Spatial Analysis of Almond Leaf Scorch Disease in the San Joaquin Valley of California: Factors Affecting Pathogen Distribution and Spread.

Almond leaf scorch (ALS) disease has emerged as a serious threat to almond (Prunus amygdalus) production areas throughout California's San Joaquin Valley. This disease is caused by the xylem-limited bacterium Xylella fastidiosa, and this pathogen is transmitted by xylophagous insects including sharpshooter leafhoppers (Hemiptera: Cicadellidae) and spittlebugs (Hemiptera: Cercopidae). Among four orchards surveyed, enzyme-linked immunosorbent assay (ELISA) and bacterial isolation followed by polymerase chain reaction (PCR) were equally effective in detecting X. fastidiosa from ALS-symptomatic trees. Disease incidence varied among almond cultivars in each orchard, with the highest mean incidence and most severe symptoms frequently encountered in 'Sonora'. X. fastidiosa isolates consisted of mixtures of grape or "G-genotype" and almond or "A-genotype" strains present in surveyed orchards. The X. fastidiosa G-genotypes characterized from each orchard were associated with the most severely affected 'Sonora' trees in three of the four orchards. Both ordinary runs and simple randomization analyses revealed aggregations of ALS in three of the four orchards. Clusters of ALSaffected trees frequently occurred in the outermost orchard rows. Plots of semivariance in ALS incidence over distance varied in shape and magnitude among cultivars. Semivariance increased over distance in 'Sonora' and 'Carmel', indicating spatial dependence or aggregations of incidence best fit by a combination of spherical and linear models. These results document both random and aggregate patterns of ALS spatial distribution in selected orchards and further illustrate how cultivar susceptibility influences the distribution patterns of ALS incidence. Following the recent introduction and establishment of the glassy-winged sharpshooter, Homalodisca coagulata, the impact upon the epidemiology and spread of ALS is unknown.

ABCA1 and amphipathic apolipoproteins form high-affinity molecular complexes required for cholesterol efflux.

Apolipoproteins, such as apolipoprotein A-I (apoA-I), can stimulate cholesterol efflux from cells expressing the ATP binding cassette transporter A1 (ABCA1). The nature of the molecular interaction between these cholesterol acceptors and ABCA1 is controversial, and models suggesting a direct protein-protein interaction or indirect association have been proposed. To explore this issue, we performed competition binding and chemical cross-linking assays using six amphipathic plasma proteins and an 18 amino acid amphipathic helical peptide. All seven proteins stimulated lipid efflux and inhibited the cross-linking of apoA-I to ABCA1. Cross-linking of apoA-I to ABCA1 was saturable and occurred at high affinity (Kd of 7.0 +/- 1.9 nM), as was cross-linking of apoA-II. After binding to ABCA1, apoA-I rapidly dissociated (half-life of 25 min) from the complex and was released back into the medium. A mutant form of ABCA1 (W590S) that avidly binds apoA-I but fails to promote cholesterol efflux released apoA-I with similar kinetics but without transfer of cholesterol to apoA-I. Thus, a high-affinity, saturable, protein-protein interaction occurs between ABCA1 and all of its amphipathic protein ligands. Dissociation of the complex leads to the cellular release of cholesterol and the apolipoprotein. However, dissociation is not dependent on cholesterol transfer, which is a clearly separable event, distinguishable by ABCA1 mutants.

Peroxisome proliferator-activated receptors in macrophage biology: friend or foe?

Peroxisome proliferator-activated receptor (PPAR)-gamma is a nuclear hormone receptor, with a well-established role in adipogenesis and glucose metabolism. Over the past 3 years several laboratories have reported that this protein can influence macrophage responses to a variety of inflammatory stimuli. The effect of PPAR-gamma activation on macrophage lipid uptake, cholesterol efflux, and cytokine production have all recently been examined in several in-vitro culture systems. In addition, PPAR-gamma ligands have been shown to influence atherosclerotic lesion formation in murine models of that disease. This review attempts to summarize and critically evaluate that work and its implications for the use of PPAR-gamma activators in understanding and treating the pathogenetic processes that contribute to atherosclerotic plaque formation.

