Expression of CCL20 and granulocyte-macrophage colony-stimulating factor, but not Flt3-L, from modified vaccinia virus ankara enhances antiviral cellular and humoral immune responses.

While modified vaccinia virus Ankara (MVA) is currently in clinical development as a safe vaccine against smallpox and heterologous infectious diseases, its immunogenicity is likely limited due to the inability of the virus to replicate productively in mammalian hosts. In light of recent data demonstrating that vaccinia viruses, including MVA, preferentially infect antigen-presenting cells (APCs) that play crucial roles in generating antiviral immunity, we hypothesized that expression of specific cytokines and chemokines that mediate APC recruitment and activation from recombinant MVA (rMVA) vectors would enhance the immunogenicity of these vectors. To test this hypothesis, we generated rMVAs that express murine granulocyte-macrophage colony-stimulating factor (mGM-CSF), human CCL20/human macrophage inflammatory protein 3alpha (hCCL20/hMIP-3alpha), or human fms-like tyrosine kinase 3 ligand (hFlt3-L), factors predicted to increase levels of dendritic cells (DCs), to recruit DCs to sites of immunization, or to promote maturation of DCs in vivo, respectively. These rMVAs also coexpress the well-characterized, immunodominant lymphocytic choriomeningitis virus nucleoprotein (NP) antigen that enabled sensitive and quantitative assessment of antigen-specific CD8(+) T-cell responses following immunization of BALB/c mice. Our results demonstrate that immunization of mice with rMVAs expressing mGM-CSF or hCCL20, but not hFlt3-L, results in two- to fourfold increases of cellular immune responses directed against vector-encoded antigens and 6- to 17-fold enhancements of MVA-specific antibody titers, compared to those responses elicited by nonadjuvanted rMVA. Of note, cytokine augmentation of cellular immune responses occurs when rMVAs are given as primary immunizations but not when they are used as booster immunizations, suggesting that these APC-modulating proteins, when used as poxvirus-encoded adjuvants, are more effective at stimulating naïve T-cell responses than in promoting recall of preexisting memory T-cell responses. Our results demonstrate that a strategy to express specific genetic adjuvants from rMVA vectors can be successfully applied to enhance the immunogenicity of MVA-based vaccines.

Effects of increasing hydrophobicity on the physical-chemical and biological properties of a class A amphipathic helical peptide.

We have recently shown that a class A amphipathic peptide 5F with increased amphipathicity protected mice from diet-induced atherosclerosis (Garber et al. J. Lipid Res. 2001. 42: 545-552). We have now examined the effects of increasing the hydrophobicity of a series of homologous class A amphipathic peptides, including 5F, on physical and functional properties related to atherosclerosis inhibition by systematically replacing existing nonpolar amino acids with phenylalanine. The peptides, based on the sequence Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-NH(2) (Ac-18A-NH(2) or 2F) were: 3F(3)(Ac-F(3)18A-NH(2)), 3F(14)(Ac-F(14)18A-NH(2)), 4F(Ac-F(3,14)18A-NH(2)), 5F(Ac-F(11,14,17) 18A-NH(2)), 6F(Ac-F(10,11,14,17)18A-NH(2)), and 7F(Ac-F(3,10,11,14,17) 18A-NH(2)). Measurements of aqueous solubility, HPLC retention time, exclusion pressure for penetration into an egg phosphatidylcholine (EPC) monolayer, and rates of EPC solubilization revealed an abrupt increase in the hydrophobicity between peptides 4F and 5F; this was accompanied by increased ability to associate with phospholipids. The peptides 6F and 7F were less effective, indicating a limit to increased hydrophobicity for promoting lipid interaction in these peptides. Despite this marked increase in lipid affinity, these peptides were less effective than apoA-I in activating the plasma enzyme, lecithin:cholesterol acyltransferase, with 5F activating LCAT the best (80% of apoA-I). Peptides 4F, 5F, and 6F were equally potent in inhibiting LDL-induced monocyte chemotactic activity. These studies suggest that an appropriate balance between peptide-peptide and peptide-lipid interactions is required for optimal biological activity of amphipathic peptides. These studies provide a rationale for the design of small apoA-I-mimetics with increased potency for atherosclerosis inhibition.

