receptors on the enhancers. 4. Important factors for apoCII gene transcription appear to be HNF-4 and RXR-alpha/T3R-beta which binds to a thyroid response element of the proximal promoter. ApoA-I/ApoCIII/ApoA-IV Gene Cluster. 1. The transcription of the apoA-I/apoCIII/apoA-IV gene cluster is controlled by a common enhancer located 590 to 790 nucleotides upstream of the apoCIII gene. 2. Important factors for the activity of the enhancer are SP1, HNF-4 and possibly other nuclear receptors. Important factors for the activity of the proximal promoters are HNF-4, and possibly other nuclear receptors. 3. The HNF-4 binding site of the apoCIII enhancer is required for the intestinal expression of apoA-I and apoCIII gene and enhances synergistically the hepatic transcription of the two genes and possibly of apoA-IV in vivo. The three SP1 sites of the enhancer are also required for the intestinal expression of apoA-I and apoCIII genes in vivo and for the enhancement of the hepatic transcription. 4. Pro-inflammatory cytokines such as TNF-alpha and IL-1 repress, and TGF-beta stimulates the apoCIII promoter activity. The TGF-beta pathway activates SMAD3/4 proteins which interact with HNF-4 bound to the apoCIII promoter and enhancer and increase its activity. 5. It appears that other factors activated by different signaling pathways (NF-kappa-B, Jun and others) interact with HNF-4 bound to the enhancer and thus repress the activity of apoCIII promoter. Understanding the transcriptional regulatory mechanism of the apolipoprotein genes may allow, in the long run, selective increase of anti-atherogenic lipoproteins and thus reduce the risk of cardiovascular disease.

Genetic polymorphism in human apolipoprotein E.

This chapter provides the methodologies employed to study the polymorphism of human apoE. These and other related studies have advanced our understanding of the structure and function of this protein as follows: The complex array of human apoE observed by two-dimensional gel electrophoresis results from genetic variation and posttranslational modification. The genetic polymorphism of apoE is explained by the existence of three common alleles (epsilon 4, epsilon 3, epsilon 2) at a single structural gene locus. Combinations of above alleles can generate three homozygous (E4/4, E3/3, E2/2) and three heterozygous (E4/3, E3/2, E4/2) apoE phenotypes. The apoE phenotype E2/2 is found in 91% of patients with type III hyperlipoproteinemia and can be used as a molecular marker for the diagnosis of this disease. However, other rare or common apoE phenotypes have been observed in patients with type III HLP. ApoE originating from E2/2 phenotype (Arg 158 to Cys 158 substitution) has reduced affinity for the LDL receptor. This property of apoE2 can account partially for the accumulation of apoE-rich lipoprotein remnants in the plasma of patients with type III HLP. However, other genetic or environmental factors are necessary for the phenotypic expression of the disease.

Intra- and extracellular modifications of apolipoproteins.

This chapter outlined the methods used to study intra- and extracellular modifications of apolipoproteins. These and other related studies have shown that several of the apolipoproteins undergo a series of intra- and extracellular modifications as follows: All apolipoproteins studied contain an 18-26 long signal peptide which is cleaved cotranslationally by the signal peptidase of the rough endoplasmic reticulum. ApoE is further modified intracellularly with carbohydrate chains containing sialic acid and is secreted in the modified form designated apoEs. The modified apoE is subsequently desialated in plasma. ApoA-I is secreted in a proapoA-I form, which consists of 249 amino acids. The N-terminal hexapeptide of proapoA-I is cleaved extracellularly by a proapoA-I to plasma apoA-I converting protease. This cleavage generates the plasma apoA-I form which consists of 243 amino acids. Other known apolipoprotein modifications include the modification of apoB, apoC-III, and apoD with carbohydrate chains that contain sialic acid and the proteolytic cleavage of the proapoA-II segment. At the present time we are able to distinguish several isoprotein forms for a particular apolipoprotein. In addition, we began to understand the biochemical changes which lead to a few of these isoproteins. Future research should be directed toward a better understanding not only of the structure but most importantly of the physiological significance of the different apolipoprotein forms.

Cryoablation of benign breast tumors: evolution of technique and technology.

We report on improvements in cryoprobe design and techniques of cryoablation as a minimally invasive alternative to open surgery for the treatment of benign breast tumors. In the study, which was conducted in 12 centers, 124 lesions in 102 patients were monitored for a period of 12 months after cryoablation. Two different treatment techniques were used: Double HI FREEZE and Tailored Freeze. In patients treated with the Tailored Freeze technique significantly better results were recorded 12 months after the procedure: the median reduction in tumor volume was 91%, 73% of all tumors treated were nonpalpable, 84% of lesions less than 2.5 cm in maximum diameter were nonpalpable, and none of the 31 mammograms performed yielded abnormal findings. Patient satisfaction was good to excellent in 92% of the patients. The safety profile of this technique was excellent; all complications were minor. Evolution of cryoablation freezing techniques, coupled with improvements in cryoprobe design, has resulted in significant improvements in both safety and effectiveness.

Laparotomy for pelvic fracture.