Evidence that dendritic cells infiltrate atherosclerotic lesions in apolipoprotein E-deficient mice.

Earlier we reported that atherosclerotic lesions of apoE-deficient mice contained cells which stained positively with anti-S-100 antibody and that cells exhibiting the ultrastructural features of dendritic cells were present in the aortic lesions. These observations suggested that dendritic cells might be involved in mouse atherosclerosis. By employing DEC-205 and MIDC-8 antibodies specific for dendritic cells, the present study has established that dendritic cells indeed accumulate in atherosclerotic lesions of apoE-deficient mice. Finding dendritic cells infiltrating atherosclerotic lesions in apoE-deficient mice offers the possibility of investigating the migratory routes of dendritic cells and their involvement in T-cell activation.

ATP-binding cassette transporter A1 contains an NH2-terminal signal anchor sequence that translocates the protein's first hydrophilic domain to the exoplasmic space.

Mutations in the ATP-binding cassette transporter A1 (ABCA1) transporter are associated with Tangier disease and a defect in cellular cholesterol efflux. The amino terminus of the ABCA1 transporter has two putative in-frame translation initiation sites, 60 amino acids apart. A cluster of hydrophobic amino acids form a potentially cleavable signal sequence in this 60-residue extension. We investigated the functional role of this extension and found that it was required for stable protein expression of transporter constructs containing any downstream transmembrane domains. The extension directed transporter translocation across the ER membrane with an orientation that resulted in glycosylation of amino acids immediately distal to the signal sequence. Neither the native signal sequence nor a green fluorescent protein tag, fused at the amino terminus, was cleaved from ABCA1. The green fluorescent protein fusion protein had efflux activity comparable with wild type ABCA1 and demonstrated a predominantly plasma membrane distribution in transfected cells. These data establish a requirement for the upstream 60 amino acids of ABCA1. This region contains an uncleaved signal anchor sequence that positions the amino terminus in a type II orientation leading to the extracellular presentation of an approximately 600-amino acid loop in which loss-of-function mutations cluster in Tangier disease patients.

The role of PPAR-gamma in macrophage differentiation and cholesterol uptake.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma), the transcription factor target of the anti-diabetic thiazolidinedione (TZD) drugs, is reported to mediate macrophage differentiation and inflammatory responses. Using PPAR-gamma-deficient stem cells, we demonstrate that PPAR-gamma is neither essential for myeloid development, nor for such mature macrophage functions as phagocytosis and inflammatory cytokine production. PPAR-gamma is required for basal expression of CD36, but not for expression of the other major scavenger receptor responsible for uptake of modified lipoproteins, SR-A. In wild-type macrophages, TZD treatment divergently regulated CD36 and class A macrophage-scavenger receptor expression and failed to induce significant cellular cholesterol accumulation, indicating that TZDs may not exacerbate macrophage foam-cell formation.

Effects of overexpression of the amino-terminal fragment of apolipoprotein B on apolipoprotein B and lipoprotein production.

In vitro studies have shown that the binding site for microsomal triglyceride transfer protein (MTP) is within the first 17% of apoB (apoB-17). Expression of apoB-48 in McArdle cells decreases endogenous lipoprotein production; however, overexpression of human apoB in transgenic mice does not decrease endogenous mouse apoB expression. To assess this inconsistency, adenoviruses expressing human apoB-17 (AdB17) or apoB-17-beta (which contains apoB-17 plus a small lipid-binding beta-sheet region of apoB, AdB-17beta) were produced. Hepatoma cells were infected with AdB17 or AdB17-beta with AdLacZ, an adenovirus expressing beta-galactosidase, as a control. Overexpression of apoB-17 and apoB-17-beta in hepatoma cells to levels 2- to 3-fold greater than that of endogenous apoB did not alter endogenous apoB production. This was also true in the presence of oleic acid and N-acetyl-leucyl-leucyl-norleucinal. High levels of apoB-17 or beta-galactosidase expression reduced apoB-100 production; however, control protein production was also reduced. To assess the effects of apoB-17 expression in vivo, mice of three different strains were injected with AdB17. Two days after injection, plasma apoB-17 was approximately 24 times the amount of endogenous apoB in the C57BL/6 mice, 2 times the apoB-100 in human apoB transgenic mice, and 4 times the apoB-48 in apoE knockout mice. Overexpression of apoB-17 did not decrease apoB-100 or apoB-48 concentrations in mouse plasma as assessed by Western blot analysis. These results demonstrate that although the apoB-17 binds to MTP in vitro, it does not alter endogenous apoB expression in mice or in hepatoma cells.