Cationic domain 141-150 of apoE covalently linked to a class A amphipathic helix enhances atherogenic lipoprotein metabolism in vitro and in vivo.

We previously showed 1 that a peptide, Ac-hE18A-NH(2), in which the arginine-rich heparin-binding domain of apolipoprotein E (apoE) [residues 141;-150] (LRKLRKRLLR), covalently linked to 18A (DWLKAFYDKVAEKLKEAF; a class A amphipathic helix with high lipid affinity), enhanced LDL uptake and clearance. Because VLDL and remnants contain more cholesterol per particle than LDL, enhanced hepatic clearance of VLDL could lead to an effective lowering of plasma cholesterol. Therefore, in the present article we compared the ability of this peptide to mediate/facilitate the uptake and degradation of LDL and VLDL in HepG2 cells. The peptide Ac-hE18A-NH(2), but not Ac-18A-NH(2), enhanced the uptake of LDL by HepG2 cells 5-fold and its degradation 2-fold. The association of the peptides with VLDL resulted in the displacement of native apoE; however, only Ac-hE18A-NH(2) but not Ac-18A-NH(2) caused markedly enhanced uptake (6-fold) and degradation (3-fold) of VLDL. Ac-hE18A-NH(2) also enhanced the uptake (15-fold) and degradation (2-fold) of trypsinized VLDL Sf 100;-400 (containing no immuno-detectable apoE), indicating that the peptide restored the cellular interaction of VLDL in the absence of its essential native ligand (apoE). Pretreatment of HepG2s with heparinase and heparitinase abrogated all peptide-mediated enhanced cellular activity, implicating a role for cell-surface heparan sulfate proteoglycans (HSPG). Intravenous administration of Ac-hE18A-NH(2) into apoE gene knockout mice reduced plasma cholesterol by 88% at 6 h and 30% at 24 h after injection. We conclude that this dual-domain peptide associates with LDL and VLDL and results in rapid hepatic uptake via a HSPG-facilitated pathway.

A new synthetic class A amphipathic peptide analogue protects mice from diet-induced atherosclerosis.

Several synthetic class A peptide analogues have been shown to mimic many of the properties of human apo A-I in vitro. A new peptide [acetyl-(AspTrpLeuLysAlaPheTyrAspLysValPheGluLysPheLysGluPhePhe)-NH2; 5F], with increased amphipathicity, was administered by intraperitoneal injection, 20 microg/day for 16 weeks, to C57BL/6J mice fed an atherogenic diet. Mouse apo A-I (MoA-I) (50 microg/day) or phosphate-buffered saline (PBS) injections were given to other mice as controls. Total plasma cholesterol levels and lipoprotein profiles were not significantly different between the treated and control groups, except that the mice receiving 5F or MoA-I had lower high density lipoprotein (HDL) cholesterol when calculated as a percentage of total cholesterol. No toxicity or production of antibodies to the injected materials was observed. When HDL was isolated from high fat diet-administered mice injected with 5F and presented to human artery wall cells in vitro together with human low density lipoprotein (LDL), there were substantially fewer lipid hydroperoxides formed and substantially less LDL-induced monocyte chemotactic activity than with HDL from PBS-injected animals. Injection of human apo A-I produced effects similar to 5F on lipid peroxide formation and LDL-induced monocyte chemotactic activity, but injection of MoA-I was significantly less effective in reducing lipid hydroperoxide formation or lowering LDL-induced monocyte chemotactic activity. Mice receiving peptide 5F had significantly less aortic atherosclerotic lesion area compared with mice receiving PBS, whereas lesion area in mice receiving MoA-I was similar to that of the PBS-injected animals. This is the first in vivo demonstration that a model class A amphipathic helical peptide has antiatherosclerotic properties. We conclude that 5F inhibits lesion formation in high fat diet-administered mice by a mechanism that does not involve changes in the lipoprotein profile, and may have potential in the prevention and treatment of atherosclerosis.