To establish criteria for laparotomy, the records of 224 patients admitted with an acute pelvic fracture were reviewed. Forty-four patients underwent laparotomy; 2 had no intraabdominal injury. The mechanisms of injury was blunt trauma in 31 patients and gunshot wound in 13. All four patients who died had blunt trauma. Major or minor pelvic fracture classification did not predict intraabdominal visceral injury, except for bilateral pubic rami fractures, which were commonly associated with bladder rupture. The accuracy of the indications for laparotomy was calcualted and criteria were established. Signs of an acute abdominal disorder, the presence of a penetrating wound, abnormal findings on pyleography or cystography, persistent shock, evisceration, and diminished distal pulses, singly or in combination, had a 90 percent accuracy in indicating correctable traabdominal injury. Peritoneal lavage was less reliable, with a 57 percent accuracy. Additional criteria to be considered are enlarging palpable abdominal hematoma, fracture or dislocation with bony fragments protruding into the pelvis, signs of persistent bleeding, and rectal injury or a large perineal wound.

The evolving practice pattern of the breast surgeon with disappearance of open biopsy for nonpalpable lesions.

BACKGROUND: Recent advances in technology have prompted growth in the surgeon's armamentarium for breast biopsy. For nonpalpable, mammographically detected lesions, the options include stereotactic needle/wire localization and open biopsy (SNL/OBx), stereotactic needle core biopsy (SNCB), and directional, vacuum-assisted biopsy (VAB; Mammotome). METHODS: A review of 372 patients with 424 breast lesions biopsied by the same surgeon between January 1993 and August 1997 was performed. RESULTS: SNCB and VAB procedures were less invasive and less morbid than SNL/OBx. Vacuum-assisted biopsy was superior to SNCB for sampling efficiency, with 74% of microcalcifications removed compared with 20% (P <0.0001). Additionally, underestimation of disease was seen with the SNCB technique, but not with VAB. Follow-up mammography found no false negative biopsies in any group. Over the 56 consecutive months, VAB progressively replaced SNL/OBx and SNCB as the procedure of choice. CONCLUSION: A breast surgeon can use VAB to replace open biopsy and core needle procedures for the initial biopsy of nonpalpable breast lesions.

Total cost comparison of 2 biopsy methods for nonpalpable breast lesions.

OBJECTIVE: To identify, quantify, and compare total facility costs for 2 breast biopsy methods: vacuum-assisted biopsy (VAB) and needle-wire-localized open surgical biopsy (OSB). STUDY DESIGN: A time-and-motion study was done to identify unit resources used in both procedures. Costs were imputed from published literature to value resources. A comparison of the total (fixed and variable) costs of the 2 procedures was done. PATIENTS AND METHOD: A convenience sample of 2 high-volume breast biopsy (both VAB and OSB) facilities was identified. A third facility (OSB only) and 8 other sites (VAB only) were used to capture variation. Staff interviews, patient medical records, and billing data were used to check observed data. One hundred and sixty-seven uncomplicated procedures (71 OSBs, 96 VABs) were observed. Available demographic and clinical data were analyzed to assess selection bias, and sensitivity analyses were done on the main assumptions. RESULTS: The total facility costs of the VAB procedure were lower than the costs of the OSB procedure. The overall cost advantage for using VAB ranges from $314 to $843 per procedure depending on the facility type. Variable cost comparison indicated little difference between the 2 procedures. The largest fixed cost difference was $763. CONCLUSIONS: Facilities must consider the cost of new technology, especially when the new technology is as effective as the present technology. The seemingly high cost of equipment might negatively influence a decision to adopt VAB, but when total facility costs were analyzed, the new technology was less costly.

An apolipoprotein E4 fragment affects matrix metalloproteinase 9, tissue inhibitor of metalloproteinase 1 and cytokine levels in brain cell lines.

Apolipoprotein (apo) E4 isoform, a major risk factor for Alzheimer disease (AD), is more susceptible to proteolysis than apoE2 and apoE3 isoforms. ApoE4 fragments have been found in AD patients' brain. In the present study, we examined the effect of full-length apoE4 and apoE4 fragments apoE4[Δ(186-299)] and apoE4[Δ(166-299)] on inflammation in human neuroblastoma SK-N-SH and human astrocytoma SW-1783 cells. Western blot and zymography analysis showed that treatment of SK-N-SH cells with apoE4[Δ(186-299)], but not full-length apoE4 or the shorter apoE4[Δ(166-299)] fragment, leads to increased extracellular levels of matrix metalloproteinase 9 (MMP9) and tissue inhibitor of metalloproteinase 1 (TIMP1). Real-time PCR showed that interleukin (IL)-1ß gene expression is also increased in SK-N-SH cells treated with apoE4[Δ(186-299)]. Treatment of SK-N-SH cells with IL-1ß leads to increased MMP9 and TIMP1 extracellular levels, suggesting that the induction of IL-1ß may be the mechanism by which apoE4[Δ(186-299)] regulates MMP9 and TIMP1 levels in these cells. In contrast to SK-N-SH cells, treatment of SW-1783 cells with apoE4[Δ(186-299)], and to a lesser extent with apoE4, leads to increased TIMP1 extracellular levels without affecting MMP9 levels. Additionally, apoE4[Δ(186-299)] leads to decreased IL-10 gene expression in SK-N-SH cells, whereas both apoE4 and apoE4[Δ(186-299)] lead to decreased TNFα gene expression without affecting IL-1ß and IL-10 gene expression in SW-1783 cells. Overall, our findings indicate that a specific apoE4 fragment (apoE4[Δ(186-299)]), with molecular mass similar that of apoE4 fragments detected in AD patients' brain, can influence the level of inflammatory molecules in brain cell lines. It is possible that these phenomena contribute to AD pathogenesis.