Episomal segregation of the adenovirus enhancer sequence by conditional genome rearrangement abrogates late viral gene expression.

We have constructed a recombinant adenovirus gene delivery system that is capable of undergoing growth phase-dependent site-specific recombination. When propagated in 293 producer cells, the vector retains its linear double-stranded form and can be propagated to high titer and purified by conventional procedures. Upon introduction into target cells, the viral chromosome undergoes cyclization to generate an autonomously replicating circular episome and a detached linear fragment. The viral enhancer and reporter gene segregate with the circular episome, which contains no adenovirus open reading frames. The effect of rearrangement of adenovirus gene expression was assessed by quantitative reverse transcription-PCR measurement of the abundance of transcripts encoding the tripartite leader sequence (TPL) of the major late promoter. Whereas nonrearranging viruses produced approximately 10(4) TPL transcripts per 10(6) infecting genomes in the HepG2 liver cell line, no transcripts were detectable in the same cells infected with comparable levels of circularizing vector. Because no helper virus is required to propagate these vectors, the problems of recombination with and contamination by helper virus are eliminated. We also present an efficient and reliable method for generating recombinant adenoviruses.

Divergent response to LPS and bacteria in CD14-deficient murine macrophages.

Gram-negative bacteria and the LPS constituent of their outer membranes stimulate the release of inflammatory mediators believed to be responsible for the clinical manifestations of septic shock. The GPI-linked membrane protein, CD14, initiates the signaling cascade responsible for the induction of this inflammatory response by LPS. In this paper, we report the generation and characterization of CD14-null mice in which the entire coding region of CD14 was deleted. As expected, LPS failed to elicit TNF-alpha and IL-6 production in macrophages taken from these animals, and this loss in responsiveness is associated with impaired activation of both the NF-kappaB and the c-Jun N-terminal mitogen-activated protein kinase pathways. The binding and uptake of heat-killed Escherichia coli, measured by FACS analysis, did not differ between CD14-null and wild-type macrophages. However, in contrast to the findings with LPS, whole E. coli stimulated similar levels of TNF-alpha release from CD14-null and wild-type macrophages at a dose of 10 bioparticles per cell. This effect was dose dependent, and at lower bacterial concentrations CD14-deficient macrophages produced significantly less TNF-alpha than wild type. Approximately half of this CD14-independent response appeared to be mediated by CD11b/CD18, as demonstrated by receptor blockade using neutrophil inhibitory factor. An inhibitor of phagocytosis, cytochalasin B, abrogated the induction of TNF-alpha in CD14-deficient macrophages by E. coli. These data indicate that CD14 is essential for macrophage responses to free LPS, whereas other receptors, including CD11b/CD18, can compensate for the loss of CD14 in response to whole bacteria.

Cellular cholesterol efflux in heterozygotes for tangier disease is markedly reduced and correlates with high density lipoprotein cholesterol concentration and particle size.