Immediate total tooth replacement.

Successful implant placement at the time of extraction has been documented. Implant placement at the time of extraction was initially performed as a two-stage procedure often with barrier membranes and sophisticated second-stage surgical uncoverings. The authors describe the next generation of this technique, including atraumatic tooth removal with simultaneous root form, implant placement, and temporization at one appointment. This technique of "Immediate Total Tooth Replacement" allows for the maintenance of the bony housing and soft-tissue form that existed before extraction, while at the same time establishing a root form anchor in the bone for an esthetic restoration.

Persistent elevated expression of cytokine transcripts in ganglia latently infected with herpes simplex virus in the absence of ganglionic replication or reactivation.

Infection of mouse trigeminal ganglia by herpes simplex virus induces cytokine expression that persists long after infectious virus or viral antigens become undetectable. To examine mechanisms underlying this phenomenon, we used a thymidine kinase mutant, dlsptk, which fails to replicate in ganglia and does not reactivate upon ganglionic explant. Using quantitative reverse transcriptase-polymerase chain reaction assays, we found that levels of interferon-gamma and tumor necrosis factor-alpha transcripts in dlsptk-infected ganglia were lower than those in wild type-infected ganglia, but were significantly (eight- to 10-fold) higher than those in mock-infected ganglia from Day 3 to Day 100 postinfection. We also studied latency-associated transcript (LAT) negative mutants that exhibit increased expression of productive cycle transcripts in ganglia. Ganglia infected with these mutants contained levels of cytokine transcripts similar to those in wild type-infected ganglia; any increases in viral antigen expression mediated by the LAT deletion were not accompanied by increased cytokine expression. Thus, neither viral replication, the ability to reactivate, nor LAT expression in ganglia is required for persistent elevated cytokine expression. The results provide indirect evidence that low-level expression of viral productive cycle genes in neurons can provide signals that elicit cytokine expression.

A sensitive and convenient method for lipoprotein profile analysis of individual mouse plasma samples.

A simple and convenient method to determine plasma cholesterol profiles in individual mouse plasma samples is not presently available. With commonly used methods, plasma samples from several animals in a study group must often be pooled and analyzed, usually by the fast phase liquid chromatography (FPLC) method. The Column Lipoprotein Profile (or CLiP) method described here is a modification of the FPLC method that provides a simple and convenient procedure for determining plasma lipoprotein cholesterol profiles in small sample volumes, allowing determination of profiles from individual animals rather than from pooled plasma. The CLiP method is reproducible; a human sample measured five times over several days produced coefficients of variation as follows: VLDL, 10.0%; LDL, 0.93%; and HDL, 2.51%. CLiP-derived total cholesterol values of five different human samples (with total cholesterol levels ranging from 198 to 263 mg/dL) differed from VAP-II by -1.88% +/- 2.57%. Linearity of differing concentrations for each of the lipoprotein classes was determined by measuring the same sample with different aliquot sizes. The linear regression from VLDL had an r value of 0.996, while LDL, HDL, and total cholesterol all had r values of greater than 0.999. We present a direct comparison of plasma cholesterol profiles from several mouse models with gene modification or expression of transgenic proteins. In conclusion, the CLiP method provides a simple, reliable, and reproducible procedure for determination of plasma cholesterol profiles from individual plasma samples with very low sample volumes, using readily available equipment and reagents.

The receptor binding domain of apolipoprotein E, linked to a model class A amphipathic helix, enhances internalization and degradation of LDL by fibroblasts.