Tangier disease (TD), caused by mutations in the ATP-binding cassette 1 (ABC-1) gene, is a rare genetic disorder characterized by severe deficiency of high density lipoproteins (HDL) in the plasma, hypercatabolism of HDL, and defective apolipoprotein (apo)-mediated cellular cholesterol efflux. In the present study, we assessed plasma lipid concentrations, HDL particle size and subspecies, and cellular cholesterol efflux in 9 TD heterozygotes from a kindred in which the proband was homozygous for an A-->C missense mutation at nucleotide 5338 of the ABC-1 transcript. Relative to age- and gender-matched controls from the Framingham Offspring Study (FOS), TD heterozygotes had significant reductions (P < 0.000) in HDL-C (-54% female; -40% male) and apoA-I (-33% female; -37% male) concentrations, as well as significantly less cholesterol (-68% female; -58% male) distributed in the largest HDL subclasses, H5 and H4. Consequently, HDL particle size (nm) was significantly smaller (P < 0.000) in TD heterozygotes (8.6 +/- 0.6 female; 8.7 +/- 0.1 male) relative to FOS controls (9.4 +/- 0.4 female; 9.0 +/- 0.3 male). Further studies demonstrated that apoA-I-mediated cellular cholesterol efflux in TD heterozygotes was essentially half that of controls (11 +/- 2 vs. 20 +/- 3% of total [(3)H]cholesterol, P < 0. 001), with strong correlations observed between cholesterol efflux and both HDL-C level (r = 0.600) and particle size (r = 0.680). In summary, our data demonstrate that apolipoprotein-mediated cholesterol efflux is aberrant in TD heterozygotes, as it is in homozygotes. This finding, along with the associations observed between HDL-C concentration, HDL particle size, and cholesterol efflux, supports the concept that plasma HDL-C levels are regulated, in part, by cholesterol efflux, which in turn influences HDL particle size and, ultimately, HDL apoA-I catabolism.

Novel mutations in the gene encoding ATP-binding cassette 1 in four tangier disease kindreds.

Tangier disease (TD) is an autosomal co-dominant disorder in which homozygotes have a marked deficiency of high density lipoprotein (HDL) cholesterol and, in some cases, peripheral neuropathy and premature coronary heart disease (CHD). Homozygotes are further characterized by cholesteryl ester deposition in various tissues throughout the body, most notably in those of the reticuloendothelial system. Several studies have demonstrated that the excess lipid deposition in TD is due to defective apolipoprotein-mediated efflux of cellular cholesterol and phospholipids. Although much progress has been made in our understanding of the metabolic basis of TD, the precise molecular defect had remained elusive until very recently. By positional cloning methods, we: 1) confirm the assignment of TD to chromosome 9q31, 2) provide evidence that human ATP-binding cassette-1 (hABC-1) maps to a 250 kb region on 9q31, and 3) describe novel deletion, insertion, and missense mutations in the gene encoding hABC-1 in four unrelated TD kindreds. These results establish a causal role for mutations in hABC-1 in TD and indicate that this transporter has a critical function in the regulation of intracellular lipid trafficking that dramatically affects plasma HDL cholesterol levels.

Lipopolysaccharide induces scavenger receptor A expression in mouse macrophages: a divergent response relative to human THP-1 monocyte/macrophages.

Gene deletion studies indicate that the macrophage scavenger receptor A (SR-A) protects mice from LPS-induced endotoxemia. Paradoxically, cultured human monocyte-derived macrophages down-regulate SR-A expression when exposed to LPS. We found that human THP-1 monocyte/macrophages decrease SR-A expression in response to LPS independent of their differentiation status. In contrast, primary and elicited mouse peritoneal macrophages as well as the J774A.1 and RAW264.7 mouse macrophage lines increase SR-A expression in response to LPS. Exposure to LPS caused J774A.1 and RAW264.7 cells to increase SR-A transcripts by 3- and 5-fold, respectively. LPS caused a concomitant 3-fold increase in SR-A protein levels and increased cell membrane expression of the receptor. RAW264.7 cells increased SR-A transcript levels in response to LPS at concentrations as low as 1 ng/ml, and the response was saturated at 10 ng/ml. The LPS induction of SR-A transcripts required continual protein synthesis and began at 8 h, peaked by 16 h, and persisted for at least 48 h. LPS induction did not increase SR-A gene transcription or affect alternative transcript splicing, but mildly increased mature transcript stability and proceeded in the presence of actinomycin D. Finally, treatment of RAW264.7 cells with TNF-alpha did not induce SR-A transcript levels, indicating that a TNF-alpha autocrine/paracrine signaling mechanism alone is not sufficient to recapitulate the LPS induction of SR-A transcripts. The induction of SR-A expression by LPS-stimulated mouse macrophages is the opposite of the down-regulation of SR-A reported in human monocyte-derived macrophages and may have implications for the observed resistance mice show toward endotoxemia.