Human apolipoprotein E (apo E) consists of two distinct domains, the lipid-associating domain (residues 192-299) and the globular domain (residues 1-191) which contains the LDL receptor (LDLR) binding site (residues 129-169). To test the hypothesis that an arginine-rich apo E receptor binding domain (residues 141-150) is sufficient to enhance low-density lipoprotein (LDL) uptake and clearance when covalently linked to a class A amphipathic helix, a peptide in which the receptor binding domain of human apo E, LRKLRKRLLR (hApoE[141-150]), is linked to 18A, a well-characterized high-affinity lipid-associating peptide (DWLKAFYDKVAEKLKEAF), we synthesized the peptide hApoE[141-150]-18A (hE18A) and its end-protected analogue, Ac-hE18A-NH(2). The importance of positively charged residues and the role of the hydrophobic residues in the receptor binding domain were also studied using four analogues. Ac-LRRLRRRLLR-18A-NH(2) [Ac-hE(R)18A-NH(2)] and Ac-LRKMRKRLMR-18A-NH(2) (Ac-mE18A-NH(2)) contained an extended hydrophobic face, including the receptor binding region. Control peptides, Ac-LRLLRKLKRR-18A-NH(2) [Ac-hE(Sc)18A-NH(2)], had the amino acid residues of the apo E receptor binding domain scrambled to disrupt the extended hydrophobic face, and Ac-RRRRRRRRRR-18A-NH(2) (Ac-R(10)18A-NH(2)) had only positively charged Arg residues as the receptor binding domain. The effect of the dual-domain peptides on the uptake and degradation of human LDL by fibroblasts was determined in murine embryonic fibroblasts (MEF1). LDL internalization was enhanced 3-, 5-, and 7-fold by Ac-mE18A-NH(2), Ac-hE18A-NH(2), and Ac-hE(R)18A-NH(2), respectively, whereas the control peptides had no significant biological activity. All three active peptides increased the level of degradation of LDL by 100%. The LDL binding and internalization to MEF1 cells in the presence of these peptides was not saturable over the LDL concentration range that was studied (1-10 microgram/mL). Furthermore, a similar enhancement of LDL internalization was observed independent of the presence of the LDL receptor-related protein (LRP), LDLR, or both. Pretreatment of cells with heparinase and heparitinase abolished more than 80% of the enhanced peptide-mediated LDL uptake and degradation by cells. We conclude that the dual-domain peptides enhanced LDL uptake and degradation by fibroblasts via a heparan sulfate proteoglycan (HSPG)-mediated pathway.

Anterior esthetic considerations when splinting teeth.

There are many techniques for stabilizing and splinting teeth. No matter which restorative technique is chosen, the technical elements of marginal fit, psychologic contour, cleansibility, and occlusion must be met. When the restoration is in the esthetic zone of the oral cavity, there is an additional element of achieving an acceptable cosmetic result. This article presents an overview of concepts to achieve acceptable esthetic results when teeth are joined together.

Crown lengthening revisited.

Over the last 37 years, crown-lengthening procedures have been used predictably to restore teeth broken down from caries, trauma, and extensive wear. With crown lengthening, the dentogingival junction is "re-created" at a more apical level on the root to accommodate the junctional epithelium and the connective tissue attachment. Forced eruption can be used in addition, or as an alternative, to tooth lengthening. The authors discuss the indications for tooth lengthening, forced eruption, and orthodontic extrusion, as well as the treatment planning for these procedures.

Fifty years of interdisciplinary site development: lessons and guidelines from periodontal prosthesis.

Just as "osseointegration" became synonymous with successful restoration of function in the fully edentulous patient during the 1980s, the term "implant site development" has become intricately associated in the 1990s with the techniques used to achieve esthetic results with implants in the partially edentulous patient. This article explores the roots of the concept of site development within the philosophy and principles of periodontal prosthesis. In addition, the myriad of techniques that are presently collectively referred to as site development are systematically classified into a sequential four-tiered approach that optimizes their efficient application as well as overall success.

The interproximal height of bone: a guidepost to predictable aesthetic strategies and soft tissue contours in anterior tooth replacement.