Stromelysin-1 (MMP-3) expression driven by a macrophage-specific promoter results in reduced viability in transgenic mice.

Macrophage expression of matrix degrading metalloproteinases (MMPs) in human atheroma has been found to occur in rupture-prone areas of plaques. To investigate the effect of metalloproteinase activity on plaque stability, we attempted to generate mice that expressed a stromelysin-1 (MMP-3) transgene specifically in macrophages. Promoter sequences taken from a macrophage-tropic lentivirus (visna) were used to drive transgene expression. The transgene construct was expressed in macrophages in vitro and its autoactivation was established by casein zymography. Transgenic mice generated with this construct died at or before birth. No gross anatomical changes were observed in these mice. Embryos arising from a second round of oocyte injections with the transgene were examined at day 16 of gestation. Of the products of conception, approximately 40% resulted in vacant conceptuses. Only one animal of 38 examined carried the transgene and its expression of MMP-3 mRNA at E16 was faintly detected by RT-PCR. When a non-toxic reporter gene, luciferase, was substituted for the MMP-3 cDNA, healthy transgenic mice were produced that expressed the reporter gene in a wide variety of tissue macrophages, including those located in the brain, testis, lung, and thymus. These studies suggest that constitutive expression of MMP-3 in diverse populations of tissue macrophages leads to prenatal or neonatal death in the mouse. It appears likely that more sophisticated transcriptional control of MMP-3 expression will be required in order to generate stromelysin-1 transgenic mice that could be useful models for studying overexpression of this metalloproteinase's activity in the lesional macrophages of atherosclerotic plaques.

Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus.

The innate immune system contributes to the earliest phase of the host defense against foreign organisms and has both soluble and cellular pattern recognition receptors for microbial products. Two important members of this receptor group, CD14 and the Toll-like receptor (TLR) pattern recognition receptors, are essential for the innate immune response to components of Gram-negative and Gram-positive bacteria, mycobacteria, spirochetes and yeast. We now find that these receptors function in an antiviral response as well. The innate immune response to the fusion protein of an important respiratory pathogen of humans, respiratory syncytial virus (RSV), was mediated by TLR4 and CD14. RSV persisted longer in the lungs of infected TLR4-deficient mice compared to normal mice. Thus, a common receptor activation pathway can initiate innate immune responses to both bacterial and viral pathogens.

Leukotriene B4 receptor transgenic mice reveal novel protective roles for lipoxins and aspirin-triggered lipoxins in reperfusion.

Polymorphonuclear neutrophil (PMN) activation is pivotal in acute inflammation and injury from reperfusion. To elucidate components controlling PMNs in vivo, we prepared novel transgenic mice with the human leukotriene (LT) B4 receptor (BLTR) for functional characterization. Overexpression of BLTR in leukocytes dramatically increased PMN trafficking to skin microabscesses and lungs after ischemia-reperfusion, whereas mice deficient in 5-lipoxygenase (5-LO) showed diminished PMN accumulation in reperfused lungs. Hence, both BLTR expression and LT biosynthesis are critical for PMN infiltration in reperfusion-initiated second-organ injury. Also, in BLTR transgenic mice, 5-LO expression and product formation were selectively increased in exudates, demonstrating that receptor overexpression amplifies proinflammatory circuits. Endogenous lipoxin (LX) A4 was produced in ischemic lungs and elevated by reperfusion. Because LXA4 and aspirin-triggered 15-epimeric LXA4 (ATL) selectively regulate leukocyte responses, they were tested in BLTR transgenic mice. Despite excessive PMN recruitment in BLTR transgenic mice, intravenous injection of ATL sharply diminished reperfusion-initiated PMN trafficking to remote organs, and topical application of LX was protective in acute dermal inflammation. These results demonstrate a direct role for BLTR with positive feedback, involving BLTR and 5-LO signaling in controlling PMNs. Moreover, LXA4 and ATL counter BLTR-amplified networks, revealing a novel protective role for LX and ATL in stress responses that has applications in perioperative medicine.