Enhanced aesthetic objectives can be achieved with precision and predictability due to recent advances in restorative materials and procedures. Although these developments have expanded the therapeutic options available to practitioners and their patients, anterior hard and soft tissue deformities in the aesthetic zone continue to represent a significant technical challenge to the reconstructive team. The objective of this article is to present diagnostic and prognostic criteria that emphasize the osseous-gingival relationship as a means to achieve predictable aesthetic results in the anterior segment with conventional or implant-supported restorations.

Quantification of HDL2 and HDL3 cholesterol by the Vertical Auto Profile-II (VAP-II) methodology.

Of the several existing methods for quantification of major subspecies of high density lipoprotein (HDL), HDL2 and HDL3, the methods based upon double precipitation are particularly useful for large-scale studies or for routine assay because of their high speed and low cost. The Vertical Auto Profile-II (VAP-II) method developed in our laboratory primarily for the direct single test measurement of cholesterol (C) in all major lipoproteins, including Lp[a] and IDL, is rapid, highly sensitive, and suitable for large-scale studies. Here we describe the modification of this procedure so as to be able to quantify both HDL2- and HDL3-C in addition to all major lipoproteins without any additional assay steps, time, or cost. The VAP-II procedure was validated by comparison with four other methods using plasma samples obtained from 35 healthy subjects: 1) HDL-VAP-II (a variation of the VAP-II procedure designed specifically to separate HDL subspecies); 2) dextran sulfate (DS)/Mg2+ double precipitation method performed at Northwest Lipid Research Laboratories (NWLRL), Seattle, WA; 3) 4-30% polyacrylamide-agarose (4/30 PAA) nondenaturing gradient gel electrophoresis (GGE); and 4) analytical ultracentrifugation (AUC), with both GGE and AUC performed at the Donner Laboratory, University of California at Berkeley. Both HDL2- and HDL3-C measurements by VAP-II correlated well with the measurements by all comparison methods (r for HDL3-C: HDL-VAP-II, 0.948; NWLRL, 0.947; GGE, 0.861; and AUC, 0.706, and r for HDL2-C: HDL-VAP-II, 0.867; NWLRL, 0.854; GGE, 0.885; and AUC, 0.721). The measurements of HDL2- and HDL3-C by the VAP-II method are reproducible, with the long-term between-rotor CV of 5.0% for HDL3-C and 9.0% for HDL2-C.

A LAT-associated function reduces productive-cycle gene expression during acute infection of murine sensory neurons with herpes simplex virus type 1.

Herpes simplex virus (HSV) persists in the human population by establishing long-term latent infections followed by periodic reactivation and transmission. Latent infection of sensory neurons is characterized by repression of viral productive-cycle gene expression, with abundant transcription limited to a single locus that encodes the latency-associated transcripts (LATs). We have observed that LAT- deletion mutant viruses express viral productive-cycle genes in greater numbers of murine trigeminal ganglion neurons than LAT+ HSV type 1 at early times during acute infection but show reduced reactivation from latent infection. Thus, a viral function associated with the LAT region exerts an effect at an early stage of neuronal infection to reduce productive-cycle viral gene expression. These results provide the first evidence that the virus plays an active role in down-regulating productive infection during acute infection of sensory neurons. The effect of down-regulation of productive-cycle gene expression during acute infection may contribute to viral evasion from the host immune responses and to reduced cytopathic effects, thereby facilitating neuronal survival and the establishment of latency.

Securing the position of ceramic veneers in dentistry.

Porcelain laminate veneers have exhibited predictability and, as a result, experienced an incredibly rapid public acceptance with widespread utilization. The introduction of a stronger, leucite-reinforced ceramic system which could be accurately pressed (IPS Empress, Ivoclar Williams, Amherst, NY), presented new avenues for this remarkable treatment modality beyond aesthetic enhancement. Porcelain veneer restorations could be utilized to accurately alter and correct occlusal relationships, and the less abrasive surface of the material offered a distinct advantage in occlusal modifications, such as altering cuspal guidance or vertical dimension. This presentation reviews the history of porcelain veneer restoration and outlines the use of a luting cement (Variolink II, Ivoclar Vivadent, Amherst, NY) for its application.