Embryonal Stem (ES) Cell-Derived Macrophages : A Cellular System that Facilitates the Genetic Dissection of Macrophage Function.

The monocyte/macrophage (Mø) contributes to atherosclerotic lesion initiation and progression through a variety of interactions with cells of the artery wall that depend on the elucidation of a host of cytokines and growth factors by cells residing in the intima. The number and complexity of these interactions make it difficult to determine which cellular functions are contributing to the progression of atherosclerosis and which might be exploited to interrupt that progression. Studies of macrophage functions in atherosclerosis have been hindered by the limitations of available macrophage cell lines and primary cultures, including poor transfectability and the transformed state of imMortal cell lines. Recent studies have demonstrated that pluripotential mouse embryonic stem cells can be differentiated down specific hematopoietic lineages in vitro, including lines that give rise to macrophages (1). This technique provides a genetically tractable cellular system for studying myeloid cell function. Macrophages arising from this differentiation system demonstrate cell surface presentation of classic macrophage markers and macrophage functions including phagocytosis and responses to inflammatory stimuli. There are several important advantages inherent in using embryonic stem (ES) cell derived macrophages as a cell culture system for studying Mø function. As the cells are not transformed, and the progenitor cells arising from ES cells are capable of reconstituting the entire hematopoietic compartment of a mouse, they represent a cell culture system that appears to retain the physiologic regulation on growth and differentiation that is absent from transformed myelomonocytic cell lines.

In vitro-differentiated embryonic stem cell macrophages: a model system for studying atherosclerosis-associated macrophage functions.

Monocytes/macrophages (Mo) appear to play a critical role in the initiation and progression of atherosclerotic lesions. In this study, we characterized in vitro-differentiated embryonic stem (ES) cell macrophages as a model system for studying atherosclerosis-associated Mo functions. Using immunofluorescence staining and Western analysis, we demonstrate that ES Mo express typical macrophage cell surface markers, as well as the known receptors for modified forms of low density lipoprotein (LDL), including the Mo scavenger receptors (SR-A type I and type II), CD36, and CD68. Differentiated ES Mo specifically bind and degrade 125I-labeled acetylated LDL with high affinity, and their incubation with acetylated LDL (15 microg/mL) for 48 hours produces characteristic "foamy" Mo, as visualized by oil red O staining. ES Mo also express matrix-degrading metalloproteinases (MMP-3, MMP-9), which have been implicated in collagen breakdown in the fibrous cap of atherosclerotic plaques, and secrete cytokines (tumor necrosis factor-alpha, interleukin-6) in response to inflammatory stimuli. Transfection experiments, using a green fluorescent protein reporter gene, driven by the myeloid-specific promoter, CD11b, demonstrated that ES Mo can also be used to study macrophage-restricted gene expression in vitro. Taken together, these data demonstrate that ES Mo exhibit many properties typical of arterial lesion macrophages. Its ease of genetic manipulation makes it an attractive system for investigations of macrophage functions in vitro.

Functional changes in scavenger receptor binding conformation are induced by charge mutants spanning the entire collagen domain.

Macrophage scavenger receptors are trimeric integral membrane proteins that bind a diverse array of negatively charged ligands. They have been shown to play a role in the pathogenesis of atherosclerosis and in host responses to microbial infections. Earlier mutational studies demonstrated that the distal segment of the collagen domain of the receptor was critically important for high affinity ligand binding activity. In this study, mutations spanning the entire collagen domain were generated and binding was assayed in transfected cells, as well as in assays employing a secreted, receptor fusion protein. Many of the distal, positively charged C-terminal residues in the type II collagen domain of the receptor, previously reported to be essential for binding at 37 degreesC, were found not to be critical for binding at 4 degreesC. Conversely, more proximally charged residues of the collagen receptor that have not been previously mutated were shown to have substantial effects on binding that were also temperature-dependent. These data suggest that scavenger receptor ligand recognition depends on more complex conformational interactions, involving charged residues throughout the entire collagen domain, than was previously recognized.